CN111632140B - Near-infrared light activated core-shell structure nanoenzyme and preparation method thereof - Google Patents

Near-infrared light activated core-shell structure nanoenzyme and preparation method thereof Download PDF

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CN111632140B
CN111632140B CN202010430252.3A CN202010430252A CN111632140B CN 111632140 B CN111632140 B CN 111632140B CN 202010430252 A CN202010430252 A CN 202010430252A CN 111632140 B CN111632140 B CN 111632140B
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陈航榕
陈潜
张衡
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Shanghai Institute of Ceramics of CAS
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Abstract

The invention relates to a near infrared light activated core-shell structure nano enzyme and a preparation method thereof, wherein the near infrared light activated core-shell structure nano enzyme comprises the following components: the up-conversion luminescent nano material is used as an inner core, the mercapto-modified dendritic structure silica formed on the surface of the inner core is used as an outer shell, and the nano CuS is distributed on the surface and in a pore channel of the mercapto-modified dendritic structure silica.

Description

Near-infrared light activated core-shell structure nanoenzyme and preparation method thereof
Technical Field
The invention relates to a near-infrared light activated core-shell structure nano enzyme and a preparation method thereof, belonging to the field of nano medicine preparation.
Background
Cancer has become one of the leading killers of human health worldwide, and according to the World Health Organization (WHO) 2015 statistics, cancer is the leading cause of death in people before the age of 70 in 172 countries. It is reported that 1810 ten thousands of new cancer cases occur in 2018 all over the world, and 960 ten thousands of deaths occur due to cancer. The current major methods for clinical treatment of cancer are surgery, chemotherapy and radiotherapy. Although these conventional methods save a part of cancer patients to a great extent, these conventional methods have the disadvantages of large trauma, incomplete tumor removal, high toxic and side effects, and easy recurrence and metastasis of cancer. Therefore, improvement of conventional therapeutic means or development of novel therapeutic techniques is urgently required. With the development of nano science and technology, the structure, morphology and surface properties of the nano material can be better regulated and controlled, so that the nano materials with different functions can be obtained to meet the requirements of different application fields. Due to their unique properties, nanomaterials have received extensive attention and research in the biomedical field.
With the rapid development of nanotechnology, nanomaterials with enzymatic catalytic properties are widely discovered, prepared and applied. This nanomaterial with enzymatic properties is defined as "nanoenzyme" by Lucia Pasquato, paolo Scrimin and their co-workers. Nanoenzymes have been used in a variety of fields including sensors, immunoassays, diagnosis and treatment of cancer, neuroprotection, stem cell proliferation, and pollutant treatment. At present, various nano materials are developed to be used as nano enzymes, and gold nanoparticles, rare earth particles, ferroferric oxide nanoparticles and the like all show unexpected enzyme-like catalytic activity. For example, the Onychii academyelination topic group in 2007, discovered and studied the enzymatic properties of ferroferric oxide nanoparticles, which are similar to horseradish peroxidase activity. And the ferroferric oxide nano-particles are used as nano-enzyme, and still maintain good catalytic activity under wider conditions. The nano enzyme is used for diagnosing and treating cancers, and brings new opportunities for overcoming the problems of cancers.
Under the excitation of 980nm near infrared, copper sulfide nano particles (CuS) convert light into heat, thus realizing the photo-thermal treatment of tumors (PTT). PTT is a novel tumor therapy and is currently receiving wide attention. Meanwhile, cuS generates photocatalytic reaction under the irradiation of ultraviolet light to catalyze H 2 O 2 Generating hydroxyl radical (. OH). OH has strong oxidizing ability, and is destructive to cell membrane structure, protein, etc., thereby causing apoptosis of tumor cells.
Although various nanoenzymes with catalytic activity and wider application conditions are developed for treating tumors, how to start, control and close the catalytic activity of the nanoenzymes ensures that toxic and side effects in a treatment process are reduced while the tumors are treated efficiently is of great importance for overcoming cancers. There is no literature report on controlling the initiation of enzyme activity.
Disclosure of Invention
In view of the above problems, the present invention is directed to a nanoenzyme that can control activation/deactivation and a method for preparing the same.
In one aspect, the invention provides a core-shell structure nanoenzyme activated by near-infrared light, comprising: the up-conversion luminescent nano material is used as an inner core, the mercapto-modified dendritic structure silica formed on the surface of the inner core is used as an outer shell, and the nano CuS is distributed on the surface and in a pore channel of the mercapto-modified dendritic structure silica.
In the disclosure, the near-infrared light activated core-shell structure nanoenzyme can excite UCNPs in the cuss-UCNPs @ dmsns core to generate ultraviolet light under near-infrared light (e.g., 980nm laser), and the ultraviolet light can further excite nano-CuS to start photocatalytic reaction, so as to realize novel photocatalytic treatment (PCT). And CuS can generate heat under 980nm laser irradiation for photothermal therapy, and can be finally used for PTT/PCT synergistic therapy of tumor function. That is, the present invention has innovatively implemented a novel photocatalytic therapy (PCT) strategy for generating active oxygen using near-infrared light-catalyzed nanoenzymes. In addition, the silicon dioxide nano materials (DMSNs) with dendritic structures have dendritic structures and larger pore channels, so that the silicon dioxide nano materials can be better used for loading proteins, macromolecules, nanoparticles and the like.
In the present invention, up-conversion luminescent nanomaterials (UCNPs), namely: anti-Stokes luminescence (Anti-Stokes) refers to that a material is excited by low-energy light to emit high-energy light, namely, the material emits light with short wavelength and high frequency after being excited by long-wavelength and low-frequency light. The upconversion luminescent nanomaterial used in the present invention may be selected from NaYF 4 :Er/Yb、NaYF 4 :Yb/Er/Tm、NaYF 4 :Yb/Gd/Er、NaYF 4 :Yb/Tm、NaYF 4 :Yb/Tm@NaGdF 4 And NaGdF 4 At least one of Yb/ErOne kind of the material is selected; the particle size of the up-conversion luminescent nano material is 10 nm-100 nm.
Preferably, the particle size of the thiol-modified dendritic silica is 50nm to 200nm, and the diameter of the pore channel is 5 nm to 20nm.
Preferably, the content of the sulfhydryl-modified dendritic structure silicon dioxide is 40-80 wt%, and preferably 40-60 wt%.
Preferably, the particle size of the nano CuS is 1 nm-20 nm.
Preferably, the loading amount of the nano CuS is 5-40 wt%, and preferably 5-20 wt%. As the dendritic structure silicon dioxide is selected to have a macroporous structure, the loading of the nano CuS can be further improved, so that the effect of photocatalytic treatment is improved.
Preferably, the particle size of the core-shell structure nanoenzyme is 50-300 nm.
On the other hand, the invention provides a preparation method of near-infrared light activated core-shell structure nano enzyme, which comprises the following steps:
(1) Adding the up-conversion luminescent nano material into an aqueous solution of a surfactant, and performing ultrasonic emulsification to obtain emulsion A;
(2) Adding sodium salicylate and organic micromolecular amine (triethanolamine) into the emulsion A, adding a silicon source, heating in a water bath, centrifuging and washing to obtain core-shell structure nano particles of the dendritic structure silicon dioxide coated up-conversion luminescent nano material, and recording the core-shell structure nano particles as UCNPs @ DMSNs;
(3) Dispersing the obtained UCNPs @ DMSNs in an organic solvent, adding 3-mercaptopropyl trimethoxy silane MPTS and ammonia water, reacting for 10-24 hours under magnetic stirring, and then carrying out centrifugal treatment on the mixture to obtain mercapto-modified UCNPs @ DMSNs;
(4) Dispersing the obtained mercapto-modified UCNPs @ DMSNs in trichloromethane, adding CuS nano particles, reacting for 1-6 hours under stirring, and then carrying out centrifugal treatment to obtain the near-infrared light activated core-shell structure nano enzyme.
Preferably, the surfactant is at least one selected from the group consisting of cetyltrimethylammonium bromide CTAB, octadecyltrimethoxysilane CTMS, cetyltrimethylammonium chloride CTAC, polyethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymer P123, sodium dodecyl sulfate SDS and fluorosurfactants.
Preferably, the mass ratio of the up-conversion luminescent nano material to the surfactant is 1 (5-25).
Preferably, the mass ratio of the sodium salicylate to the surfactant is 10 (1-5).
Preferably, the organic small-molecule amine is selected from at least one of triethanolamine TEA and ammonia water, and the mass ratio of the organic small-molecule amine to the sodium salicylate is 1: (5-50).
Preferably, the silicon source is at least one selected from the group consisting of tetraethyl orthosilicate TEOS, methyl orthosilicate TMOS, 3-aminopropyltriethoxysilane APTES and 1, 2-bistrimethoxysilyl ethane BTME.
Preferably, the dosage of the silicon source and the surfactant is 6-25 μ L:10mg.
Preferably, the water bath heating temperature is 70-95 ℃, and the time is 2-6 hours. The water bath heating process can be started when sodium salicylate and organic micromolecular amine are added, and after a period of time, a silicon source is added to continue the water bath heating for a period of time.
Preferably, the organic solvent is at least one selected from the group consisting of ethanol, chloroform, DMSO, and cyclohexane.
Preferably, the magnetic stirring speed of the magnetons is 300-800 r/min.
Preferably, the dosage ratio of the sulfydryl modified UCNPs @ DMSNs to the CuS nano-particles is 8mg: 5-100 mu moL.
On the other hand, the invention also provides PEG modified near-infrared light activated core-shell structure nanoenzyme, which is obtained by adding methoxy-polyethylene glycol-silane into a near-infrared light activated core-shell structure nanoenzyme solution, heating in water bath at 40-80 ℃ for 6-24 hours, and then performing centrifugal treatment and washing. The obtained core-shell structure nano enzyme activated by near infrared light is modified by PEG, which can play a role in stabilizing materials and further improve the biological safety of the materials.
Preferably, the mass ratio of the near-infrared light activated core-shell structure nanoenzyme to the methoxy-polyethylene glycol-silane is 1: (0.1-2).
Has the advantages that:
in the invention, the preparation method of the nano enzyme has simple and convenient reaction, is easy to control, is easy to obtain a target product, and has novel structure and novel application; in the invention, the prepared nano enzyme with the dendritic core-shell structure has the characteristic of activating enzyme activity by near infrared light (780 nm-2526 nm), and has the function of being used for PTT/PCT synergistic treatment of tumors.
Drawings
FIG. 1 is TEM (a-c), SEM (d-f) electron micrograph of UCNPs @ DMSNs prepared in example 1, wherein scale of (a) is 0.2 μm, scale of (b) is 100nm, and scale of (c) is 20nm;
FIG. 2 shows TEM (a-b), STEM dark field image (c) and element plane scan image (d) of CuS-UCNPs @ DMSNs nanoenzyme prepared in example 1;
FIG. 3 is the EDS map (b) corresponding to the element line scan (a) and the element line scan of the CuS-UCNPs @ DMSNs nanoenzyme prepared in example 1;
FIG. 4 is a UV-Vis curve of CuS-UCNPs @ DMSNs nanoenzyme prepared in example 1 in TMB aqueous solution, wherein CUD is CuS-UCNPs @ DMSNs; UD: UCNPs @ DMSNs; CD: cuS-DMSNs;
FIG. 5 is the UV-Vis absorption value of CuS-UCNPs @ DMSNs nanoenzyme prepared in example 1 at 652nm in TMB aqueous solution, wherein CUD is CuS-UCNPs @ DMSNs; UD: UCNPs @ DMSNs; CD: cuS-DMSNs;
fig. 6 is a digital photograph of TMB aqueous solution of different samples corresponding to example 1, wherein 1: h 2 O 2 +Laser；2：CUD+Laser；3：CUD+H 2 O 2 ；4：UD+H 2 O 2 +Laser；5：CD+H 2 O 2 +Laser；6：CUD+H 2 O 2 +50℃；7：CUD+H 2 O 2 +Laser；
FIG. 7 shows the photothermal curves (a) at different concentrations, photothermal curves (b) at different power densities, photothermal stability curves (c) and photothermal curves (d) at different concentrations of CuS-DMSNs prepared in example 1;
FIG. 8 is the result of cell-level treatment with CuS-UCNPs @ DMSNs nanoenzyme prepared in example 1, wherein CUD is CuS-UCNPs @ DMSNs; UDL: UCNPs @ DMSNs +980nm laser; CDL: cuS-DMSNs +980nm laser; CUDL, cuS-UCNPs @ DMSNs +980nm laser;
FIG. 9 shows in vivo photothermographic pictures of HCT116 tumor-bearing mice during laser irradiation of CuS-UCNPs @ DMSNs nanoenzyme prepared in example 1, wherein CUD is CuS-UCNPs @ DMSNs; UD: UCNPs @ DMSNs; CD: cuS-DMSNs;
FIG. 10 is the results of in vivo layer treatment of CuS-UCNPs @ DMSNs nanoenzyme prepared in example 1, temperature change curve at the tumor of mouse during laser irradiation, wherein CUD is CuS-UCNPs @ DMSNs; UD: UCNPs @ DMSNs; CD: cuS-DMSNs;
FIG. 11 is the results of in vivo layer treatment of CuS-UCNPs @ DMSNs nanoenzyme prepared in example 1, the tumor volume profile of different treatment groups, wherein CUD is CuS-UCNPs @ DMSNs; UD: UCNPs @ DMSNs; CD: cuS-DMSNs.
Detailed Description
The present invention is further illustrated by the following examples, which are to be construed as merely illustrative, and not a limitation of the present invention.
In the disclosure, dendritic-structure silicon dioxide DMSNs are used for wrapping up-conversion luminescent nano materials UCNPs, composite nano particles with dendritic core-shell structures are firstly constructed to serve as carriers, then a pore structure of the DMSNs is used for loading nano CuS, and finally the near-light activated core-shell structure nano enzyme is prepared.
In the invention, the nano enzyme with the core-shell structure based on the dendritic structure silicon dioxide coating has better water solubility, stability and dispersibility and an obvious dendritic structure; under 980nm laser irradiation, not only can heat be generated to realize photothermal therapy (PTT) tumor, but also the peroxidase property can be realized to generate free hydroxyl, so that the photocatalysis therapy (PCT) tumor is realized; the PTT/PCT synergistic treatment can obviously inhibit the growth of the tumor and is expected to be applied to the high-efficiency treatment of the tumor.
In one embodiment of the invention, the preparation method of the low-beam activated core-shell structure nanoenzyme is simple, the reaction condition is mild, the operation is easy, and the method has the prospect of industrial implementation. The preparation of the low-beam activated core-shell structured nanoenzymes is illustrated below.
Adding up-conversion luminescent nano materials (UCNPs) into an aqueous solution containing a surfactant (CTAB), and performing ultrasonic emulsification to obtain emulsion A. Wherein, the particle size of the up-conversion luminescence nano material can be 10-100 nm. Preferably, the ratio of UCNPs to CTAB is 20mg: 100-500 mg.
Adding sodium salicylate (NaSal) and Triethanolamine (TEA) into the emulsion A, and stirring for 3-7 hours under heating in a water bath at 60-90 ℃ to uniformly mix the whole solution. Then adding Tetraethoxysilane (TEOS), continuing to react for 1-6 hours, and gradually hydrolyzing the TEOS in the process to form dendritic silicon dioxide. And (3) performing centrifugal separation (namely centrifugal treatment) and cleaning treatment to obtain core-shell structure nanoparticles of dendritic structure silica-coated UCNPs, and recording the core-shell structure nanoparticles as UCNPs @ DMSNs. The obtained UCNPs @ DMSNs have dendritic and macroporous structures and present core-shell structures. Wherein, the dosage ratio of CTAB and NaSal can be 100mg: 10-50 mg. The mass ratio of TEA to NaSal may be 1:5 to 50. Preferably, the amount ratio of CTAB to TEOS may be 100mg: 60-250 mu L. In an alternative embodiment, the cleaning process comprises two steps of cleaning and extraction. For example, washing with water and ethanol is repeated 4 to 5 times, followed by extraction with 1% NaCl in methanol to remove the structure-directing agent CTAB. The content of DMSNs in the obtained UCNPs @ DMSNs is about 60-90 wt%.
Dissolving UCNPs @ DMSNs in organic solvent (such as ethanol, chloroform, DMSO, etc.), and adding 3-Mercaptopropyltrimethoxysilane (MPTS) and ammonia (NH) while magnetically stirring 3 .H 2 O), reacting for 10-24 hours, and then centrifuging to obtain the sulfhydryl modified dendritic structure silica/up-conversion luminescent nano material, which is marked as UCNPs @ DMSNs-SH. Wherein, the magnetic stirring speed of the magnetons can be 300-800 r/min, and the temperature can be room temperature, such as 25-60 ℃.
UCNPs @ DMSNs-SH is dispersed in a solvent (such as trichloromethane, cyclohexane, DMSO and the like), copper sulfide nano particles (nano CuS) are added while stirring, after 2 to 12 hours of reaction, the composite nano material loaded with CuS is obtained by centrifugal separation, and is marked as CuS-UCNPs @ DMSNs. And after the reaction is finished, centrifugally cleaning the reaction product for at least 3 times by using deionized water, and finally obtaining the near-light activated core-shell structure nano enzyme. Among them, the copper sulfide nanoparticles preferably have a particle size of 1 to 20nm. The dosage ratio of the sulfydryl modified UCNPs @ DMSNs to the nano CuS can be 8mg: 5-100 mu moL. In addition, when deionized water is adopted for centrifugation and washing, the addition amount of the deionized water can be regulated and controlled, and the CuS-UCNPs @ DMSNs aqueous solution with the concentration of less than or equal to 200mg/mL is prepared.
It should be noted that in the present invention, the surfactant may be selected from CTAC, CTMS, P123, SDS, and the like, in addition to CTAB. The triethanolamine can be replaced by other organic small molecular amines such as ammonia water. The silicon source may also be selected from TEOS, TMOS, APTES, BTME, etc.
In another embodiment of the invention, cuS-UCNPs @ DMSNs is dispersed in ethanol solution, and a certain amount of methoxy-polyethylene glycol-silane is added dropwise, and after stirring in water bath at 60 ℃ for 24 hours, centrifugal separation is carried out. Then washing with ethanol and water for 2 times respectively to obtain PEG modified CuS-UCNPs @ DMSNs (marked as CUD).
As a detailed example of preparing a low-beam activated nucleocapsid nanoenzyme, include: (4) Firstly, dissolving a certain amount of CTAB in a certain amount of aqueous solution, ultrasonically dispersing, adding a certain amount of UCNPs into the solution, emulsifying by using a cell disruptor, adding the solution into a glass bottle, and adding a certain amount of TEA and a certain amount of NaSal while stirring. (2) Heating the solution to 80 ℃ in a water bath kettle, stirring for 1 hour, adding a certain amount of TEOS into the solution, keeping stirring, and continuing to react for 2 hours. (3) Subsequently, a certain amount of anhydrous ethanol was added, followed by centrifugation. And repeatedly washing with water and ethanol for 4-5 times, and extracting with 1% NaCl methanol solution to remove structure directing agent CTAB. (4) Dissolving a certain amount of UCNPs @ DMSNs in ethanol, adding a certain amount of MPTS and a certain amount of NH into the solution 3 .H 2 O, then stirring vigorously at room temperature to obtain UCNPs @ DMSNs-SH. (5) The final product of UCNPs @ DMSNs-SH was washed 3 times with ethanol and then dispersed in chloroform. (6)Dropwise adding a certain amount of CuS solution into a certain amount of UCNPs @ DMSNs-SH chloroform solution, stirring at room temperature for 30 minutes, and centrifuging to separate out a precipitated product. Followed by washing with chloroform and ethanol 2 times, respectively, to obtain CuS-UCNPs @ DMSNs.
The present invention will be described in detail by way of 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 only 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. Unless otherwise specified, the upconversion luminescent nanomaterials used in the following examples have a particle size of 30nm and the copper sulfide nanoparticles used have a particle size of 1 to 20nm.
Example 1
First 373mg CTAB was dissolved in 6mL of an aqueous solution, and after ultrasonic dispersion, a certain amount of UCNPs (0.064g, naYF) was added to the above solution 4 Er/Yb), emulsified for 10 minutes using a cell disruptor, the above solution was put into a 20mL glass bottle, and 10mg of TEA and 150mg of NaSal were added with stirring. The solution was heated to 80 ℃ in a water bath, and after stirring for 1 hour, 0.6mL of TEOS (0.56 g) was added to the solution, and the reaction was continued for 2 hours while keeping stirring. 10mL of absolute ethanol was then added, followed by centrifugation. And repeatedly washing with water and ethanol for 4 to 5 times, and finally extracting with 1% NaCl methanol solution to remove CTAB as structure directing agent to obtain large-pore-size UCNPs @ DMSNs (UD).
First, 150mg UCNPs @ DMSNs was dissolved in 15mL ethanol, and 75. Mu.L MPTS and 375. Mu.L NH were added to the above solution 3 .H 2 O, then stirred vigorously at room temperature for 12h. The final product, ucnps @ dmsns-SH, was washed 3 times with ethanol and then dispersed in chloroform. 0.1mL of CuS solution (concentration of 0.3 mmoL/mL) was added dropwise to 4mL of UCNPs @ DMSNs-SH (2 mg/mL) in chloroform, and stirred at room temperature for 30 minutesAfter that, the precipitated product was centrifuged. Followed by washing with chloroform and ethanol 2 times, respectively, to obtain CuS-UCNPs @ DMSNs. The content of sulfydryl modified dendritic structure silicon dioxide in the CuS-UCNPs @ DMSNs is 40-60 wt%, and the load capacity of the nano CuS is about 16-20 wt%.
To 50mL of CuS-UCNPs @ DMSNs ethanol solution (20 mg), 40mg of methoxy-polyethylene glycol-silane was added dropwise, and after stirring in a water bath at 60 ℃ for 24 hours, the mixture was centrifuged. Then washing with ethanol and water for 2 times respectively to obtain PEG-modified CuS-UCNPs @ DMSNs (denoted as CUD). Wherein, the PEG content in the PEG-modified CuS-UCNPs @ DMSNs is 40-60 wt%.
Example 2
746mg CTAB was first dissolved in 12mL of aqueous solution, after ultrasonic dispersion, a certain amount of UCNPs (0.128g, naYF) was added to the above solution 4 Er/Yb), emulsified for 10 minutes using a cell disruptor, the above solution was put into a 20mL glass bottle, and 10mg of TEA and 300mg of NaSal were added with stirring. The solution was heated to 80 ℃ in a water bath, and after stirring for 1 hour, 1.2mL of TEOS (1.12 g) was added to the solution, and the reaction was continued for 2 hours while keeping stirring. Subsequently, 10mL of absolute ethanol was added, followed by centrifugation. And repeatedly washing with water and ethanol for 4 to 5 times, and finally extracting with 1% NaCl methanol solution to remove CTAB as structure directing agent to obtain large-pore-size UCNPs @ DMSNs (UD).
First, 300mg of UCNPs @ DMSNs was dissolved in 30mL of ethanol, and 150. Mu.L of LMPTS and 750. Mu.L of NH were added to the above solution 3 .H 2 O, then stirred vigorously at room temperature for 12h. The final product UCNPs @ DMSNs-SH was washed 3 times with ethanol and then dispersed in chloroform. 0.2mL of a CuS solution (concentration: 0.2 mmoL/mL) was dropwise added to a chloroform solution of 8mL of UCNPs @ DMSNs-SH (2 mg/mL), and after stirring at room temperature for 30 minutes, the precipitated product was centrifugally separated. Then washing with chloroform and ethanol for 2 times respectively to obtain CuS-UCNPs @ DMSNs finally. The content of sulfydryl modified dendritic structure silicon dioxide in the CuS-UCNPs @ DMSNs is 45-60 wt%, and the load capacity of the nano CuS is about 10-15 wt%.
To 100mL of CuS-UCNPs @ DMSNs ethanol solution (40 mg), 80mg of methoxy-polyethylene glycol-silane was added dropwise, and after stirring in a water bath at 60 ℃ for 24 hours, the mixture was centrifuged. Then washing with ethanol and water for 2 times respectively to obtain PEG modified CuS-UCNPs @ DMSNs (marked as CUD). Wherein, the PEG content in the PEG-modified CuS-UCNPs @ DMSNs is 40-60 wt%.
Example 3
746mg CTAB was first dissolved in 12mL of aqueous solution, after ultrasonic dispersion, a certain amount of UCNPs (0.128g, naYF) was added to the above solution 4 Er/Yb), emulsified for 10 minutes using a cell disruptor, the above solution was put into a 20mL glass bottle, and 10mg of TEA and 150mg of NaSal were added with stirring. The solution was heated to 80 ℃ in a water bath, and after stirring for 1 hour, 1.2mL of TEOS (1.12 g) was added to the solution, and the reaction was continued for 2 hours while keeping stirring. Subsequently, 10mL of absolute ethanol was added, followed by centrifugation. And repeatedly washing with water and ethanol for 4-5 times, and extracting with 1% NaCl methanol solution to remove CTAB as structure directing agent to obtain UCNPs @ DMSNs (UD) with small pore diameter. The content of DMSNs in the obtained UCNPs @ DMSNs is about 60wt% -70 wt%.
Comparative example 1
300mg of DMSNs were first dissolved in 30mL of ethanol and 150. Mu.L of MPTS and 750. Mu.L of NH were added to the above solution 3 ·H 2 O, then stirred vigorously at room temperature for 12h. The final product DMSNs-SH was washed 3 times with ethanol and then dispersed in chloroform. 0.2mL of a CuS solution (concentration of 0.3 mmoL/mL) was dropwise added to 8mL of a DMSNs-SH (2 mg/mL) solution in chloroform, and after stirring at room temperature for 30 minutes, the precipitated product was centrifuged. And then washing the solution for 2 times by chloroform and ethanol respectively to obtain the CuS-DMSNs. To 100mL of an ethanol solution (40 mg) of CuS-DMSNs, 80mg of methoxy-polyethylene glycol-silane was added dropwise, and after stirring in a water bath at 60 ℃ for 24 hours, the mixture was centrifuged. Then washed with ethanol and water 2 times, respectively, to obtain PEG-modified CuS-DMSNs (denoted as CD).
FIG. 1 is a TEM (a-c) and SEM (d-f) electron micrograph of UCNPs @ DMSNs prepared in example 1. As can be seen from the figure, the synthesized UCNPs @ DMSNs have uniform size, good dispersibility, average size of about 100nm and obvious dendritic structure. The silicon dioxide with dendritic structure (dendritic silicon for short) successfully coats the UCNPs.
FIG. 2 shows TEM (a-b), STEM dark field image (c) and element plane scan image (d) of CuS-UCNPs @ DMSNs nanoenzyme prepared in example 1. FIG. 3 is the EDS map (b) corresponding to the element line scan and the element line scan of CuS-UCNPs @ DMSNs nanoenzyme prepared in example 1. As can be seen from the figure, the small-sized CuS nanoparticles are successfully loaded in the channels of UCNPs @ DMSNs, and no obvious agglomeration occurs.
FIG. 4 is the UV-Vis curve of CuS-UCNPs @ DMSNs nanoenzyme prepared in example 1 in TMB aqueous solution; FIG. 5 shows the UV-Vis absorption value of CuS-UCNPs @ DMSNs nanoenzyme prepared in example 1 at 652nm in TMB aqueous solution; figure 6 is a digital photograph of TMB in water solution for different samples. From the figure, it can be seen that when CuS-UCNPs @ DMSNs and H are simultaneously present in TMB solution 2 O 2 The solution did not change color substantially indicating no ROS generation. When the solution was irradiated with a 980nm laser, the solution slowly turned blue. These results show that when 980nm laser is applied, the 980nm laser can excite UCNPs in the CuS-UCNPs @ DMSNs core to generate ultraviolet light, and the ultraviolet light can excite CuS to start photocatalytic reaction to catalyze H 2 O 2 Generating ROS. In the present invention, the Laser light is generated by a Laser (Laser).
FIG. 7 shows CuS-UCNPs @ DMSNs nanoenzyme prepared in example 1 in different concentrations of aqueous solutions (power density = 1W/cm) 2 ) Photothermal curve (a), photothermal curve (b) at different power densities (concentration of 100 ppm), photothermal stability profile (power density = 1W/cm) 2 Aqueous solution concentration 100 ppm) (c), different concentrations (power density = 1W/cm) 2 ) Photothermal curves (d) of CuS-DMSNs. As can be seen from FIG. 7, cuS-UCNPs @ DMSNs have excellent photo-thermal properties under 980nm laser irradiation. In the invention, an infrared thermal imager tests the photo-thermal stability and the like.
FIG. 8 shows the results of cell-level treatment with CuS-UCNPs @ DMSNs nanoenzyme prepared in example 1, wherein PBS is PBS buffer, only Laser is used for illumination, UDL is "UD + Laser", CDL is "CD + Laser", and CUDL is "CUD + Laser". Wherein the laser wavelength is 980nm, and the irradiation time is 5min. As can be seen from FIG. 8, cuS-UCNPs @ DMSNs can significantly inhibit the growth of cancer cells HCT116 under 980nm laser illumination.
FIG. 9 is photo-thermal imaging picture of HCT116 tumor-bearing mouse during 980nm laser irradiation as a result of in vivo layer treatment of CuS-UCNPs @ DMSNs nanoenzyme prepared in example 1. FIG. 10 is the temperature change curve of the mouse tumor during the 980nm near infrared laser irradiation, as the result of the in vivo layer treatment of CuS-UCNPs @ DMSNs nanoenzyme prepared in example 1. FIG. 11 is the tumor volume curve of different treatment groups, which shows the results of in vivo layer treatment of CuS-UCNPs @ DMSNs nanoenzyme prepared in example 1. The in vivo treatment experiment comprises the following specific steps: first, nude mice were inoculated subcutaneously with colon cancer cells, and tumors were finally formed. The mice with subcutaneous tumors were then randomly divided into 8 groups: (1) PBS; (2) 980nm laser; (3) CUD; (4) a CUDI; (5) UD +980nm laser (NIR); (6) CD +980nm laser; (7) CUD +980nm laser; (8) a CUDI +980nm laser. Firstly, injecting various materials or PBS into different groups of mice through tail veins respectively, irradiating 980nm laser on the 5 th to 8 th groups of mice for 4 hours, and recording the temperature rise condition of the tumor of the mice by a thermal imaging instrument. The mice continued to be normally maintained for 16 days, wherein the length and width of the mouse tumor and the mouse body weight were measured with a vernier caliper every two days. Tumor volume in mice was calculated using the following formula: tumor volume = tumor width 2 X tumor length 2. As can be seen from the figure, cuS-UCNPs @ DMSNs realizes PTT/PCT synergistic treatment and has better inhibition effect on the growth of tumors.
Finally, it should be noted that: although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that: modifications and equivalents may be made to the embodiments of the invention without departing from the spirit and scope of the invention, which is to be covered by the claims.

Claims (9)

1. A near-infrared light activated core-shell structure nanoenzyme is characterized by comprising: the up-conversion luminescent nano material is used as an inner core, the mercapto-modified dendritic structure silica formed on the surface of the inner core is used as an outer shell, and the nano CuS is distributed on the surface of the mercapto-modified dendritic structure silica and in a pore channel;
the preparation method of the near infrared light activated core-shell structure nano enzyme comprises the following steps:
(1) Adding the up-conversion luminescent nano material into an aqueous solution of a surfactant, and performing ultrasonic emulsification to obtain emulsion A; the mass ratio of the up-conversion luminescent nano material to the surfactant is 1 (5-25); the surfactant is selected from at least one of cetyl trimethyl ammonium bromide CTAB and cetyl trimethyl ammonium chloride CTAC;
(2) Adding sodium salicylate and organic micromolecular amine into the emulsion A, adding a silicon source, and performing water bath heating, centrifugal treatment and washing treatment to obtain core-shell structure nanoparticles of the dendritic structure silicon dioxide coated up-conversion luminescent nano material, wherein the core-shell structure nanoparticles are marked as UCNPs @ DMSNs; the dosage of the silicon source and the surfactant is 6-25 mu L:10 mg; the mass ratio of the sodium salicylate to the surfactant is 10: (1-5); the mass ratio of the organic micromolecule amine to the sodium salicylate is 1: (5-50); the organic small molecular amine is triethanolamine TEA;
(3) Dispersing the obtained UCNPs @ DMSNs in an organic solvent, adding 3-mercaptopropyl trimethoxy silane MPTS and ammonia water, reacting for 10-24 hours under magnetic stirring, and then carrying out centrifugal treatment on the mixture to obtain mercapto-modified UCNPs @ DMSNs;
(4) Dispersing the obtained mercapto-modified UCNPs @ DMSNs in trichloromethane, adding CuS nano particles, reacting for 1-6 hours under stirring, and then carrying out centrifugal treatment to obtain the near-infrared light activated core-shell structure nano enzyme.
2. The core-shell structured nanoenzyme of claim 1, wherein the upconversion luminescent nanomaterial is selected from NaYF 4 :Er/Yb、NaYF 4 :Yb/Er/Tm、NaYF 4 :Yb/Gd/Er、NaYF 4 :Yb/Tm、NaYF 4 :Yb/Tm@NaGdF 4 And NaGdF 4 At least one of Yb/Er; the particle size of the up-conversion luminescent nano material is 10 nm-100 nm.
3. The core-shell structure nanoenzyme according to claim 1, wherein the mercapto-modified dendritic structure silica has a particle size of 50nm to 200nm and a pore diameter of 5 nm to 20nm; the content of the mercapto-modified dendritic structure silica is 40-80 wt%.
4. The core-shell structure nanoenzyme according to claim 1, wherein the particle size of the nano CuS is 1nm to 20nm; the load capacity of the nano CuS is 5-40 wt%.
5. The core-shell structure nanoenzyme according to claim 1, wherein the particle size of the core-shell structure nanoenzyme is 50 to 300nm.
6. The nucleocapsid structured nanoenzyme of claim 1, wherein the silicon source is selected from at least one of tetraethoxysilane TEOS, tetraethoxysilane TMOS, 3-aminopropyltriethoxysilane APTES and 1, 2-bistrimethoxysilyl ethane BTME; the temperature of the water bath heating is 70-95 ℃, and the time is 2-6 hours.
7. The core-shell structured nanoenzyme according to claim 1, wherein the organic solvent is at least one selected from the group consisting of ethanol, chloroform, DMSO, and cyclohexane; the rotating speed of the magnetons for magnetic stirring is 300-800 revolutions per minute.
8. The nucleocapsid structured nanoenzyme of any one of claims 1-7, wherein the ratio of the amount of thiol-modified UCNPs @ DMSNs to the amount of CuS nanoparticles is 8mg: 5-100 mu moL.
9. A PEG-modified near-infrared light activated core-shell structure nanoenzyme is characterized in that methoxy-polyethylene glycol-silane is added into a solution containing the near-infrared light activated core-shell structure nanoenzyme of any one of claims 1 to 8, the solution is heated in a water bath at 40-80 ℃ for 6-24 hours, and then the PEG-modified near-infrared light activated core-shell structure nanoenzyme is obtained through centrifugal treatment and washing.
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