Composite copper selenide-halloysite-mannose drug delivery system, and preparation method therefor and use thereof

Abstract

Provided in the present invention is a composite copper selenide-halloysite-mannose drug delivery system. The drug delivery system comprises a carrier, wherein the carrier is halloysite nanotubes (HNTs), copper selenide (CuSe) nanoparticles are grown in situ on the walls of the halloysite nanotubes to form composite CuSe@HNTs, and mannose is loaded in the lumen of CuSe-coated HNTs to form composite drug delivery system CuSe@HNTs@ME. Further provided in the present invention are a method for preparing the composite copper selenide-halloysite-mannose drug delivery system, and the use thereof. The drug delivery system of the present invention can remain stable in a gastric acid environment and directly reach the site of enteritis by means of oral administration, thereby significantly alleviating the symptoms of enteritis.

Description

Copper selenide-halothite-mannose composite drug delivery system, its preparation method, and its application Technical Field

[0001] This invention relates to the field of nanomaterials technology, and more specifically to a copper selenide-halothite-mannose composite drug delivery system, its preparation method, and its application. Background Technology

[0002] Inflammatory bowel disease (IBD) is a refractory chronic disease that significantly reduces patients' quality of life and can lead to more serious complications such as colon cancer. Currently, antibiotics are commonly used clinically to suppress intestinal infections; however, antibiotics can irritate the gut, causing gut microbiota imbalance, leading to intestinal mucosal damage, and potentially triggering antibiotic resistance. As an alternative, probiotics are widely used to regulate gut microbiota to alleviate enteritis. However, probiotics are often destroyed by stomach acid as they pass through the stomach, losing their activity and failing to effectively reach the intestines to exert their effects.

[0003] Therefore, there is an urgent need to develop a new treatment strategy that can resist stomach acid and function effectively in the intestines to provide more effective treatment for IBD. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing technologies by providing a copper selenide-halogenite-mannose composite drug delivery system and its preparation method. This drug delivery system can remain stable in the acidic environment of the stomach and can directly reach the site of enteritis via oral administration, significantly alleviating the symptoms of enteritis.

[0005] According to a first aspect of the present invention, a copper selenide-halolite-mannose composite drug delivery system is provided, the drug delivery system comprising a carrier, said carrier being halloysite nanotubes (HNTs), wherein copper selenide nanoparticles (CuSe) are grown in situ on the wall of said halloysite nanotubes to form a composite CuSe@HNTs; and mannose is loaded within the lumen of CuSe@HNTs to form a composite drug delivery system CuSe@HNTs@ME.

[0006] As an optional implementation, CuSe@HNTs have a particle size range of 78.8 nm to 1990 nm and a Zeta potential of -20.3 mV to -19.6 mV.

[0007] As an optional implementation, the CuSe@HNTs@ME has a particle size range of 58.8 nm to 1280 nm and a zeta potential of -19.2 mV to -17.2 mV.

[0008] As an optional implementation, the copper selenide nanoparticles are solid spheres and / or solid flower shapes, and the particle size range of the copper selenide nanoparticles is 43.8 nm to 295 nm.

[0009] According to a second aspect of the object of the present application, a preparation method of the aforementioned copper selenide-halloysite-mannose composite drug delivery system is provided, comprising the following steps:

[0010] Polyvinylpyrrolidone (PVP) is added to ultrapure water and dissolved thoroughly to obtain a PVP solution;

[0011] Halloysite nanotubes (HNTs) are added to the PVP solution and mixed thoroughly to allow the PVP to be modified on the surface of the halloysite nanotubes, thereby obtaining a HNTs precursor solution;

[0012] A first reducing agent solution is added to the selenium source solution and mixed thoroughly to form a selenium precursor solution;

[0013] A second reducing agent solution is added to the copper source solution and mixed thoroughly to form a copper precursor solution;

[0014] The selenium precursor solution is slowly added to the HNTs precursor solution, and the reaction is carried out under controlled temperature and stirring conditions to obtain a pre-prepared solution containing selenium and HNTs;

[0015] The copper precursor solution is slowly added to the pre-prepared solution containing selenium and HNTs, and heated in a water bath to obtain a water bath product;

[0016] The water bath product is sequentially subjected to washing and drying treatment to obtain a composite of copper selenide nanoparticles generated in situ on the tubular HNTs (CuSe@HNTs);

[0017] Mannose (ME) is dissolved in ultrapure water, and a vacuum circulation device is used to load the ME into the lumen of the CuSe@HNTs material, and through centrifugation and freeze-drying, a CuSe@HNTs composite material with ME in the lumen (CuSe@HNTs@ME) is obtained.

[0018] As an optional embodiment, in the PVP solution, the mass fraction of PVP in the total volume is 0.1 w / v% to 0.5 w / v%, and the average molecular weight of PVP is 55000 to 58000.

[0019] As an optional embodiment, the mass ratio of HNTs to PVP solution is (2:1) to (1:2).

[0020] As an optional embodiment, the selenium source includes selenium dioxide (SeO2), sodium selenite (NaSeO3), or elemental selenium (Se), and the first reducing agent is ascorbic acid (Vc), citric acid, or sodium thiosulfate; the concentration of the selenium source solution is 0.1M to 0.5M, and the concentration of the first reducing agent solution is 2 to 5 times the concentration of the selenium source solution.

[0021] As an optional implementation, the copper source includes copper sulfate (CuSO4), copper nitrate (Cu(NO3)2) or copper chloride (CuCl2), and the second reducing agent is ascorbic acid (Vc), citric acid or sodium thiosulfate; the concentration of the copper source solution is 0.2M to 1M, and the concentration of the second reducing agent solution is 2 to 5 times the concentration of the copper source solution.

[0022] As an optional implementation, in the preparation of the pre-formed solution containing selenium and HNTs, the volume of the selenium precursor solution is 1 to 2 times the volume of the HNTs precursor solution.

[0023] The reaction temperature is 25℃~30℃, the stirring speed is 100rpm~300rpm, and the reaction time is 5min~20min.

[0024] As an optional implementation, in the preparation of the water bath product, the volume of the copper precursor solution is 2 to 4 times the volume of the HNT precursor solution.

[0025] The reaction temperature is 30℃~60℃, the stirring speed is 100rpm~300rpm, and the reaction time is 3h~8h.

[0026] In a third aspect of the present invention, the application of the aforementioned copper selenide-halothite-mannose composite drug delivery system in the preparation of inflammatory bowel disease drugs is also provided.

[0027] In a fourth aspect of the present invention, a pharmaceutical composition comprising the aforementioned copper selenide-halothite-mannose complex delivery system is also provided.

[0028] As can be seen from the above technical solutions of the present invention, the copper selenide-halolite-mannose composite drug delivery system proposed in this invention grows copper selenide nanoparticles in situ on the surface of halloysite nanotubes and loads mannose into the cavity of halloysite nanotubes. It cleverly utilizes mannose to achieve precise targeting of mitochondria and achieves targeted treatment of inflammation through the interaction between the negative charge of the material and the positive charge of the inflammatory area.

[0029] The CuSe@HNTs@ME composite material combines the superoxide dismutase (SOD) and catalase (CAT) activities of copper selenide, the anti-inflammatory and antidiarrheal efficacy of halloysite, and the mitochondrial protective effect of mannose. The three work synergistically to keep the drug delivery system stable in the acidic environment of the stomach, and deliver it directly to the site of enteritis via oral administration, significantly reducing enteritis symptoms.

[0030] The composite drug delivery system of this invention not only has the dual functions of scavenging reactive oxygen species (ROS) and anti-inflammation, but also effectively protects the structure and function of the intestine, providing an innovative oral treatment option for patients with enteritis, with the potential to improve clinical symptoms and enhance quality of life. Attached Figure Description

[0031] Figure 1 is a flowchart illustrating the preparation process and mechanism of the copper selenide-halolite-mannose composite drug delivery system of the present invention.

[0032] Figure 2 shows the characterization diagrams of CuSe and CuSe@HNTs in the example of this invention; wherein, Figure 2a is the TEM image of CuSe@HNTs, Figure 2b is the XRD pattern of CuSe and CuSe@HNTs (for phase analysis), Figure 2c is the thermogravimetric analysis (TGA) diagram of HNTs, CuSe and CuSe@HNTs, Figure 2d is the specific surface area (BET) diagram of HNTs, CuSe and CuSe@HNTs, and Figures 2e and 2f are the X-ray photoelectron spectroscopy (XPS) of CuSe and CuSe@HNTs, wherein 2e is the region fitting peak diagram of Cu element and 2f is the region fitting peak diagram of Se element.

[0033] Figure 3 is a characterization diagram of CuSe, CuSe@HNTs and CuSe@HNTs@ME in the example of the present invention; wherein, Figure 3a is a particle size diagram of HNTs, CuSe, ME, CuSe@HNTs and CuSe@HNTs@ME, Figure 3b is a TGA diagram of HNTs, CuSe, ME, CuSe@HNTs and CuSe@HNTs@ME, and Figure 3c is a powder XRD diagram of ME added to CuSe@HNTs solution after vacuum cycling and without vacuum cycling.

[0034] Figure 4 is a nanozyme activity analysis diagram of CuSe and CuSe@HNTs in the examples of the present invention; wherein, Figure 4a shows the amount of H2O2 produced by peroxidase-like (CAT) activity, and Figure 4b shows the ·O2 produced by superoxide dismutase (SOD)-like activity. - Elimination rate, Figure 4c shows the EPR spectrum of the ·OH scavenging ability of the material using DMPO as a rotation trap.

[0035] Figure 5 shows the electronegativity and nanozyme activity analysis of CuSe, CuSe@HNTs, and CuSe@HNTs@ME in the examples of this invention; wherein, Figure 5a is the zeta potential diagram of different material aqueous dispersions, and Figure 5b is the EPR spectrum of the ·OH scavenging ability of the material using DMPO as a rotation trapping agent.

[0036] Figure 6 shows the stability analysis results of CuSe@HNTs@ME (CSHM) of the present invention in simulated gastric juice; wherein, Figure 6a is the HNT solution diagram after untreated and after 4h simulated gastric juice treatment, Figures 6b, 6c, 6d, 6e, 6f and 6g are the SEM, zeta potential, XRD, ME release amount and BET diagram of CSHM after untreated and after 4h simulated gastric juice treatment, respectively, and Figures 6h and 6i are the comparison of CAT-like and SOD-like activities of CuSe, CuSe@HNTs and CuSe@HNTs@ME after untreated and after 4h simulated gastric juice treatment, respectively.

[0037] Figure 7 shows the targeted adhesion results of CuSe@HNTs@ME (CSHM) of the present invention in the enteritis region; wherein, Figure 7a shows the EDS mapping images of uncoated, carboxylate-coated (negatively charged, mimicking healthy epithelium) and ammonium-coated (positively charged, mimicking inflamed epithelium) polystyrene surfaces at 37°C and after 3 hours of CSHM culture; Figure 7b shows the bio-TEM images of the mucosa of healthy and inflamed mice 24 hours after oral CSHM treatment.

[0038] Figure 8 shows acoustic imaging of the mucous membranes of healthy and inflamed mice 24 hours after oral CSHM treatment.

[0039] Figure 9 shows SEM images of the mucous membranes of healthy and inflamed mice 24 hours after oral CSHM treatment.

[0040] Figure 10 shows the protection of intestinal cells (IEC-6) from ROS-induced damage by CSHM. Among them, Figure 10a shows the effect of different concentrations of DSS on the cell viability of IEC-6 cells, Figure 10b shows the effect of HNTs, CuSe, ME, CuSe@HNTs and CuSe@HNTs@ME on the 24h cell viability of IEC-6 cells to analyze their cytotoxicity, and Figures 10c and 10d are representative and statistical diagrams of ROS staining of cells treated with 2% DSS or different components by 2′,7′-dichlorofluorescein diacetate (DCFH-DA).

[0041] Figure 11 shows CSHM-treated cells protected from ROS-induced mitochondrial damage; Figure 11a shows images labeled with the following fluorescent probes: 400 nM MitoTracker Green (Ex 490 nm / Em 516 nm) and 10 μM JC-1 (a sensitive marker of mitochondrial membrane potential); Figures 11b and 11c are quantitative statistical plots of the above fluorescent images.

[0042] Figure 12 shows the in vivo therapeutic effect of oral CSHM; Figure 12a shows the change in mouse body weight after treatment with different components; Figures 12b and 12c are respectively a statistical graph of colon length and a statistical graph of disease activity index (DAI) for each group to score these symptoms; Figure 12d is a cross section H&E staining image of colon tissue of each component. Detailed Implementation

[0043] To better understand the technical content of the present invention, specific embodiments are described below in conjunction with the accompanying drawings.

[0044] Various aspects of the invention are described in this disclosure with reference to the accompanying drawings, in which numerous illustrative embodiments are shown. The embodiments of this disclosure are not necessarily intended to encompass all aspects of the invention. It should be understood that the various concepts and embodiments described above, as well as those described below in more detail, can be implemented in any of a number of ways.

[0045] Antibiotics remain the primary treatment for inflammatory bowel disease (IBD) in clinical practice. However, long-term antibiotic use can disrupt the beneficial gut microbiota, leading to intestinal health problems. To address this issue, this invention aims to utilize the reactive oxygen species (ROS) scavenging capabilities of copper selenide nanoenzymes, the anti-inflammatory and antidiarrheal functions of halloysite nanotubes, and the mitochondrial protective effects of mannose to construct an oral selenium CuSe@HNTs@ME composite drug delivery system with dual targeting of mitochondria and inflammation. This composite drug delivery system offers both precise and efficient anti-inflammatory effects and effective protection of intestinal structure and function, providing an innovative oral treatment option for the clinical treatment of IBD.

[0046] Copper selenide-haloite-mannose combined drug delivery system

[0047] Referring to Figure 1, in an exemplary embodiment of the present invention, a copper selenide-halolite-mannose composite drug delivery system is provided. The drug delivery system includes a carrier, which is halloysite nanotubes (HNTs). Copper selenide nanoparticles (CuSe) are grown in situ on the wall of the halloysite nanotubes to form a composite CuSe@HNTs. The lumen of CuSe@HNTs is loaded with mannose to form a composite drug delivery system CuSe@HNTs@ME.

[0048] As an alternative example, CuSe@HNTs have a particle size range of 78.8 nm to 1990 nm and a zeta potential of -20.3 mV to -19.6 mV.

[0049] As an alternative example, CuSe@HNTs@ME has a particle size range of 58.8 nm to 1280 nm and a zeta potential of -19.2 mV to -17.2 mV.

[0050] As an optional example, the copper selenide nanoparticles are solid spheres and / or solid flowers, and the particle size ranges from 43.8 nm to 295 nm.

[0051] As an optional example, the CuSe content accounts for 10% to 30% of the total mass of CuSe@HNTs, and is particularly preferred to be 15% to 16%.

[0052] As an optional example, ME accounts for 1% to 5% of the total mass of CuSe@HNTs@ME, and is particularly preferred to be 1% to 2%.

[0053] Copper selenide-haloite-mannose combined drug delivery system

[0054] Referring to Figure 1, in another exemplary embodiment of the present invention, a method for preparing the aforementioned copper selenide-halothite-mannose composite drug delivery system is also provided, comprising the following steps:

[0055] Polyvinylpyrrolidone (PVP) was added to ultrapure water and dissolved completely to obtain a PVP solution.

[0056] Halloysite nanotubes (HNTs) were added to a PVP solution and mixed thoroughly to allow PVP to be modified onto the surface of the halloysite nanotubes, thus obtaining an HNTs precursor solution.

[0057] Add the first reducing agent solution to the selenium source solution and mix thoroughly to form a selenium precursor solution;

[0058] A second reducing agent solution is added to the copper source solution and mixed thoroughly to form a copper precursor solution.

[0059] The selenium precursor solution was slowly added dropwise to the HNTs precursor solution, and the reaction was carried out under controlled temperature and stirring conditions to obtain a pre-prepared solution containing selenium and HNTs.

[0060] The copper precursor solution was slowly added dropwise to a pre-prepared solution containing selenium and HNTs, and then heated in a water bath to obtain the water bath product.

[0061] The water bath product was washed and dried sequentially to obtain a composite material (CuSe@HNTs) in which copper selenide nanoparticles were generated in situ on tubular HNTs.

[0062] Mannose (ME) was dissolved in ultrapure water, and ME was loaded into the cavity of CuSe@HNTs material using a vacuum circulation device. After centrifugation and freeze-drying, CuSe@HNTs composite material (CuSe@HNTs@ME) containing ME in the cavity was obtained.

[0063] As an optional example, in the PVP solution, the mass fraction of PVP in the total volume is 0.1 w / v% to 0.5 w / v, and the average molecular weight of PVP is 55,000 to 58,000.

[0064] As an optional example, the mass ratio of HNTs to PVP solution is (2:1) to (1:2).

[0065] As an optional example, the selenium source includes selenium dioxide (SeO2), sodium selenite (NaSeO3), or elemental selenium (Se), and the first reducing agent is ascorbic acid (Vc), citric acid, or sodium thiosulfate; the concentration of the selenium source solution is 0.1M to 0.5M, and the concentration of the first reducing agent solution is 2 to 5 times the concentration of the selenium source solution.

[0066] As an optional example, the copper source includes copper sulfate (CuSO4), copper nitrate (Cu(NO3)2) or copper chloride (CuCl2), and the second reducing agent is ascorbic acid (Vc), citric acid or sodium thiosulfate; the concentration of the copper source solution is 0.2M to 1M, and the concentration of the second reducing agent solution is 2 to 5 times the concentration of the copper source solution.

[0067] As an optional example, in the preparation of a pre-formed solution containing selenium and HNTs, the volume of the selenium precursor solution is 1 to 2 times the volume of the HNTs precursor solution.

[0068] The reaction temperature is 25℃~30℃, the stirring speed is 100rpm~300rpm, and the reaction time is 5min~20min.

[0069] As an optional example, in the preparation of water bath products, what is the relationship between the amounts of the copper precursor solution and the pre-prepared solution containing selenium and HNTs? Or, in other words, what is the relationship between the substances? It is recommended to provide a range for both.

[0070] The reaction temperature is 30℃~60℃, the stirring speed is 100rpm~300rpm, and the reaction time is 3h~8h.

[0071] As an alternative example, the number of vacuum cycles when ME is loaded into the cavity of CuSe@HNTs material is 1 to 5.

[0072] As an optional example, HNTs are purified HNTs obtained by fractional centrifugation and freeze-drying.

[0073] application

[0074] In another exemplary embodiment of the present invention, the application of the aforementioned copper selenide-haloite-mannose composite drug delivery system in the preparation of inflammatory bowel disease drugs is also provided.

[0075] As shown in Figure 1, when the CuSe@HNTs@ME (CSHM) composite drug delivery system is administered orally, it first passes through the stomach, where it maintains structural stability in the acidic environment. Subsequently, the negatively charged CSHM enters the intestine, where the attraction between positive and negative charges allows it to target the positively charged sites of enteritis. CSHM possesses superoxide dismutase (SOD) and catalase (CAT) activities, effectively scavenging reactive oxygen species (ROS) in the intestine and reducing ROS-induced cell damage. Halloysite nanotubes, acting as carriers, not only enhance the anti-inflammatory and antidiarrheal functions of the material but also effectively protect and load CuSe and mannose, ensuring their stable release and delivery to the enteritis sites. Mannose binds to specific receptors on the surface of cells at the site of inflammation, causing CSHM to target and accumulate around the mitochondria. Through this precise targeting, the drug can be effectively concentrated in the inflamed area for treatment.

[0076] The stability of CSHM in the gastric acid environment of this invention mainly relies on its structural design and material properties. First, halloysite nanotubes are a natural nanotube material. The binding of CuSe nanoparticles with HNTs is stabilized on the HNT surface through interfacial forces, making them less susceptible to dissociation or destruction in gastric acid. By comparing the enzyme-like activities of CSHM before and after 4 hours of gastric acid treatment, it was found that most of its enzyme activity was retained. This indicates that the system remains stable in the gastric acid environment, protecting its active ingredients from destruction and ensuring that these ingredients can successfully reach the intestines and exert their therapeutic effects.

[0077] The mannose in the CSHM of this invention can be taken up by cells and effectively localized to mitochondria by binding to specific receptors on the cell surface. At sites of inflammation or injury, mitochondrial membrane potential, ROS levels, and mitochondrial morphology are often disrupted. Through interactions with mitochondrial-related receptors or signaling pathways, mannose can preferentially accumulate on these damaged mitochondria. Once inside the mitochondria, the ME (membrane exchange mechanism) can directly act on the mitochondrial membrane, helping to restore the damaged mitochondrial membrane potential, reduce ROS levels, and repair the size and structure of mitochondria. This targeted action ensures precise drug delivery within the cell and, by restoring mitochondrial function, further reduces cellular oxidative stress and inflammatory responses, thereby effectively promoting cell recovery and alleviating enteritis symptoms. Therefore, the targeting mechanism of mannose is not merely physical localization, but rather precise treatment of enteritis by restoring normal mitochondrial function.

[0078] In other exemplary embodiments, a pharmaceutical composition comprising the aforementioned copper selenide-halothite-mannose complex delivery system is also provided for direct oral treatment of inflammatory bowel disease.

[0079] To facilitate better understanding, the present invention will be further illustrated below with several specific examples, but the preparation process is not limited to these examples, and the content of the present invention is not limited to these examples.

[0080] Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0081] Example 1

[0082] [Preparation of CuSe and CuSe@HNTs]

[0083] First, at room temperature, 30 mg of polyvinylpyrrolidone (PVP, Mw = 58000) was dissolved in 10 mL of purified water with stirring. Then, 0.5 mL of SeO2 (0.25 M) solution was slowly added dropwise, followed by 0.2 mL of ascorbic acid (VC, 0.2 g / mL), and the mixture was stirred for 15 min. Next, a mixed solution of CuSO4 (0.5 mL, 0.5 M) and ascorbic acid (0.3 mL, 0.2 g / mL) was added dropwise to the above reaction solution. The mixture was stirred continuously in a 40 °C water bath for 6 h. Finally, the mixture was centrifuged three times at 10000 rpm / min and vacuum dried to obtain copper selenide nanoparticles (CuSe).

[0084] HNTs were purified by fractional centrifugation and freeze-dried to obtain purified HNTs. 30 mg of polyvinylpyrrolidone (PVP, Mw = 58000) was dissolved in 10 mL of purified water. The HNTs and PVP solution were mixed at a 1:1 mass ratio and stirred overnight at room temperature to obtain an HNTs precursor solution. Then, 0.5 mL of SeO2 (0.25 M) solution was slowly added dropwise to the HNTs precursor solution, followed by 0.2 mL of ascorbic acid (VC, 0.2 g / mL), and the mixture was stirred for 15 min. Next, a mixed solution of CuSO4 (0.5 mL, 0.5 M) and ascorbic acid (0.3 mL, 0.2 g / mL) was added dropwise to the above reaction solution. The mixture was stirred continuously in a 40 °C water bath for 6 h. Finally, the mixture was centrifuged three times at 10000 rpm / min and freeze-dried to obtain a composite nanomaterial of halloysite and copper selenide (CuSe@HNTs).

[0085] Example 2

[0086] [Preparation of CuSe@HNTs@ME]

[0087] A saturated solution of ME (100 μM) was prepared and then mixed with an aqueous dispersion of CuSe@HNTs (0.1 wt%) (prepared according to the method in Example 1) to obtain a total volume of 50 mL of the solution to be treated.

[0088] The mixture was stirred under a magnetic stirrer, and a cyclic vacuum pump was used to pump in / out to remove air from the HNTs chamber. Then, with the assistance of the vacuum pump, the mixture was stirred under vacuum for 0.5 hours and under non-vacuum conditions for 0.5 hours. After 3 cycles, the product was washed several times with ultrapure water by centrifugation to remove excess ME, and then freeze-dried to obtain CuSe@HNTs@ME.

[0089] Example 3

[0090] [Materials Characterization]

[0091] Transmission electron microscopy (TEM), X-ray diffraction (XRD), energy dispersive X-ray spectroscopy (EDS), and Fourier transform infrared spectroscopy (FTIR) were used to characterize the phase, composition, morphology, and crystallinity of different powders. The specific surface area of ​​powders prepared under different conditions was measured using a nitrogen adsorption-desorption analyzer (BET). The content of powders was analyzed using a thermogravimetric analyzer (TG).

[0092] As shown in Figure 2a, the in-situ grown copper selenide exhibits a flower-like morphology on halloysite nanotubes; as shown in Figure 2b, XRD data indicates that the phases and functional groups of CuSe@HNTs are composed of CuSe and HNTs, respectively; the CuSe content measured in Figure 2c accounts for 15.9% of the total mass of CuSe@HNTs; as shown in Figure 2d, the specific surface area of ​​CuSe@HNTs is significantly larger than that of CuSe, indicating that HNTs provide more specific surface area for CuSe; as shown in Figures 2e and 2f, XPS data show that CuSe in CuSe@HNTs... + / Cu 2+ The proportion of O is higher than that of CuSe. Surface CuSe@HNTs are more prone to electron transfer than CuSe. The proportion of Se-O bonds in CuSe@HNTs is smaller than that in CuSe, indicating that CuSe@HNTs are more stable and less prone to oxidation than CuSe.

[0093] Figure 3a shows that the particle size distribution of each component material is relatively concentrated. The particle size range of CuSe@HNTs is 78.8 nm to 1990 nm (average particle size is 345.3 nm), the particle size range of CuSe@HNTs@ME is 58.8 nm to 1280 nm (average particle size is 243.1 nm), and the particle size range of copper selenide nanoparticles is 43.8 nm to 295 nm (average particle size is 107.9 nm). It was also found that the size of ME is less than 10 nm, which makes it possible to be loaded into the lumen of HNTs.

[0094] As shown in Figure 3b, CuSe@HNTs@ME showed greater weight loss in TG compared to CuSe@HNTs, indicating the presence of ME, and ME accounted for 1.3% of the total mass of CuSe@HNTs@ME. As shown in Figure 3c, comparing vacuum and non-vacuum cycling, the same mass of CuSe@HNTs underwent XRD analysis after loading ME through vacuum cycling (vacuum stirring for 0.5 h, non-vacuum stirring for 0.5 h, 3 cycles) and non-vacuum cycling (normal mixing for the same time). The peak of CuSe@HNTs@ME under vacuum cycling was sharper, indicating that ME was present inside the HNTs cavity rather than on the surface.

[0095] Example 4

[0096] [Antioxidant enzyme activity assay]

[0097] Aqueous dispersions of CuSe, CuSe@HNTs, and CuSe@HNTs@ME with concentrations of 25, 50, 100, 200, and 400 μg / mL were prepared. Catalase-like (CAT) activity was characterized using a portable dissolved oxygen analyzer. H2O2 solutions and dispersions of each H2O2+ component were prepared. Using PBS buffer at pH 7.4 as the solvent, the oxygen concentration of each group was measured using a portable dissolved oxygen analyzer over a certain period until the oxygen concentration remained essentially constant. Superoxide dismutase (SOD)-like activity was characterized using NBT working solution. Mixtures of different concentrations of each component were prepared using PBS buffer at pH 7.4 as the solvent, incubated at 25°C for 20 minutes, and irradiated with a 30W LED lamp for 15 minutes. Absorbance was measured at 560 nm using UV-UIS. The scavenging ability of the materials for ·OH was tested using the Fenton reaction and electron paramagnetic resonance (EPR). (The text abruptly ends here, likely due to an incomplete translation or missing information.) 2+ The Fenton reaction of the / H2O2 system generates ·OH, which is then incubated with different components for 10 minutes. Afterwards, DMPO is added to the mixed solution, and the ·OH radical scavenging ability is detected using EPR within 5 minutes.

[0098] The results are shown in Figures 4 and 5. From the aspects of catalase (CAT) activity, superoxide dismutase (SOD) activity and ·OH scavenging, CuSe@HNTs showed stronger antioxidant enzyme activity than CuSe, and CuSe@HNTs@ME also had a slightly higher ·OH scavenging ability than CuSe@HNTs.

[0099] The results show that CuSe@HNTs is significantly better than CuSe in terms of antioxidant enzyme activity, while CuSe@HNTs@ME is slightly better than CuSe@HNTs in terms of scavenging hydroxyl radicals (·OH), indicating that the introduction of ME further enhances the antioxidant properties of the composite material.

[0100] Furthermore, as shown in Figure 5a, the Zeta potential of CuSe@HNTs is -20.3mV to -19.6mV, and the Zeta potential of CuSe@HNTs@ME is -19.2mV to -17.2mV.

[0101] Example 5

[0102] [Stability test in gastric juice]

[0103] To simulate the digestive process of food in the stomach, the material was added to a simulated gastric juice solution (pH 1.2 to 1.5) and incubated with shaking at 37°C for 4 hours. Then, it was washed and neutralized with simulated colonic fluid. Finally, the material was dissolved in water or lyophilized, and SEM, XRD, ME release, BET, zeta potential measurements, and ROS scavenging activity analysis were performed to determine whether the nanomaterial could stably cross gastric juice and reach the site of enteritis.

[0104] As shown in Figure 6a, HNTs form a gel-like substance in simulated gastric juice at 37°C (right). When HNTs leave the gastric juice and enter the intestine, they reform into a suspension (left), indicating that HNTs protect CuSe and ME from being destroyed by strong acid, allowing CuSe@HNT@ME to maintain structural stability in gastric acid.

[0105] Furthermore, SEM images showed that CuSe remained loaded on the surface of HNTs (Figs. 6b and 6c). We also measured the zeta potential of CuSe@HNTs@ME after 4 hours of SGF treatment (Fig. 6d), which showed no significant difference, indicating that CuSe@HNTs@ME retained its negatively charged properties after passing through the digestive tract. XRD results showed that CuSe@HNTs@ME retained its crystal structure after SGF treatment (Fig. 6e). Fig. 6f showed that the amount of ME released after SGF treatment was not as high as that after normal PBS treatment, indicating that ME was not burst-released in gastric acid, and most of the ME was retained, allowing it to act on the intestinal portion. Fig. 6g showed that the specific surface area did not decrease significantly after SGF treatment, and a relatively high specific surface area was still retained, providing more active sites for enzyme-like catalysis. Figs. 6h and 6i showed that after SGF treatment, CuSe@HNTs@ME retained most of its SOD-like and CAT-like activities.

[0106] As demonstrated by the above tests, the CuSe@HNTs@ME prepared in this invention exhibits oral stability.

[0107] Example 6

[0108] [CuSe@HNTs@ME Targeted Enteritis Test]

[0109] (1) In vitro targeted adhesion test: Polystyrene plates modified with positively and negatively charged polymers were used to simulate inflamed and healthy epithelial cells, respectively. Based on the change of colonic epithelial cells from negative to positive charge before and after enteritis, an experiment was designed to study the targeting ability of CuSe@HNTs@ME to diseased colonic epithelium in vitro.

[0110] First, polystyrene sheets were modified with amine-rich polymer names (positively charged) and carboxylate-rich polymer names (negatively charged) to simulate inflamed and healthy epithelium, respectively. The three modified polystyrene sheets were incubated with CuSe@HNTs@ME solution at 37°C for 3 hours, and then excess CuSe@HNTs@ME was washed away with water (handled gently). Finally, the CuSe@HNTs@ME content on different surfaces of the sheets was determined using EDS mapping and spectroscopy.

[0111] The results are shown in Figure 7, indicating that CuSe@HNTs@ME aggregates more readily on positively charged polyethylene plates.

[0112] (2) In vivo targeted adhesion test: Each mouse was given CuSe@HNTs@ME suspension by gavage, and the distribution of CuSe@HNTs@ME in the intestine was observed using photoacoustic imaging equipment or TEM.

[0113] As shown in Figure 7, CuSe@HNTs@ME tended to accumulate in the inflamed intestinal region with a higher concentration in the intestinal villi. Photoacoustic imaging results (Figure 8) showed that, compared to healthy mice, CuSe@HNTs@ME targeted and adhered to the intestines of mice with enteritis.

[0114] (3) In vitro adhesion targeting test: The intestines of mice in the healthy group and IBD group were taken and incubated with CuSe@HNTs@ME suspension. After fixation with 2.5% glutaraldehyde, the mice were dehydrated by alcohol gradient, and the enrichment of CuSe@HNTs@ME in the intestines of mice in the healthy group and IBD group was photographed by SEM.

[0115] The results are shown in Figure 9. The results indicate that CuSe@HNTs@ME is more enriched in the inflamed gut than in the gut of healthy mice.

[0116] The experiments described above demonstrate that the pathophysiological characteristics of mucosal defect-inflammation co-localization and local charge alteration allow negatively charged nanoparticles to target intestinal inflammation through electrostatic interactions. The strongly negatively charged CuSe@HNTs@ME facilitates passive deposition into the positively charged inflamed colon, enabling targeted adsorption therapy at the site of IBD colitis.

[0117] Example 7

[0118] [CuSe@HNTs@ME Cytotoxicity Test]

[0119] Cell experiments were performed using the intestinal epithelial cell line (IEC-6) under humidified conditions (5% CO2) at 37°C. The complete culture medium consisted of 90% basal medium (DMEM), 10% fetal bovine serum (FBS), and 1% penicillin-streptomycin (P / S). The half-maximal inhibitory concentration (IC50) was determined using different concentrations of DSS and different treatment times. The cytotoxic effects of different components were assessed using CCK8 assay or calcein-AM / PI staining according to the manufacturer's instructions. The results are shown in Figure 10.

[0120] As shown in Figure 10a, 2% DSS caused nearly half the cell viability of IEC-6 cells, so subsequent experiments were conducted using 2% DSS to treat IEC-6 cells. Figure 10b shows the effects of HNTs, CuSe, ME, CuSe@HNTs, and CuSe@HNTs@ME on the 24-hour cell viability of IEC-6 cells. When the concentration of all materials was 25 μg / mL, they were all non-toxic to the cells, so subsequent experiments were conducted using this concentration.

[0121] Example 8

[0122] [CuSe@HNTs@ME Intracellular ROS Clearance Test]

[0123] Intracellular ROS detection was based on the peroxide-dependent oxidation of DCFH-DA, forming a fluorescent compound called dichlorofluorescein (DCF). Cells were treated with H2O2 for 4 hours, washed with PBS, and incubated at 37°C for 30 minutes after treatment with different components. Fluorescence imaging was then performed using an inverted microscope.

[0124] As shown in Figures 10c and 10d, after 4 hours of H2O2 treatment, the overall FITC fluorescence level of the cells was significantly enhanced, indicating a significant increase in ROS levels. However, after pretreatment with CuSe@HNTs@ME, the ROS content in the cells was significantly reduced. This demonstrates that CuSe@HNTs@ME can effectively reduce intracellular ROS levels, showcasing its excellent effect in scavenging reactive oxygen species.

[0125] Example 9

[0126] [CuSe@HNTs@ME Targeted Mitochondrial Repair Test]

[0127] Cells were washed twice with PBS and labeled for 30 minutes at 37°C with the following fluorescent probes: MitoTracker Green (Ex 490 nm / Em 516 nm) and JC-1 (a sensitive marker of mitochondrial membrane potential). Imaging was detected using an inverted microscope. Images were quantified using ImageJ software after appropriate thresholding to analyze the mitochondrial function of different components.

[0128] As shown in Figure 11, treatment with MitoTracker Green (Ex490nm / Em516nm) and JC-1 (a sensitive marker of mitochondrial membrane potential), and CuSe@HNTs@ME (CSHM) reversed DSS-induced mitochondrial damage, manifested as increased mitochondrial mass and increased mitochondrial membrane potential. The key point is that the reversal intensity of ME was higher than that of HNTs and CuSe, indicating that ME plays a key role in the repair of mitochondria by CuSe@HNTs@ME.

[0129] As can be seen from the above, the CuSe@HNTs@ME of the present invention helps to alleviate DSS-induced mitochondrial lesions, thereby exerting an anti-inflammatory effect.

[0130] Example 10

[0131] [Therapeutic effects of CuSe@HNTs@ME in vivo colitis model]

[0132] C57 mice were randomly assigned to six groups (n=6): a control group (saline), a DSS group (2% DSS), a DSS+CuSe@HNTs@ME group, a DSS+CuSe@HNTs group, a DSS+HNTs group, a DSS+CuSe group, and a DSS+ME group. Administered medication via gavage. Mouse weight and condition were assessed daily during the experiment. Mice were sacrificed on the last day of the experiment, and the Disease Activity Index (DAI) for each group was determined. Colon tissue was harvested for length and size observation.

[0133] The results are shown in Figure 12. The therapeutic effect of CuSe@HNTs@ME (CSHM) was tested in a DSS-induced acute IBD model. Compared with the DSS, DSS+HNT, DSS+ME, DSS+CS, and DSS+CSH groups, CSHM inhibited weight loss in IBD mice (Figure 12a) and downregulated the disease activity index (DAI) (Figure 12c). The colons of the mice were collected, photographed, and measured on day 11. As shown in Figure 12, the colon length in the CSHM group was significantly longer than in the other groups, indicating that CSHM can inhibit the colonic shortening commonly seen in DSS treatment (Figure 12b). As shown in Figure 12d, in DSS-induced colitis mice, the colonic tissue structure collapsed, and the colonic epithelium was severely damaged. After treatment, an improvement in histological appearance was observed, preserving the integrity of the villous layer structure. Furthermore, CuSe@HNTs@ME treatment showed the most significant improvement compared to HNTs, ME, and CuSe@HNTs treatment.

[0134] As can be seen from the above tests, the CuSe@HNTs@ME of the present invention protects the intestinal structure and function.

[0135] While the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the invention. Those skilled in the art can make various modifications and refinements without departing from the spirit and scope of the invention. Therefore, the scope of protection of the present invention shall be determined by the claims.

Claims

1. A copper selenide-halothite-mannose composite drug delivery system, characterized in that, The drug delivery system includes a carrier, which is halloysite nanotubes (HNTs), on which copper selenide nanoparticles (CuSe) are grown in situ to form a complex CuSe@HNTs; the lumen of CuSe@HNTs is loaded with mannose to form a composite drug delivery system CuSe@HNTs@ME.

2. The copper selenide-halothite-mannose composite drug delivery system according to claim 1, characterized in that, The particle size range of CuSe@HNTs is 78.8 nm to 1990 nm, and the zeta potential is -20.3 mV to -19.6 mV.

3. The copper selenide-halothite-mannose composite drug delivery system according to claim 1, characterized in that, The particle size range of CuSe@HNTs@ME is 58.8 nm to 1280 nm, and the zeta potential is -19.2 mV to -17.2 mV.

4. The copper selenide-halothite-mannose composite drug delivery system according to claim 1, characterized in that, The copper selenide nanoparticles are solid spheres and / or solid flower shapes, with a particle size range of 43.8 nm to 295 nm.

5. A method for preparing the copper selenide-halothite-mannose composite drug delivery system according to any one of claims 1-4, characterized in that, Includes the following steps: Polyvinylpyrrolidone (PVP) was added to ultrapure water and dissolved completely to obtain a PVP solution. Halloysite nanotubes (HNTs) were added to a PVP solution and mixed thoroughly to allow PVP to be modified onto the surface of the halloysite nanotubes, thus obtaining an HNTs precursor solution. Add the first reducing agent solution to the selenium source solution and mix thoroughly to form a selenium precursor solution; A second reducing agent solution is added to the copper source solution and mixed thoroughly to form a copper precursor solution. The selenium precursor solution was slowly added dropwise to the HNTs precursor solution, and the reaction was carried out under controlled temperature and stirring conditions to obtain a pre-prepared solution containing selenium and HNTs. The copper precursor solution was slowly added dropwise to a pre-prepared solution containing selenium and HNTs, and then heated in a water bath to obtain the water bath product. The water bath product was washed and dried sequentially to obtain a composite material (CuSe@HNTs) in which copper selenide nanoparticles were generated in situ on tubular HNTs. Mannose (ME) was dissolved in ultrapure water, and ME was loaded into the cavity of CuSe@HNTs material using a vacuum circulation device. After centrifugation and freeze-drying, CuSe@HNTs composite material (CuSe@HNTs@ME) containing ME in the cavity was obtained.

6. The preparation method according to claim 5, characterized in that, In the PVP solution, the mass fraction of PVP in the total volume is 0.1 w / v% to 0.5 w / v, and the average molecular weight of PVP is 55,000 to 58,000.

7. The preparation method according to claim 5, characterized in that, The mass ratio of HNTs to PVP solution is (2:1) to (1:2).

8. The preparation method according to claim 5, characterized in that, The selenium source includes selenium dioxide (SeO2), sodium selenite (NaSeO3), or elemental selenium (Se), and the first reducing agent is ascorbic acid (Vc), citric acid, or sodium thiosulfate; the concentration of the selenium source solution is 0.1M to 0.5M, and the concentration of the first reducing agent solution is 2 to 5 times that of the selenium source solution.

9. The preparation method according to claim 5, characterized in that, The copper source includes copper sulfate (CuSO4), copper nitrate (Cu(NO3)2) or copper chloride (CuCl2), and the second reducing agent is ascorbic acid (Vc), citric acid or sodium thiosulfate; the concentration of the copper source solution is 0.2M to 1M, and the concentration of the second reducing agent solution is 2 to 5 times the concentration of the copper source solution.

10. The preparation method according to claim 5, characterized in that, In the preparation of pre-formed solutions containing selenium and HNTs, the volume of the selenium precursor solution is 1 to 2 times the volume of the HNTs precursor solution. The reaction temperature is 25℃~30℃, the stirring speed is 100rpm~300rpm, and the reaction time is 5min~20min.

11. The preparation method according to claim 5, characterized in that, In the preparation of the water bath product, the volume of the copper precursor solution is 2 to 4 times the volume of the HNT precursor solution; The reaction temperature is 30℃~60℃, the stirring speed is 100rpm~300rpm, and the reaction time is 3h~8h.

12. The use of the copper selenide-halolite-mannose composite drug delivery system according to any one of claims 1-4 in the preparation of inflammatory bowel disease drugs.

13. A pharmaceutical composition, characterized in that, The system includes the copper selenide-halothite-mannose complex drug delivery system according to any one of claims 1-4.

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