Synthesis and Characterisation of PEGDA with QT

NANYANG TECHNOLOGICAL UNIVERSITY

Synthesis and Characterisation of PEGDA-based Hydrogels crosslinked with Pentaerythritol Tetrakis (3-Mercaptopropionate)

Toh Jia Ban

School of Materials Science & Engineering 2011

…………………………………………………………………………………………. BEng (2011) Synthesis and Characterisation of PEGDA-based Hydrogels crosslinked with Pentaerythritol Tetrakis (3Mercaptopropionate) ………………………………………………………………………………………..... Toh JB

NANYANG TECHNOLOGICAL UNIVERSITY

Synthesis and Characterisation of PEGDA-based Hydrogels crosslinked with Pentaerythritol Tetrakis (3-Mercaptopropionate)

Submitted in Partial Fulfillment of the Requirements for the Degree of Bachelor of Engineering of the Nanyang Technological University

by

Toh Jia Ban

School of Materials Science & Engineering 2011

Abstract PEGDA-based hydrogels have been synthesised successfully by photopolymerisation and Michael-type addition reaction. Before the hydrogel is prepared, degree of acrylation is determined by 1H NMR characterisation. Photo-crosslinked hydrogels has been characterised by FTIR and show successful thiol-linkage with the acrylate moieties. Selected PEGDA-based hydrogels have been put through Instron tensile and cyclic tests, and show highly elastic behaviour. The rheometry test reaffirmed the high elastic behaviour of selected PEGDA-based hydrogels through its corresponding high storage moduli for PEGDA macromers synthesized from PEG of Mw 8000 Da and 10000 Da. Equilibrium swelling test indicates the high water content and swelling ratio of photo-crosslinked hydrogels synthesized with PEGDA macromers with high degree of acrylation. Similar trend is found in Michael-type addition crosslinked hydrogels synthesized from PEG of M w 4000 Da, 8000 Da and 10000 Da.

In addition, higher wt% concentration of PEGDA involved in the preparation of Michael-type addition crosslinked hydrogels indicate better rheological property in terms of higher storage moduli. Equilibrium swelling test is performed to determine the composition of the three types of water existing in hydrogels. During degradation test, unexpected mass loss behaviour is observed in photo-crosslinked PEGDA-based hydrogels with high composition of non-freezing water indicated quantitatively by DSC test. Further research needs to be done to determine this unexpected behaviour. Stable behaviour is observed in hydrogel with high composition of free water, which depends on the high crosslink density in the hydrogel estimated using mesh size and number average molar mass between crosslinks. PEGDA-based hydrogels with high composition of non-freezing water dissolve in water highlights that strong interaction between polymer backbone and water may be detrimental to the structural stability of polymer in water.

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Acknowledgement This project is successfully completed with due respect to my supervisor Professor Emeritus Marc Jean Abadie, and my mentor researcher Dr Vitali Lipik. They have provided due guidance and advice throughout the project over an academic year to me. I will like to thank Dr Vitali Lipik for his assistance in performing the NMR experiment single handed at the School of Biological Sciences despite his busy schedule. In addition, I will like to extend his thanks researcher Dr. Lee Bee Hoon for giving his advice and ideas in contributing to the progress of the project.

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Table of Contents

ABSTRACT ............................................................................................................. I ACKNOWLEDGEMENT..................................................................................... II TABLE OF CONTENTS ..................................................................................... III LIST OF ABBREVIATIONS ............................................................................... V 1. INTRODUCTION .............................................................................................. 1 1.1 GENERAL DESCRIPTION OF HYDROGELS .................................... 1 1.2 LITERATURE REVIEW ........................................................................ 2 1.3 SCOPE AND OBJECTIVE OF THIS WORK ....................................... 4 2.

EXPERIMENTAL METHODS ..................................................................... 4 2.1 MATERIALS ........................................................................................... 5 2.2 PEGDA SYNTHESIS .............................................................................. 5 2.3 PREPARATION OF HYDROGELS ...................................................... 6 2.4 NMR CHARACTERISATION ............................................................... 7 2.5 DSC CHARACTERISATION ................................................................. 8 2.6 FTIR CHARACTERISATION ............................................................... 8 2.7 CHARACTERISATION OF MECHANICAL PROPERTY ................. 8 2.8 SWELLING BEHAVIOUR CHARACTERISATION ........................... 9 2.9 HYDROLYTIC DEGRADATION TEST ............................................. 10

3.

RESULTS AND DISCUSSION .................................................................... 10 3.1 NMR ANALYSIS OF DEGREE OF ACRYLATION OF PEG .......... 10

3.2 MECHANICAL CHARACTERISATION OF HYDROGELS BASED ON PEGDA WITH VARIOUS STARTING PEG MOLECULAR MASSES SYNTHESIZED BY 2 APPROACHES OF CROSSLINKING ......................... 13 3.2.1 RHEOLOGICAL BEHAVIOUR OF PEGDA-BASED HYDROGELS CROSSLINKED BY MEANS OF IRGACURE 2959 PHOTOINITIATOR DIRECTLY IN RHEOMETER ........................... 14

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3.2.2 MECHANICAL PROPERTY OF PEGDA-BASED HYDROGELS OBTAINED BY MEANS OF CURING UNDER UV LIGHT OUTSIDE OF RHEOMETER (RHEOMETRY TEST, INSTRON TENSILE AND CYCLIC TESTS). ..................................................................................... 16 3.2.2.1 STIFFNESS PROPERTY MEASUREMENTS OF PEGDABASED HYDROGELS OBTAINED BY MEANS OF EXTERNAL PHOTO- CROSSLINKING OUTSIDE OR RHEOMETER MEASURED BY INSTRON MICROTESTER ................................... 16 3.2.2.2 RHEOLOGICAL BEHAVIOUR OF PEGDA-BASED HYDROGELS OBTAINED BY MEANS CURING BY UV LIGHT OUTSIDE OF RHEOMETER ............................................................. 18 3.2.3 RHEOLOGICAL BEHAVIOUR OF PEGDA-BASED HYDROGEL OBTAINED BY MICHAEL-TYPE ADDITION CROSSLINKING ..................................................................................... 20 3.3 HYDROGEL STRUCTURAL CHARACTERIZATION .................... 23 3.3.1 ATR-FTIR (ATTENUATED TOTAL REFLECTANCE FOURIER TRANSFORM INFRARED SPECTROSCOPY) ................ 23 3.3.2 SWELLING TEST ........................................................................... 23 3.3.3 TYPE OF WATER COMPOSITION USING DSC ANALYSIS (PHOTO- CROSSLINKED HYDROGEL) ............................................. 27 3.3.4 MESH SIZE AND CROSSLINK DENSITY.................................. 30 3.3.5 HYDROLYTIC DEGRADATION OF PHOTO-CROSSLINKED HYDROGELS........................................................................................... 32 4.

CONCLUSION ............................................................................................. 34

5.

RECOMMENDATION ................................................................................ 34

BIBLIOGRAPHY ................................................................................................ 35 APPENDIX 1 ........................................................................................................ 39

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List of Abbreviations Abbreviation

Term

ATR ATRP CNTs DCM DI DMSO DSC EWC (%) FTIR HA IOLs Mc Mn Mw NaOH NMR PAA PAAm PBS PEG PEGDA PHEMA PMAA PNIPAAm PVA QT SEC SR (%) TREGDA UV E R T w/v wt% Δm% Ws Wd Wdo Wdf ξ ρ ν

attenuated total reflectance atom transfer radical polymerisation carbon nanotubes dichloromethane deionised deuterated dimethyl sulphoxide differential scanning calorimetry equilibrium water content in percentage Fourier Transform Infrared Hyaluronan and its derivative injectable oracular lenses number average molar mass between crosslinks in g∙mol-1 number average molecular mass in Dalton (Da) weight average molecular mass in Dalton (Da) sodium hydroxide nuclear magnetic resonance poly (acrylic acid) polyacrylamide phosphate-buffered saline poly (ethylene glycol) poly (ethylene glycol) diacrylate poly (hydroxyethyl methacrylate) poly (methacrylic acid) poly (N-Isopropylacrylamide) poly (vinyl alcohol) pentaerythritol tetrakis (3-mercaptopropionate) size exclusion chromatography swelling ratio in percentage triethlyene glycol diacrylate ultraviolet Young’s modulus molar gas constant room temperature in Kelvin (K) weight per volume weight percentage mass loss in percentage swollen mass of hydrogel dry mass of hydrogel dry mass before hydrolytic degradation test dry mass after hydrolytic degradation test mesh size in nanometre polymer density in g∙cm-1 wavenumber v

1. Introduction 1.1 General description of Hydrogels Hydrogel is a class of viscoelastic materials having proven applications as biomaterials.[1] Hydrogels has generated interest in the biomedical materials research ever since Wichterle and Lim published a proposal in the 1960 supporting its use as biomaterials. [2] It has the potential to be made for applications in cell delivery, in tissue engineering as polymer scaffold, in wound healing, in diagnostic devices as electroconductive biomaterials.[3-6] Hydrogels has the capability to imitate the functional role of extracellular matrix in tissue.[5, 7]

Synthetic polymers used for synthesis of hydrogels can be further classified into two categories, i.e., synthetic neutral and synthetic charged. There are three common neutral synthetic polymers used for synthesizing hydrogels, i.e., poly (ethylene glycol) (PEG), poly (hydroxyethyl methacrylate) (PHEMA) and poly (vinyl alcohol) (PVA).[5]

Responsive hydrogels respond to external stimuli including pH and temperature and the three common charged synthetic polymers are poly (acrylic acid) (PAA), poly (methacrylic acid) (PMAA) and polyacrylamide (PAAm). They are also known as smart hydrogels for their ability to respond to the environment. Hydrogels responding to external stimulus as temperature are known as thermosensitive hydrogel. Thermosensitive hydrogel system is one of the extensively researched systems for tissue engineering and drug delivery and one notable thermosensitive polymer is the poly (N-Isopropylacrylamide) (PNIPAAm). They swell at lower critical solution temperature (LCST) due to a phenomenon known as reversible volume-phase transition. [5, 8]

Alternatively, Natural polymers can be used to synthesise hydrogels. Natural polymers that are widely used include collagen, fibrin, hyaluronan and its derivative (HA), alginate, chitosan and agarose. They have been successfully modified to enhance their stiffness property. [4, 5, 7, 9] For hydrogels synthesised from synthetic 1

polymers, their mechanical property is generally acceptable; however, there is a need to improve its biocompatibility such as its antifouling property to protein and cell adhesion. [10]

Synthetic polymers can be formed using various mechanism methods such as Michael-type addition, free-radical copolymerisation, coupling reaction between aldehyde and hydrazide, Diels-alder click reaction, atom transfer radical polymerisation (ATRP) and Huisgen’s 1,3-dipolar azide-alkyne cycloaddition. [1115] Conditions such as the chemical environment and temperature are critical for formation of hydrogels in both physical and chemical crosslinking reaction. [5, 16]

Hydrogels is quintessentially stiff in dry state and flexible in swollen state. The increase in water content is concomitant with the decrease in stiffness as confirmed in the Instron tensile test of photo-crosslinked PEGDA-based hydrogel. There are several ways to improving the stiffness property including surface modification and insertion of bulk materials like carbon nanotubes (CNTs).[1, 15-18] 1.2 Literature Review PEG-based materials are more biocompatible compared to other materials such as PHEMA, and US Food and Drug Administration have approved the materials for clinical usage due to it being non-toxic and non-immunogenic.[5, 10, 19] It is similar to the stiffness property of tissues in the swollen state due to its capacity for high water content.[17, 20] Surface modification of PEG has been widely used to improve its surface properties.[5, 10]

Thiol-ene coupling reaction is a combined mechanism of photopolymerisation and Michael–type nucleophilic addition. It is a high-yield reaction for producing functional materials with limited environmental interference.[21] As a single mechanism, Michael-type nucleophilic addition of thiol and diacrylate has been carried out relatively recently. Recent developments include the reaction of thiolated HA, heparin and gelatine with PEGDA.[22-27] 2

Differential Scanning Calorimetry (DSC) is a measurement of energy flow difference between a sample and a reference material through a temperature control program. [28] It is used to characterise hydrogels to determine the 3 different states of water, i.e., bound water, free water and non-freezing water.[29] 1H NMR is used to characterise hydrogels using the principle of resonant frequencies of different molecular structures to determine various functional groups or moieties present in the hydrogels.[28] The information obtained is applied to determine the degree of substitution or conversion of a chemical reaction such as acrylation.[30, 31] Fourier Transform Infrared (FTIR) and Nuclear Magnetic Resonance (NMR) had been used in confirming the functional group of various hydrogels based on common materials such as Poly (hydroxyethyl methacrylate) (HEMA).[17, 32]

During a dynamic mechanical test performed in a rheological measurement, the dynamic storage modulus, G’ and dynamic loss modulus, G’’ represents the elastic response and viscous response respectively. [1, 33] They formed a relationship through the following equation where complex modulus, G*= G’ + G”.[28] Gel point can be observed during a process known as the sol-gel transition. By measuring the storage and loss modulus using a frequency and stress sweep test, power law behaviour is observed. After the sol-gel transition, the typical behaviour for gel is represented by the storage and loss modulus running in parallel. [8] G”

G* δ

G’ Figure 1 Schematic Diagram of Complex modulus (G*), Storage modulus (G’) and Loss Modulus (G”) where δ is the phase angle In a crosslinked gel, the determination of the soluble part is known as sol (s) while the insoluble part is known as gel (g). Typically, s + g = 1.This can be used to determine the proportion of a gel that is not crosslinked, otherwise known as sol and the proportion of a gel that is crosslinked, otherwise known as gel. [8]

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The understanding of hydrogels’ structure is enhanced through two methods, i.e., the equilibrium-swelling theory and the rubber-elasticity theory. At equilibrium, the thermodynamic forces keeping it stable are the retractive elastic force and the opposing force resulting from interaction between water and polymer chains, simulating the condition of a hydrogel immersed in water. The elastic nature of hydrogels to restore to original dimension after slight deformation is similar to rubber. Therefore, the rubber-elasticity theory is tied in together with the equilibrium-swelling theory. The two theories aid in the critical understanding of hydrogels’ structure such as mesh size (ξ) and molar mass between crosslinked points (Mc); the Flory-Rehner theory is thus used to understand the neutral hydrogel characteristics through the combination of the two aforementioned theories. [5, 34, 35]

The application of PEG derivatives has widespread application in emerging biomedical field such as bioprinting live cells and cell encapsulation.[26] PEGDA has both hydrophilic and hydrophobic regions at the PEG chains (CH 2CH2O) and acrylates (CH2=CH) respectively.[31, 36] It presents an opportunity in biomaterials research in understanding how the presence of both hydrophilic and hydrophobic regions affects the biomedical application of PEGDA. [20] 1.3 Scope and Objective of this work This project seeks to understand the mechanical and structural properties of both photo-crosslinked and Michael-type addition crosslinked PEGDA-based hydrogels that is synthesised from different initial PEG molecular masses through characterisation techniques like FTIR, DSC, equilibrium swelling test and timedependent hydrolytic degradation experiment. It attempts to create an extensive approach to the thiol-ene photopolymerisation used by Niu, G. G., et al. in their research on injectable oracular lenses (IOLs).[17]

2. Experimental Methods

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2.1 Materials PEG 200 Da, 4000 Da, 8000 Da, 10000 Da and 35000 Da, TREGDA and triethylamine (TEA) was ordered from Fluka (Buchs, Switzerland). Dichloromethane and Pentaerythritol tetrakis (3-mercaptopropionate) was obtained from Aldrich (Sleeze, Germany). Diethyl ether was obtained from Fisher. Acryloyl Chloride was purchased from Merck (Darmstadt, Germany). Pluronic L121 was ordered from BASF Corp. (Parsippany, NJ, USA). Irgacure 2959 (4-(2-hydroxyethoxy) phenyl-(2hydroxy-2-propyl) ketone) was obtained from Ciba Specialty Chemical Corp. (Tarrytown, NY, USA). NaOH was obtained from Aldrich (Milwaukee, WI, USA).

Figure 2 Key materials for Synthesis of PEGDA Hydrogels 2.2 PEGDA synthesis

Triethylamine & DCM, Dark at Room Temp

Figure 3 PEGDA synthesis PEG diacrylate (PEGDA) was prepared from linear PEG where PEG of Mw 4000 Da, 8000 Da, 10000 Da and 35000 Da were used. Acrylation of PEG was carried out under Argon gas by dissolving PEG in dichloromethane (DCM) solution (0.2 g 5

PEG/mL DCM) with acryloyl chloride and triethylamine at a feed molar ratio of 1:1.5:1.5 of OH-group (PEG): acryloyl chloride: triethylamine respectively. Prior to adding DCM solution, azeotropic distillation was performed in toluene solution using a Dean-Stark trap to ensure the acrylation process was carried out in dry condition. Subsequently, the mixture was stirred in the dark at room temperature overnight. The final product was precipitated in excess refrigerated diethyl ether to eliminate toluene and subsequently recovered using filtration. Finally, the recovered product was dried in vacuo overnight. 2.3 Preparation of hydrogels The synthesis for PEGDA-based hydrogels has two different crosslinking approaches, which are thiol-ene photopolymerisation and Michael-type nucleophilic addition.

For photo-crosslinking reaction, the PEGDA powder was dissolved in deionised water (DI) water at a pre-determined mass proportion (0.25g PEGDA/mL DI water) in a 80mm-diameter Petri dish. Subsequently, photoinitiator Irgacure 2959 was added to the PEGDA solution at 0.1% (w/v) and was exposed to 365nm Ultraviolet (UV) light for minimum period of 10 min, up to a maximum time span of 20 min. The Wilber Lourmat UV lamp was set at wavelength of 365nm and the 20 wt% PEGDA solution was placed under UV light for 10 – 20 min.

Figure 4 Structural formula for photoinitiator Irgacure 2959 used in photocrosslinking For Michael-type addition crosslinking, the PEGDA was dissolved in a mixture of DI water, Phosphate-buffered saline (PBS) solution at pH7.4 and Pluronic L121 surfactant (which was referred herein as precursor solutions). The mass proportion of PEGDA of initial PEG molecular masses of 4000 Da, 8000 Da and 10000 Da were at 28 wt% (0.39 g PEGDA/ mL precursor solutions), 36 wt% (0.56g PEGDA/mL 6

precursor solutions) and 20 wt% (0.25g PEGDA/mL precursor solutions) respectively. QT was added at 50% of the required amount for equal molar thiolacrylate ratio. Additional NaOH at 10% (w/v) can be added to facilitate the crosslinking reaction if necessary. The mixing of the precursor solutions, PEGDA and QT was performed through 2 interconnected syringes for 20s.[37] The interconnected syringes were left unperturbed at room temperature for 2 days.

Legend: QT in alkaline medium PEGDA Figure 5 Michael-type Addition for crosslinking of PEGDA-based hydrogels 2.4 NMR characterisation Dry sample of PEGDA of initial PEG molecular mass 4000 Da, 8000 Da 10000 Da and 35000 Da were kept at 40˚C in a EYELA VOS-201SD vacuum oven for drying over night with molecular sieves. Subsequently, each sample was injected into a septum-capped standard (5mm, Norell) NMR tube, which was swept out by nitrogen gas. It was then characterised using NMR spectrometer Bruker DRX 400 MHz The chemical shifts were performed with reference to the water miscible organic solvent, i.e., chloroform (CCl4).[30] The spectrum for each PEGDA sample was acquired, plotted and analysed using the program Topspin (Bruker, USA).

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2.5 DSC characterisation The DSC was performed on hydrogels by putting a sample of mass 5-15mg through 2 cycles. The first cycle was to cool down the sample to -70˚C from room temperature at a computer-controlled rate. The second cycle was to heat up the sample from -70˚C to 200˚C at predetermined rate of 5˚C /min. The enthalpy of the bound and free water states was measured through integrating the area of the 2 troughs of the DSC curves in the temperature region up to 0˚C using the software Universal Analysis.[29] Swollen PEGDA-based hydrogels was placed in a hermetic aluminium pan designed for volatile materials.

2.6 FTIR characterisation For the imaging of the thioether linkage of photo-crosslinked PEGDA-based hydrogels crosslinked with QT, the attenuated total reflectance-FTIR (ATR-FTIR) scan was performed with FTIR equipment Perkin Elmer Spectrum GX. For each sample, a spectrum is obtained using the ATR utilising a diamond internal reflection element mounted on a holder at a resolution of 4cm-1 in the range 4000- 400cm-1 for a total of 16 scans.[32] The peak belonging to thiol group and acrylate was then identified using the software Spectrum (Perkin Elmer, USA). 2.7 Characterisation of Mechanical Property The viscoelastic or rheological behaviour of PEGDA-based hydrogels was characterized using Anton Paar Physica MCR 501 rheometer and mechanical property was characterised using Instron Microtester 5848. It was performed with an Anton Paar PP25/TG 25-mm diameter cone plate. In-situ photo-crosslinking was performed using the rheometer and time sweep test was performed with parameters set manually using the software RheoPlus (Anton Paar, Austria). The parameters used are shear stress, τ, set at 10 Pa and frequency, f, set at 1 Hz.[37]

For crosslinked hydrogels performed externally of the rheometer, shear stress and frequency sweep tests was performed with parameters set manually using the 8

aforementioned software. The two major parameters to be set are similar to the time sweep test. However, the variables used are different in each case for both shear stress and frequency sweep tests. For shear stress sweep test, τ was set at a range of 1 to 1000 Pa and f was set at 1 Hz. For frequency sweep test, f was set at a range of 0.1 to 100 Hz and τ was set at 10 Pa.

The Instron tensile test was performed on an Instron 5848 Microtester at a tensile rate of 5.0mm/min under load of 10N. The PEGDA hydrogel sample of initial PEG molecular masses 4000 Da, 8000 Da, 10000 Da was shaped using the Schmidt ManualPress 305 attached with a die with dumbbell-shaped orifice of 15mm in length. The graphical solution and measurement was performed with software Bluehill 2 (Instron, USA). The Instron cyclic test was performed on the hydrogel sample of initial PEG molecular masses 8000 Da and 10000 Da for consecutive stretch limit of 100%, 200% and 300%. 2.8 Swelling Behaviour Characterisation A simple gravimetric method was employed for the swelling test at 37˚C. It was based on measuring the equilibrium water content in the hydrogels after immersing it in deionised (DI) water for a continuous period at a fixed temperature.

The swelling test was studied by measuring the dry mass of hydrogel and swollen mass of hydrogel using AND GR-20L beam balance accurate up to 0.1mg. The equilibrium water content was calculated using equation (1):

EWC (%) 

Ws  Wd  100 (1) Ws

Where Ws was the swollen mass of hydrogel and Wd was the dry mass of hydrogel. In addition, the swelling ratio was also calculated to provide an alternative perspective on the volume of water content in the hydrogel using equation (2). Swelling ratio was also known as degree of swelling.[37]

SR(%) =

Ws  Wd  100 (2) Wd 9

2.9 Hydrolytic Degradation Test Gravimetric measurement of mass loss was used after PEGDA-based hydrogel samples of masses between 5-20mg was immersed in DI water at 37˚C for a period up to 8 weeks.[32] Individual sample was placed in individual separable 10mL transparent glass vial for easy observation and drying of the hydrogel in weekly mass loss measurement. Each photo-crosslinked hydrogel sample of every initial PEG molecular masses of 4000 Da, 8000 Da, 10000 Da, 35000 Da was weighed after drying for 3 days in a Binder vacuum oven at 37˚C, allowing sufficient time to achieve dry mass. The post-degradation dry mass is measured using AND GR-20L beam balance accurate up to 0.1mg. The % mass loss was calculated with equation (3):

% mass loss 

Wdo  Wdf Wdo

100 (3)

Where Wdf is the final dry mass after hydrolytic degradation test and Wdo is the initial dry mass before hydrolytic degradation test.

3. Results and Discussion 3.1 NMR analysis of degree of acrylation of PEG Degree of end group conversion can be calculated through the signals ratio of 1H NMR spectra obtained. Degree of acrylation has been calculated by means of equation (4). [31]

 Acrylates ( H 1

) M PEG  6 44 100 (4) %acrylation  1  PEG Backbone ( H b ) a

4 Where

 Acrylates ( H 1

 PEG Backbone ( H 1

b

a

)

is

the

integrated

ratio

of

the

1

Ha

proton,

) is the integrated ratio of the 1H b proton and M PEG is the

10

molecular mass of PEG prior to acrylation. Degree of PEG acrylation of PEG with different initial molecular masses is presented in table 1.

Table 1 Degree of acrylation of PEGDA and its corresponding chemical shift Mn of PEGDA or Degree of PEG 1 1 N type of macromer Ha δ (ppm) Hb δ (ppm) acrylation (%) (Da) 1

4000

92.5

5.8 - 6.5

3.2 - 4.1

2

8000

79.5

5.8 - 6.5

3.2 - 4.1

3

10000

99.0

5.75 - 6.5

3.25 - 4.2

4

35000

83.5

5.8-6.5

3.2-4.2

1

H NMR spectrum of acrylated PEG of molecular masses 4000 Da, 8000 Da, 10000

Da and 35000 Da from table 1 is presented below.

b) ∙CH2CH2O∙ 1

Ha b a 1

Hb

a) ∙CH=CH2

Figure 6 1H NMR spectrum of acrylated PEG of molecular mass 10000 Da

11

b) ∙CH2CH2O∙

1

Ha b a 1

Hb

a) ∙CH=CH2

Figure 7 1H NMR spectrum of acrylated PEG of molecular mass 4000 Da

b) ∙CH2CH2O∙

a) ∙CH=CH2

1

Ha b a 1

Hb

Figure 8 1H NMR spectrum of acrylated PEG of molecular mass 8000 Da

12

b) ∙CH2CH2O∙

1

Ha b

a) ∙CH=CH2

a 1

Hb

Figure 9 1H NMR spectrum of acrylated PEG of molecular mass 35,000 Da 3.2 Mechanical characterisation of hydrogels based on PEGDA with various starting PEG molecular masses synthesized by 2 approaches of crosslinking The mechanical properties of PEGDA-based hydrogels are characterized using Anton Paar Physica MCR 501 rheometer and Instron Microtester. The information is classified according to the types of crosslinking mechanism and the state of the PEGDA materials prior to the characterisation test.

The first segment of this subsection deals with the rheological behaviour of PEGDAbased hydrogels of different molecular masses that was crosslinked in-situ in the Physica MCR 501 rheometer under Ultraviolet light. The second segment moves into the mechanical property of PEGDA-based hydrogels photo-crosslinked by Vilber Lourmat UV lamp by subjecting the hydrogels to rheometer-based shear stress and frequency sweep tests, and Instron-based tensile and cyclic tests. The third segment covers the rheological behaviour of Michael-type addition crosslinked PEGDAbased hydrogels by subjecting the crosslinked hydrogels to rheometer-based stress and frequency sweep tests.

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3.2.1 Rheological behaviour of PEGDA-based hydrogels crosslinked by means of Irgacure 2959 photoinitiator directly in rheometer Time Sweep Rheometry Test 6 selected samples of PEGDA-based hydrogels with the following initial number average molecular mass for PEG of 200 Da, 4000 Da, 8000 Da, 10000 Da, and 35000 Da including one reference material Triethylene Glycol Diacrylate (TREGDA). All data is collected in table 2. Table 2 Mechanical properties of PEGDA-based hydrogels made by means of UV curing in rheometer Mn of PEGDA or type of Storage Loss Modulus N Observations macromer (Da) modulus (Pa) (Pa) 1

200

0.0210*104

12.0

Very weak gel

2

4000

1.08*104

71.9

Good gel

3

8000

7.70*10

4

2210

Very strong gel

4

10000

6.82*104

1500

Strong gel

5

35000

0.0336*104

72.4

Weak gel

6

TREGDA

82.0*104

77.3*104

Solid polymer

There are clear maximum values of storage modulus in the parabolic time profile for each PEGDA hydrogel with initial PEG molecular masses of 200 Da, 4000 Da, 8000 Da, 10000 Da and 35000 Da and reference material TREGDA. As observed from the storage modulus, PEGDA with Mn=8000 Da formed a very strong gel. The rheometry graph indicates the storage and loss modulus while PEGDA and QT are crosslinked in-situ with the aid of Irgacure 2959 under UV lamp attached to Physica MCR 501 rheometer. Rheological behaviour of PEGDA-based hydrogels is presented in figure 10, 11 and 12 below.

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G', G''

G', G''

3

10

10

5

Pa

Pa

10

4

2

10

10

G'

G'

1

10 G''

10

3

2

G'' 10

1

0

10

10

10

-1

10

0

50

100

Average Time

150 200 tavr

A

0

-1

250 s 300

0

50 100 150 Average Time t avr

B

Anton Paar GmbH

200

250 s 300

Anton Paar GmbH

Legend: G' Storage Modulus G'' Loss Modulus

Figure 10 Time Sweep rheological profiles for 20 wt% solution in water, photoinitiator Irgacure 2959 cured in-situ under UV light at 37˚C of A) PEGDA Mn = 35000 Da, B) PEGDA Mn = 10000 Da G', G''

10

10 Pa

Pa 10

10

10

4

10

3

G'

G' G''

10

10

10

4

3

2

G''

2

10 10

G', G’’

5

5

1

10 0

0

50

100

Average Time

Legend : G' Storage Modulus

150 tavr

C

200

Anton Paar GmbH

250 s 300

10

1

0

-1

0

50

100

Average Time

150 tavr

D

200

250 s 300

Anton Paar GmbH

G'' Loss Modulus

Figure 11 Time Sweep rheological profiles for 20 wt% solution in water, photoinitiator Irgacure 2959 cured in-situ under UV light at 37˚C of C) PEGDA Mn = 8000 Da, and D) PEGDA M n = 4000 Da 15

G', G''

4

10 Pa 10

10

Pa

3

10 10

G' 1 10 ’ G''

G'

10

G'' 10

0

10 10

10

5

2

10

10

G', G''

7

4 3 2 1

-1

10 -2

0

200

400

Average Time

600

Legend:

10

-1

0

50

100

150 t avr

Average Time

tavr

E

800 s 1000

0

Anton Paar GmbH

F

200

250

s

300

Anton Paar GmbH

G' Storage Modulus G'' Loss Modulus

Figure 12 Time Sweep rheological profiles for 20 wt% PEGDA solution crosslinked with photoinitiator Irgacure 2959 in-situ under UV light at 37˚C of E) PEGDA M n = 200 Da and, reference material F) Triethylene glycol diacrylate (TREGDA) Gel points or gelling time for each PEGDA-based hydrogel is observed to be within 30s with the exception of 200 Da PEGDA-based hydrogel.[8, 34] The fast gelling time represent the useful application for in-situ gelling process under UV light.

3.2.2 Mechanical property of PEGDA-based hydrogels obtained by means of curing under UV light outside of rheometer (rheometry test, Instron tensile and cyclic tests). 3.2.2.1 Stiffness property measurements of PEGDA-based hydrogels obtained by means of external photo- crosslinking outside or rheometer measured by Instron microtester All data is collected in the table 3 for PEGDA-based hydrogels with initial PEG molecular masses 4000 Da, 8000 Da, 10000 Da and 35000 Da.

16

Table 3 Mechanical properties of photo-crosslinked PEGDA-based hydrogels Young’s Young’s Mn of PEGDA or type of Elongation at N modulus (Pa) modulus (Pa) macromer (Da) break (%) (tensile test) (cyclic test) 1 4000 1.59*104 730 4 2 8000 10.62*10 408 4 4 3 10000 2.75*10 3.91*10 430 4 4 35000 0.86*10 357 Cyclic test had been performed for two hydrogels (based on 8000 Da and 10000 Da PEGDA). Other samples of gels (4000 Da and 35000 Da) were mechanically weak for cyclic test). The selected PEGDA-based hydrogels possess good elastic property with degree of shape recovery close to 100% even after an elongation of 300%.

Method of testing is as stated where one sample was subjected to three consecutive trials of stretch limits of 100%, 200% and 300%. After each elongation, the stress was removed and sample recovered itself. Therefore three loops (or hysteresis) curves are seen (100, 200 and 300%) on the graph. It is seen from figure 13 that elasticity of PEG Hydrogel 10000 Da is much higher in comparison to the elasticity of industrial elastomer PLC 70/30.

Figure 13 Cyclic test of selected PEGDA-based hydrogel with initial PEG molecular mass 10000 Da (left) and industrial elastomer PLC 70/30 (right) PEGDA-based Hydrogel recovers immediately with limited or insignificant hysteresis. Top line of cyclic curve indicates hydrogel undergoing tensile stress and bottom line of cyclic curve indicates hydrogel recovering along similar lines.

17

Table 4 Young’s Modulus and Elongation at break of Dry Hydrogel Mn of PEGDA or Young’s Elongation at N type of macromer modulus (Pa) break (%) (Da) (tensile test) 1

8000

6.98357*105

646

2

10000

3.10214*105

895

3.2.2.2 Rheological behaviour of PEGDA-based hydrogels obtained by means curing by UV light outside of rheometer Table 5 Rheological measurement of PEGDA-based hydrogels made by means of photo-crosslinking independent of rheometer Frequency Sweep Rheometry Stress Sweep Rheometry Test Mn of PEGDA Test or type of N Storage Loss Modulus Storage Loss Modulus macromer (Da) modulus (Pa) (Pa) modulus (Pa) (Pa) 1

4000

1.56*103

2.24*102

1.65*103

2.68*102

2

8000

17.6*103

4.75*102

18.1*103

5.44*102

3

10000

12.2*103

16.9*102

14.6*103

17.6*102

4

35000

9.73*103

15.2*102

2.69*102

2.96*102

In agreement with the time sweep profile for in-situ photo-crosslinking, the shear stress sweep and frequency sweep test was performed for photo-crosslinking of PEGDA hydrogels at increasing shear stress (fixed frequency) and at increasing frequency (fixed shear stress). It highlighted the response of the hydrogel to increasing shear stress, which turns weak.

18

Shear Stress Sweep Test G’, G” 5

100,000

10

Pa

Pa

10,000

10

Storage Modulus G'

4

1,000

10

100

10

10

10

-1

10

0

10

1

τ

10

2

10

3 Loss

Modulus G''

2

1

3

Pa

10

Anton Paar GmbH

Legend G': Storage Modulus G'': Loss Modulus Dark Blue: 10K PEGDA with photoinitiator crosslinked outside of rheometer Red: 8K PEGDA with photoinitiator crosslinked outside of rheometer Green: 4K PEGDA with photoinitiator crosslinked outside of rheometer Sky Blue: 35K PEGDA with photoinitiator crosslinked outside of rheometer Orange: 10K PEGDA 50% Michael-type addition crosslinked with tetrakis required

Figure 14 Shear stress sweep rheological profiles for photo-crosslinked PEGDA-based hydrogels and a Michael-type addition crosslinked PEGDA-based hydrogel

19

Frequency Sweep Test G', G''

105

Pa 10 4

G'

3

10

G''

Storage Modulus

Loss Modulus

2

10

1

10

10

0 -1

10

0

2

1

10

10

10

Hz

f Anton Paar GmbH

Legend G': Storage Modulus G'': Loss Modulus Dark Blue: 10K PEGDA with photoinitiator crosslinked outside of rheometer Red: 8K PEGDA with photoinitiator crosslinked outside of rheometer Green: 4K PEGDA with photoinitiator crosslinked outside of rheometer Sky Blue: 35K PEGDA with photoinitiator crosslinked outside of rheometer Orange: 10K PEGDA Michael-type addition crosslinked with 50% tetrakis needed

Figure 15 Frequency sweep rheological profiles for photo-crosslinked PEGDA-based hydrogels and a Michael-type addition crosslinked PEGDA-based hydrogel

Figure 14 and 15 indicated the superiority in storage moduli of the photo-crosslinked hydrogels for PEGDA-based hydrogel at the same mass proportion of 20 wt% in comparison to Michael-type addition hydrogels. 3.2.3 Rheological behaviour of PEGDA-based Hydrogel obtained by Michaeltype addition crosslinking All data are presented in the table 6. Rheological behaviour is shown in the figure 16 and 17 for shear stress and frequency sweep test respectively.

20

Table 6 Rheological measurement of PEGDA-based hydrogels made by means of Michael-type addition crosslinking Mn of Frequency Sweep Rheometry Stress Sweep Rheometry Test PEGDA or Test N

type

of

Macromer (Da)

Storage

Loss Modulus Storage

Loss Modulus

modulus (Pa)

(Pa)

modulus (Pa) (Pa)

1

4000

5.191*103

17.75*102

4.669*103

18.02*102

2

8000

53.924*103

17.39*102

60.5*103

20.6*102

3

10000

6.150*103

2.19*102

7.14*103

1.26*102

Shear Stress Sweep Rheometry Test RheoPlus

100,000

10

Pa

10,000

G'

G''

1,000

100 0

200

τ

400

600

Pa

1,000

1

Anton Paar GmbH

Legend G': Storage Modulus G'': Loss Modulus Dark Blue: 10K PEGDA Michael-type addition crosslinked outside of rheometer Red: 8K PEGDA Michael-type addition crosslinked outside of rheometer Green: 4K PEGDA Michael-type addition crosslinked outside of rheometer

Figure 16 Shear stress sweep rheological profiles of Michael-type addition crosslinked hydrogels

21

Frequency Sweep Rheometry Test All data are summarized on table 6. RheoPlu s

100,000

1 0

Pa

10,000

1,000 G''

G '

100

10

1 0

20

40

60 Hz 80

f Anton Paar GmbH

Legend G': Storage Modulus G'': Loss Modulus Dark Blue: 10K PEGDA Michael-type addition crosslinked outside of rheometer Red: 8K PEGDA Michael-type addition crosslinked outside of rheometer Green: 4K PEGDA Michael-type addition crosslinked outside of rheometer

Figure 17 Frequency sweep rheological profiles of Michael-type addition crosslinked hydrogels The trend in the storage moduli in Michael-type addition crosslinked and photocrosslinked PEGDA-based hydrogels is similar according to their initial molecular masses. However, the increase in mass proportion of PEGDA in the preparation of

22

hydrogels helps to increase the storage moduli of hydrogels as indicated by the shear stress and frequency sweep profile in figure 16 and 17.

3.3 Hydrogel structural characterization 3.3.1 ATR-FTIR (Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy) A

B

C

D E S-H stretching

ν, cm-1

C=C stretching

Figure 18 FT-IR image showing the remnant thiol group and the depleted acrylate group of photo-crosslinked PEGDA-based hydrogels of PEG molecular masses A) 200 Da B) 4000 Da C) 8000 Da D) 10000 Da and E) 35000 Da at wavenumber, υ = 2400 - 2600 cm-1 and υ = 1620 - 1650 cm-1 respectively The peak at the range of 2400 - 2600 cm-1 indicates thiol stretching in the photocrosslinked hydrogels showing the presence of excess QT. The disappearance of the acrylate group at the range of 1620 - 1650 cm-1 indicates that acrylate group is depleted during photo-crosslinking and hence, the successful formation of thioether moiety through the combined mechanism of free radical polymerisation and Michael-type addition. [17, 38-40]

3.3.2 Swelling test The swelling test was conducted on PEGDA-based hydrogels based on initial PEG molecular masses 200 Da, 4000 Da, 8000 Da, 10000 Da and 35000 Da with a reference macromer of TREGDA.

23

Table 7 EWC (%) of photo-crosslinked PEGDA-based hydrogels EWC (%)

EWC (%)

EWC (%)

EWC (%)

EWC (%)

EWC (%)

of PEGDA

of PEGDA

of PEGDA

of PEGDA

of PEGDA

of

Hydrogels

Hydrogels

Hydrogels

Hydrogels

Hydrogels

reference

Mn = 200

Mn = 4000

Mn = 8000

Mn =

Mn =

material,

Da

Da

Da

10000 Da

35000 Da

TREGDA

5

61.67

84.32

83.47

84.02

84.05

0.00

10

61.67

88.59

81.23

88.07

84.05

0.00

15

61.67

90.02

89.30

90.61

84.05

0.00

30

61.67

92.32

90.80

92.29

84.05

0.00

60

61.67

92.59

91.25

92.87

84.05

0.00

120

61.67

92.64

91.39

93.26

84.05

0.00

240

61.67

93.12

90.99

93.35

84.05

0.00

480

61.67

93.92

91.43

93.32

84.05

0.00

900

61.67

93.79

91.52

93.38

84.05

0.00

1140

61.67

93.79

91.52

93.44

84.05

0.00

1320

61.67

93.58

91.51

93.41

84.05

0.00

Time (min)

Table 8 Maximum EWC (%) and SR (%) of photo-crosslinked PEGDA-based hydrogels Mn of photocrosslinked

EWC (%)

SR (%)

200

61.67

160.9

4000

93.79

1510.5

8000

91.52

1079.8

10000

93.44

1424.3

35000

84.05

527.0

TREGDA

0

0

PEGDA Hydrogels (Da)

24

Figure 19 EWC (%) for immersion of photo-crosslinked PEGDA-based hydrogels in DI water Table 9 EWC (%) of Michael-type addition crosslinked PEGDA hydrogels EWC (%) of MichaelEWC (%) of MichaelEWC (%) of MichaelTime

type addition crosslinked

type addition crosslinked

type addition crosslinked

(min)

PEGDA Hydrogels with

PEGDA Hydrogels with

PEGDA Hydrogels with

Mn= 4000 Da

Mn= 8000 Da

Mn = 10000 Da

10

52.74

58.76

83.31

40

74.28

72.30

90.70

60

77.70

76.28

92.77

120

83.41

80.01

93.56

720

87.59

83.63

92.75

840

88.58

84.90

92.20

1320

88.83

85.52

92.03

2760

89.27

85.98

91.96

25

Table 10 Maximum EWC (%) and SR (%) of Michael-type addition crosslinked hydrogels Mn of Michael-type addition crosslinked

EWC (%)

SR (%)

4000

89.27

832.5

8000

85.98

613.1

10000

93.56

1453.4

PEGDA Hydrogels (Da)

Figure 20 EWC (%) for Michael-type addition crosslinked hydrogels From figure 19 and 20, it is noticed that the EWC (%) stabilised at around 4 hr, and limited or negligible increase in EWC (%) was observed thereafter. The graph also indicates that hydrogels of initial PEG molecular mass of 200 Da and 35000 Da possess the lowest EWC (%) among the hydrogels formed, excluding the reference polymer TREGDA. Using the values obtained from EWC (%), the composition of three different classes of water is obtained together with DSC characterisation. This will be presented in the next subsection for both the photo-crosslinked and Michaeltype addition crosslinked hydrogels.

26

3.3.3 Type of water composition using DSC analysis (Photo- crosslinked hydrogel)

Free water ΔH Bound water ΔH

Figure 21 DSC endotherm graph showing bound and free water for photocrosslinked PEGDA-based hydrogel of Mn = 8000 Da

Figure 22 DSC endotherm graph of Michael-type addition crosslinked hydrogel of containing 36 wt% PEGDA of M n =8000 Da From figure 21 and 22, bound water is observed at lower temperature due to its weak hydrophilic interaction with the PEG backbone while free water is observed at higher temperature due to no hydrogen bonding involved. Non-freezing water is subsequently calculated from the substraction of free water and bound water mass from total water mass. [39, 41] The equilibrium swelling mass is obtained from the SR (%) or EWC (%) from the swelling test when hydrogel is observed to have reached its equilibrium condition. 27

Photo-crosslinked PEGDA-based hydrogels Table 11 Mass of three classes of water in photo-crosslinked hydrogels Mn of Swelling NonPEGDA or Bound mass at Total water Free water Freezing N type of water mass equilibrium mass (mg) mass (mg) water mass macromer (mg) (mg) (mg) (Da) 1

200

9.0500

5.5874

4.1213

0.0000

1.4661

2

4000

14.5000

13.5997

5.6222

0.8554

7.1224

3

8000

12.1200

11.0927

8.5090

0.2169

2.3668

4

10000

10.2500

9.6776

3.6766

0.6578

5.2432

5

35000

8.4000

7.0603

0.2611

1.0272

5.7720

6

TREGDA

6.0330

0.0000

0.0000

0.0000

0.0000

Figure 23 Three classes of water in photo-crosslinked PEGDA-based hydrogels characterised using DSC The proportion of the 3 major classes of water characterised in hydrogels is different for the different PEGDA-based hydrogels of different initial PEG molecular masses. From figure 23, it is observed that hydrogel of higher initial PEG molecular mass has 28

higher proportion of non-freezing water compared to hydrogel of lower initial PEG molecular mass. In contrast, hydrogel of lower initial PEG molecular mass has higher proportion of free water as compared to hydrogel of higher initial PEG molecular mass. The study of the three classes of water is important in indicating the permeability of solutes useful for potential drug delivery purposes.[41]

Michael-type addition crosslinked PEGDA-based Hydrogel Table 12 Mass of 3 classes of water in Michael-type addition crosslinked PEGDA Mn of Swelling NonPEGDA Bound mass at Total water Free water Freezing N or type of water mass equilibrium mass (mg) mass (mg) water mass macromer (mg) (mg) (mg) (Da) 1

4000

13.2700

11.8469

5.7251

0.1039

6.0179

2

8000

7.5500

6.4912

3.6099

0.3512

2.5301

3

10000

13.6600

12.7806

4.1554

0.8099

7.8153

Figure 24 Three types of water in Michael-type addition crosslinked PEGDA-based hydrogels characterised using DSC The proportion of types of water in Michael-type addition crosslinked hydrogels is similar to the proportion in photo-crosslinked hydrogels. The similar trend between 29

the mesh size (ξ) and the molar mass between crosslinks (Mc) have impact on the composition of three classes of water observed in crosslinked hydrogels, which is discussed in the next subsection.

3.3.4 Mesh Size and Crosslink Density Using the values of Mc tabulated below, degree of crosslinking is observed to be detracting from the report.[31, 41-45] Equation (5) is used as a crude calculation of crosslink density.

Mc 

3   R T (5) E

Where ρ is polymer density in g∙cm-3, R is ideal gas constant in J∙ (mol∙K)-1, T is the temperature in K and M is Young’s Modulus in MPa. The values of polymer density is obtained through open literature due to the difficulty in obtaining accurate value.[46] Table 13 Number average molar masses (Mc) between crosslinked points for selected samples Hydrogels based N

on PEGDA or type of macromer with

E (MPa)

ρ (g∙cm-3)

Mc (g∙mol-1)

following Mn (Da) 1

4000

0.0159

1.0296

4.81*105

2

8000

0.1062

1.0320

0.72*105

3

10000

0.0275

1.0323

2.79*105

4

35000

0.0086

1.0329

8.93*105

As the polymer density is measured in a crude manner using the Braun Injekt-F syringe for small mass of hydrogel, the significance of error is high. Despite the high significance of error, it shows good correlation with mesh size determined quantitatively from storage moduli. Nevertheless, the hydrolytic degradation results in the next subsection show that the polymer with the best stability is PEGDA-based hydrogels with Mn = 8000 Da and the ideal Mc for PEGDA-based hydrogel can be inferred to be 0.72*105 g∙mol-1. 30

Alternatively, the mesh size of the hydrogel can be derived from the rubber-elasticity theory using equation (6).[34, 35] 

1

 G' N A  3    (6)  RT  Where G’ is the storage modulus in Pa, NA is the Avogadro Constant in mol-1, R is the molar gas constant in J∙(mol∙K) -1 and T is the temperature in K. Table 14 Average mesh size (ξ) of photo-crosslinked PEGDA-based hydrogels Hydrogels based on PEGDA or type N G’ (Pa) ξ(nm) of macromer with following Mn (Da) 1

4000

1.65*103

13.74

2

8000

18.1*103

6.18

3

10000

14.6*103

6.64

4

35000

2.69*102

25.15

Table 15 Average mesh size (ξ) of Michael-type addition crosslinked PEGDAbased hydrogels Mn of Hydrogels based on PEGDA N G’ (Pa) ξ(nm) or type of macromer (Da) 1

4000

4.67*103

9.71

2

8000

60.5*103

4.14

3

8.43

3

10000

7.14*10

Average ξ values calculated alternatively using rubber-elasticity theory, with frequency sweep rheological data from table 5 and 6, correlates with the physical measurement for Mc in a direct linear relationship. [5, 47, 48] From table 14 and 15, it indicates that crosslinked hydrogels with large ξ have higher composition of nonfreezing water bound to the interface and crosslinked hydrogels with small ξ have higher composition of free water, which is more mobile through hydrogel membrane. [41] EWC (%) data from table 8 and 10 highlights that large ξ generally leads to higher water content in the hydrogel to a certain extent. Hydrophilicity of the PEG backbone contributes to the higher equilibrium water content, which is observed in Michael-type addition crosslinked hydrogel of initial PEG molecular mass 10000 Da 31

with higher water content despite its relatively small mesh size in comparison to that of initial PEG molecular mass 4000 Da. [45]

3.3.5 Hydrolytic Degradation of photo-crosslinked hydrogels Sol-gel fraction Prior to the hydrolytic degradation experiment, photo-crosslinked hydrogels were immersed in DI water to measure the gel fraction in the hydrogel. The hydrogel samples of different initial PEG molecular masses were immersed for 3 days in DI water, which was changed frequently and dried in vacuo in the Binder oven at 37˚C for another 3 days. Gel fraction is determined gravimetrically after measuring the dried weight of hydrogels.[49]

Degradation Test Table 16 % mass loss over 8 weeks (yellow highlighted portion on the table indicate dissolution of polymer in the DI water) Mn of PEGDA or N

type of macromer

Δm (%) Δm (%) Δm (%) Δm (%) Δm (%) Δm (%) Δm (%) Δm (%) Week 1

Week 2

Week 3

Week 4

Week 5

Week 6

Week 7

Week 8

(Da) 1

200

65.40

69.64

69.11

71.78

70.37

70.98

71.64

73.15

1

4000

37.50

42.02

42.34

39.16

52.79

58.90

64.34

67.20

2

8000

17.28

17.39

18.64

18.42

21.33

20.93

20.00

23.72

3

10000

33.99

37.46

45.17

53.66

58.20

62.28

62.15

63.92

4

35000

57.50

59.18

70.15

70.23

78.67

88.65

88.51

87.86

5

TREGDA

0

0

0

0

0

0

0

0

32

Figure 25 % mass loss over 8 weeks in DI water at 37˚C As a test for possible biomedical application in the future, photo-crosslinked PEGDA-based hydrogels is immersed in DI water at 37˚C up to 8 weeks. The degradation result was expected to be a flat line for the hydrogels of different initial PEG molecular masses as the PEGDA-based hydrogels was a stable polymer in water due to the covalent crosslinks. On the contrary, the 1st week plots obtained for the PEGDA-based hydrogels with initial PEG molecular masses 200 Da, 4000 Da, 8000 Da, 10000 Da and 35000 Da correspond to the values obtained for the sol-gel fraction. At the end of 8 weeks, there is mass loss as observed in figure 25 in hydrogels with initial PEG molecular masses 4000 Da, 8000 Da and 35000 Da.

One possible explanation of the loss in mass for certain polymers is due to the presence of remnant NaOH trapped during crosslinking interacting with hydrolytically sensitive ester groups. It may have contributed to the dissolution of PEGDA of initial PEG molecular masses of 4000 Da, 10000 Da and 35000 Da as shown in the highlighted segment of the table 16.[50] Empirical handling errors as noted in the paper by Shah et al. due to dissolution in DI water is a potential source of inconsistency in mass loss for the aforementioned PEGDA-based hydrogels. [36] The above speculation is based on personal inference; future research need to be done to determine the reason behind the unexpected behaviour. Size exclusion chromatography (SEC) can be used to detect small molecular mass of hydrolysed products if any in future degradation tests. 33

4. Conclusion The synthesis of PEGDA-based hydrogels involved a standalone Michael-type addition mechanism without the aid of a photoinitiator, or involved the combined mechanism of Michael-type addition and free radical polymerisation with the aid of a photoinitiator like Irgacure 2959. [21] The different mechanisms involved produce hydrogels with an aesthetic difference in term of material opacity. The mechanical characterisation of photo-crosslinked PEGDA-based hydrogels highlights one possible material application as a component of artificial heart valve which requires highly elastic behaviour.[51] As expected, higher wt% concentration of PEGDA macromers in the preparation of PEGDA-based hydrogels lead to higher storage moduli of the hydrogel. On the other hand, the mesh size of the hydrogel affects composition of the three classes of water in hydrogels. Free water composition is high in hydrogel with relatively small mesh size, but non-freezing water is high in hydrogel with longer PEG backbone. [29]

5. Recommendation There can also be future exploration of work into cytotoxicity, biocompatibility and biodegradability of the photo-crosslinking and Michael-type addition crosslinking reaction.[1] There is room for cytocompatibility studies for the viability of cell encapsulation since there is interest in PEGDA-based hydrogels in the biomaterials field.[26, 51-54] As regards its possible application as a component of the heart valve, flexural strength test can be performed to determine its ability to withstand bending stress that is commonly experienced in physiological condition.[51] As pH varies within the human body, potential usage as a delivery material need to be confirmed with experiment based on the pH effect on equilibrium swelling behaviour. Swelling tests can be conducted at pH 2-8 to check the impact of pH on the EWC (%) to simulate the gastrointestinal environment.[14, 55] Swelling kinetics to determine the diffusion mechanism of water through the hydrogels’ membrane can also be examined in addition to the swelling test in order to design applications in pharmacological and biomedical industry.[45]

34

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Appendix 1

Figure 26 DSC curve of Photo-crosslinked hydrogel of containing 20 wt% PEGDA of Mn = 200 Da

Figure 27 DSC curve of Photo-crosslinked hydrogel of containing 20 wt% PEGDA of Mn = 4000 Da

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Figure 28 DSC curve of Photo-crosslinked hydrogel of containing 20 wt% PEGDA of Mn =10000 Da

Figure 29 DSC curve of Photo-crosslinked hydrogel of containing 20 wt% PEGDA of Mn =35000 Da

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Figure 30 DSC curve of Photo-crosslinked reference material of containing 20 wt% TREGDA

Figure 31 DSC curve of Michael-type addition crosslinked hydrogel of containing 20 wt% PEGDA of Mn =10000 Da

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