(M0930-12-69) Phyto-Nano-Silver containing Lipo-Carriers for the Treatment of Thermal Burn Wounds

Purpose: Thermal burn wounds are conventionally treated with silver nitrate or silver sulphadiazine formulations. In particular, the second degree burn wounds require topical therapy using antibiotic containing creams and ointments, prior to bandaging. However, most products are primarily used to reduce the risk of infections, while the wounds are seldom healed with such treatments alone. Therefore, to mitigate the infections and to increase the wound healing ability, lipo-carriers were prepared incorporating the green tea extract and pectin-coated nano-silver.

Methods: The nano-silver particles were prepared using microwave irradiation of silver nitrate in the presence of pectin. The prepared pectin-nano-silver was added to the solution of green tea extract forming the phyto-nano-silver suspension (PNSS). For the preparation of lipo-carriers, the lipid phases (oleic acid, glyceryl monostearate, and cetyl alcohol) were melted at 50°C, and PNSS was added and microwaved for 2 min at 80 W. The cooled lipo-carriers were loaded into a suitable cream base and evaluated for their physicochemical, and occlusion properties. The anti-microbial study was carried out on individual strains and biofilms of S. aureus and E. coli (MTCC 3160 and 443) using the resazurin microtitre and crystal violet-acetic acid assay. The performance evaluation of the formulations was carried out on secondary thermal burn wounds inflicted on mouse models, followed by histological examinations.

Results: The resulting lipo-carriers had a particle size of < 200 nm, while the polydispersity index was 0.253 ± 0.124. The surface of the lipo-carriers was negative in the range of -24.26 to -37.55 mV indicating good colloidal stability. On the other hand, the prepared pectin-nano-silver had a particle size < 40 nm, while the surface was negative (-20.22 to -23.37 mV) indicating the successful coating of the pectin over positively charged silver ions. The creams loaded with the lipo-carriers had a pH around 6.8 to 7.2, a viscosity of ~12,300 cP, and an occlusion factor of 30.21 ± 2.21. Pectin-nano-silver and green tea extract were seen to be antimicrobial (Fig. 1) against S. aureus with an IC 50 of 25.65 µg/mL and 31.26 µg/mL respectively. However, the two samples were found to be lesser toxic to E. coli with an increased IC 50 value ( > 45.25 µg/mL). On the other hand, the effect against the biofilms (Fig. 1) were seen to be almost similar for the two strains when tested with pectin-nano-silver and green tea extract. The prominent effect was seen with the lipo-carriers with an effective IC 50 of 8.65 µg/mL and 11.35 µg/mL against E. coli and S. aureus. Interestingly, the lipo-carriers were able to mitigate the biofilms caused by both the strains, primarily due to the permeation and uptake of lipophilic materials by the bacteria (IC 50 < 18 µg/mL). The in vivo efficacy study (Fig. 2) showed the progressive damage of the untreated disease control with edematous content and pus, compared to the deranged layers seen immediately after the thermal burn wound infliction. At the end of 7 days treatment with the green tea extract loaded creams, a slight improvement in the dermal layers and the epidermis was seen, however having an extensive accumulation of neutrophils and inflammation. The treatment with the lipo-carriers showed a marked improvement in the histology of the skin tissues, with the reformation of the epidermis, and the peeling out of the dead skin tissues on the surface. The constituents of the lipo-carriers were seen to synergistically aid in the reformation of the neo-tissues, reappearance of the hair follicles, and neo-vascularization while reducing the chances of the infection in thermal burn wound inflicted animals.

Conclusion: The lipo-carriers containing pectin coated nano-silver, and green tea extract enhanced the healing process of the skin and its regrowth, after the infliction of secondary thermal burn wounds. The results indicate that the developed formulations can be a viable alternative to the conventional silver-containing formulations in the management of burn and other dermal wounds, while simultaneously targeting the infections and the healing process.

References: 1. Oaks RJ, et al. Silver sulfadiazine. In: StatPearls. Treasure Island (FL): StatPearls Publishing; 2022.
2. Fahimi S, et al. Wound healing activity of a traditionally used poly herbal product in a burn wound model in rats. Iran Red Crescent Med J. 2015;17(9):e19960.

Acknowledgements: The authors acknowledge the support from the characterization facilities and infrastructure available at the Central Instrumental Facility, Manipal Academy of Higher Education; and Manipal College of Pharmaceutical Sciences, India, and the DST-PURSE Centre, Mangalore University, India.


Fig. 1: Antimicrobial activity of the pectin-nano-silver, green tea extract and the lipo-carriers against E. coli and S. aureus, and their biofilms


Fig. 2: Histology images of the excised rat skin showing (1) the normal morphology, (2) immediately after the infliction of thermal burn wounds, (3) untreated control after 7 days, and treatment with (4) green tea extract cream and (5) cream with lipo-carriers containing pectin-nano-silver and green tea extract (Micrographs at 20X Magnification). Notations: (A) Stratum Corneum, (B) Epidermis, (C) Dermis, (D) Muscles, (a) and (b) Neo-formed stratum corneum and epidermis, (x) showing signs of edema and infection, and (y) showing the accumulation of inflammatory mediators