Preparation, Physicochemical, and Stability Studies of Chitosan-PNIPAM Micro Gels (PDF)

Iran Polym J (2015) 24:317–328 DOI 10.1007/s13726-015-0324-5 ORIGINAL PAPER Preparation, physicochemical and stability studies of chitosan‑PNIPAM based responsive microgels under various pH and temperature conditions Abbas Khan1,2 · Muhammad Bisyrul H. Othman1 · Boon Peng Chang1 · Hazizan Md A...

Iran Polym J (2015) 24:317–328 DOI 10.1007/s13726-015-0324-5 ORIGINAL PAPER Preparation, physicochemical and stability studies of chitosan‑PNIPAM based responsive microgels under various pH and temperature conditions Abbas Khan1,2 · Muhammad Bisyrul H. Othman1 · Boon Peng Chang1 · Hazizan Md Akil1 Received: 2 October 2014 / Accepted: 1 March 2015 / Published online: 10 March 2015 © Iran Polymer and Petrochemical Institute 2015 Abstract This study describes the synthesis, characteri- A transition from sol-to-gel state at temperature beyond zation and detailed physicochemical investigation on the 50 °C was also noticed in all hydrogel samples. On the stimuli-responsive behaviour of chitosan-poly (N-isopro- basis of results obtained, we observed that variables such as pylacrylamide) [chitosan-PNIPAM] copolymer microgels. NIPAM/chitosan ratio, amount of cross-linker, and temper- Six different compositions of dual-responsive chitosan- ature/pH not only affect the above mentioned properties but PNIPAM based microgels were synthesized by soapless- also affect the dual-sensitivity and stability of the micro- emulsion free radical copolymerization method in an aque- gels. Most of the chitosan-PNIPAM particles remained sol- ous medium at 70 °C. The chemical composition, swelling/ uble and maintained good stability without significant sedi- de-swelling, volume phase transitions, electrical proper- mentation in water through a wide range of pH (pH ≈ 2–8) ties, colloidal stability, and physicochemical behaviour for about 3 months at room temperature. under various pH/temperature conditions were investigated using Fourier transform infrared spectroscopy (FTIR), Keywords Light scattering · Stimuli-responsive dynamic light scattering (DLS), zeta-potential, and rheol- polymers · Phase changes · Physicochemical properties · ogy. Various functional groups present in the copolymer Colloidal stability microgels were confirmed by FTIR analysis. The hydrody- namic diameter (Dh), volume phase transition temperature (VPTT), and swelling/de-swelling behaviour of microgels, Introduction under different pH and temperature conditions, in aqueous media were investigated using DLS measurements. Zeta Due to their tunable dimensions, large surface areas, sta- potential measurements were used to evaluate the stability, ble interior network structure, and a very short response electrical and electro-kinetic properties of the gels at vari- time, polymer macro/micro/nano and hydrogels are play- ous pH/temperatures. Likewise, the influence of tempera- ing an important part in a diverse range of applications. ture and chemical composition of microgels on the parti- Polymer microgels have rapidly gained importance in the cle behaviour was investigated through rheological studies. field of materials science owing to their potential applica- tions in biomedical , pollution control enhanced oil recovery , sensors , controlled release of encapsulated * Abbas Khan drugs in response to environmental changes , the fabri- [email protected] cation of photonic crystals, glucose sensing template- * Hazizan Md Akil based synthesis of inorganic nanoparticles and chemical [email protected] separation. These numerous applications of microgels 1 arise from their ability to undergo reversible volume phase School of Materials and Mineral Resources Engineering, Engineering Campus, Universiti Sains Malaysia, transitions in response to external stimuli such as a change 14300 Nibong Tebal, Pulau Pinang, Malaysia in pH , temperature and ionic strength of the sur- 2 Department of Chemistry, Abdul Wali Khan University rounding medium. Mardan, Mardan 23200, Pakistan Iran Polymer and Petrochemical Institute 13 318 Iran Polym J (2015) 24:317–328 To prepare stimuli-responsive hydrogels for biomedical responsive copolymer gels; one has to know in detailed applications, natural biopolymers have been considered as manner, the effect of various internal and external param- suitable candidates due to their biocompatibility, low tox- eters on their physicochemical properties. Amongst the icity, excellent biodegradability, and high content of func- tuning actors, the chemical nature, feeding composition of tional groups [12–14]. Because of the unusual properties reacting species, and the physical state of the resultant gel of various biopolymers, such as chitosan, they are consid- can be considered as internal parameters. Meanwhile, the ered to be versatile materials, with extensive applications nature of solvent, co-solvent, temperature, and pH, and the in biomedical, biotechnological, and nano-biotechnological addition of electrolytes, are categorized as external parame- fields [15, 16]. Of the commonly available biopolymers, ters. Thus, to attain a microgel system with desirable prop- natural polysaccharides, such as alginate, hyaluronic acid, erties, the tuning of both internal as well as external param- dextran, and chitosan, have been proposed for biomedical eters is of the utmost importance. purposes, due to their biodegradability and biocompatibil- To increase the application capability of polymer macro/ ity [17–19]. Among the known polysaccharides, chitosan is micro/nanogels and hydrogels especially in the fields of quite unique, not only due to its antibacterial, biodegrad- biomedical and nano-technology, the adjustment of their able, biocompatible and mucoadhesive properties but also temperature and pH-sensitivity is greatly required [21, 28]. because of its glucosamine groups, cationicity, and capac- It is so because the polymer–polymer and the polymer–sol- ity to form polyelectrolyte complexes. However, chi- vent interactions can show an unexpected re-adjustment tosan is originally insoluble in some solvents and brittle in small ranges of pH or temperature changes, and this is in nature, which limits its potential industrial applications, translated into a chain transition between extended and as compared to other synthetically available polymers. To compacted coil states. For the pH-sensitive polymers, the obtain desirable properties, modification of chitosan by key element of the system is the presence of ionizable weak chemical/physical methods and compounding it with other acidic or basic moieties attached to a hydrophobic back- moieties, are of prime importance. In connection to this, bone. In case of temperature-responding polymers, usually Zhang et al. have reported the rational design of pH- there exists a fine hydrophobic–hydrophilic balance in their responsive chitosan-based microgels for biomedical appli- structure. Thus, some changes in the solution temperature cations for the first time. around the critical temperature make the chains to col- Further, to explore the applications and understand the lapse or to expand responding to the new adjustments of fundamental mechanism of stimuli-responsive behaviour the hydrophobic and hydrophilic interactions between the of chitosan-based microgels, it is important to undertake polymeric chains and the aqueous media. Among the the detailed physicochemical studies of these hydrogels in polymers sensitive to temperature changes, poly (N-isopro- aqueous media. It is known to us that most of the studies pylacrylamide) [PNIPAM] is a widely studied temperature related to chitosan-based hydrogels have focused on the responsive polymer microgel. It is because PNIPAM applied side while the basic physicochemical investigations has a low critical solution temperature (LCST) at around of these hydrogels have been less addressed. Importantly, 32 °C in water. The soluble and flexible coil of PNIPAM physicochemical properties not only affect the absorption converts into a compact globular and insoluble state at or and release processes of drugs, but also govern the interac- above its LCST. Furthermore, the most important thing tion of particles with different biological compounds (i.e. from physicochemical and application point of view is proteins or membranes) of the tissue where they are intro- that these conformational changes from coil to globular duced. Thus, successful development of polymeric particles form with solution temperature are reversible. Hence, to act as drug delivery vehicles can easily be determined by hydrogels composed of PNIPAM experience a revers- a detailed knowledge of their chemical nature and physi- ible volume change at a similar transition temperature in cal properties, which would explain how they interact with aqueous media. From application point of view, microgels their potential biological environment/system. Numer- would be much favourable if they could respond to several ous workers have reported the physicochemical charac- stimuli simultaneously. Therefore, N-isopropylacrylamide terization of chitosan homogels, but they have specifi- (NIPAM) is copolymerized with several ionic/non-ionic cally focused on macroscopic films [23, 24], microspheres monomers to produce multi-responsive microgels [28, obtained by covalent cross-linking [25, 26], and nanoparti- 30]. Chitosan-PNIPAM based copolymer hydrogels are cles achieved by the direct precipitation of chitosan chains expected to have good potential application in biomedi- without cross-linking agents. Therefore, this lack of cal field. Mani et al. have recently reviewed stimuli- information on the fundamental physicochemical studies of responsive microgels based on polysaccharides with PNI- chitosan-based responsive copolymer gel/hydrogels needs PAM as novel biomaterials. To the best of our knowledge, to be addressed. To better understand the fundamentals and most of the studies on chitosan-PNIPAM based hydrogels explore new-fangled methods in the field of chitosan-based have focused on the applied side. Though, insufficient Iran Polymer and Petrochemical Institute 13 Iran Polym J (2015) 24:317–328 319 Table 1  Summary of gel compositions, zeta potential values and hydrodynamic diameter at pH = 6 and T = 25 °C Gel designation Composition of gel (mg/100 mL)a Cross-linker MBA (mg/100 mL) Zeta potential (mV) Dh (nm)b ACG01 500:500:10 10 0.576 160 ACG02 500:500:20 20 0.082 295 ACG04 500:1000:10 10 0.187 396 ACG05 500:1000:30 30 0.301 396 ACG06 500:1500:20 20 0.188 342 ACG07 500:1000:20 20 0.288 458 a Composition of gel = Chitosan:NIPAM:MBA b Hydrodynamic diameter in the scale of nanometer studies are available that have addressed the basic physico- microgels and their respective codes are given in Table 1. chemical investigation and stability of such micro/hydro- During the preparation of microgels, approximately gels under various environmental stimuli. 500 mg of chitosan was dissolved with different amounts Therefore, to highlight, address and further explore the of NIPAM and acetic acid in 100 mL doubly distilled and fundamentals of chitosan-PNIPAM based micro/hydrogel deionized water, under constant magnetic stirring, for system, herein we report the synthesis and detailed phys- approximately 18–20 h, in a three-neck round bottom flask, icochemical study of swelling/deswelling patterns, electri- equipped with an N2 gas inlet and condenser. The purpose cal properties, colloidal stability, volume phase changes, of addition of acetic acid (3–4 mL of 0.05 M) to the reac- and rheological behaviour of chitosan-based responsive tion system was to ionize the –NH2 groups of the chitosan [chitosan-poly(N-isopropylacrylamide),(chitosan-PNI- to form the –NH3+ groups, so that the chitosan may eas- PAM)] copolymer hydrogels, at different gel compositions, ily dissolve in the aqueous reaction medium, and hence and under various pH/temperatures in water. Moreover, on increase its degree of coupling to NIPAM/PNIPAM. After the basis of experimental results, an attempt has been made 20 h stirring under N2 purging at 30 °C, MBA (used as a to explain the behaviour of these microgels under various cross-linker) was added to the reaction flask. The tempera- solution conditions using proposed theories. ture of the reaction mixture was slowly raised to 70 °C by gradual heating in a silicon filled oil bath under N2 purg- ing with continuous stirring. After 30–40 min of constant Experimental heating at 70 °C, 5 mL of APS (0.05 M) was added to the reaction mixture, to start the polymerization reaction. Sev- Materials eral minutes after the addition of the initiator, the colour of the reaction mixture turned light milky and then slightly N-Isopropylacrylamide (NIPAM), N,N-methylenebisacryla- opaque with time during the course of reaction. The polym- mide (MBA), and chitosan (Mw ≈ 160,000 g/mol, degree of erization reaction was allowed to proceed for 5 h at con- deacetylation ≈ 91 %), were purchased from Aldrich, while stant stirring under N2 purging at 70 °C. The resultant all other chemicals were obtained from Acros. NIPAM was copolymer microgels were then purified by centrifugation purified by recrystallization from toluene/n-hexane (1:3) (Thermo Electron Co. SORVALL® RC-6 PLUS super- mixture, whilst all other chemicals were used as received speed centrifuge) and decantation, and then washed with and without further purification. Deionized distilled water water. Next, the obtained microgel was further purified by (ddH2O), used for all reactions, solution preparations, and dialysis for 1 week (Spectra/Por® molecularporous mem- microgel purifications, and was obtained from a Millipore brane tubing, cut-off 12,000–14,000, the same below was Milli-Q system (Millipore, Bedford, MA, USA). used) against a diluted HCl solution in water, with a pH of ~5 ± 0.3 at room temperature (~25 °C). To maintain con- Preparation of the microgel stant pH (pH ≈ 5) of the dialysis medium and ensure better purification of the microgel, the diluted aqueous solution of Chitosan-PNIPAM based hydrogel samples were synthe- HCl was regularly changed throughout purification process. sized by free radical surfactant-free emulsion copolym- erization of chitosan, NIPAM, and MBA, using APS as an Characterization initiator, following the previously reported procedure. However, some desired modifications were made to the FTIR analysis was carried out to identify various func- earlier synthetic method. The detail of the composition of tional groups present in the microgels. A Perkins-Elmer Iran Polymer and Petrochemical Institute 13 320 Iran Polym J (2015) 24:317–328 Spectroscopy was used to record the FTIR spectra of the oven dried microgel samples. All FTIR spectra of the sam- ples were obtained in the range 500–4000 cm−1. The pH values of the hydrogel systems were measured by using a WTW-Inolab-pH-720 pH meter. Zeta potential measure- ments were performed with the help of a Malvern Zeta- sizer, NANO ZS (Malvern Instruments Limited, UK), using a He–Ne laser with a wavelength of 633 nm, and a detector angle of 173º. Original microgel samples were diluted in the desired buffer or electrolyte solution, as per the experi- mental requirement. Data was obtained from the average of six measurements at a stationary level in a cylindrical cell. Standard deviation was always below 5 %. The rheological behaviour of hydrogels in aqueous solu- tion was investigated by measuring the change in viscos- ity using a cone and plate viscometry (Physica MCR301 Rheometer, Anton Paar Austria). The rheometer was equipped with Temperature Control-Peltier Systems (−40 to 200 °C). The temperature dependence of shear stress Fig. 1  Typical FTIR spectra of pure chitosan and selected chitosan- PNIPAM microgels and hence viscosity was noted using parallel plate–plate arrangement measuring system. The distance between the plates was 0.2 mm (PP25-SN24578, d = 0.2 mm). The emulsion technique. Detailed microgel compositions with temperature range for experiment was 25–75 °C with a their given code and basic properties are shown in Table 1. heating rate of 0.033 °C per second and a fixed shear rate The chemical structures of the pure chitosan and chitosan- of 50/s. PNIPAM microgels were confirmed by FTIR spectroscopy. Likewise, dynamic laser light scattering (DLS) measure- Figure 1 shows the typical FTIR spectra of pure chitosan ments for size determination were performed on a stand- and some representative microgels. ard Malvern Zetasizer, NANO ZS (Malvern Instruments Different IR peaks at various/specific positions cor- Limited, UK), using a He–Ne laser with a wavelength of responding to different functional groups can be seen in 633 nm, and a detector angle of 90º. All microgel solutions Fig. 1. FTIR spectra of pure chitosan shows a broad band were passed through Millipore Millex-HV filters (with at approximately 3300–3400 cm−1 which was assigned to a pore size of 0.80 μm) to remove dust before the DLS the stretching vibration of hydroxyl groups; this peak over- measurements. All solutions were equilibrated at each cho- lapped the N–H stretching vibration in the same region. sen temperature/pH for approximately 10–15 min before The absorption peaks at 2872 cm−1 were associated with measurement. Experimental durations were in the range the C–H stretching of methylene and methyl groups of 5–10 min, and each was repeated three or more times. The glycol chitosan. The characteristic absorption peaks of the scattering intensities for each microgel were measured at chitosan at 1650–1560 cm−1 can be assigned to the car- pH range from 1 to 11 and temperatures (ranging from 20 bonyl stretching and the stretching vibration of the amino to 70 °C). The constrained regularized CONTIN method group (amide bands) of the aminoacetyl group of chitosan, was used to analyze the correlation functions from DLS whilst that at 1373.5 cm−1 is assigned to the vibration of and to obtain distribution decay rates and hence the hydro- C–H. The peak of 2872.35 cm−1 is a typical C–H vibration. dynamic size (Dh) of hydrogels. Further detail of these Similarly, the peaks at approximately 894.07, 1150.35, and analysis/results is discussed in the results and discussion 1560 cm−1, which correspond to the saccharide structure of section. chitosan, are also present. The broad peak at 1061.71 indi- cates C–O stretching vibration. Furthermore, the peaks at 1158.60 and 1253 cm−1 can be assigned to C–N stretch- Results and discussion ing of the amino group of chitosan. Compared to that of the pure chitosan, the FTIR pro- FTIR results files of the chitosan-PNIPAM hydrogels were individually monitored. It can be seen that in addition to the appearance The free radical copolymerization of chitosan, NIPAM of new peaks in the range of 1750–1250 cm−1, the posi- and MBA, using APS as an initiator, was used to prepare tion and intensity of some older peaks is also changed. chitosan-PNIPAM based microgels by employing soapless The peak at 1025.48 cm−1, which indicates C–O stretching Iran Polymer and Petrochemical Institute 13 Iran Polym J (2015) 24:317–328 321 vibration in the spectrum of chitosan, disappeared and the practice, intensities I(Γ), delivered by the CONTIN pro- broad peak at 1061.71 cm−1 shifted to 1071.85 cm−1 in the gram at logarithmically spaced values of decay rate, were chitosan-PNIPAM microgel spectra, and became slightly transformed to I(logΓ) = I(Γ)Γ to obtain the intensity more intense in the microgel sample. The new peaks at distributions of log(Γ), and so log(Dh,app). The normaliza- 1071–1155 cm−1 can be assigned to the C–N stretching of tion of I(log Dh,app) gave intensity fraction distributions. single bonded chains. The FTIR spectra of chitosan-PNI- Average values of Γ, delivered by the CONTIN program PAM copolymer hydrogels also show deformation of the by integration over the intensity distributions, were simi- two methyl groups on –C(CH3)2 at 1385 and 1362 cm−1, larly converted to intensity-average values of Dh,app [37, –CH3 and –CH2 deformation at 1460 cm−1, –CH3 sym- 38]. It is important to mention here, that the values of sizes metric stretching at 2800–2880 cm−1 and –CH3 asym- (Dh) given in this paper are the average values; and hence, metric stretching at 2976 cm−1 of the PNIPAM moieties. slightly overestimated, as soft particles possess lower Dh Two new stronger peaks, corresponding to amide I and II than the real hard sphere ones for frictional reasons. The groups of PNIPAM, at 1626 and 1546 cm−1, respectively, average size (Dh) of hydrogels at constant temperature also appeared in the hydrogel samples. A minor but broader (T = 25 °C) and pH ≈ 6 are given in Table 1. peak in the range 2000–2400 cm−1 can be assigned to the It can be seen from Table 1 that, at constant temperature intermolecular hydrogen bonding and hence the physical and pH, the microgel size clearly depends on the chemical/ crosslinking in the hydrogel. The FTIR spectra of all other feed composition of the microgel. This behaviour can also microgels, which are not shown here, have almost the same be further clarified from Dh-temperature profile (Fig. 3) FTIR pattern as those of the presented microgels. Shift- which is briefly discussed in the next section. On the aver- ing of the positions, shape and intensities of various peaks age, hydrogel size increases with increasing cross-linker in the microgels, as compared to pure chitosan, are indi- (MBA) content in the feed up to 20 mg per 100 mL of reac- cations of microgel formation. Further, an increase in the tion mixture while further increase in the cross-linker ratio intensity and change in the shape of the 3100–3500 cm−1 reduces the microgel size. The first increase in the microgel absorption band, with changing NIPAM to chitosan ratio in size with cross-linker can be assigned to the more entrap- the feed, also supports the incorporation and crosslinking ping of chitosan molecules into the microgel matrix by of chitosan in the PNIPAM matrix. MBA. In other words, the proportion of chitosan that could be entrapped in the PNIPAM matrix may increase with Effect of chemical composition on the hydrodynamic cross-linker ratio. However, the decrease in the microgel size of microgels size with MBA ratio, at 30 mg per 100 mL of the reaction mixture, can be due to increase in crosslinking density of Dynamic light scattering was used to investigate the hydro- the microgel. This may result in the microgel particles with dynamic size of the chitosan-PNIPAM based microgels, less swollen and tight/compact structure. To eliminate any in the form of hydrodynamic diameter (Dh) at various experimental error and confirm this special trend of the temperatures and pHs. During a typical DLS experiment, microgel size with cross-linker content, each experiment the intensity auto-correlation functions of the fluctuating was repeated many times but the same trend as reported in (2) signals G(τ ) were measured and analysed using the well- Table 1 was obtained. In a more comprehensive way, we known constrained regularized CONTIN method. Dis- can say that the increase in the crosslinking agent would tributions of decay rate, Γ were converted to distributions increase the conversion and crosslinking density. In addi- of apparent mutual diffusion coefficient : tion, the grafting ratio might increase as well. As a result, the particle size would increase when the monomer conver- Dapp = Ŵ/q2 (1) sion and grafting ratio were increased, but would decrease In Eq. (1), the scattering vector q = (4π n/) sin(θ/2), n when the crosslinking density was increased. The former is the refractive index of the solvent, and θ is the scatter- two factors dominated when the MBA was increased to ing angle. Supposing that the particles are solid, the appar- 20 mg per 100 mL but the latter factor dominated when ent diffusion coefficient was then converted to an apparent it was further increased to 30 mg per 100 mL. It can be hydrodynamic diameter [Dh,app, diameter of the hydro- further seen that the size of the microgel also varies with dynamically equivalent hard sphere, corresponding to the changing the content of NIPAM in the microgel while apparent diffusion coefficient (Dapp)], using the Stokes– keeping the amount of other moieties constant. An increase Einstein relationship : in the size of microgels can be observed with NIPAM/chi- tosan ratio ranges from 1 to 2. This situation is understand- Dh,app = kT /(3π ηDapp ) (2) able as it may increase the incorporation of number of chi- where, k is the Boltzmann constant, T is the absolute tem- tosan chains in the microgel and hence will result in higher perature, and η is the viscosity of the solvent. In actual degree of copolymerization. However, a little reduction Iran Polymer and Petrochemical Institute 13 322 Iran Polym J (2015) 24:317–328 polymer–solvent interactions as compared to polymer– polymer interactions below its LCST. It is necessary to remind here, that the discussion in this section is at a temperature (T = 25 °C) which is below the LCST of PNI- PAM. At T < LCST, both chitosan and PNIPAM are hydro- philic in nature, though PNIPAM is expecting to be more hydrophilic compared to chitosan because of its chemical nature. Therefore, on the average, variation of hydrogel size due to changing the NIPAM/chitosan ratio and amount of cross-linker can be assigned to the change in hydrophilic- hydrophobic balance of hydrogels. Temperature‑induced volume phase transitional behaviour of the hydrogels To explore the potential application of stimuli-responsive smart microgels, it is very important to investigate the effect of temperature on their volume phase transitional changes [6, 39]. For this reason we studied the effect of Fig. 2  Plots of intensity distributions of apparent hydrodynamic diameter for aqueous solutions of ACG06 at different temperatures temperature on the physicochemical properties of chitosan- and under pH ≈ 6 PNIPAM based microgel/hydrogels in an aqueous medium. Figure 2 shows the typical intensity fraction distributions of apparent Dh plots from DLS at various temperatures rang- ing from 15 to 70 °C for ACG06 in water while keeping the pH of the medium constant pH (pH ≈ 6). Average values of Dh were obtained from the maxima of each peak of such distribution plots. It was observed that the shapes and posi- tions of these peaks shifted along with the changing tem- perature of the microgel solutions. To see the effect of tem- perature and chemical composition on the hydrodynamic size and volume phase transition temperature (VPTT), we have plotted the values Dh for each microgel as a function of temperature in Fig. 3. On an overview, Dh decreases with increasing temperature, indicating the shrinking of the microgels. However, it is significant to mention that the reduction of the size with temperature for all hydrogel samples reaches a plateau somewhat once the temperature becomes close to or higher than the LCST of PNIPAM. Moreover, this deflection point in the Dh-temperature pro- file varies with changing the composition of microgels. In case of chitosan-PNIPAM polymeric hydrogels, there Fig. 3  Dependence of the average hydrodynamic diameter on tem- exists a fine hydrophobic–hydrophilic balance in their perature for different chitosan-PNIPAM microgels in aqueous solu- chemical structure. Thus, based on Dh-temperature profile tions at pH ≈ 6 outcomes, the thermo–sensitive behaviour of the microgels under study can be attributed to the alternation in hydrophi- licity/hydrophobicity balance of the network. It is because in the size of hydrogel was seen when the NIPAM/chi- for stimuli-responsive polymers, the polymer–polymer and tosan ratio was further increased to 3. As a result of this the polymer–solvent interactions can show an unexpected phenomenon, the population density or number of hydro- re-adjustment in small/specific ranges of pH or tempera- gel particles having relatively smaller size may increase ture changes, and this is translated into a chain transition instead of increasing the size of individual particle. This between extended and compacted coil states. There- is because the higher content of NIPAM can enhance the fore, some changes in the solution temperature, around hydrophilic capacity of the hydrogels which will increase the critical temperature, make the chains to collapse or to Iran Polymer and Petrochemical Institute 13 Iran Polym J (2015) 24:317–328 323 expand responding to the new adjustments of the hydro- phobic and hydrophilic interactions between the polymeric chains themselves and with the water. The earlier studies of PNIPAM homopolymer chains revealed that when the solu- tion temperature is lower than the lower critical solution temperature (LCST ≈32 °C), PNIPAM is hydrophilic and exists as individual random coil chains in water, while at higher temperatures (T > LCST), PNIPAM becomes hydro- phobic and collapses depending on the overall condition of solution. Furthermore, we were expecting that the hydrophilic chitosan moieties will not only affect the hydrodynamic size of microgels but should also have significant impact on the VPTT of our chitosan-PNIPAM based microgels. The effect of chemical composition and various contents in the feed on the Dh is already discussed in the previ- ous section. Figure 3 shows that VPTT changes by vary- ing the ratio of NIPAM/chitosan and also by the amount of cross-linker (MBA) in the reaction feed. This effect can Fig. 4  Plots of intensity distributions of apparent hydrodynamic diameter for aqueous solutions of ACG06 at different pH and be assigned to the variation of hydrophilic-hydrophobic T = 25 °C balance due to alteration of the incorporation of number chitosan chains in the microgel while changing NIPAM/ chitosan ratio. This is important to note that at T > LCST, PNIPAM becomes hydrophobic and collapses, while chi- tosan is still hydrophilic and affects the VPTT range of microgels. At T > 40 °C, there is no prominent change in the size of microgels with further increasing temperature; this effect may arise due to the presence of chitosan, which contains rigid polysaccharide chains and thereby hinders further contraction of hydrogel particles. Similarly, varying the amount of cross-linker in the hydrogels may not only change the physicochemical combination of chitosan with the PNIPAM matrix but also results in hydrogel particles with different swelling ratios. It can also be said that chi- tosan-PNIPAM hydrogels having different swelling ratios may have dissimilar particle structure/morphology and hence different LCST/VPTT. The previous studies of PNI- PAM based copolymer also showed that LCST depends on the morphology of copolymer particles. As supposed here, hydrogel particle having different structures may have different degrees of polymer–polymer and polymer–solvent Fig. 5  Plots of average hydrodynamic diameter as a function of pH for different chitosan-PNIPAM microgels in aqueous solutions at interactions which can affect their corresponding LCST/ T = 25 °C VPTT values in aqueous media. It is worth mentioning that the change of temperature-induced On/Off (swelling/de- swelling) window for chitosan-PNIPAM based hydrogels Effect of pH on volume phase changes and stability by varying the chemical composition of the microgels is of chitosan‑PNIPAMm microgels important from biomedical point of view. On an overview, it can be seen from Fig. 3 that, almost for all microgel sam- To better understand the physicochemical properties, sta- ples tested, complete de-swelling/shrinkage occurs in the bility and especially the “On/Off” behaviour of the syn- temperature range 36–39 °C. As this temperature range thesized chitosan-PNIPAM hydrogels in aqueous media, is near to human body temperature, hence it reflects the we also investigated the effect of pH on microgel size applications of these hydrogels as potential drug delivery (Dh) at room temperature. As chitosan is known as a pH- system. sensitive biomaterial and its hydrophilic character varies Iran Polymer and Petrochemical Institute 13 324 Iran Polym J (2015) 24:317–328 with change in pH of the medium. However, PNIPAM the degree of stability also depends on the chemical com- moiety of our hydrogel system is not a typical pH-sensitive position, for example the ratio of NIPAM/chitosan and polymer. The typical intensity fraction distributions of the the amount of cross-linker (MBA) in the feed composi- apparent hydrodynamic diameter plots from DLS at vari- tion. After 3 months, the hydrogels having lower NIPAM/ ous pHs and 25 °C for ACG06 in water are shown in Fig. 4. chitosan exhibited some sedimentation/aggregation at or Meanwhile, the dependence of Dh on pH for four repre- above pH ≈ 8 of the medium. This effect can be attributed sentative microgels is presented in Fig. 5. To see the effect to the decrease in hydrophilic capacity with decrease in of pH on the microgel size (Dh), we selected four different NIPAM content. compositions; one series (ACG06 and ACG07) composed of microgels having the same cross-linker content but dif- Effect of pH on the electrophoretic mobility of microgel ferent NIPAM/chitosan ratios. Whereas, the second series particles (ACG01 and ACG02) have similar NIPAM/chitosan ratio but different cross-linker (MBA) contents. It can be seen The stability, swelling/de-swelling, and physicochemical that, on the average, there is no prominent change in the information under different pH conditions can be obtained Dh versus pH profile up to pH ≈ 6. However, by further by knowing the electrical state of ionisable groups of the increasing the pH of the investigation medium, different chitosan-PNIPAM microgel particles. For this reason the behaviours of pH-dependence of Dh can be observed for electrophoretic mobility (μ) and zeta potential (ζ) were microgels having different chemical compositions. The measured as a function of pH of the medium for four rep- trend of Dh with pH of the solution, as shown in Fig. 5, resentative microgels having different compositions. The indicates that chitosan-PNIPAM hydrogels are more stable reported values of ζ-potential were calculated from the in acidic and neutral media but less so in basic medium. electrophoretic mobilities (μ) of hydrogel particles using It is understandable because the pKa value for chitosan the relationship : is approximately equal to 6.5, due to which pH-sensitive 3µx1 behaviour, in terms of Dh, varies with pH of hydrogel solu- ζ = (3) 2ε0 εr × f (ka) tion. The less prominent change in Dh-pH profile, and hence the stability in acidic and neutral pH regions, can be Here, ε0 is the permittivity of vacuum, εr is the rela- attributed to the presence of positive charges on the sur- tive permittivity of the medium, and η is the viscos- face of hydrogel particles. These charges arise due to the ity of water. The values of ε0, εr, and η were taken as protonation of amino (−NH2) groups of chitosan to a posi- 8.854 × 10−4 J−1 C2 m−1, 78.5, and 8.904 × 10−4 Nm−2 s, tively charged (−NH+ 3 ) groups when pH < pKa of chitosan. respectively. Similarly, the factor f(κa), known as the Henry This makes the hydrogels friendlier to aqueous media due factor, depends on particle shape; for a sphere with ka > 1 it to increased hydrophilicity. However, in the neutral pH is given by : region, the amino groups are not or much less protonated, 3 9 75 330 so the stability can be attributed to the presence of hydro- f (ka) = + + − 3 3 (4) philic PNIPAM. The change in microgel size in the alka- 2 2ka 2k 2 a2 k a line pH region may arise due to inter-particle interactions. In Eq. (4), k is the reciprocal of Debye length. For the Because some of the hydroxyl groups of chitosan can be present case, the product κa was approximately 2.1, which ionized/deprotonated. Further information and clarification corresponds to f(κa) ≈ 1.04. The behaviour of zeta poten- can be found from the behaviour of zeta potential with pH tial as a function of pH of the medium, for four composi- in the next section. tions tested, is shown in Fig. 6. Moreover, it is important to mention here that although As seen, for all microgels, the zeta potential changes the true solubility of pure chitosan chain is limited only along with the changing pH of the medium, indicate their to acidic media, but chitosan-PNIPAM particles remained pH-sensitivity due to the presence of chitosan moieties. It soluble and maintained their stability in water in a wide can be seen that the zeta potential values are positive for range of pH (pH ≈ 2–8) for about 3 months at room tem- all microgels in the acidic pH region (pH ≈ 1.4–6). On perature. This significant outcome, of our general/naked- the average, these values decrease with an increasing pH eye observations, indicates that chitosan chains are not of the medium until reaching neutral pH (pH ≈ 6.3–7.5). only attached physicochemically to PNIPAM but also have The positive values of zeta potential at pH < pKa (6.5) of chemical linkage with PNIPAM networks. Thus, it can be chitosan, are assigned to the presence of positive charges said that the chitosan-PNIPAM hydrogels, reported in this on the surface of hydrogel particles arise due to protonation work, resulted in improved particles with better stability in of amino (−NH2) groups of chitosan to positively charged acid, neutral, and alkaline aqueous solution, as compared (−NH+ 3 ) groups. Further, in neutral pH region, the val- to unmodified PNIPAM and chitosan only. Furthermore, ues of zeta potential become almost equal to zero, which Iran Polymer and Petrochemical Institute 13 Iran Polym J (2015) 24:317–328 325 Fig. 6  Dependence of zeta potential on the pH of the medium for aqueous solutions of different chitosan-PNIPAM microgels at T = 25 °C Fig. 7  Apparent viscosity as a function of temperature for chitosan- PNIPAM microgels at constant shear rate and pH ≈ 6, a different NIPAM contents and b different cross-linker amount reflects that the amino groups of chitosan are no more or much less protonated. Thus, this region in the zeta poten- Effect of temperature and chemical composition tial-pH profile can be assigned as the isoelectric point of on the rheological behaviour of hydrogels the hydrogels, at which the microgel particles are expected to be uncharged. Moreover, Fig. 5 also reveals that in alka- The rheological studies of stimuli-responsive polymers line pH region (pH > 7.5) hydrogels exhibit negative zeta in aqueous media, is very helpful to elucidate the phase potential values which become more negative with increas- changes and association behaviour in response to shear rate ing pH of solution. This situation arises due to ionization/ applied at different pH and temperatures [42, 43]. The tem- de-protonation of some of the hydroxyl (–OH) groups of perature-dependence of chitosan-PNIPAM hydrogel vis- chitosan and also due to some contribution of the anionic cosity, in aqueous media at a constant pH ≈ 6, is shown in nature of the initiator (APS) used in this study. There is no Fig. 7. It can be seen that, for the four selected hydrogels, prominent change in zeta potential when pH > 11, mean- there is no prominent change in viscosity with temperature ing that ionization capacity of hydroxyl (–OH) groups has up to 28–29 °C. However, at temperatures beyond these, already reached to its maximum limit. It is worth mention- the viscosity increases slowly and after reaching a cer- ing that irrespective of the sign (either +ve or −ve), the tain temperature, which can be called as VPTT, an abrupt zeta potential values are less than four which suggest the increase in viscosity is observed. The drastic increase in stability of microgels over a wide range of pH at constant viscosity at/above VPTT can be assigned to the appear- temperature. The effect of microgel composition on the ance of gelation/association process. Moreover, after behaviour of zeta potential, at pH ≈ 6 and T = 25 °C, can reaching a peak point, the viscosity starts to decrease with be seen in Table 1. Again the zeta potential values, for all further increase in temperature. This may occur due to samples, are very close to zero reflecting the uncharged of dehydration and shear thinning effects. At T > 50 °C, fewer partially charged state of hydrogels at pH ≈ 6. The slight changes in viscosity-temperature profile can be seen. Pre- change in zeta potential, for the microgels having different viously, it was observed that when swollen particles are chemical compositions, can be attributed to the variation of subjected to a temperature above LCST of PNIPAM, the hydrophilic–hydrophobic balance which indirectly affects particles shrink. The phenomenon happens as a result of the interaction with solvent and hence the charge density disruption of hydrogen bonding with water molecules and on the particles. Finally, in addition to the quality/quantity the formation of hydrophobic associations among isopropyl of microgel charge, zeta potential results also support the moieties of PNIPAM [45, 46]. Consequently, a resistance stability and swelling/de-swelling behaviour of microgel in to flow and hence increase in viscosity at low shear rates response to the pH of its environment. can be observed due to formation of compact particles. Iran Polymer and Petrochemical Institute 13 326 Iran Polym J (2015) 24:317–328 However, further increase in temperature (above 50 °C) and pH for their potential applications. DLS results show may cause dehydration of hydrogels and hence weaken the that the size (Dh) of the particle decreases with increasing association of collapsed particles which leads to decrease temperature up to 38 °C, indicating the shrinkage of the in viscosity due to an indirect shear thinning effect. It is microgels. Moreover, the size and VPTT are affected by important to mention here that de-swelling/shrinkage of the varying the chemical composition of the microgels, this hydrogels particle with temperature beyond VPTT was also effect can be assigned to the alternation in hydrophilicity/ observed in our dynamic light scattering results (Fig. 3). hydrophobicity balance of the network. Further, the com- The appearance of gelation behaviour, at/above VPTT, in plete de-swelling/shrinkage occurs in the temperature range response to temperature was also observed through visual 36–39 °C, and as this temperature range is near to human analysis of tube inversion method. At this point we also body temperature, hence it reflects the applications of these wish to stress that temperature can disrupt/disturb both hydrogels as potential drug delivery system. The size of intra- and inter-polymer association, but with opposite microgels was found to be quite stable at T > 40 °C and effects. pH 6. Though, a transition from sol-to-gel behaviour with Moreover, it can also be seen from Fig. 7a, b that chemi- temperature beyond 50 °C was also noticed through visual cal composition of chitosan-PNIPAM based hydrogels observation. It was also observed that most of chitosan- greatly affects their rheological behaviour. Both of these PNIPAM particles remained soluble and maintained good figures clarify that varying the chemical composition, such stability without significant sedimentation in water through as NIPAM/chitosan ratio and cross-linker (MBA) contents a wide range of pH (pH ≈ 2–8) for about 3 months at room of the chitosan-PNIPAM hydrogel systems, not only affect temperature. This indicates that the chitosan chains are not the values of viscosity but also alter the VPTT. Figure 7a only attached physicochemically to PNIPAM but also have shows that the values of both viscosity and VPTT are chemical linkage with PNIPAM networks. Irrespective of higher for hydrogels with greater NIPAM/chitosan ratio. the sign (either +ve or −ve), zeta potential values are less It is because that hydrogels having more PNIPAM may than four, which further reflects that these chitosan-PNI- not only enhance the hydrophilicity of hydrogels but also PAM hydrogels resulted in improved particles with better increase the incorporation of number chitosan chains in stability in acid, neutral, and alkaline aqueous solution, as the microgel. Similarly, Fig. 7b shows that the average vis- compared to unmodified PNIPAM and chitosan only. The cosity and VPTT increase with increasing the cross-linker temperature-induced sol–gel behaviour and volume phase (MBA) content in the feed. As microgels with higher cross- changes of the hydrogels were further confirmed by rheo- linker content may result in particles with less swollen/ logical studies. After detailed physicochemical investiga- compact structure, which may exhibit more resistance to tions, we conclude that most of the properties of hydrogel flow and hence increase in viscosity of the system. Moreo- such as swelling/de-swelling, volume phase transitions, ver, the proportion of chitosan that could be entrapped in electrical properties, colloidal stability, and rheological the PNIPAM matrix may also increase with cross-linker behaviour are not only influenced by changing the chemi- ratio. Importantly, from biomedical point of view ideal cal compositions of microgels but also by varying the pH/ injectable hydrogels should offer low resistance to shear temperatures of the medium. Finally, the present study sug- flow so that hydrogel can easily flow with less consump- gests that these dual-responsive microgels with adjustable tion of energy. Otherwise, they should not be suitable for properties promise important applications in the fields of biomedical applications. It can be easily realised from our biomedical and biotechnology. viscosity results that, on the average, the hydrogels under present investigation exhibit low viscosity and can be used Acknowledgments Dr. Abbas Khan is extremely grateful to the Academy of Sciences for developing countries (TWAS) and USM for potential biomedical applications. for TWAS-USM Post-Doctoral research fellowship. He also wishes to acknowledge Abdul Wali Khan University Mardan Pakistan for postdoc study leave. The authors also wish to thank the Ministry of Conclusions Science, Technology and Innovation (MOSTI) Malaysia for spon- soring the project under the Fundamental Research Grant Scheme FRGS/203/PBAHAN/6071242. The soapless emulsion free-radical copolymerization method was successfully employed to prepare six differ- ent compositions of dual-responsive chitosan-PNIPAM copolymer microgels. The swelling/de-swelling, volume References phase transitions, electrical properties, colloidal stabil- 1. 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