A liver-targeting drug delivery carrier, a preparation method and application thereof

Abstract

The application discloses a preparation method of a liver-targeting drug delivery carrier, and comprises the following steps: synthesizing bromic acid N-acetylgalactosamine ester; and synthesizing the liver-targeting drug delivery carrier. x The liver-targeting drug delivery carrier GalNAc-C x CD is designed based on bromic acid N-acetylgalactosamine ester Br-C GalNAc and beta-cyclodextrin, which can include poorly soluble drugs to be delivered to the liver and released in a targeted manner, greatly improves the targeting efficiency on the liver, and the targeting delivery rate can reach more than 70%, while effectively improving the solubility and bioavailability of the poorly soluble drugs, improving the drug treatment effect, providing a new strategy for the delivery of other poorly soluble drugs for treating liver diseases, and laying a foundation for the development of drugs and functional foods for intervening NASH.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, in particular to a liver-targeting drug delivery carrier and a preparation method and application thereof. BACKGROUND

[0002] Most natural medicinal plant-derived compounds, such as fisetin, quercetin, silybin, hyperoside, etc., have the shortcomings of poor water solubility and low bioavailability, so that the active ingredients are low in the degree of being absorbed into human circulation by small intestinal epithelial cells after oral, gastric and intestinal digestion, cannot normally play a curative effect, and cause the dosage to be continuously increased, thereby increasing unnecessary damage to the gastrointestinal tract, liver and other organs of the body, and directly causing drug development failure.

[0003] In order to solve the problem of low bioavailability, various new drug delivery systems have emerged, including cyclodextrin, liposome, nanoparticle, microemulsion, polymer nanoparticle, etc.; the drug delivery system refers to a carrier or a delivery method for delivering drugs to target cells or tissues in a targeted manner, and can release drugs, improve drug efficacy and reduce adverse reactions; the liver is a very important organ, because it participates in many metabolic activities of the human body, and the metabolism of drugs in the body often depends on liver function; however, the liver is easily damaged by various factors, such as viral infection and inflammation, which affects the metabolism of drugs and brings various troubles to treatment; therefore, the research on liver-targeting drug delivery systems is of great significance; the existing liver-targeting drug delivery systems often have complex synthesis steps, low yield, high cost, poor targeting and poor efficacy, therefore, there is an urgent need for a liver-targeting drug delivery system with good targeting, high yield and high bioavailability. SUMMARY

[0004] In view of the above shortcomings in the prior art, one of the purposes of the present application is to provide a liver-targeting drug delivery carrier to improve the liver targeting and bioavailability of drugs.

[0005] The technical solution of the present application to solve the above technical problems is as follows:

[0006] A preparation method of a liver-targeting drug delivery carrier, comprising the following steps:

[0007] (1) Synthesis of bromic acid N-acetylgalactosamine ester Br-C x -GalNAc (x = 5, 7 or 11): N-acetylgalactosamine and x-bromate (x = 5, 7 or 11) are used as raw materials, and Br-C is synthesized by enzymatic reaction x -GalNAc (x = 5, 7 or 11);

[0008] (2) Synthesis of a liver-targeting drug delivery carrier: with bromic acid N-acetylgalactosamine ester Br-C xUsing GalNAc (x = 5, 7, or 11) and β-cyclodextrin as raw materials, a liver-targeted drug delivery carrier, GalNAc-C, was synthesized. x -CD (x = 5, 7 or 11).

[0009] Furthermore, step (1) specifically includes:

[0010] N-acetylgalactosamine and bromate were reacted at a molar ratio of 1:1 to 1:4, with TLIM lipase as a catalyst and acetone as a solvent, at 55-70°C for 12-20 hours to obtain synthetic N-acetylgalactosamine bromate Br-C. x -GalNAc(x = 5, 7 or 11).

[0011] Furthermore, the N-acetylgalactosamine and bromate are reacted in a molar ratio of 1:1 at a reaction temperature of 55°C for 12 hours.

[0012] Furthermore, step (2) specifically includes:

[0013] Br-C x A solution of -GalNAc (x = 5, 7 or 11) was added to an alkaline solution of β-cyclodextrin, and the mixture was reacted at 40-60°C for 0.5-2 hours. After the reaction was completed, excess anhydrous ethanol was added, the mixture was sealed, and incubated overnight at 4-6°C. The supernatant was then removed to obtain the product.

[0014] Furthermore, in step (2), Br-C x The mass ratio of -GalNAc (x = 5, 7 or 11) to β-cyclodextrin is 1:1; the alkaline solution is a 10% hydroxide solution; the reaction temperature is 55°C and the reaction time is 1 hour.

[0015] The second objective of this invention is to provide a liver-targeted drug delivery carrier prepared using a method for preparing a liver-targeted drug delivery carrier.

[0016] A third objective of this invention is to provide a liver-targeted drug delivery system, which includes a liver-targeted drug delivery carrier and a drug.

[0017] Furthermore, the drug is a flavonoid or a polyphenol compound.

[0018] Furthermore, the drug is curcumin or flavonoid.

[0019] The fourth objective of this invention is to provide the application of the above-mentioned liver-targeting drug delivery carrier in the preparation of drugs for treating liver diseases.

[0020] Furthermore, the liver disease is non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, cirrhosis, or hepatocellular carcinoma.

[0021] This invention has the following beneficial effects: This invention is based on N-acetylgalactosamine bromate Br-C x A liver-targeted drug delivery carrier, GalNAc-C, was designed using GalNAc and β-CD. x -CD encapsulates poorly soluble drugs, delivering them to the liver for targeted release, significantly improving targeting efficiency to the liver (with a delivery rate exceeding 70%). It also effectively enhances the solubility and bioavailability of poorly soluble drugs, improving therapeutic efficacy. This provides a new strategy for delivering other poorly soluble drugs with therapeutic effects on liver diseases, laying the foundation for the development of drugs and functional foods for NASH intervention. Furthermore, its preparation process is simple, yields high-yield products, and produces products with good therapeutic effects, demonstrating significant application potential. Attached Figure Description

[0022] Figure 1 The FT-IR spectrum of Br-C7-GalNAc obtained in Example 1 of this invention;

[0023] Figure 2 The Br-C7-GalNAc prepared in Example 1 of this invention 1 H NMR spectrum;

[0024] Figure 3 The Br-C7-GalNAc prepared in Example 1 of this invention 13 C NMR spectrum;

[0025] Figure 4 The infrared spectrum of GalNAc-C7-CD obtained in Example 1 of this invention;

[0026] Figure 5 The GalNAc-C7-CD prepared in Example 1 of this invention 13 C NMR spectrum;

[0027] Figure 6 The FT-IR spectrum of Br-C5-GalNAc obtained in Example 2 of this invention;

[0028] Figure 7 The Br-C5-GalNAc prepared in Example 2 of this invention 13 C NMR spectrum;

[0029] Figure 8 The FT-IR spectrum of GalNAc-C5-CD prepared in Embodiment 2 of this invention;

[0030] Figure 9 GalNAc-C5-CD prepared in Example 2 of this invention 13 C NMR spectrum;

[0031] Figure 10 Br-C prepared for embodiment 3 of the present invention 11 -FT-IR spectrum of GalNAc;

[0032] Figure 11 Br-C prepared for embodiment 3 of the present invention 11 -C NMR spectrum of GalNAc;

[0033] Figure 12 GalNAc-C prepared in Example 3 of this invention 11 -CD FT-IR spectrum;

[0034] Figure 13 GalNAc-C prepared in Example 3 of this invention 11 -CD 3 C NMR spectrum;

[0035] Figure 14 This is a schematic diagram of cell drug administration in Example 5 of the present invention;

[0036] Figure 15 This is a comparison of cellular uptake rates of cyclodextrins with different lengths of connecting arms modified with GalNAc in Example 5 of the present invention.

[0037] Figure 16 This is a spectrum of the GalNAc competitive inhibition experiment in Example 5 of the present invention;

[0038] Figure 17 GalNAc-C in Embodiment 5 of the present invention 11 -CD-FITC(A) / GalNAc-C 11 Tissue distribution diagram of CD-Ni(B) in zebrafish;

[0039] Figure 18 GalNAc-C in Embodiment 6 of the present invention 11 SEM results of -CD-Fn;

[0040] Figure 19 GalNAc-C in Embodiment 6 of the present invention 11 Infrared spectrum results of -CD-Fn;

[0041] Figure 20 GalNAc-C in Embodiment 9 of the present invention 11 -CD-Fn cell uptake rate diagram;

[0042] Figure 21 GalNAc-C in Embodiment 9 of the present invention 11 -CD-Fn in vitro drug release diagram. Detailed Implementation

[0043] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0044] Example 1

[0045] A method for preparing a liver-targeted drug delivery carrier includes the following steps:

[0046] (1) Synthesis of N-acetylgalactosamine bromate Br-C7-GalNAc: Br-C7-GalNAc was synthesized by enzymatic reaction using N-acetylgalactosamine and methyl 7-bromate as starting materials; wherein the molar ratio of N-acetylgalactosamine to bromate was 1:1, TLIM lipase was used as the catalyst, acetone was used as the reaction solvent, and the reaction was carried out at 55℃ for 12 h, with a product yield of 78.77%; the reaction equation is as follows:

[0047]

[0048] (2) Purification of Br-C7-GalNAc

[0049] A chromatographic column was packed with silica gel (200-300 mesh). The product after the enzymatic reaction was wet-loaded and eluted with dichloromethane / methanol. The eluent was analyzed by thin-layer chromatography (TLC). The products were collected and combined, and dried in a vacuum drying oven to obtain the final product.

[0050] (3) Characterization of Br-C7-GalNAc structure

[0051] To characterize the product, the product was subjected to... 1 HNMR, 13 CNMR and FT-IR measurements:

[0052] like Figure 1 As shown in the infrared spectrum, at 1641 cm⁻¹ -1 1581cm -1 The peak at 551 cm⁻¹ is a characteristic peak for amide bonds on GalNAc. -1 The peak at 1379 cm⁻¹ is the C-Br peak on ethyl 7-bromate, while the peak at 1379 cm⁻¹ is the peak on ethyl 7-bromate. -1 1330cm -1 The peak at this location represents the characteristic peak of newly formed ester groups in the product.

[0053] 1H NMR (600MHz, DMSO-d6) δ7.54(d,J=8.5Hz,1H,H-20),6.33(d,J=4.4Hz,1H,H-15),4.93(t,J=4.0Hz,1H,H-11),4.58(q,J=6 .0,4.5Hz,1H,H-14),4.52(d,J=6.0Hz,2H,H-13),4.43(dd,J=7.0,4.3Hz,2H,H-17),3.97(td,J=10.0,8.9,3.4Hz,1H,H-2 ), 3.80(t,J=6.4Hz,1H,H-12), 3.72(t,J=3.8Hz,1H,H-19), 3.66-3.61(m,2H,H-8), 3.54(dt,J=8.6,3.2Hz,1H,H-3), 3.44-3.40(m,1H,H-6), 3.39-3.33(m,9H,H-22), 3.30(d,J=6.2Hz,1H,H-24), 1.83(s,5H,H-5,H-4,H-16); combined with N-acetylgalactosamine and ethyl 7-bromoheptanoate. 1 ¹H NMR data analysis showed that the product exhibited characteristic peaks of N-acetylgalactosamine at 6.5 ppm and 7.8 ppm, and characteristic peaks of ethyl 7-bromoheptanoate at 3.5 ppm and 4.1 ppm; the results are as follows. Figure 2 As shown;

[0054] 13 C10 NMR (151 MHz, DMSO) δ 173.34 (C-21), 172.67 (C-9), 94.07 (C-15), 78.12 (C-11), 73.48 (C-12), 71.38 (C-13), 63.77 (C-17), 57.22 (C-14), 42.90 (C-8), 42.76 (C-2), 42.62 (C-3), 42.48 (C-5), 42.34 (C-4), 26.12 (C-6), 25.86 (C-22). The carbonyl groups of the ester and amide groups of the target product were at 172 ppm and 173 ppm, respectively, indicating an transesterification reaction between the two substrates; the results are as follows. Figure 3 As shown;

[0055] The product structure characterization results showed that TLIM lipase catalyzed the transesterification reaction between N-acetylgalactosamine and ethyl 7-bromoheptanoate, indicating that the target product was successfully synthesized.

[0056] (4) Synthesis of liver-targeted drug delivery carrier: Using N-acetylgalactosamine bromate Br-C7-GalNAc and β-cyclodextrin as raw materials, the liver-targeted drug delivery carrier GalNAc-C7-CD was synthesized.

[0057] Take a 100mL clean beaker, add 6mL of 10% NaOH solution and 300mg of β-cyclodextrin, and stir until the β-cyclodextrin is completely dissolved; weigh 300mg of Br-C7-GalNAc and dissolve it in 2mL of N,N-dimethylformamide, add the Br-C7-GalNAc solution dropwise to the alkaline solution of β-cyclodextrin, stir and react at 55℃ for 1h, after the reaction is complete, add excess anhydrous ethanol, seal, incubate at 4℃ overnight, discard the supernatant, and freeze-dry the remaining part for 24 hours to obtain the crude product; the reaction equation is shown below.

[0058]

[0059] (5) Characterization of GalNAc-C7-CD structure

[0060] In the GalNAc-C7-CD infrared spectrum, such as Figure 4 As shown, at 3386cm -1 It is the broad hydroxyl peak of β-cyclodextrin, 2929 cm⁻¹ -1 These are extended peaks of β-cyclodextrin CH, at 1652 and 1383 cm⁻¹. -1 This is a characteristic peak of amides on GalNAc. A new ester bond has formed in the substance, 1320 cm⁻¹. -1 The position represents the antisymmetric stretching vibration of the ester group COC, at 1159 cm⁻¹. -1 The presence of a symmetric stretching vibration of the ester group COC at the position indicates that the target product has been synthesized.

[0061] GalNAc-C7-CD 13C NMR(151MHz,DMSO)δ169.80(C-3,C-16),105.01(C-82,71,C-60,C-49,C-38,C-27,C-93),84.6 2(C-79,C-68,C-57,C-46,C-35,C-24,C-90,C-79),76.16(C-13),75.53(C-87),75.13(C-81,C- 80, C-70, C-69, C-59, C-58, C-48, C-47, C-37, C-36, C-26, C-25, C-92, C-91), 63.01, 51.69 (C-11), 42.62, 34.39 (C-17), 32.14 (C-20), 31.80 (C-22), 28.59 (C-19), 25.19 (C-21), 17.06 (C-2). The peaks at 169 ppm and 167 ppm represent the carbonyl group of N-acetylgalactosamine and the linker arm; the peak at 76.1 ppm represents the ether bond formed by β-cyclodextrin and the linker arm; the peak between 50 ppm and 83 ppm represents cyclodextrin; and the peak between 20 ppm and 35 ppm represents the linker arm. This indicates successful reaction and synthesis of the target product, such as... Figure 5 As shown.

[0062] The structural characterization of the product confirmed the successful reaction between Br-C7-GalNAc and β-CD, indicating the synthesis of the target product GalNAc-C7-CD. GalNAc-C7-CD was subsequently synthesized using the same method. x -CD(x=5,11).

[0063] Example 2

[0064] A method for preparing a liver-targeted drug delivery carrier includes the following steps:

[0065] (1) Synthesis of N-acetylgalactosamine bromate Br-C5-GalNAc: Br-C5-GalNAc was synthesized from N-acetylgalactosamine and methyl 5-bromate via an enzymatic reaction. The molar ratio of N-acetylgalactosamine to bromate was 1:1. TLIM lipase was used as the catalyst, acetone as the reaction solvent, and the reaction was carried out at 60℃ for 125 h. The yield of the product was 79.21%. The reaction equation is as follows:

[0066]

[0067] (2) Purification of Br-C5-GalNAc

[0068] A chromatographic column was packed with silica gel (200-300 mesh). The product after the enzymatic reaction was wet-loaded and eluted with dichloromethane / methanol. The eluent was analyzed by thin-layer chromatography (TLC). The products were collected and combined, and dried in a vacuum drying oven to obtain the final product.

[0069] (3) Characterization of Br-C5-GalNAc structure

[0070] To characterize the reaction products, the products were subjected to... 1 HNMR, 13 CNMR and FT-IR measurements;

[0071] like Figure 6 As shown in the infrared spectrum, at 1642 cm⁻¹ -1 1582cm -1 The peak at 551 cm⁻¹ is a characteristic peak of the amide bond on GalNAc. -1 The peak at 1390 cm⁻¹ is the C-Br peak on ethyl 5-bromate, while the peak at 1390 cm⁻¹ is the peak on ethyl 5-bromate. -1 1328cm -1 The peak at this location represents the characteristic peak of newly formed ester groups in the product.

[0072] like Figure 7 As shown, 13 C NMR (100MHz, DMSO) δ 173.36, 172.69, 94.08, 78.14, 74.28, 73.49, 71.40, 70.52, 63.79, 63.68, 57.22, 53.36, 26.13, 25.87, 25.78; 172ppm and 173ppm represent the carbonyl groups of the ester and amide groups of the target product, respectively, indicating that a transesterification reaction occurred between the two substrates. The product structure characterization results show that TLIM lipase catalyzed the transesterification reaction between N-acetylgalactosamine and ethyl 5-bromopentanoate, indicating that the target product was successfully synthesized.

[0073] (4) Synthesis of liver-targeted drug delivery carrier: Using N-acetylgalactosamine bromate Br-C5-GalNAc and β-cyclodextrin as raw materials, the liver-targeted drug delivery carrier GalNAc-C5-CD was synthesized.

[0074] Take a 100mL clean beaker, add 6mL of 10% NaOH solution and 300mg of β-cyclodextrin, and stir until the β-cyclodextrin is completely dissolved; weigh 300mg of Br-C5-GalNAc and dissolve it in 2mL of N,N-dimethylformamide, add the Br-C5-GalNAc solution dropwise to the alkaline solution of β-cyclodextrin, stir and react at 55℃ for 1h, after the reaction is complete, add excess anhydrous ethanol, seal, incubate at 4℃ overnight, discard the supernatant, and freeze-dry the remaining part for 24 hours to obtain the crude product; the reaction equation is shown below.

[0075]

[0076] (5) Characterization of GalNAc-C5-CD structure

[0077] In the GalNAc-C5-CD infrared spectrum, such as Figure 8 As shown, at 3135cm -1 It is the broad hydroxyl peak of β-cyclodextrin, at 2835 cm⁻¹. -1 This is the extended peak of β-cyclodextrin CH, 1602 cm⁻¹ -1 1401cm -1 The peak at 1330 cm⁻¹ is a characteristic peak for amide bonds on GalNAc; ester bonds have been newly formed in the substance. -1 The position represents the antisymmetric stretching vibration of the ester group COC, at 1150 cm⁻¹. -1 The presence of a symmetric stretching vibration of the ester group COC at the position indicates that the target product has been synthesized.

[0078] like Figure 9 As shown, 3 C NMR (100MHz, DMSO) δ 169.29, 167.36, 105.06, 84.66, 76.16, 75.57, 75.16, 63.05, 52.16, 26.02, 25.50, 23.88, 22.66, 21.0; The peaks at 169 ppm and 167 ppm represent the carbonyl group of N-acetylgalactosamine and the linker arm, the peak at 76.1 ppm represents the ether bond formed by β-cyclodextrin and the linker arm, the peak between 52 ppm and 84 ppm represents the cyclodextrin, and the peak between 21 ppm and 26 ppm represents the linker arm, indicating successful reaction and synthesis of the target product. The product structure characterization results confirm the successful reaction of Br-C5-GalNAc and β-CD, indicating the synthesis of the target product GalNAc-C5-CD.

[0079] Example 3

[0080] A method for preparing a liver-targeted drug delivery carrier includes the following steps:

[0081] (1) Synthesis of N-acetylgalactosamine bromate Br-C 11 -GalNAc: Br-C is synthesized from N-acetylgalactosamine and methyl 11-bromate using an enzymatic reaction. 11 -GalNAc; wherein, the molar ratio of N-acetylgalactosamine to bromate is 1:1, TLIM lipase is used as a catalyst, acetone is used as a reaction solvent, the reaction is carried out at 60℃ for 12 h, and the product yield is 79.21%; the reaction equation is as follows:

[0082]

[0083] (2)Br-C 11 -GalNAc purification process

[0084] A chromatographic column was packed with silica gel (200-300 mesh). The product after the enzymatic reaction was wet-loaded and eluted with dichloromethane / methanol. The eluent was analyzed by thin-layer chromatography (TLC). The products were collected and combined, and dried in a vacuum drying oven to obtain the final product.

[0085] (3)Br-C 11 -GalNAc structural characterization

[0086] To characterize the reaction products, the products were subjected to... 1 HNMR, 13 CNMR and FT-IR measurements:

[0087] like Figure 10 As shown in the infrared spectrum, at 1641 cm⁻¹ -1 1562cm -1 The peak at 553 cm⁻¹ is a characteristic peak for amide bonds on GalNAc. -1 The peak at 1379 cm⁻¹ is the C-Br peak on ethyl 11-bromoundecanoate, while the peak at 1379 cm⁻¹ is the peak on ethyl 11-bromoundecanoate. -1 1328cm -1 The peak at this location represents the characteristic peak of newly formed ester groups in the product.

[0088] like Figure 11 As shown, 13 C NMR (100MHz, DMSO) δ 173.40, 172.78, 94.08, 78.13, 74, 79, 73.49, 71.38, 70.49, 63.77, 63.66, 57.22, 53.38, 26.12, 25.86, 25.77; 172ppm and 173ppm represent the carbonyl groups of the ester and amide groups of the target product, respectively, indicating that a transesterification reaction occurred between the two substrates. The product structure characterization results show that TLIM lipase catalyzed the transesterification reaction between N-acetylgalactosamine and ethyl 11-bromoundecanoate, indicating that the target product was successfully synthesized.

[0089] (4) Synthetic liver-targeted drug delivery carrier: using N-acetylgalactosamine bromate Br-C 11 Using GalNAc and β-cyclodextrin as raw materials, a liver-targeted drug delivery carrier, GalNAc-C, was synthesized. 11 -CD:

[0090] Take a 100mL clean beaker, add 6mL of 10% NaOH solution and 300mg of β-cyclodextrin, stir until the β-cyclodextrin is completely dissolved; weigh 300mg of Br-C 11-GalNAc was dissolved in 2 mL of N,N-dimethylformamide, and Br-C 11 The GalNAc solution was added dropwise to the alkaline solution of β-cyclodextrin, and the mixture was stirred at 55°C for 1 hour. After the reaction was complete, excess anhydrous ethanol was added, the mixture was sealed, and incubated at 4°C overnight. The supernatant was discarded, and the remaining portion was freeze-dried for 24 hours to obtain the crude product. The reaction equation is shown below:

[0091]

[0092] (5) GalNAc-C 11 -CD structure characterization

[0093] GalNAc-C 11 -CD infrared spectrum, such as Figure 12 As shown, the infrared spectrum indicates that at 3386 cm⁻¹... -1 It is a broad hydroxyl peak of β-cyclodextrin, at 2929 cm⁻¹. -1 These are the extended peaks of β-cyclodextrin CH, at 1655 and 1385 cm⁻¹. -1 This is a characteristic peak of amides on GalNAc; ester bonds have formed in the substance, 1350 cm⁻¹ -1 The position represents the antisymmetric stretching vibration of the ester group COC, at 1156 cm⁻¹. -1 The presence of a symmetric stretching vibration of the ester group COC at the position indicates that the target product has been synthesized.

[0094] like Figure 13 As shown, 3 C10 NMR (100MHz, DMSO) δ 169.29, 167.36, 105.06, 84.66, 76.16, 75.57, 75.16, 63.05, 52.16, 26.02, 25.50, 23.88, 22.66, 21.0; The peaks at 169 ppm and 167 ppm represent the carbonyl group of N-acetylgalactosamine and the linker arm; the peak at 76.1 ppm represents the ether bond formed by β-cyclodextrin and the linker arm; the peak between 52 ppm and 84 ppm represents cyclodextrin; and the peak between 21 ppm and 26 ppm represents the linker arm, indicating successful reaction and synthesis of the target product; the product structure characterization results show that Br-C 11 The reaction between GalNAc and β-CD was successful, indicating the synthesis of the target product GalNAc-C. 11 -CD.

[0095] Example 4

[0096] (1) Synthesis of N-acetylgalactosamine-modified β-cyclodextrin (GalNAc-C) x -CD), and perform structural characterization; using GalNAc-C x-CD inclusion of the fluorescent dye Nile Red was used to verify the relationship between the distance between GalNAc and the β-cyclodextrin surface and the recognition ability of ASGPR receptors on the hepatocyte surface. Subsequently, a competitive inhibition experiment was performed using GalNAc to verify GalNAc-C x -CD targeting is mediated by the ASGPR receptor on the surface of hepatocytes.

[0097] (2) GalNAc-C prepared using Example 3 11 -CD preparation of GalNAc-C 11 -CD laccase was characterized by SEM and FT-IR; the levels of F in different media (pH=1.6, pH=6.8, pH=7.4, 20% liver homogenate) were detected. n Drug release was verified to validate GalNAc-C. 11 -CD-F n Hepatic-targeted release characteristics of GalNAc-C; influencing factor experiments were conducted to preliminarily verify GalNAc-C. 11 -CD-F n Stability.

[0098] (3) Pharmacokinetic experiments were conducted on SD rats to determine F. n After GalNAc-C 11 -CD packaged, for F n Effects on altered bioavailability; F in mice administered the same dose via gavage n and GalNAc-C 11 -CD-F n Mice were euthanized, and their major organs (heart, liver, spleen, lungs, and kidneys) were collected for F1 content analysis. n The content of GalNAc-C was determined to identify its tissue distribution; mice were administered GalNAc-C via gavage for four consecutive months. 11 -CD-F n Detection of GalNAc-C 11 -CD-F n Nephrotoxicity in mice.

[0099] (4) An in vitro non-alcoholic steatohepatitis model was established by treating L02 cells with PO / H2O2, and drug intervention was applied simultaneously (F n GalNAc-C 11 -CD-F n ), verify GalNAc-C 11 -CD package to F n Effects of drug efficacy; an in vivo model of non-alcoholic steatohepatitis was established using a diet of HFHC + 15% Fru, with simultaneous drug intervention (F n GalNAc-C 11 -CD-Fn Silibinin (as a positive drug) was used to detect GalNAc-C. 11 -CD's inclusion pair F n The effects of drug efficacy.

[0100] Example 5 GalNAc-C x Validation and screening of hepatic targeting of CD

[0101] Weigh out GalNAc-C according to the molar ratio of 1:1. x -CD is dissolved in water; weigh out the fluorescent dye Nile Red (Ni) and dissolve it in ethanol, then add the Nile Red ethanol solution to GalNAc-C. x In an aqueous solution of -CD, the mixture was stirred at room temperature for 1 hour, filtered through a 0.45 μm microporous membrane, and the solution was freeze-dried for 24 hours to obtain a light purple powder (GalNAc-C). x -CD-Ni, x=5, 7, 11);

[0102] Weigh the GalNAc-C prepared in Example 3 according to a molar ratio of 1:1. 11 -CD dissolves in water; weigh out FITC and dissolve it in ethanol, then add the FITC ethanol solution to GalNAc-C. 11 In an aqueous solution of -CD, the mixture was stirred at room temperature for 1 hour, filtered through a 0.45 μm microporous membrane, and the solution was freeze-dried for 24 hours to obtain an orange powder (GalNAc-C). x -CD-FITC, x = 5, 7, 11);

[0103] 5.1 Effect of the length of the connecting arm between GalNAc and β-cyclodextrin on liver targeting intensity

[0104] Logarithmic growth phase cells (L02, HSC-t6) were digested, counted, and seeded into 12-well plates at 5 × 10⁶ cells / well. 5 Cells / well were cultured in a CO2 incubator for 24 hours. Unattached cells were washed away with PBS, and GalNAc-C cells were then cultured in the appropriate culture medium. x CD-Ni (calculated as Nile Red) was diluted to a working solution of 1 μg / mL and added to the well plate. The addition method was as follows: Figure 14 As shown; GalNAc-Cx-CD-Ni and Ni were co-cultured with cells for 0.5, 1 and 2 h respectively. The cells were washed twice with PBS to remove the fluorescent dyes that were not taken up by the cells. An appropriate amount of PBS was added, and the cells were observed and photographed under a fluorescence microscope.

[0105] L02 cells against GalNAc-C x The uptake rate of -CD-Ni is consistent with this report, meaning that the longer the distance between GalNAc and the packaging material β-CD, the stronger the targeted absorption. Experimental results are as follows...Figure 15 As shown.

[0106] 5.2 GalNAc Competitive Inhibition Test

[0107] L02 cells in logarithmic growth phase were digested, counted, and seeded into 24-well plates at a density of 2.5 × 10⁻⁶. 5 cells / wells, with Nile Red (Ni) and GalNAc-C 11 -CD-Ni were co-cultured separately for 1 h; then treated with 250 mM GalNAc for 0.5 h, followed by the addition of Ni and GalNAc-C respectively. 11 -CD-Ni (20 μM Nile Red) was co-cultured for 1 hour, followed by washing twice with PBS and recording the images using a fluorescence microscope.

[0108] GalNAc treatment did not affect hepatocyte uptake of Nile Red alone, but it significantly inhibited hepatocyte uptake of GalNAc-C. 11 -CD-Ni absorption, its fluorescence intensity compared to GalNAc-C without GalNAc treatment. 11 -CD-Ni group decreased by 61%, indicating that L02 cells are more effective against GalNAc-C. 11 -CD-Ni uptake is selective and dependent on the ASGPR receptor on the surface of hepatocytes, as shown in the following figure. Figure 16 As shown.

[0109] 5.3, GalNAc-C 11 -CD tissue distribution

[0110] Take 5-day-old zebrafish and add Ni / GalNAc-C to the culture water. 11 -CD-Ni (1 μg / mL, based on Ni), FITC / GalNAc-C 11 -CD-FITC (1 μg / mL, based on FITC) was co-cultured for 30 min. Zebrafish co-cultured with the dye were then taken, washed, and their dye distribution was observed under a laser confocal microscope and photographed.

[0111] Zebrafish exhibit a high degree of conservation in disease signaling pathways between humans and zebrafish. Up to 87% of human homologous genes are present in zebrafish, and some disease-related genes show up to 99% conservation with human genes, indicating that results obtained from drug trials in zebrafish are generally applicable to humans. Tissue distribution experiments in zebrafish show that GalNAc-C... 11 -CD has significant liver-targeting properties, such as Figure 17 As shown, Nile Red and FITC fluorescent dyes are distributed throughout the zebrafish, while GalNAc-C... 11-CD-Ni and GalNAc-C 11 -CD-FITC mainly accumulates in the liver, indicating that GalNAc-C 11 -CD also has liver-targeting properties in vivo.

[0112] Example 6: GalNAc-modified liver-targeting β-cyclodextrin-lactus xanthophyll (GalNAc-C) 11 -CD-F n Preparation of inclusion complexes

[0113] Weigh out 100 mg of GalNAc-C according to a molar ratio of 1:1. 11 - Dissolve CD in 50 mL of water; weigh 20 mg of flavonoid (F) n Dissolve in 10 mL of ethanol, then add the rutin ethanol solution to GalNAc-C. 11 The solution of -CD was stirred at room temperature for 1 hour, filtered through a 0.45 μm microporous membrane, and then freeze-dried for 24 hours to obtain a yellow powder (GalNAc-C). 11 -CD-F n );

[0114] SEM results show (e.g.) Figure 18 As shown), the individual F n It appears as a stack of rod-shaped crystals, GalNAc-C 11 -CD has a structure of fine, fragmented crystals, GalNAc-C 11 -CD and F n The 1:1 physical mixture exhibits a simple stacking of the two, forming GalNAc-C. 11 -CD-F n After inclusion, the appearance is completely different from that of the individual substances and physical mixtures; the morphological characteristics of lacquer xanthophyll completely disappear, indicating that lacquer xanthophyll is completely encapsulated in GalNAc-C. 11 -CD inside.

[0115] Infrared detection spectrum as shown Figure 19 As shown, F n The characteristic peaks are consistent with those reported in other literature. Rhubarb has typical hydroxyl, benzene ring, and carbonyl groups, with peaks at 3521, 1606, 1284, and 1273 cm⁻¹, respectively. -1 At this location, corresponding to OH stretching, C=C stretching, and COH bending vibrations; GalNAc-C 11 In the structure of -CD, at 3386cm -1 A broad hydroxyl peak is observed at 2929 cm⁻¹. -1 The extended peak at this location is CH; a physical mixture of GalNAc-C 11 -CD+F nThe strong characteristic peak of rutin is still present (hydroxyl group: 3521 cm⁻¹). -1 Carbonyl groups: 1284, 1273 cm -1 However, it mainly manifests as GalNAc-C. 11 -CD characteristic peaks, possibly indicating high-mass GalNAc-C 11 -CD covered up F n Characteristic absorption peaks; inclusion complex GalNAc-C 11 -CD-F n The main manifestation is GalNAc-C 11 The characteristic peaks of -CD and lacquer yellow pigment disappear, indicating that in GalNAc-C 11 -CD and F n During the formation of the inclusion complex, lacquer xanthophyll is completely masked by GalNAc-C. 11 -The inside of the CD.

[0116] Example 7 Determination of inclusion rate and drug loading

[0117] Weigh out three batches of GalNAc-C respectively. 11 -CD-F n 3 mg of the inclusion compound was dissolved in methanol to prepare a 1.5 mg / mL solution. The solution was sonicated for 20 min, cooled to room temperature, filtered through a 0.45 μm microporous membrane, and the peak area was determined by HPLC. The rutin content was calculated based on the rutin standard curve, and the inclusion rate and drug loading were calculated using the following formulas:

[0118]

[0119]

[0120] GalNAc-C 11 -CD-F n The finished inclusion complexes are shown in Table 1 below, for three batches of GalNAc-C. 11 -CD-F n The content of flavonoids was determined, and the inclusion rate and drug loading of cyclodextrin were calculated. The results are shown in Table 1. GalNAc-C 11 -CD-F n The average inclusion rate was 97.90%, and the average drug loading was 16.32%, indicating that GalNAc-C... 11 -CD has a good encapsulation efficiency for rosin;

[0121] Table 1 GalNAc-C 11 -CD-Fn inclusion ratio and drug loading

[0122]

[0123] Example 8: GalNAc-modified liver-targeting β-cyclodextrin improves the solubility of rutin.

[0124] Weigh 1g of lacquer xanthocyanin and add it to a 100mL volumetric flask. Add water to make up to the volume and let it stand at room temperature. Shake vigorously for 30s every 5 minutes. After 30 minutes, test the solubility of lacquer xanthocyanin.

[0125] Weigh 1g of GalNAc-C 11 -CD-F n Add (calculated as lacquinone) to a 100 mL volumetric flask, dilute to volume with water, place at room temperature, shake vigorously for 30 seconds every 5 minutes, and measure the solubility of lacquinone after 30 minutes.

[0126] Detection of rutin and GalNAc-C respectively 11 -CD-F n The content in aqueous solution is shown in Table 2. The solubility of rosin increased from the original 9.37 μg / mL to [a value] after GalNAc-C [processing]. 11 -CD encapsulation increased the concentration to 3.35 mg / mL, which is 357.84 times the original concentration.

[0127] Table 2. Water solubility of lacquinone

[0128]

[0129] Note: ****P<0.0001

[0130] Example 9 GalNAc-C 11 -CD-F n Cell uptake assay and in vitro drug release

[0131] L02 cells in the logarithmic growth phase were harvested, digested, counted, and 2.5 × 10⁻⁶ cells were collected. 6 Cells were seeded in a six-well plate, and flavonoids (F) were added. n ), GalNAc-C 11 -CD-F n The cells were co-cultured for 2 hours (calculated as 20 μM laccase) and washed twice with PBS. The cells were then lysed with 0.5% SDS. 100 μL of lysis buffer was added to 100 μL of methanol, vortexed for 5 min, centrifuged at 12000 r / min for 15 min, filtered through a 0.45 μm microporous membrane, and the laccase content was detected by HPLC.

[0132] L02 cells against GalNAc-C 11 -CD-F n The intake results showed (e.g.) Figure 20The β-cyclodextrin-encapsulated form of rosin, modified with GalNAc, can be taken up by hepatocytes at a rate 1.3 times higher than that of rosin alone at the same dose. This indicates that the β-cyclodextrin-encapsulated form of GalNAc can target the drug to the surface of hepatocytes, where it can be recognized by the ASGPR protein and then taken up by endocytosis.

[0133] In vitro drug release testing simulates in vivo conditions such as the digestive tract and blood (e.g., temperature, pH value, stirring rate) to test the drug release rate of the formulation. A reasonable in vitro drug release rate is then determined to monitor the product manufacturing process and control product quality. The test results show (…). Figure 21 ), GalNAc-C 11 -CD-Fn showed a cumulative release rate of less than 20% over 12 hours in media with pH = 6.8 and pH = 7.4, and virtually no release in media with pH = 1.6. However, it exhibited explosive release in 20% liver homogenate, with a cumulative release of 58.95% over 12 hours, indicating that GalNAc-C... 11 -CD can deliver the drug flavin to the liver for targeted release, whereas the original flavin drug is essentially not released in any of the four media.

[0134] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a liver-targeted drug delivery carrier, characterized in that, Includes the following steps: (1) Synthesis of N-acetylgalactosamine bromate Br-C x -GalNAc, x = 5, 7, or 11: Br-C is synthesized from N-acetylgalactosamine and x-bromate, x = 5, 7, or 11, via an enzymatic reaction. x -GalNAc, x = 5, 7 or 11; (2) Synthetic liver-targeted drug delivery carrier: using N-acetylgalactosamine bromate Br-C x Using GalNAc (x = 5, 7, or 11) and β-cyclodextrin as raw materials, a liver-targeted drug delivery carrier, GalNAc-C, was synthesized. x -CD, x = 5, 7 or 11.

2. The method for preparing the liver-targeted drug delivery carrier according to claim 1, characterized in that, Step (1) The specific steps are as follows: N-acetylgalactosamine and bromate were reacted at a molar ratio of 1:1 to 1:4, with TLIM lipase as a catalyst and acetone as a solvent, at 55-70°C for 12-20 hours to obtain synthetic N-acetylgalactosamine bromate Br-C. x -GalNAc, x = 5, 7 or 11.

3. The method for preparing the liver-targeted drug delivery carrier according to claim 2, characterized in that, The N-acetylgalactosamine and bromate were reacted in a molar ratio of 1:1 at a temperature of 55°C for 12 hours.

4. The method for preparing the liver-targeted drug delivery carrier according to claim 1, characterized in that, Step (2) specifically includes: Br-C x -GalNAc, x = 5, 7 or 11 solution is added to an alkaline solution of β-cyclodextrin and reacted at 40-60℃ for 0.5-2 hours. After the reaction is complete, excess anhydrous ethanol is added, the mixture is sealed, and incubated overnight at 4-6℃. The supernatant is then removed to obtain the product.

5. The method for preparing the liver-targeted drug delivery carrier according to claim 4, characterized in that, In step (2), Br-C x -GalNAc, x = 5, 7 or 11, is in a mass ratio of 1:1 to β-cyclodextrin; the alkaline solution is a 10% sodium hydroxide solution; the reaction temperature is 55°C and the reaction time is 1 hour.

6. A liver-targeted drug delivery carrier prepared by the method of any one of claims 1-5.

7. A liver-targeted drug delivery system, characterized in that, Includes the liver-targeted drug delivery carrier and drug as described in claim 6.

8. The liver-targeted drug delivery system according to claim 7, characterized in that, The drug is a flavonoid or polyphenol compound.

9. The liver-targeted drug delivery system according to claim 8, characterized in that, The drug is curcumin or flavonoid.

10. The use of the liver-targeting drug delivery carrier according to claim 6 in the preparation of a drug for treating liver disease; wherein the liver disease is non-alcoholic fatty liver disease, cirrhosis, or hepatocellular carcinoma.

11. The use of the liver-targeting drug delivery carrier according to claim 10 in the preparation of drugs for treating liver diseases, characterized in that, The non-alcoholic fatty liver disease mentioned above is non-alcoholic steatohepatitis.

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