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PREPARATION OF NOVEL FUNCTIONALIZED
NANOFIBERS FROM HYDROPHILIC POLYMERS AND
THEIR APPLICATIONS
NARAHARI MAHANTA
NATIONAL UNIVERSITY OF SINGAPORE
2012
PREPARATION OF NOVEL FUNCTIONALIZED
NANOFIBERS FROM HYDROPHILIC POLYMERS AND
THEIR APPLICATIONS
NARAHARI MAHANTA
M. Sc., M. Phil., (Sambalpur University, India)
M. Tech., (Indian Institute of Technology, Kharagpur, India)
A THESIS SUBMITTED
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
DEPARTMENT OF CHEMISTRY
NATIONAL UNIVERSITY OF SINGAPORE
2012
Declaration
I hereby declare that this thesis is my original work and it has been written by me in its
entirety. I have duly acknowledged all the sources of information which have been
used in the thesis.
This thesis has also not been submitted for any degree in any university previously.
_______________
Narahari Mahanta
17 September 2012
Dedicated to
My beloved parents, wife and family members who were always there with
me a source of inspiration
Name: NARAHARI MAHANTA
Degree: DOCTOR OF PHILOSOPHY
Department: CHEMISTRY
Thesis title: PREPARATION OF NOVEL FUNCTIONALIZED
NANOFIBERS FROM HYDROPHILIC POLYMERS AND THEIR
APPLICATIONS
Abstract
In this investigation, preparation of hydrophilic nanostructured membranes derived from
poly (vinyl alcohol) (PVA) and cellulose based macromer was developed for
environmental detoxification. To obtain the best extraction efficiency, surface hydroxyl
groups of PVA nanofibers were modified with functional groups, such as thiols and
amines. Apart from that cellulosic nanofibers were isolated from sugarcane bagasse and
amine group was incorporated by coating the cellulose nanofiber with chitosan. Both
PVA and cellulose nanofibers showed high extraction efficiency (more than 90%)
towards silver and gold nanoparticles from water. Similarly, Fe (III) coordinated PVA
nanofiber was developed for the removal of Arsenic from water with very high
efficiency. Also, PVA nanofibers incorporated with protein and methacrylated
PVA/cellulose nanofibers with silver nanoparticles were prepared. Similar materials were
also compared with hydrogel systems. Current investigation provides a method to design
of novel nanofibrous membranes from hydrophilic polymers for setting up large scale
water purification system.
Keywords: Polyvinyl alcohol, Cellulose, Electrospinning, Nanofiber, Water filtration,
Biomedical application
i
ACKNOWLEDGEMENTS
First and foremost, my heartfelt gratitude goes to my supervisor, Assoc. Prof. Suresh
Valiyaveettil for teaching me the discipline of science, his guidance, support and
encouragement during the course of this work. He gave me a lot of space to try things and
many helpful suggestions when things just would not seem to work right. I must be very
thankful to him for his patience and helping me in my toughest time during research with
moral support.
Many people have contributed their time and effort in helping me to accomplish this
research. I sincerely thank all the current and former members of the group for their
cordiality and friendship. Special thanks to Dr. Siva, Dr. Satyananda, Dr. Balaji, Dr.
Ankur, Dr. Sajini, Dr. Asha Rani, Dr. Jinu Poul, Dr. Vinod, Dr. Manoj, Jinuk, Pradipto,
Tanay, Kiruba, Rama, Ashok, Chunyen, Daizy for all the good times in the lab and
helping exchange knowledge skills. Special thanks to Yiwei who helped with cell growth
studies during my experimentation.
I must acknowledge the technical assistance provided by Ms Tang and Madam Loy
during the SEM and TEM studies for a lot of valuable suggestions and letting me to use
her equipments.
The gratitude that is most difficult to express in words is towards my family. I whole
heartedly thank my parents, brother, sister, brother-in-law, parents-in-law, manisha,
varsha, subham, punam, pipi, and mimi for their support and encouragement. In
particular, I wish to express gratitude to my wife Mrs. Nikasha Mohanta for her patience,
love, care and understanding during my PhD career.
I thank the National University of Singapore for granting the research scholarship.
ii
Table of Contents
Acknowledgments i
Table of contents ii
Summary viii
List of Tables x
List of Figures xi
Abbreviations and Symbols xvii
Chapter 1: Introduction
1.1 Introduction to nanofibers 2
1.2 Synthetic nanofibers by electrospinning technique 2
1.2.1 Characterization of polymeric nanofibers 5
1.2.1.1 Geometrical characterization 5
1.2.1.2 Thermo-mechanical property analysis 6
1.3 Isolation of nanofiber from natural sources 7
1.3.1 Characterization of cellulose nanofibers 9
1.4 Preparation of hydrogel from hydrophlic polymers 10
1.4.1 Characterization of hydrogel 11
1.4.1.1 Physical and geometrical analysis 11
1.4.1.2 Thermo-mechanical properties of hydrogel 11
1.5 Properties of hydrophilic polymers 12
1.6 Applications and challenges in fabrication of PVA and cellulose nanofiber
membranes
16
1.6.1 Designing of biocompatible membranes for practical aspects 14
1.6.1.1 Nanofibrous hydrogel for tissue engineering application 15
1.6.1.2 Antimicrobial agents in wound healing 16
1.6.2 Environmental membranes and filters 17
1.6.2.1 Removal of heavy metal ions from water 18
1.6.2.2 Waterborne pathogen and dye pollutants 20
1.6.2.3 Removal of micro and nanoparticulates from water 21
iii
1.7 Scope and outline of the thesis 23
1.8 References 26
Chapter 2: Nanofibrous Poly (vinyl alcohol) Membranes for
Removal of Nanoparticles from Aqueous Environment
2.1 Introduction 44
2.2 Experimental section 45
2.2.1 Materials 45
2.2.2 Synthesis of citrate-stabilized gold and silver nanoparticles 46
2.2.3 Fabrication of PVA nanofibers by electrospinning technique 46
2.2.4 Incorporation of surface thiol and amine functional by surface 47
modification technique
2.2.5 Nanoparticle extraction study using the surface modified PVA
nanofibers
48
2.2.6 Desorption study of nanoaprticles from nanofiber 48
2.3 Characterization 49
2.4 Results and discussion 49
2.4.1 Morphological and spectroscopic analysis 49
2.4.2 Adsorption of nanoparticles by using designed nanofiber 52
2.5 Summary and Conclusions 58
2.6 References 59
Chapter 3: Isolation and Fabrication of Cellulose Based Nanofibers
for Filtration of Nanoparticles from Aqueous Environment
3.1 Introduction 63
3.2 Experimental section 64
3.2.1 Materials 64
3.2.2 Isolation of cellulose nanofibers from sugarcane bagasse 65
3.2.3 Preparation of chitosan coated sugarcane nanofibers 65
3.2.4 Preparation of PVP capped Au and Ag-nanoparticles 66
3.2.5 Study on adsorption behaviour of nanoparticle 66
using chitosan coated cellulose nanofibers
iv
3.2.6 Desorption study of nanoparticles from functionalized nanofibers 66
3.2.7 Test for filtration efficiency of nanoparticles by chitosan 67
coated cellulose nanofiber
3.3 Characterization 67
3.4 Results and discussion 67
3.4.1 Isolation of cellulose nanofibers from sugarcane bagasse 67
3.4.2 Preparation of sugarcane and chitosan coated sugarcane 69
nanofibrous membranes
3.4.3 Surface morphology of chitosan coated sugarcane nanofibers 70
3.4.4 Preparation of Au and Ag-nanoparticles 71
3.4.5 Extraction of nanoparticles from aqueous environment 72
using nanofibers
3.4.6 Desorption and time-dependent readsorption efficiency analysis 77
3.4.7 Filtration of nanoparticles by using chitosan coated cellulose nanofibers 79
3.5 Summary and Conclusions 80
3.6 References 81
Chapter 4: Fabrication of Fe (III) Immobilized PVA Nanofibers as a
Sorbent for Arsenic Removal from Water
4.1 Introduction 87
4.2 Experimental section 88
4.2.1 Materials 88
4.2.2 Fabrication of PVA/Fe (III) polymeric nanofibers by 89
electrospinning technique
4.2.3 Adsorption analysis of arsenic using designed nanofibers from
water
89
4.3 Characterization 90
4.4 Results and discussion 90
4.4.1 Morphological and spectroscopic analysis 90
4.4.2 Structure property analysis of the resultant nanofibers 93
4.4.3 Filtration efficiency of arsenic using designed nanofibers 94
v
4.5 Summary and Conclusions 99
4.6 References 100
Chapter 5: In situ Preparation of Silver Nanoparticles on
Biocompatible Methacrylated Poly (vinyl alcohol) and Cellulose
Based Polymeric Nanofibers
5.1 Introduction 105
5.2 Experimental section 106
5.2.1 Materials 106
5.2.2 Synthesis of methacrylated poly(vinyl alcohol) / dextran / methyl
cellulose
107
5.2.3 Test for reduction of silver salt 108
5.2.4 Fabrication of PVA/Cellulose nanofiber by electrospinning technique 108
5.2.5 Cytocompatibility analysis of methacrylated designed nanofiber 109
5.3 Characterization 110
5.4 Results and discussion 111
5.4.1 Test for reduction of silver salt by designed polymer 111
5.4.2 Designing of methacrylated macromer 112
5.4.3 Nanofibers of methacrylated macromer by electrospinning technique 113
5.4.4 X-ray diffraction analysis of nanofibers containing silver nanoparticle 115
5.4.5 Thermal analysis of designed macromer with Ag-nanoparticles 116
5.4.6 Mechanical properties of designed macromer with Ag-nanoparticles 117
5.4.7 Cytotoxic analysis on the designed methacrylated nanofiber 119
5.5 Summary and conclusions 120
5.6 References 121
Chapter 6: Nanofibers Derived from Poly (vinyl alcohol) and Milk
protein and its Biocompatibility Evaluation
6.1 Introduction 125
6.2 Experimental section 127
6.2.1 Materials 127
6.2.2 Fabrication of nanofibers by electrospinning techniques 127
vi
6.2.3 Cell viability and cell adhesion property analysis using human
fibroblast
128
6.3 Characterization 129
6.4 Results and discussion 130
6.4.1 Morphological analysis 130
6.4.2 Effect of additives on the physico-mechanical behaviour 132
6.4.3 XRD analysis of PVA composite nanofibers 135
6.4.4 Cell adhesion and viability study on designed electrospun 136
nanofibers membranes
6.5 Summary and conclusions 139
6.6 References 141
Chapter 7: Preparation of Viscoelastic Hydrogels from Poly (vinyl
alcohol)/Fe (III) Complex and its Cytocompatibility Evaluation
7.1 Introduction 146
7.2 Experimental section 147
7.2.1 Materials 147
7.2.2 Preparation of Fe3+
coordinated PVA hydrogels 148
7.2.3 Swelling analysis of hydrogels 148
7.2.4 SEM and spectroscopic analysis of hydrogels 149
7.2.5 Thermo-mechanical behavior analysis of hydrogels 149
7.2.6 Biocompatibility evaluation using human fibroblast 150
7.2.7 Cell adhesion study on the hydrogel surface 152
7.3 Results and discussion 152
7.3.1 Spectroscopic analysis of crosslinked hydrogel 152
7.3.2 Physical behavior analysis by swelling study 153
7.3.3 Mechanical property of hydrogel 154
7.3.3.1 Stress-Strain behaviour 155
7.3.3.2 Dynamic mechanical analysis 156
7.3.3.4 Creep behavior 158
7.3.4 Thermal analysis of crosslinked hydrogel 160
7.3.5 Cell viability and cell adhesion study 161
7.4
vii
7.5 7.4 Summary and Conclusions 163
7.6 7.5 References 165
Chapter 8: Conclusions and future studies
8.1 Conclusions
8.2 Future studies
169
172
List of Publications and Presentations 173
viii
SUMMARY
Recently there has been increasing interest for the fabrication of nanostructured
polymeric scaffolds by using various techniques. For this, electrospinning is a recently
advanced technique which provides polymer in to fiber form in to nanometer range
which used for many advanced applications. The aim of this thesis was to derive
nanofibers derived from hydrophilic polymers such as poly (vinyl alcohol) (PVA) and
cellulose based macromer with novel hybrid materials, their characterization and possible
application. Here a detailed summary of each chapter is included.
In chapter one, a brief review of literature related to fabrication, characterization
and applications of the electrospun nanofibers are given. In addition, a brief summary of
the preparation and their physical property study along with the scope and outline of the
present research work was discussed.
Chapter two describes the preparation of electrospun PVA nanofibers with diameters
ranging between 300 – 500 nm and were used for the extraction of nanosized contaminants
from aqueous environment. To obtain the best extraction efficiency, surface hydroxyl
groups of PVA nanofibers were chemically modified with functional groups, such as thiols
and amines. The extraction studies revealed that functionalized nanofibers have high
extraction efficiency for dissolved nanoparticles in water and can be used for removal of
the nanocontaminants from aqueous environment.
In chapter three, cellulosic nanofibers with 30 - 40 nm in diameter were extracted
from renewable source such as sugarcane bagasse. The nanofibers were coated with
chitosan to introduce additional functional groups onto the surface. The designed
ix
cellulosic nanofibers were used to filter metal nanoparticles from water which showed
high extraction efficiency (80 - 90%) towards silver and gold nanoparticles.
The development of nanofibrous membranes prepared by electrospinning of polymer
solutions and used for the filtration of arsenic ions from water elucidates in chapter four.
Our approach was to incorporate Fe3+
ions in a hydrophilic polymer matrix and used for
the preparation of nanofiber mats for arsenic removal from water with high efficiency.
We described the preparation of ultra-fine nanofiber mats of cellulosic polymer and
PVA containing silver nanoparticles were fabricated using electrospinning technique in
chapter five. In the present study, hydrophilic biocompatible polymers with coordination
sites for silver nanoparticles. The biocompatibility of the designed methacrylated
nanofiber was carried out by cell viability analysis using human lung fibroblast IMR-90.
Chapter six describes the fabrication of porous three dimensional nanofibrous
membranes from PVA, milk protein and inorganic salts such as calcium carbonate
(CaCO3) or magnesium carbonate (MgCO3). Physico-mechanical and cytocompatibility
property study demonstrated that the hybrid nanofiber may provide significant advantages
for such materials in biomedical applications.
Metal (Fe3+
) coordinated PVA based hydrogels were prepared as scaffolding material
from PVA macromer and cytocompatibility study was investigated in chapter seven.
Enhanced physico-mechanical property and non toxic nature of these hydrogels
demonstrated that these hydrogels may find practical application for biomedical
procedures which require highly viscoelastic and mechanically stable biomaterials.
Chapter eight summarize about the conclusion and future studies that can be carried
out using our functional nanofibers.
x
LIST OF TABLES
Table No. Title of the Table Page
No.
Chapter-2
Table 2.1 Table for summary of the results obtained on 3rd
trial of adsorption
for both Ag and Au nanoparticles
55
Chapter-3
Table 3.1 Elemental analysis of concentration of Au in hydrochloric acid
(A), and silver in nitric acid (B) after desorption and
corresponding desorption efficiency
77
Table 3.2 Summary of filtration of different nanoparticles solution after passing
through a column filled with chitosan coated nanofiber.
80
Chapter-4
Table 4.1 Elemental analysis of PVAFe uncrosslinked nanofiber and PVAFe
crosslinked nanofiber
91
Table 4.2 Parameters of Freundlich and Langmuir isotherms for As (III) and
As (V) sorption on the Fe3+
loaded PVA nanofiber.
98
Chapter-5
Table 5.1 Mechanical properties of PVA/CaCO3 composite nanofiber 118
Chapter-6
Table 6.1 Elemental analysis of milk incorporated PVA nanofiber, showing
the presence of nitrogen content due to the amino acids
132
Table 6.2 DSC analysis of PVA-milk protein/CaCO3 nanofiber and PVA-
milk protein/MgCO3 nanofiber
135
Chapter-7
Table 7.1 DSC analysis of lyophilised sample of hydrogels 161
xi
LIST OF FIGURES
Figure No. Title of the Figure Page
No.
Chapter 1
Figure 1.1 Schematic diagram of electrospinning 4
Figure 1.2 FESEM image of PVA nanofibers Setup 4
Figure 1.3 Repeat unit of cellulose 8
Figure 1.4 FESEM images of cellulose nanofibers 9
Figure 1.5 Water swollen network of cross-linked hydrogel 10
Figure 1.6 Statistics on the literature published on the advanced application of
nanofibers
14
Chapter 2
Figure 2.1 Reaction scheme of surface modification of PVA-NFs with A.
MPA (PVA-MPA); B. MSA (PVA-MSA); and C. Lysine (PVA-
LYS).
47
Figure 2.2 A. FESEM image of electrospunned PVA nanofibers and B. ATR-
IR of PVA NFs and PVA NFs surface modified with MSA, MPA
and Lysine.
50
Figure 2.3 Scanning electron micrograph images of the surface modified (A).
PVA-LYS NFs; (B). PVA-MSA NFs; (C).PVA-MPA NFs.
51
Figure 2.4 TEM images of (A) citrate capped Au NPs and (B) citrate capped
Ag NPs.
52
Figure 2.5 Time-dependent adsorption efficiencies of citrate capped-AgNPs
and AuNPs (2 10-4
M solutions) on different surface modified-
PVA nanofibers.
53
Figure 2.6 Representative UV-Vis spectra (A) of the Ag NP and (B) for the
Au NP original solution and after filtration and corresponding
photographs of the nanoparticle solutions before and after
filtration.
54
Figure 2.7 Second time adsorption efficiencies of citrate capped-AgNP and 54
xii
AuNP solutions (2 10-4
M) on same nanofibers as a function of
time.
Figure 2.8 FESEM images of A. AgNP adsorbed PVA-LYS NFs, B. AuNPs
adsorbed PVA-MSA NFs and C. AuNPs adsorbed on to PVA-
MPA NFs.
56
Figure 2.9 Energy dispersive spectra of A. AgNP adsorbed PVA-LYS NFs,
B. AuNPs adsorbed PVA-MSA NFs and C. AuNPs adsorbed on to
PVA-MPA NFs.
56
Figure 2.10 : EDS spectrum of silver nanoparticles desorption from PVA-
LYS NFs (A) and PVA-MSA NFs (B) by using 2N HCl.
57
Chapter 3
Figure 3.1 SEM image of cellulose nanofibers bleached with sodium
hypochloride and acetic acid (A) and with Bleaching:HCl (10:1
vol%) (B).
68
Figure 3.2 Attenuated total reflectance-Infrared spectra of sugarcane
nanofiber (A), chitosan (B) and chitosan coated sugarcane
nanofibers (C).
70
Figure 3.3 SEM images of chitosan coated cellulose nanofiber (A) and
crosslinked chitosan (B).
71
Figure 3.4 UV-visible spectra of PVP and citrate capped Au (A) and Ag-
nanoparticles (B); TEM images of PVP capped Au-nanoparticles
(C), citrate capped Au- nanoparticles (D), PVP capped Ag-
nanoparticles (E), and citrate capped Ag- nanoparticles (F). Inserts
show the corresponding camera images of nanoparticle solutions.
72
Figure 3.5 Time-dependent adsorption efficiency of chitosan-coated
sugarcane nanofibers, crosslinked sugarcane nanofibers and
crosslinked chitosan for PVP capped Au-nanoparticles (A), PVP
capped Ag-nanoparticles (B), citrate capped Au- nanoparticles (C)
and, citrate capped Ag-nanoparticles (D); UV-visible spectra of
solutions before and after adsorption for PVP capped Au-
nanoparticles (E), PVP capped Ag- nanoparticles (F), citrate
capped Au-nanoparticles (G), and citrate capped Ag- nanoparticles
(H). Inserts show the corresponding decolourization of the
nanoparticles solutions before and after adsorption.
73
xiii
Figure 3.6 Graphs for time-dependent readsorption efficiency of chitosan-
coated sugarcane nanofibers for subsequent adsorptions of PVP
capped Au-nanoparticles (A), citrate capped Au-nanoparticles (B),
PVP capped Ag-nanoparticles (C), and Citrate capped Ag-
nanoparticles (D).
75
Figure 3.7 SEM images of Au (A) and Ag-nanoparticles (B) adsorbed
nanofiber mat.
76
Figure 3.8 EDX spectra of chitosan-coated sugarcane nanofibers after
adsorption of PVP capped Au-nanoparticles (A), PVP capped Ag-
nanoparticles (B), citrate capped Au- nanoparticles (C) and, citrate
capped Ag-nanoparticles (D). Inserts showed the corresponding
elemental mappings of the nanoparticles.
76
Figure 3.9 Time-dependent adsorption efficiency of chitosan-coated
sugarcane nanofibers for first time adsorption, second time without
and with acid treated nanofiber using PVP capped Au-
nanoparticles (A), PVP capped Ag-nanoparticles (B), citrate
capped Au-nanoparticles (C), and citrate capped Ag-nanoparticles
(D).
78
Figure 3.10 Camera images of filtration of different nanoparticle solution
using chitosan coated sugarcane nanofiber i.e. PVP capped Au-
nanoparticles (A), PVP capped Ag-nanoparticles (B), Citrate
capped Au-nanoparticles (C) and citrate capped Ag-nanoparticles
(D). Bottom portion of tube is the pure water after filtration of
nanoparticle solution.
79
Chapter 4
Figure 4.1 FESEM image shows morphology of PVA/Fe (III) composite
nanofiber and Energy-Dispersive X-ray Spectroscopy (EDX)
shows the quantitative presence of Fe in the nanofiber.
91
Figure 4.2 ATR-IR spectra of PVA nanofiber and PVA/Fe3+
crosslinked
nanofiber
92
Figure 4.3 Curves for glass transition temperature (A) and curves for melting
point (B) analyzed by DSC analysis of PVA and Fe3+
crosslinked
nanofiber.
94
Figure 4.4 Concentration dependent (20 PPM, 40 PPM, 60 PPM, 80 PPM and
100 PPM) adsorption of As (III) and As (V) ions using Fe3+
95
xiv
coordinated PVA nanofiber as a function of time.
Figure 4.5 EDX spectra of arsenic adsorbed PVAFe nanofiber. 95
Figure 4.6 Isotherm plots (Freundlich and Langmuir isotherms) for As (III)
and As (V) sorption on the Fe3+
loaded PVA nanofiber.
98
Chapter 5
Figure 5.1 Schematic procedure for the synthesis of polymer nanofibers
containing silver nanoparticles.
107
Figure 5.2 Test for reduction of AgNO3 into Ag-nanoparticle, with different
polymer solutions PVA 10 wt% (A), DEX 10 wt% (B), MC 5 wt%
(C), PVA 10 wt% + DEX 5 wt% (D), PVA 10 wt% + MC 5 wt%
(E) the time intervals of 0 , 6 , 20 and after 48 h
112
Figure 5.3 Scanning Electron Microscope (FESEM) image of PVA-Ag
nanofiber (A), PVA /DEX-Ag nanofiber (B) and PVA /MC-Ag
nanofiber (C).
114
Figure 5.4 Transmission Electron Microscope (TEM) image of PVA-Ag
nanofiber (A), PVA /DEX-Ag nanofiber (B) and PVA /MC-Ag
nanofiber (C).
115
Figure 5.5 X-ray diffraction (XRD) pattern of PVA-Ag nanofiber (A), PVA
/DEX-Ag nanofiber (B) and PVA/MC-Ag nanofiber (C).
115
Figure 5.6 Effect of Ag-nanoparticles on thermal property of PVA NF (8A),
PVA/DEX NF (8B), and PVA/MC NF (8C).
117
Figure 5.7 Optical micrograph of IMR-90 cells around nanofiber after 4 days
of co-culture with IMR 90 cells and cell viability test with IMR-90
on designed nanofiber up to 4 day of analysis. The data
represented mean and standard deviations of four samples. Control
experiment was normalized as same cell line was used with
different time scales.
119
Chapter 6
Figure 6.1 Figure 6.1: FESEM images of PVA NF (A), PVA-CC NF (B),
PVA-MGC NF (C), PVA-MP NF (D), PVA-MP-CC NF (E) and
PVA-MP-MGC NF (F).
131
Figure 6.2 Energy dispersive spectra of PVA NF (A), PVA-CC NF (B) and 131
xv
PVA-MGC (C).
Figure 6.3 TGA thermogram of PVA NF (A), PVA-MP NF (B), PVA-MP-
CC NF (C), and PVA-MP-MGC NF (D).
133
Figure 6.4 Glass transition temperature analysis of PVA NF (A), PVA-MP NF (B),
PVA-CC NF (C), PVA-MP-CC NF (D) PVA-MGC NF (E) and PVA-
MP-MGC NF (F).
134
Figure 6.5 XRD plot for PVA NF (A), CaCO3 (B), PVA-MP-CC NF (C), MgCO3
(D) and PVA-MP-MGC NF (E).
136
Figure 6.6 Cell viability assay on PVA-MP/CaCO3 and PVA-MP/MgCO3
composite nanofibers
137
Figure 6.7 SEM images of IMR-90 cell on PVA NF (A), PVA-MA NF (B),
PVA-MA-CC NF (C) and PVA-MA-MGC NF (D) after 7 days.
138
Chapter 7
Figure 7.1 ATR-IR spectra of PVA (a) and lyophilized dried sample of Fe
(III) crosslinked PVA (b) complex (A), Study on swelling ratio of
hydrogels for different degree of crosslinking (B).
153
Figure 7.2 SEM images of freeze dried lyophilised sample of PVFeGel-1 (A),
PVFeGel-4 (B).
154
Figure 7.3 Stress-strain graph for hydrogels with different crosslinker
concentration under uniaxial compression mode (A) and graph
for modulus of hydrogels with different crosslinker concentration
(B).
156
Figure 7.4 Graph of storage modulus vs frequency of different hydrogels at
biological temperature (37 °C) (A) and graph for storage modulus
vs temperature of hydrogels with different degree of crosslinkers
at constant frequency (B).
156
Figure 7.5 Creep behavior of hydrogels with different concentration of
crosslinkers at biological temperature (37 °C) (A) and Graph for
strain recovery with linear time scale after release the applied
stress to hydrogels of different crosslinker concentration (B).
159
Figure 7.6 Curves for glass transition temperature analysed by DSC analysis
of only PVA and lyophilised sample of PVA hydrogels from
different crosslinker concentration
160
xvi
Figure 7.7 Optical micrograph of IMR-90 cells around hydrogel after 5 days
of coculture (A) and cells grow on the surface of hydrogel (B).
162
Figure 7.8 Cell viability test with IMR-90 on croslinked hydrogels upto 5 day
of analysis. The data represented mean and standard deviations of
four samples. Control experiment was normalised as same cell line
was used with different proliferated time scale
163
xvii
ABBREVIATIONS AND SYMBOLS
λ Wavelength
% Percentage
nm Nanometer(s)
μm Micrometer(s)
3D Three dimensional
Ag+ Silver ions
AgNO3
NP
Silver nitrate
Nanoparticle
BSA Bovine Serum Albumin
conc. Concentration
cm Centimeter
et. al In Latin - et alii (and others)
ECM Extracellular matrix
g Gram(s)
HCl Hydrochloric acid
Min Minutes
xviii
h Hour(s)
i.e. That is (Latin id est)
kV Kilovolts
keV Kilo electron volts
MPa
kPa
Pa
Megapascals
Kilopascals
Pascals
MeOH Methanol
mg Milligram(s)
ml Milliliter(s)
mmol Milli molar
NaCl Sodium chloride
NaBH4 Sodium borohydride
MC Methyl cellulose
PCL Poly (caprolactone)
PEO Poly (ethylene oxide)
PVA Poly (vinyl alcohol)
xix
RT Room temperature
NF Nanofiber
rpm Revolutions per minute
SEM Scanning Electron Microscope
DSC Differential Scanning Calorimetry
TGA Thermo Gravimetric Analysis
EDX Energy Dispersive X-ray analysis
FT-IR Fourier Transform Infrared Spectroscopy
NMR Nuclear Magnetic Resonance
FESEM Field emission Scanning Electron Microscope
ICP-OES Inductive Coupled Plasma Atomic Emission Spectroscopy
UV-Vis Ultra-Violet Visible Spectroscopy
XRD X-ray diffraction
1
Chapter 1
Introduction
2
1.1 Introduction to nanofibers
In recent years, several methods have been proposed for the fabrication of various
architectures from different polymers. Out of these methods, nanofibers and hydrogels are
the most suitable fabrication technique owing to their high surface to volume ratio.1-3
Fibers with diameter less than 1000 nm are defined as nanofibers. Such type of nanofibers
are synthesized using a number of methods such as drawing, template synthesis, phase
separation, self assembly and electrospinning.4-8
In addition, other methods such as wet
spinning, dry spinning, melt spinning and gel spinning which are capable of producing
fibers with diameters down to the micrometer range.9-13
Apart from synthetic nanfibers,
natural fibers can be derived from various sources. This can be either by self assembly
from different natural macromers or by isolation of cellulose and protein fiber from
naturally occuring materials.14-17
1.2 Synthetic nanofiber by electrospinning technique
Out of several reported methods, electrospinning is an efficient technique to prepare
nanofibers with diameter ranging from a few nanometers to micrometer range.5 This
process is very simple, convenient, effective and widely utilized for generating
nanofibrous membrane. Fiber formation via electrospinning is based on uniaxial
stretching of a viscoelastic jet derived from a polymer solution.18,19
Electrospinning is an excellent technique where polymer can be fabricated in
nanodimensions with different assemblies.1,20
Many researchers have investigated various
parameters affecting the morphology and the diameter of the electrospun PVA nanofiber
e.g solution concentration, flow rate, applied voltage, distance between needle, the
collector and types of collector.21-23
The polymer usually dissolved in an appropriate
3
solvent and used for electrospinning. This process entails the utilization of a high voltage
source to create an electrically charged feed solution.
The schematic for electrospinning setup is shown in Figure 1.1. The electrospinning
technique consists of a very small needle through which the polymer solution can be
forces into a field with a high voltage difference.24,25
Typically the positive power supply
connects to the metallic needle portion and the negative end connected to a grounded
plate which acts as a collector. While increasing the electric field strength, it causes
repulsive interaction between the positive charges in the liquid and simultaneously gets
attracted by the oppositely charged collector. This process creates a tensile force to the
polymer solution, resulting in break the surface tension of the polymer drop and generates
a Taylor cone. By increasing the voltage beyond this point, such Taylor cone elongates
and get attracted by oppositely charged grounded plate to produce a fiber structure. An
applied voltage in the range of 5 to 30 kV is needed to form nanofibers and quality of
nanofibers depend on distance between the needle tip and collector, voltage applied,
solvent used and concentration of polymers.24,25
According to the collector configuration,
rotating plate, stationary plate gives various types of fiber.26,27
On application of these
stationary plate, it generates nonwoven fiber mats. However, woven or knitted fibers can
also be generated by using various rotating drum and translating collector.28
Packing
density and porosity of the fiber mats greatly depends on the types of collector plate used
in electrospinning process.
Electrospinning provides nanofibers in the diameter range of 10 - 2000 nm, which can
be achieved by selecting the suitable polymer-solvent system. A typical SEM image of
PVA nanofiber is shown in Figure 1.2. The diameter and morphology of nanofibers
4
generally depends on number of parameters which includes polymer properties, types of
polymer, viscosity, electrical conductivity, polarity, strength of applied electric field,
feeding rate for the polymer solution, the distance between spinneret, collector plate,
temperature and humidity.29-40
Apart from these other parameters are also important like
temperature and humidity for the formation of smooth fiber.
Figure 1.1: Schematic diagram of electrospinning Figure 1.2: FESEM image of PVA nanofibers
setup
The limitation of this technique includes broad range of fiber thickness, random
orientation of nanofibers, and low mechanical properties of the fiber meshes.
Salient features of electrospinning are:
Suitable solvent should be used for dissolving the polymer.
The vapour pressure of the solvent should be suitable enough, so that it evaporates
quickly from the fiber to maintain its integrity when it reaches to the target but not
too quickly to allow the fiber to harden before it reaches the nanometer range.
5µm
5
The viscosity and surface tension of the solvent must neither be too large to
prevent the jet from forming nor too small to allow the polymer solution to drain
freely from pipette.
The power supply should be adequate enough to overcome the viscosity and
surface tension of the polymer solution and to sustain the jet from the pipette.
The gap between the pipette and grounded surface should not be too small to
create sparks between the electrodes but should be large enough for the solvent to
evaporate in time to form fibers.
1.2.1 Characterization of polymeric nanofibers
Polymeric nanofibers derived from electrospinning technique have different applications
in many fields. The ultimate product performance solely depends on the basic properties
of nanofibers which include chemical and physical properties, morphology and thermo-
mechanical properties.
1.2.1.1 Geometrical characterization
Nanofibrous membranes with high surface area, small pore size and the possibilities of
producing three dimensional structures is the main cause for development in advanced
applications.41-43
So it is very important to study the morphology of the nanofibrous
membranes. Various techniques are used for this purpose, such as scanning electron
microscope (SEM), transmission electron microscope (TEM) and atomic force
microscopy (AFM). The SEM gives idea about the surface characteristics such as
morphology, smoothness, fiber diameter, pore size and interfiber adhesion properties.
Similarly, TEM analysis of nanofiber membrane involves passing the beam of electron
that are transmitted through an ultrathin specimen.44-47
This technique gives more idea
6
about the distribution of different additives inside the nanofiber matrix. Furthermore,
AFM gives more idea about the three dimensional surface profile and even this technique
can be used for the measurement of nanoscale contacts and atomic binding.48-50
1.2.1.2 Thermo-mechanical property analysis
The bulk property of the nanofibrous materials plays vital role in real application. It is
well known that the nanofiber have marked difference in their thermal and mechanical
properties when compared to the bulk polymers. For physical characterization of such
materials, techniques such as thermogravimetric analysis (TGA), differential scanning
claorimetry (DSC), X-ray diffarction (XRD) and dynamic mechanical property (DMA) are
used.
Thermal properties:
Thermal analysis of polymeric materials gives idea about the structural property
relationship of the designed materials.51
The common thermal properties include glass
transition temperature (Tg), Melting points (Tm) and melting point enthalpy (ΔHm). The
Tg of the nanofiber correspond to shift in segmental motion of the main polymeric chain
network. The increase in Tg indicates the restriction of polymer chain either by
crosslinking or by binding with some other additives. Similarly, increase in Tg indicates
the formation of voids in between inter-chain resulting increase in the chain segmental
motion at lower temperature and this study gives idea about the plastic or rubbery property
in application condition. Melting point gives idea about the change in phase transition at
particular temperature and correspondingly change in crystallinity of the polymer
backbone and can be easily calculated by the crystallization peak from DSC analysis.52-54
7
Mechanical properties:
It is known that the electrospinning reduces the crystallinity as compared to bulk
polymers. In real application, the mechanical property of nanofiber is the most important
parameter,55,56
which involves measurement of modulus, tensile strength and yield point.
The electrospunned nanofiber with the presence of tiny pores influences the ultimate
mechanical property of thermoplastic elastomers.45,57
It is also reported that a decrease in
mechanical property was observed at nanometer scale. So far, much information is not
available on the mechanical properties of nanofiber and their composites. Due to the small
diameter of the nanofiber, various researchers used AFM techniques to investigate the
mechanical properties of the individual nanofiber.58,59
1.3 Isolation of nanofiber from natural sources
Despite the ease of production of synthetic nanofibers, natural nanofibers find much
importance in current research. Agricultural by-products have rich cellulose contents,
making them highly suitable sources for nanofibers and since they are renewable,
biodegradable and abundantly available, their usage impose less stress on environment.
Examples include corn stover, wheat straw, rice straw, barley straw, coir, sugarcane
bagasse, pineapple leaf and banana leaf. Chemically, natural cellulose nanofibers are
extracted by alkaline treatment using sodium hydroxide, or acid treatment, such as
sulphuric acid on agricultural by-products. The quality of the extracted nanofibers depend
on chemical concentration, temperature and duration of treatment among other factors .60
Cellulose is the most abundant natural polymer. It has a repeat unit consisting of two
anhydroglucose rings linked by β-1,4-glycosidic bonds, and has a large amount of
hydroxyl groups.61
8
The three main components of naturally occurring agricultural by-products are
cellulose, hemicellulose and lignin. The composition depends on the source
with
approximate cellulose content of 32% to 48%, hemicellulose content of 27% to 32%, and
lignin content of 18% to 26%.62,63
Lignin consists of highly crosslinked polymers of
arylpropane units and it make bonds with the neighbouring cells and cellulose.
Hemicellulose is a polysaccharide based polymer generally found between cellulose and
lignin. The presence of numerous hydroxyl groups gives rise to hydrogen bonding,
making it strongly bounded to cellulose. Due to its smaller degree of polymerisation and
amorphous nature, it is not as strong as cellulose. As shown in Figure 1.3, cellulose is a
polymer with a repeat unit consisting of two anhydroglucose rings linked by β-1, 4-
glycosidic bonds.
Figure 1.3: Repeat unit of cellulose
In the extraction procedure, bleaching was mainly used to remove lignin.64
Sodium
hypochlorite can degrade cellulose, hemicelluloses and lignin.62
In sugarcane bagasse
outermost layer is made up of lignin, on bleaching it removes lignin portion.
NaOCl + H2O ⇌ HOCl + NaOH
HOCl ⇌ H+ + ClO
-
ClO- + HClO → ClO
. + Cl
. + OH
-
Sodium hypochlorite dissolved in water according to the equations above, forming
hypochlorous acid and hypochlorite ions which could react to produce chlorine radicals.
9
Such radicals are the active species in degradation process where they abstract hydrogen
from phenolic hydroxyl groups in lignin to give phenoxy and mesomeric
cyclohexadienoyl radicals, which further react to cause degradation of lignin.
Figure 1.4: FESEM images of cellulose nanofibers.
Subjecting the bleached fibres to alkaline treatment results in dissolution of
hemicellulose and produce cellulose nanofibers. The nanofibers isolated from sugarcane
bagasse is shown in Figure 1.4. Furthermore, by alkaline treatment with potassium
hydroxide, ionisation of the hydroxyl groups in cellulose and hemicellulose results in
alkoxide ions which disrupt the hydrogen bonding to remove hemicellulose and residual
lignin.65,66
Lignin was fragmented through cleavages of α-aryl and β-aryl ether bonds.
1.3.1 Characterization of cellulose nanofibers
Like synthetic polymeric annofibers, cellulose based nanofibers also possess sufficient
mechanical integrity. The presence of hydroxyl groups on the cellulose moiety with
different configuration has ability to form hydrogen bond between adjacent polymer
chains and stabilizing the structure. Hence it is very important to understand the physical
property which satisfies the product requirement. Owing to that various physico-
200 nm
10
mechanical properties of natural nanofibers can be carried out as same method discussed
in earlier section for testing electrospunned nanofibers (Section 1.2.1)
1.4 Preparation of hydrogel from hydrophlic polymers
Hydrogels are water swollen polymeric three-dimentional (3D) materials which are able to
hold sufficient amounts of water in the network structure in a swollen state. Several
hydrophilic polymers are used to make hydrogels for various applications. The hydroxyl
groups play an important role for the fabrication of network structure. The hydrophilicity
of the network helps to accommodate sufficient amount of water by forming hydrogen
bonds. Owing to that various researchers have attempted to fabricate hydrogel from PVA
and cellulose based macromers using different crosslinking methods. Such hydrogels were
fabricated by different chemical, physical, radiation and photochemical methods with
tunable desired properties.67-73
A schematic for the water swollen crosslinked network of hydrogel is shown in
Figure 1.5. Highly swollen hydrogel network structure is soft in nature, result in good
mechanical integrity which can be achieved by stabilization due to the hydrogen bonding
Water
Hydrophilic unit
Hydrophobic unit
Figure 1.5: Water swollen network of cross-linked hydrogel
11
with water.74
The water swollen 3D hydrogel network can be tuned by different
crosslinking methods which influence the final mechanical properties such ascompliance,
elasticity and mechanical stress.75-77
As a consequence, several researchers paid much
attention to the physico-mechanical behavior of different crosslinked hydrophilic
hydrogel.
1.4.1 Characterization of hydrogel
The hydrogel network has high water retention capacity in its network structure and
mechanically stable, which allow diffusion of small and macromolecules. For designing
a hydrogel, it is very important to investigate the structure-property analysis of the
material relevant to their desired properties. Prior to designing for a specific application,
the morphological, thermal, physical and mechanical properties need to be investigated at
earlier stages.
1.4.1.1 Physical and geometrical analysis
The electron microscopy provides information regarding the morphology and pore
diameter of the hydrogel network. The purpose of crosslink is to prevent the flow behavior
of the polymer solution. However in between these crosslinks, the molecular segments
remain flexible and particularly, the intrinsic chain flexibility in hydrogel network is
greatly determined by the nature and amount of crosslinker.
1.4.1.2 Thermo-mechanical properties of hydrogel
To gain a better understanding on the effect of crosslinking toward the molecular mobility
of the designed macromer, thermal analysis is a very efficient technique. Furthermore,
thermal characteristics during dynamic condition provide information for stability,
shrinkage, expansion and storage. Similarly, the mechanical properties of designed
12
hydrogel under different condition also plays an important role. On highlighting, DMA is
an useful technique for characterization of hydrogels because it helps to correlate wide
range of material properties. Common techniques for measuring mechanical properties of
swollen hydrogels are static compressive modulus, dynamic mechanical analysis and
creep behavior. All these methods give information about the viscoelastic behavior of the
hydrogel.
1.5 Properties of hydrophilic polymers
Hydrophilic polymers contain polar or charged functional groups, which makes them
soluble in water. Poly (vinyl alcohol) (PVA) and cellulsoe based macromer are important
polymers which offers interesting properties for different applications. PVA is neutral and
water soluble polymer which is commercially prepared by hydrolysis of polyvinyl acetate.
The methods of polymerization techniques offer different microcrystalline PVA by which
tacticity can be controlled giving a wide range of interesting properties. Furthermore,
hydrophilicity and water solubility of PVA can be controlled by the extent of hydrolysis
and molecular weight. Atactic PVA is a semicrystalline polymer, hydrophilic, and has
good chemical resistance, thermal stability, physical and mechanical properties and
nontoxic in nature.12
The properties such as water solubility, mechanical properties,
solvent resistivity, adhesion property depend on molecular weight, tacticity and degree of
hydrolysis.13,14,17
Since it is a water soluble polymer with excellent film and fiber forming
properties, it is considered as one of the best materials for forming nanofibrous hydrogel
for various applications.2,4
The methods of fabrication is expected to influence the ultimate
performance due to avaibility of wide range of morphologies and properties and hence
developing fabrication methods is important for tuning the product properties.
13
Cellulose
The major component in rigid cell walls of plants is cellulose. Cellulose is a linear
polysaccharide polymer consisting of ten to several thousand monosaccharide units.
Polysaccharides synthesized by plants and animals are stored in the form of food,
structural support, and metabolized for energy. The acetal linkage in the cellulose moiety
controls water solubility and degradation behavior. The structure of cellulose consists of a
long polymer chain of glucose units connected by a β- acetal linkage. All repeating units
are β-D-glucose, and all β-acetal links connect C-1 of one glucose to C-4 of next
glucose.78
However, the α-acetal linkages of repeating units results in the formation of
water soluble cellulose.79
The examples of such α-cellulose includes dextran, methyl
cellulose, hydroxyethyl cellulose and starch. Due to their hydrophilicity and low
cytotoxicity, such polymers are widely used in the medical fields to prepare scaffolds for
artificial pancreas,80
synthetic vitreous body,81,82
wound dressing, transdermal drug
delivery, artificial skin, and cardiovascular devices.83-89
1.6 Applications and challenges in fabrication of PVA and cellulsoe nanofiber
membranes
Nanofibers are the fibers with diameters less than 1000 nm and are exciting new class of
material used for a wide range of applications such as medical, filtration, barrier, wipes,
personal care, composite, garments and insulation.90-94
Special properties of nanofibers
make them suitable for a wide range of applications from medical to consumer products
and industrial to high-tech applications for aerospace, capacitors, transistors, battery
separators, energy storage, fuel cells, and information technology.95-99
Figure 1.6 shown
for a statisstics on the research paper published on the different application of
electrospunned nanofibers. Nanofibers have significant applications in the area of
14
filtration since their surface area is substantially greater and have smaller micropores
than melt blown webs. Nanofibers are ideally suited for filtering submicron particles from
air or water.
Figure 1.6: Statistics on the literature published on the advanced application of
electrospunned nanofibers. This statistical data is from the scopous website; http://www-
scopus-com.libproxy1.nus.edu.sg/home.url and key word for search was electrospinning,
nanofiber with subsequent application areas as highlighted as on January 2011.
High porous structure with high surface area makes them ideally suited for many filtration
applications. These properties make the nanofiber being applied in a wide range across
biomedical and environmental areas such as protein purification, air cleaning, and heavy
metal ions and dye removal, has been widely explored.100-106
1.6.1 Designing of biocompatible membranes for practical aspects
During the last decades, development of micro- and nanofabrication technologies of
biocompatible, water soluble polymers helped to develop new technologies. PVA and
cellulose based macromers were widely used in preparing microstructured hydrogels and
nanofibers.107-111
Due to porous structures of such materials and high hydrophilicity with
Solar Cells
(10%) Tissue Engineering
(34.7%)
Engineeringfgdgfdgd
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Filtration
(32.8%)
Batteries
(13.5 %)
Fuel Cells
(9%)
Solar Cells
(10%)
15
large surface area to volume ratio, microengineered scaffolds have potential to be used in
various applications in the emerging fields like water treatment, biomedical engineering
and biosensors.92,112-117
1.6.1.1 Nanofibrous hydrogel for tissue engineering application
Fabrications of nano-sized materials have offered unique opportunities for cell attachment,
growth and differentiation. Recently various studies have been conducted using polymeric
nanofiber membranes resulting in an ideal 3D network which provided suitable tissue
engineering scaffolds118,119
and for wound dressing materials.120,121
The inherent properties
of nanofibers, which are uniquely capable of mimicking and reorganizing the biological
microenvironment to supplement damaged or diseased tissue and to stimulate the
regeneration of neo-tissue. In addition, hydrogel was also fabricated as a soft tissue
substitute and used in drug delivery application.122
These challenges highlight the
fabrication procedure of three dimensional polymeric networks which lead to a porous
network structure. Highly porous 3D structures favor exchange of nutrients and waste
products with surrounding environments making them potential substitutes for tissue
engineering scaffolds and related biomedical applications.
Apart from the design of fabrication procedure, polymer selection is an important
criterion for the ultimate product performance for tissue regeneration. Several researchers
have focused on the fabrication of biocompatible hydrophobic polymers for their good
mechanical properties, but its main drawbacks are being prone to blood platelet adhesion
and lack of haemocompatibility.123,124
In order to overcome these issues, PVA and
cellulose based macromers were used by several researchers and reported for their
excellent properties such as sufficient hydrophilicity, good physical and mechanical
16
properties and non-toxic nature with excellent biocompatibility.125, 126
Furthermore, such
types of hydrogels were fabricated and investigated for potential application for
mechanical responses to porcine aortic tissue, artificial articular cartilages and for heart
valve applications.127-132
However, due to highly hydrophilic in nature, it results in
minimal cell adhesion and protein adsorption. As a consequence, several researchers have
studied the incorporation of natural molecules such as gelatin and collagen that can be
recognized for preliminary cell adhesion which are used for tissue growth.126,133
Such
challenges for enhancing the cell growth on the material surface are potential research
areas for development biocompatible membranes for various biomedical applications.
1.6.1.2 Antibacterial agents in wound healing
Similarly, nanostructured silver is considered as an important material due to their
potential for application in biology and are also well known for their antimicrobial
activities for many years.134
Liu and Watanabe et al. have suggested synthesis of non
agglomerated nanoparticles with polymers as template.135,136
In addition, Gupta et al.
developed silver nanoparticle loaded poly (acrylamide-co-itaconic acid) grafted cotton
fabric which was applied in the field of textile technology and explored for their excellent
antibacterial properties.137
The final products are in micrometer range, and the efficiency
can be enhanced by decreasing the fiber diameter to nanometer range due to its high
surface area. Recently Kong and Xu et al. have synthesised nanofiber containing silver
nanoparticles by radical mediated dispersion polymerization and applied as an
antibacterial agent.138,139
Nam and Park et al. provided an effective way to design the
cellulose based polymers which contain high number of hydroxyl groups and ethereal
17
linkages. Such functional groups help to make ion-dipole linkages and stabilize Ag metal
nanoparticles at the ionized selective sites without becoming coagulated.137,140
1.6.2 Environmental membranes and filters
Recently, membrane technologies are the potential solution for solving the problems
associated with wide range of waste water treatments in many industrial applications.141-
143 These membranes are semipermeable barriers which reject to specific molecules and
compounds by its specific pore size by size exclusion principle. In addition, the presence
of suitable functional groups on the membrane surface trap to specific pollutants by
chemical interaction.106,144
Due the availability of high porosity and interconnected open
pore structure, nanofibers are widely used in waster water purification; i.e. removal of
various inorganic and organic pollutants from water.145,146
These fibers are broadly
classified in two categories; such as, synthetic fibers and natural fibers. Synthetic fibers
are the material which is chemically modified from commercially available polymers or
starting from a monomer. In the case of natural polymers, which are generally extracted
from the natural sources or from plants waste products. However the immobilization of
desired functional groups by surface modification technique helps to increase the
efficiency of the nanofibers.
For the waste water purification, membrane technology received worldwide attention
in recent years in industry due to high surface area, easy to handle, recyclability and
reusability.147,148
The adsorption and desorption efficiency towards particular analytes
greatly depends on the surface functional groups present on adsorbents.149
Electrospunned
nanofibrous materials have gained considerable interest due to their unique properties,
such as high volume to surface ratio and interesting porous structures with superior
18
mechanical properties.150
This fabrication technique offers controllable fiber diameter,
pore size and orientation at lower cost. Due to high porosity ranging from sub-micron to
several micrometers and interconnected open pore structure, it allows a high permeability
for developing high-performance filtration membranes over conventional existing
materials.151
Some examples of nanofibers being used in water treatment application
especially in the area of removal of submicron sized solid particles, heavy metal ion
separation, organic compounds and waterborne pathogen are listed.
1.6.2.1 Removal of heavy metal ions from water
In recent years, industrial development and the advent of new technologies have resulted
in significant increase in the amount of heavy metal ions in wastewater. These heavy
metals are the serious problem towards the aquatic systems. Various mesoporous materials
such as silica,152-154
carbonaceous materials,155,156
magnetic nanomaterials,157, 158
and
asbestos nanoparticles159
are widely used for heavy metal removal from waste water.
The effect of magnetic nanoparticle and their composites were used to adsorb heavy
metal ions from aqueous solution and the process of sorption is known to be pH and
temperature dependent.160,161
Similarly the nanocomposite containing magnetic
nanoparticles has strong ability for the removal of As(III) from aqueous media over a wide
pH range and also unaffected by the presence of competing ions such as sulphate and
phosphate.162-164
Apart from that some other metal oxides were fabricated into
nanostructured membranes which showed good removal efficiency of heavy metal ions
from waste water.165-167
Similarly carboneous materials also used for the heavy metal ion
filtration.168-170
Carbon nanotubes incorporated polyacrylic acid incorporated nanofibers
with the presence of zero valent iron nanoparticles shown good efficiency to remove the
19
europium ions from aqueous solution.171
In the water filtration system, use of nano
particles is less preferable because of chances of contamination and difficulty in recycling
process.
Membrane technology provided solution for water purification due to low cost, high
surface area, easy handling, recyclability and reusability.147,172
Nanofibrous membranes
has attracted interest due to their unique properties, such as high volume to surface ratio
and interesting porous structures with superior mechanical properties.150,173
Due to these
inherent properties these nanofibers offer an attractive solution for the removal of heavy
metals from aqueous environment. Recently, nanofibers derived from chitosan and
blending of silk fibroin/wool keratose were used for the removal of heavy metal ions from
aqueous media.174,175
In addition, several synthetic polymers with suitable functional
groups were coated to nanofibers, such as polypyrrole coated PVDF-HFP, dithizone
nanofiber-coated membrane and chloridized polyvinyl chloride and investigated for the
adsorption of heavy metal ions from water.176-178
This implies that extraction efficiency
can be enhanced by modifying surface functional groups, which interact with toxic metal
ions from the polluted environment. In addition to that biopolymer such as chitosan and
chitosan modified synthetic macromer also have been widely used in heavy metal removal
from waste water.179-181
In water purification system, the membrane should be hydrophilic in nature. Hence,
for the hydrophobic polymers, surface can be converted into hydrophilic by various
methods like plasma treatment, chemical oxidation and grafting, and blending with
hydrophilic polymers. The surface modification technology has added advantages towards
intact mechanical integrity with suitable functionalities on the nanofiber surface. Recently
20
various researchers have been demonstrated novel methods to enhance the mechanical
properties and as well as the hydrophilicity of electrospun polyethersulfone nanofiber
membrane.182-184
This process significantly improves the mechanical integrity of
electrospun membranes without disturbing the fiber size and morphology. Another
method involves the use of simple oxidative reaction by using ammonium persulfate that
can produce high hydrophilicity on the Polyethersulfone (PES) membrane.185
polyethersulfone is a tough, durable, and temperature resistant aromatic polymer. These
membrane provides high flow rate, low extractables, and greater mechanical strength as
compared to other polymeric membranes. Hence these PES membrane materials are
widely used for commercial microfiltration and ultrafiltration. However some nanofibers
have very low mechanical properties. To overcome these issues few studies involve the
incorporation of other supportive layers which provide mechanical strength and efficient
surface functional groups on the top layers of the membrane.186
1.6.2.2 Waterborne pathogen and dye pollutants
Water contaminated with bacteria and viruses is the main cause of diseases in several
countries. Currently, chemical disinfectants such as chlorine and membrane-based water
filtration systems are employed to control microbial pathogens.187,188
Carbon nanotubes
are also used as nanofilter for the application virus removal from water.189
Researchers
prepared various functionalized nanofibers and used for the antimicrobial filters in waste
water purification. Owing to that, quarternary ammonium salts and polymers with
biguanide groups shows very good antimicrobial activity.190
In addition, silver metal
nanoparticles incorporated on to the polymer nanofiber are widely used for antimicrobial
activity.191
Such silver nanoparticle embedded nanofibers provides high surface area and
21
hence used for various antimicrobial filtration system.192
Similarly biopolymers such as
chitosan are known to be biocompatible, hydrophilic, non-antigenicity, non-toxicity and
biodegradable in nature.174, 193
Another important area of water pollution comes from contaminated dye used in
textile industries. These organic dye compounds pose health hazards from the
contaminated water source. Various functionalized nanofibers have been used for the
removal of organic pollutants from water. Recently, PMMA nanofibers functionalized
with phenylcarbonylated and azidophenylcarbonylated β-cyclodextrins are used for the
removal of phenolphthalein as a model molecule.194
This investigation suggested that
these functionalized nanofibers can find practical application in the filtration of organic
pollutants from water. The development of photocatalytic TiO2 embedded nanofibers is
widely used in the degradation of dye pollutants. Such photoactive moieties proved to be
excellent materials for the degradation of dye molecules in presence of light.189,195
Similarly, humic acid is a natural organic pollutants, commonly found in waste water. For
the removal of these oganic pollutants, nanofibers embedded with TiO2 also used by
various researchers.196
Furthermore, iron nanoparticles are also widely used for their
sorption of wide variety of pollutants such as chlorinated organic solvents, organochlorine
pesticides and polychlorinate biphenyls.
1.6.2.3 Removal of micro and nanoparticulates from water
The removal of micro and nano-sized particulate contaminants from polluted water is the
main focus for water filtration technology. Removal of micro sized solid contaminants can
be easily achieved using nanofibers membranes. Recently, several researchers have
fabricated electrospunned membranes derived from nylon-6, polyvinylidene fluoride and
22
polysulphone which were used as pre-filters for the removal of model polystyrene micro-
particles.101,26-27
In comparison to nanofiber membranes of fixed pore size has been
filtered for different polystyrene particles. The results revealed that the microfiltration
membranes were used to remove micro-particles most effectively without any damage to
the membrane.
Although several investigations have combined the usage of submicron sized
particulate filter and heavy metal removal by nanofibrous membranes, the removal of
nanoparticle has not been investigated till date. Owing to that, our target to develop
functionalized novel hybrid nanfibrous membranes from hydrophilic polymers.
The overall goals of this thesis were to fabricate nanofibers and hydrogels from
biocompatible water soluble polymer, such as PVA and cellulose and exploring their
possible applications. The overall schematic diagram for the this thesis work is shown
below.
Scheme 1: Schematic diagram for outline of our targeted research
Nanofiber
Synthetic Natural
Geometrical Physico-
mechanical Physico-
mechanical
Geometrical
Fabrication Characterization Characterization Isolation Application Application
23
1.7 Scope and outline of the thesis
The aim of this research work is: і) to prepare nanofibers from PVA macromer by
electrospinning technique and surface modified using thiols and amines. іi) to Isolate
polysaccharide based nanofibers and incorporation of amines by surface coating with
chitosan. iіі) to fabricate nanofibers from PVA//Fe3+
nanofibers, their structure property
analysis. іv) to prepare cytocompatible nanofibrous membranes by incorporation of
protein and methacrylate moiety. v) to prepare cytocompatible metal coordinated PVA
hydrogels and finally. vі) to demonstrate some these membranes for suitable applications
including environmental detoxification.
PVA and cellulose based macromers were chosen for our investigation because of
their unique properties as mentioned earlier. Chapter 2 describes the fabrication of
nanofiber from poly (vinyl alcohol) and surface modification with suitable functional
groups such as thiols and amines, their characterization and application for entrapment of
nanoparticles from water.
The incorporation of suitable functional groups on the PVA nanofiber gives idea
about good filtration efficiency, isolation of cellulosic nanofibers from raw sugarcane
bagasse and incorporation of amine groups by surface coating is explored. These
cellulosic nanofibers can be isolated from renewable sources, which have less threat to
environment after usage. Chapter 3 describes isolation of cellulosic nanofibers from
sugarcane bagasse and surface coating with chitosan to incorporate amine groups. These
functionalized nanofibers were used for the filtration of different nanoparticles (Au and
Ag) with different capping agents. The adsorption and filtration study revealed that
24
designed membranes have good efficiency toward the filtration of nanoparticles from
water.
Arsenic is a potent contaminant in waterways across the world. In chapter 4 we
explored the preparation of Fe3+
immobilized PVA nanofiber for the removal of arsenic
from water. The fiber morphology and physical behavior was investigated by various
analytical techniques. The time and concentration dependent study was carried out in
detail. This study showed that the designed nanofibrous membranes removed arsenic from
water more effectively.
Because of the biocompatibility and suitable pendent hydroxyl groups of PVA and
cellulose based macromer, we were interested to make nanofibrous structure with silver
nanoparticles. Chapter 5 describes the insitu preparation of silver nanoparticles on the
PVA and cellulose nanofiber surface. For crosslinking of designed macromer,
methacrylated moiety was incorporated to the pendent hydroxyl groups. These
methacrylated macromers are found to be cytocompatible in nature which tested with
human caucasian fetal lung fibroblast cell line (IMR-90). The electron microscopic study
and structure-property relationship study was carried out in detail using various analytical
techniques. The cytocompatible nature of the methacrylated nanofibers suggests that the
nanofiber containing silver nanoparticles may find practical applications in the field of
microbial filtration.
The cytocompatible nature of PVA nanofiber motivated us to prepare novel
membranes with the incorporation of natural protein using inorganic salts (CaCO3 and
MgCO3). Chapter 6 describes the fabrication of nanofibers derived from PVA-milk
25
protein and metal carbonates. Physical characterization of nanofibers was carried out in
detail using various analytical techniques.
Chapter 7 describes a novel approach for the preparation PVA hydrogel with a
coordination crosslinker (Fe3+
). The structure-property analysis was carried out in detail
using various analytical techniques. The static and dynamic mechanical property of these
hydrogels were studied in detail and correlated to the network structure. A detailed study
on cytocompatibility was carried out using IMR-90 cell lines and the results are discussed
in detail.
26
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43
Chapter-2
Nanofibrous Poly (vinyl alcohol) Membranes for
Removal of Nanoparticles from Aqueous
Environment
This chapter is published as a full paper in Nanoscale, 2011, 3, 4625–4631; DOI:
10.1039/c1nr10739a.
44
2.1 Introduction
The recent applications of nanotechnology in commercial products1, 2
and pharmaceutical
fields3 has increased the number of new innovative products in the market, but at the same
time is expected to pose a threat to our environment due to poor waste disposal. Improper
disposal of nanomaterials incorporated products will enhance accumulation and long time
exposure to living system, which may cause serious health concerns in the near future.
Various studies have shown that nanoparticles enter human body through multiple ways
such as inhalation, dietary route and skin contact with contaminated samples. Such
contamination poses significant hazard to humans and other living beings as the small size
of nanoparticles facilitate transport across cell membranes to various organs. It is also
known that some nanoparticles are toxic to living systems.4-7
Mesoporous materials such
as silica,8-10
carbonaceous materials,11, 12
magnetic nanomaterials, 13, 14
and asbestos
nanoparticles15
are widely used for heavy metal removal from waste water.
Membrane technology provided solution for water purification due to low cost, high
surface area, easy handling, recyclability and reusability.16, 17
Resently, nanofibrous
membranes has attracted interest in many advanced application due to their unique
properties, such as high volume to surface ratio and interesting porous structures with
superior mechanical properties.18, 19
This technique offers controllable fiber diameter, pore
size and orientation at a lower cost. Nanofibrous membranes are known to offer high
separation rates for the removal of micron to sub-micron particles from water. Several
researchers have been attempted to prepare electrospun membranes derived from nylon-6,
polyvinylidene fluoride and polysulfone were used as pre-filters for the removal of
polystyrene micro-particles.20-23
45
Recently, electrospun nanofibers derived from different biopolymers such as
chitosan24
and silk fibroin/wool keratose25
were used for the removal of heavy metal ions
from aqueous media. The extraction efficiency can be enhanced by modifying surface
functional groups, which interact with toxic metal ions. Surface modification can be
achieved in a variety of ways which includes coating of nanofibers with synthetic
polymers and covalent linking of functional groups onto the nanofiber surface. Dioxime
modified poly (acrylonitrile),26
polypyrrole coated poly (vinylidene fluoride),27
dithizone
nanofiber coated membrane,28
and chloridized polyvinyl chloride29
were investigated for
the adsorption of various heavy metal ions. All these nanofibers showed high adsorption
efficiency due to their large surface area with suitable functional groups.
In this study, we propose the development of nanofibrous membranes using
electrospinning for the removal of nanoparticles from aqueous environment. Although
several investigations have combined the usage of heavy metal removal by nanofibrous
membranes, the removal of nanoparticles from water has not been investigated in detail.
In this context, we have fabricated PVA nanofibers and chemically modified the surface to
incorporate amine and thiols, which could act as strong ligating groups for nanoparticles.
The adsorption capacity was quantified by UV-Vis spectroscopy and the adsorption of
nanoparticles on the surface of nanofiber was confirmed by FESEM.
2.2 Experimental section
2.2.1 Materials
PVA with average molecular weight of 146,000 - 186,000, silver nitrate (AgNO3),
hydrogen tetrachloroaurate trihydrate (HAuCl4·3H2O), sodium citrate tribasic dihydrate
(Na3C6H5O7·2H2O), mercaptopropionic acid (MPA) and mercaptosuccinic acid (MSA)
46
were purchased from Sigma Aldrich and used as received. Glutaraldehyde, lysine and HCl
were purchased from Merck. The solvents used were of analytical grade and purchased
from local suppliers. Ultrapure water was used for solution preparations.
2.2.2 Synthesis of citrate-stabilized gold (Au NP) and silver (Ag NP) nanoparticles
Citrate capped gold nanoparticles were synthesized following a reported procedure.30
Briefly, 10 mL aqueous solution of HAuCl4·3H2O (5 10-3
M) was diluted to 190 mL
with ultrapure water and was heated to boiling. Sodium citrate solution (0.5%, 10 mL)
was added and continued boiling until a color change was observed. Color of the boiling
solution gradually changed to wine red, indicating the formation of Au NP colloidal
solution. The final solution was cooled to room temperature and topped up to 200 mL with
water to account for boiling.
Similarly, AgNO3 solution (5 10-3
M, 25 mL) was prepared and diluted to 200 mL
with water. Solution was heated to boiling and 10 mL of 1% sodium citrate solution was
added. The boiling was continued until a color change (pale yellow) was observed, cooled
to room temperature and the solution was diluted to 250 mL for further usage.
2.2.3 Fabrication of PVA nanofibers by electrospinning technique
Aqueous solution of PVA (10 wt%) was prepared by heating at 80 °C in water under
constant stirring for 2 hrs. This solution was used for the preparation of nanofibers by
electrospinning. The electrospinning set up consists of a hypodermic syringe and a needle
with an internal diameter of 1.2 mm. A programmable syringe pump (Optrobio
Technologies Pte Ltd) was used to feed the PVA solution. Positive electrode was
connected to the syringe needle containing polymer solution and negative electrode was
connected to the grounded aluminum foil covered copper plate which served as the
47
collector. The experiment was carried out at a positive high voltage of 15 – 18 kV.
Distance between the needle tip and the collecting plate was maintained at 10 cm with a
flow rate of 8 µL/min under ambient conditions. The resultant PVA fibers were
crosslinked using gluteraldehyde in presence of acid by ionic mechanism.
2.2.4 Incorporation of surface thiol and amine functional by surface modification
technique
Surface hydroxyl groups of PVA NFs were modified with thiol groups by the reaction
with mercaptopropionic acid (MPA) in presence of hydrochloric acid.31
Typically, cross
linked PVA NFs (0.1g) in water (10 mL) was kept at 80 °C, then a mixture of MPA (0.2
mL) and HCl (0.04 mL, 7N) in water was added drop wise over 20 min.
+
(PVA) (MPA) A. (PVA-MPA)
MSA modified PVA Lysine modified PVA
B. (PVA-MSA) C. (PVA-LYS)
Figure 2.1: Reaction scheme of surface modification of PVA-NFs with A. MPA (PVA-
MPA); B. MSA (PVA-MSA); and C. Lysine (PVA-LYS).
HCH2C
OH
HO O
SH
O O
HCH2C
HCH2C
OH
SH
H2O, H+, 80 °C
O O
COOH
HCH2C
HCH2C
SH
OHO O
HCH2C
HCH2C
OH
NH2
NH2
48
The temperature was maintained at 80 °C for 8 h, cooled to room temperature, filtered,
washed with water to remove excess amount of acid and dried. A similar procedure was
also followed for the surface modification with mercaptosuccinic acid (MSA) and lysine.
2.2.5 Nanoparticle extraction study using the surface modified PVA nanofibers
To test the filtration capacity of the PVA and surface modified PVA NFs (4 - 5 mg) were
added to 10 mL of citrate stabilized Au NP or Ag NP solutions (2 10-4
M) for different
time intervals at room temperature. Then the whole mixture was kept for constant shaking
at room temperature. For all samples 3 sets of experiments was carried out to find out the
error bar during our experimentations. At a certain time intervals the nanoparticle
solutions were removed and the percentage of nanoparticle extracted was calculated from
the concentration of nanoparticles remained versus the original concentration of
nanoparticle solution. For this quantification, nanoparticle solutions were analysed by UV-
Vis spectroscopy. The time dependent adsorption efficiency was plotted from the
corresponding data obtained.
2.2.6 Desorption study of nanoparticles from nanofiber
Nanoparticle adsorbed nanofibers were kept for shaking with 2M HCl for 2 hr at room
temperature. The acid desorbed nanofiber samples were cleaned thoroughly with DI water.
But for lysine modified nanofibers, during acid washing there may be chance of
protonation on amine groups and hence are kept for shaking with dilute sodium
bicarbonate solution. Then the samples were cleaned thoroughly with DI water and used
for readsorption studies with corresponding nanoparticle solution.
49
2.3 Characterization
The resultant fiber morphology was characterized by JEOL JSM-6701F field emission
scanning electron micrograph equipped with energy dispersive X-ray spectrometer (EDS)
and surface area was analyzed using Brunauer-Emmett-Teller (BET). The electrospun
nanofibers collected on glass cover slips were mounted on copper stubs with a double
sided conducting carbon tape and carefully coated with a thin layer of platinum and
examined. As synthesized nanoparticles were deposited on carbon coated copper grids and
used for transmission electron microscopy (TEM) study using JEOL JEM 2010, operating
at 200 kV. The quantitative measurements of nanoparticles were carried out by UV-Vis
spectra recorded on a Shimadzu UV-1601 PC spectrophotometer. Surface modifications
of PVA NFs were characterized by attenuated total reflectance – infra red (ATR- IR)
spectroscopy.
2.4 Results and discussion
2.4.1 Morphological and spectroscopic analysis
Non-woven PVA nanofibrous mats were prepared using electrospinning technique. It was
observed that stable jets could be formed when applied voltage of 15 kV was used. The
electrospinning process employs high voltage to create an electrically charged jet of
polymer solution. Oppositely charged supply was connected to the needle and grounded
collector plate. As the magnitude of voltage is increased, the hemispherical surface of the
liquid lengthens to form a taylor cone and comes out as fiber with nanodimensions. After
a critical voltage is applied, the electrostatic force will overcome the surface tension and
the charged jet is ejected from the tip of the taylor cone which undergoes a process of
elongation and evaporation to produce polymer nanofibers. The polymer flow rate of 8
50
1 µm
A
µl/min and the distance between the electrode and the collecting plate was maintained at
10 cm for all samples. Figure 2.2A shows the SEM images of the membranes prepared
from PVA. The morphology of fabricated nanofibers showed smooth surface without any
bead formation. The average diameter of PVA nanofiber was between 300 and 500 nm. In
order to make the nanofibers applicable in nanoparticle adsorption from water, the
nanofibers were cross-linked by glutaraldehyde in acidic condition. In comparison to
conventional melt spinning fabrication techniques, electrospinning technique gives
ultrafine fibers with high surface area to volume ratio. The BET surface area of PVA NFs
shows that the 3D porous fiber mat has a surface area of 5.29 m2 g
-1.
The adsorption capacity of nanoparticle solely depends on the functional groups
present on the surface of the nanofiber. In order to increase the adsorption capacity,
surface hydroxyl groups of PVA NFs were modified by lysine, MSA and MPA. The acid
group of the selected monomer reacts with hydroxyl groups on the polymer backbone to
form ester linkages. The incorporation of surface functional groups was characterised by
ATR-IR spectroscopy (Figure 2.2B).
Figure 2.2: A. FESEM image of electrospun PVA nanofibers and B. ATR-IR of PVA
NFs and PVA NFs surface modified with MSA, MPA and Lysine.
4000 3500 3000 2500 2000 1500 1000
D
B
A
1646 c
m-1
2552 c
m-1
Ab
sorb
an
ce
Wavenumber (cm-1)
A. PVA-LYS NF
B. PVA-MSA NF
C. PVA-MPA NF
D. PVA NF
C
B
51
ATR-FTIR spectrum of the MPA and MSA functionalized PVA NFs showed a
characteristic absorption associated with the stretching mode of the S-H group at 2552 cm-
1. For all nanofibers, the broad absorption at 3000 - 3600 cm
-1 corresponds to the O-H
stretching vibrations. For PVA-LYS NFs, the N-H stretching vibration overlap with the
hydroxyl region. PVA-LYS NFs showed characteristic N-H bending vibration at 1646 cm-
1 and medium-weak bands (1250 - 1020 cm
-1) due to C-N stretching vibrations. Alcohols
also gave a strong and broad band due to C-O stretching in the 1000 - 1200 cm-1
region
which overlaps the amine bands.
FESEM image of surface modified-PVA NFs is shown in Figure 2.3. All nanofibers
formed a highly porous mesh-like structure with fiber diameter ranging from 300 to 500
nm. From the SEM image of PVA-MPA NFs, it was observed that the fibers were
interconnected (Figure 2.4C). It was also observed that the PVA-MPA NFs mats are
slightly rigid and shrunk compared with the unmodified PVA NFs owing to the formation
of disulphide crosslinks between the sulfur groups of MPA. Such cross-linking networks
were not observed in the case of PVA-LYS NFs.
C
1 µm
B
1 µm
A
1 µm
Figure 2.3: Scanning electron micrograph images of the surface modified (A). PVA-LYS
NFs; (B). PVA-MSA NFs; (C).PVA-MPA NFs.
52
20 nm20 nm
BA
2.4.2 Adsorption of nanoparticles by using designed nanofibers
Two metal nanoparticles (Au and Ag) were used for our investigation. Nanoparticles were
synthesized using reported procedure and fully characterized. The morphology and size of
synthesized Au NPs (15 - 20 nm) and Ag NPs (20 - 40 nm) were characterized using TEM
(Figure 2.4). UV-Vis spectra of Au and Ag NPs showed absorbance maxima at 518 nm
and at 420 nm, respectively.
Figure 2.4: TEM images of (A) citrate capped Au NPs and (B) citrate capped Ag NPs.
To investigate the nanoparticle adsorption capacity by using our functionalized
nanofiber, a known amount of nanofibers were kept for constant shaking with nanoparticle
solution. It is well understood that the adsorption of the metal nanoparticles depends on
the active functional groups on the fiber surface and the nature of the nanoparticles. For
this study, different surface modified nanofibers were added to the aqueous solutions of
nanoparticles (2 10-4
M ) and shaken at various time intervals (3 hr 20 min for Ag NPs
and 2 hr 30 min for Au NPs). At a regular intervals, aliquots were collected and quantified
using UV spectroscopy. Percentage of Ag NPs and Au NPs adsorbed on different
nanofibers from aquous solutions is shown in Figure 2.5. From this figure, it can be seen
53
0
20
40
60
80
100
0 30 60 90 120 150 180 210
Ag
NP
ad
so
rbe
d (
%)
Time (min)
PVA-LYS NF
PVA-MSA NF
PVA-MPA NF
PVA NF
0
20
40
60
80
100
0 30 60 90 120 150
Au
NP
ad
so
rbe
d (
%)
Time (min)
PVA-LYS NF
PVA-MSA NF
PVA-MPA NF
PVA NF
that PVA-LYS NFs have stronger affinity towards the Ag NPs, whereas PVA-MPA,
PVA-MSA and PVA-LYS NFs showed higher affinity towards the Au NPs. However,
with PVA-MPA and PVA-MSA NFs, only 50% of Ag NPs was adsorbed from aqueous
solution. Unmodified PVA NFs showed 20% adsorption capacity for both Ag and Au
NPs. This can be explained by the nanoparticle-nanofiber interaction which depends on
the functional group present on the nanofiber surface. According to hard-soft acid-base
(HSAB) theory, sulfur acts as a soft donor and nitrogen as a borderline donor atom.
Furthermore, Pal et al.32
explored the stronger interaction of gold towards soft sulfur atom.
Similarly, silver is comparatively less soft than gold and shows stronger interaction
toward nitrogen.
Figure 2.5: Time-dependent adsorption efficiencies of citrate capped-AgNPs and AuNPs
(2 10-4
M solutions) on different surface modified-PVA nanofibers.
PVA-MPA and PVA-MSA NFs showed strong interaction with Au NPs. Similarly,
Xie et al.33
demonstrated that interaction of Ag towards nitrogen is stronger with respect
to sulfur and the amine groups preferably coordinate to silver atoms which form normal
co-ordinate bonds whereas thiol groups showed weak interaction with silver atoms.
Higher adsorption efficiency of PVA-LYS NFs towards Ag NPs can be attributed to the
presence of free terminal –NH2 groups. Figure 2.6A represents the UV-Vis spectra of
54
400 450 500 550 600 650 700
Wavelength(nm)
Ab
so
rba
nc
e (
a.u
)
Original Filtrate
After filtration
Original Au
NP solution
B
300 350 400 450 500 550 600 650 700
Ab
so
rba
nc
e (
a.u
)
Wavelength (nm)
Original Filtrate
Original Ag
NP solutionA
After filtration
0
20
40
60
80
100
0 1 2 3 4 5 6 7 8 9 1011 12
Ag
NP
ad
so
rbe
d (
%)
Time (Hr)
PVA-LYS NF
PVA-MSA NF
PVA-MPA NF
PVA NF
0
20
40
60
80
100
0 1 2 3 4 5 6 7 8 9 1011 12
Au
NP
ad
so
rbe
d (
%)
Time (Hr)
PVA-LYS NF
PVA-MSA NF
PVA-MPA NF
PVA NF
AgNPs before and after adsorption on PVA-LYS NFs and corresponding color image of
the nanoparticle solution. The gray color of Ag NP solution became almost colorless and
color of the nanofiber gradually turns gray indicating the effective adsorption of
nanoparticles from solution to the fiber surface.
As shown in figure, the UV-Vis absorbance of solution was decreased considerably after
shaking with nanofiber membrane. A similar observation was also achieved for the case of
AuNP solution by using PVA-SH NFs (Figure 2.6B).
Figure 2.6: Representative UV-Vis spectra (A) of the Ag NP and (B) for the Au NP
original solution and after filtration and corresponding photographs of the nanoparticle
solutions before and after filtration.
Figure 2.7: Second time adsorption efficiencies of citrate capped-AgNP and AuNP
solutions (2 10-4
M) on same nanofibers as a function of time.
55
The yellow color of AgNP solution and red wine color of the AuNP solution becomes
faint and the corresponding UV-Vis spectra are given in Figure 2.6. These results indicate
that the nanoparticles are effectively adsorbed on the surface of actively functionalized
nanofibers. Same nanofibers without precleaning of adsorbed nanoparticles were used for
a second time adsorption using similar conditions and the adsorption affinity was studied
(Figure 2.7). This study was carried out to investigate the maximum adsorption capacity
towards the nanoparticle filtration using same amount of nanofiber.
For the case of PVA-LYS NFs, the adsorption of AgNPs reaches to 60% after 6 h and
the adsorption capacity was less than 70% after 12 h. Similarly for the AuNPs adsorption,
it reaches maximum up to 70% after 12 h. Furthermore, during a third trial, it was
observed that the percentage of adsorption decreased up to 30% after 12 h (Table-2.1)
which suggest that after a certain extent of surface coverage, the fiber showed very little
affinity towards nanoparticles. Such surface activated process leads to the active
adsorption of nanoparticles onto the surface as confirmed by observing the surface
structure under SEM (Figure 2.8).
Sample Percentage of
AgNP adsorbed
Percentage of
AuNP adsorbed
PVA-LYS NF 35 17
PVA-MSANF 23 18
PVA-MPANF 20 22
PVA NF 8 7
Table 2.1: Table for summary of the results obtained on 3rd
trial of adsorption
for both Ag and Au nanoparticles.
56
0.0 1.6 3.2 4.8 6.4
keV
0
400
800
1200
1600
Cou
nts
C
O Pt PtAu
Au
0.0 1.6 3.2 4.8 6.4
keV
0
400
800
1200
1600
2000
Cou
nts
C
OPt Pt
AuAu
0.0 1.6 3.2 4.8 6.40
400
800
1200
1600
Cou
nts
C
OAg
Ag
AgPtPt
keV
Figure 2.8: FESEM images of A. AgNP adsorbed PVA-LYS NFs, B. AuNPs adsorbed
PVA-MSA NFs and C. AuNPs adsorbed on to PVA-MPA NFs.
Figure 2.8A showed the SEM image of PVA-LYS NFs after the adsorption of Ag NPs on
the surface of nanofiber. Similarly, PVA-MSA NFs and PVA-MPA NFs showed the Au
NPs on the surface of nanofibers (Figure 2.8 B and C). From the figure, it was observed
that the nanoparticles were adsorbed uniformly on the nanofiber surface without any
agglomeration or aggregation and uniformly distributed throughout the nanofiber surface.
Energy dispersive spectral (EDS) data also confirms the peaks corresponding to the
presence of nanoparticles on the nanofiber surface (Figure 2.9).
Figure 2.9: Energy dispersive spectra of A. AgNP adsorbed PVA-LYS NFs, B. AuNPs
adsorbed PVA-MSA NFs and C. AuNPs adsorbed on to PVA-MPA NFs.
Desorption study of the nanoparticles from the nanofibers was carried out using dilute
HCl (2M). It was observed that the desorption efficiency was very low as confirmed by
the EDS analysis of acid washed nanofiber (Figure 2.10). This may be due to strong
1
200 nm 100 nm 100 nm
A B C
57
0.0 0.8 1.6 2.4 3.2 4.0 4.8 5.6 6.4 7.2 keV
0
3000
6000
9000
12000
15000 C
O Ag
Ag
Ag
Pt Pt
Pt
0
3000
6000
9000
12000
C
O
Pt Pt
Pt Au
Au
Au
1.6 0.8 0.0 2.4 3.2 4.0 4.8 5.6 6.4 7.2
interaction of surface functional group with noble nanoparticles whereas nanofibers
having very low adsorption efficiency get desorbed by acid treatement. Furthermore, by
increasing the acid concentration, it is expected to cause damage to the organic fiber
surface. Hence current investigation mainly focused for one time adsorption study of
nanoparticle removal by surface modified nanofibrous membranes.
Figure 2.10: EDS spectrum of silver nanoparticles desorption from PVA-LYS NFs (A)
and PVA-MSA NFs (B) by using 2 N HCl.
The above results suggest that the surface modified PVA NFs have high adsorption
efficiency for removal of only Ag and Au nanoparticles in water. Such modified
nanofibers have several advantages which include high surface area, large number of
surface functional groups, easy to handle and can be fabricated according to the desired
ZAF Method Standardless Quantitative
Analysis
Element Mass% Error% Atom%
C K 56.75 0.09 72.96
O K 25.54 0.86 24.66
Ag L 15.30 1.11 2.19
Pt 2.41 1.17 0.19
Total 100.00 100.00
ZAF Method Standardless Quantitative
Analysis
Element Mass% Error% Atom%
C K 63.99 0.11 81.80
O K 17.45 0.70 16.75
Au M 11.62 1.01 0.91
Pt M 6.94 0.97 0.55
Total 100.00 100.00
58
product properties. In addition, membrane thickness and size can be controlled by
electrospinning technique.
2.5 Summary and Conclusions
We have demonstrated an approach for the preparation of surface modified PVA NFs with
different functional groups. These NFs were used for the nanoparticle filtration from
aqueous environment and showed different affinities towards metal nanoparticles,
depending on the active surface functional groups on the fiber surface. The adsorption
analysis showed that the amine and thiol modified PVA NFs had more than 90%
adsorption efficiency for Au NP, whereas unmodified PVA NFs had less than 20%
affinity. Similarly, PVA-LYS NFs showed good adsorption behavior (more than 90%)
toward the AgNP in aqueous environment. The adsorption of nanoparticles showed
uniform distribution throughout the nanofiber without any agglomeration. The modified
synthetic nanofibers are interesting material for removing nano contaminants from
aqueous environment.
59
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62
Chapter 3
Isolation and Fabrication of Cellulose Based
Nanofibers for Filtration of Nanoparticles from
Aqueous Environment
This chapter is published as a full paper in J. Mater. Chem., 2012, 22, 1985–1993; DOI:
10.1039/c1jm15018a.
63
3.1 Introduction
The use of nanoparticles in research,1 medicine
2 and for commercial purposes
3 has been
increasing owing to the interesting properties of nanomaterials. The usage of such
nanoparticles increases the risk of contaminating air and water which ultimately cause
serious environmental problems. Recent studies have shown that such nanoparticles can
enter living systems through water4 which possess health hazard to humans. The small
sizes of nanoparticles facilitate movements across cell membranes and potential to reach
various organs.5,6
The risks associated with nanoparticle contamination has been widely
reported, which include health concerns posed by cell necrosis and apoptosis,7, 8
alter
organelle structure and affect hormonal levels in mammals.9 Recently, the levels of
toxicity caused by gold (Au) and silver (Ag) nanoparticles to living systems were
documented by several groups. 10-12
So the removal of nanoparticles from environment is a
great challenge for future research.
Various established technologies have been used for water filtration and a few low
cost conventional methods, such as sand filter, charcoal, carbon and magnetic materials
are tested as affordable source for water purification.13-15
The main limitation for such
methods are low efficiency and potential contamination from particulate filter media.
However, membrane technology has caught attention in waste water purification due to
large surface area, high efficiency and low cost.16
Membrane separations such as
microfiltration and ultrafiltration are currently used for industrial, medical and analytical
applications for waste water treatment.17, 18
Particle filtration membranes solely depend on
the ratio of particle size to membrane pore size ratio. Electrospinning is one of the
methods for preparing nanofibrous membranes, where many polymers can be used to
64
control fiber diameter, pore size and morphology.19, 20
Synthetic nanofibers, with high
surface area to volume ratio could conceivably be an effective filtration system with good
mechanical properties.21, 22
Nanofibers have been used for filtration of microparticles,23
heavy metal ions24, 25
and microbial pathogens.26
However, most of the synthetic fibers are
non-biodegradable and pose disposal problems in the future. So naturally occurring
polymers are interesting owing to their easy availability, low cost and biodegradability. 27,
28 Due to wide availability cellulose based microfibers have been used for heavy metal
ion separation 29, 30
and pollutant removal from water.31-33
Chitosan is a biopolymer with
free amino groups, derived from shells of shrimp and other sea crustaceans34, 35
used for
many biomedical applications.36
Here we introduce a new method to filter nanoparticles from aqueous environment by
using cellulose based natural nanofibers. Nanofibers were isolated from raw sugarcane
bagasse by treating with bleach and alkali solution. To improve the extraction efficiency,
isolated cellulose nanofibers were coated with chitosan and tested for nanoparticle
filtration. Natural renewable fibrous materials are used to remove nano-particulate
contaminants from the aqueous environment.
3.2 Experimental section
3.2.1 Materials
Chitosan (degree of deacetylation was around 75 to 85%), silver nitrate (AgNO3),
hydrogen tetrachloroaurate trihydrate (HAuCl4·3H2O), sodium citrate and sodium
borohydride were purchased from Sigma Aldrich. All materials were used without further
purification. Glutaraldehyde, nitric acid (HNO3) and hydrochloric acid (HCl) were
purchased from Merck. Potassium hydroxide (KOH) was purchased from GDE chemicals.
65
The solvents used were of analytical grade and purchased from commercial suppliers.
Deionised water was used for preparing stock solutions. The sugarcane bagasse used was
the fibrous waste material obtained after extraction of juice and collected from a local
market.
3.2.2 Isolation of cellulose nanofibers from sugarcane bagasse
The procedure for the isolation of cellulose from sugarcane bagasse was slightly modified
from a reported procedure.37
Typically, raw bagasse was kept for soxhlet extraction in
toluene: ethanol mixture (2:1) for 6 h. After which, the bagasse was cleaned thoroughly
with water and kept in a solution of bleach (NaOCl): acetic acid (2:1) at 70 °C for 1 h. The
bleached bagasse was cleaned in water to remove residual bleach and acetic acid. The
cleaned fibers were then kept in 6% KOH solution for 16 h at room temperature and then
heated at 80 °C for 2 h. This yielded fiber that were composed of 80 - 90% nanofibers,
which were washed repeatedly using 0.6 M HCl and water in order to neutralize residual
amount of alkali present and air dried under room temperature.
3.2.3 Preparation of chitosan coated sugarcane nanofibers
Chitosan (0.5%) was dissolved in aqueous acetic acid (1 vol%) solution. Then 10 mg of
sugarcane nanofibers were soaked in chitosan solution (0.3 ml). In order to crosslink the
designed material, glutaraldehyde (50 μl) and phosphoric acid (50 μl) were added to it.
The resulting crosslinked product was cleaned thoroughly with water to remove excess
amount of unreacted acid and glutaraldehyde and dried using lyophilisation.
66
3.2.4 Preparation of PVP and citrate capped Au and Ag-nanoparticles
Gold nanoparticles were synthesized by the reduction of gold (Au3+
) ions using sodium
borohydride as the reducing agent. Preparation of Ag-nanoparticle was performed using
similar procedure using the reduction of silver nitrate. Both nanoparticles were capped by
using PVP and citrate as the capping agent. The concentration of nanoparticles used for
filtration was maintained at 2 x 10-4
M for all samples.
3.2.5 Study on adsorption behaviour of nanoparticle using chitosan coated cellulose
nanofiber
In order to test the filtration capacity of the only sugarcane nanofiber, chitosan coated
sugarcane nanofibers, and lyophilized chitosan (20 mg) was added to a 15 ml centrifuge
tube containing Au or Ag-nanoparticle solution (10 ml, 2.0 10-4
M), and kept for
constant shaking. Aliquots were taken from the solutions at definite intervals to quantify
the amount of nanoparticles adsorbed by nanofiber and analyzed using UV-Vis
spectroscopy. A time dependant adsorption intensity curve was plotted from the data
obtained.
3.2.6 Desorption study of nanoparticles from functionalized nanofiber
Au-nanoparticle adsorbed nanofiber was shaken with dilute HCl (5 ml, 2 M) for 2 h at
room temperature. Similarly Ag-nanoparticle adsorbed nanofiber was shaken with dilute
HNO3 (5 ml, 2 M) for 2 h. Acid desorbed samples were cleaned thoroughly with water
and rinsed with dilute sodium bicarbonate to neutralize protonated amino groups of
chitosan during acid treatment. Samples were washed with water and used for
readsorption studies with the same concentration and volume of nanoparticle solution.
67
3.2.7 Test for filtration efficiency of nanoparticle by chitosan coated cellulose
nanofiber
For this study, 100 mg of chitosan coated sugarcane nanofibers were packed in a small
glass column with 2 cm diameter. Then nanoparticle solution was passed through the
nanofibrous membrane and filtrate was collected in a test tube up to 20 ml. The filtration
efficiency was calculated by measuring the absorbance intensity before and after filtration
using UV-Vis spectroscopy.
3.3 Characterization
The morphology of the cellulose nanofibers was characterised using a field emission
scanning electron microscopy (FESEM, JEOL JSM-6701F). The samples were placed on
a double sided carbon tape and coated with a thin layer of platinum for viewing under
SEM. The size of the nanoparticles was determined using transmission electron
microscope (TEM, JEOL JEM 2010). After filtration, energy dispersive X-ray
spectroscopy (EDX, JEOL JED 2300) was employed to confirm the adsorption of
nanoparticles onto the designed nanofibrous material. The quantitative measurements of
nanoparticles were carried out using UV-Vis spectroscopy (Shimadzu-1601 PC
spectrophotometer). The presence of functional groups on the surface was characterized
by attenuated total reflectance–infrared (ATR- IR) spectrophotometer.
3.4 Results and discussion
3.4.1 Isolation of cellulose nanofibers from sugarcane bagasse
Cellulose source from plant byproducts are cheap and requires very little processing.
Sugarcane bagasse has three main components with approximate cellulose content of 40%
to 45%, hemicellulose content of 28% to 30%, and lignin content of 19% to 21% and is
68
solely dependent on the source.38
Lignin consists mainly of polymers of arylpropane units
which bind the neighbouring cells and form a highly crosslinked network. Hemicellulose
is a polysaccharide polymer generally found between cellulose and lignin. Due to smaller
degree of polymerisation and amorphous nature, properties of hemicellulose are marginal
as compared to cellulose.30
Cellulose compose of anhydroglucose rings linked via β-1,4-
glycosidic bonds.
This investigation focuses mainly to extract neat cellulose fibers by the removal of
lignin and hemicelluloses from the sugarcane bagasse using various chemical methods. A
two steps chemical treatment were employed, in the first step, bagasse was treated with
sodium hypochlorite to remove lignin. Sodium hypochlorite dissolved in water to form
hypochlorous acid and hypochlorite ions which could react to produce chlorine radicals.
These radicals are the active species in the degradation of lignin to give phenoxy and
mesomeric cyclohexadienyl radicals, which led to degradation of lignin.39
In second step,
the bleached fibers were subjected to alkaline treatment which removed the hemicellulose
portion, leaving behind pure cellulose nanofibers.
Figure 3.1: SEM image of cellulose nanofibers bleached with sodium hypochloride and
acetic acid (A) and with Bleaching:HCl (10:1 vol%) (B).
200 nm 200
nm
A B
69
Alkaline treatment with KOH ionises the hydroxyl groups in cellulose and hemicelluloses,
resulting in the formation of alkoxide ions. This removes the hydrogen bonding and
enhances the stability for the removal of hemicellulose and residual lignin.40
When the solution of sodium hypochlorite and acetic acid was used as the bleaching
agent, nanofibers were obtained after one cycle of bleaching followed by KOH treatment
(Figure 3.1A). However, when HCl was used along with sodium hypochlorite, the
extraction of nanofibers was difficult and the yields were poor. A mixture (2:1) of
hypochlorite and acetic acid was used as the bleaching agent for subsequent nanofiber
extractions (Figure 3.1B). From the SEM images, it could be seen that cellulose
nanofibers with diameters of 30 - 40 nm and approximate lengths in the micrometer range
were obtained after the extraction. The isolated cellulose nanofiber showed a 3D porous
structure with a surface area of 1.17 m2 g
-1.
3.4.2 Preparation of sugarcane and chitosan coated sugarcane nanofibrous
membranes
Sugarcane nanofibrous membranes were fabricated from cellulose nanofibers extracted
from sugarcane by crosslinking with glutaraldehyde in acid medium, which led to the
formation of crosslinked network through the reaction of cellulose with glutaraldehyde to
form acetal linkages.41
Chitosan is a biopolymer generally derived from N-deacetylation
of chitin which is the second most abundant natural polymer. It is a copolymer of 2-
amino-2-deoxy-β-D-glucopyranose and 2-acetamido-2-deoxy-β-D-glucopyranose with the
repeating units linked via β-1,4-glycosidic bonds.42, 43
70
1000 1500 2000 2500 3000 3500 4000
Ab
so
rba
nce
1256 c
m-1
C
A
Wavenumber (cm-1)
B
15
80
cm
-1
The chitosan was coated on the extracted sugarcane nanofibers and crosslinked with
glutaraldehyde in an acidic medium. Two mechanisms had been proposed for this
crosslinking; via a Schiff’s base reaction mechanism and an acetal reaction mechanism.44,
45 Cellulose nanofibers and chitosan coated nanofibers were characterised using ATR-IR
as shown in Figure 3.2. It can be seen that the IR peaks of crosslinked sugarcane
nanofibers and chitosan-coated nanofibers were similar except for the peaks at 1580 cm-1
corresponds to the N-H bending mode. Similarly there was a small peak at 1256 cm-1
which is due to C-N stretching vibration of amine groups on chitosan coated sugarcane
nanofibers. Since chitosan was coated on the surface of the cellulose nanofibers, the peak
intensity was comparatively smaller.
Figure 3.2: Attenuated total reflectance-Infrared spectra of sugarcane nanofiber (A),
chitosan (B) and chitosan coated sugarcane nanofibers (C).
3.4.3 Surface morphology of chitosan coated sugarcane nanofibers
Chitosan was coated on the cellulose nanofibers which provide a high surface area
scaffold, having efficient functionality (amine group) on the surface. The crosslinked
chitosan coated nanofiber was dried using lyophilizer and SEM image showed a uniform
71
morphology for chitosan coated cellulose nanofiber (Figure 3.3A). From the figure, it can
be seen that crosslinked chitosan-coated sugarcane nanofibers are porous in nature,
whereas cross linked chitosan showed flake type structure (Figure 3.3B). Furthermore, it
was observed that the fibers had a highly interconnected network structure which
reinforced the mechanical rigidity of the material. The lyophilised crosslinked fibers used
for filtration showed good mechanical stability throughout our investigation. For all
designed fibers, there was no significant swelling in aqueous medium. As a result, these
membranes had high specific area per unit mass with suitable functional groups exposed
for the extraction of nanoparticles.
Figure 3.3: SEM images of chitosan coated cellulose nanofiber (A) and crosslinked
chitosan (B)
3.4.4 Preparation of Au and Ag-nanoparticles
Nanoparticles (Au and Ag) were synthesized using reduction of metal ions into
nanparticles.46, 47
Nanoparticles have high surface area and high surface energies, which
led to unstability and aggregation of these particles. In order to stabilise the nanoparticles
and prevent aggregation, citrate ions or poly(vinylpyrrolidone) (PVP) were used as
capping agents.48, 49
100 µm
200 nm
A B
72
PVP or citrate capped Au-nanoparticles showed absorption maxima at 520 nm and
519 nm respectively, which were similar to the reported values in literature.50
Similarly,
Ag-nanoparticles showed the absorption maxima at 397 nm and 389 nm. The values
obtained were consistent with the value reported (390 nm) in the literature.51
The
synthesized nanoparticles had diameters of 10 to 15 nm as shown by the TEM images
(Figure 3.4).
20 nm20 nm 20 nm 20 nm
Figure 3.4: UV-visible spectra of PVP and citrate capped Au (A) and Ag- nanoparticles
(B); TEM images of PVP capped Au-nanoparticles (C), citrate capped Au- nanoparticles
(D), PVP capped Ag-nanoparticles (E), and citrate capped Ag- nanoparticles (F). Inserts
show the corresponding camera images of nanoparticle solutions.
3.4.5 Extraction of nanoparticles from aqueous environment using nanofibers
To test the extraction efficiency, a known amount of nanofibers were kept in nanoparticle
solution in a plastic vial and shaken for 10 h. At particular time intervals, aliquots were
collected to find the remaining concentration of nanoparticles in the solution.
A B
D C E F
73
B
C
D
A
After Before
E
F
G
H
After Before
After Before
After Before
74
Figure 3.5: Time-dependent adsorption efficiency of chitosan-coated sugarcane
nanofibers, crosslinked sugarcane nanofibers and crosslinked chitosan for PVP capped
Au-nanoparticles (A), PVP capped Ag-nanoparticles (B), citrate capped Au- nanoparticles
(C) and, citrate capped Ag-nanoparticles (D); UV-visible spectra of solutions before and
after adsorption for PVP capped Au-nanoparticles (E), PVP capped Ag- nanoparticles (F),
citrate capped Au-nanoparticles (G), and citrate capped Ag- nanoparticles (H). Inserts
show the corresponding decolourization of the nanoparticles solutions before and after
adsorption.
The extraction efficiency of the nanofibers was determined by using changes in intensity
at absorption maximum. The standard curve for Au and Ag-nanoparticles were plotted
using the absorption intensity at λmax for standard solutions of known concentrations. This
curve was used to translate the absorption of unknown sample to its concentration. Figure
3.5 shows time dependant filtration efficiency of the cellulose nanofibers coated with
chitosan for filtration of different nanoparticles. Chitosan coated cellulose nanofibers
showed filtration efficiency of 35 - 40% for Au and 40 - 50% for Ag-nanoparticles within
30 minutes. Furthermore, filtration capacities reached ~80% for Au and ~90% for Ag-
nanoparticles after 5 h.
Comparing Au and Ag-nanoparticles capped with the same capping agent, chitosan-
coated sugarcane nanofibers had higher adsorption efficiencies toward Ag- nanoparticles.
Furthermore, as illustrated in Figure 3.5, crosslinked sugarcane nanofibers showed
negligible adsorption efficiencies for all nanoparticles. Amino groups, considered as
borderline soft bases, can interact with the Ag-nanoparticles with high efficiency.52
Chitosan was chosen as the coating material due to its biocompatibility and
biodegradability. In comparison with pure chitosan, chitosan coated sugarcane nanofibers
showed high extraction efficiency towards nanoparticles. This was due to the presence of
multiple amino functional groups on the chitosan-coated sugarcane nanofibers which
75
facilitate the extraction of metal nanoparticles.
To investigate the effect of capping agents, nanoparticle extraction analyses for both
neutral PVP capped and negatively charged citrate capped nanoparticles were compared.
From Figure 3.6, there was no significant difference in extraction efficiencies regardless
of the capping agents used. It is conceivable that the effective charges of the capping
agents were insignificant in determining the extraction efficiency of nanoparticles.
Furthermore, all nanofibers were also used for a second round extraction experiment
using fresh nanoparticles solution (2 x 10-4
M). The purpose of this study was to ensure
that nanofibers can be re-used for maximum effectiveness toward nanoparticle filtration.
As expected, the results of the second time extraction showed a substantial drop in the
efficiency i.e. decreased to ~40% for citrate and PVP capped Au-nanoparticles and
Figure 3.6: Graphs for time-dependent readsorption efficiency of chitosan-coated
sugarcane nanofibers for subsequent adsorptions of PVP capped Au-nanoparticles (A),
citrate capped Au-nanoparticles (B), PVP capped Ag-nanoparticles (C), and Citrate
capped Ag-nanoparticles (D).
A B
C D
76
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
keV
0
1500
3000
4500
6000
7500
9000
10500
12000
Co
un
ts
C
O
Ag
Ag
Ag
PtPt
Pt
Pt Pt
0.00 0.80 1.60 2.40 3.20 4.00 4.80 5.60 6.40 7.20
keV
0
800
1600
2400
3200
4000
4800
5600
Cou
nts
C
O
PtPt
PtAu
Au
Au
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
keV
0
400
800
1200
1600
2000
2400
2800
3200
3600
4000
Cou
nts
C
O
Ag
Ag
Ag
PtPt
Pt
Pt Pt
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
keV
0
400
800
1200
1600
2000
2400
2800
3200
3600
Cou
nts
C
O
PtPt
Pt
Pt Pt
Au
Au
Au
Au Au
IMG1 100 µm
Pt M 100 µm
C K 100 µm
Au M 100 µm
O K 100 µm IMG1 100 µm
Ag L 100 µm
C K 100 µm
Pt M 100 µm
O K 100 µm
~70-80% for Ag-nanoparticles. As for Au-nanoparticles, the efficiency was decreased
significantly and no attempts were made for third extraction.
100 nm
100 nm 100 nm
A B
Figure 3.7: SEM images of Au (A) and Ag-nanoparticles (B) adsorbed nanofiber mat.
A B
C D
Figure 3.8: EDX spectra of chitosan-coated sugarcane nanofibers after adsorption of PVP
capped Au-nanoparticles (A), PVP capped Ag-nanoparticles (B), citrate capped Au-
nanoparticles (C) and, citrate capped Ag-nanoparticles (D). Inserts showed the
corresponding elemental mappings of the nanoparticles.
77
But for citrate and PVP capped Ag-nanoparticles, extractions were carried out for third
time without precleaning the fibers. From the graph, it was observed that 3rd
time
extraction efficiency was low (20 - 30% after 12 h).
So in conclusion, when chitosan coated sugarcane nanofibers were reused without
surface cleaning for subsequent extraction of nanoparticles, it showed a decrease in
efficiency. Furthermore, the EDX spectra of chitosan-coated sugarcane nanofibers after
adsorption showed peaks corresponding to Au and Ag-nanoparticles (Figure 3.8).
Elemental mappings showed that nanoparticles were evenly distributed on the nanofiber
surface. Such data supports for high extraction efficiency of nanofibers during the
extraction process.
3.4.6 Desorption and time-dependent readsorption efficiency analysis
Desorption of nanoparticles from nanofiber surface was investigated by acid treatment to
examine the reusability. Cellulose and chitosan are known to undergo cleavage at β-1,4-
glycosidic bonds when subjected to treatment with dilute acids at high temperatures or
treatment with concentrated acids at low temperatures.53, 54
Table 3.1: Elemental analysis of concentration of Au in hydrochloric acid (A), and silver
in nitric acid (B) after desorption and corresponding desorption efficiency
Nanoparticles
Concentration of Au in
Acid/ ppm
Desorption Efficiency/
%
PVP capped Au 0.21 3
Citrate capped Au <0.10 <1
Nanoparticles
Concentration of Ag in
Acid/ ppm
Desorption Efficiency/
%
PVP capped Ag 0.30 7
Citrate capped Ag 0.54 13
78
In our experiment, mild conditions were used for desorption studies by socking the fibrous
mat with dilute acids (2M) for 2 h at room temperature. For effective desorption, dilute
hydrochloric acid and nitric acid were used for desorption of Au and Ag-nanoparticles,
respectively.55, 56
The time dependent extraction studies of acid treated fiber samples were compared
with untreated fibers (Figure 3.9 A-D). Mild acid treatment on chitosan-coated sugarcane
nanofibers adsorbed with nanoparticles gave rise to low desorption efficiencies.It is
conceivable that nanoparticles interact strongly with chitosan-coated sugarcane nanofibers
and hence were difficult to remove using acid treatment (Table 3.1).
Figure 3.9: Time-dependent adsorption efficiency of chitosan-coated sugarcane
nanofibers for first time adsorption, second time without and with acid treated nanofiber
using PVP capped Au-nanoparticles (A), PVP capped Ag-nanoparticles (B), citrate capped
Au-nanoparticles (C), and citrate capped Ag-nanoparticles (D).
C
A B
D
First time
Second time without acid treatment
Second time with acid treatment
79
Hence, no significant improvement in extraction efficiency was observed after acid
treatement on chitosan-coated sugarcane nanofibers. Such nanofibers could be considered
as controlled disposable adsorbent for nanomaterials, owing to the low cost of preparation
and biodegradability.
3.4.7 Filtration of nanoparticles by using chitosan coated cellulose nanofibers
In order to check the filtration efficiency, the nanoparticles were filtered by passing
through a column packed with chitosan coated sugarcane nanofiber (Figure 3.10). The
nanoparticle concentrations of the original solution and after extractions were quantified
and compared using UV-Vis spectroscopy. The filtration of 10 ml nanoparticle solution
through a column packed with 100 mg of nanofibers gave almost 98 to 99% filtration
efficiency. Repeated filtration of additional 10 ml nanoparticle solution gave about 85 to
95% nanoparticle filtration efficiency (Table 3.2).
Figure 3.10. Camera images of filtration of different nanoparticle solution using chitosan
coated sugarcane nanofiber i.e. PVP capped Au-nanoparticles (A), PVP capped Ag-
nanoparticles (B), Citrate capped Au-nanoparticles (C) and citrate capped Ag-
nanoparticles (D). Bottom portion of tube is the pure water after filtration of nanoparticle
solution.
Pure water after
filtration
Chitosan coated
cellulose nanofiber
Original nanoparticle
solution
A B
B
C D
80
Table 3.2: Summary of filtration of different nanoparticles solution after passing through
a column filled with chitosan coated nanofiber.
PVP capped Au
nanoparticle
PVP capped Ag
nanoparticle
Citrate capped
Au nanoparticle
Citrate capped
Ag nanoparticle
Time/min Efficiency/ % Efficiency/ % Efficiency/ % Efficiency/ %
45 (first 10 ml) 98.15 99.50 97.09 99.55
Next 10 ml 86.62 94.64 84.44 92.45
This decreased efficiency may be due to, an increase in surface coverage with
nanoparticles and lack of active binding sites on the nanofiber surface.
3.5 Summary and Conclusions
We have demonstrated a novel approach for the fabrication of cellulose based nanofibers
with suitable functional groups to be used for nanoparticle extraction from aqueous
medium. The coated nanofibers were found to be highly efficient in extracting Au and Ag-
nanoparticles with very high efficiency. The incorporation of chitosan on the fiber surface
was found to enhance the filtration capacity. Presence of the adsorbed nanoparticles on the
nanofibers was confirmed by SEM and EDX. Biocompatibility, biodegradability, cost
effectiveness and the ease of handling of designed nanofibers make it a viable method for
large scale filtration systems.
81
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86
Chapter-4
Fabrication Fe (III) Immobilized PVA Nanofiber
as a Sorbent for Arsenic Removal from Water
87
4.1 Introduction
The main risk of chronic arsenic poisoning comes from contaminated water resources in
many parts of the world.1-3
Arsenic exists in different oxidation states and As (III) is more
likely to occur in anaerobic ground waters, while As (V) is more prevalent in surface
water.4 Arsenic content is usually introduced into our water from different industrial
wastewater discharge and geochemical sources.5 A lethal level results in acute arsenic
poisoning and multiple organ failure, leading to mortality.6 Currently, the World Health
Organisation deems arsenic concentration below 10 μgL-1
in water as safe level for
consumption.7
Symptoms of arsenic poisoning include gastritis, nausea, partial paralysis
and blindness.8-13
Therefore, the current research focus on developing a more cost-
effective arsenic removal method which may help to reduce serious health threats to
society.
Various arsenic removal systems have been developed, such as physical, chemical
and other methods in many laboratories. Physical methods such as reverse osmosis14
and
electrofiltration15
are the most effective techniques for the arsenic removal from water
supply. Similarly, chemical methods include chemical coagulation16
and usage of ion
exchange resins.17
However, these methods are slow and required technological
knowledge, which limits many practical use and not cost effective to be applied large scale
in rural areas. Furthermore, different researchers have attempted the arsenic removal by
using metal oxides,18, 19
calcium carbonate,20, 21
silicate clays22, 23
and organic materials.24
The adsorption processes are highly sensitive to pH changes and soil conditions, where
minerals such as other oxyanions may compete with arsenic ions for adsorption sites and
reduce the extraction efficiency.25
88
Other innovative methods which involve usages of locally grown aquatic plants for
arsenic removal near abandoned mine sites and mineral deposit sites.26-29
Safe disposals of
these arsenic rich plants would also be a serious concern, as normal decomposition would
merely release their arsenic content back into the soil or water, rendering the efforts self-
defeating.
Recently, much efforts have been focused on the removal of pollutants using naturally
occurring cellulosic waste materials such as rice husk, coconut husk, oak bark, pine wood,
phosphorylated orange waste30
and other plant biomass.31
Similarly other adsorbents such
as activated carbon,32
chitosan,33
activated alumina,34
carbon char,35
titanium dioxide,36
and iron oxide-coated sand were also used for arsenic removal.37
Hence it is very
important to design a cheaper way with high arsenic removal efficiency for practical use.
In this investigation, our research efforts were focused on the development of
nanofibrous membranes prepared by electrospinning of polymer solutions and used for the
filtration of arsenic from water. Fe3+
ions incorporated PVA nanofibrous membranes were
prepared in laboratory scale. Our approach was to incorporate Fe3+
ions in a hydrophilic
polymer matrix and used for the preparation of nanofiber mats for arsenic removal from
water. Fe3+
ions act as crosslinker for PVA and an active component for extraction of
arsenic anions from water.
4.2 Experimental section
4.2.1 Materials
Poly(vinyl alcohol) (PVA) with weight average molecular weight of 146,000-186,000,
Sodium arsenite (NaAsO2) and sodium arsenate (Na2HAsO4.7H2O) were purchased from
Sigma Aldrich. FeCl3.6H2O was purchased from Biolab Int Ltd and NH4OH was
89
purchased from mallinckrodt Baker. All other chemicals used for the study were analytical
grade and ultrapure water was used for preparation of solutions. Prior to experiment, all
glasswares were thoroughly washed. PVA solution (10 wt%) was prepared by stirring
PVA granules at 80 °C in Millipore water for 2 hrs. To the PVA solution, FeCl3 was
added in the ratio of (1 : 2 wt%) and mixed properly.
4.2.2 Fabrication of PVA/Fe (III) polymeric nanofiber by electrospinning technique
The PVA/Fe3+
ion nanofibers (PVFeNF) were fabricated using electrospinning technique.
The electrospinning setup consisted of a hypodermic syringe, a needle with flat tip and a
programmable syringe pump (Optrobio Technologies Pte Ltd) were used to feed the
polymer solution.38
A positive electrode of high voltage power supply was connected to
the syringe needle and negative electrode was connected to a grounded copper plate
covered with aluminum foil. Glass cover slips for collection of fibers were placed on the
collector plate. For preparation of nanofiber, a positive high voltage supply of 16,000 V
was applied and the distance between the tip of the needle to the collector distance was
maintained at 12 cm. The feed rate of the polymer solution was set as 3 μl/min. The
resultant nanofibers were kept in a desiccator containing concentrated liquid ammonia for
6 hrs for complete cross-linking. Subsequently, the nanofibers were cleaned thoroughly
with distilled water, dried, and used for arsenic removal from water.
4.2.3 Adsorption analysis of arsenic using designed nanofiber from water
The designed PVFeNF (10 mg) was kept into different concentration of As (III) and As
(V) solution (20 ppm, 40 ppm, 60 ppm, 80 ppm and 100 ppm) in 15 ml falcon tubes and
shaked. Appropriate amounts of solution were taken from each tube after 0.5 hr, 1 hr, 2
hrs, 4 hrs, and 6 hrs of shaking. Final concentration of arsenic ions in the sample was
90
determined using Inductively Coupled Plasma – Optical Emission Spectrometer (ICP-
OES). From the results, the effectiveness of the PVFeNF for extraction of arsenic ions
from water was calculated. Arsenic adsorbed nanofibers were kept for shaking with 1M
HNO3 for 4 hrs at room temperature. The nanofibers were removed and the amount of
arsenic in acid solution was measured again by ICP-OES to calculate the desorbed amount
of arsenic.
4.3 Characterization
The morphology of the polymeric nanofiber was observed using a field emission scanning
electron microscopy (FESEM, JEOL JSM-6701F). The electrospun nanofibers were
collected on glass cover slips and were mounted on copper stubs with a double sided
conducting carbon tape and coated with platinum. All IR measurements were carried out
in attenuated total reflectance (ATR) mode (Thermo Scientific) in the range of 600 - 4000
cm-1
and the spectrum was evaluated using Ominic software. Thermal properties of each
lyophilized crosslinked samples were studied using differential scanning calorimetry (TA
instruments - DSC 2920) at a heating rate of 10 °C/min in an inert N2 atmosphere.
Quantitive determination of arsenic removal was carried out using 5300 DV ICP-OES.
4.4 Results and discussion
4.1.1 Morphological and spectroscopic analysis
To prepare highly porous nanofibrous membranes, electrospinning technique was used. A
mixture of PVA and FeCl3 in proper ratio (1 : 2 wt%) was used to prepare nonwoven
nanofibrous membranes. Electrospinning parameters were optimized properly to get
nanofibers without any bead formation. All nonwoven nanofibers were deposited on a
plate form a mat with a fiber diameter ranging from 600 to 800 nm (Figure 4.1).
91
Figure 4.1. FESEM image shows morphology of PVA/Fe (III) composite nanofiber and
Energy-Dispersive X-ray Spectroscopy (EDX) shows the quantitative presence of Fe in
the nanofiber.
For practical applications, the water soluble nanofibers must be crosslinked to reduce the
solubility and swelling. For this, a saturated atmosphere of ammonia was made inside a
desiccator and nanofibers were kept inside for 6 hrs for complete crosslinking. The
resultant fibers were cleaned thoroughly and incorporation of Fe3+
ions on the nanofiber
mat was investigated using EDX spectroscopy (Figure 4.1). From EDX, it can be seen that
the peak intensity for Fe unit is high indicating the incorporation on the nanofiber matrix.
Furthermore, the elemental analysis data revealed a high proportion of Fe3+
ion in the
uncrosslinked and crosslinked sample. However, high amount of chlorine content was
Table 4.1. Elemental analysis of PVAFe uncrosslinked nanofiber and PVAFe Crosslinked
nanofiber
Sample ID C (wt%) H (wt%) Cl (wt%) Fe (wt%)
PVFe NF Uncross 26.13 5.24 17.57 11.99
PVFe NF Cross 25.51 4.22 0.31 21.17
0
160
320
480
640
800
960
Co
un
ts
C
N
O
Cl
ClFeFe
Fe
FePt
Pt
Pt Pt
0.1 mm0.1 mm0.1 mm0.1 mm0.1 mm
10 µm
92
1000 1500 2000 2500 3000 3500
B
Ab
sorb
an
ce %
Wavenumber (cm-1)
A. PVA NF
B. PVFeNF CROSS
A
detected for noncrosslinked sample, whereas no chlorine was detected for crosslinked
nanofibers (Table 4.1). This result indicates that the coordination of Fe3+
ions with a PVA
matrix and incorporation of ammonia removes the chlorine from polymer matrix.
Crosslinking of PVA with the help of Fe3+
ions involves a coordination mechanism.
As the Fe3+
ion is a high spin d5 species, it generally forms hexacoordinate complex with
suitable ligands.39
In the coordination process Cl- can be replaced by –OH groups in basic
medium by the formation of hydroxo- and oxo-polynuclear complexes.40
To study the
coordination behavior of Fe3+
ions and PVA, IR spectroscopy was used. After
coordination, the surface of nanofibers was characterized in the ATR mode and the
spectrum is shown in Figure 4.2. A broad absorption at 3000 - 3600 cm-1
corresponds to
the -OH stretching vibrations of the hydroxyl group of PVA. As atactic PVA contain a
few acetate moiety, C=O stretching absorption peak of ester group at 1713 cm-1
was also
observed. There was a sharp peak at 1090 cm-1
corresponds to the stretching band of C-O
group of PVA which overlaps with the Fe-O-C linkage of PVFeNF system.
Figure 4.2: ATR-IR spectra of PVA nanofiber and PVA/Fe3+
crosslinked nanofiber
93
40 60 80 100 120 140 160
He
at
flo
w
Temperature (°C)
PVA NF
PVFE NF CROSS
Tg increases
160 180 200 220 240
Hea
t fl
ow
Temperature (°C)
PVA NF
PVFE NF CROSS
A B
So the formation of Fe (III)–PVA complexes did not introduce any significant changes in
the IR spectrum, which closely matched with the previous report.41
4.4.2 Structure property analysis of the resultant nanofiber
Incorporation of crosslinks to the polymeric system is to reduce the flow behavior,
solubility and swelling of the polymer in water. Crosslinks along the polymer chains
reduce the flow behavior and sliding motion between polymer chains. However, in
between the crosslinks the polymer segments remained flexible.
Figure 4.3: Curves for glass transition temperature (A) and curves for melting point (B)
analyzed by DSC analysis of PVA and Fe3+
crosslinked nanofiber.
To gain a better understanding of the effect of corosslinking towards the molecular
mobility of the PVA-Fe3+
ion complex, thermal analysis was carried out at a temperature
range from room temperature to 250 °C. The glass transition behavior was recorded which
corresponds to the second heating cycle for PVA nanofiber and Fe3+
incorporated PVA
nanofiber is shown in Figure 4.3.
94
The effect of crosslinking reflects the transition from glassy to rubbery behavior.
From the graph, it can be seen that upon crosslinking, glass transition temperature (Tg) of
PVFeNF increased to 80 °C from 71 °C for PVANF. Similarly from the melting peak
data, PVA nanofiber showed a melting point at 195 °C, whereas no significant melting
point was observed for PVFe NFs. Melting is a first order transition which occurs with
increase in entropy and enthalpy of polymeric network. However, due to highly
crosslinked network formation due to stable coordination bonds between Fe (III) ions and
hydroxyl groups of PVA, it is difficult to melt such a polymer complex.
4.4.3 Filtration efficiency of arsenic using designed nanofiber
In natural water, arsenic exists mainly in the form of both As (III) and As (V). The
distribution of As (III)/As (V) ratio depends on the pH and other parameters. Owing to
this, our study was focused on the removal of both As (III) and As (V) from water. To
investigate the arsenic extraction capacity, a known amount of designed nanofibers were
kept for constant shaking with different concentrations of arsenic solution.
Figure 4.4: Concentration dependent (20 PPM, 40 PPM, 60 PPM, 80 PPM and 100 PPM)
adsorption of As (III) and As (V) ions using Fe3+
coordinated PVA nanofiber as a function
of time.
0
20
40
60
80
100
0 1 2 3 4 5 6 7
As (III) 20 PPM As (III) 40 PPM
As (III) 60 PPM As (III) 80 PPM
As (III) 100 PPMArs
en
ic (
III)
re
mo
va
l /%
Time (h)
0
20
40
60
80
100
0 1 2 3 4 5 6 7
As (V) 20 PPM As (V) 40 PPM
As (V) 60 PPM As (V) 80 PPM
As (V) 100 PPMArs
en
ic (
V) re
mo
va
l /%
Time (h)
95
Nanofibers were removed and concentration of arsenic remained in solution was measured
and plotted against extraction time (Figure 4.4) for As (III) and As (V). The objective of
this study was to find out the equilibrium time of adsorption and the amount of arsenic
removal during the extraction with nanofibers. The adsorption profile demonstrates that
the maximum adsorption was achieved within 30 min for all concentration ranges.
Typically for As (III) solution (20 ppm), the adsorption efficiency was around 78% in 30
min time and 95% after 4 h of extraction. When a high concentration (100 ppm) was used
with same amount of fibers, it reaches around 50% adsorption efficiency in 30 min and
after 4 h, it was 54%. Similarly for the case of As (V), the percentage of arsenic removed
was low as compared to As (III) at same concentration range using same amount of
nanofibers. Furthermore, the high content of arsenic species on the nanofiber matrix after
extraction from water was examined using EDX analysis (Figure 4.5). It is well
understood that the adsorption efficiency depends on the surface charge of the adsorbent
and the charge of the arsenic anions. Complexation of arsenite or arsenate with the Fe3+
ions incorporated filters have been used by various researchers.41-43
Figure 4.5: EDX spectra of arsenic adsorbed PVAFe nanofiber
0
160
320
480
640
Co
un
ts C
O
FeFe
Fe
FeAs
AsPt
Pt Pt
ZAF Method Standard less
Quantitative Analysis
Element Mass% Error% Atom%
C K 21.08 0.12 46.10
O K 16.02 0.20 26.30
Fe K 51.75 0.60 24.34
As K 8.15 7.46 2.86
Pt M 2.99 0.59 0.40
Total 100.00 100.00
96
We have used PVA membrane to check the extraction efficiency, but it was found to
be very low (less than 10% for 20 ppm for As (III), adsorbent amount used - 10 mg),
whereas Fe3+
loaded PVA nanofiber showed more than 90% extraction efficiency at the
same working condition. As incorporation of Fe3+
increases the arsenic anion extraction in
great extent, we gave more emphasis on Fe3+
immobilized PVA nanofiber for the removal
of arsenic from water.
A mechanism was proposed using spectroscopic data which showed arsenate and
arsenite form bidentate, binuclear complexes with the Fe3+
ions. The presence of free d-
orbital on Fe (III) atom in the PVA/Fe complex has ability to form complexes and
hydroxo bridges.44
The highly electron rich arsenic species act as strong ligand for iron
(III) ions.45
It was also hypothesized that As (III) preferably binds with Fe (III) ions and
other arsenic species adsorb via physical adsorption or weak ligand exchange reaction.46
The percentage removal of arsenic ion was calculated as
((Ci –Cf)/Ci) × 100
[Ci = initial concentration, Cf = final concentration of arsenic solution]
To check the reusability of the nanofiber after extraction, desorption study of the
arsenic ions adsorbed nanofibers was carried out using dilute HNO3 (1M). From the ICP
study, a desorption efficiency of 50 - 60% was observed. At this acid concentration, the
fiber lost its normal morphology. Hence current investigation focused on developing
disposable PVFeNF membranes for arsenic removal from water. All data corresponds to
the first time adsorption experiments analyzed in terms of the linear Freundlich and
Langmuir model and corresponding parameters were evaluated.
97
1 + KaCe
qmKaCe
qe=
The Freundlich isotherm47
equation can be written as
qe = KF Ce
Linear form of the equation is; Log (qe) = Log (KF) + Log (Ce)
Where qe is the amount adsorbed (mg/g), Ce the equilibrium concentration of the
adsorbate (mg/l) and KF and n are Freundlich constants related to adsorption capacity and
adsorption intensity. The Freundlich constants KF and n, were calculated from the plots of
Log (qe) Vs Log (Ce) for both As (III) and As (V) and is shown in Figure 4.5. From the
graph, it can be seen that the adsorption of both As (III) and As (V) on PVFeNF follows
Freundlich isotherm. The calculated values for adsorption capacity (KF) and intensity (1/n)
derived from plot with the R2 values are summarized in Table 4.1.
Similarly, Langmuir equation48
was used for our study and is expressed in the
following form
The linear form of the above equation is given by :
Where qe is the amount adsorbed (mg/g), Ce the equilibrium concentration for the
adsorbate (mg/l) and qm and Ka are Langmuir constants denoting the adsorption capacity
(mg/g) and energy of adsorption (l/mg). The plot against Ce/qe Vs Ce gives a straight ine.
The Langmuir constants are calculated from the slope and intercept and given in the Table
4.2. It is observed that the value of qm is 66.67 mg/g for As (III) which is very high
1/n
=
Ce
qe
1
qm Ce +
1
Kaqm
1
n
n
n
98
0
0.4
0.8
1.2
1.6
2
2.4
2.8
0 10 20 30 40 50 60 70 80
Ce
/ q
e
Ce
Langmuir Isotherm
As(III)
As(V)0
0.4
0.8
1.2
1.6
2
0 0.4 0.8 1.2 1.6 2
Lo
g q
e
Log Ce
Freundlich isotherm
As(III)
As(V)
Table 4.2: Parameters of Freundlich and Langmuir isotherms for As (III) and As (V)
sorption on the Fe3+
loaded PVA nanofiber.
Sorbate
Freundlich isotherm Langmuir isotherm
KF
(mg/g)
n R2
qm
(mg/g)
Ka
(l/mg)
R2
As(III) 1.06 2.53 0.9514 66.67 0.0775 0.9251
As(V) 0.77 2.67 0.9611 35.97 0.0553 0.9394
Figure 4.6: Isotherm plots (Freundlich and Langmuir isotherms) for As (III) and As (V)
sorption on the Fe3+
loaded PVA nanofiber.
as compared to 35.97 mg/g for As (V). It is well understood that the extraction of the
arsenic ions depends on the active functional groups on the fiber surface and the oxidation
state of the arsenic species. As R2 value is more for Freundlich isotherm, so the adsorption
appears to be through physisorption. But from the acid desorption value of 55 - 65% due
to strong interaction, and the R2
value close to Langmuir constant, it can be concluded that
our process follows both adsorption processes. Furthermore, the Langmuir constant (Ka )
99
is in between 0 to 1 range, and Freundlich constant (n) is in the range 0 - 10, indicating the
adsorption is a favorable process.
4.5 Summary and conclusion
A novel approach for the preparation of Fe3+
coordinated PVA nanofibrous membranes
via electrospinning technique is reported. The characterization of the composite nanofiber
was carried out using FESEM and EDX. FESEM image showed the diameter of the
nanofiber as 600 - 800 nm. The presence of Fe ions in the nanofiber was examined using
the EDX analysis and found to be around 21%. The structure - property relationship was
investigated in detail using thermal analysis. It was observed that the Fe3+
coordinated
PVA enhances the glass transition temperature with the absence of m.p.. This may be due
to low interchain molecular mobility through strong Fe ion induced crosslinking between
polymer chains. Nanofibers were tested for its effectiveness in extracting arsenic (As III
and As V) contaminants from aqueous environment. From the results, it was observed that
the composite nanofiber has very high efficiency (66.67 mg/g for As (III) and 35.97 mg/g
for As (V)) towards the arsenic removal from water. Compared to other available
adsorbents such as carbon particles incorporated with Fe (III) salts, our method is more
practical due to the absence of leachable materials and easy to handle during practical
usage. Hence, the designed nanofiber could be used for the removal of arsenic ions from
water with high efficiency.
100
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Recio, M. A. L.; Merce, A. L. R. J. Mol. Struct. 2008, 877, (1-3), 89-99.
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104
Chapter 5
In situ Preparation of Silver Nanoparticles on
Biocompatible Methacrylated Poly (vinyl alcohol)
and Cellulose Based Polymeric Nanofibers
This chapter is accepted as a full paper for publication in the journal "RSC Advances"
105
5.1 Introduction
Silver-polymer nanostructured materials are considered as important in biology,1
catalysis2 and are well known for their antimicrobial activities.
3, 4 Several fabrication
techniques have been used for the fabrication of non-agglomerated nanoparticles with
polymer as the template.5, 6
Naturally-occurring fabrics such as cotton containing silver
nanoparticles have been tested for antibacterial properties.7 Silver (Ag) nanoparticles
loaded fabrics have great potential in the field of antiseptic dressing, bandage and for
biomedical applications. Recently, hydrophilic polymeric hydrogels with Ag-
nanoparticles were prepared and applied for antimicrobial applications.8, 9
Nanofibers
prepared using electrospinning has attracted attention because of highly porous polymeric
network with superior mechanical properties and high surface area to volume ratio.10
As a
result, such nanofibers were used to fabricate polymer nanofiber mats for different
applications.11-13
A few mechanically stable and biodegradable macromers containing
silver nanoparticles was used to prepare nanofibers and showed antimicrobial activities
against S. aureus and E. coli.14
Recently many researchers fabricated natural polysaccharides and applied them in
various biomedical applications15-17
because of wide useful properties such as nontoxicity,
biocompatiblity and biodegradablity.18-20
In addition cellulose based polymers were also
used for synthesis and capping agent for metal nanoparticles.4, 21, 22
Cellulose contains a
number of hydroxyl groups with lone pairs of electrons which help to create ion-dipole
linkages hence stabilizing the Ag-nanoparticles without coagulation. Poor mechanical
properties23
and easy biodegradability24
of such natural polymers limit their use in many
applications. To overcome these issues several researchers have focused on fabrication of
106
hybrids from mechanically stable polymers, such as polystyrene and polycaprolactone
with Ag-nanoparticles.25-27
Recently, PMMA nanofiber containing silver nanoparticles
was synthesized by radical mediated dispersion polymerization and applied as an
antibacterial agent.9 Silver nanoparticles incorporated polymer nanofiber showed
enhanced antibacterial efficacy compared to that of silver sulfadiazine and silver nitrate at
the same silver concentration. The hydrophobicity with lack of functional groups to
provide coordination sites for Ag-nanoparticles are the main drawbacks for commercial
polymers in many practical applications.
In the present study, hydrophilic biocompatible polymers with coordination sites for
silver and sufficient mechanical integrity was chosen as polymer matrix.
Photocrosslinkable PVA and polysaccharides based polymers (dextran and methyl
cellulose) were use to fabricate a few nanofibrous mats with different compositions via
electrospinning technique. Polysaccharides with hydroxyl groups can efficiently
coordinate the nanoparticles and by blending with atactic PVA provides good mechanical
integrity. The reduction capacity of the designed polymers was investigated by colour
changing experiment. The effect of Ag-nanoparticles on the microcrystalline structure of
the polymer matrix was investigated by thermal and mechanical analysis. The
biocompatibility of the designed methacrylated nanofiber was carried out by cell viability
analysis using human lung fibroblast IMR-90.
5.2 Experimental Section
5.2 1 Materials
Polyvinyl alcohol (PVA) (Mw ≈ 146,000 - 186,000), dextran (DEX, Mw ≈ 35,000 -
45,000), silver nitrate (99.99%) and glycidyl methacrylate (GMA) were purchased from
107
Sigma Aldrich. Methyl cellulose (MC) was purchased from BDH chemicals ltd. England.
All chemicals were used without further purification. Prior to experiment, all glasswares
were thoroughly washed and dried. Aqueous PVA solution (10 wt%) was prepared by
heating at 80 °C with continuous stirring. Similarly MC (5 wt%) and DEX (5 wt%)
solutions were prepared by dissolving in ultrapure water at room temperature with
constant stirring for 12 h. Solutions of PVA / MC (10 : 5) and PVA / DEX (10 : 5) were
prepared by mixing the prepared individual polymer solution and kept for constant stirring
for 6 h. AgNO3 was dissolved in deionised water in dark to avoid the chances of reduction
under light. Before electrospinning, silver nitrate solution was added to the polymer
solution and stirred for 5 minutes for proper homogeneity. Figure 5.1 shows the schematic
representation of the procedure followed for electrospinning of Ag/polymer nanofibers.
Electrospinning UV/Heat
Polymer-Ag+ aq. solution Nanofiber-Ag+ Nanofiber-Ag NP
Figure 5.1: Schematic procedure for the synthesis of polymer nanofibers containing silver
nanoparticles.
5.2.2 Synthesis of methacrylated poly(vinyl alcohol) / dextran / methyl cellulose
Methacrylated PVA was prepared according to the procedure proposed by Hennink et
al.28, 29
. PVA (2 g) was dissolved in DMSO (50 ml) and DMAP (1 g) was added to it.
GMA was added in the proper molar ratio with respect to the number of hydroxyl groups
(based on per monomer unit) and stirred the reaction mixture for 2 days. The reaction was
108
carried out under nitrogen atmosphere. A similar procedure was used for the preparation
of methacrylated methyl cellulose and methacrylated dextran. After the reaction has
ended, the substituted products were dialysed against water, reprecipitated with acetone
and freeze dried. The final products were stored at -20 °C to avoid self-crosslinking. The
methacrylated derivatives obtained from all designed macromers were characterized using
FTIR and NMR spectroscopy.
5.2.3 Test for reduction of silver salt
A colour changing experiment was performed to investigate the extent of reduction of
AgNO3 by 5 different polymers (and their mixture) such as PVA (10 wt%), DEX (5 wt%),
MC (5 wt%), PVA (10 wt%) + DEX (5 wt%) and PVA (10 wt%) + MC (5 wt%). AgNO3
solution (5 wt%) was added to each set of polymer mixture and mixed gentely. Colour
changes in the samples were monitored by a digital camera at different time intervals such
as 0 h, 6 h, 20 h and 48 h.
5.2.4 Fabrication of PVA/Cellulose nanofiber by electrospinning technique
For our study, aqueous AgNO3 was added to the aqueous solution of methacrylated
macromers and stirred for 5 to 10 minutes before electrospinning. AgNO3 (5 wt%)
corresponding to the total dry weight of polymer was added to all polymer composition
designed for our study. The electrospinning setup consists of a hypodermic syringe with a
flat tip and an internal diameter of 1.2 mm. A programmable syringe pump (Optrobio
Technologies Pte Ltd) was used to feed the polymer solution. The positive electrode of the
high voltage power supply was connected to the syringe needle containing polymer
solution. The negative electrode was connected to the grounded copper plate covered with
aluminium foil which served as the collector. The solutions were electrospunned at a
109
positive voltage supply of 15 – 18 kV. The distance between the needle tip and collecting
plate was 10 cm and a constant flow rate was maintained at 4 µl/min. To allow ease of
fiber removal, the glass cover slips were made hydrophobic with silylating agent. The
nonwoven electrospun nanofibers were collected on the glass cover slips placed on the
grounded aluminium foil. The whole process was carried out at ambient conditions.
During the electrospinning process, the fibers were brownish in colour, indicating
reduction of Ag ion to Ag-nanoparticles. The resultant nanofibers were kept under UV
irradiation at 365 nm for crosslinking for 6 h. The samples were further heated at 100 °C
for 6 h inside the oven for complete reduction of silver ions.
5.2.5 Cytocompatibility analysis of methacrylated designed nanofiber
Electrospun nanofiber sheets derived from methacrylated macromer (PVAMA, PVA/DEX
MA and PVA/MC MA) were collected on round glass cover slips (diameter 13 mm) and
then crosslinked under UV (365 nm, 6 h) for crosslinking. Then the fiber samples were
placed on the 24 well culture plate and conditioned in sterile PBS before seeding with
cells. IMR-90 (passage 18 ± 3) was purchased from Coriell Cell Repositories, USA and
maintained in Modified Eagles Medium with glutamine supplemented with 15% FBS
(Standard Quality, EU-approved origin PAA Laboratories), 1% each of penicillin,
streptomycin, non-essential amino acids, vitamins, and 2% essential amino acids (Gibco,
Invitrogen). Cells were subcultured at 80 - 90% confluence and maintained at 37 °C in a
humidified incubator with 5% CO2.
The viability of cells on respective nanofiber mesh was measured using CellTiter -
Glo luminescent cell viability assay (Promega) following the manufacturer’s instructions.
For the viability assay, ~5000 cells were placed along with nanofibers in a 24 - well plate
110
(Griener Bio-one). This was maintained for 4 days in 1 ml medium per well at 37 °C in a
humidified incubator with 5% CO2. At respective timings, the medium in each well was
removed and replaced with 200 µl fresh medium. CellTiter-Glo (200 µl) reagent, pre-
warmed to room temperature was then added and mixed properly. After 10 mins, the
mixture was transferred to white, flat-bottom 96 - well plates (Corning, Costar) and
luminescence readings were recorded using Tecan Infinite F200 micro-plate reader.
5.3 Characterization
FTIR spectra of methacrylated derivatives were recorded using Nicolet 5700 FTIR
(Thermo Electron Corporation) and NMR spectra were recorded using a Bruker 400 MHz
instrument with TMS as internal standard and d-DMSO as solvent. The resultant fiber
morphology of the nanofiber was observed using the field emission scanning electron
microscopy (FESEM, JEOL JSM-6701F). The electrospun nanofibers were collected on
glass cover slips and were mounted on copper stubs with a double sided conducting
carbon tape and coated with platinum. Similarly, the electrospun samples were deposited
on carbon coated copper grids and used for transmission electron microscopy (TEM)
study using JEOL JEM 2010 with accelerating voltage of 200 kV. X-ray diffraction
(XRD) patterns were taken with a Bruker-AXS D8 DISCOVER with GADDS powder X-
ray diffractometer in a 2θ scan configuration in the range from 2º to 70º. Thermal
properties of the composite nanofibers were studied by thermo gravimetric analyser (TA
instrument 2960) from room temperature to 900 °C and differential scanning calorimetry
(TA instrument 2920) at a heating rate of 10 °C/min in an inert N2 atmosphere. Static
mechanical characterization was carried out by DMA instrument (TA instrument, Q800).
111
5.4 Results and Discussion
5.4.1 Test for reduction of silver salt by designed polymer
The extent of reduction of silver nitrate was observed by colour changing experiment for
different polymers and mixtures (Figure 5.2). The extent of reduction of Ag-ions to Ag-
nanoparticle with designed polymer solution can be observed directly and depends on the
functionality present on the polymer backbone. It is conceivable that the hydroxyl groups
of PVA, DEX and methyl cellulose are the key functional groups responsible for reduction
of Ag-ion to Ag-nanoparticles.
The hydroxyl groups having lone pairs of electrons, coordinate with silver ions and
act as a reducing agent as well as a stabilizer for the Ag-nanoparticles.30
A brown
coloration was observed after a certain period of time indicating that the reduction of the
Ag-ions and subsequent formation of Ag-nanoparticles increased with time. It was
observed that the rate of particle formation was maximum for the case of methyl cellulose
as compared to PVA and dextran with same amount of AgNO3 loading. Furthermore, the
color for PVA-AgNO3 remained fairly clear throughout the experiment. Low reduction of
Ag+ ions in PVA/DEX-AgNO3 mixture was due to the low availability of hydroxyl groups
as a result of intermolecular hydrogen bonds between DEX and PVA molecules.
Photographs taken at 24 h clearly showed that the hydroxyl groups in MC-AgNO3 and
PVA/MC-AgNO3 have the highest rate of reduction.
The presence of substituted methyl groups on MC results in weak inter polymer chain
interactions in aqueous medium, but highly viscous nature with partial negative charge on
the oxygen atom of substituted hydroxyl group of the pyranose unit enhances the
reduction of Ag-ions.
112
A CB D E
A CB D E
A CB D E
A CB D EAfter 48 hr
After 20 hr
After 6 hr
After 0 hr
Figure 5.2: Test for reduction of AgNO3 into Ag-nanoparticle, with different polymer
solutions PVA 10 wt% (A), DEX 10 wt% (B), MC 5 wt% (C), PVA 10 wt% + DEX 5
wt% (D), PVA 10 wt% + MC 5 wt% (E) the time intervals of 0 , 6 , 20 and after 48 h.
Such colour changing observation provides an idea about the extent of reduction of Ag-
ions to Ag-nanoparticles by different functional groups and molecular structure of the
polymer.
5.4.2 Designing of methacrylated macromer
As our designed macromers are water soluble, it is important to make them water
insoluble in real application. A few crosslinking experiments were used to reduce the
water solubility for various hydrophilic polymers.31, 32
But many of the crosslinking agents
are highly toxic in nature.33
Owing to that, a crosslinking method was developed by
attaching methacrylated groups to the hydroxyl position of the macromer. For this, the
methacrylate group was introduced to PVA (10 DS), MC (10 DS) and DEX (30 DS) by
reacting with GMA in presence of DMAP in DMSO as the solvent.34, 35
The pendent
113
methacrylated groups of macromers were crosslinked via photopolymerization. For
crosslinking, a cytocompatible initiator, IRGACURE 2959 (1 wt%) was mixed with
solution prior to electrospinning. NMR spectrum of methacrylated - macromer showed
peaks of vinyl protons at δ 5.6 and δ 5.9 ppm,methyl group showed a peak at δ 1.8 ppm
and anomeric proton appeared at δ 5.2 ppm (SI 2).
5.4.3 Nanofibers of methacrylated macromer by electrospinning technique
Electrospinning was used to prepare highly porous, nonwoven nanofibrous mats. The
mixture of methacrylated macromers, AgNO3 and initiator were mixed in proper ratio and
electrospun into nanofibrous structure. The preparation of nanofibers by the
electrospinning process entails the utilization of a high voltage to create an electrically
charged jet of polymer solution. Oppositely charged power supply was connected to the
needle and grounded collector plate. After a critical voltage, the electrostatic force
overcomes the surface tension of the polymeric solution and the charged jet is ejected
from the tip of the needle which undergoes a process of elongation and evaporation
leaving behind nonwoven nanofiber mat. During the electrospinning process, in presence
of high voltage, Ag ions were converted to Ag-nanoparticles inside the fiber matrix with
an observable color change.
To get the optimised nanofiber without any bead formation, different polymer
concentration and blend composition were used for electrospinning. It is well known that
the properties of the polymer solutions such as viscosity, surface tension, electrical
conductivity, polymer concentrations and other parameters strongly influence the
formation of nanofibers by electrospinning process. The change in the viscosity, or
conductivity did not lead to the formation of MC nanofibers. PVA (10 wt%) was added to
114
A
10 µm 10 µm 10 µm
B C
a solution of MC (5 wt%) in the volume ratio of 1:1 (volume ratio) for getting smooth
nanofibers via electrospinning. The change in the viscosity or conductivity did not lead to
the formation of MC nanofibers. Similarly, PVA (10 wt%) was added to DEX (5 wt%)
and electrospun to get smooth nanofibers without any bead formation. The fiber
morphology of the PVA-Ag, PVA/DEX-Ag and PVA/MC-Ag mixtures were observed
under similar conditions using SEM (Figure 5.3). All nanofibers showed diameters
ranging from 300 to 500 nm. For the case of only PVA-Ag, fibers with smooth
morphology were obtained but PVA/MC-Ag and PVA/DEX-Ag fibers showed more
interconnections and not as smooth as PVA-Ag nanofibers.
Figure 5.3: Scanning Electron Microscope (FESEM) image of PVA-Ag nanofiber (A),
PVA /DEX-Ag nanofiber (B) and PVA /MC-Ag nanofiber (C).
The presence of methacrylate groups on the macromer and initiator in the nanofibers
helps to crosslink the polymer chains in the solid state. The nanofibers were irradiated
under UV (365 nm) for 6 h, and reduction of silver ions was completed by heating at 100
°C for 6 h. The formation of Ag-nanoparticles was observed under TEM (Figure 5.4). It
has been found that the distribution of spherical silver nanoparticles with an average
diameter of 10 to 20 nm was uniform throughout the nanofiber matrix.
115
10 20 30 40 50 60 70 80 90
Ag
(220)
Ag
(111)
Ag
(222)
Ag
(311)
Ag
(20
0)
2θ
Inte
nsit
y
200 nm 200 nm 200 nm
A CB
Figure 5.4: Transmission Electron Microscope (TEM) image of PVA-Ag nanofiber (A),
PVA /DEX-Ag nanofiber (B) and PVA /MC-Ag nanofiber (C).
5.4.4 X-ray diffraction analysis of nanofibers containing silver nanoparticle
The formation of silver nanoparticles in the nanofiber matrix was further confirmed by X-
ray diffraction analysis (Figure 5.5). Five peaks were observed at 2θ = 38.4º, 44.2º, 64.5º,
77.6º and 81.6º, which corresponds to the crystal faces of (111), (200), (220), (311) and
(222) of silver, consistent with the formation of face-centred-cubic silver crystallite inside
the nanofiber matrix.36, 37
Since PVA is semicrystalline and both MC and DEX are
amorphous in nature, the small intensity diffraction peaks of polymers are dominated by
the presence of sharp peaks from Ag- nanoparticles.
Figure 5.5: X-ray diffraction (XRD) pattern of PVA-Ag nanofiber (A), PVA /DEX-Ag
nanofiber (B) and PVA/MC-Ag nanofiber (C).
116
5.4.5 Thermal analysis of designed macromer with Ag-nanoparticles
The purpose of crosslinking the macromer is to make the entire fiber insoluble in water
and to enhance mechanical integrity. Thermal characteristics during dynamic
processing conditions provide information on stability, shrinkage, expansion and
storage. The intrinsic chain flexibility and temperature required to induce segmental
motion is determined by the nature and amount of crosslinks. Simultaneously the
introduction of Ag-nanoparticles in the polymer matrix also effect segmental motion of
the polymer backbone.
To gain better understanding on the effect of corosslinking and nanoparticles
towards the molecular mobility of the designed macromer, thermal properties of the
composite nanofiber were analysed using differential scanning calorimetry (DSC).
DSC traces of PVA NF, PVA-DEX NF and PVA/MC NF with and without Ag-
nanoparticles were shown in Figure 5.6. All values are recorded for the second heating
cycles. From Figure 5.6, the glass transition temperature (Tg) of the PVA nanofiber is
71.2 °C and PVA/DEX NF showed a Tg at 79.9 °C. This could be due to the hydrogen
bond interaction between PVA and DEX moiety which increase the glass transition
temperature. For the case of PVA/MC, the Tg was observed at 72.8 °C, which indicate
low interaction between PVA and MC. A decrease in Tg by the incorporation of Ag-
nanoparticles was observed for all polymeric nanofibers. The incorporation of bulky
Ag-nanoparticles led to decrease in interaction between polymer chains and increase in
free volume inside the lattice, which led to a decrease in Tg.
117
Figure 5.6: Effect of Ag-nanoparticles on thermal property of PVA NF (8A), PVA/DEX
NF (8B), and PVA/MC NF (8C).
Similarly, thermal degradation behaviour of the polymer blends mixed with Ag-
nanoparticles was carried out by TGA analysis (SI-3). The first weight loss was due to the
loss of adsorbed moisture and volatile materials inside the composite nanofibers. A second
decomposition was observed at a range of 250 °C to 370 °C due to the degradation of
PVA via dehydration reaction of polymer chains. The third weight loss between 370 °C
and 450 °C corresponds to further degradation of polymer backbone. In the case of PVA,
PVA/DEX and PVA/MC nanofibers containing Ag-nanoparticles the residual content was
maximum even after heating up to 900 °C.
5.4.6 Mechanical properties of designed macromer with Ag-nanoparticles
Mechanical properties of fibers in static condition need to be evaluated because the load
bearing capacity plays an important role during real applications. For this, uniaxial stress-
strain behavior of designed nanofiber was investigated using tensile mode. This
measurement provides information on material properties which includes the tensile
(young’s) modulus, tensile strength (TS) (ultimate or at break) and also the elongations at
break (EB). The data from mechanical studies of PVA NF and Ag-nanoparticle
incorporated nanofibers are shown in Table 5.1. From the data, the modulus, TS and EB
40 60 80 100 120 140 160
Heat
Flo
w (
W/g
)
Temperature (°C)
PVA NF
PVA-Ag NFTg decreases
40 60 80 100 120 140 160
Heat
Flo
w (
W/g
)
Temperature (°C)
PVA/DEX NF
PVA/DEX-Ag NF
Tg decreases
40 60 80 100 120 140 160
He
at
Flo
w (
W/g
)
Temperature (°C)
PVA/MC NF
PVA/MC-Ag NF
Tg decreases
A B C
118
decreases with the incorporation of Ag-nanoparticles for all nanofibers. The TS was
decreased from 12.6 MPa for PVANF to 8 MPa for PVA/DEX-Ag nanofibers. Similarly
for the case of PVA/MC-Ag nanofibers, it was decreased to 4.6 MPa. Significant
differences in modulus, TS and EB between PVA/DEX-Ag NF and PVA/MC-Ag NF
were also observed. Decrease in mechanical properties is due to the effect of nanoparticles
on the nanofiber which can be correlated with the increase in volume fraction and nature
of the nanoparticles.
During the application of tensile force to the polymer matrix, the nanoparticle polymer
interface regions pull apart physically resulting in the formation of many molecular
level cracks and voids. For the polymers, the fracture at the interface depends on the
forces of attachment of fillers, packing mechanism and any influence on molecular
orientation. As the nanoparticles are in the range of 10 - 20 nm size, with weak
interaction with the matrix it can be concluded that Ag-nanoparticle offers no
reinforcing action towards the nanofiber matrix which led to a decrease in mechanical
property.
Table 5.1: Mechanical properties of PVA/CaCO3 composite nanofiber
Composition
Modulous of
elasticity (MPa)
Tensile
Strength (MPa)
Elongation
at break (%)
PVA NF 3.02 12.6 30
PVA-Ag NF 1.99 7.9 24.1
PVA/DEX-Ag NF 2.07 8.1 18.1
PVA/MC-Ag NF 1.49 4.6 6.7
119
5.4.7 Cytotoxic analysis on the designed methacrylated nanofiber
For biomaterial applications, a few criteria such as suitable functional groups, physico-
mechanical properties, expected durability and suitable interface properties must be
considered. Apart from being biocompatible, the material should be free from toxic
leachable chemicals during the application. All methacrylated PVA, dextran and MC
nanofibers were screened for cytotoxicity analysis and cell viability assay up to 4 days
with commercially available fibroblast cell line (IMR-90). After 4 days, the cell
morphology was found to be normal (Figure 5.7A) indicating that the fibrous material
showed no cytotoxic effect towards the surrounding cells.
0
1
2
3
4
Control PVA MA PVA/DEX MA PVA/MC MA
Day 1
Day 2
Day 4
Lu
min
es
en
ce
(x
10
5)
Figure 5.7: Optical micrograph of IMR-90 cells around nanofiber after 4 days of
coculture with IMR 90 cells and cell viability test with IMR-90 on designed nanofiber up
to 4 day of analysis. The data represented mean and standard deviations of four samples.
Control experiment was normalised as same cell line was used with different time scales.
In a typical procedure, all methacrylated nanofibers (10 mm diameter) were placed over
the 24-well culture plate with IMR-90 cells. The plates were incubated at 37 °C and the
cell viability was monitored after day-1, day-2 and day-4. The cell viability data revealed
no enhancement or reduction in cell growth in presence of methacrylated nanofibers in
comparison to the control after 4 days of incubation (Figure 5.7B). These results suggest
that methacrylated nanofibers showed no cytotoxic effects towards human cells.
120
5.5 Summary and conclusion
The present investigation focused on the preparation of ultrafine nanofibrous membranes
from water soluble polymers containing Ag-nanoparticles prepared using electrospinning
technique. To make the water soluble polymer insoluble in aqueous environment,
photocrosslinkable methacrylate groups were incorporated on the polymer backbone.
These nanofibers were photocrosslinked under UV irradiation and annealed at 100 ºC for
the reduction of Ag-ions to Ag-nanoparticles. Characterization of the PVA-Ag,
PVA/DEX-Ag, and PVA/MC-Ag nanofibers was carried out using various physico-
mechanical analyses. The average diameter of the nanofibers ranged between 300 to 500
nm. TEM revealed that Ag- nanoparticles with an average diameter of 10 to 20 nm are
uniformly distributed throughout the nanofibers. Furthermore powder XRD confirms the
presence of Ag-nanoparticles in the nanofiber matrix. The effect of Ag-nanoparticles on
modifying the property of nanofibers was evaluated using DSC and stress-strain
measurements. The effectiveness of silver nitrate reduction was improved with the
addition of dextran and methyl cellulose separately to PVA solution. The
cytocompatibility nature of the methacrylated nanofibers suggests that the nanofiber
containing silver nanoparticles may find practical applications in the field of antimicrobial
application.
121
5.6 References
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H.; Kimmey, M. B.; Li, X. D.; Xia, Y. Nano Letters 2005, 5, (3), 473-477.
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3. Kong, H.; Jang, J. Biomacromolecules 2008, 9, (10), 2677-2681.
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434.
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Materials Letters 2007, 61, (8-9), 1801-1804.
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7. Gupta, P. B., M.;Bajpai, S. K. The Journal of Cotton Science 2008, 12, 280-286.
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S. J. Biomed. Mater. Res. Part A 2004, 68A, (3), 473-478.
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Biomaterialia 2010, 6, (9), 3640-3648.
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20. Wege, H. A.; Kim, S.; Paunov, V. N.; Zhong, Q. X.; Velev, O. D. Langmuir 2008,
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25. Jeon, H. J.; Kim, J. S.; Kim, T. G.; Kim, J. H.; Yu, W. R.; Youk, J. H. Applied
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28. Van Dijk-Wolthuis, W. N. E.; Franssen, O.; Talsma, H.; Van Stenbergen, M. J.;
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725-730.
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33. Ramires, P. A.; Milella, E. J. Mater. Sci.-Mater. Med. 2002, 13, (1), 119-123.
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124
Chapter 6
Nanofibers Derived form Poly (Vinyl Alcohol) and
Milk Protein and its Biocompatibility Evaluation
This chapter is published as a full paper in J. Nanosci. Nanotechnol. 2012, 12, 6156–6162;
doi:10.1166/jnn.2012.6203
125
6.1 Introduction
In recent years, developments of polymeric materials find increasing demand in
biomedical applications.1 So, designing of three dimensional (3D) biomimetic
environments is the most important parameter, which allows unique opportunities for cell
attachment, growth and differentiation.2 Polymeric scaffolds impart suitable mechanical
strength which satisfies the above criteria.3, 4
Current research mainly focoused on seeding
cells on to the 3D scaffold which allows tissue formation in both in vitro and in vivo.
Hence designing a suitable scaffold material with desired chemical composition,
microstructure, porosity and other structural behaviour are the important factor for cell
material interaction.5, 6
Various fabrication techniques have been attempted for the preparation of polymeric
scaffolds, which includes physical, chemical, thermal and electrostatic fabrication
techniques.7-10
Polymeric nanofibers fabricated by electrospinning offer the ideal 3D
networks which address the requirement of tissue engineering scaffold.11
Electrospinning
serves as an excellent technique by which different biocompatible polymers can be
fabricated to nanofibrous structures12-14
which offers unique properties such as high
surface area to volume ratio and high porosity.15
Furthermore nanofibrous membrane
provides fine pores that allow to pass nutrients and waste materials and hence widely used
as scaffold material in biomedical application.16-18
For the past few years, synthetic and natural polymers have been fabricated for the
use in tissue engineering. Although many natural polymers are widely used for this
purpose, but exhibit poor mechanical property and easy biodegradability.19, 20
To
overcome these issues several researchers focused on the use of hydrophobic polymers
126
due to their good mechanical property, but blood platelet adhesion and low
hemocompatibility hinders the wide spread usage.21, 22
To overcome such issues, poly
(vinyl alcohol) (PVA) is chosen as it is semicrystalline in nature with sufficient
hydrophilicity. PVA has good chemical resistance, thermal stability, physico- mechanical
properties and low toxicity with good biocompatibility.23-25
High hydrophilicity of PVA
led to minimal cell adhesion and protein adsorption. For that, PVA-based polymers
incorporated with natural molecules such as gelatine,26
heparin27
and collagen28
that can
be recognized by cells and enhance cell adhesion which leads to potential application as
scaffold for tissue growth.
By incorporation of protein on to the PVA matrix helps to improve in
biocompatibility with interesting physico-mechanical properties. Recently, sodium
casinate based membrane was used for cell proliferation which also induces cell adhesion,
making it suitable for biomedical applications.29
The presence of calcium leads to
flocculation via electrostatic interactions between casein and divalent calcium ions.30
Casein incorporated PVA cryogel is also reported for its enhanced biocompatibility and
anti-thrombogenic properties.31
Jayakrishnan et al.32
reported the sustained release of
progesterone from glutaraldehyde crosslinked casein microspheres. They reported that
glutaraldehyde crosslinked casein microspheres are excellent biodegradable carriers for
long term delivery of steroids. Furthermore, in situ coprecipitation of milk protein by
casinate-calcium phosphate interaction has been explored as potential candidate for bone
tissue engineering application.33
Furthermore, the incorporation of inorganic salts in the fabrication of scaffold plays
an important role. Magnesium ion is known for the acceleration of the nucleation process
127
of hydroxyapatite formation34
and also for the bone tissue reconstruction.35
Calcium salts
are used as bone-filling material and have good osteoconductivity. Guided bone
regeneration membranes fabricated from CaCO3 composite membranes is found to have
interesting cell proliferation activity.36, 37
In this study, composites of PVA-milk protein with CaCO3 or MgCO3 were used for
the preparation of nanofibers and screened for cell adhesion and proliferation. We
emphasize that although a plethora of protein composites were investigated to obtain
tissue construct, the incorporation of natural milk protein onto PVA need more
investigation in detail. For this, we fabricated the PVA nanofibers with different
compositions. The microscopic morphology, surface area analysis and the effect of
additives on to physical properties were investigated in detail. We also investigated the in
vitro biocompatibility of resultant nanofibers by cell attachment and viability study of
human lung fibroblast on the fiber surface.
6.2 Experimental Section
6.2 1 Materials
PVA with weight average molecular weight 146,000 - 186,000, MgCO3, CaCO3 and
phosphoric acid were purchased from Sigma Aldrich. Glutaraldehyde was purchased from
Alfa aesar. Commercially available fresh milk was purchased from supermarket and was
centrifuged at 4 °C at a speed of 15,000 rpm for 60 minutes to obtain milk protein
separated from fats.
6.2.2 Fabrication of nanofiber by electrospinning techniques
PVA solution (10 wt %) was prepared by heating at 80 °C in ultrapure water under stirring
for 2 h. All composite solutions were prepared by stirring PVA (10 wt %) solution with
128
the required amount of CaCO3 or MgCO3 for 24 hrs. Resultant solutions were used for
preparing nanofibrous assembly via electrospinning. For this, the polymer solution was
placed in a hypodermic syringe with an inner diameter of 1.2 mm. A programmable
syringe pump (Optrobio Technologies) was used to feed the polymer solution and a
positive high DC voltage supply (15 to 20 kV) was applied to the polymer solution. The
flow rate of the polymer solution was maintained at 5 µl/min. A grounded aluminium foil
was used as collector and placed at a distance of 12 cm below the needle. The resultant
nanofibers were collected on the glass cover slips placed above the aluminium foil. The
composite nanofiber of PVA and milk protein of 66.6/33.3 dry wt% represent as PVA-MP
NF. Similarly 66.6/33.3 dry wt % of PVA/CaCO3 and PVA/MgCO3 represents as PVA-
CC NF and PVA-MGC NF. Furthermore the mixture of PVA/milk protein/CaCO3 and
PVA/milk protein/MgCO3 (50/25/25 dry wt%) represent as PVA-MP-CC NF and PVA-
MP-MGC NF respectively.
6.2.3 Cell viability and cell adhesion property analysis using human fibroblast
Electrospun nanofiber sheets were collected on round glass cover slips (diameter 13 mm)
and crosslinked with glutaraldehyde. The fiber samples were thoroughly washed with
acetone, water and sterilised under UV irradiation, placed on the bottom of 24 - well
culture plate and conditioned in sterile PBS before seeding with cells. IMR-90 (passage 18
± 3) was purchased from Coriell Cell Repositories, USA and maintained in Modified
Eagles Medium with glutamine supplemented with 15 % FBS (Standard Quality, EU-
approved origin PAA Laboratories), 1 % each of penicillin, streptomycin, non-essential
amino acids, vitamins, and 2 % essential amino acids (Gibco, Invitrogen). Cells were
129
subcultured at 80-90 % confluence and maintained at 37 °C in a humidified incubator with
5 % CO2.
The viability of cells on respective nanofiber mesh was measured using CellTiter-Glo
luminescent cell viability assay (Promega,) following manufacturer’s instructions. This
assay is a homogeneous method for determining the number of viable, metabolically
active cells based on quantification of ATP concentration. The procedure involves
addition of an equal volume of reagent to the medium in test wells, which in a single step
generates a luminescent signal proportional to the concentration of ATP present in cells.
The assay is based on the conversion of luciferin to oxyluciferin by a recombinant
luciferase in the presence of ATP. The observed luminescence is proportional to the
quantity of ATP in cells. The reagent contains detergents to break the cell membrane,
causing ATP release into the medium with ATPase inhibitors to stabilise the released
ATP. For the ATP assay, ~5,000 cells were plated per well with nanofibers in a 24-well
plate (Griener Bio-one). This was maintained for and 7 days in 1 ml medium per well at
37 °C in a humidified incubator with 5 % CO2. At respective timings, the medium in each
well was removed and replaced with 200 µl fresh medium. 200 µl of CellTiter-Glo reagent
pre-warmed to room temperature was then added and mixed properly. After 10 mins, the
mixture was transferred to white, flat-bottom 96 - well plates (Corning, Costar).
Luminescence readings were measured using Tecan Infinite F200 micro-plate reader.
6.3 Characterization
The fiber morphology and quantitative presence of CaCO3 and MgCO3 on the composite
nanofibers were characterized by a JEOL JSM-6701F field emission scanning electron
micrograph (FESEM). Thermal properties of each composite nanofiber were studied by
130
thermo-gravimetric analysis (TA instruments - SDT 2960) with a heating rate of 10
°C/min in an inert N2 atmosphere; and differential scanning calorimetry (TA instruments)
at a heating rate 10 °C/min in an inert N2 atmosphere. X - ray diffraction (XRD) pattern of
the samples were recorded by Bruker-AXS: D8 DISCOVER with GADDS Powder X-ray
diffractometer. Scans were made from 2° to 70° (2θ). The Brunauer – Emmett – Teller
(BET) surface area was measured using N2 physical adsorption method with a surface area
analyser (micromeritics ASAP 2020). Prior to the BET measurement, samples were
degassed in a vacuum oven at 120 °C for 10 h. The relative pressure range P/P0 of 0.05 –
0.30 was used for calculating the BET surface area. To observe cell morphology using
FESEM, the nanofiber samples from cultured vials were washed thrice with phosphate
buffer saline (PBS), fixed with 2.5 % glutaraldehyde for 1 h and dehydrated using ethanol
series (50 %, 70 %, 80 %, 90 %, 95 % and 100 %). The fiber sheets were finally dried
using critical point dryer coated with platinum and observed under FESEM.
6.4 Results and discussion
6.4.1 Morphological analysis
Non-woven nanofiber of respective mixture solution was fabricated by electrospining
technique. The electrospinning process entails the utilization of a high voltage to create an
electrically charged jet of polymer solution. As the magnitude of voltage is increased, the
hemispherical surface of the liquid at the top of the needle forms Taylor cone and
ultimately comes out as fiber in the nanometer range. Figure 6.1 shows the SEM images
of the nanofibrous membranes prepared from PVA NF and with different other designed
compositions. The morphology of fabricated composite nanofibers showed smooth surface
131
Figure 6.1: FESEM images of PVA NF (A), PVA-CC NF (B), PVA-MGC NF (C),
PVA-MP NF (D), PVA-MP-CC NF (E) and PVA-MP-MGC NF (F).
morphology without any bead formation. The average fiber diameter of all nanofibers was
between 300 and 500 nm. It was observed that the surface morphology of PVA-MGC NF
and PVA-MP-MGC NF was not smooth as compared to CaCO3 composite fiber. In order
to confirm the presence of additives on nanofiber matrix, EDS analysis was carried out.
The peak of calcium and magnesium indicates the presence of the additives inside the
nanofiber matrix as shown in Figure 6.2. Similarly the incorporation of milk protein on to
the composite nanofiber matrix was confirmed by elemental analysis showing the
Figure 6.2: Energy dispersive spectra of PVA NF (A), PVA-CC NF (B) and PVA-MGC
NF (C).
A B C
132
presence of nitrogen moiety of the amino acids (Table 6.1).
Table 6.1: Elemental analysis of milk incorporated PVA nanofiber, showing the presence
of nitrogen content due to the amino acids
Carbon (%) Hydrogen (%) Nitrogen (%)
PVA NF 59.67 8.79 < 0.5
Dried Milk protein 42.97 7.99 5.71
PVA-MP NF 54.22 7.53 2.31
In comparison to conventional melt spinning fabrication techniques, electrospinning gives
ultrafine fibers with high surface area to volume ratio. The BET results of PVA nanofiber
shows that the 3D porous fiber mat has a high surface area of 5.29 m2 g
-1 with total pore
volume of 0.023 cm3 g
-1. Such surface area achieved was mainly due to the formation of
fibers with smaller diameter fibers of around 300 - 500 nm. This value is comparable to
the recently reported literature derived from PCL/Gelatine nanofiber having surface area
of 6.56 m2 g
-1 38
and PVA/tin glycolate composite fibers with surface area of 17 m2 g
-1.39
Due to such high surface area of electrospun nanofibers is the main reason for wide
applicability in current research in comparison to other conventional fabrication
techniques.
6.4.2 Effect of additives on the physico-mechanical behaviour
The thermal analysis of polymeric composite nanofibers was performed by TGA. In all
cases, weight loss was recorded from room temperature to 900 °C in N2 atmosphere.
Figure 6.3 shows the weight loss for PVA NF, PVA-MP NF, PVA-MP-CC NF and PVA-
133
MP-MGC NF. The first weight loss at around 100 °C is due to adsorbed moisture and
volatile materials such as solvent molecules present in the composite nanofiber. Another
decomposition (T-1) at the range of 250 °C to 350 °C is due to degradation of PVA by
dehydration reaction of polymer chains and second decomposition (T-2) at 350 °C to 450
°C corresponds to the degradation of polyene residues which found maximum for PVA
nanofiber. CaCO3 in the composite nanofiber undergoes degradation to emit CO2 at
temperature range of 650 °C to 750 °C and is shown as T-3. The remaining residue found
after 900 °C was CaO.
However, for PVM-MGC NF, T-4 and T-5 at the region of 320 °C to 470 °C can be
attributed to degradation of hydromagnesite and also PVA. The identity of the residue
present after 470 °C is established as MgO. To gain a better understanding on the effect of
additives toward the molecular mobility of the designed macromer, DSC analyses were
carried out. All values were recorded for second heating cycles of the analysis.
200 400 600 8000
20
40
60
80
100
T-5
T-4
T-3
T-2
T-1
Wei
gh
t (%
)
Temperature (°C)
PVA NF
PVA-MP NF
PVA-MP-CC NF
PVA-MP-MGC NF
Figure 6.3: TGA thermogram of PVA NF (A), PVA-MP NF (B), PVA-MP-CC NF (C),
and PVA-MP-MGC NF (D).
134
Figure 6.4 shows the glass transition temperature (Tg) of PVA NF and compared with all
designed composite nanofibers. It was observed that the Tg of PVA NF is at 71.7 °C. The
addition of milk and CaCO3 onto the nanofiber caused the Tg of the polymer backbone to
shift slightly towards lower temperatures. For polymers, the segmental mobility at the
interface depends greatly on the force of attachment of fillers, packing mechanism and
other factors which can influence molecular orientation. The effect of non-reinforcing
filler particles (CaCO3 and MgCO3) on the
40 60 80 100 120 140 160
Hea
t F
low
(W
/g)
Temperature (°C)
PVA NF
PVA-MP NF
PVA-CC NF
PVA-MP-CC NF
PVA-MGC NF
PVA-MP-MGC NF
Tg decreases
Figure 6.4: Glass transition temperature analysis of PVA NF (A), PVA-MP NF (B),
PVA-CC NF (C), PVA-MP-CC NF (D) PVA-MGC NF (E) and PVA-MP-MGC NF (F).
polymer composite nanofiber lowered the temperature required to start the segmental
motion in between polymer chains. From the Tg values summarised in Table 6.1 it can be
concluded that there is no reinforcing action towards the polymer when CaCO3 or MgCO3
was incorporated in the designed nanofiber.
135
Table 6.2: DSC analysis of PVA-milk protein/CaCO3 nanofiber and PVA-milk
protein/MgCO3 nanofiber
NF Sample
Glass Transition
Temperature
(Tg in °C)
Melting Point
(M.P in °C)
Melting Peak
Enthalpy
(ΔHm in J/g)
PVA NF 71.7 195 35
PVA-MP NF 69.8 194 26.2
PVA-CC NF 68.0 176 7.1
PVA-MP-CC NF 65.5 182 6.5
PVA-MGC NF 68.4 196 22
PVA-MP-MGC NF 64.1 184 4.3
Furthermore the comparative melting points for all designed nanofibers are listed in the
table. By introducing fillers, the chain interactions decreased which caused a decrease in
melting point and hence melting peak enthalpy also decreases.
6.4.3 XRD analysis of PVA composite nanofibers
X-ray powder diffraction profiles of composite nanofibers were collected at room
temperature and compared with that of CaCO3 and MgCO3 powders. Figure 6.5 shows the
XRD pattern of PVA NF, CaCO3, PVA-MP-CC NF, MgCO3 and PVA-MP-MGC NF
composites. CaCO3 incorporated fibers gave calcite peaks which were comparable with
JCPDF No-86-0174. As atactic PVA is semicrystalline in nature, the diffraction patterns
were dominated by CaCO3 lattice. Similar patterns were also observed for MgCO3
incorporated fibers. The peaks found in semicrystalline PVA were replaced by crystalline
MgCO3. The sharp reflections arise from hydromagnesite which is comparable with
JCPDF No-701177. The semicrystallinity of the PVA matrix was lost in the composite
fiber matrix.
136
10 20 30 40 50 60 70
a
Inte
nsi
ty
b
c
d
e
Figure 6.5: XRD plot for PVA NF (A), CaCO3 (B), PVA-MP-CC NF (C), MgCO3 (D)
and PVA-MP-MGC NF (E).
6.4.4 Cell adhesion and viability study on designed electrospun nanofibers
membranes
Cell proliferation and differentiation are mainly controlled by the chemical and physical
parameters of the designed scaffold. Materials used for tissue engineering applications
must be able to meet a number of requirements. Firstly, they must not have any toxic
effect towards the surrounding tissues. Apart from that, various other components
involved in the growth and differentiation of cells on the surface, which includes
adsorption of serum components onto the extracellular matrix produced by the cells40
and
surface roughness.41
Polymeric nanofibers offer good mechanical properties and a porous structure which
allow diffusion of nutrient and waste molecules. Nanofibrous materials have been studied
137
0
5
10
15
20
25
30
Lu
min
esen
ce ( x
10
5)
Day 1 Day 3 Day 7
0
20
40
60
80
TCPSLu
min
ese
nce
( 1
05)
Lu
min
esc
en
ce (
10
5)
for its superior cell attachment properties. High aspect ratio platforms can allow more
filapia projecting from the edges of the nanofibrous scaffold.42
IMR-90 was seeded in
each sample well and cultured for various time intervals (1, 3 and 7 days). Tissue culture
polystyrene (TCPS) was used as positive reference. Figure 6.6 shows the cell proliferation
on individual composite nanofiber. On day 1, Cell number and growth are similar for all
samples. From day 3 onwards, cell viability increased in almost all samples as compared
to the PVA nanofiber. Increased cell proliferation was most significant after 7 days of the
experiment.
Figure 6.6: Cell viability assay on PVA-MP/CaCO3 and PVA-MP/MgCO3 composite
nanofibers
138
the graph, PVA-MP NF did not show any cytotoxic effect with low cell proliferation
behaviour. The best enhancements in cell viability were observed on the case of PVA-MP-
CC NF and PVA-MP- MGC NF, where cell viability was improved in the order of 1.5 to 2
fold, as compared to PVA NF.
Nanofibrous materials having high surface area with incorporation of natural proteins
can provide a suitable platform for cell proliferation. Casein is a phosphoprotein which is
expected to enhance the biocompatibility and hence increases in cell proliferation.
Similarly, CaCO3 and MgCO3 are known for cytocompatibility and both were studied for
guided bone tissue regeneration.35, 43
10µm
10 um
20µm
A B
10µm
220µm 220µm
C D
Figure 6.7: SEM images of IMR-90 cell on PVA NF (A), PVA-MA NF (B), PVA-MA-
CC NF (C) and PVA-MA-MGC NF (D) after 7 days.
139
A similar behaviour was observed by Serizawa et al. for L-929 fibroblasts cell adhesion
onto a CaCO3 deposited film of PVA-casted on polyethylene.44
The combined effect of
milk protein embedded with CaCO3 and MgCO3 enhanced cell proliferation on our PVA
composite NF surface. These results indicate that extracellular matrix production on the
nanofiber surface might be enhanced by the Casein-CaCO3 and Casein-MgCO3
composites.
The enhancement in cell adhesion behaviour on both PVA-MP-CC NF and PVA-MP-
MGC NF composite fibers were further supported by the SEM images (Figure 6.7). The
SEM image of PVA NF (Figure 6.7A) shows no significant cell adhesion, but for PVA-
MP-CC NF and PVA-MP-MGC NF (Figure 6.7B and 6.7C), it was observed that a large
number of cells adhered on these surfaces. PVA fibers are highly hydrophilic in nature and
do not allow cells to proliferate on its surface. Natural proteins have peptide chains which
facilitate cellular adhesion and proliferation hence increasing its biocompatibility which is
confirmed from the cell viability study. Our work has demonstrated that mixing of milk
proteins with PVA-CaCO3 and PVA-MgCO3 can significantly improve cell viability and
cell proliferation of IMR-90 over PVA nanofiber.
6.5 Summary and conclusions
Highly porous 3D nanofibrous membranes were prepared from PVA-milk protein/CaCO3
and PVA-milk protein/MgCO3 by electrospinning technique. The average diameter of
nanofibers was 300 - 500 nm, and is highly porous in nature with very high surface area of
5.29 m2 g
-1. The existence of CaCO3 and MgCO3 on the nanofiber matrix was proved by
XRD and EDS. The addition of inorganic additives (CaCO3 and MgCO3) causes decrease
in glass transition temperature and hence acts as non-reinforcing behaviour towards the
140
PVA backbone. The results of in vitro cell culture studies revealed that the composite
nanofibers were cytocompatible. The presence of milk protein on the nanofiber matrix
favours cell proliferation and attachment. Furthermore the composite nanofiber of milk
protein incorporated with CaCO3 and MgCO3 greatly enhanced cell proliferation. Such an
enhanced effect on cell compatibility of natural protein incorporated designed nanofiber,
would be very preferable in biomedical application.
141
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145
Chapter 7
Preparation of Viscoelastic Hydrogels from Poly
(vinyl alcohol)/Fe (III) Complex and its
Cytocompatibility Evaluation
146
7.1 Introduction
With the rapidly evolving application of polymeric scaffold in biomedical field, designing
and fabrication of new materials is an important aspect towards developing new class of
biomaterials. Hydrogels are of special interest owing to the presence of large amount of
water within its network structure.1 Hydrogels with high water content and elastic in
nature are suitable for many applications such as matrices for the controlled release of
pharmaceutical agents, contact lenses and bio-adhesives.2-6
The different crosslinking
methods used for the preparing hydrogels such as chemical, physical and ionic
crosslinking, impart unique properties to the hydrogels.7
Recently, poly (vinyl alcohol) (PVA) hydrogels have been studied for potential
biomedical applications.8, 9
The porous network structure of the gels favours exchange of
nutrients and waste products with surrounding body environments making them attractive
substitutes for soft tissues10, 11
and drug delivery applications.12, 13
PVA gels are known to
be biocompatible, stable at 37 °C and its high hydrophilicity ensures minimal cell
adhesion and protein adsorption which are beneficial in biomedical applications.14-16
Furthermore, PVA hydrogels have shown to exhibit good mechanical responses to porcine
aortic tissue,17
human carotid arteries and for potential heart valve applications artificial
articular cartilages18, 19
and for intravascular applications.20
PVA is a semi-crystalline polymer with pendent hydroxyl groups. The
hydrophilicity and water solubility of PVA can be controlled by the extent of hydrolysis of
acetal protecting groups and molecular weight.21
PVA hydrogels were formed via both
chemical and physical crosslinking mechanisms. The freeze thaw technique involves
repeated cooling the hydrogel to a lower temperature of -20 °C and thawing to room
147
temperature for several cycles. By this process, the crystallinity of hydrogel increases and
the polymer chains become physically crosslinked.22
Glutaraldehyde was used as the
chemical crosslinking agent for the pendent hydroxyl groups of PVA in presence of acid
catalysts.23-25
The hydroxyl groups of PVA can react with glutaraldehyde under acidic
conditions to forms acetal rings. The glutaraldehyde crosslinking method has limitations
as traces of residual glutaradehyde makes the hydrogel cytotoxic. Anseth et al. studied
ester-acrylate and glycidyl acrylate as pendent groups on substituted PVA as
photocrosslinking sites.26, 27
The gelling behaviour of PVA-methacrylate and its potential
use in tissue replacement and drug delivery has also been explored.28
Recently acryloyl or
methacryloyl derivatized PVA hydrogel was synthesized by grafting PVA with acrylic
acid or methacrylic acid.29
Though several investigations have been carried out for crosslinking PVA using
chemical and photochemical methods in recent years, the design of suitable biomaterial
having desired mechanical property proves to be the most challenging. In this study, we
have examined crosslinking of PVA using metal ions to obtain hydrogel for potential
biomedical applications. Even though, a plethora of crosslinking techniques have been
investigated to obtain hydrogels from PVA, the fabrication of PVA-Fe (III) coordinated
gel and its detail property study has not been investigated to the best of our knowledge for
any biological applications. Here, physico-mechanical behavior, cytotoxicity, cell
adhesion and growth in PVA-Fe hydrogel are investigated in detail.
7.2 Experimental
7.2.1 Materials
Poly (vinyl alcohol) (PVA) with weight average molecular weight 146,000 - 186,000 was
purchased from Sigma Aldrich. FeCl3.6H2O was purchased from Biolab Int Ltd and
148
NH4OH was purchased from Mallinckrodt Baker. Dialysis membrane (molecular weight
cut off 3,500) was purchased from thermoscientific and prepared according to procedure.
Tissue culture polystyrene (TCPS) plates were purchased from Griener Bio-One.
Dulbecco’s Minimum Essential Medium (DMEM) was purchased from PAA
Laboratories. Phosphate buffered saline (PBS) was purchased from 1st BASE. All other
chemicals used for the study were analytical grade.
7.2.2 Preparation of Fe3+
coordinated PVA hydrogels
PVA solution (10 wt%) was prepared by heating PVA granules at 80 °C in Millipore
water with constant stirring for 2 hrs. FeCl3 was dissolved in Millipore water at
various concentrations (0.1 M, 0.15 M, 0.2 M, and 0.25 M), solution (1 ml) was added
to 5 ml of 10% PVA solution, and mixed properly using magnetic stirrer. To above
solution, NH4OH (1 ml) was added drop wise with continuous shaking for the
formation of hydrogel. According to the above four designed concentration of FeCl3
crosslinker used, the sample set was designated as PVFeGel-1, PVFeGel-2, PVFeGel-3
and PVFeGel-4, respectively.
7.2.3 Swelling analysis of hydrogel
Hydrogels were prepared on previously weighed plastic vials. For this, solution of PVA
(1ml, 10 wt%) was added with different proportion of FeCl3 (200 µl) solution. NH4OH
(200 µl) was added drop wise with constant homogenization resulting in the formation of
crosslinked gels. To these crosslinked samples, 20-fold excess amount of PBS was added
and allowed to swell in incubator at 37 °C. Surface of the hydrogel was swabbed with the
help of blotting paper to remove excess PBS and were weighed in an analytical balance in
regular time intervals to monitor the water absorption capacity inside the hydrogel
149
networks. Experiments were done in triplicates. Swelling ratio (Qm) was calculated from
the ratio of weight of PBS uptake to the weight of dried gel.
Qm = (Ws - Wd)/Wd
Where Ws is the total weight of swollen hydrogel and Wd is the weight of hydrogel
dried by lyophilizer.
7.2.4 SEM and spectroscopic analysis of hydrogel
The swollen gel was dried using freeze-dry method and the samples were cut carefully by
scalpel blade. Sample was coated with platinum and the resultant dried gel morphology
was characterized by a JEOL JSM-6701F field emission scanning electron micrograph
(Tokyo, Japan). All IR measurements were carried out in attenuated total reflectance
(ATR) mode (Thermo Scientific). The recording spectral range was fixed at 600 - 4000
cm-1
and the spectrum was evaluated using ominic software.
7.2.5 Thermo-mechanical behavior analysis of hydrogels
The mechanical property measurements were carried out using a dynamic mechanical
analyzer, DMA Q800 (TA instruments). All experiments were carried out in compression
mode. PVA hydrogels were completely immersed in PBS solution and allowed to swell in
incubator at 37 ºC. Molded circular shaped samples measuring 10 mm in diameter and 2
to 3 mm in thickness were prepared using a die. DMA controlled force mode was used
with a preload force of 0.01 N to measure the stress-strain property of the hydrogels. The
temperature of the chamber was raised to a biological temperature i.e 37 °C and was kept
constant during force ramping. The force was ramped at a rate of 0.1 N/min and resulting
mechanical properties were recorded.
For dynamic mechanical analysis, multi frequency-strain mode was used. A static
150
load of 0.001 N was employed, along with constant oscillating amplitude of 50. To
study the influence on viscoelastic behavior with change in frequency, the specimen
was held isothermally at 37 °C and deformed at constant amplitude over a range of
frequencies 1 Hz to 10 Hz and the mechanical properties were measured. To evaluate
the change in viscoelastic properties, the specimens were exposed to a series of
isothermal temperatures from 20 °C to 45 °C. At each temperature, the material was
deformed at constant amplitude (strain 1%) over a frequency of 1 Hz and the
subsequent mechanical properties were evaluated.
For creep experiment, a constant static force of 0.01 N was applied and the
temperature was kept at 37 °C throughout the experimentation. A stress of 200 Pa was
applied to the swollen hydrogel for 5 minutes and allowed to relax for 10 minutes and the
strain release from the sample was measured. From this the percentage strain developed
and the strain recovery calculated with time.
Thermal properties of each lyophilized crosslinked samples were studied by
differential scanning calorimetry (TA instruments - DSC 2920) using a heating rate 10
°C/min in an inert N2 atmosphere.
7.2.6 Biocompatibility evaluation using human fibroblast
Molded shaped (3 mm diameter and 2 mm height) swollen gel samples were
thoroughly washed with water, followed by PBS, sterilized under UV-irradiation and
placed on the bottom of a 24-well culture plate. The gels were washed and conditioned
in sterile PBS before seeding the cells. IMR-90 was purchased from Coriell Cell
Repositories, USA and maintained in Modified Eagles Medium with glutamine (MEM,
PAA Laboratories), supplemented with 15% FBS (Standard Quality, EU-approved
151
origin, PAA Laboratories GmbH), 1% each of penicillin, streptomycin, non-essential
amino acids, vitamins and 2% essential amino acids (Gibco, Invitrogen). Cells were
subcultured at 80 - 90% confluence and maintained at 37 °C in a humidified incubator
with 5% CO2.
The viability of cells on respective hydrogel was measured using CellTiter-Glo
luminescent cell viability assay (Promega) following the manufacturer’s instructions.
This assay is a homogeneous method for determining the number of viable,
metabolically-active cells in culture based on quantification of ATP concentrations.
The procedure involves addition of an equal volume of reagent to the medium in test
wells, which in a single step generates a luminescent signal proportional to the
concentration of ATP present in cells. The reagent contains detergents to break the cell
membrane, causing ATP release into the medium and ATPase inhibitors to stabilize
the released ATP. The assay is based on the conversion of beetle luciferin to
oxyluciferin by a recombinant luciferase in the presence of ATP. For the ATP assay,
~5,000 cells at passage 18 ± 3 were plated per well in a 24-well plate (Griener Bio-
one) and the gel samples of 3 mm diameter and 2-3 mm height was placed above the
cell monolayer. This was maintained for day-1, day-2 and day-5 in 1 ml medium at 37
°C. Fresh media was replaced every other day. At respective timing, the medium in
each well was removed and replaced with 200 µl fresh medium. CellTiter-Glo reagent
(200 µl) pre-warmed to room temperature was added and mixed properly. After 10
mins, the mixture was transferred to flat-bottom 96-well plates (Corning, Costar).
Luminescence readings were measured using Tecan Infinite F200 micro-plate reader.
152
7.2.7 Cell adhesion study on the hydrogel surface
IMR-90 cells were trypsinized using trypsin-EDTA solution to get cells in suspension.
Approximately 5000 cells per vial were added on to the swollen gel. Cells were
allowed to adhere on to the material for 2 hours and then sufficient medium was added
and incubated at 37 ºC. Cell growth was monitored periodically on top of hydrogel and
the morphology was observed under optical microscope (Olympus, BX61).
7.3 Results and Discussion
7.3.1 Spectroscopic analysis of crosslinked hydrogel
PVA is an uncharged, water soluble polymer which is commercially prepared by the
hydrolysis of polyvinyl acetate. PVA was selected for our study owing to hydrophilic
nature and the potential of chemically modifying free pendent hydroxyl groups on the
backbone with desired functional groups.3, 30-33
In our investigation, a novel crosslinking
method was used for in situ gelation of the aqueous PVA solution by FeCl3 in basic
medium. The crosslinking is due to the coordination process of Fe (III) with pendent
hydroxyl groups of PVA macromer. Fe (III) ion is a high spin d5 species and generally
forms hexacoordinate complex with suitable ligands.34
In the coordination process,
replacement of Cl- is dependent on pH of the medium. In basic medium, Cl
- was replaced
by –OH groups to form FeO(OH). Particularly, an increase in pH leads to the hydrolysis
of hydrated ions, forming of hydroxo- and oxo-polynuclear complexes.35
To study the coordination behavior of Fe (III) and PVA, infrared spectroscopy was
used. After coordination process, the material became insoluble in most of the common
solvents and ATR mode was used to detect the changes of functional groups. The
recorded IR spectrum for PVA and dried PVFeGel is shown in Figure 7.1A. A broad
153
4000 3200 2400 1600 800
b
Ab
so
rban
ce %
Wave number (cm-1)
a0
5
10
15
20
25
30
0 5 10 15 20 25 30 35
Time (Hr)
Sw
ell
ing
rati
o(Q
m)
5
10
15
20
25
30
0 5 10 15 20 25 30 35 40 45 50 55 60 65
Sw
elli
ng
ra
tio
n (
Q)
Time (Hr)
PVFeGel-1PVFeGel-2PVFeGel-3PVFeGel-4
absorption at 3000-3600 cm-1
corresponds to the O-H stretching vibrations of the hydroxyl
groups of PVA. As atactic PVA contain some acetate moiety, C=O stretching
absorption of ester group at 1713 cm-1
was observed. There was a sharp peak at 1090 cm-1
corresponding to the stretching band of C-O group of PVA, which overlaps with the Fe-O-
C linkage of gel system. So the formation of Fe(III)–PVA complexes does not introduce
any significant changes in the IR spectrum of PVA.36
Figure 7.1: ATR-IR spectra of PVA (a) and lyophilized dried sample of Fe (III)
crosslinked PVA (b) complex (A), Study on swelling ratio of hydrogels for different
degree of crosslinking (B).
7.3.2 Physical behavior analysis by swelling study
Gels were allowed to swell in PBS at 37 ºC and weight of the swollen gel was noted at
regular intervals. Swelling ratio (Qm) was calculated from the ratio of weight of PBS
uptake to weight of dried gel. Figure 7.1B shows the relationship between the Qm with
different amount of crosslinkers. Qm increased rapidly from 10 to 20 within the first 10
hours, thereafter, swelling was slowed down significantly.
A B
V
154
The volumetric swelling ratio for PVFeGel-1 showed a maximum up to 26 while for
PVFeGel-4 it was 16. These results illustrate that the crosslinking density is directly
related to swelling ratio. Lower crosslinked network provides high interchain distances,
while lower amount of crosslinking able to retain sufficient amount of water inside the
network. Similarly an increase in crosslink density brings different chains closure to each
other, resulting lower swelling and hence shows tight structure.
Samples with optimum swelling were lyophilized, sliced and used for SEM
analysis. The SEM images of PVFeGel-1 and PVFeGel-4 showed a highly porous
interconnected network structure with an average pore size of 500 - 800 µm as shown
in Figure 7.2. The hydrogel has high water retention capacity which allows diffusion
of both small and macromolecules during its potential applications.
Figure 7.2: SEM images of freeze dried lyophilised sample of PVFeGel-1 (A), PVFeGel-
4 (B).
7.3.3 Mechanical property of hydrogel
For designing a polymeric biomaterial, the composition and structure of the material
relevant to the desired properties should be investigated. Prior to designing a biomaterial,
the important properties such as, thermal, physical, mechanical and biological properties
A B
155
need to be thoroughly evaluated. Thermal characteristics during dynamic processing
conditions provide information for stability, shrinkage, expansion and storage.
Furthermore, sterilization methods for biomaterials include steam or irradiation before
implantation inside the body. So the mechanical properties under different conditions play
a vital role during the design of a biomaterial scaffolds. DMA is a useful technique for
characterization of biomaterials because it helps to correlate wide range of material
properties. Common techniques for measuring mechanical properties of swollen hydrogels
are static compressive modulus, dynamic mechanical analysis and creep behavior. All
these methods give an idea about the viscoelastic behavior of the hydrogel.
7.3.3.1 Stress-Strain behaviour: Mechanical property in static condition gives the idea
about the load bearing capacity during real application. For this, uniaxial stress strain
behavior of coordinated hydrogels in compression mode was carried out and shown in
Figure 7.3A. Under an applied stress of 6 Pa, PVFeGel-4 and PVFeGel-1 show strain of
47% and 62%, respectively (Figure 7.3B). When compressive stress was applied, strain
develops locally over the hydrogel network and it dissipates by deforming the hydrogel
network. So less coordinated gel shows higher strain at constant stress and the modulous
of the gel network was calculated up to the elastic limit. PVFeGel-1 showed moduli
around 7.66 ± 1.73 Pa and PVFeGel-4 showed 52.9 ± 4.4 Pa (Figure 7.3B). The
coordinated network of the physical entanglement helps to attain its original conformation
after releasing the stress, up to the elastic limit. Increasing the crosslinking density leads to
the formation of stiffer network and hence limits the capacity to dissipate stress imposed
during compression. Thus, it can be concluded that the gel modulus varies owing to
simple changes in the crosslinker concentration.
156
0
10
20
30
40
50
60
PVFeGel-1 PVFeGel-2 PVFeGel-3 PVFeGel-4
Mo
du
lus
in P
a
20 25 30 35 40 450.0
0.5
1.0
1.5
2.0
Sto
rag
e M
od
ulu
s (
kP
a)
Temperature (°C)
PVFeGel-1
PVFeGel-2
PVFeGel-3
PVFeGel-4
2 4 6 8 100.0
0.5
1.0
1.5
2.0
Sto
rag
e M
od
ulu
s (
kP
a)
Frequency (Hz)
PVFeGel-1
PVFeGel-2
PVFeGel-3
PVFeGel-4
0 10 20 30 40 50 60 700
3
6
9
12
Str
es
s (
Pa
)
Strain (%)
PVFeGel-1
PVFeGel-2
PVFeGel-3
PVFeGel-4
Figure 7.3: Stress-strain graph for hydrogels with different crosslinker concentration
under uniaxial compression mode (A) and graph for modulus of hydrogels with different
crosslinker concentration (B).
7.3.3.2 Dynamic mechanical analysis: For hydrogels used in tissue engineering, it is
important to assess the mechanical properties, especially, when they are subjected to
external deformations. Dynamic mechanical analyzer correlates the strain induced by the
application of constant dynamic stress. The storage modulus (G') was characterized to
correlate the viscoelastic behavior of the polymeric hydrogel.
Figure 7.4: Graph of storage modulus vs frequency of different hydrogels at biological
temperature (37 °C) (A) and graph for storage modulus vs temperature of hydrogels with
different degree of crosslinkers at constant frequency (B).
A B
V
A B
V
157
Values of G' of the designed hydrogels in its equilibrium swollen samples are shown in
Figure 7.4A at 37 °C at different frequencies. It was observed that by increasing the
concentration of crosslinking, the G' of the resulting gel increases. Furthermore, increase
in G' can be depicted with rise in frequency. From the graph, it shows that the G' for
PVFeGel-1 is 0.58 kPa at 1Hz and 0.78 kPa at 10 Hz. Similarly G' for PVFeGel-4 is 1.1
kPa at 1 Hz and 1.5 kPa at 10 Hz. An increase in G' reveals that elastic response is
proportional to concentration of crosslinkers and network structure. For understanding the
material performance, it is important to study the viscoelastic behavior with change in
temperature. For this study, a temperature limits up to 45 °C was employed owing to
dehydration of gel at higher temperature.
We monitored change in G' at a temperature range of 20 - 45 °C for all gels (Figure
7.4B) and only marginal changes in G' were observed. So the material has no change in
elastic response at same frequency over the temperature range. For all crosslinker
concentrations, very low dissipation factor (tan δ << 1) was observed, which indicates the
hydrogel is viscoelastic in nature. These studies predicted that swollen hydrogels can
maintain viscoelastic behavior with change in frequency and has a wide range of working
temperature.
Several hydrogels have been used for nucleus pulposus replacements, contact lenses,
targeted drug delivery application, cartilage bone and vocal cord mucosa replacement.37-39
For all these applications, it is very important to match the viscoelastic behaviour of the
synthetic hydrogels with that of the native tissues, when subjected to the dynamic loadings
under physiological conditions. The modulus of hydrogels can be controlled by changing
158
the crosslinking density. Furthermore, the mechanical stability revealed from DMA results
indicates that hydrogels are stable under ambient conditions.
7.3.3.4 Creep behavior: Creep is the fundamental test of polymeric biomaterial which
is directly applicable to product performance. This test gives an idea about the
biomaterials recovered after removal of a constant amount of load. Figure 7.5A shows
the comparative study of PVFeGel-2 to PVFeGel-4 where applied force was 200 Pa for
5 minutes, then relaxed for 10 minutes.
From the graph, it can be seen that there is a quick elastic recovery for all hydrogels.
By applying stress the viscoelastic strain reaches to 23% for PVFeGel-2 within 5 minutes,
whereas for PVFeGel-4, it was only 10%. The total strain developed for PVFeGel-4 after
5 minutes of loading was minimum, which is due to increase in crosslinking density. This
causes lowering in molecular motion and hence the strain is less at same amount of
applied stress. But for the less crosslinked gel (PVFeGel-2), the movement of chains is
easier and hence small amount of force can cause high deformation by retarding elastic
response.
Similarly upon releasing the stress, the strain recovery study was carried out for the
designed hydrogels (Figure 7.5B). From the graph it was observed that the viscoelastic
strain recovery was 57% for PVFeGel-2 and 66% for PVFeGel-4. From this study, it can
be concluded that there is quick deformation for lower crosslinked gel which has less
tendency to retrack back to its original position. Similarly, for higher crosslinked network
(PVFeGel-4), initial value of viscoelastic strain developed within 5 minutes was less.
159
5 10 15 20
0
20
40
60
80
Str
ain
re
co
very
(%
)
Time (min)
PVFeGel-2
PVFeGel-3
PVFeGel-4
66%
57%
5 10 15 200
5
10
15
20
25
S
tra
in %
Time (Min)
PVFeGel-2
PVFeGel-3
PVFeGel-4
Figure 7.5: Creep behavior of hydrogels with different concentration of crosslinkers at
biological temperature (37 °C) (A) and Graph for strain recovery with linear time scale
after release the applied stress to hydrogels of different crosslinker concentration (B).
This result indicated that the intermolecular bond strength is high which also resulted in
high percentage of strain recovery after withdrawing the stress from the hydrogel. This
behavior was in agreement with higher G' value corresponding to more elastic response
for PVFeGel-4. From the strain development by application of stress, it was observed that
there was slow increase in strain upon application of stress and similarly recovered path
also follow same route. This indicated the elastic behavior of the gel network.
Furthermore, the time lag in development of initial strain and loss in strain after recovery
indicates the flow behavior.40
Hence, the result explains an elastic nature during the application of stress but
retarded by viscous resistance. This increase in crrosslink network of the polymeric
chain which increases the elastic nature after releasing of stress from hydrogel. In this
point the interchain bond strength is high as by increasing the crosslink density the
retrack back to original conformation is increases. The result obtained from creep
study and storage modulus analysis showed a viscoelastic nature of the hydrogel.
A B
V
160
40 60 80 100 120 140 160 180
He
at
Flo
w (
W/g
)
Temperature (°C)
PVA SUPP
PVFeGel-1
PVFeGel-2
PVFeGel-3
PVFeGel-4
Tg increases
7.3.4 Thermal analysis of crosslinked hydrogel
The purpose of introducing crosslinks in the system is to prevent the flow and to
enhance mechanical integrity of the polymers. However, in between the crosslinks, the
molecular segments remain flexible. In particular, the intrinsic chain flexibility and
temperature required to induce segmental motion is determined by the nature and
amount of crosslinks. To gain a better understanding on the effect of corosslinking
toward the molecular mobility of the designed macromer, thermal analyses of the gels
were carried out from room temperature to 250 °C. The glass transition behavior (T g)
was calculated from second heating cycle for all lyophilized samples and shown in
Figure 7.6. The effect of crosslinking reflects the transition from glassy to a rubbery
state. Table-7.1 shows the summary of Tg, melting point and melting peak enthalpy of
all prepared hydrogel and PVA.
Figure 7.6: Curves for glass transition temperature analysed by DSC analysis of only
PVA and lyophilised sample of PVA hydrogels from different crosslinker concentration
161
Table-7.1 shows the summary of Tg, melting point and melting peak enthalpy of all
prepared hydrogel and PVA. Upon crosslinking, Tg of PVFeGel-4 increased to 80.4 °C
whereas for pure PVA, it was 72.6 °C and the melting temperature for PVFeGel-4 was
also increased to 230 °C.
Table 7.1: DSC analysis of lyophilised sample of hydrogels
Similarly the melting enthalpy (ΔHm) increased around two-fold for PVFeGel-4 in
comparison to PVA. The melting is an endothermic process which absorbs certain amount
of energy to heat the polymer at a particular temperature which is directly proportional to
the crosslink density. For PVA gel system, this may be due to increase in formation of
stable coordination bonds between Fe (III) ion and hydroxyl groups of PVA. Furthermore,
coordination imposes additional constraints on the segmental motion of PVA backbone
and hence resulted an increase in Tg. So it can be concluded that by increasing the
crosslinks, the chain segmental mobility starts at higher temperature.
7.3.5 Cell viability and cell adhesion study
The surface properties of biomaterials are important because the biological responses are
largely controlled by surface functional groups and topography of such materials.
Sample ID Glass Transition
Temperature (Tg
in ˚C)
Melting Point
(M.P in ˚C)
Melting Peak
Enthalpy (ΔHm in
J/g)
PVA 72.6 193 28.5
PVFeGel-1 74.2 227 36.9
PVFeGel-2 75.3 229 42.3
PVFeGel-3 77.1 230 54.2
PVFeGel-4 80.4 230 58.1
162
Figure 7.7: Optical micrograph of IMR-90 cells around hydrogel after 5 days of coculture
(A) and cells grow on the surface of hydrogel (B).
with minimal platelet adherence and no sign of accumulation or pseudopodium. Our
Hydrogels are hydrophilic polymer network and can be used as biomaterial with low
platelet adsorption, protein adsorption and blood clot formation were observed.41
The
highly hydrophilic surface results in low interfacial tension between hydrogel surface and
surrounding biological fluids. Similarly Zhang et al.20
reported the PVA based materials
investigation towards the biocompatibility of prepared PVA based hydrogel was carried
out using cytotoxicity analysis and cell viability assay up to 5 days with IMR-90 cell line.
For the cytotoxicity study, polymerized gels (PVFeGel-1 to PVFeGel-4) were seeded
with IMR-90 and monitor the cell growth and proliferation. After 5 days, the cell
morphology was found to be normal (Figure 7.7A) indicating that material has no
cytotoxic effect towards the surrounding tissue. For cell viability study, hydrogel (3 mm
dia and 2 mm in height) was placed over the 24-well culture plate having uniform IMR-90
monolayer. The cell viability data revealed that there was almost no enhancement or
decrease in cell growth by the presence of gel in comparison to the control (Figure7. 8)
after 5 day of incubation. These results suggest that the hydrogels are free from any toxic
leachable material and also material surface has also no affect on cell growth. Similarly,
A B
163
0
2
4
6
8
10
12
14
16
Lu
min
es
en
ce
(x
10
5) Day 1 Day 2 Day 5
Figure 7.8: Cell viability test with IMR-90 on crosslinked hydrogels up to 5 day of
analysis. The data represented mean and standard deviations of four samples. Control
experiment was normalised as same cell line was used with different proliferated time
scale.
for cell adhesion study, cells were directly seeded on the surface of the designed
hydrogels. Typically, different PVA gels with different degree of crosslinking were used
for cell adhesion studies. After seeding cells, plates were incubated at 37 °C and
monitored for 5 days. It was observed that the polymer does not allow cell growth on to its
surface (Figure 7B) but the cells around the gels are normal even after 5 days.
From above study, it was concluded that hydrogels are not cytotoxic in nature and do
not allow cell adhesion on their surfaces owing to the hydrophilic nature of PVA.
7.4 Summary and conclusion
The present investigation focused on the structure-property relationship study of Fe
(III) crosslinked PVA hydrogel system. Simple changes in the formulation of
hydrogels led to significant differences in final properties of the material. No leaching
164
of iron ions was observed during our repeated swelling and drying cycles. From the
swelling studies, it was observed that the Qm decreased with an increase in
crosslinking density. Thermal analysis revealed an increase in Tg, M.P and ΔHm with
increase in concentration of crosslinks. From the mechanical studies, it was observed
that the hydrogels are viscoelastic in nature. Furthermore, viscoelastic response and
relaxation behavior was greatly influenced by the amount of crosslinks in the
hydrogel. Temperature dependent dynamic mechanical study showed no significant
change in modulus up to 45 °C. In vitro cell culture study showed that hydrogels were
nontoxic towards IMR-90 fibroblast cells, and no significant cell attachment was
observed on the hydrogel surface. Extensive biological evaluation needs to be done to
use these materials for real applications.
165
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168
Chapter 8
Conclusions and future studies
169
8. 1 Conclusions
The development of nanostructured materials provide high surface area which improves
performance for many advanced applications. The porous structures derived from
polymeric nanofibers are dynamic, because the pore size and shape can be designed
according to desired application which is difficult to get from conventional rigid
structures. The aim of this thesis was to explore the preparation of functionalized
nanofibers derived from hydrophilic polymers and their potential applications. Initial
investigations were focussed on designing of functionalized PVA and cellulose nanofibers
for water filtration system. For this PVA nanofiber was prepared using electrospinning
technique and tested for the extraction of nanoparticles from water. These nanofibers were
characterized by various electron microscopic measurements. To increase the nanoparticle
filtration efficiency, surface hydroxyl groups of PVA nanofibers were modified with
different functional groups; such as thiols and amines. These nanofibers showed very high
filtration efficiency towards the removal of different designed metal nanoparticles (Au and
Ag) from water. The adsorption analysis revealed that the amine and thiol modified PVA
nanofibers showed more than 90% adsorption efficiency towards Au NPs, and amine
modified nanofibers showed good adsorption behavior (more than 90%) towards AgNPs
from aqueous environment. TEM analysis showed that nanoparticles were uniformly
distributed throughout the surface of nanofiber without agglomeration. Hence, the surface
modified PVA nanofibers are proposed to be interesting material for the removal of
nanoparticles from water.
Similarly, a new method for the fabrication of cellulose based nanofibers from
sugarcane bagasse was developed and introduced suitable functional groups for the
170
extraction of nanoparticles from water. For functionalization, chitosan was coated on the
cellulose nanofiber and used for the extraction of Au and Ag-nanoparticles from water.
The incorporation of chitosan on the surface of nanofiber helps to increase the filtration
capacity. Presence of adsorbed nanoparticles on nanofiber surface was confirmed by SEM
and EDX. Easy accessibility, biodegradability, cost effectiveness and the ease of handling
of designed nanofibers make it a viable method for large scale filtration systems.
Furthermore, a novel approach for the preparation of Fe3+
coordinated PVA
nanofibrous membranes were prepared by electrospinning technique. The characterization
of the designed nanofiber was carried out using by FESEM and EDX. Microscopic image
revealed that the diameter of the nanofibers are 600 - 800 nm. The presence of Fe-ions in
the nanofiber was examined using the EDX analysis and found to be around 21%. The
structure - property relationship was investigated in detail using thermal analysis.
Nanofibers were tested for its effectiveness in extracting arsenic contaminants from water.
From the results, it was observed that the composite nanofiber has high efficiency (67
mg/g for As (III) and 36 mg/g for As (V)) towards the arsenic removal from water.
Compared to other available adsorbents incorporated with Fe unit, our method is more
practical due to the absence of leachable materials and more practical to handle during
practical usage.
In another attempt, electrospun PVA nanofibers derived from water soluble polymers
containing Ag-nanoparticles were prepared using electrospinning technique. These
methacrylated nanofibers were photocrosslinked under UV irradiation and annealed at 100
ºC for the reduction of Ag-ions to Ag-nanoparticles. Characterization of the resultants
nanofibers were carried out using various physico-mechanical analysis. The average
171
diameter of the nanofibers ranged between 300 to 500 nm. TEM revealed that Ag-
nanoparticles with an average diameter of 10 to 20 nm are uniformly distributed on the
nanofiber surface. The effect of Ag-nanoparticles on the ultimate properties of nanofibers
was evaluated using DSC and stress-strain measurements.
Similarly, highly porous 3D nanofibrous membranes were prepared from PVA-milk
protein/CaCO3 and PVA-milk protein/MgCO3 using electrospinning technique. The
average diameter of nanofibers was 300 - 500 nm and highly porous in nature with high
surface area of 5.29 m2 g
-1. The physico-mechanical properties of the resultant nanofiber
matrix were investigated by various techniques, such as TGA, DSC, XRD and EDS
analysis. In vitro cell culture experiments revealed that the composite nanofibers were
cytocompatible in nature. The incorporation of milk protein with carbonate salt enhances
the cell proliferation and attachment on the nanofiber surface. Increased efficiency toward
the cell attachment of natural protein incorporated PVA nanofiber, find practical
application in the biomedical field.
The application of PVA nanofibers encourage to compare the preparation of
hydrogel type scaffolding material derived from PVA macromer using Fe (III) as
crosslinker. From the structure property analysis, it was observed that a simple change
in the formulation of hydrogels led to significant differences in final properties of the
material. Swelling studies revealed that a swelling ratio was decreased with an
increase in crosslinking density. Thermal analysis revealed that an increase in T g, m.p
and ΔHm by increasing in concentration of crosslinking agent added to the PVA
macromer. From the mechanical studies, it was observed that the hydrogels are
viscoelastic in nature. Overall, viscoelastic response and relaxation behavior was
172
greatly influenced by the amount of crosslinks in the hydrogel. In vitro cell culture
study showed that hydrogels were nontoxic towards fibroblast cells, and no significant
cell attachment was observed on the hydrogel surface.
8. 2 Future studies
The designed synthetic PVA nanofibers and chitosan coated cellulose nanofibers have
very large specific surface area with strong affinity towards the adsorption of
nanoparticles from water. Similarly Fe3+
nanofiber shown to be very efficient to trap
arsenic species from water. However, detail investigation such as effect of organic matter
and other competitive anions on the adsorption behavior need to be investigated in detail.
Furthermore, as these nanofibers have low mechanical properties, so for real application it
need a support materials with sufficient mechanical properties. Similarly, nanofiber with
silver nanoparticles need to screened for their antimicrobial property.
The studies of nanofiber incorporated with milk protein and metal carbonates are
promising towards the cell proliferation behavior. Similarly, highly viscoelastic hydrogels
are also fabricated by metal coordination mechanism. However, for practical application
these scaffolds must be extended to different cell growth work. Furthermore, extensive
physico-mechanical analysis and series of animal study need to be investigated for using
as scaffold in tissue engineering.
173
PUBLICATIONS
Refereed journal articles
1. Narahari Mahanta, Suresh Valiyaveettil; Surface Modified Electrospun Poly(vinyl
alcohol) Membranes for Extracting Nanoparticles from Water (Nanoscale, 3, (11),
2011, 4625-4631.
2. Sajini Vadukumpully, Jinu Paul, Narahari Mahanta, Suresh Valiyaveettil; Flexible
conductive graphene/poly (vinyl chloride) composite thin films with high mechanical
strength and thermal stability, Carbon, 49, (1), 2011, 198-205.
3. Narahari Mahanta, Leong Wai Ying, Suresh Valiyaveettil; Isolation and
Characterization of Cellulose Based Nanofibers for Nanoparticle Extraction from
Aqueous Environment, Journal of Materials Chemistry, 22 (5), 2012, 1985 - 1993.
4. Narahari Mahanta, Yiwei Teow, Suresh Valiyaveettil;
Fabrication and
Characterization of Hybrid Nanofibers from Poly (vinyl alcohol), Milk Protein and
Metal Carbonates (Journal of Nanoscience and Nanotechnology, J. Nanosci.
Nanotechnol. 12, 2012, 6156–6162.
5. Narahari Mahanta, Suresh Valiyaveettil; In situ Preparation of Silver Nanoparticles
on Biocompatible Methacrylated Poly (vinyl alcohol) and Cellulose Based Polymeric
Nanofibers (RSC Advances, under revision).
6. Narahari Mahanta, Yiwei Teow, Suresh Valiyaveettil;
A novel approach for
preparation of viscoelastic nanofibrous hydrogels from poly(vinyl alcohol)/Fe(III)
complex for biomedical application (Polymer, under revision).
7. Narahari Mahanta, Suresh Valiyaveettil; A Novel Approach for the Fabrication Fe
(III) Immobilized PVA Nanofiber as a Sorbent for Arsenic Removal from Water
(under preparation)
Invited conference and symposium presentations
1. Narahari Mahanta and Suresh Valiyaveettil. Nanofibers derived from hydroxyethyl
cellulose/PEO blend by electrospinning process. Asia nano, Singapore, 3- 7 Nov 2008.
[Poster presentation]
2. Lim Zhong Hong, Tay Jian Hua, Narahari Mahanta and Suresh Valiyaveettil.
Nanofibers derived from poly(vinyl alcohol) and cellulose-based polymers via
electrospinning. Singapore Science and Engineering Fair (SSEF) 2009, Singapore.
3. Narahari Mahanta and Suresh Valiyaveettil. Preparation of poly (vinyl alcohol) and
cellulose based polymeric nanofibers containing silver nanoparticles by
electrospinning technique, International conference on materials for advanced
technologies (ICMAT), Singapore, 28 June-3 July 2009. [Poster presentation]
174
4. Narahari Mahanta, Teow Yiwei and Suresh Valiyaveettil. Nanofibers derived from
poly(vinyl alcohol) and milk protein for biomedical application. MRS Fall, Boston,
MA , USA, 30 Nov – 4 Dec 2009. [Oral Presentation]
5. Narahari Mahanta and Suresh Valiyaveettil. Preparation and study of poly (vinyl
alcohol) and cellulose based polymeric nanofibers containing silver nanoparticles. 5th
MPSGC, Chulalonkorn University, Bangkok, Thailand, 7-9 Dec 2009. [Oral
presentation]
6. Narahari Mahanta, Teow Yiwei and Suresh Valiyaveettil. Viscoelastic behaviour of
poly(vinyl alcohol)/Fe3+
complex and its biomedical application, Singapore
International Chemistry Conference 6 (SICC-6), Singapore, 16-19 Dec 2009. [Oral
presentation]
7. Tai Jia Xing, Narahari Mahanta and Suresh Valiyaveettil. Preparation of poly (vinyl
alcohol)/Fe3O4 composite nanofibers by electrospinning technique. Singapore
International Chemistry Conference 6 (SICC-6), Singapore, 16-19 Dec 2009. [Poster
presentation]
8. Narahari Mahanta and Suresh Valiyaveettil. Fabrication of electrospun nanofibers
and their application for nanomaterial removal from aqueous environment. Singapore
International Water Week (SIWW-2010), Singapore, 28 june-02 July 2010. [Poster
presentation]
9. Jhinuk Gupta, Narahari Mahanta, Tan Wei Jun and Suresh valiyaveettil. Synthesis of
organic materials: Effective for nanowaste removal from aqueous environment, MRS
Spring, San Francisco, USA, 5 – 10 April, 2010 [Poster presentation]
10. Narahari Mahanta, Yiwei Teow and Suresh Valiyaveettil. Fabrication of poly (vinyl
alcohol) macromer for biomedical application. Pacifichem, Honolulu, Hawaii, USA,
15-20 Dec 2010. [oral presentation]
11. Sundheep Subramani, Narahari Mahanta, Suresh Valiyaveettil. Electrospun
synthetic and polysaccharide based nanofibers for adsorption of nanoparticles from
aqueous environment. Singapore Science and Engineering Fair (SSEF) 2011,
Singapore.
12. Narahari Mahanta, Yiwei Teow and Suresh Valiyaveettil. Preparation of
Viscoelastic hydrogels from poly (vinyl Alcohol) based macromer and their
biocompatibility evaluation. International conference on materials for advanced
technologies (ICMAT), Singapore, 26 June- 1July 2011. [Poster presentation].
13. Narahari Mahanta, Yiwei Teow and Suresh Valiyaveettil. Fabrication of Nanofibers
from Poly (vinyl Alcohol)/Fe3O4 Composite by Electrospinning Technique.
International conference on materials for advanced technologies (ICMAT), Singapore,
26 June- 1July 2011. [Poster presentation].