Nano-drug as well as preparation method and application thereof

Abstract

The invention relates to the technical field of drug preparation, and discloses a nano-drug, the nano-drug is prepared by loading a mixed drug in a hollow drug-loaded nano-material, and the mixed drug comprises osimertinib and ginsenoside; the invention also discloses an application of the nano-drug in antitumor drugs. According to the invention, a hollow structure of the hollow nano-material is utilized, and two drugs are loaded by using a small amount of materials to synthesize the nano-drug with an excellent treatment effect, so that the anti-cancer drug can be transported to a tumor site in a targeted manner to play a combined chemotherapy role, and the hollow drug-loaded nano-material can release manganese ions at the tumor site, so that the treatment effect is improved. And chemical kinetics and chemotherapy are combined to treat tumors, so that the use of other materials is effectively reduced, and a very good treatment effect is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of drug preparation, and in particular to a nano drug and a preparation method and application thereof. Background Art

[0002] Osimertinib is a third-generation EGFR tyrosine kinase inhibitor (EGFR-TKI) designated as first-line treatment for patients with advanced non-small cell lung cancer (NSCLC) with sensitive EGFR mutations, as well as second-line treatment for patients with resistance-associated mutations T790M after previous EGFR-TKI treatment, and as adjuvant therapy for patients with early resected NSCLC with EGFR mutations. Despite the durable responses in patients with advanced NSCLC, similar to other targeted therapies, the inevitable resistance to EGFR-TKIs greatly limits the clinical efficacy of patients with EGFR activating mutants. Therefore, it is necessary to find more concise and efficient methods to treat tumors. Currently, combining chemotherapy with other therapies such as chemodynamics, photodynamics, and sonodynamics has become a research hotspot in tumor treatment. Summary of the invention

[0003] In view of this, the present invention provides a nano drug to solve the problems of poor water solubility, low bioavailability and poor clinical efficacy of the existing anticancer drug osimertinib.

[0004] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0005] In a first aspect of the present invention, the present invention provides a nano drug, wherein the nano drug is prepared by loading a mixed drug in a hollow drug-carrying nano material, and the mixed drug includes osimertinib and ginsenoside.

[0006] Preferably, the mass ratio of the mixed drug to the hollow drug-loaded nanomaterial is 1:(0.1-1).

[0007] Preferably, the mass ratio of the mixed drug to the hollow drug-loaded nanomaterial is 1:0.2.

[0008] Preferably, the mass ratio of osimertinib to ginsenoside is (0.5-1.5):1.

[0009] Preferably, the mass ratio of osimertinib to ginsenoside is 1:1.

[0010] Preferably, the hollow drug-loaded nanomaterial is hollow manganese dioxide nanoparticles.

[0011] Preferably, the ginsenoside is ginsenoside Rg3.

[0012] In a second aspect of the present invention, the present invention provides a method for preparing the nanomedicine described in any one of the above items, comprising the following steps: uniformly mixing the hollow drug-loaded nanomaterial solution, the osimertinib solution, and the ginsenoside solution to obtain the nanomedicine.

[0013] Preferably, the osimertinib solution is obtained by dissolving osimertinib in a mixed solvent, and the ginsenoside solution is obtained by dissolving ginsenoside in a mixed solvent.

[0014] Preferably, the mixed solvent consists of dimethyl sulfoxide and water, and dimethyl sulfoxide accounts for 0.1-10% of the volume of the mixed solvent.

[0015] Preferably, the dimethyl sulfoxide accounts for 0.1-10% of the volume of the mixed solvent.

[0016] Preferably, the mixing is performed under light-proof conditions.

[0017] In the third aspect of the present invention, there is provided a use of the nanomedicine described in any one of the above items and / or the nanomedicine prepared by any one of the above methods in anti-tumor drugs.

[0018] Preferably, the anti-tumor drug includes a drug for treating or adjuvant treating non-small cell lung cancer.

[0019] The present invention provides a nano drug, which has the following beneficial effects compared with the prior art:

[0020] The present invention discloses a nano drug for treating tumors with simple preparation and outstanding efficacy. Osimertinib and ginsenoside Rg3 are loaded into a hollow manganese dioxide nano material. By selecting a hollow manganese dioxide with high safety and high bioavailability and capable of loading two drugs, glutathione (GSH) overexpressed in the tumor microenvironment can react with the hollow manganese dioxide as a reaction substrate to release Mn 2+ It can not only activate STING / STAT3 to reduce osimertinib resistance, but also react with overexpressed hydrogen peroxide (H2O2) in the tumor to produce hydroxyl radicals (·OH) to kill cancer cells. At the same time, ginsenoside Rg3 works together to alleviate osimertinib resistance, and chemotherapy synergizes with chemodynamic therapy to exert anti-tumor effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0022] Figure 1 This is a transmission electron microscope image (TEM image) obtained by the embodiment of the present invention;

[0023] Figure 2 The particle size diagram of HMnO2@OSI-Rg3 and HMnO2 prepared in the embodiment of the present invention;

[0024] Figure 3 This is a schematic diagram of the results of the cytotoxicity experiment of the embodiment of the present invention;

[0025] Figure 4 This is a schematic diagram of the results of a cell uptake experiment in an embodiment of the present invention;

[0026] Figure 5 Schematic diagram of live staining experimental results according to an embodiment of the present invention. DETAILED DESCRIPTION

[0027] Embodiments of the present invention are described in detail below, and the examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals represent the same or similar elements or elements with the same or similar functions from beginning to end. The embodiments described below by reference to the accompanying drawings are exemplary, are only used to explain the present invention, and are not to be construed as limitations of the present invention. In the embodiments, if specific techniques or conditions are not indicated, the techniques or conditions described in the literature in this area or the product specifications are carried out. The reagents used or the instruments that are not indicated by the manufacturer are all conventional products that can be obtained commercially.

[0028] In one aspect of the present invention, the present invention provides a method for preparing a nano drug, comprising the following steps: uniformly mixing a hollow drug-loaded nano material solution, an osimertinib solution, and a ginsenoside solution to obtain a nano drug.

[0029] In some embodiments of the present invention, the hollow drug-loaded nanomaterial is a hollow manganese dioxide nanoparticle. The SiO2 in the MnO2-wrapped SiO2 nanoparticles is etched using the "silicon dioxide template-assisted etching" method to obtain MnO2 nanoparticles with hollow interior, which are the hollow drug-loaded nanomaterial.

[0030] Specifically, the preparation method of the hollow drug-loaded nanomaterial is as follows: adding a manganese source to a water suspension of SiO2 nanoparticles to disperse uniformly to form a mixed solution, adding polyallylamine hydrochloride to the mixed solution, and obtaining MnO2-wrapped SiO2 nanoparticles through a light-proof reaction; adding the MnO2-wrapped SiO2 nanoparticles to a Na2CO3 aqueous solution with a molar concentration of 0.5-4M, and performing an etching reaction at 60°C-85°C to remove SiO2 in the nanoparticles, thereby obtaining MnO2 nanoparticles with hollow interiors. Wherein, the manganese source is KMnO4 or MnSO4, and the SiO2 nanoparticles are obtained by hydrolyzing ethyl orthosilicate in ethanol through ammonia catalysis.

[0031] In the present invention, hollow manganese dioxide has a high loading capacity, which can not only degrade GSH to produce manganese ions to activate the stimulator of interferon genes (STING), and synergistically inhibit transcription activator 3 (STAT3) with ginsenoside Rg3 to reduce osimertinib resistance, but also decompose H2O2 to produce reactive oxygen species, thereby realizing chemodynamic therapy and synergistic chemotherapy to treat tumors.

[0032] In some embodiments of the present invention, the osimertinib solution is obtained by dissolving osimertinib in dimethyl sulfoxide, and the ginsenoside solution is obtained by dissolving ginsenoside Rg3 in dimethyl sulfoxide; preferably, the dimethyl sulfoxide accounts for 0.1-10% of the volume of the mixed solvent; more preferably, the dimethyl sulfoxide accounts for 0.1-2% of the volume of the mixed solvent, for example, it can be 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 1.5%, 2%, etc. By limiting the amount of dimethyl sulfoxide added, not only can osimertinib and ginsenoside Rg3 be fully dissolved, but also it can be ensured that the test results will not be affected by the toxicity of dimethyl sulfoxide.

[0033] Furthermore, the mass ratio of the sum of the masses of osimertinib and ginsenoside to the mass ratio of the hollow drug-loaded nanomaterial is 1:(0.1-1), preferably 1:(0.1-0.5), for example, it can be 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, etc.

[0034] Further, the mass ratio of osimertinib to ginsenoside is (0.5-1.5):1, preferably (0.8-1.12):1, and more preferably 1:1. Further, in the nanomedicine, the hollow drug-loaded nanomaterial.

[0035] In some embodiments of the present invention, the ginsenoside is ginsenoside Rg3, which has multiple activities such as anti-tumor and anti-inflammatory, and can assist in the treatment of cancer, improving the efficacy through synergistic effects. Ginsenoside Rg3 can not only directly inhibit STAT3 itself, but also inhibit STAT3 by inhibiting interleukin-6 (IL-6), achieving the effect of dual inhibition of STAT3, greatly reducing osimertinib resistance.

[0036] In some embodiments of the present invention, the mixing is carried out under light-proof conditions for 12-24 hours, so that the mixing is more uniform and the ginsenoside Rg3 can continue to maintain a high bioavailability.

[0037] Furthermore, after the hollow drug-loaded nanomaterial solution, osimertinib solution, and ginsenoside solution are uniformly mixed, the method further includes: centrifuging at 3500-4000 rpm for 5 minutes, and then washing with polybutylene succinate for several times to obtain the nano drug HMnO2@OSI-Rg3.

[0038] In another aspect of the present invention, the present invention provides a nano drug, which is prepared by the above-mentioned method. Therefore, the nano drug has all the characteristics and advantages of the above-mentioned method, which will not be repeated here.

[0039] The osimertinib-ginsenoside Rg3@hollow manganese dioxide (HMnO2@OSI-Rg3) nanomedicine of the present invention utilizes the hollow structure of hollow nanomaterials and uses less material to load two drugs to synthesize a nanomedicine with excellent therapeutic effect. It can not only transport anticancer drugs to the tumor site in a targeted manner to exert a combined chemotherapy effect, but also enable the hollow manganese dioxide to release Mn at the tumor site. 2+ , exerting chemical dynamics to coordinate chemotherapy in treating tumors, effectively reducing the use of other materials and achieving good therapeutic effects.

[0040] In another aspect of the present invention, there is provided a use of the nanomedicine described in any one of the above items and / or the nanomedicine prepared by any one of the above methods in anti-tumor drugs; preferably, the anti-tumor drug includes a drug for the treatment or adjuvant treatment of non-small cell lung cancer.

[0041] The poor water solubility and low accumulation of anticancer drugs osimertinib and ginsenoside Rg3 in tumors lead to low bioavailability and poor efficacy; while hollow manganese dioxide kills tumor cells, normal cells around the tissue will also be damaged, and the dose of hollow manganese dioxide nanoparticles also affects normal cells. Based on this, the present invention provides a combined chemotherapy-coordinated chemokinetic treatment method based on osimertinib and ginsenoside Rg3, utilizing the high loading capacity of hollow manganese dioxide to load osimertinib and ginsenoside Rg3 into hollow manganese dioxide, adding chemokinetic therapy on the basis of the combined chemotherapy efficacy, so that the drug is effectively gathered at the tumor site, the poor water solubility of the drug is alleviated, and the bioavailability is improved, while the dose of hollow manganese dioxide is also reduced, and the combined treatment of non-small cell lung cancer is achieved through combined chemotherapy and chemokinetic therapy.

[0042] The present invention will be described below by specific examples, and it will be appreciated by those skilled in the art that the following specific examples are only for illustrative purposes, and do not limit the scope of the present invention in any way. In addition, in the following examples, unless otherwise specified, the reagents and equipment used are all commercially available. If in the following examples, specific treatment conditions and treatment methods are not clearly described, then conditions and methods known in the art can be used to process.

[0043] The hollow drug-loaded nanomaterials involved in the following embodiments of the present invention are prepared by the following method:

[0044] S1: Preparation of SiO2 nanoparticles: Take 1mL of tetraethyl orthosilicate (TEOS) and slowly add it dropwise into 10mL of deionized water containing 1.25mL EtOH and 0.32mL NH3·H2O (25%), and then stir at 100-1500r / min at room temperature for 72 hours; after the reaction is completed, the above reactants are centrifuged and washed three times with EtOH and water respectively, and the resulting precipitate is SiO2 nanoparticles. The SiO2 nanoparticles are resuspended in deionized water and stored for later use.

[0045] S2: Preparation of SiO2@MnO2 nanoparticles

[0046] An aqueous solution containing 50 mg KMnO4 was added to an aqueous suspension containing 6.5 mg SiO2 nanoparticles, and then added dropwise to a polyallylamine hydrochloride (PAH) aqueous solution (10-50 M) after ultrasonic mixing. The dropping was stopped when the reaction solution turned light yellow, and stirring was continued for 24 hours in a dark environment to obtain MnO2-coated SiO2 nanoparticles, which were recorded as SiO2@MnO2. PAH was positively charged in aqueous solution, and the amino group on PAH was connected to the negatively charged SiO2 surface. At the same time, PAH, as a reducing agent, reduced KMnO4 to MnO2, so that MnO2 was coated on the SiO2 surface, and finally MnO2-coated SiO2 nanoparticles, namely SiO2@MnO2 nanoparticles, were obtained.

[0047] S3: Preparation of hollow MnO2 nanoparticles

[0048] Take 20 mg of nanoparticles SiO2@MnO2 and add them to a Na2CO3 (0.5-4M) aqueous solution. Stir continuously at 60℃-85℃ for 12 hours to remove the silica template. After the reaction is completed, the product is centrifuged and washed three times with deionized water to obtain hollow metal oxide HMnO2 with nanozyme activity, that is, MnO2 nanoparticles with hollow interior, that is, hollow drug-loaded nanomaterials.

[0049] Example 1

[0050] This embodiment provides a method for preparing a nano drug, the steps are as follows:

[0051] (1) Weigh 1 mg of osimertinib and 1 mg of ginsenoside Rg3, respectively, and dissolve them in 20 μl of dimethyl sulfoxide until they are completely dissolved to prepare 50 mg / ml osimertinib solution and 50 mg / ml ginsenoside Rg3 solution for later use;

[0052] (2) dispersing 1 mg of the hollow drug-loaded nanomaterial in water to form a hollow manganese dioxide solution with a concentration of 1 mg / mL;

[0053] (3) Under ultrasonic conditions, the osimertinib solution and ginsenoside Rg3 solution prepared in step (1) were added to the hollow manganese dioxide solution, mixed on a rotary mixer in the dark for 12 h, and centrifuged at 3500-4000 rpm for 5 min, and then washed several times with PBS buffer to obtain HMnO2@OSI-Rg3.

[0054] like Figure 1 The figure shows the transmission electron microscope image (TEM image) prepared in this example, with a scale of 100 nm. It can be seen from the figure that the morphology of HMnO2@OSI-Rg3 is round nanoparticles.

[0055] like Figure 2 Shown is the DLS particle size diagram of HMnO2@OSI-Rg3 and HMnO2 prepared in this example. It can be concluded from the figure that the particle size of HMnO2 becomes larger after drug loading, indicating that osimertinib and ginsenoside Rg3 are successfully loaded on HMnO2.

[0056] The present invention also conducted a cytotoxicity experiment and used the CCK-8 test to evaluate the toxicity of HMnO2@OSI-Rg3. H1975 OR cells were seeded in a 96-well plate at a density of 8,000 cells per well and incubated overnight (37°C, 5% CO2). The cells were then treated with different concentrations of OSI, Rg3, HMnO2, and HMnO2@OSI-Rg3 (i.e., HMnO2@OG in the figure) and incubated for 24 hours. The cell viability was measured on an ELISA instrument using a CCK-8 test kit at 450 nm. The results are shown in Figure 2. Figure 3 As shown. Figure 3 It can be concluded that with the increase of the concentrations of OSI, Rg3, and HMnO2, the cell survival rate decreased to varying degrees, but the decreasing trend was relatively slow. When HMnO2@OSI-Rg3 was used, the cytotoxicity could be quickly reduced to about 10% with the increase of the concentration of HMnO2@OSI-Rg3. This indicates that the HMnO2@OSI-Rg3 prepared by the present invention can effectively reduce cytotoxicity.

[0057] The present invention also conducted a cell uptake experiment, adding Cy5.5 (Cyanine 5.5) to HMnO2@OSI-Rg3, mixing on a rotary mixer in the dark for 12 h, loading Cy5.5 onto the nanoparticles HMnO2@OSI-Rg3, and placing 4×10 4 H1975OR cells were seeded on a glass-bottom confocal culture dish and incubated for 12 h (all groups contained 2 μg / mL OSI and 6.25 μg / mL Rg3) at 37 °C for 1, 2, 4, and 7 h, respectively. The cells were then washed three times with PBS buffer and fixed with 100 μl of 4% formaldehyde solution for 10 min. The nucleus was stained with the nuclear dye DAPI, and the cell images were acquired using a laser confocal microscope. The results are shown in Figure 4 As shown. Figure 4 It can be concluded that as time goes by, the red fluorescence becomes more and more abundant and brighter, indicating that the cells take up more and more HMnO2@OSI-Rg3.

[0058] The present invention also conducted a live-dead staining experiment, using a calcein / PI live / dead detection kit to evaluate the viability of H1975 OR cells, and seeded H1975 OR cells at a density of 8000 cells per well in a 96-well plate and incubated overnight (37°C, 5% CO2), and then incubated the cells with HMnO2@OSI-Rg3 (OSI concentration of 8ug / ml, Rg3 concentration of 25ug / ml) for 24 hours, then washed with PBS buffer, and stained the cells with calcein-AM / PI reagent for 15 minutes, and detected live cells / dead cells on a fluorescence microscope. The results are as follows: Figure 5 As shown. Figure 5 It can be seen that compared with other materials, HMnO2@OSI-Rg3 has the best toxic effect on cells.

[0059] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A nano drug, characterized in that: The nano drug is prepared by loading a mixed drug in a hollow drug-carrying nano material, and the mixed drug includes osimertinib and ginsenoside.

2. The nano drug according to claim 1, characterized in that The mass ratio of the mixed drug to the hollow drug-loaded nanomaterial is 1:(0.1-1); The mass ratio of osimertinib to ginsenoside is (0.5-1.5):

1.

3. The nano drug according to claim 2, characterized in that The mass ratio of the mixed drug to the hollow drug-loaded nanomaterial is 1:0.2; The mass ratio of the osimertinib to the ginsenoside is 1:

1.

4. The nanomedicine according to any one of claims 1 to 3, characterized in that The hollow drug-loaded nanomaterial is hollow manganese dioxide nanoparticles; The ginsenoside is ginsenoside Rg3.

5. A method for preparing the nano drug according to any one of claims 1 to 4, characterized in that: The following steps are involved: The hollow drug-loaded nanomaterial solution, the osimertinib solution and the ginsenoside solution are evenly mixed to obtain a nano drug.

6. The method for preparing the nano drug according to claim 5, characterized in that: The osimertinib solution is obtained by dissolving osimertinib in a mixed solvent; The ginsenoside solution is obtained by dissolving ginsenoside in a mixed solvent; The mixed solvent consists of dimethyl sulfoxide and water, and the dimethyl sulfoxide accounts for 0.1-10% of the volume of the mixed solvent.

7. The method for preparing the nano drug according to claim 5, characterized in that: The mixing is performed under light-protected conditions.

8. Use of the nanomedicine according to any one of claims 1 to 4 or the nanomedicine prepared by the method according to any one of claims 5 to 7 in anti-tumor drugs.

9. The use of the nano drug according to claim 8, characterized in that: The anti-tumor drugs include drugs for treating or assisting in treating non-small cell lung cancer.

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