Preparation method of titanium monoxide nanosheet and application thereof in preparation of antibacterial and antitumor drugs

Abstract

The application discloses a preparation method of titanium monoxide nanosheets and application of the titanium monoxide nanosheets in preparation of antibacterial and antitumor drugs, and is characterized in that TiO powder is used as raw material, and TiO nanosheets are obtained through water bath ultrasonic treatment. The TiO nanosheets prepared by the application have certain killing effects on bacteria and tumors, and the TiO nanosheets have sharp edges of two-dimensional nanosheets, so that the TiO nanosheets can cut bacteria or tumor cells. In addition, the TiO nanosheets have good near-infrared light absorption, so that the TiO nanosheets can further improve the killing effects on bacteria and tumor cells in cooperation with a photothermal effect.

Description

Technical Field

[0001] This invention belongs to the field of inorganic nanomaterial preparation technology, and specifically relates to a method for preparing TiO nanosheets with anti-tumor and antibacterial properties. Background Technology

[0002] Multidrug-resistant bacteria pose a serious threat to food, health, and the environment, especially pathogenic drug-resistant bacteria. The problem of drug-resistant bacteria has sparked great interest in the development of novel antimicrobial agents. Antimicrobial agents are generally divided into two categories: organic and inorganic. Organic antimicrobial agents are currently the most widely used, but their misuse can lead to the emergence and evolution of drug-resistant bacteria. Many organic antimicrobial agents suffer from drawbacks such as short lifespan, easy decomposition, and low thermal stability, which limits their use in certain situations. Therefore, inorganic antimicrobial agents have been extensively studied due to their broad-spectrum and inherent bactericidal effects. Among the many inorganic antimicrobial agents, silver, copper, and zinc-containing materials are typical examples. However, under light or high temperatures, silver ions are easily reduced to metallic silver, causing sample discoloration. In recent years, metal oxides have been widely used as bactericides in the biological and medical fields. Nanomaterials such as zinc oxide and copper oxide have been developed into effective antimicrobial materials in various fields. Among inorganic antimicrobial agents, inorganic nanomaterials have attracted widespread attention due to their unique structure and morphology. These inorganic nanomaterials all share a common characteristic: sharp edges. Therefore, the mechanism of physical antibacterial action differs from that of other antibacterial agents, primarily due to the puncture or cleavage of bacterial membranes caused by sharp edges during physical contact. Unlike antibiotics, these nanomaterials with sharp structures do not require concern about multidrug resistance of pathogens due to their physical antibacterial mechanism. Sunaina et al. designed a mechanolytic hydrogel dressing for skin lesions infected with Staphylococcus aureus, which is a sodium alginate hydrogel loaded with gold nanostars with a pointed topology up to 120 nm, designed to penetrate bacterial membranes (ACS Applied Materials & Interfaces 2022, 14(39), 44084-44097). The first report on two-dimensional materials combating pathogenic bacteria was graphene nanosheets, which were able to cleave the E. coli cell membrane, causing many phospholipids to leak out of the bacterial membrane. Since then, many two-dimensional materials with graphene-like structures, such as MXene, Sb2Se3, MoS2, and black phosphorus, have been used as antibacterial agents. However, most two-dimensional materials have poor inherent antibacterial properties, requiring higher dosages and longer reaction times to achieve effective bactericidal effects. Therefore, developing antibacterial agents with intrinsic antibacterial properties is of great significance.

[0003] The synergistic effect of multiple antibacterial mechanisms can reduce the dosage and toxic side effects of antibacterial agents. Near-infrared lasers offer advantages in clinical applications such as good penetration, remote control capability, and high site specificity. Furthermore, bacteria are heat-sensitive, and high-temperature treatment can lead to bacterial inactivation. Near-infrared laser photothermal therapy (PTT) is an effective method for killing bacteria and has been extensively studied in recent years. Therefore, materials with photothermal effects and inherent antibacterial properties can not only kill bacteria at high temperatures under near-infrared laser irradiation but also improve antibacterial efficiency through the synergistic effect of the two antibacterial mechanisms. Developing antibacterial agents with photothermal effects and inherent antibacterial properties may be an important way to improve bactericidal efficiency. TiO2 is a metal oxide containing a large amount of oxygen and titanium vacancies and is commonly used in electronic devices. Titanium is more abundant on Earth than copper and zinc, thus its cost is lower. Titanium has been widely used in human health care, but the application of titanium monoxide in biomedicine has not yet been reported. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for preparing titanium monoxide nanosheets and their application in the preparation of antibacterial and antitumor drugs.

[0005] To achieve its objectives, the present invention employs the following technical solution:

[0006] This invention first discloses a method for preparing titanium monoxide nanosheets, characterized by: using TiO powder as raw material, obtaining TiO nanosheets by water bath ultrasonication, the specific steps being: dispersing TiO powder in a liquid solvent to obtain a TiO dispersion; ultrasonicating the TiO dispersion in a water bath, then centrifuging at low speed to remove the lower layer of particle precipitate, taking the upper uniform solution and centrifuging at high speed, washing the obtained precipitate with ethanol and water sequentially, and then freeze-drying to obtain TiO nanosheets.

[0007] Preferably, the concentration of TiO in the TiO dispersion is 0.01–200 mg / mL.

[0008] Preferably, the liquid solvent is at least one selected from water, ethanol, methanol, acetone, N-methylpyrrolidone, dimethyl sulfoxide, N,N-dimethylformamide, dichloromethane, chloroform, ethylene glycol, glycerol, isopropanol, and polyethylene glycol 200.

[0009] Preferably, the temperature of the water bath is 0–80°C, and the ultrasonic time is 0.1 h–200 h.

[0010] Preferably, the low-speed centrifugation speed is 1000–3500 rpm, and the high-speed centrifugation speed is 10000–20000 rpm. The purpose of high-speed centrifugation is to purify TiO; other purification methods such as dialysis, ultrafiltration, and rotary evaporation can also be used.

[0011] The TiO nanosheets prepared by this invention have good water dispersibility and can be dispersed in liquids such as water, physiological saline, PBS buffer, and cell culture medium, as well as in hydrogels such as sodium alginate hydrogel and F127 hydrogel.

[0012] This invention also discloses the application of TiO nanosheets in the preparation of antibacterial and antitumor drugs.

[0013] Compared with existing technologies, the beneficial effects of this invention are reflected in:

[0014] 1. The present invention produces TiO nanosheets with good dispersibility. The method is simple and the product has a uniform morphology.

[0015] 2. The TiO nanosheets prepared in this invention possess a certain killing effect on bacteria and tumors. Furthermore, due to their sharp two-dimensional nanosheet edges, they can cut bacterial or tumor cells. In addition, the TiO nanosheets exhibit good near-infrared light absorption, which can synergistically enhance the killing effect of TiO nanosheets on bacteria and tumor cells. Therefore, the TiO nanosheets of this invention have broad application prospects in the preparation of antibacterial and antitumor drugs.

[0016] 3. The TiO nanosheets prepared in this invention enhance the antibacterial and antitumor effects by combining multiple killing mechanisms during the antibacterial and antitumor process. Therefore, a lower dosage of TiO nanosheets can be used to reduce the toxicity of TiO nanosheets and improve biosafety. Attached Figure Description

[0017] Figure 1 This is a TEM image of the TiO nanosheets prepared in Example 1.

[0018] Figure 2 The image shows the XRD pattern of the TiO nanosheets prepared in Example 1.

[0019] Figure 3 XPS images of the TiO nanosheets prepared in Example 1, where A is the full spectrum of TiO, B is the fine spectrum of Ti, and C is the fine spectrum of oxygen.

[0020] Figure 4 These are the temperature rise curves and thermal images of TiO nanosheets of different concentrations prepared in Example 1 under near-infrared light irradiation at different powers (808 nm), where: A represents the temperature rise curves and thermal images of TiO aqueous dispersions of different concentrations under near-infrared light irradiation at 2 W / cm². 2 Figure A shows the temperature rise curve under near-infrared light irradiation; Figure B shows the temperature rise curve of 100 μg / mL TiO2 aqueous dispersion under near-infrared light irradiation at different powers; Figure C shows the thermal image corresponding to Figure A; and Figure D shows the thermal image corresponding to Figure B.

[0021] Figure 5The images show digital photographs of the TiO nanosheets used in Example 1 for antibacterial coating. In Example 1: A shows the anti-Escherichia coli coating data of TiO aqueous dispersions of different concentrations under (+NIR) / no (-NIR) near-infrared light irradiation; B shows the anti-MRSA bacterial coating data of TiO aqueous dispersions of different concentrations under (+NIR) / no (-NIR) near-infrared light irradiation.

[0022] Figure 6 The images show scanning electron microscope (SEM) images of TiO nanosheets prepared in Example 1 killing MRSA under near-infrared light irradiation with and without.

[0023] Figure 7 The different concentrations of TiO nanosheets prepared in Example 1 were used to treat MRSA with / without near-infrared light irradiation, resulting in MRSA protein leakage.

[0024] Figure 8 The bar graph shows the viability of 4T1 cells (mouse breast cancer cells) after irradiation with near-infrared light at 1064 nm for 5 min using TiO nanosheets of different concentrations prepared in Example 1.

[0025] Figure 9 The images show the live and dead cell staining of TiO nanosheets prepared in Example 1 that killed tumor cells (PI stains dead tumor cells, AM stains live tumor cells, and Merge represents an image of live and dead cells superimposed).

[0026] Figure 10 This is a digital photograph of the freeze-dried TiO nanosheets from Example 2.

[0027] Figure 11 This is a digital photograph of the freeze-dried TiO nanosheets dispersed in physiological saline in Example 3, with a concentration of 100 μg / mL. Detailed Implementation

[0028] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to examples. The following content is merely an example and illustration of the concept of the present invention. Those skilled in the art can make various modifications or additions to the described specific embodiments or use similar methods to replace them, as long as they do not depart from the inventive concept or exceed the scope defined by the claims, all of which should fall within the protection scope of the present invention.

[0029] Example 1

[0030] In this embodiment, TiO nanosheets were prepared according to the following steps:

[0031] 300 mg of TiO powder was weighed and dispersed in 20 mL of N-methylpyrrolidone, and sonicated in a water bath at room temperature for 24 hours. The sonicated solution was centrifuged at 1000 rpm for 10 min to remove the lower layer of precipitate. The upper homogeneous solution was then centrifuged at 10000 rpm. The precipitate was washed several times with ethanol and water, collected, and freeze-dried in a freeze dryer to obtain TiO nanosheets.

[0032] Figure 1 The image shows a TEM image of the TiO nanosheets prepared in this embodiment. As can be seen from the image, TiO exhibits a two-dimensional nanosheet structure with a particle size of approximately 100–200 nm.

[0033] Figure 2 The image shows the XRD pattern of the TiO nanosheets prepared in this embodiment. As can be seen from the image, the TiO nanosheets after ultrasonication still exhibit the TiO phase and no chemical reaction has occurred.

[0034] Figure 3 The image shows an XPS image of the TiO nanosheets prepared in this embodiment. It can be seen from the image that the Ti element in the TiO after sonication is in the +1 valence state and no valence state change has occurred.

[0035] The heating curves and thermal images of TiO nanosheets with different concentrations under near-infrared light irradiation at different powers (1064 nm) were recorded using a thermal imager. The results are as follows: Figure 4 As shown, A represents TiO2 aqueous dispersions of different concentrations at 2W / cm². 2 Figures A and B show the heating curves under near-infrared light irradiation, respectively. Figure B represents the heating curves of a 100 μg / mL TiO2 aqueous dispersion under near-infrared light irradiation at different powers. Figure C shows the thermal imaging corresponding to Figure A, and Figure D shows the thermal imaging corresponding to Figure B. It can be seen that the heating curves of a 25 μg / mL TiO2 nanosheet aqueous dispersion under near-infrared light irradiation at 2 W / cm² are... 2 Under near-infrared light irradiation, the temperature can rise to above 55℃ within 5 minutes. An aqueous dispersion of 100 μg / mL TiO2 nanosheets at 1.5 W / cm²... 2 Under near-infrared light irradiation, the temperature can rise to above 55℃ within 5 minutes. An aqueous dispersion of 50 μg / mL TiO nanosheets can reach this temperature at 2 W / cm². 2 Under near-infrared light irradiation, the temperature can rise to above 60℃ within 5 minutes. An aqueous dispersion of 100 μg / mL TiO nanosheets can reach this temperature at 2 W / cm². 2 Under near-infrared light irradiation, the temperature can rise to above 65℃ within 5 minutes. Bacteria can be killed at 55℃, indicating that TiO nanosheets have excellent photothermal properties and can kill bacteria even at low concentrations.

[0036] The in vitro antibacterial activity of TiO2 nanosheets was evaluated using the plate count method. MRSA and Escherichia coli (E. coli) were typical Gram-positive and Gram-negative bacteria, respectively. Different concentrations of TiO2 nanosheets were placed in 1.5 mL centrifuge tubes, and 1 mL of bacteria (1.0 × 10⁻⁶) was added. 7 CFU / mL). After incubation at 37℃ for 2 hours, it was treated with a 1064nm near-infrared laser (2.0W / cm²). 2 Irradiate for 5 minutes (using a sample that has been left to stand for 5 minutes without laser irradiation as a control), count the remaining viable bacteria on an agar plate, and the results are as follows. Figure 5 As shown in the figure, without laser irradiation, TiO nanosheets can only partially kill bacteria. Even at a TiO nanosheet concentration of 150 μg / mL, they cannot completely eliminate E. coli and MRSA. Under near-infrared light irradiation, the antibacterial activity of TiO nanosheets is greatly enhanced. (1064 nm laser (2.0 W / cm²)) 2 After irradiation for 5 minutes, the activity of Escherichia coli and MRSA bacteria treated with 150 μg / mL TiO2 nanosheets was almost zero.

[0037] For morphological analysis, bacteria treated with TiO nanosheets were collected by centrifugation, fixed with 2.5% glutaraldehyde, and incubated at 4°C for 12 h. The glutaraldehyde was removed by centrifugation. The bacteria were then treated sequentially with a series of ethanol solutions (30, 50, 70, 80, 90, 95, and 100%) for 10 minutes each. They were then drop-coated onto silicon wafers and freeze-dried. The morphology of the bacteria was observed using a field emission scanning electron microscope. Figure 6 The images show scanning electron microscopy (SEM) images of TiO2 nanosheets prepared in this embodiment killing MRSA cells under near-infrared light irradiation and without. The images show that some MRSA cells exhibit morphological damage, possibly partly due to membrane damage caused by the sharp edges of the TiO2 nanosheets. Subsequently, the bacteria treated with TiO2 nanosheets were tested using the BCA method; the nanosheet structure cuts and disrupts the cell membrane, leading to leakage of protein contents. Figure 7 The figure shows the protein leakage of MRSA after treatment with different concentrations of TiO nanosheets prepared in this example under near-infrared light irradiation and without irradiation. As can be seen from the figure, the protein leakage of TiO nanosheets at 150 μg / mL is the highest under near-infrared irradiation. Therefore, it can be concluded that under near-infrared light, TiO nanosheets kill bacteria by disrupting the bacterial membrane, causing leakage of internal bacterial substances.

[0038] TiO nanosheets not only kill bacteria but also have a significant killing effect on tumor cells. To verify the in vitro killing effect of TiO nanosheets on tumor cells, 4T1 cells were seeded at a concentration of 5000 / well in 96-well cell culture plates and treated with different concentrations of TiO (0–100 μg / mL) for 12 h under both laser-positive and laser-negative conditions. The relative survival rate was detected using a CCK-8 assay kit. Figure 8 The bar graph shows the viability of 4T1 cells prepared in this embodiment with and without 1064 nm near-infrared light irradiation for 5 min, at different concentrations. The graph shows that co-culturing TiO nanosheets with 4T1 cells does not induce cell death, even at a dose of 100 μg / mL. However, when treated with near-infrared laser, due to the high photothermal conversion efficiency of TiO, the increased temperature leads to tumor cell death, resulting in a large number of tumor cells dying.

[0039] Further verification of the killing effect of TiO nanosheets on tumor cells was conducted using live / dead staining of tumor cells. 4T1 cells (approximately 1 × 10⁶ cells per well) were used. 5 The inoculum was evenly distributed in a 6-well plate and incubated for 24 hours. The wells were divided into four groups according to treatment:

[0040] Control group: Mouse tumor cells 4T1 cells were not treated in any way;

[0041] Laser module: Only 2.0W / cm² 2 Near-infrared light was used to irradiate the culture medium containing tumor cells for 5 minutes;

[0042] TiO group: 100 μg / mL TiO nanosheets were added to the cell culture medium;

[0043] TiO2+Laser group: 100 μg / mL TiO2 nanosheets were added to the cell culture medium, and 2.0 W / cm² was used simultaneously. 2 Near-infrared light was used to irradiate the culture medium containing tumor cells for 5 minutes;

[0044] After treatment, the culture medium was replaced with fresh medium. Calcein AM and propidium iodide (PI) were added and incubated for 30 minutes. The cells were then gently washed three times with PBS to remove the dye. Subsequently, the different treatment groups were imaged using an inverted fluorescence microscope (ECLIPSETi2, Nikon, Japan). Figure 9 The images show the live and dead cell staining of TiO nanosheets prepared in this embodiment, indicating their ability to kill tumor cells. The images show that the Control, Laser, and TiO groups exhibited relatively low tumor cell killing activity, while the TiO+Laser group showed significant tumor cell killing activity. This demonstrates that TiO nanosheets possess good photothermal effects and can be used for photothermal therapy of tumors.

[0045] Example 2

[0046] In this embodiment, TiO nanosheets were prepared according to the following steps:

[0047] 1 mg of TiO powder was weighed and dispersed in 100 mL of water, and sonicated in a water bath at room temperature for 0.1 hours. The sonicated solution was then centrifuged at 1000 rpm for 10 min to remove the lower precipitate. The supernatant was collected and dialyzed for 3 days. After dialysis, the solution was freeze-dried to obtain TiO nanosheets, as shown below. Figure 10 As shown.

[0048] When using it, the freeze-dried TiO nanosheets can be dispersed in sodium alginate hydrogel.

[0049] Example 3

[0050] In this embodiment, TiO nanosheets were prepared according to the following steps:

[0051] 10g of TiO powder was weighed and dispersed in 50mL of dimethyl sulfoxide, and sonicated in a water bath at room temperature for 200 hours. The sonicated solution was centrifuged at 1000rpm for 10min to remove the lower layer of precipitate. The upper homogeneous solution was then centrifuged at 10000rpm. The precipitate was washed several times with ethanol and water, collected, and freeze-dried in a freeze dryer to obtain TiO nanosheets.

[0052] When using, the freeze-dried TiO nanosheets can be dispersed in physiological saline, such as... Figure 11 As shown.

[0053] The above description is merely an exemplary embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing titanium monoxide nanosheets, characterized in that: TiO powder was dispersed in a liquid solvent to obtain a TiO dispersion. The TiO dispersion was ultrasonicated in a water bath, then centrifuged at low speed to remove the lower layer of precipitated particles. The upper homogeneous solution was collected and centrifuged at high speed. The resulting precipitate was washed with ethanol and water sequentially, and then freeze-dried to obtain TiO nanosheets. The temperature of the water bath was 0~80℃, and the ultrasonication time was 0.1 h~200 h. The speed of the low-speed centrifugation was 1000~3500 rpm, and the speed of the high-speed centrifugation was 10000~20000 rpm.

2. The preparation method according to claim 1, characterized in that: The concentration of TiO in the TiO dispersion is 0.01~200 mg / mL.

3. The preparation method according to claim 1, characterized in that: The liquid solvent is at least one selected from water, ethanol, methanol, acetone, N-methylpyrrolidone, dimethyl sulfoxide, N,N-dimethylformamide, dichloromethane, chloroform, ethylene glycol, glycerol, isopropanol, and polyethylene glycol 200.

4. A TiO nanosheet prepared by the preparation method according to any one of claims 1 to 3.

5. The use of the TiO nanosheets of claim 4 in the preparation of antibacterial and antitumor drugs.

Images