2.1 Reagents and kits
N, N-Dimethyltetradecylamine (≥ 99%) was purchased from Feixiang Corporation. Fluorescein isothiocyanate I (FITC); succinimidyl-3-(2-pyridyldithio) propionate (SPDP, MW 312.37); 3-(4,5-dimethyl Thiazole-2-yl)-2,5- diphenyltetrazolium bromide (MTT); cholesterol and other molecular biology reagents were purchased from Sigma, USA. The AChE gene was deposited by the National Laboratory of Oncogenes and Related Genes of the Shanghai Cancer Institute (sequence number: NG007474.1). The micro BCA protein assay kit, the apoptosis and cell cycle assay kit, and the luciferase activity assay kit were purchased from Promega (Madison, USA). Lipofectamine 2000 (Lipo 2000) was purchased from Invitrogen, USA. 1,2-dioleoyl-sn-glyceryl-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glyceryl-3-phosphoethanolamine-N- [(polyethylene glycol)- 2000] (DSPE-PEG) was purchased from Avanti, USA. Fetal bovine serum, penicillin/streptomycin, Dulbecco's modified Eagle's medium, DMEM were purchased from Gibco, USA. RIPA lysis buffer, DMSO, and PMSF were purchased from Shanghai Shenggong Biotechnology Co., Ltd. The Transwell chamber was purchased from Corning, USA. Apoptosis Detection Kit (BD, USA), Tunel staining kit was purchased from Biyuntian Bio. Mouse anti-human AChE monoclonal antibody, mouse anti-human TfR monoclonal antibody was purchased from Santa Cruz, USA. Goat anti-mouse IgG (H + L)-HRP, goat anti-rabbit IgG (H + L)-HRP was purchased from Abcam, USA. ECL (Millipore, USA), TRIzol, SYBR MIX were purchased from TaKaRa. Epichlorohydrin (≥ 99%), absolute ethanol, isopropanol (≥ 99%), chloroform (≥ 99%), ethylenediaminetetraacetic acid (EDTA, 99.9%), epichlorohydrin and other biochemical reagents All are analytical grades and are purchased from Sinopharm. Glycidylhexadecyldimethylammonium chloride (GHDC) and other polymers are synthesized and stored by our own laboratory.
2.2 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 amplification 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.
2.3 Cell culture and transfection experiments
Human embryonic kidney cell 293T cells, liver cancer cell line SMMC-7721 were preserved by the laboratory. And cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL− 1 penicillin and 100 µg/mL streptomycin in a humidified incubator with 5% CO2 at 37 °C. SMMC-7721 cells were seeded at 2 × 105 cells/well in 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 PL/pGFP, Tf-PL/pGFP and Lipo 2000/pGFP were 25:1. The serum-free medium was then replaced with complete medium after 6 hours. Then, the cells were cultured for an additional 48 hours at 37 °C. Expression of GFP was visualized by an Olympus IX 71 inverted fluorescence microscope (Olympus Corp., Tokyo, Japan). Cell suspensions were harvested and analyzed by flow cytometry (BD Biosciences, San Jose, CA, USA) to determine transfection efficiency.
2.4 Liver cancer cell lines and animal experiments
SMMC-7721 cells in Dulbecco's Moded Eagle's medium containing 10% fetal bovine serum (FBS) (GIBCO BRL, Grand Island, NY, USA) or RPMI 1640 (GIBCO BRL, Grand Island, NY, USA) The concentration of penicillin and streptomycin in the medium was 50 µg/mL, respectively; and the cells were cultured at 37 °C in a humidified environment containing 5% CO2. Female BALB/c-nu nude mice (5–6 weeks old; body weight, 18–20 g) were obtained from Shanghai Experimental Animals Inc. (SLAC; Shanghai, China) and maintained under conditions free of specific pathogens. All animal experiments were conducted in accordance with the guidelines set by the Animal Care and Use Committee of Shanghai Jiao Tong University School of Medicine (Shanghai, China).
2.5 Synthesis and Analysis of Tf-GHDC
Tf-GHDC was prepared by modifying Tf using GHDC. The conjugate was formed by gently stirring 20 mg of Tf into 20 mg of GHDC dissolved in 20 mL of double distilled water (ddH2O). 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 ddH2O 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.
2.6 Preparation and characterization of transferrin liposomes
Transferrin liposomes were prepared by reverse evaporation method, dissolving macromolecular quaternary ammonium salt (mass concentration 1–100%) and lipid component (mass concentration 0.1–60%) in chloroform. The GHDC-modified macromolecular quaternary ammonium salt Tf-GHDC (mass concentration 0.1–39%) was dissolved in the aqueous phase, and then PEG-modified transferrin liposome (Tf-PL) was prepared by reverse evaporation. 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. Prior to 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. A 10 µL sample was taken for atomic force microscopy (AFM) analysis. Approximately 30 µL of the sample was dropped on freshly cleaved mica for 10 min and subjected to atomic force microscopy (AFM) analysis (Bioscope SPM, DI, USA). Agarose gel electrophoresis experiments were performed with different weight ratios of TfPL and DNA. X-ray photoelectron spectroscopy (XPS) analysis of the samples was performed using a PHI-1600 instrument (PerkinElmer) and a MgKα X-ray source (1253.6 eV). The prepared transferrin liposome solution was placed in an ultrafiltration centrifuge tube, centrifuged at room temperature for 15 min at 8 000 r/min, and the flow-through was removed and then using an anti-human transferrin ELISA kit to qualify the dose according to the manufacturer's instructions. The conjugation efficiency of Tf was calculated. The calculation formula is: CE% = (transferrin addition amount - transferrin outflow amount) / transferrin addition amount × 100%. In the same way, non-targeted liposomes (PL) 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 prior to the solvent evaporation step and adding Cy5.5 to ddH2O prior to the hydration step.
2.7 Characterization of the transgenic performance of proteoliposome
The appropriate amount of proteoliposome was taken and demulsified 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 efficiency (EE) of the liposome to the gene were determined according to the formula. The calculation formula is: DL% = (total amount of gene - unencapsulated free gene) / total amount of system × 100%; EE% = (total amount of gene - unencapsulated free gene) / gene × 100%. The release rate of the in vitro gene of the proteoliposome carrying the gene was determined using a dialysis method. Briefly, 2 mL of plasmid-loaded 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. A 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.
2.8 Expression of transferrin receptor and acetylcholinesterase in hepatocellular carcinoma cell lines
Expression of transferrin receptor in hepatoma cells was analyzed by Western Blot and flow cytometry. The results were compared to TfR expression levels in normal liver tissue and liver cancer samples. 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 was 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 quantification. 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.
2.9 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 fluorescence microscopy (TE2000; Nikon; Kyoto, Japan) and flow cytometry (FACS; BD Biosciences; San Jose, CA, USA), respectively. Transfection medium was replaced with normal medium and then replaced with Tf-PL-GFP and PL-GFP for uptake studies. The cells were washed three times with PBS and fixed in 4% paraformaldehyde, then DAPI stained the nuclei, and finally subjected to fluorescence microscopic observation.
2.10 Tumor spheroid uptake analysis
To form tumor cell spheroids, SMMC-7721 cells were plated in a 24-well plate (1 × 103 cells/well) in 100 µL Matrigel: DMEM medium (1:1 v/v). Incubate for 12 hours at 37 °C. The cells were then cultured for 7 days in complete growth medium (1 mL/well) and then used in subsequent experiments. The obtained spheroids were incubated with transferrin liposomes containing 4 nM-rhodamine B for 3 hours at 37 °C. They were then rinsed three times with PBS and fixed in 4% paraformaldehyde for 15 minutes. Tumor sphere uptake was analyzed by confocal laser scanning microscopy (C2; Nikon; Kyoto, Japan).
2.11 In vitro cytotoxicity assays
SMMC-7721 cells were seeded in triplicate in 96-well plate (5 × 103 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.
2.12 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 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.
2.13 Cell cycle and apoptosis analysis
SMMC-7721 cells were subjected to gene therapy intervention with transfectant liposomes, then harvested after 24, 48 and 72 hours, while untreated cells were used as controls. For cell cycle analysis, cells were fixed in 1 mL of 70% ethanol at 4 °C for 24 hours, then centrifuged, rinsed with cold PBS and stained with PI (200 µg/mL) for 10 minutes at 4 °C in the dark. Apoptosis was assessed using the Annexin V-FITC/PI Apoptosis Detection Kit. After treatment and harvest, cells were suspended in 200 µL of binding buffer and incubated with 5 µL of Annexin V-FITC and 5 µL of PI for 15 minutes in the dark. Flow cytometry analysis was performed using a BD flow cytometer (Calibur; USA) to assess cell cycle distribution and apoptosis.
2.14 In vivo imaging of mice inoculated with SMMC-7721
SMMC-7721 cells (2 × 106) were injected subcutaneously into the right dorsal skin of 4-5-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, Cy5.5-labeled non-targeted liposome (PL), Cy5.5-labeled transferrin liposome (TfPL) were injected at a dose of 100 µg/kg Cy5, respectively. Images were taken at 24 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.
2.15 Evaluation of antitumor efficiency 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). Mice were injected intravenously with saline (control) or transfected proteoliposome or proteoliposome 50 µg/kg at 10, 12, 14, 16, 18, 20, 22, 24 and 26 days after implantation. The anti-tumor efficiency 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.
2.16 HE Staining
For HE staining, tissues were fixed in 4% paraformaldehyde for more than 24 hours. Paraffin-embedded tissue sections (4 µm) were dewaxed and rehydrated. Hydration sections were stained with Mayer's hematoxylin and eosin.
2.17 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.