How to Target Small-Molecule Fluorescent Imaging Probes to the Plasma Membrane-The Influence and QSAR Modelling of Amphiphilicity, Lipophilicity, and Flip-Flop.

Many new fluorescent probes targeting the plasma membrane (PM) of living cells are currently being described. Such probes are carefully designed to report on relevant membrane features, but oddly, the structural features required for effective and selective targeting of PM often receive less attention, constituting a lacuna in the molecular design process. We aim to rectify this by clarifying how the amphiphilicity and lipophilicity of a probe, together with the tendency to flip-flop across the membrane, contribute to selective PM accumulation. A simplistic decision-rule QSAR model has been devised that predicts the accumulation/non-accumulation of small-molecule fluorescent probes in the PM. The model was based on probe log P plus various derived measures, allowing the roles of amphiphilicity, lipophilicity, and flip-flop to be taken into account. The validity and wide applicability of the model were demonstrated by evaluating its ability to predict amphiphilicity or PM accumulation patterns in surfactants, drugs, saponins, and PM probes. It is hoped that the model will aid in the more efficient design of effective PM probes.

NIR laser pointer for in vivo photothermal therapy of murine LM3 tumor using intratumoral China ink as a photothermal agent.

The photothermal effect is one of the most promising photonic procedures currently under development to successfully treat several clinical disorders, none the least some kinds of cancer. At present, this field is undergoing a renewed interest due to advances in both photothermal materials and better-suited light sources. However, scientific studies in this area are sometimes hampered by the relative unavailability of state-of-art materials or the complexity of setting up a dedicated optical facility. Here, we present a simple and affordable approach to do research in the photothermal field that relies on a commercial NIR laser pointer and a readily available everyday pigment: China ink. A proof-of-concept study is presented in which mice bearing intradermal LM3 mammary adenocarcinoma tumors were successfully treated in vivo employing China ink and the laser pointer. TUNEL and Ki-67 post-treatment tissue assessment clearly indicates the deleterious action of the photothermal treatment on the tumor. Therefore, the feasibility of this simple approach has been demonstrated, which may inspire other groups to implement simple procedures to further explore the photothermal effect.

Establishing the subcellular localization of photodynamically-induced ROS using 3,3'-diaminobenzidine: A methodological proposal, with a proof-of-concept demonstration.

The critical involvement of reactive oxygen species (ROS) in both physiological and pathological processes in cell biology makes their detection and assessment a fundamental topic in biomedical research. Established methodologies to study ROS in cell biology take advantage of oxidation reactions between the ROS and a reduced probe. After reacting the probe reveals the presence of ROS either by the appearance of colour (chromogenic reaction) or fluorescence (fluorogenic reaction). However current methodologies rarely allow for a site-specific detection of ROS production. Here we propose a colorimetric reaction driven by the oxidation of 3,3'-diaminobenzidine (DAB) by photodynamically-produced ROS that allows for fine detection of the ROS production site. The introduced methodology is fast, easy to implement and permits cellular resolution at the submicrometric level. Although the basic protocol is proved in a photodynamic model of ROS generation, the principle is applicable to many different scenarios of intracellular ROS production. As a consequence this proposed methodology should greatly complement other techniques aiming at establishing a precise subcellular localization of ROS generation.

Vehiculization determines the endocytic internalization mechanism of Zn(II)-phthalocyanine.

It is generally accepted that compounds of nanomolecular size penetrate into cells by different endocytic processes. The vehiculization strategy of a compound is a factor that could determine its uptake mechanism. Understanding the influence of the vehicle in the precise mechanism of drug penetration into cells makes possible to improve or modify the therapeutic effects. In this study, using human A-549 cells, we have characterized the possible internalization mechanism of the photosensitizer Zn(II)-phthalocyanine (ZnPc), either dissolved in dimethylformamide (ZnPc-DMF) or included in liposomes of dipalmitoyl-phosphatidyl-choline. Specific inhibitors involved in the main endocytic pathways were used. Co-incubation of cells with ZnPc-liposomes and dynasore (dinamin-mediated endocytosis inhibitor) resulted in a significant decrease of photodamage, whereas other inhibitors did not alter the photodynamic effect of ZnPc. On the contrary, cells treated with ZnPc-DMF in the presence of dynasore, genistein (caveolin-mediated endocytosis inhibitor) or cytochalasin D (macropinocytosis and caveolin-mediated endocytosis inhibitor) showed a significant decrease in ZnPc uptake and photodynamic damage. These results suggest that ZnPc-DMF penetrates into cells mainly by caveolin-mediated endocytosis, whereas ZnPc-liposomes are internalized into cells preferentially by clathrin-mediated endocytosis. We conclude that using different drug vehiculization systems, it is possible to modify the internalization mechanism of a therapeutic compound, which could be of great interest in clinical research.

Uptake and localisation of small-molecule fluorescent probes in living cells: a critical appraisal of QSAR models and a case study concerning probes for DNA and RNA.

Small-molecule fluorochromes are used in biology and medicine to generate informative microscopic and macroscopic images, permitting identification of cell structures, measurement of physiological/physicochemical properties, assessment of biological functions and assay of chemical components. Modes of uptake and precise intracellular localisation of a probe are typically significant factors in its successful application. These processes and localisations can be predicted using quantitative structure activity relations (QSAR) models, which correlate aspects of the physicochemical properties of the probes (expressed numerically) with the uptake/localisation. Pay-offs of such modelling include better understanding and trouble-shooting of current and novel probes, and easier design of future probes ("guided synthesis"). Uptake models discussed consider adsorptive (to lipid or protein domains), phagocytic and pinocytotic endocytosis, as well as passive diffusion. Localisation models discussed include those for cytosol, endoplasmic reticulum, Golgi apparatus, lipid droplets, lysosomes, mitochondria, nucleus and plasma membrane. A case example illustrates how such QSAR modelling of probe interactions can clarify localisation and mode of binding of probes to intracellular nucleic acids of living cells, including not only eukaryotic chromatin DNA and ribosomal RNA, but also prokaryote chromosomes.

Do folate-receptor targeted liposomal photosensitizers enhance photodynamic therapy selectivity?

One of the current goals in photodynamic therapy research is to enhance the selective targeting of tumor cells in order to minimize the risk and the extension of unwanted side-effects caused by normal cell damage. Special attention is given to receptor mediated delivery systems, in particular, to those targeted to folate receptor. Incorporation of a model photosensitizer (ZnTPP) into a folate-targeted liposomal formulation has been shown to lead an uptake by HeLa cells (folate receptor positive cells) 2-fold higher than the non-targeted formulation. As a result, the photocytotoxicity induced by folate-targeted liposomes was improved. This selectivity was completely inhibited with an excess of folic acid present in the cell culture media. Moreover, A549 cells (folate receptor deficient cells) have not shown variations in the liposomal incorporation. Nevertheless, the differences observed were slighter than expected. Both folate-targeted and non-targeted liposomes localize in acidic lysosomes, which confirms that the non-specific adsorptive pathway is also involved. These results are consistent with the singlet oxygen kinetics measured in living cells treated with both liposomal formulations.

Tumour cell death induced by the bulk photovoltaic effect of LiNbO3:Fe under visible light irradiation.

This work reports a pioneer application of the bulk photovoltaic effect in the biomedical field. Massive necrotic cell death was induced in human tumour cell cultures grown on a bulk photovoltaic material (iron-doped lithium niobate, LiNbO(3):Fe) after irradiation with visible light. Lethal doses (≈100% cell death) were obtained with low-intensity visible light sources (10-100 mW cm(-2) irradiances) and short exposure times of the order of minutes. The wavelength dependence to induce the lethal effect observed is consistent with that corresponding to the bulk photovoltaic effect generation in LiNbO(3):Fe. Necrosis also occurred when cultured tumour cells were exposed to LiNbO(3):Fe microparticles and visible light.

Lipid Peroxidation Assay Using BODIPY-Phenylbutadiene Probes: A Methodological Overview.

The assessment of reactive oxygen species has increasing importance in biomedical sciences, due to their biological role in signaling pathways and induction of cell damage at low and high concentrations, respectively. Detection of lipid peroxidation with sensing probes such as some BODIPY dyes has now wide application in studies using fluorescent microplate readers, flow cytometry, and fluorescence microscopy. Two phenylbutadiene derivatives of BODIPY are commonly used as peroxidation probes, non-oxidized probes and oxidized products giving red and green fluorescence, respectively. Peculiar features of lipoperoxidation and BODIPY dye properties make this assessment a rather complex process, not exempt of doubts and troubles. Color changes and fluorescence fading that are not due to lipid peroxidation must be taken into account to avoid misleading results. As a characteristic feature of lipoperoxidation is the propagation of peroxyl radicals, pitfalls and advantages of a delayed detection by BODIPY probes should be considered.

Cell uptake of Zn(II)-phthalocyanine-containing liposomes by clathrin-mediated endocytosis.

The study of uptake mechanisms of therapeutic drugs has a growing interest in biomedical research. In this work the cell uptake and phototoxicity of the photosensitizer Zn(II)-phthalocyanine (ZnPc) in dipalmitoyl-phosphatidyl-choline liposomes have been studied in the presence or absence of inhibitors of macropinocytosis (cytochalasin D), and clathrin-mediated endocytosis (dynasore). No differences in the uptake or photodynamic damage were observed in A-549 cells subjected to incubation with either ZnPc alone or in combination with cytochalasin D. On the contrary, co-incubation of A-549 cells with ZnPc and dynasore resulted in a significant decrease of photodamage as well as negligible uptake of the photosensitizer. These results indicate that ZnPc is internalized into cells preferentially by a mechanism of clathrin-mediated endocytosis.

Fluorescent redox-dependent labeling of lipid droplets in cultured cells by reduced phenazine methosulfate.

Natural and synthetic phenazines are widely used in biomedical sciences. In dehydrogenase histochemistry, phenazine methosulfate (PMS) is applied as a redox reagent for coupling reduced coenzymes to the reduction of tetrazolium salts into colored formazans. PMS is also currently used for cytotoxicity and viability assays of cell cultures using sulfonated tetrazoliums. Under UV (340 nm) excitation, aqueous solutions of the cationic PMS show green fluorescence (λem: 526 nm), whereas the reduced hydrophobic derivative (methyl-phenazine, MPH) shows blue fluorescence (λem: 465 nm). Under UV (365 nm) excitation, cultured cells (LM2, IGROV-1, BGC-1, and 3T3-L1 adipocytes) treated with PMS (5 µg/mL, 30 min) showed cytoplasmic granules with bright blue fluorescence, which correspond to lipid droplets labeled by the lipophilic methyl-phenazine. After formaldehyde fixation blue-fluorescing droplets could be stained with oil red O. Interestingly, PMS-treated 3T3-L1 adipocytes observed under UV excitation 24 h after labeling showed large lipid droplets with a weak green emission within a diffuse pale blue-fluorescing cytoplasm, whereas a strong green emission was observed in small lipid droplets. This fluorescence change from blue to green indicates that reoxidation of methyl-phenazine to PMS can occur. Regarding cell uptake and labeling mechanisms, QSAR models predict that the hydrophilic PMS is not significantly membrane-permeant, so most PMS reduction is expected to be extracellular and associated with a plasma membrane NAD(P)H reductase. Once formed, the lipophilic and blue-fluorescing methyl-phenazine enters live cells and mainly accumulates in lipid droplets. Overall, the results reported here indicate that PMS is an excellent fluorescent probe to investigate labeling and redox dynamics of lipid droplets in cultured cells.

Photothermal effect by 808-nm laser irradiation of melanin: a proof-of-concept study of photothermal therapy using B16-F10 melanotic melanoma growing in BALB/c mice.

The photothermal effect is undergoing great interest due to advances in new photosensitizing materials and better-suited light sources, but studies are frequently hampered by the need to employ exogenous photothermal agents and expensive irradiation devices. Here we present a simple strategy based on direct NIR irradiation of the melanin pigment with a commercial 808-nm laser pointer. Proof-of-concept studies showed efficient photothermal effects on melanin in vitro and in vivo. After NIR irradiation, BALB/c mice bearing B16-F10 melanotic melanoma tumors revealed severe histopathological damage and massive necrosis in melanin-containing tumor tissue, while surrounding healthy tissues showed no damage. Therefore, the feasibility of this approach may allow implementing direct procedures for photothermal therapy of pigmented tumors.

Photosensitization ability of a water soluble zinc(II)tetramethyltetrapyridinoporphyrazinium salt in aqueous solution and biomimetic reverse micelles medium.

The spectroscopic properties and the photodynamic activity of a highly water soluble zinc(II)tetramethyltetrapyridino[2,3-b:2',3'-g:2",3"-l:2"',3'''-q]porphyrazinium salt (ZnTM2,3PyPz) were investigated in aqueous homogeneous solution and in biomimetic reverse micelles medium bearing photooxidizable biological substrates. Absorption and fluorescence spectroscopic studies indicate that ZnTM2,3PyPz is dissolved as monomer in water and in n-heptane/sodium bis(2-ethylhexyl)sulfosuccinate (AOT, 0.1 M)/water (W0 = 30) micellar system. Fluorescence quantum yields (phi F) of 0.29 and 0.27 were calculated for ZnTM2,3PyPz in water and in AOT micelles, respectively. Spectroscopic analysis at different AOT concentrations showed interaction between ZnTM2,3PyPz and AOT reverse micelles with a binding constant (Kb) of 1.7 x 10(3) M(-1). The photosensitization ability of ZnTM2,3PyPz was evaluated using 9,10-dimethylanthracene (DMA). Singlet molecular oxygen, O2(1 delta g), production yielded values of phi(delta) = 0.65 for ZnTM2,3PyPz in AOT micelles. Also, ZnTM2,3PyPz induced efficiently the decomposition of the amino acid L-tryptophan (Trp) and the nucleotide guanosine 5'-monophosphate (GMP) in both media. A value of approximately 3.6 x 10(7) s(-1) M(-1) was found for the second order rate constant of Trp (k(r)(Trp)) decomposition in the AOT system, which is near to that found in pure water. Moreover, ZnTM2,3PyPz formed stable complexes with GMP with a binding constant of K(GMP) = 1.0 x 10(3) M(-1). 1H NMR studies indicated that ZnTM2,3PyPz interacts mainly with the guanine moiety more than the sugar part of GMP. On the other hand, the photodynamic activity of ZnTM2,3PyPz produced decomposition of GMP. Quantification of GMP by HPLC indicates that the nucleotide is photooxidized with a k(obs)(GMP) = 2.6 x 10(-4) s(-1) in water. Photooxidation of GMP considerably increases in deuteriated water indicating that ZnTM2,3PyPz appears to perform its photosensitizing action via the intermediacy of O2(1delta g). Also, efficient sensitized decomposition was observed in micellar media resembling that in pure water. These results provide a better understanding of the effective photodynamic action produced by ZnTM2,3PyPz like a potential phototherapeutic agent for the treatment of neoplastic diseases by photodynamic therapy.

A mechanism for the fluorogenic reaction of amino groups with fluorescamine and MDPF.

Fluorescamine and 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) react with primary amines at alkaline pH to yield highly fluorescent products, claimed to be diaryl-2-hydroxy-pyrrolinones. However, it seems to have been overlooked that these products appear as the pseudobase of coplanar and cationic diarylpyrrolones. Horse blood smears subjected to fluorogenic MDPF and fluorescamine reactions showed eosinophil granules with bright blue-white fluorescence, which required washing at neutral pH and was dependent on the presence of amino groups. The fluorescence of MDPF-butylamine product was abolished at alkaline pH and by bisulphite, suggesting that nucleophilic attacks to the pyrrolone ring (with formation of carbinol (pseudobase) and sulfonate derivatives, respectively), destroy the planarity and conjugation of the whole molecule, thus abolishing the emission at long wavelength. Analysis of the correlation between the largest conjugated fragment (LCF) values and the emission peaks of several fluorophores (fluorescamine- and MDPF-butylamino products, non-rigid fluorochromes) showed the best-fit when the cationic pyrrolones were considered. Although the pseudobases of fluorescamine- and MDPF-amino derivatives are formed at alkaline pH, a full conjugated, coplanar and cationic molecule is suggested to be the true fluorescent product.

Tetrazolium salts and formazan products in Cell Biology: Viability assessment, fluorescence imaging, and labeling perspectives.

For many years various tetrazolium salts and their formazan products have been employed in histochemistry and for assessing cell viability. For the latter application, the most widely used are 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), and 5-cyano-2,3-di-(p-tolyl)-tetrazolium chloride (CTC) for viability assays of eukaryotic cells and bacteria, respectively. In these cases, the nicotinamide-adenine-dinucleotide (NAD(P)H) coenzyme and dehydrogenases from metabolically active cells reduce tetrazolium salts to strongly colored and lipophilic formazan products, which are then quantified by absorbance (MTT) or fluorescence (CTC). More recently, certain sulfonated tetrazolium, which give rise to water-soluble formazans, have also proved useful for cytotoxicity assays. We describe several aspects of the application of tetrazolium salts and formazans in biomedical cell biology research, mainly regarding formazan-based colorimetric assays, cellular reduction of MTT, and localization and fluorescence of the MTT formazan in lipidic cell structures. In addition, some pharmacological and labeling perspectives of these compounds are also described.

Caspase-2: a possible trigger of apoptosis induced in A-549 tumor cells by ZnPc photodynamic treatment.

Photodynamic therapy applied to cell cultures represents a widely accepted experimental method to investigate molecular mechanisms that lead to apoptotic cell death. In this context the subcellular localization of photosensitizers seems to be a significant factor in order to determine the apoptotic pathway that could be activated. We have characterized the experimental conditions that induce apoptotic cell death in A-549 cells incubated with ZnPc and irrradiated with red light. Previously we have found that in this cell line the drug is localized in the Golgi apparatus after 3-h incubation. Indirect immunofluorescence analysis of the events that lead to apoptosis made possible the detection of caspase-2 activation in the Golgi region immediately after photodynamic treatments. A few minutes later, the morphology of this organelle starts to disrupt and just 6 h after treatment the nuclei appear affected showing the fragmented appearance typical of apoptotic cell death. From this results we assume that following the photodynamic treatment of A-549 cells with ZnPc, the activation of caspase-2 in the Golgi apparatus could begin to initiate immediately the apoptotic process.

Long-term regression of the murine mammary adenocarcinoma, LM3, by repeated photodynamic treatments using meso-tetra (4-N-methylpyridinium) porphine.

Photodynamic therapy (PDT) is based on the cytotoxic effect induced by a photosensitizer in the presence of light and molecular oxygen, with production of reactive oxygen species which cause cell death and tumor destruction. Here we describe the response of the murine mammary adenocarcinoma, LM3, to repeated PDT treatments using the synthetic porphyrin derivative, meso-tetra (4-N-methylpyridinium) porphine (TMPyP). Intradermal LM3 tumors in BALB/c mice were left untreated, only treated with light, only injected with 0.9% NaCl solution, or with TMPyP alone (10 microg in 0.1 ml of 0.9% NaCl). For PDT, the intratumoral TMPyP injection was followed 1 h later by blue-red light irradiation for 50 min (80 mW/cm2 total dose: 240 J/cm2). In all cases, control and PDT treatments were performed on the depilated and glycerol-covered skin over the tumor of anesthetized mice and repeated four times (every two days). In a pilot experiment, no significant differences were found in the growth rate of untreated tumors (n=4) and tumors only treated with light (n=4), 0.9% NaCl (n=3) or TMPyP (n=3). PDT-treated tumors (n=3) showed transitory regression and growth delay. In a second approach, the average diameter (mean, mm +/- SEM) of control (drug alone, n=15) vs PDT tumors (n=17) was 2.13+/-0.11 vs 2.02+/-0.10 at day 0, and 4.00+/-0.17 vs 0.20+/-0.07 at day 9, p<0.0001. At day 37 the average diameter of tumors from control vs the PDT group was 10.98+/-0.59 vs 6.31+/-0.82, p<0.0001. PDT caused partial regression of tumors in one from a total of 17 mice, long-term regression in 15, and cure in one animal. Significant differences in the survival and tumor size at death were found between control and PDT-treated mice. Histopathological analysis of LM3 tumors one day after a unique PDT treatment showed extensive hemorrhage and necrotic areas. These results indicate the considerable potential of intratumoral injection of photosensitizers and repeated PDT protocols.

DNA-binding fluorochromes: correlation between C-banding of mouse metaphase chromosomes and hydrogen bonding to adenine-thymine base pairs.

Numerous non-rigid and bow-shaped cationic fluorochromes exist which bind to chromatin DNA, but only some of them are able to selectively label pericentromeric heterochromatin (C-bands) in metaphase chromosomes. Likewise, some DNA ligands allow the recognition of adenine-thymine sequences establishing hydrogen bonds with acceptor atoms in the minor groove. In the present study, we have employed cationic fluorochromes based on a variety of chemical groups to analyze comparatively their potential to demonstrate C-bands, as well as the relationship between this feature and structural parameters of fluorochromes such as curvature radius and hydrogen-bonding ability. Only fluorochromes that bind DNA by hydrogen bonds demonstrated the characteristic C-banding pattern in mouse metaphase chromosomes (together with a weak G/Q-banding), whereas the other (non-hydrogen-bonding) cationic fluorochromes produced uniform emission without any visible banding.

In vitro human cell responses to a low-dose photodynamic treatment vs. mild H2O2 exposure.

Photodynamic treatments allow control of the amount of reactive oxygen species (ROS) produced through the photosensitizer concentration and the light dose delivered to the target. In this way low ROS doses can be achieved in situ to study cell responses related to redox regulation. In this study a comparison has been made between different cell responses to a low-dose photodynamic treatment and both low and relatively high concentrations of H2O2 in human immortalized keratinocytes (HaCaT). The obtained results show that the photodynamic treatment induces a stimulating cell response roughly equivalent to that produced by exposing cells to 10(-5)M H2O2. Higher H2O2 concentrations gave rise to concentration-dependent deleterious effects on the cell cultures. Of importance is that the photodynamic treatment did not produce genotoxic damage, as measured by micronuclei frequency, while cultures exposed to 10(-5)M H2O2 displayed a significant increase in the amount of cells with micronuclei. In summary, the low-dose photodynamic treatment promotes cell proliferation but does not incur in the excessive clastogenic lesions observed after H2O2 exposure. It is therefore proposed as a promising alternative to direct H2O2 exposure in the study of cell redox signalling.

Photoactivation of ROS Production In Situ Transiently Activates Cell Proliferation in Mouse Skin and in the Hair Follicle Stem Cell Niche Promoting Hair Growth and Wound Healing.

The role of reactive oxygen species (ROS) in the regulation of hair follicle (HF) cycle and skin homeostasis is poorly characterized. ROS have been traditionally linked to human disease and aging, but recent findings suggest that they can also have beneficial physiological functions in vivo in mammals. To test this hypothesis, we transiently switched on in situ ROS production in mouse skin. This process activated cell proliferation in the tissue and, interestingly, in the bulge region of the HF, a major reservoir of epidermal stem cells, promoting hair growth, as well as stimulating tissue repair after severe burn injury. We further show that these effects were associated with a transient Src kinase phosphorylation at Tyr416 and with a strong transcriptional activation of the prolactin family 2 subfamily c of growth factors. Our results point to potentially relevant modes of skin homeostasis regulation and demonstrate that a local and transient ROS production can regulate stem cell and tissue function in the whole organism.

Photodynamic toxicity and its prevention by antioxidative agents in Bufo arenarum embryos.

In this work we describe an experimental model to evaluate the photodynamic toxicity on amphibian embryos, as well as the protective effect of antioxidants against the lethal oxidative stress induced by photosensitization. Bufo arenarum embryos were treated with 10 mg/l methylene blue (MB) in AMPHITOX solution for 72 h and then irradiated with a red laser or white light for variable times. Both light sources affected the survival of MB-treated animals and lethal effects occurred within the initial 12 h post-irradiation. For white light irradiation, the most effective phototoxic condition in our study, the LD10, 50 and 90 at 6 h post-irradiation corresponded to 13.57, 19.87 and 29.10 J/cm2, respectively. To explore the action of antioxidants against the photogenerated oxidative stress, MB-treated embryos were incubated with 1mM glutathione (GSH) or ascorbic acid (AA) during 48 h before irradiation. For GSH and 21.6 J/cm2 irradiation, the survival increased from 20 to 90%, whereas 100% survival was achieved with AA even after 43.2 J/cm2 irradiation. These results indicate that both the lethal photodynamic effect and its prevention by antioxidants can be evaluated by means of a simple toxicity test employing amphibian embryos.