(T1030-06-38) Paving the Road to Use saRNA Loaded into Polymeric Nanoparticles as Nanovaccines Platform to Target Glioblastoma

Purpose: Glioblastoma (GBM) is a well-known, aggressive, and fast-growing brain cancer that limits the median overall survival rate to nearly 15 months after patient diagnosis. Nanovaccines (NVs) are a promising immunotherapy for GBM since they can efficiently convert the cold microenvironment of GBM into a hot microenvironment [1]. The FDA-approved poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs) have been used to deliver therapeutics effectively to the brain in a controlled release manner [2]. However, optimization of their preparation methodology is needed to be applicable for gene delivery, specifically, the challenging big in-size; self-amplifying RNA (saRNA) [3]. Optimization of the production methodology of PLGA NPs to be applicable for effective gene delivery to the brain in a controlled release manner, loaded with saRNA, which is considered the optimal choice for the vaccination application is the main aim of this work.

Methods: We have developed the production methodology of the FDA-approved poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs) to be applicable for effective gene delivery to the brain in a controlled release manner. Specifically, the challengeable big in size, self-amplifying RNA (saRNA), which is considered the optimal choice for the vaccination application. However, their toxicity related to boost translation of the gene of interest (GOI) hindered their use in clinics. Therefore, our hypothesis is that PLGA NPs with their slow and controlled release properties, can deliver the saRNA effectively and sustain its translation into the GBM microenvironment. The produced NPs with the traditional method, optimized method, and automated method were physicochemically characterized and the quantification assay of the loaded saRNA was carefully optimized. In addition, the transfection assay to quantify the translated gene of interest (GOI) was performed on 2D cultured GBM cells (U87 MG) and compared to the spheroids model. Recently, we moved to use the xenograft GBM zebrafish model to evaluate the macrophages activation.

Results: The produced NPs with the traditional, optimized, and automated methods were physicochemically characterized, and the quantification assay of the loaded saRNA was carefully optimized. The hydrodynamic diameters of all the formulations are within the range of 94-150nm. The positive zeta potential values are associated with the presence of ALC-0315 lipid. Scanning Electron Microscopy showed a spherical shape of PLGA NPs, and the well-distributed ones were automatically produced. A heparin assay was used to induce the release of saRNA from polymeric NVs, and the saRNA integrity was also evaluated. In addition, the transfection assay to quantify the translated GOI was performed on 2D cultured GBM cells (U87 MG) and compared with a 3D spheroid GBM model. The formulations did not show cytotoxicity on all tested cell lines for 24, 48, and 72 hours of incubation, and their transfection assay successfully translated into the GOI after 72 hours of incubation. Due to the high transfection levels, we are currently assessing the NVs' macrophage activation and tumor reduction efficacy in the zebrafish xenograft model.

Conclusion: We have developed RNA-friendly protocols for saRNA delivery through PLGA NPs using classical and automated techniques, known as the polymeric NVs platform. Our results showed a promising way of protecting the saRNA integrity and translation of its GOI in vitro within 72 hours of incubation. However, NVs require a long incubation time to allow the extracellular expression of GOI and consequently, the immunostimulatory effect, which will be further investigated during the in vivo studies later this year.

References: 1. Hameedat, F., Mendes, B.B., Conniot, J., Di Filippo, L.D., Chorilli, M., Schroeder, A., Conde, J. and Sousa, F., Nature Reviews Materials, 2024., pp.1-15.
2. Sousa, F., Lee, H., Almeida, M., Bazzoni, A., Rothen-Rutishauser, B. and Petri-Fink, A., Drug Delivery and Translational Research, 2023. pp.1-13.
3. Blakney, A.K., Ip, S. and Geall, A.J., Vaccines, 2021, p.97.

Acknowledgements: Thanks to the University of Fribourg and Adolphe Merkle Institution for the funding for the first year of the presenter's PhD. Also, we thank the University of Groningen for the funding of the second year of the presenter's PhD.
Authors declare no conflict of interest.


The nanovaccines characterization: (a) hydrodynamic diameters and PdI (left), and zeta potential and pH (right). (b) Microscopic images collected by SEM of all formulations. Scale bar=2um.