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Class Year

2020

School

School of Medicine

Abstract

Title: Chitosan Microparticles: A Potential Therapeutic Intervention for Alzheimer's Disease with Cerebral Amyloid Angiopathy

Introduction: Alzheimer’s Disease (AD) is an irreversible neurodegenerative disorder characterized by amyloid beta (Ab) plaques in the brain parenchyma. Cerebral Amyloid Angiopathy (CAA), a common comorbidity with AD, is defined by Ab plaques form in the walls of the cerebral vessels. The past decade has seen significant progress in plaque diagnosis without similar therapeutic gains. In patients with CAA/AD the vascular plaques activate a part of innate immune system called the complement cascade, leading to lysis of cerebrovascular smooth-muscle cells (cvSMC) and vascular fragility. Chitosan-based microparticles (CMPs) may protect vessels and slow the progression of CAA/AD by delivering a plasmid to selectively upregulate CD59, the natural defense against activated complement. Chitosan is a versatile gucosamine polysaccharide derived from arthropod exoskeletons. Purified chitosan demonstrates high biocompatibility and low cytotoxicity.

Materials and Methods: CMPs were precipitated under mildly acidic (pH 5) conditions from high molecular weight chitosan (85% deacetylated) in the presence of the cross-linker sodium triphosphate. Encapsulation of plasmids or cumarin-6 was spontaneous during precipitation. Cellular toxicity, targeting and transfection efficacy were analyzed in primary human cvSMC culture. CMPs toxicity was determined by MTT assay of cvSMC after exposure to various particle concentrations. Targeting was examined by visualizing uptake of CMPs fluorescently labeled with cumarin-6. To measure transfection efficacy, cultured cvSMC were exposed to GFP- or CD59-loaded CMPs and the levels of GFP or CD59 protein expressed were analyzed by western blot, in comparison to empty CMPs.

Results: CMPs were non-toxic even the highest concentrations. Uptake of CMPs appears to be dependent on cvSMC morphology. Fluorescently-labeled CMPs colocalized with smooth muscle alpha-actin. Particle uptake and transfection in cultured cvSMC was confirmed by changes in protein expression after cvSMC exposure to plasmid-loaded CMPs.

Conclusions: Chitosan microparticles are nontoxic and taken up by human cvSMC in a phenotype-dependent manner. Cells exposed to plasmid-loaded particles are readily transfected with the protective glycoprotein CD59. Taken together, this indicates that CMPs are a promising future candidate for therapeutic intervention to prevent vascular fragility from activated complement in AD/CAA.

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