CHAPTER 5 SURFACE TOPOGRAPHY STUDIES ON ELECTROPHORETICALLY DEPOSITED CHITOSAN ON POLYCAPROLACTONE MICRO FIBROUS SUBSTRATES

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1 89 CHAPTER 5 SURFACE TOPOGRAPHY STUDIES ON ELECTROPHORETICALLY DEPOSITED CHITOSAN ON POLYCAPROLACTONE MICRO FIBROUS SUBSTRATES 5.1 INTRODUCTION Synthetic bio polymers such as PCL, poly (glycolic acid) (PGA), poly(l-lactic acid) (PLLA), and poly(lactic-co-glycolic acid) (PLGA) offer easier processability for producing fibrous web using various fibre forming techniques. The developed fibrous web morphology is also controllable one as compared to natural bio polymers such as chitosan, collagen and alginate. Natural biopolymers are still often considered to be difficult to process directly into micro fibrous web due to their unstable nature in processing conditions such as highly toxic solvents (hexafluoroisopropanol, trifluoroacetic acid) and resulting fibrous web also having a poor mechanical, biocompatible properties (Cai et al 2010). Even though synthetic bio polymers are more biocompatible, theydo not possess good hydrophilicity and cannot secure bioactive materials. Surface modification of biomaterials is performed to enhance characteristics features such as hydrophilicity, cell attachment, cell proliferation and protein adsorption. Modulation of biomaterials topography by changing roughness and surface features enhances cell growth (Tuzlakoglu et al 2010). In order to apply micro fibrous web and textiles materials in biomedical uses, their surfaces are to be modified by chemically and

2 90 physically using different techniques such as plasma treatment, wet chemical method, surface graft polymerization and coating of bioactive materials (Yoo et al 2009). Polymeric fibrous substrates when treated with plasma in the presence of oxygen, argon, ammonia or air, the surface adhesion and hydrophillic properties get better by the alteration of the surface chemical composition. In a recent work, electro spun PCL nanofibres were modified with argon plasma, which enhance carboxylic acid groups on the nanofibrous substrate (Yoo et al 2009). Electrospun PCL fibrous web when treated with 5M NaOH, dramatically enhanced the hydrophilic property due to the highly rough surface. The modified PCL fibrous web showed better cell morphology and adhesion due to the unique hydrophilic surface topography. Surface graft polymerization was employed to enhance hydrophilicity and also used to build functional groups on the surface for strongly immobilization of bioactive materials to enhanc cell adhesion, proliferation, and differentiation (Yoo et al 2009). Simple coating on biomaterials with other biopolymers is also one of the attractive and easy process to enhance functional properties of the materials. Coatings were achieved by simple dip coating, spin coating or by electro induced deposition of ph responsive polymers on biomaterial surfaces. EPD is one of the attractive biomaterial fabrication techniques used to construct micro and nano scaled architectures (Pang et al 2009; Pishbin et al 2010; Boccaccini et al 2010). This technique is based on migration of charged particles under the influence of electric charges resulting in ordered deposition of metals, polymers, ceramics, glasses and their composites over the substrates (Guo et al 2012; Simchi et al 2009; Boccaccini et al 2010). EPD of polymers on different substrates are carried out for various applications such as biomems, biosensors, enzyme immobilizations and micro fluidic

3 91 devices (Koev et al 2010; Sheng et al 2011; Luo 2005; Luo et al 2010). The deposition of polymers by EPD is also carried out for improved cell adhesion (Meng et al 2009), anti-microbial activity and drug delivery properties (Boccaccini et al 2010). At present ph responsive polymers such as chitosan, alginates are coated on metal implants by EPD for bone tissue engineering. Further research work are being carried out by various researchers in the field of EPD. Chitosan is the second most abundant natural biopolymer, obtained by partial deacetylation of chitin which is predominantly present in the exoskeleton of shellfish and crustaceans (Jayakumar et al 2010; Wang et al 2005). Chitosan coated as thin films possess diverse properties making them suitable for extensive applications such as water purification, filtration, surgical dressings, drug encapsulation and food processing (Yoon et al 2006; Knill et al 2004; Ferrari et al 2013; Tripathi et al 2010). Another interesting property of chitosan is its ph sensitivity and is attributed to presence of abundant primary amine groups at C2 position of glucosamine residue (Tripathi et al 2010). At low ph, chitosan is readily soluble in water due to the protonation of primary amine groups. Once the ph is elevated above its ph (6.6), deprotonation of the amine group takes place and it leads to the solgel transition. This property can be advantageously used for the deposition of chitosan on substrates using electrophoresis principle. In EPD of acidic chitosan, when an electrical potential is applied between the anode and cathode, hydroxyl ions generated at the cathode deprotonate the chitosan and it gets deposited as a gel over the cathode. Further increase in duration of applied voltage leads to increased deposition of the hydrogel film due to electrophoretic motion of charged chitosan macromolecules (Pishbin et al 2010; Shi et al 2008; Cheng et al 2012; Fernandes et al 2003).The deposition of chitosan macromolecules by EPD principles has been attempted on substrate such as titanium, gold carbon and stainless steel (Simchi et al 2009;

4 92 Pang & Zhitomirsky 2005; Zhou et al 2007; Tan et al 2010). However to our knowledge, no attempt has been made to deposit chitosan on ultrafine fibrous material by EPD principle. It has been reported in recent work that the coating of chitosan on PCL fibres by drop casting method demonstrated better functional properties compared to that of blending for wound dressing applications. It has been further reported that drop casted chitosan on PCL fibres exhibited good platelet aggregation, anti-bacterial, anti-adhesive, and anti-inflammatory activities (Bai et al 2013). The advantage of this architecture is that it can offer maximum exposure of chitosan to the wound bed. In the present work it is proposed to deposit chitosan on ultrafine fibrous web by electrophoretic principle to obtain uniform coating of polymer. The ultrafine fibrous web is produced by centrifugal spinning. Centrifugal spinning is a facile and emerging technology (Shanmuganathan et al 2012; Padron et al 2013) for the production of ultrafine fibrous web and the detailed experimental set-up of centrifugal spinning is reported elsewhere (2.11.3) in the previous chapter. The polymeric solutions are converted into fibres by applying centrifugal force resulting in highly aligned fibrous web. The aligned ultrafine fibres are produced without application of high voltage in short duration which is very difficult to achieve in electrospinning process. The highly aligned PCL fibrous web produced by centrifugal spinning setup was placed in cathode and the parameters like molecular weight, concentration of polymer, voltage, duration of voltage application and ph were varied to study the weight add on % of coated fibrous matrices. In the previous attempt mentioned in Chapter 4; quicker sol-gel transition within short duration was observed and in the present study utmost care was taken to overcome the above limitation by carefully selecting the electrode and solvent for the chitosan. The developed web by the modified process parameters were analyzed for its morphology using scanning electron microscope and the results obtained are discussed in the following sections.

5 EXPERIMENTAL Materials Polycaprolactone (PCL) (M W -70,000-90,000), Chitosan with low molecular weight ( cp), (product number ), medium molecular weight ( cp) (product number ) and high molecular weight ( cp) (product number ) were purchased from Sigma Aldrich (USA). Acetic acid, Chloroform and sodium hydroxide (AR Grade) were procured from SISCO research Laboratories Pvt Ltd (India). Universal ph indicator solution was purchased from Rankem Pvt. Ltd., (India). Regulated DC power supply unit (0-30Volt, 0-2 amps capacity with accuracy of ±0.05%) was supplied by Kpos Pvt. Ltd., (India). All chemicals were used as received Development of Centrifugal Spun Fibrous Substrates PCL solution (15%) was prepared by dissolving PCL in chloroform and the solution was stirred for an hour without forming any air bubbles. PCL solution was filled into the pot type spinneret head using a syringe. The rotation speed of the pot type spinneret was fixed at 5000 rpm based on various preliminary trials. The produced micro fibrous web was collected in the round bottom collector. The collected aligned micro fibrous sheet was kept in vacuum to remove excess solvent present on the fibres and used for further study Electro Deposition of Chitosan on Centrifugal Spun Substrates Chitosan with different molecular weight solutions were prepared by dissolving chitosan flakes using acetic acid as a solvent for desired concentrations (0.25, 0.5, 0.75 w/v). The required ph (3.5, 4.5, 5.5) was adjusted using 1 M NaOH drop wise into the chitosan solution. Graphite

6 94 sheets were used as the electrodes (both anode and cathode). Centrifugal spun micro fibrous web was cut into sample size 20 20mm having an average thickness of 0.11±3 mm. The centrifugal spun web was placed on the cathodic surface with the help of an adhesive tape. The chitosan solution (50ml) was filled in the electro deposition cell with the dimensions of cm (length, width, height) and distance between the electrode was kept as 5cm. Desirable voltage with respect to time was supplied by regulated DC power supply unit. After electro deposition the micro fibrous web was dried at room temperature for 48 hours Characterization of Chitosan Coated PCL Substrates The weight add on % was calculated by precise LC-GC (Model AS 220/X) weighing balance and it was calculated using the below equation 5.1 (5.1) The surface morphology of the web was observed by using scanning electron microscope (SEM) after sputtering the samples with gold at an acceleration voltage of 10kV. 5.3 RESULTS AND DISCUSSION The major parameters that affect the EPD of chitosan on the matrices are material and process parameters. The material parameters namely molecular weight of chitosan, concentration of chitosan and the process parameters namely voltage, deposition time and ph of the cell were also altered to study their influence on weight add on % and topography of

7 95 deposition. The schematic diagram of EPD of chitosan on centrifugal spun fibrous matrices shown in Figure 5.1. Figure 5.1 Schematic diagram of EPD of chitosan on centrifugal spun PCL fibrous substrates Influence of Chitosan Molecular Weight and Concentration The chitosan concentration used in the electro deposition cell was altered from 0.25w/v to 0.75w/v for three different molecular weights of chitosan with voltage of 5Volt, ph of 5.5 and time duration of 5 minutes. The data for weight add on % as a function of chitosan concentration and molecular weight is given in Figure 5.2.

8 96 Figure 5.2 Effect of molecular weight and concentration of chitosan on weight add on (%) From the figure, it can be seen that for all molecular weight taken for study, the deposition of chitosan macro molecules on matrices increased with increase in concentration of chitosan. However, the handle ability of the deposited mat was good when 0.25w/v and 0.5w/v chitosan concentration was used. Moreover with increase in molecular weight, add on % was found to be higher for high molecular weight chitosan and topography of deposition was significantly different for all molecular weights. The topography of deposition was analyzed using SEM and they show different surface profiles for the various samples (Figure 5.3).

9 97 Figure 5.3 (a) low molecular weight chitosan deposited PCL fibrous surface (b) cross section of low molecular weight chitosan deposited matrices (c) medium molecular weight chitosan deposited matrices (d) cross section of medium molecular weight chitosan deposited matrices showing better infiltration (e) high molecular weight chitosan deposited matrices (f) cross section of high molecular weight chitosan deposited matrices showing surface deposition Better infiltration and coating of chitosan macromolecules on ultra - fine fibrous web was observed in low (Figure 5.3 a, 5.3 b) and medium

10 98 molecular weight (Figure 5.3 c, 5.3 d) chitosan compared to that of high molecular weight chitosan. In the case of high molecular weight chitosan, Figure 5.3 (e) shows the surface topography of the web and it was coated with chitosan uniformly without any space whereas Figure 5.3 (f) shows the cross section view of the matrices in which, chitosan can be observed on the surface alone and confirms the poor infiltration Influence of Voltage and Time In order to study the net effect of chitosan deposition on the matrices with respect to the voltage on the ultra-fine fibrous web; the chitosan concentration was fixed at 0.5w/v for medium molecular chitosan. The time of deposition was fixed at 5 min and the ph was maintained at 5.5. The weight add on % of chitosan shows significant variation with respect to varying applied voltage as shown in Figure 5.4. Figure 5.4 Effect of voltage, time and ph on weight add on (%)

11 99 The weight add on % increased up to 10 V and further increase of voltage to 15V led to decrease in weight add on %. These results can be better explained by the evolution of gases at cathode. The gas evolution is controlled at lower voltage but at elevated voltage of 15V, gas evolution was found to be higher. This reflected in the weight add on % leading to decreased deposition at higher voltage. The SEM micrograph confirms (Figure 5.5 a, 5.5 b) the above statement and it can be seen that the surface becomes rougher and a lot of pores in the matrices can be noticed. Moreover with increase in voltage, chitosan macromolecules quickly attain the sol -gel state and are not able to infiltrate into the ultra-fine fibrous web resulting in layered coating on the surface of fibrous web as shown in Figure 5.6. Figure 5.5 EPD of chitosan on PCL matrices (a) 10V at 5 min (b) 15V at 5 min (c) 10 minutes at 5V (d) 15 minutes at 5V

12 100 To evaluate the influence of duration of applied voltage on weight add on %, the voltage was fixed at 5V and the other parameters (ph 5.5, duration of time 5 minute, medium molecular weight chitosan(0.5 w/v)) were kept as a constant. It was observed that the weight add on % significantly varied with respect to the duration of voltage application (Figure 5.4). The weight add on % increases up to 10 minutes of voltage application and on further increase in duration of applied voltage the add on% decreased. The decrease in weight add % is attributed to the gas evolution which results in creation of pores followed by disintegration of chitosan deposited on the matrices (Figure 5.5 c, 5.5 d). Figure 5.6 Schematic diagrams showing the effect of voltage on infiltration of chitosan in PCL matrices

13 Influence of ph The effect of ph of chitosan solution in EPD was altered from 3.5 to 5.5 to study its effect on weight add on % (Other parameters such as concentration of medium molecular weight chitosan (0.5 w/v), 5V and 5 minutes duration was kept as a constant) and the results are given in Figure 5.4. As reported in literature higher deposition of chitosan macromolecules on the matrices were observed at ph 5.5 (Zhitomirsky I & Hashambhoy 2007; Altomare et al 2012). At lower ph, decreased deposition is observed due to decrease in ionization and lower net charge density as cited in literature. The SEM micrograph shows the deposition of chitosan studied at various ph (Figure 5.7). It can be seen that from the figure that uniform deposition is obtained at ph 5.5 with pores (Figure 5.7 c). The porous morphology obtained in the substrates will aid in transport of moisture and air across the web, these features makes the substrate as a suitable candidate for wound dressing application. Figure 5.7 EPD of chitosan on PCL matrices at varying ph (a) 3.5 (b) 4.5 (c) 5.5

14 CONCLUSIONS The present study demonstrated that the material parameters and process parameters have significant effect on the amount of deposition and topography of chitosan deposition on the matrices. The results indicate that ph of acidic chitosan solution play a vital role in weight add on % compared to other parameters such as molecular weight, concentration of chitosan, voltage and duration of voltage application. But the above mentioned parameters have significant effect on the topography of the coating on the PCL fibrous matrices. Better infiltration of chitosan in the matrices was observed when low and medium molecular weight chitosan was used for EPD. At higher voltage and longer duration of applied voltage, higher amount of gases were evolved from the electrodes leading to poor adhesion of the chitosan on the PCL matrices. Based on the studies conducted better deposition of chitosan on PCL matrices was observed for medium molecular weight chitosan of 0.5 w/v concentration when the EPD cell was maintained at ph of 5.5, 5 Volt for a duration of 10 minutes. The observed results provide ample scope for tailoring the deposition of chitosan with different topography for various biomedical applications such as wound dressing and tissue engineering.