Platelet extracellular vesicles are efficient delivery vehicles of doxorubicin, an anti-cancer drug: preparation and in vitro characterization.

ABSTRACT

Platelet extracellular vesicles (PEVs) are an emerging delivery vehi for anticancer drugs due to their ability to target and remain in the tumor microenvironment. However, there is still a lack of understanding regarding yields, safety, drug loading efficiencies, and efficacy of PEVs. In this study, various methods were compared to generate PEVs from clinical-grade platelets, and their properties were examined as vehicles for doxorubicin (DOX). Sonication and extrusion produced the most PEVs, with means of 496 and 493 PEVs per platelet (PLT), respectively, compared to 145 and 33 by freeze/thaw and incubation, respectively. The PEVs were loaded with DOX through incubation and purified by chromatography. The size and concentration of the PEVs and PEV-DOX were analyzed using dynamic light scattering and nanoparticle tracking analysis. The results showed that the population sizes and concentrations of PEVs and PEV-DOX were in the ranges of 120-150 nm and 1.2-6.2 × 1011 particles/mL for all preparations. The loading of DOX determined using fluorospectrometry was found to be 2.1 × 106, 1.7 × 106, and 0.9 × 106 molecules/EV using freeze/thaw, extrusion, and sonication, respectively. The internalization of PEVs was determined to occur through clathrin-mediated endocytosis. PEV-DOX were more efficiently taken up by MDA-MB-231 breast cancer cells compared to MCF7/ADR breast cancer cells and NIH/3T3 cells. DOX-PEVs showed higher anticancer activity against MDA-MB-231 cells than against MCF7/ADR or NIH/3T3 cells and better than acommercial liposomal DOX formulation. In conclusion, this study demonstrates that PEVs generated by PLTs using extrusion, freeze/thaw, or sonication can efficiently load DOX and kill breast cancer cells, providing a promising strategy for further evaluation in preclinical animal models. The study findings suggest that sonication and extrusion are the most efficient methods to generate PEVs and that PEVs loaded with DOX exhibit significant anticancer activity against MDA-MB-231 breast cancer cells.

What is the context?● Current synthetic drug delivery systems can have limitations and side effects.● Platelet extracellular vesicles (PEVs) are a natural and potentially safer alternative for delivering cancer drugs to tumors.● However, there is still a lack of understanding about how to produce PEVs and how effective they are in delivering drugs.What is new?● We compared different methods for producing PEVs from clinical-grade platelets and found that sonication and extrusion were the most effective methods.● The PEVs were loaded with a cancer drug called doxorubicin (DOX) and tested their ability to kill breast cancer cells.What is the impact?● PEVs loaded with DOX were effective at killing cancer cells, especially MDA-MB-231 breast cancer cells.● This study demonstrates that PEVs are a promising strategy for delivering cancer drugs to tumors and that sonication and extrusion are the most efficient methods for producing PEVs.● The results suggest that further evaluation of PEVs in preclinical animal models is warranted to determine their potential as a cancer drug delivery system.Abbreviations ADP adenosine diphosphate; bFGF basic fibroblast growth factor; BSA bovine serum albumin; CD41 platelet glycoprotein IIb; CD62P P-selectin; CFDASE 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester; CPLT cryopreserved platelet; CPZ chlorpromazine hydrochloride; CTC circulating tumor cell; DMSO dimethyl sulfoxide; DDS drug delivery system; DOX doxorubicin; EPR enhanced permeability and retention; EV extracellular vesicle; FBS fetal bovine serum; GMP good manufacturing practice; GF growth factor; HER2 human epidermal growth factor receptor 2; HGF hepatocyte growth factor; Lipo-DOX liposomal doxorubicin; MDR multi-drug resistance; MMP-2 matrix metalloproteinase-2; MP microparticle; MSC mesenchymal stromal cell; NP nanoparticle; NTA nanoparticle tracking analysis; PAR-1 protease activated receptor-1; PAS platelet additive solution; PBS phosphate-buffered saline; PC platelet concentrate; PEG polyethylene glycol; PEV platelet-derived extracellular vesicle; DOX-PEV doxorubicin-loaded platelet-derived extracellular vesicle-encapsulated; PFA paraformaldehyde; PF4 platelet factor 4; P-gp P-glycoprotein; PLT platelet; PS phosphatidylserine; SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis; SEM scanning electron microscopy; TCIPA tumor cell-induced PLT aggregation; TDDS targeted drug delivery system; TEG thromboelastography; TF tissue factor; TF-EV extracellular vesicle expressing tissue factor; TME tumor microenvironment; TNBC triple-negative breast cancer; TXA2 thromboxane-A2; VEGF vascular endothelial growth factor; WHO World Health Organization.