QUEENSLAND UNIVERSITY OF TECHNOLOGY...Submitted by: Karthika Prasad Master of Science (Nanoscience) Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy

QUEENSLAND UNIVERSITY OF TECHNOLOGY

NANOCARBON POLYMER COMPOSITE

FOR BREAST IMPLANTS

Submitted by: Karthika Prasad

Master of Science (Nanoscience)

Submitted in fulfilment of the requirements for the degree of

Doctor of Philosophy

School of Chemistry, Physics and Mechanical Engineering

Science and Engineering Faculty

Queensland University of Technology

2019

Nanocarbon polymer composite for breast implants Page i

KEYWORDS

Antibacterial, Bacterial infection, Breast augmentation, Breast cancer, Biocompatibility,

Carbon nanostructures, Graphene, Mastectomy, Nanoparticles, Nanowalls,

Nanodiamonds, Plasma-enabled synthesis, Silicone breast implants, Tissue-implant

interaction, Tensile test, Tear Test.

Nanocarbon polymer composite for breast implants Page ii

ABSTRACT

An increase in the incidence of mastectomy and a concomitant rise in the demand for breast

augmentation has resulted in the tremendous growth in the number of patients in receipt of

breast implants. Typical breast implant designs include silicone, saline and gummy bear

implant configurations, each optimised to deliver a specific set of qualities. Long-term use of

implants presents a range of potential issues, from implant rupture and leakage to development

of chronic infection and capsular contracture. As such, most silicone breast implants will

necessitate surgical removal at some stage, and strategies to extend their useful life are being

subject of intense investigation within the industry.

Nanomaterials have been explored extensively in the field of medicine, particularly in the areas

of targeted drug delivery, imaging, sensing and artificial implants. In parallel, they are being

actively researched as a means to improve material, e.g. bulk mechanical strength, and surface,

e.g. biocompatibility, properties of composites.

The proposed work aimed to explore the possibility of using the unique properties of

nanoscale carbon materials to enhance mechanical strength and cell-surface compatibility of

breast implants by creating novel nanocarbon-reinforced silicone composites that have

similar flexibility yet improved safety profile when compared to currently available implants.

To realise this aim, the performance of different types of nanocarbons in limiting the failure

of nanocarbon-reinforced silicone composites was investigated. The effect of synthesis

method and specific properties of nanomaterials on their mechanical and biological

performances was studied to select the most promising candidates for the composite. The

synthesis and performance of silicone composites reinforced with vertical graphene flakes

and nanodiamond were then studied, showing a significant improvement in mechanical

strength, tear resistance and biocompatibility.

Nanocarbon polymer composite for breast implants Page iii

TABLE OF CONTENTS

Contents

Keywords .................................................................................................................................. i

Abstract .................................................................................................................................... ii

Table of Contents .................................................................................................................... iii

List of Figures ......................................................................................................................... vi

List of Tables ......................................................................................................................... xii

List of Abbreviations ............................................................................................................ xiii

List of Publication ................................................................................................................. xiv

Statement of Original Authorship ........................................................................................ xvii

Acknowledgments ............................................................................................................... xviii

1 CHAPTER 1: INTRODUCTION ..................................................................................... 1

1.1 Background .....................................................................................................................1

1.2 Context ............................................................................................................................2

1.3 Purposes ..........................................................................................................................3

1.4 Significance ....................................................................................................................4

1.5 Thesis Outline .................................................................................................................5

1.6 Reference ........................................................................................................................8

2 CHAPTER 2: LITERATURE REVIEW ......................................................................... 9

2.1 Introduction ..................................................................................................................14

2.2 Brief History of Materials for Breast Augmentation ....................................................17

2.3 Implant-Associated Issues ............................................................................................21

2.3.1 Implant-related infections ...................................................................................22

2.3.2 Capsular Contracture ..........................................................................................25

2.3.3 Implant Rupture ..................................................................................................28

2.3.4 Breast Implant–Associated Cancers ...................................................................32

2.4 Nanotechnology in breast implants...............................................................................34

2.4.1 Nanomaterials to control infection .....................................................................38

2.4.2 Nano-patterned surface to control capsular contracture .....................................43

2.4.3 Nanomaterials for reinforcement ........................................................................45

2.4.4 Nanomaterials for radiation therapy enhancement .............................................51

2.4.5 Wound healing promoted by nanomaterials .......................................................55

2.4.6 Nanomaterials for drug delivery .........................................................................57

2.5 Current trends in breast augmentation ..........................................................................64

2.6 Conclusion and perspectives .........................................................................................68

2.7 Reference ......................................................................................................................70

3 CHAPTER 3: RESEARCH DESIGN ............................................................................ 92

3.1 Methodology .................................................................................................................93

Nanocarbon polymer composite for breast implants Page iv

3.2 Instruments ...................................................................................................................93

3.3 Procedure and Timeline ................................................................................................94

4 CHAPTER 4: RESEARCH FINDINGS ........................................................................ 97

4.1 Introduction ................................................................................................................101

4.2 Results ........................................................................................................................102

4.3 Discussion ...................................................................................................................109

4.4 Conclusion ..................................................................................................................115

4.5 Methods ......................................................................................................................116

4.6 Reference ....................................................................................................................119

5 CHAPTER 5: RESEARCH FINDINGS ...................................................................... 126

5.1 Introduction ................................................................................................................130

5.2 Results ........................................................................................................................131

5.2.1 Structural and Morphological Characterization of Nanomaterials ...................131

5.2.2 Antibacterial Studies ........................................................................................134

5.3 Discussion ...................................................................................................................137

5.4 Materials and Methods ...............................................................................................141

5.5 Conclusions ................................................................................................................143

5.6 References ..................................................................................................................145

6 CHAPTER 6: RESEARCH FINDINGS ....................................................................... 149

6.1 Introduction ................................................................................................................153

6.2 Materials and methods ................................................................................................155

6.2.1 CAP device and plasma treatment approach ....................................................155

6.2.2 Microorganism and culture conditions .............................................................155

6.2.3 Nanodiamond ...................................................................................................156

6.2.4 Experimental procedure....................................................................................156

6.2.5 Cell growth and cell viability ...........................................................................156

6.2.6 Scanning Electron Microscopy (SEM) and Helium Ion Microscopy (HIM) ...156

6.3 Results and discussion ................................................................................................157

6.3.1 Effect of CAP Treatment on cell morphology..................................................157

6.3.2 Effect of CAP Treatment and different concentrations of NDs on cell

growth ...............................................................................................................160

6.3.3 Effect of CAP treatment and different concentrations of NDs on cell

viability .............................................................................................................163

6.3.4 Effect of CAP treatment and different concentrations of NDs on cellular

uptake ...............................................................................................................165

6.4 Conclusion ..................................................................................................................166

6.5 References ..................................................................................................................168

7 CHAPTER 7: RESEARCH FINDINGS ....................................................................... 172

7.1 Introduction ................................................................................................................176

7.2 Experimental Section ..................................................................................................177

7.2.1 Materials ...........................................................................................................177

7.2.2 Synthesis of Nanocarbon composite ................................................................177

7.2.3 Tensile test ........................................................................................................178

Nanocarbon polymer composite for breast implants Page v

7.2.4 Tear test ............................................................................................................178

7.2.5 Nanoindendation test ........................................................................................178

7.2.6 Bacterial assessment .........................................................................................178

7.2.7 Biocompatibility tests .......................................................................................180

7.3 Results ........................................................................................................................181

7.4 Discussions .................................................................................................................186

7.4.1 Mechanical Reinforcement ...............................................................................187

7.4.2 Bacteria-surface interactions ............................................................................189

7.4.3 Biocompatibility studies ...................................................................................192

7.5 Conclusion ..................................................................................................................193

7.6 Reference ....................................................................................................................195

CHAPTER 8: CONCLUSIONS AND FUTURE RECOMMENDATION .................. 199

Nanocarbon polymer composite for breast implants Page vi

LIST OF FIGURES

CHAPTER 2

Figure 1. (a) Estimated age-specific incidence and mortality rates for breast cancer, by

sex, 2017. Reproduced with permission from ref [3] Copyright @ Australian Institute of

Health and Welfare (b) Estimated top elective surgeries in America in the year 2016, breast

augmentation is the second leading type of an invasive elective surgery. Reproduced with

permission from ref [4]. Copyright @ American Society for Aesthetic Plastic

Surgery………………………………………………………………………………..14

Figure 2. The evolution of breast reconstruction. (a)The breast augmentation started

in 3000BC. with alteration of clothing in 3000 BC, followed by using petroleum jelly,

paraffin wax, olive oil and fat and tissue grafting in 1930s [40-42]. (b)The evolution

of silicone implants started in 1950 and is still in use. (c) Additive tissue manufacturing

is the current trend in breast reconstruction. Reproduced with permission from ref

[43]…………………………………………………………………………………..19

Figure 3. The surface of a typical silicone breast implant presents a suitable ground

for bacterial growth and biofilm formation (a) [72], often leading to an acute infection

that significantly damages breast tissues (b) (c) [73]. Biofilm on a textured implant,

compared with biofilm on a smooth implant (d) (e)

[74]…………………………………………………………………………. ………23

Figure 4. Various type of bacterial species found on breast implant infections.

Reproduced with permission from reference [81] Copyright @1201-9712/ 2015 The

Authors. Published by Elsevier Ltd on behalf of International Society for Infectious

Diseases…………………………………………………………………………….24

Figure 5. Proposed mechanism of capsular contracture resulting from bacterial

contamination of implant surface……………………………………………………25

Figure 6. Capsular contracture in breast implants. (a) Implants covered with a capsule

[96]. (b) A capsule removed from the implant [97]. (c) The layered structure of the

capsule. Reproduced with permission from [96, 97]……………..…………………26

Figure 7. (a) The incidence of capsular contracture in recipients of polyurethane foam-

covered implants [103]. Examination of explanted implants shows a correlation

between degradation of polyurethane coating and capsular contracture. Beyond nine

years after implantation, where no coating was present in any patient, substantial

increase in incidence and severity of contracture is observed. Inset: Reproduced with

permission from reference [103]. Copyright © 2005 Springer Science+Business

Nanocarbon polymer composite for breast implants Page vii

Media, Inc. Thick implant capsule retrieved from a patient with severe cellular

inflammation after 156 months (b) Bleeding of silicone gel within the implant capsule

after 122 months of implantation (c) Reproduced with permission from reference

[104]……………………………………………………………………. 27

Figure 8. (a) Estimated number of PIP implant ruptures as reported to the TGA,

Australia by year of implantation and % rupture rate by year of implantation as a

fraction of implants sold in that year [114]. (b-c) MRI scans of a broken implant. Green

and orange arrows indicate sites of rupture in silicone breast implants [115].

Reproduced with permission from ref. [115]. Copyright © 2016 Lubbock Avalanche-

Journal. (d-f) Rupture of implants resulting from capsular contracture

[116]…………………………………………………………………………………29

Figure 9. A case of ALCL where the patient underwent left-sided capsulotomy with

excision of a firm capsular structure containing a seroma (right) [132]. A close up of

bacteria formation on implant (left). Reproduced with permission from Ref. 129

Copyright © 2015 Elsevier publication and ABC news net

[133]…………………………………………………………………………………32

Figure 10. Possible antibacterial mechanisms of nanomaterials. Nanomaterials

produce free radicals, e.g. reactive oxygen species (ROS) which induce oxidative

stress and irreversibly damage bacteria (e.g., their membrane, DNA, and

mitochondria), potentially leading to cell death. Reproduced with permission from

reference [168]………………………………………………………………………39

Figure 11. TEM images of E. coli and S. aureus (a) Untreated E. coli. (b) Catechin-

Cu nanoparticles (20 ppm) treated E. coli. Arrows denote rupture of cells (c) Untreated

S. aureus. (d) Catechin-Cu nanoparticles (10 ppm) treated S. aureus. Arrows denote

cell rupture. Reproduced with permission from [181] Copyright © 2015, Macmillan

Publishers Limited………………………………………………………………….41

Figure 12. The mechanism of bacterial attachment to the implant surface. Reproduced

with permission from ref [196]. Copyright © 1996-2016 MDPI AG (Basel,

Switzerland)…………………………………………………………………………45

Figure 13. (a) Atomistic model of a CNT; (b) SEM images of CNT film [227]; (c)

Photographs of a CNT solid (10 × 10 × 4 mm3) compressed by a load of 800 N (2 MPa)

with its thickness reduced by 30%, and recovered to original shape after compression

[228]. (d) TEM image of multiwalled carbon nanotube. (e)Multiwall carbon nanotube

just prior to (above) and after (below) tensile testing

[229]……………………………………………………………………………….. 49

Figure 14. Mechanism of crack propagation in epoxy matrix reinforced by graphene

flakes; (a) graphene agglomeration; (b) homogeneously-dispersed graphene.

Reproduced with permission from ref [235]………………………………………..50

Nanocarbon polymer composite for breast implants Page viii

Figure 15. Interaction of X-rays with high Z material nanoparticle. Reproduced with

permission from reference [238]. Copyright ©2009-2016Translational Cancer

Research……………………………………………………………………………..54

Figure 16. Schematic illustration of graphene oxide -wrapped Dox-loaded

mesophorous silicon nanoparticle bound with Cy5.5-labeled AS1411 aptamer and the

corresponding NIR light controlled intracellular drug release. Reproduced with

permission from ref [253]…………………………………………………………..58

Figure 17. Breast reconstruction achieved by sustained regeneration of high-volume

adipose tissue. (a) Laser scanning is used on the patient prior to mastectomy to generate

a 3D model of the beast. (b) A porous patient-specific scaffold is then created using

3D printing of poly (D,L)-lactide polymer, displaying good homogeneity of filament

thickness (B, inset) and 90% porosity across the volume of the implant (c). (d)

Fluorescence imaging of the scaffold construct in mice shows substantial increase in

adipose tissue over time. (e) and (f) Top and side views are of scaffolds as-fabricated

(left) and after 24 weeks of implantation. Explanted scaffolds show the development

of well-vascularised adipose tissue. Reproduced with permission from reference

[270]……………………………………………………………………................. 65

CHAPTER 3

Figure 1. Research Methodology………………………………………………... 93

CHAPTER 4

Figure 1. SEM images of synthesized nanomaterials. (a) rGO nanosheets with a large

number of reactive edges, (b) nAg nanoparticles of uniform size and near spherical

shape, (c) rGO−nAg composite showing uniform distribution of

nAg…………………………………………………………………………. . …….103

Figure 2. Representative HRTEM images of (a) rGO−nAg nanocomposite, (b) lattice

resolved image of nAg in rGO−nAg nanocomposite. (c) Size distribution histogram of

nAg in rGO-nAg nanocomposite presented in (a)………...………………………..103

Figure 3. Reduction of GO to rGO and subsequent incorporation of nAg was

confirmed spectroscopically: (a) FTIR spectra for GO and rGO; (b) XRD of rGO and

rGO−nAg composite; (c) UV spectrum of nAg; (d) UV−Vis spectra for rGO and

rGO−nAg composite……………………………………………………………....105

Figure 4. Well diffusion study. Representative plates of (a) P. mirabilis, (b) S. aureus,

and (c) E. coli. Red circles indicate the zone of inhibition from wells loaded with

nitrofurantoin; yellow circles indicate the zone of inhibition from wells loaded with

rGO−nAg…………………………………………………………………….……106

Figure 5. Viable count of bacteria after exposure to (a) rGO, (b) nAg, (c) rGO−nAg

composite, and (d) standard antibiotic nitrofurantoin…………………...…………108

Nanocarbon polymer composite for breast implants Page ix

Figure 6. A symbolic representation of the mechanism of process of destruction of

bacteria from the cumulative effect of cell-wrapping as well as cell - trapping

mechanisms of rGO nanosheets and cell penetration of Ag

nanomaterial…………………….………………………………………………....110

Figure 7. A symbolic representation of the mechanism by which the rGO−nAg

nanoparticles kill the bacteria. The rGO punctures cell wall and enter the cytoplasm.

Silver nanoparticles directly enter into the cell, induces oxidative stress and damage

the cell contents ………………………………………………….…………….…115

CHAPTER 5

Figure 1. Raman spectra of graphene nanosheets deposited from methane on copper

and from bagasse on porous nickel substrates…………..………………..…….….132

Figure 2. (a,b). Representative SEM images of graphene nanowalls produced from (a)

methane on copper substrate and (b) from bagasse on nickel substrate. (c,d) EDS

spectra of graphene formed from (c) methane on copper substrate and (d) from bagasse

on nickel substrat …………………………………………………………………..133

Figure 3. TEM images of the samples deposited from (a) methane on copper substrate,

and (b) bagasse on porous nickel substrate……………………………….………134

Figure 4. Representative SEM images of E. coli cell attachment on the surfaces of (a)

methane-derived (GNW_M) and (b) bagasse-derived (GNW_B) graphene, and (c)

pure copper substrate after 4 h of incubation at 22 °C. SEM images of S. aureus cell

attachment on the surfaces of (d) GNW_M, (e) GNW_B, and (f) copper after

incubation under the same conditions………………………...……………. ….…..135

Figure 5. The survival rate of (a) E. coli and (b) S. aureus bacteria when exposed to

graphene fabricated from methane (GNW_M) and bagasse (GNW_B), and a pure

copper substrate ……………………………………………………………………136

Figure 6. Schematic representation of plasma enabled synthesis of graphene. (a)

Conversion of methane gas into graphene in the presence of plasma. (b) Reforming of

sugarcane bagasse into graphene…………………………………………………..142

Figure 7. Comparative evaluation of the antibacterial efficacy of graphene nanowalls

synthesised from different precursors. High-cost, high-purity conventional, i.e.,

methane (a), and low-cost, natural carbon, i.e., sugarcane bagasse (b) sources are

converted using low-temperature plasma process (c) into high-quality graphene sheets

(d,e). When bacterial cells are exposed to thus-fabricated surface-immobilised

graphenes (f), anti-bacterial activity of graphene is observed to differ (g), expressed in

terms of cell attachment and number of colony-forming units…………….………143

Nanocarbon polymer composite for breast implants Page x

CHAPTER 6

Figure 1. Representative SEM images of yeast cells showing untreated (left) and 3-

minute plasma-treated yeast cells (right)…………………………………………...158

Figure 2. Representative HIM images of yeast cells showing untreated (a, d), plasma-

treated (b, e) and yeast cells that were exposed to NDs after receiving the plasma

treatment (c, f). Surfaces of cells that have received plasma treatment clearly show

features that are absent from surfaces of non-treated cells……………………….…159

Figure 3. Growth of plasma-treated yeast cells during 24 h period of exposure to

different concentrations of NDs. Each treatment was done in triplicate and the error

bars represent the standard deviation around the mean of the cell density at each time

point (measured at 600 nm). ………………………………………………………..161

Figure 4. Growth of yeast cells during the 24 h period, incubated with different

concentrations of NDs. Cells received no plasma treatment………………………. 162

Figure 5. Synergistic effects of plasma and NDs on viability of yeast cells. Cells were

incubated for 24 h. 3 min plasma treatment in combination with ND nanoparticles (100

µg/mL) shows significant inhibition of cell growth relative to other groups. All values

are normalized to control, plasma non-treated cells without ND. Mean values (± SE)

are given………………………………………………………………………..….164

Figure 6. Uptake of non-terminated nanodiamonds by plasma-treated yeast cells as a

function of ND concentration as visualised by fluorescent and corresponding

brightfield microscopy. Cells were incubated for 24 h. Nanoparticles are seen as green

dots entrapped within the cells, as well as outside in the medium. Micrographs show

the highest uptake of NDs at a concentration of 100 μg/ml, whereas the lowest uptake

was observed at concentration 5 μg/ml……………………………………………..165

Figure 7. Mechanism of nanotoxicity. Plasma treatment led to changes in the cell

membrane of yeast cells, which facilitated the passage of NDs into the cell and

subsequent accumulation of NDs within the cell, potentially interfering with cellular

metabolism……………………………………………………………………...… 166

CHAPTER 7

Figure 1. Mechanical test results of nanocarbon enhanced silicone. (a) Tensile test

result showing higher tensile strength of graphene reinforced silicon when compared

to that of nanodiamond reinforced silicone and normal silicone dispersion. (b) Tear

test results which show a comparative improvement for graphene- silicone dispersion.

Nanocarbon polymer composite for breast implants Page xi

(c) Nano indentation results show the improved indentation hardness of graphene

reinforced silicone………………………………………………………….………184

Figure 2. Representative CLSM images of S. aureus attachment to the a) Control, b)

Graphene, and c) Nanodiamond surfaces after an incubation periods of 18 hr. The

viability of surface attached cells for all substrates was found to be ~98%. d) Bar

graphs of the average number of surface attached cells. Error bars display the standard

deviation of the data. The statistical p-values were 0.00000006 (**) and 0.011

(***)………………………………………………………………………...……...185

Figure 3. HDF cell growth on the surface of (a) Control samples (b) Graphene coated

silicone composite and (c) Nanodiamond coated silicone composite (d) Tissue culture

plates on day 1, 3 and 7 confocal micrographs. (e) Graphical representation of optical

density different samples……………………………………………………..……187

Figure 4. (a) The bonding between silicone and graphene (b) Representation of the

mechanism by which graphene limits crack propagation…………………………..188

Figure 5. A symbolic representation of the mechanism by which the graphene kills

bacteria…………………………………………………………………..…………190

Figure 6 (a) Symbolic representation of biofilm formation silicone surface (b) possible

anti-adhesion mechanism on nanodiamond surface……………………………….191

Nanocarbon polymer composite for breast implants Page xii

LIST OF TABLES

CHAPTER 2

Table 1: A comparison between reported silicone breast implant rupture rates……..31

Table 2: Examples of recent ALCL cases related to breast implants………………..33

Table 3: Various types of silica-polymer nanocomposites. Adapted with permission

from ref [199]……………………………………………….……………………….47

Table...4: Mechanical properties of graphene/graphite-based polymer

nanocomposites. Adapted with permission from reference [237]. Copyright 2006-2016

Scientific Research Publishing Inc.…………………………….…………..……......52

Table 5: Systems for targeted delivery of bioactive agents based on polymeric

nanoparticles. Adapted with permission from ref [252]……………………….….. ..59

Table 6: Selected example of specific features of nanomaterials and their role in

enhancing performance of breast implant materials………….……………………..60

CHAPTER 4

Table 1: The average zones of inhibition (in mm) of rGO, nAg, rGO−nAg, and

nitrofurantoin. ..........................................................................................................107

CHAPTER 7

Table 1: Key results of the mechanical test data…………………………..............182

Nanocarbon polymer composite for breast implants Page xiii

LIST OF ABBREVIATIONS

PDMS Polydimethyl Siloxane

FDA Food and Drug Administration

MRI Magnetic Resonance imaging

PIP Poly Implant Prostheses

ALCL Anaplastic Large Cell Lymphoma

ROS Reactive Oxygen Species

DNA Deoxyribonucleic Acid

GO Graphene Oxide

rGO Reduced graphene oxide

Ag Silver

CNT Carbon Nanotubes

ND Nanodiamond

TEM Transmission Electron Microscope

SEM Scanning Electron Microscope

HIM Helium Ion Microscope

CAP Cold Atmospheric Plasma

CSLM Confocal Laser Scanning Microscopy

HDF Human Dermal Fibroblast

RNOS Reactive Nitrogen and Oxygen Species

Nanocarbon polymer composite for breast implants Page xiv

LIST OF PUBLICATION

First-authored papers

1. Prasad, K., Bandara, C.D., Kumar, S., Singh, G.P., Brockhoff, B., Bazaka,

K., Ostrikov, K. (2017) Effect of precursor on antifouling efficacy of

vertically-oriented graphene nanosheets. Nanomaterials, 7(7), 170 (2017)

(Journal front cover image) (Q2, IF 3.55)

2. Prasad, K., Bazaka, O., Chua, M., Rochford, M., Fedrick, L., Spoor, J.,

Symes, R., Tieppo, M., Collins, C., Cao, A., Markwell, D., Ostrikov, K.,

Bazaka, K. Metallic biomaterials: Current challenges and opportunities.

Materials, 10(8), 884 (2017) (Q1, IF 2.72)

3. Prasad, K., Lekshmi, G.S., Ostrikov, K., Lussini, V., Blinco, J., Mohandas,

M., Vasilev, K., Bottle, S., Bazaka, K., Ostrikov, K. Synergic bactericidal

effects of reduced graphene oxide and silver nanoparticles against gram-

positive and gram-negative bacteria. Scientific Reports, 7, 1591 (2017)

(Discussed in Indian media as an effectual work; Editor’s choice paper for the

year 2017) (Q1, IF 4.122)

4. Prasad, K., Zhou, R., Zhou, R., Schuessler, D., Ostrikov, K., Bazaka, K.

Cosmetic reconstruction in breast cancer patients: Opportunities for

nanocomposite materials. Acta Biomaterialia (accepted, IF Q1, 6.319)

5. Prasad, K., Recek, N., Zhou, R., Aramesh, M., Wolff, A., Speight, R.E.,

Mozetič, M., Bazaka, K., Ostrikov, K. Effect of multi-model environmental

stress on dose-dependent cytotoxicity of nanodiamonds in plasma-treated

Saccharomyces cerevisiae cells. (to be submitted to Sustainable Materials and

Technologies)

Nanocarbon polymer composite for breast implants Page xv

6. Prasad, K., Aramesh, M., Tran, P., Bazaka, K., Ostrikov, K. Mechanism of

adhesion of human osteoblast cancer cells on surfaces decorated with

hydrogen- and oxygen-terminated nanodiamond (in preparation)

7. Prasad K, Rifai A, Fox K, Schuessler D, Bazaka B, Ostrikov K Nanocarbon

polymer composite for breast reconstruction (ready to be submitted to

Biomaterials)

Co-authored Papers

1. Somanathan, T., Prasad, K., Ostrikov, K.K., Saravanan, A., Krishna,

V.M. Graphene Oxide Synthesis from Agro Waste. Nanomaterials 5, 826-

834 (2015) (the most cited paper in Nanomaterials in 2016) (Q1, IF 3.55)

2. Zhou, R., Zhou, R., Prasad, K., Fang, Z., Speight, R.E., Bazaka, K., Ostrikov,

K. Cold atmospheric plasma activated water as a prospective green

disinfectant: the crucial role of peroxynitrite. Green Chemistry (accepted Q1,

IF 9.125)

3. Zhou, R., Recek, N., Prasad, K., Wang, P., Zhou, R., Aramesh, M., Speight,

R. E., Mozetič, M., Bazaka, K., Ostrikov, K. Chemo-Radiative Stress as

Sensitiser of Size and Charge Dependent Nanodiamond Toxicity. ACS Nano

(in review)

4. Zhou, R., Wang, P., Guo, Y., Prasad, K., Zhou, R., Yu, F., Recek, N.,

Speight, R.E., Fang, Z., Bazaka, K., Ostrikov K. Prussian Blue Analogue

Nanoparticles Improve the Fermentation Efficiency by Fighting against

Oxidative Stress in Saccharomyces cerevisiae (in preparation)

Conference Presentations

1. Prasad, K., Kumar, S., Bazaka, K., Ostrikov, K. ‘Precursor Specific Effects on

Plasma Produced Graphene’ APPC-AIP Congress, 5-9 December 2016,

Brisbane, Australia (Poster Presentation)

Nanocarbon polymer composite for breast implants Page xvi

2. Prasad, K., Recek, N., Aramesh, M., Bazaka, K., Ostrikov, K. ‘Effect of

engineered nanodiamonds on the cellular structure and growth of Saccharomyces

cerevisiae’ ICBNI 27-30 September 2016, Brisbane, Australia (Oral

Presentation)

3. Prasad, K., Aramesh, M., Tran, P., Bazaka, K., Ostrikov, K. ‘Mechanism of

human osteoblast cancer cell adhesion on hydrogen and oxygen terminated

nanodiamond surfaces’ ICONN February 2018, Wollongong, Australia (Oral and

Poster Presentation)

4. Zhou, R., Recek, N., Prasad, K., Bazaka, K., Ostrikov, K. ‘Synergistic effects of

atmospheric-pressure cold plasma and charged nanodiamonds on the

fermentation efficiency of S. cerevisiae’ ICONN February 2018, Wollongong,

Australia (Poster Presentation)

Nanocarbon polymer composite for breast implants Page xvii

STATEMENT OF ORIGINAL AUTHORSHIP

The work contained in this thesis has not been previously submitted to meet

requirements for an award at this or any other higher education institution. To the best of

my knowledge and belief, the thesis contains no material previously published or written

by another person except where due reference is made.

Signature:

Date: April 2019

QUT Verified Signature

Nanocarbon polymer composite for breast implants Page xviii

ACKNOWLEDGMENTS

Firstly, I would like to express my sincere gratitude to my Principal Supervisor,

Prof. Ken Ostrikov, who is the reason why I am at QUT doing a PhD. His direction,

guidance and thoughtful discussions during my entire candidature helped me learn a lot. I

am thankful for the opportunities you have provided me, for your patience, your friendly

approach, and, above all, even with your busy schedule, you have made yourself available

every time whenever I needed you either through emails or in person. I look forward to

continuing my life as a scientist and I am forever thankful to you for this.

My PhD would never have been this successful without Dr. Kateryna Bazaka,

my Associate Supervisor. Your never-ending support and readiness to help always

inspired me in every aspect of my life, not just with my PhD. I thank you for helping me

learn the art of experimental science, the art of writing, the art of teaching and everyday

support both in and out of university. Without you this work would not have come

together, the ideas and concepts would not be as fully formulated and I would have nothing

to present here.

Many thanks to Mr. David Schuessler, R&D Director, Allergan, a leading

breast implant producer globally, for mentoring me throughout the project and providing

an industry perspective. Without you, it would have been much more challenging for us

to formulate the most pressing challenges and benchmarks within the breast implant

industry. Thank you for you strong support and your willingness to come down to visit us

at QUT all the way from the US.

I would like to express my sincere gratitude to Dr. Shailesh Kumar, Dr.

Morteza Aramesh and Dr. Nina Recek who were there for me during the initial stage of

my candidature and readily offered their guidance to me. Shailesh, you made my days at

Nanocarbon polymer composite for breast implants Page xix

CSIRO, Linfield very productive. I learned a lot about plasma-enabled techniques from

you. Morteza, I am forever thankful to you for introducing me to the world of

nanodiamonds. Thanks for giving me an exposure to this wonderful material and with

your help, I was able to learn a lot about bio techniques, which I always wanted to do.

Nina, you are more than a colleague, you are a good friend who helped me a lot and taught

me many aspects of plasma and microorganisms. I thank Prof. John Bell and Prof.

Nunzio Motta for accepting me as their student.

Thanks to our team members at CSIRO, especially Dr. Adrian Murdock, who

helped me overcome a major challenge in my project. Adrian, your graphene ink solved

many of my problems and I am always thankful for you for that. I would like to thank Dr.

Tim, Dr. Michael and Dr. Shafique, for your support during my CSIRO visits.

The friendly staff at CARF made my project easier than it could have been. Mr.

Gregory Paterson, no matter where I go, I would never forget you in my life. Greg, the

amount of effort you had put into helping me realise the most challenging aspects of the

mechanical testing is beyond words and I cannot thank you enough for your help and

support. Dr. Jamie Riches, Dr. Sanjelina Sing, Dr. Jennifer MacLeod, Dr. Josh

Lipton-Duffin, Dr. Annalena Wolff, Dr. Yanan Xu, Dr. Dilini Galpaya, Dr. Rachel,

and Dr. Jennifer - thanks a ton for all the help you have provided me with.

To all my teammates at QUT – thank you Jickson for all the inspiration,

motivation, for sharing your lab space and of course for accompanying me to all those new

restaurants. Rokon, thank you for being there ready to help anytime with anything. Special

thanks to Renwu and Rusen - you guys are amazing, with you I have learned a lot about

plasma and of course will never forget the amazing Chinese food that Renwu cooks, and

the way you write papers. Rusen, you always reminded me about finishing my works and

you were constantly helping me do a lot of my work. Yanru, even though you were only

Nanocarbon polymer composite for breast implants Page xx

there for a short span of time, I enjoyed your company a lot. Janith, you are the new

member of our team, but you got along with us so quickly and it was great knowing you.

Thank you guys, you presence made my time at QUT ever more enjoyable.

To rest of my friends at QUT, Vish - thanks for always being around with strong

motivating words and I was always happy in your company , Yachana, Fawad, Daniel,

Chaturanga, Peiyu etc. and non QUT friends, Sreejith - you were more than a friend - my

family in Brisbane and I will never forget your inspiration and all your help, Tania - you

too is a lot more than a friend, Nandhu, Kristline, Arun, Ali, Mariyam, Reshmi, Radheesh,

Eby, Nikhil, Jemin, Nevia, Greeshma, Jesly, Gloria, Mohini, etc. Thanks to each and

every one of you for making my journey memorable.

Last but above all, I would have been nothing without the love and support from

my family - my dad, who always wanted for me to reach heights, my mom, my sisters,

my grandparents, my husband, who joined me just months before my completion but still

was doing his best to help me, my in-laws. You all have supplied me with endless

encouragement, prayers, love and guidance. Thank you all.

Nanocarbon polymer composite for breast implants Page 1

1Chapter 1: Introduction

1.1 Background

According to statistics provided by the World Cancer Research Fund International

(WCRFI), breast cancer is the most common type of cancer in women worldwide and it

is estimated that 1 in 8 women are at risk, with a majority of them opting for mastectomy

as a means of preventing and treating the disease [1]. For patients who elect to undergo

mastectomy, breast reconstruction is generally offered to restore the visual appearance of

the breast without affecting the prognosis or detection of recurrence of cancer.

Approximately 70% of patients undergoing breast reconstruction elect the implant-based

treatment, with the balance undergoing autogenous tissue–based reconstruction [2].

Despite substantial innovations in the design and fabrication of breast implants,

modern implants are still faced with a number of challenges. In terms of device

strength, rupture of the implant shell remained a persistent issue. Scarring,

infection, blood clots, pain, changes in breast sensation, damage or leakage of implant

are among another issues connected with the breast implant surgery. In spite of the

significant efforts to improve long-term stability of the implant, breast augmentation

still remain a largely short-term solution, with most patients having to undergo

implant removal and replacement at some stage, from within the first few months of

implantation to a maximum of 15 years of use [3, 4]. Treatment of complications that

arise as a result of implant use often requires multiple surgeries, device removal, long-

term systemic antibiotics, and extended rehabilitation, and is frequently ineffective,

leading to worse clinical outcomes and increased financial costs for the patient and

the healthcare system. What is needed in the current scenario is implant materials or

coatings that can resist infection while simultaneously promoting strength and


flexibility of the implant. Such materials and/or devices would be particularly

advantageous the future of breast implants.

Nanocarbon materials are of great interest in biomedical industry due to their large

surface-area-to-volume ratio, ability to be dispersed in a polymer, which means that

their introduction into a polymer matrix can facilitate a noticeable enhancement in a

range of desired properties, from Young’s modulus, tensile strength, heat resistance,

to biocompatibility and resistance to bacterial attachment [5]. Proper dispersion of

nanocarbon materials within a polymer matrix can produce light weight composites

with excellent mechanical properties. Nonetheless, the cost involved in the synthesis

of high quality nanocarbon materials, and the challenges involved in achieving a

uniform dispersion of this additive within a polymer matrix make the development of

such composites non-trivial [6].

This work investigates the synthesis of silicone-nanocarbon composite materials with

the aim to overcome the major challenges faces by currently available silicone

implants, namely propensity to rupture, biofilm development and capsular contracture.

1.2 Context

Understanding the functionalities of carbon nanomaterials is critical in the design and

development of new and improved implants. Although the path to clinical translation

remains thought-provoking, the carbon nanomaterials represented in this thesis create

opportunities for the development of new and improved implants.

The advent of carbon nanomaterials has resulted in the advancement of materials and

structures that enabled the expansion of the inventory of implantable biomaterials and

medical devices. Concentrating on the real progress in the utilisation of advanced

functional nanomaterials in controlling the processes at the interface between artificial and

living systems, this thesis will fill the gap in understanding of the current development in


the breast reconstruction and the importance of new or improved materials in this

particular field. As such, this project may help the manufacturers and future researchers

engaged in the development of breast implants or other silicone-based biomedical

materials.

1.3 Purposes

The aim of this research is to explore the possibility of using the unique properties of

nanoscale carbon materials to enhance mechanical strength and cell-surface compatibility of

breast implants by creating novel nanocarbon-reinforced silicone composites that have

similar flexibility yet improved safety profile when compared to currently available implants.

The main hypothesis is that nanocarbons can improve the mechanical stability of

silicones used to produce breast implants as well as to reduce the rate of biofilm

formation on the surface of the implant and thus prevent infection and capsular

contracture.

Research is subdivided into the following specific aims:

To synthesise nanocarbon materials by various techniques;

Demonstrate feasibility of nanocarbon-silicone composite synthesis for

manufacturing of high performance composites;

Develop techniques for uniform dispersion of nanocarbons in silicone;

Investigate the reinforcing effect of nanocarbon materials, optimising the

composite;

Identify optimum conditions for manufacturing of silicone implants with

enhanced mechanical strength, bactericidal effects, and improved

biocompatibility.

Aims correspond to the following research questions:

1. What are the effects of introducing nanocarbons into the silicone?


2. How do the characteristics of nanocarbons affect the properties of silicone

composites?

3. Can strong implants be synthesised by introducing nanocarbon materials into

silicone?

1.4 Significance

Since 1963, breast implants have been used for both reconstruction and aesthetic

purposes. Since then, the demand for breast implants has increased over years and is

likely to continue increasing. The burden associated with implant-related health issues

such as inflammation, rupture, infection etc. is also going to increase. The invention

of implants signifies a great opportunity to increase the quality of life of many

patients. Nevertheless, it is accompanied with potential risks to consider. Therefore,

it has become a necessity to perform research to prevent the threats caused by the

implants to patients. The incorporation of nanomaterials into the implant may open a

way for future “lifelong” implants.

Even though there are many methods for breast reconstruction, the majority of patients

prefer to use breast implants as they are the fastest method of breast reconstruction.

Next generation advanced materials are promising for addressing the range of

challenges currently faced by existing implants, and also offer breast reconstruction

that not only minimize the likelihood of medical complications but possibly offer

more realistic, aesthetically pleasing outcomes for the patients. The significance of

this project is that it attempts to formulate a materials-based strategy that would

address all of the key challenges faced by the existing implants. It is aimed at

enhancing the properties of the already exciting breast implants through incorporating

nanomaterials such as graphene nanosheets into the currently used silicone polymer.


1.5 Thesis Outline

Chapter 1 provides a brief introduction to the thesis, giving an insight into major

problems selected for the project and the proposed ways to overcome them. The issues

related to the currently available implants and the research gap in the current area are

discussed in this chapter. The hypothesis for the project and the project aims are briefly

stated.

Chapter 2 of this thesis will give a comprehensive literature review on the history of

breast implants, issues related to breast implants and the possibility of integrating

nanoparticles into the currently available breast implant materials. The motives behind

breast reconstruction, the evolution of breast implants, the development of different

types of breast implants, the significance of the issues related to the implants, the

different types of nanomaterials available for breast reconstruction, the research gap,

and the effective outcomes are described in detail in this chapter. This chapter has been

published in Acta Biomaterialia as a review article

Chapter 3 provides a brief overview of the research methodology used for material

synthesis and characterisation.

Chapter 4 reports on the antibacterial properties of graphene-based materials in

suspension with a view of their application as an active agent in eluting systems. The

findings build towards the goal of mitigating pathogen attachment and biofilm

formation through surface functionalisation and elution of bioactive carbon-based

nanoparticles. This chapter studies the antibacterial properties of reduced graphene

oxide/silver nanocomposite as a promising material for deactivation of common

human pathogenic bacterial strains, including pathogenic multidrug resistant

Escherichia coli, Staphylococcus aureus, and Proteus mirabilis. The chapter outlines


synthesis and fundamental properties of rGO/Ag nanocomposite, and proposes a

mechanism for the observed biological activity. It shows that the nanocomposite

material has improved antibacterial ability when compared to normal systemic

antibiotics. This chapter is published in Scientific Reports.

Chapter 5 investigates antibacterial properties of surface-immobilised graphene-based

materials as potential agents for contact killing of bacteria on implant surfaces. This

chapter studies the antibacterial properties of graphene grown on different surfaces

from different precursors by means of chemical vapour deposition. The article studies

the effect of precursor and substrate on the biological activity of thus-grown graphene

nanoflakes, and proposes a likely mechanism of action against common human

pathogenic bacteria with different shape and cell wall structure. The antifouling

efficacy of graphene nanowalls, i.e., substrate-bound vertically-oriented graphene

nanosheets, is demonstrated against biofilm-forming Gram-positive and Gram-

negative. This chapter is published in Nanomaterials.

Chapter 6 reports on the potential toxicity of nanodiamonds in suspension for cells

exposed to oxidative stress, considering the potential use of these nanodiamonds for

surface functionalisation to control cell-surface interactions by controlling surface

chemistry and morphology of implants. It describes the effect of atmospheric-pressure

plasma, a tool used for cancer therapy that delivers highly-reactive ROS and RNS

species, UV light, and mild heat to affected tissues, on the interactions between inert

nanodiamond particles (NDs) and cells. The study for the first time demonstrates that

under oxidative stress conditions, the toxicity of otherwise biocompatible

nanoparticles can potentially be increased, as demonstrated in model eukaryotic

organism (Saccharomyces cerevisiae) and breast cancer cells. The chapter is to be

communicated to Sustainable Materials and Technologies.


Chapter 7 gives an insight on how the above mentioned graphene and nanodiamond

helped in reinforcing the mechanical, bactericidal and cell adhesion properties of the

currently available silicone implant material. Both graphene and nanodiamond made

significant increase in the mechanical properties of the implant shell, with graphene

showing a 68% increase in mechanical strength and nanodiamond with a 19% increase

in mechanical strength. The antibacterial properties and the cell adhesion properties

were improved by using graphene and nanodiamond as a coating. This chapter is to be

communicated to Biomaterials.

The thesis concludes with chapter 8, where major findings and future prospects are

discussed.


1.6 Reference

[1] 'Breast cancer statistics' Retrived from https://www.wcrf.org/int/cancer-facts-

figures/data-specific-cancers/breast-cancer-statistics on 12th September 2018.

[2] Quinn TT, Miller GS, Rostek M, Cabalag MS, Rozen WM, Hunter-Smith DJ.

Prosthetic breast reconstruction: indications and update. Gland surgery 2016;5:174.

[3] Risks of Breast Implants- from FDA, USA. Updated on 2013. Accessed on 16 June

2015. Available from, http://www.fda.gov/MedicalDevices/ProductsandMedical

Procedures/ ImplantsandProsthetics/BreastImplants/ucm064106.htm.

[4] Breast Implant Complications–latest from the NHS [Internet]. London: NHS Choices;

2014 Accessed on 16 June 2015 Available from:

http://www.nhs.uk/Conditions/Breastimplants/Pages/Complications.aspx.

[5] Iyer P, Mapkar JA, Coleman MR. A hybrid functional nanomaterial: POSS

functionalized carbon nanofiber. Nanotechnology 2009;20:325603.

[6] Ramanathan T, Abdala AA, Stankovich S, Dikin DA, Herrera-Alonso M, Piner RD, et

al. Functionalized graphene sheets for polymer nanocomposites. Nature

Nanotechnology 2008;3:327.

http://www.fda.gov/MedicalDevices/ProductsandMedical

http://www.nhs.uk/Conditions/Breastimplants/


2Chapter 2: Literature review

The literature review of this thesis has been accepted for publication in Acta Biomateriala as a

review article:

Cosmetic reconstruction in breast cancer patients: Opportunities for

nanocomposite materials

Karthika Prasad, Renwu Zhou, David Schuessler, Kostya (Ken) Ostrikov, Kateryna Bazaka


Statement of Contribution of Co-Authors for Thesis by Published Paper

The authors listed below have certified that:

1. they meet the criteria for authorship in that they have participated in theconception, execution, or interpretation, of at least that part of the publication intheir field of expertise;

2. they take public responsibility for their part of the publication, except for theresponsible author who accepts overall responsibility for the publication;

3. there are no other authors of the publication according to these criteria;4. potential conflicts of interest have been disclosed to (a) granting bodies, (b) the

editor or publisher of journals or other publications, and (c) the head of theresponsible academic unit, and

5. they agree to the use of the publication in the student’s thesis and its publicationon the QUT’s ePrints site consistent with any limitations set by publisherrequirements.

In the case of this chapter:

“Cosmetic reconstruction in breast cancer patients: Opportunities for nanocomposite

materials”- Accepted on Jan 2019

Contributor Statement of contribution

Karthika Prasad Conception of idea of writing a review on breast implants,

designed the review structure and wrote the manuscript

28/11/2018

Renwu Zhou Assisted in analysing and revising the manuscript, figures and

schematic representations

David Schuessler Suggested improvements and edited manuscript

Kostya Ostrikov Assisted with idea conception, article structure designing, major

editions and revisions

Kateryna Bazaka

Brought forward the idea of writing a review, proposed the concept,

designed review structure, co-wrote the manuscript, and supervised

manuscript revision

I have sighted email or other correspondence from all Co-authors confirming their certifying authorship.

(If the Co-authors are not able to sign the form please forward their email or other correspondence confirming the

certifying authorship to the RSC).

Kostya Ostrikov 28/11/2018

Name Signature Date

Principal Supervisor Confirmation




PREFACE

Literature review

Using examples from scientific literature, outlines key challenges of presently available

silicone breast implants in light of their use for post-mastectomy reconstruction:

1. Susceptibility to pathogenic fouling and biofilm formation, which may lead to acute

and chronic infections, and capsular contracture.

2. Limited surface biocompatibility, which may lead to foreign body response and

capsular contracture, with the former potentially, playing a role in cancer development

and the latter potentially leading to implant deformation (poor aesthetics) and rupture.

3. Poor mechanical strength, which may lead to implant leakage and rupture.

Introduces potential opportunities for carbon-based nanomaterials to combat these challenges

through:

1. Mitigation of pathogen attachment and biofilm formation through surface

functionalization and elution of bioactive carbon-based nanoparticles.

2. Surface functionalization to control cell-surface interactions by controlling surface

chemistry and morphology of implants.

3. Implant reinforcement to improve mechanical strength.


Abstract

The most common malignancy in women, breast cancer remains a major medical challenge that

affects the life of thousands of patients every year. With recognized benefits to body image and

self-esteem, the use of synthetic mammary implants for elective cosmetic augmentation and post-

mastectomy reconstruction continues to increase. Higher breast implant use leads to an increased

occurrence of implant-related complications associated with implant leakage and rupture, capsular

contracture, necrosis and infections, which include delayed healing, pain, poor aesthetic outcomes

and the need for revision surgeries. Along with the health status of the implant recipient and the

skill of the surgeon, the properties of the implant determine the likelihood of implant-related

complications and, in doing so, specific patient outcomes. This paper will review the challenges

associated with the use of silicone, saline and “gummy bear” implants in view of their application

in patients recovering from breast cancer-related mastectomy, and investigate the opportunities

presented by advanced functional nanomaterials in meeting these challenges and potentially

opening new dimensions for breast reconstruction.


Statement of Significance:

Breast cancer is a significant cause of morbidity and mortality in women worldwide, which is

difficult to prevent or predict, and its treatment carries long-term physiological and psychological

consequences. Post-mastectomy breast reconstruction addresses the cosmetic aspect of cancer

treatment. Yet, drawbacks of current implants contribute to the development of implant-associated

complications, which may lead to prolonged patient care, pain and loss of function.

Nanomaterials can help resolve the intrinsic biomechanical mismatch between implant and tissues,

enhance mechanical properties of soft implantable materials, and provide an alternative avenue for

controlled drug delivery. Here, we explore advances in the use of functionalized nanomaterials to

enhance the properties of breast implants, with representative examples that highlight the utility of

nanomaterials in addressing key challenges associated with breast reconstruction.

Keywords: breast cancer, mastectomy, silicone breast implants, bacterial infection, tissue-implant

interaction, nanoparticles


2.1 Introduction

The inception of modern age breast augmentation and reconstruction dates back to 1960s when

silicone gel implants were introduced [1]. Since then, there has been a rapid increase in the

demand for synthetic mammary implants for post-mastectomy breast reconstruction and

elective cosmetic augmentation [1]. According to current estimates, between 5 to 10 million

women worldwide have had the breast reconstruction or augmentation surgery, and the rate of

reconstruction continues to increase (Fig. 1) [2].

Depending on the circumstances for choosing the surgery, elective breast surgeries can be

broadly categorized as reduction, reconstruction, and augmentation. Correction of congenital

or post-mastectomy deformities is the key motive behind most reconstructive surgeries,

whereas breast augmentation generally aims to improve the cosmetic appearance (size, shape,

and position) of healthy breasts. Neither reconstructive nor cosmetic breast surgery is medically

necessary, and in fact may present considerable medical risks for the implant recipient.

Figure 1. (a) Estimated age-specific incidence and mortality rates for breast cancer, by sex,

2017. Reproduced with permission from ref [3] Copyright @ Australian Institute of Health and

Welfare (b) Estimated top elective surgeries in America in the year 2016, breast augmentation

is the second leading type of an invasive elective surgery. Reproduced with permission from

ref [4]. Copyright @ American Society for Aesthetic Plastic Surgery


Morbidity and mortality from breast cancer are increasing, with estimated 1 in 8 US women

developing the disease in their life time [5]. Similar lifetime risk of developing invasive breast

cancer has been suggested for women living in Australia [6]. Of those diagnosed with breast

cancer, approximately 70% of the patients will choose to undergo breast-conservation

treatment whereas 30% will elect to undergo mastectomy [7]. This choice will be primarily

governed by the stage of cancer and its invasiveness, with patients with larger tumor sizes,

positive lymph nodes or involving different areas of the breast generally undergoing

mastectomy or combined mastectomy and radiation treatments [8].

Mastectomy is often considered as the only effective means to prevent cancer from spreading

from the primary tumor to adjacent lymph nodes for those patients who are unable to undergo

radiation therapy due medical reasons or because of unsuccessful lumpectomy at the earlier

stage of cancer [9]. Other considerations include the fear of recurrence, for which mastectomy

is regarded as a safer approach to breast cancer therapy, removal of healthy tissues for the

purpose of cancer prevention (e.g. in individuals genetically predisposed to breast cancer), and

the concern for cosmetic result, where breast conservation therapy (e.g. lumpectomy) is thought

to provide better aesthetic outcomes [10].

Recent advancements in understanding the genetic bases of breast cancer have led to an

increase in elective prophylactic mastectomy in high-risk individuals with the view to prevent

breast cancer [11-13]. For patients who elect to undergo mastectomy, breast reconstruction is

generally offered to reinstate the visual form of the breast without negatively affecting the

efficacy of the chosen oncotherapy, patient prognosis, e.g. survival or recurrence of the disease,

or the accuracy of the diagnostics employed to detect new or recurrent cancer [14, 15]. Around

70% of patients undergoing breast reconstruction elect the implant-based treatment, with the

balance undergoing autogenous tissue–based reconstruction [15].


In recent years, the number of individuals who elect mastectomy has grown, from 35.6% in

2005 to 38.4 % in 2008 [16]. Although the reasons behind the observed increase remain

unclear, it is possible that skin-sparing mastectomy offers patients a desirable combination of

an oncologically-safe strategy for the surgical management of breast cancer at early stages of

the disease with desirable aesthetic results attained by preserving the skin envelope of the breast

[17]. Furthermore, unlike conventional radical mastectomy that often necessitates the use of

tissue expanders and nipple prosthesis, and may require multiple surgeries before the desired

cosmetic outcome is achieved; the preservation of skin flap facilitates immediate reconstruction

of the breast. Compared to delayed reconstruction, immediate reconstruction can be potentially

less surgically-challenging and traumatic for the patient. Indeed, the surgical management of

cancerous growth has recently become more considerate of the psychosocial and emotional

distress arising from mastectomy [18, 19].

Chemotherapy and post-mastectomy radiotherapy is likely to affect the timing of the

reconstructive process, since implants may interfere with such treatments [18, 20]. In one study,

68 % of individuals who were implanted with a tissue expander or an implant immediately

after primary tumor removal and subsequently underwent irradiation were subject to capsular

contracture, as opposed to 40 % in the non-irradiated group [21]. Delayed and/or multi-stage

reconstruction may also be required in cases where remaining tissue is insufficient to provide

adequate implant support and coverage. In this case, incidence of late complications such as

tissue, e.g. skin and fat necrosis, hematoma, seroma, and wound dehiscence are more likely to

occur [22].

The multi-stage reconstruction generally starts with an introduction of a tissue expander, the

role of which is to increase the size of the available skin to accommodate an implant. The

expander is gradually filled with saline until the preferred expander fill volume is attained, after

which the expander is replaced with a permanent implant. The duration of the reconstruction


process can be reduced if acellular dermal matrices are used. These biological matrices afford

structural support in the lower pole of the breast, limiting muscle dissection, and help shape

the inframammary and lower breast folds [23], contributing to better breast shape and position.

The tension of the mastectomy skin is reduced to attain higher initial volume and faster rate of

expansion [23]. The matrix promotes significant revascularization and cellular infiltration, thus

encouraging tissue regeneration. In addition to improving expander dynamics, other benefits

of acellular matrices include reduced pain, and potentially lower incidence of late

complications, such as capsular contracture [24], yet some concerns remain regarding the

immunological response to the presence of donor nucleic acid residue in patients that are

treated with these matrices [25].

Significant breast reconstruction or augmentation may be required to reshape healthy breast

tissue when it is not cosmetically pleasing for the reasons of postpartum deflation and ptosis,

congenital anomalies, e.g. tubular breasts, or post pubertal underdevelopment, e.g. strong

micromastia [26,27]. A significant underdevelopment or lack of breast tissue, micromastia can

be either bilateral, where both breasts are affected, or unilateral in which case one breast is

smaller than the other [28]. Similar to unilateral breast reconstruction, correction of unilateral

micromastia is challenging as aesthetically pleasing results, and in particular symmetry, need

to be obtained while dealing with different types of media (i.e. breast tissue and an artificial

silicone implant) that behave differently with time. Other types of breast asymmetries include

differences in breast volume and shape, position of inframammary fold, position and size of

breast base or nipple-areola complex [29].

2.2 Brief History of Materials for Breast Augmentation

Known efforts to gain a more aesthetically pleasing breast shape and volume date back to

around 3000 BC, when primitive undergarments were used by Minoan women to correct the

appearance of their breasts. The first bodice as it is known today was created in the 13th century


and was sufficiently versatile to suit a variety of body shapes and clothing styles [30]. The 18th

century heralded the advent of intrusive endeavours at breast augmentation. Early breast

implant materials included a broad range of solid-phase natural and synthetic materials,

including ivory, glass, metal, and elastic materials, however none of these produced desired

cosmetic results. Moreover, the recipients of such implants often suffered agonizing pain

during and post-implantation and sometimes serious health issues [31].

Driven by the need for an implant material that better resembled the appearance and the

softness of breast tissue, as well as the desire for the implantation technique that was less

invasive, 1890s witnessed materials such as petrolatum, paraffin and liquid fats, e.g. plant oil

being injected into the breast [32]. Later in 1930s, injection of autologous fat extracted from

the buttocks, and transplantation of autogenous tissues, such as those harvested from the

dermis, fat or fascia to breast became common. But all of these methods were found to lead to

complications and, frequently, sub-optimal aesthetic results, particularly due to the difficulty

in controlling the physical location of the injected medium [31].

Between 1950 and 1960, failure of breast augmentation techniques led numerous specialists to

attempt prostheses models (Fig. 2) produced using plastics [30-35]. Pangman pioneered the use

of polyvinyl alcohol for breast implantation. The prosthesis had a sponge-like structure that

had to be cut to shape before being cleaned with water for 24 hours and sterilized. As the

sponge-like structure of the implant had the disadvantage of being invaded by fibrous tissue,

Pangman and Wallace [36] produced a prosthesis comprised of a sponge core surrounded by

an Ivalon polyurethane which was again covered with an Ivalon layer. Ivalon provided a

protective barrier whereas the sponge allowed for the implant to remain soft and breast tissue-

like [36-39].


Figure 2. The evolution of breast reconstruction. (a)The breast augmentation started in

3000BC. with alteration of clothing in 3000 BC, followed by using petroleum jelly, paraffin

wax, olive oil and fat and tissue grafting in 1930s [40-42]. (b)The evolution of silicone implants

started in 1950 and is still in use. (c) Additive tissue manufacturing is the current trend in breast

reconstruction. Reproduced with permission from ref [43].

After the Pangman prosthesis, the next material to enter the breast implant field in 1961 was

silicone, with the first patient receiving a silicone implant in 1962 [44]. Known as the Cronin–

Gerow implant, the 1963 prosthesis model consisted of a rubber-like shell and silicone gel

filling [45], which was fastened to the chest wall by means of a polyethylene terephthalate

(Dacron) patch to reduce its rotation (Fig.2 b) [46].

Initially manufactured for the aviation industry during World War II, silicone (or polydimethly

siloxane, PDMS) was chosen for its low toxicity, biological stability and cell and tissue

compatibility, softness, inertness, elasticity and flexibility [47,48], as well as optical


transparency which enables its use as a material for contact lenses and other medical devices

[49]. Silicone gels of varying viscosity can be synthesized by controlling the length and/or the

extent of crosslinking between the polymer chains [50]. When the fraction of cross linkers is

increased, the movement of the individual polymer chains becomes restricted which in turn

affects the viscosity [51].

Over the following 50 years, silicone-based breast prostheses underwent numerous

transformations to address the evolving demands of the surgeons and patients alike. Driven by

the demand for more natural look and feel from the implants, in 1970s silicone implants were

redesigned to reduce the thickness of the outer shell and lower the viscosity of the gel filling,

which notably improved the feel and appearance of the implant. However, thinner shell was

associated with higher rupture rate.

Subsequent efforts to enhance the cosmetic appearance of the implant included the

development of double lumen shell designs, where a saline breast implant contained a smaller

silicone gel implant within. The inner compartment was filled with silicone gel to allow the

implant to hold an aesthetically pleasing shape of the breast, while the outside compartment

contained saline. Furthermore, the volume of such an implant could be easily adjusted by

controlling the volume of saline in the outer compartment [46]. They were also safer if

ruptured, as released saline would be safely absorbed by the patient’s body. The modern

version of this construction, known as the Becker breast implant, is a common choice for breast

reconstruction procedures.

In 1980s, the fourth generation of breast implant emerged which had an elastomer-coated shell

and a thicker gel filling that decreased the probability of gel leakage. The firmer texture of

these implants allowed the manufacturers to make shaped as well as anatomic models of the

implant which provided a better fit to the natural breast shape and various body types of


women. The choice of the shape dictates the surface finish, with tapered prostheses requiring

a uniformly textured surface to reduce the rotational movement of the implant within the breast

pocket. On the other hand, prosthesis with a round shape can be made to possess both textured

and smooth interfaces to accommodate individual preferences of the surgeon [46, 52, 53].

In the second half of 1990s, the “gummy bear” implants were developed. These implants were

made of a silicone gel with a higher cohesiveness that greatly reduced the risk of filler leakage

and its migration from the peri-implant milieu to other tissues and organs. These types of

prostheses were associated with reduced incidence of rupture and capsular contracture, and

regarded as a considerably safer alternative to early generation breast implant devices [54-56].

The major disadvantages with this implant type included a relatively large incision required to

insert and position the implant, and the possibility of implant fracture due to a less elastic nature

of the gummy bear silicone [57].

2.3 Implant-Associated Issues

Despite significant progress in the design and manufacturing of breast implants, a number of

challenges associated with their use remain. In terms of mechanical strength, rupture, leakage

and failure of the implant shell remain a persistent issue. Scarring, pathogen colonization

and biofilm development, blood clotting, pain, changes in breast sensation are among the

challenges of the breast implant surgery. Despite best efforts to improve long-term stability of

the implant, breast augmentation remains a largely short- to medium-term solution, with most

patients having to eventually undergo implant removal and replacement from within first few

months of the initial surgery to a maximum of 15 years [58, 59]. This report focuses on three

most common issues that may lead to implant-associated complications and may be mitigated

through the careful use of nanoscale materials, namely infection, capsular contracture and

implant rupture.


2.3.1 Implant-related infections

Implant-associated infections are not limited to breast implants [60, 61]. Whenever an artificial

material is introduced into the body, it provides microorganisms with a highly-suitable ground

for attachment, and subsequent colonization and biofilm formation [62]. In biofilm state,

pathogens show greater resistance to conventional systemic antimicrobial therapies, requiring

up to 5-10 times greater dose of antibiotics compared to that used to treat equivalent sessile

bacteria [63, 64]. Often, the antibiotic treatment fails and removal of the implant is necessary,

which is performed at a substantial cost to the patient and healthcare system [65]. An infection

has the potential to require not one, but two or more operations - one to remove the implant,

then a second surgery to place another implant several months later, after the tissues have fully

healed and regenerated and the infection has been fully eliminated.

After breast implantation, up to 2.9% of patients get infection [66], of which approximately

1.7% can be attributed to acute infections and 0.8% to delayed onset infections [67,68].

Infection of the peri-implant milieu is one of the main complications associated with breast

implant surgery in developing countries, probably because of inadequate healthcare funding

[69], reported to vary from 1% up to 53% for post-mastectomy breast reconstruction

[68,70,71].


Figure 3 The surface of a typical silicone breast implant presents a suitable ground for bacterial

growth and biofilm formation (a) [72], often leading to an acute infection that significantly

damages breast tissues (b) (c) [73]. Biofilm on a textured implant, compared with biofilm on a

smooth implant (d) (e) [74].

Endogenous microbiological flora of the human breast and pathogenic bacteria from the

ambient environment can reach the surface of the implant during the surgery, or subsequently,

via the breast ducts. According to some studies [75,76], coagulase-negative staphylococci is

among the pathogens most frequently detected in breast implant-associated infections,

although recent reports also suggest Staphylococcus aureus as a microorganism responsible for

up to 67% of breast implant infections, with methicillin resistant S. aureus detected in 68% of

these cases [77]. Furthermore, while the silicone shell of saline implants is impermeable to

Candida and Aspergillus, the injection port through which the implant is filled can provide an

access route for these pathogens to reach the implant filler [78]. Infections can also develop

later in the implant life; originating elsewhere in the body, infectious agents can be transported

by blood to the surface of the implant, where they attach and commence colonization [79].

Unlike living tissues that can sense the presence of foreign pathogenic organisms and instigate

immune response, inert synthetic implants allow uncontrolled microorganism attachment and

biofilm development [80].


Figure 4. Various type of bacterial species found on breast implant infections. Reproduced

with permission from reference [81] Copyright @1201-9712/ 2015 The Authors. Published by

Elsevier Ltd on behalf of International Society for Infectious Diseases.

The dynamics of surface-bacteria interactions is governed by both surface chemistry and

surface morphology of the implant as well as the properties of bacterial cells [82]. It is generally

accepted that an increase in surface complexity or micro-roughness increases the susceptibility

of a surface to cell attachment and biofilm growth [83], by providing physical shelter for

bacterial cells and thus preventing their detachment [84]. Figure 3d demonstrates the

attachment of bacterial cell on a textured and smooth implant surface.


In most cases, by the time the infection is detected in the peri-implant space, the biofilm has

already reached the late stages of development and might have caused capsular contracture

which frequently necessitates the removal of the implant (Fig.4) [85-87].

Figure 5. Proposed mechanism of capsular contracture resulting from bacterial contamination

of implant surface.

2.3.2 Capsular Contracture

Every device implanted in the body has the potential to incite an immune response which may

lead to the formation of a collagen fibre envelope around the implant with the intent to isolate

the foreign material from the host tissues [88]. An abnormal contraction of this envelope

around the breast implant, known as capsular contracture, may lead to significant compression,

deformation, or displacement of the implant [89]. It is among the most often encountered

complications related to breast augmentation procedures, with clinically significant capsular

contracture reported to occur in 15% to 45% of cases [90-93], with up to 92% of these reported

to occur within the first 12 months post implantation [94,95].


Figure .6. Capsular contracture in breast implants. (a) Implants covered with a capsule [96].

(b) A capsule removed from the implant [97]. (c) The layered structure of the capsule.

Reproduced with permission from [96, 97].

Although, biofilm-induced capsular contracture can potentially be minimized by the use of an

antibiotic-impregnated mesh [98] or by topical irrigation with antibiotics [99], capsular

contracture may occur following an infection, hematoma and seroma (Fig.6) [100,101]. The

placement of the implant also affects the incidence of capsular contracture, with subglandular

placement (typically used to slightly lift the breast) being more susceptible to the condition.

Symptoms of capsular contracture can vary from mild discomfort and mild breast firmness to

severe pain, distorted shape, palpability and/or movement of the implant, and in some cases, a

rupture of the implant. Corrective surgery is often necessary when pain is severe [102], and

may involve removal of the implant capsule tissue, and, in some instances, the removal and

replacement of the whole entire implant. Furthermore, even after the corrective surgery, there

is still a probability of capsular contracture [102].

(a) (b) (c)


Figure 7. (a) The incidence of capsular contracture in recipients of polyurethane foam-

covered implants [103]. Examination of explanted implants shows a correlation between

degradation of polyurethane coating and capsular contracture. Beyond nine years after

implantation, where no coating was present in any patient, substantial increase in incidence

and severity of contracture is observed. Inset: Reproduced with permission from reference

[103]. Copyright © 2005 Springer Science+Business Media, Inc. Thick implant capsule

retrieved from a patient with severe cellular inflammation after 156 months (b) Bleeding of

silicone gel within the implant capsule after 122 months of implantation (c) Reproduced with

permission from reference [104].

In the second generation silicone breast implants, capsular contracture was a major issue [46].

Attempts were made to resolve it through the addition of a polyurethane foam coating to the

surface of the implant. The coating was shown to effectively reduce the rate of capsular

contracture by minimizing the formation of scar tissue around the implant (Fig 7).


Furthermore, it was found to induce an inflammatory reaction that hindered the development

of fibrous collagen tissue in the vicinity of the breast implant. Subsequent concerns regarding

potential health effects from biodegradation of polyurethane led to the removal of these

products from the market across the United States and Canada [52, 53].

Moreover, scientific studies suggest that the biodegradation of the polyurethane envelope

starts to within ∼2 years after breast augmentation surgery [105]. Exposure of the polyurethane

envelope to bodily fluids leads to the formation of 2, 4-diaminotoluene (2, 4-toluene diamine)

(TDA), a substance known to promote carcinogenesis in animals and potentially, in humans

[106]. Studies have shown that TDA may act as a tumor promoter, encouraging hepatic

cellular proliferation and promoting cellular mutations in rats [105,107]. These observations

suggest that women with subglandular polyurethane-covered implants may be more

susceptible to the development of breast cancer during the first few years after the surgery.

Subsequently, their risk of cancer decreases with increasing follow-up [108].

In cancer patients, radiation therapy may promote capsular contracture. Rosato and Dowden

reported that in patients that received bilateral breast reconstruction and were then subjected to

unilateral radiation therapy, the irradiated reconstruction site became contracted, unlike the

non-irradiate site, which retained its softness. They found the increase in the incidence of

capsular contracture as a result of subjecting the reconstructed breast to radiation to be

statistically significant. Also, they reported that radiation therapy after implant-based breast

reconstruction positively contributes to the increase in the incidence of capsular contracture

[109].

2.3.3 Implant Rupture

It is well established that all breast implants are only temporary devices which will eventually

break down. However, the exact life-time of currently available breast implants is difficult to


estimate accurately, since in some patients they may last for decades, while in others break

down within several months post implantation. The present estimate for durability of most

implants is 7−12 years [110-112].

Common causes for implant rupture include inappropriate handling of the implant prior to or

during implantation, such as physical damage by instruments during the surgical procedure,

applying excessive mechanical force to the implant during handling, or folding or wrinkling

of the implant shell which may lead to its premature weakening [113]; iatrogenic causes; as

well as post-implantation damage, including excessive blow to the chest, compression of the

implant for the purpose of mammographic imaging; and severe compression by the fibrous

capsule [112].

Figure 8. (a) Estimated number of PIP implant ruptures as reported to the TGA, Australia by

year of implantation and % rupture rate by year of implantation as a fraction of implants sold


in that year [114]. (b-c) MRI scans of a broken implant. Green and orange arrows indicate sites

of rupture in silicone breast implants [115]. Reproduced with permission from ref. [115].

Copyright © 2016 Lubbock Avalanche-Journal. (d-f) Rupture of implants resulting from

capsular contracture [116].

According to a report published by the Unites States Food and Drug Administration agency,

the majority of silicone implant recipients will be subject to at least one ruptured implant within

11 years post-surgery, with the probability of rupture increasing with the residence time (Fig.8)

[112]. In 21% of the implant recipients, silicone migrated outside of the breast capsule, even

though most patients were unaware that this had happened [66]. FDA recommends magnetic

resonance imaging (MRI) for monitoring and early discovery of implant rupture. According to

2014 FDA guidelines, patients were recommended to undergo screening at 36 months after

implantation, and every 24 months thereafter [117]. The cost of MRI screening and the

associated secondary test recommended by FDA is also a major concern of the patients with

breast implants. If the implants are more durable less screening would be required, decreasing

the cost to health care system [118].

When the shell breaks, silicone gel filling of the implants can transform into liquid silicone at

normal body temperature, which facilitates silicone migration across the silicone shell, and

subsequently from the peri-implant milieu to the nipple ducts, auxiliary lymph nodes, pleura,

chest wall and upper arm, as well as to other organs [119]. The issue of silicone implant rupture

and leakage is not unique to breast implants, with cases showing the migration of silicone from

damaged calf implants to lungs [120]. Wear particles from silastic finger joints have reportedly

led to axillary lymphadenopathy and malignant lymphoma [121]. Considering the significantly

greater size of breast implants and their physical proximity to the lungs compared to finger

joint or calf implants, silicone breast implants may present a more serious health threat.


The reported rate of rupture can vary based on the type of implant and the methodology used

to estimate the rupture rate. In 2012, Berry and Stanek reported an implant rupture rate of

15−33% by conducting a study on 453 patients who used PIP (Poly Implant Prothèse, a

dominant global supplier of silicone gel breast implants) implants [122,123]. Another group

from Europe, Maijers and Niessen reported a rupture rate of 24% based on the study of 224

implants over 10 years [123]. An 18-month study reported by Crouzet et al. stated a rupture

rate of 3% in just 1.6 years [124]. A study conducted in France by Tropet et al in 2013 reported

a rupture rate of 8.7% in 4.7 year [125].

In a more recent study from Oulharj and colleagues, the rate of rupture of the PIP silicone

implants was estimated to be 7.7% [126]. The reported rate of rupture for the fifth generation

of silicone breast implants, which are supposed to be of the highest quality to date, was found

to be lower, at 2 % estimated for Mentor memory gel [127] and 3.8% reported for Natrelle

implants [128]. The latter value is also much lower than that reported for the current generation

Natrelle implants by Spear et al., who estimated the 10-year implant rupture rate at 7.7% [129].

The discrepancy in the reported estimates may at least in part be attributed to the limited

availability of information regarding the specific properties of each type of the implant, e.g. a

detailed description of polymer chemistry, mechanical and surface properties of the implants

used in these studies.

Table 1. A comparison between reported silicone breast implant rupture rates.

Article Year-

Published

Percentage of

rupture

Time period (in

years)

Berry and Stanek [82] 2012 15-33 % 10

Maijers and Niessen [83] 2012 24% 10

Crouzet et al. [84] 2012 3% 1.6


Tropet et al. [85] 2013 8% 4.7

Guillermo Blugerman et al.

[130]

2013 4%

12%

15%

2

4

< 6

Oulharj et al. [40] 2014 7% Not mentioned

2.3.4 Breast Implant–Associated Cancers

A rare and aggressive form of non-Hodgkin lymphoma (NHL), anaplastic large cell lymphoma

(ALCL) is a cancer of the lymphatic system, which involves an abnormal proliferation of

primarily larger in size T-cells with increased expression of the cytokine receptor CD30 and

displaying a cytotoxic immunophenotype [131]. Depending on their expression of anaplastic

lymphoma kinase, ALCL can be categorized as ALK-positive or ALK-negative. ALCL can

affect the lymph nodes and the skin [131].

Figure 9. A case of ALCL where the patient underwent left-sided capsulotomy with excision

of a firm capsular structure containing a seroma (right) [132]. A close up of bacteria formation

on implant (left). Reproduced with permission from Ref. 129 Copyright © 2015 Elsevier

publication and ABC news net [133].


An association between ALCL and breast implants was first reported in 1997 and since then

there has been an increasing awareness of this potential link and this is now identified as BIA-

ALCL (breast implant associated ALCL) [134,135]. There are at least over 500 known cases

worldwide but not all have been reported in the medical literature [136]. Textured implants

are associated with a greater number of cases of ALCL than are smooth implants [137]. It has

been suggested that textured implants provide a significantly larger surface area for bacterial

attachment, where the surface features may afford a physical protection to the cells from

detachment, aiding biofilm formation. This in turn increases the likelihood of inflammation

and infection-mediated development of scar tissue, and invokes an enhanced T-cell response

[138]. Persistent infection at the surface of the implant have been shown to lead to an increase

in the lymphocytic infiltrate dominated largely by T-cells, which suggests that a chronic

biofilm infection may lead to T-cell hyperplasia, which in turn is a possible cause of ALCL

[139]. Therefore, it is suggested that prevention of infection may reduce the risk of capsular

contracture and consequently reduce the risk of lymphocyte activation and possible conversion

to ALCL [140].

Table 2: Examples of recent ALCL cases related to breast implants

Author (year) Type of Implant Reason for

implantation

Presentation Number of

cases

Hart et. al [141]

2014

Saline textured Cosmetic Effluence 2

Talagas et. al [142]

2014

Saline Cosmetic Contracture 1

Sorensen et. al [134]

2014

Silicone textured Reconstruction Contracture 1

George et. al [143]

2013

Silicone textured Reconstruction Effusion 1


Zakhary et. al [144]

2013

Saline textured Reconstruction Effusion 1

Aladily et.al [145]

2012

Silicone textured

and Saline

Reconstruction Effusion 13

Current efforts focus on the possible treatment outcome for patients with this diagnosis and

the best form of treatment. This small group of patients is heterogeneous, which makes it

challenging to identify the most appropriate treatment modality [134]. Some of the reports

suggest that ALK-negative cases may follow a more indolent course, similar to the disease

pattern of cutaneous ALCL, rather than that of systemic ALCL, which has a worse prognosis

and mandates a far more aggressive treatment regimen [134,145].

2.4 Nanotechnology in breast implants

The emergence of nanomaterials and nanostructures, including nanocarbons such as graphene

and graphene-family composites, has revolutionized many areas of medicine and resulted in

the significant expansion of the inventory of implantable biomaterials and medical devices

[146-151]. Owning to their unique dimensions, particularly in terms of surface to volume ratio

that enables the majority of constituent atoms to be at the surface or the interface, materials at

nanoscale display rich chemistries and properties that are more “surface”-dependent compared

to their bulk counterparts [152,153].

As discussed in the previous section, the implants currently available on the market are unlikely

to last for more than 11 years, with some implants breaking with a period of few months post-

implantation. Furthermore, issues that arise from suboptimal interactions between cells and the

surface of the implant that lead to e.g. biofilm formation remain a significant healthcare

challenge for the use of these devices [154]. Infections can lead to capsular contracture and

subsequent rupture of the implant [87,155,156]. To address these issues, it is often necessary


to perform revision surgeries, e.g. to remove the capsule and/or the infected implant, and

administer a course of systemic antibiotics for prophylaxis and/or treatment. In terms of patient

outcomes, these issues may delay healing, lead to persistent pain and suboptimal aesthetic

outcomes, compromising the quality of life of the patient and increasing the financial burden

associated with this family of procedures [157]. For cancer patients, persistent infection and

chronic inflammation may compromise their recovery from mastectomy, and subsequently

affect the success of their cancer treatment and potential for cancer reoccurrence.

The aforementioned challenges have motivated industry and the scientific community to seek

novel approaches to improve the properties of breast implants, including those based on

introducing nanotechnology into various stages of breast implant manufacturing. The

hypothesis is that by incorporating nanomaterials into the already existing polymers, it may be

possible to reduce the incidence of implant rupture and biofilm development without affecting

the biocompatibility of the implant. Furthermore, this potential advancement in properties,

performance and durability could be attained at a relatively low cost with respect to material

cost (since most nanostructured are added in very small quantities), or integration into existing

process workflows. Indeed, the most effective solutions to address current implant challenges

are likely to involve an intelligent combination of nanomaterials, traditional antibiotics,

enhancers, and free drugs. Nanomaterials present an attractive set of tools that when used

wisely and appropriately may expand the current toolkit researchers have to combat implant-

associated challenges, augmenting (when used in combination with presently-available

strategies) and in certain instances providing a suitable alternative to existing strategies.

When designing a breast implant, generic number of general requirements are to be taken into

consideration, namely maximum stretch ability, minimum weight, high resistance to stress or

force, minimum cost, physical and chemical durability, excellent biocompatibility and minimal

toxicity. There is an evident need for novel or improved implant materials and coatings that


can resist rupture and pathogen colonization without compromising existing strength and

flexibility, and the process ability of these materials into aesthetically-pleasing implant shapes.

Recent discoveries have demonstrated that nanomaterials can be used to address critical issues

associated with implant-based breast reconstruction, specifically with respect to

biocompatibility, resistance to microbial colonization, longevity and mechanical strength. The

ability to use nanoparticles to address the former two issues relies on the ability of these

structures to directly interact with the target cells and/or physiological environment. This is

most commonly attainted through surface immobilization (e.g. for physical killing of attached

cells or production of reactive oxygen species in the proximity of the surface of implant) or

controlled elution of nanoparticles from the polymer matrix (e.g. where free nanoparticles

penetrate cellular membrane to induce intracellular oxidative stress). Where nanoparticles are

used realize the latter aspects of mechanical strength and durability, the benefits arise from the

unique chemical and physical interactions between the nanoparticles and polymer chains within

the composite. These benefits are most often attained by uniformly dispersing the nanoparticles

within the polymer matrix, however, concentrating the nanoparticles within the top-most layer

as a strategy to selectively enhance mechanical properties of the external shell may also be

considered. Therefore, select examples discussed in the following sections will focus on direct

cell−nanoparticle interactions when considering infection control and biocompatibility

challenges, and direct nanoparticle−polymer interactions when discussing mechanical

robustness and durability.

The idea of introducing microscopic particles in the form of fillers and other additives to

silicone implant is well-established and serves a wide range of purposes. For instance, a limited

quantity of amorphous silica (SiO2) filler is commonly added to liquid PDMS to enhance the

strength and elasticity, and thus overall performance of silicon rubber suitable for medical

applications [158]. The typical size of the particles is below 30 μm. Unlike crystalline silica,


the amorphous silica is considered to be more biocompatible. Similarly, other types of particles,

such as pigments, CaCO3, BaSO4, metal oxides etc., are introduced to enhance other

mechanical (e.g. stiffness), electrical, and biochemical properties of silicone elastomers, as well

as render the implants radiopaque. This principle can be extended even further to other

nanoscale materials with the aim to make the resultant composite material superior in terms of

key mechanical properties. However, this is not a trivial challenge since as-grown

nanostructures, e.g. carbon nanotubes, typically appear as an inhomogeneous mixture of

nanoparticles with different chiralities, inner and outer diameters, and aspect ratios, as well as

with significant differences in their chemical composition and presence of structural defects.

For example, carbon nanotubes often appear curled or twisted. Furthermore, most nanoparticles

have a propensity to aggregate. This tendency makes the preparation of uniform composites a

challenge, potentially compromising the mechanical strength and fracture resistance, the very

properties these admixtures are ought to enhance. Surface covalent and non-covalent

functionalization is often used to overcome this challenge and enable sufficient dispersion of

the nanoparticles within the polymer, creating a nanomaterial surface with greater chemical

affinity for and more efficient thermodynamic wetting with the surrounding polymer matrix.

In the field of healthcare, the interest in nanoparticles stems from the ability of researchers to

engineer them to be of the size similar to that of most biological molecules and structures, and

possess the unique size-dependent physical and chemical properties that may not be attainable

in bulk materials [159]. As such, nanomaterials can deliver significant benefits to in vivo and

in vitro research [160], spatially and temporally-controlled targeted delivery of agents for

treatment, imaging, sensing, and in modification of artificial implants to enhance treatment

outcomes and enable tissue regeneration and function restoration [87].

Despite the wealth of evidence that suggest that nanomaterials can both improve the

mechanical strength of implants and also impart bactericidal and other desirable biological


effects of materials, there is an evident lack of discussion on how these could be used to address

the specific challenges associated with the use of breast implants, particularly in the context of

post-mastectomy reconstruction and augmentation. Discussion below aims to bridge this gap,

suggesting a number of ways in which improvements can be realized through intelligent

introduction of nanoparticles into existing material systems as an adjuvant or alternative

strategy to conventional approaches.

2.4.1 Nanomaterials to control infection

Any surgical involvement associated with breast reconstruction and augmentation, such as

tissue removal, implant placement, implant filling, presents considerable medical risks, with

infection being one of the key risks for the breast implant use. There is a persistent need to

develop materials that could prevent attachment and proliferation of pathogenic

microorganisms on their surfaces without compromising the ability of these implant materials

to interact favourably with human cells and tissues [161]. After decades of intense

investigation, there is a large body of evidence regarding the antibacterial activity of

nanoparticles [162,163]. Nanomaterials generally display significantly higher chemical

reactivity in comparison with their bulk counterparts, attributed to the much higher ratio of

surface are to volume in the former [164]. This reactivity allows nanoscale materials to engage

more effectively with the target pathogen cells, i.e. a large percentage of atoms and functional

groups available on the surface of the particle can engage more effectively and/or uniquely

with the target cells, resulting in greater biological activity of these nanostructures.

Many nanoparticles such as Ag, ZnO, and CuO show excellent anti-bacterial activity and are

effective in preventing the growth and reducing the viability of a range of bacterial pathogens

responsible for nosocomial infections [165,166]. Numerous studies demonstrated their

effectiveness in preventing bacterial infection when used in conjunction with a polymer matrix,

with the composite used as a coating or as a bulk implant material. Owing to their enhanced


antimicrobial property, metal oxide nanoparticles are being introduced into a wide range of

medical coatings [167].

Figure 10. Possible antibacterial mechanisms of nanomaterials. Nanomaterials produce free

radicals, e.g. reactive oxygen species (ROS) which induce oxidative stress and irreversibly

damage bacteria (e.g., their membrane, DNA, and mitochondria), potentially leading to cell

death. Reproduced with permission from reference [168].

Antibacterial activity of the nanoparticles is generally attributed to several structural and

functional attributes, although the exact mechanism of cell-surface interactions and subsequent

nanomaterial toxicity is yet to be fully understood [169]. Most materials-based strategies rely

on highly controlled spatio-temporal elution of nanomaterials from the bulk polymer matrix or

top most coating in order to facilitate nanoparticle−cell interaction as well as facilitate chemical

changes in the nanoparticles induced by their interaction with physiological environment. Once

in their free form, nanomaterials are able to adhere to the cell membrane of the pathogenic cell

by means of electrostatic interactions, the nature of which is dependent on the compatibility

between chemical and physical characteristics of the cell and the surface [170]. Some

nanomaterials, such as graphene, may have sharp edges which disrupt the cell membrane and


facilitate physical penetration of the material into the target cell. Furthermore, some

nanomaterials, such as graphene, may induce oxidative stress by generating highly reactive

free radicals, inducing mitochondrial damage, lipid peroxidation, protein modification, and

DNA damage. At significantly high doses, these perturbations may result in irreversible

bacterial cell damage and cell death [171-173]. It should be noted that in the case of host cells,

damage from reactive species can lead to cancerogenesis. The mechanism of nanoparticle

toxicity depends on the combination of physical and chemical properties of nanoparticles,

namely their composition and surface functionalization, and the respective properties of the

interacting cells [174-177].

A number of studies have investigated the strong antibacterial property of graphene and

graphene-related materials, identifying oxidative damage as one of the possible mechanisms

responsible for the observed bactericidal and bacteriostatic effects [178]. The damage

resulting from reactive species negatively affects membrane integrity of bacterial cells, and

may result in the loss of intracellular content [179]. Recent studies show that dual polymer

functionalized graphene oxide (GO) and similar sandwich-like antibacterial structures

display an improved bactericidal activity against gram-positive S. aureus and gram-negative

E. coli in comparison with separate nanosheets or nanoparticles, or their composites [179].

These studies emphasize the improved antibacterial activity of nanocomposites in

comparison to individual nanomaterials [179,180].


Figure.11. TEM images of E. coli and S. aureus (a) Untreated E. coli. (b) Catechin-Cu

nanoparticles (20 ppm) treated E. coli. Arrows denote rupture of cells (c) Untreated S.

aureus. (d) Catechin-Cu nanoparticles (10 ppm) treated S. aureus. Arrows denote cell

rupture. Reproduced with permission from [181] Copyright © 2015, Macmillan Publishers

Limited

Coatings impregnated with nanoparticles have been successfully used in various dental and

bone implants [182,183] and as antibacterial coatings on existing biocompatible bulk materials.

While considering the anti-bacterial activity of nanoparticles, for an implant manufacturer it is

vital to consider the mechanism of action by which nanomaterials kill bacteria.

There are a number of nanoparticle materials that can be effectively kill pathogenic bacteria

upon contact without the need to penetrate into the cell, rendering these materials

particularly amenable for surface immobilisation approaches. Among these are vertically-

oriented graphene nanoflakes, which feature sharp edges oriented orthogonally to the

surface of the substrate and exceptionally large surface area. As discussed earlier, these


edges can mechanically disrupt the integrity of the membrane of sessile microorganisms,

leading to leakage of intracellular material, as well as release highly reactive oxidative

species in close proximity to implant surface to induce oxidative stress onto planktonic

pathogenic organisms. Given that vertically oriented graphenes can be engineered to have

exceedingly high contact angle, it is also possible to design super hydrophobic antifouling

surfaces that would prevent initial stages of cell attachment. Other nanomaterials in the form

of a coating can also be used to mimic the lotus effect and repel bacteria from attaching to the

implant. Though this method is effective, it is limited to the implant surfaces [184].

Furthermore, upon exposure to biological fluids, masking of nanotopography by protein

fouling can take place, limiting the efficacy of the lotus effect [185].

It should be noted that in the very similar way to traditional drug or biomolecule elution

strategies, mitigation of microbial attachment and biofilm formation using nanoparticle elution

suffers a number of challenges. These include attachment of proteins from the peri-implant

milieu and build-up of cellular debris that may prevent effective diffusion of necessary

concentrations of active agents to sustain the required level of antimicrobial efficacy, and a

closely related issue of sub-inhibitory levels leading to the development of bacterial resistance.

Considering that implants should ideally last for decades, the long-term sustainability of such

a strategy and its efficacy against late haematogenous infections, where pathogens are

introduced through blood or lymph from a distant focus of infection, e.g. dental infection, or

contiguous spread from an adjacent focus of infection, e.g. migration of pathogenic organisms

via lactiferous ducts. On the other hand, rapid release of nanomaterials can be toxic to adjacent

host tissues, and potentially accumulate within those tissues or be transported to other sites or

organs. From the translational point of view, even though nanomaterial elution may present an

attractive approach for limiting infection, it should be noted that the most suitable method for

fabrication of such composites is yet to be fully developed and optimized, as it depends on both


the desired end application and the polymer matrix used [186]. As previously mentioned, the

desired nanoparticles can be either integrated into the topmost layer of the polymer or

introduced into the matrix, however in the case of the former, that may limit the quantity of

nanomaterials that can be loaded and released from such a layer without a significant loss of

mechanical integrity of the layer. In the case of the latter, although it may be possible to attain

a greater level of total nanoparticle loading, it may be difficult to maintain the needed rate of

elution to effectively and reliably combat infectious agents over extended periods of time to

target not only sessile bacteria, but also that in the peri-implant milieu [187].

2.4.2 Nano-patterned surface to control capsular contracture

In some instances, capsular contracture is aseptic in nature that is it is caused by the natural

reaction of the tissues to the foreign body, i.e. the implant, in the absence of an infection [63].

It has previously been shown that surfaces with a higher roughness or a texture are associated

with a reduction in the amount of scar tissue formed around the breast implant [34, 35]. Indeed,

the micro environment created by the surface features of the implant is critical for cell

attachment and regeneration of breast tissue [188]. However, surfaces of presently-available

breast implants have relatively large surface features, the scale of which does not necessarily

correlates with that of biological features required for favourable cell-surface interactions.

Surfaces decorated with nano- and micro-patterns can be effectively used to control

differentiation, attachment, proliferation, and migration of a wide variety of cell types and

substrata, both in vitro and in vivo [189-192]. Numerous studies have shown nanomaterials as

excellent nano-scaffolds for cell growth and tissue regeneration [193]. For instance, the

cytoskeleton of human mesenchymal stem cells was shown to align along the 350 nm-wide

grooves on the substrate surface. Similar preferential alignment was observed in stem-cell-

derived osteoblasts grown on nanogrooves of polystyrene [194]. The attachment preferences

were influenced by the dimensions and orientation of the nanoscale features, particularly the


depth of grooves more so than their spacing or pitch. Indeed, deeper groves led to increased

cell orientation, whereas higher pitch or more frequent spacing of the groves had the opposite

effect [193]. Therefore, a coating or a pattern which can mimic that of host tissues, such as the

basal layer of the skin, can potentially provide a more suitable environment for the cells to

attach and grow, and as such significantly hinder the development of the capsule around the

implant.

On the other hand, it is important to consider the implications of introducing a nanostructured

surface on the potential interactions between said surface and bacteria. There is evidence that

certain types of features that constitute a rough surface may encourage bacterial colonization,

by providing a physical shelter for bacteria and thus preventing their physical detachment

through sheer flow (Fig. 11) [195]. For example, corrosion-associated micro-cracks on the

surface of the implant have been linked to an increased incidence of bacterial colonization.

Rougher surfaces may also provide more points of direct physical contact between the

pathogenic cell and the implant surface, facilitating bacterial attachment and promoting biofilm

formation [196]. It should be noted that ultra-smooth surfaces have also been shown to be

susceptible to a significant bacterial load, where attachment is facilitated by physical forces,

including van der Waals interactions, and a greater number of molecular attachment points

[197].

Complex surface morphologies with features at different length scales across multiple tiers

may offer a suitable solution, by having sufficiently small features that would interact with

bacterial cells preventing their attachment and settlement, as well as larger features to which

mammalian cells may respond. Indeed, a number of surface-immobilized nanoscale materials,

such as vertically oriented graphenes, have the capacity to selectively promote attachment and

govern orientation of mammalian cells while simultaneously effectively preventing adhesion

and biofilm formation by pathogenic microorganisms. From this perspective, alternations made


on the breast implant surface at nanoscale can be more effective in controlling bacterial

attachment and promoting tissue integration that chemical strategies alone, reducing the

likelihood of infection-related and aseptic “foreign body ” inflammatory responses. As such,

this strategy may be useful in minimizing clinical complications due to capsular contracture.

Figure.12. The mechanism of bacterial attachment to the implant surface. Reproduced with

permission from ref [196]. Copyright © 1996-2016 MDPI AG (Basel, Switzerland)

2.4.3 Nanomaterials for reinforcement

Rupture of the silicone breast implants remains a major issue ever since their invention. A

biocompatible material which can improve the strength without affecting the elasticity or the

biocompatibility of the implants is required. Because of the higher surface area of the

nanoparticles per unit of volume compared to bulk and microscale materials, their ability to

interact with other particles or molecules within a mixture is high. These interactions are

responsible for the increased strength and heat resistance, which is often observed in

nanoparticle-containing composite materials [198].

Silica fillers have been used from many decades as fillers in the manufacturing of implants.

Recently, silica nanoparticles have replaced the bulk silica particles in functional composites,

where they provide mechanical reinforcement or act as fillers [199]. The unique aspect of

polymer composites enhanced by silica nanoparticles lies in the ability to achieve highly-

uniform particle dispersion, which ensures the global improvement of the mechanical


properties of these materials [199]. In the case of nanomaterials, for a given volume of the

material, the number of atoms available on the outermost layer of the material decreases with

an increase in the size of the constituent particles [200]. For silica nanoparticles, more than

fifty percent of the silica atoms resides on the surface when the size of nanoparticle is less than

5 nm, which implies that they should have one or more ≡Si-OH functional moieties on their

surface, with the concentration being inversely proportional to the size of the nanoparticle

[201]. The physical, chemical and functional properties of the composites are determined by

the density and spatial distribution of these silanol groups on the surface of silica, since the

latter determines the specific surface area and the strength of the interactions between the filler

particles and the polymer matrix at their interface [199].

For instance, polyhedral oligomeric silsesquioxanes (POSS), the cage like molecules of Si and

O, are attracting significant attention for their ability to disperse in most common polymers,

notably enhancing the mechanical properties of the composite [202-206]. Of particular interest

is the propensity of these particles to connect not only to polymer chains but also to one another,

which opens up a possibility for creating fibre-like structures and one, two and three

dimensional scaffolds of crystalized particles within the bulk of the polymer [207]. Through

the reduction of interactions between the POSS particles, and formation of bonds of high

strength between the surface of filler particles and the polymer, it is possible to decrease the

viscosity of highly filled resins, while improving their mechanical properties and surface

quality [208]. Indeed, the dispersion of the POSS particles within the polymer matrix enhances

the mechanical strength, modulus, and stiffness, and reduces polymer viscosity, while

maintaining the low weight and ductility of the original polymer [209,210]. These improved

properties of the nanocomposites enables their use in a broader range of applications, for

instance, in the delivery of biologically-active molecules and therapeutics [211-213].


Apart from silica nanoparticles, nanostructures made of carbon, such as solid and hollow

nanofibers, vertically-oriented and horizontal graphene’s, nanodiamonds, and fullerenes hold

often unique and highly desirable combinations of physical, chemical, mechanical and

biological characteristics, which makes them well-suited for a range of bioengineering and

medical applications [214]. The exceptional mechanical strength and elasticity of carbon

nanostructures can be utilized to increase the mechanical strength of the implants without

affecting their flexibility [215]. Cylindrical fullers (CNTs) and graphene are of particular

interest owing to a highly-favourable and unique surface and mechanical attributes [216-218].

Table 3: Various types of silica-polymer nanocomposites. Adapted with permission from ref

[199]

Polymer matrix Silica size

(nm)

Silica

content

Major property changes (with

increasing nanofiller content)

Thermoplastic

polyurethane [219]

7 10 wt.% decreased Tg;

increased shear and storage modulus;

increased tensile and peel strength

Epoxy (DGEBA) [220] 75 and 330 1–5 wt.% increased aggregation level;

increased elastic modulus;

higher modulus for smaller particles

Epoxy (TGDDM) [221] 12.5 5 and

10 wt.%

decreased Tg, constant Tβ;

increased elastic and yield modulus

Acrylic polymer [222] 15–20 10–

50 wt.%

increased thermal stability;

enhanced hardness;

excellent optical transparency


Epoxy [223] 4000 14–

39 vol.%

increased Young’s modulus;

monotonic variation in the yield

strength and yield stress

DGEBA - Diglycidylether of bisphenol;

TGDDM- Tetraglycidyl 4-4′diaminodiphenylmethane

The elastic modulus of multi walled CNTs is estimated to be 1TPa, similar to that of diamond

[224]. In comparison with steel, the mechanical strength of CNTs is 100 times higher, whereas

the density is only 15% of the respective value for steel [225]. The compression strength of

CNTs is in the range of 100GPa. Given the outstanding properties of the CNTs, it may be

possible to use CNTs to enhance the properties of existing materials and to give rise to novel

composite materials with unique properties [226].


Figure 13. (a) Atomistic model of a CNT; (b) SEM images of CNT film [227]; (c) Photographs

of a CNT solid (10 × 10 × 4 mm3) compressed by a load of 800 N (2 MPa) with its thickness

reduced by 30%, and recovered to original shape after compression [228]. (d) TEM image of

multiwalled carbon nanotube. (e)Multiwall carbon nanotube just prior to (above) and after

(below) tensile testing [229].

Graphene, the 2D carbon nanostructure is identified to have the potential to enhance the

properties of ceramics, polymers and metal matrix composites for functional and structural

applications due to its superior Young’s modulus and tensile strength [230,231]. It is believed

that the major reason behind implant rupture is crack propagation. However, the ability of

graphene to resist crack propagation in a composite makes this material a promising candidate

for the reinforcement of breast implants. Introduction of graphenes or graphene-like materials

into the matrix of the polymer can greatly improve the fracture toughness of the composite

even when the concentration of the added nanomaterial is very low. Here, it serves to deflect

the crack and thus inhibit the propagation of the crack through the polymer matrix. The

integration of graphene into epoxy has shown to have increased the fracture toughness by


around 131% [232]. The advancement of a crack in a polymer or epoxy- graphene composite

is hindered when the crack encounters the graphene sheets, which are far more mechanically

robust when compared to the polymer (Fig.13). However, the extent of the improvement in

fracture toughness that can be gained from the addition of the graphene sheets, such as the

ability of the reinforcing phase to bridge the crack and strengthen the matrix will be

significantly affected by the dispersion of graphene within the composite, and the nature of the

interactions at the polymer-graphene interface (fig.13) [233-235].

Figure 14. Mechanism of crack propagation in epoxy matrix reinforced by graphene flakes;

(a) graphene agglomeration; (b) homogeneously-dispersed graphene. Reproduced with

permission from ref [235].

In graphene, the free movement of electrons is facilitated by Pz orbital which forms a π-bond

with a half-filled band. Graphene has a capacity to self-heal by spontaneously directing free

carbon atoms in its vicinity to fill the holes, and thus repair the graphene lattice at ambient

conditions. This property is the main reason behind graphene’s superior ability to deflect the

propagation of the cracks in the composite [235].


Even though the sp2 structure of graphene is similar to that of multi-walled CNTs (MWCNTs),

the former is significantly easier to disperse within the polymer matrix, probably due to the

differences in the nature of the interactions between these nanomaterials and polymer chains

associated with the specifics of their geometry and surface chemistry. Interestingly, microscopy

and diffraction studies have shown that the addition of graphene significantly improves the

dispersion of MWCNTs in silicone, likely due to the strong interactions between the graphene

flakes and both MWCNTs and polymer matrix, which leads to the graphene to act as a

compatibilizer. When these carbon nanostructures with exceptional mechanical strength and

elasticity of carbon nanostructures are added to the polymer matrix, this can increase the

mechanical strength of the implants without affecting their flexibility [236].

2.4.4 Nanomaterials for radiation therapy enhancement

In many cases, mastectomy is followed by a radiation therapy, where the former treatment

removes the primary tumor and any tissue likely to be cancerous, and the latter kills individual

cells which may have been left behind after the surgery or displaced, and prevent the migration

of these cells to other sites. Radiotherapy delivers precise doses of high-energy ionizing

radiation to the area likely to have cancerous tissues or cells, leading to apoptosis or necrosis

depending on the treatment dose [238]. In the treatment of breast cancer, the target sites for

radiation therapy include the chest wall and the proximal lymph nodes, including those located

in the collarbone and underarm regions. Even though most patients with both silicone and

saline breast prostheses can safely undergo radiation therapy, the physical presence of the

device within the breast pocket makes the planning of the procedure more complex.

Furthermore, since radiation therapy can increase the likelihood of inflammation and fibrous

tissue formation around the implant, it may lead to capsular contracture and breast hardening,

as well as to delayed healing [239]. Indeed, radiation treatment of chest plate may lead to

unintentional fibrosis of the tissues lining the lungs, leading to long-term symptoms, e.g. chest


pains, breathlessness, and coughing, and in some instances, permanent scarring of the tissues.

Implant-based solutions which would enhance the specificity and efficacy of the treatment

while lowering the therapeutic dose are of great interest.

Table 4: Mechanical properties of graphene/graphite-based polymer nanocomposites. Adapted

with permission from reference [237]. Copyright 2006-2016 Scientific Research Publishing

Inc.

Mechanical properties of graphene/graphite-based polymer nanocomposites

Matrix Filler type Filler loading

(wt.% a vol.% b)

Process % Increase

E

% Increase

TS

%Increase

flexural

strength

Epoxy EG 1a Sonication 8 -20

EG 1a Shear 11 -7

EG 1a Sonication

and shear

15 -6

EG 0.1a Solution

87

PMMA EG 21a Solution 21

GNP 5a Solution 133

PP GnP-1 3b Melt

26

GnP-15 3b Melt

8

Graphite 2.5b SSSP

60

LLDPE GnP 15a Solution

200

Paraffin

coated GnP

30a Solution

22

HDPE EG 3a Melt 100 4

UG 3a Melt 33

PPS EG 4a Melt

-20

PVA GO 0.7a Solution

76

Graphene 1.8b Solution

150


TPU Graphene 5.1b Solution 200

Sulfonated

Graphene

1a Solution

75

PETI EG 5a In situ 39

10a In situ 42

EG - expanded graphite; PMMA-poly(methyl methacrylate); GNP- Ionic liquid functionalized graphene;

PP-polypropylene; SSSP-solid-state shear pulverization; LLDPE -linier low density polyethylene; PPS-

poly(phenylene sulphide); PVA-poly(vinyl alcohol) ; TPU-Thermoplastic polyurethane ; PETI -

phenylethynyl-terminated polyimide.

The interest in the application of nanomaterials, particularly those containing metals, for highly

controlled spatio-temporal enhancement of specificity and efficacy of radiotherapy in treating

cancer has increased drastically over the past few years [240]. Since these densely packed metal

nanoparticles have the capacity to selectively absorb and scatter penetrating X-ray and gamma

radiation, and they can be engineered to selectively target specific tissues or cells, e.g. cancer

cells, it is possible to use them to localize and consolidate the treatment to the diseased tissues.

The use of nanoparticles can also facilitate greater likelihood of interaction between the

incident radiation and the target tissue [241,242]. The scattering of photoelectrons from the

surface of the metal particles as a result of their exposure to γ radiation can further increase the

therapeutic efficacy of the treatment. By taking advantage of these phenomena, it is possible

to reduce the treatment dose, and limit excessive organ and tissue damage [238].


Figure 15: Interaction of X-rays with high Z material nanoparticle. Reproduced with

permission from reference [238]. Copyright ©2009-2016Translational Cancer Research.

When an incident x-ray encounters a metal nanoparticle, a variety of physical events may take

place, including scattering of the x-rays and electrons, photoelectron scattering, and scattering

of fluorescence photons and Auger and Compton electrons, as shown in Figure 14. The energy

transferred from the incident way to the electron in an atom in the nanoparticle is sufficient to

eject an electron from its orbital. The ejected electron will gain a kinetic energy that equals the

energy of the incident wave less that required to remove the electron from the atom, which will

determine the distance it would be able to travel through the tissue. The likelihood of the

photoelectric effect depends on the energy of the incident radiation E, which should exceed the

energy that binds the electrons in the inner shell, and on the atomic number of the target atom

Z, expressed as Z3/E3 [243].The vacancy that arises from the removal of the inner-shell electron

can be filled by another higher-energy electron of the same atom. The excess energy is typically

released as photon emission, however, in some cases, another electron can gain this energy to

leave the atom. The energy of thus-released photons is relatively low compared to these so-

called Auger electrons, which have a significantly greater ionization density, however the


coverage range is higher in the former. As a material with large Z value of 79 and excellent

biocompatibility with host tissues, gold is well suited for the aforementioned photosensitization

reactions [238].

A coating of nanomaterial can potentially be applied to the breast implant surface to enhance

the outcomes of the radiotherapy treatment, which may be of direct benefit to patients who

have undergone cancer-related mastectomy. Among high Z particles, gold nanoparticles have

a suitable combination of photosensitization [132], chemical inertness, and excellent

biocompatibility. Since Au nanoparticles can greatly enhance the effects of radiation across

greater areas of cancerous tissues, this eliminates the need for delivering the nanoparticles to

every individual cancer cell. Furthermore, unlike iodine and other low molecular weight dyes,

the low systemic clearance of nanoparticles provides sufficient time for these particles to be

efficiently taken up by the tissues [244].

2.4.5 Wound healing promoted by nanomaterials

Biofilm development remains a serious medical complication in patients with breast implants.

In some instances, the site of surgical incision which remains after the insertion of the breast

implant heals slowly, particularly in the case of cancer patients where tissue repair is seriously

hindered by radiation therapy. Indeed, the lasting side effects of radiotherapy, such as skin

atrophy, excessive formation of fibrous connective tissue in irradiated organs and tissues, and

damage to the microvasculature responsible for the distribution of blood within tissues, are

associated with a significantly greater incidence of wounds that are difficult to heal or even

repair surgically [245,246]. These side-effects are not unique to radiation therapy, with several

types of anti-cancer drugs shown to induce an irritation to the veins. The leakage of an

intravenously-infused chemotherapy drug may induce serious, sometimes lasting severe

damage of the extravascular tissue and neurons, resulting in the development of necrotic ulcers


which are challenging to heal. In such cases, patients may suffer from pain, reduced limb

function and cosmetic disfigurement [247].

Recently, a variety of nanotechnology platforms based on 0D carbon dots and fullerenes, 1D

nanofibers, 2D graphene sheets, and 3D dendrimers, liposomes, and nanoemulsions containing

ceramic, metallic, and organic nanoparticles with a wide range of chemical, electrical, thermal,

magnetic and other properties have been developed for wound healing therapy [248]. Their

therapeutic effect and potency for wound healing is largely dependent on the chemical and

physical properties of nanomaterials, including size and shape, colloidal stability, surface

functionalization and surface charge [248]. Those nanoparticles which are both biodegradable

and biocompatible have the potential to provide additional benefits over conventional highly-

stable particles which cannot be degraded by the cell, and as such may persist in the body.

At the time of implant insertion, superior therapeutic effects can be achieved by a combination

of smart materials and technologies with conventional therapeutic strategies. Smart skins and

triboelectric nanogenerators are cable of harvesting biomechanical energy which can drive the

bioelectric dressing, the primary aim of which is to stimulate tissue regeneration and wound

healing. These could be used both as adjuvant therapy and as a stand-alone alternative to

conventional systemic antibiotics and antiseptics. In addition, electrically-controlled drug

delivery can be achieved within a dressing with the help of an electrically-responsive graphene

oxide nanocomposite film. In this approach of fast wound healing, the drug release can be

modulated by either endogenous or exogenous electrical current, which is not currently

available. Similarly, collagen or gold nanoparticle scaffolds could act as a biodegradable

structure which enables the fast migration of cells to the wound site, following which an

electrical stimulus can enhance the cellular activity [249].


2.4.6 Nanomaterials for drug delivery

Nanoparticles are rapidly emerging as a viable alternative to overcome limitations of traditional

drug delivery systems, with significant promise in the treatment of various types of cancer

[250].

The chemistry of polymer-based nanomaterial systems facilitates chemical modification,

expanding their potential in targeted oncotherapy [251]. The active drug molecule can be either

bound to the surface of the nanomaterial, or trapped within the polymeric core with the aim to

protect it from the immediate environment until the target tissue is reached. The unique

chemistry of polymers enables the generation of a wide variety of structurally and functionally

diverse systems, spanning solid polymeric nanoparticles, polymersomes and micelles,

conjugates, highly branched structures, e.g. dendrimers, ligand-conjugated polymers, and

polymer–lipid hybrid systems [251]. Their functionality can be further enhanced by

introducing specific functional groups and molecules on their surface, the addition of which

can significantly increase their residence time in blood, limit nonspecific distribution, and

enable high-specific ligand-mediated attachment to target antigens on the cell surface to

increase specificity [251]. Combination strategies, where passive targeting (via extended

residence time) is further enhanced by active targeting (via specific ligands) is considered a

promising strategy, with several methods developed and experimentally verified to shown

improved selectivity and efficacy over individual approaches [252].


Figure.16. Schematic illustration of graphene oxide -wrapped Dox-loaded mesophorous

silicon nanoparticle bound with Cy5.5-labeled AS1411 aptamer and the corresponding NIR

light controlled intracellular drug release. Reproduced with permission from ref [253]

Since the nanoparticles are already used in polymer nanocomposites to provide additional

strength, flexibility, and stiffness, it should be possible to extend their function to drug delivery

to enable tissue regeneration, e.g. as composite breast implant scaffolds for concomitant

delivery of chemotherapy agents and to sustain the growth of adipose tissue to replace excised

breast tissue [254]. For conventional silicone implants, by decorating the surface of the shell

with drug-carrying nanostructures and engineered nanotopographies, it may be possible to

guide cell attachment and proliferation [255]. These examples show that there is a possibility

of improving the surface of breast implant by incorporating nanoscale drug delivery systems,

with significant benefits for breast cancer patients.


Table 5: Systems for targeted delivery of bioactive agents based on polymeric nanoparticles.

Adapted with permission from ref [252]

Carrier

composition

Therapeutics Indication Status Targeting

Polymer-polymer micellar nanoparticles

PEG-PLGA

[256]

Lonidamine + Paclitaxel MDR breast

cancer

In

vivo

Active; EGFR

Methoxy PEG-

PLGA [257]

Doxorubicin + Paclitaxel Various

cancer

In

vitro

Passive

PEG-PLA [258,

259]

Paclitaxel, Etoposide, or

Docetaxel + 17-AAG

Various

cancer

In

vitro

Active; HSP90

PEG-PLA [259] Combretastatin A4 +

Doxorubicin

Various

cancer

In

vitro

Active;

angiogenesis

Polymer-Lipid micellar nanoparticles

P(MDS-co-

CES) [260]

Paclitaxel + Interleukin-12 or

siRNA

Breast cancer In

vivo

Active; Bcl-2

PEG-b-PHSA

[261]

Doxorubicin + Wortmannin Breast cancer In

vitro

Passive

Nonmicellar polymeric nanoparticles

PACA [262] Doxorubicin + Cyclosporine

A

Various

cancer

In

vitro

Passive

PEG-PLGA - polyethylene glycol-poly lactic acid; P (MDS-co-CES) - poly (N-

methyldietheneamine sebacate)-co-[(cholesteryl oxocarbonylamido ethyl) methyl bis

(ethylene) ammonium bromide] sebacate. PEG-b-PHSA: PEG-block-poly(N-hexyl stearate

l-aspartamide); PACA: polyalkylcyanoacrylate;


Table 6: Selected example of specific features of nanomaterials and their role in enhancing

performance of breast implant materials

Examples of

Nanomaterials

Proposed application Mechanism Key considerations References

Silver

nanoparticles

(nAg)

− control of infection − produce ROS,

− perturb function of cell wall,

− induce gene regulation

changes,

− interfere with metabolite

binding.

− nanoscale dimension

facilitate effective

transport to cells and

across cellular

membranes;

− large surface to

volume ratio enable

high level of

chemical activity;

− mechanism of

antibacterial action

different from

conventional

antibiotics reduced

development of

resistance and enable

use on antibiotic-

resistant pathogens;

− suitability for

controlled release

and surface

immobilization

applications.

[162, 263]

Metal oxide

nanoparticles

(e.g. ZnO)

− Control of infection − induce membrane

permeabilisation and

oxidative stress in

pathogenic bacteria;

− reduce cell viability and

inhibit cell propagation and

biofilm development.

− high chemical

reactivity due to high

surface to volume

ratio;

− small size promotes

cellular uptake;

− availability of

different particle

morphologies

enables tailoring of

nanoparticle-induced

biological effects;

− suitability for

controlled release

and surface

immobilization

applications.

[165,166]

Graphene

− control of infection

− control of rupture

− synergistic antibacterial

effect through contact

killing (via sharp edges of

graphene sheets penetrating

− suitable for surface

immobilization for

drug-free prevention

of biofilm formation;

[231, 264]


bacterial cell walls,

inducing leakage of

cytoplasm and intracellular

content and pathogen death)

and oxidative stress.

− chemical binding of

graphene sheets to polymer

molecules to enhance

mechanical stress and

prevent crack propagation.

− biocompatible to

human cell lines;

− unique chemical

structure enables

self-healing via

spontaneous re-

distribution of

carbon atoms to fill

and thus repair

damaged areas of

graphene lattice at

ambient conditions;

as such, superior

ability to deflect

propagation of

cracks in polymer

composites.

Gold

nanoparticles

− enhancing efficacy of

cancer radiotherapy

− selectively absorb and

scatter penetrating X-ray

and gamma radiation;

− enable controlled spatio-

temporal enhancement of

specificity and efficacy of

radiotherapy in treating

cancer;

− biocompatible to healthy

cells.

− a suitable

combination of

photosensitization,

chemical inertness,

and excellent

biocompatibility;

− increased likelihood

of interaction

between the incident

radiation and the

target tissue due to

scattering of

photoelectrons from

the surface of

particles as a result

of their exposure to γ

radiation;

− greater therapeutic

efficacy at lower

radiation dose.

[132]

Nanodiamond − promoting wound

healing after surgery

− highly biocompatible;

− large surface area

functionalized with

different ligand molecules

for conjugation of various

compounds or drugs;

− functionalized via covalent

and non-covalent bonding;

− attracts and binds insulin,

with resulting clusters in

alkaline wound areas

accelerating tissue healing

and regeneration;

− decreases incidence of

infection.

− efficient delivery of

drugs and

biomolecules to

enhance therapeutic

outcomes and

promote wound

healing;

− flexible sp2/sp3

bonds provide

particle with ability

to take on distinct

geometrical forms,

e.g. graphene plane-

like stretched face of

diamond and

[246]


diamond surface-like

puckered graphene,

giving a greater

degree of template

flexibility;

− bright fluorescence

(550−800 nm) from

nitrogen-vacant

(NV) defect centers

in nanodiamond

crystal lattice

enables biolabelling

and visualization.

Carbon

nanotubes and

nanowires

− control rupture − sp2 hybridization affords

superior elastic modulus

(for multiwalled CNT

~1TPa), mechanical

strength (100 times greater

than steel), and compression

strength (~100GPa);

− covalent attachment enabled

by radical or carbanion

additions or cycloaddition

reactions to carbon double

bonds at tube sidewalls and

edges;

− conversion of sp2 to sp3

hybridization has a low

energy barrier due to sp2

hybridized carbon atoms

being strained by the

curvature of the carbon tube.

− in the composite,

enables efficient

stress transfer from

weaker polymer to

mechanically

superior individual

nanotubes;

− improves tensile

modulus;

− limits crack

propagation;

− covalent and non-

covalent

functionalization of

nanotubes control

surface wetting by

polymer matrix;

− non-covalent

attachment enabled

by π-stacking

interactions of

graphitic sidewalls

with polymers

containing

conjugations or

heteroatoms with

free electron pair.

[224, 225]

Dendrimers − facilitating controlled

delivery and spatio-

temporal release of

drugs and

biomolecules for

breast cancer treatment

and prophylaxis

− a cage-like structure of

dendrimers enables

entrapment of therapeutic

agents via host-guest

interactions and covalent

bonding;

− large loading capacity

achieved by a high ratio of

surface groups to molecular

volume of dendrimers.

− unique shape and

small size enables

extensive drug

incorporation and

functionalization of

surface with specific

antibodies for

enhanced targeting;

− efficient delivery and

controlled release

[265]


significantly reduces

effective doses for

adjuvant cancer

therapy in patients

undergoing breast

reconstruction after

mastectomy and for

tissue regeneration.

Nano textured

surfaces

− preventing capsular

contracture

− nano topography as a

chemical-free means of

controlling cell and protein

attachment;

− nanopillars of high aspect

ratio induce high stresses in

cellular membranes upon

direct contact, leading to

cell rupture, loss of

intracellular content and cell

death;

− by controlling spacing, the

possibility to preferentially

deter settlement of specific

types of organisms;

− ability to control chemistry

while preserving physical

killing effects;

− patterns, e.g. oriented

grooves, arrays, and

plateaus provide mechanical

stimuli to promote cell

differentiation, orientation

and migration, and drive

tissue formation.

− simultaneous control

of host

tissue−implant

surface interactions

to minimise ‘foreign

body reaction’,

inflammation and

scar tissue formation,

and control of

pathogenic

colonization to

prevent implant-

associated infection;

− efficacy against

broad range of

pathogenic

organisms, including

Gram positive and

Gram negative

bacteria and fungi;

− prevention of biofilm

formation through

inhibiting first stages

of microorganism

attachment (e.g.

control of surface

energy), as well as

direct physical

killing of attached

organisms.

[266]


2.5 Current trends in breast augmentation

Although silicone remains a primary material of choice for breast reconstruction, other

biomaterials, such as nanostructured cellulose, are also attracting interest. Similar to silicone,

nanocellulose can be organized into a variety of 3D structures, with controlled thickness and

architecture. In addition to this, nanocellulose has remarkable physical properties, exceptional

surface chemistry and outstanding biocompatibility, biodegradability and low toxicity, which

makes this material a better candidate to be used in biomedical implants [267].

The main advantage of nanocellulose or other biodegradable scaffold material over silicone

lies in their ability to support tissue regeneration, which is a promising direction in the field of

breast reconstruction. This approach to breast reconstruction involves expanding stem cells in

a culture, and then introducing these cells into a biomaterial, where the latter provides a

physical framework for cell proliferation and tissue formation [268,269]. At present, adipose-

derived stem cells are the primary cells for breast reconstruction procedures due to their relative

abundance and ability to undergo differentiation into epithelial cells. By harnessing the

advances in micro- and nano-scale fabrication, including 3D printing, it may be possible to

construct autologous tissue models with high degree of control over their mechanical,

chemical, biological and geometric characteristics across macro-, micro- and nano-scales

[270].


Figure 17. Breast reconstruction achieved by sustained regeneration of high-volume adipose

tissue. (a) Laser scanning is used on the patient prior to mastectomy to generate a 3D model of

the beast. (b) A porous patient-specific scaffold is then created using 3D printing of poly (D,

L)-lactase polymer, displaying good homogeneity of filament thickness (B, inset) and 90%

porosity across the volume of the implant (c). (d) Fluorescence imaging of the scaffold

construct in mice shows substantial increase in adipose tissue over time. (e) And (f) Top and

side views are of scaffolds as-fabricated (left) and after 24 weeks of implantation. Explanted

scaffolds show the development of well-vascularised adipose tissue. Reproduced with

permission from reference [270].

In addition to cellulose, there is a range of other polymer materials that are suitable for the

fabrication of scaffolds for adipose tissue regeneration. For instance, poly(d,l)-lactide breast


scaffolds were with pore sizes of greater than 1 mm were 3D printed (via fused deposition

modelling) to follow the shape and size of the patient’s breast, with the data acquired using a

3D scanner prior to undergoing mastectomy [271]. Polycaprolactone is another common

polymer material used in 3D printing of soft-tissue scaffolds. In vivo studies of 3D printed

polycaprolactone scaffolds implanted in subglandular pockets in immunocompetent minipigs

showed suitable levels of angiogenesis and adipose tissue regeneration over 24 weeks of

observation [272]. In the study, prevascularisation and delayed fat-injection were utilized to

enable regeneration of large volumes of adipose tissue, since a simple transfer of significantly

large volumes of adipose tissue into the scaffold is frequently associated with adipose tissue

necrosis due to insufficient vascularization and consequent formation of oil cysts.

Hyaluronic acid (HA) based scaffolds are yet another category of natural materials used for

adipose tissue engineering. Owning to an attractive combination of chemical, physical and

biological properties, HA specifically interacts with endogenous receptors on cells, actively

stimulating tissue growth and remodelling. However, its strong hydrophilic nature has an

adverse effect on the mechanical properties when used as a scaffold. In addition to this,

processing and handling of unmodified HA is challenging, with chemical modifications

required to make this material more suitable for practical tissue engineering applications [273].

An alternative approach to improving mechanical properties of HA-based scaffolds is by

combining HA with other biocompatible and bioresorbable materials into composites. For

instance, in vitro performance of hybrid collagen−HA scaffolds for adipose tissue engineering

have recently been explored. The combination of these two materials results in a biocompatible

material with increased stiffness, mechanical strength and enhanced cell proliferation rate.

Unlike HA scaffolds, collagen−HA scaffolds exhibit slower water dissolution rate due to a

higher degree of crosslinking in the composite scaffold, significantly improving mechanical

strength such a scaffold can provide during tissue regeneration [274].


The possibility of using a known anti-tumor agent, e.g. tannic acid, as a collagen cross-linking

agent for fabrication of collagen breast implant scaffolds with anti-cancer property have also

been investigated [275]. A hydrolysable plant tannin, tannic acid induces collagen cross-

linking via a combination of hydrogen bonding and hydrophobic effects. The study showed

that it was in principle possible to selectively alter metabolic activity of healthy versus cancer

cells [275].

Clinical studies of 139 patients receiving an implant-based breast reconstruction using a long-

term bioresorbable silk-derived biological scaffold showed high level of patient and

investigator satisfaction, with complication rates reported to be of the same level as those for

other implantable soft-tissue support materials [276].

Scaffolds using synthetic polymers have also been widely investigated. For example, poly

(glycolic acid) fibre-based matrices reinforced with poly (L-lactic acid) showed to provide

sufficient mechanical support for regeneration and maintaining volume of adipose tissue in

vivo using a murine model [277]. Fluortex monofilament-expanded polytetrafluoroethylene

scaffolds with an average pore size of ~50μm have also been investigated in vitro, with

scaffold functionalization with collagen, albumin, and fibronectin needed to facilitate

preadipocyte attachment and proliferation [278].

Although the tissue engineering approach of fabricating customized scaffolds using 3D

scanning/printing opens a new way for breast reconstruction, its practical realization faces its

own challenges. Similar to silicone implants, the material from which the scaffold is

constructed should be non-toxic both in its intact form and as the products of its

biodegradation, mechanically flexible yet robust, and importantly it should maintain its

mechanical properties for the length of time necessary for the tissue to develop [279,280].

Furthermore, the material should allow for diagnostic imaging for early stage detection of


breast cancer. It is important that the products of polymer degradation do not elicit any

response that may lead to the recurrence or development of new cancers.

For scaffold development, nanomaterials can not only provide the necessary reinforcement but

to enable highly-controlled delivery of biologically-active agents, such as drugs or growth

factors, which can be gradually released as the scaffold degrades, providing the necessary

biochemical cues for each stage of tissue development. In addition to this, the nanoscale

scaffold materials, which possess high mechanical stability, are porous and biodegradable,

which makes this material an ideal candidate for biomedical applications [280].

2.6 Conclusion and perspectives

There is a large body of evidence that suggests that silicone breast implants have the capacity

to positively contribute to the patients recovering from breast cancer through providing an

avenue for maintaining healthy body image, improving sexuality and self-esteem, and thus

positively contributing to the overall quality of life of the patient. As both the incidence of

breast cancer and the use of implants continue to increase, the burden associated with implant-

related health issues as a result of implant leakage and rupture, bacterial infection, capsular

contracture, tissue necrosis and nerve damage, and associated delayed recovery, severe pain

and loss of function will continue to increase. This paper reviewed the potential of new and

modified materials to address these challenges, with a particular focus on the benefits that can

be delivered by nanostructured and nanoscale materials for patients undergoing or recovering

from breast cancer treatment with the goal to not only minimize the likelihood of medical

complications but possibly offer more realistic, aesthetically pleasing outcomes for the patients.

Among the various materials, polymers reinforced with nanomaterials are most promising for

long-term implantation strategies. In addition to improving the properties of the implants,

nanomaterials can also enable implant-based drug delivery for cancer patients, reduce bacterial

infection, and facilitate self-healing to mitigate tear propagation. Nanostructured biodegradable


materials may provide the necessary framework for tissue regeneration. Regardless of the

strategy selected, namely long-term stable implants for tissue replacement or biodegradable

implants for tissue regeneration, the future of the breast implants will rely on and will be driven

by the advancements in materials engineering.

However, the new or modified materials, just like any new implant surface structure or shell

material would need a clinical trial and depending on the degree of difference from current

devices it will be impossible to make any performance claims without extensive pre-clinical

and clinical data. These can significantly increase the time needed for translation of the newly

developed material or concept from laboratory into clinical setting. Typically, in breast implant

field, clinical trials can take 2-3 years, which increases to 5+ years once one considers the time

involved in trial set up and final reporting. Thus, it is not unexpected for companies to spend

5+ years to have the data ready for regulatory submission to FDA or CE.


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3Chapter 3: Research design

This chapter describes the design adopted by this research to achieve the aims and objectives stated

below.

OBJECTIVES

Key Objectives of this research work are:-

Study different synthesis techniques for nanocarbon materials.

There are different synthesis techniques available for the production of

nanocarbon materials, such as chemical vapor deposition (CVD), the epitaxial

growth on silicon carbide, liquid exfoliation of graphite crystals, chemical

reduction, detonation, etc.

The cost and quality of nanocarbon can vary dramatically depending on the

synthesis technique used. In this project, nanocarbon will be synthesized using

Hummers methods and plasma enhanced chemical vapor deposition (PECVD).

Based on the quality and properties of nanocarbons synthesized by these

methods, decision is made on their suitability for the composite.

Understand the ratio of polymer matrix to nanocarbon

For the preparation of nanocarbon-reinforced composites, it is important to

determine the optimal weight proportions of nanocarbon which can make a

composite with improved mechanical strength, without affecting its flexibility

or biocompatibility.

Normally the percentage of nanocarbons added to polymers for making

membranes are less than 5%. Using this benchmark as a guideline, this project

will aim to produce composites that contain less than 5% of nanocarbon to

minimally affect the current cost of implants.


3.1 Methodology

In order to achieve above aims and objectives, nanocarbon materials will be synthesized

through different methods, mixed with polymer in different compositions, to determine an

optimal type/fraction of nanocarbon to be used to maximize composite mechanical durability.

Then, the biocompatibility of most mechanically robust nanocarbon-polymer composites will

be examined.

Figure 1: Research Methodology

3.2 Instruments

A variety of instruments were used in this thesis, which include, not limited to ultrasonicator,

tubular furnace, scanning electron microscope, transmission electron microscope, atomic force

microscope, Instron universal tester, nanoindenter, confocal microscope, etc.


3.3 Procedure and Timeline

As the work involves material synthesis, characterization, and mechanical and biological

testing, the work will be conducted in four different phases.

Phase 1:

In the first stage, different types of carbon nanomaterials, e.g. those based on graphene, are

synthesized by different techniques. Thus synthesized materials are characterized using

scanning electron microscopy (SEM), transmission electron microscopy (TEM), confocal

micro Raman spectroscopy and x-ray photoelectron spectroscopy (XPS).

Phase 2:

The second stage involves reinforcing silicone with carbon nanomaterial in order make the

polymer material stronger. This phase includes selecting different weight proportions of

carbon nanomaterial which is to be mixed with the polymer. After synthesizing nanocarbon -

polymer composites, the composites should undergo various mechanical testing including but

not limited to:-

Tensile testing

Tensile tests are done on a mechanical tester and it measures the force necessary to break a

composite and the extent to which the sample stretches or elongates before it reaches its

breaking point. The output form the tensile tests will be stress-strain graph, which can be used

to find the Young’s modulus.

Also the tensile test can provide details regarding tensile strength, tensile strain and percentage

elongation of the sample.

Nano indentation

Nano indentation tests are used to measure hardness samples at nanoscale. It is one of the most

known ways of testing mechanical properties of any material. Nano indentation tests give an

idea about the plastic or elastic deformation of the samples.


Tear Testing

Tear tests are performed in a similar manner to tensile testing, and give a value of the tear

strength of a particular material.

The mechanical testing should be done on the samples right after synthesis and at various

intervals to determine whether the sample shows the same properties if it is stored for a long

period of time. The composite or the sample which shows the finest mechanical properties

will be taken for further studies. (Instrumental parameters were determined to obtain the best

details about the mechanical properties).

Phase 3:

Phase three involves biological testing of the sample. Here, the effects of composite properties

on bacterial and eukaryotic cell attachment were taken into consideration.

Biocompatibility

Due to the variation in cell physiology, the toxic effect of materials is highly dependent on the

type of the cell studied. In this project, we are concentrating on human dermal fibroblasts

which the most are commonly found cells in the empty pockets of human breast. In order to

identify the exact effect of the composite on the cells, in vitro studies will be done with the

sample by creating cytotoxicity assays following the protocols outlined in literature.

Surface Morphological Studies

In this project, surface morphological studies will be conducted to view the attachment of the

cells to the surface of the sample. Microscopic techniques like SEM and AFM will be used

for this purpose.

Antibacterial Studies

It is gram positive bacteria which often cause bacterial infection on the breast implant surface,

so in this study gram positive bacteria will be used to test their attachment and biofilm

formation on the surface of the composite.


Phase 4:

Then the final step involves comparing the product with the implants already available on the

market so as to evaluate the quality of the new implants.


4Chapter 4: Research Findings

This chapter of this thesis is published in Scientific Reports on 8th May 2017:

Synergistic bactericidal effects of reduced graphene oxide and silver

nanoparticles against Gram-positive and Gram-negative bacteria

Karthika Prasad, G. S. Lekshmi, Kola Ostrikov, Vanessa Lussini, James Blinco,

Mandhakini Mohandas, Krasimir Vasilev, Steven Bottle, Kateryna Bazaka &

Kostya Ostrikov

Doi: 10.1038/s41598-017-01669-5



Statement of Contribution of Co-Authors for

Thesis by Published Paper


6. they meet the criteria for authorship in that they have participated in the conception,

execution, or interpretation, of at least that part of the publication in their field of expertise;

7. they take public responsibility for their part of the publication, except for the responsible

author who accepts overall responsibility for the publication;

8. there are no other authors of the publication according to these criteria;

9. potential conflicts of interest have been disclosed to (a) granting bodies, (b) the editor or

publisher of journals or other publications, and (c) the head of the responsible academic

unit, and

10. they agree to the use of the publication in the student’s thesis and its publication on the

QUT’s ePrints site consistent with any limitations set by publisher requirements.


“Synergic bactericidal effects of reduced graphene oxide and silver nanoparticles against

Gram-positive and Gram-negative bacteria.” Published in Scientific Reports on 8th May

2017.

I have sighted email or other correspondence from all Co-authors confirming their certifying authorship. (If the Co-authors are not able to sign the form please forward their email or other correspondence confirming the

certifying authorship to the RSC).



Karthika Prasad Conception of idea. Designed and conducted the experiments,

analyse the data and wrote the manuscript.

28/11/2018

G.S LakshmiAssistance in designing, conducting antibacterial studies and

analysing data and revising manuscript

Kola Ostrikov Helped with analysing antibacterial studies

Vanessa Lussini Assisted in executing ESR study

James Blinco Assisted in interpreting the ESR study

Mandhakini Mohandas Advised on properly executing the research

Krasimir Vasilev Assisted in interpreting the antimicrobial part of the paper.

Steven Bottle Assisted in interpreting the results

Kateryna Bazaka

Supervised manuscript assembly, provided her expertise to interpret

results and propose key mechanisms of activity, helped in writing

of the article

Kostya Ostrikov Supervised the research, helped with data analysis and

interpretation and major manuscript editing

Name Signature Date




PREFACE

The focus of this chapter is on mitigation of pathogen attachment and biofilm formation

through surface functionalisation and elution of bioactive carbon-based nanoparticles.

Antibacterial properties of graphene-based materials in suspension are studies here with a view

of their application as an active agent in eluting systems.

This paper studies the antibacterial properties of reduced graphene oxide/silver nanocomposite

as a promising material for deactivation of common human pathogenic bacterial strains,

including gram positive and gram negative bacterial species. Along with giving insights into

the traditional synthesis of nanocarbon composites, the article also outlines synthesis and

fundamental properties of rGO/Ag nanocomposite, and proposes a mechanism for the observed

biological activity.


Abstract

Reduced graphene oxide (rGO) is a promising antibacterial material, the efficacy of which can be

further enhanced by the addition of silver nanoparticles (nAg). In this study, the mechanisms of

antibacterial activity of rGO−nAg nanocomposite against several important human pathogenic

multi-drug resistant bacteria, namely Gram-positive coccal Staphylococcus aureus and Gram-

negative rod-shaped Escherichia coli and Proteus mirabilis are investigated. At the same

concentration (100 μg/ml), rGO−nAg nanocomposite was significantly more effective against all

three pathogens than either rGO or nAg. The nanocomposite was equally active against P. mirabilis

and S. aureus as systemic antibiotic nitrofurantoin, and significantly more effective against E. coli.

Importantly, the inhibition was much faster in the case of rGO−nAg nanocomposite compared to

nitrofurantoin, attributed to the synergistic effects of rGO-nAg mediated contact killing and

oxidative stress. This study may provide new insights for the better understanding of antibacterial

actions of rGO−nAg nanocomposite and for the better designing of graphene-based antibiotics or

other biomedical applications.


4.1 Introduction

According to the report published by World Health Organization, bacterial resistance to

antibiotics is a major global threat to public health akin that posed by global warming and

terrorism [1]. In the European Union alone, annual health care costs and productivity losses

attributed to bacterial resistance by major health care-associated bacterial infections is

estimated to approach 2.5 million hospital days, 25,000 deaths and economic losses on the

order of €1.5 billion[2]. Unsurprisingly, there is a significant interest in the development of

novel strategies to combat the spread of resistant microorganisms, e.g. by developing new

antibiotics and other therapeutics [3-5]. Alternative therapies that positively contribute to the

rational use of conventional antibiotics are particularly highly desired [6].

Recently, graphene-based materials have emerged as promising antibacterial materials [7-11].

Originally actively researched for their excellent thermal, mechanical and electrical properties

that make them well-suited for such applications as energy devices, sensors, and field-effect

transistors [12, 13], chemically modified graphenes such as graphene oxide (GO) and reduced

GO (rGO) have been shown to inhibit the growth of several clinically-relevant pathogens,

including Escherichia coli [14-16]. The observed antibacterial activity of GO and rGO has

been attributed to the favourable combination of physical structure and chemical functionality,

[17] where the basal planes and edges of GO are decorated with exogenous functional groups

such as hydroxyl, epoxy group and carbonyl groups [18, 19]. Upon contact with such a

nanostructure, membrane stress induced by the sharp edges of graphene nanosheets has been

shown to cause significant physical damage to cell membrane, and subsequent loss of bacterial

membrane integrity and leakage of intracellular material [20]. As the case with other

nanomaterials, smaller sized nanoparticles (<10 nm) of rGO were found to exhibit higher

antibacterial activity, owing to the favourable combination of high surface area and mobility

across cell membrane [21, 22].


Stronger antibacterial activity can potentially be achieved by combining nanomaterials with

complimentary action against multiple bacterial targets [23]. The present study explores

whether it is possible to complement membranolytic and oxidative activity of rGO with the

free radical formation of silver nanoparticles. The antibacterial properties of silver and silver-

based nanomaterials are well-documented [24]’ [25, 26]. The benefits of Ag ions and Ag

nanoparticles include their efficacy against both Gram-positive and Gram-negative bacteria,

and a multifaceted mechanism of action. This multifaceted mechanism of action translates into

attacking the bacteria on several fronts (e.g. blocking respiration by binding to bacterial DNA,

binding to enzyme to block energy cycle, binding to protein disulfide bridges to disrupt

function), which makes it difficult for bacteria to develop resistance. This gives silver

advantages compared to traditional antibiotics which typically target only a single site of the

bacterium cell. Importantly, Ag nanoparticles (nAg) show low or no cytotoxicity to human

cells and are suggested in some reports that silver nanoparticles aids in reducing inflammation

[27-30]. This combination of properties makes silver and silver nanoparticles very attractive in

protecting medical devices prone to being infected.

This investigation aims to explore the mechanisms of activity of rGO−nAg nanocomposites

against pathogenic multi-drug resistant bacterial species, namely Escherichia coli (gram

negative), Staphylococcus aureus (gram positive), and Proteus mirabilis (gram negative).

4.2 Results

Structural and morphological characterization of nanomaterials: Successful formation of

rGO was confirmed by SEM microscopy. The microstructural analysis (Figure 1.a) shows a

sheet-like structure with wrinkles, and a relatively large number of reactive edges indicative of

the formation of rGO nanoflakes. SEM imaging confirmed a well dispersed solution of silver

nanomaterials having approximately spherical shape (Figure 1.b). The rGO dispersion


remained homogeneous for several days, which facilitated uniform dispersion and binding of

Ag nanoparticles to rGO sheets (Figure 1.c).

Figure 1. SEM images of synthesized nanomaterials. (a) rGO nanosheets with a large number

of reactive edges, (b) nAg nanoparticles of uniform size and near spherical shape, (c) rGO−nAg

composite showing uniform distribution of nAg.

The morphology of rGO−nAg nanocomposites was examined using high-resolution

transmission electron microscopy (HRTEM). The HRTEM images presented in Figure 2a

showed nAg with an average diameter of 5.36 nm to be uniformly distributed on the rGO . The

lattice fringes of nAg shown in Figure 2b confirm the crystalline structure of nAg. The particle

size distribution of rGO−nAg nanocomposite (Figure 2c) was estimated using Image J software

and HRTEM image shown in Figure 2a. Based on the size distribution histogram and HRTEM

images the size of the nAg was in the range of 1-15nm.


Figure 2. Representative HRTEM images of (a) rGO−nAg nanocomposite, (b) lattice resolved

image of nAg in rGO−nAg nanocomposite. (c) Size distribution histogram of nAg in rGO-nAg

nanocomposite presented in (a).

The FTIR spectra (Figure 3.a) of rGO significantly differed from that of GO. The peak at 3500

cm−1 is typically attributed to O–H stretching vibrations of adsorbed water molecules and

structural OH groups, and the peak at 1600 cm−1 is attributed to O–H bending vibrations [31,

32]. The presence of carboxyl and epoxy functional groups can also be detected at around 1734

cm−1, 1225 cm−1and 1053 cm−1, respectively [32]. Due to thermal reduction, some oxygen-

containing functional groups are partially removed. The intense absorption band at 3500 cm–1

is decreased after reduction. The carboxyl stretching vibration is also decreased. The absorption

intensity of the band at 1080 cm–1, which is assigned to epoxide (C–O–C) group, is also

weakened in reduced graphene oxide [33].


Figure 3. Reduction of GO to rGO and subsequent incorporation of nAg was confirmed

spectroscopically: (a) FTIR spectra for GO and rGO; (b) XRD of rGO and rGO−nAg

composite; (c) UV spectrum of nAg; (d) UV−Vis spectra for rGO and rGO−nAg composite.

The investigated structure diffracts the monochromatic beam of x-rays. As can be seen on the

spectrum for rGO (Figure 3.b), a new high index, strong broad peak is obtained at 2θ = 24.1°

for (002) plane and a small peak is obtained at 2θ = 42.91°for (100) plane. It is the transitional

stage between graphene oxide and graphene, as rGO is obtained with a peak value at 2θ = 23.1°

for (002) plane and a small index peak of graphene existence is observed at 2θ = 43° for (100)

plane with inter layer distance of 0.37 nm [34]. For the rGO−nAg composite, along with the

observed diffraction peaks at 2θ = 23, 43°, the XRD pattern also showed peaks at 38°, 46° and

a

d

b

c


64°, which according to the JCPDS files 04-0783 and 84-0713, correspond to (111), (200) and

(220) crystal planes of nAg. This confirms the formation of rGO−nAg composite.

The addition of nAg particles to rGO produces a characteristic absorption band at 426 nm

(Figure 3.c-d). That is, an intense longitudinal band has appeared due to the contribution from

the dipole oscillation along the long axis of the nanomaterials [35]. The rGO−nAg formation

was visually confirmed as a continuous color change of the solution from light yellow to grey.

The UV-Vis spectroscopy of rGO showed a peak red-shifted to 260 nm, confirming successful

GO reduction (Figure 3.d).

Zone of inhibition: Plates were inoculated with S. aureus, E. coli and P. mirabilis and allowed

to grow to achieve confluency. Wells containing various concentrations of rGO, nAg,

rGO−nAg, or standard antibiotic nitrofurantoin were made in the plates. Zones of inhibition

were measured after 24 hr. of incubation. Figure 4 shows representative images of plates.

Figure 4. Well diffusion study. Representative plates of (a) P. mirabilis, (b) S. aureus, and (c)

E. coli. Red circles indicate the zone of inhibition from wells loaded with nitrofurantoin; yellow

circles indicate the zone of inhibition from wells loaded with rGO−nAg.


Table 1. The average zones of inhibition (in mm) of rGO, nAg, rGO−nAg, and nitrofurantoin.

Isolates P. mirabilis E. coli S. aureus

rGO

crude

12±2

10±1

11±1

50µg/ml No zone No zone No zone

100 µg/ml No zone No zone No zone

200 µg/ml 18±2 9±1 No zone

nAg

100µg/ml No zone No zone 8±1

rGO−nAg

100µg/ml 23±2 25±2 24±1

Nitrofurantoin

100µg/ml 24±2 No zone 26±1

As expected, the zone of inhibition for rGO and nAg was concentration-dependent. The

concentrations of 50 µg/ml and 100 µg/ml of rGO and nAg, respectively, were insufficient to

inhibit the organisms tested. At these concentrations, nitrofurantoin inhibited P. mirabilis and

S. aureus, but not E. coli. rGO−nAg nanomaterial composite showed strong activity, inhibiting

all pathogens tested, including E. coli shown to be resistant to standard antibiotic.


Figure 5. Viable count of bacteria after exposure to (a) rGO, (b) nAg, (c) rGO−nAg composite,

and (d) standard antibiotic nitrofurantoin.

Survival rate vs time: The survival rate vs time was calculated for both gram positive S.aureus

and gram negative P. mirabilis. At the end of each exposure time of 24 hours, the samples

were inoculated on plate count agar (PCA) and the results were tabulated as colony forming

units (CFU/ml). Complete inhibition was detected at the end of 4 hours incubation in the

presence of rGO and nAg, while rGO−nAg demonstrated complete inhibition at the end of 2-

2.5 hours (Figures 5).


4.3 Discussion

Infections caused by multidrug resistant (MDR) isolates are usually difficult to treat. The

pharmaceutical industry is now facing a great challenge due to the evolution of multidrug

resistant and pandrug resistant organisms. The discovery of new effective antibacterial agents

is challenging, time consuming (it could take well over 10 years from discovery to obtaining

all regulatory approvals) and expensive. Nanoparticles may address this need and provide a

novel therapeutic solution to limit the problem of antibiotic resistance [36].

The antibacterial activity of rGO, nAg, and rGO−nAg composite was assessed against three

important pathogenic bacterial species, namely S. aureus, E. coli and P. mirabilis. rGO

exhibited considerable broad spectrum antibacterial activity against both Gram-positive S.

aureus and Gram-negative E. coli and P. mirabilis bacterial pathogens, however it required a

significantly higher concentration to achieve the desired level of inhibition compared to

rGO−nAg. In the agar well diffusion method, rGO exhibited only a small zone of inhibition

while rGO−nAg composite was able to achieve a zone of inhibition twice the size of rGO used

on its own. It is important to note that inhibitory activity of rGO−nAg and rGO was observed

against the multidrug resistant strain (resistant to more than three antibiotics, including

nitrofurantoin) of E. coli used in this study. Time-resolved measurement of survival showed

inhibition of P. mirabilis by rGO between 2−3 hr after exposure and complete inhibition after

3 hr of incubation. Similar results were shown for S. aureus, where significant inhibition was

observed after 3 hr. After 18 hr of incubation, no viable organisms could be detected. The

results obtained from this study correlated well with the previously published findings [37].

Time-resolved viability testing showed that nAg significantly inhibited P. mirabilis after 3 hr

and E. coli after 4 hr of exposure. Even though nAg could not inhibit the growth of either P.

mirabilis or E. coli when tested using well method, the coupling of nAg with rGO significantly

enhanced the inhibitory activity of the composite.


In addition to larger zone of inhibition, rGO−nAg composites significantly reduced time

necessary to achieve complete inhibition. Both S. aureus and P. mirabilis were completely

inhibited after 2.5 hr of incubation, with a significant reduction in the number of viable bacteria

attained after 2 hr of incubation. The required incubation time for rGO−nAg was not only

shorter than that required for either rGO or nAg, but also shorter than that required to achieve

the same reduction in viability using standard antibiotic nitrofurantoin (100 µg/ml).

Figure 6: A symbolic representation of the mechanism of process of destruction of bacteria

from the cumulative effect of cell-wrapping as well as cell - trapping mechanisms of rGO

nanosheets and cell penetration of Ag nanomaterial.

While nitrofurantoin kills the bacterial by damaging bacterial DNA [38], the mechanism of

activity of rGO−nAg is yet to be fully elucidated. Previous studies have suggested that one

potential mode of action of sheet-like graphene-based materials involves cell physical

wrapping and entrapment of bacterial cells by these nanomaterials. In addition to physical

entrapment of the cell, the direct contact between the sharp edges of rGO sheets with cells can

physically damage cell membrane, resulting in leakage of intracellular material and negatively

affecting cell metabolism. The edge of graphene nanosheets have relatively high aspect ratio

which makes them an attractive nanostructure for direct contact inactivation of microorganisms

[20]. From this standpoint, increasing the sheet area enhances the rate of inactivation [39].

n r r n


Essential to cell growth and metabolism, bacterial respiration relies on electron transport

mediated by extracellular electron acceptors [40]. An electron conduit that forms between

surface respiratory proteins of the microbial membranes and the extracellular environment

generates energy needed to support cell activity. When surface respiratory proteins that display

n-type semiconducting behaviour and a bandgap of ~ 2.6−3.1 eV[41] come into contact with

semi metallic materials such as rGO, where the oxygen percentage content is low, Shottky

barrier is formed and electrons are transferred from cell membranes to rGO electron acceptors

driven by Fermi level alignment[42]. Since bacteria strive to maintain a negative resting

membrane potential by means of proton gradient, contact with rGO may lead to steady loss of

electrons over time [42]. The value of the surface charge differs depending on the bacterial

species, with Gram-negative E. coli having a less negatively charged surface compared to

Gram-positive S. aureus, due to the former having the membrane isoelectric point pI = 4~5 and

the latter having the pI of 2~3 under culture medium conditions[42]. The differences in surface

electron states may account, at least in part, for the differences in inhibitory activity of rGO

and rGO−nAg composites against Gram-positive and Gram-negative bacterial strains.

Oxidative stress induced by rGO nanosheets and nAg also play an important role [37].

rGO−nAg causes the oxidative stress by an imbalance between the production of reactive

oxygen and the ability of the biological system (such as bacterial cell) to readily detoxify the

reactive intermediates or easily repair the resulting damage [43]. The excess formation or

insufficient removal of highly reactive molecules, such as reactive oxygen species (ROS), and

resultant oxidative stress can arise from an increase in oxidant generation, a decrease in

antioxidant protection, or a failure to repair oxidative damage [44]. This eventually leads to

significant cell damage and cell death [45].

Depending on their size and oxidation level, rGO sheets can adsorb on the surface layer of the

cell, embed and subsequently cross the lipid bilayer, or can be taken up by the cell via vesicular


structures [46]. Graphene sheets with higher degree of oxidation can enter the cell more

efficiently owing to the lower energy state that exists between an oxidized graphene sheet and

the membrane [47, 48]. It has been observed that the nature of the graphene edges, e.g. their

sharpness and chemical composition, mediated the penetration of graphene in the lipid bilayers.

The initial piercing of the cell membrane by sharp and rough edges of graphene has been shown

to lower the energy barrier for graphene penetration [49].

Treatment with nAg also contributes to oxidative stress through the formation of free radicals

[50, 51]. Among generated reactive oxygen species (ROS), superoxide, hydrogen peroxide and

hydroxyl radicals were reported to play key role in the observed oxidative activity [52, 53].

The free radicals, which are short-lived reactive chemical intermediates that contain one or

more unpaired electrons [54], induce cellular damage when they pass this unpaired electron

onto nearby cellular structures. This leads to oxidation of cell membrane lipids and amino acids

that make up proteins or nucleic acid [55]. Ag ion treatment has been shown to result in

cytoplasm membrane shrinkage and separated from the cell wall. This led to release of cellular

contents and significant cell wall degradation [56]. Similarly, reduced graphene oxide induces

ROS-dependent oxidative stress by excess accumulation of intracellular ROS, such as

hydrogen peroxide, superoxide anions, hydroxyl radicals and singlet molecular oxygen [47, 57,

58].

The synergic effect of the individual components, nAg and rGO , as shown in Table 1 is

responsible for the observed increase in antibacterial activity of rGO-nAg nanocomposite

(Figure 6) [59]. With regard to the rGO-Ag composite, physical interaction between the sharp

edges of rGO sheets disrupts the cell membrane and facilitates the transport of silver ions across

the cell membrane [60-63]. The cell entrapment property of rGO ensures high local

concentrations of Ag ions in the immediate proximity of the cell membrane. It is also possible

that rGO contributed to increased permeation of silver ions into the bacteria. Similar effects


have been observed in Ag nanoparticles encapsulated in poly lactic acid polymer matrix, where

lactic acid disrupted the bacteria cell membrane and thus facilitated entry of silver

ions/nanoparticles into the Gram-positive and Gram-negative bacteria [64].

An important characteristic of metals like silver is their capacity to participate in redox

reactions. In addition to the affinity of a metal for a donor ligand, reduction potential is a

thermodynamic parameter that determines the tendency of a metal species to acquire electrons

from a donor and become reduced. The donor species loses electrons and becomes oxidized;

thus, reduction and oxidation always occur simultaneously [65].

Another destruction mechanism of rGO nano sheets is by extracting phospholipids from lipid

membranes [49]. Graphene’s unique two-dimensional structure with all sp2 carbons facilitates

strong dispersion interactions between graphene and cell membrane lipid molecules. On the

surface of graphene cooperative movements of extracted lipid molecules were observed due to

the redistribution of the hydrophobic tails to maximize hydrophobic interactions with the

graphene surface. This lipid extraction mediated destructive method was demonstrated by

previous research for both outer and inner membranes of E. coli [49]. While exposure to rGO

results in the dose-dependent loss of membrane integrity, as characterised by progressive

extraction of adenine and protein from bacteria[66], soft acids such as Ag tend to associate

tightly with soft bases, such as the sulphhydryl (R−SH) groups that are found in proteins.

Consequently, the antibacterial toxicity of these metals which is approximately proportional to

their affinity for –S destructs the cells by protein denaturation [67]’ [68]. Moreover, pore

formation can occur when all phospholipids are oxidized and this allows reactive oxygen

species to enter the cell and cause oxidative damage to intracellular macromolecules, such as

DNA or proteins. Previous research have also found that high concentration of reactive oxygen

and nitrogen species are produced during the treatment of the cell membrane with plasma, an


ionised gas consisting of highly reactive ions, electrons, photons and neutral species, and this

can even destroy the cell membrane of cancer cells[69].

The rGO−nAg composite might also disrupt the cellular donor ligands that coordinate Fe. The

direct or indirect destruction of [4Fe–4S] clusters could result in the release of additional

Fenton-active Fe into the cytoplasm, resulting in an increased ROS formation [65]. While at

low doses, cells may be able to upregulate ROS-detoxification enzymes to withstand toxic

doses of these elements; higher doses may inflict irreversible damage on cells.

Together, the cell membrane penetrating properties of rGO sheets, the oxidative stress of rGO

and nAg and the free radicle formation of Ag nanoparticles contribute to enhanced antibacterial

efficacy of rGO−nAg nanocomposites.

Figure 7: A symbolic representation of the mechanism by which the rGO−nAg nanoparticles

kill the bacteria. The rGO punctures cell wall and enter the cytoplasm. Silver nanoparticles

directly enter into the cell, induces oxidative stress and damage the cell contents.


The morphology of the cells plays a vital role in the bactericidal effect of rGO−nAg

nanocomposite. Gram-positive and Gram-negative bacteria possess dissimilar cell wall

structure and chemical composition [70]. In comparison with the delicate thin peptidoglycan

cell membrane in Gram-negative bacteria, Gram-positive bacteria possess cell wall consisting

of multiple layers of peptidoglycan which provide better cell membrane integrity and prevent

cell disruption [71, 72]. Previously, exposure of S. aureus cells to rGO−nAg nanocomposite

has been shown to result in cell wrinkling and damage, with some cells being completely

covered by the rGO−nAg, whereas exposure of E. coli to the same concentrations of rGO−nAg

led to complete cell fragmentation [72]. In other words, for Gram-negative E. coli, the primary

mechanism of rGO−nAg bactericidal activity is through disruption of bacterial cell wall

integrity, whereas for Gram-positive S. aureus, the effect is bacteriostatic and is associated

with dramatic hindering of cell growth [72].

4.4 Conclusion

In this study, rGO and nAg nanomaterials were first synthesized using wet chemical methods,

and then combined to form rGO−nAg nanocomposites. The properties of individual materials

and the uniform distribution of nAg on rGO sheets were confirmed using microscopy and

spectroscopy techniques. The produced rGO−nAg nanomaterial composites exhibited

enhanced efficacy against all three pathogens tested. The activity of rGO−nAg nanocomposite

was also superior to that of conventional systemic antibiotic, nitrofurantoin, even for a

multidrug resistant strain of E.coli used in this study. The antibacterial activity of rGO−nAg

composite against S. aureus is even more significant, being far superior to that of

nitrofurantoin. These results suggest that rGO−nAg nanocomposite may present a viable

alternative to some conventional antibacterial agents.


4.5 Methods

Synthesis and characterization of r , n , and r −n nanocomposite: Graphene

oxide was prepared from natural graphite following Hummers method [73]. Briefly, 1g of

NaNO3 and 46 ml of H2SO4 was added to 1g of natural graphite powder and stirred

continuously in an ice bath to maintain the temperature of the mixture below 20° C. Then, 6 g

of KMnO4 was added slowly while stirring. After 1 hr, the ice bath was removed, the system

was heated to 35° C and the temperature was maintained at 35° C for 30 min; 70 ml water was

slowly added to the system and stirred for another 15 min. Then, 80 ml of hot (60° C) water

along with 30% H2O2 aqueous solution were added to reduce the residual KMnO4 until the

bubbling has disappeared. The product formed was washed several times to remove the

remaining salt impurities. After thermal reduction at 200° C for 3 hr, a black colored powder

of rGO was obtained.

AgNO3 was reduced by sodium potassium tartrate in the presence of poly vinyl pyrolidone

(PVP) (MW 40,000) by first heating 50 ml solution of 1.2 mM PVP and 0.2 mM AgNO3 to 80°

C with vigorous stirring and then gradually adding 2 mM sodium potassium tartrate solution

until complete reduction of AgNO3 had been achieved. Then the reaction mixture gradually

became turbid and a light yellow suspension was obtained indicating the reaction was

successful.

The rGO and nAg nanomaterial solutions were combined at the ratio of 9:1 using vigorous

stirring for 2 hr, yielding rGO−nAg nanocomposite.

The characterization of the synthesized composite was carried out using UV-Vis absorption

spectroscopy, Fourier Transform Infrared (FTIR) spectrometry, X-Ray Diffraction

Spectrometry (XRD), Scanning Electron Microscopy (SEM), and Transmission Electron

Microscopy (TEM).


Bacterial growth: The antibacterial activity of rGO, nAg and rGO−nAg nanocomposite was

determined by modified agar well diffusion method [74] and survival rate determination

methods [75]. Clinical bacterial isolates of S. aureus, P. mirabilis and E. coli used in this study

were obtained from Department of Microbiology, Vels University, Chennai, India, where they

were extensively tested using standard methods for antibiotic susceptibility, e.g. using the

double disc diffusion test and morphological characterization. Using Kirby-Bauer method, E.

coli cultures isolated from UTI patients of a tertiary care hospital in Chennai were shown to be

resistant to β-lactam antibiotics, such as ampicillin (10 μg/ml), attributed to the production of

extended spectrum β-lactamases, and non-β-lactam antibiotics, such as gentamycin (10 μg/ml),

co-trimoxazole (1.25/23.75 μg/ml), and ciprofloxacin (5 μg/ml)[76]. S. aureus isolates were

found to be resistant to gentamycin (10 μg/ml), tetracycline (30 μg/ml), and trimoxozole (25

μg/ml), while being susceptible to chloramphenicol and ofloxacin at 30 and 32 μg/ml,

respectively [77]. P. mirabilis was resistant to chloramphenicol (30 μg/ml), amoiclav (30

μg/ml), methicillin (30 μg/ml), and streptomycin (30 μg/ml), with susceptibility to ceffriaxone

(30 μg/ml) and nalidixic acid (30 μg/ml) [78].

The inocula for antibiogram assay were prepared following the recommendations of CLSI

(2010 guidelines). Test organisms were incubated in standard nutrient broth at 37° C for 4−6

hr. The inoculum, visual turbidity of 0.5 McFarland standards, was used to inoculate the surface

of Mueller-Hinton agar plates. Wells of approximately 6 mm in diameter were made in the

plates using a sterile borer. Each well was loaded with one of the following: undiluted rGO

(crude), rGO solution (at 50 µg/ml, 100 µg/ml, or 200 µg/ml), nAg nanomaterial, or rGO−nAg

nanocomposite. A standard antibiotic nitrofurantoin, an antibiotic clinically used for the

treatment of these pathogens, (100 µg) was loaded in the center of the well to compare the

antibacterial activity of the graphene composite. The plates were incubated at 37°C for 18 hr.


The survival rates of a Gram negative and Gram positive pathogens was determined using

spread plate method. Three sets of flasks containing 100 ml of nutrient broth were inoculated

with either S. aureus or P. mirabilis to the density of 3 × 108 CFU/ml. To each flask, an

antibacterial material, namely 0.1 g of rGO, 0.1 g of nAg, or 0.1 g of rGO−nAg was added. At

regular time intervals, few ml aliquots of bacterial suspension were taken from each flask, and

transferred onto agar plates, spread evenly and allowed to incubate for 18−24 hr at 37 °C, 5 %

CO2. The formed colonies were then counted using a plate counter


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This chapter of this thesis is published in Nanomaterials on 4th July 2017 as:

Effect of Precursor on Antifouling Efficacy of

Vertically-Oriented Graphene Nanosheets

Karthika Prasad, Chaturanga D. Bandara, Shailesh Kumar,

Gurinder Pal Singh, Bastian Brockhoff , Kateryna Bazaka, and

Kostya (Ken) Ostrikov

Doi: 10.3390/nano7070170



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“Effect of Precursor on ntifouling Efficacy of Vertically-Oriented Graphene

Nanosheets” Published in Nanomaterials on 4th July 2017


Karthika Prasad Conception of idea, designed and conducted the experiments,

analyzes the data and wrote the manuscript.

28/11/2018

Chaturanga D. Bandara Assisted with antibacterial studies and analyzing data and revising

manuscript

Shailesh Kumar Assisted with plasma enabled synthesis of graphene and revising

manuscript

Gurinder Pal Singh Contributed to the result interpretation

Bastian Brockhoff Contributed to the result interpretation

Kateryna Bazaka

Proposed the idea, co-ordinated experimental design and data

collection, helped in interpreting results and underlying mechanisms,

helped with article preparation and communication

Kostya Ostrikov Helped with conception of idea, supervised the research, helped with

data analysis and interpretation, and major manuscript editing


(If the Co-authors are not able to sign the form please forward their email or other correspondence

confirming the certifying authorship to the RSC).


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PREFACE

This chapter highlights antibacterial properties of surface-immobilised graphene-based

materials as potential agents for contact killing of bacteria on implant surfaces. This paper

studies the antibacterial properties of graphenes grown on different surfaces from different

precursors by means of chemical vapour deposition. The article studies the effect of

precursor and substrate on the biological activity of thus-grown graphene nanoflakes, and

proposes a likely mechanism of action against common human pathogenic bacteria with

different shape and cell wall structure.


Abstract

Antifouling efficacy of graphene nanowalls, i.e., substrate-bound vertically-oriented

graphene nanosheets, has been demonstrated against biofilm-forming Gram-positive

and Gram-negative bacteria. Where graphene nanowalls are typically prepared using

costly high-temperature synthesis from high-purity carbon precursors, large-scale

applications demand efficient, low-cost processes. The advancement of plasma

enabled synthesis techniques in the production of nanomaterials has opened a novel

and effective method for converting low-cost natural waste resources to produce

nanomaterials with a wide range of applications. Through this work, we report the

rapid reforming of sugarcane bagasse, a low-value by-product from sugarcane

industry, into high-quality vertically-oriented graphene nanosheets at a relatively low

temperature of 400 °C. SEM visualisation showed that graphene nanowalls fabricated

from methane were significantly more effective at preventing surface attachment of

Gram-negative rod-shaped Escherichia coli compared to bagasse-derived graphene,

with both surfaces showing antifouling efficacy comparable to copper. Attachment

of Gram-positive coccal Staphylococcus aureus was lower on the surfaces of both

types of graphene compared to that on copper, with bagasse-derived graphene being

particularly effective. Toxicity to planktonic bacteria estimated as a reduction in

colony-forming units as a result of sample exposure showed that both graphenes

effectively retarded cell replication.

Keywords: graphene; nanowalls; plasma-enabled synthesis


5.1 Introduction

Recent times have witnessed a significant increase in the use of nanomaterials,

especially graphene, for a wide range of applications, ranging from electronics to

agriculture and manufacturing [1-3]. However, incorporation of graphene into day-to-

day devices demands large-scale production of high-quality graphene which is cost

effective [3]. Hence, substantial efforts have been made to develop numerous cost-

effective ways of producing high-quality graphene [4-6].

The use of natural resources for the production of graphene and graphene-based

products has been studied widely [7, 8]. Recently, plasma-enhanced chemical vapour

deposition (PECVD) has been used for the production of high-quality graphene

nanosheets from a variety of resources [9]. In this low-temperature synthesis

technique, the graphene can be grown directly on a wide range of desired substrates

without any external heating or catalyst, and it is therefore considered a promising

method for controllable graphene synthesis [1, 3, 9-11]. Advantageously, vertically-

oriented graphene nanosheets, also known as nanowalls, can be fabricated with

excellent control over the spatial arrangement, density, and orientation of the

nanosheets [12, 13]. In addition to this, vertical orientation of surface-immobilised

graphene affords the material a number of advantageous properties in comparison with

conventional horizontal, randomly oriented graphene sheets, particularly for

applications where chemical or biological reactivity and mechanical robustness are

desired. The free-standing, self-supported rigid structure of vertically-oriented

graphene sheets prevents collapse and/or the random stacking of graphene nanosheets

associated with strong van der Waals interactions. Such a mechanically-stable, non-

agglomerated morphology ensures high specific surface area (or surface-to-volume


ratio), long reactive edges, and abundant open channels between the sheets [14]. These

graphene networks can serve as platforms for highly-sensitive and selective field-

effect transistor biosensors [15, 16].

This work investigates the antibacterial activity of graphene fabricated from raw,

multicomponent, low-cost resources, compared to that of graphene derived from high-

purity carbon precursor, against pathogenic multi-drug resistant bacterial species,

namely Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus. A

fast and reactive plasma-enabled process is used for reforming sugarcane bagasse and

methane gas into graphene. This particular graphene growth process does not involve

toxic or hazardous gases and does not require any catalyst or external substrate heating

to produce thin vertical graphene sheets on the same substrate. This method is also

environmentally friendly, does not produce any chemical residue or waste, and is

energy and material-efficient.

5.2 Results

5.2.1 Structural and Morphological Characterization of Nanomaterials

Successful formations of graphene from both precursors were confirmed by Raman

spectroscopy. The Raman spectra were collected using a Renishaw inVia spectrometer

with laser excitations of 633 nm. Figure 1 shows the characteristic bands at 1590 cm−1

(G band), 1320 cm−1 (D band) and 2600 cm−1 (2D), which confirms the formation of

graphene from different precursors.


Figure 1. Raman spectra of graphene nanosheets deposited from methane on

copper and from bagasse on porous nickel substrates.

The ratio of intensity of 2D and G bands indicates the number of layers of graphene.

The increased ratio indicates the formation of thinner graphene flakes, whereas the

ratio is smaller for thicker ones. Here, the thinnest flakes were obtained on plasma-

treated copper substrate, where methane was converted to graphene.

The morphology and elemental distribution of the graphene are characterized by Field

Emission Scanning Electron Microscopy (FESEM, ZEISS SIGMA VP) employing an

electron gun voltage of 10 kV with an energy dispersion spectrum (EDS).

The SEM images in Figure 2 confirm the findings from Raman spectra. While Figure

2a shows an SEM image of graphene nanosheets deposited on a copper foil using

methane as precursor gas, Figure 2b shows the image of graphene nanosheets on a

nickel substrate using sugarcane bagasse powder as precursor. SEM images reveal

relatively transparent nanosheets on copper foil, which suggests that the deposited

nanosheets are thinner than the graphene nanosheets deposited on porous nickel

substrate. These results correlate well with the results obtained by calculating the

intensity ratios from Raman spectra.


(d)

Figure. 2. (a,b). Representative SEM images of graphene nanowalls produced

from (a) methane on copper substrate and (b) from bagasse on nickel substrate.

(c,d) EDS spectra of graphene formed from (c) methane on copper substrate and

(d) from bagasse on nickel substrate.

The energy dispersion spectra (EDS) for the samples are shown in Figure 2. The EDS

spectra indicate the presence of carbon on copper and porous nickel substrates. The

absence of any other additional peaks confirms the contamination-free deposition of

graphene nanosheets.

Further characterization of the graphene nanosheets was performed using

Transmission electron microscopy (TEM). The crystal images of graphene nanosheets

were collected using JEOL 2100F HR-TEM. The electron beam energy used for this

analysis was 200 keV.

(b) (a)

(c)


TEM samples were prepared by placing a drop of graphene dispersed in isopropanol

on a carbon-coated copper grid and subsequently evaporating the isopropanol. Figure

3 represents the TEM images of graphene nanosheets deposited from methane and

sugarcane bagasse on copper and nickel substrates, respectively. Images clearly show

the graphitic edges and the crystalline structure of deposited graphene. On nickel

substrate, the formation of thicker graphene is evident from the greater number of

layers formed. The interplanar distance between subsequent two layers was 0.134 nm.

With an average number of layers between 6 and 9, vertically-oriented methane-

derived graphene nanowalls are notably thinner than that of bagasse-derived graphene,

with the latter typically having between 15 and 20 layers.

Figure 3. TEM images of the samples deposited from (a) methane on copper

substrate, and (b) bagasse on porous nickel substrate.

5.2.2 Antibacterial Studies

The antibacterial efficacy of two different graphene samples against Gram-negative

rod-shaped E. coli and Gram-positive coccal S. aureus bacteria were investigated in

terms of cell attachment and toxicity to planktonic cells. For this purpose, cell cultures

(a)

(b)


were incubated in the presence of different graphene samples and copper in 30 µL of

Luria broth at room temperature. After 4 h of incubation, the surfaces of the samples

were visualised using SEM. As evident from Figure 4, graphene nanowalls fabricated

from methane were significantly more effective in preventing surface attachment of E.

coli compared to bagasse-derived graphene, with both surfaces showing antifouling

efficacy comparable to copper. Attachment of S. aureus was lower on the surfaces of

both types of graphene compared to that on copper, with bagasse-derived graphene

being particularly effective.

Figure 4. Representative SEM images of E. coli cell attachment on the

surfaces of (a) methane-derived (GNW_M) and (b) bagasse-derived

(GNW_B) graphene, and (c) pure copper substrate after 4 h of incubation at

22 °C. SEM images of S. aureus cell attachment on the surfaces of (d)

GNW_M, (e) GNW_B, and (f) copper after incubation under the same

conditions.


Toxicity of the surfaces to planktonic bacteria was investigated by estimating the

number of colony-forming units (i.e., live cells) at different times during the incubation

period. The results of this study are summarized in Figure 5. Taking into consideration

the growth rate of E. coli bacteria, graphene samples fabricated from bagasse

(GNW_B) showed considerable toxicity against planktonic bacteria. Although the cell

numbers gradually increased over the period of incubation, the cell numbers were

below (at 1 h) or similar to (at 2 h) those on copper. On the other hand, graphene

derived from methane (GNW_M) effectively retarded cell replication, with cell

numbers increasing only slightly during the first hour of exposure, and then deceasing

to below the initial seed values. The efficacy of methane-derived graphene was

significantly better than that of copper, a known broad-spectrum antibacterial agent.

Cells incubated in the presence of copper surfaces first experienced limited

antibacterial action from copper, with cell numbers reaching 9.3 × 107 CFU/ml.

However, after 2 h of exposure, there was a significant reduction in the number of

viable cells, attributed to the diffusion of copper ions from the surface of the substrate.

Figure 5. The survival rate of (a) E. coli and (b) S. aureus bacteria when exposed

to graphene fabricated from methane (GNW_M) and bagasse (GNW_B), and a

pure copper substrate.

(a) (b)


In contrast to E. coli bacteria, the growth of S. aureus was effectively retarded by

graphene nanowalls from bagasse (GNW_B) and methane (GNW_M), with the latter

being characterized by slightly lower numbers of surviving organisms at 2 h. The

growth of cells incubated in the presence of copper was limited in the first hour of

incubation. However, the cell numbers increased significantly after 2 h of incubation,

and were substantially higher than those observed for cells incubated in the presence

of graphene samples. 1 x 10+9

As a broad-spectrum antifouling and antibacterial agent, GNW_M is more efficient

than GNW_B for the pathogens tested, i.e., E. coli and S. aureus.

5.3 Discussion

In this experiment, we successfully synthesised graphene using methane gas and

sugarcane bagasse as the precursors. The Raman spectra of the obtained graphene

(Figure 1) shows the characteristic graphene bands with G band at 1590 cm−1, D band

at 1320 cm−1 and 2D band at 2600 cm−1 (2D). This confirms the formation of a

graphitic structure [17]. Moreover, from the intensity ratio of 2D and G bands, which

indicates the number of layers of graphene, it is evident that the thinnest flakes were

obtained on plasma-treated copper substrate, where methane was converted to

graphene. Further, the D and G bands give an insight about the crystallinity of the

structure, which can be determined by calculating the ratio of intensities of D and G

bands (ID/IG). With a decrease in the value of ID/IG, the crystallinity of the structure

increases [10]. Therefore, in this experiment, the ratio of ID to IG was lower for

graphene formed from methane gas, which shows that the graphene formed was highly

crystalline.


The high-resolution SEM images in Figure 2 clearly show the formation of vertically-

aligned graphene nanosheets on both substrates. The images correlated well with the

findings from the intensity ratio of Raman spectra and show formation of thinner

graphene layer on copper substrate and thicker ones on nickel substrate. The absence

of any other peak in the EDS spectra (Figure 2) demonstrates the purity of the graphene

formed. The graphitic edges and the crystalline structure of deposited graphene are

shown in TEM images in Figure 3.

The morphology of graphene layers grown by means of plasma-enhanced synthesis

has been previously shown to depend on the chemistry and state of the precursor, as

well as on the properties of the catalyst. The growth of vertical graphene is considered

to be a step-flow process on the basis of nucleation at the bottom, and the thickness of

graphene will depend on the number of layers that nucleates from the bottom

[11].Transition metals, such as nickel and copper, are commonly used for graphene

production due to their ability to readily absorb and interact with carbon sources due

to their partially filled d sub-shell [18]. When compared with nickel, copper exhibits

comparatively low carbon solubility, which leads to a different mechanism of graphene

formation. Specifically, the growth of graphene on copper is dominated by the direct

deposition of carbon atoms on the catalyst surface, with limited diffusion of carbon

atoms into the copper, which enables growth of thinner graphene layers [19]. On the

other hand, when nickel is used as a catalyst, carbon atoms from the carbonaceous

source diffuse readily into the catalyst bulk during the high-temperature processing,

and precipitate to the catalyst surface during the cooling period. Since the growth

mechanism combines that of surface growth and precipitation of carbon from the

catalyst bulk, the structure of resulting graphene may differ substantially from that


deposited on Cu substrate [20, 21]. This was evident from the characterization

performed on graphene produced in this study.

The antifouling efficacy of methane- and bagasse-derived graphene surfaces was

comparable to or better than that of copper. There are several possible mechanisms

that may be responsible for the bactericidal activity of graphene [22]. The observed

differences in antifouling and bactericidal activity of graphene against phenotypically-

distinct bacteria, namely E. coli and S. aureus, shown in Figures 4 and 5 can be at least

in part attributed to differences in physico-chemical properties of these materials. The

sharp edges characteristic of vertically-oriented graphenes are one of the most

important mechanisms in terms of antibacterial activity [23], where the sharp edges of

graphene may physically disrupt cellular membranes, resulting in the loss of bacterial

membrane integrity, which may lead to leakage of intracellular substances, and

eventually to cell death [23]. Sharper edges of methane-derived graphene may thus be

more effective in compromising the integrity of the soft membrane of E. coli, resulting

in contact bacterial inactivation on the surface [24].

Attachment of S. aureus was lower on the surfaces of both types of graphene compared

to that on copper, with bagasse-derived graphene being particularly effective. The

electron transfer mechanism from a microbial membrane to graphene is another

mechanism for the destruction of bacteria [25], which may be particularly relevant in

this case. Graphene-based materials induce oxidative stress towards the endogenous

antioxidant glutathione. Here, the graphene acts as a conductor between the negatively

charged cell and the metal. This electron flow towards the graphene metal substrate

ultimately results in cell death [26].


In addition to mechanisms associated with direct cell-surface contact, oxidative stress

induced by graphene nanowalls may inhibit the bacterial metabolism and disrupt

essential cellular functions, eventually leading to cellular inactivation. Oxidative stress

can include ROS-dependent or ROS-independent pathways. In the former, the stress

is induced by excess accumulation of extracellular ROS species, such as hydrogen

peroxide, superoxide anions, hydroxyl radicals, and singlet molecular oxygen. These

ROS species induce lipid peroxidation, intercellular protein inactivation and gradual

disintegration of cell membrane, followed by the eventual cell death. ROS-

independent oxidative stress oxidizes the vital cellular structure without ROS

production, which can be induced by the charge transfer from cellular membrane to

graphene, where graphene acts as an electron pump [27, 28].

In comparison to this, the antifouling mechanism of copper involves several processes.

At the initial stages, non-enzymatic peroxidation of membrane phospholipids takes

place, leading to loss of membrane integrity. This may be followed by rapid and

extensive degradation of genomic DNA and necrotic cell death. The time of onset of

killing, the rate of cell death, and the kinetics of lipid peroxidation are inherently linked

to structural characteristics and metabolic state of the cell. Furthermore, it is possible

that S. aureus is more efficient that E. coli in terms of surface attachment and

production of extracellular polymeric substances on the surface of copper, which may

mask the surface and prevent the process of contact-mediated membrane peroxidation.

This is supported by the SEM images (Figure 4), which show significantly higher

numbers of attached S. aureus cells on the copper surface. In contrast, the surface

copper substrate exposed to E. coli cells remains minimally colonised [29].

Our results indicate that graphene nanowalls from methane and bagasse are as efficient

as copper in preventing surface colonization by bacterial strains tested, attributed to


thin, sharp edges of the thus-produced graphene, as well as the ability of graphene

walls to transfer elections and induce oxidative stress on the cell. Although methane-

derived graphene is slightly more effective against E. coli, the lower-cost bagasse-

derived graphene provides attractive antifouling and antibacterial activity for large-

scale applications.

5.4 Materials and Methods

The deposition of vertically-oriented graphene nanosheeets was carried out in a RF

inductively coupled plasma CVD system. A polycrystalline copper film was used as a

substrate for the graphene to grow from methane gas precursor, whereas a 99%-pure

porous nickel foam was used for the reforming of sugarcane bagasse. For each

deposition, 0.5 mg of sugarcane bagasse was placed evenly on the substrate prior to

being loaded into the camber. A low-temperature inductively coupled plasma was used

to both reform sugarcane bagasse, to heat the polycrystalline copper catalyst, and to

dissociate the hydrocarbon gas precursor.

In Figure 6a, the CH4 precursor was heavily diluted in hydrogen (H2) and argon (Ar).

Growth is carried out over a range of plasma exposure times, with best results obtained

at 10 min. A gas mixture of H2/Ar/CH4 at a flow rate of 85/10/5 sccm, respectively,

was fed into the chamber for deposition.

Figure 6b represents the formation of graphene nanosheets through reforming

sugarcane bagasse with plasma-enabled synthesis. Powdered sugarcane bagasse was

evenly distributed on the surface of the porous nickel substrate. A gas mixture of H2/Ar

was fed into the chamber at a flow rate of 50 and 15 sccm, respectively. In both the


experiments, the chamber pressure was maintained at 2.0 Pa and plasma was generated

using RF power of 760 W and the deposition was carried out for 10 min.

Figure.6. Schematic representation of plasma enabled synthesis of graphene. (a)

Conversion of methane gas into graphene in the presence of plasma. (b)

Reforming of sugarcane bagasse into graphene.

For the qualitative analysis of bactericidal activity of graphene against E. coli and S.

aureus, bacterial cultures were refreshed on nutrient agar plates from a stock culture

and grown overnight at 37 °C in a 5 mL of nutrient broth [30]. The culture was

collected at the logarithmic stage of growth and washed twice with 0.01 M PBS

solution (pH = 7.4). An aliquot of 1 mL of bacterial suspension from an adjusted OD600

= 0.1 bacterial suspension was placed on the surface of graphene nanowalls on

fabricated on copper and nickel substrates and was allowed to incubate for 4 h at room

(a)

(b)


temperature (22 °C) in a cell culture plate. Untreated copper and nickel substrate was

used as control. After every 1 h, a 10 μL aliquot was taken and a 10× dilution series

(10−1 to 10−8) was made, and from the resulting 100 μL solution, 30 μL was plated on

nutrient agar media, in triplicate, for each solution. Plates were incubated overnight at

37 °C, and colonies for each aliquot between 3 and 30 were counted and recorded with

their respective dilution factor. This experiment was conducted in triplicate.

5.5 Conclusions

Production of high-quality, large-area graphene sheets in a cost-effective way has

always been a challenge [31, 32]. Low-temperature plasma-enabled processing has

recently emerged as a highly-versatile family of techniques for controlled synthesis of

nanomaterials [33, 34] and modification of abiotic [35] and biological objects [36, 37].

In this paper, we have successfully demonstrated a cost-effective, single-step plasma-

enabled synthesis of graphene from methane gas and sugarcane bagasse (Figure 7).

Methane gas and sugarcane bagasse were completely transformed into high-quality

graphene within 10 min at 400 °C. We also demonstrated the antifouling efficiency of

the thus-produced graphene, and concluded that the graphene synthesised from the

methane gas was more crystalline, thinner and more effective against bacteria.


Figure 7. Comparative evaluation of the antibacterial efficacy of graphene nanowalls

synthesised from different precursors. High-cost, high-purity conventional, i.e.,

methane (a), and low-cost, natural carbon, i.e., sugarcane bagasse (b) sources are

converted using low-temperature plasma process (c) into high-quality graphene sheets

(d,e). When bacterial cells are exposed to thus-fabricated surface-immobilised

graphenes (f), anti-bacterial activity of graphene is observed to differ (g), expressed in

terms of cell attachment and number of colony-forming units.


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This chapter of this thesis is to be submitted to Sustainable Materials and

Technologies

Effect of multi-model environmental stress on dose-dependent

cytotoxicity of nanodiamonds in Saccharomyces cerevisiae cells

Karthika Prasad, Nina Recek, Renwu Zhou, Morteza Aramesh,

Annalena Wolff, Robert E. Speight, Miran Mozetič, Kateryna

Bazaka, Kostya (Ken) Ostrikov






1. they meet the criteria for authorship in that they have participated in theconception, execution, or interpretation, of at least that part of the publicationin their field of expertise;2. they take public responsibility for their part of the publication, except forthe responsible author who accepts overall responsibility for the publication;3. there are no other authors of the publication according to these criteria;4. potential conflicts of interest have been disclosed to (a) granting bodies,(b) the editor or publisher of journals or other publications, and (c) the headof the responsible academic unit, and5. they agree to the use of the publication in the student’s thesis and itspublication on the QUT’s ePrints site consistent with any limitations set bypublisher requirements.In the case of this chapter:

“Effect of multi-model environmental stress on dose-dependent cytotoxicity of

nanodiamonds in Saccharomyces cerevisiae cellsto be submitted to Sustainable

Materials and Technologies


Karthika Prasad Design and conducted the experiments, contributed towards the

conception of idea as in which nanoparticles to be used, interpreted

the data and wrote the manuscript. 28/11/2018

Nina Recek Assistance in designing, conducting experiments and analyzing data

and revising manuscript

Renwu Zhou Helped with analyzing the data and interpretation of result

Morteza Aramesh Advised on properly executing the research with nanodiamonds

Annalena Wolff Helped with capturing and interpreting HIM data

Robert E. Speight Advised on properly executing the research and interpreting the data

Miran Mozetič Advised on properly executing the research and result interpretation

Kateryna Bazaka

Conception of idea, experimental design, supervision of research,

helped in interpreting results and proposed underlying mechanisms,

helped in writing of the article and communication

Kostya Ostrikov Supervised the research, helped with data analysis and interpretation

and major manuscript editing

I have sighted email or other correspondence from all Co-authors confirming their certifying

authorship.(If the Co-authors are not able to sign the form please forward their email or other correspondence



Name Signature Date




PREFACE

This chapter deals with the potential toxicity of nanodiamonds in suspension for cells

exposed to oxidative stress. Nanodiamonds are well known for their biocompatibility and

mechanical strength and can be used as a potential candidate for enhancing the properties

of breast implants.

This article for the first time demonstrate that under oxidative stress conditions, the toxicity

of otherwise biocompatible nanoparticles can potentially be increased, as demonstrated in

model eukaryotic organism (S. cerevisiae) and the limitations of using that in breast

implants.


Abstract

A tremendous increase in the use of functionalised nanomaterials for industrial

processes and consumer products inevitably promotes their interaction with living

organisms and environment. Previous studies have investigated cell-nanoparticle

interactions under conditions otherwise favourable for cell survival and proliferation,

yet in real life, organisms are subject to environmental stresses, which may affect their

response to nanoparticles. This work investigates the effect of atmospheric-pressure

plasma, a model stress inducing environment rich in highly-reactive ROS and RNS

species, UV light, and mild heat, on the interactions between inert nanodiamond

particles (NDs) and a eukaryotic model organism, Saccharomyces cerevisiae. Plasma

treatment significantly affected nanoparticle uptake attributed to changes in membrane

properties. Accumulation of nanoparticles in larger deposits inside the cells and around

the cell wall affected cell survival and proliferation. Plasma-treated cells exposed to

100 µg ml-1 NDs for 24 h showed significant inhibition of metabolic activity and 55%

reduction in cell viability, whereas at lower concentrations (0, 5 and 50 µg ml-1) of

NDs, no significant effect on cell viability or cell growth was observed. These results

suggest that presence of intra- or extra-cellular stresses is an important determinant of

cell fate upon exposure to nanoparticles.

Keywords: nanodiamonds, cold atmospheric plasma, cell viability and growth,

cellular uptake


6.1 Introduction

Owing to its unique size dependent physicochemical properties, engineered

nanomaterials are now being used for a wide range of industrial processes and

consumer products, including applications related to medicine, e.g. for surface

functionalisation and encapsulation of implantable devices, contrast agents for

visualisation of tissues and physiological functions, diagnostics of diseases, systems

for delivery of biologically-active agents, cancer therapies, etc [1-4]. A remarkable

increase in their use and their inevitable disposal into the environment has raised some

concerns regarding their impact on human health and ecosystems. There are numerous

studies reporting cell−nanoparticle interactions, however, to our knowledge,

combination effects of plasma stress and NDs on yeast cells has not yet been reported.

Plasma closely imitates environmental stress conditions and most likely triggers

intracellular responses to nanoparticles. Furthermore, in medicine and biotechnology,

cells are often intentionally subjected to stress to induce desirable biological responses,

e.g. oxidative stress that triggers apoptosis in cancer cells [5,6] or heat pre-treatment

as the means to enhance alcohol tolerance in industrial yeast [7]. In addition to

individual stresses, treatments that can act as a source of multiple stressors have also

attracted attention both for their ability to induce desired biological responses more

effectively [8-10] as well as to serve as a model for harsh environmental exposure.

Among these treatments, cold atmospheric plasmas (CAP) have attracted a rapidly

growing interest in medicine and biotechnology as a highly-reactive environment, rich

in biologically-active species, primarily reactive oxygen and nitrogen species (RONS)

such as hydroxyl radicals, which are essential for various biological processes in

unicellular and multicellular organisms [11]. Advantageously, in addition to RONS,


CAP act as a source of energetic electrons, mild heat, UV photons, electric field

effects, and others [12,13].

The objective of this study is to examine the role of environmental stressors in the

interactions between inert nanodiamond particles (NDs) and a eukaryotic model

organism, Saccharomyces cerevisiae, using CAP as a model environment rich in

highly-reactive ROS and RNS species, UV light, and mild heat. S. cerevisiae is an

industrial unicellular microorganism, which is being used in food, pharmaceuticals,

wine and beer fermentation industries and bioethanol production [14]. The importance

of stress tolerance of this fermentation-grade microorganism in industrial processes

makes it an interesting candidate to study biological effects, induced by external agents

such as plasma and nanoparticles. Nanodiamond nanoparticles (NDs) have recently

become an exciting candidate for biomedical applications, such as drug carriers, assay

systems, implant coatings etc [15-18]. NDs exhibit a number of attractive properties,

such as desirable chemical and biological stability, biocompatibility at both cellular

and whole-organism levels, and the capacity to emit strong fluorescence when defects

are introduced into the ND structure. Additionally, the surface of NDs can be

chemically altered through various chemical routes to provide NDs with specific

functionalities. This attractive combination of the physical and chemical properties, as

well as favourable biocompatibility makes NDs an attractive material for evaluating

the stress-dependent toxicity of these nanoparticles in biological systems [19,20].


6.2 Materials and methods

6.2.1 CAP device and plasma treatment approach

For this experiments, an atmospheric pressure plasma jet (model kINPen08, neoplas)

was used to generate plasma. The device operates within a voltage range of 2–6 kV at

a frequency of 1.7 MHz and electric power of 6.5 W. The plasma was generated by a

pin-type electrode located within a thin quartz tube with the dimensions of 1.6 mm and

4mm for the inner and outer diameters, respectively. Argon (Ar) was flown at a flow

rate of 5 standard litres per minute through the quartz tube. The cells damage which

can be caused due to the use of plasma was prevented by lowering the neutral gas

temperature to below 40 °C.. The yeast colonies were kept 15 mm away from the exit

of the nozzle, with the distance being experimentally ascertained to enable the plasma

jet (~12 mm in length) to uniformly cover the treated colony. Each yeast colony

received 3 min of plasma treatment. The duration of the treatment was chosen

experimentally as not to induce significant reduction in cell viability, metabolic

activity or cell growth. To account for the effects of gas flow, the control samples

received 3min of exposure to Ar flow at the same flow rate in the absence of plasma

discharge.

6.2.2 Microorganism and culture conditions

The haploid strain, S. cerevisiae AWRI 1631, was given by the Australian Wine

Research Institute. Colonies for plasma treatment were generated by streaking cells

from the frozen stock cultures on yeast extract peptone dextrose (YPD) agar plates.

YPD was prepared using an OXOID agar, 15g, containing glucose, 20 g L-1, OXIOD

Bacteriological peptone, 20 g L-1, and OXOID Yeast extract, 10 g L-1. Cells were

incubated at 30 °C.


6.2.3 Nanodiamond

Detonation (ultradispersed) nanodiamonds of size 15 nm were purchased from

PlasmaChem GmbH and were used without any further purification. Suspensions of

nanodiamonds were prepared using MiliQ water to produce concentrations of 5, 50

and 100 µg ml-1 (in the media).

6.2.4 Experimental procedure

Individual yeast colonies were subjected to direct plasma treatment for 3 min. Right

after the plasma treatment, the colonies were moved into cell culture (YPD) and NDs

were introduced to the plasma treated cells to achieve a final concentration of 0, 5, 50

or 100 µg ml-1. A control experiment was also performed where the cells were not

exposed to plasma treatment. An aliquot of 100 µl of these suspensions was then

transferred to a 96-well plate and placed into a microplate reader with continuous

orbital shaking, for cell growth measurements.

6.2.5 Cell growth and cell viability

A microplate reader was used to measure the cell growth and viability. Cell growth

was observed over a period of 24 h with a temperature of 30 °C with constant orbital

shaking and absorbance was measured at 600 nm. After the incubation, cell viability

assay was performed using the LIVE/DEAD yeast viability kit (Thermo Fisher

Scientific), based on the supplied protocol. Laser scanning electron microscopy was

performed using Zeiss LSM 510 instrument.

6.2.6 Scanning Electron Microscopy (SEM) and Helium Ion Microscopy (HIM)

Yeast cell morphology of both plasma treated and non-treated cells were assessed

immediately following plasma exposure using scanning electron microscopy (SEM)

in order to visualise the effects of this types of environmental stress on cell shape, size


and membrane structure. Since it is possible for the finer surface features and

morphological changes to be masked by a gold coating layer typically used in SEM,

helium ion microscopy (HIM) was also performed on these cells to visualise these

surface features. The Zeiss Orion Nanofab (HIM) allows to image non-conductive

biological samples without requiring a metal coating. The charge compensation in the

helium ion microscope is achieved with a floodgun which are hidden underneath.

To prepare the samples for scanning, a drop of cell media was put on a glass slide.

The cells were fixed by means of a 30-min treatment using a 4% glutaraldehyde

(Sigma-Aldrich, USA) in PBS. Subsequently, the yeast cells were dehydrated by

applying an increasing gradient of ethanol. Finally, the cells were dried in the vacuum

environment following the critical point method. In SEM, to prevent sample charging,

the dried cells were coated with a thin (several nm-thick) layer of gold. SEM

visualisation was performed using a ZEISS Sigma Scanning electronic microscope at

10-15 kV. The HIM images were recorded using the following parameters: 25 kV

acceleration voltage, 0.6 pA blanker current, 0.5 µs dwell time, 128-line averaging,

2048-pixel resolution. Optimal contrast in the images was achieved using the floodgun

deflectors in combination with the following floodgun parameters: line mode, 634 eV

energy, 370 μs floodtime, 100 μs grid blank time.

6.3 Results and discussion

6.3.1 Effect of CAP Treatment on cell morphology

Single colonies of yeast cells were treated by CAP and SEM and HIM images of

plasma-treated and non-treated cells were taken immediately after the CAP treatment

(Fig. 1 and 2, respectively). SEM visualisation revealed that plasma treatment did not

significantly affect cell size or shape, with cells showing spherical shape typical of


haploid S. cerevisiae cells. To compare the finer surfaces features of cells that were

subjected to plasma treatment to that of original cells, numerous yeast cells were

evaluated using HIM and representative areas for the non-treated cells, the plasma

stressed cells and the treated cells with nanodiamond are shown in Fig. 2a-c.

Figure 1. Representative SEM images of yeast cells showing untreated (left) and 3-

minute plasma-treated yeast cells (right).

Remnant nanodiamonds, which were left in the surrounding cell solution, are clearly

visible in Fig. 2c. A comparison of unmodified and treated cells shows differences in

membrane morphology. The cell membrane of the untreated yeast cells appears

smooth while the membranes of the plasma treated yeast cells shows altered

morphology., with surface features that appear to be absent from the surface of

untreated yeast cells (Fig. 2d-f).

The cell membrane of S. cerevisiae contains membrane compartments through which

nanoscale particles are able to diffuse into the cell through extremely slow


Figure 2. Representative HIM images of yeast cells showing untreated (a, d), plasma-

treated (b, e) and yeast cells that were exposed to NDs after receiving the plasma

treatment (c, f). Surfaces of cells that have received plasma treatment clearly show

features that are absent from surfaces of non-treated cells.

Brownian diffusion [21]. The highly ordered bilayer structure of the S. cerevisiae is

responsible for the slow diffusion and makes yeast highly tolerant to adverse

environmental conditions. Membrane remodelling in response to plasma treatment

may alter the highly ordered cell membrane, allowing the NDs to pass through the lipid

bilayer into the cell.

Membrane remodelling in response to stress is not unusual, with reported changes in

cell wall composition, e.g. in the fatty acid and lipid content, and isoprenoid

metabolism, and in membrane composition, such as in the fraction of palmitoleic acid,

ergosterol, and oleic acid [22].


Upon plasma treatment, some changes in membrane transport may arise due to a direct

effect of the plasma (chemical reactive species, UV light). By altering the physico-

chemical properties of cell wall and membrane, the cells may attempt to regulate cross-

membrane transport and thus prevent damage or withstand forces from the stressor.

Previous studies of CAP-cell interactions have reported pore formation in the cell

membrane [23] in response to CAP-generated chemically active species which have

the capability to induce chemical and physical changes on biological surfaces [24,25],

however these changes in membrane permeability are generally considered transient.

6.3.2 Effect of CAP Treatment and different concentrations of NDs on cell

growth

In addition to the potential changes in membrane structure and transport properties,

the CAP treatment is known to induce oxidative stress in the yeast cells [26], which

will lead to changes in intracellular activity. In general, intracellular responses to a

stress may include, for example, accumulation of α-amino acids that play an important

role in protein synthesis, such as tryptophan and proline, and storage solutes, such as

mycose and glycogen [22], changes in the expression and transcription of genes

responsible for stress response, alteration of redox machinery and peroxisomal

function, and many others. Previous studies have shown CAP treatment to enhance

hexokinase activity and secondary metabolite production in S. cerevisiae, and affect

redox balance in mammalian cell lines [27].

By monitoring the cell growth over the 24 h period, the effect of plasma treatment on

cell growth and viability was studied as a function of ND concentration. Two separate

studies were performed. First, the lag growth phases of cells incubated with

nanoparticles (at concentrations of 5, 50 and 100 µg ml-1), not subjected to plasma

were compared to the control group (no NDs, no plasma). Secondly, the lag growth


phases were compared when the cells were first subjected to plasma treatment and then

incubated in the presence of the same concentrations of NDs. As a positive control,

yeast cells that received plasma treatment, but not exposed to NDs were used. Results

of both studies were compared, to evaluate the synergistic effects of plasma stress and

NDs on cell growth and viability.

Figure 3. Growth of plasma-treated yeast cells during 24 h period of exposure to

different concentrations of NDs. Each treatment was done in triplicate and the error

bars represent the standard deviation around the mean of the cell density at each time

point (measured at 600 nm).

Comparing the lag phase of cell growth between different concentrations of

nanoparticles, it is evident that the lag phase of growth was slightly prolonged with

increasing concentrations of NDs when no plasma was applied to yeast cells (Fig. 3).

Furthermore, the lag phase is extensively prolonged in plasma treated yeast cells; one

can observe elongation of lag phase with higher concentrations of NDs (Fig. 4).

The lag phase of cell growth is the phase where cells adapt to the environment

conditions, such as temperature, pH, oxygen availability and potential environmental


stress [28-30], which in this case is the presence of the nanoparticles in the growth

medium. In this phase, cells prepare to reproduce, so the presence of additional stress

would result in a prolonged lag phase.

Figure 4. Growth of yeast cells during the 24 h period, incubated with different

concentrations of NDs. Cells received no plasma treatment.

From the growth curves (see Fig. 3 and 4) it is observed that the lag phase is extended

in cells treated with plasma in a dose dependent manner, compared to non-treated

control. Furthermore, when adding the NDs in the growth medium, the synergistic

effects of plasma and NDs on lag phase of cell growth is even more pronounced (the

lag phase was more prolonged), confirming that the combination of different stress

agents significantly affect cell growth and viability.

S. cerevisiae cells are known to adapt to various stress conditions that arise during

fermentation [31-34]. For example, trehalose is a disaccharide found in yeast cells that

enhances their tolerance to the oxidative stress and causes enhanced intracellular

oxidation, thereby protecting the cells, and enabling the cell to overcome the stress and

survive [26,35,36]. Although, there are numerous studies proving that plasma is

stressful to living organisms and effects are dose dependent stress tolerance, and effect


is clearly seen from Fig. 4 [37,38]. Cells treated with plasma and not challenged with

NDs exhibit similar growth with longer lag phase to that of control group that received

neither plasma nor ND treatment (see graph on Fig. 3). A longer lag phase is a

consequence of plasma treatment, whereas the cell density after 24 h is similar in both

groups. Both groups exhibited good cell growth and shorter lag phases in comparison

to groups with synergistically combined plasma and NDs stress on the cells (Fig. 3).

Furthermore, significant deviations in cell density after 24 h clearly show dependence

of cell density on ND concentration when ND treatment is combined with plasma

stress.

6.3.3 Effect of CAP treatment and different concentrations of NDs on cell

viability

Nanodiamonds are biocompatible and are considered to be the most biocompatible

among all carbon nanoparticles presently studied [39]. However, there is a strong

tendency for the NDs to agglomerate, regardless of their surface charge [40,41].

Regardless of how biocompatible the nanodiamonds are, their accumulation within the

cell will considerably change the in vitro (e.g. cellular uptake and cytotoxicity of NDs

to cells), as well as in vivo fate (metabolism, toxicity, bio distribution) of any living

organism [3,42]. Higher concentrations of NDs, along with plasma treatment also

resulted in a significant decline in the cell viability due to the increased amount of

nanoparticle uptake into the cells (Fig. 5). Cytotoxicity was induced by the


Figure 5. Synergistic effects of plasma and NDs on viability of yeast cells. Cells were

incubated for 24 h. 3 min plasma treatment in combination with ND nanoparticles (100

µg/mL) shows significant inhibition of cell growth relative to other groups. All values

are normalized to control, plasma non-treated cells without ND. Mean values (± SE)

are given.

aggregation of a large amount of NDs, which affected metabolic activities of the cell

[3,43], and simultaneously prevented cells from effectively mitigating oxidative stress

induced by the plasma treatment. Interestingly, biocompatibility of NDs was found to

be significantly influenced by the concentration and aggregation of these particles

inside the living organism (Fig.6). Our results showed that cells exposed to 100 µg ml-

1 NDs in combination with plasma treatment exhibit significantly lower cell viability,

reduced by 55% compared to control group without NDs. However, plasma treatment

in the absence of NDs in suspension had a limited effect on cell density. Moreover,

NDs were only cytotoxic in combination with plasma at a high particle concentration

of 100 µg ml-1. Lower concentrations of NDs (at 5 µg ml-1 and 50 µg ml-1) were not

associated with cell toxicity, nor were they able to notably inhibit the growth of cells

relative to the control group.


Figure 6. Uptake of non-terminated nanodiamonds by plasma-treated yeast cells as a

function of ND concentration as visualised by fluorescent and corresponding

brightfield microscopy. Cells were incubated for 24 h. Nanoparticles are seen as green

dots entrapped within the cells, as well as outside in the medium. Micrographs show

the highest uptake of NDs at a concentration of 100 μg/ml, whereas the lowest uptake

was observed at concentration 5 μg/ml.

6.3.4 Effect of CAP treatment and different concentrations of NDs on cellular

uptake

Fluorescent and bright field images of yeast cells, incubated with NDs were acquired

by using laser scanning confocal microscopy. NDs emit green fluorescence, which

makes them easy to detect inside the cells. Plasma treatment increased permeability of

the cell membrane, resulted in easier and increased uptake of NDs into the cell (Fig.


7). Inside the cells, nanoparticles were found proportional to their concentrations,

specifically around the cell wall, as well as outside in the medium. Images show the

agglomeration of the NDs in the yeast cells.

Figure 7. Mechanism of nanotoxicity. Plasma treatment led to changes in the cell

membrane of yeast cells, which facilitated the passage of NDs into the cell and

subsequent accumulation of NDs within the cell, potentially interfering with cellular

metabolism.

6.4 Conclusion

NDs are known as inert and biocompatible in the body and thus, their extensive use in

materials for food packaging, medical implants, as a drug delivery inside the cells, has

not raised particular concerns to date. This study confirms that even at relatively high

concentrations of 100 µg ml-1, NDs alone do not significantly negatively affect the


survival of S. cerevisiae, however cell growth was slowed down. However, when cells

were subject to plasma stress, and then exposed to NDs at the same concentration, this

resulted in a significant reduction in cell viability, likely attributed to changes in

membrane transport properties which allowed enhanced uptake and accumulation on

NDs. Even though NDs are considered biologically inert, their agglomeration within

the cells and along the cell wall is likely to have interfered with cellular metabolism.

These results highlight the significance of intra- and extra-cellular stresses in

determining cell fate upon exposure to nanoparticles, which may be even more

significant in the case of chemically-reactive nanoparticles. This study also

demonstrates plasma as a useful tool for mimicking natural environmental exposure.


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This chapter of this thesis is to be submitted to Biomaterials as:

Nanocarbon polymer composite for breast

implants

Karthika Prasad, Aaqil Rifai, Kate Fox, David Schuessler, Kateryna

Bazaka, Kostya (Ken) Ostrikov






1. they meet the criteria for authorship in that they have participated in theconception, execution, or interpretation, of at least that part of the publicationin their field of expertise;2. they take public responsibility for their part of the publication, except for

the responsible author who accepts overall responsibility for thepublication;

3. there are no other authors of the publication according to these criteria;4. potential conflicts of interest have been disclosed to (a) granting bodies,

(b) the editor or publisher of journals or other publications, and (c) thehead of the responsible academic unit, and

5. they agree to the use of the publication in the student’s thesis and itspublication on the QUT’s ePrints site consistent with any limitations setby publisher requirements.


“Nanocarbon polymer composite for breast reconstruction” To be submitted to

Biomaterials on September 2018


(If the Co-authors are not able to sign the form please forward their email or other correspondence




Karthika Prasad Conception of idea. Designed and conducted the experiments,

analyze the data and wrote the manuscript.

28/09/2018

Aaqil Rifai Assisted with executing the microbial and biocompatibility studies

Kate Fox Advised on properly executing the bio part of the project

David Schuessler Industrial mentor, helped with the execution of the project to suit

industrial needs.

Kateryna Bazaka

Proposed the original concept, supervised experimental design and

data collection, helped in interpreting results and underlying

mechanisms, helped with article writing.

Kostya Ostrikov Supervised the research, helped with execution of the research, data

analysis and interpretation and major manuscript editing

Name Signature Date




PREFACE

This article investigates the mechanical properties of silicone when reinforced with

different carbon materials, i.e. graphene-based materials and nanodiamonds. It was

verified through this work that our hypothesis was right. Both graphene reinforced

silicone and nanodiamond reinforced silicone showed enhanced mechanical strength

and bactericidal properties without compromising on biocompatibility


Abstract

The use of synthetic mammary implants for post-mastectomy breast reconstruction

and elective cosmetic augmentation continues to increase, yet the issues of implant

leakage and rupture, capsular contracture and infections, and associated prolonged

patient care, pain and loss of function. In this work, carbon nanomaterials of sp2 and

sp3 hybridization are introduced into the polymer matrix and on the surface of breast

implant-grade silicone with the aim to enhance durability and biocompatibility

performance, respectively.

The significance of nanocarbon materials in enhancing the strength, bactericidal effect

and biocompatibility of the standard breast implant material is proved. This work has

led to development of polymer-nanocarbon composite which is 68% stronger and a

nanocarbon coating on shell surface which have improved bactiricidal against gram

positive S.Aureus bacteria and excellent adhesion of HDF cells. This study may

provide new insights into the better understanding of nanocarbon materials for

biomedical application.

Key words: breast augmentation, synthetic mammary implants, mastectomy, cosmetic

augmentation, nano-carbon


7.1 Introduction

Since 1962, breast implants have been used for both reconstruction and aesthetic

purposes [1]. Over the years, the demand for breast implants has been increasing

steadily, with the global breast implants market size reaching USD 1.2 billion in 2016.

The trend is likely to continue in the future [2], with silicone implants projected to

retain their position as the preferred option for breast augmentation and reconstruction

surgeries.

In spite of substantial progress in the design and manufacturing of silicone breast

implants, a number of challenges remain. For one, the material needs to be sufficiently

soft to give the appearance and feel of real tissues, yet at the same mechanically robust

to prevent rupture, leakage and failure of the implant shell [3-5]. Similarly, the

material need to be highly biocompatible, both in terms of its chemistry and surface

topography properties, to minimize foreign body response and associated formation

of hard connective tissue capsule around the implant, yet at the same time it should

resist uncontrolled protein fouling, microbial colonization and biofilm formation [6,

7]. Despite best efforts to address these often competing priorities, implantation using

silicone breast implants remains only a short- to medium- term solution, with the

majority of patients undergoing implant removal within the span of 3 months to 10

years post-implantation [8].

Nanocarbon materials are of great interest to the biomedical industry as a means of

enhancing the bulk and surface properties of polymer materials. When introduced into

a polymer matrix at very low concentrations, nanocarbons can be used to enhance

Young’s modulus, tensile strength, and thermal resistance of the material, whereas

surface decoration of polymers with nanomaterials can be used to control cell−surface


interactions and biocompatibility [9]. Nonetheless, achieving a uniform dispersion of

nanocarbons within the polymer matrix is not trivial [10].

Using graphene or nanodiamond as the nanocarbon of choice, we report the synthesis

of silicone−nanocarbon composites surface-decorated with nanocarbons with the aim

to deliver a breast implant material with increased mechanical properties and

biocompatibility.

7.2 Experimental Section

7.2.1 Materials

The polymer NUSIL MED 10-6400 (Part A and B), which is an addition cure silicone

dispersion, was purchased from Nusil technology. Few layered vertical graphene

flakes were collected from CSIRO, Linfield and nanodiamonds were purchased from

Adams Nano, respectively. The reagents for biocompatibility tests and Dulbecco's

Modified Eagle's Medium (supplemented with 10% fetal bovine serum and 1%

penicillin/streptomycin) were obtained from Sigma Aldrich. For antibacterial testing,

Staphylococcus aureus (CIP 65.8T) was purchased from the Culture Institute Pasteur,

France, and glycerol nutrient broth and nutrient agar (Oxoid) were purchased from

Thermo Fisher Scientific. Every material was used as purchased unless stated.

7.2.2 Synthesis of Nanocarbon composite

Few layer vertical graphene flakes and 120 nm nanodiamonds were dispersed in

isoproponal and ultrasonicated for 25 min at 40 kHz. Subsequent to ultrasonication,

0.8 wt% of either graphene or nanodiamond dispersion was added into 6 ml of silicone

dispersion (Part A and B) and mixed. The composite was then cured at 75 °C for 45

min. For biological studies, graphene flakes or nanodiamond were then coated onto

the polymer matrix via dip coating method.


7.2.3 Tensile test

Trotec laser cutter was used to cut “dog bone” shaped tensile test specimens. A

universal testing machine from Intron was used to perform tensile tests under

displacement control at a rate of 2 mm/min. A 250 N pneumatic grip was used for

measuring the load. The tensile strength, tensile strain, percentage elongation, distance

and load were estimated using Bluehill 3 software. All the samples were 0.34 mm thick

and the tensile specimen was 50 mm long, with a gauge length of 30 mm.

7.2.4 Tear test

Rectangular shaped samples with a cut in the middle were prepared using a Trotec

laser cutter. A universal testing machine (Intron) was used to perform tear tests at a

constant displacement rate of 2 mm/min. A 250 N pneumatic grip was used for

measuring the load. The tensile strength, tensile strain, percentage elongation, distance

and load were measured by using Bluehill 3 software. All the samples were 0.32 mm

thick and 45 mm long.

7.2.5 Nanoindendation test

Average readings of hardness were obtained for each composite specimen by

measuring five points with a Hysitron TI 950 Triboindenter using a Berkovich cube

corner tip with a radius of about 100 nm. The load applied onto the specimen was 5

mN and maximum displacement covered was 20 nm.

7.2.6 Bacterial assessment

S. aureus (CIP 65.8T) bacterial stocks were prepared in 20% glycerol nutrient broth

and stored at –80°C until needed. Prior to each experiment, the stock was refreshed

upon nutrient agar for 24 h at 37° C. S. aureus suspensions were prepared by

suspending a single loopful of bacteria in 5 mL of nutrient broth The suspensions were


then further diluted using nutrient broth to obtain an optical density OD600 = 0.1. Pure

silicone was used as the control in comparatision to the graphene and nanodiamond

coated silicone. The samples were sterilised by repeatedly washing their surface with

ethanol, then rinsed with MilliQ water and dried under a gentle N2 flow. Finally, the

samples were UV sterilised. The sterilised surfaces were then incubated in the presence

of 1 mL of bacterial suspension in a sterilised 12-well plate (In Vitro Technologies)

for 18 h at 25 °C.

Following incubation, confocal laser scanning microscopy (CSLM) was utilised to

assess the surface attachment of live and dead bacteria. Prior to CSLM imaging,

samples were removed from the bacterial suspensions and washed gently twice with

MilliQ water for 3 sec. This process removes unattached bacteria from the surface,

allowing all imaging experiments to be performed under similar conditions. Surface

bacteria were then stained for 30 min in the dark using LIVE/DEAD Baclight Bacterial

Viability Kit, L7012 (Molecular Probes, Life Technologies) according to the

manufacturer’s protocol. SYTO9 permeates both intact and damaged membranes of

the cells, and fluoresces green when bound to nucleic acids and excited by a 485 nm

wavelength laser; however, propidium iodide (PI) only enters bacteria with significant

membrane damage (non-viable cells) and binds with higher affinity to the intracellular

nucleic acids than SYTO9. The number of viable and non-viable bacterial cells was

determined by pixel counting at their respective fluorescence emission wavelengths.

CSLM images were obtained using a FV1000 Spectroscopic Confocal System

(Olympus, Tokyo, Japan) with an inverted microscope at 60x magnification. Images

were taken for 5 different randomly selected fields of view to obtain the representative

data for the entire surface. After image acquisition, the imaging software Fluoview FV


4.2 was utilised to assess the CSLM images on a frame-by-frame basis, which allowed

the total surface-bacteria count to be calculated. The experiments were performed in

triplicates and were repeated three times.

7.2.7 Biocompatibility tests

Human Dermal Fibroblasts (HDF) were used to assess the biocompatibility of the

composite materials, as this cell line is commonly used to measure cytotoxicity and

biocompatibility of materials used in biomedical applications. Prior to cell seeding, all

samples were cleaned with the standard procedure using alcohol based solvents and

UV sterilization. Samples were placed individually into a 24-well plate, then seeded

with HDF cells in Dulbecco's Modified Eagle's Medium (DMEM) (supplemented with

10% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin) at the density of

40,000 cells per well. The well plate was incubated in cell culture for 1, 3 and 7 days

under 37 °C, 5% CO2. After incubation, cells were rinsed with Phosphate Buffer

Solution (PBS). Then, paraformaldehyde was applied for 30 min to fix the cells to the

samples. The samples were stored at 4 °C in the fridge prior to imaging.

To observe cell spreading and attachment in response to the presence of graphene and

NDs on the polymer surface, the nucleus and actin filaments were stained. The cells

were firstly fixed with 4% paraformaldehyde for 1 hr at room temperature after

removing cell culture media from the well plate. The cells were permeabilized with

staining buffer (1% BSA w/v, 0.3% Triton-X 100 w/v in PBS) for 1 hr at room

temperature. During each staining and fixative process, the samples were washed with

PBS. To observe the actin filaments, the cells were stained with Alexa Fluor 594

Phalloidin (1:40 dilution) and incubated for 2 hr at room temperature, at which point


1 µl of Hoechst was added and left for 5 min. The samples were then washed with

staining buffer and stored with 1 ml PBS at 4 °C for confocal imaging.

To assess the viability and morphology of cellular growth, the Olympus Confocal

Microscope was used to obtain confocal micrographs of the merged nucleus and actin

filament maps. The nucleus data were captured using DAPI filter (350nm/470nm). The

actin filament data were captured using the TRITC filter (561nm/600nm).

Cell proliferation was assessed using MTS assay, where HDF cells in medium were

seeded on the composite material (grapheme coated silicone and nanodiamond coated

silicone) and control (pure silicone) in a 24-well plate at approximately 4 × 104

cell/cm2 . The plate was incubated in a CO2 incubator for 1, 3 and 5 days. Then, 200

µL of [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-

2H-tetrazolium, inner salt; MTS(a)] solution was added to the wells to quantify the

level of cell proliferation for different time periods. 100 µl medium from each well

was then transferred to an ELISA 96-well plate, and absorbance at 490 nm was

recorded using a SpectraMax Paradigm Multi-Mode Microplate Reader. The optical

density (OD) corresponds to the viable cell numbers.

7.3 Results

The mechanical properties of silicone−nanocarbon composites were tested using three

different types of tests, i.e. tensile test to calculate the maximum tensile strength, tear

test to calculate the tear resistance, and nanoindentation test to measure the indentation

hardness of the composite (figure 1).


The results of tensile strength, % elongation and nanoindetation hardness were

calculated directly from the data generated, whereas the tear strength was estimated as

the maximum force the sample can withhold divided by the thickness of the sample.

Table 1: Key results of the mechanical test data.

Compared to that of unmodified silicone material, graphene-reinforced silicone

composites exhibited a significant improvement in tensile strength and tear strength,

while also showing an increase in nanoindendation hardness, which is comparatively

less when considering the thickness of the sample material used. The addition of

nanodiamond led only to a slight improvement in the aforementioned properties. As

seen in table 1, the addition of graphene to silicone, increased its tensile strength from

11.8 MPa to 19.3 MPa, elongation from 758.8% to 1232.3%, tear strength from 26.2

N/mm to 46.59 N/mm, and indentation hardness from 1.04 MPa to 1.484 MPa. The

addition of the nanodiamond into silicone matrix resulted in an approximately 19.4%

increase in tensile strength, 44.3% increase in elongation, 39.05% improvement in tear

Mechanical Test Pure silicone Graphene Composite Nanodiamond

Composite

Tensile Strength (MPa) 11.8 19.3 14.1

% Elongation 758.8 1232.3 803.1

Tear Strength (N/mm) 26.2 46.59 36.43

Indentation Hardness (MPa) 1.04 1.484 1.183


strength and around 13% increase in indentation hardness compared to silicone

control.

Figure 1. Mechanical test results for nanocarbon-enhanced silicone composites. (a)

Tensile test results show higher tensile strength of graphene-reinforced silicone


compared to that of nanodiamond-reinforced silicone and silicone control. (b) Tear test

results show a comparative improvement in tear strength in graphene-reinforced

silicone. (c) Nanoindendation results show considerable changes in the indentation

hardness of graphene reinforced silicone.

Figure 2. Representative CLSM images of S. aureus attachment to the surfaces of a)

pure silicone b) silicone composite decorated with graphene, and c) silicone composite

decorated with nanodiamond after an incubation periods of 18 hr. The viability of cells

attached to surfaces for all substrates was found to be ~98%. d) Bar graphs of the

average number of attached cells. Error bars display the standard deviation of the data.

Data = Mean ± Standard deviation. The statistical p-values were 0.00000006 (**) and

0.011 (***).


The ability of composites coated with either graphene flakes or nanodiamond to reduce

attachment and colonisation by pathogenic microorganisms were studied using S.

aureus bacterial strain. The surfaces of nanocarbon-coated samples showed a

statistically significant reduction in bacterial attachment compared to control, with

graphene-coated silicone showing the lowest level of bacterial cell attachment among

the tested samples.

To assess the biocompatibility of silicone-nanocarbon composites with mammalian

cell lines, the materials were exposed to human dermal fibroblast cells to determine

the cell viability and biocompatibility of the composite over the periods of 1, 3, and 7

days, with the results shown in Figure 3. Sustained cell growth was observed on the

surfaces of all composite materials, with cells showing preferential attachment to

nanodiamond-coated surfaces when compared to graphene-coated surfaces.


Figure 3. Confocal micrographs of HDF cell growth on the surface of (a) pure silicone

(b) graphene-coated silicone composites, (c) nanodiamond-coated silicone

composites, and (d) tissue culture plates on day 1, 3 and 7. (e) Optical density of

suspensions containing cells grown in the presence of different silicone samples. Data

= Mean ± Standard deviation. The statistical p-values was < 0.048 (*).

7.4 Discussions

On the most basic level, the majority of currently used implants have a durable silicone

outer shell and a softer filler, e.g. silicone gel or saline, with multiple variations in

terms of implant design optimised to address different objectives. For example, thicker

silicone gels provide shape stable implants, whereas double lumen designs (with a

smaller inner silicone gel-filled implant within a larger-volume saline implant) aim to

reduce the negative effects of silicone gel leakage should the outer shell rupture.

Regardless of the design, however, most implants will need to be removed at some

point in time due to shell rupture, gel leakage, infection or capsular contracture, with

the properties of the shell playing a critical role in most if not all of these


complications. Extending the lifetime and improving cell-surface properties of the

silicone shell material is therefore an area of intense research and development.

7.4.1 Mechanical Reinforcement

In silicones used in the breast implant industry, the intermolecular forces between the

polymer chains are fairly week [11], which means that in order to attain desired

mechanical properties, the presently available breast implant-grade silicones have to

use microscopic fillers, such as treated silica, and coupling agents to improve adhesion

and interactions between the polymer chains [12, 13]. However, while improving some

aspects of mechanical performance, e.g. strength, these strategies may result in

materials that are harder and less elastic, which are not desirable traits for breast

implant materials.

When nanomaterials such as graphenes are used to reinforce polymer matrices, high

surface area-to-volume ratio of these nanostructures ensures a large area of interface

between these materials and polymer chains. As such, even at very low levels, these

nanomaterials can potentially deliver substantial changes in the mechanical properties

of silicones when compared to other fillers [14].

Once introduced into the polymer matrix, nanocarbon fillers interact with silicone

polymer chains at the molecular level, and these strong interfacial interactions enable

efficient stress transfer from a mechanically weaker polymer matrix to a mechanically

superior nanocarbon particles. Apart from increasing the mechanical strength, these

interactions also increase the plastic flow resistance of the polymer matrix, which in

turn leads to increased hardness. Graphene and nanodiamond represent 2D and 0D

structures of carbon, respectively, each featuring a different hybridization state [15-

17]. Due to sp2 hybridization and large number of reactive lattice defects and long


edges, graphene flakes have a tendency to form stronger bonds with silicone chains

when compared to that of sp3 hybridized nanodiamond. This may at least in part

explain higher mechanical strength of graphene-reinforced silicone when compared to

nanodiamond-reinforced silicone. Furthermore, the inherent nature of the bonding

interaction at the interface between nanocarbon additive and silicone matrix is likely

to have a notable effect on the resultant mechanical properties of the composite.

Composites produced using nanodiamond are likely to be non-covalent assemblies,

where the interactions between the polymer matrix and the nanodiamond are governed

by relatively weak dispersive forces. On the other hand, due to a large number of

defects in the lattice and the presence of highly chemically-reactive edges in vertically

oriented graphenes used in this study, the graphene-reinforced composite is more

likely to feature both covalent and non-covalent linkages between the nanoparticles

and the silicone polymer matrix, resulting in stronger interfacial bonding.

Figure 4. (a) Bonding between silicone and graphene, (b) Schematic representation

of the mechanism by which graphene limits crack propagation.


The outstanding mechanical strength of graphene is due to the stability of the sp2 bonds

that forms hexagonal lattice; these bonds restrict in-plane deformation of the lattice

[18]. Graphene-reinforced silicones show superior tensile strength because of the

cross-linking effect between the graphene sheets and the polymer matrix via π–π

stacking interactions [19]. Moreover, stacking of graphene sheets within the polymer

matrix leads to a more efficient transfer of stress under loading, which is transferred

from matrix to graphene via covalent and non-covalent bonding [19, 20]. In addition,

the unique chemical structure of graphene allows it to resist crack propagation via

spontaneous re-distribution of carbon atoms to fill and repair damaged areas of

graphene lattice at ambient conditions, which is essential for the prevention of

mechanical failure of the implant material [21].

Unlike 1D or 2D nanostructures, the small size of nanodiamond provides it with a

much greater area of interface with polymer chains in the composite [22]. Owing to

their 0D structure and spherical shape, nanodiamonds do not form stacks or bundles in

the polymer matrix, which enables their easy dispersion in the polymer matrix [23].

The covalent bonding in principle is possible given that there is a possibility that

several of the atoms on the surface of nandiamonds are in sp2 hybridization,

contributing to improved mechanical properties [24].

7.4.2 Bacteria-surface interactions

Next to mechanical failure, microbial attachment and biofilm formation are a common

issue associated with breast implant use. In addition to being a cause of acute and

chronic infection, the associated inflammation may in some instance lead to peri-

implant tissue necrosis and the formation of a capsule around the implant. Overtime,

this capsule can reduce in size due to scar formation, a process called capsular


contracture, which in turn results in the implant being subjected to deformation or

mechanical stress [25]. This deformation not only leads to poor aesthetic results, but

may cause implant rupture or leakage. Once formed, the capsule often requires surgical

removal, which is typically accompanied by the replacement of the implant.

Carbon nanomaterials are well known for their antimicrobial and antifouling activity.

The antimicrobial mechanism of carbon nanomaterials relies on the unique

combination of physical and chemical properties [26]. The antimicrobial activity of

carbon nanostructures normally involves both physical and chemical mechanisms,

which include but not limited to ROS production, structural damage to cellular

membrane, electron transfer, etc [27].

Figure 5: A schematic representation of the mechanisms by which surface-

immobilised graphene may reduce bacterial attachment and biofilm formation.

Fig. 2(b) shows that decoration of silicone composites with graphene flakes

substantially reduced attachment and proliferation of S. aureus cells, with some cells

being attached but non-viable (represented by red dots). One of the key characteristic

features of vertical graphenes are their sharp edges that play a vital role in the


antibacterial activity of these nanomaterials. The sharp edges of graphene have the

capacity to change the nanoscale topography of the surface, effectively reducing the

number of possible attachment points between the microbial cell and the surface.

Furthermore, the sharp edges can directly puncture the cell wall, resulting in loss of

cell membrane integrity, leakage of intercellular substances, and eventually death of

the bacterial cell [28, 29]. In addition to the physical mechanism of preventing

microorganism attachment, graphene can also inhibit the metabolism of bacteria by

inducing oxidative stress, which interferes with its cellular function and leads to

cellular inactivation [28].

Figure 6 (a) Schematic representation of biofilm formation on silicone surface, (b)

possible antifouling mechanism on nanodiamond-decorated surface.

The lack of co-localization between nanodiamond and bacterial formation validates

the antifouling characteristics of nanodiamond-decorated composite surfaces (fig 2).

The bacterial adhesion process is limited on the nanodiamond surface due to its

extremely smooth characteristics [30]. In addition, as an electrically active surface,

nanodiamonds can react and form chemical bonds with biomolecules in the


surrounding environment and can interact with the bacterial cell membrane, alter cell

morphology, cause disruptions in the cell memberane, and hinder the bacterial

adhesion and biofilm formation on the nanodiamond surface [31].

7.4.3 Biocompatibility studies

The biocompatibility of nanomaterials comes from their nanostructured surface which

enhances the adsorption of extracellular matrix molecules from the culture media

serum that mediate subsequent cell adhesion [32]. Even though nanocarbon materials

may display cytotoxicity [33], in this experiment both types of silicone-nanocarbon

composites exhibited superior biocompatibility when compared to that of a normal

unmodified silicone matrix.

The exact mechanism behind the biocompatibility of graphene is yet to be fully

elucidated, and may include a number of physicochemical interactions that takes place

at the interface, which include but not limited to kinetic or thermodynamic interactions

between the surface of nanomaterials and that of biological components of the cells,

such as proteins, peptides, amino acids, and phospholipids [34]. In this experiment,

the high affinity of cells to sp2 hybridised graphene may be attributed to a combination

of electrostatic interactions between peptides of the cellular membrane and

chemically-reactive edges of graphene flakes [35], as well as the capacity of graphene

to adsorb the proteins associated with the extracellular matrix, thereby providing a

suitable platform for cellular attachment.

In contrast to graphene, nanodiamonds are assumed to have the highest

biocompatibility among all known carbon nanomaterials. Nanodiamonds are known

to improve the surface hydrophilicity of surfaces to which they are applied [36], and

cellular attachment, depending on the cell properties, is often greater on hydrophilic


surface [37]. In addition, just like any other nanoparticle, the high surface-to-volume

ratio and the shape of nanodiamond may promote cell adhesion.

7.5 Conclusion

Nanocarbon-enhanced silicone composites have the potential to overcome challenges

currently faced by existing implants, and offer breast reconstruction solutions that not

only minimize the likelihood of medical complications but possibly offer more

realistic and aesthetically pleasing outcomes for the patients. The results from this

study show that silicone matrices reinforced with nanocarbons, particularly graphene

flakes, have significantly improved mechanical properties, and as such are highly

promising for long-term implantation. When decorated with nanoparticles, these

nanocomposites also show lower microbial attachment and improved compatibility

with mammalian cell lines. Importable, these notable improvements were attained by

adding less than 1%wt of nanocarbon materials, suggesting that the price of the

resulting product will not be significantly different to that of the base silicone implant.


Acknowledgement

The authors would like to thank SEF launch pad at QUT Gardens Point for giving

access to the laser cutter. The authors thank Mr. Greg Paterson for providing technical

assistance with mechanical testing at QUT. The authors thank the MicroNano

Research Facility at RMIT University for access to the PC2 laboratory and cell culture

facilities. The authors wish to acknowledge the ARC Centre for Nanoscale

Biophotonics for their help with sample preparation and use of confocal microscopy

facilities. The authors acknowledge the assistance with bacterial culturing and imaging

from Dr. Aaron Elbourne in association with Prof. Elena Ivanova and Prof. Russell

Crawford from the School of Science, RMIT University.


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Chapter 8: Conclusions and Future

Recommendation

Carbon nanomaterials offer numerous exciting possibilities for implantable materials

to be applied to solve problems in breast reconstruction, namely structural failure and

limited tissue-implant surface integration, and thus lower health care costs and deliver

improved patient outcomes. Yet, for this to happen, better understanding of how the

addition of nanomaterials challenges the physical properties, ageing behaviour and

biological performance of the implant is required.

Through this project we have emphasized the need for new or modified materials, such

as those modified by carbon nanomaterials, to advance the currently available breast

implants to provide better patient outcome. The thesis has discussed the future

directions for the use of nanomaterials in breast reconstruction, stimulating discussion

on whether reconstruction technique will rely on and will be driven by the

advancements in materials engineering, and what steps are needed to realise this future.

The findings of this thesis suggest that composite materials reinforced by certain types

of nanocarbons can demonstrate significantly improved mechanical strength and

durability. However, further studies should be performed to optimise thus-prepared

composites, as well as to study their behaviour over an extended period of time.


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QUEENSLAND UNIVERSITY OF TECHNOLOGY...Submitted by: Karthika Prasad Master of Science (Nanoscience) Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy