A mangosteen shell extract-loaded targeted release nanoparticle, and a preparation method and application thereof

Abstract

The application discloses a kind of targeted release nanoparticles of load mangosteen shell extract and its preparation method and application, the nanoparticle includes nanometer gold plugging mesoporous silica particle, and mangosteen shell extract is loaded in mesoporous silica particle, and nanometer gold is connected with the aptamer of specific recognition intracerebral amyloid oligomer by Au-S bond.The nanoparticle can be targeted to the oligomer (AβO) of Aβ protein and reach amyloid plaque, and the higher concentration of H2O2 in plaque can make borate ester bond break, release mangosteen shell extract and free nanometer gold, both can not only pass through antioxidant, free radical and reduce Aβ aggregation, deposit, and the nanoparticle drug loading is large, accurate target, release amount is high, can be used as a kind of potential drug for treating alzheimer's disease, after being made into nasal spray, it can effectively permeate blood-brain barrier, more conducive to reducing drug solution loss, fast absorption.

Description

Technical Field

[0001] This invention relates to the field of metal nanomaterial preparation, and specifically discloses a targeted release nanoparticle loaded with mangosteen shell extract, its preparation method, and its application. Background Technology

[0002] Alzheimer's disease (AD) is a neurodegenerative disease, also known as primary senile dementia. The pathological features of AD include the appearance of amyloid plaques outside brain cells and neurofibrillary tangles inside brain cells. Beta-sheet structures are recurring secondary structures ubiquitous in proteins. Amyloid plaques are mainly composed of amyloid-β polypeptide (Aβ), and Aβ aggregation is a dominant factor leading to neurodegeneration in AD, playing a crucial role in its pathogenesis. Free radicals and oxidative damage are involved in the aggregation and nucleation of Aβ; therefore, anti-oxidation and free radical scavenging can reduce Aβ aggregation and deposition, and are a possible treatment for Alzheimer's disease.

[0003] Mangosteen (Garcinia mangostana L.), also known as mangosteen fruit or mangosteen, is a tropical evergreen tree belonging to the genus Garcinia in the family Clusiaceae. The mangosteen husk, deep in color, is mostly reddish-purple or dark purple, making up three-quarters of the fresh weight of the mangosteen. Although often considered a discarded fruit shell, the mangosteen husk actually contains rich medicinal and health benefits. The main component of the mangosteen husk is xanthone (MSX), also known as oxanthones, a type of heterocyclic compound. It is characterized by containing a bis(benzopyrone) core, with various substituents such as methoxy, hydroxyl, one or more C5 units, C10 units, or cyclized furan and pyran rings. Common xanthone compounds include mangiferin and mangosteen flavonoids, etc. α- and γ-mangosteen flavonoids (also known as α- and γ-mangosteen flavonoids) are a class of compounds widely used in the food and pharmaceutical fields. Xanthones have antibacterial, antioxidant, and anti-inflammatory effects. The high reactivity of hydroxyl substitution can also scavenge free radicals. Therefore, xanthones in mangosteen shell extract can reduce the aggregation and deposition of Aβ by resisting oxidation and scavenging free radicals through this property, thereby achieving the effect of treating Alzheimer's disease.

[0004] However, there is currently a lack of drug-loaded nanoparticles that target Aβ protein oligomers (AβO) and can be released in the amyloid plaque environment, thus affecting Aβ aggregation. Meanwhile, the greatest challenge in treating neurological diseases is the blood-brain barrier, making drug crossing of the blood-brain barrier a primary hurdle to overcome. Although the emergence of nanomedicine has brought hope to the development of neuroprotective agents, current methods often utilize modified nanocarriers such as brain-targeting peptides and transferrin to assist drugs in crossing the blood-brain barrier through active transport. However, due to systemic blood circulation losses and immune system clearance, the efficiency of drug delivery to the brain remains extremely limited. Summary of the Invention

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

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

[0007] A third objective of this invention is to provide an application of the above-mentioned targeted release nanoparticles.

[0008] A fourth objective of this invention is to provide a nasal spray containing the aforementioned targeted release nanoparticles.

[0009] The above-mentioned objective of this invention is achieved through the following technical solution:

[0010] This invention provides targeted release nanoparticles, the particles comprising mesoporous silica particles blocked by gold nanoparticles, the mesoporous silica particles containing mangosteen shell extract, and aptamers specifically recognizing amyloid oligomers in the brain connected to the gold nanoparticles via Au-S bonds; the mesoporous silica is 4-carboxyphenylboronic acid modified mesoporous silica, the gold nanoparticles are monosaccharide modified gold nanoparticles, and the 4-carboxyphenylboronic acid modified mesoporous silica forms borate ester bonds with β-D-glucose on the gold nanoparticles.

[0011] Mesoporous silica nanoparticles (MSNs) are microspheres with uniform pore size and an ordered mesoporous structure. They are typically synthesized using supramolecular assembly of surfactants. When the surfactant concentration exceeds a critical micelle concentration, it polymerizes to form micelles, and the silica source condenses on the surface of the micelles. The surfactant is then removed through solvent extraction or calcination, thus forming mesopores. Mesoporous silica possesses advantages such as large specific surface area, large pore capacity, easy surface functionalization, good biocompatibility, and an ordered and stable mesoporous framework. These advantages allow for the loading of larger amounts of drugs, which, upon targeting, can enhance therapeutic efficacy by increasing the drug concentration at the target site. Furthermore, nanoporous silica has the ability to cross the blood-brain barrier, making it a highly advantageous carrier for treating Alzheimer's disease.

[0012] Gold nanoparticles (AuNPs) are biocompatible inert nanoparticles. They can induce the formation of spherical oligomers and scattered short fibers, resulting in low-toxicity substances, from Aβ monomers. The surface charge and ligand energy of gold nanoparticles target and bind to Aβ protein oligomers (AβO), influencing Aβ aggregation. In amyloid plaques, the concentration of H₂O₂ is higher than elsewhere, making it a key element for controlled sustained release. Given the good biocompatibility and tunable particle size of gold nanoparticles, they are an excellent choice for sealing mesopores. Mesoporous silica modified with 4-carboxyphenylboronic acid can form boronic acid ester bonds with β-D-glucose on gold nanoparticles to obtain MSN-AuNPs. After MSN-AuNPs loaded with mangosteen husk extract reach amyloid plaques, the high concentration of H2O2 in the plaques can break the boronic acid ester bonds, releasing the loaded mangosteen husk extract. Furthermore, the freed gold nanoparticles can also affect the aggregation of Aβ. Thus, both the freed gold nanoparticles and the released mangosteen husk 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 segment. Aptamers can bind to various targets, such as proteins, metal ions, cells, and viruses. When aptamers interact with small molecules such as amino acids, they mainly bind through the formation of pseudo-links, inner loops, and protrusions. For large molecules, the interaction mechanisms are mainly hydrogen bonds, polar groups, shape, and van der Waals forces. Compared to antibodies, nucleic acid aptamers possess high affinity and specificity for target molecules, while also offering unique advantages such as simple synthesis and strong stability, resisting harsh detection environments such as high temperatures, strong acids, and strong alkalis. This invention enhances the targeting of mesoporous silica nanoparticles by modifying aptamers that specifically recognize amyloid oligomers (AβO) in the brain onto gold nanoparticles via an Au-S reaction. The gold nanoparticles serve as a key to controlled sustained release of the nanoparticles, influence Aβ aggregation, and act as an "anchor" connecting the targeting aptamer, thus eliminating the need for additional targeting modifications on the surface of the mesoporous silica nanoparticles.

[0014] Furthermore, the nucleotide sequence of the aptamer is 5′-SH-(CH2)6-GCTGCCTGTGGTGTTGGGGCGGGTGCG-3′.

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

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

[0017] S2. Mix mangosteen shell extract with 4-carboxyphenylboronic acid modified mesoporous silica, centrifuge, and wash to obtain mesoporous silica loaded with mangosteen shell extract (MSX / MSN-BA).

[0018] S3. Mesoporous silica loaded with mangosteen shell extract was mixed with excess aptamer gold nanoparticles, centrifuged, and washed to obtain targeted release nanoparticles (MSX / MSN-Apt / AuNPs) loaded with mangosteen shell extract.

[0019] Further, the preparation method of aptamer gold nanoparticles in step S1 is as follows: β-D-glucose solution is mixed with tetrachloroauric acid trihydrate, and sodium borohydride solution is added to obtain monosaccharide-modified gold nanoparticles; the aptamer is diluted with TE buffer and a thiol reducing agent (TCEP) is added to obtain an aptamer solution; the monosaccharide-modified gold nanoparticles are mixed with the aptamer solution and incubated to obtain aptamer gold nanoparticles.

[0020] Furthermore, the method for preparing the 4-carboxyphenylboronic acid-modified mesoporous silica in step S2 is to aminate the mesoporous silica and then modify it with 4-carboxyphenylboronic acid.

[0021] Preferably, mesoporous silica is dissolved in an organic solvent and ultrasonically dispersed, followed by the addition of 3-aminopropyltriethoxysilane and refluxed for 12–24 h to obtain aminated mesoporous silica. The aminated mesoporous silica is then 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 10–20 min. Then, the dimethyl sulfoxide solution of the aminated mesoporous silica is added, and the mixture is stirred at room temperature for 12–24 h. After centrifugation and washing, mesoporous silica modified with 4-carboxyphenylboronic acid is obtained.

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

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

[0024] Furthermore, the preparation method of mangosteen shell extract in step S2 is to mix 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 mangosteen shell extract in step S2 is as follows: after crushing mangosteen shells, add the mangosteen shell powder to 70-75% ethanol at a material-to-liquid ratio of 60-65 g / mL, extract by ultrasonication for 70-75 minutes, filter, concentrate by rotary evaporation, extract the product with ethyl acetate, and then elute with petroleum ether:acetone = 5:1 to obtain the extract.

[0026] The present invention also provides the application of the above-mentioned targeted release nanoparticles in the preparation of products for the prevention and / or treatment of Alzheimer's disease.

[0027] This invention provides a nasal spray comprising the aforementioned targeted-release nanoparticles loaded with mangosteen shell extract. The cilia on the surface of the nasal mucosa, as the main defense barrier of the respiratory tract, not only clear inhaled allergens, dust, and bacteria in mucus, but also significantly affect the retention time of drugs administered via nasal administration. The nasal cavity is also filled with a 2-4 mm thick mucosa, whose surface epithelial cells possess numerous microvilli (approximately 400 microvilli per mucosal epithelial cell). These microvilli enhance the mucosal surface area and provide favorable conditions for drug absorption. Beneath the nasal epithelial cells lies a rich network of capillaries and lymphatic vessels, providing ample nutrition and immune support to the nasal mucosa. After absorption into the nasal capillaries, the drug can effectively cross the blood-brain barrier. Nasal administration allows the drug to directly enter the systemic circulation, offering the advantage of rapid onset of action. It is suitable for emergency and self-treatment use, and patients can easily self-medicate, making it suitable for long-term use in chronic diseases. The spray produces fine, uniform droplets that effectively cover the inside of the nasal cavity, reducing medication loss and promoting rapid absorption, thereby improving bioavailability. This dosage form demonstrates significant advantages for nasal administration.

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

[0029] Preferably, the concentration of 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 glycerin for injection, 0.01% benzalkonium chloride, 0.005–0.015 v / v, and disodium EDTA, 0.04–0.06 g / mL.

[0031] To improve drug absorption through the nasal mucosa, adding absorption enhancers is an important approach. However, traditional enhancers can cause damage or irritation to the nasal mucosa to varying degrees. Chitosan, on the other hand, is a product obtained by deacetylation of chitin extracted from the shells of crustaceans. Chitosan's permeation-enhancing mechanism involves increasing intercellular spaces and promoting bioadhesion, and it is non-toxic and non-irritating to the nasal mucosa. Cyclodextrin's solubilizing effect allows phospholipids in biological membranes to dissolve and be extracted, thereby increasing intercellular spaces and promoting drug absorption. Methylated β-cyclodextrin has a stronger absorption-enhancing effect than parent cyclodextrin and hydroxypropyl-β-cyclodextrin. Methylated β-cyclodextrin interacts with lipids or divalent ions on the mucosa, increasing nasal mucosal permeability. Furthermore, cyclodextrin can inhibit the degradation of drugs by enzymes on the nasal mucosa and also increases the solubility of poorly soluble drugs.

[0032] Preferably, in addition to mangosteen peel extract, each 100mL of the above nasal spray also contains 0.005g chitosan, 0.05g methyl-β-cyclodextrin, 2.56g glycerin for injection, 0.01mL of 0.01% benzalkonium chloride, and 0.05g EDTA-2Na.

[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 this invention include mesoporous silica particles blocked by gold nanoparticles. The mesoporous silica particles are loaded with mangosteen shell extract. The gold nanoparticles are connected to aptamers that specifically recognize amyloid oligomers in the brain via Au-S bonds. The mesoporous silica is 4-carboxyphenylboronic acid modified mesoporous silica, and the gold nanoparticles are monosaccharide modified gold nanoparticles. The 4-carboxyphenylboronic acid modified mesoporous silica forms borate ester bonds with β-D-glucose on the gold nanoparticles. The nanoparticles can target the oligomer of Aβ protein (AβO) to reach amyloid plaques. The high concentration of H2O2 in the plaques can break the borate ester bonds, releasing mangosteen shell extract and free gold nanoparticles. Both can not only reduce the aggregation and deposition of Aβ through anti-oxidation, free radical scavenging, and reduction of Aβ, but also have a large drug loading capacity, precise targeting, and high release rate. They can be used as a potential drug for the treatment of Alzheimer's disease. When made into a nasal spray, it can effectively cross the blood-brain barrier, help the drug enter the cerebrospinal fluid, have brain targeting, and further reduce drug loss and promote rapid absorption. Attached Figure Description

[0035] Figure 1 This is a schematic diagram of MSN synthesis.

[0036] Figure 2 The working curve for α-twistedin.

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

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

[0039] Figure 5 Optical micrograph of ciliary movement.

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

[0041] Figure 7 The relationship between GSH concentration and OD, along with standard curves, and the relationship between dopamine concentration and peak current, along with standard curves, are presented. Figure 7 In the diagram, A represents the glutathione standard curve; B represents the dopamine DPV curve; and C represents the dopamine standard curve.

[0042] Figure 8 The values ​​represent the GSH content in zebrafish from different groups. Note: * indicates a significant difference compared to the model group (p < 0.1); *** indicates an extremely significant difference compared to the model group (p < 0.001). The model group showed extremely significant differences from both the positive control group and the DMSO group.

[0043] Figure 9 The values ​​represent the DA content in zebrafish brain tissue from different groups. Note: * indicates a significant difference compared to the model group (p < 0.1); *** indicates an extremely significant difference compared to the model group (p < 0.001). The model group showed extremely significant differences from both the positive control group and the DMSO group.

[0044] Figure 10 The AChE content in zebrafish brain tissue from different groups is shown. Note: *** indicates a highly significant difference compared to the model group (p < 0.001). The model group showed highly significant differences compared to the blank group, DMSO group, and positive control group.

[0045] Figure 11 The spontaneous behavioral trajectories of zebrafish in different groups are shown. Note: a. Control group; b. Model group; c. DMSO group; d. Low-dose MSX group; e. Medium-dose MSX group; f. High-dose MSX group; g. Low-dose MSX nanoparticle group; h. Medium-dose MSX nanoparticle group; i. High-dose MSX nanoparticle group; j. Positive group.

[0046] Figure 12 The swimming distances of zebrafish in different groups are shown. Note: *** indicates that the difference is extremely significant compared to the model group (p < 0.001). The model group was significantly different from the blank group, DMSO group, and positive control group.

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

[0048] Figure 14 To detect AChE activity in the brain tissue of mice from different groups. Note: MSX1–3 were administered via gavage at MSX 150, 300, and 450 mg / kg / d, respectively; nanoparticles 1–3 were administered via gavage at MSX / MSN-Apt / AuNPs nanoparticles at 150, 300, and 450 mg / kg / d, respectively; nasal sprays 1–3 were administered via nasal application at MSX / MSN-Apt / AuNPs nasal spray at 150, 300, and 450 mg / kg / d, respectively. Note: * indicates significant difference compared to the model group (p < 0.1); ** indicates significant difference compared to the model group (p < 0.05); *** indicates highly significant difference compared to the model group (p < 0.001). The model group showed highly significant differences from both the positive control group and the DMSO group.

[0049] Figure 15 This study aimed to detect GSH activity in the brain tissue of mice from different groups. Note: MSX1–3 were administered via gavage at doses of 150, 300, and 450 mg / kg / d (MSX, MSN-Apt / AuNPs, respectively); nanoparticles 1–3 were administered via gavage at doses of 150, 300, and 450 mg / kg / d (MSX / MSN-Apt / AuNPs); and nasal sprays 1–3 were applied to the nose at doses of 150, 300, and 450 mg / kg / d (MSX / MSN-Apt / AuNPs). Note: * indicates significant difference compared to the model group (p < 0.1); *** indicates highly significant difference compared to the model group (p < 0.001). The model group showed highly significant differences from both the positive control group and the DMSO group.

[0050] Figure 16 To detect dopamine levels in the brain tissue of mice in different groups. Note: MSX1–3 were administered via gavage at MSX levels of 150, 300, and 450 mg / kg / d, respectively; nanoparticles 1–3 were administered via gavage at MSX / MSN-Apt / AuNPs nanoparticle levels of 150, 300, and 450 mg / kg / d, respectively; nasal sprays 1–3 were administered via nasal application at MSX / MSN-Apt / AuNPs nasal spray levels of 150, 300, and 450 mg / kg / d, respectively. Note: *** indicates a highly significant difference compared to the model group (p < 0.001). The model group showed highly significant differences from both the positive control group and the DMSO group. Detailed Implementation

[0051] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[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. The Aβ42 polypeptide and the aptamer 5′-SH-(CH2)6-GCTGCCTGTGGTGTTGGGGCGGGTGCG-3′ were purchased from Shanghai Gil Biochemical Co., Ltd.; HAuCl4·3H2O (>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 Corporation. 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, the α-mangostin standard was purchased from the reference substance experimental consumables center, the glutathione kit was purchased from Nanjing Jiancheng Bioengineering Institute, dimethyl sulfoxide was purchased from Tianjin Kemi Scientific Reagent Co., Ltd.; AB strain zebrafish adult fish were provided by Nanjing Yishulihua Biotechnology Co., Ltd.; 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 (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, then set aside for 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, then set aside for use.

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

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

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

[0059] After drying and pulverizing the mangosteen shells, the powder was added to 70.966% ethanol at a material-to-liquid ratio of 62.303 g / mL. The mixture was then ultrasonically extracted for 70.725 minutes, filtered, and concentrated by rotary evaporation to obtain anoxanthone extract, namely mangosteen shell extract (MSX).

[0060] The xanthonone extract obtained by rotary evaporation was mixed with water, and then an equal volume of ethyl acetate was added. The mixture was thoroughly stirred to ensure efficient transfer of the target compound to the ethyl acetate layer. The solution was then allowed to stand, allowing the immiscibility of ethyl acetate and water to allow for natural separation, with most of the total xanthonone compounds transferring to the ethyl acetate layer. To further improve extraction efficiency, the extraction steps were repeated 3 to 4 times, collecting the ethyl acetate layer separately each time. After multiple extractions, all collected ethyl acetate layers were combined to obtain a solution containing a high concentration of the target compound. The combined ethyl acetate layer was then concentrated using a rotary evaporator, with solvent removed by heating and reducing pressure to enrich the total xanthonone compounds, yielding an extract.

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

[0062] In this invention, the extraction of xanthones uses an ethanol concentration of 70.966%, a solid-liquid ratio of 62.303 g / mL, and an extraction time of 70.725 minutes. Under these conditions, the extraction rate of xanthones is 4.527%. A mixture of petroleum ether and acetone (5:1 ratio) is used to elute the ethyl acetate fraction. Under these conditions, the α-pachyrhodin content 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] MSN creation process diagram is shown below 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 vigorously stirred for 1 h. After 1 h, 0.5 mL of tetraethoxysilane (TEOS) was added dropwise, and vigorous stirring continued for another 1 h. Mesoporous silica was obtained by centrifuging at 5500 rpm for 30 min, and washed several times with ethanol, followed by centrifugation to remove the ethanol. The mesoporous silica was heated under reflux in HCl / methanol for 24 h to remove CTAB as a surfactant. Finally, the mesoporous silica was obtained by centrifuging at 5500 rpm for 30 min and then vacuum dried at 60 °C for 10 h to remove the solvent from 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 after sonication for 5 min, 1.0 mL of 3-aminopropyltriethoxysilane (APTES) was added. The mixture was then heated under reflux for 24 h to modify the surface of MSN with 3-aminopropyltriethoxysilane, thus obtaining MSN-NH2.

[0070] 240 mg of purified MSN-NH2 was dissolved in 12 mL of dimethyl sulfoxide (DMSO). 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) were added to 3.0 mL of DMSO and stirred for 15 min. This mixture was then added to the MSN-NH2 solution, and the mixture was stirred and reacted at room temperature for 24 h. The solution was then centrifuged at 5500 rpm for 30 min, and 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 all the glassware used overnight in aqua regia, then rinse with deionized water and air dry. Next, mix 50 mL of β-D-glucose solution (0.05 M) with 0.20 mL of tetrachloroauric acid trihydrate (HAuCl4·3H2O) solution (0.05 M) until homogeneous. Then, add 0.60 mL of freshly prepared sodium borohydride (NaBH4) solution (0.05 M) dropwise. When the solution gradually turns wine-red, it can be determined that gold nanoparticles (AuNPs) have formed. Pour the wine-red solution into a volumetric flask, seal it, and store it in a refrigerator at 4°C for later use.

[0073] The aptamer was diluted to 20 μM with TE-Buff buffer. 40 μL of the aptamer was mixed with 40 μL of 10 mM thiol reducing agent TCEP and incubated at 37°C for 1 hour to reduce disulfide bonds and promote Au-S bond formation. The mixture was then diluted to 400 μL with PBS. Gold nanoparticles were added to the aptamer solution and incubated at 4°C for 18 hours. Hexamethylenetetramine alcohol (MCH) blocked non-specific binding sites, yielding Apt / AuNPs.

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

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

[0076] Example 3: Determination of MSN embedding efficiency and in vitro drug release rate

[0077] (1) Construction of α-twistedin working curve

[0078] α-Dextrin working curve: ethanol standard solutions of α-dextrin with concentrations of 2.00 μg / mL, 4.00 μg / mL, 6.00 μg / mL, 8.00 μg / mL, and 10.0 μg / mL were prepared. The absorbance values ​​A of a series of standard solutions were measured using a UV spectrophotometer. The working curve was fitted with AC to obtain the linear regression equation.

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

[0080]

[0081] C 原: Concentration of xanthones in mangosteen shell extract, C 后 Xanthones content in a solution of mangosteen shell extract encapsulated in silica.

[0082] (3) Dissolve 10 mg of MSN-BA loaded with mangosteen shell extract in 10 mL of anhydrous ethanol and treat with 1 mM H2O2; dissolve 10 mg of MSN-BA loaded with mangosteen shell extract in 10 mL of anhydrous ethanol and treat with 5 mM H2O2; dissolve 10 mg of MSX / MSN-Apt / AuNPs powder in 10 mL of anhydrous ethanol and treat with 1 mM H2O2; dissolve 10 mg of MSX / MSN-Apt / AuNPs powder in 10 mL of anhydrous ethanol and treat with 5 mM H2O2; seal the above solutions and place them in a digital display constant temperature water bath shaker at 37°C for 5 hours. After each incubation period, centrifuge and collect the supernatant. Measure the xanthonone content of the mangosteen shell extract released in the supernatant using a UV spectrophotometer. The absorption peak of xanthonone is at 318 nm. Calculate the in vitro release rate of the drug according to formula (2).

[0083]

[0084] The results are as follows Figure 2 As shown in Table 1, the absorbances measured by a UV spectrophotometer for concentrations of 0.00, 2.00, 4.00, 6.00, 8.00, and 10.0 μg / mL were 0, 0.1134, 0.2382, 0.3385, 0.4599, and 0.5767A, respectively. A standard curve was fitted to the absorbance values ​​against the concentrations, yielding the linear regression equation: A = 0.0575C + 0.0004, R0. 2 =0.9995.

[0085] Table 1. Absorbance records of α-dextrin at different concentrations

[0086]

[0087] The encapsulation efficiency of α-thosomerin in mesoporous silica was calculated: the absorbance before encapsulation was 0.8546 Å, and the absorbance of the supernatant after encapsulation was 0.7593 Å, as measured by a UV spectrophotometer. After processing using the α-thosomerin working curve, the concentration of α-thosomerin in the supernatant before encapsulation was 44.57 μg / mL, and the concentration after encapsulation was 31.54 μg / mL, resulting in an encapsulation efficiency of 29.27%.

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

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

[0090]

[0091] like Figure 3 As shown in Table 2, AuNPs effectively blocked MSN. Without H2O2 treatment, almost no mangosteen husk extract was released. Comparing the release of the MSN-BA group and the MSN-AuNPs group, AuNPs achieved a slow-release effect, with the loaded mangosteen husk extract gradually and slowly released into the solution over time. Furthermore, in the MSN-BA group, since there was no AuNPs blocking effect, the release was similar regardless of whether H2O2 treatment was used. In the MSN-AuNPs group, the release rate increased with increasing H2O2 concentration.

[0092] Example 4: Preparation of mangosteen husk nasal spray and its cilia toxicity test

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

[0094] Take three portions: 0.005g chitosan, 0.05g methyl-β-cyclodextrin, 2.56g glycerin for injection, 0.01mL 0.01% benzalkonium chloride, and 0.05g EDTA-2Na. Place each of the three drug portions with 100mg, 500mg, and 1000mg MSX / MSN-Apt / AuNPs respectively in a 100mL volumetric flask, and add deionized water to the 100mL mark. This prepares nasal sprays with concentrations of 1.00mg / mL, 0.500mg / mL, and 0.100mg / mL negative MSX / MSN-Apt / AuNPs, which are then dispensed into 10mL medical spray bottles.

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

[0096] The duration of palatal cilia movement in bullfrogs was determined using an in vitro method. Bullfrogs were euthanized using a double medullary destruction method, and the palatal mucosa was removed with ophthalmic surgical scissors. Blood clots and debris were washed away with physiological saline, and a 3×3 mm sample was taken. 2The palatal mucosa was prepared by laying the mucosa, ciliated side up, on a glass slide. A 1% sodium deoxycholate aqueous solution (which has severe ciliary toxicity) was used as a positive control, and physiological saline as a negative control. 0.1 mL of the drug solution was added to the surface of the mucosa, with three mucosa samples forming a group, for a total of five groups. A coverslip was then gently placed over the samples, and the dynamic activity of the cilia was observed in detail using a 150x (15×10) optical microscope. Next, the samples were placed in a chromatography tank containing a small amount of deionized water, ensuring the tank was well-sealed to near-saturation with water vapor, while maintaining the room temperature at 22°C. During the experiment, the samples were removed at appropriate time intervals to observe ciliary movement. If ciliary movement was observed, the samples were returned to the chromatography tank, and this process was continued until ciliary movement completely ceased. The total time from the start of drug administration to the cessation of ciliary movement was recorded. To assess the effect of the drug on ciliary movement, the ratio of the duration of continuous ciliary movement in the drug-treated group to that in the control group was calculated, and this ratio was converted into a relative percentage of movement. The higher this percentage, the smaller the effect of the drug on ciliary movement, meaning that the longer the cilia remain in motion under the influence of the drug.

[0097] The results are as follows Figures 4-6 As shown, compared with the saline group in the negative control group, the relative motility percentage of the drug-treated groups was reduced. The time from drug administration to ciliary cessation in the saline group was (121±9) min, with a relative motility percentage of 100%. The time at a drug concentration of 0.1 mg / mL was (46±7) min, with a relative motility percentage of 38%. The time at a drug concentration of 0.5 mg / mL was (27±11) min, with a relative motility percentage of 22%. The time at a drug concentration of 1 mg / mL was (13±5) min, with a relative motility percentage of 11%. In contrast, the 1% sodium deoxycholate aqueous solution, serving as the positive control group, had a time from drug administration to ciliary cessation of movement of (0±0) min, with a relative motility percentage of 0%, and the cilia stopped beating immediately upon application. These results indicate that the 1 mg / mL concentration of the drug has low ciliary toxicity, and that ciliary toxicity increases with increasing drug concentration, implying increased irritation to the nasal cavity. Therefore, it can be concluded that a 0.1 mg / mL mangosteen peel extract nasal spray will not have significant ciliary toxicity; however, ciliary toxicity increases with increasing drug concentration. This result can provide basic data for drug dosage exploration and facilitate preliminary experiments for drug development and utilization.

[0098] Example 5: The repair effect of total xanthones on oxidative damage in zebrafish

[0099] I. Experimental Methods

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

[0101] The adult AB strain zebrafish were provided by Nanjing Yishu Lihua Biotechnology Co., Ltd.

[0102] Twenty-five zebrafish were taken and divided into five groups, each group consisting of five zebrafish in a 0.5 L solution. The solutions were prepared with tebuconazole at concentrations of 0.0 mg / L, 0.5 mg / L, 0.7 mg / L, 1.0 mg / L, and 2.0 mg / L, respectively. After two days of exposure, the number of dead individuals in each group and the non-lethal concentration were recorded.

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

[0104] Place the zebrafish in an ice-water mixture for 10 minutes. After observing carefully until the zebrafish stops moving, place it on a foam board, securing the eyes and tail. Starting from the pelvic fin, cut open the zebrafish's abdomen towards the head, then along the spine to the tail, cutting downwards to the ventral opening. Open the abdominal skin to clearly observe the zebrafish's internal organs. Clean the petri dish and add a few drops of physiological saline. Place the visceral mass in the saline drop in the petri dish. Use a dissecting needle and forceps to separate the visceral mass, removing only the liver. Place the separated liver into a centrifuge tube containing physiological saline. Incubate the centrifuge tube containing the liver tissue at 35 degrees Celsius to maintain tissue viability and set aside for later use.

[0105] Remove the zebrafish liver tissue from the centrifuge tube, rinse with ice-cold saline, blot dry with absorbent paper, weigh and record the data, then place it in a grinding mortar, add an appropriate amount of homogenizing medium, and manually grind for 5 minutes in an ice-water bath to homogenize the tissue. Pour the homogenate into a 1.5 mL centrifuge tube, centrifuge at 2500 rpm for 10 minutes, and collect the supernatant for analysis.

[0106] According to the instructions of the glutathione assay kit, prepare four corresponding reagents and prepare a 20 μmol / L GSH standard solution in the specified proportions. Obtain the supernatant using the previously established method for preparing 10% tissue homogenate. Specifically, take 0.5 mL of 10% tissue homogenate, mix it with 2.0 mL of reagent, centrifuge at 3500 rpm for 10 min, and retain the supernatant for testing. Add the reagents according to the kit instructions, mix thoroughly, and allow to develop color for 5 min. Measure the OD value of each test tube using a UV-Vis spectrophotometer at 420 nm, with a light path of 1 cm and double-distilled water as the zeroing condition, and record the data.

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

[0108] Cut off the zebrafish's head, and use ophthalmic scissors to cut along the spine towards the space between the zebrafish's eyes to expose the brain tissue. Use a dissecting needle to separate the brain tissue from the skull, and then place the brain tissue in a centrifuge tube containing physiological saline. Place the centrifuge tube containing the brain tissue in a constant temperature water bath at 35 degrees Celsius to ensure tissue viability and set aside for later use.

[0109] The zebrafish brain tissue obtained from the centrifuge tube was removed, rinsed with ice-cold saline, blotted dry with absorbent paper, weighed and recorded, and then placed in a grinding mortar. An appropriate amount of artificial brain tissue cerebrospinal fluid was added, and the tissue was manually ground for 5 minutes in an ice-water bath to homogenize it. The homogenate was poured into a 1.5 mL centrifuge tube, centrifuged at 2500 rpm for 10 minutes, and the supernatant was collected for analysis.

[0110] Take 1 mL of the brain tissue supernatant obtained 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 as the reference electrode, a platinum sheet as the counter electrode, and the prepared modified carbon fiber electrode as the working electrode, differential pulse voltammetry (DPV) was used as the determination method. The determination was performed under the conditions of an initial voltage of -0.3 V, an ending 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. The peak height was recorded as shown in equation (3).

[0111]

[0112] (4) Observation of zebrafish kinetic behavior

[0113] Five zebrafish were placed in each group, and each was placed in a petri dish. After transferring the zebrafish to the petri dish, they were allowed to acclimatize for 2-3 minutes before being filmed for 2 minutes to capture motion. ImageJ motion tracking software was used to measure swimming activity at 25 frames per second, capturing swimming movements within a predefined area (one area corresponds to one hole with an inner diameter of approximately 120 mm). Detection variables were set for each animation file to ensure optimal detection for all juvenile fish. Subsequently, ImageJ software was used to analyze the data, providing parameters for stillness time and movement distance to characterize individual swimming activity. The results were compared, and the experimental data were recorded.

[0114] (5) Take 60 zebrafish and divide them into 10 groups and place them in petri dishes. Each group has 6 zebrafish in 1.0L of solution. These groups are called blank group, 1% DMSO group, oxidative stress model group, positive control group and MSX low-dose group, medium-dose group and high-dose group; MSX / MSN-Apt / AuNPs (nanoparticles) low-dose group, medium-dose group and high-dose group. The blank group is placed in deionized water; the DMSO group is placed in 1% DMSO solution; the oxidative stress model group is placed in 0.5 mg / L tebuconazole solution. The water or solution is changed every 2 days for 4 days. Then the blank group and 1% DMSO group repeat the above steps. The model group is placed in 1% DMSO solution, the positive group is placed in 50 mg / L vitamin C 1% DMSO solution, and the other groups are given low (10 mg / L), medium (25 mg / L) and high (50 mg / L) doses (drug dissolved in 1% DMSO) respectively. The solution is changed every 2 days for 7 days. The administration details are shown in Table 3.

[0115] Table 3. Dosing regimens for the drug's effect on oxidative stress repair in zebrafish.

[0116]

[0117] Five fish were taken from each group, and their movement trajectory and parameters were measured for 2 minutes. 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] II. Experimental Results

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

[0120] The results are shown in Table 4. When the concentration of tebuconazole was 0.5 mg / L, the mortality rate was 0%, which was considered 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 and mortality rate

[0122]

[0123] The results are as follows Figure 7 As shown, the concentration of GSH exhibits a good linear relationship with the OD value in the range of 0 μmol / L to 100 μmol / L. The GSH regression equation is: y = 0.0037x - 0.0011, where the ordinate is the OD value and the abscissa is the glutathione concentration (μmol / L). R 2=0.9987. In a phosphate buffer solution with pH = 8.0, the scan rate was adjusted to 10 mV / s, and scans were performed from -0.4 V to 0.6 V. The dopamine concentration showed a good linear relationship with the peak current in the range of 1.0 μg / L to 20.0 μg / L. The regression equation was: y = 1.2842x - 0.53623, where the ordinate represents dopamine concentration in μg / L and the abscissa represents peak current in A. Correlation coefficient R0 2 =0.9956.

[0124] (2) The therapeutic effects of different concentrations and administration methods on oxidative damage in zebrafish

[0125] The results are as follows Figure 8 As 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 successful. The MSX administration groups at different doses increased by 12.1%, 23.1%, and 32.8% compared with the model group, respectively; the medium-dose MSX group showed a significant difference compared with the model group, and the high-dose MSX group showed a highly significant difference compared with the model group; the nanoparticle administration groups at different doses increased by 25.8%, 35.0%, and 49.6% compared with the model group, respectively, and all three groups showed highly significant differences compared with the model group; the positive group increased by 47.8%, which also showed a highly significant difference compared with the model group. The results showed that tebuconazole-induced oxidative damage in zebrafish was mitigated by both total xanthones and MSX / MSN-Apt / AuNPs (nanoparticles), with MSX / MSN-Apt / AuNPs (nanoparticles) showing superior therapeutic effects compared to the total xanthone treatment group (at the same dose). Furthermore, there was no significant difference between the DMSO group and the control group; this concentration of solvent is non-toxic and did not affect the experimental results.

[0126] The results are as follows Figure 9As shown, the dopamine content in the model group increased by 31.7% compared to the DMSO group, with a highly significant difference between the two groups, indicating successful modeling. The dopamine content in zebrafish brain tissue decreased by 3.5%, 4.5%, and 7.4% in different MSX administration groups compared to the model group, respectively; the high-dose MSX group showed a significant difference compared to the model group, while the low-dose and medium-dose groups showed no significant difference. The dopamine content in zebrafish brain tissue decreased by 12.0%, 19.6%, and 27.6% in different nanoparticle administration groups compared to the model group, respectively, with highly significant differences in all three groups compared to the model group. The positive control group showed a 21.9% decrease, also with a highly significant difference compared to the model group, indicating that the positive control drug inhibited the increase in dopamine induced by oxidative stress. The dopamine concentration in the drug-treated group was lower than that in the model group, indicating that the drug inhibited the increase in dopamine caused by oxidative stress. Furthermore, the MSX / MSN-Apt / AuNPs (nanoparticles) had a better effect on dopamine levels than the total xanthonone extract group. Additionally, there was no significant difference between the DMSO group and the blank group; this concentration of solvent is non-toxic and did not affect the experimental results.

[0127] The results are as follows Figure 10 As shown, the acetylcholinesterase (AChE) content in the model group increased by 54.8% compared to the DMSO group, indicating a highly significant difference between the two groups, signifying successful modeling. The different doses of MSX administered to the model group showed a decrease of 8.3%, 17.7%, and 27.1% in AChE content in zebrafish brain tissue, respectively. All three MSX dose groups showed highly significant differences compared to the model group, indicating that MSX inhibits the increase in AChE induced by oxidative stress. The different doses of nanoparticles administered to the model group showed a decrease of 15.6%, 28.1%, and 37.5% in AChE content in zebrafish brain tissue, respectively. All three groups showed highly significant differences compared to the model group, indicating that nanoparticles inhibit the increase in AChE induced by oxidative stress. The positive control group showed a decrease of 21.9% compared to the model group, a highly significant difference, indicating that the positive control drug inhibits the increase in AChE induced by oxidative stress. Furthermore, the MSX / MSN-Apt / AuNPs (nanoparticles) had a better effect on AChE content than the total xanthonone extract group. Additionally, there was no significant difference between the DMSO group and the normal group, suggesting that the solvent DMSO did not affect the experimental results.

[0128] The zebrafish's movement trajectory and motion parameters within 2 minutes are as follows: Figures 11-13 As shown, the zebrafish in the model group preferred to swim in the central area and were more active; while the drug-treated group was relatively quiet and less active. Figure 11It can be seen that the movement distance of the model group is the longest, and it is significantly higher than that of the blank group and the DMSO group. There is no obvious difference in the movement distance between the administration group and the blank group and the DMSO group. The stationary time of the model group is the shortest, significantly lower than that of the blank group (DMSO group), while there is no obvious difference in the stationary time of the administration group compared with the blank group (DMSO group), indicating that total xanthones and MSX / MSN-Apt / AuNPs (nanoparticles) have an obvious repair effect on oxidative stress. The results show that the zebrafish in the blank group (DMSO group) have normal activity patterns, swimming evenly and smoothly in the observation hole. The movement behavior of the zebrafish in the model group is enhanced, and the administration of total xanthones and MSX / MSN-Apt / AuNPs (nanoparticles) will restore the behavior of zebrafish. In addition, there is no obvious difference between the DMSO group and the blank group, and it can be considered that the solvent toxicity does not affect the experimental results.

[0129] Example 6 Repair Effect of Total Xanthones on Oxidative Damage in Mice

[0130] I. Experimental Method

[0131] (1) Mouse Rearing

[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 (Guangdong) 2023-001. All animal experiments were completed in an SPF-grade laboratory. Rearing 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 drug can cross the BBB. Drugs or compounds with low molecular weight (less than 400 Da) that do not ionize at physiological pH and are lipophilic can cross the BBB through a 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. A three-electrode system was used, with a graphite electrode as the working electrode, an Ag / AgCl electrode as the reference electrode, and a platinum plate electrode as the counter electrode. The three electrodes were immersed in 10 mM Fe(CN)6 3- / 4-The tests were conducted using a mixed electrolyte solution of 1.0 M KCl. Before each test, N2 was bubbled into the electrolyte solution to eliminate the influence of dissolved oxygen. In cyclic voltammetry (CV) detection, the scan rate was 0.1 V / s, and the scan voltage was controlled within the range of -1 V to 1.5 V. In electrochemical impedance spectroscopy (EIS) detection, the scan frequency was controlled between 0.1 Hz and 50 kHz. After incubation with different concentrations of acetylcholinesterase, the changes in impedance values ​​(ΔRct / Rct0) were 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 brain of each group of mice was detected according to the above method.

[0137] (3) The glutathione and dopamine content in mice was determined by the method described in Example 5.

[0138] (4) Alzheimer's disease grouping and medication

[0139] After one week of acclimatization, mice were randomly divided into 13 groups: blank control 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), and low-dose, medium-dose, and high-dose groups of MSX / MSN-Apt / AuNPs (nanoparticles) nasal spray, with 20 mice in each group. Model groups, positive control groups, and xanthonone extract groups were administered 30 minutes after gavage, followed by intraperitoneal injection of 1 mg / kg scopolamine hydrobromide to establish the model. MSX / MSN-Apt / AuNPs (nanoparticles) were administered 30 minutes after gavage, followed by intraperitoneal injection of 1 mg / kg scopolamine hydrobromide to establish the model. MSX / MSN-Apt / AuNPs (nanoparticles) nasal spray groups were applied 30 minutes after nasal application, followed by intraperitoneal injection of 1 mg / kg scopolamine hydrobromide to establish the model. Except for the blank control group which received intraperitoneal injection of 0.9% saline, the 1% DMSO group, model group, positive control group, xanthonone extract group, MSX / MSN-Apt / AuNPs (nanoparticles) group, and xanthonone nasal spray group all received intraperitoneal injection of scopolamine hydrobromide 1 mg / kg, with an injection volume of 0.1 mL / 10 g. Dosage details are shown in Table 5. During the experiment, the mice were weighed every two days, and the dosage was adjusted according to the changes in their body weight. The medication was administered continuously for 14 days.

[0140] Table 5 Grouping and Dosing Regimen

[0141]

[0142] II. Experimental Results

[0143] The levels of acetylcholinesterase in the brain tissue of mice in each group were measured, and the results are as follows: Figure 14 As shown, compared with the DMSO group, the acetylcholinesterase level in the model group increased by 75.0%, with a highly significant difference between the two groups, indicating successful modeling. There was no difference in acetylcholinesterase level between the blank group and the DMSO group, indicating that this concentration of DMSO had no effect on acetylcholinesterase level in mice. Compared with the model group, the acetylcholinesterase level in the positive control group decreased by 28.6%, with a highly significant difference between the two groups, indicating that the positive control drug was effective in reducing acetylcholinesterase level in the mouse AD model. The three doses of MSX reduced acetylcholinesterase level by 6.6%, 12.1%, and 17.6% compared with the model group, respectively. The low-dose group showed no significant difference from the model group, the medium-dose group showed a significant difference, and the high-dose group showed a highly significant difference. The three doses of MSX / MSN-Apt / AuNPs reduced acetylcholinesterase level by 14.3%, 22.0%, and 25.3% compared with the model group, respectively. The low-dose group showed a relatively significant difference from the model group, while the medium-dose and high-dose groups showed a relatively significant difference. All comparisons between the model groups showed highly significant differences. The acetylcholinesterase levels in the three doses of MSX / MSN-Apt / AuNPs nasal spray group decreased by 27.5%, 36.3%, and 45.1% compared to the model group, respectively. All three dose groups showed highly significant differences compared to the model group, indicating that they have a significant inhibitory effect on acetylcholinesterase activity. MSX / MSN-Apt / AuNPs nasal spray is superior to MSX and MSX / MSN-Apt / AuNPs treatment groups in inhibiting acetylcholinesterase activity. This invention modifies the surface of gold nanoparticles with an aptamer that specifically recognizes amyloid oligomers in the brain via an Au-S reaction. MSX is then sealed within the mesoporous silica pores by the gold nanoparticles. Simultaneously, it is prepared as a nasal spray, which further enhances the drug's targeting. Taking advantage of the high reactive oxygen species content in the brains of AD mice, the silica mesoporous pores are opened, releasing MSX. Therefore, the MSX / MSN-Apt / AuNPs nasal spray group has the lowest AChE content in the brain tissue of mice compared to the MSX group, and has a stronger inhibitory effect on AChE.

[0144] The results are as follows Figure 15As shown, the GSH content / liver weight (mg / 100g) in the liver tissue of the model group mice decreased by 36.30% compared with the DMSO group, and the difference between the two groups was highly significant, indicating that the model was successfully established. There was no difference in GSH content in 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 in the liver tissue of mice. Compared with the model group, the GSH content in the liver tissue of the positive control group increased by 48.4%, and the difference between the two groups was highly significant, indicating that the positive control drug was effective in increasing the GSH content in the liver tissue of the mouse AD model. The GSH content in the liver tissue of mice in the three MSX groups increased by 4.4%, 8.6%, and 17.6% compared with the model group, respectively. There was no significant difference between the low-dose group and the model group, while the medium-dose and high-dose groups showed significant differences. The GSH content in the three MSX / MSN-Apt / AuNPs groups increased by 13.3%, 19.9%, and 26.2% compared with the model group, respectively, and all three dose groups showed highly significant differences compared with the model group. The GSH content in the three MSX / MSN-Apt / AuNPs nasal spray groups was significantly higher than that in the model group. The dosages increased by 38.4%, 46.8%, and 50.8%, respectively, and all three dosage groups showed highly significant differences compared with the model group. The results indicate that the total xanthones, MSX / MSN-Apt / AuNPs (nanoparticles), and MSX / MSN-Apt / AuNPs (nanoparticles) nasal spray groups all have a certain therapeutic effect on AD mice, and the therapeutic effect of MSX / MSN-Apt / AuNPs nanoparticle nasal spray is better than that of the total xanthones extract and MSX / MSN-Apt / AuNPs (nanoparticles) treatment groups.

[0145] The results are as follows Figure 16As shown, the dopamine content in the cerebrospinal fluid of mice in the model group decreased by 66.2% compared with the DMSO group, and the difference between the two groups was highly significant, indicating that the model was successfully established. There was no difference in 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 mouse cerebrospinal fluid. Compared with the model group, the dopamine content in the cerebrospinal fluid of mice in the positive control group increased by 172%, and the difference between the two groups was highly significant, indicating that the positive control drug was effective in increasing the dopamine content in the cerebrospinal fluid of mice with Alzheimer's disease (AD). The dopamine levels in the cerebrospinal fluid of mice in the three MSX groups were increased by 50.0%, 64.0%, and 90.0% compared with the model group, respectively, and all three MSX groups showed highly significant differences compared with the model group. The dopamine levels in the three MSX / MSN-Apt / AuNPs groups were increased by 87.0%, 111%, and 137% compared with the model group, respectively, and all three dose groups showed highly significant differences compared with the model group. The dopamine levels in the three MSX / MSN-Apt / AuNPs nasal spray groups were increased by 107%, 143%, and 184% compared with the model group, respectively, and all three dose groups showed highly significant differences compared with the model group. The results showed that the total xanthones extract, MSX / MSN-Apt / AuNPs (nanoparticles), and MSX / MSN-Apt / AuNPs nanoparticle nasal spray all had a good therapeutic effect on increasing dopamine in AD mice, and the MSX / MSN-Apt / AuNPs nanoparticle nasal spray had a better therapeutic effect than the total xanthones and MSX / MSN-Apt / AuNPs (nanoparticles) treatment groups.

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

Claims

1. A targeted release nanoparticle, characterized in that, The particles comprise mesoporous silica particles sealed with gold nanoparticles, the mesoporous silica particles containing mangosteen shell extract, and aptamers specifically recognizing amyloid oligomers in the brain linked to the gold nanoparticles via Au-S bonds; the mesoporous silica is 4-carboxyphenylboronic acid-modified mesoporous silica, the gold nanoparticles are monosaccharide-modified gold nanoparticles, and the 4-carboxyphenylboronic acid-modified mesoporous silica forms borate ester bonds with β-D-glucose on the gold nanoparticles; the nucleotide sequence of the aptamer is 5′–SH–(CH2)6-GCTGCCTGTGGTGTTGGGGCGGGTGCG-3′; the mangosteen shell extract is α-thujone.

2. The method for preparing the targeted release nanoparticles according to claim 1, characterized in that, Includes the following steps: S1. Add monosaccharide-modified gold nanoparticles to the aptamer solution to obtain aptamer gold nanoparticles; S2. Mix mangosteen shell extract with 4-carboxyphenylboronic acid modified mesoporous silica, centrifuge, and wash to obtain mesoporous silica loaded with mangosteen shell extract. S3. Mesoporous silica loaded with mangosteen shell extract was mixed with excess aptamer gold nanoparticles, centrifuged, and washed to obtain targeted release nanoparticles loaded with mangosteen shell extract.

3. The preparation method according to claim 2, characterized in that, The method for preparing the 4-carboxyphenylboronic acid-modified mesoporous silica in step S2 is to aminate the mesoporous silica and then modify it with 4-carboxyphenylboronic acid.

4. The preparation method according to claim 2, characterized in that, In step S2, the mass ratio of mangosteen shell extract to 4-carboxyphenylboronic acid-modified mesoporous silica is 0.15–0.2:

1.

5. The preparation method according to claim 2, characterized in that, The preparation method of mangosteen shell extract in step S2 is to mix mangosteen shell with ethanol for extraction, extract with ethyl acetate, and then separate and purify the product by column chromatography.

6. The use of the targeted release nanoparticles of claim 1 in the preparation of products for the prevention and / or treatment of Alzheimer's disease.

7. A nasal spray, characterized in that, The nasal spray includes the targeted release nanoparticles as described in claim 1.

8. The nasal spray according to claim 7, characterized in that, The concentration of mangosteen shell extract in the nasal spray is 0.05–0.5 mg / mL.

9. The nasal spray according to claim 7, characterized in that, The nasal spray also contains 0.004-0.006% chitosan, 0.04-0.06% methyl-β-cyclodextrin, 2.5-2.6% glycerin for injection, 0.005-0.015% benzalkonium chloride, and 0.04-0.06% disodium EDTA.

Images