Chemicals and materials:
Doxorubicin and ε-Caprolactone were purchased from Aladdin co. (Shanghai, China). IR780 was purchased from Sigma-Aldrich co. (St. louis, USA), PEG was obtained from Peng Sheng Biological Co., Ltd (Shanghai, China). MTT Cell Proliferation and Cytotoxicity Assay Kit was obtained from Beyotime (Shanghai, China).1×Phosphate buffer solution (PBS) and deionized water were used in the experiments. All C57BL/6 female mice (18-20 g) were obtained from Yangzhou University Medical Center.
Synthesis of PCL-PEG-SS
Firstly, we synthesized HO-PCL-b-PEG114 with the terminal group of hydroxyl group. The specific synthesis steps are shown in Figure 1. The synthesis of HO-PCL-b-PEG114 is carried out by using PEG114-OH as an initiator and stannous octoate (Sn(Oct)2) as a catalyst to initiate ring-opening polymerization of monomer ε-caprolactone (ε-CL). Specifically, 2 g of dry treated CH3O-PEG114-OH (0.4 mmol) and 4 g of de-vaporized ε-CL (35.1 mmol) were weighed and dissolved in 10 mL of vacuum-distilled anhydrous toluene, followed by One drop of Sn(Oct)2. The liquid nitrogen was frozen, evacuated, purged with nitrogen, thawed, and cycled three times. After reacting for 24 h in a 110 °C oil bath, it was added dropwise to 500 mL of ice diethyl ether under stirring to precipitate. It was suction filtered, washed with diethyl ether three times, and dried in vacuo to get white solid HO-PCL-b-PEG114. The degree of polymerization of the PCL segment was calculated by nuclear magnetic resonance to be Dp=87. The block copolymer was HO-PCL87-b-PEG114. Then, to synthesis PCL-PEG-SS, 0.5g HO-PCL87-b-PEG114 dissolved in 20mL DMSO, were added 4-(dimethylamino) pyridine (DMPA, 0.2 g) solution in DMSO (3 mL) and lipoic acid anhydride (0.6 g) in DMSO (3 mL), respectively. The reaction was stirred for 48 h under nitrogen at 30 °C. The product was isolated by precipitation in cold ethanol, washed several times with ethanol, and dried in vacuo.
Preparation of DOX&IR780@PEG-PCL-SS NPs
After hydrophobization of doxorubicin, different concentrations of DOX, IR780 and polymer PEG-PCL-SS were dissolved in DMSO, added to PBS under ultrasonic conditions. Nanoparticles loaded with near-infrared photosensitizers were prepared by hydrophobic interaction of PCL and hydrophobic small molecules. The DTT was accurately weighed and the nanoparticles were reacted using DTT. Because DTT can partially hydrolyze the disulfide bond of the nanoparticles within a controllable range, the spatial structure of the nanoparticles after reflection is more compact. The nanoparticles loaded with photosensitizer and chemotherapeutic drugs were obtained by dialysis and concentration.
Characterization of DOX&IR780@PEG-PCL-SS NPs
Particle size and surface potential were measured by a Brookhaven BI-90Plus laser particle size analyzer. Transmission electron microscopy was used to observe the morphology of the nanoparticles. Sample preparation method: a suitable concentration of the nanoparticle solution was dropped on a 200 mesh copper mesh and dried overnight. Other characterizations include: nanoparticle stability, drug loading and UV spectroscopy.
Cell culture
MB49 murine bladder carcinoma cells were obtained from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. The cell line was maintained in RPMI 1640 and antimicrobial-antimycotic (Gibco/Invitrogen, Carlsbad, CA) supplemented with 10% fetal calf serum (Hyclone, Logan, UT). The cell line was transduced with the firefly luciferase gene by a lentivirus vector.
In vitro temperature curve
We prepared an internal cross-linked polymer nanoparticle containing IR780, an internal cross-linked polymer nanoparticle containing doxorubicin and IR780, and a PBS solution. The 808 nm near-infrared laser irradiation system was used for 1-3 minutes, and the temperature probe measurement system was used. The photothermal effect of the IR780-containing nanoparticles was evaluated (50μg/mL IR780).
In vitro drug release
Doxorubicin and IR780 were carried into the inner crosslinked polymer nanoparticles. After dialysis, a small amount of organic solvent was removed, and then the dialysis bag filled with nanoparticles was dropped in a PBS solution containing or without GSH, and the drug-loaded nanoparticles were continuously triggered by GSH. The internal chemical structure of the internally crosslinked polymer nanoparticles is changed, and the drug is continuously released from the drug-loaded nanoparticles. In some experimental groups, near-infrared laser irradiation can be used to irradiate the cross-linked polymer nanoparticles. The near-infrared laser can promote the photothermal reaction of IR780 in the drug-loaded nanoparticles, and further promote the release of the drug from the nanoparticles. The drug content of nanoparticles in PBS was measured at different time points under different experimental conditions, and the drug release curve was obtained.
Cellular uptake
Firstly, we used bladder tumor cells to determine the uptake of nanoparticles in tumor cells. We added free doxorubicin solution, free IR780 solution, and drug-loaded nanoparticles containing doxorubicin and IR780 to the culture system of bladder tumor cells. The cellular uptake of the nanoparticles was evaluated by observing the proportion of fluorescent cells in the tumor cells by fluorescence microscopy. As a result, the tumor targeting of the internally crosslinked polymer nanoparticles and the time during which the tumor cells uptake the drug-loaded nanoparticles can be determined.
In vitro cytotoxicity
Different concentrations of doxorubicin-containing nanoparticles, IR780-containing nanoparticles, drug-loaded nanoparticles containing doxorubicin and IR780, and nanoparticles without any drug were dissolved in the cell culture solution. After 24 hours, the number of living cells was evaluated by MTT method, and the absorbance of the solution at 570 nm was measured to obtain the in vitro cytotoxicity and optimum concentration of the drug-loaded nanoparticles.
In vitro therapeutic efficacy of DOX&IR780@PEG-PCL-SS NPs
Different concentrations of free doxorubicin solution, free IR780 solution, doxorubicin-containing nanoparticles, IR780-containing nanoparticles, doxorubicin and IR780 nanoparticles were prepared and dissolved in tumor cell culture. In the solution, after the tumor cells were fully ingested with drugs or drug-loaded nanoparticles, some experimental groups were irradiated with 808 nm near-infrared laser for 3 minutes, and then cultured for 24 hours. After 24 hours, the number of living cells was evaluated by MTT method. The absorbance of the solution at 570nm gives the experimental group with the strongest killing ability to tumor cells. Combined with the results of cytotoxicity experiments, it is used for the concentration of the dual sensitive internal cross-linked polymer nanoparticles in vitro and in vivo.
Establishment of an orthotopic bladder cancer model in C57BL/6 mice
All mice received care following the guidelines of the Care and Use of Laboratory Animals and their use followed the terms of the Institutional Animal Care regulations and Use Committee of Nanjing University. All animal experiments were approved by the Administration Committee of Experimental Animals in Jiangsu Province and the Ethic Committee of Nanjing University.
After anesthetizing C57BL/6 mice with 2% pentobarbital, the midline incision was taken to expose the bladder position of the mouse. After using the syringe to absorb the urine in the bladder, the MB49 bladder cancer cell suspension was injected into the bladder muscle layer. Then, put the bladder into the abdominal cavity and closed the abdominal cavity. After one week of successful modeling, a small animal CT examination was performed to observe whether there was a tumor in the bladder area.
In vivo bio-distribution
Construction orthotopic bladder cancer model, and the synthesis of free IR780 solution and drug-loaded nanoparticles loaded with doxorubicin and IR780. After the experimental black mice were anesthetized, the free IR780 solution and the drug-loaded nanoparticles loaded with doxorubicin and IR780 were injected into the mice by tail vein administration, and the small black mice were anesthetized at different time points for small animal imaging.
Therapeutic efficacy of DOX&IR780@PEG-PCL-SS NPs in orthotopic bladder cancer model
The luciferase plasmid was constructed and the cell line was transfected with lentivirus. The MB49-luciferase cell line was constructed to establish an orthotopic bladder cancer model. One week after modeling, the mice were initially screened with small animals and randomly divided in groups: saline, doxorubicin-containing nanoparticles (4 mg/kg IR780), IR780-containing nanoparticles (2.5 mg/kg DOX), "drug-loaded nanoparticles containing doxorubicin and IR780"(2.5 mg/kg DOX, 4 mg/kg IR780) were injected into mice by tail vein administration. Some experimental groups were irradiated with 808 nm near-infrared laser after 24 hours, and repeated administration after one week. After 3 weeks, the in vivo animal image was repeated, and the mice were sacrificed, and the heart, liver, spleen, lung, kidney and bladder of the mice were taken out.
In vivo toxicity
Different concentrations of drug-loaded nanoparticles loaded with doxorubicin and IR780 were synthesized, and the experimental mice were anesthetized. Different concentrations of drug-loaded nanoparticles loaded with doxorubicin and IR780 were injected into mice by tail vein administration. After 24 hours, the mice were sacrificed. The blood was taken by the eyeball. About 1 ml of blood was taken from each mouse, and ALT, AST, BUN, and Cr in the blood of the mice were immediately detected.
Mouse organ sectioning and staining examination
After the mice were sacrificed, the heart, liver, spleen, lung, kidney and bladder of the mice were taken out. After dehydration and fixation, the sections were stained with H&E, and the bladder and tumor tissues were stained with TUNEL to observe the apoptosis and necrosis in the bladder cancer tissues.