Synthesis, construction, and evaluation of self-assembled nano-bacitracin A as an efficient antibacterial agent in vitro and in vivo

Abstract

Bacitracin A (BA) is an excellent polypeptide antibiotic that is active against gram-positive bacteria without triggering multidrug resistance. However, BA is inactive against gram-negative bacteria because of its inability to cross the outer membrane of these cells, and it has strong nephrotoxicity, thus limiting its clinical applications. Nanoantibiotics can effectively localize antibiotics to the periplasmic space of bacteria while decreasing the adverse effects of antibiotics. In this study, biodegradable hydrophobic copolymers of poly (d,l-lactide-co-glycolide) (PLGA) were attached to the N-termini of BA to design a novel class of self-assembled nano-bacitracin A (nano-BAs), and their potential as antibacterial agents was evaluated in vitro and in vivo. Nano-BAs had a core-shell structure with a mean diameter <150 nm. Impressively, nano-BAs had strong antibacterial properties against both gram-positive and gram-negative bacteria, and the distribution of antibacterial activity as a function of PLGA block length was skewed toward longer PLGA chains. No cytotoxicity against HK-2 cells or human red blood cells (hRBCs) was observed in vitro, suggesting good biocompatibility. A high local density of BA mass on the surface promoted endocytotic cellular uptake, and hydrophobic interactions between the PLGA block and lipopolysaccharide (LPS) facilitated the uptake of nano-BAs, thereby leading to greater antibacterial activities. In addition, Nano-BA_5K was found to be effective in vivo, and it served as an anti-infective agent for wound healing. Collectively, this study provides a cost-effective means of developing self-assembling nano-polypeptide antibiotic candidates with a broader antibacterial spectrum and a lower toxicity than commercially available peptide antibiotics, owing to their modification with biodegradable copolymers.

Keywords: bacitracin A; broader antibacterial spectrum; nano-BAs; poly (d,l-lactide-co-glycolide); wound healing.

Figure 1

Schematic illustration of the synthetic steps of BA–b–PLGA–b–BA. Abbreviation: BA, bacitracin A.

Figure 2

¹H NMR spectrum of bacitracin A (A), PLGA (B), CDI–PLGA–CDI (C), and BA–PLGA–BA (D). Abbreviations: NMR, nuclear magnetic resonance; BA, bacitracin A.

Figure 2

¹H NMR spectrum of bacitracin A (A), PLGA (B), CDI–PLGA–CDI (C), and BA–PLGA–BA (D). Abbreviations: NMR, nuclear magnetic resonance; BA, bacitracin A.

Figure 3

The typical GPC spectrum of PLGA_3K, PLGA_5K, PLGA_8K (A), and BA–PLGA_3K–BA, BA–PLGA_5K–BA, and BA–PLGA_8K–BA (B). Abbreviations: GPC, gel permeation chromatography; BA, bacitracin A.

Figure 4

TEM images, particle size, and distribution of Nano-BA_3K (A), Nano-BA_5K (B), and Nano-BA_8K (C) at pH 7.4. Abbreviation: TEM, transmission electron microscopy.

Figure 5

Time-kill kinetics of nano-BAs against E. coli ATCC 25922 (A) and S. aureus ATCC 29213 (B) for various periods of time. Experiments were performed in triplicate, and the antibacterial efficiency is expressed as the mean log (CFU/mL) ± SD, with the SD shown by the error bars. Abbreviations: ATCC, American Type Culture Collection; BA, bacitracin A.

Figure 6

SEM micrographs of E. coli ATCC 25922 treated by negative control (A), BA solution (B), Nano-BA_5K (C), and polymyxin B (D) for 2 h. Abbreviations: SEM, scanning electron microscopy; BA, bacitracin A; E. coli, Escherichia coli; ATCC, American Type Culture Collection.

Figure 7

TEM micrographs of E. coli ATCC 25922 treated by negative control (A), BA solution (B), Nano-BA_5K (C), and polymyxin B (D) for 2 h. Abbreviations: TEM, transmission electron microscopy; BA, bacitracin A; E. coli, Escherichia coli; ATCC, American Type Culture Collection.

Figure 8

In vitro cytotoxicity of Nano-BA_3K and Nano-BA_5K against HK-2 human tubular epithelial cells after 48-h incubation (mean ± SD, n=6). **P<0.05: significantly different from the BA solution (A). Dose-dependent hemolytic activities of nano-B compared with that of polymyxin B. The tests were repeated six times, and the data are expressed as the mean ± SD of six replicates. SD is shown by the error bars (B). Abbreviation: BA, bacitracin A.

Figure 9

Wound closure with Nano-BA_5K- (A, B), Polysporin^® ointment- (C, D), and saline- (E, F) treated mice at different time intervals. Abbreviation: BA, bacitracin A.

Figure 10

Bacterial survival rates on wounds treated with saline, Polysporin^® ointment, and 1× MIC Nano-BA_5K on days 0, 3, and 6 after infection (A). Hydroxyproline levels in the wound tissue from saline-, Polysporin^® ointment-, and 1× MIC Nano-BA_5K-treated animals at days 3 and 6 (B). Abbreviations: MIC, minimum inhibitory concentration; BA, bacitracin A.