| Tumor is one of the lethalest diseases in the world. Due to variousinternal or external risk factors, cells lost the growth regulation and propagatewithout control. Routine strategies, including surgery, radiotherapy,chemotherapy, interventional therapy and biological therapy, lack of enoughspecificity and lead to seriously toxic and side effect. It’s extremely importantto develop an effective tumor targeting drug delivery system.Long circulation liposomal drug delivery system can passively target totumor and reduce the distribution in other tissues due to enhanced permeationand retention (EPR) effect. At the meanwhile, drug can be protected from theenzymatic degradation and promoting long circulation time. Moreover, theeffect of the liposomal therapeutics may farther increase because of the active targeting effect. It can be achieved by the specific ligand conjugation andtumor biomarker recognition.Tie2is identified as a receptor tyrosine kinase and plays essential rolesin tumor induced angiogenesis. It is mainly expressed on endothelial cells aswell as some tumor cells. It will be upregulated during tumor angiogenesis.Therefore, we designed a liposomal drug delivery system of which bindingability to Tie2protein is increased.The key component of targeting drug delivery system is the screening ofligands of tumor specific marker. Because of the advantages including lessimmunogenicity, easy manipulation, lower cost and more stable bioactivity,peptide ligands were chosen as targeting molecule. Direct coating phagedisplay method, computer-aided drug design technology and natural ligandbinding domain mimics analysis were combined to screen Tie2ligand.Finally, we found two peptides PH1and An1could specifically bind to Tie2protein. We use surface plasmon resonance (SPR) technology to calculate theaffinity constant.To estimate the potential of peptides using in targeting liposomal drugdelivery system, we use SPDP linker to ligate the peptides to the hydrophilicdistal end of DSPE-PEG lipid and measure the ligation efficiency. Finally, weuse post-insertion method for the preparation of peptide modified liposome. We mixed the peptide-lipid micelle with pre-made blank liposome solution（HSPC:Chol:DSPE-PEG=2:1:0.1）and incubated at60℃for1hour.During the in vitro experiments, cell ELISA, laser confocal microscopeand FACS were used to analyze the specific binding ability of our targetingDDS to Tie2positive cells. Peptide PH2and GE11conjugated liposome andconventional long circulation liposome were chosen as negative controls. Wefound that PH1conjugated liposome can bind to Tie2positive tumor cells(SPC-A-1, H1299) and vessel endothelial cell (HUVEC), but not Tie2negative cell (SMMC-7721). The specific cytotoxicity of cis-platin liposomewhich conjugated to PH1peptide was greatly improved. Beside, we also dosome research on peptide targeting gene delivery system and ultrasoundsonoporation mediated transfection.Compared to in vitro system, in vivo environment is much morecomplex. The physiological barriers, non-specific ineractions and internalMPS system may interfere the targeting effect. We labled the peptide An1andPH1conjugated liposome with fluorescent dye Cy5.5, injected via tail vein,and analyzed the fluorescence distribution using eXplore Optix live animalfluorescence imaging system. Compared to control group, An1peptide andPH1-liposome indeed concentrated at the tumor and retained longer time.Furthermore, we encapsulated doxorubicin in the liposome using ammonium sulphate gradient method. PH1conjugation increased the drug concentrationof the tumor site and enhanced the inhibition of the growth of tumor. |
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