Detection of early atherosclerotic plaques with TLR4-conjugated zwitterionic near-infrared fluorophore: a pilot mice study

Objectives: Atherosclerosis is a major contributor to heart disease, which is the leading cause of death worldwide. Early detection of high-risk inflamed plaques can improve patient outcomes. We propose a novel non-invasive NIRF imaging technique with toll-like receptor 4 (TLR4)-conjugated zwitterionic NIR fluorophore (ZW800-1C) targeting macrophage TLR4 that could aid in locating such plaques for the first time to the best of our knowledge.

Methods: We developed a carotid atherosclerotic model using 8-week-old male FVB/NJ mice (n=4, The Jackson Laboratory) that were on 1% high cholesterol diet (HCD) and given Streptozotocin (40mg/kg) intraperitoneally for 5 consecutive days to induce diabetes. After four weeks (excluding 1 week of acclimation) on 1% HCD, the left carotid artery was ligated surgically and the right carotid artery was kept intact as a negative control. Two weeks later, TLR4-ZW800-1C (5.55 µM of TLR4, ex/em: 753/772 nm) was injected via tail-vein before imaging. The mice were imaged with a Fluorescence-Assisted Resection and Exploration (FLARE^TM, Curadel, LLC), a NIR imaging system at 760 nm excitation wavelength and white light at various time points (baseline, 4h, 8h, and 24h). Baseline images were acquired before the contrast agent injection. The experiment was done under anesthesia according to MGH IACUC-approved protocol (2019N000104). Carotid arteries and heart tissues were collected and stained with H&E, EVG, and Trichrome for histological analysis by a trained pathologist. Analysis of histology is under review. Image analysis and processing were performed using ImageJ and MATLAB.

Results: NIR fluorescent signal could be detected over the ligation and upper part of the heart regions for both mice with an average signal to baseline ratio of 2.00±0.72, 1.91±0.92, and 1.77±0.66 at 4h, 8h, and 24h respectively. Exposed reflectance images of the aortic arch at 24h showed a localized region of fluorescent signal proximal to the left carotid artery showing signal to background ratio (SBR) of 4.79± 0.35.

Conclusions: This preliminary study shows promise that TLR4-conjugated ZW800-1C is present in mild plaque conditions. Continued investigation will include larger sample sizes and histological analyses in mice models, and potentially in large animals using intravascular NIRF catheters. Acknowledgements: The authors would like to thank the MGH Center for Comparative Medicine animal facility staff. Support for this work came from the National Heart, Lung, and Blood Institute and the National Institute of Biomedical Imaging and Bioengineering of the National Institutes of Health under award numbers R00HL127180 and T32EB013180, as well as from MGH Sundry fund 2008A055408.