Methods

PEG-PLGA nanoparticles were synthesized by the double emulsion method reported in the previous published paper (Wang et al., 2013). Briefly, 20 mg mPEG-PLGA dissolved in 1 mL dichloromethane and 200 μL deionized water or 200 μL Anlotinib hydrochloride solution or 200 μL Cy5.5 dye solution were mixed together and added into a 10 mL centrifuge tube. The mixture was emulsified by sonication via a probe ultrasonicator at 30% power for 5 min within ice-bath. Then, 2 mL 5% sodium cholate was added into the tube. The mixture was emulsified again by sonication at 35% power for another 5 min. Finally, the mixture was added dropwise into 10 mL 0.5% sodium cholate and stirred for 10 min. The dichloromethane in the solution was moved out by vacuum evaporation and the nanoparticles were collected by centrifugation at 15,000 g for 30 min and resuspended in 1 mL deionized water for further usage. For the preparation of pH-responsive nanoparticles (PEOz-NPs-A), 20 mg mPEG-PLGA was replaced by 18 mg mPEG-PLGA and 2 mg PEOz-PLGA during the above procedure.

The particle size and zeta potential of the prepared nanoparticles were measured by DLS at a concentration of 1 mg/mL. The morphology was observed by TEM. Briefly, 10 μL nanoparticle solution with a concentration of 0.1 mg/ml was dropped onto the copper grids. After incubation for 5 min, the solution was moved out by liquid-moving machine. Then, 8 μL phosphotungstic acid staining solution was used to stain the sample and TEM images were carried out.

For the drug encapsulation evaluation, the obtained nanoparticles were demulsified by acetonitrile and the amount of Anlotinib hydrochloride was detected by High Performance Liquid Chromatography at 220 nm. The following formulae were used to calculate the drug LE and drug EE:

EE = (weight of loaded drug)/(weight of initially added drug) × 100%;

LE = (weight of loaded drug in the nanoparticle)/(weight of the nanoparticle) × 100%.

The above prepared pH-responsive nanoparticles (PEOz-NPs-A) were added into two dialysis bags and, respectively, transferred into two 50 mL centrifuge tubes with 30 mL of PBS at different pH values (6.8 and 7.2). The centrifuge tubes were placed in a shaker and incubated at 37°C for different time intervals. In each time point, 1 ml of PBS outside the lysis bag was taken out and 1 ml of fresh PBS at corresponding pH value was added. Finally, the amount of Anlotinib hydrochloride in the 1 ml of PBS was determined by High Performance Liquid Chromatography at 220 nm based on the previous report (Gao et al., 2020). Then, the accumulated drug release was calculated.

Human non-small cell lung cancer cell A549, and HUVEC were purchased from the American Type Culture Collection (ATCC, Rockville, MD, United States) and cultured at 37°C and 5% carbon dioxide in DMEM supplemented with 10% FBS and 1% penicillin and streptomycin. Mouse breast cancer cell 4T1 was purchased from the American Type Culture Collection (ATCC, Rockville MD, United States) and cultured at 37°C and 5% carbon dioxide in 1,640 RPMI supplemented with 10% FBS and 1% penicillin and streptomycin.

For the cytotoxicity studies, A549, 4T1, and HUVEC were sub-cultured into 96-well plates at a density of 5 × 10³ cells per well for 24 h. Then, the tumor cells were treated with different drug formulations of PBS, PEOz-NPs, Anlotinib, NPs-A, and PEOz-NPs-A. The corresponding does of anlotinib were 10, 10, and 4 μmol for A549, 4T1, and HUVEC respectively. After 24 h incubation, the culture medium was replaced by fresh medium containing 10% CCK-8 assay buffer and incubation for another 1 h. The cell viability was detected by microplate reader at a wavelength of 450 nm.

4T1 cells were sub-cultured into confocal petri dishes at a density of 1 × 10⁵ cells in each dish for 24 h. Then, the medium was replaced by fresh medium with Cy5.5-labelled nanoparticle NPs and pH-responsive nanoparticle Cy5.5-PEOz-NPs. After incubation for 8 h, the cellular nucleus was stained using Hoechst 33342 and the cells were observed by laser scanning Confocal microscope.

For the animal imaging, the tumor volume was allowed to grow to ∼200 mm³. The mice were divided into two groups and intravenously injected with 100 μL NPs-Cy5.5 and PEOz-NPs-Cy5.5. After injected for 6 and 24 h, the mice were imaged by an optical imaging system (IVIS Spectrum, PerkinElmer).

Female BALB/c nude mice and BALB/c mice, 6–8 weeks old and with an average body weight 18 g, were purchased from Beijing Vital River Laboratories. All animal experiments were undertaken according to the China Medical University Animal Care and Use Committee. The cultured A549 and 4T1 tumor cells were collected and suspended by a mixture of PBS and matrigel (1:1) and transplanted into the subcutaneous area on the dorsal flank of each mouse. When the tumor volume grown to 100 mm³ (volume = length × width²/2, measured with a Vernier caliper), the mice were then randomly divided into different five treated groups including Saline, PEOz-NPs, Anlotinib, NPs-A, and PEOz-NPs-A. The mice were intravenously injected with 100 μL solutions with different drug formulations (equivalent to 1 mg kg^–1 bodyweight of Anlotinib) every 3 days for total six times. The tumor volumes of each mouse were measured with a Vernier caliper every 3 days.

Female BALB/c mice, 6–8 weeks old and with an average body weight 18 g, were randomly divided into four treated groups including Saline, PEOz-NPs, Anlotinib, and PEOz-NPs-A. The mice were intravenously administrated with 100 μL drug solutions (equivalent to 2 mg kg^–1 bodyweight of Anlotinib) every 2 days for total three times. Post-treatment for 7 days, the blood of mice were drawn from ocular venous plexus and the serum was separated by centrifugation at 3,000 rpm/min for 10 min. The major organs were also isolated for histologic analysis.

Statistical analyses were performed using SPSS 19.0. Student’s t-test was used for comparison between two groups. One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was used for comparison of three or more groups. All the results are presented as means ± SD. P < 0.05 was considered statistically significant.