Targeted Protein Liposomes-Mediated AChE Gene Therapy for Effective Liver Cancer Treatment


 Background: Effective methods to deliver therapeutic genes to solid tumors and improve their bioavailability are the main challenges of current medical research on gene therapy. The development of efficient non-viral gene vector with tumor-targeting has very important application value in the field of cancer therapy. Proteolipid integrated with tumor-targeting potential of functional protein and excellent gene delivery performance has shown potential for targeted gene therapy.Results: Herein, we prepared transferrin-modified liposomes (Tf-PL) for the targeted delivery of acetylcholinesterase (AChE) therapeutic gene to liver cancer. We found that the derived Tf-PL/AChE liposomes exhibited much higher transfection efficiency than the commercial product Lipo 2000 and shown premium targeting efficacy to liver cancer SMMC-7721 cells in vitro. In vivo, the Tf-PL/AChE could effectively target liver cancer, and significantly inhibit the growth of liver cancer xenografts grafted in nude mice by subcutaneous administration. Conclusion: This study proposed a transferrin-modified proteolipid-mediated gene delivery strategy for targeted liver cancer treatment, which has a promising potential for precise personalized cancer therapy.

silence disease-causing transthyretin (TTR) [31]. It is known that transferrin (Tf) is a plasma protein responsible for transporting iron into cells via the transferrin receptor (TfR) [32][33][34]. TfR is overexpressed in many human tumors, and Tf-conjugated carrier can be used to selectively target drug delivery to cancer cells [35][36][37][38]. Many literature reports the content of the transferrin receptors in liver cancer tissues is higher than normal liver tissues [39,40]. The tumor database information of Oncomine show that the expression of TfRs in liver cancer tissues was 2.780-fold higher compared to normal liver tissue (p<0.01, Supporting Information Figure S1). At the same time, the survival rate of the patients with high TfR expression was lower than the patients with low TfR expression ( Figure S2). Therefore, transferrin receptors can be a candidate active targeting site for liver cancer gene therapy.
Acetylcholine (ACh) is involved in a variety of cell biological behaviors, such as cell proliferation, cell differentiation, cell migration and cellular immune response, so the accumulation of ACh in the tumor microenvironment directly or indirectly supports tumor growth [41]. Acetylcholinesterase (AChE) plays key role in the cholinergic system, and its dysregulation is involved in a variety of human diseases [42]. AChE can degrade the ACh and reduce the malignant development risk of liver cancer, thus AChE has been identi ed as a prognostic marker in liver cancer [41,43,44]. Oncomine database shows extremely high expression of ACh in various tumor tissues including liver cancer, p=0.002 ( Figure S3), while AChE shows signi cant low expression in liver cancer p=0.012 ( Figure S4). Therefore, AChE can be used as a gene therapy target for liver cancer. In this study, we for the rst time to construct transferrin modi ed liposomes (Tf-PL) using transferrin-glycidyl hexadecyl dimethylammonium chloride (GHDC) instead of lipids, the transferrin was combined with GHDC to realize the hydrophilic and lipophilic modi cation, and then the transferrin liposome was prepared and used for gene loading. Tf-PL preparation was showed in Figure 1A, transferrin was utilized to mediate the delivery of AChE gene to the cytoplasm via transferrin receptor-mediated endocytosis ( Figure 1B). In this study, the tumor targeting ability and growth inhibitory effect of Tf-PL/AChE were evaluated through a series of in vitro and in vivo experiments.

Results And Discussion
Preparation and characterization of the protein liposomes In this study, the liposome was prepared by physically assembling method ( Figure 1A), the amphiphilic protein derivative modi ed by GHDC is different from the conventional protein-modi ed nanospheres or liposomes. In order to re ect the targeting capacity of Tf, non-Tf GHDC-liposomes (GL) were also prepared for comparison. The morphology of GL/AChE and Tf-PL/AChE was analyzed by transmission electron microscopy (TEM) (Figure 2 A and B). Dynamic light scattering (DLS) was used to evaluate the particle size and potential of GHDC-liposomes (GL) and Tf-PL. Figure 2 C and D showed the particle size and potential data of liposomes in aqueous solution. The particle size of Tf-PL/AChE was 112.9 ± 4.5 nm (PDI = 0.149), the potential was 21.8 ± 0.5 mV, and the particle size of GL/AChE was 99.82 ±2.2 nm (PDI= 0.118), and potential was 27.3 ± 0.4 mV (Table S5, Supporting Information). Both of the liposomes with double-layer skeleton structure with a regular spherical shape. The UV-vis spectrum in Figure 2E showed that the intermediate Tf-GHDC and nal Tf-PL have similar UV absorption curves with the Tf, indicating the successful Tf modi cation. The protein electrophoresis results indicated that Tf-PL has transferrin content ( Figure 2F).
The characteristic peaks of transferrin and GHDC were also observed in the nuclear magnetic H spectrum, and the long-chain methylene stretching vibration absorption peak was observed at 2922 cm -1 and 2852 cm -1 ( Figure S6). Suggesting the proteoliposome prepared in this study had a higher protein component. The optimized distribution ratio was determined by studying the effects of different distribution ratios on liposome size, charge, gene encapsulation e ciency (EL), and loading (DL) (Table S1-4, Supporting Information). The loading of Tf-PL to AChE optimized to the distribution ratio was (6.31±0.32) %, the encapsulation e ciency was (94.3±1.01) %, the load corresponding to PL (6.07±0.43) %, and the encapsulation ratio (91.2 ± 0.79) % (Table S5, Supporting Information). At the same time, the quantitative analysis of transferrin on the surface of Tf-PL performed by ELISA showed that the e ciency of transferrin modi cation on the surface of liposome was (88.7±2.31) % ( Figure S7 and Table S5, Supporting Information), suggesting most of the transferrin was distributed on the surface of proteoliposome.
In vitro gene release study and cytotoxicity of the proteoliposomes As a gene carrier, besides the high gene delivery e ciency, the Tf-PL also should have high stability during storage. The stability of GL/AChE and Tf-PL/AChE under different pH conditions was explored ( Figure S8). Figure S8 A show the total release at different time points, and Figure S8B show the gene release rate within 12 hours. Compared with the rapid release of free AChE, GL/AChE and Tf-PL/AChE showed similar sustained release of AChE at pH 7.4 and pH 5.5; the burst release was not obvious, and the stability of Tf-PL/AChE complex was higher.
The Figure S9 show the in vitro cytotoxicity of the prepared proteoliposomes. The results indicated that the relative survival rate of normal liver HepZJ cells and SMMC-7721 cells were still high when the concentration of GL and Tf-PL reached 500 μg/mL after 48h of culture. Herein, the proteolipid prepared in this study has good biocompatibility and low cytotoxicity, which lays a foundation for subsequent applications.
In vitro cellular uptake and transfection e ciency study To determine whether the TfR could be a therapeutic target for the treatment of liver cancer, we compared the expression of TfR between liver cancer (N2=225) and normal liver tissue (N1=220) by using the online database Oncomine. We found that the expression of TfR was signi cantly increased in liver cancer specimens, which was 2.780 times the normal specimens ( Figure S1). In order to further investigate the expression of TfR in cells, we selected liver cancer cells and immortalized liver cell lines currently preserved in the laboratory, and detected TfR expression by Western blot. The results in Figure S5 show that compared with normal liver cells, liver cancer cell lines express higher levels of TfR. TfR may be a potential drug delivery target for the treatment of liver cancer. Then, we compared the expression of TfR in HepZJ and SMMC-7721 cells by ow cytometry. As shown in Figure 3 A, the ow cytometry results showed that the TfR expression of SMMC-7721 was as much as 7.36 times of HepZJ cells, the expression difference between the two cells was signi cant (p<0.01), which can be used for further experimentations. To study the interaction between liposomes and cell surface receptors, and assess the ability of Tf-PL to speci cally bind to TfR and trigger receptor-mediated internalization of liposome delivery in TfR-positive cells, we compared binding e ciency of FITC-labeled GL and Tf-PL in SMMC-7721 cells. As shown in Figure 3B, SMMC-7721 cells showed signi cant higher endocytosis of Tf-PL/FITC than GL/FITC. In order to study whether Tf-PL/FITC was taken up through TfR-mediated endocytosis, the SMMC-7721 cells were treated with Tf for 6h before the Tf-PL/FITC added. Confocal microscopy shows that the FITC uorescence was signi cantly reduced, so the endocytosis of Tf-PL/FITC by SMMC-7721 cells was disturbed by the competitive combination of Tf. The confocal microscopy result indicated that Tf-PL was taken up through TfR-mediated endocytosis. We also analyzed the subcellular localization of Tf-PL after endocytosis by cells, the results of confocal experiment showed that the endocytic Tf-PL were mostly distributed in cytoplasm and partly in lysosomes ( Figure S10 Based on the above research results, it is concluded that the Tf-PL is suitable to be used as a vector for SMMC-7721 cells gene therapy. In vitro tumor cell proliferation inhibition study Figure S3 and S4 shows that an overexpression of ACh and a low expression of AChE were detected in liver cancer cells. Therefore, we measured the effect of ACh and AChE on the proliferation of SMMC-7721 cells by CCK-8 assay. As shown in Figure S11 A, ACh could signi cantly promote the proliferation of SMMC-7721, with the increased concentration and prolonged action time of ACh, the proliferation promotion effect is more obvious. On the contrary AChE could signi cantly inhibit the proliferation of SMMC-7721, and the inhibition of proliferation was more obvious with the increase concentration and prolonged action time of AChE (Figure S11 B). Therefore, this study demonstrated that the overexpression of AChE is a theoretically strategy to inhibit the proliferation of liver cancer cells. Then the effect of AChE gene therapy on liver cancer SMMC-7721 cells was studied. It was found that GL/AChE and Tf-PL/AChE inhibited the growth of SMMC-7721 cells in a concentration-and time-dependent manner. Free AChE gene had little effect on the proliferation of SMMC-7721 cells and HepZJ cells ( Figure 5). Tf-PL/AChE showed the highest cytotoxicity with IC50 values of 4.25 μg/mL (48 h) and 3.45 μg/mL (72 h), respectively ( Figure   5B). Thus, Tf modi cation could signi cantly enhance the uptake of Tf-PL/AChE by liver cancer SMMC-7721 cells, Tf-PL delivered more AChE to cells and subsequently inhibited cell growth.
In vitro cell migration and wound healing study Cell migration plays an important role in tumor growth and metastasis, so the role of Tf-PL/AChE on the migration of SMMC-7721 cells was assessed by transwell migration and scratch experiments ( Figure S12 and Figure S13). Although obvious migration of the SMMC-7721 cells without any treatment was observed. The migration of SMMC-7721 cells treated with Tf-PL/AChE and GL/AChE signi cantly reduced. and Tf-PL/AChE was the most effective, the inhibition rate was 45.1%, p<0.05. These observations indicate that Tf-PL/AChE treatment can effectively block the migration of SMMC-7721 cells.
In the wound healing experiment, 48 h after cell scratch, the control group, free AChE and GL/AChE group showed signi cant cell healing, while Tf-PL/AChE Group SMMC-7721 cell wound healing was signi cantly delayed. The above results indicate that Tf-PL/AChE treatment signi cantly affected the migration and proliferation of SMMC-7721, Tf-PL could improve the effect of gene therapy. Real-time uorescence imaging of nude mouse organs and tumors was performed by injecting a nearinfrared dye (Cy5.5)-labeled liposomes into the tumor-bearing nude mice, and the target effect of the Tf-PL/AChE was studied by the distribution of uorescent signals (Figure 8). The uorescence image of Cy5.5 showed that after 24 hours of injection, the uorescence signal was mainly found in the kidney, lung, liver, tumor, and peripheral blood. Moreover, the uorescence signal of mouse tumors in the Tf-PL group was stronger than the other groups. After 72 hours, the uorescence signals in the kidney, lung, liver, tumor, and peripheral blood were signi cantly decreased. There was still obvious Cy5.5 signal distribution in the tumor of the Tf-PL group, but the intratumoral signal of the PL group was signi cantly decreased. At the same time, green uorescent protein (GFP) expression was studied to indirectly mimic the expression of the acetylcholine ester gene to analyze the targeted delivery of the Tf-PL-carrying gene to the tumor. There was a signi cant GFP uorescence signal at the tumor site in mice, 24h or 72h. Based on the above experimental results, it can be proved that the Tf-PL group has extremely high intensity and distribution in tumors, so Tf-PL can effectively target liver cancer.
In vivo antitumor e cacy study In this study, a nude mouse xenograft model of SMMC-7721 tumor was established, and different therapy treatments were given to analyze the anti-tumor effects of different groups. Mice bearing SMMC-7721 tumors were injected with normal saline, free AChE, GL/AChE, and Tf-PL/AChE every 7 days for 4 consecutive injections. As shown in Figure 9A, free AChE had slight e ciency in inhibiting tumor growth, tumor growth in mice treated with GL/AChE and Tf-PL/AChE was signi cantly inhibited. Especially, the mice treated with Tf-PL/AChE had the most pronounced inhibitory effect on tumor growth compared to the other groups ( Figure 9A and Figure S14). The nal weight and volume of the tumor in the Tf-PL/AChE group were (0.25 ± 0.12) g and (517.14 ± 112.63) mm 3 , respectively (Figure 9 B, C). The tumor suppression effect of free AChE was 7.81%, tumor growth inhibition was ine cient. However, the tumor weight inhibition rate of Tf-PL/AChE was 77.47%, while that of GL/AChE group was only 48.21%, Tf-PL/AChE effectively inhibited the growth of liver cancer cells and reduced the weight of the tumor. These results supported the superior antitumor e cacy of Tf-PL/AChE treatment in vivo. As shown in Figure 9 D, the HE staining results showed the tumors in the GL/AChE and Tf-PL/AChE groups showed mild staining and a large area of blank, in Tf-PL/AChE group tumor tissue showed a large area of necrosis, so the tumor tissue necrosis was very serious. The tumor tissues of control and the Free AChE groups were darker, showed identi able tissues. Taken together, the Tf-PL/AChE could effectively inhibit tumor growth.
Currently, non-viral vectors have been widely used for gene therapy [43,44]. In order to achieve targeted delivery of therapeutic drugs to tumor cells, receptor-mediated active targeting has been generally adopted in recent years, such as the presence of asialoglycoprotein receptors on liver parenchymal cell membranes; mannose receptors are distributed on non-parenchymal cell membranes. According to many studies both at home and abroad, it has been suggested that the content of transferrin receptor in liver cancer tissue is signi cantly higher than that in adjacent and normal ovarian tissue [64, 65], as shown in Figure S1. Therefore, Tf-modi ed liposomes can increase the therapeutic gene uptake by liver cancer cells with high TfR expression on the cell membrane surface, thus the therapeutic genes can speci cally act on liver cancer cells, reducing the possibility of toxic side effects. Our study describes a method for easily preparing a neutral targeting gene delivery system by complexing cationic nanoparticles with a therapeutic plasmid by electrostatic interaction. The obtained neutral targeting gene delivery system provides a robust and exible non-viral platform for mediating cancer gene therapy for intravenous administration.
In this work, we attempted to directly construct protein liposomes using protein derivatives by amphiphilic modi cation of the tumor-targeting protein-transferrin, and by in vitro and in vivo delivery of the gene. The effect evaluation con rmed the signi cance of the effectiveness and targeted effect of the constructed gene/proteoliposome. In order to study the performance of Tf-modi ed AChE gene-carrying proteoliposomes, we conducted a series of cell experiments in vitro. Qualitative and quantitative analysis showed that Tf-PL enters SMMC-7721 cells more signi cantly than non-targeted liposome GL, signi cantly increasing the uorescence intensity in targeted liver cells. The TfR competition experiment showed that the uptake of Tf-PL by SMMC-7721 cells after the TfR receptor was occupied by Tf was signi cantly reduced, so Tf can competitively inhibit the targeted endocytosis mediated by the TfR receptor and Tf-PL mainly depends on the TfR mediated endocytosis into liver cancer cells. These results indicate that Tf-modi ed AChE-loaded proteoliposomes have good active targeting properties.
Then analyze the tumor cell inhibitory ability of Tf-guided targeting liposomes. Cytotoxicity test results show that compared with Free AChE and GL/AChE groups, Tf-PL/AChE has a more obvious inhibitory effect on the proliferation of liver cancer cells, and is concentration-and time-dependent, which may be signi cantly related to Tf-PL/AChE. Apoptosis and cell cycle arrest in G0/G1-S phase is related (see Figure 6 and Figure 7). Cell migration experiments and scratch repair experiments show that Tf-PL/AChE has a better therapeutic effect than Free AChE and GL/AChE treatments, and can signi cantly inhibit cell proliferation and cell migration. This may be due to the modi cation of Tf on the surface of liposomes, receptor-mediated endocytosis makes it easier for liposomes to enter cells, and the accumulation of therapeutic genes in cells is higher, thereby enhancing the inhibitory effect on tumor cells.
In vivo imaging of small animals showed that Tf-PL/Cy5.5 liposomes still had a strong uorescence intensity at the tumor site 72 hours after receiving uorescence injection; while Cy5.5 basically did not see uorescence at the tumor site. In the in vivo anti-tumor experimental study, we found that compared with Free AChE and GL/AChE, Tf-PL/AChE can effectively inhibit the growth of subcutaneous transplanted tumors in mice bearing SMMC-7721 liver cancer. At the end of the experiment, the tumor weight inhibition rate of Tf-PL/AChE was 77.47%, while that of GL/AChE group was only 48.21%. Our data suggest that the AChE gene is an effective therapy gene for the treatment of liver cancer and Tf-PL shows promising clinical applications value.

Conclusions
In this work, we put forward a strategy for the delivery of the AChE gene by the transferrin modi ed liposome targeting TfR on the surface of liver cancer cells for liver cancer therapy. This strategy provides an alternative approach to replace the conventional virus mediated gene carrier for cancer therapy, which can effectively bypass the biosafety problems that live viruses may cause. The present gene delivery system has good blood compatibility and degradability, low toxicity, good tumor targeting ability, and high transfection e ciency. Furthermore, ectopic expression of the AChE gene can signi cantly inhibit the proliferation of liver cancer cells in xenograft model. The proteolipid prepared directly from Tf enhances the therapeutic gene transfected into the hepatoma cells, suggesting a potential liver cancer delivery system. In conclusion, the strategy of combining the transferrin liposome and AChE gene provides a new idea for gene therapy of liver cancer.

Preparation of AChE plasmid
The therapeutic acetylcholinesterase (AChE) plasmid was obtained from the National Laboratory for Oncogenes and Related Genes, Cancer Institute of Shanghai JiaoTong University (sequence:NG007474.1). Plasmid ampli cation was achieved by transforming competent E. coli and enlarging the number of E. coli in large quantities, and the plasmid was extracted using the Qiagen EndoFree Plasmid Mega Kit (Qiagen, Hilden, Germany). After passing the test, the plasmid was then dissolved in sterile endotoxin-free water and stored at -20 °C for later use.

Cell culture and transfection experiments
Immortalized human liver normal cells HepZJ cells, liver cancer cell line SMMC-7721 cells were preserved by the laboratory. And cultured in Dulbecco's Modi ed Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL-1 penicillin, and 100 μg/mL streptomycin in a humidi ed incubator with 5% CO 2 at 37 °C. SMMC-7721 cells were seeded at 2 x 105 cells/well in a 6-well plate (Corning Inc., NY, NJ, USA) in 2 mL of complete medium. After 24 hours of incubation, the medium in each well was replaced with 2 mL of fresh serum-free medium. The pVAX-GFP (pGFP) reporter plasmid was maintained at 2 μg per well, while the mass ratios of GL/pGFP, Tf-PL/pGFP, and Lipo 2000/pGFP were 25:1. The serum-free medium was then replaced with a complete medium after 6 hours. Then, the cells were cultured for an additional 48 hours at 37°C. The expression of GFP was visualized by an Olympus IX 71 inverted uorescence microscope (Olympus Corp., Tokyo, Japan). Cell suspensions were harvested and analyzed by ow cytometry (BD Biosciences, San Jose, CA, USA) to determine transfection e ciency.

Synthesis of Tf-GHDC
Tf-GHDC is synthesized by conjugating Tf with GHDC. Accurately weigh 20 mg of Tf dissolved in 20 mL of double deionized water, add 20 mg of GHDC, and stir gently to form a conjugate. The resulting solution was incubated at 37 °C for 24 hours to allow the reaction to proceed. Unreacted GHDC was separated from the conjugate by dialysis against ddH 2 O for 36 hours. Nuclear magnetic resonance analyzer was performed to obtain spectral absorption peaks of Tf, CHDC, and Tf-GHDC to compare changes in Tf before and after conjugation.

Synthesize the Tf-PL/AChE nanoparticles
The main steps of preparing Tf-PL by thin lm dispersion method are as follows: (1) Accurately weigh the lipid material with a molar ratio of DOPE:CHOL: DSPE-PEG = 3:1:0.4 and dissolve it in an appropriate amount of chloroform solution; (2) Place the solution in a spherical bottle at room temperature for 5-10 minutes, and then remove the chloroform by rotary evaporation under reduced pressure to form a colorless transparent lm; (3) Vacuum the spherical ask overnight to completely remove organic solvents; (4) Take a certain amount of Tf-GHDC lyophilized powder dissolved in PBS buffer and add it to the lipid lm in (3), and hydrate it in a 35 °C water bath for 4 hours; (5) Ultrasound the above suspension in a water bath with an ultrasonic power of 100 W, with 5 S interruptions every 10 S, and repeat 100 times to obtain Tf-PL/AChE. (6) The liposome precipitate was obtained by centrifugation, resuspended in ultrapure water, and stored at 4°C.
The preparation process of transferrin liposomes without plasmids or drugs is the same as that of Tf-PL/AChE, except that only Tf-GHDC lyophilized powder PBS solution is added for hydration. The preparation of other liposomes is the same as that of Tf-PL/AchE-Dox, except that GHDC lyophilized powder is replaced by Tf-GHDC lyophilized powder. The preparation process of uorescently labeled transferrin liposomes is the same as that of Tf-PL/AchE-Dox, except that FITC-Tf-GHDC lyophilized powder is replaced by Tf-GHDC lyophilized powder.
Characterization of Tf-PL/AChE The average particle size, size distribution, and zeta potential of the proteoliposome were determined using a Malvern Zetasizer (Nano-ZS 90, Malvern Instruments Limited, UK) based on quasi-elastic light scattering at 25 °C. The morphology and shape of the liposomes were imaged by TEM. Before imaging, the liposomes were coated on a carbon-coated copper grid, stained with 4% uranyl acetate for 10 min, and allowed to dry. TEM was carried out using a 7650 TEM (Hitachi; Kyoto, Japan) at 120 kV. Agarose gel electrophoresis experiments were performed with different weight ratios of Tf-PL and DNA. The prepared transferrin liposome solution was placed in an ultra ltration centrifuge tube, centrifuged at room temperature for 15 min at 8 000 r/min, and the ow-through was removed and then using an anti-human transferrin ELISA kit to qualify the dose according to the manufacturer's instructions. The conjugation e ciency of Tf was calculated. The calculation formula is: CE% = (transferrin addition amounttransferrin out ow amount) / transferrin addition amount × 100%. In the same way, non-targeted liposomes (GL) were prepared only with GHDC and cholesterol (Chol). FITC and Cy5.5 labeled liposomes were prepared by adding the desired amount of FITC to the lipid organic solution before the solvent evaporation step and adding Cy5.5 to ddH 2 O before the hydration step.

Characterization of the transgenic performance of proteoliposome
The appropriate amount of proteoliposome was taken and demulsi ed with methanol. Nanodrop 2000 was used as the main absorption peak of nucleic acid with 260 nm ultraviolet absorption peak. The gene load (DL) of the liposome and the encapsulation e ciency (EE) of the liposome to the gene were determined according to the formula. The calculation formula is: DL% = (total amount of geneunencapsulated free gene) / total amount of system × 100%; EE% = (total amount of geneunencapsulated free gene) / gene × 100%. The stability analysis of the in vitro gene of the proteoliposome carrying the gene was determined using a dialysis method. Brie y, 2 mL of plasmidloaded liposomes were suspended in a dialysis bag with a molecular weight cut-off of 12 kDa and dialyzed against 18 mL PBS (pH 7.4) containing 0.1% Tween-80 for 7 days (v/v) On a horizontal shaker (100 rpm) at 37 °C. 2 mL aliquot was taken at predetermined intervals and replaced with an equal volume of fresh medium. The DNA content of the samples collected at each time point was measured using Nanodrop 2000.

Expression of transferrin receptor and acetylcholinesterase in hepatocellular carcinoma cell lines
The normal liver cells and three human hepatoma cell lines were selected, and the expression level of TfR on the cell membrane was analyzed by Western Blot. The expression of acetylcholinesterase in normal liver cells and three human hepatoma cell lines were analyzed. The cells were collected in 1.5 ml tubes, washed twice with PBS, then 0.1 ml RIPA lysis buffer containing 1 mM PMSF was added, and then placed on ice for 30 minutes. The supernatant was obtained by centrifugation at 13,000 rpm for 15 minutes at 4°C . Subsequently, the protein concentration was determined by BCA protein quanti cation. A total of 20 μg of protein sample was separated on a 12% SDS-PAGE gel and then transferred to a PVDF membrane which was blocked in 5% skim milk for 1 hour. Membranes were incubated with mouse anti-human AChE monoclonal antibody or mouse anti-human TfR monoclonal antibody (1:500) overnight at 4 °C and washed three times with PBST followed by goat anti-mouse IgG (H+L)-HRP was incubated for 2 hours at room temperature. Finally, ECL luminescence is used for detection.
Cellular uptake and localization of liposomes in SMMC-7721 cells SMMC-7721 cells were incubated with proteoliposomes at a series of FITC concentrations (0.33, 1, and 2 nM) for 2 hours at 37 °C and then washed three times with PBS. Cellular uptake of FITC-labeled proteoliposomes was qualitatively and quantitatively analyzed by uorescence microscopy (TE2000; Nikon; Kyoto, Japan) and ow cytometry (FACS; BD Biosciences; San Jose, CA, USA), respectively. The transfection medium was replaced with normal medium and then replaced with Tf-PL-GFP and GL-GFP for uptake studies. The cells were washed three times with PBS and xed in 4% paraformaldehyde, then DAPI stained the nuclei and nally subjected to uorescence microscopic observation.
In vitro cytotoxicity assays SMMC-7721 cells were seeded in triplicate in a 96-well plate (5 x 10 3 cells/well). After 24 hours, the medium was replaced with 100 μL of complete growth medium containing different concentrations of transferrin liposomes and incubated for an additional 24, 48, 72 hours. Cells that were not exposed to the transfected protein liposomes were used as controls. Cell viability was measured by the MTT assay according to the manufacturer's instructions.
Cell migration assay SMMC-7721 cell migration was measured using a transwell assay kit (Corning Life Sciences; Tewksbury, MA, USA) with 8 μm pores as previously described. SMMC-7721 cells were suspended in a serum-free medium containing the gene preparation and DMEM supplemented with 10% FBS as a chemoattractant.
Cells migrated after 16 hours were stained with 0.1% crystal violet and counted from 5 randomly selected regions under an inverted microscope.
Wound healing assay About 5×10 5 SMMC-7721 cells were added into a 6-well cell culture plate. The next day, scrape the cells straight with 1 mL tip, and wash off the suspended cells with PBS, and then serum-free medium was added. Incubate 6-well cell culture plate at 37 ℃ within 5% CO 2 cell incubator . Use an inverted microscope to observe cell repair and take pictures after 24 h.

Cell cycle and apoptosis analysis
Human liver cancer SMMC-7721 cells were inoculated in a 6-well plate with 1.5 × 10 5 cells /2 mL per well and cultured overnight at 37 °C in 5% CO 2 incubator. The cells were then cultured in 2.5 μg/mL AChE, GL/AChE, and Tf-PL/AChE medium for 24, 48, and 72 h, and the control group was set with medium only. 1) Cell cycle detection: Cells were collected, washed twice with pre-cooled PBS at 4 °C, added with precooled 70% ethanol, and xed at 4 °C for 24 h. The xed cells were washed twice with pre-cooled PBS at 4°C , then 0.2 mL PI staining solution (200 μg/mL) was added. The xed cells were bathed at 37°C for 20 min and then detected by ow cytometry.
Flow cytometry analysis was performed using a BD ow cytometer (Calibur; USA) to assess cell cycle distribution and apoptosis.
In vivo imaging of mice inoculated with SMMC-7721 SMMC-7721 cells (2×10 6 ) were injected subcutaneously into the right dorsal skin of 6-week-old female BALB/c nude mice. The tumor is allowed to grow for 10 days to about 100 mm3 after inoculation. Mice were randomly divided into three groups, a blank control group, a non-targeted treatment group, and a targeted treatment group, n = 3 in each group. 200 μL of physiological saline containing Cy5.5, Cy5.5labeled non-targeted liposome (GL), Cy5.5-labeled transferrin liposome (Tf-PL) were injected at a dose of 100 μg/kg Cy5, respectively. Images were taken at 24 and 72 hours after injection using the MAESTRO in vivo imaging system (Cambridge Research & Instrumentation; Hopkinton, MA, USA). The mice were then harvested and the heart, liver, spleen, lung, kidney, and tumor were dissected, washed with saline, and imaged using the MAESTRO in vitro imaging system.

Evaluation of antitumor e ciency and safety in vivo
Mice bearing SMMC-7721 liver cancer were established as described above and randomly divided into 4 groups (n=6 per group), control, AChE, GL/AChE and Tf-PL/AChE. Mice were injected intravenously with saline (control) or therapeutic agent 50 μg/kg at 10,12,14,16,18,20,22,24, and 26 days after implantation. The anti-tumor e ciency was determined according to the tumor volume using the following formula: larger diameter × (smaller diameter/2) 2 . Systemic toxicities were assessed by monitoring body weight changes and nephrotoxicity.

HE Staining
For HE staining, tissues were xed in 4% paraformaldehyde for more than 24 hours. Para n-embedded tissue sections (4 μm) were dewaxed and rehydrated. Hydration sections were stained with Mayer's hematoxylin and eosin.

Statistical Analysis
For multiple comparisons, a one-way ANOVA test was performed. The t-test (two-tailed) was used for comparison between the two groups. Data are expressed as mean ± standard deviation (S.D.). Survivors were estimated using a log-rank test. *p<0.05, **p<0.01, ***p<0.001. preparation and review. DC and JJ participated in the animal experiments. All authors approved the nal manuscript.