For protein quantify, BCA protein quantitation reagent kit (Beyotime, P0010S, Shanghai, China) was purchased from Beyotime Institute of Biotechnology, China. For western blotting, the first antibodies against β-actin (PA1-46296, Invitrogen), HA Tag Antibody (26183, Invitrogen), Flag Tag Antibody (PA1-984B, Invitrogen), Her2 Antibody (MA5-13105, Invitrogen), Granzyme B Antibody (MA1-80734, Invitrogen), Perforin Antibody (14-9392-82, Invitrogen) and TNF-α Antibody (ARC3012, Invitrogen) and goat anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody conjugated HRP (G-21234, Invitrogen) were purchased from Thermo Fisher Scientific, Inc. The ECL western-blotting substrate kit (32106, Pierce) and ELISA kits for the cell cultured supernatant immune factors: TNF-α (BMS607-3, Invitrogen) and IFN-γ (BMS606, Invitrogen) were purchased from Thermo Fisher Scientific (China) Co. Ltd., Beijing, China.
Human breast cancer cell lines SK-BR-3 and mouse breast cancer 4T1 cells were all from the American Type Culture Collection (ATCC; USA). Chinese hamster ovary (CHO) cells were obtained from the BeNa Culture Collection (Beijing, China). SK-BR-3, 4T1 and CHO cells were cultured in Dulbecco's Modified Eagle Medium (DMEM; Invitrogen, USA) supplemented with 10% fetal bovine serum (FBS; Beyotime, Shanghai, China) and 100µg/ml penicillin-chain Mycin (Beyotime, Shanghai, China).
BALB/c mice (8-weeks, female) and BALB/c-nu mice (8-weeks, female) were purchased from Changsheng Bioscience Co.inc (Liaoning, China). All experimental animals were maintained and propagated under pathogen-free conditions in the Animal Management Center of Jinan University Laboratory, with temperature of 22°C–25°C, relative humidity of 55% ± 5%, 12 h light/12 h dark cycle, adequate feed, and sterilized drinking water. All animal experiments were completed under the supervision of the Experimental Animal Ethics Committee of Jinan University.
2.3. Preparation of mesoporous silica powders (MSNs)
The MSNs were synthesized using a soft-templating method . A mixture of 1.90 g of cetyl-trimethylammonium tosylate (CTATos, MERK) and 0.35 g of triethanolamine (TEAH3) in 0.1 L of deionized water was stirred at 80°C for 1 h and 14.58 g of tetraethyl-orthosilicate (TEOS) was quickly added into the surfactant solution. The mixture was stirred at 80°C with a stirring speed of 1200 rpm for another 2 h. The synthesized C-MSNs were filtered, washed, and dried in the oven at 100°C for 20 h.
2.4. Construction of GPI-anchored CD80 and anti-Her2 scFv
GPI-CD80 cDNA was constructed by fusing cDNA encoding the extracellular domain of CD80 and the GPI-anchor signal sequence of LFA-3 [29; 30]. The chimeric CD80-LFA3 cDNA was ligated into pCMV6-AN-HA (PS100013, Origene, USA), a mammalian expression vector with N-terminal HA tag. For construction of GPI-anchored anti-Her2 scFv, the amino acid sequence of anti-Her2 scFv was further constructed by the protein sequence published by Carter et al. and which express the single-chain antibody with neutralizing Her2 activity [31; 32]. The chimeric anti-Her2 scFv-LFA3 cDNA was cloned into pCMV6-AN-DDK (PS100014, Origene, USA) a mammalian expression vector with N-terminal DDK tag. Constructed plasmid was transfected into CHO cells with lipofectamine™ 2000 (11668027, Invitrogen, USA).
2.5. Preparation of MSNs-Cell (MSNs-C)
The CHO cell membrane vesicles (CMs) were firstly prepared as previously described [20; 33]. Briefly, after harvesting CHO cells by centrifugation, the cells were washed with PBS and centrifuged, and repeated three times. The obtained cells were resuspended in pre-chilled Tris buffer (10 mM Tris, 10 mM MgCl2 and 1×EDTA free protease inhibitor, pH 7.4), and then the cells were homogenized at 22,000 rpm for 1 min in a cell homogenizer (Bioprep-24, Allsheng Life Science, China). The homogenized solution was centrifuged at 500 g for 10 min at 4°C, then the supernatant was taken, and the supernatant was centrifuged at 10,000 rpm for 10 min and centrifuged at 100,000 g for 1 h (Optima XPN-100, Beckman, USA). The obtained cell membrane precipitate fraction was washed once with 10 mM Tris (pH 7.4) buffer, and the cell membrane fraction was collected by repeated centrifugation, then resuspended, and sonicated using an ultrasonic generator (Qsonica Q700, Qsonica, USA) for 5 sec to homodisperse the cell membrane. Finally, the CM of the CHO cells were obtained by extruding the solution through a 400 nm polycarbonate membrane (LiposoFast, Avestin, Canada).
To prepare MSNs-C , 5 mg of MSN was dispersed into 10 ml of an aqueous solution of DOX (0.5 mg/mL, 6 mL) and stirring was continued for 8 h. 1 mL of the prepared CM (1 mg/mL) was added to 6 mL of the DOX-loaded MSN dispersion solution. The mixture was gently stirred for 3 h, then the mixture was centrifuged and washed to remove excess DOX and other impurities from the supernatant. Finally, the centrifuged product was suspended and stored in PBS buffer. The supernatant was combined and measured at 496 nm of a UV-vis spectrophotometer (UV759CRT-FS, YOKE instrument, China) and the calculated DOX loading capacity was calculated. The DOX loading of MSNs or MSNs-C is expressed as the mass of DOX loaded per gram of MSN or MSNs-C.
2.6. Characterization of MSNs and MSNs-C
The nanosystem was characterized by field-emission scanning electron microscopy (FESEM; Merlin, Zeiss, Germany), high-resolution transmission electron microscopy (HRTEM; JEM-2100F, JEOL, Japan), Fourier transform infra-red spectroscopy (FTIR; EQUINOX55, Bruker, Germany), Raman particle size analysis (LabRAM Arami, HJY, France), N2 adsorption-desorption isotherm (3H-2000BET, BeiShiDe instrument, China), Zeta potential analysis (Zetasizer, Malvern Ltd, UK), and X-ray photoelectron spectroscopy (XPS; Axis Ultra DLD, Kratos, UK).
2.7. Drug release
We evaluated the release of DOX in MSNs and MSNs-C by dialysis. 10 mg of MSNs-C was dispersed in PBS and the solution was transferred to a dialysis bag (Biotech membrane 14KD, Sangon Biotech, China). The dialysis bag was then placed in 30 mL of PBS buffer and stirred with a magnetic stirrer. At different time points, the content of DOX was determined by taking 0.5 mL of PBS from the buffer system, and an equal volume of fresh PBS was added to the buffer system. The cumulative drug release is calculated by the following formula:
Cumulative drug release (%) = (Mt/M0) × 100, Mt represents the amount of drug released from the nanoparticle at time t, and M0 is the amount of drug loaded into the nanoparticle.
2.8. Establishment of 4T1- Her2+ cells
Compared to the SK-BR-3 cell line, this cell continues to express Her2 gene so that the targeting effect of the Her2 gene on the drug cannot be verified alone; alternatively, the 4T1 and 4T1-Her2+ cell line can achieve.
To construct 4T1-Her2+ cells, the DNA sequence of Her2 (extracellular domain-containing signal peptide) containing 652 amino acids was cloned into a pcDNA3.1 vector. The Her2 expression vector (Her2-ORF/pcDNA3.1) or the empty pcDNA3.1 vector was transfected to 4T1 cells using Lipofectamine™ 2000 (11668027, Invitrogen, USA). After G418 selection (600 mg/ml), several stable transformants were established by the cylinder technique. The presence of 4T1-Her2+ cells were analysed by western blot analysis and quantitative real-time polymerase chain reaction (qPCR).
2.9. Colony forming assay
4T1 and 4T1-Her2+ cells were plated respectively at 2×105 cells/ml in 12-well cell culture plate. After culturing for 6 h in cell incubator, the cells were co-cultured with nanoparticles and the activated peripheral blood mononuclear cell (PBMC) for 12 h, then wash 3 times with PBS. Surviving colonies were stained with 0.4% crystal violet (3580948, Sigma, USA) in 50% methanol, and the visible colonies were counted.
The obtained mouse PBMCs were activated according to the method in the article . PBMCs and irradiated tumor cells were co-cultured in 96-well cell culture plates for 72 h in a total volume of 200µL/well Iscove's Modified Dulbecco's Medium (IMDM; Invitrogen, USA). Activated PBMC were co-cultured with 4T1 or 4T1-Her2+ cells, and nanoparticles were added to the wells at different concentration for Colony forming assay. The cell cultured supernatant were collected for Elisa analysis to determine the concentrations of TNF-α and IFN-γ. Secreted cytokines in cell culture supernatants were analyzed using a TNF-α Elisa kit and an IFN-γ Elisa kit following the manufacturer’s instructions.
2.11. Confocal microscopy
CHO cells and SK-RB-3 cells were fixed in PBS buffer containing 4% paraformaldehyde (PFA) for 30 min, then washed three times with PBS buffer, and permeabilized with PBS buffer containing 0.2% Triton X-100, blocked with donkey serum. Anti-Flag antibody (F3165, Sigma, USA) and anti-HA antibody (SAB4300603, Sigma, USA) were used as primary antibodies. Donkey anti-mouse IgG conjugated with Alexa Fluor 488 (R37119, Thermo Fisher, USA) and donkey anti-rabbit IgG conjugated with Alexa Fluor 647 (A-31573, Thermo Fisher, USA) as a secondary antibody. DAPI dye (ab104139, Abeam, USA) was used for nuclear staining. Images were captured under 63× oil objective using a confocal microscope (TCS SP8, Leica, Germany).
2.12. Isolation of immune cells and flow cytometry
The isolations of immune cells from spleen and tumor tissues were processed as previously described [36; 37]. CD8+ T cells were purified by using CD8+ T cell isolation kit (Miltenyi Biotec, Germany) . In the analysis of CD8+ T cell responses, cells were derived from splenocytes and tumor infiltrating lymphocytes of mice treated with nanoparticle drugs. Resuspend the cells in RPMI 1640 medium containing 2 mg/mL nanoparticles (DOX free), culture for 24 h, and with PMA (50 ng/mL), ionomycin (1 mg/mL) and BD Golgi plug (555029, Bioscience, USA) incubate for 6 h. After harvesting the cells, staining with PE-anti-CD8-antibody (ab28017, Abcam, USA) and intracellular APC -anti-IFNGR1-antibody (ab275700, Abcam, USA) the percentage of CD8+ IFN-γ+ cells were analyzed by flow cytometry. Mouse spleen cells and tumor infiltrating lymphocytes were stained in PBS buffer containing 3% FBS at 4°C, then treated with Red Blood Cell Lysis Buffer (00-4333-57, Invitrogen, USA) and incubated with purified Fc-block (CD16/CD32), then staining with anti-CD11b-PE (Fab1124p, R&D systems, USA) and anti-Gr1Percp (Fab1037c, R&D systems, USA).
2.13. Tumor transplantation models and groups
In order to verify the targeted effect of Her2 gene on the distribution of MSNS-C in mice, SK-BR-3 cells, 4T1 cells and 4T1-Her2+ cells were transplanted into BALB/c-nu mice, respectively. Until the tumor volume is about 150mm3, the three groups of mice were injected with MSNs-C, MSNs or Saline, respectively, and the mouse tissue was analyzed by DOX fluorescence imaging 12 hours later. Subsequently, various organs and tumor tissues of the mice were collected, and the expression level of Her2 gene in the mouse tissues was tested by Real time PCR reactions.
In the SK-BR-3 cell tumor xenograft model, 100µL of SK-BR-3 cell suspension (1 × 107 cells) was subcutaneously injected into the right inguinal region of eight-week-old female BALB/c-nu mice. After 7 days, the mice were randomly divided into 4 groups (6 mice in each group), and were treated with 200µl of GPI-modified MSNs-C, unmodified MSNs-C and MSN nanoparticle solutions or normal saline at regular intervals. The size of the tumor was measured with a vernier caliper every other day to observe the growth of the tumor, and the tumor volume (V) was calculated by the formula: V = 1/2 × large diameter × (small diameter) 2. After 29 days, the animals were euthanized, and the tumors were excised and weighed, photographed, and stored in liquid nitrogen for subsequent analysis.
4T1-Her2+ cells were transplanted into BALB/c mice in the same manner as described above. After 6 days, the mice were randomly divided into 7 groups (6 mice/group). Three groups were treated with 1 mg/Kg, 5 mg/Kg or 10mg/Kg doses of 200µl nanoparticle solution to verify the effect of different nanoparticle concentrations on tumor growth. The other 4 groups were treated by intravenous injection of 200µl 5mg/kg nanoparticle solution or normal saline to verify the effects of different nanoparticles on the immune function of mice. After 29 days, tumor cells were isolated and incubated with MSNs-C (10µg/well) for 24 hours, and then restimulated with PMA and ionomycin for 6 hours. CD8+ T cells were sorted by magnetic beads, and intracellular IFN-γ was detected by flow cytometry.
2.14. Histological analysis
Morphologic analyses were carried out on 4% paraformaldehyde-fixed for 24–48 h before being processed and embedded into paraffin wax. Deparaffinized, rehydrated sections were immunolabeled with anti-Ki67 antibody (ab15580, Abcam, USA). The antigen antibody was visualized using 3,3-diaminobenzidine. The sections were then dehydrated, washed (with xylene) and fixed (with resin). In every tumor section, the percentage of Ki67 positive cells in three different regions was counted.
2.15. Tunel assay
Apoptotic DNA fragments in cells were visualized according to the Click-iT™ TUNEL Kit (C10625, Invitrogen, USA). Cells marked with a brown nucleus were defined as TUNEL positive cells. The percentage of TUNEL positive cells in each region was estimated after averaging the values of three different regions.
2.16. RNA isolation and Real time PCR analysis
Total RNA from cells and tissues was isolated using TRIzol reagent (15596026, Invitrogen, USA) following manufacturer instructions, and total RNA was reverse-transcribed into cDNA using SuperScript III RT Kit (18080044, Invitrogen, USA). Real time PCR reactions were performed using specific primers to measure the mRNA abundance of genes. The data shown in the figure is the relative abundance of the indicated mRNAs, which are normalized to Actb and HPRT, respectively. Specific primer sequences for genes are derived from published articles. HER2 forward primer (TGCAGGGAAACCTGGAACTC) and reverse primer (ACAGGGGTGGTATTGTT CAGC), mouse Interferon gama (IFN-γ) forward primer (ATGAACGCTACACACTGCATC) and reverse primer (CCATCCTTTTGCCAGTTCCTC), mouse perforin 1 forward primer (AGCACAAGTTCGTGCCAGG) and reverse primer (GCGTCTCTC ATTAGGGAGTTTTT), mouse Granzyme B forward primer (CCACTCTCGACCCT ACATGG) and reverse primer (GGCCCCCAAAGTGACATTTATT), mouse Tumor necrosis factor alpha(TNF-α) forward primer (CCCTCACACTCAGATCATCTTCT) and reverse primer (GCTACGACGTGGGCTACAG), human HPRT forward primer (CCTGGCGTCGTGATTAGTGAT) and reverse primer (AGACGTTCAGTCCTGTC CATAA), mouse HPRT forward primer (TCAGTCAACGGGGGACATAAA) and reverse primer (GGGGCT GTACTGCTTAACCAG) were synthesized by Sangon Biotech (Guangzhou, China). The relative mRNA levels of multiple cytokine genes in each sample were displayed as 2−ΔΔCt values and were representative of at least three independent experiments.
2.17. Western blot analysis
Tissues were lysed in RIPA buffer, and total protein was extracted. Then the protein concentration was determined using BCA assay and equalized before loading. Protein samples were separated by SDS-PAGE gels, transferred to polyvinylidene fluoride membranes (Millipore, Bedford, USA), and blocked with 5% non-fat dry milk (Sigma, Germany) in TBST for 2 hours at room temperature followed by incubation with primary antibody (1:1000 dilution) at 4°C overnight. After washing 3 times with TBST, the PVDF membrane was incubated with peroxidase-conjugated secondary antibody for 1 hour at room temperature, and then washed 3 times with TBST again. After fully wetting the PVDF membrane with ECL Western Blotting Substrate Kit for 1 minute, immunolabelling was detected using Tanon 5200 Chemiluminescent Imaging System.
2.18. Statistical analysis
The data are expressed as the average ± standard deviation. All experiments were repeated three or more times, and statistical methods were used to compare the data between the analysis of each group using an unpaired two-tailed Student's t test to perform statistical difference analysis. Comparison of multiple groups using one-way analysis of variance (ANOVA), followed by LSD test were performed for comparison between groups. Analysis and graphs of all data were performed using GraphPad Prism software (version 6.0) and the data were expressed as mean ± standard deviation. Statistical significance was set to p < 0.05.