Chlorin e6 combined with albumin nanoparticles as a potential composite photosensitizer for photodynamic therapy of tumors.

AIM: To synthesize and to study for photodynamic activity a composite photosensitizer consisting of chlorin e6 and human serum albumin nanoparticles (HSA NPs). MATERIALS AND METHODS: Starting from sorption-purified HSA, the albumin nanoparticles with a different degree of lysine residues cross-linking (10; 20; 40, and 100%) were obtained by the coacervation method. The HSA NPs were used for synthesis of nanocomposites with chlorin e6 and fluorescein isothiocyanate (FITC)-labeled preparations. Malignant lymphocytes of the MT-4 (human T-cell leukemia) line and normal lymphocytes of healthy donors served as cell targets. For photodynamic treatment, a semiconductor laser was exploited as a light source, and cell viability was assessed by MTT or trypan blue dye exclusion tests. For cell imaging and HSA NPs visualization, the fluorescence microscopy and transmission electron microscopy were applied, respectively. C57Bl/6 mice were used in animal experiments. RESULTS: The absorption and fluorescence spectra of chlorin e6-HSA NPs composites were characterized, and by the electron microscopy investigation the size of NPs (nanospheres) was estimated: 100-120 nm. FITC-labeled albumin preparations allowed to establish that HSA NPs have much higher exposition and concentration dependent affinity to malignant cell surface than initial HSA. In experiments with MT-4 cells on PDT activity of chlorin e6-HSA NPs, the nanocomposite effectiveness elevated along with increasing percentage of cross-linked amino acid residues, and for the nanocomposite with 100% of albumin cross-linking it exceeded the activity of free chlorin e6. In contrast to malignant cells, the complexation of chlorin e6 with HSA NPs decreased its photodynamic effect on normal human lymphocytes. Intravenous introduction of the chlorin e6-HSA NPs composite to mice showed prolonged circulation of the nanocomposite in blood in comparison with free PS. CONCLUSION: Promising results obtained with chlorin e6-HSA NPs composites warrant conduction of full-fledged PDT studies in vivo using the nanocomposites as photosensitizers.

Photodynamic responsiveness of human leukemia Jurkat/A4 cells with multidrug resistant phenotype.

UNLABELLED: Photodynamic therapy (PDT) is considered as a possible alternative approach to overcoming multidrug resistance (MDR). Analysis of cross-resistance to PDT in cells with different MDR pathways and resistance levels seems to be advantageous for elucidating the general mechanisms of cancer cell resistance to various treatment modalities. AIM: The aim of the study was to clarify whether the Jurkat/A4 leukemia cells with MDR phenotype are cross-resistant to PDT. METHODS: Human T-cell acute lymphoblastic leukemia line Jurkat and Jurkat/A4 subline with MDR phenotype were used. 5-Aminolevulinic acid (ALA) and Photolon (a complex of chlorine-e6 and polyvinylpyrrolidone; PL) or gold nanocomposite of PL were applied as photosensitizers. The cells were pretreated with photosensitizers and exposed to laser radiation at corresponding wavelengths. The phototoxicity was assessed in trypan blue exclusion test. The hypodiploid cell fraction was analyzed by flow cytometry of propidium iodide-stained cells. Expression of genes related to PDT resistance was analyzed by microarray technique with Affymetrix U133A chips. RESULTS: ALA-mediated PDT resulted in dose-dependent cell death in both lines, the relative photodynamic efficacy in Jurkat/A4 cells being inferior to that in the parental Jurkat cells. There was no correlation between phototoxicity and apoptosis induction both in Jurkat and Jurkat/A4 cells. PL-mediated general phototoxicity in Jurkat cells amounted up to 75% at the maximal photosensitizer dose with about 40% of apoptotic death fraction. PL-phototoxicity in Jurkat/A4 cells was considerably lower. In contrast to Jurkat cells, PL-gold composite did not increase the efficacy of photosensitization as compared to free PL in Jurkat/A4 cells. CONCLUSIONS: Multidrug-resistant Jurkat/A4 cells exhibit reduced sensitivity to phototoxic effect in comparison with parental Jurkat cells independently of nature of the photosensitizer being assayed.

Chronobiological approaches to antiangiogenic photodynamic therapy of tumors: the first experimental evaluation.

UNLABELLED: In research of the last decade, rhythmic (circadian) variations of vascular endothelial growth factor (VEGF) production by tumors were discovered. The present paper authors have earlier synthesized and characterized a new derivative photosensitizer - an immunoconjugate of hematoporphyrin with antiVEGF antibodies. AIM: To elaborate and to test a novel modification of the photodynamic therapy of tumors (PDT) method, founding upon a timed introduction of the immunoconjugated photosensitizer to tumor-bearing animals, so that this coincides with a maximum content of VEGF in tumor tissues. METHODS: Circadian variations of VEGF contents in murine transplanted tumors, Lewis lung carcinoma and sarcoma 180, were determined by ELISA method. Immunoconjugated photosensitizer concentrations in tumors were estimated by spectrofluorometry. Photoirradiation of the tumors was carried out with a red light (wavelength of 635 nm) from a semiconductor laser. Light doses were chosen, calculating on a partial inhibition of tumor growth, in order that a dependence of PDT efficiency on a daily time-moment (circadian rhythm phase) of the treatment could be observed distinctly. RESULTS: Circadian variations of the VEGF levels in Lewis lung carcinoma and sarcoma 180 were demonstrated with the maximum at 14:00 h and the minimum at 02:00 h. Intra-abdominal introduction into tumor-bearing mice of the immunoconjugated photosensitizer resulted in a greater accumulation of the immunoconjugate in tumors at 14:00 h than at 02:00 h. Laser irradiation of carcinomas and sarcomas at 14:00 h or 02:00 h after introduction of the immunoconjugated photosensitizer to mice the day before at the same time points, induced a significantly enhanced inhibition of tumor growth in animals treated at day-time versus those treated at night-time. CONCLUSION: The obtained results justify further attempts to transfer principles of tumor chronochemotherapy onto photodynamic therapy.

Photodynamic activity of hematoporphyrin conjugates with gold nanoparticles: experiments in vitro.

AIM: To synthesize a conjugate of hematoporphyrin with gold nanoparticles, and to evaluate its photodynamic activity in experiments on cultures of transformed cells. METHODS: nanosized gold particles and nanocomposites synthesis methods, cell culture methods, photobiology methods, trypan blue dye exclusion test, chemiluminescence assay. RESULTS: Various hematoporphyrin-gold nanocomposites were obtained, which contained similar hematoporphyrin concentration (5 microg/ml) and varied concentrations (0.5-5 microg/ml) of gold nanoparticles with a diameter of 15 nm or 45 nm. It was established by chemiluminescence method that nanocomposites synthesized induce more efficiently the formation of photo-oxidative products than original photosensitizer. The experiments with transformed cell lines showed that photodynamic in vitro activity of synthesized hematoporphyrin-nanogold composites is much higher than that of the original photosensitizer. The better activity of the nanocomposites with gold particles of 45 nm vs such of 15 nm which was demonstrated in the experiments, can be apparently connected with the fact that bigger particles are able to transport more porphyrin molecules into malignant cells. CONCLUSION: The results obtained warrant the necessity of further studies with hematoporphyrin-gold nanocomposites in vivo on transplanted tumors of animals which have to define the real perspectives of the nanocomposites application in PDT.

Hyperflav - perspective photosensitizer for PDT: cell studies.

BACKGROUND: Application of hypericin (an alkaloid from Hypericum perforatum plants) as photodynamic agent may become the next successful step in photodynamic therapy of malignant tumors. Hyperflav - is a purified Hypericum extract designed for the purpose of photodynamic diagnosis. AIM: Present studies investigated the effectiveness in vitro of Hyperflav application as a photosensitizer for photodynamic therapy. METHODS: Hyperflav photodynamic activity was assessed in phototoxic cell tests on Jurkat, MT-4 and Namalwa leukemic cell lines. Spectroscopic measurements of Hyperflav solutions were performed. RESULTS: Hyperflav aqueous solubility was maintained in presence of polyvinylpyrrolidone with the most pronounced photodynamic activity at 1:5 (w/w) Hyperflav-PVP ratio. Hyperflav fluorescence spectrum in ethanol exhibits two main peaks around 597 and 647 nm, in accordance with the spectrum of pure hypericin. Fluorescence spectrum of aqueous solution exhibits peaks at 604 and 655 nm and indicates decreasing in fluorescence intensity. Hyperflav at drug dose range of 5 -25 µg/ml and light dose 15 J/cm(2) showed a dose-dependent cytotoxicity on tested cell cultures, while dark cytotoxicity was not observed. Light irradiation of cell samples preincubated with 15 µg/ml Hyperflav resulted in 69.9, 76.0 and 78.3% cell death of Jurkat, MT-4 and Namalwa cultures, respectively. Combined preparation of Hyperflav with gold nanoparticles showed low photocytotoxicity (24.2%) in comparison with Hyperflav alone (99.6%) on Namalwa cells. CONCLUSION: Hyperflav being solubilized in nontoxic aqueous media exhibits in vitro photodynamic activity at doses that do not have dark toxicity, and therefore it meets requirements as a perspective photosensitizer. Further studies, particularly in vivo, are warranted to fully evaluate photodynamic potential of Hyperflav.

Sensitivity of normal and malignant human lymphocytes to 5-aminolevulinic acid-mediated photodynamic damage.

AIM: To compare the sensitivity of normal and malignant human lymphocytes to 5-aminolevulinic acid (ALA) - mediated photodynamic damage. METHODS: Blood lymphocytes isolated by Ficoll-sodium metrizoate density gradient from healthy donors (6) and hematologic patients (20) with different forms of lympholeukemia, and also transformed lymphocytes of human B-cell (Raji, Namalwa) and T-cell (MT-4, HUT-78) lines were investigated. Diagnoses of chronic lymphoproliferative disorders were made on the grounds of morphological, cytochemical and immunocytochemical studies of peripheral blood and bone marrow cells, with immunophenotype determination by monoclonal antibodies to differentiation antigens of T, B lymphocytes and NK cells and immunocytochemical ABC-AP method. Cells of leukemic B- and T-cell lines were cultured in standard RPMI-1640 medium. For photodynamic treatment, the cells were incubated with ALA and then irradiated by a helium-neon laser (wavelength of 633 nm). The number of dead cells was determined in 20 h with trypan blue dye exclusion test. RESULTS: The striking difference in responsiveness to ALA-mediated photodynamic treatment (ALA-PDT) between normal lymphocytes and cells isolated from lymphatic leukemia patients was established. A bulk of leukemic cells (mean for 10 patients with B-CLL - 62.06 -/+ 4.03%) were destroyed under the lowest ALA-PDT doses tested: 1 mM ALA, irradiation dose of 25 J/cm(2). However, it was virtually impossible to attain any appreciable damage of lymphocytes from healthy donors even with the highest treatment doses (5 mM ALA, 150 J/cm(2)). High sensitivity to ALA-PDT of malignant lymphocytes was confirmed in experiments with human T- and B-cell leukemic cell lines, and in these experiments, an anomalous reaction to the treatment of Raji cells was also detected. The mechanisms of the difference between normal and malignant lymphocytes are discussed in terms of altered heme-synthesis processes in malignant cells. CONCLUSIONS: 1) It is shown for the first time that blood lymphocytes from lymphatic leukemia patients are highly sensitive to the damage with ALA-PDT while lymphocytes of normal donors are practically not damaged. 2) Transformed lymphocytes of human T-cell lines are more sensitive than lymphocytes of B-cell lines. 3) Lymphocytes of the Raji line display anomalous dose-effect dependence with ALA-PDT. 4) It is proposed to evaluate the drastic difference in ALA-PDT responsiveness of normal and malignant lymphocytes as a possible simple and low-traumatic test for B-CLL screening among the elderly people.

Circadian rhythms of cytotoxic activity in peripheral blood mononuclear cells of patients with malignant melanoma.

AIM: To study circadian rhythms (CR) of cytotoxic activity in peripheral blood mononuclear cells of patients with malignant melanoma were compared with those in healthy men. METHODS: The NK-cell and phagocyte cytotoxic activity in five patients with malignant melanoma stage I or II and 12 healthy donors has been assessed by radioimmune assay and NBT-test. RESULTS: The circadian rhythmicity in NK-cells and phagocyte activity in all cancer patients under study has been disrupted. The extent of such disruption tended to increase in patients with more advanced cancer. The most typical alterations were discoordination between the cytotoxicity rhythms of NK-cells and phagocytes (synchronized in healthy persons) and alterations in basic rhythm parameters: phase shifts and amplitude damping. CONCLUSION: In melanoma patients the significant alteration of CR in NK-cells and phagocytes cytotoxic activity was revealed. In spite of individual variations, the degree of the rhythm disruption basically depended on a disease stage. The alteration of CR phase and amplitude and discoordination between the rhythms of NK-cells and phagocyte were registered in all cases studied.

Effect of photodynamic therapy on tumor angiogenesis and metastasis in mice bearing Lewis lung carcinoma.

AIM: To investigate the influence of photodynamic therapy (PDT) on the tumor angiogenesis and metastasis. METHODS: The effect of PDT with a photosensitizer 5-aminolaevulinic acid (ALA) on tumor metastasis and angiogenesis was studied on the model of subcutaneously implanted Lewis lung carcinoma (3LL) in C57Bl/6 mice. VEGF level in blood serum of animals was evaluated by ELISA. RESULTS: It has been shown that application of ALA-PDT resulted in the lowered rate of metastatic spreading and decreased VEGF level in blood serum of 3LL-bearing mice, and morphologic alterations of vascular system in tumor tissue. CONCLUSION: The antitumor action of PDT using ALA as a photosensitizer is caused partly by antimetastatic and photodamaging effect mediated by vascularization disturbances in tumor tissue.

[Use of low-energy laser radiation in experimental and clinical oncology]. / Primenenie nizkoénergeticheskogo lazernogo izlucheniia v éksperimental'noi i klinicheskoi onkologii.

Experimental data concerning the action of low-energy (nondamaging) laser radiation on the malignant cells in vitro and on animal tumours are reviewed. It is shown that under certain conditions the helium-neon and helium-cadmium laser radiation inhibits the tumour cell growth. This inhibition may be mediated by the activation of the organism protection mechanisms. The encouraging results of the application of the low-energy laser radiation in the complex treatment of oncological patients are presented.

[Preservation of the circadian rhythm of human lymphocytes under in vitro conditions]. / Sokhranenie sutochnoi ritmiki u limfotsitov cheloveka v usloviiakh in vitro.

The proportions of E-rosette-forming cells on the 9th, 13th, 17th, 21st, 1st and 5th h were determined in the blood of healthy donors divided into 3 groups (4 men in each). I--the blood was taken at every specified time point and the rosette formation was tested immediately; II--the blood was taken at hour 9, stored at +20 degrees C throughout the experiment and used for the tests at the same time points; III--the blood taken at hour 9, the mononuclear cells were obtained and kept throughout the experiment for the determination of rosette formation at the same time points. In all the three groups similar circadian curves were obtained with a peak at 13th and a minimum at the 1st hour. It is believed that lymphocytes possess oscillator(s) of the activity of receptors that are used for cell subset identification.

[Experimental validation and the initial experience of the use of intravenous laser irradiation of the blood in oncology]. / Eksperimental'noe obosnovanie i pervyi opyt primeneniia vnutrivennogo lazernogo oblucheniia krovi v onkologii.

The treatment of some non-oncological and then oncological patients was undertaken on the basis of the results obtained by laser blood irradiation (a helium-neon laser, the wavelength of 0.633 micron) in the tumour-bearing mice. The method was applied to 25 patients with the cancer of cervix uteri after their radiotherapy and resulted in the stimulation of their hemopoietic and immunologic systems.

[Laser therapy of cervix dysplasia]. / Lazernaia terapiia displazii sheiki matki.

Irradiation from a continuous wave CO2-laser was used in the treatment of 1,234 cases of cervical epithelial dysplasia. Diagnosis was established on the findings of extended colposcopy, aiming biopsy and cytological examination of smears. Such advantages over diathermocoagulation as complication-free recovery and a low incidence of recurrences (2.6%) were shown. The end results were studied.

[Selective action of argon laser radiation in breast cancer]. / Selektivnoe deistvie izlucheniia argonovogo lazera pri rake molochnoi zhelezy.

The attempts were made to apply the argon laser radiation (the green region of light spectrum) for the treatment of breast carcinoma. The 46 post-operative skin metastases of breast carcinoma in 15 women were irradiated with no photosensitizers used. As a result of the treatment (4 to 50 sessions) the complete resolution of metastatic tumours was achieved in 13 patients. In contrast to the methods of laser coagulation treatment of tumours, used till now, the described approach provides for selective elimination of tumour cells without normal tissue alteration. The involvement of photothermic or photochemical mechanisms in this selective effect is discussed.