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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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Accumulation of cationic and neutral liposomes was observed with the scanning laser ophthalmoscope (SLO) in vivo. Cationic liposomes (CL)-indocyanine green (ICG), CL-Oregon green (OG), or neutral liposomes labeled with OG were applied intravenously at d1, d5, d10, or d14 after laser treatment. SLO images were recorded for each laser choroidal neovascularization (CNV) 60 min later. A: One representative CNV is shown for each time point and for each of the formulations tested. The CL-ICG images are taken from the same CNV in one mouse, and the corresponding infrared (IR) images are shown for comparison. The other images are taken from different animals. Although neutral liposomes did not accumulate within the CNV, CL-OG was found from d10 onwards and CL-ICG starting from d5. B: The ratio of accumulation of liposomes in the CNV to the control area was calculated for each CNV as described in the methods section. The means and the standard errors of the mean are shown. Values for CL-OG at d10 and d14 and for CL-ICG at d5, d10, and d14 were significantly higher compared to those of neutral liposomes. Data are means obtained from five mice. Error bars indicate SEM, and asterisks indicate statistical significance (p<0.05 as compared to d1). C: Absorption spectrum of mouse RPE. After the retina as removed, the RPE was scraped out, homogenized by pipetting, and diluted in water. Transmission is higher at longer wavelengths. The OG emission was detected at 530 nm and that of ICG at 830 nm. The murine RPE absorption was about twice as high at 530 nm as at 830 nm. This gives an estimate of the reduction of fluorescence emitted by fluorophores when they are located behind the RPE in vivo and is one of the reasons why ICG is more suitable for diagnostics of sub-RPE lesions than OG or fluorescein.
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f3: Accumulation of cationic and neutral liposomes was observed with the scanning laser ophthalmoscope (SLO) in vivo. Cationic liposomes (CL)-indocyanine green (ICG), CL-Oregon green (OG), or neutral liposomes labeled with OG were applied intravenously at d1, d5, d10, or d14 after laser treatment. SLO images were recorded for each laser choroidal neovascularization (CNV) 60 min later. A: One representative CNV is shown for each time point and for each of the formulations tested. The CL-ICG images are taken from the same CNV in one mouse, and the corresponding infrared (IR) images are shown for comparison. The other images are taken from different animals. Although neutral liposomes did not accumulate within the CNV, CL-OG was found from d10 onwards and CL-ICG starting from d5. B: The ratio of accumulation of liposomes in the CNV to the control area was calculated for each CNV as described in the methods section. The means and the standard errors of the mean are shown. Values for CL-OG at d10 and d14 and for CL-ICG at d5, d10, and d14 were significantly higher compared to those of neutral liposomes. Data are means obtained from five mice. Error bars indicate SEM, and asterisks indicate statistical significance (p<0.05 as compared to d1). C: Absorption spectrum of mouse RPE. After the retina as removed, the RPE was scraped out, homogenized by pipetting, and diluted in water. Transmission is higher at longer wavelengths. The OG emission was detected at 530 nm and that of ICG at 830 nm. The murine RPE absorption was about twice as high at 530 nm as at 830 nm. This gives an estimate of the reduction of fluorescence emitted by fluorophores when they are located behind the RPE in vivo and is one of the reasons why ICG is more suitable for diagnostics of sub-RPE lesions than OG or fluorescein.

Mentions: The distribution of CL labeled with ICG or OG in the fundus was followed by SLO after intravenous injection. Figure 2 shows representative images at various time points after application of labeled CL-ICG or CL-OG 14 days after laser coagulation. No specific signal was detected before CL-ICG or CL-OG was injected. Circulation of CL formulations in the blood plasma was faintly visible for 2–3 min after application based on the respective fluorescence signal (Figure 2B). After that, fast accumulation of fluorescence at the CNV site was observed. Typically, the signal due to CL-OG was visible between 20 and 120 min after application. The CL-ICG signal was stronger and therefore was visible as early as 5 min after application and was observed for several hours, in some cases even for 24 h. The CL-ICG and CL-OG peak signal was observed between 30 and 90 min so that a measuring time point of 60 min after injection was chosen in the following experiments. CL-ICG resulted in a more intense and precise image compared to CL-OG (Figure 2C). In contrast, neutral liposomes did not accumulate at the laser site but remained much longer in circulation (Figure 3) similar to ICG or fluorescein in angiography.


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)

Accumulation of cationic and neutral liposomes was observed with the scanning laser ophthalmoscope (SLO) in vivo. Cationic liposomes (CL)-indocyanine green (ICG), CL-Oregon green (OG), or neutral liposomes labeled with OG were applied intravenously at d1, d5, d10, or d14 after laser treatment. SLO images were recorded for each laser choroidal neovascularization (CNV) 60 min later. A: One representative CNV is shown for each time point and for each of the formulations tested. The CL-ICG images are taken from the same CNV in one mouse, and the corresponding infrared (IR) images are shown for comparison. The other images are taken from different animals. Although neutral liposomes did not accumulate within the CNV, CL-OG was found from d10 onwards and CL-ICG starting from d5. B: The ratio of accumulation of liposomes in the CNV to the control area was calculated for each CNV as described in the methods section. The means and the standard errors of the mean are shown. Values for CL-OG at d10 and d14 and for CL-ICG at d5, d10, and d14 were significantly higher compared to those of neutral liposomes. Data are means obtained from five mice. Error bars indicate SEM, and asterisks indicate statistical significance (p<0.05 as compared to d1). C: Absorption spectrum of mouse RPE. After the retina as removed, the RPE was scraped out, homogenized by pipetting, and diluted in water. Transmission is higher at longer wavelengths. The OG emission was detected at 530 nm and that of ICG at 830 nm. The murine RPE absorption was about twice as high at 530 nm as at 830 nm. This gives an estimate of the reduction of fluorescence emitted by fluorophores when they are located behind the RPE in vivo and is one of the reasons why ICG is more suitable for diagnostics of sub-RPE lesions than OG or fluorescein.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351413&req=5

f3: Accumulation of cationic and neutral liposomes was observed with the scanning laser ophthalmoscope (SLO) in vivo. Cationic liposomes (CL)-indocyanine green (ICG), CL-Oregon green (OG), or neutral liposomes labeled with OG were applied intravenously at d1, d5, d10, or d14 after laser treatment. SLO images were recorded for each laser choroidal neovascularization (CNV) 60 min later. A: One representative CNV is shown for each time point and for each of the formulations tested. The CL-ICG images are taken from the same CNV in one mouse, and the corresponding infrared (IR) images are shown for comparison. The other images are taken from different animals. Although neutral liposomes did not accumulate within the CNV, CL-OG was found from d10 onwards and CL-ICG starting from d5. B: The ratio of accumulation of liposomes in the CNV to the control area was calculated for each CNV as described in the methods section. The means and the standard errors of the mean are shown. Values for CL-OG at d10 and d14 and for CL-ICG at d5, d10, and d14 were significantly higher compared to those of neutral liposomes. Data are means obtained from five mice. Error bars indicate SEM, and asterisks indicate statistical significance (p<0.05 as compared to d1). C: Absorption spectrum of mouse RPE. After the retina as removed, the RPE was scraped out, homogenized by pipetting, and diluted in water. Transmission is higher at longer wavelengths. The OG emission was detected at 530 nm and that of ICG at 830 nm. The murine RPE absorption was about twice as high at 530 nm as at 830 nm. This gives an estimate of the reduction of fluorescence emitted by fluorophores when they are located behind the RPE in vivo and is one of the reasons why ICG is more suitable for diagnostics of sub-RPE lesions than OG or fluorescein.
Mentions: The distribution of CL labeled with ICG or OG in the fundus was followed by SLO after intravenous injection. Figure 2 shows representative images at various time points after application of labeled CL-ICG or CL-OG 14 days after laser coagulation. No specific signal was detected before CL-ICG or CL-OG was injected. Circulation of CL formulations in the blood plasma was faintly visible for 2–3 min after application based on the respective fluorescence signal (Figure 2B). After that, fast accumulation of fluorescence at the CNV site was observed. Typically, the signal due to CL-OG was visible between 20 and 120 min after application. The CL-ICG signal was stronger and therefore was visible as early as 5 min after application and was observed for several hours, in some cases even for 24 h. The CL-ICG and CL-OG peak signal was observed between 30 and 90 min so that a measuring time point of 60 min after injection was chosen in the following experiments. CL-ICG resulted in a more intense and precise image compared to CL-OG (Figure 2C). In contrast, neutral liposomes did not accumulate at the laser site but remained much longer in circulation (Figure 3) similar to ICG or fluorescein in angiography.

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