Materials
Ester-terminated PLGA (with inherent viscosity of 0.55-0.75 dL/g) was purchased from Durect Corporation. Lipid PEGs terminated with methoxyl groups (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)−3000] (ammonium salt), DSPE-MPEG (molecular weight (MW) of PEG, 3000 Da) were purchased from Avanti Polar Lipids. Cationic ethylenediamine core-poly(amidoamine) (PAMAM) dendrimer generation 0 (G0) were purchased from Sigma-Aldrich. CXCR4-targeting peptide CTCE-9908 (KGVSLSYRCRYSLSVGK, CTCE) and scrambled peptide (LYSVKRSGCGSRKVSYL, SCP) were custom synthesized by GL Biochem (Shanghai) Ltd. Lipofectamine 2000 (L2K) was purchased from Invitrogen. Firefly Luciferase mRNA (Luc mRNA, L-7202), Enhanced Green Fluorescent Protein mRNA (EGFP mRNA, L-7201), and Cyanine 5 Firefly Luciferase mRNA (Cy5-Luc mRNA, L-7702) were purchased from TriLink Biotechnologies (San Diego, CA). Murine p53 mRNA with chemical modification (full substitution of Pseudo-U and 5-Methyl-C, Capped (Cap 1) using CleanCap® AG, Polyadenylated (120 A)) was custom-synthesized by TriLink Biotechnologies (San Diego, CA). InVivoMAb anti-mouse PD-1 (CD279) was purchased from Bioxcell. D-luciferin-K + salt bioluminescent substrate (no. 122799) was obtained from PerkinElmer. Primary antibodies used for western blot experiments as well as immunofluorescent and immunohistochemistry staining included: anti-p53 (sc-126, Santa Cruz Biotechnology, 1:500 dilution), anti-GAPDH (Cell Signaling Technology, # 5174; 1:2000 dilution), anti-beta-Actin (Cell Signaling Technology; 1: 2,000 dilution), and anti-rabbit and anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling Technology). Secondary antibodies used in this study included: Alexa Fluor® 488 Goat-anti Rabbit IgG (Life Technologies, A-11034), and Alexa Fluor® 647 Goat-anti Mouse IgG (Life Technologies, A-28181). All other chemicals and solvents were purchased from Sigma-Aldrich and used without further purification.
Synthesis of ionizable lipid-like compounds (G0-Cn)
A series of ionizable lipid-like compounds termed G0-Cn were synthesized through ring opening of epoxides bearing different alkyl chain lengths by generation 0 of poly (amidoamine) (PAMAM) dendrimers (M1). Briefly, substoichiometric amounts of epoxide were added to increase the proportion of products with one less tail than the total possible for a given amine monomer. The amine (1 equiv, typically 1 millimole (mmol)) and epoxide (9 equiv, typically 1 millimole (mmol)) were added to a 50 mL round-bottom glass flask containing a magnetic stir bar. The flask was sealed, and the reaction was heated to 95 °C with homogeneous stirring for 2 days. The crude products were separated by chromatography on silica with gradient elution from CH2Cl2 to 15:1 CH2Cl2/MeOH. The separated product was characterized by 1H NMR spectrum.
mRNA complexation ability of G0-C8 and its stability in organic solvent
Gel electrophoresis was used to study the mRNA complexation ability of ionizable compound G0-C8 and optimize the ratio between G0-C8 and mRNA in the NPs with free EGFP-mRNA or EGFP-mRNA complexed with G0-C8. Free EGFP-mRNA was also incubated with DMF to evaluate the stability of mRNA in organic solvent (DMF). The EGFP-mRNA were first incubated with G0-C8 at different weight ratios (weight ratios of G0-C8/mRNA: 1, 2, 5, 10, and 20) or DMF for 20 min at room temperature. The volumes of samples were then adjusted with loading dye (Invitrogen) and run into an E-Gel 2% agarose (Invitrogen) gel for 30 min at 50 V. Ambion Millennium markers-Formamide (Thermo Fisher Scientific) was used as a ladder. Finally, the gel was imaged under ultraviolet and the bands were analyzed.
Synthesis of lipid-PEG-CTCE HCC targeting peptide (DSPE-PEG-CTCE) and lipid-PEG- scrambled peptide (DSPE-PEG-SCP)
We conjugated the CXCR4-targeting peptide CTCE-9908 (KGVSLSYRCRYSLSVGK, CTCE) and scrambled peptide (LYSVKRSGCGSRKVSYL, SCP) to DSPE-PEG-MAL to construct the HCC targeted NPs and the non-targeted control NPs, respectively. Synthesis of DSPE-PEG-CTCE and DSPE-PEG-SCP was achieved through the efficient thiol-maleimide Michael addition click reaction. In brief, DSPE-PEG-maleimide and the thiol-CTCE peptide (3:1) or thiol-scrambled peptide were each dissolved in dimethylsulfoxide (DMF). The peptide solution was diluted in 0.1 M sodium phosphate buffer, pH 7.4, and DSPE-PEG was then added to the mixture. The final reaction mixture was 1:1 DMF/(sodium phosphate buffer) with 5 mM peptide and 15 mM DSPE-PEG maleimide. The reaction was allowed to proceed for 2 h at room temperature and then dialyzed against DI water for purification. Lastly, the product was lyophilized to obtain white powder as the final product (DSPE-PEG-CTCE or DSPE-PEG-SCP). The chemical structures of DSPE-PEG-CTCE and DSPE-PEG-SCP were confirmed by 1H-NMR spectrum.
Optimization of the mRNA NPs: the effect of targeting ligand densities
The cellular uptake of Enhanced Green Fluorescent Protein mRNA (EGFP mRNA) NPs engineered with seven different densities of CTCE peptide (EGFP-mRNA-CTCE NPs, CTCE density: 2%, 3%, 4%, 5%, 6%, 7%, and 10%, respectively) and 5% scrambled peptide (SCP) was studied to optimize the surface chemistry and targeting efficacy of the mRNA NPs by measuring GFP expression using flow cytometry (BD Biosystems, Heidelberg, Germany) and analyzed using Flowjo software (Flowjo V10).
Preparation of mRNA NPs and the formulation optimization
An optimized and robust self-assembly technique was employed to prepare mRNA-encapsulated polymer-lipid hybrid NPs based on our previous report27, but we extensively optimized the ratios among different NPs’ components, the pH of the solution for mRNA complexation, and the sequence in which reagents were added, which affected the encapsulation, morphology, and transfection efficiency of the mRNA. Briefly, G0-C8 and PLGA were dissolved separately in anhydrous DMF to form a homogeneous solution at concentrations of 2.5 mg/ml and 5 mg/ml, respectively. DSPE-MPEG, DSPE-PEG-CTCE and DSPE-PEG-SCP were dissolved in DNase/RNase-free HyPure water (GE Healthcare Life Sciences, catalog no. SH30538) at the concentration of 1 mg/mL. All of the reagents listed above were sonicated for 5 min in a water-bath sonicator before use. Citrate buffer with pH 3.0–3.5 was first added to 80 μg of G0-C8 (in 32 μl of DMF), then 16 μg of p53 mRNA (in 16 μl of citrate buffer) was added, mixed gently (at a G0-C8/mRNA weight ratio of 5), and allowed to stay at room temperature for 15 min to ensure the sufficient electrostatic complexation. Afterwards, 250 μg of PLGA polymers (in 50 μl of DMF) was added to the mixture and gently mixed. The final mixture was added dropwise to 10 ml of DNase/RNase-free HyPure water consisting of 1 mg hybrid lipid-PEGs under uniform magnetic stirring (1000 rpm) for 30 min. An ultrafiltration device (EMD Millipore, MWCO 100 kDa) was used to remove the organic solvent and free compounds from the NP dispersion via centrifugation at 4 °C. After washing 3 times with DNase/RNase-free HyPure water, the mRNA NPs were collected and finally concentrated in pH 7.4 PBS buffer. The NPs were used fresh or stored at −80 °C for further use.
Physicochemical characterization and stability of mRNA NPs
The hydrodynamic diameter, zeta potential, and morphology of the p53-mRNA NPs were measured to assess their physicochemical properties. Sizes and zeta potentials of both CTCE- p53-mRNA NPs and SCP-p53-mRNA NPs were measured by dynamic light scattering (DLS, Brookhaven Instruments Corporation) at 20 °C. Diameters are reported as the intensity mean peak average. To prepare NPs for Transmission Electron Microscopy (TEM) to characterize their morphology and shape, CTCE-p53-mRNA NPs were negatively stained with 2% uranyl acetate and then imaged with a Tecnai G2 Spirit BioTWIN microscope (FEI Company). To verify the in vitro stability of the synthesized polymer-lipid hybrid mRNA NPs in an environment mimicking the physiological milieu, CTCE-p53-mRNA NPs were incubated in 10% serum-containing PBS solution at 37 °C in triplicate for 96 hr with constant stirring at 100 rpm. At each time point, an aliquot of NP solution was withdrawn for particle size measurement using DLS and analyzed at various time intervals to evaluate any change in size distribution. To test the encapsulation efficiency (EE%) of mRNA in the NPs, Cy5-Luc-mRNA NPs were prepared according to the aforementioned method. Dimethyl sulfoxide (DMSO, 100 μl) was added to 5 μl of the NP solution to extract the mRNA encapsulated in the NPs, and the fluorescence intensity of Cy5-Luc-mRNA was measured using a multi-mode microplate reader (TECAN, Infinite M200 Pro). The amount of loaded mRNA in the engineered NPs was calculated to be ~67.5%.
Cell culture
The p53-null murine HCC cell line RIL-175 was used throughout. RIL-175 (a p53-null/Hras mutant line syngeneic to C57Bl/6 mouse strain background, Luciferase-tagged) was kindly provided by Dr. Tim Greten (NIH). All other cells were purchased from American Type Culture Collection (ATCC). Dulbecco’s Modified Eagle’s Medium (DMEM; ATCC) was used to culture RIL-175 cells. The cell culture medium was supplemented with 10% fetal bovine serum (Hyclone, SH30071.03), Pen-Strep (100 U ml−1 and 100 μg ml−1, respectively). Cell culture and all biological experiments were performed at 37 °C in 5% CO2 conditions and the normal level of O2 in a cell culture incubator. All cell lines were routinely tested using a mycoplasma contamination kit (R&D Systems) before any in vitro cell experiments or in vivo tumor model preparation.
Cell viability and transfection efficiency of EGFP-mRNA NPs
CTCE-EGFP-mRNA NPs and SCP-EGFP-mRNA NPs were prepared for evaluated the cell viability of the mRNA NPs along with their transfection efficiency of EGFP-mRNA. For the cell viability tests, RIL-175 cells were plated in a 96-well plate at a density of 5 × 103 cells per well. After 24 h of cell adherence, cells were treated with EGFP-mRNA at various mRNA concentrations (0.0625, 0.125, 0.250, 0.500, and 0.750 μg ml−1) for 24 hr, the cells were washed with PBS buffer (pH 7.4), followed by changing the culture medium to 0.1 ml fresh complete medium per well and further incubation for another 24 hr to evaluate cell viability by the Alamar Blue assay according to the manufacturer’s protocol and a microplate reader (TECAN, Infinite M200 Pro). To test the transfection efficiency, RIL-175 cells were seeded at a density of 5 × 104 cells per well on a 6-well plate and allowed to attach and grow until ~80% confluence. Cells were transfected with EGFP-mRNA NPs at the mRNA concentration of 0.5 μg ml−1 for 24 h followed by washing with fresh complete medium and further incubated for 24 h to assess transfection efficiency by measuring GFP expression using flow cytometry (DXP11 Flow Cytometry Analyzer). The percentages of GFP-positive cells were calculated and analyzed using Flowjo software (Flowjo V10).
Establishment of CXCR4-KO RIL-175 cells
The precise gene-editing system of CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 (CRISPR associated) was performed to knock out the CXCR4 gene in RIL-175 cells. Briefly, the single guide RNA (sgRNA) targeting CXCR4 was designed on the online tool (http://www.genome-engineering.org) including sgRNA1 (forward: 5′-CACCGTCGAGAGCATCGTGCACAAG-3′, reverse:5′-AAACCTTGTGCACGATGCTCTCGAC-3′) and sgRNA 2 (forward: 5′-CACCGGGACTTACACTCACACTGAT-3′, reverse: 5′-AAACATCAGTGTGAGTGTAAGTCCC-3′), and sequentially were phosphorylated and annealed. At one time, the lentiviral expression lentiCRISPRv2 plasmid (Addgene, cat. no. 52961, USA) was digested and dephosphorylated with BsmBI enzyme (ThermoFisher, cat. No. ER0451) following by running DNA gel and gel purify the larger band leaving the 2 kb filler piece. Next, the ligation reaction of lentiCRISPRv2 and sgRNAs was established for incubating 10 min at room temperature. After finishing the process of transformation in Stbl3 bacteria and validation by DNA sequencing, the lentiCRISPv2 inserted with sgRNAs targeting CXCR4 was selected out. Then the lentivirus system including lentiCRRISPv2 and the packaging plasmids pVSVg (AddGene, cat. No.8454) and psPAX2 (AddGene, cat. No.12260) were co-transfected into HEK293T cells to produce the complete lentivirus and further transfected into RIL-175 wide type cells. The puromycin (2 μg/μl) previously included in the lentiCRISPRv2 was used to screen out the positive cells successfully transfected with the complete lentivirus. Finally, the quantitative PCR and western blotting were performed to detect the expression of CXCR4 from both transcriptional and protein levels.
Cellular uptake of dye-labeled mRNA-encapsulated NPs
To monitor the cellular uptake of the NPs, Cy5-Luc-mRNA-NPs were prepared. RIL-175 cells were first seeded in 35 mm confocal dishes (MatTek) at a density of 5 × 104 cells per well and incubated at 37 °C in 5% CO2 for 24 h. The cells were then incubated with medium (DMEM) containing Cy5-Luc-mRNA-NPs at different time intervals. The cells were then washed with PBS, counterstained with Hoechst 33342 (Thermofisher), and analyzed using an Olympus microscope (FV1200, Olympus).
In vitro cell growth inhibition assay with p53-mRNA NPs
RIL-175 or HCA-1 cells were plated in 96-well plates at a density of 5 × 103 cells per well. After 24 h of cell adherence, cells were treated with empty NPs (blank NPs), free p53 mRNA, p53-mRNA NPs at different mRNA concentrations (0.0625, 0.125, 0.250, 0.500, and 0.750 μg ml−1). After 24 h of incubation, the cells were washed with PBS buffer (pH 7.4) and further incubated in fresh medium for another 24 h. AlamarBlue cell viability was used to verify the in vitro cell growth inhibition efficacy of p53-mRNA NPs.
Immunoblotting
Protein extracts from cells taken from dissected tumors in each group were prepared using lysis buffer (1 mM EDTA, 20 mM Tris-HCl pH 7.6, 140 mM NaCl, 1% aprotinin, 1% NP-40, 1 mM phenylmethylsulphonyl fluoride, and 1 mM sodium vanadate), and supplemented with protease inhibitor cocktail (Cell Signaling Technology) and boiled at 100 °C for 10 min. Equal amounts of protein were determined with a bicinchoninic acid protein assay kit (Pierce/Thermo Scientific) according to the manufacturer’s instructions. After gel electrophoresis and protein transformation, membranes were blocked with 3% bovine serum albumin (BSA) in TBST (150 mM NaCl, 50 mM Tris-HCl at pH 7.4, and 0.1% Tween 20) for 1 h at room temperature with gentle shaking. Membranes were rinsed and then incubated overnight at 4 °C with appropriate primary antibodies. The immunoreactive bands were visualized using an enhanced chemiluminescence (ECL) detection system (Cell Signaling Technology).
Immunofluorescence staining and microscopy
For immunofluorescence staining, cells or tumor tissues from each treatment group were washed with ice-cold PBS and fixed with 4% paraformaldehyde (Electron Microscopy Sciences) in PBS for 20 min at room temperature, followed by permeabilization in 0.2% Triton X-100-PBS for 10 min. Samples were followed by blocking with PBS blocking buffer containing 2% normal goat serum, 2% BSA, and 0.2% gelatin for 1 h at room temperature. Then, the samples were incubated in primary antibodies at the appropriate concentration for 1 h at room temperature, washed with PBS and incubated in goat anti-rat-Alexa Fluor 647 (Molecular Probes) at 1:1000 dilution in blocking buffer for another 1 h at room temperature. Finally, stained cells were washed with PBS, counterstained with Hoechst 33342 (Molecular Probes-Invitrogen, H1399, 1:10000 dilution in PBS), and mounted on slides with Prolong Gold antifade mounting medium (Life Technologies). The slides were imaged under a confocal laser scanning microscope (Olympus, FV1100).
Animals
For the s.c. tumor model, all animal procedures were performed in ethical compliance and with approval by the Institutional Animal Care and Use Committees at Harvard Medical School. Immunocompetent male and female C57BL/6 mice (5-6 weeks old or 6–8 weeks old) were obtained from Charles River Laboratories and housed in a pathogen-free animal facility of Brigham and Women’s Hospital, Harvard Medical School. For each experiment, mice were randomly allocated to each group. Mice were put for at least a 72 h acclimation period prior to use in order for physiological parameters to return to baseline after shipping and transferring. All animals were housed in single-unit cages with 12-h alternate light and dark cycles and at controlled ambient temperature (68-79 °F) with humidity between 30%-70%. For the orthotopic tumor model, all animal experiments were performed after approval by the Institutional Animal Care and Use Committee of the Massachusetts General Hospital.
Pharmacokinetics study
Healthy C57Bl/6 mice (5–6 weeks old, n = 3 per group) were injected intravenously with free Cy5-Luc-mRNA, CTCE-Cy5-Luc-mRNA NPs, or SCP-Cy5-Luc-mRNA NPS through the tail vein at the mRNA dose of 350 μg per kg of animal weight. Blood was collected retroorbitally at different time points (5 min, 30 min, 1 h, 2 h, 6 h, 12 h, and 24 h) and the fluorescence intensity of Cy5-Luc-mRNA was measured using a microplate reader (TECAN, Infinite M200 Pro). Pharmacokinetics was evaluated by calculating the percentage of Cy5-Luc mRNA in blood at various time points.
HCC tumor model preparation
Two p53-null RIL-175 HCC tumor models, an ectopic (s.c.) grafted model and an orthotopic model, were developed for in vivo biodistribution, modulation of the immune microenvironment, therapeutic efficacy, and in vivo toxicity studies. An orthotopic p53-wild type HCA-1 HCC tumor model was also developed for the in vivo therapeutic efficacy study. For the s.c. grafted model, ~1 × 106 RIL-175 cells in 100 μl of culture medium mixed with 100 μl of matrigel (BD Biosciences) were implanted subcutaneously in the right flank of C57Bl/6 mice (6–8 weeks old). Mice were monitored for tumor growth every other day according to the animal protocol. To develop the RIL-175 orthotopic model, ~1 million RIL-175 cells 1:1 in Matrigel (Mediatech/Corning, Manassas, VA) were grafted into the left extrahepatic lobe of C57Bl/6 mice (6–8 weeks old). Tumor growth was monitored by high-frequency ultrasonography every 3 days according to the animal protocol. For the HCA-1 orthotopic model, approximately 1 million HCA-1 cells 1:1 in Matrigel (Mediatech/Corning, Manassas, VA) were grafted into the left extrahepatic lobe of C3H mice (6–8 weeks old). Tumor growth was monitored by high-frequency ultrasonography every 3 days according to the animal protocol. When the tumor volume reached about ~100 mm3 (for ectopic model) or ~5 mm in diameter (for orthotopic model), mice were randomly assigned to a treatment group.
Biodistribution of mRNA NPs in the RIL-175 HCC tumor model
The biodistribution and tumor accumulation of mRNA NPs were assessed in C57Bl/6 mice bearing with s.c. grafted RIL-175 tumor (~100–200 mm3) and in the RIL-175 orthotopic model (~5 mm in diameter), respectively. In brief, RIL-175 bearing C57Bl/6 mice (5–6 weeks old, n = 3 per group) were injected intravenously with free Cy5-Luc-mRNA, CTCE-Cy5-Luc NPs or SCP-Cy5-Luc NPs via the tail vein at a mRNA dose of 350 μg per kg of animal weight. After 24 h, all the mice were sacrificed, and dissected organs and tumors were visualized using a Syngene PXi imaging system (Synoptics Ltd). The data were analyzed by Image J software.
Flow cytometry and cytokine analysis
Tumor immune-environment responses were assessed in the s.c. grafted and orthotopic HCC models by cytokine detection and flow cytometry after treatment. RIL-175 tumor-bearing C57Bl/6 mice (6–8 weeks old, n = 3 per group) were systemically (i.v. via tail vein) injected with CTCE-targeted p53 mRNA NPs or control groups (i.e., PBS or CTCE-EGFP NPs) every 3 days for four injections (at the murine p53 or EGFP mRNA dose of 350 μg/kg animal body weight). For the combinatorial immunotherapy group, one day after each i.v. injection of CTCE-p53 NPs, mice underwent intraperitoneal (i.p.) administration of aPD1 (100 μg per dose). The tumor inoculation and treatment schedule are depicted in Fig. 3a and Supplementary Fig. 22a. Forty-eight hrs post treatment, mice were euthanized and tumor tissue was harvested and homogenized for flow cytometry and cytokine analysis. For flow cytometry, tumor tissues were resected and minced, and fragments were incubated in HBSS with 1.5 mg/mL of hyaluronidase and 15 µg/mL of collagenase for 30 minutes at 37 °C. Digested tissues were passed through a 70-µm cell strainer and washed twice with phosphate-buffered saline (PBS)/0.5% bovine serum albumin. Prior to immunostaining, cells were washed with the buffer and fixed and permeabilized with FoxP3/Transcription Factor Staining Buffer Set (eBioscience/Thermo Fischer Scientific) to stain the intracellular markers. Harvested cells were incubated in Dulbecco’s Modified Eagle Medium with cell activation cocktail with BD Leukocyte Activation Cocktail, with BD GolgiPlug™(1:500, Biolegend) for 6 h at 37 °C. The cells were stained with the antibodies of cell surface and intracellular marker in the buffer with brefeldin A. Cells were stained with fluorescence-labeled antibodies CD11c (Biolegend, cat. no. 117310, clone N418), CD80 (Biolegend, cat. no. 104722, clone 16-10A1), CD 86 (Biolegend, cat. no. 105005, clone clone GL-1), CD4 (Biolegend, cat. no. 100412, clone GK1.5), CD3 (Biolegend, cat. no. 100204, clone 17 A2), CD8 (Biolegend, cat. no. 140408, clone 53–5.8), CD11b (Biolegend, cat. no. 101208, clone M1/70), F4/80 (Biolegend, cat. no. 123116, clone BM8), CD206 (Biolegend, cat. no. 141716, clone C068C2), Gr-1 (Biolegend, cat. no. 108412, clone RB6-8C5), CD45 (Biolegend, cat. no. 103108, clone 30-F11), TCR (Biolegend, cat. no. 109243, clone H57-597), CD39 (Biolegend, cat. no. 143805, clone Duha59), Ki67 (Biolegend, cat. no. 652423, clone 16A8), CD11b (Biolegend, cat. no. 101243, clone M1/70), CD206 (Biolegend, cat. no. 141717, clone C068C2), Forkhead box protein P3 (FoxP3; Biolegend, cat. no. 126419, clone MF-14), IFN-γ Receptor βchain (Biolegend, cat. no. 113605, clone MOB-47), CD119 (BD Bioscience, cat. no. 740897, clone GR20), FITC (Biolegend, cat. no. 503805, clone JES6-5H4) following the manufacturer’s instructions. All antibodies were diluted 200 times, except for FoxP3 and CD119 staining, which were 1:100 dilution. The stained cells were measured on a flow cytometer (Accuri C6 Plus, BD Biosciences) and analyzed by FlowJo software (Flowjo V10). The numbers presented in the flow cytometry analysis images are percentage based. For cytokine studies, tissue samples were assayed in duplicate using the MSD proinflammatory Panel I, a highly sensitive multiplex enzyme-linked immunosorbent assay (ELISA) for quantitatively measuring 10 cytokines-IFN-γ, interleukin (IL)−1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, TNF-α, KC/GRO and IL-9, IL-15, IP-10, MCP-1, MIP-1α, MIP-2, IL-17A/F, IL-27p28/IL-30, IL-33 using electrochemiluminescence-based detection (MesoScale Discovery, Gaithersburg, MD).
In vivo therapeutic efficacy
The therapeutic effects of p53-mRNA NPs and their integrated antitumor effect with anti-PD1 were evaluated in the p53-null HCC s.c. RIL-175 tumor model, p53-null RIL-175 orthotopic tumor model, and p53-wild-type HCA-1 orthotopic tumor model. For the s.c. model, RIL-175 tumor-bearing C57Bl/6 mice (6–8 weeks old, n = 5 per group) were monitored for tumor growth every other day after tumor implantation; tumor size was measured using a digital caliper and calculated as 0.5 × length × width2. When the tumor volume reached about ~100 mm3, mice were randomly divided into five groups (n = 5), which received treatment with PBS, CTCE-EGFP NPs, CTCE-p53 NPs, aPD1, or the combination of CTCE-p53 NPs and aPD1 according to the schedule in Supplementary Fig. 22a at the mRNA dose of 350 μg/kg animal body weight, while the aPD1 were administrated by i.p. at 100 μg per dose one day after the p53-mRNA NPs treatment. Tumor growth was measured and calculated every 3 days. The body weights of all mice were recorded every three days during this period. Animals were euthanized upon showing signs of imperfect health or when the size of their accumulated tumors exceeded 1.0 cm3. For the orthotopic HCC tumor model, tumor growth was monitored by high-frequency ultrasonography every 3 days. When the tumor size reached ~5 mm in diameter, mice were randomly assigned to a treatment group (n = 12). Treatments were administered according to the schedule in Fig. 3a. For the comparison of side-by-side the in vivo survival of the combination of CTCE-p53 NPs with aPD1 against the new standard of care in HCC patients (i.e., anti-VEGFR2 antibody + aPD-L1 antibody) in the orthotopic RIL-175 tumor model, treatments were administered i.p. every 3 days for 4 doses at 10 mg/kg of aPD-L1 antibody (Bioxcell, #BE0101, clone 10F.9G2), and 10 mg/kg of anti-VEGFR-2 antibody (Bioxcell, #BE0060, clone DC101) (Supplementary Fig. 20a). For survival studies, the endpoint was moribund status, defined as signs of prolonged distress, >15% weight loss compared with the starting date, body condition score >2, or tumor size of >15 mm in diameter.
Bioluminescence
To further explore the therapeutic efficacy of our therapeutic strategy, tumors were also assessed using an in vivo bioluminescence imaging system (Bruker Xtreme scanner). Mice were monitored for tumor growth by bioluminescent in vivo imaging every 6 days (Day 0, 6, and 12); specifically, 8 minutes after intraperitoneal injection of 150 mg/kg D-luciferin substrate (PerkinElmer, Catalog#122799), mice from each treatment group (n = 3) were imaged.
Immunohistochemistry staining
The expression of p53 protein and CD8 + cells in tumor tissue sections from different in vivo treatment groups were assessed by immunohistochemistry. Tumor sections were fixed in 4% buffered formaldehyde solution and embedded in paraffin. Paraffin-embedded sections were deparaffinized, rehydrated, and washed in distilled water. In order to retrieve the antigen, tumor tissue sections were incubated in 10 mM citrate buffer (pH = 6) for 30 min, washed in PBS, and immersed in 0.3% hydrogen peroxide (H2O2) for 20 min, then incubated in blocking buffer (5% normal goat serum and 1% BSA) for 60 min. Tissue sections were then incubated with the appropriate primary antibodies (PBS solution supplemented with 0.3% Triton X-100) at 4 °C overnight in a humid chamber. After being rinsed with PBS, the samples were incubated with biotinylated secondary antibody at room temperature for 30 min, rinsed again with PBS, and incubated with the avidin-biotin-horseradish peroxidase complex (ABC kit, Vector Laboratories, Inc). After being washed again, stains were processed with the diaminobenzidine peroxidase substrate kit (Impact DAB, Vector Laboratories, Inc) for 3 min. Sections were evaluated using a Leica Microsystem after being counterstained with hematoxylin (Sigma), dehydrated, and mounted.
In vivo toxicity evaluation
The in vivo toxicity of p53-mRNA NPs was comprehensively studied in both the p53-null HCC s.c. graft tumor model and the p53-null orthotopic HCC tumor model. In brief, the major organs were harvested at the end point, sectioned, and H&E stained to evaluate the histological differences. In addition, blood was drawn, and serum was isolated at the end of the in vivo efficacy experiment. Various parameters including ALT, AST, BUN, RBC, WBC, Hb, MCHC, MCH, HCT, and LY were tested to evaluate toxicity.
Statistical analysis
A two-tailed Student’s t-test or a one-way analysis of variance (ANOVA) was performed when comparing two groups or more than two groups, respectively. Statistical analysis was carried out using Prism 8.0 (GraphPad) and Microsoft Excel. Data are expressed as standard deviation (S.D.) or standard error means (S.E.M) as described in the main text. Difference was considered to be significant if P < 0.05 (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 unless otherwise indicated). All studies were performed at least in triplicate unless otherwise stated.
Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
