Jean-Marie Devoisselle

Fluorescence properties of indocyanin green: II. In-vitro study related to in-vivo behavior

The fluorometric properties of indocyanine green has been largely described. Salts, proteins, lipoproteins are mentioned as modifying the fluorescence characteristics of ICG. We have recently observed that ICG is able to be bound at the interface of model membranes. In this study, we reinvestigated the spectral properties of ICG in biological media. The fluorescence quenching curves of ICG in the water, protein solution and whole blood are very similar but the absorption characteristics of ICG are quite different from one medium to another, ICG displays an aggregative behavior in water and serum depending on its concentration but we observed no modification of the absorption spectra in blood. This quenching property is also observed in vivo using blood sampling. These results show that the spectral behavior of ICG in biological media may be taken in account when fluorescence measurements are performed.

Fluorescence properties of indocyanin green: I. In-vitro study with micelles and liposomes

ICG is a tricarbocyanin fluorescent dye used in angiography. Several reports point out the advantage of ICG to fluoresce in the near IR wavelength range enabling the imaging of deep tissues. If the fluorescence characteristics of ICG in buffer or protein solutions are described in vitro, little is known concerning the physicochemical properties of ICG. This study aims to evaluate the physicochemical and fluorescence spectral characteristics of ICG in aqueous solution and in presence of micelles and liposomes. ICG exhibits a tensioactive property and when incubated with micelles and liposomes tends to be aggregated or embedded at the interface. The fluorescence is very low in the aggregated form and is very high when ICG is embedded at the interface and a shift of the emission peak toward longer wavelength is observed. Such in vitro study could contribute to a better understanding of some observed unusual properties of ICG in vivo.

Effect of indocyanin green formulation on blood clearance and in vivo fluorescence kinetic profile of skin

Indocyanine green has been used to measure cardiac and liver functions. More recently, it has been proposed as a contrast agent in ophthalmic angiography, tumor imaging and as an infrared absorbing dye in the context of laser-induced thermal damage of blood vessels. The aim of the study is to overcome the disadvantage of a very short blood half-time and to participate to a better confinement in blood vessels. Indocyanine green was administered intravenously to Wistar rats at a 7.5 mg/kg dose. Formulations consist in indocyanine green aqueous solution and o/w emulsion. Blood samples were collected and analyzed by spectrophotometry. Fluorescence was recorded in vivo by spectrofluorometry using an optic fiber coupled to an optical multichannel analyzer. The fiber optic was placed at a 4 mm distance from the skin surface. Results show that aqueous solution of indocyanine green leads to a rapid blood clearance. To the administration of ICG emulsion belongs the advantage of increasing the half-time and the residence time of indocyanine green in skin. It may be noted that however the formulation is, the observed blood clearance profiles are quite different from the tissue fluorescence kinetic profiles. The dye could have a longer residence time (20 - 60 min. plateau phase). Moreover, a shift of the maximum emission peak is noted after i.v. administration. The study of ICG fluorescence in the presence of model membranes shows that ICG is able to interact with phospholipid bilayers. These findings may be interesting for therapeutic applications of indocyanine green requiring a high level of dye in tissues for a great period of time and participate to the knowledge of ICG behavior in vivo.

In-vivo pharmacokinetic study of two fluorescein derivatives by fluorescence spectroscopy

We have already demonstrated the ability of fluorescence spectroscopy and imaging to measure the pH of superficial tissues using pH sensitive fluorescent probes. The purpose of this study was to investigate the in vivo behavior of such fluorescent probes. We report the monitoring of tissue fluorescence after injection of two fluorescein derivatives (carboxyfluorescein and biscarboxyethyl-carboxyfluorescein). The in vivo study was performed on anaesthetized adult Wistar rats. After laparotomy, CF or BCECF solution was injected into the penial vein. Fluorescence spectra were recorded during one hour using an optical multichannel analyzer coupled to a CCD camera. Fiber optic was placed alternatively on the liver area or on the skin. Blood samples were collected and fluorescence was measured in vitro. A clear linear relationship between dose and fluorescence intensity was found in liver for these fluorescent markers. Concerning spectral characteristics, it was found that CF and BCECF spectra show a shift compared to in vivo maximum emission peak and BCECF emission peak was different when recorded in the liver and in the skin. Differences of kinetic profiles are also observed between CF and BCECF. The BCECF derivative displays a fluorescence peak in the liver two minutes after injection, while CF fluorescence peak is observed seven minutes after injection. Clearance of skin fluorescence is slower than the plasmatic one indicating that dye elimination in superficial blood vessels does not follow the same pharmacokinetic behavior. Based on these preliminary findings, fluorescence spectroscopy appears as a tool in pharmacokinetic study in situ and in vivo.

Thermal damage assessment of blood vessels in a hamster skin flap model by fluorescence measurement of a liposome-dye system

The present study was undertaken to evaluate the feasibility of thermal damage assessment of blood vessels by using laser-induced release of liposome-encapsulated dye. Experiments were performed in a hamster skin flap model. Laser irradiation was achieved with a 300micrometers fiber connected to a 805nm diode laser after potentiation using a specific indocyanine green (ICG) formulation. Liposomes- encapsulated carboxyfluorescein were prepared by the sonication procedure. Carboxyfluorescein was loaded at high concentration in order to quench its fluorescence. The measurements were performed after i.v. injection of DSPC liposomes and lasted 40 minutes. Fluorescence emission was measured with an ultra high sensitivity intensified camera. Three different shapes of fluorescent spots were identified depending on target and energy deposition in tissue: (i) intravascular fluorescence, (ii) transient low fluorescence circular spot and (iii) persistent high intense fluorescence spot. These images are correlated with histological data. The advantages of this liposome-dye system are (1) direct measurements can be obtained, (2) several repeated readings can be made from one injection, (3) continuous monitoring of the fluorescence can be made, (4) temperature-sensitive range can be adapted using different liposomes compositions, (5) circulation times of several hours can be achieved using DSPC liposomes (6) the tissue microcirculation and the vessel macrocirculation can be investigated simultaneously, therefore changes in response to a treatment regimen and/or ICG formulations can be detected. One main constraint exists: the fluorescent dye encapsulated into the liposomes has to be carefully chosen in order to avoid any direct absorption by the dye itself. In conclusion, one of the most significant applications of this experimental technique is the evaluation of various degrees of tissue thermal damage. It could be possible to consider the application of this technique in ophthalmology and dermatology and possibly for the evaluation of burn injury.

In-vivo and ex-vivo spectrofluorometric and imaging study of liposome uptake by the liver using a pH-sensitive probe

Liposomes are known to be uptaken by the liver cells after intraveinous injection. Only few techniques are available to follow this process in vivo like nuclear magnetic resonance spectroscopy or scintigraphy. Intracellular pathway and liposomes localization in the different liver cells require sacrifice of the animals, cells separation and electronic microscopy, then little is known about liposomes kinetic uptake by the acidic intracellular compartments in vivo. We propose in this study a new method to follow liposomes uptake in the liver in vivo using a fluorescent pH sensitive probe 5,6-carboxyfluorescein and two different composition of liposomes: phospholipids DSPC/Chol and DMPC in order to evaluate the influence of the formulation on the release characteristics of liposomes in the lysosomes. We have already demonstrated the ability of the fluorescence spectroscopy and imaging using a pH dependent probe to monitor pH in living tissues. As pH of lysosomes is very low, the kinetic liposomes uptake in this intracellular acidic compartment is followed by monitoring the pH of the whole liver in vivo and ex vivo. Carboxyfluorescein is used at high concentration (100 mM) in order to quench its fluorescence. Liposomes are injected to Wistar rats into the penil vein. After laparotomy, fluorescence spectra and images are recorded during two hours. Results show a clear relationship between formulation of liposomes and stability in the acidic compartments of hepatic cells. After sacrifice and flush with cold saline solution, pH of the liver ex vivo is found to be 5.0-5.5. Data show a rapid clearance of release dye and an uptake of liposomes by the liver cells and, as liposomes penetrate in the acidic compartment, dye is released from liposomes and is delivered in lysosomes leading to the decrease of the pH.

Study of normal/tumorous tissue fluorescence using a pH-dependent fluorescent probe in vivo

The pH of interstitial fluid of malignant tumors tends to be lower than that of normal tissue and depressed by glucose administration. This study aimed to evaluate the effectiveness of dual-wavelength ratio fluorometry using a pH-dependent indicator (5,6-carboxyfluorescein: 5,6-CF) for the characterization of normal and tumoral areas in vivo. 5,6-CF has two main characteristics: it has two wavelengths of maximum absorbance (465 and 490 nm) and its fluorescence emission (maximum at 515 nm) increases as a function of pH in the physiological 6 - 7.4 pH range. The experimental study was performed on 31 CDF mice bearing lymphoid leukaemia P388 grafted subcutaneously. The tissular pH values were evaluated from the ratio of the fluorescence intensities (I490/I465) on the basis of a calibration curve linking pH measurements performed intratissularly with a microelectrode and fluorescence intensities ratio values. The fluorescence intensity reached its maximum value at 60 min after 5,6-CF and glucose administration, followed by a plateau (90 min). The ratios remain constant at 1.79 +/- 0.06 for normal tissue and 1.61 +/- 0.07 (without glucose administration) for tumoral tissue. The tumoral tissue ratios decrease down to 1.35 +/- 0.04 after 6 g/kg glucose administration. These results were correlated to the pH measurements in accordance to the calibration curve. This study validates the relevance of dual-wavelength fluorometry using a pH-dependent indicator to characterize in-vivo normal and tumoral tissues after glucose administration.

Experimental evaluation of site-specific delivery of methylene blue to the hair follicles using fluorescence imaging

This study aimed to evaluate the penetration of Methylene Blue (MB)-loaded microspheres into the follicular ducts of rats. MB was incorporated into porous nylon microspheres which have a size of 10 ?m (Orgasol 4000 Nat Cos, Elf-Atochem, France). The microspheres were dispersed into fluid silicone. Male hairless rats (NU, Iffa Credo, France; mutant hairless Sprague Dawley rat) were used to evaluate the penetration of MB into hair follicles. After formulation application, MB diffusion was induced and skin biopsies were realized immediately, 2 hours and 26 hours after MB loaded microspheres application. All samples were snap frozen in liquid nitrogen and processed stored at -80°C before embedded in TissuTek and cryostat-cut. 14?m sections were realized. MB fluorescence was observed with Eclipse E800 microscope (Nikon, Tokyo, Japan) expanded for fluorescence microscopy (with FITC, TRITC and CY5 filters) and penetration depth was quantified using image analysis software (Sigma Pro, Jandel, USA). Autofluorescence was evaluated using FITC and TRITC filter. MB was seen 200?m deep in hair follicles (2 hours after application) and at 600?m (26 hours after application). This galenical approach aims to transport MB into the hair follicle specifically and deeply.

Intestine pH measurements using fluorescence imaging: an in-vivo preliminary study

Measurement of gastrointestinal intramucosal pH has been recognized as an important factor in the detection of hypoxia-induced dysfunctions. However, current pH measurement techniques are limited in terms of time and spatial resolution. A major advance in accurate pH measurement was the development of the ratiometric fluorescent indicator dye, 2,7-bis(carboxyethyl)-4,5- carboxyfluorescein (BCECF). This study aimed to demonstrate the feasibility of fluorescence imaging technique to measure in vivo the pH of intestine. The intestine was inserted in an optical chamber placed under a microscope. Animals were injected i.v. with the pH-sensitive fluorescent dye BCECF. Fluorescence was visualized by illuminating the intestine alternately at 490 and 470 nm. The emitted fluorescence was directed to an intensified camera. The ratio of emitted fluorescence at excitation wavelengths of 490 and 470 nm was measured, corrected and converted to pH by constructing a calibration curve. The pH controls were performed with a pH microelectrode correlated with venous blood gas sampling. We concluded that accurate pH measurements of rat intestine can be obtained by fluorescence imaging using BCECF. This technology could be easily adapted for endoscopic pH measurement.

In vivo fluorescence imaging of lysosomes: a potential technique to follow dye accumulation in the context of PDT?

Lysosomes and intracellular acidic compartments seem to play an important role in the context of PDT. Some photosensitizers are localized in the lysosomes of tumor-associated macrophages. Liposomes, which are lysosomotropic drug carriers, are used to deliver photosensitizers in tumors. Liposomes are taken up by the liver cells after intravenous injection. Intracellular pathway and liposomes localization in the different liver cells require sacrifice of the animals, cell separation, and observation by electronic microscopy. Little is known about liposomes kinetic uptake by the acidic intracellular compartments in vivo. We propose in this study a new method to follow liposomes uptake in the liver in vivo using a fluorescent pH-sensitive probe. We have already demonstrated the ability of fluorescence spectroscopy and imaging using a pH-dependent probe to monitor pH in living tissues. As pH of lysosome is very low, the kinetic of liposome uptake in this intracellular acidic compartment is followed by monitoring the pH of the whole liver in vivo and ex vivo. Liposomes-encapsulated carboxyfluorescein are prepared by the sonication procedure. Carboxyfluorescein is used at high concentration (100 mM) in order to quench its fluorescence. Liposomes are injected to Wistar rats into the peinil vein. After laparotomy, fluorescence spectra and images are recorded during two hours. Results show a rapid fluorescence increase followed by a slow phase of fluorescence decrease. pH decreases from physiological value to 6.0. After sacrifice and flush with cold saline solution, pH of liver ex vivo is found to be 5.0 - 5.5. These data show a rapid clearance of released dye and an uptake of liposomes by the liver cells and, as liposomes penetrate in the acidic compartment, dye is released from liposomes and is delivered in lysosomes leading to the decrease of pH.