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Novel polymeric composites based on carboxymethyl chitosan and poly(acrylic acid): in vitro and in vivo evaluation

Novel polymeric composites based on carboxymethyl chitosan and poly(acrylic acid): in vitro and... efficient, stable, chemically crosslinked polymeric system that have pH responsive behaviour and can effectively release 5-FU in a controlled manner. Furthermore it can target colonic cancer minimizing the side effects of in vivo chemotherapy via 5-FU. Swelling and drug release studies were performed to evaluate its in vitro release behaviour. Hydrogels were also characterized by FTIR, SEM and DSC. In vitro cytocompatibility and cytotoxicity of the hydrogels were determined by MTT assay using HeLa cells. Devel- oped hydrogels were then administered to rabbits orally to evaluate its pharmacokinetic behaviour in vivo. Maximum swelling, drug loading and release were observed at pH 7.4. Similarly maximum absorption was achieved at pH 7.4 in rabbits. It is concluded that CMC-co-poly(AA) have a great potential to be used for controlled drug delivery and colonic targeting for the delivery for various anticancer drugs. 1 Introduction Electronic supplementary material The online version of this article Predominantly, the pharmaceutical researchers have been (doi:10.1007/s10856-017-5952-1) contains supplementary material, focusing to discover the novel drugs and unusual drug which is available to authorized users. administration systems, out of which controlled release * Muhammad Sohail systems have great importance. Among the diverse cate- msmarwat@gmail.com gories of polymeric systems employed by the researchers as release rate controlling barriers, hydrogels gained a Department of Pharmacy, COMSATS Institute of Information Technology, Abbottabad 22060, Pakistan remarkable attraction to be exploited for the development of a range of novel drug delivery systems [1]. The mechanisms Faculty of Pharmacy and Alternative Medicine, the Islamia University of Bahawalpur, Bahawalpur, Punjab 63100, Pakistan of drug release from the controlled drug delivery systems (DDSs) depend upon the polymeric network systems in Department of Pharmacy, University of Malakand, Lower Dir, KPK, Pakistan which the therapeutic agents are incorporated. The retention 147 Page 2 of 14 J Mater Sci: Mater Med (2017) 28:147 times of the drugs to be released from the formulations of controlled drug delivery systems. The difference of Osmotic polymeric networks are variable, which could be from few pressure between inside of the gel and its surroundings is hours to months or a year, depending upon the type of the key factor which tunes swelling of the CMC gel [17, formulations. A three-dimensional, cross-linked polymeric 18]. CMC which is a water-soluble derivative of chitosan, network system consisting of various natural or synthetic has received a noticeable attraction for its important bio- substances acquiring a high degree of adaptability due to medical and pharmaceutical applications. Additionally, high amounts of water imbibing ability are said to be as Carboxymethyl chitosan shows distinctive properties hydrogels [2]. Hydrogels exhibit the property of being including low toxicity, biocompatibility, high viscosity and flexible and soft rubbery consistency and strength, resem- better potential to be formulated as films and hydrogels bling living tissues in swollen form due to the capability to [16]. CMC hydrogel systems have shown significant swel- retain large amount of water or biological fluids under ling behaviour in basic solutions and thus are widely been various physiological conditions. Owing to the presence of studied for controlled delivery [19]. linkages both physical and chemical in hydrogel systems, Acrylic acid (AA) is a commercial polyelectrolyte super- the penetration of water to polymeric network in hydrogel, absorbent and a pH responsive monomer used in variety of the systems is only swelled up but not dissolved [2–7]. The drug delivery devices for site-specific delivery of various hydrophilic characteristic of the network is because of the drugs [20]. Polyacrylic acid formed of acrylic acid shows chemical groups present in the polymer structure which promising biocompatibility and bioadhesivness at the include hydroxyl (–OH), amidic (-–CONH–),carboxylic mucosal lining due to the presence of certain groups such as (–COOH), sulphonic (–SO H), etc. [1]. Hydrogels can be carboxylic, that interacts by hydrogen bonding with the fabricated using both the natural and synthetic polymers [6]. mucin, a glycoprotein [10]. AA is non toxic and quite The rate and extent of hydration of hydrogel systems sensitive to temperature and pH and customarily shows depend on the nature of polymeric network, aqueous swelling behaviour above pH 5. One of the main applica- environment and available hydrophilic groups in the struc- tions and potentially novel property of AA gels is its pro- tures [8]. Smart hydrogels are able to respond to the external minent role to develop sustained gastro-intestinal drug stimulus or environmental conditions like temperature and delivery system [21]. pH by using smart polymers with such potentialities [9]. 5-Fluorouracil (5-FU) the most important anti-cancerous Such hydrogel systems have been exclusively reported for agent, which has been used via oral administration for dif- the targeted and controlled delivery of a range of drug ferent types of cancers; however it has got certain severe compounds [10]. adverse effects that cannot be neglected and imminently There has been reported still an important issue and a required to be addressed. Therefore to overcome the adverse challenge for the scientists both from academia and phar- drug reactions of 5-FU, a hydrogel system is formulated to maceutical R&D sector to develop the site specific drug deliver the drug at its specific site of colon cancer which is delivery systems to effectively and in a controlled way the main purpose of study as well. This objective was deliver the active pharmaceutical ingredients to the colon achieved by pH responsive behaviour of the developed [11]. It has been determined that CRC is one of the major hydrogel and consequently controlled release of the drug cause of the death worldwide [12]. Chemotherapeutic both in vitro and in vivo. agents play a valuable role in the treatment of cancers at The main focus of the present study was to effectively different stages [13]. Paul Ehrlich, proposed an idea of deliver the most widely used anti cancer drug, 5- “magic bullet”, where the drug would only target the dis- Fluorouracil to its site of action for the treatment of colon eased cells without harming the health cells [14]. A suc- cancer and to avoid the systemic side-effects and decreased cessful colonic delivery is achieved when a drug is secured bioavailability. Owing to the most imperative pharmaceu- from the upper GIT environment and eventually releases by tical attributes of biocompatible polymers and monomers, reaching the colon [15]. this study was designed to utilise them for formulation of a Chitin, a natural polysaccharide undergoes deacetylation novel pH-sensitive carboxymethyl chitosan-co-poly(AA) and yields a derivative, chitosan, which shows better bio- hydrogel by free radical polymerization technique using N, degradability and biocompatibility. Chitosan undergoes N-methylenebisacrylamide (MBA) as a cross-inking agent. carboxymethylation yielding Carboxymethyl chitosan In this regards, by varying compositions of polymer, (CMC) by substituting some of the chitosan’s –OH groups monomer and the cross-linker, various formulation were with –CH COOH groups [16]. CMC is widely used in synthesized to investigate the swelling characteristics, controlled or sustained release and pH responsive drug thermal stability, morphological properties and drug release delivery systems. CMC is an amphoteric polyelectrolyte from the drug delivery matrices. In addition, to establish the acquiring both charges including positive and negative, and cytotoxicity level of the produced hydrogel, the cell line many researchers developed hydrogels as matrices for study on Cells (HeLa cells and Vero cells) was also J Mater Sci: Mater Med (2017) 28:147 Page 3 of 14 147 Table 1 Formulations of CMC-co-poly(AA) hydrogels performed. The comparative cell viability study on both free form of 5-FU and encapsulated in hydrogel at various Formulation code CMC (g/100 g) AA (g/100 g) MBA (g/100 g) concentrations (1, 2, 4, 6, 8, 10, 15 and 20 µg/ml) by MTT F1 0.4 20 0.4 Assay was also performed. Furthermore the developed F2 0.8 20 0.4 hydrogels CMC-co-poly(AA) was subjected to in vivo F3 1.2 20 0.4 study using animal models (rabbits) to validate the con- F4 0.8 16 0.4 trolled and targeted (pH-responsive) drug delivery. F5 0.8 24 0.4 F6 0.8 32 0.4 F7 0.8 24 0.2 2 Materials and methods F8 0.8 24 0.4 F9 0.8 24 0.6 2.1 Chemicals Carboxymethyl chitosan (CMC) was purchased from represents proposed chemical structure of developed Shangai chemicals limited, (China). Acrylic acid, Sodium hydrogel. hydroxide pellets, Potassium dihydrogen phosphate were purchased from DAEJUNG Company (Korea). Hydro- chloric acid was purchased from Scharlau, (Spain). N,N- 2.3 Fourier transform infrared spectroscopy (FT-IR) methylene(bis)acrylamide (MBA) was purchased from Fluka (Germany). Benzoylperoxide (BPO) and ethanol The produced hydrogel samples were properly crushed/ were purchased from Daejung, (Korea). Distilled water was milled for analysis. Confirmation of CMC-AA hydrogels freshly prepared in the laboratory of COMSATS institute of formation was investigated using fourier transform infrared information technology, Abbottabad. spectroscopy. FT-IR analysis of the polymer, monomer and hydrogels was performed. The FT-IR spectra were scanned 2.2 Synthesis of carboxymethyl chitosan-co-poly(AA) −1 over a range of 4500–500 cm . hydrogels Various ratios of polymer, monomer and cross-linker were 2.4 Differential scanning calorimetry (DSC) used to formulate hydrogels by chemical cross-linking method known as free radical polymerization technique. Differential scanning calorimetry (DSC) analysis of the Weighed amount of CMC was dissolved in water and produced hydrogel formulation, CMC and AA was carried stirred continuously using a magnetic stirrer until a clear out to determine the glass transition temperature (Tg) of the solution obtained. The dissolved oxygen from the polymer samples using diamond series thermal analysis system solution was removed by purging the nitrogen stream for (Perkin Elmer, USA). In the standard aluminium pan, 30 min at room temperature. Benzoyl peroxide (BPO), the sealing of 0.5 to 3 mg samples by keeping temperature initiator used was weighed and dissolved in specified between 20–500 °C at a heating rate of 20 °C/min with amount of ethanol at room temperature with continuous purging of nitrogen and the samples were analysed three stirring until the clear solution obtained and then this times. initiator solution was added slowly to the Acrylic Acid (AA), which is the monomer. Additionally, solution of N,N- methylene(bis)acrylamide (MBA), was separately prepared 2.5 Scanning electron microscopy (SEM) by adding up distilled water in specific amount. At room temperature, BPO-AA solution was slowly added to the Investigation of the structural morphology and porosity of CMC solution with continuous stirring. Finally, the MBA prepared hydrogel samples was evaluated using JEOL solution was added drop-wise to the polymer-monomer analytical SEM apparatus (JSM-5910, Japan). All the solution. The prepared solution was finally added to the samples for SEM analysis were prepared by grinding to the glass tubes which were then placed in the water bath at 55 °C optimum sized particles and then mounted on the alumi- for 4 h, followed by 60 °C for 8 h and finally 65 °C for 8 h. nium stub with double adhesive tape. Gold coating of the The glass tubes were placed at room temperature for an produced samples was carried out under argon atmosphere, hour and then the hydrogels were treated with ethanol-water using gold sputter coater. At different magnifications, (70:30) to wash un-reacted contents. The produced discs photomicrographs were obtained to carry out the morphol- were dried in vacuum oven at 40 °C for one week. Table 1 ogy studies. shows the composition of all formulations and Figure S1 147 Page 4 of 14 J Mater Sci: Mater Med (2017) 28:147 2.6 Swelling studies 2.9 Drug loading evaluation The humid weight measurements and the pH-sensitivity Extraction technique has been employed to measure the determination were conducted by immersing the weighed drug loading efficiency. Fresh buffer with pH 7.4 has been hydrogel discs into the prepared HCl solution (pH: 1.2) and used to extract the drug, 5-FU. Samples were collected and buffer solution (pH: 7.4) at room temperature. The discs analysed at different time of intervals. The process con- were drawn from the solution and tapped on the blotted tinued till the solution is left with no more drug. The cali- paper to remove excess liquid and then weighed at pre- bration curve of various 5-FU dilutions was constructed and determined time intervals from all the containers and placed used for determination of drug contents in hydrogel. The back in the same solution. Weighing process was continued analysis of drug quantification was carried out at wave- until a constant weight of the hydrogel discs was achieved. length 266 nm using UV–vis-spectrophotometer (UV-1601 Following equation was used to calculate the percent Shimadzu). All the samples were analysed in triplicate. swelling ratio 2.10 Drug release studies ðÞ Ws  Wd ð1Þ %SR ¼  100 Wd Dissolution properties were evaluated for the estimation of where, Ws is the weight of swollen disc pH-responsive targeted delivery and controlled drug release at different pH. Drug release profile was analyzed by 2.7 Sol–gel fraction immersion of each of the loaded hydrogel discs in 900 ml solutions at both low pH (1.2) and high pH (7.4) in USP Evaluation of the consumed reactants in developing the Dissolution apparatus-II (Semi-automated Dissolution Tes- CMC-co-poly(AA) hydrogels is determined by sol-gel ter with auto-sampler of Pharma Test Germany) at 37 ± 0.5 fraction. Soluble unreacted contents are generally the sol °C. Samples were withdrawn at estimated time intervals and contents of the polymerization reaction. For this purpose, assessed using UV-Spectrophotometer (UV-1601 Shi- hydrogels were cut into discs that are almost 2 mm thick madzu) at 266 nm wavelength. Maintenance of sink con- and were dried at 55 °C until the weight of the disc is dition with fresh dissolution medium was ensured after equilibrated. These dried discs were then subjected to every withdrawal of samples. extraction by placing them in the soxhlet apparatus for 4 h in deionized boiling water and again dried at same tem- 2.11 Cell cultures and cell viability studies perature until weight reaches an equilibrium. Sol and gel fraction were determined using the following equation: To perform cell cytotoxicity study, Cells (HeLa cells and Vero cells) were cultured in a medium containing RPMI- ðÞ Wi  We ð2Þ Sol fraction ¼  100 1640 supplemented with l-glutamine (2 mM), penicillin We −1 −1 (100 UmL ) and streptomycin (100 ug mL ) accom- whereas, Wi = initial weight of dried hydrogel disc before panied with 10% FBS grown in a 75 cm tissue culture flask extraction and stored in an incubator supplied with 5% CO at a We = dried hydrogel weight after extraction constant temperature of 37 C. After 80% confluency, the cells were harvested, seeded and cultured at 10,000 cells/ Gel fraction ¼ 100  solfraction ð3Þ well in a 96-well flat bottom cell culture plate and used for cell viability studies. The comparative cell viability study was conducted for 5-FU both in free form and encapsulated 2.8 Drug loading studies in hydrogel form at various concentrations (1, 2, 4, 6, 8, 10, 15 and 20 µg/ml) by MTT Assay. Cell viability study was Post-synthesis diffusion method has been adopted to load conducted in 24 well plate. 5-FU in free form was used as the drug in hydrogels. CMC/AA hydrogels discs were positive control while untreated cells were used as negative loaded with model drug 5-Fluorouracil (5-FU) by swelling control respectively. The cytotoxicity of the hydrogel was of gels in suitable medium. Immersion of the dried hydrogel determined by placing the drug loaded disk in 24 well plate discs into 1% drug solution prepared in buffer solution with containing different concentrations of 5-FU. Cell culture pH 7.4 was ensured at room temperature for 72 h. Discs medium containing RPMI-1640 supplemented with l- −1 were then collected and washed with distilled water. The glutamine (2 mM), penicillin (100 UmL ) and streptomy- −1 5FU loaded discs were initially dried at room temperature cin (100 ug mL ) was added on the top of the hydrogel followed by drying in oven at 40 °C until equilibrium is disk followed by incubation for 24 h at 37 °C. The absor- attained. bance was calculated with BioTek synergy HT (BioTek J Mater Sci: Mater Med (2017) 28:147 Page 5 of 14 147 Instruments, Inc.; Winooski, VT) at 490 nm. The cell via- concentration of the polymers and monomers in incon- bility % was calculated by using the following formula; sistent formulations. After drying in oven, formulations became light yellowish and golden yellow in colour sample Cell Viability% ¼  100 ð4Þ depending upon polymer-monomer ratios in the prepared ontrol matrices. The hydrogels with higher monomer concentra- where A and A refer to the absorbance’s of the tions were shiny, non-sticky and non-abrasive showed great sample control sample and control wells respectively. The measurements mechanical strength and the formulations with maximum were performed in triplicate. The compiled data were pre- concentration of cross-linker attained excellent strength and sented as Mean Cell Viability ± SD. stability [2]. Hydrogels with more polymeric ratio were brittle and difficult to grind. All the produced hydrogels 2.12 In vivo evaluation exhibited appropriate gelling and retained shape in swelled form as well. In vivo analysis of 5-FU in rabbit plasma was performed using an accurate, simple, sensitive and reproducible 3.2 Fourier transform infrared spectroscopy (FTIR) HPLC-UV method developed and validated by [22]. The HPLC method was used to quantify drug in rabbit plasma The FTIR spectra of polymer, monomer and developed after the administration of 5-FU loaded hydrogel discs in hydrogels are shown in Fig. 1. The FTIR spectra of car- rabbits. Healthy albino rabbits (2.0–2.6 kg) were obtained boxymethyl chitosan has shown following main peaks: the from the animal house of Faculty of Pharmacy and Alter- −1 peak at 1030 and 1063 cm represent the C–O stretch of native Medicine, the Islamia University of Bahawalpur- –CH –OH in primary alcohols and –CH–OH in cyclic Pakistan. The study protocols were evaluated and approved −1 alcohols. The peaks found at 1400 and 1600 cm show by Pharmacy Research Ethics Committee (PREC). Health symmetric and asymmetric stretch of –COO in corre- rabbits were selected and divided in to two groups of 12 sponding carboxylic acid salt. The peak existing at 2900 rabbits each (Group A and Group B). Drug solution (5-FU, −1 cm shows –C–H stretch and similar results have been 50 mg/kg) was administered to group-A (as control) using reported by [23], while working on superporous hydrogels feeding tube in the first phase. In second phase, 5-FU loa- containing poly(acrylic acid-co-acrylamide)/O-carbox- ded discs of hydrogels were administered orally to group-B ymethyl chitosan interpenetrating polymer networks. The of rabbits. After regular intervals, the blood samples (0.5 ml −1 peak appearing at 1741 cm reveals the presence of each) were drawn from the jugular vein of rabbits. Hepar- −1 –COOH group and at 1506 cm the presence of –NH inized polypropylene tubes were used for collection of group has been confirmed. Similar findings have been plasma and stored at −70 °C in ultra-low freezer (Sanyo, −1 reported by [24]. A peak at 3400 cm , occupancy of –OH Japan). After dosing in rabbits, estimation of drug concentration in rabbit plasma was performed using Microsoft Office Excel 2007 program. Pharmacokinetic parameters were calculated using Kinetica version 4.1.1 (Thermo Electron Corporation). 3 Results 3.1 Physical appearance The hydrogels were smooth in texture and upon drying a slight change in colour was observed from transparent to yellowish colour. Physical appearance of CMC-AA hydrogels synthesized by free radical polymerization is shown in Figs. S2 and S3. The polymerisation of CMC and AA occurred by crosslinking and consequently resulted in stable polymeric networks. There was observed that the few of the freshly prepared gels were transparent and few of them appeared cloudy or milky white, depending on the difference in the Fig. 1 FTIR spectra of CMC, AA and CMC-co-poly(AA) 147 Page 6 of 14 J Mater Sci: Mater Med (2017) 28:147 stretch has been unveiled, resembling with already reported shows DSC thermograms of CMC and CMC-co-poly (AA). −1 CMC spectra by [23, 25]. Peaks at 3429 cm accredited to CMC-co-poly(AA) hydrogel matrix and the polymer, car- both the hydrogen bonded (–O–H and –N–H) groups. A boxymethyl chitosan (CMC) went through DSC cycle runs −1 band at 1765 cm is assigned to the amino group (–NH to analyse the thermal behaviour at a temperature starting deformation) [26]. from 0–500 °C. In the present study, hydrogel appeared to The acrylic acid spectrum present remarkable peaks at be thermally more stable than that of individual polymer −1 1600 cm due to –C–C stretch and –C–O stretching at and monomer components. Comparatively, smaller peaks in −1 –1 1700 cm [9]. A stretching vibration at 2972 cm reveals the formulation unveiling new polymeric structure. −1 –CH presence and C–C stretch at 1296 cm . The band at In the DSC investigation, an initial endothermic peak −1 1173 cm represents –C–O stretching vibration whereas appeared at 280 °C corresponding to water loss, which has −1 –C = O stretch is represented at 1635 cm by [8]. A also been reported by [29] in CMC. The expectation of −1 broader peak at 3000 cm represents –O–H stretching and water evaporation in the endothermic peak reflects the −1 band at 2922 cm is evident of –C–H group [9, 27]. N–H physical or molecular changes in carboxymethylation. In stretching vibrations appeared between 3330 and 3060 the DSC thermogram of CMC-co-poly(AA) hydrogel, Fig. −1 −1 cm and C–N stretching at 1650 cm are the indication of 2 showed similarity of endothermic peak has been observed presence of cross-linking agent, methylene- with minor changes. Smaller endothermic peak appeared at bis-acrylamide (MBA) [27]. a slight difference from the one appeared in CMC ther- The FTIR spectrum of CMC-co-poly(AA) testifying the mogram at about 300 °C and minor fluctuations at 400 °C −1 major changes between 1200–2800 cm region, which were observed. indicates that a broad peak is formed showing interactions between CMC and AA, in which hydroxyl groups of CMC 3.4 Scanning electron microscopy (SEM) are substituted with acrylate [28] and new bonds formation between them confirming new cross-linked polymeric sys- To evaluate the morphological characteristics of the pro- tem. Thus, displaying AA grafting on the polymeric back- duced hydrogels, SEM study was carried out. Samples were bone of CMC via MBA cross-linking agent. crushed to desired size in order for better evaluation. Samples were analysed by taking micrographs ranging from 3.3 Differential scanning calorimetry (DSC) 100× to 10,000 × level. Micrographs of SEM are shown in Fig. 3. Scanning electron microscopy has been conducted DSC of pure polymer CMC and CMC-co-poly(AA) for evaluating the surface morphology of the prepared hydrogels were performed to understand the thermal beha- hydrogel formulation. Scanning Electron Microscopy is essential in regard of viour and stability of the compound and formulation. Figure 2 investigating the constitution of prepared matrices from open surfaces and cross-sectional parts by SEM. A smoother outer texture, an interconnected denser inner part has shown in the Fig. 3. With the progression of poly- merization reaction, a reduction in the solubility of poly- meric network occurs causing water molecules evaporation, leading to a compact interconnected polymeric network when the copolymerization reaction ends. Swelling cap- ability of the hydrogel matrices depends on how much the network structure is porous [22]. 3.5 Sol–gel analysis Sol–gel analysis was performed to determine the uncross- linked polymer fraction in hydrogel structure. Table S1 shows calculations for sol and gel fractions of each hydrogel formulation. The sol-gel fraction of prepared CMC-co-poly(AA) formulations were inquired to appraise the influence of increasing CMC and AA contents on sol- gel fraction shown in Table S1. The extraction process emerges the uncross-linked polymer removal of the gel Fig. 2 DSC thermograms of polymer (CMC) and hydrogel structure. The extracted gels were then dried in drying oven J Mater Sci: Mater Med (2017) 28:147 Page 7 of 14 147 Fig. 3 SEM micrographs of CMC-co-poly(AA) hydrogels at 45 °C until consistent or stable weight was achieved. Table 2 Sol–gel fraction of CMC-co-poly (AA) hydrogel Increased gel reaction reveals increasing quantity of both formulations polymer and monomer (Table 2). Serial # Formulation code Sol fraction (%) Gel fraction (%) 1 F1-A 2.44 ± 0.271 97.56 ± 2.172 3.6 Determination of drug loading efficiencies (%DLE) 2 F2-A 2.18 ± 0.191 97.82 ± 1.778 3 F3-A 1.91 ± 0.162 98.09 ± 1.872 Diffusion method was employed for entrapment of 5-FU (Table 3). The difference of weights in solutions before and 4 F4-A 1.84 ± 0.134 98.16 ± 1.694 afterwards the swelling experiments were determined by 5 F5-A 1.47 ± 0.107 98.53 ± 1.278 UV–visible spectrophotometry at 266 nm wavelength, thus 6 F6-A 1.11 ± 0.113 98.89 ± 1.008 results obtained reflect the weight of entrapped drug in the 7 F7-A 0.99 ± 0.051 99.01 ± 0.563 hydrogel. Table S2 shows the entrapped drug in the 8 F8-A 0.86 ± 0.057 99.14 ± 0.578 hydrogel discs in various formulations along with the 9 F9-A 0.45 ± 0.033 99.50 ± 0.221 release at various pH. There was observed increase in the drug loading in the formulations with increasing polymeric content. However, a decrement in the entrapment of model drug was observed by increasing both the monomer 147 Page 8 of 14 J Mater Sci: Mater Med (2017) 28:147 Table 3 Effect of reaction variables on drug loading and percent release 40 Formulation 5-FU loading g/ g % drug % release of 5- code of dry gel loading FU up to 36 h pH 1.2 pH 1.2 pH 7.4 pH 7.4 F-1 0.743 74 24.291 95.091 10 F-2 0.788 78 24.546 93.29 F-3 0.845 84 24.54 92.572 020 40 60080 100 120 140 160 T Time (hours) F-4 0.858 85 24.554 91.485 F-5 0.825 82 24.786 96.299 Fig. 4 Swelling index of F1A hydrogel at pH 1.2 and pH 7.4 F-6 0.787 78 24.912 96.747 F-7 0.799 79 21.451 93.393 12 20 F-8 0.732 73 19.103 93.456 F-9 0.656 65 17.37 89.63 10 00 80 8 and cross-linker’s content in the formulation as shown in 60 6 pH 1.2 Table S2. 40 4 pH 7.4 20 2 3.7 Effect of pH on swelling 0 10 20 30 40 Investigating the swelling behaviour of CMC-co-poly(AA) Tim me (hours) hydrogels at pH 1.2 and 7.4 indicating that hydrogel discs underwent pH dependant swelling. Studies were conducted Fig. 5 In vitro release of 5-Fluorouracil from CMC-co-poly(AA) on formulations with increasing concentrations of polymer, monomer and cross-linker. With increased concentration of polymer and keeping the other variables i.e. monomer and was observed with increasing cross-linker’s percentage as shown in Fig. S6, respectively. cross-linker ratios constant, there was observed a remark- able pH dependant swelling assigning to the ionizable functional groups. All the hydrogels formulations demon- strated a significant difference in the swelling index at both 3.9 In vitro drug release studies pH values. Dynamic swelling was evaluated with respect to time. Figure 4 shows the swelling behaviour of hydrogel Drug release was performed at pH 1.2 and pH 7.4 in order formulation at various pH. to investigate the release behaviour of 5-Fluorouracil to interpret the targeting and controlled release. Percent drug 3.8 Effect of polymer, monomer and crosslinking agent release of 5-FU from CMC-co-poly (AA) has been shown on swelling in Fig. 5 to better compare the findings. Different percen- tages of release rate were observed with increasing polymer Results showed that by increasing the concentration of CMC concentration which includes 95, 93.2 and 92.5%, respec- in hydrogel formulations, while keeping the Acrylic acid tively (Fig. S7). Likewise, percent release obtained by contents constant resulted in comparative increase in swelling increasing monomer concentration were 91.4, 96.2 and index at acidic pH 1.2. At basic pH 7.4, a drop of swelling 96.7%, whereas a decrease in drug release was observed ratio was noticed with increasing CMC concentration. This with increasing cross-linking agent in hydrogels with 93, 93 study demonstrated that hydrogel formulations exhibited and 89%, respectively (Figure S8 and S9). In vitro drug higher swelling as compared to the acidic pH 1.2. The results release behaviour of gels were carried out to predict the have been shown in Fig. S4. At higher pH values, the effect of release characteristics of CMC-co-poly(AA) hydrogels in keeping constant polymeric ratio and increasing the contents the simulated gastro-intestinal fluids [30]. 5-Fluorouracil, as of acrylic acid, the maximum swelling was observed in a model drug was loaded for evaluating its release against hydrogel formulations with higher amounts of acrylic acid as the pH stimuli. Maximum drug loading was noticed in the compared to the formulation with minimum amount of discs that showed better swelling behaviour as well. The monomer used and the results are shown in Fig. S5. A percentage release of 5-Fluorouracil studies at pH 1.2 and decrement in the swelling at both the low as well as high pH pH 7.4 has been shown in Fig. 1. Percent Release Dynamic swelling J Mater Sci: Mater Med (2017) 28:147 Page 9 of 14 147 3.10 Effect of hydrogel composition on drug release behaviour Release studies were conducted on CMC-co-poly(AA) hydrogels with varying CMC concentrations of 0.2 gm (F1), 0.4 gm (F2) and 0.6 gm (F3) in the three formulations whereas the other two variables i.e. monomer and cross- linker ratios were kept constant. Results showed a decline in the drug release percentage with increasing the polymer concentration and this phenomenon can be explained rela- tively with swelling kinetics of the hydrogel. Cumulative drug release percentage with different concentrations of carboxymethyl chitosan as a function of time is shown in Fig. S7. The release profile of 5-Fluorouracil from selected samples with increase in monomer concentration at pH 1.2 and pH 7.4 at 37 °C are presented in Fig. S8. Higher drug release percentage was observed as the AA contents were increased at both low as well as at high pH. A reduction in drug release was observed with an increase in MBA con- centration. Cumulative percent release is shown in Fig. S9. 3.11 Determination of cell viability The in vitro cytocompatibility and cytotoxicity of the hydrogels were determined by MTT assay. Figure (A) shows the in vitro cytocompatibility against Vero cells (Normal cells). Saline and distilled water (DW) were used Fig. 6 The in vitro cytocompatibility against vero cells (Normal cells) and anticancer activities of 5-FU on free and loaded form as a control with above 85% cell viability in this experi- ment. The results shown in Fig. 6 clearly represent that the S11. The Pharmacokinetic parameters of 5-FU oral solution hydrogel sample (F2, 20 µg/ml) has good cytocompatibility and hydrogels are summarized in Tables S5 and S6. The with no detectable cytotoxicity. For the determination of developed CMC based polymeric matrices could effectively cell cytotoxicity, HeLa cells previously cultured were sub- deliver the anticancer drug to the colon part of the GIT jected to MTT assay. Figure 6 shows the comparative (Tables 4–7). anticancer activities of 5-FU on free and loaded form at various concentrations. 4 Discussion 3.12 In vivo evaluation 4.1 Structural, thermal and morphological evaluation In order to evaluate the in vivo absorption of 5-FU loaded hydrogels, discs were administered to animal models (rab- FTIR spectrum of CMC-co-poly(AA) showed a different bits) and blood samples were collected up to 24 h and were pattern from carboxymethyl chitosan and acrylic acid FTIR analysed via an accurate, simple and reproducible HPLC- peaks. Appearance of new peaks in synthesized hydrogels UV method [22]. The chromatograms of 5-FU in blank and and deviation from pure ingredients spectra confirmed the spiked plasma are shown in Fig. 7. CMC-co-poly(AA) formation of new bonds in cross-linked structures. hydrogels loaded with 5-FU showed an increased plasma DSC graphs revealed that a thermally stable polymeric concentration up to 24 h. The maximum drug concentration network is synthesized by combination of carboxymethyl C observed was (Mean ± SD) (121.262 ± 5.332 μg/mL) max chitosan and acrylic acid with methylene bisacrylamide. at T of (Mean ± SD) (24.00 ± 0.00 h). The results of the max Microscopic scanning of hydrogels showed a rough and study has revealed that Cmax of hydrogel was less as wavy surface along with micropores and channels. Micro- compared to oral drug solution, so it can be expected that porous structure of hydrogel network facilitates the diffu- drug will be released in GIT up to extended period of time sion of solvent into network. Interaction of solvent (24 h). Plasma drug concentrations in rabbits are summar- molecules initiates the ioinization of functional groups at ized in Table S3 and S4 and represented in Figs. S10 and 147 Page 10 of 14 J Mater Sci: Mater Med (2017) 28:147 Fig. 7 Chromatogram of blank and spiked plasma (Rabbit) Table 4 Plasma concentrations in rabbit plasma for 5-FU solution Table 5 Plasma concentrations in rabbit plasma for hydrogel (50 mg/ (50 mg/kg) kg) Plasma concentrations of 5-FU solution Plasma concentrations in hydrogel Time Plasma Time Plasma (min.) concentration (Hrs.) concentration (Mean ± SD) (Mean ± SD) 5 0.000 ± 0.000 0.5 0.000 ± 0.000 10 73.281 ± 9.362 1 0.000 ± 0.000 15 203.672 ± 14.666 2 0.000 ± 0.000 20 253.331 ± 9.542 3 22.118 ± 23.211 25 304.251 ± 8.113 4 54.876 ± 6.362 30 236.271 ± 13.673 8 73.864 ± 7.428 40 124.671 ± 12.745 12 94.635 ± 8.263 50 85.092 ± 8.478 16 112.382 ± 6.214 60 44.876 ± 22.763 24 121.262 ± 5.332 70 0.000 ± 0.000 4.2 Sol–gel analysis various pH levels and creates repulsive forces between The study revealed that by increasing quantity of both crosslinked joints. Repulsive forces produce cavities that polymer and monomer, increased gel reaction was achieved. lead to swelling and drug release. The basis of this elevation is a polymerization reaction due J Mater Sci: Mater Med (2017) 28:147 Page 11 of 14 147 Table 6 Pharmacokinetics of 5-FU after administration of oral has been reported two general methods for loading of drugs solution to healthy rabbits of Group-A onto hydrogels, in the first method drug is added to the S. No. Pharmacokinetic parameters Oral solution (Mean ± SD) hydrogel synthesis solution; however, few serious draw- backs may occur as drug molecule with reactive sites can be 1. C (µg/ml) 304.6 ± 8.113 max chemically attached to hydrogel constituents with sub- 2. T (min) 25.00 ± 0.00 max sequent loss of efficacy. Therefore, on the basis of the 3. AUC (µg.h/ml) 155.192 ± 11.396 tot mentioned side effects which could potentially be occurred, 4. AUMC (µg.h /ml) 94.372 ± 14.63 tot the second method i.e. absorption/diffusion method was −1 5. K (min ) 0.049 ± 0.010 el employed to entrap/load 5-FU by immersing each disc in 6. t (min) 14.572 ± 1.728 1/2 1% drug solution [22]. 7. MRT (h) 0.601 ± 0.052 8. Clearance (L/min) 0.006 ± 0.0021 4.4 Effect of pH on swelling 9. V (L) 0.112 ± 0.113 It has become evident that pH has a strong effect on swelling ability due to carboxylic groups presence in monomer in the hydrogel structure. The similar swelling Table 7 Pharmacokinetics of 5-FU after administration of Hydrogels behaviour by hydrogel formulations, has also been pre- to healthy rabbits of Group-B viously reported [33]. The carboxylic groups which are S. No. Pharmacokinetic parameters Hydrogel (Mean ± SD) weak acid in nature, are mainly responsible for pH sensi- 1. C (µg/ml) 121.262 ± 5.332 max tivity of hydrogel formulations. At high pH, carboxylic 2. T (h) 24.00 ± 0.00 groups get protonated causing ionic repulsion thus leading max 3. AUC (µg.h/ml) 1996.276 ± 123.634 to swelled gels and at low pH, unprotonated carboxylic tot groups give rise to unswelled or collapsed hydrogels. 4. AUMC (µg.h /ml) 29916.372 ± 153.222 tot −1 Increase in the degree of ionization, is responsible for 5. K (h ) 0.1463 ± 0.0021 el conversion of polymeric into hydrophilic network, sup- 6. t (h) 5.2403 ± 0.2363 1/2 porting the swelling kinetics. Similar results were observed 7. MRT (h) 14.592 ± 0.236 in pH-sensitive Acrylic acid/PVA hydrogels formulations 8. Clearance (L/min) 1.162 ± 0.0362 [27]. 9. V (L) 6.625 ± 0.271 4.5 Effect of hydrogel composition on swelling to cross-linking at greater extent, thus resulting in the stable Results has shown that by increasing CMC concentration product formulation. Na-Alg/CMC hydrogels (smart super- while keeping acrylic acid contents constant, comparative absorbent) prepared using MBA as cross-linker in already increase in swelling index was observed at acidic pH 1.2. reported study by [31] showed similar findings of increasing This could be accredited to the presence of amine groups gel fraction with an increase in sodium alginate content. It which ionize at low pH, with subsequent increased swelling was observed that increasing the concentration of CMC behaviour owing to the electrostatic repulsions. It was (F1–F3), AA (F4–F6) and MBA (F7–F9), the sol fraction observed that at basic pH 7.4, a swelling ratio is dropped showed decreased whereas the gel fraction increased with increasing CMC concentration. It is assigned to the resulting in more grafting. Dergunov et al. [32] has also fact as increased number of amine groups get linked to more observed, increased in the gel fraction by increasing chit- carboxylic groups resulting in less number of free car- osan concentration in chitosan and polyvinyl pyrrolidone boxylic groups present for ionization and consequently, a hydrogel. Similarly high AA content and cross-linking decrease in swelling with increasing CMC concentration agent showed similar trend results in increased gel fraction. was observed. Similar results have been observed in [10] Similar findings were reported in pH-sensitive hydrogels of pH-sensitive chitosan-co-acrylic acid hydrogels. chitosan-co-acrylic acid for controlled release of verapamil Acrylic acid is an anionic monomer, comprising of car- by [10]. boxylic groups. It was observed that by increasing acrylic acid ratio, swelling was increased significantly at higher pH 4.3 Drug loading efficiency values is due to the presence of carboxylic groups, available for ionization, and the formulations with more acrylic acid Chemically cross-linked CMC-co-poly(AA) hydrogels were contents have shown maximum swelling due to more car- used to incorporate the drug in the network structure. There boxylic groups which after protonation causes ionic 147 Page 12 of 14 J Mater Sci: Mater Med (2017) 28:147 repulsion and increased swelling. Similar results have been higher number of ionizable groups at pH 7.4 with higher reported by [10, 27, 30, 34]. AA concentration leading to polymer chain relaxation and As crosslinking agent’s concentration is increased, a inturn providing raised swelling and drug release. Similar decrement in the swelling at both the low as well as high pH swelling and drug release behaviour was observed in pH- was observed. The reason behind this phenomenon is; sensitive cationic guar gum and poly(acrylic acid) poly- increased cross-linking causes decrease in mesh size of the electrolyte hydrogels. An increase in swelling and keto- hydrogels and reduced mesh-size conceals the carboxylic profen release with an increase in PAA component in the groups and thus hinderance in the ionization process due to gel structure was observed in the study reported by [36]. higher degree of cross-linking with decreased polymeric As discussed in swelling studies increased MBA con- chain relaxation. This gives rise to the reduced swelling centration in gels also reduced in vitro drug release. It was index with higher crosslinker;s concentration. found that increasing the cross-linking agent caused an increase in entanglement between polymer and monomer 4.6 In vitro drug release studies due to hydrogen bonding resulting in hindrance in network expansion decreasing chain relaxation eventually reduction The drug release from hydrogel formulations depends on in drug release was observed. Similar trend of cross-linker the swelling characteristics and composition of the hydrogel concentration was observed in chitosan-co-acrylic acid including polymer, monomer and cross-linking agent, hydrogel prepared by [10]. MBA being a cross-linking which in succession, is an essential parameter of chemical agent used in many polymeric networks and presented good organization of the hydrogels. Also, environmental pH biocompatibility lacking any deleterious effects on cell influences the release rate of the incorporated drug from viability and functionality. hydrogel formulations. A remarkable difference in drug release at both pH was observed; a lesser amount of the 4.8 Cell viability studies drug was released at pH 1.2 and higher amounts of release was observed at pH 7.4. There has been reported that that The results of the study demonstrates that 5-FU has dose the hydrogels showed release in phosphate buffer of pH 7.4, dependent anticancer activity and the % cell viability upto 36 h [35]. The prepared hydrogels have shown higher decreased with increasing dose concentration per well. The 5-FU release for longer period of time under sink conditions produced results also exhibited that 5-FU has high toxicity at basic pH (approximately 90% and more during 36 h) in free form as compared to the loaded form in hydrogel formulations. The cell viability study highlights the bio- which is important for anti-cancerous drug targeting to colon. As the hydrogels swell dramatically at intestinal pH compatible nature of the hydrogels. It also indicates that 5- conditions and the drug was released. Practically, these FU has retained its anticancer activity after loading into matrices could bypass the acidic gastric environment with sustained release hydrogel matrix. very low proportion of the encapsulated drug release, indicating them to be the ideal candidates for controlled and 4.9 In vivo evaluation targeted delivery system of drugs. As shown in the results of the in vivo studies in rabbits, a 4.7 Effect of hydrogel composition on in vitro drug clear difference can be observed in plasma concentrations of release oral 5-FU solution and 5-FU loaded hydrogel. The low t max value indicates the rapid absorption of pure 5-FU in solution In vitro drug release study revealed that by increasing CMC form, while t value for 5-FU loaded hydrogel was much max concentration in hydrogel composition, drug release is greater, that shows slower absorption of 5-FU from decreased. As already discussed, swelling decreases with hydrogel disc, indicating controlled release behavior. The increasing polymer content in the formulation and due to absorption of 5-FU from oral solution was rapid and less number of carboxylic groups left for ionization because achieved maximum plasma level (C ) of 304.6 ± 8.113 max they get linked to the amine groups leading to less swelling μg/mL within 25.00 ± 0.00 min. However, maximum and ultimately less release. Similar results reported by [23] plasma concentration (121.262 ± 5.332 μg/mL) after in which by increasing CMC content, a decrease in swelling administration of hydrogel containing equivalent amount of ratio of superporous hydrogels/interpenetrating networks drug was obtained after 25.00 ± 0.00 h. C of 5-FU after max was observed that ultimately accounted for a decline in administration of hydrogel containing equivalent amount of release rate. drug was less than that of oral solution. After administration It was observed that by increasing acrylic acid (mono- of hydrogel formulations, the plasma concentrations were mer) composition drug release is increased. Ranjha et al. maintained for relatively longer period of time. The elim- [10] explained that this phenomena of swelling is due to ination half-life (t ) of 5-FU loaded hydrogel and 5-FU 1/2 J Mater Sci: Mater Med (2017) 28:147 Page 13 of 14 147 8. Sohail M, Ahmad M, Minhas MU, Liaqat A, Munir A, Khalid I. oral solution was 5.2403 ± 0.2363 h and 14.572 ± 1.728 Synthesis and characterization of graft PVA composites for con- min, respectively. The elimination half-life (t ) of 5-FU 1/2 trolled delivery of Valsartan. Lat Am J Pharm. 2014;33: loaded hydrogel was comparatively greater than pure val- 1237–44. sartan solution indicating that the drug is slowly eliminated 9. Amin MCIM, Ahmad N, Halib N, Ahmad I. Synthesis and characterization of thermo-and pH-responsive bacterial cellulose/ from the body. The MRT of 5-FU in oral solution and acrylic acid hydrogels for drug delivery. Carbohydr Polym. hydrogel was 0.601 ± 0.052 h and 14.592 ± 0.236 h, 2012;88:465–73. respectively, with a large difference. AUC obtained after tot 10. Ranjha NM, Ayub G, Naseem S, Ansari MT. Preparation and administration of oral solution and hydrogel was 94.372 ± characterization of hybrid pH-sensitive hydrogels of chitosan-co- 2 2 acrylic acid for controlled release of verapamil. J Mater Sci: Mater 14.63 µg.h /ml and 29916.372 ± 153.222 µg.h /ml, respec- Med. 2010;21:2805–16. tively with a large difference. This was probably due to the 11. Paharia A, Yadav AK, Rai G, Jain SK, Pancholi SS, Agrawal GP. ability of the CMC-co-poly(AA) hydrogel to control the Eudragit-coated pectin microspheres of 5-fluorouracil for colon release of 5-FU. targeting. AAPS PharmSciTech. 2007;8:E87–E93. 12. Watanabe T, Itabashi M, Shimada Y, Tanaka S, Ito Y, Ajioka Y, et al. Japanese Society for Cancer of the Colon and Rectum (JSCCR) guidelines 2010 for the treatment of colorectal cancer. 5 Conclusion Int J Clin Oncol. 2012;17:1–29. 13. Simmonds P. Palliative chemotherapy for advanced colorectal can- cer: systematic review and meta-analysis. Br Med J. 2000;321:531. A stable cross-linked polymeric structure has been synthe- 14. Park JH, Saravanakumar G, Kim K, Kwon IC. Targeted delivery sized by a optimized solution polymerization technique. of low molecular drugs using chitosan and its derivatives. Adv Carboxymethyl chitosan based hydrogels are prepared Drug Deliv Rev. 2010;62:28–41. successfully by feed ratio of acrylic acid and methylene bis- 15. Chourasia M, Jain S. Pharmaceutical approaches to colon targeted drug delivery systems. J Pharm Pharm Sci. 2003;6:33–66. acrylamide. Significant network swelling and drug release 16. Farag RK, Mohamed RR. Synthesis and characterization of car- at higher pH while insignificant swelling and drug release at boxymethyl chitosan nanogels for swelling studies and anti- lower pH showed pH-responsive properties. Prolonged drug microbial activity. Molecules. 2012;18:190–203. release behaviour of fabricated hydrogels imparted addi- 17. Mourya V, Inamdar NN, Tiwari A. Carboxymethyl chitosan and its applications. Adv Mater Lett. 2010;1:11–33. tional benefit of controlled drug delivery at administration 18. Kumar Singh Yadav H, Shivakumar H. In vitro and in vivo eva- site. In-vivo and cell viability studies confirmed the bio- luation of ph-sensitive hydrogels of carboxymethyl chitosan for compatibility and efficacy of 5-FU loaded cross-linked intestinal delivery of theophylline. ISRN Pharm. 2012;2012:1–9 matrices. 19. Jankaew R, Rodkate N, Lamlertthon S, Rutnakornpituk B, Wichai U, Ross G, et al. “Smart” carboxymethylchitosan hydrogels crosslinked with poly (N-isopropylacrylamide) and poly (acrylic Compliance with ethical standards acid) for controlled drug release. Polym Test. 2015;42:26–36. 20. Huang Y, Lu J, Xiao C. Thermal and mechanical properties of Conflict of interest The authors declare that they have no competing cationic guar gum/poly (acrylic acid) hydrogel membranes. Polym interests. Degrad Stab. 2007;92:1072–81. 21. Hussain T, Ranjha NM, Shahzad Y. Swelling and controlled release of tramadol hydrochloride from a pH-sensitive hydrogel. Des Monomers Polym. 2011;14:233–49. References 22. Minhas MU, Ahmad M, Khan S, Ali L, Sohail M. Development and optimization of fast and new reversed-phase HPLC method for analysis of 5-fluorouracil in human and rabbit plasma. Pak Vet 1. Ganji F, Vasheghani-Farahani S, Vasheghani-Farahani E. Theo- J. 2015;35:1. retical description of hydrogel swelling: a review. Iran Polym J. 23. Yin L, Fei L, Cui F, Tang C, Yin C. Superporous hydrogels 2010;19:375–98. containing poly (acrylic acid-co-acrylamide)/O-carboxymethyl 2. Sohail M, Ahmad M, Minhas MU, Ali L, Khalid I, Rashid H. chitosan interpenetrating polymer networks. Biomaterials. Controlled delivery of valsartan by cross-linked polymeric 2007;28:1258–66. matrices: synthesis, in vitro and in vivo evaluation. Int J Pharm. 24. Chen X-G, Park H-J. Chemical characteristics of O- 2015;487:110–9. carboxymethyl chitosans related to the preparation conditions. 3. Ranjha NM, Qureshi UF. Preparation and characterization of Carbohydr Polym. 2003;53:355–9. crosslinked acrylic acid/hydroxy propyl methyl cellulose hydro- 25. Guo B, Yuan J, Yao L, Gao Q. Preparation and release profiles of gels for drug delivery. Int J Pharm Pharm Sci. 2014;6:400–10. pH/temperature-responsive carboxymethyl chitosan/P (2-(dime- 4. Hoffman AS. Hydrogels for biomedical applications. Adv Drug thylamino) ethyl methacrylate) semi-IPN amphoteric hydrogel. Deliv Rev. 2012;64:18–23. Colloid Polym Sci. 2007;285:665–71. 5. Ullah F, Othman MBH, Javed F, Ahmad Z, Akil HM. Classifi- 26. Mohamed RR, Seoudi RS, Sabaa MW. Synthesis and character- cation, processing and application of hydrogels: a review. Mater ization of cross‐linked polyethylene glycol/carboxymethyl chit- Sci Eng C. 2015;57:414–33. osan hydrogels. Adv Poly Technol. 2015;34:1–6 6. Van Tomme SR, Storm G, Hennink WE. In situ gelling hydrogels 27. Ranjha NM, Hanif M, Naz A, Shah MS, Abbas G, Afzal Z. for pharmaceutical and biomedical applications. Int J Pharm. Synthesis and characterization of cetirizine‐containing, pH‐sensi- 2008;355:1–18. tive acrylic acid/poly (vinyl alcohol) hydrogels. J Appl Polym Sci. 7. Jiang S, Liu S, Feng W. PVA hydrogel properties for biomedical 2016;133:1–8 application. J Mech Behav Biomed Mater. 2011;4:1228–33. 147 Page 14 of 14 J Mater Sci: Mater Med (2017) 28:147 28. Gao X, Cao Y, Song X, Zhang Z, Zhuang X, He C, et al. Bio- chitosan–polyvinyl pyrrolidone hydrogels. Radiat Phy Chem. degradable, pH‐responsive carboxymethyl cellulose/poly (acrylic 2005;72:619–23. acid) hydrogels for oral insulin delivery. Macromol Biosci. 33. Byun H, Hong B, Nam SY, Jung SY, Rhim JW, Lee SB, et al. 2014;14:565–75. Swelling behavior and drug release of poly (vinyl alcohol) 29. Sabaa MW, Mohamed RR, Eltaweel S, Seoudi RS. Crosslinked hydrogel cross-linked with poly (acrylic acid). Macromol Res. poly (vinyl alcohol)/carboxymethyl chitosan hydrogels for 2008;16:189–93. removal of metal ions and dyestuff from aqueous solutions. J Appl 34. Ranjha NM, Mudassir J, Sheikh ZZ. Synthesis and characteriza- Polym Sci. 2012;123:3459–69. tion of pH-sensitive pectin/acrylic acid hydrogels for verapamil 30. Minhas MU, Ahmad M, Khan S, Ali L, Sohail M. Synthesis and release study. Iranian Polym J. 2011;20:147–59. characterization of β-cyclodextrin hydrogels: crosslinked poly- 35. Minhas MU, Ahmad M, Ali L, Sohail M. Synthesis of chemically meric network for targeted delivery of 5-fluorouracil. Cellulose cross-linked polyvinyl alcohol-co-poly (methacrylic acid) hydro- Chem Technol. 2016;50:2. gels by copolymerization; a potential graft-polymeric carrier for 31. Pourjavadi A, Barzegar S, Mahdavinia G. MBA-crosslinked Na- oral delivery of 5-fluorouracil. DARU J Pharm Sci. 2013;21:44. Alg/CMC as a smart full-polysaccharide superabsorbent hydro- 36. Huang Y, Yu H, Xiao C. pH-sensitive cationic guar gum/poly gels. Carbohydr Polym. 2006;66:386–95. (acrylic acid) polyelectrolyte hydrogels: swelling and in vitro drug 32. Dergunov SA, Nam IK, Mun GA, Nurkeeva ZS, Shaikhutdinov release. Carbohydr Polym. 2007;69:774–83. EM. Radiation synthesis and characterization of stimuli-sensitive http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Materials Science: Materials in Medicine Springer Journals

Novel polymeric composites based on carboxymethyl chitosan and poly(acrylic acid): in vitro and in vivo evaluation

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Publisher
Springer Journals
Copyright
Copyright © 2017 by Springer Science+Business Media, LLC
Subject
Materials Science; Biomaterials; Biomedical Engineering; Regenerative Medicine/Tissue Engineering; Polymer Sciences; Ceramics, Glass, Composites, Natural Materials; Surfaces and Interfaces, Thin Films
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0957-4530
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1573-4838
DOI
10.1007/s10856-017-5952-1
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28823104
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Abstract

efficient, stable, chemically crosslinked polymeric system that have pH responsive behaviour and can effectively release 5-FU in a controlled manner. Furthermore it can target colonic cancer minimizing the side effects of in vivo chemotherapy via 5-FU. Swelling and drug release studies were performed to evaluate its in vitro release behaviour. Hydrogels were also characterized by FTIR, SEM and DSC. In vitro cytocompatibility and cytotoxicity of the hydrogels were determined by MTT assay using HeLa cells. Devel- oped hydrogels were then administered to rabbits orally to evaluate its pharmacokinetic behaviour in vivo. Maximum swelling, drug loading and release were observed at pH 7.4. Similarly maximum absorption was achieved at pH 7.4 in rabbits. It is concluded that CMC-co-poly(AA) have a great potential to be used for controlled drug delivery and colonic targeting for the delivery for various anticancer drugs. 1 Introduction Electronic supplementary material The online version of this article Predominantly, the pharmaceutical researchers have been (doi:10.1007/s10856-017-5952-1) contains supplementary material, focusing to discover the novel drugs and unusual drug which is available to authorized users. administration systems, out of which controlled release * Muhammad Sohail systems have great importance. Among the diverse cate- msmarwat@gmail.com gories of polymeric systems employed by the researchers as release rate controlling barriers, hydrogels gained a Department of Pharmacy, COMSATS Institute of Information Technology, Abbottabad 22060, Pakistan remarkable attraction to be exploited for the development of a range of novel drug delivery systems [1]. The mechanisms Faculty of Pharmacy and Alternative Medicine, the Islamia University of Bahawalpur, Bahawalpur, Punjab 63100, Pakistan of drug release from the controlled drug delivery systems (DDSs) depend upon the polymeric network systems in Department of Pharmacy, University of Malakand, Lower Dir, KPK, Pakistan which the therapeutic agents are incorporated. The retention 147 Page 2 of 14 J Mater Sci: Mater Med (2017) 28:147 times of the drugs to be released from the formulations of controlled drug delivery systems. The difference of Osmotic polymeric networks are variable, which could be from few pressure between inside of the gel and its surroundings is hours to months or a year, depending upon the type of the key factor which tunes swelling of the CMC gel [17, formulations. A three-dimensional, cross-linked polymeric 18]. CMC which is a water-soluble derivative of chitosan, network system consisting of various natural or synthetic has received a noticeable attraction for its important bio- substances acquiring a high degree of adaptability due to medical and pharmaceutical applications. Additionally, high amounts of water imbibing ability are said to be as Carboxymethyl chitosan shows distinctive properties hydrogels [2]. Hydrogels exhibit the property of being including low toxicity, biocompatibility, high viscosity and flexible and soft rubbery consistency and strength, resem- better potential to be formulated as films and hydrogels bling living tissues in swollen form due to the capability to [16]. CMC hydrogel systems have shown significant swel- retain large amount of water or biological fluids under ling behaviour in basic solutions and thus are widely been various physiological conditions. Owing to the presence of studied for controlled delivery [19]. linkages both physical and chemical in hydrogel systems, Acrylic acid (AA) is a commercial polyelectrolyte super- the penetration of water to polymeric network in hydrogel, absorbent and a pH responsive monomer used in variety of the systems is only swelled up but not dissolved [2–7]. The drug delivery devices for site-specific delivery of various hydrophilic characteristic of the network is because of the drugs [20]. Polyacrylic acid formed of acrylic acid shows chemical groups present in the polymer structure which promising biocompatibility and bioadhesivness at the include hydroxyl (–OH), amidic (-–CONH–),carboxylic mucosal lining due to the presence of certain groups such as (–COOH), sulphonic (–SO H), etc. [1]. Hydrogels can be carboxylic, that interacts by hydrogen bonding with the fabricated using both the natural and synthetic polymers [6]. mucin, a glycoprotein [10]. AA is non toxic and quite The rate and extent of hydration of hydrogel systems sensitive to temperature and pH and customarily shows depend on the nature of polymeric network, aqueous swelling behaviour above pH 5. One of the main applica- environment and available hydrophilic groups in the struc- tions and potentially novel property of AA gels is its pro- tures [8]. Smart hydrogels are able to respond to the external minent role to develop sustained gastro-intestinal drug stimulus or environmental conditions like temperature and delivery system [21]. pH by using smart polymers with such potentialities [9]. 5-Fluorouracil (5-FU) the most important anti-cancerous Such hydrogel systems have been exclusively reported for agent, which has been used via oral administration for dif- the targeted and controlled delivery of a range of drug ferent types of cancers; however it has got certain severe compounds [10]. adverse effects that cannot be neglected and imminently There has been reported still an important issue and a required to be addressed. Therefore to overcome the adverse challenge for the scientists both from academia and phar- drug reactions of 5-FU, a hydrogel system is formulated to maceutical R&D sector to develop the site specific drug deliver the drug at its specific site of colon cancer which is delivery systems to effectively and in a controlled way the main purpose of study as well. This objective was deliver the active pharmaceutical ingredients to the colon achieved by pH responsive behaviour of the developed [11]. It has been determined that CRC is one of the major hydrogel and consequently controlled release of the drug cause of the death worldwide [12]. Chemotherapeutic both in vitro and in vivo. agents play a valuable role in the treatment of cancers at The main focus of the present study was to effectively different stages [13]. Paul Ehrlich, proposed an idea of deliver the most widely used anti cancer drug, 5- “magic bullet”, where the drug would only target the dis- Fluorouracil to its site of action for the treatment of colon eased cells without harming the health cells [14]. A suc- cancer and to avoid the systemic side-effects and decreased cessful colonic delivery is achieved when a drug is secured bioavailability. Owing to the most imperative pharmaceu- from the upper GIT environment and eventually releases by tical attributes of biocompatible polymers and monomers, reaching the colon [15]. this study was designed to utilise them for formulation of a Chitin, a natural polysaccharide undergoes deacetylation novel pH-sensitive carboxymethyl chitosan-co-poly(AA) and yields a derivative, chitosan, which shows better bio- hydrogel by free radical polymerization technique using N, degradability and biocompatibility. Chitosan undergoes N-methylenebisacrylamide (MBA) as a cross-inking agent. carboxymethylation yielding Carboxymethyl chitosan In this regards, by varying compositions of polymer, (CMC) by substituting some of the chitosan’s –OH groups monomer and the cross-linker, various formulation were with –CH COOH groups [16]. CMC is widely used in synthesized to investigate the swelling characteristics, controlled or sustained release and pH responsive drug thermal stability, morphological properties and drug release delivery systems. CMC is an amphoteric polyelectrolyte from the drug delivery matrices. In addition, to establish the acquiring both charges including positive and negative, and cytotoxicity level of the produced hydrogel, the cell line many researchers developed hydrogels as matrices for study on Cells (HeLa cells and Vero cells) was also J Mater Sci: Mater Med (2017) 28:147 Page 3 of 14 147 Table 1 Formulations of CMC-co-poly(AA) hydrogels performed. The comparative cell viability study on both free form of 5-FU and encapsulated in hydrogel at various Formulation code CMC (g/100 g) AA (g/100 g) MBA (g/100 g) concentrations (1, 2, 4, 6, 8, 10, 15 and 20 µg/ml) by MTT F1 0.4 20 0.4 Assay was also performed. Furthermore the developed F2 0.8 20 0.4 hydrogels CMC-co-poly(AA) was subjected to in vivo F3 1.2 20 0.4 study using animal models (rabbits) to validate the con- F4 0.8 16 0.4 trolled and targeted (pH-responsive) drug delivery. F5 0.8 24 0.4 F6 0.8 32 0.4 F7 0.8 24 0.2 2 Materials and methods F8 0.8 24 0.4 F9 0.8 24 0.6 2.1 Chemicals Carboxymethyl chitosan (CMC) was purchased from represents proposed chemical structure of developed Shangai chemicals limited, (China). Acrylic acid, Sodium hydrogel. hydroxide pellets, Potassium dihydrogen phosphate were purchased from DAEJUNG Company (Korea). Hydro- chloric acid was purchased from Scharlau, (Spain). N,N- 2.3 Fourier transform infrared spectroscopy (FT-IR) methylene(bis)acrylamide (MBA) was purchased from Fluka (Germany). Benzoylperoxide (BPO) and ethanol The produced hydrogel samples were properly crushed/ were purchased from Daejung, (Korea). Distilled water was milled for analysis. Confirmation of CMC-AA hydrogels freshly prepared in the laboratory of COMSATS institute of formation was investigated using fourier transform infrared information technology, Abbottabad. spectroscopy. FT-IR analysis of the polymer, monomer and hydrogels was performed. The FT-IR spectra were scanned 2.2 Synthesis of carboxymethyl chitosan-co-poly(AA) −1 over a range of 4500–500 cm . hydrogels Various ratios of polymer, monomer and cross-linker were 2.4 Differential scanning calorimetry (DSC) used to formulate hydrogels by chemical cross-linking method known as free radical polymerization technique. Differential scanning calorimetry (DSC) analysis of the Weighed amount of CMC was dissolved in water and produced hydrogel formulation, CMC and AA was carried stirred continuously using a magnetic stirrer until a clear out to determine the glass transition temperature (Tg) of the solution obtained. The dissolved oxygen from the polymer samples using diamond series thermal analysis system solution was removed by purging the nitrogen stream for (Perkin Elmer, USA). In the standard aluminium pan, 30 min at room temperature. Benzoyl peroxide (BPO), the sealing of 0.5 to 3 mg samples by keeping temperature initiator used was weighed and dissolved in specified between 20–500 °C at a heating rate of 20 °C/min with amount of ethanol at room temperature with continuous purging of nitrogen and the samples were analysed three stirring until the clear solution obtained and then this times. initiator solution was added slowly to the Acrylic Acid (AA), which is the monomer. Additionally, solution of N,N- methylene(bis)acrylamide (MBA), was separately prepared 2.5 Scanning electron microscopy (SEM) by adding up distilled water in specific amount. At room temperature, BPO-AA solution was slowly added to the Investigation of the structural morphology and porosity of CMC solution with continuous stirring. Finally, the MBA prepared hydrogel samples was evaluated using JEOL solution was added drop-wise to the polymer-monomer analytical SEM apparatus (JSM-5910, Japan). All the solution. The prepared solution was finally added to the samples for SEM analysis were prepared by grinding to the glass tubes which were then placed in the water bath at 55 °C optimum sized particles and then mounted on the alumi- for 4 h, followed by 60 °C for 8 h and finally 65 °C for 8 h. nium stub with double adhesive tape. Gold coating of the The glass tubes were placed at room temperature for an produced samples was carried out under argon atmosphere, hour and then the hydrogels were treated with ethanol-water using gold sputter coater. At different magnifications, (70:30) to wash un-reacted contents. The produced discs photomicrographs were obtained to carry out the morphol- were dried in vacuum oven at 40 °C for one week. Table 1 ogy studies. shows the composition of all formulations and Figure S1 147 Page 4 of 14 J Mater Sci: Mater Med (2017) 28:147 2.6 Swelling studies 2.9 Drug loading evaluation The humid weight measurements and the pH-sensitivity Extraction technique has been employed to measure the determination were conducted by immersing the weighed drug loading efficiency. Fresh buffer with pH 7.4 has been hydrogel discs into the prepared HCl solution (pH: 1.2) and used to extract the drug, 5-FU. Samples were collected and buffer solution (pH: 7.4) at room temperature. The discs analysed at different time of intervals. The process con- were drawn from the solution and tapped on the blotted tinued till the solution is left with no more drug. The cali- paper to remove excess liquid and then weighed at pre- bration curve of various 5-FU dilutions was constructed and determined time intervals from all the containers and placed used for determination of drug contents in hydrogel. The back in the same solution. Weighing process was continued analysis of drug quantification was carried out at wave- until a constant weight of the hydrogel discs was achieved. length 266 nm using UV–vis-spectrophotometer (UV-1601 Following equation was used to calculate the percent Shimadzu). All the samples were analysed in triplicate. swelling ratio 2.10 Drug release studies ðÞ Ws  Wd ð1Þ %SR ¼  100 Wd Dissolution properties were evaluated for the estimation of where, Ws is the weight of swollen disc pH-responsive targeted delivery and controlled drug release at different pH. Drug release profile was analyzed by 2.7 Sol–gel fraction immersion of each of the loaded hydrogel discs in 900 ml solutions at both low pH (1.2) and high pH (7.4) in USP Evaluation of the consumed reactants in developing the Dissolution apparatus-II (Semi-automated Dissolution Tes- CMC-co-poly(AA) hydrogels is determined by sol-gel ter with auto-sampler of Pharma Test Germany) at 37 ± 0.5 fraction. Soluble unreacted contents are generally the sol °C. Samples were withdrawn at estimated time intervals and contents of the polymerization reaction. For this purpose, assessed using UV-Spectrophotometer (UV-1601 Shi- hydrogels were cut into discs that are almost 2 mm thick madzu) at 266 nm wavelength. Maintenance of sink con- and were dried at 55 °C until the weight of the disc is dition with fresh dissolution medium was ensured after equilibrated. These dried discs were then subjected to every withdrawal of samples. extraction by placing them in the soxhlet apparatus for 4 h in deionized boiling water and again dried at same tem- 2.11 Cell cultures and cell viability studies perature until weight reaches an equilibrium. Sol and gel fraction were determined using the following equation: To perform cell cytotoxicity study, Cells (HeLa cells and Vero cells) were cultured in a medium containing RPMI- ðÞ Wi  We ð2Þ Sol fraction ¼  100 1640 supplemented with l-glutamine (2 mM), penicillin We −1 −1 (100 UmL ) and streptomycin (100 ug mL ) accom- whereas, Wi = initial weight of dried hydrogel disc before panied with 10% FBS grown in a 75 cm tissue culture flask extraction and stored in an incubator supplied with 5% CO at a We = dried hydrogel weight after extraction constant temperature of 37 C. After 80% confluency, the cells were harvested, seeded and cultured at 10,000 cells/ Gel fraction ¼ 100  solfraction ð3Þ well in a 96-well flat bottom cell culture plate and used for cell viability studies. The comparative cell viability study was conducted for 5-FU both in free form and encapsulated 2.8 Drug loading studies in hydrogel form at various concentrations (1, 2, 4, 6, 8, 10, 15 and 20 µg/ml) by MTT Assay. Cell viability study was Post-synthesis diffusion method has been adopted to load conducted in 24 well plate. 5-FU in free form was used as the drug in hydrogels. CMC/AA hydrogels discs were positive control while untreated cells were used as negative loaded with model drug 5-Fluorouracil (5-FU) by swelling control respectively. The cytotoxicity of the hydrogel was of gels in suitable medium. Immersion of the dried hydrogel determined by placing the drug loaded disk in 24 well plate discs into 1% drug solution prepared in buffer solution with containing different concentrations of 5-FU. Cell culture pH 7.4 was ensured at room temperature for 72 h. Discs medium containing RPMI-1640 supplemented with l- −1 were then collected and washed with distilled water. The glutamine (2 mM), penicillin (100 UmL ) and streptomy- −1 5FU loaded discs were initially dried at room temperature cin (100 ug mL ) was added on the top of the hydrogel followed by drying in oven at 40 °C until equilibrium is disk followed by incubation for 24 h at 37 °C. The absor- attained. bance was calculated with BioTek synergy HT (BioTek J Mater Sci: Mater Med (2017) 28:147 Page 5 of 14 147 Instruments, Inc.; Winooski, VT) at 490 nm. The cell via- concentration of the polymers and monomers in incon- bility % was calculated by using the following formula; sistent formulations. After drying in oven, formulations became light yellowish and golden yellow in colour sample Cell Viability% ¼  100 ð4Þ depending upon polymer-monomer ratios in the prepared ontrol matrices. The hydrogels with higher monomer concentra- where A and A refer to the absorbance’s of the tions were shiny, non-sticky and non-abrasive showed great sample control sample and control wells respectively. The measurements mechanical strength and the formulations with maximum were performed in triplicate. The compiled data were pre- concentration of cross-linker attained excellent strength and sented as Mean Cell Viability ± SD. stability [2]. Hydrogels with more polymeric ratio were brittle and difficult to grind. All the produced hydrogels 2.12 In vivo evaluation exhibited appropriate gelling and retained shape in swelled form as well. In vivo analysis of 5-FU in rabbit plasma was performed using an accurate, simple, sensitive and reproducible 3.2 Fourier transform infrared spectroscopy (FTIR) HPLC-UV method developed and validated by [22]. The HPLC method was used to quantify drug in rabbit plasma The FTIR spectra of polymer, monomer and developed after the administration of 5-FU loaded hydrogel discs in hydrogels are shown in Fig. 1. The FTIR spectra of car- rabbits. Healthy albino rabbits (2.0–2.6 kg) were obtained boxymethyl chitosan has shown following main peaks: the from the animal house of Faculty of Pharmacy and Alter- −1 peak at 1030 and 1063 cm represent the C–O stretch of native Medicine, the Islamia University of Bahawalpur- –CH –OH in primary alcohols and –CH–OH in cyclic Pakistan. The study protocols were evaluated and approved −1 alcohols. The peaks found at 1400 and 1600 cm show by Pharmacy Research Ethics Committee (PREC). Health symmetric and asymmetric stretch of –COO in corre- rabbits were selected and divided in to two groups of 12 sponding carboxylic acid salt. The peak existing at 2900 rabbits each (Group A and Group B). Drug solution (5-FU, −1 cm shows –C–H stretch and similar results have been 50 mg/kg) was administered to group-A (as control) using reported by [23], while working on superporous hydrogels feeding tube in the first phase. In second phase, 5-FU loa- containing poly(acrylic acid-co-acrylamide)/O-carbox- ded discs of hydrogels were administered orally to group-B ymethyl chitosan interpenetrating polymer networks. The of rabbits. After regular intervals, the blood samples (0.5 ml −1 peak appearing at 1741 cm reveals the presence of each) were drawn from the jugular vein of rabbits. Hepar- −1 –COOH group and at 1506 cm the presence of –NH inized polypropylene tubes were used for collection of group has been confirmed. Similar findings have been plasma and stored at −70 °C in ultra-low freezer (Sanyo, −1 reported by [24]. A peak at 3400 cm , occupancy of –OH Japan). After dosing in rabbits, estimation of drug concentration in rabbit plasma was performed using Microsoft Office Excel 2007 program. Pharmacokinetic parameters were calculated using Kinetica version 4.1.1 (Thermo Electron Corporation). 3 Results 3.1 Physical appearance The hydrogels were smooth in texture and upon drying a slight change in colour was observed from transparent to yellowish colour. Physical appearance of CMC-AA hydrogels synthesized by free radical polymerization is shown in Figs. S2 and S3. The polymerisation of CMC and AA occurred by crosslinking and consequently resulted in stable polymeric networks. There was observed that the few of the freshly prepared gels were transparent and few of them appeared cloudy or milky white, depending on the difference in the Fig. 1 FTIR spectra of CMC, AA and CMC-co-poly(AA) 147 Page 6 of 14 J Mater Sci: Mater Med (2017) 28:147 stretch has been unveiled, resembling with already reported shows DSC thermograms of CMC and CMC-co-poly (AA). −1 CMC spectra by [23, 25]. Peaks at 3429 cm accredited to CMC-co-poly(AA) hydrogel matrix and the polymer, car- both the hydrogen bonded (–O–H and –N–H) groups. A boxymethyl chitosan (CMC) went through DSC cycle runs −1 band at 1765 cm is assigned to the amino group (–NH to analyse the thermal behaviour at a temperature starting deformation) [26]. from 0–500 °C. In the present study, hydrogel appeared to The acrylic acid spectrum present remarkable peaks at be thermally more stable than that of individual polymer −1 1600 cm due to –C–C stretch and –C–O stretching at and monomer components. Comparatively, smaller peaks in −1 –1 1700 cm [9]. A stretching vibration at 2972 cm reveals the formulation unveiling new polymeric structure. −1 –CH presence and C–C stretch at 1296 cm . The band at In the DSC investigation, an initial endothermic peak −1 1173 cm represents –C–O stretching vibration whereas appeared at 280 °C corresponding to water loss, which has −1 –C = O stretch is represented at 1635 cm by [8]. A also been reported by [29] in CMC. The expectation of −1 broader peak at 3000 cm represents –O–H stretching and water evaporation in the endothermic peak reflects the −1 band at 2922 cm is evident of –C–H group [9, 27]. N–H physical or molecular changes in carboxymethylation. In stretching vibrations appeared between 3330 and 3060 the DSC thermogram of CMC-co-poly(AA) hydrogel, Fig. −1 −1 cm and C–N stretching at 1650 cm are the indication of 2 showed similarity of endothermic peak has been observed presence of cross-linking agent, methylene- with minor changes. Smaller endothermic peak appeared at bis-acrylamide (MBA) [27]. a slight difference from the one appeared in CMC ther- The FTIR spectrum of CMC-co-poly(AA) testifying the mogram at about 300 °C and minor fluctuations at 400 °C −1 major changes between 1200–2800 cm region, which were observed. indicates that a broad peak is formed showing interactions between CMC and AA, in which hydroxyl groups of CMC 3.4 Scanning electron microscopy (SEM) are substituted with acrylate [28] and new bonds formation between them confirming new cross-linked polymeric sys- To evaluate the morphological characteristics of the pro- tem. Thus, displaying AA grafting on the polymeric back- duced hydrogels, SEM study was carried out. Samples were bone of CMC via MBA cross-linking agent. crushed to desired size in order for better evaluation. Samples were analysed by taking micrographs ranging from 3.3 Differential scanning calorimetry (DSC) 100× to 10,000 × level. Micrographs of SEM are shown in Fig. 3. Scanning electron microscopy has been conducted DSC of pure polymer CMC and CMC-co-poly(AA) for evaluating the surface morphology of the prepared hydrogels were performed to understand the thermal beha- hydrogel formulation. Scanning Electron Microscopy is essential in regard of viour and stability of the compound and formulation. Figure 2 investigating the constitution of prepared matrices from open surfaces and cross-sectional parts by SEM. A smoother outer texture, an interconnected denser inner part has shown in the Fig. 3. With the progression of poly- merization reaction, a reduction in the solubility of poly- meric network occurs causing water molecules evaporation, leading to a compact interconnected polymeric network when the copolymerization reaction ends. Swelling cap- ability of the hydrogel matrices depends on how much the network structure is porous [22]. 3.5 Sol–gel analysis Sol–gel analysis was performed to determine the uncross- linked polymer fraction in hydrogel structure. Table S1 shows calculations for sol and gel fractions of each hydrogel formulation. The sol-gel fraction of prepared CMC-co-poly(AA) formulations were inquired to appraise the influence of increasing CMC and AA contents on sol- gel fraction shown in Table S1. The extraction process emerges the uncross-linked polymer removal of the gel Fig. 2 DSC thermograms of polymer (CMC) and hydrogel structure. The extracted gels were then dried in drying oven J Mater Sci: Mater Med (2017) 28:147 Page 7 of 14 147 Fig. 3 SEM micrographs of CMC-co-poly(AA) hydrogels at 45 °C until consistent or stable weight was achieved. Table 2 Sol–gel fraction of CMC-co-poly (AA) hydrogel Increased gel reaction reveals increasing quantity of both formulations polymer and monomer (Table 2). Serial # Formulation code Sol fraction (%) Gel fraction (%) 1 F1-A 2.44 ± 0.271 97.56 ± 2.172 3.6 Determination of drug loading efficiencies (%DLE) 2 F2-A 2.18 ± 0.191 97.82 ± 1.778 3 F3-A 1.91 ± 0.162 98.09 ± 1.872 Diffusion method was employed for entrapment of 5-FU (Table 3). The difference of weights in solutions before and 4 F4-A 1.84 ± 0.134 98.16 ± 1.694 afterwards the swelling experiments were determined by 5 F5-A 1.47 ± 0.107 98.53 ± 1.278 UV–visible spectrophotometry at 266 nm wavelength, thus 6 F6-A 1.11 ± 0.113 98.89 ± 1.008 results obtained reflect the weight of entrapped drug in the 7 F7-A 0.99 ± 0.051 99.01 ± 0.563 hydrogel. Table S2 shows the entrapped drug in the 8 F8-A 0.86 ± 0.057 99.14 ± 0.578 hydrogel discs in various formulations along with the 9 F9-A 0.45 ± 0.033 99.50 ± 0.221 release at various pH. There was observed increase in the drug loading in the formulations with increasing polymeric content. However, a decrement in the entrapment of model drug was observed by increasing both the monomer 147 Page 8 of 14 J Mater Sci: Mater Med (2017) 28:147 Table 3 Effect of reaction variables on drug loading and percent release 40 Formulation 5-FU loading g/ g % drug % release of 5- code of dry gel loading FU up to 36 h pH 1.2 pH 1.2 pH 7.4 pH 7.4 F-1 0.743 74 24.291 95.091 10 F-2 0.788 78 24.546 93.29 F-3 0.845 84 24.54 92.572 020 40 60080 100 120 140 160 T Time (hours) F-4 0.858 85 24.554 91.485 F-5 0.825 82 24.786 96.299 Fig. 4 Swelling index of F1A hydrogel at pH 1.2 and pH 7.4 F-6 0.787 78 24.912 96.747 F-7 0.799 79 21.451 93.393 12 20 F-8 0.732 73 19.103 93.456 F-9 0.656 65 17.37 89.63 10 00 80 8 and cross-linker’s content in the formulation as shown in 60 6 pH 1.2 Table S2. 40 4 pH 7.4 20 2 3.7 Effect of pH on swelling 0 10 20 30 40 Investigating the swelling behaviour of CMC-co-poly(AA) Tim me (hours) hydrogels at pH 1.2 and 7.4 indicating that hydrogel discs underwent pH dependant swelling. Studies were conducted Fig. 5 In vitro release of 5-Fluorouracil from CMC-co-poly(AA) on formulations with increasing concentrations of polymer, monomer and cross-linker. With increased concentration of polymer and keeping the other variables i.e. monomer and was observed with increasing cross-linker’s percentage as shown in Fig. S6, respectively. cross-linker ratios constant, there was observed a remark- able pH dependant swelling assigning to the ionizable functional groups. All the hydrogels formulations demon- strated a significant difference in the swelling index at both 3.9 In vitro drug release studies pH values. Dynamic swelling was evaluated with respect to time. Figure 4 shows the swelling behaviour of hydrogel Drug release was performed at pH 1.2 and pH 7.4 in order formulation at various pH. to investigate the release behaviour of 5-Fluorouracil to interpret the targeting and controlled release. Percent drug 3.8 Effect of polymer, monomer and crosslinking agent release of 5-FU from CMC-co-poly (AA) has been shown on swelling in Fig. 5 to better compare the findings. Different percen- tages of release rate were observed with increasing polymer Results showed that by increasing the concentration of CMC concentration which includes 95, 93.2 and 92.5%, respec- in hydrogel formulations, while keeping the Acrylic acid tively (Fig. S7). Likewise, percent release obtained by contents constant resulted in comparative increase in swelling increasing monomer concentration were 91.4, 96.2 and index at acidic pH 1.2. At basic pH 7.4, a drop of swelling 96.7%, whereas a decrease in drug release was observed ratio was noticed with increasing CMC concentration. This with increasing cross-linking agent in hydrogels with 93, 93 study demonstrated that hydrogel formulations exhibited and 89%, respectively (Figure S8 and S9). In vitro drug higher swelling as compared to the acidic pH 1.2. The results release behaviour of gels were carried out to predict the have been shown in Fig. S4. At higher pH values, the effect of release characteristics of CMC-co-poly(AA) hydrogels in keeping constant polymeric ratio and increasing the contents the simulated gastro-intestinal fluids [30]. 5-Fluorouracil, as of acrylic acid, the maximum swelling was observed in a model drug was loaded for evaluating its release against hydrogel formulations with higher amounts of acrylic acid as the pH stimuli. Maximum drug loading was noticed in the compared to the formulation with minimum amount of discs that showed better swelling behaviour as well. The monomer used and the results are shown in Fig. S5. A percentage release of 5-Fluorouracil studies at pH 1.2 and decrement in the swelling at both the low as well as high pH pH 7.4 has been shown in Fig. 1. Percent Release Dynamic swelling J Mater Sci: Mater Med (2017) 28:147 Page 9 of 14 147 3.10 Effect of hydrogel composition on drug release behaviour Release studies were conducted on CMC-co-poly(AA) hydrogels with varying CMC concentrations of 0.2 gm (F1), 0.4 gm (F2) and 0.6 gm (F3) in the three formulations whereas the other two variables i.e. monomer and cross- linker ratios were kept constant. Results showed a decline in the drug release percentage with increasing the polymer concentration and this phenomenon can be explained rela- tively with swelling kinetics of the hydrogel. Cumulative drug release percentage with different concentrations of carboxymethyl chitosan as a function of time is shown in Fig. S7. The release profile of 5-Fluorouracil from selected samples with increase in monomer concentration at pH 1.2 and pH 7.4 at 37 °C are presented in Fig. S8. Higher drug release percentage was observed as the AA contents were increased at both low as well as at high pH. A reduction in drug release was observed with an increase in MBA con- centration. Cumulative percent release is shown in Fig. S9. 3.11 Determination of cell viability The in vitro cytocompatibility and cytotoxicity of the hydrogels were determined by MTT assay. Figure (A) shows the in vitro cytocompatibility against Vero cells (Normal cells). Saline and distilled water (DW) were used Fig. 6 The in vitro cytocompatibility against vero cells (Normal cells) and anticancer activities of 5-FU on free and loaded form as a control with above 85% cell viability in this experi- ment. The results shown in Fig. 6 clearly represent that the S11. The Pharmacokinetic parameters of 5-FU oral solution hydrogel sample (F2, 20 µg/ml) has good cytocompatibility and hydrogels are summarized in Tables S5 and S6. The with no detectable cytotoxicity. For the determination of developed CMC based polymeric matrices could effectively cell cytotoxicity, HeLa cells previously cultured were sub- deliver the anticancer drug to the colon part of the GIT jected to MTT assay. Figure 6 shows the comparative (Tables 4–7). anticancer activities of 5-FU on free and loaded form at various concentrations. 4 Discussion 3.12 In vivo evaluation 4.1 Structural, thermal and morphological evaluation In order to evaluate the in vivo absorption of 5-FU loaded hydrogels, discs were administered to animal models (rab- FTIR spectrum of CMC-co-poly(AA) showed a different bits) and blood samples were collected up to 24 h and were pattern from carboxymethyl chitosan and acrylic acid FTIR analysed via an accurate, simple and reproducible HPLC- peaks. Appearance of new peaks in synthesized hydrogels UV method [22]. The chromatograms of 5-FU in blank and and deviation from pure ingredients spectra confirmed the spiked plasma are shown in Fig. 7. CMC-co-poly(AA) formation of new bonds in cross-linked structures. hydrogels loaded with 5-FU showed an increased plasma DSC graphs revealed that a thermally stable polymeric concentration up to 24 h. The maximum drug concentration network is synthesized by combination of carboxymethyl C observed was (Mean ± SD) (121.262 ± 5.332 μg/mL) max chitosan and acrylic acid with methylene bisacrylamide. at T of (Mean ± SD) (24.00 ± 0.00 h). The results of the max Microscopic scanning of hydrogels showed a rough and study has revealed that Cmax of hydrogel was less as wavy surface along with micropores and channels. Micro- compared to oral drug solution, so it can be expected that porous structure of hydrogel network facilitates the diffu- drug will be released in GIT up to extended period of time sion of solvent into network. Interaction of solvent (24 h). Plasma drug concentrations in rabbits are summar- molecules initiates the ioinization of functional groups at ized in Table S3 and S4 and represented in Figs. S10 and 147 Page 10 of 14 J Mater Sci: Mater Med (2017) 28:147 Fig. 7 Chromatogram of blank and spiked plasma (Rabbit) Table 4 Plasma concentrations in rabbit plasma for 5-FU solution Table 5 Plasma concentrations in rabbit plasma for hydrogel (50 mg/ (50 mg/kg) kg) Plasma concentrations of 5-FU solution Plasma concentrations in hydrogel Time Plasma Time Plasma (min.) concentration (Hrs.) concentration (Mean ± SD) (Mean ± SD) 5 0.000 ± 0.000 0.5 0.000 ± 0.000 10 73.281 ± 9.362 1 0.000 ± 0.000 15 203.672 ± 14.666 2 0.000 ± 0.000 20 253.331 ± 9.542 3 22.118 ± 23.211 25 304.251 ± 8.113 4 54.876 ± 6.362 30 236.271 ± 13.673 8 73.864 ± 7.428 40 124.671 ± 12.745 12 94.635 ± 8.263 50 85.092 ± 8.478 16 112.382 ± 6.214 60 44.876 ± 22.763 24 121.262 ± 5.332 70 0.000 ± 0.000 4.2 Sol–gel analysis various pH levels and creates repulsive forces between The study revealed that by increasing quantity of both crosslinked joints. Repulsive forces produce cavities that polymer and monomer, increased gel reaction was achieved. lead to swelling and drug release. The basis of this elevation is a polymerization reaction due J Mater Sci: Mater Med (2017) 28:147 Page 11 of 14 147 Table 6 Pharmacokinetics of 5-FU after administration of oral has been reported two general methods for loading of drugs solution to healthy rabbits of Group-A onto hydrogels, in the first method drug is added to the S. No. Pharmacokinetic parameters Oral solution (Mean ± SD) hydrogel synthesis solution; however, few serious draw- backs may occur as drug molecule with reactive sites can be 1. C (µg/ml) 304.6 ± 8.113 max chemically attached to hydrogel constituents with sub- 2. T (min) 25.00 ± 0.00 max sequent loss of efficacy. Therefore, on the basis of the 3. AUC (µg.h/ml) 155.192 ± 11.396 tot mentioned side effects which could potentially be occurred, 4. AUMC (µg.h /ml) 94.372 ± 14.63 tot the second method i.e. absorption/diffusion method was −1 5. K (min ) 0.049 ± 0.010 el employed to entrap/load 5-FU by immersing each disc in 6. t (min) 14.572 ± 1.728 1/2 1% drug solution [22]. 7. MRT (h) 0.601 ± 0.052 8. Clearance (L/min) 0.006 ± 0.0021 4.4 Effect of pH on swelling 9. V (L) 0.112 ± 0.113 It has become evident that pH has a strong effect on swelling ability due to carboxylic groups presence in monomer in the hydrogel structure. The similar swelling Table 7 Pharmacokinetics of 5-FU after administration of Hydrogels behaviour by hydrogel formulations, has also been pre- to healthy rabbits of Group-B viously reported [33]. The carboxylic groups which are S. No. Pharmacokinetic parameters Hydrogel (Mean ± SD) weak acid in nature, are mainly responsible for pH sensi- 1. C (µg/ml) 121.262 ± 5.332 max tivity of hydrogel formulations. At high pH, carboxylic 2. T (h) 24.00 ± 0.00 groups get protonated causing ionic repulsion thus leading max 3. AUC (µg.h/ml) 1996.276 ± 123.634 to swelled gels and at low pH, unprotonated carboxylic tot groups give rise to unswelled or collapsed hydrogels. 4. AUMC (µg.h /ml) 29916.372 ± 153.222 tot −1 Increase in the degree of ionization, is responsible for 5. K (h ) 0.1463 ± 0.0021 el conversion of polymeric into hydrophilic network, sup- 6. t (h) 5.2403 ± 0.2363 1/2 porting the swelling kinetics. Similar results were observed 7. MRT (h) 14.592 ± 0.236 in pH-sensitive Acrylic acid/PVA hydrogels formulations 8. Clearance (L/min) 1.162 ± 0.0362 [27]. 9. V (L) 6.625 ± 0.271 4.5 Effect of hydrogel composition on swelling to cross-linking at greater extent, thus resulting in the stable Results has shown that by increasing CMC concentration product formulation. Na-Alg/CMC hydrogels (smart super- while keeping acrylic acid contents constant, comparative absorbent) prepared using MBA as cross-linker in already increase in swelling index was observed at acidic pH 1.2. reported study by [31] showed similar findings of increasing This could be accredited to the presence of amine groups gel fraction with an increase in sodium alginate content. It which ionize at low pH, with subsequent increased swelling was observed that increasing the concentration of CMC behaviour owing to the electrostatic repulsions. It was (F1–F3), AA (F4–F6) and MBA (F7–F9), the sol fraction observed that at basic pH 7.4, a swelling ratio is dropped showed decreased whereas the gel fraction increased with increasing CMC concentration. It is assigned to the resulting in more grafting. Dergunov et al. [32] has also fact as increased number of amine groups get linked to more observed, increased in the gel fraction by increasing chit- carboxylic groups resulting in less number of free car- osan concentration in chitosan and polyvinyl pyrrolidone boxylic groups present for ionization and consequently, a hydrogel. Similarly high AA content and cross-linking decrease in swelling with increasing CMC concentration agent showed similar trend results in increased gel fraction. was observed. Similar results have been observed in [10] Similar findings were reported in pH-sensitive hydrogels of pH-sensitive chitosan-co-acrylic acid hydrogels. chitosan-co-acrylic acid for controlled release of verapamil Acrylic acid is an anionic monomer, comprising of car- by [10]. boxylic groups. It was observed that by increasing acrylic acid ratio, swelling was increased significantly at higher pH 4.3 Drug loading efficiency values is due to the presence of carboxylic groups, available for ionization, and the formulations with more acrylic acid Chemically cross-linked CMC-co-poly(AA) hydrogels were contents have shown maximum swelling due to more car- used to incorporate the drug in the network structure. There boxylic groups which after protonation causes ionic 147 Page 12 of 14 J Mater Sci: Mater Med (2017) 28:147 repulsion and increased swelling. Similar results have been higher number of ionizable groups at pH 7.4 with higher reported by [10, 27, 30, 34]. AA concentration leading to polymer chain relaxation and As crosslinking agent’s concentration is increased, a inturn providing raised swelling and drug release. Similar decrement in the swelling at both the low as well as high pH swelling and drug release behaviour was observed in pH- was observed. The reason behind this phenomenon is; sensitive cationic guar gum and poly(acrylic acid) poly- increased cross-linking causes decrease in mesh size of the electrolyte hydrogels. An increase in swelling and keto- hydrogels and reduced mesh-size conceals the carboxylic profen release with an increase in PAA component in the groups and thus hinderance in the ionization process due to gel structure was observed in the study reported by [36]. higher degree of cross-linking with decreased polymeric As discussed in swelling studies increased MBA con- chain relaxation. This gives rise to the reduced swelling centration in gels also reduced in vitro drug release. It was index with higher crosslinker;s concentration. found that increasing the cross-linking agent caused an increase in entanglement between polymer and monomer 4.6 In vitro drug release studies due to hydrogen bonding resulting in hindrance in network expansion decreasing chain relaxation eventually reduction The drug release from hydrogel formulations depends on in drug release was observed. Similar trend of cross-linker the swelling characteristics and composition of the hydrogel concentration was observed in chitosan-co-acrylic acid including polymer, monomer and cross-linking agent, hydrogel prepared by [10]. MBA being a cross-linking which in succession, is an essential parameter of chemical agent used in many polymeric networks and presented good organization of the hydrogels. Also, environmental pH biocompatibility lacking any deleterious effects on cell influences the release rate of the incorporated drug from viability and functionality. hydrogel formulations. A remarkable difference in drug release at both pH was observed; a lesser amount of the 4.8 Cell viability studies drug was released at pH 1.2 and higher amounts of release was observed at pH 7.4. There has been reported that that The results of the study demonstrates that 5-FU has dose the hydrogels showed release in phosphate buffer of pH 7.4, dependent anticancer activity and the % cell viability upto 36 h [35]. The prepared hydrogels have shown higher decreased with increasing dose concentration per well. The 5-FU release for longer period of time under sink conditions produced results also exhibited that 5-FU has high toxicity at basic pH (approximately 90% and more during 36 h) in free form as compared to the loaded form in hydrogel formulations. The cell viability study highlights the bio- which is important for anti-cancerous drug targeting to colon. As the hydrogels swell dramatically at intestinal pH compatible nature of the hydrogels. It also indicates that 5- conditions and the drug was released. Practically, these FU has retained its anticancer activity after loading into matrices could bypass the acidic gastric environment with sustained release hydrogel matrix. very low proportion of the encapsulated drug release, indicating them to be the ideal candidates for controlled and 4.9 In vivo evaluation targeted delivery system of drugs. As shown in the results of the in vivo studies in rabbits, a 4.7 Effect of hydrogel composition on in vitro drug clear difference can be observed in plasma concentrations of release oral 5-FU solution and 5-FU loaded hydrogel. The low t max value indicates the rapid absorption of pure 5-FU in solution In vitro drug release study revealed that by increasing CMC form, while t value for 5-FU loaded hydrogel was much max concentration in hydrogel composition, drug release is greater, that shows slower absorption of 5-FU from decreased. As already discussed, swelling decreases with hydrogel disc, indicating controlled release behavior. The increasing polymer content in the formulation and due to absorption of 5-FU from oral solution was rapid and less number of carboxylic groups left for ionization because achieved maximum plasma level (C ) of 304.6 ± 8.113 max they get linked to the amine groups leading to less swelling μg/mL within 25.00 ± 0.00 min. However, maximum and ultimately less release. Similar results reported by [23] plasma concentration (121.262 ± 5.332 μg/mL) after in which by increasing CMC content, a decrease in swelling administration of hydrogel containing equivalent amount of ratio of superporous hydrogels/interpenetrating networks drug was obtained after 25.00 ± 0.00 h. C of 5-FU after max was observed that ultimately accounted for a decline in administration of hydrogel containing equivalent amount of release rate. drug was less than that of oral solution. After administration It was observed that by increasing acrylic acid (mono- of hydrogel formulations, the plasma concentrations were mer) composition drug release is increased. Ranjha et al. maintained for relatively longer period of time. The elim- [10] explained that this phenomena of swelling is due to ination half-life (t ) of 5-FU loaded hydrogel and 5-FU 1/2 J Mater Sci: Mater Med (2017) 28:147 Page 13 of 14 147 8. Sohail M, Ahmad M, Minhas MU, Liaqat A, Munir A, Khalid I. oral solution was 5.2403 ± 0.2363 h and 14.572 ± 1.728 Synthesis and characterization of graft PVA composites for con- min, respectively. The elimination half-life (t ) of 5-FU 1/2 trolled delivery of Valsartan. Lat Am J Pharm. 2014;33: loaded hydrogel was comparatively greater than pure val- 1237–44. sartan solution indicating that the drug is slowly eliminated 9. Amin MCIM, Ahmad N, Halib N, Ahmad I. Synthesis and characterization of thermo-and pH-responsive bacterial cellulose/ from the body. The MRT of 5-FU in oral solution and acrylic acid hydrogels for drug delivery. Carbohydr Polym. hydrogel was 0.601 ± 0.052 h and 14.592 ± 0.236 h, 2012;88:465–73. respectively, with a large difference. AUC obtained after tot 10. Ranjha NM, Ayub G, Naseem S, Ansari MT. Preparation and administration of oral solution and hydrogel was 94.372 ± characterization of hybrid pH-sensitive hydrogels of chitosan-co- 2 2 acrylic acid for controlled release of verapamil. J Mater Sci: Mater 14.63 µg.h /ml and 29916.372 ± 153.222 µg.h /ml, respec- Med. 2010;21:2805–16. tively with a large difference. This was probably due to the 11. Paharia A, Yadav AK, Rai G, Jain SK, Pancholi SS, Agrawal GP. ability of the CMC-co-poly(AA) hydrogel to control the Eudragit-coated pectin microspheres of 5-fluorouracil for colon release of 5-FU. targeting. AAPS PharmSciTech. 2007;8:E87–E93. 12. Watanabe T, Itabashi M, Shimada Y, Tanaka S, Ito Y, Ajioka Y, et al. Japanese Society for Cancer of the Colon and Rectum (JSCCR) guidelines 2010 for the treatment of colorectal cancer. 5 Conclusion Int J Clin Oncol. 2012;17:1–29. 13. Simmonds P. Palliative chemotherapy for advanced colorectal can- cer: systematic review and meta-analysis. Br Med J. 2000;321:531. A stable cross-linked polymeric structure has been synthe- 14. Park JH, Saravanakumar G, Kim K, Kwon IC. Targeted delivery sized by a optimized solution polymerization technique. of low molecular drugs using chitosan and its derivatives. Adv Carboxymethyl chitosan based hydrogels are prepared Drug Deliv Rev. 2010;62:28–41. successfully by feed ratio of acrylic acid and methylene bis- 15. Chourasia M, Jain S. Pharmaceutical approaches to colon targeted drug delivery systems. J Pharm Pharm Sci. 2003;6:33–66. acrylamide. Significant network swelling and drug release 16. Farag RK, Mohamed RR. Synthesis and characterization of car- at higher pH while insignificant swelling and drug release at boxymethyl chitosan nanogels for swelling studies and anti- lower pH showed pH-responsive properties. Prolonged drug microbial activity. Molecules. 2012;18:190–203. release behaviour of fabricated hydrogels imparted addi- 17. Mourya V, Inamdar NN, Tiwari A. Carboxymethyl chitosan and its applications. Adv Mater Lett. 2010;1:11–33. tional benefit of controlled drug delivery at administration 18. Kumar Singh Yadav H, Shivakumar H. In vitro and in vivo eva- site. In-vivo and cell viability studies confirmed the bio- luation of ph-sensitive hydrogels of carboxymethyl chitosan for compatibility and efficacy of 5-FU loaded cross-linked intestinal delivery of theophylline. ISRN Pharm. 2012;2012:1–9 matrices. 19. Jankaew R, Rodkate N, Lamlertthon S, Rutnakornpituk B, Wichai U, Ross G, et al. “Smart” carboxymethylchitosan hydrogels crosslinked with poly (N-isopropylacrylamide) and poly (acrylic Compliance with ethical standards acid) for controlled drug release. Polym Test. 2015;42:26–36. 20. Huang Y, Lu J, Xiao C. Thermal and mechanical properties of Conflict of interest The authors declare that they have no competing cationic guar gum/poly (acrylic acid) hydrogel membranes. Polym interests. Degrad Stab. 2007;92:1072–81. 21. Hussain T, Ranjha NM, Shahzad Y. Swelling and controlled release of tramadol hydrochloride from a pH-sensitive hydrogel. Des Monomers Polym. 2011;14:233–49. References 22. Minhas MU, Ahmad M, Khan S, Ali L, Sohail M. Development and optimization of fast and new reversed-phase HPLC method for analysis of 5-fluorouracil in human and rabbit plasma. Pak Vet 1. Ganji F, Vasheghani-Farahani S, Vasheghani-Farahani E. Theo- J. 2015;35:1. retical description of hydrogel swelling: a review. Iran Polym J. 23. Yin L, Fei L, Cui F, Tang C, Yin C. Superporous hydrogels 2010;19:375–98. containing poly (acrylic acid-co-acrylamide)/O-carboxymethyl 2. Sohail M, Ahmad M, Minhas MU, Ali L, Khalid I, Rashid H. chitosan interpenetrating polymer networks. Biomaterials. Controlled delivery of valsartan by cross-linked polymeric 2007;28:1258–66. matrices: synthesis, in vitro and in vivo evaluation. Int J Pharm. 24. Chen X-G, Park H-J. Chemical characteristics of O- 2015;487:110–9. carboxymethyl chitosans related to the preparation conditions. 3. Ranjha NM, Qureshi UF. Preparation and characterization of Carbohydr Polym. 2003;53:355–9. crosslinked acrylic acid/hydroxy propyl methyl cellulose hydro- 25. Guo B, Yuan J, Yao L, Gao Q. Preparation and release profiles of gels for drug delivery. Int J Pharm Pharm Sci. 2014;6:400–10. pH/temperature-responsive carboxymethyl chitosan/P (2-(dime- 4. Hoffman AS. Hydrogels for biomedical applications. Adv Drug thylamino) ethyl methacrylate) semi-IPN amphoteric hydrogel. Deliv Rev. 2012;64:18–23. Colloid Polym Sci. 2007;285:665–71. 5. Ullah F, Othman MBH, Javed F, Ahmad Z, Akil HM. Classifi- 26. Mohamed RR, Seoudi RS, Sabaa MW. Synthesis and character- cation, processing and application of hydrogels: a review. Mater ization of cross‐linked polyethylene glycol/carboxymethyl chit- Sci Eng C. 2015;57:414–33. osan hydrogels. Adv Poly Technol. 2015;34:1–6 6. Van Tomme SR, Storm G, Hennink WE. In situ gelling hydrogels 27. Ranjha NM, Hanif M, Naz A, Shah MS, Abbas G, Afzal Z. for pharmaceutical and biomedical applications. Int J Pharm. Synthesis and characterization of cetirizine‐containing, pH‐sensi- 2008;355:1–18. tive acrylic acid/poly (vinyl alcohol) hydrogels. J Appl Polym Sci. 7. Jiang S, Liu S, Feng W. PVA hydrogel properties for biomedical 2016;133:1–8 application. J Mech Behav Biomed Mater. 2011;4:1228–33. 147 Page 14 of 14 J Mater Sci: Mater Med (2017) 28:147 28. Gao X, Cao Y, Song X, Zhang Z, Zhuang X, He C, et al. Bio- chitosan–polyvinyl pyrrolidone hydrogels. Radiat Phy Chem. degradable, pH‐responsive carboxymethyl cellulose/poly (acrylic 2005;72:619–23. acid) hydrogels for oral insulin delivery. Macromol Biosci. 33. Byun H, Hong B, Nam SY, Jung SY, Rhim JW, Lee SB, et al. 2014;14:565–75. Swelling behavior and drug release of poly (vinyl alcohol) 29. Sabaa MW, Mohamed RR, Eltaweel S, Seoudi RS. Crosslinked hydrogel cross-linked with poly (acrylic acid). Macromol Res. poly (vinyl alcohol)/carboxymethyl chitosan hydrogels for 2008;16:189–93. removal of metal ions and dyestuff from aqueous solutions. J Appl 34. Ranjha NM, Mudassir J, Sheikh ZZ. Synthesis and characteriza- Polym Sci. 2012;123:3459–69. tion of pH-sensitive pectin/acrylic acid hydrogels for verapamil 30. Minhas MU, Ahmad M, Khan S, Ali L, Sohail M. Synthesis and release study. Iranian Polym J. 2011;20:147–59. characterization of β-cyclodextrin hydrogels: crosslinked poly- 35. Minhas MU, Ahmad M, Ali L, Sohail M. Synthesis of chemically meric network for targeted delivery of 5-fluorouracil. Cellulose cross-linked polyvinyl alcohol-co-poly (methacrylic acid) hydro- Chem Technol. 2016;50:2. gels by copolymerization; a potential graft-polymeric carrier for 31. Pourjavadi A, Barzegar S, Mahdavinia G. MBA-crosslinked Na- oral delivery of 5-fluorouracil. DARU J Pharm Sci. 2013;21:44. Alg/CMC as a smart full-polysaccharide superabsorbent hydro- 36. Huang Y, Yu H, Xiao C. pH-sensitive cationic guar gum/poly gels. Carbohydr Polym. 2006;66:386–95. (acrylic acid) polyelectrolyte hydrogels: swelling and in vitro drug 32. Dergunov SA, Nam IK, Mun GA, Nurkeeva ZS, Shaikhutdinov release. Carbohydr Polym. 2007;69:774–83. EM. Radiation synthesis and characterization of stimuli-sensitive

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Published: Aug 19, 2017

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