PLGA nanoparticles loaded with host defense peptide LL37 promote wound healing

Abstract

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.

Introduction

Wound treatment and its medical complications remain one of the most prevalent and economically challenging healthcare issues in the world. The annual wound care products market has reached to $15.3 billion by 2010 and still the need for post-surgical wound care is sharply on the rise. In the USA alone there are more than 100 million wounds annually, including surgical incisions, trauma, burns, blast injuries, diabetic ulcers, ostomies, bedsores, and more [1], [2]. Acute wounds heal in a very orderly, timely and efficient manner characterized by four distinct, strictly connected but overlapping phases: hemostasis, inflammation, proliferation and remodeling. Any disturbances in these phases may result in an incomplete and improper restoration of the injured tissue [3]. In these severe clinical conditions wound repair may fail to proceed, leading to chronic non-healing wounds. Current treatment options are limited, costly, and inefficient. As a result, the development of new therapeutics is absolutely necessary to satisfy the unmet clinical need.

One of the simple and pragmatic solutions to faster wound healing process was the application of exogenous lactate that accelerates angiogenesis, activation of procollagen factors and recruitment of endothelial progenitor cells in wounds. Utilization of poly (lactic-co-glycolic acid) (PLGA) is one of the strategies to supply lactate sustainably [4]. Moreover, PLGA is biodegradable, biocompatible, has versatile degradation kinetics and approved by the European Medical Agency and Food and Drug Administration as an excipient for parenteral products [5]. Thus the key advantage of PLGA drug delivery systems is the polymer can perform the dual roles: being a wound healing agent itself and its capability to release loaded drugs sustainably to the wound.

LL37 belongs to antimicrobial peptides/host defense peptides that are part of the innate immune system and represent the first line of defense against many invading pathogens [6]. hCAP-18/LL37 is up to now the only antimicrobial peptide of the cathelicidin family identified in humans. LL37 has been detected in an inactive proform in several types of cells such as neutrophils, mast cells, monocytes, macrophages, NK cells, γδ cells, B cells, dermal epithelial cells and keratinocytes, inflamed skin and blister fluids. Any infection or tissue and cell damage that occurs during the wound may activate TLR(s) and/or an alteration in the cytokine milieu, which provides a trigger that activates the cell to degranulate. This leads to the release of the inactive hCAP18 precursor in the extracellular environment, where it can be processed by specific proteases into the active 37 amino acid long LL37 peptide [7], [8], [9]. LL37 exerts different immunomodulatory functions like broad antimicrobial activity, antiviral and antifungal activity, endotoxin-binding properties, modulation of pro-inflammatory response, chemotaxis, influence on cell proliferation and differentiation, promotion of wound healing and angiogenesis, etc. [10]. The major difficulty associated with LL37 administration is its immediate degradation in the wound environment and thus the treatment requires high dose and dosing frequency of LL37 [11] or gene therapy [12] to produce the therapeutic effect.

PLGA nanoparticles (NP) have been successfully proved to be efficient carriers for large biomolecules such as vaccines and proteins for the treatment of various ailments [5], [13]. Hence, we hypothesized that the administration of LL37 encapsulated in PLGA nanoparticles (PLGA-LL37 NP) could efficiently deliver the LL37 and accelerate wound closure through different mechanisms due to the encapsulated LL37 and lactate released from PLGA. Thus, we present the PLGA-LL37 NP for the active healing of dermal wounds and study their mechanisms of action.