Materials
All mRNAs were purchased from APE×BIO or TriLink Biotechnologies. Manganese (Ⅱ) chloride tetrahydrate was obtained from Alfa Aesar (China) Chemicals Co., Ltd. DMG-PEG2K (R-PEG-0021), Cy5-DMG-PEG2K (R-PF-0170), and DSPC (LP-R4-076) obtained from Xi’an Ruixi Biological Technology Co., Ltd. DLin-MC3-DMA was purchased from AVT (Shanghai) Pharmaceutical Tech Co., Ltd. Cholesterol was purchased from Solarbio Biotech Co., Ltd. SM-102 was purchased from Xiamen Sinopeg Biotech Co., Ltd. Hoechst 33342 (62249), LysoTracker® Green DND-26 (L7526) and Lipofectamine 3000 (L3000075) were purchased from Invitrogen Trading (Shanghai) Co., Ltd. FITC anti-mouse CD11c antibody (117305, 1:100), PE-Cy7 anti-mouse CD86 antibody (105013, 1:100), APC anti-mouse CD80 antibody (104713, 1:100), APC anti-mouse CD40 antibody (124611, 1:100), APC anti-mouse H-2Kb bound to SIINFEKL antibody (141605, 1:100), PE anti-mouse H-2Kb bound to SIINFEKL antibody (141603, 1:100), were purchased from Biolegend. T-Select H-2kb OVA Tetramer-SIINFEKL-PE mouse (TS-5001-1C) were purchased from MBL life science. IFN-1α (MM-47294M1) and IFN-1β (MM-46462M1) ELISA kit assay were obtained from Meimian Industrial Co., Ltd. Mouse Anti-PEG IgM ELISA (#PEGM-1) and Mouse Anti-PEG IgG ELISA (#PEGG-1) were purchased from Life Diagnostics, Inc. The mouse IL-6 (1210602), TNF-α (1217202), IFN-γ pre-coated ELISpot kit (2210005) were obtained from Dakewe Biotech Company. OVA257–264 (SIINFEKL) were obtained from Solarbio Biotech Co., Ltd. Phenylmethanesulfonyl fluoride (PMSF) was purchased from Beijing Lablead. D-Luciferin (D1007) was purchased from Solarbio Biotech Co., Ltd. STING (D2P2F) Rabbit mAb (13647) and Anti-rabbit IgG, HRP-linked Antibody 7074 (Goat) were purchased from Cell Signaling Technology (CST). RPMI 1640 medium and Opti-MEM reduced serum medium were obtained from Thermo Fisher Scientific Inc.
Mn-mRNA and Lipid@Mn-mRNA (L@Mn-mRNA) preparation
For a reaction volume of 200 μL, we selected a 600 μL EP tube. First, 50 μL of 6 mM Mn2+ solution (manganese chloride tetrahydrate aqueous solution) was mixed with 130 μL of RNzyme-free water. Then, 20 μL of 1 mg/mL mRNA was combined with the former mixture. It was then vortexed for 6 minutes. Afterwards, the sample was heated in the metal bath at 65 °C for 5 minutes. After completion of the reaction, the sample was cooled to the room temperature. The Mn-mRNA nanoparticles were collected through centrifugation at 60000 g for 15 minutes, and the Mn-mRNA nanoparticles are dispersed in 200 μL of enzyme-free water. The mRNA and Mn2+ that were not included in Mn-mRNA nanoparticles could be removed though the centrifugation. SM-102 (or Dlin-MC3-DMA), cholesterol, DSPC, and DMG-PEG2000 were mixed according to the proportions specified in the formulation (molar ratio of 50:38.5:10:1.5) using anhydrous ethanol as the solvent. A volume ratio of anhydrous ethanol to aqueous phase (1:3) and a total lipid to mRNA mass ratio (10:1) was maintained. The aqueous phase was then rapidly injected into the ethanol phase with a pipette, followed by rapid pipetting for 150 times. It was allowed to stand at room temperature for 1 hour. Finally, excess ethanol from the system was removed using a dialysis cup with a cut-off molecular weight of 10 kDa to obtain the L@Mn-mRNA.
For the preparation of Fe-mRNA, Cu-mRNA, and Zn-mRNA, 6 mM M2+ solutions were respectively prepared using ferric chloride tetrahydrate, anhydrous copper (II) sulfate, and zinc nitrate enneahydrate. The ratios of M2+ to bases were referred from the literature33,34. The remaining steps were identical to the preparation method of Mn-mRNA. When preparing Mn-mRNA with different Mn2+ to nucleotide molar ratios, the amount of manganese ions was adjusted accordingly. When preparing L@Mn-mRNA with different total lipid-to-mRNA mass ratios, the amount of lipids was adjusted accordingly.
Mn-mRNA, L@Mn-mRNA and LNP-mRNA characterization
The morphological characterization of the particles was imaged using transmission electron microscopy (Hitachi HT7700) and cryo-electron microscopy (Thermo Fisher Titan Krios), respectively. Distribution of phosphorus (green) and manganese (red) elements of particles was detected by Energy-Dispersive X-ray Spectroscopy (EDS) element analysis from field-emission transmission electron microscope (JEOL JEM-F200). The size, polydispersity index (PDI) and zeta potentials of Mn-mRNA, L@Mn-mRNA and LNP-mRNA were measured by using dynamic light scattering (Malvern Panalytical Mastersizer). Each set of data was tested three times. Diameters are reported as the number mean peak average. The characterization of particle size and zeta potential for all particles was conducted by diluting the particles in water at a ratio of 1:50. To calculate mRNA reaction efficiency, Zn-mRNA or Mn-mRNA nanoparticles and the corresponding supernatant were dissolved by mixing them with a hydrochloric acid solution at pH 336. The mRNA content in both particles and supernatant was measured separately using the Quant-it™ RiboGreen RNA Assay Kit (n = 3 in each group). The Mn2+ content was measured using ICP-MS (Perkinelmer NexION 300X). The mRNA encapsulation efficiency of LNP-mRNA was calculated by Quant-it™ RiboGreen RNA Assay Kit according to the instructions. Three independent replicate experiments were performed.
The quantitative calculation of the Mn-mRNA composition was showed below:
1. Mn ratio involved in the reaction (by ICP-MS) equals to
“Mn in the precipitate (Mn-mRNA) (μg)” / “Mn in the precipitate (Mn-mRNA) (μg) + Mn in the supernatant solution (μg)”
2. Mn amount involved in the reaction equals to “Mn Input” × “Mn ratio involved in the reaction”
3. mRNA amount involved in the reaction equals to “mRNA Input” × “mRNA encapsulation efficiency”
4. The proportion of mRNA in Mn-mRNA equals to “mRNA involved (μg)” / “Mn involved (μg) + mRNA involved (μg)”
5. The proportion of Mn in Mn-mRNA equals to “Mn involved (μg)” / “Mn involved (μg) + mRNA involved (μg)”
Agarose gel electrophoresis
Agarose gel electrophoresis was run on a 1 % agarose gel using 1X TAE as electrophoresis buffer at 100 V for 30 minutes. The mRNA sample amount in each well was 0.5 μg. Subsequently, we utilized ultraviolet light for imaging.
Simulated calculation
The geometric and electronic properties of metal ions-mRNA have been computed using the Gaussian 09W program package at B3LYP/6-31G(d,p) level47. In order to ensure the feasibility of the calculation, we have adjusted the number of self-selected electrons in systems. After optimization, the length of bonds was measured by Mutifwn48. Utilizing the iefpcm implicit solvent model, single-point energy calculations for the structures were performed employing the wb97xd49 functional with the def2TZVP50 basis set. Frequency calculations were carried out using the 6-311Gd51 basis set to determine the binding energies of the complexes at 298K for different components.
LNP-mRNA and L@Mn-mRNA Young’s moduli test from AFM
Initially, a circular mica sheet with a diameter of 1 cm was affixed securely onto an iron plate. Subsequently, 30 μL of LNP-mRNA and L@Mn-mRNA were incubated respectively on the mica sheet for 20 minutes. After gently washing the mica sheet with PBS, the sheet was then covered with 20 μL of PBS. AFM imaging and force measurements were carried out in aqueous medium at 26 ± 1 °C by using a Bruker Multimode-8 with NanoScope Analysis software (Version 1.9). The probe used for measurements was SCANASYST-FLUID+. AFM images were recorded with the following scanning parameters: scan rate, 0.977 Hz; peak force amplitude, 40 nm; peak force frequency, 2 KHz and spring constant, 0.7 N/m. The force between the tip and the sample was carefully maintained so as not to collapse the nanoparticles. The resolution was 256 × 256 pixels per AFM. Seven data points were selected for quantitative analysis in each group.
Cells and animals
Female C57BL/6 (6–8 weeks old) were purchased from SPF Biotechnology. This study was approved by the ethics committee of the National Center for Nanoscience and Technology of China (approval number NCNST21-2103-0401). The B16-OVA cell line was purchased from Meisen Chinese Tissue Culture Collections (CTCC; cell line number CTCC-003-0259). The DC 2.4 cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The B16-OVA cells and DC 2.4 cells were cultured in RPMI 1640 containing 10% fetal bovine serum (FBS), Pen-Strep (100 U/mL and 100 μg/mL, respectively) solution at 37 ℃ with 5% CO2. Bone Marrow-Derived Dendritic Cells (BMDCs) were obtained through the following steps. After euthanizing C57BL/6 mice (6-8 weeks), they were placed in 50 mL centrifuge tubes filled with 75% ethanol for 10 minutes for sterilization. Subsequently, the leg bones were removed, muscles were stripped, and bones on both sides were clipped. Bone marrow was then flushed into new culture dishes using a needle. Red blood cell lysis buffer was added afterwards, and the lysis was terminated after 3 minutes. Finally, cells were collected by centrifugation and resuspended in culture medium containing 20 ng/mL GM-CSF (Peprotech) and 20 ng/mL IL-4 (Peprotech), with changing medium every two days. On Day 6, the nonadherent cells were aspirated and incubated in 6-well plates with fresh medium for further investigation.
In vitro transfection
DC 2.4 cells were seeded in a 96-well plate at a density of 104 cells per well. Different kinds of mRNAs were heated for different durations in a metal bath, then mixed with Lipofectamine™ 3000 Transfection Reagent according to the instructions to form complexes (n = 3 in each group). These complexes were then transfected into cells at a dose of 100 ng mRNA per well. After 24 hours, the EGFP expression level was measured using flow cytometry (ACEA NovoExpress) with 10000 live cells in FITC channel. The luciferase mRNA expression level was detected by mixing an equal volume of cell suspension and the Bright-Lite Plus Luciferase Assay System (Vazyme), and then results were measured by a plate reader.
Cellular uptake and lysosomal escape
L@Mn-Cy5-Mrna (L@Mn-Cm) and LNP-Cy5-mRNA (LNP-Cm) were synthesized using Cy5-labeled mRNA. Cy5-L@Mn-mRNA (CL@Mn-m) and Cy5-LNP-mRNA (CLNP) were synthesized using Cy5-labeled DMG-PEG2K. The particles were separately incubated with DC 2.4 cells for 4 and 10 hours. DC 2.4 cells were stained with Hoechst 33342 (1:500) for 5 minutes and washed with Opti-MEM medium three times. After that, DC 2.4 cells were stained with LysoTracker® Green DND-26 (1:10000) for 30 minutes and washed with Opti-MEM medium three times. The cellular uptake (Hoechst 33342/405 nm; Cy5/640 nm) and lysosome escape events (DND-26/488 nm) were captured using a CLSM (Olympus FV3000). The endosomal escape ratio was analyzed using an image analysis algorithm (ImageJ).
DC 2.4 cells were seeded in a 12-well plate at a density of 105 cells per well. Subsequently, LNP-mRNA and L@Mn-mRNA nanoparticles were separately incubated with DC 2.4 cells at an equal mRNA dosage for varying durations (mRNA dose: 0.5 μg/mL). Finally, cell uptake was quantitatively analyzed using flow cytometry (ACEA NovoExpress) with 10000 live cells in APC channel (n = 5 in each group).
In vitro and in vivo Bioluminescence
L@Mn-mLuc and LNP-mLuc were respectively incubated in BMDCs for 12 and 24 hours. Subsequently, an equal volume of cell suspension and the Bright-Lite Plus Luciferase Assay System (Vazyme) were mixed. Finally, the bioluminescence intensity was measured using a plate reader.
At 6 and 24 hours after the injection of L@Mn-mLuc and LNP-mLuc (mRNA dose: 5 μg per mouse), mice were injected intraperitoneally with 0.2 mL d-luciferin (15 mg/mL in DPBS). Ten minutes later, the mice were imaged with an in vivo imaging system (PerkinElmer IVIS). Bioluminescence was quantified using the Living Image software (PerkinElmer).
Western blot analysis
β-actin and STING protein expression in BMDCs were analyzed by western blot. PBS, LNP-mOVA (2 μg/well), L@Mn-mOVA (1 μg/well) and L@Mn-mOVA (2 μg/well) were separately incubated with BMDCs in a 6-well plate (3*105 cells per well) for 24 hours. Subsequently, cells were lysed on ice using cell lysis buffer containing protease and phosphatase inhibitors. The lysate was then centrifuged in a pre-chilled centrifuge. Centrifugation was conducted at 4 °C and 12,000 rpm for 20 minutes. After centrifugation, the supernatant was collected for protein quantification. The protein concentration of the samples was quantified using a bicinchoninic acid assay (BCA protein assay kit; Beyotime) according to the manufacturer’s instructions. Protein lysates (15 μg per sample) were resolved by SDS-PAGE and transferred to a polyvinylidene fluoride membrane. After being blocked in 5% BSA, the polyvinylidene fluoride membrane was incubated with primary antibodies followed by the specific secondary antibodies. Immunoreactive proteins were visualized using enhanced chemiluminescence reagents (Bio-Rad). Quantitively analysis of WB results were carried out using ImageJ software (n = 3 in each group).
Enzyme-linked immunosorbent assay (ELISA)
PBS, LNP-mOVA (2 μg/well), L@Mn-mOVA (1 μg/well) and L@Mn-mOVA (2 μg/well) were separately incubated with BMDCs in a 6-well plate (3*105 cells per well) for 24 hours. The supernatant was collected for the detection of IL-6 and TNF-α secretion from BMDCs. After diluting the supernatant five-fold, 50 µL of the test sample was added to the pre-coated plates. Subsequent steps were carried out according to the manufacturer's instructions. Finally, the results were read using a plate reader.
For the measurement of cytokines in serum, blood samples were collected from each group of mice at the endpoint of treatment (Day 17). After preparing serum from the blood samples, the serum was diluted five-fold. Then 50 µL of the test sample was added to pre-coated plates. Subsequent steps were carried out as described above (n = 4 in each group).
L@Mn-mRNA promoted maturation and antigen presentation of DCs in vitro and in vivo
BMDCs were seeded into a 12-well plate (1 × 105 cells per well) and then incubated with PBS, LNP-mOVA (mOVA dose: 1 μg/mL), L@Mn-mOVA (mOVA dose: 0.5 μg/mL) and L@Mn-mOVA (mOVA dose: 1 μg/mL) for 24 hours. The medium from each group was collected for ELISA of TNF-α, IL-6, IFN-1α and IFN-1β according to the manufacturer’s instructions. Meanwhile, cells were collected and stained with FITC anti-mouse CD11c antibody (1:100), PE-Cy7 anti-mouse CD86 antibody (1:100), APC anti-mouse CD80 antibody (1:100), APC anti-mouse CD40 antibody (1:100) and PE anti-mouse SIINFEKL-H-2kb antibody (1:100) for 30 minutes at 4 °C before the detection of flow cytometry (ACEA NovoExpress).
iLN from each mouse was harvested at 24 hours post-injection of PBS, LNP-mOVA (10 μg mOVA per mouse), L@Mn-mOVA (5 μg mOVA per mouse) and L@Mn-mOVA (10 μg mOVA per mouse) at the inguinal regions and were gently mechanically disrupted using sterile pestles in RPMI 1640 medium in a 1.5 mL tube. The resulting cell suspensions were collected and stained with FITC anti-mouse CD11c antibody (1:100), PE-Cy7 anti-mouse CD86 antibody (1:100), APC anti-mouse CD80 antibody (1:100), APC anti-mouse CD40 antibody (1:100) and APC anti-mouse SIINFEKL-H-2kb antibody (1:100) for 30 min at 4 °C before being analyzed by flow cytometry (ACEA NovoExpress).
Anti-tumour effects in a subcutaneous B16-OVA cancer model
To evaluate the anti-tumour effect of the L@Mn-mRNA vaccine in a solid tumour model, 1 × 106 murine melanoma cells B16-OVA cells were injected subcutaneously into the right flank of C57BL/6 mice on Day 0. The mice were subcutaneously administered with PBS, LNP-mOVA (10 μg mOVA per mouse), L@Mn-mOVA (5 μg mOVA per mouse) and L@Mn-mOVA (10 μg mOVA per mouse) at the right bilateral inguinal regions on Day 4, 7 and 10. Mice treated with PBS were used as a negative control group. The tumour volume was measured every other day using Vernier calipers and calculated by the following formula: tumour volume = length × 1/2 width2. The mice were euthanized on Day 17. The tumours were collected, and digested into single-cell suspensions to analyze the infiltrating immune cells by flow cytometry. The resulting cell suspensions were collected and stained with FITC anti-mouse CD3 antibody (1:100), PE-Cy7 anti-mouse CD8a antibody (1:100), APC anti-mouse CD4 antibody (1:100), PE anti-mouse F4/80 antibody (1:100) and APC anti-mouse SIINFEKL-H-2kb antibody (1:100) for 30 minutes at 4 °C before being analyzed by flow cytometry (ACEA NovoExpress). Blood and spleens were collected to analyze the proportion of specifical T cells that recognize the SIINFEKL antigen. Spleens were collected for IFN-γ ELISpot analysis. According to the manufacturer’s instructions, splenocytes were seeded in a 96 well plate (105 cells per well), pre-coated with a mouse anti-IFN-γ antibody and incubated with SIINFEKL peptide for 20 hours. A biotinylated antibody specific for IFN-γ and alkaline-phosphatase conjugated to streptavidin were subsequently used to detect the IFN-γ secreted by the re-stimulated T cells. By adding a substrate solution, visual spots were formed at the sites of captured IFN-γ, and automated spot quantification was caried out by Dakewe Biotech. Blood and the major organs, including heart, liver, spleen, lung and kidney, were collected for hematoxylin and eosin (H&E) staining to assess the safety of the L@Mn-mRNA vaccines.
Long-term immune memory test
The C57BL/6 mice (6-8 weeks, female) were subcutaneously administered with PBS, LNP-mOVA (10 μg mOVA per mouse), L@Mn-mOVA (5 μg mOVA per mouse) and L@Mn-mOVA (10 μg mOVA per mouse) at the right bilateral inguinal regions on Day 0, 3 and 6. To analyze memory T cells, splenocytes were collected at Day 70 (n = 5) and stained with FITC anti-mouse CD3, PE-Cy7 anti-mouse CD8, APC anti-mouse CD44 and PE anti-mouse CD62L antibodies. The central memory T cells (CD3+CD8+CD44highCD62Lhigh) and effector memory T cells (CD3+CD8+CD44highCD62Llow) were analyzed by flow cytometry (ACEA NovoExpress).
Anti-PEG IgG and IgM measurement
The C57BL/6 mice (6-8 weeks, female) were subcutaneously administered with PBS, LNP-mOVA (10 μg mOVA per mouse), L@Mn-mOVA (5 μg mOVA per mouse) and L@Mn-mOVA (10 μg mOVA per mouse) at the right bilateral inguinal regions on Day 0, 3 and 6. To detect Anti-peg IgM and Anti-peg IgG, blood of each group was respectively collected at Day -1, 13 and Day 20. After allowing the whole blood to stand and then centrifuging it to obtain plasma, the plasma was diluted 500-fold. ELISA assay kits are utilized to separately measure the antibody titers of Anti-peg IgM and Anti-peg IgG. Briefly, 100 μL diluted samples was dispensed into the coated wells. Then, 100 μL diluted HRP conjugate was added into each well. After dispensing 100 μL TMB reagent into each well, the plate was gently mix for 20 minutes. Finally, 100 μL stop solution was added, and the results were collected at a plate reader.
Statistical analysis
All results are presented as the mean ± S.D. from at least three independent experiments. Statistical differences between two groups were determined by two-tailed t-test, and the differences among three or more groups were determined by one-way ANOVA with the Tukey multiple comparison post-test. P values of *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 were regarded as significant. Animal survival rates were compared with the log-rank test using GraphPad Prism 9.0 (GraphPad Software, San Diego, CA, USA).