Materials
Ionizable lipid ALC-0315 was purchased from MedChemExpress (Monmouth Junction, NJ, USA). Cholesterol, DSPC and DMG-PEG were obtained from Sigma-Aldrich (St. Louis, MO, USA). 1,2-dioleoyl-sn-glycero-3-phosphate (18:1 PA) was purchased 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 complex (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), Brilliant Violet 510-conjugated anti-mouse CD69 antibody (BioLegend, 104531), and Brilliant Violet 421-conjugated anti-mouse IFN-γ antibody (BioLegend, 505829) were 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 were obtained from the Cell Bank of Type Culture Collection (Chinese Academy of Sciences). C57BL/6J mice (female, 6–8 weeks old) and BALB/c mice (female, 6–8 weeks old) were purchased from the Shanghai Laboratory Animal Center (Shanghai, China). B6; 129S6-Gt(ROSA)26Sortm14(CAG−tdTomato)Hze/J Ai14 mice (female, 6–8 weeks old, 007908) were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). All animal experiments were conducted in strict accordance with the guidelines established by the Shanghai Laboratory Animal Commission. These procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of East China Normal University (approval number: m20240814).
Ionizable lipids synthesis
The ionizable lipid library presented in this study was synthesized following the methodologies described in our patents (WO/2024/152512, WO/2024/193525, and WO/2025/011532), where detailed procedures can be found. The lipids were activated through 2-butyloctanoate esters, followed by amide coupling with a diverse range of diamines. The central pentanedioic acid scaffold was functionalized with N, N-dialkylated diamines of different chain lengths and substitution patterns. Finally, chromatographic purification was performed using silica gel chromatography with a CH₂Cl₂/MeOH gradient. Specifically, 5-(benzyloxy)−5-oxopentyl 2-butyloctanoate (64 g, 163.87 mmol) was hydrogenated using 10% Pd/C (3.49 g) in a MeOH/THF (1:1) solvent mixture. The reaction was conducted at 25 °C for 16 h under hydrogen atmosphere. The product was obtained as a colorless oil (45 g) after filtration and concentration. Subsequently, this intermediate was converted to its corresponding acyl chloride by treatment with oxalyl chloride (2.54 g) and catalytic DMF (49 mg) in DCM at 0 °C for 3 h under nitrogen. After solvent removal, 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 using K₂CO₃ and KI as catalysts at 90 °C for 16 h. The mixture was then extracted with ethyl acetate (EtOAc) and purified by silica gel chromatography using a DCM/MeOH (50:1 to 10:1). Finally, 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 15:1), the target compound was isolated as a colorless oil (410 mg).
Formulation of LNPs
To improve delivery efficiency, a series of lipids were synthesized and screened as described previously [46]. Briefly, ionizable lipids, DSPC, cholesterol, and PEG lipid were dissolved in ethanol at different molar ratios. To achieve spleen-targeting, the lipid mixture was combined with anionic lipid 18:1 PA. Notably, the ionizable cationic lipids were procured from YolTech Therapeutics (Shanghai, China). The lipid-mRNA combination was prepared using a microfluidic mixer (Precision Nanosystems, Vancouver, BC), at an aqueous-ethanol flow rate ratio of 3:1 to achieve the desired lipid-to-mRNA weight ratio of 40:1. Subsequently, the LNPs were diluted in PBS to a concentration of 0.5 ng/µl mRNA for in vitro assays. This preparation was further concentrated using Amicon Ultra-15 mL Centrifugal Filter Units (Millipore Sigma), sterilized by filtration through 0.22-µm filters, and stored at −80 °C for future use. Detailed formulation information is described in Table S1. The physicochemical properties of the LNPs, such as particle size, polydispersity index and zeta potential were systematically characterized by dynamic light scattering using the NANO ZS3600 (Malvern, Worcestershire, UK). Additionally, RNA encapsulation efficiency within the LNPs was determined with the Quant-iT Ribogreen Assay (Life Technologies, R11490).
Identification of personalized neoantigens
Candidate neoantigens were primarily derived from non-synonymous mutations. HCC neoantigens were identified through 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 each candidate was further predicted using the NetMHCpan binding affinity predictor (IC50 < 500 nM to H-2Kb), as described by Chen et al. [50]. In this study, one B16-M01 [49] and seven Hepa1-6 neoantigens (9-mer mutant) [50] were selected to prepare mRNA vaccinations (Table S2).
Design and synthesis of neoantigen mRNAs
mRNAs encoding luciferase, GFP, Cre recombinase and neoantigens were synthesized using the HiScribe T7 High Yield RNA Synthesis Kit (NEB, E2040S). The linearized DNA template encompassed 5’ and 3’ untranslated regions as well as a polyadenylated [poly(A)] tail. To enhance both stability and translational efficiency, the mRNA was capped using TriLink CleanCap Reagent AG (TriLink, N-7113), adhering to the manufacturer’s protocol. Meanwhile, uridine triphosphate (UTP) was substituted with 1-methylpseudoUTP during transcription. Subsequently, the synthesized mRNAs were purified with the RNeasy Mini Kit (QIAGEN, 74143), following the manufacturer’s protocol. RNA concentration was determined using a NanoDrop spectrophotometric measurement at 260 nm, and RNA quality was evaluated by analyzing transcript integrity and size distribution using an Agilent Bioanalyzer 2100 system.
In vivo screening of mLuc LNPs
To systematically assess the performance of different LNP formulations (as outlined in Table S1), BALB/c mice received luciferase-encoding mRNA at a dose of 0.5Â mg/kg through an intravenous tail vein injection. 6Â h after the injection, they received D-luciferin (BD Bioscience) intraperitoneally at a dose of 25Â mg/kg based on body weight. After fifteen-minute incubation, bioluminescence was captured using the In Vivo Imaging System (PerkinElmer, Waltham, MA). To further evaluate tissue-specific transfection efficiency, the liver and spleen were surgically excised and subjected to imaging analysis.
Spleen-targeted delivery of Cre mRNA in Ai14 mice
Ai14 mice received intravenous injections of Cre mRNA at a dose of 0.5 mg/kg to determine their effect on immune subsets. 72 h post-injection, mice were imaged using the In Vivo Imaging System (PerkinElmer, Waltham, MA). Subsequently, reporter protein expression was assessed via flow cytometry. Spleen cell suspensions were prepared by disruption and filtration through a 40-µm strainer. 1 × 106 cells were incubated with 100 µL of flow 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 were analyzed by flow cytometer (Fortessa, BD Biosciences). Data processing was conducted using FlowJo-v10, with gating strategies provided in Fig. S3A.
BMDC activation assay
Bone marrow-derived dendritic cells (BMDCs) were isolated from the femurs of C57BL/6 mice, and cultured in the presence of 10 ng/mL IL-4 (R&D Systems, 404-ML-010/CF) and 20 ng/mL mGM-CSF (R&D Systems, 415-ML-020/CF) to promote differentiation. Then, the cells were treated with Hepa-M01-mRNA-loaded LNPs for 24 h. To evaluate DC activation, expression of CD40 and CD86 was analyzed by flow cytometry, while the secretion of IL-12 and TNF-α was quantified using enzyme-linked immunosorbent assay (ELISA).
Subcutaneous B16F10 melanoma model
C57BL/6 mice were subcutaneously injected with 5 × 105 B16F10 cells and randomly assigned to three groups: B16M01-mL242, B16WT01-mL242, and PBS control (0.5 mg/kg; n = 5 per group). Tumor volume was measured every 3–6 days after transplantation with calipers and calculated according to the formula: (A × B 2)/2 (where A and B denote the largest and smallest tumor diameter, respectively). On day 25 post-inoculation, tumors were harvested, photographed and weighed. B16F10 cells were maintained in complete Dulbecco’s Modified Eagle’s Medium (DMEM) at 37 °C with 5% CO2.
Orthotopic Hepa1-6 HCC model
To investigate the efficacy of neoantigen mRNA treatment, an orthotopic HCC model was developed utilizing female C57BL/6 mice (aged 6–8 weeks; n = 5). On day 0, a mixture of 3 × 105 Hepa1-6-luciferase cells and Matrigel was injected into the liver subcapsular space. Subsequently, on days 7 and 14, treatment groups received intravenous tail vein injections of neoantigen mRNAs formulated with L242-20Lipo at doses of either 0.1 mg/kg or 0.5 mg/kg in a total volume of 200 µL. In contrast, control groups received either 0.5 mg/kg wild-type antigen mRNA or PBS. Tumor burden was monitored weekly using an IVIS Spectrum animal imaging system (PerkinElmer, Waltham, MA). Photon emission was quantified 15 min after D-luciferin injection, and then in vivo bioluminescence within regions of interest was calculated using IVIS Living Image 4.0 software. On day 28, tumor tissues were collected for immunofluorescence analysis, and splenic lymphocytes were isolated for immunostimulation detection.
Immunofluorescence analysis
Tumor tissues were fixed in 4% neutral-buffered paraformaldehyde (PFA) at 4 °C for 24 h. Then, the tissues were cryoprotected in 30% sucrose solution at 4 °C for 12 h. The samples were embedded in Optimal Cutting Temperature (OCT) compound (Tissue Tek, 4583), frozen at − 80 °C for 12 h, and sectioned into 10-µm slices using a cryostat microtome (Thermo Scientific). For TUNEL assay, the 10-µm slices were incubated with Alexa Fluor 546-labeled deoxyuridine triphosphate for 1 h. For immunofluorescence staining, tissue sections were subjected to antigen retrieval by heat treatment, blocked, and incubated with FITC-conjugated anti-Ki67 antibody. After washing, the slides were mounted with antifade medium containing DAPI and imaged using an Olympus BX53 upright fluorescence microscope. Statistics were performed using ImageJ software.
Flow cytometry analysis
For flow cytometry analysis, single-cell suspensions were prepared from tumors, spleens and lymph nodes. Cells were incubated with fluorochrome-conjugated antibodies in 0.5% bovine serum albumin for 30 min in the dark. Next, intracellular staining was conducted following an established protocol [54]. Briefly, cells were fixed with 0.05% glutaraldehyde (Sigma, 111-30-8) at room temperature for 10 min, followed by permeabilization with 0.1% Triton X-100 (Thermo Fisher Scientific, HFH10) for an additional 15 min. They were stained with anti-mouse IFN-γ-BV421 mAb (BioLegend, 505829) for 30 min at 4 °C in the dark. Finally, flow cytometry analysis was conducted using a flow cytometer (Fortessa, BD, USA), and the acquired data were analyzed with FlowJo v.10 to comprehensively assess cell subsets. The gating strategies are illustrated in Fig. S4.
ELISPOT assay
For BMDC stimulation, 5 × 104 cells were pulsed with 2 µg/mL of each neoantigen peptide. Subsequently, these BMDCs were 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 were visualized using the ELISPOT Analysis System (SinSage, AT-Spot-2200). Finally, images were captured and the spot number for each group was calculated automatically, facilitating the precise quantification of immune responses.
In vitro cytotoxicity assay of T cells
To assess the cytotoxic activity of splenic CD8+ T cells, cells were labeled with anti-mouse CD3-FITC, anti-mouse CD4-APC, and anti-mouse CD8-PE/Cyanine7 antibodies. Then, CD3+ CD4− CD8+ T cells were sorted by FACS on a BD FACSAria III (BD Biosciences, USA). For the cytotoxicity assay, CD8+ T cells were co-cultured with Hepa1-6 cells, and apoptosis induction was evaluated by dual staining with Annexin V-FITC (Invitrogen, A10788) and propidium iodide (PI; Invitrogen, A10788). Flow cytometric analysis was conducted on a BD Fortessa (BD Biosciences, USA). Additionally, Hepa1-6 cell apoptosis was confirmed by fluorescence microscopy.
Histopathological analysis
For histological evaluation, tissues from murine liver, heart, spleen, lungs, and kidneys were collected and fixed in 4% paraformaldehyde at 25 °C for 12 h. Fixed tissues were dehydrated through a graded ethanol-xylene gradient, and then embedded in paraffin. Then, 4-µm sections were prepared using a rotary microtome (Leica, RM2235) and stained with hematoxylin and eosin (H&E), Masson, and Sirius Red [55]. Finally, slides were examined under an upright fluorescence microscope (Olympus, BX53) to assess histopathological alterations.
Serum biochemistry
C57BL/6 mice (n = 4 per group) were administered intravenously NeoPol-mL242 (0.1 mg/kg or 0.5 mg/kg), WTPol-mL242 (0.5 mg/kg), or PBS (control). Blood samples were collected 2 h, 24 h, 72 h, 7 days, and 14 days post-injection. They were allowed to clot at room temperature for 1 h, and then centrifuged at 1,000 × g for 10 min to isolate the serum. Serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), albumin (ALB), and total protein (TP) were quantified using the Aspartate Aminotransferase Assay Kit, Alanine Aminotransaminase Assay Kit, Alkaline Phosphatase Assay Kit, Albumin Assay Kit, and Total Protein Assay Kit (Sailuofei Biotechnology, China), respectively, following the manufacturer’s protocols. To evaluate 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) were measured using the Mouse IL-6 ELISA Kit (BioLegend, 431307), Mouse TNF-α ELISA Kit (Abcam, ab208348), Mouse IP-10 ELISA Kit (Abcam, ab260067), and Mouse IL-1β Precoated ELISA Kit (Dakewe, 1210122), respectively.
RT-qPCR
Organ tissues (liver, spleen, lungs, pancreas, heart and kidneys) were homogenized using a homogenizer (TissueMaster). Total RNA was isolated using TRIzol extraction Reagent (TS424, Sigma) following the manufacturer’s protocol. RNA integrity was verified by Nanodrop prior to reverse transcription. cDNA synthesis was performed using PrimeScript RT-PCR (RR036A, TaKaRa). Quantitative real-time PCR (RT-qPCR) was conducted with SYBR Green SuperMix (11202ES08, Yeasen, Shanghai, China). All mRNA expression levels were normalized to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Primer sequences used for RT-qPCR are listed in Table S4.
Data analysis
Data were analyzed using unpaired two-tailed Student’s t-tests in GraphPad Prism V.9.0 (GraphPad, San Diego, CA). Numerical variables are reported as mean ± standard deviation (SD). Statistical significance was set as * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. To ensure robust evaluation, at least three biologically independent experiments were conducted for all animal studies and cell-based experiments.
