pH-Responsive Biocompatible Nanocomposite Hydrogels for

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pH Responsive Biocompatible Nanocomposite Hydrogels for Therapeutic Drug Delivery Rabia Kouser, Arti Vashist, Mohammad Zafaryab, Moshahid A Rizvi, and Sharif Ahmad ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00260 • Publication Date (Web): 08 Nov 2018 Downloaded from http://pubs.acs.org on November 8, 2018

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ACS Applied Bio Materials

pH Responsive Biocompatible Nanocomposite Hydrogels For Therapeutic Drug Delivery Authors: Rabia Kousera, Arti Vashista,c, Mohammed Zafaryabb, Moshahid. A. Rizvib, Sharif Ahmada* Rabia Kousera, Material Research Laboratory, Department of Chemistry, Jamia Millia Islamia, New Delhi, 110025. Orcid iD: 0000-0002-6884-1629. Arti Vashistc, Center of Personalized Nanomedicine, Institute of NeuroImmune Pharmacology, Department of Immunology, Herbert Wertheim College of Medicine, Florida International University, Miami, FL-33199, USA. Orcid iD: 0000-0002-7519-1863 Mohammed Zafaryabb, Genome Biology Lab. Department of Biosciences, Jamia Millia Islamia, New Delhi, 110025. Orcid iD: 0000-0003-3591-3371 Moshahid. A. Rizvib, Genome Biology Lab. Department of Biosciences, Jamia Millia Islamia, New Delhi, 110025. Orcid iD: 0000-0001-5744-0136. Sharif Ahmada*, Material Research Laboratory, Department of Chemistry, Jamia Millia Islamia, New Delhi. Orcid iD: 0000-0001-5799-7348. *Corresponding

Author

Email:

[email protected];

Tel:+911126947508.

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Fax:+911126840229;

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ABSTRACT Present study reports the formulations of biocompatible nanocomposite hydrogels using chitosan (CH), polyvinyl alcohol (PVA), Oleo polyol and fumed silica (SiO2) via free radical polymerization method for anticancer drug delivery. The structural, morphological and mechanical analysis were conducted using FT-IR spectroscopy, SEM, TEM and rheological techniques. The effect of SiO2 concentration on mechanical strength, swelling ratios, morphological and drug delivery behavior was investigated. The incorporation of SiO2 nanoparticles in hydrogels resulted in a significant enhancement in its properties. MTT assay of a human embryonic kidney (HEK-293) and human colon (HCT116) cancer cell lines was conducted up to 48h for biocompatibility, and cytotoxicity tests. These studies confirmed the biocompatible nature of nanocomposite hydrogels. The cisplatin loaded nanocomposite hydrogels exhibits sustained release as compare to that of free cisplatin at pH 4.0 and pH 7.4. The in vitro cytotoxicity test of cisplatin-loaded hydrogels using human colon (HCT116) cancer cell line indicates that these hydrogels successfully inhibited the growth of HCT116 cancer cells. The results of in-vitro tests for drug loading, sustained release, biodegradability, biocompatibility and anti-proliferative activity of cisplatin loaded nanocomposite hydrogels suggest that in future, it may find applications for the development of topical (in vivo, in the form of tablets) drug delivery systems. Keywords: Biocompatible, Nanocomposite hydrogels, Drug delivery, Cisplatin, SiO2. 1. Introduction Over the last few decades, the sustainable and biocompatible polymer nanocomposite hydrogels have attracted the attention of researchers due to their significant applications in the area of biomedical sciences.1


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The treatment for cancer is a great challenge to the scientists and medical experts.2 Chemotherapy has been found as a promising therapeutic alternative to clinicians.3 Among various chemotherapeutic agents, cisplatin is the most active drugs, used for the treatment of solid tumors.4 However, its application is still limited for cancer chemotherapy, due to its nonspecific bio-distribution and severe side effects.5 The tolerable doses of cisplatin have shown undesirable side effects in cancer patients.2 One of the techniques, which minimize these side effects, is the targeted delivery of the drug in a slow and prolonged manner that could be achieved by sustained hydrogels.6,7 Hydrogels act as delivery vehicles by encapsulating the small-sized drug molecules within its interstitial spaces of cross-linked network.8 Up on administration they come in contact with physiological fluids and swells, increasing the gap between the polymeric networks, thereby provide the passage for the diffusion of drugs to target site.9 Recently, the use of biodegradable polymer-based hydrogels for cisplatin delivery has attracted the attention of researchers.10 Among various biodegradable polymers, chitosan has been regularly used in the field of various biomedical applications due to its easy availability, biodegradability, biocompatibility, nontoxicity and pH sensitivity.11,12 However, the poor mechanical strength and fast absorbing capability at low pH, results in fast release of drugs that limits its application in drug delivery systems.13 In order to overcome these shortcomings, scientists modified chemical structure of chitosan using various water soluble synthetic polymers such as polypropylene, polyvinylchloride, and polyvinyl alcohol (PVA) etc.14–16,17 These compounds bear large number of functional groups that can interact with chitosan imparting extra chemical interactions within the structure, hence enhancing its physical properties and in vivo biodegradability.18,19 Among these polymers, PVA is found to provide the necessary properties required for the drug delivery systems.20’21 However, the sustained release of drugs loaded within these hydrogels could not be achieved using this method.22 Therefore, further modifications are required such 3 ACS Paragon Plus Environment


as development of nanocomposite hydrogel systems, which interact with the drug molecules, stabilizing the drug-hydrogel system and slowing the release rate.21,23–25Among the various nanofillers used in hydrogel compositions, the incorporation of fumed silica nanofillers not only provides sustained drug release but also imparts superior physicochemical and mechanical properties, thermal stability, excellent biocompatibility, optical transparency, low toxicity, and versatile functionalities to the nanocomposite hydrogels.26,27 Literature reveals that the use of fumed silica nanoparticles within the permissible limits (5-50mg/kg) of toxicity, induced highly porous and hydrophilic structure to hydrogels.28,29 Thus, the homogeneous dispersion of biocompatible fumed silica nanoparticles in sustainable nanocomposite hydrogels provide promising opportunities for strategic drug delivery system, especially for the treatment of cancer therapy30. It is interesting that these nanoparticles tend to eliminate from the body through the complementary system or urine after targeted delivery.28 Popat et. al. have reported silica nanoparticles based nanocomposite hydrogels with reference to their application in chemotherapeutic drug delivery, exhibiting reduced side effects.31 Bijay et. al. also designed hydrophilically modified chitosan-PVA and silica-based nanocomposite membrane by sol-gel method.32 However, no work has been reported on chitosan, polyvinyl alcohol, silica-based composites for chemotherapeutic drug delivery system. Keeping these in mind, we have used silica nanoparticles within permissible range in the present hydrogels. Herein, we report a novel formulation of hydrophobically modified chitosan - PVA using linseed oil derived polyol (as a cross-linking agent), and SiO2 nanoparticles based nanocomposite hydrogels via free radical polymerization technique. The structural, morphological, and physio-mechanical properties of these hydrogels were characterized by FT-IR, SEM and rheological techniques respectively. Their biocompatibility, and cytotoxicity studies were investigated with the help of HEK-293 and human colon (HCT116) 4 ACS Paragon Plus Environment

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cancer cell lines, which confirms their biocompatible behavior. Cisplatin was chosen as a model drug in the present system to prove the pH responsive behavior of resultant nanocomposite hydrogels anticancer drug delivery. The studies further confirmed that the loading and sustained release rate of cisplatin drug using fumed silica dispersed nanocomposite hydrogel (3mg,10mg) is much higher than those of such systems.22,33,34 The in vitro cytotoxicity of cisplatin-loaded hydrogels using HCT116 cancer cell line confirmed that these hydrogels successfully kills the HCT116 cancer cells. The present study further reveals that the proposed nanocomposite hydrogels show a potential scope for their application in the field of anticancer drug delivery.

2. Experimental section 2.1 Materials Chitosan (CH) C6H11NO4, (Mol. Wt. 1526.464 g/mol., Solubility-33.3 mg /mL in dilute acids,), N, N-methylenebisacrylamide (MBAAm) C7H10N2O2, (Mol. Wt. 154.17, MB005100G), N, N, No, No-tetra methyl-ethylene diamine (TEMED) C6H16N2, (Mol. Wt. 116.21, Acrylamide (GRM305-100G), CH2=CHCONH2, (Mol. Wt. 71.08, minimum assays: 99.0%), MTT assays and DMEM (Dulbecco’s modified Eagle’s medium), 0.25% trypsin and 0.02% ethylene diamine tetra acetic acid (EDTA) mixture were purchased from Himedia,(India). polyvinyl alcohol (PVA), (-CH2CHOH-)n, (Mol. Wt. 1,25,00, density 1.19-1.31 g/cm, Melting point- 200 °C, Boiling point 228 °C,) (CDH) Mumbai, (India). Fumed Silica (Silicon dioxide (SiO2) packed under nitrogen, with average particles size: 5-50nm, Specific surface: > 200 m2 /g, Bulk Density: ca.0, 048 g/cm3, Purity: > 99, 8%, excl. ca. 2% moisture), Plasma Chem GmbH Rudower Chaussee 29, D-12489 Berlin. Buffer tablets of pH 4 and pH 7.4 were purchased from S.D. Fine chemicals ltd, India. Cisplatin (cis-Diamine platinum (II) dichloride) an anticancer drug, (Mol. Wt. 300.05, ≥ 99.9% trace metals basis) and Fetal bovine serum (FBS) was procured from Gibco (South Africa). The Linseed oil polyol 5 ACS Paragon Plus Environment


(Reflective index 1.489, specific gravity 1.063, and inherent viscosity 0.9214) was prepared as per reported procedure.8 All the reagents were used as such. 2.2 Synthesis of polyvinyl alcohol/chitosan/polyol (PCB) hydrogel and polyvinylalcohol/chitosan/polyol/fumed silica nanocomposite (PCBSF) nanocomposite hydrogels The PCB and PCBSF hydrogels were prepared using 2% chitosan in 1% acetic acid solution, 2% PVA, and 1% linseed polyol. The polymer blends of these constituents were made in different ratios (Table1) via free radical polymerization technique23. Initially, mixing of chitosan and PVA was carried out by stirring in a beaker with the help of magnetic stirrer followed by the addition of polyol, MBAAm, APS, and TEMED as a crosslinker, initiator, and accelerator, respectively. Nitrogen gas was purged for 3-4 minutes in the reaction mixture to provide an inert environment during the formulation of hydrogels. The reaction mixture was kept for 5-10 minutes for gelation at room temperature (25oC). The complete gel formation took place within 30 minute. The thick PCB-3 (CH/Polyvinyl alcohol/Polyol) hydrogel blocks were cut into cubes of 1×1 cm area with the help of surgical blade. The stability of these hydrogel cubes was tested by keeping them in 10 mL double distilled water (DDW) for 24 h in the test tube. After 24h, no dissolution or any other visible changes in water-dipped cubes were observed. Similarly, the PCBSF nanocomposite hydrogel cubes containing different concentrations (3, 6 and 10 mg) of fumed silica (SiO2) nanoparticles were made following the method used for PCB-3. The SiO2 nanoparticles were homogeneously dispersed within the PCB-3 hydrogel (a pre-polymer) using wensar ultrasonic cleaner (Model LMUC-4) in a sonication up to 1h. After obtaining a homogeneous mixture, the PCBSF gels were prepared following the same procedure as described above for PCB-3 hydrogel. Table 1. Various compositions of PCB plain and PCBSF nanocomposite hydrogels.


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S.NO

Chitosan

PVA

Polyol

APS

TEMED

Fumed

(1%) (w/v)

Bis/AAm 26:4 (30%)mL

(2%)

(2%)mL

(5%) mL

(mL)

silica(mg)

(w/v)

PCB-3

10

10

3

10

1

0.2

0

PCBSF-3

10

10

4

10

1

0.2

3

PCBSF-4

10

10

6

10

1

0.2

6

PCBSF-6

10

10

8

10

1

0.2

10

Where PCB-3 (Mixture of Chitosan-Polyvinyl alcohol and 3mL of 1% Polyol in a 100mL beaker). Whereas, in case of PCBSF-3, PCBSF-4 and PCBSF-6 (Chitosan-Polyvinyl alcoholPolyol-SiO2). The PCBSF-3, PCBSF-4 and PCBSF-6 having different concentrations of SiO2 (3mg, 6mg and 10mg) in (pre-polymer) Chitosan-Polyvinyl alcohol-Polyol. 2.3 Purification and drying of PCB and PCBSF hydrogels. The PCB and PCBSF hydrogel cubes were washed 4 to 5 times by tumbling in double distilled water at room temperature (25oC) for 24h to eliminate the unreacted monomers, crosslinkers and initiators. The hydrogels of different compositions were kept on glass slides for air drying under ambient conditions for 5 to 10 days, resulted in the formation of completely dried hydrogel cubes of PCB and PCBSF. 3. CHARACTERIZATION AND MEASUREMENTS The PCB and PCBSF hydrogels were further dried at room temperature for 24 h for FT-IR analysis. The chemical structures of these dried hydrogels were investigated using FT-IR spectra in the range of 400-4000 cm-1 recorded by FT-IR spectrophotometer (Perkin Elmer Cetus Instruments, Norwalk, CT). The morphology of these dried and swelled PCB and PCBSF hydrogel samples was investigated using SEM (Environmental Scanning Electron 7 ACS Paragon Plus Environment


Microscope model FEI Quanta 200F). These hydrogels were primarily swelled in aqueous media at pH 4.0 and pH 7.4 under room temperature (25oC) for 24 h. The internal structure of these swelled specimens was examined after freeze drying by tumbling in liquid nitrogen, lyophilizing for 24h to attain the complete drying, and porous structure without any split. Transmission electron microscopy (TEM) a Model Philips Morgagni, 268 operating at 80 KV, (AIIMS, New Delhi, India) was used to characterize the size and morphology of dispersed SiO2 in PCBSF. The samples of these hydrogels for TEM analysis were prepared by crushing and dispersing in deionized water. The dilute suspensions of these samples were initially sonicated (100 W) for 10-20 minutes under the ice-water bath, thereafter, several drops of sonicated suspensions were deposited onto a standard holey carbon-coated copper grid. These grids were then stained with uranyl acetate solution (2wt %) for 30 seconds, prior to their analysis, kept overnight for drying in a vacuum oven at 30oC and then located in the TEM chamber for image attainment using ASTM method. The rheological properties (storage modulus) G' and loss modulus (G'') of PCB and PCBSF hydrogels were investigated with the help of a rheometer (MCR101, Anton Peer, Japan). The parallel plate measuring system along with 25 mm diameter plate was applied with the separation distance between the measuring plates (the gap distance) as 1 mm. The experiments were conducted at 37oC under the regulation of Peltier unit. The storage modulus (G') and Loss modulus (G") was measured at a frequency sweep over the frequency range of 0.01-10 Hz. The X-ray diffractometric analysis of PCB and PCBSF hydrogels was conducted using Siemens X-ray diffractometer, model D5000 equipped with Ni-filtered Cu Kα radiation (λ = 1.5406Ǻ) within the angle range of 2θ=10-60O. The diffractometer was operated with 1o diverging and receiving slits at 50 kW and 40mA, continuous scans of these samples were carried out. 3.1 Swelling studies 3.1.1 Swelling ratio (Sr) and equilibrium swelling ratio (%EWS) 8 ACS Paragon Plus Environment

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The swelling ratios of PCB and PCBSF hydrogels were investigated in phosphate buffer solutions (PBS) at pH 4.0 and pH 7.4. The water uptake capability was determined by gravimetric measurements using an analytical balance (Sartorius balance; model BP210S). The gels were placed in test tubes containing 25 ml PBS at room temperature. After the desired time intervals (0 min,10 min, 20 min, 30 min,…. up to 4320min.), the swelled hydrogels were wisely taken out from the stock solutions, sponged with Whatman filter paper so as to remove the extra liquid present on the exterior of hydrogel cubes. These hydrogels were then refilled in a parallel manner with the new PBS at the same pH. The swelling ratio of these hydrogels was determined using the following equation. 21 Swelling Ratio = Wt-Wo/Wo Where

and

(1)

are the weights of the sample in dry and swollen states at time t,

respectively. Further, equilibrium water content % EWS of the fully hydrated PCB and PCBSF was considered after 72h, once they attained the equilibrium, and calculated using the equation.35 %EWS = (We-Wo)/Wo× 100 Where

and

(2)

are the weights of the sample in dry and completely swollen states

(equilibrium), respectively. In addition, the % EWS of PCB and PCBSF hydrogels in various simulated physiological solutions such as urea, sodium dihydrogen phosphate (NaH2PO4), Dglucose, sodium chloride (NaCl), potassium nitrate (KNO3) and potassium iodide (KI) was also calculated. 3.2 Hydrolytic degradation The in vitro hydrolytic degradation of PCB and PCBSF hydrogels was performed in pH 7.4 and pH 4.0 at 25 oC. The weight loss of the hydrogel samples was recorded at a given time



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periods. All measurements were done in triplicate and the rate of degradation was determined by the change in their mass loss using equation.26 Wet Mass Change (%) = Wt/We×100

(3)

Where Wt and We are the weight of hydrogels in their unique equilibrium swollen states at time t at different pH conditions. 3.2.1 Soil burial degradation The Soil burial tests on PCB and PCBSF hydrogels were accomplished under adequate (average amount) soil for eight weeks. This soil was slightly acidic with a pH 6.7, found from Indian Agricultural Institute (PUSA, New Delhi, India) .36 These specimens of 1cm×1cm size were buried in the soil under 30% moist environment in separate beakers and the degradation of all the specimens was cautiously determined at various intervals. 3.3 Cell culture 3.3.1 Cell line Maintenance and MTT assays The MTT assay is generally used to validate the cytotoxicity/ biocompatibility of any biological and synthetic materials in vitro.37 Thus, the biocompatibility/ viability/ antiproliferative activity of PCB and PCBSF hydrogels was measured in in vitro condition using normal HEK-293 and HCT116 human colon cancer cell lines. The cells were procured from National Centre for Cell Sciences (NCCS, Pune, India). The technique used for the evaluation of compatibility was same as described in our previous work.26 3.4 Method used for the evaluation of biocompatibility and cytotoxicity The MTT assay test was conducted for the evaluation of biocompatibility of PCB and PCBSF hydrogels by using the method described in our previous published research work. 26, 38, 39 3.4.1 Cisplatin-loaded hydrogels The in Vitro loading of cisplatin onto the PCB and PCBSF hydrogels (PCB-3, PCBSF-3, and PCBSF-6) was carried out at pH 4.0 and pH 7.4. The dry hydrogel samples were weighed and soaked in 10 mL PBS containing cisplatin drug (5.5 mM) at given pH for 72h. The amount of 10 ACS Paragon Plus Environment

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cisplatin-loaded onto the gels was measured by UV spectroscopy (a Lambda 950 UV/Vis spectrophotometer; Perkin-Elmer). The complexed gels were washed with PBS solution before drying. The maximum loading of cisplatin was determined after 72h of treatment when gels turned into yellow color. The supernatant liquid of the drug solution was analyzed at a wavelength of 309 nm and 301 nm at pH 4.0 and pH 7.4, respectively. The % drug loading was calculated using equation.40 Drug Loading (%) = Weight of drug in nanocomposite hydrogel/Weight of nanocompositehydrogel×100

(4)

3.4.2 Cisplatin release from hydrogels In vitro release experiments on the cisplatin loaded PCB and PCBSF hydrogels (PCB-3, PCBSF-3, and PCBSF-6) were carried out by placing them in 10mL PBS solutions in a test tube at pH 4.0 and pH 7.4 for 144h at room temperature. At planned time intervals, 3ml of the solution was withdrawn and the same amount of the dissolution medium was added back to the beaker to maintain a constant volume. The amount of cisplatin released was analyzed by measuring the absorbance of the solution in UV-Vis spectrophotometer at a given time and above-mentioned wavelength range. The standard calibration curve was derived from the absorbance spectra at 309 nm and 301 nm wavelengths for different concentrations of standard solution of cisplatin drug. The drug release (%) studies were performed in triplicate and their mean average was calculated using the below given equation.40 Drug Release (%) = Concentration× Dissolution bath volume × dilution factor/1000

(5)

4. RESULTS AND DISCUSSION 4.1 Structure and stability of PCB and PCBSF hydrogels The digital pictures of swollen PCB and PCBSF hydrogels were captured using a digital camera “Canon IXUS 185 20MP at 8x Optical Zoom” (Figure S1), which revealed that the 11 ACS Paragon Plus Environment


PCB and PCBSF exhibit enhanced the integrity of hydrogels in PBS at pH 4.0 and pH 7.4. This stability can be attributed to the presence of SiO2 nanoparticles trapped within the crosslinked spaces of PCBSF and linked with hydrogen bonds between amine/ hydroxyl groups of CH, hydroxyl groups of PVA and polyol.41 Scheme 1 represents the scheme for the proposed mechanism involved in the formation of PCB and PCBSF nanocomposite hydrogels, depicting various possible interactions. Those interactions in nanocomposite hydrogel induced highly crosslinked structure, which prevented the termination of hydrogel networks up to certain interval of time.

Scheme 1. Schematic illustration for the preparation PCB and PCBSF hydrogels. Figure 1A; Showing the FT-IR spectroscopy of PCB and PCBSF hydrogels. The dispersion of SiO2 nanoparticles led to the broadening of peaks at 3296 cm-1 (-NH2/-OH stretching vibration), which is much higher in PCBSF than in PCB, which further support the TEM 12 ACS Paragon Plus Environment

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analysis of swelling behavior of these hydrogels (Figure.3). In addition, the homogeneous dispersion of SiO2 in PCBSF results in the close entanglements of polymer chains, which imparts high mechanical strength to its structure (Figure S2). Moreover, the MTT assay study on HEK-293 Cell line has shown that PCB and PCBSF cause an insignificant decline in cellular viability even after 48h treatment. The biocompatible behavior of the nanocomposite hydrogels could act as a game changer in the field of drug delivery system.42,43 Figure 1A; showing the FT-IR absorption spectra of PCB-3, PCBSF-3 and PCBSF-6. The peaks observed at 3190 cm

-1

and 3296 cm

-1

in all the three cases signifies –OH/NH2

stretching vibration, which indicates the formation of hydrogen bonding between CH-PVApolyol.44 The presence of broad peaks at 3296 cm-1 in PCB-3, PCBSF-3, and PCBSF-6, suggest strong interactions between –OH groups of polyol and -NH2/-OH groups of chitosan.23 However, in case of PCBSF-3 and PCBSF-6, the broadening at 3296 cm-1 is much higher than that of PCB-3.44 The peaks at 1649 cm−1,1539 cm−1 and 1415 cm-1 may be attributed to the carbonyl stretching, absorption of -NHCOCH3 (acetamide) groups and C-H bonds of CH, PVA polymeric chains.45,46 The peak around 1090 cm-1 in all cases is assigned to C-O stretching vibration of the chitosan moiety.47 Moreover, the absorption peaks, which were observed at 2925 cm−1 and 2876 cm−1 in all cases (PCB 3, PCBSF 3 and PCBSF-6), are due to the -CH2 asymmetric stretching vibration and the C-H bond of chitosan and polyvinyl alcohol respectively.48 However, the greater intensity of the peaks in the case of PCBSF-3 and PCBSF-6 could be an evidence for the integration of SiO2 nanoparticles, enhancing the stretching vibration of the polymeric chains. XRD patterns (Figure. 1B); of CH, PVA, fumed silica (SiO2), PCB-3, PCBSF-3 and PCBSF6 exhibit a broad peak at about 21.8o confirming amorphous nature of pure silica.49 For chitosan (CH), the major peak of chitosan (Figure 1B); was observed at 2θ=20o (maximum intensity), corresponds to the characteristic peak of CH chains aligned through 13 ACS Paragon Plus Environment


intermolecular interactions, which can be attributed to the hydrated crystalline structure, while the presence of broad peak indicates the predominant amorphous structure of CH.50 For pure PVA, X-ray pattern (Figure 1B); exhibits peaks at 2θ =19.55o and 40.95o, indicating semi-crystalline structure that can be due to the presence of hydroxyl groups in the side-chain of the PVA51. However, in case of PCB-3; the hydroxyl groups of PVA molecules might react with CH species; thus, increasing the chain mobility, which results in the decrease of its crystalline nature.52 After the incorporation of SiO2 in PCB matrix (PCBSF-3), the standard peak assigned to PCB-3 at 2θ =23° has shifted towards the higher θ values i.e. 2θ = 24° in PCBSF-3, while in the case of PCBSF-6 it is shifted to 2θ = 24.3°. The X-ray studies revealed that on increasing the concentration of SiO2, the diffraction peaks shifted towards the higher θ values, whereas the intensity of these peaks decreases after the dispersion of SiO2. These results confirm the intercalation between CS, PVA and SiO2 nanostructure. In addition, the presence of SiO2 hindered the polymer crystallization due to the steric restriction and resulted in a physically cross-linked structure.53

Figure 1. (A) FT-IR spectra of PCB-3, PCBSF-3 and PCBSF-6. (B) XRD pattern of chitosan (CH), Fumed silica, PVA, PCB-3, PCBSF-3 and PCBSF-6.


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The SEM micrographs of PCB-3, PCBSF-3 and PCBSF-6 are depicted in Figure 2A-H. The micrograph of dry PCB-3 gel revealed the formation of a smooth surface without any noticeable defects (Figure 2A-B). While the swollen surface of the same sample exhibits the presence of many globules and pores, which are distributed uniformly on the surface (Figure 2C- D); On the other hand, the surface topology of PCBSF-3 (Figure.2E-H); possesses voids and micropores along with flakes and porous structure. In PCBSF-3, at higher magnification, SiO2 nanoparticles are visible as chain/ring structure (Figure 2E-F); which is likely due to the phase separation. The difference in the polymer and inorganic phase distribution indicate the two-phase discrete structure54. In the case of PCBSF-6, with increasing the concentration of SiO2, formation of a swelled layer is observed (Figure 2G-H). It seems that the SiO2 nanoparticles are slightly connected with each other at the concentration of 10mg supporting the hypothesis of partial agglomeration of fumed silica into CH, PVA and polyol based matrix. The agglomerated structure resulted due to dipole-dipole interactions between the silica nanoparticles, due to the presence of surface hydroxyl groups of polymer matrix.53 Finally, the miscibility of these components increasing, leading to the remarkable improvement in rheological properties (Figure S2) of the polymeric materials.



Figure. 2 (A-D) SEM micrographs of dry and swollen PCB-3 (pH-4), at 2.50KX, 2.00KX, 212X, and 150X. SEM micrographs of PCBSF-3 and PCBSF-6 (E-H) at 1.50KX, 4.13KX, 3.00KX, 3.68KX respectively. The transmission electron micrographs of PCBSF-3 and PCBSF-6 (Figure.3); reveal the chain and ring distribution pattern of SiO2 nanoparticles in PCBSF matrix and show the spherical globular morphology of relatively uniform size ranging from 50 to 200 nm (Figure 3A-D). In case of PCBSF-3, linear and smooth chains of SiO2 sphere particles are visible (Figure 3A-B). The linear chains of closely well connected SiO2 nanoparticles in CH/PVA/Polyol matrix. This can be attributed to the hydrogen bonding and dipole-dipole interaction between SiO2 and CH/PVA/Polyol matrix49. While in case of PCBSF-6, the chain of silica particles with increasing concentration of fumed silica (10mg), acquired the ringshaped morphology along with linear chain (Figure.3C-D). The formation of chain and ring structure could be assigned to the electrostatic interaction among –OH groups of CH, PVA and polyol matrix with SiO2 nanoparticles. However, in both the cases negligible agglomeration is observed, this suggests that SiO2 nanoparticles act as physical fillers, which 16 ACS Paragon Plus Environment

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may further stabilize the nanocomposite hydrogel network leading to the enhanced mechanical properties of the polymeric materials.

Figure 3. TEM of PCBSF-3 at magnification 200kv×9900 (A and B), and PCBSF-6 at magnification 200kvj×19200 (C and D). 4.2 Rheological studies of PCB and PCBSF hydrogels The rheological behavior of PCB-3, PCBSF-3 and PCBSF 6 was investigated using frequency sweep test. The dynamic mechanical properties i.e. storage modulus (G') and loss modulus (G'') are plotted against angular frequency for these hydrogels at room temperature (25o) (Figure S2). The dispersion of SiO2 nanoparticles in PCBSF hydrogels resulted in the enhancement of G' as compared to PCB hydrogel. The oscillatory frequency studies under sweep test of hydrogels revealed that the presence of SiO2 (3mg, 10mg) nanoparticles in PCBSF-3 and PCBSF-6 hydrogels induces higher magnitude of G' values i.e. 128000Pa and 141000 Pa, respectively, which is found to be almost two fold higher than that of PCB-3 (66900Pa) hydrogel, confirming the significance of concentration of SiO2 nanoparticles that 17 ACS Paragon Plus Environment


influences the G'. The higher values of G' confirmed that the PCBSF-3 and PCBSF-6 hydrogels behave as a more rigid and superior elastic material compared to PCB-3. The higher G' in PCBSF-3 and PCBSF-6 might be due to the higher cross-linked density induced by the presence of strong bond in SiO2 nanoparticles within the polymer matrix, which hindered the chain mobility and deformability of polymer.55 While the lower G' for PCB-3 is attributed to the presence of large spaces within the polymer network structure that helps in imbibing the excess amount of water, enhancing the swelling ability of PCB-3 hydrogel.56 This behavior has been also reported earlier in various studies that the gel strength is inversely proportional to the swelling capacity of hydrogel56 i.e. the hydrogel of more mechanical strength should have the lower swelling capacity as observed in case of PCBSF-3 and PCBSF-6 (Figure.4A,B). On the other hand, the performance of G'' verses frequency test are shown (Figure S2). The G'' values of PCBSF-3, PCBSF-6, and PCB-3 were measured and it was found that the presence of SiO2 (3mg, 10mg) nanoparticles in PCBSF-3 and PCBSF-6 hydrogels induce higher degree of G''-values i.e. 20600 Pa and 35400 Pa, respectively than that of PCB-3 (14000 Pa) hydrogel. This implies that PCB-3 gel shows low elasticity while PCBSF-3, PCBSF-6 have higher elasticity. The flatness of curves (frequency independent behavior) and the magnitude of elastic modulus indicate that the material is hard to gel like. Further, all these data indicate that the stiffness of the gel increases considerably upon the incorporation of SiO2 into the polymer matrix.55 4.2.1 Swelling and %EWS behavior of PCB and PCBSF nanocomposite hydrogels The swelling and equilibrium swelling ratio of PCB-3, PCBSF-3 and PCBSF-6 was measured in phosphate buffer solutions at pH-4.0 and pH-7.4. The swelling behavior of nanocomposite hydrogels are depicted in Figure.4A-B. Initially, the hydrated molecules are in contact with the top surface of hydrogels. The PCB-3 hydrogel shows higher swelling ratios (9.40 and 8.05) than those of PCBSF-3 (8.77 and 7.18) and PCBSF-6 (6.80 and 6.68) at pH -4.0 and 18 ACS Paragon Plus Environment

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pH-7.4 after 72h (Figure.4A-B); which ascribed the formation of more elastic and porous assembly within the PCB-3 hydrogel. The presence of –OH and –NH group of chitosan, PVA and polyol, make the hydrogel more hydrophilic in nature, helping in the imbibing of water. However, in case of PCBSF-3 and PCBSF-6 hydrogels, the presence of SiO2 induces more densed and higher cross linked network that reduces the pore size of cagy structure and the extent of hydrophilicity of pores, walls of hydrogel, resulting in the lowering of water uptake capacity and equilibrium swelling ratio, which further enhances the stiffness and elastic modulus of swelled hydrogels. These results are well in agreement with the mechanical behavior of hydrogels. The presence of SiO2 in PCBSF-3 and PCBSF-6 hydrogels led to the formation of SiO4 tetrahedral crosslinked bonding within the hydrogel and the hydrophobic nature of SiO4 reduces the swelling ability of the hydrogel to some extent as compared to that of PCB-3 hydrogel. The % EWS of PCB-3 was found to be 756% and 642% at pH 4.0 and pH 7.4 respectively (Figure.5A). While PCBSF-3, and PCBSF-6 show the decrease in % EWS values (700%, 498% and 500%, 486%) with increasing concentration of SiO2 nanoparticles at pH 4.0 and pH 7.4, respectively. This can be due to the squeezing of pores and presence of hydrogen bonding between SiO2 and other polar functional groups. The extent of squeezing of cagy structure have a linear relation with SiO2 filler concentration. Swelling study revealed that the nanocomposite hydrogels have shown exceptionally good response for swelling and stability as compared to other such hydrogels under slightly acidic medium (pH 4) reported earlier in the literature, which facilitate their application in the field of drug loading and controlled release.21,57



Figure 4. Swelling ratios of PCB-3, PCBSF-3 and PCBSF-6 at pH 4.0 (A) and pH 7.4 (B). The p value is ≤ 0.05, indicate that the data is significance. 4.2.2 % EWS of PCB and PCBSF nanocomposite hydrogels in physiological solutions The electrochemical interactions within the constituents of PCB-3, PCBSF-3 and PCBSF-6 hydrogels with various biologically active molecules and molecular ions in physiological solutions can be found to act as a good candidature in the field of drug delivery. The effects of various physiological solutions (urea (5% w/v), D-glucose (5% w/v), KI (15% w/v), NaCl (0.1%), KNO3, and KH2PO4) on the swelling ratio of PCB-3, PCBSF-3 and PCBSF-6 hydrogels are shown in Figure 5B. These studies revealed that the nanocomposite hydrogels under physiological solutions display intermediate swelling ratio23. While in D-glucose solution, the PCB-3 and PCBSF-3 and PCBSF-6, hydrogels show highest value of the % EWS. The %EWS of PCB-3, PCBSF-3 and PCBSF-6 were 20 ACS Paragon Plus Environment

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found 638%, 537% and 525%, respectively, which can be qualified due to the existence of extra hydroxyl(-OH) groups in their swelled states. While in case of urea, NaCl, KNO3 physiological solutions, the nanocomposite hydrogels show lower swelling ratio. This could be due to the presence of salts in swelling environment, that produces transmeric effect, leading to the decrease in the repulsion activity among the cross linked pores of the polymer chains, ultimately causing their reduction in the swelling activity of hydrogels.21,23

Figure 5. (A) % EWS of PCB-3, PCBSF-3 and PCBSF-6 in PBS (pH-4.0 and pH-7.4). (B) %EWS of PCB-3, PCBSF-6 and PCBSF-6 in various physiological solutions. The p value is ≤ 0.05, indicate that the data is significance. 4.3 Biodegradability studies 4.3.1 Hydrolytic degradation in PCB and PCBSF nanocomposite hydrogels The hydrogel degradation occurs through the rupture and cleavage of cross-linked networks58. The changes in crosslinking can be analyzed by determining the extent of swelling ratios. The swelling ratio values were utilized to assess the nature of degradation in 21 ACS Paragon Plus Environment


the PBS environment at selected interval of time. The effect of pH on the degradability was measured by the determination of mass loss (%) of PCB-3, PCBSF-3, and PCBSF-6 at pH 4.0 for 50 days (Figure S3).The swelling ratios of PCB-3 hydrogel experiences a significant increase in swelling up to 72h, indicating that degradation starts after attaining the highest %EWS (756%). A insignificant change in swelling ratio was observed after attaining the said %EWS, indicating that the PCB-3 began to disintegrate physically after a certain period of time, causing hindrance in the measurement of weight of fully swollen samples, that can be attributed to the splitting of hydrogel into smaller pieces. On the other hand, the PCBSF-3 and PCBSF-6 nanocomposite hydrogels after attaining the highest % EWS (700% and 500%), retained their stability and reduced slowly in pH 4 solution without any significant increase in swelling. The enhancement in degradation stability in case of PCBSF-3 and PCBSF-6 can be attributed to the strong electrostatic interactions and hydrogen bonding among polar functional groups of the constituents of nanocomposite hydrogels.59 The prolonged stability with less degradability of nanocomposite hydrogels, make them a potential candidates for the sustain release of drugs. 4.3.2 Soil burial degradation of PCB and PCBSF nanocomposite hydrogels The biodegradability degradation was conducted on PCB and PCBSF hydrogels for 8 weeks. The hydrogel samples were charged in a beaker comprising soil of known composition over 30% humidity.21 The different composition ratios of PCB-3, PCBSF-3 and PCBSF-6 nanocomposite hydrogels of standard size (1cm×1cm) were taken in three different beakers, the extent of mass loss after degradation of these hydrogels in soil was measured on an analytical balance (with accuracy ±0.0001) at fixed time periods for 8 week (Figure S3). The weight loss Vs time graphs revealed that the loss in weight in case of PCB-3 started earlier as compared to that of PCBSF-3 and PCBSF-6. The PCB-3 hydrogel having lower mechanical strength, began to disintegrate just after 7 days of incubation within beaker containing soil, 22 ACS Paragon Plus Environment

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while PCBSF-3 starts to degrade after 15 days. The slow degradation rate of PCBSF-3 and PCBSF-6 can be attributed to the higher mechanical strength, which induces due to the formation of strong Si-O bond of SiO2 and formation of electrostatic interactions among –OH groups of CH, PVA and polyol matrix with SiO2 nanoparticle. The continuous weight loss (Figure S3) in these hydrogels demonstrated the eco-friendly behavior of PCB and PCBSF nanocomposite hydrogels. The biodegradability study was performed to insure the biodegradable nature of nanocomposite hydrogels for the given period of time (8 weeks). 4.4 In Vitro Cell Assay of PCB and PCBSF hydrogels The MTT assay studies of Cell viability have showed that the PCB and PCBSF hydrogels do not cause any effect on cellular feasibility up to 48h treatment (Figure 6A). The different concentration of hydrogels were used to investigate the toxicity on Human Embryonic Kidney (HEK-293) cells, which confirmed that these hydrogels demonstrate lower cytotoxicity than those described in other toxicological studies.60,61 In PCB-3, there is only 6% toxicity with ≥94% cell viability compared to that of control (100% cell viability) at 200μg/mL after 48h incubation. While, SiO2 modified nanocomposite hydrogels (PCBSF-3 and PCBSF-6), have ≥90% cell viability at 150 μg/mL. Whereas at 200 μg/mL, these gels showed marginal toxicity (20-21%) with 78-79.9% cell viability after 48h incubation, which demonstrated that the marginal toxicity in these hydrogels depends on the extent of loading of SiO2 nanoparticle. These hydrogels did not show any significant toxicity even at higher doses (200μg/mL). In addition, the morphology of the cell with PCB and PCBSF (CH-PVA biopolymer-modified SiO2) disks treatment was analyzed using an inverted microscope (Meotic), which scanned the entire probes, which shows the morphology of cells after treated with hydrogels was same as comparison to that of control cells morphology even at the highest dose of treatment (i.e. 200 μg/mL) after 48h. This suggest that the interaction of both PCB and PCBSFs with HEK-cells did not have any negative effect on cell life and 23 ACS Paragon Plus Environment


morphology (Figure 6B). The MTT assays indicate that these hydrogels are found to be biocompatible at these tested concentrations (i.e.50-200 μg/mL). Thus, PCB and PCBSF hydrogels exhibits promising biocompatibilities suggesting that these nanocomposite hydrogels can be act as good candidature for their safely used in the area of drug delivery.

Figure 6. (A) Cells viability of PCB-3, PCBSF-3 and PCBSF-6, evaluated by MTT assay test on HEK-293 for 48h. (B) Morphology of cells captured through inverted microscope (Meotic) at the higher dose (200µg/ml). The p value is ≤ 0.05, indicate that the data is significance. 4.5. Drug loading of PCB and PCBSF hydrogels Cisplatin was used as a standard drug to assess both the drug loading and controlled release behavior of nanocomposite hydrogels. The drug was loaded onto the hydrogels as described in previous section. In all cases, the gels showed the change in color from white to yellow after perfect loading. The loading of cisplatin drug in PCB-3 hydrogel showed a high % loading as compared to that of PCBSF-3 and PCBSF-6 after 144h at both pH 4.0 and pH 7.4 (Table S1), which can be attributed to the higher swelling behavior and interactions of drug molecules with the polar functional groups of constituent moieties of PCB-3. Whereas, the loading of cisplatin was found comparatively lower in case of PCBSF-6 and PCBSF-3 at both pH (pH 4.0 and pH 7.4). Furthermore, the extent of reduction in % loading was found higher 24 ACS Paragon Plus Environment

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at pH-7.4 than pH-4.0. It can be attributed to the crosslinked dispersion of fumed silica (SiO2) nanoparticles into the polymer matrix. The dispersion induces denser crosslinked structure with reduced pore size and reduction in hydrophilicity of the gels, which is based on higher stability of Si-C bond than C-H and other bonds.62 Moreover, the referred Si-O-Si bonds have high thermal stability and mechanical résistance. 4.5.1. Drug Release behavior of PCB and PCBSF hydrogels in vitro The in vitro cisplatin drug release behavior was investigated by putting the drug loaded dried PCB-3, PCBSF-3, and PCBSF-6 hydrogels in pH-4.0 and pH-7.4 at 25o C for certain defined period of times (Figure 7A-B); No noticeable burst release was observed in these hydrogels at both pH-4.0 and pH-7.4. The drug release data demonstrate that these nanocomposite hydrogels act as optimal platform for sustained release of anticancer drug, which can be due to the cross-linked porous network of hydrogel, achieved by the dispersion of SiO2 nanofillers and other cross linker into the polymer matrix that reduces the diffusion path for the drug entrapped within the inner part of hydrogels and function as a drug reservoir for cancer treatment. The amount of drug release was found to be 85.19%, 78.07% and 72.72% for PCB-3, PCBSF-3 and PCBSF-6 respectively, at pH-4.0 after 144h. The release pattern at pH 4.0 was found to be much higher than that at pH-7.4 (52.19% for PCB3, 43.67% for PCBSF-3 and 36.02% for PCBSF-6) for the same period. Although; these study revealed that the swelling ratios of the resultant hydrogels exhibits not much difference, but the release rate of the drug is found to be much higher in acidic medium (pH4.0), indicating the release of cisplatin from the loaded hydrogels is pH responsive. The PCB-3, PCBSF-3 and PCBSF-6 hydrogels show an increase in the release rate of drugs with the decrease in pH (i.e. at pH-4.0) (Figure 7A-B;) This can be attributed to the poor electrostatic interaction between the drug molecules and hydrogels at lower pH. The higher drug release rate of PCB-3 as compared to those of PCBSF-3 and PCBSF-6 at both pH-4.0 25 ACS Paragon Plus Environment


and pH-7.4 can be assigned to the higher swelling ability of PCB-3 hydrogel due to more porous and flexible structure, which led for the faster and higher release rate of the drug in PCB-3. Further, literature revealed that the chitosan based hydrogels exhibit poor flexibility, low degradability and poor solubility of drug at higher pH (i.e. pH-7.4). Therefore lower release rate of drug is recorded at pH-7.4.63 The slightly lower and sustained drug release rate (78.07% and 72.72%) of PCBSF-3 and PCBSF-6 as compared to that of PCB-3 (85.19%) can be due to the presence of SiO2 nanoparticles into the polymer matrix, it has been observed that the hydroxyl groups is attached with SiO2 bonds or sandwiched with in the cross linked polymer matrix, which might have reduced the water uptake capacity, as a result, low %EWS of nanocomposite hydrogels. Further, the drug release graph shows that the PCB-3 (containing highest amount of drug i.e. 24.44%) exhibited fast and higher release rates than those of PCBSF-3 and PCBSF-6, can be attributed for holding a lesser amount of drug at both the pH (pH-4 and pH-7.4). The literature revealed that the release rate of cisplatin at alkaline and acidic medium were found lower i.e. 18% and 61.7% respectively, after 48 h, than the release rate reported in the present work.34 Fang et al. reported the chitosan based hydrogels shows cisplatin drug release only 13%-20% in the first 3h and thereafter only a slight release was observed up to 5h much lower than that of our hydrogels.22 The prolonged sustained and higher efficient release rate of cisplatin in our nanocomposite hydrogels as compared to other reported works can be attributed to the selection of sustainable (biodegradable, biocompatible) polymers and fumed silica (SiO2) dispersion that showed efficient drug release behavior. In contrast, the release profile of free cisplatin in PBS showed a total release of more than 90% in the first 6h (inset in Figure 7B).


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Figure 7. Drug release graphs for PCB-3, PCBSF-3 and PCBSF-6 at pH-4 and pH-7.4 (A) and (B). The p value is ≤ 0.05, indicate that the data is significance. 4.6 In Vitro anti-cancer efficacies of nanocomposite hydrogels MTT assay on HCT116 human colon cancer cells after treatment shows that PCB and PCBSF hydrogels do not induce significant anti-cancer effect at the dose range from 10-160 µg/mL (Figure.8A). The different concentration (10 µg/mL, 20µg/mL, 40µg/mL, 60µg/mL, 80µg/mL and 160µg/mL) of (PCB-3, PCBSF-3 and PCBSF-6) the resultant hydrogels were used to investigate the anti-cancer activity on HCT116 cancer cells and it was found that no significant difference in cell viability after the addition of disks compared to control was observed (Figure 8A). Although; the PCB-3, PCBSF-3 and PCBSF-6 exhibits slight cytotoxicity (i.e.28%-29%) at higher concentration (160 µg/mL) which is maximum cytotoxicity. In our study, we have found the cell viability is approximately ≥75% at the 160 µg/mL, which was relatively high, indicating that that the resultant hydrogel films had no significant growth inhibition of HCT116 cells without cisplatin. 4.6.1 In Vitro anti-cancer efficacies of cisplatin loaded nanocomposite hydrogels The anticancer activity of free cisplatin and cisplatin loaded PCB and PCBSF hydrogels against HCT116 human colon cancer cell line was examined using an MTT assay. In order to evaluate the anti-proliferative activity of free cisplatin and cisplatin loaded PCB-3, PCBSF-3 and PCBSF-6 hydrogels, the different concentration (50µg/mL, 100µg/mL, 150µg/mL, 27 ACS Paragon Plus Environment


200µg/mL) of the resultant hydrogels were used on HCT116 cells upto 48h (Figure 8B). The MTT assay on HCT116 cells after treatment indicated that cisplatin loaded PCB and PCBSF hydrogels show significant anti-proliferative effect at the dose range from 50-200µg/mL (Figure 8B). Priors to this study, the biocompatibility of PCB and PCBSF hydrogels was examined (Figure 6A).The PCB and PCBSF nanocomposite hydrogels was found to be nontoxic material (200µg/mL), which indicate high biocompatibility that does not affect the results of the cell viability in the cisplatin carriers. The PCB-3 possess cytotoxicity (50.28%) at IC50 143.26 µg/mL. However, the PCBSF-3 and PCBSF-6 kills 59% (IC50 149.16 µg/mL) and 65% (154 µg/mL) cancer cells respectively, after being incubated for 48h. In contrast, the free cisplatin (Figure 8C) shows anticancer activity at lower doses (IC50 121.43 µg/mL) that killed 56% cancer cells (44% cell viability) and left only 18% of the cell viability at high concentration (IC50 200 µg/mL). Overall, the cytotoxic effects of the treated hydrogels (PCB and PCBSF) was lesser than those of the cisplatin free drug. The main reason that these hydrogels can show low anticancer activity compared to free cisplatin is due to the slow internalization of the polysaccharide based hydrogels into the cell and the slow release process of cisplatin.64 On the other hand, the higher cytotoxicity of the cisplatin free drug can be attributed to the fast release of cisplatin due to unmodified drug carrier.64 The results after 48h of incubation suggest that the PCB and PCBSF hydrogels show significant anticancer activity on colon HEC116 cancer cells.


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Figure 8. (%) Cell viability of PCB-3, PCBSF-3 and PCBSF-6 (A). Free cisplatin and cisplatin loaded PCB-3, PCBSF-3 and PCBSF-6 samples (B and C) at HCT116 human colon cancer cell lines estimated by MTT assay after incubation for 48h. The p value is ≤ 0.05, indicate that the data is significance. 5. Conclusion The highly efficient and sustainable PCB hydrogel and fumed silica modified PCBSF nanocomposite hydrogels were prepared by free radical polymerization method. The study revealed that the unique characteristic properties are associated with the introduction of SiO2 in the CH/PVA/polyol based hydrogel. It was observed that the addition of SiO2 in the PCB polymer matrix resulted in the enhanced stability of nanocomposite hydrogels inducing restricted hydrophilicity in swelling ratio measurement and rheological properties. The PCB hydrogel and PCBSF nanocomposite hydrogels could be well dispersed in the aqueous solution that helps in conducting in-vitro cytotoxicity test, which indicated that these nanocomposite hydrogels are highly biocompatible and non-toxic in nature, supporting its applications in drug delivery system. The in-vitro cytotoxicity of cisplatin loaded 29 ACS Paragon Plus Environment


nanocomposite hydrogels indicated that these hydrogels effectively inhibited the growth of HCT116 cancer cell line. The release rate of cisplatin in nanocomposite hydrogels was pH responsive and improved with the reduction in pH. The release rate of cisplatin in PCB-3 hydrogel was found to be 85.19% and 52.19% much higher and fast than PCBSF-3 (78.07% and 43.67%) and PCBSF-6 (72.72%, 36.01%) at both pH 4.0 and pH 7.4 after 72h. These nanocomposite hydrogels showed pH and time dependent drug release as confirmed by the in vitro drug dissolution profiles. These results can suggest that the PCB and PCBSF nanocomposite hydrogels are promising platforms to construct pH responsive controlled anticancer drug delivery system. In vitro tests of drug loading and sustained release, biodegradability, biocompatibility and anti-proliferative activity of cisplatin loaded nanocomposite hydrogels suggest that in future, it may find applications for the development of topical (in vivo, in the form of tablets) drug delivery systems. ASSOCIATED CONTENT Supporting Information The Supporting information is available free of charge on the ACS publications website. Digital pictures of swollen nanocomposite hydrogels. Storage moduli (G') and loss moduli (G'') for hydrogels. Biodegradability graphs analyzed by hydrolytic and soil burial tests. Table of loading of cisplatin in PCB and PCBSF hydrogels. ACKNOWLEDGEMENTS One of the authors Rabia Kouser appreciate the University Grants Commission, for providing financial assistance. The authors further grateful to All India Institute of Medical Sciences (Central Facility) for SEM and TEM analysis and CIF of CIRBSc, Jamia Millia Islamia for XRD analysis. Conflicts of Interest. There is no conflicts of interest REFERENCES 30 ACS Paragon Plus Environment

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Graphical Abstract


pH-Responsive Biocompatible Nanocomposite Hydrogels for

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