Cholesterol-Anchored Ac-SDKP Modified Platelet sEVs Effectively Deliver Anti-Inflammatory Peptides

Small extracellular vesicles (sEVs) released from thrombin-activated platelets carry adhesion proteins inherited from the parent cell membrane. Whether these vesicles can function as carriers for short anti-inflammatory peptides depends on a methodical evaluation of loading performance, biological compatibility, and intrinsic inflammatory potential. This protocol describes the isolation of platelet sEVs by thrombin-triggered vesiculation and differential ultracentrifugation, followed by surface decoration with the anti-inflammatory tetrapeptide Ac-SDKP via a cholesterol-polyhistidine anchor. The isolated vesicles were 90-300 nm in diameter, spherical by transmission electron microscopy, and positive for the platelet markers CD41 and CD62P. Cholesterol-directed membrane insertion gave loading efficiencies of 93.7% to 97.0% across four peptide-to-vesicle feeding ratios. The drug payload rose from 9.75% to 37.5% (w/w) as peptide input increased, without measurable change in vesicle diameter or zeta potential. Incubation with human coronary artery endothelial cells or human umbilical vein endothelial cells for 24 h produced no loss of metabolic activity at the concentrations tested. A single intravenous dose of unmodified sEVs at 20 mg/kg in mice did not prolong tail bleeding time or alter serum ALT, AST, BUN, or creatinine over a 7-day observation window. Histological examination of the lung, liver, spleen, and kidney showed no evidence of injury. Thrombin stimulation triggered substantial release of TNF-alpha, IL-6, and IL-1beta from intact platelets; the sEV fraction released negligible amounts of these cytokines, irrespective of thrombin exposure. In lipopolysaccharide-treated endothelial monolayers, Ac-SDKP-decorated sEVs suppressed cytokine secretion more effectively than an equal concentration of free Ac-SDKP. These results describe a practical method for generating peptide-functionalized platelet sEVs and provide an initial dataset on their pharmaceutical properties, supporting further investigation of this platform for anti-inflammatory delivery in vascular disorders.