Application of low-intensity pulsed ultrasound in promoting the uptake of exosomes carrying chemotherapy drugs by tumor cells

Abstract

This invention relates to the field of biomedical technology, and more particularly to the application of low-intensity pulsed ultrasound in promoting the uptake of exosomes carrying chemotherapeutic drugs by tumor cells. The invention uses an intensity of 200 mW / cm². 2 ~350mW / cm 2 Low-intensity pulsed ultrasound, without damaging exosomes carrying chemotherapeutic drugs, promotes the uptake efficiency of these drugs by tumor cells, thereby enhancing the therapeutic effect of chemotherapeutic drugs on tumor cells. This invention, through cell and animal experiments, demonstrates that low-intensity pulsed ultrasound combined with exosomes carrying chemotherapeutic drugs can enhance the therapeutic effect of chemotherapeutic drugs on tumor cells, providing new theoretical and experimental basis for tumor treatment and showing promising application prospects.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of low-intensity pulsed ultrasound in promoting the uptake of exosomes carrying chemotherapeutic drugs by tumor cells. Background Technology

[0002] In medicine, cancer refers to malignant tumors originating from epithelial tissue. The term "cancer" generally refers to all malignant tumors, a common and prevalent disease that seriously endangers people's health. Studies have shown that malignant tumors are the second leading cause of death after cardiovascular and cerebrovascular diseases. Currently, chemotherapy is one of the clinical treatments for malignant tumors. Chemotherapy for malignant tumors relies on chemotherapeutic drugs being delivered to the target tumor site through blood circulation to inhibit the proliferation of tumor cells. However, existing chemotherapeutic drugs suffer from poor drug delivery efficiency, which is one of the major challenges facing cancer treatment.

[0003] Exosomes are small vesicles with a diameter of 30-100 nm. They have been widely used due to their advantages, including wide availability, no risk of immune response, and the ability to be repeatedly administered without developing tolerance. Studies have shown that exosomes can serve as delivery carriers for chemotherapeutic drugs. Encapsulating chemotherapeutic drugs within exosomes can improve drug delivery efficiency and enhance the therapeutic effect of chemotherapy on tumor cells. However, in cancer treatment, the efficiency of exosome delivery of chemotherapeutic drugs depends on the uptake capacity of tumor cells. Due to their heterogeneity, the unique structure of their cell membranes, and the complexity of the tumor microenvironment, tumor cells limit their uptake of exosomes, resulting in low uptake efficiency of exosomes carrying chemotherapeutic drugs, thus affecting the therapeutic effect of chemotherapy on tumor cells.

[0004] Low-intensity pulsed ultrasound (LIPUS) is a type of low-intensity ultrasound with pulsed output, typically less than 3 W / cm². 2 However, there are currently no reported studies on the effect of low-intensity pulsed ultrasound on promoting the uptake of exosomes carrying chemotherapeutic drugs by tumor cells. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides the application of low-intensity pulsed ultrasound in promoting the uptake of exosomes carrying chemotherapeutic drugs by tumor cells. Extensive research by the inventors has shown that low-intensity pulsed ultrasound can improve the uptake efficiency of exosomes carrying chemotherapeutic drugs by tumor cells, thereby enhancing the therapeutic effect of chemotherapeutic drugs on tumor cells.

[0006] A first aspect of the present invention provides the use of low-intensity pulsed ultrasound in promoting the uptake of exosomes carrying chemotherapeutic drugs by tumor cells.

[0007] The application of low-intensity pulsed ultrasound as described above in promoting the uptake of exosomes carrying chemotherapeutic drugs by tumor cells, wherein the intensity of the low-intensity pulsed ultrasound is 200 mW / cm². 2 ~350mW / cm 2 ;

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

[0009] The application of low-intensity pulsed ultrasound as described above in promoting the uptake of exosomes carrying chemotherapeutic drugs by tumor cells, 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 application of low-intensity pulsed ultrasound as described above in promoting the uptake of chemotherapeutic drugs by tumor cells by exosomes, wherein the exosomes carrying chemotherapeutic drugs include turmeric exosomes.

[0011] The application of low-intensity pulsed ultrasound as described above in promoting the uptake of exosomes carrying chemotherapeutic drugs by tumor cells, wherein the chemotherapeutic drugs include epirubicin.

[0012] The application of low-intensity pulsed ultrasound as described above in promoting the uptake of exosomes carrying chemotherapeutic drugs by tumor cells, wherein the mass ratio of epirubicin to turmeric exosomes is 2:1.

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

[0014] The application of low-intensity pulsed ultrasound as described above in promoting the uptake of exosomes carrying chemotherapeutic drugs by tumor cells is achieved by a low-intensity pulsed ultrasound device.

[0015] The application of low-intensity pulsed ultrasound as described above in promoting the uptake of exosomes carrying chemotherapeutic drugs by tumor cells includes:

[0016] Obtain tumor cells;

[0017] Chemotherapy drugs encapsulated in exosomes are mixed with physiological saline to obtain exosome drug solution;

[0018] The exosome drug solution and the tumor cells are mixed in a container, and then the container is subjected to low-intensity pulsed ultrasound using an ultrasound probe in a low-intensity pulsed ultrasound device to promote the uptake efficiency of the exosomes carrying the chemotherapy drug by the tumor cells.

[0019] The application of low-intensity pulsed ultrasound as described above in promoting the uptake of exosomes carrying chemotherapeutic drugs by tumor cells includes:

[0020] Chemotherapy drugs encapsulated in exosomes are mixed with physiological saline to obtain exosome drug solution;

[0021] After administering the exosome drug solution to a subject with malignant tumors, the ultrasound probe of a 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, so as to promote the uptake efficiency of tumor cells on exosomes carrying chemotherapy drugs.

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

[0023] This invention uses a strength of 200 mW / cm 2 ~350mW / cm 2 Low-intensity pulsed ultrasound, without damaging exosomes carrying chemotherapeutic drugs, promotes the uptake efficiency of these drugs by tumor cells, thereby enhancing the therapeutic effect of chemotherapeutic drugs on tumor cells. This invention, through cell and animal experiments, demonstrates that low-intensity pulsed ultrasound combined with exosomes carrying chemotherapeutic drugs can enhance the therapeutic effect of chemotherapeutic drugs on tumor cells, providing new theoretical and experimental basis for tumor treatment and showing promising application prospects. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 The images show fluorescence patterns, bright-field plots, and fluorescence intensity statistics of tumor cells in each group (Exo+US100, Exo+US200, Exo+US250, Exo+US300, Exo+US350, Exo, Control) in Example 1 of this invention; among them, Figure 1 A represents the fluorescence image of exosomal drugs in each group of tumor cells;

[0026] Figure 1 B represents the bright field diagram of tumor cells in each group; Figure 1 C represents the fluorescence intensity statistics of tumor cells in each group;

[0027] Figure 2The graph shows 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 images show the H&E staining results of tumor tissue sections (Control, EXO, EXO+US) at 200x (200x) and 400x (400x) magnification in Example 2 of the present invention.

[0029] Figure 4 These are TUNEL staining results of tumor tissue sections from each group (Control, EXO, EXO+US) in Example 2 of this invention; wherein, Figure 4 Figure A shows the TUNEL staining results of tumor tissue sections from each group. Figure 4 B is a statistical graph of the TUNEL fluorescence ratio of tumor tissue sections in each group; where green fluorescence represents broken DNA fragments and blue fluorescence represents cell nuclei. The more broken DNA fragments there are, the more tumor cells are damaged by apoptosis.

[0030] Figure 5 This describes the therapeutic effects of each treatment group in Example 2 of the present invention on tumor proliferation in mice; wherein, Figure 5 A represents the diagram of each group of tumors; Figure 5 B is a quantitative statistical chart of the size of tumors in each group. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this art or in accordance with the product instructions. Reagents or instruments used without specified manufacturers 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 the uptake of exosomes carrying chemotherapeutic drugs by tumor cells.

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

[0034] In this invention, the exosomes are turmeric-derived exosomes, and the chemotherapy drugs include epirubicin.

[0035] The present invention does not impose any particular limitation on the source or specific preparation method of exosomes encapsulating chemotherapy drugs. They can be obtained through commercial channels, prepared according to methods well known to those skilled in the art, or prepared according to methods in the literature.

[0036] Through extensive research, the inventors have demonstrated that this invention utilizes low-intensity pulsed ultrasound to stimulate tumor cells that have been administered exosomes loaded with chemotherapeutic drugs. This promotes the uptake efficiency of the chemotherapeutic drugs by tumor cells, thereby enhancing the therapeutic effect of the chemotherapeutic drugs on tumor cells (e.g., reducing tumor cell activity). Furthermore, compared to tumor-bearing mice administered exosomes loaded with chemotherapeutic drugs alone, tumor-bearing mice receiving low-intensity pulsed ultrasound combined with exosomes loaded with chemotherapeutic drugs exhibited higher tumor cell apoptosis rates and smaller tumor volumes, demonstrating better tumor treatment efficacy.

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

[0038] In this invention, intensity refers to the intensity of ultrasound output. When the intensity of low-intensity pulsed ultrasound is within the above range, it can further promote the uptake of chemotherapeutic drugs encapsulated in exosomes by tumor cells, thereby enhancing the therapeutic effect of chemotherapeutic drugs.

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

[0040] In this 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 chemotherapy drugs encapsulated in exosomes by tumor cells and enhance the therapeutic effect of chemotherapy drugs.

[0042] In some embodiments, 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.

[0043] In this invention, duty cycle refers to the proportion of the energized time to the total time within a pulse cycle, time refers to the irradiation time of one 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 this invention can vary considerably depending on a variety of factors, such as the target cells, biological type or tissue, and the general condition of the subject to be treated.

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

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

[0047] In some embodiments, the exosomes containing the chemotherapeutic drugs include turmeric exosomes.

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

[0049] In some embodiments, the chemotherapeutic drugs included in the exosomes containing the chemotherapeutic drugs include epirubicin.

[0050] This invention does not specifically limit the 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 this invention, when the mass ratio of epirubicin to turmeric exosomes is 2:1, low-intensity pulsed ultrasound can be used to promote the uptake of exosomes carrying chemotherapy drugs by tumor cells, thereby enhancing the therapeutic effect of chemotherapy drugs.

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

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

[0055] This invention does not impose any particular limitation on the specific equipment of the low-intensity pulsed ultrasonic device, and any low-intensity pulsed ultrasonic device well known in the art can be used.

[0056] A second aspect of the present invention provides a method for using the aforementioned low-intensity pulsed ultrasound in promoting the uptake of exosomes carrying chemotherapeutic drugs by tumor cells, comprising:

[0057] Obtain tumor cells;

[0058] Chemotherapy drugs encapsulated in exosomes are mixed with physiological saline to obtain exosome drug solution;

[0059] The exosome drug solution and tumor cells were mixed in a container, and then the container was subjected to low-intensity pulsed ultrasound using an ultrasound probe in a low-intensity pulsed ultrasound device to promote the uptake efficiency of the exosomes carrying chemotherapy drugs by the tumor cells.

[0060] In some embodiments, the concentration of the above-mentioned 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%–20%, the duration is 5–10 minutes, and the modulation frequency is 1000 Hz. Furthermore, the intensity of the aforementioned low-intensity pulsed ultrasound is preferably 250 mW / cm². 2 The duty cycle can be preferably 10%, the time can be preferably 10 min, and the modulation frequency is 1000 Hz.

[0062] This invention does not impose any particular limitation on the container used; it can be selected as needed. For example, a six-hole plate can be used as the container.

[0063] A third aspect of the present invention provides a method for using the above-mentioned low-intensity pulsed ultrasound in promoting the uptake of exosomes carrying chemotherapeutic drugs by tumor cells, comprising:

[0064] Chemotherapy drugs encapsulated in exosomes are mixed with physiological saline to obtain exosome drug solution;

[0065] After administering exosome medication to subjects with malignant tumors, the ultrasound probe of a low-intensity pulsed ultrasound device is aimed at the skin area corresponding to the tumor site to perform low-intensity pulsed ultrasound, thereby promoting the uptake efficiency of exosomes carrying chemotherapy drugs by tumor cells.

[0066] In some embodiments, the concentration of the above-mentioned 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 duration 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 is preferably 20%, the duration is preferably 90s, and the modulation frequency is 100Hz.

[0068] The following detailed description, in conjunction with embodiments, illustrates the application of the low-intensity pulsed ultrasound provided by the present invention in promoting the uptake of exosomes carrying chemotherapeutic drugs by tumor cells. However, these descriptions 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, all materials and reagents used in the following examples are commercially available.

[0071] In the following embodiments, “about” means + / - five percent (5%) of the described unit of measurement.

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

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

[0074] The tumor cells used in this embodiment 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) Strength is 100mW / cm 2 Low-intensity pulsed ultrasound combined with exosomes loaded with chemotherapy drugs (Exo+US100): MDA-MB-231 breast cancer cells of passage 10 and in good growth condition were prepared into a concentration of 5×10 5 A tumor cell suspension of 1 cell / mL was added to a six-well plate and then incubated at 37°C for 24 hours. After removing the supernatant, the cells were washed twice with PBS to obtain the cultured tumor cells.

[0078] The exosomes loaded with chemotherapy drugs (curcuma exosomes loaded with epirubicin) were diluted with sterile physiological saline to obtain an exosome solution with a concentration of 6 μg / mL.

[0079] One mL of complete culture medium (prepared from RPMI-1640 medium (1640), fetal bovine serum (FBS), and penicillin-streptomycin mixture (PSS) at a mass ratio of 100:10:1), one mL of exosome solution, and cultured tumor cells were mixed and placed in a six-well plate. Then, a low-intensity pulsed ultrasound device was turned on, with a duty cycle of 10% and an intensity of 100 mW / cm². 2 The duration was 10 minutes and the modulation frequency was 1000 Hz. The ultrasound probe in the low-intensity pulse ultrasound device was applied to the bottom of a six-well plate to perform low-intensity pulse ultrasound, and tumor cells were obtained after low-intensity pulse ultrasound.

[0080] Tumor cells subjected to low-intensity pulsed ultrasound were cultured in an incubator for 24 hours, followed by fluorescence staining and tumor cell viability testing.

[0081] (2) Strength is 200mW / cm 2 Low-intensity pulsed ultrasound combined with exosomes carrying chemotherapy drugs (Exo+US200): compared with (1) an intensity of 100 mW / cm 2 The process of combining low-intensity pulsed ultrasound with exosomes carrying chemotherapy drugs is basically the same, the difference being: the intensity is 100mW / cm 2 Replace with a strength of 200mW / cm 2 .

[0082] (3) Strength is 250mW / cm 2 Low-intensity pulsed ultrasound combined with exosomes carrying chemotherapy drugs (Exo+US250): compared with (1) an intensity of 100 mW / cm 2 The process of combining low-intensity pulsed ultrasound with exosomes carrying chemotherapy drugs is basically the same, the difference being: the intensity is 100mW / cm 2 Replace with a strength of 250 mW / cm 2 .

[0083] (4) Strength is 300mW / cm 2 Low-intensity pulsed ultrasound combined with exosomes carrying chemotherapy drugs (Exo+US300): compared with (1) an intensity of 100 mW / cm 2 The process of combining low-intensity pulsed ultrasound with exosomes carrying chemotherapy drugs is basically the same, the difference being: the intensity is 100mW / cm 2 Replace with a strength of 300mW / cm 2 .

[0084] (5) Strength is 350mW / cm 2Low-intensity pulsed ultrasound combined with exosomes carrying chemotherapy drugs (Exo+US350): compared with (1) an intensity of 100 mW / cm 2 The process of combining low-intensity pulsed ultrasound with exosomes carrying chemotherapy drugs is basically the same, the difference being: the intensity is 100mW / cm 2 Replace with a strength of 350mW / cm 2 .

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

[0086] The exosomes loaded with chemotherapy drugs (curcuma exosomes loaded with epirubicin) were diluted with sterile physiological saline to obtain an exosome solution with a concentration of 6 μg / mL.

[0087] 1 mL of complete culture medium (prepared from RPMI-1640 medium (1640), fetal bovine serum (FBS) and penicillin-streptomycin mixture (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, then incubated in an incubator for 24 h, followed by fluorescence staining and tumor cell viability testing.

[0088] (7) Control group: MDA-MB-231 breast cancer cells of passage 10 and in good growth condition were prepared into a concentration of 5×10⁻⁶. 5 A tumor cell suspension of 1 cell / mL was added to a six-well plate and then incubated at 37°C for 24 hours. After removing the supernatant, the cells were washed twice with PBS to obtain the cultured tumor cells.

[0089] 1 mL of complete culture medium (prepared from RPMI-1640 medium (1640), fetal bovine serum (FBS) and penicillin-streptomycin mixture (PSS) at a mass ratio of 100:10:1), 1 mL of sterile physiological saline, and cultured tumor cells were mixed and placed in a six-well plate. After culturing in an incubator for 24 h, fluorescence staining and tumor cell viability tests were performed.

[0090] 2. Experimental Results

[0091] (1) Fluorescent staining test

[0092] Figure 1 These are the fluorescence images, bright-field images, and fluorescence intensity statistics of each group of tumor cells in Example 1 of the present invention; wherein, Figure 1 A represents the fluorescence image of exosomal drugs in each group of tumor cells; Figure 1 B represents the bright field diagram of tumor cells in each group; Figure 1 C represents the fluorescence intensity statistics of tumor cells in each group.

[0093] Depend on Figure 1 AC analysis revealed that exosomes carrying chemotherapeutic drugs entered the tumor cells; the fluorescence intensity of the exosome group carrying chemotherapeutic drugs (Exo) was significantly lower than that at 200 mW / cm². 2 Low-intensity pulsed ultrasound combined with exosomes carrying chemotherapy drugs (Exo+US200), with an intensity of 250 mW / cm 2 Low-intensity pulsed ultrasound combined with exosomes carrying chemotherapy drugs (Exo+US250), with an intensity of 300mW / cm 2 Low-intensity pulsed ultrasound combined with exosomes carrying chemotherapy drugs (Exo+US300) and an intensity of 350mW / cm 2 The fluorescence intensity of the exosome group (Exo+US350) combined with low-intensity pulsed ultrasound and chemotherapy drugs indicated an intensity of 200 mW / cm². 2 Strength is 250mW / cm 2 Strength is 300mW / cm 2 and a strength of 350mW / cm 2 The drug uptake in the low-intensity pulsed ultrasound combined with exosomes carrying chemotherapy drugs was significantly higher than that in the exosomes carrying chemotherapy drugs group (Exo), indicating that low-intensity pulsed ultrasound can promote the uptake efficiency of exosomes carrying chemotherapy drugs by tumor cells; intensity was 100 mW / cm. 2 The fluorescence intensity of the low-intensity pulsed ultrasound combined with exosomes loaded with chemotherapeutic drugs (Exo+US100) was not significantly different from that of the exosomes loaded with chemotherapeutic drugs (Exo), indicating that an intensity of 100 mW / cm² was sufficient. 2 The amount of drug introduced in the low-intensity pulsed ultrasound combined with exosomes carrying chemotherapy drugs was basically the same as that in the exosome group carrying chemotherapy drugs (Exo), indicating that the intensity was 100 mW / cm. 2 Low-intensity pulsed ultrasound at certain times cannot effectively improve the uptake efficiency of tumor cells on exosomes carrying chemotherapy drugs.

[0094] (2) Tumor cell survival rate 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 loaded with chemotherapy drugs (Exo) was significantly higher than that in the group with an intensity of 200 mW / cm 2 Low-intensity pulsed ultrasound combined with exosomes carrying chemotherapy drugs (Exo+US200), with an intensity of 250 mW / cm 2 Low-intensity pulsed ultrasound combined with exosomes carrying chemotherapy drugs (Exo+US250), with an intensity of 300mW / cm 2 Low-intensity pulsed ultrasound combined with exosomes carrying 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 carrying chemotherapeutic drugs (Exo+US350) group was improved, indicating that low-intensity pulsed ultrasound can promote the uptake of exosomes carrying chemotherapeutic drugs by tumor cells and enhance the therapeutic effect of chemotherapeutic drugs on tumor cells. Meanwhile, the cell survival rate of tumor cells in the low-intensity pulsed ultrasound combined with exosomes carrying chemotherapeutic drugs (Exo+US350) group was improved. 2 The cell survival rate of tumor cells in the low-intensity pulsed ultrasound combined with exosomes loaded with chemotherapeutic drugs (Exo+US100) was not significantly different from that in the exosomes loaded with chemotherapeutic drugs (Exo), indicating that the intensity of 100 mW / cm² was sufficient. 2 Low-intensity pulsed ultrasound at that time could not effectively promote the uptake of exosomes carrying chemotherapy drugs by tumor cells, so its therapeutic effect was not significantly improved.

[0097] Example 2 Animal Experiment

[0098] 1. Experimental Methods

[0099] (1) Constructing a mouse breast cancer model

[0100] Fifteen healthy female 8-week-old Balb / c mice (purchased from Chongqing Tengxin Biotechnology Co., Ltd.) were used to establish a mouse mammary cancer model. 0.1 mL of the solution (approximately 1 × 10⁻⁶ mL) was subcutaneously injected into the upper right hind leg of each mouse. 7 4T1 breast cancer cells of good condition with a density of 1 / mL were collected. After 7 days, the tumor cells were allowed to grow to a suitable size (approximately 80 mm²). 3 Afterwards, tumor-bearing mice were obtained; 15 tumor-bearing mice were randomly divided into 3 groups (n=5 per group) and given the following different treatments:

[0101] ① Control group:

[0102] Mice were sedated and analgesic using a gaseous anesthetic (isoflurane). 0.2 mL of sterile saline was injected into the tail vein of the mice. After the injection, the injection was repeated every 4 days for a total of 4 times. The mice were observed every two days throughout the treatment and before the last injection.

[0103] ②Exosome therapy group (EXO) containing chemotherapy drugs:

[0104] The exosomes loaded with chemotherapy drugs (curcuma exosomes loaded with epirubicin) were diluted with sterile physiological saline to obtain an exosome solution with a concentration of 0.5 mg / mL.

[0105] Mice were sedated and analgesic using a gaseous anesthetic (isoflurane). 0.2 mL of exosome solution with a concentration of 0.5 mg / mL was injected into the tail vein of the mice. After the injection, injections were given every 4 days for a total of 4 times. The mice were observed every two days throughout the treatment and before the last injection.

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

[0107] The exosomes loaded with chemotherapy drugs (curcuma exosomes loaded with epirubicin) were diluted with sterile physiological saline to obtain an exosome solution with a concentration of 0.5 mg / mL.

[0108] Mice were sedated and analgesic using a gaseous anesthetic (isoflurane). 0.2 mL of exosome solution at a concentration of 0.5 mg / mL was injected via the tail vein. After injection, medical ultrasound coupling gel (purchased from Chongqing Haifu Medical Technology Co., Ltd.) was applied to the skin area corresponding to the tumor site. A low-intensity pulsed ultrasound device was then activated: duty cycle of 20%, sound power of 1 W, duration of 90 seconds, and modulation frequency of 100 Hz. The ultrasound probe was aimed at the skin area corresponding to the tumor site for low-intensity pulsed ultrasound. Exosome injection and low-intensity pulsed ultrasound were performed every 4 days, for a total of 4 times. Throughout the entire period and before the last exosome injection and low-intensity pulsed ultrasound, the mice were observed every two days.

[0109] On day 21, mice in each group were sacrificed, and tumors were obtained and their volumes were measured. Tumors from each group were prepared into tumor tissue sections, and then hematoxylin and eosin (H&E staining) and TUNEL staining (TUNEL staining is a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method, which is a commonly used technique for detecting DNA fragmentation in apoptosis) were performed on the tumor tissue sections.

[0110] 2. Experimental Results

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

[0112] Figure 3 The images show the H&E staining results of tumor tissue sections at 200x (200x) and 400x (400x) magnification in Example 2 of this invention.

[0113] Depend on Figure 3 It was found that, compared with the exosome therapy group (EXO) and the control group, the exosome therapy group (EXO+US) with low-intensity pulsed ultrasound combined with exosomes loaded with chemotherapy drugs showed significant nuclear collapse and morphological changes in tumor cells, and an increased tumor cell apoptosis rate. This indicates that the treatment effect of the exosome therapy group (EXO+US) with low-intensity pulsed ultrasound combined with exosomes loaded with chemotherapy drugs was better than that of the exosome therapy group (EXO). This further demonstrates that low-intensity pulsed ultrasound can promote the uptake of exosomes loaded with chemotherapy drugs by tumor cells and enhance the therapeutic effect of chemotherapy drugs on tumor cells.

[0114] (2) TUNEL staining

[0115] Figure 4 These are images showing the TUNEL staining results of tumor tissue sections from each group in Example 2 of this invention; wherein, Figure 4 Figure A shows the TUNEL staining results of tumor tissue sections from each group. Figure 4 B is a statistical graph of the TUNEL fluorescence ratio of tumor tissue sections in each group; where green fluorescence represents broken DNA fragments and blue fluorescence represents cell nuclei. The more broken DNA fragments there are, the more tumor cells are damaged by apoptosis.

[0116] Depend on Figure 4 As shown in A and B, the tumor cells in the control group showed almost no apoptosis. Compared with the exosome therapy group (EXO) which contained chemotherapy drugs, the exosome therapy group (EXO+US) showed more green fluorescence, indicating that the tumor cells in the exosome therapy group (EXO+US) with low-intensity pulsed ultrasound showed more apoptosis. This further demonstrates that low-intensity pulsed ultrasound can promote the uptake of exosomes containing chemotherapy drugs by tumor cells and enhance the therapeutic effect of chemotherapy drugs on tumor cells.

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

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

[0119] Depend on Figure 5 As shown in A and B, the tumor volume in the low-intensity pulsed ultrasound combined with exosomes loaded with chemotherapy drugs (Exo+US) group was smaller than that in the control group and the exosomes loaded with chemotherapy drugs group (Exo), indicating that the low-intensity pulsed ultrasound combined with exosomes loaded with chemotherapy drugs group (Exo+US) can significantly inhibit tumor growth.

[0120] In summary, this invention demonstrates that low-intensity pulsed ultrasound can promote the uptake of exosomes carrying chemotherapeutic drugs by tumor cells, reduce tumor cell activity, promote tumor cell apoptosis, and inhibit tumor growth, resulting in better tumor treatment efficacy. This invention provides new theoretical and experimental basis for tumor treatment and has promising application prospects.

[0121] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions 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. The application of low-intensity pulsed ultrasound in promoting the uptake of exosomes carrying chemotherapeutic drugs by tumor cells, characterized in that, include: Obtain tumor cells; Chemotherapy drugs encapsulated in exosomes are mixed with physiological saline to obtain exosome drug solution; The exosome drug solution and the tumor cells were mixed in a container, and then a low-intensity pulsed ultrasound probe was used to apply low-intensity pulsed ultrasound to the container to promote the uptake efficiency of the exosomes carrying the chemotherapy drug by the tumor cells; the intensity of the low-intensity pulsed ultrasound was 200 mW / cm. 2 ~350mW / cm 2 The low-intensity pulsed ultrasound has a duty cycle of 10% to 20%, a duration of 5 to 10 minutes, and a modulation frequency of 1000 Hz; the tumor cells are breast cancer cells.

2. The application of low-intensity pulsed ultrasound according to claim 1 in promoting the uptake of exosomes carrying chemotherapeutic drugs by tumor cells, characterized in that, The exosomes carrying chemotherapy drugs include turmeric exosomes.

3. The application of low-intensity pulsed ultrasound according to claim 2 in promoting the uptake of exosomes carrying chemotherapeutic drugs by tumor cells, characterized in that, The exosomes containing chemotherapy drugs include epirubicin.

4. The application of low-intensity pulsed ultrasound according to claim 3 in promoting the uptake of exosomes carrying chemotherapeutic drugs by tumor cells, characterized in that, The mass ratio of epirubicin to turmeric exosomes is 2:1.

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