CN111920782A - Composite lipid nanocapsule composition and preparation method and application thereof - Google Patents

Abstract

The invention discloses a composite lipid nanocapsule composition, a preparation method and application thereof, and belongs to the technical field of medicine. The composite lipid nanocapsule composition is composed of a lipid core with a negative charge on the surface, a first-layer capsule wall with a positive charge, and a second-layer capsule wall with a negative charge. Adsorption combined to form, can be used for intravenous injection. Among them, the lipid core is the reservoir for loading antitumor drugs, chitosan has the functions of promoting transmembrane transport and controlling drug release, and the low molecular weight heparin coated on the outside of the nanocapsules can interact with heparinase to enrich the nanocapsules. It is collected and degraded in the malignant tumor site to achieve the function of targeting the tumor. In addition, the carrier material used in the composition has high biological safety, good tumor targeting, high drug encapsulation rate, good stability, controllable release, simple preparation method, and is used for targeted delivery of anti-tumor drugs with broadly application foreground.

Description

Composite lipid nanocapsule composition, preparation method and application thereof

technical field

The invention belongs to the technical field of medicine and nano-medicine, and relates to a composite lipid nano-capsule composition and a preparation method and application thereof.

Background technique

Malignant tumors are one of the major diseases that threaten human health and cause death of patients. Chemotherapy is a conventional treatment method for malignant tumors, but chemotherapy drugs generally have defects such as non-selective treatment, large toxic and side effects, and cancer cells are prone to develop drug resistance, and the patient's tolerance is poor. In addition, many antitumor drugs also have physical and chemical defects such as poor water solubility and poor stability. Therefore, it is necessary to improve the physicochemical properties of drugs through preparation technology, especially according to the biological characteristics of tumors and their microenvironment, use preparation technology to improve the tumor targeting of drugs, reduce the toxicity and side effects of the body, and improve the tumor treatment effect.

Nanocapsules are drug-storage nanoparticles formed by encapsulating solid or liquid drugs as capsule cores. Drug carrier system (10 ~ 1000nm). As a drug carrier, in addition to improving the solubility, dispersibility and other conventional physical and chemical properties of the drug, it also has the following advantages: 1. Improve the stability of the drug and reduce the impact of environmental factors on the drug; 2. Endow the drug with slow and controlled release 3. It has passive targeting, and the capsule material can also be positioned in specific tissues after surface modification to achieve the purpose of active targeting; 4. Due to the nano-sized particles and their tiny size, they can be used for intravenous injection. Can cause vascular embolism.

Based on the above advantages, nanocapsules have broad prospects for anti-tumor drug delivery, but there are still some problems in nanocapsule preparations that have been researched more now, such as poor biocompatibility of carrier materials and poor tumor targeting of preparations. The drug release is not ideal, the controllability of the preparation process is poor, and the quality is unstable.

A microcapsule is disclosed in the prior art, see the master's thesis "Preparation and Properties of Layer-by-Layer Self-Assembled Heparin/Chitosan Microcapsules" (Liang Yan, 2014, Jiangnan University, China). The microcapsules are prepared by repeatedly and alternately electrostatically adsorbing the negatively charged natural polysaccharide heparin and the positively charged natural polysaccharide chitosan on the surface of the core CaCO3 template by layer _- by-layer self _- assembly technology, and then removing the CaCO3 template. The research results show that the microcapsules have good biocompatibility and stability. However, because the particle size of the microcapsules prepared by this technology is about 4-5 μm, it is not suitable for intravenous administration, and the ability of targeted drug delivery is insufficient, which limits its clinical application.

SUMMARY OF THE INVENTION

The technical problem solved by the present invention is to provide a composite lipid nanocapsule composition, which has the following properties: the polymers and functional materials used have good biocompatibility, safety and non-toxicity; 1000nm), can be used for intravenous injection; has good tumor targeting; high drug encapsulation rate and good stability; the provided preparation method is simple and feasible.

Another technical problem solved by the present invention is to provide the application of the composite lipid nanocapsule composition.

The technical problem solved by the present invention is achieved through the following technical solutions:

The first aspect of the technical solution of the present invention is to provide a composite lipid nanocapsule composition, the composition comprising the following components: oil, phospholipid, ascorbyl palmitate, antitumor compound, chitosan, low molecular weight heparin. The particle size of the composite lipid nanocapsules ranges from 10 to 1000 nm, preferably between 10 and 500 nm, and more preferably between 10 and 200 nm. The particle size refers to the average particle size of light intensity (Intensity-WeightedMean Diameter, abbreviated as: particle size) measured by a laser particle size analyzer.

The complex lipid nanocapsule composition may or may not also contain cholesterol.

The composite lipid nanocapsule is composed of a negatively charged lipid core on the surface, a positively charged first layer capsule wall, and a negatively charged second layer capsule wall, which are electrostatically adsorbed between the positive and negative charged groups. Formed by action binding (see Figure 1); the surface negatively charged lipid core contains the following components: lipids, phospholipids, ascorbyl palmitate, antitumor compounds; the positively charged first layer vesicles The wall contains chitosan; the negatively charged second layer of the capsule wall contains low molecular weight heparin; the surface negatively charged lipid core may or may not also contain cholesterol.

Further, in the composite lipid nanocapsule composition, the amount of ascorbyl palmitate per gram of phospholipid is 0.001-100 grams, preferably 0.02-0.12 grams; the amount of oil per gram of phospholipids is 0.001-100 grams ml, preferably 0.8-1.2 ml; the amount of chitosan per gram of phospholipids is 0.001-100 grams, preferably 0.015-0.045 grams, more preferably 0.01875-0.03125 grams; the amount of low molecular weight heparin per gram of chitosan is 0.001-100 grams, preferably 0.42-5.25 grams.

Further, in the composite lipid nanocapsule composition, the oil is one or more oils of natural or synthetic origin, preferably including medium chain triglycerides (medium chain oil for short) or soybean oil; The phospholipid is one or more phospholipids of natural or synthetic origin, preferably including lecithin (the main component is phosphatidylcholine, PC), more preferably, the phosphatidylcholine content is greater than or equal to 80%; the Chitosan, preferably including low-molecular-weight chitosan; the low-molecular-weight heparin is a kind of lower molecular weight heparin and its salts made after depolymerization, preferably including low-molecular-weight heparin sodium, more preferably including Enoxaparin sodium.

Further, in the composite lipid nanocapsule composition, the antitumor compound includes a taxane compound, further, the taxane compound includes paclitaxel; the amount of paclitaxel per gram of phospholipids is 0 to 100 grams, preferably 0.02 to 0.03 grams.

Further, the preparation method of the composite lipid nanocapsule composition is as described in the second aspect of the technical solution of the present invention. The second aspect of the technical solution of the present invention provides the preparation method of the composite lipid nanocapsule composition of the first aspect, wherein the negatively charged lipid core on the surface comprises the following preparation steps:

(1) phospholipid and ascorbyl palmitate are dissolved in organic solvent A to obtain a homogeneous solution, and organic solvent A is removed to obtain a mixed membrane material;

(2) dissolving the mixed membrane material, grease and antitumor compound obtained in step 1 in organic solvent B to obtain a uniform solution, and removing organic solvent B to obtain a lipid membrane complex;

(3) Dispersing the lipid membrane complex obtained in step 2 in an aqueous phase, emulsification and homogenization, thereby obtaining lipid nanoparticles with negative surface charges, that is, the negatively charged lipid cores on the surface.

Among the components of the lipid core with negative surface charge, the amount of ascorbyl palmitate per gram of phospholipids is 0.001-100 grams, preferably 0.02-0.12 grams; the amount of oil and fat per gram of phospholipids is 0.001-100 grams. ml, preferably 0.8 to 1.2 ml.

Further, the antitumor compound includes a taxane compound, and further, the taxane compound includes paclitaxel; the amount of paclitaxel per gram of phospholipid is 0-100 grams, preferably 0.02-0.03 grams.

Further, the organic solvent A includes but is not limited to one or more of methanol, ethanol, and tetrahydrofuran, preferably methanol. The organic solvent B includes, but is not limited to, one or more of chloroform, dichloromethane, tetrahydrofuran, n-hexane, cyclohexane, ethyl acetate, petroleum ether, methanol, and ethanol, preferably dichloromethane.

Further, the aqueous phase for dispersing the lipid membrane complex may or may not contain glycerol.

Further, the methods for removing organic solvent A and removing organic solvent B include but are not limited to drying under reduced pressure, solvent evaporation, rotary evaporation, spray drying, and freeze drying.

The preparation method of the composite lipid nanocapsule composition, wherein the surface negatively charged lipid core, the positively charged first layer capsule wall, the negatively charged second layer capsule wall, the three through The composite lipid nanocapsule formed by electrostatic adsorption between positive and negative charged groups comprises the following preparation steps:

(1) Disperse the negatively charged lipid core in the aqueous phase containing chitosan, and after self-assembly, the chitosan is adsorbed on the surface of the lipid core, so that the lipid core is coated with the first layer of capsule wall , resulting in nanocapsules with positive surface charges.

(2) Disperse the nanocapsules with positive charges on the surface obtained in step 1 in an aqueous phase containing low molecular weight heparin, and through self-assembly, the low molecular weight heparin is adsorbed on the surface of the nanocapsules, so that the nanocapsules are covered with a second layer of capsules wall to obtain the composite lipid nanocapsules.

In the water phase containing chitosan, the chitosan concentration is 0.001-100 mg/mL, preferably 0.15-0.3 mg/mL, more preferably 0.2-0.3 mg/mL, further preferably 0.2 mg/mL; The volume ratio of the chitosan solution is 0.001-10,000 parts per part of the lipid core solution, preferably 1.5-3.0 parts, more preferably 2.0-3.0 parts, further preferably 2.5 parts. In terms of mass ratio, the amount of chitosan per gram of phospholipid is 0.001-100 grams, preferably 0.015-0.045 grams, more preferably 0.01875-0.03125 grams, further preferably 0.025 grams.

The low-molecular-weight heparin-containing aqueous phase, wherein the low-molecular-weight heparin concentration is 0.001-100 mg/mL, preferably 0.05-0.35 mg/mL, more preferably 0.08 mg/mL; The portion of the chitosan-coated surface positively charged nanocapsule solution is 0.001 to 10,000 parts, preferably 1.00 to 2.75 parts, and more preferably 1.2 parts. In terms of mass ratio, the amount of low molecular weight heparin per gram of chitosan is 0.001-100 grams, preferably 0.42-5.25 grams, more preferably 0.672 grams.

Further, the water phase containing chitosan, the medium used to adjust its acidity, includes but not limited to acetic acid, hydrochloric acid, phosphoric acid, preferably hydrochloric acid, more preferably, the concentration of hydrochloric acid in the water phase is 0.005mol/L.

The composite lipid nanocapsule composition can be made into various dosage forms containing the composite lipid nanocapsules, including but not limited to injection, lyophilized powder, and gel. Prepared by conventional methods, preferably injection or lyophilized powder.

The third aspect of the technical solution of the present invention provides the application of the above-mentioned composite lipid nanocapsule composition in the preparation of anti-tumor drugs, and the tumors include but are not limited to breast cancer, pancreatic cancer, gastric cancer, bladder cancer, brain cancer, Ovarian cancer, prostate cancer, lung cancer, liver cancer, Ewing's sarcoma, multiple myeloma, or B lymphoma.

beneficial technical effect

Compared with the prior art, the present invention has the following beneficial technical effects:

The composite lipid nanocapsule composition provided by the present invention will have various properties, including improving drug stability, being usable for intravenous injection, having slow and controlled release properties, and being able to actively target tumor sites, reduce the toxic and side effects of normal tissues, and improve tumor performance. treatment effect. The advantages of the present invention are as follows,

1. The lipid composite nanocapsules prepared by the present invention are small-sized particles with a particle size in the nanometer scale, which can improve the dispersibility, permeability and stability of the drug, and can be used for intravenous injection. retention effect), is an excellent carrier for targeted delivery of antitumor drugs.

2. The lipid core with negative charge on the surface prepared by the present invention, the core of which is oil, can be loaded with hydrophobic drugs and improve the solubility of drugs.

3. In the lipid core with negative charge on the surface prepared by the present invention, the membrane material comprises phospholipid. The main component of lecithin is phosphatidylcholine, and when it is used alone as a membrane material, the negative charge density at the interface of the prepared nanoparticle is small and uncontrollable, which is not conducive to the subsequent preparation of nanocapsules. The present invention finds that ascorbyl palmitate (Ascorbylpalmitate, AP, see Figure 2 for chemical structural formula), one side is lipophilic palmitate, the other side is ascorbic acid acid polyhydroxy group at the hydrophilic end, and is negatively charged after hydrolysis , the ascorbyl palmitate and phospholipids are made into a mixed membrane material, which can significantly enhance the negative charge density on the surface of the lipid core and improve the stability of the lipid core. .

4. The composite lipid nanocapsule prepared by the present invention has the effect of actively targeting tumors. Low-molecular-weight heparin (LMWH) is a general term for a class of heparins with lower molecular weights made after depolymerization, with a narrow average molecular weight distribution range and fewer side effects than undepolymerized heparin. Heparanase (HPA, heparinase for short) is an endogenous endo-β-D-glucuronidase that can specifically excise heparan sulfate proteoglycans on the extracellular matrix. A large number of studies have found that the overexpression of heparinase is closely related to the occurrence, development, invasion and metastasis of tumors, and is an important molecular target related to tumors. Heparin and its lysate, low molecular weight heparin, are the substrates of heparinase, which can bind to heparinase through enzyme-substrate interaction and inhibit the degradation of endogenous heparan sulfate proteoglycans. Pharmacological effects such as inflammation, anticoagulation and antithrombosis. The invention coats the low molecular weight heparin on the outer side of the composite lipid nanocapsule, and utilizes its action characteristics with heparinase to enrich and degrade the nanocapsule in the malignant tumor site, and has active targeting for the tumor and its microenvironment. effect.

5. The composite lipid nanocapsule provided by the present invention will have a multifunctional synergistic effect. The composite lipid nanocapsule is composed of three parts: the lipid core is a drug-loading reservoir, chitosan can promote transmembrane transport and control drug release, and low-molecular-weight heparin can actively target the high expression of heparinase. site of malignant tumor. After the composite lipid nanocapsules enter the systemic circulation, compared with normal tissues, they are more likely to be enriched and degraded at the tumor site, and the exposure of the chitosan-coated nanocapsules makes the nanocapsules easily taken up by tumor cells and the drug is released intracellularly. Through the synergistic effect of various functional materials, the tumor treatment effect of the drug will be enhanced, the toxic and side effects of the body will be reduced, and the patient's tolerance will be improved.

6. The composite lipid nanocapsule provided by the present invention has good drug encapsulation efficiency, stability and controllable release performance. The drug is encapsulated in the lipid core and is not disturbed by the functional material of the capsule wall and the external environment, which improves the encapsulation efficiency and stability of the drug. The present invention adopts chitosan with strong positive charge, and chitosan can be closely combined with the lipid core with negative charge on the surface and the low molecular weight heparin, which is beneficial to improve the stability of nanocapsules circulating in the body; on the other hand, because the shell The glycan has a strong positive charge and can interact with a variety of drug molecules. The present invention first prepares drug-loaded lipid nanoparticles (lipid core), and then coats chitosan, and the drug does not directly contact chitosan , which eliminates the interference to the drug, and only needs to adjust the drug release rate by changing the amount of chitosan adsorbed on the surface of the lipid core, and the drug release is more controllable.

7. The carrier material used in the composite lipid nanocapsule provided by the present invention is mainly from natural sources, has good biocompatibility, and is safe and non-toxic.

8. The preparation process of the composite lipid nanocapsule provided by the present invention is simple and feasible, is beneficial to industrial production, and meets the needs of clinical treatment.

Description of drawings

Fig. 1 Schematic diagram of the preparation method of composite lipid nanocapsules

Figure 2 Structure of ascorbyl palmitate

FIG. 3 shows the factor-level contours of the star point design-effect surface optimization method in Example 9 of the present invention.

Figure 4 shows the particle size distribution of the composite lipid nanocapsules in Example 11 of the present invention

Fig. 5 shows the transmission electron microscope image of the composite lipid nanocapsule of Example 11 of the present invention

Fig. 6 has shown the transmission electron microscope picture of the embodiment of the present invention 12 composite lipid nanocapsule lyophilized powder

Figure 7 shows the release curve of the complex lipid nanocapsules of Example 13 of the present invention

Fig. 8 shows the graph of in vivo tumor targeting distribution of tumor-bearing mice injected with complex lipid nanocapsules over time in Example 14 of the present invention, A: common solution group; B: lipid core group; C: complex lipid nanocapsules Group

Fig. 9 shows the fluorescence intensity of tumor tissue in tumor-bearing mice injected with complex lipid nanocapsules for 24 hours in Example 14 of the present invention

Detailed ways

The present invention will be further described below in conjunction with specific embodiments, but these embodiments are only exemplary and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present invention can be changed or replaced without departing from the spirit and scope of the present invention, but these changes and replacements all fall within the protection scope of the present invention.

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified. instrument:

Experimental ultra-high pressure homogenizer: Nano DeBEE, American BEE company

Laser particle size potentiometer: Nicomp 380ZLS, PSS, USA

Reagents:

Phospholipids: PC-98T, PC≥98%, Shanghai Aiwei Tuo Pharmaceutical Technology Co., Ltd.

LIPOID E80, PC: 80～85%, Lipoid Gmbh, Germany

Chitosan: CHITOSAN, Low molecular weight, Sigma-aldrich Company

Low molecular weight heparin: Enoxaparin sodium, Hebei Changshan Biochemical Pharmaceutical Co., Ltd.

DiR: DiR iodide, near-infrared fluorescent cyanine dye for cell membranes, AAT Bioquest, USA

Example 1 Adjustment of negative surface charge of ascorbyl palmitate

Lipid cores containing different ratios of ascorbyl palmitate were prepared and compared to common liposomes without ascorbyl palmitate.

Table 1 Formulation and characterization of different ratios of ascorbyl palmitate

1. The preparation method of prescription A1 (ordinary liposome):

(1) Dissolve phospholipid (PC: 80-85%) and cholesterol in chloroform-methanol (88:12) to obtain a clear and transparent solution, and remove the organic solvent by rotary evaporation to obtain a lipid film.

(2) The lipid film obtained in step 1 was placed in a water bath at 40-60° C., and 25 mL of phosphate buffer (pH=7) containing about 0.2% Tween 80 was slowly added under stirring to obtain a crude emulsion. The crude emulsion was homogenized under high pressure to obtain liposomes.

2. The preparation method of prescription A2～A5:

(1) Dissolve phospholipids (PC≥98%) and ascorbyl palmitate in methanol to obtain a clear and transparent solution, remove methanol by vacuum rotary evaporation in a water bath at 30-40°C, and collect the resulting precipitate, that is, a mixed membrane material.

(2) Dissolving the mixed membrane material and medium chain oil obtained in step 1 in dichloromethane to obtain a uniform solution, and vacuum rotary evaporation in a 30-40° C. water bath to remove dichloromethane to obtain a lipid membrane complex.

(3) The lipid membrane complex obtained in step 2 was placed in a water bath at 40-60° C., and 50 mL of phosphate buffer (pH=7) containing about 2.5% glycerol was slowly added under stirring to obtain a crude emulsion. The crude emulsion is homogenized under high pressure to obtain blank nanoparticles with negatively charged surfaces, namely blank lipid cores.

3. Characterization method: take the nanoparticles obtained from each recipe, dilute them with water to an appropriate concentration, and use a laser particle size potentiometer to measure the average particle size of light intensity (Intensity-Weighted Mean Diameter, referred to as particle size), polydispersity coefficient ( PDI) and Zeta potential (abbreviation: potential).

4. Results: The absolute value of the negative charge potential on the surface of common liposomes without adding ascorbyl palmitate was low, which was not conducive to the subsequent coating of positively charged capsule walls. With the addition of ascorbyl palmitate, the negative charge density increased significantly. When the added amount reaches a certain level (about 8% of the phospholipid), increasing the amount of ascorbyl palmitate does not change the negative charge density significantly, which is presumed to be caused by the ascorbyl palmitate on the interface film being close to saturation.

Example 2 Comparison of Different Phospholipids

The effects of using phospholipids containing different proportions of phosphatidylcholine (PC: 80%-100%) on the preparation of lipid cores were investigated.

Table 2 Formulation and characterization of different phospholipids

Preparation:

(1) Dissolve the phospholipids and ascorbyl palmitate in methanol to obtain a clear and transparent solution, remove the methanol by vacuum rotary evaporation in a water bath at 30-40°C, and collect the resulting precipitate, that is, the mixed membrane material.

(2) Dissolving the mixed membrane material and medium chain oil obtained in step 1 in dichloromethane to obtain a uniform solution, and vacuum rotary evaporation in a 30-40° C. water bath to remove dichloromethane to obtain a lipid membrane complex.

(3) The lipid membrane complex obtained in step 2 was placed in a water bath at 40-60° C., and 50 mL of phosphate buffer (pH=7) containing about 2.5% glycerol was slowly added under stirring to obtain a crude emulsion. The crude emulsion is homogenized under high pressure to obtain blank nanoparticles with negatively charged surfaces, namely blank lipid cores.

The results showed that there was no obvious difference between the lipid cores prepared by using phospholipids containing different proportions of phosphatidylcholine (PC: 80%-100%).

Example 3 Comparison of different oils and fats

The effects of different lipids on the preparation of nanocapsule lipid cores were investigated.

Table 3 Formulations and characterization of different oil phases

Preparation:

(1) Dissolve phospholipids (PC≥98%) and ascorbyl palmitate in methanol to obtain a clear and transparent solution, remove methanol by vacuum rotary evaporation in a water bath at 30-40°C, and collect the resulting precipitate, that is, a mixed membrane material.

(2) Dissolving the mixed membrane material obtained in step 1 and the oil phase in the recipe amount in dichloromethane to obtain a uniform solution, and vacuum rotary evaporation in a 30-40° C. water bath to remove dichloromethane to obtain a lipid membrane complex.

(3) Place the lipid membrane complex obtained in step 2 in a water bath at 40 to 60° C., and slowly add 50 mL of phosphate buffer (pH=7) containing about 2.5% glycerol under stirring to obtain a thick emulsion. The crude emulsion is homogenized under high pressure to obtain blank nanoparticles with negatively charged surfaces, namely blank lipid cores.

The results showed that either medium chain oil or soybean oil could be used as the oil phase to prepare lipid cores with small average particle size and rich in negative surface charges.

Example 4 Preparation and characterization of paclitaxel lipid cores

Preparation and characterization of drug-loaded lipid cores using the antitumor compound paclitaxel as a model drug

Table 4 Formulation composition and characterization

Preparation:

(1) Dissolve phospholipids (PC: 80-85%) and ascorbyl palmitate in methanol to obtain a clear and transparent solution, remove methanol by vacuum rotary evaporation in a water bath at 30-40°C, and collect the resulting precipitate, that is, the mixed membrane material .

(2) Dissolving the mixed membrane material, paclitaxel and medium chain oil obtained in step 1 in dichloromethane to obtain a uniform solution, and vacuum rotary evaporation in a water bath at 30-40° C. to remove dichloromethane to obtain a lipid membrane complex.

(3) The lipid membrane complex obtained in step 2 was placed in a water bath at 40-60° C., and 50 mL of phosphate buffer (pH=7) containing about 2.5% glycerol was slowly added under stirring to obtain a crude emulsion. The crude emulsion was homogenized under high pressure to obtain a negatively charged paclitaxel lipid core.

Example 5 Encapsulation efficiency of paclitaxel lipid core

Method for Determining the Encapsulation Efficiency of Paclitaxel Lipid Core Drugs

(1) HPLC chromatographic conditions: ZORBAX Eclipse XDB-C18 (4.6mm×150mm, 3.5μm) as the chromatographic column; the mobile phase is methanol-water-acetonitrile (23:41:36): the detection wavelength is 227nm, and the column temperature is 35 ℃, the flow rate was 1.2 mL per minute, and the injection volume was 10 μL.

(2) Separation of encapsulated and free drugs by micro-column centrifugation: take a 5mL syringe (with filter paper at the bottom), add Sephadex G 50, fill it to about 5mL mark, and naturally flow through the pressure with water To form a uniform gel column without breakage, centrifuge at 2000 rpm- ¹ for 3 minutes to dehydrate the gel column to prepare a gel micro-column for use. Take 0.5 mL of paclitaxel lipid core solution, drop it evenly on the upper end of the micro ^- column, centrifuge at 2000 rpm for 3 minutes, and collect the eluate. Then wash the gel micro ^- column with water, 0.5 mL each time, centrifuge at 2000 rpm for 3 minutes, wash three times in total, and collect the eluate. All eluates were combined, dissolved with methanol-glacial acetic acid (200:1), and diluted to an appropriate concentration, and the content of paclitaxel was determined by HPLC as the amount of encapsulated drug (W1). Another 0.5 mL of paclitaxel lipid core solution was taken, dissolved with methanol-glacial acetic acid (200:1), and diluted to an appropriate concentration. The content of paclitaxel was determined by HPLC as the total amount of paclitaxel in the lipid core solution before column centrifugation (W total)

Encapsulation rate % = W1/W total × 100%

(3) The drug encapsulation efficiency of the paclitaxel lipid core prepared by the method of Example 4: 96.0%

Example 6 Effect of adding cholesterol

The effects of adding different proportions of cholesterol on the preparation of paclitaxel lipid core were investigated.

Table 5. Formulation and characterization of cholesterol in different proportions

Preparation:

(1) Dissolve phospholipids (PC: 80-85%) and ascorbyl palmitate in methanol to obtain a clear and transparent solution, remove methanol by vacuum rotary evaporation in a water bath at 30-40°C, and collect the resulting precipitate, i.e., a mixed membrane material.

(2) Dissolving the mixed membrane material obtained in step 1, the recipe amount of paclitaxel, medium chain oil, and cholesterol in dichloromethane to obtain a uniform solution, and vacuum rotary evaporation in a water bath at 30 to 40 ° C to remove dichloromethane to obtain a lipid membrane Complex.

(3) The lipid membrane complex obtained in step 2 was placed in a water bath at 40-60° C., and 50 mL of phosphate buffer (pH=7) containing about 2.5% glycerol was slowly added under stirring to obtain a crude emulsion. The crude emulsion was homogenized under high pressure to obtain a negatively charged paclitaxel lipid core.

The results showed that adding the above different proportions of cholesterol could make the drug encapsulation rate greater than 90% and rich in negatively charged paclitaxel lipid core, indicating that adding cholesterol had no obvious effect on the preparation of paclitaxel lipid core.

Example 7 Comparison of different oil phase ratios

The effect of adding different proportions of oil on the preparation of paclitaxel lipid core was investigated.

Table 6 Formulation and characterization of different oil phase ratios

Preparation:

(1) Dissolve phospholipids (PC: 80-85%) and ascorbyl palmitate in methanol to obtain a clear and transparent solution, remove methanol by vacuum rotary evaporation in a water bath at 30-40°C, and collect the resulting precipitate, i.e., a mixed membrane material.

(2) Dissolving the mixed film material obtained in step 1, the recipe amounts of paclitaxel, medium chain oil, and cholesterol in dichloromethane to obtain a uniform solution, and vacuum rotary evaporation in a water bath at 30 to 40 ° C to remove dichloromethane to obtain a lipid film Complex.

(3) The lipid membrane complex obtained in step 2 was placed in a water bath at 40-60° C., and 50 mL of phosphate buffer (pH=7) containing about 2.5% glycerol was slowly added under stirring to obtain a crude emulsion. The crude emulsion was homogenized under high pressure to obtain a negatively charged paclitaxel lipid core.

The results showed that the above formulations with different proportions of the oil phase could produce the paclitaxel lipid core with a drug encapsulation rate greater than 90% and rich in negative charges.

Example 8 Comparison of different drug loadings

The effects of different drug loadings of paclitaxel on the encapsulation efficiency of lipid core drugs were investigated.

Table 7 Prescriptions with different drug loadings

Preparation:

(1) Dissolve phospholipids (PC: 80-85%) and ascorbyl palmitate in methanol to obtain a clear and transparent solution, remove methanol by vacuum rotary evaporation in a water bath at 30-40°C, and collect the resulting precipitate, i.e., a mixed membrane material.

(2) Dissolving the mixed membrane material obtained in step 1, the recipe amount of paclitaxel and medium chain oil in dichloromethane to obtain a uniform solution, vacuum rotary evaporation in a water bath of 30～40°C to remove dichloromethane to obtain a lipid membrane complex .

(3) The lipid membrane complex obtained in step 2 was placed in a water bath at 40-60° C., and 50 mL of phosphate buffer (pH=7) containing about 2.5% glycerol was slowly added under stirring to obtain a crude emulsion. The crude emulsion was homogenized under high pressure to obtain a negatively charged paclitaxel lipid core.

The results showed that the paclitaxel lipid core of prescription Z3 had drug precipitation after being placed at room temperature for 24 hours. The paclitaxel lipid cores of formulation Z1 and formulation Z2 have good stability and high encapsulation efficiency (greater than 80%).

The consumption of embodiment 9 chitosan

The star point design-response surface optimization method was used to investigate the effect of the amount of chitosan, including different chitosan concentrations and solution volumes, on the preparation of nanocapsules coated with the first layer of the capsule wall. The results were analyzed with Design-Expert 8.0 software.

(1) Preparation method of paclitaxel lipid core:

Table 8 Formulation composition and characterization

a) Dissolve the prescribed amount of phospholipids (PC: 80-85%) and ascorbyl palmitate in methanol to obtain a clear and transparent solution, remove methanol by vacuum rotary evaporation in a water bath at 30-40°C, and collect the resulting precipitate, that is, the mixed membrane material .

b) Dissolving the obtained mixed membrane material, paclitaxel and medium chain oil in dichloromethane to obtain a uniform solution, and vacuum rotary evaporation in a water bath at 30-40° C. to remove dichloromethane to obtain a lipid membrane complex.

c) The obtained lipid membrane complex was placed in a water bath at 40-60° C., and 50 mL of phosphate buffer (pH=7) containing about 2.5% glycerol was slowly added under stirring to obtain a crude emulsion. The crude emulsion was homogenized under high pressure to obtain a negatively charged paclitaxel lipid core.

(2) Preparation method of nanocapsules coated with the first layer of capsule wall: take an appropriate amount of paclitaxel lipid core solution with negative charge on the surface, and slowly dropwise add an appropriate amount of chitosan solution (0.005mol/L hydrochloric acid) of a certain concentration under stirring. After self-assembly, chitosan is adsorbed on the surface of the lipid core to obtain nanocapsules with positive charges on the surface.

(3) Prescription design and effect index of star point design-response surface optimization method:

Taking the central composite method (CCD) of the star point design-response surface optimization method as the model, two factors and five levels were used to design the prescription. Taking the chitosan concentration (X1) and the volume ratio of the chitosan solution to the lipid core solution (V chitosan: V lipid core) (X2) as the two factors investigated, five levels were selected for each factor, respectively. A total of 13 experiments were carried out, including 8 non-central points and 5 central points,

The factor level table is as follows:

Table 9 Star point design factor level table

Effect index and analysis method: The overall normalized value (OD) obtained by mathematical transformation of the particle size, PDI and surface potential of the prepared nanocapsules was used as the effect index, and the results were analyzed by Design-Expert 8.0 software.

Calculation of the overall normalized value (OD): First, the Hassan method is used to calculate the d value for the effect that the smaller the value is better and the effect that the larger the value is, the better. The smaller the particle size and PDI value, the better, and the surface potential The higher the value, the better. The overall normalized value (OD) is the geometric mean of the individual effects:

d _{particle size} =(y _max -y _i )/(y _max -y _min )

d _PDI =(y _max -y _i )/(y _max -y _min )

d _potential =(y _i -y _min )/(y _max -y _min )

Among them, y _max is the maximum value of the effect, y _min is the minimum value of the effect, and y _i is the current measurement value

Overall rating normalized value (OD) = geometric mean (d _{particle size} , d _PDI , d _potential )

(4) Results:

Table 10 Nanocapsule self-assembly prescription combination coated with chitosan

The results showed that the higher the concentration of chitosan in the formulation combination and the larger the volume ratio of the chitosan solution to the lipid core solution, the smaller the particle size of the prepared nanocapsules and the higher the positive surface charge. Therefore, the amount of chitosan can be changed according to the needs, including changing the concentration of chitosan and the volume of the solution to obtain nanocapsules with the desired particle size and potential that coat the first layer of the capsule wall.

A quadratic polynomial fitting was performed on the results obtained from 13 experiments, and the fitting mathematical model (Actual Equation) was:

_{OD = -6.601 + 32.072X1 +} ^{3.241X2-7.001X1X2-33.994X12-0.328X22 .}

The coefficient evaluation model (Coded Equation) is:

_{OD =} ^0.826 _{+ 0.224X1 + 0.266X2-0.175X1X2-0.085X12-0.082X22} ^.

The regression coefficient of quadratic polynomial fitting is R ² =0.9848, r=0.9924, and the P value of F test is less than 0.01, which is a better fitting model. Drawing the contour map of X ₁ , X ₂ with OD with Design-Expert 8.0 software, see Figure 3.

Comprehensive experimental measurements and contour maps, the preferred range is: the chitosan concentration is 0.15-0.3 mg/mL, and the volume ratio of the chitosan solution to the lipid core solution is 1.5-3.0. A more preferred range is: the chitosan concentration is 0.2-0.3 mg/mL, and the volume ratio of the chitosan solution to the lipid core solution is 2.0-3.0.

After conversion, the preferred range in terms of mass ratio is: the amount of chitosan per gram of phospholipid is 0.015-0.045 grams, more preferably 0.01875-0.03125 grams.

Example 10 Consumption of low molecular weight heparin

The effect of the dosage of low molecular weight heparin, including the concentration and solution volume of different low molecular weight heparin, on the preparation of composite lipid nanocapsules coated with the second layer of capsule wall was investigated.

(1) the preparation method of the nanocapsule covering the first layer of capsule wall:

Table 11 Formulation composition and characterization

a) Dissolve the prescribed amount of phospholipids (PC: 80-85%) and ascorbyl palmitate in methanol to obtain a clear and transparent solution, remove methanol by vacuum rotary evaporation in a water bath at 30-40°C, and collect the resulting precipitate, that is, the mixed membrane material .

b) Dissolving the obtained mixed membrane material, paclitaxel and medium chain oil in dichloromethane to obtain a uniform solution, and vacuum rotary evaporation in a water bath at 30-40° C. to remove dichloromethane to obtain a lipid membrane complex.

c) The obtained lipid membrane complex was placed in a water bath at 40-60° C., and 50 mL of phosphate buffer (pH=7) containing about 2.5% glycerol was slowly added under stirring to obtain a crude emulsion. The crude emulsion was homogenized under high pressure to obtain a negatively charged paclitaxel lipid core.

d) Take 1 part of paclitaxel lipid core solution, slowly dropwise add 2.5 parts of 0.20mg/mL chitosan solution (0.005mol/L hydrochloric acid as solvent) under stirring, after self-assembly, chitosan is adsorbed on On the surface of the lipid core, the lipid core is coated with the first layer of capsule wall, thereby obtaining the surface-charged nanocapsules (CS-NCs) coated with the first layer of capsule wall.

(2) Preparation method of composite lipid nanocapsules coated with the second layer of capsule wall: take an appropriate amount of CS-NCs solution with positive charges on the surface, and slowly dropwise add an appropriate amount of low molecular weight heparin solution (pH 7.5) with a certain concentration under stirring ), after self-assembly, low molecular weight heparin is adsorbed on the surface of the nanocapsules, so that the nanocapsules are covered with a second layer of capsule wall, thereby obtaining paclitaxel composite lipid nanocapsules.

(3) Prescription design and effect index of star point design-response surface optimization method:

Taking the central composite method (CCD) of the star point design-response surface optimization method as the model, two factors and five levels were used to design the prescription. Taking the concentration of low molecular weight heparin and the volume ratio of low molecular weight heparin solution to CS-NCs nanocapsule solution (V _{low molecular weight heparin} : V _CS-NCs ) as two factors, five levels were selected for each factor, and a total of 13 experiments were carried out. In the experiment, there are 8 non-central points and 5 central points. The particle size, PDI and potential of the prepared composite lipid nanocapsules were used as effect indicators.

The factor level table is as follows:

Table 12 Star point design factor level table

(4) Investigation results of star point design-effect surface optimization method

Table 13. Recipe combination for self-assembly of composite lipid nanocapsules coated with low molecular weight heparin

The results showed that different concentrations of low molecular weight heparin and different volume ratios of low molecular weight heparin solution and CS-NCs nanocapsule solution in the formula combination designed by Star Point could produce composites with smaller particle size and higher negative surface charge. For lipid nanocapsules, there was no significant difference between the formulations. Therefore, the dosage of low-molecular-weight heparin can be changed according to requirements, including changing the concentration and solution volume of low-molecular-weight heparin to prepare the desired composite lipid nanocapsules coating the second layer of capsule wall.

The investigated low molecular weight heparin concentration was 0.05-0.35 mg/mL, and the volume ratio of the low molecular weight heparin solution to the chitosan-coated nanocapsule solution was 1.25-2.75.

(5) Continue to change the concentration and solution volume of low molecular weight heparin:

Table 14. Recipe combination for self-assembly of composite lipid nanocapsules coated with low molecular weight heparin

The results showed that when the concentration of low molecular weight heparin was 0.06-0.08 mg/mL, and the volume ratio of low molecular weight heparin solution to chitosan-coated nanocapsule solution was 1.0-1.2, smaller particle size and higher surface band could still be obtained. Negatively charged complex lipid nanocapsules.

Based on the experimental results of (4) and (5), the investigated low molecular weight heparin concentration is 0.05-0.35 mg/mL, and the volume ratio of low molecular weight heparin solution to chitosan-coated nanocapsule solution is 1.00-2.75. After conversion, the preferred range according to the mass ratio is: the amount of low molecular weight heparin per gram of chitosan is 0.42-5.25 grams.

Example 11 Preparation and characterization of paclitaxel complex lipid nanocapsules

Formulation composition of table 15 complex lipid nanocapsules

(1) Preparation method:

(1) Dissolve the phospholipids (PC: 80～85%) and ascorbyl palmitate of the recipe amount in methanol to obtain a clear and transparent solution, remove the methanol by vacuum rotary evaporation in a water bath at 30～40°C, and collect the obtained precipitate, that is, a mixed membrane material.

(2) Dissolving the mixed membrane material, paclitaxel and medium chain oil obtained in step 1 in dichloromethane to obtain a uniform solution, and vacuum rotary evaporation in a water bath at 30-40° C. to remove dichloromethane to obtain a lipid membrane complex.

(3) The lipid membrane complex obtained in step 2 was placed in a water bath at 40-60° C., and 50 mL of phosphate buffer (pH=7) containing about 2.5% glycerol was slowly added under stirring to obtain a crude emulsion. The crude emulsion was homogenized under high pressure to obtain a negatively charged paclitaxel lipid core.

(4) Take 1 part of paclitaxel lipid core solution, slowly dropwise add 2.5 parts of 0.20 mg/mL chitosan solution (0.005 mol/L hydrochloric acid as solvent) under stirring, after self-assembly, chitosan adsorption On the surface of the lipid core, the lipid core is coated with the first layer of vesicle wall to obtain the surface-positively charged nanocapsules (CS-NCs)

(5) Take 1 part of the CS-NCs solution with a positive charge on the surface, and slowly add it dropwise to 1.2 parts of a 0.08 mg/mL low molecular weight heparin solution (pH 7.5) under stirring. After self-assembly, the low molecular weight heparin adsorbs On the surface of the nanocapsules, the nanocapsules are coated with a second layer of capsule wall, thereby obtaining the paclitaxel complex lipid nanocapsules (LH-NCs).

(2) Encapsulation rate determination method:

The drug encapsulation efficiency of nanocapsules (CS-NCs) and composite lipid nanocapsules (LH-NCs) coated with the first layer of capsule wall was determined by low-speed centrifugation, as follows:

(1) HPLC chromatographic conditions: ZORBAX Eclipse XDB-C18 (4.6mm×150mm, 3.5μm) as the chromatographic column; the mobile phase is methanol-water-acetonitrile (23:41:36): the detection wavelength is 227nm, and the column temperature is 35 ℃, the flow rate was 1.2 mL per minute, and the injection volume was 50 μL.

(2) Separation of encapsulated and free drugs by low-speed centrifugation: take 0.5 mL of CS-NCs solution or paclitaxel complex lipid nanocapsule solution, dilute to 10 mL with water, shake well, take about 8 mL, and place at 1000 rpm- ¹ Centrifuge for 10 minutes to pellet unencapsulated paclitaxel crystal aggregates. Then take 2mL of supernatant to a 25mL volumetric flask, add 12mL of methanol-glacial acetic acid (200:1) to complete the dissolution, then dilute to the mark with mobile phase, shake well, and measure the content of paclitaxel by HPLC, which is taken as the amount of encapsulated drug E1 . Another 2 mL of the solution before centrifugation was taken, and the same method was used to determine the paclitaxel content by HPLC, which was taken as the total amount of paclitaxel in the nanocapsule solution before centrifugation E0. The encapsulation rate was calculated according to the following formula.

Encapsulation rate%=E1/E0×100%

(3) Results:

Table 16 Characterization of composite lipid nanocapsules

The results show that in the preparation process of the composite lipid nanocapsules, with the increase of the number of layers of the lipid core coating the capsule wall, the particle size increases slightly, the surface charge of the particle is in the form of alternating positive and negative charges, and the drug encapsulation efficiency is greater than 90%. , showing that paclitaxel complex lipid nanocapsules can be successfully prepared by electrostatic adsorption of positive and negative charges. The particle size distribution diagram and transmission electron microscope diagram of the composite lipid nanocapsules are shown in Figure 4 and Figure 5, respectively.

Example 12 Preparation and characterization of paclitaxel composite lipid nanocapsule lyophilized powder

Preparation method: Take the paclitaxel composite lipid nanocapsule solution (LH-NCs) prepared by the method in Example 11 of the present invention, add 6 g of freeze-dried proppant trehalose per 100 mL to dissolve completely, shake well, and freeze-dry to obtain a composite Lyophilized powder of lipid nanocapsules.

The freeze-drying process is shown in the table below.

Table 17 Freeze drying process

Characterization method: Take an appropriate amount of paclitaxel complex lipid nanocapsule lyophilized powder, redissolve it in normal saline and measure it.

Results: The average particle size was 159.2±15.6nm, the polydispersity coefficient (PDI) was 0.273±0.013, the Zeta potential was -53.96±5.67mV, and the drug encapsulation efficiency was 99.47±1.04%. The TEM image is shown in Figure 6.

Example 13 Release degree of composite lipid nanocapsules

Take the paclitaxel composite lipid nanocapsule lyophilized powder prepared by the method of Example 12 of the present invention to investigate the drug release characteristics

Using the shaking dialysis method, take an appropriate amount of paclitaxel complex lipid nanocapsule lyophilized powder, reconstitute it with physiological saline, and shake well to prepare a complex lipid nanocapsule solution with a paclitaxel concentration of about 0.12 mg/mL, and take 1 mL of it and place it in a dialysis bag , seal. The dialysis bag was placed in 50 mL of release medium at 37°C with shaking at a rate of 100 times per minute. Take out 1 mL of release medium at 0.5, 1, 2, 4, 8, 12, 24, 36, and 48 hours, respectively, and add 1 mL of fresh medium at the same time. The drug content of the taken-out release medium was determined by the HPLC method described in Example 11 of the present invention, and the cumulative drug release rate was calculated. The release curves of the composite lipid nanocapsules in two release media were investigated respectively. The release media were: pH 5.0 phosphate buffer (containing 0.5% Tween 80) and pH 7.4 phosphate buffer (containing 0.5% Tween 80). temperature 80).

Drug cumulative release rate = (cumulative drug release/total drug amount) × 100%

The results showed that the cumulative drug release rate of the paclitaxel composite lipid nanocapsules was 91.5% in pH 5.0 medium and 82.2% in pH 7.4 medium. The release curve is shown in Figure 7.

DDSolver 1.0 software was used to fit the mathematical model of the release curve. The release curves at pH 5.0 and pH 7.4 were the most consistent with the classical Higuchi diffusion equation. The results are shown in the following table.

Table 18 Fitting of Release Curves

The results showed that the drug was mainly affected by its own diffusion behavior during the drug release process of the paclitaxel composite lipid nanocapsules, which belonged to the diffusion-controlled release mechanism.

Example 14 In vivo tumor targeting distribution of complex lipid nanocapsules in tumor-bearing mice

Composite lipid nanocapsules were prepared with fluorescent dye DiR, and the in vivo tumor targeting distribution of composite lipid nanocapsules was investigated by in vivo imaging technology of small animals.

(1) Preparation of DiR composite lipid nanocapsules

Table 19 Formulation composition of DiR composite lipid nanocapsules

(1) Dissolve phospholipids (PC≥98%) and ascorbyl palmitate in methanol to obtain a clear and transparent solution, remove methanol by vacuum rotary evaporation in a 30-40°C water bath, and collect the resulting precipitate, i.e., the mixed membrane material.

(2) Dissolve the mixed membrane material, DiR and medium chain oil obtained in step 1 in dichloromethane to obtain a uniform solution, and remove dichloromethane by vacuum rotary evaporation in a 30-40° C. water bath to obtain a lipid membrane complex.

(3) The lipid membrane complex obtained in step 2 was placed in a water bath at 40-60° C., and 50 mL of phosphate buffer (pH=7) containing about 2.5% glycerol was slowly added under stirring to obtain a crude emulsion. The crude emulsion was homogenized under high pressure to obtain a solution of DiR lipid nanoparticles with negative surface charges, namely the lipid core.

(4) Take 1 part of lipid core solution, slowly dropwise add 2.5 parts of 0.20 mg/mL chitosan solution (0.005 mol/L hydrochloric acid as solvent) under stirring, after self-assembly, chitosan is adsorbed on lipid The surface of the cytoplasmic core was coated with the first layer of vesicle wall to obtain the surface-positively charged nanocapsules (DiR-CS-NCs).

(5) Take 1 part of DiR-CS-NCs solution with a positive charge on the surface, and slowly add it dropwise to 1.2 parts of 0.08 mg/mL low molecular weight heparin solution (pH 7.5) under stirring. After self-assembly, low molecular weight Heparin was adsorbed on the surface of the nanocapsules, so that the nanocapsules were coated with the second layer of capsule wall, thereby obtaining DiR composite lipid nanocapsules (DiR-LH-NCs) with negative charges on the surface.

Characterization results:

Table 20 Characterization of DiR complex lipid nanocapsules

(2) In vivo tumor targeting distribution in tumor-bearing mice

Establishment of 4T1 tumor-bearing mouse model: Balb/c mice, female, body weight 17±2g. Each mouse was in situ inoculated with 1×10 ⁶ 4T1 breast cancer cells in the fat pad of the fourth mammary gland, and fed normally. After the tumor volume grew to about 800 mm ³ , it was used for the experiment.

Experimental group: The 4T1 model mice were randomly divided into 3 groups, one in each group, namely DiR solution group (2% DMSO-water as solvent), DiR lipid core group and DiR composite lipid nanocapsule group. Each group was administered by tail vein injection at a dose of 0.1 mg/kg DiR. After administration, IVIS imaging was performed using a small animal in vivo imaging system at 2, 4, 8, 12, and 24 hours, respectively, and the tumor was quickly removed after 24 hours. Tissue, luminescence detection was performed, and the tumor luminescence intensity of each group was recorded.

The results showed that the fluorescence intensity of tumor tissue in the DiR composite lipid nanocapsule group and the lipid core group was significantly higher than that in the solution group, as shown in Figure 8. After the 24-hour test, the tumor tissue was taken out, as shown in Figure 9, the luminescence values of each group They are: DiR solution group (8.314×10 ⁸ p/s), DiR lipid core group (8.764×10 ⁹ p/s), DiR complex lipid nanocapsule group (1.073×10 ¹⁰ p/s), showing Good tumor targeting distribution of composite lipid nanocapsules and lipid cores.

Claims (10)

1. A composite lipid nanocapsule composition comprising the following components: oil, phospholipid, ascorbyl palmitate, antitumor compound, chitosan, low molecular weight heparin; the composite lipid nanocapsule The diameter ranges from 10 to 1000 nm. 2 . The composite lipid nanocapsule composition according to claim 1 , wherein the composite lipid nanocapsule composition can also contain cholesterol. 3 . 3. The composite lipid nanocapsule composition according to any one of claims 1-2, wherein the composite lipid nanocapsule composition is composed of a negatively charged lipid core on the surface, a positively charged first A layer of capsule wall and a second layer of capsule wall with negative charges are formed by electrostatic adsorption between positive and negative charged groups; the lipid core with negative charges on the surface includes the following components: lipids, phospholipids, Ascorbyl palmitate, an antitumor compound; the positively charged first layer capsule wall comprises chitosan; the negatively charged second layer capsule wall comprises low molecular weight heparin; the surface negatively charged lipid The core may also contain cholesterol. 4. The composite lipid nanocapsule composition according to claim 1, wherein the amount of ascorbyl palmitate per gram of phospholipid is 0.001 to 100 grams; the amount of oil and fat per gram of phospholipid is 0.001 to 100 milliliters; The amount of chitosan per gram of phospholipids is 0.001-100 grams; the amount of low molecular weight heparin per gram of chitosan is 0.001-100 grams. 5. The composite lipid nanocapsule composition according to claim 1, wherein the oil is one or more oils of natural or synthetic origin, including medium chain triglycerides or soybean oil; the phospholipid is One or more phospholipids of natural or synthetic origin, including lecithin; the low-molecular-weight heparin is a kind of heparin with lower molecular weight and its salts prepared after depolymerization; the anti-tumor compound includes taxus Alkane compounds. 6 . The composite lipid nanocapsule composition according to claim 5 , wherein the taxane compound comprises paclitaxel; the amount of paclitaxel per gram of phospholipid is 0-100 grams. 7 . 7. the preparation method of the composite lipid nanocapsule composition described in any one of claim 1-6, is characterized in that, described lipid core with negative surface charge comprises the following preparation steps: (1) phospholipid and ascorbyl palmitate are dissolved in organic solvent A to obtain a homogeneous solution, and organic solvent A is removed to obtain a mixed membrane material; (2) dissolving the mixed membrane material, grease and antitumor compound obtained in step 1 in organic solvent B to obtain a uniform solution, and removing organic solvent B to obtain a lipid membrane complex; (3) Dispersing the lipid membrane complex obtained in step 2 in an aqueous phase, emulsification and homogenization, thereby obtaining lipid nanoparticles with negative surface charges, that is, the negatively charged lipid cores on the surface. 8. the preparation method of the composite lipid nanocapsule described in any one of claim 1-6, it is characterized in that, described by the lipid core with negative charge on the surface, the first layer capsule wall with positive charge, the The second layer of the negatively charged capsule wall, and the composite lipid nanocapsule formed by the combination of the three through electrostatic adsorption between the positive and negative charged groups includes the following preparation steps: (1) Disperse the negatively charged lipid core in the aqueous phase containing chitosan, and after self-assembly, the chitosan is adsorbed on the surface of the lipid core, so that the lipid core is coated with the first layer of capsule wall , so as to obtain nanocapsules with positive charges on the surface; (2) Disperse the nanocapsules with positive charges on the surface obtained in step 1 in an aqueous phase containing low molecular weight heparin, and through self-assembly, the low molecular weight heparin is adsorbed on the surface of the nanocapsules, so that the nanocapsules are covered with a second layer of capsules wall to obtain the composite lipid nanocapsules. 9. preparation method according to claim 7, is characterized in that, described organic solvent A includes but not limited to one or more in methanol, ethanol, tetrahydrofuran; Described organic solvent B includes but not limited to chloroform, dichloromethane, One or more of tetrahydrofuran, n-hexane, cyclohexane, ethyl acetate, petroleum ether, methanol and ethanol. 10. The application of the composite lipid nanocapsule composition according to any one of claims 1-6 in the preparation of antitumor drugs.

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