Poster J2 - Pharmacokinetics of nerve-regenerative polymersomes post sciatic nerve injury

Friday, October 25, 2024

3:30 PM - 4:30 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Vanessa Marahrens (she/her/hers)

Undergraduate Researcher
Clemson University
Savage, Minnesota, United States
Sophia Fetten (she/her/hers)

Undergraduate Researcher
Clemson University
Charleston, United States

Primary Investigator(s)

Jessica Larsen, PhD

Carol and John Cromer '63 Family Endowed Associate Professor
Clemson University Department of Chemical and Biomolecular Engineering, South Carolina, United States

Introduction: When an injury occurs to a peripheral nerve, the damage is often irreversible and attempted repair is abysmally slow, causing the patient to experience permanent disability or neuropathy. The injury can be caused by a traumatic crush or severance of the nerve and causes the axons to undergo Wallerian degeneration. In the peripheral nervous system (PNS), the axon segments next to the injury site can regrow slowly, but the growth and development of these nerves are constrained by stress granules which interfere with nerve regeneration by inhibiting protein synthesis and disrupting cellular homeostasis. Full recovery of injured nerves is only successful when there is a growth distance of a few centimeters from the target. If the distance is larger than a few centimeters, nerve regeneration is rarely successful and often needs surgical intervention. Currently, the blood-nerve barrier (BNB) limits non-invasive routes of therapeutic administration to the nerve. . The BNB is the endoneurial microenvironment that interfaces the neurons and the vasculature of the PNS. There are tight junctions that serve to protect the peripheral nerves by limiting access to harmful substances to enter from the bloodstream. To bypass the BNB and allow the option for a non-invasive therapy for patients, targeted nanoparticles called polymersomes were created to deliver payloads to nerves.

Materials and

Methods: Polymersomes are vesicles made from amphiphilic diblock co-polymers. In our work we use polyethylene glycol (PEG, 1000 Da) polylactic acid (PLA, 5000 Da) (PEG-PLA) as the polymersome backbone. A hydrophobic ratio of 25-40% leads to the formation of a vesicle, which contains a hydrophobic core, capable of encapsulating hydrophobic molecules, an aqueous center, capable of encapsulating hydrophilic molecules, and an exterior layer of PEG that helps shield the vesicle by drawing in water molecules and promoting specific protein absorption.Targeted delivery is achieved by attaching a variety of ligands to the exterior PEG layer. The two ligands chosen were Apolipoprotein E3 (ApoE), to target inflammation, and Rabies virus glycoprotein-based peptide (RVG, to target nicotinic acetylcholine receptors (nAchR) and neural cell adhesion molecules (NCAM)). Polymersomes were created and characterized to confirm successful ligand attachment via dynamic light scattering. They were confirmed to be non-toxic in neural cell lines via MTS assay. A sciatic nerve injury (ScNI) was created in Sprague Dawley rats, followed by either intranerve (IN) or intramuscular (IM) injection of 100 mM polymersomes with near-IR dye (Alexa Fluor 647). The animals were monitored over a 48-hour period, during which they were imaged using IVIS, and blood plasma samples were collected at 1, 4, 24, and 48 hours post-injection.

Results, Conclusions, and Discussions: Sprague Dawley (SD) rats underwent unilateral ScNI, followed by either an IN or IM injection of untagged (n=3 per route), ApoE-tagged (n=3 per route), and RVG-tagged (n=3 per route). In Figure 1, the results of dye contained in the injury site were observed via IVIS at the time periods of 1, 4, 24, and 48 hours post injection. With IN injection, both ApoE and RVG tagged polymersomes enabled high AF647 retention in the nerve at one hour post-injury. With IM injection, only RVG tagging enabled neural fluorescence at one hour post-injury. RVG led to the overall highest detectable fluorescence in the nerve likely due to specificity towards neural receptors. For IM injections, reduced retention was expected due to the non-invasive treatment route and needing to overcome the BNB barrier, which could improve with increased polymersome doses, as 100 mM is a very low dose. The nAch and NCAM receptors are more nerve-specific than the lipoprotein-related receptors that are upregulated during inflammation. Overall based on our in vivo analysis, ApoE appears to lead to longer polymersome retention in the injured nerve, while RVG enables increased nerve penetration of polymersomes. SD rats were euthanized and their organs were collected for a post-mortem analysis. Figure 2 depicts the IVIS imaging of the kidneys, livers, spleens and sciatic nerves to get a better understanding of how the polymersomes are excreted. In general, the excretory organs from the SD rats that were injected with polymersomes IN experienced a higher AF647 concentration at 48 hours than those rats who were injected with polymersomes IM. This is likely due to the fact that polymersomes take longer to enter the bloodstream when injected IN and therefore are eliminated more slowly by the excretory organs than IM-injected counterparts. Hematoxylin and Eosin (H&E) staining was performed to ensure no damage was done to the excretory organs and that they were in a healthy condition at the end of the experiment. The overall experiment gave significant insight to the possibilities of drug delivery to the peripheral nervous system using polymersomes and either ApoE or RVG targeting ligands.

Acknowledgements (Optional):