Time-resolved in-vivo fluorescence of photosensitizing porphyrins

doi:10.1016/1011-1344(93)80176-A

Journal of Photochemistry and Photobiology B: Biology

Volume 21, Issues 2–3, December 1993, Pages 143-147

https://doi.org/10.1016/1011-1344(93)80176-A Get rights and content

Abstract

Various components of photosensitizing porphyrins (e.g. monomers, aggregates, ionic species) have been recently localized in single cells by time-resolved fluorescence microscopy. Novel time-resolving techniques, based on picosecond laser diodes, a frequency-doubled Nd:YAG laser and time-gated microscopic equipment, were used for in-vivo measurements of the chick chorioallantoic membrane (CAM) exhibiting a pronounced vasculature. Changes of the fluorescence decay kinetics after light exposure were correlated with the formation of a photoproduct (Photosan, aminolaevulinic acid) or changes of the intracellular binding sites (tetraphenyl-porphyrins). Fluorescent components with different decay times were shown to be distributed differently within the tissue.

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Spectroscopic characterization and fluorescence imaging of Helicobacter pylori endogenous porphyrins
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Citation Excerpt :
Most of the reported studies in cell concern ALA-induced porphyrin production [21]. It is shown that ALA-induction leads to the production of large amounts of monomeric porphyrin showing long-lived lifetime [22]. In our case, no induction is performed, and the detected fluorescence is only due to spontaneously produced porphyrins under physiological conditions.
Conventional antimicrobial strategies have become increasingly ineffective due to the rapid emergence of antibiotic resistance among pathogenic bacteria. In order to overcome this problem, antimicrobial PhotoDynamic Therapy (PDT) is considered a promising alternative therapy. PDT has a broad spectrum of action and low mutagenic potential. It is particularly effective when microorganisms present endogenous photosensitizing pigments. Helicobacter pylori (Hp), a pathogen notoriously responsible of severe gastric infections (chronic gastritis, peptic ulcer, MALT lymphoma and gastric adenocarcinoma), produces and accumulates the photosensitizers protoporphyrin IX and coproporphyrin, thus it might be a suitable target of antimicrobial PDT. With the aim to design and develop an ingestible LED-based robotic pill for intragastric phototherapy, so that irradiation can be performed in situ without the use of invasive endoscopic light, photophysical studies on the Hp endogenous photosensitizers were carried out. These studies represent an important prerequisite in order to select the most effective irradiation conditions for Hp eradication. The photophysical characterization of Hp porphyrins, including their spectroscopic features in terms of absorption, steady-state and time-resolved fluorescence, was performed on bacterial extracts as well as within planktonic and biofilm growing Hp cells.
Critical Role of ABCG2 in ALA-Photodynamic Diagnosis and Therapy of Human Brain Tumor
2015, Advances in Cancer Research
Citation Excerpt :
ALA-induced PpIX biosynthesis has many other advantages. PpIX is an essentially monomeric compound with a high fluorescence yield (Schneckenburger, König, Kunzi-Rapp, Westphal-Frösch, & Rück, 1993) and photosensitizing capability due to its good singlet oxygen quantum efficiency (Pottier & Truscott, 1986; Weishaupt, Gomer, & Dougherty, 1976) and is rapidly metabolized in vivo (Loh et al., 1993). Animal and human studies have shown that ALA induces PpIX clearance from the skin within 24 h after systemic, topical, or intradermal administration (Kennedy & Pottier, 1992), whereas HpDs cause prolonged skin photosensitivity (1–2 months).
Primary brain tumors occur in around 250,000 people per year globally. Survival rates in primary brain tumors depend on the type of tumor, patient's age, the extent of surgical tumor removal, and other factors. Photodynamic diagnosis (PDD) is a practical tool currently used in surgical operation of aggressive brain tumors, such as glioblastoma and meningiomas, whereas clinical application of photodynamic therapy (PDT) to brain tumor therapy has just recently started. Both PDD and PDT are achieved by a photon-induced physicochemical reaction, which is induced by the excitation of porphyrins exposed to light. In fluorescence-guided gross-total resection, PDD can be achieved by the administration of 5-aminolevulinic acid (5-ALA) as the precursor of protoporphyrin IX (PpIX). Exogenously administered ALA induces biosynthesis and accumulation of PpIX, a natural photosensitizer, in cancer cells. However, ATP-binding cassette transporter ABCG2 plays a critical role in regulating the cellular accumulation of porphyrins in cancer cells and thereby its expression and function can affect the efficacy of PDD and PDT. In response to the photoreaction of porphyrins leading to oxidative stress, the nuclear factor erythroid-derived 2-related transcription factor can transcriptionally upregulate ABCG2, which may reduce the efficacy of PDD and PDT. On the other hand, certain protein kinase inhibitors potentially enhance the efficacy of PDD and PDT by blocking ABCG2-mediated porphyrin efflux from cancer cells. In this context, it is of great interest to develop ABCG2 inhibitors that can be applied to PDD or PDT for the therapy of brain tumor and other tumors.
Non-invasive imaging of skin physiology and percutaneous penetration using fluorescence spectral and lifetime imaging with multiphoton and confocal microscopy
2011, European Journal of Pharmaceutics and Biopharmaceutics
New multiphoton and confocal microscope technologies and fluorescence lifetime imaging techniques are now being used to non-invasively image, in space (three dimensions),in time, in spectra, in lifetime and in fluorescence anisotropy (total of 7 dimensions), fluorescent molecules in in situ and in vivo biological tissue, including skin. The process involves scanning a 2D area and measuring fluorescence at a given tissue depth below the surface after excitation by a laser beam with a wavelength within the one-photon or two-photon absorption band of the fluorophores followed by the stacking together of a series of 2D images from different depths to reconstruct the full spatial structure of the sample. Our aim in this work is to describe the principles, opportunities, limitations and applications of this new technology and its application in defining skin morphology, disease and skin penetration in vitro and in vivo by drugs, chemicals and nanoparticles. A key emphasis is in the use of fluorescence lifetime imaging to add additional specificity and quantitation to the detection of the various exogenous chemicals and nanoparticles that may be applied to the skin as well as endogenous fluorescent species in the skin. Examples given include equipment configuration; components in skin autofluorescence in various skin strata; imaging and quantification of coexisting drugs and their metabolites; skin pH; nanoparticle zinc oxide skin penetration; liposome delivery of drugs to deeper tissues; and observations in skin ageing and in various skin diseases.
Self-aggregation of free base porphyrins in aqueous solution and in DMPC vesicles
2008, Biophysical Chemistry
Free base porphyrin (PPhe), derivatized with aminosulfonyl groups linked to the aromatic amino acid phenylalanine at the meso-positions, was mixed with DMPC vesicles. The resulting interaction was studied by absorption, steady-state and transient state fluorescence, at different pHs.
At pH = 2 to pH = 9, the aforementioned porphyrin predominates as an aggregated species, with a co-facial arrangement of the molecules taking into account the blue shift of the Soret band (414 nm for the monomer and 401 nm for the aggregate). Upon interaction with DMPC vesicles, the competing hydrophobic interactions with the bilayer destabilize the aggregated species in favor of monomer incorporation. Fluorescence lifetimes also show that the long component assigned to the monomer contributes only 30% to the overall decay in solution (e.g. pH = 7.0) whereas in DMPC vesicles this contribution increases up to 85% independent of the solution pH, which confirms a location of the probe in an environment “protected” from free water.
The picture changes completely in the case of TSPP, an anionic porphyrin which does not incorporate in DMPC vesicles. Remarkably, at pH = 2.5 all the experimental findings point to the self-assembling of the porphyrin units in J-aggregates induced at the surface of the DMPC vesicle. In fact, upon removal of the aqueous solvent, we could define by fluorescence lifetime imaging microscopy (FLIM) regions where the fluorescence lifetime is that characteristic of the J-aggregate (<τ_F> 0.11 ns).
Fluorescence lifetime imaging in PDT. An overview
2005, Medical Laser Application
Various problems arising during molecular imaging of different fluoroprobes and metabolites used in photodynamic therapy (PDT) could be circumvented by focusing on time-resolved detection. For this, an interesting new method seems to be time-correlated single photon counting, where a time-to-amplitude converter determines the temporal position and a scanning interface connected to the scanning unit of a laser microscope determines the spatial location of a signal. In combination with spectral resolved detection (spectral lifetime imaging) the set-up achieves the features of highly sophisticated lifetime imaging systems.
The potential of time-resolved detection is evident for the precursor 5-ALA, where the photosensitizing porphyrins are synthesized in the tumor cells. Protoporphyrin IX, which is produced in mitochondria, induces high phototoxicity but also different metabolites could be involved. During cell differentiation various enzymes, which are active in varying degrees control the ALA metabolism. It is therefore of main interest to localize 5-ALA metabolites during cell differentiation and cell growth. Subcellular differentiation of those metabolites without extensive extraction procedures is not trivial, because of highly overlapping spectral properties. Measuring the fluorescence lifetime on a subcellular level could be a successful alternative (fluorescence lifetime imaging, FLIM). As it will be discussed FLIM and SLIM in principal are powerful techniques to improve selectivity of fluorescence guided diagnosis and in fact PDT. Usage in clinics will depend at least on the availability of easy and fast detection systems, which can be realized in different ways. For excitation, relatively cheap laser diodes or simple custom-made Ti:Saphir laser systems are now on the market.
Im Rahmen der photodynamischen Therapie ist die laserinduzierte Fluoreszenzdiagnostik ein wichtiges Hilfsmittel, um Tumorbegrenzungen oder Tumorreste sichtbar zu machen und so eine vollständige Resektion zu gewährleisten. Das Problem jedoch ist neben der Sensitivität eine häufig zu geringe Selektivität, die besonders im Bereich von Hirntumoren zu unbefriedigenden Ergebnissen führt. Um die Selektivität zu verbessern, bieten sich zeitaufgelöste Methoden an, mit deren Hilfe es möglich wird, verschiedene Fluorophore und Metaboliten eindeutig zu unterscheiden. Neu und interessant ist in diesem Sinne die zeitkorrelierte Einzelphotonenzählung, auch time-correlated single photon counting (TCSPC) genannt, die in Kombination mit einer Scanningeinheit und kurzgepulster Anregung die bildgebende Darstellung der Fluoreszenzabklingzeit (sogenanntes fluorescence lifetime imaging (FLIM)) ermöglicht.
An Hand der für die Klinik zugelassenen 5-Aminolevulinsäure (5-ALA) lässt sich das Potential zeitaufgelöster Fluoreszenzmessungen besonders gut demonstrieren. Nach der Applikation von 5-ALA entstehen in den Zellen unterschiedliche Porphyrine, die alle mehr oder weniger stark phototoxisch sind. Protoporphyrin IX, das in den Mitochondrien synthetisiert wird, ist dabei das wirksamste Porphyrin. Es können jedoch auch andere Metaboliten, wie Uroporphyrin und Koproporphyrin entstehen. Die Synthese dieser Porphyrine wird durch Enzyme gesteuert, die in Abhängigkeit des Diffenzierungsgrades der Zelle mehr oder weniger aktiv sind. Wie im Rahmen dieser Arbeit diskutiert wird, können durch FLIM die verschiedenen Metaboliten mit subzellulärer Auflösung unterschieden werden. Zeitaufgelöste Methoden können damit allgemein die Selektivität der Fluoreszenzdiagnostik erhöhen und damit die PDT signifikant verbessern.
Fluorescence spectroscopy of normal mouse skin exposed to 5-aminolaevulinic acid and red light
2001, Journal of Photochemistry and Photobiology B: Biology
Photobleaching and phototransformation of protoporphyrin IX (PpIX) was investigated in normal mouse skin. The PpIX was induced by topical application of 5-aminolaevulinic acid (ALA). Exposure to laser light (635 nm) caused photobleaching of PpIX fluorescence and formation of fluorescent products. Analysis of the fluorescence spectra revealed appearance of new fluorescent photoproducts during light exposure. The main photoproduct, supposedly chlorin-type photoprotoporphyrin (PPp), exhibited fluorescence with an emission maximum at 675 nm. The other products exhibited main fluorescence peaks at around 588 and 623 nm that can presumably be attributed to an endogenous metallo-porphyrin and water-soluble porphyrin(s), respectively. Our results indicate that light exposure causes alterations in the enzymatic pathway of PpIX synthesis from ALA and leads to accumulation of intermediate water-soluble porphyrins. ALA-induced porphyrins are transported away from the treated area and partly deposited in remote skin sites.

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^☆: Paper presented at the First International Lithuanian-Italian Workshop on Photosensitized Tumour Therapy, Vilnius, May 25–31, 1992.

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Journal of Photochemistry and Photobiology B: Biology

Abstract

References (16)

Time-resolved fluorescence in Photobiology

J. Photochem. Photobiol. B: Biol.

Time-resolved fluorescence spectroscopy of hematoporphyrin derivative in micelles

Chem. Phys. Lett.

Intracellular localization of meso-tetraphenylporphine tetrasulfonate probed by time-resolved and microscopic fluorescence spectroscopy

J. Photochem. Photobiol. B: Biol.

The chick chorioallantoic membrane (CAM) as an in vivo model for photodynamic therapy

J. Photochem. Photobiol. B: Biol.

Photodynamic therapy with endogeneous protoporphyrin IX: basic principles and present clinical experience

J. Photochem. Photobiol. B: Biol.

Fluorescence formation during photodynamic therapy in the nucleus of cells incubated with cationic and anionic water-soluble photosensitizers

J. Photochem. Photobiol. B: Biol.

Use of time-gated fluorescence imaging for diagnosis in biomedicine

J. Photochem. Photobiol. B: Biol.

Picosecond fluorescence spectroscopy on incorporation processes of hematoporphyrin derivative into malignant tumour cells in vitro

Photochem. Photobiol.

Cited by (58)

Spectroscopic characterization and fluorescence imaging of Helicobacter pylori endogenous porphyrins

Critical Role of ABCG2 in ALA-Photodynamic Diagnosis and Therapy of Human Brain Tumor

Non-invasive imaging of skin physiology and percutaneous penetration using fluorescence spectral and lifetime imaging with multiphoton and confocal microscopy

Self-aggregation of free base porphyrins in aqueous solution and in DMPC vesicles

Fluorescence lifetime imaging in PDT. An overview

Fluorescence spectroscopy of normal mouse skin exposed to 5-aminolaevulinic acid and red light

Time-resolved in-vivo fluorescence of photosensitizing porphyrins☆

Abstract

J. Photochem. Photobiol. B: Biol.

Chem. Phys. Lett.

J. Photochem. Photobiol. B: Biol.

J. Photochem. Photobiol. B: Biol.

J. Photochem. Photobiol. B: Biol.

J. Photochem. Photobiol. B: Biol.

J. Photochem. Photobiol. B: Biol.

Picosecond fluorescence spectroscopy on incorporation processes of hematoporphyrin derivative into malignant tumour cells in vitro

Photochem. Photobiol.