Human colon cancer cell line: SW620, CW-2, LoVo and HCT116 purchased from ATCC (90% DMEM + 10% FBS) 0.1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000(ammonium salt) (DSPE-PEG2000-NH2) were purchased from Ananti Polar Lipids, Inc. (Beijing, China). Cetuximab, a monoclonal antibody anti-EGFR, was purchased from Shanghai TheraMabsBiotech (product number TM-Beva-00002). DMSO (D8371, Solarbio), dialysis bag (3.5KD, F132590, BBI), DMEM medium (Cat. No. 11965-126), fetal bovine serum (FBS) (product number 16000-044), trypsin and double antibodies were purchased from Gibco company. MTT powder was purchased from Azerbaijan Latin (Cat. No. T100896-1g), irinotecan (Irinotecan, CPT-11), purchased from Solarbio (SI8480-20 mg), EDC purchased from Sigma (D8740-500 mg), NHS purchased from Sigma (D8740-500 mg) Balb/c nude mice were purchased from Weitong Lihua.
Main instruments: ordinary microscope (OLYMPUS company in Japan), small desktop refrigerated centrifuge (Technology University Zhongjia), cell incubator (American Thermoelectric), heating magnetic stirrer (C-MAG HS 10, IKA) CM-H2DCFDA were obtained from ThermoFisher.
Cell and cell culture
CCD-18Co primary colon cell lines and HCT116, SW620, CW-2 and LoVo colon cancer cells and were all purchased from American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured in DMEM supplemented with 10% FBS (No-16000-044/Gibco, USA) in the humid chamber, maintaining 37 °C and 5% CO2. The concentrations of 1, 5, 10, 50, and 100 of μg/mL CPT-11 (SI8480-20 mg; Solarbio) were, respectively, added into the cell’s medium for 48 h.
In vivo experiments were performed using female BALB/c nude mice (4–6 weeks old) obtained from Shanghai SLAC laboratory animal Co., Ltd (Shanghai, China). The whole animal experiment was conducted following the Animal Care Guidelines of Rongchang District People’s Hospital of Chongqing. All the nudes were housed, fed with a regular diet, given acidified water without antibiotics. We injected subcutaneously with 100 μL SW620 cell suspensions (1 × 106 cells/mL) into the right flank after 1 week. SW620 were cultured, digested, centrifuged, collected, and diluted to 5 * 107/mL; then, the suspension was subcutaneously injected on the right limbs of 6-week-old mice morphologically similar (1 week in advance). When the tumor formation was observed in all mice after 2 weeks of inoculation, the mice were randomly divided into four groups, with three mice in each group, including controlling groups treated with PBS (C), groups treated with CPT-11-loaded liposome (Lipo-CPT-11), free CPT-11 (CPT-11), and CPT-11-loaded EGFR-targeted DSPE-PEG2000 liposome (EGFR-Lipo-CPT-11). When the tumor volume increased to about 100 mm3, the mice were injected intravenously once a week for 3 weeks. The tumor volumes were measured every 3–4 days; then, after 4 weeks of treatment, the mice were quickly dislocated and killed to collect and weigh the tumor. The tumor volume (V) was calculated as follows: V = (L × W2)/2, where L and W were the longest and the shortest diameter of the tumor.
Preparation of CPT-11-loaded DSPE-PEG2000 targeting EGFR liposome
2 mg of the DSPE-PEG2000-NH2 purchased from Ananti Polar Lipids, Inc. (Beijing, China) was dissolved into 1 mL of DMSO (D8371, Solarbio), to obtain 2 mg/mL solution A, and 2 mg of irinotecan (SI8480-20 mg, Solarbio) was dissolved into 1 mL of DMSO to form 2 mg/mL solution B. solutions A and B were well mixed according to the 1:1 mass ratio and slowly stirred at room temperature for 2 h in the dark. Then, 2 mL of the mixed solution was transferred to a dialysis bag (3.5KD, F132590, BBI) to perform overnight dialysis in 18.2 MΩ deionized water and 2.62 mL of CPT-11-loaded DSPE-PEG2000 liposome (Lipo-CPT-11). After the characterization of particle size and the stability of the loaded liposome nanoparticles, the coupling of EGFRs to CPT-11-loaded DSPE-PEG2000 liposomes were prepared using EDC and NHS as coupling reagents. Briefly, 200 μL of CPT-11-loaded DSPE-PEG2000 liposome suspension in PBS buffer were added to a prepared solution of 100 μL of 0.25 M EDC and 100 μL of 0.25 M NHS, and the mixture was incubated for 10 min at room temperature with a pH adjusted to 7.5 using 1 M NaOH. Then, 20 μL of antibody anti-EGFR (product number TM-Beva-00002) (100 mg/mL) was added to the mixture, and the solution was gently stirred for 8 h at 4 °C. Finally, EGFR-coated CPT-11-loaded DSPE-PEG2000 liposomes (EGFR-Lipo-CPT-11) were separated from unbound antibody anti-EGFR using a saline pre-equilibrated Sepharose CL-4B column, and the upper solution containing EGFR-coated CPT-11-loaded DSPE-PEG2000 liposomes was collected, pooled, sterilized and stored under nitrogen at 4 °C.
The morphology and particle size of CPT-11-loaded DSPE-PEG2000 liposome
The shapes and surface morphologies of CPT-11-loaded EGFR-targeted DSPE-PEG2000 liposomes were observed via a transmission electron microscope (H-6009IV, Hitachi, Japan). The particle sizes of these liposomes were also determined by dynamic light scattering (Malvern Nano-ZS 90) at room temperature, and all measurements were repeated three times. To quantify the EGFR overexpressed tumor, western blot analysis was applied on four different CRC cell lines, including CW-2, LoVo, SW620 and HCT116 cells. Briefly, All the specimens were heated in sample buffer and sorted using SDS-PAGE with 10% gradient. The recovered proteins were then moved to a PVDF membrane (Millipore, Bedford, MA, USA) blocked in a 5% skim milk solution (Becton–Dickinson & Co., Sparks, MD, USA). After 2 h in blocking buffer (TBS containing 5% skim milk and 0.1% Tween20), the PVDF membrane was incubated with the primary antibody. Then, anti-IgG at 1:1500 was used to detect the specific secondary antibody, and the chemiluminescence system (ECL, Pierce) was used to identify relative proteins.
The physical stability, drug loading, and cumulative release profile in vitro of CPT-11-loaded DSPE-PEG2000 targeting EGFR liposome
The physical stability of liposomes was primarily determined by the critical micelle concentration in an aqueous solution at 25 °C. Micellar diameter changes as a function of time and scattering intensities were evaluated by DLS as mentioned above. 10 mg of CPT-11-loaded DSPE-PEG2000 targeting EGFR liposome was dissolved into 0.1 mL acetonitrile, and the amount of irinotecan was measured by HPLC. The drug loading was calculated as follows: drug/(polymer + drug) × 100%. The lyophilized powder of CPT-11-loaded DSPE-PEG2000 targeting EGFR liposome was dissolved into the deionized water, and 5 mL of them were put into a dialysis bag (molecular weight cut-off 3500 g/mol). Then, the bag was immersed in 30 mL of phosphate-buffered saline (PBS, pH 7.4) containing Tween-80 (0.5% w/w), and the medium was stirred at 70 rpm at 37 °C. Samples were collected at 6th, 12th, 24th, 36th h, 48th h, and 72nd h, and the same volume of fresh PBS was added to maintain the buffer volume unchanged. The concentration of the released irinotecan in the dialysis media was determined by HPLC (LC-10ATvp, Shimadzu) with a C18 column (Symmetry shield TM RP18, 3.9 mm × 150 mm, from Waters) at 25 °C. The standard curve was evaluated at 10, 25, 50, 100, 150, and 200 ug/mL, while the drug concentrations were calculated at each time point. Next, the accumulative release amount of irinotecan was calculated using a calibration curve and expressed as the Percentage of released concentration through the following formulation: Cumulative percentage release (%) = Volume of sample withdrawn (mL)/bath volume (v) × P (t − 1) + Pt. Where Pt = Percentage released at time t and P (t − 1) = Percentage release before ‘t’.
In vitro cytotoxicity of free CPT-11 and the antitumor activity of CPT-11-loaded DSPE-PEG2000 targeting EGFR liposome
SW620 cells were exposed to the different concentrations of irinotecan (1, 5, 10, 50, and 100 μg/mL), and cell viability was measured to explore the IC50 of irinotecan through MTT assay. Then, cells were separated into four groups, including the control group (C), cells treated with free CPT-11 (CPT-11), CPT-11 loaded liposome (Lipo-CPT-11), and CPT-11-loaded DSPE-PEG2000 targeting EGFR liposome (EGFR-Lipo-CPT-11), respectively. Then, MTT assays were performed to evaluate the antitumor activities produced by the four groups.
According to the grouping, control, EGFR liposome, CPT-11-loaded DSPE-PEG2000 particles and CPT-11-loaded DSPE-PEG2000 targeting EGFR liposome groups were cultured in a 37 °C, 5% CO2 saturated humidity incubator for 48 h. Add 20 µL MTT solution (Sigma-Aldrich, MO, USA) to each well and continue to incubate for 4 h, discard the supernatant, add 150 µL DMSO to each well and shake for 15 min to dissolve the whole crystals. An enzyme-linked immunoassay detected the absorbance value (A570) of each well at 570 nm, and the growth inhibition rate of each group of cells was calculated. The calculation formula was applied as follows: Cell viability (%) = (experimental group absorbance value/control group absorbance value) × 100%.
Reactive oxygen species (ROS) analysis in vitro
Reactive oxygen species from cellular was determined using the conversion of non-fluorescent 5, 6-Chloromethyl-2V, 7V dichloro dihydro fluorescein diacetate (CM-H2DCFDA) to its fluorescent derivative (DCF) by Reactive Oxygen Species Assay Kit (Abcam, USA) according to the manufacturer’s recommendation. Four groups of SW620 cells, respectively, treated with free CPT-11, CPT-11-loaded DSPE-PEG2000 particles, CPT-11-loaded DSPE-PEG 2000 targeting EGFR liposome, and the control group. Then, 50 μmol/L DCFH2-DA were added for 30 min, washed with PBS, and centrifuged. Furthermore, PBS containing 1% Triton X-100 was added, and the intensity of DCF fluorescence (OD) was assessed via a microplate reader at 485 nm of excitation and 530 nm of emission.
Cell apoptosis assay
The SW620 cells from different treatment groups were collected by centrifugation at 1500 rpm and were stained with Annexin V-FITC for 30 min and propidium iodide (PI) for 5 min after being washed with pre-cooling PBS. Finally, the samples were subjected to a fluorescence-activated cell-sorting (FACS) flow cytometer (BD Biosciences, San Jose, CA, USA). Flow cytometry was performed to measure cell apoptosis.
Western blot analysis
Total protein was extracted from lysed cells, and the equivalent amount of protein was separated with 10% SDS-PAGE, then transfected to a PVDF membrane (Millipore). After being blocked with 5% nonfat milk (Cell Signaling Technology), the membranes were incubated with primary antibodies against cleaved-caspase-9 (Abcam, ab2324) and cleaved-caspase-3 (Abcam, ab208003) and GAPDH at 4 °C overnight followed by secondary antibody incubation. The protein bands were visualized using CLARITYTM Western ECL substrate (Bio-Rad), and the protein level was quantified and normalized with GAPDH.
SPSS version 18.0 was used for statistical analysis through the one-way ANOVA method. All values were presented as the mean ± SD differences with p < 0.05 were considered statistically significant.