Donepezil-loaded transferrin chitosan-cyclic peptide RGD nanoparticles, preparation method and application thereof

Abstract

The present invention relates to the field of biotechnology, and in particular to a kind of donepezil-loaded transferrin chitosan-cyclic peptide RGD nanoparticles, a preparation method and an application thereof. The donepezil-loaded transferrin chitosan-cyclic peptide RGD nanoparticles provided by the present invention have excellent solubility and dispersibility in water, small particle size, high encapsulation rate and good sustained-release effect. Chitosan-cyclic peptide cRGD can significantly improve the drug targeting ability. When delivered to the nose and brain, chitosan prolongs nasal retention and reduces nasal mucosal toxicity, while transferrin helps cross the blood-brain barrier, and cyclic peptide RGD can accurately target and locate the lesion site. In the nanoparticles provided by the present invention, both the chitosan-cyclic peptide RGD linker and transferrin have the advantages of good biocompatibility, natural degradability and safety. It is expected to improve bioavailability by comprehensively optimizing the drug release and absorption process, and provide a new dosage form and delivery strategy for the treatment of brain diseases.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to donepezil-loaded transferrin chitosan-cyclic peptide RGD nanoparticles, a preparation method and applications thereof. Background Art

[0002] Donepezil belongs to the hexahydropyridine oxide class and acts as a second-generation, specific, reversible central acetylcholinesterase (AChE) inhibitor. Its mechanism of action is to inhibit AChE activity, slowing the decomposition of acetylcholine (ACh) in the synaptic cleft, thereby increasing ACh content and improving cognitive function in patients with Alzheimer's disease (AD). In clinical treatment, cholinesterase inhibitors are the first-line medication for the treatment of mild to moderate AD, primarily used to improve patients' cognitive function, enhance their daily living abilities, and promote overall clinical changes. Donepezil can also be used to treat moderate to severe AD on this basis.

[0003] However, donepezil itself has many drawbacks, including poor water solubility, slow absorption after oral administration, low average bioavailability, a small amount of drug that reaches the brain after oral administration, and severe adverse reactions. These problems combined have greatly limited the effective application of donepezil in the clinical treatment of Alzheimer's disease. In addition, studies have shown that as the dosage of donepezil gradually increases, the probability of patients experiencing adverse reactions also increases accordingly. Based on the above situation, it is particularly necessary to develop a safe and efficient sustained-release nanodelivery system for the delivery of donepezil. Such a system can not only improve the therapeutic effect of donepezil, but also overcome its existing limitations, thereby expanding its application in the medical field.

[0004] Transferrin (Tf) is a plasma protein with unique properties. It is non-toxic, has extremely low immunogenicity, and possesses excellent biocompatibility and biodegradability. According to relevant research reports, the Tf molecular structure contains 679 amino acid residues, 256 of which are hydrophobic amino acids. Tf can specifically bind to the Tf receptor (TfR). In the process of crossing the blood-brain barrier (BBB) ​​and tumor cell membranes, transcytosis mediated by the active targeting transferrin receptor (TfR) pathway is a critical transport mechanism. Notably, TfR is highly expressed in brain capillary endothelial cells (BCEC).

[0005] In the conventional treatment of brain diseases, drugs are usually administered orally or intravenously. The blood-brain barrier is a necessary link for drugs to enter the brain and reach the lesions. The blood-brain barrier is composed of brain capillary endothelial cells, glial cells and choroid plexus. It has the characteristics of selective permeability, allowing only specific molecules to enter the brain and other surrounding cells. It plays an important role in blocking harmful substances in the blood and protecting the safety of brain tissue. But at the same time, this characteristic also makes it difficult for most small molecule drugs, large molecule peptides and proteins to enter the brain. This phenomenon seriously limits the effective treatment of central nervous system diseases. Due to the existence of the blood-brain barrier, it is difficult for drugs to fully reach their intended site of action, which greatly reduces the actual therapeutic effect of the embedded drugs. These unfavorable factors have led to a significant restriction on the application of related drugs.

[0006] Therefore, there is an urgent need to develop a new drug delivery system that is expected to enhance the targeted drug therapeutic efficacy of donepezil, improve the drug's water dispersion properties, enhance its bioavailability, and prolong the drug's action period, thereby providing strong support and possible directions for the optimization of brain disease treatment strategies. Summary of the Invention

[0007] In view of this, the present invention provides donepezil-loaded transferrin chitosan-cyclic peptide RGD nanoparticles, a preparation method and applications thereof.

[0008] The technical solution of the present invention is achieved as follows:

[0009] In the first aspect, the present invention provides a kind of transferrin / chitosan-cyclic peptide RGD nanoparticles loaded with donepezil.

[0010] Including core structure and surface modification structure;

[0011] The core structure comprises: transferrin nanostructures coated with donepezil formed by depolymerizing transferrin at a pH of 2-3, interacting with donepezil molecules after depolymerization, and then recombining under neutral conditions;

[0012] The surface modified structure active ingredient comprises a chitosan-cyclic peptide RGD linker;

[0013] The chitosan portion of the chitosan-cyclic peptide RGD linker is adsorbed on the surface of the transferrin nanostructure;

[0014] The cyclic peptide RGD portion in the chitosan-cyclic peptide RGD connector is exposed at the outermost layer of the chitosan-cyclic peptide RGD connector.

[0015] In a second aspect, the present invention provides the use of the donepezil-loaded transferrin / chitosan-cyclic peptide RGD nanoparticles in the preparation of a nose-to-brain delivery system.

[0016] In a third aspect, the present invention provides a method for preparing the donepezil-loaded transferrin / chitosan-cyclic peptide RGD nanoparticles.

[0017] The steps include:

[0018] S1. Take transferrin and dissolve it in distilled water at 37°C to obtain a 0.5 mg / mL transferrin aqueous solution;

[0019] S2. Dissolve donepezil in anhydrous ethanol to obtain an anhydrous ethanol solution of 0.9-1.1 mg / mL donepezil;

[0020] S3, adjusting the pH value of the transferrin aqueous solution to 2-3 to depolymerize the cage structure; after depolymerization, adding the donepezil anhydrous ethanol solution to the transferrin aqueous solution using a microsyringe; then adjusting the pH value of the transferrin aqueous solution to 7.0 to reorganize the cage structure to form a transferrin nanostructure encapsulated with donepezil;

[0021] S4, stirring the solution obtained in step S3 for 1-2 hours, and removing ethanol from the solution by rotary evaporation;

[0022] S5. Add 1 mg / mL chitosan-cyclic peptide RGD linker aqueous solution to the solution prepared in step S4 and stir for 30-60 minutes;

[0023] S6. Freeze-drying the solution obtained in step S5 to obtain donepezil-loaded transferrin / chitosan-cyclic peptide RGD nanoparticles.

[0024] In a fourth aspect, the present invention provides the application of the preparation method in the preparation of a nose-brain delivery system.

[0025] The beneficial effects of the present invention include at least the following:

[0026] The transferrin chitosan-cyclic peptide RGD nanoparticles loaded with donepezil provided by the present invention have excellent solubility and dispersibility in water, small and uniform particle size distribution, high drug encapsulation rate and good sustained release. Its preparation is based on the principle of hydrophobic and electrostatic interaction. Donepezil is loaded on transferrin to form a core through hydrophobic interaction, and the chitosan-cyclic peptide RGD linker is precisely attached to the surface of transferrin nanoparticles by electrostatic interaction, thereby constructing a stable overall structure. Among them, chitosan-cyclic peptide cRGD can significantly enhance drug targeting ability. During nose-brain delivery, chitosan and transferrin work together. The former prolongs the nasal retention time with bioadhesion and reduces nasal mucosal toxicity with good biocompatibility. Transferrin assumes the transport function across the blood-brain barrier. The cyclic peptide RGD accurately locates the brain lesion site with high affinity to enhance targeting. As natural biomacromolecules, chitosan-cyclic peptide cRGD and transferrin both have good biocompatibility, natural degradability and safety, providing a guarantee for drug delivery.

[0027] The present invention constructs a transferrin chitosan-cyclic peptide RGD nanoparticle loaded with donepezil for nasal-brain delivery. The nanoparticles not only significantly enhance stability but also successfully achieve the goal of bypassing the blood-brain barrier. Through nasal administration, the nanoparticles can significantly improve the stability and sustained-release efficiency of the drug on the nasal mucosa, thereby optimizing the drug's action process in the body and significantly improving the effectiveness and durability of the treatment. In addition, by comprehensively optimizing the drug's release and absorption mechanism, the nanoparticles effectively improve bioavailability. It is expected that while ensuring the therapeutic effect, the required drug dose can be reduced, thereby reducing the risk of toxic side effects. Therefore, the present invention provides a new drug dosage form and delivery strategy with great potential and broad prospects for the treatment of brain diseases.

[0028]

Term Explanation

[0029] In some specific embodiments of the present invention, the blood-brain barrier (BBB) ​​represents a highly selective semipermeable boundary composed of the end feet of endothelial cells, pericytes and astrocytes, which controls the exchange of substances between the blood and the central nervous system and prevents harmful substances from entering the brain.

[0030] In some specific embodiments of the present invention, donepezil (DPZ) is a drug used to treat Alzheimer's disease; it belongs to the class of cholinesterase inhibitors, which increases the concentration of acetylcholine in the brain by inhibiting acetylcholinesterase, thereby improving cognitive function.

[0031] In some embodiments of the present invention, Alzheimer's disease (AD) refers to a progressive neurodegenerative disease characterized by the appearance of amyloid plaques and neurofibrillary tangles in the brain, leading to a gradual decline in cognitive functions, including memory, language, and thinking abilities.

[0032] In some specific embodiments of the present invention, transferrin (Tf) represents the major iron-containing protein in plasma, is a β-globulin, is mainly synthesized by the liver, and its main function is to transport iron ions.

[0033] In some specific embodiments of the present invention, a cyclic peptide (also known as a cyclic polypeptide) refers to a cyclic polymer formed by covalently linking two or more amino acids through peptide bonds.

[0034] In some specific embodiments of the present invention, the cyclic peptide-chitosan conjugate (CPC) refers to a complex in which the cyclic peptide and chitosan are bound together by chemical covalent bonds or other interactions.

[0035] In some specific embodiments of the present invention, the cyclic peptide RGD (cRGD) represents a cyclic peptide formed by cyclizing the RGD sequence to enhance its stability and binding ability.

[0036] In some specific embodiments of the present invention, Arginine-Glycine-Aspartic acid (RGD) represents a short peptide sequence that can specifically bind to integrin receptors on the cell surface.

[0037] In some embodiments of the present invention, chitosan (CS) refers to a natural polysaccharide derived from deacetylated chitin.

[0038] In some specific embodiments of the present invention, Donepezil@Transferrin nanoparticles (Donepezil@TfNPs) represent nanoparticles formed by the combination of donepezil and transferrin, wherein “@” indicates that donepezil is combined with transferrin.

[0039] In some specific embodiments of the present invention, donepezil@transferrin / chitosan-cyclic peptide RGD (Donepezil@Tf / CS-cRGD) represents a complex comprising donepezil, transferrin, chitosan and cyclic peptide RGD, wherein “@” indicates that donepezil is combined with transferrin to form an integral structure, which then forms a more complex complex with chitosan and cyclic peptide RGD.

[0040] In some embodiments of the present invention, the "@" symbol is used in these expressions to concisely represent a binding or complex relationship between different components, indicating that one component is encapsulated, adsorbed, or otherwise combined with one or more other components to form an integrated structure. For example, in the expression "donepezil@transferrin nanoparticles," the "@" symbol indicates that donepezil is bound to transferrin.

[0041] In some embodiments of the present invention, the "@" symbol is often used in the naming of nanoparticles, etc., to indicate a loading relationship. Here, "donepezil@transferrin / chitosan-cyclopeptide RGD nanoparticles" intuitively reflects that the donepezil drug is loaded onto nanoparticles composed of transferrin and chitosan-cyclopeptide RGD. This is consistent with the meaning of "donepezil-loaded transferrin chitosan-cyclopeptide RGD nanoparticles," both of which illustrate the fact that the nanoparticles serve as carriers for loading the drug donepezil. Therefore, in some embodiments of the present invention, the donepezil-loaded transferrin chitosan-cyclopeptide RGD nanoparticles provided by the present invention can also be represented by "donepezil@transferrin / chitosan-cyclopeptide RGD nanoparticles." BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0043] Figure 1 Schematic diagram of the preparation process of donepezil@transferrin / chitosan-cyclic peptide RGD nanoparticles in an embodiment of the present invention;

[0044] Figure 2 TEM transmission electron microscopy results of donepezil@transferrin / chitosan-cyclic peptide RGD nanoparticles in the embodiment of the present invention;

[0045] Figure 3 : is the in vitro release curve in pure water in the embodiment of the present invention;

[0046] Figure 4Fluorescence intensity of SH-SH5Y cells treated with three different groups of drug-loaded nanoparticles containing Coumarin 6 and Donepezil, according to the present invention: (A) Confocal laser scanning microscopy images (scale bar: 50 μm): a. Donepezil / Coumarin6@TfNPs; b. Donepezil / Coumarin 6@Tf / CS NPs; c. Donepezil / Coumarin 6@Tf / CS-cRGD NPs. (B) Flow cytometry analysis results.

[0047] Figure 5 The free drug with different concentrations and three groups of different drug-loaded nanoparticles in the embodiment of the present invention have an effect on Aβ 25-35 Inhibitory effect of induced SHSY-5Y cytotoxicity;

[0048] Figure 6 The free drug and three groups of different drug-loaded nanoparticles in the embodiment of the present invention are shown in Figure 2. 25-35 Fluorescence intensity of Aβ protein aggregation induced in SH-SY5Y cells;

[0049] Figure 7 The free drug and three groups of different drug-loaded nanoparticles in the embodiment of the present invention are shown in Figure 2 for the effect of free drug and three groups of different drug-loaded nanoparticles on Aβ 25-35 Effects of induced ROS levels in SH-SY5Y cells;

[0050] Figure 8 is the protein expression level of SH-SY5Y cells treated with free drugs or different drug-loaded nanoparticles for 48 hours in the embodiment of the present invention: a. is Aβ 25-35 , b. Aβ 25-35 +FreeDonepezil, c. for Aβ 25-35 +Donepezil@TfNPs, d.

[0051] Aβ 25-35 +Donepezil@Tf / CS NPs, e. is Aβ 25-35 +Donepezil@Tf / CS-cRGD NPs;

[0052] Figure 9 These are the permeability results of the drug-loaded nanoparticles across the in vitro BBB model at different time points in the examples of the present invention. DETAILED DESCRIPTION

[0053] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be described clearly and completely below. Obviously, the embodiments described are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. If the specific conditions are not specified in the embodiments, they shall be carried out according to conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially. The methods of obtaining various biological materials described in the following embodiments only provide an experimental method to achieve the specific disclosed purpose, and should not be a limitation on the source of the biological materials of the present invention. In fact, the sources of the biological materials used are extensive, and any biological materials that can be obtained without violating laws and moral ethics can be replaced and used according to the prompts in the embodiments.

[0054] In some specific embodiments of the present invention, preferably, donepezil (purity of 98%) was purchased from Anage Chemical Co., Ltd.; transferrin (purity of 98%) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; chitosan-cyclic arginine-glycine-aspartic acid (chitosan-cyclic peptide RGD linker) was purchased from Xi'an Qiyue Biotechnology Co., Ltd.; chitosan (deacetylation degree of 80%, molecular weight MW = 500000) was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0055] Example 1 Preparation of Donepezil@Transferrin / Chitosan-Cyclic Peptide RGD Nanoparticles (Donepezil@Tf / CS-cRGD NPs) and Determination of Their Pharmaceutical Characterization

[0056] (1) Preparation method

[0057] In this embodiment, a method for preparing donepezil@transferrin / chitosan-cyclic peptide RGD nanoparticles is provided, and the specific steps are as follows (schematic diagram as shown in FIG. Figure 1 (shown) indicates

[0058] 1. Accurately weigh 5 mg of transferrin using an analytical balance, disperse it in 10 mL of 37°C distilled water, and stir continuously at 600 rpm to fully dissolve.

[0059] 2. Accurately weigh 2.5 mg of donepezil using an analytical balance, add 2.5 mL of anhydrous ethanol, sonicate for 3 minutes to completely dissolve it, and then stir for 10 minutes.

[0060] 3. Under stirring, the pH value of the transferrin aqueous solution is adjusted to about 2.5 to achieve the disaggregation of the cage structure. Then, the solution prepared in step 2 is slowly added dropwise to the transferrin solution using a syringe. The pH value of the transferrin solution is then adjusted to a neutral pH of 7.0 to achieve the reorganization of the cage structure, and the small molecule drug package is loaded into the transferrin nanocage structure.

[0061] 4. After stirring for 1 hour, remove the ethanol by rotary evaporation in a water bath at 40°C, and add distilled water to the original volume before rotary evaporation.

[0062] 5. Under stirring, dissolve 1 mg of chitosan-cyclic peptide RGD linker in 1 mL of distilled water, add 1 mL of the solution dropwise to the solution prepared in step 4, and stir for 30 minutes.

[0063] 6. After freeze-drying the solution in step 5, donepezil@transferrin / chitosan-cyclic peptide RGD nanoparticles can be obtained.

[0064] (II) Pharmaceutical characterization

[0065] For the preparation Donepezil@transferrin / chitosan-cyclic peptide RGD nanoparticles (hereinafter referred to as nanoparticles) Conduct pharmaceutical characterization tests, including the following:

[0066] 1. Determination of particle size, polydispersity coefficient, and ζ-potential

[0067] After the nanoparticle suspension was diluted with an appropriate amount of water, the particle size, polydispersity coefficient and ζ-potential of the nanoparticles in deionized water were measured using a Malvern laser particle size analyzer.

[0068] The measurement results showed that the average particle size of the nanoparticles was 247.3 nm, the polydispersity coefficient was 0.202, and the ζ-potential was +9.6 mV.

[0069] 2. Surface morphology observation

[0070] The nanoparticle suspension diluted with an appropriate amount of water was dropped onto a copper grid with a carbon film to prepare a sample, and negatively stained with 2% phosphotungstic acid, and then dried at room temperature.

[0071] The morphology of the loaded nanoparticles was observed using a transmission electron microscope. The results showed that their surface morphology was spherical and the size distribution was relatively uniform.

[0072] 3. Determination of Encapsulation Efficiency and Drug Loading

[0073] The freeze-dried nanoparticles were dissolved in 3 mL of deionized water, and the concentration of donepezil was measured using a UV-visible spectrophotometer at a wavelength of 312 nm.

[0074] The calculation formulas for drug loading and encapsulation efficiency are as follows:

[0075] Drug loading = (mass of drug encapsulated in nanoparticles / total mass of different drug-loaded nanoparticles) × 100%

[0076] Encapsulation efficiency = (mass of drug encapsulated in nanoparticles / total mass of drug input) × 100%

[0077] The measurement results showed that the encapsulation efficiency of donepezil in the nanoparticles was 72.84% and the drug loading was 12.84%.

[0078] (3) TEM observation

[0079] like Figure 2 As shown in Figure 2, TEM observations of the donepezil@transferrin / chitosan-cyclic peptide RGD nanoparticles prepared in this example revealed spherical structures with a relatively uniform size distribution. Most nanoparticles had a diameter of approximately 200 nm, which is close to the average particle size of 247.3 nm previously measured by a Malvern laser particle size analyzer. This size uniformity helps ensure consistent and predictable drug release, which is crucial for the performance of drug delivery systems.

[0080] At the same time, the nanoparticles showed good dispersibility and no obvious agglomeration phenomenon, which indicated that the nanoparticles had good stability in the solution and were beneficial for in vivo transportation and drug release.

[0081] Example 2

[0082] In this example, donepezil@transferrin / chitosan-cyclic peptide RGD nanoparticles were first prepared, and then their pharmaceutical characteristics were determined. The specific method is as follows:

[0083] (1) Preparation method

[0084] The difference from the preparation method of Example 1 is that 5 mg of transferrin in step 1 is changed to 10 mg of transferrin, and the rest remain unchanged.

[0085] (II) Pharmaceutical characterization

[0086] The determination method is the same as the pharmaceutical characterization determination method in Example 1, and the determination results are as follows:

[0087] 1. The average particle size is 291.7 nm, the polydispersity coefficient is 0.281, and the ζ-potential is +8.48 mV.

[0088] 2. The surface morphology is spherical and the size distribution is relatively uniform.

[0089] 3. The encapsulation efficiency of donepezil in the nanoparticles prepared in this example was 60.84%, and the drug loading was 9.06%.

[0090] Example 3

[0091] (1) Preparation method

[0092] The difference from the preparation method of Example 1 is that, in step 1, 5 mg of transferrin is changed to 6 mg of transferrin; in step 2, 2.5 mg of donepezil is changed to 2 mg of donepezil; and the rest remain unchanged.

[0093] (II) Pharmaceutical characterization

[0094] The determination method is the same as the pharmaceutical characterization determination method in Example 1, and the determination results are as follows:

[0095] 1. The average particle size is 269.8 nm, the polydispersity coefficient is 0.302, and the ζ-potential is +10.5 mV.

[0096] 2. The surface morphology is spherical and the size distribution is relatively uniform.

[0097] 3. In the nanoparticles prepared in this example, the encapsulation efficiency of donepezil was 65.21%, and the drug loading was 8.76%.

[0098] Comparative Example 1 Preparation of Donepezil@Transferrin Nanoparticles (Donepezil@TfNPs) and Determination of Its Pharmaceutical Characterization

[0099] (1) Preparation method

[0100] In this comparative example, a method for preparing donepezil@transferrin nanoparticles is provided, and the specific steps are as follows:

[0101] Steps 1-4 are the same as steps 1-4 in Example 1;

[0102] Step 5. The solution obtained in step 4 is freeze-dried to obtain donepezil@transferrin nanoparticles.

[0103] (II) Pharmaceutical characterization

[0104] 1. The average particle size is 121.9 nm, the polydispersity coefficient is 0.214, and the ζ-potential is -17.3 mV.

[0105] 2. Surface morphology: spherical, with relatively uniform size distribution.

[0106] 3. In donepezil@transferrin nanoparticles, the encapsulation efficiency of donepezil was 67.08% and the drug loading was 10.81%.

[0107] Comparative Example 2 Preparation of Donepezil@Transferrin / Chitosan Nanoparticles (Donepezil@Tf / CS NPs) and Determination of Its Pharmaceutical Characterization

[0108] (1) Preparation method

[0109] In this comparative example, a method for preparing donepezil@transferrin / chitosan nanoparticles is provided, and the specific steps are as follows:

[0110] Steps 1-3 are the same as steps 1-3 in Example 1.

[0111] Step 4: Dissolve 1 mg of chitosan in 0.1 mol / L acetic acid solution while stirring. After dissolution, adjust the pH of the chitosan acetic acid solution to between 5.0 and 6.0 with NaOH solution. Add 1 mL of the solution dropwise to the solution prepared in step 3 and stir for 30 minutes.

[0112] Step 5: After stirring, remove ethanol by rotary evaporation in a water bath at 40°C, and add distilled water to the volume before rotary evaporation.

[0113] Step 6. After freeze-drying the solution in step 5, donepezil@transferrin / chitosan nanoparticles can be obtained.

[0114] (II) Pharmaceutical characterization

[0115] 1. The average particle size is 191.5 nm, the polydispersity coefficient is 0.268, and the ζ-potential is +13.5 mV.

[0116] 2. The surface morphology is spherical and the size distribution is relatively uniform.

[0117] 3. In donepezil@transferrin / chitosan nanoparticles, the encapsulation efficiency of donepezil was 70.42% and the drug loading was 12.24%.

[0118] The data statistics are as follows:

[0119] Table 1

[0120]

[0121] As shown in Table 1, regarding the preparation principle, the present invention successfully prepared drug-loaded transferrin nanoparticles using hydrophobic interactions and surface-modified them using chitosan-cyclopeptide. Specifically, by leveraging the pH stability of transferrin, depolymerization is achieved under acidic conditions. Specifically, under acidic conditions, specific domains of transferrin undergo conformational changes, causing them to depolymerize into monomeric or oligomeric forms, exposing internal hydrophobic regions. Upon addition of donepezil, the hydrophobic portions of donepezil bind to the hydrophobic regions of transferrin through hydrophobic interactions. Subsequently, the pH is adjusted to neutral, and transferrin refolds and assembles, encapsulating the donepezil within to form the nanoparticle core. The chitosan-cyclopeptide adheres to the nanoparticle surface through electrostatic adsorption or other chemical interactions, increasing the overall size of the particles and thereby increasing the average particle size.

[0122] The charge characteristics of the nanoparticles show that the surface of the donepezil@transferrin / chitosan-cyclic peptide RGD nanoparticles is positively charged. This is the result of the successful adsorption of anionic transferrin molecules onto the surface of cationic chitosan molecules, and is also a key indicator of the successful formation of the nanostructure. When the nanoparticles are not coated with chitosan-cyclic peptide, their ζ-potential is negative. However, after the chitosan-cyclic peptide molecules are successfully adsorbed onto the nanoparticle surface, the ζ-potential becomes positive.

[0123] Regarding particle size and stability, polydispersity index and potential measurements indicate that the nanoparticles prepared by the present invention have a relatively narrow size distribution and good uniformity. This good uniformity, combined with the appropriate surface charge (positive charge), results in high stability of the nanoparticles in solution, reducing interparticle aggregation and improving their performance in applications such as drug delivery.

[0124] Furthermore, the donepezil encapsulation efficiency in the various drug-loaded nanoparticles was consistently above 60%, and the drug loading efficiency was consistently above 8%. These data strongly demonstrate the excellent drug loading capacity of transferrin-based nanoparticles, allowing for easy co-loading of the drug within the nanoparticles. A higher encapsulation efficiency means more drug can be effectively encapsulated within the nanoparticles, minimizing drug loss during preparation and storage, while also improving drug stability.

[0125] Higher drug loading rate ensures that sufficient amount of drug can be carried in the nanoparticles per unit mass, which is of great significance for improving the therapeutic effect of the drug. In drug delivery systems, good drug loading capacity is one of the key factors to ensure that the drug can effectively reach the target site and play a role. Therefore, the nanoparticles prepared by the present invention show good performance in terms of structural characteristics (particle size, charge, uniformity and stability) and drug loading capacity, which provides strong support for its application in the field of biomedicine, especially in drug delivery.

[0126] Example 1 exhibits relatively balanced and ideal properties in terms of average particle size, polydispersity coefficient, zeta potential, encapsulation efficiency, and drug loading. Although other examples may have more optimal values ​​for certain individual parameters, considering all parameters comprehensively, Example 1 achieves a good balance across multiple key indicators and is therefore considered a preferred solution.

[0127] Transferrin receptors, one of the most common receptors on brain cells, interact with hydrophobic donepezil to form water-soluble core-shell nanoparticles, enhancing drug targeting while overcoming the hydrophobic nature of the drug. Chitosan, a commonly used sustained-release agent, is linked to cRGD. This cross-linking with negatively charged proteins creates a double-layer structure, ensuring nanoparticle stability and delaying drug release. It also enhances the nanoparticle's targeting ability, enabling smooth drug delivery to the brain. Furthermore, the negatively charged cRGD enhances its targeting during treatment.

[0128] Example 4

[0129] For the drug-loaded nanoparticles prepared in Example 1, Comparative Example 1, and Comparative Example 2 (for ease of description, they are collectively referred to as three groups of different drug-loaded nanoparticles in the examples and figures of the specification. Among them, the nanoparticles prepared in Example 1 are Donepezil@Tf / CS-cRGD NPs, the nanoparticles prepared in Comparative Example 1 are Donepezil@TfNPs, and the nanoparticles prepared in Comparative Example 2 are Donepezil@Tf / CS NPs.), the cumulative drug release percentages were measured. The specific measurement method is as follows:

[0130] Drug-loaded nanoparticles were dispersed in 2 ml of PBS (0.01 M, pH 7.4) and placed in a dialysis bag. The bag was then immersed in a pure water release medium and incubated in a shaker at 37°C. Samples were removed at predetermined time intervals. The drug concentration was determined by measuring the absorbance at 312 nm using a UV-visible spectrophotometer. The cumulative drug release percentage was calculated using the following formula:

[0131] Cumulative drug release percentage = amount of drug released / total amount of drug entrapped × 100%;

[0132] Determination The results are as follows Figure 3 shownThe drug is released slowly and continuously from the transferrin nanoparticles. Due to the presence of chitosan, which is positively charged and bioadhesive, cross-linking with the negatively charged protein carrier to construct nanoparticles prolongs the retention time of the nanoparticles in the nasal cavity. Therefore, the release rate of Donepezil@Tf / CS and Donepezil@Tf / CS-cRGD drug-loaded nanoparticles is relatively slow. This indicates that the transferred iron-chitosan-cyclic peptide RGD nanoparticles loaded with donepezil provided by the present invention can, on the one hand, increase the drug's residence time in the body, targeting the brain to improve efficacy; on the other hand, the slow release can reduce the frequency of drug administration.

[0133] Example 5

[0134] In the examples, the drug-loaded nanoparticles prepared in Example 1, Comparative Example 1, and Comparative Example 2 (i.e., the "three groups of different drug-loaded nanoparticles" mentioned in Example 4) were used as test objects, and the specific test steps were as follows:

[0135] (1) In vitro biological evaluation

[0136] To accurately assess the uptake of drug-loaded nanoparticles by neural cells, this example employed the method of encapsulating the fluorescent dye Coumarin 6 in a nanoparticle delivery system and observing the intracellular fluorescence intensity using confocal laser scanning microscopy (CLSM) and flow cytometry. The steps are as follows:

[0137] 1. Flow cytometry

[0138] SH-SY5Y cells (purchased from Wuhan Punosai Life Science Technology Co., Ltd., catalog number CL-0208) were seeded in six-well plates at a density of approximately 2×10 5 / well, after culturing in the incubator for 24 hours, the old culture medium was removed and replaced with fresh culture medium containing coumarin 6-labeled free drugs or nanoparticles. After 4 hours, the culture medium was removed, and the cells were washed three times with PBS. Trypsin was added for digestion, and the cells were blown with PBS to terminate the digestion. The cells were centrifuged at 1500 rpm for 5 minutes, the supernatant was removed, and paraformaldehyde was added to fix the cells. After the cells were resuspended, the fluorescence intensity of Donepezil in the cells was measured by flow cytometry.

[0139] 2. Confocal laser scanning microscopy observation:

[0140] SH-SY5Y cells were seeded in a confocal culture dish and cultured in an incubator for 24 hours. The old culture medium was removed and replaced with fresh culture medium containing coumarin 6-labeled free drugs or different nanoparticles. After incubation for 4 hours, the culture medium was removed and washed three times with PBS. Culture medium containing Hoechst33342 was added to stain the cells for 15 minutes, and then the cells were washed three times with PBS, fresh culture medium was added, and observation was performed using a confocal laser scanning microscope.

[0141] 3. Results

[0142] Evaluate the uptake of drug-loaded nanoparticles by neural cells and the accumulation of drugs within the cells (drug concentration):

[0143] After treatment with three groups of different drug-loaded nanoparticles, the results like Figure 4 As shown, a certain intensity of fluorescence can be detected in the cells. Statistical analysis found that due to the modification of cRGD cyclic peptide, the fluorescence intensity in the cells was significantly enhanced compared with the unmodified nanoparticle group, indicating that it significantly increased the accumulation of drugs in cells, thereby improving the bioavailability of drugs. Figure 4 The data show that cRGD-modified drug-loaded nanoparticles (Donepezil / Coumarin6@Tf / CS-cRGDNPs) exhibit significant advantages in cellular uptake. Both confocal laser scanning microscopy images and flow cytometry analysis show that they can significantly enhance intracellular drug accumulation, thereby improving drug bioavailability.

[0144] The unmodified nanoparticles (Donepezil / Coumarin6@TfNPs) performed the worst in terms of cellular uptake, while the uptake effect of nanoparticles modified with chitosan alone (Donepezil / Coumarin6@Tf / CS NPs) was between the two.

[0145] These results provide strong data support for further research and development of nanoparticle-based drug delivery systems, indicating that cRGD cyclic peptide modification plays an important role in improving drug efficacy.

[0146] (2) Aβ 25-35 Inhibition of SH-SY5Y cytotoxicity induced by

[0147] 1. Method

[0148] In order to determine the potential protective effect of drug-loaded nanoparticles on SH-SY5Y neural cells, the CCK-8 method was used in this example to investigate the protective effect of nanoparticles on Aβ 25-35 Inhibitory effect of SH-SY5Y cell cytotoxicity induced by cytotoxicity. The specific steps are as follows:

[0149] a. SH-SY5Y cells were seeded into 96-well plates, and 100 μL of culture medium was added to each well. The cells were cultured according to the incubation conditions for the SH-SY5Y cytotoxicity assay. After 24 hours, different concentrations of free drug or three different groups of drug-loaded nanoparticles were added, and the cells were incubated for an additional 6 hours.

[0150] b. Add Aβ to each well at a final concentration of 25 μM 25-35 The cells were then cultured in an incubator for 24 h.

[0151] c. Add 10 μL of CCK-8 solution to each well and incubate the cells in the cell culture incubator for 2 h.

[0152] d. Use a microplate reader to measure the OD value of each well at a wavelength of 450 nm to calculate the cell viability.

[0153] 2. Results

[0154] like Figure 5 shown , in the presence of Aβ 25-35 After treatment with Aβ (25 μM) for 24 h, the survival rate of SH-SY5Y cells was significantly decreased compared with the blank group, indicating that Aβ 25-35 Has significant neurotoxicity.

[0155] After 24 hours of treatment with free drugs and three groups of different drug-loaded nanoparticles (concentrations of 5 μg / mL and 10 μg / mL, respectively), the survival rate of SH-SY5Y cells was significantly increased. Moreover, compared with free drugs, nanoparticles can more effectively improve the survival rate of nerve cells. These data indicate that nanoparticles have a significant effect on Aβ 25-35 The results showed that the drug-loaded nanoparticles can better deliver drugs into cells, thereby more effectively reducing Aβ expression. 25-35 Toxic damage to cells, thereby increasing cell survival rate.

[0156] It can be seen that the transferrin chitosan-cyclic peptide RGD nanoparticles loaded with donepezil provided by the present invention have the effect of inhibiting Aβ 25-35 It has a significant effect on the induced SH-SY5Y cytotoxicity, and differently modified nanoparticles have differences in protecting cells. cRGD-modified nanoparticles show better cell protection performance.

[0157] (III) Aβ in SH-SY5Y cells 25-35 Gathering

[0158] 1. Method

[0159] To evaluate the effect of nanoparticles on intracellular Aβ25-35 In this study, confocal laser scanning microscopy and fluorescence microplate reader were used to analyze the Aβ aggregation inhibition effect in SH-SY5Y cells. 25-35 The specific operations are as follows:

[0160] a. Seed SH-SY5Y cells in a 96-well plate, add 100 μL of culture medium to each well, and incubate for 24 hours under appropriate conditions.

[0161] b. Adding free drugs or different nanoparticles (including Donepezil@Tf / CS, Donepezil@Tf and

[0162] Donepezil@Tf / CS-cRGD) and continue incubation for 12 h.

[0163] c. Add Aβ25-35 solution with a final concentration of 25 μM to each well and incubate for another 24 h.

[0164] d. After 24 h, add 20 μM ThT dye solution to each well and mix thoroughly.

[0165] e. Measure using a fluorescence microplate reader with an excitation wavelength of 450 nm and an emission wavelength of 490 nm. Ensure the reader is preheated and calibrated before measurement. Measure each sample at least three times and take the average value.

[0166] 2. Results

[0167] like Figure 6 shown , via Aβ 25-35 The treated cells showed strong green fluorescence, which indicated that Aβ 25-35 Protein aggregation. When cells were incubated with free drug and drug-loaded nanoparticles, green fluorescence was weakened. Statistical analysis revealed that:

[0168] The free drug group (Aβ + FreeDonepezil) had a certain effect on weakening green fluorescence. Among the three groups of drug-loaded nanoparticles: After treatment with Comparative Example 1 (Aβ + Donepezil@Tf), green fluorescence was weakened. After treatment with Comparative Example 2 (Aβ + Donepezil@Tf / CS), green fluorescence was further weakened. Green fluorescence in the group treated with Example 1 (Aβ + Donepezil@Tf / CS-cRGD) almost completely disappeared, and the difference was significant compared to the free drug group and other nanoparticle groups.

[0169] The results showed that nanoparticles can inhibit intracellular Aβ 25-35 Aggregation, wherein the nanoparticles modified with cRGD (Example 1) have an effect on Aβ 25-35The inhibitory effect on aggregation was the most significant. This may be because cRGD-modified nanoparticles can more effectively deliver drugs into cells, thereby more successfully interfering with Aβ 25-35 aggregation process, thereby reducing intracellular Aβ 25-35 The fluorescence intensity generated by aggregation. It can be seen that the transferrin chitosan-cyclic peptide RGD nanoparticles loaded with donepezil provided by the present invention have the effect of inhibiting intracellular Aβ 25-35 The results showed that cRGD modification has a positive effect on aggregation, and cRGD modification can enhance this inhibitory effect.

[0170] (IV) Determination of intracellular ROS

[0171] 1. Method

[0172] Oxidative stress is considered one of the key mechanisms leading to cognitive aging and neurodegenerative diseases such as Alzheimer's disease (AD). Oxidative stress caused by the accumulation of reactive oxygen species (ROS) can induce neurotoxicity. The present invention uses a DCFH-DA probe to measure intracellular ROS levels. To determine intracellular ROS levels, this study performed the following procedures:

[0173] SH-SY5Y cells were seeded on 6-well plates with 2 mL of culture medium per well. The incubation conditions were consistent with the cytotoxicity test of SH-SY5Y cells. After 24 h of incubation, fresh culture medium containing free drugs or nanoparticles (Donepezil@Tf / CS, Donepezil@Tf, and Donepezil@Tf / CS-cRGD) was added. After incubation for 12 h, Aβ was added at a final concentration of 25 μM. 25-35 The solution was incubated for 24 h, and then the ROS assay was performed according to the procedure of the ROS reagent kit. Each sample was measured at least 3 times to obtain the average value.

[0174] 2. Results

[0175] like Figure 7 shown The fluorescence intensity of the model group was significantly enhanced after treatment with Aβ protein, indicating that the level of ROS in the cells was significantly increased after protein induction; the fluorescence intensity of SH-SY5Y cells was significantly reduced after treatment with three groups of different drug-loaded nanoparticles. The results show that nanoparticles can inhibit Aβ 25-35 The induced ROS level in SH-SY5Y cells increased. The mechanism may be that nanoparticles can effectively deliver drugs and reduce Aβ 25-35 The oxidative stress response induced by the drug can reduce the intracellular ROS level, alleviate the oxidative damage of the cells, and protect the cells from oxidative stress damage. 25-35It has a positive effect on induced cellular oxidative stress damage, and differently modified nanoparticles show different degrees of effect in reducing intracellular ROS levels, among which cRGD-modified nanoparticles perform the most outstandingly.

[0176] (5) Western blot

[0177] 1. Method

[0178] In this example, the Western blot method was used to determine the effect of nanoparticles on Aβ 25-35 The effects of induced Tau protein hyperphosphorylation in SH-SY5Y cells were investigated, and the expression of p-GSK-3β, GSK-3β, Tau, Bcl-2, Bax, and Cleaved caspase-3 proteins in cells after specific treatment were detected. The specific procedures were as follows:

[0179] SH-SY5Y cells were seeded in six-well plates and incubated for 12 h before addition of 25 μM Aβ 25-35 The solution was incubated for another 12 hours. The old medium was aspirated and replaced with fresh medium containing free drug or different drug-loaded nanoparticles (Donepezil@Tf / CS, Donepezil@Tf, and Donepezil@Tf / CS-cRGD). After 24 hours of incubation, the cells were washed with PBS and gently shaken. The cells were then digested with trypsin. Finally, the digestion process was terminated with PBS and gently pipetted to create a cell suspension. The cells were then centrifuged at 1500 rpm for 5 minutes and the cell pellet was resuspended in PBS.

[0180] First, extract protein from the sample, add protease inhibitors and then add an appropriate amount of total protein extractant, and shake in an ice bath for 30 minutes. Then centrifuge at 9000rpm for 5 minutes to separate the supernatant, and determine its concentration using a BCA kit. Use SDS-PAGE gel electrophoresis to separate the total protein according to molecular weight. Transfer the protein on the gel to a PVDF membrane by electroblotting. After blocking at room temperature for 1 hour, remove the blocking solution, add a specific primary antibody, incubate at 4°C overnight, recover the primary antibody, wash the membrane three times with TBST, then add a labeled secondary antibody, incubate at room temperature for 30 minutes, wash the membrane four times with TBST, and expose to light. The expression level of specific proteins in cells is evaluated by measuring the expression of the internal control GAPDH.

[0181] 2. Results

[0182] Studies have shown that GSK-3 contributes to the hyperphosphorylation of tau protein, which is one of the hallmarks of AD. The present invention also measured the expression levels of GSK-3β and p-GSK-3β in SH-SY5Y cells.

[0183] like Figure 8 shown Compared with the control group, Aβ 25-35 The expression level of Tau protein in SH-SY5Y cells was significantly upregulated, which was consistent with Aβ 25-35 Compared with the control group, the expression level of Tau protein in SH-SY5Y cells was significantly downregulated after nanoparticle treatment. These results indicate that nanoparticles can inhibit Aβ 25-35 Induced Tau protein expression in SH-SY5Y cells.

[0184] To further explore the Aβ 25-35 The effects of inducing cell apoptosis were determined by measuring Bcl-2, Bax and cleaved caspase-3. 25-35 The expression of anti-apoptotic protein Bcl-2 was down-regulated and the expression of pro-apoptotic protein Bax was up-regulated in SH-SY5Y cells. 25-35 It can induce cell apoptosis. At the same time, after the drug-loaded nanoparticles are treated, Aβ 25-35 The up-regulated level of Bax and the down-regulated level of Bcl-2 were inhibited, and the expression of cleaved caspase-3 was reduced. 25-35 It has a positive effect on inducing hyperphosphorylation of Tau protein and cell apoptosis in SH-SY5Y cells.

[0185] (VI) Establishment and evaluation of in vitro blood-brain barrier model

[0186] 1. Method

[0187] Brain microvascular endothelial cells hCMEC / D3 were cultured at 1.0×10 3 The cells were seeded into the chamber of a 24-well Transwell plate at a density of 100 cells / mL. After 24 h of growth, the cells were fused. The BBB model was evaluated by a 4-h leakage test, TEER test, and FLU permeability test to confirm that it met the requirements of subsequent experiments.

[0188] A monolayer of hCMEC / D3 cells was seeded in the upper chamber of the Transwell, and 600 μL of DMEM medium was added to each well of the lower chamber. 200 μL of free drug or nanoparticles (Donepezil@Tf / CS, Donepezil@Tf, and Donepezil@Tf / CS-cRGD) were added to the upper chamber 6 or 12 h in advance. 400 μL of solution was aspirated from the lower layer of the Transwell chamber at 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h, respectively. The lower layer culture medium solution was replenished with PBS buffer solution each time. The UV absorbance was measured at 312 nm, and the transmittance of Donepezil in the lower chamber was calculated.

[0189] The percentage of drug-loaded nanoparticles crossing the blood-brain barrier is calculated according to the formula: Percentage of crossing the blood-brain barrier (%) = absorbance value of lower layer nanoparticles / absorbance value of added nanoparticles × 100% (formula).

[0190] 2. result

[0191] like Figure 9 shown The blood-brain barrier model established by the transwell chamber in the present invention was used to calculate the permeability of free drugs and three groups of different drug-loaded nanoparticles. The permeability of free drugs and nanoparticles gradually increased with time. Compared with free drugs, the permeability of nanoparticles was significantly higher. At 24 hours, the permeability of the Donepezil@Tf / CS-cRGD NPs group was 16.21%, which was higher than that of the free drug group by 6.102%. The results showed that the transferrin chitosan-cyclic peptide RGD nanoparticles loaded with donepezil provided by the present invention can increase the permeability of Donepezil to the blood-brain barrier model.

[0192] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A donepezil-loaded transferrin / chitosan-cyclic peptide RGD nanoparticle, characterized in that: Including core structure and surface modification structure; The core structure is: transferrin is depolymerized under pH 2-3, interacts with donepezil molecules after depolymerization, and then reassembled under neutral conditions to form a transferrin nanostructure encapsulated with donepezil; The surface modification structure is a chitosan-cyclic peptide RGD linker; The chitosan portion of the chitosan-cyclic peptide RGD linker is adsorbed on the surface of the transferrin nanostructure; The cyclic peptide RGD portion in the chitosan-cyclic peptide RGD connector is exposed at the outermost layer of the chitosan-cyclic peptide RGD connector.

2. The donepezil-loaded transferrin / chitosan-cyclic peptide RGD nanoparticles according to claim 1, characterized in that: in, The mass ratio of the transferrin, donepezil and chitosan-cyclic peptide RGD linker is 5:2.5:

1.

3. Use of the donepezil-loaded transferrin / chitosan-cyclic peptide RGD nanoparticles according to any one of claims 1 to 2 in the preparation of a drug for nose-to-brain delivery for the treatment of Alzheimer's disease.

4. The method for preparing donepezil-loaded transferrin / chitosan-cyclic peptide RGD nanoparticles according to claim 1, characterized in that: The steps include: S1. Take transferrin and dissolve it in 10 ml of distilled water at 37°C to obtain a 0.5 mg / mL transferrin aqueous solution; S2. Dissolve donepezil in anhydrous ethanol to obtain an anhydrous ethanol solution of 0.9-1.1 mg / mL donepezil; S3, adjusting the pH value of the transferrin aqueous solution to 2-3 to depolymerize the cage structure; after depolymerization, adding the donepezil anhydrous ethanol solution to the transferrin aqueous solution using a microsyringe; then adjusting the pH value of the transferrin aqueous solution to 7.0 to reorganize the cage structure to form a transferrin nanostructure encapsulated with donepezil; S4, stirring the solution obtained in step S3 for 1-2 hours, and removing ethanol from the solution by rotary evaporation; S5. Add 1 mg / mL chitosan-cyclic peptide RGD linker aqueous solution to the solution prepared in step S4 and stir for 30-60 minutes; S6. Freeze-drying the solution obtained in step S5 to obtain donepezil-loaded transferrin / chitosan-cyclic peptide RGD nanoparticles.

5. The preparation method according to claim 4, characterized in that Also includes: The volume ratio of the transferrin aqueous solution, the donepezil anhydrous ethanol solution and the chitosan-cyclic peptide RGD linker aqueous solution is 10:2.5:

1.

6. The preparation method according to claim 4, characterized in that Also includes: In step S3, a micro syringe is used to dropwise add the solution to the transferrin aqueous solution at a rate of 1 drop / second.

7. The preparation method according to claim 4, characterized in that Also includes: Stirring speed 500-800rpm.

8. The preparation method according to claim 4, characterized in that Also includes: In the step S6, freeze drying is performed for 40-60 hours. 9 . Use of donepezil-loaded transferrin / chitosan-cyclic peptide RGD nanoparticles prepared according to the method of any one of claims 4 to 8 in the preparation of a drug for nose-to-brain delivery for the treatment of Alzheimer's disease.

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