The Synthesis, Characterization, and Release of Poly(D,L-lactide-co-glycolide) Microspheres For Delivery Of Nogo-66(1-40) Antagonist Peptide and Chondroitinase ABC

Alexander Tatara and Shelly Sakiyama-Elbert

Department of Biomedical Engineering, Washington University in St. Louis

 

Spinal cord injuries devastate the lives of millions, victims and families alike. Unfortunately, regeneration of spinal nerve tissue is limited due to the inhibitory environment created by cells in the central nervous system following injury. The Nogo-66 receptor, expressed on the surface of neurons, interacts with inhibitory factors in the injured spinal tissue causing growth cone collapse and preventing axonal regeneration. Astrocytes surround the cyst that forms in an injury and become reactive, releasing chondroitin sulphate proteoglycans (CSPGs), molecules inhibitory to axon growth. This inhibitory environment prevents regeneration of the spinal cord and functional recovery from injury. The goal of my project was to create a drug delivery system to administer two target drugs in order to alleviate the inhibitory environment within the spinal cord following injury and encourage regeneration of damaged axons. The chosen drugs were Nogo-66(1-40) antagonist peptide, a protein which inhibits the Nogo-66 receptor, and Chondroitinase ABC, an enzyme which deactivates CSPGs. Poly(D,L-lactide-co-glycolide) (PLGA) microspheres were chosen as an ideal vehicle to deliver the drug, due to their inexpensive production and ability to release a drug in a slow, linear manner for up to two months after an initial large release. I designed a protocol to enhance encapsulation efficiency, reduce burst release, and produce an appropriately sized sphere. PLGA microspheres were synthesized, characterized for relative shape and diameter using bright field microscopy, and loaded with a model protein (Aprotinin) at different ratios of target drug to polyethylene glycol (a porogen) to test in vitro burst release profiles as analyzed with a whole protein concentration assay (micro-bicinchoninic acid assay). Plans for future testing involve using Lipoxidase as a second model protein, followed by testing the actual target drugs, Nogo-66(1-40) antagonist peptide and Chondroitinase ABC, with an assay to measure retained functionality after encapsulation and in vivo testing in animal spinal cord injury models.