Detection of Intimal Macrophages in Atherosclerotic Lesions Using Biocompatible CD36‐Targeted Ligand Containing Nanoparticle
Authors: Dhanasekara Chathurika Samudani , Zhang Jia , Nie Shufang , Wang Shu 
Published: November 2023

Atherosclerosis provides the pathological background for cardiovascular disease, which is the leading cause of death in the United States. Intimal macrophages are determinant cells in lesion initiation and progression. They not only secrete mediators that amplify the lesion inflammatory response, but also take up oxidized low‐density lipoproteins (oxLDL) and subsequently become lipid‐laden macrophages (foam cells), the hallmark of the early stage of atherosclerosis. Oxidized phospholipids (oxPL) on oxLDL are responsible for their binding to intimal macrophages via their scavenger receptors, primarily the CD36 receptor. We synthesized biocompatible and biodegradable nanoparticles (NP) and incorporated an oxPL as a CD36 ligand on the surface of NP to make ligand‐NP (L‐NP). Our objectives were to compare the target specificity of NP and L‐NP to macrophages in vitro and to aortic lesions in LDL receptor null (LDLr−/−) mice, and to investigate the targeting mechanisms.MethodsBinding assays and competitive binding assays using a CD36 antibody or human oxLDL were conducted using primary mouse macrophages and THP‐1 derived macrophages. Binding assays were also conducted using CD36 knockdown macrophages. Macrophages were incubated with florescence dye labeled NP and L‐NP. Images were taken using an EVOS® fluorescence microscope. The cellular fluorescence signal intensity was analyzed using a NIH ImageJ software. Six 6‐week old male LDLr−/− mice developed atherosclerotic lesions on feeding with an atherogenic diet for 22 weeks. Near infrared dye labeled NP or L‐NP were given to the mice via the intravenous route. After 20 hours, mice were imaged using the IVIS® Lumina XR imaging system. Upon sacrifice, hearts were perfused with saline, and images of longitudinally opened aortas were taken using the fluorescence microscope to demonstrate aortic lesions and nanoparticle signals. The dissected aortas were embedded in O.C.T. compound and serially sectioned. Images of cross‐sections of aortas were taken using the fluorescence microscopy. The nanoparticle signal intensity on the cross‐sections of aortic lesions was analyzed. Macrophages and their CD36 receptors on the cross‐sections of aortas were stained by F4/80 and CD36 antibodies, respectively.ResultsL‐NP had a higher macrophage binding affinity and uptake than NP in vitro as evidenced by a significantly higher cellular fluorescence intensity. CD36 antibody and oxLDL competitively inhibited binding and uptake of L‐NP by macrophages. CD36 knockdown significantly decreased macrophage binding and uptake of L‐NP. L‐NP had a higher fluorescence signal intensity in 1) in‐situ aorta images as well as isolated hearts and aortas and 2) the cross‐sections of aortic lesions than NP (p<0.001). Moreover, L‐NP co‐localized with intimal macrophages and their CD36 receptors in aortic lesions.ConclusionOur L‐NP target intimal macrophages via a CD36‐mediated mechanism. This molecular target approach enables detection of lesion macrophage content and distribution, which may facilitate noninvasive imaging of atherosclerotic lesions and disclose lesion inflammation.Support or Funding InformationGrant Funding Source: NIH 1R15AT007013