Copper nanoparticles capable of regulating intestinal flora as well as preparation method and application of copper nanoparticles

Abstract

The invention relates to a copper nanoparticle capable of regulating and controlling intestinal flora and a preparation method and application thereof.The copper nanoparticle comprises a copper nanoparticle carrier and a medicine, the medicine comprises medicine active ingredients used for regulating and controlling the intestinal flora, and preparation raw materials of the copper nanoparticle carrier comprise copper salt, sodium hydroxide and hydrogen peroxide. The copper-containing component in the copper nanoparticle can consume H2S overexpressed at a tumor site in situ, and can react with H2S to generate a product with a good photothermal conversion effect in situ, and the product can mediate the photothermal conversion reaction under the irradiation of near-infrared light, so that the tumor is treated; meanwhile, the copper nanoparticles are cracked after reacting with H2S, the loaded medicine is released, and the intestinal flora is synchronously regulated and controlled.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and particularly to a copper nanoparticle capable of regulating intestinal flora, a preparation method thereof, and an application thereof. Background Art

[0002] Colorectal cancer is the most common malignant tumor in the digestive system. In normal tissues, the concentration of H2S is usually at the micromolar (μM) level, and it mainly has two major sources. Exogenous H2S is produced by H2S-producing intestinal flora such as sulfate-reducing bacteria by decomposing sulfur-containing amino acids or sulfates, while endogenous H2S is synthesized by the enzymatic reactions of cells themselves, and the main enzymes involved are cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3-MST). In patients with colorectal cancer, due to the increased abundance of H2S-producing bacteria and the overexpression of related enzymes such as CBS, the concentration of H2S at the intestinal tumor site is significantly increased, and the specific concentration varies depending on the tumor stage, location, and individual differences. Overexpressed H2S can promote the proliferation of tumor cells, induce angiogenesis, and promote tumor invasion and metastasis, playing an important role in the occurrence and development of colorectal cancer. Therefore, inhibiting the occurrence and development of tumors by consuming the overexpressed H2S at the tumor site is a potential treatment strategy.

[0003] At the same time, intestinal microorganisms play an indispensable role in the digestion, nutrition absorption, immune regulation, and metabolic regulation of the body. The homeostasis of intestinal microorganisms is of great significance for human health, and the disorder of intestinal microorganisms is closely related to the occurrence, development, and treatment of colorectal cancer. In patients with colorectal cancer, the abundance of pathogenic bacteria such as Fusobacterium nucleatum increases, which can promote the occurrence and metastasis of colorectal cancer, induce chemotherapy resistance, and reduce the efficacy of immunotherapy. The abundance of probiotics such as Lactobacillus decreases. Probiotics can often play an active role in inhibiting the progression of colorectal cancer and enhancing the anti-tumor efficacy by producing metabolites such as short-chain fatty acids. Therefore, reshaping the homeostasis of intestinal microorganisms will be beneficial to enhancing the anti-tumor efficacy and has considerable practical application prospects. Summary of the Invention

[0004] In view of the above problems, the present invention provides a copper nanoparticle capable of regulating intestinal flora. The copper-containing component in the copper nanoparticle can in-situ consume the overexpressed H2S at the tumor site and in-situ generate a product with good photothermal conversion effect by reacting with H2S, which can mediate a photothermal conversion reaction under near-infrared light irradiation to treat tumors. At the same time, the copper nanoparticle lyses after reacting with H2S and releases the loaded drug to synchronously regulate the intestinal flora.

[0005] To achieve the above object, the present invention provides a copper nanoparticle capable of regulating the intestinal flora, comprising a copper nanoparticle carrier and a drug, wherein the drug comprises a drug active ingredient for regulating the intestinal flora, and the preparation raw materials of the copper nanoparticle carrier comprise a copper salt, sodium hydroxide, and hydrogen peroxide.

[0006] Reacting with the above raw materials can, on the one hand, prepare a copper nanoparticle carrier with a mesoporous structure, facilitating the subsequent drug loading process. On the other hand, after the copper nanoparticle carrier reacts with H2S subsequently, a copper sulfide product with a photothermal effect can be smoothly 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 drug active ingredient comprises short-chain fatty acids.

[0011] In one embodiment, the drug active ingredient comprises at least one of sodium butyrate, sodium acetate, sodium propionate, and butyryl glycerol.

[0012] The present invention also provides a preparation method of the copper nanoparticle, comprising the following steps:

[0013] Preparing the copper nanoparticle carrier: mixing the copper salt, sodium hydroxide, and hydrogen peroxide evenly and standing to obtain the copper nanoparticle carrier;

[0014] Preparing the copper nanoparticle: mixing the copper nanoparticle carrier and the drug evenly and stirring overnight to obtain the copper nanoparticle.

[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 drug for consuming H2S, regulating the intestinal flora, and / or anti-tumor, comprising an active ingredient and a pharmaceutically acceptable excipient, wherein the active ingredient comprises the above-mentioned copper nanoparticle.

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

[0018] The above H2S can be produced by overexpression in the tumor site or by using NaHS in vitro. When producing H2S by NaHS in vitro, 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 / cm 2 .

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

[0021] A copper nanoparticle capable of regulating the intestinal flora, its preparation method and application thereof according to the present invention. The copper-containing component in the copper nanoparticle can in-situ consume the overexpressed H2S in the tumor site and in-situ generate a product with a good photothermal conversion effect by reacting with H2S. Under near-infrared light irradiation, it can mediate a photothermal conversion reaction, so that the temperature of the tumor site rises to achieve the purpose of thermal ablation of the tumor; at the same time, the copper nanoparticle cracks after reacting with H2S and releases the loaded drug in a responsive manner to synchronously regulate the intestinal flora. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is the particle size diagram of copper nanoparticles Cu@NaBu NPs;

[0023] Figure 2 is the transmission electron microscope (TEM) diagram of copper nanoparticles Cu@NaBu NPs;

[0024] Figure 3 is the in vitro H2S-responsive release diagram of copper nanoparticles Cu@RB NPs loaded with rhodamine B;

[0025] Figure 4 is the in vitro temperature rise diagram of copper nanoparticles Cu@NaBu NPs;

[0026] Figure 5 is the relative cell viability diagram of copper nanoparticles Cu@NaBu NPs acting on intestinal cancer cells;

[0027] Figure 6 is the in vivo intestinal flora Shannon Index diagram of copper nanoparticles Cu@NaBu NPs;

[0028] Figure 7It is the in vivo intestinal flora health index diagram of copper nanoparticles Cu@NaBu NPs;

[0029] Figure 8 It is the schematic structural diagram of copper nanoparticles Cu@NaBu NPs before and after reacting with H2S. Specific implementation manners

[0030] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below with reference to the relevant drawings. The preferred embodiments of the present invention are shown in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0032] Source:

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

[0034] Example 1

[0035] The copper nanoparticles Cu@NaBu NPs that can regulate the intestinal flora are prepared by the following method:

[0036] I. Preparation of copper nanoparticle carriers.

[0037] (1) Take 127.5 mg of CuCl2·2H2O and dissolve it in 150 mL of absolute ethanol, and stir evenly.

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

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

[0040] (4) After standing for 48 h, centrifuge the obtained precipitate, which 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 the intestinal flora.

[0043] (5) Mix the copper nanocarrier and sodium butyrate at a mass ratio of 1:1, stir for 12 h, and then dialyze in water for 12 h to remove free sodium butyrate.

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

[0045] Comparative Example 1

[0046] Copper nanoparticles loaded with rhodamine B Cu@RB NPs are used to simulate the release of sodium butyrate by loading rhodamine B. The specific preparation method is as follows:

[0047] I. Prepare the copper nanoparticle carrier.

[0048] (1) Take 127.5 mg of CuCl2·2H2O and dissolve it in 150 mL of absolute ethanol, and stir evenly.

[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) After standing for 48 h, centrifuge the obtained precipitate, which is the copper nanoparticle carrier.

[0052] II. Prepare copper nanoparticles loaded with rhodamine B.

[0053] (5) Mix the copper nanocarrier and rhodamine B at a mass ratio of 1:1, stir for 12 h, and then dialyze in water for 24 h.

[0054] (6) The obtained copper nanoparticles loaded with rhodamine B are stored at room temperature for later use.

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

[0056] Experimental Example 1

[0057] In vitro characterization.

[0058] Use the dynamic light scattering (DLS) method to measure the particle sizes of the Cu@NaBu NPs prepared in Example 1 and the nanoparticles after reaction with H2S.

[0059] The specific operation of the reaction between Cu@NaBu NPs and H2S includes: Mix Cu@NaBu NPs and NaHS with a concentration of 1.78 mmol / L in equal volume, and vortex for 5 min. NaHS can rapidly dissolve and release H2S by itself.

[0060] The detection results are as Figure 1 、Figure 2 As shown Figure 1 It shows that the average particle size in Example 1 is 287.5 nm, and the average particle size after the reaction is 123 nm. Figure 2 Indicating similar results Figure 2 The left figure of... is the copper nanoparticles before the reaction Figure 2 The right figure of... is the copper nanoparticles after the reaction. After the reaction, the copper nanoparticles are cracked and reorganized. The above results all illustrate that copper nanoparticles can react with H2S and consume H2S. The schematic structural diagrams of Example 1 of the present invention before and after the reaction with H2S are as Figure 8 shown

[0061] Experimental Example 2

[0062] In vitro release test

[0063] Take 1 mL of the copper nanoparticles loaded with rhodamine B prepared in Comparative Example 1 and 1 mL of the product after the reaction of Comparative Example 1 with H2S, respectively, put them into a dialysis bag (MWCO: 3500 Da), immerse them in 50 mL of PBS solution with pH = 7.4, and stir magnetically at 100 r / min. And take out 1 mL of the release solution at time points 0.5, 1, 2, 4, 8, 12, 24 h, and replenish 1 mL of the release solution. Measure the absorption peak at 560 nm by an ultraviolet absorption spectrometer, and calculate the drug content in the release solution according to the standard curve.

[0064] Figure 3 It is indicated that the release before the reaction of Comparative Example 1 with H2S is relatively slow, and the drug is released rapidly after the reaction of Comparative Example 1 with H2S.

[0065] Experimental Example 3

[0066] In vitro heating test

[0067] Respectively take 0.5 mL of Example 1 and the product after the reaction of Example 1 with H2S, irradiate with near-infrared light (1064 nm, 1 Wcm -2 ) for 10 min, and record the temperature change situation by an infrared thermal imager. The results are shown in Figure 4 .

[0068] Figure 4 It shows that Example 1 has no photothermal conversion ability, and the product after the reaction of Example 1 with H2S has a significant photothermal effect.

[0069] Experimental Example 4

[0070] In vitro cytotoxicity test

[0071] Dilute Example 1 and the product after the reaction of Example 1 with H2S to the required concentration with cell culture medium respectively, add 100 μL and act on intestinal cancer cells (CT26). The group with near-infrared light irradiation is irradiated with near-infrared light (1064 nm, 1 W / cm once2 , 1 min), the MTT assay was performed, and the test results are shown in Figure 5 .

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

[0073] Experimental Example 5

[0074] Detection of intestinal flora in vivo.

[0075] A mouse orthotopic intestinal cancer model was established using conventional methods. After successful modeling, normal saline, Example 1, and Example 1 combined with near-infrared light irradiation (1064 nm, 1 W / cm 2 ) were used for treatment. After 14 days of treatment, the mice to be sampled were placed in a clean cage lined with sterile filter paper. After the mice defecated, fecal samples were immediately collected, and new filter paper was used for sampling different mice. The samples were stored and transported using dry ice and tested on the machine. The results are shown in Figure 6 .

[0076] Figure 6 The result is the Shannon Index of the flora. The higher the value, the higher the species diversity, that is, the higher the richness and the more uniform the distribution. The species diversity of the mice in the PBS group decreased, and the flora diversity was effectively improved and restored after treatment, indicating enhanced stability and significant improvement in flora health.

[0077] Figure 7 It shows that the flora health index of the mice in the PBS treatment group is negative, representing flora imbalance and being in a diseased state. The flora health index of the mice treated with Example 1 changed from negative to positive, and there was a significant difference between the two, indicating that the intestinal flora of the mice was close to the healthy state after treatment with Example 1, and the flora disorder was effectively improved.

[0078] In summary, the copper nanoparticles of the present invention can consume overexpressed H2S in tumors to achieve the purpose of reducing H2S to inhibit tumor progression. Moreover, after the copper nanoparticles of the present invention react with H2S, a photothermal effect product CuS is generated in situ, which can kill tumor cells by heating under laser irradiation. At the same time, the copper nanoparticles of the present invention can achieve H2S-induced responsive drug release, improving the targeting of drugs. The sodium butyrate released by the copper nanoparticles can regulate the intestinal flora to make it closer to the healthy state and reshape the intestinal microecological balance.

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

[0080] The above-described embodiments merely represent several implementation manners of the present invention. The description thereof is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. A copper nanoparticle capable of regulating intestinal flora, characterized in that: The invention comprises a copper nanoparticle carrier and a drug, wherein the drug comprises a drug active ingredient for regulating intestinal flora, and raw materials for preparing the copper nanoparticle carrier comprise copper salt, sodium hydroxide and hydrogen peroxide.

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; the molar ratio of the copper salt, sodium hydroxide and hydrogen peroxide is (0.5-1.5):(0.75-1.25):(6-10).

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 any one of claims 1 to 4, characterized in that The pharmaceutically active ingredients include short-chain fatty acids.

6. The copper nanoparticles according to claim 5, characterized in that The active pharmaceutical ingredient comprises at least one of sodium butyrate, sodium acetate, sodium propionate and butyric acid glyceride.

7. The method for preparing copper nanoparticles according to any one of claims 1 to 6, characterized in that: The following steps are involved: Preparation of copper nanoparticle carrier: copper salt, sodium hydroxide and hydrogen peroxide are uniformly mixed and allowed to stand to obtain copper nanoparticle carrier; Preparation of copper nanoparticles: The copper nanoparticle carrier and the drug are mixed evenly, and stirred overnight to obtain copper nanoparticles.

8. The preparation method according to claim 7, characterized in that: The molar ratio of the copper salt, sodium hydroxide and hydrogen peroxide is (0.5-1.5):(0.75-1.25):(6-10).

9. A drug for consuming H2S, regulating intestinal flora and / or anti-tumor, characterized in that: The invention comprises an active ingredient and a pharmaceutically acceptable auxiliary material, wherein the active ingredient comprises the copper nanoparticles according to any one of claims 1 to 6.

10. The method for using the copper nanoparticles for non-therapeutic purposes according to any one of claims 1 to 6, characterized in that: The following steps are involved: The copper nanoparticles are used to consume H2S to generate products with photothermal effect. The copper nanoparticles are cracked to release drugs for regulating intestinal flora. Near-infrared light is used to irradiate the products to trigger photothermal conversion reaction.

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