Glycyrrhetinic acid-modified block copolymer and use thereof as a liver-targeting drug carrier

Abstract

The application discloses a glycyrrhetinic acid modified block copolymer and application thereof as a liver-targeting drug carrier. The block copolymer provided by the application is proposed for the first time and comprises a hydrophilic segment, a glycyrrhetinic acid modified hydrophobic segment, wherein the hydrophilic segment is polyethylene glycol or polyethylene glycol monomethyl ether, and the hydrophobic segment is a polyester with a terminal hydroxyl group modified by glycyrrhetinic acid esterification. The block copolymer provided by the application can be applied as a drug carrier material, self-assembled into a drug-loaded nanomicelle in an aqueous medium, and significantly improves the drug loading capacity, solubility and stability of a hydrophobic drug molecule; and has an active liver targeting property, can direct drug delivery to liver tissues, improves the drug treatment effect, and effectively reduces the systemic toxic side effects.

Description

Technical Field

[0001] This invention relates to glycyrrhetinic acid-modified block copolymers and their application as liver-targeting drug carriers, belonging to the field of biomedical technology. Background Technology

[0002] Polymer micelles are a promising new drug delivery system, with amphiphilic block copolymers exhibiting excellent biocompatibility and biodegradability finding wide application. A large number of amphiphilic block copolymer molecular chains are oriented to form nanomicelles with a typical core-shell structure. Drug molecules are encapsulated within the hydrophobic core through weak interactions with hydrophobic segments, while the hydrophilic chains form a stable outer shell. This unique structure allows for the encapsulation of drugs with varying properties, increasing drug solubility and preventing degradation and inactivation. This makes them widely used for delivering hydrophobic anticancer drugs, metal complexes, siRNA, and more. Furthermore, the size of polymeric material micelles is typically less than 100 nm, enabling them to circulate stably in the bloodstream for extended periods and accumulate effectively in target tissues such as tumors based on the EPR effect, demonstrating significant advantages as a drug delivery system.

[0003] Glycyrrhetinic acid (GA) is a pentacyclic triterpenoid saponin found in licorice, possessing a wide range of pharmacological activities including anti-inflammatory, anticancer, and antibacterial effects. It has been confirmed that the cell membrane fraction of isolated rat hepatocyte homogenates contains numerous glycyrrhetinic acid-specific binding sites, exhibiting the highest activity. The binding of glycyrrhetinic acid to these sites is saturable and highly specific, making it suitable as a specific receptor for liver-targeted delivery.

[0004] Liver-targeted delivery systems using glycyrrhetinic acid as a ligand have attracted widespread attention. Chinese patent CN101254308A discloses a glycyrrhetinic acid-polyethylene glycol / chitosan liver-targeted composite drug delivery system and its preparation method. This composite drug delivery system uses the liver-targeting compound glycyrrhetinic acid-polyethylene glycol and chitosan or chitosan derivatives as carrier materials, and is prepared by ion-crosslinking to encapsulate drugs for treating liver diseases and anticancer drugs. This patent connects glycyrrhetinic acid and polyethylene glycol to form a liver-targeting carrier material for preparing composite nanoparticles. The entire process involves multi-step synthesis, making the preparation method complex. Furthermore, glycyrrhetinic acid is a hydrophobic group, and modifying it with hydrophilic groups such as polyethylene glycol would affect its hydrophilic properties, limiting its applications. Chinese patent CN 104725631A discloses a polyethylene glycol monomethyl ether material modified with glycyrrhetinic acid as a liver-targeting drug carrier; Chinese patent CN102558546A discloses a method for esterifying polyethylene glycol or polyethylene glycol monomethyl ether using the hydroxyl or carboxyl groups of glycyrrhetinic acid and its use in preparing composite drug-loaded micelles. These two patents only use polyethylene glycol as the hydrophilic segment and glycyrrhetinic acid as the hydrophobic segment to construct the carrier material for preparing composite drug-loaded micelles, but they limit the drug loading capacity (maximum drug loading is only 2.5%) and stability, making practical applications impossible. Summary of the Invention

[0005] To address the aforementioned technical problems, the present invention aims to provide glycyrrhetinic acid-modified block copolymers and their application as liver-targeting drug carriers.

[0006] To achieve the above-mentioned objectives, the technical solution of the present invention is as follows:

[0007] The application of glycyrrhetinic acid-modified block copolymers as liver-targeting drug carriers, wherein the glycyrrhetinic acid-modified block copolymers include hydrophilic and hydrophobic segments.

[0008] The hydrophilic segment is polyethylene glycol or polyethylene glycol monomethyl ether with a number average molecular weight between 400 and 20,000; the hydrophobic segment is a polyester with terminal hydroxyl groups modified by glycyrrhetinic acid esterification, and the polyester is selected from one of polylactide, polyglycolic acid, polyglycolic acid, polycaprolactone, polycarbonate or polydioxanone with a number average molecular weight between 500 and 100,000.

[0009] Preferably, the hydrophilic segment is polyethylene glycol monomethyl ether with a molecular weight between 750 and 5000, and the hydrophobic segment is selected from polylactide with a number average molecular weight between 1000 and 50000.

[0010] The glycyrrhetinic acid-modified block copolymer of this invention is a first-time proposal. Experimental data show that the grafting rate of glycyrrhetinic acid modification in the block copolymer of this invention is between 20% and 70%. Furthermore, when used as a carrier material, the glycyrrhetinic acid-modified block copolymer of this invention can significantly improve drug loading and stably encapsulate hydrophobic drug molecules. Meanwhile, this invention possesses active liver-targeting capabilities, including but not limited to nanomicelles.

[0011] The preparation method of the above-mentioned glycyrrhetinic acid modified block copolymer includes the following steps:

[0012] (1) Take glycyrrhetinic acid, 4-dimethylaminopyridine and carbodiimide hydrochloride in a molar ratio of 1:(0.5~2):(0.5~2), add anhydrous dichloromethane until completely dissolved, stir at room temperature, and activate;

[0013] (2) After activation, add 0.25 to 2.5 times the molar amount of triethylamine and 0.25 to 5 times the molar amount of mPEG-PLA respectively;

[0014] (3) Remove anhydrous dichloromethane by depressurization, dissolve the residue in acetone, dialyze to remove small molecule impurities in an aqueous medium, and then centrifuge and freeze dry to obtain the final product.

[0015] Preferably, the molar ratio of glycyrrhetinic acid to the block copolymer mPEG-PLA is 1.5:1.

[0016] Beneficial effects

[0017] The block copolymer provided by this invention is a novel concept. Furthermore, experimental data shows that the block copolymer provided by this invention can significantly increase the drug loading capacity of hydrophobic drug molecules. It also significantly increases drug uptake and exhibits active liver-targeting properties. Therefore, the block copolymer of this invention can be used as a liver-targeting drug carrier.

[0018] The block copolymer provided by this invention can be used as a drug carrier material. It can self-assemble into drug-loaded nanomicelles in an aqueous medium, which significantly improves the drug loading capacity, solubility and stability of hydrophobic drug molecules. It also has active liver-targeting properties, which can deliver drugs to liver tissue in a targeted manner, thereby improving the therapeutic effect of drugs and effectively reducing systemic toxic side effects. Attached Figure Description

[0019] Appendix Figure 1 The 1H NMR spectrum of the glycyrrhetinic acid-modified block copolymer is shown.

[0020] Appendix Figure 2 Transmission electron microscopy image of C6@mPEG-PLA-GA liver-targeted micelles.

[0021] Appendix Figure 3To measure the fluorescence intensity of C6 in HepG2 cells by flow cytometry. Detailed Implementation

[0022] The present invention will be further illustrated below with reference to specific embodiments. The purpose of these embodiments is to provide a better understanding of the invention and to demonstrate its essential characteristics. Therefore, the examples given should not be considered as limitations on the scope of protection of the present invention. It is also specifically noted that, unless otherwise specified, the specific experimental methods and equipment involved in the embodiments are conventional methods or implemented according to the conditions recommended in the manufacturer's instructions, and the reagents involved are commercially available unless otherwise specified. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods.

[0023] Example 1

[0024] Preparation of glycyrrhetinic acid-modified block copolymer 1

[0025] Dissolve 0.075 mmol of GA, 0.075 mmol of DMAP, and 0.075 mmol of EDCI in 5 mL of anhydrous dichloromethane and activate at room temperature by stirring at 500 rpm for 4 hours. Then add 0.075 mmol of triethylamine and mPEG. 2000 -PLA 1500 The reaction mixture was stirred at 500 rpm for 48 h at room temperature under argon protection (0.05 mmol). After the reaction was complete, the solvent was removed by rotary evaporation under reduced pressure. The residue was dissolved in 5 mL of acetone, and the solution was placed in a dialysis bag with a molecular weight cutoff of 3500, clamped at both ends, and dialyzed with pure water for 24 h to remove unreacted starting material and acetone. The solution was then lyophilized to obtain the final product. The yield of the prepared reaction was calculated to be 81.5% by weighing.

[0026] The proton NMR spectrum of the product was determined using deuterated chloroform as a solvent (Bruker AV400 NMR spectrometer). The spectrum is attached. Figure 1 δH: 5.66 (d, J = 37.2 Hz, OH), 2.45–2.14 (m, OH), 1.62–1.53 (m, 4H), 1.37 (s, OH), 1.26 (d, J = 4.0 Hz, OH), 1.21 (s, OH), 1.15–1.11 (m, OH), 1.00 (d, J = 1.3 Hz, OH), 0.86 (s, OH), 0.83 (s, OH), 0.81 (d, J = 1.6 Hz, OH). The calculated glycyrrhetinic acid modification grafting rate of the product was 23%.

[0027] Preparation of glycyrrhetinic acid-modified block copolymer 2

[0028] Dissolve 0.075 mmol of GA, 0.075 mmol of DMAP, and 0.075 mmol of EDCI in 5 mL of anhydrous dichloromethane and activate at room temperature by stirring at 500 rpm for 4 hours. Then add 0.075 mmol of triethylamine and mPEG. 2000 -PLA 2000 The reaction mixture was stirred at 500 rpm for 48 h at room temperature under argon protection (0.05 mmol). After the reaction, the solvent was removed by rotary evaporation under reduced pressure. The residue was dissolved in 5 mL of acetone, and the solution was placed in a dialysis bag with a molecular weight cutoff of 3500, clamped at both ends, and dialyzed with pure water for 24 h to remove unreacted raw materials and acetone. The solution was then lyophilized to obtain the final product. The yield of the prepared reaction was calculated to be 73.3% by weighing.

[0029] The proton NMR spectrum of the product was determined using deuterated chloroform as solvent (Bruker AV400 NMR spectrometer). δH: 5.69(s,OH), 2.34(s,OH), 1.37(s,OH), 1.26(d,J=4.0Hz,OH), 1.21(s,OH), 1.17~1.11(m,1H), 1.00(d,J=1.4Hz,OH), 0.86(s,OH), 0.85~0.77(m,1H). The glycyrrhetinic acid modification grafting rate of the product was calculated to be 35%.

[0030] Preparation of glycyrrhetinic acid-modified block copolymer 3

[0031] Dissolve 0.075 mmol of GA, 0.075 mmol of DMAP, and 0.075 mmol of EDCI in 5 mL of anhydrous dichloromethane and activate at room temperature by stirring at 500 rpm for 4 hours. Then add 0.075 mmol of triethylamine and mPEG. 2000 -PLA 5000 The reaction mixture was stirred at 500 rpm for 48 h at room temperature under argon protection (0.05 mmol). After the reaction, the solvent was removed by rotary evaporation under reduced pressure. The residue was dissolved in 5 mL of acetone, and the solution was placed in a dialysis bag with a molecular weight cutoff of 3500, clamped at both ends, and dialyzed with pure water for 24 h to remove unreacted starting material and acetone. The solution was then lyophilized to obtain the final product. The yield of the prepared reaction was calculated to be 67.1% by weighing.

[0032] The proton NMR spectrum of the product was determined using deuterated chloroform as solvent (Bruker AV400 NMR spectrometer). δH: 5.61 (s, OH), 2.32 (s, OH), 1.37 (s, OH), 1.26 (d, J = 1.6 Hz, OH), 1.20 (d, J = 3.9 Hz, OH), 1.12 (s, OH), 1.00 (d, J = 2.3 Hz, OH), 0.86 (d, J = 1.9 Hz, OH), 0.80 (d, J = 2.1 Hz, OH), 0.70 (d, J = 11.6 Hz, OH). The glycyrrhetinic acid modification grafting rate of the product was calculated to be 50%.

[0033] Example 2 Pharmacological Evaluation

[0034] Evaluation of drug loading in glycyrrhetinic acid-modified block copolymers

[0035] Coumarin-6 (C6) has physicochemical properties similar to hydrophobic drugs and exhibits fluorescence. Therefore, C6 was chosen to replace hydrophobic drugs for the preparation and subsequent evaluation of polymer micelles.

[0036] Glycyrrhetinic acid-modified block copolymers (mPEG) were taken separately. 2000 -PLA 1500 -GA, mPEG 2000 -PLA 2000 -GA, mPEG 2000 -PLA 5000 45 mg of GA and 5 mg of C6 were dissolved in a 500 mL round-bottom flask with 5 mL of acetone. The mixture was then rotary evaporated in a 45 °C water bath (50–100 rpm × 5 min) to ensure thorough mixing and homogeneous formation of a uniform film. After complete solvent removal, 4 mL of preheated physiological saline was added. Once the film was hydrated, it was filtered through a 0.45 μm polyethersulfone membrane to obtain C6-loaded micelles C6@mPEG. 2000 -PLA 1500 -GA, C6@mPEG 2000 -PLA 2000 -GA, C6@mPEG 2000 -PLA 5000 -GA.

[0037] The particle size and distribution (polydispersity index, PDI) of the micelles were determined using a laser particle size analyzer, and the morphology of the micelles was observed using a transmission electron microscope. The results are as follows: Figure 2 As shown, the average particle size of the three drug-loaded micelles prepared was ~200 nm, with a PDI of 0.1–0.2, and the particle size distribution was uniform. C6@mPEG was quantitatively determined by fluorescence spectroscopy. 2000 -PLA 1500 -GA had an encapsulation efficiency of 96.4% and a drug loading of 15.6%; C6@mPEG2000 -PLA 2000 -GA had an encapsulation efficiency of 94.2% and a drug loading of 14.8%. C6@mPEG 2000 -PLA 5000 -GA had an encapsulation efficiency of 93.1% and a drug loading of 14.2%.

[0038] Experimental data show that, compared with previously disclosed polyethylene glycol copolymers or liver-targeting copolymers with glycyrrhetinic acid as the hydrophobic end, the drug loading capacity of the glycyrrhetinic acid-modified block copolymer of the present invention is increased by approximately 6 times. Therefore, the block copolymer provided by the present invention can be used as a drug carrier material, significantly improving the drug loading capacity of hydrophobic drug molecules.

[0039] Furthermore, subsequent stability and targeting tests have shown that the glycyrrhetinic acid-modified block copolymer of the present invention has excellent micelle stability and liver targeting, making it very suitable for use as a liver-targeting drug carrier.

[0040] Stability study of glycyrrhetinic acid-modified block copolymers

[0041] drug-loaded micelles C6@mPEG 2000 -PLA 1500 -GA as the target, drug-loaded micelles C6@mPEG prepared from unmodified materials 2000 -PLA 1500 As a control, the particle size change of micelles was measured at 4℃ and room temperature to understand their micelle stability under different storage conditions.

[0042] Take 2 mL of micelle solution and place it at 4 °C and room temperature in the dark to determine the particle size and PDI of the micelles at different times (2 h, 4 h, 6 h, 8 h, 12 h, 24 h, 48 h, 72 h).

[0043] The results showed that: C6@mPEG 2000 -PLA 1500 After being placed at 4℃ and room temperature for 2 hours, the particle size of the micelles increased from 40 nm to 500 nm, and the PDI changed from 0.15 to 0.49. Under incubation conditions of 37℃ and 10% BSA, the particle size reached 1000 nm within 2 hours, and the PDI was approximately 0.52, indicating that the control drug-loaded micelles prepared with the unmodified material had poor stability. The drug-loaded micelles C6@mPEG prepared with the modified material... 2000 -PLA 1500 -GA gels, after being placed at 4℃ and room temperature for 72 hours, showed RSD of particle size and PDI changes of <1%, indicating that the targeted modification of the material can significantly improve the stability of drug-loaded micelles.

[0044] Experiments show that, compared with the control drug-loaded micelles prepared from unmodified materials, the RSD of particle size and PDI variation of the present invention is <1%, which can significantly improve the stability of drug-loaded micelles.

[0045] Liver-targeting study of glycyrrhetinic acid-modified block copolymers

[0046] Drug-loaded micelles C6@mPEG prepared from free C6 and unmodified materials 2000 -PLA 1500 Targeted drug-loaded micelles C6@mPEG 2000 -PLA 1500 Using GA as the experimental subject, this study investigated the uptake of C6 in three different formulations of HepG2 liver cancer cells to determine the target drug-loaded micelles C6@mPEG. 2000 -PLA 1500 - The liver-targeting effect of GA.

[0047] HepG2 cells in the logarithmic growth phase were seeded into 12-well plates and incubated at 37°C with 5% CO2 for 24 hours. After removing the plates and observing good cell adhesion under a microscope, the culture medium was aspirated from the wells, and 500 μL of C6@mPEG was added to each well. 2000 -PLA 1500 -GA drug-loaded micelle solution, with the same content of C6 solution and C6@mPEG 2000 -PLA 1500 As a control group, each group had 3 replicates. After 24 hours of culture, the drug solution was discarded, the cells were washed twice with PBS, and then the uptake of cells was quantitatively analyzed by flow cytometry.

[0048] like Figure 3 As shown, the fluorescence intensity of free C6 was the lowest, and although it increased slightly with time, it was still significantly lower than that of C6@mPEG. 2000 -PLA 1500 and C6@mPEG 2000 -PLA 1500 -GA indicates that micellar formulations can significantly increase C6 absorption, suggesting a significant increase in uptake. (Compared to C6@mPEG) 2000 -PLA 1500 In comparison, C6@mPEG 2000 -PLA 1500 -GA significantly increased the uptake of C6 by HepG2 liver cancer cells, and the difference in uptake became more pronounced with prolonged treatment time. At 6 hours, C6@mPEG 2000 -PLA 1500 -GA intake reached C6@mPEG 2000 -PLA 1500 More than twice as much. The results show that C6@mPEG 2000 -PLA1500 -GA has a targeting effect on HepG2 liver cancer cells and can significantly increase the uptake of C6, enabling the drug to accumulate in liver cancer cells.

[0049] The above pharmacological evaluation data show that the glycyrrhetinic acid-modified block copolymer of the present invention can significantly increase the drug loading of hydrophobic drug molecules and significantly increase drug uptake, and has active liver-targeting characteristics. Therefore, the block copolymer of the present invention can be used as a liver-targeting drug carrier.

[0050] The block copolymer provided by this invention is a novel concept. Its structure differs from existing polyethylene glycol copolymers. The block copolymer of this invention has fixed hydrophobic ends, and the terminal hydroxyl groups of the hydrophobic polyester segments are modified with glycyrrhetinic acid. The glycyrrhetinic acid-modified block copolymer of this invention significantly improves the drug loading capacity of hydrophobic drug molecules. This is because, compared to existing mPEG-PLA copolymers, the glycyrrhetinic acid-modified block copolymer of this invention can significantly reduce the critical micelle concentration, making it easier to form micelles and exhibiting stronger encapsulation properties. Therefore, it can greatly improve the drug loading capacity and stability of the prepared drug-loaded nanomicelles. Furthermore, the determination of the critical micelle concentration in this invention also confirms the above viewpoints.

[0051] Determination of critical micelle concentration of glycyrrhetinic acid-modified block copolymers

[0052] The critical micelle concentration (CMC) of the copolymer was determined using the pyrene fluorescent probe method, and the fluorescence intensity was measured using an ELISA reader. The concentration was then calculated using the ratio of the two fluorescence intensities.

[0053] Accurately weigh 1 mg of the probe compound pyrene, dissolve it in acetone to prepare a 0.1 mg / mL solution. Transfer several 50 μL portions of the solution into separate test tubes, allow to stand until the solvent evaporates completely to dryness, and then add an appropriate amount of the copolymer material (mPEG). 2000 -PLA 1500 mPEG 2000 -PLA 1500 -GA, mPEG 2000 -PLA 2000 mPEG 2000 -PLA 2000 -GA, mPEG 2000 -PLA 5000 Add 5 mL of water to GA to make up to the mark, preparing solutions with a polymer concentration of 0.05 mg / mL to 1 mg / mL. Disperse the pyrene completely using low-frequency sonication. Let stand overnight, and measure the fluorescence intensity of each solution at 373 nm and 384 nm. Calculate the ratio I. 373 / I 384 The CMC value of the material is calculated by fitting the result to logC.

[0054] The results showed that mPEG was measured. 2000 -PLA 1500 mPEG 2000 -PLA 1500 The CMCs of -GA were 66.9 μg / mL and 0.76 μg / mL, respectively, and the mPEG... 2000 -PLA 2000 mPEG 2000 -PLA 2000 The CMCs of -GA were 89.7 μg / mL and 0.93 μg / mL, respectively, and the mPEG content was... 2000 -PLA 5000 mPEG 2000 -PLA 5000 The CMC values ​​of -GA were 102.9 μg / mL and 1.28 μg / mL.

[0055] The above results show that the critical micelle concentration of the copolymer material of the present invention is significantly reduced by nearly 100 times after being modified with hydrophobic segments of glycyrrhetinic acid. This indicates that the glycyrrhetinic acid-modified block copolymer of the present invention is more likely to form micelles and has stronger encapsulation properties, thus greatly improving the drug loading capacity and stability of the prepared drug-loaded nanomicelles.

[0056] The present invention has been illustrated with the above embodiments to explain the detailed method of the present invention. However, the present invention is not limited to the detailed method described above, that is, it does not mean that the present invention must rely on the detailed method described above to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., are all included within the protection scope and disclosure scope of the present invention.

Claims

1. The application of glycyrrhetinic acid-modified block copolymers as liver-targeting drug carriers, characterized in that, Glycyrrhetinic acid-modified block copolymers include hydrophilic and hydrophobic segments. The hydrophilic segment is polyethylene glycol monomethyl ether with a number average molecular weight of 2000; the hydrophobic segment is a polyester modified with glycyrrhetinic acid by hydroxyl-terminated esterification, and the polyester is selected from polylactide with a number average molecular weight between 1500 and 5000.

2. The application according to claim 1, characterized in that, The hydrophilic segment of the glycyrrhetinic acid-modified block copolymer is selected from polyethylene glycol monomethyl ether with a number average molecular weight of 2000, and the hydrophobic segment is selected from polylactide with a number average molecular weight of 1500.

3. A method for preparing glycyrrhetinic acid-modified block copolymers, characterized in that, The steps are as follows: (1) Take glycyrrhetinic acid, 4-dimethylaminopyridine and carbodiimide hydrochloride in a molar ratio of 1:(0.5-2):(0.5-2), add anhydrous dichloromethane until completely dissolved, stir at room temperature and activate; (2) After activation, add 0.25 to 2.5 times the molar amount of triethylamine and 0.25 to 5 times the molar amount of mPEG-PLA respectively, wherein the number average molecular weight of mPEG in mPEG-PLA is 2000 and the number average molecular weight of PLA is between 1500 and 5000. (3) Remove anhydrous dichloromethane by depressurization, dissolve the residue in acetone, dialyze to remove small molecule impurities in an aqueous medium, and then centrifuge and freeze dry to obtain the final product.

4. The preparation method according to claim 3, characterized in that, The molar ratio of glycyrrhetinic acid to mPEG-PLA is 1.5:1.

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