| Objective:As a biomarker for angiogenesis, integrin αvβ3 is considered important in the angiogenesis associated diseases. In the present study, we prepared a doxorubin hydrochloride-loaded and PEGylated liposome of which the surface was decorated with a novel P1c peptide. It was expected that the prepared P1c-modified sterically stable liposomes (SSL) could specially target tumor via enhancing drug delivery to tumor cells and neovascular where integrin αvβ3 expression was upregulated. This study provide a basis for the clinical therapy for angiogenesis associated diseases.Methods:1. The liposomes were prepared by thin film hydration method and doxorubicin (DOX) was encapsulated into liposomes by the ammonium sulfate gradient method, and the particle size, morphology, zeta potential, encapsulation efficiency, the conjugation ratio of the P1c decoration and the in vitro DOX relaease were evaluated.2. Human glioblastoma U87MG cells with high αvβ3 expression and human breast cancer MCF-7 cells with low αvβ3 expression were used for evaluating the tumor targeting potential in vitro. Cellular uptake of doxorubicin was determined by flow cytometry and confocal laser-scanning microscopy. The cytotoxicity of Plc-modified targeting liposome (P1c-DOXL), non-targeting liposome (DOXL) and free doxorubicin (DOX) on U87MG and MCF-7 tumor cells was investigated by CCK-8 assay.3. The in vivo tumor targeting potential and antitumor effect of Plc-modifid targeting liposome were studied in glioblastoma U87MG tumor-xgrafted BALB/c nude mice model by methods of tumor volume determination, immunohistochemical analysis and immunofluorescence staining. DOXL group, DOX group and saline group were used as control.Results:1. The mean particle sizes were 131.2 and 128.4 nm for Plc-modified targeting liposome (P1c-DOXL) and non-targeting liposome (DOXL), respectively. The zeta potentials for P1c-DOXL and DOXL were -19.7±2.8 mV and -33.1±1.6 mV, respectively. The transmission electron microscope image demonstrated the liposome of a sphere shape and well dispersed. The encapsulation efficiencies of two types of liposome were more than 95%. The conjugation ratio for P1c decoration was 66.8±1.6%. The release profile of doxorubicin in phosphate buffer solution (pH7.4) at 37℃ within 72 hours demonstrated liposomal encapsulation could effectively delay the release of doxorubicin.2. The flow cytometry and confocal laser-scanning microscopy experiments consistently showed that the intracellular fluorescence intensity of Plc-modified targeted liposome group was stronger than that of non-targeted liposome group and αvβ3-mAb group in U87MG cells (P<0.05), while there was no significant differences between the tested groups in MCF-7 cells (P> 0.05). CCK-8 assay showed that the anti-tumor activity of P1c-modified targeted liposome was superior to non-targeted liposome in U87MG cells, whereas there was no significant diffirences between the liposome groups in MCF-7 cells (P>0.05). However, the data demonstrated that free DOX had the highest cellular uptake and cytotoxic activity on both tumor cell lines.3. In vivo targeting and antitumor activitiy of P1c-modifid targeting liposome were evaluated using U87MG tumor-xgrafted BALB/c nude mice. The inhibitory rates of tumor growth were 48.08%,31.03% and 22.9% for the Plc-modified targeting liposome, non-targeting liposome and DOX groups, respectively. The results from immunohistochemical and immunofluorescence analysis indicated that the Plc-modified targeting liposome group of the lowest expression of integrin αvβ3 and CD31 compared to treatment with non-targeting liposome and DOX groups implying the superior activity of the P1c-modifid targeting liposome.Conclusions:Plc-modified targeting liposome exhibiting sustained release, enhanced the antitumor effect of DOX through targeting tumor cells and neovascular where integrin αvβ3 was overexpressed. The results indicated that the P1c-modified drug delivery system might be a promising solution for active targeting delivery. |
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