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 inestigated. 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/cm2. 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/cm2). 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.Цель: изучить чувствительность к фотодинамическому воздействию, опосредованному 5-аминолевулиновой кислотой (АЛК), нормальных и малигнизированных лимфоцитов человека. Методы: объектом исследования служили лимфоциты, выделенные с помощью градиента плотности фиколл-верографина из крови здоровых доноров (6) и гематологических больных (20) с различными формами лимфолейкозов, а также трансформированные лимфоциты В-клеточных (Raji, Namalwa) и Т-клеточных (МТ-4, HUT-78) линий человека. Диагноз хронического лимфопролиферативного заболевания ставили на основании морфологического, цитохимического и иммуноцитохимического исследования периферической крови и клеток костного мозга; иммунофенотипирование проводили с использованием панели моноклональных антител к дифференцировочным антигенам Т-, В-лимфоцитов, NK-клеток и АВС-АР-метода. Трансформированные лимфоциты клеточных линий культивировали в стандартной RPMI-1640 среде с 10% эмбриональной телячьей сывороткой. Для фотодинамического воздействия клетки инкубировали с 1,0–5,0 мМ АЛК в течение 4 ч и облучали гелий-неоновым лазером (длина волны – 633 нм), варьируя дозу от 10 до 150 Дж/см2 . Количество погибших клеток после дополнительной 20-часовой инкубации определяли с помощью теста с трипановым синим. Результаты: показано, что нормальные и малигнизированные лимфоциты человека резко отличаются по чувствительности к АЛК-опосредованному фотодинамическому действию (АЛК-ФД). Значительная часть лимфоцитов крови больных лейкозом (в среднем 62,06 ± 4,03% у 10 пациентов с В-клеточным хроническим лимфолейкозом, В-ХЛЛ) погибала при минимальных параметрах воздействия (1 мМ АЛК, доза облучения 25 Дж/см2 ), тогда как даже наивысшая доза (5 мМ АЛК, 150 Дж/см2 ) не влияла на жизнеспособность нормальных лимфоцитов. В опытах in vitro в Т-клеточных линиях отмечали больший процент гибели клеток, чем в В-клеточных. При этом линия Raji отличается парадоксальным отсутствием зависимости количества погибших клеток от дозы воздействия, что, по-видимому, связано с известным для этой линии дефектом сигнальных путей апоптоза. Обсуждаются вероятные механизмы установленных отличий между нормальными и малигнизированными лимфоцитами в контексте альтерации процессов синтеза клетками гема при малигнизации. Выводы: 1) впервые показано, что малигнизированные лимфоциты крови больных лимфолейкозом проявляют высокую чувствительность к АЛК-ФД при фактическом отсутствии такой чувствительности у лимфоцитов здоровых доноров; 2) озлокачествленные лимфоциты Т-клеточных линий более чувствительны к АЛК-ФД по сравнению с В-клеточными; 3) для клеток линии Raji установлена аномальная зависимость доза/эффект; 4) на основе полученных результатов предложен простой и малотравматичный тест для скрининга пожилых людей на наличие В-ХЛЛ

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 μg/ml) and varied concentrations (0.5–5 μg/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

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

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

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.Цель: изучить циркадианные ритмы (ЦР) цитотоксической активности мононуклеарных клеток периферической кро- ви (МКПК) больных злокачественной меланомой по сравнению с таковыми здоровых доноров. Методы: активность НК-клеток и МКПК, выделенных из крови 5 больных со злокачественной меланомой I и II стадий и 12 здоровых доноров, оценивали при помощи радиоиммунного метода и NBT-теста. Результаты: установлено, что у онкологических больных нарушена нормальная циркадианная ритмичность активности НК-клеток и МКПК, причем степень выраженности таких нарушений возрастает параллельно с прогрессией заболевания. К наиболее типичным изменениям относятся дискоорди- нация между цитотоксическими ритмами НК-клеток и мононуклеарными фагоцитами (синхронизированными у здоровых доноров) и изменения основных параметров ритма: сдвиг фаз и смещения амплитуды. Выводы: у больных меланомой выяв- лены значительные изменения ЦР НК-клеток и МКПК, причем степень нарушения ритма зависит от стадии заболевания. Во всех изученных случаях выявлены изменения фаз и амплитуды ЦР, декоординация ритмов НК-клеток и МКПК. Ключевые слова: злокачественная меланома, циркадианный ритм, активность НК-клеток, цитотоксическая активность мононуклеарных клеток

Hyperflav — perspective photosensitizer for PDT: cell studies

Present studies investigated the effectiveness in vitro of Hyperflav application as a photosensitizer for photodynamic therapy

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

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. Key Words: photodynamic therapy, leukemia cells, multidrug resistance, apoptosis

Water soluble, multifunctional antibody-porphyrin gold nanoparticles for targeted photodynamic therapy

Photodynamic therapy (PDT) is a treatment of cancer by which tumour cells are destroyed using reactive oxygen species produced by photosensitizers following activation with visible or near infrared light. Successful PDT depends on the solubility and the targeting ability of the photosensitizers. In this work, the synthesis of a porphyrin-based water soluble nanoparticle conjugate containing a targeting agent that recognizes the erbB2 receptor overexpressed on the surface of particular cancer cells is reported. The nanoparticle conjugates were synthesized following two different protocols, viz. a biphasic and a monophasic method, with the aim to determine which method yielded the optimal nanosystem for potential PDT applications. The nanoparticles were characterized using UV–Vis absorption and fluorescence spectroscopies together with transmission electron microscopy and zeta potential measurements; and their ability to produce singlet oxygen following irradiation was investigated following the decay in absorption of a singlet oxygen probe. The nanoparticles synthesized using the monophasic method were shown to produce the highest amount of singlet oxygen and were further functionalized with anti-erbB2 antibody to target the erbB2 receptors expressed on the surface of SK-BR-3 human breast cancer cells. The water soluble, antibody-porphyrin nanoparticle conjugates were shown to elicit targeted PDT of the breast cancer cells

Photosensitiser-gold nanoparticle conjugates for photodynamic therapy of cancer

Gold nanoparticles (AuNPs) have been extensively studied within biomedicine due to their biocompatibil- ity and low toxicity. In particular, AuNPs have been widely used to deliver photosensitiser agents for photodynamic therapy (PDT) of cancer. Here we review the state-of-the-art for the functionalisation of the gold nanoparticle surface with both photosensitisers and targeting ligands for the active targeting of cancer cell surface receptors. From the initial use of the AuNPs as a simple carrier of the photosensitiser for PDT, the field has significantly advanced to include: the use of PEGylated modification to provide aqueous compatibility and stealth properties for in vivo use; gold metal-surface enhanced singlet oxygen generation; functionalisation of the AuNP surface with biological ligands to specifically target over- expressed receptors on the surface of cancer cells and; the creation of nanorods and nanostars to enable combined PDT and photothermal therapies. These versatile AuNPs have significantly enhanced the efficacy of traditional photosensitisers for both in vitro and in vivo cancer therapy. From this review it is apparent that AuNPs have an important future in the treatment of cancer