Keratin based nanofibresnanofibres by by electrospinning ... · Coffee-Talk, 4 th February 2014 Keratin based nanofibresnanofibres by by electrospinning: fabrication and applications

Coffee-Talk, 4th February 2014

KeratinKeratin basedbased nanofibresnanofibres byby

electrospinningelectrospinning: :

fabricationfabrication and and applicationsapplications

[email protected]

fabricationfabrication and and applicationsapplications

Annalisa Annalisa AluigiAluigi

High Textile Technology laboratory (LATT):High Textile Technology laboratory (LATT):

NanofibresSurface

modification and

polymer deposition

Low Temperature Plasma Electrospinning

BIELLA

INTRODUCTIONINTRODUCTION OFOF MYSELFMYSELF

Research activities:

� Design of new polymeric fibres;

� Set-up of new textile processes;

� Study of handle, wear properties and comfort of textiles;

� Development of analytical methods for the quality assessment of fibres and fabrics.

Section of Biella Wet and melt spinning

apparatus

New polymer-based

microfibres

My main research topic:Processing of biopolymers Processing of biopolymers

from textile materialsfrom textile materials

a non food protein at high sulphur content

Wool

Feather

KERATINKERATIN

KERATINKERATIN

Feather

Horns & Nails

Hair and stratum corneum of the skin

Keratin is distinguished from other structural proteins (fibroin

or collagen) by the number of cysteinecysteine aminoamino acidacid residuesresidues in

the protein molecules (7-20% of the total amino acid residues)

KERATINKERATIN

The disulphide covalent bonds confer to keratin a high resistance

to chemical and enzymatic attaks

KERATINKERATIN WASTESWASTES a renewable source of biopolymers

Millions of tons/year Millions of tons/year of keratin wastes of keratin wastes fromfrom

1. Poor quality raw wool not fit for spinning

1

2

Moreira et al., Mycopathologia (2007) 163, 153-160

KERATINKERATIN

2. Hair and feathers from butchery

3. By-products of the textile industry

2

3

Disposal of keratin wates � environmentalenvironmental problemproblem!!!!

KERATINKERATIN WASTESWASTES Industrial applications

KERATINKERATIN

FireFire FightingFighting FoamsFoamsAnimalAnimal FeedstuffFeedstuff

HydrolyzedHydrolyzed KeratinKeratin

PowderPowder

CosmeticsCosmetics

FertilizerFertilizer

PowderPowder

Intermediate Intermediate filaments filaments

o Low-sulphur content

keratin (2-3 %wt) (LSLS)

o MW: 60, 45 kDa

o α-helix structure

WOOLWOOL

• Keratins (84%)

WOOLWOOL

CuticleCuticle

o High-sulphur content

keratin (8% wt) (HSHS)

o MW: 28-11 kDa

o Disordered structure s/βsheet

MatrixMatrix

o High-sulphur content

keratin (8% wt) (HSHS)

o MW: 28-11 kDa

o Disordered structure

• Other proteins,

lipids (16%)

CO

HC

NH

CO

CO

CH

NH

CO

CH2

CH2

NH2

� Reducing agents

� Thioglycolic acid

� DTT (dithiothreitol)

� 2-Mercaptoethanol

� Oxidizing agents

� Peracetic acid

� Performic acid

KERATINKERATIN EXTRACTIONEXTRACTION

WOOLWOOL

CO

HC

NH

CO

HC

NH

CO

CH

NH

CO

HC

NH

C=O

NH2

CH2HO

CH2S S CH2

� Performic acid

� Sulphitolysis (SO32-, HSO3

-, S2O52-)

Protein denaturing agent: UREA

WoolWool--SS--SS--Wool + SOWool + SO3322-- �� WoolWool--SS-- + Wool+ Wool--SS--SOSO33

--

CysteineCysteine--SS--sulphonatesulphonateCysteineCysteine

Freeze-dried Keratin

Molecular weight distribution

Isoelectric Point

Amino acid compositionAmino acid Mol (%)

CYA 0.36

ASP 8.31

SER 8.66

GLU 14.50

GLY 7.71

HIS 0.78

ARG 6.26

(pI) = 4-4.5Hydrophilic

Amino Acids

WOOLWOOL KERATINKERATIN PROPERTIESPROPERTIES

WOOLWOOL KERATINKERATIN

THR 5.36

ALA 5.26

PRO 6.71

LANT 1.02

CYS + CYS-SO3- 8.45

TYR 3.41

VAL 6.30

MET 0.46

LYS 3.54

ILE 3.37

LEU 7.44

PHE 2.07

Low sulphur

content keratin

High sulphur

content keratin

Hydrophobic

Amino Acids

C. Tonin, A. Aluigi, C. Vineis, A. Varesano, A. Montarsolo and F. Ferrero,

Journal of Thermal Analysis and Calorimetry, 89 (2007) 601-608

Amino acid composition

Amino acid Mol (%)

CYA 0.36

ASP 8.31

SER 8.66

GLU 14.50

GLY 7.71

HIS 0.78

AdsorbentAdsorbent forfor heavyheavy--metalsmetals or or

cationiccationic dyesdyes

KERATINKERATIN FORFOR WASTEWATERWASTEWATER TREATMENT AND AIR TREATMENT AND AIR CLEANINGCLEANING

Functional groups able to bind cations

KeratinKeratin can can fixfix formaldehydeformaldehyde

APPLICATIONSAPPLICATIONS

HIS 0.78

ARG 6.26

THR 5.36

ALA 5.26

PRO 6.71

LANT 1.02

CYS + CYS-SO3- 8.45

TYR 3.41

VAL 6.30

MET 0.46

LYS 3.54

ILE 3.37

LEU 7.44

PHE 2.07

Wool-S-SO3H

� A. Aluigi , C. Tonetti, C. Vineis, C. Tonin, R. Casasola, F. Ferrero, Journal of Biobased Material and

Bioenergy, 6 (2012) 230-236.

�A.Aluigi, A. Corbellini, F. Rombaldoni and G. Mazzuchetti, Textile Research Journal, 83 (2013) 1574-1586

KeratinKeratin can can fixfix formaldehydeformaldehyde

Microparticles

Concentration-dependent and time-dependent effect of RK-based microparticles on LDH release by THP-1 monocytic

cell line

WOOLWOOL KERATINKERATIN FORFOR BIOMEDICALBIOMEDICAL APPLICATIONSAPPLICATIONS

�� Biodegradable (in vitro and in vivo) (Yamouchi K. et al., J. Biomed. Mater. Res. 1996, 31, 439-44)

� Fibroblast and osteoblast cell growth (Yamouchi K.; Mniwa M.; Mori T., J. Biomater. Sci. Polym. Edn. 1998, 9, 259-70)

Cytotoxicity test


Microparticles

Spry-drying process

F. Cilurzo, F. Selmin, A. Aluigi, S. Bellosta, Polym. Adv. Technol. 24 (2013) 1025-1028

Wool

Scaffold sponges

Keratin/Ceramides Film Biodegradable Vessel

Keratin powder

K. Katoh et al. Biomaterials 25 (2004) 4255–4262

Nanofibrous Membrane

KERATINKERATIN PROCESSINGPROCESSINGF. Selmin, F. Cilurzo, A. Aluigi, S. Franzé, P.

Minghetti, Results in Pharma Science 2 (2012)

72-78C. Vineis, A. Aluigi, A. Varesano, C. Tonetti, G.

Mazzuchetti, Alimenta XXI 3 2013

Hydrogels

Microspheres

Nanofibrous Membrane

A. Aluigi et al. Journal of

Biobased Material and

Bioenergy, 6 (2012) 230-236

F. Cilurzo, F. Selmin, A. Aluigi, S. Bellosta,

Polym. Adv. Technol. 24 (2013) 1025-1028

Research project funded by Pfizer International Operations

NANOFIBROUSNANOFIBROUS MEMBRANESMEMBRANES

� very high surface to volume ratio

� highly porous, interstitional pore size within nano range

� design flexibility for chemical/physical functionalization

� unique physical, chemical, electrical, optical properties

Nanofibers

NANOFIBRESNANOFIBRES

Nanofibers

PP microfibres (5 micron) and

electrospun nylon nanofibers

(200 nm)

Wound Dressing• prevent scars

• bacterial shieldingTissue Engineering Scaffolds• adjustable biodegradation rate

• better cell attachement

• controllable cell directional growth

Drug delivery• increased dissolution rate

• drug nanofibre interface

Hemostatic Devices• higher effiiciency in fluid absorption

Electrical conductors• ultra small devices

NANOFIBRESNANOFIBRES

Cosmetics• higher transfer rate Filter media

• higher efficiency filtration

Protective Clothing• chemical barrier

• lightness

• comfort (transpiration properties

Sensor devices• higher sensitivity

Technical Textiles• acustic insulation

• thermal insulation

NANOFIBRENANOFIBREAPPLICATIONSAPPLICATIONS

KERATINKERATIN NANOFIBRESNANOFIBRES: : WHYWHY??

KeratinKeratin PropertiesProperties

� Biodegradable (in vitro and in vivo) [1]

� Fibroblast and osteoblast cell growth [2]

� Heavy-metals and formaldeyde absorption [3]

NanofibrousNanofibrous Membrane Membrane PropertiesProperties

� High surface to volume ratio

CNR-ISMAC (Institute for Macromolecular Studies) BIELLA

� High surface to volume ratio

� High porosity

� High surface functionality

[1] Yamouchi K.; Yamauchi A.; Kusunoki T.; Kohda A.; Konishi Y., J. Biomed. Mater. Res. 1996, 31, 439-44.

[2] Yamouchi K.; Mniwa M.; Mori T., J. Biomater. Sci. Polym. Edn. 1998, 9, 259-70.

[3] A. Aluigi, C. Vineis, C. Tonin, C. Tonetti, A. Varesano,, Journal of Biobased Materials and Bioenergy, 2009,3(3), 311–319

BiomedicalBiomedical ApplicationsApplications

�� Scaffolds for cell growth

� Medical Textiles

�� Drug delivery systems

FiltrationFiltration SystemsSystems

�Air Filters

�Water depuration systems from

heavy-metals or cationic dyes

NANOFIBERSNANOFIBERS BYBY ELECTROSPINNINGELECTROSPINNING PROCESSPROCESS

ELECTROSPINNINGELECTROSPINNING

• Polymer solution

• Polymer melt

BasicBasic setupsetup forfor anan electrospinningelectrospinning apparatusapparatus

High voltage supplier [10 ÷ 30 KV DC or AC]

Syringe with a small diameter needle [0.2 ÷ 1.5 mm]

Collecting screen• Polymer melt

ElectrospinningElectrospinning ProcessProcessSurface tension

Electrostatic force

Taylor cone

Whipping

Nanofibers

CONTROLCONTROL OFOF THE THE NANOFIBRENANOFIBRE MORPHOLOGYMORPHOLOGY AND AND STRUCTURESTRUCTURE

Intrinsic properties of the polymer fluid

• Viscosity

• Conductivity

• Surface tension

Operational parameters

ELECTROSPINNINGELECTROSPINNING

Operational parameters

• Applied voltage

• Tip to collector distance

• Fluid flow rate

Environmental conditions

• Temperature

• Humidity

KERATINKERATIN--BASEDBASED NANOFIBRENANOFIBRE MEMBRANESMEMBRANES

PolymerPolymer SolventSolvent ApplicationsApplications

Keratin/PEO [1-2] WaterTissue Engineering

Air filters

Keratin/Fibroin [3] Formic Acid

Tissue Engineering

Water depuration

Air filters

KERATINKERATIN NANOFIBRESNANOFIBRES

Electronic

Air filters

Keratin/PA 6 [4] Formic AcidWater depuration

Air filters

Keratin [5] Formic Acid

Tissue Engineering

Water depuration

Air filters [1] Aluigi, A., Varesano, A., Montarsolo, A., Vineis, C., Ferrero, F., Mazzuchetti, G., Tonin, C., Journal of Applied Polymer Science 104 (2007),

863-870

[2] Aluigi, A., Vineis, C., Varesano, A., Mazzuchetti, G., Ferrero, F., Tonin, C. , European Polymer Journal 44 (2008), 2465-2475

[3] Zoccola, M., Aluigi, A., Vineis, C., Tonin, C., Ferrero, F., Piacentino, M.G., Biomacromolecules 9 (2008), 2819-2825

[4] Aluigi, A., Vineis, C., Tonin, C., Tonetti, C., Varesano, A., Mazzuchetti, G., Journal of Biobased Materials and Bioenergy 3 (2009), 311-319

[5] Aluigi, A., Corbellini, A., Rombaldoni, F., Zoccola, M., Canetti, M., International Journal of Biological Macromolecules 57 (2013), 30-37

KERATINKERATIN//FIBROINFIBROIN BLENDBLEND NANOFIBRESNANOFIBRES

BombixBombix Mori Mori SilkSilk

Fibroin (75% wt)

(∼300) kDa


WoolWool

Keratin (84% wt)

(60-18) kDa

β-sheetα-helix

Electronic

AppliedApplied VoltageVoltage ((kVkV)) 30

Flow Rate (ml/min)Flow Rate (ml/min) 0.005

Work Work DistanceDistance (cm)(cm) 10

CapillaryCapillary DiameterDiameter (mm)(mm) 0.2

DepositionDeposition TimeTime (min)(min) 20

ElectrospinningElectrospinning ConditionsConditions

KeratinKeratin in Formic Acid

15% wt

FibroinFibroin in Formic Acid

15% wt

100100 0

9090 10

7070 30

5050 50

3030 70

1010 90

00 100

BlendBlend SolutionsSolutions

β-sheetα-helix

KERATINKERATIN//FIBROINFIBROIN BLENDBLEND SOLUTIONSSOLUTIONS

Additivity Rule

∑=i

iiT lnwln ηη

component i ofviscosity solution

itycosvis ltheoreticacomponent i the of fraction weightw

thi

T

thi

=

==

ηη

ViscosityViscosityEnhanced

interactions

between the

two proteins


Electronic

� experimental data; - theoretical viscosity

ConductivityConductivity

MORPHOLOGYMORPHOLOGY OFOF KERATINKERATIN//FIBROINFIBROIN BLENDBLEND NANOFIBRESNANOFIBRES


Electronic

Keratin/Fibroin 0/100Diameter: 945±483 nm

Keratin/Fibroin 10/90Diameter: 518 ±205 nm


Keratin/Fibroin 50/50Diameter: 207 ±66




PURE PURE KERATINKERATIN NANOFIBRESNANOFIBRES

“Bottom-up” configuration of

electrospinning apparatus


A. Aluigi, A. Corbellini, F. Rombaldoni, G. Mazzuchetti, Textile

PARAMETERS VALUES

Solute Keratin powder

Solvent Formic Acid (98%)

Protein concentration 15% wt

Viscosity 0.18 ± 0.01 Pa s

Conducibility 2.1 mS/cm

PARAMETERS VALUES

Applied voltage 25 kV

Flow - rate 3 µl/min

Tip to target distance 20 cm

Deposition time 60 min

Temperature 21-25 °C

Relative Humidity 45-50%

SolutionSolution PropertiesProperties ElectrospinningElectrospinning ConditionsConditions

A. Aluigi, A. Corbellini, F. Rombaldoni, G. Mazzuchetti, Textile

Research Journal 83 (2013) 1574-1586

PROPERTIESPROPERTIES OFOF KERATINKERATIN NANOFIBRENANOFIBRE MEMBRANESMEMBRANES

FlexibileFlexibile!!!!


PROPERTIES VALUES

Fibre diameter (nm) 223±74

Thickness (µm) 50

Porosity (%) 90

Specific surface area (m2/g) 13.59

KERATINKERATIN NANOFIBRENANOFIBRE MEMBRANESMEMBRANES ASAS ADSORBENTSADSORBENTS FOR…FOR…

CationicCationic DyesDyes [2][2]

((MethyleneMethylene BlueBlue))

HeavyHeavy--metalsmetals [3][3]


FormaldehydeFormaldehyde [1][1]

HeavyHeavy--metalsmetals [3][3]

(Cu(Cu2+2+))

AdsorptionAdsorption FiltrationFiltration

ACTIVE FILTER MEDIA

[1] A. Aluigi, C. Vineis, C. Tonin, C. Tonetti, A. Varesano, G. Mazzuchetti, J. Biobased Mater Bio 3(2009) 311-319

[2] A. Aluigi, F. Rombaldoni, C. Tonetti, L. Jannoke, J. Hazard. Mater. 268 (2014) 156-165

[3] A. Aluigi. A. Corbellini, F. Rombaldoni, G. Mazzuchetti, Text. Res. J. 83 (2013) 1574-1586

FORMALDEHYDEFORMALDEHYDE ADSORPTIONADSORPTION


Decrease of formaldehyde concentration with an initial concentration of 0.6

ppm (100%) during time in the presence of filters

Tests performed at 20°C and 65% r.h.

A. Aluigi , C. Vineis, C. Tonin, et al., Journal of Biobased Material and Bioenergy, 3 (2009) 311-319.

METHYLENEMETHYLENE BLUEBLUE AND AND Cu(Cu(IIII)) ADSORPTIONADSORPTION

Properties Keratin

Nanofibres

Wool

Fabric

Fibre diameter (nm) 223 ± 74 19 µm

Thickness (µm) 50 500

Porosity (%) 90 69.7

Specific surface area (m2/g

1) 13.59 0.16

MethyleneMethylene BlueBlue

Adsorption CapacityAdsorption Capacity

adsorptionafter solutionstock theinamount )(/

adsorption before solutionstock theinamount )(/

mg/g)

1

0

10

IICuMBq

IICuMBq

m

qqq(

=

=

−=

Cu(II)Cu(II)


The higher adsorption capacities of keratin nanofibrous memebranes compared to wool fabrics are due to their

higher porosity and higher surface area per unit mass.

MethyleneMethylene BlueBlue Cu(II)Cu(II)

COMPARISONCOMPARISON WITHWITH OTHEROTHER NANOSTRUCTUREDNANOSTRUCTURED MEMBRANESMEMBRANES

Membrane C0(mg/L)Adsorption Capacity

(mg/g)References

Keratin nanofibre membrane 50 11.3 A. Aluigi et al. Text. Research J. 83 (2013) 1574-1586

Keratin nanofibre membrane 4 5.8 A. Aluigi et al. Text. Research J. 83 (2013) 1574-1586

Cu(II) adsorption


nanofibre membrane 4 5.8 A. Aluigi et al. Text. Research J. 83 (2013) 1574-1586

Keratin/Fibroin nanofibre membrane 4 2.88 C.S. Ki et al., J. Mem. Sci. 302 (2007) 20-26.

Fibroin nanofibre membrane 4 1.65 C.S. Ki et al., J. Mem. Sci. 302 (2007) 20-26

Activated nylon-basedmembrane 50 10.8

Z.Y. He et al., Bioresour. Technol. 99 (2008) 7954-7958

CNR

CNR-ISMAC, Biella

• Alessio Varesano

• Claudia Vineis

• Fabio Rombaldoni

• Cinzia Tonetti

• Marina Zoccola

• Claudio Tonin

• Giorgio Mazzuchetti

• Alessandro Corbellini• Raffaella Casasola

• Ludovika Jannoke

• Prof. Franco Ferrero

[email protected]

• Giorgio Mazzuchetti

Documents

Keratin based nanofibresnanofibres by by electrospinning ... · Coffee-Talk, 4 th February 2014 Keratin based nanofibresnanofibres by by electrospinning: fabrication and applications