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Preparation and Practical Applications of 2 ′ ,7 ′ -Dichlorodihydrofluoresceinin Redox Assays

View Article: PubMed Central - PubMed

ABSTRACT

Oxidative stress,a state in which intra- or extracellular oxidantproduction outweighs the antioxidative capacity, lies at the basisof many diseases. DCFH2-DA (2′,7′-dichlorodihydrofluoresceindiacetate) is the most widely used fluorogenic probe for the detectionof general oxidative stress. However, the use of DCFH2-DA,as many other fluorogenic redox probes, is mainly confined to thedetection of intracellular oxidative stress in vitro. To expand theapplicability of the probe, an alkaline hydrolysis and solvent extractionprocedure was developed to generate high-purity DCFH2 (2′,7′-dichlorodihydrofluorescein)from DCFH2-DA using basic laboratory equipment. Next, theutility of DCFH2 was exemplified in a variety of cell-freeand in vitro redox assay systems, including oxidant production bytransition metals, photodynamic therapy, activated macrophages, andplatelets, as well as the antioxidative capacity of different antioxidants.In cells, the concomitant use of DCFH2-DA and DCFH2 enabled the measurement and compartmentalized analysis ofintra- and extracellularly produced oxidants, respectively, usinga single read-out parameter. Furthermore, hepatocyte-targeted liposomeswere developed to deliver the carboxylated derivative, 5(6)-carboxy-DCFH2, to hepatocytes in vivo. Liposome-delivered 5(6)-carboxy-DCFH2 enabled real-time visualization and measurement of hepatocellularoxidant production during liver ischemia-reperfusion. The liposomal5(6)-carboxy-DCFH2 can be targeted to other tissues whereoxidative stress is important, including cancer.

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Real-timeanalysis of hepatocellular oxidative stress using liposome-deliveredCDCFH2 during hepatic ischemia-reperfusion (IR) and shamoperation in mouse livers. (A) Uptake of NBD-labeled GM1 and GM1 +lactosyl-PE (LPE) liposomes by hepatocytes (HCs), Kupffer cells (KCs),and endothelial cells (ECs), which was analyzed using flow cytometry.Oxidant formation during IR was analyzed by intravital fluorescence(flu) microscopy (D) and spectroscopy (B) in a standardized mousemodel of liver IR (60 min ischemia) using CDCFH2-encapsulatingGM1 liposomes. (C) Cumulative fluorescence formation during 20 minreperfusion, which was significantly higher in the IR group comparedto sham controls.
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fig3: Real-timeanalysis of hepatocellular oxidative stress using liposome-deliveredCDCFH2 during hepatic ischemia-reperfusion (IR) and shamoperation in mouse livers. (A) Uptake of NBD-labeled GM1 and GM1 +lactosyl-PE (LPE) liposomes by hepatocytes (HCs), Kupffer cells (KCs),and endothelial cells (ECs), which was analyzed using flow cytometry.Oxidant formation during IR was analyzed by intravital fluorescence(flu) microscopy (D) and spectroscopy (B) in a standardized mousemodel of liver IR (60 min ischemia) using CDCFH2-encapsulatingGM1 liposomes. (C) Cumulative fluorescence formation during 20 minreperfusion, which was significantly higher in the IR group comparedto sham controls.

Mentions: Lastly, intracellular oxidativestress was measured in vivo usingliposome-encapsulated CDCFH2, a more hydrophilic form ofDCFH2 that is oxidized to the highly fluorescent CDCF (λex = 495 nm, λem = 525 nm) by oxidants.19 The alkaline hydrolysis and two-phase liquidextraction method was used to prepare CDCFH2 from CDCFH2-DA. Next, CDCFH2 was encapsulated in hepatotargetedliposomes. The liposomes, composed of DPPC, cholesterol, lactosyl-phosphatidylethanolamine(LPE) and monosialotetrahexosylganglioside (GM1) in varying molarratios, were optimized in vitro (S–XVI through S–XIX) and investigated in vivo in terms ofintrahepatic accumulation (S–XX and S–XXI) and distribution to hepatocytes and nonparenchymal cells (Figure 3A). CDCFH2 was subsequently encapsulated into the optimal formulation for hepatocellulartargeting (i.e., DPPC:cholesterol:GM1 in a 55:40:5 molar ratio) tovisualize oxidative stress in vivo. Hepatocellular oxidative stresswas induced using a standardized mouse model of hepatic ischemia-reperfusioninjury,20 which is associated with extensiveoxidative stress in the reperfusion phase. Accordingly, hepatocellularCDCF formation was observed in the ischemia-reperfusion group butnot in sham-operated control animals (Figure 3B–D). These data are the first toshow selective hepatocellular oxidant formation during early reperfusion.Proof-of-concept was provided only in the liver, where redox perturbationslie at the basis of numerous disorders. Nevertheless, the applicabilityof the method is expandable to other redox-pertinent tissues to whichliposomes can be targeted, such as solid tumors21 and atherosclerotic plaques.22


Preparation and Practical Applications of 2 ′ ,7 ′ -Dichlorodihydrofluoresceinin Redox Assays
Real-timeanalysis of hepatocellular oxidative stress using liposome-deliveredCDCFH2 during hepatic ischemia-reperfusion (IR) and shamoperation in mouse livers. (A) Uptake of NBD-labeled GM1 and GM1 +lactosyl-PE (LPE) liposomes by hepatocytes (HCs), Kupffer cells (KCs),and endothelial cells (ECs), which was analyzed using flow cytometry.Oxidant formation during IR was analyzed by intravital fluorescence(flu) microscopy (D) and spectroscopy (B) in a standardized mousemodel of liver IR (60 min ischemia) using CDCFH2-encapsulatingGM1 liposomes. (C) Cumulative fluorescence formation during 20 minreperfusion, which was significantly higher in the IR group comparedto sham controls.
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getmorefigures.php?uid=PMC5382573&req=5

fig3: Real-timeanalysis of hepatocellular oxidative stress using liposome-deliveredCDCFH2 during hepatic ischemia-reperfusion (IR) and shamoperation in mouse livers. (A) Uptake of NBD-labeled GM1 and GM1 +lactosyl-PE (LPE) liposomes by hepatocytes (HCs), Kupffer cells (KCs),and endothelial cells (ECs), which was analyzed using flow cytometry.Oxidant formation during IR was analyzed by intravital fluorescence(flu) microscopy (D) and spectroscopy (B) in a standardized mousemodel of liver IR (60 min ischemia) using CDCFH2-encapsulatingGM1 liposomes. (C) Cumulative fluorescence formation during 20 minreperfusion, which was significantly higher in the IR group comparedto sham controls.
Mentions: Lastly, intracellular oxidativestress was measured in vivo usingliposome-encapsulated CDCFH2, a more hydrophilic form ofDCFH2 that is oxidized to the highly fluorescent CDCF (λex = 495 nm, λem = 525 nm) by oxidants.19 The alkaline hydrolysis and two-phase liquidextraction method was used to prepare CDCFH2 from CDCFH2-DA. Next, CDCFH2 was encapsulated in hepatotargetedliposomes. The liposomes, composed of DPPC, cholesterol, lactosyl-phosphatidylethanolamine(LPE) and monosialotetrahexosylganglioside (GM1) in varying molarratios, were optimized in vitro (S–XVI through S–XIX) and investigated in vivo in terms ofintrahepatic accumulation (S–XX and S–XXI) and distribution to hepatocytes and nonparenchymal cells (Figure 3A). CDCFH2 was subsequently encapsulated into the optimal formulation for hepatocellulartargeting (i.e., DPPC:cholesterol:GM1 in a 55:40:5 molar ratio) tovisualize oxidative stress in vivo. Hepatocellular oxidative stresswas induced using a standardized mouse model of hepatic ischemia-reperfusioninjury,20 which is associated with extensiveoxidative stress in the reperfusion phase. Accordingly, hepatocellularCDCF formation was observed in the ischemia-reperfusion group butnot in sham-operated control animals (Figure 3B–D). These data are the first toshow selective hepatocellular oxidant formation during early reperfusion.Proof-of-concept was provided only in the liver, where redox perturbationslie at the basis of numerous disorders. Nevertheless, the applicabilityof the method is expandable to other redox-pertinent tissues to whichliposomes can be targeted, such as solid tumors21 and atherosclerotic plaques.22

View Article: PubMed Central - PubMed

ABSTRACT

Oxidative stress,a state in which intra- or extracellular oxidantproduction outweighs the antioxidative capacity, lies at the basisof many diseases. DCFH2-DA (2′,7′-dichlorodihydrofluoresceindiacetate) is the most widely used fluorogenic probe for the detectionof general oxidative stress. However, the use of DCFH2-DA,as many other fluorogenic redox probes, is mainly confined to thedetection of intracellular oxidative stress in vitro. To expand theapplicability of the probe, an alkaline hydrolysis and solvent extractionprocedure was developed to generate high-purity DCFH2 (2′,7′-dichlorodihydrofluorescein)from DCFH2-DA using basic laboratory equipment. Next, theutility of DCFH2 was exemplified in a variety of cell-freeand in vitro redox assay systems, including oxidant production bytransition metals, photodynamic therapy, activated macrophages, andplatelets, as well as the antioxidative capacity of different antioxidants.In cells, the concomitant use of DCFH2-DA and DCFH2 enabled the measurement and compartmentalized analysis ofintra- and extracellularly produced oxidants, respectively, usinga single read-out parameter. Furthermore, hepatocyte-targeted liposomeswere developed to deliver the carboxylated derivative, 5(6)-carboxy-DCFH2, to hepatocytes in vivo. Liposome-delivered 5(6)-carboxy-DCFH2 enabled real-time visualization and measurement of hepatocellularoxidant production during liver ischemia-reperfusion. The liposomal5(6)-carboxy-DCFH2 can be targeted to other tissues whereoxidative stress is important, including cancer.

No MeSH data available.


Related in: MedlinePlus