FIGURE 6.

Generation of EVs expressing targeting peptides for endothelial cells. (A) Amino acid sequence of TP1, TP2, TP-FLAG, which was included as negative control, and TP-9R. (B) Schematic of constructs expressed on EVs consisting of a specific targeting peptide (TPx) connected to transmembrane protein Lamp2b, flanked by a N-terminal signal peptide (SP) and GNSTM glycosylation sequence, and C-terminal HA-tag. (C) Representative western blot analysis showing expression of HA-tag and PalmGFP in cell lysate (CL) harvested from CPC lines stably expressing PalmGFP⁺ and specific TPs. CPCs transduced with GFP were included as negative control. GAPDH and beta-actin (ß-ACT) were included as house-keeping proteins. (D) Representative NTA plots showing the size distribution and particle concentration of concentrated conditioned medium (CM) derived from PalmGFP⁺ TP-expressing CPCs. (E) GFP fluorescence per 10¹⁰ particles determined in CM derived from PalmGFP⁺ TP-expressing CPCs (n = 2). Data are displayed as mean ± SD. (F) Representative western blot analysis showing expression of HA-tag, PalmGFP, CD81, Syntenin-1 (SYNT) and AnnexinA1 (ANXA1) in CM. CL of PalmGFP⁺ CPCs stably expressing TP-FLAG were included as control. CM derived from CPCs transduced with GFP was included as negative control (cntrl). Calnexin (CNX) was only present in CL. Uncut blots are included in Supplementary Figure S10.