Wound healing potential of green synthesized silver ...

Materials Express 2158-5849/2015/5/159/006

Copyright © 2015 by American Scientific Publishers All rights reserved. Printed in the United States of America

doi:10.1166/mex.2015.1225

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Wound healing potential of green synthesized silver nanoparticles prepared from Lansium domesticum fruit peel extract Shiv Shankar1 , Lily Jaiswal1 , Rangabhatla Sai Laxmi Aparna2 , Rangabhatla Gunneswara Subramanya Vara Prasad2, ∗ , Govindappa Prem Kumar3 , and Channakeshava Murthy Manohara3 1

Delivered by Publishing Technology to: Guest User IP: 110.225.24.222 On: Tue, 10 Mar 2015 16:12:35 ABSTRACT Copyright: American Scientific Publishers Silver nanoparticles (AgNPs) are proved to be potential wound healing materials for normal and burn related wounds due to their antibacterial and antifungal properties. Although, the wound healing properties of AgNPs through green synthesis were less reported. In the present study, green synthesized AgNPs were incorporated in Pluronic F127 gels as a delivery system to evaluate their wound healing potentiality. Interestingly, the wound healing activity of 0.1% w/w (AgNPs) in Pluronic F127 gels was enhanced to a considerable extent which could be corroborated by increased amount of hydroxylproline content (2.39 ± 0.28 g/mL), wound tensile strength (33.41 ± 2.38 N/cm2 , and wound closure time, which were quite higher than other treated groups. Histopathology and biochemical tests did not show any inflammation, however, the amount of collagen production was increased in the group treated by 0.1% w/w AgNPs in Pluronic F127 gels. Keywords: Green Synthesis, Wound Healing, Silver Nanoparticles, Biochemical Parameters.

1. INTRODUCTION Since ancient times, silver has been used for the treatment of various microbial infections. Recently, silver nanoparticles (AgNPs) in various forms are finding its applications in medical and pharmaceutical fields.1 AgNPs based medical products like wound healing materials and coating on biomedical implants were proven to be effective in reducing bacterial infections.2 AgNPs in a dosedependent manner can promote wound healing and reduce scar appearance. Besides, AgNPs silver nanoparticles elicit no inflammatory responses and no toxicity on the liver and kidneys. The potential biomedical benefits of AgNPs is, ∗

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therefore, enormous.3 There has been an upsurge of interest in developing green technology for the synthesis of these metal nanoparticles. Green synthesized nanoparticles possess a combination of the relevant antibacterial properties with enhanced biocompatibility.4–7 There are various reports on green synthesis of AgNPs, but their biological and pharmaceutical applications were least documented. In this attempt, we formulated a gel based product containing green synthesized AgNPs using Lansium domesticum (LD) fruit peel extract from our previous reports7 and pluronic F127. Pluronic F127 was found to be a potential wound healing material by enhancing VEGF (Vascular endothelial growth factor) and TGF- during inflammatory phases of the healing process.8 The wound healing

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School of Applied Sciences, RMIT University, Melbourne, 3001, Australia Faculty of Pharmaceutical Sciences, Asian Metropolitan University, Kualalumpur 43200, Malaysia 3 Department of Veterinary Pharmacology and Toxicology, Veterinary College, Hebbal, Bangalore 585401, Karnataka State, India 2

Materials Express potential of AgNPs loaded gels was evaluated in the animal model.

2. MATERIALS AND METHODS

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All chemicals were procured from Sigma Chemicals, USA, unless otherwise stated. The preparation of AgNPs was based on our previously reported work and the same

Wound healing potential of green synthesized silver nanoparticles Shankar et al.

AgNPs were used for the following animal experimentation as an extension of our previous work.9 Pluronic F127 (20 wt%) was dissolved in cold water and then transferred to 8 C and kept overnight for complete solubilization. The solution temperature was brought to 37 C for gelation. Then AgNPs were added and thoroughly mixed for uniform distribution of AgNPs in the gel.

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Fig. 1. Graph representing relation between wound closure time and wound diameter. Photographs showing wound healing activity from day 1 to day 12.

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Female Sprague–Dawley rats that were eight weeks old and weighing about 200–250 g were used for the study. All animals were maintained in accordance with the Masterskill University guidelines for the welfare of experimental animals (MUCH/AEC/HS/2012/12). All the animals were divided into four groups; Group 1: untreated group, Group II: treated with pluronic AgNPs, Group III: treated with 20% pluronic and Group IV: treated with povidone iodine (inset of Fig. 2) and each group containing six animals randomly for the experimentation. The animals were anesthetized with 10% chloral hydrate. The hair was shaved using electronic shaver, and the exposed skin was washed with sterile water. A full-thickness skin excision wounds were made (2 cm in diameter). All the gels were locally applied at the wound site every day. The dorsal skin of each animal, including the incision site, was excised for wound tensile strength determinations at 12-day post wounding protocol. The excised dorsal skin contained a

Materials Express portion of the experimental incision was made into strips (20 mm × 6 mm). The strips were individually mounted on a tensometer with a cross head speed of 25 mm/min, and the wound tensile strength was determined. The resulting strength values for the three strips of each wound were averaged to get a mean determination for each animal. Hydroxyproline content and wound tensile strength were measured by previously described method.10 For histological examination of the wound, samples of the tissues were processed in automatic tissue processor unit for light microscopy followed by procedures like fixating, dehydrating, embedding, and cutting using microtome (SLEE 9911). The sections made from wound were stained with hematoxylin-eosin (H&E—basic staining).11 Biochemical parameters like aspartate aminotransferase (AST or SGOT), alanine aminotransferase (ALT or SGPT), creatinine, and blood urea nitrogen (BUN) were measured as markers of inflammatory responses.

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Fig. 2.

Graphs showing (a) hydroxyproline content; (b) skin tensile strength after 12 days. Figure inset showing TEM image of AgNPs.


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3. RESULTS AND DISCUSSION

biocompatability of the AgNPs prepared by LD fruit peel extract can be attributed to the presence of triterpene glyIn vivo model showed enhanced wound closure time in the cosides (Lansioside A, B and C) in the extract. Among group treated with Pluronic AgNPs (Fig. 1). Neocollagen the triterpene glycosides, Lansioside A contains N -acetylproduction by the native sub dermal tissue is an indirect D-glucosamine as sugar moiety that could enhance the measure of wound healing activity, and can be estimated biocompatibility and wound healing activity.9 17 AgNPs by measuring wound tensile strength. In our present study, we tested wound tensile strength at day 12 post-wounding prepared from this extract showed the presence of these protocol. Figure 2(b) depicts the tensile strength measurecompounds on the surface of NPs. Both triterpenes and ments of various groups. The gel of 0.1% w/w AgNPs amino sugars are potent phytochemicals that enhance cell with 20% Pluronic F127 showed higher tensile strength viability.9 Reports reveal that triterpenes can protect var(33.41 ± 2.38 N/cm2 when compared to other treatment ious cells from oxidative damage caused by free radicals and control groups. The results are closely aligned with Kwan et al.,11 who performed similar experiments on wound healing potential of AgNPs. They reported that the increase in tensile strength of AgNPs treated sample could be due to organized collagen fibers during the wound healing process.11 One of the key enzymatic modification in wound healing process is hydroxylation of proline that is a precursor for collagen. Enhanced hydroxyproline content is a marker of collagen deposition. Highest hydroxyproline content of 3.59 ± 0.61 g/mL was seen in samples treated with 0.1% AgNPs followed by 2.39 ± 0.28 g/mL for standard and 1.19 ± 0.05 g/mL for Pluronic alone. Control group has low hydroxyproline content shown in Figure 2(a). Figure 3 depicts repby Publishing resentative micrographs of H&EDelivered stained sections of the Technology to: Guest User 110.225.24.222 wound area after 7th day (Figs.IP:3(a)–(d)) and 12th On: day Tue, 10 Mar 2015 16:12:35 Copyright: American Scientific Publishers (Figs. 3(e)–(h)). Histopathological examination revealed marked exudate, consisting of inflammatory cells trapped, edema, more fat cells, and area of hemorrhages on day 7 in the control group in which granulated tissue was not fully formed (Fig. 3(b)). Whereas, AgNPs Pluronic treated wound showed remarkable organization with increased epithelization and marked fibroblast proliferation (Fig. 3(d)). On day 12 AgNPs Pluronic (Fig. 3(h)) treated wound showed complete epithelization with keratinisation and also fibrous connective tissue proliferation, whereas, standard and Pluronic gel demonstrated epithelization and cystic cavities containing exudate (Figs. 3(e) Fig. 3. Histopathological observation of wounds treated with different and (g)). Most of the toxicological studies on AgNPs are samples (a)–(d) after 7 days and (e)–(h) after 12 days of wound healing 12–14 restricted to oral or inhalational administration but protocol; (a) standard treated group, showing fibroblasts proliferation the most rationale route of entry of 90% AgNPs is via (light green arrow), and thick scab formation (black arrow) in the wound topical/ dermal with undetermined systemic and topical area and note the epithelial cell proliferation below the scab (yellow toxicity potentials.15 In our study, we elucidated the bioarrow). (b) Control (untreated group), showing marked exudate, consisting of inflammatory cells (black arrow) and also fibroblast proliferation chemical markers of liver and kidneys at time intervals in the wound area. (c) Pluronic treated group showing scab formation of 0, 7 and 12 days. The results (Fig. 4) revealed that (yellow arrow), neovascularization (light green arrow), and epithelial there were no significant differences in AST, ALT, creaticell proliferation (black arrow). (d) AgNPs Pluronic treated group shownine, and BUN between the control and treatment groups ing epithelization (yellow arrow), and marked fibroblast proliferation (black arrow). (e) Standard treated group, showing epithelization (black which clearly indicate that the wound healing material arrow) and cystic cavities containing exudate (yellow arrow). (f) Conof the present study did not induce any toxic effects in trol (untreated group), showing scab formation (black arrow) and fibrous liver and kidney. Our results align with Vinay et al. who connective tissue formation (yellow arrow). (g) Pluronic treated group reported that Pluronic F127 did not produce any toxicoshowing complete epithelization (black arrow) and cystic spaces (yellow logical symptoms on the rat model. Similar results were arrow). (h) AgNPs Pluronic treated group showing complete epithelization with keratinisation (yellow arrow) and also fibrous connective tissue also obtained by Garg et al.16 who used green syntheproliferation (black arrow). sized AgNPs for wound healing activity. Finally, enhanced 162


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Fig. 4. Graphs showing biochemical parameters (a) AST, (b) ALT, (c) creatinine, and (d) BUN.

and radiation.18 Triterpenes from Azadirachta indica have shown to protect the cell from starvation up to 70 days.19 Besides the protective effect of triterpenes, amino sugars also enhance the cell viability in various cell lines.20 So based on the above mentioned results and literature, it is clearly evident that the gels containing AgNPs synthesized using aqueous extract of LD fruit peel possess low cytotoxic profiles and can be effectively used in biomedical applications.

4. CONCLUSION In vivo wound healing tests showed enhanced wound closure time, hydroxyproline content, tensile strength, and excellent histocompatibility in group treated with 0.1% w/w AgNPs Pluronic gel. This investigation provides us with evidence that AgNPs obtained from LD fruit peel extract may be ideal candidates for future studies exploring their use in pharmaceutical and biomedical applications. Acknowledgments: Authors are thankful for the facilities provided by RMIT University, Australia and Asian Metropolitan University, Malaysia. Mater. Express, Vol. 5, 2015

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Received: 4 October 2014. Revised/Accepted: 23 December 2014.


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