An oligosaccharide-small molecule drug nano selenium compound and a preparation method and application thereof

Abstract

This invention relates to the field of selenium composite nanomaterials technology, and particularly to an oligosaccharide-small molecule drug@nano-selenium composite, its preparation method, and applications. The composite includes selenium nanoparticles and an oligosaccharide-small molecule drug conjugate layer encapsulating the selenium nanoparticles. The conjugate layer is formed by grafting small molecule drugs and oligosaccharide molecules; the molecular weight of the small molecule drug is 100-3000 Daltons. This invention covalently binds small molecule drugs and oligosaccharide molecules to generate amphiphilic oligosaccharide derivatives, and encapsulates the selenium nanoparticles as a stabilizer, thereby obtaining a multifunctional composite selenium nanoparticle with good water dispersibility. This achieves improved solubility of small molecule drugs and enhanced stability of selenium nanoparticles. The three components together form a stable nano-formulation, enabling efficient delivery of the composite drug system and reducing toxic side effects, thus enhancing the therapeutic effects on neurodegenerative diseases, cardiovascular diseases, diabetes, bone-related diseases, and tumors.

Description

Technical Field

[0001] This invention relates to the field of selenium composite nanomaterials technology, and in particular to an oligosaccharide-small molecule drug@nano-selenium complex, its preparation method and application. Background Technology

[0002] Currently, oligosaccharides such as chitosan oligosaccharides, isomaltooligosaccharides, chitosan oligosaccharides, alginate oligosaccharides, galacto-oligosaccharides, polygalacturonic acid oligosaccharides, and mannan oligosaccharides have shown important potential functions in the treatment of neurodegenerative diseases, bone-related diseases, and tumors. These oligosaccharides possess biological activities such as immunomodulation, antioxidation, anti-inflammation, and tissue regeneration promotion, and are being studied for the prevention and treatment of various diseases, including diabetes, cardiovascular diseases, liver diseases, and cancer. In addition, small molecule drugs such as icariin and its glycosides, squalamine, tea polyphenols, tannic acid, triptolide, hesperidin, resveratrol, resveratrol glycoside, curcumin, quercetin, and quercetin have been shown to have significant therapeutic potential in neurodegenerative diseases, bone-related diseases, cardiovascular diseases, and anti-cancer applications. However, these molecules are generally hydrophobic, have extremely low water solubility, and poor bioavailability, which greatly limits their direct application.

[0003] Selenium, an essential trace element for the human body, possesses multiple biological functions, including antioxidant, anti-inflammatory, and immunomodulatory effects. Nano-selenium, due to its high bioactivity and low toxicity, has become a research hotspot in recent years and is considered to have potential applications in the treatment of neurodegenerative diseases, osteoporosis, cardiovascular diseases, diabetes, and tumors. Nano-selenium can be prepared efficiently and conveniently via chemical reduction. In the preparation process, using amphiphilic molecules with strong emulsifying abilities—such as polysaccharides, proteins, peptides, and DSPE-PEG—as stabilizers is key to obtaining nano-selenium with good water dispersibility and ideal efficacy. These stabilizers often synergistically enhance the efficacy of nano-selenium, allowing it to exert a greater therapeutic effect. However, due to the small molecular weight and poor emulsifying ability of oligosaccharides, they are difficult to use directly as stabilizers in the preparation of nano-selenium, limiting their application in nanomedicine preparation.

[0004] Therefore, existing technologies still need to be improved and developed. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide an oligosaccharide-small molecule drug@nano-selenium complex, its preparation method and application, in order to solve the problem that oligosaccharides cannot directly stabilize nano-selenium.

[0006] The technical solution of the present invention is as follows:

[0007] An oligosaccharide-small molecule drug@nano-selenium complex includes nano-selenium particles and an oligosaccharide-small molecule drug conjugate layer encapsulating the nano-selenium particles.

[0008] The oligosaccharide-small molecule drug conjugate layer is formed by grafting small molecule drugs and oligosaccharide molecules; the molecular weight of the small molecule drugs is 100-3000 Daltons.

[0009] The oligosaccharide-small molecule drug@nano-selenium complex, wherein the oligosaccharide molecule includes one or more of chitosan oligosaccharide, isomalt oligosaccharide, chitosan oligosaccharide, alginate oligosaccharide, galactooligosaccharide, polygalacturonic acid oligosaccharide, and mannan oligosaccharide.

[0010] The oligosaccharide-small molecule drug@nano-selenium complex, wherein the small molecule drug contains at least one of amino, hydroxyl, and carboxyl groups; and / or, the small molecule drug includes one or more of icariin, icariin, squalamine, tea polyphenols, tannic acid, triptolide, hesperidin, resveratrol, resveratrol glycoside, curcumin, quercetin, and quercetin.

[0011] The oligosaccharide-small molecule drug@nano-selenium complex, wherein the small molecule drug contains only hydroxyl or amino groups, the oligosaccharide molecule contains only hydroxyl or amino groups, and the small molecule drug and the oligosaccharide molecule are grafted together via a first linker molecule; or, the small molecule drug contains only carboxyl groups, the oligosaccharide molecule contains only carboxyl groups, and the small molecule drug and the oligosaccharide molecule are grafted together via a second linker molecule.

[0012] The oligosaccharide-small molecule drug@nano-selenium complex, wherein the first linker molecule is a carboxylic acid derivative containing two or more carboxyl groups or producing two or more carboxyl groups after hydrolysis; and the second linker molecule is a compound containing two or more amino groups.

[0013] A method for preparing an oligosaccharide-small molecule drug@selenium nanocomplex includes the following steps:

[0014] Small molecule drugs, oligosaccharide molecules and reaction catalysts are mixed to obtain oligosaccharide-small molecule drug conjugates;

[0015] The oligosaccharide-small molecule drug conjugate was dissolved in water and mixed with an aqueous selenium source solution to obtain a mixed solution;

[0016] A reducing agent was added to the mixed solution, and after dialysis, an oligosaccharide-small molecule drug@nano-selenium complex was obtained.

[0017] The method for preparing the oligosaccharide-small molecule drug@selenium nanocomplex, wherein the small molecule drug contains only hydroxyl or amino groups, and the oligosaccharide molecule contains only hydroxyl or amino groups, includes the following steps in preparing the oligosaccharide-small molecule drug conjugate:

[0018] The first linker molecule is subjected to esterification or amidation reaction with the small molecule drug to obtain a first product; then the first product is subjected to amidation or esterification conjugation with the oligosaccharide molecule to obtain an oligosaccharide-small molecule drug conjugate.

[0019] Alternatively, the first linker molecule can be conjugated with the oligosaccharide molecule to obtain a second product; the second product can then be reacted with the small molecule drug via amidation or esterification to obtain an oligosaccharide-small molecule drug conjugate.

[0020] The method for preparing the oligosaccharide-small molecule drug@selenium nanocomplex, wherein the small molecule drug contains only a carboxyl group, the oligosaccharide molecule contains only a carboxyl group, and the steps for preparing the oligosaccharide-small molecule drug conjugate include:

[0021] The second linker molecule is subjected to an amidation reaction with the small molecule drug to obtain a third product; the third product is then subjected to an amidation conjugate with the oligosaccharide molecule to obtain an oligosaccharide-small molecule drug conjugate.

[0022] Alternatively, the second linker molecule can be conjugated with the oligosaccharide molecule to obtain a fourth product; then, the fourth product can be subjected to an amidation reaction with the small molecule drug to obtain an oligosaccharide-small molecule drug conjugate.

[0023] The method for preparing the oligosaccharide-small molecule drug@nano-selenium complex, wherein the reaction catalyst comprises one or more of N,N'-dicyclohexylcarbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N,N'-diisopropylcarbodiimide, 1,1'-carbodiimide diimidazole, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, and 4-dimethylaminopyridine; and / or, the reducing agent comprises one or more of ascorbic acid and sodium bisulfite.

[0024] Application of an oligosaccharide-small molecule drug@nanose selenium complex in the preparation of drugs for neurodegenerative diseases, bone-related diseases, diabetes, cardiovascular diseases, and tumor treatment.

[0025] Beneficial Effects: This invention provides an oligosaccharide-small molecule drug@selenium nanocomposite, its preparation method, and its application. The oligosaccharide-small molecule drug@selenium nanocomposite comprises selenium nanoparticles and an oligosaccharide-small molecule drug conjugate layer encapsulating the selenium nanoparticles. The oligosaccharide-small molecule drug conjugate layer is formed by grafting small molecule drugs and oligosaccharide molecules. The molecular weight of the small molecule drug is 100-3000 Daltons. This invention covalently binds small molecule drugs and oligosaccharide molecules to generate amphiphilic oligosaccharide derivatives, and encapsulates the selenium nanoparticles as a stabilizer, thereby obtaining a multifunctional composite selenium nanoparticle with good water dispersibility. This achieves improved solubility of small molecule drugs, enhanced stability of selenium nanoparticles, and solves the problem that oligosaccharides cannot directly stabilize selenium nanoparticles. Specifically, the covalent binding of oligosaccharide molecules and small molecule drugs forms amphiphilic oligosaccharide derivatives, improving the water solubility and stability of small molecule drugs. After binding, the oligosaccharide derivatives act as stabilizers for selenium nanoparticles, ensuring good dispersibility of the selenium nanoparticles. Together, these three components form a stable nanocarrier, enabling efficient drug delivery, enhancing therapeutic effects, and reducing side effects. Meanwhile, the combination of the immunomodulatory effects of oligosaccharide molecules, the cytotoxicity of small molecule anticancer drugs, and the apoptosis-inducing properties of nano-selenium can exert multiple anticancer mechanisms, thereby improving the efficiency and effectiveness of antitumor treatment. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of the process flow for preparing an oligosaccharide-small molecule drug@nanoselenium complex according to the present invention;

[0027] Figure 2 This is a schematic diagram illustrating the preparation process of ICA-Cho@Se NPs in Example 1;

[0028] Figure 3 TEM image of ICA-Cho@Se NPs obtained in Example 1;

[0029] Figure 4 This is a diagram showing the particle size distribution of ICA-Cho@Se NPs obtained in Example 1;

[0030] Figure 5 The graph shows the effects of ICA-Cho@SeNPs on pole climbing and rotarod behavior disorders in PD mice.

[0031] Figure 6 A graph showing data on how ICA-Cho@SeNPs improved movement distance and immobility time in PD mice;

[0032] Figure 7This is a characterization diagram of the effect of different contents of ICA-Cho@SeNPs on the motor behavior of PD model mice, including experimental records of its effects on behavioral indicators such as mouse movement speed, cadence, stride length period, stride length, standing time and swing speed.

[0033] Figure 8 The graph shows the analytical data on how different ICA-Cho@SeNPs contents improved movement speed, rhythm, stride duration, stride length, standing speed, and swing speed in PD mice.

[0034] Figure 9 The figure shows the effects of ICA-Cho@SeNPs on dopaminergic neurons and glial cells in the substantia nigra region of PD mice. Detailed Implementation

[0035] This invention provides an oligosaccharide-small molecule drug@selenium nanocomposite, its preparation method, and its application. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0036] It will be understood by those skilled in the art that, unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. It should also be understood that terms such as those defined in general dictionaries should be understood to have the same meaning as in the context of the prior art, and should not be interpreted in an idealized or overly formal sense unless specifically defined as herein.

[0037] Combining oligosaccharides, small molecule drugs, and nano-selenium to prepare composite nano-selenium with more complete and powerful functions and excellent colloidal stability would be an ideal strategy to comprehensively leverage the effects of each component.

[0038] Based on this, the present invention provides an oligosaccharide-small molecule drug@nano-selenium complex, comprising nano-selenium particles and an oligosaccharide-small molecule drug conjugate layer encapsulating the nano-selenium particles.

[0039] The oligosaccharide-small molecule drug conjugate layer is formed by grafting small molecule drugs and oligosaccharide molecules; the molecular weight of the small molecule drugs is 100-3000 Daltons.

[0040] In this embodiment, by covalently binding small molecule drugs with oligosaccharide molecules to form oligosaccharide-small molecule drug conjugates (i.e., amphiphilic oligosaccharide derivatives), and then coating the nano-selenium particles as a stabilizer, a multifunctional composite nano-selenium with good water dispersibility is obtained. This achieves improved solubility of small molecule drugs, enhanced stability of nano-selenium, and solves the problem that oligosaccharides cannot directly stabilize nano-selenium. Specifically, the covalent binding of oligosaccharide molecules with small molecule drugs forms amphiphilic oligosaccharide derivatives, improving the water solubility and stability of small molecule drugs. After binding, the amphiphilic oligosaccharide derivatives act as stabilizers for nano-selenium particles, ensuring good dispersibility. Together, these three components form a stable nanocarrier, enabling efficient drug delivery, enhanced therapeutic effects, and reduced side effects. Simultaneously, the immunomodulatory effects of oligosaccharide molecules, the cytotoxicity of small molecule drugs, and the apoptosis-inducing properties of nano-selenium, combined, exert multiple anti-cancer mechanisms, improving the efficiency and effectiveness of anti-tumor therapy.

[0041] It should be noted that the small molecule drugs refer to hydrophobic functional molecules with disease treatment functions or potential, with a molecular weight between 100 and 3000 Daltons.

[0042] Specifically, this invention organically combines oligosaccharide molecules, hydrophobic small-molecule drugs, and nano-selenium, overcoming the limitations of their respective applications. Traditionally, oligosaccharides cannot be directly used as stabilizers for nano-selenium due to insufficient emulsifying ability, while hydrophobic small-molecule drugs have limited clinical applications due to poor water solubility and low bioavailability. Therefore, effective stabilizers are needed to improve the water dispersibility and stability of nano-selenium during its preparation process, namely, by covalently binding small-molecule drugs with oligosaccharide molecules to generate amphiphilic oligosaccharide derivatives as stabilizers. In addition, this invention, by covalently binding small-molecule hydrophobic drugs with oligosaccharide molecules to generate amphiphilic oligosaccharide derivatives, not only improves the water solubility and stability of small-molecule drugs but also endows them with the ability to act as stabilizers for nano-selenium, thereby enhancing the water dispersibility and stability of nano-selenium. At the same time, the bioactivity of oligosaccharide molecules, small-molecule drugs, and nano-selenium is fully utilized in the composite material, producing a synergistic effect and enhancing the therapeutic effect. Compared with existing technologies, the oligosaccharide-small molecule drug@nano-selenium complex provided by this invention achieves a synergistic effect greater than the sum of its parts (1+1+1>3). It solves key problems such as the inability of oligosaccharides to directly stabilize nano-selenium, the poor water solubility of small molecule drugs, and insufficient dispersibility and stability of nano-selenium, maximizing the potential medical value of each component. Furthermore, the complex has a simple preparation process, strong scalability, and good prospects for industrialization.

[0043] In some embodiments, the oligosaccharide molecules include one or more of chitosan oligosaccharides, isomaltooligosaccharides, chitosan oligosaccharides, alginate oligosaccharides, galacto-oligosaccharides, polygalacturonic acid oligosaccharides, and mannosaccharides. These oligosaccharides possess biological activities such as immunomodulatory, antioxidant, anti-inflammatory, and tissue regeneration-promoting effects, and can be used to prepare drugs for the treatment or prevention of various diseases such as diabetes, cardiovascular disease, liver disease, and cancer.

[0044] Specifically, in the treatment of neurodegenerative diseases, oligosaccharide molecules have neuroprotective and nerve regeneration-promoting effects, small molecule drugs such as squalamine and resveratrol can improve nerve function, while nano-selenium provides powerful antioxidant and anti-inflammatory capabilities. The organic structure of these three components effectively improves the water solubility and bioavailability of small molecule drugs, and also enhances the stability of nano-selenium. Their overall synergistic effect can be used to treat neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease. In the treatment of bone-related diseases, oligosaccharides promote osteoblast proliferation and differentiation, small molecule drugs such as icariin promote bone formation, and the anti-inflammatory and antioxidant properties of nano-selenium help reduce bone tissue damage. The combined effect of these three components improves the bioactivity and function of the material, significantly enhancing the therapeutic effect on diseases such as osteoporosis and osteoarthritis. In multi-synergistic anti-cancer therapy, the immunomodulatory effects of oligosaccharides, the cytotoxicity of small molecule anticancer drugs, and the apoptosis-inducing properties of nano-selenium, when organically combined, can exert multiple anti-cancer mechanisms, improving the efficiency and effectiveness of anti-tumor treatment.

[0045] In some embodiments, the small molecule drug contains at least one of amino, hydroxyl, and carboxyl groups; and / or, the small molecule drug includes one or more of icariin, icariin, squalamine, tea polyphenols, tannic acid, tripterygium, hesperidin, resveratrol, resveratrol glycoside, curcumin, quercetin, and quercetin. The groups contained on the small molecule drug can be grafted onto groups on oligosaccharide molecules to form oligosaccharide-small molecule drug conjugates; and all of the above-mentioned small molecule drugs have disease therapeutic functions and can be used to treat various diseases.

[0046] In some embodiments, the small molecule drug contains only hydroxyl or amino groups, the oligosaccharide molecule contains only hydroxyl or amino groups, and the small molecule drug and the oligosaccharide molecule are grafted together via a first linker molecule; or, the small molecule drug contains only carboxyl groups, the oligosaccharide molecule contains only carboxyl groups, and the small molecule drug and the oligosaccharide molecule are grafted together via a second linker molecule.

[0047] In some embodiments, for small molecule drugs containing hydroxyl or amino groups, they can be directly grafted with oligosaccharides containing carboxyl groups (such as alginate oligosaccharides, polygalacturonic acid oligosaccharides); or grafted with oligosaccharide molecules containing only hydroxyl or amino groups through a first linker molecule.

[0048] In some embodiments, for small molecule drugs containing carboxyl groups, they can be directly combined with oligosaccharide molecules through amidation or esterification to form chitosan oligosaccharide-small molecule drug conjugates; or they can be grafted with oligosaccharide molecules containing carboxyl groups (such as alginate oligosaccharides, polygalacturonic acid oligosaccharides) through a second linker molecule.

[0049] In some embodiments, the first linker molecule is a carboxylic acid derivative containing two or more carboxyl groups or producing two or more carboxyl groups upon hydrolysis; the second linker molecule is a compound containing two or more amino groups. The first or second linker molecule acts as a bridge to connect the oligosaccharide molecule to a small molecule drug.

[0050] In some embodiments, the molecular weight of the first linker molecule is 100-2000 Daltons, including but not limited to dicarboxylic compounds and succinimide esters of dicarboxylic compounds; the dicarboxylic compounds include but are not limited to oxalic acid, malonic acid, dithiodipropionic acid, PEG-diacid, succinic acid, succinic anhydride, glutaric acid, and glutaric anhydride. The aforementioned first linker molecule can undergo esterification or amidation with a small molecule drug followed by amidation or esterification with an oligosaccharide molecule; or, it can first be conjugated with an oligosaccharide molecule and then subjected to esterification or amidation with a small molecule drug to form an oligosaccharide-small molecule drug conjugate.

[0051] In some embodiments, the molecular weight of the second linker molecule is 100-2000 Daltons, including but not limited to one or more of ethylenediamine, 1,6-hexanediamine, 1,2-propanediamine, piperazine, o-phenylenediamine, dithiodiphenylamine, p-phenylenediamine, 4,4'-diaminodiphenyl ether, polyethyleneimine, diaminopolyoxypropylene, piperidinediamine, and diaminoimidazolidine. The aforementioned second linker molecule can undergo an amidation reaction with a small molecule drug followed by an amidation conjugation reaction with an oligosaccharide molecule; or, it can first undergo a conjugation reaction with an oligosaccharide molecule, and then undergo an amidation reaction with a small molecule drug to form an oligosaccharide-small molecule drug conjugate.

[0052] In addition, such as Figure 1 As shown, the present invention also provides a method for preparing oligosaccharide-small molecule drug@nanoselenium complex, comprising the following steps:

[0053] Step S10: Mix the small molecule drug, oligosaccharide molecule and reaction catalyst to obtain oligosaccharide-small molecule drug conjugate;

[0054] Step S20: Dissolve the oligosaccharide-small molecule drug conjugate in water and mix it with a selenium source aqueous solution to obtain a mixed solution;

[0055] Step S30: Add a reducing agent to the mixed solution, and after dialysis, obtain oligosaccharide-small molecule drug@nano-selenium complex.

[0056] In this embodiment, by covalently binding small molecule drugs with oligosaccharide molecules to generate amphiphilic oligosaccharide derivatives, and then coating the selenium nanoparticles as a stabilizer, a multifunctional composite selenium nanoparticle with good water dispersibility is obtained. This achieves improved solubility of small molecule drugs, enhanced stability of selenium nanoparticles, and solves the problem that oligosaccharides cannot directly stabilize selenium nanoparticles. Specifically, the covalent binding of oligosaccharide molecules with small molecule drugs forms amphiphilic oligosaccharide derivatives, improving the water solubility and stability of small molecule drugs. After binding, the oligosaccharide derivatives act as stabilizers for selenium nanoparticles, ensuring good dispersibility of selenium nanoparticles. Together, these three components form a stable nanocarrier, enabling efficient drug delivery, enhancing therapeutic effects, and reducing side effects. Simultaneously, the immunomodulatory effects of oligosaccharide molecules, the cytotoxicity of small molecule anticancer drugs, and the apoptosis-inducing properties of selenium nanoparticles combine to exert multiple anticancer mechanisms, improving the efficiency and effectiveness of antitumor therapy.

[0057] Specifically, the oligosaccharide-small molecule drug@selenium nanocomplex prepared using this method has the following advantages:

[0058] 1) Improved efficacy: The three work synergistically to enhance biological functions such as anti-oxidation, anti-inflammation and cell regeneration, significantly improving the treatment effect on neurodegenerative diseases, bone-related diseases and so on.

[0059] 2) Improved drug performance: It improved the water solubility and bioavailability of small molecule hydrophobic drugs, and reduced the dosage and side effects.

[0060] 3) Optimize material properties: Enhance the water dispersibility and stability of nano-selenium, thereby improving the functional stability of the composite material.

[0061] 4) Expanding application areas: It provides new ideas for developing multifunctional nanomedicine carriers, which can be applied to the treatment of various diseases and have broad clinical application value.

[0062] In some embodiments, in step S10, when the small molecule drug contains a hydroxyl or amino group and the oligosaccharide molecule contains a carboxyl group, the small molecule drug can be directly grafted onto the oligosaccharide molecule to obtain an oligosaccharide-small molecule drug conjugate.

[0063] In some embodiments, in step S10, when the small molecule drug contains only hydroxyl or amino groups, and the oligosaccharide molecule contains only hydroxyl or amino groups, the step of preparing the oligosaccharide-small molecule drug conjugate includes:

[0064] The first linker molecule is subjected to esterification or amidation reaction with the small molecule drug to obtain a first product; then the first product is subjected to amidation or esterification conjugation with the oligosaccharide molecule to obtain an oligosaccharide-small molecule drug conjugate.

[0065] Alternatively, the first linker molecule can be conjugated with the oligosaccharide molecule to obtain a second product; the second product can then be reacted with the small molecule drug via amidation or esterification to obtain an oligosaccharide-small molecule drug conjugate.

[0066] Specifically, when the small molecule drug contains only hydroxyl or amino groups, the first linker molecule is first esterified or amidated with the small molecule drug, and the product is then purified and amidated or esterified with oligosaccharide molecules. For example, in the reaction between the first linker and the small molecule drug, taking a linker molecule containing two carboxyl groups as an example, one end of the carboxyl group is covalently linked to the hydroxyl or amino group in the small molecule drug, while the other carboxyl group or its derivative remains unchanged. The reaction product is separated and purified by column chromatography, HPLC, extraction, recrystallization, etc. After the grafting of the small molecule drug-first linker covalent product with the oligosaccharide molecule is completed, purification is carried out by recrystallization, that is, a solvent that can dissolve the small molecule drug-first linker covalent product but cannot dissolve the final oligosaccharide-small molecule drug conjugate is added to the reaction solvent, so that the product is washed with the solvent after precipitation. Alternatively, the first linker molecule may first conjugate with the oligosaccharide molecule, and then the product may be grafted with a small molecule drug through esterification or amidation. In this case, the product obtained in both steps is purified by recrystallization, that is, a solvent that can dissolve the linker or the small molecule drug-first linker covalent product, but cannot dissolve the oligosaccharide-first linker conjugate or the final oligosaccharide-small molecule drug conjugate, is added to the reaction solvent, and the product is washed with the solvent after precipitation.

[0067] In some embodiments, in the route of first covalently binding a small molecule drug and a first linker arm, the molar ratio of the small molecule drug to the first linker arm molecule is 1:3-1:5, and the molar ratio of the first product to the oligosaccharide molecule is 1:1-1:50, wherein the molar amount of the oligosaccharide molecule is calculated based on its average molecular weight. Alternatively, in the route of first grafting the first linker arm and the oligosaccharide molecule, the molar ratio of the oligosaccharide molecule to the first linker arm is 1:1-1:50, wherein the molar amount of the oligosaccharide molecule is calculated based on its average molecular weight; the molar ratio of the second product (chitosan oligosaccharide-linker arm molecule) to the small molecule drug is 1:1-1:50, and the molar amount of the chitosan oligosaccharide-linker arm molecule is based on the molar amount of chitosan oligosaccharide therein.

[0068] In some embodiments, step S10, mixing the small molecule drug, oligosaccharide molecule and reaction catalyst, further includes a reaction solvent; the reaction solvent is one of pure DMSO or a mixture of DMSO with water, DMF, methanol, ethanol, dichloromethane, chloroform, ethyl acetate, etc.; the volume ratio of the mixed solvent is DMSO: other solvents = 9:1 to 1:9.

[0069] In some embodiments, the reaction in step S10 is carried out at 0°C-70°C, the molar ratio of the oligosaccharide molecule to the small molecule drug is 1:1-1:50, and the molar amount of the oligosaccharide molecule is calculated based on its average molecular weight.

[0070] In some embodiments, in step S10, when the small molecule drug contains a carboxyl group, the small molecule drug can directly bind with the oligosaccharide molecule through amidation or esterification to form an oligosaccharide-small molecule drug conjugate.

[0071] In some embodiments, in step S10, the small molecule drug contains only a carboxyl group, and the oligosaccharide molecule contains only a carboxyl group. The step of preparing the oligosaccharide-small molecule drug conjugate includes:

[0072] The second linker molecule is subjected to an amidation reaction with the small molecule drug to obtain a third product; the third product is then subjected to an amidation conjugate with the oligosaccharide molecule to obtain an oligosaccharide-small molecule drug conjugate.

[0073] Alternatively, the second linker molecule can be conjugated with the oligosaccharide molecule to obtain a fourth product; then, the fourth product can be subjected to an amidation reaction with the small molecule drug to obtain an oligosaccharide-small molecule drug conjugate.

[0074] Specifically, when the small molecule drug contains a carboxyl group and is grafted with a carboxyl-containing oligosaccharide molecule, the second linker molecule is first subjected to an amidation reaction with the small molecule drug, and the product is purified and then subjected to amidation conjugation with the oligosaccharide molecule. As an example, in the reaction between the second linker and the small molecule drug, taking a linker molecule containing two amino groups as an example, one of its amino groups is covalently linked to the carboxyl group of the small molecule, while the other amino group remains unchanged. The reaction product is separated and purified by column chromatography, HPLC, extraction, recrystallization, etc. After the grafting of the small molecule drug-second linker covalent product with the oligosaccharide molecule is completed, it is generally purified by recrystallization. That is, a solvent that can dissolve the small molecule drug-second linker covalent product but cannot dissolve the final oligosaccharide-small molecule drug conjugate is added to the reaction solvent, so that the product is washed with the solvent after precipitation. Alternatively, the second linker molecule may first conjugate with the oligosaccharide molecule, and then the product may undergo an amidation reaction with a small molecule drug for grafting. In this case, both products are generally purified by recrystallization, that is, a solvent that can dissolve the second linker or the small molecule drug-second linker covalent product, but cannot dissolve the oligosaccharide molecule-second linker conjugate or the final oligosaccharide-small molecule drug conjugate, is added to the reaction solvent, so that the product is washed with the solvent after precipitation.

[0075] In some embodiments, in the route of first covalently binding a small molecule drug and a second linker, the molar ratio of the small molecule drug to the second linker molecule is 1:3-1:5, and the molar ratio of the third product to the oligosaccharide molecule is 1:1-1:50, wherein the molar amount of the oligosaccharide molecule is calculated based on its average molecular weight. Alternatively, in the route of first grafting a second linker and an oligosaccharide molecule, the molar ratio of the oligosaccharide molecule to the second linker is 1:1-1:50, wherein the molar amount of the oligosaccharide molecule is calculated based on its average molecular weight; the molar ratio of the fourth product (chitosan oligosaccharide-linker molecule) to the small molecule drug is 1:1-1:50, and the molar amount of the chitosan oligosaccharide-linker molecule is based on the molar amount of chitosan oligosaccharide therein.

[0076] In some embodiments, the reaction catalyst comprises one or more of N,N'-dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N,N'-diisopropylcarbodiimide (DIC), 1,1'-carbodiimide diimidazole (CDI), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU), benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU), and 4-dimethylaminopyridine (DMAP); and / or, the reducing agent comprises one or more of ascorbic acid (Vc) and sodium bisulfite (NaHSO3).

[0077] In some embodiments, in step S10, after the small molecule drug, oligosaccharide molecule and reaction catalyst are mixed and reacted, the resulting product is dried or freeze-dried at 30℃-80℃ and then stored in an environment of -80℃-25℃ in the dark.

[0078] In some embodiments, the selenium source in the aqueous selenium source solution includes, but is not limited to, one or more of selenite, selenate, and selenium salt, such as Na2SeO3, Na2SeO4, and Na2Se.

[0079] In some embodiments, the molar ratio of selenium in the selenium source aqueous solution to the oligosaccharide-small molecule drug conjugate (calculated as oligosaccharide) is 50:1 to 1:50.

[0080] In some embodiments, the dialysis involves stirring the mixture containing the reducing agent at room temperature for 2-24 hours, then dialyzing it with pure water. The dialysis product is then filtered through a filter membrane with a thickness of 0.22 μm or 0.45 μm and stored at 4°C.

[0081] In addition, the present invention also provides the application of oligosaccharide-small molecule drug@nano-selenium complex in the preparation of drugs for neurodegenerative diseases, bone-related diseases, diabetes, cardiovascular diseases, and tumor treatment.

[0082] The following examples further illustrate the present invention in detail. It should also be understood that the following examples are only for further explanation of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above description of the present invention are within the scope of protection of the present invention.

[0083] Example 1

[0084] This embodiment prepares ICA-Cho@Se NPs, and the preparation process is illustrated in the schematic diagram below. Figure 2 As shown, the specific steps are as follows:

[0085] (1) Synthesis of chitosan oligosaccharide (Cho) and icariin (Ica) conjugate: 70 mg of chitosan oligosaccharide with an average molecular weight of 2000 and 150 mg of dithiobis(succinimide propionate) were dissolved in 5 mL of DMSO. After stirring at 70 °C for 24 h, 50 mL of dichloromethane was added to the reaction solution. The precipitate was then centrifuged and washed three times with dichloromethane to obtain chitosan oligosaccharide-dithio-succinimide ester (Cho-NHS). Then, 300 mg of Ica was weighed and dissolved in 5 mL of DMSO with the above Cho-NHS. 50 mg of DMAP was added, and the reaction was carried out at room temperature for 8 h. 50 mL of ethyl acetate was added to the reaction solution, and the precipitate was then centrifuged and washed three times with ethyl acetate to obtain icariin-dithio-chitosan oligosaccharide conjugate (ICA-Cho). The product was dried at 50 °C and stored away from light.

[0086] (2) Preparation of ICA-Cho@Se NPs: 5 mL of 40 mM Na₂SeO₃ was mixed with 70 mL of 1.5 mg / mL ICA-Cho aqueous solution. 25 mL of freshly prepared 40 mM Vc solution was slowly added to the mixture while stirring at room temperature. Stirring was continued at room temperature for 2 h. The mixture was then dialyzed with ultrapure water, and the dialysate was filtered through a 0.22 μm syringe filter to obtain the product. The product was sealed and stored at 4 °C, or freeze-dried after adding 3% mannitol. The freeze-dried product was stored at room temperature. Its morphology and hydrated particle size information are as follows: Figure 3 and Figure 4 As shown.

[0087] Example 2

[0088] The specific steps for preparing Cho-Cel@Se NPs in this embodiment are as follows:

[0089] (1) Synthesis of chitosan oligosaccharide (Cho) and tripterygium sorbitol (Cel) conjugate: 100 mg of chitosan oligosaccharide with an average molecular weight of 3000 and 250 mg of tripterygium sorbitol were dissolved together in 5 mL of DMSO:H2O = 9:1 mixed solution. Then, 120 mg of EDC and 70 mg of NHS were added to the reaction system. After stirring at 30 °C for 5 h, 50 mL of dichloromethane was added to the reaction solution. After centrifugation, the precipitate was obtained and washed three times with dichloromethane to obtain the chitosan oligosaccharide-tripterygium sorbitol conjugate (Cho-Cel). The product was dried naturally at room temperature in the dark and then stored in the dark at 4 °C.

[0090] (2) Preparation of Cho-Cel@Se NPs: Mix 5 mL of 40 mM Na2SeO3 with 70 mL of 3 mg / mL Cho-Cel aqueous solution. While stirring at room temperature, slowly add 25 mL of freshly prepared 40 mM Vc solution to the above system, and continue stirring at room temperature for 3 h. Then dialyze with ultrapure water and filter the dialysate through a 0.22 μm syringe filter to obtain the product. Store the product in a sealed container at 4 °C, or freeze-dry after adding 3% mannitol. Store the freeze-dried product at room temperature.

[0091] Example 3

[0092] The behavioral disorders in PD mice were investigated using ICA-Cho@Se NPs prepared in Example 1, as detailed below:

[0093] Different concentrations (0.01, 0.1, 1 mg / kg) of ICA-Cho@SeNPs were administered to C57BL / 6 mice via gavage. The effects of ICA-Cho@SeNPs on pole climbing and rotarod behavior disorders in PD mice were investigated. Figure 5 The data are shown as mean ± standard error, n = 6. Compared with the control group, # This indicates that P < 0.05. ## This indicates that P < 0.01. ### This indicates that P < 0.001; compared with the MPTP group, * This indicates that P < 0.05. ** This indicates that P < 0.01. *** (P < 0.001) The results showed that 0.1 mg / kg and 1 mg / kg of ICA-Cho@SeNPs significantly shortened the head-turning and dismounting time during pole climbing in PD mice. Figure 5 (A in the text); effectively prolongs the rotator dwell time in PD mice ( Figure 5 (B in the text). Data on ICA-Cho@SeNPs improving movement distance and immobility time in PD mice are shown in the figure below. Figure 6 The data are shown as mean ± standard error, n = 6. Compared with the control group,# This indicates that P < 0.05. ## This indicates that P < 0.01. ### This indicates that P < 0.001; compared with the MPTP group, * This indicates that P < 0.05. ** This indicates that P < 0.01. *** (Indicates P < 0.001), where Figure 6 In the equation, A and B represent the distance traveled, and C represents the time spent stationary. It can be seen that increasing the distance traveled in the open field reduces the time spent stationary. Figure 7 and Figure 8 The study demonstrated that different levels of ICA-Cho@SeNPs improved kinetic speed in PD mice. Figure 7 and Figure 8 A) rhythm ( Figure 7 and Figure 8 B) Step size period ( Figure 7 and Figure 8 C in the middle), step size ( Figure 7 and Figure 8 D in the middle), standing ( Figure 7 and Figure 8 E in the middle), swing speed ( Figure 7 and Figure 8 The original data records and data analysis charts (F in the text) Figure 8 Data are expressed as mean ± standard error, n = 6. Compared with the control group, # This indicates that P < 0.05. ## This indicates that P < 0.01. ### This indicates that P < 0.001; compared with the MPTP group, * This indicates that P < 0.05. ** This indicates that P < 0.01. *** (P < 0.001) indicates that ICA-Cho@SeNPs improve gait disorder in PD mice.

[0094] The verification in this embodiment shows that ICA-Cho@SeNPs can improve the behavioral disorders induced by MPTP in PD mice.

[0095] Example 4

[0096] The effects of ICA-Cho@Se NPs prepared in Example 1 on dopaminergic neuron damage and glial cell activation in the substantia nigra region of PD mice were studied, as detailed below:

[0097] Western blot analysis of TH protein expression, a marker molecule for dopaminergic neurons in the substantia nigra of mice, and the effects of ICA-Cho@SeNPs on dopaminergic neurons and glial cells in the substantia nigra of PD mice are shown in the following results. Figure 9Data are presented as mean ± standard error. In Western blot experiments, n = 6; in immunofluorescence staining experiments, n = 3. Compared to the control group, # This indicates that P < 0.05. ## This indicates that P < 0.01. ### This indicates that P < 0.001; compared with the MPTP group, * This indicates that P < 0.05. ** This indicates that P < 0.01. *** (P < 0.001) indicates that 0.1 mg / kg and 1 mg / kg of ICA-Cho@SeNPs significantly improved MPTP-induced upregulation of TH expression in the substantia nigra region of PD mice (e.g., P < 0.001). Figure 9 (As shown in A and B in the diagram). Immunofluorescence staining was used to detect TH in the substantia nigra region of mice (as shown in A and B in the diagram). Figure 9 The expression of microglia marker Iba1 and astrocyte marker GFAP (as shown in C and D in the figure) Figure 9 As shown in C, E, and F in the figure, the results indicate that MPTP can significantly induce the activation of microglia and astrocytes, and 0.1 mg / kg and 1 mg / kg of ICA-Cho@SeNPs can significantly reverse the above changes.

[0098] In summary, this invention provides an oligosaccharide-small molecule drug@selenium nanocomposite, its preparation method, and its application. The oligosaccharide-small molecule drug@selenium nanocomposite comprises selenium nanoparticles and an oligosaccharide-small molecule drug conjugate layer encapsulating the selenium nanoparticles. The oligosaccharide-small molecule drug conjugate layer is formed by grafting small molecule drugs and oligosaccharide molecules. The molecular weight of the small molecule drug is 100-3000 Daltons. This invention covalently binds small molecule drugs and oligosaccharide molecules to generate amphiphilic oligosaccharide derivatives, and encapsulates the selenium nanoparticles as a stabilizer, thereby obtaining a multifunctional composite selenium nanoparticle with good water dispersibility. This achieves improved solubility of small molecule drugs, enhanced stability of selenium nanoparticles, and solves the problem that oligosaccharides cannot directly stabilize selenium nanoparticles. Specifically, the covalent binding of oligosaccharide molecules and small molecule drugs forms amphiphilic oligosaccharide derivatives, improving the water solubility and stability of small molecule drugs. After binding, the oligosaccharide derivatives act as stabilizers for selenium nanoparticles, ensuring good dispersibility of the selenium nanoparticles. Together, these three components form a stable nanocarrier, enabling efficient drug delivery, enhancing therapeutic effects, and reducing side effects. Meanwhile, the combination of the immunomodulatory effects of oligosaccharide molecules, the cytotoxicity of small molecule anticancer drugs, and the apoptosis-inducing properties of nano-selenium can exert multiple anticancer mechanisms, thereby improving the efficiency and effectiveness of antitumor treatment.

[0099] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. An oligosaccharide-small molecule drug nanoseleium complex, characterized in that, It includes selenium nanoparticles and an oligosaccharide-small molecule drug conjugate layer encapsulating the selenium nanoparticles; The oligosaccharide-small molecule drug conjugate layer is formed by grafting small molecule drugs and oligosaccharide molecules; the molecular weight of the small molecule drugs is 100-3000 Daltons.

2. The oligosaccharide-small molecule drug nano selenium complex according to claim 1, characterized in that, The oligosaccharide molecules include one or more of chitosan oligosaccharides, isomaltooligosaccharides, chitosan oligosaccharides, alginate oligosaccharides, galactooligosaccharides, polygalacturonic acid oligosaccharides, and mannan oligosaccharides.

3. The oligosaccharide-small molecule drug nano selenium complex according to claim 1, characterized in that, The small molecule drug contains at least one of amino, hydroxy, and carboxyl groups; and / or, the small molecule drug includes one or more of icariin, icariin, squalamine, tea polyphenols, tannic acid, tripterygium oleracea, hesperidin, resveratrol, resveratrol glycoside, curcumin, quercetin, and quercetin.

4. The oligosaccharide-small molecule drug@selenium nanocomplex according to claim 1, characterized in that, The small molecule drug contains only hydroxyl or amino groups, the oligosaccharide molecule contains only hydroxyl or amino groups, and the small molecule drug and the oligosaccharide molecule are grafted together via a first linker molecule; or, the small molecule drug contains only carboxyl groups, the oligosaccharide molecule contains only carboxyl groups, and the small molecule drug and the oligosaccharide molecule are grafted together via a second linker molecule.

5. The oligosaccharide-small molecule drug@nano-selenium complex according to claim 4, characterized in that, The first linker molecule is a carboxylic acid derivative containing two or more carboxyl groups or producing two or more carboxyl groups after hydrolysis; the second linker molecule is a compound containing two or more amino groups.

6. A method for preparing the oligosaccharide-small molecule drug nano selenium complex according to any one of claims 1-5, characterized in that, Including the following steps: Small molecule drugs, oligosaccharide molecules and reaction catalysts are mixed to obtain oligosaccharide-small molecule drug conjugates; The oligosaccharide-small molecule drug conjugate was dissolved in water and mixed with an aqueous selenium source solution to obtain a mixed solution; A reducing agent was added to the mixed solution, and after dialysis, an oligosaccharide-small molecule drug@nano-selenium complex was obtained.

7. The method for preparing the oligosaccharide-small molecule drug@selenium nanocomplex according to claim 6, characterized in that, The small molecule drug contains only hydroxyl or amino groups, and the oligosaccharide molecule contains only hydroxyl or amino groups. The steps for preparing the oligosaccharide-small molecule drug conjugate include: The first linker molecule is subjected to esterification or amidation reaction with the small molecule drug to obtain a first product; then the first product is subjected to amidation or esterification conjugation with the oligosaccharide molecule to obtain an oligosaccharide-small molecule drug conjugate. Alternatively, the first linker molecule can be conjugated with the oligosaccharide molecule to obtain a second product; the second product can then be reacted with the small molecule drug via amidation or esterification to obtain an oligosaccharide-small molecule drug conjugate.

8. The method for preparing the oligosaccharide-small molecule drug@selenium nanocomplex according to claim 6, characterized in that, The small molecule drug contains only a carboxyl group, the oligosaccharide molecule contains only a carboxyl group, and the steps for preparing the oligosaccharide-small molecule drug conjugate include: The second linker molecule is subjected to an amidation reaction with the small molecule drug to obtain a third product; the third product is then subjected to an amidation conjugate with the oligosaccharide molecule to obtain an oligosaccharide-small molecule drug conjugate. Alternatively, the second linker molecule can be conjugated with the oligosaccharide molecule to obtain a fourth product; then, the fourth product can be subjected to an amidation reaction with the small molecule drug to obtain an oligosaccharide-small molecule drug conjugate.

9. The method for preparing the oligosaccharide-small molecule drug@selenium nanocomplex according to claim 6, characterized in that, The reaction catalyst comprises one or more of N,N'-dicyclohexylcarbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N,N'-diisopropylcarbodiimide, 1,1'-carbodiimide diimidazole, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, and 4-dimethylaminopyridine; and / or, the reducing agent comprises one or more of ascorbic acid and sodium bisulfite.

10. The use of the oligosaccharide-small molecule drug@nano-selenium complex as described in any one of claims 1-5 in the preparation of drugs for neurodegenerative diseases, drugs for bone-related diseases, drugs for diabetes, drugs for cardiovascular diseases, and drugs for cancer treatment.

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