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Objective: A new material: trimethyl-palmitoyl-chitosan (TPCS) was synthesized and modified with lactobionic acid to obtain an active liver tumor-targeting carrier.The self-assembling loaded harmine (HM) micelles were prepared with three polymers (TPCS1, TPCS2 and Lac-TPCS2).The cellular uptake mechanism was investigated.The antitumor activity and liver tumor-targeting effect of the novel micelles were evaluated for exhibiting anti-tumor activity with less adverse effects in vitro and in vivo.(Fig.1A)Fig.1.(A) Schematic illustration of three HM-loaded micelles for anti-cancer tnerapy (B)Synthetical procedure of three polymers(TPCS 1, TPCS2 and Lac-TPCS2) Methods: Three polymers were synthesized and characterized by IR, 1HNMR and element analyzer.The self-assembled micelles loaded with Harmine, an anti-tumor ingredient for liver and esophageal cancer, were prepared with the thin film-hydration method.Cytotoxicity and cellular uptake were measured.Endocytosis inhibitors such as chlorpromazine, wortmannin, cytochalasin B, methyl-β-cyclodextrin and genistein were used to study the precise cellular uptake mechanism.In vivo antitumor activity was evaluated on H22 tumor-bearing mice.Liver tumor-targeting effect was investigated by near-infrared fluorescence imaging.Results: The FT-IR spectra of the three polymers are shown in Fig.2, indicating that PA was successfully grafted to TMC and the introducing of Lac extended the carbon chain.Fig.2 FT-IR spectra of (a)CS, (b) TMC, (c) TPCS 1, (d) TPCS2, (e) Lac-TPCS2.The critical micellar concentration (CMC) of three polymers (2.7-5.6×10-3 mg/mL) was apparently lower than common surfactants of small molecular weight.HM-loaded micelles (TPCS 1/HM, TPCS2/HM and Lac-TPCS2/HM) presented satisfactory particle size (about 200 nm) and drug release characteristics in vitro (Fig.3).TPCS2/HM had a high encapsulation efficiency and drug loading amount of(91.15% ± 0.14%) and (14.58% ± 0.23%), respectively.Fig.3 (A) Stem-Volmer curve of HM and TPCS2/HM.(B) Release of TPCS2/HM under pH5.3 and 7.4.(C)Release of three HM loaded micelles(TPCS 1/HM, TPCS2/HM and Lac-TPCS2/HM) under pH7.4.Morphology of (D)TPCS 1/HM; (E)TPCS2/HM by transmission electron microscope.(F)Lac-TPCS2/HM observed by atomic force microscope(AFM).HM and HM-loaded micelles significantly inhibited the growth of HepG2 cell in a dose-dependent manner.Furthermore, Lac-TPCS2/HM showed the strongest inhibition effect.HM uptake amount of the three blank FITC-labeled micelles in HepG2 (Fig.4A) followed the order: Lac-TPCS2 > TPCS2 > TPCS1.HM uptake of Lac-TPCS2/HM (129.4%) was clearly increased as compared with that of TPCS2 in HepG2 cells, indicating that the endocytosis process was mediated by ASGP-R (Fig.4B).And it's also proved that Lac-TPCS2/HM may enter HepG2 cell through endocytosis route affected by chlorpromazine, methyl-β-cyclodextrin, and genistein, thereby indicating that endocytosis was mainly a clathrin-and caveolae-mediated process (Fig.4C).Fig.4 (A) Fluorescence intensities after HepG2 treated with three FITC-labeled micelles measured by flow cytometry (B) Intracellular fluorescence intensities (n=5) after A549 and HepG2 were treated with FITC-labeled micelles (C) Intracellular fluorescence intensities (n=5) after HepG2 were treated with FITC-Lac-TPCS2 at 4 ℃ or 37 ℃ or in the presence of different inhibitors including 7μg/mL chlorpromazine, 500nM wortmannin, 10μg/mL cytochalasin B, 3nM methyl-β-cyclodextrin (M-β-CD), and 200 nM genistein.(*P<0.05, vs.37℃ group; **P<0.01, vs.37℃ group;***P<0.001, vs.37℃ group.) (D) FITC-TPCS2 distribution in cells at different time points Antitumor experiments in vivo revealed that three types of HM-loaded micelles (TPCS1/HM, TPCS2/HM, and Lac-TPCS2/HM) could significantly inhibit tumor growth and extend the life time of mice bearing H22 tumors after intravenous administration.Next in vivo near-infrared fluorescence imaging results demonstrated a satisfactory liver tumor-targeting effect of Lac-TPCS2/HM (Fig.5).Fig.5 In vivo distribution of Lac-TPCS2 in female Balb/c nude mice bearing H22 tumor after intravenous injection.(A) Typical in vivo real-time imaging.(B) Ex vivo imaging of tumor and organs.Treatment of saline was used as a control.Conclusion: In this work, a novel liver-targeting drug delivery system with high drug-loading efficacy was prepared.The release of three HM-loaded polymers was relatively slower than that of free HM.Compared with TMC, TPCS1, TPCS2, and Lac-TPCS2 all displayed their improved safety and compatibility with HepG2 cells.Lac-TPCS2 particularly had a significant anti-tumor effect with tumor-targeting efficacy and satisfactory safety in hepatoma-bearing mice, which may enter the cells through clathrin-and caveolae-mediated endocytosis.In all, the novel polymers hold great potential in the development of nanomedicine for tumor-targeting therapy.