Periodic Reporting for period 1 - OncoNanoR (Engineering the immunosuppressive microenvironment in mesothelioma with targeted LipidNanoParticles encapsulating self-amplifying RNA)
Reporting period: 2023-01-01 to 2025-06-30
Malignant pleural mesothelioma (MPM) is a highly aggressive disease and standard chemotherapy treatment achieves an overall survival of 12 months. Anti–programmed cell death 1 protein immunotherapy has a low response rate and does not improve the patient's survival due to the immunosuppressive tumor microenvironment. The EU-funded OncoNanoR project aimed to develop innovative immunotherapy for the treatment of MPM, involving activation of the innate and adaptive tumor microenvironment. The general goal was to achieve an intracellular delivery of potentially therapeutic RNA molecules using intravenous injection of MPM-targeted lipid nanoparticles. The specific objective for this period was to obtain and characterize an saRNA-LNP system with appropriate features for biomedical administration; with the ability to target MPM cells.
In particular, during the last period, we were able to design, formulate under reproducible conditions, and characterize lipid nanoparticles (LNPs) containing mRNA. In order to standardize the procedures, three methods were used: NanoAssemblr® Ignite microfluidic platform from Precision Nanosystems Inc.’s (PNI, Canada), vortex mixing, and pipette mixing. Different ionizable lipids, RNA working concentration, total volume, volume rate ratios (aqueous to ethanol phase) and phase addition order, N/P ratios, mixing times, and speed were tested when possible; and the resulting LNPs were analyzed to determine the amount of encapsulated RNA, the amount of cholesterol and total lipids, particle size, polydispersity index (PdI), and zeta potential. Two different buffers were used for particle dilution and conservation, and possible changes in particle size and PdI were analyzed for several weeks.
Similar and positive results were achieved by the three methods used for particle production. In general, particle size was between 80-160 nm, polydispersity index presented values under 0,15; and zeta potential was always close to neutrality, as expected due to the presence of pegylated molecules on particle surface. Encapsulation efficiency was over 70% and up to 87% in some cases; and the particles produced presented high stability over time when stored at 4 0C, in terms of size, PdI, and encapsulated RNA.
Partial results of the project were included in a poster presentation at the 6th ASPIC International Congress 2024 (Faro, Portugal), and we also participated in a collaborative review article (in preparation), which includes aspects related to the generation of RNA-LNPs systems to fight cancer.
Conclusions:
mRNA-LNP systems with relevant features for biomedical applications were efficiently generated using three different methods. The systems proved to be stable over time when stored at 4 0C, and the mRNA was encapsulated inside LNPs at high percentages. Having an extensive standardization phase allowed us to select the best conditions for subsequent production and functionalization steps.
Group URL: https://cibb.uc.pt/en/research-group/tumor-microenvironment-and-targeted-therapies(opens in new window)
Partial results of the project were included in a poster presentation at the 6th ASPIC International Congress 2024 (Faro, Portugal), and we also participated in a collaborative review article (in preparation), which includes aspects related to the generation of RNA-LNPs systems to fight cancer.
Conclusions:
mRNA-LNP systems with relevant features for biomedical applications were efficiently generated using three different methods. The systems proved to be stable over time when stored at 4 0C, and the mRNA was encapsulated inside LNPs at high percentages. Having an extensive standardization phase allowed us to select the best conditions for subsequent production and functionalization steps.
Group URL: https://cibb.uc.pt/en/research-group/tumor-microenvironment-and-targeted-therapies(opens in new window)