Study On The Co-delivery Of Doxorubicin And MiR-122 With Nanosystem For Hepatocellular Carcinoma Therapy

Objective: Hepatocellular carcinoma(HCC)is the third leading cause of malignancies death in the world,and its incidence is increasing year by year.Although surgery is the first choice in HCC,it is a pity that most patients had lost the opportunity of operation.Therefore,it is essential to develop a safe and effective treatment for the advanced patients.Recently,owing to their advantages of easy degradation,low toxicity and sustained release,nanometer materials have become the research focus in the field of drug delivery carriers.Chemotherapeutic drug-loaded nanoparticles can not only achieve a better antitumor effect,but also reduce the systemic toxicity of chemotherapeutic drugs.Further study showed that combination of gene and chemotherapy drug can effectively enhance the efficacy of drugs,and achieve synergistic antitumor effects by regulating cell signal pathway.Micro RNA-122(mi R-122)is a liver specific mi RNA,which is frequently down-regulated in HCC,and is closely related to the occurrence and development of liver tumors.Mi R-122 can interfere directly or indirectly with the expression of multi-drug resistance related genes and proteins,so as to improve the chemosensitivity of tumor cells.In this study,a nanosystem based on ?-cyclodextrin-cored star polymer(s CDP)was prepared to co-deliver antitumor drug doxorubicin(DOX)and mi RNA-122 and form drug/gene co-loaded nanoparticles.Moreover,the properties of nanoparticles were characterized and its combination effects were further detected in vivo and in vitro.Methods: 1)Using DOX as model drug,a series of different feed ratios of s CDP/DOX nanoparticles were prepared by dialysis method,and their sizes and zeta potentials were characterized by dynamic laser scattering(DLS);the drug loading content(LC)and encapsulation efficiency(EE)were both determined by UV/vis spectrometry so as to confirm the optimum preparation ratio;transmission electron microscope(TEM)was used to observe the morphology of s CDP/DOX nanoparticles,the in vitro release rate of drug was investigated by dynamic dialysis method;2)Agarose gel retardation assay was used to evaluate the ability of s CDP/DOX condensing mi R-122;DLS was used to determine the particle sizes and potentials of s CDP/DOX/mi R-122 nanoparticles.The morphology of the optimal nanoparticles was further observed by TEM.3)The expression level of mi R-122 was detected by q RT-PCR in Hep G2 cells treated with s CDP/DOX/mi R-122;the cellular uptake of DOX and mi R-122 was detected by flow cytometry and confocal laser scanning microscopy(CLSM).4)CCK-8 kit was employed to evaluate the cytotoxicity of s CDP/DOX/mi R-122 nanoparticles on Hep G2 cells;Annxin V-APC/7-AAD cell apoptosis analysis kit was to detect the cell apoptosis rate;expression of p53 and casepase3 protein were detected by western blot;5)The expression of mi R-122 target genes Bcl-w,CCNG1,MDR1 and MRP were determined by q RT-PCR and western blot in m RNA and protein levels respectively;6)The nude mice liver cancer model was constructed and the tumor growth in nude mice bearing the tumor was observed.Results: Section 1: 1)s CDP/DOX nanoparticles with different feed ratio were successfully prepared;when the feed ratio was 10/3,the average size of s CDP/DOX nanoparticles is 183.8 nm with the polydispersity index(PDI)of 0.169 and the zeta potential is +20.3 m V;the drug LC is 12.62% and EE is 48.16%,respectively;the morphology of s CDP/DOX nanoparticle is irregular;the drug release from s CDP/DOX nanoparticles showed p H-sensitivity.2)Both s CDP and s CDP/DOX can complex mi R-122 when N/P?10;the average size of s CDP/DOX/mi R-122 nanoparticle(N/P=10/1)is 160.4 nm with the PDI of 0.177 and the zeta potential is +20.4 m V;the morphology of s CDP/DOX/mi R-122 nanoparticle is regular with spherical structure,and the stability in aqueous solution is better.Second 2: 1)s CDP/DOX/mi R-122 nanoparticles can be successfully uptake by Hep G2 cell,the expression level of mi R-122 is significantly increased;the cellular uptake of s CDP/DOX/mi R-122 nanoparticle is in a time-dependent manner,which gradually release DOX into the nucleus.2)s CDP has good biocompatibility;s CDP/DOX/mi R-122 nanoparticles can significantly inhibit Hep G2 cell proliferation,promote cell apoptosis and increase the expression of p53 and cleaved caspase-3;s CDP/DOX/mi R-122 nanoparticles can down regulate the expression of Bcl-w and CCNG1 leading to irreversible cell apoptosis,decrease the expression of MDR1,MRP and P-gp,and improve the sensitivity to DOX of Hep G2 cells,thus realizing the synergistic antitumor effect in vitro.3)s CDP/DOX/mi R-122 nanoparticles can slow down the weight loss of nude mice bearing tumor and significantly inhibit tumor growing,thus realizing a synergistic anti-tumor effect in vivo.Conclusions: In this study,?-cyclodextrin-cored star cationic polymer was applied as carrier to co-deliver DOX and mi R-122;s CDP/DOX/mi R-122 nanoparticles with uniform and stable properties were successfully prepared;the expression level of mi R-122 in Hep G2 cells significantly increased after incubation of s CDP/DOX/mi R-122 nanoparticles;s CDP/DOX/mi R-122 nanoparticle could significantly inhibit tumor growth not in vitro and in vivo;the mechanism of tumor suppression were investigated by cytological level,we found that s CDP/DOX/mi R-122 could play a synergistic antitumor effects by inducing apoptosis,reducing the expression of multidrug resistance proteins,and interfering cycle regulation and apoptosis pathway.This study provides a theoretical basis for the development of drug/gene co-loaded nanosystem for the treatment of liver cancer,which has a great potential in HCC therapy.