A vitamin A-functionalized polyphenol-phospholipid complex, its preparation method and application

Abstract

This invention discloses a vitamin A-functionalized polyphenol-phospholipid complex, its preparation method, and its applications, belonging to the field of pharmaceutical formulation and nanodelivery technology. The phospholipid complex comprises a polyphenol drug, a phospholipid material, and a vitamin A phospholipid ligand. The vitamin A phospholipid ligand is formed by vitamin A or its derivatives and a phospholipid derivative containing amino or other reactive groups. The phospholipid complex forms a phospholipid composite structure through non-covalent interactions between the polyphenol drug and the phospholipid material, and the vitamin A phospholipid ligand is inserted into or assembled into the surface or interior of the phospholipid composite structure. Animal experiments show that this phospholipid complex can reduce serum transaminase levels and alleviate the degree of liver fibrosis, and has good blood compatibility and tissue safety. This invention provides an industrially achievable targeted nano-formulation solution for early intervention in liver fibrosis, with promising application prospects.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical formulation and nanodelivery technology, specifically relating to a polyphenol-phospholipid complex functionalized with vitamin A or its derivatives, its preparation method, and its application in the preparation of drugs for the prevention and / or treatment of liver fibrosis. Background Technology

[0002] Liver fibrosis (LF) is a common pathological process in various chronic liver injuries (such as viral hepatitis, alcoholic liver disease, non-alcoholic fatty liver disease, and metabolic disorders), characterized by excessive accumulation of extracellular matrix (ECM) and remodeling of liver structure. Without timely intervention, liver fibrosis will irreversibly progress to cirrhosis, liver failure, and even hepatocellular carcinoma (HCC). Liver cancer remains a serious health threat worldwide, and epidemiological analyses predict a significant increase in its incidence in the coming years. Studies have shown that liver fibrosis is theoretically reversible; therefore, active treatment intervention during the fibrotic stage is of great significance in reducing the incidence of cirrhosis and liver cancer, and alleviating the social burden of healthcare.

[0003] In the development and progression of liver fibrosis, hepatic stellate cells (HSCs) are widely recognized as core effector cells. Under normal physiological conditions, HSCs are in a resting state, primarily responsible for storing vitamin A. When the liver is stimulated by injury, HSCs are activated and transform into a myofibroblast-like phenotype. Activated HSCs produce large amounts of reactive oxygen species (ROS), triggering oxidative stress, enhancing cell migration and proliferation, and secreting excessive amounts of extracellular matrix (ECM) components such as collagen. Excess ECM accumulates in the liver microenvironment, forming fibrotic scar tissue, which not only disrupts the liver's normal blood supply but also further induces hepatocyte damage, creating a vicious cycle. Therefore, inhibiting HSC activation, promoting their return to a resting state, or inducing apoptosis has become a key strategy in anti-liver fibrosis research.

[0004] However, the pathological changes accompanying liver fibrosis (such as capillary formation of hepatic sinusoidal endothelial cells, tissue hypoxia, and dense ECM deposition) create an extremely complex tissue microenvironment, forming natural physical barriers that severely hinder the delivery and accumulation of traditional antifibrotic drugs at the lesion site. Natural polyphenolic compounds, such as resveratrol (RES), have been proven to possess excellent antioxidant, anti-inflammatory, and antifibrotic activities, capable of inhibiting HSC activation by scavenging ROS. However, resveratrol's extremely poor water solubility, chemical instability, and very low bioavailability in vivo pose significant challenges to its clinical translation.

[0005] While existing technologies have attempted to develop various nanocarriers (such as liposomes and micelles) to improve drug delivery, they often suffer from problems such as insufficient targeting, poor carrier stability, or poor biocompatibility. Therefore, developing an intelligent drug delivery system that can both improve the physicochemical stability of resveratrol and precisely target and activate hepatic stellate cells to achieve an effective therapeutic concentration at the lesion site is a pressing technical challenge in the prevention and treatment of liver fibrosis. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a vitamin A-functionalized polyphenol-phospholipid complex. By combining phospholipids and nano-assembly, the water dispersibility and stability of the polyphenol drug are improved, and active targeted delivery to hepatic stellate cells is achieved through vitamin A phospholipid ligand modification.

[0007] The vitamin A-functionalized polyphenol-phospholipid complex (hereinafter referred to as the phospholipid complex) of this invention comprises: a polyphenol drug, a phospholipid material forming a nanocarrier, and a vitamin A phospholipid ligand. The vitamin A phospholipid ligand is distributed on the surface of the phospholipid complex or embedded in its structure, endowing the phospholipid complex with a specific targeting and uptake advantage for hepatic stellate cells. Based on the mediated recognition between the vitamin A phospholipid ligand and receptors on the surface of hepatic stellate cells, the enrichment concentration of the phospholipid complex in target cells is further increased, thereby exerting antioxidant and anti-fibrotic effects.

[0008] The phospholipid complex forms a phospholipid complex structure through non-covalent interaction between the polyphenol drug and the phospholipid material, and the vitamin A phospholipid ligand is inserted or assembled into the surface or interior of the phospholipid complex structure, thereby achieving active targeted delivery to hepatic stellate cells.

[0009] The polyphenolic drugs include resveratrol, quercetin, curcumin, epigallocatechin gallate (EGCG), luteolin, rutin, kaempferol, apigenin, puerarin, hesperidin, genistein, chlorogenic acid, ferulic acid, polygalactoside, silybin, etc.; resveratrol is preferred.

[0010] The phospholipid material is one or more pharmaceutically acceptable phospholipids or their derivatives, including lecithin, egg yolk phosphatidylcholine, hydrogenated lecithin, soybean phosphatidylcholine, phosphatidylethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), and combinations thereof.

[0011] The vitamin A phospholipid ligand is a ligand formed by covalently linking vitamin A or its derivatives with a phospholipid derivative containing amino or other reactive groups through chemical bonds such as amide bonds; wherein, the vitamin A or its derivatives are one of vitamin A, retinol, retinoic acid, retinal, or pharmaceutically acceptable derivatives thereof; preferably, retinoic acid containing a carboxyl group; the phospholipid derivatives containing amino or other reactive groups include 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), dipalmitoylphosphatidylethanolamine (DPPE), distearatelphosphatidylethanolamine (DSPE), phosphatidylethanolamine (PE), or polyethylene glycol-modified derivatives thereof; the vitamin A phospholipid ligand is preferably a ligand formed by linking vitamin A or its derivatives with DOPE through amide bonds.

[0012] The mass ratio of the polyphenol drug to the phospholipid material is 1:(1~15), preferably 1:(3~6).

[0013] The vitamin A phospholipid ligand accounts for 0.5% to 30% of the total phospholipid complex, preferably 1% to 5%.

[0014] The average particle size of the phospholipid complex is 20 nm to 800 nm, preferably 50 nm to 200 nm; the polydispersity index (PDI) is not greater than 0.50; and the zeta potential is -5 mV to -50 mV, preferably -20 mV to -30 mV, to obtain better colloidal stability.

[0015] The present invention also provides a method for preparing the phospholipid complex, comprising the following steps:

[0016] Step 1: Dissolve the polyphenol drug, phospholipid material and vitamin A phospholipid ligand in an organic solvent, heat and stir to mix thoroughly;

[0017] Step 2: Remove the organic solvent to obtain a film-like or solid residue;

[0018] Step 3: Add an aqueous medium to the obtained residue for hydration to obtain a primary emulsion or a nano-dispersion system;

[0019] Step 4: Perform ultrasonic dispersion or high-pressure homogenization on the obtained system to form a nano-dispersion with uniform particle size;

[0020] Step 5: The obtained nano-dispersion is subjected to impurity removal and unencapsulated drug removal treatment to obtain the phospholipid complex.

[0021] In step 1, the mass ratio of polyphenol drug to phospholipid material is 1:(1~15); preferably 1:(3~6). The molar fraction of vitamin A phospholipid ligand in the total phospholipid complex is 0.5%~30%; preferably 1%~5%.

[0022] In step 1, the preparation of vitamin A phospholipid ligands involves activating vitamin A or its derivatives in a solvent using an EDC / NHS system, followed by reaction with phospholipid derivatives containing amino or other reactive groups. After the reaction is complete, small molecule impurities are removed by dialysis or centrifugation, and the product is obtained by drying.

[0023] In step 1, the organic solvent includes one or more of anhydrous ethanol, methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide (DMF), chloroform, acetone, acetonitrile, and toluene; the stirring temperature is 30℃~50℃.

[0024] In step 3, the aqueous medium is deionized water or PBS buffer.

[0025] In step 5, the impurity removal and unencapsulated drug removal process includes one or more of filtration, centrifugation, and dialysis; preferably, filtration is carried out using a 0.22 μm filter membrane.

[0026] This invention also provides the use of the phospholipid complex in the preparation of antioxidant products and in medicaments for the prevention and / or treatment of liver fibrosis, chronic liver injury, fatty liver disease, and liver diseases related to abnormal activation of hepatic stellate cells. The administration methods of the medicaments include intravenous injection, oral administration, and local administration; preferably, intravenous injection. Through the recognition mediated by vitamin A phospholipid ligands and receptors on the surface of hepatic stellate cells, the uptake and accumulation of the phospholipid complex in hepatic stellate cells are enhanced, thereby exerting antioxidant and anti-fibrotic effects.

[0027] Compared with the prior art, the present invention has at least the following beneficial effects:

[0028] (1) By combining polyphenol drugs with phospholipid materials and nano-assembly, the water dispersibility and in vivo stability of polyphenol drugs can be improved, and sustained drug release can be achieved while slowing down the burst release effect.

[0029] (2) Through the modification of vitamin A phospholipid ligand, active targeted uptake of hepatic stellate cells is achieved, and the amount of drug accumulation in the target cells is increased; (3) The antioxidant defense capacity in the target cells is enhanced, the levels of reactive oxygen species and lipid peroxidation are reduced, thereby inhibiting the activation of hepatic stellate cells and the excessive production of extracellular matrix.

[0030] (4) The phospholipid complex of the present invention has good blood compatibility and tissue safety. Animal experiments have confirmed that it can significantly reduce serum transaminase levels and alleviate the degree of liver fibrosis. Moreover, the formulation can be industrialized and provides a novel targeted nano-formulation solution for the early intervention of liver fibrosis, with good application prospects. Attached Figure Description

[0031] Figure 1The particle size distribution (a) and morphology diagram (b) of the vitamin A-modified resveratrol-phospholipid complex prepared in Example 3 of this invention are shown.

[0032] Figure 2 This is an in vitro drug release diagram of the vitamin A-modified resveratrol-phospholipid complex in this invention;

[0033] Figure 3 This is a schematic diagram of the in vitro evaluation results of intracellular ROS changes in this invention; **P<0.01, ****P<0.0001;

[0034] Figure 4 The graph shows the serum ALT levels in mice in each treatment group for liver fibrosis in this invention; *P<0.05, **P<0.01, ****P<0.0001, ns indicates no statistical difference;

[0035] Figure 5 The graph shows the serum AST levels in mice in each treatment group for liver fibrosis in this invention; *P<0.05, ****P<0.0001, ns indicates no statistical difference;

[0036] Figure 6 The graph shows the HYP levels in mouse tissues in each treatment group of liver fibrosis in this invention; *P<0.05, **P<0.01, ****P<0.0001, ns indicates no statistical difference. Detailed Implementation

[0037] To more clearly illustrate the purpose, technical solution, and beneficial effects of this invention, the following embodiments are provided for further explanation. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of protection of this invention. Those skilled in the art can make various modifications or equivalent substitutions to the embodiments without departing from the spirit of this invention.

[0038] In the following embodiments, reagents, materials and equipment not specifically mentioned are all commercially available; experimental conditions not specifically mentioned are all conventional conditions in the art.

[0039] Example 1

[0040] Preparation of vitamin A phospholipid ligand (VA-DOPE):

[0041] Retinic acid was reacted with N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) in a molar ratio of VA acid:NHS:EDC:DOPE = 1:(0.5~5):(0.5~5):(0.5~5) in the polar aprotic solvent N,N-dimethylformamide. The mixture was activated at 20℃~30℃ for 0.5 h~2 h, followed by the addition of a solution containing dioleoylphosphatidylethanolamine (DOPE) in the same solvent. The reaction was carried out in the dark at 40℃~70℃ for 6 h~24 h to form vitamin A phospholipid ligands. After the reaction, the reaction solution was placed in a dialysis bag with a molecular weight cutoff of 300 Da~1000 Da and purified by dialyzing in a methanol / water mixture (1:1 volume ratio). The dialysis was then repeated with distilled water. After dialysis, the mixture was centrifuged, and the precipitate was washed 2~3 times with distilled water and dried to obtain a yellow solid product.

[0042] Example 2

[0043] Preparation of resveratrol phospholipid complex (RES-PC):

[0044] Resveratrol and egg yolk lecithin were mixed at a mass ratio of 1:4 and added to anhydrous ethanol. The mixture was stirred at 50°C for 30 min until completely dissolved. The ethanol was then evaporated to remove the film-like residue. Distilled water was slowly added to the residue at 30°C and hydrated for 10 min to obtain an initial dispersion. The dispersion was then homogenized by ultrasonic dispersion (25% amplitude, 6 min). Finally, the unencapsulated drug was removed by filtration through a 0.22 μm filter membrane to obtain the RES-PC dispersion.

[0045] Example 3

[0046] Preparation of Vitamin A-functionalized resveratrol-egg yolk lecithin complex (VA-RES-PC):

[0047] Resveratrol and egg yolk lecithin were mixed at a mass ratio of 1:4, and VA-DOPE (3% phospholipid content) was added. The mixture was then added to anhydrous ethanol and stirred at 50°C for 30 min until completely dissolved. The ethanol was then evaporated to remove the film-like residue. Distilled water was slowly added to the residue at 30°C and hydrated for 10 min to obtain an initial dispersion. This dispersion was then homogenized using ultrasonic dispersion (25% amplitude, 6 min). Finally, the mixture was filtered through a 0.22 μm filter to remove unencapsulated drug, yielding a VA-RES-PC dispersion. Figure 1 ).

[0048] Example 4

[0049] Preparation of Vitamin A-functionalized resveratrol-soybean lecithin complex (VA-RES-SPC):

[0050] Resveratrol and soybean lecithin were mixed at a mass ratio of 1:2, and VA-DOPE (3% phospholipid content) was added to tetrahydrofuran. The mixture was stirred at 45°C for 20 min until completely dissolved. The tetrahydrofuran was then evaporated to remove the film-like residue. Distilled water was slowly added to the residue at 25°C and hydrated for 10 min to obtain an initial dispersion. The dispersion was then extruded five times using a liposome extruder. Finally, the unencapsulated drug was removed by filtration through a 0.22 μm filter membrane to obtain the VA-RES-SPC dispersion.

[0051] Example 5

[0052] Preparation of Vitamin A-functionalized resveratrol-hydrogenated lecithin complex (VA-RES-HSPC):

[0053] Resveratrol and hydrogenated soybean lecithin were mixed at a mass ratio of 1:3, and VA-DOPE (3% phospholipid content) was added. The mixture was then added to anhydrous ethanol and stirred at 35°C for 40 min until completely dissolved. The ethanol was then evaporated to remove the film-like residue. Distilled water was slowly added to the residue at 40°C and hydrated for 6 min to obtain an initial dispersion. The dispersion was then homogenized by ultrasonic dispersion (25% amplitude, 6 min). Finally, the unencapsulated drug was removed by filtration through a 0.22 μm filter membrane to obtain the VA-RES-HSPC dispersion.

[0054] Example 6

[0055] In vitro release studies:

[0056] The release behavior of resveratrol was investigated using dialysis: equal volumes of resveratrol aqueous solution, RES-PC dispersion (resveratrol phospholipid complex without 3% VA-DOPE), and VA-RES-PC dispersion were placed in dialysis bags (MWCO 3500) and shaken at 37℃ and pH 7.4 PBS. Samples were taken at preset time points, and the absorbance at 306 nm was measured by UV spectrophotometry to calculate the cumulative release rate. The results are as follows: Figure 2 This indicates that free resveratrol exhibits a significant burst release in the early stages; while the release of RES-PC and VA-RES-PC is significantly delayed, with a cumulative release amount at 48 h that is significantly lower than that of free resveratrol, suggesting that the phospholipid complex can effectively reduce burst release and achieve sustained release.

[0057] Example 7

[0058] Antioxidant capacity evaluation:

[0059] 1×10 6HSC-T6 cells were seeded in 6-well plates and incubated for 4 h with 25 μg / ml RES, RES-PC, and VA-RES-PC. An equal volume of culture medium was used as a control. The drug-containing and control media were aspirated, washed three times with PBS, and then the DCFH-DA probe was diluted 1:1000 with the culture medium and added to the wells. After incubation for 4 h, the culture medium was discarded, and the cells were washed three times with PBS. Intracellular ROS levels were quantitatively detected by flow cytometry. Results are as follows: Figure 3 As shown, compared to the control group, RES, RES-PC, and VA-RES-PC all reduced intracellular ROS levels to varying degrees. RES-PC significantly reduced ROS levels compared to RES, demonstrating that the phospholipid complex can increase the water solubility of RES, which is beneficial for drug accumulation in cells. VA-RES-PC more significantly reduced intracellular ROS levels, demonstrating that the vitamin A-modified phospholipid complex can actively target hepatic stellate cells, exhibiting more significant antioxidant capacity compared to the passively targeted unmodified phospholipid complex.

[0060] Example 8

[0061] Pharmacodynamic experiments:

[0062] Male KM mice were randomly divided into 5 groups: saline group (ip 0.9% NaCl for 4 weeks + iv 0.9% NaCl for 2 weeks), liver fibrosis model group (ip 1% DMN for 4 weeks + 0.9% NaCl for 2 weeks), liver fibrosis + RES treatment group (ip 1% DMN for 4 weeks + RES for 2 weeks), liver fibrosis + RES-PC treatment group (ip 1% DMN for 4 weeks + RES-PC for 2 weeks), and liver fibrosis + VA-RES-PC treatment group (ip 1% DMN for 4 weeks + VA-RES-PC for 2 weeks). Except for the normal control group, a liver fibrosis model was established by intraperitoneal injection of dimethylnitrosamine (DMN): 1% DMN, 10 mg / kg, 3 times a week for 4 weeks; subsequently, each treatment group received intravenous injection of the corresponding preparation (10 mg / kg as RES), 3 times a week for 2 weeks. Mice were sacrificed 48 hours after the last administration for blood and organ collection. Serum ALT, AST, and other liver function indicators, as well as hydroxyproline (HYP) fibrosis markers, were measured. Results are as follows: Figure 4 , Figure 5 and Figure 6 As shown, the levels of ALT, AST, and HYP in the serum of fibrotic mice were significantly elevated. After treatment with different preparations, the levels in each group decreased to varying degrees. Among them, VA-RES-PC could more effectively improve liver function indicators and reduce collagen deposition, suggesting that it has a strong anti-liver fibrosis effect.

[0063] The above is merely a description of preferred embodiments of the present invention. Any changes, equivalent substitutions, or improvements made by those skilled in the art without departing from the concept of the present invention should fall within the protection scope of the present invention.

Claims

1. A vitamin A-functionalized polyphenol-phospholipid complex, characterized in that, The phospholipid complex includes a polyphenol drug, a phospholipid material, and a vitamin A phospholipid ligand; the vitamin A phospholipid ligand is formed by vitamin A or its derivatives and a phospholipid derivative containing amino or other reactive groups; the phospholipid complex forms a phospholipid complex structure through non-covalent interaction between the polyphenol drug and the phospholipid material, and the vitamin A phospholipid ligand is inserted into or assembled into the surface or interior of the phospholipid complex structure.

2. The vitamin A-functionalized polyphenol-phospholipid complex according to claim 1, characterized in that, The polyphenol drugs mentioned include resveratrol, quercetin, curcumin, epigallocatechin gallate, luteolin, rutin, kaempferol, apigenin, puerarin, hesperidin, genistein, chlorogenic acid, ferulic acid, polygalactoside, and silymarin.

3. The vitamin A-functionalized polyphenol-phospholipid complex according to claim 1, characterized in that, The phospholipid materials include lecithin, egg yolk phosphatidylcholine, hydrogenated lecithin, soybean phosphatidylcholine, phosphatidylethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine, and combinations thereof.

4. The vitamin A-functionalized polyphenol-phospholipid complex according to claim 1, characterized in that, The vitamin A or its derivative is one of retinoic acid, retinol, retinaldehyde or a pharmaceutically acceptable derivative thereof; the phospholipid derivative containing amino or other reactive groups includes 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine, dipalmitoylphosphatidylethanolamine, distearateylphosphatidylethanolamine, phosphatidylethanolamine or its polyethylene glycol derivative.

5. A method for preparing the vitamin A-functionalized polyphenol-phospholipid complex according to any one of claims 1-4, characterized in that, Includes the following steps: Step 1: Dissolve the polyphenol drug, phospholipid material and vitamin A phospholipid ligand in an organic solvent, and heat and stir. Step 2: Remove the organic solvent to obtain a film-like or solid residue; Step 3: Add an aqueous medium to the obtained residue for hydration to obtain a primary emulsion or a nano-dispersion system; Step 4: Perform ultrasonic dispersion or high-pressure homogenization on the obtained system to form a nano-dispersion with uniform particle size; Step 5: The obtained nano-dispersion is subjected to impurity removal and unencapsulated drug removal treatment to obtain phospholipid complex.

6. The preparation method according to claim 5, characterized in that, The ratio of polyphenol drug to phospholipid material is 1:(1~15), preferably 1:(3~6); the molar fraction of vitamin A phospholipid ligand in the total phospholipid complex is 0.5%~30%, preferably 1%~5%.

7. The preparation method according to claim 5, characterized in that, Preparation method of vitamin A phospholipid ligand: Vitamin A or its derivatives are activated in a solvent using an EDC / NHS system, and then reacted with phospholipid derivatives containing amino or other reactive groups. After the reaction is completed, small molecule impurities are removed by dialysis or centrifugation and the product is obtained by drying.

8. The preparation method according to claim 5, characterized in that, The organic solvent includes one or more of anhydrous ethanol, methanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, chloroform, acetone, acetonitrile, and toluene; the stirring temperature is 30℃~50℃; the aqueous phase medium is deionized water or PBS buffer; the obtained phospholipid complex has a particle size of 20nm~800nm, preferably 50nm~200nm; a polydispersity index (PDI) of not more than 0.50; and a zeta potential of -5mV to -50mV, preferably -20mV~-30mV.

9. The use of the vitamin A-functionalized polyphenol-phospholipid complex according to any one of claims 1-4 in the preparation of a medicament for the prevention and / or treatment of liver fibrosis, chronic liver injury, fatty liver disease, and liver diseases related to abnormal activation of hepatic stellate cells, characterized in that... The routes of administration include intravenous injection, oral administration, and topical administration.

10. The use of the vitamin A functionalized polyphenol-phospholipid complex according to any one of claims 1-4 in the preparation of antioxidant products.

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