Cerium dioxide nano-drug delivery system as well as preparation method and application thereof

Abstract

The invention discloses a cerium dioxide nano-drug delivery system and a preparation method and application thereof, and relates to the technical field of drug delivery, the cerium dioxide nano-drug delivery system comprises cerium dioxide nano-particles, the surfaces of the cerium dioxide nano-particles are modified with multifunctional peptides with targeting integrin and transmembrane functions, and the cerium dioxide nano-particles are modified with the multifunctional peptides with targeting integrin and transmembrane functions. The multifunctional peptide is connected with the cerium dioxide nanoparticles through a polyethylene glycol derivative containing a thioketal connecting arm. According to the application, cerium dioxide nanoparticles are taken as a carrier, and the surface of the cerium dioxide nanoparticles is connected with multifunctional peptides with targeting integrin and transmembrane functions through a polyethylene glycol derivative containing a thioketal connecting arm; on one hand, the targeting function of the multifunctional peptide is utilized to locate the integrin, so that the targeting ability of the cerium dioxide nano-drug delivery system to target cells with high-expression integrin on the surface such as macrophages is improved, and on the other hand, the cerium dioxide nano-particles serving as the carrier are beneficial to locating the inflammatory microenvironment; and the targeting effect of the cerium dioxide nano-drug delivery system is improved under the comprehensive action.

Description

Technical Field

[0001] The present application relates to the field of drug delivery technology, and in particular to a cerium dioxide nano-drug delivery system and a preparation method and application thereof. Background Art

[0002] Nano drug delivery system uses nanomaterials to load drugs for drug delivery, thereby increasing drug loading, drug efficacy and reducing its 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 nano-drug delivery systems. However, drug delivery systems loaded with nucleic acid drugs have problems such as susceptibility to nuclease degradation, high immunogenicity, and low biomembrane penetration efficiency. Summary of the Invention

[0004] In view of the shortcomings of the above-mentioned related art, the present application provides a ceria nano drug delivery system and its preparation method and application. The present application uses ceria nanoparticles as a carrier, and connects a multifunctional peptide with targeting integrin and membrane-penetrating function on its surface through a polyethylene glycol derivative comprising a thioketal linker arm. On the one hand, the targeting ability of the ceria nano drug delivery system is improved, and on the other hand, the internalization efficiency of the ceria nano drug delivery system to target cells is improved; when used to load RNA drugs, on the one hand, it is beneficial to improve the targeting ability and membrane-penetrating ability of the loaded RNA drugs, and on the other hand, the macromolecular structure of the multifunctional peptide shields the RNA drugs loaded on the carrier during the delivery process, reducing the degree of degradation of the RNA drugs during the delivery process, and during the drug action process, the thioketal linker arm selectively breaks under the high ROS level of the inflammatory microenvironment, timely eliminating the barrier of the multifunctional peptide, improving the action efficiency of the RNA drugs, and under the comprehensive effect, improving the targeting in the drug delivery process and the effective utilization rate of the drug.

[0005] In the first aspect, the present application provides a cerium dioxide nano drug delivery system using the following technical solutions: A ceria nano drug delivery system comprises ceria nanoparticles, the surfaces of which are modified with a multifunctional peptide having integrin targeting and membrane-penetrating functions, and the multifunctional peptide is connected to the ceria nanoparticles via a polyethylene glycol derivative containing a thioketal linker arm.

[0006] Preferably, the multifunctional peptide is formed by connecting an RGDC targeting peptide and a TAT cell-penetrating peptide.

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

[0008] In a second aspect, the present application provides a method for preparing a cerium dioxide nano-drug delivery system using the following technical solutions: A preparation method of a ceria nano drug delivery system comprises the following steps: mixing a cerium source, a solvent and a ligand, preparing by a high-temperature pyrolysis method under an inert atmosphere, separating, washing and purifying to obtain ceria nanoparticles modified with ligands, mixing the ceria nanoparticles, deionized water, a multifunctional peptide and a polyethylene glycol derivative, ultrasonically treating the mixture and stirring for reaction, completing ligand exchange and purifying the product to obtain the ceria nano 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 Pei source to the ligand is 1:2.9-3.1.

[0012] Preferably, the molar ratio of the Pei source to 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 condition of the high-temperature pyrolysis method is 250-270° C., and the reaction time is 2-4 hours.

[0015] Preferably, the temperature condition of the high-temperature pyrolysis method is 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, phosphinate and amide phosphonate.

[0021] Preferably, the ligand is oleylamine.

[0022] In a third aspect, the present application provides the application of the above-mentioned ceria nano drug delivery system in loading RNA drugs.

[0023] Preferably, the RNA drug includes one or more of 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 drug loading comprises the following steps: preparing a mixed solution of the ceria nano drug delivery system and the RNA drug, stirring for 12-36 hours, and fixing the RNA drug to the surface of the ceria nanoparticles through 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 ceria nanoparticles as carriers, and connects a multifunctional peptide with integrin targeting and membrane-penetrating functions to its surface via a polyethylene glycol derivative containing a thioketal 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 ceria nano-drug delivery system to target cells such as macrophages that highly express integrins on their surfaces. On the other hand, the ceria nanoparticles as carriers are conducive to targeting the inflammatory microenvironment. The combined effect improves the targeting effect of the ceria nano-drug delivery system. 2. Through the membrane-penetrating ability of multifunctional peptides, the ceria nano-drug delivery system loaded with macromolecules can be mediated to cross the cell membrane and enter the cytoplasm through various pathways such as electrostatic interaction, membrane fusion or macromolecular pinocytosis, thereby improving the intracellular delivery efficiency of the ceria nano-drug delivery system and drugs; 3. When the cerium dioxide nano-drug delivery system of the present application is applied to loaded RNA drugs, on the one hand, it is beneficial to improve the targeting ability and membrane penetration ability of the loaded RNA drugs. On the other hand, during the delivery process, the macromolecular structure of the multifunctional peptide shields the RNA drugs loaded on the carrier, reducing the degree of degradation of the RNA drugs during the delivery process. During the drug action process, the thioketal linker arm selectively breaks under the high ROS level of the inflammatory microenvironment, promptly eliminating the obstruction of the multifunctional peptide and improving the efficiency of the RNA drug. Under the combined effect, the targeting of the drug delivery process and the effective utilization rate of the drug are improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is a transmission electron micrograph of the cerium dioxide nano drug delivery system of Example 1; Figure 2 This is the EDS element distribution diagram of the ceria nano drug delivery system loaded with RNA drug in Application Example 1; Figure 3This is a comparison chart of the targeting rates of the ceria nano-drug delivery system loaded with RNA drugs in Application Example 1 to different cells; Figure 4 is the adsorption-desorption isotherm of the ceria nano drug delivery system of Example 1; Figure 5 This is a BET specific surface area analysis diagram of the ceria nano drug delivery system of Example 1; Figure 6 This is the pore size distribution curve of the cerium dioxide nano drug delivery system in Example 1. DETAILED DESCRIPTION

[0027] The present application is described in further detail below in conjunction with Examples. The following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. In the following examples, if specific conditions are not specified, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. The methods used are conventional methods well known in the art unless otherwise specified, and the consumables and reagents used are commercially available unless otherwise specified. Unless otherwise indicated, the professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any method or material similar or equivalent to the described content may also be applied to the present invention.

[0028] The raw materials used in the examples and comparative examples can all be obtained commercially.

[0029] Example 1 Example 1 of the present application provides a cerium dioxide nano 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), 0.802 g (3.0 mmol) of oleylamine (Oleylamine, C 18 H 37 NH2) and 4g of 1-octadecene (1-Octadecene, C 18 H 36) was added to a 250 mL three-necked flask. The flask was equipped with a condenser, an argon inlet, and a thermometer. The reactants were stirred at room temperature for 1 hour using a magnetic stirrer to ensure thorough mixing. Next, high-purity argon (99.99%) was continuously purged through the gas inlet for 30 minutes to remove air and moisture. The reaction system was then 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 was complete, the system was cooled to room temperature and the product was isolated by centrifugation (10,000 rpm, 30 minutes). The precipitate was washed three times with ethanol to remove unreacted products and impurities. Finally, the precipitate was washed three times with deionized water, each time suspending the product in an appropriate amount of deionized water. After centrifugation, the supernatant was discarded and purified to obtain oleylamine-modified ceria nanoparticles.

[0030] Preparation of ceria nanodrug delivery system: Ceria nanoparticles, deionized water, a 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 penetrating peptide sequence, synthesized and purified by Shanghai Sangon Biotechnology Co., Ltd.), and a polyethylene glycol derivative containing a thioketal linker arm (TK) (PEG, mPEG-TK-COOH, purchased from Xi'an Ruixi Biotechnology Co., Ltd., catalog number: R-PEG-5975) were mixed to obtain a mixture. The concentration of the ceria nanoparticles, the multifunctional peptide, and the polyethylene glycol derivative in the mixture was 2.8 mg / mL, 0.1 mg / mL, and 0.1 mg / mL, respectively. The mixture was placed in a bath ultrasonicator at a frequency of 40-60 kHz for 30 minutes to promote ligand exchange between the oleylamine ligand on the surface of the ceria nanoparticles and the multifunctional peptide and polyethylene glycol derivative. The sonicated mixture was then stirred at room temperature for 24 hours to ensure sufficient binding of the multifunctional peptide and polyethylene glycol derivative to the ceria nanoparticle surface. 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, each time suspending the precipitate in an appropriate amount of deionized water. After centrifugation, the supernatant was discarded to obtain the ceria nanodrug delivery system.

[0031] Application Example 1 Application Example 1 of the present application provides an application of a ceria nano drug delivery system in loading RNA drugs. The steps of loading RNA drugs are as follows: taking a miRNA-mimics drug as an RNA drug, using DEPC water as a solvent, and configuring a mixed solution with a miRNA-mimics drug concentration of 2 μg / mL, 200 μg of the ceria nano drug delivery system obtained in Example 1 is added to 10 mL of the mixed solution and stirred at room temperature for 12 hours. During this process, the RNA drug is fixed to the surface of the ceria nanoparticles by van der Waals forces and macromolecular adsorption. After stirring, the unbound RNA drug is separated by ultracentrifugation (20,000 rpm, 90 minutes), and the precipitate is collected. Subsequently, the precipitate is washed 3 times with deionized water, and the precipitate is suspended in an appropriate amount of deionized water each time. After centrifugation, the supernatant is discarded to obtain a ceria nano drug delivery system loaded with RNA drugs.

[0032] RNA drug loading onto ceria nanoparticle drug delivery systems is primarily achieved through covalent bonding and non-covalent interactions. For covalent attachment, the thioketal (C=S) group plays a key role. This highly reactive functional group can form stable linkages with a variety of reactive groups on nucleic acid molecules: it can undergo nucleophilic addition reactions with amino groups on nucleic acids (such as those on modified bases) to form stable thioamide bonds (-CSNH-); or it can react with hydroxyl groups on nucleic acids (such as the 2', 3', or 5' terminal hydroxyl groups on the ribose ring) to form thioester bonds (-COS-). This linkage not only exhibits excellent in vitro stability but also has the potential to respond to the intracellular reducing environment, enabling intelligent release. Non-covalent interactions are also utilized. Van der Waals forces primarily occur between the oxygen atoms in the PEG molecule and the nucleic acid bases, with cumulative effects enhancing stability in multi-molecular systems. Macromolecular adsorption, based on the flexible structure of the PEG chain, forms multiple 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 the negatively charged nucleic acid backbone), and hydrophobic interactions are also important forces that enhance the binding of nucleic acids to delivery systems. The synergistic effect of multiple attachment mechanisms ensures the stability of nucleic acid drugs during in vivo delivery while retaining the ability to be controlled and released at the target site.

[0033] Test and Inspection (1) Transmission electron microscopy imaging of the cerium dioxide nano drug delivery system of Example 1 was performed, and the results were as follows: Figure 1 shown.

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

[0035] (3) The ceria nano drug delivery system loaded with RNA drugs in Example 1 was used to verify its targeting efficiency on macrophages, dendritic cells, fibroblasts and epithelial cells by targeted flow cytometry. After macrophages, dendritic cells, fibroblasts and epithelial cells were cultured to the logarithmic growth phase, they were co-cultured with the fluorescently labeled RGDC-TAT peptide-modified nucleic acid chimeric ceria nano delivery system (concentration of 5 μg / mL) for 3 hours. A control group was set up in which ceria nanoparticles modified with disordered peptide segments loaded with RNA drugs were co-cultured with macrophages as the control group. After the culture, the unbound nanoparticles were washed with PBS to remove them, and the single cell suspension was prepared by trypsin digestion. The uptake efficiency of the nano delivery system by each cell line was detected by flow cytometry. The targeting performance was evaluated by analyzing the fluorescence intensity and the percentage of positive cells. The results are as follows: Figure 3 As shown, Figure 3 A is a group of ceria nano drug delivery system loaded with RNA drugs and co-cultured with macrophages (Macrophage), Figure 3 B is the co-culture group of disordered peptide-modified cerium dioxide nanoparticles loaded with RNA drugs and macrophages (Macrophage+Control), Figure 3 C is a group of ceria nano drug delivery system loaded with RNA drugs and co-cultured with dendritic cells (Dendritic cell), Figure 3 D is a group of fibroblasts co-cultured with a ceria nano drug delivery system loaded with RNA drugs. Figure 3 E is a group of co-cultured cerium dioxide nano drug delivery system loaded with RNA drugs and epithelial cells (Epithelial cell), Figure 3 F is a comparison chart of the targeting rates of each group.

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

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

[0038] Reference Figure 1,Depend on Figure 1 A It can be observed that the ceria nano drug delivery system of Example 1 shows clear lattice fringes and the particle size is about 7-12 nm; Figure 1 B It can be observed that the morphology of the cerium dioxide nano drug delivery system in Example 1 is uniformly dispersed without agglomeration, and its size distribution is consistent with Figure 1 A is consistent.

[0039] Reference Figure 2 The results show uniform coverage of Ce (red) and O (green) signals; localized enrichment of N (nitrogen) and P (phosphorus) signals (from the amino acids of the multifunctional peptide); and detection of S (sulfur) signals (from the thioketal bond of mPEG-TK-COOH). This verifies that the ceria nanodrug delivery system of Example 1 is a structure composed of ceria nanoparticles as the core, with a multifunctional peptide modified on the surface via mPEG-TK-COOH.

[0040] Reference Figure 3 The results showed that the targeting rate of the cerium dioxide nano drug delivery system of Example 1 to macrophages was the best, reaching 19.02%, compared with the targeting rate of the cerium dioxide nano drug delivery system of Example 1 to dendritic cells, fibroblasts and epithelial cells. Figure 3 A ceria nanoparticle drug delivery system loaded with RNA drugs and co-cultured with macrophages (Macrophage) and Figure 3 Comparison of the results of the co-culture group (Macrophage+Control) of ceria nanoparticles modified with disordered peptide segments loaded with RNA drugs in B shows that the design of the multifunctional peptide is beneficial to improving the targeting and internalization efficiency of the ceria nanodrug delivery system of Example 1 to macrophages.

[0041] Reference Figure 4 The results show that there is a type IV isotherm and an H1 hysteresis loop feature in the adsorption-desorption isotherm diagram, namely a steep adsorption branch and a gentle desorption branch, indicating that the pore size of the cerium dioxide nanodrug delivery system in Example 1 is concentratedly distributed, which is conducive to the adsorption and loading of RNA drugs.

[0042] Reference Figure 5 The results showed that the structural design of the ceria nano-drug delivery system of Example 1 is conducive to increasing the specific surface area of ​​ceria nanoparticles, thereby providing sufficient drug loading sites for RNA drugs and enhancing the RNA adsorption capacity.

[0043] Reference Figure 6The results show that the narrow peak is concentrated at the 3-7 nm position, and the uniform mesopore size matches the molecular size of the RNA drug, which is beneficial to improving the loading stability of the cerium dioxide nano-drug delivery system of Example 1 for RNA drugs and the release rate of RNA drugs in response to ROS.

[0044] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed, but as long as they are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. A cerium dioxide nano drug delivery system, characterized in that: The invention comprises cerium dioxide nanoparticles. The surface of the cerium dioxide nanoparticles is modified with a multifunctional peptide having integrin targeting and membrane-penetrating functions. The multifunctional peptide is connected to the cerium dioxide nanoparticles via a polyethylene glycol derivative containing a thioketal connecting arm.

2. The cerium dioxide nano drug delivery system according to claim 1, characterized in that: The multifunctional peptide is formed by connecting an RGDC targeting peptide and a TAT cell-penetrating peptide.

3. The cerium dioxide nano drug delivery system according to claim 2, characterized in that: The amino acid sequence of the multifunctional peptide is SEQ ID NO:

1.

4. A method for preparing the cerium dioxide nano drug delivery system according to claims 1-3, characterized in that: The following steps are involved: A cerium source, a solvent and a ligand are mixed, prepared by a high-temperature pyrolysis method under an inert atmosphere, and then separated, washed and purified to obtain ligand-modified ceria nanoparticles. The ceria nanoparticles, deionized water, a multifunctional peptide and a polyethylene glycol derivative are mixed and ultrasonically treated and stirred for reaction. After completing the ligand exchange, the product is purified to obtain the ceria nanodrug delivery system.

5. The method for preparing the cerium dioxide nano drug delivery system according to claim 4, wherein: The weight ratio of the cerium dioxide nanoparticles to the multifunctional peptide is 20-30:

1.

6. The method for preparing the cerium dioxide nano drug delivery system according to claim 4, wherein: The molar ratio of the Pei source to the ligand is 1:2.9-3.

1.

7. The method for preparing the ceria nano drug delivery system according to claim 4, wherein: The weight ratio of the cerium source to the solvent is 1:9-10.

8. The method for preparing the cerium dioxide nano drug delivery system according to claim 4, wherein: The temperature condition of the high-temperature pyrolysis method is 250-270° C., and the reaction time is 2-4 hours.

9. Use of the ceria nano drug delivery system according to any one of claims 1 to 3 in loading RNA drugs.

10. The use according to claim 9, characterized in that: The RNA drug loading comprises the following steps: The ceria nano drug delivery system and the RNA drug are prepared into a mixed solution, which is stirred for 12-36 hours, and the RNA drug is fixed to the surface of the ceria nanoparticles through van der Waals force and macromolecular adsorption.

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