CN115475255A - Enzyme response type silicon dioxide release nano preparation, preparation method and application - Google Patents

Enzyme response type silicon dioxide release nano preparation, preparation method and application Download PDF

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CN115475255A
CN115475255A CN202210795969.7A CN202210795969A CN115475255A CN 115475255 A CN115475255 A CN 115475255A CN 202210795969 A CN202210795969 A CN 202210795969A CN 115475255 A CN115475255 A CN 115475255A
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enzyme
simvastatin
preparation
msn
mesoporous silica
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王晓琳
宋柯辰
肖翊
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Macau Univ of Science and Technology
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Abstract

The invention belongs to the technical field of pharmaceutical preparation application, and provides an enzyme-responsive silicon dioxide nano preparation, a preparation method and application thereof. The invention adopts simvastatin as a model drug, and then modifies polyethyleneimine and hyaluronic acid on the outer surface of mesoporous silica nanoparticles layer by layer to form an enzyme-responsive silica nano preparation. The preparation method comprises the following steps: 1) Synthesizing mesoporous silica nanoparticles under alkaline conditions; 2) Loading simvastatin into the pore channels of the nanoparticles; 3) Respectively coating polyethyleneimine and hyaluronic acid on the outer surface of the mesoporous silica nano-particles layer by layer to finally obtain the enzyme-responsive silica nano-preparation. The enzyme-responsive silicon dioxide nano preparation has the characteristics of uniform particle size, good dispersibility, high drug loading capacity and low toxicity, and can trigger an enzyme-responsive property to release a drug under a high-concentration enzyme environment at a focus part to improve the anti-atherosclerosis effect of the preparation.

Description

Enzyme response type silicon dioxide release nano preparation, preparation method and application
Technical Field
The invention relates to the technical field of pharmaceutical preparations, in particular to an enzyme-responsive silicon dioxide-releasing nano preparation, a preparation method and application thereof.
Background
Atherosclerosis (atheroclosis) is one of the leading causes of cardiovascular disease. Atherosclerosis is a chronic inflammatory disease, and is characterized in that lipid is retained in the wall of middle and large arteries, mononuclear cells are infiltrated to form plaques, and the plaques can finally cause fatal events such as myocardial infarction, ischemic stroke and the like along with the development of the disease. Conventional drug therapies including lipid-lowering drugs (e.g., statins, fibrates), platelet aggregation inhibitors, antihypertensive drugs, and antidiabetic drugs (e.g., thiazolidinediones) are generally low in efficacy and serious in side effects, and thus development of novel drug carriers is required for precise treatment of atherosclerosis. Thus, a variety of functionalized organic or inorganic nanoparticles are used to combat atherosclerosis.
Mesoporous Silica Nanoparticles (MSNs) have received much attention because of their highly ordered pore channels, large surface area, high pore volume, multiple surface functions and good biocompatibility. They can achieve high loading of therapeutic drugs on internal or external surfaces while releasing the drugs in a controlled manner. In addition, the release of the drug can be further manipulated by modifying the "switch" of the stimulus response, which can be removed under a particular environmental stimulus to release the encapsulated drug. In addition, the particle surface can be coupled with various targeting ligands to realize accurate drug delivery to target cells.
A large amount of hyaluronidase (HAase) exists in the atherosclerotic plaque part and the macrophage, and can trigger enzyme response characteristics aiming at the nano preparation modified by Hyaluronic Acid (HA), so that the leakage of a medicament from the inside of a pore canal is accelerated; in addition, a large number of CD44 receptors on the surfaces of macrophages can be specifically combined with the nano preparation modified by hyaluronic acid, enter the macrophages through endocytosis and release drugs. A layer of Polyethyleneimine (PEI) is modified on the surface of the nanoparticle through electrostatic adsorption, so that a large number of amino groups are provided for chemical modification of hyaluronic acid.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide an enzyme-responsive silicon dioxide nano preparation which has the characteristics of high drug loading capacity, low toxicity, macrophage targeting property and enzyme-responsive drug release at plaque parts, the silicon dioxide nano preparation with the enzyme responsiveness is formed by loading simvastatin into mesoporous silicon dioxide nanoparticles and then modifying polyethyleneimine and hyaluronic acid on the outer surfaces of the mesoporous silicon dioxide nanoparticles layer by layer, and the hydration particle size of the prepared nanoparticles is 100-220 nm.
The second purpose of the invention is to provide a preparation method of an enzyme-responsive silicon dioxide nano preparation, which comprises the following steps:
1) Preparing mesoporous silica nanoparticles in an alkaline environment, and drying and calcining to obtain mesoporous silica nanoparticle powder;
2) Dissolving mesoporous silica nanoparticles and simvastatin in a solvent, reacting completely, and collecting a sample to obtain the simvastatin-entrapped mesoporous silica nanoparticles;
3) Adding polyethyleneimine into the simvastatin-encapsulated mesoporous silica nanoparticles obtained in the step 2), and collecting a sample after complete reaction to obtain polyethyleneimine-modified simvastatin-encapsulated mesoporous silica nanoparticles;
4) Adding the activated sodium hyaluronate into the mesoporous silica nanoparticles coated with simvastatin and modified by polyethyleneimine obtained in step 3) to react, and collecting a sample after the reaction is completed to finally obtain an enzyme-responsive silica nano preparation;
preferably, the anti-atherosclerosis drug in 2) is simvastatin.
Preferably, the solvent in 2) is dichloromethane.
Preferably, the mass ratio of the mesoporous silica nanoparticles to the simvastatin in the step 2) is = 2; the concentration of the simvastatin in the solvent is 15-30 mg/ml.
Preferably, the concentration of the polyethyleneimine in the step 3) is 2mg/ml.
Preferably, the solvent in 3) is a PBS buffer solution of pH = 7.4; 4) Wherein the solvent is ultrapure water.
Preferably, the activator in 4) is an amide condensing agent, and the amide condensing agent is EDC/NHS. Preferably, in the step 1), hexadecyl ammonium bromide and sodium hydroxide are dissolved in ultrapure water, the solution is stirred and heated to 80 ℃, after the solution is completely clarified, absolute ethyl alcohol is added, the solution is stirred for 5min, then ethyl orthosilicate solution is slowly dropped in, the solution reacts for 2 hours at 80 ℃, the solution is centrifuged and washed by distilled water for 3 times and methanol for 1 time, then the product is transferred to an evaporation dish, dried in a constant-temperature oven for one night, after the product is completely dried, the product is transferred to a muffle furnace, and calcined for 5 hours at 550 ℃, and finally white powder is obtained, and the product is recorded as mesoporous silica nanoparticle MSN;
2) In the method, 30mg of MSN and 15mg of simvastatin SIM are mixed in 2ml of dichloromethane, the mixture is subjected to ultrasonic treatment for 10min to be uniformly dispersed and then stirred for 24 hours at room temperature, then the supernatant is removed by centrifugation, and the residual solid is dried in a vacuum oven at room temperature for 24 hours, and then the product is marked as simvastatin-loaded mesoporous silica nanoparticle SIM @ MSN;
3) Weighing SIM @ MSN, dispersing in PBS, slowly dropwise adding 100 mu l of PEI solution into the nano solution at the rotating speed of 500rpm, stirring at normal temperature for 30min, centrifuging the product, washing with water for 3 times, storing at 4 ℃, and recording as simvastatin-loaded polyethyleneimine-coated mesoporous silica nanoparticle SIM @ PEI-MSN;
4) In the method, HA, NHS and EDC are dissolved in ultrapure water and stirred for 1h to activate carboxyl, then SIM @ PEI-MSN is added, ultrasonic dispersion is carried out for 10min, stirring is continued for 24h, then the product is centrifuged, washed for 3 times and freeze-dried overnight, and the product is recorded as an enzyme-response silicon dioxide release nano preparation SIM @ HA-MSN.
In the technical scheme of the invention, the amide condensing agent in the step 4) is selected from EDC/NHS.
The polyethyleneimine used in the invention is a commercial product, and the molecular weight of the polyethyleneimine is preferably 10kDa; the sodium hyaluronate used in the present invention is a commercially available product, and the molecular weight thereof is preferably 330kDa.
The third purpose of the invention is to provide the application of the enzyme-responsive silicon dioxide nano preparation, and the enzyme-responsive silicon dioxide nano preparation is applied to the treatment of atherosclerosis.
Compared with the prior art, the invention has the beneficial effects that:
the preparation method of the enzyme response type silicon dioxide nano preparation is simple, low in cost and free of pollution; the nanoparticles have the characteristics of uniform particle size, good dispersibility, high drug loading rate, high biocompatibility and low toxicity, can specifically target a CD44 receptor on the surface of macrophage while improving the bioavailability of simvastatin, and can trigger an enzyme response characteristic to release a drug to improve the anti-atherosclerosis effect of the simvastatin under the high-concentration enzyme environment at a focus part.
Drawings
FIG. 1 is a process flow diagram of an enzyme responsive mesoporous silica nano-formulation prepared in the present invention;
FIG. 2 is a transmission electron microscope image of an enzyme-responsive mesoporous silica nano-formulation prepared in the present invention;
FIG. 3 is a diagram illustrating a hydrated particle size of an enzyme-responsive mesoporous silica nanoparticle formulation prepared in the present invention;
FIG. 4 is a Zeta potential diagram of an enzyme-responsive mesoporous silica nano-formulation prepared in the present invention;
FIG. 5 is a nitrogen adsorption/desorption curve of an enzyme-responsive mesoporous silica nano-formulation prepared in the present invention;
FIG. 6 is an in vitro drug release curve of an enzyme-responsive mesoporous silica nano-formulation prepared in the present invention;
FIG. 7 is a graph showing the cytotoxicity evaluation results of an enzyme-responsive mesoporous silica nano-formulation prepared in the present invention;
FIG. 8 is a diagram illustrating the result of evaluating the blood compatibility of an enzyme-responsive mesoporous silica nano-formulation prepared in the present invention;
FIG. 9 is a diagram illustrating the results of the evaluation of cellular uptake of an enzyme-responsive mesoporous silica nanoparticle formulation prepared in accordance with the present invention;
FIG. 10 is a graph showing the in vitro anti-inflammatory evaluation results of an enzyme-responsive mesoporous silica nanoparticle formulation prepared in the present invention;
fig. 11 is a graph showing the result of evaluating the anti-macrophage foaming of the enzyme-responsive mesoporous silica nano-formulation prepared in the present invention.
Detailed Description
In order to make the technical solutions of the present invention easier to understand, the technical solutions of the present invention are now clearly and completely described by using specific embodiments in conjunction with the accompanying drawings, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments.
Example 1
A preparation method of an enzyme-responsive silicon dioxide nano preparation comprises the following steps:
1) 0.4g of cetylammonium bromide and 1.44ml of 2M sodium hydroxide were dissolved in 200ml of ultrapure water, and heated to 80 ℃ with stirring. After the solution is completely clarified, 0.4ml of absolute ethyl alcohol is added, the mixture is stirred for 5min, then 2ml of tetraethoxysilane solution is slowly dripped, and the reaction is carried out for 2h at the temperature of 80 ℃. After centrifugation (10200rpm, 15 minutes), the mixture was washed 3 times with distilled water and 1 time with methanol. The product was then transferred to an evaporation dish and dried overnight in a constant temperature oven. And transferring the product to a muffle furnace after the product is completely dried, calcining the product at 550 ℃ for 5 hours to finally obtain white powder, and recording the product as the mesoporous silica nanoparticle MSN.
2) 30mg of MSN and 15mg of simvastatin SIM were mixed in 2ml of dichloromethane, and dispersed uniformly by sonication for 10min, followed by stirring at room temperature for 24 hours. Next, the supernatant was removed by centrifugation, and after drying the remaining solid in a vacuum oven at room temperature for 24 hours, the product was recorded as simvastatin-loaded mesoporous silica nanoparticle sim @ msn.
3) 30mg SIM @ MSN was weighed and dispersed in 4ml PBS, 100. Mu.l PEI solution (75 mg/ml) was slowly dropped into the nano solution at 500rpm, and stirred at room temperature for 30min. And then centrifuging the product, washing with water for 3 times, storing at 4 ℃, and marking as simvastatin-loaded polyethyleneimine-coated mesoporous silica nanoparticles SIM @ PEI-MSN.
4) 15mg HA, 40mg NHS and 30mg EDC are dissolved in 10ml of ultrapure water and stirred for 1h to activate carboxyl, then 30mg SIM @ PEI-MSN is added, stirring is continued for 24h after ultrasonic dispersion is carried out for 10min, then the product is centrifuged, washed for 3 times by water and freeze-dried overnight, and the product is recorded as an enzyme-responsive silicon dioxide release nano preparation SIM @ HA-MSN. Please refer to fig. 1 for a process flow chart of the enzyme-responsive mesoporous silica nano-formulation.
As shown in fig. 2, which is a transmission electron microscope image of an enzyme-responsive silica nanoparticle preparation prepared in the present invention, it can be seen that the nanoparticles are oval, two-dimensional pores distributed on the surface are difficult to distinguish due to the modification of the polymer, and the hydrated particle size result shown in fig. 3 is 189.1 ± 5.8nm.
As shown in FIG. 4, the Zeta potentials of MSN, SIM @ PEI-MSN and SIM @ HA-MSN prepared in example 1 were-25.1. + -. 2.8mV, + 30.1. + -. 3.0mV and-22.6. + -. 3.4mV, respectively, and the absolute values of the surface charges of the three were high, and the repulsive interaction between particles was strong, so that they could be stably dispersed in the solution.
As shown in FIG. 5, the BET specific surface area, the pore volume and the pore size of the SIM @ HA-MSN are significantly reduced compared with the MSN, which is a nitrogen adsorption-desorption curve diagram and a pore size distribution diagram, and indicates that the nano surface is successfully modified.
Example 2
A method for measuring drug loading and encapsulation efficiency of mesoporous silica nanoparticles comprises the following steps:
and (3) measuring the drug loading rate and the encapsulation rate of the simvastatin-loaded mesoporous silica nanoparticle SIM @ MSN by using an ultraviolet spectrophotometer. 30mg of MSN and 15mg of SIM are precisely weighed and mixed in 2ml of dichloromethane, and the mixture is subjected to ultrasonic treatment for 10min to be dispersed uniformly and then stirred at room temperature for 24 hours. Then the supernatant is collected by centrifugation, and is filtered by a 0.22 μm filter membrane, and then the simvastatin content is measured by an ultraviolet spectrophotometer, and the detection wavelength is set to be 238nm.
The calculation is made according to the equation set out below:
drug loading rate = (nano Chinese medicine content)/(nano total amount) × 100%
Encapsulation ratio = (actual drug loading)/(initial drug loading) × 100%
Table 1 shows the drug loading and encapsulation efficiency of the simvastatin loaded mesoporous silica nanoparticle sim @ msn prepared in example 1.
Table 1:
drug loading (%) Encapsulation efficiency (%)
21.32±1.31 55.21±2.23
As can be seen from Table 1, the simvastatin loaded mesoporous silica nanoparticle SIM @ MSN prepared in example 1 has high drug loading rate and high encapsulation efficiency.
Example 3
An in vitro enzyme response release research of an enzyme response type silicon dioxide nano preparation SIM @ HA-MSN comprises the following steps:
briefly, an amount of nanoparticles was uniformly dispersed in 40mL of PBS (10 mM, 0.2% sds, ph = 7.4). To investigate the responsiveness of the enzyme, the addition of hyaluronidase to the medium was chosen. All samples were shaken at 37 ℃ at 100 rpm. At the indicated time intervals, the samples were centrifuged and 2ml of buffer was collected and the same volume of fresh buffer was replaced. The amount of released SIM was measured by high performance liquid chromatography (HPLC, agilent 1200 series) at a wavelength of 238nm using an Agilent C18 column (4.6 mm. Times.250mm, 5 μm). The mobile phase was acetonitrile: 0.025M sodium dihydrogen phosphate solution (76 24V), flow rate 1.0mL/min, sample size 20. Mu.L.
As shown in fig. 6, the in vitro release results for simvastatin of example 3. The slow release amount of SIM @ MSN in 48h is 70.11 +/-4.64%, while the slow release amount of SIM @ HA-MSN is 28.02 +/-4.11%, which shows that the HA coating can effectively prevent the release of SIM. To mimic the plaque environment with high levels of hyaluronidase (HAase) expression, HAase (100U/mL) was added to the buffer. Compared with the low drug release of blank PBS, the drug release rate of SIM @ HA-MSN is 62.24 +/-5.82% in 48h, because the HA layer is rapidly broken after HAase degradation. Generally speaking, the results show that the HA modification on the SIM @ HA-MSN can be used as a 'gatekeeper', can effectively block the drug leakage of normal tissues, and HAs the effect of in vitro slow release compared with the SIM @ MSN.
Example 4
An enzyme-responsive silica nano preparation for researching cytotoxicity comprises the following steps:
as shown in FIG. 7, the results of the cytotoxicity of the nanoparticles on mouse macrophage Raw264.7 cells and human umbilical vein epithelial cells HUVEC cells are shown, and the toxicity of the nanoparticles on the cells is determined by using an Alma blue reagent. The method comprises the following specific steps: raw264.7 cells and HUVEC cells were seeded into 96-well plates at a density of 5X 10 per well, respectively 3 Cells, at a temperature of 37 ℃ and a humidity of 5% 2 The culture was carried out overnight in an incubator. Different concentrations (0, 10,50,200, 400. Mu.g/mL) of SIM @ HA-MSN were added to 96-well plates as an experimental group and a blank medium as a control group, respectively. After 24h the medium was discarded and 100. Mu.L of Alamar blue reagent was added to each well for an additional 4h of incubation. The analysis was then carried out using a microplate reader (SpectraMax ID5, bio-format) with the wavelengths set at 570nm and 600nm. Calculating and reporting the percentage of cell viability of the experimental and control groups
The cytotoxicity experiment result shows that after cells are incubated for 24 hours at the concentration of 20-400 mu g/mL by SIM @ HA-MSN, the survival rates of Raw264.7 cells and HUVECs are higher than 80%, and the particles are proved to have good cell compatibility. The reduction in cell viability resulting from high doses of SIM @ HA-MSN may be due to the inhibition of macrophage proliferation and inhibition of plaque inflammatory responses by released SIM.
Example 5
An enzyme-responsive silica nano-preparation for researching the blood compatibility comprises the following steps:
the hemocompatibility of the nanoparticles was evaluated as shown in fig. 8. Since damage to erythrocytes induces the release of hemoglobin, the rate of hemolysis is determined by measuring the absorbance of hemoglobin in the supernatant at a wavelength of 576 nm. The hemolysis rate of MSN is dose-dependent, and the hemolysis rate is as high as 46.24 +/-3.92% at 800 mug/mL, and erythrocytes are obviously hemolyzed. In contrast, hemolysis rate (< 5%) of sim @ ha-MSN was negligible. This result indicates that SIM @ HA-MSN HAs better blood compatibility than naked MSN due to the shielding effect of HA.
Example 6
An enzyme-responsive silica nano preparation for researching the cellular uptake comprises the following steps:
CD44 mediated cellular uptake was studied as shown in figure 9. Raw264.7 cells were cultured at 5X 10 4 Density of/well inoculated in 24-well plates at 37 ℃ with humidity 5% 2 The culture was carried out overnight in an incubator. 100ng/mL LPS was added to each well and incubated for 24h. After PBS washing, FITC-modified HA-MSN (FITC-HA-MSN) and MSN (FITC-MSN) were incubated with Raw264.7 cells for 2h, respectively, at a final concentration of 50. Mu.g/mL per group. After PBS washing, the wells were fixed with 100. Mu.L of 4% PFA solution for 10min per well and stained with DAPI for 10min. Finally, the cells were observed under a confocal laser scanning microscope (CLSM, leica Stellaris). In the competitive inhibition assay, HA-containing medium (10 mg/mL) was used in place of the blank medium, and FITC-labeled HA-MSN (50 mg/mL) was co-cultured with Raw264.7 cells in the same manner as described above.
FITC-MSN treated cells exhibited faint green fluorescence. In contrast, the FITC-HA-MSN group captured significant green fluorescence. Notably, raw264.7 cells were treated with 10mg/mL free HA prior to FITC-HA-MSN treatment, which blocked HA-MSN recognition and internalization by the cells, due to blocking of CD44 receptors on macrophages, thereby exhibiting very weak green fluorescence. Thus, the above data indicate that HA can act to target inflammatory macrophages through CD 44-mediated internalization.
Example 7
A research of influence of an enzyme-responsive silicon dioxide nano preparation on macrophage inflammatory factor levels comprises the following steps:
the effect of SIM @ HA-MSN on the typical inflammatory factors secreted by Raw264.7 cells is shown in FIG. 10. Briefly, raw264.7 grown in log phase was set at 10 5 Per mL into a 24-well plate, 5% CO at 37% 2 Incubate overnight in the incubator. The positive control group was treated with 100ng/mL LPS for 24h, and the remaining groups were treated with the same amount of LPS and the equivalent amount of free SIM, SIM @ MSN or SIM @ HA-MSN for 24h. The concentrations of TNF-alpha and IL-6 protein were determined using ELISA kits. TNF-alpha and IL-6 levels were significantly reduced after SIM, SIM @ MSN and SIM @ HA-MSN treatment compared to control groups stimulated with LPS only. Notably, the minimum levels of TNF- α and IL-6 for the SIM @ HA-MSN group were 514.12 and 273.62pg/mL, respectively. The results show that SIM @ HA-MSN has strong anti-inflammatory effect on inflammatory macrophages.
Example 8
A research of the influence of an enzyme response type silicon dioxide nano preparation on macrophage foaming comprises the following steps:
the inhibitory effect of SIM @ HA-MSN on Raw264.7 cell-induced foam cells is shown in FIG. 11. Briefly, at 5 × 10 4 Density per well cells were seeded in 24-well plates, 5% CO at 37 ℃ 2 Incubate overnight in the incubator. The positive control group was treated with 100ng/mL LPS for 24h, and the remaining groups were treated with the same amount of LPS and the equivalent amount of free SIM, SIM @ MSN or SIM @ HA-MSN for 24h. Subsequently, attachment of PBS to each group of macrophages was washed 3 times, fixed to 4% PFA for 10 minutes, stained with freshly filtered 0.3% oil red O for 15min. Finally, the mixture was placed in 60% isopropanol for 5min and imaged by an optical microscope. We evaluated the inhibitory effect of SIM @ HA-MSN on oxLDL induced foam macrophage formation, with the significant reduction of oil red staining areas after SIM, SIM @ MSN and SIM @ HA-MSN treatment. The inhibition effect of SIM @ HA-MSN is the most remarkable, and the staining area is only 6.77 +/-0.85%. These results demonstrate the potent inhibitory effect of SIM @ HA-MSN on macrophage foam, whichMay help to remove foam cells from the plaque.
From the above-mentioned examples 3 to 8, it can be confirmed that the silica nano-formulation having enzyme responsiveness has a good therapeutic effect on atherosclerosis.
It should be noted that the embodiments described herein are only some embodiments of the present invention, and not all implementations of the present invention, and the embodiments are only examples, which are only used to provide a more intuitive and clear understanding of the present invention, and are not intended to limit the technical solutions of the present invention. Other embodiments, as well as other simple alternatives and variations to the embodiments of the present invention, which will occur to persons skilled in the art without inventive faculty, are within the scope of the invention.

Claims (10)

1. A silica nano-formulation having enzyme responsiveness, characterized in that: the silica nano preparation with enzyme responsiveness is formed by loading simvastatin into mesoporous silica nanoparticles and then modifying polyethyleneimine and hyaluronic acid on the outer surfaces of the mesoporous silica nanoparticles layer by layer, and the hydration particle diameter of the prepared nanoparticles is 100-220 nm.
2. A method for preparing the silica nano-formulation having enzyme responsiveness according to claim 1, wherein: the preparation method of the silicon dioxide nano preparation with enzyme responsiveness comprises the following steps:
1) Preparing mesoporous silica nanoparticles in an alkaline environment, and drying and calcining to obtain mesoporous silica nanoparticle powder;
2) Dissolving mesoporous silica nanoparticles and simvastatin in a solvent, reacting completely, and collecting a sample to obtain simvastatin-entrapped mesoporous silica nanoparticles;
3) Adding polyethyleneimine into the simvastatin-encapsulated mesoporous silica nanoparticles obtained in the step 2), and collecting a sample after complete reaction to obtain polyethyleneimine-modified simvastatin-encapsulated mesoporous silica nanoparticles;
4) Adding the activated sodium hyaluronate into the mesoporous silica nanoparticles coated with simvastatin and modified by polyethyleneimine obtained in the step 3) to react, and collecting a sample after the reaction is completed to finally obtain the enzyme response type silica nano preparation.
3. The method for preparing an enzyme-responsive silica nano-formulation according to claim 2, wherein: 2) The anti-atherosclerosis medicine is simvastatin.
4. The method for preparing a silica nano-formulation having an enzyme responsiveness according to claim 3, wherein: 2) The solvent in (1) is dichloromethane.
5. The method for preparing an enzyme-responsive silica nano-formulation according to claim 4, wherein: 2) The mass ratio of the mesoporous silica nanoparticles to the simvastatin is =2 and is 1-2; the concentration of the simvastatin in the solvent is 15-30 mg/ml.
6. The method for preparing an enzyme-responsive silica nano-formulation according to claim 5, wherein: 3) Wherein the concentration of the polyethyleneimine in the solution is 2mg/ml.
7. The method for preparing an enzyme-responsive silica nano-formulation according to claim 6, wherein: 3) The solvent is PBS buffer solution of pH = 7.4; 4) Wherein the solvent is ultrapure water.
8. The method for preparing an enzyme-responsive silica nano-formulation according to claim 7, wherein: 4) The activating agent is amide condensing agent which is EDC/NHS.
9. The method for preparing an enzyme-responsive silica nano-formulation according to claim 2, wherein: 1) Dissolving hexadecyl ammonium bromide and sodium hydroxide in ultrapure water, stirring and heating to 80 ℃, adding absolute ethyl alcohol after the solution is completely clarified, stirring for 5min, slowly dripping ethyl orthosilicate solution, reacting for 2h at 80 ℃, centrifuging, washing for 3 times with distilled water, washing for 1 time with methanol, transferring the product to an evaporation dish, drying overnight in a constant-temperature oven, transferring to a muffle furnace after the product is completely dried, and calcining for 5h at 550 ℃, so as to obtain white powder, wherein the product is recorded as mesoporous silica nanoparticle MSN;
2) Mixing MSN and simvastatin SIM in dichloromethane, performing ultrasonic treatment for 10min to uniformly disperse the MSN and simvastatin SIM, stirring at room temperature for 24 hours, centrifuging to remove supernatant, and drying the residual solid in a vacuum oven at room temperature for 24 hours to obtain a product, namely simvastatin-loaded mesoporous silica nanoparticle SIM @ MSN;
3) Weighing SIM @ MSN, dispersing in PBS, slowly dropwise adding 100 mu l of PEI solution into the nano solution at the rotating speed of 500rpm, stirring at normal temperature for 30min, centrifuging the product, washing with water for 3 times, storing at 4 ℃, and recording as simvastatin-loaded polyethyleneimine-coated mesoporous silica nanoparticle SIM @ PEI-MSN;
4) Dissolving HA, NHS and EDC in ultrapure water, stirring for 1h to activate carboxyl, adding SIM @ PEI-MSN, ultrasonically dispersing for 10min, continuing stirring for 24h, centrifuging the product, washing with water for 3 times, and freeze-drying overnight, wherein the product is marked as an enzyme-responsive silica-releasing nano preparation SIM @ HA-MSN.
10. Use of the silica nanoformulation with enzymatic responsiveness according to claim 1, characterized in that it is used in the treatment of atherosclerosis.
CN202210795969.7A 2022-06-14 2022-07-07 Enzyme response type silicon dioxide release nano preparation, preparation method and application Pending CN115475255A (en)

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