American Society for Photobiology

ASP Conference 2016: 21-26 May 2016
Tampa Marriott Waterside Hotel & Marina


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P2 - Poster Session 2

Florida 6   18:00 - 20:00

 
P2-1   Comparison of photoactivation and photorepair of T(6-4)T and T(6-4)C photoproducts by Xenopus (6-4) photolyase on FTIR study M Kumagai*, Nagoya Institute of Technology ; D Yamada, Nagoya Institute of Technology; T Iwata, Nagoya Institute of Technology; J Yamamoto, Osaka University; ED Getzoff, The Scripps Research Institute; S Iwai, Osaka University; H Kandori, Nagoya Institute of Technology

Abstract: Photolyases (PHRs) are DNA repair proteins that revert UV-induced photoproducts. Two types of PHRs have been reported: CPD PHR repairs cyclobutane pyrimidine dimers (CPDs), while (6-4) PHR repairs pyrimidine-pyrimidine (6-4) photoproducts ((6-4) PPs). Flavin adenine dinucleotide (FAD) is the chromophore of PHRs. The oxidized and neutral radical forms are resting states, and fully reduced (FADH^-) form is enzymatically active. Upon the illumination to the FADH^- form, the repair of photoproduct is achieved as a trigger for the electron transfer from FADH^- to the photoproduct.We have measured the photoactivation and photorepair processes of Xenopus (6-4) PHR by light-induced difference Fourier-transform infrared (FTIR) spectroscopy [1-5]. When the photoactivation process was compared between substrate-bound and unbound (6-4) PHR, different structural changes were observed in the α-helices and neutral His residue(s). In addition, hydrogen-bonding environment of C=O groups from (6-4) PP was influenced upon photoactivation. Thus, FTIR spectroscopy can detect faint changes which cannot be detected by X-ray crystallography.(6-4) PHR can repair both T(6-4)T and T(6-4)C, where the OH and NH2 groups are bound at C5 position of 5' side, respectively. Here, to investigate structural perturbation of the enzyme by (6-4) PP binding, photoactivation and photorepair process of T(6-4)T and T(6-4)C were compared by FTIR spectroscopy. We observed the spectral difference between presence of T(6-4)T and T(6-4)C PP in the photoactivation and photorepair processes. Identification of vibrational bands by use of isotope labeling and mutants is now in progress. Molecular mechanisms of substrate recognition, repair by (6-4) PHR will be discussed based on the observation. [1] Zhang Y. et al. Biochemistry 50, 3591-3598 (2011) [2] Zhang Y. et al. J. Phys. Chem. Lett. 2, 2774-2777 (2011) [3] Yamada D. et al. Biochemistry 51, 5774-5783 (2012). [4] Yamada D. et al. Biophys. Physicobiol. 12, 139-144 (2015). [5] Yamada D. et al. Biochemistry 55, 715-723 (2016).

P2-4   Photochemistry of Cholestatrienol - a Fluorescent Analogue of Cholesterol S Gupta*, Florida State University ; S Chakraborty, Florida State University; RJ Clark, Florida State University; J Saltiel, Florida State University

Abstract: Cholestatrien-3beta-ol (CTL) is a natural product(ref.1) that has found considerable use as a fluorescent probe for cholesterol intracellular trafficking(ref.2). Although known since 1930, the photochemical reactivity of dehydroergosterol(ref.3), an analogue of CTL, has been ignored. We encountered CTL as a trace impurity in 7-dehydrocholesterol, the precursor in the synthesis of vitamin D3. As it is highly fluorescent, it interfered with our initial emission studies in the vitamin D3 field. Its use in biochemical applications prompted us to investigate its photoproducts. Irradiation of CTL (313 nm) in ethanol indicated the formation of a photoproduct, whose UV spectrum is consistent with an s-trans-diene moiety that could form by photoaddition of ethanol to one of CTL's double bonds. Subsequent irradiation of CTL in tetrahydrofuran gave the same product as in ethanol proving that photoaddition is not the major photoreaction. 1D 1H, 2D COSY NMR and UV-Vis. and MALDI spectra of this major photoproduct are consistent with CTL photorearrangement to pentacyclic: (3aR,9aS)-3-(6-hydroxy-6-methylheptan-2-yl)-3a,9a-dimethyl-1,2,3a,4,6,7,8,9,9a,9b,10b-dodecahydrobenzo[2,3]cyclopropa[1,2-b]-asindacen-7-ol. The structure assignment of this pentacyclic product was definitively established by X-ray crystallography. References: 1.Wassif, CA; Brownson, KE; Sterner, AL; Forlino, A; Zerfas, PM; Wilson, WK; Starost, MF; Porter, FD (2007) Hum. Mol. Genet. 16, 1176-1187. 2.Scheidt, HA; Muller, P; Herrmann, A; Huster, D (2003) J. Biol. Chem. 278, 45563"45569. 3.Morton, RA; Heilbron, IM; Spring, FS (1930) Biochem. J. 24, 136-140. (Moved from P1.14)

P2-5   A broader filtration of UVA1 wavelengths improves skin photoprotection, in vitro C MARIONNET*, L'Oreal Research and Innovation ; C PIERRARD, L'Oreal Research and Innovation; C GOLEBIEWSKI, L'Oreal Research and Innovation; D CANDAU, L'Oreal Research and Innovation; F BERNERD, L'Oreal Research and Innovation

Abstract: Sun exposure can lead to photoaging and photocarcinogenesis. It involves UVB (290-320 nm) and UVA (UVA2, 320-340 nm and UVA1, 340-400 nm). UVA1 rays represent majority of UV that reach the Earth surface. They generate reactive oxygen species and contribute to harmful effects including immunosuppression and carcinogenesis in vivo. At the molecular and cellular level we previously showed in a model of reconstructed skin, that UVA1 exposure led to epidermal and dermal damage, such as fibroblast apoptosis and a strong alteration of gene and protein expression involving essential biological pathways. However, sunscreen formulas that efficiently filter UV wavelengths up to 370 nm lack, up to now, a sufficient absorption in the range of 370-400 nm UVA1 wavelengths. The present study aimed at investigating if an enlargement of spectral absorption could improve the protection against UVA1-induced biological damage. Three filtrating formulations were used: a state of the art formulation (SAF) (absorbing 280-370 nm UV rays), and two other formulations including prototype UVA1 filters, allowing a gain of absorption in the UVA1 range (280-385nm, 280-395 nm, respectively). The efficiency of these three formulations was tested after topical application onto reconstructed skin prior UVA1 exposure. At different time points post UVA1 exposure, cell and tissue morphology, as well as gene and soluble protein expression were assessed. Results show that, as compared to an unprotected human reconstructed skin, SAF could prevent UVA1-induced cell and tissue damage as well as the modulation of gene and protein expression, although the protection was not complete. The use of formulations including UVA1 filters provided a superior protection than the SAF. On most of the tested endpoints, the broadening of UVA1 absorption spectrum led to a better protection. These results show that a broader filtration of UVA1 wavelengths can afford a significant better biological protection against UVA1-induced damage.

P2-6   Tuning the photophysical properties of fluorinated porphyrin derivatives: effect on biological activity and its application in design of efficient anticancer and antimicrobial PDT agents B Pucelik*, Jagiellonian University, Krakow, Poland ; R Paczynski, Jagiellonian University and Malopolska Centre of Biotechnology, Krakow, Poland; CS Vinagreiro, University of Coimbra, Coimbra, Portugal; NPF Gonzalves, Luzitin SA Ed. Bluepharma,Coimbra, Portugal; LG Arnaut, University of Coimbra, Coimbra, Portugal; MM Pereira, University of Coimbra, Coimbra, Portugal; JM Dabrowski, Jagiellonian University, Krakow, Poland

Abstract: Tetrapyrrolic macrocycles have been recently explored as scaffolds for the development of more efficient photosensitizers for photodynamic therapy of cancer (PDT) as well as photodynamic inactivation (PDI) of microorganisms. Herein, we perform the comparison of physicochemical and photophysical properties of the series of structurally related fluorinated sulphonamide porphyrin derivatives designed for PDT and PDI application. Our studies report that the chemical modification of the macrocycle by tunable substituents provides an access to the desired biological properties needed for efficient treatment. Thus, we evaluate the broad-spectrum photodynamic activity of investigated photosensitizers against a variety of cancer cell lines and a panel of pathogenic microbes consisting of the Gram-positive and Gram-negative bacteria. All of tested photosensitizers showed favorable characteristics due to strong absorption in visible or near infrared region of spectrum and high singlet oxygen quantum yields. Moreover, selected compounds was found to be a highly effective antimictrobial agents against Gram-positive (S. aureus, B. subtilis) and Gram-negative (E.coli, P. aeruginosa, P. putida) bacteria which are most resistant to antimicrobial PDT. The death of prevalent pathogens was dependent on the PS concentration and illumination time. Significantly, completed eradication of the initial population of microorganisms was observed after photodynamic inactivation mediated porphyrin-based photosensitizer. On the other hand, in the following studies investigated porphyrins and bacteriochlorins indicated the lack of dark cytotoxicity and strong photodynamic effect towards cancer cells (A549, CT26, 2H11, B16F10). The improved photodynamic activity of selected compounds reveal that fluorinated porphyrins and their tetrahydro- derivatives are worth further in vivo investigations not only on animal tumor models but also might prove beneficial to treat the localized wound infections.

P2-7   The Austrian UVA-Network A.W. Schmalwieser*, University of Veterinary Medicine, Institute of Physiology and Biophysics ; M. Blumthaler, Div. of Biomedical Physics, Medical University Innsbruck; B. Klotz, Div. of Biomedical Physics, Medical University Innsbruck; M. Schwarzmann, Div. of Biomedical Physics, Medical University Innsbruck; D. Baumgartner, University of Graz, Kanzelhoehe Observatory; G. Schauberger, University of Veterinary Medicine, Institute of Physiology and Biophysics

Abstract: In 2012 the Austrian UVB-Network (www.uv-index.at) was enlarged by UVA-broadband instruments. Three out of 12 stations were equipped with detectors type J033 (CMS-Schreder, Austria) which responds to 310 nm to 400 nm (r50%=330 " 375 nm) and possess a cosine-like response. At two stations both global and diffuse (using a shadow-ring) UVA radiation is measured. The stations are located in Vienna (16.4°E, 48.3°N, 153 m), Innsbruck (11.4°E, 47.3N, 577m) and Mt. Gerlitzen (13.9°E, 46.7°N, 1526 m). The devices are cared according international recommendations for UV-Index measurements. Measurements are corrected by a calibration matrix in respect to solar elevation and total ozone column. Here we present the global and diffuse UVA measurements during a period of up to 4 years as well as a comparison of global and diffuse and a comparison to erythemally effective UV and to total global radiation. At Vienna (153 m asl) the daily global UVA dose reaches 12 kJ/m^2 in January and 70 kJ/m^2 in June and July while the corresponding values of the diffuse component are 9 kJ/m^2 and 40 kJ/m^2 respectively. The ratio between diffuse and global shows a clear annual cycle whereas in winter it is between 0.75 and 0.9 while in summer it can be within 0.4 and 0.8. The ratio between UV-Ery and UVA is highest in August reaching a value of 0.12 and lowest in January with 0.04. The ratio between UVA global and total global is relative constant during the year varying around 0.055 with a higher variability in winter which may be partly driven by the higher uncertainties due to low irradiances. At the alpine station Mt. Gerlitzen (1526 m) the daily global UVA dose reaches higher values than at Vienna. The ratio of daily global UVA doses from these two stations is not constant over the year but depends on season.

P2-8   PUVA pretreatment down-modulates key inflammatory cytokines and leads to reduced activation of the signal transducer and activator of transcription (STAT1/3) pathway in the imiquimod model of psoriasis NP Shirsath*, Research Unit of Photodermatology, Department of Dermatology and Venereology, Medical University of Graz, Graz, Austria ; J Ober, Center for Medical Research, Medical University of Graz, Graz, Austria; R Frei, Institute of Experimental and Clinical Pharmacology, Medical University of Graz, Graz, Austria; G Mayer, Research Unit of Photodermatology, Department of Dermatology and Venereology, Medical University of Graz, Graz, Austria; A Heinemann, Institute of Experimental and Clinical Pharmacology, Medical University of Graz, Graz, Austria; P Wolf, Research Unit of Photodermatology, Department of Dermatology and Venereology, Medical University of Graz, Graz, Austria

Abstract: Psoriasis is a chronic disease for which PUVA (psoralen+ultraviolet-A) is one of the most effective treatment options. Here by using the IMQ (Imiquimod) model we dissect the early cellular events and signals promoting psoriasis. We left mouse skin untreated (group1) or pretreated it with 0.25J/cm2 PUVA followed by IMQ at 48 and 72h (group2). In group3, IMQ was applied after a gap of 8 days post PUVA. PUVA pretreatment significantly reduced the macroscopic and microscopic psoriasiform skin changes and decreased influx of innate immune cells. Bone marrow neutrophiles showed less responsiveness towards CXCL1 in a migration assay. Moreover, significant downmodulation of key inflammatory cytokines was achieved with reduced expression of phospho STAT1/3 levels in skin sections (in group2) after PUVA treatment. However, this effect of PUVA was lost when a gap of 8 days was kept to apply IMQ, pointing to an indirect effect of PUVA modulating psoriatic manifestation.

P2-9   PhotochemCAD 3. Diverse Modules for Photophysical Calculations with Accompanying Spectral Databases M Taniguchi*, North Carolina State University ; H Du, North Carolina State University; JS Lindsey, North Carolina State University

Abstract: PhotochemCAD is a software program and accompanying spectral database for use in photochemistry and photobiology. The development of "PhotochemCAD" was motivated by the desire to have at one's fingertips spectral data (absorption spectra with molar absorption coefficients, emission spectra with quantum yields, references to the original photochemical literature) for a wide variety of representative compounds and the ability to perform photochemically relevant calculations using such spectral data. The program enables calculations (Förster energy transfer, oscillator strength, fluorescence lifetime, multicomponent analysis, blackbody radiator, transmission) and simulations (energy transfer, artificial spectrum creation based on Lorentzian and Gaussian distributions). PhotochemCAD 1 and 2 were released in 1998 and 2005, respectively. PhotochemCAD 3 contains the following revisions: (1) expanded spectral database, (2) multiple database handling capability, and (3) user interface enhancements. In addition to the original spectral database (~150 compounds), over 1000 new spectral data have been incorporated, which includes (i) ~400 commercial fluorophores (cyanine dyes, BODIPYs, Q-dot, Alexa Fluor, fluorescence proteins, etc.) and (ii) ~650 tetrapyrrole macrocycles (porphyrins, phthalocyanines, chlorins, bacteriochlorins). The spectral data in the PhotochemCAD database can be exported to other programs, and spectral data recorded by the user can be easily imported to the PhotochemCAD database. The availability of this program and database was anticipated to facilitate the design and analysis of a wide variety of photochemical systems (e.g., energy-transfer dyads, artificial light-harvesting architectures, molecular photonic devices, etc.). PhotochemCAD 3 will be available in due course for free downloading at http://www.photochemcad.com

P2-10   Investigating the Effects of Blue Light Irradiation on Retinal Pigment Epithelial (RPE) Cell Death in Relation to Age Related Macular Degeneration (AMD) JC Tournear*, NIU ; K Denius, NIU; ER Gaillard, NIU

Abstract: Age related macular degeneration (AMD) is a common retinal disorder found in the elderly and is the leading cause of blindness in the Western world. Cell death is a symptom of age related macular degeneration (AMD), but it is unclear the mechanism in which RPE cells undergo for this symptom to occur. A2E is a widely studied fluorophore that has long been correlated with AMD and when exposed to blue light acts as a photosensitive generator of singlet oxygen. This study aims to modify ARPE-derived extra cellular matrix (ECM) using A2E and blue light irradiated A2E in order to investigate the ARPE-19 cell death mechanism. Post modification, healthy ARPE-19 cells were seeded onto the ECM and allowed to attach for 30 min. Unattached cells are removed and fixed for further study. Attached cells were allowed to grow prior to viability analysis. After 24 hours of growth, both unattached and attached cells were stained with Annexin-V and PI then subjected to analysis using flow cytometry to investigate apoptosis versus necrosis as mechanisms of cell death. Blue light irradiated A2E modified ECM showed not only a loss in viability, but also a change in proliferation and cell morphology. When analyzing both the unattached and attached simultaneously, a further decrease in cell viability was observed suggesting difficulty attaching to modified ECM. Both necrotic and apoptotic cells were observed supporting the idea that cells have difficulty in both cell attachment and proliferation. This study showed implications for the symptom of AMD of cell death and can help to further understand the pathogenesis of the disease.

P2-11   Synthesis and characterization of long chain pterin derivatives: O vs N substitution M Vignoni, Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas (INIFTA) and UNLP-CONICET ; N Walalawela, Brooklyn College and Graduate Center of the City University of New York; AA Ghogare*, Brooklyn College and Graduate Center of the City University of New York; SM Bonesi, Centro de Investigaciones de Hidratos de Carbono (CIHIDECAR) and FCEN-CONICET; AH Thomas, Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas (INIFTA) and UNLP-CONICET; A Greer, Brooklyn College and Graduate Center of the City University of New York

Abstract: We describe a synthetic process for the N- and O-alkylation of 2-aminopteridin-4-(3H)-one (pterin) by nucleophilic substitution (SN2). A decane chain was introduced either at N or O of the amide leading to two new alkylated pterins. Dialkylation of both N and O amide pterin sites was not observed. However, two adducts from N-amine condensation of dimethylformamide (DMF) were obtained as by-products since DMF was used as solvent. The new pterin derivatives were characterized by NMR and mass spectrometry, and also absorption and emission spectra, fluorescence lifetime and singlet oxygen production were measured. The use of pH changes and comparison to the N-methylated pterin (2-amino-3-methylpteridin-4-(3H)-one), which exists in the keto form lacking the acidic NH, was key to assessing the alkylation at N and O positions in the new pterins. Unlike typical pterins, our new long chain pterins show lipophilic solubility, making them potential fluorescent probes in cell membranes.

P2-12   Alkyne photochemistry for double DNA-clevage and aldehyde release IV Alabugin*, Florida State University ; S Roy, Florida State University; B Breiner, Florida State University; WY Yang, Florida State University; R Mohamed, Florida State University; T De Faria, Florida State University

Abstract: We present two C1-C5 cycloaromatization reactions enabled by the high energy of alkyne functionality. The C1-C5 photocyclization of enediynes leads to four H-atom transfers from the environment and, thus, can be utilized for the design of efficient reagents for double-strand DNA photocleavage. The ratio of double- (ds) to single-strand (ss) DNA damage by hybrid molecules that combine enediynes with lysine and related basic peptides and amino acids exceeds the ds:ss ratios provided by the natural enediynes, the most potent family of anticancer agents in Nature. Furthermore, the reactivity towards DNA and the ds:ss ratio are increased dramatically at the lower pH of hypoxic cancer tissues. This increase is based on the new photophysical approach to selective DNA photodamage based on pH-gated photoinduced electron transfer between an appropriately positioned amino group and the DNA-damaging "warhead".[1] On the other hand, the newly discovered "self-terminating" C1-C5 photocyclization of enynes leads to photorelease of formaldehyde and related carbonyl compounds.[2] 1. Breiner, B.; Kaya, K.; Roy, S.; Yang, W.-Y.; Alabugin, I. V. Hybrids of Amino Acids and Acetylenic DNA-Photocleavers: Optimizing Efficiency and Selectivity for Cancer Phototherapy. Invited "Perspective". Org. Biomol. Chem. 2012, 10, 3974-3987. 2. The Missing C1-C5 Cycloaromatization Reaction: Triplet State Antiaromaticity Relief and Self-Terminating Photorelease of Formaldehyde for Synthesis of Fulvenes from Enynes. Mohamed, R. K.; Mondal, S.; Jorner, K.; Faria Delgado, T.; Ottosson, H. J. Am. Chem. Soc., 2015, 137, 15441"15450.

P2-13   Mechanisms of Photoisomerization of the Vitamin D3 isomers Pre-, Provitamin-D3 and Lumisterol in EPA Glass at 77 K Bayda M*, Florida State University, Tallahassee, FL; Present address: Faculty of Chemistry, Adam Mickiewicz University, Poznan, PL ; Redwood C, Florida State University, Tallahassee, FL; Gupta S, Florida State University, Tallahassee, FL; Dmitrenko O, University of Delaware; Saltiel J, Florida State University

Abstract: We recently measured fluorescence spectra in the course of the photoisomerizations of previtamin and provitamin D3 (Pre and Pro) to tachysterol (Tachy) at 77 K in EPA glass. Our analysis using singular value decomposition with self-modeling led to the conclusion that Pre exists as the s-cis,s-cis-conformer (cZc-Pre) which gives, exclusively, the unstable s-cis,s-cis-conformer of Tachy (cEc-Tachy) and Pro gives tEc-Tachy, the stable s-trans,s-cis-conformer, as the major photoproduct. The major Pre photoproduct from Pro is tZc-Pre and not the expected cZc-Pre. Accordingly, the Pre to Tachy cis-trans photoisomerization proceeds via a conformer specific one bond twist process as proposed by Havinga. We extended these studies to lumisterol (Lumi), whose structure differs substantially from that of its stereoisomer, Pro. Initially, the light induced ring openings of Pro and Lumi are expected to give (-)cZc(-)-Pre and (+)cZc(+)-Pre, respectively. In the case of Pro, most of the initially formed cZc-Pre proceeds to tZc-Pre, the precursor of tEc-Tachy. In contrast, our new results show that under the same conditions most cZc-Pre formed from Lumi retains its conformation and isomerizes to cEc-Tachy. We detected no (+)cZc(+)-Pre fluorescence. Its formation as an intermediate from the ring-opening of Lumi was established by UV absorption measurements.

P2-14   Metal Chelation Modulates Phototherapeutic Properties of Mitoxantrone-Loaded Porphyrin-Phospholipid Liposomes KA Carter*, University at Buffalo, State University of New York ; S Wang, University at Buffalo, State University of New York; J Geng, University at Buffalo, State University of New York; D Luo, University at Buffalo, State University of New York; S Shao, University at Buffalo, State University of New York; JF Lovell, University at Buffalo, State University of New York

Abstract: An ongoing goal of drug delivery systems is to deliver therapeutic payloads while reducing the total exposure of the drug to healthy tissues and organs. To this end, nanoparticles such as liposomes have shown promise as delivery vehicles and have achieved several examples of clinical translation. Liposomes incorporating porphyrin-phospholipid (PoP) can be formulated to release entrapped contents in response to nearinfrared (NIR) laser irradiation. Here, we examine effects of chelating copper or zinc into the PoP. Cu(II) and Zn(II) PoP liposomes, containing 10 molar % HPPH-lipid, exhibited unique photophysical properties and released entrapped cargo in response to NIR light. Cu-PoP liposomes exhibited minimal fluorescence and reduced production of reactive oxygen species upon irradiation. Zn-PoP liposomes retained fluorescence and singlet oxygen generation properties; however, they rapidly self-bleached under laser irradiation. Compared to the free base form, both Cu- and Zn-PoP liposomes exhibited reduced phototoxicity in mice. When loaded with mitoxantrone and administered intravenously at 5 mg/kg to mice bearing human pancreatic cancer xenografts, synergistic effects between the drug and the light treatment (for this particular dose and formulation) were realized with metallo-PoP liposomes. The drug-light-interval affected chemophototherapy efficacy and safety.

P2-15   Carbohydrate Coated Gold Nanoparticles as SERS probes for Confocal Raman Microscopy of Cancer Cells MC Gomes*, University of Aveiro ; A Cunha, University of Aveiro; JPC Tome, University of Lisbon; T Trindade, University of Aveiro; Zheng G, University Health Network Toronto, University of Toronto

Abstract: Amongst the wide variety of imaging techniques used in Biology, Confocal Raman Microscopy (CRM) has been used as one of the most relevant techniques considering its high sensitivity and non-destructive nature. This technique combines the Raman spectroscopy features, allowing the characterization of specimens in terms of their chemical composition, with the Confocal Microscopy. As a result, a mapping image can be acquired, assessing the distribution of a specific specimen inside a cell without the need for staining. Here, Gold Nanoparticles (AuNP) has earned an outstanding role being used as transducers for Surface-Enhanced Raman Scattering (SERS). Due to their easy surface functionalization, AuNP can be coated with a Raman reporter molecule and at the same time functionalized with several biomolecules, for increased selectivity. During the last years, AuNP-based SERS probes have been used to monitor several cellular responses to outside stimulus or even to detect cancer distribution in tissues. Intending to develop novel AuNP-based SERS probes with specificity to cancer cells, we synthetized a carbohydrate (glucose and galactose) coated 60 nm AuNP (AuNP@Glu and AuNP@Gal) and tested their ability as SERS probes using CRM. For this purpose, we used Rat 9Lluc glioma as cancer cells model and evaluated the AuNP surface nature in terms of uptake efficiency and as SERS probes for Bioimaging. The experimental uptake extent, unveiled that AuNP@Gal presented higher uptake over the glucose ones. This can be explained by the higher levels of galectin-1 observed by Western blot, on the 9Lluc glioma cells membrane, when compared with the GLUT-1 levels. However, due to the high sensibility of CRM and the surface enhancement signal provided by the gold, we were able to observe both AuNP SERS signal and mapping it inside the 9Lluc glioma cells. Moreover, it was possible to observe the AuNP distribution inside the cells, being notorious their absence in the nucleus. This work showed that carbohydrates coated AuNP can be used as SERS probes for cancer cells, allowing us to take advantages from a new type of microscopy, and opening new prospects for the use of these SERS probes for in vivo tumour identification. M. C. Gomes thanks FCT for the grant SFRH/BD/88334/2012. Thanks are due to FCT and FEDER, for funding the project PTDC/CTM/101538/2008, QOPNA (Pest-C/QUI/UI0062/2011), CICECO (Pest-C/CTM-LA0011/2011) and CESAM (PEst-C/MAR/LA0017/2011) research units. Thanks are also due to Terry Fox Research Institute, the Princess Margaret Cancer Foundation, the Canadian Institutes of Health Research, the Ontario Institute for Cancer Research, the Natural Sciences and Engineering Research Council of Canada, the Canada Foundation for Innovation.

P2-16   Keeping Tabs on Your Lab: Recognition and Detection of Data Manipulation HZ Hill*, Rutgers NJ Medical School

Abstract: Over the last 10 years, there has been a marked increase in retractions of articles in the scientific literature. Even more unsettling is the fact that well over half of these retractions are due to scientific misconduct. Scientists are increasingly becoming aware that not all data generated in their laboratories is reflective of the truth. In order to make certain that results have not been influenced by the experimenter(s), the wise chief can employ certain tests to ascertain that no tampering of results has occurred. Data alteration can fall into at least 3 categories: plagiarism, image manipulation and numerical fabrication. Commercial products are available to test for plagiarism and image alteration. An excel spreadsheet has been developed that allows for the easy testing of three types of number manipulation that might be encountered when counting cells in suspension or colonies. Examples will be shown to demonstrate image rearrangements and suspicious numbers. Ways to avoid these pitfalls will be discussed.

P2-17   Indomethacin-Derived Mitochondrial Reactive Oxygen Species Accelerated Cancer Specific Porphyrin Accumulation to Enhance Photodynamic Therapeutic Effect in Gastric Epithelial Cells H Ito, University of Tsukuba ; M Tamura, University of Tsukuba; Y Nagano, University of Tsukuba; H Matsui*, University of Tsukuba; HP Indo, Kagoshima University; HJ Majima, Kagoshima University

Abstract: Photodynamic therapy is useful for the treatment of cancer because it is minimally invasive for patients. Certain porphyrin compounds and their derivatives have been used as the photosensitizer because they accumulate specifically in cancerous tissues. However, the detailed mechanism of this phenomenon has not been clarified. We previously reported that a proton-coupled folate transporter, HCP1, transported porphyrins and that regulation of the protein was associated with cancer-specific reactive oxygen species from mitochondria (mitROS) (Hiyama K et al. J Porph Phtal. 2013; 17: 36-43). Therefore, over-generation of mitROS could increase HCP1 expression and the effect of photodynamic therapy. We investigated whether pretreatment with indomethacin influenced photodynamic therapy by using a rat normal gastric mucosal cell line, RGM1, its cancer-like mutated cell line, RGK1, and a manganese superoxide dismutase (MnSOD)-overexpressed RGK cell line, RGK-MnSOD. Indomethacin promotes the generation of cellular mitROS by inhibiting the electron transport chain, and MnSOD scavenges the mitROS. We elucidated that indomethacin enhanced cancer-specific mitROS generation and increased HCP1 expression. Furthermore, RGK1 cells showed higher cellular incorporation of hemato-porphyrin and better therapeutic effect with indomethacin treatment whereas RGK-MnSOD cells did not show a difference. Thus, we concluded that indomethacin improved the effect of photodynamic therapy by inducing increased mitROS generation in cancer cells.

P2-18   Selective Cell Targeting In Zebrafish Embryos RN Jones*, Loyola University Chicago ; S Erhard, Loyola University Chicago; A Gen, Loyola University Chicago; M Malham, Loyola University Chicago; KW Olsen, Loyola University Chicago; R Dale, Loyola University Chicago

Abstract: To attack cancer cells in a specific manner, we have developed a folate-targeted, photodynamic therapy (PDT) agent. The folate (FA) and the chlorin e6 (Ce6) photosensitizer were attached to bovine serum albumin (BSA) using carbodiimide chemistry. Since over 50% of cancers overexpress folate receptors, FA-BSA-Ce6 can be taken up by them and is an effective PDT agent. Cancer cells are not alone in expressing many folate receptors. Embryos need folate and have more folate receptors than adult cells. This suggested that FA-BSA-Ce6 could be used to study development in zebrafish. Zebrafish are good candidates for these studies because they share a high degree of sequence and functional homology with humans and their embryos and larvae are transparent making it easy to see the impact of treatment without invasive techniques. The folate receptor has ~50% amino acid identity between zebrafish and humans. To map the expression of the zebrafish folate receptor, we used reverse transcription-polymerase chain reaction (RT-PCR) on total RNA prepared from 9 embryonic stages. Expression was seen before the zebrafish maternal-zygotic transition, suggesting that folate receptor mRNA is maternally loaded. Whole-mount in situ hybridization was performed and all of the stages tested with the antisense probe demonstrated the presence of folate receptor mRNA. Stages through 1 day post fertilization (dpf) express the folate receptor globally. By 2 dpf, expression is seen in the head and heart of the embryo. To confirm the presence of the folate receptor protein, 3 dpf embryos were treated with florescent-tagged folate. Fluorescent-tagged folate embryos demonstrated florescence in the head and cloaca, which is where the mRNA was observed. Embryos at 4 dpf were exposed to 1, 2.5, 5 and 10 µM FA-BSA-Ce6 and then treated with 1 to 6 min of 660 nm light. The lower concentrations and light times allowed the embryos to recover while the higher ones resulted in non-viable embryos.

P2-19   Photodynamic Elimination of Melanoma and Nonmelanoma Skin Cancer Cells using the Endogenous Tryptophan Photoproduct 6-Formylindolo[3,2-b]carbazole (FICZ) R Justiniano*, University of Arizona, UA Cancer Center and College of Pharmacy ; SL Park, University of Arizona, UA Cancer Center and College of Pharmacy; JD Williams, University of Arizona, UA Cancer Center and College of Pharmacy; DR Wieland, University of Arizona, UA Cancer Center and College of Pharmacy; GT Wondrak, University of Arizona, UA Cancer Center and College of Pharmacy

Abstract: Recently, we have demonstrated that 6-formylindolo[3,2-b]carbazole (FICZ), a tryptophan-derived photoproduct and endogenous high affinity aryl hydrocarbon receptor (AhR) agonist generated in skin as a result of UV exposure, displays activity as a nanomolar UVA-photosensitizer. Here, we report that the endogenous photosensitizer FICZ can be used for the photodynamic elimination of human melanoma and nonmelanoma skin cancer (NMSC) cells. Employing a panel of cultured human squamous cell carcinoma (SCC-25) and melanoma skin cancer cells (A375, G361, LOX), photodynamic induction of apoptosis was achieved by the combined action of solar simulated UVA (6.6 J/cm2) and FICZ (10 nM), whereas exposure to the isolated action of UVA or FICZ did not impair cell viability. Comet analysis revealed the introduction of Fpg-sensitive oxidative base lesions following combined FICZ/UVA exposure, a genotoxic photodynamic effect observed also upon FICZ photoexcitation using a blue light source (LED 460 nm). Photooxidative effects were substantiated by detection of glutathione depletion, MitoSOX Red fluorescence microscopy, and upregulation of stress response gene expression (HMOX1, HSPA1A, HSPA6), all of which were antagonized by antioxidant cotreatment (NAC, sodium azide). In SKH1 mouse skin, an experimental photodynamic regimen (involving topical application of FICZ combined with UVA exposure) blocked the tumorigenic progression of UVB-induced mouse papillomas suggesting the preclinical utility of the endogenous tryptophan-derived photosensitizer FICZ for the photodynamic elimination of cutaneous malignant cells.

P2-20   BSA Based, Folate Directed Photodynamic Therapy Agent's Effectiveness In HeLa Cells KW Olsen*, Loyola University Chicago ; RN Jones, Loyola University Chicago; S Hira, Loyola University Chicago; K Kiernan, Loyola University Chicago; H Khaleeluddin, Loyola University Chicago; R Kooistra, Loyola University Chicago; K Sullivan, Loyola University Chicago; S Kanzok, Loyola University Chicago

Abstract: We have developed a novel photodynamic therapy (PDT) agent that specifically targets cancer cells. We used carbodiimide chemistry to attach folate (FA) and chlorin e6 (Ce6) to bovine serum albumin (BSA). A typical synthesis had 1 FA and 10 Ce6 per BSA. More than 50% of cancers overexpress the folate receptor. The folate containing conjugate can be taken up by the cell into the cytoplasm via receptor mediated endocytosis. Therefore, FA-BSA-Ce6 has double selectivity due folate-targeting plus limited-area light exposure. Ce6 has a large extinction coefficient of 59,000 M-1cm-1 at 660 nm (Lee et al., 2012). Both FA and Ce6 are hydrophobic. Covalently attaching them to BSA increased their solubility, which should allow more efficient delivery to tumors. A singlet oxygen sensor (p-nitroso-N,N'-dimethylaniline) was used to demonstrate reactive oxygen species production when illuminated at 660 nm. FA-BSA-Ce6 was specifically taken up into the cells via receptor mediated endocytosis. Hela cells exposed to FA-BSA-Ce6 in the dark showed no cytotoxicity. Upon exposure to light, the conjugate was dose (time and concentration) dependent. Concentrations less than or equal to 2 µM were not effective at killing Hela cells but concentrations of at 5 µM and above lead to 95% cell death with a 4 min exposure to 660 nm light.

P2-21   Selective Oxidations In Biological Samples By Atomic Oxygen JT Petroff, Saint Louis University ; SM Baumann, Saint Louis University; E Solomon, Washington University in St. Louis; JM Jez, Washington University in St. Louis; RD McCulla*, Saint Louis University

Abstract: Early investigations into the reactivity between atomic oxygen [O(3P)] and biomolecules revealed that O(3P) was more selective than other reactive oxygen species (ROS). The oxidation of biomolecules by endogenous or environmental ROS generates a myriad of different oxidation products that may have important roles in disease pathology and redox signaling in cells. The photodeoxygenation of various aromatic heterocyclic oxides, such as dibenzothiphene S-oxide (DBTO), has been suggested to generate ground state atomic oxygen [O(3P)] as an oxidant. Using DBTO derivatives as a source of O(3P), initial investigation revealed O(3P) can selectively oxidize biomolecules in complex environments. For example, O(3P) has been shown to selectively oxidize plasmalogens in low-density lipoprotein, quantitatively oxidize cysteine residues in proteins, and induce strand-scission in DNA. To achieve selective oxidations in complex mixtures of biomolecules, current work has focused on preparing DBTO derivatives whose properties allow the targeting of specific biomolecules for oxidation in a biological milieu. For example, the ability of lipophilic DBTO derivatives to affect the redox state of the RubisCO large subunit in Arabidopsis leaf extract was explored.

P2-23   Electronic excitation energy transfer in DNA and DNA-Ag cluster complexes. ZV Reveguk*, Saint-Petersburg State University ; EO Ikonnikov, Saint-Petersburg State University; AI Kononov, Saint-Petersburg State University

Abstract: We report on the electronic excitation transfer in single-stranded and double-stranded cytosine oligonucleotides and also in the complexes of DNA duplexes with Ag nanoclusters studied by steady-state and time-resolved femtosecond spectroscopy. Fluorescence up-conversion spectra of the cytosine oligonucleotides are discussed in the frame of electronic excitation transfer to the low-lying exciton states of stacked cytosines with reduced inter-base distance that have been predicted by QM calculations (R. R. Ramazanov, D. A. Maksimov, A. I. Kononov, J. Am. Chem. Soc. 2015, 137, 11656). Steady-state fluorescence excitation spectra of the DNA-Ag cluster complexes show that the energy is transferred from 10-30 DNA bases to Ag cluster. Fluorescence decay curves for the cytosine oligonucleotides and the DNA-Ag cluster complexes show that the processes of energy transfer occur within <100 fs. The obtained results are explained by coherent excitonic mechanism of the transfer. The observed efficient energy transport along DNA strand can affect the distribution of UV-induced lesions in DNA.

P2-24   Resistance of oral cancer cells to 5-aminolevulinic acid-mediated photodynamic therapy FCP Rosin, School of Dentistry, University of Sao Paulo ; L Corrêa*, School of Dentistry, University of Sao Paulo

Abstract: Oncologic photodynamic therapy (PDT) has been used as an adjuvant treatment for oral squamous cells carcinoma (OSCC). The aim of this study was to investigate whether OSCC (SCC9 cells) develop resistance after several cycles of 5-aminolevulinic acid-mediated PDT (5-ALA"PDT), and to verify whether the expression of markers associated with cell survival (NFB, Bcl-2, iNOS, mTOR, and Akt) is altered during this process. SCC9 cell line was subjected to one cycle of PDT (630 nm, 5.86 J/cm2, 150 mW, 150 s). Survival cells after this first PDT cycle were cultured again and submitted to more five cycles, increasing the radiation dose. Four resistant cell populations were obtained. The resistant populations showed significant increase on pNFB, iNOS, pmTOR, and pAkt. In conclusion, OSCC cells exhibited increased viability after five cycles of 5-ALA-PDT and overexpression of proteins associated with resistance. This fact must be carefully considered when initiating PDT for OSCC.

P2-25   Tumor-selective photodynamic therapy using copper-free click-conjugated liposomes ZS Silber*, Northeastern University ; G Obaid, Massachusetts General Hospital; J Kuriakose, Massachusetts General Hospital; M Broekgaarden, Massachusetts General Hospital; Y Wang, Massachusetts General Hospital; J Hui, University of Pennsylvania; A Tsourkas, University of Pennsylvania; T Hasan, Massachusetts General Hospital

Abstract: Photodynamic therapy (PDT) utilizes the photoirradiation of a photosensitizer (PS) to convert available oxygen into a cytotoxic reactive molecular species. Liposomal formulations of PSs retain their photoactivity and promote PS uptake in tumors through the enhanced permeability and retention effect. By conjugating tumor-targeting moieties at the surface of liposomal PSs, tumor cell selectivity is increased and off-target phototoxicity is reduced, allowing higher PDT doses to be tolerated. We have developed a liposomal platform that utilizes copper-free click chemistry to surface-conjugate antibodies modified with an azide moiety through two distinct methods. This reaction occurs bioorthogonally, without the need for toxic copper catalysts and yields no side products. The first method utilizes a Protein Z molecule that is site-specifically photocrosslinked to the Fc region of an anti-EGFR antibody, Cetuximab. The second method utilizes a stochastically conjugated azide linker that has no control over antibody orientation at the liposome surface. The site-specific formulation allows complete control over the antibody's positioning at the liposome surface without interfering with cellular antigen binding. The site-specific antibody-liposome formulation also has higher conjugation efficiency than the stochastic method. However, in vitro binding assays show that the stochastic antibody liposomes have a higher rate of binding to high-EGFR expressing cells. We have characterized the size and zeta potential of the antibody liposomes using dynamic light scattering following conjugation. We are currently examining the selective phototoxicity and pharmacokinetic behavior of the two conjugation methods in vitro and in vivo. The developed clicked antibody liposomal construct is a flexible and reproducible platform for targeted drug delivery, allowing a variety of targeting ligands to be attached and multiple hydrophobic and hydrophilic PDT agents to be used in combination.



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