Cancer treatment or diagnostic agent

Abstract

The present invention addresses the problem of providing a therapeutic drug and a diagnostic drug for cancer that have high selectivity for cancer, low toxicity and little side effect. The present invention provides a therapeutic drug or diagnostic drug for cancer, said drug comprising a photosynthetic bacterium.

Description

[Technical Field]

[0001] The present invention relates to a therapeutic or diagnostic agent for cancer using photosynthetic bacteria. [Background technology]

[0002] Unlike normal vascular endothelial cells, cancer tissues and inflammatory sites have wide gaps (approximately 200 nm) between them. Nanoparticles with size-controlled nanoparticles (10–100 nm) are known to specifically accumulate in cancer tissues, known as the enhanced permeation and retention (EPR) effect. The EPR effect has been cited extensively in papers related to nanoparticle-based drug delivery systems and is a key pillar of selective cancer chemotherapy. However, there are also many doubts and criticisms about the EPR effect. In fact, for solid tumors, several environmental factors, such as heterogeneous blood flow in tumor tissues and high tissue pressure at the tumor center, have been found to be fatally ineffective against the EPR effect (e.g., Masayuki Yokoyama, Drug Delivery System, 33(2), pp. 89–97 (2018)). Furthermore, despite many years and significant investment, drug carriers utilizing the EPR effect have not been successful in human clinical trials.

[0003] On the other hand, strategies to improve tumor targeting have been adopted, such as loading cancer cell-specific antibodies onto drug carriers (e.g., Hisataka Kobayashi, Drug Delivery System, 29(4), p.274-284(2014)). However, antibodies have difficulty penetrating the interstitial barrier surrounding cancer, and sufficient selectivity and efficacy have not been achieved. Furthermore, because different cancer patients express different biomarkers (antigens), it is necessary to create custom (owner)-made antibodies suitable for each cancer patient. However, the antibody production itself requires a great deal of effort and cost, and the resulting enormous medical costs are a problem.

[0004] In recent years, attention has been focused on targeted cancer therapy using anaerobic microorganisms that can selectively accumulate, grow, and proliferate within hypoxic tumors (Shibin Zhou et al. Nature Reviews Cancer, 18, pp. 727-743 (2018)). However, conventional cancer bacteriotherapy is essentially a drug delivery system, which transports anticancer drugs. Furthermore, to exert anticancer activity, the microorganisms must be genetically engineered to manipulate or modify them. This can result in unpredictable and uncontrollable problems, such as the development of drug resistance in the bacteria. Furthermore, the bacteria used are often genetically attenuated strains of Salmonella or Escherichia coli, which always carry the risk of reactivating their virulence in the body. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Masayuki Yokoyama, Drug Delivery System, 33(2), pp.89-97(2018) [Non-patent document 2] Hisataka Kobayashi, Drug Delivery System, 29(4), p.274-284(2014) [Non-patent document 3] Shibin Zhou et al. Nature Reviews Cancer, 18, p.727-743(2018) Summary of the Invention [Problem to be solved by the invention]

[0006] Nanomedicine has had the following problems: (1) it relies on the EPR effect, which has an unclear mechanism, resulting in low selectivity for cancer; (2) it uses anticancer drugs with strong side effects; (3) its synthesis requires a great deal of effort and cost; and (4) photodynamic therapy-type nanomedicine, which utilizes oxygen, is ineffective because the inside of a tumor is oxygen-deficient.

[0007] Antibody therapy has several problems: (1) it is necessary to create custom-made antibodies for biomarkers that are expressed at different levels among cancer patients; (2) it is unable to penetrate the cancer stromal barrier (it only binds to the surface of the tumor), so even though it uses immune cells, it is difficult to eliminate cancer cells deep within the tumor; and (3) it requires a great deal of effort and cost to produce.

[0008] Conventional bacterial therapy has the following problems: (1) it uses genetically modified Salmonella, Listeria, and Escherichia coli bacteria that have been weakened, which carries the risk of them becoming virulent again (reverting mutation) in the body, and the genetic modification can lead to the acquisition of drug resistance; and (2) it is a passive drug delivery method, which requires complex genetic design.

[0009] An object of the present invention is to provide a cancer therapeutic agent and a cancer diagnostic agent that are highly selective for cancer and have little toxicity or side effects. [Means for solving the problem]

[0010] As a result of intensive research to solve the above-mentioned problems, the present inventors have discovered that by using photosynthetic bacteria that are capable of highly selective accumulation, growth, and proliferation in hypoxic tumor environments and that function using near-infrared light, which is highly biotransparent, it is possible to provide cancer therapeutic and diagnostic agents that are highly selective for cancer and have few toxicity or side effects, and have thereby completed the present invention.

[0011] That is, the present invention is as follows. <1> A cancer therapeutic or diagnostic agent comprising photosynthetic bacteria. <2> The photosynthetic bacteria are photosynthetic bacteria having bacteriochlorophyll. <1> A therapeutic or diagnostic agent for cancer according to the above. <3> The photosynthetic bacteria are purple photosynthetic bacteria or green photosynthetic bacteria. <1> or <2> A therapeutic or diagnostic agent for cancer according to the above. <4> The photosynthetic bacteria are Rhodopseudomonas bacteria, Blastochloris bacteria, Afifella bacteria, Rhodobacter bacteria, Pararhodospirillum bacteria, Rhodomicrobium bacteria, Rhodovulum bacteria, or Marichromatium bacteria; <1> from <3> 10. The cancer therapeutic or diagnostic agent according to any one of the preceding claims. <5> Used in combination with light irradiation, <1> from <4> 1. A therapeutic or diagnostic agent for cancer according to any one of the above. <6> 6. The cancer therapeutic or diagnostic agent according to claim 5, wherein the light irradiation is near-infrared light irradiation. [Effects of the Invention]

[0012] The cancer therapeutic and diagnostic agents of the present invention have the following advantages: High selectivity for tumors can be achieved through distinct mechanisms derived from hypoxia, immune evasion, and chemotaxis. No anticancer drugs with strong side effects are used, and the bacteria used are of low toxicity, so side effects and toxicity are low. The bacteria used can replicate themselves indefinitely and are cheap to produce. It is possible to use not only the photodynamic mode, which uses oxygen, but also the thermal mode, which does not require oxygen.Furthermore, it is effective in degenerating cancer cells simply by administering bacteria without using light. Bacteria can highly selectively accumulate, grow, and proliferate in the microenvironment common to solid tumors. Bacteria can accumulate, grow, and proliferate deep within tumors. No genetic modification is required and high selectivity against tumors can be expressed. [Brief explanation of the drawings]

[0013] [Figure 1] Figure 1 shows a conceptual diagram of purple photosynthetic bacteria driven by near-infrared light. [Figure 2]Figure 2 shows the chemical structure of bacterial chlorophyll (BChla). [Figure 3] Figure 3 shows the UV-Vis-NIR light absorption characteristics of each bacterium. [Figure 4] FIG. 4 shows the fluorescence spectrum of R. Palustris (excitation wavelength: 805 nm). [Figure 5] Figure 5 shows the temperature change of each bacterial dispersion when irradiated with a near-infrared laser [wavelength: 808 nm, output: 1.2 W (ca. 61.1 mW / mm2), irradiation time: 5 min]. [Figure 6] Figure 6 shows the generation behavior of reactive oxygen species (ROS) singlet oxygen from an R. palustris dispersion when irradiated with a near-infrared laser. [Figure 7] FIG. 7 shows the cytotoxicity evaluation of the purple photosynthetic bacterium R. Palustris. [Figure 8] Figure 8 shows the survival rate evaluation when various cancer cells were irradiated with near-infrared laser in a control (DMEM medium without bacteria) and in the presence of different concentrations of R. palustris. [Figure 9] Figure 9 shows the measurement of tumor surface temperature in a Colon26 tumor-bearing model mouse during near-infrared laser irradiation. [Figure 10] FIG. 10 shows the evaluation of antitumor activity using near-infrared-activated R. Palustris. [Figure 11] FIG. 11 shows photographs of mice after each treatment (arrows indicate each tumor that was irradiated with the laser). [Figure 12] FIG. 12 shows photographs of tumors excised 34 days after various treatments. [Figure 13] FIG. 13 shows the survival rate of mice over 34 days for each treatment. [Figure 14] Figure 14 shows in vivo near-infrared region I (NIR-I) fluorescence bioimaging of Colon26 tumor-bearing model mice. [Figure 15] FIG. 15 shows a photograph of red colonies derived from R. Palustris that grew specifically within the tumor. [Figure 16]FIG. 16 shows the number of viable R. Palustris bacteria in various organs and tumors. [Figure 17] FIG. 17 shows the fluorescence intensity derived from R. Palustris in various organs and tumors. [Figure 18] FIG. 18 shows the viable counts of R. palustris in various organs and tumors. [Figure 19] FIG. 19 shows a photograph of green colonies derived from Blastochloris viridis that grew specifically within the tumor. [Figure 20] FIG. 20 shows the number of live Blastochloris viridis bacteria in various organs and tumors. [Figure 21] FIG. 21 shows NIR-I fluorescence microscopy images of mouse macrophages (RAW264.7) co-cultured with R. palustris (1×10 8 CFU / mL) for 4 hours. [Figure 22] Figure 22 shows photoacoustic (PA) imaging of mouse tumors using R. Palustris. [Figure 23] FIG. 23 shows angiography by near-infrared region II (NIR-II) fluorescence bioimaging using Blastochloris viridis. [Figure 24] FIG. 24 shows the UV-Vis-NIR light absorption characteristics of each bacterium. [Figure 25] FIG. 25 shows the UV-Vis-NIR light absorption characteristics of each bacterium. [Figure 26] FIG. 26 shows the fluorescence spectrum of each bacterium (bacterial concentration: 2.5E+07 CFU / mL). [Figure 27] FIG. 27 shows the fluorescence spectrum of each bacterium (bacterial concentration: 2.5E+07 CFU / mL). [Figure 28] FIG. 28 shows the temperature change of each bacterial dispersion when irradiated with a near-infrared laser [wavelength: 808 nm, irradiation time: 2 min]. [Figure 29] FIG. 29 shows the cytotoxicity evaluation of each bacterium. [Figure 30] Figure 30 shows the cytotoxicity assessment of each bacterium. [Figure 31] FIG. 31 shows the cytotoxicity evaluation of each bacterium. DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention relates to a cancer therapeutic or diagnostic agent comprising photosynthetic bacteria. The photosynthetic bacteria used in the present invention are preferably those having bacteriochlorophyll. Examples of bacteriochlorophyll include bacteriochlorophyll a, bacteriochlorophyll b, bacteriochlorophyll c, bacteriochlorophyll d, bacteriochlorophyll f, and bacteriochlorophyll g, and photosynthetic bacteria having one or more of these can be used. An example is photosynthetic bacteria having bacteriochlorophyll a or bacteriochlorophyll b. More specifically, purple photosynthetic bacteria or green photosynthetic bacteria can be used. Figure 1 shows a conceptual diagram of purple photosynthetic bacteria powered by near-infrared light. Figure 2 shows the chemical structure of bacterial chlorophyll (BChl a).

[0015] Examples of photosynthetic bacteria include Rhodopseudomonas bacteria, Blastochloris bacteria, Afifella bacteria, Rhodobacter bacteria, Rubrivivax bacteria, Pararhodospirillum bacteria, Rhodocista bacteria, Marichromatium bacteria, Phaeochromatium bacteria, Rhodoferax bacteria, Rhodomicrobium bacteria, Thermochromatium bacteria, Chlorobaculum bacteria, and Rhodovulum bacteria. Among the above, bacteria of the genus Rhodopseudomonas, Blastochloris, Afifella, Rhodobacter, Pararhodospirillum, Rhodomicrobium, Rhodovulum, or Marichromatium are preferred.

[0016] Specific examples of purple photosynthetic bacteria include Rhodopseudomonas Palustris, Blastochloris viridis, Afifella marina, Blastochloris sulfoviridis, Rhodobacter blasticus, Rhodobacter capsulatus, Rhodobacter sphaeroides, Rhodopseudomonas pseudopalustris, Rubrivivax gelatinosus, Pararhodospirillum oryzae, Pararhodospirillum sulfurexigens, Rhodocista centenaria, Marichromatium litoris, Phaeochromatium fluminis, Rubrivivax gelatinosus, Rhodoferax fermentans, Rhodomicrobium udaipurense, Rhodomicrobium vannielii, and Rhodovulum sulfidophilum. Specific examples of green photosynthetic bacteria include Thermochromatium tepidum and Chlorobaculum tepidum. Among the above, Rhodopseudomonas Palustris, Blastochloris viridis, Pararhodospirillum oryzae, Pararhodospirillum sulfurexigens, Rhodomicrobium udaipurense, Rhodomicrobium vannielii, Rhodovulum sulfidophilum, Afifella marina, Rhodobacter sphaeroides, Marichromatium litoris, Rhodobacter capsulatus, and Blastochloris sulfoviridis are particularly preferred.

[0017] The photosynthetic bacteria mentioned above can be purchased from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) or ATCC (American Type Culture Collection), etc. In Japan, photosynthetic bacterial strains are preserved and distributed at the National Bioresource Center (NBRC) of the National Institute of Technology and Evaluation (which took over from IFO), and the RIKEN BioResource Center, and can be obtained from these institutions. DSMZ https: / / www.dsmz.de / collection / catalogue / microorganisms ATCC https: / / www.atcc.org / Advanced%20Search.aspx NBRC https: / / www.nite.go.jp / nbrc / cultures / nbrc / index.html RIKEN BRC https: / / jcm.brc.riken.jp / ja / In addition to the above, there are some bacteria that can be obtained from domestic university laboratories in some cases.

[0018] The cancer therapeutic or diagnostic agent of the present invention can be used in combination with light irradiation. That is, photosynthetic bacteria can be administered to a subject, allowed to accumulate in the affected area where cancer is present, and then the affected area can be irradiated with light. When irradiated with light, photosynthetic bacteria generate fluorescence, heat, or singlet oxygen (reactive oxygen species: ROS), which can kill cancer cells (Figure 1).

[0019] The light irradiation can be carried out by laser irradiation, preferably by near-infrared laser irradiation. Near-infrared light is an electromagnetic wave having a wavelength of about 0.7 to 2.5 μm, and has a wavelength close to that of red visible light. The wavelength of the near-infrared light is not particularly limited, but is preferably 700 nm to 2000 nm, and may be 700 nm to 1400 nm, 750 nm to 1200 nm, 750 nm to 1000 nm, or 750 nm to 900 nm. One example is 808 nm.

[0020] The output of the near-infrared light may be appropriately selected depending on the wavelength of the light used, and may be, for example, 0.5 W or more, preferably 0.7 W or more, and more preferably 1.0 W or more. There is no particular upper limit, but it is generally 20 W or less, and preferably 10 W or less. The duration of light irradiation is not particularly limited as long as the effects of the present invention can be obtained, but is generally 1 to 30 minutes, preferably 1 to 20 minutes. The light irradiation may be carried out once or twice or more.

[0021] When using photosynthetic bacteria as a diagnostic agent, near-infrared fluorescence images can be observed in subjects to which the bacteria have been administered. For example, fluorescence wavelengths of 810 to 1500 nm can be observed using an excitation wavelength of 740 to 1200 nm.

[0022] The subject to which the cancer therapeutic or diagnostic agent of the present invention is administered is a human or a non-human mammal (for example, a laboratory animal such as a mouse), preferably a subject suffering from cancer.

[0023] Specific examples of cancer include, but are not limited to, breast cancer, lung cancer, uterine cancer, ovarian cancer, pancreatic cancer, adrenal cortical cancer, non-Hodgkin's lymphoma, multiple myeloma, leukemia, Kaposi's sarcoma, Ewing's sarcoma, soft tissue sarcoma, nephroblastoma, glioblastoma, prostate cancer, liver cancer, bone cancer, chondrosarcoma, kidney cancer, bladder cancer, stomach cancer, colon cancer, rectal cancer, thyroid cancer, head and neck cancer, and skin cancer (such as melanoma).

[0024] Methods of administration to a subject include, but are not limited to, administration by injection (subcutaneous injection, intramuscular injection, intradermal injection, intraperitoneal injection, intratumoral injection, intravenous injection, etc.) or local administration, etc. Preferably, administration by intravenous injection.

[0025] Injections can be prepared by conventional methods using pharmaceutically acceptable carriers (eg, physiological saline, suitable buffer solutions, etc.).

[0026] The dosage of photosynthetic bacteria can be appropriately determined depending on the condition of the subject, but the number of bacteria is generally 1 × 10 7 CFU / kg ~ 1 x 10 13 CFU / kg, preferably 1 x 10 8 CFU / kg ~ 1 x 10 12 CFU / kg. The dosages described above may be administered in one or more divided doses (such as 2, 3, or 4 doses) or in a single formulation.

[0027] The present invention further provides the following: <a1>A method for treating cancer, comprising administering photosynthetic bacteria to a subject. <a2>Further comprising irradiating the subject to which the photosynthetic bacteria has been administered with light. <a1>A method for treating cancer as described in the above. <a3>A method for diagnosing cancer, comprising administering photosynthetic bacteria to a subject. <b1>A photosynthetic bacterium for use in the treatment or diagnosis of cancer. <b2>Used in combination with light irradiation, <b1>The photosynthetic bacteria described in. <c1>Use of photosynthetic bacteria for the manufacture of a therapeutic or diagnostic agent for cancer.

[0028] The present invention will be explained in more detail with reference to the following examples, but the present invention is not limited to these examples. [Example]

[0029] <Bacterial culture> The bacteria used in this study were obtained from the National Institute of Technology and Evaluation Biological Resource Center (NBRC) and the American Type Culture Collection (ATCC). Rhodopseudomonas Palustris (NBRC16661), Blastochloris viridis (NBRC 102659), Pararhodospirillum oryzae (NBRC107573), Pararhodospirillum sulfurexigens (NBRC104433), Rhodomicrobium udaipurense (NBRC109057), Rhodomicrobium vannielii (NBRC100050), Rhodovulum sulfidophilum (ATCC35886), Afifella marina (NBRC100434), Rhodobacter sphaeroides (NBRC12203), Marichromatium litoris (NBRC104939), Rhodobacter capsulatus (NBRC16435), Blastochloris S. sulfoviridis (NBRC103805) was anaerobically cultured in 543 ATCC medium at 26-30°C under tungsten lamp irradiation. Bifidobacterium bifidum (NBRC 100015) was anaerobically cultured in 385 NBRC medium at 37°C.

[0030] Reagents used for bacterial culture were obtained from FUJIFILM Wako Pure Chemical.

[0031] <Analysis of the optical properties of bacteria> The absorbance spectra of the bacterial suspensions were measured at room temperature using a UV-Vis-NIR spectrophotometer (V-730 BIO; Jasco), and the fluorescence of the bacterial suspensions was measured using fluorescence spectrometers (FP-8600 NIR Spectrofluorometer; Jasco or Fluorolog-3; HORIBA Jobin Yvon).

[0032] The UV-Vis-NIR light absorption characteristics of each bacterium ((a) Rhodopseudomonas palustris, (b) Blastochloris viridis, (c) Bifidobacterium bifidum) are shown in Figure 3. The UV-Vis-NIR light absorption characteristics of each bacterium are also shown in Figures 24 and 25. These are the results of measurements at a fixed bacterial concentration. The fluorescence spectrum of R. palustris (excitation wavelength: 805 nm) is shown in Figure 4. The fluorescence spectra of each bacterium when the excitation wavelength and fluorescence wavelength were changed are shown in Figures 26 and 27.

[0033] <Temperature measurement> The temperature change of the bacterial dispersion after laser irradiation was investigated as follows. A fiber-coupled continuous-wave laser with a wavelength of 808 nm (laser spot diameter, approximately 5 mm; output, 1.2 W, approximately 61.1 mW mm) was applied to PBS buffer solution (100 μL) containing bacteria or PBS buffer solution (100 μL) without bacteria. -2 The temperature change of the solution during laser irradiation was measured using a temperature sensor (AD-5601A; A&D). Near-infrared laser irradiation [wavelength: 808 nm, output: 1.2 W (ca. 61.1 mW / mm 2 ), irradiation time: 5 min]. The temperature change of the dispersion of each bacterium ((a) Rhodopseudomonas Palustris, (b) Blastochloris viridis, (c) Bifidobacterium bifidum) is shown in Figure 5.

[0034] As above, near-infrared laser irradiation [wavelength: 808 nm, output: 1.2 W (ca. 61.1 mW / mm 2 ), irradiation time: 2 min]. The temperature change of the dispersion of each bacteria is shown in Figure 28.

[0035] <Reactive oxygen species (ROS) detection> For ROS analysis, a 96-well plate with a clear bottom and a black main body (Thermo Fisher Scientific) and the singlet oxygen detection reagent, singlet oxygen sensor green (SOSG) (Invitrogen) were used. R. palustris was dispersed in PBS buffer (100 μL, 5 × 10 9 CFUml -1 ) was diluted with PBS buffer containing SOSG. The final concentrations of R. palustris and SOSG in the system were 1.3 × 10 9 CFU ml -1 The sample was then exposed to a near-infrared laser with a wavelength of 808 nm and an output of 1.2 W (ca. 61.1 mW mm -2 ) and irradiated for 5 minutes. PBS buffer without R. palustris was used as a control. Green fluorescence associated with ROS generation was measured using a microplate reader (Infinite 200 PRO M Plex) (excitation wavelength: 485 nm, emission wavelength: 535 nm). Near-infrared laser irradiation [wavelength: 808 nm, output: 1.2 Wca. 61.1 mW / mm 2 Figure 6 shows the generation behavior of reactive oxygen species (ROS) singlet oxygen from R. palustris dispersions under conditions of [irradiation time: 5 min].

[0036] <Cell culture and cytotoxicity evaluation> Mouse colon cancer cells (Colon26) and human normal diploid fibroblasts (MRC5) were obtained from the Japanese Collection of Research Bioresources Cell Bank. Human alveolar basal epithelial adenocarcinoma cells (A549) and human colon adenocarcinoma (HT29) were purchased from DS Pharma Biomedical. Mouse macrophages (RAW264.7) were obtained from the Riken Bio Resource Center. Colon26 cells were cultured in 10% fetal bovine serum, 2 mM l-glutamine, 1 mM sodium pyruvate, gentamycin, and penicillin-streptomycin (100 IU ml -1 The other cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 Medium (Gibco) containing 10% fetal bovine serum, 2 mM l-glutamine, 1 mM sodium pyruvate, gentamycin, penicillin-streptomycin (100 IU ml -1 The cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco) containing Hank's balanced salt solution (Life Technologies) at 37°C in a humidified chamber under a 5% CO2 atmosphere.

[0037] Cell viability was assessed using the Cell Counting Kit (CCK)-8 (Dojindo Laboratories) according to its manual. 3 cells well -1 ) were seeded into 96-well plates and incubated overnight. The cells were then exposed to bacterial dispersion solution for 4 hours, washed with fresh culture medium, and then incubated in CCK-8 solution. Finally, cell viability was calculated by measuring the absorbance at 450 nm using a microplate reader (Infinite 200 PRO M Plex; Tecan).

[0038] The cytotoxicity of the purple photosynthetic bacterium R. palustris was evaluated by measuring the viability of mouse colon cancer cells (Colon 26), human alveolar basal epithelial adenocarcinoma cells (A549), human colon adenocarcinoma cells (HT29), and human normal diploid fibroblasts (MRC5) after co-cultivation with different concentrations of R. palustris for 4 hours. The graphs in Figure 7 show, from left to right, MRC5, Colon 26, A549, and HT29.

[0039] As described above, the cytotoxicity of each bacterium was evaluated using mouse colon cancer cells (Colon 26). The results are shown in Figures 29 to 31. The survival rate of mouse colon cancer cells (Colon 26) was measured when different concentrations of each bacterium were co-cultured for 4 hours.

[0040] <Evaluation of cell viability by laser irradiation> Colon26, A549, and HT29 cells (5 × 10 3 Cells were seeded at various concentrations (0.16, 0.31, 0.63, 1.25 × 10 cells / well) into a 96-well plate and incubated overnight. 9 CFU ml -1 After treatment with cell culture medium (100 μL) containing R. palustris or cell culture medium (100 μL) without R. palustris, the cells were exposed to a laser (wavelength 808 nm, output 1.2 W, ~61.1 mW mm -2 ) for 5 minutes. After laser irradiation, the cells were washed and incubated in fresh medium. Cell viability was assessed using a CCK-8 kit immediately after laser irradiation and after 24 hours of incubation.

[0041] When various cancer cells were irradiated with a near-infrared laser [wavelength: 808 nm, output: 1.2 W (ca. 61.1 mW / mm ]] on a control (DMEM medium without bacteria) and on cancer cells coexisting with different concentrations of R. palustris, 2 ) Survival rate evaluation at an irradiation time of 5 min [Cell survival rate was measured by the WST-8 method 24 hours after laser irradiation] is shown in Fig. 8.

[0042] <Fluorescence observation of R. Palustris in macrophage cells> RAW264.7 cells (2.5×10 5 cells / well -1 ) were seeded in a 24-well plate and incubated overnight. The cells were exposed to a cell culture medium (1×10 8 CFU) in which R. Palustris was dispersed or a cell culture medium without R. Palustris, and then cultured for 2 hours under the conditions of 37 °C and 5% CO₂. The cells were washed and incubated in fresh medium for 4 hours, and observed at room temperature using a fluorescence microscope system (IX73; Olympus) equipped with a near-infrared fluorescence mirror unit (IRDYE800-33LP-A-U01; Semrock) and an objective lens (×60 magnification, aperture 1.35; UPLSAPO60X, Olympus).

[0043] NIR-I fluorescence microscope images of mouse macrophages (RAW264.7) co-cultured with R. Palustris (1×10 8 CFU / mL) for 4 hours (The white arrow indicates R. Palustris that is phagocytosed by macrophages and still retains near-infrared fluorescence) are shown in Fig. 21.

[0044] <In vivo anti-cancer experiment and toxicity evaluation> All animal experiments were carried out with the approval of the Animal Experiment Committee of the Japan Advanced Institute of Science and Technology. Four-week-old female wild-type mice (n = 10; average weight = 15 g; BALB / cCrSlc) were purchased from Japan SLC, Inc. and acclimated for one week. A mixed solution (v / v, 1:1) consisting of a Colon26 cell dispersion (1×10 6 cells) (100 μL) and culture medium / matrigel (Corning) was administered subcutaneously at two sites on the lateral abdomen of the mice to create colon cancer model mice. Approximately two weeks later, solid cancer (~400 mm 3 ) were injected into the tail vein of mice with R. palustris (1 × 10 9 CFU ml -1 ) or PBS buffer solution (200 μL each). Near-infrared laser (wavelength 808 nm, output (713 mW, 36.3 mW mm )) was administered to the solid tumor on the right side only once every two days. -2 The tumor surface temperature was measured during laser irradiation using an IR thermography device (i7; FLIR, Nashua).

[0045] Near-infrared laser irradiation [wavelength: 808 nm, output: 0.9 W (ca. 45.9 mW / mm 2 ), irradiation time: 3 min]. 9 The results are shown in Figure 9. From the left, each graph in Figure 9 shows PBS, R.Palustris, PBS + Laser, and R.Palustris + Laser.

[0046] The behavior and characteristics of the mice, measurement of the solid tumor size, and changes in mouse weight were monitored every two days. The size of the solid tumor was calculated using the following formula. V = L × W 2 / 2 Here, V represents the volume of the solid tumor, L represents the length of the solid tumor, and W represents the width of the solid tumor.

[0047] Meanwhile, the in vivo toxicity of R. palustris was evaluated by conducting a mouse blood test. Specifically, R. palustris dispersion (1 × 10 9 CFU ml -1 ) or PBS buffer was administered in 200 μL each into the tail vein of 10-week-old female mice (n = 5; average weight = 21 g, BALB / cCrSlc, Japan SLC), and blood was collected from the abdominal aorta 7 and 30 days later. Complete blood cell counts (CBC) and biochemical tests were analyzed by Japan SLC, Inc. and Oriental Yeast Co., Ltd. The results of the CBC and biochemical tests are shown in Tables 1 and 2.

[0048] [Table 1]

[0049] [Table 2]

[0050] <Colony Count> Colon26-inoculated colon cancer model mice (female, 8 weeks; n = 5; average weight = 19 g; average tumor size ~ 400 mm 3 ;BALB / cCrSIc; Japan SLC) was injected into the tail vein of a mouse (5 × 10 9 CFU ml -1 ), B. viridis (5 × 10 9 CFU ml -1 ) and bacterial culture medium (200 μL each). After 1, 24, 48, and 144 hours, each tissue and tumor was excised, cut, and weighed. After homogenizing in PBS buffer at 4°C using a pestle, the mixture was stirred for 20 minutes at 15°C, 380 rpm, and min. -1 The supernatant was diluted 10-fold with PBS buffer, and each sample (100 μL) was plated on an agar medium and cultured anaerobically for 7 days. The number of bacterial colonies formed was manually counted.

[0051] Antitumor activity evaluation using near-infrared-activated R. palustris [PBS (200 μL) or R. palustris dispersion (200 μL, 1 × 10 9 CFU were administered via the tail vein of the mice, and the laser was irradiated only on the tumor on the right side of the mice (wavelength: 808 nm, output: 0.9 W (ca. 45.9 mW / mm 2 ), irradiation time: 3 min); the black arrows in the graph indicate the days when bacteria or PBS were administered, and the red arrows indicate the days when laser irradiation was performed] are shown in Figure 10. Photographs of the mice after each treatment (arrows indicate the tumors irradiated with the laser) are shown in FIG. Photographs of tumors excised 34 days after various treatments are shown in FIG. The survival rates of mice over 34 days for each treatment are shown in FIG.

[0052] A photograph of a red colony derived from R. palustris that grew specifically within the tumor (R. palustris (200 μL, 1 × 10 9 CFU / mL) was administered into the tail vein of a Colon 26 cancer-bearing mouse model. Two days later, various organs and tumors were removed, and the extract was applied to an agar medium and cultured under anaerobic conditions. Figure 15 shows the results. The number of viable R. palustris bacteria in various organs and tumors [R. palustris (200 μL, 1 × 10 9 CFU / mL) was administered into the tail vein of a Colon 26 cancer-bearing mouse model. Seven days later, various organs and tumors were removed, and the extracts were cultured on agar medium under anaerobic conditions. Figure 16 shows the results. Fluorescence intensity derived from R. palustris in various organs and tumors [R. palustris (200 μL, 1 × 10 9 CFU / mL) was administered via the tail vein to a Colon26 cancer-bearing model mouse, and fluorescence bioimaging analysis was performed over time to measure the fluorescence intensity. Viable counts of R. palustris in various organs and tumors [R. palustris (200 μL, 1 × 10 9 (CFU / mL) was administered to Colon26-bearing cancer model mice via the tail vein. Various organs and tumors were removed over time, the extract was applied to agar medium, and then cultured under anaerobic conditions. The number of colonies formed was measured, as shown in Fig. 18.

[0053] Photograph of green colonies derived from Blastochloris viridis that grew specifically in the tumor [Blastochloris viridis (200 μL, 1×10 9 CFU / mL) was administered to Colon26-bearing cancer model mice via the tail vein. Two days later, various organs and tumors were removed, the extract was applied to agar medium, and then cultured under anaerobic conditions, as shown in Fig. 19. Viable cell count of Blastochloris viridis in various organs and tumors [Blastochloris viridis (200 μL, 1×10 9 CFU / mL) was administered to Colon26-bearing cancer model mice via the tail vein. Seven days later, various organs and tumors were removed, the extract was applied to agar medium, and then cultured under anaerobic conditions, as shown in Fig. 20.

[0054] <In vivo fluorescence bioimaging> To measure the in vivo distribution of bacteria using NIR-I fluorescence, a mouse colon cancer model mouse consisting of Colon26 (female; 8 weeks; n = 3; average weight = 19 g; average tumor size = 430 mm 3 ; BALB / cCrSIc; Japan SLC) was administered a bacterial culture medium containing R. Palustris (200 μL, 1×10 9 CFU) or PBS buffer without bacteria via the tail vein. Near-infrared fluorescence images of the mice and major biological tissues were observed using VISQUE TM InVivo Smart-LF (Vieworks). The excitation wavelength and fluorescence wavelength were λ ex = 740 - 790 nm and λ em = 810 - 860 nm, respectively. Image analysis was performed using CleVue TM software (Vieworks). In vivo near-infrared I region (NIR-I) fluorescence bioimaging (Ex: 740 nm - 790 nm, Em: 810 nm - 860 nm) of Colon26-bearing cancer model mice [Image was taken 5 days after injecting R. Palustris (200 μL, 1 × 10 9 CFU / mL) or PBS (200 μL) into the tail vein of mice, and the arrow indicates the location of the tumor] is shown in Fig. 14.

[0055] NIR-II fluorescence bioimaging was performed by Summit Pharmaceuticals International. A human colon adenocarcinoma model mouse consisting of HT29 (female; 10 weeks; n = 3; average weight = 21 g; average tumor size = 100 mm 3 ; BALB / cSlc-nu / nu; Japan SLC) with solid cancer was administered a PBS buffer containing B. viridis (20 μL, 1 × 10 8 CFU). NIR-II fluorescence derived from B. viridis was observed using SAI-1000 (Shimazu) under a laser output of 2 W and an exposure time of 200 msec.

[0056] Angiography using NIR-II fluorescence of B. viridis was measured using the same system (laser output = 10 W, exposure time = 400 msec) after injecting a PBS buffer containing B. viridis (20 μL, 1 × 10 9 CFU) into the tail vein of human colon adenocarcinoma model mice consisting of the same HT29 (n = 3).

[0057] Angiography by near-infrared II region (NIR-II) fluorescence bioimaging using Blastochloris viridis (Ex: 980 nm, Em: 1000 nm - 1700 nm) [Image was taken after injecting B. viridis (20 μL, 10 9 CFU) into the tail vein of nude mice] is shown in Fig. 23.

[0058] <In vivo photoacoustic (PA) imaging> PA imaging was performed by Summit Pharmaceuticals International using a multispectral optoacoustic tomography (MSOT) inVision 256-TF system (SYS-MSOTiV256TF; iThera Medical). R. palustris (20 μL, 1 × 10 8 The PBS buffer solution containing the dispersed CFU was injected into HT29 human colon adenocarcinoma model mice (female; 10 weeks; n=3; average weight=21g; average tumor size=100mm). 3 PA images were obtained by administering the antibody to solid tumors of BALB / cSlc-nu / nu mice (Japan SLC).

[0059] Photoacoustic (PA) imaging of mouse tumors using R.Palustris [PBS (20 μL) (left side of photo), R.Palustris (20 μL, 10 8 CFU / mL) (right side of the photo) was administered and photographed] is shown in Figure 22.

[0060] <Statistical analysis of data> In the data, ± indicates standard deviation, and n indicates the number of samples used. Statistical analysis of the data was performed using Student's t-test. *, **, and *** indicate p values ​​of < 0.05, < 0.005, and < 0.001, respectively.

Claims

1. A therapeutic or diagnostic agent for cancer, comprising a photosynthetic bacterium, wherein the photosynthetic bacterium is a bacterium of the genus Rhodopseudomonas.

2. The cancer therapeutic or diagnostic agent according to claim 1 , which is used in combination with light irradiation.

3. The cancer therapeutic or diagnostic agent according to claim 2 , wherein the light irradiation is near-infrared light irradiation.

Images