Preparation of a nitric oxide imaging agent from gelatin derivative micelles

Abstract

Introduction: Nitric oxide (NO) is an intracellular and intercellular messenger that plays an important role in cellular events in physiological and pathophysiological processes. NO is one of the inflammation markers and macrophages of an inflammatory cell produce a large amount of NO compared with other cells. Non-invasive detection system of NO is highly required to realize an early therapeutic treatment considering the process of pathophysiological changes. The objective of this study is to develop an imaging agent of nitric oxide (NO).

Methods: A water-insoluble DAR-4M of fluorescent dye for NO was solubilized in water through the micelle formation with gelatin grafted with l-α-phosphatidylethanolamine distearoyl (DAR-4M micelles). Physicochemical and biological properties of DAR-4M micelles were investigated by using cultured cells and animals.

Results: The DAR-4M micelles responded to NO secreted from a NO donors, in contrast to the same concentration of free DAR-4M. When RAW264.7 of a macrophage cell line was stimulated by lipopolysaccharide (LPS) to allow them to generate NO, the DAR-4M micelles could detect NO of the cells to a significant great extent compared with free DAR-4M. After the intravenous injection of DAR-4M micelles or free DAR-4M to a mouse model of aristolochic acid (AA) induced acute interstitial nephritis, the DAR-4M micelles enhanced the fluorescence intensity from the kidneys to a significant great extent compared with the free DAR-4M injection. In case of DAR-4M micelles injection into normal mice, such an enhanced kidney fluorescence was not observed. A body distribution experiment demonstrated that the kidney accumulation of DAR-4M micelles was not modified by the AA-induced inflammation. After the AA injection, the number of CD11b-positive cells increased with time, indicating the increased number of inflammatory macrophages.

Conclusion: DAR-4M micelles are effective in imaging NO generated from macrophages accompanied with inflammation.

Keywords: Fluorescent dye; Gelatin; Macrophages; Nitric oxide; Polymer micelles; Water-solubilization.

Fig. 1

Effect of DAR-4M concentration on the reactivity of DAR-4M micelles (■) or free DAR-4M (□) to NO donors. The DAR-4M micelles were prepared by DSPE-10. The fluorescent intensity ratio was calculated as the fluorescence intensity of 1.0 for the group in NO-donor-free PBS at the corresponding DAR-4M concentration. *p < 0.05; significance between the two groups.

Fig. 2

Effect of the DAR-4M micelles concentration on the viability of RAW264.7 cells 3 h after incubation. The DAR-4M micelles were prepared at DAR-4M concentrations of 0.5, 1, 2, 5, 10, and 20 μM. The viability of cells cultured without DAR-4M micelles (DAR-4M concentration = 0) is indicated as 100%. *, p < 0.05; significant against the viability of cells cultured without DAR-4M micelles.

Fig. 3

Confocal laser microscopic pictures of RAW264.7 cells 1 h after incubation with (A, E) free DAR-4M, (B, F) DSPE-10, (C, G) the mixture of free DAR-4M and DSPE-10, and (D, H) DAR-4M micelles. Cells were stimulated for 24 h by 100 ng/mL of LPS (A, B, C, D) and not (E, F, G, H). The DAR-4M micelles were prepared by DSPE-10. Scale bar is 50 mm.

Fig. 4

Fluorescent intensity of LPS-stimulated cells 1 h after incubation with free DAR-4M (■), the mixture of free DAR-4m and DSPE-10 (□), and DAR-4M micelles (). The LPS concentration is 100 ng/ml. The DAR-4M micelles were prepared by DSPE-10. *p < 0.05; significance between the two groups.

Fig. 5

Confocal laser microscopic pictures of cells 1 h after incubation with DAR-4M micelles. Red: DAR-4M. Green: lysosome. Scale bar is 50 μm.

Fig. 6

IVIS analysis of isolated kidneys of mice 1 h after intravenous injection of free DAR-4M and DAR-4M micelles. Mice were treated with AA for 1, 4, 7, 21, and 35 days. The DAR-4M micelles were prepared by DSPE-10. (A) IVIS images of isolated kidney. (B) Total photon flux of isolated kidneys after intravenous injection of free DAR-4M (■) and DAR-4M micelles (□).

Fig. 7

Percentage of CD11b-positive cells to the total live cells in kidneys of AA-treated mice after injection of DAR-4M micelles. The DAR-4M micelles were prepared by DSPE-10. *p < 0.05; significance between the two groups.

Fig. 8

Body distribution of mice 1 h after intravenous injection of ¹²⁵I–DAR-4M micelles. ¹²⁵I–DAR-4M micelles were intravenously injected to the normal (0 day after injection) (■) and AA-treated mice 1 day (□), 4 days (), 7 days (), 21 days () and 35 days () after injection.