In the present study, we have successfully investigated the in vitro anti-bacterial, anti-fungal and in vivo wound healing activity of silver nanoparticles (15 nm ± 2 nm) synthesized by green route using porphyran, a polysaccharide isolated from marine red algae. These nanoparticles showed significant antibacterial and antifungal activity against Bacillus Subtilis and Candida albican. This may be attributed to the rapid internalization of silver nanoparticles by an endocytosis mechanism through the bacterial and fungal cell walls leading to the alteration of the internal structure of the cell and subsequent cell death. Furthermore, 200 µg/gm silver nanoparticles gel formulation was evaluated for in vivo wound healing activity using an excision wound model in Wistar rats. The silver nanogel formulation exhibited a significant reduction in wound excision within 16 days as compared to the control group. This improvement in wound healing process may be due to the antibacterial property, reduction in wound inflammation or the modulation of cytokines. These results establish the potential of porphyran reduced silver nanoparticles for effective antibacterial, antifungal and in vivo wound healing activity.
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Key words: Porphyran, silver nanoparticles, antibacterial activity, antifungal activity, wound healing activity.
Silver salts are well established as antibacterial agents. However, silver colloids exhibit strong antibacterial and antifungal activity due to the high specific surface area and a high fraction of surface atoms compared to bulk silver metal . Moyer et al. reported the use of silver nitrate solution for burn wound healing process . Interest in this research field was increased dramatically with the discovery of several potential therapeutic applications for colloidal silver. In 1968, Fox launched 1% silver sulfadiazine cream in the market as a topical antimicrobial agent for the treatment of several wound infections . Demling and DeSanti reported that the toxicity of silver was mostly associated with the carrier as compared to silver ions e.g. nitrate from silver nitrate or sulphadiazine from the silver sulphadiazine creams . However, Jain et al. reported that silver nitrate and silver sulfadiazine, like silver compounds, may get neutralized by anions like chloride and bicarbonate in body fluids causing argyria upon prolonged use of these topical preparations which could delay the healing process . Therefore, research focused on synthesizing silver nanoparticles (AgNps) by several approaches based on chemical, electrochemical and photochemical methods. Unfortunately, most of these utilized harsh reducing and stabilizing agents for the synthesis of AgNps making them unsuitable for therapeutic applications.
Several methods using biological approaches for the synthesis AgNps using micro-organisms, fungi and plant extracts have been reported [6-17]. Apart from these reports, few researchers utilized naturally occurring components such as starch and chitosan for the rapid synthesis of AgNps. [18,19] A green synthesis-based biological approach to obtain biocompatible colloidal silver, especially for medicinal and therapeutic applications, is much sought after.
In the present study, we have successfully investigated in vitro antibacterial activity against Bacillus Subtilis (B. Subtilis) and antifungal activity against Candida albicans (C. albicans) of porphyran (sulfated polysaccharide isolated from marine red algae) reduced AgNps. The in vivo wound healing activity of 200 µg/gm silver nanoparticles gel formulation was carried out in Wister rat and compared with control using excision wound model.
Materials and methods
Silver nitrate was purchased from Sigma Aldrich Ltd. India. B. Subtilis and C. Albicans were gifted by National Chemical Laboratory, Pune, India. The components of the Luria-Bertani (LB) medium and Sabouraud dextrose (SD) were supplied by HiMedia Laboratories, India. Analytical grade reagents from Merck India Ltd., India, were used for the synthesis of AgNps. Carbopol® 980 NF (poly acrylic acid polymer) was a gift sample from Noveon, India. Milli-Q grade deionized water (Millipore) was used for the preparation of the solutions.
Synthesis of silver nanoparticles using Porphyran
The silver nanoparticles were synthesized using porphyran, as reported earlier by our group . Briefly, the 0.01% w/v of porphyran was added to 100 ml aqueous solution of AgNO3 (10−3 M) and the pH of the solution was adjusted to 11 followed by heating for 5 min at 70 oC on water bath. The color of the solution changed from colorless to dark yellow on heating. The AgNps dispersion was dialyzed using dialysis tubing (12 kDa cut off) for 12 h to remove the ionic impurities. After dialysis, the pH of the AgNps solution was found to be 7. The concentration of silver in the above nano dispersion was determined by atomic absorption spectrophotometry (AA 201, Chemito, India). The subsequent nanodispersion was characterized by UV visible spectroscopy (Jasco, Japan), high resolution transmission microscopy (HRTEM) and zeta potential (Zetasizer, Malvern, UK)
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2.3. Preparation of AgNps containing gel (AgNps-Gel)
In order to use AgNps in the form of a topical formulation, the 1% w/w Carbopol® 980 NF based gel formulations containing 200 µg/gm AgNps was prepared. Briefly, the required amount of Carbopol® 980 NF was added to water and kept 12 h for complete humectation of polymer chains. AgNps dispersion was added to the hydrated carbopol solution with stirring to give a final concentration of 200 µg AgNps per gram of gel. Gelling was induced by neutralizing the dispersions to pH 6.8-7.0 using sodium hydroxide solution (0.5M). This gel formulation was stored at room temperature for further use.
2.4. In vitro antibacterial and antifungal activity
The in vitro anti-bacterial and anti-fungal activity of the synthesized AgNps was carried out on B. Subtilis and C. albicans respectively using a variety of concentrations of AgNps (5, 10 and 15 μg/ml) by culturing the organisms on LB agar and Sabouraud dextrose (SD) plates (106 colony forming units (CFU) of each strain per plate) respectively. Plates without AgNps were used as controls. Plates were incubated for 24 h for B. Subtilis and 48 h C. albicans at 37 °C and the numbers of colonies were counted.
In another experiment, the fresh colonies were taken from the agar plate and inoculated in 100 ml Luria broth (LB) culture and Sabouraud dextrose (SD) broth culture for Bacillus Subtilis and Candida albicans respectively. Growth was allowed until the optical density was reached at 0.1 at 600 nm (OD of 0.1 corresponds to a concentration of 1 CFU/ml of medium). Subsequently, 2-108 CFU from above were added to 100 ml liquid LB and SD media with the addition of 5, 10 and 15 μg/ml of AgNps separately. Control broths were used without nanoparticles. These AgNps treated and control solutions were incubated at 37oC±2. The growth rate of the colonies was determined by measuring the optical density at 600 nm at regular intervals (for B. Subtilis- 0, 6, 12, 18 and 24 and C. albicans - 0, 6, 12, 18, 24 and 48 h).
2.5. In vivo wound healing activity of AgNps-Gel formulation
The In vivo wound healing activity of AgNps was performed at Poona College of Pharmacy, Pune, India. Animal handling was performed according to Good Laboratory Practice. The study protocol was approved by the IAEC (Institutional Animal Care and Ethics Committee), constituted as per guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India.
Twelve to thirteen weeks old Wistar male rats weighing 180-200 g each were provided by Lupin Research Park, Pune, India. The animals were housed under standard conditions of temperature (25 oC), in 12/12 h light and dark cycles and fed with a standard pellet diet and water ad libitum. The animals were divided into groups containing six animals per group. The rats were then anesthetized by administering ketamine (0.5 ml/kg b. w. i.p.). A full thickness of the excision wound of circular area (approx. 500 mm2) and 2mm depth was made on the shaved back of the rats 30 min later the administration of ketamine injection. The wounding day was considered as day 0. Group I served as the control (untreated), Group II received standard cream (1% w/w Framycetin Sulphate) as a positive control and Group III served as test group, receiving 200 µg/gm AgNps gel formulation. 1 gm of cream was applied daily for up to 16 days to the wounds. The wounds were covered with the appropriate cotton dressings. The dressings were changed daily. The progressive change in the wound area was monitored planimetrically by tracing the wound margin on graph paper at day 8 and 16. The change in the healing of the wound, i.e. the measurement of the wound area on graph paper was expressed as unit (mm2) and the mean % wound closer was measured by following formula detailed below:
Where n = number of days 8th and 16th day.
Results and discussion
Evaluation of silver nanoparticles
In this study, we have utilized isolated porphyran for the size-controlled synthesis of AgNps at optimized pH and temperature conditions . The reduction of AgNO3 occurred on boiling for 2-5 min as indicated by the formation of dark yellow color (Inset picture Fig. 1) and the UV/Visible spectra of 0.01% w/v porphyran reduced AgNps recorded SPR centered at 405 nm (Fig. 1). HRTEM images revealed that porphyran capped AgNps were spherical in shape with a narrow size distribution and average particle size of 14±6 nm (Fig.2). The zeta potential of the AgNps was found to be - 32.05 mV. This showed that the obtained AgNps were capped with the anionic porphyran that helps the nanoparticles attain stability by electrostatic means.
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In vitro antibacterial and antifungal activity
In this experiment, bacterial as well as fungal growth was studied by incorporating increasing concentrations of AgNps in LB and SD broth plates that were inoculated with 106 CFU from B. substilis and C. albicans strains respectively. In case of B. substilis, it was observed that as concentration of AgNps increased the bacterial growth was decreased. However, low, medium and high concentration of AgNps inhibited 89, 95 and 99% of bacterial growth after 24 h incubation period (Fig. 3). In case of C. albicans, higher dose of AgNps (15 μg/ml) inhibited 65% of fungal growth (Fig. 3A) after 48 h incubation period.
In another experiment, the progressive growth inhibition of B. substilis was observed at lower (5 µg/ml) concentration of AgNps (Fig. 3A). The lag phase was found to be more prolonged (up to 24 h). In the case of C. albicans, the control group showed fungal growth began at 6h and continued up to 48 h. However, in the presence of AgNps, inhibition of fungal growth was observed at 15 µg/ml for up to 48 h (Fig. 3B). These result justified that lower and higher concentration of synthesized AgNps were more effective against B. substilis and C. albicans respectively. It was interesting to note that the negatively charged AgNps showed effective antibacterial activity against the gram positive bacteria. This may be due to the electrostatic attraction between the negatively charged nanoparticles and the positively charged bacterial cell with the rapid internalization of AgNps through the bacterial cell wall leading to impaired cell structure. But, previous report suggested that silver ions are less effective against S. aureus (gram positive bacteria) due to thicker peptidoglycan containing cell wall that prevented the uptake of silver ions in the cytoplasm [31 ML]. However, exact mechanism was still unclear and it may change according to bacterial species. Panacek et al., cited that sodium dodycyl sulfate stabilized AgNps inhibited the growth of C. albicans at 50 µg/ml concentration . In our study, 15 µg/ml concentration of AgNps exhibit controlled inhibitory activity against C. albicans.
In vivo wound healing activity
We have successfully explored the porphyran reduced AgNps gel formulation for the healing of excision wound in Wistar rats. The AgNps gel was applied to the wound once a day for 16 days. Fig. 5 and 6 represent the percentage wound closed at 8 and 16 days and the images of wound contraction following treatment with the marketed cream and AgNps gel formulation compared to the control group at 0, 8 and 16 day respectively. It was observed that wound contracting ability of animals treated with AgNps gel formulation was found to be significantly higher on day 8 (% wound closure values - 94% ±12) as compared to the marketed cream (86% ±5) and control group (19% ±7). These results show that AgNps gel formulation increases the speedy healing process of the wound as compared to the control group. Lower concentration of AgNps was required for healing of wound as compared to positive controlled marketed cream. This speedy wound healing after AgNps application may be due to the antimicrobial property or the increasing and decreasing the cytokines levels during wound healing process responsible for wound repair (Wright et al., 2002). The improvement in wound healing using AgNps gel formulation may also be attributed to the hydrophilic nature, which maintains a moist healing environment (27).
Previous reports justified the improved wound healing property of nanocrystalline silver dressings due to potent anti-inflammatory activity (Wright et al., 2002) and it occur due to the small size (Bhol et al., 2004). Also, some reports revealed that AgNps has unique dissolution behavior which release Ag0 in to solution and this Ag0 nanoparticles exhibited anti-inflammatory activity was confirmed through animal study (Demling and Burrell, 2002; Fan and Bard, 2002, Mizushima et al., 1965). Abraham and Himmel reported suppression of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) after application of Ag0 nanoparticles relieving rheumatoid arthritis symptoms (Abraham and Himmel 1997). Tian et al. reported the excised wounds treated with AgNps (477 µg/dressing) completely healed in 16±0.41 days after injury, whereas mice in the control group required 18.5±0.65 days . Also they have justified the decreased level of TGF-β responsible for the scarring of wounds and increased level of Interferon-γ that help to inhibit the proliferation of fibroblast and matrix production in the wound after treatment of AgNps. Wright et al. showed AgNps promote wound healing by the inhibition of matrix metalloproteases. Matrix metalloproteases are well known for prevent wound healing and are produced by the most common wound pathogen such as S. aureus . Rakel et al. reported that a moist healing environment was associated with the fastest healing times e.g. gel formulations using gelling agents, transparent films etc as compared to air dry wound .
In this study, we have successfully utilized porphyran reduced silver nanoparticles for in vitro antibacterial, antifungal and in vivo wound healing activities. Our findings not only confirmed the efficient antimicrobial, antifungal property of silver nanoparticles, but also implicate the ability of a silver nanoparticulate gel formulation to increase the speed of healing a wound as compared to the control group. This may be due to the antimicrobial activity and modulation of cytokines involved in wound healing process.
Vinod Venkatpurwar would like to thank All India Council for Technical Education (AICTE), Delhi, India for providing National doctoral fellowship (NDF).