All animal experiments including pharmacokinetics, tissue distribution, and pharmacodynamics were carried out by relevant guidelines that were developed by the China Council on Animal. In addition, these studies were conducted and monitored in accordance with the ethical and scientific principles required by the Declaration of Helsinki. All the procedures were ethically and scientifically approved by the Ethics committee of China Medical University (No 2018PS206K, 2018-02-28).

The pharmacokinetics of BBR-10-HCPT-loaded LM, 10-HCPT injection, and BBR injection in SD rats were evaluated in this study (n = 6). Briefly, BBR-10-HCPT-loaded LM, 10-HCPT injection and BBR injection were injected respectively. The dose of BBR and 10-HCPT was 3.0 mg/kg and 1.0 mg/kg respectively. Then, 0.2 mL blood was collected at 5.0 min, 15.0 min, 30.0 min, 60.0 min, 2.0 h, 4.0 h, 6.0 h, 8.0 h, 12.0 h, 24.0 h, 48.0 h, 72.0 h, and 96.0 h, respectively after administration. The plasma concentration of BBR and 10-HCPT was measured using a validated HPLC-MS/MS method. The analytical column was poroshell 120 poroshell-C18 column, the mobile phase consisted of acetonitrile and 0.1% formic acid. Here, tetrahydropalmatine was chosen as IS and the concentration was 20.0 ng/mL. The ion ionization of BBR, 10-HCPT, and IS were positive ion ionization. Specific chromatographic and mass spectrometric conditions were shown in Tables 1 and and2.2. 100.0 μL plasma samples and 20.0 μL IS solution were placed in a 10.0 mL EP tube and allowed to mix for 1.0 min. Then, 4.0 mL of extraction solvent (dichloromethane:diethyl ether = 3:2) was added and mixed for 5.0 min by vortex continually, followed by centrifugation at 5000 rpm for 10.0 min. The supernatant was then transferred to a clean tube and evaporated to dryness under nitrogen at 40.0 °C. The residue was reconstituted with 300.0 μL of methanol. After centrifugation at 15,000 rpm for 10 min, a 2.0 μL aliquot of the solution was injected into the HPLC-MS/MS system for analysis.

Gradient condition of HPLC.

aAcetonitrile; b0.1% formic acid water.

MS parameters of BBR, 10-HCPT, and IS.

Tissue distribution of BBR-10-HCPT loaded LM was evaluated in SD rats. BBR injection and 10-HCPT injection were used as a reference. BBR-10-HCPT loaded LM and injections were administrated via the tail vein. The dose of BBR and 10-HCPT was 3.0 mg/kg and 1.0 mg/kg, respectively. Rats were killed at 0.5 h, 1 h, 4 h, 8 h, 24 h, 48 h, 72 h, respectively, post-administration, and then the liver, heart, lung, spleen, kidney, and tumor were collected and weighed. Then, 300.0 μL tissue homogenate and 20.0 μL IS solution were placed in a 3.0 mL EP tube and allowed to mix for 1.0 min. The BBR and 10-HCPT existed in each tissue were extracted with the mixed solvent mentioned before and analyzed by HPLC-MS/MS method.

HIF-1α is determined by using a commercial rat enzyme-linked immunosorbent assay (ELISA) kit. Firstly, rat tissue would be cut into pieces, weighed, and homogenized with pH 7.4 PBS for 3 min and then the supernatant was transferred to an EP tube after centrifugation at 2500 rpm for 25 min. Secondly, the calibration solution was prepared by diluting the stock solutions with standard diluent. The calibration concentrations were 5 ng/L, 10 ng/L, 20 ng/L, 40 ng/L, and 80 ng/L. Thirdly, a blank hole without samples and enzyme reagents, standard hole adding 50 μL calibration solution, and test hole adding 40 μL standard diluent and 10 μL tissue sample solution were essential during the test. Then, an enzyme-labeled plate sealed with the membrane was incubated at 37 °C. After 30 min, the solutions were removed, and enough detergent was added to each hole. The detergent was removed and added fresh detergent after 30 s, repeat the above operation 5 times. Fourthly, 50 μL of enzyme-labeled reagent was added to each hole in addition to the blank holes. Similarly, the enzyme-labeled plate was incubated again at 37 °C for 30 min and was washed 5 times with detergent. Finally, color developing reagents A and B were added to each hole in order and finally incubated at 37 °C in dark. After 10 min, 50 μL of termination fluid was added to each hole to terminate the reaction. OD values were measured at a wavelength of 450 nm after setting the blank hole to zero.

The anti-tumor effect of BBR-10-HCPT loaded LM was evaluated in nude mice bearing Hep-3B tumor xenograft. The mice were treated with BBR-10-HCPT loaded LM solution, BBR injections, 10-HCPT injection, and saline, respectively. The dose of BBR and 10-HCPT was 3.0 mg/kg and 1.0 mg/kg. Mice were administered preparations twice per week for four weeks. Tumor volume and body weight were measured twice a week. Tumor weight were measured at the end of the experiment. Tumor growth inhibition rate was measured by the following equation:

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