T17 - Deoxyribonuclease I-Loaded Fibrin Specific Nanogels for Treatment of Immunothrombosis

Friday, October 10, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

KC

Kevin M. Cole, II

Student
North Carolina State University, United States

Co-Author(s)

Ethan James Pozy

Graduate Student
Lampe Joint Department of Biomedical Engineering at UNC Chapel Hill and NC State University
Durham, North Carolina, United States
MP

Megan G. Palmisano

Post Doctoral Research Fellow
North Carolina State University, United States
Rosemary L. Bayless, DVM, MS, PhD, DACVIM (LAIM)

Assistant Professor
North Carolina State University
Raleigh, North Carolina, United States

Last Author(s)

Ashley C. Brown

Lampe Distinguished Professor
North Carolina State University and University of North Carolina at Chapel Hill
Raleigh, North Carolina, United States

Primary Investigator(s)

Ashley C. Brown

Lampe Distinguished Professor
North Carolina State University and University of North Carolina at Chapel Hill
Raleigh, North Carolina, United States

Introduction: : Sepsis is a condition that is characterized by a dysregulated immune response to an infection. Septic patients have an increased risk of developing immunothrombosis. Immunothrombosis is the process of blood clot formation driven by activated white blood cells, particularly neutrophils. These clots obstruct blood flow, which can damage tissues and organs, and ultimately contribute to an increased mortality rate. Neutrophils directly contribute to immunothrombosis via the release of neutrophil extracellular traps (NETs). The NETs are composed of nuclear DNA that is released in a web-like formation to trap and neutralize pathogens. These NETs also contribute to a more prothrombotic environment and cause clots to be more resistant to degradation with limited therapeutics. This study investigates the use of fibrin-specific nanogels (FSNs) dual-loaded with tissue plasminogen activator (tPA), which breaks down clots, and deoxyribonuclease I (DNase), which degrades the nuclear DNA content in NETs, to serve as an effective treatment for neutrophil-mediated immunothrombosis. We hypothesize that DNase will increase the rate of clot degradation in the presence of neutrophils.

Materials and

Methods: : Core gels were synthesized using 10% Bis and 90% NIPAM. Then, a shell comprised of 93% NIPAM, 2% Bis, and 5% acrylic acid was synthesized on top of the cores to form core-shell gels. These were functionalized using N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide (EDC), N-hydroxysuccinimide (NHS) chemistry with a fibrin-specific antibody fragment. Unconjugated antibody fragments and remaining EDC + NHS were removed by dialysis. FSNs were non-covalently loaded with tPA and DNase to allow for drug delivery at dosages of 300 ng/mL for tPA alone or in combination with DNase at 3 and 30 U/mL. Whole blood was collected in sodium citrate anticoagulant from six healthy university-owned horses via jugular venipuncture. Horse blood was used as it served as a more representative model to humans, and their blood coagulates more, compared to the traditionally used mouse blood. Neutrophils were isolated via Ficoll density centrifugation and were stimulated with 12-myristate 13-acetate (PMA) to induce NET release. Ex vivo fibrin clots were created for clot absorbance assays to evaluate clot degradation in the presence or absence of stimulated neutrophils. FSNs containing tPA alone, tPA and DNase at 3 U/mL, or tPA and DNase at 30 U/mL, respectively. were added to these clots endogenously (during the clot formation process) or exogenously (as an overlay on top of a formed clot). Absorbance (350 nm) was measured every 45 seconds for 12 hours to monitor clot degradation.

Results, Conclusions, and Discussions:: The presence of stimulated neutrophils reduced the rate and extent of clot degradation by FSNs loaded with tPA alone (Fig. 1A). When DNase was included with tPA in the FSNs, clot degradation returned to a similar level as that observed when tPA FSNs were delivered in the absence of neutrophils. These trends were observed in both exogenous and endogenous clots (Fig. 1B). Area under the curve (AUC) analysis showed that endogenous 3 U/mL and 30 U/mL DNase tPA FSNs in the presence of neutrophils had significantly smaller AUC than fibrin-thrombin clots. This finding was consistent with exogenous clot fibrinolysis, with the inclusion of 3 U/mL tPA FSNs in the absence of neutrophils resulting in significant decreases in AUC from fibrin-thrombin clots. Clot degradation rates showed that endogenous 3 U/mL DNase tPA FSNs in the presence or absence of stimulated neutrophils had significantly higher degradation rates than our fibrin-thrombin clots, but were not significantly different between treatment groups. All exogenous treatments exhibited significantly higher degradation rates than fibrin-thrombin clots, but there were no significant differences between treatment groups. Time to half of peak clot formation showed no differences between treatments. The presence of stimulated neutrophils diminished the action of tPA on clot degradation. When stimulated neutrophils were present, FSNs loaded with tPA alone did not affect the AUC of clots, while FSNs loaded with both tPA and DNase significantly reduced clot AUC compared to control fibrin-thrombin clots. The results from this study show promise for DNase as a treatment for immunothrombosis when delivered via FSNs, preventing off-target anti-coagulant action and subsequent bleeding in critically ill patients.

Acknowledgements and/or References (Optional): :