Synthesis and Evaluation of pH- and Temperature-Responsive Chitosan-p(MAA-co-NIPAM) Hydrogels PDF

International Journal of Biological Macromolecules 108 (2018) 367–375 Contents lists available at ScienceDirect International Journal of Biological Macromolecules...

International Journal of Biological Macromolecules 108 (2018) 367–375 Contents lists available at ScienceDirect International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac Synthesis and evaluation on pH- and temperature-responsive chitosan-p(MAA-co-NIPAM) hydrogels S.Z.M. Rasib a , Z. Ahmad a , A. Khan c , H.M. Akil a,∗ , M.B.H. Othman b , Z.A.A. Hamid a , F. Ullah a a School of Materials and Mineral Resources Engineering, Universiti Sains Malaysia, Engineering Campus, 14300 Nibong Tebal, Penang, Malaysia b School of Chemical Sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia c Department of Chemistry, Abdul Wali Khan University Mardan, 23200 Pakistan a r t i c l e i n f o a b s t r a c t Article history: In this study, chitosan-poly(methacrylic acid-co-N-isopropylacrylamide) [chitosan-p(MAA-co-NIPAM)] Received 10 August 2017 hydrogels were synthesized by emulsion polymerization. In order to be used as a carrier for drug delivery Received in revised form systems, the hydrogels had to be biocompatible, biodegradable and multi-responsive. The polymerization 20 November 2017 was performed by copolymerize MAA and NIPAM with chitosan polymer to produce a chitosan-based Accepted 4 December 2017 hydrogel. Due to instability during synthesis and complexity of components to produce the hydrogel, Available online 6 December 2017 further study at different times of reaction is important to observe the synthesis process, the effect of end product on swelling behaviour and the most important is to find the best way to control the hydrogel Keywords: Chitosan synthesis in order to have an optimal swelling behaviour for drug release application. Studied by using pH-responsive Fourier transform infra-red (FTIR) spectroscopy found that, the synthesized was successfully produced Temperature-responsive stable chitosan-based hydrogel with PNIPAM continuously covered the outer surface of hydrogel which influenced much on the stability during synthesis. The chitosan and PMAA increased the zeta potential of the hydrogel and the chitosan capable to control shrinkage above human body temperature. The chitosan-p(MAA-co-NIPAM) hydrogels also responses to pH and temperature thus improved the ability to performance as a drug carrier. © 2017 Elsevier B.V. All rights reserved. 1. Introduction toxicity during the testing, and this improved the efficiency of the drug loaded. The released profile of PNIPAM with chi- Hydrogels are unique materials that have the capability to tosan also showed that the sol-gel transition temperature was absorb water to respond to either a physical, chemical or biochemi- dragged to below human body temperature. Furthermore, chi- cal environment [1–4]. A significant contribution is made by the use tosan cross-linked with PMAA hydrogels is able to respond to ionic of hydrogels as a drug carrier, especially since they can be used for strength, which changes drastically due to the osmotic pressure of an extended period of time in the body system without any health the ambient solution [10–13]. The recent synthesis of chitosan in risks. Chitosan, as a natural polymer, is selected for the hydrogels the copolymerization of PMAA and PNIPAM has improved the func- synthesis in order to achieve the biodegradable and biocompat- tions of the p(NIPAM-co-MAA) hydrogels, which are dependent on ible characteristics which enable the carrier to survive longer in the swelling behaviour of the monomers and also the widespread the body system [6–8]. Chitosan has been modified by crosslinking application in the biological pH body. This synthesis technique with other moieties/monomers for pH and temperature sensitivity succeeded in releasing drugs with a higher release in an acidic envi- to improve the performance of the chitosan as a drug carrier. ronment. However, this system has been developed by coating the Studies on chitosan cross-linked with PNIPAM and PMAA sepa- chitosan with p(NIPAM-co-MAA), while in this paper the system rately, have attracted more intention since chitosan cross-linked was synthesized by random co-polymerization to ensure that the with PNIPAM was found to have a low level of toxicity or no chitosan, PMAA, and PNIPAM were located in the whole system for establishing fast-acting hydrogels. From our previous work, Chitosan-p(MAA-co-NIPAM) hydro- ∗ Corresponding author. gels were synthesized by using free-emulsion polymerization. The E-mail address: [email protected] (H.M. Akil). synthesized has succeeded however the particles sizes were not https://doi.org/10.1016/j.ijbiomac.2017.12.021 0141-8130/© 2017 Elsevier B.V. All rights reserved. 368 S.Z.M. Rasib et al. / International Journal of Biological Macromolecules 108 (2018) 367–375 homogeneous and aggregate at the end of the synthesis. As Table 1 The main composition of chitosan-p(MAA-co-NIPAM) hydrogel. a solution for the inhomogeneity on particles, the synthesis was improved by applying an emulsifier which is SPAN 80 to control Chemical Composition (g) the size. However, after four hours reaction, the aggregation still Chitosan 0.5 produced and again affect the homogeneity of the particles. Some NIPAM 1.0 studies found that the insolubility of the PNIPAM network during MAA 0.5 polymerization causes the instability on micelle due to the growing MBA 0.06 polymer chains and the phase separation of the hydrogel particles. Fernandez-Nieves et al. suggested that only the monodis- persed particles at a low monomer conversion could be considered and NIPAM were added under constant stirring for approximately for further application. For implementation, we were stopped the 20 min in a three-neck round-bottom flask equipped with a N2 gas synthesis for a certain period before the instability of micelle is inlet and condenser. The amount of raw materials was shown in achieved. Since the hydrogels contained polymer and monomers Table 1. After 30 min of stirring under N2 purging at 30 ◦ C, MBA (chitosan, MAA, and NIPAM), therefore the hydrogels produced (which was used as the cross-linker), with a span of 80, was added could not be easily estimated since the reactivity of each monomer as an emulsifier to the reaction flask. The clear solution changed to was different and the polymerization cannot be stopped randomly. become a milky solution. Our hypothesis was that the hydrogels before the aggregation After 1 h, the temperature of the reaction mixture was slowly also have unwanted characteristic due to the primary aggregation raised to 70 ◦ C under N2 purging, along with continuous stirring which lead to lower swelling and lower stability of hydrogels. Fur- and gradual heating in a silicon-filled oil bath. After 30 min of con- ther understanding hydrogel needs to be considered at different stant heating at 70 ◦ C, 5 mL of APS (0.05 M) were added to the time of reaction so that the effect on pH and temperature respon- reaction mixture in order to start the polymerization reaction. The sive on hydrogels can be understood very well. The investigation polymerization reaction was allowed to proceed and stop after on hydrogels for each time of reaction also necessary as a ways to 30 min (RT30) at constant stirring under N2 purging at 70 ◦ C. The find a better solution to avoid the aggregation during synthesis. copolymer hydrogels that were obtained were then purified by Therefore, it is important to observe the structural changes centrifugation (Sorvall RC-6 Plus superspeed centrifuge, Thermo beyond the reaction time and the effect of the network formed Electron Co., Waltham, MA, USA) and decantation, and were then toward pH and temperature responsive behaviour of the hydrogels. washed with water. Next, each resultant hydrogel was further puri- Herein, a detailed study on reaction via emulsion polymerization on fied by dialysis through a Spectra/Por molecular porous membrane the structure, swelling behaviour, and zeta potential/electrokinetic tubing using distilled water with a pH of 5 ± 0.3 at room tempera- of the chitosan-p(MAA-co-NIPAM) hydrogels are presented. In ture (25 ◦ C) that was frequently changed for 1 week. The purified order to understand the structural changes, and the effects of hydrogels were then freeze–dried for 48 h with a LABCONCO freeze the reaction, pH and temperature variations on the swelling/de- dry system after freezing overnight at −40 ◦ C. Different hydrogel swelling behaviour, the synthesized hydrogel samples were with similar compositions were prepared by varying time to stop characterized using FTIR, FESEM, zeta potential and dynamic light the polymerization reaction to 60 (RT60), 120 (RT120), and 180 min scattering techniques. (RT180). Time of polymerization is limited to 180 min before the phase start to be separated as prolong the time. Propose synthesis 2. Experimental was shown in Fig. 1. 2.1. Materials 2.3. Hydrogel characterization N-Isopropylacrylamide (NIPAM), N,N-Methylenebisacrylamide The morphological examination of the chitosan-p(MAA-co- (MBA) (purity >95.0%) and chitosan (Mw ≈ 700–1000 kDa, degree of NIPAM) hydrogels was carried out using field emission scanning deacetylation ≈ 90.0%) were purchased from Zhejiang Golden-Shell electron microscopy (FESEM) (LEO SUPRA 35VP, Carl Zeiss, Pharmaceutical Co., Ltd (Zhejiang, China), SpanTM 80 (Croda Inter- Germany). The freeze-dried hydrogels were stubbed lightly on national Plc., UK), and acetic acid (≥99.85%) from Sigma–Aldrich. double-sided carbon tapes, and were then sputter-coated with gold NIPAM was purified by recrystallization from a toluene/n-hexane to cover the hydrogels with a thin layer of conducting material. (1:3) mixture. Methacrylic acid (MAA) (purity >95.0%) was further The freeze-dried hydrogels were used as the FTIR sample prepa- purified by distillation under reduced pressure, while all the other ration to identify the presence of various functional groups in the chemicals were used as received and without further purification. hydrogels. All the FTIR spectra of the hydrogels were obtained Deionised distilled water (DDH2 O) was used for all the reactions within the range of 4000–800 cm−1 using a Perkins-Elmer Spec- and solution preparations, and the hydrogels were purified by ® trum One apparatus. The 5 mg of freeze-dried hydrogels were press using the membrane filter, Spectra/Por molecularporous mem- 80% on the spectrometer and were scan by 4 scans at a resolu- brane tubing, which was obtained from Spectrum Laboratories, Inc., tion of 2 cm−1. The peaks at 1648 cm−1 , 1716 cm−1 , 1155 cm−1 and Rancho Dominguez, CA, USA; cut-off 12,000–14,000. 2930 cm−1 were chosen, which corresponded to the peaks of the PMAA, PNIPAM, chitosan and C H bond stretching, respectively. 2.2. Preparation of chitosan-p(MAA-co-NIPAM) hydrogels A baseline was constructed by connecting the lowest data points on each side of the peak. The area between the baseline and the All the hydrogels were synthesized by the free-radical copoly- top of the peak represented the peak area. The ratios of the peak merization of chitosan, MAA, NIPAM and MBA using ammonium area values for the PMAA, PNIPAM and chitosan peaks against the persulfate (APS) as an initiator. The whole procedure was almost methyl peaks as the reference peaks indicated the relative changes the same as with the previous paper, with some modifications on in the composition of the structural components in relation to each the dissolution of the chitosan and emulsifier. Briefly, 1% of other. FTIR results were supported by the solid state 13 C NMR mea- glacial acetic acid was prepared in 100 mL of deionized water. The surement, carried out by using a Bruker Avance 400 MHz at the same amount of chitosan was added slowly into the solution until resonance frequency of 100.62 MHz, 47.546 W with spin rate was the whole solution was dissolved to form a clear solution. Then MAA 8 KHz.  S.Z.M. Rasib et al. / International Journal of Biological Macromolecules 108 (2018) 367–375 369 Fig. 1. Proposed mechanism for synthesis of chitosan-p(MAA-co-NIPAM) hydrogels. The stability of the hydrogels was obtained based on the zeta the free radical was activated. By prolonging the reaction to 60 min, potential values. The zeta potential measurements were performed the homogeneous hydrogels were formed and no prominent effect using a NANO ZS Zetasizer (Malvern Instruments, Malvern, UK) on shape beyond the time. At this stage, the SPAN 80 acted as a with a wavelength of 633 nm and a detector angle of 173◦. The controller to stabilize the emulsion by the formation of monolayer freeze-dried hydrogels were diluted into different buffer solutions film at the oil/water (o/w) interface. The highly hydrophobic sur- with pH 1.68, 4.01, 7.4 and 10.01 to be used to verify the effect of factant tail from the SPAN 80 interacted with increased physical the zeta potential on each hydrogel. The temperature of the sample entanglement and cross-linking chain length of polymer, while the was also varied from 25 to 55 ◦ C to study the effect of the tem- hydrophilic head was effective for separating the layer with the perature on the stability of the hydrogels. Before that, the diluted aqueous solvent. The polymer grew and reached a limitation after hydrogels were passed through PTFE/L filters (with a pore size of one hour reaction. The progress in the formation of distinct spher- 0.80 ␮m). The average of three readings was taken for each pH and ical beads are similarly observed in the work of Liu et al. and temperature applied to the hydrogels. The hydrogels were equi- Elaissari, A.. librated at each chosen temperature/pH for approximately 120 s before starting the measurement. The swelling of the hydrogels was examined in terms of the par- 3.2. Fourier transform infra-red (FTIR) ticle size. Similar to the preparation of the zeta potential sample, the freeze-dried hydrogels were diluted into different pH solutions and The FTIR spectra of the chitosan and chitosan-p(MAA-co- were passed through PTFE/L filters (with a pore size of 0.80 ␮m). NIPAM) at different times are shown in Fig. 3. Based on the chitosan The temperature was also varied to study the effect of temperature spectrum, the broad stretching peak at 3000–3600 cm−1 could be on the swelling behaviour of the hydrogels. Each sample was equi- attributed to the hydrogen bonds (O H), overlapping with the librated approximately 120 s before starting the measurement, and stretching vibration of the N H (primary amines) bonds of chi- each measurement was repeated three or more times. tosan. The medium peaks at 1655 and 1598 cm−1 were referred to the C O of the acetyl groups , while the peaks at 1421 and 1384 cm−1 indicated the alkane C H bending. Furthermore, 3. Results and discussion the C O C symmetric and anti-symmetric group of chitosan could be in the range of 1000–1155 cm−1. 3.1. Scanning electron microscopy The significant functional groups involved in the synthesis of the chitosan-p (MAA-co-NIPAM) hydrogels were peaks at 1689 cm−1 Fig. 2 shows the SEM of hydrogel beyond the reaction. After which contributed to the carboxylic carbonyl group of MAA 30 min of reaction, the hydrogels were formed inhomogeneous in and at the double bond ( C C ) within the MAA and NIPAM shape. The rapid reaction occurred in the monomer droplets once monomers at the peaks of 1638 and 1619 cm−1 , respectively. The 370 S.Z.M. Rasib et al. / International Journal of Biological Macromolecules 108 (2018) 367–375 Fig. 2. FESEM microscopy of freeze-dried chitosan-p(MAA-co-NIPAM) hydrogel; magnification 10 kX; (a) RT30, (b) RT60, (c) RT120, and (d) RT180. Fig. 3. FTIR spectra of chitosan and different reaction time of chitosan-p(MAA-co-NIPAM) hydrogel. chitosan-p(MAA-co-NIPAM) hydrogels spectrum at the region of the PMAA and PNIPAM were successfully crosslinked by copoly- 1700–1740 cm−1 referred to C O of MAA which involved in electro- merization since the double bond peaks of NIPAM and MAA were static interaction with ammonium cation of chitosan. These peaks not observed throughout the reaction time as confirmed by the were progressively shifted to lower wavenumber as the reaction absence in peaks of 1638 and 1619 cm−1. While the crosslinker time being extended as shown in Fig. 3 from 1740 to 1716 cm−1. MBA form a hydrogel network within both of the monomer and chi- This indicated that the interaction increased with the time. While, tosan crosslinked within the network. Since most of the peaks were  S.Z.M. Rasib et al. / International Journal of Biological Macromolecules 108 (2018) 367–375 371 13 Fig. 4. C NMR spectrum of the chitosan-p(MAA-co-NIPAM) hydrogel highlight an absence alkene group of MAA and NIPAM monomers. almost similar beyond the time, this shows that the formation of PMAA can be clearly observed at 21.39 (e), 28.78 (a), 41.0 (b), 44.3 chitosan-p(MAA-co-NIPAM) hydrogels was successfully produced (d), 21.3 (c), 175.3 (c), and 180.0 (f) ppm respectively. From and the structure was close to the proposed structure shown in the signals showed that the two co-monomers were successfully Fig. 1. incorporated into the network since there are no signal at region The extended formation of the crosslinked network was moni- 120–140 ppm which referred to the signal of double bond carbon tored at 30, 60, 120, and 180 min by using FTIR to investigate the atom from both of the monomers. Thus the 13 C NMR also strongly conversion of monomers and chitosan during the reaction. Three supported the FTIR result with the presence of PMAA, PNIPAM and peaks were recorded by the peak at 1716 cm−1 , 1648 cm−1 and chitosan signals 1155 cm−1 which were corresponded to C O of PMAA, C O of PNI- PAM, and C O C of chitosan, respectively. The monitoring was made by comparing the normalized integrated peaks area for each 3.4. Response of chitosan-p(MAA-co-NIPAM) hydrogels to pH peak as shown in Fig. 5 which represented the contents of PMAA, PNIPAM, and chitosan at a different time of reaction. The chitosan In order to have better understanding on the effect of showed higher concentration at the initial reaction. High molec- chitosan-p(MAA-co-NIPAM) hydrogels networks formed at differ- ular weight of chitosan rapidly crosslinked into the network and ent reaction time in response to pH, a number of experiments were the concentration was controlled by micelle after one hour reac- carried out at a fixed temperature corresponding to the human tion. For both of the monomers, there was initial abroad increased body temperature (37 ◦ C). The swelling/de-swelling behaviour was from 30 to 60 min and slowly achieving constant change in rate studied in terms of the hydrodynamic size using the dynamic light beyond 180 min. The addition reaction catalyzed by radical addition scattering (DLS) technique. A series of buffer solutions, with the pH became effective after 30 min. Saturation occurred beyond 60 min ranging from 1.68 to 10, were used to study the swelling behaviour for both monomers as most monomers have been converted into under different pH environments. It should be noted here that the the crosslinked network and diffused in the micelles system. The value of the hydrodynamic diameter was referred to as the max- PMAA reached a constant normalized integrated peaks area faster imum value of each peak. The effect on the swelling behaviour at than PNIPAM due to the less concentration of MAA feed at the ini- different pH levels is shown in Fig. 6. Generally, by prolonging the tial synthesis compared to NIPAM monomer and the propagation reaction time, all the hydrogels showed almost the same pattern of of PMAA was faster rather than PNIPAM [23,24]. Since PNIPAM con- swelling, except for the hydrogels after 30 min of reaction. The same tinuously increased in the area, PNIPAM chain kept growing in the network present in the hydrogels behaved consistently although aqueous solution and thus created a potential to induce precipi- the crosslinking within the time was increased. tation of chain aggregation. Based on the behaviour, it could The hydrogels prepared at various times showed difference be estimated that after prolonging the polymerization, the surface in hydrodynamic diameter, which indicated that the time during layer of particles was covered mostly by PNIPAM, while the inner polymerization influenced the degree of swelling. For a short reac- part was randomly cross-linked by PMAA, PNIPAM, and chitosan. tion time, the swelling was less mostly due to the unfavourable amount of soluble polymer in the structure, which enabled the hydrogels to swell more. The higher content of chitosan influenced 3.3. 13 C nuclear magnetic resonance (13C NMR) analysis the hydrogels to swell more in an acidic environment, while less content of PMAA in the network restricted the hydrogel to swell The chemical structure of the synthesized chitosan-p(MAA-co- at high basic pH. Meanwhile, prolonging the polymerization time NIPAM) hydrogel was verified using 13C NMR as shown in Fig. 4. made the hydrogel swelled more and the swelling behaviour was The resonance signals characterized for chitosan were 55.30 (2), restricted even more. As shown in the normalized integrated peak 61.8 (6), 70.3 (5), 74.2 (3), 81.6 (4), and 98.3 (1) ppm respectively, area in Fig. 5, by prolonging the reaction time, the contents of which is close to the chemical shift from some studies related to PMAA and PNIPAM increased. This also increased the crosslinking chitosan [25–27]. The characteristic resonances of the PNIPAM and points among the polymer chains and hence, led to an increase 372 S.Z.M. Rasib et al. / International Journal of Biological Macromolecules 108 (2018) 367–375 Fig. 5. Normalized integrated peak area referring to PMAA PNIPAM and chitosan peaks at different time with estimating end polymer product with surface layer covered with PNIPAM. intramolecular of hydrogen bonds presented in the chitosan chain and network obstructed the swelling. Since the temperature during experiment was 37 ◦ C lead to weakening of intermolecular hydrogen bonds, therefore there are high possibility is due to the degree of crosslinking. Generally, the chitosan-p(MAA-co-NIPAM) match with p(NIPAM-co-MAA) hydrogels swelling behaviour from most other researchers [11,34–37] especially in a basic environment. However, the presence of chitosan in the hydrogels improved the swelling in an acidic environment. From the graph, swelling was observed in an acidic pH and reached maximum swelling around pH 7. As the pH was increased further to basic condition, de-swelling was noticed. Based on the ionization of chitosan in the acidic solution, the amino group in the chitosan, NH2 , pro- tonated to form NH3 +. This excess NH3 + ions induced coulomb repulsion between the similar-charged ions, and hence, the swelling behaviour was imparted to the hydrogels at lower/acidic pH environment. Referring to the pKa of the MAA, which was ∼4.65, changing the environment from acidic to pH closer to that of value resulted in the deprotonation of the COOH group to COO− , while the chitosan, with a pKa of ∼6.5, was protonated to become an NH2 group in basic environment. It could be seen from the graph that the hydrogels slowly de-swelled once they approached pH 4. The COOH groups started to deprotonate to partial negatively-charged COO− groups and the existing NH3 + groups produced an electrostatic attraction between both charges. Fig. 6. Dependence of the average hydrodynamic diameter on various pH for dif- ferent reaction time of polymerization on chitosan-p(MAA-co-NIPAM) hydrogels Since more COO− groups were deprotonated, there were more (T = 37 ◦ C). attractions between the COO− and NH3 + groups. Under these conditions, the NH3 + groups that were not attracted became less in the cross-linking density. As a result, the space between and reduced the hydrodynamic diameter of the hydrogels. the polymer chains was reduced, which restricted the structure With a further increase in the pH of the solution, most of the to swell. The hydrodynamic diameter in the acidic environ- COO− groups produced were above pH 4.65, while the NH3 + ment was lower compared to that of in basic environment which group was continuously deprotonated. The swelling at this stage gave an idea that chitosan was restricted to swell. The chitosan has depended on the repulsion force between the same carboxyl higher degree of crosslinking and thus restricted repulsion within charges. Since amide charges were still present, the repulsion the chitosan to overcome the force to swell the overall hydrogel between them also contributed to an increase in the hydrodynamic network. Additional, the stronger force of intermolecular and diameter of the hydrogels. The hydrodynamic diameter reached  S.Z.M. Rasib et al. / International Journal of Biological Macromolecules 108 (2018) 367–375 373 Fig. 7. Dependence of zeta potential on the pH of the medium aqueous solutions for Fig. 8. Dependence of hydrodynamic diameter on the temperature for different different reaction time of polymerization chitosan-p(MAA-co-NIPAM) hydrogels at reaction time of polymerization chitosan-P(MAA-co-NIPAM) hydrogels at pH 7.4. T = 37 ◦ C. 3.5. Response of chitosan-p(MAA-co-NIPAM) hydrogels to temperature In order to study the effect of the polymerization time on the its maximum swelling at pH 7, which was an indirect indication temperature-responsive behaviour of the hydrogels, the volume of the complete deprotonation of the amide group. Under these phase transition temperature (VPTT) was investigated based on the conditions, the hydrogel swelling was attributed to the repulsion maximum peak of hydrodynamic diameter of the hydrogels. The between the charged carboxyl groups of the gels. High concentra- existence of the NIPAM, which was a temperature sensitive moiety tion of the carboxyl charges increased the hydrodynamic diameter, within the structure of the hydrogels changed the hydrophobic- and the hydrogels swelled more compared to the hydrogels with a hydrophilic balance in their chemical structure. This gave rise to lower reaction time. the swelling and collapse of the hydrogels when the tempera- The swelling and de-swelling of the hydrogels were further ture passed through the lower critical solution temperature (LCST explained by a study on the zeta potential at different pH levels ≈32 ◦ C) of the PNIPAM. The effect on the VPTT of the hydrogels as shown in Fig. 7. The positive potential values at low pH levels was shown in the graph of the hydrodynamic diameter against indicated the potential produced by the NH3 + group on the chi- temperature in Fig. 8. Generally, the pattern of VPTT at different tosan chain. Those values decreased with an increase in the pH until temperature conditions was almost the same, except for 30 min of the zeta potential reached zero, which indicated that the charges reaction time, and the hydrodynamic diameters were changed with between the NH3 + and COO− were balanced. The isoelectric points time of reaction accordingly. were found to be different for each of the reactions performed at As temperature increased, hydrogels showed swollen configu- different times. This was due to the different extent of the con- ration before the hydrogels collapsed. According to Khan et al. , sumption of OH− to neutralise the charges since the composition the diameter of hydrogel increases due to the interaction of acetyl of each of the hydrogels was also different. At higher pH levels group from the chitosan that becomes hydrophobic and results in (pH ≥ 4), the zeta potential values became negative due to the nega- an aggregation. Furthermore, hydrogen bonding between PMAA tive charges on the hydrogels. These negative charges arose because and PNIPAM weaken and expand the hydrogels. This behaviour was of the deprotonation of the COOH groups to the COO− groups. also well explained by Giussi et al. in their research on MAA- Under these conditions, the amide groups lost their charges and NIPAM microgel which mentioned that the binding of the microgel became neutral. This was reflected in the slightly plateau region in between MAA-NIPAM gets weaken and increases the swelling of the zeta potential versus the pH curve at pH levels above the neutral microgel into a certain distance within the charges. The hydrody- pH level of 7. It could be concluded that the reaction time during namic size increased to high temperature proportional to the time the polymerization of hydrogels affected the zeta potential once of reaction since more dissociate MAA present in the hydrogels (the the hydrogels were applied to various pH environments. The value pH was greater above their pKa), thus requiring more spaces to rest of zeta potential for 30 min reaction was high at low pH indicated the energy. In contrast to the hydrogel synthesized within 60 min, that many chitosan surrounded the surface layer, while the value less crosslinked within the structure allowed the network to swell decreased as prolonged time due to the cross-linked covered by more compared to the other two hydrogels synthesized for 120 and PNIPAM. Similar situation at higher pH (basic condition), the zeta 180 min. potential for hydrogels reacted for 30 min was higher compared to The hydrogels prepared at 30 min collapsed at about 32 ◦ C, while other time of reaction since the surface layer mostly was covered the hydrogels prepared at 60 min of reaction started to collapse by PMAA. at/above ∼35 ◦ C. When the reaction time was prolonged to 120 min, 374 S.Z.M. Rasib et al. / International Journal of Biological Macromolecules 108 (2018) 367–375 produced high electrostatic repulsion within the structures and obstructed the PNIPAM for further shrinkage. The measurement of the zeta potential at different temperatures is given in Fig. 9. The variation in the values of the zeta potential was referred to the repulsion of the PMAA chains. As shown in the figure, high zeta potential values were obtained when the reaction time was 120 min. High zeta potential values indicated an increase in the swelling of the hydrogels under these conditions. However, the hydrodynamic diameter, as shown in the previous figure (Fig. 8), did not match with this zeta potential results where the hydrody- namic diameter at 180 min reaction was greater compared to the hydrodynamic diameter at 120 min reaction. By extending the reac- tion time, the PNIPAM content in the structure covered the outer surface and thus lowering the zeta potential of hydrogels reacted at 180 min. The increase in temperature has slightly increased the zeta potential which indicated that the ionization of the PMAA did not affect the stability of the hydrogels and no structural changed within the temperature except on breaking the hydrogen bond which did not affect the zeta potential value. The ionization only disturbed the shrinkage process where the shrinkage diameter was only 50 percent from the maximum swelling before collapsed. 4. Conclusion The emulsion polymerization method was successfully applied to synthesize chitosan-p(MAA-co-NIPAM) hydrogels. Based on the Fig. 9. Dependence of zeta potential on the various temperature for different reac- overall investigation on different reaction time during synthesis tion time of polymerization chitosan-P(MAA-co-NIPAM) hydrogels at pH 7.4. on the hydrogel, as the reaction continued, chitosan, PMAA, and PNIPAM were continuously cross-linked and at the end, the outer surface of hydrogels was covered mostly by PNIPAM. Hydrogels the hydrogels collapsed slightly lower than the hydrogel at 60 min with short reaction time were less swollen, but prolonging the reac- at ∼30 ◦ C and then increased in the LCST/VPTT to ∼34 ◦ C as the tion would increase the crosslinked density and thus restricted the reaction was further prolonged to 180 min. This showed that differ- hydrogels to swell. Therefore, intermediate reaction time hydrogels ent concentrations of the actual amount of hydrogels also affected were most suitable to be used for further application. The existence the degree of polymer–polymer and polymer-solvent interaction of chitosan in the network improved the zeta potential of hydrogels changes in the LCST/VPTT values. The presence of a hydrophilic in an acidic environment however, the rigidity of chitosan reduced moiety, PMAA in the hydrogels structure played an important role the ability of hydrogels to shrink after applying the body tem- in the alteration of the LCST. For the sample collected at 30 min, perature. The shrinkage of hydrogels was also limited due to the low content of PMAA present allowed the hydrogel to collapse electrostatic repulsion from dissociated PMAA in pH 7.4. However, near to the LCST value. Meanwhile, after one hour of reaction the dissociated PMAA is important to stabilize the hydrogels in a time, the repulsive interactions caused by the PMAA were found basic environment. Once hydrogels were applied at human body to be more effective compared to the hydrophobic forces of the temperature, degradation of chitosan will take over along the time NIPAM. Thus, a higher temperature was required to overcome the and the hydrogels have a tendency to shrink smaller than the ear- hydrophilic/hydrophobic balance to allow the volume phase tran- lier diameter. Since PNIPAM covered the outer surface of hydrogels, sition to occur. It was believed that by prolonging the reaction, it lowered the zeta potential value to be less than 30 mV either more and more PNIPAM were incorporated and cross-linked in the in acidic or in basic environment. Therefore, improvisation was hydrogel structure. Under these conditions, the LCST temperature given to the hydrogels by dropwise the PMAA and chitosan during was around 32 ◦ C, meaning that the hydrophobic force of the PNI- synthesis to ensure the crosslink within PMAA, PNIPAM and chi- PAM exceeded the repulsive contribution of the PMAA because the tosan will be more homogeneous especially at the outer surface of concentration of PMAA was increasing relatively less compared to hydrogels. The chitosan-p(MAA-co-NIPAM) hydrogels performed the formation/crosslinking of PNIPAM. However, the VPTT for well in various pH environment and kept collapsing with tempera- the hydrogels at 120 and 180 min reduced to lower than the VPTT ture. In conclusion, the swelling and shrinkage for all the hydrogels of hydrogels at 60 min reaction because of increment on PNIPAM occurred within the human body temperature and as a result, the content in the network. chitosan-p(MAA-co-NIPAM) hydrogels are suitable to be used for It was further observed that there was a reduction in the hydro- drug delivery. dynamic diameter when the temperature rose above the LCST of the hydrogels. This reduction then reached a plateau at a higher temperature in the hydrodynamic diameter against the tempera- Conflicts of interest ture profile. Under such circumstances, it can be said that the linear thermo-sensitivity of the chitosan-p(MAA-co-NIPAM) hydrogels There are no conflicts to declare. were not disturbed by the temperature. However, the hydrody- namic diameter was higher proportional to the time of reaction. Acknowledgements It occurred due to high crosslinking within the structures added with rigidity structures of chitosan chain thereby hindered the net- This research was financially supported by the Ministry of work to shrink. This is an advantage to control large amount of Higher Education Malaysia [FRGS-6071325 and FRGS-6071337] drug bursting in the early release. Furthermore, conjugated PMAA and School of Materials and Mineral Resources Engineering,  S.Z.M. Rasib et al. / International Journal of Biological Macromolecules 108 (2018) 367–375 375 Engineering Campus Universiti Sains Malaysia for facilities and M. Ray, K. Pal, A. Anis, A. Banthia, Development and characterization of technical support. chitosan-based polymeric hydrogel membranes, Des. Monomers Polym. 13 (3) (2010) 193–206. S.M. Silva, C.R. Braga, M.V. Fook, C.M. Raposo, L.H. Carvalho, E.L. 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