ICAM-1-Targeted Antibody-Nanocarrier System for Selective Delivery to Ischemic Muscles: A Biodistribution Study in PAD

Introduction: : Peripheral arterial disease (PAD) is a chronic and progressive vascular condition that primarily affects elderly and diabetic populations. Despite available treatments such as stents, bypass surgery, and gene or stem cell therapies, these interventions often yield limited success due to poor targeting and restricted delivery to ischemic tissues. While conventional nanoparticles offer potential for drug and gene delivery, non-targeted systems frequently suffer from poor tissue specificity and off-target distribution, especially in poorly perfused ischemic environments. To address these limitations, we developed an actively targeted nanocarrier system using poly(lactic-co-glycolic acid) (PLGA) nanoparticles conjugated with anti-intercellular adhesion molecule-1 (αICAM-1) antibodies. This antibody-functionalization enables selective binding to ICAM-1, a molecule significantly upregulated in inflamed and ischemic vasculature, thereby enhancing localization, retention, and therapeutic payload delivery to diseased tissues in PAD.

Materials and

Methods: : Antibody-conjugated PLGA nanoparticles were designed to actively target ICAM-1, a molecule overexpressed in ischemic tissues. PLGA nanoparticles were synthesized using a double emulsion solvent evaporation method and covalently conjugated with αICAM-1 antibodies via NHS/EDC chemistry. Nanoparticles were characterized for size, zeta potential, morphology, and antibody conjugation efficiency. In vitro binding assays with TNF-α-stimulated endothelial cells confirmed ICAM-1-mediated targeting. Preliminary biodistribution studies demonstrated that αICAM-1-conjugated nanoparticles showed significantly enhanced accumulation in ischemic muscles compared to non-targeted controls. Building on these promising results, we are now conducting an expanded in vivo biodistribution study with a larger animal cohort to validate targeting efficiency and assess consistency across time points. Nanoparticles were characterized for physicochemical properties and antibody conjugation efficiency. Targeting specificity was confirmed in vitro using TNF-α-activated endothelial cells. For in vivo analysis, IR-820-loaded αICAM-1 nanoparticles were intravenously administered into a murine PAD model, and tissue accumulation was assessed through in vivo and ex vivo imaging as well as immunohistochemistry of tissue sections.

Results, Conclusions, and Discussions:: Antibody-conjugated PLGA nanoparticles exhibited a uniform size distribution (~150–200 nm) and appropriate surface charge for systemic delivery. Successful antibody functionalization was confirmed through conjugation efficiency analysis. In vitro binding assays demonstrated that αICAM-1-conjugated nanoparticles showed significantly higher binding to TNF-α-activated endothelial cells compared to non-targeted controls, confirming the role of ICAM-1 in mediating targeted interactions. In vivo biodistribution studies further supported these findings: IR-820-labeled αICAM-1 nanoparticles preferentially accumulated in ischemic muscle tissues of PAD mice, with signal intensity correlating strongly with regions of elevated ICAM-1 expression confirmed via immunohistochemistry. Fluorescence signals in ischemic limbs remained consistently elevated at 4, 24, and 72 hours post-injection, indicating sustained tissue retention and enhanced targeting. These results validate the targeting strategy and support the potential of antibody-conjugated nanocarriers for selective delivery in PAD.
This study demonstrates that αICAM-1 antibody-conjugated PLGA nanoparticles can selectively accumulate in ischemic tissues due to ICAM-1 overexpression, validating their potential for targeted delivery in PAD. Our biodistribution data supports the feasibility of antibody-functionalized nanocarriers for future therapeutic applications, and ongoing work will assess therapeutic payload delivery and long-term efficacy

Acknowledgements and/or References (Optional):: funding support of this research: the American Heart Association Institutional Research Enhancement Award 25AIREA1357216 (TN)