Biodegradable nanoparticles for restenosis following PTA: a feasibility study

INTRODUCTION Percutaneous transluminal angioplasty (PTA) is known to effectively improve the prognosis of patients with vascular diseases1. However, poor reendothelialization, and excessive migration and proliferation of vascular smooth muscular cells in the tunica media, can result in obstructive neointimal hyperplasia, and are the major mechanisms involved in restenosis following PTA2. Many animal studies indicate that local delivery of intimal hyperplasia inhibiting drugs can enhance vascular reendothelialization and prevent restenosis2, although this remains controversial. This may be due to inadequate drug concentrations or to the short period that the effective drug concentrations is available locally. The local administration of drug by biodegradable nanoparticles could enhance the drug concentrations in vessel wall and, thereby, yield the desired therapeutic effects. Recently, anti-oxidants (AA) grafted on poly(lactide-co- glycolide) (g-AA-PLGA) were proposed as novel biodegradable materials stable to gamma irradiation, characterized by different surface properties and suitable for drug delivery3,4. In this work, we investigated the feasibility to prepare g-AA-PLGA nanoparticles as carriers for the local administration of drugs in the vessel wall during PTA. To achieve this goal, the rate and extent of cellular uptake of such nanoparticles in comparison with the naïve PLGA was studied using macrophages, smooth muscular and endothelial cells as representative of the cells present in the vessel wall. EXPERIMENTAL SET-UP g-AA-PLGA synthesis and characterization Caffeic acid (CA) or resveratrol (RV) were grafted to PLGA (L/G ratio 50/50, Mw=26 KDa, Tg=47.1±0.5 °C) by a free radicals-induced strategy3,4. g-AA-PLGA were characterized by the DPPH assay, GPC analysis and DSC. Nanoparticles preparation and characterization Surfactant-free nanoparticles (NPs) were prepared by the solvent displacement method, adding dropwise 1 mL of 1% polymeric solution in organic solvent to 10 mL of MilliQ® water. Hydrodynamic diameter (Dh) and zeta potential () were determined by a Zetasizer Nano ZS (Malvern Instrument, UK). Fluorescent NPs with 10% w/w of a FITC- PLGA conjugate were also prepared. Cellular uptake and exocytosis Macrophages, smooth muscular and endothelial cells were exposed to the fluorescent nanosuspension (100 μg/mL) over a 24 h period. At predetermined times, media were removed and NP uptake was determined by measuring the intracellular fluorescence. The uptake efficiency was calculated normalizing the observed fluorescence intensity in each well (IOBS) for the mean fluorescence intensity of the negative control (INC) according to equation 1: Uptake efficiency = (IOBS - INC)/INC eq.1 The exocytosis of nanoparticles was followed for up to 7 days. The absolute number of detected photons (i.e. the derived count rate, DCR), Dh and correlogram shape were considered to qualitatively establish NPs released by the cells. The cytotoxicity of the tested formulation was also evaluated by the MTT assay5. Statistical analysis One-way and two-way ANOVA followed by Tukey’s test (=0.05) were performed using OriginPro 2015 (USA). Outliers were discarded according to Dixon’s T-test. RESULTS AND DISCUSSION Polymer synthesis and characterization g-AA-PLGA were synthesized by performing a two-step grafting procedure at room temperature. In the former step, the oxidation of ascorbic acid by H2O2 led to the formation of hydroxyl radicals able to activate the PLGA backbone; then, the reactive sites on the preformed PLGA macroradicals react with the AA molecules resulting in CA or RV insertion. This approach avoids the degradation of the AA due to high temperature and the generation of toxic by- products. Both types of PL