Román-Martínez, Angélica

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    Polymeric nanocarriers for the enhancement of enzyme replacement therapy in lysosomal storage diseases: A proof of concept study
    (2016) Román-Martínez, Angélica; Latorre-Esteves, Magda; College of Engineering; Almodovar, Jorge; Torres-Lugo, Madeline; Department of Chemical Engineering; Cortes-Figueroa, José E.
    Lysosomal Storage Diseases (LSDs) are a group of inheritable genetic diseases caused by mutant lysosomal enzymes, leading to the accumulation of undigested macromolecules in the lysosomes and causing increases in lysosome size and number, cellular dysfunction, clinical abnormalities and premature death. These LSDs can be treated with Enzyme Replacement Therapy (ERT) through intravenous administration of a recombinant enzyme in replacement of the defective enzyme. However, this is an expensive and inefficient method with adverse side effects associated with the high enzyme amounts required for the treatment, the need of posttranslational modification of the enzyme and the host immune system response. Nanoparticle drug delivery systems (DDSs) are a promising alternative to enclose the therapeutic cargo, then overcoming the drawbacks associated with ERT. As building blocks of those DDSs, biodegradable synthetic polymers are considered an attractive alternative for protein delivery applications because they can be designed to obtain desirable properties like low immune response, stimuli response, specific circulation time and affinity to certain drugs and environments. This research project aims to develop biodegradable and bioresponsive polymersomes, composed of amphiphilic block copolymers of Polyethylene glycol and Polycaprolactone (PEGPCL), suitable for the encapsulation and delivery of protein therapeutics as proof of concept for ERT applications. This goal was achieved through the synthesis and optimization of protein loaded polymersomes using a Water in-Oil-in Water (WOW) double emulsion method, and the addition of a protein corona composed of Fetal Bovine Serum (FBS) components. First, a group of copolymers were synthesized by Ring Opening Polymerization, using either Stannous Octoate (SO) or Hydrochloric Acid (HCl) as catalysts. Characterization was carried out by Proton Nuclear Magnetic Resonance (1H-NMR) and Gel Permeation Chromatography (GPC). Results demonstrated that SO was a better catalyst for achieving the synthesis of block copolymers of the desired molecular weight. This catalyst was used to synthesize the copolymers used in the formation of bioresponsive polymersomes. The second part of the project was to prepare a series of Water-in-Oil in Water (WOW) emulsion formulations by varying copolymer molecular weight, solvent evaporation pressure, copolymer: stabilizer ratio, emulsification technique and protein concentration in the aqueous core. The in vitro performance of the polymersomes was assessed in order to obtain ideal properties in terms of behavior under biologically relevant buffers, size, encapsulation efficiency and protein release profile. The optimal formulation consisted of using PEG–PCL with a molecular weight of the polymeric chain of 2000g/mol and 5000g/mol, respectively (PEG2000-PCL5000); a ratio of 1:1 of PEG–PCL:PVA, and reduced pressure (generated by a vacuum pump) for solvent evaporation to generate the desired polymersomes through the WOW emulsion method. In the aqueous core, the addition of model protein with a concentration of 10mg/mL (compared to concentrations of 25 and 50 mg/mL) gave the best entrapment efficiency. These polymersomes were found to be nontoxic at concentrations of up to 2mg/mL. A protein corona was added to the polymersomes to further fine-tune the desired protein release behavior. This led to the formulation of a system that completely suppresses protein release in physiological conditions (Phosphate Buffer Saline Solution, PBS, pH 7.4) and shows sustained release in acidic lysosomal environment (Artificial Lysosomal Fluid, ALF, pH 4.5). The size of these particles was determined to be theoretically appropriate for cellular internalization through measurement by Dynamic Light Scattering techniques (221 ± 21 nm in PBS and 190 ± 66 nm in ALF). We conclude that we have developed a system that warrants further investigation for the development of polymeric nanocarriers that are suitable for the enhancement of Enzyme Replacement Therapy for the treatment of Lysosomal Storage Diseases.