Preparation method and application of composite nano-drug capable of relieving liver fibrosis
By preparing manganese diselenide@polydopamine-pPB nanomedicine, the diagnostic and treatment challenges of liver fibrosis have been solved, enabling precise diagnosis and treatment of liver fibrosis, with significant therapeutic potential and biosafety.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHANDONG JIANZHU UNIV
- Filing Date
- 2026-04-10
- Publication Date
- 2026-06-26
AI Technical Summary
Existing technologies are insufficient to effectively alleviate liver fibrosis, which leads to damage to liver structure and function, increases the risk of liver cancer, and has no significant therapeutic effect.
A manganese diselenide@polydopamine-pPB nanomedicine was prepared. It releases Mn2+ to achieve MRI imaging through responsive degradation in a weakly acidic liver fibrosis microenvironment, and releases selenium to scavenge reactive oxygen species and inhibit the activation of hepatic stellate cells.
It enables precise diagnosis and treatment of liver fibrosis, reduces side effects, and has significant therapeutic potential and biocompatibility.
Smart Images
Figure CN122272841A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, and in particular to a method for preparing and applying a composite nanomedicine that can alleviate liver fibrosis. Background Technology
[0002] Liver fibrosis is a serious liver disease characterized by excessive accumulation of connective tissue in the liver, often caused by viral hepatitis, chronic alcoholism, and non-alcoholic fatty liver disease. Liver fibrosis leads to irreversible damage to the structure and function of the liver, potentially progressing to cirrhosis and liver failure. This not only reduces patients' quality of life but also increases the risk of liver cancer. Patients with cirrhosis often face serious complications such as ascites, esophageal variceal bleeding, and hepatic encephalopathy, resulting in a high mortality rate. Therefore, early intervention and effective treatment are crucial for improving prognosis.
[0003] In recent years, nanomaterials have seen rapid development in the medical field, particularly demonstrating great potential in disease diagnosis and treatment. Due to their unique physicochemical properties, such as responsive release, suitable particle size for endocytosis, and ease of surface functionalization, nanomaterials have become an ideal choice for drug delivery systems. In the treatment of liver fibrosis, nanomaterials offer significant advantages. First, nanomaterials readily accumulate in the liver, as it is the body's primary detoxification organ and possesses a rich reticuloendothelial system. Second, the microenvironment of liver fibrotic cells is more acidic than that of normal cells due to increased metabolites, especially lactic acid and other organic acids, during chronic inflammation and extracellular matrix remodeling. This acidic environment promotes the degradation and release of active ingredients from responsive nanomaterials, thereby enhancing therapeutic efficacy and reducing side effects.
[0004] This invention prepares a composite nanomedicine that can alleviate liver fibrosis. The synthesis method is simple and highly operable. The synthesized product is stable and reproducible. The manganese diselenide-coated polydopamine nanomaterial synthesized in this invention can be used to alleviate liver fibrosis and also relieve chronic inflammation caused by liver fibrosis, which is beneficial for clinical application. Summary of the Invention
[0005] To address the aforementioned problems, this invention provides a method for preparing and applying a composite nanomedicine that can alleviate liver fibrosis. The preparation method provided by this invention is simple, mild, and highly operable. The prepared nanomedicine has both good biocompatibility and biodegradability, and can effectively scavenge reactive oxygen species and inhibit the activation of hepatic stellate cells, thereby alleviating liver fibrosis and showing excellent clinical application prospects.
[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a method for preparing a composite nanomedicine that can alleviate liver fibrosis, comprising the following steps: S1. Preparation of manganese diselenide nanoparticles: 1) Dissolve sodium selenite and manganese acetate in pure water and stir until homogeneous to obtain solution A; 2) Add hydrazine hydrate to solution A and stir the reaction at room temperature to obtain solution B; 3) Transfer solution B to a reaction vessel and carry out a solvothermal reaction to obtain solution C. After the reaction is completed, adjust the pH of solution C to 7.0 to obtain solution D. Centrifuge and wash the product to obtain manganese diselenide nanoparticles. S2. Preparation of manganese diselenide@polydopamine nanomaterials (MSP): The manganese diselenide nanoparticles obtained in step S1 were first stirred with SH-PEG-NH2 in pure water in an ice bath in the dark to obtain solution E. Then, solution E was centrifuged and the product was washed. After removing the supernatant, precipitate H was obtained. Then, it was mixed with dopamine hydrochloride in Tris-HCl and stirred at room temperature in the dark. After the reaction was completed, the product was centrifuged and washed to obtain manganese diselenide@polydopamine nanomaterial, namely MnSe2@PDA, abbreviated as MSP. S3. Preparation of manganese diselenide@polydopamine-pPB nanoprobes (MSPp): The MSP and pPB cyclic peptide obtained in step S2 were mixed in Tris-HCl buffer and stirred in an ice bath in the dark. After the reaction was completed, the product was centrifuged and washed to obtain manganese diselenide@polydopamine-pPB nanomedicine (MnSe2@PDA-PPB, abbreviated as MSPp), which is a composite nanomedicine for relieving liver fibrosis.
[0007] Preferably: in step S1, 1) Add sodium selenite powder and manganese acetate powder to pure water at a mass ratio of 1:2, mix and stir until completely dissolved to obtain solution A; wherein the mass of sodium selenite is 0.35g and the mass of manganese acetate is 0.70g. 2) Add 9 mL of hydrazine hydrate to solution A obtained in step 1) above, and stir continuously for 30 minutes to obtain a homogeneous solution B; 3) Transfer the solution B obtained in step 2) to a high-pressure reactor, seal it and place it in an oven. Perform a solvothermal reaction at 100°C for 24 hours. After the reaction is completed, allow it to cool naturally to room temperature, collect the product, adjust the pH of the product to 7.0 with citric acid to prevent the material from agglomerating under alkaline conditions, and wash and dry it to obtain manganese diselenide nanomaterials. More preferably, in step 1), the amount of pure water added is sufficient to completely dissolve the sodium selenite and manganese acetate powder, and the stirring process is carried out at room temperature. The amount of pure water used is 20 mL / 0.35 g sodium selenite.
[0008] More preferably, in step 2), the hydrazine hydrate is a 50% analytical grade reagent, and the amount of hydrazine hydrate added is 9 mL / 0.35g sodium selenite; the stirring speed is 500-800 rpm, and the stirring reaction time is 30 minutes to ensure that the solution is mixed evenly without precipitation.
[0009] Preferably, in step 3), the high-pressure reactor is a polytetrafluoroethylene-lined reactor. During the solvothermal reaction, the oven temperature is kept constant. After the reaction, the pH value is adjusted to 7.0 to prevent material agglomeration. Then, centrifugation is performed at a speed of 10,000-14,000 rpm for 8-15 minutes. The product is then washed sequentially with deionized water and anhydrous ethanol, with each washing being done 2-3 times with pure water and anhydrous ethanol. After centrifugation and vacuum drying, pure manganese diselenide nanomaterials are obtained.
[0010] Preferably, in step S2, the mass ratio of manganese diselenide nanoparticles, SH-PEG-NH2 and dopamine hydrochloride is 10:1:10; the volume ratio of pure water, manganese diselenide nanoparticles and Tris-HCl is 20 mL:20 mg:30 mL.
[0011] Preferably, in step S2, the stirring is magnetic stirring at a speed of 400-600 rpm for 12-24 hours; the centrifugation is performed at a speed of 10000-14000 rpm for 8-15 minutes; and the washing is performed with pure water 2-3 times.
[0012] Preferably, the molecular weight of SH-PEG-NH2 in step S2 is 2000-5000 to ensure the hydrophilicity and biocompatibility of the nanoparticles.
[0013] Preferably, in step S3, the pPB modification occurs through a Michael addition reaction between the thiol groups (-SH) on the surface of the pPB molecule and the highly reactive o-benzoquinone groups on the surface of PDA under weakly alkaline conditions, forming a stable thioether bond (CSC). Ice bath stirring in the dark is used to suppress non-specific reactions, protect the active groups, and prevent dopamine oxidation. The mass ratio of MSP to pPB cyclic peptide is 15:1-10:1; the buffer solution is Tris-HCl buffer with a pH of 8.5; the stirring is magnetic stirring at 300-500 rpm for 8-12 hours. This invention creatively uses a Tris-HCl buffer system with a pH of 8.5 in step S3. This weakly alkaline environment effectively promotes the dissociation of the terminal thiol groups of the pPB cyclic peptide, significantly improving the efficiency of its Michael addition reaction with the quinone groups on the surface of polydopamine, ensuring stable and high-density modification of the targeting ligand. This is a technical effect that is difficult to achieve using a neutral buffer solution alone.
[0014] After the reaction is completed, the centrifugation speed is 10000-14000 rpm and the time is 8-15 minutes; the washing is performed by washing with pure water and ethanol 2-3 times each.
[0015] Preferably, the washing reagents are all sterile deionized water and anhydrous ethanol.
[0016] The present invention also provides a manganese diselenide@polydopamine-PPB nanomedicine prepared by the above-described preparation method, which is a composite nanomedicine for alleviating liver fibrosis.
[0017] This invention also provides the application of manganese diselenide@polydopamine-PPB nanomedicine prepared by the above-described preparation method, or the application of the above-described manganese diselenide@polydopamine-PPB nanomedicine in the preparation of drugs for the diagnosis or treatment of liver fibrosis. Compared with the prior art, the beneficial effects of the present invention are: In summary, this invention develops a pPB cyclic peptide-modified PDA-coated manganese diselenide nanomaterial. This nanomedicine, upon entering the liver fibrosis microenvironment, undergoes responsive degradation under weakly acidic pH conditions, releasing Mn. 2+ This nanomedicine enables T1-weighted MRI imaging for precise diagnosis of liver fibrosis lesions. Simultaneously, the released selenium exhibits antioxidant activity similar to GPx, effectively scavenging reactive oxygen species (ROS) at the lesion site and inhibiting hepatic stellate cell activation. It synergistically alleviates the progression of liver fibrosis through both oxidative stress regulation and cellular pathway inhibition. Furthermore, this nanomedicine demonstrates excellent biocompatibility and exhibits no significant cytotoxicity. Therefore, the manganese diselenide@PDA-PPB nanocomposite shows significant potential in the treatment of liver fibrosis, providing a novel and highly effective therapeutic strategy. Attached Figure Description
[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.
[0019] Figure 1 Transmission electron microscopy (TEM) images of the prepared manganese diselenide@PDA; Figure 2 Fourier transform infrared spectra of the prepared manganese diselenide and manganese diselenide@PDA; Figure 3 The hydrated particle size of the prepared manganese diselenide@PDA; Figure 4 Analysis of the T1 imaging capability of the prepared manganese diselenide@PDA; Figure 5 The results of the in vitro hemolysis experiment of the prepared MSPp nanospheres were determined; Figure 6The biocompatibility of the prepared MSPp nanospheres in 3T3 cells and HUVEC cells (at different concentrations). Figure 7 The scavenging effect of the prepared MSPp nanospheres on reactive oxygen species; Figure 8 The concentration uptake of the prepared MSPp nanospheres; Figure 9 The expression of liver fibrosis-related proteins in different groups. Detailed Implementation
[0020] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, unless otherwise specified, the following embodiments and features described therein can be combined with each other.
[0021] The sources of some of the raw materials for this invention are shown in Table 1: Table 1 This invention provides a method for preparing a composite nanomedicine that can alleviate liver fibrosis, comprising the following steps: S1. Preparation of manganese diselenide nanoparticles: 1) Sodium selenite, manganese acetate, and pure water are mixed to obtain solution A; 2) Mix solution A obtained in step 1) with hydrazine hydrate, stir and react to obtain solution B; 3) Transfer the solution B obtained in step 2) to the reaction vessel for solvothermal reaction to obtain solution C. Then adjust the pH value to 7.0 with citric acid to obtain solution D. After washing and drying, manganese diselenide nanoparticles are obtained. S2. Preparation of manganese diselenide@polydopamine nanomaterials (MSP): The manganese diselenide nanoparticles obtained in step S1 were first stirred with SH-PEG-NH2 in a pure water system in an ice bath in the dark for 4-6 hours. After centrifugation and washing, they were mixed with dopamine hydrochloride in a Tris-HCl (pH=8.5) system and reacted at room temperature in the dark to obtain manganese diselenide@polydopamine nanomaterials (MSP). S3. Preparation of manganese diselenide@polydopamine-pPB nanoprobes (MSPp): The MSP obtained in step S2 was mixed with the pPB cyclic peptide in Tris-HCl buffer (pH=8.5) and reacted in an ice bath in the dark for 6-12 hours with stirring to obtain manganese diselenide@polydopamine-pPB nanomedicine (MnSe2@PDA-pPB).
[0022] This invention involves mixing sodium selenite, manganese acetate, and purified water to obtain solution A. In this invention, the preferred mass ratio of sodium selenite to manganese acetate is 1:2. The preferred volume ratio of purified water to sodium selenite is 20 mL:0.35 g.
[0023] In this invention, solution A is mixed with hydrazine hydrate and stirred to obtain solution B. Preferably, the amount of hydrazine hydrate added is 9 mL. Stirring is preferably done using magnetic stirring at a speed of 500-800 rpm. The stirring reaction time is preferably 30 minutes. In this invention, the hydrazine hydrate acts as a reducing agent, reducing sodium selenite and manganese acetate to manganese diselenide.
[0024] In this invention, solution B is transferred to a reaction vessel for a solvothermal reaction to obtain manganese diselenide nanoparticles. Preferably, the temperature of the solvothermal reaction is 100°C, and the reaction time is 24 hours. The reaction is further preferably followed by pH adjustment, centrifugation, and washing to obtain the manganese diselenide nanoparticles. Preferably, the centrifugation speed is 10000-12000 rpm, and the centrifugation time is 10-15 minutes. Preferably, the precipitate obtained by centrifugation is washed 2-3 times each with pure water and anhydrous ethanol.
[0025] This invention involves mixing manganese diselenide nanoparticles with SH-PEG-NH2 and dopamine hydrochloride in a Tris-HCl system (pH=8.5) and reacting the mixture with stirring to obtain manganese diselenide@polydopamine nanomaterials (MSP). In this invention, the preferred mass ratio of the manganese diselenide nanoparticles to SH-PEG-NH2 and dopamine hydrochloride is 10:1:10. The preferred dosage ratio of Tris-HCl to manganese diselenide nanoparticles is 30 mL:20 mg. In this invention, SH-PEG-NH2 improves the dispersibility of the nanomaterial and enhances its safety, while dopamine hydrochloride forms a polydopamine coating layer. In this invention, the mixing is preferably carried out under stirring, with a preferred stirring speed of 400-600 rpm and a preferred stirring time of 12-24 hours. After stirring, centrifugation and washing are also preferred, with a preferred centrifugation speed of 12000 rpm and a preferred centrifugation time of 10 minutes. In this invention, the precipitate obtained by centrifugation is preferably washed 2-3 times with pure water.
[0026] In this invention, the obtained MSP and PPB cyclic peptide are mixed in a buffer solution and reacted with stirring to obtain manganese diselenide@polydopamine-PPB nanomedicine (MnSe2@PDA-PPB). In this invention, the mass ratio of MSP to pPB cyclic peptide is preferably 15:1-10:1. The buffer solution is preferably Tris-HCl buffer (pH=8.5). In this invention, the pPB cyclic peptide targets and activates the PDGFR-β receptor on the surface of hepatic stellate cells. In this invention, the mixing is preferably carried out with stirring, the stirring speed is preferably 300-500 rpm, and the stirring time is preferably 12 hours. After stirring, centrifugation and washing are also preferred, the centrifugation speed is preferably 12000 rpm, and the centrifugation time is preferably 10 minutes; the precipitate obtained by centrifugation is preferably washed 2-3 times each with pure water and ethanol.
[0027] This invention also provides the application of the anti-liver fibrosis nanomedicine described in the above-mentioned technical solution in the preparation of drugs for the diagnosis or treatment of liver fibrosis. This invention does not impose any particular limitations on the dosage form or preparation method of the drug; those skilled in the art can determine the appropriate dosage form and preparation method based on pharmaceutically acceptable formulations of manganese diselenide@polydopamine-pPB nanomedicine.
[0028] To further illustrate the present invention, the following detailed description is provided in conjunction with embodiments, but these should not be construed as limiting the scope of protection of the present invention. Example 1
[0029] A method for preparing a manganese diselenide@polydopamine-PPB nanomedicine (MnSe2@PDA-PPB, i.e., an anti-liver fibrosis nanomedicine) includes the following steps: 1. Preparation of manganese diselenide nanoparticles: 1) Dissolve 0.35 g sodium selenite and 0.70 g manganese acetate in 20 mL of pure water and stir until homogeneous to obtain solution A; 2) Add 9 mL of hydrazine hydrate to solution A and stir at 600 rpm for 30 minutes at room temperature to obtain solution B; 3) Transfer solution B to a Teflon-lined high-pressure reactor and maintain at 100°C for 24 hours. After the reaction is complete, centrifuge at 11,000 rpm for 10 minutes to collect the precipitate, and wash it twice with pure water and twice with anhydrous ethanol to obtain manganese diselenide nanoparticles (MnSe2).
[0030] 2. Preparation of manganese diselenide@polydopamine nanomaterials (MSP): Take 20 mg of manganese diselenide nanoparticles and mix them with 2 mg of SH-PEG-NH2 in 20 mL of pure water in an ice bath in the dark for 4-6 hours. After centrifugation and washing, add 20 mg of dopamine hydrochloride and dissolve it in 30 mL of Tris-HCl (pH=8.5). Stir at 500 rpm in the dark at room temperature for 6-12 hours. After stirring, collect the mixture by centrifugation at 12000 rpm for 10 minutes and wash it twice with pure water to obtain manganese diselenide@polydopamine nanomaterials, abbreviated as MSP.
[0031] 3. Preparation of manganese diselenide@polydopamine-pPB nanomedicine (MSPp): Take 10 mg of MSP, add 1 mg of PPB cyclic peptide, dissolve in 5 mL of Tris-HCl buffer (pH=8.5), and stir at 400 rpm for 12 hours under ice bath conditions protected from light. After stirring, centrifuge at 12000 rpm for 10 minutes to collect the sample, and wash twice with pure water and twice with ethanol to obtain manganese diselenide@polydopamine-pPB nanomedicine, abbreviated as MSPp.
[0032] Experimental Example 1 Performance Analysis: (1) such as Figure 1 As shown, an appropriate amount of manganese diselenide@polydopamine nanomaterial (MSP) was prepared into a 1 mg / mL solution with pure water. 10-20 µL of the solution was pipetted onto a copper grid, dried, and used to prepare a transmission electron microscopy (TEM) sample. The results showed that the prepared MnSe2@PDA exhibited a uniform spherical shape and a distinct core-shell structure (MnSe2 core and PDA shell).
[0033] (2) For example Figure 2 As shown, appropriate amounts of manganese diselenide (MnSe2) and manganese diselenide@polydopamine nanomaterials (MnSe2@PDA) were taken, and their infrared absorption spectra were measured using a Fourier transform infrared spectrometer. The results showed that, compared with pure MnSe2, MnSe2@PDA exhibited significantly higher absorption spectra at 3600 cm⁻¹. -1 A broad peak for the phenolic hydroxyl group appears nearby, at 1650 cm⁻¹. -1 A characteristic C=C peak of the aromatic ring appears nearby, at 1280 cm⁻¹. -1 The presence of CN characteristic peaks nearby indicates that PDA was successfully coated on the MnSe2 surface.
[0034] (3) such as Figure 3 As shown, manganese diselenide@polydopamine nanomaterials were placed in pure water and sonicated for 2 min. The hydrated particle size distribution of the nanoparticles was then measured and calculated using a particle size analyzer. The results showed that the hydrated particle size of the prepared MnSe2@PDA was approximately 150-200 nm and was uniformly distributed.
[0035] (4) As Figure 4 shown, first, the MnSe2@PDA-pPB composite nanomaterial was prepared into a MSPp stock solution with a manganese concentration of 20 μg / mL based on the manganese concentration. Then, at least 5 concentration gradients from 20 μg / mL to 0 μg / mL were obtained by the serial dilution method. For each concentration point, at least 300 μL of sample solution was finally prepared to ensure that 200 μL of the sample was used for each measurement. 200 μL of the sample at each concentration point was transferred separately into a chamber compatible with a 0.5T magnetic resonance imaging instrument, and then measurements were carried out to obtain the T1 values at different concentrations. Subsequently, a graph was plotted with the manganese concentration as the abscissa and the reciprocal of T1 as the ordinate to obtain a fitting curve. The results showed that the longitudinal relaxation rate (1 / T1) of the sample showed a good linear upward trend, and the slope of the fitting straight line was the longitudinal relaxation rate r1 of the material. The calculated value was approximately 14.121 mM -1 ·s -1 , and the intercept of the fitting straight line at a manganese concentration of 0 approached 0, indicating that the background relaxation of the pure water system had little interference on the test results. This result indicated that the composite nanomaterial MnSe2@PDA-pPB had a significant concentration-dependent T1 relaxation enhancement ability at a 0.5T magnetic field strength, could effectively shorten the longitudinal relaxation time of the surrounding water protons, and had the application potential as a T1-weighted magnetic resonance imaging contrast agent.
[0036] (5) As Figure 5 shown, MnSe2@PDA-pPB was added to a 2% (v / v) red blood cell suspension, and red blood cell suspensions with final concentrations of 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, and 100 μg / mL were prepared respectively. The red blood cell suspension diluted with PBS was used as a negative control (-), representing the normal state without hemolysis, and was used to judge whether hemolysis occurred in the experimental group. The supernatant should be clear and all red blood cells should sink. The red blood cell suspension diluted with ultrapure water was used as a positive control (+), representing the complete hemolysis state (all red blood cells ruptured), and was used to calculate the hemolysis rate. The supernatant should be red and transparent. Each group of solutions was incubated at 37 °C for 4 h, and then the solutions were centrifuged at 3000 rpm for 15 min. The hemolysis phenomenon was photographed, and the absorbance of the samples at 542 nm was detected with an enzyme-labeled instrument. Finally, through the formula: Hemolysis rate (%) = [(A 样品 - A 阴性对照 ) / (A 阳性对照 - A 阴性对照The hemolysis rate was calculated by multiplying the result by 100%. The results showed that when MnSe2@PDA-PPB was co-incubated with erythrocytes at different concentrations, almost all erythrocytes sank, and the supernatant showed no significant change compared to the negative control group; the hemolysis rate was consistently below 5%. This indicates that the prepared MnSe2@PDA-PPB did not cause significant hemolysis and has good blood compatibility.
[0037] (6) For example Figure 6 As shown, human umbilical vein endothelial cells (HUVECs) and mouse embryonic fibroblasts (3T3) with controllable passage numbers (5-10 passages) were selected, at a ratio of 1×10⁻⁶. 4 Cells were cultured at a density of cells / well in 96-well plates and incubated for 24 h in an incubator containing 5% CO2. After cell stabilization, different concentrations (0, 2.5, 5, 10, 20, 40 μg / mL) of MnSe2@PDA-pPB were added to the wells and co-incubated with the cells for 24 h. Afterward, the culture medium containing the material was aspirated, and medium containing 5 mg / mL MTT was added and reacted for 4 h. Then, DMSO was added and the cells were reacted in a shaker for 15 min. The absorbance of the cells at each concentration was measured using a microplate reader, and cell viability was calculated. The results showed that within the experimental concentration range, MnSe2@PDA-PPB had no significant cytotoxicity to normal human umbilical vein endothelial cells (HUVECs) and mouse embryonic fibroblasts (3T3), and the cell viability was above 85%, indicating that the material has good biocompatibility with normal cells and meets the safety requirements for in vivo application.
[0038] (7) For example Figure 7 As shown, prepare a confocal dish and add 2×10⁻⁶ ppm of the solution to each well. 5LX-2 cells, incubated with TGF-β-containing medium, were cultured for 24 h in a 37°C incubator containing 5% CO2. Afterward, each well was treated with a reactive oxygen species (ROSup:DMEM=1:1000) positive control reagent for 30 min. Then, wells containing 10 μg / mL MnSe2@PDA (MSP) and MnSe2@PDA-PPB (MSPp) were incubated for 6 h. The positive control group received the ROS positive control reagent for 30 min, followed by medium replacement. The negative control group received no treatment. Four groups were formed: negative control, positive control, MnSe2@PDA (MSP) treatment group, and MnSe2@PDA-PPB (MSPp) treatment group. MnSe2@PDA-PPB was co-incubated with activated LX-2 cells. After 6 h, intracellular ROS levels were detected using the DCFH-DA fluorescent probe. The results showed that, compared with the co-incubation results of the MnSe2@PDA group, the ROS fluorescence signal of the MnSe2@PDA-pPB treatment group was significantly reduced, indicating that the nanomedicine modified with pPB cyclic peptide was more effective in scavenging intracellular reactive oxygen species.
[0039] (8) such as Figure 8 As shown, cy5.5-labeled MnSe2@PDA-PPB was co-incubated with LX-2 cells at concentrations of 0, 10, and 40 µg / mL for 4 h. The uptake of the nanomaterial by the cells was detected using an inverted fluorescence microscope or a confocal microscope. The results showed that the red fluorescence captured by the microscope gradually increased with increasing concentration, indicating that the prepared MnSe2@PDA-PPB could be effectively taken up by LX-2 cells in a concentration-dependent manner.
[0040] (9) such as Figure 9 As shown, LX-2 cells incubated with TGF-β-containing medium were seeded at a density of 2 x 10⁻⁶ cells per well. 5Cells were seeded into six-well plates and cultured for 24 h in a 37°C incubator containing 5% CO2. Different concentrations (0, 5, 10, 20 μg / mL) of MnSe2@PDA-pPB material were then added and cultured for another 24 h. Cells were then scraped off and lysed using a cell scraper, and protein concentration was determined using a BCA protein quantification kit. Subsequently, 20 μg of protein was loaded per well, and the proteins were separated by SDS-PAGE electrophoresis (upper gel constant voltage 90V, lower gel constant voltage 150V) and transferred to a PVDF membrane. After blocking with 5% skim milk powder, antibodies against α-SMA, COL1A1, and GAPDH (1:1000-2000) were sequentially applied. Finally, protein bands were visualized using an ECL assay kit. The results showed that as the concentration of the nanomaterial increased, the protein expression levels of α-SMA and COL1A1 significantly decreased, confirming the anti-liver fibrosis ability of MnSe2@PDA-PPB.
[0041] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.
Claims
1. A method for preparing a composite nanomedicine that can alleviate liver fibrosis, characterized in that, The preparation method includes the following steps: S1. Dissolve sodium selenite and manganese acetate in pure water and stir until homogeneous to obtain solution A; add hydrazine hydrate to solution A and stir at room temperature to obtain solution B; transfer solution B to a reaction vessel and carry out a solvothermal reaction. After the reaction is completed, adjust the pH of the solution to 7.0, centrifuge and wash the product to obtain manganese diselenide nanoparticles. S2. The manganese diselenide nanoparticles obtained in step S1 are first stirred with SH-PEG-NH2 in a pure water system in an ice bath in the dark for 4-6 hours. After centrifugation and washing, they are mixed with dopamine hydrochloride in a Tris-HCl system at pH=8.
5. The mixture is stirred at room temperature in the dark. After the reaction is completed, the product is centrifuged and washed to obtain manganese diselenide@polydopamine nanomaterials, namely MnSe2@PDA, abbreviated as MSP. S3. Mix the MSP obtained in step S2 with the pPB cyclic peptide in a buffer solution, stir the mixture in an ice bath in the dark, and centrifuge and wash the product after the reaction is complete to obtain manganese diselenide@polydopamine-pPB nanomedicine, namely MnSe2@PDA-pPB, abbreviated as MSPp.
2. The preparation method according to claim 1, characterized in that, In step S1, during the preparation of solution A, the mass ratio of sodium selenite to manganese acetate is 1:2; the amount of pure water used is 20 mL / 0.35 g sodium selenite.
3. The preparation method according to claim 1, characterized in that, In step S1, during the preparation of solution B, the amount of hydrazine hydrate added is 9 mL / 0.35 g sodium selenite; the stirring is magnetic stirring at a speed of 500-800 rpm; and the stirring reaction time is 30 minutes.
4. The preparation method according to claim 1, characterized in that, In step S1, the temperature of the solvothermal reaction is 100°C and the time is 24 hours.
5. The preparation method according to claim 1, characterized in that, In step S1, after the reaction is completed, the centrifugation speed is 10000-14000 rpm and the time is 8-15 minutes; the washing is performed by washing with pure water and anhydrous ethanol 2-3 times each.
6. The preparation method according to claim 1, characterized in that, In step S2, the mass ratio of manganese diselenide nanoparticles, SH-PEG-NH2 and dopamine hydrochloride is 10:1:10; the volume ratio of pure water, manganese diselenide nanoparticles and Tris-HCl is 20 mL:20 mg:30 mL.
7. The preparation method according to claim 1, characterized in that, In step S2, the stirring is magnetic stirring at a speed of 400-600 rpm for 12-24 hours; the centrifugation is performed at a speed of 10000-14000 rpm for 8-15 minutes; and the washing is performed with pure water 2-3 times.
8. The preparation method according to claim 1, characterized in that, In step S3, the mass ratio of MSP to pPB cyclic peptide is 15:1-10:1; the buffer solution is a Tris-HCl buffer solution with pH=8.5; the stirring is a magnetic stirrer with a speed of 300-500 rpm for 8-12 hours. After the reaction is completed, the centrifugation speed is 10000-14000 rpm and the time is 8-15 minutes; the washing is performed by washing with pure water and ethanol 2-3 times each.
9. The manganese diselenide@polydopamine-pPB nanomedicine prepared by the preparation method according to any one of claims 1-8.
10. The use of the manganese diselenide@polydopamine-pPB nanomedicine prepared by the preparation method according to any one of claims 1-8 or the manganese diselenide@polydopamine-pPB nanomedicine according to claim 9 in the preparation of drugs for the diagnosis or treatment of liver fibrosis.