CN114939162A

CN114939162A - Multifunctional nano catalyst for ultrasonic-mediated bio-orthogonal reaction and preparation method and application thereof

Info

Publication number: CN114939162A
Application number: CN202210294854.XA
Authority: CN
Inventors: 夏丽丽; 陈蒙; 冯炜; 陈雨; 董彩虹
Original assignee: University of Shanghai for Science and Technology
Current assignee: University of Shanghai for Science and Technology
Priority date: 2022-03-24
Filing date: 2022-03-24
Publication date: 2022-08-26
Anticipated expiration: 2042-03-24
Also published as: CN114939162B

Abstract

The invention discloses a multifunctional nano catalyst for ultrasonic-mediated bio-orthogonal reaction and a preparation method and application thereof. The multifunctional nano catalyst of the invention is prepared by Cu-containing ²⁺ The compound and the macromolecular polymer are obtained by a hydrothermal synthesis method, and the compound and the macromolecular polymer are subjected to ultrasonic induction catalysis bio-orthogonal reaction. The multifunctionThe nano-catalyst can be used as a sonosensitizer to generate a large amount of active oxygen under ultrasound while selectively controlling in-vivo in-situ drug synthesis through ultrasound, and can also be used as a contrast agent for Magnetic Resonance Imaging (MRI) and Photo Acoustic Imaging (PAI), so that a visual biological orthogonal and Sonodynamic (SDT) synergistic anti-tumor effect is realized.

Description

Multifunctional nano catalyst for ultrasonic-mediated bio-orthogonal reaction and preparation method and application thereof

Technical Field

The invention relates to the technical field of biomedicine, in particular to a subminiature multifunctional nano catalyst for ultrasonic-mediated biological orthogonal reaction, a preparation method and biological application thereof.

Background

Cancer has been one of the most fatal diseases among various diseases in the world, and seriously threatens the life and health of human beings. Chemotherapy is widely used clinically as a cornerstone of a variety of anti-cancer strategies. However, chemotherapy is prone to cause some toxic and side effects (fatigue, nausea, vomiting, alopecia, weight loss, bleeding, susceptibility to infection, etc.) due to its non-specific targeting, poor bioavailability, rapid inactivation, drug resistance, poor pharmacokinetics, etc. In order to solve these problems, the development of a reliable therapeutic regimen and a feasible drug synthesis strategy with promising clinical application prospects is urgently needed.

The bio-orthogonal reaction is a biological process that is carried out in vivo in a living organism and is not interfered by other spontaneous reactions in the organism. Bio-orthogonality is currently widely used in the field of nanomedicine due to its possessing some bio-functional applications. For example: marking biomolecules such as proteins and nucleic acids, selectively activating inactive substances in biological environments, cell surface functionalization, in vivo imaging monitoring and the like. These bioorthogonal reactions are satisfactorily achieved in living systems.

In recent years, bioorthogonal transformations mediated by biocompatible metal catalysts have shown great potential for applications in synthetic biology and medicinal chemistry, which can promote or accelerate reactions and, in addition, can achieve new chemical reactions in organisms that cannot be catalyzed by natural enzymes. Currently, the most widely used metal catalysts for such conversion applications are mainly Cu, Pd, Ru, Ir, Fe and Au. These catalysts promote the bioorthogonal reactions to proceed smoothly, mainly by participating in bond cleavage, cross-coupling, and cycloaddition reactions. However, the reported metal catalysts have limited their application in complex organisms due to their potential biological toxicity, poor water solubility, low dispersibility, difficult metabolism and poor stability.

With the continuous development of the bioorthogonal reaction in the field of nano medicine, the controllable regulation and the on-demand catalysis of the bioorthogonal reaction in organisms and the like are further explored. For example, the use of visible, ultraviolet and near-infrared light as external stimuli for controlling the occurrence of bioorthogonal reactions has been reported, but inevitably, the limited penetration of the light source limits its further use in the body.

Disclosure of Invention

The invention designs and synthesizes a subminiature multifunctional nano-catalyst for ultrasonic-mediated bio-orthogonal reaction, which overcomes the defects of potential biological toxicity, poor water solubility, low dispersibility, difficult metabolism, poor stability and the like of the traditional metal catalyst and also endows the catalyst with some new functions, such as: the nano-catalyst can realize MR and PA imaging while selectively ultrasonically controlling in-vivo in-situ drug synthesis, and can also be used as a sonosensitizer to generate a large amount of Reactive Oxygen Species (ROS), so that a visualized biological orthogonal and Sonodynamic (SDT) synergistic antitumor effect is realized.

In a first aspect, the present invention provides a subminiature multifunctional nanocatalyst for ultrasound-mediated bio-orthogonal reactions, which catalyzes bio-orthogonal reactions by ultrasound induction, the multifunctional nanocatalyst being composed of Cu-containing nanocatalyst ²⁺ The compound and the macromolecular polymer are obtained by a hydrothermal synthesis method; the macromolecular polymer is sodium polyacrylate.

The multifunctional nano catalyst is characterized in that metal nano particles of the multifunctional nano catalyst are obtained by modifying sodium polyacrylate, so that the metal nano particles can exist stably in organisms, and in a preferable scheme, the average molecular weight Mw of the sodium polyacrylate is 1200.

The multifunctional nano catalyst (Cu @ P NCs for short) can realize the generation of ultrasonic mediated bio-orthogonal reaction, has the effect of triggering SDT treatment, and has the MR and PA imaging capability.

In a second aspect, the invention provides a preparation method of the multifunctional nano catalyst Cu @ P NCs, which comprises the following steps:

(1) solution preparation:

preparation of Cu (NO) at a concentration of 0.3M ₃ ) ₂ ·3H ₂ Aqueous solution of O: 0.7248g of Cu (NO) were weighed out ₃ ) ₂ ·3H ₂ Dissolving O in 10mL of deionized water, and ultrasonically dispersing and dissolving the O;

preparation of Na at a concentration of 0.3M ₂ HPO ₄ ·12H ₂ O aqueous solution: 1.0744g of Na were weighed ₂ HPO ₄ ·12H ₂ Dissolving O in 10mL of deionized water, and ultrasonically dispersing and dissolving the O;

preparation of a 1M aqueous NaOH solution: 0.4g of NaOH was weighed out and dissolved in 10mL of deionized water, and the solution was dispersed by sonication and allowed to dissolve.

(2) The synthesis steps are as follows:

adding Cu (NO) ₃ ) ₂ ·3H ₂ O (4mL) aqueous solution and sodium polyacrylate (0.36g) were dissolved in deionized water (40mL), and the mixture was stirred to obtain a uniform mixture, followed by adding Na to the mixture ₂ HPO ₄ ·12H ₂ Continuously stirring an O (2mL) solution, then adjusting the pH value to 7.0 by using a NaOH solution, and finally transferring the reaction solution to a 100mL polytetrafluoroethylene reaction kettle, lining the reaction kettle, and heating for 6 hours at 120 ℃; after cooling to room temperature, extraction with cyclohexane followed by centrifugation with ethanol precipitation gave a blue product, which was washed several times with water and ethanol to remove unreacted reagents. Finally obtaining the ultra-small multifunctional nano catalyst Cu @ P NCs.

Specifically, Cu (NO) ₃ )2·3H ₂ The mass ratio of O to sodium polyacrylate is 1: 1.25; in the process of precipitating the reaction solution by using ethanol, absolute ethanol is needed, and the volume of the absolute ethanol is more than 10 times of that of the reaction solution; the precipitate was washed with water and ethanol in a volume ratio of (3: 7).

In a third aspect, the multifunctional nanocatalyst catalyzes bio-orthogonal reaction by ultrasonic induction during tumor therapy according to the application of the multifunctional nanocatalyst in tumor therapy, and is used as a sound sensitive agent to realize SDT, and is also used as a contrast agent material for MRI and PAI.

In a fourth aspect, the present invention provides a composition for tumor therapy by bioorthogonal reaction, comprising a precursor and the multifunctional nanocatalyst as described above, wherein the multifunctional nanocatalyst catalyzes the precursor to bioorthogonal reaction by ultrasonic induction.

In a preferable scheme, the composition is used for ultrasonic-mediated fluorescent molecule synthesis, the precursors are fluorescent precursor combinations (drug 1 and drug 2), the multifunctional nano catalyst can trigger the synthesis of a fluorescent reporter molecule by ultrasonic, the occurrence of bioorthogonal reaction in cells is shown in real time through a fluorescent reporting strategy, and the synthetic route of the drug 1 and the drug 2 is as follows.

A

B

anhydrous NaOAc: anhydrous sodium acetate; ac of ₂ O: acetic acid; HCl: hydrochloric acid; ethanal: ethanol; NaNO ₂ : sodium nitrate; NaN ₃ : sodium azide; DMF: n-dimethylformamide; HBTU is O-benzotriazole-tetramethyluronium hexafluorophosphate; DIPEA: diisopropylethylamine.

In another preferred scheme, the composition is used for in-situ synthesis of active drugs, the precursor is a drug precursor combination (drug 4 and drug 5), the ultra-small multifunctional nano catalyst can trigger in-situ synthesis of the active drugs by ultrasound, and the synthesis routes of the drug 4 and the drug 5 are as follows.

C

D

NaN ₃ : sodium azide; HCl: hydrochloric acid; pd (PPh3) 4: palladium tetrakistriphenylphosphine; and (3) CuI: copper iodide; THF: tetrahydrofuran; et 3N: triethylamine; TABF: tetrabutylammonium fluoride.

In the composition for treating tumor, the multifunctional nano-catalyst can be used as a sound-sensitive agent to induce the generation of ROS, so that the combined treatment of bio-orthogonal reaction and SDT is realized, and the effect of inhibiting the activity of tumor is excellent.

In the composition for tumor treatment, the multifunctional nano catalyst also has MR and PA imaging capabilities, and realizes the purpose of diagnosis and treatment integration.

The invention has the following beneficial effects:

the invention successfully designs and synthesizes the microminiature multifunctional high-efficiency catalyst Cu @ P NCs by utilizing the sodium polyacrylate modified copper nanoparticles. The catalyst can trigger the generation of Cu (I) active sites in Cu @ P NCs through ultrasound under the condition of hypoxia in tumors, further realize Cu (I) mediated azide-alkyne cycloaddition reaction, generate active drugs at tumor sites, avoid the problems of toxic and side effects on normal cells and the like caused by low targeting of traditional therapeutic drugs, and effectively kill tumor cells. On the other hand, the generation of the time-space control bio-orthogonal reaction can be realized through ultrasound, and the problem that the traditional bio-orthogonal reaction catalyst is lack of selective on-demand synthesis of active drugs is solved. Meanwhile, separation of electrons and holes in the Cu @ P NCs is caused in the ultrasonic process, the Cu @ P NCs is endowed with the function of a sound-sensitive agent, a large amount of ROS can be generated, oxygen can be consumed, the performance of biological orthogonal reaction is positively promoted, and the SDT treatment is cooperated, so that the effect of efficiently amplifying the anti-tumor effect is realized. In addition, due to the special properties of Cu @ P NCs, the purpose of diagnosis and treatment integration is achieved through MR and PA imaging. More importantly, compared with other metal catalysts, the Cu @ P NCs have the advantages of good water solubility, high bioavailability, capability of being discharged out of a body through kidney metabolism, no toxic or side effect on an organism and the like.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained from the drawings without inventive effort.

FIG. 1 is a Transmission Electron Microscope (TEM) photograph of the multifunctional nanocatalyst Cu @ P NCs prepared in example 1 of the present invention;

FIG. 2 shows the generation of Cu (I) active sites by the multifunctional nanocatalyst Cu @ P NCs under ultrasonic induction in example 2;

FIG. 3 shows the ultrasonic mediated bio-orthogonal catalytic synthesis of fluorescent reporter group by the multifunctional nanocatalyst Cu @ P NCs in example 3;

FIG. 4 shows the ultrasonic mediated bio-orthogonal catalytic synthesis of active drug molecules by the multifunctional nanocatalyst Cu @ P NCs in example 4;

FIG. 5 shows that multifunctional nanocatalyst Cu @ P NCs as sonosensitizers can generate large amounts of ROS;

figure 6 shows the results of in vitro treatment evaluation of ultrasound-mediated bio-orthogonal synergistic SDT in example 6;

FIG. 7 shows the test results of multi-functional nanocatalyst Cu @ P NCs multi-modal imaging in example 7.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The copper nano-composite is modified by sodium polyacrylate to synthesize an ultra-small multifunctional nano-catalyst Cu @ P NCs, and the controllable performance of high-efficiency catalytic bioorthogonal reaction is realized by ultrasonic triggering. Under the stimulation of exogenous ultrasonic waves, the catalytic activity of the Cu @ P NCs can be regulated and controlled through reversible valence conversion between Cu (II) and Cu (I). After the Cu @ P NCs are internalized into cells, the generation of Cu (I) active sites in the Cu @ P NCs is triggered by ultrasound, and then the azide-alkyne cycloaddition reaction is catalyzed through typical Cu (I) to locally catalyze the drug synthesis. Meanwhile, electrons and holes in the Cu @ P NCs are separated in the ultrasonic catalysis process, so that the Cu @ P NCs is endowed with the function of a sound-sensitive agent, and the generation of active oxygen is triggered by ultrasonic waves, so that the Cu @ P NCs can be used as the sound-sensitive agent to generate a large amount of active oxygen and consume oxygen, the oxygen-deficient biological orthogonal reaction is greatly promoted, and the aim of efficiently killing tumor cells is fulfilled. Meanwhile, the Cu @ P NCs have strong near infrared absorption and unpaired electron pairs on the outermost Cu layer rail, so that the photoacoustic imaging and nuclear magnetic resonance imaging capabilities are endowed, and the visualization of the treatment process is realized.

The present invention will be described in further detail with reference to examples. It is also to be understood that the following examples are illustrative of the present invention and are not to be construed as limiting the scope of the invention, and that insubstantial modifications and adaptations of the invention by those skilled in the art based on the foregoing description are intended to be included within the scope of the invention. The specific process parameters and the like of the following examples are also merely one example of suitable ranges, i.e., those skilled in the art can select the appropriate ranges through the description herein, and are not limited to the specific values exemplified below.

EXAMPLE 1 preparation of multifunctional Nanocatalysts Cu @ P NCs 4mL of Cu (NO) ₃ ) ₂ ·3H ₂ O (0.3M) waterThe solution and 0.36g of sodium polyacrylate were dissolved in 40mL of deionized water, and the mixture was stirred at room temperature for 15min to obtain a uniform solution. Then, 2mL of Na was added to the mixture ₂ HPO ₄ ·12H ₂ The O solution was stirred at room temperature for 2h and then the pH was adjusted to 7.0 with NaOH solution. And finally, transferring the reaction solution to a 100mL polytetrafluoroethylene reaction kettle, and heating the lining in the high-pressure reaction kettle at 120 ℃ for 6 hours. After cooling to room temperature, centrifugation (13000rpm, 10min) with ethanol precipitation gave a blue product, which was washed several times with water and ethanol to remove unreacted reagents. Finally obtaining the ultra-small multifunctional nano catalyst Cu @ P NCs.

FIG. 1 is a TEM image of the Cu @ PNCs multifunctional nanocatalyst of the present example. As can be seen from FIG. 1, the Cu @ PNCs have a uniform spherical distribution and a particle size of about 3 nm.

Example 2

Ultrasonic induction of the subminiature multifunctional nano catalyst Cu @ P NCs to generate Cu (I) active sites: a new cuprous reagent is selected as an indicator for detecting the generation of Cu (I) active sites by Cu @ P NCs under ultrasonic induction. First, 1.04mg of the reagent for cuprous oxide was weighed out and dissolved in 5mL of absolute ethanol, and then diluted to 5 times with deionized water for use. Then buffer solution with pH 6.2 is prepared for standby, and KH is used ₂ PO ₄ And NaOH to adjust pH. 5mg of Cu @ P NCs were weighed out and dissolved in 10mL of deionized water at a concentration of 500. mu.g/mL for use. The final test working solution contained 0.75mL buffer, 1.0mL Cu @ P NCs solution and 0.8mL cuprous reagent, and the working solution was exposed to ultrasound at 1MHz, 1.5W/cm ² And carrying out ultrasonic treatment for 10min under the condition that the duty ratio is 50%, and finally measuring the absorbance of the working solution at the wavelength of 450nm by using an ultraviolet-visible absorption spectrum. FIG. 2 shows the UV absorption spectrum of the Cu @ P NCs generated by ultrasonic excitation of the Cu (I) active sites in the example. From the spectra we can see that with increasing ultrasound time and ultrasound power, more and more cu (i) active species are generated.

Example 3

Ultrasonic mediated fluorescent molecule synthesis: firstly, a metal catalyst Cu @ P NCs (1mg/mL) and drug molecules 3-azido-7-hydroxycoumarin (drug 1, 100 mu M) and 6-oxo-6- (propynyl-2-Alkene-1-oxyammonia) hexyl) triphenylphosphine (drug 2, 100 mu M) mixed solution is placed in a six-hole plate, and the mixed solution is subjected to ultrasonic treatment for 10min (1MHz, 1.5W/cm) by using an ultrasonic probe ² Duty 50%) and then the mixture was transferred to a three-necked flask and the reaction was stirred continuously at 37 ℃ under nitrogen. During the reaction, 0.5, 1.0, 1.5 and 2.0h of reaction solution were collected respectively, and fluorescence intensities (E) of the reaction solutions at different time points were monitored by fluorescence spectroscopy _x /E _m :＝347/482nm)。

FIG. 3 shows the results of ultrasound-mediated synthesis of fluorescent molecules. As can be seen from FIG. 3, the reaction solution emitted a strong fluorescence signal after the ultrasonic treatment.

Example 4

Ultrasonic mediated drug molecule synthesis: a pair of safe and nontoxic small molecules are converted into toxic small molecules, so that the antitumor therapy is realized. In the first step, we have verified the in vitro synthesis of active drug molecules by high performance liquid chromatography. The specific implementation steps are as follows: firstly, a mixed solution of a metal catalyst Cu @ P NCs (1mg/mL) and drug molecules of 5-azide-1, 2, 3-trimethoxybenzene (drug 4, 100 mu M) and 5-acetylene-2-methoxyaniline (drug 5, 100 mu M) is placed in a six-hole plate, and the mixed solution is subjected to ultrasonic treatment for 10min (1MHz, 1.5W/cm) by using an ultrasonic probe ² Duty 50%) and then the mixture was transferred to a three-necked flask and the reaction was stirred continuously at 37 ℃ under nitrogen atmosphere. During the reaction, 2,4,8 and 24h of reaction solution were collected, purified by centrifugation (13000rpm, 10min), and then the newly formed compound was monitored by high performance liquid chromatography.

FIG. 4 shows the formation of active drug molecules by HPLC, and from FIG. 4 we can see that new peaks appear, and the yield of the product is higher and higher with longer reaction time, and the content of the reactant is reduced accordingly.

Example 5

And (3) testing the generation of ROS by using the subminiature multifunctional nano catalyst Cu @ P NCs as a sonosensitizer.

FIG. 5 shows the use of Electron paramagnetic resonance (ESR) monitoringAnd measuring the generation condition of the ROS. As can be seen from FIG. 5, under the condition of ultrasonic stimulation, Cu @ P NCs can remarkably generate singlet oxygen (C) ¹ O ₂ ) Superoxide anion (O) ^- ₂ ) And hydroxyl radicals (. OH); however, without sonication, there is little production of singlet oxygen, superoxide anions, and hydroxyl radicals. The Cu @ P NCs are stimulated by ultrasound to cause separation of electrons and holes in the Cu @ P NCs, so that the Cu @ P NCs can be used as an efficient sonosensitizer to absorb energy released by the ultrasound, further generate a large amount of ROS, consume oxygen, enhance the treatment effect of the SDT and greatly improve the catalytic efficiency under the bioorthogonal hypoxic condition.

Example 6 ultrasound-mediated bio-orthogonal responses in conjunction with testing of SDT for anti-tumor proliferation inhibitory activity in vitro, demonstrated therapeutic levels of synthetic active drug in vitro at the cellular level. First, 4T1 cells were cultured at 5X 10 cells per well ³ The density of (2) was inoculated in a 96-well plate and cultured for 24h before starting the treatment. The complete culture solution was replaced with a mixed culture solution containing drug 4 (20. mu.M) and drug 5 (20. mu.M), a mixed culture solution of drug 4 (20. mu.M), drug 5 (20. mu.M) and Cu @ P NCs (100. mu.g/mL) as a metal catalyst, respectively. Then, they were sonicated and not sonicated, respectively, and they were co-cultured in an anaerobic culture bag for 12 hours, and the complete medium culture and non-sonicated groups were used as negative control groups. After the end of the incubation, the cells were washed twice with PBS buffer, then 100. mu.L of fresh medium containing the cell counting reagent cck-8 (10. mu.L) was added per well and incubated with the cells for 1h, and finally their absorbance at 450nm was recorded by a microplate reader.

FIG. 6 shows the results of the study of the proliferation inhibitory activity of mouse breast cancer cells 4T1 by ultrasound-mediated bio-orthogonal reaction in combination with SDT. As can be seen from FIG. 6(a-b), the cytotoxicity of drug 4, drug 5 and Cu @ P NCs was almost negligible, and the survival rate of cells reached more than 90% even at drug concentrations of 80. mu.M or 80. mu.g/mL. However, as can be seen from FIG. 6(b), the viability of 4T1 cells was significantly decreased with increasing concentrations of Cu @ P NCs under Ultrasound (US) stimulation, with cell viability of 58.1% when the concentration reached 80. mu.g/mL. In addition, we found that the cell viability of Cu @ P NCs, drug 4 and drug 5 was significantly reduced to 36.8% after US treatment in the presence of them compared to SDT triggered by Cu @ P NCs alone. The result shows that the SDT combined bioorthogonal reaction triggered by Cu @ P NCs further enhances the killing effect on tumor cells.

Example 7 testing of multi-functional nanocatalyst Cu @ P NCs multimodal imaging capabilities.

We examined MR and PA imaging of Cu @ P NCs in mice. The 4T1 tumor cells were first seeded at a density of 1X 10 ⁷ Inoculating to leg or oxter of mouse, and allowing tumor volume to reach 250mm ³ Left and right, tumor-bearing mice were treated for MR and PA imaging. For PA and MR imaging, we intratumorally injected the nanocatalyst Cu @ P NCs into mice, 2h later, the mice were scanned and imaged with a clinical MRI scanner (Siemens prism MR 3.0T, germany) and a small animal PA imaging system (VEVO LAZR-X, usa), respectively, and in addition, images of the mice before injection of the nanocatalyst served as negative controls.

FIG. 7 shows bimodal imaging results of Cu @ P NCs in tumor-bearing mice. For PA imaging, from fig. 7(a), PA signals at tumor sites were significantly enhanced after Cu @ P NCs injection, demonstrating its ability to PA image. In addition, as for MR imaging, it can be seen from fig. 7(b) that the contrast of MR imaging at the tumor site is significantly enhanced compared to other tissues after Cu @ P NCs injection. The above results indicate that Cu @ P NCs can be used as a new and promising MR/PA bimodal imaging contrast agent.

In the two groups of compositions for treating tumors through bioorthogonal reaction, the multifunctional nano catalyst Cu @ P NCs is respectively combined with the fluorescent drug precursor and the active drug precursor, and cell and animal experiments in the embodiment verify that the precursor compounds can be selectively activated in tumor cells, so that bioorthogonal reaction is cooperated with SDT treatment to achieve high-efficiency anti-tumor effect.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. The multifunctional nano catalyst for the ultrasonic-mediated bio-orthogonal reaction is characterized in that the ultrasonic-induced catalysis of the bio-orthogonal reaction is carried out, and the multifunctional nano catalyst is prepared from Cu-containing nano catalyst ²⁺ A compound of (a) and a macromolecular polymer.

2. The multifunctional nanocatalyst of claim 1, wherein the macromolecular polymer is sodium polyacrylate.

3. A method of preparing the multifunctional nanocatalyst of any of claims 1-2, comprising the steps of:

adding Cu (NO) ₃ ) ₂ ·3H ₂ Dissolving O water solution and sodium polyacrylate in deionized water, stirring to obtain uniform mixed solution, and adding Na into the mixed solution ₂ HPO ₄ ·12H ₂ And continuously stirring the O solution, then adjusting the pH value to 7.0 by using a NaOH solution, finally transferring the reaction solution to a 100mL polytetrafluoroethylene reaction kettle, lining the reaction kettle, heating the reaction solution in the high-pressure reaction kettle at 120 ℃ for 6h, cooling the reaction solution to room temperature, extracting the reaction solution by using cyclohexane, then precipitating and centrifuging the reaction solution by using ethanol to obtain a blue product, washing the blue product by using water and ethanol for several times to remove unreacted reagents, and finally obtaining the multifunctional nano catalyst Cu @ PNCs.

4. The method as claimed in claim 3, wherein Cu (NO) ₃ ) ₂ ·3H ₂ The mass ratio of O to sodium polyacrylate is 1: 1.25; when the reaction solution is precipitated by ethanol, the volume of the ethanol is more than 10 times of that of the reaction solution, and when the precipitate is washed by the ethanol and the water, the volume ratio of the ethanol to the water is 7: 3.

5. the use of the multifunctional nanocatalyst of any of claims 1-2 for the treatment of tumors, wherein the multifunctional nanocatalyst catalyzes the bioorthogonal reaction by ultrasound induction during the treatment of tumors and as a sonosensitizer for the sonodynamic treatment of tumors and as a contrast agent material for MRI and PAI.

6. A composition for tumor treatment by bio-orthogonal reaction, comprising a precursor and the multifunctional nanocatalyst of claim 1, wherein the multifunctional nanocatalyst catalyzes the precursor to perform bio-orthogonal reaction by ultrasound induction.

7. The composition of claim 6, wherein the precursor comprises 3-azido-7-hydroxycoumarin (drug 1) and 6-oxo-6- (propynyl-2-en-1-oxyamino) hexyl) triphenylphosphine (drug 2) for use in ultrasound-mediated fluorescent molecular synthesis.

8. The composition of claim 6, wherein the precursors 5-azido-1, 2, 3-trimethoxybenzene (drug 4) and 5-ethynylene-2-methoxyaniline (drug 5) are used for in situ synthesis of ultrasound mediated active drugs.

9. The composition of any one of claims 6-8, wherein the multifunctional nanocatalyst catalyzes a bio-orthogonal reaction of the precursor through ultrasound induction, and is simultaneously used as a sonosensitizer material for SDT therapy, achieving a bio-orthogonal reaction in combination with SDT therapy, and is also used as a contrast agent material for MRI and PAI.