A Novel Bibranched Fructose Modified Liposome For Paclitaxel Delivery In Breast Cancer Therapy

In this study,we designed a novel Fru-Fru-Chol ligand decorated PTX-loaded liposomes,targeting to GLUT 5 over-expressing breast cancer cells.Model drug paclitaxel was released through the high affinity of bibranched fructose-cholesterol with GLUT 5.We aimed to verify the idea of Fru-Fru-Chol ligand improving the binding between breast cancer cell and the drug delivery sysdem based on the Fru-Chol ligand and Fru+Fru-Chol ligand.Methods:1)The synthesis route of Fru-Fru-Chol ligand was designed.The bibranched fructose residue was covalently linked to cholesterol and the Fru-Fru-Chol ligand was synthesized.2)The PTX/Lip?PTX/Lip-Fru-Chol?PTX/Lip-Fru+Fru-Chol andPTX/Lip-Fru-Fru-Chol was prepareded by the thin film dispersion method.We usedtransmission electron microscope(TEM),dynamic laser particle size analyzer(DIS),zetapotentiometer,HPLC,dynamic dialysis and turbidity assay to measure the particle size,Zeta potential,morphology,entrapment efficiency,drug release and in vitro stability of liposomes.3)MTT assay was used to evaluate the cellular cytotoxicity of PTX/Lip?PTX/Lip-Fru-Chol?PTX/Lip-Fru+Fru-Chol and PTX/Lip-Fru-Fru-Chol to mice breast cancer cells(4T-1)and Human embryonic kidney cells(293T).4)The cellular uptake of CFPE/Lip?CFPE/Lip-Fru-Chol?CFPE/Lip-Fru+Fru-Chol and CFPE/Lip-Fru-Fru-Chol in 4T-1 cells and 293T cells were measured quantitatively and qualitatively by flow cytometry and laser confocal microscope.5)The accumulation of DID?DID/Lip?DID/Lip-Fru-Chol?DID/Lip-Fru+Fru-Chol and DID/Lip-Fru-Fru-Chol in tumor was in vivo observed by near infrared imaging system.Results:1)The Fru-Fru-Chol ligands were successfully synthesized in this study.The structures of important compound and T.M.in the synthesis route were confirmed by nuclear magnetic resonance spectroscopy(~1H-NMR)and high resolution mass spectrometry(HRMS).The molecular weight of Fru-Fru-Chol was 1171.43g/mol and the yield was 63%.2)Paclitaxel was respectively encapsulated in liposomes and liposomes modified by Fru-Chol?Fru+Fru-Chol and Fru-Fru-Chol ligand,then prepared and characterized.The diameter of the particle was between 100 nm and 110 nm,The potential was negative.The dispersion index(PDI)was less than 0.3.The morphology of Lip-Fru-Fru-Chol was regularly spherical in shape and uniform.The entrapment efficiency of paclitaxel was more than 86%.The in vitro release curve of PTX/Lip-Fru-Fru-Chol was consistent with that of PTX/Lip?PTX/Lip-Fru-Chol and PTX/Lip-Fru+Fru-Chol.And the liposomes were stable in vitro.3)The results of the cellular cytotoxicity study demonstrated that Lip-Fru-Fru-Chol had the highest toxicity to positive cell line mice breast cancer cells(4T-1),but lower toxicity to negative cell line Human embryonic kidney cells(293T)compared with Lip,Lip-Fru-Chol and Lip-Fru+Fru-Chol.And the toxicity of different lipid materials to 4T-1 cells was low in each group,indicating that Lip-Fru-Fru-Chol is an anti-tumor drug delivery system with high efficiency and low toxicity.Not only Lip-Fru-Fru-Chol could target breast cancer cells efficiently but was not toxic to normal cells.4)The results of in vitro activity study showed that the fluoresense intensisty of CFPE/Lip-Fru-Fru-Chol was 2.52,2.76 and 3.52 times higher than that of CFPE/Lip-Fru+Fru-Chol?CFPE/Lip-Fru-Chol and CFPE/Lip,respectively,indicating that Fru-Fru-Chol ligand further enhanced the in vitro binding between the drug delivery system and breast cancer cell compared with Fru-Chol and Fru+Fru-Chol ligand.5)The in vivo imaging study indicated that the fluorescence intensity of DID/Lip-Fru-Fru-Chol accumulated in tumor tissues was higher than that of DID?DID/Lip?DID/Lip-Fru-Chol and DID/Lip-Fru+Fru-Chol at the same time.indicating that Fru-Fru-Chol ligand can effectively improve the in vivo binding between the drug delivery system and breast cancer cell compared with Fru-Chol and Fru+Fru-Chol ligand.Conclusion:This study constructed a novel?safe and effective drug delivery system(PTX/Lip-Fru-Fru-Chol),which improved the binding between the drug delivery system and breast cancer cell compared with Fru-Chol and Fru+Fru-Chol ligand in vivo and in vivo.All of these studies provide new ideas and new methods for the design and preparation of tumor targeting liposomes.