Work

BS15 In vivo whole-body fluorescence molecular imaging of experimental atherosclerosis using anti-malondialdehyde-modified low-density lipoprotein humanised antibody fragment targeted nanoparticles

Year: 2022

Type: conference-paper

Abstract: Introduction Oxidative modification of low-density lipoprotein (LDL), for example by malondialdehyde (MDA) adduction, with subsequent uptake by foam cells and lipid necrotic core formation, is central in the evolving atherosclerotic plaque. Moreover, greater necrotic core size is a key feature of vulnerable atherosclerotic lesions, that is plaques that have an increased risk of rupturing and precipitating a myocardial infarction or stroke, depending on the vascular bed affected. Thus, MDA-LDL is an attractive target, for both the molecular imaging and therapeutics of vulnerable atherosclerotic plaques. Methods LO1 is a spontaneously arising IgG3k monoclonal murine antibody that reacts with MDA-LDL. Humanised LO1Fab fragments have been engineered to reduce immunogenicity whilst maintaining antigen specificity and improving lesion penetration. Fluorescent poly(lactic-co-glycolic acid) (PLGA) - polyethylene glycol (PEG) nanoparticles (NP) were formulated and functionalised with LO1Fab fragments. NP function was assessed on ELISA and fluorescence microscopy, prior to in vivo imaging in atherosclerotic LDL-receptor-null mice fed a high-fat diet for 40-weeks. Mice were imaged 4-hours after iv injection of NP using fluorescence molecular tomography. Tissue-specific uptake was confirmed ex vivo using fluorescence reflectance imaging. Results PLGA-PEG NPs functionalised with LO1Fab were formulated with size 210nm, polydispersity index <0.2 index (variability of nanoparticle size around the average) and conjugation efficiency of 30%. In vitro function was confirmed on ELISA with MDA-LDL on solid phase, with detection via: LO1Fab presence, using an anti-CH1 antibody; nanoparticle presence, using an anti-PEG antibody; as well as direct fluorescence on an infrared scanner. Fluorescence microscopy on cryosections from atherosclerotic mice confirmed binding to fatty aortic lesions. Following in vitro evaluation, the NPs were assessed in vivo. Construct half-life was estimated at 90-minutes for both the targeted and untargeted NPs in a two-phase decay model, based on fluorescence analysis of serial tail vein blood samples. There was significantly greater uptake in the background-corrected region of interest (heart and aortic arch vessels) in mice injected with LO1Fab-NP versus untargeted NP (mean ± SD) (64.7±22.9 vs 25.2±26.5pmol; n=7; p=0.02). Ex vivo fluorescence analysis of the aortae confirmed these findings (1.0±0.3 vs 0.5±0.2pmol; n=7; p=0.002; Figure 1). Conclusions Humanised antibody Fab fragment fluorescent NPs have been developed that successfully target MDA-LDL and localise to atherosclerotic plaques in experimental atherosclerosis. These targeted NPs have the potential to amplify fluorescent signal for imaging and carry a therapeutic cargo for targeted drug delivery to atherosclerotic plaques.

Source: Heart

DOI: 10.1136/heartjnl-2022-bcs.195