Development of novel ultrashort antimicrobial peptide nanoparticles with potent antimicrobial and antibiofilm activities against multidrug-resistant bacteria

Abstract

Conventional antibiotics are facing strong microbial resistance that has recently reached critical levels. This situation is leading to significantly reduced therapeutic potential of a huge proportion of antimicrobial agents currently used in clinical settings. Antimicrobial peptides (AMPs) could provide the medical community with an alternative strategy to traditional antibiotics for combating microbial resistance. However, the development of AMPs into clinically useful antibiotics is hampered by their relatively low stability, toxicity, and high manufacturing costs. In this study, a novel in-house-designed potent ultrashort AMP named RBRBR was encapsulated into chitosan-based nanoparticles (CS-NPs) based on the ionotropic gelation method. The encapsulation efficacy reported for RBRBR into CS-NPs was 51.33%, with a loading capacity of 10.17%. The release kinetics of RBRBR from the nanocarrier exhibited slow release followed by progressive linear release for 14 days. The antibacterial kinetics of RBRBR-CS-NPs was tested against four strains of Staphylococcus aureus for 4 days, and the developed RBRBR-CS-NPs exhibited a 3-log decrease in the number of colonies when compared to CS-NP and a 5-log decrease when compared to control bacteria. The encapsulated peptide NP formulation managed to limit the toxicity of the free peptide against both mammalian cells and human erythrocytes. Additionally, the peptide NPs demonstrated up to 98% inhibition of biofilm formation when tested against biofilm-forming bacteria. Loading RBRBR into CS-NPs could represent an innovative approach to develop delivery systems based on NP technology for achieving potent antimicrobial effects against multidrug-resistant and biofilm-forming bacteria, with negligible systemic toxicity and reduced synthetic costs, thereby overcoming the obstructions to clinical development of AMPs.

Keywords: antibiofilm; drug delivery; nanoparticles; ultrashort antimicrobial peptides.

Figure 1

Release kinetics of RBRBR from RBRBR-CS-NPs in PBS, pH 7.4, 37°C. Abbreviations: CFU, colony forming unit; CS-NPs, chitosan nanoparticles; PBS, phosphate buffered saline; RBRBR, novel in-house-designed potent ultrashort antimicrobial peptide.

Figure 2

Antibacterial activity of RBRBR-CS-NPs against control-strain Staphylococcus aureus (ATCC 29213). Notes: Data expressed as mean ± SEM of three independent experiments. *P<0.05 compared to RBRBR-CS-NPs. Abbreviations: ATCC, American Type Culture Collection; CFU, colony forming unit; CS-NPs, chitosan nanoparticles; RBRBR, novel in-house-designed potent ultrashort antimicrobial peptide.

Figure 3

Antibacterial activity against MRSA strains. Notes: Data expressed as mean ± SEM of three independent experiments. *P<0.05 compared to RBRBR-CS-NPs. Abbreviations: ATCC, American Type Culture Collection; CS-NPs, chitosan nanoparticles; MRSA, methicillin-resistant Staph ylococcus aureus; RBRBR, novel in-house-designed potent ultrashort antimicrobial peptide.

Figure 4

Antibiofilm activity of RBRBR-CS-NPs represented as % of viable bacterial cells/positive control after treatment. Notes: Results are the mean values ± SEM of three independent experiments. *P<0.05 compared to control. Abbreviations: CFU, colony forming unit; CS-NPs, chitosan nanoparticles; RBRBR, novel in-house-designed potent ultrashort antimicrobial peptide.

Figure 5

Hemolytic activity of RBRBR-CS-NPs against human red blood cells. Notes: Data expressed as means ± SEM of three independent experiments. *P<0.05 compared to RBRBR-CS-NPs. Abbreviations: CS-NPs, chitosan nanoparticles; RBRBR, novel in-house-designed potent ultrashort antimicrobial peptide.

Figure 6

Cytotoxicity range of free RBRBR concentrations after treatment with Vero mammalian cell line. Note: Free RBRBR IC₅₀ 187.2 µg/mL after 24 hours of incubation. Abbreviations: CS-NPs, chitosan nanoparticles; IC₅₀, half maximal inhibitory concentration; RBRBR, novel in-house-designed potent ultrashort antimicrobial peptide.

Figure 7

Cytotoxicity of RBRBR-CS-NPs vs free RBRBR. Note: *P<0.05 compared to RBRBR-CS-NPs. Abbreviations: CS-NPs, chitosan nanoparticles; RBRBR, novel in-house-designed potent ultrashort antimicrobial peptide.