Human in vivo fluorescence microimaging of the alveolar ducts and sacs during bronchoscopy.

The aim of the present study was to assess fibred confocal fluorescence microscopy (FCFM) as a tool for imaging the alveolar respiratory system in vivo during bronchoscopy. A 488-nm excitation wavelength FCFM device was used in 41 healthy subjects including 17 active smokers. After topical anaesthesia, the 1.4-mm miniprobe was introduced into the bronchoscope working channel and advanced distally to the alveoli. Morphometric and cellular analyses were performed on selected frames harbouring a minimal compression effect. In vivo acinar microimaging was obtained from each lung segment except for the apical and posterior segments of both upper lobes. Reproducible patterns, corresponding to the elastic framework of the axial and peripheral interstitial systems, were recorded from 192 separate acini. The mean+/-sd thickness of the acinar elastic fibres was 10+/-2.7 microm. Alveolar mouth diameters (mean+/-sd 278+/-53 microm) were normally distributed but appeared smaller in the right upper lobe and right medial basal segment. Lobular microvessels (median diameter 90 microm) were equally distributed throughout the lungs. Alveolar macrophages were not detectable in nonsmokers, whereas a specific tobacco-tar-induced fluorescence was observed in smoking subjects, providing fine details of the alveolar walls and macrophages. A strong correlation was found between the number of cigarettes smoked per day and the amount of large and mobile macrophages observed in vivo, as well as with the intensity of the macrophage alveolitis. Fibred confocal fluorescence microscopy enables accurate exploration of the peripheral lung in vivo in both smokers and nonsmokers.

Targeting of HIV gp120 by oligonucleotide-photosensitizer conjugates. Light-induced damages.

Some guanine-rich oligonucleotides inhibit HIV infectivity through interaction with the gp120 glycoprotein. Besides, photoinactivation of viruses attracts attention for blood decontamination. The feasibility of targeting a red light-absorbing chlorin-type photosensitizer to gp120 through covalent coupling with 8-mer phosphodiester oligodeoxynucleotides is investigated. Some conjugates inhibit binding of antibodies directed to gp120. Inhibition is significantly increased upon red light activation. The activity of the conjugates correlates with their ability to self-associate, a process strongly favored by the propensity of the hydrophobic chlorin moiety to dimerize. Thus, the photosensitizer moiety both promotes structures with a higher affinity for gp120 and, upon light activation, can induce site-directed damages to the protein.

Photodynamic inactivation of cell-free HIV strains by a red-absorbing chlorin-type photosensitizer.

We have investigated the photodynamic activity of a new chlorin-type photosensitizer on a reference human immunodeficiency virus type 1 (HIV-1) strain, two wild-type HIV-1 isolates and two drug-resistant HIV-1 isolates. This chlorin was highly effective for the inactivation of free viruses, as assessed by two different quantitative cell culture assays. In the absence of blood components, all the HIV strains, including wild-type and drug-resistant mutant isolates, were totally inactivated using 30 micrograms ml-1 of chlorin and 0.75 J cm-2 of 661 nm light. Successful killing of HIV-1 strains in either plasma or whole blood was also obtained by increasing the chlorin concentration moderately. Our results demonstrate the antiviral efficiency of this chlorin, suggesting the potential application of dye-sensitized photoirradiation to decontaminate blood products.

Kinetics of incorporation of porphyrins into small unilamellar vesicles.

The kinetics of hematoporphyrin or deuteroporphyrin incorporation in egg phosphatidylcholine small unilamellar vesicles have been investigated by fluorescence stopped-flow measurements. The processes can be described by a fast equilibrium. The on-rate constant is nearly diffusion controlled regardless of the compound used and the pH. The affinity of these porphyrins for the vesicles is merely governed by the exit rate which depends on the structure of the porphyrin and on its charge determined by pH.

Interactions of dicarboxylic porphyrins with membranes in relation to their ionization state.

The interactions of dicarboxylic porphyrins with membrane systems are discussed with particular emphasis on the effect of the charge of the porphyrin and the nature of the side-chains. The incorporation of hematoporphyrin or related dicarboxylic porphyrins within small unilamellar vesicles as membrane models is favored by a decrease of the pH in the range of physiological pH values. This effect might play an important role in the retention of porphyrins by tumors, which are more acidic than normal tissues. Kinetics studies also show that the partition of the porphyrin between the lipidic bilayer and the aqueous phase is governed by its release rate rather than by its incorporation rate.

In vitro uptake of dicarboxylic porphyrins by human atheroma. Kinetic and analytical studies.

Human atheromatous aorta segments as well as presumably disease-free control aorta were obtained at autopsy. They were incubated with solutions of various purified dicarboxylic porphyrins including hematoporphyrin (HP) and hydroxyethylvinyldeuteroporphyrin (HVD), and with solutions of Photofrin. Selective labelling of the atheroma was shown by macroscopic and microscopic observations of the characteristic porphyrin fluorescence associated with the atheromatous plaques. The time dependence of the uptake, monitored by absorption spectrophotometry or by high performance liquid chromatography, was inferred from the disappearance of the porphyrins in the incubation medium. Significant binding was observed in the absence of albumin or serum proteins. The uptake of HP was less than that of the more hydrophobic compounds HVD or Photofrin when these porphyrins were used alone. The presence of albumin or serum drastically reduces atheroma labelling. Some competition between HP and HVD for binding sites is also seen. The present results do indicate that hydrophobic porphyrins have an intrinsic affinity for atheroma and that they can be taken up through passive processes. Taking into account previous data on animal models (Photochem. Photobiol. (1989), 731-737), it appears however that, in vivo, interactions with proteins and pharmacokinetics will primarily determine plaque labelling.

Uptake and photodynamic efficiency of hematoporphyrin, hydroxyethylvinyldeuteroporphyrin and hematoporphyrin derivative (Photofrin II): a study with isolated mitochondria.

The uptake of Photofrin II (PFII), hematoporphyrin (Hp) and hydroxyethylvinyldeuteroporphyrin (HVD) by isolated mitochondria was studied using the high performance liquid chromatography (HPLC) technique. The various PFII components show a high affinity for mitochondria. At 5.75 micrograms/ml PFII, their ratio of incorporation was found to be very similar, except for Hp which is about two times less incorporated. These results were reproduced with pure Hp and pure HVD. The uptake of Hp and HVD increases with concentration but, while that of Hp reaches a plateau, the uptake of HVD continues to increase. At a high porphyrin concentration (approximately 10(-5) M), the loss of respiratory control is obtained with the same light dose for Hp and PFII. Taking into account the uptake and the known photophysical parameters of the various porphyrins, the photodynamic efficiency of HVD seems equivalent to that of Hp. The present results and known data on cell photoinactivation suggest that the activity of these porphyrins is mainly dependent on their incorporation.

Singlet molecular oxygen quenching by saturated and unsaturated fatty-acids and by cholesterol.

The rate constants of molecular singlet oxygen quenching by saturated and unsaturated fatty-acids and by cholesterol-membrane critical components - membrane critical components - have been measured by time resolved detection of the 1270 nm phosphorescence of singlet molecular oxygen [O2(1deltag)]. We have determined (i) an increment of 5.7 x 10(2)M(-1)s(-1) per -CH2- in C6D6 and CD3OD for saturated fatty acids between C4 and C20, (ii) an increment of 3 x 10(4)M(-1)s(-1) per non-conjugated cis-double bond for C18 unsaturated fatty acids, identical in C6D6 and DC3OD, (iii) a lower quenching rate constant by a factor of 2.7 for the trans-C16 and trans-C18 as compared to the corresponding cis-monounsaturated fatty acids, (iv) a rate constant of O2x(1deltag) quenching by cholesterol of 5.7 x 10(4)M(-1)s(-1) in benzene. These rate constants are compared to those obtained for other membrane cellular components.

Photofrin II uptake by atheroma in atherosclerotic rabbits. Fluorescence and high performance liquid chromatographic analysis on post-mortem aorta.

Atherosclerotic lesions were induced in normal and Watanabe rabbits by atherogenic diet and stripping of aorta endothelium. The rabbits were injected with Photofrin II and sacrificed two days later. Atheromatous aorta as well as normal aorta from control animals were characterized by their fluorescence spectra using front face excitation. Characteristic emission peaks at 631 and 694 nm were displayed at atheromatous plaques. The excitation spectrum shows a strong band at 394 nm and weaker bands at 446, 504, 536 and 574 nm. Although no fluorescence of normal aorta can be seen by visual inspection, emission with a maximum at 626 nm was detected by spectrofluorimetry. Normal phase high performance liquid chromatography analysis of extracts from atheroma and control aorta were also carried out. The specific labelling of atheroma involves mainly protoporphyrin, hematoporphyrin and also minor components of Photofrin II which are accumulated. Some other components are accumulated but do not appear to be specifically retained by atheroma.

Fundamental aspects in tumor photochemotherapy: interactions of porphyrins with membrane model systems and cells.

Some molecular aspects underlying photochemotherapy and photodiagnosis of tumors with porphyrins are reviewed. The nature of the clinically used photosensitizer HpD is first presented along with structures of molecules found to be efficient in vitro. The possible role of pH in the preferential retention of dicarboxylic porphyrins by tumors is discussed in light of results obtained with membrane models. The uptake of dicarboxylic porphyrins by cells most likely involves passive mechanisms. Cell photoinactivation using a purified porphyrin does not depend upon the incubation time but only on the intracellular concentration of the dye. This likely reflects a poor specificity of the photoinactivation processes with regard to the cellular localization of the dye. The properties which should be presented by more efficient photosensitizers are discussed.

Spectrofluorimetric study of porphyrin incorporation into membrane models--evidence for pH effects.

The effects of hydrophobicity and charges of dicarboxylic porphyrins upon their interactions with membrane model systems are investigated. Four protonation steps are evidenced from fluorescence emission studies of hematoporphyrin IX and its more hydrophobic parent compound lacking of alcoholic chain, deuteroporphyrin IX. They are attributed to the successive protonations of the inner nitrogens of the porphyrin cycle (pK = 4.7 and 2.9 for hematoporphyrin and 4.4 and 2.7 for deuteroporphyrin) and successive deprotonations of propionic groups (pK approximately equal to 5.0 and 5.5 for hematoporphyrin and 5.4 and 6.0 for deuteroporphyrin). These porphyrins, as well as their dimethyl ester forms, are shown to incorporate as monomers into the hydrophobic bilayer of egg phosphatidylcholine small unilamellar vesicles, although the esterified forms are highly aggregated in aqueous solutions. In the case of the non-esterified forms, the incorporation of the porphyrins into the lipidic bilayer is reversible and strongly pH-dependent. A theoretical model is presented which takes into account the various protonation steps and the partition equilibria of the porphyrin between the vesicle lipidic phase and the water medium. The neutral form of the porphyrin (i.e., carboxylic groups protonated) presents the higher affinity, with constants of K approximately equal to 2 X 10(5) and K approximately equal to 6 X 10(6) M-1 (relative to lipid concentration) for hematoporphyrin and deuteroporphyrin, respectively. Protonation of one inner nitrogen leading to the monocationic form is sufficient to prevent incorporation into the hydrophobic bilayer. On the other hand, deprotonation of the peripheral propionic chains leading to anionic forms is less effective. These interactions between vesicles and porphyrins lead to shifts of the apparent pK of nitrogens and carboxylic groups, the latter one being now in the range of physiological pH. These results are discussed with regards to the hypothesis of a possible role of pH in the preferential uptake of porphyrins by tumors.