In Vitro And In Vivo Evaluation Of Liposomes Laoding Paclitaxel For Integrin Targeting

In modern pharmaceutical sciences,it is an important task to develop the active targeting drug delivery system especially to tumor region.The objective of the present study is to develop the novel RGD peptide modified sterically stable liposomes（RGD-SSL） loading paclitaxel（PTX） with the use of thin film dispersion method and membrane extrusion technique,and it is hoped that the prepared RGD-SSL is to be used for imporove the tumor targeting effects through the high affinity between the RGD peptide and integrin receptors overexpressed on the cell membrane surface of tumor and neovasculature.First,the binding affinity and molecular mechanism between RGD peptides（RGD,GRGDS and GCRGDCS） and integrin receptors（αvβ3,αvβ5 andα5β1） were explored respectively by computer-aided molecular simulation studies.As a result,receptor binding affinity can be changed depending on the proper modification of amino acid sequence in the peptides.Second,three kinds of leading compounds,DSPE-PEG₂₀₀₀-RGD,DSPE-PEG₂₀₀₀-GRGDS and DSPE-PEG₂₀₀₀-GCRGDCS,were synthesized by active ester method.RGD,GRGDS and GCRGDCS were coupled with DSPE-PEG₂₀₀₀-NHS respectively and characterized by MALDI-TOF mass.The yield of the DSPE-PEG₂₀₀₀-RGD,DSPE-PEG₂₀₀₀-GRGDS and DSPE-PEG₂₀₀₀-GCRGDCS were determined to be 46.99%,43.07%and 31.57%by fluorescence intensity method, respectively.Third,the SSL-PTX,RGD-SSL-PTX,GRGDS-SSL-PTX and GCRGDCS-SSL-PTX were prepared by the thin film dispersion method and membrane extrusion technique with the post-insertion of DSPE-PEG₂₀₀₀,DSPE-PEG₂₀₀₀-RGD,DSPE-PEG₂₀₀₀-GRGDS and DSPE-PEG₂₀₀₀-GCRGDCS,and their encapsulation efficiency,mean particle size,morphology and in vitro release were also evaluated.As a result,the RGD peptides modification didn’t affect the above physicochemical characters.The encapsulation efficiencies of 100.14%,97.94%,96.97%,94.82% and the average particle size 115.5±2.2,117.3±0.6,117.5±1.3,121.2±2.0nm were characterized for the preparation of SSL-PTX,RGD-SSL-PTX,GRGDS-SSL-PTX and GCRGDCS-SSL-PTX, respectively,and they were all in spherical shape.The release profile of paclitaxel in phosphate buffer solution containing 0.1%Tween-80（pH7.4） within 8h has demonstrated that liposomal encapsulation could effectively delay the release of paclitaxel. Fourth,two kinds of tumor cells,human ovarian cancer SKOV-3 and human breast cancer MDA-MB-231,were used for evaluating tumor targeting ability in vitro.It was shown from the cell adhesion experiment that the ability of cell adhesion was dependent on the concentration of RGD peptide up to 0.2μmol lipid per well.From the results,RGD was shown the strongest binding affinity to SKOV-3 cell and GCRGDCS was the best one for MDA-MB-231 cells.Rhod-PE fluorescence labeled blank RGD-SSL was tested for cell adhesion and uptake in vitro by flow cytometry and confocal microscopy.The intracellular fluorescence intensity associated with RGD-SSL,GRGDS-SSL and GCRGDCS-SSL observed for SKOV-3 cells were 6.31,3.85 and 5.82 fold than that of SSL,while for MDA-MB-231,it was 6.18,2.23 and 14.40 fold respectively, determined by flow cytometry following incubation for 1h.Confocal microscopy experiments also showed that the red fluorescence intensity increased after 1,2 and 4h incubation.At each time point, the intracellular flourescence intensity of *RGD*-SSL was stronger than that of SSL which in accordance with the results of the flow cytometry.In vitro cytotoxicities of various liposome formulations on SKOV-3 and MDA-MB-231 cells also indicated that *RGD*-SSL-PTX were superior to SSL-PTX.Cell apoptosis of SKOV-3 proved that *RGD*-SSL increased the apoptosis compared with SSL-PTX but didn’t change normal cell cycle.Above all,it was suggested that RGD and its derivates modification could significantly enhance the cell adhesion and intracellular uptake of liposomal paclitaxel in our research work in terms of tumor cell targeting in vitro.At last,in vivo anti-tumor activities were evaluated on BALB/c nude mice xenografted SKOV-3 and MDA-MB-231 solid tumor separately.For SKOV-3 group,RGD-SSL-PTX showed greatly increased antitumor efficacy compared to Taxol^? which is present-day chemotherapy（P < 0.01） in accordance with the in vitro experiments,but for MDA-MB-231 group,the GCRGDCS-SSL-PTX might have an advantage over Taxol^? but no obviously differences and correlations with in vitro results.In conclusion,as a carrier for antiproliferative drug,*RGD*-SSL can greatly improve the cellular uptake and increase the cytotoxicity under the expectation.However,the in vivo antitumor efficacy is still need to be improved.