Dataset DOI: 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 < .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 < .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.