Preparation method and application of size-controllable black phosphorus quantum dots

Abstract

The invention discloses a preparation method and application of size-controllable black phosphorus quantum dots, and the preparation method of the black phosphorus quantum dots comprises the following steps: mixing black phosphorus with deoxygenated water, then carrying out ice-bath ultrasonic treatment, and centrifuging to obtain the black phosphorus quantum dots. According to the preparation method of the black phosphorus quantum dot, the black phosphorus quantum dot with low toxicity and good biocompatibility can be controllably prepared, the size of BP QDs is accurately controlled, the stability problems of rapid degradation, oxidation and the like are solved, and the application of the black phosphorus quantum dot in the field of organisms is expanded. The black phosphorus quantum dots are modified by cationic polymer polyethyleneimine, so that the black phosphorus quantum dots can be protected from being oxidized and degraded into phosphate, and the black phosphorus quantum dots can be adsorbed on cell membranes through electrostatic adsorption and have important application significance in the field of biology.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a method for preparing size-controllable black phosphorus quantum dots and applications thereof. Background Art

[0002] In 2015, Zhang et al. first reported a simple top-down method to generate 0D BP QDs from bulk BP in solution. Since then, various synthetic strategies have been categorized as top-down or bottom-up routes, such as ultrasonic exfoliation, electrochemical conversion, solvent thermal treatment, pulsed laser ablation (PLA), and substrate epitaxial growth to fabricate high-quality scalable BP QDs. However, these organic solvents are all toxic and have limitations in the biomedical field.

[0003] BP QDs have good biocompatibility due to their adjustable band gap (0.3-2.0 eV). BP is further reduced to quantum dots (QDs) to obtain a larger surface area, which has more active sites than large-mass phosphorus and can load more drugs. Under physiological conditions, it can be converted into non-toxic phosphite / phosphate ions and rapidly degraded into non-toxic PO4 in the body. 3- and acidic H + , making BP QDs a better biodegradable nanomaterial. However, BP QDs are easily oxidized in air, resulting in oxidation defects on their surface, which seriously affects their performance. Therefore, appropriate modification of BP QDs to improve stability is a necessary condition for further application.

[0004] Current research has found that the sizes of the prepared BP QDs are all non-uniform, and the size problem of BP QDs has a certain impact in applications. For example, large-size quantum dots: generally refer to BP QDs with diameters ranging from tens of nanometers to hundreds of nanometers. These quantum dots have relatively high photothermal conversion efficiency and are suitable for applications such as photothermal therapy. Medium-size quantum dots: with a diameter between 10 nanometers and 50 nanometers. This type of quantum dot exhibits good optical properties and can be better dispersed and positioned in the body, making it suitable for applications in bioimaging and drug carriers. Small-size quantum dots: BP QDs with a diameter of less than 10 nanometers. These quantum dots usually have stronger quantum confinement effects and higher biocompatibility, making them ideal choices for fields such as cell imaging and targeted drug delivery.

[0005] In biomedical research and clinical applications, delivering functional biomacromolecules (such as nucleic acids, proteins, etc.) into living cells is a key step in achieving gene editing, cell therapy, and drug development. For example, in gene therapy, normal genes need to be delivered to diseased cells to correct or supplement defective genes; in drug development, drug molecules need to be delivered into cells to achieve the treatment of diseases. Traditional delivery methods include viral vectors, chemical transfection, and physical methods (such as electroporation), but these methods have certain limitations. Viral vectors may trigger immune responses and potential gene integration risks; chemical transfection has low delivery efficiency and may be toxic to cells; although electroporation can achieve high delivery efficiency, it causes greater damage to cells and may affect the physiological function and activity of cells. As an emerging physical delivery method, photoporation technology uses laser pulses to form instantaneous pores in the cell membrane, thereby achieving efficient delivery of macromolecules. Compared with traditional methods, photoporation technology has the following advantages: First, it can achieve precise delivery to specific cells or cell regions, reducing the impact on other cells; second, it can achieve higher delivery efficiency without damaging cells; third, by adjusting laser parameters such as energy density and pulse width, the size and formation time of cell membrane pores can be precisely controlled, thereby achieving the delivery of biomacromolecules of different sizes and properties.

[0006] Therefore, precise control of the size, shape, and surface chemistry of BP QDs is a major challenge in their synthesis. Current methods also lack reproducibility, resulting in a wide size distribution. Furthermore, stability issues such as rapid degradation and oxidation limit their long-term functionality and applications. Summary of the Invention

[0007] The present invention aims to solve at least one of the above-mentioned technical problems existing in the prior art. To this end, the present invention aims to provide a method for preparing size-controllable black phosphorus quantum dots and its application.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is:

[0009] The first aspect of the present invention provides a method for preparing black phosphorus quantum dots, comprising the following steps:

[0010] Black phosphorus was mixed with deoxygenated water and then ultrasonicated in an ice bath, and then centrifuged to obtain black phosphorus quantum dots.

[0011] Black phosphorus quantum dots are easily oxidized in an aerobic environment, causing changes in their structure and performance, or even degradation. For example, the layered structure of black phosphorus is stacked through van der Waals forces, and the oxidation process will gradually penetrate from the surface to the inside. Degradation will lead to a weakening of the interlayer bonding force, and ultimately cause the quantum dots to agglomerate, decompose, or change in size unevenly. For example, the originally uniformly dispersed BPQDs may form large aggregates after oxidation, losing the size effect and high specific surface area advantages of the nanomaterial. Therefore, the use of deoxygenated water for ultrasonic exfoliation in the present invention can effectively reduce the oxidative effect of dissolved oxygen on black phosphorus quantum dots, thereby maintaining the integrity of its structure and the stability of its performance; further, compared with other organic solvents, deoxygenated water is non-toxic, which can eliminate the potential hazards brought by organic solvents, reduce toxicity to organisms, and expand its application in organisms.

[0012] In some embodiments of the present invention, the black phosphorus may be in any shape, such as flake black phosphorus and / or block black phosphorus.

[0013] As used herein, “flake-like black phosphorus” refers to a thin flake-like substance having a two-dimensional layered structure.

[0014] As used herein, “bulk black phosphorus” refers to a substance having a three-dimensional macroscopic solid structure formed by stacking multiple black phosphorus crystal layers.

[0015] In some embodiments of the present invention, the mass volume ratio of the black phosphorus to the deoxygenated water is (50-70) mg: (40-50) mL.

[0016] In some embodiments of the present invention, the temperature of the ice bath is below 10°C, such as 0-10°C, 4-8°C, etc.

[0017] In some embodiments of the present invention, the power of the ultrasound is 550-650W, such as 580-620W, 590W, 600W, 610W, 620W, etc.; the time of the ultrasound is 6-10h, such as 7-9h, 8h, etc.

[0018] In some embodiments of the present invention, the ultrasound is set to be on for 2 to 4 seconds and off for 1 to 3 seconds, such as on for 3 seconds and off for 2 seconds.

[0019] In some embodiments of the present invention, the centrifugal force of the centrifugation is 4000-10000g; such as 4000-4100g, 4000-6000g, 5500-5800g, 5000-10000g, 9000-9100g, etc., and the centrifugation time is 10-60min, such as 10min, 20min, 30min, 40min, 50min, 60min. In the present invention, the higher the centrifugal force, the more conducive it is to the exfoliation and dispersion of black phosphorus quantum dots. In the present invention, black phosphorus quantum dots of different particle sizes can be obtained by controlling different centrifugal conditions.

[0020] In some embodiments of the present invention, the centrifugation includes a first centrifugation and a second centrifugation; the centrifugal force of the first centrifugation is 4000~6000g, and the time of the first centrifugation is 1~30min, such as 10min, 20min, etc.; the centrifugal force of the second centrifugation is 5000~10000g, and the time of the second centrifugation is 10~40min, such as 20min, 30min, etc.

[0021] In some embodiments of the present invention, the centrifugal radius is 6 to 8 cm.

[0022] In some embodiments of the present invention, the centrifugation includes performing a first centrifugation on the ultrasonic product, taking the supernatant and performing a second centrifugation, and collecting the second centrifugal precipitate to obtain black phosphorus quantum dots.

[0023] In some embodiments of the present invention, the method for preparing black phosphorus quantum dots further comprises dispersing the prepared black phosphorus quantum dots in deoxygenated water to prepare a dispersion of black phosphorus quantum dots. In the present invention, dispersing the black phosphorus quantum dots in deoxygenated water can prevent the black phosphorus quantum dots from being oxidized.

[0024] In some embodiments of the present invention, the mass volume ratio of the black phosphorus quantum dots to deoxygenated water is (30-50) mg:100 mL, such as (30-45) mg:100 mL, (32-43) mg:100 mL.

[0025] The second aspect of the present invention provides black phosphorus quantum dots prepared by the black phosphorus quantum dot preparation method.

[0026] In some embodiments of the present invention, the average particle size of the black phosphorus quantum dots is 2.0 to 4.0 nm, such as 2.5 to 3.5 nm, or 2.55 nm to 3.19 nm.

[0027] In some embodiments of the present invention, the black phosphorus quantum dots may be in the shape of at least one of nanosheets and dots.

[0028] The third aspect of the present invention provides a polymer black phosphorus quantum dot composite, comprising the aforementioned black phosphorus quantum dots and polyethyleneimine encapsulating the black phosphorus quantum dots.

[0029] In the present invention, polyethyleneimine (PEI) is adsorbed through the van der Waals force between its cationic amino groups and the unsaturated P bonds on the surface of black phosphorus quantum dots to form a stable PEI modification layer. It also forms covalent bonds with the surface of black phosphorus quantum dots through chemical bonding, further enhancing the stability of the modification layer.

[0030] In some embodiments of the present invention, the weight average molecular weight (Mw) of the polyethyleneimine is 8k to 50k, such as 10k, 15k, 20k, 25k, 30k, 35k, 40k, 45k, or 50k.

[0031] In some embodiments of the present invention, the mass ratio of the polyethyleneimine to the black phosphorus quantum dots is 0.05 to 0.30, such as 0.05 to 0.28, or 0.052 to 0.278.

[0032] In some embodiments of the present invention, the average particle size of the polymer black phosphorus quantum dot composite is 1.0 to 10.0 nm, such as 1.0 nm, 2.0 nm, 3.0 nm, 4.0 nm, 5.0 nm, 6.0 nm, 7.0 nm, 8.0 nm, 9.0 nm, and 10.0 nm.

[0033] In some embodiments of the present invention, the polyethyleneimine wraps the black phosphorus quantum dots through electrostatic adsorption and / or chemical bonding.

[0034] In some embodiments of the present invention, the absorption band of the polymer black phosphorus quantum dot composite in visible light is 310 to 350 nm, such as 313 to 345 nm.

[0035] In some embodiments of the present invention, the energy band gap of the polymer black phosphorus quantum dot composite is 2.8 to 4.0 eV, such as 2.9 to 3.85 eV.

[0036] The fourth aspect of the present invention provides a method for preparing the polymer black phosphorus quantum dot composite, comprising the following steps:

[0037] The black phosphorus quantum dot dispersion is mixed with a polyethyleneimine solution to prepare the polymer black phosphorus quantum dot composite.

[0038] In some embodiments of the present invention, the black phosphorus quantum dots are dispersed in deoxygenated water to prepare a dispersion of black phosphorus quantum dots.

[0039] In some embodiments of the present invention, the mass volume ratio of the black phosphorus quantum dots to deoxygenated water is (30-50) mg:100 mL, such as (30-45) mg:100 mL, (32-43) mg:100 mL.

[0040] In some embodiments of the present invention, the solvent used in the polyethyleneimine solution includes any one of water, ethanol, and anhydrous ethanol.

[0041] In some embodiments of the present invention, the pH of the polyethyleneimine solution is 6.0 to 7.0, such as 6.5 to 7.0, 6.8 to 6.9. In the present invention, controlling the pH of the polyethyleneimine solution within this range can prevent PEI precipitation.

[0042] In some embodiments of the present invention, the mass concentration of the polyethyleneimine solution is 0.5 to 2.0 mg / mL, such as 0.8 to 1.5 mg / mL.

[0043] In some embodiments of the present invention, the mixing operation includes stirring after ultrasound; the ultrasound time is 1 to 3 minutes; the ultrasound power is 40 to 50 Hz; the stirring rate is 30 to 50 rpm; and the stirring time is 8 to 24 hours, such as 8 to 12 hours.

[0044] The fifth aspect of the present invention provides an application of the polymer black phosphorus quantum dot complex in drug delivery carriers, cell imaging, and the preparation of tumor treatment drugs.

[0045] In some embodiments of the present invention, the tumor treatment includes at least one of photothermal therapy, photodynamic therapy, and targeted therapy.

[0046] In some embodiments of the present invention, the polymer black phosphorus quantum dot complex is used in the preparation of a tumor photothermal therapeutic agent.

[0047] In some embodiments of the present invention, the polymer black phosphorus quantum dot complex is used in the preparation of a tumor photothermal bubble generator.

[0048] In some embodiments of the present invention, the polymer black phosphorus quantum dot complex is used in the preparation of a drug for delivering an agent through a target biological membrane, and the polymer black phosphorus quantum dot complex is in physical contact with one or more pores on the target biological membrane.

[0049] In some embodiments of the present invention, the one or more holes are formed using at least one of a perforator selected from the group consisting of a thermal perforator, a mechanical perforator, a laser perforator, and a water perforator.

[0050] A sixth aspect of the present invention provides a system for delivering the polymer black phosphorus quantum dot complex, comprising:

[0051] The perforator and the polymer black phosphorus quantum dot composite.

[0052] In some embodiments of the present invention, the perforator comprises at least one of a thermal perforator, a mechanical perforator, a laser perforator, and a water perforator.

[0053] Using photoperforation technology to achieve photothermal bubble generation requires the use of simulated tissue to simulate deep tissue to observe bubble formation. During this process, the nanoparticles must be fixed in a certain position. This allows the laser to be fixedly irradiated at the location where the nanoparticles are located, resulting in targeted bubble formation and improved efficiency. In the present invention, the perforator in the delivery system delivers the polymer black phosphorus quantum dot complex after perforation. The polymer black phosphorus quantum dot complex can be stably fixed in a specific location in the tissue, increasing its probability of bubble formation in deep tissue.

[0054] The beneficial effects of the present invention are:

[0055] The preparation method of black phosphorus quantum dots of the present invention can controllably prepare black phosphorus quantum dots with low toxicity and good biocompatibility, accurately control the size of BP QDs, solve their stability problems such as rapid degradation and oxidation, and expand their application in the field of organisms.

[0056] In the polymer black phosphorus quantum dot complex of the present invention, the black phosphorus quantum dots are modified with the cationic polymer polyethyleneimine, which can protect the black phosphorus quantum dots from being oxidized and degraded into phosphates, and can also be adsorbed on the cell membrane through electrostatic adsorption, which has important application significance in the biological field.

[0057] The polymer black phosphorus quantum dot complex of the present invention can be applied to tumor photoperforation therapy. The polymer black phosphorus quantum dot complex is positioned at a specific location of the tissue, subjected to photothermal action, and formed into bubbles, which are applied to cell delivery. It is expected that the application will be expanded to organisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 This is a schematic diagram of the preparation process of black phosphorus quantum dots in Example 1 of the present invention.

[0059] Figure 2 Schematic diagram of the preparation process of PEI-modified black phosphorus quantum dot nanoparticles in Example 2 of the present invention.

[0060] Figure 3These are the transmission electron microscope images and electron diffraction patterns of the black phosphorus quantum dots of Example 1 of the present invention, wherein A1 is the transmission electron microscope image of BP QDs (1), and B1 is the electron diffraction pattern of BP QDs (1); A2 is the transmission electron microscope image of BP QDs (2), and B2 is the electron diffraction pattern of BP QDs (2); A3 is the transmission electron microscope image of BP QDs (3), and B3 is the electron diffraction pattern of BP QDs (3).

[0061] Figure 4 These are the transmission electron microscopy images and electron diffraction patterns of the PEI-modified black phosphorus quantum dot nanoparticles in Example 2 of the present invention, wherein C1 is the transmission electron microscopy image of PEI (Mw 40k) -BP QDs (1), and D1 is the electron diffraction pattern of PEI (Mw 40k) -BP QDs (1); C2 is the transmission electron microscopy image of PEI (Mw 40k) -BP QDs (2), and D2 is the electron diffraction pattern of PEI (Mw 40k) -BP QDs (2); C3 is the transmission electron microscopy image of PEI (Mw 40k) -BP QDs (3), and D3 is the electron diffraction pattern of PEI (Mw 40k) -BP QDs (3).

[0062] Figure 5 These are the transmission electron microscope image and Zeta diagram of PEI (Mw 10k) -BP QDs (3) in Example 3 of the present invention; wherein, C4 is the transmission electron microscope image of PEI (Mw 10k) -BP QDs (3), D4 is the electron diffraction pattern of PEI (Mw 10k) -BP QDs (3); and D5 is the Zeta potential diagram of PEI (Mw 10k) -BP QDs (3).

[0063] Figure 6 It is the particle size distribution diagram of BP QDs (3) (A3-1), PEI (Mw 10k)-BP QDs (3) (C4-1), and PEI (Mw 40k)-BPQDs (2) (C2-1) of the present invention.

[0064] Figure 7 E1 is the absorption spectrum of BP QDs (2) and BP QDs (3) measured by UV-visible spectrophotometer; F1 is the absorption spectrum of BP QDs (2) and PEI (Mw 40k)-BP QDs (2) measured by UV-visible spectrophotometer; F2 is the absorption spectrum of BP QDs (3) and PEI (Mw 40k)-BP QDs (3) measured by UV-visible spectrophotometer; G1 is the Zeta potential diagram of BP QDs (3) and PEI (Mw 40k)-BP QDs (3).

[0065] Figure 8H1 and H4 correspond to the Raman spectra of BP QDs (1) and PEI (Mw 40k)-BP QDs (1) on the first day and the 30th day, respectively; H2 and H5 correspond to the Raman spectra of BP QDs (2) and PEI (Mw 40k)-BP QDs (2) on the first day and the 30th day, respectively; H3 and H6 correspond to the Raman spectra of BP QDs (3) and PEI (Mw 40k)-BP QDs (3) on the first day and the 30th day, respectively.

[0066] Figure 9 I1, I2, and I3 are the energy band gap diagrams of BP QDs (1), BP QDs (2), and BP QDs (3), respectively; I4, I5, and I6 are the energy band gap diagrams of PEI (Mw 40k)-BP QDs (1), PEI (Mw 40k)-BP QDs (2), and PEI (Mw 40k)-BP QDs (3), respectively.

[0067] Figure 10 From left to right are the simulated tissue sample images of the product of step S2 of Example 4, the product of step S2-1 of Example 5, and the product of step S2-2.

[0068] Figure 11 From left to right are the chromatograms of the simulated tissues of the product of step S2 of Example 4, the product of step S2-1 of Example 5, and the product of step S2-2. DETAILED DESCRIPTION

[0069] The present invention is further described in detail below using specific examples. Unless otherwise specified, the raw materials, reagents, and apparatus used in the examples and comparative examples are available from conventional commercial sources or by conventional methods. Unless otherwise specified, all experimental or testing methods are conventional in the art.

[0070] Polyethylenimine (Mw 40000) was purchased from Maokang Bio: POLYETHYLENIMINE, LINEAR, AVERAGE Mw 40000;

[0071] Polyethyleneimine (Mn 10000) was purchased from Sigma: POLYETHYLENIMINE, LINEAR, AVERAGE MN10000;

[0072] Black phosphorus was purchased from the Chinese Academy of Sciences.

[0073] Example 1

[0074] This embodiment prepares a black phosphorus quantum dot. Figure 1 The schematic diagram of the preparation process of black phosphorus quantum dots is shown. The specific process is as follows:

[0075] Black phosphorus quantum dots were prepared by liquid phase exfoliation method, using ultrasonic (below 10℃, 3s / 2s on / off interval, 600w / 8h). 50mg black phosphorus wafer was added to 40mL deoxygenated water, and ultrasonic crushing was performed to obtain a mixed solution of black phosphorus quantum dots, and a black phosphorus quantum dot dispersion was obtained by centrifugation. Three sets of ultrasonic parameters were set for centrifugation, as follows:

[0076] (1) The first centrifugation parameters: height control 2.5cm, about 4524rcf, 1min, take the supernatant part (the sediment can be reserved for use); the second centrifugation parameters: height control 2.5cm, about 4071rcf, 10min, take the second centrifugation sediment part, collect the second centrifugation sediment part and disperse it in new deoxygenated water to obtain 430μg / mL black phosphorus quantum dot dispersion, recorded as BP QDs(1).

[0077] (2) The first centrifugation parameters: height control 2.5cm, about 4071rcf, 10min, take the supernatant part (the sediment can be reserved for use); the second centrifugation parameters: height control 2.5cm, about 5655rcf, 20min, take the second centrifugation sediment part, collect the second centrifugation sediment part and disperse it in new deoxygenated water to obtain 410μg / mL black phosphorus quantum dot dispersion, recorded as BP QDs(2).

[0078] (3) The first centrifugation parameters: height control 2.5cm, about 5655rcf, 20min, take the supernatant part (the sediment can be reserved for use); the second centrifugation parameters: height control 1.5cm, about 9048rcf, 30min, take the second centrifugation sediment part, collect the second centrifugation sediment part and disperse it in new deoxygenated water to obtain 320μg / mL black phosphorus quantum dot dispersion, recorded as BP QDs(3).

[0079] Example 2

[0080] In this embodiment, a PEI modified black phosphorus quantum dot nanoparticle was prepared, Figure 2 The preparation process is shown in the schematic diagram, and the specific process is as follows:

[0081] S1: In a 20mL glass beaker, 10mg PEI (Mw 40k) powder was added to 9mL ultrapure water, and stirred uniformly on a magnetic stirrer. After the PEI was completely dissolved, 1mol / L sodium hydroxide (NaOH) solution was used to adjust the pH to 6.80-6.90 to prevent the PEI powder from precipitating again. The solution was transferred to a graduated cylinder and diluted to 10mL with water. A disposable 0.2μm PES vacuum filter was used to filter and sterilize, and a 1mg / mL storage solution was obtained, which was stored at 4℃ for 3 months.

[0082] S2: Add the PEI (Mw 40k) stock solution to the BP QDs dispersion prepared in Example 1 (volume ratio of stock solution to dispersion: 1:9). Sonicate for 1–2 minutes and stir overnight in the dark. Centrifuge the solution at 10,000 rpm for 30 minutes to remove free PEI. Repeat at least three times.

[0083] The PEI-modified black phosphorus quantum dot nanoparticles prepared from different BP QDs dispersions in Example 1 were respectively denoted as PEI (Mw 40k)-BP QDs (1), PEI (Mw 40k)-BP QDs (2), and PEI (Mw 40k)-BP QDs (3).

[0084] Example 3

[0085] This example prepares PEI-modified black phosphorus quantum dot nanoparticles. The specific process is as follows:

[0086] S1: In a 10 mL glass beaker, add 5 mg of PEI (Mn 10k) powder to 5 mL of anhydrous ethanol to obtain a 1 mg / mL PEI (Mn 10k) solution.

[0087] S2: Add the PEI (Mn 10k) stock solution to the BP QDs dispersion prepared in Example 1 (volume ratio of stock solution to dispersion: 1:9). Sonicate for 1–2 minutes and stir overnight in the dark. Centrifuge the solution at 10,000 rpm for 30 minutes to remove free PEI. Repeat at least three times.

[0088] The PEI-modified black phosphorus quantum dot nanoparticles prepared from different BP QDs dispersions in Example 1 were respectively denoted as PEI (Mw10k)-BP QDs (1), PEI (Mw 10k)-BP QDs (2), and PEI (Mw 10k)-BP QDs (3).

[0089] Example 4

[0090] In this embodiment, black phosphorus quantum dots are fixed in the simulated tissue. The specific process is as follows:

[0091] S1: Prepare the simulated tissue: Prepare 2 g agar, 2.5 g gelatin powder, and 2.5 mL milk per 100 mL water. Place the mixture in a beaker in a water bath with a heated stirrer, heat to above 90°C, and then pour into an inverted 12-well plate.

[0092] S2: Prepare the simulated tissue first. When the temperature drops but it has not solidified, mix 0.3-0.4 mL of BP QDs (3) with 1.9-2 mL of simulated tissue and wait for solidification. The thickness is about 3-5 mm.

[0093] Example 5

[0094] In this embodiment, black phosphorus quantum dots are fixed in the simulated tissue. The specific process is as follows:

[0095] S1: Prepare simulated tissue: Prepare 2g agar, 2.5g gelatin powder, and 2.5mL milk per 100mL water. Place the mixture in a beaker in a water bath with agitator and heat to above 90°C. Pour the mixture into an inverted 12-well plate to obtain simulated tissue.

[0096] S2-1: First, add 0.5-0.6 mL of simulated tissue to solidify a layer, then add 0.3-0.4 mL of a mixture of BP QDs (3) and 0.7-0.6 mL of simulated tissue to form a layer, and finally add 0.8-0.7 mL of simulated tissue to form a layer. A total of three layers are added, and the total thickness is maintained at approximately 3-5 mm (when fixed to the bottom).

[0097] S2-2: First, add 0.8-0.7 mL of simulated tissue to solidify a layer, then add 0.3-0.4 mL of a mixture of BP QDs (3) and 0.7-0.6 mL of simulated tissue to form a layer, and finally add 0.5-0.6 mL of simulated tissue fluid to form a layer. A total of three layers are added, and the total thickness is maintained at approximately 3-5 mm (when fixed on the surface).

[0098] Test Example 1

[0099] The morphology, crystal form and particle size of the prepared black phosphorus quantum dots were detected using transmission electron microscopy, electron diffraction, UV-visible spectrophotometer and Zeta potential.

[0100] Figure 3 The transmission electron microscope image and electron diffraction pattern of black phosphorus quantum dots in Example 1 are shown, wherein: Figure 3 A1 in the figure is a transmission electron micrograph of BP QDs(1). Figure 3 Figure B1 is the electron diffraction pattern of BP QDs (1). Figures A1 and B1 show that the bare black phosphorus quantum dots exhibit distinct nanosheets, and their electron diffraction patterns show distinct rings, indicating polycrystalline structures with crystal planes (020), (021), (040), and (060) (PDF#47-1626). Because the quantum dots are small and disorderly stacked, the black phosphorus quantum dots may become polycrystalline.

[0101] Figure 3 A2 in the figure is a transmission electron micrograph of BP QDs(2). Figure 3 B2 in the figure is the electron diffraction pattern of BP QDs(2). Figure 3 As can be seen from A2, the bare black phosphorus quantum dots are in the form of nanosheets, and quantum dots are precipitated.

[0102] Figure 3 A3 in the figure is a transmission electron micrograph of BP QDs(3). Figure 3 B3 in FIG is the electron diffraction pattern of BP QDs (3). Figure 3 It can be seen from A3 that the black phosphorus quantum dots with the third set of parameters show an obvious uniform point distribution.

[0103] Figure 4 The transmission electron microscopy and electron diffraction patterns of PEI-modified black phosphorus quantum dot nanoparticles in Example 2 are shown, wherein: Figure 4 C1 in the figure is a transmission electron micrograph of PEI (Mw 40k)-BP QDs (1). Figure 4 D1 in FIG is the electron diffraction pattern of PEI (Mw40k)-BP QDs (1). Figure 4 As can be seen from C1 and D1, the surface of the nanosheets appears as black dots, indicating that the quantum dots are attached to the nanosheets after precipitation. After modification, their dispersibility is enhanced. The electron diffraction is obviously ring-shaped, indicating that the black phosphorus quantum dots modified with PEI are also polycrystalline.

[0104] Figure 4 C2 in the figure is a transmission electron micrograph of PEI (Mw 40k)-BP QDs (2). Figure 4 D2 in is the electron diffraction pattern of PEI (Mw40k)-BP QDs (2). Figure 4 As can be seen from C2 and D2, the modified black phosphorus quantum dots show an obvious uniform circular distribution, and the electron diffraction shows an obvious ring shape.

[0105] Figure 4 C3 in the figure is a transmission electron micrograph of PEI (Mw 40k)-BP QDs (3). Figure 4 D3 in FIG is the electron diffraction pattern of PEI (Mw40k)-BP QDs (3). Figure 4 As can be seen from C3 and D3, the modified black phosphorus quantum dots show agglomeration phenomenon. The reason may be that the PEI modification with a larger molecular weight will affect its dispersibility and cause agglomeration. There is no obvious ring in the electron diffraction, indicating that the crystallinity is poor and the structure may be relatively loose.

[0106] Figure 5 The transmission electron microscopy and Zeta diagram of PEI (Mw 10k)-BP QDs (3) in Example 3 are shown; wherein, Figure 5 C4 in the figure is a transmission electron micrograph of PEI (Mw 10k)-BP QDs (3). Figure 5 D4 in FIG is the electron diffraction pattern of PEI (Mw 10k)-BP QDs (3). Figure 5As can be seen from C4 and D4, the modified black phosphorus quantum dots show obvious uniform dot-like distribution, good dispersion, and obvious ring-shaped electron diffraction.

[0107] Figure 5 Figure D5 is the Zeta potential diagram of PEI (Mw 10k)-BP QDs (3). It can be seen that, theoretically, the surface of black phosphorus quantum dots is negatively charged, while PEI is positively charged. Through electrostatic adsorption, the two combine to make the surface of black phosphorus quantum dots positively charged. The experimental Zeta potential shows that the theory is consistent with the experiment, and the material preparation is successful.

[0108] Figure 6 The particle size distribution diagrams of BP QDs(3)(A3-1), PEI(Mw 10k)-BP QDs(3)(C4-1), and PEI(Mw 40k)-BPQDs(2)(C2-1) are shown. It can be seen from the figure that the average particle size of BP QDs(3) is 2.55±0.64817nm; the average particle size of PEI(Mw10k)-BP QDs(3) is 3.03±0.81642nm; and the average particle size of PEI(Mw 40k)-BP QDs(2) is 5.06±3.2473nm.

[0109] As can be seen from the above figures, the present invention can enhance the dispersibility of black phosphorus quantum dots after modification. For example, the black phosphorus quantum dots in BP QDs (2) are effectively and evenly dispersed after PEI modification, and the dispersion of smaller-sized black phosphorus quantum dots is even better when modified with smaller molecular weight PEI. The present invention successfully and accurately prepares black phosphorus quantum dots of different uniform sizes through relevant centrifugation parameters, achieving controllable preparation.

[0110] Figure 7 The UV-visible absorption spectra of BP QDs(2), BP QDs(3), PEI(Mw 40k)-BP QDs(2), PEI(Mw 40k)-BPQDs(3) and the Zeta potential diagram of BP QDs(3) and PEI(Mw 40k)-BP QDs(3) are shown. Figure 7 It can be seen from E1, F1, and F2 that BP QDs and PEI-BP QDs have uniform absorption in the visible and near-infrared bands. In the visible light range, the highest absorption band of BP QDs appears near a wavelength of about 320nm, while the highest absorption band of PEI-BP QDs appears near a wavelength of about 320-330nm. The absorption of PEI-BP QDs is more stable and broader than that of BP QDs. This shows that PEI modification can improve the stability of bare black phosphorus quantum dots. Figure 7The zeta potential diagram in G1 shows that, theoretically, the black phosphorus quantum dots have a negative surface charge, while PEI has a positive charge. Through electrostatic adsorption, the two combine to impart a positive charge to the black phosphorus quantum dots. Experimental zeta potential results show that the theory agrees with the experiment, indicating successful material preparation.

[0111] Figure 8 BP QDs(1) and PEI(Mw 40k)-BP QDs(1)( Figure 8 H1 is the first day, H4 is the 30th day), BP QDs (2) and PEI (Mw 40k)-BP QDs (2) ( Figure 8 H2 is the first day, H5 is the 30th day), BP QDs (3) and PEI (Mw 40k)-BP QDs (3) ( Figure 8 H3 is the first day and H6 is the 30th day) The Raman spectra were measured at a temperature of 23.7°C and a power of about 9.6mW on the first day and 30 days after placement. As shown in Figures H1, H2, and H3, the bare black phosphorus quantum dots have a peak at 373.5cm -1 、448.69cm -1 and 476.9cm -1 There are three prominent peaks at 374.2 cm, which are an out-of-plane phonon mode A1g and two in-plane modes B2g and A2g. -1 、450.1cm -1 and 478.3cm -1 There are three prominent peaks at 100 nm, representing an out-of-plane phonon mode A1g and two in-plane modes B2g and A2g. The blue shift caused by PEI modification may be due to the transfer of lone pairs of electrons from the amino groups in PEI to the empty orbitals of black phosphorus, resulting in P-type doping of the black phosphorus quantum dots. This increases the electron cloud density of the P-P bond in black phosphorus and increases the bond vibration frequency. Figure 8 H4, H5, and H6 are the Raman spectra of six samples measured after 30 days. It can be seen from the figure that the characteristic peaks of the PEI-modified black phosphorus quantum dots are still retained after 30 days, and the intensity is lower than that before 30 days. However, the bare black phosphorus quantum dots basically have no characteristic peaks after 30 days. In summary, PEI modification can enhance the stability of black phosphorus quantum dots.

[0112] Black phosphorus quantum dots are direct bandgap semiconductors, and modified black phosphorus quantum dots are also direct bandgap semiconductors. The bandgap of various parameters can be obtained by analyzing the absorption spectrum measured by UV-visible spectrophotometer. The optical bandgap is one of the optical parameters and can be measured by optical techniques based on transmission and reflection (such as the Dow plot). The Dow relationship is:

[0113] (αhν) γ =B(hν-Eg)

[0114] Among them, α: represents the absorption coefficient, which has the physical meaning of describing the degree to which light is absorbed when propagating in a material. Unit: usually cm -1 . hν: represents the photon energy, physical meaning: the energy of the incident photon, which is directly related to the frequency ν of the light. Unit: usually expressed in electron volts (eV). γ: represents the exponential factor, physical meaning: the parameter that characterizes the type of semiconductor transition. B: represents the Tauc coefficient, physical meaning: the proportional constant related to material properties (such as effective mass, dielectric constant, etc.). Eg: represents the optical band gap, physical meaning: the energy difference between the top of the semiconductor valence band and the bottom of the conduction band. Unit: eV.

[0115] Figure 9 The band gap diagrams of BP QDs and PEI (Mw 40k)-BP QDs based on absorption spectra are shown; Figure 9 I1, I2, and I3 are the band gap diagrams of BP QDs (1), BP QDs (2), and BP QDs (3), respectively. Figure 9 The extension of the intercept on the x-axis is shown to determine the band gap value. When (αhν) 2 = 0, the energy band gap Eg is obtained by linear extrapolation of the axis. Figure 9 It can be clearly seen from I1-I3 that the maximum absorption peak of BP QDs (1) is at 326.08nm, the maximum absorption peak of BP QDs (2) is at 319.95nm, and the maximum absorption peak of BP QDs (3) is at 309.99nm. The band gap of bare BP QDs (1) is 2.09eV, the band gap of BP QDs (2) is 2.26eV, and the band gap of BPQDs (3) is 2.75eV. The larger the band gap, the smaller the wavelength of the maximum absorption peak, because the energy required for the transition is greater. Moreover, as the size decreases, the band gap gradually increases, that is, the measured data is consistent with the theoretical report.

[0116] Figure 9 I4, I5, and I6 are the energy band gap diagrams of PEI (Mw 40k)-BP QDs (1), PEI (Mw 40k)-BP QDs (2), and PEI (Mw 40k)-BP QDs (3), respectively. Figure 9In I4-J6, the maximum absorption peak of PEI (Mw 40k)-BP QDs (1) is at 314.79nm, the maximum absorption peak of PEI (Mw 40k)-BP QDs (2) is at 322.54nm, and the maximum absorption peak of PEI (Mw 40k)-BP QDs (3) is at 344.37nm. The larger the band gap, the smaller the wavelength of the maximum absorption peak, because the energy required for the transition is large. The band gaps are 3.04eV, 2.94eV, and 3.84eV, respectively. The band gap change after PEI modification of black phosphorus quantum dots may be due to the formation of charge transfer complexes (CTCs) between the cationic properties of polyethyleneimine and black phosphorus quantum dots, and black phosphorus quantum dots may accept electrons. Therefore, black phosphorus quantum dots are likely to form donor-acceptor complexes with polymer materials.

[0117] Test Example 2

[0118] This experimental example uses a photothermal OCT system to capture the tomographic images of the simulated tissue of Example 4.

[0119] Figure 10 From left to right are the simulated tissue sample images of the product of step S2 of Example 4, the product of step S2-1 of Example 5, and the product of step S2-2. Figure 10 It can be seen that BP QDs (3) were successfully fixed at any position, near the bottom, and near the surface. It has been verified that black phosphorus quantum dots modified with PEI of different molecular weights have similar effects as black phosphorus quantum dots in simulated tissues and can be fixed at different positions in the simulated tissues.

[0120] Figure 11 From left to right are the chromatograms of the simulated tissue of the product of step S2 of Example 4, the product of step S2-1 of Example 5, and the product of step S2-2. In the figure, J-1, J-2, and J-3 are respectively a mixed gel of BP QDs (3) and simulated tissue, a mixed gel of BP QDs (3) and simulated tissue on the upper surface (simulated tissue blank is in the lower layer), and a mixed gel of BP QDs (3) and simulated tissue on the lower surface (simulated tissue blank is in the upper layer). It can be seen from Figure J-1 that black phosphorus quantum dots are evenly distributed in the simulated tissue; in Figure J-2, the mixed gel of black phosphorus quantum dot dispersion and simulated tissue (upper layer) is clearly stratified from the blank gel; in Figure J-3, the mixed gel of black phosphorus quantum dot dispersion and simulated tissue (lower layer) is also clearly stratified from the blank gel.

[0121] The above demonstrates that the present method for distributing black phosphorus quantum dots within simulated tissue can effectively control the fixed layering of black phosphorus quantum dots. This allows for subsequent pre-photoperforation experiments to compare the effects of laser irradiation on black phosphorus quantum dots at different depths, when the laser is irradiated from the upper or lower layers.

[0122] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A method for preparing black phosphorus quantum dots, characterized by: The following steps are involved: Black phosphorus was mixed with deoxygenated water and then ultrasonicated in an ice bath, and then centrifuged to obtain black phosphorus quantum dots.

2. The method for preparing black phosphorus quantum dots according to claim 1, wherein: The mass volume ratio of the black phosphorus to the deoxygenated water is (50-70) mg: (40-50) mL.

3. The method for preparing black phosphorus quantum dots according to claim 1, wherein: The centrifugal force of the centrifugation is 4000-10000 g; preferably, the centrifugation time is 10-60 min.

4. The method for preparing black phosphorus quantum dots according to claim 3, wherein: The centrifugation includes a first centrifugation and a second centrifugation; the centrifugal force of the first centrifugation is 4000-6000g, and the time of the first centrifugation is 1-30 minutes; the centrifugal force of the second centrifugation is 5000-10000g, and the time of the second centrifugation is 10-40 minutes.

5. Black phosphorus quantum dots prepared by the method for preparing black phosphorus quantum dots according to any one of claims 1 to 4.

6. A polymer black phosphorus quantum dot composite, characterized by: The method comprises the black phosphorus quantum dots according to claim 5 and polyethyleneimine encapsulating the black phosphorus quantum dots.

7. The polymer black phosphorus quantum dot composite according to claim 6, characterized in that: The mass ratio of the polyethyleneimine to the black phosphorus quantum dots is 0.05 to 0.30; preferably, the weight average molecular weight of the polyethyleneimine is 8k to 50k.

8. A method for preparing the polymer black phosphorus quantum dot composite according to claim 6 or 7, characterized in that: The following steps are involved: The black phosphorus quantum dot dispersion is mixed with a polyethyleneimine solution to prepare the polymer black phosphorus quantum dot composite.

9. Use of the polymer black phosphorus quantum dot complex according to claim 6 or 7 in drug delivery carriers, cell imaging, and preparation of tumor treatment drugs.

10. A system for delivering the polymer black phosphorus quantum dot complex according to claim 6 or 7, comprising: A perforator and the polymer black phosphorus quantum dot composite according to claim 6 or 7; preferably, the perforator comprises at least one of a thermal perforator, a mechanical perforator, a laser perforator and a water perforator.

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