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In vivo imaging of choroidal angiogenesis using fluorescence-labeled cationic liposomes.

Hua J, Gross N, Schulze B, Michaelis U, Bohnenkamp H, Guenzi E, Hansen LL, Martin G, Agostini HT - Mol. Vis. (2012)

Bottom Line: The best signal was obtained with CL-ICG.These results establish fluorophore-labeled CL as high affinity markers to selectively stain active CNV.Labeling of angiogenic vessels using CL can be of interest not only for functional imaging in ophthalmology but also for other conditions where localization of active angiogenesis is desirable.

View Article: PubMed Central - PubMed

Affiliation: University Eye Hospital, Albert-Ludwigs University of Freiburg, Killianstrasse 5, Freiburg im Breisgau, Germany

ABSTRACT

Purpose: Precise monitoring of active angiogenesis in neovascular eye diseases such as age-related macular degeneration (AMD) enables sensitive use of antiangiogenic drugs and reduces adverse side effects. So far, no in vivo imaging methods are available to specifically label active angiogenesis. Here, we report such a technique using fluorophore-labeled cationic liposomes (CL) detected with a standard clinical in vivo scanning laser ophthalmoscope (SLO).

Methods: C57Bl/6 mice underwent laser coagulations at day 0 (d0) to induce choroidal neovascularization (CNV). Liposomes labeled with Oregon green, rhodamine (Rh), or indocyanine green (ICG) were injected into the tail vein at various time points after laser coagulation, and their fluorescence was observed in vivo 60 min later using an SLO, or afterwards in choroidal flatmounts or cryosections.

Results: SLO detected accumulated fluorescence only in active CNV lesions with insignificant background noise. The best signal was obtained with CL-ICG. Choroidal flatmounts and cryosections of the eye confirmed the location of retained CL in CNV lesions. Neutral liposomes, in contrast, showed no accumulation.

Conclusions: These results establish fluorophore-labeled CL as high affinity markers to selectively stain active CNV. This novel, non-invasive SLO imaging technique could improve risk assessment and indication for current intraocular antiangiogenic drugs in neovascular eye diseases, as well as monitor therapeutic outcomes. Labeling of angiogenic vessels using CL can be of interest not only for functional imaging in ophthalmology but also for other conditions where localization of active angiogenesis is desirable.

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Cationic liposomes (CL) formulations labeled with indocyanine green (ICG) or Oregon green (OG) were internally quenched. Liposomes composed of 60 mol% 1,2 dioleoyl-3-trimethylammonium propane (DOTAP), 40 - x mol% 1,2 dioleoyl-sn-glycero-3-phosphocholine (DOPC), and x mol% ICG-DOPE or OG-DOPE were prepared and diluted 1:50 (CL-ICG) or 1:2,000 (CL-OG). Intraliposomal quenching reduces the fluorescence in the liposomal membrane at concentrations of more than 0.5 mol% ICG or 5 mol% OG. The fluorescence signal of ICG is ten times lower than that of OG if the different dilutions are taken into account.
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f1: Cationic liposomes (CL) formulations labeled with indocyanine green (ICG) or Oregon green (OG) were internally quenched. Liposomes composed of 60 mol% 1,2 dioleoyl-3-trimethylammonium propane (DOTAP), 40 - x mol% 1,2 dioleoyl-sn-glycero-3-phosphocholine (DOPC), and x mol% ICG-DOPE or OG-DOPE were prepared and diluted 1:50 (CL-ICG) or 1:2,000 (CL-OG). Intraliposomal quenching reduces the fluorescence in the liposomal membrane at concentrations of more than 0.5 mol% ICG or 5 mol% OG. The fluorescence signal of ICG is ten times lower than that of OG if the different dilutions are taken into account.

Mentions: The fluorophores OG, ICG, and Rh were stably linked to CL as they were covalently bound to phospholipids. This was confirmed with high-performance liquid chromatography analysis that did not detect any unbound fluorophore at any time during the study. Fluorescence intensity was optimal at 1 mol% ICG and 5 mol% OG in total liposomes due to intraliposomal quenching at higher fluorophore ratios (Figure 1). The inset in Figure 1 shows the respective molecular structure; OG differs from fluorescein only by having two F atoms at the aromatic ring system. In both structures, “R” indicates where the linker to the lipid moiety has been attached. Quenching of Rh was similar to OG in CL. Therefore, Rh was also used at a concentration of 5 mol%.


In vivo imaging of choroidal angiogenesis using fluorescence-labeled cationic liposomes.

Hua J, Gross N, Schulze B, Michaelis U, Bohnenkamp H, Guenzi E, Hansen LL, Martin G, Agostini HT - Mol. Vis. (2012)

Cationic liposomes (CL) formulations labeled with indocyanine green (ICG) or Oregon green (OG) were internally quenched. Liposomes composed of 60 mol% 1,2 dioleoyl-3-trimethylammonium propane (DOTAP), 40 - x mol% 1,2 dioleoyl-sn-glycero-3-phosphocholine (DOPC), and x mol% ICG-DOPE or OG-DOPE were prepared and diluted 1:50 (CL-ICG) or 1:2,000 (CL-OG). Intraliposomal quenching reduces the fluorescence in the liposomal membrane at concentrations of more than 0.5 mol% ICG or 5 mol% OG. The fluorescence signal of ICG is ten times lower than that of OG if the different dilutions are taken into account.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3351413&req=5

f1: Cationic liposomes (CL) formulations labeled with indocyanine green (ICG) or Oregon green (OG) were internally quenched. Liposomes composed of 60 mol% 1,2 dioleoyl-3-trimethylammonium propane (DOTAP), 40 - x mol% 1,2 dioleoyl-sn-glycero-3-phosphocholine (DOPC), and x mol% ICG-DOPE or OG-DOPE were prepared and diluted 1:50 (CL-ICG) or 1:2,000 (CL-OG). Intraliposomal quenching reduces the fluorescence in the liposomal membrane at concentrations of more than 0.5 mol% ICG or 5 mol% OG. The fluorescence signal of ICG is ten times lower than that of OG if the different dilutions are taken into account.
Mentions: The fluorophores OG, ICG, and Rh were stably linked to CL as they were covalently bound to phospholipids. This was confirmed with high-performance liquid chromatography analysis that did not detect any unbound fluorophore at any time during the study. Fluorescence intensity was optimal at 1 mol% ICG and 5 mol% OG in total liposomes due to intraliposomal quenching at higher fluorophore ratios (Figure 1). The inset in Figure 1 shows the respective molecular structure; OG differs from fluorescein only by having two F atoms at the aromatic ring system. In both structures, “R” indicates where the linker to the lipid moiety has been attached. Quenching of Rh was similar to OG in CL. Therefore, Rh was also used at a concentration of 5 mol%.

Bottom Line: The best signal was obtained with CL-ICG.These results establish fluorophore-labeled CL as high affinity markers to selectively stain active CNV.Labeling of angiogenic vessels using CL can be of interest not only for functional imaging in ophthalmology but also for other conditions where localization of active angiogenesis is desirable.

View Article: PubMed Central - PubMed

Affiliation: University Eye Hospital, Albert-Ludwigs University of Freiburg, Killianstrasse 5, Freiburg im Breisgau, Germany

ABSTRACT

Purpose: Precise monitoring of active angiogenesis in neovascular eye diseases such as age-related macular degeneration (AMD) enables sensitive use of antiangiogenic drugs and reduces adverse side effects. So far, no in vivo imaging methods are available to specifically label active angiogenesis. Here, we report such a technique using fluorophore-labeled cationic liposomes (CL) detected with a standard clinical in vivo scanning laser ophthalmoscope (SLO).

Methods: C57Bl/6 mice underwent laser coagulations at day 0 (d0) to induce choroidal neovascularization (CNV). Liposomes labeled with Oregon green, rhodamine (Rh), or indocyanine green (ICG) were injected into the tail vein at various time points after laser coagulation, and their fluorescence was observed in vivo 60 min later using an SLO, or afterwards in choroidal flatmounts or cryosections.

Results: SLO detected accumulated fluorescence only in active CNV lesions with insignificant background noise. The best signal was obtained with CL-ICG. Choroidal flatmounts and cryosections of the eye confirmed the location of retained CL in CNV lesions. Neutral liposomes, in contrast, showed no accumulation.

Conclusions: These results establish fluorophore-labeled CL as high affinity markers to selectively stain active CNV. This novel, non-invasive SLO imaging technique could improve risk assessment and indication for current intraocular antiangiogenic drugs in neovascular eye diseases, as well as monitor therapeutic outcomes. Labeling of angiogenic vessels using CL can be of interest not only for functional imaging in ophthalmology but also for other conditions where localization of active angiogenesis is desirable.

Show MeSH
Related in: MedlinePlus