Antimicrobial peptide series(DermaGen)

In the present study, we investigate degradable anionic dendritic nanogels (DNG) as carriers for antimicrobial peptides (AMPs). In such systems, the dendritic part contains carboxylic acid-based anionic binding sites for cationic AMPs, whereas linear poly(ethylene glycol) (PEG) chains form a shell for promotion of biological stealth. In order to clarify factors influencing membrane interactions of such systems, we here address effects of nanogel charge, cross-linking, and degradation on peptide loading/release, as well as consequences of these factors for lipid membrane interactions and antimicrobial effects. The DNGs were found to bind the AMPs LL-37 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES) and DPK-060 (GKHKNKGKKNGKHNGWKWWW). For the smaller DPK-060 peptide, loading was found to increase with increasing nanogel charge density. For the larger LL-37, on the other hand, peptide loading was largely insensitive to nanogel charge density. In line with this, results on the secondary structure, as well as on the absence of stabilization from proteolytic degradation by the nanogels, show that the larger LL-37 is unable to enter into the interior of the nanogels. While 40-60% nanogel degradation occurred over 10 days, promoted at high ionic strength and lower cross-linking density/higher anionic charge content, peptide release at physiological ionic strength was substantially faster, and membrane destabilization not relying on nanogel degradation. Ellipsometry and liposome leakage experiments showed both free peptide and peptide/DNG complexes to cause membrane destabilization, indicated also by antimicrobial activities being comparable for nanogel-bound and free peptide. Finally, the DNGs were demonstrated to display low toxicity towards erythrocytes even at peptide concentrations of 100 µM.

Bacterial infections are mostly due to bacteria in their biofilm-mode of growth, while penetrability of antimicrobials into infectious biofilms and increasing antibiotic resistance hamper infection treatment. In-vitro, monolaurin lipid nanocapsules (ML-LNCs) carrying adsorbed antimicrobial peptides (AMPs) displayed synergistic efficacy against planktonic Staphylococcus aureus, but it has not been demonstrated, neither in-vitro nor in-vivo, that such ML-LNCs penetrate into infectious S. aureus biofilms and maintain synergy with AMPs. This study investigates the release mechanism of AMPs from ML-LNCs and possible antimicrobial synergy of ML-LNCs with the AMPs DPK-060 and LL-37 against S. aureus biofilms in-vitro and in a therapeutic, murine, infected wound-healing model. Zeta potentials demonstrated that AMP release from ML-LNCs was controlled by the AMP concentration in suspension. Both AMPs demonstrated no antimicrobial efficacy against four staphylococcal strains in a planktonic mode, while a checkerboard assay showed synergistic antimicrobial efficacy when ML-LNCs and DPK-060 were combined, but not for combinations of ML-LNCs and LL-37. Similar effects were seen for growth reduction of staphylococcal biofilms, with antimicrobial synergy persisting only for ML-LNCs at the highest level of DPK-060 or LL-37 adsorption. Healing of wounds infected with bioluminescent S. aureus Xen36, treated with ML-LNCs alone, was faster when treated with PBS, while AMPs alone did not yield faster wound-healing than PBS. Faster, synergistic wound-healing due to ML-LNCs with adsorbed DPK-060, was absent in-vivo. Summarizing, antimicrobial synergy of ML-LNCs with adsorbed antimicrobial peptides as seen in-vitro, is absent in in-vivo healing of infected wounds, likely because host AMPs adapted the synergistic role of the AMPs added. Thus, conclusions regarding synergistic antimicrobial efficacy, should not be drawn from planktonic data, while even in-vitro biofilm data bear little relevance for the in-vivo situation.