Green Reduced Graphene Oxide Toughened Semi- IPN Monolith

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B: Biophysics; Physical Chemistry of Biological Systems and Biomolecules

Green Reduced Graphene Oxide Toughened Semi-IPN Monolith Hydrogel as Dual Responsive Drug Release System: Rheological, Physico-Mechanical and Electrical Evaluations Sayan Ganguly, Poushali Das, Priti Prasanna Maity, Subhadip Mondal, Sabyasachi Ghosh, Santanu Dhara, and Narayan Chandra Das J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b02919 • Publication Date (Web): 03 Jul 2018 Downloaded from http://pubs.acs.org on July 5, 2018

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The Journal of Physical Chemistry

Green Reduced Graphene Oxide Toughened SemiIPN Monolith Hydrogel as Dual Responsive Drug Release System: Rheological, Physico-Mechanical and Electrical Evaluations Sayan Ganguly1, Poushali Das2, Priti Prasanna Maity3, Subhadip Mondal1, Sabyasachi Ghosh1, Santanu Dhara3, Narayan Ch. Das1,2* 1

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3

Rubber Technology Centre, Indian Institute of Technology, Kharagpur 721302 School of Nanoscience and Technology, Indian Institute of Technology, Kharagpur 721302 School of Medical Science and Technology, Indian Institute of Technology, Kharagpur 721302

ACS Paragon Plus Environment

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Abstract

Macroporous

hydrogel

monoliths

having

tailor-made

features

viz.

conductivity,

superstretchability, excellent biocompatibility, and biodegradability become the most nurtured field of interest in soft biomaterials. Green method assisted reduced graphene oxide has been inserted by in situ free radical gelation in semi-IPN hydrogel matrix to fabricate conducting hydrogel. Mechanical toughness has been implemented for the graphene-polymer physisorption interactions with graphene basal planes. Moreover, the as-prepared 3D scaffold type monolith hydrogel has been rheologically superior regarding their high elastic modulus, and delayed gel rupturing. κ-carragenaan, being one of the component of the hydrogel, it has biodegradable nature. The most significant outcome is their low electrical percolation threshold and reversibly ductile kind of nature. Reversible ductility provides them rubber like consistency in flow conditions. Surprising, the hydrogels showed dual stimuli responsiveness i.e. environmental pH and external electrical stimulation. Electro-stimulation has been adopted here for the first time in semi-IPN systems which could be ideal alternative for iontopheretic devices and pulsatile drug release through skin. Regarding this the hydrogel also has been passed in biocompatibility assay; they are non-cytotoxic and cell-proliferation without/negligible cell death in live-dead assay. Porosity of the nanocomposite scaffold-like gels was also analyzed by micro-computed tomography (µ-CT) which exhibited their connectivity in cell/voids inside matrix. Thus, the whole experimentations are the on the support of biocompatible soft material for dual-responsive tunable drug delivery.


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Introduction Carbon nanomaterials draw attention due to their unique features like high surface area (~2630 m2g-1), strengthening effect (Young’s modulus ~1.0 TPa) in polymer matrix, high thermal conductivity (~5000 Wm-1K-1), electronic mobility (~2.5×105 cm2v-1s-1), good optical clarity (transmittance ~97.7%) and obviously intrinsic conductivity

1,2

. The most widely nurtured

conducting nanoparticle with outstanding features is graphene. Graphene is a monolayer thick catenated sp2 hybridized honeycomb nanostructure 3. The extraordinary properties of graphene or their functionalzed forms can make them applicable towards high strength composites, gas sensors, actuators, medical electrodes, conducting biomaterials, transistors and so on

4-6

. The

extensive applications of graphene based materials; there is immense demand of low cost graphene based materials to fulfill the abovementioned devices and applications 7. There are plenty of methods proposed by several researchers about the preparation of reduced graphene oxide (RGO) in order to fulfillment of the graphene. Among all the methods to prepare RGO, ecofriendly or green method development is a common practice which is very much compatible to the polymer matrix as well as electrically conducting. Now if the conductivity of the RGO nanoparticles be used as an enhancer of electronic transport inside a polymer matrix, then we can get electro-conductive polymer nanocomposites. Thus our aim was to fabricate such a hydrogel with conductive nanoparticles which can actuates after external electric stimuli and also supports the strengthening criteria of nanocomposite hydrogels. Hydrogels are typical crosslinked mass of polyelectrolyte chains. They are either physically crosslinked or chemically. In general hydrogels are swellable in water medium and insoluble in any media 8-10. Electro-conductive hydrogels can be prepared either by incorporating intrinsically conducting polymer or carbonaceous conducting nanoparticles. Here conducting polymers


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normally cannot enhance the mechanical properties and toughness. Thus it is better to go with the carbonaceous nanoparticles especially graphene which not only withstands external loads but also tailored some specific attributes into the fabricated hydrogels. But till now there are several limitations of electro-conductive nanoparticles filled hydrogels, like easily deformability, withstanding specific shape for a long period of time and lack of rubber-like toughness. To overcome such demerits, the acceptability of the covalent crosslinking reaction cannot be ruled out. Nanoparticles associated with covalent crosslinker have profound effect on the mechanical strengthening of nanocomposite hydrogels 11. Another problem which is well known that pristine graphene has severe toxicity over human physiological activities which can also be overcome by fabrication of in situ nanocomposite hydrogel. Now one question may arise that why kappa carrageenan has been employed here to fabricate the hydrogel. Kappa-carrageenan is a wellknown thixotropic biopolymer used in several food grade items as a viscosity enhancer 12. These are naturally occurring polysaccharides obtained from red seaweeds and composed of sulfated Dgalactose. Kappa-carrageenan is an anionic biopolymer due to the presence of sulphonate moieties. In this report we developed an electro-conducting nanocomposite hydrogel which involved twostage synthesis and fabrication method. In the first stage of synthesis we developed reduced graphene oxide (RGO) by a green method using L-cysteine as the green reducing agent. The second stage was based on the in situ free radical gelation technique between kappa-carrageenan and acrylic acid using a divinylic crosslinking agent. The incorporation of RGO into the gel matrix was studied in the context of conductivity and electrical percolation theory. The rheological and mechanical characteristics were also investigated to disclose the effect of RGO inside gel matrix.


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Experimental details Materials Graphite powder and L-cysteine (~97%) were purchased from Sigma. Kappa-carrageenan (mucopolysaccharides from the cell walls of the red algae), acrylic acid (AA, 99%), 2Acrylamido-2-methyl-1-propanesulfonic acid (AMPS, 99%) and N, N-methylenebisacrylamide (MBA) were procured from Sigma Aldrich. The redox initiator system; potassium persurfate and sodium metabisulfite and other chemicals were procured from Merck, Germany. All chemicals used here are of analytical grade. Preparation of water-dispersible graphene oxide (GO) Graphene oxide (GO) was prepared by the modified Hummer’s method from graphite flakes as reported on earlier literature

13

. The method has been briefed in supporting information named

section ‘1.1’. Reduction of GO by L-cysteine Aqueous dispersion of GO (20 mL) having concentration of 0.1 g/100 mL was taken in a glass vial. Then the dispersion was mixed with 4 mM of L-cysteine solution and ultrasonicated for 45 min. after that the glass vial was heated at ~90oC for 12 h and a dark blackish dispersion was obtained. This dark dispersion pH was adjusted at ~9.0 after addition of ~30% ammonia solution to enhance the colloidal stability of the dispersion. This colloidal dispersion is reduced graphene oxide (RGO). Then the whole solution was successively washed thoroughly by ethanol and water followed by centrifugation at 10000 rpm for 20 min. The final solid mass was collected and dried overnight at ~45oC. Synthesis of in situ nanocomposite hydrogel


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For preparing the in situ NCHG, initially the RGO was ultrasonicated in water for 30 min. Then definite amount of kappa-carrageenan aqueous dispersion was added into the RGO colloidal dispersion which stabilized the system. The system was again sonicated for 30 min. The monomer acrylic acid (AA) and 2-Acrylamido-2-methyl-1-propanesulfonic acid (AMPS), was poured into the system and stirred vigorously for 15 min. The AA to AMPS ratio was taken 1: 0.088 (mol ratio). After that MBA and redox initiator couple was added accordingly and kept the temperature in between 35-38oC. The mixture was sealed with Parafilm M stretch wrap film (ETA-352, Edutek) and prior to pre-gel state the total system was carefully transferred to a Teflon Petridis and sealed with a lid and kept in the room temperature for complete gelation. The system was kept for overnight and the gelled sheet was taken out from the Petridis and dipped into ethanol-phosphate buffer solution (initially used 70% ethanol aqueous solution) to leach out the unreacted monomer fractions and initiator fragments. Then the gelled mass was dried under room temperature and taken for further characterizations. Material characterizations Swelling experiment Water uptake behavior and grafting of monomers on to kappa-carrageenan skeleton have been elucidated in detail in supporting information named section ‘1.2’14. X-ray photoelectron spectroscopy (XPS) XPS analysis was carried out using a commercial Kratos Axis Ultra DLD spectrometer with AlKrα radiation (1486.7eV). Emission current of the X-ray source was fixed at 20 mA for an anode voltage of 15 kV. XPS samples were prepared by spreading the powder sample on a conducting copper tape. To remove the surface contamination occurred; samples were locally sputtercleaned using low energy argon ions under the ultra-high vacuum condition prior to the XPS


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measurements. At the same time, Ar+ beam induced any kind of degradation of the samples has been eliminated by comparing the XPS spectra before and after the sputter cleaning. High resolution XPS spectra were collected using pass energy of 20 eV, with a step size of 0.02 eV. The UHV chamber base pressure was maintained < 10-9 mbar throughout the measurements. To compensate any kind of charging effect, O 1s binding energy peak at 532.5 eV has been used as a reference here. Fourier Transform Infrared (FTIR) Spectroscopy FTIR spectra of the pure hydrogel and drug loaded hydrogel samples were executed by a FTIR spectrophotometer (Perkin Elmer, Spectrum-2, Singapore) in ATR mode. The range of study was fixed at 500-4000 cm-1 with a 16 scans and 4 cm-1 resolution. Thermal Analysis Thermogravimetric analysis (TGA) of the hydrogel sample and composite gel were performed in nitrogen atmosphere with 35 mL/min volumetric flow rate in Perkin Elmer instrument (TGA50, Shimadzu, Japan) in 25 to 600oC temperature range and scan rate of 10oC/min. Field emission scanning electron microscopic (FESEM) studies Surface morphology of the hydrogel was performed in field emission scanning electron microscope (FESEM, MERLIN with tungsten filament; Carl ZEISS, SMT, Germany) with the accelerating voltage set to 15 kV. Initially the hydrogel sample was swollen in pH=7.4 (phosphate buffer saline, PBS media, Sigma; containing 0.01 (M) phosphate buffer, 0.0027 (M) KCl and 0.137 (M) NaCl). Then the swollen hydrogels were sudden quenched in liquid nitrogen followed by lyophilization in Vitris Benchtop lyophilizer for 30 h. Swelling and deswelling experiments


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Water uptake/Swelling sttudy was performed gravimetrically on a definite quantity of dried discshaped hydrogel (xerogel) pieces. At first the considered amount of specimens were plunged into 100 mL borosilicate glass container with 103 times volume of DI water at ambient temperature ( ̴ 30oC). After ~ 48 h the specimens were moved out and soaked to tissue paper without pressing. The swelling ratio (SR) of the hydrogels was calculated by the following equation: =

(1)

Where Ws and Wd are the weight of swollen hydrogel and dried hydrogel respectively. At equilibrium point, Ws = Weq where Weq is the mass of the swollen gel at equilibrium point. Equilibrium swelling ratio or ESR was calculated by replacing Ws with We in eq (1). The buffer solutions was prepared by H3PO4, KH2PO4, K2HPO4 and NaOH in DI water. 0.1 (M) NaCl aqueous solution was imparted to keep the proper ionic strength of the buffer. For kinetic study of water uptake the above mentioned experiment was done with time. Rheological characterization Rheological study of the hydrogels were executed in swollen state at phosphate buffer (pH ̴ 7.4) using a rheometer (Bohlin Gemini-2, Malvern, UK). Shear sweep was performed at 1 Hz in the shear range from 1-100 Pa using parallel plate geometry with 1 mm clearance. Frequency sweep experiments were carried out in the range of 1-10 Hz at 25 Pa constant stress. The gel strength (elastic modulus; Gʹ and loss modulus; Gʺ) was calculated as per literature reported elsewhere.15 Mechanical property study Tensile testing was performed by using universal tensile testing machine (Hounsfield) with a 500 N load cell. The specimen dimension was 35 mm × 12 mm × 12 mm. Both ends of the specimen was gripped by flint paper to avoid slippage and pulled at a constant crosshead speed of 50


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mm/min at 24oC. The ultimate tensile strength was obtained from the point of rupture of the hydrogel. Micro-computed tomography (µ-CT) Microstructural analysis and porosity calculation were carried out through micro CT imaging. Samples were scanned using X-ray radiation for 3D imaging using micro CT (GE phoenix v|tome|x, Germany) with the X-ray source voltage 110 kV and beam current 100 µA. The scanning resolution was achieved up to 16 µm for analyzing defined region of interest (ROI). VGStudio MAX (Volume Graphics, Germany) software was used for total porosity and pore architecture. The 3D images processed here were inverted to analyze connectivity in micro-pores in quantitative fashion. Biodegradation study by hen egg lysozyme Biodegradation of the kappa-carrageenan based hydrogels were studied by using hen egg lysozyme as reported in literature

16

. The detailed procedure has been given in supporting

information named section ‘1.3’. In vitro Cytotoxicity assay and live-dead assay The in vitro cytotoxicity assay was performed for the RGO-filled hydrogel to evaluate the system as an efficient soft biomaterial. The live-dead study was also performed to monitor the extent of living character of cells in hydrogel matrix. The detailed working procedures were elaborated in supporting information named section ‘1.4 and 1.5’. pH-responsive drug release In vitro drug release study was performed at 33oC±0.5oC taking diltiazem hydrochroride as a model drug. The detail procedure is given in supporting in supporting information named section ‘1.6’.


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Electric-stimulation for drug release Electric pulse driven release was also performed as the detailed experimental procedure is given in supporting information under the section ‘1.7’. Effect of drug release on temperature Temperature dependent drug release was performed at 30oC, 37oC and 44oC. The drug loaded specimens were immerged into DI water maintaining 40 rpm speed in a steady state. The release data were plotted as per the procedure given above named ‘pH-responsive drug release’. Results and discussion Morphological analysis of RGO Transmission electron microscopic (TEM) images of GO and RGO provided information about the sheet like morphology. Figure 1a is the corrugated randomly dispersed nanosheets of GO. In inset of the Figure 1a showed the SAED pattern of GO which revealed the prominent polycrystalline ring patterns. The rings are from the diffraction of (1100) and (1120) crystalline planes of GO. This result implied that the crystalline array of GO was not destroyed before reduction. The wrinkled single sheet of RGO was also depicted in Figure 1b. The typical polycrystalline diffraction pattern was depicted in Figure 1b (inset). The XRD patterns of pristine graphite, GO and reduced graphene oxide (RGO) has been presented in Figure 1c. The graphite powder showed a strong and sharp peak at 2θ value of 26o which corresponds to the (002) plane. This result has resemblance with graphitic structure as per ICDD-PDF # 411487. In case of GO the 2θ value of the diffraction peak was evaluated at 11.3o. The increment in d-spacing for GO with respect to the pristine graphite can imply the enhancement of gallery gap. The inter-sheet distance or gallery gap was enhanced because of the insertion of water molecules and other oxygenated moieties generated by oxidation of graphite.


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After reduction of GO by amino acid, the plot showed no such characteristic strong peak in the XRD pattern. This can corroborates the transformation of GO to RGO, rather say single layer of RGO 17. The chemical reduction method was also supported by the Raman spectra of the GO and RGO in Figure 1d. The chemical reduction of GO to RGO can be supported by assessing the two characteristic peaks in Raman spectra; one is D-band at 1350 cm-1 and another one is G band at 1575 cm-1

18

. The D and G bands correspond to the structural anomalies and the graphitic

architectures respectively. The D and G bands for GO before and after reduction were at 1361 and 1622 cm-1 respectively. After reduction of GO by L-cysteine the intensity ratio of the D and G band peaks (ID/IG) was increased from 0.92 to 1.06. Such increment in intensity ratio implies enhancement of sp2 domains after reduction of GO. Another cause of this increment of intensity ratio can be supported by the presence of unrepaired disparities that persists even after removal of oxygenated moieties from GO surface 19. The UV-vis absorption spectra of GO and RGO in aqueous medium depicted in Figure 1e. GO has a characteristic peak at 230 nm associated with a small shoulder peak at ~300 nm which were due to the ᴨ-ᴨ* transition of the aromatic C=C plasmon and n-ᴨ* transition of carbonyl groups 20

. The red shift of the Plasmon peak (230 nm) i.e. shifting of 230 nm peak to 270 nm peak of

RGO confirms better ᴨ-stacking and higher ᴨ-electron concentration

21

. This result implies that

GO might be chemically reduced with restoring their gallery gap dimension as of graphite. Thus lowering of gallery gap of RGO with respect to GO infers better aromatic interaction among the RGO sheets. Similar kind of result was also reported elsewhere 22. Chemical reduction of GO is still an open question though a plausible mechanistic approach has been given in Figure 1f. We hypothesized SN2 nucleophilic reaction followed by thermal


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elimination. L-cysteine is an S-containing (because of its thiol group) amino acid. Thiol groups are very much prone to nucleophilic reactions and readily oxidized into L-cystine. It means Lcysteine and GO takes part in a redox type nucleophilic reaction where L-cystine is a byproduct. To remove out the L-cystine, the whole product mixture was shanked with mild alkaline medium (0.1 M). Side by side the addition of NaOH into the RGO dispersion also helps to partically stabilize the nanosheet dispersion. The less amount of carboxylic acid groups present in the GO surface might be turned into sodium carboxylate salt. The carboxylated nanosheets repelled each other because of the surface electrostatic negative charges and the nanosheets dispersion was stabilized. Thus another point of choosing this chemical reduction is its stabilizer free one-pot synthesis by gentle reaction condition. Chemical reduction of GO can be confirmed by the XPS where the chemical abundance of the surface functionalities can be evaluated. Complete reduction to eradicate the oxygenated functionalities from GO surface is very difficult; that’s why XPS is necessary to evaluate the relative amount of carbon and oxygen before and after reduction (Figure S1a). The high resolution C1s spectra of GO (Figure S1b) and RGO (Figure S1c) imply the characteristic elemental peaks at 284.6 eV, 285.4 eV, 286.2 eV, 288.2 eV and 289.1 eV for C-C, C-OH, Cepoxy, C=O and O-C=O respectively. It is evident form the plots that after chemical reduction the intensities of the oxygenated functionalities (C=O, C-O, O-C=O) present in RGO became weaker than GO. This suggests removal of oxygenated groups from the GO surface to some extent. Besides this, the intensity of the C=C (sp2) peak radically increased in C1s spectra of RGO which suggests restoration of graphitic array after reduction of GO. After reduction of GO, a small peak of Na1s was also observed at 1070 eV which can be due to the presence of residual NaOH during reduction.


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Physical and morphological analysis of in situ nanocomposite hydrogel The preparation of semi-IPN was schematically represented in Figure 2. The reaction mechanism showed grafting of monomers onto kappa-carrageenan as well covalent crosslinking by divinylic MBA. The initiation was a redox triggered reaction which produced sulfate and bisulfate free radicals which latterly produced hydroxyl free radical. Hydroxyl free radical abstracted one hydrogel from the pendant hydroxyl group resulting formation of kappa-carrageenan macro radical. This macro radical was then very prone to Michael type grafting with vinylic monomers. Here acrylic acid (AA) and 2-Acrylamido-2-methyl-1-propanesulfonic acid (AMPS) were used to prepare copolymeric hydrogel where MBA acts as a divinylic crosslinker. Thus it could also be proposed that being a divinylic crosslinking ability of MBA, the prepared hydrogel is basically a terpolymer hydrogel. The FTIR result in the terms of evidence was also given in Figure S2(A). The characteristics absorption peaks at 840, 916, 1020 and 1224 cm-1 corresponds to the D-galactose-4-sulfate, 3,6-anhydro-D-galactose, glycosidic linkage and ester sulfate stretching respectively 23. The broad absorption peak at 3200-3600 cm-1 was due to the stretching vibration of hydroxyl groups. In the FTIR spectrum of hydrogel, absoption peaks at 1724, 1550, 1654, 1216 and 3452 cm-1 implied the grafting reaction. These all peaks are appeared due to carbonyl, carboxylic acid group stretching and symmetric and asymmetric stretching vibrations of carboxylates, stretching modes of amine, sulfate and hydroxyl groups respectively. For grafted hydrogel, the hydroxyl peak broadening was noticed as an outcome of Michael type grafting. Similar kind of Michael type grafting phenomenon was also evidenced elsewhere

14, 24

. For in

situ nanocomposite hydrogels the similar type of peak broadening was seen due to the formation of physical interaction and H-bonding in between basal planes’ and residual groups present in RGO and gel matrix. The evidence of partial reduction of GO was also showed in Figure S2(B).


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The characteristic hydroxyl peaks at 3200-3400 cm-1 was drastically diminished for the RGO specimen. But complete was not taken place which affect slight absorption peak of RGO in the peak. The FTIR spectra of RGO did not show any characteristic peaks at ~2900 cm-1 (-NH2 stretching) and 2550 cm-1 (S-H stretching) which could imply that there were no L-cysteine residue in RGO nanoparticles. For clear understanding of the grafting, similar kind of free radical polymerization reaction was carried out in absence of the divinylic crosslinker (here MBA). Grafted kappa-carrageenan was estimated after extracting the un-grafted homopolymers, oligomeric fractions and unreacted monomers by using a dialysis bag (D9402). This resulted around 78% grafted product. The calculated grafted percentages were 62.08%, 63.11%, 64.74%, 65.01%, 69.57%, 72.65% and 77.28% for 0.1, 0.3, 0.5, 0.7, 1.0 and 1.5 wt% κ-carrageenan contents respectively. The grafting efficiency was also measured and those are 44.85%, 45.23%, 46.04%, 49.77%, 53.36% and 56.79% for the aforementioned concentrations of kappa carrageenan respectively. The effect of kappa-carrageenan on grating is depicted in Figure S3. The prepared gels were also monitored by XRD which revealed the semi-crystalline nature of the hydrogel as showed in Figure S4. Microstructural evaluation of the synthesized nanocomposite semi-IPN hydrogel was performed in electron micrograph in Figure 3a. The freeze dried hydrogel showed highly porous microstructure with a thin cell wall. Such porosity could be an outcome of crosslinking and formation of 3D network inside the gel matrix. Porous hydrogels also have superior water uptake behavior. Water uptake of hydrogels normally depends on their microstructure or better says their porous type of architecture. The microstructure of the hydrogels can be controlled by the graphene loading concentration. Polymer chains have the proneness to physisorbed over the graphene


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sheets because of high surface energy of graphene sheets. Such morphological implies the greater physical crosslinking inside the gel matrix which results lower porosity. Lowering of porosity drastically deteriorates the water uptake behavior i.e. swelling. The swelling data showed in Figure 3b. In case of unfilled pure copolymer hydrogel, i.e. poly(AA-co-AMPS) the swell ratio enhanced to 9.41 whereas for κ-carrageenan-g-poly(acrylic acid) gel the SR increased to 16.42. The incorporation of κ-carrageenan, a highly hydrophilic natural bio-macromolecule, improves the swelling around three fold. With increasing the κ-carrageenan content the water uptake value improves drastically. This improvement can be implied as the electrostatic repulsive effect of the sulphonate groups present in the κ-carrageenan. But after 1.5 wt% concentration, the swelling ratio decreased gradually. This could be a cause of extensive grafting of monomers onto κ-carrageenan backbone which implicate more compact microstructure. Compact microstructure diminished the pore size resulting low water imbibition to gel 3D network. Thus for better swelling property the 1 wt% κ-carrageenan content hydrogel can be a desirable choice. Besides this, RGO content in feed concentration during in situ gelation also has significant effect in water uptake behavior (Figure 3c). As per the aforementioned result on swelling, 1 wt% κ-carrageenan was chosen for further synthesis of nanocomposite semi-IPN. Incorporation of RGO as filler for gel matrix, the swelling ratio decreased gradually. The polymer to RGO basal plane physisorption phenomenon is very much stringent for the in situ nanocomposite hydrogels. Physisorption implicates physical crosslinking which cut down the water uptake. Thermal stability of the hydrogel and RGO filled nanocomposite hydrogels was evaluated by TGA analysis (Figure 3d). The unfilled semi-IPN gel showed 3-stage thermal degradation which has resemblance to native κ-carrageenan. The 1st stage degradation (~30-190oC) implied to the


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water loss. The 2nd stage (~190-250oC) and 3rd stage (250-500oC) weight loss were justified to the chain degradation and carbonization to ashes. For semi-IPN hydrogel the weight loss characteristic is almost similar to κ-carrageenan. For filled nanocomposite hydrogels, i.e. RGO loaded hydrogels showed higher thermal stability than unfilled hydrogel. This result may be due to the high thermal conductivity of the graphene nanosheets and compact microstructure of physisorbed graphene sheets inside gel matrix.

Swelling and mechanism of diffusion

Kinetics of water uptake The swelling data were plotted (St vs t) and also fitted directly to the following pseudo second order rate Equation and shown in Figure S5 and Figure S6,14

=

=

(2)

Here, kS2 corresponds to 2nd order rate constant, Secal is calculated ESR and r0 is initial swelling rate. The statistical parameters such as r2, χ2, F and the equilibrium swelling ratio (ESR), both experimental (Swexpt) and calculated (Swcal) , rate of swelling (r0), 2nd order rate constant (kS2) of the swelling are shown in Table S1. From Table S1 the values of Swexpt and Swcal are also noticed to be very close and data fittings prove the values of r2 close to unity, low values of χ2 and high values of F. Herein, kappa-carrageenan content and RGO filler content were varied separately. Kappa-carrageenan, being a highly hydrophilic biopolymer it has sufficient water tolerance character. The kappa-carrageenan content up to 1.0 wt% showed linear increment in water uptake behavior but beyond 1.0 wt% the swelling was decreased. The cause was the indirect effect of high medium viscosity. High viscosity restricted the monomer mobility during


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grafting reaction results hindrance in diffusion of monomers. If the monomers were not allowed to be grafted at the active sites of kappa-carrageenan then crosslinking or gelation would not take place properly which might be the cause of low water uptake at high kappa-carrageenan concentration. The swelling results are showed in Figure S5-S7. In case of RGO-filler in situ systems, the water uptake was controlled by the physisorbed RGO nanosheets’ organization. RGO normally makes physical interactions with the polymer chains resulting crosslinking points (Figure S8-S10). Such crosslinking points drastically diminished the water imbibition through the gel matrix. Thus RGO-filled hydrogels showed low water uptake behavior. Diffusion The study of diffusion of water molecules through intertwined networks of semi-IPN type hydrogel is substantial to investigate the release and uptake behavior of small molecules. The time dependent water uptake data was fitted into the eq. (3) and (4) to calculate the diffusion characteristics viz. diffusion coefficient (D), diffusion exponent (n), and diffusion constant (kD) of the hydrogels. The fitted plot is depicted in Figure S7. The working equations mentioned above are given below, =

=π

=

(3)

&

! # ' " % $

(4)

Here F corresponds to fractional water uptake and r is radius of cylindrical hydrogel sample. The nonlinear

regression

and

data

fitting

were

carried

on

to

evaluate

diffusion

characteristics/parameters of hydrogels. The values of diffusion characteristics, i.e., kD, n and D of the hydrogels are also summed up in Table S1. The prepared semi-IPN hydrogels showed the diffusion exponent value in the range of 0.4-0.62 which signifies non-Fickian anomalous


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diffusion behavior. Swelling of hydrogel is a continuous ‘in-line’ process where un-solvated glass/partially rubbery system enjoys a transition toward relatively rubbery state. Ideal Fickian transportation is occurred for those polymer systems where glass transition temperature (Tg) is far below the ambient temperature. This happens due to the deviation of rate of polymer chain relaxation and diffusion rate. In case of Fickian diffusion the rate of diffusion is always lower than the rate of polymer chain relaxation in the gel matrix. In our case the diffusion exponent data implied anomalous diffusion. Anomalous diffusion is a special class of hypothesis where the rate of diffusion and the rate of chain relaxation are assumed to be comparable 25. Rheological analysis of in situ nanocomposite hydrogel Rheology is choice characterization procedure in viscoelasticity analysis of hydrogels. Figure 4(a) is frequency sweep experiment of the prepared hydrogels. In case of pure κ-carrageenan the storage/elastic modulus (Gˊ) goes below of the loss/viscous modulus (G˝) which signifies the high flowability of the biopolymer dispersion (Figure S11). For the prepared hydrogel i.e. κcarrageenan-g-poly(acrylic acid-co-AMPS), the Gˊ value was much higher than G˝ which implied the stable gel formation. The gel strength of the prepared hydrogels was given in Table 1. The sufficient gradient between the elastic and viscous modulus corresponds to the gel strength (Gˊ/G˝) which is clearly seen in the κ-carrageenan-g-poly(acrylic acid-co-AMPS). The flow behavior with κ-carrageenan concentration also significantly provides information of gel strength in case of semi-IPN. The improvement in gel strength was seen when κ-carrageenan concentration was enhanced. This could be a reflection of higher percentage of Michael type grafting of poly(acrylic acid-co-AMPS) onto κ-carrageenan. Thus it can be inferred that grafting could be a major strategy to improve gel strength as well as sustaining dimensional stability. Michael type grafting offers superior elastic nature of hydrogels. The in situ RGO toughened


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nanocomposite hydrogels were also tested under same conditions (frequency sweep) (Figure 4b). The flow property of the hydrogels was restricted after insertion of RGO as filler. In case of toughened hydrogels the polymer chains in the gel matrix were anchored covalently as well as physically. Physical attachment is due to the physisorption of the matrix polymer chains onto basal plane of the RGO nanosheets. Physisorption enhanced the secondary crosslinking inside the gel matrix. Such secondary crosslinking improves the inherent stress withstanding feature of the hydrogel. In case of higher RGO loaded hydrogel the monotonous steep rise (higher slop) has been observed which implied the better elastic response of RGO filled hydrogels than unfilled hydrogels. The stress sweep experiment was also conducted for in situ RGO toughened nanocomposite hydrogels (Figure 4c). The plot showed the gradual drop of modulus with increasing the shear stress. The decreasing trend of modulus also showed a sudden fall after a definite amount of stress, i.e. drastic change in slope of the modulus plot. This sudden change in slope is called the ‘critical stress’ for network rupture. This critical stress is normally termed as yield stress (σ). For unfilled semi-IPN hydrogel system the σ value was calculated to be 1066 Pa. After addition of small amount of RGO, the in situ nanocomposite hydrogel showed a right shift to higher σ value. This implied the strengthening phenomenon of the nanofiller reinforced semiIPN hydrogels. As a matter of fact, high aspect ratio nanoparticles (L/D >100) always improves mechanical strength of nanocomposites. Here RGO, being high aspect ratio filler plays the role very crucially. It can be hypothesized that polymer anchored graphene sheets restricts the movement of polymer chains inside the gel matrix which provided another physical crosslinking. These physical crosslinking junctions sometimes also called as ‘fix points’. The gel strength of a nanocomposite gel is directly proportional to the no of fix points. That implies delayed network


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rupture of gel matrix and that’s why the point of rupture right shifted. Similar decaying trend was also noticed for shear viscosity measurement. In case of Figure 4d the shear viscosity gradually decreased with increasing shear rate. It quite resemblances to the special type shear thinning behavior. Shear thinning is a time-dependent behavior for non-Newtonian fluids. When high shear rate was applied for the hydrogel specimens the network breakage was accelerated and a sudden drop of shear viscosity was noticed. This can also be termed as thixotropy. The thixotropy was also seen in case of RGO filled nanocomposites but is a delayed fashion. Uniaxial tensile behavior of in situ nanocomposite hydrogels Uniaxial stretching of hydrogels and nanocomposite hydrogels reflect their internal morphology. Herein, two variable parameters were selected for uniaxial tensile measurements i.e. κcarrageenan and RGO content. As shown in Figure 5a, the uniaxial tensile strength increased monotonically with κ-carrageenan content. Increasing the κ-carrageenan content is directly proportional to the grafting percentage. This grafting percentage provides more compact network inside the gel matrix. The κ-carrageenan macro-radical produced during the redox initiation reaction have more propensity to couple covalently with the poly (acrylic acid-co- 2Acrylamido-2-methylpropane sulfonic acid) copolymer chain. This coupling drastically enhances crosslink density which boosted the externally applied stress. Thus the tensile stress was noticed an enhancement after addition of κ-carrageenan. In case of in situ RGO nanocomposite hydrogels, the uniaxial tensile property increased up to 2.0 wt% of filler loading. After 2.0 wt% loading the tensile strength decreased which was shown in Figure 5b. The initial increment in tensile strength was purely the reflection of reinforcing character of RGO nanosheets inside gel matrix. The elongation was also improved after RGO loading. For unfilled hydrogel the elongation was 191% whereas for 2.0 wt% loaded


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nanocomposite hydrogel it reaches to 914%. Thus a monotonic improvement in tensile property as well elongation has been monitored here. This improvement is due the microstructural alteration by RGO-gel matrix interactions. The load withstanding capability for RGO filled hydrogels is preferentially high for other unfilled hydrogels. A brief comparative study was also given in Table 1 regarding the tensile strength of the hydrogels. Now another cause of such superstretchablity of RGO loaded hydrogels could be a proper stress distribution phenomenon which cannot be ruled out. The primary hypothetical diagnosis for stress distribution requires superior dispersion of RGO nanoplatelets throughout the gel matrix. Stress transfer from RGO nanosheets to polymer chains are normally occurred from their interfaces. For highly dispersed anisotropic nanofillers the exposed interfaces towards the polymer chains is quite high resulting fast stress transfer evenly throughout the nanocomposite hydrogel matrix. Such thing is very much clear in the elongation data of the nanocomposite hydrogels. But after 2.0 wt%, the RGO loaded hydrogel showed deterioration of elongation which could be an agglomeration effect of RGO nanosheets. RGO nanosheets have high surface area, that’s why they are highly prone to adhered together and forms a special network formation. This microstructure of RGO nanosheets cannot mitigate the proper stress transfer from high elastic domain (RGO nanosheets) to highly viscoelastic gel matrix. As a result the nanocomposite hydrogel ruptured before going to high elongation. Thus it is termed as stress-concentration behavious of high surface area nanofillers (here RGO)

26

. Additionally, RGO sheets have dual character of polarity like amphiphilic

macromolecules. The polarity lies high in the edges and least in the basal planes of nanosheets. Such gradient type polar configuration makes these RGO nanosheets attractive nano-building blocks. It is worth mentioning that in terms of amphiphilic nature, RGO is more superior to the conventional amphiphilic macromolecules because of their relatively high lateral dimensions.


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Lateral dimension of anisotropic nanoparticles have a stringent effect on gelation. Such lateral conjugated basal planes turn RGO into stiff and stable network forming agent. This network formation was better when polymer chains are physisorped onto the basal planes of the RGO nanosheets (Figure 5c). Another thing is also attracting in the stress-strain plot which is the necking of the RGO filled hydrogels. Necking is a reflection of rubbery nature and ductility imposed on specimens. The mechanism is graphically represented in Figure 5d. A hypothetical consecutive orientation has been observed for the RGO nanosheets inside the matrix during uniaxial stretching. For purely chemical crosslinking the network rupture normally held instantaneously but in case of RGO loaded gels, the physical adsorption of polymer chains onto RGO nanosheets provides another secondary interaction which is typically a physical bonding. This physical interaction could be the cause of this necking type plastic deformation. For better understanding of the comparison between loading and elongation at break, Figure 5(e) and 5(f) are given. Now the question of rubber-like elasticity imposed on in situ nanocomposite hydrogels. Rubber-like elasticity is best understood for the reversible ductility of the specimens. Herein, the cyclic loading-unloading cycle for the unfilled hydrogel and the in situ RGO nanocomposite hydrogels was performed as shown in Figure 5(g). For unfilled gel the unloading curves intercepted at ~77% strain which implied inferior reversibility of the unloaded semi-IPN hydrogels. But RGO as a filler provides reversible ductility into the nanocomposite semi-IPN hydrogels. For RGO loaded samples the permanent set were seen from ~48% to ~16% which implied ‘rubber-like elastic’-type character. This could be a reflection of elastic nature of RGO nanofiller. The aforementioned stress-transfer phenomenon is again dominant factor here. High content of RGO loaded nanocomposite hydrogels showed better ductility than low content of


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graphene loaded hydrogels. This could be implied as a increment in interfacial area for high content graphene filled hydrogels which promotes fast stress transfer from relatively elastic RGO nanosheets to viscoelastic gel matrix. Stress relaxation behavior of in situ RGO nanocomposite hydrogels Stress relaxation is a ‘stress-decay’ phenomenon observed majorly in case of viscoelastic systems. This physical transformation normally occurs after application of finite amount of stresses externally applied to the viscoelastic body. This happens primarily due to withstand the internal microstructure even after application of plastic strain. Herein, the gel specimens were subjected to plastic strain at 100% for 5 min and then stress release in a decaying manner has been monitored for 15 min (Figure 6a-f). Though the unfilled hydrogel is a covalently crosslinked semi-IPN it showed stress relaxation behavior. But after incorporation of RGO filler into the gel matrix the stress relaxation behaviors was superior to unfilled gel. The superiority of stress relaxation could be evaluated from the residual stress. Residual stress localized inside gel matrix could be hampered its rubber-like elasticity as well as its cyclic loading unloading behavior indirectly. As shown in the respective figure, for the G0.5 nanocomposite hydrogel the residual stress was little bit lowered than the unfilled hydrogel. This residual stress was measured to be belittled with RGO content. For clear understanding the residual stress versus RGO content has been plotted in Figure 6g. It is an obvious fact of formation of secondary physical crosslinking between the RGO basal plane and the gel matrix polymer chains by virtue of physisorption. The significant load bearing agent, RGO acts as a nano-building block for the hydrogel systems. When the hydrogel specimens were subjected to stretch up to a definite amount (here 100%), the RGO nano-building blocks were prone to axially oriented in machine direction. It can be hypothesized that the highly elastic nanosheets of RGO might help to


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promote further load bearing capability which was absent in case of unfilled semi-IPN system. This kind of mechanism might also provide toughening feature of nanocomposite hydrogels which is already discussed previously. The orientation of RGO nanosheets was more profound for higher anisotropic filler loading. Proper dispersion of anisotropic nanosheets during stretching provides better stress distribution than low filler loaded hydrogels. On the verge of stress relaxation study the RGO nanosheets dissipates localized stress to the viscoelastic gel matrix and this might happened when large amount of filler-loaded system because of the generation of enormous surface area. Similar kind of stress relaxation behavior was also studied for varying the κ-carrageenan concentration. It is shown from the Figure 6h that the κ-carrageenan content has a positive impact on stress relaxation phenomenon. It is already been discussed that monomers were grafted onto κ-carrageenan during gelation. Grafting was increased with increasing the κcarrageenan concentration. The mechanical strengthening was accounted by means of grafting on to κ-carrageenan due to the more compact microstructure. The residual stress was measured to be increased monotonically with the κ-carrageenan concentration. This implied the promotion of elastic nature of semi-IPN after κ-carrageenan grafting. It can be inferred that polysaccharide based grafted semi-IPN could be an ideal choice to prepare tough hydrogels with superior rubber-like elasticity. Figure 6i is the comparative study of κ-carrageenan content, grafting percentage and residual stress for the respective semi-IPN hydrogels. Micro-computed tomography (µ-CT) analysis The porosity is a significant feature of a cryo-hydrogel which can reflect its physic-mechanical quality, water imbibition features and diffusion controlling attributes. The porosity of a hydrogel normally depends up on their monomer concentration. In our case the hydrogel formatting


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components are monomers (AA and AMPS), kappa-carrageenan biopolymer and RGO nanoparticles. It has been stated previously that the fabricated hydrogel monolith was facilitated by coupling of physisorption interaction (between RGO nano-sheets and gel matrix) and analogous covalent crosslinking (with divinylic comonomer, MBA). The porous microstructure is clearly observed in FESEM images but the limitation of FESEM is to quantify the porosity inside cryo-gel. Pore connectivity for a gel is better understood in case of micro-computed tomography (µ-CT) which can clearly elaborated pictographically the connected and nonconnected pores. µ-CT addresses this issue in a non-destructive and non-time consuming fashion. The incident X-ray scans the specimen in cross-sectional 2D way. 2D images are used by furnishing software to reconstruct the complete 3D spatial analysis. Micro-CT is comparatively new tool to analyze the porous structured materials with respect to others 27. Micro-CT analysis as showed in Figure 7a revealed macro-porous structure of RGOfilled semi-IPN. The porosity was calculated of ~52% having pore size of 80-110 µm. Micro-CT provides information regarding the pore connectivity status inside the hydrogel. The horizontal and vertical slices were taken by the software showed homogeneity of pores through the samples (Figure 7b-7i). Connected pores are directly related to their water uptake behaviors as well deswelling characteristics. Conventional hydrogels have high amount of porosity than RGO hydrogels. This phenomenon can be implied as the dual cross-linkability among the gel matrix; one is covalent crosslinking and another one is physisorption of polymer chains onto basal planes of RGO nano-sheets. Figure 7j is the 3D view of spatial porosity already generated during lyophilizing. The image showed two colors for clear understanding. The red ones are the connected pores and the blue colors designates the non-connected pores. This showed high number of connected pores which could provide an idea of diffusion small molecules (like water,


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dyes, drugs and other analytes etc.) through the channels. Besides this high amount of porosity also has a relationship with the physico-mechanical signatures of the prepared cryo-gel. Uniaxial tensile testing result will be dropped when porosity will be much higher fraction. In our work the mechanical property showed super-stretchable character of the hydrogel which is quite supportive with the micro-CT diagnosis. The highly porous gels will fail easily due to their facile rupture of thin cell-walls. But in our case the crosslinked hydrogel withstand stress with a high elongation at break. Thus micro-CT is more attractive from the view of handling a material before service where porosity is an essential criterion. Biodegradability of the hydrogels Polymer derived products are always suffer from biological decay. Biodegradation is a process of accelerated molecular weight breakdown assisted with hen egg lysozyme. Figure S12 is the biodegradation study of kappa-carrageenan based semi-IPN hydrogels. The plot showed a gradual mass loss of the prepared hydrogel which was monitored after 0, 7, 14, and 21 days in hen egg lysozyme medium. Hen egg lysozyme was chosen due to its high similarity to human lysozyme because both have analogous binder ingredients

28

. For control experiment, PBS was

taken as control medium for parallel study. Lysozyme is a glycoside hydrolase; it can propagate the enzymatic chain scission the 1, 4-β-glycosidic bond present in the polysaccharides. In our system, kappa-carrageenan is also a polysaccharide which has a tendency to degrade in presence of lysozyme medium. The degradation of kappa-carrageenan might be assumed as an enzymatic scission of the polysaccharide backbone. This enzymatic reaction evolves typical glucopyranose sugars as outputs. The rate of degradation is always dependent of active degradation sites. In a similar parallel experiment there was no mass loss of the hydrogel in control experiment. Electrical conductivity and percolation analysis of nanocomposite hydrogels


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Electrical conductivity of polymer composites is normally increased after addition of intrinsically conducting fillers. Herein, we proposed an in situ RGO nanocomposite hydrogel which could undergo insulator to conductor transition. Conducting hydrogels is very much significant on account of their electric stimuli responsive drug delivery, iontopheretic devices, RFID systems and so on. Transition from insulating hydrogel to conducting gel the system undergoes a physical phenomenon called percolation. It is a typical 3D pathway formation inside a polymer matrix which promotes electron conduction by hopping mechanism

29

. Percolation

threshold for a polymer composite vehemently depends over the shape, geometry, dispersion status and aspect ratio of fillers. For anisotropic fillers viz. CNTs, modified graphene and nanowires, the percolation threshold were reported elsewhere 30-32. Figure 8a showed the electrical conductivity of RGO-filled nanocomposite hydrogels as a function of RGO content. The conductivity of unfilled (neat) semi-IPN and RGO were around 10-13 S/cm and 0.126 S/cm respectively. The electrical conductivity increased proportionally with increasing the filler loading. After 0.5 wt% of RGO loading the conductivity value increased around 2 fold. But at 1 wt% filler loading the electrical conductivity was elevated to ~10-6 S/cm. The electrical conductivity showed ~10-3 S/cm value at 2.0 wt% and went almost saturated beyond this loading. The percolation threshold was evaluated after the power law equation: 33 σ = σ0 (m-mc)t

(5)

Where σ is the electrical conductivity of the composites, m and mc corresponds to the filler mass content and percolation threshold respectively, t stands for the critical resistance exponent related to the conductive network formation mechanism. The most abrupt change in conductivity was noticed in between 0.5 wt% and 1.0 wt%. As per the equation mentioned above, the percolation


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threshold concentration was calculated to be 0.69 wt%. The linear fitting in the Figure 8a-inset showed the t-value of 2.74 with goodness of fit or around 0.97. The statistical theory based on percolation infers that for randomly distributed spatially oriented filers the percolation exponent lies on the range of 2 to 3 which is quite similar in our study 34. Percolation is that specific point where the conducting fillers get connected with each other to form a continuous 3D network via which electron hopping can take place. Herein, the RGO nanosheets were well dispersed in such a way that the inter-connection was prominent throughout the gel matrix. Figure 8(b, c) is a pictorial illustration of percolation model occurred in hydrogel matrix. Hypothetical interparticle electronic transfer was also elaborated by Polley and Boonstra where they proposed that the electron can traverse even in insulating matrix if the inter-conductive surface will be less than their critical distance. Critical distance is scientifically stated as the maximum distance between two consecutive conducting inclusions for electron mobility 35. At 0.68 wt% of RGO content the conducting pathway formation was initiated and after a certain limit the network formation was saturated and that’s why the conductivity was negligible increased. The frequency dependent characteristics of the hydrogels were also carried out and shown in Figure 8d. Electrical conductivity is a summation of DC and AC conductivity which can be written as: σ(total) = σ(DC) + σ(AC); where σ(total), σ(DC) and σ(AC) correspond to the total conductivity, DC conductivity and AC conductivity respectively. The DC conductivity is an outcome of interconnected conductive RGO nanosheets whereas AC conductivity is a result of both the polyelectrolyte chains inside the gel matrix and the RGO nanosheets. As shown in the figure, the unfilled gel showed a progressive enhancement of AC conductivity with frequency. The nanocomposite hydrogels also followed the similar kind of steep rise with frequency, but the slope of the rise was gradually lowered with filler loading. The rising of the AC conductivity was


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due to the entrapped ions present in the hydrogels (i.e. H+ from acrylic acid segments and Na+ from AMPS segments). These ions were become mobile after application of external electric field and actively traversed throughout the gel matrix. These provide extra AC conductivity inside the hydrogels. In vitro cytocompatibility assessment of the hydrogel To calculate the cell viability quantitatively, the tissue culture plate (TCP) and the hydrogel disc surface cell proliferation experimentation was executed. The absorbance values found from MTT assay and converted into the cell population rate by using standard curve represented as shown in Figure 9a. After 5th day of the study, the cell population was measured to be 4.62±0.52×105 and 5.16±0.58×105 for control and gel surface respectively. These results expose that cell growth on hydrogel surface was comparatively higher than TCP. This higher population of cell over hydrogel surface might be the porous type morphology of the hydrogels which provide high surface area for cellular attachment. This cell grown is a support of biocompatibility of the nanocomposite hydrogel. In vitro cytotoxicity provides information regarding the human cell compatibility of the prepared hydrogel (in situ RGO based semi IPN). Cellular compatibility has significant necessity for a material to be used as a biomaterial. Figure 9b is the qualitative estimation of cellular attachment over the gel surface and the control experimentation. The cells were labeled by rhodamine-phalloidin and DAPI at particular time intervals. The timed growth in cell population after 1st day of the culture unveiled that the cells with low population did not display any wellextended self-distinguished cytoplasm skeletons. But on the 3rd and 5th day of study, the grown cells exhibited quite distinct cytoplasm skeleton over the hydrogel surface as well as control


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study. The uninterrupted growth of cell population could lay down that the as-prepared gel was non-cytotoxic and can be a suitable choice for biomaterial. Live-dead assay of the hydrogel Cell viability is a significant and most trust-worthy feature of any system to be used as an effective biomaterial. Live-dead assay is an in vitro cell assay which can provide ideas of cytotoxicity. Herein, live-dead assay of the hydrogel and TCP was performed to investigate the adhesion and cell-viability towards MG 63 cell-lines (Figure 9c). After 1st day, 3rd day and 5th day of culture, it was found out that cell adhesion and proliferation were gradually increasing for hydrogel disc surface compared to TCP. The Figure 9c also evidenced that there were very negligible amount of dead cells (red stained in figure) and dense live-cell sheet (green stained in figure) on hydrogel disc and TCP. These results can imply that the synthesized hydrogel samples were cell viable and again it can be inferred that the hydrogel can be employed as a promising candidate for controlled release study. pH-responsive in vitro drug release pH-responsiveness of hydrogel is normally generated from the interaction chemistry among the functional groups which are in close proximity. In this system the key role playing groups are carboxylic acid and sulfonate moieties present in the kappa-carrageenan/poly(AA-co-AMPS) semi-IPN network (Figure 10). To evaluate the nature of in vitro drug release, three different pHmedia were taken for consideration; one is acidic (pH 2.2), physiological simulated fluid (pH 7.4) and alkaline (pH 10.2). The cumulative drug release data obtained from the different time intervals of drug release was plotted an fitted into the renowned Ritger-Peppas model; 36 (

= () = +, *

(6)


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Where Mt and Mα imply the drug release at finite time ‘t’ and at equilibrium respectively. KP is proportionality constant (or Peppas constant) and n is the diffusional coefficient which has a direct correspondence to the mechanistic pathway of drug release behavior. The drug normally diffused out from the hydrogel monolith obeying the hypothetical molecular chain relaxation phenomenon. According to this theory when drug entrapped hydrogel specimens were immerged into the aforementioned pH-media, there was a swelling governed phenomena and concentration gradient which played the driving forces for drug release. Initially drug loaded samples have higher drug concentration inside the gel matrix. But after interacting to the aqueous front the drug molecules get mobile and diffuse out from the matrix showing a time dependent exponential growth pattern. The solvent molecules (here water) imbibed towards the gel matrix which results lowering of glass transition temperature (Tg). Lowering of Tg after solvent can be inferred as a transition of glassy to rubbery state. In the dried xerogel matrix (high Tg) the drug release is normally difficult due to lack of mobility of drug molecules. But after decrement of Tg, the drug release becomes more feasible. Such drug release characteristics could be tuned by controlling the intrinsic gel microstructure by manipulating the synthesis parameters. In acidic environment the carboxylic acid groups (-COOH) and sulfonate (-OSO3-) both have high tendency to get protonated. These protonation generates extensive inter-molecular Hbonding inside the matrix to tighten the gel microstructure. This affected to collapsing of gels in acidic environment and formation of more compact network. These features can restrict the release of drug molecules from the hydrogel matrix. But with gradual increment in pH, the cumulative drug release feature was promoted by raising the pore size of the hydrogels’ matrix. At psychological pH (i.e. 7.4), the drug release showed a stiff increment comparative to the release at lower pH. The initial stiff rising of cumulative plot could be defined as ‘burst release’.


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‘Burst release’ is a synergistic outcome of pore size and pH-media. High pore size and high pH are two determining factors for this kind of release. In higher pH, the carboxylic acid groups and sulfonate groups became de-protonated to form anions which follows the rule of electrostatic repulsion. This repulsive force dominated over their elastic force of attraction resulting swelling of gel matrix enormously. This high swelling can be correlated to the ‘burst release’ phenomenon inevitably. Electric stimuli responsive drug release The electric pulsatile drug release behavior was studied after taking diltiazem hydrochloride as a model drug (Figure 11a). The drug release was executed at 6 volt in PBS medium (pH =7.4). The electric pulsatile release plot has resemblance to the pH-responsive drug release profiles as shown previously. Electric pulse acted as an ‘on-off’ stimulant in the drug release behavior just like fluctuating medium pH. In situ RGO-filled semi-IPN hydrogels show better drug release behavior outcomes than unfilled nanocomposite hydrogels. Our semi-IPN system has poly(AAco-AMPS) and kappa-carrageenan as polyelectrolytes assembled with divynilic crosslinker forming spatial architecture. The collapsing of polyelectrolyte gels under electric pulse dates back on 1982 by Tanaka and his coworkers

37

. This collapsing of polyelectrolyte 3D system is

also terminologically attributed as ‘reversible volume change’ phenomenon. The collapsing of gels can be implied as a phase transition phenomenon of the gel network, counter ions and the solvent front (swelling media). The phase transition of the hydrogel is an outcome of two competitive forces; one is the positive osmotic pressure gradient of the counter ions, rubber-like elasticity of the polyelectrolyte gel, and lastly the negative pressure due to the inter-polymer macromolecular chain affinity

38

. In case of unfilled semi-IPN hydrogel, the diltiazem release

was extremely fast which corresponds to the result of stationary current. After application of


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external electric field, the mobile protons were free enough to loiter inside polyelectrolyte matrix which causes generation of stationary current. Thus a positive pulling force has been generated which pulled the hydrogel toward the positive electrode. Such internal pulling affected the pore size and burst release of drug molecules was occurred. This burst release was throttled after incorporation of nano-filler which might act as physical barrier of the nanocomposite hydrogels. The electric pulse triggered was more significant in composite hydrogels. Mild alkaline condition (here physiological pH=7.4) made the gel into partially deprotonated. This could turn the gel slightly anionic in nature. When external electric field was applied, the lack of H-bonding yielded a repelling effect and the present counter cations like H+, Na+, K+ migrate to negative electrode. This migration was occurred with association of water and drug molecules. This induces drug release from the hydrogel matrix which is essentially a synergistic consequence of electro-osmosis and cataphoresis

39

. Meanwhile, the dispersed RGO nanosheets were pulled to

positive electrode side resulting anisotropic feature in the hydrogels. Electrical percolation also has an effect over drug release. The release profile has a resemblance to G0.5 hydrogel with and without electro-stimulation. This means 0.5 wt% RGO loaded hydrogel has no significant effect in drug release behavior. But in case of G1.0 hydrogel the drug release was raised ~ 0.98% with respect to G1.0 hydrogel without electro-stimulation which implies the external applied electric field could stimulate the composite hydrogel. The calculated electric percolation threshold for RGO was in between 0.5 and 1.0 wt% as calculated above. Before percolation concentration there was no significant effect of conducting RGO in drug release, but beyond percolation, the drug release could be tuned by regulating the electric pulse. At higher filler concentration, i.e. beyond electric threshold concentration, the ‘switch on-off’ drug release profile was more striking and drastically differed from the lower RGO reinforced hydrogel. Thus, these results


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inferred that tunable control release would be more prominent after a critical concentration of RGO and this critical concentration could be named as the threshold concentration. The electro-stimulated cumulative release was again investigated after fitting the release data in Peppas model as shown in eq. (6). The calculated Peppas constant values were reduced with increasing the RGO content without electric pulse which corresponds to the slow release of drug molecules from the gel matrix. Such typical slow release can be hypothesized by the tortoise pathway formation by the RGO nanosheets resulting restriction of the diffusion channels for drug molecules from the matrix to media. Increment in Peppas constant was observed after application of voltage which means relatively faster drug release. Besides this, another Peppas parameter is noticeable; the exponent value, ‘n’ also showed a gradual decrement with increasing the RGO concentration without application of voltage. But after application of electric field, the microstructural deformation may result monotonous growth in ‘n’ values. Figure 11b depicted the voltage dependent timed release profile of the nanocomposite hydrogels. Herein, G2.5 hydrogel has been chosen on the basis of most electro-responsive system. The DC voltage was varied from 0 V to 10 V throughout the experiment as per the diagram depicted in Figure 11b. It is clearly observed that G2.5 showed gradual increment in drug release with application of external voltage and the release profile became stiffer with increasing the voltage. It is due to effect of extensive pull-out effect exhibited by the positive electrode where RGO nano-sheets were pulled toward the electrode. The plausible molecular interaction played inside the gel matrix has been illustrated graphically in Figure 11c. The plot showed that the release was improved with enhancement of voltage. These experimental outcomes can be assigned to the microstructural tuning and charge adjustment of the colloidal RGO nanosheets in gel matrix which are liable for the lowering of drug-filler interaction inside


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matrix (Figure 11d). Side by side, the synergistic effect of electro-osmosis and cataphoresis cannot be ruled out to promote release of drug molecules from gel matrix. Electro-stimulation characteristics could be better understood after calculating the order of electric sensitivity (ES). Electric sensitivity or ES was computed after taking the difference of electric field induced release (Rel) and nonelectric field induced release (Rnonel). ES could be a determining parameter to choice ideal hydrogel for electro-stimulation purposes. The experiment was executed at 6 V for 1 min duration. Below the electric percolation threshold concentration of RGO, there was no as such significant ES value (Figure 11e). But it was increased after incorporation of RGO nano-filler. For G2.5 the ES value showed maximum among the prepared hydrogels. These data again support the electric pulsatile behavior of hydrogels. Figure 11f is the timed release profile for all RGO filled nanocomposite hydrogels (G0.5, G1.0, G1.5, G2.0 and G2.5) keeping the voltage constant at 10 volt. It has been shown that the release was enhanced with increasing the RGO content. This means the electro-responsive pull out effect on RGO nanosheets became more prominent when external RGO loading was varied. With increasing the RGO content, the pull out became more efficiently toward positive electrode and the release of drug molecules were increased. The most important thing is that, this electro-stimulating feature of nanocomposite hydrogels was observed even at 2 volt. This implied our prepared system could be also applicable at moderate voltage which could be propagating its applicability towards transdermal drug delivery. Effect of temperature on drug release Temperature-dependent drug release is exclusively happened up on their diffusion character or diffusion co-efficient. The temperature dependent drug release profile is shown in Figure S13. We were taking two extreme concentration of RGO reinforced hydrogels, named; G0.5 and


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G2.5. The diffusion coefficient (kD) for drug (dilatiazem hydrochloride) showed gradual decrement with increasing the medium temperature raising from 28oC to 45oC. Such trend corroborates faster release of drug molecules from the hydrogel matrix than relatively low temperature. But in case of higher loading of RGO, the network became more compact due to extensive physisorption onto RGO nanosheets. It restricts the free release of drug molecules from matrix. Moreover, RGO content also has significance on burst release phenomenon. It was seen that G0.5 gel showed burst release whereas G2.5 showed no such burst release at elevated temperature. High amount of RGO could delay the drug and water release due to tortoise mechanism. But in case of low RGO loading, this typical tortoise pathway has not been that much stringent comparative to low high RGO loading. Hence, it can be inferred that fillerpolymer ratio is a major determining synthetic parameter to regulate the drug release behavior and diffusion characteristics of the hydrogel matrix. The calculated exponent (n) also revealed that the drug release obeyed anomalous Fickian type release kinetics. Conclusions In this study, biopolymer based semi-IPN reinforced with RGO was prepared to study its rheological, mechanical and electrical characters. The main aim was pulsatile drug delivery after application of external electric field which was served by the gel assembly very efficiently. The nanocomposites exhibited very low electrical percolation and modulated electro-stimulation behavior with varying the RGO content. Repetitive ‘on-off’ in drug release was clearly revealed for high loading of RGO. Besides this, the hydrogels also exhibited pH-responsive drug release behavior up on varying the environmental pH. Moreover, the in situ RGO hydrogels were tested as superstretchable. The cyclic stress-strain experiment showed the RGO filled gels had minimal amount of residual stress than unfilled hydrogels. Rheological results showed a delayed network


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rupture for nanocomposite hydrogels with increasing the RGO content. The acceptability of the as-prepared RGO based nanocomposite hydrogel as biomaterials were supported by cytotoxicity and live-dead assay. These bio-assays represented that our prepared RGO based semi-IPN could be used as electrically induced, long standing, robust, physiologically reliable soft biomaterial for drug delivery. Our belief in near future the in vivo study will also be published elsewhere to manifest the physiological acceptance of animal tissue. Our data potently affirm that the presented hypothesis of insertion of RGO for electrically conductive semi-IPNs can successfully shunt the stereotyped electro-stimulating drug release system.

ASSOCIATED CONTENT

Supporting Information 1.1 In vitro cytotoxicity experiment 1.2 Live-dead assay of the nano-composite hydrogel 1.3 pH-responsive drug release experiment 1.4 Electric stimuli responsive drug release experiment Figure S1 Figure S1 Figure S1 (a) XPS spectra of GO and RGO. High resolution C1s survey scan of (b) GO and (c) RGO Figure S2 FTIR spectra of (a) pure κ-carrageenan (b) RGO filled hydrogel (c) unfilled hydrogel Figure S3 Effect of kappa-carrageenan on grafting percentage and grafting efficiency. Figure S4 XRD of pure gel (unfilled gel), 0.5 wt% RGO content nanocomposite hydrogel (G 0.5) and 2.0 wt% RGO content nanocomposite hydrogel (G 2.0) Figure S5 Swelling data for variation of kappa-carrageenan concentrations Figure S6 Fitting of swelling data to pseudo 2nd order kinetic equation for varying of kappacarrageenan content hydrogels Figure S7 Plot of swell ratio vs time and fitted to ‘allometric 1’ model to evaluate diffusion parameters (hydrogels with kappa-carrageenan content variation)


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Figure S8 Time dependent water uptake behavior of RGO filled nanocomposite hydrogels Figure S9 Fitting of swelling data to pseudo 2nd order kinetic equation for RGO-filled nanocomposite hydrogels Figure S10 Plot of swell ratio vs time and fitted to ‘allometric 1’ model to evaluate diffusion parameters (hydrogels with varying the RGO-content) Figure S11 Stress-sweep of kappa-carrageenan aqueous dispersion Figure S12 Biodegradation study of hydrogel by hen egg lysozyme (in PBS as media) Figure S13 Temperature dependent release of drug from (a) low RGO loaded hydrogel (G0.5) and (b) high RGO loaded hydrogel (release is studied in PBS medium, pH~7.4) Table S1 Swelling and diffusion parameters obtained from kinetics study of the hydrogels

Corresponding Author *E-mail: [email protected] Acknowledgements Narayan Ch. Das thanks to the Science and Engineering Research Board (SERB), Department of Science and Technology (DST), Ministry of Science and Technology, Govt. of India (ECR/2016/000048) for financially supporting this work sincerely. The authors are thankful to Indian Institute of Technology, Kharagpur; West Bengal, India for providing all types of research facilities.

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Figure 1 (a) TEM image of GO in aqueous dispersion (b) TEM image of RGO (c) XRD of pristine graphite, GO and RGO (inset the aqueous dispersion of GO and RGO in glass vial) (d) Raman spectra of GO and RGO (e) UV-visible spectra of GO and RGO (f) Plausible mechanism of reduction of GO by amino acid (here L-cysteine).


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Figure 2 Schematic illustration of redox imitator assisted gelation.


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Figure 3(a) FESEM image of in situ RGO-nanocomposite semi-IPN (sample code: G1.0) (b) Effect of swelling on κ-carrageenan content (c) Effect of RGO filler in swelling; the inset diagrams are the pictorial representation of pristine semi-IPN and RGO filler semi-IPNs. With increasing the RGO content the 3D network formation restricts the water uptake due to physical crosslinking. (d) Gradual weight loss profile of unfilled gel and RGO filled gel. (inset: weight loss profile of GO and chemically reduced GO).


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Figure 4 (a) Frequency sweep plot for semi-IPNs varying the biopolymer (kappa-carrageenan) content (b) Frequency sweep plot for semi-IPNs varying the RGO content (c) Stress sweep experiment for RGO reinforced semi-IPNs (d) effect over shear viscosity after varying the RGO content.


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Figure 5(a) Typical stress-strain plot for varying κ-carrageenan content (wt%) (b) Effect of RGO concentration as in situ filler and its toughening feature in gel matrix (c) Graphical representation of RGO nanosheets and their physiroped polymer attachments which affects the mechanical robustness of the nanocomposite hydrogels (d) Cartoon of consecutive steps during stretching and plausible mechanistic pathway for RGO assisted toughening (e) Effect of κ-carrageenan content in elongation at break (EB) and Tensile strength of semi-IPNs (f) Effect of RGO content in elongation at break (EB) and Tensile strength of nanocomposite semi-IPNs (g) Typical cyclic loading-unloading plot for RGO nanocomposite semi-IPN hydrogels.


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Figure 6 (a-f) Stress relaxation behavior of unfilled hydrogel and RGO-filled hydrogels (g) Gradual decrement in residual stress with RGO content (h) Stress relaxation study of without kappa-carrageenan and 1.0 wt% kappa-carrageenan semi-IPN (i) effect of grafting in residual stress.


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Figure 7 (a) 3D view of RGO-hydrogel monolith (b-e) vertical slice view after reduction by software (f-h) horizontal slice view (i) side view (j) 3D porous structure with connected (red portions) and non-connected (blue domains) pores.


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Figure 8 (a) Electrical conductivity of in situ RGO/kappa-carrageenan-g-poly(acrylic acid-co-2Acrylamido-2-methylpropane sulfonic acid) nanocomposite hydrogels, where 0.69 wt% represents a critical concentration at the percolation threshold. (b) and (c) are the graphical illustration of formation of conductive network before and after percolation. (d) AC conductivity of pure AA hydrogel and graphene loaded hydrogels.


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Figure 9 (a) Cell proliferation results of semi-IPN and control experiment results (b) Fluorescent microscopic images for 1st, 3rd and 5th day of assay after fluorescent imaging. Hydrogel surface showed better cell population over control both due to high surface area of gels for its porous morphology (c) Live-dead staining images of MG63 cells showing abundant cell growth on to hydrogel surface.


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Figure 10 Timed release profile for RGO-reinforced semi-IPNs in (a) acidic environment; i.e. pH~2.2 (b) physiological pH~7.4 (c) alkaline environment; i.e. pH~10.4.


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Figure 11 (a) Electric pulsatile release profile of diltiazem drug from RGO based semi-IPNs and unfilled semi-IPN (b) Cumulative release of drug with voltage as a function (c) graphical of typical set-up for electric responsive drug release measurement (d) Pictorial illustration of plausible chemical interactions among the component of the semi-IPN (e) effect of RGO nanoparticles in electrostimulation and electrical sensitivity (f) Time-dependent cumulative release of diltiazem hydrochloride from the all RGO-reinforced semi-IPNs at 10 V.


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Table 1 Gel strength parameters evaluated from rheological and mechanical study

Polymer code

Gel strength (G’/G”)

Yield stress (kPa)

Elongation at break (%)

κ-carrageenan hydrogels car0.0

10.34

121.0

186

car0.1

11.63

124.7

274

car0.3

11.96

133.7

385

car0.5

12.37

142.1

429

car0.7

13.85

148.9

515

car1.0

16.54

158.4

561

car1.5

17.88

165.2

615

In situ RGO nanocomposite hydrogels G0.5

12.68

124.7

389

G1.0

13.70

161.8

635

G1.5

15.08

255.4

761

G2.0

16.11

292.7

914

G2.5

18.26

314.1

352


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Table 2 Comparative study of graphene based hydrogels and their swelling and mechanical property Name of hydrogel

Fabrication method

Filler loading (wt%)

Swelling behavior

Mechanical strength*

Ref.

Poly(acrylamide)/GO Poly(acrylic acid)/GO/Fe(+3) PVA/RGO Poly(acrylic acid)/graphene Poly(acrylamide)/graphen e Poly(acrylamide)/Pristine Graphene Poly(acrylic acid)/GO Poly(acrylic acid)/GO

FR FR

0.03 0.5

23.8 (g/g) -

65.5 kPat 777.3 kPat

40

FT FR

8.11 3.0

8.09 MPat 19.03 MPac

42

FR

3.0

1.31 MPac

44

FR

0.5

.006 MPac

45

FR FR

0.073 2.0

25 kPat

46

Poly(acrylic acid -coacrylamide)/GO Poly(acrylamide)/GO Poly(acrylic acid)/GO/gelatin Poly(acrylic acid)/GO

FR

0.5

43.47% ~ 55 (swell ratio) 43.5 (swell ratio) ~ 45 (swell ratio) 508 (g/g) 48 (swell ratio) ~ 400 (g/g)

-

48

FR FR

3.0 0.3 wt%

~ 16 (g/g) -

32 kPat 0.25 MPat

49

FT

0.1

4 MPat

51

PAA-g-amylose/GO

U

3.0

200 (swell ratio) -

42.47 MPat

52

41

43

47

50

53 PAA/water dispersible FR 2.0 7.5 (%) 411.9 kPat graphene 54 Poly(acrylamide)/ Physical 0.15 5.8 (swell 0.945 MPat Fe3O4/RGO ratio) 55 PVA/PEG/GO FT 1.5 4.2 MPat Poly(AA-coFR 2.5 6.6 (swell 488.5 kPat Present AMPS)/Carrageenan/RG ratio) work O * tTensile strength; cCompressive strength; FR=Free radical gelation, FT=Freeze thaw method,

U=UV irradiation method.


58

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TOC Graphic 85x58mm (220 x 220 DPI)


Green Reduced Graphene Oxide Toughened Semi- IPN Monolith

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