Materials
Borocaptate Sodium (BSH) was purchased from Katchem Co., Ltd. (Prague, Czechoslovakia). Doxorubicin hydrochloride (DOX-HCl) and ixazeomib was purchased from Dalian Meilun Biotechnology Co., Ltd. (Dalian, China). iRGD (CRGDKGPDC) were customized from GL Biochem Co., Ltd. (Shanghai, China). Plasmid was customized from GenePharma Co., Ltd. (Shanghai, China), and its sequence is showed in Additional file 1: Fig. S22. 1-Bromomethyl-o-carborane (CB) was purchased from YL Biochem Co., Ltd. (Zhengzhou, China). 1,2-Dioleoyl-3-trimethylammonium-propane chloride (DOTAP), 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), N-(carbonyl-methoxypolyethyleneglycol 5000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG5000) was purchased from CordenPharma Co., Ltd. (Switzerland). Lipo 2000 from Life Technologies was purchased from Thermo Fisher Scientific Co., Ltd. (Shanghai, China). DPEC water was purchased from TianGen Biochem Co., Ltd. (Beijing, China). Polyethyleneimine (PEI, MW 25KDa) was purchased from Sigma-Aldrich (USA). DAPI, Lyso-Tracker Red, Firefly Luciferase Reporter Gene Assay Kit and BCA Protein Assay Kit were purchased from Beyotime Co., Ltd. (Shanghai, China). APC anti-mouse CD47 antibody (MIAP301, Santa Cruz, USA). EasyPure® HiPure PlasmidmaxiPrep Kit was purchased from TransGen Biotechnology Co., Ltd. (Beijing, China). Nuclear Extraction Kit was purchased from keygene Biochem Co., Ltd. (Nanjing, China). The NT50 D-T neutrongenerator for BNCT was kindly kindly provided by Shiwei Jing’s lab, School of Physics, Northeast Normal University. All other materials and solvents were of reagent grade and used as received.
Synthesis of DOX-CB
DOX-HCl (300 mg, 0.5172 mmol) and triethylamine (TEA, 215 μL, 1.5517 mmol) were completely dissolved with 10 mL anhydrous DMSO and then kept stirring in vacuum for 2 h at room temperature. DOX was obtained by alkalization. Weigh 0.5172 mmol of CB and put it into the above reaction system, stir it for 48 h at room temperature. The mixture was dialyzed (the MWCO of dialysis bag was 500) for 36 h in DMSO until the dialysate became clear, then freeze-dried to obtain the solid powder of DOX-CB.
Preparation of DOX-CB@lipo, lipo-pDNA and DOX-CB@lipo-pDNA
The plasmid was was extracted and purified by EasyPure®hiPure PlasmidmaxiPrep Kit under the guidance of the instructions. Liposomes comprising DOTAP, DOPE, CHOL and DSPE-PEG5000 in the molar ratio of 0.8:1:0.5:0.023 were prepared using the lipid film hydration method; 30 mg of all lipids were mixed with 50 mL chloroform. The lipid film was prepared in a rotary evaporator. The lipid film was then hydrated with 5 mL 5% sucrose solution (containing DEPC) for 30 min at 50 °C under shaking. The resulting liposomes were sonicated in a bath-type sonicator for 30 min and then prepared by the probe supersonic method at ice water bath (3 min, 30% power). Weigh the 3 mg DOX-CB and 30 mg of all lipids mentioned above, fully dissolved with 50 mL chloroform and repeat the procedure of liposome synthesis above. Then the DOX-CB@lipo was obtained.
The plasmid was diluted with DEPC water, and mixed with the empty cationic liposome solution (containing DEPC) in equal volume. After continuous blowing for 1 min and standing at room temperature for 20 min, the cationic liposome-plasmid complex (lipo-pDNA) was formed. DOX-CB@lipo-pDNA was obtained in this method analogously.
Preparation of complex of nanoliposome-peptide
The iRGD was accurately weighed and prepared into solution, then mixed with the prepared nanoliposome solution, and stand at room temperature for 30 min. The complex of nanoliposome-peptide (lipo-iRGD, lipo-pDNA-iRGD, DOX-CB@lipo-iRGD, DOX-CB@lipo-pDNA-iRGD) can be obtained. The molar ratio of iRGD and nanoliposome is 5%.
Characterization of multifunctional nanoliposomes
According to the above procedures, lipo-pDNA with different lipo/pDNA(/N/P) ratios (0, 1, 2, 3, 4, 5, 10, 20, 30, 40 and 50) were prepared. The particle size, zeta, PDI and other characteristic parameters of each sample were measured by DLS (Zetasizer90, Malvern Instruments Ltd). Each sample were measured three times in parallel.
Liposome, DOX-CB@lipo, DOX-CB@lipo-iRGD, DOX-CB@lipo-pDNA(N/P = 2), DOX-CB@lipo-pDNA-iRGD (N/P = 2) were also characterized according to the above method. The appearance characteristic was observed by TEM (H-7650, Hitachi Corporation).
Cell lines
The cancer cell lines GL261 was purchased from Beina Chuanglian Biotechnology Institute (Beijing, China) and the luciferase transfected GL261 (Luc-GL261) were cultured in complete growth media (DMEM medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin) at 37 °C in a humid atmosphere maintained of 5% CO2.
Cell viability assay
GL261 Cells were seeded in 96-well plates with a concentration of 3.0 × 103 cells/well, respectively. After 24 h hatch in cell culture incubator, different polymeric formulations (lipo-pDNA, lipo-pDNA-iRGD, DOX-CB@lipo-pDNA, DOX-CB@lipo-pDNA-iRGD with different N/P ratio 0.4, 1, 1.6, 2, 4, 10, 20, 40) were added into each well in various concentrations for 48 h incubation followed by Cell Counting Kit-8 (CCK8, Beyotime, Shanghai, China). And cell viability was calculated as percentages by compared with control (lipo2000 with plasmid with different N/P ratio 2, 3, 4, 5 referring to the Life Technologies). The optical density at 450 nm (OD450 nm) was measured using a multiwell plate reader (ELX800, Biotek, USA). Each well was repeated at least 6 times, and cell proliferation was presented as mean ± SD.
Gene recombination assay
A series of lipo-pDNA (N/P ratio 1, 1.6, 2, 4) and lipo2000-pDNA (2) were prepared according to the above experimental procedures. Plasmid solution (100 μg/mL) and PEI solution (130 μg/mL) were mixed by equal volume to obtain the PEI-pDNA complex.
GL261 Cells were seeded in 24-well plates with a concentration of 5.0 × 104 cells/well, respectively. After 18 h hatch in cell culture incubator, PEI-pDNA, lipo2000-pDNA (2), lipo-pDNA (N/P = 2) were added into each well for 6 h incubation. Then incubate for 48 h in fresh growth media and the expression of GFP was observed under inverted fluorescence microscope.
Western blot analysis
To examine the gene transfection efficiency of different preparations, GL261 Cells were seeded in 6-well plates with a concentration of 1.0 × 105 cells/well and incubated for 18 h. 160 μL naked pDNA, lipo2000-pDNA (2), lipo-pDNA (N/P = 2) and lipo-pDNA (N/P = 4) of an equivalent pDNA dose of 2.5 μg/μL with 2 mL serum-free medium were added into each well for 6 h incubation. Fresh serum containing complete culture medium was used to continue the culture for 72 h. Then we collected GL261 cells and obtained cellular proteins using T-PER Tissue Protein Extraction Reagent (Thermo Pierce). The total protein concentration was then quantified by a BCA protein assay kit. Western blot tests were processed in the standard fashion, and analyzed quantitatively protein expressions by the ImageJ software. The following antibodies were used in the procedure: CD47 (ab175388, Abcam), GAPDH (ab181602, Abcam), and goat anti-rabbit IgG (H + L) secondary antibody (31210, Thermo Pierce).
Determination of gene transfection efficiency
GL261 Cells were seeded in 6-well plates with a concentration of 5.0 × 104 cells/well and incubated for 18 h. Then washed by PBS three times and then coincubated for another 6 h, respectively, with 40 μL lipo2000-pDNA (2), lipo-pDNA (N/P = 2), lipo-pDNA (N/P = 4) of an equivalent pDNA dose of 2.5 μg/μL and 500 μL serum-free medium. Fresh serum containing complete culture medium was used to continue the culture for 24 h. In the experiment, pure culture medium blank control group and plasmid incubation control group were set.
After transfection, the cells were washed by PBS for 3 times, then 200 μL cell lysis solution was added in each well and shaked for 40 min at room temperature. After carefully blowing the lysate repeatedly, the liquid was centrifuged at 4 °C for 20 min, and the rotating speed was 14,000 rpm. The supernatant was moved into the new EP tube. Add the equal volume of Firefly Luciferase Reporter Gene Assay Kit into EP tube, mix evenly and determined the relative light unit (RLU) by the chemiluminescence instrument (E6080, Promega, USA). In the same time, BCA Protein Assay Kit was used to determine the corresponding protein concentration. The relative light intensity per mg protein (RLU per mg protein) was used to evaluate the transfection efficiency of each sample.
Flow cytometry assay
To detect the expression of CD47 after transfection, GL261 Cells were seeded in 6-well plates with a concentration of 1.0 × 105 cells/well and incubated for 18 h. 160 μL naked pDNA, lipo2000-pDNA (2), lipo-pDNA (N/P = 2) and lipo-pDNA (N/P = 4) of an equivalent pDNA dose of 2.5 μg/μL with 2 mL serum-free medium were added into each well for 6 h incubation. Compared with control (without polymers incubation and with only plasmid incubation). Then incubate for 72 h in fresh growth media. The experiments were repeated 3 times.
After digestion and centrifugation, the cells were incubated with anti-mouse CD16/32 on ice for 10 min, washed with PBS for 3 times, and incubated with APC anti-mouse CD47 antibody on ice for 30 min, then washed with PBS for 3 times. The expression of CD47 in each sample was detected by flow cytometry.
Cellular uptake and distribution
To determine the cellular uptake of DOX, GL261 cells were seeded into glass bottom dishes (20 mm, Nest) at a cell density of 1.0 × 104 cells/dish, incubated for 24 h, washed by PBS and then coincubated for another 6 h, respectively, with free DOX and DOX-CB of an equivalent DOX dose of 3.5 μM. After washing with cold PBS for three times, the cells were fixed in 4% paraformaldehyde, incubated with DAPI for 10 min, and rinsed with PBS for three times. The DAPI and intracellular DOX were intuitively observed by laser scanning confocal microscope (LSCM, Zeiss LSM 800, Germany). The fluorescence channels for DOX were kept at the same laser intensity so as to quantify cellular uptake of DOX by an US National Institutes of Health-ImageJ software. For cellular distribution of different preparations, the cells were seeded into dishes, and incubated with free DOX (1 μg/mL), DOX + CB, DOX-CB, DOX-CB@lipo, DOX-CB@lipo-pDNA-iRGD of the equivalent DOX dose, treating with or without ixazeomib for 2, 4, and 8 h. Samples were then fixed, stained with DAPI, and visualized by LSCM eventually.
Determination of boron content ratio in nucleus and cytoplasm
The tumor cells GL261 in logarithmic growth phase were evenly seeded in a 150 mm diameter cell culture dishes, incubated for 16 h, washed by PBS and then coincubated for another 3 h, respectively, with free DOX, free CB, DOX + CB, DOX-CB@lipo, DOX-CB@lipo-pDNA-iRGD of the equivalent boron dose of 3.5 μg/mL. After washing with cold PBS for three times, the cells were counted and collected, at the same time part of the samples were handled with the Nuclear Extraction Kit at low temperature, and treated with nuclear staining agent. Finally, add an appropriate amount of concentrated nitric acid and hydrogen peroxide mixed solution (V: V = 3:1) to the obtained cells and nuclei, heat them with wet method at 70 °C, and digest them for more than 24 h. The boron concentration was determined by ICP-MS and the ratio of boron content in nucleus and cytoplasm was calculated.
Phagocytic assay
GL261-GFP was transfected with lipo2000-pDNA (2) and lipo-pDNA (N/P = 2) as the previous procedure and collected. In the experiment, pure culture medium blank control group and plasmid incubation control group were set. The primary mouse BMDMs were extracted from C57BL/6 mice using the previous method [53, 54] and then resuspended in complete culture medium and evenly inoculated in confocal dish. After 5–7 days of stimulation and differentiation, they were replaced with fresh culture medium. The cells were fixed by 4% paraformaldehyde for 20 min, handled with 0.25% Triton X-10 for 20 min, then blocked with goat serum for 25 min. The samples were stained with primary antibodies CD47 (ab175388, Abcam) conjugated to F4/80 in 1:30 dilution. Secondary antibody goat anti-mouse IgG (ab31160, Abcam) was utilized in 1:200 dilution and stained with DAPI before observation. The fluorescence images were taken by inverted confocal microscope to observe the phagocytosis of macrophages to tumor cells.
Mice and animal models
C57BL/6 mice (male, 5-week-old) purchased from Slaccas (Shanghai, China) were adaptive fed for more than 1 week for subsequent experiments. The animals were maintained under standard housing conditions and all related experiments were conducted followed by the guidelines which has been evaluated and approved by the Ethics Committee of Zhejiang University.
Orthotopic Glioma Mice Model: To establish the glioma model, 5 week-old male C57BL/6 mice (about 20 g) were anesthetized by intraperitoneal injection of 0.08 mL/10 g 1% pentobarbital solution. After the mice were fixed and the surgical area was exposed, 3 × 105 cells/5 μL Luc-GL261 cells were trypsinized and resuspended in sterile PBS were slowly injected into the left corpus striatum (2 mm lateral, 1 mm posterior to the bregma, and 3.0 mm in depth) by a stereotactic fixation device using a mouse adaptor. Six days later, the orthotopic glioma mice model were imaged and evaluated by IVIS spectrum (PerkinElmer, USA) after 5 min postintraperitoneal injection of d-luciferin (150 mg∙kg−1, Gold Biotechnology, USA). The MRI (4.7 T/30 cm Bruker Biospec scanner, Germany) was observed too. The T/N ratios and BNCT experiments were executed in the orthopotic glioma mice model. The T/N ratios were the ratios of the boron concentration in the tumor to that in the surrounding normal tissue measured by ICP-MS.
In vivo anti tumor effect of BNCT combined immunochemotherapy
The orthotopic glioma mice model were randomly divided into nine groups: (1) DOX-CB@lipo-iRGD-in situ-N (+): On day 6 to day 8, the mice were treated with DOX-CB@lipo-iRGD by intratumoral injection, after the injection for about 24 h, all the mice were irradiated with the thermal neutron beam for 2 h at the neutron irradiator (The neutron generator ion source voltage is 1879 V, the ion source current is 0.208 mA, the accelerator voltage is 90 kV, and the thermal neutron yield is about 1.58% × 108/s). (2) DOX-CB@lipo-pDNA-iRGD-in situ-N (+): On day 6 to day 8, the mice were treated with DOX-CB@lipo-pDNA-iRGD by intratumoral injection, after the injection for about 24 h, all the mice were irradiated with the thermal neutron beam for 2 h at the neutron irradiator. On the day 13, day 16, day 19, the mice were in situ injected with 10 μL lipo-pDNA-iRGD. (3) DOX-CB@lipo-iRGD-iv-N (+): On day 6 to day 8, the mice were treated with DOX-CB@lipo-iRGD by intravenous injection, after the injection for about 24 h, all the mice were irradiated with the thermal neutron beam for 2 h at the neutron irradiator. (4) lipo-pDNA-iRGD-in situ-N (+): On day 6 to day 8, the mice were treated with lipo-pDNA-iRGD by intratumoral injection, after the injection for about 24 h, all the mice were irradiated with the thermal neutron beam for 2 h at the neutron irradiator. On the day 13, day 16, day 19, the mice was in situ injected with 10 μL lipo-pDNA-iRGD. (5) DOX + CB@lipo-iRGD-iv-N (+): On day 6 to day 8, the mice were treated with DOX + CB@lipo-iRGD by intravenous injection, after the injection for about 24 h, all the mice were irradiated with the thermal neutron beam for 2 h at the neutron irradiator. (6) BSH-N (+): On day 9, the mice were treated with 100 μL BSH by intravenous injection, after the injection for about 2 h, all the mice were irradiated with the thermal neutron beam for 2 h at the neutron irradiator. (7) N (+): On day 9, all the mice were irradiated with the thermal neutron beam for 2 h at the neutron irradiator. (8) N (−): Blank control group was made after modeling, without irradiation. (9) sham: 5 μL PBS intracranial injection was used as the sham operation control group, without irradiation. The concentrations of DOX-CB, DOX + CB was 0.3 mg/mL, the concentrations of BSH was 20 mg/mL, the concentrations of plasmid was 100 μg/mL, and the ratio of N/P was 2. The survival curve and average weight change curve of each experimental group were recorded and drawn.
Histopathological evaluation
For histological analysis, the brain, heart, liver, spleen, lung, and kidney samples of the mice model in the different groups were fixed in 4% paraformaldehyde for 24 h and washed twice with PBS. Paraffin-embedded tissue sections were stained with H&E and observed on a microscope. The brain tumors were also detected by immunohistochemistry.
Statistics
Statistical analysis was performed using GraphPad Prism 8 (GraphPad Software, CA, USA). Data were presented as means ± S.D. Statistical evaluation of differences between experimental groups was performed by one-way ANOVA followed by Tukey’s multiple comparisons test. Statistical significance was considered at least at p < 0.05.