In Situ Silver Nanowire Deposited Cross-Linked Carboxymethyl

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In-situ Silver Nanowires Deposited Cross linked Carboxymethyl Cellulose: A Potential Transdermal Anticancer Drug Carrier Barun Mandal, Arun Prabhu Rameshbabu, Saundray Raj Soni, Animesh Ghosh, Santanu Dhara, and Sagar Pal ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b10716 • Publication Date (Web): 26 Sep 2017 Downloaded from http://pubs.acs.org on September 28, 2017

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ACS Applied Materials & Interfaces

In-situ Silver Nanowires Deposited Cross linked Carboxymethyl Cellulose: A Potential Transdermal Anticancer Drug Carrier Barun Mandal†, Arun Prabhu Rameshbabu‡, Saundray Raj Soni┴, Animesh Ghosh ┴, Santanu Dhara‡* and Sagar Pal†*

†

Polymer Chemistry Laboratory, Department of Applied Chemistry, Indian Institute of

Technology (ISM), Dhanbad - 826004, India.

‡

Biomaterials and Tissue Engineering Laboratory, School of Medical Science and Technology,

Indian Institute of Technology, Kharagpur- 721302, India.

┴

Department of Pharmaceutical Sciences, Birla Institute of Technology, Mesra, Ranchi –

835215, India.

Corresponding Author: †*

E-mail: [email protected]; [email protected]; Tel: +91-326-2235769

‡*

E-mail: [email protected]; Tel: +91-3222-282306

1 ACS Paragon Plus Environment


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Abstract: Recently a novel biopolymeric nanocomposite hydrogel comprised of in-situ formed silver nanowires (AgNWs) deposited chemically crosslinked carboxymethyl cellulose (CMC) has been developed, which demonstrates superior efficacy as anti-cancer drug-curcumin carrier. The crosslinked polymer has been prepared by grafting poly [2-(methacryloyloxy) ethyl trimethyl-ammonium chloride] on CMC using diethylene glycol dimethacrylate crosslinker. The nanocomposite hydrogel has the capability to encapsulate both hydrophobic/hydrophilic transdermal drugs. With variation of reaction conditions/parameters, several composite materials have been synthesised and depending on lower swelling/ higher crosslinking and greater gel strength, optimised grade of nanocomposite hydrogel has been selected. The developed

nanocomposite

hydrogel

is

characterized

with

FTIR/NMR

spectra,

FESEM/XRD/TGA/AFM/XPS analyses and UV-visible spectroscopy. Rheological study has been performed to enlighten the gel strength of the composite material. The synthesised nanocomposite hydrogel is biodegradable and non-toxic to mesenchymal stem cells (hMSCs). In-vitro release of curcumin suggests that in-situ incorporation of AgNWs on crosslinked CMC enhanced the penetration power of nanocomposite hydrogel and released the drug in sustained way (~62% for curcumin released in 4 days). Ex-vivo rat skin permeation study confirms that the drug from both the crosslinked and nanocomposite hydrogel was permeable through the rat skin in controlled fashion. Additionally the curcumin loaded composite hydrogel can efficiently kill the MG 63 cancer cells, which has been confirmed by apoptosis study and therefore, probably be a suitable carrier for curcumin delivery towards cancer cells.

Keywords: Carboxymethyl cellulose; Curcumin; Nanocomposite; Silver nanowire; Transdermal drug delivery.


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Introduction From last few decades, it is believed that the stratum corneum of skin is an impermeable barrier for most substances, except gases to penetrate. Recent studies reveal that skin is also being considered for the systematic drug circulation. Transdermal or topical administration offers several clinical benefits over oral drug delivery such as it requires a lower daily dose, it has direct access to the target site and there is probability of major drug interactions as well as gastrointestinal concerns.1-3 It also has other advantages like ease of dose termination, minimize the pain and prevent systemic side-effects.4-8 Though stratum corneum of epidermis performs as an impermeable barrier towards several substances for transdermal drug administration, still in last few decades, numerous efforts have been made to improve the transdermal drug administration.4 A number of attempts have been made to expand the skin permeability such as microneedle,9,10 chemical/lipid enhancers,11-13 laser ablation,14 electric fields using iontophoresis and electroporation15,16 as well as pressure waves generated by ultrasound or photoacoustic effects.17,18 However, all these processes accomplish with several disadvantages.19-22 In this respect nanoparticles (NPs)/nanowires (NWs) based polymeric nanocomposites raise a significant attention owing to their pronounced effect on local penetration into the skin for transdermal drug delivery.4,23 There are several reports, which showed that the NPs/NWs and particulate materials have been used for the percutaneous delivery of drugs, although it is difficult for the particles to penetrate some of dermal drugs and clinical cosmetic products through the skin.24-27 Amongst various nanoparticles/nanowires, AgNPs/AgNWs are extremely useful for biomedical application as they are biocompatible in nature and also can be synthesised easily to its desired size and shape for precise biomedical purposes.28-30 AgNPs/AgNWs recently received a great attention owing to their significant contributions towards clinical practice, as silver nanomaterials show optical chemical reactivity that can functionalize biologically active moieties (such as targeting and



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therapeutic molecules). Also it possesses characterized surface plasmon modes, which in turn can be used to develop tunable contrast agents as well as thermal ablation therapies. Moreover, silver nanomaterials release Ag+ ions that can interact with the thiol groups containing cell cytoplasm protein as well as inner mitochondrial membrane, which result perturbation of normal cells activity.31-35 Moreover, due to the surface functionality of NPs/NWs, it plays a crucial role towards cell attachment and biological distributions.36, 37 NPs/NWs-based anticancer transdermal delivery systems are one of the most powerful tools for efficiently carry and deliver therapeutic agents for dermal and systemic administration. In this concern, herein we have developed functionalized carboxymethyl cellulose (CMC) and AgNWs based biodegradable and biocompatible nanocomposite material for potential use in control transdermal drug delivery.38 The nanocomposite hydrogel has been synthesized by insitu deposition of AgNWs onto chemically crosslinked CMC. The chemically crosslinked CMC based hydrogel was synthesized by free radical polymerization (FRP) through grafting of [2-(methacryloyloxy) ethyl] trimethyl-ammonium chloride] (METAC) on CMC in presence of diethylene glycol dimethacrylate (DEGDMA) crosslinker. CMC was chosen for nanocomposite hydrogel synthesis owing to its enormous applications in clinical biomedical field.39-45 Also use of METAC and DEGDMA make the hydrogel structure more porous and rigid that in turn facilitate the drug loading efficiency and sustained release characteristics as well. The main advantage of crosslinked hydrogel as drug carrier is the availability of various binding sites (mainly due to the presence of different functional groups) that form physical/covalent attachment with the drug molecules in its three dimensional network. The drug molecules can be attached to the crosslinked network through hydrogen bonding or physical interactions. In swollen state of hydrogel, the drug molecules are usually released in the media through diffusion. Additionally, the combination of AgNWs with chemically crosslinked CMC would further enhance the drug loading efficiency and controlled release


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characteristics.

Henceforth,

it

would

probably

serve

as

a

good

substituted

biodegradable/biocompatible nanocomposite hydrogel for transdermal drug administration into the skin. AgNWs have been considered here, since the probability of formation of agglomeration is less during synthesis and also it possesses higher accessible area for binding the drug molecules. Curcumin was used as a model transdermal drug because of its easy availability (as it is main constituent of turmeric and extracted from the roots of curcuma longa), cost effective and mainly it is natural transdermal anticancer drug for skin administration that can be applied to the skin defence, control of dermal blood flow, and wound healing.46 EXPERIMENTAL SECTION Chemicals Carboxymethyl cellulose (CMC) with 0.6 degree of substitution was supplied by Hindustan Gum and Chemicals Ltd., Haryana, India. DEGDMA (from TCI Pvt. Ltd., Tokyo, Japan), METAC (from Sigma-Aldrich, USA), AgNO3 (Sigma-Aldrich, USA), potassium persulfate (from Glaxo Smith Kline Pharmaceuticals Ltd., Mumbai, India), acetone (from E. Merck (I) Pvt. Ltd., Mumbai, India), sodium borohydride (Spectrochem, India), hydroquinone (S. D. Fine chemicals, Mumbai, India) and egg white lysozyme hydrochloride (from TCI Pvt. Ltd., Tokyo, Japan) were utilized as obtained. Milli Q water has been used for experimental works. Synthesis Synthesis of crosslinked hydrogel (cl-CMC-pMETAC): CMC (1 g) was mixed with 80 mL Milli Q water in 250 mL round-bottom (RB) flask. The resulting solution was then put on an oil bath for constant heating at 50 °C and continuous stirring using magnetic stirrer. The reaction temperature was increased to 75 °C in inert atmosphere (N2). After addition of initiator solution under stirring, the monomer (METAC) was added to the solution. When the reaction mixture was turned into milky white, then



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crosslinker (DEGDMA) was added and the crosslinking reaction was allowed to continue for three hours. Then the polymerization reaction was stopped with addition of saturated hydroquinone solution. To remove excess crosslinker/unreacted monomer/homopolymer, the reaction mixture was washed with acetone. The synthesized hydrogel was then dried at 50 °C under vacuum. Different grades of crosslinked hydrogel were synthesized with variation of reaction parameters/conditions and optimised grade (cl-CMC-pMETAC 6) was selected with higher crosslinking and lower equilibrium swelling (Table S1, Supporting Information). clCMC-pMETAC 6 was further used for in-situ deposition of AgNWs. [1H NMR (400 MHz, DMSO d6, δ, ppm): 1.86 (-CH3 proton from cross linker unit), 2.03 (NCH3 proton), 2.01 (from monomer unit), 3.2-3.8 (CMC ring proton, -OH proton from CMC unit and -CH2 proton of monomer and crosslinker unit merge together), 3.95 (-CH2 proton from CMC unit), 4.50 and 4.60 (-CH2 proton from crosslinker unit), 4.75 and 4.83 (-CH2 proton from monomer unit), 5.72 (anomeric proton). C NMR (400 MHz, solid state, δ, ppm): 19.4, 24.0, 32.3, 38.4, 46.0, 55.3, 60.9, 65.5, 71.3,

13

74.7, 76.9, 78.3, 103.3, 178.3]. In-situ synthesis of nanocomposite hydrogel (cl-CMC-pMETAC/AgNWs): The crosslinked hydrogel was synthesized in the same method as described in above section. 0.5 mmol silver nitrate (AgNO3) solution was added drop wise during the formation of crosslinked hydrogel and then NaBH4 (reducing agent) was added slowly. The colour of the solution became grey white, which indicates the formation of AgNWs and then reaction was terminated with hydroquinone solution. Finally, the reaction mixture was cooled, precipitated using acetone and dried at 50 °C. Here also, AgNO3 concentration was varied to obtain various nanocomposite hydrogels (Table S1, Supporting Information) and the optimized one (cl-CMCpMETAC/AgNWs 2) was selected with lower equilibrium swelling.


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[13C NMR (500 MHz, solid state, δ, ppm): 19.7, 24.5, 32.9, 40.0, 46.0, 55.3, 61.2, 65.7, 73.4, 74.8, 78.1, 80.0, 104.7, 178.6]. For comparison, only AgNWs was synthesised using the same reaction condition (i.e. reaction temperature 75 °C, reaction time – three hours) as discussed in the previous sections, but in absence of crosslinked CMC. Synthesis of CMC and poly (METAC) based graft copolymer (CMC-g-pMETAC): Also CMC and poly (METAC) based graft copolymer was prepared in absence of DEGDMA in the same way as discussed in the synthesis of crosslinked hydrogel section. [1H NMR (400 MHz, DMSO d6, δ, ppm): 2.00 (-CH3 proton from monomer unit), 2.06 (NCH3 proton from monomer unit), 3.2-3.8 (CMC ring proton, -OH proton from CMC unit and CH2 proton from monomer unit merge together), 3.95 (-CH2 proton from CMC unit), 4.61 and 4.83 (-CH2 proton from monomer unit), 5.71 (anomeric H from CMC unit)]. The digital photographs of all the synthesised materials are shown in Fig. S1, Supporting Information. Characterization Crosslinked hydrogel and nanocomposite were characterized using FTIR spectroscopy (Cary 600 series, Agilent Technologies FTIR spectrophotometer); 1H-NMR spectroscopy (in liquid state, 400 MHz, µZEOL Spectrophotometer),

13

C NMR spectroscopy (solid state, 500 MHz

Bruker spectrophotometer), XRD (PANanalytical ‘X’ Pert PRO, Netherlands), XPS (PHI 5000 Versa Probe II, FEI Inc.), TGA/DTG (Perkin Elmer Pyris Diamond TG-DTA), FESEM (FESEM Supra 55, Zeiss, Germany), AFM (Bruker Dimension Icon Nanoscope V, Germany), UV-visible spectroscopy (UV-1800, Shimadzu, Japan) and UV-visible Diffuse Reflectance analysis (UV-VIS-NIR Spectrophotometer, Agilent Cary 5000). The detailed experimental procedure and sample preparation techniques are discussed in Supporting Information.



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Swelling study The three dimensional chemically crosslinked hydrogel has a greater tendency to imbibe water. The water absorption capacity of developed graft copolymer, crosslinked hydrogel and nanocomposite hydrogel was studied by determining equilibrium swelling using phosphate buffer (pH: 5.65) at physiological temperature (37 °C). The detailed experimental procedure is given in Supporting Information. Rheological properties The

rheological

features

of

CMC-g-pMETAC,

cl-CMC-pMETAC

and

cl-CMC-

pMETAC/AgNWs were performed in swell state (PBS buffer, pH 5.65). The measurement was carried out using a Rheometer (Bohlin Gemini-2, Malvern, UK). The experimental technique has been described in Supporting Information. Biodegradation study Biodegradable nature of the nanocomposite hydrogel (cl-CMC-pMETAC/AgNWs) was studied with hen egg lysozyme hydrochloride solution as reported before.47,48 The experimental method has been elucidated in Supporting Information. Cytotoxicity test and morphological assessment The morphological assessment and cytotoxicity results of cl-CMC-pMETAC/AgNWs nanocomposite hydrogel was investigated using previously reported method.49 Here, the number of viable cells was calculated using Vybrant® MTT Cell Proliferation Assay Kit (Invitrogen). Results represented here are mean ± standard deviation (n = 5). The experimental process has been reported in Supporting Information. In vitro cell uptake study Development of fluorescence labelled cl-CMC-pMETAC/AgNWs nanocomposite hydrogel To prepare the fluorescence labelled cl-CMC-pMETAC/AgNWs nanocomposite hydrogel, FITC was used. The nanocomposite hydrogel (2 mg/mL) was dispersed to FITC solution (0.2


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mg/mL in DMSO) and was then kept in dark for six hours with constant stirring. Afterward, the FITC loaded composite was centrifuged at 8000 rpm for 20 min.50-51 To eliminate the unloaded FITC, the precipitate was rinsed with water repeatedly.50-51 Finally, FITC loaded clCMC-pMETAC/AgNWs nanocomposite hydrogel was dried under vacuum (45 °C) and employed for intracellular uptake study. Intracellular uptake study The FITC loaded cl-CMC-pMETAC/AgNWs nanocomposite hydrogel was used for intracellular uptake study. The experimental process is given in the Supporting Information. Apoptosis analysis by Tunel Assay Apoptosis of MG 63 cancer cells was investigated after exposing cells to curcumin-loaded nanocomposite using Tunel Assay. The detailed experimental procedure has been explained in the Supporting Information. In-vitro drug release study Drug loading and entrapment efficiency The entrapment and loading efficiency of curcumin were performed by mixing nanocomposite hydrogel with drug solution (curcumin was dissolved in alcohol-water mixture - 70:30). The experimental results suggest that maximum entrapment and loading were attained with drug and composite hydrogel ratio of 1:5. After mixing of drug and hydrogel, it was stirred for 1-3 days. Then the drug loaded composite hydrogel was separated by centrifuging the mixture at 10000 rpm for 15 min and decanted. To remove the free curcumin from the mixture, the same procedure was repeated several times. The entrapment efficiency (eq. 2, Supporting Information) and loading efficiency (eq. 3, Supporting Information) of curcumin were calculated using UV-vis spectrophotometer. In-vitro transdermal delivery of curcumin



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In-vitro transdermal delivery of curcumin was carried out with Franz Diffusion Cell Apparatus (Orchid Scientific & Innovative India Pvt. Ltd., India) at 32 °C and pH 5.65. The transdermal delivery of curcumin loaded graft copolymer/hydrogel and nanocomposite was carried out using cellulose acetate dialysis membrane (LA390, average flat width - 25.27 mm, average diameter - 15.9 mm and capacity approximate - 1.99 mL/cm).52 The diffusion cell apparatus mainly

comprises

two

parts,

upper

part

contains

drug

loaded

copolymer/hydrogel/nanocomposite, which is called donor compartment and lower part contains PBS buffer (pH 5.65). The pH is comparable with human skin pH (i.e. 5.4–6.9). As human skin temperature remains more or less ~32 °C, so the drug release study was performed at 32±0.5°C.53 The released curcumin was quantified using UV-vis spectrophotometer at 332 nm by collecting aliquots after different time intervals. Korsemeyer-Peppas model54 was used to investigate the transdermal drug release mechanism (details are given in Supporting Information). Ex-vivo rat skin permeation study Ex-vivo skin permeation study was performed on healthy male adult Wistar strain albino rats (weighing approximately 240 g, 5-8 weeks of age). The details methodology of ex-vivo skin permeation study has been demonstrated in Supporting Information. RESULTS AND DISCUSSION Preparation and characterization of nanocomposite hydrogel Chemically crosslinked CMC based nanocomposite hydrogel was synthesized by free radical polymerisation. At first, radical site was generated on CMC backbone in presence of potassium persulphate under inert atmosphere (N2). The radical form of CMC was then reacted with METAC to form grafted hydrogel macroradical (R1, Scheme 1). Owing to the presence of two polymerization sites (i.e. two double bonds) in DEGDMA, it reacts with macroradical R1 to form three dimensional polymeric network (cl-CMC-pMETAC, Scheme 1). Once the


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crosslinked hydrogel formed, it was then used as a template for the uniform deposition of AgNWs that was formed by the addition of AgNO3 solution in presence of NaBH4. Here crosslinked hydrogel works as stabilizer for the growth and fabrication of AgNWs. Because of the presence of chelate effect that was exerted by various functional groups of polymer network, AgNWs were successfully deposited on the crosslinked CMC (Scheme 1).



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Scheme 1: Proposed mechanism for synthesis of cl-CMC-pMETAC/AgNWs. UV-vis study was performed to investigate the deposition of AgNWs onto the hydrogel matrix. Fig. 1a shows the UV-vis spectra of cl-CMC-pMETAC and cl-CMC-pMETAC/AgNWs. It is obvious that hydrogel does not show any characteristics peak, while cl-CMC12 ACS Paragon Plus Environment

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pMETAC/AgNWs showed absorption bands at 353 and 381 nm, which are appeared due to the transverse plasmon resonance of AgNWs.55,56 The successful incorporation of AgNWs on hydrogel was mainly occurred due to the physical interactions between the polymeric network and NWs. UV-vis DRS of curcumin and curcumin loaded with nanocomposite hydrogel are demonstrated in Fig. S2, Supporting Information. From the spectra, it is apparent that curcumin showed absorption bands at 327 and 465 nm, which were shifted to higher region for composite hydrogel (i.e. red shift; from 327 to 330 nm and 465 to 491 nm). This is probably due to the hydrogen bonding as well as physical interactions between drug and nanocomposite hydrogel that in turn extrapolate the conjugation of curcumin with nanocomposite hydrogel.

Fig. 1: (a) UV-vis spectra of cl-CMC-pMETAC, cl-CMC-pMETAC/AgNWs, (b) XRD pattern of CMC, cl-CMC-pMETAC, cl-CMC-pMETAC/AgNWs, and (c) XRD pattern of as synthesized AgNWs.

Crystalline nature of AgNWs can be analysed from XRD pattern. Fig. 1b shows the XRD pattern of CMC, cl-CMC-pMETAC and cl-CMC-pMETAC/AgNWs. The peaks are indexed with face centred cubic silver (JCPDS Card File No04-0783).57 The typical XRD patterns confirm the amorphous nature of CMC and cl-CMC-pMETAC. While XRD pattern of cl-



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CMC-pMETAC/AgNWs shows the presence of different peaks of metallic Ag at 35.25°, 43.65°, 62.96° and 74.72°, which correspond to (111), (200), (220) and (311) planes, respectively along with traces of impurity as AgCl with (200) plane. The above observations suggest that AgNWs were successfully deposited on crosslinked CMC. Fig. 1c represents the XRD pattern of as synthesized AgNWs, which possesses all the characteristics peaks of metallic silver indexed at 38.11°, 44.27°, 64.61°, 77.30° and 81.54° corresponds to (111), (200), (220), (311) and (222) planes. The elemental composition has been verified using quantitative spectroscopic technique XPS. Fig. 2a represents the XPS spectra of all compositions that illustrate the main characteristic peaks of C (1s), N (1s), O (1s) and Ag (3d). The deconvolution XPS peaks of independent materials like C (1s), N (1s), O (1s), and Ag (3d) are explained in details. Fig. 2b represents that the deconvolution XPS peaks of carbon were appeared at different regions as 282.96, 284.55, 285.86 and 286.94 eV. The XPS peak observed at 282.96 eV is mainly due to C-C and C-H bonds. Whereas peaks at 284.55, 285.86 and 286.94 eV were found for C-OH, C-O-C and C=O, respectively. The deconvolution XPS peaks of oxygen are shown in Fig. 2c. The Shirley backline correction of O1s peaks provides mainly three Gaussian components, which are 529.21, 530.92 and 532.49 eV corresponding to the bonds of C=O, C-OH and O in H2O molecule, respectively. The deconvolution XPS spectrum of nitrogen show only two different peaks at 397.19 and 400.58 eV that are mainly due to the bonds for N-C and quaternary Natom (Fig. 2d). Fig. 2e shows the Ag (3d) spectrum in which doublet peak emerged at 372.08 and 366.08 eV for Ag (3d3/2) and Ag (3d5/2). The difference between two humps of XPS peak of Ag is 6.0 eV, which confirmed the presence of metallic silver that has been synthesized successfully in presence of cl-CMC-pMETAC hydrogel as a template and NaBH4 as reducing agent. After deconvolution of Ag (3d5/2), three different peaks were perceived at 365.86, 366.12 and 367.30 eV, which are due to Ag2O, Ag and AgO, respectively. The deconvolution results


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of Ag (3d) clearly suggest that most of the silver remains in zero chemical state. In comparison with the XPS spectra of nanocomposite hydrogel, the crosslinked hydrogel (Fig. S3, Supporting Information) shows all characteristics peaks of C (1s), N (1s), O (1s). The individual deconvolution results demonstrate the following observed peaks for C (1s) -283.61, 284.85, 285.98 and 287.63 eV; O (1s) – 529.71, 531.17 and 532.58 eV; N (1s) – 400.59 and 401.21 eV. This observations suggest that all the peaks were shifted to the lower region in case of nanocomposite hydrogel, which authenticates the physical as well as hydrogen bonding interactions between the hydrogel network and AgNWs as projected in Scheme 1.58

Fig. 2: XPS spectra of (a) cl-CMC-pMETAC/AgNWs and deconvolution peaks of (b) C (1s), (c) O (1s), (d) N (1s), and (e) Ag (3d).



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To investigate the surface morphology, FESEM analysis was performed. Fig. 3 represents the FESEM micrographs of CMC, cl-CMC-pMETAC and cl-CMC-pMETAC/AgNWs. FESEM micrograph of CMC (Fig. 3a) reveals that the morphology is particle type. After modification of CMC through crosslinking, the morphology became porous and continuous in nature (Fig. 3b and Fig. S4, Supporting Information). Fig. 3c-d demonstrate the FESEM micrographs of clCMC-pMETAC/AgNWs, which confirmed that AgNWs are finely dispersed on crosslinked hydrogel. For pristine AgNWs, the Ag particles were agglomerated and bigger in size than that of composite hydrogel (Fig. S5, Supporting Information). This is due to the stabilization of AgNWs by the polymeric moiety and corresponding functional groups in composite hydrogel, which restrict the respective growth of AgNWs. This observation authenticates that hydrogel matrix favours the uniform growth and fabrication of AgNWs. Further cross sectional FESEM (Fig. S6, Supporting Information) analysis was performed to verify whether the AgNWs were deposited only on the surface or on bulk. The micrographs (i.e. Fig. 3c-d and Fig. S6, Supporting Information) clearly reveal that AgNWs were deposited on the surface of the hydrogel as well as on bulk. EDAX analysis provides the supplementary information about the presence of Ag in nanocomposite, which in turn further supports the successful deposition of AgNWs on crosslinked CMC (Fig. S7, Supporting Information).


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Fig. 3: FESEM micrographs of (a) CMC, (b) cl-CMC-pMETAC, and (c)-(d) cl-CMCpMETAC/AgNWs.

The topographic AFM images of cl-CMC-pMETAC/AgNWs in two and three dimensions are shown in Fig. S8, Supporting Information. Two shades of colours are clearly visible from the phase images of cl-CMC-pMETAC/AgNWs, the light colour is responsible for the presence of AgNWs, while the bright portion is owing to the existence of crosslinked hydrogel. This observation further suggests that formation of AgNWs took place successfully and remain intact with the crosslinked CMC through physical interaction. The incorporation of monomer and crosslinker units on CMC backbone as well as the interactions between the drug molecule and composite hydrogel were studied using FTIR spectroscopy. Fig. S9, Supporting Information shows the FTIR spectra of CMC, cl-CMCpMETAC, cl-CMC-pMETAC/AgNWs, curcumin and curcumin loaded with cl-CMCpMETAC/AgNWs. FTIR spectrum of CMC demonstrated distinctive vibrational bands at 17 ACS Paragon Plus Environment


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3336, 2905, 1587, 1408, 1311 and 1035 cm-1, which are corresponding to –OH stretching, CH stretching, carbonyl stretching, –CH2 scissoring, –OH bending and C-O-C stretching vibrations, respectively (Fig. S9a, Supporting Information). cl-CMC-pMETAC shows additional vibrational band at 1721 cm-1, which is responsible for the carbonyl stretching vibrations of ester group (Fig. S9b, Supporting Information). This suggests the successful incorporation of METAC and DEGDMA on CMC backbone. FTIR spectrum of cl-CMCpMETAC/AgNWs (Fig. S9c, Supporting Information) displays all the characteristics peaks of cl-CMC-pMETAC, while the bands were shifted to lower region. This indicates the probable physical interactions between the crosslinked CMC and AgNWs as represented in Scheme 1. Fig. S9d, Supporting Information represents the FTIR spectrum of curcumin. The spectrum clearly shows the characteristic –OH stretching band at 3604 cm-1 and other bands at 1698 and 1601 cm-1 corresponding to carbonyl stretching frequency and aromatic C=C bond stretching frequency, respectively. FTIR spectrum of curcumin loaded with cl-CMC-pMETAC/AgNWs (Fig. S9e, Supporting Information) exhibits that it contains the characteristic bands of curcumin. However the bands were shifted to lower region, which is probably owing to the Hbonding and physical interactions that present between the drug molecule and nanocomposite hydrogel network. The possible interaction between curcumin and synthesized crosslinked hydrogel is shown in Fig. S10, Supporting Information. To confirm the covalent attachment of monomer and crosslinker units with CMC, 1H NMR and 13C NMR spectra were recorded. The 1H NMR spectra of CMC, CMC-g-pMETAC and clCMC-pMETAC are demonstrated in Fig. S11-S13, Supporting Information. The anomeric proton of CMC shows δ value in the range of 5.71 ppm and H7 at 3.94 ppm. Whereas all other ring protons i.e. H2, H3, H4, H5, H6 and OH (2, 3) display chemical shifts in the range of 3.23.8 ppm (Fig. S11). In case of CMC-g-pMETAC (Fig. S12), apart from the peaks of CMC, some additional peaks were observed at δ (ppm) =2.00, 2.06, 4.61 and 4.83. These additional


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peaks are due to the presence of methyl proton (H9 and H12) and methylene protons (H11 and H10). This clearly recommends that METAC was successfully grafted onto the CMC backbone. In case of crosslinked CMC (Fig. S13), few additional peaks were found at δ = 1.86, 4.50 and 4.60 ppm. These peaks are responsible for the presence of methyl (H14) and methylene protons (H15 and H16) of crosslinker (i.e. DEGDMA). These observations confirmed the successful incorporation of crosslinker onto the grafted hydrogel network. The solid state 13C NMR spectrum of CMC shows chemical shifts at δ (ppm) =177.1, 102.7, 80.8, 73.1 and 60.2,59 that correspond to carbonyl carbon of –CH2COO–, anomeric carbon, C4 carbon, overlapping of C2, C3, C5 carbons and C6 carbon, respectively.59 Fig. 4a shows the 13

C NMR spectrum of cl-CMC-pMETAC. Compared to CMC, few additional peaks were

observed for cl-CMC-pMETAC. The peaks at 19.4 and 24.0 ppm were found due to the –CH3 carbon of DEGDMA and METAC, respectively. The peaks at 32.3 and 38.4 ppm are attributed to tertiary carbon of crosslinker and monomer units. Moreover peaks at 46.0, 55.3, 60.9 and 65.5 ppm are due to methylene carbon of crosslinker and monomer that confirmed the successful incorporation of METAC and DEGDMA onto the CMC backbone. The carbonyl peak was observed to be broad due to the presence of carbonyl groups in both monomer as well as crosslinker unit. In case of cl-CMC-pMETAC/AgNWs (Fig. 4b), all the characteristic peaks were present, although the peak positions were shifted to higher δ value. This is mainly because of physical interactions that present between AgNWs and crosslinked polymer network as projected in Scheme 1.



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Fig. 4: 13C NMR spectra of (a) cl-CMC-pMETAC and (b) cl-CMC-pMETAC/AgNWs.

The TGA/DTG analyses of CMC revealed that two degradation zones were appeared at 45-85 °C and 256-325 °C for the loss of moisture and the decarboxylation of CMC, respectively.59 Fig. S14a and b, Supporting Information demonstrate the TGA/DTG analyses of cl-CMCpMETAC and cl-CMC-pMETAC/AgNWs, respectively. In cl-CMC-pMETAC (Fig. S14a, Supporting Information), compared to the degradation pattern of CMC,

59

two additional

weight loss regions were observed. The first zone was found between 234-352 °C, which is responsible for the degradation of crosslinker unit (poly DEGDMA), while the other degradation was observed in the range of 356-425 °C. This is as a result of the degradation of poly (METAC) present on CMC backbone. This further confirms the successful attachment of METAC and DEGDMA units onto the CMC backbone. The TGA/DTG curves of cl-CMCpMETAC/AgNWs illustrate the similar degradation pattern as of cl-CMC-pMETAC, however


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it is obvious that the thermal stability was enhanced after incorporation of AgNWs (Fig. S14b, Supporting Information). Rheological characteristics: Fig. 5a represents the frequency sweep measurement of cl-CMC-pMETAC/AgNWs in which G' and G'' were varied with frequency. In the frequency range of 0.1-10 Hz, viscous modulus (G') was greater than loss modulus (G''), which ensured the gel characteristics and elastic behaviour of the fabricated nanocomposite hydrogel (cl-CMC-pMETAC/AgNWs). In oscillation mode, the amplitude sweep measurement was performed with variation of frequency (1-10 Hz), which has been represented in Fig. 5b-d. From the results, it is evident that G' remains greater than that of G'' upto a definite shear stress. This behaviour confirmed the gel nature of the developed nanocomposite hydrogel. However, after definite shear stress (yield stress, σ), both G' and G'' decreased sharply, which is probably because of the breakage of H-bonding interactions of the crosslinked gel network.



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Fig. 5: Plots of (a) G', G" vs. frequency, and (b, c, d) G', G" vs. shear stress using cl-CMCpMETAC/AgNWs nanocomposite hydrogel.

From Fig. 5b-d, it has been noticed that with increase in frequency, yield stress was increased as well. This is because the three dimensional crosslinked network vibrates rapidly and became tough to rearrange under the executed motion,50 which in turn results the rigidity of nanocomposite hydrogel network. This confirms the rigid like behaviour of the composite hydrogel with higher gel strength at high frequency compare to lower frequency (Table 1). This phenomenon implies that more stress is required to break the gel network at higher frequency compare to lower frequency. From Fig. S15 a-b, Supporting Information and Table 1, it is obvious that the yield stress as well as gel strength of cl-CMC-pMETAC/AgNWs is greater compared to both of CMC-g-pMETAC and cl-CMC-pMETAC. Fig. S15c, Supporting


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Information shows the variation of shear viscosity with shear rate. This is evident that shear viscosity was decreased invariably with increase in shear rate, which indicates the shear thinning behaviour of graft copolymer, various crosslinked CMC and nanocomposite hydrogels in the considered range of shear rate.50,59,60,61

Table 1: Rheological properties of cl-CMC-pMETAC and cl-CMC-pMETAC/AgNWs Composite

Applied

Yield stress

Gel

frequency

(Pa)

strength

(Hz)

(G'/ G'')

CMC-g-pMETAC

1

45

3.48

cl-CMC-pMETAC

1

56

4.17

1

72

4.34

5

99

4.49

10

133

4.71

cl-CMC-pMETAC/AgNWs

Swelling properties Swelling properties of hydrogel is very important due to the direct correlation with absorption of drug and its release from the hydrogel matrix. It has been well explained that sustained release property of a hydrogel was enhanced with decrease in its equilibrium swelling.62 It depends on various factors not limited to network structure, environmental condition and crosslinking density.63 The swelling characteristics of CMC-g-pMETAC, crosslinked hydrogels and nanocomposite hydrogels were investigated (Fig. S16, Supporting Information). It has clearly been observed from Table S1 and Fig. S16, Supporting Information that the pure graft copolymer demonstrates maximum equilibrium swelling. While in case of hydrogels, with increase in crosslinking, equilibrium swelling was decreased considerably. This can be



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explained by the fact that with incorporation of crosslinker, the hydrogel network became more rigid and compact that ensures lower swelling. After deposition of AgNWs, further decrease in swelling was witnessed for nanocomposite hydrogel. This is because of the additional interactions that arise due to the presence of AgNWs, which binds with polymer framework that in turn restrict the formation of more H-bonding with the media. Biodegradation study: The biodegradable nature of cl-CMC-pMETAC/AgNWs was investigated using hen egg lysozyme hydrochloride solution. It is known that lysozyme hydrochloride mainly degrades the glycosidic linkage of polysaccharide backbone.47 Fig. S17a, Supporting Information shows the continuous weight loss during the study period, which suggests the biodegradable nature of cl-CMC-pMETAC/AgNWs. This was further ascertained by FESEM study (Fig. S17b and c, Supporting Information). FESEM images of cl-CMC-pMETAC/AgNWs before and after degradation clearly suggest that after biodegradation, the smooth and porous surface of nanocomposite hydrogel became deformed. In vitro cytotoxicity experiment: The human mesenchymal stem cells (hMSCs) were grown on normal medium (control) and clCMC-pMETAC/AgNWs containing media for comparison. The cellular viability assessment (for 1-5 days) was performed by assessing growth of cells that seeded in normal medium and cl-CMC-pMETAC/AgNWs containing medium. It is evident from Fig 6a that cells grown on normal media proliferated at a slower rate than cl-CMC-pMETAC/AgNWs containing medium. After 5 days, the number of cells appeared on cl-CMC-pMETAC/AgNWs containing medium and normal medium were 3.78×105 and 2.57×105, respectively. The cell population observed on the cl-CMC-pMETAC/AgNWs containing medium was higher as nanocomposite hydrogel provides superior metabolic activity for the cells.


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Fig. 6: (a) Cell proliferation results on control medium and cl-CMC-pMETAC/AgNWs containing medium by MTT assay (mean ± SD, n = 3) and (b) cellular attachment of hMSCs by rhodamine-phalloidin and DAPI assay at 1, 3 and 5 days.

The cellular assessment of hMSCs grown in cl-CMC-pMETAC/AgNWs and control media was appraised by rhodamine-phalloidin and DAPI assay with time (Fig. 6b). From the morphology it is evident that cells grown on cl-CMC-pMETAC/AgNWs containing media were similar to that of the cells cultured in control media. Fig. 6b suggests that on day 1, number of cells was few that was increased on day 3 and day 5 along with bigger in size. The hMSCs grown on day 5 in cl-CMC-pMETAC/AgNWs containing media displayed enlarged F-actin filaments and enhanced cell to cell association, since F-actin signify the presence of thin filaments in cells and allows the cells to interact with surroundings.49, 51 The fluorescent images visibly establish that cells grown on cl-CMC-pMETAC/AgNWs containing media were providing a favorable atmosphere for cell to cell connections, confirming the cytocompatibility and nontoxic nature of cl-CMC-pMETAC/AgNWs.



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Intracellular uptake study The intracellular release of curcumin from curcumin-loaded cl-CMC-pMETAC/AgNWs nanocomposite hydrogel was verified using MG 63 cancer cell lines. The viability of the curcumin-loaded cl-CMC-pMETAC/AgNWs nanocomposite hydrogel was analyzed. Effective cellular internalization of composite hydrogel is essential for intracellular release of curcumin and competent therapy. To identify the intracellular passage of the nanocomposite, FITC was internalized into the cl-CMC-pMETAC/AgNWs nanocomposite hydrogel. The fluorescence image of FITC-loaded nanocomposites were taken using fluorescence microscopy after certain time of intervals (2 h and 4 h) (Fig. S18, Supporting Information). The green fluorescent colored FITC-loaded nanocomposite (Fig. S18, Supporting Information) confirmed that the nanocomposite was successfully incorporated through endocytosis or macropinocytosis into MG 63 cancer cells.64 With increasing incubation time, the emergent green fluorescent intensity of FITC-loaded nanocomposite was also increased, which suggest that large number of FITC-loaded nanocomposites have been endorsed by MG 63 cells. This also supports that loaded nanocomposites were circulated and up taken through the cytoplasm into the MG 63 cancer cells. Using fluorescence microscopy, the release property of intracellular curcumin was examined from curcumin-loaded nanocomposite. The MG 63 cells were incubated with different concentrations of curcumin loaded nanocomposite and control (20, 40, 60, 80 and 100 μg/mL). The fluorescence images of MG 63 cells treated with curcumin-loaded nanocomposites were captured after 24 hours and shown in Fig. 7 (i).


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Fig. 7: Fluorescence images of MG 63 cancer cells with different concentrations of curcumin-loaded nanocomposite over incubation: (i) (a): control, (b): 20 μg/mL, (c): 40 μg/mL, (d): 60 μg/mL, (e): 80 μg/mL, (f): 100 μg/mL and (ii) Cell viability results with different concentrations of the curcumin-loaded nanocomposite.

The red fluorescence pictures of the Mg 63 cells signify that curcumin loaded composite hydrogel was endocytized into the cells [Fig. 7i (b-f)]. It has also noticed that with increase in dosing of the nanocomposite, the number of cancer cells were decreased (Fig. 7ii), which ensures that curcumin-loaded nanocomposite validate considerable growth inhibition. The cytotoxicity of curcumin-loaded cl-CMC-pMETAC/AgNWs nanocomposite was deliberated using MTT assay when it was incubated with MG 63 cancer cells with different concentrations (20-100 μg/mL). It is obvious that number of MG 63 cells were decreased after incubation with curcumin loaded nanocomposite, which confirm its cytotoxicity behaviour (Fig. 7ii). These observations thus authenticate that the curcumin-loaded cl-CMCpMETAC/AgNWs nanocomposite could efficiently devastate the MG 63 cancer cells and probably be an outstanding carrier for curcumin delivery towards cancer cells. We have further performed TUNEL assays to determine whether curcumin loaded clCMC-pMETAC/AgNWs nanocomposite has the ability to kill MG 63 cells by inducing apoptosis. As observed from Fig. 8, MG 63 cells exposed to curcumin loaded cl-CMC27 ACS Paragon Plus Environment


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pMETAC/AgNWs nanocomposite for 24 hours showed high number of TUNEL-positive cells. On the other hand, no notable TUNEL-positive populations was observed in control (absence of curcumin loaded cl-CMC-pMETAC/AgNWs nanocomposite), suggesting the killing ability of the curcumin loaded nanocomposite hydrogel towards MG 63 cells.

Fig. 8: Tunel assay images of MG 63 cancer cells with control and curcumin-loaded nanocomposite hydrogel treated after 24 hours of incubation.

Transdermal delivery of curcumin In-vitro Curcumin is a major constituent of turmeric and also it is an important natural anticancer drug as it extracted from turmeric (root of curcuma longa). Curcumin shows major interaction with nanocomposite hydrogel as it possess –C=O as well as –OH that can bind with –OH and –C=O groups present into nanocomposite hydrogel (cl-CMC-pMETAC/AgNWs). The interaction between drug molecule and nanocomposite hydrogel is mainly through hydrogen bonding/physical interactions (as demonstrated in Fig. S10, Supporting Information). Due to the presence of AgNWs onto crosslinked CMC backbone, the interaction becomes more 28 ACS Paragon Plus Environment

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prominent and it shows sustained release characteristics. Curcumin loading was carried out in cl-CMC-pMETAC/AgNWs with variation of time (1-3 days) and the results are shown in Fig. S19 and Table S2, Supporting Information. It is obvious that maximum loading and entrapment were observed after 3 days of loading. The loading efficiency and entrapment efficiency of curcumin were 18.11% and 78.63%, respectively. Fig. 9a represents the in-vitro release profile of curcumin loaded CMC-g-pMETAC, cl-CMCpMETAC and cl-CMC-pMETAC/AgNWs upto 4 days. From drug release profiles, it is apparent that nanocomposite hydrogel released the drug at controlled rate in comparison to crosslinked polymer and graft copolymer. The graft copolymer released the entire drug within 48 hours whereas crosslinked hydrogel shows more sustained release behaviour owing to the presence of crosslinked network on the polymer backbone. Finally, nanocomposite hydrogel, which has maximum gel strength demonstrates the release of curcumin in most sustained manner. The reason behind this may be the presence of AgNWs that interact with the crosslinked polymer and make the network more compact. Besides, from the Korsemeyer Peppas model, it is obvious that release mechanism is Fickian diffusional in nature (Table S3). Ex-vivo skin permeation study The cumulative drug permeation of curcumin through rat skin versus time profile are shown in Fig. 9 (b). From the result it was observed that continuous, linear and steady state permeation release rate profile of drug from both nanocomposite and hydrogel were obtained. The ex-vivo permeation of the drug was controlled and the release was gradually increased as observed from Fig. 9 (b). The steady state flux and apparent permeability coefficient was found to be 93.34 ± 6.37 µg/cm2.h and 74.65 ± 3.54 cm h-1, respectively from curcumin loaded crosslinked hydrogel network, while from curcumin loaded nanocomposite hydrogel steady flux and apparent permeability coefficient were found to be 76.83 ± 8.65 µg/cm2.h and 27.30 ± 2.76 cm



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h-1, respectively. These results thus illustrate that the prepared nanocomposite hydrogel could be an ideal formulation for the delivery of curcumin through transdermal route.

Fig. 9: (a) Release pattern of curcumin loaded with graft copolymer, crosslinked hydrogel and nanocomposite hydrogel. Results are mean ± SD (n=3); (b) Ex-vivo release of curcumin form crosslinked hydrogel and nanocomposite hydrogel. Conclusion It is thus evident that a novel biocompatible and non-toxic nanocomposite hydrogel was successfully fabricated. Various characterizations confirm the formation of crosslinked network as well as uniform deposition of AgNWs. The rheological analyses demonstrate the excellent gel characteristics of the nanocomposite hydrogel. The composite is non-cytotoxic towards hMSCs and MG 63 cancer cells, while the curcumin loaded composite hydrogel destroys the cancer cells, which has been confirmed by triggered apoptosis test. Finally, the incorporation of AgNWs enhanced the sustained release characteristic of transdermal drug


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curcumin in both in-vitro and ex-vivo pathway and the developed nanocomposite hydrogel (clCMC-pMETAC/AgNWs) seems to be an excellent alternate as transdermal drugs carrier. Acknowledgements Authors earnestly acknowledge the financial support from SERB, Department of Science & Technology, New Delhi, India in form of a research grant (File No. - EMR/ 2014/000471). Details of Supporting Information: Detailed discussion of characterization techniques; Experimental procedure for rheological study; Experimental procedure for swelling study; Experimental procedure for biodegradation study; Experimental procedure for In vitro cytocompatibility study; Experimental procedure for Intracellular uptake study; Experimental procedure for Apoptosis analysis by Tunel Assay; Drug release mechanism models; Experimental procedure for Ex-vivo skin permeation study; Synthesis details; UV-Vis-NIR result, FTIR and NMR spectra; EDAX analysis; AFM analysis; TGA/DTG results; Result of rheological parameters; Swelling results; Biodegradation results; Confocal images of intra cellular uptake study; drug release mechanism data.



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In Situ Silver Nanowire Deposited Cross-Linked Carboxymethyl

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