Controlling keratinocyte activity by LL-37 conjugated nanoparticles
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1
Coimbra University, CNC- Centre of Neuroscience and Cell Biology, Portugal
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2
Université Catholique de Louvain, Louvain Drug Research Institute, Pharmaceutics and Drug Delivery, Belgium
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3
Coimbra University, CIEPQPF, Department of Chemical Engineering, Portugal
Introduction: LL37 is an antimicrobial peptide (AMP) predominantly found in human skin that acts at different levels for the homeostasis of the skin. LL37 acts as first line of defense against bacteria, virus and fungi, but, very importantly, it plays a role in immunomodulation, angiogenesis and wound healing properties. Angiogenic properties of LL37 have been associated with binding via formyl peptide receptor-like 1 (FPRL1) in endothelial cells, while the immunomodulatory and chemotactic properties have been associated with binding via P2X7, epidermal growth factor receptor (EGFR) or FPRL1. Although the wound healing properties of LL37 peptide have been demonstrated, it is unknown the bioactivity of permanently immobilized LL37. Here, we investigated the wound healing potential of LL37-conjugated NPs that have a gold (Au) core and a hydrophilic cationic LL37 peptide shell. We have selected Au NPs because it is relatively easy the immobilization of high concentrations of LL37 per surface area, the modification of their properties (including size, charge and morphology), and they have been used in the clinic for many years. We hypothesize that LL37-conjugated NPs have enhanced wound-healing properties than LL37 peptide because they prolong in time the biological activity of the peptide and they might act differently in terms of mechanism. So far no study has compared the in vivo performance of soluble AMP relatively to chemically immobilized AMP formulations in terms of their regenerative potential. To verify this hypothesis we have evaluated initially the physico-chemical properties of LL37-Au NPs and the binding process of LL37 peptide to the NP formulation by molecular dynamic studies. The cytotoxicity and antimicrobial properties as well as their stability against proteases have been also studied. To show the unique properties of this formulation we have evaluated their pro-migratory properties against keratinocytes. Finally, we have evaluated in vivo the regenerative potential of LL37-Au NPs in a splinted mouse full thickness excisional model.
Results and Discussion: The formulation LL37- Au NPs used in this work has high density of LL37 peptide (30% of peptide per mass of gold), an average diameter of 21 nm and positive Z-potential (14 mV). LL37-Au NPs showed antimicrobial activity against E. coli similar to soluble LL37, they are rapidly internalized in epithelial cells by scavenger receptor- and clathrin- mediated endocytosis. No significant cytotoxic effect was observed in HUVECs and HaCaT treated with soluble LL37 and LL37- Au NPs up to a concentration of 30 μg/mL and 400 μg/mL (ca. 100 μg /mL of LL37), respectively. We demonstrated that LL37 GNPs induce a prolonged EGFR phosphorylation and ERK pathway activation, which enhances the migratory properties of HaCaT cells as compared to soluble LL37 peptide. The enhanced migratory properties of HaCaT cells, after exposure to LL37-Au NPs, was mediated by the activation of ADAM17 metalloproteinase and P2X7 receptor. We further show that mice wounds treated with LL37-Au NPs have a faster wound closure than the ones treated with soluble LL37.
EC (Marie Curie ITN, project no 289454, “Nanodrug”); ERC project no 307384, “Nanotrigger”; COMPETE funding (Project “Stem cell based platforms for Regenerative and Therapeutic Medicine”, Centro- 07-ST24-FEDER-002008)
Keywords:
Regenerative Medicine,
nanoparticle,
Bioactive molecule,
Cell modulation
Conference:
10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016.
Presentation Type:
General Session Oral
Topic:
Cellular migration and biomaterials
Citation:
Comune
M,
Rai
A,
Pinto
S,
Aday
S,
Chereddy
KK,
Ferreira
AF,
Simões
PN,
Préat
V and
Ferreira
L
(2016). Controlling keratinocyte activity by LL-37 conjugated nanoparticles.
Front. Bioeng. Biotechnol.
Conference Abstract:
10th World Biomaterials Congress.
doi: 10.3389/conf.FBIOE.2016.01.00618
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Received:
27 Mar 2016;
Published Online:
30 Mar 2016.