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1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD), ε-caprolactone, 6-bromo-1-hexanol, p-toluenesulfonyl chloride, methoxyl polyethylene glycol (mPEG-OH, Mw = 2 kDa) were purchased from Sigma–Aldrich. Solvents were either employed as purchased or dried according to procedures described in the literature. Millipore ultrapure water was obtained on a Milli-Q purification system. Transmission electron microscopy (TEM) investigations were carried out on a HT-7700 instrument. UV–vis absorption spectra were recorded by using a Hitachi U-3010 spectrophotometer. Confocal laser scanning microscopy (CLSM) images was recorded on a LSM710META (Zeiss) microscope. Gel permeation chromatography (GPC) was conducted on a Waters Chromatography, Inc. (Milford, MA) system using THF containing 0.05 M LiBr as eluent. The sizes of the nanoformulations were determined by a DLS analyzer (Zetasizer Nano ZS90 Malvern Instruments, Malvern) with a detection angle of 90° at 25 °C using an incident He–Ne laser (λ = 633 nm). ITC experiments were carried out with a Microcal VP-ITC calorimeter at 298.1 K. The anticancer efficacy was determined by using Cell Counting Kit-8 (CCK-8, Solarbio, Beijing) according to the instructions of the manufacturer. The absorbance of the bioreduced soluble formazan product was measured at 450 nm using a TECAN Infinite F200 PRO. H&E tissue and cell staining was performed by BBC Biochemical (Mount Vernon, WA) and the images were collected using a BX41 bright field microscopy (Olympus).
Synthesis of PCL-MV
The synthetic routes of PCL-MV and Nap-PEG were illustrated in Additional file 1: Scheme S1. To a solution of 6-bromo-1-hexanol (181 mg, 1.00 mmol) and ε-caprolactone (4.56 g, 40.0 mmol) in anhydrous CH2Cl2 (10 mL), TBD (139 mg, 1.00 mmol) was added and the mixture was stirred at room temperature. After 20 min, the reaction was quenched by adding 1 mL of acetic acid. The resulting solution was precipitated into an excess of diethyl ether. After filtration, the sediments was dissolved in CH2Cl2 and precipitated into an excess of diethyl ether; the above dissolution–precipitation cycle was repeated three times. After drying in a vacuum oven overnight at room temperature, PCL-Br was obtained as a white solid (4.45 g, yield: 93.9%). The molecular weight and composition of PCL-Br were determined by 1H NMR spectroscopy (Additional file 1: Fig. S1) and GPC (Additional file 1: Fig. S2).
A mixture of PCL-Br (1.15 g) and excessive MVI (700 mg) in DMF (10 mL) was stirred at 85 °C overnight. After reaction, the solvent was poured into 150 mL of H2Cl2, and the mixture was washed with water for three times. The solvent was evaporated and the residue was dissolved in 10 mL of THF. The resulting solution was precipitated into an excess of diethyl ether. The above dissolution–precipitation cycle was repeated three times. The solid was dried overnight in a vacuum to give a pale yellow powder with a yield of 92.7%. The molecular weight and composition of PCL-MV were determined by 1H NMR spectroscopy (Additional file 1: Fig. S3) and GPC (Additional file 1: Fig. S4).
Synthesis of Nap-PEG
mPEG-OH (10.0 g, 5.00 mmol) and NaOH (4.00 g, 100 mmol) were dissolved in the mixture of THF and H2O (150 mL, THF/H2O = 1/1, v/v). p-Toluenesulfonyl chloride (5.70 g, 30.0 mmol) was dissolved in 30 ml of THF, and the solution was droply added into the mPEG-OH solution at 0 °C. The mixture was further stirred at room temperature overnight. The organic solvent was evaporated, and the solution was extracted by CH2Cl2 (3 × 50 mL). The organic phase was further washed with water for three times to eliminate the excessive NaOH and p-toluenesulfonyl chloride. The solvent was concentrated into 10 mL, and the resulting solution was precipitated into an excess of diethyl ether. The above dissolution–precipitation cycle was repeated three times to afford mPEG-OTs without further purification.
A mixture containing mPEG-OTs (2.15 g), 6-methoxy-2-naphthol (1.74 g, 10.0 mmol) and K2CO3 (2.76 g, 20.0 mmol) in CH3CN (50 mL) was added to a round-bottom flask under nitrogen atmosphere and heated at reflux for 12 h. The organic phase was obtained after filtration and the solvent was removed by rotary evaporation to afford the crude product. The residue was dissolved in 5 mL of CH2Cl2, and the resulting solution was precipitated into an excess of diethyl ether. The above dissolution–precipitation cycle was repeated three times. The solid was dried overnight in a vacuum to give a dark green powder with a yield of 87.4%. The molecular weight and composition of PCL-MV were determined by 1H NMR spectroscopy (Additional file 1: Fig. S5) and GPC (Additional file 1: Fig. S6).
Synthesis of Nap-DFO
The synthetic route of Nap-DFO was illustrated in Additional file 1: Scheme S2. A mixture containing tert-butyl N-(2-bromoethyl)carbamate (2.24 g, 10 mmol), 6-methoxy-2-naphthol (0.87 g, 5.00 mmol) and K2CO3 (2.76 g, 20.0 mmol) in CH3CN (50 mL) was added to a round-bottom flask under nitrogen atmosphere and heated at reflux for 12 h. The organic phase was obtained after filtration and the solvent was removed by rotary evaporation to afford the crude product, which was isolated by flash column chromatography to give Nap-Boc as a gray solid with a yield of 76.8%. 1H NMR (Additional file 1: Fig. S7), 13C NMR (Additional file 1: Fig. S8) and mass (Additional file 1: Fig. S9) spectra were utilized to confirm the preparation of Nap-Boc.
Trifluoroacetic acid (2.00 mL) was added to the solution of Nap-Boc (0.63 g, 2 mmol) in CH2Cl2 (15 mL), and the mixture was stirred at room temperature for 8 h. The solvent was removed by rotary evaporation and the compound was washed by methanol for three times to give Nap-NH2 as a brown solid with a yield of 64.5%.1H NMR (Additional file 1: Fig. S10), 13C NMR (Additional file 1: Fig. S11) and mass (Additional file 1: Fig. S12) spectra were utilized to confirm the preparation of Nap-NH2. Nap-DFO was obtained by labelling Nap-NH2 with NCS-DFO, and the successful preparation of Nap-DFO was verified by 1H NMR (Additional file 1: Fig. S13), 13C NMR (Additional file 1: Fig. S14) and mass spectrum (Additional file 1: Fig. S15).
Preparation of supramolecular nanomedicine
DOX (8.00 mg), PCL-MV (18.5 mg) and Nap-PEG (11.2 mg) were dissolved in DMSO (10 mL), 10 mL of aqueous solution containing CB[8] (1.00 mg/mL) was droply added into the mixture solution. After stirring in the dark for 2 h, the resulting mixture was sealed in dialysis bags with a molecular weight cut-off of 3.5 kDa and dialyzed against DI water for 12 h to remove free DOX and CB[8]. The drug loading content was estimated to be 18.4% by using UV spectroscopy. For the preparation of 89Zr labelled nanomedicine, DOX (8.00 mg), PCL-MV (18.5 mg), Nap-DFO (0.300 mg), and Nap-PEG (10.5 mg) were dissolved in DMSO (10 mL), 10 mL of aqueous solution containing CB[8] (1.00 mg/mL) was droply added into the mixture solution. After stirring in the dark for 2 h, the resulting mixture was sealed in dialysis bags with a molecular weight cut-off of 3.5 kDa and dialyzed against DI water for 12 h to remove Nap-DFO, free DOX and CB[8]. SNM@DOX was labelled with 89Zr by mixing the nanomedicine with radioactive isotope at 37 °C for 1 h under constant stirring.
Drug release studies
In vitro released profiles of DOX from SNM@DOX at different pH value were monitored using the dialysis method. The SNM@DOX was dissolved in 25 mL of distilled water and sealed in dialysis bags with a molecular weight cut-off of 2 kDa at pH 7.4, 6.0, and 5.0, respectively. The dialysis apparatus was agitated on an orbital shaker at 100 rpm at 37 °C. At designated time intervals, 1 mL of medium was taken out from the 25 mL solution out of the dialysis bag for UV detection and was then put back to the original system. The DOX concentration was calculated with a standard curve calibrated with DOX samples of known concentrations.
Cell cultures
HepG2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Cells grew as a monolayer and were detached upon confluence using trypsin (0.5% w/v in PBS). The cells were harvested from the cell culture medium by incubating in a trypsin solution for 5 min. The cells were centrifuged, and the supernatant was discarded. A 3 mL portion of serum-supplemented DMEM was added to neutralize any residual trypsin. The cells were resuspended in serum-supplemented DMEM at a concentration of 1 × 104 cells/mL. Cells were cultured at 37 °C and 5% CO2.
Evaluation of cytotoxicity
The cytotoxicities of CB[8], PCL-MV, Nap-PEG, DOX·HCl, and SNM@DOX against HepG2 cells were determined by MTT or CCK-8 assay in a 96-well cell culture plate. All solutions were sterilized by filtration with a 0.22 μm filter before tests. HepG2 cells were seeded at a density of 1 × 104 cells/well in a 96-well plate, and incubated for 24 h for attachment. Cells were then incubated with CB[8], PCL-MV, Nap-PEG, DOX·HCl, and SNM@DOX at various concentrations for 24 h. After washing the cells with PBS buffer, 20 μL of a MTT solution (5 mg/mL) was added to each well. After 4 h of incubation at 37 °C, the MTT solution was removed, and the insoluble formazan crystals that formed were dissolved in 100 μL of dimethylsulfoxide (DMSO). The absorbance of the formazan product was measured at 570 nm using a spectrophotometer (Bio-Rad Model 680). For CCK-8 assay, WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium, monosodium salt] solution was added and cells were incubated for 4 h at 37 °C under 5% CO2. The absorbance of each well was measured with a luminescence microplate reader (Bio-Rad 680) at 450 nm. Untreated cells in media were used as a control. All experiments were carried out with five replicates.
Cellular internalization studies by CLSM
HepG2 cells were treated with SNM@DOX (the concentration of DOX was 2.00 μM) in the culture medium at 37 °C for 4 and 9 h, respectively. The cells were washed three times with PBS and fixed with fresh 4.0% formaldehyde at room temperature for 15 min. After washing with PBS, the cells were stained with DAPI (1 μg/mL) for 15 min. The images were taken using a LSM-510 confocal laser scanning microscope (Zeiss, Germany) (100 × oil objective, 405/488 nm excitation).
Animals and tumor models
Female nude mice (4 weeks old, ~ 20 g body weight) were purchased from Zhejiang Academy of Medical Sciences and maintained in a pathogen-free environment under controlled temperature (24 °C). Animal care and handling procedures were in agreement with the guidelines evaluated and approved by the ethics committee of Zhejiang University of Technology. Study protocols involving animals were approved by the Zhejiang University of Technology Animal Care and Use Committee. The female nude mice were injected subcutaneously in the right flank region with 200 μL of cell suspension containing 2 × 106 HepG2 cells. The tumors were allowed to grow to ~ 100 mm3 before experimentation. The tumor volume was calculated as (tumor length) × (tumor width)2/2. Relative tumor volumes were calculated as V/V0 (V0 was the tumor volume when the treatment was initiated).
Pharmacokinetics and biodistribution
For pharmacokinetic studies, the mice were randomly divided into two groups (n = 3). The aqueous solutions of free DOX·HCl, and SNM@DOX were i.v. injected via tail vein at a dose of 10.0 mg DOX/kg. The blood samples (0.1 mL) were taken from the eye socket at the different time points post injection. The plasma was obtained by centrifugation at 3000 rpm for 15 min, and the amount of DOX in the plasma was assayed by HPLC. The DOX concentrations in the tumor tissues and organs were analyzed by HPLC. 200 μL of 10% (w/v) tissue homogenate was added with 100 μL of PBS. The above mixture was subsequently extracted with chloroform/isopropanol (4:1, v/v) by vortex mixing for 3 min. After centrifugation at 10,000 rpm for 5 min, the organic phase was separated and evaporated to dryness under a stream of nitrogen. The residue was dissolved in 200 μL of mobile phase (methanol/water/acetic acid = 65:35:2, v/v/v). After centrifugation at 10,000 rpm for 5 min, the supernatant was collected for HPLC analysis. The mice bearing HepG2 tumors were i.v. injected 89Zr SNM@DOX and anesthetized with isoflurane (1.0 ~ 2.0%) in oxygen delivered at a flow rate of 1.0 L/min. All PET imaging scans were conducted on a micro-PET/CT at different time post injection.
In vivo anti-tumor evaluation
The mice were divided into three treatment groups randomly (n = 5), when the mean tumor volume reached about 100 mm3 and this day was set as day 0. Mice were administered intravenously with PBS, SNPs, free DOX·HCl (5.00 mg DOX/kg), and SNM@DOX (5.00 mg DOX/kg), respectively every 3 days for four times. Tumor volume and body weight were measured every 3 days. The tumor inhibition study was stopped on the 18th day. In the histological assay, the tissues were fixed in 4% paraformaldehyde for 24 h. The specimens were dehydrated in graded ethanol, embedded in paraffin, and cut into 5 mm thick sections. The fixed sections were deparaffinized and hydrated according to a standard protocol and stained with hematoxylin and eosin (H&E) for microscopic observation.
Statistical analysis
Data are presented as the mean ± standard deviation. Statistical analysis of data was performed with one-way analysis of variance. The level of significance was defined at *p < 0.05, **p < 0.01, ***p < 0.001.
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