Targeted Glutamine-Decorated Chitosan Nanoparticles With Enhanced Synergism Of Chemotherapeutic And Natural Medicine For Hepatic Carcinoma Therapy

Hepatocellular carcinoma（HCC）is one of the severe cancers that seriously threaten human health,and its incidence is increasing yearly.Doxorubicin（Dox）is a first-line chemotherapeutic drug for the treatment of liver cancer,but it has the disadvantages of narrow therapeutic window,high cardiotoxicity,and strong drug resistance,which make the therapeutic effects poor.Considering that it is difficult to achieve sufficient therapeutic effect with a single drug or means,in recent years,the way of combining anticancer drugs with natural products has attracted much attention.Modern pharmacological studies have shown that tetrandrine（Tet）is an effective anti-tumor drug with therapeutic effects on various tumors such as liver cancer,breast cancer,and colon cancer,but its poor water solubility limits its clinical application.In order to achieve active targeting to tumor sites and combined application,a novel pH-responsive nanogel drug release system modified with glutamine（Gln）was constructed in this study.Nanogels decorated by Gln could actively target to tumor sites,and pH-sensitive hydrazone bonds could responsively release drugs under the acidic conditions of lysosomes.In addition,the combined therapeutic effect of Dox and Tet synergistically enhanced the effect of tumor inhibition.In this study,the matrix materials of nanogel were prepared by condensation reaction:glutamine modified chitosan（CS-g-Gln）and carboxymethyl chitosan/adriamycin hydrochloride graft with acid-sensitive hydrazone bond（CS-g-Dox）.The chemical structure of the modified polymer was characterized by ¹NMR and FT-IR.Eight nanogels including blank nanogels（NPs）,Dox nanogels（DNPs）,tetrandrine nanogels（TNPs）,Dox and Tet co-loaded nanogels（DTNPs）,glutamine-modified nanogels（GNPs）,glutamine-modified Dox nanogels（GDNPs）,glutamine-modified Tet nanogels（GTNPs）,glutamine-modified Dox and Tet co-loaded nanogels（GDTNPs）were prepared by reverse emulsification method.The microstructure,chemical structure and material characteristics of the nanogels were characterized and analyzed by mean particle size,polydispersity index（PDI）,Zeta-potential,atomic force microscopy and FT-IR.The drug loading rate,entrapment efficiency and in vitro acid-responsive release behavior of the nanogels were determined by high performance liquid chromatography（HPLC）.And the pharmaceutical parameters and in vitro drug release characteristics of the nanogels were studied to provide a reference for in vivo and in vitro studies.The in vitro antitumor effect of the nanogels was evaluated using human hepatoma cell lines Bel-7402,Hep G-2,and human hepatocyte cell line L-02.The tumor cell uptake of the nanogel and the cell cycle arrest effect was determined by flow cytometry;the intracellular distribution of the nanogel and the mitochondrial targeting of the gel were analyzed by confocal microscopy,the effect of the combination on cell viability was verified by MTS assay,the gene and protein expressions of Bcl-2,Bax,and mitochondrial inner membrane protein cytochrome C（Cyt-C）in the apoptotic pathway were detected by RT-PCR assay and Western-blot assay to carry out the molecular mechanism study of GDTNPs anti-tumor.The mouse model of hepatocellular carcinoma was established using the mouse hepatocellular carcinoma cell line H22.After the model was successful,DNPs,GDNPS,and free Dox were intravenously injected,respectively.The enrichment of Gln-modified nanogels in the tumor tissues of tumor-bearing mice was investigated using small animal imaging techniques to analyze the active targeting ability of Gln-modified nanogels.PBS was used as the blank control group,and free drug,nanogel without Gln modification,and nanogel loaded with a drug were used as controls to evaluate the pharmacodynamics and pharmacological effects of GDTNPs by monitoring the tumor volume,tumor weight,HE staining of tumor tissues,and observation of immunofluorescence staining in tumor-bearing mice.The FT-IR and ¹NMR spectroscopy results of CS-g-Gln and CS-g-Dox demonstrated that the polymer modification was successful.Atomic force microscopy photographs of nanogels prepared by the inverse microemulsion method showed that the nanogel surface was smooth,non-adherent,and round in shape.The average particle size of the gel was（50–60）nm and PDI was<0.3,and all nanogels presented electronegativity.In GDTNPs,the drug loading rate of Tet was（11.1±0.89）%,the encapsulation rate was（45.11±2.03）%,the drug loading rate of Dox was（1.21±0.11）%,the encapsulation rate was（92.36±4.10）%,and the Zeta-potential was（-23.1±2.068）m V.The in vitro release results showed that the release behavior of Dox and Tet was pH-dependent in different types of nanoparticles,the release was poor at pH 7.4,the release was accelerated at pH 5.5,and showed stable continuous release characteristics,which could be used as a good release carrier for Dox and Tet in combination chemotherapy.Cell uptake experiments showed that Gln-modified nanogel GDTNPs had more uptake in hepatoma cells than free drugs or DNPs,while normal hepatocytes had little uptake,demonstrating the effectiveness of Gln modification.Confocal microscopy analysis revealed that the nanogels were mainly distributed in the cytoplasm after entering the cells and had mitochondrial targeting.Cell cycle experiments revealed that Dox,as a non-specific cycle inhibitor,can affect cells at all times,especially in S phase.Significant S phase arrest occurred in the group treated with Tet alone,and S phase and G₂ phase arrest were significantly enhanced in the combined treatment,indicating that the cytotoxic effect of Dox combined with Tet was enhanced.MTS experiments also demonstrated the effect of Dox combined with Tet on cell viability.RT-PCR and WB experiments demonstrated that GDTNPs can promote the expression of Cyt-C by promoting the expression of Bax,inhibiting the expression of Bcl-2,and affecting mitochondrial function,ultimately exerting anti-tumor effects.The results of in vivo fluorescence tracing in mice showed that after intravenous administration,the amount of GDNPs enriched in the tumor site increased compared with DNPs and free drug Dox.In vivo pharmacodynamic results indicate that GDTNPs can significantly inhibit tumor growth by inhibiting the proliferation of tumor cells and promoting tumor cell apoptosis.The above results suggest that Gln-modified GDTNPs can actively target to the tumor site,deliver the two drugs Dox and Tet simultaneously to mitochondria in tumor cells,release drugs in a pH-responsive manner,improve bioavailability,and exert the therapeutic advantages of synergistic treatment of liver cancer with chemotherapeutic drugs and natural products.