Self-quenching synthesis of coordination polymer pre-drug nanoparticles for selective photodynamic therapy.

The design and preparation of a photoactive coordination polymer nanoplatform with tumor-related stimuli-activatability and biodegradability is highly desirable for achieving highly precise photodynamic therapy (PDT). Herein, novel "pre-photodynamic" nanoparticles (Fe-IBDP NPs) with a tumor microenvironment (TME)-activatable PDT and good biodegradability were synthesized by carrying out facile coordination assembly of an IBDP photosensitizer with an Fe3+ quenching agent. After being taken up by cancer cells, our "inactive" Fe-IBDP NPs were activated by the TME and as a result decomposed and released the photoactive carboxyl-functionalized diiodo-substituted BODIPY (IBDP) photosensitizer, which generated cytotoxic singlet oxygen (1O2) under light irradiation. By contrast, these NPs showed relatively low cytotoxicity in normal cells. This work also provided a feasible method for preparing the next generation of photoactive nanomedicines for use in precise phototherapy.

"Off-on" switching of intracellular singlet oxygen release under biocompatible conditions.

Precise spatiotemporal control of singlet oxygen generation is of immense importance considering its involvement in photodynamic therapy. In this work, we present a rational design for an endoperoxide which is highly stable at ambient temperatures yet, can rapidly be converted into a highly labile endoperoxide, thus releasing the "stored" singlet oxygen on demand. The "off-on" chemical switching from the stable to the labile form is accomplished by the reaction with fluoride ions. The potential utility of controlled singlet oxygen release was demonstrated in cell cultures.

Perfluorocarbon-based O2 nanocarrier for efficient photodynamic therapy.

Tumor hypoxia is considered as one of the major factors that limit the efficiency of photodynamic therapy (PDT), in which oxygen (O2) is needed to generate singlet oxygen (1O2) for cell destruction. Inspired by the excellent O2 carrying ability of perfluorocarbon molecules in artificial blood, we prepared a series of polymer micelles with a perfluorocarbon core to carry both photo-sensitizer and O2 to the tumor site, aiming to improve PDT efficiency. We found that the accelerated generation of 1O2 correlated with the increased perfluorocarbon amount in solution. In vitro cell study further showed that the new perfluorocarbon formulation not only improved the production of 1O2, leading to enhanced photodynamic therapy efficiency, but also significantly reduced cell toxicity when compared with the one without these perfluoro units. This work provides a new option for improving PDT efficiency with the new perfluorocarbon-incorporated nanoplatform.

Lanthanide-doped upconversion nanoparticles electrostatically coupled with photosensitizers for near-infrared-triggered photodynamic therapy.

Lanthanide-doped upconversion nanoparticles (UCNPs) have recently shown great promise in photodynamic therapy (PDT). Herein, we report a facile strategy to fabricate an efficient NIR-triggered PDT system based on LiYF4:Yb/Er UCNPs coupled with a photosensitizer of a ß-carboxyphthalocyanine zinc (ZnPc-COOH) molecule via direct electrostatic interaction. Due to the close proximity between UCNPs and ZnPc-COOH, we achieved a high energy transfer efficiency of 96.3% from UCNPs to ZnPc-COOH, which facilitates a large production of cytotoxic singlet oxygen and thus an enhanced PDT efficacy. Furthermore, we demonstrate the high efficacy of such a NIR-triggered PDT agent for the inhibition of tumor growth both in vitro and in vivo, thereby revealing the great potential of the UCNP-based PDT systems as noninvasive NIR-triggered PDT agents for deep cancer therapy.

Fungicidal photodynamic effect of a twofold positively charged porphyrin against Candida albicans planktonic cells and biofilms.

BACKGROUND: Antimicrobial photodynamic therapy is an interesting alternative for the treatment of superficial mucocutaneous mycoses. In immunodeficient patients, these infections are frequently recurrent and resistant to the most commonly used antifungal medications. Candida albicans biofilms frequently cause such infections that can even evolve to deep-seated mycoses. MATERIALS & METHODS: The efficiency of a photodynamic therapy was investigated against C. albicans using a twofold positively charged porphyrin (XF-73) in comparison with the well-known fourfold positively charged porphyrin (5,10,15,20-tetrakis(1-methyl-4-pyridyl)-21H,23H-porphine, tetra-p-tosylate salt). RESULTS: After incubation with 0.5 µM of XF-73 for 15 min and irradiation with blue light (12.1 J/cm(2)), the viability of C. albicans planktonic cells decreased by over 6 log10. For biofilm cells, a longer incubation time (4 h) with 1 µM of XF-73 and a light dose of 48.2 J/cm(2) was necessary to achieve over 5 log10 cell killing. Cell killing was mediated by singlet oxygen that was directly detected via its luminescence at 1270 nm in XF-73-incubated C. albicans biofilms for the first time. Antimicrobial photodynamic therapy yielded better results for XF-73 compared with 5,10,15,20-tetrakis(1-methyl-4-pyridyl)-21H,23H-porphine, tetra-p-tosylate salt when using the same conditions. CONCLUSION: This study provides evidence that XF-73 is a highly efficient photosensitizer to kill C. albicans and it would be worthwhile to test this photosensitizer in clinical studies for both prophylaxis and treatment of infections caused by this microorganism, preventing the spread of C. albicans throughout the bloodstream.

Comparative analysis of four oxidized guanine lesions from reactions of DNA with peroxynitrite, singlet oxygen, and γ-radiation.

Oxidative damage to DNA has many origins, including irradiation, inflammation, and oxidative stress, but the chemistries are not the same. The most oxidizable base in DNA is 2-deoxyguanosine (dG), and the primary oxidation products are 8-oxodG and 2-amino-imidazolone. The latter rapidly converts to 2,2-diamino-oxazolone (Ox), and 8-oxodG is further oxidized to spiroiminodihydantoin (Sp) and guanidinohydantoin (Gh). In this study, we have examined the dose-response relationship for the formation of the above four products arising in calf thymus DNA exposed to gamma irradiation, photoactivated rose bengal, and two sources of peroxynitrite. In order to carry out these experiments, we developed a chromatographic system and synthesized isotopomeric internal standards to enable accurate and precise analysis based upon selected reaction monitoring mass spectrometry. 8-OxodG was the most abundant products in all cases, but its accumulation was highly dependent on the nature of the oxidizing agent and the subsequent conversion to Sp and Gh. Among the other oxidation products, Ox was the most abundant, and Sp was formed in significantly greater yield than Gh.

Ex vivo assessment of protective effects of carvacrol against DNA lesions induced in primary rat cells by visible light excited methylene blue (VL+MB).

Carvacrol belongs to frequently occurring phenolic components of essential oils (EOs) and it is present in many kinds of plants. Biological effect of this phenol derivative on human beings is however not sufficiently known. The present study was undertaken to evaluate the level of VL+MB-induced oxidative DNA lesions in hepatocytes and testicular cells (freshly isolated from control or carvacrol-watered rats) by the modified single cell gel electrophoresis (SCGE). The results showed that carvacrol significantly reduced the level of VL+MB-induced oxidized bases (EndoIII- and Fpg-sensitive sites) only in hepatocytes but not in testicular cells. Chromosomal aberration assay of primary hepatocytes, isolated from control or carvacrol-watered rats did not testify any genotoxic activity of carvacrol. We suggest that in vivo applied synthetic carvacrol, whose antioxidative activity was confirmed by DPPH assay, exhibits primarily a strong hepatoprotective activity against oxidative damage to DNA.

Responsive and mitochondria-specific ruthenium(II) complex for dual in vitro applications: two-photon (near-infrared) induced imaging and regioselective cell killing.

A mitochondria-permeable ruthenium(II) complex has been designed as a responsive probe which may be used to sensitize the formation of singlet oxygen (Phi(Delta) = 0.93) and cause local damage in cellulo when exposed to UV and near-infrared laser excitation.

Agrobacterium tumefaciens iron superoxide dismutases have protective roles against singlet oxygen toxicity generated from illuminated Rose Bengal.

Singlet oxygen is a highly reactive form of molecular oxygen that is harmful to biological systems. Here, the role of three iron-containing superoxide dismutase (sodB) genes is clearly shown in protecting Agrobacterium tumefaciens against singlet oxygen toxicity. A sodBI mutant was more sensitive to singlet oxygen than both wild-type bacteria and a double sodBII-sodBIII mutant strain. Moreover, a sodBI-sodBII double mutant had higher sensitivity to singlet oxygen than a single sodBI mutant, although the double mutant was comparable to a sodB null mutant. High-level expression of sodBI and sodBII fully complemented the singlet oxygen hypersensitivity phenotype of the sodB null mutant, while high-level expression of sodBIII encoding a periplasmic SOD only partially restored the phenotype. Taken together, our data suggest that SodBI and SodBII have novel protective roles against singlet oxygen toxicity through unknown mechanisms.

Impaired activation of caspase cascade during cell death induced by newly synthesized singlet oxygen generator, 1-buthylnaphthalene-4-propionate endoperoxide.

Endoperoxides of naphthalene derivatives generate singlet oxygen under physiological conditions. Here we have synthesized a new endoperoxide of a naphthalene derivative, 1-buthylnaphthalene-4-propionate endoperoxide (BNPE), and studied its cytotoxic properties on HepG2 and HaCaT cells. BNPE induced cell death at much lower concentration than 1-methylnaphthalene-4-propionate endoperoxide (MNPE) and naphthalene dipropionate endoperoxide (NDPE). A positive correlation exists between the amount of endoperoxide incorporated into cells and its cytotoxic ability. The cytotoxic effect of BNPE was attenuated by alpha-tocopherol but not by sodium azide. In contrast, the effects of MNPE and NDPE were attenuated by both alpha-tocopherol and sodium azide. The caspase cascade in cells treated with endoperoxide was impaired. Caspase activity in a soluble protein fraction were inhibited similarly by the above three endoperoxides. These results suggest an abortive apoptotic pathway due to the suppression of caspase activation is a general feature of cell death induced by singlet oxygen.

Cellular prion protein protects against reactive-oxygen-species-induced DNA damage.

Although the cellular form of the prion protein (PrPC) is critical for the development of prion disease through its conformational conversion into the infectious form (PrPSc), the physiological role of PrPC is less clear. Using alkaline single-cell gel electrophoresis (the Comet assay), we show that expression of PrPC protects human neuroblastoma SH-SY5Y cells against DNA damage under basal conditions and following exposure to reactive oxygen species, either hydroxyl radicals following exposure to Cu2+ or Fe2+ or singlet oxygen following exposure to the photosensitizer methylene blue and white light. Cells expressing either PrPDeltaoct which lacks the octapeptide repeats or the prion-disease-associated mutants A116V or PG14 had increased levels of DNA damage compared to cells expressing PrPC. In PrPSc-infected mouse ScN2a cells there was a significant increase in DNA damage over noninfected N2a cells (median tail DNA 2.87 and 7.33%, respectively). Together, these data indicate that PrPC has a critical role to play in protecting cells against reactive-oxygen-species-mediated DNA damage; a function which requires the octapeptide repeats in the protein, is lost in disease-associated mutants of the protein or upon conversion to PrPSc, and thus provide further support for the neuroprotective role for PrPC.

Plasmid DNA acquires immunogenicity on exposure to singlet oxygen.

In the present study, the effect of singlet oxygen (1O2) (generated by ultraviolet (UV) irradiation of methylene blue) on plasmid DNA has been analyzed by UV spectroscopy, fluorescence spectroscopy, and S1 nuclease digestibility. Both native and 1O2-modified plasmid DNA were treated with a number of restriction enzymes to map out the sites damaged by 1O2. It was also observed that, on exposure to 1O2, native plasmid DNA that is non-immunogenic acquired the ability to elicit an immune response in experimental animals. However, the induced antibodies exhibited appreciable cross reactivity with various polynucleotides and nucleic acids. The data indicate that the antibodies, though cross-reactive, preferentially bind 1O2-modified epitopes on plasmid DNA. Gel retardation assay further substantiated the enhanced recognition of 1O2-modified plasmid DNA over the native form. The antibodies developed were then subjected to competition ELISA with sera from various diseases such as systemic lupus erythematosus, rheumatoid arthritis, and cancer. These results suggest that upon exposure of DNA to 1O2, neo-epitopes are generated, which may be one of the factors for the induction of circulating autoantibodies in the three diseases.

Oxidative and alkylating damage in DNA.

Modification of cellular DNA upon exposure to reactive oxygen and nitrogen species is the likely initial event involved in the induction of the mutagenic and lethal effects of various oxidative stress agents. Evidence has been accumulated for the significant implication of singlet oxygen (1O(2)), generated as the result of UVA activation of endogenous photosensitizers as porphyrins and flavins. 7,8-Dihydro-8-oxo-2'-deoxyguanosine (8-oxodGuo) has been shown to be the exclusive product of the reaction of 1O(2) with the guanine moiety of cellular DNA, in contrast to the hydroxyl radical, which reacts almost indifferently with all the nucleobases and the sugar moiety of DNA. Furthermore 8-oxodGuo is also produced by other oxidants and can be used as an ubiquitous biomarker of DNA oxidation but can not be a specific marker of any particular species. The role of DNA etheno adducts in mutagenic and carcinogenic processes triggered by known occupational and environmental carcinogens has also been studied. Much interest in etheno adducts resulted from the detection of increased levels of 1,N(6)-etheno-2'-deoxyadenosine and 3,N(4)-etheno-2'-deoxycytidine in DNA from human, rat and mouse tissues under pathophysiological conditions associated with oxidative stress. A method involving on-line HPLC with electrospray tandem mass spectrometry detection has been developed for the analysis of 1,N(2)-etheno-2'-deoxyguanosine (1,N(2)-epsilondGuo) in DNA. This methodology permits direct quantification of 20 fmol (7.4 adducts/10(8) dGuo) of the etheno adduct from approximately 350 microg of crude DNA hydrolysates. This method provides the first evidence of the occurrence of 1,N(2)-epsilondGuo as a basal endogenous lesion and may be utilized to better assess the biological consequences of etheno DNA damage under normal and pathological conditions. This work addresses the importance of isotope labeling associated with mass spectrometry technique for biomolecule damage studies.

Reduced joining of DNA ends correlates with chromosomal instability in three melanoma cell lines.

Chromosomal instability plays a pivotal role in multistep carcinogenesis by facilitating the acquisition of the multiple genetic alterations necessary for malignant transformation. In order to study the role of abnormal DNA repair in malignant melanoma, we measured the ability of cell lines from malignant melanoma and that of primary melanocytes to process 4 different kinds of DNA damage (pyrimidine dimers, oxidative DNA lesions, replication errors, and DNA double-strand breaks) using 4 different plasmid assays. Based on the number of chromosomes, the DNA index, and the rates of spontaneous micronuclei, the chromosomal stability in primary melanocytes and the melanoma line LIBR was characterized as being high, intermediate in M1, and low in MeWo. Repair of UVB photoproducts, of oxidative DNA damage, and of replication errors was not impaired in any melanoma line. Using linearized shuttle vector plasmid pZ189, LIBR cells and primary melanocytes exhibited a high efficiency of joining overlapping DNA ends, reflecting proficient repair of DNA double-strand breaks. Joining efficiency was reduced slightly in M1 and 2.9-fold in MeWo. This indicates that in the melanoma cell lines studied here, an increase in chromosomal instability is accompanied by a pronounced impairment in the ability to join DNA ends. Although a small sample was studied, this raises the possibility that defects in DNA end joining may also contribute to genetic instability and chromosome aberrations in melanoma.

Site-specific hydroxylation at polyguanosine in double-stranded DNA by nickel(II) in the presence of SH compounds: comparison with singlet oxygen-induced DNA damage.

We examined the mechanism of DNA damage induced by carcinogenic Ni(II) in the presence of SH compounds. In the presence of model endogenous SH compounds, dithiothreitol (DTT), 1,4-dithio-L-threitol, and dithioerythritol, Ni(II) induced damage to (32)P-5'-end-labeled DNA fragments obtained from the human c-Ha-ras-1 protooncogene and the p53 tumor suppressor gene. The intensity of Ni(II)-mediated DNA damage induced by DTT was stronger than that by other model endogenous SH compounds, 1,4-dithio-L-threitol and dithioerythritol. DNA damage induced by Ni(II) plus DTT was observed only when the DNA was treated with piperidine, suggesting that Ni(II) plus DTT caused only base damage. Formamidopyrimidine-DNA glycosylase, which is known to recognize 8-oxodG as well as Fapy residues, treatment induced cleavage sites, mainly guanine residues, particularly at the 5'-GG-3', 5'-GGG-3', and 5'-GGGG-3' sequences, in DNA incubated with Ni(II) in the presence of DTT. SOD and catalase inhibited the DNA damage, suggesting that DNA damage involved superoxide anion and hydrogen peroxide. Sodium azide, a potent and relatively specific scavenger of (1)O(2), inhibited DNA damage by Ni(II) in the presence of DTT, whereas the sequence specificity of DNA damage was different from that obtained by (1)O(2) generating agent. The formation of 8-oxodG in calf thymus DNA by Ni(II) was observed with the physiological thiols, dihydrolipoic acid and mercaptopyruvate, as well as with DTT. These results suggest that Ni(II) and DTT form a reactive species, which may be responsible for causing guanine-specific DNA damage. Endogenous SH compounds, which have similar chemical structures to DTT, would participate in nickel carcinogenesis through causing oxidative DNA damage.

Cytotoxicity and mutagenesis induced by singlet oxygen in wild type and DNA repair deficient Escherichia coli strains.

Singlet oxygen ((1)O(2)) is a product of several biological processes and can be generated in photodynamic therapy, through a photosensitization type II mechanism. (1)O(2) is able to interact with lipids, proteins and DNA, leading to cell killing and mutagenesis, and can be directly involved with degenerative processes such as cancer and aging. In this work, we analyzed the cytotoxicity and mutagenesis induced after direct treatment of wild type and the DNA repair fpg and/or mutY deficient Escherichia coli strains with disodium 3,3'-(1,4-naphthylidene) diproprionate endoperoxide (NDPO(2)), which releases (1)O(2) by thermodissociation. The treatment induced cell killing and mutagenesis in all strains, but the mutY strain showed to be more sensitive. These results indicate that even (1)O(2) generated outside bacterial cells may lead to DNA damage that could be repaired by pathways that employ MutY protein. As (1)O(2) is highly reactive, its interaction with cell membranes may generate secondary products that could react with DNA, leading to mutagenic lesions.