A comparison of different strategies for antimicrobial peptides incorporation onto/into lipid nanocapsules.

Aim: Over the last decade, antimicrobial peptides (AMPs) have emerged as a promising alternative for the treatment of various infections. The aim of this work is to explore the potential of lipid nanocapsules for the delivery of AMPs. Three approaches were compared in terms of encapsulation efficiency, peptide activity and protection against proteases: peptide encapsulation, surface adsorption or covalent attachment of three selected AMPs. Results: A potentiation of the antimicrobial activity and a partial protection of the peptides after adsorption were demonstrated compared with native peptides. Conversely, encapsulation allowed better peptide stability, correlated with higher encapsulation efficiencies and a preservation of the activity. Finally, the covalent attachment strategy turned out to be less conclusive due to peptide inactivation. Conclusion: In brief, a lipid nanocapsule-based platform appears suitable to deliver AMPs.

Reverse micelle-lipid nanocapsules: a novel strategy for drug delivery of the plectasin derivate AP138 antimicrobial peptide.

INTRODUCTION: Resistance to traditional antibiotics is an increasingly serious problem. Antimicrobial peptides (AMPs) have emerged as a new therapeutic class with great potential against infectious diseases, as they are less prone to induce resistance. Nanotechnology-based delivery strategies can improve the efficiency and stability of AMPs, particularly against proteolytic degradation. Lipid nanocapsules (LNCs) are a new generation of biomimetic nanocarriers and were used in this study to deliver peptides. METHODS: AMP-loaded reverse micelles (RM) were developed and incorpo rated into LNCs by the phase inversion process and the antimicrobial activity of the AMPs-loaded LNC was evaluated by the minimum inhibitory concentration method. We studied the activity of AMP solutions and AMP-loaded LNCs against Gram-positive and Gram-negative bacterial strains and then evaluated the encapsulation of a new cationic AMP called AP138. Finally, we analyzed the effect of enzymatic attack on AP138 and AP138-RM-LNCs after incubation with trypsin. RESULTS: AP138 was efficiently encapsulated in the LNCs (encapsulation efficiency = 97.8% at a drug loading of 0.151%), resulting in protection against degradation by proteases and the preservation of antimicrobial activity against Staphylococcus aureus, including methicillin-resistant Staphylococcus aureus. CONCLUSION: This study shows that RM-LNCs are an excellent candidate system to deliver AMPs.

Synergistic Effect of Combinations Containing EDTA and the Antimicrobial Peptide AA230, an Arenicin-3 Derivative, on Gram-Negative Bacteria.

The worldwide occurrence of resistance to standard antibiotics and lack of new antibacterial drugs demand new strategies to treat complicated infections. Hence, the aim of this study was to examine the antibacterial activities of an antimicrobial peptide, arenicin-3 derivative AA230, and ethylenediaminetetraacetic acid (EDTA) as well as the two compounds in combination against Gram-negative bacteria. AA230 showed strong antibacterial activity against all of the studied standard strains and clinical isolates, with minimum inhibitory concentrations ranging between 1 µg/mL and 8 µg/mL. AA230 exhibited a bactericidal mode of action. EDTA inhibited the growth of Acinetobacter baumannii at 500⁻1000 µg/mL. Strains of Acinetobacter baumannii were found to be more susceptible to EDTA than Pseudomonas aeruginosa or Escherichia coli. The antibacterial effects of both AA230 and EDTA were independent of the antibiotic resistance patterns. Indifference to synergistic activity was observed for AA230 and EDTA combinations using checkerboard titration. In time-kill studies, a substantial synergistic interaction between AA230 and EDTA was detected against all of the tested strains. The addition of EDTA enabled a 2⁻4-fold decrease in the AA230 dose. In conclusion, AA230 could have potential applications in the treatment of infections caused by Gram-negative organisms, and its effect can be potentiated by EDTA.

Synergistic interactions between antimicrobial peptides derived from plectasin and lipid nanocapsules containing monolaurin as a cosurfactant against Staphylococcus aureus.

Development of effective antibacterial agents for the treatment of infections caused by Gram-positive bacteria resistant to existing antibiotics, such as methicillin-resistant Staphylococcus aureus (MRSA), is an area of intensive research. In this work, the antibacterial efficacy of two antimicrobial peptides derived from plectasin, AP114 and AP138, used alone and in combination with monolaurin-lipid nanocapsules (ML-LNCs) was evaluated. Several interesting findings emerged from the present study. First, ML-LNCs and both plectasin derivatives showed potent activity against all 14 tested strains of S. aureus, independent of their resistance phenotype. Both peptides displayed a considerable adsorption (33%-62%) onto ML-LNCs without having an important impact on the particle properties such as size. The combinations of peptide with ML-LNC displayed synergistic effect against S. aureus, as confirmed by two methods: checkerboard and time-kill assays. This synergistic interaction enables a dose reduction and consequently decreases the risk of toxicity and has the potential of minimizing the development of resistance. Together, these results suggest that ML-LNCs loaded with a plectasin derivative may be a very promising drug delivery system for further development as a novel antibacterial agent against S. aureus, including MRSA.

Cubosomes post-loaded with antimicrobial peptides: characterization, bactericidal effect and proteolytic stability.

Novel antibiotics, such as antimicrobial peptides (AMPs), have recently attended more and more attraction. In this work, dispersed cubic liquid crystalline gel (cubosomes) was used as drug delivery vehicles for three AMPs (AP114, DPK-060 and LL-37). Association of peptides onto cubosomes was studied at two cubosome/peptide ratios using high performance liquid chromatography, ζ-potential and circular dichroism measurements. AMPs impact on the cubosome structure was investigated using small angle x-ray scattering and cryogenic transmission electron microscopy. The antimicrobial effect of the AMP loaded cubosomes was studied in vitro by minimum inhibitory concentration and time-kill assays. Proteolytic protection was investigated by incubating the formulations with two elastases and the antimicrobial effect after proteolysis was studied using radial diffusion assay. Different association efficacy onto the cubosomes was observed among the AMPs, with LL-37 showing greatest association (>60%). AP114 loaded cubosomes displayed a preserved antimicrobial effect, whereas for LL-37 the broad spectrum bacterial killing was reduced to only comprise Gram-negative bacteria. Interestingly, DPK-060 loaded cubosomes showed a slight enhanced effect against S. aureus and E. coli strains. Moreover, the cubosomes were found to protect LL-37 from proteolytic degradation, resulting in a significantly better bactericidal effect after being subjected to elastase, compared to unformulated peptide.

Antibacterial action of lipid nanocapsules containing fatty acids or monoglycerides as co-surfactants.

Lipid nanocapsules (LNCs) are a new generation of biomimetic nanocarriers obtained via a phase inversion temperature method and have an oily core of medium-chain triglycerides that is surrounded by a shelf containing a lipophilic surfactant (lecithin) and a hydrophilic surfactant macrogol 15-hydroxystearate. The aim of the present study was to produce LNCs with antibacterial activity by replacing lecithin with other lipophilic surface active compounds, namely medium-chain fatty acids and their 1-monoglycerides, which are known to have antimicrobial properties. Fatty acids and monoglycerides were found to affect the properties of LNCs, such as particle size and zeta potential. Incorporation of a co-surfactant decreased significantly particle size (p⩽0.0039). Furthermore, incorporation of either lecithin or fatty acids with at least 10 carbon atoms yielded LNCs with the zeta potential significantly more negative than that of LNCs composed solely of triglycerides and macrogol 15 hydroxystearate (p⩽0.0310). Moreover, they were capable of decreasing the phase inversion temperature. The activity of the LCNs against Gram-positive S. aureus, including a methicillin-resistant strain, increased with increases in the length of the hydrocarbon tail. Monoglyceride-LNCs were found to be more active than the corresponding fatty acids. The opposite behaviour was observed for Gram-negative bacteria, whereby only caproic acid- and caprylic acid-LNCs were found to be active against these organisms. The monoglyceride-LNCs were bactericidal, and they killed in a time-dependent manner. Fatty acid-LNCs killed in a concentration-dependent manner. A haemolysis assay was performed to obtain preliminary information on the safety of the tested LNCs. In the case of fatty acid-LNCs, the concentrations at which bacterial growth was inhibited were similar to the haemolytic concentrations. However, monoglyceride-LNCs showed antibacterial action at concentrations much lower than those at which haemolysis was observed. In conclusion, monoglyceride-LNCs are promising candidates as carriers for the encapsulation of antibacterial agents, particularly against S. aureus.

Understanding the adsorption of salmon calcitonin, antimicrobial peptide AP114 and polymyxin B onto lipid nanocapsules.

The adsorption of therapeutic molecules, e.g., peptides, onto nanocarriers is influenced by the properties of the carrier, adsorbed molecule and continuous phase. Hence, through changes in the composition of the nanocarrier and the medium, it should be possible to tune the system to make it capable of efficiently adsorbing peptides. The adsorption of calcitonin, antimicrobial peptide AP114 and polymyxin B onto lipid nanocapsules was investigated. The adsorption data were fitted to a Langmuir isotherm. Dynamic light scattering and laser Doppler velocimetry were used to investigate the changes in the hydrodynamic diameter and zeta potential, respectively, of the nanocarrier. The peptide adsorption was primarily governed by electrostatic forces; however, even without the presence of an ionisable surfactant, a significant amount of each tested molecule was adsorbed due to the enormous surface area of the nanocarriers and to peptide-nanocarrier interactions. The addition of an ionisable lipophilic surfactant, lecithin, improved the adsorption yield, which reached values of up to 100%. The adsorption yield and the properties of the nanocarrier, particularly the zeta potential, depended on the carrier and peptide concentrations and their mixing ratio. The adsorption of all tested molecules obeyed the Langmuir model over a limited concentration range.

Lipid-based nanoformulations for peptide delivery.

Nanoformulations have attracted a lot of attention because of their size-dependent properties. Among the array of nanoformulations, lipid nanoformulations (LNFs) have evoked increasing interest because of the advantages of their high degree of biocompatibility and versatility. The performance of lipid nanoformulations is greatly influenced by their composition and structure. Therapeutic peptides represent a growing share of the pharmaceutical market. However, the main challenge for their development into commercial products is their inherent physicochemical and biological instability. Important peptides such as insulin, calcitonin and cyclosporin A have been incorporated into LNFs. The association or encapsulation of peptides within lipid-based carriers has shown to protect the labile molecules against enzymatic degradation. This review describes strategies used for the formulation of peptides and some methods used for the assessment of association efficiency. The advantages and drawbacks of such carriers are also described.