Hollow hybrid nano drug-loaded silica spheres, preparation method and application thereof

Abstract

The present invention provides hollow hybrid nano-drug-loaded silica spheres, a preparation method thereof, and an application thereof. The preparation method comprises: (1) dissolving a biological enzyme in water to form an aqueous phase, and adding the aqueous phase to an oil phase to form an oil-in-water microemulsion; (2) adding a silica precursor, a disulfide-bonded bridging organosilicon precursor, and a silane coupling agent to the microemulsion, and then adding an alkaline solution to react to form nano-drug-loaded silica spheres; (3) dispersing the nano-drug-loaded silica spheres in water to obtain hollow nano-drug-loaded silica spheres; (4) reacting the hollow nano-drug-loaded silica spheres with a copper-based precursor to obtain copper-based MOF-coated nano-drug-loaded silica spheres; and (5) loading the copper-based MOF-coated nano-drug-loaded silica spheres with a sonosensitizer to obtain hollow hybrid nano-drug-loaded silica spheres. The preparation method has a simple process and mild conditions. The prepared silica spheres can be completely degraded in tumor tissues, are chemically stable under other conditions, and have good targeting properties. The method is also conducive to the fixation and activity maintenance of enzymes, sonosensitizers, and metal ions.

Description

Technical Field

[0001] The present invention belongs to the field of drug carriers, and in particular relates to a hollow hybrid nano drug-loaded silicon sphere, a preparation method and application thereof. Background Art

[0002] Malignant tumors pose a serious threat to human health. Traditional cancer therapies, such as surgery, radiotherapy, and chemotherapy, can damage normal cells and produce varying degrees of toxic side effects. Therefore, targeted drug delivery and reduced drug toxicity are crucial to overcoming the limitations of cancer treatment. The development of nanomaterials has brought new technological innovations to the field of drug delivery. Nanocarriers possess unique properties, such as nanoscale size, high surface area, and excellent physicochemical properties, which can modulate the pharmacokinetic and pharmacodynamic profiles of drugs, enhancing therapeutic efficacy. Compared with systemic administration, drug delivery using nanocarriers can enhance the water solubility of lipid-soluble drugs, improving their in vivo stability and blood circulation time. Furthermore, specific properties within tumor tissue, such as low pH, high GSH content, and abnormally expressed proteins, can serve as endogenous stimuli to regulate drug release from nanomaterials. Furthermore, some emerging materials can simultaneously respond to exogenous stimuli, such as light, sound waves, and magnetic fields, to meet diverse needs.

[0003] Chemodynamic therapy (CDT) has attracted widespread attention in recent years. CDT is primarily based on the Fenton / Fenton-like reaction mediated by metal ions such as iron (II), copper (I), manganese (III), and cobalt (II), which generates hydroxyl radicals (·OH), which are toxic to tumor cells. Due to the high heterogeneity and complexity of solid tumor tissue, the use of CDT alone has limited anti-tumor effects. One important reason for this is the abnormally elevated levels of glutathione (GSH) in tumor tissue. GSH is a reducing substance that can scavenge the generated ·OH, thus limiting the efficacy of CDT.

[0004] Sonodynamic therapy (SDT) induces tumor cell apoptosis through appropriate ultrasound exposure and the reactive oxygen species generated by sonosensitizers under ultrasound conditions. It boasts advantages such as high accuracy, minimal side effects, and good patient compliance, making it a highly clinically transformative cancer therapy. However, existing porphyrin sonosensitizers have poor hydrophilicity, low bioavailability, and rapid clearance from the body, making them difficult to accumulate in tumors. Furthermore, sonosensitizers are easily quenched by the external environment, resulting in poor SDT efficacy.

[0005] In recent years, enzymatic-based cancer therapies have gained popularity. Unlike traditional treatments, enzymes are mostly naturally occurring proteins that catalyze specific reactions under mild conditions, exhibiting high activity, biocompatibility, and safety. However, enzymes are easily inactivated in the external environment, significantly reducing their catalytic efficiency and significantly limiting their application in cancer treatment. Furthermore, proteins used alone are easily phagocytosed by macrophages while circulating in the body, preventing them from effectively accumulating in tumor sites.

[0006] Solid tumor tissues are characterized by high heterogeneity and complexity. It is of great significance and value to study targeted drug delivery and combine various therapies to improve the tumor cell killing effect. Summary of the Invention

[0007] The present invention addresses the targeting defects of existing tumor drugs and combines enzymatic reaction / CDT / SDT to improve the killing effect of tumor cells. It provides a hybrid nano-drug-loaded silica sphere, its preparation method and application. The preparation method has a simple process, mild conditions, stable chemical properties, and is conducive to the fixation and activity maintenance of enzymes, sonosensitizers and metal ions.

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

[0009] The preparation method of hybrid nano drug-loaded silicon spheres comprises the following steps:

[0010] (1) A surfactant, a co-surfactant and an organic solvent are mixed to prepare an oil phase, a biological enzyme is dissolved in water to form an aqueous phase, and the aqueous phase is added to the oil phase to form a water-in-oil (W / O) reverse microemulsion; the surfactant can be selected from hexadecyltrimethylammonium bromide (CTAB), polyoxyethylene (5) nonylphenyl ether (CO-520), polyethylene glycol octylphenyl ether (Triton X-100), etc., the co-surfactant can be selected from n-propanol, n-butanol, n-hexanol, etc., and the organic solvent can be selected from n-decane, n-octane, cyclohexane, etc. The biological enzyme is one or more of catalase, superoxide dismutase, lactate oxidase and horseradish peroxidase, all of which have a killing effect on tumor cells.

[0011] (2) Adding a silica precursor, a disulfide bond-containing bridging organosilicon precursor, and an aminosilane coupling agent to the reverse microemulsion, and then adding an alkali solution to form solid nano-drug-loaded silica spheres.

[0012] Silica precursors are synthesized into porous silica through hydrolysis and polycondensation to form a silica shell framework. Orthosilicates or silane compounds can be used. Bridging organosilicon precursors containing disulfide bonds can form disulfide bonds within the silica sphere skeleton, which is stable in normal tissues. However, upon reaching the tumor, they undergo bond exchange reactions with overexpressed GSH in the tumor microenvironment, causing degradation of the outer layer of the nano-silica carrier. Examples include bis(trimethoxysilylpropyl) disulfide (BTEPDS), bis(triethoxysilylpropyl) disulfide (BTMPDS), and 3-triethoxysilylpropyl) tetrasulfide (BTES). Aminosilane coupling agents can provide particle amination reaction sites, greatly improve the rigidity and strength, and enhance the stability of silicon nanoparticles under normal physiological conditions. Optional: γ-aminopropyltriethoxysilane, γ-aminopropyltrimethoxysilane, N-β (aminoethyl) -γ-aminopropyltrimethoxysilane, N-β (aminoethyl) -γ-aminopropyldimethoxysilane, N-β (aminoethyl) -γ-aminopropyltriethoxysilane, phenylaminomethyltriethoxysilane, phenylaminomethyltrimethoxysilane, etc.

[0013] (3) The solid drug-loaded nano-silica spheres are dispersed in deionized water and stirred at 35-45°C for 0.5-1 hour. The mixture is cooled and centrifuged, and then washed with deionized water to obtain hollow drug-loaded nano-silica spheres, which are dispersed in water to form a hollow drug-loaded nano-silica sphere solution. The reaction mechanism is that the middle part of the silica spheres is silicate oligomers, and the degree of condensation is low in the center of the silica shell. This can be removed by washing with stirring and centrifuging, and the silica spheres become hollow.

[0014] (4) The hollow nano-drug-loaded silica sphere solution is mixed with an ethanolic solution of copper acetate [Cu(AcO)2·H2O] and ultrasonically treated for 10 to 30 minutes. After washing with water, the solution is resuspended in deionized water. An ethanolic solution of an organic ligand 1,3,5-benzenetricarboxylic acid (TMA) is added to the resuspended solution and the ultrasonic treatment is repeated. The above steps are repeated to obtain copper-based MOF-coated hollow nano-drug-loaded silica spheres. The copper-based precursor can be selected from copper acetate monohydrate, copper sulfate pentahydrate, copper nitrate trihydrate, copper chloride dihydrate, etc., and the organic ligand can be selected from 1,3,5-benzenetricarboxylic acid (TMA).

[0015] (5) The copper-based MOF-coated hollow nano-drug-loaded silica spheres and the sonosensitizer are mixed and dispersed in an ethanol solvent, and the mixture is stirred thoroughly to obtain hollow hybrid nano-drug-loaded particles. The sonosensitizer can be selected from dihydrochlorin e6 (Ce6), hematoporphyrin monomethyl ether (HMME), protoporphyrin IX (PPIX), meso-tetra-(4-carboxyphenyl)porphyrin (TCPP), etc.

[0016] As a further improvement to the technical solution, in step (1), the surfactant is CO-520, the co-surfactant is n-hexanol, and the organic solvent is n-decane; the volume ratio of the surfactant, co-surfactant, and organic solvent in step (1) is 2-4:1:30-40. CO-520 is an alkylphenol polyoxyethylene ether, a nonionic surfactant with high surface activity, good chemical stability, and good biodegradability. n-hexanol is a fat-soluble alcohol used as a co-surfactant to increase the polarity of the oil, has low solubility in water, low toxicity, and good solvency. n-Decane has a high boiling point and good stability in air.

[0017] As a further improvement to the technical solution, the reaction temperature in step (2) is 10-30°C and the reaction time is 6-12 hours. The alkali solution in step (2) is 1-3% (v / v) ammonia water. Ammonia water, as a catalyst, is a weak base and is not easy to destroy the structure of other components in the system. It can also provide OH-, promoting the hydrolysis and condensation of TEOS to form a porous silica shell. Ammonia water within this concentration range forms the thickest shell. Exceeding this concentration, the concentration of free OH- in the solution increases, and a portion of TEOS will hydrolyze and condense in the solution to produce free silica particles, making the obtained HSN shell thinner and also increasing the particle size of the generated solid silicon sphere particles.

[0018] As a further improvement of the technical solution, in step (2), the silica precursor is tetraethyl silicate, the bridging organosilicon precursor containing a disulfide bond is 3-triethoxysilylpropyl) tetrasulfide, and the silane coupling agent is 3-aminopropyltrimethoxysilane; the volume ratio of the bridging organosilicon precursor containing a disulfide bond to the silica precursor is 1 to 2:1 to 2, and the volume ratio of the silane coupling agent to the silica precursor is 1:20 to 25.

[0019] As a further improvement of the technical solution, in step (1), the mixture is mixed into a water-in-oil (W / O) reverse microemulsion under stirring in a water bath at 15-30°C.

[0020] As a further improvement of the technical solution, the mass ratio of the silicon spheres to the sonosensitizer in step (5) is 2:1, the mixing temperature of the silicon spheres and the sonosensitizer is 20-30°C, and the stirring speed is 300-400 rpm.

[0021] As a further improvement to the technical solution, the enzyme is glucose oxidase (GOx). GOx consumes glucose in tumor tissue to produce gluconic acid and H2O2. It also synergizes with copper (II), which scavenges GSH within tumor cells and then reacts with existing and newly generated H2O2 to produce highly cytotoxic ·OH. These two enzymes synergistically enhance the drug's toxicity to tumor cells.

[0022] As a further improvement of the technical solution, the copper-based precursor is copper acetate.

[0023] The invention also discloses hybrid nano drug-loaded silicon spheres prepared according to the preparation method.

[0024] An application of the hybrid nano-drug-loaded silica spheres. The hybrid nano-drug-loaded silica spheres have the characteristics of GSH response. In tumor tissues with high GSH concentrations, the silica sphere skeleton is programmed to degrade and release internal molecules, achieving efficient drug delivery. Therefore, the hybrid nano-drug-loaded silica spheres can be used to prepare drugs for treating solid tumors.

[0025] The present invention has outstanding substantive features and significant progress compared to the prior art. Specifically, the hollow hybrid nano-drug-loaded silica spheres prepared by the method of the present invention utilize the dual-responsiveness of the tumor microenvironment to deliver enzyme-related preparations. On the one hand, they can protect the high activity of the enzyme for a long time. On the other hand, when the nano-silicon spheres circulate to the tumor site, the endogenous stimulation of the tumor microenvironment causes the silica spheres to lyse and release the enzyme and the sonosensitizer they carry, achieving the effect of starvation therapy / CDT / SDT synergistic therapy based on enzymatic reactions. Furthermore, the hollow hybrid nano-drug-loaded silica spheres prepared by the method of the present invention have the advantages of uniform particle size, regular shape and good dispersibility. Furthermore, the hollow hybrid nano-drug-loaded silica spheres prepared by the method of the present invention have a high enzyme loading capacity and can maintain high enzyme activity and efficient and controllable release of biological enzymes. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Transmission electron microscope images of drug-loaded silicon spheres in different states.

[0027] Figure 2 This is the particle size distribution diagram of GOx@HSN@HKUST-1-TCPP.

[0028] Figure 3 Energy spectrum of GOx@HSN@HKUST-1.

[0029] Figure 4 UV spectrum of GOx@HSN containing CY7.

[0030] Figure 5 This is the UV spectrum of GOx@HSN@HKUST-1-TCPP. Figure 6 This is a graph showing the change of glucose consumption by GOx@HSN@HKUST-1 over time.

[0031] Figure 7 This is a real-time pH change chart.

[0032] Figure 8 This is a diagram of the generation of H2O2 during the reaction of GOx and glucose.

[0033] Figure 9 This is the cumulative release curve of Si.

[0034] Figure 10 Transmission electron microscopy image of the degradation process of GOx@HSN@HKUST-1.

[0035] Figure 11 Cu 2+ Graph showing the change in DTNB absorption wavelength during the induced GSH consumption process.

[0036] Figure 12 Figure 2 is the TMB absorption wavelength diagram at different GOx@HSN@HKUST-1 concentrations.

[0037] Figure 13 For the generated 1 Absorbance changes at 400nm as O2 increases.

[0038] Figure 14 This is the result diagram of the hydrated particle size stability of GOx@HSN@HKUST-1-TCPP.

[0039] Figure 15 This is the distribution diagram of GOx@HSN@HKUST-1-TCPP in tumor-bearing mice. DETAILED DESCRIPTION

[0040] The technical solution of the present invention is further described in detail below through specific implementation methods.

[0041] Experimental methods in the examples of this disclosure that do not specify specific conditions are generally based on conventional conditions or the conditions recommended by the raw material or product manufacturers. Reagents without specific sources are conventional reagents purchased from the market.

[0042] Example 1: (1) Synthesis of GOx@HSN

[0043] 1) Place 20 ml of n-decane in a 20°C constant temperature water bath, then add 1.63 ml of polyoxyethylene (5) nonylphenyl ether (CO-520) dropwise, and stir at 20°C / 200 rpm for 30 min. Then, add 550 μL of n-hexanol and 500 μL of an 8 mg / ml GOx aqueous solution dropwise, followed by stirring at 20°C / 200 rpm for 30 min to prepare a reverse microemulsion.

[0044] 2) 100 μL of bis-(γ-triethoxysilylpropyl) tetrasulfide (BTES) and 100 μL of tetraethyl silicate (TEOS) were mixed and added to the reverse microemulsion. Then, 25 μL of 3-aminopropyltrimethoxysilane ethanol solution (APTMS ethanol solution was prepared by adding 200 μL of APTMS to 1.4 ml of anhydrous ethanol) was added and stirred at 200 rpm for 10 minutes at a constant temperature of 20°C. Finally, 250 μL of 28 wt% ammonia water was added and the reaction was stirred at 200 rpm for 8 hours at a constant temperature of 20°C. Then, 3 ml of ethanol was added and the mixture was centrifuged at 13,000 rpm for 20 minutes to obtain solid nano-silicon spheres loaded with GOx. The transmission electron microscope image is shown in FIG. Figure 1 Middle a.

[0045] 3) The solid nanoparticles loaded with drug from step 2) were resuspended in 70 ml of deionized water, stirred at 400 rpm in a 40°C water bath for 50 min, and then centrifuged at 13,000 rpm for 20 min. The product was washed once and centrifuged to obtain 28 mg of hollow nanoparticles loaded with GOx, GOx@HSN. The transmission electron microscope image is shown in FIG. Figure 1 Middle b.

[0046] (2) Synthesis of GOx@HSN@HKUST-1-TCPP

[0047] 4) Resuspend 15 mg of GOx@HSN in 8 ml of deionized water to form a GOx@HSN resuspension. Mix the GOx@HSN resuspension with 2 ml of 15 mM copper acetate [Cu(AcO)2·H2O] ethanol solution and sonicate at 300 W for 20 min. Centrifuge at 13,000 rpm for 15 min, rinse the centrifuged product with water, and resuspend it in 8 ml of deionized water. Add 2 ml of 10 mM 1,3,5-benzenetricarboxylic acid (TMA) ethanol solution to this resuspension and sonicate at 300 W for 25 min. Centrifuge at 13,000 rpm for 15 min, rinse with water, and resuspend it in 8 ml of deionized water. Repeat the above steps twice to obtain copper-based MOF-coated hollow nano-drug-loaded silica spheres.

[0048] GOx@HSN@HKUST-1 12mg.

[0049] 5) The copper-based MOF-coated nano-drug-loaded silica sphere solution (2 mg / mL) was mixed and dispersed with a 1 mg / ml sonosensitizer TCPP ethanol solution, and stirred at 400 rpm for 24 h to obtain hollow hybrid nano-drug-loaded particles. The transmission electron microscope image is shown in FIG. Figure 1 Middle c.

[0050] 1. Experimental results are as follows Figure 1 and Figure 2The results clearly demonstrate that the hollow hybrid nano-drug-loaded silica spheres of the present invention possess a core-shell structure. Compared to solid silica spheres, the interior of the spheres is hollow, differing from the solid outer layer in transparency. Transmission electron microscopy images clearly reveal the presence of cavities. Scanning electron microscopy results and particle size distribution plots indicate that the prepared silica spheres exhibit uniform particle size, regular shape, and good dispersion. Furthermore, after loading the copper-based MOF and sonosensitizer, their morphology is well maintained. The hydrated particle size distribution ranges from 40 to 70 nm. Figure 3 The energy spectrum of GOx@HSN@HKUST-1 scanned by electron microscope and the energy spectrum mapping diagram prove that the silicon spheres are loaded with metal ions and contain S inside the silicon spheres.

[0051] 2. Observe the elemental distribution of the nanomaterial using an electron microscope, comprising the following steps: resuspend 100 μg of the nanomaterial GOx@HSN@HKUST-1 described in Example 1 in 1 mL of anhydrous ethanol, drop the suspension onto a molybdenum mesh, and observe and photograph the distribution of the main elements using an electron microscope.

[0052] The experimental results are as follows Figure 3 It shows that Cu and S are uniformly distributed in the nanomaterial GOx@HSN@HKUST-1.

[0053] 3. Detecting the loading amount of the biological enzyme in the nanomaterial using a fluorescent labeling method, including the following steps:

[0054] 1) Add 1 mg of CY7 to 1.0 ml of an aqueous solution containing 2 mg of GOx. Stir for 24 h and then dialyze using a 3.5 kD dialysis bag to remove free dye to obtain a GOx-CY7 solution.

[0055] 2) Concentrate the GOx-CY7 solution to about 500 μL using a 10 kd ultrafiltration tube;

[0056] 3) In the synthesis step (1) of GOx@HSN, the GOx aqueous solution was changed to a GOx-CY7 enzyme solution, and the other steps remained unchanged to obtain fluorescently labeled hollow nano-drug-loaded silica spheres GOx-CY7@HSN.

[0057] 4) In the synthesis step (1) of GOx@HSN, the GOx aqueous solution was changed to an equal volume of water, and the other steps remained unchanged, thereby obtaining fluorescently labeled hollow nano-drug-loaded silica spheres HSN without encapsulating biological enzymes.

[0058] 5) Measure the UV-visible spectra of GOx@HSN and HSN solutions of the same concentration from 500 nm to 800 nm, calculate the absorbance difference at 748 nm, and calculate the fluorescent dye concentration in GOx-CY7@HSN based on the CY7 standard curve to estimate the corresponding drug loading capacity and loading efficiency.

[0059] The experimental results are as follows Figure 4 As shown in the figure, it is proved that the silica spheres are loaded with GOx. 1mgGOx@HSN is loaded with 56μg GOx, the GOx encapsulation efficiency is 45%, and the silica sphere drug loading rate is 5.6%.

[0060] 4. Using UV quantitative analysis to analyze the loading of TCPP in the nanomaterial, including the following steps:

[0061] 1) Take the nanomaterials GOx@HSN@HKUST-1 and GOx@HSN@HKUST-1-TCPP in Example 1

[0062] Aqueous solutions of GOx@HSN@HKUST-1-TCPP and GOx@HSN@HKUST-1 with the same concentration were prepared, and the UV-visible spectra were detected from 300 nm to 800 nm. The absorbance difference at 416 nm was calculated, and the TCPP concentration in GOx@HSN@HKUST-1-TCPP was calculated based on the TCPP standard curve, and the corresponding TCPP loading capacity and loading rate were deduced.

[0063] The experimental results are as follows Figure 5 As shown in the figure, it is proved that TCPP is loaded on the silica sphere. 1mg GOx@HSN@HKUST-1-TCPP loaded with 106.08μg TCPP, the encapsulation efficiency of TCPP is 21.22%, and the loading efficiency of GOx@HSN@HKUST-1 is 10.61%.

[0064] Example 2

[0065] The use of DNS reagent to detect GOx activity in nanomaterials includes the following steps:

[0066] 1) Resuspending the GOx@HSN@HKUST-1 prepared in Example 1 in a PBS buffer solution at pH 5.5;

[0067] 2) Add the above materials to a glucose solution at pH 5.5, with the final concentration of GOx@HSN@HKUST-1 and the glucose solution both at 1 mg / ml, and place in a 37°C incubator;

[0068] 3) Take 100 μL of the reaction supernatant at 0.5 h, 1 h, 2 h, 4 h, 6 h, 12 h, 24 h, and 48 h, mix with 400 μL of DNS reagent, incubate in an oil bath at 100°C for 5 minutes, and then in an ice bath for 20 minutes.

[0069] 4) Then add 1 ml of PBS buffer (pH = 5.5) respectively, mix well, and measure the absorbance at 514 nm.

[0070] The experimental results are as follows Figure 6As shown in the figure, in the system containing GOx@HSN@HKUST-1, the glucose consumption rate is extremely high, with about 50% consumed within 4 h.

[0071] Example 3

[0072] Using a pH meter to detect changes in pH in the system includes the following steps:

[0073] 1) The nanomaterial GOx@HSN@HKUST-1 prepared in Example 1 was added to a prepared 1 mg / ml glucose solution to a concentration of 50 μg / ml.

[0074] 2) Use a pH meter to measure real-time pH changes.

[0075] The experimental results are as follows Figure 7 As shown in the figure, in the system containing GOx@HSN@HKUST-1, the generated gluconic acid causes the pH of the system to drop rapidly, and the pH value drops from 7.4 to 4.8 within 5 h.

[0076] Example 4

[0077] 3,3',5,5'-tetramethylbenzidine (TMB) was used to detect the generation of H2O2 during the reaction of GOx and glucose, including the following steps:

[0078] 1) The nanomaterial GOx@HSN@HKUST-1 in Example 1 was resuspended in a PBS buffer solution at pH 5.5;

[0079] 2) Three portions of the above materials were added to a glucose solution at pH 5.5 to obtain three experimental groups with a final glucose concentration of 1 mg / ml and GOx@HSN@HKUST-1 concentrations of 10, 20, and 50 μg / ml, respectively. The mixture was then placed in a 37°C incubator.

[0080] 3) 100 μL of the supernatant from the three experimental groups (1, 3, 5, 10, 20, 30, 40, 50, and 60 minutes) was mixed with 1.5 mL of PBS (pH 5.5) and 200 μL of horseradish peroxidase (HRP, 1 U / mL) and incubated at room temperature for 10 min.

[0081] 4) Add 200 μL of 1 mM TMB solution and measure the absorbance at 652 nm using UV light after 5 minutes.

[0082] The experimental results are as follows Figure 8 As shown, adding 50 μg / ml GOx@HSN@HKUST-1 to the system can make the H2O2 concentration in the solution reach 0.9 mM within 1 h.

[0083] The experimental phenomena and data of Examples 2-4 show that the GOx@HSN@HKUST-1 provided by the present invention has high stability, can maintain the catalytic activity of the biological enzyme loaded inside the silica sphere to a great extent, and can quickly catalyze the decomposition of glucose.

[0084] Example 5

[0085] This embodiment uses an inductively coupled plasma optical emission spectrometer to detect the degradation of nanomaterials, including the following steps:

[0086] 1) 2 mg of the nanomaterial GOx@HSN@HKUST-1 from Example 1 was resuspended in 2 ml of a 10 mM glutathione solution at pH 5.5, transferred to a dialysis bag with a molecular weight cutoff of 3.5 kD, and then placed in a centrifuge tube containing 18 ml of a glutathione solution at pH 5.5;

[0087] 2) The Si content in the system was detected by inductively coupled plasma optical emission spectrometry at time points of 6, 12, 24, 48, 72, 96, and 168 hours, and the cumulative release was calculated and a cumulative release curve was drawn.

[0088] The experimental results are as follows Figure 9 As shown in the figure, GOx@HSN@HKUST-1 was completely degraded in a system simulating the tumor microenvironment, and the Si release rate reached 80% within 168 h.

[0089] Example 6

[0090] The degradation of nanomaterials was observed using a transmission electron microscope, which included the following steps:

[0091] 100 μg of the nanomaterial GOx@HSN@HKUST-1 in Example 1 was resuspended in 2 ml of glutathione solution (10 mM) at pH 5.5. Points were taken at 6, 12, 24, 48, 72, 96, and 168 hours, and dropped onto a copper grid. The degradation was observed and photographed using a transmission electron microscope.

[0092] The experimental results are as follows Figure 10 As shown in the figure, GOx@HSN@HKUST-1 gradually breaks down in a system simulating the tumor microenvironment and is eventually completely degraded, demonstrating that this drug delivery system has good biosafety.

[0093] Example 7

[0094] Detection of Cu using 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) 2+ The induced GSH consumption includes the following steps:

[0095] 1) The nanomaterial GOx@HSN@HKUST-1 prepared in Example 1 was resuspended in a PBS buffer solution at pH 5.5 to a final concentration of 500 μg / ml and incubated at 37°C for 12 h;

[0096] 2) adding the suspension obtained in step 1) to the GSH solution to make the final GSH concentration 1 mM;

[0097] 3) 100 μL of the reaction supernatant after 4 h, 8 h, and 12 h was mixed with 897 μL of PBS buffer solution at pH 7.4, and then 3 μL of DTNB solution (DTNB dissolved in DMSO at a concentration of 10 mg / ml) was added. The mixture was mixed and the absorbance was measured using a UV spectrophotometer in the wavelength range of 270-550 nm.

[0098] The experimental results are as follows Figure 11 As shown, DTNB alone has a measurable absorbance peak at approximately 320 nm, but it can react with GSH to produce a yellow derivative, 5'-thio-2-nitrobenzoic acid (TNB), which has a measurable absorbance peak at approximately 412 nm. Initially, the mixed solution of DTNB and GSH shows a clear TNB absorbance peak at 412 nm, corresponding to the initial GSH level.

[0099] After the addition of GOx@HSN@HKUST-1, the absorbance peak at 412 nm decreased significantly with time, and the absorbance peak of DTNB at 320 nm increased, indicating that the Cu released by GOx@HSN@HKUST-1 2+ Induced GSH consumption and decreased GSH concentration.

[0100] Example 8

[0101] The generation of ·OH was detected using 3,3',5,5'-tetramethylbenzidine (TMB), comprising the following steps:

[0102] 1) The nanomaterial GOx@HSN@HKUST-1 prepared in Example 1 was resuspended in a 10 mM GSH solution at pH 5.5 to give experimental groups with final concentrations of 0, 50, 100, and 200 μg / ml, respectively, and incubated at 37°C for 12 h;

[0103] 2) Take 100 μL of the reaction supernatant from different experimental groups and mix it with 100 μL of 50 mM H2O2 and 740 μL of pH = 7.4 PBS buffer solution, and react at room temperature for 4 hours;

[0104] 3) Add 60 μL of 1 mM 3,3',5,5'-tetramethylbenzidine (TMB) ethanol solution to the solutions of the different experimental groups prepared in 2), incubate at room temperature for 20 min, and measure the absorbance using a UV spectrophotometer in the wavelength range of 500–800 nm.

[0105] After reacting with GSH, Cu 2+ Can be reduced to Cu + , Cu + It can react with H2O2 to generate toxic ·OH. After ·OH oxidizes TMB, the mixed solution changes from colorless to blue-green, and an absorbance peak appears at 652nm. The experimental results are as follows Figure 12 As shown, the absorbance of TMB at 652 nm increased with the concentration of GOx@HSN@HKUST-1.

[0106] Example 9

[0107] Detection with 9,10-anthryl-bis(methylene)dimalonic acid (ABDA) 1 The generation of O2 includes the following steps:

[0108] 1) 100 μg of the nanomaterial GOx@HSN@HKUST-1-TCPP prepared in Example 1 was resuspended in 900 μL of H2O;

[0109] 2) Add 100 μL of ABDA solution (100 μg / ml) to the solution prepared in 1), mix well, and measure the absorbance using a UV spectrophotometer in the wavelength range of 320–420 nm.

[0110] 3) Ultraviolet absorption spectra were measured after ultrasound for 1 min, 5 min, 10 min, and 15 min, respectively. The ultrasound power was 2 W / cm 2 , the ultrasonic frequency is 1 MHz and the duty cycle is 50%.

[0111] ABDA and 1 O2 reacts to form the corresponding endoperoxide, which is detected by the decrease in absorbance at 400nm. Figure 13 As shown in the figure, the concentration of ABDA at 400 nm decreases with the increase of ultrasound duration, indicating that the generated 1 O2 increases.

[0112] Example 10

[0113] The stability of nanomaterials is tested using a dynamic scattering nanoparticle size analyzer, including the following steps:

[0114] 1) 1 mg of GOx@HSN@HKUST-1-TCPP was dispersed in 5 mL of PBS (pH 7.4) and RMPI 1640 complete medium containing 10% fetal bovine serum, respectively, and incubated at 37°C. The size of GOx@HSN@HKUST-1-TCPP was measured at different time points (0, 6, 12, 24, 48, 72, and 168 hours) using a dynamic scattering nanoparticle size analyzer.

[0115] The experimental results are as follows Figure 14 As shown in Figure 3, GOx@HSN@HKUST-1-TCPP maintained good stability under simulated physiological conditions (non-tumor microenvironment).

[0116] Example 11

[0117] The biodistribution of nanomaterials using a small animal in vivo imaging system includes the following steps:

[0118] 1) Take 5 million 4T1 cells in the logarithmic growth phase and inoculate them subcutaneously at the thigh root of three 5-6 week old balb / c female mice. After 7-10 days, they grow to 60-100 mm. 3 Subcutaneous tumors. 100 μL of 12 mg / ml GOx@HSN@HKUST-1-TCPP (the dosage for mice after TCPP dissociates from the silica spheres is approximately 5 mg / kg) was injected into tumor-bearing mice via the tail vein. Fluorescence imaging of the mice was performed at 0, 1, 2, 4, 8, 12, 24, 48, and 72 hours (fluorescence instrument Ex = 660 nm, Em = 710 nm).

[0119] 2) After 72 hours, the mice were sacrificed, and the tumors and major organs were removed and imaged (fluorescence instrument Ex = 660 nm, Em = 710 nm).

[0120] The experimental results are as follows Figure 15 As shown, Figure 15 Figure a shows the distribution of GOx@HSN@HKUST-1-TCPP in mice, and the circle in the figure is the mouse tumor. Figure 15 Figure b is the average fluorescence intensity of GOx@HSN@HKUST-1-TCPP distributed in mouse tumors. The fluorescence first increases and then decreases, indicating that GOx@HSN@HKUST-1-TCPP gradually accumulates and is eventually metabolized in mouse tumors. The metabolic trend is good and it has good biosafety. Figure 15 Figure c shows the distribution of GOx@HSN@HKUST-1-TCPP in isolated mouse organs, which is mainly distributed in the mouse tumor site, indicating that the drug-loaded material has good aggregation effect and biosafety at the tumor site.

[0121] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to preferred embodiments, ordinary technicians in the field should understand that the specific implementation methods of the present invention can still be modified or some technical features can be replaced by equivalents without departing from the spirit of the technical solutions of the present invention. They should all be included in the scope of the technical solutions for which protection is sought.

Claims

1. A method for preparing hollow hybrid nano-drug-loaded silica spheres, comprising the following steps: (1) A surfactant, a co-surfactant and an organic solvent are mixed to prepare an oil phase, a biological enzyme is dissolved in water to form an aqueous phase, and the aqueous phase is added to the oil phase to form a water-in-oil (W / O) reverse microemulsion; (2) adding a silica precursor, a disulfide-bonded bridging organosilicon precursor, and a silane coupling agent to the reverse microemulsion, and then adding solid nano-drug-loaded silica spheres formed by the reaction of alkaline solution; (3) dispersing the solid nano drug-loaded silica spheres in deionized water, stirring and reacting at 40-60° C. for 0.5-1 hour, cooling to room temperature, centrifuging, and then washing with deionized water to obtain hollow nano drug-loaded silica spheres, which were dispersed in water to form a hollow nano drug-loaded silica sphere solution; (4) Mixing the hollow nano-drug-loaded silica sphere solution with the copper-based precursor solution and dispersing them by ultrasonic treatment; The solution was then washed with water and resuspended in deionized water. An organic ligand was added to the resuspended solution and ultrasonicated to obtain copper-based MOF-coated hollow nano-drug-loaded silica spheres. (5) The copper-based MOF-coated nano-drug-loaded silica spheres and the sonosensitizer are mixed and dispersed in an ethanol solvent, and the hollow hybrid nano-drug-loaded silica spheres are obtained after thorough stirring and mixing; The biological enzyme is glucose oxidase; The organic ligand is 1,3,5-benzenetricarboxylic acid.

2. The preparation method according to claim 1, characterized in that In step (1), the surfactant is CO-520, the cosurfactant is n-hexanol, the organic solvent is n-decane, and the volume ratio of the surfactant, the cosurfactant and the organic solvent is 2-4:1:30-40. The mixture is stirred in a water bath at 15-30°C to form a water-in-oil (W / O) reverse microemulsion.

3. The preparation method according to claim 2, characterized in that In step (2), the reaction temperature is 10-30° C., and the alkali solution is 1-3% v / v ammonia water.

4. The preparation method according to claim 1, characterized in that In step (2), the silica precursor is tetraethyl silicate, the disulfide-containing bridging organosilicon precursor is 3-(triethoxysilylpropyl) tetrasulfide, and the silane coupling agent is 3-aminopropyltrimethoxysilane; the volume ratio of the disulfide-containing bridging organosilicon precursor to the silica precursor is 1-2:1-2, and the volume ratio of the silane coupling agent to the silica precursor is 1:20-25.

5. The preparation method according to claim 1, characterized in that The copper-based MOF-coated nano-drug-loaded silicon spheres obtained in step (4) are subjected to step (4) operation repeatedly several times and then to step (5) operation.

6. The preparation method according to claim 4, characterized in that In step (5), the mass ratio of the copper-based MOF-coated nano-drug-loaded silicon spheres to the sonosensitizer is 2:1, the mixing temperature of the MOF-coated nano-drug-loaded silicon spheres and the sonosensitizer is 20-30°C, and the stirring speed is 300-400 rpm.

7. The preparation method according to claim 1, characterized in that The copper-based precursor is copper acetate.

8. Hybrid nano drug-loaded silica spheres prepared according to the preparation method according to any one of claims 1 to 7.

9. The use of the hybrid nano drug-loaded silicon spheres according to claim 8, characterized in that: The hybrid nano drug-loaded silicon spheres are used for preparing drugs for treating solid tumors.

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