Supplies
Ionizable lipid ALC-0315 was bought from MedChemExpress (Monmouth Junction, NJ, USA). Ldl cholesterol, DSPC and DMG-PEG had been obtained from Sigma-Aldrich (St. Louis, MO, USA). 1,2-dioleoyl-sn-glycero-3-phosphate (18:1 PA) was bought from MilliporeSigma (Burlington, MA, USA). Purified fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD3 antibody (BioLegend, 100203), allophycocyanin (APC)-conjugated anti-mouse CD4 antibody (BioLegend, 116013), peridinin-chlorophyll-protein advanced (PerCP)/Cyanine5.5-conjugated anti-mouse CD19 antibody (BioLegend, 152405), phycoerythrin (PE)-conjugated anti-mouse NK1.1 antibody (eBioscience, 12-5941-82), PE/Cyanine7-conjugated anti-mouse CD8 antibody (BioLegend, 100722), APC/Cyanine7-conjugated anti-mouse F4/80 antibody (BioLegend, 157315), APC-conjugated anti-mouse CD11c antibody (BioLegend, 117309), PE-conjugated anti-mouse CD40 antibody (eBioscience, 12-0401-82), PerCP/Cyanine5.5-conjugated anti-mouse CD86 antibody (BioLegend, 159211), FITC-conjugated anti-mouse CD62L antibody (eBioscience, 11-0621-82), PE-conjugated anti-mouse CD44 antibody (eBioscience, 12-0441-82), Good Violet 510-conjugated anti-mouse CD69 antibody (BioLegend, 104531), and Good Violet 421-conjugated anti-mouse IFN-γ antibody (BioLegend, 505829) had been obtained from Thermo Fisher Scientific (Waltham, MA, USA). Anti-mouse MHC-Tetramer-PE (Ptpn2376−384 (RWLYWQPTL): H-2Kb) was obtained from AtaGenix (Wuhan, China).
Cells and animals
Murine B16F10 cells and Hepa1-6 cells had been obtained from the Cell Financial institution of Kind Tradition Assortment (Chinese language Academy of Sciences). C57BL/6J mice (feminine, 6–8 weeks outdated) and BALB/c mice (feminine, 6–8 weeks outdated) had been bought from the Shanghai Laboratory Animal Heart (Shanghai, China). B6; 129S6-Gt(ROSA)26Sortm14(CAG−tdTomato)Hze/J Ai14 mice (feminine, 6–8 weeks outdated, 007908) had been bought from The Jackson Laboratory (Bar Harbor, ME, USA). All animal experiments had been carried out in strict accordance with the rules established by the Shanghai Laboratory Animal Fee. These procedures had been authorised by the Institutional Animal Care and Use Committee (IACUC) of East China Regular College (approval quantity: m20240814).
Ionizable lipids synthesis
The ionizable lipid library offered on this examine was synthesized following the methodologies described in our patents (WO/2024/152512, WO/2024/193525, and WO/2025/011532), the place detailed procedures may be discovered. The lipids had been activated via 2-butyloctanoate esters, adopted by amide coupling with a various vary of diamines. The central pentanedioic acid scaffold was functionalized with N, N-dialkylated diamines of various chain lengths and substitution patterns. Lastly, chromatographic purification was carried out utilizing silica gel chromatography with a CH₂Cl₂/MeOH gradient. Particularly, 5-(benzyloxy)−5-oxopentyl 2-butyloctanoate (64 g, 163.87 mmol) was hydrogenated utilizing 10% Pd/C (3.49 g) in a MeOH/THF (1:1) solvent combination. The response was carried out at 25 °C for 16 h below hydrogen environment. The product was obtained as a colorless oil (45 g) after filtration and focus. Subsequently, this intermediate was transformed to its corresponding acyl chloride by remedy with oxalyl chloride (2.54 g) and catalytic DMF (49 mg) in DCM at 0 °C for 3 h below nitrogen. After solvent removing, 2 g 5-chloro-5-oxopentyl 2-butyloctanoate was produced. The intermediate (1 g) was coupled with N, N-diethylpropane-1,3-diamine (870 mg) in acetonitrile utilizing K₂CO₃ and KI as catalysts at 90 °C for 16 h. The combination was then extracted with ethyl acetate (EtOAc) and purified by silica gel chromatography utilizing a DCM/MeOH (50:1 to 10:1). Lastly, the product was synthesized by coupling the above intermediate (500 mg) with 5-chloro-5-oxopentyl 2-butyloctanoate (310 mg) in DCM. After silica gel chromatography (40:1 to fifteen:1), the goal compound was remoted as a colorless oil (410 mg).
Formulation of LNPs
To enhance supply effectivity, a collection of lipids had been synthesized and screened as described beforehand [46]. Briefly, ionizable lipids, DSPC, ldl cholesterol, and PEG lipid had been dissolved in ethanol at totally different molar ratios. To attain spleen-targeting, the lipid combination was mixed with anionic lipid 18:1 PA. Notably, the ionizable cationic lipids had been procured from YolTech Therapeutics (Shanghai, China). The lipid-mRNA mixture was ready utilizing a microfluidic mixer (Precision Nanosystems, Vancouver, BC), at an aqueous-ethanol move charge ratio of three:1 to attain the specified lipid-to-mRNA weight ratio of 40:1. Subsequently, the LNPs had been diluted in PBS to a focus of 0.5 ng/µl mRNA for in vitro assays. This preparation was additional concentrated utilizing Amicon Extremely-15 mL Centrifugal Filter Models (Millipore Sigma), sterilized by filtration via 0.22-µm filters, and saved at −80 °C for future use. Detailed formulation info is described in Desk S1. The physicochemical properties of the LNPs, corresponding to particle measurement, polydispersity index and zeta potential had been systematically characterised by dynamic gentle scattering utilizing the NANO ZS3600 (Malvern, Worcestershire, UK). Moreover, RNA encapsulation effectivity inside the LNPs was decided with the Quant-iT Ribogreen Assay (Life Applied sciences, R11490).
Identification of personalised neoantigens
Candidate neoantigens had been primarily derived from non-synonymous mutations. HCC neoantigens had been recognized via genomic and transcriptomic sequencing of liver tissue from wild-type C57BL/6 mice and the Hepa1-6 cell line (variant allele frequency ≥ 10%, sequencing depth ≥ 20). The immunogenicity of every candidate was additional predicted utilizing the NetMHCpan binding affinity predictor (IC50 < 500 nM to H-2Kb), as described by Chen et al. [50]. On this examine, one B16-M01 [49] and 7 Hepa1-6 neoantigens (9-mer mutant) [50] had been chosen to arrange mRNA vaccinations (Desk S2).
Design and synthesis of neoantigen mRNAs
mRNAs encoding luciferase, GFP, Cre recombinase and neoantigens had been synthesized utilizing the HiScribe T7 Excessive Yield RNA Synthesis Equipment (NEB, E2040S). The linearized DNA template encompassed 5’ and three’ untranslated areas in addition to a polyadenylated [poly(A)] tail. To boost each stability and translational effectivity, the mRNA was capped utilizing TriLink CleanCap Reagent AG (TriLink, N-7113), adhering to the producer’s protocol. In the meantime, uridine triphosphate (UTP) was substituted with 1-methylpseudoUTP throughout transcription. Subsequently, the synthesized mRNAs had been purified with the RNeasy Mini Equipment (QIAGEN, 74143), following the producer’s protocol. RNA focus was decided utilizing a NanoDrop spectrophotometric measurement at 260 nm, and RNA high quality was evaluated by analyzing transcript integrity and measurement distribution utilizing an Agilent Bioanalyzer 2100 system.
In vivo screening of mLuc LNPs
To systematically assess the efficiency of various LNP formulations (as outlined in Desk S1), BALB/c mice acquired luciferase-encoding mRNA at a dose of 0.5 mg/kg via an intravenous tail vein injection. 6 h after the injection, they acquired D-luciferin (BD Bioscience) intraperitoneally at a dose of 25 mg/kg based mostly on physique weight. After fifteen-minute incubation, bioluminescence was captured utilizing the In Vivo Imaging System (PerkinElmer, Waltham, MA). To additional consider tissue-specific transfection effectivity, the liver and spleen had been surgically excised and subjected to imaging evaluation.
Spleen-targeted supply of Cre mRNA in Ai14 mice
Ai14 mice acquired intravenous injections of Cre mRNA at a dose of 0.5 mg/kg to find out their impact on immune subsets. 72 h post-injection, mice had been imaged utilizing the In Vivo Imaging System (PerkinElmer, Waltham, MA). Subsequently, reporter protein expression was assessed through move cytometry. Spleen cell suspensions had been ready by disruption and filtration via a 40-µm strainer. 1 × 106 cells had been incubated with 100 µL of move cytometry staining buffer (eBioscience) containing a panel of fluorescent antibodies: FITC anti-CD3, PerCP-Cy5.5 anti-CD19, APC-Cy7 anti-F4/80 and APC anti-CD11c. After 1 h of incubation at 4 °C, cells had been analyzed by move cytometer (Fortessa, BD Biosciences). Knowledge processing was carried out utilizing FlowJo-v10, with gating methods supplied in Fig. S3A.
BMDC activation assay
Bone marrow-derived dendritic cells (BMDCs) had been remoted from the femurs of C57BL/6 mice, and cultured within the presence of 10 ng/mL IL-4 (R&D Programs, 404-ML-010/CF) and 20 ng/mL mGM-CSF (R&D Programs, 415-ML-020/CF) to advertise differentiation. Then, the cells had been handled with Hepa-M01-mRNA-loaded LNPs for twenty-four h. To judge DC activation, expression of CD40 and CD86 was analyzed by move cytometry, whereas the secretion of IL-12 and TNF-α was quantified utilizing enzyme-linked immunosorbent assay (ELISA).
Subcutaneous B16F10 melanoma mannequin
C57BL/6 mice had been subcutaneously injected with 5 × 105 B16F10 cells and randomly assigned to a few teams: B16M01-mL242, B16WT01-mL242, and PBS management (0.5 mg/kg; n = 5 per group). Tumor quantity was measured each 3–6 days after transplantation with calipers and calculated in accordance with the formulation: (A × B 2)/2 (the place A and B denote the biggest and smallest tumor diameter, respectively). On day 25 post-inoculation, tumors had been harvested, photographed and weighed. B16F10 cells had been maintained in full Dulbecco’s Modified Eagle’s Medium (DMEM) at 37 °C with 5% CO2.
Orthotopic Hepa1-6 HCC mannequin
To analyze the efficacy of neoantigen mRNA remedy, an orthotopic HCC mannequin was developed using feminine C57BL/6 mice (aged 6–8 weeks; n = 5). On day 0, a combination of three × 105 Hepa1-6-luciferase cells and Matrigel was injected into the liver subcapsular area. Subsequently, on days 7 and 14, remedy teams acquired intravenous tail vein injections of neoantigen mRNAs formulated with L242-20Lipo at doses of both 0.1 mg/kg or 0.5 mg/kg in a complete quantity of 200 µL. In distinction, management teams acquired both 0.5 mg/kg wild-type antigen mRNA or PBS. Tumor burden was monitored weekly utilizing an IVIS Spectrum animal imaging system (PerkinElmer, Waltham, MA). Photon emission was quantified 15 min after D-luciferin injection, after which in vivo bioluminescence inside areas of curiosity was calculated utilizing IVIS Residing Picture 4.0 software program. On day 28, tumor tissues had been collected for immunofluorescence evaluation, and splenic lymphocytes had been remoted for immunostimulation detection.
Immunofluorescence evaluation
Tumor tissues had been fastened in 4% neutral-buffered paraformaldehyde (PFA) at 4 °C for twenty-four h. Then, the tissues had been cryoprotected in 30% sucrose resolution at 4 °C for 12 h. The samples had been embedded in Optimum Reducing Temperature (OCT) compound (Tissue Tek, 4583), frozen at − 80 °C for 12 h, and sectioned into 10-µm slices utilizing a cryostat microtome (Thermo Scientific). For TUNEL assay, the 10-µm slices had been incubated with Alexa Fluor 546-labeled deoxyuridine triphosphate for 1 h. For immunofluorescence staining, tissue sections had been subjected to antigen retrieval by warmth remedy, blocked, and incubated with FITC-conjugated anti-Ki67 antibody. After washing, the slides had been mounted with antifade medium containing DAPI and imaged utilizing an Olympus BX53 upright fluorescence microscope. Statistics had been carried out utilizing ImageJ software program.
Movement cytometry evaluation
For move cytometry evaluation, single-cell suspensions had been ready from tumors, spleens and lymph nodes. Cells had been incubated with fluorochrome-conjugated antibodies in 0.5% bovine serum albumin for 30 min at nighttime. Subsequent, intracellular staining was carried out following a longtime protocol [54]. Briefly, cells had been fastened with 0.05% glutaraldehyde (Sigma, 111-30-8) at room temperature for 10 min, adopted by permeabilization with 0.1% Triton X-100 (Thermo Fisher Scientific, HFH10) for an extra 15 min. They had been stained with anti-mouse IFN-γ-BV421 mAb (BioLegend, 505829) for 30 min at 4 °C at nighttime. Lastly, move cytometry evaluation was carried out utilizing a move cytometer (Fortessa, BD, USA), and the acquired knowledge had been analyzed with FlowJo v.10 to comprehensively assess cell subsets. The gating methods are illustrated in Fig. S4.
ELISPOT assay
For BMDC stimulation, 5 × 104 cells had been pulsed with 2 µg/mL of every neoantigen peptide. Subsequently, these BMDCs had been co-incubated with 5 × 105 splenic T cells in 96-well Multiscreen plates pre-coated with anti-IFN-γ antibody (Abcam, ab64029). The co-culture was maintained at 37 °C with 5% CO2 for 48 h. IFN-γ spot-forming cells had been visualized utilizing the ELISPOT Evaluation System (SinSage, AT-Spot-2200). Lastly, pictures had been captured and the spot quantity for every group was calculated routinely, facilitating the exact quantification of immune responses.
In vitro cytotoxicity assay of T cells
To evaluate the cytotoxic exercise of splenic CD8+ T cells, cells had been labeled with anti-mouse CD3-FITC, anti-mouse CD4-APC, and anti-mouse CD8-PE/Cyanine7 antibodies. Then, CD3+ CD4− CD8+ T cells had been sorted by FACS on a BD FACSAria III (BD Biosciences, USA). For the cytotoxicity assay, CD8+ T cells had been co-cultured with Hepa1-6 cells, and apoptosis induction was evaluated by twin staining with Annexin V-FITC (Invitrogen, A10788) and propidium iodide (PI; Invitrogen, A10788). Movement cytometric evaluation was carried out on a BD Fortessa (BD Biosciences, USA). Moreover, Hepa1-6 cell apoptosis was confirmed by fluorescence microscopy.
Histopathological evaluation
For histological analysis, tissues from murine liver, coronary heart, spleen, lungs, and kidneys had been collected and stuck in 4% paraformaldehyde at 25 °C for 12 h. Fastened tissues had been dehydrated via a graded ethanol-xylene gradient, after which embedded in paraffin. Then, 4-µm sections had been ready utilizing a rotary microtome (Leica, RM2235) and stained with hematoxylin and eosin (H&E), Masson, and Sirius Purple [55]. Lastly, slides had been examined below an upright fluorescence microscope (Olympus, BX53) to evaluate histopathological alterations.
Serum biochemistry
C57BL/6 mice (n = 4 per group) had been administered intravenously NeoPol-mL242 (0.1 mg/kg or 0.5 mg/kg), WTPol-mL242 (0.5 mg/kg), or PBS (management). Blood samples had been collected 2 h, 24 h, 72 h, 7 days, and 14 days post-injection. They had been allowed to clot at room temperature for 1 h, after which centrifuged at 1,000 × g for 10 min to isolate the serum. Serum ranges of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), albumin (ALB), and complete protein (TP) had been quantified utilizing the Aspartate Aminotransferase Assay Equipment, Alanine Aminotransaminase Assay Equipment, Alkaline Phosphatase Assay Equipment, Albumin Assay Equipment, and Complete Protein Assay Equipment (Sailuofei Biotechnology, China), respectively, following the producer’s protocols. To judge systemic inflammatory responses, serum concentrations of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interferon gamma-induced protein 10 (IP-10), and interleukin-1β (IL-1β) had been measured utilizing the Mouse IL-6 ELISA Equipment (BioLegend, 431307), Mouse TNF-α ELISA Equipment (Abcam, ab208348), Mouse IP-10 ELISA Equipment (Abcam, ab260067), and Mouse IL-1β Precoated ELISA Equipment (Dakewe, 1210122), respectively.
RT-qPCR
Organ tissues (liver, spleen, lungs, pancreas, coronary heart and kidneys) had been homogenized utilizing a homogenizer (TissueMaster). Complete RNA was remoted utilizing TRIzol extraction Reagent (TS424, Sigma) following the producer’s protocol. RNA integrity was verified by Nanodrop previous to reverse transcription. cDNA synthesis was carried out utilizing PrimeScript RT-PCR (RR036A, TaKaRa). Quantitative real-time PCR (RT-qPCR) was carried out with SYBR Inexperienced SuperMix (11202ES08, Yeasen, Shanghai, China). All mRNA expression ranges had been normalized to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Primer sequences used for RT-qPCR are listed in Desk S4.
Knowledge evaluation
Knowledge had been analyzed utilizing unpaired two-tailed Scholar’s t-tests in GraphPad Prism V.9.0 (GraphPad, San Diego, CA). Numerical variables are reported as imply ± customary deviation (SD). Statistical significance was set as * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. To make sure strong analysis, not less than three biologically impartial experiments had been carried out for all animal research and cell-based experiments.