Manuscript Details
Manuscript number MOLLIQ_2020_1339_R1
Title Silver Chloride Nanoparticles Embedded in Self-Healing Hydrogels with Biocompatible and Antibacterial Properties
Article type Full length article Abstract
The incorporation of inorganic materials into soft matter has drawn considerable attention of researchers particularly for the development of hydrogels with self-healing ability in biomedical applications. In our study, the self-healing hydrogels formed by cross-linking reaction between poly(acrylic) acid and aluminium (Al3+) ions incorporated with gelatin-stabilized silver chloride nanoparticles (AgCl NPs) were successfully fabricated in a single step. The self- healing process of such hydrogels was facilitated by dynamic ionic interaction that occurred between –COOH from PAA and Al3+ ions. The effect by introducing gelatin and varying the concentration of silver in the PAA-Al3+ hydrogel were carried out. With the addition of gelatin, the hydrogel exhibited rather poor mechanical properties than the free- gelatin hydrogel. The evidence of gelatin in stabilizing AgCl NPs in the hydrogel was proved by SEM analysis and the crystallinity of AgCl NPs in the hydrogels was confimed by XRD analysis as well. Furthermore, the antibacterial efficacy on escherichia coli increased with increasing concentration of silver in the hydrogel. In addition, the results of cell viability (L929 mouse fibroblast cells) determined by LDH method revealed that gelatin in hydrogel could enhance the proliferation of cells. Nonetheless, the overuse of silver in the hydrogel led to toxicity of cells.
Keywords Self-healing; Hydrogel; Biocompatible; Antibacterial Manuscript category Simple organic liquids and mixtures
Corresponding Author Mimpin Ginting Corresponding Author's
Institution
Department of Chemistry
Order of Authors Subur Pasaribu, Mimpin Ginting, Indra Masmur, Jamaran Kaban, Hestina Hestina
Suggested reviewers Alfin Kurniawan, Timbangen Sembiring, Suryadi Ismadji
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March 7th, 2020 Schroer, W. Ph.D.
Editor-in-chief, Journal of Molecular Liquids University of Bremen, Germany
Manuscript Title: Silver Chloride Nanoparticles Embedded in Self-Healing Hydrogels with Biocompatible and Antibacterial Properties
Manuscript type: Research paper Dear Professor Schroer,
Enclosed please find a copy of the manuscript entitled “Silver Chloride
Nanoparticles Embedded in Self-Healing Hydrogels with Biocompatible and Antibacterial Properties”. In this study, we demonstrated the fabrication of polyacrylic acid-Al3+ self-healing hydrogels incorporated with gelatin-stabilized silver chloride nanoparticles (AgCl NPs). In addition, we also studied the effect by varying silver concentration. Scanning electron microscopy (SEM) analysis were employed to study the surface morphology of the resultant hydrogels. To the best of our knowledge, this is the first study demonstrating the fabrication of such biocompatible hydrogel with metal nanoparticles for antibacterial application in one step preparation. The obtained hydrogels demonstrated satisfying mechanical properties and self-healing ability as well. Moreover, the antibacterial activity of the prepared hydrogels was investigated toward
Escherichia coli via disk diffusion method and measurement of absorbance of OD600 value. To this end, the hydrogels also possessed good biocompatibility indicated by increasing cell viability tested on L929 mouse fibroblast cells.
The prepared manuscript was not previously submitted or rejected. Moreover, this manuscript was not previously published by any of the authors and the content is not under consideration for publication in another journal at the time of submission. We hope that this original full paper fits the journal scope and its standard quality to be considered for publication in Journal of Molecular Liquids. I am looking forward to hearing from you.
Sincerely yours,
Dr. Mimpin Ginting Associate Professor Department of Chemistry
Faculty of Mathematics and Natural Sciences University of Sumatera Utara
Email: [email protected]
Dear Prof. Wolffram Schroer,
We would like to thank the editor for very careful review and for the constructive suggestions on our manuscript. We also appreciate the time and efforts by the editor and reviewers in reviewing this manuscript. We have adopted the suggestions and the appropriate changes have been introduced to the main text of the revised manuscript (highlighted in yellow). Moreover, we also added more information in each analyses (including their figures) in the results and discussion part. Therefore, hopefully you find our responses satisfactory and that the revised manuscript is now completely acceptable for publication in Journal of Molecular Liquids.
We look forward to receive further communications.
Sincerely, Mimpin Ginting
We would like to thank the reviewer for careful and thorough reading of this manuscript and for the thoughtful comments and suggestions. Comments and Responses to your comments are given below.
Response to Reviewer #1:
This manuscript is well prepare and can be accepted for publication in Journal of Molecular Liquids, however, prior to its acceptance, the authors need to provide sufficient responses to my following comments:
1. Line 201: how long the self healing process of hydrogel took place?
Response: Thanks for the thoughtful comments and suggestions. The self-healing process occurred in 12 h at 37C. Further, we have added the information regarding the time needed for the self-healing of hydrogel in Line 201 in the revised manuscript.
2. The mechanism of self healing process should be given in the manuscript (Schematic diagram) Response: Thanks for the suggestion. As in the revised manuscript, we have provided the self- healing mechanism of the hydrogel as shown in Fig. 2a.
3. The indexing of the wavenumber should be shown in Figure 3.
Response: Thanks for the comment. We have modified Fig. 3 by indicating the wavenumber of the corresponding peak.
Response to Reviewer #2
The manuscript reported by Pasaribu et al. demonstrates the fabrication of healable, biocompatible, and anti-bacterial PAA-gelatin hydrogels embedded with AgCl particles. The experiments are well designed along with the characterization of the hydrogel materials. However, some of the results in this study are poorly discussed and the formation and self-healing mechanisms of the hydrogels lack detailed explanation. This manuscript can be accepted for publication in Journal of Molecular Liquids after the authors do some modifications and properly address all the following comments:
1. In my opinion, the effect of AlCl3 concentration is more important to be studied instead of AgNO3 since this parameter will affect the cross-link density and the corresponding mechanical and self-healing properties of the hydrogels. From Figure 5, it is obvious that the variation of AgCl content in the hydrogels shows minor or no effect on the mechanical and self-healing properties.
Response: Thanks for the critical suggestion. We have conducted more experiment to study the effect of AlCl3 concentration particularly on their cross-link density and mechanical and self-
healing properties of the fabricated hydrogel samples. The concentrations of AlCl3 were varied from 0.1 – 0.9 mol% of AA. The cross-link density was determined using gel fraction determination method while the mechanical and self-healing properties was examined by means of tensile test. The results of swelling degree, gel fraction, mechanical properties and self-healing efficiency including their explanations were described in detail in the revised manuscript.
2. More detailed discussion of the FTIR characterization results should be given. For example, the strong vibrational peak of the acrylic acid monomer at around 1400 cm-1 disappears in all hydrogel samples. In addition, please also highlight the peaks in the IR spectra that involve in the polymerization of AA.
Response: Thanks for the constructional advice. In the revised manuscript, we have modified the discussion of FTIR analysis and highlighted the wavenumber of the peaks in Fig. 3 as well.
3. From the XRD pattern, the broad Bragg reflection at 2-theta of ~21° seems to be shifted for hydrogel samples containing higher silver content. What contributes to this reflection shift?
Response: Thanks for the comment. In our opinion, the peak shifting probably due to the technical issue. For example, the position of samples on the stage somehow might be a little bit higher or lower when doing the XRD analysis, therefore might affect and cause shifted peaks.
4. Please provide the crystallite size information of AgCl particles embedded in each hydrogel sample
Response: Thanks for the insightful comment. The crystal size of AgCl embedded in hydrogel was obtained from XRD data by Scherrer equation calculated using Diffrac.Eva V.4.3.0.2 software. The AgCl crystal size slightly increased from 29.4 to 35.4 nm with increasing silver concentration. However, the hydrogel with the absence of gelatin (Gel0/Ag0.3) exhibited bigger AgCl nanoparticles which was found to be 46.4 nm.
5. There is no statistical analysis for the quantitative antibacterial activity (Figure 8c) and the cell culture results (Figure 9)
Response: Thanks for the suggestion. In the revised manuscript, we have provided the statistical analysis for the results of antibacterial activity and cell viability using One-way analysis of variance (ANOVA) by Origin 9.0 software. The differences with a p-value of 0.05 or less were assumed statistically significant.
6. In the cell culture results, the authors observed that Gel10/Ag0.4 and Gel10/Ag0.5 samples induce cytotoxicity towards L-929 cells. How much the concentration of AgCl particles loaded in these hydrogel samples?
Response: Thanks for drawing our attention to this study. To address this issue, we have performed ICP-AES analysis to quantitatively determine AgCl NPs loaded in the hydrogels. In brief, we found that the loading of silver in the hydrogel was 42.5 and 50.1 µg/L for Gel10/Ag0.4
and Gel10/Ag0.5, respectively and the result of ICP analysis was shown in Figure 8 as in the revised manuscript.
Highlights
PAA-Al3+ self-healing hydrogels formed by physical cross-linking (ionic interaction) between carboxylic groups and Al3+ ions
The hydrogels network of PAA-Al3+ is interpenetrated by AgCl-stabilized gelatin
Relatively high amount of AgCl nanoparticles causes toxic to L929 mouse fibroblast cell
1
Silver Chloride Nanoparticles Embedded in Self-Healing Hydrogels with
2
Biocompatible and Antibacterial Properties
3 Subur P. Pasaribu1, Mimpin Ginting2*, Indra Masmur2, Jamaran Kaban2 and Hestina3
4 1Department of Chemistry, Faculty of Mathematics and Natural Sciences, Mulawarman University, 5 Samarinda-75123, Indonesia.
6 2Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Sumatera 7 Utara, Medan-20155, Indonesia.
8 3Department of Chemistry, Universitas Sari Mutiara Indonesia, Medan-20123, Indonesia.
9 *Corresponding Author: Mimpin Ginting, Department of Chemistry, Faculty of Mathematics and 10 Natural Sciences, Universitas Sumatera Utara, Medan-20155, Indonesia.
11 Phone number: +628126528852 12 e-mail: [email protected]
14 Abstract
15 The incorporation of inorganic materials into soft matter has drawn considerable attention of 16 researchers particularly for the development of hydrogels with self-healing ability in biomedical 17 applications. In our study, the self-healing hydrogels formed by cross-linking reaction between 18 poly(acrylic) acid and aluminium (Al3+) ions incorporated with gelatin-stabilized silver chloride 19 nanoparticles (AgCl NPs) were successfully fabricated in a single step. The self-healing process 20 of such hydrogels was facilitated by dynamic ionic interaction that occurred between –COOH from 21 PAA and Al3+ ions. The effect by introducing gelatin and varying the concentration of silver in the 22 PAA-Al3+ hydrogel was carried out. With the addition of gelatin, the hydrogel exhibited rather 23 poor mechanical properties than the free-gelatin hydrogel. The evidence of gelatin in stabilizing 24 AgCl NPs in the hydrogel was proved by SEM analysis and the crystallinity of AgCl NPs in the 25 hydrogels was confimed by XRD analysis as well. Furthermore, the antibacterial efficacy on 26 Escherichia coli increased with increasing concentration of silver in the hydrogel. In addition, the 27 results of cell viability (L929 mouse fibroblast cells) determined by LDH method revealed that 28 gelatin in hydrogel could enhance the proliferation of cells. Nonetheless, the overuse of silver in 29 the hydrogel led to toxicity of cells.
30
31 Keywords: Self-healing; Hydrogel; Biocompatible; Antibacterial
33 1. Introduction
34 Extracellular matrix is a complex macromolecular network that acts as 3D physical scaffold 35 where biological cell resides. As known, the three-dimensional materials are more 36 preferable for cell growth compared to 2D materials[1]. In the past few years, self-healing 37 hydrogels have gained significant interest mainly due to their unique properties which 38 resemble the native extracellular matrix (ECM) of human as well as repairing or restoring 39 themselves autonomously when subjected to damage. The self-healing hydrogel can be 40 prepared either by dynamic covalent bonds (e.g. boronate ester bond, coordination bond, 41 Diels-Alder bond, etc.) or non-covalent interactions (electrostatic interaction, hydrogen 42 bond, hydrophobic interaction, etc.)[2]. In general, the self-healing phenomena of both 43 ways of preparation was due to the dynamic equilibrium between dissociation and 44 recombination.
45 Thereupon, such hydrogels are widely used for biomedical related fields such as tissue 46 engineering. On the other hand, the physiochemical and mechanical properties of hydrogel 47 can be adjusted for the desired applications such as polymer, initiator, and cross-linker 48 selection and controlling the cross-linking density[3, 4].
49 Several types of silver particularly silver halide (e.g. silver chloride, silver bromide, etc.) 50 possess superior antibacterial properties. Silver works on bacterial cells by binding and 51 damaging their DNA replication which also affect the metabolism pathway of bacterial 52 cells[5, 6]. Among all silver type materials, silver chloride (AgCl) is more preferable due 53 to its properties such as the slow release of Ag+ and the ease of its preparation as well. As 54 reported elsewhere, many methods have been developed to fabricate AgCl[7-9]. However, 55 the simplest way to obtain AgCl is by introducing chloride (Cl-) ions together with Ag in
56 which lead the Ag instantly precipitate in its chloride form. In spite of the modest 57 preparation, there is still remaining issue regarding the agglomeration of AgCl particulate 58 during the synthesis. In addition to address the agglomeration issue of AgCl NPs, gelatin 59 was employed. It is because gelatin can stabilizes silver particulate through the reaction 60 between its amine (-NH2) functional groups and Ag[10].
61 Incorporation of metal nanoparticles into hydrogel has brought hydrogels to broader scope.
62 The so-called hydrogels-metal nanoparticles composite provides more advantages such as 63 the antibacterial properties of silver metal, the magnetic properties of Fe3O4, etc. For 64 instance, Zhang et. al. (2012) has demonstrated hydrogels incorporated with Fe3O4 which 65 are self-healable and possess magnetic properties[11]. Recently, Nesovic (2019) 66 synthesized chitosan-based hydrogel incorporated with silver nanoparticles for wound 67 dressings[12].
68 On the whole, the aforesaid challenges have motivated us to solve such issues by creating 69 a new material through a simple method. Hence, in this study, we synthesized the self- 70 healing hydrogel incorporated with AgCl NPs in a simple single step preparation. The 71 networks of hydrogel were mainly composed of polymer chain of acrylic acid (PAA) to 72 cross-link with Al3+ through ionic interaction. Acrylic acid was chosen because its polymer 73 exhibited superior mechanical properties while Al3+ was utilized due to the low cytotoxicity 74 to mamalian cells[13, 14]. The resulted hydrogel demonstrated good antibacterial 75 properties toward Escherichia coli as well as enhanced the L929 mouse fibroblast cells 76 proliferation. To the best of our knowledge, the antibacterial and non-toxic PAA-Al3+ self- 77 healing hydrogel incorporated with gelatin-stabilized AgCl NPs has not been reported 78 before.
79
80 2. Materials and methods 81 2.1. Chemicals
82 Acrylic acid (CH2CHCOOH, 99.1%, Echo Chemical), Aluminium (III) chloride anhydrous 83 (AlCl3, 99%, Sigma-Aldrich), gelatin (9000-70-8, Showa Chemical), silver nitrate 84 (AgNO3, 99.8%, Aencore Chemical), and ammonium persulfate ((NH4)2S2O8, 98%, 85 Sigma-Aldrich) were analytical grade and used as obtained without further purification. All 86 solutions were prepared using 18.2 M deionized water (Millipore).
87
88 2.2. Preparation of hydrogels
89 The preparation of hydrogels was formulated as given in Table 1 with different 90 composition of AlCl3 and AgNO3. Firstly, AlCl3 was dissolved in deionized water then 91 added by AA with continuous stirring. Prior to the addition of silver nitrate, gelatin was 92 added followed by heating at 50C and stirred for 24 h. Moreover, the mixture solution was 93 purged by N2 to remove the oxygen from the solution. Immediately, the polymerization of 94 AA was conducted by adding APS with a known amount and performed for 24 h at 37C.
95 Afterwards, the contaminants were washed out from the obtained hydrogels by deionized 96 water. In this study, the nomenclature of hydrogels was written as Alx/Agz for variation of 97 AlCl3 concentration and Gely/Agz for variation of silver concentration where x, y, and z 98 represent concentration of AlCl3, gelatin and silver, respectively.
99
100 Table 1. Formula of hydrogels (AA: 30 w/v%, and APS: 0.0225 g)
Samples AlCl3
(mol% of AA)
AgNO3
(mol% of AA)
Gelatin (wt%) Al1/Ag0.3 0.1
Al3/Ag0.3 0.3 Al7/Ag0.3 0.7 Al9/Ag0.3 0.9
0.3 10
Gel0/Ag0.3 0.3 -
Gel10/Ag0.1 0.1
Gel10/Ag0.2 0.2
Gel10/Ag0.3 0.3
Gel10/Ag0.4 0.4
Gel10/Ag0.5
0.5
0.5
10
101 102
103 2.3. Characterization of the hydrogels
104 2.3.1. Fourier transform infrared (FTIR) spectroscopy analysis
105 Samples were analysed using KBr method in which a tiny part of samples was mixed and 106 grinded with KBr powder. The FTIR analysis was analysed using a Bio-Rad model FTS- 107 3500GX spectrometer. All spectra were recorded in the wavenumber of 4000-400 cm-1 and 108 a scan number of 128.
109
110 2.3.2. Degree of swelling (DS) and gel fraction (GF)
111 Hydrogels were allowed to swell in deionized water at 37C until equilibrium swollen was 112 reached. Then, the excess surface water was blotted and the hydrogels were weighed as Ws, 113 whereas the initial weight denoted as Wd. The degree of swelling was calculated using eqn 114 (1):
115 DS = Ws -W Wd (1)
d × 100%
116 Subsequently, the equilibrium swollen hydrogels were dried and weighed as Wd. The gel 117 fraction of hydrogels was calculated by eqn (2):
118 GF = WWd' (2)
d × 100%
119 Each determination was run in triplicate and the results were expressed as mean ± standard 120 deviation (SD).
121
122 2.3.3. Self-healing efficiency
123 Initially, the hydrogels were cut into dog bone shape like with the size of 6 cm 1 cm 124 0.5 cm (LWH). The pristine hydrogels were tested using Testometric M500-25AT 125 equipped with a 100 N load cell and a crosshead speed of 50 mm/min. At the same time, 126 the interface of torn hydrogels was healed by gently put in contact to each other for 12 h at 127 37C followed by tensile testing. Then, the self-healing efficiency () was determined by 128 eqn (3):
129 = (3)
× 100 %
130 Where and denoted as the tensile stress of initial hydrogel and healed hydrogel, 131 respectively. The tests were run in triplicate for each specimen and expressed the results as 132 mean ± standard deviation (SD).
133
134 2.3.4. Scanning electron microscope (SEM) analysis
135 In order to prevent the shrinking of hydrogel during SEM analysis, the hydrogels were 136 firstly freeze-dried. Thereafter, the tiny pieces of samples were platinum-sputtered and 137 observed using JEOL (JSM-6500F, Tokyo, Japan) FE-SEM at an accelerating voltage of 138 10 kV.
139
140 2.3.5. X-ray diffraction (XRD) analysis
141 A Bruker D2 Phaser X-ray diffractometer was used to investigate the crystallinity of 142 hydrogels (Cu K radiation) with lambda ()= 0.151418 nm at 30kV and 10 mA. The 143 diffraction patterns were collected in the 2 range from 10-70. The crystal size was 144 calculated using Scherrer eqn (4):
145 𝐷= 𝑊 𝐶𝑜𝑠0.9 (4)
146
147 2.3.6. Silver loading measurement
148 To obtain the loading of AgCl NPs in the hydrogel, an inductively coupled plasma-atomic 149 emission spectroscopy (ICP-AES) analysis using a JY2000-2 (Horiba) was carried out.
150 Firstly, the hydrogel samples were freeze-dried for 2 days and calcined at 500C for two 151 hours. The resulted ash was subsequently dissolved in 1 M HCl followed by filtration using 152 a nylon filter (= 0.22 µm). the analyses were run in triplicate and the results were 153 expressed as mean ± standard deviation (SD).
154
155 2.3.7. Antibacterial activity
156 The antibacterial experiment was performed via disk diffusion method on Escherichia coli.
157 The Luria-Bertani (LB) broth containing E.coli with a concentration of OD600= 0.05 was 158 added on LB agar plate and spreaded by glass beads. Thereafter, the samples including the 159 positive control (H2O2 2500 ppm on filter paper) were cut into round-shaped with a 160 diameter of 4 mm and adhered on the surface of agar. Furthermore, such agar plate was 161 placed in incubator at 37C for 12h with a face down-position. Likewise, to quantitatively 162 measure the antibacterial activity of the hydrogels, samples (with same weight) were 163 introduced in a tube filled with E.coli containing LB broth and incubated for 12h at 37C 164 with a rotation shake at 200 rpm. Finally, the OD600 of the suspension was measured by 165 JASCO V-630 UV/VIS spectrophotometer.
166
167 2.3.8. In vitro cell culture
168 The in vitro cell culture study was conducted using L-929 mouse fibroblast cell (American 169 Type Culture Collection CCL-1). An -modified minimum essential (MEM) with 1%
170 antibiotics and 10% horse serum was used as the medium to grow the cells in a T75 tissue 171 culture flasks. Afterward, the confluent cells were rinsed carefully using 0.25% trypsin in 172 PBS (pH 7.4). Prior to the cell seeding on the sample, the hydrogels were cut into round 173 shape with diameter of 1 cm then put in 24-well plates followed by sterilization using UV 174 light (265 nm) for at least 30 min. As the control group, tissue culture polystyrene (TCPS) 175 was utilized with the cell density of 2 104 cells/mL. The experiment was run (incubated) 176 for 24, 48 and 72 h in a 37C incubator which can control the CO2 at 5%. Correspondingly, 177 lactate dehydrogenase (LDH) assay was employed to determine the L929 cell viability 178 followed the procedure reported by Tsai (2011). After the determined period of incubation,
179 the cells were rinsed with PBS (pH 7.4) and the lysis of the cells was performed using 180 Triton X-100 (1% in PBS) for at least 30 min. Afterward, the lysates were moved to a new 181 sterilized 96-well plate with the addition of same volume of LDH reaction mixture. Then, 182 oxamate solution (16 mg/mL) was utilized to cease the reaction. At last, enzyme-linked 183 immunosorbent assay (ELISA) Bio-Rad iMark reader was used to measure the 184 absorbance value at OD490. The experiment was conducted in triplicate and expressed the 185 results as mean ± standard deviation (SD).
186
187 2.3.9. Statistical evaluation
188 The statistical analysis was obtained from three independent replicate experiments and 189 expressed as mean ± standard deviation (SD) using Origin 9.0 software. One-way analysis 190 of variance (ANOVA) method was used to evaluate the statistical significance where the 191 differences with a p-value of 0.05 or less were assumed statistically significant.
192
193 3. Results and discussion 194 3.1. Preparation of hydrogels
195 Initially, the purpose of heating the mixture solution of AA and AlCl3 is to dissolve the 196 gelatin. Further, AgCl was precipitated when AgNO3 was introduced due to the reaction 197 with AlCl3. However, the utilization of gelatin is to stabilize or prevent the agglomeration 198 of AgCl through the reaction of amine (-NH2) functional groups from gelatin with Ag as 199 reported elsewhere[15]. In Fig. 1a, the photographs clearly showed that in the absence of 200 gelatin, a transparent solution with tiny particulate was obtained. Meanwhile, the mixture 201 solution turned to greyish-blue color by adding gelatin. In addition, Fig. 1b displayed that
202 the color of mixture solution changed from transparent to white, grey and then greyish-blue 203 as a function of time indicating that the reaction between gelatin and Ag continuously 204 occurred.
205
206 Figure 1. Photograph of mixture solution (a) with different composition after stirred for 207 24 h; (b) stirred as a function of time from 0 – 24 h at 37C.
208
209 Previous to the polymerization of AA initiated by APS, the mixture solution was degassed 210 using N2 to remove the oxygen which could interfere the polymerization. This 211 polymerization resulted in polyacrylic acid (PAA) in which its carboxylic (-COOH) 212 functional group subsequently cross-linked with Al3+ ions through ionic interaction forming 213 the hydrogel networks which contributed to the self-healing ability of hydrogels. At the 214 same time, the stabilized AgCl particles were also entrapped in the hydrogel networks. Fig.
215 2a depicted the schematic illustration for self-healing mechanism of hydrogel whereas the
216 actual self-healing processes of hydrogel were photographed and displayed in Fig. 2b in 217 which the autonomous self-healing was carried out at 37C for 12 h.
218
219 Figure 2. (a) Schematic illustration for the self-healing mechanism of the hydrogel.
220 (b) Photographs of self-healing process of (i) initial hydrogel; (ii) ruptured-off hydrogel;
221 (iii) healed hydrogel; and (iv) stretching on healed hydrogel 222
223 3.2. Chemical Functionalities
224 The FTIR spectra of acrylic acid, gelatin and hydrogels analysed was shown in Fig. 3. In 225 the spectrum of AA, the hydroxyl (OH), carbonyl (C=O), symmetric carboxylic ions (COO- 226 ) and alkene (C=C-H) functional groups were located at wavelength of ~3150 cm-1, 1730 227 cm-1, 1407 cm-1 and 985 cm-1, respectively. The spectra of all hydrogels confirmed that the 228 polymerization of AA was occurred indicated by decreasing peak of the bending vibration 229 of C=C-H at wavelength of 985 cm-1 and the vibration peak shifted from 1730 cm-1 to 1714
230 cm-1 revealed the physical cross-linking between –COOH functional groups from AA and 231 Al3+ which formed the networks of hydrogel. The peak correlated with symmetric 232 carboxylic ions at 1407 cm-1 is disappeared in all hydrogel sample and it is due to that the 233 carboxylic ions (COO-) interacted ionically with aluminium ions (Al3+) created the 234 aluminium carboxylate formation[16, 17]. Meanwhile, the presence of gelatin in the 235 hydrogels was proved by the peaks at 1532 cm-1 which is assigned to N-H bending amide 236 II[18]. The absorption peak of N-H stretching from gelatin was overlapped with OH 237 stretching in the wavelength ~3380 cm-1. Other than that, there is no significant difference 238 among the hydrogels.
239
240 Figure 3. FTIR spectra of acrylic acid, gelatin and hydrogels.
241
242 3.3. Degree of swelling and gel fraction
243 Degree of swelling indicated the ability of hydrogel to retain water inside the network of 244 hydrogel while gel fraction somehow indicated the cross-linking degree which was 245 determined by gravimetric method. The reason of retaining water in the hydrogel networks 246 is due to the hydrophilicity of carboxylic (COOH) functional groups of PAA[19, 20]. Fig.
247 4a showed that the prepared hydrogels exhibited greater water absorption ability with low 248 concentration of AlCl3. The degree of swelling was calculated to be 183.5, 97.6, 67.2, 59.3, 249 and 52.9% for Al1/Ag0.3, Al3/Ag0.3, Al5/Ag0.3, Al7/Ag0.3,and Al9/Ag0.3, respectively. On the 250 other hand, the gel fraction increased from 15.9 % to 93.9% with increasing AlCl3
251 concentration. It described that when low AlCl3 concentration is used, hydrogel with low 252 cross-linking density is obtained and subsequently allow the network to absorb greater 253 amount of water because the absorption is not limited by the cross-link junctions.
254 Meanwhile, a lot of cross-link junctions are created at higher concentration of AlCl3 which 255 limit its network to absorb water. Furthermore, the swelling degree of Gel0/Ag0.3 was 256 127.6% whereas with the presence of gelatin in the hydrogel, the DS decreased drastically 257 to ~65% proved that the presence of gelatin caused the water absorption ability of hydrogel 258 degraded (Fig. 4b). On contrary, with the presence of gelatin, the gel fraction of hydrogels 259 increased relatively high to ~84% from 55.7% which might be due to the interlacement 260 between gelatin and PAA-Al3+ network chain. Overall, by varying the silver concentration 261 in the hydrogel did not affect both results of degree of swelling and gel fraction.
262
263 Figure 4. The Equilibrium swelling degree and gel fraction of hydrogels (a) with 264 variation of AlCl3 concentration, and (b) with variation concentration of silver.
265
266 3.4. Self-healing efficiency
267 The determination of self-healing efficiency of the hydrogels was based on the tensile test 268 results of initial and healed hydrogel. The results of mechanical properties including their 269 self-healing efficiency was represented in Fig. 5. As in variation of AlCl3 concentration, 270 particularly from 1% to 5%, the , and of hydrogels enhanced drastically and their 271 corresponding self-healing efficiency certainly upgraded from ~34% to 75% as well (Fig.
272 5a). However, the self-healing efficiency was not significantly improved for Al7/Ag0.3, and 273 Al9/Ag0.3. This result points that the saturation of AlCl3 as a cross-linker is occurred at 5%
274 of AlCl3. On the other side, the of hydrogel with gelatin and variation of silver 275 concentration showed no significant alteration which is ~0.7 MPa while Gel0/Ag0.3
276 exhibited better mechanical properties (0.79 MPa). Likewise, similar trend was obtained 277 for the tensile stress of healed hydrogels with the : 0.64 MPa for Gel0/Ag0.3 and ~0.5 MPa 278 for hydrogels with gelatin. Correspondingly, the self-healing efficiency of gelatin-free and
279 with gelatin hydrogels was found to be 80.2% and ~70%, respectively. This results 280 confirmed that the ionic interaction between –COOH from PAA and Al3+ played important 281 role for the self-healing as aforementioned and regardless, gelatin possesses poor 282 mechanical properties therefore reduces the self-healing efficacy[21, 22].
283
284 Figure 5. The mechanical properties composed of tensile stress of pristine and healed and 285 self-healing efficiency of hydrogels (a) with variation of AlCl3 concentration, and (b) with
286 variation concentration of silver.
287
288 3.5. Surface morphology of hydrogels
289 The surface morphology of hydrogels observed at magnification of x500 and x3000 using 290 SEM analysis depicted clearly the difference between with and without gelatin hydrogel in 291 silver chloride nanoparticles (AgCl NPs) distribution. As can be observed in Fig. 6a, a lump 292 of silver was observed in Gel0/Ag0.3 hydrogels whereas gelatin proved its function as 293 stabilizing agent for silver indicated by well distribution of AgCl NPs in the hydrogels (Fig.
294 6b-f)[10]. Besides, the amount of AgCl NPs roughly increased with increasing silver 295 concentration which can be observed visually in the SEM images.
296
297 Figure 6. SEM images of hydrogel samples: (a) Gel0/Ag0.3; (b) Gel10/Ag0.1; (c) 298 Gel10/Ag0.2; (d) Gel10/Ag0.3; (e) Gel10/Ag0.4; and (f) Gel10/Ag0.5 observed at magnification
299 of x500 and x3000. (scale bar: 10 mm)
300
301 3.6. Crystallinity of hydrogels
302 X-ray diffraction (XRD) analysis was used to examined the crystallinity of silver in the 303 hydrogels. All of the hydrogels exhibited six diffraction peaks at 2 = 27.8, 32.2, 46.2, 304 54.8, 57.5, and 67.5 (Fig. 7), which can be assigned to AgCl (111), (200), (220), (311), 305 (222), and (400) planes, respectively (JCPDS #85-1355). Meantime, the broad peak located 306 at 2= ~21.5 ascribed for the PAA-Al3+ networks of hydrogel and gelatin as well which is 307 overlapped each other. The crystal size of AgCl embedded in hydrogel was obtained from 308 the diffractogram by Scherrer equation calculated using Diffrac.Eva V.4.3.0.2 software.
309 The AgCl NPs crystal size slightly increased from 29.4 to 35.4 nm with increasing silver 310 concentration. However, the absence of gelatin in the hydrogel (Gel0/Ag0.3) lead AgCl NPs
311 to have bigger size which was found to be 46.4 nm. Thereafter, the intensity of AgCl peaks 312 increased with increasing the concentration of silver from 0.1 to 0.5 mol% of AA.
313
314 Figure 7. The XRD spectra of hydrogels.
315
316 3.7. Silver loading in hydrogel
317 In this case, the purpose of calcination of hydrogel is to burn all the organic compound therefore 318 the resulted residue is Ag. The results in Fig. 8 demonstrated that the amount of Ag embedded in 319 the hydrogel consistently increased as more AgNO3 was introduced. The concentration of silver in 320 hydrogels was increased from 21.35 µg/L to 50.07 µg/L as increased AgNO3 from 0.1 to 0.5 %.
321 However, wide error bar was obtained for Gel0/Ag0.3 because the embedded AgCl NPs in the 322 hydrogels were not distributed uniformly.
323
324 Figure 8. The loading amount of Ag in hydrogel with various concentration of silver.
325
326 3.8. Effect of AgCl NPs on antibacterial activity
327 Fig. 9a showed the antibacterial experiment after attaching the samples on bacteria 328 containing agar plate. Then, after incubation for 12 h at 37C, the zone of inhibition was 329 observed clearly around the samples indicated that the bacteria was unable to grow 330 (inhibited) around the area of samples. The measured average zone of inhibition was found 331 to be 8.1 cm, 11.7 cm, 10.4 cm, 11.1 cm, 11.5 cm, 12.3 cm, and 13.1 cm for H2O2 on filter 332 paper, Gel0/Ag0.3, Gel10/Ag0.1, Gel10/Ag0.2, Gel10/Ag0.3, Gel10/Ag0.4, and Gel10/Ag0.5
333 hydrogels, respectively (Fig. 9b). Correspondingly, we also investigated the antibacterial 334 ability of hydrogel by measuring the OD600 value before and after incubation for 12 h at 335 37C as shown in Fig. 9c. An OD600 value of 0.89 was obtained when E.coli was inoculated 336 in LB-broth medium while the OD600 of the suspension with Gel0/Ag0.3 is 0.12. Similar to
337 the result determined by disk diffusion method, the OD600 value decreased from 0.24 to 338 0.04 with increasing silver concentration. Both of these results revealed that AgCl NPs 339 significantly inhibited the growth of E.coli by damaging the cell membrane and disrupting 340 the metabolism of bacteria cells[23, 24]. Even though, in this regard, the uniform 341 distribution of AgCl NPs enhanced by gelatin did not perform any significant effect on 342 antibacterial properties of the hydrogels.
343
344 Figure 9. The images of antibacterial experiments of hydrogels tested on E.coli using disk 345 diffusion method with the condition: (a) initial; and (b) incubated for 12 h at 37C. (c) the 346 absorbance of OD600 value measured using UV-Vis spectroscopy of samples. Asterisks indicate 347 the significant differences in OD600 value (*p < 0.05, **p < 0.01, ***p < 0.001 and ns denotes
348 not statistically significant).
349
350 3.9. Effect of gelatin and AgCl NPs on cell viability
351 The results of cell viability determined by LDH assay showed that the L929 fibroblast cells 352 cultured on Gel0/Ag0.3 are less biocompatible compared to the gelatin containing hydrogel 353 (Gel10/Ag0.1 – Gel10/Ag0.5) after culture for 24 h as can be seen in Fig. 10. This is due to the 354 utilization of gelatin which is very biocompatible therefore could enhance the cell 355 proliferation[25].
356 The cells number maintained on Gel0/Ag0.3, Gel10/Ag0.1, Gel10/Ag0.2, and Gel10/Ag0.3
357 hydrogels keep increasing gradually as prolonging the time of culture and demonstrated no 358 significant difference in cell viability. Meanwhile, for Gel10/Ag0.4 and Gel10/Ag0.5
359 hydrogels, the cells number are found to be relatively low and keep decreasing with 360 increasing time of culture. The incorporation of AgCl NPs in the hydrogel is actually non- 361 toxic towards mamalian cells[26]. However, based on ICP results, relative high 362 concentration of AgCl NPs in such hydrogel (Gel10/Ag0.4 and Gel10/Ag0.5) could lead to 363 toxicity toward L929 mouse fibroblast cells as indicated by reducing viable cells as 364 increasing the culture time. Likewise, this results also agreed with the study reported by 365 Mishra (2018)[27].
366
367 Figure 10. Cell viability of L929 fibroblast cells cultured on samples at 24, 48, and 72 h 368 determined by LDH assay. Asterisks indicate the significant differences in cell viability 369 (*p < 0.05, **p < 0.01, ***p < 0.001 and ns denotes not statistically significant).
370
371 3. Conclusion
372 In this study, PAA/Al3+ self-healing hydrogel incorporated with AgCl NPs stabilized using 373 gelatin has been successfully fabricated in single step synthesis. The formation of such 374 hydrogels is emphasized by FTIR analysis. The autonomous self-healing of hydrogel is 375 owing to ionic interaction between carboxylic (-COOH) functional groups and Al3+ ions 376 formed the reversible interaction between them. However, the efficiency of self-healing 377 reduces when gelatin is introduced in the hydrogel. Furthermore, the observation using 378 SEM displays the role of gelatin in stabilizing the AgCl NPs in the hydrogel. Then, XRD 379 analysis ensures the type of silver which corresponded to AgCl NPs in the hydrogel. The
380 biocompatibility tests through LDH assay method to determine the cell viability revealed 381 that hydrogel with relative high concentration of AgCl NPs can cause toxic to L929 mouse 382 fibroblast cell. Lastly, this synthesized hydrogel could be suitable for biomedical 383 applications.
384
385 Acknowledgements
386 The authors would thank both Universitas Mulawarman and Universitas Sumatera Utara 387 (USU) for the facilities to conduct the research.
388
389 References
390 1. Geckil, H., et al., Engineering hydrogels as extracellular matrix mimics. Nanomedicine 391 (Lond), 2010. 5(3): p. 469-84.
392 2. Liu, Y. and S.H. Hsu, Synthesis and Biomedical Applications of Self-healing Hydrogels. Front 393 Chem, 2018. 6: p. 449.
394 3. Rivest, C., et al., Microscale hydrogels for medicine and biology: synthesis, characteristics 395 and applications. Journal of Mechanics of Materials and Structures, 2007. 2(6): p. 1103-1119.
396 4. Wenger, M.P., et al., Mechanical properties of collagen fibrils. Biophys J, 2007. 93(4): p.
397 1255-63.
398 5. Yakabe, Y., et al., Kinetic Studies of The Interaction Between Silver Ion and Deoxyribonucleic 399 Acid. Chem. Lett., 1980. 9: p. 373-376.
400 6. Sim, W., et al., Antimicrobial Silver in Medicinal and Consumer Applications: A Patent 401 Review of the Past Decade (2007(-)2017). Antibiotics (Basel), 2018. 7(4).
402 7. Dhas, T.S., et al., Facile synthesis of silver chloride nanoparticles using marine alga and its 403 antibacterial efficacy. Spectrochim Acta A Mol Biomol Spectrosc, 2014. 120: p. 416-20.
404 8. Husein, M.M., E. Rodil, and J.H. Vera, A novel method for the preparation of silver chloride 405 nanoparticles starting from their solid powder using microemulsions. J Colloid Interface Sci, 406 2005. 288(2): p. 457-67.
407 9. Kang, Y.O., et al., Antimicrobial Silver Chloride Nanoparticles Stabilized with Chitosan 408 Oligomer for the Healing of Burns. Materials (Basel), 2016. 9(4).
409 10. Nguyen, N.T.-P., et al., Stabilization of silver nanoparticles in chitosan and gelatin hydrogel 410 and its applications. Materials Letters, 2019. 248: p. 241-245.
411 11. Zhang, Y., et al., A magnetic self-healing hydrogel. Chem Commun (Camb), 2012. 48(74): p.
412 9305-7.
413 12. Nešović, K., et al., Chitosan-based hydrogel wound dressings with electrochemically 414 incorporated silver nanoparticles – In vitro study. European Polymer Journal, 2019. 121.
415 13. Wataha, J.C., C.T. Hanks, and Z. Sun, Effect of Cell Line on in vitro metal ion cytotoxicity.
416 Dent Mater, 1994. 10: p. 156-161.
417 14. Faturechi, R., et al., Influence of Poly(acrylic acid) on the Mechanical Properties of 418 Composite Hydrogels. Advances in Polymer Technology, 2015. 34(2): p. n/a-n/a.
419 15. Nellist, D.R. and J.W. Janus, The Reaction of Silver Ion with Gelatin. The Journal of 420 Photographic Science, 2016. 9(5): p. 269-272.
421 16. Max, J.P. and C. Chapados, Infrared Spectroscopy of Aqueous Carboxylic Acids: Comparison 422 between Different Acids and Their Salts. J. Phys. Chem. A, 2004. 108: p. 3324-3337.
423 17. Ginting, M., et al., Self-healing composite hydrogel with antibacterial and reversible 424 restorability conductive properties. RSC Advances, 2020. 10(9): p. 5050-5057.
425 18. Satapathy, M.K., et al., Microplasma-assisted hydrogel fabrication: A novel method for 426 gelatin-graphene oxide nano composite hydrogel synthesis for biomedical application. PeerJ, 427 2017. 5: p. e3498.
428 19. Akala, E.O., P. Kopeckova, and J. Kopecek, Novel pH-Sensitive Hydrogels with Adjustable 429 Swelling Kinetics. Biomaterials, 1998. 19: p. 1037-1047.
430 20. Elliott, J.E., et al., Structure and swelling of poly(acrylic acid) hydrogels: effect of pH, ionic 431 strength, and dilution on the crosslinked polymer structure. Polymer, 2004. 45(5): p. 1503-
432 1510.
433 21. Ginting, M., et al., A simple one-pot fabrication of silver loaded semi-interpenetrating 434 polymer network (IPN) hydrogels with self-healing and bactericidal abilities. RSC Advances, 435 2019. 9(67): p. 39515-39522.
436 22. Pan, J., et al., An antibacterial hydrogel with desirable mechanical, self-healing and 437 recyclable properties based on triple-physical crosslinking. Chemical Engineering Journal, 438 2019. 370: p. 1228-1238.
439 23. Clement, J.L. and P.S. Jarrett, Antibacterial Silver. Met. Based Drugs., 1994. 1: p. 467-482.
440 24. Slavin, Y.N., et al., Metal nanoparticles: understanding the mechanisms behind antibacterial 441 activity. J Nanobiotechnology, 2017. 15(1): p. 65.
442 25. Su, K. and C. Wang, Recent advances in the use of gelatin in biomedical research. Biotechnol 443 Lett, 2015. 37(11): p. 2139-45.
444 26. Sanyasi, S., et al., Polysaccharide-capped silver Nanoparticles inhibit biofilm formation and 445 eliminate multi-drug-resistant bacteria by disrupting bacterial cytoskeleton with reduced 446 cytotoxicity towards mammalian cells. Sci Rep, 2016. 6: p. 24929.
447 27. Mishra, M., et al., Antibacterial Efficacy of Polysaccharide Capped Silver Nanoparticles Is 448 Not Compromised by AcrAB-TolC Efflux Pump. Front Microbiol, 2018. 9: p. 823.
449
Figure 1. Photograph of mixture solution (a) with different composition after stirred for 24 h; (b)
stirred as a function of time from 0 – 24 h at 37C.
Figure 2. (a) Schematic illustration for the self-healing mechanism of hydrogel. (b) Photographs of self-healing process of (i) initial hydrogel; (ii) ruptured-off hydrogel; (iii) healed hydrogel; and
(iv) stretching on healed hydrogel
Figure 3. FTIR spectra of acrylic acid, gelatin and hydrogels.
Figure 4. The Equilibrium swelling degree and gel fraction of hydrogels (a) with variation of AlCl
3concentration, and (b) with variation concentration of silver.
Figure 5. The mechanical properties composed of tensile stress of pristine and healed and self- healing efficiency of hydrogels (a) with variation of AlCl
3concentration, and (b) with variation
concentration of silver.
Figure 6. SEM images of hydrogel samples: (a) Gel
0/Ag
0.3; (b) Gel
10/Ag
0.1; (c) Gel
10/Ag
0.2;(d) Gel
10/Ag
0.3;(e) Gel
10/Ag
0.4;and (f) Gel
10/Ag
0.5observed at magnification of x500 and x3000. (scale
bar: 10 mm)
Figure 7. The XRD spectra of hydrogels.
Figure 8. The loading amount of Ag in hydrogel with various concentration of silver
Figure 9. The images of antibacterial experiments of hydrogels tested on E.coli using disk diffusion method with the condition: (a) initial; and (b) incubated for 12 h at 37 C. (c) the absorbance of OD
600value measured using UV-Vis spectroscopy of samples. Asterisks indicate the significant differences in OD
600value (*p < 0.05, **p < 0.01, ***p < 0.001 and ns denotes not
statistically significant).
Figure 10. Cell viability of L929 fibroblast cells cultured on samples at 24, 48, and 72 h determined by LDH assay. Asterisks indicate the significant differences in cell viability
(*p < 0.05, **p < 0.01, ***p < 0.001 and ns denotes not statistically significant).
Samples AlCl
3(mol% of AA)
AgNO
3(mol% of AA)
Gelatin (wt%) Al
1/Ag
0.30.1
0.3 10
Al
3/Ag
0.30.3 Al
7/Ag
0.30.7 Al
9/Ag
0.30.9 Gel
0/Ag
0.30.5
0.3 -
Gel
10/Ag
0.10.1
10
Gel
10/Ag
0.20.2
Gel
10/Ag
0.30.3
Gel
10/Ag
0.40.4
Gel
10/Ag
0.50.5
Table 1. Formula of hydrogels (AA: 30 w/v%, and APS: 0.0225 g)
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:
Declaration of interests: none
Subur P. Pasaribu: Methodology, Investigation, Analysis, Writing – Original draft preparation.
Mimpin Ginting: Investigation, Analysis, Writing – Reviewing & Editing. Indra Masmur:
Data Analysis, Writing - Reviewing & Editing. Jamaran Kaban: Data Analysis, Reviewing &
Editing. Hestina: Data & Statistical Analysis, Reviewing & Editing.