Genipin crosslinked gelatin-diosgenin-nanocellulose hydrogels for potential wound dressing and healing applications

Journal Pre-proof Genipin crosslinked gelatin-diosgenin-nanocellulose hydrogels for potential wound dressing and healing applications

Sevinc Ilkar Erdagi, Fahanwi Asabuwa Ngwabebhoh, Ufuk Yildiz PII:

S0141-8130(19)38898-1

DOI:

https://doi.org/10.1016/j.ijbiomac.2020.01.279

Reference:

BIOMAC 14584

To appear in:

International Journal of Biological Macromolecules

Received date:

1 November 2019

Revised date:

28 January 2020

Accepted date:

28 January 2020

Please cite this article as: S.I. Erdagi, F.A. Ngwabebhoh and U. Yildiz, Genipin crosslinked gelatin-diosgenin-nanocellulose hydrogels for potential wound dressing and healing applications, International Journal of Biological Macromolecules(2018), https://doi.org/ 10.1016/j.ijbiomac.2020.01.279

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© 2018 Published by Elsevier.

Journal Pre-proof Research Paper

Genipin crosslinked gelatin-diosgenin-nanocellulose hydrogels for potential wound dressing and healing applications

Sevinc Ilkar Erdagi*, Fahanwi Asabuwa Ngwabebhoh*, Ufuk Yildiz Department of Chemistry, Kocaeli University, Umuttepe campus, 41380 Kocaeli, Turkey

ABSTRACT

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The present study focuses on the synthesis and evaluation of neomycin-loaded hydrogels as

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potential substrate for wound healing application. Herein, genipin crosslinked gelatin interpenetrated diosgenin-modified nanocellulose (DGN-NC) hydrogels were synthesized.

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The hydrogels’ chemical structures as well as surface morphology, mechanical property, and thermal behavior were characterized. Swelling analysis and gelation kinetics of the hydrogels

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were studied and the results obtained showed good swelling capacity as well as high gel yield.

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In addition, the prepared loaded hydrogels were evaluated for antibacterial activity against human pathogenic E. coli and S. aureus bacteria with inhibition capacity determined in the

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range of 50–88%. In vitro cytocompatibility and drug release studies were also explored under simulated physiological conditions achieving high cell viability and release percentage >80% and >90% after 24 h, respectively. In effect, the design hydrogels in the present study

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possess adequate incorporated antibacterial properties with significant potentials towards wound dressing and healing applications. Keywords:

Hydrogel Diosgenin-nanocellulose Gelatin Genipin Antibacterial activity

*Corresponding authors: E-mail addresses: [email protected] (S.I. Erdagi) [email protected] (F.A. Ngwabebhoh)

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Journal Pre-proof 1. Introduction Over the last decades, a substantial number of interesting wound dressing materials from naturally derived polymers, such as chitosan, cellulose, alginate, gelatin, and their derivatives have been used in the fabrication of hydrocolloids, skin substitutes and hydrogel dressings [13]. Amongst all, hydrogels have been widely considered as the most suitable choice for wound dressings attributed to its fascinating features of biocompatibility, high water retention ability, as well as flexibility [4, 5]. In effect, this class of ever-evolving innovative functional polymeric materials has always been of great research interest in the biomedical sector, particularly in the pharmaceutical drug control release fields including cardiology, oncology,

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eye contact lenses, cosmetics, wound healing, etc [6-8]. In addition, hydrogels as three-

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dimensional network material prepared from natural or synthetic polymers have also demonstrated to be biodegradable with low cytotoxicity, making them ideal candidates for

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application biomedicine.

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Nanocellulose (NC), a polysaccharide extracted mostly via acid hydrolysis of pristine cellulose is composed of repeating β-D-glucopyranose units and has been commonly applied

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in the preparation of hydrogels [9, 10]. Several studies on NC-based hydrogels have proven to be very suitable as drug delivery systems, tissue engineering scaffolds and wound dressing

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materials with good mechanical properties and dimensional stability. A recent study by Poonguzhali et al. reported the fabrication of a chitosan/poly(vinylpyrrolidone)/nanocellulose

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hydrogel for wound dressing application. Results obtained showed that the material possessed high water retention abilities, good blood compatibility and high level of antibacterial properties [11]. On the other hand, gelatin a natural polymer derived from the hydrolysis of collagen has also been widely investigated and commercialized as a good polymeric material with a wide range of application in biomedicine, food and the chemical industry [12]. However, hydrogel materials formed solely from gelatin particularly in the dry state, are partially brittle, less flexible and show rapid degradation. But over the years, the chemical and physical stabilization of the gel features has been achieved using common covalent crosslinkers such as formaldehyde, glutaraldehyde and epoxy compounds. Nonetheless, these crosslinkers exhibit a certain level of physiological toxicity due to the presence of unreacted moieties both from the crosslinker and by-products generated during the synthesis reaction [13, 14]. In comparison, genipin a natural extract from Gardenia jasminoides Ellis has recently attracted the attention of researchers as a potential chemical crosslinker. Hence has been effectively and extensively employed for the crosslinking of various amino-containing 2

Journal Pre-proof polymeric molecules [15, 16]. This plant extract has shown reduced cytotoxicity and antiinflammatory properties when compared to the aforementioned crosslinking agents. Recently, steroid compounds particularly from plants have demonstrated high significance and potential in the development of novel drugs for a wide variety of diseases. From this viewpoint, diosgenin (DGN) also known as 3β-hydroxy-5-spirostene, a steroidal saponin mainly isolated from therapeutic herbs, is a well-known starting material for the formulation of several synthetic steroidal drugs in various pharmaceutical industries [17]. For example, a study by Quiñones et al. revealed that the conjugation of DGN on the backbone of cellulose

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via hydrolysable ester bonds enhanced the bioavailability of diosgenin [18]. In addition, reports on DGN depicts this compound as an antitumor drug with high anticancer ability

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against a variety of cancer cells and has proven to be non-toxic on normal cells. In essence, this compound exhibits considerable antibacterial property and wound migration inhibition

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[19-21]. However, due to its hydrophobicity and poor solubility, its bioavailability in the

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clinical application has been limited. Conversely, neomycin an aminoglycoside antibiotic isolated from Streptomyces fradiae is a highly water-soluble drug that is widely used to treat

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bacterial infections due to its efficient antibacterial activity against both Gram (−) and (+) bacteria [22-24]. So far, few studies on loaded neomycin hydrogel systems have been

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investigated in order to enhance its systematic effect and residence time as well as bioavailability [25, 26]. Thus, the present study investigates the preparation of

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gelatin/diosgenin-nanocellulose antibiotic based hydrogels via a combined drug effect of neomycin and diosgenin following an in vitro release and cytocompatibility study as well as antibacterial activity evaluation. The drug release behavior of the in situ loaded hydrogels was systematically studied under the human body simulated physiological pH conditions. In vitro cytocompatibility tests were also performed using human dermal fibroblast cell lines, which depicted the hydrogels to promote cell proliferation. In addition, antibacterial activity against Gram (−) (E. coli) and Gram (+) (S. aureus) bacteria was tested. The physiochemical properties of the hydrogels, such as thermal, surface morphology, mechanical, swelling and gelation kinetics were examined. To the best of our knowledge, this is the first report on the preparation of bioinspired drug-loaded antibiotic delivery systems based on genipin crosslinked gelatin/diosgenin-nanocellulose hydrogels with synergistic drug effect for potential application as wound dressing/healing substrate.

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2. Material and methods 2.1. Materials Microcrystalline cellulose was supplied by Alfa Aesar chemicals (Karlsruhe, Germany). Sodium hydroxide (NaOH), ammonium persulfate ((NH4)2S2O8, ≥98%), neomycin trisulfate salt (C23H46N6O13·3H2SO4), diosgenin (C27H42O3, ≥93%), 4-dimethylaminopyridine (DMAP, 99%), N, N'-dicyclohexylcarbodiimide (DCC, 60 wt% in xylenes), dimethyl sulfoxide (DMSO, ˃99%) and gelatin (from porcine skin Type A, lyophilized powder) were all supplied

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by Sigma Aldrich (Darmstadt, Germany). Genipin (>97% purity) used as the crosslinking

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agent was purchased from TCI chemicals. All chemicals were of analytical grade and used without further purification. Distilled water (dH2O) was used for the preparation of all

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solutions. S. aureus (ATCC 29213) and E. coli (ATCC 25922) were purchased from RTA

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Laboratories in Gebze, Turkey.

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2.2. Preparation of carboxylated nanocellulose (C-NC) C-NC was prepared via the oxidation method using ammonium persulfate (APS, a strong

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oxidizing agent of low long-term toxicity and high water solubility) as previously described with slight moderations [27, 28]. In brief, 5 g of pristine cellulose was added to 500 mL of 1M

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APS aqueous solution. The mixture was heated to 65 °C for 15 h under continuous stirring to generate a suspension of carboxylated cellulose. The suspension was diluted with ≈500 mL of cold dH2O to stop the reaction, then followed centrifugation for 10 min at 12000 rpm. Subsequently, centrifugation/washing cycles were repeatedly performed until a pH 4 solution pH was reached. The sample ultra-sonicated for 30 min using an ultrasonic homogenizer (HD2070 Bandelin SonoPuls) to produce C-NCs. Finally, the obtained nano-suspension was vacuum freeze-dried at -80 ℃ using a Labconco freeze-dry/shell freeze lyophilizer system (Labconco Corp., Kansas City, USA) to yield white powder of C-NC particles. 2.3. Synthesis of diosgenin-conjugated nanocellulose (DGN-NC) DGN-NC was synthesized via the Steglich esterification method in the presence of DCC and DMAP as catalysts [29]. Briefly, 1 g (6.1 mmol for glucose unit) of C-NC was homogeneously dispersed in 50 mL of DMSO for 12 h under magnetic stirring. To this homogeneous solution, 0.10 g of DCC (0.5 mmol) and 0.004 g of DMAP (0.3 mmol) were 4

Journal Pre-proof added and stirred for another 1 h at room temperature under nitrogen atmosphere to activate the carboxylic acid groups of C-NC. Subsequently, 2.5 g of diosgenin (, 6.1 mmol) dissolved in 15 mL of DMSO was added to the above prepared solution and stirred for 6 h in the temperature range of 75–80 oC. The resulting solution was then cooled, dialyzed first for a day against DMSO followed by three days dialysis against water to remove any unreacted diosgenin. The obtained purified DGN-NC was lyophilized and stored under refrigeration for further usage. 2.4. Preparation of hydrogel

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Initially, gelatin was dissolved in 4.25 mL dH2O at 50 ℃ under continuous mechanical stirring

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on a hot plate to obtain a complete dissolution of 3.5% w/v initial concentrated solution. Varying concentrations of DGN-NC were also prepared by dispersion in 5 mL dH2O at room

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temperature for 5 min using an ultrasonic homogenizer to obtain clear and homogeneous varying concentrations of 1.5%, 3% and 6% w/v. The various prepared DGN-NC samples

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were then added to gelatin solutions under vigorous stirring producing different mixture

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weight ratios of gelatin: DGN-NC (1:0, 1:0.5, 1:1 and 1:2). To these mixtures, a constant volume of 0.75 mL genipin solution of stock concentration 5 mM was slowly added under continuous mechanical stirring. The obtained gel solutions were poured into glass petri dishes

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and allowed overnight to undergo gelation at 40 ℃. The crosslinked gel samples were then vacuum freeze-dried for 24 h and the obtained samples stored for further analyses. The

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detailed composition and designations of the gels are outlined in Table 1. Table 1 Compositions of genipin crosslinked gelatin: DGN-NC hydrogels. Sample code Gel-1 Gel-2 Gel-3 Gel-4

DGN-NC (%w/v) 0.00 1.50 3.00 6.00

Gelatin (%w/v) 3.50 3.50 3.50 3.50

Genipin (mL) 0.75 0.75 0.75 0.75

Molar ratio DGN-NC: Gel: Gen 0.00: 1.00: 0.005 0.35: 0.60: 0.003 0.55: 0.45: 0.002 0.70: 0.30: 0.001

2.5. Gelation study The gelation study also known as the extent of crosslinking of the hydrogels was determined via UV/vis spectrophotometer colorimetric absorbance analysis. The colorimetric method was based on reading the color change intensity of prepared samples as a function of time. In 5

Journal Pre-proof effect, homogeneous mixtures of the gel samples were transferred into 3 mL cuvettes and stored at a pre-determined temperature of 40 ℃. The gel samples were then analyzed at varying time intervals (0-24 h) using a dual-beam spectrophotometer (Cary 60 Scan, Agilent, Turkey) in the wavelength range of 0-800 nm to determine the change in color intensity to blueish, which is the typical color formed during genipin crosslinking reactions. The average gelation time for the different gel samples was determined based on the maximum absorbance color intensity value reached. It was assumed that the extent of complete genipin molecules reaction/consumption in the samples is proportional to the absorbance values of the

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hydrogels’ color intensity.

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2.6. Swelling and gel yield measurements

The swelling behavior of the hydrogels was performed by immersing determined amounts of

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dried gel samples in 25 mL of phosphate saline buffer (PBS) solutions of different initial pH (4.5, 5.5, 6.5, 7.5, 8.5 and 9.5) at 37 °C for 24 h, in order to reach equilibrium swelling. Once

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equilibrium swelling was reached, the samples were removed from the aqueous medium,

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blotted with tissue paper to absorb excess water on the gel surfaces, weighed in triplicates and the average value recorded. The equilibrium swelling percentages (ES%) were then calculated

WS - Wd ×100 Wd

(1)

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ES%=

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using the following mathematical formulation;

Where, Ws is the weight of the swollen hydrogel samples (g) and Wd is the weight of the dried samples (g). In addition, at the end of the swelling analysis, the swollen gel samples were washed for several cycles with dH2O and dried in a vacuum oven at 50 ℃ to reach constant weight. The gel yield percentage was then calculated following the equation below. Gel yield (%)=

Wf ×100 Wi

(2)

Where, Wi is the initial dry weight of the hydrogels after synthesis and Wf is the final dry weight of the samples after washing with dH2O. 2.7. Loading efficiency of neomycin in hydrogels Neomycin, a hydrophilic model antibiotic drug, was loaded into the hydrogels via the swelling diffusion method. In detail, a stock solution was prepared by dissolving 0.05 g 6

Journal Pre-proof neomycin sulfate in 50 mL dH2O of pH 7 under mechanical stirring to ensure complete dissolution. Thereafter, pre-weighed disc shape dried hydrogels were immersed into the neomycin solution and kept overnight at room temperature to reach equilibrium uptake. The swollen loaded hydrogels were removed from the drug solution and rinsed with dH2O. The hydrogels were then dried in an oven at 37 oC for 24 h to achieve constant weight and the concentration of the residual drug in solution via determined spectrophotometric analysis [30]. The quantification of the loaded neomycin in the hydrogels proceeded via generation of a standard curve using the prepared neomycin stock solution. Basically, different concentrations of neomycin solutions were prepared via serial dilution to obtain various

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concentrations ranging from 5 to 500 ppm. In order to perform UV/vis spectrophotometric readings, a mixture of 0.5 mL neomycin solution and 0.1 mL copper sulfate solution (0.0475

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g CuSO4 in 50 mL methanol) was prepared and completed with methanol to the total volume

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of 3 mL and then employed for absorbance reading at 253 nm wavelength [31]. The absorbance of the residual neomycin solution was measured and the concentration of

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neomycin in the hydrogels estimated from the standard curve and the mathematical

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formulations below.

mg )] +0.0769 mL

R²=0.998

(3)

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Absorbance = 0.0008× [drug (

The loading efficiency percentage (LE%) was also calculated using the equation: Amount of drug loaded ×100 Initial drug amount

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LE (%) =

(4)

2.8. Characterization methods

The absorbance spectra were measured on a UV/vis spectrophotometer (Agilent, Cary 60) in the wavelength range 200–800 nm. Fourier Transform Infrared (FTIR) spectra were acquired on Bruker Tensor 27 FTIR spectrophotometer under a 4 cm−1 resolution over the wavenumber range from 4000 to 400 cm−1. Structural characterization following the liquid nucleic magnetic resonance (1HNMR) method was performed using Bruker Avance 300 MHz spectrophotometer. The surface morphology of the hydrogels was analyzed with a scanning electron microscope (SEM, QUANTA 400F Field Emission SEM). Prior to analyses, the samples were attached to aluminum holders using a silver-based adhesive. Sample conductivity was achieved by sputter coating with a 3 nm Au-Pd layer. The unconfined compression analysis was performed using a Testometric analyzer series M350-5CT (Labor 7

Journal Pre-proof Machine s.r.o., Czech Republic). Before analyses, wet cylindrical-shaped samples (10 mm in diameter and 6 mm in thickness) were rehydrated in distilled water at 37 °C overnight. The samples were compressed at 70% between two impermeable parallel plates at a crosshead speed of 10 mm/min using a 100 N load cell. The compression modulus (stiffness) was determined from the uniaxial slope region of the compressive stress-strain curve within the range of 5–20% strain. Thermal properties of the hydrogels were investigated by thermogravimetric analysis (TGA) using the Mettler Toledo TGA-SDTA 851, under nitrogen atmosphere at 20 mL/min flow rate and a heating rate of 10 °C/min (within 25–700 °C temperature range; sample mass: 4.4–5.1 mg). The operating parameters were kept constant

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for all the samples in order to obtain comparable data. The hydrogels’ transitions were confirmed by differential scanning calorimetry (DSC) analysis (Mettler Toledo DSC 4000

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calorimeter) under nitrogen atmosphere in the temperature range of 25–300 °C, at a constant

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2.9. In vitro cytocompatibility studies

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rate of 10 °C/min.

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In vitro cytotoxicity test of the drug-loaded hydrogels was performed against human dermal fibroblast (HDF; ATCC PCS‐ 201–012) cell lines, measured using MTT assay. In brief, HDF cells were maintained in Dulbecco's modified Eagle's medium (DMEM), supplemented with

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10% fetal bovine serum (FBS) and 1% antibiotic solution, which was then cultured at 37 °C in a humidified incubator containing 5% CO2. Prior to the assay, cells were grown in 96-well

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plates (flat bottom) at a density of 1 × 104 cells per well with 100 µL of the medium. The hydrogels were sterile via immersion in 70% ethanol solution for 1 h and subsequently washed 3 times with sterile PBS. The sterilized hydrogels of average diameter 15 mm and thickness 3 mm were immersed in DMEM and swelled to an equilibrium state. In order to determine the viability of the HDF cells, 100 µL of the MTT solution (5 mg/mL) was diluted in serum-free DMEM medium, mixed and incubated at 37 °C for 4 h. After incubation, purple colored crystals were formed followed by dissolution in DMSO and the absorbance measured at 570 nm using as Elisa reader. All experiments were independently performed in triplicates and stored for 1, 3 and 5 days. The cell viabilities were then evaluated by MTT assay. 2.10. Antibacterial activity The antibacterial property of neomycin loaded hydrogels was tested via agar disc diff usion using spread plates and a viable cell count method against S. aureus (ATCC 29213) and E. 8

Journal Pre-proof coli (ATCC 25922) as the model bacteria was performed. A piece of each hydrogel of diameter 5 mm and a thickness of 3 mm was cut, washed and sterilized severally. Before the disc diff usion experiments, the bacteria were inoculated on tryptic soy broths (TSA) and incubated at 37 °C for 4 h. After incubation, the turbidity of the suspensions was controlled with a 0.5 McFarland standard (108 CFU/mL). Following incubation, several colonies were selected and suspended in TSA for the disc diff usion tests. Inoculation was performed using a cotton swab. The entire plate was covered by streaking back and forth from edge to edge. Swabbing was repeated 3 times while rotating the plate at 60°. The Circular disc hydrogel (control) and neomycin loaded hydrogels were placed on the bacteria lawn. After 30 min, the

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discs were placed on the inoculated plate while pressing each disc down firmly. When the incubation was completed at 37 °C for 24 h, zone diameters around the discs were measured.

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The maintenance inhibition ratio was calculated using Eq. (5). All tests were performed in

T ×100 C

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Maintenance inhibition ratio=

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triplicate and the average value recorded.

(5)

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Where, T is the inhibition zone diameter of the test sample and C the inhibition zone diameter

2.11. In vitro release studies

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of the control.

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The release of neomycin and diosgenin from the hydrogels was performed at two different pH of 7.4 and 5.5 for a period of 48 h as previously described [32]. In brief, dried 5 mg circular disc shape neomycin loaded hydrogels were immersed in 100 mL bottle vials containing 50 mL of 0.1M phosphate buff er solution of pH 5.5 and 7.4. The bottle vials were then tightly closed and placed in an agitating water bath of 37 °C at a shaking speed of 100 rpm. At varying time intervals, 0.25 mL of the volume extracts were collected and replaced with an equal volume of fresh phosphate buff er solution. The collected samples were analyzed using UV/vis spectrophotometry from the above obtained calibration curve and the release amount of neomycin calculated using Eq. (6). Readings were conducted in triplicate for each sample and the average value recorded. Release (%) =

Amount of drug released × 100 Total amount loaded

2.12. Statistical analysis 9

(6)

Journal Pre-proof All measurements were conducted in triplicates and the results reported as the mean ± standard deviation. Statistical analysis was carried out using OriginLab 9 software (OriginLab Northampton, MA, USA). P-values <0.05 were considered statistically significant.

3. Results and discussion 3.1. Hydrogel synthesis In order to synthesize DGN-NC, carboxylated NC (average particle size in the range of 50 – 250 nm determined using a Zetasizer, Malvern Instruments, UK) and the bioactive molecule

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diosgenin (obtained from natural plants) were used. The synthesis of DGN-NC occurred via the Steglich esterification method under mild and quantitative conditions between the

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carboxyl groups of NC and the hydroxyl groups of diosgenin under the aid of DCC as the

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coupling agent. DMAP was used to remove water produced during the esterification reaction [33]. The yield of the reaction produced 92% DGN-NC. The schematic representations of the

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different reactions are summarized in Fig. 1A and B. Genipin is well known to be highly reactive with primary amino groups of any given moiety [3]. Thus in this study, genipin

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reacted with the amino groups of gelatin while incorporating DGN-NC in the gelatin crosslinked network via physical entanglement to form a semi-interpenetrating hydrogel

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structure as presented in Fig. 1C [34, 35].

The prepared genipin crosslinked gelatin/DGN-NC produced blueish hydrogels of which the

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change in coloration was associated with the reaction of genipin to amino groups of gelatin [36]. In essence, the crosslinking reaction between gelatin and genipin can occur via two different pathways under mild acidic or neutral milieu which includes; firstly, a nucleophilic attack of the amino groups of gelatin on the olefinic carbon atom (C3) of genipin, followed by the opening of the carbonyl and dihydropyran ring. Secondly, a nucleophilic attack of the amino groups of gelatin to the carboxyl group of genipin leading to an amide formation. Further reactions involve oxygen radical-induced polymerization of genipin that might be generated amongst genipin molecules already crosslinked with amino groups of gelatin. This in essence may lead to genipin copolymers that possess high conjugation of C–C double bond, plus might possibly be responsible for the blueish color of the gel samples. According to literature [13], gels formed under acidic pH differ from those fabricated under neutral or basic pHs. The lower the pH value (acidic), the slower the crosslinking rate and vice versa. Thus,

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Journal Pre-proof following the results from pre-trial experiments, the crosslinking medium in the present

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preparation step was maintained at pH 5.5 for optimum genipin reactivity.

Fig. 1. Schematic illustration for the synthesis of (a) carboxylated nanocellulose, (b) diosgenin-conjugated nanocellulose and (c) formation of hydrogel system. 3.2. Gelation time and gel yield analysis As observed in Fig. 2A, a typical crosslinked system demonstrated a stable solid hydrogel system was formed as no flow of gel noticed when cuvette was tilted. Fig. 2B shows the maximum wavelength absorbance of genipin crosslinked gelatin/DGN-NC gelling systems. As observed, the maximum wavelength (λmax) color absorbance of the crosslinked systems was determined at 602 nm. The maximum blueish color intensity of hydrogel systems resulted from an irreversible crosslinking process as well as the oxygen radical‐ induced 11

Journal Pre-proof polymerization of genipin that caused the hydrogels to assume the blue color [37, 38]. According to Fig. 2C, the investigation of the gelling systems as a function of time depicted the optimum gelling time for all hydrogels to be t = 15 h. However, the absorbance intensity of hydrogel samples decreased with an increasing amount of DGN-NC. Gel-1 as the control showed the highest blueish color intensity, which was due to the absence of DGN-NC. A study by Damida et al. investigated the reaction kinetics of a genipin crosslinked chitosan hydrogel as a function of time and temperature. Results showed that increasing temperature from 25 to 50 ℃ significantly decreased the time of gelation [13]. Therefore, in the present study all prepared gelling systems were crosslinked at 40 ℃ to allow for optimum genipin

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crosslinking with gelatin, while incorporating DGN-NC to form stable gel systems. On the other hand, high gel yield percentages of 83.67±2.18%, 84.98±2.25%, 89.65±2.08%, and

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90.17±3.51% were determined for the prepared hydrogel systems Gel-1, Gel-2, Gel-3, and

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Gel-4 samples, respectively, as shown in Fig. 2D. The increase in gel yield from Gel-1 to Gel4 was associated with the addition of DGN-NC, which significantly incorporated hydrophobic

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property in the hydrogel structure, thereby decreasing the leaching or increasing the

3.3. Swelling studies

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insolubility of gelatin polymer chains and to a lesser extent genipin.

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Swelling in hydrogel systems occurs when water molecules are gradually absorbed into its network structure. Generally, this process occurs by hydrogen bond formation of water

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molecules with hydrophilic groups in the polymer chains that orientates bound water to form cage-like structures or clusters, allowing excess water to enter freely into the gel network, thus results in swelling [15]. In this case, the crosslinked hydrogel systems were allowed to swell in 25 mL PBS solutions of pH in the range 4.5 to 9.5 solution at 37 ℃ (Fig. 2E). All hydrogels revealed good water uptake capacity and reached equilibrium swelling within 24 h. The genipin-crosslinked gelatin system (Gel-1) without DGN-NC swelled the highest with maximum swelling at acidic conditions (pH 4.5). This was due to the protonation of free primary amino groups (–NH3+) on gelatin backbone. Compared to the other hydrogel systems, the swelling percentage was in the magnitude of Gel-1>Gel-2>Gel-3>Gel-4. The decrease in swelling percentage was attributed to the incorporation of DGN-NC, which increased the hydrophobic property of the gel systems as the amount of DGN-NC increased, thereby affecting the hydrophilic property that in tend reduced the swelling degree of hydrogels [39]. Thus, the swelling of the hydrogels was found to be inversely proportional to DGN-NC concentration. On the other hand, this in tend increased the mechanical stability of the 12

Journal Pre-proof hydrogel systems where the more DGN-NC incorporated the more mechanically stable the crosslinked network of the hydrogels. Considerably, this indicated the feasibility of our gelatin/DGN-NC hydrogel samples to be suited as drug delivery patches, given the water inside the gel samples was high enough to act as a natural penetration enhancer by simple hydration. This phenomenon is believed to ease the diffusion of hydrophilic drug molecules to

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the target or application site.

Fig. 2. (a) Images showing the change in color associated with crosslinked formed hydrogels. (b) UV absorbance of crosslinked gels at t = 15 h, (c) gelation rate of prepared hydrogels, (d) gel yield and (e) equilibrium swelling of prepared gelatin/DGN-NC hydrogels.

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Journal Pre-proof 3.4. FTIR and 1HNMR analysis The representative FTIR spectra of the pure DGN, the carboxylated NC, and the DGNconjugated NC (depicted in Fig. 3A) were analyzed in order to elucidate their structure. The characteristic peaks of DGN and carboxylated NC were observed in the spectra range of 3650 to 3080 cm−1 (broad-OH stretching band), 3010 to 2770 cm−1 (aliphatic C-H stretching bands), 1150 to 1057 cm−1 (skeletal vibrations of C-O and C-C stretching, C-O-C bridge stretch). Meanwhile, a new absorption peak highlighted at 1720 cm−1 (C=O) in the spectrum of DGN-NC was assigned to the carbonyl group of the ester linkage. This confirmed the

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esterification between carboxylated NC and DGN. The peaks observed in pure diosgenin were also noticed in DGN-NC confirming that DGN group was successfully conjugated to the

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backbone of NC. A peak at 1630 cm−1 was also observed in the spectrum of DGN-NC, which is probably due to absorbed water on cellulose surface [18]. Further investigation of the

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functional groups on the hydrogels and the interaction of the model drug neomycin with the hydrogels, neomycin and neomycin-loaded hydrogels were characterized by FTIR analysis.

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The characteristic bands at 1640 cm−1, 1540 cm−1 as well as 1446 cm−1 corresponded to the

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amide (C=O stretching), amino group (N–H stretching) and C-N stretching of the amide in the gelatin, respectively [40]. In the dried hydrogel spectrum, the peak assignment was as

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follows: 3730 cm−1 and 3000 cm−1 (the stretching of the -OH groups, 2920 and 2950 cm−1 (CH- stretching) and 1300-1000 cm−1 (C-O stretch peak of alcohol groups). The FTIR

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spectrum of neomycin was also included for comparison. Fig. 3B shows the interaction of neomycin with the hydrogels leading to the shifting of some peaks to lower wavelength while overlapping with related hydrogels’ band. A new peak was observed at about 1050 cm−1, which can be assigned to the ether frequencies of neomycin. The changes in the absorption intensity displayed that hydrogen bonds may have form between -OH and -NH2 groups in neomycin and the hydrogels [41, 42]. Fig. 3C illustrates the 1HNMR spectra of DGN, carboxylated NC and DGN-NC. The synthesis of DGN-NC was confirmed by comparing its 1HNMR with that of DGN and carboxylated NC. The characteristic peaks were observed as follows: at 1.7–4.1 ppm corresponding to carboxylated NC, at 3.36 and 3.47 ppm relating to m, 2H and Hd, 3.52 ppm attributed to m, 1H, and Hc, at 4.38 ppm ascribed to s, 1H and Hb, and at 5.32 ppm relating to s, 1H, and Ha for DGN [43, 44]. In the DGN-NC spectrum, contributions from both DGN and carboxylated NC were visible, which allow for possible determination of DGN parts based on the intense peaks related to them. The signals from the protons of methine groups on a 14

Journal Pre-proof cellulose backbone, vinylic proton (a) and methine proton (b) of the DGN were still present after the reaction. The structure of DGN-NC was also confirmed by the proton shift denoted as (c) and the appearance of the new peaks at around 2.1 ppm. In the DGN spectrum, the Hc proton was first observed at 3.47 ppm and then shifted to a downfield of 4.95 ppm upon esterification, which was attributed to the esterified nature of the hydroxyl group [45]. This evidence also implied that DGN had been successfully conjugated onto carboxylated NC via an ester linkage. The degree of substitution (DS) of DGN-NC is defined as the average number of diosgenyl groups ester-linked to hexuronic acid residues. The characteristic peaks observed at 0.74-1 ppm (CH3 protons) and at 5.35 ppm (vinyl proton) for DGN as well as at

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2.60 ppm (H2 glucose proton) and 4.34 ppm (the anomeric proton signal) for NC were employed to calculate the degree of substitution (DS) [18, 46]. The DS value of DGN-NC was

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0.08, indicating that, there were approximately 8 units of diosgenin esterified to every 100

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hexuronic acid residues.

Fig. 3. FTIR spectra of (a) DGN, carboxylated NC and DGN-NC, (b) hydrogels and (c) NMR spectra of DGN-NC.

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Journal Pre-proof 3.5. Thermal behavior TGA and DSC were used to evaluate thermal stability and to determine the decomposition temperature of the gels. The analysis results are summarized in Table 2. As can be seen in Fig. 4A, two-stage weight loss was recorded for the hydrogels. The first thermal degradation appeared between 50 and 160 °C, which was due to the loss of moisture and bonded water [47]. The second weight loss occurred between 300 °C and 430 °C which was attributed to the thermal degradation of cellulose and DGN. This is associated with the results of the previously reported study [48]. The amount of the first loss was in the range of 4 and 6%.

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However, a major loss was achieved in the second stage determined to be about 60%. The temperature related to the maximum weight loss was in the magnitude as follows; Gel-1 (355 C)
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for the maximum decomposition temperatures of the gels in the first and second stages when

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DGN-NC content was higher. A residue of ca. 20 wt% remained after heating Gel-1 to 800

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°C. Upon introduction of DGN-NC into the hydrogels, a residue of ca. 9-15 wt% remained after heating the gels (Gel-2, Gel-3, and Gel-4) to 800 °C. This is in agreement with data

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reported in similar previous studies [22, 49], indicating the significance of incorporated hydrophobic groups in hydrogels’ structure. The decrease in thermal stability of the gels

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based on DGN-NC (Gel-2, Gel-3, and Gel-4) compared to the control gel (Gel-1) was attributed to the nature of DGN such as molecular structure, hydrogen bonds interaction and

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degree of substitution. The results showed that the hydrogels were suitable for practical uses due to their thermal stability of up to 300 °C with a 50% weight loss above 400 °C [50]. DSC curves of the hydrogels are depicted in Fig. 4B. The peak at around 90 oC was related to the denaturation temperature (Td) (helix-to-coil transition) of the gelatin [51]. The Td values of the conjugated gels shifted towards higher temperatures compared to the value of the control gel as seen in Fig. 4B. As mentioned in a similar previous study [52], the improved in Td values are important for the applications of hydrogels as biomaterials where they may be exposed to heat in the preparation or consumption process. The melting transition temperature (Tm) of the present hydrogels and the associated enthalpies of the endothermic phase transitions are presented in Table 2 [53]. One endothermic phase transition was observed for the control gel (Gel-1) at 221.4 oC whereas three endothermic phase transitions were observed for the DGN-NC based gels (Gel-2, Gel-3, and Gel-4). The first appeared in the range of 120126 oC, the second in the range of 210-212 oC, and the third in the range of 223-225 oC. The second endothermic peak corresponded to the melting temperature of DGN observed for Gel16

Journal Pre-proof 2, 3, and 4 [54]. This was also confirmed from the Hm2 values increasing when DGN content in the hydrogels increased.

Table 2 The denaturation temperature (Td), melting phase transition (Tm) and associated enthalpies (∆H) of the hydrogels.

Gel-1 Gel-2 Gel-3 Gel-4

Tmax (oC) 362.8 368.2 380.6 384.6

Residue at Td* 700°C (%) (oC)

20.11 15.99 9.83 9.13

89.82 90.17 123.2 125.8

∆Hd* (j/g) 110.7 73.36 1.37 3.62

Tm1* (oC) 221.4 224.2 222.9 223.2

∆Hm1* (j/g) 8.78 4.19 1.80 0.75

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Samples

Tm2* (oC) 210.7 211.2 211.8

∆Hm2* (j/g) 2.60 13.34 21.54

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Tmax, the temperature related to the maximum weight loss determined by TGA. Td, the denaturation temperature of gelatin. Tm1, the melting temperature of the hydrogels. Tm2, the melting temperature of the diosgenin. * determined by DSC.

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3.6. Mechanical properties

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Fig. 4C shows typical compression stress-strain curves of the hydrogels compressed to a maximum of 70% strain of their original heights. The compression modulus of the hydrogels

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was obtained as 3.04±0.15 kPa, 3.85±0.36 kPa, 5.15±0.15 kPa and 8.04±0.31 kPa for Gel-1, Gel-2, Gel-3 and, Gel-4, respectively. While the ultimate compressive stress at 70%

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compression was determined as 14.06±1.38 kPa, 24.09±1.08 kPa, 64.27±2.01 kPa and 184.78±1.84 kPa for Gel-1, Gel-3, and Gel-4, respectively. According to deduced results, the gradual incorporation of DGN-NC significantly increased the mechanical integrity of the hydrogels. This increase in compressive modulus and strength of hydrogels with increasing amount of DGN-NC, ascertains the relevance of the materials to be suitable for the desired application in wound healing.

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Fig. 4. (a) TGA thermograms, (b) DSC curves and (c) typical compressive stress-strain curves and modulus of Gel-1, Gel-2, Gel-3 and Gel-4 hydrogels.

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Journal Pre-proof 3.7. Morphological properties The morphological properties of the freeze-dried hydrogels were evaluated using SEM micrographs and EDX analysis, as given in Fig. 5. The surface of Gel-1 appeared to be rough and uneven [53]. However, the hydrogels with different DGN-NC ratio had porous morphology with interconnected porosity [21]. The pore interconnections and the size of the pores depended on the ratio of DGN-NC. It was observed that as the ratio of DGN-NC in the hydrogel increased, the size of the pores slightly decreased. Similar results based on the pore size have been obtained in previous studies [55, 56]. Three-dimensional porous structures

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were observed with the pore size ranging from 10 to 30 m. This type of pore structure may provide a platform for cell adhesion and proliferation [57]. Furthermore, EDX (energy-

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dispersive X-ray) analysis was conducted for carbon, oxygen, and nitrogen. The percentages of the detected element on the surface of the hydrogels were calculated and given in Fig. 5.

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The decreased percentage of nitrogen with increasing DGN-NC ratio in hydrogels was

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assigned to the decrease of gelatin ratio in the hydrogels due to leaching.

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3.8. Loading efficiency of neomycin in the hydrogels

Loading efficiency (LE) is an important parameter to evaluate drug content in hydrogels. LE

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for neomycin was calculated as 80.1%, 87.6%, 91%, and 95.5% for Gel-1, Gel-2, Gel-3 and Gel-4, respectively. The high LE obtained for loaded hydrogels was due to the hydrogen bond

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interaction between the polar groups of hydrogels and neomycin [32]. It could also implied that the three-dimensional network structure of the hydrogels provided a barrier causing neomycin molecules to be trapped. However, the hydrophilic parts of the gel structure enhanced the solubility of neomycin within the lipophilic matrix and provided the entrapment of the drug in the hydrogel [58].

3.9. In vitro antibacterial studies of drug loaded hydrogels The in vitro antibacterial study was tested to evaluate the efficiency of the drug-loaded hydrogels for expected antibacterial effects. Neomycin has a broad antibiotic spectrum and is active against a variety of Gram (+) and Gram (−) bacteria [59]. The inhibitory effect of neomycin loaded hydrogels was expressed in terms of inhibition zone diameter as shown in Fig. 6A and maintenance inhibition ratio as calculated in Table 3.

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Fig. 5. SEM micrographs with EDX analysis of (a) Gel-1, (b) Gel-2, (c) Gel-3, and (d) Gel-4. Scale bar indicates 20 and 100 μm, respectively. An illustration of the antibacterial mechanism is given in Fig. 6B. The action mechanism of the antibiotic drugs is not clear, but it is considered to be in this order; inactivating the enzymes, adhering to the cell wall, damaging, penetrating inside the cell, bounding to the DNA and killing the organisms [32, 60]. The drug-loaded hydrogels showed good antibacterial activity against Gram (+) and Gram (−) bacteria. Among the studied hydrogels, the DGN-NC based formulations were found to possess higher inhibition against both types of bacteria. The microbial zone inhibition of neomycin loaded hydrogels as compared to non20

Journal Pre-proof loaded can be related to the fact that drug combination increases the eff ect of the drugs in hydrogels (Fig. S1 and Table S1). This was due to the synergistic antibacterial effect of neomycin in combination with DGN, since previous studies have proven DGN to possess antibacterial capacity on bacterial surface, thus resulting in better membrane permeability [41, 61]. It can also be revealed that the increase of DGN-NC ratio in the hydrogels enhanced the antibacterial effect towards Gram (+) bacteria. The maintenance inhibition ratio against S.aureus was observed to be higher than that against E. coli. This was due to the diff erent cell membrane constituent and structure of bacteria. Gram (+) bacteria contain an outer

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peptidoglycan layer while Gram (−) bacteria contain an outer phospholipid membrane [32].

Fig. 6. (a) A visual demonstration of inhibition zones and (b) antibacterial action mechanism of the neomycin loaded hydrogels. 21

Journal Pre-proof Table 3 In vitro T50 release values and microbial zone inhibition of the loaded-hydrogels. Samples

Gel-1 Gel-2 Gel-3 Gel-4

T50 values (min) pH 5.5 pH 7.4 14 ± 0.5 12 ± 0.4 34 ± 0.8 27 ± 0.6 58 ± 0.1 56 ± 0.1 258 ± 0.1 195 ± 0.2

Diameter of inhibition zones (mm) S.aureus (G+) E.coli (G−) 24 ± 0.2 16 ± 0.2 26 ± 0.1 20 ± 0.2 27 ± 0.3 26 ± 0.1 27.3 ± 0.1 28 ± 0.2

Maintenance Inhibition ratio (%) S.aureus (G+) E.coli (G−) 75 50 82 63 84 81 85 88

3.10. In vitro cytocompatibility studies of drug loaded hydrogels

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The cytocompatibility of the hydrogels was evaluated using MTT assay with human dermal

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fibroblast (HDF) cells to further test relevance for potential applications as wound dressing substrates [2, 62]. Although the different formulation compositions the hydrogels were

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prepared, the cytocompatibility test was only studied for non-loaded and loaded Gel-4 containing since it demonstrated the best mechanical integrity and contained the highest ratio

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of diosgenin. The results in Fig. 7A illustrates the cell viability at regular time intervals (24,

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48, and 72 hours) of the non-loaded and loaded sample at a concentration of 0.1 mg/mL. After 24 h incubation, cell viability 88.3%, 80.1%, and 58% was observed for the non-loaded Gel-4,

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loaded gel Gel-4, and DGN, respectively. Following a longer incubation time of 48 and 72 hours, the cell viability of the hydrogels was obtained higher than 94%. This increase in the viability was attributed to cell multiplication in both the non-loaded and loaded samples

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indicating the hydrogel as safe for biomedical applications [63]. In addition, the high cytocompatibility of the hydrogels was contributed by the presence of diosgenin, a natural compound abundantly found in plants. Moreover, the choice of the polymers and genipin all from natural sources to a certain degree depict the hydrogels to be non-toxic towards normal human cells [64]. 3.11. In vitro release study of neomycin from hydrogels The release of in situ loaded neomycin from the hydrogels was investigated in simulated physiological solutions of pH 7.4 plasma fluid and pH 5.5 for the skin. The release studies were evaluated over a period of 12 h and the results are presented in Fig. 7B and C. Results revealed that the release of the drug from the hydrogels majorly depended on solution pH and the ratio of DGN-NC in the formulated hydrogels. However, the time for 50% release of the drug (T50) was also determined as a response parameter. As observed for two investigated 22

Journal Pre-proof pHs, a fast release of neomycin occurred in the first hour and then a sustained/stable release up to 12 h. The release percentage of neomycin from Gel-1 after 15 min was estimated at 60% and 40% in pH 5.5 and pH 7.4, respectively. The DGN-NC based hydrogels (Gel-2, Gel-3, and Gel-4) released percentage after 15 min was in the range of 30% to 23% and 25% to 18% for pH 5.5 and pH 7.4, respectively. The higher release obtained for the medium of pH 5.5 was attributed to protonation of the free amines (-NH3+) on the backbone of gelatin, which led to the fast swelling of the hydrogel network and promoting the leaching of the drug molecules into the release medium [65]. Thus, depicting a higher drug release behavior in acidic pH compared to the neutral pH. In essence, neomycin release was influenced by the pH of release

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media, which was in good agreement to a similar previous report [22]. Also, an increase in the ratio of DGN-NC in the hydrogels contributed significantly to the release rate of the

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hydrogels. By increasing the amount of DGN-NC, the release rate of the hydrogels was

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delayed for about 2-3 h compared to Gel-1 as control. This may be attributed to the gradual increase in the hydrophobic character of the hydrogels, incorporated via the modified NC with

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DGN. This in tend to decrease the swelling of the hydrogels decreases the release efficiency of the drug. Furthermore, the increase in DGN-NC may have also limited the absorption of

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neomycin during the drug loading process due to a low degree of swelling of the hydrogels. [50]. However, all investigated hydrogels showed high release percentages of approximately

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100% after 24 h, attributed to the high solubility of neomycin in water. The achieved results in the present study were in close agreement with the initial release rates of similarly prepared

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hydrogels from previous studies [22, 26]. Further analysis, estimated T50 values for the hydrogels calculated using the release curves and results are given in Table 3. The T50 values in pH 7.4 were higher than that of pH 5.5, indicating that the hydrogels were physically stable in the neutral medium, which was in accordance with the previous study [65] that reported the T50 values were shorter for acidic medium compared to that of neutral.

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4. Conclusions

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Fig. 7. (a) Time-dependent cell viability of hydrogels for HDF cells, (b) drug release profiles pH 7.4 and (c) drug release at pH 5.5.

In this study, we aimed to formulate biocompatible hydrogels with sustain release drug rates by limiting the fast initial release phase. Thus, diosgenin derived from natural plants was conjugated on the backbone carboxylated modified nanocellulose to form DGN-NC. Genipin crosslinked gelatin incorporated DGN-NC hydrogels were successfully synthesized via formation of an interpenetrating network structure. The optimum gelation time for the crosslinked hydrogel systems was determined to be t = 15 h. All prepared hydrogel samples showed good swelling abilities for which the swelling behaviors can be controlled through variation of DGN-NC concentrations. The chemical structures of the crosslinked gelatin/DGN-NC based hydrogels were assessed with FTIR and 1HNMR. SEM micrographs depicted three-dimensional porous structures with pore sizes ranging from 10 to 30 m. The thermal properties of the hydrogels were also evaluated by DSC and TGA techniques. Mechanical property analysis was performed via unconfined compression and the results depicted the hydrogel to possess good mechanical integrity with Gel-4 showing the highest compression strength. In vitro cytotoxicity studies showed that the hydrogels were safe and biocompatibility to human healthy dermal fibroblast cell lines. Cell viabilities of hydrogels were close to 100% after 3 days’ incubation. Neomycin loaded hydrogels showed good 24

Journal Pre-proof antibacterial activity against both Gram (−) and Gram (+) bacteria. The inhibitory effect of the drug-loaded DGN-NC containing hydrogels (Gel-2, Gel-3, and Gel-4) was significantly higher than that of the control (Gel-1). It was revealed that DGN enhanced inhibition via combination with the antibacterial properties of neomycin, which in combination showed high annihilation ability of bacterial organisms. Owing to high inhibition against bacterial penetration, the hydrogels could be applied to wounds to protect against infections and accelerate the healing process. The release studies showed a two-manner release profile attributed to a rapid release within the initial 15 min and then sustained release for 9-12 h. These results clearly suggest that the crosslinked gelatin/DGN-NC based hydrogels could be suitable as a potential polymeric antibiotic substrate in wound dressing applications.

Conflict of interest

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The authors declare no conflict of interest.

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Acknowledgements

The authors Asabuwa N. Fahanwi and Sevinc I. Erdagi wish to thank TUBITAK 2215 and

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2211 fellowship programs and Kocaeli University for the financial support (BAP) provided in

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completing this work. In addition, we extend our thanks to Assoc. Prof. Dr. Güralp Özkoç (Chemical Engineering Department, Kocaeli University) for allowing us to use the Mettler

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Toledo TGA-SDTA 851 and Mettler Toledo DSC 4000 instruments for thermogravimetric analysis and differential scanning calorimetry measurements. In addition, a great thanks to Dr.

References

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antibacterial activity study.

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Arda Acemi (Biology Department, Kocaeli University) for the aid in growth media for

[1] X. Shen, J.L. Shamshina, P. Berton, G. Gurau, R.D. Rogers, Hydrogels based on cellulose and chitin: fabrication, properties, and applications, Green Che. 18 (2016) 53-75. [2] N.S. Capanema, A.A. Mansur, A.C. de Jesus, S.M. Carvalho, L.C. de Oliveira, H.S. Mansur, Superabsorbent crosslinked carboxymethyl cellulose-PEG hydrogels for potential wound dressing applications, Int. J. Biol. Macromol. 106 (2018) 1218-1234. [3] M. Ubaid, G. Murtaza, Fabrication and characterization of genipin cross-linked chitosan/gelatin hydrogel for ph-sensitive, oral delivery of metformin with an application of response surface methodology, Int. J. Biol. Macromol. 114 (2018) 1174-1185. [4] S.C. Chen, Y.C. Wu, F.L. Mi, Y.H. Lin, L.C. Yu, H.W. Sung, A novel pH-sensitive hydrogel composed of N,O-carboxymethyl chitosan and alginate cross-linked by genipin for protein drug delivery, J Control Release 96 (2004) 285-300. [5] L.J. Del Valle, A. Díaz, J. Puiggali, Hydrogels for Biomedical Applications: Cellulose, Chitosan, and Protein/Peptide Derivatives, Gels 3 (2017) 27. [6] R. Narayanaswamy, V.P. Torchilin, Hydrogels and Their Applications in Targeted Drug Delivery, Molecules 24 (2019) 603. 25

Journal Pre-proof [7] J. Hoque, R.G. Prakash, K. Paramanandham, B.R. Shome, J. Haldar, Biocompatible injectable hydrogel with potent wound healing and antibacterial properties, Mol. Pharm. 14 (2017) 1218-1230. [8] F.A. Ngwabebhoh, U. Yildiz, Nature‐ derived fibrous nanomaterial toward biomedicine and environmental remediation: Today's state and future prospects, J. Appl. Polym. 136 (2019) 47878. [9] H. Du, W. Liu, M. Zhang, C. Si, X. Zhang, B. Li, Cellulose nanocrystals and cellulose nanofibrils based hydrogels for biomedical applications, Carbohydr Polym 209 (2019) 130144. [10] N. Saba, M. Jawaid, Recent advances in nanocellulose-based polymer nanocomposites, Cellulose-Reinforced Nanofibre Composites, first edition, Woodhead Publishing, 2017.

of

[11] R. Poonguzhali, S.K. Basha, V.S. Kumari, Synthesis and characterization of chitosanPVP-nanocellulose composites for in-vitro wound dressing application, Int. J. Biol. Macromol. 105 (2017) 111-120.

ro

[12] L. Fan, H. Yang, J. Yang, M. Peng, J. Hu, Preparation and characterization of chitosan/gelatin/PVA hydrogel for wound dressings, Carbohydr. Polym. 146 (2016) 427-434.

re

-p

[13] S. Dimida, C. Demitri, V.M. De Benedictis, F. Scalera, F. Gervaso, A. Sannino, Genipin‐ cross‐ linked chitosan‐ based hydrogels: Reaction kinetics and structure‐ related characteristics, J. Appl. Polym. 132 (2015) 42256.

lP

[14] W. Zhang, G.L. Ren, H. Xu, J.L. Zhang, H.D. Liu, S.S. Mu, X. Cai, T. Wu, Genipin cross-linked chitosan hydrogel for the controlled release of tetracycline with controlled release property, lower cytotoxicity, and long-term bioactivity, J. Polym. Res. 23 (2016) 9.

na

[15] J.R. Khurma, D.R. Rohindra, A.V.J.P.B. Nand, Swelling and Thermal Characteristics of Genipin Crosslinked Chitosan and Poly(vinyl pyrrolidone) Hydrogels, Polym. Bull. 54 (2005) 195-204.

Jo ur

[16] S.A.A. Ghavimi, E.S. Lungren, T.J. Faulkner, M.A. Josselet, Y. Wu, Y. Sun, F.M. Pfeiffer, C.L. Goldstein, C. Wan, B.D. Ulery, Inductive co-crosslinking of cellulose nanocrystal/chitosan hydrogels for the treatment of vertebral compression fractures, Int. J. Biol. Macromol. 130 (2019) 88-98. [17] T. Moses, K.K. Papadopoulou, A. Osbourn, Metabolic and functional diversity of saponins, biosynthetic intermediates and semi-synthetic derivatives, Crit. Rev. Biochem. Mol. Biol. 49 (2014) 439-462. [18] J.P. Quiñones, C.C. Mardare, A.W. Hassel, O. Brüggemann, Self-assembled cellulose particles for agrochemical applications, Eur. Polym. 93 (2017) 706-716. [19] S. Ghosh, P. More, A. Derle, R. Kitture, T. Kale, M. Gorain, A. Avasthi, P. Markad, G.C. Kundu, S. Kale, Diosgenin functionalized iron oxide nanoparticles as novel nanomaterial against breast cancer, J Nanosci Nanotechnol. 15 (2015) 9464-9472. [20] J.P. Quiñones, K.V. Gothelf, J. Kjems, Á.M.H. Caballero, C. Schmidt, C.P. Covas, N, O6-partially acetylated chitosan nanoparticles hydrophobically-modified for controlled release of steroids and vitamin E, Carbohydr. Polym. 91 (2013) 143-151. [21] C. Yang, H. Wu, J. Wang, Effect of steroidal saponins-loaded nanobioglass/phosphatidylserine/collagen bone substitute on bone healing, Biomed. Tech. 62 (2017) 487-491.

26

Journal Pre-proof [22] S. Jabeen, A. Islam, A. Ghaffar, N. Gull, A. Hameed, A. Bashir, T. Jamil, T. Hussain, Development of a novel pH sensitive silane crosslinked injectable hydrogel for controlled release of neomycin sulfate, Int. J. Biol. Macromol. 97 (2017) 218-227. [23] P. Sun, F. Yu, J. Lu, M. Zhang, H. Wang, D. Xu, L. Lu, In vivo effects of neomycin sulfate on non-specific immunity, oxidative damage and replication of cyprinid herpesvirus 2 in crucian carp (Carassius auratus gibelio), Aquaculture and Fisheries 4 (2019) 67-73. [24] J. Zheng, Y. Li, H. Guan, J. Zhang, H. Tan, Enhancement of neomycin production by engineering the entire biosynthetic gene cluster and feeding key precursors in Streptomyces fradiae CGMCC 4.576, Appl. Microbiol. Biotechnol. 103 (2019) 1-13. [25] A.H. Patel, R.M. Dave, Formulation and evaluation of sustained release in situ ophthalmic gel of neomycin sulphate, Bull. Pharm. Res. 5 (2015) 1-5.

of

[26] J.S. Choi, D.W. Kim, D.S. Kim, J.O. Kim, C.S. Yong, K.H. Cho, Y.S. Youn, S.G. Jin, H.-G. Choi, Novel neomycin sulfate-loaded hydrogel dressing with enhanced physical dressing properties and wound-curing effect, Drug Deliv. 23 (2016) 2806-2812.

-p

ro

[27] M. Cheng, Z. Qin, Y. Liu, Y. Qin, T. Li, L. Chen, M. Zhu, Efficient extraction of carboxylated spherical cellulose nanocrystals with narrow distribution through hydrolysis of lyocell fibers by using ammonium persulfate as an oxidant, J. Mat. Chem. A 2 (2014) 251258.

re

[28] A.C.W. Leung, S. Hrapovic, E. Lam, Y. Liu, K.B. Male, K.A. Mahmoud, J.H.T. Luong, Characteristics and Properties of Carboxylated Cellulose Nanocrystals Prepared from a Novel One-Step Procedure, Comm. 7 (2011) 302-305.

na

lP

[29] P. Sarika, N.R. James, P.A. Kumar, D.K. Raj, T. Kumary, Gum arabic-curcumin conjugate micelles with enhanced loading for curcumin delivery to hepatocarcinoma cells, Carbohydr. Polym. 134 (2015) 167-174.

Jo ur

[30] D. Pathania, D. Gupta, N. Kothiyal, G. Eldesoky, M. Naushad, Preparation of a novel chitosan-g-poly (acrylamide)/Zn nanocomposite hydrogel and its applications for controlled drug delivery of ofloxacin, Int. J. Biol. Macromol. 84 (2016) 340-348. [31] M. Jeżowska‐ Bojczuk, W. Szczepanik, S. Mangani, E. Gaggelli, N. Gaggelli, G. Valensin, Identification of copper (II) binding sites in the aminoglycosidic antibiotic neomycin B, Eur. J. Inorg. Chem. 15 (2005) 3063-3071. [32] F.A. Ngwabebhoh, S.I. Erdagi, U. Yildiz, Pickering emulsions stabilized nanocellulosicbased nanoparticles for coumarin and curcumin nanoencapsulations: In vitro release, anticancer and antimicrobial activities, Carbohydr. Polym. 201 (2018) 317-328. [33] L. Yang, B. Zhang, L. Wen, Q. Liang, L.-M. Zhang, Amphiphilic cholesteryl grafted sodium alginate derivative: Synthesis and self-assembly in aqueous solution, Carbohydr. Polym. 68 (2007) 218-225. [34] L. Cui, J. Jia, Y. Guo, Y. Liu, P. Zhu, Preparation and characterization of IPN hydrogels composed of chitosan and gelatin cross-linked by genipin, Carbohydr. Polym. 99 (2014) 3138. [35] H.-T. Lu, W.-T. Chang, M.-L. Tsai, C.-H. Chen, W.-Y. Chen, F.L. Mi, Development of injectable fucoidan and biological macromolecules hybrid hydrogels for intra-articular delivery of platelet-rich plasma, Mar. Drugs 17 (2019) 236. [36] D.M. Kirchmajer, C.A. Watson, M. Ranson, M.i.h. Panhuis, Gelapin, a degradable genipin cross-linked gelatin hydrogel, RSC Adv. 3 (2013) 1073-1081. 27

Journal Pre-proof [37] M.F. Butler, Y.-F. Ng, P.D.A. Pudney, Mechanism and kinetics of the crosslinking reaction between biopolymers containing primary amine groups and genipin, J. Polym. Sci. Pol. Chem. 41 (2003) 3941-3953. [38] G. Gorczyca, R. Tylingo, P. Szweda, E. Augustin, M. Sadowska, S. Milewski, Preparation and characterization of genipin cross-linked porous chitosan–collagen–gelatin scaffolds using chitosan CO2 solution, Carbohydr. Polym. 102 (2014) 901-911. [39] H. T. Lu, T.-W. Lu, C.-H. Chen, F.-L. Mi, Development of genipin-crosslinked and fucoidan-adsorbed nano-hydroxyapatite/hydroxypropyl chitosan composite scaffolds for bone tissue engineering, Int. J. Biol. Macromol. 128 (2019) 973-984. [40] W. Wan Ishak, I. Ahmad, S. Ramli, M. Mohd Amin, Gamma Irradiation-Assisted Synthesis of Cellulose Nanocrystal-Reinforced Gelatin Hydrogels, Nanomaterials 8 (2018) 749.

ro

of

[41] I.P. Merlusca, D.S. Matiut, G. Lisa, M. Silion, L. Gradinaru, S. Oprea, I.M. Popa, Preparation and characterization of chitosan–poly (vinyl alcohol)–neomycin sulfate films, Polym. Bull. 75 (2018) 3971-3986.

-p

[42] R. Preethika, R. Ramya, M. Ganesan, S. Nagaraj, K. Pandian, Synthesis and characterization of neomycin functionalized chitosan stabilized silver nanoparticles and study its antimicrobial activity, Nanosystems: Physics, Chemistry, Mathematics 7 (2016) 583-591.

re

[43] Y. Dong, K.J. Edgar, Imparting functional variety to cellulose ethers via olefin crossmetathesis, Polym. Chem. 6 (2015) 3816-3827.

lP

[44] C. Li, L. Dai, K. Liu, L. Deng, T. Pei, J. Lei, A self-assembled nanoparticle platform based on poly (ethylene glycol)–diosgenin conjugates for co-delivery of anticancer drugs, RSC Adv. 5 (2015) 74828-74834.

na

[45] A. Sethi, R.P. Singh, D. Shukla, P. Singh, Synthesis of novel pregnane-diosgenin prodrugs via Ring A and Ring A connection: A combined experimental and theoretical studies, J. Mol, Struct. 1125 (2016) 616-623.

Jo ur

[46] M. Bagheri, S. Shateri, Synthesis and characterization of novel liquid crystalline cholesteryl-modified hydroxypropyl cellulose derivatives, J. Polym. Res. 19 (2012) 9842. [47] Y.Y. Zhao, Z.T. Sun, Effects of gelatin-polyphenol and gelatin-genipin cross-linking on the structure of gelatin hydrogels, Int. J. Food Prop. 20 (2018) 2822-2832. [48] M. Bagheri, S. Shateri, Synthesis and characterization of novel liquid crystalline cholesteryl-modified hydroxypropyl cellulose derivatives, J. Polym. Res. 19(3) (2012) 13. [49] B.O. Jung, S.J. Chung, S.B. Lee, Preparation and characterization of eugenol‐ grafted chitosan hydrogels and their antioxidant activities, J. Appl. Polym. Sci. 99 (2006) 3500-3506. [50] A.T. Zafalon, V.J. dos Santos, F. Esposito, N. Lincopan, V. Rangari, A.B. Lugão, D.F. Parra, Synthesis of polymeric hydrogel loaded with antibiotic drug for wound healing applications, TMS Annual Meeting & Exhibition, Springer, (2018), 165-176. [51] A.L. Daniel-da-Silva, A.M. Salgueiro, T. Trindade, Effects of Au nanoparticles on thermoresponsive genipin-crosslinked gelatin hydrogels, Gold Bull. 46 (2013) 25-33. [52] Y. Zhao, Z. Sun, Effects of gelatin-polyphenol and gelatin–genipin cross-linking on the structure of gelatin hydrogels, Int. J. Food Prop. 20 (2017) 2822-2832.

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Journal Pre-proof [53] S. Parvez, M.M. Rahman, M.A. Khan, M.A.H. Khan, J.M. Islam, M. Ahmed, M.F. Rahman, B. Ahmed, Preparation and characterization of artificial skin using chitosan and gelatin composites for potential biomedical application, Polym. Bull. 69 (2012) 715-731. [54] C. Liu, J. Chang, L. Zhang, H. Xue, X. Liu, P. Liu, Q. Fu, Preparation and evaluation of diosgenin nanocrystals to improve oral bioavailability, AAPS PharmSciTech 18 (2017) 20672076. [55] W. Treesuppharat, P. Rojanapanthu, C. Siangsanoh, H. Manuspiya, S. Ummartyotin, Synthesis and characterization of bacterial cellulose and gelatin-based hydrogel composites for drug-delivery systems, Biotechnology reports 15 (2017) 84-91. [56] Y. Jiang, X. Xv, D. Liu, Z. Yang, Q. Zhang, H. Shi, G. Zhao, J. Zhou, Preparation of Cellulose Nanofiber-reinforced Gelatin Hydrogel and Optimization for 3D Printing Applications, BioResources 13 (2018) 5909-5924.

ro

of

[57] X. Zheng, Q. Zhang, J. Liu, Y. Pei, K. Tang, A unique high mechanical strength dialdehyde microfibrillated cellulose/gelatin composite hydrogel with a giant network structure, RSC Adv. 6 (2016) 71999-72007.

-p

[58] P. Sriamornsak, J. Nunthanid, K. Cheewatanakornkool, S. Manchun, Effect of drug loading method on drug content and drug release from calcium pectinate gel beads, AAPS Pharmscitech. 11 (2010) 1315-1319.

re

[59] B. Findlay, G.G. Zhanel, F. Schweizer, Neomycin–phenolic conjugates: Polycationic amphiphiles with broad-spectrum antibacterial activity, low hemolytic activity and weak serum protein binding, Bioorg. Med. Chem. 22 (2012) 1499-1503.

na

lP

[60] I. Muhamad, S. Asgharzadehahmadi, D. Zaidel, E. Supriyanto, Characterization and Evaluation of Antibacterial Properties of Polyacrylamide Based Hydrogel Containing Magnesium Oxide Nanoparticles, Int. J. Biol. Biomed. Eng 7 (2013) 108-113.

Jo ur

[61] H. Deng, D. McShan, Y. Zhang, S.S. Sinha, Z. Arslan, P.C. Ray, H. Yu, Mechanistic study of the synergistic antibacterial activity of combined silver nanoparticles and common antibiotics, Environ. Sci. Technol. 50 (2016) 8840-8848. [62] A. Basu, J. Lindh, E. Ålander, M. Strømme, N. Ferraz, On the use of ion-crosslinked nanocellulose hydrogels for wound healing solutions: Physicochemical properties and application-oriented biocompatibility studies, Carbohydr. Polym. 174 (2017) 299-308. [63] R. Rakhshaei, H. Namazi, A potential bioactive wound dressing based on carboxymethyl cellulose/ZnO impregnated MCM-41 nanocomposite hydrogel, Mater. Sci. Eng. C 73 (2017) 456-464. [64] Y. Yuan, B. Chesnutt, G. Utturkar, W. Haggard, Y. Yang, J. Ong, J. Bumgardner, The effect of cross-linking of chitosan microspheres with genipin on protein release, Carbohydr. Polym. 68(3) (2007) 561-567. [65] A. Singh, A.K. Kureel, P. Dutta, S. Kumar, A.K. Rai, Curcumin loaded chitin-glucan quercetin conjugate: synthesis, characterization, antioxidant, in vitro release study, and anticancer activity, Int. J. Biol. Macromol. 110 (2018) 234-244.

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Journal Pre-proof AUTHORSHIP STATEMENT

Manuscript title: Genipin crosslinked gelatin-diosgenin-nanocellulose hydrogels for potential wound dressing and healing applications All persons who meet authorship criteria are listed as authors, and all authors certify that they have participated sufficiently in the work to take public responsibility for the content, including participation in the concept, design, analysis, writing, or revision of the manuscript.

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Furthermore, each author certifies that this material or similar material has not been and will

International Journal of Biological Macromolecules.

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Authorship contributions

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not be submitted to or published in any other publication before its appearance in the

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Category 1

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Conception and design of study: Ilkar Erdagi, Ngwabebhoh, Yildiz;

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Acquisition of data: Ilkar Erdagi, Ngwabebhoh;

Category 2

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Analysis and/or interpretation of data: Ilkar Erdagi, Ngwabebhoh, Yildiz.

Drafting the manuscript: Ilkar Erdagi, Ngwabebhoh, Yildiz; Revising the manuscript critically for important intellectual content: Ilkar Erdagi, Ngwabebhoh; Category 3 Approval of the version of the manuscript to be published (the names of all authors must be listed): Sevinc Ilkar Erdagi, Fahanwi Asabuwa Ngwabebhoh, Ufuk Yildiz.

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Graphical abstract

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Research Highlights  Genipin crosslinked gelatin/diosgenin-nanocellulose hydrogels were developed  The hydrogels exhibited suitable moist environment based on water retention ability  Drug loading and release efficiency of hydrogels was investigated  The hydrogels showed excellent antibacterial effect towards Gram + and − bacteria.

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 Results depict hydrogels as suitable antibacterial drug carriers for usage in wound healing

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