S. A. Hilton1, C. Zgheib1, L. C. Dewberry1, M. M. Hodges1, S. Singh3, S. Seal3, G. Sener2, M. Krebs2, K. W. Liechty1 1University of Colorado,Laboratory For Fetal And Regenerative Biology, Department Of Surgery,Aurora, CO, USA 2Colorado School of Mines,Department Of Chemical And Biological Engineering,Golden, CO, USA 3University of Central Florida,Department Of Material Science Engineering, AMPAC And NSTC Center,Orlando, FL, USA
Introduction
Impairments in wound healing and wound strength are a significant clinical problem in diabetic wounds. We have previously shown that local injection of cerium oxide nanoparticles conjugated to MicroRNA-146a (CNP-miR146a) improves diabetic wound healing in a murine model through decreased inflammation and improved angiogenesis. We hypothesized that topical delivery of CNP-miR146a in a zwitterionic gel would be more clinically relevant, result in improved healing and improved wound strength.
Methods
12 week old female mice that are breed homozygous diabetic (Db/Db) were used. A single 8mm full thickness punch wound was made on the dorsal neck skin of each mouse. Zwitterionic gels were prepared by dissolving [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (SBMA) and 2-hydroxyethyl methacrylate (HEMA) in water. To load CNPs into the zwitterionic cryogels, CNP-miR146a was added in the gelation solution. Polymerization was initiated using APS and TEMED, then the reaction mixtures were poured into a plastic mold with inner diameter of 0.5 cm and polymerized at -20 ºC for 24 hr. Cryogels were thawed at room temperature. To remove unreacted monomers and other unbound ingredients, the cryogels were washed with PBS several times.
Wounds were treated with one time administration of zwitterionic gel only (n=5) or gel impregnated with CNP-miR146a (n=5, ~10ng Wounds were photographed over time to closure, and animals were euthanized 4 weeks after wound closure for biomechanical testing. A dumbbell shaped sample was taken from cranial to caudal on each mouse with the healed wound in the center. The Instron 5942 testing unit with Bluehill 3 Software was used for examining maximum load, extension, tensile strain.
Results
Mice wounds treated with CNP-miR146a gel demonstrated a significant improvement in time to complete wound healing. Untreated diabetic mouse wounds typically heal at day 22-24 post healing. Wounds treated with control gel healed at day 20 and wounds treated with CNP-miR146a impregnated gel healed at day 14 (P-value=0.002). Wounds also showed improved strength after healing with increased maximum load of 3.24N compared to 2.04N (P-value = 0.03). Elastic modulus measures resistance to being deformed when stress is applied. We see improved modulus with CNP-146a gel compared to control gel – 22.26MPa compared to 14.68MPa (P-value = 0.02). Tensile stress at maximum load is also improved – 1.63MPa in control gel compared to 2.59 in treatment gel (P-value = 0.03).
Conclusions
Diabetic mice wounds treated with zwitterionic gel impregnated with CNP-miR146a demonstrated improved time to complete wound healing, strength, elasticity, and resistance to stress after healing. This study shows feasibility of topical delivery of a therapeutic for diabetic wound healing via zwitterionic gel, as well as no adverse effects on wound strength.
