Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Jun 7;5(8):1800397.
doi: 10.1002/advs.201800397. eCollection 2018 Aug.

Rapid Response Fluorescence Probe Enabled In Vivo Diagnosis and Assessing Treatment Response of Hypochlorous Acid-Mediated Rheumatoid Arthritis

Huan Feng  1 , Zhiqiang Zhang  1 , Qingtao Meng  1 , Hongmin Jia  1 , Yue Wang  1 , Run Zhang  2
Affiliations

Rapid Response Fluorescence Probe Enabled In Vivo Diagnosis and Assessing Treatment Response of Hypochlorous Acid-Mediated Rheumatoid Arthritis

Huan Feng et al. Adv Sci (Weinh). .

Abstract

Diagnosis and early assessment of the treatment response of rheumatoid arthritis (RA) necessitates a reliable bioanalytical method for rapid, sensitive, and specific detection of the hypochlorous acid (HOCl) biomarker in inflammatory diseases. Herein, two fluorescence probes, Probe-1 and Probe-2 are developed for quantitative monitoring and visualization of inflammatory response-related HOCl levels in vitro and in vivo. In the presence of HOCl, fluorescence "OFF-ON" response is obtained for both the probes as a result of specific HOCl-triggered C=N bond cleavage reaction. Probe-1 and Probe-2 feature rapid response (<4 s), a high degree of sensitivity and selectivity toward HOCl, which allow them to be used for quantification of HOCl in a simulated physiological condition. Using Probe-2 as the probe, fluorescence imaging and flow cytometry analysis of HOCl levels in lysosome of inflammatory mimic cells, visualization of HOCl generation in endotoxin-induced inflammation of adult zebrafish and RA of mice are possible. Probe-2 exhibits high effectiveness for early assessment of the treatment response of HOCl-mediated RA in mice with an antiarthritic drug, methotrexate (MTX). The results demonstrate that Probe-2 is a powerful tool for future studies on diagnosis and monitoring treatment efficiency in a broad range of inflammatory diseases, including RA.

Keywords: fluorescence probes; hypochlorous acid; inflammatory diseases; rheumatoid arthritis; treatment response.

PubMed Disclaimer

Figures

Scheme 1
Scheme 1
A) Schematic illustration of the design strategy and the sensing mechanism of the probes for monitoring of HOCl‐mediated RA. Chemical structure of B) Probe‐2 and C) Probe‐1 and its reaction with HOCl.
Figure 1
Figure 1
A) UV–vis absorption spectra of Probe‐1 (10 × 10−6 m) in PBS buffer (DMSO:H2O = 3:7, v/v; pH 7.4) in the presence of different concentrations of HOCl (0–120 × 10−6 m), inset: plot of the absorbance at 367 nm of Probe‐1 against the concentration of HOCl. B) UV–vis absorption spectra of Probe‐2 (10 × 10−6 m) in PBS buffer (DMSO:H2O = 3:7, v/v; pH 7.4) upon the addition of different concentrations of HOCl (0–100 × 10−6 m), inset: the color change of Probe‐2 in the presence of HOCl. C) UV–vis absorption spectra of Probe‐1 (10 × 10−6 m) in PBS buffer (DMSO:H2O = 3:7, v/v; pH 7.4) in the presence of various ROS and anion species (120 × 10−6 m). D) UV–vis absorption spectra of Probe‐2 (10 × 10−6 m) in PBS buffer (DMSO:H2O = 3:7, v/v; pH 7.4) in the presence of various ROS and anion species (100 × 10−6 m).
Figure 2
Figure 2
Fluorescence response of Probe‐1 and Probe‐2 toward HOCl. Fluorescence spectra of A) Probe‐1 (10 × 10−6 m) and B) Probe‐2 (10 × 10−6 m) in PBS buffer (DMSO:H2O = 3:7, v/v; pH 7.4) upon the addition of increasing amounts of HOCl. Enhanced fluorescence intensity factor (F/F 0) of C) Probe‐1 emission at 427 nm and D) Probe‐2 emission at 575 nm in the presence of various ROS and anion species in PBS buffer (DMSO:H2O = 3:7, v/v; pH 7.4). The competitive species include: (1) Cl−, (2) Br−, (3) I−, (4) SO4 2−, (5) HCO3 −, (6) PO4 3−, (7) inorganic phosphates (Pi), (8) NO3 −, (9) NO2 −, (10) 1O2, (11) H2O2, (12) ·OH, (13) ONOO−, (14) HOCl, and (15) mixed species. Excitations were performed at 370 and 490 nm for Probe‐1 and Probe‐2, respectively.
Figure 3
Figure 3
A,B) Fluorescence enhancement time profiles of Probe‐1 (10 × 10−6 m) and Probe‐2 (10 × 10−6 m) for the addition of HOCl in PBS buffer (DMSO:H2O = 3:7, 20 × 10−3 m, pH 7.4). Inset: time courses of fluorescence intensity changes of Probe‐1 (10 × 10−6 m) and Probe‐2 (10 × 10−6 m) after addition of HOCl within seconds. The excitation and emission wavelength (λex/λem) are 370/427, 490/575 nm for Probe‐1 and Probe‐2, respectively.
Figure 4
Figure 4
Bright‐field, fluorescence, and merged images of endogenous HOCl generation in LPS‐induced inflammatory mimic J774A.1 macrophage. A–C) The macrophage was incubated with 4 × 10−6 m Probe‐2 for 30 min. D–F) J774.1 macrophage cells were treated with LPS (1.0 µg mL−1) for 4 h, and then incubated with Probe‐2 for another 30 min. G) Fluorescence imaging of endogenous HOCl generation in macrophage cells with LPS stimulation. H,I) The cells of interest shown in (a) and (b) of (G). J–O) Intracellular colocalization analysis of Probe‐2 with LysoSensor Green in living J774A.1 macrophage cells. (J) The lysosome of J774A.1 cells was stained by LysoSensor Green. (K) Cells were stimulated with LPS (1.0 µg mL−1) for 4 h, and then incubated with Probe‐2 for another 30 min. (L) Merged imaging of (J), (K), and the cell nucleus stained with Hochest 33342, the area of interest (a, d, h): intercellular space, (b, e): lysosome, (d, g): cell nucleus, (f): cytoplasm. (M) Zoom‐in fluorescence image of selected area in (L). (N) Fluorescence intensity profiles of the linear region of interest across macrophage cells in (L). (O) Merged image of (L) and corresponding bright‐field image. Scale bars are 20 µm.
Figure 5
Figure 5
A) Fluorescence imaging of endogenous HOCl production in zebrafish. Zebrafish only (a); zebrafish was stimulated with LPS (2 µg mL−1) for 3 h (b); then stained with Probe‐2 (10 × 10−6 m) for 1 min (c); 17 min (d); 34 min (e); 51 min (f); 78 min (g); 108 min (h); and 138 min (i), respectively. B) Mean fluorescence intensity of zebrafish treated at different conditions shown in (A).
Figure 6
Figure 6
Visualization of HOCl‐mediated inflammatory response in RA of mice. A) Control group 1 (mice only); B) group 2 (left hind limbs were stimulated with λ‐carrageenan in PBS for 4 h); the Probe‐2 was then injected and then the mice were imaged after C) 1 min, D) 5 min, E) 10 min, F) 15 min, G) 20 min. The right hind limbs were injected with Probe‐2 only as the control group. H) Mean fluorescence intensities were recorded for the interested areas of control group, (1) left hind limbs and (2) right hind limbs. Values are the mean ± SD for each group of three experiments; *p < 0.05.
Figure 7
Figure 7
Monitoring of HOCl‐mediated RA treatment response. A) Control group; B) both left and right hind limbs were stimulated with λ‐carrageenan in PBS for 4 h; C) left hind limbs were administered with MTX for another 6 h; D) Probe‐2 was injected locally into both right and left hind limbs. E) Mean fluorescence intensities were recorded for the interested areas of control group, (1) left hind limbs and (2) right hind limbs. Values are the mean ± SD for each group of three experiments; *p < 0.05.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Joo Y.‐B., Park K.‐S., N. Engl. J. Med. 2017, 377, e27. - PubMed
    1. a) Filippin L. I., Vercelino R., Marroni N. P., Xavier R. M., Clin. Exp. Immunol. 2008, 152, 415; - PMC - PubMed
    2. b) Chen Z., Liu Z., Li Z., Ju E., Gao N., Zhou L., Ren J., Qu X., Biomaterials 2015, 39, 15. - PubMed
    1. Brand D. D., Latham K. A., Rosloniec E. F., Nat. Protoc. 2007, 2, 1269. - PubMed
    1. Herman S., Zurgil N., Deutsch M., Inflammation Res. 2005, 54, 273. - PubMed
    1. Firestein G. S., Paine M. M., Boyle D. L., Arthritis Rheum. 1994, 37, 193. - PubMed

LinkOut - more resources