(F-202) Lipid nanoparticles design for delivery of mRNA to cancer cells and cells in the tumor microenvironment

Introduction:: The malignant mass is not only comprised of cancerous cells, but also numerous resident cells in the tumor microenvironment (TME), such as endothelial cells and fibroblasts. Cancer growth and dissemination are significantly affected by the interplay between the cancer cells with their counterparts in the TME. Therefore, not only cancer cells but the cells in the TME can represent viable targets for therapy to arrest the malignant growth. Since the pandemics, mRNAs vaccines and therapeutics gained significant attention as the next generation of the efficient therapies. Understanding the efficacy of RNAs delivery using lipid nanoparticles (LNP) in different origin cancer cells and the tumor microenvironment cells will help to develop better cancer treatments. To efficiently encapsulate negatively charged mRNA, LNP should contain positively charged lipids, which can be either ionizable at certain pH or constantly cationic.



The objective of this work was to assess the kinetics of LNP cellular uptake and mRNA expression in breast, colon and brain cancer cells and tumor microenvironment cells: fibroblasts, endothelial cells, and macrophages, while studying the mechanisms involved in differential behaviors of LNP formulated with cationic and ionizable lipids. For this purpose, we designed ionizable, DLin-MC3 LNP or cationic, DOTAP LNP and assessed them for cellular uptake and mRNA delivery in vitro and in vivo.


Materials and Methods:: mRNA-LNP containing ionizable lipids (LNP-A) and cationic lipids (LNP-B) were formulated using microfluidics mixer (Precision NanoSystems). mRNA-LNP were characterized for size, zeta potential using dynamic light scattering (DLS) and mRNA encapsulation efficiency using RiboGreen assay. Breast cancer cell lines studied included murine 4T1 and human MCF7, MDA-231, SUM-159 and T-47D cells, brain cancer cell lines murine N2A neuroblastoma and human SY5Y neuroblastoma cells, and colon cancer cell line human HT-29 cells, while non-cancer TME cell lines were BJ fibroblasts, and HUVEC and HDMVEC endothelial cells. LNP were tagged with rhodamine lipid to investigate the cellular uptake kinetic and a reporter GFP mRNA to evaluate mRNA expression. We have also assessed the delivery and mRNA expression in a co-culture of cancer cell spheroids and human primary macrophages (isolated from human blood). IncuCyte S3®, was used to determine the LNP uptake and GFP mRNA expression. Mechanisms of LNP uptake were assessed in various cells using inhibitors of specific pathways. Additionally, expression of mRNA delivered from LNP in tumor microenvironment was evaluated in vivo in a syngeneic 4T1 breast cancer model using mRNA luciferase and IVIS imaging.


Results, Conclusions, and Discussions:: In vitro results demonstrated that LNP-B performed significantly better in terms of uptake and mRNA expression in fibroblasts, endothelial cells and human neuroblastoma, while LNP-A performed better in the breast, colon and mouse neuroblastoma cancer cells. In vivo performance of the systems following intratumoral injection into orthotopic breast tumors in syngeneic mice was not in line with in vitro behavior. The results demonstrated that LNP A and LNP B delivered mRNA with similar efficacy in the tumor, while pointing out that TME cells can be involved. These findings are important to further understand the design parameters of various mRNA LNP systems for potential clinical applications.


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