Development, characterization and cellular evaluation of mRNA-LNPs for single co-delivery of mRNA encoding immunomodulatory and reporter proteins
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Date
2025-06-23
Authors
López López, Alice Montserrat
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Abstract
mRNA-based therapies encoding immunotherapeutic proteins have emerged as a potential for cancer treatment, with advantages such as transient protein expression and potential to encode multiple therapeutic targets. Lipid nanoparticles (LNPs) are the most popular carrier of mRNA due to the success of COVID-19 vaccines. This study focuses on the formulation and in vitro evaluation of an LNP system designed for co-delivery of mRNA encoding the immunostimulatory interleukin-12 (IL-12), in combination with reporter mRNAs, with the aim of enabling combinatory immunotherapies for gastric cancer. Initially, LNPs encapsulating individual mRNAs (mFLuc, eGFP, mCherry and mIL-12) were tested. All formulations showed successful protein expression. Subsequently, the capacity of LNPs to co-encapsulate two different mRNAs was tested with the marker proteins eGFP and mCherry. Co-expression was observed at single-cell level through confocal microscopy and flow cytometry in both HEK293 and NCI-N87 cells, a gastric cancer cell line. This finding supported the feasibility of dual protein expression from a single LNP formulation. Finally, the co-encapsulation of IL-12 and eGFP mRNA was tested in NCI-N87 cells. ELISA quantification of IL-12 secretion by co-encapsulated LNPs demonstrated that the presence of a second mRNA does not compromise the expression of the cytokine. These findings demonstrate the feasibility of co-encapsulating mRNA encoding for immunomodulatory proteins in a single formulation, supporting its potential application in combinatory mRNA-based immunotherapy for gastric cancer. Future studies should focus on in vivo validation, exploration of cytokine synergy and evaluation of therapeutic efficiency in relevant models.
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Keywords
Lipid nanoparticles, mRNA delivery, Gastric cancer, Co-encapsulation, Combined immunotherapy