Mixed-metal Ru(II)-Pt(II) supramolecular complexes having the [(Ph₂phen)₂Ru(BL)PtCl₂]₂+ (Ph₂phen = 4,7-diphenyl-1,10-phenanthroline, and BL (bridging ligand) = dpp = 2,3-bis(2-pyridyl)pyrazine, or dpq = 2,3-bis(2-pyridyl)quinoxaline) structural motif were synthesized and their redox, photophysical, and photochemical properties studied. Subsequently the application of the Ru(II)-Pt(II) bimetallic complexes in light activated DNA modification and cytotoxicity were evaluated. The supramolecular design entails covalently coupling an efficient Ru(II) chromophore for photodynamic therapy (PDT) activity through a polyazine bridging ligand (dpp or dpq) to a cis-PtCl₂ bioactive site for covalent binding to biological substrates. The bioactive site is comparable to the first generation Pt-based chemotherapy agent cisplatin, cis-[PtCl₂(NH₃)₂]. The Ph₂phen ligand is known in [Ru(Ph₂phen)₃]²+ to provide enhanced excited state lifetime and increase quantum efficiency for singlet oxygen generation in comparison to the phen analog (Φ₁₀₂ = 0.97, Ph₂phen and Φ₁₀₂ = 0.54, phen). The redox and photophysical properties were analyzed at each synthetic step providing systematic evaluation of the complex properties. The [(Ph₂phen₂2Ru(BL)PtCl₂](PF₆)₂ complexes display reversible RuII/III oxidations at +1.61 (dpp) and +1.63 (dpq) V vs. Ag/AgCl with an irreversible PtII/IV oxidation occurring prior at +1.51 V vs. Ag/AgCl. Four reversible ligand reductions occur at -0.45 (dpp0/-), -1.15 (dpp-/2-), -1.33 (Ph₂phen0/-), and -1.52 (Ph₂phen0/-) V vs. Ag/AgCl. For the [(Ph₂phen)₂Ru(dpq)PtCl₂](PF₆)₂ complex, the first two reductions shift to more positive potentials at -0.19 and -0.95 V vs. Ag/AgCl, while the TL reductions remain generally unperturbed. The electronic absorption spectroscopy for the [(Ph₂phen)₂Ru(dpq)PtCl₂](PF₆)₂, BL = dpp or dpq, complexes is dominated in the UV region by Ph₂phen (274 nm) and BL-based (310-320 nm) π⟶ π* transitions and in the visible region by metal-to-ligand charge transfer (MLCT) transitions at 424 nm (Ru(dπ)→ Ph₂phen(π*) 1CT) and 517 nm (Ru(dπ)→ dpp(π*) 1CT) or 600 nm (Ru(dπ)→ dpq(π*) 1CT). Steady-state and time-resolved emission spectroscopy shows that upon attaching Pt to the Ru monometallic precursor the λmaxem shifts from 664 nm for [(Ph₂phen)2Ru(dpp)](PF₆)₂ to 740 nm for [(Ph₂phen)₂Ru(dpp)PtCl₂](PF₆)₂ and the excited state lifetime is reduced from 820 ns to 44 ns in accordance with the energy gap law. The τ = 44 ns for the Ru(dπ)→ dpp(π*) 3CT excited state was somewhat unexpected upon TL variation given the lack of formal involvement of Ph₂phen in the emissive state. This likely results from the Ph₂phen contribution to the formally Ru(dπ) donor orbital. Although not typically done, given the complexity of the study the Φ₁₀₂ was quantified for the [(Ph₂phen)₂Ru(BL)PtC₂]Cl₂ (BL = dpp, Φ₁₀₂ = 0.07 or dpq, Φ₁₀₂ = 0.03) complexes supporting 1O2 generation via energy transfer from the 3MLCT excited state. The thermal and photochemical interactions of the [(Ph₂phen₂2Ru(BL)PtCl₂]Cl₂ (BL = dpp or dpq) supramolecular complexes were studied in the presence of DNA and U87MG cancer cells. Thermal binding at the cis-PtCl₂ BAS in the Ru(II)-Pt(II) architecture was compared to cisplatin displaying similar reduced migration through the gel attributed to covalent binding to DNA. DNA photocleavage studies provided evidence of efficient strand cleavage when excited at 455 nm likely enhanced by producing 1O2 locally at the DNA target. DNA photobinding by the [(Ph₂phen)₂Ru(dpp)PtCl₂]Cl₂ complex was observed utilizing low energy light where typical Pt(II) agents do not absorb. This is the first example of MLCT excitation of a Ru(II)-Pt(II) complex to induce a photobinding event. MLCT excitation enhances electron density on the dpp making the Pt(II) a weaker Lewis acid and promoting halide loss. In addition, this system is photoactivated with low energy red light in the therapeutic window. These studies validate the supramolecular design and show that coupling a Ru(II) chromophore for PDT activity and a cis- PtCl₂ binding moiety for covalent DNA targeting affords a complex applicable in photochemotherapies. Analysis of cytotoxicity in the dark for [(Ph₂phen)₂Ru(dpp)PtCl₂]Cl₂ and cisplatin afforded LC50 values of 100 μM, which are confirmed by previous reports for cisplatin and the currently used chemotherapy, TMZ in U87MG cells. Photolysis of the [(Ph₂phen)₂Ru(dpp)PtCl₂]Cl₂ resulted in substantial reduction in the observed LC50 values to approximately 5 μM. The enhanced cytotoxicity via excitation into the formally Ru(dπ)→ BL(π*) CT excited state of [(Ph₂phen)2Ru(dpp)PtCl2]Cl2 indicates that the bimetallic complex undergoes an efficient light activated mechanism of action. The Ru(II)-Pt(II) complex displays substantially lower LC50 values through PDT action than currently used clinical treatments with LC50 values of 100 μM. The [(Ph₂phen)₂Ru(BL)PtCl₂]₂+ (BL = dpp or dpq) mixed-metal supramolecules utilizing the Ph₂phen TL have displayed surprising results. The direct coupling of the cis-PtCl₂ moiety to the (Ph₂phen)₂Ru(BL) chromophore display dramatically enhanced photophysical properties, relative to the bpy and phen systems with a longer excited state lifetime and improved light activated interactions with DNA, which was not previously observed for directly coupled Ru(II)- Pt(II) systems. The Ph₂phen TL positively influence the bioactivity compared to the typical deactivation observed in the bpy and phen systems. Probing the [(Ph₂phen)₂Ru(BL)PtCl₂]₂+ (BL = dpp or dpq) biological interactions confirms the importance of coupling an efficient light absorbing and 1O2 generating PDT-type unit with a cis-PtCl2 DNA binding unit for applications in covalent DNA photomodification, DNA photocleavage, and photocytotoxicity. It is proposed that excitation using visible light into the formally Ru(dπ)→ BL(π*) CT excited state leads to enhanced electron density on the BL and weakened Lewis acidity at the Pt(II) center, which facilitates halide loss for efficient biological substrate modification. Upon coordination of the Ru(II)-Pt(II) complexes at the biological substrate, 1O2 is localized providing effective targeting of the highly reactive oxygen species. The visible light induced activity of the [(Ph₂phen)₂Ru(BL)PtCl₂]₂+ (BL = dpp or dpq) supramolecules suggests a new mode of action in relation to cisplatin, which was further supported by the enhanced photocytotoxicity observed in the presence of U87MG cells. The results indicate that the Ru(II)-Pt(II) supramolecular structural motif hold great promise as a future photochemotherapy agent. / Ph. D.

A series of mixed-metal bimetallic complexes [(TL)2M(dpp)RhCl2(TL)]3 (M = Ru and Os, terminal ligands (TL) = phen, Ph2phen, Me2phen and bpy, terminal ligands (TL) = phen, bpy and Me2bpy ), which couple one Ru or Os polyazine light absorber (LA) to a cis-RhIIICl2 center through a dpp bridging ligand (BL), were synthesized using a building block method. These are related to previously studied trimetallic systems [{(TL)2M(dpp)2RhCl2]5+, but the bimetallics are synthetically more complex to prepare due to the tendency of RhIII halide starting materials to react with diimine ligands to form cis-[Rh(NN)2Cl2]+ motifs. The bimetallic complexes, [(phen)2Ru(dpp)RhCl2(bpy)]3+, [(phen)2Ru(dpp)RhCl2(phen)]3+, [(Ph2phen)2Ru(dpp)RhCl2(phen)]3+, [(Me2phen)2Ru(dpp)RhCl2(phen)]3+, [(bpy)2Ru(dpp)RhCl2(bpy)]3+, [(bpy)2Ru(dpp)RhCl2(Me2bpy)]3+ and [(bpy)2Os(dpp)RhCl2(phen)]3+, were characterized and studied by electrochemistry, electronic absorption spectroscopy, ESI-mass spectrometry, steady-state and time-resolved emission spectroscopy. Ï¿" ï¿" The electrochemical properties of bimetallic complexes with polyazine ligands exhibit a reversible one-electron metal-based oxidation, a quasi-reversible RhIII/IICl2 overlapped with a small amount of RhII/ICl and an irreversible RhII/ICl2 �reductions prior to the reversible bridging ligand dpp0/- �reduction. ï¿" ï¿" The title bimetallic complexes are efficient light absorbers due to the [(TL)2MII(dpp)] light absorber subunit. The bimetallics display ligand-based ï¿"'ï¿"* transitions in the UV region and metal-to-ligand charge transfer (MLCT) transitions in the visible region of the spectrum with approximately half the absorption extinction coefficient values relative to the trimetallics in the spectrum. The Os,Rh bimetallic complex, [(bpy)2Os(dpp)RhCl2(phen)]3+, displays Os(dï¿")'dpp(ï¿"*) CT transition at 521 nm and a low energy absorption band at 750 nm in the near-infrared region representing direct 1GS'3MLCT excitation due to the high degree of spin orbital coupling in Os complexes. The bimetallic complexes [(phen)2Ru(dpp)RhCl2(bpy)]3+, [(phen)2Ru(dpp)RhCl2(phen)]3+, [(Ph2phen)2Ru(dpp)RhCl2(phen)]3+, [(Me2phen)2Ru(dpp)RhCl2(phen)]3+, [(bpy)2Ru(dpp)RhCl2(bpy)]3+ and [(bpy)2Ru(dpp)RhCl2(Me2bpy)]3+ display Ru(dï¿")'dpp(ï¿"*) MLCT transitions centered at 505, 508, 515, 516, 510 and 506 nm, respectively. The bimetallic complex [(Ph2phen)2Ru(dpp)RhCl2(phen)]3+ displays enhanced absorption. Ï¿" ï¿" The photophysical properties of Ru,Rh bimetallic complexes are close to those of trimetallic analogues. In room temperature acetonitrile, both bimetallic and trimetallic complexes display a weak and short-lived emission from the Ru(dï¿")'dpp(ï¿"*) 3MLCT excited state. For example, the bimetallic complex [(phen)2Ru(dpp)RhCl2(bpy)]3+ emits at 766 nm and the trimetallic complex [{(phen)2Ru(dpp)}2RhCl2]5+ emits at 760 nm. At 77 K in 4:1 ethanol/methanol glass, the bimetallics, as well as trimetallics, exhibit a more intense blue-shifted emission with a longer lifetime, which is from the same 3MLCT excited state. At 77 K, the low temperature emission from the same 3MLCT state of [{(phen)2Ru(dpp)}2RhCl2]5+ blue-shifts to 706 nm with the emission lifetime of 1.8 ms and the bimetallic [(phen)2Ru(dpp)RhCl2(bpy)]3+ emits at 706 nm (t = 1.8 ms). The Ru,Rh complexes 3MLCT excited states can populate Ru(dï¿")'Rh(ds*) triplet metal-to-metal charge transfer (3MMCT) excited states through intramolecular electron transfer at room temperature, which is impeded in the rigid matrice at 77 K due to the large reorganizational energy and restricted molecular motion. The emission of Os,Rh bimetallic complex [(bpy)2Os(dpp)RhCl2(phen)]3+ could not be detected by our instruments likely due to its expected red-shifted emission which lies outside our detector window. ï¿" ï¿" �The Ru,Rh bimetallics display interesting and efficient photo-reactivity with DNA activated by visible light. The DNA gel shift assay, selective precipitation, ESI-mass spectrometry and polymerase chain reaction (PCR) studies suggest that Ru,Rh bimetallic complexes photobind to DNA following visible light excitation. This reactivity is not observed for analogous Ru,Rh,Ru trimetallics due to the steric protection of the Rh site in that motif. The bimetallic [(TL)2Ru(dpp)RhCl2(TL)]3+ systems can photobind and photocleave DNA through low-lying 3MMCT excited states when excited by the low energy visible light, with or without molecular oxygen. This is unusual but desirable reactivity for photodynamic therapy (PDT) drug development. The Os,Rh bimetallic complex [(bpy)2Os(dpp)RhCl2(phen)]3+ photobinds and photocleaves DNA under red therapeutic light excitation without molecular oxygen, an unprecedented result. Polymerase chain reaction experiments were used to evaluate the impact on DNA amplification of the DNA photo-modification and photo-damage induced by [(bpy)2Os(dpp)RhCl2(phen)]3+ under red light irradiation. Either photobinding or photocleavage induced by red light excitation of [(bpy)2Os(dpp)RhCl2(phen)]3+ on DNA inhibits amplification via PCR methods, a model for in vivo replication. Moreover, significant thermal stability of DNA photo-modification over 90 "C is required for PCR. A red light-activated drug that acts in an oxygen-independent mechanism to impede DNA amplification is unique in this field and desirable for study as a new class of PDT drugs. / Ph. D.

Cancer, the second leading cause of death worldwide, is characterized by uncontrolled and abnormal cell growth. Even though researchers have made significant progress in its treatment over the past several decades, innovative therapeutic approaches that both improve patient survival and lessen the many debilitating side effects of conventional cancer treatments are vital. Accordingly, we first investigated the mechanism of interaction of a bimetallic complex, Ru(II)-Rh(III), with DNA. Non-covalent binding of Ru(II)-Rh(III) is strong and involves electrostatic and, potentially, groove binding interactions. Ru(II)-Rh(III) photobinds and photocleaves DNA through an O2-independent, metal-center mediated mechanism that could be beneficial in hypoxic tumors. Furthermore, the extent of covalent binding and cleavage of DNA, which inhibit PCR amplification, is dependent upon the strength of the non-covalent interactions. These results suggest that the toxicity of Ru(II)-Rh(III) could be selectively generated in tissues irradiated with light (e.g., a tumor). Secondly, we identified protein combinations selectively present in melanoma, which could be utilized in heteromultivalency. Heteromultivalent scaffolds display higher affinity towards cells that express a protein combination in comparison to those with only one of the proteins, which facilitates cell discrimination. Using an empirically-optimized threshold-based screening method and expression profiles of melanoma patients and normal tissues, we identified surface proteins and protein combinations that are selectively found in melanoma patients and not in normal tissues. After a preliminary validation process using the scientific literature, we used immunofluorescence to confirm differential expression of some of these combinations in established melanoma cell lines in comparison to immortalized keratinocytes controls. Finally, we investigated the resazurin assay, a method used for the evaluation of proliferation and cytotoxicity in more than 2,000 publications. We found that only ~14% of these utilized validated assay conditions, while ~40% failed to report essential analytical parameters needed for their replication. We evaluated assay parameters needed for accurate estimation of cell number in eight cell lines, and found that these are highly variable and independent of tissue type, growth kinetics, and energetic parameters. Furthermore, we obtained some insights into the biochemical reduction of resazurin and proposed minimum reporting standards, along with a sample protocol for assay validation. / PHD / Cancer, a group of diseases characterized by uncontrolled and abnormal cell growth, is the second-leading cause of death worldwide. Even though researchers have made significant progress in its treatment over the past several decades, innovative therapeutic approaches that both improve survival outcome and lessen the many debilitating side-effects of conventional cancer treatments are vital. First, we investigated the mechanism of interaction of a particular molecule, Ru(II)- Rh(III), with DNA. We found that Ru(II)-Rh(III) is strongly attracted to DNA due to its charge and an interaction with the indentations along its helix. Upon light activation only, Ru(II)-Rh(III) binds to and cleaves DNA without the need for molecular oxygen, which is scarce in tumors and can limit the activity of other drugs, and to an extent that is affected by the concentration of ions in the solution. Thus, the cytotoxic effect of Ru(II)-Rh(III) might be selectively activated in those tissues that are irradiated with light (e.g., a tumor). Secondly, we identified protein combinations selectively present in melanoma, which could be utilized in heteromultivalency. Heteromultivalent scaffolds bind strongly to cells that express a combination of proteins rather than one protein at a time, making them excellent candidates for delivering a payload in a selective manner. Using expression profiles of melanoma and normal tissues, we identified surface proteins and protein combinations that are selectively found in melanoma patients and not in normal tissues. After a preliminary validation process using the scientific literature, we used confirmed differences in the expression intensities of some of these combinations in melanoma cell lines in comparison to normal skin controls. Finally, we investigated the resazurin assay, a method used for the evaluation of cell growth and drug candidates in more than 2,000 publications. We found that only ~14% of these utilized validated assay conditions, while ~40% failed to report essential analytical parameters needed for their replication. We evaluated assay conditions for eight cell lines, and found that these are highly variable and independent of tissue type and some metabolic parameters. Furthermore, we obtained insights into the mechanism through which cells react with resazurin and proposed minimum reporting standards for publications, along with a protocol for assay validation.

Cyanine dyes that absorb light in the near infrared to far red region of the electromagnetic spectrum are desirable as photosensitizers for photodynamic cancer therapy. Light of wavelengths in this range is able to deeply penetrate tissue allowing for practical in vivo use of these dyes. A series of three structurally similar pentamethine cyanine dyes that absorb light ~800 nm to ~500 nm was tested to determine the effects of structural influences on the yields of supercoiled DNA photo-converted to nicked or linear forms. Possible mechanisms and optimal parameters for near- quantitative DNA photocleavage with a symmetrical quinoline pentamethine cyanine dye are discussed.

Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department: Department of Biophysics and Physical Chemistry Candidate: Pavel Cermak Supervisor: Assoc. Prof. Veronika Novakova, PhD. Title of Thesis: Preparation and photophysical evaluation of tetra-3,4- pyridoporphyrazines suitable for the photodynamic therapy Tetra-3,4-pyridoporphyrazines (TPyPz) are aza-analogues of phthalocyanines. Their large system of conjugated bonds enables them to absorb light in the red part of the absorption spectrum. Due to their ability to produce singlet oxygen, they can be potentially used as photosensitizers in photodynamic therapy (PDT). Its mechanism is based on co-functioning of three elements - photosensitizer, light and oxygen. Photosensitizer excited by light absorption transfers its energy into tissue oxygen, thus, creating cytotoxic singlet oxygen. This method is beneficial for its high selectivity, low toxicity, minimal invasion and fast effect. The aim of this work was to synthetize and study water-soluble TPyPz suitable for PDT. Water solubility was achieved by quarternized amines, forming of salts or using suitable delivery systems (hydrophilic emulsion). Hydrophilicity was also increased by introduction of hydrophilic non-charged substituents (OH). At first, appropriate precursors for...

Master of Science / Department of Anatomy & Physiology / Deryl L. Troyer / Breast cancer ranks second as a cause of cancer death in women in the USA. Detection of early tumors and tumor-targeted treatments could decrease the problems associated with breast cancer management. Photodynamic therapy (PDT) is a cancer treatment that uses a photosensitizer and a specific wavelength of light and is currently in clinical trials for breast cancer. When tumor cells which have absorbed photosensitizer are exposed to the correct wavelength of light, reactive oxygen species are generated, resulting in tumor cell death. Poor tissue penetration of light is a major limitation in PDT, restricting its use to treatment of localized tumors. Light generation at the tumor area might increase the effectiveness of PDT. Polymorphonuclear neutrophils (PMNs) are known to often infiltrate breast adenocarcinoma, and their activatation in tumor stroma produces luminescence in the presence of luminol. Here, we hypothesized that luminol can be used as a theranostic agent for luminescence-based early tumor detection (diagnosis) and in situ PDT (treatment). BALB/c mice were transplanted with 4T1 mammary adenocarcinoma cells to establish a breast adenocarcinoma model. The early tumor detection objective was tested by daily intraperitoneal injection of luminol and in vivo luminescence imaging. To test the PDT treatment objective,the photosensitizer 5-aminolevulinic acid (ALA) and luminol were administered to mice through intraperitoneal and intravenous routes, respectively. This treatment regimen was repeated six times and ALA alone/luminol alone/saline treated tumor-bearing mice were used as controls. Results demonstrated that luminol allowed detection of activated PMNs only two days after 4T1 cell transplantation, even though tumors were not yet palpable. Relative differences in the increase of tumor volume and final tumor weights were analyzed to test the in situ PDT. Analysis of the data showed luminol treatments resulted in breast adenocarcinoma tumor growth attenuation. In conclusion this study provides evidence that luminol can be a theranostic agent for breast adenocarcinoma.

La thérapie photodynamique est fondée sur l’activation sélective par la lumière de médicaments appelés photosensibilisateurs. Non toxique en l’absence d’excitation lumineuse, le photosensibilisateur est capable, une fois excité, de transférer son énergie pour former de l’oxygène singulet qui induit la mort de la cellule. L’utilisation d’une excitation biphotonique offre de nouvelles perspectives pour la thérapie photodynamique du cancer. En effet, la dépendance quadratique de l’absorption à deux photons (ADP) avec l’intensité du laser permet une très grande sélectivité spatiale, ce qui rend possible un traitement plus sélectif des tumeurs, et la lumière infra-rouge utilisée permet le traitement de tumeurs plus profondes. Dans ce travail, nous avons développé une famille de nano-objets tout organiques pour la thérapie photodynamique biphotonique. Ils présentent des sections efficaces d’ADP très élevées dans la gamme spectrale d’intérêt biologique et leur efficacité a été démontrée in vitro. / Photodynamic therapy is based on the selective activation by light of drugs called photosensitizers. Non-toxic in the absence of excitation light, the photosensitizer is able, upon excitation, to transfer energy to produce singlet oxygen, which induces cell death. Two-photon excitation offers new perspectives for photodynamic therapy of cancer. Indeed, the quadratic dependence of two-photon absorption (TPA) with the laser intensity allows high spatial selectivity, which enables a more selective treatment of tumors, and the use of infrared light allows treatment of deeper tumors. In this work, we have developed a family of fully organic nano-objects for two-photon photodynamic therapy. They exhibit very high TPA cross-sections in the biological spectral range of interest and their efficiency has been demonstrated in vitro.

Objetivo: Avaliar, in vivo, o efeito do tratamento endodôntico em sessão única utilizando a Terapia Fotodinâmica Antimicrobiana (aPDT) no reparo de lesões periapicais induzidas em dentes de cães, por meio da avaliação histopatológica e imunohistoquímica da angiogênese e de marcadores de formação óssea. O tratamento endodôntico em duas sessões com curativo de demora à base de hidróxido de cálcio (CH) foi utilizado como controle. Métodos: Lesões periapicais foram induzidas em 48 pré-molares superiores e inferiores de 6 cães, com 12 meses de idade. Após instrumentação dos canais radiculares, os dentes foram divididos, aleatoriamente, em 4 grupos: CH/120dias (d) e CH/180dias (d): canais radiculares preenchidos com curativo à base de CH; aPDT/120d e aPDT/180d: canais radiculares condicionados com fotossensibilizador à base de fenotiazina (10 mg/mL), por 1 minuto e irradiados com laser de diodo em toda a extensão dos canais, conforme as recomendações do fabricante. Em seguida, todos os canais radiculares foram obturados com cimento AH Plus e, após 120 ou 180 dias, os animais foram eutanasiados e os blocos contendo dentes e tecido ósseo foram submetidos ao processamento histotécnico e à coloração de hematoxilina e eosina (HE) para a análise descritiva da região periapical e mensuração das lesões periapicais, em microscopia convencional, e contagem de vasos sanguíneos sob luz convencional e no modo fluorescente. A análise imunohistoquímica foi realizada para avaliação dos marcadores da formação óssea osteopontina (OPN) e fosfatase alcalina (ALP). Os dados obtidos foram analisados estatisticamente utilizando os testes two-way ANOVA e qui-quadrado, com nível de significância de 5%. Resultados: Aos 120 dias, os dentes do grupo CH/120d apresentaram processo de reparo avançado, com ligamento periodontal apenas ligeiramente aumentado, presença abundante de fibras colágenas e escassas células inflamatórias. Os dentes do grupo aPDT/120d apresentaram o ligamento periodontal moderadamente aumentado e o infiltrado inflamatório era moderado. Poucas fibras colágenas foram observadas. Aos 180 dias, o mesmo padrão foi observado. As lesões periapicais nos grupos tratados com curativo à base de hidróxido de cálcio foram menores que as lesões nos grupos tratados com aPDT (p<0,001), e apresentaram maior número de vasos sanguíneos (p<0,0001), independentemente dos períodos de avaliação. Além disso, os dentes dos grupos tratados com pasta à base de hidróxido de cálcio apresentaram imunomarcação significativamente mais intensa para ALP e OPN (p<0,001), em ambos os períodos. Conclusões: Embora o tratamento com a aPDT tenha estimulado a angiogênese e a expressão dos marcadores da formação óssea, o tratamento endodôntico realizado em duas sessões empregando curativo à base de hidróxido de cálcio estimulou mais intensamente esses processos e promoveu melhor reparo das lesões periapicais / Aim: The aim of this study was to evaluate the in vivo effect of one-session endodontic treatment with antimicrobial photodynamic therapy (aPDT) in the repair of apical periodontitis, in dogs\' teeth, by histopathologic evaluation and imunohistochemistry for angiogenesis and bone formation markers. Two-session treatment with a calcium hydroxide (CH) dressing was used as control. Methods: Apical periodontitis were induced in 48 upper and lower premolars of six 12-monthold dogs. After root canals instrumentation, teeth were randomly divided into 4 groups: CH/120days (d) and CH/180days (d): root canals filled with CH-based dressing; aPDT/120d and aPDT/180d: root canals conditioned with phenothiazinebased photosensitizer (10 mg/mL) for 1 minute and irradiated with diode laser throughout the canals and according to the manufacturer\'s recommendations. Root canals were filled with AH Plus cement, and after 120 or 180 days, the animals were euthanized and teeth were submitted to histotechnical processing and HE staining for description of the periapical region and measurement of apical periodontitis in conventional microscopy and for counting blood vessels under conventional and fluorescent microscopy. Immunohistochemical analysis was performed to evaluate the bone formation markers osteopontin (OPN) and alkaline phosphatase (ALP). Data were statistically analyzed using two-way ANOVA and chi-square test with a significance level of 5%. Results: At 120 days, teeth in Group CH/120d presented the periodontal ligament only slightly enlarged with advanced repair and abundant collagen fibers. Inflammatory cells were scarce. Teeth in group aPDT/120d presented the periodontal ligament moderately enlarged and the inflammatory infiltrate was moderate. Few collagen fibers were observed. At 180 days, the same pattern was observed. Apical periodontitis in CH-treated groups were smaller than the lesions in aPDT-treated groups (p<0.001) and had a greater number of blood vessels (p <0.0001), regardless of evaluation periods. The teeth treated with calcium hydroxide showed significantly more intense immunostaining for ALP and OPN (p<0.001), in both periods. Conclusions: Although aPDT has stimulated angiogenesis and the expression of bone formation markers, the two-session endodontic treatment with a calcium hydroxide-based dressing stimulates them more intensely and promoted better apical periodontitis repair

O objetivo do presente estudo foi avaliar, in vitro, o efeito de diferentes tempos de pré-irradiação do fotossensibilizador na terapia fotodinâmica em biofilmes formados por Streptococcus mutans e Candida albicans, por meio da avaliação da carga microbiana. Os fatores em estudo foram: tempos de pré-irradiação do fotossensibilizador em 3 níveis (1, 2 ou 5 minutos). Para o controle do biofilme dentário cariogênico com aPDT foi utilizado o azul de metileno (0,01%) associado ao laser de diodo (&lambda;=660 nm). O digluconato de clorexidina (CHX a 0,12%) e a solução salina foram utilizados como controle positivo e negativo, respectivamente. O delineamento do estudo foi realizado em blocos completos e casualizados, sendo a amostra composta por 15 culturas de biofilmes de S. mutans, divididas aleatoriamente em 5 grupos e 15 culturas de C. albicans, também divididas em 5 grupos. O experimento foi realizado em triplicata (n=3) e as variáveis de resposta foram obtidas por meio de análise quantitativa da viabilidade bacteriana, expressa em unidades formadoras de colônia (UFC) por mm2 da área do espécime. Os dados obtidos foram analisados com o auxílio do teste one-way ANOVA e pós-teste de Tukey. Todas as análises foram efetuadas por meio do programa Graph Pad Prism 4.0, com nível de significância de 5%. Para o grupo de S. mutans, apenas a solução salina apresentou diferença estatisticamente significante quando comparada aos demais tratamentos (p<0.05), ou seja, o tratamento com aPDT, independentemente do tempo de irradiação aplicado, foi semelhante ao tratamento com CHX e ambos foram mais eficazes na redução do biofilme cariogênico, em comparação à solução salina. Para o grupo de C. albicans não houve diferença estatística entre os grupos (p>0.05). Portanto, pode-se concluir que o tratamento com aPDT diminuiu o número de UFCs de S. Mutans de forma semelhante à CHX, independentemente do tempo de pré-irradiação aplicado. Não foi possível constatar nenhum efeito desta terapia e dos diferentes tempos de pré-irradiação sobre o biofilme de C. albicans. Desta forma, o tempo de pré-irradiação de 1 minuto pode ser utilizado com o objetivo de reduzir a carga microbiana de S. Mutans. / The aim of the present study was to evaluate, in vitro, the effect of different pre-irradiation times of the photosensitizer in photodynamic therapy in biofilms formed by on by Streptococcus mutans and Candida albicans, through the evaluation of the microbial load. The factors under study were: times of pre-irradiation of the photosensitizer in 3 levels (1, 2 or 5 minutes). For the control of the cariogenic dental biofilm with aPDT, methylene blue (0.01%) was used in association with the diode laser (&lambda;=660 nm). Chlorhexidine digluconate (0.12% CHX) and saline were used as positive and negative controls, respectively. The study design was carried out in complete and randomized blocks. The sample consisted of 15 S. mutans biofilms cultures, randomly divided into 5 groups and 15 C. albicans cultures, also divided into 5 groups. The experiment was performed in triplicate (n = 3) and the response variables were obtained through quantitative analysis of bacterial viability, expressed in colony forming units (CFU) per mm2 of the specimen area. The data were analyzed with the aid of the ANOVA one-way test and Tukey\'s post-test. All analyzes were performed using the Graph Pad Prism 4.0 program, with a significance level of 5%. For the S. mutans group, only the saline solution presented a statistically significant difference when compared to the other treatments (p <0.05), that is, the treatment with aPDT, irrespective of the irradiation time applied, was similar to the treatment with CHX and both were more effective in reducing cariogenic biofilm compared to saline. For the group of C. albicans there was no statistical difference between the groups (p> 0.05). Therefore, it can be concluded that the treatment with aPDT reduced the number of CFUs of S. Mutans in a similar way to CHX, independently of the pre-irradiation time applied. No effect of this therapy or of the different pre-irradiation times on the C. albicans biofilm could be observed. In this way, the pre-irradiation time of 1 minute can be used to reduce the microbial load of S. mutans.

<p>I. Gallium-hemoglobin Coated Silver Nanoparticles for Antimicrobial Photodynamic Therapy Against Bacterial Pathogens One of the mechanisms for bacterial pathogens’ hemin acquisition is through cell-surface hemin receptors (CSHRs), which are responsible for rapid hemin recognition. GaPpIX, as a hemin analog, can be rapidly taken up by CSHR-expressing bacteria, such as <i>Staphylococcus aureus</i> (<i>S.aureus)</i>. Previous works shown that GaPpIX has aPDI activity at micromolar level of concentration following 10 seconds of 405-nm light exposure using LED array. The photosensitizing ability of GaPpIX can be further enhanced by incorporating with hemoglobin (GaHb) and 10 nm silver nanoparticles (AgNP). The results suggested a higher aPDI activity of GaHb-AgNP than any of its components against MRSA strains and neglectable cytotoxicity against keratinocytes. GaHb-AgNPs were also found having aPDI activity against intracellular MRSA and <i>Mycobacterium abscessus </i>but not effective against <i>S. aureus</i> biofilm. GaHb-AgNPs have no significant toxicity toward macrophages with concentrations lower than 22.64 μg/mL.</p> <p> </p> <p>II. Synthesis and Properties of Magnetic Gold Nanoparticles</p> <p> Superparamagnetic gold nanoparticles support hybrid magnetic and plasmonic properties that can be exploited for a variety of applications. In this paper we present new insights on the synthesis of magnetic gold nanoparticles (MGNPs) with an emphasis on efficiency, scalability, and waste reduction, supported by a comprehensive analysis of their physical and materials properties. Aqueous suspensions of colloidal Fe₃O₄ are conditioned with 5-kDa polyethylene glycol and L-histidine to mediate the nucleation and growth of gold by a mild reducing agent. Isotropic MGNPs on the order of 100 nm can be synthesized using scalable reaction conditions with Au:Fe mole ratios as low as 1:2 and cleansed with generally regarded as safe (GRAS) chemicals for the removal of residual iron oxide. High-resolution energy-dispersive x-ray imaging of individual MGNCs revealed these to be ultrafine composites of gold and SPIO rather than core–shell structures. The attenuated total reflectance infrared (ATR-IR) spectroscopy and Raman spectroscopy indicated that the cleansing step does change the optical properties of the synthesized MGNPs. Magnetometry of MGNCs in bulk powder form confirmed their superparamagnetic nature, with bulk moments between 6 to 7 emu/g.</p>