光动力作用对铜绿假单胞杆菌杀伤效应的研究

目的:探讨不同的光动力剂量下光动力疗法(photodynam ic therapy,PDT)对体外培养的铜绿假单胞杆菌的杀伤效应。方法:以耐药性较强的铜绿假单胞杆菌(Pseudom onas aeruginos,P.aeruginosa)为研究对象,采用亚甲基蓝(m ethylene b lue,MB)作为光敏剂,用656 nm的激光作为光源(m axoutput=300 mW),对不同系列浓度的MB进行不同剂量的光照,用菌落计数的办法来观测PDT对铜绿假单胞杆菌的杀伤作用;同时利用血培养基检测铜绿假单胞杆菌致病性的改变。结果:在光照剂量相同的情况下,浓度适中(131.7 m ol)的亚甲基蓝溶液能够有效地杀伤铜绿假单胞杆菌,使其致病性降低;而浓度较高(1 317 m ol)或较低(13.17 m ol)的亚甲基蓝溶液对铜绿假单胞杆菌的杀伤作用相对较弱。结论:光动力作用对体外培养的铜绿假单胞杆菌具有明确的杀伤作用,但是其效果和剂量关系密切,所以在治疗过程中必须寻找合适的光敏剂剂量。     

Methylene blue-mediated photodynamic therapy induces mitochondria-dependent apoptosis in HeLa cell

Methylene blue (MB), a widely studied reagent, is investigated in this work for its usage in photodynamic therapy (PDT). PDT has been proved to be highly effective in the treatment of different types of cancers. Previous studies showed MB has both high affinity for mitochondria and high photodynamic efficiency. To elucidate the effects of MB in PDT, we analyzed PDT-induced apoptosis in HeLa cells by introducing different doses of MB into the culture media. Our data showed that MB-mediated PDT triggered intense apoptotic cell death through a series of steps, beginning with photochemical generation of reactive oxygen species. The release of cytochrome c and activation of caspase-3 indicated that MB-PDT-mediated apoptosis in HeLa cells was executed by the mitochondria-dependent apoptotic pathway. Importantly, proteomic studies confirmed that expression levels of several mitochondrial proteins were altered in MB-PDT-induced apoptosis, including TRAP1, mitochondrial elongation factor Tu and peroxiredoxin 3 isoform b. Western blot data showed that phosphorylation of ERK1/2 and PKA were reduced in MB-PDT treated cells, indicating several signal molecules participating in this apoptotic cascade. Moreover, MB-PDT induced an increase in the strength of interaction between Bcl-xL and dephosphorylated Bad. This led to loss of the pro-survival function of Bcl-xL and resulted in mitochondria-mediated apoptosis. This study provides solid evidence of a strong induction by MB-PDT of a mitochondria-dependent apoptosis cascade in HeLa cells.     

Graphene oxide-methylene blue nanocomposite in photodynamic therapy of human breast cancer

The interaction of methylene blue (MB) as a photosensitizer with graphene oxide nano-sheets (GO) was examined in aqueous solution using UV-vis spectrophotometric techniques. MB–GO composites were prepared by mixing the solutions of GO nano-sheets and methylene blue due to interacting of the cationic methylene blue photosensitizer via electrostatic and π–π stacking or hydrophobic cooperative interactions. The cell killing potential of nanocomposite was examined on the MDA-MB-231 breast cancer cells in the absence and presence of red LED irradiation. The results demonstrated that the MB-GO nanocomposite has good performance in photodynamic therapy (PDT) during red LED irradiation. The cytotoxicity of nanocomposite caused reducing cell viability up to 20%. These effects would be due to the nano size structure of composite that could lead to effective cellular penetration. Also the significant difference has seen in lower concentrations of MB and MB-GO nanocomposite. The results show more than 40% increases in cell killing potential in lower concentrations of nanocomposite by using 2.5 μg/mL of each compound. The ratio of GO/MB can affect the interaction and higher ratios of graphene oxide (GO/MB > 1) can induce dimerization of MB. In lower concentrations and ratios of (GO/MB < 1) the free MB concentration increases and the electron shuttling effect of GO in photo activity decreases – which could affect the photocatalytic yield in PDT. The cell viability measurements confirm these effects on cancer cell killing potential of nanocomposite. According to microscopic and PDT assay results, the nanocomposite distribution and diffusion in cells enhanced the photochemical reaction yield in photodynamic therapy of MDA-MB-231 breast cancer cell line.     

Attaching NorA efflux pump inhibitors to methylene blue enhances antimicrobial photodynamic inactivation of Escherichia coli and Acinetobacter baumannii in vitro and in vivo

Resistance of bacteria to antibiotics is a public health concern worldwide due to the increasing failure of standard antibiotic therapies. Antimicrobial photodynamic inactivation (aPDI) is a promising non-antibiotic alternative for treating localized bacterial infections that uses non-toxic photosensitizers and harmless visible light to produce reactive oxygen species and kill microbes. Phenothiazinium photosensitizers like methylene blue (MB) and toluidine blue O are hydrophobic cations that are naturally expelled from bacterial cells by multidrug efflux pumps, which reduces their effectiveness. We recently reported the discovery of a NorA efflux pump inhibitor-methylene blue (EPI-MB) hybrid compound INF55-(Ac)en–MB that shows enhanced photodynamic inactivation of the Gram-positive bacterium methicillin-resistant Staphylococcus aureus (MRSA) relative to MB, both in vitro and in vivo. Here, we report the surprising observation that INF55-(Ac)en–MB and two related hybrids bearing the NorA efflux pump inhibitors INF55 and INF271 also show enhanced aPDI activity in vitro (relative to MB) against the Gram-negative bacteria Escherichia coli and Acinetobacter baumannii, despite neither species expressing the NorA pump. Two of the hybrids showed superior effects to MB in murine aPDI infection models. The findings motivate wider exploration of aPDI with EPI-MB hybrids against Gram-negative pathogens and more detailed studies into the molecular mechanisms underpinning their activity.     

Photodynamic inactivation of Lasiodiplodia theobromae: lighting the way towards an environmentally friendly phytosanitary treatment

The fungus Lasiodiplodia theobromae is one of the main causal agents of trunk canker and dieback of grapevine. The objective of this work was to evaluate the efficiency of photodynamic inactivation (PDI) of L. theobromae with synthetic and natural photosensitizers and irradiation with either sunlight or artificial photosynthetically active radiation. Although the growth of the mycelium could not be completely prevented with natural sunlight irradiation, phenothiazine dyes (methylene blue, MB; toluidine blue O, TBO), riboflavin and a cationic porphyrin (Tetra-Py⁺-Me) caused complete inhibition under continuous irradiation with artificial light. Free radicals were the main cytotoxic agents in the PDI with MB, indicating the predominance of the type I mechanism. PDI with MB or Tetra-Py⁺-Me may represent a promising approach for the sanitation of vine material in greenhouse nurseries, in order to reduce the risk of infection upon grafting.     

Photodynamic therapy for American cutaneous leishmaniasis: the efficacy of methylene blue in hamsters experimentally infected with Leishmania (Leishmania) amazonensis

The aim of this study was to investigate the effectiveness of Photodynamic Therapy (PDT) using Methylene Blue (MB) as the photosensitizing compound and a Light-Emitting Diode (LED) in American cutaneous leishmaniasis (ACL). Hamsters were experimentally infected with Leishmania (Leishmania) amazonensis. After the development of the lesions in the footpad, the animals were treated with MB three times a week for 3 months. Ten minutes after each application of MB, the lesions were irradiated with LED for 1 h. The lesions were evaluated weekly by the measurement of the hamster footpad thickness. At the end of the treatment the parasitic load was quantified in the regional lymph node of the hamsters. The treatment promoted a decrease in the thickness of infected footpad (P = 0.0001) and reduction in the parasitic load in the regional lymph node (P = 0.0007) of the animals from group treated with MB + LED. PDT using MB + LED in ACL caused by L. amazonensis shows a strong photodynamic effect. This therapy is very promising, once it is an inexpensive system and the own patient can apply it in their wound and in their house without the need of technical assistance.     

Induction of base-pair substitution and prameshift mutations in wild-type and repair-deficient strains of Salmonella typhimurium by the photodynamic action of methylene blue

Induction of back mutations to prototrophy by methylene blue (MB)-sensitized photodynamic (PD) treatment has been studied in wild-type and repair-deficient strains of Salmonella typhimurium carrying either the base-pair substitution mutation hisG46 or the frameshift mutation hisD3052. We found that reversion of the hisG46 mutation was increased in a strain carrying a uvrB deletion and decreased in a strain carrying a recA-type mutation. Reversion of the hisD3052 (frameshift) mutation, on the other hand, was decreased in both uvrB deletion and recA-type strains. The former results are consistent with the hypothesis that the majority of MB-sensitized PD-induced base-pair substitution mutations arise by a mechanism similar to that currently believed to be involved in UV mutagenesis. The latter results suggest that PD-induced frameshift mutations may arise in some other way, and two possible mechanisms involving sequential action of the excision repair and recombinational repair pathways are considered.     

Differential effects of N-TiO2 nanoparticle and its photo-activated form on autophagy and necroptosis in human melanoma A375 cells

The manipulation of autophagy provides a new opportunity for highly effective anticancer therapies. Recently, we showed that photodynamic therapy (PDT) with nitrogen-doped titanium dioxide (N-TiO₂) nanoparticles (NPs) could promote the reactive oxygen species (ROS)-dependent autophagy in leukemia cells. However, the differential autophagic effects of N-TiO₂ NPs in the dark and light conditions and the potential of N-TiO_2-based PDT for the treatment of melanoma cells remain unknown. Here we show that depending on the visible-light condition, the autophagic response of human melanoma A375 cells to N-TiO₂ NPs switches between two different statuses (ie., flux or blockade) with the opposite outcomes (ie., survival or death). Mechanistically, low doses of N-TiO₂ NPs (1-100 µg/ml) stimulate a nontoxic autophagy flux response in A375 cells, whereas their photo-activation leads to the impairment of the autophagosome-lysosome fusion, the blockade of autophagy flux and consequently the induction of RIPK1-mediated necroptosis via ROS production. These results confirm that photo-controllable autophagic effects of N-TiO₂ NPs can be utilized for the treatment of cancer, particularly melanoma.     

A non-aggregated silicon(IV) phthalocyanine-lactose conjugate for photodynamic therapy

To develop a highly efficient photosensitizer for photodynamic therapy (PDT), we have designed and synthesized a phthalocyanine-lactose conjugate (Pc-Lac) through axial modification of silicon(IV) phthalocyanine with lactose moieties. With the lactose substituents, Pc-Lac is highly hydrophilic and non-aggregated with efficient reactive oxygen species (ROS) generation in aqueous media. With these desirable properties, Pc-Lac shows high photocytotoxicity and cellular uptake toward HepG2 cells. In addition, in vivo fluorescence imaging shows that Pc-Lac could selectively remain at tumor site, leading to its enhanced photodynamic efficacy against H22 tumor-bearing mice. Therefore, Pc-Lac shows a great potential as a highly efficient molecular photosensitizer for PDT.     

Photodynamic and Antibiotic Therapy Impair the Pathogenesis of Enterococcus faecium in a Whole Animal Insect Model

Enterococcus faecium has emerged as one of the most important pathogens in healthcare-associated infections worldwide due to its intrinsic and acquired resistance to many antibiotics, including vancomycin. Antimicrobial photodynamic therapy (aPDT) is an alternative therapeutic platform that is currently under investigation for the control and treatment of infections. PDT is based on the use of photoactive dye molecules, widely known as photosensitizer (PS). PS, upon irradiation with visible light, produces reactive oxygen species that can destroy lipids and proteins causing cell death. We employed Galleria mellonella (the greater wax moth) caterpillar fatally infected with E. faecium to develop an invertebrate host model system that can be used to study the antimicrobial PDT (alone or combined with antibiotics). In the establishment of infection by E. faecium in G. mellonella, we found that the G. mellonella death rate was dependent on the number of bacterial cells injected into the insect hemocoel and all E. faecium strains tested were capable of infecting and killing G. mellonella. Antibiotic treatment with ampicillin, gentamicin or the combination of ampicillin and gentamicin prolonged caterpillar survival infected by E. faecium (P = 0.0003, P = 0.0001 and P = 0.0001, respectively). In the study of antimicrobial PDT, we verified that methylene blue (MB) injected into the insect followed by whole body illumination prolonged the caterpillar survival (P = 0.0192). Interestingly, combination therapy of larvae infected with vancomycin-resistant E. faecium, with antimicrobial PDT followed by vancomycin, significantly prolonged the survival of the caterpillars when compared to either antimicrobial PDT (P = 0.0095) or vancomycin treatment alone (P = 0.0025), suggesting that the aPDT made the vancomycin resistant E. faecium strain more susceptible to vancomycin action. In summary, G. mellonella provides an invertebrate model host to study the antimicrobial PDT and to explore combinatorial aPDT-based treatments.     

Photodynamic effect of light-emitting diode light on cell growth inhibition induced by methylene blue

The aim of this study was to propose the use of red light-emitting diode (LED) as an alternative light source for methylene blue (MB) photosensitizing effect in photodynamic therapy (PDT). Its effectiveness was tested against Staphylococcus aureus (ATCC 26923), Escherichia coli (ATCC 26922), Candida albicans (ATCC 90028) and Artemia salina. The maximum absorption of the LED lamps was at a wavelength of 663 nm, at intensities of 2,4,6 and 12 J.cm-2 for 10, 20, 30 and 60 min of exposure, respectively. Assays with and without LED exposure were carried out in plates containing MB at concentrations of 7 to 140.8 (micro) M for microorganisms and 13.35 to 668.5 (micro) M for microorganisms or microcrustaceans.The LED exposure induced more than 93.05%, 93.7% and 93.33% of growth inhibition for concentrations of 42.2 (micro)M for S.aureus (D-value=12.05 min) and 35.2 (micro)M for E.coli (D-value=11.51 min) and C.albicans (D-value=12.18 min), respectively after 20 min of exposure. LED exposure for 1 h increased the cytotoxic effect of MB against A.salina from 27% to 75%.Red LED is a promising light device for PDT that can effectively inhibit bacteria, yeast and microcrustacean growth.     

Synergic Effect of Photodynamic Therapy with Methylene Blue and Surfactants in the Inhibition of Candida albicans

Photodynamic therapy (PDT) has been originally developed for the treatment of cancer, but it has been successfully employed in the treatment of infectious diseases, including fungal infections. Surfactants are amphiphilic compounds that also have antifungal properties. The present work demonstrates the synergic effect of PDT with methylene blue (MB) and LED combined with four different surfactants in the killing of Candida albicans. Subinhibitory concentrations of CTAC, HPS, SDS and Triton X-100 were tested with MB PDT. The combined therapies proved to be more efficient than PDT or surfactants separately. The best results were obtained with CTAC and HPS and PDT with MB at the concentration of 32 μg/mL. In conclusion, the combination of surfactants and PDT is an alternative antifungal treatment that can achieve more effective performance with minimal discomfort to the patient.     

Liposomal temocene (m-THPPo) photodynamic treatment induces cell death by mitochondria-independent apoptosis

Background

The cell death pathway activated after photodynamic therapy (PDT) is controlled by a variety of parameters including the chemical structure of the photosensitizer, its subcellular localization, and the photodynamic damage induced. The present study aims to characterize a suitable m-THPPo liposomal formulation, to determine its subcellular localization in HeLa cells and to establish the cell death mechanisms that are activated after photodynamic treatments.

Methods

Liposomes containing m-THPPo were prepared from a mixture of DPPC and DMPG at a 9:1 molar ratio. In order to procure the best encapsulation efficiency, the m-THPPo/lipid molar ratio was considered. HeLa cells were incubated with liposomal m-THPPo and the subcellular localization of m-THPPo was studied. Several assays such as TUNEL, annexin V/propidium iodide and Hoechst-33258 staining were performed after photodynamic treatments. The apoptotic initiation was assessed by cytochrome c and caspase-2 immunofluorescence.

Results

m-THPPo encapsulated in liposomes showed a decrease of the fluorescence and singlet oxygen quantum yields, compared to those of m-THPPo dissolved in tetrahydrofuran. Liposomal m-THPPo showed colocalization with LysoTracker® and it induced photoinactivation of HeLa cells by an apoptotic mechanism. In apoptotic cells no relocalization of cytochrome c could be detected, but caspase-2 was positive immediately after photosensitizing treatments.

Conclusions

Photodynamic treatment with liposomal m-THPPo leads to a significant percentage of apoptotic morphology of HeLa cells. The activation of caspase-2, without the relocalization of cytochrome c, indicates a mitochondrial-independent apoptotic mechanism.

General significance

These results provide a better understanding of the cell death mechanism induced after liposomal m-THPPo photodynamic treatment.     

A possible theranostic approach of chitosan-coated iron oxide nanoparticles against human colorectal carcinoma (HCT-116) cell line

Iron oxides have become increasingly popular for their use as a diagnostic and therapeutic tool in oncology. This study aimed to improve pharmacological valuable of Fe₃O₄, which may be use to diagnosis colorectal cancers (CRC). Here, we have developed chitosan (CS) coated Fe₃O₄ through a cost-effective procedure. First, we determined the characterization of OA-C-Fe₃O₄ by FTIR, UV–Vis spectra, and TEM. Then, we evaluated the photodynamic therapeutic (PDT) activity of OA-C-Fe₃O₄ in human colorectal carcinoma cell lines (HCT 116). Current results revealed that the light-induced enhanced reactive oxygen species (ROS) activity of the nanoparticles (NPs) and caused cell death via the activity of caspase 9/3. The in vitro magnetic resonance imaging (MRI) experiments in (HCT 116) and human embryonic kidney cells (HEK 293) illustrated that nanohybrid is an effective MRI contrasting agents for the diagnosis of colorectal cancer.     

Real-time molecular recognition between protein and photosensitizer of photodynamic therapy by quartz crystal microbalance sensor

Real-time investigation of molecular recognition between protein and the photosensitizer of photodynamic therapy (PDT) was carried out by a quartz crystal microbalance (QCM) sensor integrated into a flow injection analysis (FIA) system. The photosensitizer meso-tetrakis(4-hydroxyphenyl)porphyrin (p-THPP) was immobilized on the gold electrode of the QCM chip by combining the sol-gel and self-assembly methods. Such a rapid screen analysis of molecular recognition showed that the p-THPP-immobilized sensor exhibited sensitive and specific interaction only with hemoglobin (Hb). The kinetic rate constants (k_ass and k_diss) and the equilibrium association constant (K_A) for p-THPP-Hb interaction were calculated by linear regression. The sensing performance characteristics of the proposed sensor were investigated. The sensor showed excellent selectivity, reproducibility, and repeatability for the detection of Hb. A linear calibration plot was obtained over a range from 0.2 to 1.0 μM with a detection limit (signal/noise ratio = 3) of 0.15 μM. The response mechanism of the sensor is discussed in detail. Due to its low cost and simple manipulation, this QCM-FIA system was shown to be a highly effective method for the investigation of interaction between biomacromolecules and the PDT photosensitizer. It also provides a potential strategy for screening an efficient and less harmful photosensitizer for PDT application.     

Synthesis and biological evaluation of 173-dicarboxylethyl-pyropheophorbide-a amide derivatives for photodynamic therapy

Three novel 17³-dicarboxylethyl-pyropheophorbide-a amide derivatives as photosensitizers for photodynamic therapy (PDT) were synthesized from pyropheophorbide-a (Ppa). Their photophysical and photochemical properties, intracellular localization, photocytotoxicity in vitro and in vivo were investigated. All target compounds exhibited low cytotoxicity in the dark and remarkable photocytotoxicity against human esophageal cancer cells. Among them, 1a showed highest singlet oxygen quantum yield. Upon light activation, 1a exhibited significant photocytotoxicity. After PDT treatment, the growth of Eca-109 tumor in nude mice was significantly inhibited. Therefore, 1a is a powerful and promising antitumor photosensitizer for PDT.     

In vitro and in vivo matrix metalloproteinase expression after photodynamic therapy with a liposomal formulation of aminolevulinic acid and its methyl ester

Photodynamic therapy (PDT) is a well-known method for the treatment of malignant tumors, and its principles have been well established over the past 30 years. This therapy involves the application of a chemical called a photosensitizer and its subsequent excitation with light at the appropriate wavelength and energy. Topical photodynamic therapy with aminolevulinic acid (5-ALA) is an alternative therapy for many malignant processes, including nonmelanoma skin cancers such as basal-cell carcinoma (BCC). Our novel approach for this study was to use a liposomal formulation of 5-ALA and its methyl ester (commercially available as metvix) both in vitro and in vivo, and to check whether the liposome-entrapped precursors of photosensitizers can induce the expression of metalloproteinases (MMPs) in animal tumor cells and in other tissues from tumor-bearing rats and in selected cell lines in vitro. We also checked whether the application of tissue inhibitors of matrix metalloproteinases (TIMPs) has any effect on MMPs in the above-mentioned experimental models, and if they can cause complete inhibition of MMP expression. Immunohistochemical studies revealed that after the PDT, the intensity of expression of MMPs in healthy animals was very low and seen in single cells only. After the PDT in tumor-bearing rats, MMP-3 was expressed in the tumor cells with the highest intensity of staining in the tissues directly adjacent to the tumors, while MMP-2 and -9 were not found. In the control groups, there was no observed expression of MMPs. In vitro studies showed that MMP-3 was expressed in MCF-7 cells after PDT, but MMP-9 was not observed and MMP-2 was only seen in single cases. Our studies confirmed that the application of an MMP-3 inhibitor may block an induction of MMP-3 expression which had previously been initiated by PDT. The preliminary data obtained from cancer patients revealed that new precursors are effective in terms of PDT, and that using MMP inhibitors should be considered as a potential enhancing factor in clinical PDT.     

Embelin (2,5-dihydroxy-3-undecyl-p-benzoquinone): A bioactive molecule isolated from Embelia ribes as an effective photodynamic therapeutic candidate against tumor in vivo

The present study was carried out to assess the photosensitizing potential of embelin, the biologically active natural product isolated from Embelia ribes in photodynamic therapy (PDT) experiments in vivo. In vitro PDT clearly indicated that embelin recorded significant cytotoxicity in Ehrlich's Ascites Carcinoma (EAC) cells, which is superior to 5-aminolevulinic acid, a known photodynamic compound. For in vivo experiments solid tumor was induced using EAC cells in the male Swiss albino mice of groups I, II, III and IV. Group I served as the control (without solid tumor), group II served as tumor bearing mice without treatment and groups III and IV served as treatments. At the completion of 4 weeks of induction, the tumor bearing mice from group III and IV were given an intraperitoneal injection with embelin (12.5 mg/kg body weight). After 24 h, tumor area in the Group III and IV animals was exposed to visible light from a 1000 W halogen lamp. The mice from groups I to III were sacrificed 2 weeks after the PDT treatment and the marker enzymes (myeloperoxidase [MPO], β-d-glucuronidase, and rhodanese) were assayed and expression of Bcl-2 and Bax were analyzed in normal and tumor tissues. Animals from group IV were sacrificed after 90 days of PDT treatment and the above mentioned parameters were recorded. Reduction in tumor volume and reversal of biochemical markers to near normal levels were observed in the treated groups. This is the first report on PDT using a natural compound for solid tumor control in vivo. The uniqueness of the mode of treatment lies in the selective uptake of the nontoxic natural compound, embelin from the medicinal plant E. ribes used in Indian system of medicine, by the solid tumor cells and their selective destruction using PDT without affecting the neighboring normal cells, which is much advantageous over radiation therapy now frequently used.     

Lead Structures for Applications in Photodynamic Therapy. 6. Temoporfin Anti-Inflammatory Conjugates to Target the Tumor Microenvironment for In Vitro PDT

Due to the ongoing development of clinical photodynamic therapy (PDT), the search continues for optimized photosensitizers that can overcome some of the side effects associated with this type of treatment modality. The main protagonists being: post-treatment photosensitivity, due to only limited cellular selectivity and post-treatment tumor regrowth, due to the up-regulation of pro-inflammatory agents within the tumor microenvironment. A photosensitizer that could overcome one or both of these drawbacks would be highly attractive to those engaged in clinical PDT. Certain non-steroidal anti-inflammatory drugs (NSAIDs) when used in combination with PDT have shown to increase the cytotoxicity of the treatment modality by targeting the tumor microenvironment. Temoporfin (m-THPC), the gold standard chlorin-based photosensitizer (PS) since its discovery in the 1980’s, has successfully been conjugated to non-steroidal anti-inflammatory compounds, in an attempt to address the issue of post-treatment tumor regrowth. Using a modified Steglich esterification reaction, a library of “iPorphyrins” was successfully synthesized and evaluated for their PDT efficacy.     

Towards PDT with Genetically Encoded Photosensitizer KillerRed: A Comparison of Continuous and Pulsed Laser Regimens in an Animal Tumor Model

The strong phototoxicity of the red fluorescent protein KillerRed allows it to be considered as a potential genetically encoded photosensitizer for the photodynamic therapy (PDT) of cancer. The advantages of KillerRed over chemical photosensitizers are its expression in tumor cells transduced with the appropriate gene and direct killing of cells through precise damage to any desired cell compartment. The ability of KillerRed to affect cell division and to induce cell death has already been demonstrated in cancer cell lines in vitro and HeLa tumor xenografts in vivo. However, the further development of this approach for PDT requires optimization of the method of treatment. In this study we tested the continuous wave (593 nm) and pulsed laser (584 nm, 10 Hz, 18 ns) modes to achieve an antitumor effect. The research was implemented on CT26 subcutaneous mouse tumors expressing KillerRed in fusion with histone H2B. The results showed that the pulsed mode provided a higher rate of photobleaching of KillerRed without any temperature increase on the tumor surface. PDT with the continuous wave laser was ineffective against CT26 tumors in mice, whereas the pulsed laser induced pronounced histopathological changes and inhibition of tumor growth. Therefore, we selected an effective regimen for PDT when using the genetically encoded photosensitizer KillerRed and pulsed laser irradiation.