Effect of CCR2 inhibitor-loaded lipid micelles on inflammatory cell migration and cardiac function after myocardial infarction

Abstract

Background: After myocardial infarction (MI), inflammatory cells infiltrate the infarcted heart in response to secreted stimuli. Monocytes are recruited to the infarct via CCR2 chemokine receptors along a CCL2 concentration gradient. While infiltration of injured tissue with monocytes is an important component of the reparatory response, excessive or prolonged inflammation can adversely affect left ventricular remodeling and worsen clinical outcomes.

Materials and methods: Here, we developed poly(ethylene glycol) (PEG)-distearoylphos-phatidylethanolamine (PEG-DSPE) micelles loaded with a small molecule CCR2 antagonist to inhibit monocyte recruitment to the infarcted myocardium. To specifically target CCR2-expressing cells, PEG-DSPE micelles were further surface decorated with an anti-CCR2 antibody.

Results: Targeted PEG-DSPE micelles showed eight-fold greater binding to CCR2-expressing RAW 264.7 monocytes than plain, non-targeted PEG-DSPE micelles. In a mouse model of MI, CCR2-targeting PEG-DSPE micelles loaded with a CCR2 small molecule antagonist significantly decreased the number of Ly6C^high inflammatory cells to 3% of total compared with PBS-treated controls. Furthermore, CCR2-targeting PEG-DSPE micelles significantly reduced the infarct size based on epicardial and endocardial infarct arc lengths.

Conclusion: Both non-targeted and CCR2-targeting PEG-DSPE micelles showed a trend toward improving cardiac function. As such, PEG-DSPE micelles represent a promising cardiac therapeutic platform.

Keywords: CCR2; inflammatory monocytes; micelles; myocardial infarction.

Figure 1

Structure and size of PEG-DSPE Micelles. (A) Schematics of CCR2-targeting and non-targeted but loaded micelles. (B) Hydrodynamic diameter of non-targeted (plain) and (C) CCR2-targeting micelles by dynamic light scattering. Abbreviation: PEG-DSPE, poly(ethylene glycol)-distearoylphosphatidylethanolamine.

Figure 2

(A) The small molecule CCR2 antagonist inhibits migration of RAW 264.7 cells. (B) The molecular structure of the BMS CCR2 small molecule antagonist with a molecular weight of 593.66 Da.

Figure 3

Binding of CCR2-targeting and non-targeted (plain) micelles to RAW 264.7 cells as analyzed by (A) flow cytometry and (B) MFI, and (C, D) fluorescence microscopy with nuclei stained in blue (DAPI) and DiD-labeled CCR2-targeting micelles shown in red. Scale bar =50 μm. Data are presented as mean ± SD, ^****P<0.0001 by one-way ANOVA followed by Dunnett’s test. (E) MTT assay of CCR2-targeting and non-targeted (plain) micelles in RAW 264.7 cells. Abbreviation: MFI, mean fluorescence intensity.

Figure 4

Effect of CCR2-targeting and non-targeted micelles on the number of inflammatory monocytes (Ly6C^high Mo) in the spleen (A–C) and in the infarcted heart (D–F), n=4–6. Data are presented as mean ± SD, ^*P<0.05, ^***P<0.001 by one-way ANOVA followed by Dunnett’s test compared with PBS-treated mice. Abbreviation: FITC, fluorescein isothiocyanate.

Figure 5

(A) Schematic depicting the timeline of MI induction and treatment with CCR2-targeting and plain micelles containing a CCR2 small molecule antagonist. (B) Infarct area quantified by length, ^*P<0.05 by one-way ANOVA followed by Dunnett’s test. (C) Infarct area expressed as percentage of LV area and total area. (D) Masson trichrome staining (12 days after MI) of sequential heart sections of mice treated with PBS, non-targeted micelles, and CCR2-targeting micelles. (E) EF % and FS % 12 days post MI of mice treated with PBS (n=6), non-targeted micelles (NT, n=6), and CCR2-targeting micelles (T, n=4). Sham-treated mice served as controls. Data are presented as mean ± SD. Abbreviations: EF, Ejection fraction; FS, fractional shortening; LV, left ventricular; MI, myocardial infarction.