Phospholipid complex delivery system and preparation method and application thereof

Abstract

The invention discloses a phospholipid complex delivery system and a preparation method and application thereof. The preparation method comprises the following steps: mixing and stirring active ingredients of traditional Chinese medicines and phospholipid for reaction to prepare a phospholipid compound; the traditional Chinese medicine active component comprises saikoside A; the preparation method comprises the following steps: carrying out a reaction on a mixed reaction system containing hyaluronic acid, 1-ethyl-(3-dimethylaminopropyl) carbodiimide, cyclodextrin and oligomeric histidine to prepare an oligomeric histidine modified hyaluronic acid grafted cyclodextrin compound; and mixing the phospholipid compound with the oligomeric histidine modified hyaluronic acid grafted cyclodextrin compound, and incubating under the condition of a low-frequency alternating electric field to prepare the phospholipid compound delivery system. According to the phospholipid complex delivery system, the oral bioavailability of the traditional Chinese medicine active ingredients can be obviously improved, aggregation of the active ingredients in tumor cells can be enhanced through the active targeting effect, and the anti-tumor effect of the active ingredients is improved.

Description

Technical Field

[0001] The invention relates to a phospholipid complex delivery system and a preparation method and application thereof, and belongs to the field of traditional Chinese medicine and biopharmaceutical technology. Background Art

[0002] Colorectal cancer is the third most common malignant tumor in the world. The current treatment methods for colorectal cancer mainly include systemic treatment, local treatment, surgical treatment and postoperative adjuvant treatment. However, the disease is prone to recurrence and metastasis after surgery, has great toxicity and side effects of chemotherapy, has many complications, and has a heavy economic burden on patients and low quality of life. Therefore, finding a drug for the prevention and treatment of colon cancer with reliable efficacy and few side effects is still an important direction in the field of colorectal cancer research and has important practical significance. Studies have shown that traditional Chinese medicine can complement the advantages of surgery, radiotherapy and chemotherapy, and prevent and treat CRC recurrence and metastasis through the advantages of multi-component, multi-target and multi-link effects. It has now become an important part of the comprehensive diagnosis and treatment of CRC.

[0003] Bupleurum is one of the most commonly used APIs in many natural products and is often used together with other APIs to treat diseases. Bupleurum has a 2,000-year history of medicine and was included in the Shennong Bencao Jing in the Eastern Han Dynasty. It is one of the most important Chinese herbs and is used in traditional Chinese medicine prescriptions for its effects of relieving external symptoms, clearing away heat, regulating liver qi, and raising yang qi, such as Xiao Chaihu Tang and Chaihu Shugan San. With the development of modern pharmacology, many valuable activities of Bupleurum have been discovered, such as anti-inflammatory, anti-tumor, liver protection, and immunomodulatory activities. Bupleurum saponin content is as high as about 7% of the total dry weight of Bupleurum root, which is the key secondary metabolite for Bupleurum to exert its efficacy. Bupleurum saponin A (SSA) is a glycosylated oleanane-type saponin and one of the main components of Bupleurum saponins. In recent years, it has attracted attention due to its multiple biological activities. It has anti-inflammatory, anti-tumor, antiviral and liver protection activities both in vivo and in vitro. However, due to its poor gastrointestinal absorption when taken orally, its bioavailability is extremely low. As a new drug delivery technology, phospholipid complex delivery system has gradually emerged in the research of active ingredients of traditional Chinese medicine in recent years. As the main component of cell membrane, phospholipids have good biocompatibility and degradability, and can form stable complexes with various drug molecules through hydrogen bonds or van der Waals forces. This complex can not only improve the solubility and stability of the drug, but also show good effects in absorption and penetration. However, natural drug phospholipid complexes have the disadvantages of easy aggregation, hydrolysis and oxidation, and poor stability. Therefore, the development of efficient, safe and stable traditional Chinese medicine drug delivery system has become an important direction of current traditional Chinese medicine research. Summary of the invention

[0004] The main purpose of the present invention is to provide a phospholipid complex delivery system and a preparation method and application thereof, so as to overcome the deficiencies in the prior art.

[0005] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention includes: An embodiment of the present invention provides a method for preparing a phospholipid complex delivery system, which comprises: The active ingredients of traditional Chinese medicine and phospholipids are mixed and stirred to react to obtain a phospholipid complex; the active ingredients of traditional Chinese medicine include saikosaponin A; wherein the mass ratio of the active ingredients of traditional Chinese medicine to the phospholipids is 1:2.5-20; A mixed reaction system comprising hyaluronic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, cyclodextrin and oligohistidine is reacted to obtain a hyaluronic acid-grafted cyclodextrin complex modified with oligohistidine; wherein the molar ratio of the hyaluronic acid, cyclodextrin and oligohistidine is 2:1:0.1-0.3; And, the phospholipid complex is mixed with the oligohistidine-modified hyaluronic acid-grafted cyclodextrin complex and incubated under the condition of a low-frequency alternating electric field to prepare a phospholipid complex delivery system; wherein the frequency of the low-frequency alternating electric field is 20-200 Hz, and the electric field strength is 1-15 V / cm.

[0006] The embodiment of the present invention also provides a phospholipid complex delivery system prepared by the aforementioned preparation method, which comprises: a phospholipid complex and a carrier loaded with the phospholipid complex; wherein the carrier comprises a hyaluronic acid-grafted cyclodextrin complex modified with oligohistidine.

[0007] The embodiment of the present invention also provides the use of the aforementioned phospholipid complex delivery system in the preparation of targeted delivery cancer drugs; wherein the cancer includes colorectal cancer, and the colorectal cancer includes the HCT116 colon cancer cell line.

[0008] Compared with the prior art, the present invention has the following beneficial effects: (1) Compared with phospholipid complexes, the phospholipid complex delivery system provided by the present invention has a hyaluronic acid grafted cyclodextrin complex modified with oligohistidine, which exposes more hyaluronic acid to the outside through oligohistidine, and the complex has better targeting. The interaction between hyaluronic acid and CD44 enhances the cell aggregation in tumor cells that overexpress CD44. The phospholipid complex delivery system can improve the oral bioavailability of active ingredients of traditional Chinese medicine, enhance the accumulation of active ingredients in tumor cells through active targeting, and improve its anti-tumor effect. In addition, the hyaluronic acid grafted cyclodextrin complex exhibits pH-sensitive properties, can disintegrate faster in a slightly acidic environment, and can quickly release the active ingredients of the drug after entering the cancer cells, and has better efficacy. (2) The small molecules used in the present invention are natural active ingredients in Chinese herbal medicines and have low toxicity to normal cells; (3) The particle size of the phospholipid complex delivery system in the present invention is concentrated in the range of 100-200 nm, and the particle size uniformity reaches PDI≤0.2; it exhibits higher toxicity to tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0010] Figure 1-Figure 4 These are the effects of the drug-lipid ratio, reaction temperature, solvent ratio and reaction time of the phospholipid complex on the complexing rate in Experimental Example 1 of the present invention; Figure 5 FTIR spectra of saikosaponin A, phospholipids, physical mixtures and phospholipid complexes in Experimental Example 3 of the present invention; Figure 6 is saikosaponin A, phospholipids and phospholipid complexes in Test Example 3 of the present invention 1 H NMR spectrum; Figure 7-Figure 8 The particle size and Zeta potential spectrum of the phospholipid complex delivery system nanoparticles in Experimental Example 5 of the present invention; Fig. 9 This is a transmission electron microscopy image of the phospholipid complex delivery system nanoparticles in Experimental Example 5 of the present invention; Figure 10-Figure 14 These are the inhibitory effect diagrams of SSA in the present invention, SSA-SPC prepared in Example 8, His-HA-CD NPs prepared in Example 18, HA-CD-1 NPs prepared in Comparative Example 1, and His-HA-CD-2 NPs prepared in Comparative Example 2 on the growth of colon cancer HCT 116 cells; Fig.15 This is a diagram of the cellular uptake of FITC, FITC-SSA-SPC, FITC-His-HA-CDNPs, FITC-HA-CD-1 NPs, and FITC-His-HA-CD-2 NPs by HCT116 cells in Experimental Example 7 of the present invention. DETAILED DESCRIPTION

[0011] In view of the defects of the prior art, the inventor of this case has proposed the technical solution of the present invention after long-term research and extensive practice. In order to facilitate the understanding of the present application, the present application will be described in more detail as follows. However, it should be understood that the present invention can be implemented in many different forms and is not limited to the embodiments or examples described herein. On the contrary, the purpose of providing these embodiments or examples is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0012] Specifically, as one aspect of the technical solution of the present invention, a method for preparing a phospholipid complex delivery system involves: The active ingredients of traditional Chinese medicine and phospholipids are mixed and stirred to react to obtain a phospholipid complex; the active ingredients of traditional Chinese medicine include saikosaponin A; wherein the mass ratio of the active ingredients of traditional Chinese medicine to the phospholipids is 1:2.5-20; A mixed reaction system comprising hyaluronic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), cyclodextrin and oligohistidine is reacted to obtain a hyaluronic acid-grafted cyclodextrin complex modified with oligohistidine; wherein the molar ratio of the hyaluronic acid, cyclodextrin and oligohistidine is 2:1:0.1-0.3; And, the phospholipid complex is mixed with the oligohistidine-modified hyaluronic acid-grafted cyclodextrin complex and incubated under the condition of a low-frequency alternating electric field to prepare a phospholipid complex delivery system; wherein the frequency of the low-frequency alternating electric field is 20-200 Hz, and the electric field strength is 1-15 V / cm.

[0013] In some preferred embodiments, the preparation method specifically comprises: The active ingredients of traditional Chinese medicine are mixed with phospholipids, and then a mixed organic solvent is added to form a mixed solution, so that the concentration of the active ingredients of traditional Chinese medicine in the mixed solution is 1-10 mg / mL.

[0014] Furthermore, the mixed solution is stirred at 20-60° C. in the dark for 0.5-4 h, and then subjected to reduced pressure distillation, vacuum drying, dichloromethane redissolution, filtration, and drying treatment to obtain a phospholipid complex.

[0015] Furthermore, the mass ratio of the active ingredients of the traditional Chinese medicine to the phospholipids is 1:10-20, and the effect is better when the mass ratio is 1:10.

[0016] Furthermore, the mixed organic solvent includes methanol and chloroform, wherein the volume ratio of methanol to chloroform is 4:3-3:4, and the effect is better when the volume ratio of methanol to chloroform is 2:3.

[0017] Furthermore, the temperature of the light-shielded stirring is 40-60 °C, and the effect is better when the temperature is 50 °C.

[0018] Furthermore, the light-proof stirring time is 2 hours.

[0019] Furthermore, the stirring speed used in the light-shielded stirring is 300-800 rpm.

[0020] Furthermore, the pore size of the filter membrane used for the dichloromethane re-dissolution filtration is 0.1-1.0 μm.

[0021] In some preferred embodiments, the preparation method specifically comprises: dissolving hyaluronic acid in anhydrous formamide, adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide and ice bathing for 0.5-2 h, then adding cyclodextrin and oligohistidine and reacting at 20-40 °C for 24-48 h, and then dialyzing and drying to obtain an oligohistidine-modified hyaluronic acid grafted cyclodextrin complex.

[0022] In the present invention, when preparing the oligohistidine-modified hyaluronic acid-grafted cyclodextrin complex, adding oligohistidine will expose more hyaluronic acid in the complex to the outside, and the complex will have better targeting. At the same time, the complex shows pH-sensitive characteristics and can disintegrate faster in a slightly acidic environment. The tumor microenvironment is weakly acidic, and the histidine-modified nanoparticles are more stable in a neutral or slightly alkaline environment and are easier to release in an acidic environment. Therefore, after entering the cancer cells, the active ingredients of the drug can be quickly released, and the drug efficacy is better.

[0023] Furthermore, the molecular weight of the hyaluronic acid is 1-10 kDa, and the effect is better when the molecular weight is 3 kDa.

[0024] Furthermore, the cyclodextrin includes any one or more combinations of α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin; wherein the β-cyclodextrin includes hydroxypropyl-β-cyclodextrin.

[0025] Furthermore, the oligohistidine is an oligohistidine with 15-30 residues.

[0026] In some preferred embodiments, the preparation method specifically includes: dissolving the oligohistidine-modified hyaluronic acid grafted cyclodextrin complex in water to form an oligohistidine-modified hyaluronic acid grafted cyclodextrin complex aqueous solution, then adding an ethanol solution of a phospholipid complex to the oligohistidine-modified hyaluronic acid grafted cyclodextrin complex aqueous solution and incubating under low-frequency alternating electric field conditions to obtain a suspension of the phospholipid complex delivery system, and then centrifuging, resuspending the precipitate in distilled water, and freeze-drying to obtain the phospholipid complex delivery system.

[0027] Furthermore, the mass ratio of the phospholipid complex to the oligohistidine-modified hyaluronic acid-grafted cyclodextrin complex is 1:1-5, and the effect is better when the mass ratio of the phospholipid complex to the oligohistidine-modified hyaluronic acid-grafted cyclodextrin complex is 1:1.

[0028] Furthermore, the incubation temperature is 30-50 °C and the time is 0.5-2 h.

[0029] The incubation in the present invention is carried out under the condition of a low-frequency alternating electric field, so that the particle size of the prepared phospholipid complex delivery system is concentrated in the range of 100-200 nm, and the particle size uniformity reaches PDI≤0.2; it exhibits higher toxicity to tumor cells and its empty loading rate is significantly reduced.

[0030] In some more specific embodiments, the method for preparing the phospholipid complex delivery system comprises the following steps: S1, mixing saikosaponin A and phospholipids in a mass ratio of 1:2.5-1:20; S2, adding an organic solvent, wherein the volume ratio of the organic solvent is methanol:chloroform is 4:3-3:4, so that the drug concentration is 1-10 mg / mL; S3, magnetically stir at 30-60 °C in dark for 0.5-4 h, stirring speed 300-800 rpm; S4, evaporating the reaction solution under reduced pressure at 40 °C to remove the organic solvent, drying in a reduced pressure vacuum oven for 12 h, re-dissolving in dichloromethane, filtering through a 0.22 μm filter membrane, and drying to obtain saikosaponin A phospholipid complex; S5, dissolving 0.02 mmol of cyclodextrin in anhydrous formamide, adding 0.2 g of EDC and placing in an ice bath for 2 h, dropping 0.04 mmol of hyaluronic acid solution and 0.001-0.003 mmol of oligohistidine, reacting for 48 h, dialyzing and freeze-drying to obtain a hyaluronic acid-grafted cyclodextrin complex modified with oligohistidine; S6. Dissolve the oligohistidine-modified hyaluronic acid grafted cyclodextrin complex in an aqueous solution, add the saikosaponin A phospholipid complex ethanol solution dropwise into the aqueous solution, and incubate under conditions of a low-frequency alternating electric field to obtain a phospholipid complex delivery system suspension, centrifuge, resuspend the precipitate in distilled water, and then freeze-dry to obtain the phospholipid complex delivery system.

[0031] Preferably, in step S1, the mass ratio of saikosaponin A to phospholipid is 1:10.

[0032] Preferably, the organic solvent in step S2 is methanol:chloroform in a volume ratio of 2:3.

[0033] Preferably, the drug concentration in step S2 is 2 mg / mL.

[0034] Preferably, the reaction temperature in step S3 is 50 °C.

[0035] Preferably, the reaction time in step S3 is 2 h.

[0036] Preferably, the cyclodextrin in step S5 is hydroxypropyl-β-cyclodextrin.

[0037] Preferably, the molecular weight of the hyaluronic acid in step S5 is 3 kDa.

[0038] Preferably, in step S6, the feed ratio (mass ratio) of saikosaponin A phospholipid complex to oligohistidine-modified hyaluronic acid grafted cyclodextrin complex is 1:1.

[0039] Preferably, the frequency of the low-frequency alternating electric field is 20-200 Hz, and the electric field strength is 1-15 V / cm. As another aspect of the technical solution of the present invention, it relates to a phospholipid complex delivery system prepared by the aforementioned preparation method, which comprises: a phospholipid complex and a carrier loaded with the phospholipid complex; wherein the carrier comprises a hyaluronic acid grafted cyclodextrin complex modified with oligohistidine.

[0040] In some preferred embodiments, the particle size of the phospholipid complex delivery system is 100-200 nm, and the particle size uniformity PDI ≤ 0.2.

[0041] As another aspect of the technical solution of the present invention, it also relates to the use of the aforementioned phospholipid complex delivery system in the preparation of targeted delivery cancer drugs; wherein, the cancer includes colorectal cancer, and the colorectal cancer includes the HCT116 colon cancer cell line.

[0042] The present invention is further described by the following examples: The present invention can be better understood according to the following examples. However, it is easy for those skilled in the art to understand that the specific material ratios, process conditions and results described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0043] Unless otherwise specified, the various raw materials, reaction equipment, testing equipment and testing methods used in the following examples are all well known in the art.

[0044] Experimental Example 1 Preparation of phospholipid complex 1. Test methods (including Examples 1 to 17) 1.1. Preparation of phospholipid complexes at different drug-lipid ratios According to the mass ratio of saikosaponin A (SSA) and soybean lecithin (SPC) (Aladdin Reagent (Shanghai) Co., Ltd., phosphatidylcholine content> 96%) of 1:2.5, 1:5, 1:10, 1:15 and 1:20, an organic solvent with a volume ratio of methanol: chloroform of 2:3 was added to make the drug concentration 2 mg / mL. After complete dissolution, it was magnetically stirred in a water bath at 40 °C in the dark for 2 h, with a stirring speed of 500 rpm. After the reaction was completed, the reaction solution was evaporated under reduced pressure at 40 °C to remove the organic solvent, dried in a vacuum oven under reduced pressure for 12 h, redissolved in dichloromethane, filtered through a 0.22 μm organic filter membrane, and dried to obtain saikosaponin A phospholipid complex (SSA-SPC).

[0045] 1.2 Preparation of phospholipid complexes at different reaction temperatures After mixing saikosaponin A and soybean lecithin (Aladdin Reagent (Shanghai) Co., Ltd., phosphatidylcholine content> 96%) in a mass ratio of 1:10, an organic solvent with a volume ratio of methanol: chloroform of 2:3 was added to make the drug concentration 2 mg / mL. After complete dissolution, the mixture was magnetically stirred in a water bath at 20 °C, 30 °C, 40 °C, 50 °C and 60 °C for 2 hours in a dark place, with a stirring speed of 500 rpm. After the reaction was completed, the reaction solution was evaporated under reduced pressure at 40 °C to remove the organic solvent, dried in a vacuum oven under reduced pressure for 12 hours, redissolved in dichloromethane, filtered through a 0.22 μm organic filter membrane, and dried to obtain saikosaponin A phospholipid complex (SSA-SPC).

[0046] 1.3 Preparation of phospholipid complexes at different solvent ratios After mixing saikosaponin A and soybean lecithin (Aladdin Reagent (Shanghai) Co., Ltd., phosphatidylcholine content>96%) in a mass ratio of 1:10, organic solvents with a volume ratio of methanol:chloroform of 3:2, 4:3, 1:1, 2:3 and 3:4 were added to make the drug concentration 2 mg / mL. After complete dissolution, the drug was magnetically stirred in a water bath at 40 °C in the dark for 2 h, with a stirring speed of 500 rpm. After the reaction was completed, the reaction solution was evaporated under reduced pressure at 40 °C to remove the organic solvent, dried in a vacuum oven under reduced pressure for 12 h, redissolved in dichloromethane, filtered through a 0.22 μm organic filter membrane, and dried to obtain saikosaponin A phospholipid complex (SSA-SPC).

[0047] 1.4. Preparation of phospholipid complexes at different reaction times After mixing saikosaponin A and soybean lecithin (Aladdin Reagent (Shanghai) Co., Ltd., phosphatidylcholine content>96%) in a mass ratio of 1:10, an organic solvent with a volume ratio of methanol:chloroform of 2:3 was added to make the drug concentration 2 mg / mL. After complete dissolution, it was magnetically stirred in a water bath at 40 °C in the dark for 0.5h, 1h, 2h, 3h and 4h, wherein the stirring speed was 500 rpm. After the reaction was completed, the reaction solution was evaporated under reduced pressure at 40 °C to remove the organic solvent, dried in a vacuum oven under reduced pressure for 12 h, redissolved in dichloromethane, filtered through a 0.22 μm organic filter membrane, and dried to obtain saikosaponin A phospholipid complex (SSA-PC).

[0048] The complexation rate (%) of the above saikosaponin A phospholipid complex was calculated according to the following formula: ; 2. Test results The complex ratios of various phospholipid complexes obtained in this test example are shown in Table 1 and Figure 1-Figure 4 shown.

[0049] Table 1 The complexing rate of phospholipid complexes prepared under different conditions of the present invention ; The experimental results showed that SSA-SPC was prepared by solvent evaporation method. The compound rate was used as the evaluation index to investigate the effects of different drug-lipid ratios, reaction temperature, solvent ratio and reaction time on the compound rate. The optimal preparation process of SSA-SPC was determined, that is, the drug-lipid ratio was 1:10, the reaction solvent was a mixed solution of methanol and chloroform 2:3, and magnetic stirring was performed in a 50 °C water bath for 2 h.

[0050] Experimental Example 2 Preparation of phospholipid complex delivery system nanoparticles and determination of their physicochemical properties 1. Test methods (including Examples 18 to 22, Comparative Examples 1 and 2) 1.1. Preparation of phospholipid complex The phospholipid complex was prepared according to Example 8.

[0051] 1.2. Preparation of oligohistidine-modified hyaluronic acid-grafted cyclodextrin complex First, hyaluronic acid HA (0.02 mmol) was dissolved in 6 mL of anhydrous formamide, then 0.2 g of EDC was added, the solution mixture was stirred in an ice bath for 2 h, and 6 mL of hydroxypropyl-β-cyclodextrin HPCD (0.04 mmol) dissolved in anhydrous formamide and 0.002 mM oligohistidine were added dropwise for reaction, the reaction mixture was stirred for another 48 h, and the solution was dialyzed with excess double distilled water for two days. The dialyzate was freeze-dried to obtain a white powder oligohistidine-modified hyaluronic acid grafted cyclodextrin complex (denoted as: His-HA-CD).

[0052] 1.3. Preparation of phospholipid complex delivery system nanoparticles The oligohistidine-modified hyaluronic acid grafted cyclodextrin complex (His-HA-CD) was dissolved in an aqueous solution, and an ethanol solution of saikosaponin A phospholipid complex (SSA-SPC) was added dropwise, wherein the weight ratio of SSA-SPC to His-HA-CD was 1:1-1:5, and the mixture was incubated at 37 °C for 2 h under the conditions of a low-frequency alternating electric field with a frequency of 100 Hz and an electric field strength of 8 V / cm to obtain a saikosaponin A phospholipid complex delivery system nanoparticle suspension, centrifuged at 3500 rpm for 10 min, and the precipitate was resuspended in a small amount of pure water and freeze-dried to obtain saikosaponin A phospholipid complex delivery system nanoparticles (referred to as: His-HA-CD NPs). The nanoparticles prepared in each example were taken in water to obtain a 1 mg / mL suspension, and the particle size, PDI and potential thereof were measured respectively.

[0053] Comparative Example 1: The method is the same as Example 18, except that oligohistidine is lacking when preparing the oligohistidine-modified hyaluronic acid grafted cyclodextrin complex, and the prepared nanoparticles are recorded as HA-CD-1 NPs.

[0054] Comparative Example 2: The method was the same as Example 18, except that the incubation was not carried out under the condition of a low-frequency alternating electric field, and the prepared nanoparticles were recorded as His-HA-CD-2 NPs.

[0055] 2. Experimental results The average particle size, PDI and potential of various nanoparticles obtained in each step of this test example are shown in Table 2 below.

[0056] Table 2 Average particle size, PDI and potential of phospholipid complexes with different formulations of the present invention ; The test results show that when the weight ratio of the phospholipid complex to the oligohistidine-modified hyaluronic acid-grafted cyclodextrin complex changes from 1:1 to 1:5, the particle size of the His-HA-CD NPs prepared under the condition of a low-frequency alternating electric field is concentrated in the range of 100-200 nm, and the particle size uniformity reaches PDI≤0.2. In the absence of oligohistidine and a low-frequency alternating electric field, the size and PDI of the prepared nanoparticles increase. In view of the high permeability and retention effect (EPR effect) of tumor tissue, an embodiment with a small particle size and a low PDI is selected for subsequent research, that is, the His-HA-CD NPs with a weight ratio of 1:1 in Example 18 are selected for subsequent research.

[0057] Experimental Example 3 FTIR and 1 HNMR determination The potassium bromide pellet method was used and Fourier transform infrared spectrometer was used at 4000 cm -1 ~400 cm -1 Infrared spectra of SSA, SPC, SSA-SPC (from Example 8) and the physical mixture of saikosaponin A and soybean lecithin (PM) were scanned in the range of 1.5 Å. 1 HNMR spectra of SSA, SPC and SSA-SPC (from Example 8) were recorded. 1 HNMR spectrum, the solvent of SSA is D2O, and the solvent of SPC and SSA-SPC is CDCl3. The experimental results show that by FTIR, 1 HNMR measurements confirmed the formation of the SSA-SPC complex (e.g. Figure 5 and Figure 6 ).

[0058] Experimental Example 4 Determination of drug loading and encapsulation efficiency of phospholipid complex delivery system nanoparticles 1. Test methods Take the His-HA-CD NPs prepared in Example 18, the HA-CD-1 NPs prepared in Comparative Example 1, and the His-HA-CD-2 NPs prepared in Comparative Example 2, centrifuge the nanoparticle suspension at 14000 rpm for 10 min, measure the concentration of free SSA in the supernatant, and calculate the drug loading and encapsulation efficiency of the nanoparticles according to the following formula: ; 2. Test results The test results show that the encapsulation efficiency of saikosaponin A in the nanoparticles prepared in Example 18 is 98.20%, and the drug loading is 4.46%; the encapsulation efficiency of saikosaponin A in the nanoparticles prepared in Comparative Example 1 is 95.75%, and the drug loading is 4.35%; the encapsulation efficiency of saikosaponin A in the nanoparticles prepared in Comparative Example 2 is 92.40%, and the drug loading is 4.20%.

[0059] Experimental Example 5 Transmission electron microscopy determination of nanoparticles of phospholipid complex delivery system First, the His-HA-CD NPs suspension prepared in Example 18 was gently sonicated to ensure uniform dispersion.

[0060] Weigh 0.02 g of phosphotungstic acid and dissolve it in 1 mL of water. Use tweezers to pick up the copper mesh and place it on the wet filter paper. Take about 50 μL of the sample and gently drop it on the copper mesh. After staining for about 2 minutes, use a transmission electron microscope to observe the morphology of the nanoparticles and take pictures. Fig. 9 As shown, the morphology of the nanoparticles is shown to be monodisperse spherical nanoparticles. In addition, the particle length shown by the TEM ruler is relatively consistent with the measured particle size (e.g. Figure 7-Figure 8 ).

[0061] Experimental Example 6 In vitro antitumor activity of phospholipid complex delivery system nanoparticles 1. Test methods HCT116 cells were seeded into a 96-well plate and incubated for 24 h until adherence and fusion, and then a series of total drug concentrations of 22 μM, 24 μM, 26 μM, 28 μM, 30 μM, 32 μM, 34 μM, 36 μM, 38 μM, and 40 μM SSA, SSA-SPC prepared in Example 8, His-HA-CD NPs prepared in Example 18, HA-CD-1 NPs prepared in Comparative Example 1, and His-HA-CD-2 NPs prepared in Comparative Example 2 were added, incubated for 24 h, the culture medium was discarded, and then 100 μL of culture medium and 20 μL of MTT (5 mg / mL) were added to each well. After incubation at 37 ° C in the dark for 4 h, the culture medium was discarded, 150 μL of DMSO was added to each well, shaken for 10 min, and the absorbance was measured at 490 / 630 nm on a microplate reader.

[0062] 2. Test results Compared with SSA, SSA-SPC prepared in Example 8, HA-CD-1 NPs prepared in Comparative Example 1, and His-HA-CD-2 NPs prepared in Comparative Example 2, the His-HA-CD NPs prepared in Example 18 showed enhanced cytotoxicity, indicating that the nanoparticles resulted in the accumulation of saikosaponin A in HCT116 cells, which may be due to the cancer cell targeting selectivity and pH-responsive release properties of HA ( Figure 10-Figure 14 ). Based on the above data, it can be seen that the His-HA-CD NPs nanoparticles prepared in Example 18 have a significant effect on reducing the cell viability of cancer cells HCT116.

[0063] Experimental Example 7 In vitro cellular uptake of phospholipid complex delivery system nanoparticles 1. Test methods Quantitative uptake analysis was performed using flow cytometry. First, fluorescein isothiocyanate (FITC) was used to fluorescently label the nanoparticles SSA-SPC in Example 8, the His-HA-CD NPs prepared in Example 18, the HA-CD-1 NPs prepared in Comparative Example 1, and the His-HA-CD-2 NPs prepared in Comparative Example 2. In short, FITC-modified SPC, HACD, ​​and His-HA-CD were used to replace SPC, HACD, ​​and His-HA-CD, respectively, and FITC-SSA-SPC and FITC-His-HA-CD NPs, FITC-HA-CD-1 NPs, and FITC-His-HA-CD-2 NPs were prepared by referring to the methods of Examples 8 and 18 and Comparative Examples 1-2. HCT116 cells were seeded into 6-well plates, and 24 hours later, labeled nanoparticles (FITC, FITC-SSA-SPC, FITC-His-HA-CD NPs, FITC-HA-CD-1 NPs, FITC-His-HA-CD-2 NPs) and free HA were administered to HCT116 cells for 30 minutes before pretreatment. After incubation in an incubator for 4 hours, the culture medium was discarded, and the cells were collected by trypsin digestion after rinsing with PBS. The cells were suspended in PBS and centrifuged at 1800 rpm for 5 minutes, and quantitative uptake analysis was performed by flow cytometry. At the same time, HCT116 cells without any treatment were used as the control group.

[0064] 2. Test results Compared with FITC-SSA-SPC, FITC-HA-CD-1 NPs, and FITC-His-HA-CD-2 NPs, FITC-His-HA-CD NPs showed higher cellular uptake. When free hyaluronic acid pretreated cells, it competitively bound to the CD44 receptor on the cell surface, thereby blocking the receptor binding channel of subsequent FITC-His-HA-CD NPs nanoparticles. The uptake rate of nanoparticles decreased after pretreatment (e.g. Fig.15As shown), indicating that cells rely on CD44 receptor-mediated endocytosis to take up His-HACDNPs. Combined with the cytotoxicity results of Experimental Example 6, it is confirmed that the enhanced cytotoxicity of His-HA-CD NPs compared with unmodified SSA-SPC is due to the increased drug uptake caused by hyaluronic acid modification.

[0065] In addition, the inventors of this case also referred to the aforementioned embodiments and conducted experiments with other raw materials, process operations, and process conditions described in this specification, and obtained relatively ideal results.

[0066] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.

Claims

1. A method for preparing a phospholipid complex delivery system, characterized in that: include: The active ingredients of traditional Chinese medicine and phospholipids are mixed and stirred to react to obtain a phospholipid complex; the active ingredients of traditional Chinese medicine include saikosaponin A; wherein the mass ratio of the active ingredients of traditional Chinese medicine to the phospholipids is 1:2.5-20; A mixed reaction system comprising hyaluronic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, cyclodextrin and oligohistidine is reacted to obtain a hyaluronic acid-grafted cyclodextrin complex modified with oligohistidine; wherein the molar ratio of the hyaluronic acid, cyclodextrin and oligohistidine is 2:1:0.1-0.3; And, the phospholipid complex is mixed with the oligohistidine-modified hyaluronic acid-grafted cyclodextrin complex and incubated under the condition of a low-frequency alternating electric field to prepare a phospholipid complex delivery system; wherein the frequency of the low-frequency alternating electric field is 20-200 Hz, and the electric field strength is 1-15 V / cm.

2. The preparation method according to claim 1, characterized in that: Specifically include: The active ingredients of traditional Chinese medicine are mixed with phospholipids, and then a mixed organic solvent is added to form a mixed solution, so that the concentration of the active ingredients of traditional Chinese medicine in the mixed solution is 1-10 mg / mL; And, the mixed solution is stirred at 20-60° C. in the dark for 0.5-4 h, and then distilled under reduced pressure, dried in vacuum, re-dissolved in dichloromethane, filtered, and dried to obtain a phospholipid complex.

3. The preparation method according to claim 2, characterized in that: The mixed organic solvent includes methanol and chloroform, wherein the volume ratio of methanol to chloroform is 4:3-3:4; And / or, the stirring speed used in the light-shielded stirring is 300-800 rpm; And / or, the pore size of the filter membrane used for the dichloromethane re-dissolution and filtration is 0.1-1.0 μm.

4. The preparation method according to claim 1, characterized in that: Specifically include: Hyaluronic acid was dissolved in anhydrous formamide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide was added and ice-bathed for 0.5-2 h, then cyclodextrin and oligohistidine were added and reacted at 20-40 °C for 24-48 h, and then dialyzed and dried to obtain the oligohistidine-modified hyaluronic acid grafted cyclodextrin complex.

5. The preparation method according to claim 4, characterized in that: The molecular weight of the hyaluronic acid is 1-10 kDa; And / or, the cyclodextrin includes any one or more combinations of α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin; wherein the β-cyclodextrin includes hydroxypropyl-β-cyclodextrin; And / or, the oligohistidine is an oligohistidine with 15-30 residues.

6. The preparation method according to claim 1, characterized in that: Specifically include: The oligohistidine-modified hyaluronic acid grafted cyclodextrin complex is dissolved in water to form an oligohistidine-modified hyaluronic acid grafted cyclodextrin complex aqueous solution, and then the ethanol solution of the phospholipid complex is added dropwise to the oligohistidine-modified hyaluronic acid grafted cyclodextrin complex aqueous solution and incubated under a low-frequency alternating electric field to obtain a suspension of the phospholipid complex delivery system, which is then centrifuged, the precipitate is resuspended in distilled water, and freeze-dried to obtain the phospholipid complex delivery system.

7. The preparation method according to claim 6, characterized in that: The mass ratio of the phospholipid complex to the oligohistidine-modified hyaluronic acid-grafted cyclodextrin complex is 1:1-5; And / or, the incubation temperature is 30-50 °C and the time is 0.5-2 h.

8. The phospholipid complex delivery system prepared by the preparation method according to any one of claims 1 to 7, characterized in that: include: Phospholipid complex and carrier loaded with phospholipid complex; wherein the carrier comprises a hyaluronic acid grafted cyclodextrin complex modified with oligohistidine.

9. The phospholipid complex delivery system according to claim 8, characterized in that: The particle size of the phospholipid complex delivery system is 100-200 nm, and the particle size uniformity PDI is ≤0.

2.

10. Use of the phospholipid complex delivery system according to claim 8 or 9 in the preparation of targeted cancer drug delivery; wherein, The cancer includes colorectal cancer, and the colorectal cancer includes the HCT116 colon cancer cell line.

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