BrSPR-20-P1 peptide isolated from Brevibacillus sp. developed into liposomal hydrogel as a potential topical antimicrobial agent

Abstract

A novel BrSPR-20-P1 antimicrobial peptide (P1-AMP; NH₂-VVVNVLVKVLPPPVV-COOH) isolated from Brevibacillus sp. SPR-20 was encapsulated in a liposome containing varying proportions of L-α-phosphatidylcholine (PC) and cholesterol (CH). P1-AMP liposomes were incorporated into a chitosan hydrogel to achieve a peptide concentration of 0.02%. P1-AMP has been tested for its antibacterial and in vitro wound healing activities. The physicochemical characteristics of liposomes and hydrogel were investigated, including in vitro drug release, permeability, cell toxicity, antimicrobial activities, and stability studies. P1-AMP showed higher antimicrobial and wound-healing activities than the negative control. A toxicity test of P1-AMP in keratinocyte cell lines revealed cell viability of 100% at a concentration range of 1.96–1000 μg mL⁻¹. The empty liposomes exhibited an average particle size ranging from 324.5 ± 8.6 to 1823.7 ± 288.2 nm. The size range of P1-AMP liposomes was 378.6 ± 14.0 to 2363.0 ± 255.6 nm. The zeta potential of the blank liposome ranged from −40.43 ± 2.51 to −60.17 ± 0.93 mV and it decreased to −57.33 ± 0.72 to −70.33 ± 0.15 mV of the liposome loaded with peptide. SEM images showed liposomes were ovoid spheres with smooth surfaces. The chosen formulation, composed of PC to CH in an 18 : 1 ratio (formulation F3), had the highest entrapment effectiveness with small particle size and possessed an acceptable zeta potential. The developed P1-AMP liposome-loaded hydrogels exhibited a yellowish-clear appearance with a viscosity of 758.0 ± 149.8 cPs. The P1-AMP was rapidly released from the P1-AMP-loaded liposome hydrogel formulation. The P1-AMP-loaded liposome showed high permeability compared to P1-AMP alone or P1-AMP in hydrogel without the incorporation of liposomes. The minimum inhibitory concentration (MIC) against Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) of P1-AMP-loaded liposome hydrogel was 2 μg mL⁻¹, equivalent to P1-AMP. It completely killed S. aureus at 10× and 5× MIC after 6 and 12 h of incubation, respectively. The formulation did not induce cytotoxicity to the tested keratinocyte cell and remained stable for at least 6 months under the studied conditions.