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Objective To optimize the formulation of a new kind of ultrasound contrast agents carrying the sensitizer of hematoporphyrin(HP)with[Poly(lactic-co-glycolic acid),PLGA]for material.Methods The technique of double emulsion was applied to produce HP loaded PLGA ultrasound microbubbles,which was optimized through orthogonal test using encapsulation efficiency for the detected index.Then morphology and distribution of HP-PLGA microbubbles were observed through light microscope and scaning electron microscope.The size,Zeta potential and the properties of releasing behavior and ultrasound imaging in vitro of Hp-PLGA contrast agents were detected.Results The optimization parameters were picked out as 10 mg/ml for concentration of HP,40 mg for PLGA,and 1/5 for volume ratio of water inside to dichloromethane.The optimized HP-PLGA contrast agents were spheric with the mean size of 602.3 nm,and Zeta potentiaI of-(17.1±1.6)mV.The drug loading and encapsulation efficiency of HP-PLGA were(2.15±0.15)%and(63.5±2.6)%,respectively.And the releasing behavior of HP-PLGA contrast agents in vitro was that after an obvious release of about 35.1%in former 24 h,there were 86.5%HP-PLGA released within 14 days.The ultrasound imaging of HP-PLGA could be enhanced obviously in vitro.Conclusions The self-made HP-PLGA ultrasound microbubble might be a useful tool for delivering sensitizer and thus provide a novel strategy for sonodynamic therapy on tumor.
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O surgimento da resistência antifúngica aos tratamentos convencionais tem proporcionado o desenvolvimento de novas modalidades terapêuticas para o tratamento da candidose bucal. Nesse contexto, a utilização da PDT vem sendo sugerida como método alternativo para a inativação de microrganismos patogênicos. Este estudo avaliou a efetividade da PDT na inativação de C. albicans e C. glabrata, ATCC e resistente a fluconazol, por meio da utilização do agente fotossensibilizador Photogem® e da iluminação com LEDs de comprimento de onda azul. Inicialmente, os microrganismos avaliados foram inoculados em tubos de ensaio contendo meio de cultura líquido e incubados overnight a 37ºC. Em seguida, foram obtidas suspensões celulares das espécies de Candida avaliadas. Essas suspensões foram transferidas para placas de 96 orifícios, tratadas com cinco diferentes concentrações de Photogem® (2,5; 5; 10; 25 e 50mg/L) e expostas a quatro doses de luz (10,5; 18; 25,5 e 37,5J/cm2). Suspensões adicionais foram tratadas somente com as cinco concentrações do fotossensibilizador ou apenas com as quatro doses de luz. Cada condição experimental foi realizada três vezes. Após a realização desses experimentos, foram obtidas diluições seriadas de cada amostra (10-1 a 10-3), e alíquotas de 25 µL dessas diluições foram plaqueadas, em triplicatas, em Sabouraud Dextrose Agar. Adicionalmente, alíquotas de 25 µL foram removidas das cavidades das placas de orifícios e transferidas diretamente para um quadrante da placa de Petri, sem a realização de diluição. As placas foram incubadas a 37ºC por 48 horas. Após a incubação, foi realizada a contagem das colônias viáveis (ufc/mL), e os valores obtidos foram analisados com o teste t de Student (p < 0,05). Os 16 resultados demonstraram que a inativação de Candida spp. ocorreu de forma concentração/dose dependente, que resultou na completa inativação desses microrganismos em determinadas condições experimentais. A dose de luz mínima que promoveu a completa inativação das duas origens de C. albicans foi 18 J/cm2, em associação com 50mg/L de Photogem® . Após a aplicação de 25,5 e 37,5 J/cm2, baixas concentrações de fotossensibilizador foram requeridas para a inativação total dessa espécie, sendo que diferenças estatisticamente significantes foram apontadas entre os valores obtidos para as duas origens de C. albicans. Também foram observadas diferenças estatisticamente significantes na obtenção da inativação total das duas origens de C. glabrata. Para a ATCC, não houve crescimento de colônias viáveis após o tratamento com 10, 25 e 50 mg/L de Photogem® seguido de iluminação a 37,5 J/cm2 . No entanto, somente as concentrações de 25 e 50 mg/L foram capazes de eliminar a C. glabrata resistente a fluconazol, nas doses de 25,5 e 37,5 J/cm2. Assim, a fotoativação do Photogem® pela luz do LED demonstrou efetividade na inativação das duas espécies de Candida avaliadas, ATCC e resistente a fluconazol
Oral candidosis is an opportunistic infection that affects a significant percentage of the population. The oral infection caused by Candida spp. is usually treated with topical and systemic antifungal drugs. However, the widespread use of these agents has resulted in an alarming increase in the rate of antifungal resistance. Recently, the photodynamic therapy (PDT) has been studied as an alternative modality of killing microorganisms, including viruses, fungi and bacteria. The aim of this study was to determine whether Candida albicans and C. glabrata, ATCC and fluconazole-resistant strains, could be photosensitized by Photogem® in combination with blue Light Emitted Diode (LED). Suspensions of each Candida strain, containing viable cells per milliliter, were treated with five concentrations of Photogem® (2.5, 5, 10, 25 and 50 mg/l), followed by LED irradiation in four light doses (10.5, 18, 25.5 and 37.5 J/cm2). Each experimental condition was carried out in triplicate and repeated tree times. From each sample, serial dilutions were obtained and aliquots of 25 µl of each dilution were plated on Sabouraud Dextrose Agar. All plates were incubated at 37°C for 48 hours. After incubation, colonies were counted (CFU/ml) and the data were statistically analyzed by the Student's t test (p < 0.05). The results demonstrated a concentration/dose-dependent pattern of inactivation, that resulted in complete elimination of all Candida evaluated. The minimal light dose for the complete inactivation of both C. albicans source was 18 J/cm2 , in conjunction with 50 mg/l of Photogem®. After 25.5 and 37.5 J/cm2 , a lower concentration of Photogem® was required to totally inactivate C. albicans ATCC (5 and 2.5 mg/l) in comparison with C. albicans fluconazole-resistant (10 and 5 mg/l). There were statistically significant differences in the log (CFU/ml) for the minimal light dose to complete inactivation of both C. glabrata sources. For C. glabrata ATCC, no viable cells were detected after treatment with 10, 25.5 or 50 mg/l of Photogem® followed by 37.5 J/cm2 , while the association of 25 mg/l with 25.5 J/cm2 was sufficient to totally inactivate the C. glabrata fluconazole-resistant. The photoactivation of Photogem® by blue LED light proved to be effective for the inactivation of fluconazole-resistant and ATCC strains of C. albicans and C. glabrata
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Fluconazole , Candida albicans , Candida glabrata , Photochemotherapy , Hematoporphyrins , Drug Resistance, Fungal , Data Interpretation, StatisticalABSTRACT
Objective To study the in vitro effects of sonodynamic management on human ovarian cancer cell line HRA. Methods Hematoporphyrin was selected as a sonosensitizer and ultrasound with certain intensity was used to activate hematoporphyrin.Then MTT assay and clony-forming efficiency assay were applied to determine the growth inhibitory effects of ovarian cancer cells. Electron microscope was applied to detect the morphological changes of the cells. Cell apoptosis was analyzed using flow cytometry. Results Hematoporphyrin had no significant inhibitory effects on the cells. However, when hematoporphyrin was used before ultrasound,it could enhance the killing effects of ultrasound(P
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AIM: To investigate the characterization of absorption of hematoporphyrin monomerthyl ether (HMME), a domestic new generation photosensitizer product, by activated T cells from human peripheral blood. METHODS: Evaluation was performed by flow cytometry on the effects of incubating concentration and time of HMME on absorption by activated T cells. Lymphocytes were separated from human peripheral blood by density gradient centrifugation with Ficoll and T cells were activated with polyclonal stimulators PHA and PDB+Ion. To analyze the effects of HMME incubating doses on the absorption of activated T cells, the cultural lymphocytes were incubated with a serial doses of HMME for 1 h and HMME absorption were measured by FACS after immuno-staining with anti-CD3 antibody. To test the impact of HMME incubating time on the absorption of activated T cells, the cultural lymphocytes were incubated with HMME for various times and HMME absorption were measured by FACS after immuno-staining with anti-CD3 antibody. RESULTS: The HMME absorption-dose curve and absorption-time curve were shifted to right and up in the activated T cells as compared to resting T cells. HMME absorptions of activated T cells were statistic significantly larger than that of resting T cells in the doses between 5 mg/L to 20 mg/L. HMME absorptions of either activated T cells or resting T cells underwent a gradual increase with the incubation-time in HMME at concentration of 10 mg/L. HMME absorptions of activated T cells were statistic significantly larger than that of resting T cells in the incubation-time between 15 to 60 min. CONCLUSION: The differences of HMME absorption between activated T cells and resting T cells depend on the incubation times and doses of HMME. HMME absorption of activated T cells are significantly larger than that of resting T cells in certain incubation-times and doses. These results suggest that incubation time and dose associated with HMME-PDT therapeutic windows will be created for selective deletion of activated T cells.
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The separation and analysis of hematoporphyrin photosensitizers PSD-001, photocarcinorin,photofrin II and other HPD preparations in comparison with authentic samples protoporphyrin (PP), 3-(8)-(1-hydroxyethyl) -8-(3-)-vinyldeuteroporphyrin ( HVD)and hematoporphyrin(HP) using thin layer chromatography(TLC) under different conditions are described here.It has been shown that there are 3 and 2 main spots in PSD-001 and photocarcinorin by common TLC, but 8-9 spots by high performance TLC.It has been found by comparison with authentic HP,HVD and PP that the amount of PP in PSD-001 or photocarcinorin is less than 3% and HVD,about 2.5% and 6.3% respectively,the HP content of photocarcinorin is much lower than other photosensitizers mentioned above and there is not any component near the origin spot.The Rf of one of the major components of photocarcinorin is 0.65.This component seems to be hardly found in other photosensitizers.These suggest that photocarcinorin is a new photosensitizing agent differing from HPD and photofrin II.
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The preparation,chemical composition and physicochemical properties of photocar-cinorin (PsD-007),a new drug of photolocalizaion and photochemotherapy for human malignancies,are reported here.It has been shown by the resul s of thin layer and high performance liquid chromatographic (TLC & HPLC) analyses that about 85% of its total weight are relatively hydrophobic porphyrins of unknown structure in which a fraction with the same retention time as .hematoporphyrin(Hp)monoacetate constituts over 60%.There are also 4.2% of Hp,8.6% of 3(8) hydroxycthy lS-(3)-vinyldcutero-porphyrin (HVD) and 0.6% of portoporphyrin (Pp) in this drug.The presence and amount of HVD in PsD-007 were determined by an authentic sample prepared in our laboratory.The changes of the composition of PsD-007 on exposure to different doses of argon laser (488+ 514.5nm) were detected by HPLC.The variations in Soret peak of UV absorption spectra of PsD-007 protected from light at a constant temperature were also determined
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Department of Pathological Anatomy of Second Military Medical College Photosensitive reaction of HPD and its cytotoxic action were studied, focussing on the spectral effect of the irradiating light. A human hepatocarcinoma cell line(SMMC-7721)with HPD was used. Killing effects on tumor cells after irradiation of light in three different wave-bands were observed, both qualitatively and quantitatively.Results were compared with those of Kinsey and Matsushima.