Photo-thermal preparation and preparation method and application thereof

Abstract

The invention discloses a photo-thermal preparation and a preparation method and application thereof. The photo-thermal preparation comprises an exosome and two-dimensional molybdenum disulfide quantum dots, and the two-dimensional molybdenum disulfide quantum dots are located on the surface and / or inside the exosome; the surfaces of the two-dimensional molybdenum disulfide quantum dots are modified with polyvinylpyrrolidone. The tumor enrichment capacity of the two-dimensional molybdenum disulfide quantum dots is enhanced through biological active targeting of the natural exosome, local high heat is generated under irradiation of near-infrared light to kill tumor cells, and damage to normal tissue is reduced. The preparation process is simple, the biological safety is high, the accuracy and the curative effect of photo-thermal therapy can be remarkably improved, and the preparation method has a clinical application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a photothermal preparation and a preparation method and application thereof. Background Art

[0002] Photothermal therapy (PTT) is a novel, temporally and spatially controllable tumor treatment. It utilizes photothermal agents to convert light energy into heat at specific locations, increasing the local tumor temperature and achieving thermal ablation. Because biological tissue exhibits a "water window effect," near-infrared light, as a non-invasive light source, can better penetrate skin, blood, and soft tissue, reaching deeper tissues without damaging normal tissue, making it an ideal light energy source for PTT. To achieve minimally invasive, painless, and cost-effective PTT with high tumor suppression, the key lies in developing highly effective, biosafe, and tumor-targeted PTT agents. In recent years, a large number of PTT agents have been developed for PTT, including gold nanomaterials, carbon nanotubes, two-dimensional materials, and organic dyes. Two-dimensional materials have garnered significant attention due to their unique layered structure and physicochemical properties, ultrathin structure, high specific surface area, and unique optoelectronic properties. Two-dimensional molybdenum disulfide quantum dots (referred to as MoS2 in this paper), a two-dimensional transition metal sulfide, have particularly attracted research attention.

[0003] Molybdenum disulfide (MoS2) is a novel two-dimensional transition metal sulfide. Two-dimensional MoS2 quantum dots (QDs) exhibit excellent photothermal conversion efficiency, biocompatibility, and biodegradability, demonstrating significant potential for application in tumor PTT. However, their lack of active biological targeting limits their further application in tumor photothermal therapy. To this end, researchers both domestically and internationally have explored various approaches to improve their performance. Some studies have shown that adjusting the size of the MoS2 to 50-300 nm improves the tumor targeting of photothermal materials. However, this passive targeting strategy is susceptible to material clearance by the endothelial reticular system, rendering it ineffective. Alternatively, researchers have directly introduced minimally invasive interventional techniques, but these approaches are sensitive to the tumor site. Consequently, increasing research has focused on surface modification of MoS2, specifically the addition of chemical / biological molecules (such as aptamers, human serum albumin, and folic acid) that selectively bind to tumor cell-specific receptors, thereby increasing their accumulation in tumor tissue and reducing their uptake in normal tissue. Chemical modification methods still face challenges such as stability, biocompatibility, and lack of targeting capabilities. While biomolecular modification offers high specificity, it is accompanied by difficulties in synthesis and preservation of the original chemical structure and properties of two-dimensional materials. Its complex design also greatly limits its application in tumor-targeted drug delivery systems. Therefore, it is necessary to develop a new type of photothermal agent to address these issues. Summary of the Invention

[0004] To address the issues raised in the aforementioned background technology, the present invention provides a photothermal preparation, its preparation method, and its application. This invention leverages the bio-active targeting properties of natural exosomes to enhance the tumor-accumulating capacity of two-dimensional molybdenum disulfide quantum dots. Under near-infrared light irradiation, it generates localized hyperthermia that kills tumor cells while minimizing damage to normal tissue. This invention boasts a simple preparation process and high biosafety, significantly improving the precision and efficacy of photothermal therapy and possessing promising clinical applications.

[0005] In order to achieve the above-mentioned objectives, the technical solution adopted by the present invention is as follows: On the one hand, the present invention provides a photothermal preparation, comprising: exosomes and two-dimensional molybdenum disulfide quantum dots, wherein the two-dimensional molybdenum disulfide quantum dots are located on the surface and / or inside the exosomes; the surface of the two-dimensional molybdenum disulfide quantum dots is modified with polyvinyl pyrrolidone (PVP).

[0006] Surface modification of two-dimensional MoS2 quantum dots with polyvinylpyrrolidone can alter their surface hydrophobicity, improve their water solubility and biocompatibility, and reduce their likelihood of being pumped out of the cell by efflux pumps. Polyvinylpyrrolidone also forms a polymer layer on the surface of the two-dimensional MoS2 quantum dots, creating a steric hindrance effect that hinders direct contact between the efflux pumps and the two-dimensional MoS2 quantum dots, thereby inhibiting the efflux pumps' effect on the MoS2 quantum dots and retaining them within tumor cells to function. During the preparation of two-dimensional MoS2 quantum dots, as the MoS2 quantum dots nucleate and grow, polyvinylpyrrolidone reacts with the MoS2 surface, modifying the MoS2 quantum dots. This reduces surface energy, minimizes aggregation, and improves the material's dispersibility, resulting in a smaller, more dispersed, and more uniform particle size. This not only allows for uniform heating and enhances light absorption, but also facilitates metabolic excretion and reduces potential biotoxicity.

[0007] Furthermore, by modifying the surface of two-dimensional MoS2 quantum dots to render them hydrophilic, they promote internalization of the quantum dots. Polyvinylpyrrolidone (PVP) surface-modified MoS2 quantum dots reduces direct contact between exosomal enzymes and the quantum dots in lysosomes. The polymer layer formed on the surface of the quantum dots is highly stable, maintaining relative stability within the lysosome and providing a shielding effect on the MoS2 quantum dots, preventing degradation and destruction in the lysosome.

[0008] Furthermore, the diameter of the two-dimensional molybdenum disulfide quantum dots is 1 to 30 nm;

[0009] The diameter of the exosomes is 30 to 200 nm.

[0010] Furthermore, the source of the exosomes is at least one of the centrifugal supernatant after culturing cells, solid tumor tissue, biological fluid, and Chinese medicinal materials.

[0011] Furthermore, the cells in the centrifugal supernatant after culturing the cells include tumor cells, immune cells, mesenchymal stem cells, HEK-293 cells, CHO cells, and Vero cell lines;

[0012] Preferably, the biological fluid includes blood and milk;

[0013] Preferably, the Chinese medicinal materials include fresh herbal medicine cell wall-broken liquid or dry herbal medicine decoction extract.

[0014] Furthermore, pharmaceutically acceptable excipients are also included.

[0015] Furthermore, the dosage forms of the photothermal preparation include injections, lyophilized powders, gels, transdermal patches, sprays, suppositories, and implants.

[0016] In another aspect, the present invention provides a method for preparing any of the above-mentioned photothermal preparations, comprising the following steps:

[0017] The molybdenum disulfide powder is ball-milled, then immersed in an n-butyl lithium cyclohexane solution, the n-butyl lithium cyclohexane solution is removed after sufficient immersion, and the powder is washed with n-hexane, then added to water, subjected to water bath ultrasonic treatment and centrifugal separation to obtain a two-dimensional molybdenum disulfide quantum dot dispersion; polyvinyl pyrrolidone is added to the two-dimensional molybdenum disulfide quantum dot dispersion, and then stirred or shaken to obtain two-dimensional molybdenum disulfide quantum dots with surfaces modified with polyvinyl pyrrolidone;

[0018] The two-dimensional molybdenum disulfide quantum dots modified with polyvinyl pyrrolidone are mixed with exosomes in phosphate buffer or physiological saline, and then sonicated, frozen and thawed, or extruded, and then incubated at 37° C. or 4° C. for 14 to 18 hours to obtain the photothermal preparation;

[0019] or,

[0020] The molybdenum disulfide powder is ball-milled, then immersed in an n-butyl lithium cyclohexane solution, the n-butyl lithium cyclohexane solution is removed after sufficient immersion, and the powder is washed with n-hexane, then added to water, subjected to water bath ultrasonic treatment and centrifugal separation to obtain a two-dimensional molybdenum disulfide quantum dot dispersion; polyvinyl pyrrolidone is added to the two-dimensional molybdenum disulfide quantum dot dispersion, and then stirred or shaken to obtain two-dimensional molybdenum disulfide quantum dots with surfaces modified with polyvinyl pyrrolidone;

[0021] When the cell confluence is 70% to 80%, two-dimensional molybdenum disulfide quantum dots modified with polyvinyl pyrrolidone are dissolved in cell culture medium and incubated with the cells in a CO2 incubator at 37°C for 4 to 24 hours. The supernatant is then discarded and replaced with fresh cell culture medium for a further 24 to 48 hours. The cell supernatant is collected and the exosomes are extracted to obtain the photothermal preparation.

[0022] Furthermore, the mass ratio of the polyvinyl pyrrolidone to molybdenum disulfide powder is 1:(0.1-10);

[0023] Preferably, the ball milling frequency is 20 to 60 Hz, and the ball milling time is 2 to 6 hours;

[0024] Preferably, the water bath ultrasound time is 0.5 to 2 hours;

[0025] Preferably, the stirring time is 6 to 24 hours;

[0026] Preferably, the oscillation time is 6 to 24 hours;

[0027] Preferably, the mass ratio of the two-dimensional molybdenum disulfide quantum dots modified with polyvinyl pyrrolidone to the exosomes is 1:(1-100); preferably 1:1.25, 1:3.3, 1:10, 1:12.5, 1:20, 1:33, 1:100;

[0028] Preferably, the frequency of the ultrasound is 20 to 60 kHz, and the time of the ultrasound is 10 to 30 minutes;

[0029] Preferably, the freeze-thaw step is to incubate the mixture at room temperature for 10 to 30 minutes, then transfer it to -80°C or liquid nitrogen for rapid freezing, and then thaw it at room temperature, and repeat this step 3 to 10 times;

[0030] Preferably, the extrusion is performed by repeatedly passing the mixture through a 100-200 nm polycarbonate membrane 5-15 times.

[0031] Furthermore, the mass ratio of the polyvinyl pyrrolidone to molybdenum disulfide powder is 1:(0.1-10);

[0032] Preferably, the ball milling frequency is 20 to 60 Hz, and the ball milling time is 2 to 6 hours;

[0033] Preferably, the water bath ultrasound time is 0.5 to 2 hours;

[0034] Preferably, the stirring time is 6 to 24 hours;

[0035] Preferably, the oscillation time is 6 to 24 hours;

[0036] Preferably, the ratio of the mass of the two-dimensional molybdenum disulfide quantum dots modified with polyvinyl pyrrolidone on the surface to the volume of the cell culture medium is (0.03-0.10 mg):1 mL;

[0037] Preferably, the supernatant exosomes are extracted by ultraspeed differential centrifugation, density gradient centrifugation or polyethylene glycol precipitation, and then dispersed in phosphate buffer, and then high-purity exosomes are obtained by magnetic bead immunoassay, size exclusion chromatography or ultrafiltration.

[0038] Ultrasonic treatment of the mixed solution can produce cavitation and mechanical vibration. The localized high temperature, high pressure, and strong shear force generated by the cavitation effect can increase the membrane permeability of exosomes or temporarily create tiny pores or cracks in the exosomes. It can also improve the dispersion of the two-dimensional MoS2 quantum dots modified with polyvinylpyrrolidone (PVP), increase the contact area between the two-dimensional MoS2 quantum dots and the exosomes, and facilitate the MoS2 quantum dots modified with polyvinylpyrrolidone to enter the exosomes or adsorb on the exosome surface.

[0039] Freeze-thaw mixing refers to the process of alternating freezing and thawing of a mixed solution. During the freezing process, water in the solution forms ice crystals. The growth of these ice crystals may squeeze and damage the membrane structure of the exosomes, causing minor damage to the membrane or increasing permeability. During the thawing process, the exosome membrane structure recloses, encapsulating the free-flowing two-dimensional MoS2 quantum dots (MoS2) with polyvinylpyrrolidone (PVP) on their surfaces. Furthermore, the freeze-thaw process may alter the surface properties of the MoS2 particles. Surface defects expose active sites and polar groups, surface oxidation alters hydrophilicity, and mechanical cutting and breaking of aggregates by ice crystals results in smaller particle size, increased specific surface area, and a reduction in active sites. This strengthens the electrostatic interaction between the MoS2 quantum dots (MoS2) with polyvinylpyrrolidone and the exosomes, leading to a more compatible hydrophobic interaction.

[0040] Extrusion of a mixed solution involves using a specialized instrument to pass the mixed solution through a filter membrane or microporous device with a defined pore size. This process applies external force to the exosomes, causing them to deform and pass through the micropores. During this process, the exosome membrane structure is stretched and squeezed, potentially creating temporary openings or gaps that facilitate the entry of molybdenum disulfide quantum dots modified with polyvinylpyrrolidone into the exosomes. Furthermore, extrusion can make the exosomes more uniform in size and other parameters, preventing aggregation.

[0041] Incubation involves maintaining the mixed solution at a specific temperature for a period of time. Incubation at 37°C, close to the physiological temperature of the human body, simulates the in vivo environment, allowing recombination to more closely resemble real physiological conditions. This optimizes electrostatic adsorption, allowing the electrostatically adsorbed 2D MoS2 quantum dots (MoS2) modified with PVP to reach dynamic equilibrium adsorption within the biological environment, thereby reducing premature desorption and ineffectiveness after entry into the human body. Incubation at 4°C slows the movement of exosomes and MoS2 quantum dots (MoS2) modified with PVP, achieving a high recombination rate and low aggregation of MoS2 quantum dots. This inhibits thermal aggregation of MoS2 quantum dots, preventing them from aggregating from 1-30 nm quantum dots to large micron-sized particles. Even when desorbed within the human physiological environment, they desorb as individual, free small particles rather than aggregated large particles, making them more easily excreted.

[0042] In another aspect, the present invention provides a use of any of the above-mentioned photothermal preparations in the preparation of a drug for photothermal therapy of tumors.

[0043] Furthermore, the tumors include breast cancer, skin cancer, prostate cancer, lung cancer, brain tumor, melanoma, head and neck tumor, bladder cancer, liver cancer and esophageal cancer.

[0044] Furthermore, any of the above-mentioned photothermal preparations is administered intravenously or locally to the tumor site, and the tumor cells are killed by the photothermal conversion effect of molybdenum disulfide under near-infrared light irradiation.

[0045] Furthermore, based on the molybdenum disulfide quantum dots with polyvinyl pyrrolidone modified on the surface, the dosage of the photothermal preparation is 5 to 10 mg / kg.

[0046] Furthermore, the near-infrared light used in the photothermal therapy has a wavelength of 700 to 1200 nm and a power density of 0.4 to 1.0 W / cm 2 The irradiation time is 5 to 15 minutes.

[0047] Compared with the prior art, the present invention has the following beneficial effects:

[0048] (1) The photothermal preparation of the present invention has good photothermal conversion efficiency, biocompatibility and degradability.

[0049] (2) Natural exosomes carry a large amount of biological information of parent cells. Their introduction gives photothermal preparations tumor targeting, which can improve the accuracy and effectiveness of photothermal therapy and reduce damage to normal tissues. At the same time, the membrane structure and biological origin of exosomes can reduce immune rejection and improve in vivo safety.

[0050] Currently, there are no patent reports on the use of exosome vesicles as targeted carriers in combination with molybdenum disulfide for anti-tumor treatment. The significant advantage of this invention lies in overcoming the challenge of improving the targeted delivery of molybdenum disulfide without affecting its surface properties. This method can achieve high accumulation of the photothermal material molybdenum disulfide in tumor tissue, while triggering the release of the photothermal material through membrane rupture under near-infrared laser irradiation. This is expected to achieve the dual effects of targeted and controlled release, ultimately significantly improving the precision and efficacy of tumor photothermal therapy.

[0051] (3) The preparation method of the present invention is simple and easy to implement, and can be easily scaled up for production. It can also simultaneously load genes or small molecule drugs to achieve multimodal treatment.

[0052] (4) This application uses two-dimensional MoS2 quantum dots. Compared with conventional two-dimensional MoS2 nanosheets, quantum dots have narrow band gap characteristics, which make them have stronger light absorption ability at specific wavelengths. At the same time, the quantum confinement effect of quantum dots can effectively separate photogenerated electrons and holes, reduce their recombination, and thus improve the photothermal conversion efficiency; while conventional nanosheets are prone to rapid recombination of photogenerated electron-hole pairs, thereby reducing the photothermal conversion efficiency, especially in low light intensity or application scenarios requiring continuous thermal effect. In addition, small-sized MoS2 quantum dots have a larger specific surface area and higher surface activity, which makes them easy to interact with exosomes and form composite materials with exosomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments of the present invention. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0054] Figure 1 Schematic diagram of the preparation process of photothermal preparation;

[0055] Figure 2 Visual appearance of two-dimensional molybdenum disulfide quantum dots modified with polyvinyl pyrrolidone (MoS2-PVP) dissolved in ultrapure water (UPW), MoS2-PVP dissolved in phosphate buffer (PBS), two-dimensional molybdenum disulfide quantum dots modified with polyethylene glycol (MoS2-PEG) dissolved in ultrapure water (UPW), and MoS2-PEG dissolved in phosphate buffer (PBS);

[0056] Figure 3The following are visual appearance images of two-dimensional molybdenum disulfide quantum dots (MoS2-PVP) with surface modification of polyvinyl pyrrolidone, exosomes (ME), and photothermal preparation (MoS2-PVP@ME) dissolved in phosphate buffer (PBS) prepared in Example 1 of the present invention, and transmission electron microscopy images and dynamic light scattering test images of the photothermal preparation prepared in Example 1 of the present invention, wherein Figure 3 A in the figure is the visual appearance of two-dimensional molybdenum disulfide quantum dots modified with polyvinyl pyrrolidone, exosomes, and photothermal preparations dissolved in phosphate buffered saline (PBS), respectively. Figure 3 B in the figure is a transmission electron microscope image of the photothermal preparation. Figure 3 C in the figure is the dynamic light scattering test graph of the photothermal preparation;

[0057] Figure 4 The thermal imaging images of the two-dimensional molybdenum disulfide quantum dots, exosomes, photothermal preparations and PBS modified with polyvinyl pyrrolidone on the surface prepared in Example 1 of the present invention under near-infrared light irradiation and the photothermal stability results of the two-dimensional molybdenum disulfide quantum dots and photothermal preparations modified with polyvinyl pyrrolidone on the surface prepared in Example 1 of the present invention are shown. Figure 4 A in the figure is a thermal image of two-dimensional molybdenum disulfide quantum dots modified with polyvinyl pyrrolidone, exosomes, photothermal preparations, and PBS under near-infrared radiation (NIR). Figure 4 Figure B shows the photothermal stability of two-dimensional molybdenum disulfide quantum dots and photothermal preparations modified with polyvinyl pyrrolidone.

[0058] Figure 5 This is a storage stability test chart of the photothermal preparation prepared in Example 1 of the present invention, wherein Figure 5 A in the figure is the visual appearance of the photothermal preparation at 0, 1, 3, 5, and 7 days after being placed at 4°C. Figure 5 B in the figure is the protein staining result of the exosomes, MoS2-PVP and photothermal preparation prepared in Example 1 at 0, 1, 3, 5 and 7 days at 4°C. Figure 5 C in the figure is the thermal imaging image of the photothermal preparation at 0, 1, 3, 5, and 7 days under NIR irradiation;

[0059] Figure 6 This is a flow chart for the application of photothermal preparations in photothermal therapy;

[0060] Figure 7 Flow cytometry plots of the photothermal preparation prepared in Example 1 labeled with PKH26 dye after co-incubation with human mammary epithelial cells MCF-10A, human breast cancer cells MCF-7, and mouse breast cancer cells 4T1;

[0061] Figure 8 This is a graph showing the results of cell death and viability staining after co-incubation of two-dimensional molybdenum disulfide quantum dots modified with polyvinyl pyrrolidone, exosomes, a photothermal preparation, and PBS with human breast cancer MCF-7 cells prepared in Example 1 of the present invention;

[0062] Figure 9 This is a diagram showing the application effect of two-dimensional molybdenum disulfide quantum dots modified with polyvinyl pyrrolidone, a photothermal preparation, and PBS in photothermal therapy, prepared in Example 1 of the present invention. Figure 9 A in the figure is the thermal imaging result of two-dimensional molybdenum disulfide quantum dots modified with polyvinyl pyrrolidone, photothermal preparation and PBS during photothermal treatment. Figure 9 B in the figure is a graph showing the changes in tumor volume of 4T1 tumor-bearing mice 20 days after photothermal treatment with two-dimensional molybdenum disulfide quantum dots modified with polyvinyl pyrrolidone, photothermal preparations, and PBS. PBS is a group of mice injected with PBS solvent and not irradiated with near-infrared light NIR. PBS+NIR is a group of mice injected with PBS solvent and irradiated with near-infrared light NIR. MoS2-PVP+NIR is a group injected with two-dimensional molybdenum disulfide quantum dots modified with polyvinyl pyrrolidone and irradiated with near-infrared light NIR. MoS2-PVP@ME+NIR is a group injected with photothermal preparations and irradiated with near-infrared light NIR. Figure 9 Figure C is a statistical graph of the weight changes of 4T1 tumor-bearing mice during photothermal treatment with two-dimensional molybdenum disulfide quantum dots modified with polyvinyl pyrrolidone, photothermal preparations, and PBS. DETAILED DESCRIPTION

[0063] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0064] It should be noted that all raw materials in the embodiments of the present invention can be purchased on the market or prepared according to conventional methods well known to those skilled in the art; in addition, the "and / or" in the full text includes three schemes, taking A and / or B as an example, including technical scheme A, technical scheme B, and technical schemes that satisfy both A and B; in addition, the technical schemes between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in this field to implement them. When the combination of technical schemes is contradictory or cannot be implemented, it should be deemed that such a combination of technical schemes does not exist and is not within the scope of protection required by the present invention.

[0065] The experimental methods in the following examples, unless otherwise specified, are conventional methods. The experimental materials and reagents used in the following examples, unless otherwise specified, are commercially available. The quantitative experiments in the following examples were performed in triplicate, and the data are presented as the mean or mean ± standard deviation of the three replicates.

[0066] Example 1: Preparation of photothermal preparation

[0067] The schematic diagram of the preparation process of photothermal preparation is as follows Figure 1 shown.

[0068] Preparation of two-dimensional molybdenum disulfide quantum dots with surface modification of polyvinyl pyrrolidone or polyethylene glycol: molybdenum disulfide powder is ball-milled (40 Hz, 4 h), and then 200 mg is soaked in 10 mL of 2.5 mol / L n-butyl lithium cyclohexane solution. After soaking for 2 days, the n-butyl lithium cyclohexane solution is removed, and the mixture is washed three times with n-hexane. Then, it is added to 100 mL of water, subjected to 40 kHz water bath ultrasound for 30 min and centrifuged to obtain a two-dimensional molybdenum disulfide quantum dot dispersion; 200 mg of polyvinyl pyrrolidone or polyethylene glycol is added to the two-dimensional molybdenum disulfide quantum dot dispersion, and then stirred at 500 r / min for 12 h. Finally, unbound polyvinyl pyrrolidone or polyethylene glycol is removed by dialysis to obtain two-dimensional molybdenum disulfide quantum dots (MoS2-PVP, MoS2-PEG) with surface modification of polyvinyl pyrrolidone or polyethylene glycol.

[0069] MoS2-PVP and MoS2-PEG were dissolved in phosphate buffered saline (PBS) and ultrapure water (UPW), respectively, and their appearances were recorded. Figure 2 As shown. Figure 2 It can be seen that both MoS2-PVP and MoS2-PEG have a brown-black appearance and are stored in UPW and PBS, respectively. Under these conditions, MoS2-PEG has poor stability in both solvents and quickly aggregates and precipitates after addition, which cannot meet the use requirements. MoS2-PVP has good dispersibility.

[0070] Exosome extraction and purification: Exosomes were extracted from the supernatant of human breast cancer MCF-7 cells after culture using polyethylene glycol precipitation. The cell culture supernatant was centrifuged at 4°C at 300×g for 10 minutes, 2000×g for 10 minutes, and 10,000×g for 30 minutes. The supernatant was collected and mixed with half the volume of total exosome extraction reagent overnight (16 hours). The next day, the supernatant was centrifuged at 10,000×g for 30 minutes, discarded, and the pellet resuspended in 1 mL of PBS. Highly purified exosomes (ME) were obtained by ultrafiltration (100 kDa centrifugation for 10 minutes).

[0071] Preparation of the photothermal preparation: Two-dimensional molybdenum disulfide quantum dots (MoS2) surface-modified with polyvinylpyrrolidone (PVP) and exosomes were mixed in phosphate buffer at a mass ratio of 1:33. Self-assembly and electrostatic adsorption were performed under 40kHz water bath ultrasound for 30 minutes. The mixture was then incubated at 4°C for 16 hours to allow the two to bind tightly together, forming the photothermal preparation. Unbound MoS2-PVP@ME and exosomes were then removed by centrifugation to obtain a pure composite (MoS2-PVP@ME) for use as the photothermal preparation.

[0072] Two-dimensional MoS2 quantum dots modified with polyvinyl pyrrolidone, exosomes and photothermal preparations were dissolved in phosphate buffered saline (PBS) and their appearances were recorded. Figure 3 As shown in A, from Figure 3 It can be seen from A in Figure 1 that the appearance of MoS2-PVP@ME is brown-black, similar to the appearance of MoS2-PVP, with good dispersion and no obvious aggregation or precipitation.

[0073] The photothermal preparation MoS2-PVP@ME was scanned by transmission electron microscopy, and the results were as follows: Figure 3 As shown in B. Figure 3 As can be seen from B in the figure, the microscopic morphology of the photothermal preparation is spherical, which is consistent with the "cup-disc" morphology of exosomes. The photothermal preparation MoS2-PVP@ME was tested by dynamic light scattering, and the results are as follows: Figure 3 As shown in C. Figure 3 As can be seen from Figure C, the particle size of the preparation is 145.9±7.5nm, which is still in the nanoscale range.

[0074] Example 2: Photothermal performance test of photothermal preparation

[0075] PBS, two-dimensional molybdenum disulfide quantum dots MoS2-PVP with surface modification of polyvinyl pyrrolidone prepared in Example 1, exosomes ME and photothermal preparation MoS2-PVP@ME were placed under 1064 nm near-infrared laser at 0.4 W / cm 2 Irradiate for 5 minutes, the results are as follows Figure 4 As shown in A. Figure 4 It can be seen from A in that the photothermal preparation has a high photothermal conversion efficiency and a significant temperature increase, which basically overlaps with the temperature increase trend of two-dimensional molybdenum disulfide quantum dots modified with polyvinyl pyrrolidone on the surface, indicating that the photothermal preparation retains the excellent photothermal properties of two-dimensional molybdenum disulfide quantum dots.

[0076] After the photothermal preparation cooled to room temperature, the 1064nm laser was turned on again for 5 minutes. The above steps were repeated 4 times while the thermal imager monitored the real-time temperature. The temperature changes of the 5 heating and cooling cycles were observed to investigate the photothermal stability. The results are as follows: Figure 4 As shown in B. Figure 4 As can be seen from B, after four repeated "on-off" irradiations of near-infrared light, the photothermal preparation can still reach the highest temperature during the initial heating when it is irradiated with laser again, which proves that repeated laser irradiation has no obvious effect on the photothermal properties of two-dimensional molybdenum disulfide quantum dots, and the photothermal preparation has good photothermal stability and the potential for repeated photothermal use.

[0077] Example 3: Storage stability test of photothermal preparation

[0078] The photothermal preparation prepared in Example 1 was placed in a 4°C refrigerator for 0, 1, 3, 5, and 7 days, and then returned to room temperature. The appearance of the photothermal preparation at each time point (PBS, ME, and MoS2-PVP as controls), marker protein (ME and MoS2-PVP as controls), and photothermal conversion efficiency (PBS as a negative control) were observed to test the storage stability of the photothermal preparation.

[0079] The appearance of the photothermal preparation at each time point was recorded. The results are as follows: Figure 5 As shown in A, from Figure 5 A in Figure 1 shows that the appearance of the photothermal preparation did not change significantly after being stored at 4°C for 7 days. SDS-PAGE gel electrophoresis was used to separate the exosome ME, MoS2-PVP, and proteins in the photothermal preparation at each time point, and the Coomassie brilliant blue staining method was used to visualize the color. The results are shown in Figure 1. Figure 5 As shown in B, from Figure 5 As can be seen from B in the figure, the protein composition of the photothermal preparation and the exosome at each time point is basically the same, indicating that the photothermal preparation retains the protein composition of the exosome and retains the material basis for targeting the tumor. 2 Irradiate the photothermal preparation at each time point for 5 minutes. The results are as follows Figure 5 As shown in C, from Figure 5 It can be seen from C in that the photothermal conversion performance of MoS2-PVP@ME has almost no change compared with day 0. The above results show that the photothermal preparation has good storage stability at 4°C.

[0080] The application flow chart of photothermal preparations in photothermal therapy is as follows Figure 6 As shown. Figure 6 It can be seen that after using MoS2-PVP@ME, local high heat can be generated under near-infrared light irradiation to kill tumor cells.

[0081] Example 4: In vitro targeting test of photothermal preparations

[0082] The photothermal preparation prepared in Example 1 was labeled with PKH26 dye and then incubated with human mammary epithelial cells MCF-10A, human breast cancer cells MCF-7, and mouse breast cancer cells 4T1 for 1 hour (the control group consisted of cells not incubated with the photothermal preparation, i.e., blank controls). After trypsin digestion, the cell pellet was collected by centrifugation at 300×g for 5 minutes at 4°C, washed and resuspended in PBS, and analyzed by flow cytometry. The results are shown in Figure 2. Figure 7 As shown. Figure 7 It can be seen that the uptake of photothermal preparations in human cancer cells MCF-7 and mouse cancer cells 4T1 cells is similar, and both are higher than that in human normal cells MCF-10A, proving that photothermal preparations have a universal tumor cell tropism.

[0083] Example 5: In vitro toxicity and killing effect test of photothermal preparation

[0084] Photothermal preparations, two-dimensional MoS2 quantum dots modified with polyvinyl pyrrolidone, exosomes, or PBS were used in in vitro photothermal therapy experiments on MCF-7 tumor cells. The cytotoxicity and photothermal killing effects were evaluated by live-death staining. 24 hours after the MCF-7 cells were inoculated, the original culture medium was replaced with photothermal preparations, two-dimensional MoS2 quantum dots modified with polyvinyl pyrrolidone, exosomes, or PBS dissolved in the culture medium. After incubation for another 4 hours, the cells were exposed to 0.4W / cm 2 After washing the cells with PBS, 200 μL of detection reagent (Calcein AM / PI cell viability and cytotoxicity detection kit) was added to each well and the cells were observed and imaged using a live cell imaging device. The results were as follows: Figure 8 As shown. Figure 8 As can be seen from the figure, under the condition of no NIR, cells can be labeled with Calcein AM (Calcein Acetoxymethyl Ester) and show green fluorescence. At the same time, the cell morphology is intact, indicating that under this condition, the photothermal agent has no significant effect on cell viability. At 0.4W / cm 2 Under NIR, all cells in the field of view corresponding to the two-dimensional MoS2 quantum dots modified with polyvinyl pyrrolidone and the photothermal agent were labeled with red fluorescence by propidium iodide (PI), indicating that under these conditions, the two-dimensional MoS2 quantum dots modified with polyvinyl pyrrolidone and the photothermal agent can cause late apoptosis or necrosis of tumor cells, and the photothermal agent can achieve efficient photothermal killing. These results show that the photothermal agent has light-triggered response capabilities, has a significant tumor killing effect, and retains a certain degree of biocompatibility even in the absence of NIR.

[0085] Example 6: In vivo anti-tumor effect and toxicity evaluation of photothermal preparations

[0086] A 4T1 subcutaneous transplant tumor model was established using Balb / c mice. The molybdenum disulfide quantum dots with surface modification of polyvinyl pyrrolidone and the photothermal preparation prepared in Example 1 were dissolved in phosphate buffer and injected into the tumor-bearing mice via the tail vein (8 mg / kg based on molybdenum disulfide quantum dots with surface modification of polyvinyl pyrrolidone). 200 μL of PBS was injected into the tail vein as a control experiment. 24 hours after injection, the photothermal preparation can specifically target the tumor tissue under the guidance of exosomes. Then, 0.48W / cm 2 The tumor was locally irradiated with 1064nm NIR for 10 minutes at a time, and the temperature changes of the tumor site of the mouse were monitored using an infrared thermal imager. Figure 9 As shown in A. Figure 9 As shown in A, as the laser irradiation time increased, the temperature of the tumor site in the photothermal preparation group gradually increased, and within 2 minutes, the temperature was higher than that in the MoS2-PVP group. This shows that the photothermal preparation has a significant ability to heat solid tumors, which indirectly reflects that the photothermal preparation has a more prominent tumor targeting ability. After the photothermal treatment, the tumor volume and weight of the mice were recorded daily to investigate the treatment effect and toxicity. The results are shown in the figure below. Figure 9 B and Figure 9 As shown in C. Figure 9 B and Figure 9 As can be seen from Figure C, after 20 days, the tumor volume of mice in the photothermal preparation group was significantly reduced, and no obvious toxic side effects were observed. In addition, tumor tissues were separated at 20 days, and the tumor inhibition rates of each group were compared (tumor inhibition rate = tumor weight of NIR experimental group / tumor weight of PBS control group × 100%). In the NIR irradiation experimental group, the tumor inhibition rates of PBS, MoS2-PVP group, and MoS2-PVP@ME group were 0.87%, 16.23%, and 75.92%, respectively. The tumor inhibition rate of the photothermal preparation group was significantly better than that of the other groups. The above results show that the photothermal preparation prepared in Example 1 has a significant solid tumor killing effect and good in vivo safety.

[0087] The above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit the present application. Although the present application has been described in detail with reference to the aforementioned embodiments, a person of ordinary skill in the art should understand that the technical solutions described in the aforementioned embodiments can still be modified, or some or all of the technical features therein can be replaced by 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 present application.

Claims

1. A photothermal preparation, characterized in that: include: Exosomes and two-dimensional molybdenum disulfide quantum dots, wherein the two-dimensional molybdenum disulfide quantum dots are located on the surface and / or inside the exosomes; and the surface of the two-dimensional molybdenum disulfide quantum dots is modified with polyvinyl pyrrolidone.

2. The photothermal preparation according to claim 1, characterized in that The diameter of the two-dimensional molybdenum disulfide quantum dots is 1 to 30 nm.

3. The photothermal preparation according to claim 1, characterized in that The diameter of the exosomes is 30 to 200 nm.

4. The photothermal preparation according to claim 1, characterized in that The exosomes are derived from at least one of the centrifugal supernatant after culturing cells, solid tumor tissue, biological fluids, and Chinese medicinal materials.

5. The photothermal preparation according to claim 4, characterized in that: The cells in the centrifugal supernatant after culturing the cells include tumor cells, immune cells, mesenchymal stem cells, HEK-293 cells, CHO cells, and Vero cell lines; Preferably, the biological fluid includes blood and milk; Preferably, the Chinese medicinal materials include fresh herbal medicine cell wall-broken liquid or dry herbal medicine decoction extract.

6. The method for preparing the photothermal preparation according to any one of claims 1 to 5, characterized in that: The following steps are involved: The molybdenum disulfide powder is ball-milled, then immersed in an n-butyl lithium cyclohexane solution, the n-butyl lithium cyclohexane solution is removed after sufficient immersion, and the powder is washed with n-hexane, then added to water, subjected to water bath ultrasonic treatment and centrifugal separation to obtain a two-dimensional molybdenum disulfide quantum dot dispersion; polyvinyl pyrrolidone is added to the two-dimensional molybdenum disulfide quantum dot dispersion, and then stirred or shaken to obtain two-dimensional molybdenum disulfide quantum dots with surfaces modified with polyvinyl pyrrolidone; The two-dimensional molybdenum disulfide quantum dots modified with polyvinyl pyrrolidone are mixed with exosomes in phosphate buffer or physiological saline, and then sonicated, frozen and thawed, or extruded, and then incubated at 37° C. or 4° C. for 14 to 18 hours to obtain the photothermal preparation; or, The molybdenum disulfide powder is ball-milled, then immersed in an n-butyl lithium cyclohexane solution, the n-butyl lithium cyclohexane solution is removed after sufficient immersion, and the powder is washed with n-hexane, then added to water, subjected to water bath ultrasonic treatment and centrifugal separation to obtain a two-dimensional molybdenum disulfide quantum dot dispersion; polyvinyl pyrrolidone is added to the two-dimensional molybdenum disulfide quantum dot dispersion, and then stirred or shaken to obtain two-dimensional molybdenum disulfide quantum dots with surfaces modified with polyvinyl pyrrolidone; When the cell confluence is 70% to 80%, two-dimensional molybdenum disulfide quantum dots modified with polyvinyl pyrrolidone are dissolved in cell culture medium and incubated with the cells in a CO2 incubator at 37°C for 4 to 24 hours. The supernatant is then discarded and replaced with fresh cell culture medium for a further 24 to 48 hours. The cell supernatant is collected and the exosomes are extracted to obtain the photothermal preparation.

7. The method for preparing the photothermal preparation according to claim 6, characterized in that: The mass ratio of the polyvinyl pyrrolidone to molybdenum disulfide powder is 1:(0.1-10); Preferably, the ball milling frequency is 20 to 60 Hz, and the ball milling time is 2 to 6 hours; Preferably, the water bath ultrasound time is 0.5 to 2 hours; Preferably, the stirring time is 6 to 24 hours; Preferably, the oscillation time is 6 to 24 hours; Preferably, the mass ratio of the two-dimensional molybdenum disulfide quantum dots whose surface is modified with polyvinyl pyrrolidone to the exosomes is 1:(1-100); Preferably, the frequency of the ultrasound is 20 to 60 kHz, and the time of the ultrasound is 10 to 30 minutes; Preferably, the freeze-thaw step is to incubate the mixture at room temperature for 10 to 30 minutes, then transfer it to -80°C or liquid nitrogen for rapid freezing, and then thaw it at room temperature, and repeat this step 3 to 10 times; Preferably, the extrusion is performed by repeatedly passing the mixture through a 100-200 nm polycarbonate membrane 5-15 times.

8. The method for preparing the photothermal preparation according to claim 6, characterized in that: The mass ratio of the polyvinyl pyrrolidone to molybdenum disulfide powder is 1:(0.1-10); Preferably, the ball milling frequency is 20 to 60 Hz, and the ball milling time is 2 to 6 hours; Preferably, the water bath ultrasound time is 0.5 to 2 hours; Preferably, the stirring time is 6 to 24 hours; Preferably, the oscillation time is 6 to 24 hours; Preferably, the ratio of the mass of the two-dimensional molybdenum disulfide quantum dots whose surface is modified with polyvinyl pyrrolidone to the volume of the cell culture medium is (0.03-0.10 mg):1 mL.

9. Use of the photothermal preparation according to any one of claims 1 to 5 in the preparation of a drug for photothermal therapy of tumors.

10. The use according to claim 9, characterized in that These tumors include breast cancer, skin cancer, prostate cancer, lung cancer, brain tumor, melanoma, head and neck cancer, bladder cancer, liver cancer, and esophageal cancer.

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