Nano mechanical characterisation of a single electrospun nanofiber

Summary Mechanical characterization of a single electropsun polymeric nanofiber is crucial in order to predict the deformation behavior and failure mechanism of fibrous scaffolds when us

Acknowledgements

The author would like to express sincere appreciation for the unconditional and

invaluable guidance given by the following people throughout the course of the research project

- A/Prof Lim Chwee Teck for his continuous guidance, support and encouragement

- A/Prof Sow Chorng Haur for sharing his precious knowledge

- Ms Eunice Tan Phay Shing from Nano Biomechanics lab for her mentorship and sharing of invaluable experience on mechanical characterization of nanofibers

- Dr Tua Puat Siong from Structural lab for his kind support in fabricating the

structural frame needed for the research project

- Mr Lim Soon Huat from CICFAR lab for his assistance in operating the

environmental scanning electron microscope on in situ observation of nanofiber deformation

- Mr Kazutoshi Fujihara, Mr Teo Wee Eong and Mr Ryuji Inai from Nanofiber Processing/ Surface Modification lab for their knowledge on fabrication of

nanofibers

- Ms Satinderpal Kaur from Physical & Chemical Characterization lab for her

assistance for the use of the differential scanning calorimetry

Table of Contents

Acknowledgements i

Table of Contents ii

Summary v

List of Tables vii

List of Figures viii

CHAPTER 1 Introduction 1

1.1 Background 1

1.2 Objectives 4

1.3 Scope 4

1.3.1 Material and structural characterization 4

1.3.2 Mechanical characterization 5

CHAPTER 2 Literature Review 6

2.1 Nanofiber fabrication via electrospinning 6

2.2 Collection of aligned nanofibers 9

2.3 Mechanical test of single nanofibers 11

2.4 In situ observation of deformation 15

2.5 Deformation of electrospun nanofibers 17

CHAPTER 3 Fabrication of Nanofibers 19

3.1 Electrospinning 19

3.2 Morphological properties of electrospun non woven mat 20

CHAPTER 4 Characterization of Electrospun PCL Nanofibers 22

4.1 Background of PCL 22

4.2 Crystal structure 23

4.3 Thermal properties & crystallinity 24

4.4 Tensile test of single electrospun PCL nanofibers 28

4.4.1 Collection of aligned nanofibers 28

4.4.2 Isolation of single nanofibers 29

4.4.3 Tensile test specimen 30

4.4.4 Results 32

4.5 Discussion 35

4.5.1 Concentration effect 35

4.5.2 Diameter dependency 38

CHAPTER 5 Deformation of Single Electrospun PCL Nanofibers 43

5.1 In situ observation on morphological change 43

5.2 Ex situ observation on morphological change & nanostructural rearrangement45 5.3 Results & discussion 46

5.3.1 Morphological changes 46

5.3.2 Nanostructural rearrangement 50

5.4 Summary 54

CHAPTER 6 Conclusions 58

CHAPTER 7 Recommendations 61

References……… 62

Appendix A 71

Appendix B 73

Appendix C 75

Appendix D 78

Summary

Mechanical characterization of a single electropsun polymeric nanofiber is crucial in order to predict the deformation behavior and failure mechanism of fibrous scaffolds when use in tissue engineering The relationship between nanostructure and mechanical property of single electrospun nanofiber is still unknown although it was reported that crystallinity and molecular orientation determines the strength of a fiber Our objective is

to unravel the structure-property relationship of a single electrospun polymeric nanofiber undergoing tensile loading

Based on tensile test results, we show that ductility of single electropsun PCL

(Polycaprolactone) nanofibers increases with increasing concentration but decreases with increasing crystallinity Furthermore, mechanical properties of a single electrospun PCL nanofiber are dominated by its diameter due to the nanostructure formation during and after electrospinning For crystallization which occurs during electrospinning, there will

be formation of fibrillar structures which results in higher crystallinity due to the higher degree of molecular orientation; however for crystallization which occurs after the

electrospinning jet reaches the collector, fibers will have the form of lamellar structures

In-situ observation on deformation of single electrosun PCL nanofiber undergoing tensile loading was achieved by using field emission scanning electron microscope (FESEM) It was observed that nanofibers failed by multiple necking phenomenon This study also provides direct visualization of nanostructural rearrangement of nanofiber by using

behavior of single nanofibers can be elucidated by a series of nanostructural

rearrangements during critical stages of tensile loading, namely linear elastic

deformation, yielding, plateau and strain hardening

In conclusion, nanofibers with fibrillar structures exhibited brittle behavior as they do not experience plateau in view of the fact that fibrils have already been established In contrast, nanofibers with lamellar structures exhibited ductile behavior as strain are required to transform lamellar structures into fibrillar structures and that explains the existence of the plateau

List of Tables

Table 2.1 Summary of the effects of electrospinning parameters 8 Table 2.2 Schematics of different configurations for the collection of aligned nanofibers

10 Table 2.3 Overview of mechanical tests for mechanical properties measurement of

Table 2.4 Summary of the use of various microscopes and the coupled mechanical test

device for in situ observation of deformation 16 Table 3.1 Electrospinning parameters used for fabrication of nanofibers 20 Table 4.1 Thermal properties of electrospun PCL at various concentrations 25

List of Figures

Figure 2.1 Schematic diagram of eletrospinning set up 7

Figure 2.2 Fiber deposition mechanism for a frame collector with two parallel strips [13]

Reprinted from Biomaterials, 26, Tan, E P S., Ng, S Y Lim, C T., Tensile testing of a single ultrafine polymeric fiber, 1453-1456, Copyright (2005), with

Figure 2.3 Nanofibers suspended over etched grooves of silicon wafer: (a) Electron

micrograph of PLLA nanofibers deposited onto the silicon wafer (b) AFM contact mode image of a single nanofiber (300 nm diameter) suspended over an etched groove (c) schematic diagram of a nanofiber with mid-span deflected by

an AFM tip [6] Reused with permission from E P S Tan, Applied Physics Letters, 84, 1603 (2004) Copyright 2004, American Institute of Physics 14 Figure 2.4 (a) AFM phase image of a PLLA nanofiber revealing the fibrillar structure

(b) a closeup view of the surface of the nanofiber showing the shish-kebab morphology [6] Reused with permission from E P S Tan, Applied Physics Letters, 84, 1603 (2004) Copyright 2004, American Institute of Physics 16

Figure 2.5 Electron micrographs of multiple neck formation in electrospun nanofibers

(a) and (b) 7 wt% PEO electrospun nanofibers (c) and (d) 4 wt% PEO

electrospun nanofibers [56] Reused with permission from E Zussman,

Applied Physics Letters, 82, 3958 (2003) Copyright 2003, American Institute

Figure 3.1 SEM images of PCL non woven mats at various concentrations (a) 8 wt% PCL

(b) 10 wt% PCL (c) 12 wt% PCL (d) 14 wt% PCL Scale bars represent 50 μm

21

Figure 4.2 XRD profile of electrospun PCL scaffolds at various polymer concentrations

23

Figure 4.8 (a) Primary collector with parallel strips (b) Side view of primary collector 29 Figure 4.9 Secondary collector which consists of upper and lower frame 30 Figure 4.10 Nanofiber span across the perforated lower frame was shown at (a) lower and

Figure 4.15 Plot of stress-strain curves for different concentrations at nanofiber diameter

Figure 4.16 Concentration effect on Young’s modulus (E), Yield stress (σy), Ultimate

tensile stress (σu), and Ultimate strain (εu) 37 Figure 4.17 Diameter dependency of Young’s modulus (E) for different concentrations

40 Figure 4.18 Diameter dependency of Yield stress (σy) for different concentrations 40 Figure 4.19 Diameter dependency of Ultimate tensile stress (σu) for different

Figure 4.20 Diameter dependency of Ultimate strain (εu) for different concentrations 41 Figure 4.21 AFM phase image showing the surface morphology of (a) 150 nm (scan size

= 600 nm) (b) 450 nm (scan size = 1 μm) nanofibers fabricated from 10 wt%

Figure 5.1 Slideable collector was used for observation on nanofibers deformation 44 Figure 5.2 Experimental setup for in situ observation on morphological change: Top layer

of slideable collector was mounted where bottom layer was glued on stage 45 Figure 5.3 Schematic of mica substrate for AFM imaging 46 Figure 5.4 Sequential deformation behavior of nanofibers undergoing tensile loading 49

Figure 5.5 Nanostructures of nanofibers revealed by AFM phase imaging (a) Neck region

showing aligned and misaligned lamellae as well as surface depressions (b) Interlamellar fragmentation (scan size = 2 μm) (c) Crystallites connecting two fibrils at high strain levels (scan size = 1 μm) (d) Developed and developing fibrils at high strain levels (scan size = 1 μm) (e) Fibrillar structures of a near-failure fiber (scan size = 2 μm) Scale bars represent 250 nm 56 Figure 5.6 Schematics of nanostructural rearrangement of nanofiber undergoing tensile

CHAPTER 1 Introduction

1.1 Background

The increasing demand of biodegradable polymeric scaffolds that is analogous to nature’s extracellular matrix generates interest for the mechanical characterization of single

nanofibers in order to ensure the mechanical compatibility of scaffolds An

understanding of the structural and nano mechanical properties of individual nanofibers is crucial in order to predict the deformation behavior and failure mode of tissue-engineered scaffolds under various loading conditions during cell proliferation [1], cell

differentiation [2], and cell migration [3]

In view of the small size of nanofibers and the small load required for deformation, mechanical characterization of these fibers has not been widely performed until recently Atomic force microscope (AFM) based three-point bend test and nanoindentation are the two commonly used methods for mechanical characterization of single polymeric

nanofibers However, three-point bend test [4-6] and nanoindentation [7] only allow the characterization of elastic properties, which is the measurement of Young’s modulus

Of all the mechanical characterization tests, tensile test remains the most direct method in determining the mechanical properties of nanofibers Tensile test not only determines Young’s modulus, yield characteristics, ultimate tensile stress, and maximum draw ratio (ultimate strain) of single nanofibers, it also provides a clue on how nanostructures

rearrange themselves under tensile loading This consequently determines the failure mechanism of polymeric nanofibers

Literature shows that the fabrication methods of polymeric nanofibers include drawing [8], self assembly [9], template synthesis [10], phase separation [6, 7] and electrospinning [4, 5, 11-13] Electrospinning being one of the most popular fabrication methods for nanofibers, has been studied extensively because of its simple and economical setup The parameters that affect the electrospinning process of nanofibers include applied voltage, feed rate, solution conductivity, needle tip to collector distance, and ambient conditions like temperature and humidity [14-16] These parameters together determine the final form of fibers, including diameter and fiber morphology Conventional electrospinning process always results in non woven mat formation because of the whipping instability [14] However, it was demonstrated that aligned electrospun nanofibers can be collected

by using frame collector with parallel conductive strips [13], which enables the transfer

of nanofibers onto other substrates for further processing

It is commonly believed that the whipping instability during electrospinning results in the stretching and acceleration of electrospinning jet, known as the drawing process [17] This results in the formation of oriented polymer chains in the nanofibers [18, 19] Higher draw ratio (the ratio of the cross-sectional area of the undrawn material to that of the drawn material) will result in more oriented polymer chains being formed in the nanofibers, and this leads to higher strength nanofibers However, there is no direct evidence in literature to support this unique characteristic of electrospun nanofibers The

purpose of this study is to provide quantitative and qualitative analysis on the mechanical properties of nanofibers obtained from the tensile test of single electrospun polymeric nanofibers Polycaprolactone (PCL) nanofibers are chosen due to its extensive

applications in areas of tissue engineering [20-22] Under physiological conditions, PCL

is degraded by the hydrolysis of its ester linkages and thus, makes it a suitable candidate for use as an implantable biomaterial In order to evaluate the mechanical properties of PCL fibrous scaffold, it is important to first obtain the tensile properties of single PCL nanofibers Once the fundamental property of fibrous scaffolds is resolved, engineered structure of fibrous scaffolds can be achieved to suit various cell loading conditions

Atomic force microscopy (AFM) was utilized to investigate the nanostructures of

electrospun nanofibers in order to provide a correlation between mechanical properties and nanostructures The deduction of nanostructural change associated with deformation

of polymer were normally performed by wide-angle X-ray diffraction (WAXD) and small-angle X-ray scattering (SAXS) [23-25] However, these methods do not permit direct visualization of deformation of bulk polymers in single polymeric nanofibers AFM, on the other hand, allows direct visualization of nanostructural rearrangement of electrospun nanofibers undergoing tensile loading Based on the tensile test results and deformation behavior, the deformation and failure mechanisms of electrospun a

polymeric nanofiber is elucidated

The deformation behavior and failure mode of single electrospun nanofibers either morphologically or microstructurally will then be evaluated by using scanning electron microscopy (SEM) and atomic force microscopy (AFM)

1.3 Scope

1.3.1 Material and structural characterization

Mechanical properties of polymeric materials are dependent on their physical properties such as thermal properties, crystallinity, crystal structure and morphology Thus the following material properties of electrospun nanofibers using the accompanying

techniques will be studied:

- Thermal properties and crystallinity using differential scanning calorimetry (DSC)

- Crystal structure using X-ray diffraction (XRD)

- Nanostructures of nanofibers using atomic force microscope (AFM)

- Random mat morphology using scanning electron microscope (SEM)

1.3.2 Mechanical characterization

Mechanical characterization of single polymeric nanofibers will be performed using a nano tensile tester Based on the tensile test results, stress-strain behavior of single nanofibers will be obtained and this will be used to correlate the deformation behavior and failure mode of nanofibers

CHAPTER 2 Literature Review

2.1 Nanofiber fabrication via electrospinning

Electrospinning fabricates superfine fibers with diameter ranging from 10 μm down to 10

nm [16] via the application of electric field that is capable of drawing polymer solution through a spinneret [14] The schematic diagram of electrospinning set up is shown in Figure 2.1 It consists of four components: a high voltage power supply, a syringe pump,

a spinneret (metallic needle), and a grounded collector (counter electrode) In an

electrospinning process, a voltage is applied to the polymer solution and charges

accumulate on the surface of the polymer solution Due to the electrostatic attraction of the opposite electrode, suspended polymer solution from the tip of the needle forms a Taylone cone [26] when critical voltage is reached under the influence of electric field Once the applied voltage exceeds the critical voltage, charges accumulated on the surface

of the Taylor cone will overcome surface tension to induce the formation of

electrospinning jet that is subsequently accelerated towards the grounded collector

It is shown by several studies that the key process in the formation of sub-micron fibers is the whipping instability [18, 19] The whipping instability is controlled by the

electrostatic interaction between the external electric field and the surface charges on the jet [17] Electrospinning jet becomes bent and stretched under the influence of whipping instability, which results in large area reduction of the electrospinning jet The stretching process is accompanied by rapid solvent evaporation: Subsequently, a solidified fiber is collected as a non woven mat on the collector

The parameters affecting the electrospinning process can be broadly classified into three groups [14]:

- Polymer solution parameters: viscosity (determined by concentration and molecular weight), conductivity (determined by solvent used) and surface tension (determined

by concentration and molecular weight)

- Process parameters: applied voltage, feed rate and distance between tip and collector

- Ambient parameters: temperature and humidity

Table 2.1 summarizes the effects of the above mentioned parameters during

electrospinning except for ambient parameters (including temperature and humidity) These parameters affect material properties such as viscosity and surface tension [27], which is in fact a subset under polymer solution parameters Polymer concentration effect and variation of applied voltage are the two most commonly studied

electrospinning parameters among the above mentioned parameters

Figure 2.1 Schematic diagram of eletrospinning set up

Syringe Syringe pump

Table 2.1 Summary of the effects of electrospinning parameters

Surface tension

[16, 35]

Polymer concentration and molecular weight determine the surface tension as well When surface tension dominates, beads are obtained instead of homogeneous fibers In some cases, surfactants are used to lower the surface tension of the medium in which they are dissolved in order to reduce the formation of bead-like structures

Applied voltage

[16, 28-31]

Higher applied voltage may give rise to stronger electric field and acceleration of charges in the jet and instability region, which results in increased elongation of the fibers, and hence formation

of finer fibers

Feed rate

[15, 30]

Higher flow rate tends to stabilize electrospinning instability

However, lower flow rate favors formation of finer fibers An optimum feed rate can be obtained to achieve both criteria: stable electrospinning jet and the formation of finer fibers

2.2 Collection of aligned nanofibers

Due to the chaotic motion (whipping instability) of the highly charged electrospinning jet, conventional electrospinning process always results in the formation of non woven mat with randomly oriented fibers However, as electrospinning is a process dictated by the electric field, it was suggested that the deposition of electrospun fibers can be

controlled by manipulating the electric field [29] It was identified that the use of a conductive frame-like structure allowed the collection of aligned nanofibers [13, 36] by altering the macroscopic electric field It was also demonstrated that collection of aligned nanofibers could be achieved by using a pair of parallel conductive strips as counter electrodes [37, 38] For these two mechanisms, air gap was utilized Another method of collecting aligned nanofibers is the use of a rotating disk [39, 40] with tapered edge as this enabled the control of deposition and alignment of the electrospun nanofibers Table 2.2 provides the schematics of different configurations used to collect aligned nanofibers

The fiber deposition mechanism for a frame collector with two parallel conductive strips

is illustrated in Figure 2.2 [13] The fibers are collected perpendicular to the parallel strips due to the fact that the electrostatic forces near the strips are pointed towards the strips (Figure 2.2a) As the polymer jet goes through whipping instability during its travel towards the collector, part of the descending jet is attached to one of the strips (Figure 2.2b) The remaining portion of the jet will be pulled towards the opposite strip (Figure 2.2c) These processes are repeated (Figure 2.2d) and a membrane of aligned fibers is eventually formed across the strips

Table 2.2 Schematics of different configurations for the collection of aligned nanofibers

Conductive frame collector

Elsevier

60 oAir gap

Regions where electric force towards parallel strips are prominent

Whipping instability

(a) (b)

One part of jet attached

to strip

The process is repeated

Remaining portion of jet pulled to opposite strip

Air gap

Rotating disk

θ Tapered edge

ω

2.3 Mechanical test of single nanofibers

There are various kinds of mechanical tests being conducted at the microscale: bulge, bend, resonance, nanoindentation and tension Mechanical characterization will involve three main components: specimen preparation, force application and measurement, and displacement or strain measurement Due to the difficulty in measuring the small load required to deform a single nanofiber, different innovative and sophisticated systems had been developed to determine the mechanical properties of single nanofibers

Characterization of hard materials’ such as carbon and quartz is relatively easier compare

to soft materials like polymers as manipulation of soft materials might alter its

mechanical properties significantly since thermal history influences greatly the

mechanical properties of polymers

AFM based resonance test has been used for characterizing nanorods/nanowires made of hard materials such as quartz (silicon dioxide) and carbon [41-43] Measurement of Young’s modulus on electrospun polymeric nanofibers was also carried out in which the nanofibers were reinforced by carbon [11] There was also AFM based bend test [12] reported for mechanical characterization of single electrospun polymeric nanofibers All these methods are capable of determining the Young’s modulus (E) of the tested

materials Nonetheless, as the length of nanofibers for AFM based resonance test and bend test is not a constant and hence may affect the Young’s modulus obtained Besides,

it is difficult to make a comparison based on the results since the length of nanofibers is also a variable

On the other hand, AFM based three-point bend test [4-6] and nanoindentation test [7] are the two commonly used methods for characterizing single polymeric nanofibers AFM three-point bend test on gold nanowire was also reported [44] Three-point bend test is a modified version of conventional bend test in which the nanofibers are first suspended over microsized groove An AFM cantilever is then used to apply a small deflection at the midspan of the nanofiber Figure 2.3 shows the SEM image, AFM image, and schematic diagram of the suspended nanofiber for three-point bend test Young’s modulus was determined by beam bending theory [6] Similarly, these tests are able to determine the Young’s modulus of nanofibers The fact that AFM was used in conjunction with all these tests is because of the capability of AFM to attach a single rod/wire/fiber, impose small loads, and measure small displacements

Apart from the above mentioned tests, tensile test of single polymeric nanofibers was also reported [13, 40] Comparatively, tensile test has the advantage of obtaining more than just the elastic property of the materials In view of the capability of tensile test in

determining Young’s modulus, yield characteristics, ultimate tensile stress, and

maximum draw ratio (ultimate strain) of single nanofibers, tensile test was chosen to be the test method for conducting mechanical characterization of single electrospun PCL nanofibers Table 2.3 gives an overview of all the above mentioned mechanical tests and their respective characterized materials

Table 2.3 Overview of mechanical tests for mechanical properties measurement of

nanoscale materials

Mechanical tests [Reference] Materials Fabrication methods properties Measured

bend test PAN

1 nanofibers [12] Electrospinning PAN-derived

carbon nanofibers [11]

Electrospinning

resonance test Silicon dioxide

(SiO2) and carbon nanorods/nanowires [41-43]

solid technique

Vapor-liquid-PEO2 and glass nanofibers [4] Electrospinning Titanium dioxide

Gold nanowires [44] Electrodeposition AFM based

indentation

nano-PLLA nanofibers

[7] Phase separation

Young’s modulus

PCL4 nanofibers [13] Electrospinning Tensile test

PLLA nanofibers [40] Electrospinning

Young’s modulus, yield characteristics, ultimate tensile stress, and ultimate strain

1 PAN = polyacrylonitrile

2 PEO = polyethylene oxide

3 PLLA = poly (L-lactic acid)

4 PCL = polycaprolactone

Figure 2.3 Nanofibers suspended over etched grooves of silicon wafer: (a) Electron micrograph of PLLA nanofibers deposited onto the silicon wafer (b) AFM contact mode image of a single nanofiber (300 nm diameter) suspended over an etched groove (c) schematic diagram of a nanofiber with mid-span deflected by an AFM tip [6] Reused with permission from E P S Tan, Applied Physics Letters, 84, 1603 (2004) Copyright

2004, American Institute of Physics

2.4 In situ observation of deformation

In order to predict material response accurately, it is important to understand the

fundamental deformation behavior and failure mode of materials However, it is always

a great challenge to trace the in situ deformation behavior of nano scale materials as the resolution capacity of microscope must be high enough to capture the changes at the nano scale Three most commonly used microscopy techniques for the in situ observation of deformation at nano scale is the transmission electron microscopy (TEM), atomic force microscopy (AFM), and scanning electron microscopy (SEM)

For viewing under TEM and SEM, special pretreatment of materials is usually needed, especially for observation of polymeric materials All three microscopes can be used to observe the morphological changes of materials However, only AFM allow the

observation of nanostructures of as-prepared polymeric materials It is known that AFM phase imaging (tapping mode with detection of the phase shift) is able to reveal the difference in surface elasticity of crystalline and amorphous phases [45] because phase image exhibits high contrast for surface features and for compositional variations in heterogeneous materials [46] AFM image on nanostrutures of a single nanofiber is shown in Figure 2.4

Generally, in situ observation of deformation for nano scale materials is accomplished by coupling the mechanical test device with the high resolution microscope Table 2.4 presents the in situ observation of deformation that had been carried out using TEM, SEM, and AFM coupled with specific mechanical test device

Table 2.4 Summary of the use of various microscopes and the coupled mechanical test

device for in situ observation of deformation

Microscope Mechanical test device Material Type [Reference]

Tensile test Thin film [47]

Nanoindentation Thin film [48]

Resonance test Nanobelts/nanotubes [49, 50] TEM

Tensile test (induced by thermal stress) Nanotube [51]

AFM Melt extrusion and drawing

Thin film [55]

Figure 2.4 (a) AFM phase image of a PLLA nanofiber revealing the fibrillar structure (b) a closeup view of the surface of the nanofiber showing the shish-kebab morphology [6] Reused with permission from E P S Tan, Applied Physics Letters, 84, 1603 (2004)

Copyright 2004, American Institute of Physics

2.5 Deformation of electrospun nanofibers

Researchers observed that some electrospun nanofibers collected by rotating disk

revealed multiple necks, as shown in Figure 2.5 Hence, it was reported that electrospun nanofibers failed by a multiple necking mechanism due to stretching induced by the rotating disk[56] Another study carried out on electrospun composite nanoporous fiber also observed necking phenomenon for nanoporous fiber undergoing tensile loading induced by thermal stress [57] It was observed that the fiber deformed by necking once the strain reached a certain critical value, followed by propagation of necking along the fiber until the fiber completely failed There was no mention of observation on multiple necking phenomenon in the above study

For both studies, only morphological change of electrospun nanofiber undergoing

stretching was provided Furthermore, there was no correlation for stress-strain behavior and nanostructures of electrospun nanofibers Hence, the structure-property relationship remains unclear

Figure 2.5 Electron micrographs of multiple neck formation in electrospun nanofibers (a) and (b) 7 wt% PEO electrospun nanofibers (c) and (d) 4 wt% PEO electrospun nanofibers [56] Reused with permission from E Zussman, Applied Physics Letters, 82,

3958 (2003) Copyright 2003, American Institute of Physics

CHAPTER 3 Fabrication of Nanofibers

3.1 Electrospinning

Electrospun nanofibers were fabricated from three concentrations of polycaprolactone (PCL) (Mn = 80,000) (Aldrich) solution, namely 10, 12, and 14 wt% PCL Detailed calculation of polymer solution weight percentage is shown in Appendix A Nanofibers fabricated from 8 wt% PCL had a beads-on-string morphology and hence were not

considered for tensile testing PCL was dissolved in a solvent mixture of

dichloromethane (DCM) (Fisher scientific) and N, N-dimethylformamide (DMF)

(MERCK) in the ratio of 4:1(w/w)

Although DMF is a non-solvent for PCL, it has high dielectric constant and hence favors the formation of fibers with finer diameter during electrospinning [34] The dominant factor controlling fiber formation is the surface charge of electrospinning jet By adding

in DMF, conductivity of polymer solution is increased, which in turn increases the

surface charge density [58], leading to a stable whipping motion of elongational flow and hence finer fibers are obtained A common set of electrospinning parameters was used to fabricate different concentrations of nanofibers in order to assess the concentration effect Electrospinning parameters used are shown in Table 3.1

Table 3.1 Electrospinning parameters used for fabrication of nanofibers

Tip to collector distance (cm) 15 ± 0.5

3.2 Morphological properties of electrospun non woven mat

The solution concentration plays an important role in determining the resultant fiber morphology [30, 31] Dilute polymer solutions are normally found to produce beads and/or beads-on-string structures instead of the desired fibrous morphology It was observed that the lowest workable concentration for PCL solution with the proposed solvent system and electrospinning parameters is 10 wt% PCL Non woven mats of 8 wt% PCL revealed a beads-on-string morphology As a result, nanofibers fabricated from 8 wt% PCL are not suitable for tensile test since the cross-section of nanofibers is non uniform The SEM images of PCL non woven mat fabricated at various

concentrations are shown in Figure 3.1

The morphological changes were significant when concentration was varied 8 wt% PCL non woven mats possessed the finest diameter nanofibers; however its beaded structures are unfavorable Electrospun nanofibers fabricated from 10 wt% PCL on the other hand displayed uniform diameter due to the increased degree of chain entanglements The mean diameter of nanofibers fabricated from different concentrations are 630 ± 200 nm for 10 wt% PCL, 660 ± 105 nm for 12 wt% PCL and 765 ± 95 nm for 14 wt% PCL Hence, the mean diameter of nanofibers increased with increasing concentrations with 14

wt% PCL showing the maximum mean diameter Occasionally, diameter of nanofibers was found to be non uniform and this could be due to the instability of solvent

evaporation Nonetheless, non uniform nanofibers collected were discarded in order not

to affect the accuracy of tensile test results

Figure 3.1 SEM images of PCL non woven mats at various concentrations (a) 8 wt% PCL (b) 10 wt% PCL (c) 12 wt% PCL (d) 14 wt% PCL Scale bars represent 50 μm

CHAPTER 4 Characterization of Electrospun PCL

Nanofibers

4.1 Background of PCL

Poly (ε –caprolactone) (PCL) is a semicrystalline, biodegradable polymer having a melting point (Tm ) of ~ 60°C and a glass transition temperature (Tg) of ~ -60°C [59] The repeating molecular structure of PCL homopolymer consists of five nonpolar

methylene groups and a single relatively polar ester group, as shown in Figure 4.1 This combination offers the compatibility of PCL with numerous other polymers to form polymer blends [60] Due to the slow degradation rate, PCL is used for the design of long-term, implantable drug delivery system [61] PCL behaves rubbery at room

temperature due to its extremely low Tg Hence, PCL is highly permeable to many low molecular weight drugs [62] In addition, PCL has been used extensively in the area of tissue engineering [20-22]

Figure 4.1 Chemical structure of PCL

prominent peaks were observed The intensity profile of the peak agreed well with the values reported in the literature [63] The interesting thing to note is the reduction in peak intensity when polymer concentration is increased This is most likely due to the lower degree of molecular orientation resulted from electrospinning process for higher concentration electrospun PCL scaffolds

4.3 Thermal properties & crystallinity

The thermal properties of PCL random mats with different concentrations were studied using differential scanning calorimetry (DSC) (Pyris 6 DSC, Perkin Elmer) at a heating rate of 10 °C/min under nitrogen atmosphere within a heating temperature range of 20 to

80 °C As-received semicrystalline PCL pellets was examined to ensure that there was

no recrystallization occurred by operating with a heating temperature range of -80 to 80

°C as the reported glass transition temperature (Tg) of PCL was -60°C [64] The melting temperature (Tm) and the degree of crystallinity (χc) were obtained based on the results of thermogram Tm was taken to be the peak temperature and χc was calculated as ΔHm /

ΔHm° The ΔHm is the enthalpy of melting of PCL and ΔHm° is the enthalpy of melting

of fully crystalline PCL, which is 139.5 J/g [64] ΔHm was estimated from the area under the melting peak of the thermogram Samples of 2–3 mg were used for thermal analysis

DSC results showed the presence of both amorphous and crystalline regions on

eletrospun PCL nanofibers, suggesting PCL nanofibers are semicrystalline materials Table 4.1 shows the results of the thermal properties obtained from DSC The

thermograms of all the samples are found in Figure 4.3 – 4.7 The DSC results for all the samples are presented in Appendix B The melting peaks exhibit a shoulder which is likely due to the presence of varying lamellar thickness

The χc and Tm of as-received semicrystalline PCL pellets are comparable to reported values in the literature [65] By comparing the χc and Tm for different concentrations of electrospun PCL with the as-received PCL, it was noted that all the concentrations

exhibited lower χc and Tm The decrease in χc and Tm could be attributed to the fact that electrospinning is a rapid solidification process accompanied with significant solvent evaporation which hinders the formation of crystallites, thereby resulting in higher

content of amorphous regions

As mentioned earlier, 8 wt% PCL revealed a beads-on-string morphology, and thus it was expected that its χc is even lower than 14 wt% PCL since it does not have continuous fiber morphology Uniform fibrous structures started to form at 10 wt% PCL solution due to the optimum density of polymer chains within the solution that enables formation

of uniform diameter fibers 10 wt% PCL has the highest χc whereas 14 wt% PCL has the lowest χc Based on DSC results, χc decreased with increasing concentration

Table 4.1 Thermal properties of electrospun PCL at various concentrations

Figure 4.7 DSC thermograms for 14 wt% PCL

4.4 Tensile test of single electrospun PCL nanofibers

In view of the advantages of tensile test over other mechanical characterization tests, several preparation steps were carried out for the tensile test of nanofibers in order to characterize the mechanical properties of single electrospun nanofiber Hence, Young’s modulus (E), yield stress (σy), ultimate tensile stress (σu), and ultimate strain (εu) of single nanofibers were obtained

4.4.1 Collection of aligned nanofibers

A primary collector with parallel conductive strips was placed 60o to the horizontal axis for the deposition of aligned nanofibers [13], as shown in Figure 4.8 The configuration

of primary collector enables the transfer of nanofibers onto secondary collector or other substrates for other purposes

Figure 4.8 (a) Primary collector with parallel strips (b) Side view of primary collector

4.4.2 Isolation of single nanofibers

A secondary collector which consists of upper and lower frames was used to isolate single strand nanofibers for tensile testing, as illustrated in Figure 4.9 The gap between the parallel strips is equal to the length of secondary collector Single strand nanofibers were isolated by passing the secondary collector through the gap of primary collector so that the same strand of nanofiber was deposited onto the upper and lower frames which served different purposes: upper frame served as tensile test specimen whereas lower frame was provided for sample diameter measurement The carbon tapes were used to hold the polymeric nanofibers in place

60 ° Parallel strips

Figure 4.9 Secondary collector which consists of upper and lower frame

4.4.3 Tensile test specimen

The gauge length used was 10 mm [66] Lower frame of secondary collector was gold coated in order to determine the diameter of nanofibers using scanning electron

microscopy (SEM) (Quanta FEG 200, FEL, Netherlands) The SEM image for single strand nanofibers collected on lower frame was shown in Figure 4.10 at lower and higher magnification Diameter of each collected nanofibers was then measured by using

ImagePro software SEM images at higher magnification were used to investigate the diameter uniformity of nanofibers Nanofibers with non-uniform diameter were

discarded Figure 4.11 shows that the upper frame was mounted on the nano tensile tester (Nano Bionix System, MTS, TN, USA) and stretched to failure at a low strain rate

of 1 %/s at room temperature The alignment of the nanofibers was checked with an intense light source

Carbon tapes