Preparation and application of shikonin-loaded nanoparticles for inhibiting aerobic glycolysis

Abstract

The invention discloses a preparation method of an SHK-loaded albumin nano-drug, and relates to the technical field of drug preparation. Comprising the following steps: weighing BSA (Bovine Serum Albumin), dissolving the BSA in 2mL of water, and adjusting the pH value to 8.0-9.0 by using a 3mol / L sodium hydroxide solution; dissolving SHK by using absolute ethyl alcohol, then dropwise adding the SHK into the BSA solution at the speed of 0.2 mL / min, then supplementing and dropwise adding absolute ethyl alcohol, and continuously stirring during the period; adding a certain amount of a cross-linking reagent BAC solution dissolved by absolute ethyl alcohol, continuously stirring at 50 DEG C for 24 hours, and further wrapping with dextran, so as to form the nano-drug with tumor targeting property and controlled release capability. The dextran in the nanoparticles is hydrolyzed in the intestinal tract to form glucose conjugate nanoparticles, then the glucose conjugate nanoparticles are specifically taken in by tumor cells with high sugar requirements, SHK is released in the tumor cells in a targeted mode, the Warburg effect in the colorectal cancer is effectively blocked, tumors are killed, and the targeting performance, safety and bioavailability of the SHK are remarkably improved.

Description

Technical Field

[0001] The invention relates to the technical field of drug preparation, and in particular to a method for preparing an albumin nano drug loaded with SHK. Background Art

[0002] Colorectal cancer (CRC) is a malignant tumor that originates in the colon or rectum and is a major health problem worldwide. Colorectal cancer ranks third in prevalence in both men and women and is the second leading cause of cancer-related deaths. Metabolomics studies have shown that in order to meet the needs of their growth and proliferation, colorectal cancer cells undergo significant metabolic reprogramming and exhibit a unique metabolic signature of high glucose uptake. The Warburg effect is a hallmark of colorectal cancer metabolism, in which increased glycolysis and lactate production acidify the tumor microenvironment, promote immune escape, and affect tumor angiogenesis and cellular metabolism.

[0003] At present, the treatment of CRC is still based on the principle of individualization, with surgical resection as the main method, supplemented by comprehensive treatments such as adjuvant chemotherapy, chemoradiotherapy, targeted therapy, and immunotherapy. However, the drug resistance of tumors seriously limits the efficacy. Although great progress has been made in the international field in recent years in drug targeted therapy and immunotherapy for CRC, the results of mutation detection such as KRAS, NRAS, BRAF, MSI / MMR, PIK3CA / PIK3CB in CRC patients suggest that less than one-third of patients can benefit from targeted drug therapy and immunotherapy, and less than 8% of patients can benefit from PD-1. Clinically, most patients with stage III and IV CRC still lack effective targeted and immunotherapy drugs.

[0004] SHK is a natural active compound derived from the root of the natural plant Zongfu. A large number of studies have confirmed that SHK, as a classic inhibitor of the PKM2 pathway, can effectively block the glycolysis pathway in tumor cells, thereby inhibiting their growth and proliferation. In addition, SHK also regulates tumor cell apoptosis and angiogenesis through multiple mechanisms, thereby achieving the effect of inhibiting tumors. However, SHK has poor water solubility and insufficient bioavailability. When it reaches the colon, a large amount of drug loss may occur, resulting in insufficient drug uptake by colorectal cancer cells. Albumin is the main protein in plasma. Due to its excellent biocompatibility, long half-life and ability to bind to multiple drugs, it is widely used as a drug delivery carrier. In addition, colorectal cancer cells express specific albumin-related receptors (such as GP60), which can promote the cellular uptake of albumin-drug conjugates. For example, albumin-paclitaxel conjugates, which are mainly used for the treatment of breast cancer, non-small cell lung cancer and pancreatic cancer, have shown good therapeutic effects in clinical trials of colorectal cancer, suggesting a new direction for the treatment of colorectal cancer. Therefore, albumin-mediated SHK delivery has the potential to enhance therapeutic effects and reduce side effects. Summary of the invention

[0005] The object of the present invention is to provide a method for preparing an albumin nanomedicine loaded with SHK to solve the problems raised in the above background technology.

[0006] To achieve the above object, the present invention provides the following technical solution: a method for preparing an albumin nanomedicine loaded with SHK, comprising the following steps: Weigh BSA and dissolve it in 2 mL of water. Adjust the pH to 8.0-9.0 with 3 mol / L sodium hydroxide solution. Dissolve SHK in anhydrous ethanol, then add it to the BSA solution at a rate of 0.2 mL / min, and then add anhydrous ethanol (the volume ratio of water to anhydrous ethanol is 1:4) while stirring continuously (600-800 rpm); A certain amount of cross-linking reagent BAC solution dissolved in anhydrous ethanol (the mass ratio of BSA to BAC was 10:1) was added, stirred continuously at 50°C for 24 h, and further encapsulated with dextran to form a nanodrug with tumor targeting and controlled release capabilities.

[0007] As a specific solution of the technical solution of the present application, the pH value of the BSA solution is 8.0-9.0.

[0008] As a specific solution of the technical solution of the present application, the speed of adding SHK to the BSA solution is 600-800 rpm, and / or the volume ratio of water to anhydrous ethanol in step S2 is 1:4.

[0009] As a specific solution of the technical solution of the present application, the cross-linking reagent BAC solution is dissolved with anhydrous ethanol, and / or the mass ratio of BSA to BAC is 10:1.

[0010] As a specific solution of the technical solution of the present application, the BAC solution needs to be encapsulated with dextran to form a nanomedicine with tumor targeting and controlled release capabilities.

[0011] An albumin nanomedicine loaded with SHK, prepared by the method described in any one of claims 1 to 5.

[0012] As a specific solution of the technical solution of the present application, the nanoparticles can inhibit the growth of colon cancer cells by inhibiting aerobic glycolysis.

[0013] As a specific solution of the technical solution of the present application, the nanoparticles can inhibit the growth of colon cancer cells by inhibiting aerobic glycolysis.

[0014] A preparation and application of nanoparticles loaded with shikonin for inhibiting aerobic glycolysis, using the method for preparing the SHK-loaded albumin nanoparticle and the SHK-loaded albumin nanoparticle as described in any one of claims 1 to 8.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The preparation and application of nanoparticles loaded with shikonin that inhibit aerobic glycolysis, through the hydrolysis of dextran in the nanoparticles in the intestine to form glucose-conjugated nanoparticles, which are then specifically taken up by tumor cells with a high demand for sugar. The targeted release of SHK in tumor cells effectively blocks the Warburg effect in colorectal cancer, kills tumors, and significantly improves the targeting, safety and bioavailability of SHK. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is the transmission electron microscope image of DCMS-SHK of the present invention; Figure 2 is the particle size distribution of DCMS-SHK of the present invention; Figure 3 Screening of pharmacological targets for COAD of the present invention; Figure 4 This is a graph showing the results of CT26 cells taking up CMS-SHK and DCMS-SHK according to the present invention; Figure 5 The ability of DCMS-SHK to inhibit the proliferation of colorectal cancer cell line CT26 in the CCK8 experiment of the present invention; Figure 6 The Calcein / PI of the present invention detects the effect of DCMS-SHK on the activity of colorectal cancer cell line CT26 cells; Figure 7 The clone formation experiment of the present invention proves that DCMS-SHK inhibits the proliferation of colorectal cancer cell line CT26; Figure 8 The pyruvate and lactate assays of the present invention confirm that DCMS-SHK kills tumor cells by interfering with aerobic glycolysis; Fig. 9 The hemolysis experiment of the present invention verifies that DCMS-SHK has no toxic effect on normal cells; Fig.10 Schematic diagram of the synthesis and action mechanism of the nanoparticles of the present invention. DETAILED DESCRIPTION

[0017] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0018] like Figure 1-Figure 10 As shown, the present invention provides a technical solution: a method for preparing an albumin nanomedicine loaded with SHK. It should be understood that the experimental materials and equipment used in the following embodiments are commercially available unless otherwise specified, wherein the experimental cells include: CT26 cells were purchased from Wuhan Prosai Life Science Co., Ltd.

[0019] Example 1: Preparation and characterization of DCMS-SHK S1. Weigh a certain amount of BSA and dissolve it in 2 mL of water. Adjust the pH value to 8.0-9.0 with 3 mol / L sodium hydroxide solution.

[0020] S2. Dissolve SHK in anhydrous ethanol and then add it to the BSA solution at a rate of 0.2 mL / min. Then add anhydrous ethanol (the volume ratio of water to anhydrous ethanol is 1:4) while stirring continuously (600-800 rpm) S3. Add a certain amount of cross-linking reagent BAC solution dissolved in anhydrous ethanol (the mass ratio of BSA to BAC is 10:1), stir continuously at 50°C for 24 h, and further encapsulate it with dextran to form a nanodrug with tumor targeting and controlled release capabilities, namely DCMS-SHK.

[0021] The morphology of the nanoparticles was examined using a transmission electron microscope (TEM, Thermo Figure 1 The particle size distribution was determined using a particle size analyzer (Malvern Panalytical). Figure 2 ). It can be seen that the particle size is concentrated around 156.6nm. The difference in the size of drug particles is small, which can ensure its stable bioavailability and efficacy.

[0022] Example 2: Determining therapeutic targets through bioinformatics analysis To identify targets associated with colorectal adenocarcinoma (COAD), transcriptome data of COAD patients were extracted from The Cancer Genome Atlas (TCGA) database. Batch correction and differential gene expression (DEG) screening were performed using the “limma” package in R, with the criteria set as |log2(fold change)|>1 and P value <0.05 to identify genes that were significantly different in the COAD dataset. Figure 3 As shown in the figure, the levels of PKM2 and SLC2A1 in tumor tissues are significantly increased compared with normal tissues. The protein GLUT1 encoded by the SLC2A1 gene can activate the activity of D-glucose transmembrane transporter and dehydroascorbic acid transmembrane transporter. It is involved in dehydroascorbic acid transport and glucose transmembrane transport. It plays a role in the upstream or cellular response of glucose starvation. The uptake of glucose in malignant tumor cells is mainly regulated by GLUT1. Therefore, this experiment designed dextran-coated nanoparticles to enhance the targeting ability of uptake by tumor tissues. The PKM2 gene is highly associated with the Warburg effect in tumors. The upregulation of PKM2 or the replacement of PKM1 by PKM2 triggers aerobic glycolysis, a process observed in many cancers. Unlike PKM1, which retains high constitutive PK activity, the low activity of PKM2 enables cancer cells to accumulate various glycolytic intermediates, which are necessary for the biosynthesis of nucleic acids, amino acids and phospholipids, and promote anabolism through the pentose phosphate pathway. In addition, PKM2 accumulated in the cytoplasm can translocate to the nucleus, where it acts as a protein kinase and transcriptional coactivator to regulate the transcription of tumor genes and metabolic genes, leading to rapid cell proliferation and tumor formation. Therefore, this study identified PKM2 as a therapeutic target and planned to use SHK drugs for experimental treatment.

[0023] Example 3: DCMS-SHK targeting colorectal cancer cells CT26 cells in the logarithmic growth phase were seeded in 6 cm culture dishes and cultured for 24 h to allow cell attachment. Then, DCMS-SHK labeled with fluorescein isothiocyanate (FITC) and nanoparticles (CMS-SHK) without dextran were added for 4 h. After washing with PBS, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.25% Triton X-100, and finally stained with Hoechst33342 (Beyotech, China) for nuclear staining and Actin-Tracker Red-594 (Beyotech, China) for cytoskeleton staining. After washing with PBS, the slides were sealed and the fluorescence intensity in the cells was examined under a fluorescence microscope.

[0024] Direct observation by confocal microscopy ( Figure 4 ), blue fluorescence represents the cell nucleus stained with Hoechst 33342, and green fluorescence represents FITC-labeled nanoparticles. Compared with CMS-SHK, higher intensity green fluorescence can be observed in CT26 cells after 4 hours of co-incubation with DCMS-SHK, indicating that the ability of DCMS-SHK to target colon tumor cells is significantly better than that of CMS-SHK without dextran coating.

[0025] Example 4: DCMS-SHK inhibits proliferation of colorectal cancer cell line CT26 In order to further verify the biosafety and tumor killing effect of DCMS-SHK prepared in the above example, CT26 cells in the logarithmic growth phase were seeded into 96-well plates at a density of 10,000 per well. After culturing for 24 hours, the cells were treated with the specified drug concentration and cultured for another 48 hours. Cell Counting Kit-8 (CCK8) was used to evaluate cell proliferation. The results showed that ( Figure 5 ), the inhibitory ability of DCMS-SHK at different concentrations on cell proliferation was better than that of the SHK group and the blank vector group (hereinafter referred to as the Blank-NPs group), and Blank-NPs at different concentrations had no obvious inhibitory effect on cells.

[0026] Example 5: DCMS-SHK inhibits the activity of colorectal cancer cell line CT26 CT26 cells in the logarithmic growth phase were cultured for 24 hours and then treated with the specified drugs (PBS, SHK, and DCMS-SHK) for 24 hours. The cell viability was evaluated using the Calcein / PI cell viability / cytotoxicity assay kit (Beyotech, China). The Calcein / PI results showed ( Figure 6 ), compared with the PBS group and SHK group, the activity of cells was significantly decreased after DCMS-SHK treatment.

[0027] Example 6: DCMS-SHK inhibits cloning of colorectal cancer cell line CT26 Cells in the logarithmic growth phase were seeded into 12-well plates at a density of 1,000 cells per well. After 2 days of culture, the cells were treated with the designated drugs (PBS, SHK, and DCMS-SHK). After 14 days, the cells were washed with PBS, fixed with anhydrous ethanol, and stained with crystal violet, followed by colony counting, and the experiment was repeated three times. PKM2 is a downstream molecule of hypoxia-inducible factor-1α (HIF-1α) and a key enzyme for aerobic glycosidase in cancer cells. Inhibition of PKM2 can inhibit the proliferation of cancer cells by reducing intracellular ATP levels. The clone formation assay was used to evaluate the proliferation ability of cells under drug treatment conditions. After drug treatment, the number of clones was significantly reduced ( Figure 7 ), indicating the effectiveness of the drug in inhibiting the proliferation of CT26 cells. The above experiments (Examples 4-6) all show the effectiveness of DCMS-SHK in inhibiting the proliferation of CT26 cells.

[0028] Example 7: DCMS-SHK kills tumor cells by interfering with aerobic glycolysis CT26 cells in the logarithmic growth phase were cultured in 6-well culture plates for 24 hours and then treated with appropriate drug concentrations for 24 hours. The cells were collected and divided into two parts. The first part was washed with PBS and detected using the CheKine™ Micro Pyruvate (PA) Assay Kit (Abbkine, China); the second part was analyzed for PA content by measuring the optical density (OD) value at 520 nm. The samples were processed using the CheKine™ Micro Lactate Assay Kit (Abbkine, China) and the optical density (OD) value at 450 nm was measured to analyze the lactate metabolism level.

[0029] As mentioned above, the Warburg effect is a hallmark of CRC metabolism, which leads to increased glycolysis and lactate production, acidifies the tumor microenvironment, promotes immune escape, and affects tumor angiogenesis and cell metabolism. Glycolysis is the process of converting glucose into pyruvate to provide energy to cells. Therefore, measuring the concentrations of pyruvate and lactate in CT26 cells after drug treatment can clarify whether the nanomaterials inhibit glycolysis of tumor cells. Compared with the control group ( Figure 8 ), the levels of pyruvate and lactate in the DCMS-SHK and SHK treatment groups were significantly reduced, indicating that SHK can effectively inhibit the aerobic glycolysis pathway. It also makes it clear that the mechanism by which SHK-loaded nanoparticles kill tumor cells is also achieved by interfering with aerobic glycolysis.

[0030] Example 9 DCMS-SHK has no obvious toxic side effects on normal cells Fresh mouse blood was collected using K2-EDTA anticoagulant tubes, and red blood cells were separated by centrifugation, purified, and diluted to a concentration of 1×10 8 cells / mL. Different concentrations of DCMS-SHK were incubated with red blood cells at 37°C for 2 hours. The absorbance of the supernatant of each group at 480 nm was measured using a spectrophotometer. The 1% Triton-100-treated group served as the positive control, and its hemoglobin release rate was 100%. The calculation formula for the hemolysis percentage is as follows: Hemolysis percentage = (OD480 of the sample) / OD480 of the positive control × 100%. Compared with the positive control, no concentration of DCMS-SHK caused significant hemolysis, demonstrating the safety of DCMS-SHK ( Fig. 9 ).

[0031] In summary, the present invention prepares the nano drug DCMS-SHK. In the intestine, dextran is hydrolyzed into glucose, and tumor cells take up the nanoparticles by taking up glucose. SHK then kills tumor cells and inhibits proliferation by inhibiting aerobic glycolysis, thereby treating colorectal cancer ( Fig.10 ).

[0032] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is limited by the attached embodiments and their equivalents.

Claims

1. A method for preparing an albumin nanodrug loaded with SHK, characterized in that: The following steps are involved: Weigh BSA and dissolve it in 2 mL of water. Adjust the pH to 8.0-9.0 with 3 mol / L sodium hydroxide solution. Dissolve SHK in anhydrous ethanol, then add it to the BSA solution at a rate of 0.2 mL / min, and then add anhydrous ethanol (the volume ratio of water to anhydrous ethanol is 1:4) while stirring continuously (600-800 rpm); A certain amount of cross-linking reagent BAC solution dissolved in anhydrous ethanol (the mass ratio of BSA to BAC was 10:1) was added, stirred continuously at 50°C for 24 h, and further encapsulated with dextran to form a nanodrug with tumor targeting and controlled release capabilities.

2. The method for preparing a SHK-loaded albumin nanomedicine according to claim 1, characterized in that: The pH value of the BSA solution is 8.0-9.

0.

3. The method for preparing a SHK-loaded albumin nanomedicine according to claim 1, characterized in that: The speed of adding SHK to the BSA solution is 600-800 rpm, and / or the volume ratio of water to anhydrous ethanol in step S2 is 1:

4.

4. The method for preparing a SHK-loaded albumin nanodrug according to claim 1, characterized in that: The cross-linking reagent BAC solution is dissolved in anhydrous ethanol, and / or the mass ratio of BSA to BAC is 10:

1.

5. The method for preparing a SHK-loaded albumin nanomedicine according to claim 1, characterized in that: The BAC solution needs to be encapsulated with dextran to form a nano drug with tumor targeting and controlled release capabilities.

6. An albumin nanodrug loaded with SHK, characterized in that: The method is prepared by any method described in claim 1 to claim 5.

7. The albumin nanodrug loaded with SHK according to claim 6, characterized in that: The nanoparticles can inhibit the growth of colon cancer cells by inhibiting aerobic glycolysis.

8. The albumin nanodrug loaded with SHK according to claim 6, characterized in that: The nanoparticles can inhibit the growth of colon cancer cells by inhibiting aerobic glycolysis.

9. Preparation and application of shikonin-loaded nanoparticles for inhibiting aerobic glycolysis, characterized in that: The method for preparing the SHK-loaded albumin nanomedicine and the SHK-loaded albumin nanomedicine are used as described in any one of claims 1 to 8.

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