The Synergistic Anticancer Mechanism Of Sorafenib-loaded GPC3-targeted Nanoparticles Specific To HCC

Objective:Sorafenib approved by FDA for the treatment of advanced hepatocellular carcinoma(HCC)is a multi-kinase inhibitor on multi-target.It is necessary to be administered continuously for sustained inhibition on its targets.The PCL-TPGS/Pluronic P123 nanocarryer we previously developed with high capacity of drug loading and sustained drug release has a controlled drug release rate and can reduce drug administration frequency;however the drug targeting distribution or the anti-HCC efficacy has not been enchanced.In this project,a targeted drug dilivery system based on anti-glypican-3 humanized monoclonal antibody(hGC33)modified and sorafenib loaded PCL-TPGS/Pluronic P123 nanoparticles was constructed,which has such characteristics as positioning adhesion of HCC cells,controlled drug release in tumor body and enhanced anti-HCC efficacy through joint inhibitory effect by hGC33 and sorafenib.The result of this study is helpful to improve the basic research level of our drug delivery system specific to HCC and presents a new approach and idea for the treatment of HCC.Methods:(1)The pathological tissue samples with definitely diagnosis were collected and further analyzed by immunohistochemical method to verify the expression,localization,positive expression rate and specificity of GPC3 in HCC.(2)The SFB-loaded nanoparticle NP-SFB was prepared with an improved nanoprecipitation method.First,caprolactone reacts with water-soluble vitamin derivative TPGS to form TPGS-PCL under the action of stannous octoate;under the action of dichloromethane and pyridine,Pluronic P123 reacts with p-toluenesulfonyl chloride to form P123-p-toluenesulfonate(P-toluenesulfonyl Pluronic P123,Pluronic P123-OTs,P123-OTs reacted with potassium phthalimide to give phthalimide-Pluronic P123(Pluronic P123-PI),Pluronic P123-PI Synthesis of P123(Pluronic P123-NH2),P123-NH2 and 1.2 N Crosslinker(SMMP)(3-Maleimidopropionic acid hydroxysuccinimide)with hydrazine hydrate under anhydrous ethanol Ester,N-succinimidyl 3-maleimidopropionate,SMMP)reaction to obtain maleimide-polypropylene glycol-polyethylene glycol-polypropylene glycol(Pluronic P123-Mal);P123-Mal,TPGS-PCL and SFB in acetone at a certain ratio Stirring reaction was performed under ambient conditions to obtain SFB-loaded nanoparticles(SFB-TPGS-PCL/P123-Mal,NP-SFB).(3)Construction of antibody-modified nanoparticles(Ab-SFB-NP).The use of traut’s reagent and antibody molecules,the primary amine group(-NH2)in the antibody molecule is converted to sulfhydryl(-SH)to obtain thiolated antibody(Ab-SH),and then the NP-SFB and Ab-SH are mixed at room temperature The antibody-modified nanoparticles(SFB-TPGS-PCL/P123-Ab,Ab-SFB-NP)were obtained after 2 hours of reaction.(4)The drug-loaded nanoparticles were characterized by nuclear magnetic,infrared,transmission electron microscopy,and particle size potential analyzers;the drug loading and entrapment efficiency of the nanoparticles were determined by high performance liquid chromatography(HPLC);GPC3-positive cells were used.HepG2 cells were used to determine the targeting of NP-SFB-Ab;MTT assay was used to detect the biocompatibility of nanomedicines;animal dosing experiments were used to evaluate the toxicity of nanomedicines to the major tissues and organsof the body.(5)MTT technique to assess the biological effects of Ab-SFB-NP on hepatoma cells HepG2;Transwell and scratch experiments to observe the impact of Ab-SFB-NP on the invasion and migration of HepG2 cells;flow cytometry,Modfit software analysis Ab-Effect of SFB-NP on cell cycle;WB detection of effects of Ab-SFB-NP on HepG2 cell-associated pathway molecules and regulatory mechanisms;Further establishment of a mouse model of HCC,testing of anti-HCC effects of the prepared nanomedicine in vivo,assessment In vivo anti-tumor effects of Ab-SFB-NP.Results:(1)High expression of GPC3 in HCC tissues.GPC3 is expressed in almost all HCC(32/35;91.4%),but only one case of non-HCC cancer GPC3 expression,paracancerous tissues do not express GPC3;Arg-1 and HepPar-1,GPC3 are more specific and have higher positive expression rates for HCC.GPC3 is an ideal marker of HCC and can act as a biological target for HCC treatment to facilitate targeted drug delivery.(2)Successful construction of SFB-NP drug-loaded nanoparticles.1.The characterization and detection of Ab-SFB-NPs prepared in this study showed that the encapsulation efficiency and loading rate of Ab-SFB-NPs were 75.9% and9.8%,respectively,which all achieved higher efficiency.2.The nuclear magnetic proton spectra of TPGS and TPGS-PCL are 4.08 ppm and2.37 ppm are characteristic peaks of-OCCH2-and-CH2OOC-,respectively,on polycaprolactone,and the peak single peak at 3.66 ppm is attributed to TPGS oxyethylene units.The homologue of methylene protons.It was confirmed that the amphiphilic TPGS-PCL copolymer was successfully synthesized;the elemental analysis of the two-terminal amino group P123-NH2 showed that the nitrogen content was 0.37%,which was similar to the expected result,demonstrating thatalmost all of the P123 was attached to the amino group;The 3.57,3.42,and 1.15 ppm of the NMR spectrum of the bundle are the characteristic peaks of P123,and3.66 ppm is the characteristic peak of the water-soluble vitamin E,which proves that P123-Mal and TPGS-PCL are successfully combined,and 7.28 ppm are both deuterated chloroform solvents.Peak;the absorption peak at 2978 cm-1 in the FTIR diagram is attributed to the(-CH2)methyl stretching vibration of PCL,the absorption peak at 1732 cm-1 is the carbonyl vibration peak of TPGS,and the absorption peak at 1107 cm-1 is P123-Mal.Due to the CO bond stretching vibration of the molecule and the vibration of the COC skeleton,the above main absorption peaks are all present in the copolymer,which proves that P123-Mal and TPGS-b-PCL bind well together.The uptake efficiency of different GPC3+ HepG2 cells to different nanoparticles showed that the uptake efficiency of Ab-SFB-NP was higher than that of non-antibody modified NP(SFB-NP).Ab-SFB-NP specifically targeted cell surface of the function of GPC3.5.The biocompatibility test showed that Ab-SFB-NP had good compatibility with normal blood cells and vascular endothelial cells,and there was no obvious damage to the main tissues and organs of animals,suggesting that Ab-SFB-NP is compatible with good histocompatibility.(3)The anti hepatocarcinoma effect and mechanism of the Ab-SFB-NP nano delivery system.Ab-SFB-NP has a significant inhibitory effect on GPC3+ hepatoma cells,and its efficiency is positively correlated with the expression of GPC3 on the surface of tumor cells;Ab-SFB-NP up-regulates the ability of SFB to induce HepG2 cell apoptosis;Ab-SFB-NP nanomedicine It can effectively inhibit the invasion and migration ability of tumor cells;WB assay results show that Ab-SFB-NP inhibits thec-Maf/MEK/ERK signaling pathway in HepG2 cells of GPC3+ more effectively than SFB-NP and free SFB and inhibits it.The effect was significantly higher than the inhibitory effect on the corresponding pathway in HLF cells of GPC3-negative cells;further WB results showed that Ab-SFB-NP did not affect the expression level of anti-apoptotic protein XIAP,but down-regulated the Mcl-1 and C-FLIP in HepG2 cells.The level of anti-apoptotic protein showed that SFB induced caspase-dependent apoptosis and down-regulated the expression of Mcl-1 and c-FLIP.(4)Anti-tumor effect of Ab-SFB-NP in vivo.Tumor growth was significantly inhibited in the Ab-SFB-NP group,and the results were far superior to SFB and SFB-NP groups.Conclusion:This study shows that GPC3 is an ideal marker of HCC and can act as a biological target for HCC treatment to facilitate targeted drug delivery.The prepared GPC3antibody-modified Ab-SFB-NPs nanoparticles were successfully constructed.The characterization and detection showed that the SFB encapsulation efficiency and drug loading were good,indicating that Ab-SFB-NPs have GPC3 molecular targeting.Simultaneous detection of Ab-SFB-NPs nanomedicine has good biocompatibility,no obvious toxicity was found in vivo and in vitro.Ab-SFB-NP mainly down-regulates phosphorylation of MEK1/2 and ERK by inhibiting Raf kinase activity,thereby inhibiting RAF/MEK/ERK cascade signaling pathway;in addition,Ab-SFB-NP down-regulates Mcl-1,leading to Bax and Bak The mitochondrial membrane polymerization to enhance mitochondrial permeability and promote mitochondria release cytochrome C and promote cell apoptosis;Therefore,Ab-SFB-NP can effectively inhibit the growth of HCC tumors.The new SFB nanoparticles Ab-SFB-NP prepared in this study were successfully developed for thetargeted treatment of liver cancer.