Materials, cell culture, and animals
TPL (purity ≥ 98%) was obtained from Shanghai Yuanye Biotech Co., Ltd. (Shanghai, China). Coumarin-6 (C6) and pharmaceutical grade γ-CD were supplied by Sigma-Aldrich Co. (St.Louis, MO, USA). Potassium hydroxide, methanol, ethanol, polyethylene glycol 20000 (PEG 20000), and other reagents with analytical grade were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Cell counting kit-8 (CCK-8) was obtained from Beyotime Biotechnology (Shanghai, China). Fetal bovine serum (FBS) and Dulbecco’s modified Eagle’s medium (DMEM) were obtained from Gibco Inc. (Grand Island, NY, USA). TUNEL apoptosis assay kit was obtained from Roche Pharmaceutical Co., Ltd. (Basel, Switzerland). All other chemicals used were of analytical grade.
The Huh-7 cell line and Caco-2 cell line were purchased from the Cell Bank of Typical Culture Preservation Committee of the Chinese Academy of Sciences (Shanghai, China). Huh-7 cells and Caco-2 cells were cultured in DMEM supplemented with 10% FBS, 1% penicillin/streptomycin in a humidified incubator with 5% CO2 at 37 °C.
Healthy male Sprague–Dawley (SD) mice (200 ± 20 g) and Balb/c-nu mice (18 ± 2 g) were randomly assigned to different groups. The experiment was approved by the Ethics Committee of Ninth People’s Hospital, affiliated with Shanghai Jiao Tong University School of Medicine before the research.
High-performance liquid chromatography (HPLC) analysis
The detection of TPL was performed on a Waters e2695 HPLC system (Waters Technologies, USA) with an Agilent TC-C18 column (250mm × 4.6 mm, 5 µm) and acetonitrile:water = 70:30 as the eluent. The flow rate was set at 1.0 mL·min− 1 and the column temperature was set at 30 °C. The detection wavelength was 218 nm with an injection volume of 20 µL.
Synthesis and activation of CD-MOF
The CD-MOF was synthesized according to the previously published protocols . Briefly, γ-CD (3.24 g) and potassium hydroxide (1.12 g) were dissolved in 100 mL distilled water with a 20 min incubation at 50 ℃. Then, the obtained solution was mixed with 60 mL methanol. Thereafter, 160 mL PEG20000 methanolic solution (8 mg·mL− 1) was added and the solution was kept in cold water for about 12 h to collect the precipitates. To remove the residual potassium hydroxide, γ-CD, and PEG20000, the precipitates were washed 3 times with ethanol (60 mL). To activate the obtained CD-MOF, the precipitates were immersed in dichloromethane for 72 h (refreshing dichloromethane every 24 h). At last, the blank CD-MOF was obtained by centrifugation and dried under vacuum at 50 ℃ overnight.
As shown in Fig. 1, TPL was loaded into CD-MOF with solvent adsorption method : 80 mg TPL was dissolved in 2 mL acetonitrile under ultrasonication. 20 mg Blank CD-MOF were then immersed in the TPL solution, stirring for 24 h on a magnetic stirrer (500 rpm) at room temperature. The [email protected] was collected by centrifugation, followed by washing 3 times with acetonitrile and drying for 24 h under vacuum at room temperature. [email protected] was prepared for the cellular uptake study. For C6 loading, 2 mg C6 was dissolved in 2 mL acetonitrile under ultrasonication. Other steps were the same as mentioned above. The loading capacity of TPL was calculated with the following equation :
Where WTPL and W[email protected]−MOF are weights of TPL and [email protected], respectively.
The surface morphology of the blank CD-MOF and [email protected] was characterized with a Hitachi S-4800 Scanning Electron Microscopy (SEM) (Tokyo, Japan). The nitrogen adsorption-desorption isotherms of CD-MOF and [email protected] at 77 K were determined by a Quadrasorb 2 analyzer. X-ray diffraction (XRD) patterns of the free TPL, physical mixture (PM) and [email protected] were conducted on a D8 Advance X-ray diffractometer (Karlsruhe, Germany).
Equilibrium solubility and in vitro release
Solubility of the pure TPL and [email protected] was determined in pure water with the published method . In brief, an excess amount of pure TPL or [email protected] was added into pure water, shaking (500 rpm) at 25 °C for 72 h to reach the equilibrium. After centrifugation (10000 rpm), the supernatant was analysed by HPLC to measure the concentration of TPL in the solutions.
The in vitro release behaviour of the free TPL and [email protected] was evaluated. To simulate the fate of [email protected] after oral administration, release media with pH 1.2, 7.4 and 6.8 were employed to simulate the gastric fluid, the intestinal fluid and the colonic fluid, respectively. The paddle speed was set at 100 rpm and the temperature was set at 37 ℃. 1 mL release media was then withdrawn at predetermined time points and analyzed by HPLC. In the meantime, 1 mL fresh medium was replenished.
The cell uptake of [email protected] by Caco-2 cells was evaluated by a Leica TCS-SP8 confocal laser scanning microscopy (CLSM) (Wetzlar, Germany). 1 mL Caco-2 cells were seeded into confocal dishes at the density of 5 × 104·mL− 1 12 h prior to the experiment. Then the culture medium was removed and the cells were washed twice with PBS and incubated with free C6 or [email protected] solution for 2 h. Thereafter, the cells were rinsed three times with PBS and fixed with 4% paraformaldehyde. After being fixed, the cells were washed three times with PBS and stained with Hoechst 33258 for 5 min. Finally, the cells were washed three times with PBS again and observed by CLSM.
Cell viability assay
The Cell Counting Kit-8 (CCK-8) was utilized to investigate the cell viability after the treatment with free TPL and [email protected] In brief, 150 µL Huh-7 cells were seeded in 96-well plates at the density of 5 × 104·mL− 1 overnight. Then, the cells were treated with TPL or [email protected] After treatment for 48 h, 20 µL CCK-8 was added into each well incubating for 2 h. At last, an Infinite M200 PRO microplate reader (Männedorf, Switzerland) was used to assess cell viability by measuring the absorbance at 450 nm.
In vivo pharmacokinetic studies
Ten healthy male SD mice (200 ± 20 g) were randomly divided into two groups with five mice in each group. They were orally administered with free TPL or [email protected] at a dose of 1.5 mg·kg− 1. Blood samples were collected from the retro-orbital plexus into heparinized tubes at predetermined time points centrifuging immediately (3,000 rpm for 10 minutes) to obtain plasma. Then, 0.1 mL plasma was extracted with 1.2 mL ethyl acetate for 3 times. The top organic layers were combined and dried. The residue was redissolved in 0.1 mL of acetonitrile and centrifuged. The supernatant was analyzed by HPLC.
In vivo anti-tumor activity
For establishing xenografts, 2 × 106 Huh-7 cells were injected subcutaneously into the left axillary region of male Balb/c-nu mice . The tumor volumes and weights of the mice were measured every 2 days. When the tumor volume growed up to 50 mm3 , the mice were then randomized to 3 groups and administered by gavage with saline, free TPL or [email protected] at a dose of 1.5 mg·kg− 1 TPL everyday for 10 days. All mice were sacrificed one day after the last administration. The tumors were harvested and weighed, followed by TUNEL and hematoxylin & eosin (H&E) staining assay.
Data were expressed as mean ± standard deviation (SD). The pharmacokinetic parameters were analysed with DAS 2.0 software (BioGuider Co, Shanghai, China). All data analysis of variance were performed with the SPSS version 19.0 software (SPSS Inc., Chicago, USA). P < 0.05 was considered statistically significant.