Supplementary Materials

This PDF file includes:

  • Materials and Methods
  • Fig. S1. Study summary.
  • Fig. S2. The structure schematic of synthetic mRNA.
  • Fig. S3. The chemical structure of 3′-O-Me-m7G(5′)ppp(5′)G ARCA cap.
  • Fig. S4. Chemicals for NP synthesis.
  • Fig. S5. Characterization of the engineered hybrid mRNA NPs.
  • Fig. S6. Size of EGFP-mRNA NPs and Luc-mRNA NPs with various formulations.
  • Fig. S7. Encapsulation efficiency of EGFP-mRNA NPs and Luc-mRNA NPs with various formulations.
  • Fig. S8. Normalized luminescence intensity of Hep3B cells after treatment with various Luc-mRNA NP formulations at the mRNA dose of 0.830 μg/ml.
  • Fig. S9. Endosomal escape of mRNA NPs.
  • Fig. S10. Transfection efficacy verified by CLSM imaging.
  • Fig. S11. Transfection efficacy verified by flow cytometry.
  • Fig. S12. Transfection efficacy after quenching intracellular GSH.
  • Fig. S13. In vitro toxicity of the synthetic EGFP-mRNA NPs.
  • Fig. S14. IF staining of p53 in p53-null H1299 cells.
  • Fig. S15. WB analysis of p53 protein expression.
  • Fig. S16. In vitro therapeutic efficacy of the synthetic p53-mRNA NPs in p53-null H1299 cells.
  • Fig. S17. Apoptosis of p53-null H1299 cells as determined by flow cytometry after different treatments.
  • Fig. S18. G1-phase cell cycle arrest induced by p53-mRNA NPs.
  • Fig. S19. WB analysis of apoptotic signaling pathway in p53-null H1299 cells after different treatments.
  • Fig. S20. TEM images of mitochondrial morphology in p53-null H1299 cells after different treatments.
  • Fig. S21. In vitro toxicity of the mutant p53-R175H-mRNA NPs.
  • Fig. S22. Cytotoxicity of everolimus in p53-null H1299 cells.
  • Fig. S23. Effect of everolimus on autophagy activation in p53-null H1299 cells.
  • Fig. S24. WB analysis of autophagy and apoptotic signaling pathways in p53-null H1299 cells.
  • Fig. S25. Analysis of the autophagosomes and swollen mitochondria in p53-null H1299 cells after different treatments.
  • Fig. S26. In vitro therapeutic efficacy of the combination of p53-mRNA NPs with everolimus in p53-null H1299 cells.
  • Fig. S27. In vitro apoptosis of p53-null H1299 cells after different treatments.
  • Fig. S28. In vitro toxicity of the combination of everolimus with venetoclax.
  • Fig. S29. In vitro toxicity of the combination of everolimus with siBcl-2.
  • Fig. S30. The relative mRNA expression of p53.
  • Fig. S31. The relative mRNA expression of ULK1, ATG7, BECN1, and ATG12.
  • Fig. S32. The relative mRNA expression of DRAM1, ISG20L1, and SESN1.
  • Fig. S33. The relative mRNA expression of TIGAR.
  • Fig. S34. WB analysis of AMPK and TIGAR pathways.
  • Fig. S35. Schematic representation of the possible mechanism by which p53 tumor suppressor inhibits protective autophagy and sensitizes tumor cells to everolimus.
  • Fig. S36. BioD of different mRNA NPs in HCC xenograft tumor model.
  • Fig. S37. BioD of different mRNA NPs in NSCLC xenograft tumor model.
  • Fig. S38. Blood vessel staining in tumor sections.
  • Fig. S39. Efficacy and safety of different treatments in HCC xenograft model.
  • Fig. S40. Antitumor effects of p53-mRNA NPs are synergistic with everolimus in NSCLC xenograft model.
  • Fig. S41. Murine p53 restoration in p53-null murine liver cancer RIL-175 cells.
  • Fig. S42. Therapeutic efficacy of murine p53-mRNA NPs in immunocompetent mice bearing p53-null RIL-175 tumors.
  • Fig. S43. Expression of p53 protein in HCC xenograft model after treatment with p53-mRNA NPs.
  • Fig. S44. Expression of p53 protein in NSCLC xenograft model after treatment with p53-mRNA NPs.
  • Fig. S45. IHC images from tumor sections of H1299 tumor–bearing mice before and after treatment with p53-mRNA NPs.
  • Fig. S46. In vivo toxicity of the p53-mRNA NP–mediated strategy for everolimus rescue assessed by histopathological and hematological analysis.
  • Fig. S47. IHC images from major organs and tumor sections of the HCC xenograft model.
  • Fig. S48. Evaluation of immune responses after the treatment with mRNA NPs.
  • Fig. S49. Scans of the liver metastases from different treatment groups in Fig. 6.
  • Table S1. Compositions of different NP formulations.
  • Table S2. Different p53-mRNA sequences used in this study.
  • Table S3. Primer sequences for qRT-PCR.

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