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Probing the wound healing potential of biogenic silver nanoparticles Objective: Silver nanoparticles (AgNPs) are known for their antimicrobial profile and wound healing activities. However, cytotoxicity and cosmetic abnormalities associated with silver pose a major challenge in their translation for therapeutic applications. Our objective was to develop biogenic AgNPs, using a singlestep green synthesis, and to investigate their in vitro and in vivo behaviour as wound-healing agents. l Method: AgNPs were prepared using the green synthesis approach with aqueous Bryonia laciniosa leaves extract. The AgNPs were then evaluated for physicochemical properties, stability, and antimicrobial and in vivo wound healing activities. l Results: Stable AgNPs with characteristic absorption at 408nm and 15±3nm particle size were generated via the active involvement of Bryonia laciniosa. No loss of stability was detected after 6 months at room temperature. Antibacterial activity was observed against both Gram-negative and Gram-positive bacteria with no cytotoxicity observed in vitro at a concentration of 200 μg/mL and effective cytokine modulation. In vivo wound healing experiments showed improved wound contracting ability in rats where, after 14 days, wound alleviation was 47.1±2.2% in the control groups, compared with 78.1±1.4% and 92.6±6.7% for a silver-based marketed cream and the AgNPs, respectively. l Conclusion: The developed AgNPs proved to be superior wound healing agents owing to scarless healing with insignificant inflammation and toxicity. l Declaration of interest: There were no external sources of funding for this study. The authors have no conflicts of interest to declare. l
silver, nanoparticles, antimicrobial, wound healing, cytocompatibility
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particles (AgNPs). AgNPs display distinguishing physicochemical and biological properties on account of tailored surface and size characteristics.4 AgNPs have a higher surface area and can markedly increase the release rate of singly charged silver, the soluble and most biologically active form of silver. The AgNPs have multiple antimicrobial mechanisms. On exposure to moisture or wound fluid at the wound surface, AgNPs are oxidised and release charged silver ions. The charged form of silver causes cell membrane damage by interacting with cell surface proteins of pathogenic micro-organisms5 and penetrating inside the cell. Intracellular charged AgNPs prevent DNA replication, inactivate the bacterial respiratory enzymes and impede cell division, resulting in cell lysis. Additionally, charged AgNPs are capable of generating free radicals with remarkable bactericidal potential.6,7 This leads to enhanced antimicrobial efficacy demonstrated by AgNPs. On the other hand, the improved surface features of AgNPs also carry the risk of aggregation owing to instability. Unstable AgNPs interact with or accumulate in normal cell organelles, leading to cytotoxicity.8 Thus, it becomes imperative to balance the wound healing efficacy of AgNPs against their toxicity. The activity and toxicity of nanoparticulate systems are associated with their synthesis method, reducing agent, nature, concentration and physicochemical characteristics.9 Nanoparticles are synthesised and stabilised via physicochemical methods:
V. Dhapte,1 MPharm; S. Kadam,2 PhD; A. Moghe,3 PhD; V. Pokharkar,1 PhD; 1Department of Pharmaceutics, Bharati Vidyapeeth University, Poona College of Pharmacy, Pune, India 2 Bharati Vidyapeeth University, Bharati Vidyapeeth Bhavan, Lal Bahadur Shastri Marg, Pune, India 3 Department of Cell and Molecular Biology, Bharati Vidyapeeth University, Rajiv Gandhi Institute of IT and Biotechnology, Katraj, Pune, India. Email: vbpokharkar@ yahoo.co.in
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W
ound healing is a multifaceted process, comprised of coagulation, inflammation, tissue formation, and epithelialisation.1 The normal wound-healing process involves inflammation at the injury site, angiogenesis followed by granulation, tissue growth, connective tissue and epithelium repair along with remodelling to heal the wound completely. Inflammatory pathways and cytokine modulation influence the healing process, and a number of immunological mediators and microbial colonisation can slow the repair of an injured site.2 While early inflammatory effects help in abolishing the microbial infection, prolonged inflammatory responses can lead to tissue damage.3 Microbial pathogens in the wound fluid produce toxins and enzymes that prolong the inflammatory responses and hence delay the wound recovery. Rapid wound repair with minimal scarring is essential to avoid these complications. Topical antimicrobials offer a prophylactic answer to some of these issues but, with the risk of resistance, are less helpful in wound therapy. Conventionally, silver-based products containing silver compounds were recommended in wound management for their bactericidal properties. However, high application frequency has led to reports of inflammatory effects and cosmetic defects.2 With the aid of nanotechnology the solubility and surface properties of silver can be improved by the formation of silver nano-
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research
a
b 408nm
1.25
Absorbance
1 0.75 0.5 0.25 0
300
c
400 500 Wavelength (nm)
600
50nm
AgNPs
%T
3227
1369 1541
Bryonia laciniosa
1986
3289
1647
2809 2917
4000
3500
3000
2000
Wavenumbers (cm-1)
432
1002
1324
2500
1500
Green synthesis and characterisation of AgNPs Various physicochemical characterisation tests were performed to ascertain the complete green synthesis of AgNPs using aqueous Bryonia laciniosa leaf extract along with AgNP charge and stability. Using our earlier reported method,13 0.1% w/v aqueous Bryonia laciniosa leaf extract was prepared and employed for reduction of 0.01M aqueous silver nitrate solution (Sigma Aldrich, USA) to AgNPs under magnetic stirring for 1 hour at room temperature. Subsequently, the AgNP dispersion was dialysed using 12kDa cut-off membrane for 24 hours to remove the unreacted ions and retain AgNPs only.15 The retained AgNPs were examined for silver content using atomic absorption spectrophotometry (AA 201, Chemito, India).15 In order to investigate the wound-healing applications of AgNPs, hydrophilic gels were prepared in gellan gum. A weighed amount of gellan gum was sprinkled onto the AgNP dispersion and allowed to form AgNP gel. The amount of silver in the AgNP gel was found using atomic absorption spectrophotometry to be 0.18 mg/g. The surface plasmon resonance (SPR) of the AgNP dispersion was analysed by UV/visible spectrophotometry (Jasco, Model V-570, Japan) operating at a resolution of 2nm. Fourier transformed infrared spectroscopy (FTIR) analysis of Bryonia laciniosa leaf extract and AgNPs was conducted (Thermo Scientific Nicolet iS50R). These aqueous dispersions were scanned from 500–4000 cm-1 for comparison of overlap regions and functional group identifications. The sample for X-ray diffraction (XRD) measurement was prepared by drop-coating a film of AgNPs on glass substrate. XRD patterns were recorded on X-ray Brucker AXS diffractometer (D8 Advance, USA) in the 2q angle range of 5–80°. Average crystalline size (D) was calculated according to the DebyeScherer’s formula: D=
1402
1633 1538
Methods
1015 1000
500
Kλ βCosθ
where b is the full width at half-maximum (FWHM) intensity of a peak at an angle q; K is the constant, depending on the line shape profile; and l is the wave length of the X-ray source. Transmission electron microscopic (TEM) analysis to determine size and morphology of AgNPs was performed on a TECHNAI G2 F30 S-TWIN instrument. The sample was prepared by placing a drop of AgNP dispersion on a carbon-coated copper grid and then air-drying it at room temperature. Measurements
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Fig 1. UV/visible absorption spectrum (a), TEM image (b) and FTIR spectrum (c) of AgNPs.
traction ability, inflammatory responses, and cytotoxicity of AgNPs synthesised with Bryonia laciniosa leaf extract in wound management using various in vitro and in vivo tests.
© 2014 MA Healthcare
electrochemical, reduction, vapour deposition and a microwave-assisted process. However, contamination from precursors, use of toxic solvents, hazardous byproducts and environmental concerns limit the application of these techniques. Green synthesis is a wellestablished route of nanoparticulate synthesis that is safe because it eliminates the toxicity arising from physicochemical approaches.10 Green synthesis employs plants, algae, fungi, yeasts, bacteria and their products to synthesise nanoparticles. Plants and plant extracts are preferred over others as they are easy to handle, readily available and economical.11 In Indian medicine the Bryonia laciniosa (Linn.) plant is a bitter tonic and mild laxative, which is reported to have extensive medicinal uses including as an analgesic and antipyretic, a treatment for asthma and snake bites, and a fertility aid.12 Several researchers have investigated organic extracts of Bryonia laciniosa fruit, seeds and leaves for antimicrobial, antioxidant, anti-inflammatory, cathartic and antitumour activities.13 Herbal preparations containing organic solvent extracts of Bryonia laciniosa remained less explored owing to the associated adverse side effects arising from the presence of residual organic solvents. Recently, aqueous Bryonia laciniosa extracts showed higher biological activity than its organic extracts.14 Here we assess and correlate the antimicrobial profile, wound con-
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research ty of AgNPs against the Gram-negative bacterium Escherichia coli (ATCC 25922) and the Gram-positive bacterium Staphylococcus aureus (ATCC 6538) was investigated using a well-diffusion assay.16 The autoclaved Luria-Bertani agar (Himedia Laboratories, India) plates were inoculated with 106 colony forming units (CFU), of one bacterial strain per plate, using a glass spreader. Uniform well-like cavities were bored and supplemented with various concentrations of AgNPs. These inoculated and supplemented plates were incubated aerobically at 37°C in a bacteriological incubator. After 24 hours incubation the zone of inhibition (ZOI) was calculated as the difference between the total inhibition zone diameter and the well diameter.
b
Bryonia laciniosa
5
20
35 50 20 (degree)
65
80
Wound healing activity l In vivo studies The IAEC (Institutional Animal
b Ag
Ag Bryonia laciniosa
Ag++NO3 Silver nitrate
Magnetic stirring Ag
Ag
Redution and capping
Anionic (negatively charged) silver nanoparticles (AgNPs)
Absorbance
0.9 0.6 0.3 0 300
1.2 0.9 0.6
pH2 pH4 pH6 pH8 pH10
0.3 0
400 500 600 Wavelength (nm)
Stability: up to 6 months
300 400 500 600 Wavelength (nm) pH stability
Absorbance
0 day 15 days 1 month 2 months 3 months 6 months
1.2
Absorbance
c 10-1 M 10-2 M 10-3 M 10-4 M 10-5 M 10-6 M
1.2 0.9 0.6 0.3 0 300
400 500 600 Wavelength (nm)
Electrolytic stability
were performed at an accelerated voltage of 300kV with a lattice resolution of 0.14nm and point image resolution of 0.20nm. The surface charge of the AgNPs (without any dilution) was assessed from the zeta potential measurements using the Malvern zetasizer Nano series ZS90 (Malvern Instruments, UK). The stability of the AgNPs was studied with respect to time, variable pH and electrolytic conditions. At regular time intervals, AgNP stability was reported at ambient temperature conditions up to 6 months. The effect of pH variation on the AgNP stability was studied between pH 2–10 after 24 hours. Electrolytic stability was assessed by adding 10−1–10−6M NaCl to the AgNP dispersion for 24 hours. AgNP stability was evaluated from the SPR obtained using UV/visible spectroscopy.
Antibacterial activity The antibacterial effect of the AgNPs was studied in the presence of increasing concentrations of AgNPs, above their reported minimum inhibitory concentration (MIC) of 2.5μg/ml.10 The antibacterial activi434
Ethics Committee) approved in vivo wound healing study protocol was performed following the guidelines of the committee for the purpose of control and supervision of experiments on animals (CPCSEA, India). During the experiments, animals were handled according to Good Laboratory Practice. In an experimental rat model, male Wistar rats were anaesthetised and a circular excision wound of 530.66mm2 and 2mm depth was made on their shaved back. There were three treatment groups with four animals per group: l Group 1 control (blank gellan gum gel) l Group 2 applied marketed cream (containing silver sulfadiazine) l Group 3 AgNPs gel. Equal amounts of control, marketed cream and AgNP gel (0.5g) were used for the treatment once a day. However, the applied silver content varied as the marketed cream contained silver sulfadiazine compound (Ag content=3.18mg/g) whereas the AgNP gel contained stabilised Ag in nano size (Ag content=0.18mg/g). Progressive changes in wound area were monitored planimetrically, photographed at defined intervals and further processed for the scratch assay. The area of the wounds was measured on days 0, 7 and 14 post-wounding. Percent wound alleviation was measured using following formula: % Wound = Wound Area(Day 0) - Wound Area (Day n) x 100 Wound Area (Day 0) alleviation
Where ‘n’ indicates number of days, either 7 or 14.
In vitro studies Serum levels of human interleukin-6 (IL-6) and human interleukin-10 (IL10) were determined in triplicate with cytokinespecific ELISA kits. Cytotoxicity of AgNPs was evaluated in the TE 353.Sk cell line derived from normal human skin fibroblasts. Adherent TE 353.Sk cells were cultured l
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a
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(220)
(200)
Intensity
a
(311)
(111)
Fig 2. XRD patterns (a), scheme for negatively charged AgNP synthesis (b), and stability of AgNPs (c).
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Journal of Wound Care.Downloaded from magonlinelibrary.com by 157.211.003.015 on January 8, 2015. For personal use only. No other uses without permission. . All rights reserved.
research Fig 3. Antibacterial activity of AgNPs against Staphylococcus aureus (a) and Escherichia coli (b) and related comparison of zone of inhibition (c). b
c
10μg/ml 7.5μg/ml
10μg/ml
0μg/ml
0μg/ml 2.5μg/ml 2.5μg/ml
7.5μg/ml
Zone of inhibition (mm)
a
5μg/ml
5μg/ml
16
S. aureus
AgNPs E. coli
E. coli 12
8
4
0
AgNPs S. aureus 0
5
10
15
Statistical analysis Statistical significance was determined using analysis of variance (ANOVA). A value of p10-2 M (Fig 2c). Under strong acidic conditions or higher electrolytic concentration, the negatively charged, Bryonia laciniosa-capped AgNPs tend to form complexes or undergo ionization, thereby disturbing the surface charges on the stable AgNPs. This causes a lowering of electrostatic repulsion and increases in aggregation and instability.15 AgNPs were stable for up to 6 months, as evident from their SPR, which was monitored at regular intervals (Fig 2c). On the whole, AgNPs proved to be highly stable with a good shelf life.
The effect of AgNPs on bacteria The antibacterial effect of AgNPs in increasing concentrations is shown in Fig 3. As depicted from the clear zones around inoculated wells, AgNPs were capable of inhibiting both Gram-positive Staphylococcus aureus as well as Gram-negative Escherichia coli bacteria (Fig 3a and 3b). The ZOI in the plate containing Escherichia coli increased exponentially up to a concentration of 7.5μg/ml after which the inhibitory effect levelled off irrespective of further increases in the concentration of AgNPs (Fig 3c). On the other hand, there was gradual increase in ZOI with Staphylococcus aureus up to 5μg/ml AgNPs. Beyond this, AgNPs showed a steep rise in the inhibitory effect, followed by a steady levelling effect at concentrations above 7.5μg/ml. Inhibitory potential for Staphylococcus aureus and Escherichia coli was similar at and above 10μg/ml AgNPs (Fig 3c) but at lower concentrations there was a difference in the antibacterial potential. 438
Marketed cream
AgNP gel
This discrepancy could be attributed to structural variance between Gram-positive and Gram-negative bacteria. In Gram-positive bacteria like Staphylococcus aureus, the outer cell wall is thicker (Fig 3c) than in Gram-negative bacteria (Escherichia coli ) owing to more peptidoglycan layers.17 As a result, uptake of AgNPs across the thin cell wall of Escherichia coli was faster, leading to rapid inactivation of cellular components followed by cell death. Conversely, the thicker peptidoglycan layer resisted the initial internalisation of AgNPs into Staphylococcus aureus and thus delayed the cell lysis.
In vivo wound healing All three treatment groups demonstrated minor wound contraction with a weak inflammatory response during the first 7 days on account of initial marginal influx of neutrophils.4 At day 14 the control group showed 47.07±2.19% wound alleviation, the marketed preparation containing silver sulfadiazine cured 78.13±1.35% of the wound while AgNP gel healed 92.54±3.67% of the wound (Fig 4a). Therefore, the AgNP gel-treated group showed a rapid wound epithelialisation compared with control and marketed cream-treated animal wounds. On comparing the appearance of healed wounds, the control group showed scarring as well as rubor of a considerable size, even after 14 days (Fig 4b). As a rule, scar formation and characteristic inflammatory responses are inherently associated with wound recovery caused by discrepancy among immune response or hypersensitivity.22 Bioactive materials including silver-based compounds, capable of altering the cytokine cascade, could improve wound appearance through immunomodulation.23 On application of silver sulfadiazine-containing marketed cream for 14 days, there was noteworthy
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14 days
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7 days
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Journal of Wound Care.Downloaded from magonlinelibrary.com by 157.211.003.015 on January 8, 2015. For personal use only. No other uses without permission. . All rights reserved.
research Fig 5. Schematic representation of wound healing mechanism. AgNP gel
Intact skin
Marketed cream Microbial infection Wound due to injury
Influx of neutrophils Control
Marketed cream
Release of proinflammatory mediators
AgNP gel
Stimulates fibroblasts, keratinocytes and macrophages Scar formation and inflammation
Release of anti-inflammatory mediators
Significant rubor and scar
Reduced rubor and scar
No Rubor, no Scar
reduction in the scar area along with significant inflammation. Such silver compounds are known to be toxic to fibroblast and epithelial cells, giving rise to an inflammatory response.24 However, treatment with synthesised AgNP gel for 14 days completely repaired the wound with negligible scarring and significant loss of rubor. The relative wound healing mechanism is represented in Fig 5 and supported by scratch assays and ELISA. In the quantitative analysis via scratch assays, wound alleviation was represented by the percentage of cell covered area while swelling, erythema and inflammation were reflected from the percent scratch area. As observed in Table 1, AgNP geltreated wound showed considerable decrease in scratch area with rapid upsurge in the cell covered area compared to the control and marketed cream at 14 days. Thus, formulations containing AgNPs reduce scar formation and inflammation through cytokine modulation.4,24
in cellular proliferation, keratinocyte migration and extracellular matrix production, which sequentially leads to scar formation.25 As a result, lower IL-6 levels (23.79ng/ml) induced by AgNPs reduced inflammation along with scarless wound recovery. In response to inflammation, IL-10, a key cytokine, is produced by keratinocytes and inflammatory cells to neutralise the effects of pro-inflammatory cytokines like IL-6.26 Its expression is regulated by the magnitude of inflammation and pro-inflammatory cytokine levels (Fig 5). Thus, lower IL-6 levels could stimulate relatively minuscule IL-10 levels
Cytokine-specific ELISA measurements of synthesised AgNPs revealed their wound-healing mechanism from the significantly lower levels of cytokines IL-6 and IL-10. Pro-inflammatory cytokine IL-6 stimulates fibroblast proliferation and monocyte activation, which in turn give rise to inflammatory responses. Moreover, IL-6 initiates release of neutrophils and macrophages to the wound, resulting J O U R N A L O F WO U N D C A R E V O L 2 3 , N O 9 , S E P T E M B E R 2 0 1 4
Treatment
Days
Scratch area (%)
Cell covered area (%)
Control
0
85.59±5.79
12.67±2.17
7
66.98±7.23
31.91±2.33
14
28.51±6.41
70.67±4.91
0
86.11±7.31
12.23±3.07
7
54.37±3.67
44.33±2.77
14
17.48±1.92
82.50±1.69
0
84.72±6.47
13.41±1.33
7
40.02±4.33
59.13±2.86
14
4.67±0.73
93.72±3.45
Marketed cream
AgNP gel
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Effect of AgNPs on cytokine levels
Table 1: Quantitative analysis of wounds by scratch assays
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research (153.8pg/ml) suggestive of an overall decrease in inflammation and scarless wound healing on application of AgNP gel.
Fig 6. Cytotoxicity evaluation of various AgNP concentrations in TE 353.Sk cell line. 100
440
139-148. 3. Wright, J.B., Hansen, D.L., Burrell, R.E. The comparative efficacy of two antimicrobial barrier dressings: in vitro examination of two controlled release of silver dressings. Wounds. 1998; 10:6, 179–188. 4. Rai, M., Yadav, A., Gade, A. Silver nanoparticles as a new generation of antimicrobials.
50 25 0 0
50
100
150
200
Concentration of Ag (μg/ml)
ble interleukin levels) without any major cytotoxicity. Such cytocompatible AgNP formulations with better efficacy would definitely be acceptable in wound healing, management of burns, delayed impaired wound treatment in diabetes and other such topical ailments. Certainly, these cytocompatible silver nanoparticles would emerge as effective and safe alternatives to the existing conventional treatments. However, the findings of this study are restricted to topical injuries and wounds. Future studies should explore the potential of these biogenic silver nanoparticles in diabetic delayed wound healing and special clinical ailments.
Conclusion A simple, single-step reduction process mediated by aqueous Bryonia laciniosa extract generated stable, crystalline, anionic (negatively charged) silver nanoparticles of 15±3nm size. These nanoparticles were highly stable up to 6 months and under varying pH and electrolyte concentrations. Biosynthesised silver nanoparticles demonstrated their enhanced antimicrobial potential against Gram-positive as well as Gram-negative bacteria at concentrations where no cytotoxicty was observed. They proved to be superior wound healing candidates that offered rapid, scarless wound recovery without any major inflammation owing to effective cytokine modulation. Thus, the biosynthesised silver nanoparticles proved to be safe, prophylactic-therapeutic answer for successful wound recovery. n Biotechnol Adv 2009; 27, 76-83. 5. Wong, K., Liu, X. Silver nanoparticles-the real ‘‘silver bullet’’ in clinical medicine? MedChemCommun. 2010; 1, 125-131. 6. Anisha, B.S., Biswas, R., Chennazhi, K., Jayakumar, R. Chitosan–hyaluronic acid/nano silver composite sponges for drug resistant bacteria infected
diabetic wounds. Int J Biol Macromol 2013; 62, 310-320. 7. Liu, L., Yang, J., Xie, J. et al. The potent antimicrobial properties of cell penetrating peptideconjugated silver nanoparticles with excellent selectivity for Gram-positive bacteria over erythrocytes. Nanoscale 2013; 5, 3834-3840. 8. Moulton, M.C., Braydich-Stolle,
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References 1. Tian, J., Wong, K.K., Ho, C.M. et al. Topical delivery of silver nanoparticles promotes wound healing. ChemMedChem 2007; 2, 129-136. 2. Atiyeh, B.S., Costagliola, M., Hayek, S.N., Dibo, S.A. Effect of silver on burn wound infection control and healing: review of the literature. Burns 2007; 33:2,
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Additionally, lack of IL-10 overexpression indicated absence of cytotoxicity for synthesised AgNPs.27This was further substantiated from the in vitro cytotoxicity studies in normal human skin fibroblasts, TE 353.Sk cell line. Percent cell viability was determined taking untreated cells as 100% viable. There was an insignificant fall in cell viability up to 200µg/ ml Ag concentrations (p>0.05), indicating negligible toxicity of AgNPs for normal cells (Fig 6). Charged inorganic chemicals are toxic because they generate reactive oxygen species (ROS).9 In other silver reduction reactions, chemicals used as reducing or capping agents possess their own toxic potential that triggers the production of ROS and causes toxicity.8 Unlike chemicals, bioresources like Bryonia laciniosa are safe to use.5 Bare AgNPs are unstable and highly reactive species that induce generation of ROS and, thus, toxicity. Capping bare AgNPs with a Bryonia laciniosa coat improved their stability, precluded the formation of ROS and, hence, overcame the AgNP toxicity. No significant change in percent cell viability was observed up to 200µg/ml Ag, implying cytocompatibility of AgNPs. As the synthesised AgNP dispersion and gel contained Ag concentrations less than 200µg/ml, which is sufficient to impart desirable antimicrobial and wound healing activities, no cytotoxicity concerns were associated with AgNP gel application. With the development of nanotechnology, the antibacterial efficiency of AgNPs has been revived and reclaimed.3,7,9 Outcomes from numerous studies imply that nanosilver helps to modify the inflammatory activities in wounds and assists in the preliminary phases of wound healing.6,24 Several in vitro studies demonstrated various inflammatory responses of AgNPs accountable for enhanced activity as well as toxicity.4,28,29 Thus, it is important to assess the prophylactic-therapeutic action and toxicity of developed AgNPs. In this study, stable AgNPs with improved surface properties developed via a green synthetic approach possessed a significant prophylactic antimicrobial effect for rapid, scarless wound repair (as depicted from rat excision model, scratch assays and negligi-
% Cell viability
Cytotoxicty
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research L.K., Nadagouda, M.N. et al. Synthesis, characterization and biocompatibility of ‘‘green’’ synthesized silver nanoparticles using tea polyphenols. Nanoscale 2010; 2, 763-770. 9. Mijnendonckx, K., Leys, N., Mahillon, J. et al. Antimicrobial silver: uses, toxicity and potential for resistance. Biometals. 2013; 26, 609-621. 10. He, Y., Du, Z., Lv, H. et al. Green synthesis of silver nanoparticles by Chrysanthemum morifolium Ramat. extract and their application in clinical ultrasound gel. Int J Nanomedicine 2013; 8, 1809-1815. 11. Iravani, S. Green synthesis of metal nanoparticles using plants. Green Chem. 2011; 13, 2638-2650. 12. Singh, V., Malviya, T., Tripathi, D., Naraian, U. An Escherichia coli antimicrobial effect of arabinoglucomannan from fruit of Bryonia lacinosa. Carbohydr Polym. 2009; 75, 534-537. 13. Sivakumar, T., Perumal, P., Kumar, R.S. et al. Evaluation of analgesic, antipyretic activity and toxicity study of Bryonia laciniosa in mice and rats. Am J
Chin Med. 2004; 32:4, 531-539. 14. Moghe, A.S., Gangal, S.G., Shilkar, P.R. In vitro cytotoxicity of Bryonia laciniosa (Linn.) Naud. on human cancer cell lines. Ind J Nat Products and Resources 2011; 2:3, 322-329. 15. Dhar, S., Murawala, P., Shiras, A. et al. Gellan gum capped silver nanoparticle dispersions and hydrogels: cytotoxicity and in vitro diffusion studies. Nanoscale. 2012; 4, 563-567. 16. Kora, A.J., Beedu, S.R, Jayaraman, A. Size-controlled green synthesis of silver nanoparticles mediated by gum ghatti (Anogeissus latifolia) and its biological activity. Org Med Chem Lett 2012; 2:1, 17-27. 17. Venkatpurwar, V., Pokharkar, V. Green synthesis of silver nanoparticles using marine polysaccharide: study of in-vitro antibacterial activity. Mater Lett 2011; 65:6, 999-1002. 18. Silverstein, R., Bassler, G.C., Morrill, T.C. Spectrometric identification of organic compounds. John Wiley and Sons, 1981. 19. Khan, M., Khan, M., Adil, S.F. et al. Green synthesis of silver nanoparticles mediated by
Pulicaria glutinosa extract. Int J Nanomedicine 2013; 8, 1507-1516. 20. Khanna, P., Kulkarni, D., Beri, R. Synthesis and characterization of myristic acid capped silver nanoparticles. J Nanopart Res 2008; 10, 1059-1062. 21. Mishra, P.R., Shaal, L., Müller, R.H., Keck, C.M. Production and characterization of hesperetin nanosuspensions for dermal delivery. Int J Pharm 2009; 371, 182-189. 22. Wong, K.K, Cheung, S.O, Huang, L. et al. Further evidence of the anti-inflammatory effects of silver nanoparticles. ChemMedChem 2009; 4:7, 1129-1135. 23. Edwards-Jones, V. Nanocrystalline silver: use in wound care. In: Slevin, M. (eds). Current advances in the medical application of nanotechnology. Bentham Science Publishers, 2012. 24. Jain, J., Arora, S., Rajwade, J.M. et al. Silver nanoparticles in therapeutics: development of an antimicrobial gel formulation for topical use. Mol Pharm 2009; 6:5, 1388-1401. 25. Barrientos, S., Stojadinovic,
O., Golinko, M. et al. Growth factors and cytokines in wound healing. Wound Repair Regen. 2008; 16, 585-601. 26. Be´cherel, P., LeGoff, L., Ktorza, S. et al. Interleukin-10 inhibits IgE-mediated nitric oxide synthase induction and cytokine synthesis in normal human keratinocytes. Eur J Immunol 1995; 25, 2992-2995. 27. Asadullah, K., Sterry, W., Volk, H. Interleukin-10 TherapyReview of a New Approach. Pharmacol Rev 2003; 55:2, 241-269. 28. Yilma, A.N., Singh, S.R., Dixit, S., Dennis, V.A. Anti-inflammatory effects of silver-polyvinyl pyrrolidone (Ag-PVP) nanoparticles in mouse macrophages infected with live Chlamydia trachomatis. Int J Nanomedicine 2013; 8, 2421-2432. 29. Haberl, N., Hirn, S., Wenk, A. et al. Cytotoxic and proinflammatory effects of PVP-coated silver nanoparticles after intratracheal instillation in rats. Beilstein J Nanotechnol. 2013; 4, 933-940.
Trends in Wound Care Volume V
field has progressed in recent years,
About the author
and helps busy clinicians keep
on of chapters shows how this appraised of important researc h.
Keith Cutting It offers something for those with practical as well as is Principaa l Lecture r in Tissue Viability infocus the Faculty of Society and Health, Buckinghamshire New Univers ity. He has been involved in tissue viability for a number of years and worked in what has now become covered science and theoretical debate. Topics include: the Wound Healing Researc h Unit in Cardiff. Apart from lecturing on wound care manage
ment he has maintained clinical and research roles and has supported these activities via a number of publications. Keith is also Clinical Editor of Wound Journal and is a member of a number s-UK
of wound healing societies. He is a Fellow of the Academy of Higher Education and a Regiona • Wound survey and auditwith l Fellow of the Royal Society of Medicine, and he works closely various international medical as an independent consultant.
• Bacterial profiling and biofilms
device, pharmaceutical, biotech nology and publishing compan ies
Trends in Wound Care Volume V
This highly reputable source monographs has become a Aboutof the book This highly reputable source of up-to-date monographs has become a standar seeking to keep in d text forareas standard text for those seeking to touch with key those touchkeep with key areasin of clinical and scientific research. This volume eclectic miscellany of chapters, contains an each based upon published (and so, peer-reviewed) articles from the Journal of Wound Care. Where importa of clinical and scientific research. Edited by Keith Cutting, this nt new informa tion has been published, chapter been updated accordingly. Topics s have included in this volume are: wound survey/ audit, topical negative pressure, bacterial profiling and biofilms, wound pH, scar assessm ent, volume maintains the established fi role of nitric oxide, andstandard. broblas t senesce nce, the theories on wound contraction. This collecti
Trends in Wound Care Volume V Edited by Keith Cutting
• Wound pH • Scar assessment • Fibroblast senesence • The role of nitric oxide
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ISBN-13: 978-1-85642-374-8; 234 x 156 mm; paperback; 120 pages; published 2009; £29.99 9
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• Wound contraction theories
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Probing the wound healing potential of biogenic silver nanoparticles Objective: Silver nanoparticles (AgNPs) are known for their antimicrobial profile and wound healing activities. However, cytotoxicity and cosmetic abnormalities associated with silver pose a major challenge in their translation for therapeutic applications. Our objective was to develop biogenic AgNPs, using a singlestep green synthesis, and to investigate their in vitro and in vivo behaviour as wound-healing agents. l Method: AgNPs were prepared using the green synthesis approach with aqueous Bryonia laciniosa leaves extract. The AgNPs were then evaluated for physicochemical properties, stability, and antimicrobial and in vivo wound healing activities. l Results: Stable AgNPs with characteristic absorption at 408nm and 15±3nm particle size were generated via the active involvement of Bryonia laciniosa. No loss of stability was detected after 6 months at room temperature. Antibacterial activity was observed against both Gram-negative and Gram-positive bacteria with no cytotoxicity observed in vitro at a concentration of 200 μg/mL and effective cytokine modulation. In vivo wound healing experiments showed improved wound contracting ability in rats where, after 14 days, wound alleviation was 47.1±2.2% in the control groups, compared with 78.1±1.4% and 92.6±6.7% for a silver-based marketed cream and the AgNPs, respectively. l Conclusion: The developed AgNPs proved to be superior wound healing agents owing to scarless healing with insignificant inflammation and toxicity. l Declaration of interest: There were no external sources of funding for this study. The authors have no conflicts of interest to declare. l
silver, nanoparticles, antimicrobial, wound healing, cytocompatibility
J O U R N A L O F WO U N D C A R E V O L 2 3 , N O 9 , S E P T E M B E R 2 0 1 4
particles (AgNPs). AgNPs display distinguishing physicochemical and biological properties on account of tailored surface and size characteristics.4 AgNPs have a higher surface area and can markedly increase the release rate of singly charged silver, the soluble and most biologically active form of silver. The AgNPs have multiple antimicrobial mechanisms. On exposure to moisture or wound fluid at the wound surface, AgNPs are oxidised and release charged silver ions. The charged form of silver causes cell membrane damage by interacting with cell surface proteins of pathogenic micro-organisms5 and penetrating inside the cell. Intracellular charged AgNPs prevent DNA replication, inactivate the bacterial respiratory enzymes and impede cell division, resulting in cell lysis. Additionally, charged AgNPs are capable of generating free radicals with remarkable bactericidal potential.6,7 This leads to enhanced antimicrobial efficacy demonstrated by AgNPs. On the other hand, the improved surface features of AgNPs also carry the risk of aggregation owing to instability. Unstable AgNPs interact with or accumulate in normal cell organelles, leading to cytotoxicity.8 Thus, it becomes imperative to balance the wound healing efficacy of AgNPs against their toxicity. The activity and toxicity of nanoparticulate systems are associated with their synthesis method, reducing agent, nature, concentration and physicochemical characteristics.9 Nanoparticles are synthesised and stabilised via physicochemical methods:
V. Dhapte,1 MPharm; S. Kadam,2 PhD; A. Moghe,3 PhD; V. Pokharkar,1 PhD; 1Department of Pharmaceutics, Bharati Vidyapeeth University, Poona College of Pharmacy, Pune, India 2 Bharati Vidyapeeth University, Bharati Vidyapeeth Bhavan, Lal Bahadur Shastri Marg, Pune, India 3 Department of Cell and Molecular Biology, Bharati Vidyapeeth University, Rajiv Gandhi Institute of IT and Biotechnology, Katraj, Pune, India. Email: vbpokharkar@ yahoo.co.in
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W
ound healing is a multifaceted process, comprised of coagulation, inflammation, tissue formation, and epithelialisation.1 The normal wound-healing process involves inflammation at the injury site, angiogenesis followed by granulation, tissue growth, connective tissue and epithelium repair along with remodelling to heal the wound completely. Inflammatory pathways and cytokine modulation influence the healing process, and a number of immunological mediators and microbial colonisation can slow the repair of an injured site.2 While early inflammatory effects help in abolishing the microbial infection, prolonged inflammatory responses can lead to tissue damage.3 Microbial pathogens in the wound fluid produce toxins and enzymes that prolong the inflammatory responses and hence delay the wound recovery. Rapid wound repair with minimal scarring is essential to avoid these complications. Topical antimicrobials offer a prophylactic answer to some of these issues but, with the risk of resistance, are less helpful in wound therapy. Conventionally, silver-based products containing silver compounds were recommended in wound management for their bactericidal properties. However, high application frequency has led to reports of inflammatory effects and cosmetic defects.2 With the aid of nanotechnology the solubility and surface properties of silver can be improved by the formation of silver nano-
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research
a
b 408nm
1.25
Absorbance
1 0.75 0.5 0.25 0
300
c
400 500 Wavelength (nm)
600
50nm
AgNPs
%T
3227
1369 1541
Bryonia laciniosa
1986
3289
1647
2809 2917
4000
3500
3000
2000
Wavenumbers (cm-1)
432
1002
1324
2500
1500
Green synthesis and characterisation of AgNPs Various physicochemical characterisation tests were performed to ascertain the complete green synthesis of AgNPs using aqueous Bryonia laciniosa leaf extract along with AgNP charge and stability. Using our earlier reported method,13 0.1% w/v aqueous Bryonia laciniosa leaf extract was prepared and employed for reduction of 0.01M aqueous silver nitrate solution (Sigma Aldrich, USA) to AgNPs under magnetic stirring for 1 hour at room temperature. Subsequently, the AgNP dispersion was dialysed using 12kDa cut-off membrane for 24 hours to remove the unreacted ions and retain AgNPs only.15 The retained AgNPs were examined for silver content using atomic absorption spectrophotometry (AA 201, Chemito, India).15 In order to investigate the wound-healing applications of AgNPs, hydrophilic gels were prepared in gellan gum. A weighed amount of gellan gum was sprinkled onto the AgNP dispersion and allowed to form AgNP gel. The amount of silver in the AgNP gel was found using atomic absorption spectrophotometry to be 0.18 mg/g. The surface plasmon resonance (SPR) of the AgNP dispersion was analysed by UV/visible spectrophotometry (Jasco, Model V-570, Japan) operating at a resolution of 2nm. Fourier transformed infrared spectroscopy (FTIR) analysis of Bryonia laciniosa leaf extract and AgNPs was conducted (Thermo Scientific Nicolet iS50R). These aqueous dispersions were scanned from 500–4000 cm-1 for comparison of overlap regions and functional group identifications. The sample for X-ray diffraction (XRD) measurement was prepared by drop-coating a film of AgNPs on glass substrate. XRD patterns were recorded on X-ray Brucker AXS diffractometer (D8 Advance, USA) in the 2q angle range of 5–80°. Average crystalline size (D) was calculated according to the DebyeScherer’s formula: D=
1402
1633 1538
Methods
1015 1000
500
Kλ βCosθ
where b is the full width at half-maximum (FWHM) intensity of a peak at an angle q; K is the constant, depending on the line shape profile; and l is the wave length of the X-ray source. Transmission electron microscopic (TEM) analysis to determine size and morphology of AgNPs was performed on a TECHNAI G2 F30 S-TWIN instrument. The sample was prepared by placing a drop of AgNP dispersion on a carbon-coated copper grid and then air-drying it at room temperature. Measurements
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Fig 1. UV/visible absorption spectrum (a), TEM image (b) and FTIR spectrum (c) of AgNPs.
traction ability, inflammatory responses, and cytotoxicity of AgNPs synthesised with Bryonia laciniosa leaf extract in wound management using various in vitro and in vivo tests.
© 2014 MA Healthcare
electrochemical, reduction, vapour deposition and a microwave-assisted process. However, contamination from precursors, use of toxic solvents, hazardous byproducts and environmental concerns limit the application of these techniques. Green synthesis is a wellestablished route of nanoparticulate synthesis that is safe because it eliminates the toxicity arising from physicochemical approaches.10 Green synthesis employs plants, algae, fungi, yeasts, bacteria and their products to synthesise nanoparticles. Plants and plant extracts are preferred over others as they are easy to handle, readily available and economical.11 In Indian medicine the Bryonia laciniosa (Linn.) plant is a bitter tonic and mild laxative, which is reported to have extensive medicinal uses including as an analgesic and antipyretic, a treatment for asthma and snake bites, and a fertility aid.12 Several researchers have investigated organic extracts of Bryonia laciniosa fruit, seeds and leaves for antimicrobial, antioxidant, anti-inflammatory, cathartic and antitumour activities.13 Herbal preparations containing organic solvent extracts of Bryonia laciniosa remained less explored owing to the associated adverse side effects arising from the presence of residual organic solvents. Recently, aqueous Bryonia laciniosa extracts showed higher biological activity than its organic extracts.14 Here we assess and correlate the antimicrobial profile, wound con-
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research ty of AgNPs against the Gram-negative bacterium Escherichia coli (ATCC 25922) and the Gram-positive bacterium Staphylococcus aureus (ATCC 6538) was investigated using a well-diffusion assay.16 The autoclaved Luria-Bertani agar (Himedia Laboratories, India) plates were inoculated with 106 colony forming units (CFU), of one bacterial strain per plate, using a glass spreader. Uniform well-like cavities were bored and supplemented with various concentrations of AgNPs. These inoculated and supplemented plates were incubated aerobically at 37°C in a bacteriological incubator. After 24 hours incubation the zone of inhibition (ZOI) was calculated as the difference between the total inhibition zone diameter and the well diameter.
b
Bryonia laciniosa
5
20
35 50 20 (degree)
65
80
Wound healing activity l In vivo studies The IAEC (Institutional Animal
b Ag
Ag Bryonia laciniosa
Ag++NO3 Silver nitrate
Magnetic stirring Ag
Ag
Redution and capping
Anionic (negatively charged) silver nanoparticles (AgNPs)
Absorbance
0.9 0.6 0.3 0 300
1.2 0.9 0.6
pH2 pH4 pH6 pH8 pH10
0.3 0
400 500 600 Wavelength (nm)
Stability: up to 6 months
300 400 500 600 Wavelength (nm) pH stability
Absorbance
0 day 15 days 1 month 2 months 3 months 6 months
1.2
Absorbance
c 10-1 M 10-2 M 10-3 M 10-4 M 10-5 M 10-6 M
1.2 0.9 0.6 0.3 0 300
400 500 600 Wavelength (nm)
Electrolytic stability
were performed at an accelerated voltage of 300kV with a lattice resolution of 0.14nm and point image resolution of 0.20nm. The surface charge of the AgNPs (without any dilution) was assessed from the zeta potential measurements using the Malvern zetasizer Nano series ZS90 (Malvern Instruments, UK). The stability of the AgNPs was studied with respect to time, variable pH and electrolytic conditions. At regular time intervals, AgNP stability was reported at ambient temperature conditions up to 6 months. The effect of pH variation on the AgNP stability was studied between pH 2–10 after 24 hours. Electrolytic stability was assessed by adding 10−1–10−6M NaCl to the AgNP dispersion for 24 hours. AgNP stability was evaluated from the SPR obtained using UV/visible spectroscopy.
Antibacterial activity The antibacterial effect of the AgNPs was studied in the presence of increasing concentrations of AgNPs, above their reported minimum inhibitory concentration (MIC) of 2.5μg/ml.10 The antibacterial activi434
Ethics Committee) approved in vivo wound healing study protocol was performed following the guidelines of the committee for the purpose of control and supervision of experiments on animals (CPCSEA, India). During the experiments, animals were handled according to Good Laboratory Practice. In an experimental rat model, male Wistar rats were anaesthetised and a circular excision wound of 530.66mm2 and 2mm depth was made on their shaved back. There were three treatment groups with four animals per group: l Group 1 control (blank gellan gum gel) l Group 2 applied marketed cream (containing silver sulfadiazine) l Group 3 AgNPs gel. Equal amounts of control, marketed cream and AgNP gel (0.5g) were used for the treatment once a day. However, the applied silver content varied as the marketed cream contained silver sulfadiazine compound (Ag content=3.18mg/g) whereas the AgNP gel contained stabilised Ag in nano size (Ag content=0.18mg/g). Progressive changes in wound area were monitored planimetrically, photographed at defined intervals and further processed for the scratch assay. The area of the wounds was measured on days 0, 7 and 14 post-wounding. Percent wound alleviation was measured using following formula: % Wound = Wound Area(Day 0) - Wound Area (Day n) x 100 Wound Area (Day 0) alleviation
Where ‘n’ indicates number of days, either 7 or 14.
In vitro studies Serum levels of human interleukin-6 (IL-6) and human interleukin-10 (IL10) were determined in triplicate with cytokinespecific ELISA kits. Cytotoxicity of AgNPs was evaluated in the TE 353.Sk cell line derived from normal human skin fibroblasts. Adherent TE 353.Sk cells were cultured l
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a
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(220)
(200)
Intensity
a
(311)
(111)
Fig 2. XRD patterns (a), scheme for negatively charged AgNP synthesis (b), and stability of AgNPs (c).
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Journal of Wound Care.Downloaded from magonlinelibrary.com by 157.211.003.015 on January 8, 2015. For personal use only. No other uses without permission. . All rights reserved.
research Fig 3. Antibacterial activity of AgNPs against Staphylococcus aureus (a) and Escherichia coli (b) and related comparison of zone of inhibition (c). b
c
10μg/ml 7.5μg/ml
10μg/ml
0μg/ml
0μg/ml 2.5μg/ml 2.5μg/ml
7.5μg/ml
Zone of inhibition (mm)
a
5μg/ml
5μg/ml
16
S. aureus
AgNPs E. coli
E. coli 12
8
4
0
AgNPs S. aureus 0
5
10
15
Statistical analysis Statistical significance was determined using analysis of variance (ANOVA). A value of p10-2 M (Fig 2c). Under strong acidic conditions or higher electrolytic concentration, the negatively charged, Bryonia laciniosa-capped AgNPs tend to form complexes or undergo ionization, thereby disturbing the surface charges on the stable AgNPs. This causes a lowering of electrostatic repulsion and increases in aggregation and instability.15 AgNPs were stable for up to 6 months, as evident from their SPR, which was monitored at regular intervals (Fig 2c). On the whole, AgNPs proved to be highly stable with a good shelf life.
The effect of AgNPs on bacteria The antibacterial effect of AgNPs in increasing concentrations is shown in Fig 3. As depicted from the clear zones around inoculated wells, AgNPs were capable of inhibiting both Gram-positive Staphylococcus aureus as well as Gram-negative Escherichia coli bacteria (Fig 3a and 3b). The ZOI in the plate containing Escherichia coli increased exponentially up to a concentration of 7.5μg/ml after which the inhibitory effect levelled off irrespective of further increases in the concentration of AgNPs (Fig 3c). On the other hand, there was gradual increase in ZOI with Staphylococcus aureus up to 5μg/ml AgNPs. Beyond this, AgNPs showed a steep rise in the inhibitory effect, followed by a steady levelling effect at concentrations above 7.5μg/ml. Inhibitory potential for Staphylococcus aureus and Escherichia coli was similar at and above 10μg/ml AgNPs (Fig 3c) but at lower concentrations there was a difference in the antibacterial potential. 438
Marketed cream
AgNP gel
This discrepancy could be attributed to structural variance between Gram-positive and Gram-negative bacteria. In Gram-positive bacteria like Staphylococcus aureus, the outer cell wall is thicker (Fig 3c) than in Gram-negative bacteria (Escherichia coli ) owing to more peptidoglycan layers.17 As a result, uptake of AgNPs across the thin cell wall of Escherichia coli was faster, leading to rapid inactivation of cellular components followed by cell death. Conversely, the thicker peptidoglycan layer resisted the initial internalisation of AgNPs into Staphylococcus aureus and thus delayed the cell lysis.
In vivo wound healing All three treatment groups demonstrated minor wound contraction with a weak inflammatory response during the first 7 days on account of initial marginal influx of neutrophils.4 At day 14 the control group showed 47.07±2.19% wound alleviation, the marketed preparation containing silver sulfadiazine cured 78.13±1.35% of the wound while AgNP gel healed 92.54±3.67% of the wound (Fig 4a). Therefore, the AgNP gel-treated group showed a rapid wound epithelialisation compared with control and marketed cream-treated animal wounds. On comparing the appearance of healed wounds, the control group showed scarring as well as rubor of a considerable size, even after 14 days (Fig 4b). As a rule, scar formation and characteristic inflammatory responses are inherently associated with wound recovery caused by discrepancy among immune response or hypersensitivity.22 Bioactive materials including silver-based compounds, capable of altering the cytokine cascade, could improve wound appearance through immunomodulation.23 On application of silver sulfadiazine-containing marketed cream for 14 days, there was noteworthy
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14 days
© 2014 MA Healthcare
7 days
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Journal of Wound Care.Downloaded from magonlinelibrary.com by 157.211.003.015 on January 8, 2015. For personal use only. No other uses without permission. . All rights reserved.
research Fig 5. Schematic representation of wound healing mechanism. AgNP gel
Intact skin
Marketed cream Microbial infection Wound due to injury
Influx of neutrophils Control
Marketed cream
Release of proinflammatory mediators
AgNP gel
Stimulates fibroblasts, keratinocytes and macrophages Scar formation and inflammation
Release of anti-inflammatory mediators
Significant rubor and scar
Reduced rubor and scar
No Rubor, no Scar
reduction in the scar area along with significant inflammation. Such silver compounds are known to be toxic to fibroblast and epithelial cells, giving rise to an inflammatory response.24 However, treatment with synthesised AgNP gel for 14 days completely repaired the wound with negligible scarring and significant loss of rubor. The relative wound healing mechanism is represented in Fig 5 and supported by scratch assays and ELISA. In the quantitative analysis via scratch assays, wound alleviation was represented by the percentage of cell covered area while swelling, erythema and inflammation were reflected from the percent scratch area. As observed in Table 1, AgNP geltreated wound showed considerable decrease in scratch area with rapid upsurge in the cell covered area compared to the control and marketed cream at 14 days. Thus, formulations containing AgNPs reduce scar formation and inflammation through cytokine modulation.4,24
in cellular proliferation, keratinocyte migration and extracellular matrix production, which sequentially leads to scar formation.25 As a result, lower IL-6 levels (23.79ng/ml) induced by AgNPs reduced inflammation along with scarless wound recovery. In response to inflammation, IL-10, a key cytokine, is produced by keratinocytes and inflammatory cells to neutralise the effects of pro-inflammatory cytokines like IL-6.26 Its expression is regulated by the magnitude of inflammation and pro-inflammatory cytokine levels (Fig 5). Thus, lower IL-6 levels could stimulate relatively minuscule IL-10 levels
Cytokine-specific ELISA measurements of synthesised AgNPs revealed their wound-healing mechanism from the significantly lower levels of cytokines IL-6 and IL-10. Pro-inflammatory cytokine IL-6 stimulates fibroblast proliferation and monocyte activation, which in turn give rise to inflammatory responses. Moreover, IL-6 initiates release of neutrophils and macrophages to the wound, resulting J O U R N A L O F WO U N D C A R E V O L 2 3 , N O 9 , S E P T E M B E R 2 0 1 4
Treatment
Days
Scratch area (%)
Cell covered area (%)
Control
0
85.59±5.79
12.67±2.17
7
66.98±7.23
31.91±2.33
14
28.51±6.41
70.67±4.91
0
86.11±7.31
12.23±3.07
7
54.37±3.67
44.33±2.77
14
17.48±1.92
82.50±1.69
0
84.72±6.47
13.41±1.33
7
40.02±4.33
59.13±2.86
14
4.67±0.73
93.72±3.45
Marketed cream
AgNP gel
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Effect of AgNPs on cytokine levels
Table 1: Quantitative analysis of wounds by scratch assays
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research (153.8pg/ml) suggestive of an overall decrease in inflammation and scarless wound healing on application of AgNP gel.
Fig 6. Cytotoxicity evaluation of various AgNP concentrations in TE 353.Sk cell line. 100
440
139-148. 3. Wright, J.B., Hansen, D.L., Burrell, R.E. The comparative efficacy of two antimicrobial barrier dressings: in vitro examination of two controlled release of silver dressings. Wounds. 1998; 10:6, 179–188. 4. Rai, M., Yadav, A., Gade, A. Silver nanoparticles as a new generation of antimicrobials.
50 25 0 0
50
100
150
200
Concentration of Ag (μg/ml)
ble interleukin levels) without any major cytotoxicity. Such cytocompatible AgNP formulations with better efficacy would definitely be acceptable in wound healing, management of burns, delayed impaired wound treatment in diabetes and other such topical ailments. Certainly, these cytocompatible silver nanoparticles would emerge as effective and safe alternatives to the existing conventional treatments. However, the findings of this study are restricted to topical injuries and wounds. Future studies should explore the potential of these biogenic silver nanoparticles in diabetic delayed wound healing and special clinical ailments.
Conclusion A simple, single-step reduction process mediated by aqueous Bryonia laciniosa extract generated stable, crystalline, anionic (negatively charged) silver nanoparticles of 15±3nm size. These nanoparticles were highly stable up to 6 months and under varying pH and electrolyte concentrations. Biosynthesised silver nanoparticles demonstrated their enhanced antimicrobial potential against Gram-positive as well as Gram-negative bacteria at concentrations where no cytotoxicty was observed. They proved to be superior wound healing candidates that offered rapid, scarless wound recovery without any major inflammation owing to effective cytokine modulation. Thus, the biosynthesised silver nanoparticles proved to be safe, prophylactic-therapeutic answer for successful wound recovery. n Biotechnol Adv 2009; 27, 76-83. 5. Wong, K., Liu, X. Silver nanoparticles-the real ‘‘silver bullet’’ in clinical medicine? MedChemCommun. 2010; 1, 125-131. 6. Anisha, B.S., Biswas, R., Chennazhi, K., Jayakumar, R. Chitosan–hyaluronic acid/nano silver composite sponges for drug resistant bacteria infected
diabetic wounds. Int J Biol Macromol 2013; 62, 310-320. 7. Liu, L., Yang, J., Xie, J. et al. The potent antimicrobial properties of cell penetrating peptideconjugated silver nanoparticles with excellent selectivity for Gram-positive bacteria over erythrocytes. Nanoscale 2013; 5, 3834-3840. 8. Moulton, M.C., Braydich-Stolle,
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References 1. Tian, J., Wong, K.K., Ho, C.M. et al. Topical delivery of silver nanoparticles promotes wound healing. ChemMedChem 2007; 2, 129-136. 2. Atiyeh, B.S., Costagliola, M., Hayek, S.N., Dibo, S.A. Effect of silver on burn wound infection control and healing: review of the literature. Burns 2007; 33:2,
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Additionally, lack of IL-10 overexpression indicated absence of cytotoxicity for synthesised AgNPs.27This was further substantiated from the in vitro cytotoxicity studies in normal human skin fibroblasts, TE 353.Sk cell line. Percent cell viability was determined taking untreated cells as 100% viable. There was an insignificant fall in cell viability up to 200µg/ ml Ag concentrations (p>0.05), indicating negligible toxicity of AgNPs for normal cells (Fig 6). Charged inorganic chemicals are toxic because they generate reactive oxygen species (ROS).9 In other silver reduction reactions, chemicals used as reducing or capping agents possess their own toxic potential that triggers the production of ROS and causes toxicity.8 Unlike chemicals, bioresources like Bryonia laciniosa are safe to use.5 Bare AgNPs are unstable and highly reactive species that induce generation of ROS and, thus, toxicity. Capping bare AgNPs with a Bryonia laciniosa coat improved their stability, precluded the formation of ROS and, hence, overcame the AgNP toxicity. No significant change in percent cell viability was observed up to 200µg/ml Ag, implying cytocompatibility of AgNPs. As the synthesised AgNP dispersion and gel contained Ag concentrations less than 200µg/ml, which is sufficient to impart desirable antimicrobial and wound healing activities, no cytotoxicity concerns were associated with AgNP gel application. With the development of nanotechnology, the antibacterial efficiency of AgNPs has been revived and reclaimed.3,7,9 Outcomes from numerous studies imply that nanosilver helps to modify the inflammatory activities in wounds and assists in the preliminary phases of wound healing.6,24 Several in vitro studies demonstrated various inflammatory responses of AgNPs accountable for enhanced activity as well as toxicity.4,28,29 Thus, it is important to assess the prophylactic-therapeutic action and toxicity of developed AgNPs. In this study, stable AgNPs with improved surface properties developed via a green synthetic approach possessed a significant prophylactic antimicrobial effect for rapid, scarless wound repair (as depicted from rat excision model, scratch assays and negligi-
% Cell viability
Cytotoxicty
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research L.K., Nadagouda, M.N. et al. Synthesis, characterization and biocompatibility of ‘‘green’’ synthesized silver nanoparticles using tea polyphenols. Nanoscale 2010; 2, 763-770. 9. Mijnendonckx, K., Leys, N., Mahillon, J. et al. Antimicrobial silver: uses, toxicity and potential for resistance. Biometals. 2013; 26, 609-621. 10. He, Y., Du, Z., Lv, H. et al. Green synthesis of silver nanoparticles by Chrysanthemum morifolium Ramat. extract and their application in clinical ultrasound gel. Int J Nanomedicine 2013; 8, 1809-1815. 11. Iravani, S. Green synthesis of metal nanoparticles using plants. Green Chem. 2011; 13, 2638-2650. 12. Singh, V., Malviya, T., Tripathi, D., Naraian, U. An Escherichia coli antimicrobial effect of arabinoglucomannan from fruit of Bryonia lacinosa. Carbohydr Polym. 2009; 75, 534-537. 13. Sivakumar, T., Perumal, P., Kumar, R.S. et al. Evaluation of analgesic, antipyretic activity and toxicity study of Bryonia laciniosa in mice and rats. Am J
Chin Med. 2004; 32:4, 531-539. 14. Moghe, A.S., Gangal, S.G., Shilkar, P.R. In vitro cytotoxicity of Bryonia laciniosa (Linn.) Naud. on human cancer cell lines. Ind J Nat Products and Resources 2011; 2:3, 322-329. 15. Dhar, S., Murawala, P., Shiras, A. et al. Gellan gum capped silver nanoparticle dispersions and hydrogels: cytotoxicity and in vitro diffusion studies. Nanoscale. 2012; 4, 563-567. 16. Kora, A.J., Beedu, S.R, Jayaraman, A. Size-controlled green synthesis of silver nanoparticles mediated by gum ghatti (Anogeissus latifolia) and its biological activity. Org Med Chem Lett 2012; 2:1, 17-27. 17. Venkatpurwar, V., Pokharkar, V. Green synthesis of silver nanoparticles using marine polysaccharide: study of in-vitro antibacterial activity. Mater Lett 2011; 65:6, 999-1002. 18. Silverstein, R., Bassler, G.C., Morrill, T.C. Spectrometric identification of organic compounds. John Wiley and Sons, 1981. 19. Khan, M., Khan, M., Adil, S.F. et al. Green synthesis of silver nanoparticles mediated by
Pulicaria glutinosa extract. Int J Nanomedicine 2013; 8, 1507-1516. 20. Khanna, P., Kulkarni, D., Beri, R. Synthesis and characterization of myristic acid capped silver nanoparticles. J Nanopart Res 2008; 10, 1059-1062. 21. Mishra, P.R., Shaal, L., Müller, R.H., Keck, C.M. Production and characterization of hesperetin nanosuspensions for dermal delivery. Int J Pharm 2009; 371, 182-189. 22. Wong, K.K, Cheung, S.O, Huang, L. et al. Further evidence of the anti-inflammatory effects of silver nanoparticles. ChemMedChem 2009; 4:7, 1129-1135. 23. Edwards-Jones, V. Nanocrystalline silver: use in wound care. In: Slevin, M. (eds). Current advances in the medical application of nanotechnology. Bentham Science Publishers, 2012. 24. Jain, J., Arora, S., Rajwade, J.M. et al. Silver nanoparticles in therapeutics: development of an antimicrobial gel formulation for topical use. Mol Pharm 2009; 6:5, 1388-1401. 25. Barrientos, S., Stojadinovic,
O., Golinko, M. et al. Growth factors and cytokines in wound healing. Wound Repair Regen. 2008; 16, 585-601. 26. Be´cherel, P., LeGoff, L., Ktorza, S. et al. Interleukin-10 inhibits IgE-mediated nitric oxide synthase induction and cytokine synthesis in normal human keratinocytes. Eur J Immunol 1995; 25, 2992-2995. 27. Asadullah, K., Sterry, W., Volk, H. Interleukin-10 TherapyReview of a New Approach. Pharmacol Rev 2003; 55:2, 241-269. 28. Yilma, A.N., Singh, S.R., Dixit, S., Dennis, V.A. Anti-inflammatory effects of silver-polyvinyl pyrrolidone (Ag-PVP) nanoparticles in mouse macrophages infected with live Chlamydia trachomatis. Int J Nanomedicine 2013; 8, 2421-2432. 29. Haberl, N., Hirn, S., Wenk, A. et al. Cytotoxic and proinflammatory effects of PVP-coated silver nanoparticles after intratracheal instillation in rats. Beilstein J Nanotechnol. 2013; 4, 933-940.
Trends in Wound Care Volume V
field has progressed in recent years,
About the author
and helps busy clinicians keep
on of chapters shows how this appraised of important researc h.
Keith Cutting It offers something for those with practical as well as is Principaa l Lecture r in Tissue Viability infocus the Faculty of Society and Health, Buckinghamshire New Univers ity. He has been involved in tissue viability for a number of years and worked in what has now become covered science and theoretical debate. Topics include: the Wound Healing Researc h Unit in Cardiff. Apart from lecturing on wound care manage
ment he has maintained clinical and research roles and has supported these activities via a number of publications. Keith is also Clinical Editor of Wound Journal and is a member of a number s-UK
of wound healing societies. He is a Fellow of the Academy of Higher Education and a Regiona • Wound survey and auditwith l Fellow of the Royal Society of Medicine, and he works closely various international medical as an independent consultant.
• Bacterial profiling and biofilms
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Trends in Wound Care Volume V
This highly reputable source monographs has become a Aboutof the book This highly reputable source of up-to-date monographs has become a standar seeking to keep in d text forareas standard text for those seeking to touch with key those touchkeep with key areasin of clinical and scientific research. This volume eclectic miscellany of chapters, contains an each based upon published (and so, peer-reviewed) articles from the Journal of Wound Care. Where importa of clinical and scientific research. Edited by Keith Cutting, this nt new informa tion has been published, chapter been updated accordingly. Topics s have included in this volume are: wound survey/ audit, topical negative pressure, bacterial profiling and biofilms, wound pH, scar assessm ent, volume maintains the established fi role of nitric oxide, andstandard. broblas t senesce nce, the theories on wound contraction. This collecti
Trends in Wound Care Volume V Edited by Keith Cutting
• Wound pH • Scar assessment • Fibroblast senesence • The role of nitric oxide
ltd
ISBN-13: 978-1-85642-374-8; 234 x 156 mm; paperback; 120 pages; published 2009; £29.99 9
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© 2014 MA Healthcare
ISBN 1-85642-374-3
Edited by Keith Cutting
• Wound contraction theories
J O U R N A L O F WO U N D C A R E V O L 2 3 , N O 9 , S E P T E M B E R 2 0 1 4
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Journal of Wound Care.Downloaded from magonlinelibrary.com by 157.211.003.015 on January 8, 2015. For personal use only. No other uses without permission. . All rights reserved.
