Targeted multifunctional nano diagnosis and treatment agent of glucomannan modified mesoporous dopamine as well as preparation and application of targeted multifunctional nano diagnosis and treatment agent

Abstract

The invention relates to a gulcomannan modified mesoporous dopamine targeted multifunctional nano diagnosis and treatment agent and preparation and application thereof.The targeted multifunctional nano diagnosis and treatment agent comprises a carrier and lovastatin, the carrier is a manganese ion coordinated mesoporous polydopamine carrier, and the surface of the carrier is modified with gulcomannan. The diagnosis and treatment agent has a good drug loading rate and a good delivery effect, overcomes the defect of low oral absorption rate of single lovastatin, can carry out MRI imaging monitoring, and provides a new thought for integration of diagnosis and treatment of liver cirrhosis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical engineering materials, and particularly relates to a targeted multifunctional nano-diagnostic and therapeutic agent of glucomannan-modified mesoporous dopamine, and the preparation and application thereof. Background Art

[0002] Liver cirrhosis is a terminal disease that develops from chronic liver disease. Its pathological characteristics include liver fibrosis, nodular regeneration, and diffuse destruction of liver tissue structure. It is often caused by viral hepatitis (such as hepatitis B and C), alcoholic liver disease, non-alcoholic fatty liver disease (NAFLD), etc. Some genetic diseases such as hemochromatosis and Wilson's disease may also lead to cirrhosis. During the progression of the disease, chronic inflammation and oxidative stress are key driving factors. Overactivated macrophages release pro-fibrotic factors that aggravate collagen deposition, while large amounts of reactive oxygen species (ROS) generated by oxidative stress further damage liver cells and accelerate the process of fibrosis. Patients with cirrhosis often face serious complications such as ascites, esophageal variceal bleeding, hepatic encephalopathy, and hepatocellular carcinoma. Existing treatment methods mainly rely on etiology control and symptomatic support, and lack efficient targeted treatment and real-time monitoring methods. Therefore, the development of an integrated diagnosis and treatment platform that can target chronic inflammation and oxidative stress while also having imaging monitoring capabilities has become an important need for the treatment of cirrhosis.

[0003] Lovastatin is primarily used to lower cholesterol and prevent cardiovascular disease. In recent years, its potential role in the treatment of liver cirrhosis has gradually attracted attention. However, due to its strong lipophilicity, its oral absorption rate is low and it requires hydrolysis in the liver to form its active open-ring hydroxy acid form before it can exert its pharmacological effects. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a targeted multifunctional nano-diagnostic agent with both treatment and imaging monitoring functions, and its preparation and application, based on the drug lovastatin, so as to better exert the effect of lovastatin.

[0005] The first aspect of the present invention provides a targeted multifunctional nano-diagnostic and therapeutic agent of glucomannan-modified mesoporous dopamine, comprising a carrier and lovastatin. The carrier is a mesoporous polydopamine carrier coordinated by manganese ions, and the surface of the carrier is modified with glucomannan.

[0006] Dopamine and its polymers have good biocompatibility, can exist stably in the body, and have low cytotoxicity. Through chemical modification, dopamine-based carriers can be given different functions, such as targeting, pH responsiveness, etc. 2+ ) is an excellent MRI contrast agent that enhances T1-weighted imaging signals and is widely used in clinical liver imaging. The imaging effect is achieved by coordinating manganese ions in the carrier.

[0007] Glucomannan (β-Glucan) is a natural polysaccharide that, through modification, can bind to the Dectin-1 receptor on the surface of macrophages, achieving immune activation and specific targeted accumulation in liver macrophages. Furthermore, after glucomannan modification, the dopamine carrier becomes pH-responsive, allowing rapid drug release in acidic environments, increasing local drug concentrations and reducing toxicity to normal tissues, thereby significantly enhancing therapeutic efficacy. Furthermore, glucomannan modification can improve the carrier's hydrophilicity, facilitating drug delivery.

[0008] The diagnostic and therapeutic agent of the present invention is constructed by loading lovastatin on a polydopamine carrier, and the carrier is modified with glucomannan. This multifunctional nano-diagnostic and therapeutic agent can effectively load lovastatin, has pH responsiveness, good delivery effect, and can be monitored by MRI imaging, providing a new idea for the integrated diagnosis and treatment of cirrhosis.

[0009] Preferably, the glucomannan is prepared by enzymatic hydrolysis of konjac glucomannan, has a molecular weight of 70,000, and a degree of deacetylation greater than 85%.

[0010] Preferably, the mass ratio of the carrier to lovastatin is 1-5:1, such as 1:1, 2:1, 3:1, 4:1, 5:1, etc.

[0011] The second aspect of the present invention further provides a method for preparing the targeted multifunctional nano-diagnostic and therapeutic agent, comprising the steps of:

[0012] (1) Mesoporous dopamine nanoparticles were prepared using a soft film method;

[0013] (2) β-1,4-glucanase and konjac glucomannan were dissolved in PBS solution with pH=7.4 respectively, stirred at 37°C, and after the reaction was completed, the degraded glucomannan was obtained by centrifugation.

[0014] (3) Using HEPES buffer at pH = 7.4 as a solvent, the dopamine nanoparticles in (1) and the glucomannan obtained in (2) were used to prepare a mesoporous dopamine nanoparticle solution and a manganese chloride solution, respectively. Then, under vigorous stirring in a water bath at 25°C, the manganese chloride solution was dropped into the mesoporous dopamine solution for reaction. The mesoporous polydopamine particles MnPDA coordinated with manganese ions were collected by centrifugation, and then washed with HEPES buffer at pH = 7.4 and resuspended in deionized water to obtain a MnPDA solution.

[0015] (4) dissolving glucomannan in sodium acetate buffer at pH 4.5 to obtain a glucomannan solution, and then adding the MnPDA solution obtained in (3) to the glucomannan solution, stirring overnight, centrifuging, and washing to obtain the carrier MnPDA-Glu;

[0016] (5) Lovastatin was dispersed in ultrapure water, and the MnPDA-Glu solution obtained in (4) was added dropwise while stirring, and the mixture was stirred at room temperature. The mixture was collected by centrifugation to obtain the targeted multifunctional nano-diagnostic and therapeutic agent LS@MnPDA-Glu.

[0017] Preferably, the mesoporous dopamine nanoparticles are prepared by a soft template method in step (1), specifically comprising the steps of: mixing an ethanol solution of Pluronic F-127 with an aqueous solution of dopamine hydrochloride, and then adding 1,3,5-trimethylbenzene; then ultrasonically dispersing and mixing until completely clear, adding ammonia water and continuing to stir to initiate a dopamine polymerization reaction, reacting at room temperature for 60 minutes, centrifuging to remove Pluronic F127, 1,3,5-trimethylbenzene and unreacted dopamine molecules, and then washing the precipitate to obtain mesoporous dopamine nanoparticles;

[0018] According to the mass ratio, Pluronic F-127: dopamine hydrochloride: 1,3,5-trimethylbenzene: ammonia water = 30-60 mg: 10-25 mg: 8-26 mg; the volume ratio of ammonia water to the mixed solution is 0.05-0.1: 2.06-3.13.

[0019] Preferably, in step (2), the enzymatic hydrolysis reaction is carried out according to the mass ratio of β-1,4-glucanase:konjac glucomannan=0.03-0.05:2-5; and the stirring reaction time is 12-24 hours.

[0020] Preferably, in step (3), the volume ratio of mesoporous dopamine nanoparticle solution: manganese chloride solution: resuspended deionized water is 2-8:1-5:5-15; the mass ratio of mesoporous dopamine: manganese chloride is 3-6:0.6; and the reaction time is 30-150 min.

[0021] Preferably, in step (4), the glucomannan concentration in the glucomannan solution is 0.02-0.1 g / mL.

[0022] Preferably, in step (4), the glucomannan solution is prepared by dissolving glucomannan in sodium acetate buffer at pH = 4.5 and heating at 50-70° C. for 0.5-1.5 h.

[0023] The third aspect of the present invention also provides the use of the targeted multifunctional nano-diagnostic and therapeutic agent in the preparation of drugs for magnetic resonance imaging of the liver.

[0024] Compared with the prior art, the present invention has the following beneficial effects: The mesoporous polydopamine carrier has a good specific surface area and pore structure. Modification with manganese ions enhances imaging effects, while modification with glucomannan improves its hydrophilicity, facilitating oral drug delivery. It also enables pH responsiveness and release in acidic environments. Loading lovastatin on this carrier ensures both a good drug loading rate and a good delivery effect, overcoming the low oral absorption rate of lovastatin alone. Furthermore, the preparation method is simple, the reaction conditions are mild, the process is easy to operate, and the cost is low. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 The drug release curves of LS@MnPDA-Glu in different pH environments are shown. Figure 2 The particle size changes of MnPDA-Glu in different pH environments are shown. Figure 3 The cytotoxicity of MnPDA-Glu on L929 and raw264.7 cells is shown.

[0025] The mesoporous polydopamine carrier has a good specific surface area and pore structure. Modification with manganese ions enhances imaging effects, while modification with glucomannan improves its hydrophilicity, facilitating oral drug delivery. It also enables pH responsiveness and release in acidic environments. Loading lovastatin on this carrier ensures both a good drug loading rate and a good delivery effect, overcoming the low oral absorption rate of lovastatin alone. Furthermore, the preparation method is simple, the reaction conditions are mild, the process is easy to operate, and the cost is low. DETAILED DESCRIPTION

[0026] The present invention is further described below with reference to specific examples, which, however, are not intended to limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the art.

[0027] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0028] Example 1:

[0029] (1) Preparation of mesoporous polydopamine nanoparticles (MPDANPs)

[0030] Mesoporous polydopamine nanoparticles were prepared using the "soft template method".

[0031] To a clean 15 mL reaction vial, add 1.5 mL of Pluronic F-127 (50 mg) in ethanol and 1.5 mL of dopamine (15 mg) in water. Add 20 μL of 1,3,5-trimethylbenzene (TMB). Disperse the mixture using an ultrasonic grinder and mix until completely clear. Then, add 90 μL of NH₃·H₂O and continue stirring to initiate dopamine polymerization. After reacting at room temperature for 60 minutes, centrifuge (15,000 rpm for 20 minutes) to remove any "soft mold" F127, TMB, and unreacted dopamine molecules. The precipitate is washed three times with water and ethanol and finally resuspended in 10 mL of ultrapure water for use and concentration determination.

[0032] (2) Preparation of coordination of manganese ions and mesoporous dopamine nanoparticles

[0033] 4 mL of the mesoporous dopamine resuspension obtained in step (1) was diluted to 10 mL with HEPES buffer (10 mM, pH 7.4), 2.5 mg of manganese chloride tetrahydrate was dissolved in 2 mL of HEPES buffer, and the manganese chloride tetrahydrate solution was dropped into the mesoporous dopamine solution under vigorous stirring in a 25 ° C water bath. The reaction was continued for 1 h, and then the MnPDA nanoparticles were collected by centrifugation (15000 rpm, 20 min), washed twice with HEPES buffer (10 mM, pH 7.4), and resuspended with 10 mL of deionized water. It was stored at 4 ° C for use. The product was recorded as MnPDA NPs.

[0034] (3) Degradation of Konjac Glucomannan

[0035] 40 mg of β-1,4-glucanase and 4 g of konjac glucomannan were dissolved in PBS (pH 7.4). The enzyme solution was then added to the konjac glucomannan solution and stirred at 37°C for 24 hours. After the reaction, the degraded glucomannan was centrifuged (3500 rpm, 8 minutes). The supernatant was collected, the product was lyophilized, and used later. Testing showed that the degraded glucomannan had a molecular weight of 70,000 Daltons and a degree of deacetylation of 89%.

[0036] (4) Preparation of glucomannan-modified MnPDA (MnPDA-Glu)

[0037] First, 0.5 g of glucomannan (0.5 g) obtained in step (3) was dissolved in 10 mL of sodium acetate buffer (pH 4.5) and heated at 60°C for 1 hour. Then, 10 mL of the MnPDA-GM NPs solution was added to the cooled glucomannan solution and stirred overnight. The MnPDA-GM NPs were then washed three times with deionized water by centrifugation (14,000 rpm, 10 min, 4°C) and dispersed in 5 mL of ultrapure water for further use.

[0038] (5) Preparation of LS@MnPDA-Glu

[0039] Weigh 3 mg of lovastatin and dissolve it in 5 mL of ultrapure water. Add the solution dropwise to 15 mL of MnPDA-Glu (9 mg) while stirring. Stir at room temperature for 12 h, and collect the product, Gal-LS@MnPDA-Glu, by centrifugation.

[0040] Test Example 1 Encapsulation Efficiency and Drug Loading Rate of Lovastatin

[0041] The absorbance of lovastatin was measured at 238 nm using an ultraviolet spectrophotometer, and the lovastatin content was calculated based on the measured standard curve. The encapsulation efficiency (1) and drug loading efficiency (2) were calculated according to the following formulas, where w1 is the actual loading amount of the drug, w2 is the total amount of drug input, and w3 is the total amount of carrier and drug.

[0042] EE (%) = w1 / w2×100% (1)

[0043] DLC (%) = w1 / w3×100% (2)

[0044] Finally, the encapsulation efficiency and drug loading rate of MnPDA-Glu for lovastatin were calculated to be 75.32% and 24.51%.

[0045] Test Example 2: Drug Release Curve of LS@MnPDA-Glu

[0046] First, 2 ml of LS@MnPDA-Glu solution (1 mg / mL) was added to a dialysis bag and immersed in 20 ml of phosphate-buffered saline (PBS) containing 0.5% Tween 80 (pH = 5.5, 7.4). The bag was then transferred to a thermostatic shaker (37°C, 100 rpm) for incubation. Subsequently, 2 ml of the PBS solution was removed at different time points and 2 ml of fresh PBS solution was added. The absorbance of the removed solution was measured at 240 nm, and the drug release rate was calculated according to Equation (3).

[0047] Drug release (%) = (Cn × V0 + ∑C n-1 ×V1) / m0×100%(3)

[0048] Where Cn is the concentration of the nth sample, V0 is the total volume of the buffer, V1 is the volume of each sample, and m0 represents the total amount of drug in the sample.

[0049] like Figure 1As shown, LS@MnPDA-Glu releases faster in an acidic environment at pH 5.0 than in a neutral environment, demonstrating its excellent release in the inflammatory microenvironment. At 12 hours, the drug release rate in an acidic environment reaches over 60%, indicating that upon reaching the inflammatory site, the drug can be rapidly released to exert its effects while simultaneously clearing excess ROS in the inflammatory environment. In a neutral environment, the release rate is only 25% after 72 hours, demonstrating that the nanoparticles can significantly reduce their toxic effects on normal cells.

[0050] Test Example 3: Particle Size Changes of MnPDA-Glu in Different pH Environments

[0051] An appropriate amount of MnPDA-Glu was dispersed in PBS with a pH of 5.0 and 7.4, respectively, and placed at 37°C for 4 hours before being taken out for DLS detection.

[0052] The results are as follows Figure 2 As shown in the figure, after 4 hours in a pH 5.0 environment, the particle size of MnPDA-Glu changed significantly and became non-uniform, while in a pH 7.4 PBS environment, the particle size remained relatively uniform and did not change significantly. This indicates that MnPDA-Glu will undergo significant biodegradation in an acidic environment, thereby rapidly releasing the loaded lovastatin. Figure 1 The results also proved this.

[0053] Test Example 4: Cytotoxicity

[0054] The cell activity was detected by CCK-8 assay to evaluate the cytotoxicity of MnPDA-Glu obtained in step (4) on L929 and raw264.7 cells.

[0055] The specific operation steps are as follows: First, L929 or raw264.7 cells are seeded in a 96-well plate at a density of 5000 cells / well, and then placed in a carbon dioxide incubator to culture overnight. Subsequently, the original culture medium is aspirated and replaced with fresh complete culture medium containing different concentrations of MnPDA-Glu. The selected concentration range of MnPDA-Glu is 0-1000μg / mL, and each concentration has 5 parallels. Then culture in an incubator for 24h. After culture, the cells are washed once with PBS and 100μL of fresh culture medium (containing 10% CCK-8) is added to each well. Place in an incubator and incubate for a period of time. Finally, use a microplate reader to detect and record the absorbance at a wavelength of 450nm, and calculate the cell survival rate by the following formula:

[0056] Cell viability (%) = (absorbance of experimental group - absorbance of blank group) / (absorbance of negative control group - absorbance of blank group) × 100%.

[0057] like Figure 3 As shown in the results, when the concentration of Gal-AMn-MPDA reached 1000 μg / mL, the cell viability of both L929 and raw264.7 cells exceeded 95%, indicating that even high concentrations of nanoparticles did not affect the proliferation of normal cells. This demonstrates the excellent biocompatibility of the nanoparticles.

[0058] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0059] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A multifunctional nano-diagnostic agent for targeting mesoporous dopamine modified with glucomannan, characterized in that: The invention comprises a carrier and lovastatin. The carrier is a mesoporous polydopamine carrier coordinated by manganese ions, and the surface of the carrier is modified with glucomannan.

2. The targeted multifunctional nano-diagnostic and therapeutic agent according to claim 1, characterized in that The glucomannan is prepared by enzymatic hydrolysis of konjac glucomannan, has a molecular weight of 70,000, and a deacetylation degree greater than 85%.

3. The targeted multifunctional nano-diagnostic and therapeutic agent according to claim 1, characterized in that The mass ratio of the carrier to lovastatin is 1-5:

1.

4. The method for preparing the targeted multifunctional nano-diagnostic and therapeutic agent according to any one of claims 1 to 3, characterized in that: Including steps: (1) Mesoporous dopamine nanoparticles were prepared using a soft film method; (2) β-1,4-glucanase and konjac glucomannan were dissolved in PBS solution with pH=7.4 respectively, stirred at 37°C, and after the reaction was completed, the degraded glucomannan was obtained by centrifugation. (3) Using HEPES buffer at pH = 7.4 as a solvent, the dopamine nanoparticles in (1) and the glucomannan obtained in (2) were used to prepare a mesoporous dopamine nanoparticle solution and a manganese chloride solution, respectively. Then, under vigorous stirring in a water bath at 25°C, the manganese chloride solution was dropped into the mesoporous dopamine solution for reaction. The mesoporous polydopamine particles MnPDA coordinated with manganese ions were collected by centrifugation, and then washed with HEPES buffer at pH = 7.4 and resuspended in deionized water to obtain a MnPDA solution. (4) dissolving glucomannan in sodium acetate buffer at pH 4.5 to obtain a glucomannan solution, and then adding the MnPDA solution obtained in (3) to the glucomannan solution, stirring overnight, centrifuging, and washing to obtain the carrier MnPDA-Glu; (5) Lovastatin was dispersed in ultrapure water, and the MnPDA-Glu solution was added dropwise during stirring. The mixture was stirred at room temperature and collected by centrifugation to obtain the targeted multifunctional nano-diagnostic and therapeutic agent LS@MnPDA-Glu.

5. The preparation method according to claim 4, characterized in that Step (1) specifically includes: An ethanol solution of Pluronic F-127 and an aqueous solution of dopamine hydrochloride were mixed, and 1,3,5-trimethylbenzene was added; the mixture was then ultrasonically dispersed and mixed until completely clear, and then ammonia water was added and stirred continuously to initiate a dopamine polymerization reaction. After the reaction was allowed to proceed at room temperature for 60 minutes, the Pluronic F127, 1,3,5-trimethylbenzene, and unreacted dopamine molecules were removed by centrifugation, and the precipitate was washed to obtain mesoporous dopamine nanoparticles. According to the mass ratio, Pluronic F-127: dopamine hydrochloride: 1,3,5-trimethylbenzene: ammonia water = 30-60 mg: 10-25 mg: 8-26 mg; the volume ratio of ammonia water to the mixed solution is 0.05-0.1: 2.06-3.

13.

6. The preparation method according to claim 4, characterized in that In step (2), the mass ratio of β-1,4-glucanase to konjac glucomannan is 0.03-0.05:2-5; and the stirring reaction time is 12-24 hours.

7. The preparation method according to claim 4, characterized in that In step (3), the volume ratio of mesoporous dopamine nanoparticle solution: manganese chloride solution: resuspended deionized water is 2-8:1-5:5-15; the mass ratio of mesoporous dopamine: manganese chloride is 3-6:0.6; and the reaction time is 30-150 min.

8. The preparation method according to claim 4, characterized in that In step (4), the glucomannan concentration in the glucomannan solution is 0.02 to 0.1 g / mL.

9. The preparation method according to claim 4, characterized in that In step (4), the glucomannan solution is prepared by dissolving glucomannan in a sodium acetate buffer solution with a pH of 4.5 and heating the solution at 50-70° C. for 0.5-1.5 h.

10. Use of the targeted multifunctional nano-diagnostic and therapeutic agent according to any one of claims 1 to 3 in the preparation of drugs for magnetic resonance imaging of the liver.

Images