RU2519936C2 - Method of photodynamic therapy of tumours - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method involves using a photosensitiser (PS) pre-detected in the tumour by its fluorescence that is followed by tumour irradiation by light emission at a wave length in the spectral range of PS peak absorption. The PS is presented by the genetically coded protein KillerRed by providing its tumour expression by gene insertion into the tumour cells. The tumour is exposed to the light emission at energy density of 180-270 J/cm² 3 times every second day or 7 times daily. The protein KillerRed is preferentially localised either in mytochondria and nuclei, or in nuclei.

EFFECT: method provides high-selectivity PS effect on the tumour, reduced toxic load on the intact organs and tissues, the absence of PS redistribution in the tumour, with no need for the constant radiation control.

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Description

The present invention relates to medicine, namely to Oncology, and can be used for photodynamic therapy of tumors. Despite the successes of modern medicine, cancer remains one of the main causes of death. Therefore, one of the main tasks of medicine is the development of effective methods of treating tumors.

One effective treatment for tumors is photodynamic therapy (PDT). The PDT method is based on the ability of photoactive compounds introduced into the tumor, photosensitizers, to generate reactive oxygen species under local exposure to optical radiation of a specific wavelength that cause the death of tumor cells (1). Traditionally, porphyrin-type chemical compounds are used as photosensitizers, which are introduced into the body systemically, where they accumulate in the tumor in view of its structural and functional features (2, 3).

Currently, PDT is successfully used both in the treatment of small superficial tumors of various localization, in particular cancer of the skin, bladder, bronchi, vulva, early stages of lung cancer, cervix, Barrett's esophagus, bile ducts, and interstitially in prostate cancer, head tumors brain, peritoneal carcinomatosis (1, 4-6).

The antitumor effect of PDT is based on three mechanisms (7):

- direct phototoxic damage to tumor cells;

- damage to blood vessels;

- activation of a non-specific immune response.

The relative contribution of each of them depends on many factors: the chemical nature of the photosensitizer, its localization in the tumor, the degree of vascularization and the content of macrophages in the tumor, the time from administration of the photosensitizer to irradiation. The predominance of the cellular mechanism should be expected with a high content of the photosensitizer in the tumor cells with a long period between injection and irradiation. The subcellular localization of the photosensitizer largely determines the pathway of cell death (apoptosis or necrosis) and depends mainly on its physicochemical properties. It is known that photosensitizers localized in mitochondria induce apoptotic cell death, in contrast to photosensitizers located in lysosomes or plasma membranes (8). The nucleus is considered to be the most sensitive target in the cell for reactive oxygen species; therefore, much attention is paid to the development of nuclear localization photosensitizers (9).

However, in practice, for many photosensitizers, the vascular mechanism is more characteristic: photodynamic reactions caused by the sensitizer located in the vessels of the tumor lead to vascular stasis, thrombosis, hemorrhage, hypoxia, and, as a consequence, the death of tumor cells (10).

It is characteristic that the distribution of the photosensitizer introduced into the body from the outside changes in the tumor over time, so that the same drug can act according to a different mechanism depending on the time after administration. In connection with the redistribution and, in addition, removal of the photosensitizer from the tumor, repeated irradiation in the case of standard PDT is problematic, and repeated administration of the drug will create difficulties with dosimetry.

The selectivity of the accumulation of chemically synthesized photosensitizers in the tumor is due to a number of its structural and functional features, such as increased permeability of blood vessels, decreased drainage function of the lymphatic system, lower pH, a large number of macrophages that are effective traps for hydrophobic drugs, an abnormal structure of the tumor stroma characterized by increased intercellular space and increased production of collagen that binds porphyrin s, a high level of expression of low density lipoprotein receptors (1, 4, 5). Nevertheless, the selectivity of the accumulation in the tumor of existing photosensitizers introduced exogenously into the body is quite low. According to experimental data, the concentration of drugs in the tumor exceeds the concentration in the surrounding normal tissues (skin, muscles) by only 2-3 times (11-12). The highest level of accumulation is observed in the organs of the reticuloendothelial system (liver, kidneys, spleen). This significantly reduces the effectiveness of treatment of other organs, requires the use of high doses of the drug, and increases the pharmacological burden on the body. A serious side effect when using photosensitizers associated with non-specific accumulation of the drug is their phototoxicity to the skin and eyes as a result of the occurrence of a photodynamic reaction under the influence of sunlight.

Thus, a method for the photodynamic therapy of patients with basal cell skin cancer is known (13). In this method, a derivative of the tetrapyrrole macrocycles of the chlorine series “Radachlorin” is used as a photosensitizer, the photosensitizer solution is administered intravenously 2-3 hours before irradiation, the tumor is irradiated with laser radiation with a wavelength of 662 nm at an energy density of 200 or 300 J / cm ² .

As a result, in 96% of patients, complete tumor regression was observed, in 4% - partial. However, as a result of PDT, all patients showed the appearance of edema in the irradiation zone, followed by whitening and cyanosis of the tumor and hyperemia of the surrounding soft tissues. Subsequently, dry necrosis and scab formed in the irradiated zone, rejected within 3-10 weeks.

A known method of photodynamic therapy of tumors localized on the thigh in mice is using aluminum phthalocyanine as a photosensitizer and 24 hours after intravenous administration of the photosensitizer, the tumor is irradiated with light from a xenon lamp at a wavelength of 650-700 nm at a radiation power of 200 mW / cm ² and energy density 400 J / cm ² (14).

Complete tumor regression is observed in 30% of animals with a Colo-26 tumor and in 75% of animals with an EMT-6 tumor. However, there is an accumulation of the photosensitizer in normal organs and tissues (liver, spleen, lungs, kidneys, skin, muscles). And as a result, there is a photodamage of muscle tissue around the tumor, which leads to a restriction of the mobility of 30% of the animals for at least 35 days after PDT, which is a serious side effect.

Also known is a method of photodynamic therapy of a tumor (M-1 sarcoma) in rats using the photosensitizer Photoditazine, the drug is administered intravenously and 1.5 hours after the injection, the tumor is irradiated with laser radiation with a wavelength of 661 nm at an energy density of 150, 300, and 600 J / cm ² (fifteen).

The main mechanism of tumor destruction in this method is direct coagulation necrosis caused by the destruction of tumor cells and ischemic necrosis resulting from damage to the blood vessels of the tumor. In this case, the antitumor effect of PDT is accompanied by a pronounced inflammatory reaction in the tumor tissue.

The closest to the proposed method for the combination of essential features and technical result is the known method of photodynamic therapy (PDT) of tumors described in the source (16), which the authors selected as a prototype. This method consists in the fact that 24 hours before the start of therapy, a solution of the photosensitizer, which is a mixture of sodium salts of sulfonated aluminum phthalocyanine ("Photosens") in the form of a liposomal dosage form (LLF), is intravenously injected into the body of an animal, a mouse with an inoculated Ehrlich tumor. once at a dose of 4 and 1 mg / kg, while the accumulation of the photosensitizer in the tumor is monitored by its fluorescence in vivo by the spectral-fluorescence method, and with the maximum accumulation of the photosensitizer in Pujol it is irradiated once at the wavelength of 633 nm laser radiation close to the absorption maximum of the photosensitizer, an energy density of 90 J / cm ² and the irradiation time of 10 min.

The effectiveness of PDT is assessed by inhibition of tumor growth (SRW), which is 80% for both doses.

However, this method has side effects such as:

- low accumulation of the photosensitizer in the tumor, which does not provide a high selectivity of its effect;

- high toxic load on normal vital organs and tissues (liver, lungs, kidneys, spleen);

- the gradual accumulation, redistribution of the photosensitizer in the tumor and its removal from the tumor over time requires constant dosimetric monitoring.

The objective of the invention is to develop an effective method for the photodynamic treatment of tumors, which provides high selectivity of the action of the photosensitizer on the tumor, with a low toxic load on normal vital organs and tissues and does not require constant dosimetric monitoring during therapy.

The problem is solved by the proposed method of photodynamic therapy of the tumor, including the use of a photosensitizer, a preliminary assessment of its presence in the tumor by its fluorescence, followed by irradiation of the tumor with optical radiation with a wavelength in the spectral range of the maximum absorption of the photosensitizer, according to the invention, a genetically encoded KillerRed protein is used as a photosensitizer, why ensure its expression in the tumor by embedding the corresponding gene in the tumor left cells, irradiating the tumor with optical radiation is carried out at an energy density of 180-270 J / cm ² 3 times every other day or 7 times daily.

Preferably, the localization of the KillerRed protein is mitochondrial and nuclear or nuclear.

The technical result of the proposed method is to increase the selectivity of the effect of the photosensitizer on the tumor, reduce the toxic load on normal organs and tissues, the lack of redistribution of photosensitizer in the tumor and the absence of the need for dosimetric control.

This technical result is achieved by the fact that the photosensitizer in the tumor is the genetically encoded KillerRed protein, for this it is expressed in the tumor by embedding the corresponding gene in the tumor cells, the tumor is irradiated with optical radiation at an energy density of 180-270 J / cm ² 3 times through day or 7 times daily.

The KillerRed protein is a red fluorescent protein, a dimer (17). The monomer of a protein molecule has a structure typical of proteins of the GFP family (green fluorescent protein) (18). The maximum absorption of the KillerRed protein lies at a wavelength of 585 nm (extinction coefficient 45000 M ^-1 cm ^-1 ), fluorescence at 610 nm (quantum yield 0.25) (19). KillerRed protein expression in tumor cells is achieved by embedding the corresponding gene in the cell genome. The KillerRed protein can be localized in a given cell compartment as a result of its fusion with another target protein. Irradiation of tumor cells expressing KillerRed protein in an in vitro culture with optical radiation leads to protein burnout and arrest of cell division or cell death (20).

It is preferable to take the genetically encoded KillerRed protein of mitochondrial and nuclear or nuclear localization, since localization of this protein in other cellular compartments (cytoplasm, cytoplasmic membrane, lysosomes, mitochondria) is not effective for photodynamic therapy of tumors, which is empirically proven by the authors of the presented application.

At a given radiation energy density (180-270 J / cm ² ), the KillerRed protein burns out in the tumor, which is manifested in a decrease in tumor fluorescence in the fluorescence image. Since protein burnout accompanies a photodynamic reaction, observation of this effect made it possible to evaluate the effectiveness of the selected therapeutic regimen. When implementing the proposed method, an increase in the temperature of the tumors is only ~ 1-2 ° C.

This technical result is due to the fact that the genetically encoded photosensitizer is a KillerRed protein, the gene of which is previously integrated into the tumor cells. The tumor produces this KillerRed protein by expression in the mitochondria and nuclei or nuclei of tumor cells.

This KillerRed protein has a pronounced phototoxic effect on animal tumors due to the effective localization of the protein in the cell, causing significant damage to tumor cells - in the mitochondria and nuclei or nuclei of cells. The photodynamic therapy of tumors by the proposed method leads to pronounced pathomorphological disorders in the structure of the tumor tissue: dystrophic changes in the form of vacuolization of the cytoplasm, nuclear edema, an increase in the size of the nucleus or its compression, rupture of the nuclear and cytoplasmic membranes, as well as activation of apoptosis in the cells. The phototoxic effects of the KillerRed protein in treated tumors are noted exclusively in tumor cells at the subcellular level. In this case, the genetically encoded photosensitizer acts precisely and specifically on those organelles of the cell in which it is expressed. This means that the antitumor effect of the genetically encoded photosensitizer is realized through the direct destruction of tumor cells without negative effects on healthy tissues surrounding the tumor and side effects in the form of phototoxicity to skin and mucous membranes, toxicity to vital organs and tissues.

At the same time, tumor cells stably express phototoxic protein in a given location during the growth of the neoplasm, and the localization of the protein does not change over time, so there is no need for constant dosimetric monitoring, which is crucial for the implementation of the treatment method.

The proposed method is as follows.

In an animal with a tumor no larger than 5 mm in diameter, grafted to it by subcutaneous transplantation of a suspension of tumor cells into which the corresponding KillerRed protein gene is preliminarily inserted and which express the KillerRed protein, then the presence of a photosensitizer - a genetically encoded KillerRed protein - in the tumor is assessed by its fluorescence, after which the tumor is irradiated with optical radiation, in particular a laser, LED or xenon lamp with a wavelength in the spectral range of maximum absorption of photosensitizer An ora with an energy density of 180-270 J / cm ² 3 times every other day or 7 times daily, while the localization of the genetically encoded KillerRed protein is mitochondrial and nuclear or nuclear.

The proposed method was carried out photodynamic therapy of 50 animals with an inoculated tumor (human cervical cancer). In 26 animals, the genetically encoded KillerRed protein was produced in mitochondria and nuclei (the first group), in 24 animals - in the nuclei of tumor cells (second group).

In the first group, tumor irradiation with optical radiation was carried out under the following conditions:

- energy density 180 J / cm ² , 3 times every other day (4 animals),

- energy density 180 J / cm ² , 7 times daily (4 animals),

- energy density 225 J / cm ² , 3 times every other day (4 animals),

- energy density 225 J / cm ² , 7 times daily (4 animals),

- energy density 270 J / cm ² , 3 times every other day (5 animals),

- energy density of 270 J / cm ² , 7 times daily (5 animals).

In the second group, tumor irradiation with optical radiation was carried out under the following conditions:

- energy density 180 J / cm ² , 3 times every other day (4 animals),

- energy density 180 J / cm ² , 7 times daily (4 animals),

- energy density 225 J / cm ² , 3 times every other day (4 animals),

- energy density 225 J / cm ² , 7 times daily (4 animals),

- energy density of 270 J / cm ² , 3 times every other day (4 animals),

- energy density of 270 J / cm ² , 7 times daily (4 animals).

Tumors were irradiated with optical radiation with a wavelength in the spectral range of the maximum absorption of the genetically encoded KillerRed protein (540-600 nm). These conditions are met, for example, by a solid-state laser module with diode pumping model MGL-III-593 (CNI, China) with a wavelength of 593 nm, a LED of the model BL-HP20EUYC (Betlux, China) with a wavelength of 590 nm, a xenon lamp is a white light source Thorlabs HPLS-30-04 with optical filters with a wavelength of 520-590 nm.

The presence of KillerRed protein in tumors and the change in its fluorescence intensity were monitored by in vivo surface (epiluminescent) fluorescence imaging. Fluorescence excitation is performed at a wavelength in the range of 570-590 nm, signal reception at a wavelength in the range of 610-730 nm. These conditions are satisfied, for example, by the IVIS-Spectrum setup (Caliper, USA) and the setup for fluorescence imaging, designed at the Institute of Applied Physics of the Russian Academy of Sciences (IAP RAS) (Nizhny Novgorod), described in source (21).

The result of the treatment of tumors are significant qualitative and quantitative changes in their histological structure. Marked degenerative changes in the tumor tissue are noted, such as destruction of the cytoplasmic and nuclear membranes, a decrease in the size of the nuclei or their enlargement, chromatin homogenization or chromatolysis, and pronounced cytoplasm vacuolization. The activation of apoptosis in cells is also noted. As a result of therapy, the proportion of dystrophically altered tumor cells increases from 9.9 to 68.6%, the proportion of cells with signs of apoptosis from 6.3 to 14% in the absence of side effects such as photodamage to surrounding normal tissues, toxic load on vital organs, and phototoxicity in relation to skin and mucous membranes and without the need for constant dosimetric monitoring of the photosensitizer in the tumor.

In addition, the proposed method was performed treatment of a control group of animals whose tumors did not contain KillerRed protein. Pathomorphological analysis did not reveal significant changes in the structure of these tumors.

Another control group was animals with an inoculated tumor, the cells of which expressed the KillerRed protein in mitochondria and nuclei or nuclei, but were not subjected to photodynamic therapy by the proposed method. Histological examination showed that HeLa non-irradiated with optical radiation and expressing KillerRed tumor tissue has a compact dense structure and consists of large polymorphic densely located cells. Tumors were poorly vascularized. The cells had large round or oval nuclei. A slightly basophilic cytoplasm was located around the nucleus in the form of a thin ring. Tumor cells formed complexes surrounded by a thin layer of connective tissue with small blood vessels. At the same time, the cell population in the tumors was heterogeneous. In particular, there was a small number of cells (9.9%) with swaculated cytoplasm. Some of the cells (6.3%) had signs of apoptosis, such as chromatin condensation, fragmentation of the nucleus and cytoplasm, and eosinophilia.

Examples of specific uses of the proposed method

Example 1

Animal - immunodeficient mouse, female, weighing 20 g with a tumor inoculated with human cervical cancer, the tumor was inoculated by transplantation of 2.5 million HeLa tumor cells into which the KillerRed protein gene was previously inserted, tumor cells expressed the genetically encoded KillerRed protein in mitochondria and nuclei , the tumor size was 3.2 mm (5 days after cell grafting), immediately before photodynamic therapy, a fluorescence image of the tumor was obtained in vivo using an IVIS-Spectrum setup (Caliper Life Sciences, USA) in epilumin mode stsentsii (excitation of fluorescence was made at a wavelength of 570 nm, taking the fluorescent signal at a wavelength of 620 ± 10 nm, exposure 5), then the tumor was irradiated with laser radiation at a wavelength of 593 nm with an energy density of 270 J / ^cm2, ray irradiation was performed daily, once a day for 7 days.

During the irradiation procedure, the animal was fixed for 4 limbs on a special stand.

Immediately after irradiation, a fluorescence image of the tumor was re-acquired in vivo. 24 hours after the last exposure, a histological examination of the tumor was performed. Analysis of tumor fluorescence from in vivo images showed that after laser irradiation, the intensity of the tumor fluorescence signal decreases by 27% compared to the initial value, which indicates the burnout of the KillerRed protein and the effectiveness of the exposure regimen used. Fluorescence images of the animal before and after irradiation are shown in figure 1 (A - before, B - after). The tumor in the image is indicated by a dotted line.

A pathological study revealed extensive morphological changes in the irradiated tumor expressing the KillerRed protein. The vast majority of tumor cells with pronounced vacuolization of the cytoplasm, sometimes up to the rupture of the cell membrane. The nuclei of cells enlarged due to edema, round or irregular in shape. Nuclei with a loss of integrity of the karyolemma, signs of homogenization of chromatin and chromatolysis are observed, or, conversely, hyperchromic decrease in size. In the tissue, an increased number of apoptotic cells was found with such characteristic features as chromatin condensation, fragmentation of nuclei and cytoplasm, and pronounced eosinophilia of the cytoplasm. The histological image of the tumor tissue after photo-dynamic therapy is presented in figure 2. Morphometric analysis of irradiated tissue revealed an increase in the proportion of dystrophically altered cells in tumors compared with unirradiated tissue from 9.9% to 68.6%. The proportion of cells with signs of apoptosis increased 6.3 to 14%.

Example 2

The animal is an immunodeficient mouse, female, weighing 18 g with a tumor inoculated with human cancer of the cervix uteri, a tumor of 3.7 mm in size (12 days after inoculation of the cells) is located subcutaneously in the thigh area, while the KillerRed protein gene and genetically encoded were previously inserted into the tumor cells KillerRed protein was expressed by tumor cells in mitochondria and nuclei.

Example 2 was carried out as in example 1. In this case, irradiation of the tumor with laser radiation was performed 3 times every other day. The diameter of the light spot was 8 mm.

Histological examination of the tumor, conducted after photodynamic therapy, showed the presence of dystrophic changes and apoptosis in the cells. The histological picture of the irradiated tumor tissue and the nature of the pathomorphological changes were similar to those described in Example 1. Most of the cells had vacuolated cytoplasm and destroyed cytoplasmic membranes. The nuclei had a round or irregular shape, were swollen, with tearing karyolemma. Counting the proportion of dystrophic and apoptotic cells showed an increase in this indicator to 62.3% and 12.1%, respectively.

Example 3

Animal - immunodeficient mouse, female, weighing 19 g with a tumor inoculated with human cervical cancer. A 4.5 mm tumor was located subcutaneously in the thigh area. The KillerRed protein gene was previously integrated into the tumor cells; the genetically encoded KillerRed protein was expressed by tumor cells in the mitochondria and nuclei. Registration of fluorescence images of the tumor was carried out immediately before irradiation and immediately after irradiation. To obtain fluorescence images, a setup constructed at the IAP RAS (Nizhny Novgorod) is used. To obtain an image, the animal was fixed on a stand and placed in a dark installation chamber. An LED with a radiation maximum at 585 nm was used as a radiation source. The signal was recorded using a cooled digital CCD camera (Hamamatsu ORCA2). The exposure time was 5 seconds. A filter with a passband Δλ = 645-730 nm was used to separate the pump and emission spectra. Tumor irradiation was performed using an LED at a wavelength of 590 nm 7 times daily. During the treatment session, the energy density was 180 J / cm ² , the power density was 150 mW / cm ² .

An analysis of the tumor fluorescence intensity revealed a signal decrease of 11.5% after irradiation, indicating the burnout of the KillerRed protein. A photograph of the animal in white light (A) and fluorescence images of the tumor before (B) and after irradiation (C) are shown in Fig. 3. Tumor localization is indicated by an arrow.

As a result of the pathomorphological analysis of tumors after photodynamic therapy, changes in cells similar to Example 1 were detected. The vast majority of tumor cells were in a state of sharp vacuolization of the cytoplasm, often with a violation of the integrity of the cell membrane. In some cells, a decrease in the size of nuclei is noted, condensation of chromatin (karyopicnosis), in others, on the contrary, swelling of the nucleus up to the rupture of the karyolemma. The content of cells with signs of dystrophy was 65%, with signs of apoptosis - 13.2%. Whereas in unirradiated tissue, these indices were 9.9 and 6.3%, respectively.

Example 4

Animal - immunodeficient mouse, female, weighing 20.2 g with a tumor inoculated with human cervical cancer, 3.5 mm in size, located subcutaneously in the thigh area, the KillerRed protein gene was previously inserted into the tumor cells, the genetically encoded KillerRed protein was expressed by tumor cells in mitochondria and nuclei . Registration of fluorescence images of the tumor and its therapy was carried out analogously to example 3. Irradiation of the tumor was performed 3 times every other day.

Immediately after laser irradiation, a decrease in the level of tumor fluorescence by 12% was detected.

The pathomorphological analysis of tumors after photodynamic therapy revealed changes similar to example 1. The content of cells with signs of dystrophy was 57.4%, with signs of apoptosis - 11.9%. Whereas in unirradiated tissue, these indices were 9.9 and 6.3%, respectively.

Example 5

The animal is an immunodeficient mouse, female, weighing 21 g with a tumor inoculated with human cancer of the cervix uteri, 3 mm in size, located subcutaneously in the thigh area, the KillerRed protein gene was previously inserted into the tumor cells, the genetically encoded KillerRed protein was expressed in the nuclei of tumor cells.

Registration of fluorescence images of the tumor and its therapy was carried out analogously to example 1. As a source of optical radiation, a xenon lamp with a filter at a wavelength of 520-590 nm was used.

Histological analysis of tumor tissue showed that the proportion of cells with dystrophic changes was 66.5%, and that of cells with signs of apoptosis was 12.2%.

Example 6

The animal is an immunodeficient mouse, female, weighing 20.5 g with a tumor inoculated with human cancer of the cervix, 3.2 mm in size, located subcutaneously in the thigh, the KillerRed protein gene was previously inserted into the tumor cells. The genetically encoded KillerRed protein was expressed in the nuclei of tumor cells.

Registration of fluorescence images of the tumor and its therapy was carried out analogously to example 2.

The decrease in tumor fluorescence intensity as a result of irradiation was 25%.

The proportion of cells with dystrophic changes increased to 61.8%, from cells with signs of apoptosis - 10.2%.

Example 7

Animal - immunodeficient mouse, female, weighing 19.6 g with a tumor inoculated with human cervical cancer. A tumor 3.5 mm in size grows subcutaneously in the thigh area, the KillerRed protein gene was previously inserted into the tumor cells. The genetically encoded KillerRed protein was expressed in the nuclei of tumor cells.

Registration of fluorescence images of the tumor and its therapy was carried out analogously to example 3.

The decrease in the intensity of tumor fluorescence as a result of irradiation was 16%.

The proportion of cells with dystrophic changes increased to 59.1%, from cells with signs of apoptosis 11.8%.

Example 8

The animal is an immunodeficient mouse, female, weighing 20.5 g with a tumor inoculated with a human cervical cancer 3.2 mm in size, located subcutaneously in the thigh area, the KillerRed protein gene was previously inserted into the tumor cells, the genetically encoded KillerRed protein was expressed in the nuclei of tumor cells.

Registration of fluorescence images of the tumor and its therapy was carried out analogously to example 4.

The decrease in the intensity of tumor fluorescence as a result of irradiation was 14.2%.

The histological image of the tumor tissue after treatment is shown in Fig.4. The proportion of cells with dystrophic changes was 56.1%, from cells with signs of apoptosis - 11.3%.

Example 9

The animal is an immunodeficient mouse, female, weighing 20.2 g with a tumor inoculated with human cancer of the cervix, 3.6 mm in size, located subcutaneously in the thigh region. The KillerRed protein gene was previously inserted into the tumor cells, as a result, the genetically encoded KillerRed protein was expressed in the nuclei and mitochondria of tumor cells.

Registration of fluorescence images of the tumor was carried out analogously to example 1. In this case, the tumor was irradiated with laser radiation at a wavelength of 593 nm with an energy density of 225 J / cm ² , radiation was performed daily, once a day for 7 days.

The decrease in tumor fluorescence intensity as a result of irradiation amounted to an average of 13%.

After photodynamic therapy, a histological examination of the tumor tissue was performed, which showed the presence of pathomorphological changes similar to example 1 (figure 2).

Example 10

The animal is an immunodeficient mouse, female, weighing 19.5 g with a tumor inoculated with a human cervical cancer 4.1 mm in size, the KillerRed protein gene was previously inserted into the tumor cells, the genetically encoded KillerRed protein was expressed in the nuclei of tumor cells. Registration of fluorescence images of the tumor was carried out analogously to example 1. The tumor was irradiated with laser radiation at a wavelength of 593 nm with an energy density of 225 J / cm ² , radiation was performed 3 times every other day.

The decrease in tumor fluorescence intensity as a result of irradiation was 27% after the first irradiation, 18% after the second, 8% after the third, and an average of 17.6%. The change in the intensity of tumor fluorescence during photodynamic therapy is shown in Fig.6. A pathomorphological study of the tumor after photodynamic therapy revealed dystrophic tissue changes, such as vacuolization of the cytoplasm in most cells, a change in the size of the nucleus, nuclear edema, and numerous ruptures of the nuclear and cytoplasmic membranes.

Example 11

The animal is an immunodeficient mouse, female, weighing 19.3 g with a tumor inoculated with human cancer of the cervix, 3.9 mm in size, located subcutaneously in the thigh. The tumor cells were not genetically modified; as a result, the tumor did not contain a photosensitizer, a genetically encoded KillerRed protein.

Fluorescence observation of the tumor and photodynamic therapy was performed analogously to example 1.

No tumor was identified on the resulting fluorescence images.

In the irradiated tumor without the introduced gene and not expressing the KillerRed protein, no significant structural changes were detected. The tissue of the tumor node remained dense, only a slight increase in the proportion of dystrophically altered cells was noted. Morphometric analysis of tumor tissue after treatment showed that the tumor tissue contains a small amount (18%) of dystrophically altered cells, however, these changes are different from cases when the tumor contains KillerRed protein (examples 1-8). In a treated tumor without protein, dystrophic changes are mainly vacuolization of the cytoplasm and a slight change in cell size, which is not critical for cell activity and can be reversible. Presumably, this may be due to photoactivation of endogenous chromophores or weak thermal effects (the increase in tumor temperature immediately after irradiation was about 1.3 ° C). A histological image of tumor tissue not expressing KillerRed protein after photodynamic therapy is shown in FIG. 5.

As can be seen from the obtained results, the proposed method is effective, providing high selectivity of the effect of the photosensitizer on the tumor, with a low toxic load on normal vital organs and tissues, and does not require constant dosimetric monitoring during therapy.

Information sources:

1. Kessel D. Photodynamic therapy of neoplastic disease / CRC Press, 1990.

2. ISBN: 9780849358166, p. 280.

2. Berg K., Selbo P.K., Weyergang A. et al. Porphyrin-related photosensitizers for cancer imaging and therapeutic applications /// Journal of Microscopy. 2005, Vol. 218, p. 133.

3. Josefsen L. B., Boyle R. W. Photodynamic therapy and the development of metal-based photosensitisers / Metal-Based Drugs. 2008, Vol. 2008, p.23.

4. T. Hasan, B. Ortel, A. C.E. Moor, and B.W. Pogue, Chapter 40 Photodynamic Therapy of Cancer in Holland-Frei Cancer Medicine, 6th edition, Edited by Donald W Kufe et al. Hamilton (ON): Sun Decker; 2003. ISBN-10: 1-55009-213-8, p.605.

5. Gelfond M.L. Photodynamic therapy in oncology // Practical Oncology, 2007, 8 (4), p.204.

6. Chissov V.I., Sokolov V.V., Bulgakova N.N., Filonenko E.V. Fluorescence endoscopy, dermoscopy and spectrophotometry in the diagnosis of malignant tumors of the main localizations // RBZh., 2003, 2 (4), p.45.

7. P. Mroz, A. Yaroslavsky, G. B. Kharkwal and M.R. Hamblin Cell Death Pathways in Photodynamic Therapy of Cancer // Cancers, 2011, 3, p. 2516.

8. N.L. Oleinick, R.L. Morris and A.L. Nieminen Chapter 27. Photodynamic therapy-induced apoptosis in Apoptosis, Senescence and Cancer, Gewirtz, David A .; Holt, Shawn E .; Grant, Steven (Eds.) 2nd ed., 2007, XVII, p. 599.

9. Zalessky B.H., Dynnik O.B. Molecular medicine: transformation of the processes of intracellular relocalization of photosensitizers as a reserve for the effectiveness of their phototoxic effects / Ukrainian Medical Journal, 1 (45), 2005, p. 92.

10. D. Nowis, M. Makowski, T. ł Stokłosa, et al Direct tumor damage mechanisms of photodynamic therapy // Acta Biochim Pol. 2005; 52 (2), p. 339.

11. C.J. Gomer, A. Ferrario Tissue distribution and photosensitizing properties of mono-L-aspartyl chlorin e6 in a mouse tumor model // Canc. Res. 1990. 50, p. 3985.

12. B.W. Pogue, T. Hasan, Targeting in Photodynamic Therapy and Photo-Imaging // Optics & Photonics News, Aug. 2003, p. 36.

13. E.V. Kochneva, B.A. Privalov, A.B. Lappa “Results of the second phase of a clinical study of the second generation photosensitizer“ Radachlorin® ”solution for intravenous administration of 0.35% in patients with basal cell skin cancer” // Bulletin of the Chelyabinsk Scientific Center, issue 2 (23), 2004, p.167.

14. N. Brasseur, R. Ouellet, C. La Madeleine and JE van Lier Water-soluble aluminum phthalocyanine-polymer conjugates for PDT: photodynamic activities and pharmacokinetics in tumor-bearing mice British Journal of Cancer (1999) 80 (10), p .1533.

15. Yu. S. Romanko, A.F. Tsyb, M.A. Kaplan, and V.V. Popuchiev Relationship between Antitumor Efficiency of Photodynamic Therapy with Photoditasine and Photoenergy Density / Bulletin of Experimental Biology and Medicine, Vol. 139, No.4, 2005, p. 460.

16. The prototype. Smirnova Z.S., Oborotova H.A., Makarova O.A., Orlova O.L., Polozkova A.P., Kubasova I.Yu., Lukyanets E.A., Meerovich G.A., Zimakova N. .I., Kuzmin S.G., Vorozhtsov G.N., Baryshnikov A.Yu. The effectiveness and pharmacokinetics of the liposomal dosage form of the photosensitizer "Photosens" based on aluminum sulfophthalocyanine // Chemical and Pharmaceutical Journal, No. 7, 2005, p.3.

17. M.E. Bulina, D.M. Chudakov, O.V. Britanova et al. A genetically encoded photosensitizer // Nat Biotechnol. 2006, 24 (1), p. 95.

18. S. Pletnev, N.G. Gurskaya, N.V. Pletneva et al. Structural Basis for Phototoxicity of the Genetically Encoded Photosensitizer KillerRed // J. Biol. Chem. 2009, 284, 46, p. 32028.

19. R.B. Vegh, K.M. Solntsev, M.K. Kuimova et al. Reactive oxygen species in photochemistry of the red fluorescent protein "Killer Red" // Chem. comm., 2011, 47 (17), p. 487.

20. M.E Bulina, D.M. Chudakov, O.V. Britanova, Y.G. Yanushevich, D.B. Staroverov, T.V. Chepurnykh, E.M. Merzlyak, M.A. Shkrob, S. Lukyanov, K.A. Lukyanov, A genetically encoded photosensitizer // Nat. Biotech 24 (1), 2006, p. 95.

21. M.V. Shirmanova, E.O. Serebrovskaya, K.A. Lukyanov, L. B. Snopova, M.A. Sirotkina, N.N. Prodanetz, M.L. Bugrova, E.A. Minakova, I.V. Turchin, V.A. Kamensky, S.A. Lukyanov, and E.V. Zagaynova Phototoxic effects of fluorescent protein Killer Red on tumor cells in mice / J. Biophotonics, 2013, 6 (3), p. 283.

Claims (2)

1. A method of photodynamic therapy of a tumor, including the use of a photosensitizer, a preliminary assessment of its presence in the tumor by its fluorescence, followed by irradiation of the tumor with optical radiation with a wavelength in the spectral range of the maximum absorption of the photosensitizer, characterized in that a genetically encoded KillerRed protein is used as the photosensitizer, why ensure its expression in the tumor by embedding the corresponding gene in the tumor cells, irradiating the tumor with optical zlucheniem carried out at an energy density of 180-270 J / cm ^{2 to} 3 times a day or seven times daily. 2. The method according to claim 1, characterized in that the localization of the KillerRed protein is mitochondrial and nuclear or nuclear.

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