Preparation method and application of cinobufagin and pentaline albumin nano-drug

Abstract

The invention relates to the technical field of preparation of albumin nano-drugs, in particular to a preparation method and application of a cinobufagin and pentaline albumin nano-drug. When the cinobufagin and pentaline albumin nano-drug is prepared, albumin and cinobufagin and pentaline are stably cross-linked through a chemical cross-linking method to form the cinobufagin and pentaline albumin nano-drug with controllable particle size and stable release, and the invention further discloses application of the cinobufagin and pentaline albumin nano-drug in preparation of anti-pulmonary fibrosis drugs.

Description

Technical Field

[0001] This invention relates to the field of nanomedicine technology, and in particular to a method for preparing a bufotalin albumin nanomedicine and its uses. Background Technology

[0002] Cinobufotalin, the single active ingredient in bufotalin (CAS No. 1108-68-5), belongs to the indole alkaloid class. In recent years, bufotalin has been used in numerous clinical and experimental studies and has been proven to possess anti-cancer, heat-clearing and detoxifying, swelling-reducing and analgesic, anti-inflammatory, myocardial contractility-enhancing, antiviral, and edema- and dyspnea-relieving effects. Previous studies have found that bufotalin exhibits significant anti-cancer effects against malignant cancers including lung cancer, colorectal cancer, gastric cancer, and liver cancer. Lung cancer is a common complication of pulmonary fibrosis, and multiple studies have shown a common link between pulmonary fibrosis and lung cancer. Currently, bufotalin is mainly used clinically in the comprehensive treatment of tumors such as liver cancer, lung cancer, and esophageal cancer, but research on traditional Chinese medicine treatments related to pulmonary fibrosis is limited.

[0003] The applicant discovered that while bufotalin alone has good therapeutic effects in the treatment of pulmonary fibrosis, it also found that its drug targeting is poor and its oral application has significant limitations, including cardiotoxicity, low bioavailability, poor water solubility, and lack of targeting. To improve the pharmacokinetic properties of bufotalin and reduce its toxicity, we combined it with albumin to construct a nanomedicine delivery system, which can significantly improve solubility, bioavailability, and targeting ability, thereby enhancing efficacy and reducing toxic side effects. In this regard, the inventors proposed a method for preparing bufotalin albumin nanomedicine and its uses. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing bufotalin albumin nanomedicine and its applications, in order to improve the solubility, bioavailability and targeting ability of bufotalin alone in the treatment of pulmonary fibrosis, thereby enhancing the efficacy and reducing toxic side effects.

[0005] To achieve the above objectives, the present invention provides the following technical solution: Preparation of bovine serum albumin solution: Add bovine serum albumin powder to buffer solution and stir with a magnetic stirrer at 300-400 rpm until completely dissolved to obtain bovine albumin solution.

[0006] Preparation of bufotalin solution: Dissolve bufotalin powder in an organic solvent and stir with a magnetic stirrer at 300-400 rpm for 30-40 minutes until bufotalin is completely dissolved to obtain bufotalin solution.

[0007] Addition of dithiothreitol and sodium deoxycholate: Dithiothreitol and sodium deoxycholate were added to the albumin solution in (1) to achieve albumin unfolding, resulting in a mixed albumin solution, which was then incubated at 37°C for 1 hour.

[0008] Mix bufotalin and albumin: Slowly add the bufotalin solution from (2) to the mixed albumin solution from (3), stir and mix using a magnetic stirrer, and incubate at 37°C for 10 minutes.

[0009] Dialysis treatment: The solution obtained in (4) was dialyzed for 24 hours in a buffer solution using a regenerable cellulose dialysis bag with a molecular weight cutoff of 3.5 kDa to remove unbound bufotalin, dithiothreitol and sodium deoxycholate.

[0010] Freeze-drying: The dialysis solution obtained in (5) was freeze-dried to obtain the albumin nanoparticle drug powder of bufotalin compound.

[0011] Characterization and analysis: Particle size analysis was performed using dynamic light scattering (DLS) and scanning electron microscopy (SEM); the loading of bufotalin was determined using high performance liquid chromatography (HPLC); and drug release studies were conducted using in vitro release assays.

[0012] Compared with the prior art, the beneficial effects of the present invention are: This invention employs a simple process to prepare synthetic bufotalin albumin nanoparticles with high yield and good biocompatibility, showing promise for industrialization. The bufotalin albumin nanoparticles prepared by this invention are uniform in size and dispersion, exhibiting good colloidal stability and biocompatibility, significant anti-pulmonary fibrosis effects, and potential application value. Attached Figure Description

[0013] Figure 1 This is a flowchart of the preparation method of the bufotalin albumin nanomedicine of the present invention; Figure 2 The scanning electron microscope image (scale bar 300 nm) of the bufalin albumin nanomedicine of the present invention. Figure 3 The particle size distribution diagram of the Huachansu Talin albumin nanomedicine of the present invention using dynamic light scattering (DLS); Figure 4 This is a zeta potential diagram of the bufotalin albumin nanomedicine of the present invention; Figure 5 This is a seven-day average particle size diagram of the Huachansutaling albumin nanomedicine of the present invention.

[0014] Figure 6This invention relates to the biocompatibility of the Huachansutaling albumin nanomedicine of the present invention with mouse fibroblast L929 cells.

[0015] Figure 7 The immunofluorescence icon (scale bar 25 μm) shows the expression of α-SMA in TGF-β-stimulated L929 cells by the bufotalin albumin nanomedicine of the present invention.

[0016] Figure 8 This is an immunofluorescence image of the expression of CollagenI in TGF-β-stimulated L929 cells by the bufotalin albumin nanomedicine of the present invention. The scale bar is 25 μm. Detailed Implementation

[0017] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0018] Example 1: Chemical cross-linking preparation method of Huachansu Tadalafil albumin nanomedicine Preparation of bovine serum albumin solution: Add bovine serum albumin powder to buffer solution and stir with a magnetic stirrer at 300-400 rpm until completely dissolved to obtain bovine albumin solution.

[0019] Preparation of bufotalin solution: Dissolve bufotalin powder in an organic solvent and stir with a magnetic stirrer at 300-400 rpm for 30-40 minutes until bufotalin is completely dissolved to obtain bufotalin solution.

[0020] Addition of dithiothreitol and sodium deoxycholate: Dithiothreitol and sodium deoxycholate were added to the albumin solution in (1) to achieve albumin unfolding, resulting in a mixed albumin solution, which was then incubated at 37°C for 1 hour.

[0021] Mix bufotalin and albumin: Slowly add the bufotalin solution from (2) to the mixed albumin solution from (3), stir and mix using a magnetic stirrer, and incubate at 37°C for 10 minutes.

[0022] Dialysis treatment: The solution obtained in (4) was dialyzed for 24 hours in a buffer solution using a regenerable cellulose dialysis bag with a molecular weight cutoff of 3.5 kDa to remove unbound bufotalin, dithiothreitol and sodium deoxycholate.

[0023] Freeze-drying: The dialysis solution obtained in (5) was freeze-dried to obtain the albumin nanoparticle drug powder of bufotalin compound.

[0024] Characterization and analysis: Particle size analysis was performed using dynamic light scattering (DLS) and scanning electron microscopy (SEM); the loading of bufotalin was determined using high performance liquid chromatography (HPLC); and drug release studies were conducted using in vitro release assays.

[0025] Particle size analysis: The DLS instrument was activated, and the sample was injected into its sample chamber. Measurement was initiated, and the laser beam scattered the particles in the sample. The DLS instrument calculated the dynamic size distribution of the particles based on the intensity and time delay of the scattered light, generating a particle size distribution map, including the average particle size and polydispersity index, thus providing information on particle size and distribution. According to the DLS data, the average particle size of the Huachansu Taling albumin nanoparticles was 115.8 nm, the relative dispersion index was 0.375, and the average zeta potential was -2.3 mV, indicating that the nanoparticles were uniform in size and had good dispersibility. Furthermore, a seven-day stability experiment showed that the nanoparticles maintained their uniform size over a long period.

[0026] First, attach the conductive adhesive to the sample stage. Then, attach a clean, thin copper sheet to the conductive adhesive. Use a small, repeated dripping method to apply the liquid sample to the copper sheet, allowing the sample to dry completely at room temperature as the solvent evaporates. Allow the sample to dry completely at room temperature until the solvent evaporates. After drying, perform a sputtering process on the thin copper sheet (along with the sample stage) containing the sample. Then, place the sample into the SEM sample chamber and start the SEM instrument. Turn on the SEM instrument and initialize it. Adjust the SEM instrument according to the needs of the sample, including focusing, magnification, working distance, and accelerating voltage. Observe the sample using the SEM microscope and take images. Based on the SEM images, observe the aggregation state of the particles and obtain information on the particle morphology.

[0027] The loading of bufotalin was determined by high performance liquid chromatography (HPLC). The nanomedicine sample was added to an appropriate amount of methanol to release the bufotalin from the sample. Ultrasonic treatment was used to accelerate the release, ensuring thorough mixing of the sample. The sample was then filtered through a 0.2-micron filter membrane to remove any residual particles or impurities, ensuring sample purity. HPLC analysis: Samples were analyzed using an HPLC instrument: chromatographic column: C18 reversed-phase column, mobile phase: a mixture of methanol and water, pH adjusted to 6.0, flow rate: 0.5 to 1.0 mL / min, detection wavelength: 200 to 400 nm.

[0028] Calculate the loading: Using data from the standard curve, calculate the content of bufotalin in the sample by comparing the peak area or peak height of the unknown sample with the corresponding value on the standard curve.

[0029] In vitro release experiments were conducted to study drug release: A certain amount of nanomedicine sample (e.g., 10 mg) was placed in a centrifuge tube; 5 mL of PBS was added to the centrifuge tube containing the nanomedicine sample, and release was initiated; at different time points in the release experiment (e.g., 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, and 24 hours), a certain volume of sample was taken from the centrifuge tube; after each sampling, centrifugation was performed to separate the sample into a supernatant (containing the released drug) and residue; Sample analysis: Using a spectrophotometer, the supernatant at each time point was analyzed to measure the concentration of bufalin released, and release curves were plotted. The bufalin concentration data at each time point were plotted as a release curve showing the relationship between bufalin concentration and time. Based on the release curves, the release rate and drug release kinetic parameters can be calculated. The release mechanism is analyzed using the Korsmeyer-Peppas model, which facilitates the adjustment and optimization of the formulation based on the results to obtain better drug release performance.

[0030] Example 2: Biocompatibility of Huachansu Tadalafil albumin nanomedicine This invention selects bovine serum albumin as a basic component to ensure the excellent biocompatibility of the nanocarrier. L929 cells were placed in 96-well plates, 2 x 10 cells per well. 3 Cells were cultured in quadruplicates overnight. After incubation with different reagents for 48 hours, CCK-8 assays were performed according to the manufacturer's instructions. Figure 4 As shown, the Huachansutalin albumin nanomedicine showed no significant cytotoxicity to L929 cells at concentrations ranging from 12.5 to 800 nM, indicating that the Huachansutalin albumin nanomedicine has good biocompatibility.

[0031] Example 3: Inhibitory effect of Huachansu Tadalafil albumin nanomedicine on pulmonary fibrosis cell model in vitro. Preparation of bovine serum albumin solution: Add bovine serum albumin powder to buffer solution and stir with a magnetic stirrer at 300-400 rpm until completely dissolved to obtain bovine albumin solution.

[0032] Preparation of bufotalin solution: Dissolve bufotalin powder in an organic solvent and stir with a magnetic stirrer at 300-400 rpm for 30-40 minutes until bufotalin is completely dissolved to obtain bufotalin solution.

[0033] Addition of dithiothreitol and sodium deoxycholate: Dithiothreitol and sodium deoxycholate were added to the albumin solution in (1) to achieve albumin unfolding, resulting in a mixed albumin solution, which was then incubated at 37°C for 1 hour.

[0034] Mix bufotalin and albumin: Slowly add the bufotalin solution from (2) to the mixed albumin solution from (3), stir and mix using a magnetic stirrer, and incubate at 37°C for 10 minutes.

[0035] Dialysis treatment: The solution obtained in (4) was dialyzed for 24 hours in a buffer solution using a regenerable cellulose dialysis bag with a molecular weight cutoff of 3.5 kDa to remove unbound bufotalin, dithiothreitol and sodium deoxycholate.

[0036] Freeze-drying: The dialysis solution obtained in (5) was freeze-dried to obtain the albumin nanoparticle drug powder of bufotalin compound.

[0037] Characterization and analysis: Particle size analysis was performed using dynamic light scattering (DLS) and scanning electron microscopy (SEM); the loading of bufotalin was determined using high performance liquid chromatography (HPLC); and drug release studies were conducted using in vitro release assays.

[0038] The blank group, TGF-β treatment group, bufalin group, bufalin albumin nanomedicine group, and positive control group (pirfenidone) were applied to in vitro experiments on pulmonary fibrosis. Immunofluorescence experiments were conducted. After TGF-β treatment of fibroblasts for more than 24 hours, bufalin, bufalin albumin nanomedicine, and pirfenidone were added. Under laser confocal microscopy, it was observed that the fluorescence of α-SMA and Collagen I in the bufalin albumin nanomedicine group was the weakest, while the fluorescence of α-SMA and Collagen I in the negative control cells was the strongest, indicating that bufalin albumin nanomedicine has a good therapeutic effect on pulmonary fibrosis cell models.

[0039] As can be seen from the above, the present invention uses a simple process to prepare synthetic bufotalin albumin nanoparticles with high yield and good biocompatibility, and has the prospect of industrialization. The bufotalin albumin nanoparticles prepared by the present invention are uniform in size and uniformly dispersed, have good colloidal stability and biocompatibility, have significant anti-pulmonary fibrosis effects, and have potential application value.

[0040] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. This invention uses chemical cross-linking, dithiothreitol and sodium deoxycholate to achieve the unfolding and reconstruction of albumin; the albumin is applicable to various types of proteins (including human serum albumin, collagen, bovine albumin and recombinant and semi-recombinant proteins).

2. The albumin nanoparticles of the bufotalin compound according to claim 1 have a particle size range of 1-400 nm. When the concentration of dithiothreitol is 1 mM, the concentration of sodium deoxycholate ranges from 5-30 mM; when the concentration of sodium deoxycholate is 5 mM, the concentration of dithiothreitol ranges from 1-10 mM.

3. Bufotalin is soluble in organic solvents, including various alcohols, methanol or ethanol.

4. A method for preparing bufotalin albumin nanomedicine, characterized in that... The process includes the following steps: Bovine serum albumin powder is stirred at 300-400 rpm with a magnetic stirrer until completely dissolved. Dithiothreitol and sodium deoxycholate are added to the resulting solution and incubated at 37°C for 1 hour. Simultaneously, bufotalin powder is stirred at 300-400 rpm in an organic solvent for 30-40 minutes until completely dissolved. This dissolved powder is then slowly added dropwise to the aforementioned mixture. After magnetic stirring, the mixture is incubated at 37°C for 10 minutes. Subsequently, the mixture is dialyzed in buffer solution using a 3.5 kDa regenerable cellulose dialysis bag. Finally, the mixture is freeze-dried to obtain bufotalin albumin nanoparticle powder. Particle size analysis, bufotalin loading determination, and drug release determination are performed using DLS, SEM, HPLC, and in vitro release experiments, respectively. The structural formula of bufotalin is as follows: 。 5. The method for preparing the bufotalin-based albumin nanomedicine according to claim 1, characterized in that, The molar ratio of the bufotalin to albumin is 3-50:

1.

6. The method for preparing the bufotalin-albumin nanomedicine according to claim 1, characterized in that, The concentration range of the dithiothreitol is 1-10 mM, and the concentration range of sodium deoxycholate is 5-30 mM.

7. The method for preparing the bufotalin-albumin nanomedicine according to claim 1, characterized in that, The proteins include human serum albumin, collagen, bovine albumin, and recombinant and semi-recombinant proteins.

8. The method for preparing the bufotalin-albumin nanomedicine according to claim 1, characterized in that, The drug is applicable to poorly soluble drugs, including but not limited to bufotalin.

9. The use of Huachansutaling compound albumin nanomedicine in the treatment of pulmonary fibrosis.

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