An organic afterglow nanoparticle combination agent, its preparation method and application

Abstract

The present invention belongs to the fields of nanomaterials and biomedical applications, and specifically relates to a combined agent of organic afterglow nanoparticles and its preparation method and application. The combined agent of the present invention includes chlorin E6 nanoparticles and cyanobacteria. The chlorin E6 nanoparticles in the combined agent can be excited and activated by ultrasound to generate afterglow, and the generated afterglow can stimulate cyanobacteria to produce oxygen. At the same time, the activated chlorin E6 nanoparticles can also convert the surrounding oxygen into singlet oxygen, enabling the combined agent of the present invention to generate oxygen in situ at the tumor site and convert it into singlet oxygen, improving the therapeutic efficacy of the combined agent against tumors, solving the problems of poor light penetration effect of existing photosensitizing drugs and insufficient oxygen production at the tumor site, improving the treatment effect, and having mild treatment conditions, reducing the discomfort of patients.

Description

Technical Field

[0001] The present invention belongs to the fields of nanomaterials and biomedicine. Specifically, it relates to a combined medicament based on photoacoustic dynamic therapy and its application in treating tumors. Background Art

[0002] In recent decades, the incidence and mortality of cancer have been steadily rising, seriously threatening human health. Therefore, the development of cancer treatment methods has always been the focus of the medical community. The current anti-tumor treatment strategies are not optimistic. The main reasons are that tumors are resistant to treatment, the treatment methods have large side effects on normal human tissues, poor prognosis, high costs, etc.

[0003] As a new cancer treatment method, sonodynamic therapy (SDT) has been favored because of its obvious curative effect, small toxic and side effects, strong targeting, simple operation, strong repeatability, etc. The process of sonodynamic treatment of malignant tumors is mainly through oral or injection of sonosensitizers by tumor patients. The sonosensitizers are distributed throughout the body through the circulatory system and can specifically accumulate in tumor cells. Then, the tumor location is irradiated with ultrasonic waves of a certain frequency and intensity. The acoustic energy activates the sonosensitizer to cause a series of chemical reactions, such as converting the surrounding molecular oxygen into singlet oxygen, resulting in irreversible damage to tumor cells, thereby achieving the treatment purpose. Among them, a class of substances mediating sonodynamic therapy is defined as sonosensitizers.

[0004] However, in the tumor microenvironment, due to the imbalance between rapid tumor growth and new blood vessels, an obvious hypoxic environment appears in the tumor site. The hypoxic microenvironment of tumors will affect the generation of singlet oxygen, and thus seriously affect the efficacy of SDT, making tumors resistant to various anti-cancer treatments. Therefore, choosing an effective strategy to relieve tumor hypoxia has become a key step in tumor treatment and is of great significance for improving the treatment effect.

[0005] Benefiting from the development of nanotechnology, there are currently two main strategies to address the problem of tumor hypoxia: "in-situ oxygen-producing nanomaterials" and "oxygen-carrying nanomaterials". The former decomposes hydrogen peroxide highly expressed in tumors through catalysis to produce oxygen, such as manganese dioxide nanoparticles; the latter carries oxygen to the tumor site through its own high dissolved oxygen characteristics, such as perfluorocarbon. However, these two types of materials also have their obvious disadvantages: uncontrolled oxygen production and insufficient oxygen-carrying capacity.

[0006] As a photosynthetic microorganism commonly existing in nature, cyanobacteria have formed a complex photosynthetic oxygen-producing system during the evolution process and have the ability of photocatalytic oxygen production. Therefore, they are considered natural oxygen producers. A large number of studies have proved that cyanobacteria can produce oxygen through light irradiation at the lesion site to relieve the hypoxic environment inside tumors, so as to improve the treatment effect, and their biosafety is good.

[0007] For example, Chinese Patent CN202010553755.X discloses a near-infrared-driven self-sustaining composite and its preparation method and application, which involves loading a photosensitizer and upconversion nanoparticles onto cyanobacteria. Under the irradiation of near-infrared light, the cyanobacteria are stimulated to produce oxygen, which is converted into singlet oxygen under the action of the photosensitizer.

[0008] Another example is Japanese Patent JP2005246013A, which discloses a photochemotherapy method that utilizes photolyase produced by cyanobacteria to generate oxygen.

[0009] Most of the above patents focus on using photosensitizers for tumor treatment under the action of light. However, the current research results are usually easily restricted by the penetration ability of external light sources, and there are safety hazards for patients under long-term laser exposure, which limits the development and application of cyanobacteria in the field of treatment.

[0010] Therefore, it is necessary to develop a safer and more effective method and thus develop a drug to improve the treatment effect of tumors and reduce the pain of patients during the treatment process. Summary of the Invention

[0011] The object of the present invention is to propose an organic sonoluminescence nanoparticle combination agent based on photoacoustic dynamic therapy, and provide a preparation method and application of the agent to solve the problems raised in the above background technology. The agent, preparation method and application disclosed in the present invention solve the disadvantages of poor light penetration effect of existing photosensitive drugs and insufficient oxygen production at the tumor site. The combination agent of the present invention generates oxygen in situ at the tumor site and converts it into singlet oxygen, which can efficiently kill tumor cells, improve the treatment efficacy of the combination agent on tumors, and relieve the discomfort and pain of patients.

[0012] In the first aspect, the present invention provides an organic sonoluminescence nanoparticle combination agent, which comprises chlorin E6 nanoparticles and cyanobacteria. The concentration of chlorin E6 nanoparticles in the combination agent is 100-500 μg / mL, and the concentration of the cyanobacteria is 2.5×10 7 ~12.5×10 7 cell / mL.

[0013] In the present invention, for the first time, chlorin E6 nanoparticles are used as sonoluminescence agents and used as therapeutic drugs for tumor photoacoustic dynamic therapy under the action of ultrasound. Chlorin E6 nanoparticles have excellent sonodynamic therapy performance and can eliminate cancer cells by generating singlet oxygen. Specifically, under the action of ultrasound, the chlorin E6 particles aggregated in the tumor can be activated to produce sonoluminescence, thereby stimulating the cyanobacteria to produce oxygen. The activated chlorin E6 particles then transfer energy to convert the surrounding oxygen into reactive oxygen species mainly singlet oxygen, and kill tumor cells under the synergistic action of ultrasound.

[0014] Preferably, the particle size of the chlorophyllide E6 nanoparticles is 20-50 nm, and the length of the cyanobacteria is 1-2 μm.

[0015] Preferably, the concentration of the chlorophyllide E6 nanoparticles is 500 μg / mL, the particle size of the chlorophyllide E6 nanoparticles is 50 nm, and the concentration of the cyanobacteria is 5×10 7 cell / mL.

[0016] In a second aspect, the present invention provides a method for preparing the above-mentioned combined medicament of organic afterglow nanoparticles. Specifically, the preparation method includes the following steps:

[0017] In a tetrahydrofuran medium, chlorophyllide E6 is modified using a polyether to obtain modified chlorophyllide E6; the modified chlorophyllide E6 is self-assembled in water to form chlorophyllide E6 nanoparticles;

[0018] The chlorophyllide E6 nanoparticles and cyanobacteria are mixed in water to obtain the combined medicament of organic afterglow nanoparticles.

[0019] Preferably, the chlorophyllide E6 nanoparticles are prepared by a bottom-up reaction. Specifically, it includes the following steps:

[0020] After dissolving the polyether in tetrahydrofuran, chlorophyllide E6 is added thereto to form a suspension. The suspension is dropped into water and stirred and dispersed. Tetrahydrofuran is removed by rotary evaporation, and then the chlorophyllide E6 nanoparticles are obtained by ultrafiltration purification.

[0021] Since chlorophyllide E6 itself is hardly soluble in water, in the present invention, chlorophyllide E6 is hydrophilically modified using a polyether in a reaction medium of tetrahydrofuran, so that the hydrophilically modified chlorophyllide E6 can self-assemble into nanoparticles in an aqueous solution.

[0022] Since the reaction medium tetrahydrofuran is cytotoxic, tetrahydrofuran is removed by rotary evaporation, and the rotary evaporation solution after removing tetrahydrofuran is ultrafiltered and purified to obtain a concentrated solution of chlorophyllide E6 nanoparticles.

[0023] Preferably, the dosage ratio of the polyether to tetrahydrofuran is (10-18) mg:1 mL, and the mass ratio of the polyether to chlorophyllide E6 is (20-50):1.

[0024] Preferably, the polyether is polyether F127.

[0025] Preferably, to make the polyether dissolve more fully and uniformly in tetrahydrofuran, an ultrasonic dissolution method is used with an ultrasonic cleaner, wherein the ultrasonic time is 0.5-1 h.

[0026] Preferably, the stirring is magnetic stirring, the rotation speed is 450 - 550 rpm, and the stirring time is 1 - 1.5 h.

[0027] Preferably, the rotary evaporation temperature is 15 - 20 °C, the rotary evaporation speed is 50 - 60 rpm, and the rotary evaporation time is 10 - 12 h.

[0028] Preferably, the above ultrafiltration is centrifugal ultrafiltration using a 30KD ultrafiltration tube, the centrifugation speed is 3500 - 4500 g, and the time is 10 - 15 minutes.

[0029] In a third aspect, the present invention provides an application of the above-mentioned organic sonoluminescence nanoparticle combined medicament in the preparation of a medicament for treating tumors.

[0030] Preferably, the above tumor is a cutaneous tumor.

[0031] Specifically, when in use, the organic sonoluminescence nanoparticle combined medicament of the present invention is used by intratumoral injection. Under the stimulation of ultrasound, the chlorophyllide E6 particles aggregated in the tumor are converted into an activated state to produce afterglow, which stimulates the oxygen production of cyanobacteria. The activated chlorophyllide E6 particles can also convert the surrounding oxygen into singlet oxygen through energy transfer, and kill tumor cells under the synergistic effect of ultrasound.

[0032] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0033] The organic sonoluminescence nanoparticle combined medicament disclosed by the present invention, compared with the method of exciting cyanobacteria to produce oxygen using an external light source, the in-situ luminescent chlorophyllide E6 particles adopted by the present invention have the effects of directly photoexciting cyanobacteria to produce oxygen and converting the oxygen produced by cyanobacteria into singlet oxygen, improving the ability to kill tumor cells. When using the organic sonoluminescence nanoparticle combined medicament of the present invention to treat tumors, the treatment conditions are more friendly and the drug efficacy is exerted more rapidly.

[0034] The organic sonoluminescence nanoparticle combined medicament disclosed by the present invention has unique afterglow properties, good biocompatibility, excellent oxygen production ability and remarkable sonodynamic efficacy. As a highly lethal anti-tumor medicament, this medicament combines the advantages of endogenous luminescence, alleviating hypoxia and ultrasonic catalysis, effectively solving the problem of reduced treatment effect caused by hypoxia. In addition, the problem of poor penetration ability of the external light source and the safety problem of long-term laser irradiation are further overcome through endogenous afterglow luminescence. Therefore, the technical solution disclosed by the present invention is more friendly to patients, has obvious treatment effects, and has good application prospects and far-reaching significance. Description of the Drawings

[0035] To more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the accompanying drawings required for the embodiments. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as limiting the scope. For those of ordinary skill in the art, without creative efforts, other relevant drawings can also be obtained based on these drawings.

[0036] Figure 1 It is the transmission electron microscope (TEM) photograph of chlorophyllide E6 particles in Example 1;

[0037] Figure 2 It is the transmission electron microscope (TEM) photograph of cyanobacteria in Example 1;

[0038] Figure 3 It is the particle size distribution diagram of chlorophyllide E6 nanoparticles in water in Example 1;

[0039] Figure 4 It is the particle size distribution diagram of cyanobacteria in water in Example 1;

[0040] Figure 5 It is the afterglow spectrum diagram of chlorophyllide E6 nanoparticle solutions with different concentrations in Example 1;

[0041] Figure 6 It is the ultraviolet absorption spectrum diagram of cyanobacteria suspensions with different concentrations in Example 1;

[0042] Figure 7 It is the change curve of dissolved oxygen in the aqueous solution after cyanobacteria in Example 2 are excited by chlorophyllide E6 particle solutions with different concentrations;

[0043] Figure 8 It is the change diagram of the ultraviolet characteristic absorption peak of DPBF after three different treatments in Example 3;

[0044] Figure 9 It is the electron spin resonance diagram of three different medicaments mixed with TEMP spin trap agent under ultrasonic in Example 3;

[0045] Figure 10 It is for the aqueous solution of chlorophyllide E6 nanoparticles in Example 4 with a power of 1.2 W / cm 2 The afterglow intensity changes with time after ultrasonic treatment for 1 min, and the afterglow intensity changes with time after ultrasonic treatment for another 1 min with the same power;

[0046] Figure 11 (a) and Figure 11 (b) respectively show the aqueous solution of chlorophyllide E6 nanoparticles in Example 5 with a power of 1.2 W / cm 2The afterglow intensity of the aqueous solution of chlorin E6 nanoparticles under ultrasonic treatment at different ultrasonic times and the afterglow intensity of the aqueous solution of chlorin E6 nanoparticles under ultrasonic treatment for 1 minute at different ultrasonic powers;

[0047] Figure 12 For the aqueous solution of chlorin E6 nanoparticles in Example 6, the afterglow intensities after ultrasonic treatment at a power of 1.2 W / cm 2 for 1 min and blue light irradiation for 1 min;

[0048] Figure 13 (a) and Figure 13 (b) respectively show the graphs of the changes in cell viability after incubating different concentrations of chlorin E6 nanoparticle solutions and different concentrations of cyanobacteria with tumor cells for 12 hours in Example 7;

[0049] Figure 14 (a) and Figure 14 (b) respectively show the control graphs of the killing effects of different ultrasonic times and different treatment methods on tumor cells detected by the CCK-8 method in Example 8;

[0050] Figure 15 (a) and Figure 15 (b) respectively show the levels of ROS in tumor cells in different treatment groups detected by the DCFH detection method in Example 9;

[0051] Figure 16 (a) shows the relationship between the tumor volume of nude mice and time after different treatments, Figure 16 (b) shows the digital photos of tumor treatments in different groups in Example 10. Detailed implementation manners

[0052] To better illustrate the purpose, technical solution and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. Those skilled in the art should understand that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. The test methods used in the embodiments are all conventional methods unless otherwise specified; the materials, reagents, etc. used are all commercially available unless otherwise specified.

[0053] Example 1 Synthesis of chlorin E6 nanoparticles, cultivation of cyanobacteria and preparation of combined medicaments

[0054] Add 45 mg of polyether to 3 mL of tetrahydrofuran and sonicate for 1 h to completely dissolve the polyether in tetrahydrofuran. Then add 1.5 mg of chlorin e6 powder and dissolve it completely. Next, slowly drop the obtained mixed solution into 27 mL of deionized water and continuously stir magnetically for 1 h at a rotation speed of 450 - 550 rpm. Then perform rotary evaporation at a low temperature of 15 - 20 °C in the dark, with a rotary evaporation speed of 50 - 60 rpm and a rotary evaporation time of 10 - 12 h until the solution turns yellow and the tetrahydrofuran in the solution is completely removed. Further purify the concentrated solution after rotary evaporation by centrifuging at 4500 g for 10 min through a 30 KD ultrafiltration tube, collect the solution, and obtain the concentrated solution of the chlorin e6 nanoparticles described in the present invention, which is stored in a refrigerator at 4 °C for standby.

[0055] Culture cyanobacteria in a triangular flask containing BG11 liquid medium at a culture temperature of 27 °C, a light intensity of 25 mol / (m 2 ·s), irradiate for 24 h, with a shaker speed of 150 - 200 rpm, and seal it with a sterile sealing film for standby.

[0056] The combined agent is composed of an aqueous solution of chlorin e6 nanoparticles and cyanobacteria. The concentration of chlorin e6 nanoparticles in the combined agent is 100 - 500 μg / m, and the content of cyanobacteria is 2.5×10 7 ~12.5×10 7 cell / mL.

[0057] For example, if a chlorin e6 nanoparticle solution with a concentration of 1000 μg / mL is mixed with a cyanobacteria aqueous suspension with a content of 1×10 8 cell / mL in a volume ratio of 1:1, the concentration of chlorin e6 nanoparticles in the obtained combined agent is 500 μg / mL, and the content of cyanobacteria is 5×10 7 cell / mL.

[0058] The transmission electron microscope (TEM) images of the chlorin e6 nanoparticles prepared in this example are as shown in Figure 1 , and the TEM images of the cyanobacteria cultured in the present invention are as shown in Figure 2 . It can be seen from Figure 1 and Figure 2 that spherical chlorin e6 nanoparticles are formed by a bottom-up reaction in the present invention, and the cyanobacteria cultured in the present invention are rod-shaped.

[0059] The particle size distribution diagrams of the chlorin e6 nanoparticles and cyanobacteria prepared in this example in water are respectively as shown in Figure 3 and Figure 4As shown, it can be seen that the chlorin E6 nanoparticles prepared by the present invention have good stability and dispersibility and can be stored for a long time. The particle size range of the chlorin E6 nanoparticles is 20-50 nm, and the average length of cyanobacteria is 1-2 μm.

[0060] In this example, the afterglow spectra of chlorin E6 nanoparticle solutions with different concentrations (200 μg / mL, 300 μg / mL, 400 μg / mL) after ultrasonic excitation are as Figure 5 shown. The afterglow peak is at 670 nm. The chlorin E6 nanoparticle solutions with different concentrations are obtained by dispersing the concentrated chlorin E6 nanoparticle solution prepared by the above method in deionized water; in this example, the ultraviolet absorption spectra of cyanobacteria aqueous suspensions with different concentrations (2.5×10 7 ~15×10 7 cell / mL) are as Figure 6 shown. The absorption peaks are at 450 nm and 660 nm. Figure 5 and Figure 6 The results shown indicate that cyanobacteria can absorb the afterglow emission of chlorin E6 after ultrasonic excitation.

[0061] Example 2 Testing the oxygen production performance of chlorin E6 nanoparticles by exciting cyanobacteria

[0062] The chlorin E6 nanoparticles excited by ultrasound (1.2 W / cm 2 , 1 min) were placed in a beaker, and cyanobacteria were added thereto. The concentration of chlorin E6 nanoparticles in the mixture of chlorin E6 nanoparticles and cyanobacteria was adjusted to 100, 200, and 300 μg / mL in sequence by deionized water. The content of cyanobacteria was 5×10 7 cell / mL, and the total volume of the cyanobacteria and chlorin E6 nanoparticle solution was 20 ml. The probe of the dissolved oxygen tester was inserted into the solution, and the detection value was recorded every 1 min. The results are as Figure 7 shown. The afterglow generated by the chlorin E6 nanoparticles after ultrasonic excitation can effectively stimulate cyanobacteria to produce oxygen, and the oxygen production efficiency of cyanobacteria increases with the increase in the concentration of chlorin E6 nanoparticles.

[0063] Example 3 Testing the ultrasonic performance of chlorin E6 nanoparticles

[0064] The aqueous solution of chlorin E6 nanoparticles was filled into a cuvette. The concentration of chlorin E6 nanoparticles was 100 μg / mL, and the total volume was 2 mL. Then DPBF (1,3-diphenylisobenzofuran) was added, and the concentration of DPBF was 10 μM. Then it was ultrasonically treated at 1.2 W / cm 2 . The ultraviolet absorption spectrum was detected every 1 min of ultrasonic treatment. At the same time, a pure DPBF solution without adding chlorin E6 nanoparticles and an additional solution with a final concentration of 1×107 Cyanobacteria at a concentration of Figure 8 cells / mL were used for comparative experiments under the same conditions, and the results are as

[0065] shown. Figure 8 It can be seen from

[0066] that singlet oxygen is significantly generated after ultrasonic treatment after adding chlorophyllide E6 nanoparticles, and after adding cyanobacteria, the generation of singlet oxygen increases, and the characteristic peak decreases most significantly. Moreover, the longer the ultrasonic treatment time, the more obvious the effect. 7 1 mL of chlorophyllide E6 nanoparticles (concentration: 100 μg / mL) was mixed with 1 μL of 2,2,6,6-tetramethyl-4-piperidine (TEMP), and cyanobacteria with a final concentration of 1×10 2 cells / mL were additionally added. Then, ultrasonic treatment was carried out at 1.2 W / cm Figure 9 for 3 minutes, and the results are as Figure 9 shown. It can be known from

[0067] Example 4: Test on the afterglow performance of chlorophyllide E6 nanoparticles

[0068] The chlorophyllide E6 nanoparticle solution was added to a 48-well plate, with the concentration of chlorophyllide E6 nanoparticles being 400 μg / mL and the total volume being 500 μL. After ultrasonic excitation at 1.2 W / cm 2 for 1 minute, the afterglow intensity was detected every 2 minutes using a small animal in vivo imaging system. At the 16th minute, the chlorophyllide E6 nanoparticles were ultrasonically excited again at 1.2 W / cm 2 for 1 minute, and the afterglow intensity was detected for a period of time.

[0069] The results are as Figure 10 shown. It can be seen from Figure 10 that after ultrasonic excitation, the chlorophyllide E6 nanoparticles have a detectable afterglow lasting up to 16 minutes, and after ultrasonic re-excitation, the afterglow intensity increases.

[0070] Example 5

[0071] The chlorophyllide E6 nanoparticle solution was added to a 48-well plate, with the concentration of chlorophyllide E6 nanoparticles being 400 μg / mL and the total volume being 500 μL. Ultrasonic treatment was carried out at 1.2 W / cm 2)After excitation, the afterglow intensity of chlorophyllide E6 nanoparticles was detected using a small animal in vivo imaging system.

[0072] Figure 11 (a) Under ultrasonic excitation with a power density of 1.2 W / cm 2 the afterglow intensity of chlorophyllide E6 nanoparticles was investigated at different ultrasonic times; Figure 11 (b) Under ultrasonic excitation for 1 min, the afterglow intensity of chlorophyllide E6 nanoparticles was investigated at different ultrasonic powers. It can be seen from Figure 11 that the afterglow intensity of chlorophyllide E6 nanoparticles increases with the increase of ultrasonic time and ultrasonic power.

[0073] Example 6

[0074] The chlorophyllide E6 nanoparticle solution was added to a 48-well plate with a chlorophyllide E6 nanoparticle concentration of 400 μg / mL and a total volume of 500 μL. Using chicken breast meat to simulate tissue penetration, it was excited by ultrasound and blue light respectively, and the afterglow intensity of chlorophyllide E6 nanoparticles was detected using a small animal in vivo imaging system. The results are as Figure 12 shown.

[0075] It can be seen from Figure 12 that at a thickness of 1 mm, the afterglow intensities under ultrasonic and light excitations are similar. As the thickness of the chicken breast meat increases (1 mm, 3 mm, 5 mm), the decline trend of the afterglow intensity under light excitation is more obvious compared to ultrasound. This is because as the tissue thickness increases, the penetration ability of light is relatively weak, while the tissue penetration ability of ultrasound is strong, indicating that ultrasound excitation has better tissue penetration ability for afterglow.

[0076] Example 7 Material Biocompatibility Detection

[0077] The CCK-8 method was used to evaluate the cytotoxicity of chlorophyllide E6 nanoparticles and cyanobacteria at different concentrations. 4T1 cells were seeded in a 96-well plate and cultured overnight. The RPMI-1640 culture medium containing chlorophyllide E6 nanoparticle solutions at different concentrations (0, 100, 200, 300, 400, 500 μg / mL) and cyanobacteria solutions (0, 2.5, 5, 7.5, 10, 12.5×10 7 cell / mL) was loaded onto the 96-well plate and incubated with the cells for another 8 hours. Then, after co-incubation with the CCK-8 solution for 30 min, the cell viability was evaluated using a microplate reader at an absorbance of 450 nm. The results are as Figure 13 (a) and Figure 13(b), it can be seen that after incubating with 4T1 cells for 8 hours with different concentrations of chlorophyllide E6 nanoparticles and cyanobacteria, the cell viability always remains at a high level, indicating that the chlorophyllide E6 nanoparticles and cyanobacteria of the present invention have little toxicity to cells themselves, and show good biocompatibility within the experimental concentration range.

[0078] Example 8 Evaluation of the killing effect of organic afterglow nanoparticle combination agents on tumor cells by CCK-8 method

[0079] In order to evaluate the killing effect of organic afterglow nanoparticle combination agents on 4T1 tumor cells under different treatment methods by CCK-8 method, the experimental method is as follows:

[0080] Inoculate 4T1 tumor cells into 96-well plates and culture overnight. Divide them into six groups according to different treatment methods. Load 100 μl of experimental solution onto 96-well plates respectively and incubate with the cells for 4 h. Finally, after co-incubating with CCK-8 solution for 30 min, evaluate the cell viability by measuring the absorbance at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader. Among them, the six different treatment methods are as follows:

[0081] Blank control: The experimental solution used is RPMI-1640 medium;

[0082] Ultrasound group: The experimental solution used is RPMI-1640 medium, and ultrasound treatment is carried out during the co-incubation with the cells. The ultrasound power is 1.2 W / cm 2 , and the ultrasound time is 3 min;

[0083] Chlorophyllide E6 group: The experimental solution used is RPMI-1640 medium containing 400 μg / mL chlorophyllide E6 nanoparticles;

[0084] Chlorophyllide E6 + cyanobacteria group: The experimental solution used is RPMI-1640 medium containing 400 μg / mL chlorophyllide E6 nanoparticles and 5×10 7 cells / mL cyanobacteria;

[0085] Chlorophyllide E6 + ultrasound group: The experimental solution used is RPMI-1640 medium containing 400 μg / mL chlorophyllide E6 nanoparticles, and ultrasound treatment is carried out during the co-incubation with the cells. The ultrasound power is 1.2 W / cm 2 , and the ultrasound time is 3 min;

[0086] Chlorophyllide E6 + cyanobacteria + ultrasound group: The experimental solution used is RPMI-1640 medium containing 400 μg / mL chlorophyllide E6 nanoparticles and 5×10 7RPMI-1640 medium with cyanobacteria at a density of 2 cell / mL, and ultrasonic treatment was carried out during co-incubation with the cells. The ultrasonic power was 1.2 W / cm

[0087] The results are as Figure 14 shown. In the figure, "Control", "Ultrasound", "Chlorophyllide E6", "Chlorophyllide E6 + Cyanobacteria", "Chlorophyllide E6 + Ultrasound" and "Chlorophyllide E6 + Cyanobacteria + Ultrasound" represent the blank control group, the ultrasound group, the Chlorophyllide E6 group, the Chlorophyllide E6 + Cyanobacteria group, the Chlorophyllide E6 + Ultrasound group, and the Chlorophyllide E6 + Cyanobacteria + Ultrasound group, respectively.

[0088] Figure 14 (a) shows that the simple ultrasound group (even with long-term ultrasound) did not inhibit the viability of tumor cells, while the Chlorophyllide E6 + Cyanobacteria + Ultrasound group showed a significant decrease in the viability of tumor cells with the increase of ultrasound time; Figure 14 (b) shows that the Chlorophyllide E6 + Ultrasound group could reduce the viability of tumor cells, while the Chlorophyllide E6 + Cyanobacteria + Ultrasound group, due to the production of oxygen by cyanobacteria under afterglow excitation, further reduced the viability of tumor cells, proving that the ability to inhibit tumor cells can be improved after cyanobacteria produce oxygen under the afterglow excitation of Chlorophyllide E6.

[0089] Example 9 Evaluation of the ROS level in tumor cells after different treatments by DCFH assay and evaluation of the performance of tumor cells producing ROS after different treatments

[0090] 4T1 tumor cells were divided into 6 groups according to different treatment methods in Example 8 (blank control, ultrasound group, Chlorophyllide E6 group, Chlorophyllide E6 + Cyanobacteria group, Chlorophyllide E6 + Ultrasound group, Chlorophyllide E6 + Cyanobacteria + Ultrasound group). The tumor cells were inoculated in a 6-well plate (2×10 5 cells) and attached to the culture dish, and cultured overnight. According to the treatment methods of each group described in Example 8, 100 μL of the experimental solution was added to the well plate and co-incubated with the cells for 2 hours. The results are as Figure 15 shown. The results show that the Chlorophyllide E6 + Cyanobacteria + Ultrasound group produced strong ROS, proving that under ultrasonic excitation, the combined medicament of the present invention can significantly increase the ROS level in tumor cells.

[0091] A 4T1 tumor model was established by subcutaneously injecting 4T1 cells (1×10 6 cells, in 100 μL of serum-free cell culture medium) into Balb / c nude mice. When the tumor size reached about 50 - 60 mm 3 , treatment was started, and 30 mice were randomly divided into different groups for different treatments (n = 5). The grouping is as follows:

[0092] (1) PBS as the control group, (2) ultrasound only group (1.2 W / cm 2 , 3 min), (3) chlorin e6 + cyanobacteria only group, (4) chlorin e6 + ultrasound group (1.2 W / cm 2 , 3 min), (5) chlorin e6 + cyanobacteria + ultrasound group (1.2 W / cm 2 , 3 min), (6) chlorin e6 + cyanobacteria + ultrasound group (1.2 W / cm 2 , 3 min, penetrating a depth of 3 mm). In the (6)th group, the penetration depth was achieved by placing a 3-mm-thick chicken breast on the ultrasound probe. The concentration of chlorin e6 nanoparticles was 400 μg / mL, and the concentration of cyanobacteria was 5×10 7 cell / mL. 100 μL of the solution from each group was injected in situ into Balb / c mice with 4T1 tumors. The treatment was carried out 3 times, and the tumor size and weight of each mouse were measured every 2 days. On the 14th day, the tumors of different treatment groups were dissected and weighed.

[0093] The results are as Figure 16 (a) and Figure 16 (b) show. The experimental results indicate that the growth of tumors in tumor-bearing mice injected with the combined agent of organic sonoluminescence nanoparticles is significantly inhibited. While in the control group injected with only PBS buffer under the same conditions, the tumor growth of tumor-bearing mice is obvious.

Claims

1. An organic afterglow nanoparticle combination medicament, characterized in that, The combined medicament contains chlorin e6 nanoparticles and cyanobacteria; the concentration of chlorin e6 nanoparticles in the combined medicament is 100-500 μg / mL, and the concentration of the cyanobacteria is 2.5×10 7 ~12.5×10 7 cell / mL; The combined medicament of organic acoustic afterglow nanoparticles is prepared by the following method, and the preparation method includes the following steps: in a tetrahydrofuran medium, purpurin E6 is modified by using a polyether to obtain modified purpurin E6; the modified purpurin E6 self-assembles in water to form purpurin E6 nanoparticles; the purpurin E6 nanoparticles and cyanobacteria are mixed in water to obtain the combined medicament of organic acoustic afterglow nanoparticles; The preparation method of the purpurin E6 nanoparticles is as follows: after dissolving the polyether in tetrahydrofuran, purpurin E6 is added thereto to form a suspension, the suspension is dropped into water and stirred for dispersion, tetrahydrofuran is removed by rotary evaporation, and then the purpurin E6 nanoparticles are obtained by ultrafiltration purification; The polyether is polyether F127.

2. The combined medicament of organic acoustic afterglow nanoparticles according to claim 1, wherein The particle size of the purpurin E6 nanoparticles is 20-50 nm, and the length of the cyanobacteria is 1-2 μm.

3. A preparation method of the combined medicament of the organic acoustic afterglow nanoparticles described in claim 1, characterized in that, The preparation method includes the following steps: in a tetrahydrofuran medium, purpurin E6 is modified by using a polyether to obtain modified purpurin E6; the modified purpurin E6 self-assembles in water to form purpurin E6 nanoparticles; the purpurin E6 nanoparticles and cyanobacteria are mixed in water to obtain the combined medicament of organic acoustic afterglow nanoparticles.

4. The preparation method of the organic afterglow nanoparticle combination medicament according to claim 3, wherein The preparation method of the purpurin E6 nanoparticles is as follows: after dissolving the polyether in tetrahydrofuran, purpurin E6 is added thereto to form a suspension, the suspension is dropped into water and stirred for dispersion, tetrahydrofuran is removed by rotary evaporation, and then the purpurin E6 nanoparticles are obtained by ultrafiltration purification.

5. The preparation method of the organic afterglow nanoparticle combination medicament according to claim 4, characterized in that, The dosage ratio of the polyether to the tetrahydrofuran is (10-18) mg:1 mL, and the mass ratio of the polyether to the purpurin E6 is (20-50):

1.

6. The preparation method of the organic afterglow nanoparticle combination medicament according to any one of claims 3 to 5, characterized in that, The polyether is polyether F127.

7. The preparation method of the organic acoustic afterglow nanoparticle combination medicament according to claim 4, wherein The temperature of the rotary evaporation is 15-20 °C, the rotation speed is 50-60 rpm, and the time is 10-12 h.

8. Use of the combined medicament of organic acoustic afterglow nanoparticles according to claim 1 in the preparation of a medicament for treating tumors.

9. The application according to claim 8, wherein The tumor is a cutaneous tumor.

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