DOI: 10.5061/dryad.05qfttffw
title: CD47-targeted nano-immunotherapy for treatment of atherosclerosis

description: In this ongoing project funded by the American Heart Association, we
 pursued the development of targeted nanotherapeutics to inhibit the
 CD47/SIRPα immune checkpoint axis, a promising strategy for enhancing
 macrophage-mediated clearance of diseased tissue in atherosclerosis. We
 evaluated multiple nanoparticle and small-molecule combinations,
 ultimately selecting a ferumoxytol (FMX) nanoparticle loaded with the
 potent CD47 downregulator RRx-001 emerged as the lead nanotherapeutic
 candidate. In vitro and in vivo imaging studies demonstrated that FMX
 nanoparticles preferentially accumulate in macrophage-rich regions of
 atherosclerotic plaques. Using fluorescently labeled FMX in ApoE−/− mice,
 we showed selective deposition of nanoparticles in inflamed aortic
 lesions. Fluorescence microscopy revealed colocalization of FMX with
 macrophages (MAC-3+), confirming their cellular targeting specificity.
 Quantitative analysis indicated significantly greater FMX uptake by
 macrophages compared to endothelial and smooth muscle cells (p &lt;
 0.0001), validating the macrophage-targeting potential of this
 nanoplatform. Preliminary therapeutic efficacy studies were conducted in
 ApoE−/− mice fed a high-fat diet and treated weekly with saline, FMX
 alone, or FMX loaded with CD47 inhibitors (FMX-CD47). After two months of
 treatment, aortic plaque burden was evaluated via Oil Red O staining.
 These pilot data suggest a potential role for FMX-CD47 formulations in
 attenuating atherosclerotic progression, supporting further investigation
 into this nanotherapeutic strategy.
description: # CD47-targeted nano-immunotherapy for treatment of atherosclerosis
 Dataset DOI: [10.5061/dryad.05qfttffw](10.5061/dryad.05qfttffw) This
 preliminary data report describes the development of targeted
 nanotherapeutics to inhibit the CD47/SIRPα immune checkpoint and enhance
 macrophage-mediated clearance in atherosclerosis. Ferumoxytol (FMX)
 nanoparticles loaded with the CD47 downregulator RRx-001 showed
 preferential accumulation in macrophage-rich regions of atherosclerotic
 plaques in vitro and in vivo. Imaging and quantitative analysis confirmed
 selective uptake by macrophages compared to other vascular cell types.
 Preliminary efficacy studies in ApoE−/− mice suggest that FMX-CD47
 formulations may reduce plaque formation. ## Description of the data and
 file structure **Figure_1.jpg description:** **Fig. 1 A, B:** Design and
 synthesize small-molecule inhibitors targeting the CD47/SIRPα axis. As
 shown in the figure, ferumoxytol (FMX) was chosen as the platform for drug
 loading. FMX was loaded with small-molecule inhibitors of CD47/SIRPα. The
 commercially available small molecule RRx-001 is a downregulator of CD47
 expression and was identified as the most promising lead compound.
 Attempted synthesis of several analogues of RRx-001, such as the
 difluorinated bioisostere, were unsuccessful, presumably due to the high
 ring-strain of the azetidine ring and its tendency toward ring-opening
 oligomerization. Other formulations were also conducted with other CD47
 inhibitors like NCGC00138783. This compound and its known analogues were
 synthesized according to a previously developed protocol (Burgess, T. L.
 et al. *PLOS One*, **2020**, *15*, e0226661.). To summarize, small
 molecule drug inhibitors were loaded into FMX-CyAl5 by a solvent diffusion
 method. Briefly, stock solutions of FMX-CyAl5 (30 mMiron in PBS) were
 prepared. Stock solutions of CD47 inhibitors (10mg/mL in DMSO) were also
 prepared. The DMSO solution (200 μL) was added dropwise to either
 FMX-CyAl5 solutions(1800 μL) while vortexing, and then each sample was
 mixed overnight at room temperature to prepare the corresponding
 drug-loaded FMX-CyAl5 solution with 1mg/ml concentration of desired
 inhibitors. **Fig. 1 C, D, E, F** shows the advantage of carboxymethyl
 dextran (CMD)-coated FMX as drug delivery vehicles. When utilized in vivo,
 fluorescently labeled iron oxide nanoparticles (NPs) have been shown to
 localize mainly to areas of inflammation (Fig. 1C- F). This avidity has
 proven useful in the localization and imaging of atherosclerotic lesions
 using fluorescently labeled FMX in a murine model of atherosclerosis. Iron
 oxide NPs preferentially target macrophages, and FMX deposition in murine
 atheroma can be noninvasively detected by NIR FRI. Apolipoprotein E gene
 deletion mouse was employed (apoE−/−) as a model of atherosclerosis.
 Quantitative analysis demonstrated a significantly higher number of
 FMX-positive macrophages per high-power field (hpf) compared to
 endothelial cells and smooth muscle cells (p &lt; .0001).⁠ Representative
 fused fluorescence microscopic images of the FMX (red) and
 immunofluorescently labeled cell antigens (green). A focal NIRF signal was
 noted in aortic plaque sections of apo E−/− mice that received the FMX
 agent (Figure 1). Signal was detected in various cellular-rich areas of
 atherosclerotic lesions and the adventitia adjacent to the atheroma. On
 correlative fluorescence microscopy, NPs were readily identified within
 immunofluorescent cells in atherosclerotic lesions (Figure 1). Macrophages
 were the predominant FMX-positive cell type (20.6 ± 7.3/hpf) compared to
 endothelial cells (6.6 ± 1.8/hpf) and smooth muscle cells (4.3 ±
 1.8/hpf, p &lt; .0001). Macrophages represented the majority of
 FMX-positive cells and demonstrated yellow-orange NIRF and
 immunofluorescent signal colocalization (arrowheads). Histological
 examination of the lesions by fluorescence microscopy revealed that the
 fluorescent signal from the nano agent colocalized with macrophages
 immunostained for MAC-3. These studies are supportive of our hypothesis
 and thus suggest that using FMX NPs for novel targeted nanomaterials
 targeting macrophages in atherosclerosis. **Fig. 1G. Studies in mice.** 
 The therapeutic efficacy of different formulations (saline, FMX, FMX-CD47)
 against atherosclerotic development were examined. After receiving a
 high-fat diet for 1 month, 6-week-old female ApoE−/− mice were randomly
 and investigator-blindly divided into 3 groups (n = 6 in each group from
 two batches of studies), and intravenously administered with different
 formulations, respectively, once a week, in combination with high-fat food
 for another 2 months (Fig. 1G). At the endpoint of the experiment, the
 aorta will be collected and stained by ORO, and the resultant red region
 of plaque area will be evaluated. Description of Data Tables for Studies
 in Mice (8 weeks of treatment and weight measurement; 3 weeks of
 post-treatment weight measurement; sacrifice on week 12):
 APOE_Mice_Groups.xlsx – listing of cage and tag numbers for three
 treatment groups of mice (Group-I PBS; Group-II FMX-Cyal5; Group III
 FMX-Cyal 5-RRX). Week_1_APOE_mouse_weight_032624.xlsx – listing of
 dosages, weight of mice, and cage/tag numbers for three treatment groups
 of mice (Group-I PBS; Group-II FMX-Cyal5; Group III FMX-Cyal 5-RRX) for
 Week 1 of the study (day before treatment).
 Week_1_RRX_treatment_032724.xlsx – listing of dosages, weight of mice, and
 cage/tag numbers for three treatment groups of mice (Group-I PBS; Group-II
 FMX-Cyal5; Group III FMX-Cyal 5-RRX) for Week 1 of the study (day of
 treatment). Week_2_APOE_mouse_weight_040124.xlsx – listing of dosages,
 weight of mice, and cage/tag numbers for three treatment groups of mice
 (Group-I PBS; Group-II FMX-Cyal5; Group III FMX-Cyal 5-RRX) for Week 2 of
 the study (day before treatment). Week_2_RRX_treatment_040224.xlsx –
 listing of dosages, weight of mice, and cage/tag numbers for three
 treatment groups of mice (Group-I PBS; Group-II FMX-Cyal5; Group III
 FMX-Cyal 5-RRX) for Week 2 of the study (day of treatment).
 Week_3_APOE_weight_040924.xlsx – listing of dosages, weight of mice, and
 cage/tag numbers for three treatment groups of mice (Group-I PBS; Group-II
 FMX-Cyal5; Group III FMX-Cyal 5-RRX) for Week 3 of the study (day before
 treatment). Week_3_RRX_treatment_041024.xlsx – listing of dosages, weight
 of mice, and cage/tag numbers for three treatment groups of mice (Group-I
 PBS; Group-II FMX-Cyal5; Group III FMX-Cyal 5-RRX) for Week 3 of the study
 (day of treatment). Week_4_APOE_weight_041624.xlsx – listing of dosages,
 weight of mice, and cage/tag numbers for three treatment groups of mice
 (Group-I PBS; Group-II FMX-Cyal5; Group III FMX-Cyal 5-RRX) for Week 4 of
 the study (day before treatment). Week_4_RRX_treatment_041724.xlsx –
 listing of dosages, weight of mice, and cage/tag numbers for three
 treatment groups of mice (Group-I PBS; Group-II FMX-Cyal5; Group III
 FMX-Cyal 5-RRX) for Week 4 of the study (day of treatment).
 Week_5_APOE_mouse_weight_042324.xlsx – listing of dosages, weight of mice,
 and cage/tag numbers for three treatment groups of mice (Group-I PBS;
 Group-II FMX-Cyal5; Group III FMX-Cyal 5-RRX) for Week 5 of the study (day
 before treatment). Week_5_RRX_treatment_042424.xlsx – listing of dosages,
 weight of mice, and cage/tag numbers for three treatment groups of mice
 (Group-I PBS; Group-II FMX-Cyal5; Group III FMX-Cyal 5-RRX) for Week 5 of
 the study (day of treatment). Week_6_APOE_weight_043024.xlsx – listing of
 dosages, weight of mice, and cage/tag numbers for three treatment groups
 of mice (Group-I PBS; Group-II FMX-Cyal5; Group III FMX-Cyal 5-RRX) for
 Week 6 of the study (day before treatment).
 Week_6_RRX_treatment_050124.xlsx – listing of dosages, weight of mice, and
 cage/tag numbers for three treatment groups of mice (Group-I PBS; Group-II
 FMX-Cyal5; Group III FMX-Cyal 5-RRX) for Week 3 of the study (day of
 treatment). Week_7_APOE_weight_050724.xlsx – listing of dosages, weight of
 mice, and cage/tag numbers for three treatment groups of mice (Group-I
 PBS; Group-II FMX-Cyal5; Group III FMX-Cyal 5-RRX) for Week 7 of the study
 (day before treatment). Week_7_RRX_treatment_050824.xlsx – listing of
 dosages, weight of mice, and cage/tag numbers for three treatment groups
 of mice (Group-I PBS; Group-II FMX-Cyal5; Group III FMX-Cyal 5-RRX) for
 Week 7 of the study (day of treatment). Week_8_APOE_weight_051424.xlsx –
 listing of dosages, weight of mice, and cage/tag numbers for three
 treatment groups of mice (Group-I PBS; Group-II FMX-Cyal5; Group III
 FMX-Cyal 5-RRX) for Week 8 of the study (day before treatment).
 Week_8_treatment_051524.xlsx – listing of dosages, weight of mice, and
 cage/tag numbers for three treatment groups of mice (Group-I PBS; Group-II
 FMX-Cyal5; Group III FMX-Cyal 5-RRX) for Week 8 of the study (day of
 treatment). Week_9_APOE_mouse_weights_052124.xlsx – listing of dosages,
 weight of mice, and cage/tag numbers for three treatment groups of mice
 (Group-I PBS; Group-II FMX-Cyal5; Group III FMX-Cyal 5-RRX) for Week 9 of
 the study (1 week following the initial 8-week treatment).
 Week_10_APOE_mouse_weights_052824.xlsx – listing of dosages, weight of
 mice, and cage/tag numbers for three treatment groups of mice (Group-I
 PBS; Group-II FMX-Cyal5; Group III FMX-Cyal 5-RRX) for Week 10 of the
 study (2 weeks following the initial 8-week treatment).
 Week_11_FMX-CyAL5_treatment_060524.xlsx – listing of dosages, weight of
 mice, and cage/tag numbers for three treatment groups of mice (Group-I
 PBS; Group-II FMX-Cyal5; Group III FMX-Cyal 5-RRX) for Week 11 of the
 study (3 weeks following the initial 8-week treatment).
 APOE_Mouse_Sac_ROIs_061024.xlsx – listing of cage and tag numbers for mice
 sacrificed in Week 12 of the study.