CN112915213B - Response type NO nano-drug, preparation method and application thereof - Google Patents

Response type NO nano-drug, preparation method and application thereof Download PDF

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CN112915213B
CN112915213B CN202110141514.9A CN202110141514A CN112915213B CN 112915213 B CN112915213 B CN 112915213B CN 202110141514 A CN202110141514 A CN 202110141514A CN 112915213 B CN112915213 B CN 112915213B
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雷苑
范文培
宋毛毛
孙兴怀
卢奕
陈小元
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Eye and ENT Hospital of Fudan University
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Abstract

The invention provides a response type NO nano-drug, which takes hollow mesoporous organic silicon nano-particles as a carrier material to load nitric oxide donors JS-K and L-arginine simultaneously. The invention also provides a preparation method and application of the response type NO nano-drug. The response type NO nano-drug can be activated to slowly release nitric oxide in the anterior segment redox environment to generate the effect of reducing intraocular pressure. The invention has simple preparation process, convenient operation, no need of complex and expensive equipment and easy realization of industrial production, thereby having good application prospect in the field of glaucoma treatment.

Description

Response type NO nano-drug, preparation method and application thereof
Technical Field
The invention relates to the field of medical nano materials, in particular to a nano material for treating glaucoma, and specifically relates to a response type NO nano medicament, a preparation method and an application thereof.
Background
Glaucoma is the first irreversible blinding eye disease in the world, and more particularly, glaucoma is a optic neuropathy (optic nerve disease) which is usually a condition in which aqueous humor in the eye drains poorly, resulting in an increase in intraocular pressure. The pressure in the eyes is increased, the optic nerve is damaged, and the visual field range is reduced. Blindness can result when patients present with sustained high pressure. Thus, ocular hypertension is the most dangerous factor for glaucoma, and ocular tension lowering is the most effective method for treating glaucoma.
The eye is a hollow structure containing a transparent fluid called "aqueous humor". Aqueous humor is formed in the posterior chamber of the eye by the ciliary body at a rate of about 2.5 microliters per minute. Fluid produced at a fairly constant rate then passes around the lens and into the anterior chamber of the eye through the pupillary opening in the iris. Once in the anterior chamber, the fluid exits the eye through two different routes. Aqueous outflow pathways include both non-traditional outflow pathways and traditional pathways. Among these, the non-traditional outflow pathway is the "uveoscleral tunnel route". In this route, fluid is filtered between the muscle fibers of the ciliary body, which is approximately ten percent of the aqueous humor flowing out of the human body. The traditional aqueous outflow pathway is the "canalicular" route, which involves the Trabecular Meshwork (TM) and Schlemm's Canal (SC), through which about 90% of the aqueous flows, i.e. produced by the ciliary body, through the posterior chamber, through the pupil to the anterior chamber, and out through the trabecular meshwork adjacent to the Schlemm canal.
Elevated intraocular pressure is primarily due to increased resistance to aqueous outflow from the conventional pathway, and nitric oxide is a small molecule drug that can act on the ocular hypotensive properties of the conventional pathway. Us FDA approval of 0.024% latanoprostenebund (lbn) eye drops in 2017, under the trade name VYZULTA, for the treatment of open angle glaucoma and ocular hypertension. The medicine can enhance the outflow of aqueous humor in the traditional pathway and increase the curative effect of lowering intraocular pressure by adding monobutyldiol ester serving as a nitric oxide donor part into the traditional glaucoma medicine latanoprost.
Various nitric oxide donor drugs are available, such as metallic nitroso compounds, s-nitrosothiols (SNOs), n-dinitrodioate (NONOates), and the like. The nitric oxide donor medicaments can effectively release nitric oxide molecules under the activation of internal and external stimuli. However, these donors cannot release any molecule in a controlled manner upon activation by endogenous and exogenous stimuli. Exogenous stimuli such as light, X-ray radiation, ultrasound, etc., while controlling the release of nitric oxide, are inevitably associated with potentially toxic effects on the surrounding tissues. In contrast, endogenous stimuli-responsive nitric oxide donors can produce nitric oxide under specific physiological conditions, opening the possibility of site-specific nitric oxide release at the target site, and do not require external stimuli and produce side effects. For example, endothelial nitric oxide synthase (eNOS) in a TM/SC environment can catalyze the oxidation of L-arginine to L-citrulline, producing NO, and thereby lowering intraocular pressure. However, for mice without the NOS3 gene, eNOS protein was produced too little to play a catalytic role.
In addition, the therapeutic efficacy of glaucoma is also dependent on the effectiveness of NO delivery to the target site. Free NO donors are generally too small to pass through the cornea, which is a major obstacle to conventional outflow pathways. Therefore, there is a need to develop a nano-drug carrier with good biocompatibility for loading an NO donor to pass through the cornea and release nitric oxide at a targeted site of the eye to achieve the effect of reducing intraocular pressure.
Disclosure of Invention
The invention aims to solve the technical problem of providing a response type NO nano-medicament, wherein the response type NO nano-medicament is a mesoporous organic silicon dioxide hollow nano-capsule loaded with two medicaments, and the nano-capsule prepared by the method can be activated by the oxidation reduction environment of the anterior segment of the eye to release NO and reduce intraocular pressure.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
a response type NO nano-drug takes hollow mesoporous organic silicon nano-particles (HOS) as a carrier material, and JS-K and L-arginine are simultaneously loaded on the carrier material.
In some embodiments of the present invention, the hollow mesoporous organosilicon nanoparticles have a diameter of 45.4 ± 5.2nm, a cavity size of 33.4 ± 5.2nm, and a pore size of 3-4.5 nm.
The JS-K and the L-arginine are loaded in the cavity and the mesoporous pore channel of the hollow mesoporous organic silicon nano-particles, and the loading amounts of the JS-K and the L-arginine in the hollow mesoporous organic silicon nano-particles are respectively 3-8% by mass and 8-20% by mass.
In some embodiments of the present invention, the pore size of the hollow mesoporous silicone nanoparticle is 3.8nm, and the loadings of the JS-K and the L-arginine in the hollow mesoporous silicone nanoparticle are 4% by mass and 10% by mass, respectively.
The preparation method of the response type NO nano-drug comprises the following steps:
(a) solid SiO 2 Preparation of nanoparticles (MS): mixing cetyltrimethylammonium chloride (CTAC), water and triethylMixing alcohol amine (TEA), stirring uniformly, and then dropwise adding tetraethyl orthosilicate (TEOS) to react to obtain an MS solution;
(b) mesoporous organosilicon coated solid SiO 2 Preparation of nanoparticles (MS @ MOS): adding bis- [ r- (triethoxysilyl) propyl ] into the MS solution obtained in the step (a)]-a mixed silicon source of tetrasulfide (BTES) and tetraethyl orthosilicate (TEOS) to obtain a suspension of MS @ MOS after sufficient reaction;
(c) preparation of hollow mesoporous organosilicon nanoparticles (HOS): centrifuging the suspension of MS @ MOS obtained in the step (b), washing the obtained solid with ethanol, dispersing the washed solid in an ethanol solution of concentrated hydrochloric acid, and heating for reaction to remove CTAC serving as a template; selectively etching the MS inner core by using ammonia water to obtain hollow mesoporous organic silicon nano particles HOS;
(d)HOS-J R the preparation of (1): HOS is dispersed into DMSO solution of JS-K by adopting a vacuum infusion method; then adding water into the solution under ultrasonic treatment; obtaining HOS-J by centrifuging and finally dissolving the product in water R ；
(e)HOS-L O Preparation of
Dispersing HOS and L-arginine in purified water, stirring at room temperature, centrifuging, and dissolving the final product in water to obtain HOS-L O ；
(f)HOS-J R L O Preparation of
HOS-J is added R And L O Or HOS-L O And J R Dispersing in pure water, and stirring at room temperature; obtaining HOS-J by centrifuging and finally dissolving the product in water R L O 。
Preferably, in the step (a), the volume ratio of the hexadecyl trimethyl ammonium chloride to the water to the triethanolamine to the tetraethyl orthosilicate is (1.8-2.0) to (20-25) to (0.06-0.1) to (0.8-1.0); the reaction temperature is 95 ℃, and the reaction time is 1-2 hours.
Preferably, in the step (b), the volume ratio of the MS solution to the BTES solution to the TEOS solution is 2:1 to-1: 1.
Preferably, in step (c), the mass percentage of the concentrated hydrochloric acid in the ethanol solution is 5-15%, and more preferably, 10%.
Preferably, in the step (d), the mass ratio of the HOS to the JS-K is 1:1-4:1, the concentration of the JS-K in the DMSO solution is 5-10g/L, the volume ratio of the DMSO to the water is 1:1-1:2, and the time of ultrasonic treatment is 5-15 minutes.
Preferably, in step (e), the mass ratio of HOS to L-arginine is 1:10 to 1: 20.
Preferably, in step (f), HOS-J R And L O (or HOS-L) O And J R ) The mass ratio of (A) to (B) is 1:10-1: 20.
The invention relates to application of a response type NO nano-drug in preparing a drug for treating eye diseases. In some embodiments of the invention, the ocular disease is glaucoma or ocular hypertension.
The concentrated hydrochloric acid refers to a hydrochloric acid aqueous solution with the mass percentage concentration of 37%; the indoor temperature of the laboratory is in the range of 20-25 ℃.
Has the advantages that:
the response type NO nano-drug provided by the invention can be activated in the anterior segment redox environment to release nitric oxide molecules and generate the effect of reducing intraocular pressure.
The responsive NO nano-drug provided by the invention can controllably release nitric oxide molecules at a target site of an eye part, does not stimulate surrounding tissues, has NO potential toxic effect, and has good biological safety.
The invention has simple preparation process, convenient operation, no need of complex and expensive equipment and easy realization of industrial production, thereby having good application prospect in the field of glaucoma treatment.
Drawings
FIG. 1: a) HOS-J R L O A synthetic scheme; b) HOS, HOS-J R ,HOS-J R L O Thermogravimetric plot; c) HOS-J R L O Electron micrographs of (A).
FIG. 2 is a schematic diagram: the CCK8 assay evaluates the toxicity of drugs to HCE cells. (a)0.15mg/mL for 6h, (b)0.3mg/mL for 6h, (c)0.15mg/mL for 12h, (d)0.3mg/mL for 12h, (e)0.15mg/mL for 24h, (f)0.3mg/mL for 24h.
FIG. 3: eye surface photograph (a), eyeball structure (b) and cornea photograph (c) of the mice after the drug action.
FIG. 4: (a) HOS-J R Ocular hypotensive effect on CAV1 mice; (b) HOS-L O Ocular hypotensive effect on CAV1 mice; (c) HOS-J R L O Ocular hypotensive effect on CAV1 mice.
FIG. 5: a) different concentrations of HOS, HOS-J R ,HOS-L O ，HOS-J R L O Comparison of in vitro production of NO; b) the different nano-drugs act on the NO production amount in the aqueous outflow tissue behind the ocular surface.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, which are not specified under specific conditions, are generally carried out under conventional conditions.
The starting materials or reagents used in the examples of the present invention are commercially available unless otherwise specified. The main reagents used included cetyltrimethylammonium chloride solution (25 wt.% aqueous solution), triethanolamine, tetraethylorthosilicate, bis [3- (triethoxysilyl) propyl ] tetrasulfide, ammonium hydroxide, L-ascorbic acid, sodium hydrogen carbonate, sodium hydrogen carbonate, sodium hydrogen carbonate, sodium hydrogen carbonate, sodium,
O2- (2, 4-dinitrophenyl) 1- [ ((4-ethoxycarbonyl) piperazin-1-yl ] diazo-1-1, 2-diol ester,
Ammonia solution (28 wt.% aqueous solution), dimethyl sulfoxide, sodium chloride, fluorescein isothiocyanate, mainly purchased from sigma.
The abbreviations used in the present invention have the usual meaning in the art, for example the following abbreviations have the following meanings:
Figure BDA0002928807340000051
Figure BDA0002928807340000061
in describing and claiming the present invention, the following terminology will be used:
JS-K is a nitric oxide prodrug, the chemical structure of which is shown as the following formula,
Figure BDA0002928807340000062
in the present invention, J is also represented by R Denoted JS-K.
In the present invention, Lo represents L-arginine, i.e., L-arginin.
The "vacuum infusion" method in the present invention means: a large pressure differential was created inside and outside the HOS cavity and JS-K or L-argine was loaded into the HOS cavity.
Nano Drug Carriers (Nanoscale Drug Carriers) are a submicron Drug carrier delivery system that belongs to the Nanoscale micro-category. The drug is encapsulated in submicron particles, so that the release speed can be adjusted, the permeability of a biological membrane is increased, the distribution in a body is changed, the bioavailability is improved, and the like.
Nanoparticles (NPs), also known as nanoparticles, are solid colloidal particles of 10-1000nm in size, generally composed of natural or synthetic polymeric substances, and can be used as carriers for conducting or delivering drugs. Due to differences in materials and manufacturing processes, nanospheres (nanospheres) and nanocapsules (nanocapsules), both collectively referred to as nanoparticles, can be formed.
The nano-drug is a drug formed by loading a drug on a nano-drug carrier, or various nano-particles in which the drug is dissolved or dispersed. The response type NO nano-drug means that the nano-drug releases NO under exogenous or endogenous stimulation.
The HOS nano-drug refers to a drug formed by loading a drug in a HOS cavity or a mesoporous channel. HOS-J in the invention R ，HOS-L o ，HOS-J R L o All are HOS nano-drugs.
Enos, Enos all refer to nitric oxide synthase.
Schlemm's canal (Schlemm's canal) is a circular canal-like aqueous humor drainage channel surrounding the anterior chamber angle for one circle, the inner wall is separated from trabecular meshwork by only one layer of endothelial cells, 25-35 liquid collecting tubes are arranged on the outer side wall, and aqueous humor flows into intrascleral veins (aqueous humor veins) through the channel and finally flows into anterior ciliary veins.
The "vacuum infusion" process is described in the literature: fan W, Lu N, Huang P, et al. glucose-responsive sequence generation of hydrogen peroxide and nitrile oxide for synthetic cancer stage-like/gas therapy [ J ]. Angewandte Chemie,2017,129(5):1249-1253.
Methods of cytotoxicity assessment and methods of use of the Griess assay kit are described in the literature: hu C, Sun J, Zhang Y, Lei Y, Sun X, Deng Y. local Delivery and Sustained-Release of nitrile Oxide Loaded in meso Silica Particles for Efficient Treatment of Primary Open-Angle Glaucoma. adv healthcare Mater 2018; and (7) (23) e1801047.
Mouse model for ocular hypertension-CAV 1 knockout (CAV1 KO) mice were obtained as described in the literature: y Lei, M Song, J Wu, X sun. enos activity in CAV1 knock out mouse eyes. invest optthalmol Vis sci.2016; 57(6):2805-13.
Example 1: nano medicine HOS-J R L O Preparation of
(a) Preparation of solid silica nanoparticles (MS): after 2g of hexadecyltrimethylammonium chloride (CTAC) aqueous solution and 0.1g of Triethanolamine (TEA) are uniformly stirred in a water bath at 95 ℃, 1mL of tetraethyl orthosilicate (TEOS) is added dropwise and the reaction is carried out for 1 h.
(b) Preparation of mesoporous silicone coated solid nanoparticles (MS @ MOS): and adding a mixed silicon source of bis- [ r- (triethoxysilyl) propyl ] -tetrasulfide (BTES) and TEOS into the solution, and reacting for 4 hours to obtain Mesoporous Organosilicon (MOS) -coated MS (MS @ MOS).
(c) Preparation of HOS: the product is centrifuged, washed by ethanol, dispersed in a mixed solution of 100mL ethanol and 10mL concentrated hydrochloric acid (37%), heated to 78 ℃ and reacted for 12h to remove the template CTAC. Repeat the above steps 3 times. After washing, the mixture was dispersed again in 20mL of water. And adding 0.4mL of ammonia water into 1mL of the solution to react for 3h at 95 ℃, and selectively etching the MS core to finally obtain the HOS.
(d)HOS-J R Preparation of (JS-K-Supported HOS): then by using"vacuum infusion" method, 10mg HOS was dispersed in 1mL of 5mg JS-K (denoted J) R ) In DMSO. Then 1mL of water was added dropwise to the solution under sonication for 10 minutes. Obtaining HOS-J by centrifuging and finally dissolving the product in water R 。
(e)HOS-L O The preparation of (1): 20mg of HOS and 200mg L of the mixture O Dispersed in 10mL of purified water and stirred at room temperature for 24 hours. By centrifugation, the final product is dissolved in water to obtain HOS-L O
(f) 20mg of HOS-J was added R And 200mg L O Dispersed in 10mL of purified water and stirred at room temperature for 24 hours. Obtaining HOS-J by centrifuging and dissolving the final product in water R L O 。
Synthesis of HOS-J R L O As shown in fig. 1, fig. 1a shows loading J in the HOS cavity R Synthesis of HOS-J R Then loading L in the mesoporous pore canal O Thereby obtaining HOS-J R L O . FIG. 1b shows HOS, HOS-J R And HOS-J R L O Calculating to obtain HOS-J R L O The loadings of JS-K and L-arginine in the mixture are 4% and 10%, respectively. FIG. 1c shows HOS-J R L O The transmission electron microscope image of (2) shows that the nano-drug has good dispersibility.
Example 2: evaluation of cytotoxicity
HOS Nanoparticulates were ocular surface medications, and HOS-J was evaluated accordingly R L O Effect on survival of corneal epithelial cells (HCE). HCE cells were seeded into 96-well cell culture plates at a density of 5000 cells per well. After overnight incubation at 37 ℃, cells were washed with PBS and then 100ul of fresh cell culture medium containing different concentrations (0.15 and 0.3mg/mL) of drug was added thereto and incubated for 6, 12 or 24 hours. After each time point was reached, 10ul of CCK-8 solution (10%) was added and incubation continued for 1 hour. The absorbance at 450nm was measured using a microplate reader, and the cell activity of the experimental group was calculated based on the blank group (no cell inoculated), the control group (cell plus medium containing no drug) and the experimental group (cell plus medium containing drug).
The results are shown in FIG. 2: fig. 2a shows the effect of 0.15mg/mL nano-drug incubation for 6h on cell survival rate, fig. 2b shows the effect of 0.3mg/mL nano-drug incubation for 6h on cell survival rate, fig. 2c shows the effect of 0.15mg/mL nano-drug incubation for 12h on cell survival rate, fig. 2d shows the effect of 0.3mg/mL nano-drug incubation for 12h on cell survival rate, fig. 2e shows the effect of 0.15mg/mL nano-drug incubation for 24h on cell survival rate, fig. 2f shows the effect of 0.3mg/mL nano-drug incubation for 24h on cell survival rate, and the results show that the effect of different concentrations of drugs for 6h, and the survival rate of HCE cells is not significantly affected by 12h to 24h.
Example 3: evaluation of ocular surface toxicity
2 mice were taken, the ocular surface of the first mouse was dropped with PBS (1 drop, 2. mu.L/drop) and the ocular surface of the second mouse was dropped with nano-drug (1 drop, 2. mu.L/drop, 15mg/mL) for 48h, and then photographed with a slit-lamp microscope system. Immediately after that, the mice were sacrificed by dislocation of cervical vertebrae, the eyeballs were taken, fixed with 4% paraformaldehyde, and stained with hematoxylin-eosin.
The results are shown in FIG. 3, in which FIG. 3a shows the eye pattern of the mouse after the drug action, FIG. 3b shows the eye pattern of the mouse after the drug action, and FIG. 3c shows the cornea pattern of the mouse after the drug action. The result shows that the nano-drug does not stimulate the ocular surface of the mouse after being dripped into the ocular surface of the mouse for 48 hours, does not influence the structure of the whole eyeball, and has no obvious toxic effect on corneal 5-layer cells of the mouse.
Example 4: pharmacological experiments
Intraocular pressure measurements were made at the same time period, while the mouse was awake, using a tonometer (model: tonolab) specific to Finnish animals. When measuring the intraocular pressure, the left hand gently grabs neck skin behind the mouse ear, makes lying prone of relaxing of mouse cover the cage, and the tonometer is held to the right hand and is measured the intraocular pressure. Three measurements were made and the average was taken as one intraocular pressure measurement. In the animal experiments, the experimental operation was carried out according to the animal use and health care system of the clinical center of the national institutes of health, U.S. committee for animal care and use. A mouse model for ocular hypertension, CAV1 knockout mouse, was used in the experiment. In order to familiarize and habituate the mice to tonometry, the mice were subjected to pharmacological testing 1 week prior to the studyIntraocular pressure was measured multiple times. The basal intraocular pressure of three mice was first measured one to two days before the start of the pharmacological experiment. The pharmacological protocol was right eye drop (1 drop, 2. mu.L/drop) and left eye drop PBS (1 drop, 2. mu.L/drop). HOS-J R ：15mg/mL；HOS-L O :15mg/mL；HOS-J R L O 15 mg/mL. Intraocular pressure in both eyes of the mouse was measured using a tonolab tonometer before and 5, 24, and 48 hours after instillation.
The results of the experiment are shown in FIG. 4, and HOS-J is shown in FIG. 4a, FIG. 4b and FIG. 4c, respectively R 、HOS-L O And HOS-J R L O The effect on the intraocular pressure of a mouse shows that the three nano-drugs can obviously reduce the intraocular pressure of the mouse, the effect of reducing the intraocular pressure can last for 48 hours after one-time administration, and the effect is similar to that of HOS-J R 、HOS-L O Compared with HOS-J R L O The blood pressure reduction amplitude of (1) is larger, and in 48 hours, HOS-J R L O The percentage of intraocular pressure reduction is 24%, higher than HOS-J R 5% and HOS-L O 11% of the total.
Example 5: mechanism of action of nano-drugs
The detection of nitric oxide was performed using a commercial Griess assay kit according to the instructions. First, the nitric oxide release amount of the in vitro nano-drug was measured, and the result is shown in FIG. 5a, HOS-J R L O The release amount of nitric oxide is obviously higher than that of HOS-J R And HOS-L O And the nitric oxide release amount is increased along with the increase of the concentration of the nano-drug. Then detecting the release amount of nitric oxide in the aqueous outflow tissue after the nano-drug is applied to the ocular surface, killing the mouse by cervical dislocation 5 hours after the nano-drug is applied to the ocular surface, taking the eyeball, separating the aqueous outflow tissue, and then obtaining the concentration of the nitric oxide according to the operation of the kit instruction, wherein the result is shown in figure 5b, and the result shows that HOS-J R L O The generation amount of nitric oxide is obviously higher than HOS-J R And HOS-L O 。
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and such equivalents also fall within the appended claims.

Claims (11)

1. A response type NO nano-medicament is characterized in that: the preparation method comprises the following steps of taking hollow mesoporous organic silicon nano particles as a carrier material, and simultaneously loading JS-K and L-arginine on the carrier material.
2. The responsive NO nano-drug of claim 1, wherein the hollow mesoporous organosilicon nanoparticles have a diameter of 45.4 + 5.2nm, a cavity size of 33.4 + 5.2nm, and a pore size of 3-4.5 nm.
3. The responsive NO nano-drug of claim 1 or 2, wherein the JS-K and the L-arginine are loaded in the hollow cavity and the mesoporous pore canal of the hollow mesoporous organosilicon nano-particle respectively, and the loading amounts of the JS-K and the L-arginine in the hollow mesoporous organosilicon nano-particle are 3-8% by mass and 8-20% by mass, respectively.
4. The method for preparing a responsive NO nano-drug according to any one of claims 1 to 3, characterized by comprising the steps of:
(a) solid SiO 2 Preparing nano particles: mixing hexadecyl trimethyl ammonium chloride, water and triethanolamine, stirring uniformly, dropwise adding tetraethyl orthosilicate, and reacting to obtain solid SiO 2 A nanoparticle solution;
(b) mesoporous organosilicon coated solid SiO 2 Preparation of nanoparticles solid SiO obtained in step (a) 2 Adding di- [ r- (triethoxy silicon) propyl group into nano particle solution]A mixed silicon source of tetrasulfide and tetraethyl orthosilicate, and fully reacting to obtain the mesoporous organic silicon coated solid SiO 2 A suspension of nanoparticles;
(c) preparing hollow mesoporous organic silicon nano particles by coating the mesoporous organic silicon obtained in the step (b) with solid SiO 2 Centrifuging suspension of nanoparticles to obtainWashing the solid with ethanol, dispersing the washed solid in an ethanol solution of concentrated hydrochloric acid, and heating to react to remove hexadecyl trimethyl ammonium chloride serving as a template; selectively etching solid SiO with ammonia water 2 The core of the nano-particles is used for obtaining hollow mesoporous organic silicon nano-particles;
(d)HOS-J R the preparation of (1):
dispersing hollow mesoporous organic silicon nano particles into a DMSO solution of JS-K by adopting a vacuum infusion method; then adding water into the solution under ultrasonic treatment; obtaining HOS-J by centrifuging and finally dissolving the product in water R ；
(e)HOS-L O Preparation of
Dispersing hollow mesoporous organic silicon nano particles and L-arginine in pure water, stirring at room temperature, centrifuging, and dissolving the final product in water to obtain HOS-Lo;
(f)HOS-J R L O preparation of
HOS-J is added R And L-arginine or HOS-L O And JS-K are dispersed in pure water and stirred at room temperature; obtaining HOS-J by centrifuging and finally dissolving the product in water R L O 。
5. The method for preparing responsive NO nano-drug according to claim 4, wherein in the step (a), the volume ratio of the hexadecyltrimethylammonium chloride, the water, the triethanolamine and the tetraethylorthosilicate is (1.8-2.0): (20-25): (0.06-0.1): (0.8-1.0); the reaction temperature is 95 ℃, and the reaction time is 1-2 hours.
6. The method for preparing responsive NO nano-drug according to claim 4, wherein in the step (b), the solid SiO is 2 Nanoparticle solution, bis- [ r- (triethoxysilyl) propyl ] n]The volume ratio of the tetrasulfide to the tetraethyl orthosilicate is 2:1 to-1: 1.
7. The method for preparing a responsive NO nano-drug according to claim 4, wherein in the step (c), the mass percentage of the concentrated hydrochloric acid in the ethanol solution is 5-15%.
8. The preparation method of the response type NO nano-drug according to claim 4, wherein in the step (d), the mass ratio of the hollow mesoporous organosilicon nano-particles to JS-K is 1:1-4:1, the concentration of JS-K in DMSO solution is 5-10g/L, the volume ratio of DMSO to water is 1:1-1:2, and the time of ultrasound is 5-15 minutes.
9. The preparation method of the responsive NO nano-drug according to claim 4, wherein in the step (e), the mass ratio of the hollow mesoporous organosilicon nanoparticles to the L-arginine is 1:10-1: 20.
10. The method for preparing responsive NO nano-drug according to claim 4, wherein in the step (f), HOS-J R And L-arginine or HOS-L o And JS-K is in a mass ratio of 1:10-1: 20.
11. Use of a responsive NO nano-drug according to any of claims 1 to 10 for the preparation of a medicament for the treatment of an ocular disease; the ocular disease is glaucoma or ocular hypertension.
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