Development of Nano-Sized Delivery Systems to Improve Protein Pharmaceuticals in Cancer Applications
Morales Cruz, Moraima
Griebenow, Kai H. (Consejero)
Proteins possess a highly specific biological activity that can make them potent therapeutics, but their physical and chemical instability during formulation, storage, and delivery has limited their use. Engineering nano-sized vehicles to stabilize protein therapeutics and to allow targeted treatment of complex diseases, such as cancer, is of considerable interest. In this study we selected Cytochrome c (Cyt c) as the therapeutic protein to enable the design of a tumor targeted delivery system for apoptotic proteins. Cyt c is an essential mediator of apoptosis when it is released from the mitochondria to the cytoplasm, but in many cancer cells this mechanism is disabled. It has been demonstrated that the targeted delivery of Cyt c directly to the cytoplasm of cancer cells selectively initiates apoptosis in many cancer cells. Chapter 2 shows the capability of a two-step nanoprecipitation method to obtain Cyt c-loaded poly(lactic-co-glycolic) acid nanoparticles (PLGA NPs) without irreversible loss in protein function. PLGA NPs carrying encapsulated Cyt c were not efficient in causing apoptosis, presumably because of the slow sustained release or the fact that PLGA NPs are not taken up effectively. A novel smart-release Cyt c-based NPs was then design in Chapter 3 to enhance the apoptosis in cancer cells. Cyt c was precipitated as in Chapter 2 with a solvent-displacement method to obtain protein NPs. Instead of encapsulation into PLGA, Cyt c NPs were decorated with PLGA-SH through a redox-sensitive bond to prevent premature dissolution during delivery and allow subsequent Cyt c release under intracellular reducing conditions. Our results demonstrated that the coating with a hydrophobic polymer stabilizes Cytochrome c NPs allowing for their delivery to the cytoplasm of target cells. After smart release of Cytochrome c into the cytoplasm, it induced programmed cell death. In Chapter 4 we extend this study by transforming the system into an active delivery system. The results clearly demonstrate that the folate-decorated NPs were able to improve the Cyt c stabilization upon formulation of a smart drug delivery system and to increase the tumor specificity of the stabilized Cyt c-based NPs. This study confirms the relevance of incorporating additional tumor targeting strategies to enhance the benefit associated with the use of nano-sized delivery systems.