Nanoparticle Delivery of Keap1 siRNA Accelerates Diabetic Wound Healing
Zachary M. Borab, BA, William J. Rifkin, BS, Joshua A. David, BS, Anna Zhou, MD, Joseph Frezzo, PhD, Sophia Hameedi, BS, Jin K. Montclare, PhD, Piul S. Rabbani, PhD, Daniel J. Ceradini, MD.
New York University, New York, NY, USA.
Purpose: Delayed diabetic wound healing is a prevalent and unsolved clinical problem. We have previously described that the Nrf2/Keap1 redox pathway is dysfunctional in diabetics and contributes to the delayed wound healing phenotype. We engineered a self-assembling lipoproteoplex, with Keap1 siRNA to aid in the restoration of Nrf2 and to enhance the rate of diabetic wound healing.
Methods: We designed a novel formulation of DOTAP Sodium Cholate and a super-charged protein, which was used to package siRNA and then applied to standardized wounds on diabetic mice. Wound beds were collected at several time points for analysis of tissue histology and gene expression. Rate of wound closure was calculated using photometric analysis.
Results: In the wound bed, Keap1 siRNA treated mice decreased Keap1 gene expression (55.4±22.9%; p=0.004) and protein expression (94±0.42%; p=0.038) compared to controls. Downstream protein expression of Nrf2 increased (3.27±1.63 fold; p=0.039). The expression of antioxidant genes, NQO-1 and MnSOD-1 increased (4.99±0.1 fold; p=0.04 and 3.66±0.2 fold; p=0.01, respectively). The expression of the chemokine, SDF-1 increased (7.51±0.12 fold; p=0.01). Additionally, the expression of EGF increased (6.48±0.15 fold; p=0.005). At day 10, the epithelial gap of Keap1 treated mice decreased compared to control mice (3.95±0.21 mm vs 8.07±1.37mm; p=0.004), granulation tissue increased (2.15±0.65 mm2 vs 1.15±0.44 mm2; p= 0.049), and CD31+ cells/hpf increased (109.8±17.96 vs 71.2±27.32; p=0.02). Keap1 siRNA treated mice resulted in accelerated time to wound closure (22.3±1.974 days vs 31 days; p=0.0213). Wound burden was decreased markedly by 57% compared to controls.
Conclusions: Our novel formulated drug vehicle effectively delivered siRNA to the wound bed and resulted in a clinically relevant acceleration of wound healing. Acceleration was accomplished through downstream changes in redox homeostasis, growth factors, and blood vessel formation. This novel therapy could be rapidly translated for clinical use.
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