Targeted release nanoparticles loaded with mangosteen shell extract as well as preparation method and application of targeted release nanoparticles

Abstract

The invention discloses targeted release nanoparticles loaded with mangosteen shell extract and a preparation method and application of the targeted release nanoparticles loaded with the mangosteen shell extract, the nanoparticles comprise mesoporous silica particles blocked by nanogold, and the mesoporous silica particles are loaded with the mangosteen shell extract in a wrapped mode. The nanogold is connected with an aptamer for specifically recognizing an amyloid protein oligomer in the brain through an Au-S bond. The nano-particles can target oligomer (AbetaO) of A beta protein to reach amyloid plaque, H2O2 with high concentration in the plaque can break boric acid ester bonds, the mangosteen shell extract and free nano-gold are released, the mangosteen shell extract and the free nano-gold can resist oxidation, scavenge free radicals and reduce aggregation and deposition of A beta, and the mangosteen shell extract and the free nano-gold can be used for preparing the nano-particles. Moreover, the nano-particles are large in drug loading capacity, accurate in targeting and high in release amount, can be used as a potential drug for treating the Alzheimer's disease, can effectively penetrate through a blood brain barrier after being prepared into a nasal spray, and is more beneficial to reduction of liquid medicine loss and rapid absorption.

Description

Technical Field

[0001] The invention relates to the field of metal nano material preparation, and specifically discloses a targeted release nanoparticle loaded with mangosteen shell extract, and a preparation method and application thereof. Background Art

[0002] Alzheimer's disease (AD) is a degenerative disease of the nervous system, also known as primary senile dementia. The pathological characteristics of AD are the appearance of amyloid plaques outside brain cells and neurofibrillary tangles inside brain cells. The β-sheet structure is one of the recurring secondary structures that is ubiquitous in proteins. Amyloid plaques are mainly composed of amyloid β polypeptide (Aβ). Aβ aggregation is the dominant factor leading to neurodegeneration in AD and plays an important role in the pathogenesis. Free radicals and peroxidation damage are involved in the process of Aβ aggregation and nucleation. Therefore, anti-oxidation and scavenging free radicals can reduce the aggregation and deposition of Aβ, which is a way to treat Alzheimer's disease.

[0003] Mangosteen (Garcinia mangostana L.), also known as mangosteen and phoenix fruit, is a tropical evergreen tree belonging to the genus Garcinia of the family Garcinia. The mangosteen shell, which has a deep color, mostly red-purple or dark purple, accounts for three-quarters of the fresh weight of mangosteen. Although it is often regarded as a discarded fruit shell, mangosteen shell actually contains rich medicinal and health values. The main component of mangosteen shell is xanthone (MSX), also known as xanthones, which is a heterotricyclic compound. It is characterized by containing a diphenylpyrrolidone mother nucleus, and the substituents are various, such as methoxy, hydroxyl, one or more C5 units, CIO units or cyclized furan rings and pyran rings. Common xanthone compounds include mangoside and mangostin, etc. α-, γ-mangostin (also known as α-, γ-mangostin) is a class of compounds widely used in the fields of food and medicine. Xanthones have antibacterial, antioxidant, and anti-inflammatory effects. The high reactivity of hydroxyl substitution can also phagocytize free radicals. Therefore, the xanthones in mangosteen shell extract can use this characteristic to resist oxidation and scavenge free radicals, which can reduce the aggregation and deposition of Aβ, thereby achieving the effect of treating Alzheimer's disease.

[0004] However, there is still a lack of drug-loaded nanoparticles that target Aβ protein oligomers (AβO) and can be targeted and released in the environment of amyloid plaques to affect Aβ aggregation. At the same time, the biggest challenge facing the treatment of neurological diseases is the obstruction of the blood-brain barrier, and solving the problem of drug crossing the blood-brain barrier has become the first problem to be overcome. Although the emergence of nanomedicine has brought hope to the development of neuroprotective agents, currently, brain-targeted peptides, transferrin and other modified nano-delivery carriers are mostly used to assist drugs in crossing the blood-brain barrier through active transport. However, due to systemic blood circulation loss and immune system clearance, the efficiency of drug entry into the brain is still extremely limited. Summary of the invention

[0005] The purpose of the present invention is to overcome the above-mentioned defects and shortcomings in the prior art and provide a targeted release nanoparticle.

[0006] The second object of the present invention is to provide a method for preparing the above-mentioned targeted release nanoparticles.

[0007] The third object of the present invention is to provide an application of the above-mentioned targeted release nanoparticles.

[0008] The fourth object of the present invention is to provide a nasal spray comprising the above-mentioned targeted release nanoparticles.

[0009] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0010] The invention provides a targeted release nanoparticle, which comprises a mesoporous silica particle blocked by nano-gold, wherein a mangosteen shell extract is encapsulated in the mesoporous silica particle, and an aptamer that specifically recognizes amyloid protein oligomers in the brain is connected to the nano-gold via an Au-S bond; the mesoporous silica is mesoporous silica modified with 4-carboxyphenylboronic acid, the nano-gold is monosaccharide-modified nano-gold, and the mesoporous silica modified with 4-carboxyphenylboronic acid forms a borate ester bond with beta-D-glucose on the nano-gold.

[0011] Mesoporous silica nanoparticles (MSNs) are microspheres with uniform pore size and ordered mesoporous structure. Monodisperse silica microspheres are usually synthesized by supramolecular assembly of surfactants. Surfactants will polymerize to form micelles when the critical micelle concentration is exceeded, and then the silicon source will condense on the surface of the micelles. The surfactant is then removed by solvent extraction or calcination to form mesopores. Mesoporous silica has the advantages of large specific surface area, large pore capacity, easy functional modification of the surface, good biocompatibility, ordered mesopores and stable skeleton structure. Mesoporous silica can load more drugs by virtue of its advantages. After targeting to the location, it can improve the therapeutic effect by increasing the concentration of the drug at the targeted location. In addition, nano-mesoporous silica has the ability to cross the blood-brain barrier, which is a very favorable carrier for the treatment of Alzheimer's disease.

[0012] Gold nanoparticles (AuNPs) are inert nanoparticles with good biocompatibility. Gold nanoparticles can make Aβ monomers form spherical oligomers and scattered short fibers with low toxicity. The surface charge and ligand of gold nanoparticles can target and bind to Aβ protein oligomers (AβO), which can affect the aggregation of Aβ. In amyloid plaques, H 2 O 2 The concentration in the middle is higher than that in other places, which can be used as the key to control the sustained release. Nanogold has good biocompatibility and can adjust the size of nanogold particles, so using nanogold as nanoparticles to block mesopores is a good choice. Mesoporous silica modified with 4-carboxyphenylboronic acid can form a boronate bond with β-D-glucose on nanogold to obtain MSN-AuNPs. After MSN-AuNPs loaded with mangosteen shell extract reach amyloid plaques, the higher concentration of H in the plaques 2 O 2 The borate ester bond can be broken to release the loaded mangosteen shell extract, and the freed gold nanoparticles can also affect the aggregation of Aβ, so that the free gold nanoparticles and the released mangosteen shell extract can achieve the purpose of treating Alzheimer's disease.

[0013] Aptamers, also known as nucleic acid aptamers, are composed of a single-stranded RNA or DNA. Aptamers can bind to different targets, such as proteins, metal ions, cells, viruses, etc. When aptamers act on small molecule compounds such as amino acids, they mainly bind by forming pseudo-connections, inner rings and protrusions, while for macromolecular compounds, the main forms of action are hydrogen bonds, polar groups, shapes and van der Waals forces. Compared with antibodies, nucleic acid aptamers have high affinity and high specificity for target molecules, and have the unique advantages of simple synthesis and strong stability, and can resist harsh detection environments such as high temperature, strong acid and strong alkali. The present invention modifies the aptamer that specifically recognizes amyloid oligomers (AβO) in the brain onto nanogold through the Au-S reaction to enhance the targeting of mesoporous silica nanoparticles. Nanogold is the key to controlling the sustained release of nanoparticles, and can affect the aggregation of Aβ. At the same time, it serves as an "anchor point" for connecting targeted aptamers, so there is no need for additional targeted modification on the surface of mesoporous silica nanoparticles.

[0014] Furthermore, the nucleotide sequence of the aptamer is 5′-SH-(CH 2 ) 6 -GCTGCCTGTGGTGTTGGGGCGGGTGCG-3′.

[0015] The present invention provides a method for preparing the above-mentioned targeted release nanoparticles, comprising the following steps:

[0016] S1. adding monosaccharide-modified gold nanoparticles into the aptamer solution to obtain aptamer gold nanoparticles (Apt / AuNPs);

[0017] S2. The mangosteen shell extract and the mesoporous silica modified with 4-carboxyphenylboronic acid were mixed, centrifuged, and washed to obtain mesoporous silica loaded with mangosteen shell extract (MSX / MSN-BA);

[0018] S3. The mesoporous silica loaded with mangosteen shell extract and the excess aptamer nanogold are mixed, centrifuged, and washed to obtain targeted release nanoparticles loaded with mangosteen shell extract (MSX / MSN-Apt / AuNPs).

[0019] Furthermore, the preparation method of the aptamer nanogold in step S1 is to mix the β-D-glucose solution and tetrachloroauric acid trihydrate, and add sodium borohydride solution to obtain monosaccharide-modified nanogold; dilute the aptamer with TE buffer, and add a thiol reducing agent (TCEP) to obtain an aptamer solution; mix the monosaccharide-modified nanogold with the aptamer solution, and incubate to obtain the aptamer nanogold.

[0020] Furthermore, the preparation method of the mesoporous silica modified with 4-carboxyphenylboronic acid in step S2 is to modify the mesoporous silica by amino modification and then modifying it with 4-carboxyphenylboronic acid.

[0021] Preferably, mesoporous silica is dissolved in an organic solvent and ultrasonically dispersed, and then 3-aminopropyltriethoxysilane is added and refluxed for 12 to 24 hours to obtain amino-modified mesoporous silica; the amino-modified mesoporous silica is dissolved in dimethyl sulfoxide for later use, 4-carboxyphenylboronic acid, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide are dissolved in dimethyl sulfoxide and stirred for reaction for 10 to 20 minutes, and then the dimethyl sulfoxide solution of the amino-modified mesoporous silica is added, and the reaction is stirred for 12 to 24 hours at room temperature, and the reaction is centrifuged and washed to obtain mesoporous silica modified with 4-carboxyphenylboronic acid.

[0022] Furthermore, in step S2, the mass ratio of the mangosteen shell extract to the mesoporous silica modified with 4-carboxyphenylboronic acid is 0.15-0.2:1.

[0023] Preferably, in step S2, the mass ratio of the mangosteen shell extract to the mesoporous silica modified with 4-carboxyphenylboronic acid is 0.17:1.

[0024] Furthermore, the preparation method of the mangosteen shell extract in step S2 is to mix the mangosteen shell with ethanol for extraction, extract with ethyl acetate, and then separate and purify the product by column chromatography.

[0025] Preferably, the preparation method of the mangosteen shell extract in step S2 is as follows: after crushing the mangosteen shell, according to the solid-liquid ratio of 60-65 g / mL, taking the mangosteen shell powder and adding it to 70-75% ethanol, ultrasonic extraction for 70-75 minutes, filtering, rotary evaporation and concentration, extracting the product with ethyl acetate, and then eluting with petroleum ether: acetone = 5:1 to obtain.

[0026] The present invention also provides a use of the above-mentioned targeted release nanoparticles in preparing a product for preventing and / or treating Alzheimer's disease.

[0027] The present invention provides a nasal spray, which includes the above-mentioned targeted release nanoparticles loaded with mangosteen shell extract. The cilia on the surface of the nasal mucosa serve as the main defense barrier of the respiratory tract. While removing inhaled allergens, dust and bacteria in mucus, they also have a significant impact on the retention time of drugs during nasal administration. The inside of the nasal cavity is also covered with a mucosa with a thickness of 2 to 4 mm, and its surface epithelial cells have a large number of microvilli (each mucosal epithelial cell contains about 400 microvilli). These microvilli enhance the surface area of ​​the mucosa and provide favorable conditions for drug absorption. There are abundant capillaries and lymphatic networks distributed below the nasal epithelial cells, and these structures provide sufficient nutrition and immune support for the nasal mucosa. After the drug is absorbed into the nasal capillaries, it can effectively penetrate the blood-brain barrier. After the drug is absorbed by the nasal administration, it can directly enter the systemic circulation, has the advantage of fast onset, is suitable for use in first aid and self-help, and the patient can take the medicine by himself, which is very convenient and suitable for long-term medication for chronic diseases. The droplets of the spray are small and uniform, which can effectively cover the inside of the nasal cavity, reduce the loss of liquid medicine, and promote rapid absorption, thereby improving bioavailability. This dosage form shows significant advantages in nasal administration.

[0028] Furthermore, the concentration of the mangosteen shell extract in the nasal spray is 0.5 to 0.05 mg / mL.

[0029] Preferably, the concentration of the mangosteen shell extract in the nasal spray is 0.1 mg / mL.

[0030] Furthermore, the nasal spray also contains 0.004-0.006 (g / mL)% chitosan, 0.04-0.06 (g / mL)% methyl-β-cyclodextrin, 2.5-2.6 (g / mL)% glycerol for injection, 0.01% chlorhexidine, 0.005-0.015 (v / v)%, and 0.04-0.06 (g / mL)% disodium edetate.

[0031] In order to improve the absorption rate of drugs by nasal mucosa, adding absorption enhancers is an important way. However, traditional enhancers can cause damage or irritation to the nasal mucosa to varying degrees. Chitosan is a product obtained by deacetylation of chitin extracted from the shells of crustaceans. The mechanism of chitosan's permeation promotion is the combined effect of increasing the intercellular space and biological adhesion, and it is non-toxic and non-irritating to the nasal mucosa. The solubilization effect of cyclodextrin can dissolve and extract the phospholipids in the biological membrane, thereby increasing the space between cells and promoting the absorption of drugs. Methylated-β-cyclodextrin has a stronger absorption-promoting effect than the parent cyclodextrin and hydroxypropyl-β-cyclodextrin. Methylated-β-cyclodextrin interacts with lipids or divalent ions on the mucosa to increase the permeability of the nasal mucosa. Cyclodextrin can inhibit the degradation of drugs by enzymes on the nasal mucosa, and at the same time has the effect of increasing the solubility of poorly soluble drugs.

[0032] Preferably, in addition to the mangosteen shell extract, the above nasal spray also includes 0.005g chitosan, 0.05g methyl-β-cyclodextrin, 2.56g glycerol for injection, 0.01mL 0.01% chlorhexidine, and 0.05g EDTA-2Na per 100mL.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] The targeted release nanoparticles loaded with mangosteen shell extract (MSX) provided by the present invention include nano-gold-blocked mesoporous silica particles, the mangosteen shell extract is encapsulated in the mesoporous silica particles, the nano-gold is connected to an aptamer that specifically recognizes amyloid protein oligomers in the brain through an Au-S bond, the mesoporous silica is 4-carboxyphenylboronic acid-modified mesoporous silica, the nano-gold is monosaccharide-modified nano-gold, and the 4-carboxyphenylboronic acid-modified mesoporous silica forms a boronate ester bond with β-D-glucose on the nano-gold. The nanoparticles can target oligomers (AβO) of Aβ protein to reach amyloid plaques, and the higher concentration of H in the plaques 2 O 2 It can break the borate ester bond to release mangosteen shell extract and free gold nanoparticles, both of which can not only resist oxidation, scavenge free radicals and reduce the aggregation and deposition of Aβ, but also have large drug loading capacity, precise targeting and high release amount. It can be used as a potential drug for the treatment of Alzheimer's disease. After being made into a nasal spray, it can effectively penetrate the blood-brain barrier and help the drug enter the cerebrospinal fluid. It has brain targeting, which is more conducive to reducing the loss of drug solution and rapid absorption. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Schematic diagram of MSN synthesis.

[0036] Figure 2 This is the working curve of α-mangostin.

[0037] Figure 3 In vitro release rate of MSN-AuNPs loaded with mangosteen shell extract.

[0038] Figure 4 is the relative motion percentage of cilia.

[0039] Figure 5 Optical micrograph of ciliary movement.

[0040] Figure 6 Optical micrograph showing the disappearance of ciliary motility.

[0041] Figure 7 The relationship between GSH concentration and OD and the standard curve and the relationship between dopamine concentration and peak current and the standard curve. Figure 7 A is the glutathione standard curve; B is the dopamine DPV curve; C is the dopamine standard curve.

[0042] Figure 8 The GSH content in zebrafish of different groups. Note: * indicates that compared with the model group, p < 0.1, the difference is significant; *** indicates that compared with the model group, p < 0.001, the difference is extremely significant. The model group is significantly different from the positive control group and the DMSO group.

[0043] Fig. 9 The DA content in the brain tissue of zebrafish in different groups. Note: * indicates that compared with the model group, p < 0.1, the difference is significant; *** indicates that compared with the model group, p < 0.001, the difference is extremely significant. The model group is significantly different from the positive control group and the DMSO group.

[0044] Fig.10 The AChE content in the brain tissue of zebrafish in different groups. Note: *** indicates that the difference is extremely significant compared with the model group, p<0.001. The model group is significantly different from the blank group, DMSO group and positive control group.

[0045] Fig.11 The spontaneous behavior trajectories of zebrafish in different groups. Note: a. Blank group; b. Model group; c. DMSO group; d. MSX low-dose group; e. MSX medium-dose group; f. MSX high-dose group; g. MSX nanoparticle low-dose group; h. MSX nanoparticle medium-dose group; i. MSX nanoparticle high-dose group; j. Positive group.

[0046] Fig.12 The swimming distance of zebrafish in different groups. Note: *** indicates that compared with the model group, p<0.001, the difference is extremely significant. The model group is significantly different from the blank group, DMSO group and positive control group.

[0047] Fig.13The immobility time of zebrafish in different groups. Note: *** indicates that compared with the model group, p<0.001, the difference is extremely significant. The model group is significantly different from the blank group, DSMO group and positive control group.

[0048] Fig.14 Detection of AChE activity in brain tissue of mice in different groups. Note: MSX1-3 are MSX 150, 300, 450 mg / kg / d gavage, nanoparticles 1-3 are MSX / MSN-Apt / AuNPs nanoparticles 150, 300, 450 mg / kg / d gavage, nasal sprays 1-3 are MSX / MSN-Apt / AuNPs nasal sprays 150, 300, 450 mg / kg / d nasal application. Note: * indicates that compared with the model group, p < 0.1, the difference is significant; ** indicates that compared with the model group, p < 0.05, the difference is relatively significant; *** indicates that compared with the model group, p < 0.001, the difference is extremely significant. The model group is significantly different from the positive control group and the DMSO group.

[0049] Fig.15 Detection of GSH activity in brain tissue of mice in different groups. Note: MSX1-3 are MSX 150, 300, 450 mg / kg / d gavage, nanoparticles 1-3 are MSX / MSN-Apt / AuNPs nanoparticles 150, 300, 450 mg / kg / d gavage, nasal sprays 1-3 are MSX / MSN-Apt / AuNPs nasal sprays 150, 300, 450 mg / kg / d nasal application. Note: * indicates that compared with the model group, p < 0.1, the difference is significant; *** indicates that compared with the model group, p < 0.001, the difference is extremely significant. The model group is significantly different from the positive control group and the DMSO group.

[0050] Fig.16 Detection of dopamine content in brain tissue of mice in different groups. Note: MSX1-3 are MSX 150, 300, 450 mg / kg / d gavage, nanoparticles 1-3 are MSX / MSN-Apt / AuNPs nanoparticles 150, 300, 450 mg / kg / d gavage, nasal sprays 1-3 are MSX / MSN-Apt / AuNPs nasal sprays 150, 300, 450 mg / kg / d nasal application. Note: *** indicates that compared with the model group, p<0.001, the difference is extremely significant. The model group is significantly different from the positive control group and the DMSO group. DETAILED DESCRIPTION

[0051] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0052] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0053] Experimental reagents: Petroleum ether (Ⅱ) and acetone were purchased from Guangzhou Chemical Reagent Factory; methanol was purchased from Merck KGaA; ethyl acetate was purchased from Tianjin Fuyu Fine Chemical Co., Ltd.; column chromatography silica gel (200 - 300 mesh) and thin layer chromatography silica gel plate (GF254) (50 * 200 mm) were purchased from Qingdao Ocean Chemical Co., Ltd. Aβ42 polypeptide and aptamer 5′-SH-(CH 2 ) 6 -GCTGCCTGTGGTGTTGGGGCGGGTGCG-3′ was purchased from Shanghai Gil Biochemical Co., Ltd.; HAuCl 4 ·3H 2 O (>99.9%), β-D-glucose, NaBH4, cetyltrimethylammonium bromide (CTAB), tetraethyl orthosilicate (TEOS), 3-aminopropyltriethoxysilane (APTES), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), N-hydroxysuccinimide (NHS), rhodamine B-methyl-β-cyclodextrin, chitosan, glycerol for injection, benzalkonium bromide, disodium ethylenediaminetetraacetate, absolute ethanol and sodium deoxycholate were all purchased from Sigma Company. 4-carboxyphenylboronic acid was purchased from Aladdin Reagent (Shanghai) Co., Ltd. Mangosteen was purchased from Guangzhou Panyu Fruit Market, tebuconazole powder was purchased from Bayer AG, α-mangostin standard was purchased from the reference substance experiment consumables center, glutathione kit was purchased from Nanjing Jiancheng Bioengineering Institute, dimethyl sulfoxide was purchased from Tianjin Kemi Scientific Reagent Co., Ltd.; adult AB strain zebrafish were provided by Nanjing Yishulihua Biotechnology Co., Ltd.; SPF-grade male KM mice, purchased from the Experimental Animal Center of Sun Yat-sen University, with a body weight of 20 ± 4 g and a batch number of: SCXK (Guangdong) 2023 - 001.

[0054] PBS buffer solution: Weigh 3.80 g of sodium dihydrogen phosphate and 0.504 g of disodium hydrogen phosphate, transfer them to a 100 mL volumetric flask, and make up to 100 mL with deionized water and let it stand for later use.

[0055] Normal saline: Weigh 4.5 g of sodium chloride, transfer it to a 500 mL volumetric flask, and make up to 500 mL with deionized water that has been heated to boiling and then cooled, and let it stand for later use.

[0056] 1% aqueous solution of sodium deoxycholate: Weigh 0.1 g of sodium deoxycholate and dissolve it in 10 mL of water.

[0057] All water used in the experiment is deionized water.

[0058] Example 1 Preparation of mangosteen shell extract (MSX)

[0059] The mangosteen shell was dried and crushed, and the mangosteen shell powder was added to 70.966% ethanol at a solid-liquid ratio of 62.303 g / mL, and ultrasonic extraction was performed for 70.725 minutes, and the mixture was filtered and concentrated by rotary evaporation to obtain a xanthone extract, namely, mangosteen shell extract (MSX).

[0060] The xanthone extract obtained by rotary evaporation is mixed with water, and then an equal volume of ethyl acetate is added, and the mixture is fully mixed by stirring to ensure that the target compound can be efficiently transferred to the ethyl acetate layer. Then, the mixed solution is allowed to stand, and the immiscibility of ethyl acetate and water is utilized to naturally separate the two, and most of the total xanthone compounds are transferred to the ethyl acetate layer. In order to further improve the extraction efficiency, the above extraction steps are repeated 3 to 4 times, and the ethyl acetate layer is collected separately each time. After multiple extractions are completed, all the collected ethyl acetate layers are combined to obtain a solution containing a high concentration of the target compound. Then, the combined ethyl acetate layer is evaporated and concentrated using a rotary evaporator, and the solvent is removed by heating and decompression, so that the total xanthone compounds are enriched to obtain an extract.

[0061] The obtained extract was separated and purified by column chromatography: petroleum ether: acetone = 5:1 was used for elution to obtain a solution containing the target product α-mangostin, the solution was evaporated and crystallized, dissolved in methanol for chromatography, passed through a microporous filter membrane, and finally stored in a refrigerator at 4°C.

[0062] When extracting xanthone, the ethanol concentration used is 70.966%, the solid-liquid ratio is 62.303 g / mL, and the extraction time is 70.725 minutes. Under this condition, the extraction rate of xanthone is 4.527%. A mixed solution of petroleum ether and acetone, i.e., petroleum ether:acetone=5:1, is used to elute the ethyl acetate part. Under this condition, the content of alpha-mangostin in the eluent is 95.8%.

[0063] Example 2 Preparation of targeted release nanoparticles (MSN-AuNPs) loaded with mangosteen shell extract (MSX)

[0064] Preparation process of MSN-AuNPs:

[0065]

[0066] (1) Preparation of mesoporous silica (MSN)

[0067] The schematic diagram of MSN preparation process is as follows Figure 1As shown, 0.1 g of hexadecyltrimethylammonium bromide (CTAB) and 3.5 mL of 0.2 M NaOH were added to 48 mL of water, the solution was heated to 85 ° C, and then stirred vigorously for 1 hour using a stirring paddle. After 1 hour, 0.5 mL of tetraethoxysilane (TEOS) was added dropwise and continued to stir vigorously for 1 hour. Mesoporous silica was obtained by centrifuging at 5500 rpm for 30 minutes, washed several times with ethanol and then removed by centrifugation. The mesoporous silica was heated to reflux in HCl / methanol for 24 hours to remove CTAB as a surfactant. Finally, the mesoporous silica was obtained by centrifuging at 5500 rpm for 30 minutes and then vacuum dried at 60 ° C for 10 hours to remove the solvent in the mesopores.

[0068] (2) Preparation of 4-Carboxyphenylboronic acid modified mesoporous silica (MSN-BA)

[0069] 1.0 g of mesoporous silica was added to 100 mL of anhydrous ethanol, and 1.0 mL of 3-aminopropyltriethoxysilane (APTES) was added after ultrasonication for 5 min, and then heated under reflux for 24 h to modify 3-aminopropyltriethoxysilane on the surface of MSN to obtain MSN-NH 2 .

[0070] 240 mg purified MSN-NH 2 Dissolve in 12 mL of dimethyl sulfoxide (DMSO). Add 0.090 g of 4-carboxyphenylboronic acid, 0.060 g of N-hydroxysuccinimide (NHS) and 0.120 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) to 3.0 mL of DMSO, then stir and react for 15 min, then add to the above MSN-NH 2 The solution was stirred at room temperature for 24 hours, and then centrifuged at 5500 rpm for 30 minutes, and then washed with DMSO, anhydrous ethanol and deionized water to obtain MSN modified with 4-carboxyphenylboronic acid (MSN-BA).

[0071] (3) Preparation of aptamer gold nanoparticles (Apt / AuNPs)

[0072] Soak the glassware needed in aqua regia overnight, then rinse with deionized water and dry. Then, mix 50 mL of β-D-glucose solution (0.05 M) with 0.20 mL of tetrachloroauric acid trihydrate (HAuCl 4 ·3H 2 O) solution (0.05 M) was mixed evenly, and then 0.60 mL of freshly prepared sodium borohydride (NaBH 4) solution (0.05M). When the solution gradually turns into wine red, it can be determined that gold nanoparticles (AuNPs) are formed. The wine red solution is placed in a volumetric flask, sealed, and placed in a 4°C refrigerator for later use.

[0073] The aptamer was diluted to 20 μM with TE-Buff buffer solution, and 40 μL was placed with 40 μL of 10 mM thiol reducing agent TCEP at 37°C for 1 hour to reduce disulfide bonds and promote the formation of Au-S bonds. The mixture was diluted to 400 μL with PBS. Nanogold was dropped into the above aptamer solution and incubated at 4°C for 18 hours. Hexamercaptohexanol (MCH) blocked nonspecific binding sites to obtain Apt / AuNPs.

[0074] (4) Preparation of MSN-Apt / AuNPs loaded with mangosteen shell extract (MSX)

[0075] 0.034 g of mangosteen shell extract was dissolved in 100 mL of anhydrous ethanol, and 50 mL was taken and stirred with 100 mg of MSN-BA for 24 h. The MSN-BA loaded with mangosteen shell extract was centrifuged at 5500 rpm for 30 min, and the supernatant was retained. The solid MSX / MSN-BA loaded with mangosteen shell extract was washed several times with anhydrous ethanol to wash the MSX adsorbed on the surface. The MSX / MSN-BA was dissolved in 50 mL of anhydrous ethanol, and an excess of Apt / AuNPs solution was added and stirred for 12 h. The precipitate was washed with anhydrous ethanol several times, and the precipitate was dried with nitrogen to obtain MSX / MSN-Apt / AuNPs particles.

[0076] Example 3 Determination of MSN Embedding Rate and Drug Release Rate in Vitro

[0077] (1) Preparation of α-mangostin working curve

[0078] Working curve of α-mangostin: prepare 2.00μg / mL, 4.00μg / mL, 6.00μg / mL, 8.00μg / mL, and 10.0μg / mL ethanol standard solutions of α-mangostin, use an ultraviolet spectrophotometer to measure the absorbance values ​​A of a series of standard solutions, fit the working curve with AC, and obtain the linear regression equation.

[0079] (2) Weigh 0.017 g of MSX / MSN-Apt / AuNPs particles and dissolve them in 50 mL of anhydrous ethanol. Take 5 mL of the above solution and dilute it with anhydrous ethanol to 1 / 3 of the original concentration. Use ultraviolet spectrophotometry at a wavelength of 318 nm to measure the concentration of xanthone. After the supernatant left for preparing MSX / MSN-Apt / AuNPs is diluted to 1 / 3 of the original concentration, the concentration of xanthone in the dilution is measured by ultraviolet spectrophotometer. The encapsulation rate of mesoporous silica for xanthone is calculated according to formula (1).

[0080]

[0081] C 原 : Xanthones concentration in mangosteen shell extract, C 后 : Xanthones content in the solution of mangosteen shell extract embedded in silica.

[0082] (3) 10 mg of MSN-BA loaded with mangosteen shell extract was dissolved in 10 mL of anhydrous ethanol and 1 mM H 2 O 2 Treatment: 10 mg of MSN-BA loaded with mangosteen shell extract was dissolved in 10 mL of anhydrous ethanol and treated with 5 mM H 2 O 2 Treatment: 10 mg of MSX / MSN-Apt / AuNPs powder was dissolved in 10 mL of anhydrous ethanol and treated with 1 mM H 2 O 2 Treatment: 10 mg of MSX / MSN-Apt / AuNPs powder was dissolved in 10 mL of anhydrous ethanol and 5 mM H 2 O 2 Treatment: The above solution was sealed and placed in a digital constant temperature water bath oscillator at 37°C for continuous shaking for 5 hours. After each incubation period, the supernatant was centrifuged and the content of xanthones in the mangosteen shell extract released from the supernatant was determined by ultraviolet spectrophotometer. The absorption peak of xanthones was at 318 nm. The in vitro release rate of the drug was calculated according to formula (2).

[0083]

[0084] The results are as follows Figure 2 As shown in Table 1, the absorbances of 0.00, 2.00, 4.00, 6.00, 8.00, and 10.0 μg / mL concentrations were measured by UV spectrophotometer to be 0, 0.1134, 0.2382, 0.3385, 0.4599, and 0.5767 A, respectively. The standard curve was fitted with absorbance values ​​versus concentrations, and the linear regression equation was A=0.0575C+0.0004, R 2 =0.9995.

[0085] Table 1 Absorbance record of different concentrations of α-mangostin

[0086]

[0087] Calculation of the embedding rate of α-mangostin by mesoporous silica: The result measured by UV spectrophotometer was that the absorbance value before loading was 0.8546A, and the absorbance value of the supernatant after loading was 0.7593A. After processing with the α-mangostin working curve, it was found that the concentration of α-mangostin in the supernatant before embedding was 44.57μg / mL, and the concentration after embedding was 31.54μg / mL, and the embedding rate was calculated to be 29.27%.

[0088] The in vitro release rate of the drug is: The data was measured using an ultraviolet spectrophotometer and processed and calculated. The results are shown in Table 2.

[0089] Table 2 In vitro release rate of MSN-AuNPs loaded with mangosteen shell extract

[0090]

[0091] like Figure 3 As shown in Table 2, AuNPs can effectively block MSNs without using H 2 O 2 In the case of treatment with HCl, almost no mangosteen shell extract was released. Comparing the release of the MSN-BA group and the MSN-AuNPs group, AuNPs can achieve a sustained release effect, and the loaded mangosteen shell extract is gradually released into the solution over time. In addition, because there is no AuNPs blocking in the MSN-BA group, regardless of whether H 2 O 2 The release effects of the MSN-AuNPs group were similar when treated with H 2 O 2 As the concentration of treatment increases, the rate of release also increases.

[0092] Example 4 Preparation of mangosteen shell nasal spray and cilia toxicity test

[0093] (1) Preparation of mangosteen shell nasal spray

[0094] Take 3 portions, chitosan 0.005g, methyl-β-cyclodextrin 0.05g, glycerol for injection 2.56g, 0.01% chlorhexidine 0.01mL, 0.05g EDTA-2Na, and then place the three portions of drugs with 100mg, 500mg, 1000mg MSX / MSN-Apt / AuNPs in a 100mL volumetric flask, add deionized water to the 100mL mark. Prepare nasal sprays with 1.00mg / mL, 0.500mg / mL, and 0.100mg / mL negative MSX / MSN-Apt / AuNPs concentrations, and then each is divided into 10mL medical direct spray bottles.

[0095] (2) Cilia toxicity test of mangosteen shell nasal spray

[0096] The in vitro method was used to determine the duration of the movement of the bullfrog palate cilia. The bullfrog was killed by the double myelination method. The palate mucosa was removed with ophthalmic surgical scissors, and blood clots and debris were washed with physiological saline. A 3×3 mm 2 The palatal mucosa was placed flat on a glass slide with the ciliary surface facing up. A 1% sodium deoxycholate aqueous solution with severe ciliary toxicity was used as a positive control group, and saline was used as a negative control group. 0.1 mL of the drug solution was dripped on the surface of the mucosa. Every 3 mucosa were used as a group, for a total of 5 groups. Then the coverslip was gently covered, and then a 150x (15×10) optical microscope was used to observe the dynamic activity of the cilia in detail. Next, the sample was placed in a chromatography cylinder containing a small amount of deionized water, and the chromatography cylinder was ensured to be well sealed so that the internal water vapor reached a state close to saturation, while maintaining the room temperature at 22°C. During the experiment, the sample was taken out at appropriate time intervals to observe the movement of the cilia. If the cilia were still observed to be moving, they were put back into the chromatography cylinder, and this process was continued until the cilia stopped moving completely. At this time, the total time from the start of drug administration to the cessation of cilia movement was recorded. In order to evaluate the effect of drugs on ciliary motion, the ratio of the duration of ciliary motion in the drug group to that in the control group was calculated, and this ratio was converted into a relative motion percentage. The higher this percentage is, the smaller the effect of the drug on ciliary motion is, that is, the longer the duration of ciliary motion under the action of the drug is.

[0097] The results are as follows Figures 4 to 6As shown, compared with the saline solution of the negative control group, the relative movement percentage of the drug administration group was reduced. The duration from the start of drug administration to the cessation of cilia movement in the saline group was (121±9) min, and the relative movement percentage was 100%. The duration of the drug concentration of 0.1 mg / mL was (46±7) min, and the relative movement percentage was 38%. The duration of the drug concentration of 0.5 mg / mL was (27±11) min, and the relative movement percentage was 22%. The duration of the drug concentration of 1 mg / mL was (13±5) min, and the relative movement percentage was 11%. The duration of the 1% sodium deoxycholate aqueous solution as the positive control group from the start of drug administration to the cessation of cilia movement was (0±0) min, and the relative movement percentage was 0%. The cilia stopped swinging immediately when the drug was dripped. The results show that the drug concentration of 1 mg / mL has less ciliary toxicity, and as the drug concentration increases, the ciliary toxicity also increases, which means that the irritation to the nasal cavity also increases. Therefore, it can be considered that 0.1 mg / mL mangosteen shell extract nasal spray will not have obvious ciliary toxicity, but with the increase of drug concentration, ciliary toxicity increases. This result can provide basic data for drug dosage exploration and conduct preliminary experiments for drug development and utilization.

[0098] Example 5 Repair effect of total xanthone on oxidative damage in zebrafish

[0099] 1. Experimental Methods

[0100] (1) Establishment of zebrafish oxidative damage model

[0101] Adult zebrafish of the AB strain were provided by Nanjing Yishulihua Biotechnology Co., Ltd.

[0102] Take 25 zebrafish and divide them into 5 groups and put them into culture dishes. Each group of 5 zebrafish is exposed to 0.0mg / L, 0.5mg / L, 0.7mg / L, 1.0mg / L, and 2.0mg / L of tebuconazole in 0.5L solution. After 2 days of exposure, the number of dead individuals in each group is recorded and the non-lethal concentration is recorded.

[0103] (2) Determination of glutathione content in zebrafish liver tissue

[0104] Place the zebrafish in the ice-water mixture for 10 minutes. After carefully observing and seeing that the zebrafish stops moving, place it on a foam board, fix the zebrafish's eyes and tail, start from the zebrafish's pelvic fin, cut the zebrafish's abdomen to the head, then cut along the spine to the tail, cut down to the excretory hole, and pick open the abdominal skin to clearly observe the zebrafish's visceral structure. After cleaning the watch glass, drip a few drops of saline into it, then place the visceral mass in the saline drop in the watch glass, use a dissecting needle and tweezers to separate the visceral mass, take only the liver part, and place the separated liver in a centrifuge tube with saline added. Place the centrifuge tube with liver tissue in a constant temperature water bath at 35 degrees Celsius to ensure tissue activity, and place it for later use.

[0105] Take out the prepared zebrafish liver tissue from the centrifuge tube, rinse it with ice salt water, dry it with absorbent paper, weigh it and record the data, put it in a grinding mortar, add an appropriate amount of homogenate transmitter, and grind it manually in an ice water bath for 5 minutes to homogenize the tissue. Pour the homogenate into a 1.5mL centrifuge tube, centrifuge it at 2500 rpm for 10 minutes, and take the supernatant for measurement.

[0106] According to the instructions of the glutathione detection kit, prepare four corresponding reagents and configure a 20μmol / L GSH standard solution in proportion. According to the previously established preparation method of 10% tissue homogenate, obtain the supernatant. Specifically, take 0.5mL of 10% tissue homogenate, mix it with 2.0mL of reagent 1, centrifuge it at 3500r / min for 10min, and retain the supernatant for testing. Add each reagent according to the instructions of the kit, mix well and let it develop color for 5min. Use a UV-visible photometer at 420nm, 1cm light path and double distilled water to zero the conditions, measure the OD value of each test tube, and record the data.

[0107] (3) Determination of dopamine (DA) content in zebrafish brain tissue

[0108] Cut off the zebrafish's head, use ophthalmic scissors to cut along the spine toward between the zebrafish's eyes to expose the brain tissue, use a dissecting needle to separate the brain tissue from the skull, and place the brain tissue in a centrifuge tube with saline added after separation; place the centrifuge tube with brain tissue in a constant temperature water bath at 35 degrees Celsius to ensure tissue activity, and set aside for later use.

[0109] Take out the prepared zebrafish brain tissue from the centrifuge tube, rinse with ice salt water, dry with absorbent paper, weigh and record the data, put it in a grinding mortar, add an appropriate amount of artificial cerebrospinal fluid, and grind it manually in an ice water bath for 5 minutes to homogenize the tissue. Pour the homogenate into a 1.5mL centrifuge tube, centrifuge at 2500 rpm for 10 minutes, and take the supernatant for measurement.

[0110] Take 1 mL of the brain tissue supernatant prepared in the above steps into a beaker, and add 19 mL of artificial brain tissue as the test sample. Using a CHI660E electrochemical workstation, a saturated calomel electrode reference electrode, a platinum sheet counter electrode, and the prepared modified carbon fiber electrode as the working electrode, differential pulse voltammetry (DPV) was used as the determination method, and the determination was performed under the conditions of a starting voltage of -0.3 V, a termination voltage of 0.6 V, a pulse amplitude of 50 mV, a pulse width of 0.2 s, and a pulse period of 0.5 s, and the peak height was recorded, as shown in formula (3).

[0111]

[0112] (4) Observation of zebrafish movement behavior

[0113] Take 5 zebrafish from each group and place each in a culture dish. After the zebrafish is transferred to the culture dish, wait for it to adapt to the culture dish environment for 2-3 minutes, and then take a motion capture picture with a video length of 2 minutes. Use ImageJ motion tracking software to measure swimming activity at a speed of 25 frames per second, and capture swimming movements in a predefined venue (one venue corresponds to one hole with an inner diameter of approximately 120 mm). Set detection variables for each animation file to ensure optimal detection of all fry. Subsequently, use ImageJ software to analyze the data and provide static time and movement distance parameters to characterize the swimming activity of individuals. Compare the results and record the experimental data.

[0114] (5) Take 60 zebrafish and divide them into 10 groups and place them in a culture dish, with 6 zebrafish in each group in 1.0L solution, which are respectively called blank group, 1% DMSO group, oxidative stress model group, positive control group and its low-dose, medium-dose and high-dose groups of MSX; low-dose, medium-dose and high-dose groups of MSX / MSN-Apt / AuNPs (nanoparticles). The blank group was placed in deionized water; the DMSO group was placed in 1% DMSO solution; the oxidative stress model group was placed in 0.5 mg / L tebuconazole solution, and the water or solution was changed every 2 days for a period of 4 days; then the blank group and 1% DMSO group repeated the above steps, the model group was placed in 1% DMSO solution, the positive group was placed in 50 mg / L vitamin C 1% DMSO solution, and the remaining groups were respectively given drugs according to low (10 mg / L), medium (25 mg / L), and high (50 mg / L) doses (the drug was dissolved in 1% DMSO), and the solution was changed every 2 days for a dosing period of 7 days. The drug administration conditions are shown in Table 3.

[0115] Table 3 Dosage regimen of drugs for repairing oxidative stress in zebrafish

[0116]

[0117] Five fish were selected from each group to measure their 2-min motion trajectory and its parameters, and the data were recorded. The glutathione content in the liver tissue and the dopamine and acetylcholinesterase content in the brain tissue of each group of zebrafish were measured according to the above method as indicators of oxidative damage repair.

[0118] 2. Experimental Results

[0119] (1) Establishment of zebrafish oxidative damage model

[0120] The results are shown in Table 4. When the concentration of tebuconazole was 0.5 mg / L, the number of deaths was 0 and the mortality rate was 0%, reaching a non-lethal concentration. Therefore, 0.5 mg / L of tebuconazole was used for subsequent detection of oxidative damage indicators.

[0121] Table 4 Tebuconazole dosage concentration and its lethality

[0122]

[0123] The results are as follows Figure 7 As shown in the figure, the concentration of GSH showed a good linear relationship with the OD value in the range of 0 μmol / L to 100 μmol / L. The regression equation of GSH was: y=0.0037x-0.0011, the ordinate was the OD value, the abscissa was the glutathione concentration (μmol / L), R 2 =0.9987. In a phosphate buffer solution with pH=8.0, the scan rate was adjusted to 10mv / s and scanned from -0.4v to 0.6v. The concentration of dopamine showed a good linear relationship with the peak current in the range of 1.0μg / L to 20.0μg / L. The regression equation was: y=1.2842x-0.53623, the ordinate was the dopamine concentration in μg / L, and the abscissa was the peak current in A. The correlation coefficient R 2 =0.9956.

[0124] (2) Therapeutic effects of different concentrations and administration forms on oxidative damage in zebrafish

[0125] The results are as follows Figure 8As shown, the GSH mass / liver weight (mg / 100g) of zebrafish liver tissue in the model group decreased by 34.9% compared with the DMSO group, and the difference between the two groups was extremely significant, indicating that the model was successfully established. The different doses of MSX administration groups increased by 12.1%, 23.1% and 32.8% respectively compared with the model group; there was a significant difference between the MSX medium dose group and the model group, and there was an extremely significant difference between the MSX high dose group and the model group; the different doses of nanoparticle administration groups increased by 25.8%, 35.0% and 49.6% respectively compared with the model group, and there were extremely significant differences between the three groups and the model group; the positive group increased by 47.8%, which was also extremely significant compared with the model group. The results showed that total xanthone and MSX / MSN-Apt / AuNPs (nanoparticles) can improve the decrease of GSH caused by oxidative damage in zebrafish caused by tebuconazole, and the therapeutic effect of MSX / MSN-Apt / AuNPs (nanoparticles) is better than that of the total xanthone treatment group (compared with the same dose group). In addition, there is no significant difference between the DMSO group and the blank group. This concentration of solvent is non-toxic and will not affect the experimental results.

[0126] The results are as follows Fig. 9 As shown, the dopamine content in the model group increased by 31.7% compared with the DMSO group, and there was a very significant difference between the two groups, indicating that the model was successfully established. The dopamine content in the zebrafish brain tissue of the MSX administration groups with different doses decreased by 3.5%, 4.5% and 7.4% respectively compared with the model group; there was a significant difference between the MSX high-dose group and the model group, while there was no significant difference between the low-dose and medium-dose groups; the dopamine content in the zebrafish brain tissue of the nanoparticle administration groups with different doses decreased by 12.0%, 19.6% and 27.6% respectively compared with the model group, and there were very significant differences between the three groups and the model group; the positive group decreased by 21.9%, which was also very significantly different from the model group, indicating that the positive drug has an inhibitory effect on the increase of dopamine caused by oxidative stress. The dopamine concentration in the drug-treated group decreased compared with the model group, indicating that the drug has an inhibitory effect on the increase of dopamine caused by oxidative stress, and the effect of MSX / MSN-Apt / AuNPs (nanoparticles) on dopamine content is better than that of the total xanthone extract group. In addition, there is no significant difference between the DMSO group and the blank group. This concentration of solvent is non-toxic and does not affect the experimental results.

[0127] The results are as follows Fig.10As shown, the acetylcholinesterase content in the model group increased by 54.8% compared with the DMSO group, and there was a very significant difference between the two groups, indicating that the model was successfully established. The acetylcholinesterase content in the zebrafish brain tissue of the MSX administration groups with different doses decreased by 8.3%, 17.7% and 27.1% respectively compared with the model group, and there were very significant differences between the three MSX dose groups and the model group, indicating that MSX had an inhibitory effect on the increase of AChE caused by oxidative stress; the acetylcholinesterase content in the zebrafish brain tissue of the nanoparticle administration groups with different doses decreased by 15.6%, 28.1% and 37.5% respectively compared with the model group, and there were very significant differences between the three groups and the model group, indicating that nanoparticles had an inhibitory effect on the increase of AChE caused by oxidative stress; the positive group decreased by 21.9% compared with the model group, and there was a very significant difference between the two groups, indicating that positive drugs had an inhibitory effect on the increase of AChE caused by oxidative stress. The effect of MSX / MSN-Apt / AuNPs (nanoparticles) on AChE content was better than that of the total xanthone extract group. In addition, there was no significant difference between the DMSO group and the normal group, which can be considered that the solvent DMSO did not affect the experimental results.

[0128] The movement trajectory and movement parameters of zebrafish within 2 minutes are as follows Figures 11 to 13 As shown in the figure, the zebrafish in the model group preferred to swim in the central area and were more active, while the zebrafish in the drug-treated group were relatively quiet and less active. Fig.11 It can be seen that the model group has the longest movement distance, and its movement distance is significantly higher than that of the blank group and the DMSO group. There is no significant difference in the movement distance of the drug group, the blank group and the DMSO group; the model group has the shortest immobility time, which is significantly lower than that of the blank group (DMSO group), while the immobility time of the drug group is not significantly different from that of the blank group (DMSO group), indicating that total xanthone and MSX / MSN-Apt / AuNPs (nanoparticles) have a significant repair effect on oxidative stress. The results show that the zebrafish in the blank group (DMSO group) have normal activity patterns and swim in the observation hole at a constant speed. The movement behavior of zebrafish in the model group is enhanced, and the administration of total xanthone and MSX / MSN-Apt / AuNPs (nanoparticles) will restore the behavior of zebrafish. In addition, there is no significant difference between the DMSO group and the blank group, and it can be considered that solvent toxicity will not affect the experimental results.

[0129] Example 6 Repair effect of total xanthone on oxidative damage in mice

[0130] 1. Experimental Methods

[0131] (1) Mouse breeding

[0132] SPF-grade male KM mice were purchased from the Experimental Animal Center of Sun Yat-sen University, with a body weight of 20±4 g and a batch number of: SCXK(Yue) 2023-001. All animal experiments were completed in an SPF-grade laboratory. Breeding conditions: temperature was 24±2 °C, and relative humidity was 45%±10%.

[0133] (2) Determination of acetylcholinesterase content in mouse brain tissue

[0134] AChE is mainly synthesized in neuron cell bodies. Cholinergic fibers and cholinesterase in the brain are mainly present in the hippocampal-limbic system. The blood-brain barrier (BBB) in the healthy human brain is a major obstacle to drug treatment in neurological diseases. The physicochemical properties of drugs, such as lipophilicity and molecular weight, determine whether the drugs can cross the BBB. Drugs or compounds with low molecular weight (less than 400 Da) that do not ionize at physiological pH and have lipophilicity can cross the BBB through the diffusion mechanism. Other essential compounds, such as amino acids, neuropeptides, sugars, and hexoses, usually require special carriers to cross the BBB.

[0135] All electrochemical performance tests were carried out in an electrochemical workstation CHI660E, using a three-electrode system. A graphite electrode was used as the working electrode, an Ag / AgCl electrode was used as the reference electrode, and a platinum plate electrode was used as the counter electrode. The three electrodes were immersed in a mixed electrolyte solution of 10 mM Fe(CN) 6 3- / 4- and 1.0 M KCl for testing. Before each test, nitrogen was introduced into the electrolyte solution 2 to exclude the influence of dissolved oxygen. In the cyclic voltammetry (CV) detection, the scan rate was 0.1 V / s each time, and the scanning voltage was controlled in the range of -1 V to 1.5 V; while in the electrochemical impedance spectroscopy (EIS) detection, the scanning frequency was controlled in the range of 0.1 HZ - 50 kHz each time. After incubation with different concentrations of acetylcholinesterase, the change in impedance value (△Rct / Rct0) was recorded and used to evaluate the concentration of acetylcholinesterase.

[0136] Mouse brain tissue homogenate was prepared, centrifuged at 2500 rpm for 10 min, and the supernatant was diluted with artificial cerebrospinal fluid as the test solution. The content of acetylcholinesterase in the brains of mice in each group was detected by the above method.

[0137] (3)Determine the content of glutathione and dopamine in mice by the method of measuring glutathione and dopamine in Example 5.

[0138] (4)Grouping and administration for Alzheimer's disease

[0139] After one week of adaptive feeding, the mice were randomly divided into 13 groups, namely blank group, 1% DMSO group, Alzheimer's disease model group, positive control group (huperzine A), low-dose, medium-dose and high-dose groups of xanthone extract, low-dose, medium-dose and high-dose groups of MSX / MSN-Apt / AuNPs (nanoparticles), low-dose, medium-dose and high-dose groups of MSX / MSN-Apt / AuNPs (nanoparticles) nasal spray, low-dose, medium-dose and high-dose groups, with 20 mice in each group. The model group, positive group and xanthone extract group were intraperitoneally injected with 1 mg / kg scopolamine hydrobromide 30 minutes after daily gavage; the MSX / MSN-Apt / AuNPs (nanoparticles) were intraperitoneally injected with 1 mg / kg scopolamine hydrobromide 30 minutes after daily gavage; the MSX / MSN-Apt / AuNPs (nanoparticles) nasal spray group was intraperitoneally injected with 1 mg / kg scopolamine hydrobromide 30 minutes after nasal application. Except for the blank group, which was intraperitoneally injected with 0.9% saline, the 1% DMSO group, model group, positive group, xanthone extract, MSX / MSN-Apt / AuNPs (nanoparticles) and xanthone nasal spray groups were intraperitoneally injected with 1 mg / kg scopolamine hydrobromide, with an injection volume of 0.1 mL / 10 g. The dosing conditions are shown in Table 5. During the experiment, the mice were weighed every two days, and the corresponding dosage was adjusted according to the changes in their body weight, and the drug was administered continuously for 14 days.

[0140] Table 5 Grouping and dosing regimen

[0141]

[0142] 2. Experimental Results

[0143] The acetylcholinesterase content in the brain tissue of each group of mice was detected. Fig.14As shown, compared with the DMSO group, the acetylcholinesterase content in the model group increased by 75.0%, and there was a very significant difference between the two groups, indicating that the model was successfully established. There was no difference in the acetylcholinesterase content between the blank group and the DMSO group, indicating that this concentration of DMSO had no effect on the acetylcholinesterase content in mice. Compared with the model group, the acetylcholinesterase content in the positive group decreased by 28.6%, and there was a very significant difference between the two groups. The positive drug is effective in reducing the acetylcholinesterase content in the mouse AD model. The acetylcholinesterase content in the three doses of MSX group was reduced by 6.6%, 12.1% and 17.6% respectively compared with the model group, among which the low dose group had no significant difference with the model group, the medium dose group had a significant difference with the model group, and the high dose group had a very significant difference; the acetylcholinesterase content in the three doses of MSX / MSN-Apt / AuNPs was reduced by 14.3%, 22.0% and 25.3% respectively compared with the model group, among which the low dose group had a significant difference with the model group, and the medium dose group and the high dose group had a very significant difference. There were extremely significant differences compared with the model group; the acetylcholinesterase content in the three doses of MSX / MSN-Apt / AuNPs nasal spray group was reduced by 27.5%, 36.3% and 45.1% respectively compared with the model group. There were extremely significant differences between the three dose groups and the model group, indicating that it has the effect of significantly inhibiting the activity of acetylcholinesterase. MSX / MSN-Apt / AuNPs nasal spray inhibits acetylcholinesterase activity better than MSX and MSX / MSN-Apt / AuNPs treatment groups. The present invention modifies the aptamer that specifically recognizes amyloid oligomers in the brain onto the surface of nanogold particles through the Au-S reaction, and MSX is blocked in the mesopores of mesoporous silica by nanogold, and is prepared into a nasal spray at the same time, which further enhances the targeting of the drug. The high content of active oxygen in the brain of AD mice is used to open the mesopores of silica and release MSX. Therefore, the AChE content in the brain tissue of mice in the MSX / MSN-Apt / AuNPs nasal spray group is the lowest compared with the MSX group, and the inhibitory effect on AChE is stronger.

[0144] The results are as follows Fig.15As shown, the GSH mass / liver weight (mg / 100g) of the liver tissue of mice in the model group decreased by 36.30% compared with the DMSO group, and there was a very significant difference between the two groups, indicating that the model was successfully established. There was no difference in the GSH content of the liver tissue of mice in the blank group and the DMSO group, indicating that this concentration of DMSO had no effect on the GSH content of the liver tissue of mice. Compared with the model group, the GSH content of the liver tissue of mice in the positive group increased by 48.4%, and there was a very significant difference between the two groups. The positive drug is effective in increasing the GSH content of the liver tissue of the mouse AD model. The GSH content in the liver tissue of mice in the three doses of MSX group was increased by 4.4%, 8.6% and 17.6% respectively compared with that in the model group. There was no significant difference between the low dose group and the model group, while there were significant differences between the medium dose group and the high dose group. The GSH content in the three doses of MSX / MSN-Apt / AuNPs group was increased by 13.3%, 19.9% ​​and 26.2% respectively compared with that in the model group. There were extremely significant differences between the three dose groups and the model group. The GSH content in the three doses of MSX / MSN-Apt / AuNPs nasal spray group was increased by 13.3%, 19.9% ​​and 26.2% respectively compared with that in the model group. The amounts of MSX / MSN-Apt / AuNPs (nanoparticles) and MSX / MSN-Apt / AuNPs (nanoparticles) nasal sprays were increased by 38.4%, 46.8% and 50.8% respectively. There were extremely significant differences between the three dosage groups and the model group. The results showed that total xanthone, MSX / MSN-Apt / AuNPs (nanoparticles) and MSX / MSN-Apt / AuNPs (nanoparticles) nasal spray groups all had certain therapeutic effects on AD mice, and the therapeutic effect of MSX / MSN-Apt / AuNPs nanoparticle nasal spray was better than that of total xanthone extract and MSX / MSN-Apt / AuNPs (nanoparticles) treatment groups.

[0145] The results are as follows Fig.16As shown in the figure, the dopamine content in the cerebrospinal fluid of mice in the model group decreased by 66.2% compared with the DMSO group, and there was a very significant difference between the two groups, indicating that the model was successfully established. There was no difference in the dopamine content in the cerebrospinal fluid of mice in the blank group and the DMSO group, indicating that this concentration of DMSO had no effect on the dopamine content in the cerebrospinal fluid of mice. Compared with the model group, the dopamine content in the cerebrospinal fluid of mice in the positive group increased by 172%, and there was a very significant difference between the two groups. The positive drug is effective in increasing the dopamine content in the cerebrospinal fluid of the mouse AD model. The dopamine content in the cerebrospinal fluid of mice in the three doses of MSX groups was increased by 50.0%, 64.0% and 90.0% respectively compared with that in the model group, and there were extremely significant differences between the three doses of MSX groups and the model group; the dopamine content in the three doses of MSX / MSN-Apt / AuNPs was increased by 87.0%, 111% and 137% respectively compared with that in the model group, and there were extremely significant differences between the three doses of MSX / MSN-Apt / AuNPs nasal spray groups and the model group; the dopamine content in the three doses of MSX / MSN-Apt / AuNPs nasal spray groups was increased by 107%, 143% and 184% respectively compared with that in the model group, and there were extremely significant differences between the three doses of MSX and the model group. The results showed that total xanthone extract, MSX / MSN-Apt / AuNPs (nanoparticles) and MSX / MSN-Apt / AuNPs nanoparticle nasal spray groups all had good therapeutic effects on increasing dopamine in AD mice, and the therapeutic effect of MSX / MSN-Apt / AuNPs nanoparticle nasal spray was better than that of total xanthone and MSX / MSN-Apt / AuNPs (nanoparticles) treatment groups.

[0146] In summary, MSX / MSN-Apt / AuNPs can overcome a major obstacle to drug treatment in neurological diseases - the blood-brain barrier (BBB). The direct interaction between nanoparticles and cell membranes causes the nanoparticles to be encapsulated into cells, which can be used to transport active molecules across the BBB, thereby reducing the toxicity of drug molecules and improving therapeutic efficacy.

Claims

1. A targeted release nanoparticle, characterized in that: The particles include mesoporous silica particles blocked by nano-gold, the mesoporous silica particles contain mangosteen shell extract, and the nano-gold is connected to an aptamer that specifically recognizes amyloid protein oligomers in the brain through an Au-S bond; the mesoporous silica is mesoporous silica modified with 4-carboxyphenylboronic acid, the nano-gold is monosaccharide-modified nano-gold, and the mesoporous silica modified with 4-carboxyphenylboronic acid forms a borate ester bond with β-D-glucose on the nano-gold.

2. The targeted release nanoparticle according to claim 1, characterized in that: The nucleotide sequence of the aptamer is 5′-SH-(CH2)6-GCTGCCTGTGGTGTTGGGGCGGGTGCG-3′.

3. The method for preparing the targeted release nanoparticles according to claim 1 or 2, characterized in that: The following steps are involved: S1. adding monosaccharide-modified gold nanoparticles into the aptamer solution to obtain aptamer gold nanoparticles; S2. The mangosteen shell extract and the mesoporous silica modified with 4-carboxyphenylboronic acid were mixed, centrifuged, and washed to obtain mesoporous silica loaded with mangosteen shell extract; S3. The mesoporous silica loaded with mangosteen shell extract and the excess aptamer nanogold are mixed, centrifuged, and washed to obtain targeted release nanoparticles loaded with mangosteen shell extract.

4. The preparation method according to claim 3, characterized in that: The preparation method of the mesoporous silica modified with 4-carboxyphenylboronic acid in step S2 is to modify the mesoporous silica by amino modification and then modify it with 4-carboxyphenylboronic acid.

5. The preparation method according to claim 3, characterized in that: In the step S2, the mass ratio of the mangosteen shell extract to the mesoporous silica modified with 4-carboxyphenylboronic acid is 0.15-0.2:

1.

6. The preparation method according to claim 3, characterized in that: The preparation method of the mangosteen shell extract in step S2 is to mix the mangosteen shell with ethanol for extraction, extract with ethyl acetate, and then separate and purify the product by column chromatography.

7. Use of the targeted release nanoparticles according to claim 1 or 2 in the preparation of products for preventing and / or treating Alzheimer's disease.

8. A nasal spray, characterized in that: The nasal spray comprises the targeted release nanoparticles according to claim 1 or 2.

9. The nasal spray according to claim 8, characterized in that: The concentration of the mangosteen shell extract in the nasal spray is 0.05-0.5 mg / mL.

10. The nasal spray according to claim 8, characterized in that: The nasal spray further comprises 0.004-0.006% chitosan, 0.04-0.06% methyl-β-cyclodextrin, 2.5-2.6% glycerol for injection, 0.005-0.015% 0.01% chlorhexidine, and 0.04-0.06% disodium ethylenediaminetetraacetate.

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