Doctoral defence: Maria Maloverjan “Optimizing Cell-Penetrating Peptide-Based Nanoparticles for Delivery of Nucleic Acid Therapeutics”

On 26 August at 13:15, Maria Maloverjan defends her doctoral thesis “Optimizing Cell-Penetrating Peptide-Based Nanoparticles for Delivery of Nucleic Acid Therapeutics” for obtaining the degree of Doctor of Philosophy (Biomedical Engineering).

Supervisiors: 
Professor Margus Pooga, University of Tartu
Professor Ana Rebane, University of Tartu

Opponent:
Professor Dr. Roland Brock, Medical Center of the Radboud University (The Netherlands)

Summary: 
Nucleic acids (NAs) are therapeutics with high potential, enabling the treatment of hereditary and acquired diseases. However, delivering NAs into target cells is extremely challenging. The large size and highly negative charge of NAs result in poor serum stability and hinder their ability to penetrate cellular membranes effectively. Furthermore, if cellular internalization occurs, most NA molecules are degraded by lysosomal enzymes after becoming entrapped in acidic endosomes.

To tackle these issues, various NA delivery systems have been developed. Among these, cell-penetrating peptides (CPPs) are highly promising candidates. These up to 30 amino acid-long peptides exhibit an inherent ability to cross biological membranes, carrying cargo molecules into cells. Positively charged CPPs readily form non-covalent complexes with NAs based on electrostatic interactions, and the forming nanoparticles have been shown to effectively deliver various NAs in vitro and in vivo.

The current thesis's focus was to understand better the factors determining the efficiency of CPP-based NA delivery and explore methods for improvement. As a model CPP, we used PepFect14 (PF14), an amphipathic stearylated CPP with a charge of +5. Firstly, we analyzed the protein corona of CPP/NA complexes – a layer of proteins forming around nanoparticles once exposed to a serum-containing environment, e.g., the bloodstream. We showed that the protein corona composition is highly dependent on the serum used and affects the nanoparticles' properties, cellular association, and resulting biological effect. Secondly, we investigated the impact of added Ca2+ and Mg2+ ions on the properties and functionality of the PF14/NA nanoparticles. We showed that these ions, especially Ca2+, greatly enhance the biological effect of NAs delivered by PF14, most likely facilitating the endosomal escape of the cargo. Finally, we investigated the ability of PF14 to transfect messenger RNA into cell lines, primary cells, and in vivo. We supplemented the nanoparticles with different excipients to achieve high mRNA transfection efficiency in cultured cells and in vivo.

In summary, our findings provide novel insights into the challenges associated with developing CPP/NA nanoparticles for clinical use and open potential avenues for enhancing their efficiency.

Defence can be followed in Zoom: https://ut-ee.zoom.us/j/9530588152?pwd=ZzgzMjY4YytzUkZ5aVRCd2pOdVNQQT09 (meeting ID: 953 058 8152, passcode: kaitsmine).