Preparation And Experiment Of P-Selectin Targeted Ultrasound Contrast Agent Arrying Gene And Cell-Penetrating Peptides

Objective To prepare a kind of P-selectin targeted ultrasound contrast agent(UCA) carrying gene and cell-penetrating peptides(CPPs), and to study its characteristics.Methods The phospholipid with carboxyl group was used as the membrane material. Mechanical vibration was used to prepare UCA carrying gene and CPPs, then anti-P-selectin antibody was covalently linked to the surface of UCA carrying gene and CPPs using a carbodiimide technique to prepare P-selectin targeted UCA carrying gene and CPPs. Its physical characteristic was measured. Agarose gel electrophoresis was used to detect the bonding force and protection ability of the UCA carrying gene and CPPs for DNA. The distribution of gene and CPPs on the UCA was observed using confocal laser scanning microscopy(CLSM). The encapsulation efficiencies of gene and CPPs on the UCA were investigated using fluorospectrophotometer. The connection of anti-P-selectin antibody with UCA carrying gene and CPPs was detected by immunofluorescent assay.Results The average diameter of P-selectin targeted UCA carrying gene and CPPs was (2.15±0.36)μm. The appearance of the P-selectin targeted UCA carrying gene and CPPs was spherical. The size distribution was uniform. There were no significant adhesion and aggregation. Compare with non-targeted UCA, the appearance had no significant difference. The UCA carrying gene and CPPs had a strong bonding force with DNA and could protect DNA from being degraded by DNase Ⅰ. The results of CLSM showed that gene and CPPs were distributed on the shell of UCA carrying gene and CPPs. The encapsulation efficiency of gene and CPPs on the UCA was28%and25%, respectively. Red fluorescence was observed on the surface of P-selectin targeted UCA carring gene and CPPs using fluorescent microscopy.Conclusions P-selectin targeted UCA carring gene and CPPs was prepared successfully. Objective To explore the feasibility of P-selectin targeted UCA carrying gene and CPPs transfering hypoxia human umbilical vein endothelial cell (HUVEC).Methods HUVEC were cultured in vitro and hypoxia HUVEC model was prepared using hydrogen peroxide (H2O2). Targeting specifity of P-selectin targeted UCA carrying gene and CPPs to hypoxia HUVEC was observed using light microscope and flow cytometry (FCM). Hypoxia HUVEC were randomly assigned into P-selectin targeted and non-targeted UCA carrying gene and CPPs group. Transfection effect of green fluorescent protein in the two groups was observed using fluorescent microscopy and flow cytometry at24h post transfection.Results Specific binding to hypoxia HUVEC was observed in the targeted UCA group, while binding was rare in the non-targeted UCA group. The result of FCM showed that about73.80%of the cell surface combined with the targeted UCA, but only1.39%of the cell surface combined with the non-targeted UCA. At24h post transfection, EGFP expression was observed using fluorescent microscopy both in the targeted UCA group and non-targeted UCA group. The result of FCM showed that transfection efficiency of the targeted UCA group was higher than that of the non-targeted UCA group [(18.74±0.47)%vs (15.34±0.22)%], and significant difference was found between the two groups (t=10.923, P<0.001).Conclusions The P-selectin targeted UCA carrying gene and CPPs could bing to hypoxia HUVEC effectively in vitro, and it could enhance gene expression in hypoxia HUVEC. It might provide a new approach for targeted gene delivery.