A polysaccharide-modified phospholipid complex and a method for preparing the same

Abstract

The present application relates to a polysaccharide modified drug phospholipid complex, wherein the drug phospholipid complex is prepared from a drug and a phospholipid, the drug includes oxymatrine and glycyrrhizin, and the polysaccharide modification is that the drug phospholipid complex is first modified by chitosan, and then the drug phospholipid complex coated with chitosan is modified by hyaluronic acid. The polysaccharide modified drug phospholipid complex of the present application can enhance the drug accumulation in tumor cells through synergistic effect and active targeting, and significantly improve the anti-tumor effect of the drug.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical preparations, specifically to a polysaccharide-modified phospholipid complex and its preparation method. Background Technology

[0002] According to a report by the International Agency for Research on Cancer, there are more than 10 million new cancer cases and more than 6 million cancer deaths worldwide each year. It is estimated that by 2030, there will be 21.4 million new cases and 13.2 million deaths. Chemotherapy is the most common treatment for malignant tumors; however, even when combined with surgery or radiotherapy, chemotherapy often fails. One of the main reasons for chemotherapy failure is that tumor cells develop resistance to cytotoxic drugs, or even multidrug resistance (MDR). This resistance greatly hinders the clinical application of chemotherapy drugs.

[0003] In clinical practice, multiple chemotherapy drugs with different mechanisms of action are often combined to improve drug resistance and enhance efficacy, but the outcomes remain unsatisfactory. The combined delivery of anticancer drugs using a single carrier and nanotechnology has been shown in vitro and in vivo to reverse MDR (Multiple Drug Reaction), proving more effective than combining free drugs or delivering individual drugs separately using nanocarriers. This strategy of delivering multiple drugs through a single carrier allows for precise and controlled drug delivery, such as maintaining optimal drug proportions upon reaching the target organ. Drug combinations are crucial for effective combination therapy; the combined delivery of anticancer drugs with chemosensitizers has shown better efficacy than combinations of multiple anticancer drugs.

[0004] Sophora flavescens has a long history of use in China and has a wide range of pharmacological effects, such as anti-tumor, anti-inflammatory, and immunostimulatory effects. Compound Sophora flavescens injection (CKI) was approved by the State Food and Drug Administration (SFDA) in 1995 for the treatment of breast cancer, liver cancer, and lung cancer. Oxymatrine (OMT) is one of the main components of Compound Sophora flavescens injection, and it exhibits anti-tumor activity in gastric cancer, liver cancer, non-small cell lung cancer, and prostate cancer through multiple mechanisms, including inhibiting cancer cell proliferation and metastasis and inducing apoptosis.

[0005] High levels of ATP-binding cassette (ABC) transporter-mediated efflux lead to reduced drug delivery to tumor cells or difficulty in reaching targets within tumor cells, which is currently the most widely studied mechanism of drug resistance. ABC transporter inhibitors, also known as chemosensitizers or multidrug resistance modulators, inhibit ABC transporter function, thereby reducing drug resistance and restoring drug sensitivity. Chemosensitizers have long been a hot topic in cancer research, from first to third generation, but due to their toxicity to vital organs, none have yet been approved for human use. Therefore, the development of reversal agents from natural drugs has received widespread attention in recent years. Natural drugs, due to their low toxicity, high efficacy, and multi-target advantages, are called "fourth-generation MDR reversal agents." Many naturally derived compounds have been found to inhibit the expression activity of ABC transporters such as P-gp and MRP1, such as curcumin, silymarin, glycyrrhizic acid, icariin, resveratrol, quercetin, luteolin, and apigenin.

[0006] Licorice is used as an adjuvant in many traditional Chinese medicine prescriptions, harmonizing the effects of various drugs. Glycyrrhizin (GL), the main compound isolated from licorice, has been found to enhance the therapeutic effects of various drugs. Glycyrrhizin is an MDR modulator, increasing drug uptake by promoting cell membrane permeability and inhibiting drug efflux, and inhibiting ABC transporters by increasing NO production.

[0007] Active ingredients extracted from natural plants can bind with phospholipids through hydrogen bonds or van der Waals forces to form nanoscale phospholipid complexes, which exhibit excellent absorption and penetration. Phospholipid complexes have a structure similar to liposomes, but with higher drug encapsulation efficiency and drug loading capacity compared to liposomes.

[0008] The main drawbacks of natural drug phospholipid complexes are particle aggregation, hydrolysis / oxidation of ester bonds, and poor stability under physiological conditions. Constructing core-shell nanocarriers with phospholipid complexes as the core and polymeric materials as the shell can improve the stability of phospholipid complexes. Chitosan (CS) has many advantages, such as good compatibility and permeability, and possesses various biological properties, such as antibacterial and anticancer effects. Hyaluronic acid (HA), a negatively charged polysaccharide, is often used to modify carriers to enhance the effective targeting of chemotherapeutic drugs within tumor cells.

[0009] Currently, there are no literature reports on oxymatrine / glycyrrhizin phospholipid complexes modified with polymer materials. Summary of the Invention

[0010] The purpose of this invention is to provide a polysaccharide-modified drug phospholipid complex that can enhance drug accumulation in tumor cells through synergistic and active targeting effects, thereby significantly improving the anti-tumor effect of the drug.

[0011] To achieve the above objectives, the present invention adopts the following technical solution:

[0012] A first aspect of the present invention is to provide a polysaccharide-modified pharmaceutical phospholipid complex, the pharmaceutical phospholipid complex being prepared from a drug and a phospholipid, the drug including oxymatrine and glycyrrhizin, and the polysaccharide used to modify the pharmaceutical phospholipid complex being a combination of chitosan and hyaluronic acid.

[0013] Preferably, the drug is composed of oxymatrine and glycyrrhizin.

[0014] Preferably, the polysaccharide modification involves first modifying the drug phospholipid complex with chitosan, and then modifying the chitosan-coated drug phospholipid complex with hyaluronic acid.

[0015] Preferably, the weight ratio of phospholipids to drugs added during the preparation process is 1:1 to 1:0.5. More preferably, the weight ratio of phospholipids to drugs is 1:1 to 1:0.6. Even more preferably, the weight ratio of phospholipids to drugs is 1:1.

[0016] Preferably, the weight ratio of oxymatrine to glycyrrhizin added during the preparation process is 1:3-3:1. More preferably, the weight ratio of oxymatrine to glycyrrhizin is 1:2-2:1. Even more preferably, the weight ratio of oxymatrine to glycyrrhizin is 1:1.

[0017] Preferably, the weight ratio of chitosan to the drug phospholipid complex in the preparation of the polysaccharide-modified drug phospholipid complex is 1:1 to 1:8. More preferably, the weight ratio of chitosan to the drug phospholipid complex is 1:4 to 1:8. Even more preferably, the weight ratio of chitosan to the drug phospholipid complex is 1:4.

[0018] Preferably, the weight ratio of hyaluronic acid to chitosan in the preparation of the polysaccharide-modified drug phospholipid complex is 0.5:1 to 1.5:1. More preferably, the weight ratio of hyaluronic acid to chitosan is 1:1.

[0019] Preferably, the weight ratio of hyaluronic acid, chitosan, and drug phospholipid complex in the preparation of polysaccharide-modified drug phospholipid complex is 1:1:4.

[0020] Preferably, the chitosan is a low molecular weight chitosan with a degree of deacetylation >75% and a viscosity of 20-300 cps at 1%. More preferably, the low molecular weight chitosan has a molecular weight of 50-500 kDa. Even more preferably, the low molecular weight chitosan has a molecular weight of 50-190 kDa.

[0021] Preferably, the hyaluronic acid has a molecular weight of 40-80 kDa. More preferably, the hyaluronic acid has a molecular weight of 60 kDa.

[0022] Preferably, the average particle size of the polysaccharide-modified drug phospholipid complex is less than 400 nm. More preferably, the average particle size is less than 350 nm. Even more preferably, the average particle size is less than 300 nm.

[0023] Preferably, the PDI of the polysaccharide-modified drug phospholipid complex is less than 0.2. More preferably, the PDI is less than 0.18. Even more preferably, the PDI is less than 0.16.

[0024] Preferably, both oxymatrine and glycyrrhizin in the polysaccharide-modified drug phospholipid complex exist in an amorphous form.

[0025] Preferably, the encapsulation efficiency of both oxymatrine and glycyrrhizin in the polysaccharide-modified drug phospholipid complex is greater than 40%. More preferably, the encapsulation efficiency is greater than 45%.

[0026] Preferably, the drug loading ratio of oxymatrine to glycyrrhizin in the polysaccharide-modified drug phospholipid complex is 1.2:1-0.8:1. More preferably, the drug loading ratio is 1.1:1-0.9:1. Even more preferably, the drug loading ratio is 1:1.

[0027] Preferably, the oxymatrine in the polysaccharide-modified drug phospholipid complex exhibits rapid release characteristics, while glycyrrhizin exhibits sustained release characteristics.

[0028] More preferably, the pH of the release medium for the polysaccharide-modified drug phospholipid complex is 5.0.

[0029] A second aspect of the present invention is to provide a method for preparing the above-mentioned polysaccharide-modified drug phospholipid complex, comprising the following steps:

[0030] (1) Dissolve phospholipids in an organic solvent, add oxymatrine and glycyrrhizin respectively, stir the reaction under heating, and then evaporate the filtered solution under vacuum to obtain drug phospholipid complex OGP.

[0031] (2) Dissolve chitosan in an acidic solution, add OGP suspension to the chitosan solution, and incubate to obtain chitosan-coated drug phospholipid complex CS-OGP suspension;

[0032] (3) Dissolve hyaluronic acid in water, add the hyaluronic acid solution to the CS-OGP suspension, stir, and obtain the hyaluronic acid modified chitosan-coated drug phospholipid complex HA-CS-OGP suspension. Centrifuge, dry the precipitate, and obtain the final product.

[0033] Preferably, the organic solvent in step (1) is tetrahydrofuran.

[0034] Preferably, the phospholipid concentration in step (1) is 10 mg / ml.

[0035] Preferably, the weight ratio of phospholipids added in step (1) to the total drug is 1:1 to 1:0.5. More preferably, the weight ratio of phospholipids added in step (1) to the total drug is 1:1 to 1:0.6. Even more preferably, the weight ratio of phospholipids added in step (1) to the total drug is 1:1.

[0036] Preferably, the weight ratio of oxymatrine to glycyrrhizin added in step (1) is 1:3-3:1. More preferably, the weight ratio of oxymatrine to glycyrrhizin added in step (1) is 1:2-2:1. Even more preferably, the weight ratio of oxymatrine to glycyrrhizin added in step (1) is 1:1.

[0037] Preferably, the heating temperature in step (1) is 50-70°C. More preferably, the heating temperature in step (1) is 60°C.

[0038] Preferably, the stirring time in step (1) is 4-6 hours. More preferably, the stirring time in step (1) is 5 hours.

[0039] Preferably, in step (1), vacuum evaporation is performed at 30°C for 1 hour.

[0040] Preferably, the chitosan in step (2) is a low molecular weight chitosan with a degree of deacetylation >75% and a viscosity of 20-300 cps at 1%. More preferably, the low molecular weight chitosan in step (2) has a molecular weight of 50-500 kDa. Even more preferably, the low molecular weight chitosan in step (2) has a molecular weight of 50-190 kDa.

[0041] Preferably, the acidic solution in step (2) is an acetic acid solution. More preferably, the concentration of the acetic acid solution in step (2) is 0.1M.

[0042] Preferably, the concentration of the chitosan solution in step (2) is 0.25-2 mg / ml. More preferably, the concentration of the chitosan solution in step (2) is 0.25-0.5 mg / ml. Even more preferably, the concentration of the chitosan solution in step (2) is 0.5 mg / ml.

[0043] Preferably, the concentration of the OGP suspension in step (2) is 2 mg / ml.

[0044] Preferably, the weight ratio of chitosan to OGP in step (2) is 1:1 to 1:8. More preferably, the weight ratio of chitosan to OGP in step (2) is 1:4 to 1:8. Even more preferably, the weight ratio of chitosan to OGP in step (2) is 1:4.

[0045] Preferably, in step (2), the chitosan solution and the OGP suspension are mixed in equal volumes.

[0046] Preferably, the reaction solution in step (2) is incubated at room temperature for 2-6 hours. More preferably, the reaction solution in step (2) is incubated at room temperature for 4 hours.

[0047] Preferably, the molecular weight of the hyaluronic acid in step (3) is 40-80 kDa. More preferably, the molecular weight of the hyaluronic acid in step (3) is 60 kDa.

[0048] Preferably, the concentration of the hyaluronic acid aqueous solution in step (3) is 1 mg / ml.

[0049] Preferably, the weight ratio of hyaluronic acid in step (3) to chitosan in step (2) is 0.5:1-1.5:1. More preferably, the weight ratio of hyaluronic acid in step (3) to chitosan in step (2) is 1:1.

[0050] Preferably, the reaction solution in step (3) is stirred at room temperature for 2-6 hours. More preferably, the reaction solution in step (3) is stirred at room temperature for 4 hours.

[0051] A third aspect of the present invention provides the use of the above-described polysaccharide-modified drug phospholipid complex in the preparation of a medicament for treating cancer.

[0052] Preferably, the cancer is liver cancer, lung cancer, or leukemia.

[0053] More preferably, the cancer is liver cancer.

[0054] More preferably, the liver cancer is the HepG2 liver cancer cell line.

[0055] A fourth aspect of the present invention provides a pharmaceutical composition for synergistic treatment of cancer, the pharmaceutical composition comprising oxymatrine and glycyrrhizin in a weight ratio of 1:3 to 3:1.

[0056] Preferably, the pharmaceutical composition comprises oxymatrine and glycyrrhizin in a weight ratio of 1:2 to 2:1. More preferably, the pharmaceutical composition comprises oxymatrine and glycyrrhizin in a weight ratio of 1:1.

[0057] Preferably, the concentrations of oxymatrine and glycyrrhizin in the pharmaceutical composition are both 1 μg / ml to 100 μg / ml. More preferably, the concentrations of oxymatrine and glycyrrhizin in the pharmaceutical composition are 1 μg / ml, 10 μg / ml, 50 μg / ml, or 100 μg / ml.

[0058] Preferably, the cancer is liver cancer, lung cancer, or leukemia.

[0059] A fifth aspect of the invention provides the use of the above-described pharmaceutical composition in the preparation of a medicament for treating cancer, said pharmaceutical composition comprising oxymatrine and glycyrrhizin in a weight ratio of 1:3 to 3:1.

[0060] Preferably, the pharmaceutical composition comprises oxymatrine and glycyrrhizin in a weight ratio of 1:2 to 2:1. More preferably, the pharmaceutical composition comprises oxymatrine and glycyrrhizin in a weight ratio of 1:1.

[0061] Preferably, the concentrations of oxymatrine and glycyrrhizin in the pharmaceutical composition are both 1 μg / ml to 100 μg / ml. More preferably, the concentrations of oxymatrine and glycyrrhizin in the pharmaceutical composition are 1 μg / ml, 10 μg / ml, 50 μg / ml, or 100 μg / ml.

[0062] Preferably, the cancer is liver cancer, lung cancer, or leukemia.

[0063] More preferably, the cancer is liver cancer.

[0064] More preferably, the liver cancer is the HepG2 liver cancer cell line.

[0065] A sixth aspect of the present invention provides a pharmaceutical preparation for synergistic treatment of cancer, the pharmaceutical preparation being a drug phospholipid complex modified or unmodified with polysaccharides, wherein the drug in the phospholipid complex comprises oxymatrine and glycyrrhizin in a weight ratio of 1:3 to 3:1.

[0066] Preferably, the drug comprises oxymatrine and glycyrrhizin in a weight ratio of 1:2 to 2:1. More preferably, the drug comprises oxymatrine and glycyrrhizin in a weight ratio of 1:1.

[0067] Preferably, the concentrations of oxymatrine and glycyrrhizin in the pharmaceutical composition are both 0.1 μg / ml to 50 μg / ml. More preferably, the concentrations of oxymatrine and glycyrrhizin in the pharmaceutical composition are 0.1 μg / ml, 1 μg / ml, 10 μg / ml, or 50 μg / ml.

[0068] Preferably, the cancer is liver cancer, lung cancer, or leukemia.

[0069] Preferably, the polysaccharide modification involves first modifying the drug phospholipid complex with chitosan, and then modifying the chitosan-coated drug phospholipid complex with hyaluronic acid.

[0070] Preferably, the weight ratio of hyaluronic acid, chitosan, and drug phospholipid complex in the preparation of polysaccharide-modified drug phospholipid complex is 1:1:4.

[0071] Preferably, the chitosan is a low molecular weight chitosan with a degree of deacetylation >75% and a viscosity of 20-300 cps at 1%. More preferably, the low molecular weight chitosan has a molecular weight of 50-500 kDa. Even more preferably, the low molecular weight chitosan has a molecular weight of 50-190 kDa.

[0072] Preferably, the hyaluronic acid has a molecular weight of 40-80 kDa. More preferably, the hyaluronic acid has a molecular weight of 60 kDa.

[0073] A seventh aspect of the present invention provides the use of the above-described pharmaceutical preparation in the preparation of a medicament for treating cancer, wherein the pharmaceutical preparation is a phospholipid complex modified with or without polysaccharide modification, and the phospholipid complex contains oxymatrine and glycyrrhizin in a weight ratio of 1:3 to 3:1.

[0074] Preferably, the drug comprises oxymatrine and glycyrrhizin in a weight ratio of 1:2 to 2:1. More preferably, the drug comprises oxymatrine and glycyrrhizin in a weight ratio of 1:1.

[0075] Preferably, the concentrations of oxymatrine and glycyrrhizin in the phospholipid complex are both 0.1 μg / ml to 50 μg / ml. More preferably, the concentrations of oxymatrine and glycyrrhizin in the phospholipid complex are 0.1 μg / ml, 1 μg / ml, 10 μg / ml, or 50 μg / ml.

[0076] Preferably, the cancer is liver cancer, lung cancer, or leukemia.

[0077] Preferably, the polysaccharide modification involves first modifying the drug phospholipid complex with chitosan, and then modifying the chitosan-coated drug phospholipid complex with hyaluronic acid.

[0078] Preferably, the weight ratio of hyaluronic acid, chitosan, and drug phospholipid complex in the preparation of polysaccharide-modified drug phospholipid complex is 1:1:4.

[0079] Preferably, the chitosan is a low molecular weight chitosan with a degree of deacetylation >75% and a viscosity of 20-300 cps at 1%. More preferably, the low molecular weight chitosan has a molecular weight of 50-500 kDa. Even more preferably, the low molecular weight chitosan has a molecular weight of 50-190 kDa.

[0080] Preferably, the hyaluronic acid has a molecular weight of 40-80 kDa. More preferably, the hyaluronic acid has a molecular weight of 60 kDa.

[0081] This invention has positive and beneficial effects:

[0082] 1. The present invention selects oxymatrine and glycyrrhizin to be co-loaded into phospholipid complexes through interaction with phospholipids for combination therapy of cancer. The drug loading of oxymatrine and glycyrrhizin in phospholipid complexes and polysaccharide-modified phospholipid complexes is almost consistent with a fixed 1:1 ratio, which can maintain a fixed optimized ratio.

[0083] 2. Compared with the free mixture of oxymatrine and glycyrrhizin, the oxymatrine / glycyrrhizin phospholipid complex (OGP) has a synergistic therapeutic effect on HepG2 cells.

[0084] 3. In vitro release experiments showed that the hyaluronic acid-modified chitosan-coated phospholipid complex exhibited sequential release characteristics, and this nano-formulation could improve efficacy and reduce toxicity.

[0085] 4. Compared to the chitosan-coated phospholipid complex without modified hyaluronic acid, the hyaluronic acid-modified chitosan-coated phospholipid complex enhanced cell aggregation in CD44-overexpressing tumor cells through the interaction between hyaluronic acid and CD44. Therefore, the co-delivery system of oxymatrine and glycyrrhizin can enhance drug aggregation in tumor cells through synergistic and active targeting effects, significantly improving the anti-tumor efficacy of the drug. Attached Figure Description

[0086] Figure 1 These are the P-XRD patterns of the oxymatrine / glycyrrhizin phospholipid complex (1A), phospholipid (1B), glycyrrhizin (1C), and oxymatrine (1D) of the present invention.

[0087] Figure 2 These are the release curves of the polysaccharide-modified and unmodified drug phospholipid complexes of this invention in a release medium at pH 7.4. 2A and 2B are the release curves of oxymatrine and glycyrrhizin in different carriers, respectively.

[0088] Figure 3 These are the release curves of the polysaccharide-modified and unmodified drug phospholipid complexes of this invention in a release medium at pH 5.0. 3A and 3B are the release curves of oxymatrine and glycyrrhizin in different carriers, respectively.

[0089] Figure 4 The toxicity of different formulations of this invention to HepG2 cells;

[0090] Figure 5 This describes the cellular uptake of the polysaccharide-modified drug phospholipid complexes CS-OGP and HA-CS-OGP of this invention. Detailed Implementation

[0091] The present invention will be further described below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods.

[0092] Unless otherwise specified, in the following experimental examples of this invention, OGP refers to the oxymatrine / glycyrrhizin phospholipid complex, CS-OGP refers to the chitosan-coated oxymatrine / glycyrrhizin phospholipid complex, and HA-CS-OGP refers to the hyaluronic acid-modified chitosan-coated oxymatrine / glycyrrhizin phospholipid complex. The proportions of drugs and excipients used in the preparation of OGP, CS-OGP, and HA-CS-OGP are all by weight ratio.

[0093] Experimental Example 1: Preparation and Physicochemical Properties Determination of Phospholipid Complexes with Different Drug Ratios

[0094] 1. Test Methods

[0095] 0.6 g of phospholipid (Shanghai Taiwei Pharmaceutical Co., Ltd., phosphatidylcholine content >96%) was dissolved in tetrahydrofuran. After clarification, oxymatrine and glycyrrhizin were added separately (phospholipid to total drug weight ratio 1:1, with oxymatrine and glycyrrhizin weight ratios of 2:1 and 1:2, respectively). The reaction temperature was controlled at 60℃ using a water bath, and the mixture was stirred at 600 rpm for 5 hours using a magnetic stirrer. The suspension was filtered through a 0.22 μm organic membrane to remove excess unreacted drug. The clarified solution was evaporated under vacuum at 30℃ for 1 hour to obtain a pale yellow powder, OGP. The OGP was dispersed in water to obtain an OGP suspension of 1 mg / ml. The particle size, PDI, and potential of OGP suspensions of samples 1-2 were determined.

[0096] 2. Test Results

[0097] The average particle size, PDI, and potential of the various nanoparticles obtained in this experiment are shown in Table 2 below.

[0098] Table 1. Average particle size, PDI, and potential of phospholipid complexes prepared with different drug ratios according to the present invention.

[0099]

[0100] The experimental results in the table above show that, with the weight ratio of phospholipids to total drugs fixed at 1:1, the drug phospholipid complexes obtained by varying the weight ratio of oxymatrine to glycyrrhizin within the range of 1:2 to 2:1 all meet the formulation requirements.

[0101] Experimental Example 2: Preparation and Physicochemical Properties Determination of Polysaccharide-Modified Phospholipid Complexes

[0102] 1. Test Methods

[0103] 1.1 Preparation of drug phospholipid complex (OGP)

[0104] 1 g of phospholipid was dissolved in tetrahydrofuran, with a phospholipid concentration of 10 mg / ml. After clarification, oxymatrine and glycyrrhizin were added respectively (phospholipid to total drug weight ratios of 1:1.2, 1:1, 1:0.8, and 1:0.6, where the weight ratio of oxymatrine and glycyrrhizin was 1:1). The reaction temperature was controlled at 60°C using a water bath, and the mixture was stirred at 600 rpm for 5 hours using a magnetic stirrer. The suspension was filtered through a 0.22 μm organic membrane to remove excess unreacted drug. The clarified solution was evaporated under vacuum at 30°C for 1 hour to obtain a pale yellow powder, OGP. The dried residue was placed in a desiccator overnight and then stored in a glass bottle for later use. For testing, the OGP prepared in each example was dispersed in water to obtain an OGP suspension of 1 mg / ml. The particle size, PDI, and potential of the OGP suspensions in Examples 1-4 were determined. The total drug yield present in the phospholipid complex was calculated according to the following formula:

[0105] Yield (%) = (W1-W2) / W1 × 100%;

[0106] Wherein, W1 is the total weight of the drug added during the preparation of the phospholipid complex, and W2 is the total weight of the free drug not included in the phospholipid complex.

[0107] 1.2 Preparation of chitosan-coated drug phospholipid complex (CS-OGP)

[0108] Chitosan (Sigma-Aldrich, Sigma 448869, molecular weight 50-190 kDa, degree of deacetylation >75%, 1% viscosity 20-300 cps) was dissolved in 0.1 M acetic acid solution to prepare a 2 mg / ml solution for later use. The OGP suspension prepared in Example 2 was diluted with water to 2 mg / ml. According to different weight ratios of chitosan to OGP (1:1, 1:2, 1:4 and 1:8), it was added to an equal volume of diluted chitosan solution (concentrations of 2 mg / ml, 1 mg / ml, 0.5 mg / ml and 0.25 mg / ml, respectively). The solution was incubated at room temperature for 4 h to obtain CS-OGP suspension. The particle size, PDI and potential of the CS-OGP suspensions in Examples 5-8 were measured respectively.

[0109] 1.3 Preparation of hyaluronic acid-modified chitosan-coated drug phospholipid complex (HA-CS-OGP)

[0110] Hyaluronic acid (Bloomage Biotechnology Co., Ltd., MW = 60 kDa) was prepared into a 1 mg / ml solution using distilled water. Different volumes of hyaluronic acid solution were added to the CS-OGP suspension prepared in Example 7 (chitosan concentration 0.25 mg / ml, OGP concentration 1 mg / ml) according to the weight ratio of hyaluronic acid to chitosan of 0.5:1, 1:1, 1.5:1, and 2:1, respectively. The mixture was stirred at room temperature for 4 h to obtain HA-CS-OGP suspension. The particle size, PDI, and potential of HA-CS-OGP from Examples 9-12 were measured.

[0111] 2. Test Results

[0112] The average particle size, PDI, and potential of the various nanoparticles obtained in each step of this experiment are shown in Table 2 below.

[0113] Table 2. Average particle size, PDI, and potential of phospholipid complexes with different formulations in this invention.

[0114]

[0115] Experimental results showed that OGP prepared by mixing phospholipids and total drug (with an oxymatrine and glycyrrhizin weight ratio of 1:1) at weight ratios of 1:1, 1:0.8, and 1:0.6 all yielded clear solutions with a total drug yield of over 95%. As the weight ratio of phospholipids to total drug increased from 1:1 to 1:0.6, the particle size and PDI of the phospholipid complex tended to increase; however, when the weight ratio decreased from 1:1 to 1:1.2, the prepared OGP produced a turbid solution with a total drug yield of only 44.7%. Considering the requirements of small particle size, low PDI, and high drug loading, OGP prepared with a phospholipid and total drug weight ratio of 1:1 (Example 2) was selected for the subsequent chitosan coating step.

[0116] When the weight ratio of chitosan to the drug phospholipid complex OGP changed from 1:1 to 1:8, the particle size of CS-OGP decreased from 1308±59.19 nm to 344.03±3.17 nm, and the Zeta potential decreased from 59.57±1.50 mV to 34.37±0.7 mV. This shows that the average particle size and positively charged Zeta potential of the phospholipid complex significantly increased after coating with chitosan; the higher the chitosan ratio, the larger the particle size and the higher the Zeta potential. Furthermore, when the weight ratio of chitosan to the phospholipid complex OGP changed from 1:4 to 1:8, the PDI of CS-OGP began to increase. Considering both the requirements for small particle size and low PDI, CS-OGP (Example 7) prepared with a weight ratio of chitosan and OGP of 1:4 was selected for the subsequent hyaluronic acid modification step.

[0117] The higher positive charge of CS-OGP allows for better interaction with the negatively charged hyaluronic acid, which is adsorbed onto the surface of CS-OGP nanoparticles through electrostatic interactions with chitosan. The Zeta potential of HA-CS-OGP particles decreases with increasing hyaluronic acid content, indicating that HA is coated on the particle surface. Compared to CS-OGP, hyaluronic acid-modified nanoparticles have smaller particle sizes and more uniform distribution. When the weight ratio of hyaluronic acid to chitosan is 0.5:1 to 1.5:1, the addition of hyaluronic acid to CS-OGP produces a pale blue-white solution, while a weight ratio of 2:1 results in precipitation. The particle size of HA-CS-OGP varies with different ratios of hyaluronic acid and chitosan; the smallest particle size is observed when the weight ratio is 1:1. Therefore, HA-CS-OGP prepared with a 1:1 weight ratio of hyaluronic acid and chitosan (Example 10) was selected for subsequent experiments.

[0118] Experimental Example 3: Determination of the crystal form of phospholipid complexes

[0119] The crystallization states of oxymatrine, glycyrrhizin, phospholipids, and OGP (from Example 2 in Experimental Example 2) were detected by P-XRD. The results showed that some strong diffraction peaks were visible in the XRD spectra of oxymatrine and glycyrrhizin. Figure 1 D and 1C) show a broad peak in phospholipids ( Figure 1 B) indicates that oxymatrine and glycyrrhizin exist in crystalline form, while phospholipids exist amorphously. However, these crystalline signals disappear in the XRD spectrum of OGP ( Figure 1 A) This indicates that oxymatrine and glycyrrhizin no longer maintain a crystalline form in the phospholipid complex, but are uniformly dispersed in the phospholipid matrix in an amorphous form.

[0120] Experimental Example 4: Determination of drug loading and encapsulation efficiency of polysaccharide-modified phospholipid complexes

[0121] 1. Test Methods

[0122] Take OGP prepared in Example 2 and HA-CS-OGP prepared in Example 10, and centrifuge the nanoparticle suspension at 12,000 rpm for 10 minutes. Detect the concentration of free oxymatrine and glycyrrhizin in the supernatant by HPLC. Calculate the drug loading and encapsulation efficiency of the two drugs in the phospholipid complex.

[0123] The HPLC detection conditions were as follows: An Agilent 1260 series HPLC instrument was used, with a Zorbax SBC18 column (250 mm × 4.6 mm, 5 μm) selected. The column temperature was 30℃, and the flow rate was 1.0 mL / min. For the determination of oxymatrine, the mobile phase was methanol:0.01 M KH₂PO₄ solution with a volume ratio of 15:85, adjusted to pH 3.5. For the determination of glycyrrhizin, the mobile phase was acetonitrile:0.01 M H₃PO₄ solution with a volume ratio of 40:60. The detection wavelengths for oxymatrine and glycyrrhizin were 220 nm and 250 nm, respectively.

[0124] 2. Test Results

[0125] The experimental results showed that the OGP solution with a phospholipid-to-total-drug weight ratio of 1:1 was clear and free of precipitate, with drug loadings of oxymatrine and glycyrrhizin in OGP of 26.57% and 27.13%, respectively. In HA-CS-OGP with a hyaluronic acid, chitosan, and OGP weight ratio of 1:1:4, the encapsulation rates of oxymatrine and glycyrrhizin were 54.26% and 49.32%, respectively, with drug loadings of 10.44% and 9.51%, respectively. This indicates that the drug loadings of oxymatrine and glycyrrhizin in OGP and HA-CS-OGPs were almost identical to the initial 1:1 weight ratio, suggesting that OGP and HA-CS-OGP can provide a fixed ratio of oxymatrine and glycyrrhizin to produce a synergistic anticancer effect, which is of great significance for combination therapy.

[0126] Experimental Example 5: In vitro release assay of polysaccharide-modified phospholipid complex

[0127] 1. Test Methods

[0128] In vitro release was performed using dialysis bags with a molecular weight cutoff of 1000. 1 ml of OGP prepared in Example 2, Example 2, CS-OGP prepared in Example 7, and HA-CS-OGP prepared in Example 10 were placed into the dialysis bags (total drug concentration 1 mg / ml). PBS solutions with pH values ​​of pH 5.5 and pH 7.4 were used as the release medium, respectively, and the release temperature was 37°C. At set time points, 1 ml samples were taken, and the release curves of oxymatrine and glycyrrhizin were determined by HPLC.

[0129] 2. Test Results

[0130] Compared with the free drug, the in vitro release curves of oxymatrine and glycyrrhizin from OGP, CS-OGP, and HA-CS-OGP at pH 7.4 are as follows: Figure 2As shown, the rapid release of free oxymatrine and glycyrrhizin can be attributed to their excellent solubility in water. Compared with the unmodified phospholipid complex OGP, the release curves of oxymatrine and glycyrrhizin in CS-OGP and HA-CS-OGP are slower, indicating that the chitosan and hyaluronic acid coating hinders the diffusion of the drug from CS-OGP and HA-CS-OGPs, and the release rate follows a first-order model.

[0131] like Figure 3 As shown, oxymatrine is rapidly released from the phospholipid complex at pH 5.0, similar to the drug solution. Glycyrrhizin exhibits a sustained release profile in CS-OGP and HA-CS-OGP, significantly different from the release profiles of the pure drug solution and OGP. Combining the release profiles of oxymatrine and glycyrrhizin at pH 5.0, it can be seen that CS-OGP and HA-CS-OGP show a sequential drug delivery trend in vitro.

[0132] Due to the toxicity of anticancer drugs, most chemotherapy guidelines recommend sequential administration of anticancer drugs rather than simultaneous administration. However, the distribution of free drugs delivered sequentially exhibits a non-fixed ratio in tissues. Nanoparticle-mediated drug combination delivery can maintain a fixed ratio of the two drugs during biodistribution, thereby inducing higher therapeutic effects.

[0133] HA-CS-OGP provides a fixed ratio and sequential release of oxymatrine and glycyrrhizin at pH 5.0, indicating its ability to control the release of two different drugs in cancer cells. Utilizing these drug release characteristics of HA-CS-OGP, oxymatrine, as a chemical drug, can be rapidly released from HA-CS-OGP to kill cancer cells. Subsequently, the release of glycyrrhizin continuously inhibits ABC transporters, enhancing membrane permeability, thereby increasing the accumulation of oxymatrine and reducing efflux.

[0134] Experimental Example 6: In vitro antitumor activity of polysaccharide-modified phospholipid complexes

[0135] 1. Test Methods

[0136] HepG2 cells were seeded into 96-well plates and incubated for 24 hours until they adhered and fused. Then, a series of total drug concentrations of 0.1, 1, 10, 50, and 100 μg / mL of oxymatrine, glycyrrhizin, a mixture of glycyrrhizin and oxymatrine (weight ratio 1:1), OGP prepared in Example 2, CS-OGP prepared in Example 7, and HA-CS-OGP prepared in Example 10 were added to each well. After incubation for 24 hours, the culture medium was aspirated, and then 100 μl of MTT was added to each well. After 4 hours, the culture medium was aspirated, and 100 μl of DMSO was added to each well. The cells were shaken for 10 minutes, and the absorbance was measured at 490 nm using a microplate reader to calculate the cell viability.

[0137] 2. Test Results

[0138] Figure 4 A shows the survival rate of HepG2 cells after exposure to glycyrrhizin, oxymatrine, a mixture of glycyrrhizin and oxymatrine (1:1), and OGP for 48 hours. Glycyrrhizin at the studied concentration did not affect the cell viability of HepG2 cells, while oxymatrine at a concentration of 100 μg / ml showed significant toxicity compared to the control group. The toxicity of the mixture was significantly increased at concentrations of 1 μg / ml and 100 μg / ml compared to the free drug (P<0.05), suggesting a synergistic effect between oxymatrine and glycyrrhizin in the treatment of cancer cells, with the cytotoxicity further increased after the formation of a phospholipid complex between oxymatrine and glycyrrhizin.

[0139] Compared with OGP, CS-OGP and HA-CS-OGP showed significantly enhanced cytotoxicity, especially at low concentrations of 0.1 μg / ml and 1 μg / ml. Figure 4 (B) This indicates that modifying the phospholipid complex with chitosan increased drug accumulation in cells. More importantly, HA-CS-OGP exhibited superior cytotoxicity compared to other formulations. Hyaluronic acid-modified chitosan nanoparticles enhanced tumor cell killing efficiency, possibly due to receptor-mediated internalization via CD44 receptor overexpression in HepG2 cells.

[0140] Experimental Example 7: In vitro cellular uptake of polysaccharide-modified phospholipid complexes

[0141] 1. Test Methods

[0142] Chitosan was prepared into a 5 mg / ml solution using 1% acetic acid. The pH was adjusted to 6 with sodium hydroxide. A 2 mg / ml FITC-methanol solution (chitosan:FITC = 10:1) was added, and the mixture was stirred for 24 h. After centrifugation at 4000 rpm for 30 min, the precipitate was resuspended in methanol / water (70 / 30) and centrifuged again at 3000 rpm for 20 min. This process was repeated 4-5 times. The absorbance of the washing solution at 443 nm was measured to be below 0.03 or without absorption. The FITC-modified chitosan precipitate was dried and stored in a light-proof glass bottle for later use. Following the method in Example 2, the FITC-modified chitosan was used to replace chitosan in the preparation of CS-OGP and HA-CS-OGP, with the remaining reaction conditions the same as in Example 2.

[0143] HepG2 cells were seeded into 6-well plates at 1 million cells per well and incubated overnight in a cell culture incubator for 24 hours until the cells were fully adhered. After washing with PBS, a series of concentrations of CS-OGP prepared according to Example 7 of Experiment 2 and HA-CS-OGP prepared according to Example 10 were added. After incubation for 4 hours, the cells were washed with PBS and then digested with trypsin to collect the cells. The cells were resuspended in PBS and centrifuged at 1000 rpm for 5 minutes for flow cytometry analysis.

[0144] 2. Test Results

[0145] Compared to CS-OGP, HA-CS-OGP exhibited higher cellular uptake and MFI. CS-OGP can enter cells via a non-specific pathway across the lipid bilayer of the cell membrane, depending on particle size, zeta potential, and the composition of phospholipids and chitosan. Conversely, cells can take up HA-CS-OGP via CD44 receptor-mediated selective ligand-receptor endocytosis. Combined with the cytotoxicity results of Example 5, this confirms that the enhanced cytotoxicity of HA-CS-OGP compared to unmodified chitosan nanoparticles is due to increased drug uptake resulting from hyaluronic acid modification.

[0146] Although specific embodiments of the invention have been described, those skilled in the art will recognize that various changes and modifications can be made to the invention without departing from its scope or spirit. Therefore, the invention is intended to cover all such changes and modifications falling within the scope of the appended claims and their equivalents.

Claims

1. A polysaccharide-modified drug phospholipid complex, characterized in that, The drug phospholipid complex is prepared from a drug and a phospholipid, the drug includes oxymatrine and glycyrrhizin, the weight ratio of oxymatrine to glycyrrhizin added in the preparation process is 1:1, the weight ratio of the phospholipid to the drug added in the preparation process is 1:1, and the polysaccharide used for modifying the drug phospholipid complex is a combination of chitosan and hyaluronic acid; the polysaccharide modification is first chitosan modification of the drug phospholipid complex, and then hyaluronic acid modification of the drug phospholipid complex coated with chitosan.

2. The polysaccharide-modified drug phospholipid complex according to claim 1, characterized in that, In the preparation of the polysaccharide-modified drug phospholipid complex, the weight ratio of chitosan to the drug phospholipid complex is 1:1-1:

8.

3. The polysaccharide-modified drug phospholipid complex according to claim 1, characterized in that, In the preparation of the polysaccharide-modified drug phospholipid complex, the weight ratio of hyaluronic acid to chitosan is 0.5:1-1.5:

1.

4. The polysaccharide-modified pharmaceutical phospholipid complex according to any one of claims 1 to 3, characterized in that, The chitosan is low-molecular-weight chitosan, and the molecular weight of the low-molecular-weight chitosan is 50-500 kDa.

5. The polysaccharide-modified pharmaceutical phospholipid complex according to any one of claims 1 to 3, characterized in that, The molecular weight of the hyaluronic acid is 40-80 kDa.

6. A process for the preparation of a polysaccharide-modified pharmaceutical phospholipid complex as claimed in any one of claims 1 to 5, characterized in that, The method comprises the following steps: (1) dissolving the phospholipid in an organic solvent, adding oxymatrine and glycyrrhizin respectively, stirring under heating conditions, filtering the solution, and then performing vacuum rotary evaporation to obtain a drug phospholipid complex OGP; (2) dissolving chitosan in an acid solution, adding the OGP suspension to the chitosan solution, incubating, and obtaining a chitosan-coated drug phospholipid complex CS-OGP suspension; (3) dissolving hyaluronic acid in water, adding the hyaluronic acid solution to the CS-OGP suspension, stirring, obtaining a hyaluronic acid-modified chitosan-coated drug phospholipid complex HA-CS-OGP suspension, centrifuging, drying the precipitate, and obtaining the polysaccharide-modified drug phospholipid complex.

7. Use of the polysaccharide-modified drug phospholipid complex of any one of claims 1-5 in the preparation of a drug for treating cancer, and the cancer is liver cancer.

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