(291g) Enzymatic Protection and Biocompatibility Screening of Enzyme-Loaded Polymeric Nanoparticles for Neurotherapeutic Applications.

Introduction: Drug delivery of enzyme therapeutics encounters many obstacles, including systemic clearance, protease degradation, endothelial barriers to target organ entry, and poor penetration in local tissue parenchyma. The tightly regulated blood-brain barrier (BBB) is a further hindrance specifically for neurological disease and inhibits 98% of small molecule and ~100% of enzyme and other large-molecule therapeutics from entering the brain from the bloodstream. Leveraging polymeric nanoparticles for encapsulating and delivering therapeutic cargo can help overcome several of these barriers. However, nanoparticle platforms must be carefully engineered to ensure biocompatibility. An important consideration that could influence drug delivery and efficacy is how disease conditions alter the tissue microenvironment. The presence of inflammation or oxidative stress could affect the susceptibility of cells to inadvertent damage. Using organotypic whole hemisphere brain slice models, we can screen nanoparticles for biocompatibility under different disease conditions in high-throughput fashion.

Materials and Methods: We formulated enzyme-encapsulating poly(lactic-co-glycolic acid)-block-poly(ethylene glycol) (PLGA-PEG) polymeric nanoparticles with catalase as a model enzyme. Comparing double emulsion and nanoprecipitation formulation methods, and cholic acid (CHA) and poly(vinyl alcohol) (PVA) surfactants, we evaluated the nanoparticles’ ability to protect encapsulated catalase from extracellular protease degradation to extend enzymatic activity. To study nanoparticle biocompatibility under neurological disease conditions, we developed organotypic whole hemisphere brain slice models of neuroinflammation with lipopolysaccharide (LPS) exposure, and excitotoxicity with monosodium glutamate (MSG) or oxygen glucose deprivation (OGD) exposure. Upon application of blank nanoparticles without catalase on our different disease models, we evaluated cytotoxicity via lactate dehydrogenase (LDH) release to assess nanoparticle biocompatibility. To explore the potential role of oxidative stress in cell death, we determined reduced glutathione (GSH) concentrations.

Results: All formulated nanoparticles exhibited a near-neutral surface charge and sub-100 nm size, requisite characteristics for effective diffusion in brain parenchyma. Regardless of surfactant, nanoprecipitation provided no extension of enzyme activity compared to free catalase, while CHA-emulsified double emulsion nanoparticles provided significant increases in catalase activity. For our brain slice models of neurological disease, we observed that MSG and OGD exposure elicited a 71.1% and 41.1% increase in cell death, respectively, and application of superoxide dismutase (SOD) on both MSG and OGD models fully inhibited cell death. LPS exposure did not induce an increase in cell death but did elicit an increase in inflammatory cytokine mRNA expression. We used our ex vivo slice models to screen our nanoparticles for toxicity to develop a biocompatible formulation. Surprisingly, double emulsion nanoparticles induced significant toxicity when applied to MSG- and OGD-exposed treated slices, but not for non-treated or LPS-exposed brain slices, while nanoprecipitation nanoparticles showed no toxicity for all slice treatment conditions. Double emulsion nanoparticle toxicity was consistently observed on excitotoxicity-induced slices for specifically PLGA-PEG nanoparticles made with dichloromethane as the organic solvent during formulation. Toxic nanoparticle application to MSG-exposed slices did not elicit a lower GSH concentration compared to MSG exposure alone, indicating nanoparticles did not exacerbate cell death through affecting the oxidant/antioxidant. Toxicity was eliminated when chloroform was used as the organic solvent, or with the addition of free PEG during the sonication steps of the double emulsion formulation process.

Conclusions: Our work demonstrates that polymeric nanoparticles provide promise for enzyme delivery. Double emulsion formulated polymeric nanoparticles can provide protection of enzymatic cargo from degradative in vivo conditions, but also showed toxicity in slices. Double emulsion PLGA-PEG nanoparticles are capable of enzyme protection from proteases, and are biocompatible with diseased brain slices, when free PEG is present during sonication. However, these findings demonstrate that nanoparticle therapeutics must be stringently evaluated and optimized for biocompatibility for effective progression into preclinical in vivo studies. Additionally, our results encourage the systematic approach of screening therapeutics on ex vivo brain slice models to isolate, understand, and overcome neurological disease processes to aid in preclinical neurotherapeutic development.