The Pennsylvania State University

The Graduate School

Eberly College of Science

BIOINSPIRED MINERALIZING MICROENVIRONMENTS BASED ON LIQUID-

LIQUID PHASE COEXSISTANCE

A Thesis in

Chemistry

by

Morgan L. Gulley

© 2019 Morgan L. Gulley

Submitted in Partial Fulfillment

of the Requirements

for the Degree of

Master of Science

August 2019

ii

The thesis of Morgan L. Gulley was reviewed and approved* by the following:

Christine D. Keating

Professor of Chemistry

Thesis Adviser

Raymond Schaak

DuPont Professor of Materials Chemistry

Elizabeth Elacqua

Assistant Professor of Chemistry

Philip Bevilacqua

Distinguished Professor of Chemistry and Biochemistry and Molecular Biology

Head of the Department of Chemistry

*Signatures are on file in the Graduate School.

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Abstract

Inspired by biological mineralization, this thesis focuses on ways to control synthesis through

use of microenvironments. The general approach of previous research was through performing

reactions in all-aqueous emulsion droplets of an aqueous two-phase system (ATPS). Calcium

carbonate in the amorphous morphology was formed through biomineralization with this

emulsion. As the system was looked at further, tunability of produced minerals came from the

existence of a third phase in the system produced from neutralization of a negatively charge

polymer with a cation. This phase is highly hydrated, causing an aqueous three-phase system

(A3PS) to become the general approach of mineralization for the following thesis. This third

aqueous phase is termed the coacervate. Coalescence of the original two phases was prevented

through introduction of large unilamellar vesicles (LUVs, ~110 nm diameter). These vesicles self-

assemble at the aqueous/aqueous interface with and without a coacervate present in the system.

These biogenetic minerals often contain organics, which are important for optical and mechanical

properties. The approach for this thesis uses coacervates droplets of concentrated organics for

mineralization.

In Chapter 1, an overview of the importance of the biomineral, CaCO3, production is discussed,

the importance of control over the system is conferred, along with factors to help control the

system. Chapter 2 discusses the system of mineralization and the multiple analytical techniques

used for analysis. Chapters 3 & 4 go over the changes to the system for tunability and the

conversion of stable CaCO3 minerals for further use. In Chapter 3, the concentrations of the

reactants were tested in the presence of a coacervate to see stability and tunability of the system.

Analysis of results through fluorescence confocal microscopy and scanning electron microscopy

show the effect of starting materials on the size of mineral particles formed. Chapter 4 indicates

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the importance of using the system for more than one mineral type. The conversion of the stable

amorphous calcium carbonate (ACC) minerals to calcium phosphate indicates the broad expansion

of amorphous precursors in biomineralization. The minerals produced are analyzed through x-ray

diffraction, Fourier transform infrared spectroscopy, and Raman spectroscopy to show conversion.

Chapter 5 gives an overall conclusion for the thesis with a look into the future direction of the

project.

v

TABLE OF CONTENTS

LIST OF FIGURES…………………………………………………………………………..…..vii

LIST OF TABLES………………………………………………………………………………xiv

LIST OF ABBRIVIATIONS.......………………………………………………………………..xv

ACKNOWLEDGEMENTS………………………………………………………………….....xvii

Chapter 1. INTRODUCTION…………………………………………………………………......1

1.1 Research Motivations…..…………………………………………………………..….1

1.2 Compartmentalization of Cells…...…………………………………………..…....…..2

1.3 Morphologies of Minerals……………………………………………….………….…4

1.3.1 Biological Properties………………………………………………………...6

1.3.2 Morphology Changes..……………………………………………………....7

1.4 Biomimetic Mineralization in Aqueous Three Phase Systems…..………………….....8

1.4.1 Liquid-Liquid Phase Separation.………………………………………….....9

1.4.2 Stabilization of Aqueous Two Phase Systems……………………………...11

1.4.3 Introduction of Coacervate-Containing AMVs...…………………………..14

1.5 Chapter Overviews…………………………………………………………………...19

Chapter 2. METHODS…………………………………………………………………………...21

2.1.1 Buffer Preparation………………………..………………………………………...21

2.1.2 Aqueous Two Phase Systems ……….……………………………………………...21

2.1.3 Preparation of Stock Solutions…………………………………….………………..22

2.1.4 Preparation of Lipid Vesicles……………………………………………………….22

2.1.5 Silanization of Glass Slides………………………………………………………...24

2.2 Methods of Making the System…..…………………………………………………..24

2.2.1 Formation of Artificial Mineralizing Vesicles ……….…………………….24

2.2.2 Synthesis of Amorphous Calcium Carbonate ……..……………………….25

2.2.3 Phosphate Buffer Wash…………………………………………………….26

2.3 Instrumentation of Analysis……………………………………………………...…...27

2.3.1 Confocal Microscopy Analysis…………………………………………….27

2.3.2 Scanning Electron Microscopy Analysis…………………………………...27

2.3.3 Image Analysis……………………………………………………………..28

2.3.4 X-Ray Diffraction Analysis ………………………………………………..28

2.3.5 Raman Spectroscopy……………...………………………………………..28

2.3.6 Fourier Transformed Infrared Spectroscopy………………………………..28

Chapter 3. MODIFICATION OF ARTIFICAL MINERALIZING VESICLE SYSTEMS………30

3.1 Artificial Mineralizing Vesicle System………………………………………………30

3.2 Order of Addition……………………………………………..……………………...31

vi

3.3 Effect of LUV Concentration on AMV, Coacervate, and Mineral Size……..………..33

3.3.1 Analysis…………….………………………………………………………34

3.4 Dextran:Poly(ethylene) Glycol Volume Ratio……………………….……………….40

3.4.1 Analysis……………………………………………...……………………..40

3.5 Conclusion……………………………………………………………………………44

Chapter 4: CALCIUM PHOSPHATE SYSTEM………………………………………………...45

4.1 Importance of Versatility……..………………………………………………………45

4.2 Formation of Calcium Phosphate……..……………………...……………………….46

4.3 Instrumentational Analysis………………………...…………………………………48

4.3.1 Raman Spectroscopy……………………………………….………………49

4.3.2 ATR-FTIR Spectroscopy…………………………………………………..51

4.3.3 XRD Analysis………………………………………………………………53

4.4 Conclusion……………………………………………………………………………55

Chapter 5: CONCLUSION………………………………………………………………………56

5.1 Conclusion………………………………………………..……………...…………...56

5.2 Future Direction……..……………………………………………………………….57

REFERENCES….……………………………………………………………………………….59

vii

LIST OF FIGURES

Figure 1-1. Illustration of packed macromolecules inside a mycoplasma mycoides cell (~300 nm

diameter) showing compartmentalization of cells based on color. The different light greens

represent membrane proteins, dark greens are lipids and lipoglycans, yellow is DNZ, the

multiple blues are enzymes for energy productions, and the pink/purple are aspects used in

protein synthesis such as RNA, ribosomes, and DNA polymerase. Illustration by David S.

Goodsell, the Scripps Research Institute; watercolor………………………………………….2

Figure 1-2. Different coexistences of liquid-liquid systems used as artificial cells: (a) non-

stabilized aqueous/aqueous phases, (b) aqueous/aqueous phases stabilized by a lipid bilayer,

(c) aqueous/aqueous phases stabilized by lipid vesicles in the form of an all-aqueous emulsion

droplet. Adapted with permission from Crowe, C.D.; Keating, C.D. Liquid-liquid phase

separation in artificial cells. Interface Focus. 2018, 8, 1-17……………………………………4

Figure 1-3. SEM images of biogenic CaCO3: (a) polycrystalline aggregates from poly(4-

styrenesulfonate-co-maleic acid grown minerals; scales on each image (b)microparticles from

CaCl2 and Na2CO3; scale bar 1 μm. (a) Reprinted with permission from Song, R.Q.; Cölfen,

H.; Xu, A.W.; Hartmann, J.; Antonietti, M. Polyelectrolyte-Directed Nanoparticle

Aggregation: Systematic Morphogenesis of Calcium Carbonate by Nonclassical

Crystallization. ACS Nano 2009, 3, 1966-1978. Copyright (2019) American Chemical Society.

(b) Adapted with permission from Sukhorukov, G.B.; Volodkin, D.V.; Günther, A.M.; Petrov,

A.I.; Shenoy, D.B.; Möwald, H. Porous calcium carbonate microparticles as templates for

encapsulation of bioactive compounds. J. Mater. Chem. 2004, 14, 2073-2081………………..5

Figure 1-4. SEM images of CaCO3 made under similar conditions to my samples (150 μg/mL

urease; 20 mM Ca2+; 30 mM urea), (a-c)in different liquid phases of artificial mineralizaing

vesicles (AMVs) or (d-f) different Dx:PEG aqueous two phase systems (ATPS) volume ratios

(Vd:Vp), with no coacervate present. Scale bars 2μm for all. CaCO3 forms thought to be calcite,

vaterite, and ACC. Reprinted with permission from Cacace, D.N.; Keating, C.D. Biocatalyzed

mineralization in an aqueous two-phase system: effect of background polymers and enzyme

partitioning. J. Mater. Chem. B 2013, 1, 1794-1803…………………………………………...6

Figure 1-5. SEM images of CaCO3 found in skeleton. (a-d) is stable ACC, (e-f) is transient ACC.

(a&b) Spicule body and cross-section from a marine ascidian, (c) Cystolith from a leaf of Ficus

microcarpa, (d) granule of storage structure from Orchestia cavimana, (e) spiculae from sea

urchin, (f) larva of mollusk. Scale bars were enhanced and labelled on each image. Adapted

with permission from Addadi, L.; Raz, S.; Weiner, S. Taking Advantage of Disorder:

Amorphous Calcium Carbonate and Its Roles in Biomineralization. Adv. Mater. 2003, 15, 959-

970……………………………………………………………………………………………..7

Figure 1-6. SEM images of 5 day old adult sea urchin regenerated spine via deposition of ACC

precursor phase. Image b is a higher magnification of image a. Adapted with permission from

Politi, Y.; Arad, T.; Klein, E.; Weiner, S.; Addadi, L. Sea Urchin Spine Calcite Forms via a

Transient Amorphous Calcium Carbonate Phase. Science, 2004, 306, 1161-1164…………….7

viii

Figure 1-7. SEM images of a) biomimetic apatite with plate-like morphology or ‘flowering’ and

b) hydroxyapatite. Scale bar 1 μm for both images. a) Adapted with permission from Creative

Commons Attribution License: Christophe Drouet, “Apatite Formation: Why It May Not Work

as Planned, and How to Conclusively Identify Apatite Compounds,” BioMed Research

International, vol. 2013, Article ID 490946, 12 pages,

2013. https://doi.org/10.1155/2013/490946. b) Adapted with permission from Koutsopoulos,

S. Synthesis and characterization of hydroxyapatite crystals: A review study on the analytical

methods. Wiley Periodicals, Inc. J Biomed Mater Res, 2002, 62, 600-612…………………….8

Figure 1-8. Enzymatic Mineralization of ACC within an Artificial Mineralizing Vesicle. Urea

hydrolysis of urease occurs in a LUV stabilized enzyme containing Dx-rich droplet in a

continuous PEG-rich phase. The resulting carbonate ions interact with Ca2+ in the PAA-rich

coacervate. After the solution is allowed to react, ACC minerals are formed within the

coacervate inside the Dx-rich droplet………………………………………………………….9

Figure 1-9. Phase diagram for two neutral polymers. For my system, the upper tie line (a) indicates

that at points 2, 3, & 4 that the concentration of each polymer within the solution is constant

no matter the volume of polymer-rich solution used. Adapted with permission from Keating,

C.D. Aqueous Phase Separation as a Possible Route to Compartmentalization of Biological

Molecules Accounts of Chemical Research 2011, 45, 2114-2124…………………………….10

Figure 1-10. Formation of Specific Volume Ratio Aqueous Two Phase System. Two polymers,

Dx and PEG, are dissolved in Tris buffer solution, where they form an ATPS. The two phases

are then separated into individual containers and re-combined at desired volume

ratios……………………………….…………………………………………………………11

Figure 1-11. Liposome concentration determines droplet size. (a) Optical microscopy images

showing decreased droplet size with increased liposome concentration. Scale bar, 25μm. (b)

Dependency of droplet size on liposome concentration. Blue lines indicate predicted size from

hexagonally close-packed liposomes in varying numbers of planar layers while inset shows

visual of packing possibilities. Black dots indicate results of measured droplet sizes. Adapted

by permission from [Springer Nature]: [Springer Nature][Nature Communication][ Bioreactor

droplets from liposome-stabilized all- aqueous emulsions, Dewey, D.C.; Strulson, C.A.;

Cacace, D.N.; Bevilacqua, P.C.; Keating, C.D.][2014]……………………………………….13

Figure 1-12. Stabilization of Dx:PEG ATPS through use of liposomes to indicate process of water-

in-water emulsion stabilized by lipid vesicles. Presences of lipid vesicles shows no coalescence

through suspension of droplets in continued cloudiness of solution. Reprinted by permission

from [Springer Nature]: [Springer Nature][Nature Communication][ Bioreactor droplets from

liposome-stabilized all- aqueous emulsions, Dewey, D.C.; Strulson, C.A.; Cacace, D.N.;

Bevilacqua, P.C.; Keating, C.D.][2014]……………………………………………………...14

Figure 1-13. Depiction of enzymatic mineralization of calcium carbonate through use of AMVs

(a) Schematic of artificial mineralizing vesicles used to produce CaCO3 (b) Dark field optical

microscope images of mineralization with 500mM urea (c) Brightfield (DIC) and fluorescence

overlay images of CaCO3 (d) Scanning electron microcopy image of CaCO3 (a,b,c) Adapted

with permission from Cacace, D.N.; Rowland, A.T.; Stapleton, J.J.; Dewey, D.C.; Keating,

ix

C.D. Aqueous Emulsion Droplets Stabilized by Lipid Vesicles as Microcompartment for

Biomimetic Mineralization. Langmuir 2015, 31, 11329-11338. Copyright 2019 American

Chemical Society…………………………………………………………………………….15

Figure 1-14. Depictions of Artificial Mineralizing Vesicles made up of poly(ethylene) glycol and

dextran with a large unilamellar vesicle barrier to keep from coalescence of the phases. a)

Original AMVs used in ACC mineralization. b) AMVs containing a PAA/Ca2+ coacervate for

new form of ACC mineralization…………………………………………………………….16

Figure 1-15. Stages and feature of the polymer induced liquid precursor (PILP) process. Critical

concentration of Ca2+ is observed during phase separation (a) followed by coalescence of

isotropic film droplets (b). These droplets have birefringent areas that are caused by crystal

tablet formation (c), that continue to spread laterally (d). The final produce is composed of

single-crystalline patches of calcite. Reprinted with permission from Gower, L.B.; Odom, D.J.

Deposition of calcium carbonate films by a polymer-induced liquid-precursor (PILP) process.

Journal of Crystal Growth 200, 210, 719-734………………………………………………..17

Figure 1-16. Depiction of Polymer Induced Liquid Precursor Formation. To form a PILP, the

ATPS has to undergo the same steps for the formation of a PAA/Ca2+ coacervate, but the

addition of a calcium counter ion in solution must be at a critical concentration with an increase

in supersaturation……………………………………………………………..……………...18

Scheme 1-1. Depiction of Polyaspartic Acid and Calcium Coacervate. The calcium in the ATPS

binds to the carboxyl groups of the PAA, reducing it’s negative charge to make it less solvated

and creates a coacervate………………………………………………………………………18

Figure 1-17. Still image from prepared ACC mineralization video at 0 min. This shows the

fluorescence of the A3PS before addition of urea into the solution. PAA is labelled with Alexa-

488 dye, the Dx-rich phase is labelled with Alexa-647, and the LUVs are labelled with

RhDOPE-dye. The urease enzyme and PEG-rich phase are not labelled. Adapted with

permission from Cacace, D. N. (May 2014) “Biomimetic Mineralization of Calcium Carbonate

in Aqueous Biphasic Systems” PhD diss., The Pennsylvania State University, 2014………...19

Figure 1-18. Still image from ACC mineralization video at 16 min. This shows the fluorescence

of the A3PS after addition of urea into the solution. The urea has hydrolyzed the urease enzyme

to create carbonate that interacted with the Ca2+ in the previous PAA-rich phase. This created

a mineral and released PAA into the Dx-rich phase. The labels are the same as in Figure 1-17.

Adapted with permission from Cacace, D. N. (May 2014) “Biomimetic Mineralization of

Calcium Carbonate in Aqueous Biphasic Systems” PhD diss., The Pennsylvania State

University, 2014…………………………………………………………………………...…20

Figure 2-1. Chemical structures of neutral polymers, a) dextran (TRC Inc.) and b) polyethylene

glycerol (Sigma-Aldrich), used for this ATPS………………………………………………..21

Figure 2-2. Chemical structure of poly(α,β)-DL-aspartic acid…………………………………...22

x

Figure 2-3. Chemical representation of lipids used in lipid vesicles. All images from Avanti Polar

Lipids, Inc. a) RhDOPE, b) DOPE-PEG-2K, c) Egg-PC, d) Egg-PG………………………...23

Scheme 2-1. Gentle Hydration Method allowing formation of liposomes to be used in LUV

fabrication……………………………………………………………………………………24

Scheme 2-2. Depiction of Amorphous Calcium Carbonate Mineralization. The introduction of a

urea substrate in an A3PS of PEG-rich phase, Dx-rich phase, and a PAA/Ca2+ coacervate with

a urease enzyme allows for the hydrolysis of urea by urease to generate carbonate ion. The ion

interacts with the Ca2+ found in the coacervate for form an ACC mineral that is washed with

base, dried at room temperature, and then analyzed…………………………………………..26

Figure 2-4. Confocal Microscopy Images of 1.500 mg/mL LUV concentration and 1:49 Vd:Vp

prepared coacervate formation. a) RhDOPE-dyed LUV fluorescent image, b) Alexa-488

labelled coacervate fluorescent image, c) DIC image………………………………………...27

Figure 3-1. Enzymatic Mineralization of ACC within an Artificial Mineralizing Vesicle. Urea

hydrolysis via urease occurs in LUV-stabilized Dx-rich droplets in a continuous PEG-rich

phase. The resulting carbonate ions interact with Ca2+ in the PAA-rich coacervate. ACC

minerals are formed within the coacervate inside the Dx-rich droplets. (Reprinted from Chapter

1)……………………………………………………………………………………………..30

Figure 3-2. Order of Addition for Artificial Mineralizing Vesicles. (A) indicates the formation of

a coacervate before a LLPS of Dx-rich phase and PEG-rich phase is produced. This is the order

of addition used in previous works. (B) depicts the new order of addition in which PAA and

Dx-rich phase form a phase before the addition of calcium into the system. At equilibrium both

solutions should give the same coacervate-containing AMV used in the mineralization process

of our system…………………………………………………………………………………32

Figure 3-3. Order of Addition in ACC Mineralization. The addition of Ca2+ into the ATPS for

ACC mineral formation allows for the creation of a PAA/Ca2+ coacervate. Both systems have

1.500 mg/mL LUV and 1:49 Dx:PEG. (a) depicts addition of Ca2+ after the addition of PAA,

PEG-rich phase, and Dx-rich phase. (b) depicts addition of Ca2+ after the addition of PAA,

before the creation of ATPS. All scale bars 25 μm. (c) show the histograms of the AMVs

formed and (d) shows the histograms of the coacervates formed for each order of addition….32

Figure 3-4. Effect of Change in Large Unilameller Vesicle (LUV) Concentration on AMV,

Coacervate, and Mineral Appearance. a) fluorescence images showing DOPE-Rhodamine-

labelled liposomes(RhDOPE); b) fluorescence images showing Alexa-488-labeled PAA; all

fluorescence images have 25 μm scale bars. c) SEM images of prepared amorphous calcium

carbonate minerals; all SEM images have 50 μm scale bars. Row 1 indicates a smaller amount

of LUVs present in the solution. Row 2 is the standard prepared ACC. Row 3 has the largest

amount of LUVs present in the solution……………………………………………………...36

Figure 3-5. Effect of LUV Concentration Change on Diameter of ACC Minerals. The red

indicates AMV diameter (a), and green indicates coacervate diameter (b), and blue indicates

mineral diameter(c). All samples have the same initial materials present, but change the

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concentration of LUVs. Top line: 1.125 mg/mL LUVs. Middle line: 1.500 mg/mL LUVs;

standard prepared ACC minerals. Bottom line: 1.875 mg/mL LUVs. This is ‘raw’ data used

for the analysis because the original images are analyzed using the diameter of the mineral,

AMV, and coacervate. .………………………………………………………………………38

Figure 3-6. Effect of LUV Concentration Change on Volume of ACC Minerals. This figure

shows the same change in LUV concentration as that found in Fig. 3-5. (a) indicates AMV

volume, (b) coacervate volume, (c) mineral volume. Top line: 1.125 mg/mL LUVs. Middle

line:1.500 mg/mL LUVs; standard prepared ACC minerals. Bottom line: 1.875 mg/mL LUVs.

The volume was calculated from the diameter of the solutions analyzed. The x-axis for all

histograms is a log scale. ……………………………………………………………………..39

Figure 3-7. Change in Dx:PEG Volume Ratio. a) Fluorescence images showing RhDOPE

labelled LUVs b) fluorescence images showing Alexa-488-labelled coacervate; all

fluorescence images have 25 μm scale bars. c) SEM images of prepared amorphous calcium

carbonate minerals; all SEM images have 50 μm scale bars. Row one is the least amount of

Dx-rich phase present; a time of 5 minutes is needed to create a coacervate large enough to see

under confocal microscopy. Row two is the standard for all systems. Row three is the most

amount of Dx-rich phase present in the solution……………………………………………...41

Figure 3-8. Effect of change in Dx:PEG Volume Ratio on Diameter f Prepared ACC Minerals.

The red indicates AMV size (a), green indicates coacervate size(b), and blue indicates mineral

size(c). All samples have the same initial materials present, but change the Dx:PEG volume

ratio. Top line: 1:99 Dx:PEG volume ratio. Middle line: 1:49 Dx:PEG volume ratio; standard.

Bottom line: 1:32.2 Dx:PEG volume ratio. This is ‘raw’ data used for the analysis because the

original images are analyzed using the diameter of the mineral, AMV, and coacervate. ..........42

Figure 3-9. Effect of Dx:PEG Volume Ratio Change on Volume of Prepared ACC Minerals.

This figure shows the same change in Dx:PEG volume ratio as Fig. 3-9. Top line: 1:99 Dx:PEG

volume ratio. Middle line: 1:49 Dx:PEG volume ratio. Bottom line: 1:32.2 volume ratio. The

volume was calculated from the diameter of the solutions analyzed. The x-axis for all

histograms is a log scale. ……………………………………………………………………..43

Scheme 4-1. Depiction of Calcium Phosphate Formation Through Buffer Washing. The same

process of ACC mineralization is followed, with an additional step of washing with different

concentrations of sodium phosphate buffer solutions to displace carbonate ions with phosphate

ions in the hope of creating hydroxyapatite…………………………………………………..46

Figure 4-1. Analysis of 50 mM Sodium Phosphate Buffer Wash of prepared ACC Minerals.

SEM images (TLD detector) of converted minerals. Scale bar 5 μm for each. Along with ATR-

FTIR spectra of each timed trial compared to as prepared ACC. Peaks are normalized by PO4

v4a band 602 cm-1.The characteristic peak shape and position of the v3 (~1390 cm-1) modes of

CO32- peak intensity was compared with the characteristic peak assigned to the v3 (~1020 cm-

1) stretching vibration band of PO43- peak for each trial.14,38,39 The change in peak intensity

shows an increase for the PO43- with a decrease for CO3

2-……………………………………47

xii

Figure 4-2. Analysis of 100 mM Sodium Phosphate Buffer Wash of Prepared ACC Minerals.

SEM images (TLD detector) of supposed Ca Phosphate minerals after each timed wash

compared to as prepared ACC minerals. Scale bar 5 μm for each. Along with ATR-FTIR

spectra of timed trials (one hour, six hour, and 12 hour) compared to standard ACC minerals.

Peaks are normalized by PO4 v4a band 602 cm-1.The characteristic peak position of the v3

(~1390 cm-1) mode of CO32- peak intensity was compared with the characteristic peak of the

v3 (~1020 cm-1) stretching vibration band of PO43- peak for each trial.A,38,39 As the minerals

were washed for increased amounts of time, one hour, six hours, and 12 hours, the PO43- peak

became more defined…………………………………………………………………………47

Figure 4-3. Analysis of 500 mM Sodium Phosphate Buffer Wash of Prepared ACC Minerals.

SEM images (TLD detector) of supposed Ca Phosphate minerals after each timed wash

compared to normal ACC minerals. Scale bar 5 μm for each. Peaks are normalized by PO4 v4a

band 602 cm-1.Along with ATR-FTIR spectra of timed trials (one hour, six hour, and 12 hour)

compared to standard ACC minerals to show peak intensity change. Again, the characteristic

peak position of the v3 (~1390 cm-1) mode of CO32- peak intensity was compared with the

characteristic peak of the v3 (~1020 cm-1) stretching vibration band of PO43- for each trial.14,38,39

As the minerals were washed for increased amounts of time (1,6,12 hours) the phosphate peak

has increased in intensity as well as becoming stronger………………………………………48

Figure 4-4. Raman spectrum of 500 mM Sodium Phosphate Buffer Washed Amorphous

Calcium Carbonate. Prepared ACC minerals washed for 1 hour, 4 individual minerals

analyzed; washed for 6 hours, 5 individual minerals analyzed; ACC minerals washed for 12

hours, 5 individual minerals analyzed. Calcium phosphate minerals (v1 phosphate peak) are

indicated by a strong band ~960 cm-1 that differs depending on the mineral standard created,

HAP, OCP, ACP, DCPD.39,40 Carbonate apatites (v1 carbonate peak) have peaks between

1060-1080 cm-1.41,42………………………………………………………………………….50

Figure 4-5. ATR-FTIR spectrum of prepared ACC minerals washed for 12 hours in

physiological pH buffers. Depiction of decrease in CO32- peak intensity (~1350 cm-1) and

increase of PO43- peak intensity (~1000 cm-1) with increased concentration of phosphate

present in buffer, as well as sharpening of OCP/Nanocrystalline apatite/HAP peak region (400-

700 cm-1). Peaks are normalized by PO4 v4a band 602 cm-1…………………………………52

Figure 4-6. FTIR spectra of calcium phosphate forms. Comparison of OCP, nanocrystalline

apatite, HA, and ACP are used against my samples. Reprinted with permission by Creative

Commons Attribution License: Christophe Drouet, “Apatite Formation: Why It May Not Work

as Planned, and How to Conclusively Identify Apatite Compounds,” BioMed Research

International, vol. 2013, Article ID 490946, 12 pages,

2013. https://doi.org/10.1155/2013/490946.............................................................................53

Figure 4-7. Effect of PO4 Washing on XRD of Prepared ACC Samples. XRD analysis of

amorphous calcium carbonate after being washed in different physiological pH buffers for

twelve hours compared to calcium carbonate forms: calcite, aragonite, and vaterite {Jade

Software for XRD material data 98-000-0141, 98-000-0098, 98-000-0451, respectively}, and

calcium phosphate form: hydroxyapatite, {Jade Software for XRD materials data 98-000-

0251}. Washing the buffer in Tris buffer had no effect on the amorphic nature of the ACC

xiii

minerals. The area from 10-30⁰ Theta for the three buffer washes was caused by small sample

size compared to sample prep requirements. Increase in background signal at about 50⁰ is from

sample holder….……………………………………………………………………………..54

Figure 4-8. XRD spectra of calcium phosphate forms. Peaks relative to CuKα1 wavelength used

for comparison of OCP, nanocrystalline apatite, HA, and ACP. Reprinted with permission

by Creative Commons Attribution License: Christophe Drouet, “Apatite Formation: Why It

May Not Work as Planned, and How to Conclusively Identify Apatite Compounds,” BioMed

Research International, vol. 2013, Article ID 490946, 12 pages,

2013. https://doi.org/10.1155/2013/490946.............................................................................55

xiv

LIST OF TABLES

Table 1-1. Gibbs Free Energy of calcium carbonate morphologies related to amorphous calcium

carbonate.16…………………………………………………………………………………....5

Table 3-1. Order of Addition for AMV Formation. The change between adding the calcium stock

solution after the addition of the Dx-rich phase versus before the addition of the droplet phase

was to increase likelihood of the formation of coacervate into the interior of the AMV. Original

Order of Addition is equivalent to order of addition for previous works (Cites 14, 16, 19)

whereas New Order of Addition is used for the remainder of this

thesis…………………………………………………………………...31

xv

LIST OF ABBRIVIATIONS

2D two dimensional

A3PS aqueous three-phase system

ACC amorphous calcium carbonate

ACP amorphous calcium phosphate

AMV artificial mineralizing vesicles

ATPS aqueous two- phase system

ATR-FTIR attenuated total reflectance Fourier transform infrared spectroscopy

DCPD dicalcium phosphate dihydrate

DI H2O 18.2 MΩ Nanopure water

DIC differential image contrast

DOPE-PEG-2K 1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine-N-

[Methoxy(Polyethylene glycol)-2000]

Dx dextran

EDDS Ethylenediamine-N,N'-disuccinic acid

HA hydroxyapatite

LLPS liquid-liquid phase separation

LUV large unilamellar vesicles

NCA nanocrystalline apatite

OCP octacalcium phosphate

PAA poly(α,β)-DL-aspartic acid

PC L-a-Phosphatidylcholine

PEG poly(ethylene glycerol)

xvi

PG L-a-Phosphatidyl-DL-Glycerol

PILP polymer induced liquid precursor

RhDOPE 1,2-dioleoyl-sm-glycero-3-phosphoethanolamine-N-(lissamine

rhodamine B sulfonyl)

SEM scanning electron microscopy

TLD through the lens detector

TRC Toronto Research Chemicals, INC

Vd:Vp Dx:PEG ATPS volume ratio

XRD x-ray diffraction

xvii

ACKNOWLEDGEMENTS

The work in this thesis would not be possible with the valuable contribution of many people.

First off I would like to acknowledge by thesis advisor Christine Keating for all the support that

she gave, not only in helping me write this paper and prepare for my defense, but for being a

wonderful advisor with helpful hints and an easy going manner. She allowed me to join her group,

expand my knowledge, and encouraged me every step of the way. I will forever be greatful for the

help that she gave me and for always being available when I felt like I couldn’t succeed in grad

school. I would not have been able to do any of this without your help and your support, so thank

you.

To my committee members, Elizabeth Elacqua and Raymond Schaak, thank you for first

listening to me during my first year meeting when I felt like I knew nothing, and now again. Thank

you for being encouraging when I asked for your presence as a Master’s Thesis committee instead

of for my orals; it was nice to have people offer to discuss it with me.

To the Department of Energy for funding. This work was supported by the Department of

Energy, Basic Energy Sciences, through the Biomolecular Materials program under grant # DE-

SC0008633.

To my group members, you have all been so supportive and offering me advise and help along

the way; I do not know what I would do without seeing some of you everyday or talking during

our lunch break in the lunch room. I will miss discussing all the random questions that were

brought up and enjoying all the food that everyone brings in. To all of those that helped read

through this paper, as well as all the others I have written, your reviews made me a better writer. I

would especially like to thank Andrew Rowland and Nuerxida Pulati for their help. Andrew, you

showed me all the ropes to being in the biomineralization group and you were one of the best

xviii

mentors that I have ever had. You were always encouraging after any presentation that I gave, and

always validated my confusion by telling me that it was okay. Nuerxida, I know you’re technically

not in the lab anymore, but I don’t know what I would have done if I couldn’t have just turned

around at my desk multiple times a day to talk with you and ask you questions.

To my friends that I have made while at Penn State, I couldn’t have done it without you.

Albanie, Nicole, and Liz, I really enjoyed our craft nights, wine tours, and nights just hanging out

and talking. You were always there when I needed it, and I appreciate every part of our friendship.

My game night group, thank you for the laughs and eye opening view of the world. Hannah, I

never thought I wanted anything to ever do with Pi Phi again, but I am elated that I decided to join

AAC. You have been one of the best people I could ask for in my life, and I hope that this is just

the start of our friendship. Most of all, I want to thank you, Alexis. I know I wouldn’t have made

it through these last two years if I hadn’t have had you as a mentor on my visit. You have been the

person that I can call any time during the day to just talk or go grab food with me at un-godly hours

of the night because I just can’t get Taco Hell off my mind. I don’t think I would have lasted

without you being there for me, so thank you from the bottom of my heart.

To my friends that aren’t physically with me. Our snapchats, FaceTimes, and random texts let

me keep going. I thank you all. I especially want to thank Andrea, and along with her comes

Einstein, for all the FaceTime chats talking about anything and everything and nothing all at the

same time, the texts throughout the day that keep me smiling, and the random letters that contain

pictures for my desk. I always have you to turn to no matter what and I am so glad that I get to call

you my maid of honor.

Mama and daddy, I want to thank you for always supporting me, not just through this

processes, but in my life in general. I do not know what I would do if I didn’t have you as my

xix

parents. Thank you for the random letters, the chats when I needed to vent about my life, and the

trips home. I’d like to say I did this for you, but let’s be real, I did it for me. But, I want to thank

you for always encouraging me and trying to help, even though most of the time you didn’t know

what was going on.

To my best friend, I love you and thank you for always being on my side. Alan, you have

supported me through thick and thin and I wouldn’t be where I am now without you. I came to

Penn State knowing that you supported me 100% and that never went away. You calm me down

when I need it and try to make me happy no matter what. I can’t wait to do the rest of our lives

together.

Finally, to my other half: Brittany, I wouldn’t have been able to do any of this without you.

You have been there for me from the beginning; literally the beginning. Your supporting and

complaining and being are what allows me to get through. You are my older sister and I always

try to make you proud. Thank you for calling me when I needed it, listening to me talk, visiting

me, and just being there for me always. I try to imagine what my life would be without your

support, and all I can think is all the other people in the world must really hate their lives because

they don’t have you in their corner. Thank you for being you and encouraging me and making me

the best person that I could hope to be. Love you long time.

1

Chapter 1

Introduction

1.1 Research Motivations

The aspects of artificial mineralization come from biology and characteristics of creation in

different cells. Inorganics and organics take a role in the production of minerals, and understanding

the properties of resulting minerals can give insight into how biological processes work.

Amorphous calcium carbonate (ACC) has been used in collagen studies and in incorporated

proteins.1-4 Biominerals made up of ACC have biological and optical properties such as aquatic

life with exoskeletons made of carbonate minerals of different crystal morphologies or convert

phosphate minerals, and the use in microarray lenses.3-14 These minerals can also be applicable in

the medical field through bone fiber development and bone formation.3-14 The process of

generating these minerals is of importance in the usage that they provide. Understanding how to

control local mineralization environments such as the local generation of mineralization

precursors, organic/inorganic content, tunability, and conversion is an important step in using these

minerals to understand the biological world.

In this introduction, I detail the properties of artificial mineralization that result in stability

and tunability of the resulting mineral. First, I discuss minerals formed by living organisms,

focusing on ACC and calcium phosphate morphologies (Ca-Phosphate). The characteristics for

mineral production through liquid-liquid phase separation (LLPS) are discussed followed by

stabilization of the phases. Finally, addition of a third phase for greater tunability of the system is

described.

2

1.2 Compartmentalization of Cells

Macromolecular crowding, with components such as proteins, DNA, and other organelles, is

a common occurrence in many cells.15,16 To accommodate the crowding, cells function in

compartments to allow each part of the cell the ability to operate correctly (Figure 1-1). This

compartmentalization is therefore an important aspect when considering cellular environments in

scientific study.14-17 Through compartmentalization, localization of chemical reactions, or

biochemical processes, can be identified and tracked.16 Using this same approach, artificial cell

compartmentalization will allow for control over a system. Knowing where a process is occurring

allows for tunability of the processes for further analysis.

Figure 1-1. Illustration of packed macromolecules inside a mycoplasma mycoides cell (~300 nm diameter) showing

compartmentalization of cells based on color. The different light greens represent membrane proteins, dark greens are

lipids and lipoglycans, yellow is DNA, the multiple blues are enzymes for energy productions, and the pink/purple

3

are aspects used in protein synthesis such as RNA, ribosomes, and DNA polymerase. Illustration by David S. Goodsell,

the Scripps Research Institute; watercolor.

When mimicking aspects of cells in synthetic cellular synthesis, compartmentalization of the

sample is the first step. To compartmentalize a solution, an interface must first exist. For this thesis,

the interface of interest is an aqueous/aqueous interface resulting from two polymer-rich phases

that are not coalesced. The driving force for this interface is based on physical interactions between

the polymers, resulting in a LLPS through surface tension.14-19 This aqueous/aqueous interface

must be stabilized to prevent droplet coalescence.

As in organelles, there are both membrane-bound and membrane-less compartmentalization.

Generally for a membrane-bound organelle, a lipid barrier is created between the phases for

stability whereas membrane-less organelles are stoichiometrically stable compounds.19 In LLPS,

the artificial equivalence of a membrane-less organelle would depend on phases that do not

coalesce with each other, while a membrane-bound organelle would require a physical barrier to

stay stable. Within this thesis, for the polymer-rich phases used as an artificial cell model, a barrier

between the phases of the LLPS needs to be considered because the two phases create an unstable

interface. The stabilization of the two phases could ensue from a bilayer of lipids or lipid vesicles

at the interface (Figure 1-2). With the stabilization of the two phases using a lipid barrier, selective

permeability of the system is achieved.14,15,21 For this thesis, an aqueous/aqueous emulsion

stabilized by lipid vesicles, such as Fig. 1-2c, was utilized.

4

Figure 1-2. Different coexistences of liquid-liquid systems used as artificial cells: (a) non-stabilized aqueous/aqueous

phases, (b) aqueous/aqueous phases stabilized by a lipid bilayer, (c) aqueous/aqueous phases stabilized by lipid

vesicles in the form of an all-aqueous emulsion droplet. Adapted with permission from reference 19.

1.3 Morphologies of Minerals

Calcium carbonate (CaCO3) has four common morphologies, calcite, aragonite, vaterite, and

ACC that are found as solid particles, aggregates, and microparticles (Figure 1-3). The formation

of CaCO3 can follow a biomimetic mineralization system that allows for the crystal structures of

the four morphologies to be produced in different locations in the system.15 Reitveld refinement

of XRD patterns was used to determine the formation of stable calcite and vaterite minerals in

Figure 1-4.15 This mineralization process can also be used in the formation of ACC minerals that

are stable and tunable.15

These morphologies retain different crystal lattices and orientations that benefit different

aspects of biological life. The process of conversion to other morphologies follows the Gibb’s free

energy of the system (Equation 1-1); unstable ACC takes on the intermediate forms of aragonite

and vaterite, which then can be converted into stable calcite due to activation energies that are

overcome during transformation to activation energies (Table 1-1).7,16,22 However, in the presence

of other organic/inorganic substances, an exergonic reaction takes place that produces different

5

minerals; as an example, calcium phosphate is produced in the presence of phosphates in the

system at certain activation energies.7

∆𝐺 = ∆𝐻 − 𝑇∆𝑆 (𝑬𝒒. 𝟏 − 𝟏)

Figure 1-3. SEM images of biogenic CaCO3: (a) polycrystalline aggregates from poly(4-styrenesulfonate-co-maleic

acid grown minerals; scales on each image (b)microparticles from CaCl2 and Na2CO3; scale bar 1 μm. (a) Reprinted

with permission from reference 1. (b) Adapted with permission from reference 23.

Polymorph ΔG (kJ/mol)

ACC --

Calcite -1033.9

Aragonite -1032.8

Vaterite -1030.6

Table 1-1. Gibbs Free Energy of calcium carbonate morphologies related to amorphous calcium carbonate.16

6

Figure 1-4. SEM images of CaCO3 made under similar conditions to my samples (150 μg/mL urease; 20 mM Ca2+;

30 mM urea), (a-c)in different liquid phases of artificial mineralizaing vesicles (AMVs) or (d-f) different Dx:PEG

aqueous two phase systems (ATPS) volume ratios (Vd:Vp), with no coacervate present. Scale bars 2μm for all. CaCO3

forms thought to be calcite, vaterite, and ACC. Reprinted with permission from reference15.

1.3.1 Biological Properties

CaCO3 makes up exoskeletons of particular aquatic life and is used for bone fiber development

of those spines for growth and reattachment.5,6, 24-28 The CaCO3 used for sea urchin and mollusk

exoskeleton spines is made up of stable and transient ACC, that transforms into other stable CaCO3

morphologies (Figure 1-5&6). For larval spicules in sea urchins, a calcite crystal from a transient

ACC phase is formed, whereas mollusks are found in the form of aragonite that comes from a

transient ACC phase.5,6,24-28 The stable form of ACC is found in fully grown exoskeletons of sea

creatures (Figure 1-5(a-d)), while transient ACC can be found in the areas that are in the transient

precursor phase (Figure 1-5e&f and Figure 1-6).5,6,24-28

7

Figure 1-5. SEM images of CaCO3 found in skeleton. (a-d) is stable ACC, (e-f) is transient ACC. (a&b) Spicule body

and cross-section from a marine ascidian, (c) Cystolith from a leaf of Ficus microcarpa, (d) granule of storage structure

from Orchestia cavimana, (e) spiculae from sea urchin, (f) larva of mollusk. Scale bars were enhanced and labelled

on each image. Adapted with permission from reference 5.

Figure 1-6. SEM images of 5 day old adult sea urchin regenerated spine via deposition of ACC precursor phase.

Image b is a higher magnification of image a. Adapted with permission from reference 6.

1.3.2 Morphology Changes

In the transition of unstable ACC into other polymorphs, if a nonenzymatic change occurs from

organics found in the ACC, a new mineral can also be formed. In the presence of phosphate ions,

8

an exchange reaction of carbonate and phosphate ions can occur in unstable ACC, aragonite, or

vaterite minerals to produce calcium phosphate morphologies based on the activation energy

reached.7 However, regarding calcite, no such transformation occurs because there is no distinct

activation energy barrier that can be overcome to convert calcite to a calcium phosphate

polymorph.7 The nonenzymatic exchange can produce a mineral in one of the eleven known

calcium phosphate polymorphs, including apatite and hydroxyapatite (Figure 1-7). The process of

conversion of ACC to calcium phosphate is seen in some aquatic creatures through evolution over

time (millions of years) as well as on a species level.7 If CaCO3 polymorphs can be used to create

Ca-Phosphate, then the conversion of biomimetic mineralized ACC to Ca-Phosphate may also be

possible.

Figure 1-7. SEM images of a) biomimetic apatite with plate-like morphology or ‘flowering’ and b) hydroxyapatite.

Scale bar 1 μm for both images. a) Adapted with permission from reference 37. b) Adapted with permission from

reference 38.

1.4 Biomimetic Mineralization in Aqueous Three Phase Systems

In this thesis, an aqueous three phase system (A3PS) consisting of a Dextran (Dx)-rich phase,

poly(ethylene) glycol (PEG)-rich phase, and polyaspartic acid (PAA)-rich phase was used for the

mineralization of ACC. To form the ACC, the coacervate phase (PAA-rich) was dissolved by the

9

removal of the Ca2+ by a carbonate group. The hydrolysis of urea with urease produces carbonate

(Equation. 1-2) that interacts with the Ca2+ allowing for the creation of a mineral (Figure 1-8).

(𝑁𝐻2)2𝐶𝑂(𝑎𝑞) + 𝐶𝑎2+(𝑎𝑞) + 2𝐻2𝑂(ℓ)

𝑈𝑟𝑒𝑎𝑠𝑒, 𝑝𝐻>8→ 2𝑁𝐻4

+(𝑎𝑞) + 𝐶𝑎𝐶𝑂3(𝑠) (𝑬𝒒. 𝟏 − 𝟐)

The partitioning coefficient of each reactant in the mineralization was found in earlier works for a

Dx:PEG aqueous two phase system (ATPS) termed the artificial mineralizing vesicle (AMV).14-

17,21 Urease was 8.5 time more likely to be in the Dx-rich phase than the PEG-rich phase; whereas

the relative equilibrium concentration of the Ca2+ and urea is the same for the Dx-rich phase and

the PEG-rich phase.15,16 Because the urease was found in the Dx-rich phase, the hydrolysis takes

place in this interior AMV phase. To produce minerals, the knowledge of the individual parts of

biomimetic mineralization needs to be understood.

Figure 1-8. Enzymatic Mineralization of ACC within an Artificial Mineralizing Vesicle. Urea hydrolysis of urease

occurs in a LUV stabilized enzyme containing Dx-rich droplet in a continuous PEG-rich phase. The resulting

carbonate ions interact with Ca2+ in the PAA-rich coacervate. After the solution is allowed to react, ACC minerals are

formed within the coacervate inside the Dx-rich droplet.

1.4.1 Liquid-Liquid Phase Separation

LLPS can occur by complex coacervation when two charged polymers are mixed or by

coacervation when two neutral polymers are combined. For this thesis, equilibrated Dx-rich and

10

PEG-rich phased in a 10%/10% by weight solution were utilized. These two polymers coexist

thermodynamically and create an ATPS with a Dx-rich droplet and a PEG-rich continuous phase

that exists as one phase when the volume of solution is low, but as two separate phases when the

volume is high.18 Dx and PEG undergo a non-associative phase change to form the ATPS. This is

represented on the phase diagram, and shows the relative concentrations of each polymer within

the other (Figure 1-9).

Figure 1-9. Phase diagram for two neutral polymers. For my system, the upper tie line (a) indicates that at points 2,

3, & 4 that the concentration of each polymer within the solution is constant no matter the volume of polymer-rich

solution used. Adapted with permission from reference 18.

Following the phase diagram in Fig. 1-9, the top tie line (a) shows the relative concentration

of each polymer within each phase. The partitioning coefficient of each phase (Equation 1-3) is

determined by the phase separation of the two phases. This equation was manipulated to give

relative concentrations of each phase (Equation 1-4) for analysis. It was concluded that anywhere

along the tie line for the ATPS used in this thesis and previous work done by the Keating group,

the Dx-rich phase has a 2.7% PEG and 29% Dx by weight concentration and the PEG-rich phase

has a 13% PEG and 4% Dx by weight concentration.15,16 These concentrations do not change based

11

on the volume of either solution that is added to the system (Figure 1-10). Even if the relative

volume of Dx-rich phase added into the system is small compared the amount of PEG-rich phase

added, the percent by weight of each polymer will remain the same for the overall system when

adding the pre-equilibrated phases.14,15,17,18,21

𝐾 =[𝐴𝑛𝑎𝑙𝑦𝑡𝑒]𝑇𝑂𝑃[𝐴𝑛𝑎𝑙𝑦𝑡𝑒]𝐵𝑂𝑇𝑇𝑂𝑀

=𝐶𝑇𝑂𝑃𝐶𝐵𝑂𝑇

=

𝑚𝑜𝑙𝑇𝑂𝑃𝑉𝑜𝑙𝑢𝑚𝑒𝑇𝑂𝑃⁄

𝑚𝑜𝑙𝐵𝑂𝑇𝑉𝑜𝑙𝑢𝑚𝑒𝐵𝑂𝑇⁄

=𝑚𝑜𝑙𝑇𝑂𝑃𝑉𝑇𝑂𝑃

×𝑉𝐵𝑂𝑇𝑚𝑜𝑙𝐵𝑂𝑇

(𝑬𝒒. 𝟏 − 𝟑)

𝐶𝐵𝑂𝑇 =𝑚𝑜𝑙𝑇𝑂𝑇𝐴𝐿

(𝐾 × 𝑉𝑇𝑂𝑃) − 𝑉𝐵𝑂𝑇 (𝑬𝒒. 𝟏 − 𝟒)

Figure 1-10. Formation of Specific Volume Ratio Aqueous Two Phase Systems. Two polymers, Dx and PEG, are

dissolved in Tris buffer solution, where they form an ATPS. The two phases are then separated into individual

containers and re-combined at desired volume ratios.

1.4.2 Stabilization of Aqueous Two Phase Systems

Figure 1-10 depicts that after formation of the original ATPS, each polymer-rich phase is

separated out and then added together in a specific volume ratio. Allowing the solution to set for

a certain amount of time will cause coalescence of the Dx-rich phase droplets because each droplet

12

is identical and the system will minimize the Dx:PEG interface. For the system to be used as an

artificial cell model with compartmentalization, the aqueous/aqueous interface needs to stay stable.

In a previous study of a Dx:PEG ATPS under the same conditions as the system used in this thesis,

it was concluded that the use of liposomes, in the form of large unilamellar vesicles (LUVs,

~110nm diameter), allowed for the stabilization of the ATPS.17

In a typical ATPS, some form of stabilization is needed to keep coalescence of droplets from

the continuous phase. Liposomes act as a barrier between the aqueous/aqueous interfaces of

biological cells.1,6,8,14-19,23-29 To prevent coalescence in ATPS, a phenomenon similar to that of a

Pickering Emulsion for stabilization takes place (Equation 1-5) with the use of LUVs.

∆𝐸 = −𝜋𝑅2𝛾(1 − |cos 𝜃|)2 (𝑬𝒒. 𝟏 − 𝟓)

ΔE is the energy required to remove a particle from the interface that relies on the particle radius

(R), interfacial tension (γ), and the contact angle (θ). The LUVs self-assemble at the interface

based on electrostatics of the repulsion of their negative headgroups. In this thesis, the lipids used

in the creation of LUVs are PEG-elated lipids, which further increases the electrostatics at the

interface when creating a barrier between the Dx-rich and PEG-rich phases.

Previous work increased concentration of liposomes in the solution with the results of Dx-rich

droplet size decreasing due to more surface area stabilized at higher liposome concentrations

(Figure 1-11).17 The results (black dots in Fig. 1-11b) of the droplet size versus the liposome

concentration compared to prediction of droplet size for liposome layers (blue lines in Fig. 1-11b)

indicated that between a monolayer and half-layer of liposomes surrounded each droplet for the

ATPS to account for the electrostatic between liposomes as well as the two polymer-rich phases.17

13

Figure 1-11. Liposome concentration determines droplet size. (a) Optical microscopy images showing decreased

droplet size with increased liposome concentration. Scale bar, 25μm. (b) Dependency of droplet size on liposome

concentration. Blue lines indicate predicted size from hexagonally close-packed liposomes in varying numbers of

planar layers while inset shows visual of packing possibilities. Black dots indicate results of measured droplet sizes.

Adapted by permission from [Springer Nature]: [Springer Nature][Nature Communication][ Bioreactor droplets from

liposome-stabilized all- aqueous emulsions, Dewey, D.C.; Strulson, C.A.; Cacace, D.N.; Bevilacqua, P.C.; Keating,

C.D.][2014].

In a previous study from this group, two Dx:PEG ATPS systems were allowed to sit for 24

hours: (1) a control (no liposomes) and (2) one with liposomes present.17 In Figure 1-12, it can be

seen that for no liposomes present in solution, there is no turbidity after 24 hours indicating the

Dx-rich phase and PEG-rich phases separated, with the Dx-rich phase sinking to the bottom

because of its greater density. However, having liposomes present in the solution allows for the

continuity of the turbidity, presenting that the Dx-rich phase droplets are still suspended in the

PEG-rich continuous phase. In this thesis, the use of liposomes as a barrier between the two

polymer-rich phases of the LLPS will be considered for its stability and permeability.

14

Figure 1-12. Stabilization of Dx:PEG ATPS through use of liposomes to indicate process of water-in-water emulsion

stabilized by lipid vesicles. Presences of lipid vesicles shows no coalescence through suspension of droplets in

continued cloudiness of solution. Reprinted by permission from [Springer Nature]: [Springer Nature][Nature

Communication][ Bioreactor droplets from liposome-stabilized all- aqueous emulsions, Dewey, D.C.; Strulson, C.A.;

Cacace, D.N.; Bevilacqua, P.C.; Keating, C.D.][2014].

1.4.3 Introduction of Coacervate-Containing AMVs

The study of mineralization previously conducted in the Keating group involved the production

of ACC via an ATPS consisting of a Dx-rich droplet phase and a PEG-rich continuous phase.14-

17,21 In this system, the Dx-rich droplet was encased with a LUV layer at the interface of the

polymer-rich phases. The introduction of excess urea into the system created carbonate, which

interacted with calcium cations present in the Dx-rich droplet. Figure 1-13 depicts a cartoon of

the AMV used (a) for mineralization followed by a dark field optical image of the mineralization

(b) and visual images (confocal (c) and scanning electron microscopy (SEM) (d)) of the resulting

minerals. In the dark field images, after addition of urea, the bright white spots indicate mineral,

while in the confocal images, the darker gray spots indicate mineral.14

15

Figure 1-13. Depiction of enzymatic mineralization of calcium carbonate through use of AMVs (a) Schematic of

artificial mineralizing vesicles used to produce CaCO3 (b) Dark field optical microscope images of mineralization

with 500mM urea (c) Brightfield (DIC) and fluorescence overlay images of CaCO3 (d) Scanning electron microcopy

image of CaCO3 (a,b,c) Adapted with permission from reference 14.

In the previous works on the mineralization of ACC through use of AMVs, the mineral formed

was dependent on the equilibrium concentration of Ca2+ located in the Dx-rich phase at the time

of mineralization. With a partitioning coefficient of 1.08 ± 0.01, the Ca2+ was distributed uniformly

across both polymer-rich phases. To overcome this limitation, the addition of a third phase into

the system allows for increased local concentration of Ca2+ to the Dx-rich phase, as well as control

over the shape of mineral formed (Figure 1-14).

To create a third phase in the ATPS two paths have been described in literature and previous

group work.1-4,8-11,21,29-33 Both paths involve the addition of a third polymer, normally negatively

charged, that partitions into the Dx-rich phase and interacts with free Ca2+ in solution. The first

path encompasses the creation of a polymer induced liquid precursor (PILP), which only exists

after the addition of Ca2+ counterion at a critical condition. The second path comprises the

formation of a coacervate in the system with the binding of the polymer to the Ca2+ before the

addition of a counterion. For this thesis, a coacervate made up of poly(α,β)-DL-aspartic acid (PAA)

and Ca2+ is used for mineralization.

16

Figure 1-14. Depictions of Artificial Mineralizing Vesicles made up of polyethylene glycol and dextran with a large

unilamellar vesicle barrier to keep from coalescence of the phases. a) Original AMV used in ACC mineralization. b)

AMV containing a PAA/Ca2+ coacervate for new form of ACC mineralization.

Laurie Gower coined the term PILP as the start to mineralization of carbonate minerals in her

systems.1-4,8-11,21,29-33 The process of forming a PILP in early literature starts with an ATPS

containing two neutral polymer-rich phases, free negatively-charged polymer, and a cation. The

solution reaches a critical concentration of cation counterion species and causes a phase separation

of the negatively-charged polymer, cation, and counterion as an aggregate. These aggregates are

termed droplets (Figure 1-15a). These droplets coalesced into a continuous isotropic film (Fig. 1-

15b), crystallized in ‘patches’ of nucleated birefringent crystals that form along crystallographic

planes (Fig. 1-15c), and eventually formed a continuous film (Fig. 1-15d). This process had to

take place on a glass cover slip or along the side of a glass beaker.29

17

Figure 1-15. Stages and feature of the polymer induced liquid precursor (PILP) process. Critical concentration of Ca2+

is observed during phase separation (a) followed by coalescence of isotropic film droplets (b). These droplets have

birefringent areas that are caused by crystal tablet formation (c), that continue to spread laterally (d). The final produce

is composed of single-crystalline patches of calcite. Reprinted with permission from reference 29.

In this A3PS, the PILP is considered a third aqueous phase because it is highly hydrated, so it

has more of a ‘droplet’ morphology than a particle morphology, even though a crystal is formed,

and it behaves more like a liquid than a solid.8,11,29 For an ACC mineralizing system, the PAA was

added right before Ca2+ counterions were introduced to interact with the Ca2+ present. As the

counterion for Ca2+ was added in excess, a critical concentration, after gradual supersaturation of

the ATPS, was reached at which a PAA/Ca2+-rich coacervate phase appears (Figure 1-16).8,11,29

For this thesis, phase separation occurs before the addition of a counterion, distinguishing our

mineralization pathway from that of a PILP.14-17,21

18

Figure 1-16. Depiction of Polymer Induced Liquid Precursor Formation (PILP). To form a PILP, the ATPS has to

undergo the same steps for the formation of a PAA/Ca2+ coacervate, but the addition of a calcium counter ion in

solution must be at a critical concentration with an increase in supersaturation.

PAA polymers and calcium ions make up the coacervate in our AMV coacervate containing

system (Scheme 1-1).21 The coacervate is a polymer-rich phase, composed of PAA and Ca2+, while

the remaining phase (supernatant) is PAA-poor. The supernatant in this system would be both the

PEG-rich phase and the Dx-rich phase. For this thesis, the creation of a coacervate as the third

phase of the A3PS is noted and will be designated a coacervate-containing AMV.

Scheme 1-1. Depiction of Polyaspartic Acid and Calcium Coacervate. The calcium in the ATPS binds to the carboxyl

groups of the PAA, reducing it’s negative charge to make it less solvated and creates a coacervate.

In previous works, during the formation of the mineral, confocal microscopy was used to

observe the dissolution of the PAA-rich phase upon creation of minerals (Figure 1-17&18).16

When no urea is present in the solution, an aqueous third phase composed of PAA polymer and

19

Ca2+ is present (Fig. 1-17 green channel). After addition of urea into the system, the PAA from the

coacervate is released into the Dx-rich phase (teal coloring Fig. 1-18) while a mineral is formed in

place of the third phase, taking its shape. This A3PS produced spherical minerals that were tunable

based on the initial reactant concentrations present. This coacervate-containing AMV system, for

the mineralization of ACC, is used through the rest of this thesis.

Figure 1-17. Still image from prepared ACC mineralization video at 0 min. This shows the fluorescence of the A3PS

before addition of urea into the solution. PAA is labelled with Alexa-488 dye, the Dx-rich phase is labelled with

Alexa-647, and the LUVs are labelled with RhDOPE-dye. The urease enzyme and PEG-rich phase are not labelled.

Adapted with permission from Cacace, D. N. (May 2014) “Biomimetic Mineralization of Calcium Carbonate in

Aqueous Biphasic Systems” PhD diss., The Pennsylvania State University, 2014.

1.5 Chapter Overviews

In this introduction, I explained the importance of compartmentalization in artificial mineral

formation. The inclusion of ATPS and A3PS in the process of mineralization with the stabilization

from LUVs allows for tunability of product. Chapter 2 goes into details on how LLPS is used in

artificial mineralization of CaCO3, the conversion to Ca-Phosphate morphologies, and analysis

techniques used for quantification of minerals. Chapter 3 details the changes in ACC

mineralization to show tunability of the system with inclusion of a coacervate. Chapter 4 discusses

20

Figure 1-18. Still image from ACC mineralization video at 16 min. This shows the fluorescence of the A3PS after

addition of urea into the solution. The urea has hydrolyzed the urease enzyme to create carbonate that interacted with

the Ca2+ in the previous PAA-rich phase. This created a mineral and released PAA into the Dx-rich phase. The labels

are the same as in Figure 1-17. Adapted with permission from Cacace, D. N. (May 2014) “Biomimetic Mineralization

of Calcium Carbonate in Aqueous Biphasic Systems” PhD diss., The Pennsylvania State University, 2014.

the transformation of stable ACC minerals into forms of Ca-Phosphate. Chapter 5 gives a summary

of the previous two chapters and discusses future directions that could be taken in the use of the

coacervate-containing AMV system as well as with the Ca-Phosphate minerals.

The compartmentalization of the artificial minerals mimic that of biological life. Having the

understanding of how to control and manipulate the compartments will allow for more

understanding on how biological cells work and are used. I manipulated the size of the Dx-rich

droplets in my system to see if control over the size of the location for mineralization had an effect

on the resulting mineral. I also converted ACC minerals into Ca-Phosphate morphologies to see a

broader context for the application of biomimetic mineralization.

21

Chapter 2

Methods

2.1.1 Buffer Preparation

Stock solutions of Tris acid and base were made at a 10 mM concentration in 18.2 MΩ

Nanopure water (DI H2O). Trisma hydrochloride (10mM; pH=5.56) and Trisma base (10 mM;

pH=10.02) were mixed together to form a Tris buffer at pH=7.41. The acid, base, and buffer were

kept at room temperature and used as needed.

2.1.2 Aqueous Two Phase Systems

A 10%wt/10%wt poly(ethylene) glycol (PEG)/dextran (Dx) ATPS was made by mixing Tris

buffer (10mM; pH=7.4) with PEG (8 kDa; Sigma-Aldrich, St. Louis, MO) and Dx (10 kDa;

Toronto Research Chemicals, Inc., Toronto, Canada) for 20 minutes on a VWR tube rotator and

Rotisserie (18 RPM). The solution was allowed to equilibrate for 6 hours while it separated into a

Figure 2-1. Chemical structures of neutral polymers, a) dextran (TRC Inc.) and b) polyethylene glycerol (Sigma-

Aldrich), used for this ATPS.

PEG-phase and a Dx-rich phase. The top, PEG-rich phase, of the equilibrated solution was pipetted

first and put into a new container. The bottom, Dx-rich phase, was pipetted out by leaving a little

22

air in the pipette that was slowly pushed out as the tip went through any remaining PEG-rich phase

to avoid cross contamination (Figure 1-10). The tip was dried with a kimwipe before the Dx-rich

phase was put into a new container.

2.1.3 Preparation of Stock Solutions

The following stock solutions were made on the day of experimentation for best results. The

polyamino acid, poly(α,β)-DL-aspartic acid sodium salt (2-11K MW; PAA; Sigma-Aldrich, St.

Louis, MO), was dissolved in PEG-rich phase to form a concentration of 50 mg/mL. The salt

solution, calcium chloride, was mixed dropwise with PEG-rich phase (1 M). The enzyme, urease,

with a concentration of 50 mg/mL, and the substrate, urea, with a concentration of 5 M were

dissolved in PEG-rich phase dropwise. All solutions are vortexed until no solid was present. If

time permits, the solutions can also be sonicated for one hour for even mixing. The solutions were

kept at room temperature for use.

Figure 2-2. Chemical structure of poly(α,β)-DL-aspartic acid.

2.1.4 Preparation of Lipid Vesicles

Large unilamellar vesicles (LUVs, ~110 nm diameter) were formed through a gentle hydration

method. A suspension of 1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine-N-[Methoxy

(Polyethylene glycol)-2000] (Ammonium Salt) (DOPE-PEG-2K; 1.6 mg/mL), L-a-Phosphatidyl-

DL-Glycerol (Egg,Chicken) (Sodium Salt) (EggPG; 7.5 mg/mL), 99% L-a-Phosphatidylcholine

23

(Egg,Chicken) (EggPC; 7.5 mg/mL), and 1,2-dioleoyl-sm-glycero-3-phosphoethanolamine-N-

(lissamine rhodamine B sulfonyl) (ammonium salt) (RhDOPE; 0.026 mg/mL), all in a physical

state of chloroform from Avanti Polar Lipids, Inc, were added together (Figure 2-3). The solution

went through gentle hydration with first being dried under argon to form a thin layer around the

edges of the container and then put under vacuum to remove all excess chloroform for 30 minutes

(Scheme 2-1). PEG-rich phase was used to hydrate the liposomes and the solution was incubated

for 48 hours. The solution was extruded using an Avanti micro-extruder with two 0.2 μm

membranes. The liposomes were kept at 37⁰C devoid of light until needed.

Figure 2-3. Chemical representation of lipids used in lipid vesicles. All images from Avanti Polar Lipids, Inc. a)

RhDOPE, b) DOPE-PEG-2K, c) Egg-PC, d) Egg-PG.

24

Scheme 2-1. Gentle Hydration Method allowing formation of liposomes to be used in LUV fabrication.

2.1.5 Silanization of Glass Slides

VWR micro cover glass (24x30 mm) were silanized to create a hydrophobic surface for AMV

analysis. The slides were soaked in a potassium hydroxide saturated isopropanol solution for 30

minutes. These were washed thoroughly with DI H2O (3-5 washes) and then dried overnight at

70⁰C. The slides were covered with a silane solution (3 mg PEG-Silane per 1 mL anhydrous

toluene) and left to react for 4 hours. The slides were washed with ethanol (3-5 times) and then

washed with DI H2O. They were left to dry overnight at 70⁰C. The glass slides were moved to a

closed container for further use.

2.2 Methods of Making the System

The system for this thesis involved the creation of coacervate-containing AMVs that were

converted into an amorphous calcium carbonate mineral (Figure 1-8). The resulting mineral was

analyzed and then used as a starting point for conversation into another mineral. The aspect of

creating the ACC mineral followed the same method for all parts of the thesis.

2.2.1 Formation of Artificial Mineralizing Vesicles (AMVs)

Stock solutions of PAA, PEG, Dx, calcium chloride, and LUVs were added together. The

concentrations of PAA and CaCl2 were the same in all experiments, 10.5 mg/mL and 50 mM

respectively, and standard AMVs include a 1:49 Dx:PEG ATPS volume ratio (Vd:Vp) with a 1.500

25

mg/mL LUV concentration. Alexa-488 dye labelled PAA was added first. The PEG, Dx, and CaCl2

solutions were added in succession followed by the LUVs. The whole solution was vortexed for

10-20 seconds. The Ca2+ and PAA partitioned to the Dx-rich phase with the LUVs at aqueous-

aqueous polymer-rich phases interface. Ca2+ interacted with the PAA to create a coacervate

(Scheme 1-1), which resulted in an A3PS of PEG-rich phase, Dx-rich phase, and a PAA-rich

phase. The solution was pipetted onto a silanized glass slide for analysis.

2.2.2 Synthesis of Amorphous Calcium Carbonate

The mineral formation follows the same order of addition as the AMVs with the addition of an

enzyme and chelator. PAA (10mg/mL final concentration) was added first, followed by PEG, Dx,

a urease enzyme (~1.5 mg/mL final concentration; Fluka Analytical, Sigma-Aldrich, St. Louis,

MO), CaCl2 (~50 mM final concentration), and LUVs (1.500 mg/mL final concentration). The

solution was vortexed for 10-20 seconds. The same partitioning as the AMVs occurred with the

addition of urease partitioning into the Dx-rich phase. Urease has a partitioning coefficient of

0.117±0.003, 8.5 times higher in Dx-rich phase than the PEG-rich phase, which allows the

partitioning to occur.14,15,16,21 Finally, the addition of a urea chelator (~100 mM, Sigma-Aldrich,

St. Louis, MO) with another 10 second vortex completed the solution mixing. The enzyme urease

hydrolyzes urea and releases ammonia and carbonic acid, which makes carbonate and bicarbonate

through deprotonation and then gives a net pH increase and calcium carbonate (Equation 2-1).

(𝑁𝐻2)2𝐶𝑂(𝑎𝑞) + 𝐶𝑎2+(𝑎𝑞) + 2𝐻2𝑂(ℓ)

𝑈𝑟𝑒𝑎𝑠𝑒, 𝑝𝐻>8→ 2𝑁𝐻4

+(𝑎𝑞) + 𝐶𝑎𝐶𝑂3(𝑠) (𝑬𝒒. 𝟐 − 𝟏)

The solution was left on a benchtop for 1 hour to mineralize. The solution was spun down in a

Centrifuge 5415 R (13.2 RPM; Eppendorf, Hamburg, Germany) for 20 minutes to create a

supernatant of ATPS and a pellet made of ACC. The supernatant was pipetted out and the mineral

was washed three times with Tris base (pH=10.02). The final wash was removed and the minerals

26

were dried in a speed vacuum (DNA120 Speed Vac, Thermo Savant; 1725 RPM) for 2 hours. The

resulting minerals were stored at room temperature until analysis (Scheme 2-2).

Scheme 2-2. Depiction of Amorphous Calcium Carbonate Mineralization. The introduction of a urea substrate in an

A3PS of PEG-rich phase, Dx-rich phase, and a PAA/Ca2+ coacervate with a urease enzyme allows for the hydrolysis

of urea by urease to generate carbonate ion. The ion interacts with the Ca2+ found in the coacervate for form an ACC

mineral that is washed with base, dried at room temperature, and then analyzed.

2.2.3 Phosphate Buffer Wash

Sodium phosphate buffers made up of sodium phosphate dibasic dihydrate (Sigma-Aldrich, St.

Louis, MO) and sodium phosphate monobasic (Sigma-Aldrich, St. Louis, MO) were made at

concentrations of 50, 100, and 500 mM at physiological pH. Prepared ACC minerals were washed

at room temperature for 1, 6, and 12 hours while rotating on VWR Tube Rotator and Rotisserie

(18 RPM) with each sodium phosphate buffer concentration. The solutions are thought to go

through the process of changing from ACC to a form of calcium phosphate (Equation 2-2).7 The

5 𝐶𝑎𝐶𝑂3 (𝑠) + 3 𝑃𝑂43− + 𝑂𝐻− ⇌ 𝐶𝑎3(𝑃𝑂4)3𝑂𝐻 (𝑠) + 5 𝐶𝑂3

2− (𝑬𝒒. 𝟐 − 𝟐)

samples were washed three times with DI H2O and then a final ethanol wash before drying in the

speed vac for 2 hours. The dried samples were analyzed by FTIR spectroscopy, XRD, and SEM.

The 500 mM washed solutions were also analyzed by Raman spectroscopy.

27

2.3 Instrumentation of Analysis

Both quantitative and qualitative forms of analysis were completed for coacervate-containing

AMVs, ACC minerals, and Ca-Phosphate minerals.

2.3.1 Confocal Microscopy Analysis

Confocal images were obtained using a Leica TCS SP5 PL confocal microscope (Wetzlar,

Germany) using a 63x oil immersion objective and LAS-AF software. Fluorescent images were

completed with a 543 nm laser excitation (30% from a HeNe543 laser) for RhDOPE-dyed LUVs

(emission bands: 560-600 nm) and a 488 nm laser excitation (30% from an Argon laser) for Alexa-

488 labelled PAA rich-phase (emission bands: 490-525 nm). A Differential Interference Contrast

(DIC) image was taken to see the contrast between the different phases present in the solution.

Raw images (Figure 2-4) were analyzed using an open source Java image program, ImageJ.

Figure 2-4. Confocal Microscopy Images of 1.500 mg/mL LUV concentration and 1:49 Vd:Vp prepared coacervate

formation. a) RhDOPE-dyed LUV fluorescent image, b) Alexa-488 labelled coacervate fluorescent image, c) DIC

image.

2.3.2 Scanning Electron Microscopy (SEM) Analysis

SEM images were acquired with a FESEM: NanoSEM 630, operated at high vacuum mode,

3-6 mm working distance, and a 5 keV accelerating voltage under FEI Nova software. Samples

28

needed to be coated for conductivity, so a 10 nm iridium layer was applied using a Leica EM

ACE600 at room temperature.

2.3.3 Image Analysis

The program ImageJ was used for the determination of AMV, coacervate, and mineral size

from fluorescent confocal and SEM imaging, respectively. The raw scale bar from the

instrumentation was used. Multiple sets of images were analyzed for each data set. The average

was found from one solution, yet comparison between trials was completed.

2.3.4 X-Ray Diffraction Analysis (XRD)

A PANalytical XPert Pro MPD Theta-Theta Difractometer (Almelo, The Netherlands) was

used for mineral sample analysis across a 10-70⁰ 2θ MPSS with 0.5 fix slit, 10 beam mask, 0.04

rad, 45 kV, and 40mA Cu Kα over an analysis time of about 17 minutes. The data was analyzed

using MDI-Jade+9 software (Livermore, CA).

2.3.5 Raman Spectroscopy

A Horiba Lab Ram Raman microspectroscope (Kyoto, Japan) was utilized for sample analysis

of 500 mM sodium phosphate washed prepared ACC minerals. The instrument contained a 532

nm laser light source operated at 40 mW with the application of a x100/1.0 NA air microscope

objective (Horiba, Kyoto, Japan). A 300 lines per mm grating spectrograph was used and spectra

were acquired with a back illuminated detector (2048x512 pixel) with a spectral resolution of ~4

cm-1. Each individual spectrum had an acquisition time of 10 seconds. The image collection was

conducted by Andrew Rowland (PhD candidate).

2.3.6 Fourier Transformed Infrared (FTIR) Spectroscopy

Infrared spectroscopy was completed using a Bruker V70 FTIR, Vertex (Bruker Optics,

Billerica, MA). A Harrick MVP Pro attenuated total reflection (ATR) accessory with a diamond

29

crystal, operated under N2-rich conditions, was employed because of previous knowledge of

sample perturbation and spectrum comparison between transmission and ATR-FTIR.14 The

spectra was acquired between 400-4000 cm-1 with an average of 100 scans using an DLaTGS

detector and recorded using Opus software. For all images shown, intensities are from -log(R/R0),

where R is the sample reflectance and R0 is the reference reflectance (diamond crystal).

30

Chapter 3

Modification of Artifical Mineralizing Vesicle Systems

3.1 Artificial Mineralizing Vesicle System

The formation of amorphous calcium carbonate (ACC) has been achieved through the use of

chelator stabilizing molecules.1-4,8-11,14-16,29,32 In this chapter, an aqueous three phase system

(A3PS) made up of AMV-encapsulating coacervates as depicted in Figure 3-1 will be utilized.

The ACC mineral forms in coacervate-containing AMVs via hydrolysis of urea (Equation 3-1).

(𝑁𝐻2)2𝐶𝑂(𝑎𝑞) + 𝐶𝑎2+(𝑎𝑞) + 2𝐻2𝑂(ℓ)

𝑈𝑟𝑒𝑎𝑠𝑒, 𝑝𝐻>8→ 2𝑁𝐻4

+(𝑎𝑞) + 𝐶𝑎𝐶𝑂3(𝑠) (𝑬𝒒. 𝟑 − 𝟏)

Figure 3-1. Enzymatic Mineralization of ACC within an Artificial Mineralizing Vesicle. Urea hydrolysis via urease

occurs in LUV-stabilized Dx-rich droplets in a continuous PEG-rich phase. The resulting carbonate ions interact with

Ca2+ in the PAA-rich coacervate. ACC minerals are formed within the coacervate inside the Dx-rich droplets.

(Reprinted from Chapter 1)

This chapter will focus on how changing the order of addition, LUV concentration, and

Dx:PEG ATPS volume ratio (Vd:Vp) affects the overall mineral formation in coacervate-

containing AMVs. With a change in order of addition to have the ATPS form before the coacervate

31

is expect to give a more homogeneous formation of AMVs and coacervates. I changed the

concentration of LUVs by 25% in both directions. By increasing the concentration of LUVs

present in the solution, the anticipated results would give smaller AMV, coacervate, and mineral

volumes, while decreasing the concentration would give the opposite. With a change in Vd:Vp, I

would expect that having more Dx-rich phase in the solution, smaller droplets will occur, causing

smaller coacervates and, therefore, smaller minerals.

3.2 Order of Addition

In previous works, the order of addition for the A3PS was used to help limit the interactions

between the positively charged calcium and the negatively charged LUVs during the creation of

the AMVs.14,21 In those experiments, the coacervate formed prior to the addition of any Dx-rich

phase. We hypothesized that sample heterogeneity might be improved by first distributing the PAA

within Dx-rich droplets, prior to the coacervate formation. Therefore, in the new order of addition

(Table 3-1), the calcium is added after the addition of Dx-rich phase.

Original Order of Addition New Order of Addition

PAA and label PAA and label

CaCl2 PEG

PEG Dx

Dx CaCl2

LUVs LUVs Table 3-1. Order of Addition for AMV Formation. The change between adding the calcium stock solution after the

addition of the Dx-rich phase versus before the addition of the droplet phase was to increase likelihood of the formation

of coacervate into the interior of the AMV. Original Order of Addition is equivalent to order of addition for previous

works (Cites 14, 16, 19) whereas New Order of Addition is used for the remainder of this thesis.

32

Figure 3-2. Order of Addition for Artificial Mineralizing Vesicles. (A) indicates the formation of a coacervate before

a LLPS of Dx-rich phase and PEG-rich phase is produced. This is the order of addition used in previous works. (B)

depicts the new order of addition in which PAA and Dx-rich phase form a phase before the addition of calcium into

the system. At equilibrium both solutions should give the same coacervate-containing AMV used in the mineralization

process of our system.

Figure 3-3. Order of Addition in ACC Mineralization. The addition of Ca2+ into the ATPS for ACC mineral formation

allows for the creation of a PAA/Ca2+ coacervate. Both systems have 1.500 mg/mL LUV and 1:49 Dx:PEG. (a) depicts

addition of Ca2+ after the addition of PAA, PEG-rich phase, and Dx-rich phase. (b) depicts addition of Ca2+ after the

addition of PAA, before the creation of ATPS. All scale bars 25 μm. (c) show the histograms of the AMVs formed

and (d) shows the histograms of the coacervates formed for each order of addition.

33

In (Figure 3-2b), PAA is already partitioned and accumulates somewhat in the Dx-rich phase,

while in (a) a coacervate is made first, then a Dx-rich phase droplet is formed. At equilibrium the

two methods should give the same result, but we hypothesized we might get better uniformity of

coacervate-containing AMVs by (b) because the coacervate forms inside the droplet rather than

bumping into and being engulfed by the droplet. So, it was tested to see if we got better uniformity

with changing the order of addition for coacervate-containing AMVs.

The confocal fluorescent images of these two order of additions (Figure 3-3 a&b) gives the

impression that the new order of (a) has less heterogeneity of the AMV and coacervate sizes

produced. However, based on the histograms (Fig. 3-3 c&d), which combine sizing data from

multiple images across both samples, we concluded that the order of addition has little impact on

AMV or coacervate volume size distribution because there is no shift in the overall range.

3.3 Effects of LUV Concentration on AMV, Coacervate, and Mineral Size

Without a coacervate, ACC minerals still form within the Dx-rich phase droplets of the AMVs.

In previous works, the Ca2+ was bound by the monomeric chelator, Ethylenediamine-N,N'-

disuccinic acid (EDDS), with a slight preference for the Dx-rich phase.14,16,21 The large unilamellar

vesicles (LUVs) absorb at the aqueous/aqueous interface of the Dx:PEG-rich phases. The LUVs

stabilize Dx-rich droplets within the continuous PEG-rich phase. Without the presence of LUVs

in the system, coalescence of the Dx-rich phase droplets occur. Previously, it was concluded that

LUVs create between a monolayer and half layer at the interface of the Dx:PEG ATPS emulsion

(black dots on Figure 1-11b). LUVs take up the same amount of space at the interface for this

thesis. Others in the lab have found a strong correlation between coacervate size and ACC particle

size. I sought to control coacervate and ACC particle size by varying the amount of LUVs present

in the system. In principle, the amount of LUVs available control AMV size by determining how

34

much surface is stabilized. In previous work, the LUV stabilized ATPS emulsions with no

coacervates or mineral products and increasing the LUV concentration decreased average Dx-rich

phase droplet size (Figure 1-11a). This occurred because of the larger surface area stabilized when

more LUVs were available. In that system, LUVs had very low solubility in either the Dx-rich or

the PEG-rich phase and were extremely localized at the interface.

With the addition of the coacervate phase the LUVs still adsorb to the Dx:PEG-rich phase

interface. However, changing the concentration of LUVs in AMV systems has not been studied

and may not be as straight forward due to reduced LUV stability at the interface because of the

presence of other charges. Based on the influence of LUV concentration of Dx-droplet size, it

stands to reason that the size of the ACC minerals formed could be controlled by the changing

concentration of LUVs.17 The increased concentration of liposomes present in AMVs, excluding

coacervates, showed a decrease in droplet size. Therefore, with the addition of the coacervate in

the system, this section of the chapter will focus on changing LUV concentration to determine size

of resulting minerals.

At a standard concentration of all parts for ACC mineralization, the resulting minerals made

inside AMVs were all found to be spherical with a core/shell morphology.21 Changing the LUV

concentration should not change the shape or morphology of the minerals, but should effect the

size of the spherical minerals produced.

3.3.1 Analysis

The standard of making ACC minerals through AMVs for the use in this thesis was a 1:49

Vd:Vp with 1.500 mg/mL LUVs at the aqueous/aqueous interface. Coacervates were comprised of

10 mg/mL PAA and 50 mM calcium ions from salt. Mineralization occurred upon hydrolysis of

100 mM urea via 1.5 mg/mL urease. The concentration of LUVs was increased and decreased by

35

25% to see if the resulting emulsions would produce differing sizes of spherical minerals. All other

concentrations and preparation methods were not changed. Resulting emulsions were analyzed

using fluorescent confocal microscopy, while scanning electron microscopy (SEM) was used to

analyze minerals.

If we assume that dextran droplet size for a given Dx-phase volume is determined solely by

stabilized surface area then the expected results would be that an increase in LUV concentration

would give smaller Dx-rich droplets, i.e. the AMV size would decrease. By changing the LUV

concentration of my system, I would expect the AMV sizes to change proportionally. If the LUV

concentration increases by 25% (1.500 mg/mL to 1.875 mg/mL) I would theoretically make AMVs

that are 2/3R in volume or ~17% smaller; the opposite being true for a decrease in LUV

concentration. I found that decreased amount of LUVs in the system resulted in less vesicle

aggregation at the interface (Figure 3-4); the presence of LUV aggregation, and changes in the

amount of aggregation between samples, indicates that changes in LUV concentration did not

correspond directly to changes in the amount of surface stabilized.

However, the LUV concentration did impact the resulting AMV size distribution. Confocal

fluorescent images of the AMVs are shown in Figure 3-4a. The samples showed variability in

AMV size in the images and I plotted histograms of the size populations, which showed diameters

ranging from 0.25 to 60. μm and volumes ranging from 0.0080 to 110000 μm3 for the standard

prepared AMVs (Figure 3-5 & 6 middle lines). The coacervates also showed a large range for the

standard prepared AMVs in diameter (0.25 to 56 μm) and volume (0.0082 to 91000 μm3). From

the histograms the large distribution can be seen. From SEM images (Fig. 3-4c) the heterogeneity

of resulting minerals is also apparent. I measured the mineral sizes and found a diameter range

much smaller than the range of the AMV or coacervate sizes (0.20 to 8.2 μm and 0.0042 to 280

36

μm3). Though the distribution of sizes is not as large, the heterogeneity of the sample is still present

in mineral formation, as can be seen by these histograms (Fig 3-5&6c).

Figure 3-4. Effect of Change in Large Unilameller Vesicle (LUV) Concentration on AMV, Coacervate, and

Mineral Appearance. a) fluorescence images showing DOPE-Rhodamine-labelled liposomes(RhDOPE); b)

fluorescence images showing Alexa-488-labeled PAA; all fluorescence images have 25 μm scale bars. c) SEM images

of prepared amorphous calcium carbonate minerals; all SEM images have 50 μm scale bars. Row 1 indicates a smaller

37

amount of LUVs present in the solution. Row 2 is the standard prepared ACC. Row 3 has the largest amount of LUVs

present in the solution.

From the heterogeneity of the AMVs, I was not be able to predict the expected value of AMV

volume change with change in LUV concentration, but the histograms of my AMVs should shift

left for an increase in LUVs present, or right for a decrease in LUV concentration. The resulting

coacervate and mineral histograms are anticipated to make the same shift.

From looking at the confocal fluorescent images of AMVs and coacervates, as well as the SEM

images of minerals (Fig. 3-4) for an increase (bottom line) and a decrease (top line) in LUV

concentration, the AMV sizes appear to increase with a decrease in LUVs present. The same is

true for the coacervate and mineral sizes. The histograms for these changes (Fig. 3-5&6) do show

a relative shift.

With a decreased LUV concentration the AMV distribution broadened for diameter and

volume, so a shift in the histogram peak is not visible. The same effect occurs for the coacervates.

The mineral size distribution is relatively sharper and shifts right based off the standard prepared

minerals (Fig. 3-5&6 top line). The overall distribution of the AMV, coacervate, and mineral sizes

for an increased LUV concentration had a larger number range, but the peak distribution is

relatively the same as the standard prepared distributions. From the histograms, it can be observed

that there is less broadness in all peaks, AMV, coacervate, and mineral, for both the diameter and

volume of increased LUV concentration (Fig. 3-5&6 bottom line). A shift left from the standard

prepared samples is also visible for all three channels. From the histograms, it appears that an

increase in LUV concentration across the board caused a decrease in AMV, coacervate, and

mineral volume size.

Taken together, these data illustrate the greater heterogeneity of AMVs as compared to simple

all-aqueous emulsions,17 which is consistent with the greater complexity of this system and likely

38

results primarily from both presence of Ca2+, which can bind lipid headgroups, and the increased

total ionic strength, which reduces electrostatic repulsions between the LUVs. Nonetheless, I was

able to achieve changes in the distribution of AMV, coacervate, and mineral sizes by varying the

LUVs present.

Figure 3-5. Effect of LUV Concentration Change on Diameter of ACC Minerals. The red indicates AMV diameter

(a), and green indicates coacervate diameter (b), and blue indicates mineral diameter(c). All samples have the same

initial materials present, but change the concentration of LUVs. Top line: 1.125 mg/mL LUVs. Middle line: 1.500

mg/mL LUVs; standard prepared ACC minerals. Bottom line: 1.875 mg/mL LUVs. This is ‘raw’ data used for the

39

analysis because the original images are analyzed using the diameter of the mineral, AMV, and coacervate.

Figure 3-6. Effect of LUV Concentration Change on Volume of ACC Minerals. This figure shows the same change

in LUV concentration as that found in Fig. 3-5. (a) indicates AMV volume, (b) coacervate volume, (c) mineral volume.

Top line: 1.125 mg/mL LUVs. Middle line:1.500 mg/mL LUVs; standard prepared ACC minerals. Bottom line: 1.875

mg/mL LUVs. The volume was calculated from the diameter of the solutions analyzed. The x-axis for all histograms

is a log scale.

40

3.4 Dextran:Poly(ethylene) Glycol Volume Ratio

While the change in LUV concentration did have an effect on the overall mineral size, the

LUVs are not the only component that affects the AMVs. The composition of the AMVs ATPS

phases of the AMV could also have an effect on the minerals formed. With a correlation to Dx-

rich droplet size, a change in the relative Dx-rich phase and PEG-rich phase volumes could result

in a change to the resulting mineral size. I changed the Dx:PEG relative volumes from a 1:99 Vd:Vp

to a 1:32.2 Vd:Vp expecting the droplet size to decrease (shown as AMV size), but the mineral size

to increase based on local concentration changes.

3.4.1 Analysis

Excluding the addition of PAA in the system, a 1:1, 1:3, and 1:9 Vd:Vp has been tested for

ATPS compartment size.15 In these systems, the change in amount of Dx-rich phase present (1:1

to 1:9) produced an increase in local urease concentration in the Dx-rich phase without addition of

more urease into the system.15 The local urease concentration increase caused the rate of formation

of minerals to increase as well.15 The change in local urease concentration induces a change in the

rate of mineral formation and could influence the mineral size. Changing the Dx:PEG ATPS

volume ratio to cause an increase or decrease in Dx-rich phase droplets should also cause a change

in the local urease concentration of the system even with the addition of the coacervate.

Changing the Dx:PEG ATPS volume ratio from 1:49 to 1:99 showed signs of no coacervate

formation in confocal microscopy; however, after experimentation, it was determined that a wait

time of at least 5 minutes would show a coacervate via confocal imaging. This is indicative of

coalescence of miniscule coacervates within Dx-rich droplets overtime. Emulsions were examined

under fluorescent confocal imaging and mineral formation was seen from SEM images for the

Vd:Vp from 1:99 to 1:32.2 (Figure 3-7a-b).

41

Figure 3-7. Change in Dx:PEG Volume Ratio. a) Fluorescence images showing RhDOPE labelled LUVs b)

fluorescence images showing Alexa-488-labelled coacervate; all fluorescence images have 25 μm scale bars. c) SEM

images of prepared amorphous calcium carbonate minerals; all SEM images have 50 μm scale bars. Row one is the

least amount of Dx-rich phase present; a time of 5 minutes is needed to create a coacervate large enough to see under

confocal microscopy. Row two is the standard for all systems. Row three is the most amount of Dx-rich phase present

in the solution.

42

Figure 3-8. Effect of change in Dx:PEG Volume Ratio on Diameter f Prepared ACC Minerals. The red indicates

AMV size (a), green indicates coacervate size(b), and blue indicates mineral size(c). All samples have the same initial

materials present, but change the Dx:PEG volume ratio. Top line: 1:99 Dx:PEG volume ratio. Middle line: 1:49

Dx:PEG volume ratio; standard. Bottom line: 1:32.2 Dx:PEG volume ratio. This is ‘raw’ data used for the analysis

because the original images are analyzed using the diameter of the mineral, AMV, and coacervate. Using a two

population mean it was determined that the minerals increased in size by a 95% confidence interval.

43

Figure 3-9. Effect of Dx:PEG Volume Ratio Change on Volume of Prepared ACC Minerals. This figure shows the

same change in Dx:PEG volume ratio as Fig. 3-9. Top line: 1:99 Dx:PEG volume ratio. Middle line: 1:49 Dx:PEG

volume ratio. Bottom line: 1:32.2 volume ratio. The volume was calculated from the diameter of the solutions

analyzed. The x-axis for all histograms is a log scale. A confidence interval of 95% was used for all samples and found

that the volumes increased with an increase in Dx:PEG volume ratio.

Preliminary analysis of minerals formed from standard A3PS systems was conducted in the

same manor used for LUV concentration change. Changing the Vd:Vp for the system from 1:99 to

44

1:32.2 should result in an increased AMV and coacervate size, with a decreased mineral size. The

initial quantitative analysis of diameter of particles showed that as Vd:Vp changed the AMV and

coacervate sizes increased while mineral size decreased (Figure 3-8).

Calculating the values based on volume of the minerals gave a more definite conclusion about

the Vd:Vp change. AMV and coacervate sizes for the 1:99 Vd:Vp change were decreased, while for

the 1:32.2 Vd:Vp change they increased. Figure 3-9 shows the increase in AMV and coacervate

volumes with a slight broadening in the size distribution (a,b) as well as the decrease in mineral

size with a narrowing of size distribution (c). Increasing the relative Dx-rich phase volume caused

the AMV and coacervate volumes to increase and the mineral volumes to decrease.

3.5 Conclusion

The order of addition for the system was determined to not have an effect on the heterogeneity

of the AMV size. However, the formation of the coacervate-containing AMVs an ATPS present

before the addition of the coacervate was used for the remaining changes to the system. The effect

of varying the amount of LUVs and the Dx:PEG ATPS volume ratio on AMV size, coacervate

size, and mineral size were evaluated. With an increase in LUV concentration there was an increase

in AMV, coacervate, and mineral size. With a change in Dx:PEG ATPS volume ratio from 1:99

to 1:32.2 there was an increase in AMV and coacervate size, and a decrease in mineral size. The

broad distribution of sizes from AMVs, coacervates, and minerals reflects the reduced emulsion

stability of these mineral AMV compositions compared to simple, salt-free, all-aqueous emulsions

reported previously.

45

Chapter 4

Calcium Phosphate System

4.1 Importance of Versatility

Amorphous calcium carbonate (ACC) has biological, optical, and mechanical properties, such

as making up different shells/spines of marine life, use in microlens arrays, controlled nucleation,

hardness limits, and templating deposition.1,5,6,14-17,21,23-28 Understanding how to produce calcium

carbonate in the amorphous form, as well as the crystalline polymorphs, is well known. 1,5,6,14-

17,21,23-28 In this chapter, I investigated whether the organic-rich ACC formed in chapter 3 could be

chemically converted to calcium phosphate.

ACC is a precursor for semi-stable forms of CaCO3, aragonite and vaterite, that convert into

the stable form of calcite. However, in some biological settings, the CaCO3 polymorphs can

convert into other materials. In sea sponges, the starting inorganic material of its scaffolds was

found to be silica that underwent an evolutionary change to carbonate scaffolds of calcite and

aragonite.5,6,7,24-28 As time went on, the scaffolds convert their inorganic components to calcium

phosphate in the form of hydroxyapatite (HA). From this evolution, it was concluded that inorganic

CaCO3 existed before polymorphs of calcium phosphate (Ca-Phosphate) based inorganics.7

Because of this phenomenon, it could be possible that CaCO3 might also contribute to the presence

of inorganic sources of phosphate in skeletal formation.

This chapter will focus on the conversion of ACC minerals made inside artificial mineralizing

vesicles (AMVs) to hydroxyapatite-like material. Sodium phosphate buffers of different

concentrations at different wash intervals were used for the transformation to Ca-Phosphate.

46

Analysis was doing using attenuated total transmission-Fourier transform infrared spectroscopy

(ATR-FTIR), Raman spectroscopy, and x-ray diffraction (XRD).

4.2 Formation of Calcium Phosphate

ACC minerals were made under standard conditions found in Chapter 2. As prepared ACC

minerals are washed with sodium phosphate buffers at physiological pH (~7.4) to displace

carbonate ions with phosphate ions through the process depicted in Equation & Scheme 4-1.

Na3PO4 buffer concentrations of 50, 100, and 500 mM were tested and compared. The resultant

minerals were examined using SEM and compared to Ca-Phosphate, which have a flower-like

appearance caused by scaffolding of phosphate ions (Figure 1-7). The minerals were analyzed to

determine Ca-Phosphate morphology.

5 𝐶𝑎𝐶𝑂3 (𝑠) + 3 𝑃𝑂43− + 𝑂𝐻− ⇌ 𝐶𝑎3(𝑃𝑂4)3𝑂𝐻 (𝑠) + 5 𝐶𝑂3

2− (𝑬𝒒. 𝟒 − 𝟏)

Scheme 4-1. Depiction of Calcium Phosphate Formation Through Buffer Washing. The same process of ACC

mineralization is followed, with an additional step of washing with different concentrations of sodium phosphate

buffer solutions to displace carbonate ions with phosphate ions in the hope of creating hydroxyapatite.

After 1 hour, 6 hours, and 12 hours in each buffer concentration, the resulting minerals showed

a cumulative increase in flowering, which indicates increased phosphate presence in minerals

(Figure 4-1, 4-2, 4-3, respectively). The results were also compared using ATR-FTIR, and show

a diminution in the out-of-plane bending mode of carbonate in the 1500-1300 cm-1 region (orange)

47

but an intensification in the asymmetric P-O stretch of phosphate in the 1050-1000 cm-1 region

(purple). This change is indicative of increased phosphate in sample, with decreased carbonate.

Figure 4-1. Analysis of 50 mM Sodium Phosphate Buffer Wash of prepared ACC Minerals. SEM images (TLD

detector) of converted minerals. Scale bar 5 μm for each. Along with ATR-FTIR spectra of each timed trial compared

to as prepared ACC. Peaks are normalized by PO4 v4a band 602 cm-1.The characteristic peak shape and position of the

v3 (~1390 cm-1) modes of CO32- peak intensity was compared with the characteristic peak assigned to the v3 (~1020

cm-1) stretching vibration band of PO43- peak for each trial.14,38,39 The change in peak intensity shows an increase for

the PO43- with a decrease for CO3

2-.

Figure 4-2. Analysis of 100 mM Sodium Phosphate Buffer Wash of Prepared ACC Minerals. SEM images (TLD

detector) of supposed Ca Phosphate minerals after each timed wash compared to as prepared ACC minerals. Scale bar

48

5 μm for each. Along with ATR-FTIR spectra of timed trials (one hour, six hour, and 12 hour) compared to standard

ACC minerals. Peaks are normalized by PO4 v4a band 602 cm-1.The characteristic peak position of the v3 (~1390 cm-

1) mode of CO32- peak intensity was compared with the characteristic peak of the v3 (~1020 cm-1) stretching vibration

band of PO43- peak for each trial.14,38,39 As the minerals were washed for increased amounts of time, one hour, six

hours, and 12 hours, the PO43- peak became more defined.

Figure 4-3. Analysis of 500 mM Sodium Phosphate Buffer Wash of Prepared ACC Minerals. SEM images (TLD

detector) of supposed Ca Phosphate minerals after each timed wash compared to normal ACC minerals. Scale bar 5

μm for each. Peaks are normalized by PO4 v4a band 602 cm-1.Along with ATR-FTIR spectra of timed trials (one hour,

six hour, and 12 hour) compared to standard ACC minerals to show peak intensity change. Again, the characteristic

peak position of the v3 (~1390 cm-1) mode of CO32- peak intensity was compared with the characteristic peak of the v3

(~1020 cm-1) stretching vibration band of PO43- for each trial.14,38,39 As the minerals were washed for increased

amounts of time (1,6,12 hours) the phosphate peak has increased in intensity as well as becoming stronger.

4.3 Instrumentational Analysis

The longer wash times of as prepared ACC minerals with increased concentrations of sodium

phosphate buffer showed increased evidence of phosphate conversion in both SEM and IR. These

observations are further supported by Raman, ATR-FTIR, and XRD analysis. In corresponding

literature, the process of converting CaCO3 to Ca-Phosphate was done using all four forms of

CaCO3 with sodium phosphate buffers (50 mM) at different time intervals for use in gene

49

expression.7 The results indicated that a conversion did take place for all forms of CaCO3. To

further quantify the ability of this phenomenon, more concentrated sodium phosphate buffers were

used to convert as prepared ACC minerals into Ca-Phosphate and analyzed. The mineral

conversion for increased concentrated Na3PO4 gave faster conversion than a lower concentration.

4.3.1 Raman Spectroscopy

The presence of phosphate in a system can be seen from the presence of active peaks at 435 cm-1

(O-P-O bending mode, v2), 590 cm-1 (O-P-O bending mode, v4), and 1075 cm-1 (P-O asymmetric

stretch, v3). However, the asymmetric stretch peak of PO43- is in the symmetric stretch peak region

(1060-1080 cm-1) of CO32- used for analysis. Because of this overlap, the peak found at ~1070-

1080 cm-1 is not identifiable as phosphate or carbonate. A peak in the 950-990 cm-1 range can be

indicative of several forms of Ca-Phosphate, HA, Ca3(PO4)2, amorphous calcium phosphate

(ACP), octacalcium phosphate (OCP), and dicalcium phosphate dihydrate (DCDP).40 However,

the exact location of this peak (relatively between 950-960 cm-1) provided inconclusive data for

the Ca-Phosphate formed. Any peaks past about 1550 cm-1 are not considered in literature for Ca-

Phosphate or CaCO3 spectra. Figure 4-4 shows the comparison of individual mineral particles at

specific wash times. The results show an increase in the v1 phosphate peak intensity as

concentration and time increase. The relative decrease of the v1 carbonate peak (excluding the first

mineral sample of 6hr buffer wash) is indicative of a decrease in the carbonate present in the

solution, which was expected.

50

Figure 4-4. Raman spectrum of 500 mM Sodium Phosphate Buffer Washed Amorphous Calcium Carbonate.

Prepared ACC minerals washed for 1 hour, 4 individual minerals analyzed; washed for 6 hours, 5 individual minerals

analyzed; ACC minerals washed for 12 hours, 5 individual minerals analyzed. Calcium phosphate minerals (v1

phosphate peak) are indicated by a strong band ~960 cm-1 that differs depending on the mineral standard created,

HAP, OCP, ACP, DCPD.39,40 Carbonate apatites (v1 carbonate peak) have peaks between 1060-1080 cm-1.41,42

51

Comparison of intensities between the carbonate apatite peak and the calcium phosphate peak for each wash time was

conducted.

4.3.2 ATR-FTIR Spectroscopy

Comparison of the twelve hour washes in all sodium phosphate concentrations gave the best

overall conclusion for the conversion of as prepared ACC. In previous works, a peak at ~1050 cm-

1 indicated the presences of ACC, while a peak at 1390 cm-1 indicated asymmetric stretching (v3)

mode of a CaCO3 for as prepared ACC.14 Figure 4-5 shows the ATR-FTIR spectra of as prepared

ACC minerals compared to different buffer samples washed for 12 hours. For presence of

converted ACC minerals, a decreased peak intensity of carbonate peaks was expected, with an

appearance of phosphate peaks or sharpening of phosphate peak regions. The relative peak

sharpness, position, and intensity were observed for conversion to Ca-Phosphate.

For ATR-FTIR, a doublet band at 1405/1450 cm-1 indicates a v3 mode and a peak between

870-890 cm-1 indicates out-of-plane bending of carbonate.7,19,25,39,43 Phosphate peaks can be found

at 460-470 cm-1 (v2), 952 cm-1 (v1), and 1000-1050 cm-1 (v3) with a peak range between 500-600

cm-1 (v4).7,25,37-39,43 The presence of a sharp peak at 602 cm-1, indicating a triply degenerate (v4a)

bending mode of phosphate, is a strong indicator of presence of Ca-Phosphate. Pairing this peak

with two other sharp peaks in the v4 phosphate region would indicate HA. However, the bending

mode region of phosphate, including the 602 cm-1 indicating peak, is overlaid with the translational

mode of hydroxide which was unavoidable in samples.

52

Figure 4-5. ATR-FTIR spectrum of prepared ACC minerals washed for 12 hours in physiological pH buffers.

Depiction of decrease in CO32- peak intensity (~1350 cm-1) and increase of PO4

3- peak intensity (~1000 cm-1) with

increased concentration of phosphate present in buffer, as well as sharpening of OCP/Nanocrystalline apatite/HAP

peak region (400-700 cm-1). Peaks are normalized by PO4 v4a band 602 cm-1.

While the peak positions are all relative to the phosphate regions, the exact Ca-Phosphate

morphology still needs to be determined. Figure 4-6 specifies the peaks for six different Ca-

Phosphate morphologies common in literature.37 From the peak positions of the phosphate treated

samples, the conversion to monetite, brushite, and amorphous calcium phosphate (ACP) can be

counted out. The relative peak positions and intensities of the v3 and v4 modes are indicative of

nanocrystalline apatite more so than stoichiometric HA.

53

Figure 4-6. FTIR spectra of calcium phosphate forms. Comparison of OCP, nanocrystalline apatite, HA, and ACP are

used against my samples. Reprinted with permission by Creative Commons Attribution License: from reference 37.

4.4.3 XRD Analysis

The conversion of ACC into other mineral forms was further investigated using XRD. ACC

naturally lacks a crystal structure and therefore no XRD pattern is observed. After 12 hour washes

in different buffers, the conversion of ACC into crystalline particles can be observed (Figure 4-

7).

Based on thermodynamics and kinetics, the conversion of ACC into aragonite or vaterite

followed by the transformation to calcite is the natural process that occurs.7,37-39 However, the as

prepared ACC minerals have been found to be stable for up to 12 months without any further

treatment.21 With the introduction of phosphate buffer washes, the formation of other CaCO3

morphologies is possible, but with comparison of XRD of calcite, aragonite, and vaterite to the

washed samples, conversion was to a different material. From the XRD spectra, the only

conversion looks to take place for the 500 mM sodium phosphate wash even though IR showed

54

some conversion with 50 mM sodium phosphate. Compared to HA, the peaks for 500 mM Na3PO4

are broad and undefined. In comparison to other forms of Ca-phosphate, Figure 4-8, the broadness

of the peak around 20⁰ 2θ CuKα1 wavelength and the peak range between 30-35⁰ 2θ CuKα1

wavelength of the 500 mM sodium phosphate wash indicates nanocrystalline apatite.37

Figure 4-7. Effect of PO4 Washing on XRD of Prepared ACC Samples. XRD analysis of amorphous calcium

carbonate after being washed in different physiological pH buffers for twelve hours compared to calcium carbonate

forms: calcite, aragonite, and vaterite {Jade Software for XRD material data 98-000-0141, 98-000-0098, 98-000-0451,

respectively}, and calcium phosphate form: hydroxyapatite, {Jade Software for XRD materials data 98-000-0251}.

Washing the buffer in Tris buffer had no effect on the amorphic nature of the ACC minerals. The area from 10-30⁰

55

Theta for the three buffer washes was caused by small sample size compared to sample prep requirements. Increase

in background signal at about 50⁰ is from sample holder.

Figure 4-8. XRD spectra of calcium phosphate forms. Peaks relative to CuKα1 wavelength used for comparison of

OCP, nanocrystalline apatite, HA, and ACP. Reprinted with permission by Creative Commons Attribution License:

from reference 37.

4.4 Conclusion

My data shows evidence of ACC conversion to Ca-Phosphate with exposure to phosphate

buffers. One analytical technique is not sufficient to determine the exact Ca-Phosphate

morphology. The qualitative comparison of as prepared ACC minerals to converted particles

showed flowering of the mineral, indicating scaffolding caused by displacement of carbonate with

phosphate. Spectroscopic analysis of the same minerals proved that the converted particle did

contain phosphate with lesser amounts of carbonate present. Through the use of analytical

techniques for quantitative and qualitative analysis we can reasonably conclude that the conversion

of as prepared ACC in sodium phosphate buffer produces nanocrystalline apatite.

56

Chapter 5

Conclusions and Future Directions

5.1 Conclusion

In this thesis, I defined biomimetic mineralization through the involvement of artificial

mineralizing vesicles that included microcompartments for mineral formation. An aqueous three

phase system made up of a Dx:PEG ATPS with a PAA-rich coacervate was utilized. Incorporation

of LUVs allowed for microcompartments and stability of the system. Compartmentalization of

enzyme into the Dx-rich phase by partitioning coefficients permitted localized enzyme activity.

Amorphous calcium carbonate was precipitated in the coacervate after hydrolyzed enzymes

reacted with Ca2+ within the PAA-rich coacervate. Finally, the produced ACC was allowed to react

with phosphate buffer to produce a polymorph of calcium phosphate.

Chapter 2 introduced the methods of compartmentalization, conversion, and analysis used

throughout the paper. A standard ACC mineral was formed from an AMV comprised of a 1:49

Dx:PEG ATPS with a 1.500 mg/mL LUV concentration at the interface, holding an interior

coacervate made of 10.5 mg/mL PAA chelated with 50 mM Ca2+ from stock solution. The

hydrolysis of excess urea (100mM) via urease (1.5 mg/mL) produced carbonate that interacted

with the calcium in the coacervate.

Chapter 3 focused on optimization of the system through order of addition, concentration

change of stabilizer, and ATPS volume ratio changes. The order of addition of the system was

concluded to have no effect on AMV homogeneity or localized coacervate formation. Changing

the concentration of stabilizer of the ATPS, the LUV concentration, with a coacervate present in

the system, showed that an increase in concentration resulted in decreased AMV, coacervate, and

mineral volumes, while a decrease in concentration showed the opposite. Changing the relative

57

volume ratios of the Dx:PEG ATPS indicated larger AMV and coacervate volumes with smaller

minerals as the Vd:Vp went from 1:32.2 to 1:99. This work showed the dependence of final mineral

volume on the concentration of LUVs for ATPS stabilization and volume ratio of the respected

ATPS.

Chapter 4 focused on the conversion of as prepared ACC minerals in the standard production

to morphologies of calcium phosphate. Phosphate buffer washes were used to displace carbonate

ions with phosphate ions in the inorganic phase of the mineral. The first indication of conversion

was through the scaffolding of the minerals, or flower like appearance, that is common in Ca-

Phosphate polymorphs. The resulting minerals were analyzed through ATR-FTIR, Raman, and

XRD and are indicative of a nanocrystalline apatite morphology.

The work in this thesis was the first to show the dependency on local concentrations for mineral

formation through a coacervate-containing AMV as well as the conversion of as prepared ACC

minerals to calcium phosphate. In conclusion, this research gives the background for

compartmentalization as a way of producing minerals as well as inducing mineral conversion

through CaCO3 biomineralization.

5.2 Future Direction

This work successfully showed the understanding of ACC mineralization through

compartmentalization with an A3PS stabilized with LUVs. However, the homogeneity of the

resulting minerals has not been accomplished, and more work on finding the concentrations and

volume ratios needed for optimization of the system is needed. Changing the values of the LUVs

present in the system or the relative Dx:PEG ATPS volume ratios by different amounts as well as

getting a better understanding of the effects of the coacervate will allow for understanding the

58

relationship between the systems localized concentrations and the homogeneity of the resulting

mineral size.

The conversion of ACC minerals into a Ca-Phosphate polymorph through phosphate buffer

washes was successfully provided in this thesis. The exact morphology of the resulting mineral

still needs to be understood with larger quantities of minerals produced for better quantitative

analysis results. The conversion of mineral through phosphate buffer is not the only path for Ca-

Phosphate production, so other forms of mineralization should be looked at.27,28,44 The formation

of calcium phosphate has been shown through the use of alkaline phosphatase.27,28,44 Preliminary

studies with the introduction of alkaline phosphatase as the enzyme in the A3PS produced a

mineral that did not take on the form of calcium carbonate, but has not been quantitatively

analyzed. Through this experimentation, it is expected that Ca-Phosphate would form in the

polymorph of amorphous calcium phosphate (ACP), in coacervate-containing AMVs exposed to

alkaline phosphatase.

59

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