C6 cells, HT22 cells, human THP-1 cells (the Chinese Academy of Sciences Cell Bank); Garcinia mangostana L. (Thailand); poly (lactic-co-glycolic acid) (PLGA, Lactel Absorbable Polymers); platelet-rich plasma (provided by volunteers), BALB/Nude mice (Shanghai SLAC Laboratory Animal Co., Ltd.); mannitol, sucrose, Tris/HCL, MgCl2, KCL, DiD, DiO, DiI, phorbol myristate acetate (Sigma Aldrich); protease inhibitor cocktail (Bimake, Shanghai); phosphate buffered saline (PBS, Sigma Aldrich); BCA protein kit, CCK8 kit (Beyotime, China); paraformaldehyde (Sigma, USA); FBS, penicillin–streptomycin (Gibco, USA); DMEM, RPMI 1640 (Hyclone, Shanghai).
2.2 Preparation of β-Mangostin-Loaded NPs
Ten kilograms of fresh Garcinia mangostana L. were separated and chopped, and dried for a week. The peel was pressed into a powder, and β-mangostin (13.4 mg) was obtained by chromatography. β-NPs were prepared using the ultrasonic emulsification solvent evaporation method. PLGA (20 mg) dissolved in ethyl acetate (500 µL), and β-mangostin (2 mg) dissolved in 200 µL DMSO, were used as the organic phase. The mixture was poured into 2 ml 1.5% polyvinyl alcohol (PVA) for ultrasonic treatment on ice for 10 min to form colostrum. Then, 2 ml of 0.5% PVA was added to the mixture for ultrasonic treatment on ice for another 10 min to form a well-dispersed compound emulsion. The mixture was stirred for 5 h at room temperature (1000 rpm) until the organic solvent was completely volatilized. The collected NPs were centrifuged at 4°C and 18000 g for 15 min, and washed three times, and then the obtained yellow NPs were stored at − 80°C.
2.3 Extraction of Platelet Membrane and C6 Cell Membrane
Human platelet membranes were derived from platelet-rich plasma provided by volunteers. To obtain the purified platelet membrane, the obtained platelets was centrifuged at 4°C and 100 g for 15 min. The supernatant was centrifuged at 4 ℃ and 800 g for 20 min. Then, the lysis buffer (25 mM sucrose, 75 mM mannitol, 1 mM KCl, 10 mM Tris/HCl, 1 mM MgCl2) and protease inhibitor (1000×) were added, followed by resting on ice for 15 min. The platelets were repeatedly frozen and thawed five times. After the last freeze–thaw cycle, the platelets were processed in an ultrasonic crusher for 5 s, and the process was repeated five times at intervals of 5 s. The platelets were then centrifuged at 21000 g for 10 min, re-suspended in PBS, and stored in a refrigerator at − 40°C.
The C6 cells were incubated and collected, and then cells at a density of 1×107 were suspended in 5 mL of lysis buffer containing 50 µL protease inhibitor cocktail. After being placed in an ice bath for 20 min, the C6 cells were repeatedly frozen and thawed five times. Later, the cell lysate was centrifuged at 2000 g for 15 min at 4°C, and the collected supernatant was further centrifuged at 21000 g for 30 min. Finally, the white precipitate containing the plasma membrane was stored at − 40°C for the subsequent experiments.
2.4 Preparation and Characterization of Hybrid Membrane Coated NPs
DiD (10 mg/mL) and DiO (10 mg/mL) were added to PLTM and C6M suspensions, respectively, and incubated for 1 h at room temperature on a shaking table. After centrifugation at 4°C and 21000 g for 10 min, the free dye was removed, and DiD-labeled PLTM and DiO-labeled C6M were obtained. Five hundred microliters of each solution was mixed and pushed 11 times using the liposome extruder, and another 500 µL of each solution was mixed to obtain PLTM&C6M solution. Finally, the two solutions were placed on glass slides and imaged under a confocal laser scanning microscope (CLSM).
The composite cell membrane solution PLT-C6M obtained by the above method was mixed with the β-NPs and kept overnight. The liposome extruder was used to push the solution 11 times through polycarbonate membranes of 400 nm and 200 nm. Thus, membrane-coated NPs loaded with β-mangostin were obtained. The sizes and zeta potentials of the prepared membrane-coated NPs were characterized using a Zetasizer. For transmission electron microscopy (TEM), 10 µL of 0.2 mg/mL TEM sample was added onto the carbon-coated grid after being cleaned by plasma. Then the grid was rinsed with deionized water. 10 µL of 2% uranyl acetate solution in water was dropped on the grid for 2 min and removed via filter paper. The staining step was repeated three times prior to the sample imaging with TEM operating at 200 KV.
2.5 In Vitro Stability of NPs and Drug Release of β-PCNPs
To evaluate the stability of the NPs, the PCNPs were suspended in water, 1×PBS, and 10% fetal bovine serum at a final concentration of 1 mg/mL. One week after the set time point, the sample size was determined using the Zetasizer to test the aggregation. The prepared β-NPs and β-PCNPs were placed in a dialysis bag (MWCO = 12–14 kDa), and 30 ml of the solution (H2O, PBS, and 10% FBS) containing 0.1% Tween 80 was added to a 50 ml centrifuge tube. The physiological environment in vivo was simulated on a shaker at 37°C and 200 rpm. After a predetermined time (0, 2, 4, 8, 24, 48, 72, 96, 120, 144, and 168 h), all the solutions were extracted and replaced with 30 ml of the same solution. The absorbance of the released β-mangostin was measured at 360 nm using a microplate reader.
2.6 Active Targeting and Immune Escape Characteristics of PCNPs
The cellular uptake of C6 cells and macrophages was quantitatively analyzed by laser scanning confocal microscopy (LSCM) and flow cytometry. C6 cells were seeded on a 6-well plate at a density of 5 × 105 cells per well and cultured for 12 h. THP-1 cells were maintained in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin–streptomycin at 37% and 5% CO2. The cells were differentiated into macrophage-like phenotype cells by culturing them with 100 ng/mL phorbol myristate acetate(PMA) for 48 h. Fluorescently labeled NPs were synthesized by incorporating 0.1 wt% DiD into the PLGA cores. The cell culture media were then replaced with fresh media containing NPS, PNPs, CNPs, and PCNPs. At 4 h after incubation, the cells were washed twice with PBS and fixed with pre-cooled 4% paraformaldehyde in PBS for 15 min, labeled with DAPI for 15 min, and then washed thrice with PBS. Finally, 10 µL of an anti-fluorescence quenching agent was added to the cell slide. Cellular fluorescence was observed using CLSM. After internalization, the adherent cells were further digested into a single cell suspension by trypsin digestion, suspended in 0.5 mL of PBS (0.01M, pH7.4), and analyzed by flow cytometry.
2.7 In Vitro Anti-tumor Performance
We evaluated the cytotoxicity of the nanoscale systems against C6 cells and mouse hippocampal neurons HT22 by using an in vitro CCK8 kit assay. First, we evaluated the toxicity of PCNPs on HT22 cells. Briefly, the HT22 cells were inoculated in 96 well plates at a density of 1.0 × 104 cells per well and cultured in a CO2 incubator for 24 h. The old culture medium was discarded, and 100 µL of PCNPs of different concentrations (2, 4, 8, and 16 mg/ml) was added into the well plate and incubated for 24 h. The well without the sample was considered the control group, and the well without cell and sample was considered the blank group. Before the CCK8 experiment, the medium in each well was removed and washed three times with 1×PBS. CCK8 (10 µL) was added to each well, and the plates were incubated in an incubator for 1–4 h. The absorbance at 562 nm was measured using a microplate, and the cell survival was calculated. The CCK8 kit was also used to evaluate the cytotoxicity of β-mangostin-loaded NPs coated with different cell membranes on the C6 cells. The C6 cells were seeded in 96-well plates at a density of 1.0 × 104 cells per well and cultured for 24 h. Then, the NP solutions (NPs, PNPs, CNPs, PCNPs) loaded with different concentrations of β-mangostin (5, 10, 15, 20, and 25 µg/ml) was added to the corresponding pore plates and incubated for 24 h; the pores without samples were considered the control group, and the pores without cells and samples were considered the background group. Cell viability was evaluated as described above.
2.8 In vivo Anti-tumor Performance in Subcutaneous Glioma Model
All animals were raised in a specific pathogen free environment. This project was approved by the Medical Ethics Committee of Zhejiang Provincial People's Hospital. We acknowledge that the project was performed according to international, national, and institutional rules for animal experiments, clinical studies, and biodiversity rights. C6 cell suspension in good condition was collected and used to construct a subcutaneous tumor model in mice. The cell suspension was diluted to 5 × 106 cells/mL according to the volume ratio of the cell suspension : matrix gel = 2:1. Sixty 4-week male BALB/Nude mice (18–22 g) were randomly divided into six groups of 10 mice each. A C6 cell suspension (200 µL) was injected into the subcutaneous tissue of the right back of each nude mouse. When the tumor volume reached approximately 80–100 mm3, the mice were treated with PBS, β-mangostin, β-NPs, β-PNPs, β-CNPs, and β-PCNPs every other day at the same dose (5 mg/kg, 200 µL). Totally, seven doses were administered. The weight and tumor growth of the nude mice were monitored every two days after the first injection. The tumor volume was measured using a Vernier caliper, and the volume was calculated according to the formula V = (length×width2)/2. On the 18th day, the mice were sacrificed for biochemical and pathological analyses and toxicological evaluation. The tumor was stripped, weighed, and photographed.
2.9 In Vivo Imaging of the Animals
We used the Xplore Optix MX animal optical molecular imaging system to image the healthy BALB/C nude mice to obtain the distribution of the NPs in vivo and their accumulation in the brain regions. The imaging system used 580 nm excitation and 692 nm emission wavelengths. To establish the C6 intracranial orthotopic glioblastoma mouse model, the mice were anesthetized using 3.5% chloral hydrate (1.5 mL/100 g body weight). The head (the area to be inoculated) was cleaned with 70% ethanol and then opened with a sterilized scalpel. Five microliters of the C6 cell suspension (1×105 in 10% DMEM) was injected into the striatum at a depth of 3 mm from the dural surface. Twenty days after the orthotropic glioma model was established, the mice were randomly divided into four groups and each nude mouse was injected with 200 µL of DiD-labeled NPs, PNPs, CNPs, and PCNPs via the tail vein. At 24 h after injection, the mice were anesthetized with isoflurane and imaged from the back.
2.10 In Vivo Anti-tumor Performance in Orthotopic Glioma Model
All animal experiments were conducted in accordance with the guidelines approved by the Laboratory Animal Management and Ethics Committee of Zhejiang Provincial People's Hospital. An orthotropic glioma model was constructed according to the above method. Seven days after the model was established, the mice were randomly divided into six groups (PBS, β-mangostin, β-NPs, β-PNPs, β-CNPs, β-PCNPs) and administered the same dose and injection as the mice with the subcutaneous tumor. The overall survival was monitored in all groups. On the 20th day after modeling, one mouse was randomly selected from each group, and the brains and major organs were sampled for H&E staining. The remaining mice in each group were observed until their death.
2.11 Statistical Analysis
All statistical tests were analyzed by GraphPad Prism 8 software, and the comparison between the groups was performed by unpaired t-test; the results of more than three independent repeated experiments were expressed as mean ± SEM. P < 0.05 was the statistical difference between the groups, denoted by *; P < 0.01 was the statistically significant difference, denoted by **; P < 0.001 was the statistically significant difference, denoted by ***.