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

Abstract

The invention relates to the technical field of biology, in particular to donepezil-loaded transferrin chitosan-cyclopeptide RGD (arginine-glycine-aspartic acid) nano-particles as well as a preparation method and application of the donepezil-loaded transferrin chitosan-cyclopeptide RGD nano-particles. The donepezil-loaded transferrin chitosan-cyclic peptide RGD nanoparticles provided by the invention have the advantages of excellent resolubility and dispersibility in water, small particle size, high encapsulation efficiency and good slow release effect. Chitosan-cyclopeptide cRGD can significantly improve the drug targeting ability, during nasal-brain delivery, chitosan prolongs nasal retention and reduces nasal mucosa toxicity, transferrin assists in striding over a blood-brain barrier, and cyclopeptide RGD can accurately target and locate a diseased region. In the nanoparticles provided by the invention, both the chitosan-cyclopeptide RGD linker and the transferrin have the advantages of good biocompatibility, natural degradability, safety and the like, and the bioavailability is expected to be improved by comprehensively optimizing the drug release and absorption process, so that a novel dosage form and a delivery strategy are provided for the treatment of brain diseases.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to donepezil-loaded transferrin chitosan-cyclic peptide RGD nanoparticles, a preparation method and application 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 down the decomposition rate of acetylcholine (ACh) in the synaptic cleft, thereby increasing the ACh content, thereby improving the cognitive function of patients with Alzheimer's disease (AD). In clinical treatment, cholinesterase inhibitors are the first-line drugs for the treatment of mild to moderate AD, mainly used to improve patients' cognitive function, improve their daily living ability, and promote overall clinical changes. On this basis, donepezil can also be used to treat moderate to severe AD.

[0003] However, donepezil itself has many defects, such as poor water solubility, slow absorption after oral administration, low average bioavailability, and less amount of drug that can reach the brain after oral administration, and more serious adverse reactions. These problems work together to greatly limit the effective application of donepezil in clinical anti-Alzheimer's disease treatment. In addition, studies have shown that as the dosage of donepezil increases, the probability of adverse reactions in patients also increases accordingly. Based on the above situation, it is particularly necessary to develop a safe and efficient sustained-release nanodelivery system for delivering donepezil. Such a system can not only improve the therapeutic effect of donepezil, but also improve its existing limitations, thereby expanding its application range in the medical field.

[0004] Transferrin (Tf) is a plasma protein with special properties. It is non-toxic and has very low immunogenicity. It has good biocompatibility and biodegradability. According to relevant studies, the Tf molecular structure contains 679 amino acid residues, of which 256 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 membrane, endocytosis mediated by the active targeting transferrin receptor (TfR) pathway is one of the most critical transport mechanisms. It is worth noting that 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 and only allows 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 encapsulated drugs. These unfavorable factors have jointly led to great restrictions on the application of related drugs.

[0006] Therefore, it is urgent to develop a new drug delivery system, which 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 a donepezil-loaded transferrin chitosan-cyclic peptide RGD nanoparticle, a preparation method and application thereof.

[0008] The technical solution of the present invention is achieved in this way:

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

[0010] Including core structure and surface modification structure;

[0011] The core structure comprises: depolymerizing transferrin under a pH value of 2-3, interacting with donepezil molecules after depolymerization, and then recombining under neutral conditions to form a transferrin nanostructure encapsulating donepezil;

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

[0013] The chitosan part in the chitosan-cyclic peptide RGD connector is adsorbed on the surface of the transferrin nanostructure;

[0014] The cyclic peptide RGD part 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 transferrin / chitosan-cyclic peptide RGD nanoparticles loaded with donepezil in the preparation of a nose-brain delivery system.

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

[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, taking donepezil, dissolving it 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 with 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 min;

[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 application of the preparation method in preparing 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 particle size and uniform distribution, high drug encapsulation rate and good sustained release. Its preparation is based on the principle of hydrophobic and electrostatic action, and donepezil is loaded on transferrin to form a core through hydrophobic action, and the chitosan-cyclic peptide RGD connector is accurately attached to the surface of transferrin nanoparticles by electrostatic action, thereby constructing a stable overall structure. Among them, chitosan-cyclic peptide cRGD can significantly improve drug targeting ability. During nose-brain delivery, chitosan and transferrin work together, the former prolongs the nasal retention time with biological adhesion and reduces the nasal mucosal toxicity with good biocompatibility, transferrin assumes the transport function across the blood-brain barrier, and cyclic peptide RGD accurately locates the brain lesion site with high affinity to improve targeting, and as natural biomacromolecules, chitosan-cyclic peptide cRGD and transferrin both have good biocompatibility, natural degradability and safety, providing guarantee for drug delivery.

[0027] The present invention constructs a transferrin chitosan-cyclic peptide RGD nanoparticle loaded with donepezil for nose-brain delivery, and the nanoparticle not only significantly enhances stability, but also successfully achieves the goal of bypassing the blood-brain barrier. Through nasal administration, the nanoparticles can greatly improve the stability and sustained-release efficacy 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 release and absorption mechanism of the drug, the nanoparticles effectively improve the bioavailability. It is expected that while ensuring the therapeutic effect, the required drug dose can be reduced, thereby reducing the risk of toxic and 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 specific 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 main 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, cyclic peptide (also known as 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, cyclopeptide-chitosan conjugate (CPC) refers to a complex in which cyclopeptide 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) refers to a cyclic peptide formed by cyclization modification of 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 “@” represents the combination of donepezil and 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, and then forms a more complex complex with chitosan and cyclic peptide RGD.

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

[0041] In some specific embodiments of the present invention, the "@" symbol is often used to indicate a loading relationship in the naming of nanoparticles, etc. Here, "Donepezil@Transferrin / Chitosan-Cyclic Peptide RGD Nanoparticles" intuitively reflects that the donepezil drug is loaded on nanoparticles composed of transferrin and chitosan-cyclopeptide RGD, and is consistent with "Transferrin Chitosan-Cyclic Peptide RGD Nanoparticles Loaded with Donepezil" in meaning, both of which illustrate the fact that the nanoparticles are used as carriers to load the drug donepezil. Therefore, in some specific embodiments of the present invention, the transferrin chitosan-cyclopeptide RGD nanoparticles loaded with donepezil provided by the present invention can also be represented by "Donepezil@Transferrin / Chitosan-Cyclic Peptide 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 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 The results of TEM transmission electron microscopy observation of donepezil@transferrin / chitosan-cyclic peptide RGD nanoparticles in the embodiment of the present invention are shown;

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

[0046] Figure 4The fluorescence intensity of SH-SH5Y cells treated with three different groups of drug-loaded nanoparticles containing Coumarin 6 and Donepezil in the present invention is shown in the figure below: (A) Confocal laser scanning microscopy image (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 present invention show the effects of different concentrations of free drug on Aβ 25-35 Inhibition 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. 25-35 The effect 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] Fig. 9 The permeability results of 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 scheme and advantages of the present invention clearer, the technical scheme in the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. If the specific conditions are not specified in the embodiments, they are carried out under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially. The acquisition methods of various biological materials described in the following embodiments only provide an experimental acquisition 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 Its Pharmaceutical Characterization

[0056] (I) 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. Use an analytical balance to accurately weigh 5 mg of transferrin, 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 min to completely dissolve it, and then stir for 10 min.

[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, and then the solution prepared in step 2 is slowly added drop by drop to the transferrin solution using a syringe, and then the pH value of the transferrin solution is adjusted to 7.0 to achieve neutral cage structure reorganization, 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.

[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 min.

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

[0064] (II) Pharmaceutical characterization assay

[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] (III) TEM observation

[0079] like Figure 2 As shown, the TEM transmission electron microscopy results of the donepezil@transferrin / chitosan-cyclopeptide RGD nanoparticles prepared in this example show that the nanoparticles present a spherical structure with a relatively uniform size distribution. The diameter of most nanoparticles is around 200 nm, which is close to the average particle size of 247.3 nm previously measured by the Malvern laser particle size analyzer. The uniformity of size helps to ensure the consistency and predictability of drug release, which is crucial to the performance of the drug delivery system.

[0080] At the same time, the nanoparticles showed good dispersibility without obvious agglomeration, 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] (I) Preparation method

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

[0085] (II) Pharmaceutical characterization assay

[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] (I) 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; 2.5 mg of donepezil in step 2 is changed to 2 mg of donepezil; and the rest remain unchanged.

[0093] (II) Pharmaceutical characterization assay

[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] (I) 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 assay

[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] (I) 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. Same as steps 1-3 in Example 1.

[0111] Step 4: Dissolve 1 mg of chitosan in 0.1 mol / L acetic acid solution under stirring, and 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 min.

[0112] Step 5: After stirring, remove ethanol by rotary evaporation at 40°C in a water bath, 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 assay

[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 statistics are as follows:

[0119] Table 1

[0120]

[0121] As shown in Table 1, with regard to the preparation principle, the present invention successfully prepared the drug-loaded transferrin nanoparticles by hydrophobic interaction, and used chitosan-cyclopeptide to modify its surface. Specifically, by means of the characteristics of transferrin pH stability, depolymerization is achieved under acidic conditions, that is, under acidic conditions, a specific domain of transferrin undergoes conformational changes, causing it to depolymerize into a monomer or oligomer form, exposing the internal hydrophobic region. After donepezil is added, the hydrophobic part of donepezil is combined with the hydrophobic region of transferrin by hydrophobic interaction. Subsequently, pH is adjusted to neutral, and transferrin is refolded and assembled, and donepezil is wrapped inside to form a nanoparticle core. Chitosan-cyclopeptide is attached to the surface of the nanoparticles by electrostatic adsorption or other chemical interactions, which increases the overall size of the particles, thereby increasing the average particle size.

[0122] From the charge characteristics of the nanoparticles, the surface of donepezil@transferrin / chitosan-cyclopeptide RGD nanoparticles is positively charged, which is the result of the successful adsorption of anionic transferrin molecules onto the surface of cationic chitosan molecules, and is also an important sign of the successful formation of the nanostructure. When the nanoparticles are not wrapped with chitosan-cyclopeptide, their ζ-potential is negative, and after the chitosan-cyclopeptide molecules are successfully adsorbed on the surface of the nanoparticles, the ζ-potential turns positive.

[0123] Regarding particle size and stability, the polydispersity index and potential measurement results show that the nanoparticles prepared by the present invention have a relatively narrow size distribution and good uniformity. This good uniformity and suitable surface charge characteristics (positive charge) work together to make the nanoparticles have high stability in solution, reduce the agglomeration phenomenon between particles, and are beneficial to their performance in applications such as drug delivery.

[0124] In addition, the encapsulation rate of donepezil in different drug-loaded nanoparticles was higher than 60%, and the drug loading rate was higher than 8%. These data fully demonstrate that transferrin-based nanoparticles have good drug loading capacity, and drugs can be easily co-loaded in nanoparticles. A higher encapsulation rate means that more drugs can be effectively encapsulated inside the nanoparticles, reducing the loss of drugs during preparation and storage, and also helping to improve the stability of the drugs.

[0125] Higher drug loading rate ensures that a 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 the drug delivery system, 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 shows relatively balanced and ideal characteristics in terms of average particle size, polydispersity coefficient, ζ-potential, encapsulation efficiency and drug loading. Although other examples may have better values ​​for some single parameters, considering all parameters comprehensively, Example 1 can achieve a better balance in multiple key indicators, so it can be considered that Example 1 is a preferred solution.

[0127] Transferrin receptor, one of the most common receptors on brain cells, reacts with hydrophobic donepezil to form water-soluble core-shell nanoparticles, which improves the targeting of the drug while solving the hydrophobic disadvantage. Chitosan, as a commonly used sustained-release substance, can be connected with cRGD to form a double-layer structure by cross-linking with negatively charged proteins, ensuring the stability of the nanoparticles and delaying the release of the drug. On the other hand, it can enhance the targeting ability of the nanoparticles and smoothly transport the drug to the brain. At the same time, the negatively charged cRGD will play a better targeting role during the treatment process.

[0128] Example 4

[0129] For the drug-loaded nanoparticles prepared in Example 1, Comparative Example 1 and Comparative Example 2 (for ease of description, the examples and drawings in the specification are collectively referred to as three groups of different drug-loaded nanoparticles. 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 respectively. The specific measurement method is as follows:

[0130] The drug-loaded nanoparticles were dispersed in 2 ml of PBS solution (0.01 M, pH 7.4) and placed in a dialysis bag, which was then immersed in a release medium of pure water and then incubated in a shaker at 37°C. The samples were taken out at predetermined time intervals. The absorbance at 312 nm was measured using a UV-visible spectrophotometer to determine the drug concentration. The cumulative drug release percentage was calculated according to the following formula:

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

[0132] Determination The results are as follows Figure 3 Shown, the drugs can be released slowly and continuously from the transferrin nanoparticles. Due to the presence of chitosan, which is positively charged and has bioadhesive properties, the cross-linking with the negatively charged protein carrier to construct nanoparticles prolongs the retention time of the nanoparticles in the nasal cavity, so the release rate of Donepezil@Tf / CS and Donepezil@Tf / CS-cRGD drug-loaded nanoparticles is relatively slow, indicating that the transferrin chitosan-cyclic peptide RGD nanoparticles loaded with donepezil provided by the present invention can increase the residence time of the drug in the body on the one hand, and the brain targeting effect improves the efficacy; on the other hand, the slow release can reduce the frequent use of the drug.

[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] (I) In vitro biological evaluation

[0136] In order to accurately evaluate the uptake capacity of drug-loaded nanoparticles by nerve cells, the fluorescent dye Coumarin 6 was encapsulated in the nano drug delivery system in this embodiment, and the intracellular fluorescence intensity was observed by confocal laser scanning microscopy (CLSM) and flow cytometry. The steps are as follows:

[0137] 1. Flow cytometry detection

[0138] SH-SY5Y cells (purchased from Wuhan Pronocell Life Science Co., Ltd., catalog number CL-0208) were seeded in a six-well plate 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, the culture medium was removed after 4 hours, the cells were washed three times with PBS, trypsin was added for digestion, the cells were blown with PBS to terminate the digestion, and the cells were centrifuged at 1500rpm 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 h. 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 h, the culture medium was removed and the cells were washed three times with PBS. Culture medium containing Hoechst33342 was added to stain the cells for 15 min, and then the cells were washed three times with PBS, fresh culture medium was added, and observed using a confocal laser scanning microscope.

[0141] 3. Results

[0142] Evaluation of the neuronal cell uptake of drug-loaded nanoparticles and intracellular drug accumulation (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 cyclic peptide-modified drug-loaded nanoparticles (Donepezil / Coumarin6@Tf / CS-cRGDNPs) show obvious advantages in cell uptake. Both confocal laser scanning microscopy images and flow cytometry analysis results show that it can significantly increase the accumulation of drugs in cells, thereby improving the bioavailability of drugs.

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

[0147] 1. Methods

[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 Inhibition of induced SH-SY5Y cell toxicity. The specific steps are as follows:

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

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

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

[0152] d. Use an ELISA 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 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 It 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 increased significantly. Moreover, compared with free drugs, nanoparticles can more effectively increase the survival rate of neurons. These data indicate that nanoparticles have a significant effect on Aβ 25-35 The induced SH-SY5Y cell death was inhibited, and this inhibitory effect was most prominent in Example 1 (cRGD-modified drug-loaded nanoparticles) among the drug-loaded nanoparticles. This indicates that the nanoparticles can better deliver drugs into cells, thereby more effectively reducing Aβ 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 Gather

[0158] 1. Methods

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

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

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

[0162] Donepezil@Tf / CS-cRGD) fresh medium 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. Use a fluorescence microplate reader for measurement, set the excitation wavelength to 450nm and the emission wavelength to 490nm. Make sure the microplate reader has been preheated and the parameters have been calibrated before measurement. Measure each sample at least 3 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, indicating that Aβ 25-35 Protein aggregation. When cells were incubated with free drugs and drug-loaded nanoparticles, the green fluorescence weakened. Statistical analysis found that:

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

[0169] The results showed that nanoparticles can inhibit intracellular Aβ 25-35 Aggregation, wherein the cRGD-modified nanoparticles (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 The 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 cRGD modification can enhance the inhibitory effect.

[0170] (IV) Determination of intracellular ROS

[0171] 1. Methods

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

[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 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 tested according to the steps of the ROS reactive oxygen species kit. Each sample was measured at least 3 times to take the average value.

[0174] 2. Results

[0175] like Figure 7 Shown The fluorescence intensity of the model group after Aβ protein treatment was significantly enhanced, indicating that the level of ROS in the cells was significantly increased after protein induction; the fluorescence intensity of SH-SY5Y cells after treatment with three groups of different drug-loaded nanoparticles was significantly reduced. 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 induced oxidative stress response reduces the intracellular ROS level, reduces the oxidative damage of the cells, and protects the cells from oxidative stress damage. It can be seen that the transferrin chitosan-cyclic peptide RGD nanoparticles loaded with donepezil provided by the present invention have the effect of combating Aβ 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 performed the most prominently.

[0176] (V) Western blot

[0177] 1. Methods

[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 expressions of p-GSK-3β, GSK-3β, Tau, Bcl-2, Bax, and Cleavedcaspase-3 proteins in cells after specific treatment were detected. The specific operations were as follows:

[0179] SH-SY5Y cells were seeded in six-well plates and incubated for 12 h before adding 25 μM Aβ 25-35 The solution was incubated for 12 hours. The old medium was removed and replaced with fresh medium containing free drugs or different drug-loaded nanoparticles (Donepezil@Tf / CS, Donepezil@Tf and Donepezil@Tf / CS-cRGD). After 24 hours of culture, PBS was added and slowly shaken for washing, and digested with trypsin. Finally, the digestion process was terminated with PBS and gently pipetted to make a cell suspension. After centrifugation at 1500rpm for 5 minutes, the cell pellet after centrifugation was collected by resuspending with 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 use the BCA kit to determine its concentration. Use SDS-PAGE gel electrophoresis to separate the total protein according to molecular weight. Transfer the protein on the gel to the PVDF membrane by electrotransfer technology. After blocking at room temperature for 1 hour, remove the blocking solution, add the specific primary antibody, incubate overnight at 4°C, recover the primary antibody, wash the membrane three times with TBST, then add the labeled secondary antibody, incubate at room temperature for 30 minutes, wash the membrane four times with TBST, and expose it to light. And evaluate the expression level of specific proteins in cells by measuring the expression of internal reference 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 measures 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 apoptosis in cells were measured by 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, indicating that Aβ 25-35 Can induce cell apoptosis. At the same time, after treatment with drug-loaded nanoparticles, Aβ 25-35 The up-regulated Bax level and the down-regulated Bcl-2 level were inhibited, and the expression of cleaved caspase-3 was reduced. It can be seen that the transferrin chitosan-cyclic peptide RGD nanoparticles loaded with donepezil provided by the present invention are effective in combating Aβ 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. Methods

[0187] Brain microvascular endothelial cells hCMEC / D3 were cultured at 1.0×10 3 The cells were inoculated into the chamber of a Transwell 24-well plate at a density of 100 cells / mL and allowed to fuse after 24 h of growth. 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 inoculated in the upper chamber of Transwell, and 600 μL of DMEM medium was added to each well of the lower chamber. 200 μL of free drugs or nanoparticles (Donepezil@Tf / CS, Donepezil@Tf, and Donepezil@Tf / CS-cRGD) were added to the upper chamber 6 h 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 absorption 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 Fig. 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 6.102% higher than that of the free drug group. 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 protection scope 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 comprises: depolymerizing transferrin under a pH value of 2-3, interacting with donepezil molecules after depolymerization, and then recombining under neutral conditions to form a transferrin nanostructure encapsulating donepezil; The surface modified structure active component comprises a chitosan-cyclic peptide RGD linker; The chitosan part in the chitosan-cyclic peptide RGD connector is adsorbed on the surface of the transferrin nanostructure; The cyclic peptide RGD part 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 transferrin, donepezil and chitosan-cyclic peptide RGD connector is 5:2.5:

1.

3. The donepezil-loaded transferrin / chitosan-cyclic peptide RGD nanoparticles according to claim 1, characterized in that: The chitosan-cyclic peptide RGD connector includes chitosan-cRGD.

4. Use of the donepezil-loaded transferrin / chitosan-cyclic peptide RGD nanoparticles according to any one of claims 1 to 3 in the preparation of a nose-brain delivery system.

5. The method for preparing the 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, taking donepezil, dissolving it 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 with 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 min; S6. Freeze-drying the solution obtained in step S5 to obtain donepezil-loaded transferrin / chitosan-cyclic peptide RGD nanoparticles.

6. The preparation method according to claim 5, 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.

7. The preparation method according to claim 5, characterized in that: Also includes: In the step S3, a microsyringe is used to drop the solution into the transferrin aqueous solution at a rate of 1 drop / second.

8. The preparation method according to claim 5, characterized in that: Also includes: Stirring speed 500-800rpm.

9. The preparation method according to claim 5, characterized in that: Also includes: In the step S6, freeze drying is performed for 40-60 hours.

10. Use of the preparation method according to any one of claims 5 to 9 in the preparation of a nose-brain delivery system.

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