Development and Application of a Self-Oxygen-Producing Sonosensitizer from Living Organisms

Abstract

The present invention provides a sonosensitizer with a simple preparation process, which utilizes the characteristic of cyanobacteria producing oxygen through photosynthesis after illumination to overcome the hypoxic microenvironment of tumors, and can exert a better sonodynamic effect in combination with specific ultrasound conditions to carry out effective sonodynamic treatment of targeted tumors.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a tumor-targeting sonosensitizer that can be economically and massively propagated and amplified without relying on external oxygen, and is used for sonodynamic therapy. Background Art

[0002] Photodynamic therapy (PDT) and sonodynamic therapy (SDT) are promising cancer treatment methods. They respectively generate reactive oxygen species (ROS) such as singlet oxygen and free radicals through chemical reactions between photosensitizers or sonosensitizers and water molecules or oxygen molecules in the environment under light or ultrasonic excitation, thereby killing tumor cells. Compared with traditional cancer treatment methods (such as surgery, chemotherapy, radiotherapy, etc.), PDT has the advantages of high selectivity for target tissues, low toxicity and side effects, and no damage to internal organs, but has poor penetration; compared with PDT, SDT also has the advantages of strong penetration and non-invasiveness, but has lower selectivity for target tissues. Sonodynamic therapy uses the strong tissue penetration ability of ultrasound to generate ROS or cavitation effects of sonosensitizers aggregated in deep tumors to treat tumors, which has successfully attracted wide attention. At present, tumor sonodynamic therapy has been used clinically, and three patients with advanced breast cancer have been successfully cured in combination with immunotherapy and endocrine therapy.

[0003] Although sonodynamic therapy has great advantages in the research field of malignant tumors and has achieved remarkable achievements, there are still two major bottleneck problems restricting its wide application in clinical treatment:

[0004] First, the low bioavailability of traditional sonosensitizers. As one of the three major elements of sonodynamic therapy, the ultrasonic absorption characteristics, sonodynamic activity, and targeting of sonosensitizers largely determine the sonodynamic efficacy and the clinical availability and scope of application of sonodynamic therapy. Currently, commonly used sonosensitizer molecules include: porphyrins, porphyrin derivatives, chemotherapy drug DOX, acid yellow, methylene blue, polyhydroxy fullerenes and other organic molecules. However, these traditional organic sonosensitizer molecules have problems of fast metabolism, poor targeting, and oxygen dependence, resulting in low sonodynamic therapy efficiency and seriously hindering the clinical promotion of this method.

[0005] Second, tumor hypoxia seriously restricts the improvement of sonodynamic efficacy. Hypoxia is a common symptom of most solid tumors such as breast cancer, liver cancer, and pancreatic cancer. It is mainly caused by increased oxygen consumption caused by rapid tumor proliferation and insufficient blood supply to tumor vessels. Oxygen is one of the most important reaction substrates involved in the sonodynamic treatment process. Therefore, the amount of oxygen content in tumor tissue plays a key role in the sonodynamic efficacy of tumors. Sonodynamic oxygen consumption aggravates local hypoxia in the tumor, thereby further reducing the efficacy of sonodynamic therapy. At the same time, tumor hypoxia will further induce genetic instability of tumor cells and activate some tumor survival factors, prompting the rapid formation of tumor neovascularization, causing tumor tolerance to chemotherapy and radiotherapy, promoting tumor recurrence, invasion and metastasis, and becoming one of the fundamental reasons why malignant tumors are difficult to cure.

[0006] To improve the problem of tumor hypoxia, the most common approach is to design nanoparticles using the basic characteristics of the tumor microenvironment that are different from normal tissues. Shi Jianlin's research group used a redox method to generate MnOx nanoparticles in situ on an organic mesoporous silicon channel and constructed a nanoprotease. The enzyme catalyzes the decomposition of excess H in the tumor microenvironment. 2 O 2 , effectively improving tumor hypoxia and significantly enhancing the efficiency of sonodynamic therapy. However, the efficiency of tumor oxygen production in this method depends on the concentration of nanomedicines and reaction substrates (such as hydrogen peroxide and water) in the tumor, and the final amount of oxygen increase is limited by the degree of oxygen-producing chemical reactions in the tumor. Secondly, the use of nanomaterials with good biocompatibility and degradability to encapsulate oxygen-carrying molecules such as perfluorocarbons and hemoglobin to prepare artificial nano oxygen carriers is another research idea. However, due to the inherent disadvantages of poor water solubility and short circulation time in the body, perfluorocarbons or fluorocarbon compounds need breakthroughs in size control and stability for their clinical application. Cai Lintao's research group used degradable polymers to encapsulate photosensitizer indocyanine green (ICG) and oxygen-carrying hemoglobin, and coated the surface of nanoparticles with a phospholipid layer similar to the red blood cell membrane to construct nano artificial red blood cells with oxygen-carrying and oxygen-releasing functions, which can break through the obstacles of tumor hypoxia microenvironment and insufficient oxygen supply to photodynamic therapy; laser triggering can produce cytotoxic singlet oxygen and high-valent iron hemoglobin, significantly enhancing the effect of photodynamic therapy. However, the oxygen-carrying capacity of hemoglobin is limited, and there are problems such as insufficient tumor oxygenation capacity.

[0007] In recent years, bacteria, which are the most widely distributed in nature, have the largest number of individuals, and have extremely strong reproductive abilities, have received increasing attention in the medical field. Due to their many advantageous characteristics, bacteria are well applied as "star carriers" in anti-cancer treatment: ① Bacteria are like small "robots" that can carry imaging probes to specifically target tumor cells, providing us with precise information about the tumor state and treatment effect, etc.; ② Bacteria can carry anti-tumor drug targets to tumor cells and are dispersed throughout the tumor tissue, which can greatly improve the delivery efficiency of therapeutic molecules; ③ The specific targeting effect of bacteria on tumors improves the treatment efficiency and reduces damage to normal tissues. The reason why bacteria can become a carrier for a new anti-tumor treatment strategy is closely related to their ability to target tumors and colonize in the hypoxic regions of tumors. Among them, cyanobacteria are a type of oxygen-producing photosynthetic bacteria that contain chlorophyll a, use water as a hydrogen donor and an electron donor, and produce oxygen through photocatalytic water. More importantly, the chlorophyll in cyanobacteria is a magnesium porphyrin compound with a sonodynamic effect.

[0008] In the prior art, CN109568577B discloses a targeted nanoparticle used as a photosensitizer / sonosensitizer, its preparation method and application. This patent uses PLGA to wrap the ZnCe6 complex as the core and modifies it to obtain a complex nanoparticle targeted to tumor cells. The structure and preparation process are complex; CN107670040B discloses a gold nanocage-manganese dioxide composite nanoparticle and its preparation. This patent needs to introduce manganese dioxide to degrade and release oxygen in the tumor microenvironment, thereby improving tumor hypoxia and enhancing the photodynamic therapeutic effect, and requires a more complex process. Although CN110812482A and CN110755457A disclose the use of the photosynthesis of cyanobacteria to produce oxygen, they only disclose its use as a photosensitizer for photodynamic therapy. The above prior arts are all the previous research results of this research group, mainly for photodynamic therapy, while the present invention is directed to sonodynamic therapy, especially for the improvement measures for the widespread technical defects existing in the prior art, such as the single type of sonosensitizer, lack of tumor targeting, poor stability, lack of good water solubility, and the innate hypoxia of solid tumors greatly reducing the efficacy of sonodynamic therapy. Although there are many methods to increase oxygen and enhance sonodynamic therapy, they all need to introduce other substances besides the sonosensitizer itself, which not only increases the insecurity but also requires a more complex preparation process. The present invention discovers that only by simply amplifying and culturing cyanobacteria in large quantities, with low cost and simple procedures, cyanobacteria can supply oxygen by themselves by giving specific light, overcome tumor hypoxia and exert a sonodynamic effect. Summary of the Invention

[0009] An object of the present invention is to provide a preparation method of a sonosensitizer, comprising the following steps:

[0010] (1) Pour photosynthetic bacteria and a bacterial culture medium into a culture flask or a culture dish, and culture under light;

[0011] (2) Place the photosynthetic bacteria obtained in step (1) under light of different wavelengths, and monitor the oxygen content in the solution with an oxygen meter to screen for the cyanobacteria that can produce the largest oxygen content in the cyanobacteria culture solution after light irradiation, and prepare them into a sonosensitizer; the photosynthetic bacteria are cyanobacteria, the wavelength in step (2) is 600 - 900 nm, and the light in step (2) includes laser light and LED light.

[0012] Preferably, the wavelength in step (2) above is 660 - 808 nm, and more preferably 660 nm.

[0013] Preferably, the density of viable cyanobacteria in the sonosensitizer is 1×10 6 CFU / mL to 1×10 10 CFU / mL, preferably 1 - 5×10 8 CFU / mL, further preferably 1.35 - 4.5×10 8 CFU / mL, preferably 2×10 8 CFU / mL.

[0014] The present invention also provides a sonosensitizer prepared by the above preparation method.

[0015] The present invention also provides an application of cyanobacteria in the preparation of a sonosensitizer with tumor targeting, and the sonosensitizer is prepared by the above preparation method.

[0016] The present invention also provides an application of a sonosensitizer in the preparation of a sonodynamic therapy drug. The sonodynamic therapy drug includes the sonosensitizer prepared by the above method. The sonodynamic therapy uses ultrasound. The sonosensitizer includes viable cyanobacteria that can produce oxygen under laser irradiation, and the laser wavelength is 660 - 808 nm; preferably, the laser wavelength is 660 nm.

[0017] Preferably, the conditions of the ultrasound include: 1.0 MHz, 2.0 W·cm -2 , 50% cycle, and treat for 5 - 30 minutes; preferably treat for 10 minutes.

[0018] The present invention also provides an in vitro sonodynamic tumor killing method for non - diagnostic or therapeutic purposes, and the method includes the following steps:

[0019] (1) Cultivate and amplify cyanobacteria in vitro;

[0020] (2) Evaluate the photosynthetic oxygen - producing ability of the cyanobacteria obtained in step (1) after light irradiation in an in vitro hypoxic environment.

[0021] (3) Screen for cyanobacteria that can produce the largest amount of oxygen in the cyanobacteria culture solution after irradiation with 660 - 808 nm laser, and prepare them into a sonodynamic tumor-killing reagent;

[0022] (4) Co-culture the sonodynamic tumor-killing reagent prepared in step (3) with a tumor cell line, perform ultrasonic treatment after irradiation with a laser of wavelength 660 - 808 nm, continue culturing, then add CCK8 into each well and continue incubation, and then detect the absorbance at 450 nm with an enzyme-labeled instrument to evaluate the growth of the cells.

[0023] Preferably, the density of cyanobacteria in the sonodynamic tumor-killing reagent described in step (4) is 1×10 6 CFU / mL to 1×10 10 CFU / mL, preferably 1 - 5×10 8 CFU / mL, more preferably 1.35 - 4.5×10 8 CFU / mL, preferably 2×10 8 CFU / mL.

[0024] Preferably, the ultrasonic treatment conditions described in step (4) include: 1.0 MHz, 22.0 W·cm -2 , 50% cycle, treat for 5 - 30 minutes, preferably treat for 10 minutes.

[0025] Preferably, the tumor cell line is from any one or more of the following: basal cell carcinoma, squamous cell carcinoma, esophageal cancer, malignant glioma, bladder cancer, cervical cancer, breast cancer, lung cancer, liver cancer, gastric cancer, colon cancer, rectal cancer, nasopharyngeal cancer, pancreatic cancer, thyroid cancer, prostate cancer, leukemia, lymphoma, kidney tumor, sarcoma, blastoma; more preferably, the tumor cell line is 4T1.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The present invention develops a new function of cyanobacteria that naturally exist in nature, aiming to provide a sonosensitizer with a simple preparation process, which targets tumors and exerts an effective sonodynamic effect. Specifically as follows:

[0028] (1) Utilize the oxygen-producing effect of cyanobacteria's photosynthesis and, under specific ultrasonic conditions, act as a sonosensitizer for sonodynamic therapy.

[0029] (2) Without the need to combine with other substances, only cyanobacteria itself can exert a sonodynamic therapy effect under specific ultrasonic conditions. The specific ultrasonic conditions are 1.0 MHz, 2.0 W·cm -2 , 50% cycle, ultrasonic for 10 minutes.

[0030] (3) Cyanobacteria can produce oxygen and singlet oxygen under 660 nm laser irradiation and can act as a sonosensitizer to combine with ultrasound to exert sonodynamic therapy. Cyanobacteria are living organisms that control oxygen production by controlling light. They can multiply as long as light is given in vivo, can reproduce in large numbers, and have a simple process and low cost.

[0031] (4) Without light, cyanobacteria will not grow, are easy to control, and can ensure safety in vivo. The specific targeting effect of cyanobacteria on tumors can improve the treatment efficiency and reduce damage to normal tissues. Cyanobacteria can carry anti-tumor drug targets to tumor cells and are dispersed throughout the tumor tissue, which can greatly improve the delivery efficiency of therapeutic molecules. Brief Description of the Drawings

[0032] Figure 1 The growth inhibition of cyanobacteria on 4T1. Among them, the Control group is the single 4T1 group without any treatment; the Syne group is the co-incubation group of cyanobacteria and 4T1; the Syne+L group is the group treated with 660 nm laser after co-incubation of cyanobacteria and 4T1; the Syne+US group is the group treated with ultrasound after co-incubation of cyanobacteria and 4T1; the Syne+L+US group is the group treated with 660 nm laser first and then ultrasound after co-incubation of cyanobacteria and 4T1. Detailed Embodiments

[0033] The following further elaborates on the present invention through specific embodiments, so that those skilled in the art can better understand the present invention and implement it, but the embodiments do not limit the present invention.

[0034] Unless otherwise specified, the experimental methods used in the following embodiments are all conventional methods. The materials, reagents, etc. used, unless otherwise specified, can all be obtained from commercial channels.

[0035] Embodiment

[0036] ① Prepare a 500 ml Erlenmeyer flask, pour photosynthetic bacteria and sterilized BG11 medium into it in a sterile operating table, seal the bottle mouth with an air-permeable sealing film, and place it under LED light for culture and amplification;

[0037] ② Place cyanobacteria under different wavelengths of light (dark, LED, 660 nm laser, 808 nm laser) and different colony densities, and monitor the oxygen content in the solution with an oxygen meter. It is found that cyanobacteria with the same density produce the most oxygen under 660 nm laser irradiation. And when under the same 660 nm laser irradiation, the higher the density of cyanobacteria, the more oxygen is produced;

[0038] ③ In an oxygen-deficient BG11 medium solution, where the density of cyanobacteria is 2*10 8CFU / mL (colony - forming units per milliliter), add the probe (SOSG) for detecting singlet oxygen into each well in advance, and then after irradiating with a 660 - nm laser for 5 minutes, ultrasound (1.0 MHz, 2.0 W·cm -2 , 50% cycle, 10 min) triggers the sonodynamic effect of cyanobacteria. The production level of singlet oxygen is quantified by detecting the absorbance of the solution with an enzyme - linked immunosorbent assay (ELISA) reader to evaluate the sonodynamic effect of cyanobacteria in an in vitro hypoxic environment;

[0039] ④ Co - culture cyanobacteria and the tumor cell line 4T1, after irradiating with a 660 - nm laser for 5 minutes, ultrasound (1.0 MHz, 2.0 W·cm -2 , 50% cycle) for 10 minutes, continue culturing for 24 hours, then add CCK8 into each well and continue incubating for 1 hour, and then detect the absorbance at 450 nm with an ELISA reader to evaluate the growth of 4T1 cells, further verifying the antitumor effect of the sonodynamic action of cyanobacteria.

[0040] The above - mentioned are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Use of a cyanobacterium in the preparation of a sonosensitizer targeting tumors, and a method for preparing the sonosensitizer, comprising the following steps: (1) Pour photosynthetic bacteria and a bacterial culture medium into a culture flask or a culture dish, and culture under light. (2) Place the photosynthetic bacteria obtained in step (1) under light of different wavelengths, monitor the oxygen content in the solution with an oxygen meter, screen the cyanobacterium that can produce the largest oxygen content in the cyanobacterium culture solution after light irradiation, and prepare it into a sonosensitizer; the photosynthetic bacteria are cyanobacteria, the wavelength in step (2) is 600 - 900 nm, and the light in step (2) includes laser light and LED light.

2. The use according to claim 1, wherein, The density of live cyanobacteria in the photosensitizer is 1×10 6 CFU / mL to 1×10 10 CFU / mL.

3. The use according to claim 1, wherein, the wavelength in step (2) is 660 - 808 nm.

4. The use according to claim 1, wherein, the wavelength in step (2) is 660 nm.

5. The use according to claim 1, wherein, The density of live cyanobacteria in the photosensitizer is 1-5×10 8 CFU / mL.

6. The use according to claim 1, wherein, The density of live cyanobacteria in the photosensitizer is 1.35 - 4.5×10 8 CFU / mL.

7. The use according to claim 1, wherein, The density of live cyanobacteria in the photosensitizer is 2×10 8 CFU / mL.

8. Use of a sonosensitizer in the preparation of a sonodynamic therapy drug, the sonodynamic therapy drug comprising the sonosensitizer prepared by the preparation method according to any one of claims 1 to 7, the sonodynamic therapy using ultrasound, the sonosensitizer comprising live cyanobacteria that can produce oxygen under laser irradiation, and the laser wavelength is 660 - 808 nm.

9. The use according to claim 8, wherein, the laser wavelength is 660 nm.

10. The use according to claim 8, wherein, the conditions of the ultrasound are: 1.0 MHz, 2.0 W·cm-2, 50% cycles, and treated for 5 - 30 minutes.

11. The use according to claim 8, wherein, the conditions of the ultrasound are: 1.0 MHz, 2.0 W·cm-2, 50% cycles, and treated for 10 minutes.

12. An in vitro sonodynamic tumor killing method for non-diagnostic or therapeutic purposes, the method comprising the following steps: (1) In vitro culture and amplification of cyanobacteria; (2) Evaluate the photosynthetic oxygen production ability of the cyanobacteria obtained in step (1) after light irradiation in an in vitro hypoxic environment; (3) Screen the cyanobacterium that can produce the largest oxygen content in the cyanobacterium culture solution after laser irradiation at 660 - 808 nm, and prepare it into a sonodynamic tumor killing reagent; (4) Co-culture the sonodynamic tumor killing reagent prepared in step (3) with a tumor cell line, perform ultrasonic treatment after laser irradiation at a wavelength of 660 - 808 nm, continue to culture, then add CCK8 to each well and continue to incubate, and then detect the absorbance at 450 nm with an enzyme-linked immunosorbent assay to evaluate the cell growth.

13. The method according to claim 12, wherein, The density of cyanobacteria in the sonodynamic tumor-killing reagent described in step (4) is 1×10 6 CFU / mL to 1×10 10 CFU / mL.

14. The method according to claim 12, wherein, The density of cyanobacteria in the sonodynamic tumor-killing reagent described in step (4) is 1 - 5×10 8 CFU / mL.

15. The method according to claim 12, wherein, The density of cyanobacteria in the sonodynamic tumor-killing reagent described in step (4) is 1.35 - 4.5×10 8 CFU / mL.

16. The method according to claim 12, wherein, The density of cyanobacteria in the sonodynamic tumor-killing reagent described in step (4) is 2×10 8 CFU / mL.

17. The method according to claim 12, wherein, The ultrasonic treatment conditions in the step (4) are: 1.0 MHz, 2.0 W·cm -2 , 50% cycle, and the treatment is carried out for 5 - 30 minutes.

18. The method according to claim 12, wherein, The ultrasonic treatment conditions described in step (4) are: 1.0 MHz, 2.0 W·cm -2 , 50% cycle, for 10 minutes.

19. The application according to any one of claims 1 to 7 or the method according to any one of claims 12 to 18, wherein, the tumor is selected from any one or more of the following: basal cell carcinoma, squamous cell carcinoma, esophageal cancer, malignant glioma, bladder cancer, cervical cancer, breast cancer, lung cancer, liver cancer, gastric cancer, colon cancer, rectal cancer, nasopharyngeal carcinoma, pancreatic cancer, thyroid cancer, prostate cancer, leukemia, lymphoma, kidney tumor, sarcoma, blastoma.

20. The application according to any one of claims 1 to 7 or the method according to any one of claims 12 to 18, wherein, the tumor is the tumor cell line 4T1.

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