Copper selenide-halloysite-mannose composite drug delivery system as well as preparation method and application thereof

Abstract

The invention provides a copper selenide-halloysite-mannose composite drug delivery system which comprises a carrier, the carrier is halloysite nanotubes (HNTs), copper selenide nanoparticles (CuSe) grow on the tube wall of the halloysite nanotubes in situ, and a compound CuSe-coated HNTs is formed; mannose is loaded in a tube cavity of the CuSe-coated HNTs, so that a composite drug delivery system CuSe-coated HNTs-coated ME is formed. The invention further provides a preparation method and application of the copper selenide-halloysite-mannose composite drug delivery system. The drug delivery system can keep stable in a gastric acid environment and directly reach an enteritis part through an oral administration way, so that the enteritis symptom is remarkably relieved.

Description

Technical Field

[0001] The invention relates to the technical field of nanomaterials, and in particular to a copper selenide-halloysite-mannose composite drug delivery system and a preparation method and application thereof. Background Art

[0002] Inflammatory bowel disease (IBD) is a refractory chronic disease that significantly reduces the quality of life of patients and may lead to more serious complications such as colon cancer. Currently, antibiotics are commonly used clinically to suppress intestinal infections, but antibiotics can irritate the intestines, cause intestinal flora imbalance, lead to intestinal mucosal damage, and may cause antibiotic resistance problems. As an alternative, probiotics are widely used to regulate intestinal flora to relieve enteritis. However, probiotics are often destroyed by gastric acid when passing through the stomach, lose their activity, and cannot effectively reach the intestines to exert their effects.

[0003] Therefore, there is an urgent need to develop novel therapeutic strategies that can withstand gastric acid and function effectively in the intestine to provide more effective IBD treatment. Summary of the invention

[0004] The purpose of the present invention is to provide a copper selenide-halloysite-mannose composite drug delivery system and a preparation method thereof in view of the deficiencies in the prior art. The drug delivery system can remain stable in a gastric acid environment, directly reach the site of enteritis through an oral route, and significantly alleviate the symptoms of enteritis.

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

[0006] As an optional embodiment, 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.

[0007] As an optional embodiment, the particle size range of CuSe@HNTs@ME is 58.8nm to 1280nm, and the Zeta potential is -19.2mV to -17.2mV.

[0008] As an optional implementation, the copper selenide nanoparticles are solid spherical and / or solid flower-shaped, and the particle size of the copper selenide nanoparticles ranges from 43.8 nm to 295 nm.

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

[0010] Add polyvinyl pyrrolidone (PVP) into ultrapure water and fully dissolve it to obtain a PVP solution;

[0011] Adding halloysite nanotubes (HNTs) into the PVP solution and mixing them thoroughly to modify the surface of the halloysite nanotubes with PVP, thereby obtaining a HNTs precursor solution;

[0012] Adding the first reducing agent solution to the selenium source solution and mixing thoroughly to form a selenium precursor solution;

[0013] Adding a second reducing agent solution to the copper source solution and mixing thoroughly to form a copper precursor solution;

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

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

[0016] The water bath product was washed and dried in sequence to obtain a composite of in-situ generated copper selenide nanoparticles on tubular HNTs (CuSe@HNTs);

[0017] Mannose (ME) was dissolved in ultrapure water, and ME was loaded into the lumen of the CuSe@HNTs material using a vacuum circulation device. By centrifugation and freeze-drying, a CuSe@HNTs composite material containing ME in the lumen (CuSe@HNTs@ME) was obtained.

[0018] As an optional implementation, 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.

[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~0.5M, and the concentration of the first reducing agent solution is 2~5 times the concentration of the selenium source solution.

[0021] As an optional embodiment, 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~1M, and the concentration of the second reducing agent solution is 2~5 times the concentration of the copper source solution.

[0022] As an optional embodiment, in the preparation of the prefabricated 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°C to 30°C, the stirring speed is 100rpm to 300rpm, and the reaction time is 5min to 20min.

[0024] As an optional embodiment, 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°C to 60°C, the stirring speed is 100rpm to 300rpm, and the reaction time is 3h to 8h.

[0026] According to the third aspect of the present invention, there is also provided a use of the aforementioned copper selenide-halloysite-mannose composite drug delivery system in the preparation of inflammatory bowel disease drugs.

[0027] According to a fourth aspect of the present invention, a pharmaceutical composition is also provided, comprising the aforementioned copper selenide-halloysite-mannose composite drug delivery system.

[0028] It can be seen from the above technical solutions of the present invention that the copper selenide-halloysite-mannose composite drug delivery system proposed by the present invention in-situ grows copper selenide nanoparticles on the surface of halloysite nanotubes and loads mannose into the cavity of halloysite nanotubes, ingeniously 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] CuSe@HNTs@ME composite material combines the superoxide dismutase (SOD) and catalase (CAT) activities of copper selenide, the anti-inflammatory and antidiarrheal effects of halloysite, and the protective effect of mannose on mitochondria. The three work synergistically to keep the drug delivery system stable in the gastric acid environment, reach the site of enteritis directly through the oral route, and significantly alleviate the symptoms of enteritis.

[0030] The composite drug delivery system of the present invention not only has the dual functions of scavenging reactive oxygen species (ROS) and anti-inflammation, but also can effectively protect the intestinal structure and function, providing an innovative oral treatment plan for patients with enteritis, and has the potential to improve clinical symptoms and enhance the quality of life. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is the preparation process and mechanism diagram of the copper selenide-halloysite-mannose composite drug delivery system of the present invention.

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

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

[0034] Figure 4 is a nanozyme activity analysis diagram of CuSe and CuSe@HNTs in the example of the present invention; wherein, Figure 4 a is the amount of H2O2 produced by peroxidase-like (CAT) activity, Figure 4 b is superoxide dismutase (SOD)-like activity. 2- Elimination rate, Figure 4 c is the EPR spectrum of the ·OH scavenging ability of the material using DMPO as the spin scavenger.

[0035] Figure 5 is an analysis diagram of the electronegativity and nanozyme activity of CuSe, CuSe@HNTs and CuSe@HNTs@ME in the examples of the present invention; wherein, Figure 5 a is the zeta potential diagram of water dispersions of different materials, Figure 5b is the EPR spectrum of the ·OH scavenging ability of the material using DMPO as the spin scavenger.

[0036] Figure 6 is the stability analysis result of CuSe@HNTs@ME(CSHM) of the present invention in simulated gastric fluid; wherein, Figure 6 a is the HNT solution without treatment and after 4h simulated gastric fluid treatment, Figure 6 b, 6c, 6d, 6e, 6f, and 6g are the SEM, zeta potential, XRD, ME release and BET diagrams of CSHM without treatment and after 4 h of simulated gastric fluid treatment, respectively. Figure 6 h and 6i are the comparisons of CAT-like and SOD-like activities of CuSe, CuSe@HNTs and CuSe@HNTs@ME without treatment and after 4h of simulated gastric fluid treatment, respectively.

[0037] Figure 7 This is the result of the targeted adhesion of CuSe@HNTs@ME (CSHM) in the intestinal inflammation part of the present invention; wherein, Figure 7 a is the EDS mapping images of uncoated, carboxylate-coated (negatively charged, mimicking healthy epithelium) and ammonium-coated (positively charged, mimicking inflamed epithelium) polystyrene surfaces after incubation with CSHM at 37 °C for 3 h. Figure 7 b BioTEM images of healthy and inflamed mouse mucosa 24 h after oral CSHM treatment.

[0038] Figure 8 Sonographic images of healthy and inflamed mouse mucosa 24 hours after oral CSHM treatment.

[0039] Fig. 9 SEM images of the mucosa of healthy and inflamed mice 24 hours after oral CSHM treatment.

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

[0041] Fig.11 To protect CSHM-treated cells from ROS-induced mitochondrial damage; Fig.11a is an image labeled with the following fluorescent probes: 400 nM MitoTracker Green (Ex490 nm / Em516 nm), and 10 μM JC-1 (a sensitive marker of mitochondrial membrane potential). Fig.11 b and 11c correspond to the quantitative statistical graphs of the above fluorescence images, respectively.

[0042] Fig.12 The in vivo therapeutic effect of oral CSHM; Fig.12 a is the weight change of mice after treatment with different components. Fig.12 b and Fig.12 c are the colon length statistics and the disease activity index (DAI) of each group to score these symptoms. Fig.12 d is the H&E staining image of the cross-section of colon tissue of each component. DETAILED DESCRIPTION

[0043] In order to better understand the technical content of the present invention, specific embodiments are given and described as follows in conjunction with the accompanying drawings.

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

[0045] Antibiotics are still the main means of clinical treatment of inflammatory bowel disease, but long-term use of antibiotics will destroy the probiotic flora in the human intestine, leading to intestinal health problems. To address this problem, the present invention intends to use the ability of copper selenide nanoenzymes to remove reactive oxygen species (ROS), the anti-inflammatory and antidiarrheal functions of halloysite nanotubes, and the protective effect of mannose on mitochondria to construct an oral selenium CuSe@HNTs@ME composite drug delivery system with dual effects of targeting mitochondria and inflammation. This composite drug delivery system has both precise and efficient anti-inflammatory effects and can effectively protect intestinal structure and function, providing an innovative oral treatment solution for the treatment of clinical inflammatory bowel disease.

[0046] Copper selenide-halloysite-mannose composite drug delivery system

[0047] Combination Figure 1 As shown, in an exemplary embodiment of the present invention, a copper selenide-halloysite-mannose composite drug delivery system is provided, which includes a carrier, the carrier is halloysite nanotubes (HNTs), copper selenide nanoparticles (CuSe) are in situ grown on the tube 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 optional example, 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.

[0049] As an optional example, 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.

[0050] As an optional example, the copper selenide nanoparticles are solid spheres and / or solid flowers, and the particle size of the copper selenide nanoparticles 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 preferably 15% to 16%.

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

[0053] Copper selenide-halloysite-mannose composite drug delivery system

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

[0055] Add polyvinyl pyrrolidone (PVP) into ultrapure water and fully dissolve it to obtain a PVP solution;

[0056] Adding halloysite nanotubes (HNTs) into the PVP solution and mixing them thoroughly to modify the surface of the halloysite nanotubes with PVP, thereby obtaining a HNTs precursor solution;

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

[0058] Adding a second reducing agent solution to the copper source solution and mixing thoroughly to form a copper precursor solution;

[0059] Slowly drop the selenium precursor solution into the HNTs precursor solution, and react under controlled temperature and stirring conditions to obtain a prefabricated solution containing selenium and HNTs;

[0060] The copper precursor solution is slowly added dropwise to the prefabricated solution containing selenium and HNTs, and heated in a water bath to obtain a water bath product;

[0061] The water bath product was washed and dried in sequence to obtain a composite of in-situ generated copper selenide nanoparticles on tubular HNTs (CuSe@HNTs);

[0062] Mannose (ME) was dissolved in ultrapure water, and ME was loaded into the lumen of the CuSe@HNTs material using a vacuum circulation device. By centrifugation and freeze-drying, a CuSe@HNTs composite material containing ME in the lumen (CuSe@HNTs@ME) 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~0.5M, and the concentration of the first reducing agent solution is 2~5 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~1M, and the concentration of the second reducing agent solution is 2~5 times the concentration of the copper source solution.

[0067] As an optional example, in the preparation of the prefabricated 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°C to 30°C, the stirring speed is 100rpm to 300rpm, and the reaction time is 5min to 20min.

[0069] As an optional example, in the preparation of the water bath product, the reaction temperature is 30°C to 60°C, the stirring speed is 100rpm to 300rpm, and the reaction time is 3h to 8h.

[0070] As an optional example, the number of vacuum cycles when loading ME into the lumen of the CuSe@HNTs material is 1 to 5 times.

[0071] As an optional example, the HNTs are purified HNTs after purification by fractionated centrifugation and freeze-drying.

[0072] application

[0073] In another exemplary embodiment of the present invention, there is also provided a use of the aforementioned copper selenide-halloysite-mannose composite drug delivery system in the preparation of inflammatory bowel disease drugs.

[0074] Combination Figure 1 As shown in the figure, when the composite drug delivery system containing CuSe@HNTs@ME (CSHM) enters the body through the oral route, it first passes through the stomach and maintains its structural stability in the gastric acid environment; then, the negatively charged CSHM enters the intestine, and due to the mutual attraction between positive and negative charges, it can be targeted to the positively charged intestinal inflammation site; CSHM has superoxide dismutase (SOD) and catalase (CAT) activities, which can effectively remove reactive oxygen species (ROS) in the intestine and reduce cell damage caused by ROS; as a carrier, halloysite nanotubes not only enhance the anti-inflammatory and antidiarrheal functions of the material, but also effectively protect and load CuSe and mannose, ensuring that they can be stably released and reach the intestinal inflammation site; mannose binds to specific receptors on the surface of cells in the inflammatory site, causing CSHM to aggregate around the mitochondria. Through this precise targeting effect, the drug can be effectively concentrated in the inflammatory area for treatment.

[0075] The stability of the CSHM of the present invention in the gastric acid environment mainly depends on its structural design and material properties; first, the halloysite nanotube is a natural nanotubular material, and the combination of CuSe nanoparticles and HNTs is stabilized on the surface of HNTs through interfacial forces, making it difficult to dissociate or destroy in gastric acid. By comparing the enzyme-like activity of CSHM before and after 4h gastric acid treatment, the study found that most of its enzyme activity was retained, indicating that the system is still stable in the gastric acid environment and can protect its effective ingredients from being destroyed, thereby ensuring that these ingredients can smoothly reach the intestines and play a therapeutic role.

[0076] The mannose in the CSHM of the present invention can be taken up by cells and effectively positioned in mitochondria by binding to specific receptors on the cell surface; at the site of inflammation or damage, mitochondrial membrane potential, ROS level and mitochondrial morphology are often damaged. Mannose can preferentially accumulate on these damaged mitochondria through the interaction with mitochondrial-related receptors or signal pathways. After entering the mitochondria, ME can directly act on the mitochondrial membrane to help restore the damaged mitochondrial membrane potential, reduce ROS levels, and repair the size and structure of mitochondria. This targeting effect ensures the precise delivery of drugs in cells, and by restoring mitochondrial function, it further reduces the oxidative stress response and inflammatory response of cells, thereby effectively promoting the recovery of cells and the relief of enteritis symptoms. Therefore, the targeting mechanism of mannose is not only physical positioning, but also the precise treatment of enteritis by restoring the normal function of mitochondria.

[0077] In other exemplary embodiments, a pharmaceutical composition is also provided, comprising the aforementioned copper selenide-halloysite-mannose composite drug delivery system, which can be directly orally administered to treat inflammatory bowel disease.

[0078] For better understanding, the present invention is further described below in conjunction with several specific examples, but the preparation process is not limited thereto, and the content of the present invention is not limited thereto.

[0079] Unless otherwise specified, the materials in the examples were prepared according to existing methods or directly purchased from the market.

[0080] Example 1

[0081] [Preparation of CuSe and CuSe@HNTs]

[0082] First, 30 mg of polyvinyl pyrrolidone (PVP, Mw = 58000) was stirred and dissolved in 10 mL of pure water at room temperature, 0.5 mL of SeO2 (0.25 M) solution was slowly added dropwise, and then 0.2 mL of ascorbic acid (VC, 0.2 g / mL) was added and stirred for 15 min. Then, 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, and the mixture was continuously stirred for 6 h in a 40°C water bath, and finally centrifuged three times at 10000 rpm / min and vacuum dried to obtain copper selenide nanoparticles CuSe.

[0083] The HNTs were purified by segmented centrifugation and freeze-dried to obtain purified HNTs; 30 mg of polyvinyl pyrrolidone (PVP, Mw = 58000) was stirred and dissolved in 10 mL of pure water; HNTs and PVP solution were mixed in a mass ratio of 1:1 and stirred at room temperature overnight to obtain HNTs precursor solution. Then, 0.5 mL of SeO2 (0.25 M) solution was slowly added to the HNTs precursor solution, and then 0.2 mL of ascorbic acid (VC, 0.2 g / mL) was added and stirred for 15 min. Then, a mixed solution of CuSO4 (0.5 mL, 0.5 M) and ascorbic acid (0.3 mL, 0.2 g / mL) was added to the above reaction solution, and the mixture was stirred for 6 h in a 40 ° C water bath. Finally, it was centrifuged three times at 10000 rpm / min and freeze-dried to obtain a composite nanomaterial of halloysite and copper selenide (CuSe@HNTs).

[0084] Example 2

[0085] [Preparation of CuSe@HNTs@ME]

[0086] 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 of Example 1) to obtain a total volume of 50 mL of the solution to be treated.

[0087] The mixture was stirred under a magnetic stirrer and cyclically vacuum pumped in / out to expel the air from the HNTs cavity. Then, with the assistance of a vacuum pump, the mixture was stirred under vacuum for 0.5 h and under non-vacuum conditions for 0.5 h. After three cycles, the product was washed several times by centrifugation with ultrapure water to remove excess ME, and then freeze-dried to obtain CuSe@HNTs@ME.

[0088] Example 3

[0089] [Material Characterization]

[0090] Transmission electron microscopy (TEM), X-ray diffraction (XRD), energy dispersive X-ray spectroscopy (EDS) and infrared spectroscopy (FTIR) were used to characterize the phase, composition, morphology and crystallinity of different powders. N2 adsorption-desorption tester (BET) was used to measure the specific surface area of ​​powders prepared under different conditions. The content of powders was analyzed by thermogravimetric analyzer (TG).

[0091] like Figure 2 As shown in a, the in situ grown copper selenide presents a flower-like morphology on the halloysite nanotubes; Figure 2 As shown in b, XRD data show that the phase and functional groups of CuSe@HNTs are composed of CuSe and HNTs, respectively; Figure 2 c The CuSe content measured accounts for 15.9% of the total mass of CuSe@HNTs; Figure 2 As shown in Figure d, the specific surface area of ​​CuSe@HNTs is significantly larger than that of CuSe, which indicates that HNTs provide more specific surface area for CuSe. Figure 2 e and 2f, as shown in the XPS data, Cu in CuSe@HNTs + / Cu 2+ The proportion of Se-O bonds in CuSe@HNTs is higher than that in CuSe, and the surface CuSe@HNTs is easier to transfer electrons than CuSe. The proportion of Se-O bonds in CuSe@HNTs is smaller than that in CuSe, which indicates that CuSe@HNTs is more stable and less prone to oxidation than CuSe.

[0092] Figure 3a shows that the particle size distribution of each component material is relatively concentrated. The particle size range of CuSe@HNTs is 78.8nm~1990nm (average particle size is 345.3nm), the particle size range of CuSe@HNTs@ME is 58.8nm~1280nm (average particle size is 243.1nm), and the particle size range of copper selenide nanoparticles is 43.8nm~295nm (average particle size is 107.9nm). It is found that the size of ME is below 10nm, which is possible to be loaded into the HNTs lumen.

[0093] like Figure 3 As shown in b, it was found that CuSe@HNTs@ME lost more weight than CuSe@HNTs in TG, indicating the presence of ME, and ME accounted for 1.3% of the total mass of CuSe@HNTs@ME; Figure 3 As shown in Figure c, from the comparison between vacuum cycle and non-vacuum cycle, the same mass of CuSe@HNTs was loaded with ME through vacuum cycle (vacuum stirring for 0.5h, non-vacuum stirring for 0.5h, and cycled 3 times) and non-vacuum cycle (normal mixing and stirring for the same time) and then subjected to XRD measurement. The peak of CuSe@HNTs@ME in vacuum cycle was sharper, indicating that ME existed in the HNTs lumen rather than on the surface.

[0094] Example 4

[0095] [Antioxidant enzyme activity detection]

[0096] CuSe, CuSe@HNTs and CuSe@HNTs@ME aqueous dispersions were prepared at concentrations of 25, 50, 100, 200 and 400 μg / mL, respectively. A portable dissolved oxygen meter was used to characterize the catalase (CAT) activity. H2O2 solution and H2O2+ dispersions of each component were prepared: PBS buffer with a pH of 7.4 was used as the solvent, and a portable dissolved oxygen meter was used to detect the oxygen concentration of each group within a certain period of time until the oxygen concentration remained basically unchanged; NBT working solution was used to characterize the superoxide dismutase (SOD) activity. PBS buffer with a pH of 7.4 was used as the solvent to prepare mixed solutions of various components with different concentrations, incubated at 25°C for 20 minutes, irradiated with a 30W LED lamp for 15 minutes, and tested the absorbance at a wavelength of 560nm UV-UIS; Fenton reaction and electron paramagnetic resonance (EPR) were used to test the material's ability to scavenge ·OH. By Fe 2+ The Fenton reaction of the / H2O2 system generated ·OH and incubated with different components for 10 minutes. After that, DMPO was added to the mixed solution, and the ·OH radical scavenging ability was detected by EPR within 5 minutes.

[0097] The results are as follows Figure 4and Figure 5 As shown in the figure, from the three aspects of catalase (CAT) activity, peroxidase (SOD) activity and scavenging ·OH, it was found that CuSe@HNTs had stronger antioxidant enzyme activity than CuSe, and the ability of CuSe@HNTs@ME to scavenging ·OH was also slightly higher than that of CuSe@HNTs.

[0098] From the results, it can be seen that CuSe@HNTs is significantly better than CuSe in antioxidant enzyme activity, and CuSe@HNTs@ME is slightly better than CuSe@HNTs in scavenging hydroxyl radicals (·OH), indicating that the introduction of ME further improves the antioxidant properties of the composite material.

[0099] And, from Figure 5 As can be seen from a, the Zeta potential of CuSe@HNTs is -20.3mV~-19.6mV, and the Zeta potential of CuSe@HNTs@ME is -19.2mV~-17.2mV.

[0100] Example 5

[0101] [Stability test in gastric juice]

[0102] To simulate the digestion process of food in the stomach, the material was added to a gastric fluid simulation liquid (pH 1.2 to 1.5) and incubated at 37°C for 4 hours. Then, it was washed and neutralized with simulated colon fluid. Finally, the material was dissolved in water or freeze-dried, and SEM, XRD, ME release, BET, zeta potential measurement and ROS scavenging activity analysis were performed to determine whether the nanomaterial can stably pass through gastric fluid to reach the site of enteritis.

[0103] like Figure 6 As shown in a, 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 re-form into a suspension state (left), indicating that HNTs have the ability to protect CuSe and ME from being destroyed by strong acid, allowing CuSe@HNT@ME to maintain structural stability in gastric acid.

[0104] In addition, SEM images show that CuSe is still loaded on the surface of HNTs ( Figure 6 b and 6c), we also measured the zeta potential of CuSe@HNTs@ME after SGF treatment for 4 h ( Figure 6 d), there is not much difference, CuSe@HNTs@ME can still maintain its negative charge characteristics after passing through the digestive tract. The XRD results show that CuSe@HNTs@ME retains its crystal structure after SGF treatment ( Figure 6 e); Figure 6f shows that the amount of ME released by SGF treatment is not as high as that by normal PBS treatment, indicating that in gastric acid, ME is not suddenly released, and most of the ME is retained and can act on the intestinal part; Figure 6 g shows that the specific surface area does not decrease significantly after SGF treatment, and still retains a high specific surface area, providing more active sites for enzyme-like catalysis; Figure 6 h and 6i show that after SGF treatment, CuSe@HNTs@ME retained most of the SOD-like and CAT-like activities.

[0105] It can be seen from the above tests that the CuSe@HNTs@ME prepared in the present invention has oral stability.

[0106] Example 6

[0107] [CuSe@HNTs@ME targeted enteritis test]

[0108] (1) In vitro targeting adhesion test: Positively and negatively charged polymers were used to modify polystyrene plates to simulate inflamed and healthy epithelial cells, respectively. Based on the change of colon 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 colon epithelium in vitro.

[0109] First, polystyrene plates were modified with amine-rich polymer names (positively charged) and carboxylate-rich polymer names (negatively charged) to simulate inflammation and healthy epithelium, respectively. The three modified polystyrene plates were incubated with CuSe@HNTs@ME solution at 37°C for 3 hours, and then the excess CuSe@HNTs@ME was washed off with water (gentle operation required). Finally, EDS mapping and spectroscopy were used to detect the content of CuSe@HNTs@ME on the sheets on different surfaces.

[0110] The results are as follows Figure 7 As shown, the results indicate that CuSe@HNTs@ME is more easily aggregated on the polyethylene sheet rich in positive charges.

[0111] (2) In vivo targeted adhesion test: Each mouse was given a CuSe@HNTs@ME suspension by oral gavage, and the distribution of CuSe@HNTs@ME in the intestine was observed using a photoacoustic imaging device or TEM.

[0112] The results are as follows Figure 7 As shown, CuSe@HNTs@ME tends to accumulate in the inflamed intestinal area with positive charge and accumulates more in the intestinal villi. The photoacoustic imaging results show that ( Figure 8 As shown in Figure 3, CuSe@HNTs@ME was targeted and adsorbed on the intestines of mice with enteritis compared to healthy mice.

[0113] (3) In vitro adhesion targeting test: The intestines of mice in the healthy group and the IBD group were taken and incubated with the CuSe@HNTs@ME suspension. After fixation with 2.5% glutaraldehyde and dehydration with alcohol gradient, SEM was used to photograph the enrichment of CuSe@HNTs@ME in the intestines of mice in the healthy group and the IBD group.

[0114] The results are as follows Fig. 9 As shown, the results showed that CuSe@HNTs@ME was more enriched in the inflamed intestine compared with the healthy mouse intestine.

[0115] From the above experiments, we can see that these pathophysiological characteristics of mucosal defect-inflammation co-localization and local charge changes allow negatively charged nanoparticles to target intestinal inflammation through electrostatic interactions. The strong negative charge of CuSe@HNTs@ME facilitates passive deposition into the positively charged inflamed colon, providing targeted adsorption therapy for IBD intestinal inflammation sites.

[0116] Example 7

[0117] [Cytotoxicity test of CuSe@HNTs@ME]

[0118] Intestinal epithelial cell line (IEC-6) was used for cell experiments, and the culture conditions were 37°C and a humidified atmosphere (5% CO2). The complete culture medium included 90% basal medium (DMEM), 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P / S). The half inhibitory concentration (IC50) was first analyzed using different concentrations of DSS and different treatment times. The cytotoxic effects of different components were evaluated using CCK8 analysis or calcein-AM / PI staining according to the manufacturer's instructions. The results are shown in Fig.10 shown.

[0119] like Fig.10 As shown in a, 2% DSS damaged the cell viability of IEC-6 by nearly half, so the subsequent experiments treated IEC-6 with 2% DSS; Fig.10 b shows the effects of HNTs, CuSe, ME, CuSe@HNTs and CuSe@HNTs@ME on the 24h cell viability of IEC-6 cells. When the concentration of all materials was 25 μg / mL, they were non-toxic to the cells, and subsequent experiments were treated according to this concentration.

[0120] Example 8

[0121] [CuSe@HNTs@ME ability to remove intracellular ROS]

[0122] The detection of intracellular ROS is based on the peroxide-dependent oxidation of DCFH-DA to form a fluorescent compound called dichlorofluorescein (DCF). The cells were treated with H2O2 for 4 hours, washed with PBS, and the cells treated with different components were incubated at 37°C for 30 minutes. Fluorescence imaging was detected on an inverted microscope.

[0123] The results are as follows Fig.10 As shown in Figures c and 10d, after 4 h of H2O2 treatment, the fluorescence level of FITC in the whole cell was significantly enhanced, indicating that the level of ROS increased significantly. However, after pretreatment with CuSe@HNTs@ME, the ROS content in the cell was significantly reduced. This shows that CuSe@HNTs@ME can effectively reduce the level of ROS in the cell, demonstrating its good effect in scavenging reactive oxygen species.

[0124] Example 9

[0125] [CuSe@HNTs@ME targeted mitochondrial repair test]

[0126] The cells were washed twice with PBS and labeled with the following fluorescent probes for 30 minutes at 37°C: MitoTrackerGreen (Ex490nm / Em516nm) and JC-1 (a sensitive marker of mitochondrial membrane potential). The imaging was detected on an inverted microscope. The images were quantified using ImageJ software after appropriate thresholding to analyze the mitochondrial function of different components.

[0127] The results are as follows Fig.11 As shown in the figure, MitoTracker Green (Ex490nm / Em516nm) and JC-1 (sensitive marker of mitochondrial membrane potential), CuSe@HNTs@ME (abbreviated as CSHM) treatment reversed DSS-induced mitochondrial damage, which was manifested as increased mitochondrial mass and mitochondrial membrane potential. The key point is that the reversal intensity of ME is higher than that of HNTs and CuSe, indicating that ME plays a key role in the repair of mitochondria by CuSe@HNTs@ME.

[0128] It can be seen from the above that the CuSe@HNTs@ME of the present invention helps to alleviate DSS-induced mitochondrial pathology, thereby playing an anti-inflammatory effect.

[0129] Example 10

[0130] [Therapeutic effects of CuSe@HNTs@ME on in vivo colitis model]

[0131] C57 mice were randomly divided into groups (n=6), including control group (normal saline), DSS group (2% DSS), DSS+CuSe@HNTs@ME group, DSS+CuSe@HNTs group, DSS+HNTs group, DSS+CuSe group, and DSS+ME group. Drugs were administered by intragastric gavage, and the body weight and mouse status were evaluated every day during the experiment. The mice were sacrificed on the last day of the experiment, and the disease activity index (DAI) of each group was determined, and the colon tissue was taken to observe the length.

[0132] The results are as follows Fig.12 As shown, the therapeutic effect of CuSe@HNTs@ME (referred to as CSHM) was tested in the DSS-induced acute IBD model. Compared with the DSS, DSS+HNT, DSS+ME, DSS+CS, and DSS+CSH groups, CSHM suppressed the weight loss of IBD mice ( Fig.12 a), and lowered the disease activity index (DAI) ( Fig.12 c) The colon of the mice was collected, photographed and measured on day 11. Fig.12 As shown in Figure 2, the colon length of the CSHM group was significantly longer than that of the other groups, indicating that CSHM can inhibit the colon shortening commonly seen in DSS treatment ( Fig.12 b) If Fig.12 As shown in (d), for DSS-induced colitis mice, the colon tissue structure collapsed and the colon epithelium was severely damaged. After treatment, an improvement in histological appearance was observed, and the integrity of the tissue's villus layer structure was preserved. In addition, compared with HNTs, ME, and CuSe@HNTs treatments, CuSe@HNTs@ME treatment showed the most significant improvement.

[0133] It can be seen from the above tests that the CuSe@HNTs@ME of the present invention protects the intestinal structure and function.

[0134] Although the present invention has been disclosed as above with preferred embodiments, it is not intended to limit the present invention. A person with ordinary knowledge in the technical field to which the present invention belongs may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be determined by the definition of the claims.

Claims

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

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

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

4. The copper selenide-halloysite-mannose composite drug delivery system according to claim 1, characterized in that: The copper selenide nanoparticles are solid spherical and / or solid flower-shaped, and the particle size of the copper selenide nanoparticles ranges from 43.8 nm to 295 nm.

5. A method for preparing the copper selenide-halloysite-mannose composite drug delivery system according to any one of claims 1 to 4, characterized in that: The following steps are involved: Add polyvinyl pyrrolidone (PVP) into ultrapure water and fully dissolve it to obtain a PVP solution; Adding halloysite nanotubes (HNTs) into the PVP solution and mixing them thoroughly to modify the surface of the halloysite nanotubes with PVP, thereby obtaining a HNTs precursor solution; Adding the first reducing agent solution to the selenium source solution and mixing thoroughly to form a selenium precursor solution; Adding a second reducing agent solution to the copper source solution and mixing thoroughly to form a copper precursor solution; Slowly drop the selenium precursor solution into the HNTs precursor solution, and react under controlled temperature and stirring conditions to obtain a prefabricated solution containing selenium and HNTs; The copper precursor solution is slowly added dropwise to the prefabricated solution containing selenium and HNTs, and heated in a water bath to obtain a water bath product; The water bath product was washed and dried in sequence to obtain a composite of in-situ generated copper selenide nanoparticles on tubular HNTs (CuSe@HNTs); Mannose (ME) was dissolved in ultrapure water, and ME was loaded into the lumen of the CuSe@HNTs material using a vacuum circulation device. By centrifugation and freeze-drying, a CuSe@HNTs composite material containing ME in the lumen (CuSe@HNTs@ME) 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 55000 to 58000.

7. The preparation method according to claim 5, characterized in that: The mass ratio of HNTs and 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~0.5M, and the concentration of the first reducing agent solution is 2~5 times the concentration 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~1M, and the concentration of the second reducing agent solution is 2~5 times the concentration of the copper source solution.

10. The preparation method according to claim 5, characterized in that: In the preparation of the prefabricated solution 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°C to 30°C, the stirring speed is 100rpm to 300rpm, and the reaction time is 5min to 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°C to 60°C, the stirring speed is 100rpm to 300rpm, and the reaction time is 3h to 8h.

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

13. A pharmaceutical composition, characterized in that The invention comprises the copper selenide-halloysite-mannose composite drug delivery system as described in any one of claims 1 to 4.

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