Preparation Of Reduction-response Lenvatinib Nanomedicine And Its Application For Treatment Of Hepatocellular Carcinoma

BackgroundHepatocellular carcinoma(HCC)is a common malignant tumor that seriously threatens human life and health.Surgical resection is currently the preferred treatment for HCC with low overall survival rate and high recurrence rate.Chemotherapy also fails to significantly inhibit the progression of advanced HCC due to the low efficacy and severe side effects of chemotherapeutic drugs.Molecular targeted therapy has become the first-and second-line therapy for clinical HCC treatment.Lenvatinib(LEN)is a novel oral multiple receptor tyrosine kinase inhibitor,which has been approved as the drug of choice for the treatment of HCC with better antitumor efficacy.However,the side effects of these molecularly targeted drugs have hindered their widespread application to some extent.Therefore,it is urgent to explore new strategies to improve the efficacy and safety of molecularly targeted drugs in vivo.Nanomedicines have better properties than traditional antitumor drugs,including excellent biocompatibility and bioabsorbability,long circulation time in blood,sensitivity to tumor microenvironment,high permeability and retention effect.In addition,stimuli-responsive nanogels can undergo performance transformation and self-assembly in the tumor environment,promoting anti-tumor drugs to break through multiple biological barriers in the body,achieving precise delivery and release of antitumor drugs,and improving the efficacy of anti-tumor drugs.,and reduce the toxic and side effects of antitumor drugs on other tissues and organs of the body.Purpose:The purpose of this study is to design and prepare nano-drug carriers to load antiHCC drug LEN,which can be used to promote the release of LEN in tumor cells,improve the effect of LEN in the treatment of HCC,and alleviate the toxic and side effects caused by anti-tumor drugs.Method:1.Using amino-terminated polyethylene glycol ether methyl ether(mPEG-NH2)as a macromolecular initiator,L-phenylalanine-N-carboxyl-cyclic acid anhydride(LPhe NCA),L-cystine-Synthesis of methoxypoly(ethylene glycol)-poly(Lphenylalanine-co-L-cystine)[mPEG-P(LP-co-LC)].After the synthesis of mPEGP(LP-co-LC),nuclear magnetic resonance spectroscopy(1H Nuclear magnetic resonance spectroscopy,1H NMR),dynamic light scattering(DLS),transmission electron microscope(Transmission eletron microscope,TEM),Fu Liye transform infrared spectroscopy,elemental analysis and gel permeation chromatography were used to analyze the particle size,morphology,element composition and chemical structure of the polymer,respectively.2.LEN-loaded nanogels(NG/LEN)were prepared by nanoprecipitation,and the drug loading content(DLC)and drug loading efficiency(DLE)of NG/LEN were detected by high performance liquid chromatography),the particle size and morphology of NG/LEN were analyzed by 1H NMR,DLS and TEM.In vitro release of NG/LEN was determined using a dialysis method.3.Use mouse HCC H22 cells to construct subcutaneous and orthotopic HCC animal models respectively.Mice were randomly divided into four groups: control(PBS),Free NG,Free LEN and NG/LEN groups.Then,the changes of tumor volume and body weight of mice in each group were monitored,and the necrosis of tumor cells in each group was observed by Hematoxylin-eosin staining(HE)staining,and the mice in each group were observed Other organs,including the heart,liver,spleen,and kidneys,are affected by drug toxicity.The serum levels of Alpha fetoprotein(AFP),Alanine aminotransferase(ALT)and Aspartate aminotransferase(AST)in the serum of mice in each group were measured by enzyme-linked immunosorbent assay(ELISA).Level.Results1.In this study,the mPEG-P(LP-co-LC)polymer was successfully synthesized.The molecular structure of mPEG-P(LP-co-LC)was detected by 1H NMR.The results indicated that 7.26 ppm(e)was proton resonance of LP(C6H5-,5H)in the benzene ring.The peaks at 4.85-4.31 ppm(c+f)were proton resonances of the polypeptide backbones(-C(O)CH(CH2C6H5)NH-,1H and-C(O)CH(CH2S-)NH-,1H).While the signals at 3.90(b)and 3.58 ppm(a)were assigned to the resonances of methylene and terminal methoxy protons in mPEG(-CH2CH2-,4H and CH3-,3H),respectively.Characteristic peaks of protons at 3.26-2.94 ppm(d+g)(C6H5CH2-,2H and-SCH2-,2H)for methylene protons in side groups of polypeptides.Elemental analysis showed that in the mPEG-P(LP-co-LC)polymer,N,C,H and S accounted for 2.53%,53.08%,8.015% and 3.368%,respectively.In the FT-IR spectrum results,the peaks at 1,636cm-1(vC=O)and 1,468 cm-1(vC(O)-NH)represent the vibrational peaks of the amide bond in LP-co-LC.And 1,149 cm-1(vC-O-C)represents the ether bond in mPEG.TEM results showed that mPEG-P(LP-co-LC)could self-assemble to form nanogels in aqueous solution.The mPEG-P(LP-co-LC)were all displayed as spheres with an apparent size of about 63 nm.According to the DLS results,the hydrodynamic diameter of NG was 81 nm.2.NG/LEN was successfully prepared by nanoprecipitation method.TEM results showed that NG/LEN can still maintain a regular spherical appearance,and the apparent size of the sphere is 80 nm.DLS results found that the hydrodynamic diameter of NG/LEN was 96 nm.Dialysis detection of NG/LEN nanogel DLC and DLE was 8.0%and 48.0%,respectively.The in vitro release of NG/LEN was found in PBS medium without glutathione(GSH)within 12 h of NG/LEN.The cumulative release of LEN was 65.4%.At the same time point,the cumulative amount of LEN released by NG/LEN increased to 91.6% in PBS with 10.0 n M GSH.3.The subcutaneous and orthotopic HCC animal model were successfully constructed.In the two HCC animal models,monitoring of tumor volume in mice found that NG/LEN significantly inhibited tumor growth(P < 0.001).The results of mice body weight showed that the weight of mice given LEN alone decreased,while the weight of mice in the NG/LEN group did not change significantly.The results of HE staining showed that the tumor cells of mice in the LEN and NG/LEN groups were destroyed,while the necrotic area of the tumor tissue in the NG/LEN group was larger.In addition,the results in orthotopic HCC animal model showed that compared with the NG/LEN group,the mice in the Free LEN group had more severe organ damage.The results of blood biochemical analysis showed that compared with the Free LEN group,NG/LEN could significantly improve liver function.Conclusions1.In this study,mPEG-P(LP-co-LC)copolymers were successfully designed and prepared,which are nanogel-like in aqueous solution,and the particle size and morphology characteristics meet the requirements of nanocarriers,which can be used for the next anti-HCC tumor research..2.The particle size and morphological characteristics of LEN encapsulated by mPEG-P(LP-co-LC)copolymer still meet the nano-pharmaceutical standards,and NG/LEN has a good sustained release ability under the condition of high concentration of GSH,and responsively release LEN in HCC cells.3.The subcutaneous,orthotopic HCC animal model successfully constructed by H22 cells shows that NG/LEN has better curative effect in anti-HCC tumor growth,can maintain the weight of mice and reduce the toxic and side effects caused by LEN.