Cell culture
HGCCs were obtained from surgical specimens of patients who suffered from gastric cancer. In briefly, the tissues were soaked in ethyl alcohol (95%), repeatedly washed with phosphate-buffered saline (PBS) contains of 1% penicillin-streptomycin, and then cultured with the culture medium of DMEM (Thermofisher Scientific, MA, USA), 10% fetal bovine serum (FBS, Thermofisher Scientific, MA, USA) at 37 ℃, 5% CO2 in a humidified atmosphere. All procedures were conducted in compliance with protocols approved by the Nanchang University Ethics Committee, and written informed consents were received from all participants.
Mtt Assay
The optimum concentration of DOC was evaluated by using MTT assay. Logarithmic growth cells were randomly allocated to 9 groups, including 1 control group and 8 groups with different concentration gradient that consist of 5×10− 7, 1×10− 7, 5×10− 8, 1×10− 8, 5×10− 9, 1×10− 9, 5×10− 10, 1×10− 10 mol/ L, respectively. MTT was performed to measure optical density (OD) at 24 h, 48 h, and 72 h time point after dealing with each group. The optimum concentration of DOC was ascertained according to the rate of cell proliferation inhibition [(OD in control group - OD in experimental group) / OD in control group] ×100%].
Preparation Of Targeting Dllms
An appropriate amount of lipid mixture (Dipalmitoyl phosphatidylcholine, Distearyl phosphatidylethanolamine and Diphenylphosphoryl azide with 1:1:1 ratio) was dissolved in glycerol and phosphate-buffered saline (PBS; pH 7.4), reaction in a condition of 45°C, 30min. The solution was transferred to a round bottom flask, and the solvent was removed by rotary vacuum evaporation and the milky white liquid was a final lipid with neutral charge. After that, polyethylene glycol (PEG) was added into the liquid. Finally, the cationic lipid microbubbles (LMs+) were obtained. DOC was suspended in a water-based solvent in an appropriate proportion to the film-forming material, and was separated into 1mL vials in vials. After lyophilization, 1mL hydration solution were added into bottles and rehydrate. The dispersion was mechanically vibrated at 3,000 rpm for 45 seconds and then the DLLMs were obtained. They were washed with PBS for three times and free drug (not incorporated into the LMs) was separated by centrifugal flotation. The unloaded LMs (nondrug loaded) were similarly prepared but without the addition of DOC.
Treatment Of Dllm Combined With Utmd
The HGCCs were divided into 6 groups: control (C), DOC-loaded lipid microbubbles alone (DLLM), DOC alone (DOC), DOC combined with ultrasound (US), LM combined with UTMD, and DLLM combined with UTMD. Among them, all the groups were administrated with the functional dose of DOC except C and LM combined with UTMD groups. The DOC combined with US, LM combined with UTMD and DLLM combined with UTMD groups were irradiated by ultrasound. Based on the previous research, the low frequency of the treatment meter was fixed at 1 MHz; and the intensity was set at 0.5w /cm2 at 30 seconds continuous irradiation, and then apoptosis was observed after culture for 48 h.
Construction Of Recombinant Plasmid Rc / Cmv-p16
The plasmids UT651 contain β-galactosidase-labeled E. coli LAZ gene, upstream of that is the human cytomegalovirus (CMV) promoter with the terminal transcription and polyadenylation signal from simian virus 40 in the downstream. The plasmids were amplified in E. coli DH5 medium and purified with plasmid DNA purification kits. Therefore, the recombinant plasmid RC / CMV (PRC / CMV) contained NEO, a neomycin-resistance gene which is function as in the control of CMV. The tumor suppressor gene p16 was cloned into PRC / CMV, and sequencing was performed to identify the insert orientation.
Transfection And Allocation
HGCCs were seeded into each well of a 6-well plate (5 × 105 per well). The culture medium was aspirated followed washed with PBS after 80%~90% of cell confluence. The cells were transfected with the appropriate construct by Lipofectamine 2000 following the manufacturer’s protocol. In brief, 4mg of plasmid DNA (in 10µL ddH2O) was mixed with 250µL of serum-free medium (SFM) and incubated for 20 min at room temperature with a mixture of 10µL Lipofectamine 2000 and 250 µL SFM. After then, the resultant complex mixture was added to a monolayer of pre-confluent cells seeded in a 6-well plate. The medium was replaced with fresh and complete one 6 h after transfection. The cells were exposed to irradiation 36 h after transfection.
All cells were separated into 4 groups: blank control group, LM+ combined with UTMD, empty plasmid group (PRC / CMV-Neo), and experimental group (PRC / CMV-p16). Of note, both of PRC / CMV-Neo and PRC / CMV-p16 were loaded in LM+ and treated with UTMD. The HGCCs were collected by centrifugation, resuspended in serum-free DMEM medium, and then seeded into 12-well plates at a density of 1x105/ cm2. Before ultrasonic radiation, 3ml PBS solution was mixed with microbubbles, and well distributed by oscillation. The density of microbubbles was approximately (5 ~ 10) ×108/ mL, and the diameter of microbubbles was about 2 ~ 5um. The plasmid-microbubble mixture was placed in the gastric cancer cell suspension after fully mixing 30u L of the plasmid solution and 100u L of microbubble. UTMD was as mentioned above. Finished irradiation, apoptosis was observed after 48h culture.
Cell Proliferation Assay
Cell proliferation was determined by MTT assay as described above. The OD values of each group were measured at 24 h, 48 h, and 72 h time point.
Transmission Electron Microscopy
Logarithmic growth cells were collected after 24h culture (the number of cells should be no less than 1×106 cells in each group), and then stained with lead acetate and observed under a transmission electron microscope.
Quantitation Of Apoptosis And Detection Of Cell Cycle Distribution
Cellular apoptosis was evaluated through flow cytometry by the Annexin V-FITC/PI staining kit ( Abcam, Cambridge, GB, USA). In brief, collected cells (cell volume is no less than 1×106 cells in each group) were fixed with 1% paraformaldehyde in PBS for 5 ~ 15 min at room temperature, washed twice with PBS, and stored in 70% ethanol at -20°C. Flow cytometry analysis was carried out by an EPICS flow cytometer (Coulter, Hialeah, FL, USA). Histograms of logarithmic fluorescence intensity were qualified as 10,000 cells per sample. Cell cycle distribution was detected by a flow cytometer and analyzed by Modifit LT2.0.
Animals
12-week-old male nude mice (25 ~ 27 g) were supplied by the laboratory of animal center of Nanchang University and maintained according to guidelines from the animal care committee. HGCCs in the logarithmic growth phase were harvested, added in the serum-free medium, made the proper concentration (107 cells in 1mL) and inoculated subcutaneously into the dorsal flank area of the mice (2.5×107 cells per mouse). Tumors were allowed to grow until they reached 10 mm at the 14th day after inoculation. After 14 days, 12 mice were randomized allocated to 3 groups: control group treated with normal saline (NS), LDLM+ combined with UTMD and PRC / CMV-P16 combined with UTMD. All animal experiments were performed with permission from the Animal Ethical Commission of the Nanchang University.
Mice were sacrificed 1 week after ultrasound irradiation, and the tumors were excised, measured and stored in liquid nitrogen. The frozen tumor tissues were made into 5um sections and the HE staining technique was performed. The quantitation of apoptosis was evaluated using an apoptosis Kit though Terminal deoxynucleotide transferase-mediated dUTP notch terminal marker and presented with percentage of positive nucleus staining by 200 times view of microscope.
Statistical analysis
All data are presented as mean ± standard deviation (SD) and statistical analysis was performed using SPSS 13.0 (SPSS 13.0 software, USA). LSD-t and Dunnett’s were used as appropriate for group variables, and one-way analysis of variance was used for different groups. The comparison of inhibition rate of HGCCs at different time points was detected by multivariate analysis of variance. A P value less than 0.05 indicates a statistically significant difference.