Synthesis of a hybrid nanomaterial and its application in tumor photothermal therapy

Abstract

The present invention provides a method for synthesizing a hybrid nanomaterial and its application in tumor photothermal therapy, belonging to the field of biomedical materials. The method of the present invention comprises the following steps: (a) preparing a mixed solvent of ethylene glycol / deionized water; (b) adding CuCl2·2H2O and Zn(NO3)2·6H2O into the solvent obtained in step (a), stirring and mixing, then adding thioacetamide and continuing to stir and mix; (c) transferring the mixed solution obtained in step (b) into a high-pressure autoclave lined with Teflon, sealing the autoclave in an oven at 180 °C, reacting for 3 h and then naturally cooling to room temperature; then centrifuging to collect the precipitate, washing it several times with distilled water and ethanol, and finally drying it at 60 °C for 12 h to obtain CuS / ZnS. After the present invention composes CuS and ZnS to form a CuS / ZnS hybrid nanomaterial, the recombination of electron-hole pairs can be significantly reduced, and the photothermal conversion efficiency can be greatly improved.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and specifically to the synthesis of a hybrid nanomaterial and its application in photothermal therapy of tumors. Background Art

[0002] Photothermal therapy (PTT) is a treatment method that artificially increases tissue temperature. It uses the sensitivity of cells to heat to induce cell apoptosis or increase their sensitivity to radiotherapy or chemotherapy. Compared with traditional cancer treatments (such as surgery, radiotherapy and chemotherapy), PTT has higher inherent specificity and lower invasive burden, and it only produces thermal effects in the presence of photothermal agents. Therefore, through proper design, photothermal agents can exhibit excellent photothermal performance under short-term and low-power experimental conditions. To date, most research work has focused on inorganic materials triggered by near-infrared light, including transition metal nanoparticles, gold nanoparticles and platinum nanoparticles. Although these nanomaterials have good absorbance, they lack biodegradability and have potential long-term toxicity. ZnS, as a semiconductor in the present invention, has strong visible light absorption ability. When it is modified with hydrophilic groups, it exhibits good water solubility, can directly contact cells or biological tissues in the body, and has good biocompatibility. However, ZnS is a wide bandgap semiconductor. Since most of the energy it absorbs is re-emitted in the form of light, it is easy to recombine electron-hole pairs, thereby reducing the photothermal conversion efficiency. Among metal chalcogenide compounds, CuS not only has semiconductor properties and low cytotoxicity, but also exhibits significant plasmon effects, suitable energy band size, and excellent electronic and optical properties, and can absorb near-infrared light and generate heat. So far, there has been no report on the composite of CuS and ZnS to form CuS / ZnS hybrid nanomaterials. Summary of the invention

[0003] To solve the above problems, the present invention discloses the synthesis of a hybrid nanomaterial and its application in tumor photothermal therapy. After the present invention forms a CuS / ZnS hybrid nanomaterial by compounding CuS and ZnS, the recombination of electron-hole pairs can be significantly reduced, greatly improving the photothermal conversion efficiency. Therefore, the CuS / ZnS hybrid nanomaterial is an excellent photothermal material that can be used for tumor treatment. Optimizing the concentration of the CuS / ZnS hybrid nanomaterial can increase its killing rate of cancer cells and enhance the effect of its photothermal therapy.

[0004] To achieve the above object, the technical solution of the present invention is as follows:

[0005] The present invention first provides a method for synthesizing a hybrid nanomaterial, the method comprising the following steps:

[0006] (a) preparing a ethylene glycol / deionized water mixed solvent;

[0007] (b) Adding CuCl2·2H2O and Zn(NO3)2·6H2O to the solvent obtained in step (a), stirring and mixing, and then adding thioacetamide and continuing to stir and mix; wherein the amount ratio of the solvent obtained in step (a), CuCl2·2H2O, Zn(NO3)2·6H2O, and thioacetamide is 30 mL: 85 mg: 148 mg: 150 mg;

[0008] (c) The mixed solution obtained in step (b) is transferred to a Teflon-lined autoclave, which is sealed in an oven at 180°C and naturally cooled to room temperature after reacting for 3 hours; the precipitate is then collected by centrifugation, washed several times with distilled water and ethanol, and finally dried at 60°C for 12 hours to obtain CuS / ZnS.

[0009] Furthermore, in step (a), the volume ratio of ethylene glycol / deionized water is 3:1.

[0010] Furthermore, the stirring and mixing time in step (b) is 10 minutes.

[0011] The invention also provides a CuS / ZnS hybrid nano material prepared by the method.

[0012] Another object of the present invention is to provide an application of CuS / ZnS hybrid nanomaterials in photothermal therapy, for photothermal therapy of tumor cells under laser irradiation with a wavelength of 808 nm.

[0013] The beneficial effects of the present invention are:

[0014] After the present invention forms CuS / ZnS hybrid nanomaterial by compounding CuS and ZnS, the recombination of electron-hole pairs can be significantly reduced, greatly improving the photothermal conversion efficiency. The photothermal agent CuS / ZnS prepared by the present invention exhibits excellent photothermal performance under the excitation of a light source with a wavelength of 808 nm, achieving the ability to convert light into heat at low power (0.27 W·cm -2 ) and a short time (2 min) to achieve good tumor treatment effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is the X-ray diffraction pattern of the nanomaterial CuS / ZnS described in Example 1;

[0016] Figure 2 This is a transmission electron microscope image of the nanomaterial CuS / ZnS described in Example 1;

[0017] Figure 3a is a photothermal heating curve of CuS / ZnS under irradiation of near-infrared light (808 nm) in Example 2, and b is a temperature change diagram of the heating-cooling cycle of CuS / ZnS;

[0018] Figure 4 They are respectively the MTT experimental diagrams of the treatment of MCF-7 cells in Example 3, wherein a is CuS / ZnS at different concentrations; b is CuS / ZnS under irradiation of near-infrared light (808 nm);

[0019] Figure 5 They are respectively laser confocal imaging images of MCF-7 cells in Example 4, wherein a is the control group, b is CuS / ZnS, c is the control group under near-infrared light irradiation, and d is CuS / ZnS under near-infrared light irradiation. Implementation

[0020] The present invention will be further explained below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the following specific embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. Example

[0021] Preparation and Characterization of CuS, ZnS and CuS / ZnS

[0022] Test instruments: transmission electron microscope JEOL-2100F, D / max-RC diffractometer.

[0023] First, 30 mL of ethylene glycol / deionized water (volume ratio of 3:1) mixed solvent was prepared, and then 85 mg CuCl2·2H2O and 148 mg Zn(NO3)2·6H2O were added to the mixed solvent. After stirring for 10 min, 150 mg thioacetamide was added and stirred for 10 min. Then, the reaction solution was transferred to a 50 mL Teflon-lined autoclave, which was sealed in an oven at 180°C. After reacting for 3 h, it was naturally cooled to room temperature. The precipitate was collected by centrifugation, washed several times with distilled water and ethanol, and finally dried at 60°C for 12 h to obtain CuS / ZnS. ZnS was prepared by not adding CuCl2·2H2O in the above steps. CuS was prepared by adding 1 mL sodium sulfide solution (Na2S, 1 M) to 1000 mL of CuCl2 (1 mM) and sodium citrate (0.68 mM) aqueous solution and stirring the reaction at room temperature. After 5 min, the reaction mixture was heated to 90 °C and stirred at 1000 rpm for 15 min until a dark green solution was obtained. The precipitate was collected by centrifugation, washed several times with distilled water and ethanol, and dried at 60 °C for 12 h to obtain CuS. According to the position of the characteristic diffraction peaks of the sample (Figure); CuS corresponds to (103), (102), (101); ZnS corresponds to (111), (220), (311), which can confirm the successful preparation of CuS / ZnS. In addition, from TEM ( Figure 2 ) It can be seen that the CuS / ZnS hybrid material has a uniform morphology and good dispersibility, and the particle size is about 104 nm, which can further confirm the successful preparation of CuS / ZnS. Example

[0024] Determination of photothermal properties of nanomaterials

[0025] Test equipment: solid laser-808 nm (MDL-808-10W), FOTRIC 220s thermal imager. Experimental method: using infrared laser, at a light power of 0.27 W·cm -2 Under the experimental conditions, a solution with a concentration of 0.005M was irradiated, and then the change in solution temperature was recorded, such as Figure 2 The results show that CuS can be heated to 66.8 ℃, which is significantly better than ZnS (34.8 ℃ Figure 3 a). In a short period of time, the temperature of the CuS / ZnS solution increased rapidly and reached the ideal temperature (40 °C) required for photothermal therapy in a short period of time, reaching a maximum temperature of 87.5 °C at 320 s ( Figure 3b). It can be seen from the photothermal heating curve that the temperature rise caused by the photothermal effect of CuS / ZnS is positively correlated with time. Therefore, the CuS / ZnS hybrid material can be better applied to photothermal therapy by controlling the heating time. At the same time, it is worth noting that after several heating-cooling cycles, the photothermal performance of CuS / ZnS has not been weakened ( Figure 3 b), indicating that the CuS / ZnS hybrid material has good photothermal stability. Example

[0026] Study on Photothermal Therapy of CuS / ZnS Hybrid Nanomaterials

[0027] Test instrument: Thermo Fisher Multiskan FC microplate reader. Implementation method: The methyl tetrazolium blue (MTT) method was used to determine the killing effect of CuS / ZnS on MCF-7 cells. First, the MCF-7 cells were placed in a high-glucose cell culture medium (DMEM) containing 10% fetal bovine serum and 1% double antibody, and then incubated in a cell culture incubator (37°C, 5% CO2). After the cells matured, they were inoculated into a 96-well plate six times (density was about 7000 / well), and then placed in an incubator for incubation for 24 hours to allow the cells to adhere to the well wall. Next, the original culture medium in the well was carefully aspirated, and a mixture of CuS / ZnS (0, 8, 16, 32, 64, 128 μg·mL) mixed with different concentrations of CuS / ZnS was added. -1 ) and wrapped with tin foil. In order to allow CuS / ZnS to have enough time to enter the MCF-7 cells, the MCF-7 cells were placed in the incubator and incubated for 24 h. After that, MTT (10 μL, 5 mg mL -1 ) was added to each well, and after incubation for another 4 h, the MTT solution was removed and 150 μL of dimethyl sulfoxide was added to each well to dissolve the methoxazole crystals. The 96-well plate was gently shaken for 10 min. After the methoxazole crystals were completely dissolved, the absorbance at a wavelength of 490 nm was measured using an ELISA reader ( Figure 4 a). Photothermal therapy was performed using an exciter with a wavelength of 808 nm. -2 The cancer cell killing experiment was carried out under the condition of 2 min illumination time. After the illumination was completed, the MCF-7 cells were wrapped with tin foil and placed in the incubator for another 4 h. Next, 10 μL MTT solution was added to the MCF-7 cells. After incubation for 4 h, the MTT solution was removed and 150 μL dimethyl sulfoxide was added. After gently shaking for 10 min, the absorbance at a wavelength of 490 nm was measured using an enzyme marker. Figure 3 b. The results show that without near-infrared light irradiation, CuS / ZnS (128 μg·mL -1) into MCF-7 cells, the cell survival rate was higher than 93%, confirming that CuS / ZnS has low cytotoxicity ( Figure 4 a). However, under 808 nm near-infrared light irradiation, CuS / ZnS showed its excellent photothermal performance. Since tumor cells have a weak tolerance to high temperatures, after irradiation, CuS / ZnS (128 μg mL -1 The thermal radiation produced by the radiation kills nearly 80% of cancer cells. Figure 4 b). The photothermal treatment produced by CuS / ZnS inactivates enzymes and proteins in cells, accelerates apoptosis of tumor cells, and has a good therapeutic effect. Example

[0028] Image analysis of photothermal therapy effects

[0029] Test instrument: Nikon A1 confocal microscope. Implementation method: Calcein (AM) and propidium iodide (PI) double staining reagents were used to perform staining experiments on live and dead cells respectively, and the therapeutic effects on MCF-7 cells under different conditions were further evaluated. The live cell stain is a green fluorescent dye, and the dead cell stain is a red fluorescent dye. Specifically as follows: MCF-7 cells were inoculated in a 20 mm confocal culture dish and cultured for 24 h to allow them to adhere to the wall. Then, the original culture medium was replaced with a fresh culture medium containing CuS / ZnS and cultured in an anaerobic environment for 4 h. Among them, the CuS / ZnS+light irradiation group was irradiated with light for 2 min (808 nm, 0.27 W·cm -2 ). Subsequently, the treated cells were stained with AM / PI. Finally, the live and dead cells were imaged using a laser confocal microscope. Figure 5 Compared with the confocal images of MCF-7 cells without CuS / ZnS and light ( Figure 5 a) Compared with the addition of CuS / ZnS, the green fluorescence of the lipase in the living cells stained with AM can still be observed, while the red fluorescence is almost unobservable ( Figure 5 b), which indicates that CuS / ZnS has low cytotoxicity, allowing a large number of cells to survive. In addition, after applying 808 nm light, a large amount of green fluorescence can still be observed without the presence of CuS / ZnS ( Figure 5 c), which shows that single near-infrared light irradiation cannot kill cancer cells. However, when CuS / ZnS was added and 808 nm near-infrared light was applied, a large amount of red fluorescence of dead cells stained with PI was observed ( Figure 5 d), indicating that most of the MCF-7 cells have undergone apoptosis, thus showing that this method has a significant effect on the photothermal treatment of tumors.

[0030] It should be noted that the above content only illustrates the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications all fall within the protection scope of the claims of the present invention.

Claims

1. A method for synthesizing a hybrid nanomaterial, characterized in that: The method comprises the following steps: (a) preparing an ethylene glycol / deionized water mixed solvent; (b) adding CuCl2·2H2O and Zn(NO3)2·6H2O to the solvent obtained in step (a), stirring and mixing, and then adding thioacetamide and continuing to stir and mix; wherein the amount ratio of the four substances, namely, the solvent obtained in step (a), CuCl2·2H2O, Zn(NO3)2·6H2O, and thioacetamide, is 30 mL: 85 mg: 148 mg: 150 mg; (c) transferring the mixed solution obtained in step (b) to an autoclave lined with Teflon, the autoclave is sealed in an oven at 180°C, reacting for 3 hours and then naturally cooling to room temperature; then collecting the precipitate by centrifugation, washing it with distilled water and ethanol several times, and finally drying it at 60°C for 12 hours to obtain CuS / ZnS.

2. The method for synthesizing a hybrid nanomaterial according to claim 1, characterized in that: The volume ratio of ethylene glycol / deionized water in step (a) is 3:

1.

3. The method for synthesizing a hybrid nanomaterial according to claim 1, characterized in that: The stirring and mixing time in step (b) is 10 minutes.

4. A CuS / ZnS hybrid nanomaterial prepared by the method according to any one of claims 1 to 3.

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