Preparation method of pegylated black phosphorus loaded bio-enzyme nano-particles with acoustic catalysis performance

Abstract

The invention discloses a preparation method of a pegylation acoustic catalysis effect black phosphorus loaded biological enzyme nano particle, which is a novel nano composite material based on material chemistry and pharmaceutical chemistry, and bacterial keratitis is treated by using the unique acoustic catalysis property of the nano particle. Nanoparticle synthesis mainly comprises the following steps: 1) pretreatment of commercial black phosphorus; (2) uploading biological enzyme iron-curcumin to black phosphorus in situ; and (3) synthesizing hyaluronic acid coupled phospholipid polyethylene glycol (DSPE-coated PEG2000-HA) and modifying the biological enzyme black phosphorus nanoparticles. The finally obtained nano composite material has good biocompatibility, ocular surface adhesion, biological enzyme activity and acoustic catalytic treatment effect on bacterial biofilm infection of eyes.

Description

Technical Field

[0001] The invention belongs to the field of nanomaterial synthesis, and particularly relates to the preparation and use of polyethylene glycol-modified black phosphorus-loaded bioenzyme nanoparticles with sonocatalytic effect. Background Art

[0002] In recent years, sonodynamic therapy (SDT) has attracted much attention due to its spatiotemporal controllability and non-invasiveness. It has broken through the penetration depth that is difficult for light to achieve and has become an alternative treatment to photothermal and photodynamic therapy. In addition, sonocatalytic therapy, which integrates ultrasound as a stimulus source and sonosensitizers with catalytic ability, has become a current research hotspot. Specifically, sonocatalytic therapy refers to the use of sonosensitizers with catalytic properties to produce reactive oxygen species that are highly toxic to pathogens through ultrasonic chemical reactions under the stimulation of endogenous signal molecules of the disease and the action of exogenous ultrasound, or to use sonosensitizers with catalytic effects to remove reactive oxygen species over-expressed in the disease microenvironment, such as hydrogen peroxide, singlet oxygen, hydroxyl free radicals, superoxide anions, etc., and convert them into oxygen that is harmless to the body, so as to achieve the purpose of alleviating disease hypoxia and enhancing sonodynamic therapy. Currently, a large number of inorganic sonosensitizers are being developed for the diagnosis and treatment of various diseases. Barium titanate (BaTiO3), titanium dioxide (TiO2), bismuth hypochlorite nanosheets (BiOCl), and zinc oxide (ZnO) have been shown to have promising clinical potential for sonocatalytic therapy. However, drawbacks such as limited reactive oxygen species production, poor biocompatibility, and weak targeting have limited their further clinical application.

[0003] Nanocatalytic medicine, with catalytic chemistry as its core, has flourished and is considered to be a key technology that is expected to truly solve the problem from materials to clinical practice. Black phosphorus is an important piezoelectric material with an orthorhombic structure. Its lattice is an interconnected six-membered ring, and each atom is connected to three other atoms. This makes black phosphorus highly anisotropic within the layer. This anisotropy stems from the asymmetry of the black phosphorus crystal structure. This non-centrosymmetric lattice structure will cause the atomic positions in the lattice to shift slightly when black phosphorus is subjected to external mechanical stress, resulting in asymmetric charge distribution, which in turn undergoes redox reactions with air and water in the environment to produce various reactive oxygen species. However, black phosphorus has a weak ability to generate reactive oxygen species under the action of ultrasound, making it difficult to play an important role in the complex disease environment. More importantly, under the action of oxygen, water and external light, the black phosphorus surface will produce more reactive superoxide anions (O2 -), the generated superoxide anions will be adsorbed on the phosphorus atoms on the surface of black phosphorus, and react with them to form phosphorus-oxygen bonds, and eventually degraded into phosphate ions. Therefore, how to regulate the band structure of black phosphorus to further regulate electronic behavior and improve the stability of black phosphorus to exert better antibacterial effects has become a key scientific problem that needs to be solved urgently. Specifically, iron doping significantly reduces the band gap of black phosphorus by introducing intermediate energy levels. This narrow band gap design improves the excitation efficiency of photogenerated carriers and provides energy for the antibacterial reaction. On the other hand, the enzyme-active iron-curcumin forms a Type II heterojunction with black phosphorus nanoparticles. Its band structure prompts the photogenerated electrons to migrate from the conduction band of black phosphorus to the LUMO orbit of curcumin, while the holes are transferred in the opposite direction to the valence band of black phosphorus. This spatial separation effectively inhibits electron-hole recombination, prolongs the carrier lifetime, and enhances ROS generation. More importantly, the piezoelectric polarization field and the Type II band arrangement of the iron-curcumin heterojunction work synergistically to form a directional electron pumping channel: the piezoelectric field drives the electrons of black phosphorus to accelerate their migration to the curcumin interface, while the iron (Fe 3+ / Fe 2+ ) acts as a redox medium, further transferring electrons to O2 to generate O2 - In this patent, the enzyme iron-curcumin was modified in situ on black phosphorus nanoparticles, and hyaluronic acid-coupled polyethylene glycol was modified on the outside of BP@Fe-Cur to prolong the ocular surface adhesion time and biostability of black phosphorus-loaded enzyme nanoparticles, and was successfully applied to ultrasound treatment of ocular bacterial biofilm infection. Summary of the Invention

[0004] The shortcomings of inorganic sonosensitizers currently used in sonocatalytic antibacterial treatment, such as poor sonodynamic effect, single treatment mode, and poor water solubility, have limited the further application of black phosphorus in biomedicine. This invention aims to synthesize black phosphorus-loaded enzyme nanoparticles that are responsive to disease microenvironment, have excellent biocompatibility, controllable biodegradability, and can exert good sonocatalytic effects. By in situ loading the enzyme iron-curcumin on the surface of uniformly sized black phosphorus, DSPE@PEG is finally modified. 2000 -HA and its application in the treatment of corneal bacterial biofilm infection of the eye. Specifically: Iron-curcumin has multiple biological enzyme activities, such as catalase, superoxide dismutase, glutathione peroxidase activity, etc. After being loaded on black phosphorus nanoparticles, on the one hand, iron-curcumin can respond to the high expression of hydrogen peroxide at the bacterial biofilm level to decompose and obtain a large amount of oxygen, which is used to alleviate hypoxia and enhance black phosphorus piezoelectric sonodynamic therapy; on the other hand, compared with black phosphorus nanoparticles alone, black phosphorus nanoparticles loaded with iron-curcumin have long-term stability; finally, the hyaluronic acid-modified phospholipid-polyethylene glycol coated on the surface of the nanoparticles gives the black phosphorus enzyme-loaded nanoparticles the ability to stay on the ocular surface for a long time and corneal penetration performance.

[0005] The technical solutions of the present invention are as follows:

[0006] The preparation method of black phosphorus-loaded enzyme nanoparticles with polyethylene glycol-based sonocatalytic effect comprises the following steps:

[0007] (1) Screening of commercial black phosphorus nanoparticles: Commercial black phosphorus nanoparticles (100 μg / mL) were centrifuged at 12,000 rpm and filtered through a 0.2 μm aqueous microporous filter membrane to obtain uniformly sized single-layer black phosphorus nanoparticles with a particle size of approximately 100 nm ± 5 nm. (2) Loading of bioenzyme iron-curcumin on black phosphorus nanoparticles: Iron trichloride and curcumin in a molar ratio of 1:1 were added to the screened commercial black phosphorus nanoparticles at a concentration of 100 μg / mL for in-situ loading. The mixture was stirred overnight under nitrogen protection and centrifuged at 12,000 rpm for 10 min to obtain BP@Fe-Cur nanoheterojunctions.

[0008] (3)DSPE@PEG 2000 -HA synthesis: hyaluronic acid, DSPE@PEG 2000 -NH2 and EDC / NHS were mixed in deionized water at a certain molar ratio and stirred for 24 h. The sample was dialyzed through a dialysis bag (3000 Da) for 48 h and centrifuged at 10000 rpm for 10 min.

[0009] (4)DSPE@PEG 2000 -HA modification: After vacuum drying overnight, the BP@Fe-Cur nanoheterojunction obtained in step (2) was mixed with DSPE@PEG 2000 -HA were stirred (500 rpm) overnight at a certain mass ratio to obtain black phosphorus-loaded enzyme nanoparticles with polyethylene glycol-based sonocatalytic effect;

[0010] Furthermore, the concentration of the commercial black phosphorus nanoparticle solution in step (1) is 100 μg / mL.

[0011] Furthermore, in step (2), the molar ratio of ferric chloride to curcumin is 1:1.

[0012] Furthermore, in step (3), hyaluronic acid, DSPE@PEG 2000 The molar ratio of -NH2 is 10:1.

[0013] Furthermore, in step (4), the mass ratio of BP@Fe-Cur nanoheterojunction and DSPE@PEG2000-HA is 1:1.

[0014] The main advantages of the present invention are:

[0015] The black phosphorus-based inorganic sonosensitizers reported so far have problems such as poor stability, insufficient targeting, limited sonodynamic effect, and poor biocompatibility. The present invention uses the method of constructing nano-heterojunctions to simply synthesize black phosphorus nanoparticles loaded with biological enzyme iron-curcumin with good sonodynamic effect. By modifying DSPE@PEG 2000 -HA enhances the material's adhesion to the ocular surface and corneal permeability. Compared to a single black phosphorus nanosolution, this nanocomposite material exhibits improved water dispersibility, stability, and the ability to scavenge overexpressed free radicals at the site of infection. More importantly, iron-curcumin acts as an electron acceptor, improving the inherent electron transfer pathway of black phosphorus, enhancing the efficiency of electron-hole separation, and promoting the generation of more singlet oxygen by black phosphorus under ultrasound, synergistically enhancing the sonodynamic effect, thereby rapidly clearing bacterial biofilms colonizing the eye. This nanocomposite has potential clinical application in overcoming and diagnosing ocular biofilm infections caused by drug-resistant bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to make the purpose, technical solutions and beneficial effects of the present invention more clear, the present invention provides the following drawings:

[0017] Figure 1 This is a transmission electron microscopy image of Fe-cur in BP@Fe-Cur@DSPE@PEG-HA nanoparticles in Example 1 of the present invention.

[0018] Figure 2 This is the AFM data of black phosphorus nanoparticles obtained after screening from BP@Fe-Cur@DSPE@PEG-HA nanoparticles in Example 1 of the present invention.

[0019] Figure 3 This is a diagram of the hydrated particle size of BP@Fe-Cur@DSPE@PEG-HA nanoparticles, BP, and Fe-Cur in Example 1 of the present invention.

[0020] Figure 4 This is the scanning electron microscopy element distribution map and element abundance quantitative map of BP@Fe-Cur@DSPE@PEG-HA nanoparticles in Example 1 of the present invention.

[0021] Figure 5 This is a crystal violet staining quantitative diagram and experimental flow chart of Example 1 of the present invention for the removal of bacterial biofilm by BP@Fe-Cur@DSPE@PEG-HA nanoparticles under ultrasonic conditions.

[0022] Figure 6 This is a diagram of corneal epithelial cell compatibility at different concentrations of BP@Fe-Cur@DSPE@PEG-HA nanoparticles in Example 1 of the present invention.

[0023] Figure 7This is a physical picture of Example 1 of the present invention, showing BP@Fe-Cur@DSPE@PEG-HA nanoparticles, BP, Fe-Cur, and Cur dispersed in water.

[0024] Figure 8 This is the UV-absorption spectrum of BP@Fe-Cur@DSPE@PEG-HA nanoparticles, BP, and Fe-Cur in Example 1 of the present invention.

[0025] Figure 9 This is the XPS peak spectrum of iron element in BP@Fe-Cur@DSPE@PEG-HA nanoparticles in Example 1 of the present invention.

[0026] Figure 10 This is the piezoelectric-amplitude response curve and corresponding detection principle diagram of BP@Fe-Cur@DSPE@PEG-HA nanoparticles in Example 1 of the present invention after being measured by piezoelectric force microscopy.

[0027] Figure 11 This is a graph showing the clinical scores of different groups after BP@Fe-Cur@DSPE@PEG-HA nanoparticles were used to treat the keratitis model of MRSA-infected mice in Example 1 of the present invention.

[0028] Figure 12 This is a schematic diagram of the preparation and application of BP@Fe-Cur@DSPE@PEG-HA nanoparticles for the treatment of bacterial keratitis in Example 1 of the present invention. DETAILED DESCRIPTION

[0029] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0030] Example

[0031] (1) Screening of black phosphorus nanoparticles and loading of the bioenzyme iron-curcumin: First, 100 μg / mL commercial black phosphorus nanoparticles were centrifuged at 12,000 rpm and filtered through a 0.2 μm filter membrane for later use. Subsequently, under nitrogen protection, ferric chloride and curcumin were added in a molar ratio of 1:1 to load the bioenzyme iron-curcumin in situ. The mixture was stirred at 500 rpm overnight and dialyzed for 48 hours for later use.

[0032] (2) Functional polyethylene glycol (DSPE@PEG 2000 -HA) synthesis: hyaluronic acid, DSPE@PEG 2000 -NH2, EDC / NHS were ultrasonically dissolved in deionized water, hyaluronic acid, DSPE@PEG 2000-NH2 was mixed at a molar ratio of 10:1, EDC / NHS was added at a mass ratio of 1:2, and stirred for 24 h. The sample was passed through a dialysis bag (3000 Da molecular weight), dialyzed for 48 h, and centrifuged at 10000 rpm for later use;

[0033] (3) Functional polyethylene glycol (DSPE@PEG 2000 -HA) modification: Mixed with iron-curcumin loaded black phosphorus nanoparticles at a mass ratio of 1:1 and stirred (500 rpm) overnight to obtain polyethylene glycol-coated sonocatalytic black phosphorus loaded enzyme nanoparticles. Figure 1 As shown, it is confirmed that the bioenzyme iron-curcumin has good dispersion and the particle size is about 3.5nm. The enlarged image shows that the iron-curcumin has a crystal plane spacing of 0.22nm. This feature is consistent with the characteristics of carbonized polymer dots, proving the successful synthesis of the bioenzyme iron-curcumin. Figure 2 This is the atomic force microscopy image of commercial black phosphorus nanoparticles after screening and the corresponding quantitative image of nanosheet thickness. The image confirms that the surface of the black phosphorus nanoparticles is smooth and has a flaky structure, proving that after centrifugal screening, a black phosphorus aqueous dispersion of uniform size was obtained, and the thickness of the black phosphorus nanoparticles is 4.54nm. Figure 3 This is the DLS hydrodynamic diameter distribution diagram of BFCH (BP@Fe-Cur@DSPE@PEG-HA), BP, and Fe-Cur. The particle size of the final product BP@Fe-Cur@DSPE@PEG-HA (BFCH) is 146.1 nm, and the PDI polydispersity index is 0.150, verifying that the black phosphorus nanoparticles loaded with iron-curcumin and modified with functional polyethylene glycol retain good dispersibility and uniform particle size distribution. Figure 4 This is the scanning electron microscope element distribution map of the final product BFCH. We can observe that Fe, P, C, and O elements are evenly distributed on the nanomaterial. The element abundance quantitative map on the right also verifies the presence of the above elements, further verifying the successful synthesis of the final product. Figure 5 The figure shows the quantitative graph of the crystal violet staining of the final product against biofilm at different concentrations at OD570 nm. The OD value of the final product at a concentration of 50 μg / mL is the lowest, indicating that the final product has the best anti-biofilm ability of drug-resistant bacteria under this concentration condition. Figure 6 In the experiment, the biocompatibility of the final product was verified by cytotoxicity experiments, and it was observed that the final product maintained a corneal epithelial cell (HCEC) cell survival rate of more than 90% at 12h and 24h and under different concentration conditions (0, 12.5, 25, 50, 100μg / mL). Figure 7 We observed that after 10 days at room temperature, the final product BFCH did not undergo obvious degradation, showing good stability, laying the foundation for subsequent antibacterial applications in the eyes. Figure 8The UV absorption spectra of the final product BFCH, BP alone, and Fe-Cur were observed. The successful loading of Fe-Cur onto black phosphorus was verified by observing the characteristic absorption peaks of BFCH and Fe-Cur at 450nm. Figure 9 The peak of iron element in the final product BFCH is shown in the data. 2+ / Fe 3+ The characteristic absorption spectrum of the product reflects that the iron element exists in a variable valence state in the final product. Figure 10 The PFM amplitude-phase curve of the final product at a 10V tip-substrate voltage and a schematic diagram of the PFM detection process show that when an electric field is locally applied through the PFM tip, hysteresis loops are observed in the amplitude and phase. When the applied 10V voltage is reversed, a phase change of approximately 180° occurs, proving that the final product BFCH retains the characteristics of piezoelectric materials. Figure 11 The figure shows the clinical scoring of the MRSA-infected bacterial keratitis mouse model after different treatments. Group G1 is the PBS group and group G5 is the final product + US treatment group. The data verifies the good therapeutic effect of the final product in animals.

[0034] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. Application of PEGylated sonocatalytic effect Black phosphorus-loaded enzyme nanoparticles with excellent sonocatalytic performance and in vitro antibacterial ability, especially for corneal bacterial infection of the eye, is characterized by: The PEGylated sonocatalytic black phosphorus-loaded enzyme nanoparticles have good water / lipid solubility and long-term stability. While having good sonocatalytic effects, they also prolong the retention of nanoparticles in the cornea. The preparation of PEGylated sonocatalytic black phosphorus-loaded enzyme nanoparticles includes the following steps: (1) Screening of commercial black phosphorus: First, under nitrogen protection, a 200 nm commercial black phosphorus solution (100-800 μg / mL) was centrifuged at 12000 rpm to remove unevenly sized black phosphorus nanoparticles and impurities. The solution was then filtered using a 0.2 μm microporous filter membrane and then sealed with deionized water filled with nitrogen and stored in a refrigerator at 4°C until use. (2) Enzyme loading on black phosphorus nanoparticles: Under nitrogen protection, anhydrous ferric chloride, curcumin, and black phosphorus nanoparticles (100 μg / mL, 10 mL) at a certain molar ratio (1:1) were mixed and stirred (500 rpm) overnight. Finally, the mixture was washed three times with ultrapure water at 12,000 rpm, and the precipitate was collected to obtain BP@Fe-Cur nanoparticles. The precipitate was resuspended in nitrogen-filled deionized water for later use. (3) Hyaluronic acid coupled with polyethylene glycol (DSPE@PEG 2000 -HA) synthesis: hyaluronic acid, amino polyethylene glycol (DSPE@PEG 2000 -NH2) in a certain molar ratio and EDC\NHS (1 mg / mL) in a certain mass ratio were dissolved in deionized water, mixed and stirred at room temperature for 24 h, and then the sample was dialyzed through a dialysis bag (3000 Da) for 48 h and washed three times by centrifugation at 10000 rpm; (4)DSPE@PEG 2000 -HA surface modification: Under nitrogen protection, the BP@Fe-Cur nanoparticles obtained in step (2) were mixed with DSPE@PEG 2000 -HA (1 mg / mL) was stirred overnight at a certain mass ratio. Finally, ultrapure water was used for centrifugal washing three times, and the precipitate was collected and dialyzed overnight.

2. The method for preparing polyethylene glycol-modified black phosphorus-loaded enzyme nanoparticles with sonocatalytic effect according to claim 1, characterized in that: The concentration of the commercial black phosphorus nanoparticle solution in step (1) is 100-800 μg / mL, and the centrifugal speed in step (1) is 12000 rpm.

3. The method for preparing polyethylene glycol-modified black phosphorus-loaded enzyme nanoparticles with sonocatalytic effect according to claim 1, characterized in that: The EDC / NHS mass ratio in the step (2) is 1:

2.

4. The method for preparing polyethylene glycol-modified black phosphorus-supported enzyme nanoparticles with sonocatalytic effect according to claim 1, characterized in that: In the step (2), DSPE@PEG 2000 The molar ratio of -NH2 to hyaluronic acid is 10:

1.

5. The method for preparing polyethylene glycol-treated black phosphorus-loaded enzyme nanoparticles with sonocatalytic effect according to claim 1, characterized in that: The molar ratio of anhydrous ferric chloride to curcumin in step (3) is 1:

1.

6. The method for preparing polyethylene glycol-treated black phosphorus-loaded enzyme nanoparticles with sonocatalytic effect according to claim 1, characterized in that: In the step (4), DSPE@PEG 2000 The mass ratio of -HA to black phosphorus nanoparticles loaded with biological enzyme iron-curcumin is 1:1.

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