agar-agar hybrid hydrogel: A multifunctional avenue to tackle wound healing

Journal Pre-proof Fumaric acid incorporated Ag/agar-agar hybrid hydrogel: A multifunctional avenue to tackle wound healing

Ilias Basha, Somnath Ghosh, K. Vinothkumar, B. Ramesh, P. Hema praksh kumari, K.V. Murali Mohan, E. Sukumar PII:

S0928-4931(19)33140-6

DOI:

https://doi.org/10.1016/j.msec.2020.110743

Reference:

MSC 110743

To appear in:

Materials Science & Engineering C

Received date:

24 August 2019

Revised date:

11 February 2020

Accepted date:

12 February 2020

Please cite this article as: I. Basha, S. Ghosh, K. Vinothkumar, et al., Fumaric acid incorporated Ag/agar-agar hybrid hydrogel: A multifunctional avenue to tackle wound healing, Materials Science & Engineering C (2020), https://doi.org/10.1016/ j.msec.2020.110743

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© 2020 Published by Elsevier.

Journal Pre-proof

Fumaric acid incorporated Ag/Agar-Agar hybrid hydrogel: A multifunctional avenue to tackle wound healing

llias Basha1, Somnath Ghosh2,*, K. Vinothkumar3, B. Ramesh1, P. Hema praksh kumari4, K. V. Murali Mohan5, E. Sukumar6,*

Mr Ilias Basha and Dr. B Ramesh, GITAM Institute of Medical science and Research (GIMSR),

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Department of Pharmacology, Visakhapatnam, Andhra Pradesh, 530045, India. Dr Somnath Ghosh, Indian Institute of Petroleum and Energy (IIPE), Department of Chemistry,

Dr K Vinothkumar, Meenakshi Academy of Higher Education and Research (Deemed University),

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Visakhapatnam, Andhra Pradesh, 530003, India. E-mail: [email protected]

Chennai-600 078, India

Dr P Hema prakash Kumari, GITAM Institute of Medical science and Research (GIMSR), Department

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of Microbiology, Visakhapatnam, Andhra Pradesh, 530045, India. Dr K V Murali Mohan, GITAM Institute of Medical science and Research (GIMSR), Department of

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5.

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Pathology, Visakhapatnam, Andhra Pradesh, 530045, India. Dr. E. Sukumar, Saveetha Institute of Medical and Technical Sciences, Thandalam, Chennai, Tamilnadu, 602105, india. E-mail: [email protected]

Abstract: Wound and its treatment is one of the major health concerns throughout the globe. Various extrinsic and intrinsic factors can influence the dynamics of healing mechanism. One such extrinsic factor is moist environment in wound healing. The advantages of optimum hydration in wound healing are enhanced autolytic debridement, angiogenesis and accelerated cell proliferation and collagen formation. But hydrated wounds often end up with patient’s uncomfortability, associated infection, and tissue lipid peroxidation. Healing process prefers antimicrobial, anti-inflammatory and optimum moist microenvironment.

Here, we have synthesized

fumaric acid incorporated agar-silver hydrogel (AA-Ag-FA); characterized by UV-Visible spectroscopy, FTIR spectroscopy and TEM. The surface morphology is evaluated through SEM. The size of the silver nanoparticles

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Journal Pre-proof (Ag NPs) was found to be 10-15 nm. The hydrogel shows potential antibacterial effect against Escherichia coli, Staphylococcus aureus and Psuedomonus aeruginosa which are predominantly responsible for wound infection. The gel shows reasonable antioxidant property evaluated through 2, 2-diphenyl-1-picrylhydrazyl (DPPH) assay. Topical application of the gel on the wound site heals the wound at much faster rate even compared to standard (Mega heal, Composition: Colloidal silver 32 ppm Hydrogel) gel. Histological analysis reveals better tissue proliferation (i.e. epithelialization), more granulation tissue formation, neovascularisation, fibroblast and mature collagen bundles. The lipid peroxidation of wound tissue estimated through malondialdehyde (MDA) assay was found to be reasonably less when treated with AA-Ag-FA hydrogel compared to standard (Mega heal). Cytotoxicity of the samples tested through MTT assay and live-dead cell staining shows its nontoxic

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biocompatibility nature. In our hydrogel scaffold, the bio-degradable agar-agar provides the moist

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environment; the Ag NPs inside the gel acts as bactericidal agent and fumaric acid facilities the angiogenesis path implicitly.

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Keywords: Agar-silver hydrogel • Antibacterial • Wound healing• Antioxidant • Collagen-Growth-Factor

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Introduction

Skin, the largest organ of a body, comprised mainly of epidermis, dermis, and hypodermis, is the first layer of

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defence to internal tissues, prevents pathogen raid, and maintains the body temperature, synthesizes vitamin D, serves excretory functions [1, 2]. Wound is an injury to living tissue or skin caused by various insults like

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chemical, mechanical, thermal, and ionizing radiation etc. Wound healing is the repair of injured tissues by replacing devitalized and missing cellular structures with tissue layers through organized interconnected cascade of cellular events: hemostasis, inflammation, proliferation, and remodelling [2, 3]. Hemostasis begins instantaneously after vascular injury which comprises of platelet adhesion and platelet aggregation leading to formation of primary hemostatic/platelet plug with subsequent activation of coagulation cascade with the formation of thrombin as critical event which in turn converts soluble fibrinogen to insoluble fibrin to accomplish clotting [4]. After coagulation, white blood cells (neutrophils) migrate into the inflamed tissue, engulf and degrade the bacteria and cellular debris. Subsequently release of growth factors lead to the attraction of fibroblasts. Soon after inflammatory phase, proliferation of fibroblasts leads to the formation of granular tissue. Fibroblasts synthesize and secrete collagen, the principal component of the extracellular matrix, which is deposited in the injured area to replace the initial provisional matrix comprised of fibrin [5-7].

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Journal Pre-proof On the early stage of skin recovery, tissue experiences damaged hemostasis and cell apoptosis caused by inflammation, ischemia and hypoexia. In the proliferation period, the cells proliferate rapidly after endothelial cells exhibit adequate vascularization. Finally, during the remodelling period, the tissue undergoes structure and mature vascular networking. Any of the above mentioned stage impeded by any external or internal factors generally lead to impaired healing and improper functioning [4, 8]. Being a complex and dynamic process, wound healing is a clinical challenge to the medical practitioner throughout the globe [9-12]. The major challenges of wound healing are frequent bacterial infection, sloth cell migration and proliferation and hence decelerated impaired collagen deposition on wound site [13, 14]. Out of several types of wounds, chronic wounds not only cause suffering but also invite pathogenic infection which

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ultimately may lead to amputations some time [15-17]. Several microbes (viz. Psuedomonus aeruginosa,

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Escherichia coli, Staphylococcus aureus etc), isolated from the wound bed are the major causes of wound infection which impede the healing process [18-20]. Bacterial colonization and hence spread of pathogenic

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infections on wound is the major impeder to wound healing process. On the other hand, excessive irrational

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usage of antibiotics may lead to multidrug resistant (MDR) bacteria and a series of side effects consequently [21-23]. The severity of chronic wound is so fatal that even developed countries are bound to allocate 2% to 5%

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funds of their total health budget every year [24, 25] and developing or underdeveloped counties face higher mortality [26] due to inaccessibility of resources and facilities.

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Even though, several unmanageable intrinsic factors of patients like health status, nutritional status, age factors etc. [11] affect the healing process; the extrinsic factors like low mechanical stress, debris cleaning, optimum

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moist environment, and temperature etc. can be maintained externally through proper wound dressing [27-30]. Due to high water content in human tissue; cell migration, proliferation and collagen deposition are usually accelerated in an optimal aqueous environment provided no pathogenic infection is present in the wound site [31-33]. In addition, avoiding infection is an important issue for wound care, in particular for chronic wound healing. On this quest, there is an urge to develop a multipurpose wound healing material which will (a) provide moist environment, (b) maintain temperature, (c) act as antimicrobial and antioxidant (c) facilitate the collagen formation and also (d) be biocompatible and economic [34-36]. Hydrogels are capable to encapsulate different drugs within the matrix and may stimulate the physical structure of the extracellular matrix to promote cell proliferation and tissue regeneration [37-39, 76-78].

In recent past,

polysaccharides such as dextran, cellulose, alginate, chitosan, agarose etc. have been exploited as hydrogel for wound dressing purpose [40-45]. There are several advantages of using hydrogel: may absorb wound exudates, maintain wound temperature, provide moist environment [40]. Y. Tang et al [40] have synthesized cross-linked

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Journal Pre-proof polysaccharide hydrogel without incorporation of antibiotics or any other antibacterial agents. The material was used as wound dresser. S. Pal et al [41] have reduced (in-situ) silver ion to Ag NPs inside bacterial cellulose matrix which shows promising antibacterial activity against E. coli only. The studies do not show biodegradability or cytotoxicity effects. Agarose based bioplastic was formulated by A. Awadhiya et al [42] to incorporate antibiotics and antiseptics. The bioplastic shows good haemo-compatibility. An injectable hydrogel cross-linked by Zn2+ and Ca2+ was used by Y. Li et al [43] for chronic wound healing purpose. However, the studies do not show the biocompatibility or biodegradability of the said material. Recently, C. Mao et al [44] have used hybrid nanostructures, Ag/Ag@AgCl/ZnO embedded in carboxymethyl cellulose which shows potential antibacterial activity with accelerated wound healing activity.

Therefore, it is tough to have all four

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characteristics as suggested above for healthy wound healing.

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In this report, we have synthesized silver nanoparticles (Ag NPs) in agar-agar hydrogel matrix and modified with fumaric acid. Fumaric acid (FA), a natural organic acid, first isolated from plant Fumaria officinalis is a

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key intermediate of citric acid cycle of many organisms. FA is generally used in small amounts in preparation of

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various foods i.e. as an acidulant and antioxidant [46]. In medical field, use of FA as antibacterial, antiinflammatory and analgesic is already known [47-49]. In addition, FA as a medicine is used to treat psoriasis, a

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neuro inflammatory skin condition [50, 51]. It has already been proved that fumaric acid ester (FAE) promotes gene factor, Nrf2 [52-54], which regulates antioxidant pathways minimizing the oxidative stress arises during

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chronic wounds like in diabetics. The agar-agar hydrogel provides the moist environment. The Ag NPs inside

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the gel acts as bactericidal agent and fumaric acid facilities the angiogenesis path. The hydrogel shows efficient bactericidal action against P. aeruginosa, E. coli and S. aureus. Topical application (~ 1.0 mL each time) of the gel on wound bed created through excision on wistar rat demonstrated faster wound healing rate (50% healing @ day5) compared to standard Megaheal gel (50% healing @ day8), a prescribed medicine generally used for wound treatment. Histological analysis of the tissue samples reveals higher collagen deposition. The formulation shows minimal cell inhibition against 3T3-L1 fibroblast cells evaluated through MTT assay. The gel so formulated is spreadable and easy to apply on the wound bed. Bio-degradability with slow silver-release fits AA-Ag-FA as long lasting antimicrobial wound healer. Experimental Section Materials Silver nitrate (AgNO3, Fischer Scientific, 99.9%), N, N-dimethyl formamide (DMF, Finar Ltd.

99.0%),

Polyvinyl pyrrolidone (PVP, Mw~40000, Sisco Research Laboratory Pvt. Ltd.), and agar–agar (Bacterial grade type-I, Hi-Media Laboratories Ltd, India) were used as such without any further purification for the preparation

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Journal Pre-proof of Ag NPs. For the purpose of antimicrobial assay of fumaric acid/agar-silver nanaoparticle hydrogel, nutrient broth obtained from Hi-Media Laboratories Ltd, India was used. Standard reference strains, P. aeruginosa ATCC 27853, E. coli ATCC 25922 and S. aureus ATCC25923 (supplied by GITAM Institute of medical science and research, Department of Microbiology, Visakhapatnam) were used. For lipid peroxidation assay; Thiobarbituric acid (TBA, Fluka, 99%), trichloroacetic acid (TCA, Fluka, 99%) were used.

Synthesis of silver nanoparticles in agar-agar matrix (AA-Ag) Synthesis of Ag NPs in agar-agar matrix was done as per our earlier report with little modification [55]. In a typical reaction, 0.45g of chloride free agar-agar (AA) was dissolved in 100 mL of Millipore water under

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boiling condition till a clear solution appeared. After obtaining the clear solution, 0.1 g PVP and 1.0 mL of DMF was added immediately. To this, 0.169g of AgNO 3 was added and heated at 90°C under stirring for 5h.

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The solution turned to golden yellow confirming the formation of Ag NPs.

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Incorporation of fumaric acid (FA) to Agar-silver (AA-Ag) nanoparticle hydrogel (AA-Ag-FA)

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FA was incorporated by adding 1.0 g of it into the so obtained AA-Ag hot (~90 °C) solution under stirring for

UV-visible absorption study

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10 minutes. The sample was preserved at room temperature which on time turned to gel.

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UV-vis absorption studies were carried out to ensure the reduction of Ag+ ion to Ag0 in agar-gar by taking 1 mL of solution in 3mL of Millipore water and scanned over the wavelength range 400-800 nm in a (Cary 60 UV-Vis

background.

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spectrophotometer, Agilent Technologies) spectrophotometer using a quartz cuvette with Millipore water as

FTIR spectrophotometry

The formation or evolution of new bonds and surface modification of nanoparticles can be assessed by FTIR spectroscopy. IR absorption spectra were recorded in a FT-IR SPECTRUM 1000 PERKIN ELMER spectrometer. Transmission electron microscopy (TEM) The size and morphology of Ag NPs were analysed by TEM using a TECNAI F 30 transmission electron microscope. Care was taken to prepare and mount the samples on the Cu grid (300 mesh) each time to avoid any possible agglomeration of particles. All samples were prepared by similar conditions, by placing a drop of wellsonicated samples (0.25 gm of gels) dissolved in water (3.0 mL) on a carbon-coated copper grid and

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Journal Pre-proof subsequently dried in air before transferring to the electron microscope operated at an accelerated voltage of 200kV.

In-vitro antioxidant study through DPPH test One of the desirable quality possessed by a potent wound healer is its antioxidant nature. Therefore, evaluation of antioxidant property of test samples is important here. In-vitro antioxidant study of test samples was carried out as per standard 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay [56]. Typically, 200µL of each gel samples

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were added to 2.96 mL of 10 mM DPPH present in 5mL test tube, incubated at room temperature in dark for 24h, absorbance was recorded at 517 nm by using UV-Visible Spectrophotometer (Cary 60 UV-Vis

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spectrophotometer, Agilent Technologies) and compared with the blank (without adding any test samples except

and

was the absorbance at 517nm of blank and test samples.

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, where

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ethanol). The radical scavenging activity (RSA) was calculated using the following formula:

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Zone of inhibition (ZOI) test

ZOI test is a qualitative method to evaluate the bactericidal ability of material through the inhibition of

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microbial growth. The bacterial (P. aeruginosa ATCC 27853, E. coli ATCC 25922 and S. aureus ATCC25923) lawn was prepared on sterile Muller Hinton separate agar plates by using a sterile cotton swab. Four 5mm

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diameter sterile filter paper coated with 100 µL of each of the samples (AA-Fa, AA-Ag, AA-Ag-FA and standard antibiotic: Cefoxitin) were placed equidistance to each other at the lawn and incubated at 37 °C for 24 h. The zone around the sample was then photographed on subsequent day. Cytotoxicity assay

3T3-L1 fibroblast cells were obtained from National Center for Cell Sciences, Pune (India) and cultured in 5% CO2 incubator at 37 °C using DMEM supplemented with FBS (10%), penicillin (100 U/mL) and streptomycin (100 μg/mL). The cells were sub-cultured at 75% confluence by total media replacement using 0.25% (w/v) trypsin – 0.53 mM EDTA every 2 - 3 days. The cytotoxic effect was analyzed by using MTT assay. The cells were seeded at 5 x 103 cells/well into a 96-well plate and incubated for 24 h. Spent media were then replaced with fresh media containing test materials (50 - 200 µg/mL). The plates were incubated in CO2 incubator for 24 h. Then the spent media were replaced with sterile MTT solution (5 mg/mL in PBS at pH - 7.4; 100 µL/well) and incubated for another 3 h. The unreacted dye was removed, the formazan crystals were dissolved with

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Journal Pre-proof DMSO (200 µL/well) and kept in dark for 30 min. The resulting purple colour was quantified by measuring absorbance at 570 nm. The assays were performed thrice and the results were expressed in percentage in comparison to vehicle treated cells. The following formula was used for calculation. i

(

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IC50 values were calculated using sigmoidal-growth model. Cell apoptosis by double (AO/EB) study Normal Adult Human Primary Dermal Fibroblasts (NHDF) cells were purchased from ATCC (PCS-201-012). NHDF cells were maintained in cultures in Dulbecco’s Modified Eagle’s Media (1:1) containing 10% fetal

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bovine serum and 1% antibiotic. The cells (105 cells/mL) were cultured in a 12-well plate, followed by treatment with samples AA-Ag, AA-Ag-FA, and AA-FA (5 μL/mL) for 48 hours. Later, both adherent and cells in

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suspension were collected and centrifuged. The pellet was resuspended in a solution of 25 μL PBS and 2 μL

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AO/EB dye (100 μg/mL). Slides were prepared and fluorescence was observed with the help of a Nikon eclipse Ti fluorescence microscope at 200x magnification. Tests were done in triplicate, counting a minimum of 100

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total cells each.

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Nano indentation

Nanoindentation experiments were carried out on pure agar-agar (AA), AA-Ag and AA-Ag-FA film (15 mm x

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15 mm) of ~20 μm thickness coated over glass slide. A Berkovich tip (a three-sided pyramidal diamond tip) of diameter 100 nm was used for carrying out the nanoindentation experiments in Hysitron Triboindenter

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(Minneapolis, USA). Since the mechanical properties extracted from nano-indentation technique are sensitive to the tip geometry, the tip area function has to be calibrated accurately. This was done by using a standard quartz sample and by following a standard practice. Because the loads used in indenting agar-agar films are very small, the tip area function is calibrated in low depth ranges for precise determination of the modulus and hardness. Using this area function, nanoindentation experiments were performed on single-crystal Al to cross check the standard elastic modulus, , and hardness,

, values as prescribed by the manufacturer (75.1 ± 5% GPa and 360

± 10% MPa, respectively). The standard deviation is within 5% for both these values, validating the tip calibration process. A peak load of 400 μN was applied in all the cases, with loading and unloading rates of 40 μN/s. A pause time of 10 s was allowed at the peak load to find out time-dependent plastic deformation, if any. Ten indentations were made for each sample. Degradation of AA-Ag-FA

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Journal Pre-proof The degradation of AA-Ag-FA scaffold was determined as the mass loss percentage of the gel after immersion into PBS buffers (pH 7.4) at 37 °C. First, AA-Ag-FA samples were soaked into PBS buffers for 24 h, and then air dried for 24 h. The dried samples were weighted as

. Then, the samples were immersed into PBS buffers

at 37 °C, removed at given time points and again air dried for 24 h to obtain the residual mass (

). The mass

loss rate was calculated as follows: Mass loss rate (%)

Release of Silver The release of silver from AA-Ag-FA was measured on an Inductively coupled Plasma-Optical Emission

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Spectrometer (ICP-OES, Perkin-Elmer optima 2100). 1.0 g of AA-Ag-FA was immersed into 25ml of 0.01M

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PBS buffer (pH 7.4) at 37 °C. Then, 500μL of that PBS solution was collected for ICP-OES measurement at regular intervals. A fresh PBS buffer of equal volume (500μL) was added after each measurement to maintain

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the volume constant. The amount of silver present in sample before immersing into PBS buffer was taken as . The cumulative release of

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and the amount of silver in PBS after immersion at regular interval was taken as

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silver was calculated as follows:

% Release of silver at time

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Animals ethics committee approval and surgical procedure The research protocol involving the use and handling of animal was according to the Committee for Purpose of

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Control and Supervision of Experiments on Animals (CPCSEA, Govt. of India) and was approved by the Institutional Animal Ethics Committee (IAEC), GITAM University (IAEC/GIP/-128-1287-S/approved/4/201516).The healthy male experimental albino Wistar rats (Inbred strain) weighing between 200-250g were procured from the Central animal House, Gitam University (CPCSEA Registration Number: 1287/PO/Re/S/09/CPCSEA). All the animals were caged in sterile polypropylene cages maintained at temperature 25 ± 2 °C and fed with standard pellets (supplied by M/s. Vyas Labs, Hyderabad-500 039) and water ad libitum. The studies and surgeries of animals were executed in a sterilized animal experimentation area. Male albino (36 number) Wistar rats were divided into six groups, each having six animals, allotted with unique identification number. The animal’s fur was removed with a sterile electrical clipper (Philips Norelco BT1200) carefully to avoid the skin abrasion one day prior to the creation of wound. After 6h of fasting, rats were anesthetised by intraperitoneal ketamine at a dose of 100mg/kg body weight. A full thickness of dorsal interscapular region skin of diameter of 2.5cm was excised by using a sterilized surgical dissection set and the day was considered as day zero (day 0).

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Journal Pre-proof The created wound bed of the rats of Groups (Gr) II, III, IV, V and VI was treated with standard drug (Megaheal gel), agar-agar (AA), Agar-silver (AA-Ag), fumaric acid incorporated AA-Ag (AA-Ag-FA) and fumaric acid incorporated agar-agar (AA-FA) gel respectively. The untreated wound bed of Group I rats was considered as negative control (NC). The application of the test and standard gels were carried out uniformly over the wound bed once a day from day one to the day till wound healed. The observations were recorded on day 0, 4, 8, 12 & 16 for the wound healing parameters: wound contraction, inflammatory cells, fibroblasts, re-epithelialisation, granulation tissue, and presence of collagen bundles etc.

Harvesting of tissue material for study

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Wound tissues on 0th, 4th, 8th and 16th postoperative days were collected from anesthetised animals and preserved

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(5mL, 0.1M) at -50°C for malondialdehyde (MDA) analysis.

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in 10% formaldehyde for histopathological analysis. The 8th day tissues were preserved in phosphate buffer

MDA assay

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Lipid peroxidation refers to the oxidative degradation of lipids. Quantification of lipid peroxidation is essential

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to assess oxidative stress. Extent of lipid peroxidation was estimated by assaying the amount of MDA in 8th-day wound tissue samples. First, 10% tissue homogenate was prepared by adding 500mg of tissue to 5mL of 0.1M

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phosphate buffer solution, homogenised for few minutes, centrifuged and supernatant was used for estimation. TCA-TBA-HCl solution was prepared by dissolving 15g TCA and 0.375g TBA to 100mL of 0.25N HCl at 40°C

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and stored at 4°C for future use. MDA standard plot was constructed by taking absorbance a, λ=527nm of different concentration of MDA solution treated with 1mL of TCA-TBA-HCl solution. Then, 0.5mL of homogenised tissue supernatant was added to 1mL of TCA-TBA-HCl solution, kept at boiling water-bath for 15 minutes, and centrifuged (~3000 rpm, 15minutes). The absorbance at λ= 527nm of pink colour solution was measured and compared with MDA- standard graph. Histological processing The wound tissue samples were fixed in 10% buffered (pH=7.4) formalin for light microscopic image capturing. Wound sections were then dehydrated along perpendicular direction to the anterior-posterior axis of the wound

with

absolute ethanol and embedded in paraffin. Serial sections of 5.0 µm thick were mounted on

glass slides, dewaxed, rehydrated and stained with Hematoxylin and Eosin (H&E) staining and Massion’s Trichome (MT) staining with one glass slide to each staining on all post wounding days. Data analysis

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Journal Pre-proof The values for all measurements are expressed as the mean ± the standard deviation (SD). The error bars denote ± SD. T-test, one-way or two-way analysis of variance (ANOVA) with post-test, where appropriate, was used to

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examine the differences among variables. A p value less than 0.05 was considered to be statistically significant.

Results and Discussion

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Synthesis, characterization of AA-Ag hydrogel

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Univalent silver (Ag+) ion was reduced inside the agar-agar gel to Ag0 using DMF. The advantages of choosing DMF are mild reducing agent [57], completely miscible with water [58], does not interfere with the gelling

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mechanism of agar-agar [55]. The formation of Ag NPs was characterized by the UV-Visible spectra as shown

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in Fig. 1a

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-----------------------------------------------------------------Fig. 1--------------------------------------------------------------The presence of absorbance maxima at λmax=415nm for both AA-Ag and AA-Ag-FA sample is the characteristic

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Surface Plasmon Resonance (SPR) band for spherical Ag NPs. However, the absorbance intensity was slightly decreased upon addition of FA to AA-Ag without changing λmax indicates the preserved shape and size of Ag NPs inside the gel. TEM micrographs (Fig.1b and 1c) confirm the above fact. The shape of the Ag NPs in AAAg and AA-Ag-FA was found to be spherical. Particle size distribution of AA-Ag and AA-Ag-FA (Fig 1e, f) show average size of (12.70±2.59) nm and (11.55±1.56) nm with polydispersity of ~20% and ~14% respectively. The energy dispersive X-ray absorption (EDX) pattern (Figure 1d) confirms the presence of silver in both AA-Ag and AA-Ag-FA samples. FA is one of the important ingredients of the hydrogel which acts as antimicrobial, antioxidant and facilitate the angiogenesis path when it is free FA not combined or bonded with other component of the scaffold. Therefore, it is important to ensure no covalent bond was formed when FA acid was added to AA-Ag or AA gel as we propose mentioned in the introduction. We performed a preliminary study through FTIR spectroscopy. The FTIR spectra of AA, AA-Ag, AA-Ag-FA and AA-FA are depicted in figure 1g. The FTIR spectra of AA shows

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Journal Pre-proof significant troughs centered at 3410 cm-1, 2920 cm-1 and 1638 cm-1 attributed to O-H, C-H and C-C stretching [59]. It is observed that trough-centers do not change their position and also no new peak or trough appeared for the sample AA-Ag, AA-Ag-FA and AA-FA indicating no new bond formation upon addition of FA to AA-Ag or AA. Probably, there is short-range, non-covalent interaction between –OH groups of agar-agar with metal silver nanoparticles present as depicted in figure 1h. The surface morphology of the samples was investigated by SEM (Figure S1, see supporting information). AA and AA-FA hydrogel had a smooth but flakey surface (Figure S1 a, d) indicating FA and AA are well blended but forms film on drying in SEM stab. AA-Ag and AAAg-FA also showed same surface morphology with irregular white patches (Figure S1 b, c) confirming the

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distribution of Ag NPs inside agar-agar gel.

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In-vitro antioxidant and Lipid peroxidation

In-vitro antioxidant activity of our test samples was evaluated through DPPH radical scavenging activity in

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solution.

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-------------------------------------------------------------Fig. 2------------------------------------------------------------------DPPH free radical method is an antioxidant assay based on electron-transfer that produces violet colour in

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solution [56]. This free radical, stable at room temperature, is reduced in the presence of an antioxidant molecule, giving rise to pale yellow to colourless solution. According to figure 2a, AA-Ag-FA (~%RSA=36)

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shows maximum antioxidant activity, followed by AA-FA (%RSA~29).

AA-FA showed RSA due to the

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presence of well-known antioxidant FA [46, 60]. The highest scavenging activity offered by AA-Ag-FA due to the slow leaching or diffusion of both antioxidant FA and Ag NPs [61-63] which reduces DPPH. The release profile (Fig.6b) of Ag from AA-Ag-FA gel further support the above fact. Tissue lipid peroxidation produces a variety of products; among which the most mutagenic product malondialdehyde (MDA) has been used as biomarker for assessing lipid peroxidation [64]. It is generally accepted that higher the MDA level in tissue, higher is the lipid peroxidation [64]. Figure 2b shows the MDA level in day 8 tissue sample collected from different groups of rat. The MDA level in wound tissue on day 8 of Gr-VI (AA-FA treated) was found to be minimum i.e., 14.28 ± 3.21 nM/mg tissue whereas for Gr-I (negative control) it was found to be maximum i.e., 133.40 ± 30.02nM/mg tissue. It was also observed that the MDA level for Gr-II (Mega heal treated) was more compared to Gr-V (AA-Ag-FA treated). Even though estimation of antioxidant activity through RSA (DPPH-assay) and MDA-level in the tissue of rats of different groups follow two different mechanisms; in principle, their estimate should be corroborative to each other.

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Journal Pre-proof Expectedly, RSA was more for AA-FA hydrogel. MDA-level in Gr-VI (wounds treated with AA-FA) tissue was less indicating high antioxidant activity due to the presence of FA [46, 60]. Contradictorily, RSA of AA-Ag and MDA-level in Gr-IV (wounds treated with AA-Ag) tissue was not supportive to each other. This anomaly may be due to the formation of reactive oxygen species (ROS) by Ag NPs [65-68] which oxidizes lipid leading to the formation of MDA and reduces DPPH radical [69] as proposed in the figure 2c. The possible way, Ag NPs may produce ROS, proposed and identified by Liu and Hurt et. al. [70] by monitoring the production of H2O2 over a time scale of hours to days following the addition of Ag NPs to aqueous solutions saturated with air and concluded that O2.-, a precursor of H2O2, is formed via the oxidation of Ag NPs by oxygen. O2 (DO) + Ag NPs + H+ → Ag+ + [peroxide intermediate]

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Further, the intracellular ROS detection by DCFHDA test confirmed (Figure S2, see supporting information) the

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generation of ROS and supports the proposed mechanism depicted in figure 2c.

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Since the reduction of DPPH, the pointer for the in-vitro antioxidant activity, was driven by both FA and ROS produced by Ag NPs in the aqueous environment, % RSA was maximum for AA-FA-Ag. Whereas lipid

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peroxidation is natural in wounds due to inflammation and could be enhanced or suppressed if ROS or

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antioxidant would be present respectively. In case of Gr-VI (AA-FA treated) tissue FA was there as suppressor and the MDA-level was found to be minimum. On the other hand Gr-IV (AA-Ag treated) tissue showed

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maximum MDA level due to absence of suppressor like FA but presence of ROS generator like Ag-NPS. Gr-V (AA-Ag-FA treated) tissue shows moderate MDA-level due to counter actions of Ag NPs and FA on lipid-

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peroxidation. Antibacterial performance

Antibacterial activity of AA-Ag, AA-Ag-FA and AA-FA was evaluated using zone of inhibition test against E. coli, S. aureus and P. aeruginosa. The sizes of the zone so formed (Fig. 3a-c) were compared with that of the standard antibiotics, Cefoxitin. It has been found that AA-Ag-FA showed antibacterial activity similar to the antibiotics used against the microbes. Interestingly, AA-FA also formed zone against each of the microbes confirming its antibacterial nature. AA-Ag showed larger zone (Fig. 3d) compared to than that of AA-FA but smaller than AA-Ag-FA (Fig. 3d). The antibacterial Ag NPs and FA shows additive effect with respect to bactericidal action of AA-Ag-FA sample against each of the microbes tested. The minimum inhibition concentration (MIC) was evaluated using serial dilution techniques. The data (Table S1) of MICs are corroborative to zone radius for each of the sample. ------------------------------------------------------Fig. 3--------------------------------------------------------------------------

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Journal Pre-proof Toxicity assay The cytotoxicity assay was evaluated against 3T3-L1 fibroblast cells and the results are shown in figure 4. -----------------------------------------------------------------Fig. 4----------------------------------------------------------The results show minimal cytotoxicity offered by each samples to fibroblast cells as their IC 50 values are greater than 1mg/mL. Topical application of AA-Ag-FA hydrogel shows no external toxicity appeared on skin. DMF on reaction with Ag+ converted to less toxic carbamic acid following the reaction: 2Ag+ + HCONMe2 + H2O → 2Ag0 + Me2NCOOH + 2H+ and Me2NCOOH → CO2 + Me2NH. During healing process, 1.0 mL AA-Ag-FA gel which contain 1.108 mg/mL of active ingredient (i.e. Ag + FA)

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was applied each time. Therefore, at such applied concentration (1.108 mg/mL), the samples would show no

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toxicity to the skin.

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Assessment of apoptosis by double staining (AO/EtBr)

Nuclear morphology of the cells was visualized by fluorescence microscopy after staining with acridine

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orange/ethidium bromide (AO/EB) double dye. In general, upon treatment with AO/EB dyes, the viable cells show round and green nuclei, while early apoptotic cells have fragmented DNA with green coloured nuclei and

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late apoptotic and necrotic cells show fragmented DNA with orange and red nuclei. From figure 5a-c and plot 5d, it is clear that the percentage of Live cell (of NHDF cell) on treatment with AA-Ag-FA is maximum (~

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70%) with minimum necrotic (~5%) and apoptotic (~25%) cell compared to others. Assessment of cell apoptosis further support and corroborate the MTT assay and Histological analysis. Interestingly, the number of apoptosis cell for negative control sample was quite high. However, Messadi et. al. [71] reported that for human dermal cell, apoptosis found to participate in the transition between granulation tissue and the development of definitive scar. Quantitative analysis of apoptotic cells using an Annexin-V-FITC binding assay by them showed that normal skin fibroblast cultures were found to have a two-fold higher percentage of apoptotic cells. So we presumed here that the excess apoptosis for the negative control sample was normal here for NHDF cells. -----------------------------------------------------------Fig. 5--------------------------------------------------------------------Mechanical Properties The elastic modulus (E) and hardness (H) of AA, AA-Ag and AA-Ag-FA was calculated using Oliver-Pharr44 method [72], are listed in Table 1. The elasticity of AA-Ag has increased almost five fold compared to the pure

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Journal Pre-proof AA. Similarly, the hardness has increased by almost eight times. This is due to reinforcement by Ag NPs into agar-agar. -------------------------------------------------------------Table 1----------------------------------------------------------------Degradation study One of the pivotal features of environment friendly biomaterials is its biodegradability. The controllable degradation of AA-Ag-FA is favourable for the release of Ag for the long-term bactericidal effects. Figure 6a showed ~ 29% degradation even after 40 days which suggest moderate to slow degradation nature of agar-agar. ----------------------------------------------------------------Fig. 6---------------------------------------------------------------Release of silver

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AA-Ag-FA hydrogel showed antibacterial activity due to the release of silver from the sample to the

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environment. To evaluate the same, ICP-OES was performed. Figure 6b showed release profile of Ag from AAAg-FA in PBS buffer. The release profile revealed that the rate of release of silver from the sample decreases as

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time goes on. The release rate for the first 6h was relatively high than that of the next 6h and so on. The high

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release rate at the beginning may be due to the fact that the Ag at the periphery of the sample releases easily in PBS with in short time. However, even after 72h (3days), only ~35% of the total silver released from the sample

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Wound healing activity

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indicating its long lasting antibacterial biomaterial.

Figure 7a, b shows the optical (i.e. camera) images and wound closure at different intervals of time respectively.

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The percentage of wound closure was calculated as per the formula:

, where

and

are

the size of the wound at day 0 and other days (i.e. Day 4, 8, 12 and 16), measured using ImageJ software calibrated to the scale bar given in each image and

is the wound closure at day d (d=4, 8, 12, and 16). Figure

7c is sigmoidal-logistic fitted model for wound closure vs. time as suggested by Cukjati et al. [73]. -------------------------------------------------------------Fig. 7------------------------------------------------------------------It is clear that the extent and rate of wound closure for Gr-V (i.e. treated with AA-Ag-FA) was much faster compared to other groups. As per the logistic wound healing model (Fig. 7c), the time required for 50% of healing was minimum (5.24 days) for AA-Ag-FA treated wound and maximum (7.93 days) for negative control wound (i.e. wound treated with nothing). Noticeably, the wound healing performance was even good for AA-FA sample (5.96 days). The common component in both the gel (AA-Ag-FA and AA-FA) is FA which shows considerable in-vitro antioxidant activity (Figure 2). It was also observed that on day 12, the wound treated with AA-Ag-FA was ~95% healed while in the other wounds clear defects were observed with less % of wound

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Journal Pre-proof contraction. On 16th day, complete healing was observed for wound treated with AA-Ag-FA (Gr-V) but not for other wounds.

Histological analysis The degree of healing was further assessed at the microscopic level using histological staining: H&E and MT through the quantitative scoring of several parameters like inflammatory cells, angiogenesis, granulation tissue, epidermal lining and collagen. The scoring of above mentioned parameters were given according to the criteria mentioned in Table S2 (see supporting information) and shown in figure 8b. The figure 8a shows the

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microscopic images of different tissue sections stained with H&E at different time line. The results observed on

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predetermined days i.e., on day 4, 8 and 16 are summarised in Table 2.

--------------------------------------------------------------Fig. 8------------------------------------------------------------------

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On day 4, inflammatory cells were found to be significant for all the groups and decreased gradually with time

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but the extent of decline was noteworthy (Fig. 8a, b) for Gr-V and Gr-VI indicating the potential role of fumaric

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acid in preventing the inflammation in later stages for healthy wound healing. --------------------------------------------------------------Table2-----------------------------------------------------------------

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On day 4, no granulation tissue and/or blood vessels were seen for all the groups except for Gr-VI where mild granulation tissue and tiny blood vessels were located at multiple sites (Fig. 8a) indicating early beginning of

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healing.

Considerable amount of granulation tissue was formed by 8 th day and started to decrease gradually for the next 8 days i.e., by 16th day due to subsequent conversion of granulation tissue to collagen through remodeling. Noticeably, on day 16, the decrease in granulation tissue was highest and 2nd highest for Gr-VI and Gr-V respectively indicates higher degree of collagen formation i.e. complete remodeling with regenerated adnexa. On day 16, in spite of complete remodeling, the presence of tiny to moderate size multiple blood vessels in tissues of Gr-VI (AA-FA treated) reveal the angiogenic property of AA-FA which needs to be evaluated further. On day 4, no epidermal cell proliferation was observed in Gr-I to Gr-IV skin tissue; but an intact survived epidermal lining was observed in Gr-V, and a monolayer epidermal lining proliferation was noted in group VI. On day 8, the epidermal lining was noted in all the groups with highest cell proliferation in Gr-V and Gr-VI. On day 16, incomplete epithelialisation was observed in Gr-I, Gr-II, Gr-IV and Gr-VI whereas complete thinned out lining was found in Gr-III and Group-V.

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Journal Pre-proof Collagen Estimation Masson’s trichrome staining, which can stain collagen as blue, was used to quantify collagen using the ColourThreshold in ImageJ software as described by G. F. Caetano et al [74, 75] with little modification. Five images per slide were captured at 10x magnification and deconvoluted using ImageJ software to separate blue colour from other colours present in the image. The quantity of collagen was measured in terms of number of pixels and expressed as percentage of total pixels found in an image. The figure 8c shows the collagen formation in wound tissue of a rat from each group on day 16. The collagen density so formed was found to be very less for Gr-I (NC) compared to other groups (Fig. 8c).

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Histological analysis of collagen deposition by image analysis from the skin on day 16 revealed that minimum (~20% area) collagen was formed in Gr-I (NC) skin. In all other groups, the collagen deposition was more (>

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60% area), out of which collagen was found highest (~75%) in Gr-VI skin (Fig. 8d).

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We have adopted multifunctional approach to empower the hydrogel: agar-agar towards efficient wound healing

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by introducing Ag NPs as antibacterial agent and antioxidant FA to facilitate angiogenesis, epithelialisation and collagen deposition. Our results demonstrate that Gr-V (AA-Ag-FA treated) and Gr-VI (AA-FA treated) show

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better performance towards wound healing activity. The common ingredient present in both AA-FA and AAAg-FA hydrogel is FA which is well known for its antioxidant property. In general, ROS production is involved

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in all phases of wound healing which impeded the rate of healing. Thus FA maintains the balance between oxidative and antioxidative forces necessary for a healthy wound healing. Ag NPs, the other component of the

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hydrogel, does not allow bacteria to grow on the wound bed.

Conclusions In summary, we have successfully synthesized an easy spreadable AA-Ag-FA biodegradable hydrogel ideal for topical application, well characterized through UV-Visible, FTIR spectroscopy and TEM. The hydrogel not only inhibit growth of microbes but also shows accelerated healing rate with promising epithelialisation, angiogenesis, less lipid peroxidation and considerable organized collagen deposition with minimal to no cytotoxicity. Both AA-FA and AA-Ag-FA hydrogel could be used as wound healer but AA-Ag-FA would be the ideal candidate as it shows better antibacterial activity apart from the other parameters like thickened

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Journal Pre-proof epidermal, angiogenesis, moderate lipid peroxidation, collagen deposition etc. with highest rate of wound healing.

Acknowledgements The authors thank Nano Centre (IISc, Bangalore) for electron microscope facility. The authors are indebted to

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Dr. Sandip Bhowmik for his valuable scientific suggestions.

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[73] D. Cukjati, S. Rebersek, R. Karba, D. Miklavcic, Modelling of chronic wound healing dynamics, Med. Boil Eng. Comput. 2000 (38) 339-347. [74] G. F. Caetano, M. Fronza, M. N. Leite, A. Gomes, M. A. C. Frade, Comparison of collagen content in skin wounds evaluated by biochemical assay and by computeraided histomorphometric analysis, Pharm. Biol. 11 (2016) 2555-2559. [75] G. F. Caetano, M. A. C. Frade, T. A. M. Andrade, M. N. Leite, C. Z. Bueno, A. M. Moraes, J. T. Ribeiro-Paes, Chitosan-alginate membranes accelerate wound healing, J. Biomed. Mater. Res. Part B: Appl. Biomater 103 (2015) 1013-1022. [76] K. Huang, J. Wu, Z. Gu, Black Phosphorus Hydrogel Scaffolds Enhance Bone Regenerationvia a Sustained Supply of Calcium-Free Phosphorus, ACS Appl. Mater Interfaces. 11 (2019) 2908-2916. 23

Journal Pre-proof [77] Red Jujube-Incorporated Gelatin Methacryloyl (GelMA) Hydrogels with AntiOxidation and Immunoregulation Activity for Wound Healing, Journal of Biomedical Nanotechnology 14 (2019) 1357-1370. [78] Tofu-Based Hybrid Hydrogels with Antioxidant and Low Immunogenicity Activity for Enhanced Wound Healing, Journal of Biomedical Nanotechnology 13 (2019)

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Figure 1. Formation and characterization of hydrogels. (a) UV-visible spectra of AA, AA-Ag, AA-Ag-FA and AA-FA, presence of characteristic SPR peak at 415nm confirms the formation of Ag NPs inside the gel. (b) and (c) TEM micrographs of AA-Ag and AA-Ag-FA respectively (Scale bar=50nm). The average size of the particles is more or less same. (d) EDX pattern of AA-Ag and AA-Ag-FA further confirm the presence of silver. (e) and (f) Histogram of particle size of AA-Ag and AA-Ag-FA respectively. (g) FTIR spectra of (i) AA, (ii) AA-Ag, (iii) AA-Ag-FA and (iv) AA-FA taken in diffuse reflectance mode, neither change in position of peaks nor evolution of new peak reveals no possible bonding between FA and Ag in AA-Ag-FA. (h) Schematic representation of possible interaction between Ag NPs and agar-agar. (For interpretation in regard to colour in this figure legend, the reader is referred to check the web version of the article.)

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Figure 2. In-vitro antioxidant assay by DPPH. (a) Radical scavenging activity of AA, AA-Ag, AA-Ag-FA, and AA-FA hydrogel incubated for 24h period, In-vivo lipid peroxidation assay through MDA analysis. (b) Plot of MDA level in tissue of rats of different groups after day 8 of wound healing. (c) Schematic representation of mechanisms of lipid peroxidation and in-vitro antioxidant activity. (For interpretation in regard to colour in this figure legend, the reader is referred to check the web version of the article.)

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Figure 3. Antibacterial activity: Photographs showing disc diffusion assay. Clear zone formed against the growth of (a) E. coli (b) S. aureus and (c) P. aeruginosa by the sample AA-Ag-FA (4 on the Petri dish). AA-FA (2 on the Petri dish) shows no zone formation against all the microbes used. AA-Ag (3 on Petri disc) shows zone formation against E. coli and S. aureus with lesser zone size compared to AA-Ag-FA. The standard antibiotic (1 on petri dish) used here is cefoxition, (d) Bar chart showing average zone diameter for different samples tested against different microbes. ( ) . (For interpretation in regard to colour in this figure legend, the reader is referred to check the web version of the article.)

Figure 4. Cytotoxicity assay through MTT. (a) Bar chart of % cell survival vs different concentrations of AAAg-FA (b) Plot of % cell inhibition vs log of concentrations of AA, AA-Ag and AA-Ag-FA fitted to growthsigmodal function to estimate IC50. Inset table shows the IC50 of different samples. (For interpretation in regard to colour in this figure legend, the reader is referred to check the web version of the article.)

Figure 5. Apoptosis assay through AO/EB staining. Fluorescence images (after merging) of NHDF cells treated with (a) NC (not treated) (b) AA-Ag and (c) AA-Ag-FA (Scale bar =10μm) and (d) data showing % Live, Necrotic and Apoptotic cells upon exposure with different samples ( ). (For interpretation in regard to colour in this figure legend, the reader is referred to check the web

Figure 6. (a) Degradability and (b) silver release profile of AA-Ag-FA hydrogel in

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Journal Pre-proof Figure 7. Wound healing activity: (a) Optical images of wound at different stages of healing (scale bar= 1.0 cm), (b) Bar-chart of % wound closure vs. time ( ), and (c) Sigmoidal wound healing fitted to logistic model (inset: 50% healing time as per model for different test samples). (For interpretation in regard to colour in this figure legend, the reader is referred to check the web version of the article.)

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Figure 8. Microscopic histological images (a) of H&E stained wound tissues of day 4, 8 and 16 from a rat of each group. Here, different wound parameters are indicated by arrows of different colours in which red dotted line: inflammatory exudate, double headed orange arrow: scab, green arrow: inflammatory cells/macrophages, purple arrow: infiltrated red blood cells (RBCs), dark red arrow: capillaries/blood vessels, golden arrow: fibroblasts/collagen, double headed red arrow: epithelium lining, black arrow: necrotic debris, light blue (aqua) arrow: keratinization. scale bar 50 μm, (b) Score charts for (i) angiogenesis, (ii) inflammatory cells, (iii) granulation tissue and (iv) epithelialisation for each group on day 4, day8 and day16 ( ). Microscopic images of (c) of MT stained wound tissues of day 16 from a rat of each group. Here, green arrow indicates formed collagen in each image, scale bar 20 μm. (d) Plot of collagen accumulation in terms of area (%) on day-16 of each group. Collagen content was measured by digital densitometry using ImageJ in sections of wound tissue stained with MT. The results show the collagen content in each group as a percentage of total area. (For interpretation in regard to colour in this figure legend, the reader is referred to check the web version of the article.)

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Hardness (MPa) 36.60 ± 0.08 281.30 ± 0.03 273.33 ± 0.03 47.33 ± 0.03

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AA AA-Ag AA-Ag-FA AA-FA

Elastic modulus (GPa) 1.231 ± 0.027 5.125 ± 0.126 4.983 ± 0.013 1.347 ± 0.019

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Table 1. Mechanical properties of samples evaluated through Nano-indentation

Groups Gr-I

Day 4  Significant inflammatory cells and necrotic debris  Inflammatory exudates at multiple sites  No granulation tissue  No blood vessel or angiogenesis  No proliferation of epidermal lining

Day 8  Significant inflammatory cells and necrotic debris  Significant granulation tissue with 2-3 blood vessels per site  Moderate proliferative epidermal lining  No collagen

Day 16  Mild inflammatory cells  Thinned (~0.08μm) out epidermal lining.  Negligible granulation tissue with very few (2-3 per site) tiny blood vessels.  Negligible collagen  Poor remodelling

Gr-II

 Moderate inflammatory cells and Necrotic debris.  Inflammatory exudate at one site  No granulation tissue and angiogenesis  No proliferation of epidermal lining

 Occasional/mild inflammatory cells and negligible necrotic debris  No inflammatory exudates  Moderate granulation tissue seen with numerous number (>10 per site) of tiny blood vessels  Proliferative epidermal lining  Mild collagen

Gr-III

 Significant inflammatory cells

 Moderate

 Very negligible inflammatory cells  Thinned (~0.08μm) out epidermal lining  Moderate amount of granulation tissue with moderate amount (4-6 per site) of tiny blood vessels  Regenerated appendages  Coarse collagen and moderate number of fibroblasts  Mild inflammatory cells

inflammatory

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 significant inflammatory cells  Mild inflammatory exudates at 2-3 sites  Negligible granulation tissue and mild number of tiny blood vessel or angiogenesis  Mild proliferation of epidermal lining

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Gr-VI

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 Significant inflammatory cells  Mild inflammatory exudates at 2-3 sites  No granulation tissue and No blood vessel or angiogenesis  Mild proliferation of epidermal lining

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Gr-V

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 Moderate inflammatory cells  Negligible inflammatory exudates at one site  No granulation tissue and No blood vessel or angiogenesis  mild proliferation of epidermal lining

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Gr-IV

necrotic debris  No inflammatory exudates  Significant Young granulation tissue and fibroblasts with Numerous (>10 per site) blood vessels of moderate size  Moderate proliferation of epidermal lining  Few collagen  mild or negligible inflammatory cells and necrotic debris  No inflammatory exudates  Moderate Young granulation tissue and fibroblasts with moderate number (4-6 per site) of blood vessels of moderate size.  Moderate proliferation of epidermal lining  Few collagen  Moderate inflammatory cells and necrotic debris  No inflammatory exudates  significant Young granulation tissue and fibroblasts with numerous number (> 10) of tiny blood vessels  Moderate proliferation of epidermal lining  Few collagen  Moderate inflammatory cells and necrotic debris  No inflammatory exudates  significant Young granulation tissue and fibroblasts with numerous number (>10) of tiny blood vessels (more than the group V)  significant proliferation of epidermal lining  Few collagen

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 Inflammatory exudates at multiple sites  No granulation tissue and No blood vessel or angiogenesis  Mild () proliferation of epidermal lining

 Very thinned (~0.05μm) out epidermal lining  Significant granulation with moderate number (4-6 per site) of tiny blood vessels  Regeneration of appendages  Few collagen  Mild inflammatory cells  very thinned out (~0.05μm) epidermal lining  moderate granulation tissue with few or mild number (2-3 per site) of tiny blood vessels  Regeneration of appendages  Few collagen  No inflammatory cells  Normal to hyper-proliferated (~0.10-0.15μm) epidermal lining  Negligible granulation tissue with few or mild number (2-3 per site) of tiny blood vessels  Regeneration of appendages  Few collagen  No/very negligible inflammatory cells  Normal to hyper-proliferated (~0.10-0.12μm) epidermal lining  No / Negligible granulation tissue with moderate number (4-6 per site) of tiny blood vessels at different sites  regeneration of appendages  Few collagen

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Table 2. Day wise summarised observations of histological analysis through H&E staining

Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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Highlights 

AA-Ag-FA hydrogel, a multifunctional approach to tackle wound healing.

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Fumaric acid d

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The sample synergistic antibacterial effect against microbes.

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The hydrogel shows faster rate of wound healing.

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The formulation shows enhanced collagen deposition, angiogenesis etc.

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g size and shape of Ag nano-particle inside hydrogel.

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