Intracellularly dissolving chromophore-decorated protein-based nanoparticles for photodynamic cancer therapy.
Molina-Calzada, Anna M.
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Cancer has proven to be extremely complex to treat and cure due to its heterogeneity. Current treatments rely mainly on chemotherapeutic drugs, which typically result in multidrug resistance and the generation of additional genetic mutations. In this context, protein-based therapeutics are a promising alternative. In general, proteins are natural and biocompatible macromolecules with highly specific biological activities. However, their physical and chemical instability make their formulation, storage and delivery very challenging. The emerging field of engineering nano-sized delivery systems provides exciting new opportunities to formulate proteins into nanoparticles. In this dissertation, intracellularly dissolvable protein-based nanoparticles are used as carrier for photosensitizer drugs. Photosensitizers induce cell death after irradiation by the generation of reactive oxygen species in a treatment known as photodynamic therapy (PDT). In Chapter 3, the redox-sensitive albumin nanoparticles for the delivery of the photosensitizer drug Chlorin e6 (Ce6) is systematically studied. The reversible inactivation of Ce6 as a consequence of immobilization on the nanoparticle surface was evidenced. Furthermore, its activation upon the nanoparticle dissolution in a reducing environment, such as the cell interior, was evaluated. In vitro experiments show that the synthesized redox-sensitive system enhances the function of albumin nanoparticles as carriers for photosensitizer drugs, as it prevents phototoxicity during delivery, and allows for drug activation only after cell uptake. In Chapter 4, the redox-sensitive protein-based nanoparticles are prepared with Cytochrome c (Cyt c), a pro apoptotic protein, and decorated with Ce6 for low-dose PDT. Low dose PDT has been associated with the induction of apoptosis whereas high dose PDT is associated with the induction of necrosis, a type of cell death that produces inflammation and affects neighboring tissue. The experimental results show that, upon modification, the Cyt c loses some of its pro apoptotic activity, but that a high level of function can be retained at a low drug:protein ratio. Additionally, in vitro experiments results showed that the protein nanoparticles were internalized by the cells, even without the use of a targeting moiety. The combination of both therapeutic agents into a single nanoparticle allowed for a greater induction of cell death. Evidence showed that the death induced was via apoptosis instead of necrosis.