Application of low-intensity pulsed ultrasound in promoting tumor cells to take in exosome entrapped with chemotherapeutic drugs

Abstract

The invention relates to the technical field of biological medicines, in particular to application of low-intensity pulsed ultrasound in promoting tumor cells to take in exosome entrapped with chemotherapeutic drugs. According to the method, low-intensity pulse ultrasound with the intensity of 200 mW / cm < 2 > to 350 mW / cm < 2 > is adopted, on the basis that the exosome entrapped with the chemotherapeutic drug is not damaged, the uptake efficiency of tumor cells on the exosome entrapped with the chemotherapeutic drug is promoted, and the treatment effect of the chemotherapeutic drug on the tumor cells is improved. Cell experiments and animal experiments prove that the low-intensity pulsed ultrasound combined with the exosome entrapped with the chemotherapeutic drug can enhance the treatment effect of the chemotherapeutic drug on tumor cells, new theoretical basis and experimental basis are provided for tumor treatment, and the exosome has a good application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the application of low-intensity pulsed ultrasound in promoting tumor cells to take up exosomes encapsulating chemotherapeutic drugs. Background Art

[0002] In medicine, cancer refers to malignant tumors that originate from epithelial tissues. The term "cancer" generally refers to all malignant tumors. It is a common and frequently occurring disease that seriously endangers people's health. Studies have shown that malignant tumors are the second leading cause of death in humans after cardiovascular and cerebrovascular diseases. Currently, chemotherapy is one of the methods for clinical treatment of malignant tumors. Chemotherapy of malignant tumors relies on the delivery of chemotherapy drugs to the target tumor site through the blood circulation, thereby inhibiting the proliferation of tumor cells. However, existing chemotherapy drugs have the problem of poor drug delivery efficiency, which is one of the important challenges facing tumor treatment.

[0003] Exosomes are small vesicles with a diameter of 30-100nm. They are widely used because of their wide range of sources, no risk of immune response, and the ability to achieve repeated administration without tolerance. Studies have shown that exosomes can be used as a delivery carrier for chemotherapy drugs. Chemotherapy drugs are encapsulated into exosomes to prepare exosomes encapsulating chemotherapy drugs, thereby improving the drug delivery efficiency of chemotherapy drugs and improving the therapeutic effect of chemotherapy drugs on tumor cells. However, in tumor treatment, the efficiency of exosome delivery of chemotherapy drugs depends on the uptake of exosomes by tumor cells. Due to their heterogeneity, the special structure of the cell membrane, and the complexity of the tumor microenvironment, tumor cells limit their uptake of exosomes. There is a problem of low efficiency of tumor cells in uptaking exosomes encapsulating chemotherapy drugs, which affects the therapeutic effect of chemotherapy drugs on tumor cells.

[0004] Low-intensity pulsed ultrasound (LIPUS) is a low-intensity ultrasound with a pulsed output, and its intensity is generally less than 3W / cm 2 However, there are no reports on the effect of low-intensity pulsed ultrasound on promoting the uptake of exosomes loaded with chemotherapeutic drugs by tumor cells. Summary of the invention

[0005] To solve the above technical problems, the present invention provides the application of low-intensity pulsed ultrasound in promoting tumor cells to take up exosomes encapsulating chemotherapy drugs. The inventors have shown through a large number of studies that the use of low-intensity pulsed ultrasound can promote the uptake efficiency of tumor cells into exosomes encapsulating chemotherapy drugs and enhance the therapeutic effect of chemotherapy drugs on tumor cells.

[0006] A first aspect of the present invention provides the use of low-intensity pulsed ultrasound in promoting tumor cells to take up exosomes encapsulating chemotherapeutic drugs.

[0007] The use of the low-intensity pulsed ultrasound as described above in promoting tumor cells to take up exosomes containing chemotherapeutic drugs, wherein the intensity of the low-intensity pulsed ultrasound is 200 mW / cm 2 ~350mW / cm 2 ;

[0008] Or, the acoustic power of the low-intensity pulsed ultrasound is 1W.

[0009] The use of low-intensity pulsed ultrasound as described above in promoting tumor cells to take up exosomes encapsulating chemotherapeutic drugs, wherein the duty cycle of the low-intensity pulsed ultrasound is 10% to 40%, the duration is 1 min to 10 min, and the modulation frequency is 100 Hz to 1000 Hz.

[0010] The use of low-intensity pulsed ultrasound as described above in promoting tumor cells to take up exosomes encapsulating chemotherapeutic drugs, wherein the exosomes encapsulating chemotherapeutic drugs include turmeric exosomes.

[0011] The use of low-intensity pulsed ultrasound as described above in promoting tumor cells to take up exosomes encapsulating chemotherapeutic drugs, wherein the chemotherapeutic drugs in the exosomes encapsulating chemotherapeutic drugs include epirubicin.

[0012] In the application of low-intensity pulsed ultrasound in promoting tumor cells to take up exosomes encapsulating chemotherapeutic drugs as described above, the mass ratio of epirubicin to the turmeric exosomes is 2:1.

[0013] The use of low-intensity pulsed ultrasound as described above in promoting the uptake of exosomes encapsulating chemotherapeutic drugs by tumor cells, including breast cancer cells.

[0014] As described above, the application of low-intensity pulsed ultrasound in promoting tumor cells to take up exosomes encapsulating chemotherapeutic drugs is achieved by a low-intensity pulsed ultrasound device.

[0015] The method for using low-intensity pulsed ultrasound as described above in promoting tumor cells to take up exosomes encapsulating chemotherapeutic drugs comprises:

[0016] Obtaining tumor cells;

[0017] Mixing the exosome-encapsulated chemotherapy drug with normal saline to obtain an exosome drug solution;

[0018] The exosome drug solution and the tumor cells are mixed in a container, and then a low-intensity pulsed ultrasound probe is used to act on the container to perform low-intensity pulsed ultrasound to promote the uptake efficiency of the tumor cells for the exosomes encapsulating the chemotherapy drug.

[0019] The method for using low-intensity pulsed ultrasound as described above in promoting tumor cells to take up exosomes encapsulating chemotherapeutic drugs comprises:

[0020] Mixing the exosome-encapsulated chemotherapy drug with normal saline to obtain an exosome drug solution;

[0021] After administering the exosome solution to a subject suffering from a malignant tumor, the ultrasound probe in the low-intensity pulsed ultrasound device is aimed at the skin area corresponding to the tumor site of the subject to perform low-intensity pulsed ultrasound to promote the uptake efficiency of the tumor cells for the exosomes encapsulating the chemotherapy drug.

[0022] In summary, the solution of the present invention has at least the following effects:

[0023] The present invention adopts an intensity of 200mW / cm 2 ~350mW / cm 2 The low-intensity pulsed ultrasound, without destroying the exosomes encapsulating the chemotherapy drugs, promotes the uptake efficiency of the exosomes encapsulating the chemotherapy drugs by the tumor cells, and improves the therapeutic effect of the chemotherapy drugs on the tumor cells. The present invention has confirmed through cell experiments and animal experiments that the low-intensity pulsed ultrasound combined with the exosomes encapsulating the chemotherapy drugs can enhance the therapeutic effect of the chemotherapy drugs on the tumor cells, providing a new theoretical basis and experimental basis for tumor treatment, and has a good application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 The fluorescence images, cell bright field images, and fluorescence intensity statistical graphs of tumor cells in each group (Exo+US100, Exo+US200, Exo+US250, Exo+US300, Exo+US350, Exo, and Control) in Example 1 of the present invention are shown; wherein, Figure 1 A is the fluorescence image of exosome drugs in tumor cells of each group;

[0026] Figure 1 B is the bright field image of tumor cells in each group; Figure 1 C is the statistical graph of the fluorescence intensity of tumor cells in each group;

[0027] Figure 2It is a graph showing the cell survival rate test results of tumor cells in each group (Exo+US100, Exo+US200, Exo+US250, Exo+US300, Exo+US350, Exo, Control) in Example 1 of the present invention;

[0028] Figure 3 The results of H&E staining of tumor tissue sections of each group (Control, EXO, EXO+US) at 200 times (200x) and 400 times (400x) in Example 2 of the present invention are shown;

[0029] Figure 4 The results of TUNEL staining of tumor tissue sections of each group (Control, EXO, EXO+US) in Example 2 of the present invention are shown; wherein, Figure 4 A is the TUNEL staining result of tumor tissue sections in each group; Figure 4 B is a statistical diagram of the TUNEL fluorescence ratio of tumor tissue sections in each group; green fluorescence represents broken DNA fragments, and blue fluorescence represents cell nuclei. The more broken DNA fragments there are, the more apoptotic and damaged tumor cells there are;

[0030] Figure 5 The therapeutic effects of each group of treatments in Example 2 of the present invention on tumor proliferation in mice; wherein, Figure 5 A is the graph of each group of tumors; Figure 5 B is a quantitative statistical chart of the tumor size in each group. DETAILED DESCRIPTION

[0031] To make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme in the embodiment of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the art without making creative work are within the scope of protection of the present invention. If the specific technology or conditions are not indicated in the embodiments, the technology or conditions described in the literature in this area or the product instructions are carried out. The reagents or instruments used that do not indicate the manufacturer are all conventional products that can be obtained commercially.

[0032] A first aspect of the present invention provides the use of low-intensity pulsed ultrasound in promoting tumor cells to take up exosomes encapsulating chemotherapeutic drugs.

[0033] In the present invention, low-intensity pulsed ultrasound (LIPUS) refers to a low-intensity ultrasound with a pulsed output.

[0034] In the present invention, the exosomes are exosomes derived from turmeric, and the chemotherapeutic drug includes epirubicin.

[0035] The present invention does not particularly limit the source or specific preparation method of the exosomes encapsulating the chemotherapeutic drug, which can be purchased through commercial channels, or prepared according to methods well known to those skilled in the art, or prepared according to methods in the literature.

[0036] The inventors have shown through a large number of studies that the present invention uses low-intensity pulsed ultrasound to stimulate tumor cells that have been administered with exosomes containing chemotherapeutic drugs, which promotes the uptake efficiency of tumor cells into exosomes containing chemotherapeutic drugs, and improves the therapeutic effect of chemotherapeutic drugs on tumor cells (for example, reducing tumor cell activity). In addition, compared with tumor-bearing mice that only administer exosomes containing chemotherapeutic drugs, tumor-bearing mice that are administered with low-intensity pulsed ultrasound combined with exosomes containing chemotherapeutic drugs have a higher apoptosis rate of tumor cells and a smaller tumor volume, and have a better tumor treatment effect.

[0037] In some embodiments, the intensity of the low-intensity pulsed ultrasound is 200 mW / cm 2 ~350mW / cm 2 .

[0038] In the present invention, intensity refers to the ultrasonic output intensity. When the intensity of low-intensity pulsed ultrasound is within the above range, it can further promote the uptake of exosome-encapsulated chemotherapeutic drugs by tumor cells and enhance the therapeutic effect of chemotherapeutic drugs.

[0039] In some embodiments, the acoustic power of the low-intensity pulsed ultrasound is 1W.

[0040] In the present invention, sound power refers to the total energy of sound radiated into space by a sound source per unit time.

[0041] When the acoustic power of low-intensity pulsed ultrasound is 1W, it can promote the uptake of exosome-encapsulated chemotherapy drugs by tumor cells and enhance the therapeutic effect of chemotherapy drugs.

[0042] In some embodiments, the low-intensity pulsed ultrasound has a duty cycle of 10% to 40%, a duration of 1 min to 10 min, and a modulation frequency of 100 Hz to 1000 Hz.

[0043] In the present invention, duty cycle refers to the proportion of power-on time to total time in a pulse cycle, time refers to the irradiation time of a low-intensity pulse ultrasound, and modulation frequency refers to the number of repetitions of low-intensity pulse ultrasound per unit time.

[0044] It is understood that the specific number of low-intensity pulsed ultrasounds performed in the present invention may vary greatly depending on a variety of factors, such as the target cells, the type of organism or its tissue, the general condition of the subject to be treated, etc.

[0045] In some embodiments, when the intensity of the low-intensity pulsed ultrasound is 200 mW / cm 2 ~350mW / cm 2 The duty cycle of low-intensity pulse ultrasound is 10% to 20%, the time is 5 minutes to 10 minutes, and the modulation frequency is 1000 Hz.

[0046] In some embodiments, when the acoustic power of the low-intensity pulsed ultrasound is 1 W, the duty cycle of the low-intensity pulsed ultrasound is 20% to 40%, the duration is 90 s to 3 min, and the modulation frequency is 100 Hz.

[0047] In some embodiments, the exosomes encapsulating the chemotherapeutic drug include turmeric exosomes.

[0048] The present invention does not particularly limit the specific source of turmeric exosomes, which can be purchased through commercial channels or prepared according to methods well known in the art.

[0049] In some embodiments, the chemotherapeutic drug-encapsulated exosomes include epirubicin.

[0050] The present invention does not particularly limit the specific source of epirubicin, which can be purchased through commercial channels.

[0051] In some embodiments, the mass ratio of epirubicin to turmeric exosomes is 2:1.

[0052] In the present invention, when the mass ratio of epirubicin to turmeric exosomes is 2:1, low-intensity pulsed ultrasound can be used to promote tumor cells to take up exosomes encapsulating chemotherapeutic drugs, thereby improving the therapeutic effect of chemotherapeutic drugs.

[0053] In some embodiments, the tumor cells include breast cancer cells.

[0054] In some embodiments, the low-intensity pulsed ultrasound process is achieved by a low-intensity pulsed ultrasound device.

[0055] The present invention does not impose any particular limitation on the specific low-intensity pulsed ultrasonic device, and any low-intensity pulsed ultrasonic device known in the art may be used.

[0056] The second aspect of the present invention provides a method for using the above-mentioned low-intensity pulsed ultrasound in promoting tumor cells to take up exosomes encapsulating chemotherapeutic drugs, comprising:

[0057] Obtaining tumor cells;

[0058] Mixing the exosome-encapsulated chemotherapy drug with normal saline to obtain an exosome drug solution;

[0059] The exosome drug solution and tumor cells are mixed in a container, and then a low-intensity pulsed ultrasound probe is used to act on the container for low-intensity pulsed ultrasound to promote the uptake efficiency of tumor cells into exosomes encapsulating chemotherapy drugs.

[0060] In some embodiments, the concentration of the exosome solution is 6 μg / mL.

[0061] In some embodiments, the intensity of the low-intensity pulsed ultrasound is 200 mW / cm 2 ~350mW / cm 2 The duty cycle is 10% to 20%, the duration is 5 min to 10 min, and the modulation frequency is 1000 Hz. Furthermore, the intensity of the low-intensity pulsed ultrasound can be preferably 250 mW / cm 2 The duty cycle may be preferably 10%, the time may be preferably 10 minutes, and the modulation frequency may be 1000 Hz.

[0062] The present invention does not specifically limit the container to be used, and the container can be selected as required. For example, a six-well plate can be selected as the container.

[0063] The third aspect of the present invention provides a method for using the above-mentioned low-intensity pulsed ultrasound in promoting tumor cells to take up exosomes encapsulating chemotherapeutic drugs, comprising:

[0064] Mixing the exosome-encapsulated chemotherapy drug with normal saline to obtain an exosome drug solution;

[0065] After administering the exosome solution to a subject with a malignant tumor, the ultrasound probe in the low-intensity pulsed ultrasound device is directed at the skin area corresponding to the tumor site of the subject to perform low-intensity pulsed ultrasound to promote the efficiency of tumor cells in taking up the exosomes encapsulating the chemotherapy drug.

[0066] In some embodiments, the concentration of the exosome solution is 0.5 mg / mL.

[0067] In some embodiments, the acoustic power of the low-intensity pulsed ultrasound is 1W, the duty cycle is 20% to 40%, the time is 90s to 3min, and the modulation frequency is 100Hz. Further, the acoustic power of the low-intensity pulsed ultrasound is 1W, the duty cycle may be preferably 20%, the time may be preferably 90s, and the modulation frequency is 100Hz.

[0068] The application of low-intensity pulsed ultrasound provided by the present invention in promoting tumor cells to take up exosomes encapsulating chemotherapeutic drugs is described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0069] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0070] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0071] In the following examples, "about" means + / - five percent (5%) of the stated measurement unit.

[0072] The exosomes encapsulating the chemotherapy drug used in this embodiment are turmeric exosomes encapsulating epirubicin (the mass ratio of epirubicin to turmeric exosomes is 2:1, purchased from Shenzhen Shifangjie Technology Co., Ltd.).

[0073] The low-intensity pulsed ultrasonic equipment used in this embodiment belongs to Ronghai Ultrasonic Medical Engineering Research Center.

[0074] The tumor cells used in this example include breast cancer cells MDA-MB-231 (purchased from Shanghai Fuheng Biotechnology Co., Ltd.) and breast cancer cells 4T1 (purchased from Shanghai Fuheng Cell Bank).

[0075] Example 1 Cell experiment

[0076] 1. Experimental methods

[0077] (1) Intensity: 100 mW / cm 2 Low-intensity pulsed ultrasound combined with exosomes loaded with chemotherapeutic drugs (Exo+US100): MDA-MB-231 breast cancer cells with a cell number of 10 and good growth status were prepared into a concentration of 5×10 5 1 mL of tumor cell suspension was added to a six-well plate, and then placed in an incubator at 37°C for 24 hours (h). After removing the supernatant, the plate was washed twice with PBS to obtain cultured tumor cells.

[0078] The exosomes encapsulating the chemotherapeutic drug (turmeric exosomes encapsulating epirubicin) were diluted with sterile saline to obtain an exosome drug solution with a concentration of 6 μg / mL;

[0079] 1 mL of complete culture medium (a culture medium prepared by RPMI-1640 culture medium (1640), fetal bovine serum (FBS) and penicillin-streptomycin mixed solution (PSS) at a mass ratio of 100:10:1), 1 mL of exosome solution and cultured tumor cells were mixed and placed in a six-well plate, and then the low-intensity pulsed ultrasound device was turned on: the duty cycle was set to 10% and the intensity was set to 100 mW / cm 2 , the time is 10 minutes, and the modulation frequency is 1000 Hz; the ultrasonic probe in the low-intensity pulsed ultrasonic device is applied to the bottom of the six-well plate to perform low-intensity pulsed ultrasound to obtain tumor cells after low-intensity pulsed ultrasound;

[0080] The tumor cells after low-intensity pulsed ultrasound were placed in an incubator and cultured for 24 hours, and then fluorescent staining tests and tumor cell activity tests were performed.

[0081] (2) Intensity: 200 mW / cm 2 Low-intensity pulsed ultrasound combined with exosomes loaded with chemotherapeutic drugs (Exo+US200): (1) intensity of 100 mW / cm 2 The process of low-intensity pulsed ultrasound combined with exosomes loaded with chemotherapy drugs was basically the same, except that the intensity was 100 mW / cm 2 Replaced with an intensity of 200mW / cm 2 .

[0082] (3) Intensity: 250mW / cm 2 Low-intensity pulsed ultrasound combined with exosomes loaded with chemotherapeutic drugs (Exo+US250): (1) intensity of 100 mW / cm 2 The process of low-intensity pulsed ultrasound combined with exosomes loaded with chemotherapy drugs was basically the same, except that the intensity was 100 mW / cm 2 Replaced with an intensity of 250mW / cm 2 .

[0083] (4) Intensity: 300mW / cm 2 Low-intensity pulsed ultrasound combined with exosomes loaded with chemotherapeutic drugs (Exo+US300): (1) intensity of 100 mW / cm 2 The process of low-intensity pulsed ultrasound combined with exosomes loaded with chemotherapy drugs was basically the same, except that the intensity was 100 mW / cm 2 Replaced with an intensity of 300mW / cm 2 .

[0084] (5) Intensity: 350mW / cm 2Low-intensity pulsed ultrasound combined with exosomes loaded with chemotherapeutic drugs (Exo+US350): (1) intensity of 100 mW / cm 2 The process of low-intensity pulsed ultrasound combined with exosomes loaded with chemotherapy drugs was basically the same, except that the intensity was 100 mW / cm 2 Replaced with an intensity of 350mW / cm 2 .

[0085] (6) Exosomes loaded with chemotherapeutic drugs (Exo): MDA-MB-231 breast cancer cells with a cell number of 10 and good growth status were prepared into a concentration of 5×10 5 1 mL of tumor cell suspension was added to a six-well plate, and then placed in an incubator at 37°C for 24 hours. After removing the supernatant, the plate was washed twice with PBS to obtain cultured tumor cells.

[0086] The exosomes encapsulating the chemotherapeutic drug (turmeric exosomes encapsulating epirubicin) were diluted with sterile saline to obtain an exosome drug solution with a concentration of 6 μg / mL;

[0087] 1 mL of complete culture medium (a culture medium prepared by RPMI-1640 culture medium (1640), fetal bovine serum (FBS) and penicillin-streptomycin mixed solution (PSS) in a mass ratio of 100:10:1), 1 mL of exosome solution and cultured tumor cells were mixed and placed in a six-well plate, and then cultured in an incubator for 24 hours, and then fluorescent staining test and tumor cell activity test were performed respectively.

[0088] (7) Blank group (Control): MDA-MB-231 breast cancer cells with a cell number of 10 and good growth status were prepared into a concentration of 5×10 5 1 mL of tumor cell suspension was added to a six-well plate, and then placed in an incubator at 37°C for 24 hours. After removing the supernatant, the plate was washed twice with PBS to obtain cultured tumor cells.

[0089] 1 mL of complete culture medium (a culture medium prepared by RPMI-1640 culture medium (1640), fetal bovine serum (FBS) and penicillin-streptomycin mixture (PSS) in a mass ratio of 100:10:1), 1 mL of sterile saline and cultured tumor cells were mixed and placed in a six-well plate, and then cultured in an incubator for 24 hours, and then fluorescent staining test and tumor cell survival rate test were performed respectively.

[0090] 2. Experimental results

[0091] (1) Fluorescent staining test

[0092] Figure 1 The fluorescence images, cell bright field images, and fluorescence intensity statistical images of each group of tumor cells in Example 1 of the present invention are shown; wherein, Figure 1 A is the fluorescence image of exosome drugs in tumor cells of each group; Figure 1 B is the bright field image of tumor cells in each group; Figure 1 C is the statistical graph of the fluorescence intensity of tumor cells in each group.

[0093] Depend on Figure 1 AC shows that the exosomes containing chemotherapy drugs have entered the tumor cells; the fluorescence intensity of the exosome group (Exo) containing chemotherapy drugs is significantly lower than that of the group with an intensity of 200 mW / cm 2 Low-intensity pulsed ultrasound combined with exosomes loaded with chemotherapy drugs (Exo+US200), with an intensity of 250mW / cm 2 Low-intensity pulsed ultrasound combined with exosomes loaded with chemotherapy drugs (Exo+US250), with an intensity of 300mW / cm 2 Low-intensity pulsed ultrasound combined with exosomes loaded with chemotherapy drugs (Exo+US300) and an intensity of 350mW / cm 2 The fluorescence intensity of low-intensity pulsed ultrasound combined with exosomes loaded with chemotherapy drugs (Exo+US350) shows that the intensity is 200mW / cm 2 , intensity is 250mW / cm 2 , intensity is 300mW / cm 2 and an intensity of 350mW / cm 2 The drug uptake in the low-intensity pulsed ultrasound combined with exosomes loaded with chemotherapy drugs was significantly higher than that in the exosomes loaded with chemotherapy drugs (Exo), indicating that low-intensity pulsed ultrasound can promote the uptake efficiency of tumor cells into exosomes loaded with chemotherapy drugs; the intensity was 100mW / cm 2 There was no significant difference in the fluorescence intensity of the low-intensity pulsed ultrasound combined with exosomes loaded with chemotherapeutic drugs (Exo+US100) and the fluorescence intensity of the exosomes loaded with chemotherapeutic drugs (Exo), indicating that the intensity of 100 mW / cm 2 The amount of drug in the low-intensity pulsed ultrasound combined with the exosomes loaded with chemotherapy drugs was basically the same as that in the exosomes loaded with chemotherapy drugs (Exo), indicating that the intensity of 100mW / cm 2 Low-intensity pulsed ultrasound cannot effectively improve the uptake efficiency of exosomes containing chemotherapeutic drugs by tumor cells.

[0094] (2) Tumor cell survival test

[0095] Figure 2This is a graph showing the cell survival rate test results of each group of tumor cells in Example 1 of the present invention.

[0096] Depend on Figure 2 It can be seen that the cell survival rate of tumor cells in the exosome group (Exo) containing chemotherapy drugs is significantly higher than that in the group with an intensity of 200mW / cm 2 Low-intensity pulsed ultrasound combined with exosomes loaded with chemotherapy drugs (Exo+US200), with an intensity of 250mW / cm 2 Low-intensity pulsed ultrasound combined with exosomes loaded with chemotherapy drugs (Exo+US250), with an intensity of 300mW / cm 2 Low-intensity pulsed ultrasound combined with exosomes loaded with chemotherapy drugs (Exo+US300) and an intensity of 350mW / cm 2 The cell survival rate of tumor cells in the low-intensity pulsed ultrasound combined with exosomes loaded with chemotherapy drugs (Exo+US350) group shows that low-intensity pulsed ultrasound can promote tumor cells to take up exosomes loaded with chemotherapy drugs and improve the therapeutic effect of chemotherapy drugs on tumor cells. 2 There was no significant difference in the cell survival rate of tumor cells in the low-intensity pulsed ultrasound combined with exosomes loaded with chemotherapeutic drugs (Exo+US100) group and the cell survival rate of tumor cells in the exosomes loaded with chemotherapeutic drugs group (Exo), indicating that the intensity of 100 mW / cm 2 Low-intensity pulsed ultrasound cannot effectively promote tumor cells to take up exosomes containing chemotherapeutic drugs, so its therapeutic effect is not significantly improved.

[0097] Example 2 Animal Experiment

[0098] 1. Experimental methods

[0099] (1) Construction of mouse breast cancer model

[0100] Fifteen female balb / c mice (purchased from Chongqing Tengxin Biotechnology Co., Ltd.) aged 8 weeks and in good condition were used to construct a mouse breast cancer model. 0.1 mL of 1×10 7 After 7 days, when the tumor cells grow to a suitable size (volume is about 80mm 3 ) to obtain tumor-bearing mice; 15 tumor-bearing mice were randomly divided into 3 groups (5 mice in each group), and the tumor-bearing mice were given the following different treatments:

[0101] ①Blank group (Control):

[0102] The mice were sedated and analgesic using gas anesthetic (isoflurane), and 0.2 mL of sterile saline was injected through the tail vein of the mice. After the injection, the injection was repeated every 4 days, for a total of 4 times; the condition of the mice was observed every two days during the entire period and before the last injection.

[0103] ②Exosomes loaded with chemotherapy drugs (EXO):

[0104] The exosomes encapsulating the chemotherapy drug (turmeric exosomes encapsulating epirubicin) were diluted with sterile saline to obtain an exosome solution with a concentration of 0.5 mg / mL;

[0105] The mice were sedated and analgesic using gas anesthetic (isoflurane), and a 0.2 mL exosome solution with a concentration of 0.5 mg / mL was injected through the tail vein of the mice. After the injection, the injection was repeated every 4 days, for a total of 4 times; the condition of the mice was observed every two days during the entire period and before the last injection.

[0106] ③ Low-intensity pulsed ultrasound combined with exosomes loaded with chemotherapy drugs (EXO+US):

[0107] The exosomes encapsulating the chemotherapy drug (turmeric exosomes encapsulating epirubicin) were diluted with sterile saline to obtain an exosome solution with a concentration of 0.5 mg / mL;

[0108] The mice were sedated and analgesic using gas anesthetic (isoflurane), and an exosome solution with a volume of 0.2 mL and a concentration of 0.5 mg / mL was injected through the tail vein of the mice. After the injection, a medical ultrasound coupling agent (purchased from Chongqing Haifu Medical Technology Co., Ltd.) was applied to the skin area corresponding to the tumor site of the mice, and then the low-intensity pulsed ultrasound device was turned on: the duty cycle was set to 20%, the sound power was 1 W, the time was 90 S, and the modulation frequency was 100 Hz; the ultrasound probe in the low-intensity pulsed ultrasound device was aimed at the skin area corresponding to the tumor site of the mouse for low-intensity pulsed ultrasound, and the exosome solution injection and low-intensity pulsed ultrasound were performed once every 4 days, for a total of 4 times; during the entire period and before the last exosome solution injection and low-intensity pulsed ultrasound, the mouse status was observed every two days.

[0109] On the 21st day, the mice in each group were killed, and the tumors in each group were obtained and their volumes were measured. The tumors in each group were prepared into tumor tissue sections, and then the tumor tissue sections in each group were stained with hematoxylin and eosin (H&E staining) and TUNEL staining (TUNEL staining, i.e., terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, is a commonly used technique for detecting DNA fragmentation in cell apoptosis).

[0110] 2. Experimental results

[0111] (1) Hematoxylin and eosin staining (H&E staining)

[0112] Figure 3 200 times (200x) and 400 times (400x) of the H&E staining results of tumor tissue sections in each group in Example 2 of the present invention.

[0113] Depend on Figure 3 It can be seen that compared with the exosome treatment group (EXO) and the blank group (Control), the nuclear collapse and morphological changes of tumor tissue cells in the low-intensity pulsed ultrasound combined with exosome treatment group (EXO+US) were obvious, and the apoptosis rate of tumor cells increased, indicating that the therapeutic effect of low-intensity pulsed ultrasound combined with exosome treatment group (EXO+US) was better than that of exosome treatment group (EXO) with chemotherapy drugs, further indicating that low-intensity pulsed ultrasound can promote the uptake of exosomes encapsulating chemotherapy drugs by tumor cells, and enhance the therapeutic effect of chemotherapy drugs on tumor cells.

[0114] (2) TUNEL staining

[0115] Figure 4 The results of TUNEL staining of tumor tissue sections in each group in Example 2 of the present invention are shown; wherein, Figure 4 A is the TUNEL staining result of tumor tissue sections in each group; Figure 4 B is a statistical diagram of the TUNEL fluorescence ratio of tumor tissue sections in each group; among them, green fluorescence represents broken DNA fragments, and blue fluorescence represents cell nuclei. The more broken DNA fragments there are, the more apoptotic and damaged tumor cells there are.

[0116] Depend on Figure 4 As can be seen from A and B, there is almost no apoptosis in the tumor cells of the blank group (Control). Compared with the green fluorescence in the exosome treatment group (EXO) loaded with chemotherapy drugs, there is more green fluorescence in the low-intensity pulsed ultrasound combined with exosome treatment group (EXO+US) loaded with chemotherapy drugs, indicating that there is more apoptosis in the tumor cells in the low-intensity pulsed ultrasound combined with exosome treatment group loaded with chemotherapy drugs, further indicating that low-intensity pulsed ultrasound can promote tumor cells to take up exosomes loaded with chemotherapy drugs and enhance the therapeutic effect of chemotherapy drugs on tumor cells.

[0117] (3) The therapeutic effect of each group of treatments on tumor proliferation in mice

[0118] Figure 5 The therapeutic effects of each group of treatments in Example 2 of the present invention on tumor proliferation in mice; wherein, Figure 5 A is the graph of each group of tumors; Figure 5B is a quantitative statistical chart of the tumor size in each group.

[0119] Depend on Figure 5 From A and B, we can see that the tumor volume of the low-intensity pulsed ultrasound combined with exosomes loaded with chemotherapeutic drugs (Exo+US) treatment group is smaller than that of the blank group (control) and the exosomes loaded with chemotherapeutic drugs treatment group (Exo), indicating that low-intensity pulsed ultrasound combined with exosomes loaded with chemotherapeutic drugs (Exo+US) treatment group can significantly inhibit tumor growth.

[0120] In summary, the present invention confirms that low-intensity pulsed ultrasound can promote tumor cells to take up exosomes containing chemotherapeutic drugs, reduce tumor cell activity, promote tumor cell apoptosis, inhibit tumor growth, and has a better tumor treatment effect. The present invention provides a new theoretical basis and experimental basis for tumor treatment and has good application prospects.

[0121] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of low-intensity pulsed ultrasound in promoting tumor cells to uptake exosomes loaded with chemotherapeutic drugs.

2. The use of low-intensity pulsed ultrasound in promoting tumor cells to take up exosomes encapsulating chemotherapeutic drugs according to claim 1, characterized in that: The intensity of the low-intensity pulsed ultrasound is 200 mW / cm 2 ~350mW / cm 2 ; Or, the acoustic power of the low-intensity pulsed ultrasound is 1W.

3. The use of low-intensity pulsed ultrasound in promoting tumor cells to take up exosomes encapsulating chemotherapeutic drugs according to claim 1, characterized in that: The duty cycle of the low-intensity pulse ultrasound is 10% to 40%, the duration is 1 min to 10 min, and the modulation frequency is 100 Hz to 1000 Hz.

4. The use of low-intensity pulsed ultrasound in promoting tumor cells to take up exosomes encapsulating chemotherapeutic drugs according to claim 1, characterized in that: In the exosomes carrying the chemotherapeutic drug, the exosomes include turmeric exosomes.

5. The use of low-intensity pulsed ultrasound in promoting tumor cells to take up exosomes encapsulating chemotherapeutic drugs according to claim 4, characterized in that: In the exosomes encapsulating the chemotherapeutic drug, the chemotherapeutic drug includes epirubicin.

6. The use of low-intensity pulsed ultrasound in promoting tumor cells to take up exosomes encapsulating chemotherapeutic drugs according to claim 5, characterized in that: The mass ratio of the epirubicin to the turmeric exosomes is 2:

1.

7. The use of low-intensity pulsed ultrasound in promoting tumor cells to take up exosomes encapsulating chemotherapeutic drugs according to claim 1, characterized in that: The tumor cells include breast cancer cells.

8. The use of low-intensity pulsed ultrasound in promoting tumor cells to take up exosomes encapsulating chemotherapeutic drugs according to claim 1, characterized in that: The low-intensity pulsed ultrasound process is achieved by a low-intensity pulsed ultrasound device.

9. A method for using low-intensity pulsed ultrasound according to any one of claims 1 to 8 in promoting tumor cells to take up exosomes encapsulating chemotherapeutic drugs, characterized in that: include: Obtaining tumor cells; Mixing the exosome-encapsulated chemotherapy drug with normal saline to obtain an exosome drug solution; The exosome drug solution and the tumor cells are mixed in a container, and then a low-intensity pulsed ultrasound probe is used to act on the container to perform low-intensity pulsed ultrasound to promote the uptake efficiency of the tumor cells for the exosomes encapsulating the chemotherapy drug.

10. A method for using low-intensity pulsed ultrasound according to any one of claims 1 to 8 in promoting tumor cells to take up exosomes encapsulating chemotherapeutic drugs, characterized in that: include: Mixing the exosome-encapsulated chemotherapy drug with normal saline to obtain an exosome drug solution; After administering the exosome solution to a subject suffering from a malignant tumor, the ultrasound probe in the low-intensity pulsed ultrasound device is directed toward the skin area corresponding to the tumor site of the subject to perform low-intensity pulsed ultrasound to promote the uptake efficiency of the tumor cells for the exosomes encapsulating the chemotherapeutic drug.

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