DOI: 10.6084/m9.figshare.23950474.v1
title: Platelet extracellular vesicles are efficient delivery vehicles of doxorubicin, an anti-cancer drug: preparation and in vitro characterization

description: 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 × 10<sup>11</sup> particles/mL for all preparations. The loading of DOX determined using fluorospectrometry was found to be 2.1 × 10<sup>6</sup>, 1.7 × 10<sup>6</sup>, and 0.9 × 10<sup>6</sup> 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. <b>What is the context?</b> ● 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. <b>What is new?</b> ● 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. <b>What is the impact?</b> ● 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. <b>Abbreviations:</b> 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.