Antimicrobial and pro-angiogenic properties of soluble and nanoparticle-immobilized LL37 peptides.

Antimicrobial peptides (AMPs), such as LL37 peptides, may be immobilized on the surface of medical devices to render them with antimicrobial and angiogenic properties. However, little is known about LL37 properties after immobilization. Here, we have studied the antimicrobial and pro-angiogenic activity of soluble and immobilized LL37 peptides (conjugated to gold nanoparticles). Both soluble and immobilized LL37 peptides have potent antimicrobial activity against Gram-positive and Gram-negative bacteria in the presence of 10% human serum (HS). However, the immobilized LL37 peptides showed less cytotoxicity to endothelial cells (ECs) at a concentration that was able to kill bacteria. Interestingly, although both soluble and immobilized LL37 peptides showed pro-angiogenic activities in Matrigel and chorioallantoic membrane (CAM) assays, they induce different signalling pathways in ECs. The results described here highlight the importance of investigating the properties of immobilized AMPs that might act as a new entity.

UV Light-Responsive Peptide-Based Supramolecular Hydrogel for Controlled Drug Delivery.

Low-molecular-weight self-assembled peptides may serve as promising hydrogelators for drug delivery applications by changing their structural network in response to external stimuli. Herein, inspired by the well-studied low-molecular-weight peptide hydrogelator, fluorenyl-methoxycarbonyl-diphenylalanine (Fmoc-FF), a novel peptide is designed and synthesized to include an ultraviolet (UV)-sensitive phototrigger. Similar to Fmoc-FF, 6-nitroveratryloxycarbonyl-diphenylalanine (Nvoc-FF) self-assembles to form a 3D, self-supporting, nanofibrous hydrogel. The Nvoc-FF hydrogel exhibits good mechanical properties with a storage modulus of 40 kPa. UV irradiation of the Nvoc-FF hydrogel encapsulating insulin-fluorescein isothiocyanate (insulin-FITC) results in the cleavage of Nvoc-FF peptide to produce unmasked FF, thereby facilitating the degradation of the hydrogel and the release of insulin-FITC. This release is in linear correlation to the irradiation time. In the present study, a first insight into this rigid, fibrous, light-responsive hydrogel is provided, allowing the fabrication of a novel drug delivery system for controlled release of large molecules.

Atomistic-Level Investigation of a LL37-Conjugated Gold Nanoparticle By Well-Tempered Metadynamics.

LL37 is a cathelicidin-derived antimicrobial peptide (AMP) with a broad spectrum of antimicrobial activity and wound-healing potential. The enhancement of these characteristics was recently demonstrated for a cysteine (CYS)-modified cathelicidin-derived LL37-SH conjugated with gold nanoparticles (AuNPs). Considering the potential of this peptide, we hereby report a computational study in which well-tempered metadynamics was applied to unveil the interaction of LL37-SH and LL37 with a AuNP with atomistic detail. A structural analysis combined with the free energy surface (FES) characterization allowed the assessment of the role of CYS residue during the formation of the conjugate, as well as to understand how the AuNP improves the antimicrobial activity of the peptide. It was found that CYS promotes a lower conformational entropy (before and after adsorption onto the AuNP) and a faster adsorption process when compared to the LL37 without CYS. The FES for LL37-SH is characterized by one global minimum, while for LL37 a potential metastable state was found. The presence of the AuNP leads to an elongation of the peptides along with the adsorption, which translates into the increase of the solvent-accessible surface area. This elongation combined with the greater availability of positively charged residues upon adsorption rationalizes the observed enhancement of the activity of the LL37-SH/AuNP conjugate.

Antimicrobial peptide-gold nanoscale therapeutic formulation with high skin regenerative potential.

Chronic skin wounds affect ≈3% of persons aged >60years (Davies et al., 2007) [1]. These wounds are typically difficult to heal by conventional therapies and in many cases they get infected making even harder the regeneration process. The antimicrobial peptide (AMP) LL37 combines antimicrobial with pro-regenerative properties and thus represents a promising topical therapy to address both problems. Here, we investigated the wound healing potential of soluble and immobilized LL37 (LL37-conjugated gold nanoparticles, LL37-Au NPs), both in vitro (migration of keratinocytes) and in vivo (skin wound healing). Our results show that LL37-Au NPs, but not LL37 peptide, have the capacity to prolong the phosphorylation of EGFR and ERK1/2 and enhance the migratory properties of keratinocytes in a large in vitro wound model. We further report that both LL37 and LL37-Au NPs promote keratinocyte migration by the transactivation of EGFR, a process that seems to be initiated at the P2X7 receptor, as confirmed by chemical and genetic inhibition studies. Finally, we show in vivo that LL37-Au NPs have higher wound healing activity than LL37 peptide in a splinted mouse full thickness excisional model. Animal wounds treated by LL37-Au NPs have higher expression of collagen, IL6 and VEGF than the ones treated with LL37 peptide or NPs without LL37. Altogether, the conjugation of AMPs to NPs offers a promising platform to enhance their pro-regenerative properties.

One-step synthesis of high-density peptide-conjugated gold nanoparticles with antimicrobial efficacy in a systemic infection model.

The increase in antibiotic drug resistance and the low number of new antibacterial drugs approved in the last few decades requires the development of new antimicrobial strategies. Antimicrobial peptides (AMPs) are very promising molecules to fight microbial infection since they kill quickly bacteria and, in some cases, target bacterial membrane. Although some AMPs may be stable against proteolytic degradation by chemical modification, in general, low AMP activity and stability in the presence of serum and proteolytic enzymes as well as their cytotoxicity have impaired their clinical translation. Here, we describe a one-step methodology to generate AMP-conjugated gold nanoparticles (Au NPs), with a high concentration of AMPs (CM-SH) (≈240 AMPs per NP), controlled size (14 nm) and low polydispersity. AMP-conjugated Au NPs demonstrated higher antimicrobial activity and stability in serum and in the presence of non-physiological concentrations of proteolytic enzymes than soluble AMP, as well as low cytotoxicity against human cells. Moreover, the NPs demonstrated high antimicrobial activity after in vivo administration in a chronic wound and in an animal model of systemic infection.

PLGA nanoparticles loaded with host defense peptide LL37 promote wound healing.

Wound treatment remains one of the most prevalent and economically burdensome healthcare issues in the world. Poly (lactic-co-glycolic acid) (PLGA) supplies lactate that accelerates neovascularization and promotes wound healing. LL37 is an endogenous human host defense peptide that modulates wound healing and angiogenesis and fights infection. Hence, we hypothesized that the administration of LL37 encapsulated in PLGA nanoparticles (PLGA-LL37 NP) promotes wound closure due to the sustained release of both LL37 and lactate. In full thickness excisional wounds, the treatment with PLGA-LL37 NP significantly accelerated wound healing compared to PLGA or LL37 administration alone. PLGA-LL37 NP-treated wounds displayed advanced granulation tissue formation by significant higher collagen deposition, re-epithelialized and neovascularized composition. PLGA-LL37 NP improved angiogenesis, significantly up-regulated IL-6 and VEGFa expression, and modulated the inflammatory wound response. In vitro, PLGA-LL37 NP induced enhanced cell migration but had no effect on the metabolism and proliferation of keratinocytes. It displayed antimicrobial activity on Escherichia coli. In conclusion, we developed a biodegradable drug delivery system that accelerated healing processes due to the combined effects of lactate and LL37 released from the nanoparticles.