Copper nanoparticles for regulating intestinal flora, and preparation method and application thereof

Abstract

The present application relates to a kind of copper nanoparticles that can regulate intestinal flora and its preparation method and application, and the copper nanoparticles include copper nanoparticle carrier and drug, drug includes the active ingredient for regulating intestinal flora, the preparation raw material of copper nanoparticle carrier includes copper salt, sodium hydroxide, hydrogen peroxide.The copper component in the copper nanoparticles can in situ consume the H2S overexpressed in tumor site, and in situ product with good photo-thermal conversion effect is generated by the reaction with H2S, can mediate photo-thermal conversion reaction under near-infrared light, to treat tumor;Meanwhile, the copper nanoparticles are cleaved after reaction with H2S, and the drug loaded is released, and intestinal flora is regulated simultaneously.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to a copper nanoparticle that can regulate gut microbiota, its preparation method, and its application. Background Technology

[0002] Colorectal cancer is the most common malignant tumor of the digestive system. In normal tissues, H2S concentration is typically at the micromolar (μM) level, primarily originating from two sources: exogenous H2S produced by H2S-producing gut microbiota, such as sulfate-reducing bacteria, through the breakdown of sulfur-containing amino acids or sulfates; and endogenous H2S synthesized by the cell's own enzymatic reactions, mainly involving enzymes such as cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE), and 3-mercaptopyruvate-thiotransferase (3-MST). In colorectal cancer patients, the increased abundance of H2S-producing bacteria and overexpression of CBS and related enzymes lead to a significantly elevated H2S concentration at the intestinal tumor site, with the specific concentration varying depending on the tumor stage, location, and individual differences. Overexpressed H2S can promote tumor cell proliferation, induce angiogenesis, and promote tumor invasion and metastasis, playing a crucial role in the development and progression of colorectal cancer. Therefore, inhibiting tumor development and progression by depleting overexpressed H2S at the tumor site is a promising therapeutic strategy.

[0003] Meanwhile, gut microbiota play an indispensable role in the body's digestion and nutrient absorption, immune regulation, and metabolic control. Gut microbial homeostasis is of great significance for human health, and gut microbial dysbiosis is closely related to the occurrence, development, and treatment of colorectal cancer. In colorectal cancer patients, an increased abundance of pathogenic bacteria such as nucleic acid-producing bacilli can promote the occurrence and metastasis of colorectal cancer, induce chemotherapy resistance, and reduce the efficacy of immunotherapy. Conversely, a decreased abundance of beneficial bacteria such as lactobacilli can play a positive role in inhibiting the progression of colorectal cancer and enhancing anti-tumor efficacy by producing metabolites such as short-chain fatty acids. Therefore, reshaping gut microbial homeostasis will be beneficial for enhancing anti-tumor efficacy and has considerable practical application prospects. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides copper nanoparticles that can regulate gut microbiota. The copper-containing component in these nanoparticles can in situ consume H2S overexpressed at tumor sites and generate products with good photothermal conversion effects in situ through reaction with H2S. Under near-infrared light irradiation, these products can mediate photothermal conversion reactions, thereby treating tumors. Simultaneously, the copper nanoparticles lyse after reacting with H2S, releasing the loaded drug and synchronously regulating gut microbiota.

[0005] To achieve the above objectives, the present invention provides copper nanoparticles that can regulate gut microbiota, comprising a copper nanoparticle carrier and a drug, wherein the drug comprises a pharmaceutically active ingredient for regulating gut microbiota, and the raw materials for preparing the copper nanoparticle carrier include copper salt, sodium hydroxide, and hydrogen peroxide.

[0006] Using the above-mentioned raw materials for reaction, on the one hand, copper nanoparticle carriers with mesoporous structures can be prepared, which facilitates subsequent drug loading processes. On the other hand, after the copper nanoparticle carriers react with H2S, copper sulfide products with photothermal effects can be successfully generated.

[0007] In one embodiment, the mass ratio of the copper nanoparticle carrier to the drug is 1:(0.5-1.5).

[0008] In one embodiment, the copper salt comprises copper chloride; the molar ratio of the copper salt, sodium hydroxide, and hydrogen peroxide is (0.5-1.5):(0.75-1.25):(6-10).

[0009] In one embodiment, the copper nanoparticle carrier has a mesoporous structure.

[0010] In one embodiment, the active pharmaceutical ingredient includes short-chain fatty acids.

[0011] In one embodiment, the active pharmaceutical ingredient includes at least one of sodium butyrate, sodium acetate, sodium propionate, and glyceryl butyrate.

[0012] The present invention also provides a method for preparing the copper nanoparticles, comprising the following steps:

[0013] Preparation of copper nanoparticle support: copper salt, sodium hydroxide and hydrogen peroxide are mixed evenly and allowed to stand to obtain copper nanoparticle support;

[0014] Preparation of copper nanoparticles: The copper nanoparticle carrier and the drug were mixed evenly and stirred overnight to obtain copper nanoparticles.

[0015] In one embodiment, the molar ratio of the copper salt, sodium hydroxide, and hydrogen peroxide is (0.5-1.5):(0.75-1.25):(6-10).

[0016] The present invention also provides a medicament for consuming H2S, regulating gut microbiota and / or fighting tumors, comprising an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient comprises the copper nanoparticles.

[0017] The present invention also provides a method for using the copper nanoparticles for non-therapeutic purposes, comprising the following steps: using the copper nanoparticles to consume H2S to generate a product with a photothermal effect; the copper nanoparticles lyse to release a drug for regulating intestinal flora; and irradiating the product with near-infrared light to trigger a photothermal conversion reaction.

[0018] The H2S mentioned above can be produced by overexpression at the tumor site or by in vitro production using NaHS. When H2S is produced in vitro using NaHS, the concentration of NaHS is 0.3-3.4 mmol / L.

[0019] In one embodiment, the wavelength of the near-infrared light is 808-1064 nm, and the power density of the near-infrared light is 0.5-1.5 W / cm². 2 .

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] This invention discloses a copper nanoparticle capable of regulating gut microbiota, its preparation method, and its application. The copper-containing component in the copper nanoparticle can in situ consume H2S overexpressed at the tumor site and generate a product with good photothermal conversion effect in situ through reaction with H2S. Under near-infrared light irradiation, it can mediate the photothermal conversion reaction, thereby increasing the temperature of the tumor site to achieve the purpose of thermal ablation of the tumor. At the same time, the copper nanoparticle cleaves after reacting with H2S, responsively releasing the loaded drug and simultaneously regulating the gut microbiota. Attached Figure Description

[0022] Figure 1 The particle size distribution of copper nanoparticles Cu@NaBu NPs is shown.

[0023] Figure 2 Transmission electron microscopy (TEM) image of copper nanoparticles Cu@NaBu NPs;

[0024] Figure 3 The in vitro H2S response release diagram of copper nanoparticles (Cu@RB NPs) loaded with rhodamine B;

[0025] Figure 4 The in vitro temperature rise diagram of copper nanoparticles Cu@NaBu NPs is shown.

[0026] Figure 5 The graph shows the relative cell activity of copper nanoparticles Cu@NaBu NPs against colorectal cancer cells.

[0027] Figure 6 Shannon Index diagram of gut microbiota in Cu@NaBu NPs;

[0028] Figure 7A graph showing the gut microbiota health index of copper nanoparticles Cu@NaBu NPs in vivo;

[0029] Figure 8 This is a schematic diagram of the structure of copper nanoparticles Cu@NaBu NPs before and after the reaction with H2S. Detailed Implementation

[0030] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0032] source:

[0033] Unless otherwise specified, all reagents, materials, and equipment used in this embodiment are commercially available; unless otherwise specified, all experimental methods are conventional experimental methods in this field.

[0034] Example 1

[0035] Copper nanoparticles (Cu@NaBu NPs) capable of regulating gut microbiota were prepared by the following method:

[0036] I. Preparation of copper nanoparticle carriers.

[0037] (1) Dissolve 127.5 mg CuCl2·2H2O in 150 mL of anhydrous ethanol and stir until homogeneous.

[0038] (2) Slowly and evenly add 1 mL of NaOH (40 mg / mL).

[0039] (3) Quickly and evenly add 1 mL of H2O2 (30%).

[0040] (4) The precipitate obtained by centrifugation after standing for 48 hours is the copper nanoparticle carrier.

[0041] In this embodiment, the molar ratio of copper salt, sodium hydroxide, and hydrogen peroxide is 1:1:8.

[0042] II. Preparation of copper nanoparticles that can regulate gut microbiota.

[0043] (5) After mixing the copper nanocarrier with sodium butyrate at a mass ratio of 1:1 for 12 hours, dialyze in water for 12 hours to remove free sodium butyrate.

[0044] (6) The obtained controllable gut microbiota copper nanoparticles Cu@NaBu NPs were stored at room temperature for later use.

[0045] Comparative Example 1

[0046] Rhodamine B-loaded copper nanoparticles (Cu@RB NPs) were used to simulate the release of sodium butyrate by loading rhodamine B. The specific preparation method is as follows:

[0047] I. Preparation of copper nanoparticle carriers.

[0048] (1) Dissolve 127.5 mg CuCl2·2H2O in 150 mL of anhydrous ethanol and stir until homogeneous.

[0049] (2) Slowly and evenly add 1 mL of NaOH (40 mg / mL).

[0050] (3) Quickly and evenly add 1 mL of H2O2 (30%).

[0051] (4) The precipitate obtained by centrifugation after standing for 48 hours is the copper nanoparticle carrier.

[0052] II. Preparation of Rhodamine B-loaded copper nanoparticles.

[0053] (5) After mixing copper nanocarriers with Rhodamine B at a mass ratio of 1:1 for 12 hours, the mixture was dialyzed in water for 24 hours.

[0054] (6) The obtained Rhodamine B copper nanoparticles were stored at room temperature for later use.

[0055] Unless otherwise specified, the controllable gut microbiota copper nanoparticles Cu@NaBu NPs used in the following experimental examples were all prepared in Example 1.

[0056] Experimental Example 1

[0057] In vitro characterization.

[0058] The particle size of Cu@NaBu NPs prepared in Example 1 and the nanoparticles after reacting with H2S were determined by dynamic light scattering (DLS).

[0059] The specific procedure for the reaction of Cu@NaBu NPs with H2S includes: mixing Cu@NaBu NPs with an equal volume of NaHS at a concentration of 1.78 mmol / L, and then vortexing for 5 minutes. NaHS itself can quickly dissolve and release H2S.

[0060] Test results as follows Figure 1 , Figure 2 As shown, Figure 1 The average particle size of Example 1 was 287.5 nm, and the average particle size after the reaction was 123 nm. Figure 2 Prompt similar results, Figure 2 The left image shows copper nanoparticles before the reaction. Figure 2 The right figure shows the copper nanoparticles after the reaction, which exhibit fragmentation and reorganization. These results demonstrate that copper nanoparticles can react with H₂S, consuming H₂S. The structural diagrams of Example 1 of this invention before and after the reaction with H₂S are shown below. Figure 8 As shown.

[0061] Experiment Example 2

[0062] In vitro release test.

[0063] One mL of the rhodamine B copper nanoparticles prepared in Comparative Example 1 and one mL of the product from the reaction of Comparative Example 1 with H2S were placed in dialysis bags (MWCO: 3500 Da) and immersed in 50 mL of pH 7.4 PBS solution with magnetic stirring at 100 r / min. One mL of the release solution was collected at time points of 0.5, 1, 2, 4, 8, 12, and 24 h, and 1 mL of the release solution was added back. The absorption peak at 560 nm was measured using a UV-Vis absorption spectrometer, and the drug content in the release solution was calculated based on the standard curve.

[0064] Figure 3 The results indicate that the release of the drug in Comparative Example 1 was relatively slow before reacting with H2S, but rapid after reacting with H2S.

[0065] Experimental Example 3

[0066] In vitro temperature rise test.

[0067] Take 0.5 mL of each of Example 1 and the product of the reaction between Example 1 and H2S, and irradiate them with near-infrared light (1064 nm, 1 W cm⁻¹). -2 Temperature changes were recorded using an infrared thermal imager over 10 minutes. The results are shown below. Figure 4 .

[0068] Figure 4 This indicates that Example 1 has no photothermal conversion capability, while the product of Example 1 after reacting with H2S has a significant photothermal effect.

[0069] Experiment Example 4

[0070] In vitro cytotoxicity assay.

[0071] The products from the reaction of Example 1 and Example 1 with H2S were diluted to the required concentrations with cell culture medium, and 100 μL was added dropwise to each product to act on colon cancer cells (CT26). Each product was then subjected to one near-infrared light irradiation (1064 nm, 1 W / cm²).2 MTT assay was performed (1 min), and the results are shown in [the table]. Figure 5 .

[0072] from Figure 5 It can be seen that the copper nanoparticles synthesized in Example 1 are weakly toxic, but their toxicity increases after reacting with H2S. After being irradiated with near-infrared light, their killing effect on colon cancer cells is further enhanced.

[0073] Experimental Example 5

[0074] In vivo gut microbiota testing.

[0075] A mouse orthotopic colon cancer model was established using existing conventional methods. After successful modeling, mice were administered physiological saline, Example 1, and Example 1 combined with near-infrared light irradiation (1064nm, 1W / cm²). 2 After 14 days of treatment, mice were placed in clean cages lined with sterile filter paper. Fecal samples were collected immediately after defecation, and new filter paper was used for each mouse sample. Dry ice was used for preservation and transportation before instrumental testing. Results are shown below. Figure 6 .

[0076] Figure 6 The result is the Shannon Index of the gut microbiota. The higher the value, the higher the species diversity, that is, the richness and even distribution. The species diversity of mice in the PBS group was reduced. After treatment, the gut microbiota diversity was effectively improved and restored, indicating enhanced stability and significantly improved gut health.

[0077] Figure 7 The results showed that the gut microbiota health index of mice in the PBS treatment group was negative, indicating a gut microbiota imbalance and a disease state. After treatment with Example 1, the gut microbiota health index of mice changed from negative to positive, showing a significant difference between the two groups. This indicates that after treatment with Example 1, the gut microbiota of mice was close to a healthy state, and the gut microbiota disorder was effectively improved.

[0078] In summary, the copper nanoparticles of this invention can consume overexpressed H2S in tumors, thereby reducing H2S and inhibiting tumor progression. Furthermore, after reacting with H2S, the copper nanoparticles of this invention generate CuS, a product with photothermal effects, in situ, which can kill tumor cells under laser irradiation. Simultaneously, the copper nanoparticles of this invention can achieve H2S-induced responsive drug release, improving drug targeting. The sodium butyrate released by the copper nanoparticles can regulate the gut microbiota, bringing it closer to a healthy state and reshaping the gut microecological balance.

[0079] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0080] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A copper nanoparticle capable of regulating gut microbiota, characterized in that, The invention includes copper nanoparticle carriers and drugs, wherein the drugs include pharmaceutically active ingredients for regulating gut microbiota, and the pharmaceutically active ingredients include short-chain fatty acids; The raw materials for preparing the copper nanoparticle carrier include copper salt, sodium hydroxide, and hydrogen peroxide; the molar ratio of copper salt, sodium hydroxide, and hydrogen peroxide is (0.5-1.5):(0.75-1.25):(6-10). The preparation method of the copper nanoparticles includes the following steps: Preparation of copper nanoparticle support: copper salt, sodium hydroxide and hydrogen peroxide are mixed evenly and allowed to stand to obtain copper nanoparticle support; Preparation of copper nanoparticles: The copper nanoparticle carrier and the drug were mixed evenly and stirred overnight to obtain copper nanoparticles.

2. The copper nanoparticles according to claim 1, characterized in that, The mass ratio of the copper nanoparticle carrier to the drug is 1:(0.5-1.5).

3. The copper nanoparticles according to claim 1, characterized in that, The copper salt includes copper chloride.

4. The copper nanoparticles according to claim 1, characterized in that, The copper nanoparticle carrier has a mesoporous structure.

5. The copper nanoparticles according to claim 1, characterized in that, The active pharmaceutical ingredient includes at least one of sodium butyrate, sodium acetate, sodium propionate, and glyceryl butyrate.

6. The method for preparing copper nanoparticles according to any one of claims 1-5, characterized in that, Includes the following steps: Preparation of copper nanoparticle carrier: copper salt, sodium hydroxide, and hydrogen peroxide are mixed evenly and allowed to stand to obtain copper nanoparticle carrier; the molar ratio of copper salt, sodium hydroxide, and hydrogen peroxide is (0.5-1.5):(0.75-1.25):(6-10). Preparation of copper nanoparticles: The copper nanoparticle carrier and the drug were mixed evenly and stirred overnight to obtain copper nanoparticles.

7. A drug for consuming H2S, regulating gut microbiota, and / or fighting tumors, characterized in that, It includes an active ingredient and pharmaceutically acceptable excipients, said active ingredient including copper nanoparticles according to any one of claims 1-5.

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