A Supramolecular Nanoparticle System Co-deliver Chemotherapy And Gene Agent Targeting Hepatocellular Carcinoma

Objective Hepatocellular carcinoma(HCC)is a common malignant tumor which accounts for the third leading cause of cancer-related death worldwide.Most patients are diagnosed as advanced HCC and lose the opportunity of resection while traditional chemotherapy has limited efficacy and often cause severe side effects.Thus,it is critical to develop novel therapeutic strageties to improve the current status of HCC therapy.Many investigations have confirmed that the initiation,development and metastasis of HCC are a multigene-involving process,thus gene therapy using p DNA,si RNA or micro RNA as therapeutic agents is believed to be an attractive strategy for HCC treatment.Moveover,the combination of chemotherapy and gene therapy can produce synergistic effects through differrent mechanisms.However,one of the major challenges is the requirement of a safe and efficient carrier system for the simultaneous co-delivery of genes and drugs targeting HCC.Fortunately,multifunctional nanocarriers provide great potentials for co-delivering the two therapeutic agents.In our study,a novel supramolecular nanoparticle system was designed to co-deliver doxorubicin(DOX)and oligo RNA and its HCC targeting ability was evaluated both in vitro and in vivo as well,aiming at providing a new strategy for the treatment of HCC.Methods PLCDs with different degree of substitution were synthesized by the conjugation of monoaldehyde activated β-cyclodextrin(β-CD)with poly-L-lysine(PLL)via the Shiff’s base reaction.The chemical structures of PLCDs were characterized by f Fourier infrared analysis(FT-IR)and nuclear magnetic resonance hydrogen spectrum(1H NMR).Using pyrene as a fluorophore,the loading ability of PLCDs towards hydrophobic molecules was determined by fluorescence study.Thegene-condensing ability was evaluated by agarose gel retardation assay.PLCD/DOX nanocomplexes were prepared using dialysis method and further complexed with oligo RNA to form PLCD/DOX/oligo RNA complexes,named PDR.Hyaluronic acid(HA),was coated on the surface of the above nanoparticles by incubation method to construct HCC-targeted supramolecular nanoparticle system,named HPDR.The in vitro DOX release from PDR and HPDR nanoparticles at different p H was performed using dynamic dialysis method.The expression of CD44 molecules on the surface of HCC cell lines MHCC-97 H and Hep G2 was detected using flow cytometry and confocal laser scan microscopy(CLSM).The ability of HPDR nanoparticles co-delivering DOX and FAM-RNA targeting HCC cells was also evaluated by flow cytometry and CLSM.The cell viability assays were performed to assess the biocompatibility of PLCD and HA/PLCD and to compare the cytotoxicity of PDR and HPDR nanoparticles towards HCC cells using CCK-8 reagents.MHCC-97 H tumor-bearing mice model was constructed and further injected with HPDR nanoparticles via tail vein.In vivo imaging system was employed to detect the in vivo distribution of HPDR nanoparticles.Results1.PLCDs were successfully prepared with the DS of β-CD 7.3%,12.9% and 25.1%,respectively.2.DOX could be effectively loaded into PLCD to form PLCD/DOX nanocomplexes with the drug content 11.0% and encapsulation efficiency 41.2%.3.PLCD/DOX/oligo RNA nanoparticles were stable when the N/P ratio was 30/1with an average size of 168.9 nm and zeta potential +38.7 m V.4.HA could be successfully coated on the surface of PDR nanoparticles and formed a negative-charged shell;HPDR nanoparticles have an an average size of 195.8 nm and zeta potential-22.7 m V when the HA/PDR weight ratio was 3/6.5.CD44 receptors were highly expressed on the surface of HCC cell lines MHCC-97 H and Hep G2.6.HPDR nanoparticles could co-deliver DOX and FAM-RNA into HCC cells via CD44-mediated endocytosis and showed strong cytotoxicity towards HCC cells.7.In vivo experiment showed that HPDR nanoparticles were mainly accumulated in the tumor but fewer in the liver compared with PDR nanoparticles,indicating that HPDR nanoparticles exhibited obvious HCC-targeting ability.Conclusions The novel supramolecular nanoparticle system HPDR has a promising potential for combining gene therapy and chemotherapy to treat HCC with its stable structure and strong HCC-targeting ability both in vitro and in vivo.