Materials
Reagents and kits
N, N-Dimethyltetradecylamine (≥99%) was purchased from Feixiang Corporation. Fluorescein isothiocyanate I (FITC); 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 Sangon Biotech Co., Ltd. The Transwell chamber was purchased from Corning, USA. 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). 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. Lyso-Tracker Red and DAPI were purchased from Shanghai Beyotime Biotechnology. Glycidyl hexadecyl dimethylammonium chloride (GHDC) and other polymers are synthesized and stored by our laboratory.
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.
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 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 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 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.
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). 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 (6-8 weeks old; body weight, 18-20g) 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).
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 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.
Synthesize the Tf-PL/AChE nanoparticles
The main steps of preparing Tf-PL by thin film 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 film;
(3) Vacuum the spherical flask 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 film 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 fluorescently 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 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 (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 ddH2O before the hydration step.
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 stability analysis 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. 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 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.
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. 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 fixed in 4% paraformaldehyde, then DAPI stained the nuclei and finally subjected to fluorescence microscopic observation.
In vitro cytotoxicity assays
SMMC-7721 cells were seeded in triplicate in a 96-well plate (5 x 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.
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 5×105 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% CO2 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 × 105 cells /2 mL per well and cultured overnight at 37 °C in 5% CO2 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 pre-cooled 70% ethanol, and fixed at 4 °C for 24 h. The fixed cells were washed twice with pre-cooled PBS at 4 °C, then 0.2 mL PI staining solution (200 μg/mL) was added. The fixed cells were bathed at 37°C for 20 min and then detected by flow cytometry.
2) Apoptosis detection: The cells were collected and washed with 4°C pre-cooled PBS twice. The cells were resuspended with 200 μL binding buffer. Annexin V-FITC (5 μL) and PI (10 μL) were added.
Flow cytometry analysis was performed using a BD flow cytometer (Calibur; USA) to assess cell cycle distribution and apoptosis.
In vivo imaging of mice inoculated with SMMC-7721
SMMC-7721 cells (2×106) 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, Cy5.5-labeled 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 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), 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 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.
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.
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.