Chemicals and Reagents: All solvents and reagents were used as received without further purification. Cisplatin was obtained from Sino-Platinum Metals (Guiyang, Guizhou, China). mPEG2000-silane, DOTAP, TritonX-100, hexanol, and cyclohexane were purchased from Sigma-Aldrich (St. Louis, MO, USA). The Bmi1 siRNA sequences for mice were as follows: sense strand 5'-CCA GAC CAC UCC UGA ACA UTT-3' and anti-sense strand 5'-AUG UUC AGG AGU GGU CUG GTT-3'. For human, the sequences were: sense strand 5'-CCA GAC CAC UAC UGA AUA UAA-3' and anti-sense strand 5'-UUA UAU UCA GUA GUG GUC UGG UU-3'.
Cell Culture and Cisplatin-Resistant Cells
The human tumor cell line HepG2 was obtained from the China Center for Type Culture Collection at Wuhan University (Wuhan, China). HepG2 and HepG2/MDR cells were cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 1% streptomycin, 1% penicillin, and 10% fetal bovine serum (FBS) in a 5% CO2 incubator at 37°C. MDR/HepG2 cells were generated by treating HepG2 cells with 7.5 µg/ml (25 µM) of free cisplatin for 24 hours, followed by culture in medium containing 3 µg/ml cisplatin (10 µM) for 10 days. Subsequently, the cells were treated with 10 µg/ml free cisplatin for an additional 24 hours. Surviving cells were cultured for 10–14 days with 7.5 µg/ml of cisplatin until the cells maintained steady growth for five consecutive passages. It is important to note that HepG2/MDR cells were washed for at least two passages before experiments to reduce cisplatin accumulation in cells.
Synthesis the cisplatin precursor of cis-[Pt(NH3)2(H2O)2](NO3)2
The synthesis of the cisplatin precursor, cis-[Pt(NH3)2(H2O)2](NO3)2, was conducted following a procedure described in the literature. Initially, a suspension of 0.20 mmol cisplatin (1 mL) was prepared, and then 0.39 mmol of AgNO3 was added to the suspension under dark conditions. The mixture was subsequently protected from light and heated at 60°C for 3 hours. After the reaction, the resulting AgCl precipitate was removed through centrifugation at 8000 rpm for 15 minutes. The concentration of the synthesized compound was determined using inductively coupled plasma mass spectrometry (ICP-MS) using an Agilent 7500 instrument (CA, USA).
Preparation of lipo-coated CaCO 3 /cisplatin hybrid watermelon-shaped Nanoparticles (LCa/C@B): The synthesis of LCa/C is schematically illustrated in Fig. 1A. First, two copies of a 4 mL inverse microemulsion were prepared using Triton X-100: Hexanol: Cyclohexane: H2O at a ratio of 1.02:1.04:3.94:0.3 (v/v) and stirred for 5 minutes until uniform. Subsequently, 200 µL of 200 mM CaCl2 or 200 µL of 200 mM cisplatin prodrug and 200 µL of 200 mM Na2CO3 were added to the respective inverse microemulsion, and DOPA (100 µL, 20 mM) was then added to the microemulsion containing the cisplatin precursor. The mixture was stirred for 20 minutes. The two types of emulsions were then mixed and reacted for 24 hours. After that, 16.0 mL of ethanol was added to the microemulsion, and the mixture was centrifuged at 12,000 × g for at least 15 minutes to remove the cyclohexane and surfactants. After being extensively washed with ethanol 2–3 times, the pellets were re-dispersed in 3.0 mL of chloroform to obtain DCa/C nanoparticles. To prepare LCa/C, 28 mg of DOTAP, 21.3 mg of cholesterol, and 12.6 mg of mPEG-DSPE (molar ratio 40:55:5) and 1.0 mL of DCa/C nanoparticle core were dispersed in 5.0 mL of chloroform. After chloroform was removed by rotary evaporation, residual lipids were dispersed in 2.0 mL of ddH2O to generate LCa/C. To prepare LCa/C@B, LCa/C and Bmi1 siRNA were mixed at a w/w (weight LCa/C/weight siRNA) ratio of more than 200:1 in RNase-free H2O by adding a stock solution of LCa/C into a Bmi1 siRNA solution. The samples were vortexed for 2–3 minutes and then incubated at room temperature for 30 minutes to ensure the formation of LCa/C@B nanocapsules.
Drug loading determination
The concentration of cisplatin was determined using Inductively Coupled Plasma Mass Spectrometry (ICP-MS) after lysing the sample in a Hydrofluoric acid (HF) solution.
Nanoparticle characterization
The particle size and zeta potential of the nanoparticles were measured in their aqueous solution using Zeta PALS (Zeta Potential Analyzer, Brookhaven Instruments Corporation, Austin, TX) following the manufacturer's instructions. All measurements were conducted at room temperature. Each parameter was measured three times, and the average values and standard deviations were calculated. The shape of the nanoparticles was analyzed using transmission electron microscopy (TEM, JEOL 1010, Japan). For TEM analysis at 100 kV, the powder sample of nanoparticles was dispersed in ethanol and dried on a copper grid. Elemental analysis was performed using techniques such as Energy Dispersive X-ray (EDX).
In vitro acid-responsiveness release of cisplatin under simulated conditions
To examine the acid-responsiveness, one milliliter of LCa/C was placed inside a dialysis bag with a molecular weight cut-off of 2000 Da. The dialysis bag was then immersed in 20 mL of PBS containing 0.5% tween 80 at different pH values (pH 6.5, 6.8, and 7.4) and maintained at a temperature of 37°C. The released platinum (Pt) was quantified using ICP-MS.
Intracellular uptake of nanoparticles
To investigate the intracellular uptake of nanoparticles, HepG2 and HepG2/MDR cells were cultured in 24-well plates and treated with different groups (equivalent to 2.5 µg/ml cisplatin) for 48 hours. After treatment, the cells were digested with 70% HNO3 and diluted in water to achieve a final acid concentration of 2%. The concentration of cisplatin in the solution was determined using ICP-MS. To detect the fluorescence of LCa/C@B (Bmi1 siRNA labeled with FITC), we incubated the nanoparticles for 24 hours after adsorption, and then incubated them with tumor cells for 1, 4, and 8 hours, respectively. Following that, the medium was replaced with fresh medium, and the fluorescence was observed under a fluorescence microscope.
Cytotoxicity determination
To assess the cytotoxicity of the drugs, tumor cells were cultured in 96-well plates and treated with different concentrations of the respective drugs. Following a 24-hour incubation period, cell survival was measured using a CCK-8 kit (Dojindo Laboratories, Kumamoto, Japan) in accordance with the manufacturer's instructions. Cell viability was calculated based on the assay results.
Cell cycle analyses
For cell cycle analysis by FACS, HepG2 and HepG2/MDR cells were seeded at a density of 4 × 105 cells per well or 70% confluency in DMEM with 10% FBS in 6-well plates and allowed to attach overnight. The culture medium was then replaced with fresh DMEM containing 10% FBS, and the cells were treated with PBS, free cisplatin (5 µM), LC (5 µM cisplatin), LCa/C (5 µM cisplatin), or LCa/C@B (5 µM cisplatin and 50 nM Bmi1 siRNA) for 24 hours. After the treatment, the cells were harvested by trypsinization, collected, and fixed in 70% ethanol at 4°C overnight. Following fixation, the cells were washed and suspended in 200 µL PBS. Subsequently, 5 µL of RNase (20 mg/mL) was added to the cell suspension and incubated at 37°C for 30 min. The cells were then stained with 20 µL of propidium iodide (500 µg/mL, KeyGen Biotech Co. Ltd., Nanjing, China) at 4°C for 30 min.
Western Blotting Analysis: Following treatment with different formulations, HepG2/MDR cells were collected and lysed using a cell lysis solution. The resulting lysates were subjected to high-speed centrifugation (12,000 rpm), and the supernatant containing the proteins was collected. Separation of the proteins was performed using a 10% polyacrylamide gel. Subsequently, the separated proteins were transferred onto a PVDF membrane, which was then blocked using 5% skim milk for 1 hour to prevent non-specific binding. Monoclonal antibodies against specific proteins were used for immunoblotting. The antibodies used included Bax (1:1000, Cell Signaling, Danvers, MA, USA), Caspase 3 (1:1000, Cell Signaling), Caspase 9 (1:1000, Cell Signaling), Bcl 2 (1:1000, Cell Signaling), Cyclin D (1:1000, Cell Signaling), Cyclin E (1:1000, Cell Signaling), CD133 (1:1000, Cell Signaling), Bmi1 (1:1000, Cell Signaling), and Tubulin (1:1000, Abcam). The membrane was incubated with the primary antibodies overnight at a specified dilution. Following incubation, the membrane was washed three times with TBST (PBS with 0.1% Tween-20) to remove unbound antibodies. Then, the membrane was incubated with the appropriate secondary antibody for 1 hour. After another round of washing with TBST, an enhanced chemiluminescence system (Perkin Elmer, Waltham, MA, USA) was used for image development and visualization of the protein bands.
Flow Cytometry
For the analysis of CSC marker FACS detection, HepG2 and HepG2/MDR cells treated with different drugs were incubated with Hoechst 33342 (20 µg/mL) for 1.5 hours at 37°C. To analyze the expression of CD133, phycoerythrin-conjugated CD133/1 (Miltenyi Biotec) was added to the cells. After eliminating the dead cells with propidium iodide, the cells were analyzed using FlowJo software.
Mice
C57BL/6J mice (4–6 weeks old) were obtained from Beijing Huafukang Bioscience Technology Co., Ltd. (Beijing, China). The C57BL/6J mice were housed in filter-topped cages with standard rodent chow and water available ad libitum, under a 12-hour light/dark cycle. The experimental protocol was approved by the Committee on Ethical Animal Experiment at Huazhong University of Science and Technology.
Primary HCC Mouse Model
To establish the primary HCC mouse model, a double overexpression gene of Akt/Ras was generated to induce primary HCC, and the anti-tumor effect of different drugs was evaluated. All gene constructs of hyperactive transposase (pCMV/SB), AKT (myr-AKT/pT3EF1α), and Ras (pCaggs-RasV12) were provided by Professor Xin Chen (University of California at San Francisco). The GenElute Endotoxin-free Plasmid Maxiprep Kit (Sigma, USA) was used to purify the plasmids before injecting them into the mice.
The high-pressure injection technique via the tail vein was performed following the protocol described in previous reports. Briefly, C57BL/6J mice were injected with 2 ml of saline containing NRasV12/pT2-CAGGS (5 µg), myr-AKT/pT3EF1α (5 µg), and pCMV/SB (0.4 µg) within 5–10 seconds via the tail vein. After approximately six weeks, HCC tumors were formed in the liver due to the up-regulation of AKT/Ras gene expression, and confirmation was obtained by bimanual palpation and dissection.
Mice were anesthetized by isoflurane gas (2%, inhalation) and given an i.p. injection of D-firefly luciferin (240 mg/kg) (Gold Biotechnology, St. Louis, MO). Mice were placed into the chamber of a Xenogen IVIS 200 imaging system and bioluminescence images were obtained under isoflurane anesthesia using 1 minute exposures beginning 10 minute after D-luciferin injection. The images were quantitatively analyzed by Living Image version 4.3.1 image analysis software (Caliper Life Sciences, Hopkinton, MA). Total bioluminescence measurements (photon/s) were quantified over a contour drawn around brain and coronal slices. Results were repeated two to three times in independent animals. All in vivo images are scaled to maximum intensity of 1×105 photons/s/cm2/sr.
Statistical Analysis
Student's t-test (SPSS Software, Chicago, IL) was used for comparisons between two groups. One-way ANOVA (GraphPad Prism 5.0, San Diego, CA, USA) with Dunnett's post-test was used to compare multiple groups.
