Iron fumarate nanoparticles as carriers for functional biomacromolecules

Mirzazadeh Dizaji, Negar (2024): Iron fumarate nanoparticles as carriers for functional biomacromolecules. Dissertation, LMU München: Fakultät für Chemie und Pharmazie

Abstract

In recent decades, biomacromolecules like proteins, mRNA, and the CRISPR-Cas system have become indispensable in medical and biotechnological fields, paving the way for new therapeutic strategies and advancements. While proteins play essential roles in cellular processes, driving interest in them for therapy and drug development, mRNA acts as a versatile intermediary between DNA and protein synthesis, finding applications from vaccines to gene therapy. Similarly, the CRISPR-Cas system has transformed genetic manipulation, offering precise genome editing for therapeutic purposes. However, challenges like poor stability, rapid degradation, and limited cellular uptake often hinder the effective utilization of these biomacromolecules. To address these obstacles, nanoparticles (NPs) emerge as promising solutions to overcome these challenges and unlock innovative healthcare approaches. Among the diverse array of NPs, the hybrid metal-organic nanoparticles have garnered significant attention due to their intriguing and unique properties, including tunable size, biocompatibility, and versatile surface chemistry. These characteristics render metal-organic NPs highly suitable for encapsulating and delivering biomacromolecules, enhancing their stability, promoting cellular uptake, and enabling targeted delivery. Iron-fumarate nanoparticles (Fe-fum NPs), a subclass of metal-organic NPs, possess unique characteristics stemming from their composition, which includes iron ions coordinated with fumarate ligands. These NPs demonstrate magnetic behavior, offer tunable sizes, and can encapsulate diverse cargoes. The controlled synthesis and modification of Fe-fum NPs have paved the way for their potential application across various domains in medicine and biotechnology. Although the capacity of Fe-fum NPs to store therapeutic small molecules and effectively release them to cancer cells is well established, their potential for delivering functional biomacromolecules, such as proteins and RNAs, has been relatively underexplored. The inherent pore sizes of Fe-fum nanoparticles prove insufficient to accommodate the dimensions of numerous proteins and RNA molecules, thus posing challenges for their effective entrapment within the nanoparticle pores. To overcome this limitation, in situ encapsulation of biomacromolecules in Fe-fum NPs emerges as a promising strategy, in which the nanoparticles form around biomacromolecules via a biomimetic mineralization process, ensuring a notable loading efficiency and minimal premature leakage of the biomacromolecule. The in situ encapsulation of biomacromolecules within Fe-fum NPs needs meticulous attention to synthesis conditions. This involves transitioning to water-based synthesis methods to eliminate harmful solvents, maintaining a pH near the physiological range of around 7.4, and avoiding high temperatures. Such measures are essential for preserving these delicate biomacromolecules' structural and functional integrity, thus optimizing the efficacy of Fe-fum NPs as carriers for such molecules. Initial efforts in our study to reevaluate the synthesis according to the conditions mentioned above encountered a novel challenge: either the nanoparticles increased in size or underwent aggregation. Achieving small, uniformly sized particles is crucial to optimizing their distribution within the body and facilitating cellular uptake. Thus, the first part of this thesis focuses on investigating the impact of various synthesis parameters, including solvents, modulators, and coatings, on the size, aggregation, and degradation of Fe-fum NPs synthesized using a biomimetic mineralization approach that preserves protein integrity. Our findings reveal specific conditions that facilitate the production of colloidally stable Fe-fum NPs capable of incorporating proteins. Applying a lipid coating after the synthesis of nanoparticles stabilized the size of Fe-fum in an aqueous buffer, allowing for cellular applications. Another crucial aspect of nanoparticles is their ability to release their cargo into the cytoplasm of cells after cellular internalization. Often, internalization is mediated via endocytosis, and cytosolic release implies escape from the endosome. The endosomal release ensures the payload reaches its target, facilitating the desired biological effect. In this study, we demonstrate that lipid-coated Fe-fum NPs are internalized by cells, and intracellular release of the loaded proteins and small molecules can be triggered externally via glucose shock. Furthermore, we showed that small molecule cargo can be released from the endosome by leveraging the internal trigger mechanism provided by histidine, offering an additional method for controlled release within the cellular environment. The second part of this thesis explores the versatility of the introduced biomineralization method involving Fe-fum NPs. It further investigates their capacity to maintain the structural integrity and operational effectiveness of diverse proteins throughout the synthesis and subsequent release process. For this, we initially demonstrated the ability of the Fe-fum NPs to encapsulate four distinct model proteins, including bovine serum albumin (BSA), horse radish peroxidase (HRP), green fluorescent protein (GFP), and Cas9/sgRNA ribonucleoproteins (RNPs). These Fe-fum NPs exhibited notably high loading efficiency for proteins, and facilitated their successful delivery into cells while maintaining protein activity. The delivery of RNPs via Fe-fum NPs resulted in efficient gene knockout in HeLa cells. Furthermore, we explored the potential of Fe-fum NPs in safeguarding proteins against harsh environmental conditions. Our findings revealed that integration into Fe-fum NPs effectively preserved and shielded the activity of RNPs even under conditions of pH 3.5 exposure and two months of storage at 4°C, conditions known to compromise the functionality of unprotected RNPs severely. The last part of this project delved into the potential of Fe-fum NPs as carriers for RNA, with a specific focus on mRNA delivery. Using a biomineralization technique during synthesis, RNA molecules were successfully integrated into the Fe-fum NPs, facilitating their efficient delivery into cells. Moreover, the utilization of mCherry-encoding mRNA as a model RNA confirmed the successful translation and production of mCherry protein within the cells upon glucose shock, indicating the intact delivery of mRNA and subsequent translation process. These findings underscore the potential utility of Fe-fum NPs as highly effective carriers for RNA molecules, thereby contributing to the advancement of RNA-based therapeutic and biotechnological applications. In summary, our study introduces an innovative room-temperature synthesis technique for producing Fe-fum NPs in mildly acidic aqueous settings. We demonstrate the ability to form Fe-fum NPs via biomimetic mineralization around biomacromolecules, encompassing diverse model proteins and large RNA molecules. This establishes an effective platform for delivering such molecules while preserving their functionality. Moreover, Fe-fum NPs were very efficient in safeguarding proteins from degradation during storage and against challenging environmental conditions.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: Biomacromolecules, proteins, mRNA, CRISPR-Cas, gene therapy, nanoparticles, metal-organic nanoparticles, iron-fumarate nanoparticles, Fe-fum NPs, biomimetic mineralization, drug delivery, cellular uptake, protein encapsulation, RNA delivery, endosomal release, lipid coating, gene editing, nanomedicine, controlled release.
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 540 Chemie
• Fakultäten: Fakultät für Chemie und Pharmazie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 20. Dezember 2024
• 1. Bericht­erstatter:in: Bein, Thomas