Folate-conjugated Macromolecular Prodrug Self-assembled Nanoparticles For HCC Precise Therapy

Efficient hepatocellular carcinoma(HCC)therapy remains a significant challenge due to the unsatisfactory targeting efficiency of nanoparticles(NPs)with either a passive targeting or a single active targeting property.Although a dual targeting mechanism-based strategy can promote somewhat the targeting efficiency,most of the reported NPs with dual-targeting properties generally suffer from multistep chemical design,synthesis,and purification procedures,leading to batch-to-batch variation and difficulties in scalable production.To develop a facile yet efficient strategy toward dual targeting ligands-based NPs for precise HCC therapy and potential clinical translation,folic acid(FA)was readily introduced as a hydrophobic and targeting segment to a hydrophilic macromolecular prodrug,galactosylated chitosan-5-fluorouracil acetic acid(GC-FU)to afford FA-GC-FU formulation that can self-assemble into NPs driven by the solubility variation of FA and polymer moieties without the necessity of previously used physical crosslinking.The resulting nanoparticles(FA-GC-FU)can target the overexpressed ASGPRs and FRs on the surface of HCC cells,respectively via the FA and LA residues exposed on the surface of NPs,leading to maximized targeting efficiency of HCC and minimized nonspecific uptake of normal hepatocytes in vitro and in vivo.Therefore,this study not only developed a simple yet efficient strategy toward a facile fabrication of NPs with dual targeting ligands,but also presented a precise therapeutic platform for HCC with great potential for clinical translation.FA was conjugated to a hydrophilic macromolecular prodrug,GC-FU via amide condensation reaction to afford FA-GC-FU formulation that can self-assemble into NPs driven by the solubility variation of FA and polymer moieties.The chemical structure of FA-GC-FU was identified by ~1H-NMR.The morphological characteristics of FA-GC-FU were observed by transmission electron microscopy(TEM)and the particle size distribution of FA-GC-FU was detected by nanometer particle size analyzer,which confirmed that FA and GC-FU were successful conjugation and the introduction of FA driven the formation of spherical nanoparticles.The drug load measured by ultraviolet spectrophotometry was 21.22±1.10%.Finally,the stability and drug release characteristics of FA-GC-FU were evaluated.It was confirmed that FA-GC-FU has good stability and continuous drug release characteristics.The biocompatibility of FA-GC-FU was evaluated.The blood compatibility of FA-GC-FU was detected by hemolysis test and BSA protein adsorption test,respectively,and the cytocompatibility of FA-GC-FU was evaluated by proliferation inhibition experiments of human normal liver cells L02 cells and human umbilical vein endothelial cells HUVEC cells.The results confirmed that FA-GC-FU has excellent biocompatibility compared with free 5-FU.To verify the anti-tumor effect and targeting characteristics of FA-GC-FU in vitro,cell proliferation inhibition experiments,competition binding experiments,cell apoptosis and cell cycle experiments,and cell anti-metastasis experiments using SMMC-7721 hepatoma carcinoma cell overexpressed with FR and ASGPR,and cell uptake experiments on SMMC-7721 cells and A549 human non-small cell lung cancer cells(low expression of FR and ASGPR)were performed.The results showed that FA-GC-FU exhibited the highest inhibition effect on the proliferation of HCC,confirming that FA induced the formation of FA-GC-FU nanoparticles while still had the targeting property and also proves that FA-GC-FU plays a cooperative targeting role in increasing cell uptake via mediation of FR and ASGPR.At the same time,compared with free 5-FU,FA-GC-FU can significantly induce apoptosis and block the cell cycle,and has anti-cancer cell metastasis effects at non-cytotoxic concentrations.The in vivo anti-tumor efficacy of FA-GC-FU was evaluated by the SMMC-7721 xenograft BALB/c nude mice model.The results showed that FA-GC-FU had the highest inhibition rate of tumor growth.At the same time,the biotoxicity of FA-GC-FU was evaluated using tissue section experiments and biochemical indicators.It is proved that FA-GC-FU can reduce the toxic and side effects of 5-FU.Therefore,FA-GC-FU has been proven to be a more effective and safer method for the treatment of HCC.Conclusion:We was developed nanoparticles(FA-GC-FU)by conjugating FA to hydrophilic macromolecular prodrug GC-FU.The preparation process is simple and easy.The prepared FA-GC-FU shows good biocompatibility and has better anti-tumor effects in vitro and in vivo than free 5-FU and single ligand-targeted GC-FU.