A ceria nano-drug delivery system, a preparation method and application thereof

Abstract

The application discloses a cerium dioxide nano drug delivery system and a preparation method and application thereof, relates to the technical field of drug delivery, and comprises cerium dioxide nanoparticles, the surface of the cerium dioxide nanoparticles is decorated with a multifunctional peptide with the functions of targeting integrin and cell penetration, and the multifunctional peptide is connected with the cerium dioxide nanoparticles through a polyethylene glycol derivative containing a sulfur ketal connecting arm. The application takes cerium dioxide nanoparticles as a carrier, and connects a multifunctional peptide with the functions of targeting integrin and cell penetration to the surface of the cerium dioxide nanoparticles through a polyethylene glycol derivative containing a sulfur ketal connecting arm. On the one hand, the targeting function of the multifunctional peptide is used to position integrin, so that the targeting ability of the cerium dioxide nano drug delivery system to target cells such as macrophages with high expression of surface integrin is improved; on the other hand, the cerium dioxide nanoparticles as the carrier are beneficial to positioning an inflammatory microenvironment, and the comprehensive action improves the targeting effect of the cerium dioxide nano drug delivery system.

Description

Technical Field

[0001] This application relates to the field of drug delivery technology, and in particular to a cerium dioxide nanoparticle drug delivery system, its preparation method, and its application. Background Technology

[0002] Nanomedicine delivery systems use nanomaterials to load drugs for delivery, thereby increasing drug loading capacity, drug efficacy, and reducing side effects.

[0003] Currently, lipid-based nanomaterials are commonly used as carriers to load small molecules, proteins, nucleic acids, and other types of drugs, thereby improving the biocompatibility, bioavailability, and stability of nanomedicine delivery systems. However, drug delivery systems loaded with nucleic acid drugs suffer from problems such as easy degradation by nucleases, high immunogenicity, and low biomembrane penetration efficiency. Summary of the Invention

[0004] In view of the shortcomings of the aforementioned related technologies, this application provides a cerium dioxide nanoparticle drug delivery system, its preparation method, and its application. This application uses cerium dioxide nanoparticles as a carrier and links a multifunctional peptide with targeting integrin and membrane-penetrating capabilities to its surface via a polyethylene glycol derivative containing a thioketene linker. This improves both the targeting ability of the cerium dioxide nanoparticle drug delivery system and its internalization efficiency in target cells. When used for loading RNA drugs, it enhances both the targeting and membrane-penetrating capabilities of the loaded RNA drug. Furthermore, during delivery, the macromolecular structure of the multifunctional peptide masks the RNA drug loaded on the carrier, reducing its degradation. During drug action, the thioketene linker selectively cleaves under the high ROS levels of the inflammatory microenvironment, promptly eliminating the barrier of the multifunctional peptide and improving the efficiency of RNA drug action. These combined effects improve the targeting and effective utilization of the drug during delivery.

[0005] Firstly, the cerium dioxide nanomedicine delivery system provided in this application adopts the following technical solution: A cerium dioxide nanoparticle drug delivery system includes cerium dioxide nanoparticles, the surface of which is modified with a multifunctional peptide having targeting integrin and membrane-penetrating functions, the multifunctional peptide being linked to the cerium dioxide nanoparticles via a polyethylene glycol derivative comprising a thioacetate linker arm.

[0006] Preferably, the multifunctional peptide is composed of an RGDC-targeting peptide and a TAT cell-penetrating peptide linked together.

[0007] Preferably, the amino acid sequence of the multifunctional peptide is SEQ ID NO:1.

[0008] Secondly, the preparation method of the cerium dioxide nanomedicine delivery system provided in this application adopts the following technical solution: A method for preparing a cerium dioxide nanoparticle drug delivery system includes the following steps: mixing a cerium source, a solvent, and a ligand, preparing the cerium dioxide nanoparticles by high-temperature pyrolysis under an inert atmosphere, followed by separation, washing, and purification to obtain ligand-modified cerium dioxide nanoparticles; mixing the cerium dioxide nanoparticles, deionized water, a multifunctional peptide, and a polyethylene glycol derivative, treating with ultrasound, and stirring to react; purifying the product after ligand exchange to obtain the cerium dioxide nanoparticle drug delivery system.

[0009] Preferably, the weight ratio of the cerium dioxide nanoparticles to the multifunctional peptide is 20-30:1.

[0010] Preferably, the weight ratio of the cerium dioxide nanoparticles to the multifunctional peptide is 28:1.

[0011] Preferably, the molar ratio of the source and the ligand is 1:2.9-3.1.

[0012] Preferably, the molar ratio of the source and the ligand is 1:3.

[0013] Preferably, the weight ratio of the cerium source to the solvent is 1:9-10.

[0014] Preferably, the temperature conditions for the high-temperature pyrolysis method are 250-270℃, and the reaction time is 2-4h.

[0015] Preferably, the temperature conditions for the high-temperature pyrolysis method are 260°C and the reaction time is 3 hours.

[0016] Preferably, the cerium source includes one or more of cerium nitrate, cerium nitrate hexahydrate, cerium acetate, cerium chloride, and cerium citrate.

[0017] Preferably, the cerium source is cerium nitrate hexahydrate.

[0018] Preferably, the solvent includes one or more of 1-octadecene, diphenyl ether, and n-hexadecane.

[0019] Preferably, the solvent is 1-octadecene.

[0020] Preferably, the ligand comprises one or more of oleylamine, oleic acid, trioctylphosphine oxide, dodecylamine, phosphonite, and amide phosphonate.

[0021] Preferably, the ligand is oleylamine.

[0022] Thirdly, this application provides the application of the above-mentioned cerium dioxide nanomedicine delivery system in RNA-loaded drugs.

[0023] Preferably, the RNA drug includes one or more of the following: miRNA-155 for treating osteoarthritis, miRNA-144 for treating atopic dermatitis, siRNA-TNF-α for treating ulcerative colitis, and circular RNA-circ-0000479 for treating psoriasis.

[0024] Preferably, the RNA-loaded drug comprises the following steps: preparing a mixed solution of the cerium dioxide nanoparticle drug delivery system and the RNA drug, stirring for 12-36 hours, wherein the RNA drug is fixed to the surface of the cerium dioxide nanoparticles by van der Waals forces and macromolecular adsorption.

[0025] In summary, this application includes at least one of the following beneficial technical effects: 1. This application uses cerium dioxide nanoparticles as a carrier and links a multifunctional peptide with targeting integrin and membrane-penetrating function to the surface of the nanoparticles via a polyethylene glycol derivative containing a thioacetate linker arm. On the one hand, the targeting function of the multifunctional peptide is used to locate integrins, thereby improving the targeting ability of the cerium dioxide nanoparticle drug delivery system to target cells such as macrophages that highly express integrins on their surface. On the other hand, the cerium dioxide nanoparticles as a carrier are beneficial for locating the inflammatory microenvironment. The combined effect improves the targeting effect of the cerium dioxide nanoparticle drug delivery system. 2. By leveraging the membrane-penetrating capabilities of multifunctional peptides, cerium dioxide nanoparticle drug delivery systems loaded with macromolecules can be mediated to cross the cell membrane and enter the cytoplasm through various pathways such as electrostatic interactions, membrane fusion, or macromolecular pinocytosis, thereby improving the intracellular delivery efficiency of cerium dioxide nanoparticle drug delivery systems and drugs. 3. When the cerium dioxide nanoparticle drug delivery system of this application is applied to RNA-loaded drugs, it is beneficial to improve the targeting and membrane-penetrating ability of the RNA-loaded drugs. On the other hand, during the delivery process, the macromolecular structure of the multifunctional peptide masks the RNA drug loaded on the carrier, reducing the degree of degradation of the RNA drug during delivery. During drug action, the thioacetate linker selectively breaks under the high ROS level of the inflammatory microenvironment, which promptly eliminates the blockage of the multifunctional peptide and improves the efficiency of RNA drug action. Under the combined effect, the targeting and effective utilization rate of the drug are improved during the drug delivery process. Attached Figure Description

[0026] Figure 1 This is a transmission electron microscope image of the cerium dioxide nanodrug delivery system of Example 1; Figure 2 This is the EDS elemental distribution diagram of the cerium dioxide nanodrug delivery system loaded with RNA drugs in Application Example 1. Figure 3This is a comparison of the targeting rates of the cerium dioxide nanomedicine delivery system loaded with RNA drugs in Example 1 to different cells; Figure 4 This is the adsorption-desorption isotherm of the cerium dioxide nanomedicine delivery system in Example 1; Figure 5 This is a BET surface area analysis diagram of the cerium dioxide nanomedicine delivery system in Example 1; Figure 6 This is the pore size distribution curve of the cerium dioxide nanodrug delivery system in Example 1. Detailed Implementation

[0027] The present application will be further described in detail below with reference to the embodiments. The following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified, specific conditions in the following embodiments were performed under conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the methods used are conventional methods known in the art, and the consumables and reagents used are commercially available. Unless otherwise stated, the technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods or materials similar to or equivalent to those described herein may also be applied to the present invention.

[0028] The raw materials used in the examples and comparative examples are all commercially available.

[0029] Example 1 Example 1 of this application provides a cerium dioxide nanoparticle drug delivery system, the preparation method of which is as follows: Preparation of cerium dioxide nanoparticles: 0.43 g (1.0 mmol) of cerium nitrate hexahydrate (Ce(NO3)3·6H2O) and 0.802 g (3.0 mmol) of oleylamine (C 18 H 37 NH2) and 4g of 1-octadecene (C 18 H 36The reactants were added to a 250 mL three-necked flask equipped with a condenser, an argon gas inlet device, and a thermometer. The mixture was stirred with a magnetic stirrer at room temperature for 1 hour to ensure thorough mixing. Next, the system was continuously purged with high-purity argon (99.99%) through the gas inlet device for 30 minutes to remove air and moisture. Subsequently, the reaction system was heated to 80°C and maintained for 2–4 hours to further remove residual moisture. Finally, the system was heated to 260°C and stirred for 3 hours. After the reaction, the system was allowed to cool to room temperature, and the product was separated by centrifugation (10,000 rpm, 30 minutes). The precipitate was washed three times with ethanol to remove unreacted substances and impurities. Finally, the precipitate was washed three times with deionized water, suspending the product in an appropriate amount of deionized water each time. After centrifugation, the supernatant was discarded, and the product was purified to obtain cerium dioxide nanoparticles modified with oleylamine.

[0030] Preparation of cerium dioxide nanoparticle drug delivery system: Cerium dioxide nanoparticles, deionized water, multifunctional peptide (RGDC-TAT peptide, amino acid sequence SEQ ID NO:1: RGDCGGSLYYPKVPRYGRKKRRQRRR, where RGDCGGSLYYPKVPR is the RGDC targeting peptide sequence and YGRKKRRQRRR is the TAT cell membrane penetration peptide sequence, synthesized and purified by Shanghai Sangon Biotech Co., Ltd.) and polyethylene glycol derivative (PEG, mPEG-TK-COOH, purchased from Xi'an Ruixi Biotechnology Co., Ltd., catalog number: R-PEG-5975) containing a thioketone linker arm (TK) were mixed to obtain a mixture. The concentration of cerium dioxide nanoparticles in the mixture was 2.8 mg / mL, the concentration of multifunctional peptide was 0.1 mg / mL, and the concentration of polyethylene glycol derivative was 0.1 mg / mL. The mixture was placed in a bath sonicator with an ultrasonic power set to 40-60 kHz for 30 minutes to promote ligand exchange between the oleamine ligands on the surface of cerium dioxide nanoparticles and the multifunctional peptides and polyethylene glycol derivatives. Subsequently, the sonicated mixture was stirred at room temperature for 24 hours to ensure that the multifunctional peptides and polyethylene glycol derivatives were fully bound to the surface of the cerium dioxide nanoparticles. After ligand exchange, the product was purified by ultracentrifugation (20,000 rpm, 90-120 minutes) to remove unbound ligand molecules. The product was washed three times with deionized water, with the precipitate resuspended in an appropriate amount of deionized water each time. After centrifugation, the supernatant was discarded, yielding the cerium dioxide nanoparticle drug delivery system.

[0031] Application Example 1 Application Example 1 of this application provides the application of a cerium dioxide nanoparticle drug delivery system in loading RNA drugs. The steps for loading RNA drugs are as follows: miRNA-mimics drugs are used as the RNA drug, and DEPC water is used as the solvent to prepare a mixed solution with a miRNA-mimics drug concentration of 2 μg / mL. 200 μg of the cerium dioxide nanoparticle drug delivery system obtained in Example 1 is added to 10 mL of the mixed solution, and the mixture is stirred at room temperature for 12 h. During this process, the RNA drug is immobilized on the surface of the cerium dioxide nanoparticles through van der Waals forces and macromolecular adsorption. After stirring, unbound RNA drugs are separated by ultracentrifugation (20,000 rpm, 90 min), and the precipitate is collected. Subsequently, the precipitate is washed three times with deionized water, each time suspending the precipitate in an appropriate amount of deionized water. After centrifugation, the supernatant is discarded, yielding the cerium dioxide nanoparticle drug delivery system loaded with RNA drugs.

[0032] RNA drugs are loaded onto cerium dioxide nanoparticle drug delivery systems primarily through two pathways: covalent bonding and non-covalent interactions. In terms of covalent bonding, the thioketal (C=S) group plays a crucial role. This highly reactive functional group can form stable connections with various reactive groups on nucleic acid molecules: it can undergo nucleophilic addition reactions with amino groups on nucleic acids (such as amino groups on modified bases) to form stable thioamide bonds (-CSNH-); or react with hydroxyl groups on nucleic acids (such as 2', 3' hydroxyl groups on the ribose ring, or the 5' terminal hydroxyl group) to form thioester bonds (-COS-). This connection not only exhibits good in vitro stability but may also respond to intracellular reducing environments, enabling intelligent release. Simultaneously, some non-covalent interactions are utilized. Van der Waals forces mainly occur between oxygen atoms in the PEG molecule and nucleic acid bases, and their cumulative effect enhances the stability of the multi-molecule system; macromolecular adsorption, based on the flexible structure of the PEG chain, forms multi-point contacts around the nucleic acid, providing additional stability. Hydrogen bonds (formed between PEG oxygen atoms and hydrogen bond donors in nucleic acids), electrostatic interactions (especially between cationic PEG and negatively charged nucleic acid backbones), and hydrophobic interactions are also important forces enhancing the binding of nucleic acid-delivery systems. The synergistic effect of multiple linkage mechanisms ensures the stability of nucleic acid drugs during in vivo delivery while retaining the ability to be released controllably at the target site.

[0033] Testing and Inspection (1) Transmission electron microscopy imaging was performed on the cerium dioxide nanodrug delivery system of Example 1, and the results are as follows: Figure 1 As shown.

[0034] (2) Energy dispersive X-ray spectroscopy (EDS) imaging was performed on the cerium dioxide nanomedicine delivery system of Example 1, and the results are as follows: Figure 2 As shown.

[0035] (3) The targeting efficiency of the RNA-loaded cerium dioxide nanoparticle drug delivery system corresponding to Example 1 on macrophages, dendritic cells, fibroblasts, and epithelial cells was verified by targeted flow cytometry. Macrophages, dendritic cells, fibroblasts, and epithelial cells were cultured to the logarithmic growth phase and then co-cultured with a fluorescently labeled RGDC-TAT peptide-modified nucleic acid chimeric cerium dioxide nanoparticle delivery system (concentration 5 μg / mL) for 3 hours. A control group was set up by co-culturing disordered peptide-modified cerium dioxide nanoparticles loaded with RNA drugs with macrophages. After culture, unbound nanoparticles were washed with PBS to remove unbound nanoparticles, and single-cell suspensions were prepared by trypsin digestion. The uptake efficiency of each cell line to the nanoparticle delivery system was detected by flow cytometry. The targeting performance was evaluated by analyzing fluorescence intensity and the percentage of positive cells. Figure 3 As shown, where Figure 3 A represents the co-culture group of cerium dioxide nanomedicine delivery system loaded with RNA drugs and macrophages (Macrophage). Figure 3 B represents the co-culture group of cerium dioxide nanoparticles modified with disordered peptides loaded with RNA drugs and macrophages (Macrophage+Control). Figure 3 C represents the co-culture group of cerium dioxide nanomedicine delivery system loaded with RNA drugs and dendritic cells. Figure 3 D represents the co-culture group of cerium dioxide nanomedicine delivery system loaded with RNA drugs and fibroblasts (Fibroblast). Figure 3 E represents the co-culture group of cerium dioxide nanomedicine delivery system loaded with RNA drugs and epithelial cells. Figure 3 F is a comparison chart of the targeting rates of each group.

[0036] (4) The cerium dioxide nanomedicine delivery system of Example 1 was vacuum dried overnight at 120°C to remove surface-adsorbed moisture. Then, nitrogen adsorption-desorption tests were performed using a specific surface area analyzer at liquid nitrogen temperature (77K). The relative pressure range was P / P0 = 0.01-0.99. The relationship between adsorption amount and relative pressure was recorded, and adsorption-desorption isotherms were plotted. Figure 4 As shown. The BET specific surface area of ​​the sample was analyzed, and the analysis curve is shown in Figure 1. Figure 5 As shown, the pore size distribution characteristics are analyzed, and the pore size distribution curve is obtained as follows. Figure 6 As shown.

[0037] Results Analysis The following combination Figure 1-6 The experimental data provided will be used to explain this application in detail.

[0038] Reference Figure 1,Depend on Figure 1 As can be observed, the cerium dioxide nanoparticle drug delivery system of Example 1 exhibits clear lattice fringes with a particle size of approximately 7–12 nm; Figure 1 B. It can be observed that the morphology of the cerium dioxide nanomedicine delivery system in Example 1 is uniformly dispersed without agglomeration, and its size distribution is consistent with... Figure 1 Consistent with A.

[0039] Reference Figure 2 The results showed uniform coverage of Ce (cerium, red) and O (oxygen, green) signals; local enrichment of N (nitrogen, blue) and P (phosphorus, yellow) signals (from amino acids in the multifunctional peptide); and detection of S (sulfur, fuchsin) signal (derived from thioketal bonds in mPEG-TK-COOH). This validates that the cerium dioxide nanoparticle drug delivery system of Example 1 is a structure with cerium dioxide nanoparticles as the core and a surface modified with mPEG-TK-COOH to form a multifunctional peptide.

[0040] Reference Figure 3 The results showed that the cerium dioxide nanoparticle drug delivery system of Example 1 achieved the best targeting rate of 19.02% for macrophages compared to the cerium dioxide nanoparticle drug delivery system of Example 1 for dendritic cells, fibroblasts, and epithelial cells. Figure 3 A's cerium dioxide nanodrug delivery system loaded with RNA drugs was co-cultured with macrophages. Figure 3 A comparison of the results of co-culturing cerium dioxide nanoparticles loaded with disordered peptides modified with RNA drugs and macrophages (Macrophage+Control) shows that the design of multifunctional peptides is beneficial to improving the targeting and internalization efficiency of the cerium dioxide nanodrug delivery system of Example 1 on macrophages.

[0041] Reference Figure 4 The results showed that the adsorption-desorption isotherm diagram contained type IV isotherms and type H1 hysteresis loop characteristics, namely steep adsorption branches and gentle desorption branches, indicating that the cerium dioxide nanodrug delivery system of Example 1 has a concentrated pore size distribution, which is beneficial to the adsorption and loading of RNA drugs.

[0042] Reference Figure 5 The results showed that the structural design of the cerium dioxide nanoparticle drug delivery system in Example 1 was beneficial to increasing the specific surface area of ​​cerium dioxide nanoparticles, thereby providing sufficient drug loading sites for RNA drugs and enhancing RNA adsorption capacity.

[0043] Reference Figure 6The results showed that the narrow peaks were concentrated in the 3-7 nm position, and the uniform mesopore size matched the molecular size of the RNA drug, which is beneficial to improving the loading stability of RNA drug and the release rate of RNA drug in the cerium dioxide nanodrug delivery system of Example 1.

[0044] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A cerium dioxide nanomedicine delivery system, characterized in that: The invention includes cerium dioxide nanoparticles, the surface of which is modified with multifunctional peptides that have targeting integrin and membrane-penetrating functions, and the amino acid sequence of the multifunctional peptides is SEQ ID NO:1; The preparation method includes the following steps: mixing a cerium source, solvent, and ligand, preparing the cerium dioxide nanoparticles by high-temperature pyrolysis under an inert atmosphere, followed by separation, washing, and purification to obtain ligand-modified cerium dioxide nanoparticles; mixing the cerium dioxide nanoparticles, deionized water, multifunctional peptide, and polyethylene glycol derivative containing a thioketal linker, ultrasonically treating, and stirring the reaction; purifying the product after ligand exchange to obtain the cerium dioxide nanoparticle drug delivery system; the weight ratio of the cerium dioxide nanoparticles to the multifunctional peptide is 20-30:

1.

2. A method for preparing a cerium dioxide nanomedicine delivery system as described in claim 1, characterized in that: Includes the following steps: Cerium source, solvent and ligand are mixed and prepared by high temperature pyrolysis under an inert atmosphere. After separation, washing and purification, ligand-modified cerium dioxide nanoparticles are obtained. Cerium dioxide nanoparticles, deionized water, multifunctional peptide and polyethylene glycol derivative containing thioketal linker are mixed, ultrasonically treated and stirred to complete ligand exchange and then purified to obtain the cerium dioxide nanodrug delivery system.

3. The method for preparing the cerium dioxide nanomedicine delivery system according to claim 2, characterized in that: The weight ratio of the cerium dioxide nanoparticles to the multifunctional peptide is 20-30:

1.

4. The method for preparing the cerium dioxide nanomedicine delivery system according to claim 2, characterized in that: The molar ratio of the cerium source to the ligand is 1:2.9-3.

1.

5. The method for preparing the cerium dioxide nanomedicine delivery system according to claim 2, characterized in that: The weight ratio of the cerium source to the solvent is 1:9-10.

6. The method for preparing the cerium dioxide nanomedicine delivery system according to claim 2, characterized in that: The temperature conditions for the high-temperature pyrolysis method are 250-270℃, and the reaction time is 2-4h.

7. A cerium dioxide nanodrug delivery system loaded with RNA drugs, characterized in that: The invention includes the cerium dioxide nanomedicine delivery system of claim 1 or the cerium dioxide nanomedicine delivery system prepared by any one of the preparation methods of claims 2-6, as well as RNA drugs.

8. A method for preparing a cerium dioxide nanoparticle drug delivery system loaded with RNA drug as described in claim 7, characterized in that: Includes the following steps: The cerium dioxide nanoparticle drug delivery system was mixed with an RNA drug to form a solution, and stirred for 12-36 hours. The RNA drug was then immobilized on the surface of the cerium dioxide nanoparticles through van der Waals forces and macromolecular adsorption.

Images