A layered double hydroxide material system, its preparation method and application

Abstract

This invention is a divisional application of application number 202010500967.1. The invention relates to a layered double hydroxide material system, its preparation method, and its application, specifically the application of LDH / EDTA layered double hydroxide materials in the preparation of antitumor drugs. The LDH / EDTA layered double hydroxide material is a zinc-aluminum layered double hydroxide loaded with ethylenediaminetetraacetic acid (EDTA); in the EDTA-loaded zinc-aluminum layered double hydroxide, EDTA is anion-exchange-intercalated into the interlayer space of the zinc-aluminum layered double hydroxide; the surface of the EDTA-loaded zinc-aluminum layered double hydroxide is positively charged.

Description

[0001] This invention is a divisional application of the invention patent application with application number 202010500967.1, application date June 4, 2020, entitled "A layered double hydroxide material system and its preparation method and application". Technical Field

[0002] This invention relates to a layered double hydroxide material system, its preparation method, and its application. Specifically, it relates to an NT-LDH layered double hydroxide material and an NT-LDH / EDTA layered double hydroxide material, their preparation methods, and applications. In particular, it relates to a zinc-aluminum layered double hydroxide (LDH) material system with surface-targeted modification and ethylenediaminetetraacetic acid (EDTA) loading, its preparation method, and the application of the surface-targeted modified material system in the preparation of drugs for treating bladder cancer. This invention belongs to the field of medical bionanomaterials technology. Background Technology

[0003] Bladder cancer is one of the most common malignant tumors of the genitourinary system worldwide, ranking 12th in incidence among malignant tumors, and its incidence has been gradually increasing in recent years. Clinically, 75%–85% of newly diagnosed bladder cancer patients have non-muscle-invasive bladder cancer (NMIBC). The common treatment for NMIBC is transurethral resection of the bladder tumor, followed by immediate intravesical chemotherapy based on the pathological classification. However, the treatment effect still has certain problems. First, bladder cancer resection surgery is very risky, especially for most elderly patients, and bladder cancer is difficult to completely remove, resulting in a high recurrence rate. Studies have shown that 15%–61% of NMIBC patients experience recurrence within one year post-surgery, and the recurrence rate can be as high as 31%–78% within five years. Furthermore, some patients' conditions may rapidly progress to invasive or metastatic bladder cancer. More importantly, the clinical efficacy of postoperative intravesical chemotherapy is not ideal because the production and excretion of urine in the bladder dilutes the instilled chemotherapy drugs and shortens their retention time. Furthermore, infusion chemotherapy lacks adhesion to tumor tissue, has no targeted effect, and exhibits low permeability. This necessitates numerous, large-volume infusions, which can easily lead to drug resistance and result in significant toxic side effects that reduce quality of life. Therefore, the need to develop a gentler, safer, and more promising treatment strategy has become increasingly urgent. Summary of the Invention

[0004] In view of the shortcomings and needs of the existing technology, the purpose of this invention is to provide a zinc-aluminum layered double hydroxide (LDH) that can load and release EDTA and has surface-linked antibodies targeting bladder cancer (neurotensin NT), and to develop it safely and effectively for use in the preparation of non-invasive therapeutic drugs for bladder cancer.

[0005] In a first aspect, the present invention provides an NT-LDH / EDTA layered double hydroxide material, comprising: a zinc-aluminum layered double hydroxide loaded with ethylenediaminetetraacetic acid and a neurotensin antibody NT modified on the surface of the zinc-aluminum layered double hydroxide.

[0006] The inventors of this invention are based on the premise of achieving a safe and gentle therapeutic effect by disrupting cell junctions to induce the detachment of bladder tumor cells from the tissue and their excretion in urine. This is considering that cell junctions mainly consist of tight junctions, adhesive junctions, and desmosomes and hemidesmosomes. Some key proteins expressed in these junctions are calcium-dependent, such as E-cadherin in typical adhesive junctions, and desmosome core protein and desmosome glial protein in desmosome structures, whose conformations are only stable in the presence of calcium ions. EDTA, as a classic metal ion chelator, can chelate calcium ions and disrupt the expression of calcium-dependent proteins. In related studies, it has been used to gently disrupt intercellular junctions non-enzymatically to detach cells from the tissue. However, due to the similar drawbacks of free EDTA to chemotherapy drugs, instillation therapy for bladder cancer does not achieve satisfactory results. In particular, if small molecules of free EDTA are not rapidly excreted in urine, they are likely to enter the cells instead of remaining on the cell membrane to chelate calcium ions. Therefore, to overcome these drawbacks, it is necessary to design and prepare a loading and delivery material system for EDTA.

[0007] Furthermore, LDH is a type of anionic layered dihydroxyl metal oxide with a positively charged surface. Due to its strong anion exchange capacity, LDH can adsorb many inorganic and organic ions in solution, thus it is widely used in various industrial fields. In addition, LDH possesses good biocompatibility and low toxicity, providing safety assurance for applications in the biomedical field. LDH exhibits high interlayer drug loading capacity and pH-responsive drug release performance. Moreover, the hydroxyl groups suspended on the surface of LDH can bind to specific targeting antibody molecules, enabling it to rapidly reach the lesion area from the cell surface and reduce the impact on normal tissues. Therefore, LDH has broad application prospects as a multifunctional drug delivery system. In this study, the inventors selected LDH as the material carrier.

[0008] Based on the above, the inventors, for the first time, used a zinc-aluminum layered double hydroxide loaded with ethylenediaminetetraacetic acid as the main component, and then achieved the connection with the neurotensin antibody NT through surface amylation modification, obtaining an NT-LDH / EDTA layered double hydroxide material. Because the large size of this material system prevents it from entering cells and allows it to continuously adsorb onto the cell membrane surface, it releases EDTA in the acidic microenvironment of tumors to chelate calcium ions, inhibiting the expression of calcium-dependent proteins, thereby disrupting cell junctions and causing a large number of adherent cells to detach over time. Most importantly, these detached, dispersed cells are still tightly encapsulated by the material system, inhibiting their ability to adhere to and migrate.

[0009] Preferably, the zinc-aluminum layered double hydroxide has a mass of 100 wt%, and the ethylenediaminetetraacetic acid (EDTA) content is ≥25 wt%, preferably 25 wt% to 40 wt%. Specifically, the EDTA loading mass is between 25 wt% and 40 wt% of the zinc-aluminum layered double hydroxide mass.

[0010] Preferably, the content of the neurotensin antibody NT is 3-7 wt%, for example, 3.9 wt%, with the zinc-aluminum layered double hydroxide having a mass of 100 wt%. Specifically, this means that the loading mass of the neurotensin antibody NT is 3-7 wt% of the mass of the zinc-aluminum layered double hydroxide.

[0011] Preferably, the molar ratio of Zn to Al in the zinc-aluminum layered double hydroxide is 2 to 3:1, for example, 2:1.

[0012] Preferably, the particle size of the NT-LDH / EDTA layered double hydroxide material is 700–900 nm.

[0013] Preferably, the surface amination modification includes: hydrolyzing the hydroxyl groups on the surface of the zinc-aluminum layered double hydroxide supported on ethylenediaminetetraacetic acid using the Si-OH of a silane to form suspended amino groups on the surface; wherein the silane is selected from 3-aminopropyltriethoxysilane (APTES).

[0014] Preferably, in the zinc-aluminum layered double hydroxide loaded with ethylenediaminetetraacetic acid (EDTA), EDTA is intercalated into the interlayer of the zinc-aluminum layered double hydroxide via anion exchange.

[0015] Secondly, the present invention also provides a method for preparing the NT-LDH / EDTA layered double hydroxide material as described above, comprising:

[0016] (1) The zinc-aluminum layered double hydroxide solution loaded with ethylenediaminetetraacetic acid is stirred at 70-90°C (e.g., 78°C) and refluxed under a protective atmosphere. After adding silane solution, the reaction is carried out for 3-10 hours (e.g., 4 hours). Then, the solution is filtered, washed and dried to obtain NH2-LDH / EDTA.

[0017] (2) 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were added to the aqueous solution of neurotensin antibody NT and mixed. Then NH2-LDH / EDTA was added and reacted at room temperature (25-35°C) for 12-24 hours (e.g., 12 hours). After filtration, washing and drying, the NT-LDH / EDTA layered double hydroxide material was obtained.

[0018] Preferably, the ratio of the zinc-aluminum layered double hydroxide loaded with ethylenediaminetetraacetic acid to silane is 1 mg:20 μL to 1 mg:30 μL.

[0019] Preferably, the mass ratio of the neurotensin antibody NT to NH2-LDH / EDTA is 2:5 to 4:5, for example, 2:5.

[0020] Preferably, the mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, and neurotensin antibody NT is 0.153g:0.230g:20mg to 0.306g:0.460g:20mg, for example, 0.153g:0.230g:20mg.

[0021] Preferably, the method for preparing the zinc-aluminum layered double hydroxide loaded with ethylenediaminetetraacetic acid includes:

[0022] (1) Dissolve a zinc source, an aluminum source, an anion source and cyclohexamethylenetetramine in water to obtain a mixed solution; the anion source is at least one of sodium nitrate, sodium chloride and sodium phosphate;

[0023] (2) The resulting mixed solution is stirred at 100–140°C (e.g., 120°C) and refluxed with cold water under a protective atmosphere for 1–4 hours (e.g., 2 hours). Then, Na₂H₂EDTA solution is added, and the reaction is continued at 70–100°C (e.g., 80°C) for 20–23 hours (e.g., 22 hours). After filtration, washing, and drying, zinc-aluminum layered double hydroxides loaded with ethylenediaminetetraacetic acid are obtained. All the above synthesis reactions must be carried out under an argon protective atmosphere to prevent carbon dioxide from entering the system and forming carbonate ions that insert into the LDH interlayer and are difficult to displace. Under high-temperature conditions, hexamethylenetetramine hydrolyzes to release ammonia, making the solution alkaline and providing a suitable environment for LDH nucleation and growth.

[0024] Furthermore, preferably, the zinc source is selected from at least one of zinc nitrate and zinc chloride; the aluminum source is selected from at least one of aluminum nitrate and aluminum chloride; and the molar ratio of the zinc source to the aluminum source is 2 to 3:1, for example, 2:1.

[0025] Furthermore, preferably, the mass ratio of the zinc source to the anion source (e.g., sodium nitrate) is 594–891:85–170, for example, 594:85; and the mass ratio of the zinc source to cyclohexamethylenetetramine is 99–198:117–234, for example, 99:117.

[0026] Furthermore, preferably, the mass ratio of Na2H2EDTA to zinc source is 0.6–1.2 g : 0.594–0.891 g, for example, 1.2 g : 0.594 g.

[0027] Thirdly, the present invention provides an application of the above-mentioned NT-LDH / EDTA layered double hydroxide material in the preparation of antitumor drugs. In the acidic microenvironment of urine or tumors, the NT-LDH / EDTA layered double hydroxide material releases EDTA to chelate calcium ions, inhibiting the expression of calcium-dependent proteins, thereby disrupting tumor cell adhesion and causing adherent tumor cells to detach in a time-dependent manner; moreover, the detached dispersed tumor cells are tightly wrapped by the NT-LDH / EDTA layered double hydroxide material to inhibit their ability to adhere to and metastasize.

[0028] The NT-LDH / EDTA layered double hydroxide material is preferred, and the tumor includes colon cancer and bladder cancer.

[0029] Preferably, taking a drug used to treat bladder cancer as an example, its operation process may include:

[0030] (1) NT-LDH / EDTA layered double hydroxide material was introduced into the bladder using a bladder instillation method;

[0031] (2) The NT-LDH / EDTA layered double hydroxide material rapidly identifies the tumor region and targets and accumulates outside the tumor cell membrane, starting to disrupt cell connections and causing tumor cells to peel off layer by layer from the tumor tissue;

[0032] (3) The NT-LDH / EDTA layered double hydroxide material tightly wraps the free tumor cells obtained by peeling off layer by layer, preventing them from metastasizing to the muscular layer or distant sites through nearby lymph nodes, and carrying them out of the body with the periodic generation and excretion of urine.

[0033] Fifthly, the present invention provides an NT-LDH layered double hydroxide material, comprising: a zinc-aluminum layered double hydroxide and a neurotensin antibody (NT) modified on the surface of the zinc-aluminum layered double hydroxide. Since NTS1 receptors are overexpressed on the surface of tumor cell membranes in bladder and colorectal cancers and can specifically bind to NT, the NT-LDH layered double hydroxide material obtained in this invention uses LDH as the main component, and the surface modification of NT enables the material to possess specific targeting functions.

[0034] Preferably, the zinc-aluminum layered double hydroxide has a mass of 100 wt%, and the content of the neurotensin antibody NT is 3 to 7 wt%, for example, 3.9 wt%.

[0035] Preferably, the molar ratio of Zn to Al in the zinc-aluminum layered double hydroxide is 2 to 3:1, more preferably 2:1.

[0036] Preferably, the particle size of the NT-LDH layered double hydroxide material is 700-900 nm.

[0037] In a sixth aspect, the present invention provides a method for preparing the above-mentioned NT-LDH layered double hydroxide material, comprising:

[0038] (1) The zinc-aluminum layered double hydroxide solution was stirred at 70-90℃ (78℃) and refluxed with cold water under a protective atmosphere. After adding silane solution, the reaction was carried out for 3-10 hours (4 hours). Then, after filtration, washing and drying, NH2-LDH was obtained. (2) 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were added to the aqueous solution of neurotensin antibody NT and mixed. Then, NH2-LDH was added and the reaction was carried out at room temperature (25-35℃) for 12-24 hours (12 hours). Then, after filtration, washing and drying, the NT-LDH layered double hydroxide material was obtained.

[0039] Preferably, the ratio of the zinc-aluminum layered double hydroxide to silane is 1 mg: 20-30 μL, more preferably 50 mg: 100 μL.

[0040] Preferably, the mass ratio of the neurotensin antibody NT to NH2 / LDH is 2 to 4:5, more preferably 2:5.

[0041] Preferably, the mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, and neurotensin antibody NT is 0.153g:0.230g:20mg to 0.306g:0.460:20mg, for example, 0.153g:0.230g:20mg.

[0042] Beneficial effects:

[0043] This invention provides a simple, easy-to-implement, non-invasive, and highly effective new strategy for treating bladder cancer. Specifically, it disrupts cell junctions, causing tumor cells to detach from cancerous tissue and ultimately be excreted in urine.

[0044] This invention provides a novel NT-LDH / EDTA layered double hydroxide material. This material system can efficiently target tumor regions and adhere to tumor cell membranes. The released EDTA then binds calcium ions between cells, inhibiting calcium-dependent protein expression and disrupting cell junctions. Furthermore, the material system tightly surrounds the surface of detached cells, preventing metastasis. Simultaneously, this material system exhibits relatively ideal therapeutic effects. Results also show that NT-LDH / EDTA has minimal impact on normal tissues and low systemic toxicity, which provides a guarantee for further clinical research. In summary, this invention provides a convenient, efficient, and safe innovative method for treating bladder cancer, and also has certain reference value for the treatment of other gastrointestinal tumors such as colon cancer.

[0045] In this invention, the NT-LDH / EDTA layered double hydroxide material ensures good cell shedding effect without significant cell killing ability, thus providing a guarantee of good biosafety and low toxicity. Attached Figure Description

[0046] Figure 1 This is a transmission electron microscope (TEM) image of the NT-LDH / EDTA material system prepared in Example 1 dispersed in water.

[0047] Figure 2 Here are scanning electron microscope (SEM) images of the NT-LDH / EDTA material system prepared in Example 1;

[0048] Figure 3 The XRD patterns of the material systems LDH, LDH / EDTA, NH2-LDH / EDTA and NT-LDH / EDTA prepared in Example 1 are shown below.

[0049] Figure 4 The FI-IR spectra of the material systems LDH, LDH / EDTA, NH2-LDH / EDTA and NT-LDH / EDTA prepared in Example 1 are shown.

[0050] Figure 5 The image shows the elemental mapping of the NT-LDH / EDTA material system obtained in Example 1 using transmission electron microscopy.

[0051] Figure 6The material system prepared in Example 1 is shown in the NT-LDH / EDTA transmission electron microscopy energy dispersive spectroscopy (EDS) and the elemental ratios measured by ICP-OES.

[0052] Figure 7 Zeta potential analysis of the material systems LDH / EDTA, NH2-LDH / EDTA and NT-LDH / EDTA prepared in Example 1;

[0053] Figure 8 The release behavior of EDTA in the NT-LDH / EDTA material system prepared in Example 1 was detected in slightly acidic PBS (pH 6.5).

[0054] Figure 9 Confocal images of the EDTA-free material systems NH2-LDH and NT-LDH prepared in Example 2, evaluating their targeted adhesion to T24 bladder cancer cells.

[0055] Figure 10 (a) SEM image of untreated suspended T24 cells, and SEM image of bladder cancer cells T24 detached from the supernatant using the NT-LDH / EDTA material system prepared in Example 1, and the corresponding (b) elemental energy spectrum mapping image, demonstrating that the surface of the detached cells is still coated with the material.

[0056] Figure 11 A bar chart showing the proportion of detached bladder cancer cells T24 shed using the NT-LDH / EDTA material system prepared in Example 1.

[0057] Figure 12 A bar chart showing the proportion of detached colorectal cancer cells CT26 shed using the NT-LDH / EDTA material system prepared in Example 1.

[0058] Figure 13 To evaluate the treatment process of SD rats with orthotopic bladder cancer, ultrasound imaging was used to detect the instillation of PBS, EDTA and the material systems NH2-LDH / EDTA and NT-LDH / EDTA prepared in Example 1 into the bladder. The solid yellow circle represents the tumor.

[0059] Figure 14 The images show bladder tissue (opened bladders) after bladder instillation into SD rats with orthotopic bladder cancer using PBS, EDTA, and the material systems NH2-LDH / EDTA and NT-LDH / EDTA prepared in Example 1. The blue dashed circle represents the tumor.

[0060] Figure 15The study included bladder instillation of NT-LDH / EDTA, a material system prepared in Example 1, into SD rats with orthotopic bladder cancer. After treatment, renal function was evaluated and anatomical diagrams were obtained.

[0061] Figure 16 Tissue sections of organs such as heart, liver, spleen, lung, and kidney after bladder instillation of the material system NT-LDH / EDTA (100 mg / kg) prepared in Example 1 into healthy SD rats. Detailed Implementation

[0062] The present invention will be further illustrated by the following embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the present invention.

[0063] In this field, developing convenient, rapid, specific, and non-invasive new strategies for treating bladder cancer, and constructing an LDH material system that can target and adhere to the surface of tumor cells and release EDTA with good biocompatibility, is crucial. This system can accumulate in the bladder cancer area and disrupt cell junctions, tightly encapsulating detached cells and blocking their metastasis, ultimately leading to their excretion in urine. This is of significant importance and value.

[0064] Therefore, this disclosure provides a zinc-aluminum layered double hydroxide LDH material system (or NT-LDH / EDTA layered double hydroxide material) that can load and release EDTA and has surface-linked antibodies targeting bladder cancer (neurotensin NT), and develops a new method for non-invasive, safe and effective treatment of bladder cancer.

[0065] In this invention, the NT-LDH / EDTA layered double hydroxide material can be further labeled as NT-LDH / EDTA. It is primarily composed of zinc-aluminum layered double hydroxides (LDH) with intercalated ethylenediaminetetraacetic acid (EDTA), and has a neurotensin (NT) antibody targeting bladder cancer linked to its surface. The preparation method of the NT-LDH / EDTA layered double hydroxide material is illustrated below by way of example.

[0066] In this invention, the LDH material in the main body is composed of zinc and aluminum ions, which are relatively safe for the body. To ensure that LDH continuously adheres to the surface of tumor cells, thereby disrupting cell junctions and preventing LDH from being taken up by cells and exerting a chelating effect, this invention prepares LDH with a particle size of approximately 0.8 μm. In fact, the particle size of the obtained NT-LDH / EDTA layered double hydroxide material is also approximately 0.8 μm.

[0067] In an optional embodiment, the host is prepared using a homogeneous alkaline solution method. Specifically, a mixed solution containing a zinc source (zinc metal salt), an aluminum source (aluminum metal salt), hexamethylenetetramine (HMT), and sodium chloride is placed in an oil bath at 120°C. After reflux in the oil bath for 2 hours under an argon protective atmosphere, disodium ethylenediaminetetraacetate is added to replace the interlayer anions, thus preparing EDTA-loaded LDH, labeled as LDH / EDTA. The oil bath temperature can be 80°C, and the reflux time can be 12 hours. EDTA extensively inserts into the LDH interlayer through anion exchange and can subsequently be released in a slightly acidic environment (pH 6.5).

[0068] In optional embodiments, the mass ratio of disodium ethylenediaminetetraacetate (EDTA) to the zinc source can be 1.2 g: 0.594 g. The zinc source is preferably selected from zinc nitrate and zinc chloride, etc. The aluminum source is preferably selected from aluminum nitrate and aluminum chloride, etc. The molar ratio of the zinc source to the aluminum source can be 2:1. The mass ratio of sodium nitrate to the zinc source can be 594:85. The mass ratio of cyclohexamethylenetetramine to the zinc source can be 99:117.

[0069] To link the target molecule, the LDH / EDTA surface is first aminated, and the resulting product is labeled NH2-LDH / EDTA. Specifically, the Si-OH in 3-aminopropyltriethoxysilane (APTES) silane is used to hydrolyze the hydroxyl groups on the LDH surface to obtain suspended amino groups on the host surface.

[0070] As an example, a zinc-aluminum layered double hydroxide solution loaded with ethylenediaminetetraacetic acid (EDTA) was stirred at 78°C and refluxed with cold water under a protective atmosphere. After adding a silane solution, the reaction was carried out for 4 hours, followed by filtration, washing, and drying to obtain NH₂-LDH / EDTA. The ratio of the EDTA-loaded zinc-aluminum layered double hydroxide to silane can be 50 mg: 100 μL.

[0071] The aminated LDH surface was bonded to NT antibody via peptide bonds and labeled NT-LDH / EDTA. Specifically, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were added to an aqueous solution of neurotensin antibody (NT) and mixed to activate the amino group. NH2-LDH / EDTA was then slowly added and reacted at room temperature (25°C) for 12 hours to obtain the NT-LDH / EDTA layered double hydroxide material, denoted as NT-LDH / EDTA.

[0072] In an optional embodiment, the mass ratio of neurotensin antibody NT to NH2-LDH / EDTA can be 20 mg: 50 mg. The mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, and neurotensin antibody NT can be 0.153 g: 0.230 g: 20 mg.

[0073] In this invention, NT (QLYENKPRRPYIL) is selected as the targeting antibody to target NT receptor 1 (NTS1) on the cell membrane, which is overexpressed in tumor cells such as colon cancer, prostate cancer, and bladder cancer. The amino-modified LDH surface utilizes peptide bonds to bind the NT antibody, which on the one hand enriches the NT-LDH / EDTA material system in cancerous tissues and targets and adheres to the surface of tumor cells; on the other hand, it promotes the conversion of the LDH surface from positive to negative charge, reducing adsorption and impact on normal tissues.

[0074] The synthesis reactions of LDH / EDTA, NH2-LDH / EDTA, and NT-LDH / EDTA described above all need to be carried out under an argon protective atmosphere to prevent carbon dioxide from entering the system and forming carbonate ions that insert into the LDH interlayer and are difficult to displace.

[0075] In this invention, all LDH / EDTA, NH2-LDH / EDTA, and NT-LDH / EDTA material systems were prepared, filtered and washed three times with water, and then the powders were stored at 4°C after being air-dried. Subsequent preparations were made fresh and dispersed into cell culture medium or physiological saline at the specified concentrations. The concentration of NT-LDH / EDTA added to the cell culture medium or physiological saline could be 60 mg / kg. This NT-LDH / EDTA material system showed no significant acute or chronic pathological toxicity or abnormalities to major tissues and blood indicators, demonstrating good systemic in vivo safety.

[0076] In this invention, the NT-LDH / EDTA material system, due to its large size, cannot enter cells and continuously adsorb onto the cell membrane surface. In the acidic microenvironment of urine or tumors, it releases EDTA to chelate calcium ions, inhibiting the expression of calcium-dependent proteins, thereby disrupting cell junctions and causing a large number of adherent cells to detach over time. Importantly, these detached, dispersed cells remain tightly encapsulated by the material system, inhibiting their ability to adhere to and migrate.

[0077] In this invention, the preparation process of the NT-LDH layered double hydroxide material is basically the same as that of the NT-LDH / EDTA layered double hydroxide material, the difference being whether or not disodium ethylenediaminetetraacetate is added during the reaction. This invention prepared LDH with a particle size of approximately 0.8 μm. In fact, the particle size of the obtained NT-LDH layered double hydroxide material system is also approximately 0.8 μm.

[0078] The following examples further illustrate the present invention in detail. It should also be understood that the following examples are only for further explanation of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above description of the present invention are within the scope of protection of the present invention. The specific process parameters, etc., in the following examples are merely examples within a suitable range; that is, those skilled in the art can make appropriate selections within the appropriate range based on the description herein, and are not intended to be limited to the specific values ​​in the examples below.

[0079] Example 1

[0080] The preparation process of EDTA-loaded zinc-aluminum layered double hydroxide (LDH / EDTA) is as follows: A certain amount of Zn(NO3)2·6H2O (0.594g), Al(NO3)3·9H2O (0.375g), NaNO3 (0.085g) and cyclohexamethylenetetramine (HMT) (0.702g) were weighed and dissolved in 200mL of deionized water. The solution was magnetically stirred for 20min under a high-purity argon atmosphere to remove air. Then, the solution was transferred to an oil bath at 120℃ and stirred. Under the protection of high-purity argon as a protective gas, the solution was refluxed for 2h. Then, Na2H2EDTA solution (1.2mg dissolved in 50mL of deionized water) was slowly added and the reaction was continued at 80℃ for 22h. Finally, the precipitate was filtered and collected and thoroughly washed with deionized water to obtain LDH / EDTA.

[0081] NH2-LDH / EDTA was obtained by surface amination modification of LDH / EDTA. The surface of LDH was modified into suspended amino groups by hydrolytic bonding of suspended hydroxyl groups with silanol groups in APTES. Specifically, 50 mg of LDH / EDTA was dispersed in 90 mL of deionized water. The solution was transferred to a 78°C oil bath and stirred, then refluxed under high-purity argon as a protective gas. 100 μL of APTES was added to 10 mL of ethanol solution, followed by slow addition to the above solution. After reacting for 4 h, the mixture was filtered, and the precipitate was washed sequentially with deionized water and ethanol to obtain NH2-LDH / EDTA.

[0082] NH2-LDH / EDTA surface-linked neurotensin (NT)-targeting antibody (NT-LDH / EDTA). The main mechanism involves NT binding to the amino groups on the NH2-LDH / EDTA surface via amide interaction. The specific process includes: dispersing NT (20 mg) in 20 mL of deionized water, adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, 0.153 g) and N-hydroxysuccinimide (NHS, 0.230 g), and stirring the resulting mixture at room temperature for 15 min to activate the carboxyl groups on the NT peptide. Then, adding 30 mL of an aqueous solution of NH2-LDH / EDTA (50 mg). The reaction was carried out with stirring at room temperature and protected by a high-purity argon purging system. After 12 hours, the NT-LDH / EDTA precipitate was collected by filtration, thoroughly washed with deionized water, and finally freeze-dried. The resulting powder was stored at 4°C to obtain NT-LDH / EDTA. In NT-LDH / EDTA, the loading mass of ethylenediaminetetraacetic acid is greater than 25.8 wt% of the mass of zinc-aluminum layered double hydroxide, and the loading mass of neurotensin is 3.9 wt% of the mass of zinc-aluminum layered double hydroxide.

[0083] Example 2

[0084] The preparation process of zinc-aluminum layered double hydroxide (LDH) is as follows: A certain amount of Zn(NO3)2·6H2O (0.594g), Al(NO3)3·9H2O (0.375g), NaNO3 (0.085g), and cyclohexamethylenetetramine (HMT) (0.702g) were weighed and dissolved in 200mL of deionized water. The solution was magnetically stirred for 20min under a high-purity argon atmosphere to remove air. The solution was then transferred to a 120℃ oil bath and stirred. Under high-purity argon as a protective gas, the solution was refluxed with cold water for 2h. After 2h, the temperature was changed to 80℃, and the reaction continued for 22h. Finally, the precipitate was filtered, collected, and thoroughly washed with deionized water to obtain LDH. The surface amination modification and NT-linking process in Example 2 are basically the same as in Example 1, except that NT-LDH without EDTA loading was used. The loading mass of neurotensin in NT-LDH is 3.9wt% of the mass of the zinc-aluminum layered double hydroxide.

[0085] Figure 1 This is a transmission electron microscope (TEM) image of the NT-LDH / EDTA material system prepared in Example 1 dispersed in water. Figure 1 It can be seen that the morphology of the prepared material is two-dimensional lamellar, uniformly dispersed, and relatively large in size, with a diameter of about 0.8 μm. Figure 2 The image shown is a scanning electron microscope (SEM) image of the NT-LDH / EDTA material system prepared in Example 1. Figure 2It can be seen that the morphology of the prepared material is a distinct layered structure with a uniform particle size of about 0.8 μm. The cross-sectional image shows that the thickness of a single particle can reach about 0.3 μm.

[0086] Figure 3 The XRD patterns of the material systems LDH, LDH / EDTA, NH2-LDH / EDTA, and NT-LDH / EDTA prepared in Example 1 are shown below. Figure 3 It can be seen that the fundamental diffraction peaks (003), (006), and (009) of LDH are characteristic peaks with a layered structure, but their peak positions differ with and without EDTA intercalation. Compared to LDH, the characteristic peaks of LDH / EDTA shift towards smaller angles because EDTA has a longer van der Waals end length, resulting in a layer spacing d. 003 The increase in the number of cells confirmed that Na2H2EDTA successfully inserted into the interlayer of LDH via anion exchange. On the other hand, the diffraction peak positions of NH2-LDH / EDTA and NT-LDH / EDTA were basically consistent with those of LDH / EDTA, indicating that surface modification did not alter the layered structure.

[0087] Figure 4 The FI-IR spectra of the material systems LDH, LDH / EDTA, NH2-LDH / EDTA, and NT-LDH / EDTA prepared in Example 1 are shown below. Figure 4 It can be seen that in the LDH spectrum, 3450 cm⁻¹ -1 and 1620cm -1 The broad absorption band is due to the hydrogen bond stretching vibrations in LDH and the bending vibrations of water. At 1385 cm⁻¹ -1 The sharp absorption band at 1602 cm⁻¹ is caused by the stretching vibrational mode of nitrate ions in the LDH interlayer. In the LDH / EDTA spectrum, this band is located at 1602 cm⁻¹. -1 and 1390cm -1 The strong absorption band at this point is due to the symmetric and antisymmetric vibrations of the COO group in EDTA, proving that EDTA is inserted into LDH. In the NH2-LDH / EDTA spectrum, the band is approximately 1000-1200 cm⁻¹. -1 The two absorption bands at 1265 cm⁻¹ are caused by the -O-Si vibration of APTES, which proves the successful surface amination of LDH / EDTA. In the NT-LDH / EDTA spectrum, at 1265 cm⁻¹... -1 The appearance of a new amide bond indicates that the NT molecule has successfully linked with the amino group on the surface of the NH2-LDH / EDTA material.

[0088] Figure 5 and Figure 6The figures show the elemental mapping diagram and corresponding energy dispersive spectroscopy (EDS) diagram of the NT-LDH / EDTA material system prepared in Example 1, as well as the quantitative elemental analysis by ICP-OES. As can be seen from the figures, within the framework of the prepared NT-LDH / EDTA material system, the uniform distribution of Zn, Al, O, and Si on the NT-LDH / EDTA surface can be observed through point mapping and line scanning of Zn, Al, O, and Si elements using PES. This indicates the successful preparation of zinc-aluminum LDH, and the presence of Si also demonstrates that APTES successfully modified the surface of LDH. Quantitative elemental analysis by ICP-OES shows that the zinc-aluminum molar ratio in the NT-LDH / EDTA material is 2:1.

[0089] Figure 7 Zeta potential analysis of the material systems LDH / EDTA, NH2-LDH / EDTA, and NT-LDH / EDTA prepared in Example 1 was performed by... Figure 7 It can be seen that the surfaces of LDH / EDTA and NH2-LDH / EDTA are positively charged, while the surface of NT-LDH / EDTA becomes negatively charged, which also proves that NT is successfully attached to the surface of the material system.

[0090] Figure 8 The release behavior of EDTA in the NT-LDH / EDTA material system prepared in Example 1 was detected in slightly acidic PBS (pH 6.5). Figure 8 It can be seen that EDTA is released rapidly within the first hour, and then released slowly, with a total release of 25.8 wt% over 16 hours. The release curve shows that the final loading of EDTA exceeds 25.8 wt%, but based on the release curve, it can be roughly judged that its loading will not exceed 40 wt%.

[0091] A. Cell application effect experiment

[0092] 1. Material targeting evaluation

[0093] 1.1 Experimental Materials and Instruments: The unloaded EDTA material systems NH2-LDH and NT-LDH prepared in Example 2; Rhodamine RITC dye; nuclear DAPI dye; and cell membrane DIO dye.

[0094] 1.2 Experimental Methods: RITC was stained with NH2-LDH and NT-LDH materials. Bladder cancer cells T24 were seeded on confocal dishes and then co-incubated with 200 ppm RITC-conjugated NH2-LDH and NT-LDH for 30 min, respectively. Cell membrane and nuclear staining was then performed, and the cells were observed under a 60x oil immersion microscope (FluoView FV1000).

[0095] 1.3 Experimental Results: Figure 9 This is a confocal image evaluating the adhesion of the EDTA-free material systems NH2-LDH and NT-LDH prepared in Example 2 to T24 bladder cancer cells. Figure 9 As can be seen, the red fluorescence represents the RITC-stained material system, the green fluorescence represents the DIO-stained cell membrane, and the blue fluorescence represents the DAPI-stained cell nucleus. Under the same material concentration and incubation time, NT-LDH was more enriched on the T24 cell membrane surface than NH2-LDH. The material mainly accumulated on the cell membrane rather than inside the cell.

[0096] 2. Evaluation of the material's adhesion to detached suspended cells.

[0097] 2.1 Experimental materials: The material system NT-LDH / EDTA prepared in Example 1.

[0098] 2.2 Experimental methods: Bladder cancer T24 cells were seeded in six-well plates and allowed to adhere for 24 hours. Then, the cells were treated with NT-LDH / EDTA at 200 ppm in the culture medium for 10 minutes. The supernatant was then collected and centrifuged to obtain cells for SEM energy dispersive spectroscopy (EDS) and elemental mapping.

[0099] 2.3 Experimental Results: Figure 10 To illustrate the exfoliation of T24 bladder cancer cells using the NT-LDH / EDTA material system prepared in Example 1, SEM images were obtained from the exfoliated cells in the suspension. Figure 9 As can be seen from electron microscopy morphology analysis, the exfoliated cells treated with NT-LDH / EDTA were tightly encapsulated by the material. EDS results also showed that 1.9% of the Si on the cell membrane came from NT-LDH / EDTA, and the corresponding mapping of O, Zn, Al, and Si elements on the cell membrane further confirmed that NT-LDH / EDTA did not detach from the cell membrane of the suspended single cells, indicating its good adhesion properties.

[0100] 3. Performance evaluation of materials that disrupt cell connections, leading to cell detachment.

[0101] 3.1 Experimental materials: The material system NT-LDH / EDTA prepared in Example 1.

[0102] 3.2 Experimental Methods: Bladder cancer T24 cells and colorectal cancer CT26 cells were seeded into culture flasks at a density of 1×10⁻⁶ cells. 7After 24 hours of adherent growth, cells were treated with NT-LDH / EDTA dispersed in the culture medium at 200 ppm for 15 min, 30 min, and 60 min, respectively. Untreated cells served as the control group. Subsequently, detached cells and still-adhering cells were collected from the suspension, and the cell count was determined using a cell counter to calculate the proportion of detached cells.

[0103] 3.3 Experimental Results: Figure 11 and Figure 12 The figures show the proportion of detached cells, specifically bladder cancer cells T24 and colorectal cancer cells CT26, induced by the NT-LDH / EDTA material system prepared in Example 1. As can be seen from the figures, NT-LDH / EDTA can induce a large number of detached bladder cancer and colorectal cancer cells, and the detachment ratio is time-dependent. After 60 minutes of treatment, the proportion of detached cells is greater than 70%.

[0104] In summary, the NT-LDH / EDTA material system prepared in this invention exhibits good targeting properties against bladder cancer T24 cells.

[0105] Adhesion can disrupt cell connections, causing cells to detach from the cell wall.

[0106] B. In vivo evaluation experiment

[0107] 1. Evaluation of the efficacy of treatment for orthotopic bladder cancer in SD rats.

[0108] 1.1 Experimental Materials: The material systems NH2-LDH / EDTA and NT-LDH / EDTA prepared in Example 1. PBS, EDTA, N-methyl-N-nitrosourea (MNU), isoflurane, matrix metalloproteinase 9 (MMP-9), and sodium citrate buffer.

[0109] 1.2 Experimental Methods: MNU-induced orthotopic bladder cancer modeling in rats: MNU was dissolved in citrate buffer (0.1 mmol / L) to prepare a 10 g / L MNU solution. Female Sprague-Dawley (SD) rats weighing approximately 220 g were anesthetized using a 5% isoflurane gas anesthesia machine and fixed in a supine position. Anesthesia was maintained with a 1.5% isoflurane concentration. The perineum was disinfected by wiping three times with 1% benzalkonium chloride solution. Then, a lubricated catheter was slowly inserted into the urethra and then into the bladder. After the rats urinated, 0.2 mL of MNU solution was instilled into the bladder. After completion, the catheter and the external urethral orifice were clamped for 20 minutes, and then the catheter was slowly withdrawn. Instillation was performed every two weeks for a total of four times, and tumor formation was allowed for one month after the instillation was completed.

[0110] Evaluation of the efficacy of instillation therapy for bladder cancer in rats: After the modeling period, ultrasound imaging was used to detect tumor formation in the rats. Successful model rats were randomly divided into four groups (PBS, EDTA, NH2-LDH / EDTA, and NT-LDH / EDTA). Before each treatment, the rats were withheld from food and water for 12 hours. The rats were anesthetized using the above method and instilled 2 mL of PBS, 15 mg / kg EDTA, 60 mg / kg NH2-LDH / EDTA, and 60 mg / kg NT-LDH / EDTA into the bladder, respectively. The catheter and urethral orifice were then clamped and maintained for 4 hours before the catheter was slowly withdrawn. Instillation was performed once a week for a total of six times. Ultrasound imaging was performed after the third instillation and after the end of the treatment to detect changes in the bladder tumor trend. After the treatment, one rat from each of the PBS and NT-LDH / EDTA groups was randomly selected for ultrasound imaging to detect any abnormalities in the kidneys.

[0111] After the treatment cycle ended, the rats were euthanized, and the bladders of all rats were removed to observe the final tumor size and take photographs. Simultaneously, the kidneys, livers, spleens, and lungs of the PBS and NT-LDH / EDTA groups were removed to observe for metastasis.

[0112] 1.3 Experimental Results: Figure 13 To evaluate the treatment process of SD rats with orthotopic bladder cancer, ultrasound imaging was used to detect the bladder instillation of NH2-LDH / EDTA and NT-LDH / EDTA material systems prepared in Example 1 using PBS, EDTA, and materials prepared in Example 1. Figure 11 As can be seen, the tumors in the PBS group rats gradually increased in size and even filled the entire bladder. In rats treated with EDTA perfusion, the tumors shrank somewhat, but because free EDTA has no adhesion and is easily diluted and excreted by urine, it could not effectively control tumor growth, ultimately forming multiple small, diffuse tumors. In rats treated with NH2-LDH / EDTA perfusion, tumor growth was significantly inhibited; however, due to its better targeting and enrichment, NT-LDH / EDTA perfusion was more effective, and no obvious tumor area was visible on ultrasound.

[0113] Figure 14 Bladder tissue photographs were taken after bladder instillation of SD rats with orthotopic bladder cancer using PBS, EDTA, and the material systems NH2-LDH / EDTA and NT-LDH / EDTA prepared in Example 1. Figure 12 The results show consistency with ultrasound findings. Compared to other groups of rats, the bladders of rats instilled with NT-LDH / EDTA showed no visible tumor tissue, and the surface was smooth with no significant difference in appearance or color from the bladders of normal healthy rats.

[0114] Figure 15The NT-LDH / EDTA material system prepared in Example 1 was used for bladder instillation in SD rats with orthotopic bladder cancer. Renal function was evaluated and anatomical images were obtained after treatment. Figure 15 The results show that the bladder tumors in the PBS group were large, obstructing the ureters and leading to severe bilateral hydronephrosis in the later stages. In contrast, the kidneys of the NT-LDH / EDTA treatment group did not show significant abnormalities. Furthermore, in the dissected kidney images, one rat in the PBS group showed significant tumor metastasis, with upregulated expression of the tumor metastasis-related protein MMP-9. However, the rats in the NT-LDH / EDTA group did not show distant metastasis of bladder cancer.

[0115] 2. In vivo tissue toxicity test.

[0116] 2.1 Experimental materials: The material system NT-LDH / EDTA prepared in Example 1.

[0117] 2.2 Experimental Methods: Healthy female SD rats (approximately 200g in weight) were randomly divided into three groups. Two groups of rats were then intravesically instilled with a high dose of NT-LDH / EDTA (120mg / kg) as described above. The remaining group of rats served as the control group without any treatment. Rats were sacrificed on days 7 and 30, and major tissues (heart, liver, kidney, spleen, and lung) were collected for hematoxylin and eosin (H&E) staining and histological analysis.

[0118] 2.3 Experimental Results: Figure 14 Histological sections of various organs, including the heart, liver, spleen, lung, and kidney, after bladder instillation of the NT-LDH / EDTA (120 mg / kg) material system prepared in Example 1 into healthy SD rats. Figure 16 It is evident that, compared with healthy rats in the control group, no significant toxic reactions were observed in any organ (heart, liver, spleen, lungs, kidneys) after perfusion of NT-LDH / EDTA; there was neither acute stress toxicity nor long-term tissue toxicity, indicating that the material has good biocompatibility at the in vivo level.

[0119] In summary, the NT-LDH / EDTA material system provided by this invention exhibits good targeting and adhesion. After bladder instillation, it can accumulate in the tumor region, disrupting cell junctions and causing tumor cells to detach from the tissue and ultimately be excreted in the urine. Furthermore, because the material remains tightly bound to the detached cell membranes, it inhibits distant metastasis of tumor cells. In addition, this material has low toxicity and side effects, making it of significant value and importance for the development and application of bladder cancer medical treatment techniques.

[0120] Finally, it is necessary to state that the above embodiments are only used to further illustrate the technical solution of the present invention in detail, and should not be construed as limiting the scope of protection of the present invention. Any non-substantial improvements and adjustments made by those skilled in the art based on the above content of the present invention shall fall within the scope of protection of the present invention.

Claims

1. Use of an NT-LDH / EDTA layered double hydroxide material for the preparation of a medicament for the treatment of bladder cancer or colorectal cancer, characterized in that: The NT-LDH / EDTA layered double hydroxide material comprises a zinc-aluminum layered double hydroxide loaded with ethylenediaminetetraacetic acid and a neurotensin antibody NT modified on the surface thereof; the ethylenediaminetetraacetic acid is intercalated into the interlayer of the zinc-aluminum layered double hydroxide by anion exchange; and the zinc-aluminum layered double hydroxide loaded with ethylenediaminetetraacetic acid has a positive electric property on the surface.

2. Use according to claim 1, characterized in that, The content of the ethylenediaminetetraacetic acid is ≥25wt% based on 100wt% of the zinc-aluminum layered double hydroxide.

3. Use according to claim 1, characterized in that, The content of the ethylenediaminetetraacetic acid is 25wt%-40wt% based on 100wt% of the zinc-aluminum layered double hydroxide.

4. Use according to claim 1, characterized in that, The size of the LDH / EDTA layered double hydroxide material is 700-900nm.

5. The use according to claim 1, wherein the tumor comprises colon cancer and bladder cancer.

Images