OC 69.1 - SERPIN-Inspired Nanomedicine Targeting to Activated Neutrophils Provides Safe and Effective Thromboprotection

Tuesday, July 12, 2022

2:45 PM – 3:00 PM

Location: ExCel Center, ICC Capital Suite Room 1

Presentation Format: In-Person

Presenting Author(s)

Dillon Bohinc

Research Associate II
Case Western Reserve University
Cleveland, Ohio, United States

Co-Author(s)

MC

Michelle Cruz

PhD Candidate
Case Western Reserve University
Cleveland, Ohio, United States
EA

Elizabeth A. Andraska

Vascular Surgery Integrated Resident
University of Pittsburgh
Pittsburgh, Pennsylvania, United States
JA

Jurgis Alvikas

Assistant Professor
UPMC Montefiore
Pittsburgh, Pennsylvania, United States
SR

Shruti Raghunathan

PhD Candidate
Case Western Reserve University
Cleveland, Ohio, United States
NM

Nicole Masters

PhD Candidate
Case Western Reserve University
Cleveland, Ohio, United States
KB

Kara Bane

Research scientist
CWRU School of Medicine
Cleveland, Ohio, United States
Nv

Nadine D. van Kleef

Research technician
University Medical Center Utrecht
Utrecht, Utrecht, Netherlands
MN

Marvin Nieman

Associate Professor
Case Western Reserve University
Cleveland, Ohio, United States
Coen Maas, PhD

Associate Professor
Department of CDL Research, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands
Utrecht, Utrecht, Netherlands
Steven de Maat, PhD

Assistant Professor
Department of CDL Research, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands.
Utrecht, Utrecht, Netherlands
Keith B. Neeves, PhD

Professor
University of Colorado Anschutz Medical Campus
Aurora, Colorado, United States
Matthew D. Neal, MD

Associate Professor
University of Pittsburgh
Pittsburgh, Pennsylvania, United States
AS

Anirban Sen Gupta, PhD

Professor of Biomedical Engineering
Case Western Reserve University
Cleveland, Ohio, United States
Evi X. Stavrou, MD

Associate Professor
Department of Medicine, Hematology and Oncology Division, CWRU School of Medicine, Cleveland, OH, USA.
Cleveland, Ohio, United States

Background: Neutrophils are the first responders to tissue damage and play a critical role in host defense against infection. However, unrestricted recruitment and function of activated neutrophils can prolong inflammation and contribute to the development of conditions such as thrombosis, tumor progression, autoimmunity, and chronic wounds. Functional characterization of neutrophils from individuals with chronic inflammatory states identified a distinct pathogenic sub-population that exhibits baseline activation, prolonged lifespan and susceptibility to form NETs.

Aims: We previously designed a nanoparticle (NP) system that simultaneously targets activated neutrophils and platelets. Here, we aimed to develop and characterize a stand-alone therapeutic strategy that exclusively targets activated neutrophils without the ubiquitous inhibition of resting cells, and independently of activated platelets.

Methods: We created a nanomedicine system that uniquely utilizes an α₁-antitrypsin (AAT)-derived peptide to confer binding specificity to neutrophil elastase (HNE) on activated neutrophils.

Results: We employed enzyme-sensitive probes and FRET to characterize the interaction of NEBP with free and membrane-bound HNE. NEBP showed dose-dependent binding to free HNE (Fig 1A) and importantly, a higher affinity for membrane-bound HNE compared to AAT (Fig 1B-C). Moreover, NEBP did not interact with Proteinase 3, Cathepsin G (Fig 1D-F) or non-neutrophil proteases, including tPA and plasmin (Fig 1G-J).

In vitro visualization of NEBP-assembled NPs showed specific targeting to activated neutrophils only, with partial trafficking to lysosomes (Fig 2A-C). In vivo efficacy studies with a model drug (hydroxychloroquine-HCQ) showed that drug-loaded NEBP-NPs significantly reduced inferior vena cava thrombus weights using considerably lower HCQ concentrations (Fig 2E).

Conclusion(s): We describe for the first time a novel nanoplatform that exclusively binds to activated neutrophils and demonstrate that targeting primed neutrophils, alone can be highly efficacious and safer than systemic drug treatment. These studies establish a mechanistic roadmap for cell- and activation state-specific targeting that can be applied to several neutrophil-driven pathologies.