International Journal of Biological Macromolecules 65 (2014) 524–533

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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac

Polysaccharide based hydrogels as controlled drug delivery system for GIT cancer Baljit Singh ∗ , R. Bala Department of Chemistry, Himachal Pradesh University, Shimla 171005, India

a r t i c l e

i n f o

Article history: Received 14 August 2013 Received in revised form 31 January 2014 Accepted 3 February 2014 Available online 11 February 2014 Keywords: Methotrexate Slow drug delivery Hydrogels Polysaccharide

a b s t r a c t Keeping in view the anticancer nature of psyllium and methotrexate, psyllium, if suitably tailored to prepare the hydrogels, can act as the potential candidate for the slow drug delivery systems for GI tract. Therefore, the present study is an attempt to modify the psyllium for developing the hydrogels meant for delivery of methotrexate in controlled and sustained manner. The present article discusses the synthesis and characterization of the psy-cl-poly(AAm-co-AAc) hydrogels prepared by chemical crosslinking methods. These hydrogels have been characterized with SEMs, FTIR, TGA, XRD and swelling studies. The swelling and drug release behavior of hydrogels has been determined in solution of different pH. The results indicate that the drug release from the polymeric matrix follows Fickian diffusion mechanism in pH 7.4 buffer. © 2014 Elsevier B.V. All rights reserved.

1. Introduction The human epidemiology indicates an inverse correlation between high fiber consumption and lower colon cancer rate [1]. Both, dietary fiber psyllium and methotrexate exert beneficial effects, in case of colon cancer [1,2]. However, the higher doses of methotrexate can cause toxic effects to cells of gastrointestinal mucosa. Hence, some slow delivery system, loaded with anticancer agent is strongly admirable in cancer therapy. Psyllium based hydrogels will not only reduce the side effects of the drug but matrix degradation in the colon will enhance the therapeutic potential [3]. In the present studies, psyllium-crosslinked-poly(acrylamide-coacrylic acid) [psy-cl-poly(AAm-co-AAc)] hydrogels were prepared and used to study the slow release profile of methotrexate from the drug loaded hydrogels. The hydrogel based drug delivery carrier and mode of action of psyllium/methotrexate as anticancer agent is briefly discussed. Hydrogels are a class of crosslinked polymers that, due to their hydrophilic nature, can absorb large quantities of water. Since the equilibrium swelling capacity of a hydrogel is a balance between swelling and elastic forces, hydrogels with different swelling capacities can be designed by modulating the contribution of individual forces. Certain hydrogels respond to the changes in environmental factors by altering their swelling behavior [4]. The volume phase transitions as a response to different stimuli make these materials

∗ Corresponding author. Tel.: +91 1772830944; fax: +91 1772633014. E-mail address: [email protected] (B. Singh). http://dx.doi.org/10.1016/j.ijbiomac.2014.02.004 0141-8130/© 2014 Elsevier B.V. All rights reserved.

interesting objects of scientific observations and useful materials for use in drug delivery systems [5]. The hydrogels can be prepared by grafting/crosslinking of vinyl monomers onto polysaccharides to deliver drugs locally to the specific sites in the gastrointestinal tract (GIT) [6,7]. The condition of grafting reactions can be controlled and graft copolymer with desired properties may be obtained [8,9]. Hence, this technique of graft copolymerization provides an opportunity to prepare tailor-made grafted chains of desired properties by using suitable monomers. Graft copolymerization of vinyl monomers from their binary mixtures has many advantages. It incorporates different types of polymer chains containing various functional groups in the structure of backbone polymers. The grafting of binary monomer mixture of acrylamide (AAm) and acrylic acid (AAc) onto natural polymers enhanced hydrophilicity many times. Grafting opens up the cellulose matrix and grafted monomers on the backbone surfaces, improves its water interaction and molecular retention characteristics significantly by alone or more dramatically by grafting their binary mixture. Grafted functional groups onto cellulose afford efficient absorbent sites for small molecules and ions [10,11]. Both AAm and AAc are hydrophilic monomers. Poly(AAc) based hydrogels are hydrophilic, pH responsive and have potential to deliver the drug to the colon. These hydrogels have special bioadhesive property which makes them to stick the mucosal lining of small intestine which after swelling releases the loaded drug. These hydrogels have also been reported to have biocompatibility and antibacterial properties [12]. Psyllium is mainly used as a dietary fiber. It is a complex mixture of polysaccharides with different functions and activities during passage through the gastrointestinal tract. It has various

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Table 1 Optimum reaction parameters for the synthesis of psy-cl-poly(AAm-co-AAc) hydrogels by chemical method. S. No.

[AAc] × 101 (mol/L)

[AAm] × 101 (mol/L)

[APS] (mmol/L)

[N,N -MBAAm] (mmol/L)

Psyllium (g)

Thickness of the sample (cm)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

1.46 2.92 4.38 5.84 7.30 2.92 2.92 2.92 2.92 2.92 2.92 2.92 2.92 2.92 2.92 2.92 2.92 2.92 2.92 2.92

7.05 7.05 7.05 7.05 7.05 1.41 2.82 4.23 5.64 7.05 7.05 7.05 7.05 7.05 7.05 7.05 7.05 7.05 7.05 7.05

4.38 4.38 4.38 4.38 4.38 4.38 4.38 4.38 4.38 4.38 4.38 8.76 13.14 17.52 21.90 8.76 8.76 8.76 8.76 8.76

6.49 6.49 6.49 6.49 6.49 6.49 6.49 6.49 6.49 6.49 6.49 6.49 6.49 6.49 6.49 6.49 12.98 19.47 25.96 32.45

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

0.487 0.568 0.580 0.610 0.642 0.531 0.558 0.533 0.552 0.568 0.568 0.650 0.568 0.542 0.545 0.650 0.612 0.632 0.528 0.640

pharmaceutical and food applications [13–17]. Possible mechanisms by which psyllium fibers may inhibit colon tumorigenesis include dilution and adsorption of any carcinogens and/or promoters contained within the intestinal lumen, the modulation of colonic microbial metabolic activity, and biological modification of intestinal epithelial cells [18,19]. Dietary fibers not only bind carcinogens, bile acids, and other potential toxins but also essential nutrients, such as minerals, which can inhibit the carcinogenic process. Fermentation of fibers within the large bowel results in the production of n-butyrate [20–22]. The presence of n-butyrate in the distal colon may be important in the prevention of colon cancer because the majority of tumors in both humans and experimentally induced rodent cancer models occur in the distal colon [23,24]. Methotrexate is an anti-metabolite and anti-folate drug which is used as an antineoplastic agent. It acts by inhibiting the metabolism of folic acid. It competitively and reversibly inhibits dihydrofolate reductase (DHFR), an enzyme that participates in the tetrahydrofolate synthesis. Also, folate is needed for purine base synthesis, so all purine synthesis will be inhibited. Methotrexate, therefore, inhibits the synthesis of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), thymidylates, and proteins [2]. In our earlier work, our research group has reported the psyllium based hydrogels meant for metal ion sorption study and drug delivery applications by using single vinylic monomer [25,26]. However in the present study an attempt has been made to use the binary monomer mixture for making the hydrogels. It is pertinent to mention here that the release of drug is closely related to the swelling characteristics of the hydrogels, which in turn, is a key function of chemical architecture of the hydrogels. The properties of the hydrogel, which make it favorable for use in various applications, arise mostly from its chemical composition. The advantage of binary monomer based hydrogels system is that the swelling and drug release properties of gel can be better controlled by varying the relative concentration of the monomers involved. Further, by choosing the suitable monomers, the drug delivery systems can be made site specific in gastrointestinal tract (GIT). 2. Experimental 2.1. Materials and methods Plantago psyllium mucilage was obtained from Sidhpur Sat Isabgol Factory, Gujarat, India. Acrylic acid (AAc) was obtained from Merck-Schuchardt, Germany. Acrylamide (AAm) was obtained

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.006 0.003 0.0 0.005 0.008 0.003 0.003 0.003 0.003 0.003 0.003 0.017 0.008 0.008 0.005 0.017 0.003 0.010 0.28 0.01

Amount of water uptake after 24 h (g/g of gel) 18.95 24.19 18.80 16.58 16.24 29.79 31.24 29.56 24.57 24.19 24.19 18.53 16.84 15.21 10.32 18.53 8.62 9.42 8.74 8.50

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.23 0.72 0.77 0.86 0.14 2.01 1.06 0.40 1.19 0.72 0.72 1.31 1.16 1.90 0.26 1.31 0.35 1.05 0.48 0.23

from Sisco Research Laboratories, Mumbai, India. Ammonium persulfate (APS) and N,N -methylenebisacrylamide (N,N -MBAAm) were obtained from S.D. Fine, Mumbai, India. Methotrexate drug was obtained from Cipla Limited, Goa, India. 2.2. Synthesis of psy-cl-poly(AAm-co-AAc) hydrogels The polymerization reaction was carried out with 1 g of psyllium, definite concentration of monomers (AAm and AAc), initiator (APS) and crosslinker (N,N -MBAAm) in the aqueous reaction system at 65 ◦ C temperature for 2 h. When the reaction completed, the polymers thus formed, were stirred in 1:1 mixture of distilled water and ethanol for 2 h to remove the soluble fractions left in the polymers. These polymers were then dried in oven at 40 ◦ C. These polymers were named as [psy-cl-poly(AAmco-AAc)] hydrogels/polymers. On the basis of swelling of the hydrogels and surface consistency maintained by the hydrogels after 24 h swelling, the optimum reaction parameters were evaluated for the synthesis of psy-cl-poly(AAm-co-AAc) hydrogels by varying [AAm] from 1.41 × 10−1 to 7.05 × 10−1 mol/L, [AAc] from 1.46 × 10−1 to 7.30 × 10−1 mol/L, [APS] from 4.38 to 21.90 mmol/L, and [N,N -MBAAm] from 6.49 to 32.45 mmol/L. The optimum reaction parameters for the synthesis of hydrogels were obtained as: 1 g of psyllium, 2.92 × 10−1 mol/L of AAc, 7.05 × 10−1 mol/L of AAm, 8.76 mmol/L of APS, and 6.49 mmol/L of N,N -MBAAm (Table 1). The psy-cl-poly(AAm-co-AAc) hydrogels prepared at optimum reaction conditions through chemical were used for further studies such as swelling behavior of hydrogels in solution of different pH, salt and temperature and in vitro release dynamics of model drug methotrexate from the drug loaded hydrogels. 2.3. Characterization Cryo SEMs were taken on Zeiss LEO 435 VP Microscope. Before the Cryo SEMs analysis, the samples were immersed in distilled water at 37 ◦ C for 24 h. After this, the samples were immediately frozen by immersing in liquid nitrogen and were kept in it for 30 min. Thereafter, the frozen samples were lyophilized on a freeze dryer at −40 ◦ C for 8 h. The freeze dried samples were coated with gold by using a sputter coater of JEOL (Fine Coat Ion Sputter, JFC1100), for reducing the charging effects while the SEM visualization and then examined by SEM. FTIR spectra of polymer samples were recorded in KBr pellets on Nicolet 5700 FTIR THERMO. TGA of

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B. Singh, R. Bala / International Journal of Biological Macromolecules 65 (2014) 524–533

Fig. 1.1. Cryo SEMs of psyllium.

polymer samples were recorded on Perkin Elmer (Pyris Diamond) apparatus at heating rate of 10 ◦ C/min in air. XRD patterns of powder samples were obtained Siemens Diffractometer D5000, using Cu K␣ radiation. 2.4. Swelling studies The swelling studies of the psy-cl-poly(AAm-co-AAc) polymers were carried out in distilled water by gravimetric method [27]. Known weight of polymers were taken and immersed in excess of solvent for different time intervals at 37 ◦ C and then polymers were removed, wiped with tissue paper to remove excess of solvent and weighed immediately. The difference in weight gave the gain in weight at different time intervals. The swelling of the hydrogels were studied in distilled water, buffer solution of pH 2.2 and pH 7.4, and 0.9% NaCl solution. The buffer solutions of pH 2.2 and pH 7.4 were prepared by the method reported in Pharmacopeia of India [28]. 2.5. Drug release studies The release profile of model drug methotrexate from the drug loaded psy-cl-poly(AAm-co-AAc) hydrogels was determine in distilled water, pH 2.2 buffer and pH 7.4 buffer. All the studies were carried out in triplicate. Preparation of calibration curves, drug loading to hydrogels, and drug release from the drug loaded hydrogels is discussed elsewhere [27]. The calibration curves were prepared by taking the absorbance of number of the standard solutions of references substance at the concentrations encompassing the sample concentrations were measured on the UV Visible Spectrophotometer (Carry 100 Bio, Varian) and calibration graphs were constructed. The calibration graphs for model drug methotrexate were prepared at max 302 nm in distilled water, at max 303 nm in pH 2.2 buffer, and at max 302 nm in pH 7.4 buffer. The loading of a drug into the hydrogels was carried out by swelling equilibrium method. In vitro release studies of the drug were carried out by placing dried and drug loaded sample in definite volume of releasing medium for 45 min at 37 ◦ C and then sample was transferred to the fresh releasing medium. The release of drug from the polymer samples was measured from the calibration graphs. The swelling of the polymers and drug release profile from the drug loaded polymers have been classified into three types of diffusion mechanisms on the basis of relative rate of diffusion of water into polymer matrix and rate of polymer chain relaxation [29–32]. These mechanisms can be determined by using Eq. (1). Mt = kt n M∞

(1)

where, ‘k’ and ‘n’ are constants. The normal Fickian diffusion is characterized by ‘n’ = 0.5, while Case II diffusion by ‘n’ = 1.0. In case, where value of ‘n’ is between 0.5 and 1.0, it indicates a mixture of Fickian and Case II diffusion, which is usually called non-Fickian or anomalous diffusion. Mt /M∞ is the fractional release of drug in time ‘t’, ‘k’ is the constant characteristic of the drug–polymer system, and ‘n’ is the diffusion exponent characteristic of the release mechanism. Mt and M∞ are drug released at time ‘t’ and at equilibrium swelling respectively. 3. Results and discussion 3.1. Characterization 3.1.1. Cryo-scanning electron microscopy The cryo SEM images at different magnifications have shown the change in porosity and morphology of the psy-cl-poly(AAmco-AAc) hydrogels (Figs. 1.1 and 1.2). Low-temperature scanning electron microscopy has been used to investigate the microstructure of gel samples. By using the cryo-technique, nucleation and crystallization of water can be prevented, and the spatial structure of the gels can be preserved for further investigation [33]. The microstructure of psyllium gel has been analyzed by using cryoSEM for detailed understanding of the properties of the psyllium gel. The microstructure of gel investigated by cryo-SEM at different calcium ion concentrations by Guo and co-workers [34]. They have found that after adding Ca2+ , the gel structure was composed of small aggregates and changed to particulate gel from fine stranded gel. 3.1.2. Fourier transform infrared (FTIR) spectroscopy FTIR spectra of psyllium and psy-cl-poly(AAm-co-AAc) hydrogels are presented in Fig. 2. In FTIR spectra of psyllium, the broad absorption band around 3430.6 cm−1 has been observed due to OH stretching along with some complex bands in the region 1200–1030 cm−1 due to C O and C O C stretching vibrations which are the characteristic of the natural polysaccharide. In addition, bands at about 1742 cm−1 is due to the C O stretching of galacturonic acid present in the side chains of psyllium polysaccharides. In case of FTIR spectra of chemically prepared psy-cl-poly(AAm-co-AAc) polymers, besides the absorption bands present in the psyllium, absorption bands at 1731.5 cm−1 and 1664.3 cm−1 have been observed due to C O stretching of carboxylic and amide groups respectively present in the polymer matrix. The absorption bands at 1600 cm−1 due N–H in plane bending, at 1456.2 cm−1 due to CH2 scissor vibrations have been observed. The bands at 2925.8 and 2843.8 cm−1 due to CH2 asymmetric and symmetric vibrations have also been observed. These

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Fig. 1.2. Cryo SEMs of psy-cl-poly(AAm-co-AAc) hydrogels prepared by chemical method.

Fig. 2. FTIR of (a) psyllium and (b) psy-cl-poly(AAm-co-AAc) hydrogels prepared by chemical method.

peaks have indicated the presence of poly(AAm) and poly(AAc) in the composition of the hydrogels [35,36]. 3.1.3. Thermogravimetric analysis (TGA) The results of thermogravimetry/differential thermal analysis (TG/DTA) of psyllium and psy-cl-poly(AAm-co-AAc) polymers prepared by chemical method are presented in Fig. 3. The decomposition temperature per 10% weight loss along with the initial decomposition temperature (IDT) and final decomposition temperature (FDT) for each sample is given in Table 2. In each case, weight loss due to entrapped moisture was ignored and IDT was taken as the temperature where the actual degradation of the polymer matrix started. In case of psyllium, initial 7% weight loss occurred between 28 ◦ C and 100 ◦ C temperature. This shows that

the psyllium has 7% bounded water. The IDT and FDT are observed at 220 ◦ C and 548 ◦ C (residue left = 1.80%). In general, decomposition of polysaccharides consists of four phases; each phase corresponds to the characteristic decomposition pattern of that polysaccharide. These include desorption of physically absorbed water, removal of structural water (dehydration reactions), depolymerization accompanied by the rupture of C O and C C bonds in the ring units resulting in the evolution of CO, CO2 and H2 O and finally the formation of polynuclear aromatic and graphitic carbon structures. In case of psyllium, these decomposition processes occur between 28 ◦ C and 548 ◦ C temperature. Three stages decomposition mechanism has been observed from the TGA of psyllium. These stages started at 220 ◦ C (residue left = 89%), 308 ◦ C (residue left = 39%) and 441 ◦ C (residue left = 15%) respectively. It is

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TGA psyllium DTA psyllium TGA hydrogel DTA hydrogel

100

500

(a)

150

400

60 90 40

% Intensity

120

DTA (μV)

Weight loss (%)

80

180

60

300

200

30

20

(b) 100

0 0 0

100

200

300

400

500

600

0

Temperature ( C)

10

20

Fig. 3. TGA and DTA of (a) psyllium and (b) psy-cl-poly(AAm-co-AAc) hydrogels.

30

Angle in degree (2θ)

40

50

Fig. 4. XRD pattern of (a) psyllium and (b) psy-cl-poly(AAm-co-AAc) hydrogels.

also observed from this observation that about 50% of the polymer decomposition occurred during the first stage of decomposition. The IDT and FDT of psy-cl-poly(AAm-co-AAc) polymers are found at 200 ◦ C and 626 ◦ C (residue left = 0.2%), respectively. In case of these polymers, initial 10.2% weight loss occurred between 28 ◦ C and 100 ◦ C temperature. The decomposition of polymer matrix observed in three stages, where the first stage of decomposition started at 200 ◦ C (residue left = 86%) and second and third stage started at 313 ◦ C (residue left = 60%) and at 539 ◦ C (residue left = 23.7%) respectively. The thermal behavior at temperatures higher than 340 ◦ C is mainly attributed to the process accompanying the main chain scission [37]. DTA curves of psyllium and psy-cl-poly(AAm-co-AAc) polymers showed endothermic peaks at 80 ◦ C and 64 ◦ C respectively, due to endothermic release of water from polymers. Additionally, presence of exothermic peaks, for psyllium (at 440◦ C and 548 ◦ C) and for psy-cl-poly(AAm-coAAc) polymers (at 617.5 ◦ C), have been observed which suggested exothermic decomposing of the polymers [38]. DTG analysis of polymers was studied as a function of rate of weight loss (␮g/min) vs temperature (◦ C). It gives maximal rates of samples degradation in air environment. The temperature of maximum rate of weight loss (Tmax ) have been observed at 299 ◦ C (7780 ␮g/min) and 275 ◦ C (559.5 ␮g/min) and 278 ◦ C (1372.9 ␮g/min), respectively for psyllium and psy-cl-poly(AAm-co-AAc) polymers. DTG analysis showed that the thermal degradation psyllium occurred at higher rate than the hydrogels.

The properties of the hydrogels, which make them favorable for use in various pharmaceutical as well as medicinal purposes, arise mostly from their crosslinked structure. The crosslinked structure of the gel is determined by the nature of monomers, method of preparation and nature of crosslinking agent. In order to understand the crosslinked structure of the gel, the most common approach is to study the swelling behavior of the hydrogels. Knowledge of the swelling characteristics of the gel is the first step in understanding the network structure of the gel and its capacity to function as a drug delivery carrier. The transfer of the solute is controlled by the swelling of the gel. Once this information is known, the gel can be designed by varying mesh size, which enable diffusion of required drug in specific manner. In the present work, swelling studies of the hydrogels have been carried out in order to find out the optimum reaction parameters for the synthesis of hydrogels meant for drug delivery applications. The optimum reaction parameters for the synthesis of hydrogels have been determined by varying the feed concentration of monomers, initiator, and crosslinker during grafting/crosslinking copolymerization reactions. The amount of water uptake by the polymer matrix was studied in distilled water at 37 ◦ C after fixed interval of 45 min for 450 min and thereafter that the swelling was taken after 24 h.

3.1.4. X-ray diffraction (XRD) analysis The XRD spectra of psyllium, psy-cl-poly(AAm-co-AAc) polymers are presented in Fig. 4. In all the cases broad peaks of less intensity have been observed which indicates the amorphous nature of the polymers. Similar observation has been made by Xiao and co-workers [39] in case of XRD of natural polysaccharide konjac glucomannan and its blend with poly(AAm). Mishra and coworkers [40] have also reported amorphous nature of okra gum modified with poly(AAm), that is, okra-cl-poly(AAm) crosslinked hydrogels.

3.2.1. Effect of feed [AAc] on swelling of hydrogels The effect of feed [AAc] on the structure of polymeric networks and crosslinking density was determined by studying the swelling behavior of hydrogels synthesized with different feed [AAc] in the reaction system during polymerization reaction. The results of swelling of hydrogels are presented in Fig. 5.1. The swelling of the hydrogels first increased and then decreased with increase in feed AAc concentration during the polymerization reaction. This may be due to the reason that after formation of networks of optimum pore size, further increase in monomer concentration enhances the homopolymerization reaction over the graft copolymerization

3.2. Swelling studies

Table 2 Thermogravimetric analysis of psy-cl-poly(AAm-co-AAc) hydrogels prepared by chemical methods. Sample

Psyllium Psy-cl-poly (AAm-co-AAc) hydrogels

IDT (◦ C)

220 200

FDT (◦ C)

548 626

DT (◦ C) at every 10% weight loss

Residue (%)

10

20

30

40

50

60

70

80

90

100

192 98

278 244

294 275

298 315

301 366

306 420

369 493

424 563

460 604

548 626

1.80 0.2

B. Singh, R. Bala / International Journal of Biological Macromolecules 65 (2014) 524–533

Amount of water uptake (g/g of the gel)

24

-1

[AAc] 1.46×10 mol/L -1 [AAc] 2.92×10 mol/L -1 [AAc] 4.38×10 mol/L -1 [AAc] 5.84×10 mol/L -1 [AAc] 7.30×10 mol/L

20

16

12

8

4

0 0

45

90

135 180 225 270 315 360 405 450

Time (min.)

1440

Fig. 5.1. Effect of [AAc] on swelling of psy-cl-poly(AAm-co-AAc) hydrogels. [Psyllium = 1 g, [AAm] = 7.05 × 10−1 mol/L, [APS] = 4.38 mmol/L, [N,N MBAAm] = 6.49 mmol/L].

529

reaction. It also increases the viscosity of the reaction medium which hinders the movement of free radicals in the reaction system that led to increase in crosslinking density and decrease in pore size which has decreased the amounts of water uptake by the hydrogels. The increase in monomer concentration in the reaction has also enhanced the chances of chain transfer to monomer molecule that is also responsible for increase in homopolymeriztion. Pourjavadi and co-workers [41] have reported the similar trends in swelling of hydrogels prepared by graft copolymerization of AAc onto kappa-carrageenan in an aqueous medium using a persulfate initiator and a bifunctional hydrophilic crosslinker. The hydrogels prepared with 2.92 × 10−1 mol/L of [AAc] have shown highest water uptake (24.19 ± 0.72 g/g of gel). This [AAc] was taken as the optimum concentration for further synthesis of hydrogels. The values of diffusion exponent ‘n’ and gel characteristics constant ‘k’ have been evaluated from the slope and intercept of the plot ln Mt /M∞ versus ln ‘t’ to determine the mechanism of swelling. The swelling of polymeric networks occurred through a non-Fickian diffusion mechanism. The values of diffusion coefficients for swelling of polymers prepared with different [AAc] are given in Table 3.

3.2.2. Effect of feed [AAm] on swelling of hydrogels Similarly, the effect of [AAm] on hydrogel structure was determined by taking the swelling of psy-cl-poly(AAm-co-AAc) hydrogels prepared with different [AAm]. The results of swelling

Table 3 Results of diffusion exponent ‘n’, gel characteristic constant ‘k’ and various diffusion coefficients for the swelling kinetics of psy-cl-poly(AAm-co-AAc) hydrogels prepared by chemical method. S No.

Parameters

Thickness of the sample (cm)

Diffusion exponent ‘n’

Gel characteristic constant ‘k’ × 102

Diffusion coefficients (cm2 /min) Initial Di × 103

Late time DL × 103

Effect of [AAc] 1 2 3 4 5

1.46 × 10−1 mol/L 2.92 × 10−1 mol/L 4.38 × 10−1 mol/L 5.84 × 10−1 mol/L 7.30 × 10−1 mol/L

0.487 0.568 0.580 0.610 0.642

± ± ± ± ±

0.006 0.003 0.0 0.005 0.008

0.733 0.759 0.716 0.722 0.705

0.584 0.584 0.467 0.599 0.650

3.91 5.00 5.18 5.73 5.90

3.61 4.58 4.78 6.04 5.85

Effect of [AAm] 1 2 3 4 5

1.41 × 10−1 mol/L 2.82 × 10−1 mol/L 4.23 × 10−1 mol/L 5.64 × 10−1 mol/L 7.05 × 10−1 mol/L

0.531 0.558 0.533 0.552 0.568

± ± ± ± ±

0.003 0.003 0.003 0.003 0.003

0.731 0.754 0.743 0.694 0.759

0.505 0.452 0.437 0.590 0.584

3.48 4.14 3.21 3.45 5.00

3.45 4.11 2.88 3.71 4.58

Effect of [APS] 1 2 3 4 5

4.38 mmol/L 8.76 mmol/L 13.14 mmol/L 17.52 mmol/L 21.90 mmol/L

0.568 0.650 0.568 0.542 0.545

± ± ± ± ±

0.003 0.017 0.008 0.008 0.005

0.759 0.748 0.714 0.729 0.671

0.584 0.535 0.635 0.592 0.884

5.00 7.00 4.96 4.85 4.90

4.58 6.43 4.58 4.47 4.52

Effect of [N,N - MBAAm] 6.49 mmol/L 1 12.98 mmol/L 2 19.47 mmol/L 3 25.96 mmol/L 4 32.45 mmol/L 5

0.650 0.612 0.632 0.528 0.640

± ± ± ± ±

0.017 0.003 0.010 0.28 0.01

0.748 0.607 0.630 0.612 0.597

0.535 1.398 1.146 1.349 1.448

7.00 6.61 6.14 4.60 6.76

6.43 6.46 6.08 4.52 6.65

Effect of pH 1 2 3

Distilled water pH 2.2 buffer pH 7.4 buffer

0.650 ± 0.017 0.570 ± 0.0 0.572 ± 0.003

0.748 0.557 0.667

0.535 1.756 0.767

7.00 4.65 4.01

6.43 4.94 3.98

Effect of salt 1 2

Distilled water 0.9% NaCl

0.650 ± 0.017 0.572 ± 0.003

0.748 0.573

0.535 1.611

7.00 4.69

6.43 4.50

± ± ± ± ±

0.665 0.677 0.704 0.748 0.718

0.954 0.732 0.689 0.535 0.567

5.99 4.06 4.91 7.00 5.61

5.84 4.36 4.53 6.43 4.48

Effect of temperature 22 ◦ C 1 27 ◦ C 2 32 ◦ C 3 37 ◦ C 4 42 ◦ C 5

0.582 0.575 0.565 0.650 0.583

0.008 0.005 0.005 0.017 0.006

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25

-1

Amount of water uptake (g/g of gel)

25

Amount of water uptake (g/g of gel)

[AAm] 1.41×10 mol/L -1 [AAm] 2.82×10 mol/L -1 [AAm] 4.23×10 mol/L -1 [AAm] 5.64×10 mol/L -1 [AAm] 7.05×10 mol/L

30

20

15

10

5

[APS] 4.38 mmol/L [APS] 8.76 mmol/L [APS] 13.14 mmol/L [APS] 17.52 mmol/L [APS] 21.90 mmol/L

20

15

10

5

0 0

45

90 135 180 225 270 315 360 405 450

1440

Time (min.) Fig. 5.2. Effect of [AAm] on swelling of psy-cl-poly(AAm-co-AAc) hydrogels. [AAc] = 2.92 × 10−1 mol/L, [Psyllium = 1 g, [APS] = 4.38 mmol/L, [N,N’MBAAm] = 6.49 mmol/L].

of hydrogels are presented in Fig. 5.2. The hydrogels prepared with increase in feed acrylamide concentration in binary monomer mixture of AAm/AAc, present in the reaction system, have shown the similar trends for the swelling of hydrogels, as shown in case acrylic acid variation. The swelling of hydrogels first increased and then decreased after attaining the optimum value. These trends may also be explained on the basis of similar reason as discussed in case of acrylic acid. The increase in homopolymerization, increase in viscosity and increase in chances of chain transfer to monomer led to decrease in pore size and thereafter swelling of the hydrogels. It means more networks of poly(AAm) chains are formed in the gel and the free volume available to water molecules significantly decreased which accounts for low swelling [42]. The maximum amount of water uptake (31.24 ± 1.06 g/g of gel) after 24 h was observed for hydrogels prepared with 2.82 × 10−1 mol/L of [AAm] although these hydrogels eroded within 24 h. However, the polymers prepared with 7.05 × 10−1 mol/L of [AAm], have shown the more strength and this AAm concentration was taken as the optimum concentration, for further hydrogels synthesis, during the variation of other reaction parameters. The values of diffusion exponent ‘n’ and gel characteristics constant ‘k’ are presented in Table 3. The values of ‘n’ are greater than 0.5 and less than 1, which show that the swelling of polymeric network prepared with different acrylamide concentration occurred through a non-Fickian diffusion mechanism. The values of diffusion coefficients for swelling of polymers with different [AAm] are given in Table 3. In case of non-Fickian diffusion mechanism during polymer swelling the rate of solvent diffusion is comparable to the rate of polymer chains relaxation. The solvent enhances the mobility of polymer chains by converting the glassy matrix to a swollen rubbery material. In glassy polymers, large change in composition occurs during transport of penetrant into the polymer. As a result, nonlinear “viscoelastic” diffusion is encountered, showing striking deviations from Fick’s law. The viscoelastic nature of the polymers and cracks are said to be the factors leading to non-Fickian diffusion. The physical reason for the deviation from Fickian behavior is the time-dependent properties of a glassy polymer due to the finite rate of adjustment of the polymer chains in the presence of the solvent [43–45]. 3.2.3. Effect of feed [APS] on swelling of hydrogels The effect of initiator on the network structure was studied by observing the swelling behavior of polymer networks prepared by

0 0

45

90 135 180 225 270 315 360 405 450

Time (min.)

1440

Fig. 5.3. Effect of [APS] on swelling of psy-cl-poly(AAm-co-AAc) hydrogels. [Psyllium = 1 g), [AAc] = 2.92 × 10−1 mol/L, [AAm] = 7.05 × 10−1 mol/L), [N,N’MBAAm] = 6.49 mmol/L].

varying the [APS] during polymerization reaction (Fig. 5.3). The polymers of different [APS] (varied from 4.38 to 21.90 mmol/L) were prepared and their swelling was determined. The swelling of the psy-cl-poly(AAm-co-AAc) hydrogels decreased with increase in feed [APS] in the reaction system. At higher feed [APS], the rate of free radicals formation in the reaction system increases which results in faster rate of termination of growing macroradical chains via bimolecular collision that further leads to increase in crosslinking density of the crosslinked networks thus decreasing the swelling of hydrogels [46]. The values of diffusion exponent ‘n’ and gel characteristics constant ‘k’ are presented in Table 3. The values of ‘n’ are greater than 0.5 which show a non-Fickian diffusion mechanism for the swelling of hydrogels prepared with different [APS]. The values of the diffusion coefficients are shown in Table 3. 3.2.4. Effect of feed [N, N -MBAAm] on swelling of hydrogels In the present studies the N,N -MBAAm was used as crosslinker. The feed crosslinker concentration was varied from 6.49 to 32.45 mmol/L in reaction system during the synthesis of hydrogels, and swelling of the hydrogels was determined (Fig. 5.4). It has been observed that swelling of hydrogels decreased with increase in crosslinker concentration. This may be due to increase in crosslinking density of the hydrogels with increase in crosslinker concentration in the reaction system. The values of the diffusion coefficients are present in Table 3. In gels, concentration of monomer, nature of monomer and crosslinker play an important role in formation of the polymer networks. In dilute solution, the long chain polymer molecules are in the form of coils, each having certain mobility. As the concentration increases, the mobility of coils decreases. At a critical concentration, known as gel point, the coils no longer move as units and can no longer interchange their places. Even though all of polymerizing material still exists as monomers, dimers, trimers, and so forth, a three dimensional network will appear. After the gel point is reached, more and more of these loose groups become attached to network. The transition from a concentrated solution to a gel corresponds to the transition of a liquid to a solid. In general, the formation of hydrogel network occurs by crosslinking in the existing polymer chain. If the initial composition contains not only the vinyl monomer but also even a small amount of two or multifunctional additives, e.g., crosslinker, polymerization reaction of the main monomer is accompanied by

B. Singh, R. Bala / International Journal of Biological Macromolecules 65 (2014) 524–533

20

16

18

14 12 10 8 6 4

14 12 10 8 6

2

0 0

45

90 135 180 225 270 315 360 405 450

0

1440

0

Time (min.)

20

Distilled water pH 2.2 buffer pH 7.4 buffer

18 16 14 12 10 8 6 4 2 0 45

90

135 180 225 270 315 360 405 450

45

90 135 180 225 270 315 360 405 450

1440

Time (min.)

Fig. 5.4. Effect of [N,N -MBAAm] on swelling of psy-cl-poly(AAm-co-AAc) hydrogels. [Psyllium = 1 g, [AAc] = 2.92 × 10\+ 10−1 mol/L, [AAm] = 7.05 × 10−1 mol/L,) [APS] = 8.76 mmol/L].

Swelling (g/g of gel)

16

4

2

0

Distilled water 0.9% NaCl solution

20

Amount of water uptake (g/g of gel)

18

Amount of water uptake (g/g of gel)

22

[N,N'-MBAAm] 6.49 mmol/L [N,N'-MBAAm] 12.98 mmol/L [N,N'-MBAAm] 19.47 mmol/L [N,N'-MBAAm] 25.96 mmol/L [N,N'-MBAAm] 32.45 mmol/L

531

1440

Time (min.) Fig. 5.5. Effect of pH of swelling medium on swelling of psy-cl-poly(AAm-co-AAc) (hydrogels. [Psyllium = 1 g, [AAc] = 2.92 × 10−1 mol/L, [AAm] = 7.05 × 10−1 mol/L, [APS] = 8.76 mmol/L, [N,N -MBAAm] = 6.49 mmol/L].

formation of some bridges between the newly formed chains, constructed with this multifunctional species and results the formation of gel. In the present studies, with increase in [N,N -MBAAm] in the reaction system, the hydrogels of higher crosslinking density were formed and swelling the hydrogels decreased. 3.2.5. Effect of pH, [NaCl] and temperature of swelling medium on swelling of hydrogels The hydrogels prepared at the optimum conditions were used to study the effect of pH, salt and temperature of the swelling medium on the swelling of the hydrogels. This study gives the information about the stimuli-responsive behavior of the hydrogels which can further be exploited to design the site specific drug delivery devices based on the hydrogels. The swelling of the hydrogels were carried out in distilled water, pH 2.2 and 7.4 buffer solution. The results of swelling of the hydrogels in different medium are presented in Fig. 5.5. The swelling of the hydrogels has been observed higher in the pH 7.4 buffer as compared to the pH 2.2 buffer. This may be due to partial hydrolysis of the polymer matrix and at higher pH, some of COOH and CONH2 functional groups present in the polymers

Fig. 5.6. Effect of [NaCl] on swelling of psy-cl-poly(AAm-co-AAc) hydrogels. Psyllium = 1 g, [AAc] = 2.92 × 10−1 mol/L, [AAm] = 7.05 × 10−1 mol/L, [APS] = 8.76 mmol/L, [N,N -MBAAm] = 6.49 mmol/L.

get ionized and the electrostatic repulsion between COO− groups comes into play. This repulsive force would push the network chain segments apart which expand the polymer networks and attract more water into the hydrogels, consequently, swelling increased. Under acidic pH values, most of the functional groups remained unionized and there is no anion-anion repulsive forces and network remained in the collapsed stage and consequently swelling decreased [47]. The effect of salt solution concentration on the swelling behavior of the psy-cl-poly (AAm-co-AAc) hydrogels is shown in Fig. 5.6. The swelling of hydrogel in salt solution was 5.91 ± 0.17 g/g of gel while in case of distilled water it was 18.53 ± 1.31 g/g of gel. The increase of ionic strength in the solution leads to a decrease in the swelling of the hydrogels. The behavior can be explained on the basis of osmotic pressure developed due to unequal distribution of ions in the medium and polymer networks. The ions attached to the polymer network are immobile and considered to be separated from the external solution by a semipermeable membrane. When the hydrogels are placed in water, there is maximum osmotic pressure developed and hence the maximum swelling. When the polymer is in NaCl solution the osmotic pressure developed is much lower because the external solution contains Na+ and Cl− . So the swelling is drastically reduced in salt solution. The effect of temperature of swelling medium on the swelling of psy-cl-poly(AAm-co-AAc) hydrogels was determined by varying the temperature of the swelling medium and determining the swelling of the hydrogels (Fig. 5.7). The increase in swelling has been observed with increase in temperature of the swelling medium up to 37 ◦ C and then swelling slightly decreased at 42 ◦ C. The values of diffusion exponent ‘n’ and gel characteristics ‘k’ are shown in Table 3. These values indicate that the diffusion of water molecules into hydrogels occurred through non-Fickian diffusion mechanism in solution of different pH, [NaCl], and temperature. The values of diffusion coefficients are shown in Table 3. 3.3. Drug release studies The mechanism of drug release is dependent on the rate of the swelling and drug solubility. For a successful drug delivery system, it is imperative to predict the mechanism of drug release from the drug delivery device. This is most challenging fields in drug delivery. Over the years researchers have predicted the release of drugs as a function of time, using both simple and sophisticated

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B. Singh, R. Bala / International Journal of Biological Macromolecules 65 (2014) 524–533

Table 4 Results of diffusion exponent ‘n’, gel characteristic constant ‘k’ and various diffusion coefficients for the release of methotrexate from drug loaded psy-cl-poly(AAm-co-AAc) hydrogels in different medium at 37 ◦ C. Drug releasing medium

Distilled water pH 2.2 buffer pH 7.4 buffer

Diffusion exponent ‘n’

0.558 0.713 0.531

Gel characteristic constant ‘k’ × 102

2.05 0.675 2.49

2.8

o

22 C o 27 C o 32 C o 37 C o 42 C

16

12

8

Initial Di × 103

Late time DL × 103

5.90 6.24 5.59

6.10 5.70 6.18

Distilled water pH 2.2 buffer pH 7.4 buffer

2.4

Amount of drug release (mg/20mL/g of gel)

Amount of water uptake (g/g of gel)

20

Diffusion coefficients (cm2 /min)

2.0 1.6 1.2 0.8 0.4

4

0.0 0

0 0

45

90 135 180 225 270 315 360 405 450

45

Time (min.) Fig. 5.7. Effect of temperature of the swelling medium on the swelling of psy-cl-poly(AAm-co-AAc) hydrogels. [Psyllium = 1 g, [AAc] = 2.92 × 10−1 mol/L, [AAm] = 7.05 × 10−1 mol/L),S [APS] = 8.76 mmol/L, [N,N -MBAAm] = 6.49 mmol/L].

mathematical models which give us an insight into mass transport and the effect of design parameters, such as device geometry and drug loading, on the release mechanism of the active agent in question. In vitro release dynamics of the drug from the polymer matrix in different pH solution will provide information regarding the suitability of hydrogels for site specific drug delivery. Therefore, in vitro drug release studies of the model drugs methotrexate from the drug loaded psy-cl-poly(AAm-co-AAc) hydrogels were carried out in distilled water, pH 2.2 buffer and pH 7.4 buffer and results of drug release studies are presented in Fig. 6. These pH buffers are simulated to the physiological conditions of the fluid of the stomach and colon. The release of methotrexate is observed more in solution of pH 7.4 buffer as compared to pH 2.2 buffer. This observation may be explained on the basis of swelling of the hydrogels which has also been observed higher in pH 7.4 buffer due to the partial hydrolysis of the polymer matrix. Diffusion is a phenomenon largely governed by the swelling behavior of the hydrogel which is further dependent on the nature of the polymer network, presence of hydrophilic/hydrophobic groups, crosslinking density and elasticity of the copolymer networks [48]. In the present case the matrix composed of poly(AAm) and poly(AAc) which is pH responsive and has shown more swelling at higher pH, due to COO− ions repulsion present in the polymer matrix and has expanded the polymer chains. Hence, the release has been observed more in buffer solution of pH 7.4 [49]. These observations indicate that psy-cl-poly(AAm-co-AAc) hydrogels can be used for the colon specific delivery of anticancer drug. From the slope and intercept of the plot of ln Mt /M∞ versus ln t, the values of the diffusion exponent ‘n’ and gel characteristic constant ‘k’ have been obtained for the release of methotrexate from the hydrogels. The values of diffusion exponent ‘n’ and gel characteristics are given in Table 4. The results indicate that the drug

90 135 180 225 270 315 360 405 450

1440

Time (min.)

1440

Fig. 6. Release profile of methotrexate from drug loaded psy-cl-poly(AAm-co-AAc) hydrogels. [Psyllium = 1 g, [AAc] = 2.92 × 10−1 mol/L, [AAm] = 7.05 × 10−1 mol/L), [APS] = 8.76 mmol/L, [N,N -MBAAm] = 6.49 mmol/L].

release from the polymeric matrix follows Fickian diffusion mechanism in pH 7.4 buffer, while a non Fickian diffusion mechanism has been followed in case of pH 2.2 buffer. In Fickian diffusion mechanism, the rate of penetrant diffusion is significantly slower than the rate of relaxation of polymeric chains and the mechanism is diffusion controlled. In non-Fickian diffusion, the rate of penetrant diffusion is comparable to the rate of polymer chains relaxation. Diffusion coefficients for the drug release from hydrogels in different medium are presented in Table 4. 4. Conclusions From the foregone discussion, it is concluded that both, structure and compositions of hydrogels, are influenced by the synthetic reaction parameters which is evident from the results of the swelling studies. The nature of swelling medium has also influenced the swelling of the polymer networks. Hence, AAm/AAc based hydrogels could be exploited for developing the site specific drug delivery systems to the colon. The swelling of the hydrogels and release of drugs from drug loaded hydrogels, in most of the cases, occurred through non-Fickian diffusion mechanism. In this mechanism, the rate of drug diffusion from the polymer matrix and rate of polymer chain relaxation are comparable. Further, the release of drug during initial stages was higher than the later stages. It means after maintaining certain concentration, the release of drug has occurred in a controlled and sustained manner and hence, these hydrogels can be used as slow drug delivery devices. It is further concluded that the drug delivery system developed from the modification of the psyllium may have the enhanced potential. Here enhanced potential of these delivery systems is due to the therapeutic importance of psyllium for the treatment of the colon cancer on the one hand and release of their curative agents (methotrexate) from drug loaded hydrogels on the other hand.

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