Construction And In Vitro Antitumor Efficiency Of Hyaluronic Acid Modified Liposomes Co-encapsulating Doxorubicin And Paclitaxel

Liposomes are micro-vesicles similar to biomembrane which formed by self-assembly of phospholipid in water.They have good histocompatibility,high cell affinity,sustained-release and targeting properties,and have been widely used in antitumor therapy.Tumor targeted drug delivery is one of the effective ways to improve the level of tumor chemotherapy,and researchers have developed active-targeting drug delivery system to improve the tumor targeting of chemotherapy drugs.Studies have shown that nanocarriers modified with hyaluronic acid(HA)as target molucule are effective for targeting CD44 receptors which overexpressed in most of cancer cells.In addition,the long-term application of a single chemotherapeutic drug could easily cause drug resistance in tumor cells,thus reducing the antitumor efficacy of drugs,and the combined drug regimen could exert synergistic effects according to different anti-tumor mechanisms between different drugs to improve the efficacy of chemotherapy and reduce the toxic and side effects.In this study,the specific ligand HA of CD44 receptor overexpressed on the surface of tumor cells was used as a target molecule,and small molecule cholesterol(Chol)was coupled with polymer HA to form cholesterol-hyaluronic acid conjugate(Chol-HA)with butanediamine as a connecting arm.The structure of Chol-HA conjugate was confirmed by Fourier Transform Infrared Spectroscopy(FT-IR)and Nuclear Magnetic Resonance Spectroscopy(1H-NMR).Doxorubicin hydrochloride(DOX·HCl)and paclitaxel(PTX)were used as model drugs,the cholesterol segment of Chol-HA conjugate was inserted into lipid bilayer by post-insertion method to prepare HA modified active targeted liposomal delivery system to co-delivery of both DOX and PTX(HA-DOX/PTX-Lip).The liposomes were characterized by Nano Zetasizer,transmission electron microscopy and fluorescence analysis.HA-DOX/PTX-Lip,had a suitable particle size of(125.5±0.79)nm with negative surface charge of(-9.56±0.62)mV,and acceptable encapsulateion efficacy of(93.6±0.51)%(DOX)and(70.4±1.46)%(PTX).Quantitative determination of HA on HA-DOX/PTX-Lip by carbazole method is about 9.5%mol of total lipid.The in vitro drug release study was investigated by dialysis method.The results showed that HA-DOX/PTX-Lip had a significant sustained release effect compared with drug solutions,and the cumulative release rates of both drugs in pH 5.5 PBS were much higher than those in pH 7.4 PBS.The biocompatibility of Chol-HA conjugate and blank HA-modified liposome(HA-Lip)was investigated by hemolysis assay and in vitro cytotoxicity assay.The results showed that Chol-HA and HA-Lip were less cytotoxicity and had good biocompatibility.The in vitro antitumor activity and synergistic effect of free drug and drug-loaded liposome were investigated by MTT assay.The results showed that compared with other groups(free drug group,non-modified liposome group),HA-DOX/PTX-Lips group had better in vitro anti-tumor effect and obvious synergistic effect,and the inhibition rate of MCF-7 cells with high expression of CD44 was higher than that of HepG2 cells with lower expression of CD44.The active targeting of HA is preliminarily verified.The in vitro cellular uptake and uptake mechanisms of HA-DOX/PTX-Lip were qualitatively and quantitatively analyzed by laser scanning confocal microscopy(CLSM)and flow cytometry(FCM),respectively.The results showed that the fluorescence intensity of DOX in MCF-7 cell group was higher than that in HepG2 cell group,which suggested that HA-DOX/PTX-Lips can be more ingested by MCF-7 cells with high expression of CD44 receptor by receptor-ligand mediated endocytosis.The uptake mechanism results indicated that HA-DOX/PTX-Lip entered into MCF-7 cells mainly by clathrin-mediated endocytosis,caveolin-mediated endocytosis and receptor-ligand mediated endocytosis.In summary,HA-modified active-targeting liposomes have good biocompatibility and may be effective targeting carriers for improving tumor therapeutic effects.