Supplies
Ionizable lipid ALC-0315 was bought from MedChemExpress (Monmouth Junction, NJ, USA). Ldl cholesterol, DSPC and DMG-PEG have 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 complicated (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), Sensible Violet 510-conjugated anti-mouse CD69 antibody (BioLegend, 104531), and Sensible Violet 421-conjugated anti-mouse IFN-γ antibody (BioLegend, 505829) have 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 have been obtained from the Cell Financial institution of Sort Tradition Assortment (Chinese language Academy of Sciences). C57BL/6J mice (feminine, 6–8 weeks outdated) and BALB/c mice (feminine, 6–8 weeks outdated) have been bought from the Shanghai Laboratory Animal Heart (Shanghai, China). B6; 129S6-Gt(ROSA)26Sortm14(CAG−tdTomato)Hze/J Ai14 mice (feminine, 6–8 weeks outdated, 007908) have been bought from The Jackson Laboratory (Bar Harbor, ME, USA). All animal experiments have been performed in strict accordance with the rules established by the Shanghai Laboratory Animal Fee. These procedures have 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 have 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 performed at 25 °C for 16 h underneath hydrogen ambiance. 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 therapy with oxalyl chloride (2.54 g) and catalytic DMF (49 mg) in DCM at 0 °C for 3 h underneath 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 have been synthesized and screened as described beforehand [46]. Briefly, ionizable lipids, DSPC, ldl cholesterol, and PEG lipid have been dissolved in ethanol at totally different molar ratios. To realize spleen-targeting, the lipid combination was mixed with anionic lipid 18:1 PA. Notably, the ionizable cationic lipids have 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 movement fee ratio of three:1 to realize the specified lipid-to-mRNA weight ratio of 40:1. Subsequently, the LNPs have 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 Items (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, akin to particle dimension, polydispersity index and zeta potential have 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 have been primarily derived from non-synonymous mutations. HCC neoantigens have 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] have been chosen to organize mRNA vaccinations (Desk S2).
Design and synthesis of neoantigen mRNAs
mRNAs encoding luciferase, GFP, Cre recombinase and neoantigens have 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 have 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 dimension 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 obtained luciferase-encoding mRNA at a dose of 0.5Â mg/kg via an intravenous tail vein injection. 6Â h after the injection, they obtained D-luciferin (BD Bioscience) intraperitoneally at a dose of 25Â 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 have been surgically excised and subjected to imaging evaluation.
Spleen-targeted supply of Cre mRNA in Ai14 mice
Ai14 mice obtained 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 have been imaged utilizing the In Vivo Imaging System (PerkinElmer, Waltham, MA). Subsequently, reporter protein expression was assessed through movement cytometry. Spleen cell suspensions have been ready by disruption and filtration via a 40-µm strainer. 1 × 106 cells have been incubated with 100 µL of movement 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 have been analyzed by movement cytometer (Fortessa, BD Biosciences). Knowledge processing was performed utilizing FlowJo-v10, with gating methods supplied in Fig. S3A.
BMDC activation assay
Bone marrow-derived dendritic cells (BMDCs) have been remoted from the femurs of C57BL/6 mice, and cultured within the presence of 10 ng/mL IL-4 (R&D Methods, 404-ML-010/CF) and 20 ng/mL mGM-CSF (R&D Methods, 415-ML-020/CF) to advertise differentiation. Then, the cells have been handled with Hepa-M01-mRNA-loaded LNPs for twenty-four h. To judge DC activation, expression of CD40 and CD86 was analyzed by movement 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 have 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 keeping with the components: (A × B 2)/2 (the place A and B denote the biggest and smallest tumor diameter, respectively). On day 25 post-inoculation, tumors have been harvested, photographed and weighed. B16F10 cells have 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 therapy, an orthotopic HCC mannequin was developed using feminine C57BL/6 mice (aged 6–8 weeks; n = 5). On day 0, a mix of three × 105 Hepa1-6-luciferase cells and Matrigel was injected into the liver subcapsular area. Subsequently, on days 7 and 14, therapy teams obtained 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 obtained 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 have been collected for immunofluorescence evaluation, and splenic lymphocytes have been remoted for immunostimulation detection.
Immunofluorescence evaluation
Tumor tissues have been mounted in 4% neutral-buffered paraformaldehyde (PFA) at 4 °C for twenty-four h. Then, the tissues have been cryoprotected in 30% sucrose answer at 4 °C for 12 h. The samples have been embedded in Optimum Chopping 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 have been incubated with Alexa Fluor 546-labeled deoxyuridine triphosphate for 1 h. For immunofluorescence staining, tissue sections have been subjected to antigen retrieval by warmth therapy, blocked, and incubated with FITC-conjugated anti-Ki67 antibody. After washing, the slides have been mounted with antifade medium containing DAPI and imaged utilizing an Olympus BX53 upright fluorescence microscope. Statistics have been carried out utilizing ImageJ software program.
Movement cytometry evaluation
For movement cytometry evaluation, single-cell suspensions have been ready from tumors, spleens and lymph nodes. Cells have been incubated with fluorochrome-conjugated antibodies in 0.5% bovine serum albumin for 30 min at nighttime. Subsequent, intracellular staining was performed following a longtime protocol [54]. Briefly, cells have been mounted 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 a further 15 min. They have been stained with anti-mouse IFN-γ-BV421 mAb (BioLegend, 505829) for 30 min at 4 °C at nighttime. Lastly, movement cytometry evaluation was performed utilizing a movement cytometer (Fortessa, BD, USA), and the acquired knowledge have 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 have been pulsed with 2 µg/mL of every neoantigen peptide. Subsequently, these BMDCs have 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 have been visualized utilizing the ELISPOT Evaluation System (SinSage, AT-Spot-2200). Lastly, pictures have been captured and the spot quantity for every group was calculated robotically, 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 have been labeled with anti-mouse CD3-FITC, anti-mouse CD4-APC, and anti-mouse CD8-PE/Cyanine7 antibodies. Then, CD3+ CD4− CD8+ T cells have been sorted by FACS on a BD FACSAria III (BD Biosciences, USA). For the cytotoxicity assay, CD8+ T cells have 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 performed 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 have been collected and stuck in 4% paraformaldehyde at 25 °C for 12 h. Fastened tissues have been dehydrated via a graded ethanol-xylene gradient, after which embedded in paraffin. Then, 4-µm sections have been ready utilizing a rotary microtome (Leica, RM2235) and stained with hematoxylin and eosin (H&E), Masson, and Sirius Pink [55]. Lastly, slides have been examined underneath an upright fluorescence microscope (Olympus, BX53) to evaluate histopathological alterations.
Serum biochemistry
C57BL/6 mice (n = 4 per group) have 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 have been collected 2 h, 24 h, 72 h, 7 days, and 14 days post-injection. They have 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 whole protein (TP) have 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) have 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) have 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 performed with SYBR Inexperienced SuperMix (11202ES08, Yeasen, Shanghai, China). All mRNA expression ranges have 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 have been analyzed utilizing unpaired two-tailed Pupil’s t-tests in GraphPad Prism V.9.0 (GraphPad, San Diego, CA). Numerical variables are reported as imply ± commonplace deviation (SD). Statistical significance was set as * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. To make sure sturdy analysis, no less than three biologically impartial experiments have been performed for all animal research and cell-based experiments.
