Biopolymer-Hybrid Nanomaterials for Vaccine Formulation

During the last three decades, genetic vaccines have emerged as promising therapeutic strategy to tackle cancers, autoimmune and allergic diseases as well as viral infections. Until recently, the clinical breakthrough of the technique was significantly constrained by stability issues of the administered genes and low transfection efficacies. To stabilise the fragile genes, prevent their premature degradation and systemic clearance upon administration, a variety of carrier materials, including liposomes, virosomes, peptides, dendrimers and polymers, were developed. Among these, biodegradable polydopamine nanoparticles (PDA NPs) represent a particularly interesting delivery platform, due to their tunability, chemical versatility and economical synthesis. However, the material possesses an inherent negative surface potential and therefore requires the introduction of positively charged moieties to render the NPs suitable for efficient nucleic acid immobilisation. Within this thesis, the preparation, functionalisation and characterisation of well-defined PDA NPs containing cationic amino acids, peptides and polymers, as well as their application as non-viral gene delivery vectors were explored (Figure 1). Figure 1: The modification of biopolymer NPs with functional moieties, such as cationic amino acids, peptides or polymers, enables the formulation of novel gene delivery materials. The first studies aimed at introducing positive charges to PDA NPs by using L-arginine or arginine analogues. Although co-polymerisation of dopamine with a guanidino-modified co-monomer was conducted, post-modification of PDA NPs by L-arginine surface grafting resulted in the formation of the desired material (Arg-PDA NPs). Arg-PDA NPs were thoroughly characterised and assessed on their ability to bind and protect messenger ribonucleic acid (mRNA) and plasmid deoxyribonucleic acid (pDNA) against nuclease degradation. When gene@Arg-PDA NPs were administered to human embryonic kidney cells (HEK-293), the pDNA-based formulation performed more efficiently than its mRNA counterpart. However, the resulting pDNA transfection efficacy was only 21 % of the commercial standard Lipofectamine 2000. To improve nucleic acid binding and boost the transfection, PDA NPs were modified with three bioactive peptides, an endosomal-escape sequence (R2H2), a nuclear localisation signal (NLS) and decaarginine (R10). Although the novel materials immobilised pDNA 2x more efficiently than Arg-PDA NPs, they failed to deliver it to HEK-293 cells, most likely due to the neutralisation of the surface charge upon gene binding, resulting in reduced colloidal stability. These results required additional redesign of the nanocarriers to further enhance pDNA binding, cell entry and endosomal escape of the formulation, which was achieved by modification of PDA NPs with poly-L-arginine (polyR). Despite improved colloidal stability and strong nucleic acid binding, no expression of the immobilised gene was observed upon cell administration, prompting further investigations of the delivery process. Confocal microscopy studies with fluorescently labelled pDNA highlighted that the pDNA@polyR-PDA NP formulations were constrained by inefficient gene-carrier dissociation upon cell entry. To improve the intracellular gene release, and further boost the uptake and endosomal escape of the complexes, pDNA@polyR-PDA NPs were co-formulated with CaCl2. This resulted in significantly increased expression of the cargo pDNA, indicating potential synergistic effects of polyR-PDA NPs and Ca2+ ions for gene delivery, as the transfection efficacies exceeded those of Lipofectamine and plain CaCl2 by more than 3x and 2.5x respectively. In summary, this thesis explored the potential of PDA NPs as nucleic acid carriers by modifying the material with cationic amino acids, peptides and polymers. It provides the foundation for a broader scope of PDA-mediated gene delivery in the near future and highlights the importance of colloidal stability, cellular uptake and efficient gene-carrier dissociation in this context.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2022

Author dc:creator

• Franck, Christoph

Advisor dc:contributor.advisor

• Fruk, Ljiljana

• Gene Delivery
• Polydopamine
• Nanotechnology
• Biopolymers

Licence

• http://purl.org/NET/rdflicense/allrightsreserved

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.89416

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/341998