Materials
1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 2-Dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000), SM-102 were purchased from Shochem Biotech (Shanghai, CHN), 1,2-Dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000- folate (Folate-DMG-PEG2000, FA) was purchased from Xi’an ruixi Biological (Xian CHN). Cholesterol was purchased from MilliporeSigma. Negative control (miR-NC), twa-miR152-5p (mimics), and has-miR-155-5p (mimics) were synthesized from the Gemma gene. PsiCHECK-2 and psiCHECK-2-3’-mRNA were synthesized from Sangon Biotech (Shanghai, CHN). Coumarin 6 (C6) and Cytoxan (CTX) were purchased from Aladdin (CHN). Doxorubicin (DOX) was purchased from Solarbio Science & Technology (Beijing, CHN).
Animal and cell lines
We adopt 4T1, B16, THP-1, HEK293T, and NIH3T3 cells in this study(Cell Resource Centre, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences). Animal experiments were performed using female BALB/c mice, 6-8 weeks of age, weighing 20 ± 2 g. (SPF Beijing Biotechnology, CHN). Mice were fed with standard chow for one week at 25°. Culturing was performed at 37°C with 5% CO2 in either RPMI 1640 or high-glucose DMEM medium. The culture media were enriched with 10% fetal bovine serum, accompanied by 100 U/mL penicillin and streptomycin. (Gibco, St. Louis, Missouri, USA). The animal experiments were conducted in accordance with the National Regulations for the Care and Use of Laboratory Animals in China and the National Research Council's Guide for the Care and Use of Laboratory Animals. It was approved by the Professional Committee of Animal Experimentation of Peking Union Medical College (Approval No. SLXD-20230425013).
Bioinformatic analysis
Prediction of target sites between mRNA and miRNAs. We used four classic tools to predict potential target sites of mRNA and miRNA, including Targetscan v8.0[47], RNAhybrid v2.1.2[48], miRanda v1.9-i686[49] (http://www.microrna.org/microrna/home.do), and PITA[50]. Except for miRanda, the parameters were set to default for the predictions. For miRanda, the energy threshold was set to -20.0 kcal/mol and the score threshold was set to 180. We integrated the results from these four tools and then took the intersection. Ultimately, we predicted a total of target sites between the 3′UTR of Genes in the cytotoxic T-lymphocyte protein 4 (Untranslated Regions) and twa-miR152-5p(miRNAs).
Dual-luciferase reporter assay
Vector sequences were synthesized by Sangon Biotech (Shanghai, CHN). The 3'UTRs of CTLA-4’s mRNA was cloned into the Not I and Xho I sites of the psiCHECK-2 vectors (Promega. USA). All constructs underwent sequencing verification. HEK293T cells in 96-well plates were transiently transfected with miR-152 mimics or scrambled negative control (miR-NC) using Lipofectamine 3000 (Invitrogen, USA). After 24 hours of incubation, 100 ng of luciferase vector psiCHECK2 or psiCHECK2-3'-mUTR was transfected into cells using Lipofectamine 3000 (Invitrogen, USA). After 24 hours of transfection, firefly luciferase and renilla luciferase were added sequentially to obtain fluc and rluc readings, and normalization was carried out (Solarbio Science & Technology)[51].
The activity of miR152 mimics in vitro
The logarithmically grown 4T1 or B16 cells were inoculated in 96-well culture plates with the concentration of 1.0 × 104 cells per well. After incubation for 24 hours, miR152 was transfected by Lipofectamine 3000 (Invitrogen). Suspension cells THP-1 were inoculated with 3 ~ 4 × 104 per well, and miR-155 was also transfected by Lipofectamine 3000 (Invitrogen). The final concentration of nucleic acid transfection was set at 0, 10, 50, 100, 200, 300, 400, 500, and 1000 nM, respectively, and all of them were set in a blank control group (acellular pore). Three parallel wells in each group were incubated for 48 hours. The adherent cells were replaced with a medium containing 10 % CCK8 (Solarbio Science & Technology), and the suspended cells were added with 10 % CCK8 solution, and incubated at 37°C for 2 ~ 4 hours. The absorbance value was measured using a microplate reader at the wavelength of 450 nm.
Preparation of LNP
The lipids DSPC, DMG-PEG2000, SM-102, Folate-DMG-PEG2000, and Cholesterol were dissolved in anhydrous ethanol according to the ratio (Table S2, the total concentration of lipids was 5 mg/mL), and miR152 mimics or miR-NC (6 μM) was dissolved in citrate buffer at pH 4.5 with a concentration of 0.1 M. LNP@DIR was dissolved in aqueous phase buffer with DIR (1 mg/mL) instead of miR, respectively. The preparation was carried out using a microfluidic system (NanoAssemblr, CHN) with a total flow rate (TFR) of 15 mL/min and a flow rate ratio (FRR) of 3: 1. Mature LNPs were also dialyzed and stirred in a PBS solution with pH 7.4 of 4 ℃ for at least 2 hours.
Particle Size and Zeta Potential
The particle size, zeta potential, and polydispersity indices (PDIs) of LNPs were assessed at room temperature through Dynamic Light Scattering (DLS) using Zeta Sizer Nano ZS (Malvern Instruments, UK). Each LNP sample underwent three consecutive measurements.
Storage stability of LNP
After LNP is prepared and mature, it is stored at 4 ℃, and its particle size and PDI changes on days 0, 2, 5, and 9 are monitored by Dynamic Light Scattering, parallel for 3 times (n = 3).
Cryogenic electron microscopy (Cryo-EM)
Lloyd's violet powder was prepared into 5mM working solution 0.22 filter membrane filtration, 10 % added to LNP, (i.e., 100 μL dye + 900 μL LNP) incubated at room temperature for 30 min, and the sample was prepared on the machine[52]. To prepare cryoEM grids, 2.5 μL of sample was applied to a Quantifoil 200 mesh grid, which was subsequently vitrified at 8°C using a Vitrobot Mark IV (ThermoFisher Scientific) immersed in liquid ethane. The image was acquired on a Talos Arctica using the EPU software with an accelerating voltage of 200 kV. The instrument is equipped with a 16-megapixel CCD camera using a nominal magnification of 59,000.
Encapsulation efficiency (EE) and drug loading (DL)
MiR-152 levels were determined utilizing the QubitTM 4 Fluorometer (Invitrogen, USA) and the Qubit miR Assay Kit (Invitrogen, USA). The excitation wavelength is 545nm, emission wavelength is 595nm. Adding the LNP suspension encapsulating miRNA into 10-fold volume of DMSO to destroy the LNP and release miRNA in LNP for quntifying the content of miRNA. Encapsulation efficiency (EE) and drug loading (DL) were computed using the following equations.
In vitro cell safety experiments of LNP
1.0 × 104 logarithmically proliferated 4T1 cells or NIH3T3 cells were cultured into per well of 96-well culture plates, respectively. Following a 24-hour incubation period, the culture medium containing the LNP vector (at concentrations of 0, 18.8, 37.5, 50, 75, 375, and 750 μg/mL) was replaced. Additionally, a blank control group (acellular pore) was established. Each group consisted of four replicates. After 24 or 48 hours of incubation, 10 μL of CCK-8 solution was added and the absorbance value was recorded at 450 nm after another 4 hours.
In vitro hemolysis
4 mL of whole blood was collected from SD rats by abdominal aortic sampling and added to 8 mL of normal saline. 3000 g centrifugation was performed for 5 min to separate the erythrocytes from the whole blood. Then the supernatant was washed with saline to show a transparent and colorless state, and 1 mL of the lower layer of erythrocytes was added to 50 mL of saline and mixed homogeneously to obtain a 2 % erythrocyte suspension. To determine the positive and negative controls, take 1 mL of erythrocyte suspension and mix it with 1 mL of double-distilled water or saline. Add it to 1 mL of gradient concentration nanoparticle solution as experimental group respectively. The concentrations of the experimental groups were 1000, 500, 250, 125, 62.5, 31.25, and 15.625 μg/mL, respectively. Following vortexing, each set of solutions underwent a 1-hour incubation at 37 °C on a shaker, followed by centrifugation. Subsequently, 100 μL of the supernatant was transferred to a 96-well plate, and the absorbance of hemoglobin at 570 nm was measured with 540 nm as the reference wavelength.
In vitro antitumor assay
Logarithmically proliferated 4T1 or B16 cells were harvested and seeded into 96-well culture plates at a density of 1.0 × 104 cells per well. After incubation for 24 hours, the solution containing LNP@miR152 or LNP@miR-NC (miR experimental concentration was 200 nM) was replaced, and the next day was replaced with a gradient concentration of the chemotherapeutic drug DOX or CTX. where the gradient concentration of CTX was 0, 1, 10, 15, 20 mM, and the gradient concentration of DOX was 0, 0.05, 0.1, 0.2, 0.5 μg/mL[53]. A blank control group was also set up (acellular pore). 4 parallel wells in each group were incubated for 48 hours. The blank medium was replaced and each well was incubated with 10 μL of CCK8 solution for 2 ~ 4 hours. The absorbance values were measured at 450 nm using an enzyme meter.
The in vitro antitumor activity of the combination at DOX of 0.5 μg/mL was observed individually. Inoculate 4T1 or B16 cells in 96-well culture plates at 1.0 × 104 cells per well. After the cells were cultured for 24 hours until they were completely attached to the wall, they were replaced with the solution containing LNP@miR-NC, LNP@miR152, DOX, LNP@miR152/DOX (miR was 200 nM), and six blank control groups were set up in each group, and then incubated for 48 hours. The activity of the cells was detected by CCK8.
In vitro cellular uptake and lysosome escape
LNP@C6 was prepared using coumarin 6 (C6) as an aqueous phase model drug, in which 5 mg/mL of lipid percentage DSPC: DMG-PEG 2000: SM-102: Folate-DMG-PEG2000: Cholesterol was 10: 3: 40: 2: 50, and C6 was dissolved in 125 μg/mL of citrate buffer as previously mentioned LNP@C6 was prepared. Logarithmic growth phase 4T1 or NIH3T3 cells were inoculated in 48-well plates at a concentration of 1.3 × 105 cells/mL. After the cells were cultured for 24 hours until they were completely attached to the wall, they were replaced with 200 μL of fresh medium, and 20 μL of LNP@C6 solution was added to each well, respectively (where the experimental concentration of C6 was determined by HPLC as 2.9 μg/mL). The spots were taken at 6, 4, 2, and 1 hours for lysosome-tracker-red staining for 30 min according to the concentration suggested in the instruction manual, PBS washed once, 2 μg/mL DAPI staining for 10 min, PBS washed again, and the cells were fixed with 4 % paraformaldehyde, and observed under the fluorescence microscope.
In vivo distribution
Animal model construction was performed using female BALB/c mice, 7 weeks of age. 100 μL of 4T1 cell suspension (3 × 106 cells/mL) was inoculated under the left breast pad. After the tumor size reached 300 mm3, three groups of mice were randomly assigned, each including five mice. These groups were then treated with 200 μL of either free DIR or LNP@DIR (20 μg of DIR per mice) through tail vein injection. After 48 hours, the mice were euthanized and their hearts, livers, spleens, lungs, kidneys, and tumors were dissected and collected. The drug distribution in vivo was visualized using a small animal optical imaging system. (Hopkinton, MA, USA). The distribution of the drug in the body was calculated from the fluorescence intensity values.
Quantification of the miRNA/mRNA expression levels by qPCR
miRNA quantification - reverse transcription was performed using MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) according to the manufacturer's protocol. mRNA quantification - reverse transcription was performed using HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Nanjing Vazyme Biotech, CHN) according to the manufacturer's protocol. To analyze gene expression, quantitative PCR analyses were conducted using cDNAs extracted from cellular samples, along with optimized primer pairs targeting CTLA-4. (forward, 5′-GTGCCACGACATTCACAGAG-3′; reverse, 5′-CGTCCCGTTGCCCATG-3′); IL-10 (forward, 5′-ACTCCTTGGAAAACCTCG-3′; reverse, 5′-AAGAACCCCTCCCATCAT-3′). Relative gene expression of the target gene was calculated by ∆∆Ct method using a reference housekeeping gene ACTIN (forward, 5′- CCATCTACGAGGGCTATGCT-3′; reverse, 5′CTTTGATGTCACGCACGATT-3′). Twa-miR152-5p mimics (forward, 5′- TTATTTGTTTGGACATTTGG-3′; reverse, 5′- GGCCAACCGCGAGAAGATG-3′). Relative expression of miRNAs was calculated by ∆∆Ct method using a reference housekeeping miRNA U6 (forward, 5′-CTGCGCAAGGATGACACGCAAATT-3’; reverse, 5′-AACGCTTCACGAATTTGCGT-3′). Experiments were repeated three times.
In vivo antitumor assay
100 μL of 4T1 cell suspension (3 × 106 cells/mL) was inoculated under the left breast pad of BALB/c mice, and the tumor size was observed until 50-90 mm3 ( approximately 10 d). Then 72 mice were randomly divided into 6 groups[54]. Mice were injected tail vein using saline, miR152, LNP@miR152, DOX, and LNP@miR152/DOX at a dose of 2 mg/kg (doxorubicin-based) or 13.5 nmol/kg (based on miR152)[40] per mice every day. Tumor size was calculated by measuring the longest diameter of the tumor (a) and the perpendicular transverse diameter of the tumor (b) using vernier calipers every two days after drug administration. The volume of the displaced tumor was then calculated using the formula V = 1/2 × ab2, and a tumor growth curve was generated. The mice were administered continuously for 14 days, and orbital blood was taken and euthanized 24 hours after the last administration. Serum was centrifuged, hearts, livers, spleens, lungs, kidneys, and tumors were taken, tumor sizes were determined, immersed in tissue fixative for immunophenotyping, and paraffin-embedded sections (3 - 5 μm) were made 24 hours later. Then, the tissues were soaked for 24 hours using tissue fixative, paraffin-embedded sections (3 - 5 μm) were made and colored with hematoxylin-eosin (H&E). Tumor sections were colored based on the recommended doses in TUNEL, immunohistochemistry (IHC) and immunofluorescence (IF). Then observing using a fluorescence microscope (PSC603-076).
Flow cytometry evaluation (FCM)
Fresh tumor tissue was taken as 50 mg, mechanically separated to about 1 mm3, and lysed with 1 mg/mL Collagenase II and 0.5 mg/mL DNase in a water bath at 37 ℃. Cells were filtered through 100 μm nylon membranes, and single-cell suspensions were washed three times with PBS. To obtain the cells for analysis, 500-600 μL of blood was collected from the mouse eyeballs and placed in an anticoagulation tube. The tube was rotated to prevent blood clumping, and erythrocyte lysate was added in a 10:1 ratio. After centrifugation (5 min,1,000 r/min), the supernatant was discarded, and the procedure was repeated once. The precipitate was aspirated into an EP tube, and 1 mL of PBS was added while gently blowing to avoid cell damage. Add CD3-PE, CD4-APC, CD8-FITC, CD45-percp-cy5.5, and CD25-BV42 antibodies, and the cells were incubated for 25 ~ 30 minutes in dark and washed before analysis using flow cytometry. The intracellular fluorescence intensity was measured, and the lymphocyte subpopulation was analyzed.CD45+ CD3+ CD4+ indicates CD4+ T cells, CD45+ CD3+ CD8+ indicates CD8+ T cells, and CD45+ CD3+ CD4+ CD25+ indicates Treg cells.
Enzyme linked immunosorbent assay (ELISA)
Tumor tissues were separated into bite-sized pieces and rinsed in sterile PBS (0.02 mol/L, pH 7.4) at a temperature of about 4 ℃ to remove the residual blood in it, drained of excess water and weighed. The homogenizing solution, protease inhibitor (Thermo Scientific, USA) and small steel beads were added and thoroughly ground in a homogenizer. Centrifuge (3000 rpm for 15 min) the already processed tissue mixure and retain the supernatant. The levels of tumor necrosis factor α (TNF-α), IL-2, and IL-10 were measured by ELISA (Invitrogen, USA) according to the kit instructions. The protein concentration of each group was also quantified by BCA kit, and the ELISA results were analyzed after protein equilibration.
Statistical Analysis.
All data are shown as mean ± SEM. Differences were analyzed using two-tailed t-tests. (* or # p < 0.05; ** or ## p < 0.01; *** or ### p < 0.001; **** or #### p < 0.0001). Data analysis and plotting were performed using GraphPad Prism 8.