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Tuning The Local Dose Of Fk506 For Applications In Peripheral Nerve Regeneration Of Various Injury Types
Jenny Cheung1, Katelyn Chan1,2, Marina Manoraj1,2, Jennifer Zhang1, Konstantin Feinberg3, Kaveh Mirmoeini3, Tessa Gordon1,4, Gregory H. Borschel1,2.
1Division of Plastic and Reconstructive Surgery, The Hospital for Sick Children, Toronto, ON, Canada, 2Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON, Canada, 3The Hospital for Sick Children, Toronto, ON, Canada, 4Division of Plastic and Reconstructive Surgery, Department of Surgery, University of Toronto, Toronto, ON, Canada.

PURPOSE: Following peripheral nerve injury, endogenous regeneration mechanisms are slow, and quality of recovery is often sub-optimal. FK506 is an FDA-approved immunosuppressant, but studies have shown its additional use as a neurotrophic agent. However, systemic administration of FK506 causes harmful side effects. To mitigate these, we previously developed a local FK506 delivery nerve wrap by encapsulating FK506 within biodegradable polycarbonate urethane (PCNU) fibers. These PCNU/FK506 nerve wraps are easy to synthesize, apply, and are geometrically tunable for use in a variety of clinical cases. In this work, we aimed to study the effects of different local FK506 dosages based on various nerve wrap dimensions and corresponding drug loadings.
METHODS: Coaxial electrospinning was used to encapsulate FK506 in PCNU fibers by electrically charging the inner core (PCNU/FK506) and outer shell (PCNU only) solutions flowing at a constant rate, creating a fibrous matrix. Nerve wraps were sectioned from the fibrous matrices with the following dimensions: small (1cm x 0.5cm), medium (1.5cm x 2cm), and large (2cm x. 2.5cm) from four separate mats (n=4). Encapsulation efficiency and FK506 loading were determined using liquid chromatography tandem mass spectrometry (LC-MS). In vitro FK506 release profiles from each group were determined by incubating wraps at physiological temperature, collecting release media over 31 days, and analyzing with LC-MS. Neurotrophic effect of FK506 was compared amongst groups. Dorsal root ganglia (DRGs) were harvested from 15-day-old rat embryos and incubated in early timepoint FK506 release media from all nerve wrap groups, and extended time points for medium wraps. Positive and negative controls contained 100 ng FK506/mL and media alone, respectively. After 48 hours, DRGs were fixed, stained for 3-tubulin, and imaged to analyze neurite extension and density.
RESULTS: Encapsulation efficiency was up to 97% for all groups, comparable to other FK506 delivery systems. Mean drug loadings (ng FK506/g nerve wrap standard deviation) for small, medium, and large nerve wraps were: 69.7 9.7, 116.5 27.6, and 163.8 40.1, respectively. Mean FK506 loading for small is significant compared to medium and large wraps; however, this is not so between medium and large. This indicates that surface area of nerve wraps may not directly correlate to FK506 loading; thickness or wrap weight should also be considered. In vitro drug release profiles show burst release across all groups; however, 20% of FK506 still remained in large nerve wraps after 31-days. No significant neurite extension differences were visually observed across all groups compared to the positive control, indicating FK506 bioactivity is retained. The positive and negative controls had a neurite density of 97% and 80% respectively. All nerve wrap groups had a % density within this range indicating presence of FK506 was better than none at all.
CONCLUSION: We successfully synthesized a PCNU nerve wrap for local and bioactive delivery of FK506. PCNU nerve wraps almost completely encapsulated FK506. Future work will aim to extend the FK506 release period and apply PCNU/FK506 nerve wraps to various in vivo injury models for optimization of FK506 doses to these injury types.


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