Two-photon irradiation of an intracellular singlet oxygen photosensitizer: Achieving localized sub-cellular excitation in spatially-resolved experiments

Abstract

The response of a given cell to spatially-resolved sub-cellular irradiation of a singlet oxygen photosensitizer (protoporphyrin IX, PpIX) using a focused laser was assessed. In these experiments, incident light was scattered over a volume greater than that defined by the dimensions of the laser beam as a consequence of the inherent inhomogeneity of the cell. Upon irradiation at a wavelength readily absorbed by PpIX in a one-photon transition, this scattering of light eliminated any advantage accrued to the use of focused irradiation. However, upon irradiation at a longer wavelength where PpIX can only absorb light under non-linear two-photon conditions, meaningful intracellular resolution was achieved in the small spatial domain where the light intensity was high enough for absorption to occur.     

Two-photon irradiation of an intracellular singlet oxygen photosensitizer: achieving localized sub-cellular excitation in spatially-resolved experiments

The response of a given cell to spatially-resolved sub-cellular irradiation of a singlet oxygen photosensitizer (protoporphyrin IX, PpIX) using a focused laser was assessed. In these experiments, incident light was scattered over a volume greater than that defined by the dimensions of the laser beam as a consequence of the inherent inhomogeneity of the cell. Upon irradiation at a wavelength readily absorbed by PpIX in a one-photon transition, this scattering of light eliminated any advantage accrued to the use of focused irradiation. However, upon irradiation at a longer wavelength where PpIX can only absorb light under non-linear two-photon conditions, meaningful intracellular resolution was achieved in the small spatial domain where the light intensity was high enough for absorption to occur.     

Ultraviolet-A irradiation but not ultraviolet-B or infrared-A irradiation leads to a disturbed zinc homeostasis in cells

Changes of the redox balance in cells alter the availability of intracellular free Zn(2+). Here, cells were exposed to ultraviolet (UV)-A, UV-B, or infrared (IR)-A light irradiation, and the intracellular free zinc pool was monitored. Under sublethal conditions only UV-A irradiation resulted in a transient cytoplasmic and nuclear increase of intracellular free Zn(2+). Likewise, tert-butyl hydroperoxide and singlet oxygen, but not H(2)O(2) or intracellular generation of O(2)(*-) by redox cyclers, mimicked the effects of UV-A irradiation, while disulfide stress by diamide only led to a transient cytoplasmic zinc release. These results show that only certain types of subtoxic cellular stress massively disturb the zinc homeostasis in cells.     

Preparation of poly(ethylene glycol)-attached dendrimers encapsulating photosensitizers for application to photodynamic therapy

Photodynamic therapy (PDT) is a noninvasive treatment of some diseases including cancer. We have developed poly(ethylene glycol) (PEG)-attached dendrimers as a drug-carrier candidate. In this study, we prepared nanocapsules of photosensitizers using PEG-attached dendrimers for application to PDT. Two PEG-attached dendrimers derived from poly(amido amine) (PAMAM) and poly(propylene imine) (PPI) dendrimers (PEG-PAMAM and PEG-PPI) were synthesized, and rose bengal (RB) and protoporphyrin IX (PpIX) were used as photosensitizers. Results showed that fewer PpIX molecules were encapsulated by both PEG-attached dendrimers than RB, but the complexes were more stable under physiological conditions. Furthermore, we demonstrated that PEG-PPI held photosensitizers in a more stable manner than PEG-PAMAM because of their inner hydrophobicity. We described the cytotoxicity of the complexes of photosensitizers induced by light irradiation in vitro. The complex of PpIX with PEG-PPI exhibited efficient cytotoxicity, compared with free PpIX. It was suggested that the cytotoxicity was caused by the high level of singlet oxygen production and the efficient delivery to mitochondria. Our results suggest that these PEG-attached dendrimers are a promising vehicle for PDT.     

Photobleaching kinetics, photoproduct formation, and dose estimation during ALA induced PpIX PDT of MLL cells under well oxygenated and hypoxic conditions.

Fluorescence photobleaching and photoproduct formation were investigated during delta-aminolevulinic acid (ALA) induced protoporphyrin IX (PpIX) PDT of MLL cells in vitro. Cells were incubated in either 0.1 or 1.0 mM ALA for 4 h and were treated with 532 nm or 635 nm light under well oxygenated or hypoxic conditions. Fluorescence spectra were acquired during treatment. Photobleaching and photoproduct formation were quantified using singular value decomposition fitting of fluorescence spectra to experimentally determined basis spectra for PpIX, photoprotoporphyrin (Ppp), product II (peak at 655 nm), and product III (peak at 618 nm). PpIX photobleaching occurred under both normal and hypoxic conditions. The photobleaching kinetics could not be explained by purely first- or second-order photobleaching kinetics, and were attributed to differences in PpIX binding at the two ALA incubation concentrations. Ppp was the main photoproduct and accumulated in higher levels in the absence of oxygen, likely a result of reduced Ppp photobleaching under hypoxia. Increases in product II fluorescence occurred mainly in the presence of oxygen. To assess potential fluorescence based PDT dose metrics, cell viability was measured at select times during treatment using a colony formation assay. Cell survival correlated well to changes in product II fluorescence, independent of oxygenation, sensitizer concentration, and treatment wavelength, suggesting that this product is primarily a result of singlet oxygen mediated reactions and may potentially be useful to quantify singlet oxygen dose during PDT.     

Pyridone-containing phenalenone-based photosensitizer working both under light and in the dark for photodynamic therapy

Photosensitizer attracts great attentions and has potential applications in cancer treatment. We developed here a novel pyridone-containing phenalenone-based (PPN-PYR) photosensitizer with excellent singlet oxygen generating ability. Upon light irradiation, PPN-PYR can produce singlet oxygen and transform to its endoperoxide form which in turn release singlet oxygen via thermal cycloreversion at dark. The ability of PPN-PYR to generate reactive oxygen species (ROS) in cell culture and induce corresponding apoptosis both at dark and under light was demonstrated. The efficient PDT performance of PPN-PYR was further verified on cancer cell in vitro. Our study indicate that PPN-PYR can alleviate tumor hypoxia problem and enhance the availability of intermittent photodynamic therapy.     

Nanoscale spatial induction of ultraviolet photoproducts in cellular DNA by three-photon near-infrared absorption

The high-resolution spatial induction of ultraviolet (UV) photoproducts in mammalian cellular DNA is a goal of many scientists who study UV damage and repair. Here we describe how UV photoproducts can be induced in cellular DNA within nanometre dimensions by near-diffraction-limited 750 nm infrared laser radiation. The use of multiphoton excitation to induce highly localized DNA damage in an individual cell nucleus or mitochondrion will provide much greater resolution for studies of DNA repair dynamics and intracellular localization as well as intracellular signalling processes and cell–cell communication. The technique offers an advantage over the masking method for localized irradiation of cells, as the laser radiation can specifically target a single cell and subnuclear structures such as nucleoli, nuclear membranes or any structure that can be labelled and visualized by a fluorescent tag. It also increases the time resolution with which migration of DNA repair proteins to damage sites can be monitored. We define the characteristics of localized DNA damage induction by near-infrared radiation and suggest how it may be used for new biological investigations.     

Measuring the lifetime of singlet oxygen in a single cell: addressing the issue of cell viability.

Singlet molecular oxygen, O(2)(a(1)Delta(g)), has been detected from single neurons and HeLa cells in time-resolved optical experiments by its 1270 nm phosphorescence (a(1)Delta(g)--> X(3)Sigma(-)(g)) upon irradiation of a photosensitizer incorporated into the cell. The cells were maintained in a buffered medium and their viability was assessed by live/dead assays. To facilitate the detection of singlet oxygen, intracellular H(2)O was replaced with D(2)O by an osmotic de- and rehydration process. The effect of this insult on the cells was likewise assessed. The data indicate that, in the complicated transition from a "live" to "dead" cell, the majority of our cells have the metabolic activity and morphology characteristic of a live cell. Quenching experiments demonstrate that the singlet oxygen lifetime in our cells is principally determined by interactions with intracellular water and not by interactions with other cell constituents. The data indicate that in a viable, metabolically-functioning, and H(2)O-containing cell, the lifetime of singlet oxygen is approximately 3 micros. This is consistent with our previous reports, and confirms that the singlet oxygen lifetime in a cell is much longer than hitherto believed. This implies that, in a cell, singlet oxygen is best characterized as a selective rather than reactive intermediate. This is important when considering roles played by singlet oxygen as a signaling agent and as a component in events that result in cell death. The data reported herein also demonstrate that spatially-resolved optical probes can be used to monitor selected events in the light-induced, singlet-oxygen-mediated death of a single cell.     

Photocytotoxic activity of a nitrosyl phthalocyanine ruthenium complex--a system capable of producing nitric oxide and singlet oxygen

The synthesis, structural aspects, pharmacological assays, and in vitro photoinduced cytotoxic properties of [Ru(NO)(ONO)(pc)] (pc = phthalocyanine) are described. Its biological effect on the B16F10 cell line was studied in the presence and absence of visible light irradiation. At comparable irradiation levels, [Ru(NO)(ONO)(pc)] was more effective than [Ru(pc)] at inhibiting cell growth, suggesting that occurrence of nitric oxide release following singlet oxygen production upon light irradiation may be an important mechanism by which the nitrosyl ruthenium complex exhibits enhanced biological activity in cells. Following visible light activation, the [Ru(NO)(ONO)(pc)] complex displayed increased potency in B16F10 cells upon modifications to the photoinduced dose; indeed, enhanced potency was detected when the nitrosyl ruthenium complex was encapsulated in a drug delivery system. The liposome containing the [Ru(NO)(ONO)(pc)] complex was over 25% more active than the corresponding ruthenium complex in phosphate buffer solution. The activity of the complex was directly proportional to the ruthenium amount present inside the cell, as determined by inductively coupled plasma mass spectroscopy. Flow cytometry analysis revealed that the photocytotoxic activity was mainly due to apoptosis. Furthermore, the vasorelaxation induced by [Ru(NO)(ONO)(pc)], proposed as NO carrier, was studied in rat isolated aorta. The observed vasodilation was concentration-dependent. Taken together, the present findings demonstrate that the [Ru(NO)(ONO)(pc)] complex induces vascular relaxation and could be a potent anti-tumor agent. Nitric oxide release following singlet oxygen production upon visible light irradiation on a nitrosyl ruthenium complex produces two radicals and may elicit phototoxic responses that may find useful applications in photodynamic therapy.     

PDT with genetically encoded photosensitizer miniSOG on a tumor spheroid model: A comparative study of continuous-wave and pulsed irradiation

BackgroundTherapeutic effects of PDT depend on many factors, including the amount of singlet oxygen, localization of photosensitizer and irradiation protocol. The present study was aimed to compare the cytotoxic mechanisms of PDT under continuous-wave (CW) and pulsed irradiation using a tumor spheroid model and a genetically encoded photosensitizer miniSOG.Methods¹O₂ detection in miniSOG and flavin mononucleotide (FMN) solutions was performed. Photobleaching of miniSOG in solution and in HeLa tumor spheroids was analyzed. Tumor spheroid morphology and growth and the cell death mechanisms after PDT in CW and pulsed modes were assessed.ResultsWe found a more rapid ¹O₂ generation and a higher photobleaching rate in miniSOG solution upon irradiation in pulsed mode compared to CW mode. Photobleaching of miniSOG in tumor spheroids was also higher after irradiation in the pulsed mode. PDT of spheroids in CW mode resulted in a moderate expansion of the necrotic core of tumor spheroids and a slight inhibition of spheroid growth. The pulsed mode was more effective in induction of cell death, including apoptosis, and suppression of spheroid growth.ConclusionsComparison of CW and pulsed irradiation modes in PDT with miniSOG showed more pronounced cytotoxic effects of the pulsed mode. Our results suggest that the pulsed irradiation regimen enables enhanced ¹O₂ production by photosensitizer and stimulates apoptosis.General significanceOur results provide more insights into the cellular mechanisms of anti-cancer PDT and open the way to improvement of light irradiation protocols.     

Photodamage of lipid bilayers by irradiation of a fluorescently labeled cell-penetrating peptide

Background

Fluorescently labeled cell-penetrating peptides can translocate into cells by endocytosis and upon light irradiation, lyse the endocytic vesicles. This photo-inducible endosomolytic activity of Fl–CPPs can be used to efficiently deliver macromolecules such as proteins and nucleic acids and other small organic molecules into the cytosol of live cells. The requirement of a light trigger to induce photolysis provides a more spatial and temporal control to the intracellular delivery process.

Methods

In this report, we examine the molecular level mechanisms by which cell-penetrating peptides such as TAT when labeled with small organic fluorophore molecules acquire a photo-induced lytic activity using a simplified model of lipid vesicles.

Results

The peptide TAT labeled with 5(6)-carboxytetramethylrhodamine binds to negatively charged phospholipids, thereby bringing the fluorophore in close proximity to the membrane of liposomes. Upon light irradiation, the excited fluorophore produces reactive oxygen species at the lipid bilayer and oxidation of the membrane is achieved. In addition, the fluorescent peptide causes aggregation of photo-oxidized lipids, an activity that requires the presence of arginine residues in the peptide sequence.

Conclusions

These results suggest that the cell-penetrating peptide plays a dual role. On one hand, TAT targets a conjugated fluorophore to membranes. On the other hand, TAT participates directly in the destabilization of photosensitized membranes. Peptide and fluorophore therefore appear to act in synergy to destroy membranes efficiently.

General significance

Understanding the mechanism behind Fl–CPP mediated membrane photodamage will help to design optimally photo-endosomolytic compounds.     

Fast and Effective Photodynamic Inactivation of Multiresistant Bacteria by Cationic Riboflavin Derivatives

Photodynamic inactivation of bacteria (PIB) proves to be an additional method to kill pathogenic bacteria. PIB requires photosensitizer molecules that effectively generate reactive oxygen species like singlet oxygen when exposed to visible light. To allow a broad application in medicine, photosensitizers should be safe when applied in humans. Substances like vitamin B2, which are most likely safe, are known to produce singlet oxygen upon irradiation. In the present study, we added positive charges to flavin derivatives to enable attachment of these molecules to the negatively charged surface of bacteria. Two of the synthesized flavin derivatives showed a high quantum yield of singlet oxygen of approximately 75%. Multidrug resistant bacteria like MRSA (Methicillin resistant Staphylococcus aureus), EHEC (enterohemorrhagic Escherichia coli), Pseudomonas aeruginosa, and Acinetobacter baumannii were incubated with these flavin derivatives in vitro and were subsequently irradiated with visible light for seconds only. Singlet oxygen production in bacteria was proved by detecting its luminescence at 1270 nm. After irradiation, the number of viable bacteria decreased up to 6 log₁₀ steps depending on the concentration of the flavin derivatives and the light dosimetry. The bactericidal effect of PIB was independent of the bacterial type and the corresponding antibiotic resistance pattern. In contrast, the photosensitizer concentration and light parameters used for bacteria killing did not affect cell viability of human keratinocytes (therapeutic window). Multiresistant bacteria can be safely and effectively killed by a combination of modified vitamin B2 molecules, oxygen and visible light, whereas normal skin cells survive. Further work will include these new photosensitizers for topical application to decolonize bacteria from skin and mucosa.     

Porphyrin-induced photooxidation of conjugated bilirubin

Visible light irradiation of 18 microM bilirubin ditaurate (BR-DT) at pH 7.0 for 30 min showed a 10% decrease in absorbance at 445 nm. When the reaction was carried out in the presence of a trace amount of uroporphyrin (UP), the spectrum of BR-DT disappeared without a concomitant formation of biliverdin. Photooxidation products were confirmed to be dipyrrole-containing compounds. Photo-bleaching of BR-DT was accelerated by the increasing concentration of UP and was inhibited, when UP was replaced by Cu2+UP. Formation of 2,2,6,6-tetramethylpiperidine N-oxyl through the irradiation of UP was diminished by sodium azide, a potent scavenger of singlet oxygen. The efficiency of singlet oxygen formation through visible light irradiation was in the order UP, coproporphyrin > Cu2+UP. Both bilirubin and BR-DT bound to human serum albumin (HSA) were photooxidized effectively in the presence of UP. The results indicate that irradiation of UP produces singlet oxygen with high efficiency which then rapidly oxidizes free and conjugated bilirubin.     

Cycloimide bacteriochlorin p derivatives: photodynamic properties and cellular and tissue distribution

Reactive oxygen species generated by photosensitizers are efficacious remedy for tumor eradication. Eleven cycloimide derivatives of bacteriochlorin p (CIBCs) with different N-substituents at the fused imide ring and various substituents replacing the 3-acetyl group were evaluated as photosensitizers with special emphasis on structure-activity relationships. The studied CIBCs absorb light within a tissue transparency window (780-830 nm) and possess high photostability at prolonged light irradiation. The most active derivatives are 300-fold more phototoxic toward HeLa and A549 cells than the clinically used photosensitizer Photogem due to the substituents that improve intracellular accumulation (distribution ratio of 8-13) and provide efficient photoinduced singlet oxygen generation (quantum yields of 0.54-0.57). The substituents predefine selective CIBC targeting to lipid droplets, Golgi apparatus, and lysosomes or provide mixed lipid droplets and Golgi apparatus localization in cancer cells. Lipid droplets and Golgi apparatus are critically sensitive to photoinduced damage. The average lethal dose of CIBC-generated singlet oxygen per volume unit of cell was estimated to be 0.22 mM. Confocal fluorescence analysis of tissue sections of tumor-bearing mice revealed the features of tissue distribution of selected CIBCs and, in particular, their ability to accumulate in tumor nodules and surrounding connective tissues. Considering the short-range action of singlet oxygen, these properties of CIBCs are prerequisite to efficient antitumor photodynamic therapy.     

Synthesis and biological evaluation of glucose conjugated phthalocyanine as a second-generation photosensitizer

Photodynamic therapy (PDT) is a treatment method using light and photosensitizers (PSs), which is categorized as a non-invasive surgery treatment for cancers. When the tumor is exposed to a specific light, the PSs become active and generate reactive oxygen species (ROS), mainly singlet oxygen which kills nearby cancer cells. PDT is becoming more widely recognized as a valuable treatment option for localized cancers and pre-cancers of skin as it has no long-term effects on the patient. But, due to the limited penetration rate of light into the skin and other organs, PDT can’t be used to treat large cancer cells or cancer cells that have grown deeply into the skin or other organs. Hence, in this study, our focus centers on synthesizing glucose-conjugated phthalocyanine (Pc) compatible with near-infrared (NIR) irradiation as second-generation photosensitizer, so that PDT can be used in a wider range to treat cancers without obstacles.     

The two-photon laser-induced fluorescence of the tumor-localizing photosensitizer hematoporphyrin derivative. Resonance-enhanced 750 nm two-photon excitation into the near-UV Soret band

The tumor-localizing photosensitizer hematoporphyrin derivative (HPD) is shown to undergo a simultaneous two-photon excitation into the near-ultraviolet Soret band system upon intense laser irradiation at 750 nm, a spectral region where there is no significant HPD one-photon absorbance in aqueous solution. Subsequent to this excitation, internal conversion and vibrational relaxation occur, resulting in the population of the vibrationless level of the first electronically excited singlet state. This state relaxes by two channels, the emission of fluorescence in the spectral region 600-700 nm and intersystem crossing into the triplet manifold, followed by near-resonant electronic energy transfer with surrounding oxygen to result in the generation of highly reactive singlet molecular oxygen (1 delta g). Evidence for the two-photon excitation consists in the observation both of the HPD fluorescence spectrum in the region of 615 nm as a result of 750 nm excitation and the quadratic dependence of this fluorescence emission intensity upon the excitation laser intensity. Since, in general, the penetration depth of ultraviolet and visible light into tissue varies directly with wavelength (red penetrating more deeply than blue), these studies suggest the possibility that two-photon-induced localization of tumor-bound HPD might facilitate the detection of deeper lying tumors than allowed by the current one-photon photolocalization method.     

Targeted Green-Red Photoconversion of EosFP, a Fluorescent Marker Protein

EosFP is a novel fluorescent protein from the stony coral Lobophyllia hemprichii. Its gene was cloned in Escherichia coli to express the tetrameric wild-type protein. The protein emits strong green fluorescence (516 nm) that shifts toward red (581 nm) upon near-ultraviolet irradiation at ∼390 nm due to a photo-induced modification that involves a break in the peptide backbone next to the chromophore. Using site-directed mutagenesis, dimeric (d1EosFP, d2EosFP) and monomeric (mEosFP) variants were produced with essentially unaltered spectroscopic properties. Here we present a spectroscopic characterization of EosFP and its variants, including room- and low-temperature spectra, fluorescence lifetime determinations, two-photon excitation and two-photon photoconversion. Furthermore, by transfection of a human cancer (HeLa) cell with a fusion construct of a mitochondrial targeting sequence and d2EosFP, we demonstrate how localized photoconversion of EosFP can be employed for resolving intracellular processes.     

Carbon nanodots with a controlled N structure by a solvothermal method for generation of reactive oxygen species under visible light

Carbon nanodots can function as photosensitizers that have the ability to generate reactive oxygen species such as singlet oxygen, hydroxy (OH) radicals, and superoxide ions. However, most of these can only be generated upon ultraviolet light excitation. Additionally, the mechanism of reactive oxygen species generation by carbon nanodots remains unclear. The development of carbon nanodots that can photosensitize under visible light irradiation is desirable for applications such as photodynamic therapy and pollutant decomposition under visible light. Here, we report novel carbon nanodot-based photosensitizers that generate reactive oxygen species under visible light; they were synthesized using a solvothermal method with two solvents (formamide and water) and amidol as the carbon source. Carbon nanodots from the solvothermal synthesis in formamide showed blue fluorescence, while those obtained in water showed green fluorescence. The photo-excited blue-fluorescent carbon nanodots produced OH radicals, superoxide ions, and singlet oxygen, and therefore could function as both type I and type II photosensitizers. In addition, photo-excited green-fluorescent carbon nanodots generated only singlet oxygen, therefore functioning as type II photosensitizers. It is proposed that the two photosensitizers have different origins of reactive oxygen species generation: the enrichment of graphitic N for blue-fluorescent carbon nanodots and molecular fluorophores for green-fluorescent carbon nanodots.     

Apoptosis of vascular smooth muscle cells induced by photodynamic therapy with protoporphyrin IX

Photodynamic therapy (PDT) had been shown effective in the treatment of intimal hyperplasia, which contributes to restenosis, by eradicating cells in the vessel wall. This study is designed to evaluate the effects of PDT with protoporphyrin IX (PpIX) on the viability of vascular smooth muscle cells (SMCs) and to define the cell-death pathway. Fluorescence microscopy and laser-induced fluorescence spectroscopic detection showed that SMCs selectively uptake PpIX, and the intracellular PpIX concentration increases with the amount of PpIX in the incubation solution. PDT with PpIX impaired cellular viability from 93 ± 3.4% to 36 ± 3.9% when the light intensity increases from 2 to 9 J/cm² and intracellular PpIX concentration increases from 0.5 to 20 μg/ml. Although PDT induced both apoptosis and necrosis, the ratio of apoptotic cells increased with light dosage or intracellular PpIX concentration. The loss of mitochondrial membrane potential coincided with the apoptotic ratio. Our results indicated that the induction of apoptosis of SMCs may be one of the mechanisms by which PDT inhibits restenosis in vivo.     

Antioxidant enzyme inhibitors enhance singlet oxygen-induced cell death in HL-60 cells

Singlet oxygen is a highly reactive form of molecular oxygen that may harm living systems by oxidizing critical cellular macromolecules and it also promotes deleterious processes such as cell death. The protective role of antioxidant enzymes against singlet oxygen-induced oxidative damage in HL-60 cells was investigated in control and cells pre-treated with diethyldithiocarbamic acid, aminotriazole and oxlalomalate, specific inhibitors of superoxide dismutase, catalase and NADP⁺-dependent isocitrate dehydrogenase, respectively. Upon exposure to rose bengal (20 μM)/light (15 min), which generates singlet oxygen, to HL-60 cells, the viability was lower and the lipid peroxidation and oxidative DNA damage were higher in inhibitor-treated cells as compared to control cells. We also observed the significant increase in the endogenous production of reactive oxygen species as well as the significant decrease in the intracellular GSH level in inhibitor-treated HL-60 cells exposed to singlet oxygen. Upon exposure to rose bengal (3 μM)/light (15 min), which induced apoptotic cell death, a clear inverse relationship was observed between the control and inhibitor-treated HL-60 cells in their susceptibility to apoptosis. These results suggest that antioxidant enzymes play an important role in cellular defense against singlet oxygen-induced cell death including necrosis and apoptosis.