Development and application of a nanocomposite derived from crosslinked HPMC and Au nanoparticles for colon targeted drug delivery

Abstract

Herein, we report a novel route for the synthesis of a poly(acrylamide) (PAAm) crosslinked hydroxypropyl methyl cellulose/Au nanocomposite where chemically crosslinked HPMC (c-HPMC) works as a reducing agent. At first, the crosslinked polymer was developed by grafting PAAm chains onto the HPMC backbone using ethylene glycol dimethacrylate (EGDMA) crosslinker and potassium persulfate (K₂S₂O₈) initiator. Afterwards, AuNPs have been incorporated in situ on the surface of the crosslinked hydrogel, where the hydrogel itself reduces the tetrachloroauric acid (HAuCl₄) in the reaction medium to form the nanocomposite. Different grades of nanocomposites (c-HPMC/Au) have been synthesized by altering the reaction parameters and the best one was optimized with the help of UV-visible spectroscopy. The nanocomposites synthesized, have been characterized by FTIR spectroscopy, ¹³C NMR spectroscopy, XRD studies, FESEM/EDAX/elemental mapping analyses, HR-TEM analysis and TGA analysis. HR-TEM analysis reveals the uniform distribution of spherical AuNPs on the surface of c-HPMC. Rheological characteristics disclose that the nanocomposite demonstrates higher gel strength than that of the crosslinked polymer, mainly because of the enhanced interactions between the organic matrix and inorganic fillers. The pH responsive behaviour of crosslinked hydrogel/composites has been confirmed by measuring the equilibrium swelling ratio in various buffer solutions (pH 1.2 and 7.4) at 37 °C. Biodegradability of the hydrogel/nanocomposite has been verified using hen egg lysozyme. The synthesized nanocomposite also demonstrates non-cytotoxic behaviour towards human mesenchymal stem cells (hMSCs). The in vitro drug release profiles indicate that both ornidazole and 5-amino salicylic acid (5-ASA) are released from the nanocomposite matrix in a controlled fashion. This confirms that the c-HPMC/Au nanocomposite is likely be an excellent alternative for the controlled release of colonic drugs. The release kinetics and mechanism of ornidazole and 5-ASA from the nanocomposite material has been explained using various linear and non-linear mathematical models.

---

1. Introduction

In the last decade, targeted drug delivery into colonic regions has achieved significant attention in the treatment of several bowel diseases such as ulcerative colitis, Crohn's disease, amoebiasis and colorectal cancer.^1,2 A high concentration of colonic drugs is needed for the treatment of the diseases associated with the colon.³ Besides, the colonic region has less diversity and intensity of enzymatic activities than the stomach and small intestine.⁴ Therefore, versatile strategies are required to deliver the drugs to the colon for effective therapy. Thus the design of matrices for colon targeted drug delivery should be based on the concept of preventing the burst effect in the stomach and small intestine as well as on the drug release being triggered by pH or the high activity of certain enzymes in comparison to non-target tissues.⁴

With the recent advances in synthesis of macromolecules, incorporation of nano-size particles, such as gold,^5,6 silver nanoparticles,^7,8 quantum dots,^9,10 and magnetic particles¹¹ of different shapes have been immensely studied for drug delivery applications. These drug delivery systems encapsulate the drug to manage the poor distribution and stability of the therapeutic agents.¹² Encapsulation efficiency can be optimized by the enlargement of nanoparticles into the polymer networks which increases the therapeutic efficiency by many folds.¹² In addition, gold nanoparticle (AuNP) is an effective candidate for transport and release of therapeutic agents such as drugs, proteins and peptides in their appropriate target positions.^13,14 It has numerous fascinating properties like inertness, non-cytotoxicity, biocompatibility, strong absorption capacity, scattering power, high surface area to volume ratio which provide the dense loading of therapeutic materials and make the nanoparticle as an outstanding candidates for drug delivery matrix.^15,16

Recently, research efforts have been focussed towards developing the in situ synthesis of metal nanoparticles within polymeric network architectures.¹⁷ Concerning the synthesis of metal nanoparticles, particularly gold nanoparticles, many methods have reported in the literature.¹⁸ In recent years, biomolecules and bioorganisms including Verticillium fungus, β-D-glucose, chitosan, sucrose were used in the synthesis of gold nanoparticle.¹⁸ But to the best of our knowledge, this is probably the first report with the use of a modified natural polysaccharide based chemically crosslinked hydrogel as reducing and stabilizing agent for in situ formation AuNPs on the surface of crosslinked hydrogel. Since, modified polysaccharides based physically/chemically crosslinked hydrogels have drawn a considerable interest towards pharmaceutical and biopharmaceutical field, especially in drug delivery because of their outstanding properties such as less expensive, stimulus-responsiveness, soft tissue like behaviour, excellent swelling nature, non-toxicity, biodegradability and most importantly controlled release behaviour. As the drug release behaviour from the hydrogels are not only depending on amount of crosslinking but also on different factors, including swelling properties, pH of the release media and finally biocompatibility of the hydrogel. Especially, for colon specific drug delivery, pH-sensitive hydrogels have attracted increasing attention in a stimuli-responsive manner based on pH changes in GI tract.^19–22 Whereas, the stimuli-responsive hydrogels exhibit remarkable changes in their swelling behaviour, network structure, permeability and mechanical strength in response to a number of external stimuli, including pH,²³ ionic strength,²⁴ temperature²⁵ etc.

Keeping in view, the pharmacological importance of modified polysaccharide based hydrogel/nanocomposites, this article reports the first example of in situ development of nanocomposite derived from PAAm crosslinked HPMC, where crosslinked HPMC acts as reducing and stabilizing agent for the formation of uniform and well dispersed AuNPs. Since hydroxypropyl methyl cellulose based crosslinked hydrogel/Au nanocomposite is not still reported in the literature where crosslinked hydrogel itself acts as reducing and stabilizing agent, the present study is an attempt for the in situ formation of nanocomposite for colon targeted drug delivery device. Interestingly, this nanocomposite holds hydrogel behaviour as well as characteristics of gold nanoparticle; which makes it a perfect matrix for colon targeted drug delivery. Besides, the observed characteristics of c-HPMC/Au nanocomposite such as excellent stimuli-responsive swelling behaviour, biodegradability, non-cytotoxicity, proficient drug stability and the controlled release behaviour of both ornidazole and 5-ASA opens a new and alternative route in the field of controlled drug delivery applications.

2. Experimental

2.1 Materials

Hydroxypropyl methyl cellulose (HPMC) was purchased from Lancaster, UK. Ethylene glycol dimethacrylate (EGDMA) was procured from TCI, Tokyo, Japan. Acrylamide (AAm) was acquired from E. Merck, Germany. Gold(III) chloride hydrate (HAuCl₄·XH₂O) and 5-amino salicylic acid (AR Grade) were supplied by Spectrochem Pvt. Ltd. Mumbai, India. Potassium persulfate (KPS) was supplied by Glaxo Smith Kline Pharmaceuticals Ltd., Mumbai, India. Acetone was obtained from E-Merck (I) Pvt. Ltd., Mumbai, India. Lysozyme hydrochloride from egg white was purchased from TCI Pvt. Ltd., Tokyo, Japan. Ornidazole was a gift sample from Endoc Pharma, Rajkot, Gujarat, India. Polyvinyl pyrrolidone (PVP-K30) was supplied by Spectrochem Pvt. Ltd., Mumbai, India. Double distilled water was used in all experimental works.

2.2 Synthesis

2.2.1 In situ synthesis of chemically crosslinked HPMC and gold nanocomposite (c-HPMC/Au). Chemically crosslinked hydrogel based on HPMC grafted with polyacrylamide in presence of ethylene glycol dimethacrylate (EGDMA) crosslinker followed by nanocomposite formation was carried out using free radial polymerization technique. A typical experimental procedure for copolymerization and crosslinking reaction is as follows:

1 g of HPMC was slowly dissolved in 100 mL of distilled water in a three necked round bottom flask. The flask was fitted with an electrically operated magnetic stirrer (Tarsons, Model: Spinot Digital) and kept in an oil bath maintained at a temperature of 70 °C, with constant stirring (having stirring speed of 400 rpm). Afterwards, an aqueous solution of 0.92 × 10⁻⁵ mole initiator (i.e. KPS) followed by required amount (i.e. 0.17 mole) of monomer (i.e. acrylamide) was introduced in the reaction mixture at same temperature and stirring speed. The reaction was carried out in inert atmosphere of nitrogen and was continued for 1 h. Afterwards, 2.65 × 10⁻³ mole crosslinker i.e. EGDMA was added slowly to the copolymer solution. The graft copolymerization along with crosslinking reaction was allowed to proceed at 70 °C for 2 h. Subsequently, an aqueous solution of HAuCl₄ (0.1 mM) was mixed to the reaction medium and the mixture was kept at a constant reaction temperature of 70 °C. The reduction of the gold metal ions (Au³⁺) to gold nanoparticles (Au⁰) was confirmed by the color change of the reaction mixture from yellow to wine red. Once reddish color was appeared, the composite formation reaction was continued for desired time period (2–6 h, Table 1), after which the reaction was stopped with addition of hydroquinol solution. The resultant product was cooled to room temperature. Finally the nanocomposite was collected, and dried in a vacuum oven at 40 °C, until and unless constant weight was accomplished.

Table 1 Synthesis details and equilibrium swelling of c-HPMC/Au^a

Hydrogels	Reaction parameters		Equilibrium swelling (%) (15 h)
	Monomer conc. (mole)	Reaction time (h)	pH = 1.2	pH = 7.4
a Amount of HPMC = 1.0 g (0.0062 mole) for all hydrogel synthesis. Initiator concentration 0.92 (mole × 10^−5). Crosslinker concentration 2.65 (mole × 10^−3). Amount of gold(III) chloride taken 0.1 (mole × 10^−3). Reaction temperature maintain at 70 °C.
c-HPMC/Au 1	0.17	2	305.2 ± 27.5	486.8 ± 29.6
c-HPMC/Au 2	0.085	2	214.9 ± 23.2	316.2 ± 23.6
c-HPMC/Au 3	0.0425	2	107.5 ± 10.3	188.9 ± 15.3
c-HPMC/Au 4	0.17	4	342.5 ± 23.6	517.4 ± 26.5
c-HPMC/Au 5	0.085	4	240.2 ± 17.3	331.5 ± 21.3
c-HPMC/Au 6	0.0425	4	128.2 ± 13.2	221.5 ± 26.3
c-HPMC/Au 7	0.17	6	345.8 ± 19.6	526.9 ± 23.2
c-HPMC/Au 8	0.085	6	246.9 ± 21.3	337.5 ± 26.3
c-HPMC/Au 9	0.0425	6	133.7 ± 12.3	225.7 ± 21.3

---

2.3 Characterization

UV visible spectra of nanocomposites in aqueous solution were carried out using UV-visible Spectrophotometer (Model UV-1800, Shimadzu, Japan). The scan range was 190 to 1100 cm⁻¹.

FTIR spectra of the c-HPMC and c-HPMC/Au 3 hydrogel were recorded using KBr pellet method (Model IR-Perkin-Elmer, Spectrum 2000). The scan range was 400 to 4000 cm⁻¹.

Solid state ¹³C nuclear magnetic resonance (NMR) spectra of c-HPMC and c-HPMC/Au 3 hydrogel were recorded at 500 MHz on a Bruker Avance II-500 NMR spectrophotometer.

Surface morphology, EDAX analysis and elemental mapping of neat HPMC, c-HPMC and nanocomposite were performed using Field Emission Scanning Electron Microscopy (Model: Zeiss Ultra 55cv FESEM, Make Zeiss Germany). The powder samples were coated with platinum for microscopic measurement.

Thermogravimetric analysis of c-HPMC and crosslinked HPMC/Au nanocomposite were carried out using TGA analyzer (Shimadzu DTG-60) at a heating rate of 10 °C min⁻¹ in nitrogen atmosphere.

Surface microstructures of c-HPMC/Au nanocomposite was performed using HR-TEM analyses (Model: JEM 2100, JEOL, Japan).

2.3.1 Biodegradation study. Enzymatic biodegradation study of crosslinked hydrogel/gold nanocomposite film was performed using hen egg lysozyme as described in literature.^26–28 For this purpose, the pre-weighted nanocomposite film (10 × 10 × 0.1 mm³) was kept in pH 7.4 phosphate-buffer solution with 1.5 μg mL⁻¹ of lysozyme powder at 37 °C. To ensure the enzyme activity onto hydrogel/nanocomposite film, after a certain time intervals (3, 7, 14 and 21 days), films were removed from buffer media, washed carefully with distilled water, dried in vacuum oven and reweighed. The lysozyme solution was replaced by freshly prepared lysozyme solution daily to maintain enzymatic degradation at a constant rate. The extent of in vitro degradation was determined as % weight loss of the dried films. To differentiate between dissolution and biodegradation, another film was also immersed into the PBS solution without any lysozyme.

2.3.2 Rheological study. The rheological characteristics of c-HPMC and c-HPMC/Au 3 were performed using Bohlin Gemini-2 rheometer (Malvern, UK). The shear viscosities of 1 wt% c-HPMC and c-HPMC/Au 3 in PBS media at gel state were measured at different shear rates ranging from 0.1–10 S⁻¹. The frequency sweep measurements were executed in the frequency range from 0.01 to 10 Hz at a constant stress of 1.0 Pa. The gel strength of the crosslinked hydrogel and nanocomposite were assessed by measuring the storage modulus (G′) and loss modulus (G′′) at a constant frequency of 0.5 Hz and different shear stress ranging from 1 to 6000 Pa.

2.3.3 In vitro cytocompatibility study. Equivalent weight of powder c-HPMC/Au 3 nanocomposite was made into pellets and was put in 24 well plates for in vitro studies. The details methodology for preparation of pellets has been explained in ESI.† The pellets were sterilized using 70% ethanol and UV followed by frequent washing with sterilized PBS (pH 7.4). After aspiration of PBS, pellets were incubated overnight with complete medium consisting of α-MEM (Gibco) supplemented with 10% FBS, 1% penicillin/streptomycin solution and 3.7% sodium bicarbonate at 37 °C in a 5% CO₂ atmosphere. Media was then removed and seeded with 2 × 10⁵ human mesenchymal stem cells/pellet. The hMSCs were procured from Advanced Neuro-Science Allies, Bangalore, India. Media changes were performed after 24 h and every 48 h thereafter. Cells were also seeded on lysine coated slides. After 1, 3 and 7 days, cell morphology was assessed by rhodamine–phalloidin (catalog no. R415, Invitrogen) and DAPI (4′,6-diamidino-2-phenylindole, catalog no. D1306, Invitrogen) according to the manufacturer's instructions. In brief, the cells were fixed, permeabilized using Triton-X-100 followed by blocking the non-specific sites using bovine serum albumin (Sigma). The cells were then stained with the florescent dyes and imaged using a fluorescence microscope (Zeiss Axio Observer Z1, Carl Zeiss, Germany). For cell proliferation, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay of the adherent cells on the pellets was examined after 1, 3 and 7 days. After predetermined time intervals, culture medium was discarded, washed thoroughly with PBS and incubated with 5 mg mL⁻¹ MTT solution (Sigma, US) at 37 °C for 4 h. The insoluble formazan crystals formed were dissolved in dimethyl sulfoxide and absorbance was recorded in 96-well plates at 570 nm on a microplate reader (RMS Instruments, India). Cells were also plated on tissue culture plate for positive control. For repeatability, the test was performed in triplicates.

2.3.4 Measurements of equilibrium swelling ratio and swelling kinetics. The pH-sensitivity of the hydrogel/nanocomposite were determined using the measurement of equilibrium swelling ratio (ESR) of crosslinked hydrogel (c-HPMC) and various nanocomposites (c-HPMC/Au) at 37 °C in buffer solutions of pH similar to that of gastric (pH 1.2) and intestinal fluids (pH 7.4). Briefly, a small dried pre-weighed piece of polymeric/nanocomposite material was immersed in 100 mL of buffer solution at 37 °C for 24 h to reach equilibrium swelling. The swollen hydrogel was withdrawn after every 2 h; the excess water was blotted off carefully using tissue paper and then reweighed. The equilibrium was reached at ∼15 h. Equilibrium swelling ratio was calculated by using eqn (1):

To evaluate the rate of swelling, Voigt model (eqn (2)) has been used.¹⁹

S_t = S_e(1 − e^−t/τ) (2)

where S_t (g g⁻¹) is the swelling at time t, S_e (g g⁻¹) is the equilibrium swelling, t is the time (min) for swelling and τ (min) stands for the rate parameter. Rate parameter (τ) has been evaluated using eqn (2), which is a measure of the swelling rate i.e. lower will be the rate parameter value (τ), higher will be the swelling rate.¹⁹

2.3.5 Salt effect on swelling. The equilibrium swelling ratio of nanocomposite was investigated at different salt solutions of monovalent (LiCl, NaCl and KCl), divalent (MgCl₂, CaCl₂ and BaCl₂) and trivalent (AlCl₃). The concentration of the salt solution was also varied to measure the % equilibrium swelling.

2.4 In vitro 5-amino salicylic acid and ornidazole release studies

2.4.1 Preparation of tablets. c-HPMC and c-HPMC/Au (450 mg) were finely ground in a blender, followed by the addition of PVP (50 mg) and drugs (5-ASA and ornidazole) (500 mg). The mixture was wet with ethanol and mixed further. The paste was dried at 40 °C to a constant weight. Then a mixture of silicon-di-oxide and magnesium stearate (2 : 1 ratios) was added as a lubricant, in amount not exceeding 3% of the ground powder. After mixing and sieving (20 meshes), tablets of 1 g each were prepared by compression in a tablet punching machine at a pressure of 2–3 t cm⁻².

2.4.2 Drug release study. The in vitro release of entrapped drugs (i.e. ornidazole and 5-amino salicylic acid) from crosslinked hydrogel/nanocomposite matrices were determined using standard Dissolution Test Apparatus (Lab India, Model: DS 8000), under a constant rotation of 60 rpm at 37 ± 0.5 °C using 900 mL simulated gastric fluid (SGF, pH 1.2) for 2 h, phosphate buffer having pH 6.8 for next 3 h and subsequently in 900 mL of simulated intestinal fluid (SIF, pH 7.4) for further 7 h. At definite time intervals (every 1 h), an aliquot was withdrawn and its absorbance was measured (λ_max: 278 nm at pH 1.2, λ_max: 318.5 nm at pH 6.8, and λ_max: 320 nm at pH 7.4) for ornidazole tablets and (λ_max: 303 nm at pH 1.2 and λ_max: 331 nm at pH 7.4) for 5-amino salicylic acid tablets, spectrophotometrically (using UV-visible Spectrophotometer-Model UV-1800, Shimadzu, Japan). During release study, to avoid dilution effect, equal amount of fresh buffer solution was added after every 1 h to maintain the sink condition. The % cumulative drug release from matrices (in tablet formulation) at any time was determined with the help of calibration curve of the drug solution. To evaluate the release kinetics of the copolymer matrix, the in vitro drug release data were fitted to various kinetic models such as zero order,²⁹ first order,³⁰ Higuchi³¹ and Hixson–Crowell³² models. Korsmeyer–Peppas³³ and Kopcha³⁴ models have also been used to find out the release mechanism of the entrapped drug from copolymer matrix.
Determination of erosion rate. During drug release, some tablet formulations of polymer/nanocomposite matrix disintegrated partially. The degree of erosion (D) was calculated as stated ESI.†

2.4.3 Drug stability study. The stability study of the ornidazole/5-ASA tablets were performed up to 3 months to observe the effect of environmental factors on release characteristics. Tablet was kept in a glass bottle and located in the humidity chamber where temperature was fixed at 40 ± 2 °C and relative humidity (RH) was maintained at 75 ± 5% during the entire study period.

3. Results and discussions

3.1 Synthesis of c-HPMC/Au nanocomposite

Herein, well dispersed gold nanoparticles (AuNPs) were deposited in situ on the surface of crosslinked hydroxypropyl methyl cellulose, which was synthesised by grafting of polyacrylamide chains on HPMC backbone in presence EGDMA crosslinker. Here it is presumed that the crosslinked hydrogel acts as a precursor for production and growth of AuNPs in the network. The reduction of HAuCl₄ occurred through the transfer of electrons from the –OH/–OCH₃/–CONH₂ groups of c-HPMC to the Au³⁺ ion, leading to the formation of Au⁰.^35,36 This metallic gold then nucleates and grows to form gold nanoparticles, and is subsequently capped and stabilized by the crosslinked hydrogel. The crosslinked hydrogel is oxidized to a positive radical owing to the transfer of an electron to the gold ion. The proposed reaction scheme is shown in Scheme 1.

Scheme 1 Schematic representation for the formation of c-HPMC/Au nanocomposite.

It is believed that c-HPMC networks play an important role to stabilize newly formed AuNPs in the reaction medium. In general, the stabilization of formed nanoparticles is achieved by either attaching them to large protecting crosslinked polymer chains or protecting macromolecules will encapsulate or cover the nanoparticles. First of all, the main advantage of this technique is that it provides controlled size and uniform distribution of nanoparticles on the surface of crosslinked hydrogel without using any stabilizer during the reaction. The porous nature of crosslinked hydrogel suppresses the aggregation of the nanoparticles during the formation. Such type of porosity creates an opportunity to rapid diffusion of small nano sized molecule into its cavity by different types of intermolecular attraction of crosslinked hydrogel, which make the nano sized particle stable as well as well dispersed throughout the surface of c-HPMC.

It was noticed during the reaction that increasing the reaction time, more and more AuCl₄⁻ ions were reduced to zero valent Au⁰ and deposited onto the surface of c-HPMC, resulting enlargement of the size of the Au particles. For the successful synthesis and optimization of c-HPMC/Au, concentration of acrylamide was varied. It was observed (Table 1) that with relatively low concentration of acrylamide, uniform distribution as well as smaller size of AuNPs were obtained, which was difficult with higher monomer concentration.

This is due to the formation of large number of polyacrylamide chains (with more monomer concentration), which entrap each other to form a giant molecular structure. This would result in the disruption of the formation of small and uniform AuNPs.

It has been assumed that the presence of –OH/–OCH₃/–CONH₂ groups along with the porous morphology of the hydrogel stabilizes the gold nanoparticle and also inhibit the aggregation of the nano particles during the formation. This porosity provides an opportunity to rapid diffusion of small nano sized molecules into the cavity. Thus, the particles remain stable and well dispersed within the crosslinked networks.³⁷ It has been also noticed that during the reaction with progress of time after 2 h, the formed Au particles agglomerate to produce bigger particle.

3.2 Characterization

3.2.1 UV-vis spectra. The presence of embedded Au nanoparticles on the surface of crosslinked hydrogel networks is confirmed by UV-visible spectral analysis. Fig. 1 shows the UV-visible spectra of various nanocomposites after reduction from auric chloride. All the composites showed a distinct characteristic peaks between 503.5 nm to 536 nm.^38–41 It was observed that the spectrum of c-HPMC/Au 3 demonstrated sharp UV peak in compare to other composite solutions and also peak value is much lower than that of other solutions. Broader UV peak of nanocomposite solution is an indication of aggregation of gold nanoparticles on the crosslinked hydrogel surface at this reaction condition. The variation in the optical properties of the colloidal gold particles incorporated hydrogel solution is demonstrated in the inset of Fig. 1. From Fig. 1, it is apparent that the reddish colour of the gold based nanocomposite increased gradually from (a–c). This is because, with lower acrylamide concentration, the formation of colloidal gold particles is more favourable in compared to higher acrylamide concentration. However, in case of (d–i), the colour of the nanocomposite solutions were found to be is light to deep violet. The change in colour from reddish to deep violet is an indication of aggregation of AuNPs.⁴²

Fig. 1 Colour change profile and UV-vis spectra of c-HPMC/Au nanocomposites.

The formation of colloidal gold particle is more favourable in lower concentration of acrylamide compare to higher concentration. However, in case of 4 h and 6 h reaction time (d–i) the colour of the gold nanocomposites solutions change from light violet to deep violet which indicates the aggregation of the gold nanoparticles within the hydrogel/gold nanocomposites.⁴²

3.2.2 FTIR spectra. In comparison to FTIR spectrum of HPMC,²⁰ chemically crosslinked HPMC (i.e. c-HPMC) showed few additional peaks (Fig. S1, ESI†). It is evident that all the characteristics absorption peaks of HPMC are present in c-HPMC. However, the additional peaks at 1667 cm⁻¹, 1467 cm⁻¹ and 1327 cm⁻¹ are present, which are assigned to amide-I, amide-II and C–N stretching vibrations of grafted PAAm chains, respectively. Besides, one additional peak was observed at 1730 cm⁻¹, which is the characteristics peak of ester group that is generated from the crosslinker. The presence of these additional peaks suggests the formation of crosslinked hydrogel based on HPMC and polyacrylamide in presence of EGDMA crosslinker. For c-HPMC/Au 3 nanocomposite (Fig. S1b, ESI†), all the corresponding peaks are present, however, the peaks of –OH group (3427 to 3367 cm⁻¹), amide-I (1667 to 1654 cm⁻¹), amide-II (1467 to 1447 cm⁻¹) shifted towards lower value, indicating the interactions between the –OH, –OCH₃ groups as well as amide groups of c-HPMC and AuNPs as proposed in Scheme 1.

3.2.3 ¹³C NMR spectra. Fig. 2 demonstrates the ¹³C NMR spectrum of crosslinked hydroxypropyl methyl cellulose (c-HPMC). Compared to the spectrum of HPMC,²⁰ c-HPMC showed few additional peaks. Peaks at 19.6 ppm and 170.1 ppm arise from the methyl carbon and ester carbon of crosslinker, respectively. In addition, peak at 179.2 ppm corresponds to the amide carbon. Besides, another additional peak was observed at 41.3 ppm, which is responsible for the sp³ carbon atom (i.e. –(CH₂–CH)_n units of polyacrylamide as well as crosslinker in the crosslinked hydrogel, as shown in Scheme 1). The presence of these additional peaks in the spectrum confirmed the formation of c-HPMC. The nanocomposite (c-HPMC/Au 3) contained all the corresponding peaks of c-HPMC, however, the peaks corresponding to amide, primary –OH and secondary –OH, –OCH₃ groups shifted towards higher value, indicating the interactions between these functional groups with Au⁰, as proposed in Scheme 1. Moreover, one extra peak was obtained in c-HPMC/Au 3 at 208.9 ppm, which is because of the keto carbon that is probably formed due to the oxidation of –OH groups present in the crosslinked polymer.

Fig. 2 ¹³C NMR spectra of (a) c-HPMC and (b) c-HPMC/Au 3 nanocomposite.

3.2.4 XRD analysis. Fig. 3 represents the X-ray diffraction pattern of crosslinked hydrogel (i.e. c-HPMC) and gold nanoparticle incorporated c-HPMC (c-HPMC/Au 3). In the X-ray diffraction pattern of pure hydrogel, a broad peak from 20–50° indicates its amorphous nature. Whereas in the X-ray diffraction pattern of gold nanoparticle incorporated c-HPMC in the vicinity of broad peak, two distinct peaks at 2θ = 38.5° and 44.7° can be indexed to the (111) and (200) planes of the cubic gold⁴³ and confirmed the formation of crystalline gold nanoparticles. Here it is essential to mentioned that the other low instance peaks of gold are not identifiable owing to the presence of large amount of amorphous polymer matrix.

Fig. 3 XRD profile of (a) c-HPMC and (b) c-HPMC/Au 3 nanocomposite.

3.2.5 TGA analysis. Fig. S2, ESI† represents the TGA curve of crosslinked HPMC (c-HPMC) and nanocomposite derived from c-HPMC and AuNPs (c-HPMC/Au 3). From TGA curve of c-HPMC, it is apparent that hydrogel showed two extra regions of weight loss in compared to HPMC,²⁰ which are responsible for the loss of crosslinker and polyacrylamide chains present on the hydrogel network. However, in case of c-HPMC/Au 3, one extra weight loss zone was observed in the region of 610–800 °C, which probably due to the presence of AuNPs. This authenticates the presence of Au nanoparticles on the surface of c-HPMC. Besides, from the thermograms, it is apparent that the nanocomposite is thermally more stable than that of c-HPMC.

3.2.6 FESEM, EDAX and Elemental mapping. Fig. 4a and b demonstrate the FESEM images of c-HPMC and c-HPMC/Au nanocomposite, respectively. The FESEM image of c-HPMC (Fig. 4a) clearly indicates about the porous morphology of crosslinked hydrogel, while the nanocomposite image (Fig. 4b) suggests that AuNPs are uniformly distributed on the surface of crosslinked hydrogel. Besides the presence of Au NPs were further confirmed through EDAX analysis (Fig. 4d).

Fig. 4 FESEM and EDAX analysis of (a)/(c) c-HPMC and (b)/(d) c-HPMC/Au 3.

Fig. S3, ESI† represents the EDAX elemental mapping images of C, O, N, and Au for c-HPMC and c-HPMC/Au 3 nanocomposite. In compared to c-HPMC, c-HPMC/Au 3 (Fig. S3, ESI†) shows one extra element Au, which is homogeneously distributed on the surface of c-HPMC (represented as violet points) matrix.

3.2.7 HRTEM analysis. Fig. 5 represents the TEM and HR-TEM images of the synthesized Au NPs incorporated c-HPMC (c-HPMC/Au 3). In the images, the dark spots are supposed to be the Au nanoparticles. From the image it is evident that Au nanoparticles are homogeneously distributed in throughout the polymer matrix, instead of segregated particles. The formed particles are nearly monodispersed with a particle size range of 2–5 nm. In the HR-TEM image (inset) the distinct lattice planes with an interplanar distance of 0.23 nm, correspond to (111) planes of cubic Au, confirmed that synthesized Au nanoparticles are crystalline and dark sports are nothing but Au nanoparticles.

Fig. 5 TEM and HR-TEM (INSET) image of synthesized c-HPMC/Au 3 nanocomposite.

3.3 Biodegradation study

The % weight loss during the biodegradation study is shown in Fig. S4, ESI.† It is obvious that with progress of time the weight of nanocomposite gradually decreased. This is may be because of the enzymatic hydrolysis of β-(1-4) glycosidic linkage of the composite material due to the lysozyme activity. Lysozyme generally degrades the polysaccharide backbone through enzymatic hydrolysis of the β-(1-4) glycosidic linkage through the hexameric sugar ring binding site.^26–28 Initially lysozyme breaks down the suitable glycosidic linkage of the polysaccharide back bone faster rate due to higher availability of the active site. After 1 week the rate of mass loss declined because of lower rate of enzymatic hydrolysis (Fig. S4, ESI†) of the β-(1-4) glycosidic linkage. The constant weight of the nanocomposite (Fig. S4, ESI†) in absence of lysozyme confirmed that the weight loss of the c-HPMC/Au took place only because of the enzymatic degradation of lysozyme.

3.4 Rheological characteristics

From the Fig. 6a, it has been observed that shear viscosity of both c-HPMC and c-HPMC/Au 3 gradually declined which indicates the non Newtonian-shear thinning behavior at all shear rates. Again, from the frequency sweep measurements (Fig. 6b), it has been seen that both G′, G′′ are independent of frequency and the magnitude of G′ is always greater than that of G′′, which suggest the gelation behaviour of the materials.⁴⁴

Fig. 6 (a) Shear viscosity vs. shear rate, (b) G′, G′′ vs. frequency and (c) G′, G′′ vs. shear stress.

Further, the gel strength of the crosslinked hydrogel and nanocomposite were investigated by measuring the respective storage (G′) and loss (G′′) modulus via amplitude sweep measurements within the linear visco-elastic regime (Fig. 6c).

It is apparent that beyond a critical stress value (i.e. yield stress σ_y) both modulii (G′ & G′′) declined sharply to the lower value, which suggests the flow behaviour of the hydrogel. This is attributed to the breakdown of the crosslinked network. The yield stress value of c-HPMC/Au 3 (σ_y = 3416 Pa) is much higher than that of c-HPMC (σ_y = 2664 Pa), indicating the higher gel strength of c-HPMC/Au 3 than that of c-HPMC.

3.5 In vitro cytocompatibility study and cell proliferation

The cell attachment, viability and proliferation studies signify cellular compatibility of a biomaterial intended for drug delivery applications. The cellular attachment on pellet of c-HPMC/Au 3 and control (lysine coated slides) was measured by rhodamine–phalloidin and DAPI assay at various time periods (Fig. 7a).

Fig. 7 Cell viability study of c-HPMC/Au 3 nanocomposite.

It is obvious that the morphology of the cells cultured on control was analogous to that of the cells grown on c-HPMC/Au 3. On day 7, the cells reached confluency and displayed extended actin filaments. This was further supported by MMT reduction assay (Fig. 7b).

The c-HPMC/Au 3 nanocomposite and the tissue culture plate (TCP) maintained viable population of mesenchymal stem cells throughout the study period. The absorbance values obtained from MTT assay were converted to the rate of cell proliferation using a standard curve. After 7 days, the no. of cells on composite material and TCP were 10.9 ± 2.33 × 10⁵ and 4.4 ± 0.30 × 10⁵, respectively (Fig. 7b). The higher number of cell population on c-HPMC/Au 3 may be because of the three dimensional network of the hydrogel that assist more surface area for cells attachment and proliferation. The results confirmed cytocompatibility and non-toxic nature of c-HPMC/Au 3.

3.6 Swelling and deswelling characteristic of c-HPMC and c-HPMC/Au nanocomposites

Fig. 8 and Table S1, ESI† represent the equilibrium swelling ratio (ESR) of crosslinked hydrogel (c-HPMC) and various nanocomposites (c-HPMC/Au) at pH 1.2 and 7.4. The % ESR of nanocomposites (c-HPMC/Au) is much lower than that of graft c-HPMC. This is because of the incorporation of Au NPs on the surface of crosslinked hydrogel, which results the composite structure more rigid and compact, ensuing absorption of less amount of water in compared to c-HPMC. During synthesis of various c-HPMC/Au, we were altered the monomer (i.e. acrylamide) concentration, which is hydrophilic in nature and also responsible for water absorption in hydrogel materials. Thus, from the Table S1, ESI† as well as from Fig. 8, it is obvious that with decrease in monomer concentration from 0.17 moles to 0.0425 moles, the % of equilibrium swelling declined sharply. Moreover, both c-HPMC as well as c-HPMC/Au showed pH dependent swelling behaviour and higher % of equilibrium swelling was observed at alkaline medium. Besides, the equilibrium swelling was attained at 15 h. To determine the swelling rate, the swelling values were fitted in Voigt model (eqn (2)) and the rate parameter (τ) was calculated and reported in Table S1, ESI.†

Fig. 8 Swelling study of c-HPMC and c-HPMC/Au nanocomposite (a) at pH 1.2 and (b) pH 7.4.

It is obvious that the rate parameter (τ) values (Table S1, ESI†) of c-HPMC/Au is higher in both pH media which signify that the rate of water diffusion into the c-HPMC/Au is lower than that of c-HPMC. Besides, among the various nanocomposites c-HPMC/Au 3 has higher τ value, which suggests the lower rate of swelling. Besides, it has also been observed that the rate parameter (τ) values of crosslinked hydrogel and nanocomposites are lower in alkaline medium (pH 7.4) than that of the acidic (pH 1.2) medium, which signifies that the rate of swelling in alkaline medium is higher in compared to acidic medium.

3.6.1 Deswelling study. In order to investigate the water retention behavior, it is important to investigate the deswelling characteristics. It is obvious from Fig. S5, ESI and Table S1, ESI† that the deswelling property of hydrogel/nanocomposite declined sharply and finally reached a plate. It was also observed that higher the temperature, the faster the deswelling rate (Table S1, ESI†), which should be the case. Besides, it was observed that with increase in pH, the deswelling rate gradually increased.

3.6.2 Salt effect on % of equilibrium swelling. The equilibrium swelling characteristics of nanocomposite depends mainly on media characteristics such as solution pH, ionic strength of the solution i.e. on salt concentration.^45,46 It has been observed that the swelling of the absorbents in salt solutions is substantially decreased comparing to the value measured in deionised water. The effect of salt concentration on swelling characteristics of c-HPMC/Au 3 is shown in Fig. S6, ESI.† It is obvious that with increase the charge of the cations, percentage of swelling gradually declined and the water uptake ability of composite material in the investigated salt solutions is in the order of monovalent > divalent > trivalent cations.⁴⁷ This experimental observation suggests that the swelling characteristic of nanocomposite material in different salt solutions depend more precisely on charge density (charge/radius ratio) of the cations of salts. Higher the charge density of the cation of the salt, stronger would be the interactions between the hydrophilic groups of the copolymer matrix and cationic species, which would make the copolymer more resistant towards absorption of solvent molecule. Thus experimental results depict that the equilibrium swelling is lowest in case of trivalent salt solutions, compared to divalent and monovalent. This is because the charge density decreased with increasing the size of cation.

3.7 In vitro release of ornidazole and 5-ASA

The cumulative percentage of drugs release (5-ASA and ornidazole) from HPMC, c-HPMC and different grades of c-HPMC/Au nanocomposites based tablet formulations as a function of time is shown in Fig. 9. From the release profiles, it has been noticed that the rate of release of both drugs were higher at pH 7.4 in compare to pH 1.2. This may be due to fact that at pH 1.2 the nanocomposite is in collapsed state. Thus the diffusion of drugs molecule from tablet formulations to dissolution media is very low, while in alkaline pH, the rate of swelling was higher, resulting faster rate of drug release.

Fig. 9 Release profile of HPMC, c-HPMC and c-HPMC/Au nanocomposite (a) 5-ASA release (b) ornidazole release (results are mean ± SD; n = 3).

From the release profiles, it is perceptible that nanocomposites showed more sustained release behaviour than that of pure HPMC and crosslinked HPMC, which is mainly because of their lower % swelling (Table S1, ESI†) as well as lower rate of erosion (Tables S2 and S3, ESI†). Besides, c-HPMC/Au 3 composite demonstrated most sustained release behaviour. This may be because of the well distributed gold nanoparticles onto the surface of crosslinked polymer, as well as its lowest rate of erosion (Tables S2 and S3, ESI†) and lowest % equilibrium swelling (Table S1, ESI†).

3.7.1 Drug release kinetics. To evaluate the drug release kinetics, the release data were analysed by zero order²⁹ and first order³⁰ kinetics model. It was observed that release of both drugs obeys first order kinetic model rather than zero order model (Tables S2 and S3, ESI†).

3.7.2 Release mechanism. The release data were fitted into the Higuchi model,³¹ Hixson–Crowell model,³² Korsmeyer–Peppas model³³ and nonlinear Kopcha model.³⁴ Among all above models, Korsmeyer–Peppas model is the most fundamental model for determination of the drug release mechanism. The Korsmeyer–Peppas equation³³ is given below:where M_t/M_∞ is the fractional release of drug at time t, ‘k’ is the constant characteristic of drug–polymer system, and ‘n’ is the diffusion exponent characteristic of the release mechanism.³³ The value of ‘n’ plays an important role to define the release mechanism of drug molecules from polymer matrix. When, the value of ‘n’ is n ≤ 0.45 indicates Fickian diffusion, in which the rate of diffusion is less than that of relaxation. The value of ‘n’ in the range of 0.45 < n < 0.89 suggests the mechanism is non-Fickian diffusion or anomalous diffusion, where the diffusion and relaxation rates are comparable. When n > 0.89, the major mechanism of drug release is Case II diffusion (relaxation-controlled transport), where relaxation process is very rapid in compared to diffusion of polymer.³³ In case of 5-ASA and ornidazole release from the nanocomposite, n value lies between 0.45 and 0.89 (Tables S2 and S3, ESI†) which suggest the non-Fickian diffusion mechanism of the drugs release. This implies that drug release depends on both diffusion as well as polymer relaxation.

This is further supported by Higuchi model and Hixson–Crowell models (Tables S2 and S3, ESI†); where the release data well fitted with Higuchi model that supports that the diffusion process is responsible for the drug release.

Finally from the non-linear Kopcha model, it has been observed that, the ratio of diffusional exponent (‘A’) and erosional exponent (‘B’) i.e. A/B value is less than 1 (Table S4, ESI†) which further confirms the non-Fickian diffusion mechanism of drug release. Besides from the values of ‘A’ and ‘B’, it has been concluded that drugs are released from the matrices by both diffusion and erosion process.

3.7.3 Stability study. To investigate the efficacy of c-HPMC/Au 3 nanocomposite as carrier for both colonic drugs, the stability study was performed in accelerated condition till 3 months [Fig. S7† (UV-VIS-NIR and FESEM analysis for tablet formulation), Fig. S8† (release profile of both drugs at initial and after 3 months), and Table S5, ESI†]. It is confirmed from the results that the stability of 5-ASA in c-HPMC/Au 3 is ∼97.5% and ∼98.5% for ornidazole drug.

4. Conclusions

A novel nanocomposite derived from crosslinked HPMC and AuNPs has been successfully synthesized where crosslinked hydrogel acts as reducing agent as well as stabilizing agent. The colour profile and UV-vis spectra of various grades of nanocomposites confirm the reduction of HAuCl₄ and successfully incorporation of gold nanoparticles (Au⁰) onto the surface of crosslinked hydrogel. HR-TEM analysis supports the well distribution of AuNPs on the surface of c-HPMC. Biodegradation study using hen egg lysozyme confirms the biodegradability and hMSCs cell study proves the non-cytotoxicity of the c-HPMC/Au nanocomposite. The swelling characteristics in different phosphate buffer media indicate the pH responsive behaviour of the hydrogel/nanocomposite. The synthesized hydrogel/nanocomposite demonstrates release of colonic drugs (i.e. ornidazole and 5-ASA) in a sustained way.

Acknowledgements

Authors earnestly acknowledge the financial support from Department of Science and Technology, New Delhi, India in form of a research grant (no.: SR/FT/CS-094/2009) to carry out the reported investigation.

References

1. P. L. Lakatos and L. Lakatos, Pharmacol. Res., 2008, 58, 190–195.
2. U. Klotz, Dig. Liver Dis., 2005, 37, 381–388.
3. B. Singh, N. Sharma and N. Chauhan, Carbohydr. Polym., 2007, 69, 631–643.
4. M. R. Saboktakin, R. Tabatabaie, A. Maharramov and M. A. Ramazanov, Carbohydr. Polym., 2010, 81, 631–643.
5. A. N. Shirazi, D. Mandal, R. K. Tiwari, L. Guo, W. Lu and K. Parang, Mol. Pharm., 2013, 10, 500–511.
6. H. Park, H. Tsutsumi and H. Mihara, Biomaterials, 2014, 35, 3480–3487.
7. A. Hebeish, M. Hashem, M. M. Abd El-Hady and S. Sharaf, Carbohydr. Polym., 2013, 92, 407–413.
8. P. S. K. Murthy, Y. M. Mohan, K. Varaprasad, B. Sreedhar and K. M. Raju, J. Colloid Interface Sci., 2008, 318, 217–224.
9. C. E. Probst, P. Zrazhevskiy, V. Bagalkot and X. Gao, Adv. Drug Delivery Rev., 2013, 65, 703–718.
10. X. He and N. Ma, Colloids Surf., B, 2014, 124, 118–131.
11. L. Momtazi, S. Bagherifam, G. Singh, A. Hofgaard, M. Hakkarainen, W. R. Glomm, N. Roos, G. M. Mælandsmo, G. Griffiths and B. Nyström, J. Colloid Interface Sci., 2014, 433, 76–85.
12. D. Pissuwan, T. Niidome and M. B. Cortie, J. Controlled Release, 2011, 149, 65–71.
13. A. M. Alkilany, L. B. Thompson, S. P. Boulos, P. N. Sisco and C. J. Murphy, Adv. Drug Delivery Rev., 2012, 64, 190–199.
14. C. J. Bishop, S. Y. Tzeng and J. J. Green, Acta Biomater., 2015, 11, 393–403.
15. A. Kumar, X. Zhang and X. J. Liang, Biotechnol. Adv., 2013, 31, 593–606.
16. P. Ghosh, G. Han, M. De, C. K. Kim and V. M. Rotello, Adv. Drug Delivery Rev., 2008, 60, 1307–1315.
17. Y. Murali Mohan, T. Premkumar, K. Lee and K. E. Geckeler, Macromol. Rapid Commun., 2006, 27, 1346–1354.
18. H. Huang and X. Yang, Biomacromolecules, 2004, 5, 2340–2346.
19. D. Das, R. Das, P. Ghosh, S. Dhara, A. B. Panda and S. Pal, RSC Adv., 2013, 3, 25340–25350.
20. R. Das, A. B. Panda and S. Pal, Cellulose, 2012, 19, 933–945.
21. D. Das, R. Das, J. Mandal, A. Ghosh and S. Pal, J. Appl. Polym. Sci., 2014, 131, 40039.
22. R. Das, D. Das, P. Ghosh, A. Ghosh, S. Dhara, A. B. Panda and S. Pal, Cellulose, 2015, 22, 313–327.
23. J. O. Karlsson and P. Gatenholm, Polymer, 1997, 38, 4727–4731.
24. K. Pal, A. K. Banthia and D. K. Majumdar, Des. Monomers Polym., 2009, 12, 197–220.
25. N. A. Peppas, P. Bures, W. Leobandung and H. Ichi, Eur. J. Pharm. Biopharm., 2000, 50, 27–46.
26. R. Justin and B. Chen, Carbohydr. Polym., 2014, 103, 70–80.
27. T. Freier, H. S. Koh, K. Kazazian and M. S. Shoichet, Biomaterials, 2005, 26, 5872–5878.
28. K. Tomihata and Y. Ikada, Biomaterials, 1997, 18, 567–575.
29. M. Donbrow and Y. Samuelov, J. Pharm. Pharmacol., 1980, 32, 463–470.
30. M. Gibaldi and S. Feldman, J. Pharm. Sci., 1967, 56, 1238–1242.
31. T. Higuchi, J. Pharm. Sci., 1961, 50, 874–875.
32. A. W. Hixson and J. H. Crowell, Ind. Eng. Chem., 1931, 23, 923–931.
33. R. W. Korsmeyer, R. Gurny, E. Doelker, P. Buri and N. A. Peppas, Int. J. Pharm., 1983, 15, 25–35.
34. M. Kopcha, N. G. Lordi and K. J. Toja, J. Pharm. Pharmacol., 1991, 43, 382–387.
35. R. Chen, Q. Chen, D. Huo, Y. Ding, Y. Hu and X. Jiang, Colloids Surf., B, 2012, 97, 132–137.
36. N. Wangoo, K. K. Bhasin, S. K. Mehta and C. R. Suri, J. Colloid Interface Sci., 2008, 323, 247–254.
37. Y. M. Mohan, T. Premkumar, K. Lee and K. E. Geckeler, Macromol. Rapid Commun., 2006, 27, 1346–1354.
38. Y. W. Cao, R. Jin and C. A. Mirkin, J. Am. Chem. Soc., 2001, 123, 7961–7962.
39. H. Huang and X. Yang, Biomacromolecules, 2004, 5, 2340–2346.
40. S. Link, Z. L. Wang and M. A. El-Sayed, J. Phys. Chem. B, 1999, 103, 3529–3533.
41. P. Mulvaney, Langmuir, 1996, 12, 788–800.
42. K. S. Mayya, V. Patil and S. Sastry, Langmuir, 1997, 13, 3944–3947.
43. J. Zhang, D. Han, H. Zhang, M. Chaker, Y. Zhao and D. Ma, Chem. Commun., 2012, 48, 11510–11512.
44. A. Pal and J. K. Dey, Langmuir, 2011, 27, 3401–3408.
45. P. Pal, K. Mehran, M. R. Gholam and H. Hossein, Polym. Bull., 2006, 57, 813–826.
46. V. B. Bueno, R. Bentini, L. H. Catalani and D. F. S. Petri, Carbohydr. Polym., 2013, 92, 1091–1099.
47. Q. Li, Z. Ma, Q. Yue, B. Gao, W. Li and X. Xu, Bioresour. Technol., 2012, 118, 204–209.

---

Footnote

† Electronic supplementary information (ESI) available: Methodology for preparation of nanocomposite pellets for cell proliferation study, determination of % erosion, FTIR spectra (Fig. S1), TGA analysis (Fig. S2), elemental mapping (Fig. S3), biodegradation study (Fig. S4), swelling and deswelling kinetics parameters (Table S1), deswelling study (Fig. S5), effect of salt concentration on % swelling (Fig. S6), release parameters (Tables S2–S4), drug–matrix interaction (Fig. S7), release profile at initial and after 3 months (Fig. S8), stability study results (Table S5). See DOI: 10.1039/c5ra02672e

---