Nanosized Core-Shell Protein Drug Delivery Systems

Abstract

Proteins have the complexity and specificity to be effective therapeutics. However, the same complexity that confer proteins with high specificity makes them susceptible to stability problems, immunogenicity, or rapid clearance, which shorten their circulation time in the blood stream and reduce their efficacy. The use of drug delivery systems (DDS) is usually required to better exploit proteins’ therapeutic potential safely. Nanotechnology has shown to be a promising tool to overcome limitations encountered when working with proteins. Advantages of nano-sized DDS include passive targeting, larger payloads, and when combined with other features they can be conferred with active targeting and stimuli-triggered responses like drug release under specific conditions. Despite nanotechnology having been proven to enhance drug’s efficacy and lower their secondary effects, there is still need for improvement. So far only around 1% of nano-sized formulations reach the tumors in cancer therapy.1 One way to ameliorate this limitation is to increase the loading capacity of the systems, which can enhance the treatments’ outcome. Nanoprecipitation has shown to be a reliable method to obtain nanoparticles (NPs) made completely out of protein, which allows to construction of DDS with higher payloads. Due to the hydrophilicity of most proteins, the protein NPs obtained via nanoprecipitation are not stable in aqueous media and would dissociate if exposed directly to physiological conditions. To overcome this, the protein NPs obtained by nanoprecipitation are stabilized by crosslinking or surface modifications. Cytochrome c (Cyt c) is a protein that induces apoptosis when delivered directly to the cell’s cytosol and has been used as a therapeutic protein in preclinical studies for cancer therapy. A DDS for Cyt c was previously developed: Cyt NPs to which the amphiphilic block copolymer thiol-poly(lactic-co-glycolic acid)- poly (ethylene glycol)- folate (SH-PLGA-PEG-FA) was covalently linked to the Lys residues exposed on the surface of the Cyt c NPs. Cyt c-PLGA-PEG-FA NPs are stable under physiological conditions and showed specific cytotoxicity towards cancer cells over expressing folate receptor (FR). However, the reported Cyt c-PLGA-PEG-FA NPs diameter of 338 nm is on the large side of what is considered an optimal size for NPs to be used in cancer therapy. Moreover, the superiority of a nano-sized formulation for Cyt c vs Cyt c molecules individually modified has not been proven. It is known that the covalent modification of proteins may negatively impact their activity. Additionally, some protein NPs do not have enough residues exposed and available to perform covalent modification to stabilize themselves.

From here the two aims of this thesis were designed and executed. The first aim was to compare Cyt c-PLGA-PEG-FA NPs vs Cyt c-PEG-FA, reduce the diameter of the reported Cyt c-PLGA-PEG-FA NPs and test them in Lewis Lung Carcinoma (LLC). The second aim was to develop a DDS in which no covalent modification of the protein was necessary. Thus, a method to encapsulate negatively charged proteins in a chitosan (CS) nanocapsule using α-casein as model protein was developed. For the first aim, a NP-free formulation (Cyt c-PEG-FA) was synthesized by conjugating Cyt c to poly (ethylene glycol)- folate (PEG-FA) using an amine-to-sulfhydryl crosslinker. Cyt c-PEG-FA was characterized using UV-Vis spectroscopy. Cyt c-PLGA-PEG-FA NPs were obtained by Cyt c nanoprecipitation followed by surface decoration with the co-polymer SH-PLGA-PEG-FA. Different conditions during the nanoprecipitation step were explored to optimize the NPs diameter. The efficacy of both DDS for Cyt c was tested in a cell free system, and in-vitro in Lewis Lung Carcinoma (LLC) and HeLa cells. Cyt c-PLGA-PEG-FA NPs were tested in-vivo using an LLC mouse model. For the second aim, the protein NPs were obtained via nanoprecipitation and then coated with CS electro-static self-assembly (ESA). Diameter, morphology, and surface charge of the NPs in both aims were studied using dynamic light scattering (DLS), scanning electron microscopy (SEM), and zeta potential determination, respectively. The results of the first aim show that the diameter of Cyt c-PLGA-PEG-FA NPs was reduced to 253 nm, 100 nm less than the previously reported system. Cyt c in the optimized NPs retained 88-96% of its caspase activation activity. The superiority of Cyt c-PLGA-PEG-FA NPs Vs Cyt c-PEG-FA was confirmed: in-vitro, IC50: 49.2 versus 129.5 μg/ml, respectively in LLC cells. Cyt c-PLGA-PEG-FA NPs showed specific glutathione (GHS) triggered release and specific toxicity towards cancer cells. The in-vivo studies confirmed their accumulation in LLC tumors 5 minutes after injection. On the other hand, the second aim resulted in coated protein NPs with diameters from 146±9 nm to 508±64 nm. The diameter was dependent on the CS concentration during the coating step. A maximum encapsulation efficiency of 92.7% was achieved. Optimum pH of 7.4 triggered sustained release of 82.4% of the protein in 10 h. The generality of the encapsulation method was verified with bovine serum albumin (BSA). The results shown in this thesis demonstrate the potential of nanotechnology and specifically nanoprecipitation to construct DDS for proteins and exploit their therapeutic potential. Two DDS with high loading capacity are presented in this thesis, each one with different features and applications.