The use of targeted nanoparticles (NPs) as a platform for loading photosensitizers enables selective accumulation of the photosensitizers in the tumor area, while maintaining their photodynamic therapy (PDT) effectiveness. Here two novel kinds of methylene blue (MB)-conjugated polyacrylamide (PAA) nanoparticles, MBI-PAA NPs and MBII-PAA NPs, based on two separate MB derivatives, are developed for PDT. This covalent conjugation with the NPs (i) improves the loading of MB, (ii) prevents any leaching of MB from the NPs and (iii) protects the MB from the effects of enzymes in the biological environment. The loading of MB into these two kinds of NPs was controlled by the input amount, resulting in concentrations with optimal singlet oxygen production. For each of the MB-NPs, the highest singlet oxygen production was found for an MB loading of around 11 nmol mg−1. After attachment of F3 peptide groups, for targeting, each of these NPs was taken up, selectively, by MDA-MB-435 tumor cells, in vitro. PDT tests demonstrated that both kinds of targeted NPs resulted in effective tumor cell kill, following illumination, while not causing dark toxicity.
Ultraviolet (UV) irradiation from the sun has been epidemiologically and mechanistically linked to skin cancer, a spectrum of diseases of rising incidence in many human populations. Both non-melanoma and melanoma skin cancers are associated with sunlight exposure. In this review, we discuss the UV wavelength-dependent formation of the major UV-induced DNA damage products, their repair and mutagenicity and their potential involvement in sunlight-associated skin cancers. We emphasize the major role played by the cyclobutane pyrimidine dimers (CPDs) in skin cancer mutations relative to that of (6-4) photoproducts and oxidative DNA damage. Collectively, the data implicate the CPD as the DNA lesion most strongly involved in human cancers induced by sunlight.
UV; cyclobutane pyrimidine dimer; (6-4) photoproduct; skin cancer; melanoma
Photodynamic therapy (PDT) has been used as a cancer therapy for forty years but has not advanced to a mainstream cancer treatment. Although it has been shown to be an efficient way to destroy local tumors by a combination of non-toxic dyes and harmless visible light, it is its additional effects in mediating the stimulation of the host immune system that gives PDT great potential to become more widely used. Although the stimulation of tumor-specific cytotoxic T-cells that can destroy distant tumor deposits after PDT has been reported in some animal models, it remains the exception rather than the rule. This realization has prompted several investigators to test various combination approaches that could potentiate the immune recognition of tumor antigens that have been released after PDT. This review will cover these combination approaches using immunostimulants including various microbial preparations that activate Toll-like receptors and other receptors for pathogen-associated molecular patterns, cytokines growth factors, and approaches that target regulatory T-cells. We believe that by understanding the methods employed by tumors to evade immune response and neutralizing them, more precise ways of potentiating PDT-induced immunity can be devised.
Photodynamic therapy (PDT) is a promising novel therapeutic procedure for the management of a variety of solid tumors and many non-malignant diseases. PDT has been described as having a significant effect on the immune system, which may be either immunostimulatory or, in some circumstances, immunosuppressive. The immunosuppressive effects of PDT have nearly all been concerned with the suppression of the contact hypersensitivity reaction in mice. Here, we review the immunosuppressive aspects of PDT treatment and discuss some additional mechanisms that may be involved.
Most applications of photoremovable protecting groups have used o-nitrobenzyl compounds and their (often commercially available) derivatives that, however, have several disadvantages. The focus of this review is on applications of the more recently developed title compounds, which are especially well suited for time-resolved biochemical and physiological investigations, because they release the caged substrates in high yield within a few nanoseconds or less. Together, these two chromophores cover the action spectrum for photorelease from >700 nm to 250 nm.
Photochromic fulgides and fulgimides have been identified as promising materials for applications in optical memory media, optical switches, and sensors. For applications in humid environments or biological systems, hydrolytic stability is crucial. A new photochromic methyl carboxylic acid indolylfulgimide was synthesized to improve hydrolytic stability in aqueous solution. The UV-vis spectra, extinction coefficient, thermal stability, and photochemical stability of the fulgimide were characterized in 50 mM sodium phosphate buffer (pH 7.4). The open and closed forms were both stable in buffer. At 37 °C after 500 h, the open forms of the fulgimide showed no degradation within experimental error (1–2%) by 1H NMR and 2.3% decomposition by UV-vis spectroscopy. The closed form degraded 22% and 11% after 500 h at 37 °C in buffer by UV-vis and 1H NMR data, respectively. In addition, the fulgimide cycled back and forth between the open and closed forms 80 times before degrading by 20% in buffer. The methyl group at the bridging position of the fulgimide significantly increased the thermal stability by overcoming the rapid hydrolysis of the trifluoromethyl group.
This study describes the use of methylene blue (MB) plus light (photodynamic inactivation, PDI) in the presence of hydrogen peroxide (H2O2) to kill Staphylococcus aureus, Escherichia coli, and Candida albicans. When H2O2 was added to MB plus light there was an increased antimicrobial effect, which could be due to a change in the type of ROS generated or increased microbial uptake of MB. To clarify the mechanism, the production of ROS was investigated in the presence and absence of H2O2. It was observed that ROS production was almost inhibited by the presence of H2O2 when cells were not present. In addition, experiments using different sequence combinations of MB and H2O2 were performed and MB optical properties inside the cell were analyzed. Spectroscopy experiments suggested that the amount of MB was higher inside the cells when H2O2 was used before or simultaneously with PDI, and ROS formation inside C. albicans cells confirmed that ROS production is higher in the presence of H2O2. Moreover enzymatic reduction of MB by E. coli during photosensitizer uptake to the photochemically inactive leucoMB could be reversed by the oxidative effects of hydrogen peroxide, increasing ROS formation inside the microorganism. Therefore, the combination of a photosensitizer such as MB and H2O2 is an interesting approach to improve PDI efficiency.
Feeding broccoli sprout extracts providing daily doses of 10 μmol of glucoraphanin to SKH-1 hairless mice with prior chronic exposure to UV radiation (30 mJ cm-2 of UVB, twice a week, for 17 weeks) inhibited the development of skin tumors during the subsequent 13 weeks; compared to the controls, tumor incidence, multiplicity, and volume were reduced by 25, 47, and 70%, respectively, in the animals that received the protective agent.
Through tailored oligonucleotide scaffolds, Ag nanocluster syntheses have yielded thermally and cell culture stable silver cluster-based emitters. Optimizing ssDNA stability has enabled creation of highly concentrated and spectrally pure nanocluster emitters with strong intracellular emission. Both fixed and live-cell staining become possible, and intracellular delivery is demonstrated both through conjugation to cell penetrating peptides and via microinjection.
Skin cancer is the most common cancer in the United States. Ultraviolet B (UVB) radiation in sunlight is the major environmental factor causing skin cancer. p21, a p53-inducible protein, plays an important role in cell cycle, DNA repair, and apoptosis. Here we have investigated the effect of UVB radiation on p21 and its molecular mechanisms and function in human HaCaT keratinocytes, which we used as a premalignant cellular model because normal skin harbors numerous clones of p53-mutated keratinocytes. We found that in human HaCaT keratinocytes UVB induces rapid p21 down-regulation via a proteasomal degradation mechanism. In p53-defective HaCaT cells, the p21 protein levels remain decreased at a later time post-UVB, but in normal human and mouse epidermal keratinocytes with wild-type p53 the p21 levels are initially reduced but later increase post-UVB. These findings indicate that loss of p53 function leads to sustained p21 down-regulation in response to UVB damage. Degradation of p21 following UVB radiation does not require ATR, ATM, or both, because either the ATR/ATM inhibitor caffeine or siRNA knockdown of ATR, ATM, or both failed to reverse p21 degradation. However, inhibiting MDM2 or GSK3β partially reduced UVB-induced p21 degradation, while inhibiting both enzymes completely prevented it. Restoring the p21 protein levels in UVB-irradiated keratinocytes reduced apoptosis. Although at the molecular level increasing p21 expression has no effect on the protein levels of the Bcl-2 family members, it enhances the activation of AKT, a critical survival pathway to protect cells from apoptosis. Our results suggest a distinct mechanism of p21 degradation in keratinocytes by UVB, and this p21 degradation may significantly enhance UVB-induced apoptosis of premalignant keratinocytes with a p53 defect to eliminate damaged cells and therefore prevent skin cancer development.
Orange autofluorescence from lipofuscin in the lysosomes of the retinal pigment epithelium (RPE) is a hallmark of aging in the eye. One of the major components of lipofuscin is A2E, the levels of which increase with age and in pathologic conditions, such as Stargardt disease or age-related macular degeneration. In vitro studies have suggested that A2E is highly phototoxic and, more specifically, that A2E and its oxidized derivatives contribute to RPE damage and subsequent photoreceptor cell death. To date, absorption spectroscopy has been the primary method to identify and quantitate A2E. Here, a new mass spectrometric method was developed for the specific detection of low levels of A2E and compared to a traditional method of analysis. The new mass spectrometry method allows the detection and quantitation of approximately 10,000-fold less A2E than absorption spectroscopy and the detection and quantitation of low levels of oxidized A2E, with localization of the oxidation sites. This study suggests that identification and quantitation of A2E from tissue extracts by chromatographic absorption spectroscopyoverestimates the amount of A2E. This mass spectrometry approach makes it possible to detect low levels of A2E and its oxidized metabolites with greater accuracy than traditional methods, thereby facilitating a more exact analysis of bis-retinoids in animal models of inherited retinal degeneration as well as in normal and diseased human eyes.
A2E; bis-retinoids; lipofuscin; mass spectrometry; oxidation
All-trans retinol is formed in the outer segments of vertebrate rod photoreceptors from the reduction of the all-trans retinal released by photoactivated rhodopsin. The reduction requires NADPH and is therefore dependent on metabolic input. In metabolically intact photoreceptors, a large increase in rod outer segment fluorescence, attributed to the fluorescence of all-trans retinol, follows rhodopsin photoactivation. The fluorescence increase is biphasic, including a rapid and a slow component. In metabolically compromised cells, there is a much smaller fluorescence increase following rhodopsin photoactivation, but it too contains a rapid component. We have measured the fluorescence signal in single living frog and mouse rod photoreceptors, and have characterized its dependence on the wavelengths of light selected for excitation and for collecting emission. We find that in metabolically intact cells, the excitation and emission properties of both the rapid and slow components of the fluorescence signal are in close agreement with those of all-trans retinol fluorescence. In metabolically compromised cells however, the signal can only partially be due to all-trans retinol, and most of it is consistent with all-trans retinal. The results suggest that in the outer segments of living rod photoreceptors there is rapid release of all-trans retinal, which in metabolically intact cells is accompanied by rapid conversion to all-trans retinol.
Photodynamic therapy (PDT) is an FDA-approved modality for the treatment of early-stage disease and palliation of late-stage disease. Pre-clinical studies using mouse models and clinical studies in patients have demonstrated that PDT is capable of influencing the immune system. The effect of PDT on the generation of anti-tumor immunity is regimen-dependent and is tightly linked to the degree and nature of inflammation induced by PDT. However, the precise mechanism underlying PDT regulated adaptive anti-tumor immunity remains unclear. This review will focus on the current knowledge of immune regulation by PDT.
The fluorescence of silver clusters encapsulated by single stranded oligo-DNA (24 cytosine base pairs, C24:Agn) was used to monitor the transfection of this new silver/DNA fluorophore inside living HeLa cells. For this, the C24:Agn molecules were complexed with a commercially available transfection reagent Lipofectamine and the internalization of C24:Agn was followed with confocal fluorescence microscopy. Bright near-infrared fluorescence was observed from inside the transfected HeLa cells, when exciting with 633 nm excitation, opening up the possibility for the use of these C24:Agn clusters for biological labelling and imaging of living cells and for monitoring the transfection process with limited harm to the living cells.
In this report, we describe an effect of photodynamic therapy (PDT) on membrane trafficking in murine 1c1c7 hepatoma cells. A brief exposure of 1c1c7 cells to a 20 nM concentration of the phosphatidylinositol kinase class-3 antagonist wortmannin led to the rapid appearance of cytoplasmic vacuoles. Fluorescence monitoring of plasma membrane-associated 1-[4-(trimethylamino)-phenyl]-6-phenylhexa-1,3,5-triene (TDPH) over time demonstrated that the wortmannin-induced vacuoles were derived from endocytosed plasma membrane. Low-dose photodamage catalyzed by the lysosomal photosensitizer NPe6, prior to the addition of wortmannin, prevented formation of these vacuoles. NPe6 was found to suppress for several hours the normal trafficking of TDPH-labeled plasma membrane to the cytosol, and the formation of punctate TDPH-labeled cytoplasmic vesicles. The ability of NPe6-induced photodamage to suppress wortmannin-induced vacuolization occurred under conditions that did not disrupt lysosomes and were at or below the threshold of cytostatic/cytotoxic effects. Furthermore, the suppressive effects of NPe6-PDT were not prevented by inclusion of an agent that stabilized lysosomal membranes, or by E64d, an inhibitor of lysosomal cathepsin proteases. Mitochondrial photodamage was less effective at preventing wortmannin-induced vacuole formation and PDT directed against the ER had no effect. The role of photodamage to the endocytic pathway may be a hitherto unexplored effect on cells that selectively accumulate photosensitizing agents. These results indicate that photodamage directed against endosomes/lysosomes has effects independent of the release of lysosomal proteases.
The worldwide rise in antibiotic resistance necessitates the development of novel antimicrobial strategies. Although many workers have used photodynamic therapy (PDT) to kill bacteria in vitro, the use of this approach has seldom been reported in vivo in animal models of infection. We have previously described the first use of PDT to treat excisional wound infections by Gram-(−) bacteria in living mice. However, these infected wound models involved a short timespan between infection (30 min) and treatment by PDT. We now report on the use of PDT to treat an established soft-tissue infection in mice. We used Staphylococcus aureus stably transformed with a Photorhabdus luminescens lux operon (luxABCDE ) that was genetically modified to be functional in Gram-(+) bacteria. These engineered bacteria emitted bioluminescence, allowing the progress of the infection to be monitored in both space and time with a low light imaging charge-coupled device (CCD) camera. One million cells were injected into one or both thigh muscles of mice that had previously been rendered neutropenic by cyclophosphamide administration. Twenty-four hours later, the bacteria had multiplied more than one hundredfold; poly-L-lysine chlorin e6 conjugate or free chlorin e6 was injected into one area of infected muscle and imaged with the CCD camera. Thirty minutes later, red light from a diode laser was delivered as a surface spot or by interstitial fiber into the infection. There was a light dose dependent loss of bioluminescence (to <5% of that seen in control infections) not seen in untreated infections or those treated with light alone, but in some cases, the infection recurred. Treatment with conjugate alone led to a lesser reduction in bioluminescence. Infections treated with free chlorin e6 responded less well and the infection subsequently increased over the succeeding days, probably due to PDT-mediated tissue damage. PDT-treated infected legs healed better than legs with untreated infections. This data shows that PDT may have applications in drug-resistant soft-tissue infections.
The rise of multiply antibiotic resistant bacteria has led to searches for novel antimicrobial therapies to treat infections. Photodynamic therapy (PDT) is a potential candidate; it uses the combination of a photosensitizer with visible light to produce reactive oxygen species that lead to cell death. We used PDT mediated by meso-mono-phenyl-tri(N-methyl-4-pyridyl)-porphyrin (PTMPP) to treat burn wounds in mice with established Staphylococcus aureus infections The third degree burn wounds were infected with bioluminescent S. aureus. PDT was applied after one day of bacterial growth by adding a 25% DMSO/500 μM PTMPP solution to the wound followed by illumination with red light and periodic imaging of the mice using a sensitive camera to detect the bioluminescence. More than 98% of the bacteria were eradicated after a light dose of 210 J cm−2 in the presence of PTMPP. However, bacterial re-growth was observed. Light alone or PDT both delayed the wound healing. These data suggest that PDT has the potential to rapidly reduce the bacterial load in infected burns. The treatment needs to be optimized to reduce wound damage and prevent recurrence.
Photodynamic therapy (PDT) employs a non-toxic dye, termed a photosensitizer (PS), and low intensity visible light which, in the presence of oxygen, combine to produce cytotoxic species. PDT has the advantage of dual selectivity, in that the PS can be targeted to its destination cell or tissue and, in addition, the illumination can be spatially directed to the lesion. PDT has previously been used to kill pathogenic microorganisms in vitro, but its use to treat infections in animal models or patients has not, as yet, been much developed. It is known that Gram-(−) bacteria are resistant to PDT with many commonly used PS that will readily lead to phototoxicity in Gram-(+) species, and that PS bearing a cationic charge or the use of agents that increase the permeability of the outer membrane will increase the efficacy of killing Gram-(−) organisms. All the available evidence suggests that multi-antibiotic resistant strains are as easily killed by PDT as naïve strains, and that bacteria will not readily develop resistance to PDT. Treatment of localized infections with PDT requires selectivity of the PS for microbes over host cells, delivery of the PS into the infected area and the ability to effectively illuminate the lesion. Recently, there have been reports of PDT used to treat infections in selected animal models and some clinical trials: mainly for viral lesions, but also for acne, gastric infection by Helicobacter pylori and brain abcesses. Possible future clinical applications include infections in wounds and burns, rapidly spreading and intractable soft-tissue infections and abscesses, infections in body cavities such as the mouth, ear, nasal sinus, bladder and stomach, and surface infections of the cornea and skin.
Photodynamic therapy (PDT) can lead to the creation of heterogeneous, response-limiting hypoxia during illumination, which may be controlled in part through illumination fluence rate. In the present report we consider 1) regional differences in hypoxia, vascular response, and cell kill as a function of tumor depth and 2) the role of fluence rate as a mediator of depth-dependent regional intratumor heterogeneity. Intradermal RIF murine tumors were treated with Photofrin-PDT using surface illumination at an irradiance of 75 or 38 mW/cm2. Regional heterogeneity in tumor response was examined through comparison of effects in the surface vs. base of tumors, i.e. along a plane parallel to the skin surface and perpendicular to the incident illumination. 75 mW/cm2-PDT created significantly greater hypoxia in tumor bases relative to their surfaces. Increased hypoxia in the tumor base could not be attributed to regional differences in Photofrin concentration nor effects of fluence rate distribution on photochemical oxygen consumption, but significant depth-dependent heterogeneity in vascular responses and cytotoxic response were detected. At a lower fluence rate of 38 mW/cm2, no detectable regional differences in hypoxia or cytotoxic responses were apparent, and heterogeneity in vascular response was significantly less than that during 75 mW/cm2-PDT. This research suggests that the benefits of low-fluence-rate-PDT are mediated in part by a reduction in intratumor heterogeneity in hypoxic, vascular and cytotoxic responses.
photodynamic therapy; fluence rate; hypoxia; EF3; blood flow
Fullerenes are a class of closed-cage nanomaterials made exclusively from carbon atoms. A great deal of attention has been focused on developing medical uses of these unique molecules especially when they are derivatized with functional groups to make them soluble and therefore able to interact with biological systems. Due to their extended π-conjugation they absorb visible light, have a high triplet yield and can generate reactive oxygen species upon illumination, suggesting a possible role of fullerenes in photodynamic therapy. Depending on the functional groups introduced into the molecule, fullerenes can effectively photoinactivate either or both pathogenic microbial cells and malignant cancer cells. The mechanism appears to involve superoxide anion as well as singlet oxygen, and under the right conditions fullerenes may have advantages over clinically applied photosensitizers for mediating photodynamic therapy of certain diseases.
Here we describe the detection and identification of a yellow chlorophyll catabolite (Cj-YCC) in fresh extracts of senescent leaves of Cercidiphyllum japonicum. In addition, we report its partial synthesis by oxidation of Cj-NCC-1, the major (colourless) “nonfluorescent” chlorophyll catabolite (NCC) found in degreened leaves of C. japonicum. The spectroscopic analysis and structural characterization indicated Cj-YCC to be a simple dehydrogenation product of Cj-NCC-1 (by formal removal of a hydrogen atom at the C(20)- and C(1)-positions). Indeed, NCCs are easily oxidized and were first called “rusty pigments”, as they had a tendency to turn brown upon storage on a dry silica gel plate. The yellow tetrapyrrole Cj-YCC may thus come about by oxidation of Cj-NCC-1 in the leaves. Its presence in the yellow leaves of a deciduous tree provides the first evidence for the contribution of a coloured chlorophyll catabolite to the fall colours.
Chlorophyll metabolism probably is the most visible manifestation of life. Total annual turnover of chlorophyll has been estimated to involve more than 1000 million tons. Surprisingly, chlorophyll catabolism has remained an enigma until less than twenty years ago, when a colorless chlorophyll catabolite from senescent plant leaves was identified and its structure was elucidated. In the meantime, chlorophyll breakdown products have been identified in a variety of plant leaves and their structural features have been elucidated. Most recently, chlorophyll breakdown products have also been identified in some ripening fruit. Chlorophyll breakdown in vascular plants only fleetingly involves enzyme-bound colored intermediates. The stage of fluorescent catabolites is also passed rapidly, as these isomerize further to colorless nonfluorescent tetrapyrrolic catabolites. The latter accumulate in the vacuoles of de-greened leaves and are considered the final products of controlled chlorophyll breakdown. The same tetrapyrroles are also found in ripening fruit and are effective antioxidants. Chlorophyll breakdown leads to tetrapyrroles that appear to have physiologically beneficial chemical properties, and it may thus not merely be a detoxification process.
The effect of divalent cations (calcium and magnesium) and a permeabilizing agent (EDTA) on the uptake of a cationic photosensitizer (PS), methylene blue (MB), and two anionic PSs, rose bengal (RB) and indocyanine green (ICG), by Gram-positive Enterococcus faecalis and Gram-negative Actinobacillus actinomycetemcomitans was examined. The possible roles of multidrug efflux pumps and protein transporters in photosensitizer uptake were assessed in E. faecalis cells by studies using an efflux pump inhibitor (verapamil) and trypsin treatment respectively. Divalent cations enhanced the uptake and photodynamic inactivation potential of both RB and ICG in E. faecalis and A. actinomycetemcomitans, while they decreased the uptake and bacterial killing by MB. Verapamil increased the uptake of RB (possibly due to efflux pump inhibition), whereas trypsin treatment resulted in significant decrease in RB and ICG uptake. The results suggested that the uptake of anionic PSs by bacterial cells may be mediated through a combination of electrostatic charge interaction and by protein transporters, while the uptake of cationic PSs, as previously reported, is mediated by electrostatic interactions and self promoted uptake pathways.
Ultraviolet (UV) radiation is a globally important abiotic factor influencing ecosystem structure and function in multiple ways. While UV radiation can be damaging to most organisms, several factors act to reduce UV exposure of organisms in aquatic ecosystems, the most important of which is dissolved organic carbon (DOC). In alpine lakes, very low concentrations of DOC and a thinner atmosphere lead to unusually high UV exposure levels. These high UV levels combine with low temperatures to provide a fundamentally different vertical structure to alpine lake ecosystems in comparison to most lowland lakes. Here, we discuss the importance of water temperature and UV transparency in structuring alpine lake ecosystems and the consequences for aquatic organisms that inhabit them. We present transparency data on a global data set of alpine lakes and nearby analogous subalpine lakes for comparison. We also present seasonal transparency data on a suite of alpine and subalpine lakes that demonstrate important differences in UV and photosynthetically active radiation (PAR, 400–700 nm) transparency patterns even within a single region. These data are used to explore factors regulating transparency in alpine lakes, to discuss implications of future environmental change on the structure and function of alpine lakes, and ways in which the UV transparency of these lakes can be used as a sentinel of environmental change.
We evaluated the effect of photochemical alterations of chromophoric dissolved organic matter (CDOM) on bacterial abundance, activity and community composition in a coastal lagoon of the Atlantic Ocean with high dissolved organic carbon concentration. On two occasions during the austral summer, bacteria-free water of the lagoon was exposed to different regions of the solar spectrum (full solar radiation, UV-A + PAR, PAR) or kept in the dark. Subsequently, dilution cultures were established with bacterioplankton from the lagoon that were incubated in the pre-exposed water for 5 h in the dark. Cell abundance, activity, and community composition of bacterioplankton were assessed before and after incubation in the different treatments. Changes in absorption, fluorescence, and DOC concentration were used as proxies for CDOM photoalteration. We found a significant CDOM photobleaching signal, DOC loss, as well as a stimulation of bacterial activity in the treatments pre-exposed to UV radiation, suggesting increased bioavailability of DOM. Bacterial community analysis by fluorescence in situ hybridization revealed that this stimulation was mainly accompanied by the specific enrichment of Alpha- and Betaproteobacteria. Thus, our results suggest that CDOM photoalteration not only stimulates bacterioplankton growth, but also induces rapid changes in bacterioplankton composition, which can be of relevance for ecosystem functioning, particularly considering present and future changes in the input of terrestrial CDOM to aquatic systems.