Antimicrobial peptides (AMPs) can be used as alternative therapeutic agents to traditional antibiotics. These peptides have abundant natural template sources and can be isolated from animals, plants, and microorganisms. They are amphiphilic and mostly net positively charged, and they have a broad-spectrum inhibitory effect on bacteria, fungi, and viruses. AMPs possess significant rapid killing effects and do not interact with specific receptors on bacterial surfaces. As a result, drug resistance is rarely observed with treatments. AMPs, however, have some operational problems, such as a susceptibility to enzymatic (protease) degradation, toxicity in vivo, and unclear pharmacokinetics. However, nanodelivery systems loaded with AMPs provide a safe mechanism of packaging such peptides before they exert their antimicrobial actions, facilitate targeted delivery to the sites of infection, and control the release rate of peptides and reduce their toxic side effects. However, nanodelivery systems using AMPs are at an early stage of development and are still in the laboratory phase of development. There are also some challenges in incorporating AMPs into nanodelivery systems. Herein, an insight into the nanotechnology challenges in delivering AMPs, current advances, and remaining technological challenges are discussed in depth.

Temporin is an antimicrobial peptide (AMP) family discovered in the skin secretion of ranid frog that has become a promising alternative for conventional antibiotic therapy. Herein, a novel temporin peptide, Temporin-PF (TPF), was successfully identified from Pelophylax fukienensis. It exhibited potent activity against Gram-positive bacteria, but no effect on Gram-negative bacteria. Additionally, TPF exhibited aggregation effects in different solutions. Three analogs were further designed to study the relationship between the aggregation patterns and bioactivities, and the MD simulation was performed for revealing the pattern of the peptide assembly. As the results showed, all peptides were able to aggregate in the standard culture media and salt solutions, especially CaCl₂ and MgCl₂ buffers, where the aggregation was affected by the concentration of the salts. MD simulation reported that all peptides were able to form oligomers. The parent peptide assembly depended on the hydrophobic interaction via the residues in the middle domain of the sequence. However, the substitution of Trp/D-Trp resulted in an enhanced inter-peptide interaction in the zipper-like domain and eliminated overall biological activities. Our study suggested that introducing aromaticity at the zipper-like domain for temporin may not improve the bioactivities, which might be related to the formation of aggregates via the inter-peptide contacts at the zipper-like motif domain, and it could reduce the binding affinity to the lipid membrane of microorganisms.

Host-defence antimicrobial peptides (AMPs) from amphibians are usually considered as one of the most promising next-generation antibiotics because of their excellent antimicrobial properties and low cytotoxicity. In this study, one novel Brevinin-1 type peptide, Brevinin-1GHd, was isolated and characterized from the skin secretion of the frog, Hylarana guentheri. Brevinin-1GHd was found to possess a wide range of antimicrobial activity through penetrating the bacterial membrane within a short time while showing low hemolysis at bactericidal concentrations, even against the resistant strains. It also inhibited and eradicated biofilms which are thought to be closely related to the rise in resistance. Meanwhile, Brevinin-1GHd exhibited wide-spectrum anti-proliferation activity toward human cancer lines. Taken together, these results indicate that Brevinin-1GHd with its excellent antimicrobial and anticancer activities, is a promising candidate for a novel antibiotic agent, and study of its structure-activity relationships also provided a rational template for further research and peptide analog design.

DMPC-10A (ALWKKLLKK-Cha-NH₂) is a 10-mer peptide derivative from the N-terminal domain of Dermaseptin-PC which has shown broad-spectrum antimicrobial activity as well as a considerable hemolytic effect. In order to reduce hemolytic activity and improve stability to endogenous enzymes, a D-amino acid enantiomer (DMPC-10B) was designed by substituting all L-Lys and L-Leu with their respective D-form amino acid residues, while the Ala¹ and Trp³ remained unchanged. The D-amino acid enantiomer exhibited similar antimicrobial potency to the parent peptide but exerted lower cytotoxicity and hemolytic activity. Meanwhile, DMPC-10B exhibited remarkable resistance to hydrolysis by trypsin and chymotrypsin. In addition to these advantages, DMPC-10B exhibited an outstanding antibacterial effect against Methicillin-resistant Staphylococcus aureus (MRSA) and Klebsiella pneumoniae using the Galleria mellonella larva model and displayed synergistic activities with gentamicin against carbapenem-resistant K. pneumoniae strains. This indicates that DMPC-10B would be a promising alternative for treating antibiotic-resistant pathogens.

Brevinins are an important antimicrobial peptide (AMP) family discovered in the skin secretions of Ranidae frogs. The members demonstrate a typical C-terminal ranabox, as well as a diverse range of other structural characteristics. In this study, we identified a novel brevinin-2 peptide from the skin secretion of Sylvirana guentheri, via cloning transcripts, and identifying the expressed mature peptide, in the skin secretion. The confirmed amino acid sequence of the mature peptide was designated brevinin-2GHk (BR2GK). Moreover, as a previous study had demonstrated that the N-terminus of brevinin-2 is responsible for exerting antimicrobial activity, we also designed a series of truncated derivatives of BR2GK. The results show that the truncated derivatives exhibit significantly improved antimicrobial activity and cytotoxicity compared to the parent peptide, except a Pro¹⁴ substituted analog. The circular dichroism (CD) analysis of this analog revealed that it did not fold into a helical conformation in the presence of either lipopolysaccharides (LPS) or TFE, indicating that position 14 is involved in the formation of the α-helix. Furthermore, three more analogs with the substitutions of Ala, Lys and Arg at the position 14, respectively, revealed the influence on the membrane disruption potency on bacteria and mammalian cells by the structural changes at this position. Overall, the N-terminal 25-mer truncates demonstrated the potent antimicrobial activity with low cytotoxicity.

A novel dermaseptin peptide, dermaseptin-PT9 (DPT9), was isolated and identified from Phyllomedusa tarsius by the combination of molecular cloning and LC-MS analysis. Chemically synthesised DPT9 was broadly effective against the tested microorganisms through the disruption of cell membranes and showed weak haemolytic activity towards horse erythrocytes. It also exhibited anti-proliferative effect against various human cancer cells. Moreover, an analogue with enhanced cationicity, K^{8, 23}-DPT9, in which Asp⁸ and Glu²³ were substituted by lysine residues, had a markedly increased antimicrobial effect against all tested microorganisms and disrupted microbial cell membranes. This analogue also showed no haemolysis at its effective antimicrobial concentrations. In addition, K^{8, 23}-DPT9 displayed an enhanced anti-proliferative effect against cancer cells, while displayed weak activity against the normal human cell line, HMEC-1.