A phospholipid complex delivery system, its preparation method and application

Abstract

The present invention discloses a phospholipid complex delivery system, a preparation method thereof and an application. The preparation method includes: mixing and stirring a traditional Chinese medicine active ingredient and phospholipids for reaction to obtain a phospholipid complex; the traditional Chinese medicine active ingredient includes saikosaponin A; reacting a mixed reaction system containing hyaluronic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, cyclodextrin, and oligohistidine to obtain an oligohistidine-modified hyaluronic acid grafted cyclodextrin complex; and mixing the phospholipid complex with the oligohistidine-modified hyaluronic acid grafted cyclodextrin complex and incubating under the condition of a low-frequency alternating electric field to obtain a phospholipid complex delivery system. The phospholipid complex delivery system in the present invention can significantly increase the oral bioavailability of traditional Chinese medicine active ingredients, enhance the aggregation of active ingredients in tumor cells through active targeting, and improve the anti-tumor effect of active ingredients.

Description

Technical Field

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

[0002] Colorectal cancer is the third most common malignancy globally. Currently, the main treatment methods for colorectal cancer include: systemic treatment, local treatment, surgical treatment and postoperative adjuvant treatment. However, due to problems such as easy recurrence, easy metastasis, large chemotherapeutic side effects, many complications, heavy economic burden on patients and low quality of life after the disease. Therefore, finding a reliable and less side-effect drug for the prevention and treatment of colon cancer is still an important direction in the field of colorectal cancer research and has important practical significance. Research shows 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 action. It has now become an important part of the comprehensive diagnosis and treatment of CRC prevention.

[0003] Bupleurum is one of the important raw materials with a high utilization rate among many natural products and is often used in the treatment of diseases together with other raw materials. Bupleurum has a 2000-year medical history and was included in the "Shennong Ben Cao Jing" in the Eastern Han Dynasty. It is one of the most important herbs in China and is applied in traditional Chinese medicine prescriptions such as Minor Bupleurum Decoction and Bupleurum Liver-Regulating Powder due to its effects of relieving exterior syndromes, clearing heat, regulating liver qi and lifting yang qi. 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. The content of saikosaponin in Bupleurum roots is about 7% of the total dry weight, which is the key secondary metabolite for Bupleurum to exert its medicinal effects. Saikosaponin A (SSA) is a glycosylated oleanane-type saponin and is one of the main components of saikosaponins. In recent years, it has attracted attention due to its various biological activities and has anti-inflammatory, anti-tumor, antiviral and liver protection activities both in vivo and in vitro. However, due to its poor absorption ability in the gastrointestinal tract when taken orally, its bioavailability is extremely low. As a new type of drug delivery technology, the phospholipid complex delivery system has gradually emerged in the research of active ingredients of traditional Chinese medicine in recent years. Phospholipids, as the main components of cell membranes, have good biocompatibility and biodegradability and can form stable complexes with a variety of drug molecules through hydrogen bonds or van der Waals forces. This complex can not only improve the solubility and stability of drugs, but also show good effects in absorption and penetration. However, natural drug phospholipid complexes have disadvantages such as easy aggregation, hydrolysis and oxidation, and poor stability. Therefore, developing an efficient, safe and stable traditional Chinese medicine drug delivery system has become an important direction in 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, a preparation method and an application thereof to overcome the deficiencies in the prior art.

[0005] To achieve the aforementioned invention objective, the technical solutions adopted by the present invention include:

[0006] The embodiment of the present invention provides a preparation method of a phospholipid complex delivery system, which includes:

[0007] Mix and stir the traditional Chinese medicine active ingredient and phospholipid to react to obtain a phospholipid complex; the traditional Chinese medicine active ingredient includes saikosaponin A; wherein, the mass ratio of the traditional Chinese medicine active ingredient to phospholipid is 1:2.5 - 20;

[0008] React a mixed reaction system containing hyaluronic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, cyclodextrin, and oligohistidine to obtain an oligohistidine-modified hyaluronic acid grafted cyclodextrin complex; wherein, the molar ratio of hyaluronic acid, cyclodextrin, and oligohistidine is 2:1:0.1 - 0.3;

[0009] And, mix the phospholipid complex and the oligohistidine-modified hyaluronic acid grafted cyclodextrin complex and incubate under the condition of a low-frequency alternating electric field to obtain 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.

[0010] The embodiment of the present invention also provides a phospholipid complex delivery system prepared by the aforementioned preparation method, which includes: a phospholipid complex and a carrier loaded with the phospholipid complex; wherein, the carrier includes an oligohistidine-modified hyaluronic acid grafted cyclodextrin complex.

[0011] The embodiment of the present invention also provides the use of the aforementioned phospholipid complex delivery system in the preparation of a cancer drug for targeted delivery; wherein, the cancer includes colorectal cancer, and the colorectal cancer includes the HCT116 colon cancer cell line.

[0012] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0013] (1) Compared with the phospholipid complex, in the phospholipid complex delivery system provided by the present invention, the oligohistidine-modified hyaluronic acid grafted cyclodextrin complex makes more hyaluronic acid exposed through oligohistidine, and the complex has better targeting. The interaction between hyaluronic acid and CD44 enhances cell aggregation in tumor cells with overexpression of CD44. This phospholipid complex delivery system can improve the oral bioavailability of traditional Chinese medicine active ingredients, enhance the accumulation of active ingredients in tumor cells through active targeting, and improve its anti-tumor effect; in addition, this hyaluronic acid grafted cyclodextrin complex exhibits pH-sensitive characteristics, can disintegrate faster in a slightly acidic environment, and can quickly release drug active ingredients after entering cancer cells, with better drug efficacy;

[0014] (2) The small molecules used in the present invention are natural active ingredients in Chinese herbal medicines, and have low toxicity to normal cells.

[0015] (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 shows higher toxicity to tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments recorded in the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0017] Figures 1 - 4 Respectively, they are the graphs showing the effects of the drug-lipid ratio, reaction temperature, solvent ratio, and reaction time of the phospholipid complex in Test Example 1 of the present invention on the complexation rate;

[0018] Figure 5 It is the FTIR spectra of saikosaponin A, phospholipid, physical mixture, and phospholipid complex in Test Example 3 of the present invention;

[0019] Figure 6 It is for saikosaponin A, phospholipid, and phospholipid complex in Test Example 3 of the present invention 1 1H NMR spectra;

[0020] Figures 7 - 8 It is the particle size and Zeta potential spectra of the nanoparticles of the phospholipid complex delivery system in Test Example 5 of the present invention;

[0021] Figure 9 It is the transmission electron microscopy spectrum of the nanoparticles of the phospholipid complex delivery system in Test Example 5 of the present invention;

[0022] Figures 10 - 14 Respectively, they are the graphs showing the inhibitory effects of 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 on the growth of colon cancer HCT 116 cells in the present invention;

[0023] Figure 15 It is the cell uptake situation graph of HCT116 cells for FITC, FITC-SSA-SPC, FITC-His-HA-CDNPs, FITC-HA-CD-1 NPs, and FITC-His-HA-CD-2 NPs in Test Example 7 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0024] In view of the deficiencies of the prior art, the inventors of this case, through long-term research and a large number of practices, have been able to propose the technical solution of the present invention. For the convenience of understanding this application, the following will describe this application in more detail. 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.

[0025] Specifically, as an aspect of the technical solution of the present invention, a preparation method of a phospholipid complex delivery system involves:

[0026] Mixing and stirring the traditional Chinese medicine active ingredient with phospholipids to react to obtain a phospholipid complex; the traditional Chinese medicine active ingredient includes saikosaponin A; wherein, the mass ratio of the traditional Chinese medicine active ingredient to phospholipids is 1:2.5 - 20;

[0027] Reacting a mixed reaction system containing hyaluronic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), cyclodextrin, and oligohistidine to obtain an oligohistidine-modified hyaluronic acid grafted cyclodextrin complex; wherein, the molar ratio of hyaluronic acid, cyclodextrin, and oligohistidine is 2:1:0.1 - 0.3;

[0028] And, mixing the phospholipid complex with the oligohistidine-modified hyaluronic acid grafted cyclodextrin complex and incubating under the condition of a low-frequency alternating electric field to obtain 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.

[0029] In some preferred embodiments, the preparation method specifically includes:

[0030] Mixing the traditional Chinese medicine active ingredient with phospholipids, and then adding a mixed organic solvent to form a mixed solution, such that the concentration of the traditional Chinese medicine active ingredient in the mixed solution is 1 - 10 mg / mL.

[0031] And, stirring the mixed solution in the dark at 20 - 60 °C for 0.5 - 4 h, then performing reduced pressure distillation, vacuum drying, re-dissolving and filtering with dichloromethane, and drying treatment to obtain a phospholipid complex.

[0032] Furthermore, the mass ratio of the traditional Chinese medicine active ingredient to phospholipids is 1:10 - 20, and the effect is better when the mass ratio is 1:10.

[0033] 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.

[0034] Further, the temperature for light-shielded stirring is 40 - 60 °C, and the effect is better when the temperature is 50 °C.

[0035] Further, the time for light-shielded stirring is 2 h.

[0036] Further, the stirring speed for light-shielded stirring is 300 - 800 rpm.

[0037] Further, the pore size of the filter membrane used for dichloromethane redissolution and filtration is 0.1 - 1.0 μm.

[0038] In some preferred embodiments, the preparation method specifically includes: dissolving hyaluronic acid in anhydrous formamide, adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide and performing ice bath for 0.5 - 2 h, then adding cyclodextrin and oligohistidine and reacting at 20 - 40 °C for 24 - 48 h, and then performing dialysis and drying treatments to obtain an oligohistidine-modified hyaluronic acid grafted cyclodextrin complex.

[0039] In the present invention, adding oligohistidine during the preparation of the oligohistidine-modified hyaluronic acid grafted cyclodextrin complex will expose more hyaluronic acid in the complex, and the complex has better targeting; at the same time, the complex exhibits pH-sensitive characteristics, can disintegrate faster in a slightly acidic environment, while the tumor microenvironment is weakly acidic, and the histidine-modified nanoparticles are more stable in a neutral or slightly alkaline environment and are more likely to release in an acidic environment. Therefore, after entering cancer cells, the drug active ingredients can be quickly released, and the drug effect is better.

[0040] Further, the molecular weight of the hyaluronic acid is 1 - 10 kDa, and the effect is better when the molecular weight is 3 kDa.

[0041] Further, the cyclodextrin includes any one or a combination of more than one of α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin; among them, the β-cyclodextrin includes hydroxypropyl-β-cyclodextrin.

[0042] Further, the oligohistidine is oligohistidine with 15 - 30 residues.

[0043] In some preferred embodiments, the preparation method specifically includes: dissolving the oligohistidine-modified hyaluronic acid grafted cyclodextrin complex in water to form an aqueous solution of the oligohistidine-modified hyaluronic acid grafted cyclodextrin complex, and then dropping an ethanol solution of the phospholipid complex into the aqueous solution of the oligohistidine-modified hyaluronic acid grafted cyclodextrin complex and incubating under the condition of a low-frequency alternating electric field to obtain a suspension of the phospholipid complex delivery system, and then performing centrifugation, resuspending the precipitate with distilled water, and freeze-drying treatment to obtain the phospholipid complex delivery system.

[0044] 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.

[0045] Furthermore, the incubation temperature is 30 - 50 °C and the time is 0.5 - 2 h.

[0046] 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, the particle size uniformity reaches PDI ≤ 0.2; it shows higher toxicity to tumor cells and its empty loading rate is significantly reduced.

[0047] In some more specific embodiments, the preparation method of the phospholipid complex delivery system includes the following steps:

[0048] S1. Mix according to the mass ratio of saikosaponin A to phospholipid of 1:2.5 - 1:20.

[0049] S2. Add an organic solvent, and the organic solvent is methanol:chloroform with a volume ratio of 4:3 - 3:4, so that the drug concentration is 1 - 10 mg / mL.

[0050] S3. Stir magnetically in the dark at 30 - 60 °C for 0.5 - 4 h, and the stirring speed is 300 - 800 rpm.

[0051] S4. Evaporate the reaction solution under reduced pressure at 40 °C to remove the organic solvent, dry it in a vacuum drying oven under reduced pressure for 12 h, redissolve it in dichloromethane, filter it through a 0.22 μm filter membrane, and dry it to obtain the saikosaponin A phospholipid complex.

[0052] S5. Dissolve 0.02 mmol of cyclodextrin in anhydrous formamide, add 0.2 g of EDC and ice-bath for 2 h, dropwise add a hyaluronic acid solution containing 0.04 mmol and 0.001 - 0.003 mmol of oligohistidine, dialyze and lyophilize after reacting for 48 h to obtain the oligohistidine-modified hyaluronic acid grafted cyclodextrin complex.

[0053] S6. Dissolve the oligohistidine-modified hyaluronic acid grafted cyclodextrin complex in an aqueous solution, dropwise add the saikosaponin A phospholipid complex ethanol solution to the aqueous solution, and incubate it under the condition of a low-frequency alternating electric field to obtain a phospholipid complex delivery system suspension, centrifuge, resuspend the precipitate with distilled water and then freeze-dry to obtain the phospholipid complex delivery system.

[0054] Preferably, the mass ratio of saikosaponin A to phospholipid in step S1 is 1:10.

[0055] Preferably, the organic solvent in step S2 is methanol: chloroform with a volume ratio of 2:3.

[0056] Preferably, the drug concentration in step S2 is 2 mg / mL.

[0057] Preferably, the reaction temperature in step S3 is 50 °C.

[0058] Preferably, the reaction time in step S3 is 2 h.

[0059] Preferably, the cyclodextrin in step S5 is hydroxypropyl-β-cyclodextrin.

[0060] Preferably, the molecular weight of hyaluronic acid in step S5 is 3 kDa.

[0061] Preferably, the feeding ratio (mass ratio) of saikosaponin A phospholipid complex to oligohistidine-modified hyaluronic acid grafted cyclodextrin complex in step S6 is 1:1.

[0062] 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 includes: a phospholipid complex and a carrier loaded with the phospholipid complex; wherein the carrier includes an oligohistidine-modified hyaluronic acid grafted cyclodextrin complex.

[0063] 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.

[0064] 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 a cancer drug for targeted delivery; wherein, the cancer includes colorectal cancer, and the colorectal cancer includes the HCT116 colon cancer cell line.

[0065] The present invention is further illustrated by the following examples: According to the following examples, the present invention can be better understood. However, those skilled in the art can easily understand that the specific material ratios, process conditions and their 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.

[0066] Unless otherwise specified, the various raw materials, reaction equipment, testing equipment and testing methods used in the following examples are well-known in the art.

[0067] Experimental Example 1 Preparation of Phospholipid Complex

[0068] 1. Test methods (including Examples 1 - 17)

[0069] 1.1 Preparation of phosphatidylcholine complexes at different drug - lipid ratios

[0070] After mixing saikosaponin A (SSA) and soy lecithin (SPC) (Aladdin Reagent (Shanghai) Co., Ltd., phosphatidylcholine content > 96%) at mass ratios 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 ended, the reaction solution was evaporated under reduced pressure at 40 °C to remove the organic solvent, dried in a vacuum drying 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 phosphatidylcholine complex (SSA - SPC).

[0071] 1.2 Preparation of phosphatidylcholine complexes at different reaction temperatures

[0072] After mixing saikosaponin A and soy lecithin (Aladdin Reagent (Shanghai) Co., Ltd., phosphatidylcholine content > 96%) at 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 20 °C, 30 °C, 40 °C, 50 °C, and 60 °C in the dark for 2 h, with a stirring speed of 500 rpm. After the reaction ended, the reaction solution was evaporated under reduced pressure at 40 °C to remove the organic solvent, dried in a vacuum drying 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 phosphatidylcholine complex (SSA - SPC).

[0073] 1.3 Preparation of phosphatidylcholine complexes at different solvent ratios

[0074] After mixing saikosaponin A and soy lecithin (Aladdin Reagent (Shanghai) Co., Ltd., phosphatidylcholine content > 96%) at a mass ratio of 1:10, an organic solvent with volume ratios of methanol:chloroform of 3:2, 4:3, 1:1, 2:3, and 3:4 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 ended, the reaction solution was evaporated under reduced pressure at 40 °C to remove the organic solvent, dried in a vacuum drying 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 phosphatidylcholine complex (SSA - SPC).

[0075] 1.4 Preparation of phospholipid complex at different reaction times

[0076] After mixing saikosaponin A and soybean lecithin (Aladdin Reagent (Shanghai) Co., Ltd., phosphatidylcholine content > 96%) at 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 0.5 h, 1 h, 2 h, 3 h, and 4 h, with a stirring speed of 500 rpm. After the reaction ended, the reaction solution was evaporated under reduced pressure at 40 °C to remove the organic solvent, dried in a vacuum drying 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).

[0077] Calculate the complexation rate (%) of the above saikosaponin A phospholipid complex according to the following formula:

[0078] ;

[0079] 2. Test results

[0080] The complexation rates of various phospholipid complexes obtained in this test example are shown in Table 1 below and Figures 1 - 4 as follows.

[0081] Table 1 Complexation rates of phospholipid complexes prepared under different conditions of the present invention

[0082] ;

[0083] The test results show that SSA-SPC was prepared by the solvent evaporation method. Using the complexation rate as the evaluation index, the effects of different drug-lipid ratios, reaction temperatures, solvent ratios, and reaction times on the complexation rate were investigated. 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 it was magnetically stirred at 50 °C in a water bath for 2 h.

[0084] Test Example 2 Preparation of phospholipid complex delivery system nanoparticles and determination of their physicochemical properties

[0085] 1. Test method (including Examples 18 - 22, Comparative Example 1, and Comparative Example 2)

[0086] 1.1 Preparation of phospholipid complex

[0087] The phospholipid complex was prepared according to Example 8.

[0088] 1.2 Preparation of oligohistidine-modified hyaluronic acid grafted cyclodextrin complex

[0089] First, dissolve hyaluronic acid HA (0.02 mmol) in 6 mL of anhydrous formamide, then add 0.2 g of EDC, stir the solution mixture in an ice bath for 2 h, and add 6 mL of hydroxypropyl-β-cyclodextrin HPCD (0.04 mmol) dissolved in anhydrous formamide and 0.002 mM of oligohistidine dropwise for reaction. Stir the reaction mixture for another 48 h, and dialyze the solution with an excess of double distilled water for two days. Freeze-dry the dialysate to obtain a white powder of oligohistidine-modified hyaluronic acid grafted cyclodextrin complex (denoted as: His-HA-CD).

[0090] 1.3. Preparation of phospholipid complex delivery system nanoparticles

[0091] Dissolve the oligohistidine-modified hyaluronic acid grafted cyclodextrin complex (His-HA-CD) in an aqueous solution, and dropwise add the ethanol solution of saikosaponin A phospholipid complex (SSA-SPC). The weight ratio of SSA-SPC to His-HA-CD in the feed is 1:1 - 1:5. 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, incubate at 37 °C for 2 h to obtain a suspension of saikosaponin A phospholipid complex delivery system nanoparticles. Centrifuge at 3500 rpm for 10 min, resuspend the precipitate with a small amount of pure water and then freeze-dry to obtain the saikosaponin A phospholipid complex delivery system nanoparticles (denoted as: His-HA-CD NPs). Take the nanoparticles prepared in each example to obtain a 1 mg / mL suspension in water, and measure their particle size, PDI, and zeta potential respectively.

[0092] Comparative Example 1: The method is the same as that in Example 18, except that oligohistidine is missing when preparing the oligohistidine-modified hyaluronic acid grafted cyclodextrin complex. The prepared nanoparticles are denoted as HA-CD-1 NPs.

[0093] Comparative Example 2: The method is the same as that in Example 18, except that the incubation is not carried out under the condition of a low-frequency alternating electric field. The prepared nanoparticles are denoted as His-HA-CD-2 NPs.

[0094] 2. Experimental results

[0095] The average particle size, PDI, and zeta potential of various nanoparticles obtained in each step of this test example are shown in Table 2 below.

[0096] Table 2 Average particle size, PDI, and zeta potential of phospholipid complexes with different formulations of the present invention

[0097] ;

[0098] The test results showed that when the weight ratio of the phospholipid complex to the oligohistidine-modified hyaluronic acid grafted cyclodextrin complex changed from 1:1 to 1:5, the particle size of His-HA-CD NPs prepared under the condition of low-frequency alternating electric field was concentrated in the range of 100-200 nm, and the particle size uniformity reached PDI ≤ 0.2. In the absence of oligohistidine and low-frequency alternating electric field, the size and PDI of the prepared nanoparticles increased. Given the enhanced permeability and retention (EPR) effect of tumor tissues, examples with small particle size and low PDI were selected for subsequent research, that is, His-HA-CD NPs with a weight ratio of 1:1 in Example 18 were selected for subsequent research.

[0099] Test Example 3 FTIR and 1 HNMR determination of phospholipid complex

[0100] Using the potassium bromide tablet method, Fourier transform infrared spectrometer was used to scan the infrared spectra of SSA, SPC, SSA-SPC (from Example 8) and the physical mixture (PM) of saikosaponin A and soybean lecithin in the range of 4000 cm -1 ~400 cm -1 respectively. The 1 HNMR spectra of SSA, SPC and SSA-SPC (from Example 8) were recorded, the solvent of SSA was D 1 O, and the solvents of SPC and SSA-SPC were CDCl 2 . The experimental results showed that the formation of SSA-SPC complex was confirmed by FTIR and 3 HNMR tests (as shown in 1 and Figure 5 and Figure 6 ).

[0101] Test Example 4 Determination of drug loading and encapsulation efficiency of phospholipid complex delivery system nanoparticles

[0102] 1. Test method

[0103] Take 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. The nanoparticle suspension was centrifuged at 14000 rpm for 10 min, and the concentration of free SSA in the supernatant was measured. The drug loading and encapsulation efficiency of the nanoparticles were calculated according to the following formulas:

[0104] ;

[0105] 2. Test results

[0106] The test results showed that the encapsulation efficiency of saikosaponin A in the nanoparticles prepared in Example 18 was 98.20%, and the drug loading was 4.46%; the encapsulation efficiency of saikosaponin A in the nanoparticles prepared in Comparative Example 1 was 95.75%, and the drug loading was 4.35%; the encapsulation efficiency of saikosaponin A in the nanoparticles prepared in Comparative Example 2 was 92.40%, and the drug loading was 4.20%.

[0107] Test Example 5 Transmission Electron Microscopy Determination of Nanoparticles of Phospholipid Complex Delivery System

[0108] First, the His-HA-CD NPs suspension prepared in Example 18 was slightly sonicated to disperse evenly.

[0109] Weighed 0.02 g of phosphotungstic acid and dissolved it in 1 mL of water. Picked up the copper grid with forceps and placed it on the moist filter paper, sucked about 50 μL of the sample and gently dropped it onto the copper grid. After staining for about 2 min, the morphology of the nanoparticles was observed using a transmission electron microscope and photographed. As Figure 9 shown, the morphology of the nanoparticles showed monodisperse spherical nanoparticles. In addition, the particle size length shown by the scale of TEM was relatively consistent with the measured particle size (as Figures 7 - 8 ).

[0110] Test Example 6 In Vitro Antitumor Activity of Nanoparticles of Phospholipid Complex Delivery System

[0111] 1. Test Method

[0112] HCT116 cells were seeded into 96-well plates and incubated for 24 h until adherent and confluent. Then, a series of 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 with a total drug concentration of 22 μM, 24 μM, 26 μM, 28 μM, 30 μM, 32 μM, 34 μM, 36 μM, 38 μM, and 40 μM were added respectively. After incubation for 24 h, the culture medium was discarded. Then, 100 μL of culture medium and 20 μL of MTT (5 mg / mL) were added to each well. After incubation in the dark at 37 °C 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 an enzyme-linked immunosorbent assay (ELISA) reader.

[0113] 2. Test Results

[0114] 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, His-HA-CD NPs prepared in Example 18 showed enhanced cytotoxicity, indicating that the nanoparticles enabled the accumulation of saikosaponin A in HCT116 cells, which may be due to the cancer cell-selective targeting and pH-responsive release characteristics of HA ( Figures 10 - 14 ). Based on the above data, it can be seen that the His-HA-CD NPs nanoparticles prepared in Example 18 significantly reduced the cell viability of cancer cells HCT116.

[0115] Test Example 7 In vitro cellular uptake of phospholipid complex delivery system nanoparticles

[0116] 1. Test method

[0117] Quantitative uptake analysis was performed using a flow cytometer. First, the nanoparticles SSA-SPC 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 fluorescently labeled with fluorescein isothiocyanate (FITC). Briefly, 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 according to the methods of Examples 8 and 18 and Comparative Examples 1-2. HCT116 cells were seeded in a 6-well plate. After 24 h, the 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. After pretreating HCT116 cells with the labeled nanoparticles FITC-His-HA-CD NPs for 30 min, the cells were incubated in an incubator for 4 h. Then, the culture medium was discarded, and the cells were rinsed with PBS and digested with trypsin for collection. After the cells were suspended in PBS and centrifuged at 1800 rpm for 5 min, quantitative uptake analysis was performed using a flow cytometer, and untreated HCT116 cells were used as the control group.

[0118] 2. Test results

[0119] 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 cells were pretreated with free hyaluronic acid, it competitively bound to the CD44 receptors on the cell surface, thus blocking the receptor-binding channels of subsequent FITC-His-HA-CD NPs nanoparticles. The uptake rate of the nanoparticles decreased after pretreatment (as Figure 15 shown), indicating that cells take up His-HA CD NPs through CD44 receptor-mediated endocytosis. Combining with the cytotoxicity results in Test Example 6, it was confirmed that the increased cytotoxicity of His-HA-CD NPs compared with unmodified SSA-SPC was due to the increased drug uptake caused by hyaluronic acid modification.

[0120] In addition, the inventors of this case also referred to the foregoing embodiments and conducted tests with other raw materials, process operations, and process conditions described in this specification, and all obtained relatively ideal results.

[0121] 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 by the above embodiments. What is described in the above embodiments and the specification only illustrates the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed.

Claims

1. A method for preparing a phospholipid complex delivery system, characterized in that: include: The active ingredients of traditional Chinese medicine are mixed with phospholipids for reaction, and the phospholipid complex is prepared; the active ingredients of traditional Chinese medicine are selected from saikosaponin A; wherein the mass ratio of the active ingredients of traditional Chinese medicine to the phospholipids is 1:10-20; 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 cyclodextrin is hydroxypropyl-β-cyclodextrin; 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 is 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 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: A phospholipid complex and a carrier loaded with the phospholipid complex; wherein the carrier is 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 is colorectal cancer, and the colorectal cancer is HCT116 colon cancer cell line.

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