Complex lipid nanoparticle compositions, methods of making and using the same

Abstract

The application discloses a kind of composite lipid nanocapsule compositions and its preparation method and application, belong to medical technical field.The composite lipid nanocapsule composition is by the lipid core with negative charge on the surface, the first layer of capsule wall with positive charge, the second layer of capsule wall with negative charge, three are combined by electrostatic adsorption between positive and negative charge group Formed, can be used for intravenous injection.Lipid core is the depot for loading antitumor drug, chitosan has the effect of promoting transmembrane transport and controlling drug release, and the low molecular heparin coated outside nanocapsule can be reacted with heparinase, so that nanocapsule is enriched and degraded in malignant tumor site, realizes the function of targeting tumor.In addition, the carrier material used in the composition has high biological safety, good tumor targeting, high drug encapsulation efficiency, good stability, controllable release, and the preparation method is simple, and it has wide application prospect when used for the targeted delivery of antitumor drugs.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical and nanomedicine technology, and relates to a composite lipid nanocapsule composition, its preparation method and application. Background Technology

[0002] Malignant tumors are among the leading causes of death and threats to human health. Chemotherapy is a conventional treatment for malignant tumors, but chemotherapy drugs generally suffer from drawbacks such as lack of selectivity, significant toxic side effects, and the easy development of drug resistance in cancer cells, resulting in poor patient tolerance. Furthermore, many antitumor drugs also exhibit poor water solubility and instability. Therefore, it is necessary to improve the physicochemical properties of drugs through formulation technology, particularly by leveraging the biological characteristics of tumors and their microenvironment to enhance tumor targeting, reduce toxic side effects, and improve the efficacy of tumor treatment.

[0003] Nanocapsules are drug reservoir-type nanoparticles that encapsulate solid or liquid drugs as a core. They are a class of small-sized nanoscale drug carrier systems (10–1000 nm) consisting of a thin layer of natural or synthetic polymer film encapsulating an oily or aqueous core. As a drug carrier, in addition to improving the conventional physicochemical properties of drugs such as solubility and dispersibility, they also have the following advantages: 1. Improved drug stability and reduced impact of environmental factors on drugs; 2. Imparted sustained-release properties to drugs, effectively controlling drug release; 3. Passive targeting capability; after surface modification, the capsule material can also be positioned in specific tissues to achieve active targeting; 4. Due to the extremely small size of nanoparticles, they can be used for intravenous injection without causing vascular embolism.

[0004] Based on the above advantages, nanocapsules have broad prospects for anti-tumor drug delivery. However, the nanocapsule formulations that have been studied extensively still have some problems, such as poor biocompatibility of carrier materials, poor tumor targeting of formulations, unsatisfactory drug release, poor controllability of preparation processes, and unstable quality.

[0005] A microcapsule has been disclosed in the prior art, as described in the master's thesis "Preparation and Performance Study of Layer-by-Layer Self-Assembled Heparin / Chitosan Microcapsules" (Liang Yan, 2014, Jiangnan University, China). This microcapsule is prepared by repeatedly and alternately electrostatically adsorbing negatively charged natural polysaccharide heparin and positively charged natural polysaccharide chitosan onto the surface of a core CaCO3 template using a layer-by-layer self-assembly technique, followed by removal of the CaCO3 template. The results show that this microcapsule exhibits good biocompatibility and stability. However, because the microcapsule particle size obtained by this technique is approximately 4–5 μm, it is not suitable for intravenous injection and has insufficient targeted drug delivery capability, limiting its clinical application. Summary of the Invention

[0006] The technical problem solved by this invention is to provide a composite lipid nanocapsule composition with the following properties: the polymer and functional materials used have good biocompatibility and are safe and non-toxic; the particle size is in the nanoscale (10-1000 nm), which can be used for intravenous injection; it has good tumor targeting; the drug encapsulation rate is high and the stability is good; and the preparation method provided is simple and feasible.

[0007] Another technical problem solved by the present invention is to provide the application of the composite lipid nanocapsule composition.

[0008] The technical problem solved by this invention is achieved through the following technical solution:

[0009] The first aspect of this invention is to provide a composition of composite lipid nanocapsules, the composition comprising the following components: oils, phospholipids, ascorbate palmitate, antitumor compounds, chitosan, and low molecular weight heparin. The composite lipid nanocapsules have a particle size range of 10–1000 nm, preferably between 10–500 nm, and more preferably between 10–200 nm. The particle size refers to the intensity-weighted mean diameter (IMD) measured using a laser particle size analyzer.

[0010] The composite lipid nanocapsule composition may or may not contain cholesterol.

[0011] The composite lipid nanocapsules consist of a negatively charged lipid core, a positively charged first layer of capsule wall, and a negatively charged second layer of capsule wall, which are bonded together by electrostatic adsorption between positively and negatively charged groups (see Appendix). Figure 1 (as shown); the negatively charged lipid core comprises the following components: oils, phospholipids, ascorbate palmitate, and antitumor compounds; the positively charged first capsule wall comprises chitosan; the negatively charged second capsule wall comprises low molecular weight heparin; the negatively charged lipid core may or may not contain cholesterol.

[0012] Furthermore, in the composite lipid nanocapsule composition, the amount of ascorbate palmitate relative to each gram of phospholipid is 0.001-100 g, preferably 0.02-0.12 g; the amount of oil relative to each gram of phospholipid is 0.001-100 mL, preferably 0.8-1.2 mL; the amount of chitosan relative to each gram of phospholipid is 0.001-100 g, preferably 0.015-0.045 g, more preferably 0.01875-0.03125 g; and the amount of low molecular weight heparin relative to each gram of chitosan is 0.001-100 g, preferably 0.42-5.25 g.

[0013] Furthermore, in the aforementioned composite lipid nanocapsule composition, the oil is one or more oils of natural or synthetic origin, preferably including medium-chain triglycerides (referred to as medium-chain oils) or soybean oil; the phospholipid is one or more phospholipids of natural or synthetic origin, preferably including lecithin (the main component of which is phosphatidylcholine, PC), more preferably having a phosphatidylcholine content of greater than or equal to 80%; the chitosan is preferably low molecular weight chitosan; the low molecular weight heparin is a class of heparin and its salts prepared by depolymerization, preferably including low molecular weight heparin sodium, more preferably including enoxaparin sodium.

[0014] Furthermore, in the composite lipid nanocapsule composition, the antitumor compound includes taxane compounds, and more specifically, the taxane compounds include paclitaxel; the amount of paclitaxel relative to each gram of phospholipid is 0 to 100 grams, preferably 0.02 to 0.03 grams.

[0015] Furthermore, the composite lipid nanocapsule composition is prepared as described in the second aspect of the present invention. The second aspect of the present invention provides a method for preparing the composite lipid nanocapsule composition described in the first aspect, wherein the negatively charged lipid core comprises the following preparation steps:

[0016] (1) Dissolve phospholipids and ascorbate palmitate in organic solvent A to obtain a homogeneous solution, remove organic solvent A to obtain a mixed membrane material;

[0017] (2) Dissolve the mixed membrane material, oil and antitumor compound obtained in step 1 in organic solvent B to obtain a homogeneous solution, remove organic solvent B to obtain lipid membrane complex;

[0018] (3) The lipid membrane complex obtained in step 2 is dispersed in an aqueous phase, emulsified and homogenized to obtain lipid nanoparticles with negative surface charge, namely the lipid core with negative surface charge.

[0019] In the negatively charged lipid core component, the amount of ascorbate palmitate relative to each gram of phospholipid is 0.001 to 100 grams, preferably 0.02 to 0.12 grams; and the amount of oil relative to each gram of phospholipid is 0.001 to 100 milliliters, preferably 0.8 to 1.2 milliliters.

[0020] Furthermore, the antitumor compound includes taxane compounds, and more specifically, the taxane compound includes paclitaxel; the amount of paclitaxel relative to each gram of phospholipid is 0 to 100 grams, preferably 0.02 to 0.03 grams.

[0021] Furthermore, 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.

[0022] Furthermore, the aqueous phase used to disperse the lipid film complex may or may not contain glycerol.

[0023] Furthermore, the methods for removing organic solvent A and organic solvent B include, but are not limited to, vacuum drying, solvent evaporation, rotary evaporation, spray drying, and freeze drying.

[0024] The method for preparing the composite lipid nanocapsule composition, wherein the composite lipid nanocapsule formed by the combination of a negatively charged lipid core, a positively charged first layer of capsule wall, and a negatively charged second layer of capsule wall through electrostatic adsorption between positively and negatively charged groups includes the following preparation steps:

[0025] (1) A negatively charged lipid core is dispersed in an aqueous phase containing chitosan. After self-assembly, chitosan is adsorbed onto the surface of the lipid core, so that the lipid core is coated with the first layer of capsule wall, thereby obtaining a positively charged nanocapsule.

[0026] (2) The positively charged nanocapsules obtained in step 1 are dispersed in an aqueous phase containing low molecular weight heparin. After self-assembly, the low molecular weight heparin is adsorbed onto the surface of the nanocapsules, so that the nanocapsules are coated with a second layer of capsule wall, thereby obtaining the composite lipid nanocapsules.

[0027] The aqueous phase containing chitosan has a chitosan concentration of 0.001–100 mg / mL, preferably 0.15–0.3 mg / mL, more preferably 0.2–0.3 mg / mL, and even more preferably 0.2 mg / mL. By volume ratio, the chitosan solution is 0.001–10000 parts per part of lipid core solution, preferably 1.5–3.0 parts, more preferably 2.0–3.0 parts, and even more preferably 2.5 parts. By mass ratio, the amount of chitosan per gram of phospholipid is 0.001–100 g, preferably 0.015–0.045 g, more preferably 0.01875–0.03125 g, and even more preferably 0.025 g.

[0028] The aqueous phase containing low molecular weight heparin has a low molecular weight heparin concentration of 0.001–100 mg / mL, preferably 0.05–0.35 mg / mL, and more preferably 0.08 mg / mL. By volume ratio, the low molecular weight heparin solution is 0.001–10,000 parts per part of positively charged chitosan nanocapsule solution, preferably 1.00–2.75 parts, and more preferably 1.2 parts. By mass ratio, the low molecular weight heparin is 0.001–100 g per gram of chitosan, preferably 0.42–5.25 g, and more preferably 0.672 g.

[0029] Furthermore, the aqueous phase containing chitosan, and the medium used to adjust its acidity, include, but are not limited to, acetic acid, hydrochloric acid, and phosphoric acid, preferably hydrochloric acid, and more preferably, the concentration of hydrochloric acid in the aqueous phase is 0.005 mol / L.

[0030] The composite lipid nanocapsule composition can be formulated into various dosage forms containing composite lipid nanocapsules, including but not limited to injections, lyophilized powders, and gels. The drugs in all the above dosage forms can be prepared according to conventional methods in the pharmaceutical field, preferably injections or lyophilized powders.

[0031] The third aspect of the present invention is to provide the application of the above-mentioned composite lipid nanocapsule composition in the preparation of antitumor drugs, wherein 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-cell lymphoma.

[0032] Beneficial technical effects

[0033] Compared with the prior art, the present invention has the following beneficial technical effects:

[0034] The composite lipid nanocapsule composition provided by this invention possesses multiple properties, including improved drug stability, suitability for intravenous injection, sustained-release properties, active targeting of tumor sites, reduced toxicity to normal tissues, and enhanced tumor treatment efficacy. The advantages of this invention are as follows:

[0035] 1. The lipid composite nanocapsules prepared by this invention are small-sized particles with a particle size in the nanometer range, which can improve the dispersibility, permeability and stability of drugs, and can be used for intravenous injection. They have the EPR effect (enhanced permeability and retention effect) of solid tumors and are excellent carriers for targeted delivery of anti-tumor drugs.

[0036] 2. The lipid core with a negatively charged surface prepared by the present invention is an oil, which can load hydrophobic drugs and improve the solubility of the drugs.

[0037] 3. The lipid core with a negatively charged surface prepared in this invention contains phospholipids as the membrane material. Lecithin's main component is phosphatidylcholine; using it alone as the membrane material results in a low and uncontrollable negative charge density at the nanoparticle interface, which is detrimental to the subsequent preparation of nanocapsules. This invention discovers that ascorbyl palmitate (AP, chemical structure shown in Appendix)... Figure 2 One side of the membrane is a lipophilic palmitate, and the other side is a hydrophilic ascorbic acidic polyhydroxy group. After hydrolysis, it carries a negative charge. When ascorbate palmitate is mixed with phospholipids to form a membrane material, it can significantly enhance the negative charge density on the surface of the lipid core and improve the stability of the lipid core. This is of great importance for the subsequent electrostatic adsorption and coating of the positively charged capsule wall.

[0038] 4. The composite lipid nanocapsules prepared in this invention exhibit active tumor-targeting activity. Low-molecular-weight heparin (LMWH) is a general term for a class of heparins with lower molecular weights produced by depolymerization. It has a narrow average molecular weight distribution range and fewer side effects compared to undepolymerized heparin. Heparanase (HPA) is an endogenous endo-β-D-glucuronide endonuclease that can specifically cleave heparan sulfate proteoglycans on the extracellular matrix. Numerous studies have found that the overexpression of heparanase is closely related to tumor development, invasion, and metastasis, making it an important molecular target associated with tumors. Heparin and its cleavage product, low-molecular-weight heparin, as substrates for heparanase, can bind to heparanase through enzyme-substrate interactions, inhibiting the degradation of endogenous heparan sulfate proteoglycans and exhibiting pharmacological effects such as anti-tumor metastasis, anti-inflammation, anticoagulation, and antithrombosis. This invention encapsulates low molecular weight heparin on the outer side of a composite lipid nanocapsule. By utilizing the interaction characteristics of heparin with heparinase, the nanocapsule is enriched and degraded at the site of malignant tumors, thus exhibiting an active targeting effect on tumors and their microenvironment.

[0039] 5. The composite lipid nanocapsules provided by this invention possess multifunctional synergistic effects. These composite lipid nanocapsules consist of three combined parts: a lipid core serving as a drug reservoir; chitosan promoting transmembrane transport and controlling drug release; and low-molecular-weight heparin actively targeting malignant tumor sites with high heparinase expression. After entering systemic circulation, these composite lipid nanocapsules are more readily accumulated and degraded at tumor sites compared to normal tissues. The exposed chitosan-coated nanocapsules facilitate uptake by tumor cells, leading to intracellular drug release. Through the synergistic effect of multiple functional materials, the therapeutic effect on tumors is enhanced, toxic side effects are reduced, and patient tolerance is improved.

[0040] 6. The composite lipid nanocapsules provided by this invention exhibit excellent drug encapsulation efficiency, stability, and controllable release performance. The drug is encapsulated within the lipid core, unaffected by the functional materials of the capsule wall or external environmental interference, thus improving drug encapsulation efficiency and stability. This invention utilizes chitosan, which has a strong positive charge. Chitosan can tightly bind to the negatively charged lipid core and low-molecular-weight heparin, which is beneficial for improving the stability of the nanocapsules in vivo circulation. Furthermore, because chitosan carries a strong positive charge, it can interact with various drug molecules. This invention first prepares drug-loaded lipid nanoparticles (lipid core) and then coats them with chitosan. The drug does not directly contact the chitosan, eliminating interference with the drug release. The drug release rate can be adjusted simply by changing the amount of chitosan adsorbed on the lipid core surface, making drug release more controllable.

[0041] 7. The carrier material used in the composite lipid nanocapsules provided by this invention is mainly of natural origin, has good biocompatibility, and is safe and non-toxic.

[0042] 8. The composite lipid nanocapsule preparation process provided by this invention is simple and feasible, which is conducive to industrial production and meets the needs of clinical treatment. Attached Figure Description

[0043] Figure 1 Schematic diagram of the preparation method of composite lipid nanocapsules

[0044] Figure 2 The structure of ascorbyl palmitate

[0045] Figure 3 The figure shows the factor-level contour lines of the star point design-effect surface optimization method in Embodiment 9 of the present invention.

[0046] Figure 4 The particle size distribution of the composite lipid nanocapsules of Example 11 of the present invention is shown.

[0047] Figure 5 The image shown is a transmission electron microscope (TEM) image of the composite lipid nanocapsules of Example 11 of the present invention.

[0048] Figure 6 The image shown is a transmission electron microscope (TEM) image of the lyophilized powder of the composite lipid nanocapsules of Example 12 of the present invention.

[0049] Figure 7 The release curve of the composite lipid nanocapsules of Example 13 of the present invention is shown.

[0050] Figure 8 The graph shows the in vivo tumor-targeting distribution over time in tumor-bearing mice injected with composite lipid nanocapsules according to Example 14 of the present invention. A: Ordinary solution group; B: Lipid core group; C: Composite lipid nanocapsule group.

[0051] Figure 9The fluorescence intensity of tumor tissue in tumor-bearing mice 24 hours after injection of the composite lipid nanocapsules is shown in Example 14 of this invention. Detailed Implementation

[0052] The present invention will be further described below with reference to specific embodiments, but these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that changes or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such changes and substitutions fall within the protection scope of the present invention.

[0053] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0054] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. Instruments:

[0055] Experimental Ultra-High Pressure Homogenizer: Nano DeBEE, BEE Corporation, USA

[0056] Laser particle size potential analyzer: Nicomp 380ZLS, PSS Corporation, USA

[0057] Reagents:

[0058] Phospholipids: PC-98T, PC≥98%, Shanghai Aivito Pharmaceutical Technology Co., Ltd.

[0059] LIPOID E80, PC: 80-85%, Lipoid GmbH, Germany

[0060] Chitosan: Low molecular weight, Sigma-Aldrich

[0061] Low molecular weight heparin: Enoxaparin sodium, Hebei Changshan Biochemical Pharmaceutical Co., Ltd.

[0062] DiR: DiR iodide, a near-infrared fluorescent anthocyanin dye for cell membranes, developed by AAT Bioquest, USA.

[0063] Example 1: The effect of ascorbate palmitate on regulating surface negative charge

[0064] Lipid cores containing different proportions of ascorbyl palmitate were prepared and compared with ordinary liposomes without ascorbyl palmitate.

[0065] Table 1. Formulations and characterizations of different ascorbyl palmitate ratios

[0066]

[0067] 1. Preparation method of prescription A1 (ordinary liposomes):

[0068] (1) Dissolve the prescribed amount of phospholipids (PC: 80-85%) and cholesterol in chloroform-methanol (88: 12) to obtain a clear and transparent solution. Remove the organic solvent by rotary evaporation to obtain a lipid film.

[0069] (2) The lipid membrane 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 with stirring to obtain a crude emulsion. The crude emulsion was homogenized under high pressure to obtain liposomes.

[0070] 2. Preparation methods of prescriptions A2 to A5:

[0071] (1) Dissolve the prescribed amount of phospholipid (PC≥98%) and ascorbic acid palmitate in methanol to obtain a clear and transparent solution. Remove the methanol by vacuum rotary evaporation in a water bath at 30-40℃ and collect the resulting precipitate, which is the mixed membrane material.

[0072] (2) Dissolve the mixed membrane material and medium-chain oil obtained in step 1 in dichloromethane to obtain a homogeneous solution. Remove the dichloromethane by vacuum rotary evaporation in a water bath at 30-40°C to obtain the lipid membrane complex.

[0073] (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 with stirring to obtain a crude emulsion. The crude emulsion was homogenized under high pressure to obtain blank nanoparticles with negative surface charge, i.e., blank lipid cores.

[0074] 3. Characterization method: Take the nanoparticles prepared from each formulation, dilute them with water to an appropriate concentration, and use a laser particle size potential meter to measure the intensity-weighted mean diameter (PMD), polydispersity index (PDI), and zeta potential (Zeta potential).

[0075] 4. Results: The absolute value of the negative charge potential on the surface of ordinary liposomes without ascorbate palmitate was low, which was not conducive to subsequent coating of the positively charged capsule wall. With the addition of ascorbate palmitate, the negative charge density increased significantly. When the amount added reached a certain level (approximately 8% of phospholipids), further increasing the amount of ascorbate palmitate had no significant effect on the change in negative charge density, presumably because the ascorbate palmitate on the interfacial membrane was close to saturation.

[0076] Example 2 Comparison of different phospholipids

[0077] The effect of using phospholipids containing different proportions of phosphatidylcholine (PC: 80%–100%) on the preparation of lipid cores was investigated.

[0078] Table 2. Formulations and characterizations of different phospholipids

[0079]

[0080] Preparation method:

[0081] (1) Dissolve the prescribed amount of phospholipid and ascorbic acid 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, which is the mixed membrane material.

[0082] (2) Dissolve the mixed membrane material and medium-chain oil obtained in step 1 in dichloromethane to obtain a homogeneous solution. Remove the dichloromethane by vacuum rotary evaporation in a water bath at 30-40°C to obtain the lipid membrane complex.

[0083] (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 with stirring to obtain a crude emulsion. The crude emulsion was homogenized under high pressure to obtain blank nanoparticles with negative surface charge, i.e., blank lipid cores.

[0084] The results showed that there was no significant difference in lipid cores prepared using phospholipids containing different proportions of phosphatidylcholine (PC: 80%–100%).

[0085] Example 3: Comparison of different oils

[0086] The effects of using different oils on the preparation of the lipid core of nanocapsules were investigated.

[0087] Table 3. Formulations and characterizations of different oil phases

[0088]

[0089] Preparation method:

[0090] (1) Dissolve the prescribed amount of phospholipid (PC≥98%) and ascorbic acid palmitate in methanol to obtain a clear and transparent solution. Remove the methanol by vacuum rotary evaporation in a water bath at 30-40℃ and collect the resulting precipitate, which is the mixed membrane material.

[0091] (2) Dissolve the mixed membrane material obtained in step 1 and the prescribed amount of oil phase in dichloromethane to obtain a homogeneous solution. Remove the dichloromethane by vacuum rotary evaporation in a water bath at 30-40°C to obtain the lipid membrane complex.

[0092] (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 blank nanoparticles with negative surface charge, i.e. blank lipid core.

[0093] The results showed that lipid cores with small average particle size and rich surface negative charge could be prepared using medium-chain oil or soybean oil as the oil phase.

[0094] Example 4: Preparation and characterization of paclitaxel lipid core

[0095] Using the antitumor compound paclitaxel as a model drug, a drug-loaded lipid core was prepared and characterized.

[0096] Table 4. Prescription Composition and Characterization

[0097]

[0098] Preparation method:

[0099] (1) Dissolve the prescribed amount of phospholipid (PC: 80-85%) and ascorbic acid 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, which is the mixed membrane material.

[0100] (2) Dissolve the mixed membrane material, paclitaxel and medium-chain oil obtained in step 1 in dichloromethane to obtain a homogeneous solution. Remove the dichloromethane by vacuum rotary evaporation in a water bath at 30-40°C to obtain the lipid membrane complex.

[0101] (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 with stirring to obtain a crude emulsion. The crude emulsion was homogenized under high pressure to obtain a paclitaxel lipid core with a negatively charged surface.

[0102] Example 5 Encapsulation efficiency of paclitaxel lipid core

[0103] Method for determining the encapsulation efficiency of paclitaxel lipid core drug

[0104] (1) HPLC chromatographic conditions: ZORBAX Eclipse XDB-C18 (4.6mm×150mm, 3.5μm) column; mobile phase: methanol-water-acetonitrile (23:41:36); detection wavelength: 227nm; column temperature: 35℃; flow rate: 1.2mL / min; injection volume: 10μL.

[0105] (2) Separation of encapsulated and free drugs using microcolumn centrifugation: Take a 5mL syringe (with filter paper at the bottom), add dextran gel (Sephadex G 50) to approximately the 5mL mark, allow water to flow naturally through and compact to form a uniform gel column without breaks, and centrifuge at 2000 rpm. -1 Centrifuge for 3 minutes to dehydrate the gel column and obtain a gel microcolumn for later use. Add 0.5 mL of paclitaxel lipid core solution evenly to the top of the microcolumn and incubate at 2000 rpm. -1 Centrifuge for 3 minutes and collect the eluent. Wash the gel microcolumn with 0.5 mL of water at 2000 rpm each time. -1 Centrifuge for 3 minutes, wash 3 times, and collect the eluent. Combine all eluents, dissolve in methanol-glacial acetic acid (200:1), and dilute to an appropriate concentration. Determine the paclitaxel content by HPLC, which is taken as the amount of encapsulated drug (W1). Separately, take 0.5 mL of the paclitaxel lipid core solution, dissolve in methanol-glacial acetic acid (200:1), and dilute to an appropriate concentration. Determine the paclitaxel content by HPLC, which is taken as the total amount of paclitaxel in the lipid core solution before column centrifugation (Wtotal).

[0106] Encapsulation rate % = W1 / Wtotal × 100%

[0107] (3) The drug encapsulation efficiency of the paclitaxel lipid core prepared using the method in Example 4 was 96.0%.

[0108] Example 6: Effect of Adding Cholesterol

[0109] The effect of adding different proportions of cholesterol on the preparation of paclitaxel lipid cores was investigated.

[0110] Table 5. Prescriptions and characterizations of cholesterol at different ratios.

[0111]

[0112] Preparation method:

[0113] (1) Dissolve the prescribed amount of phospholipid (PC: 80-85%) and ascorbic acid 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, which is the mixed membrane material.

[0114] (2) Dissolve the mixed membrane material obtained in step 1, the prescribed amount of paclitaxel, medium chain oil and cholesterol in dichloromethane to obtain a homogeneous solution. Remove the dichloromethane by vacuum rotary evaporation in a water bath at 30-40°C to obtain the lipid membrane complex.

[0115] (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 with stirring to obtain a crude emulsion. The crude emulsion was homogenized under high pressure to obtain a paclitaxel lipid core with a negatively charged surface.

[0116] The results showed that adding different proportions of cholesterol could produce paclitaxel lipid cores with a drug encapsulation rate of over 90% and rich in negative charges, indicating that adding cholesterol had no significant effect on the preparation of paclitaxel lipid cores.

[0117] Example 7: Comparison of different oil phase ratios

[0118] The effect of adding different proportions of oil on the preparation of paclitaxel lipid cores was investigated.

[0119] Table 6. Formulations and characterizations of different oil phase ratios.

[0120]

[0121] Preparation method:

[0122] (1) Dissolve the prescribed amount of phospholipid (PC: 80-85%) and ascorbic acid 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, which is the mixed membrane material.

[0123] (2) Dissolve the mixed membrane material obtained in step 1, the prescribed amount of paclitaxel, medium chain oil and cholesterol in dichloromethane to obtain a homogeneous solution, and remove the dichloromethane by vacuum rotary evaporation in a water bath at 30-40°C to obtain the lipid membrane complex.

[0124] (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 with stirring to obtain a crude emulsion. The crude emulsion was homogenized under high pressure to obtain a paclitaxel lipid core with a negatively charged surface.

[0125] The results showed that all the above formulations with different proportions of oil phase could produce paclitaxel lipid cores with a drug encapsulation rate of greater than 90% and rich in negative charge.

[0126] Example 8: Comparison of different drug loading capacities

[0127] The effect of different paclitaxel loading amounts on the encapsulation efficiency of lipid core drugs was investigated.

[0128] Table 7 Prescriptions with different drug loading capacities

[0129]

[0130] Preparation method:

[0131] (1) Dissolve the prescribed amount of phospholipid (PC: 80-85%) and ascorbic acid 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, which is the mixed membrane material.

[0132] (2) Dissolve the mixed membrane material obtained in step 1, the prescribed amount of paclitaxel and medium-chain oil in dichloromethane to obtain a homogeneous solution, and remove the dichloromethane by vacuum rotary evaporation in a water bath at 30-40°C to obtain the lipid membrane complex.

[0133] (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 with stirring to obtain a crude emulsion. The crude emulsion was homogenized under high pressure to obtain a paclitaxel lipid core with a negatively charged surface.

[0134] The results showed that the paclitaxel lipid core of formulation Z3 precipitated drug after being left at room temperature for 24 hours. The paclitaxel lipid cores of formulations Z1 and Z2 showed good stability and high encapsulation efficiency (greater than 80%).

[0135] Example 9: Amount of chitosan

[0136] The design-response surface methodology was employed to investigate the effect of chitosan dosage, including different chitosan concentrations and solution volumes, on the preparation of nanocapsules coating the first layer of the capsule wall. The results were analyzed using Design-Expert 8.0 software.

[0137] (1) Preparation method of paclitaxel lipid core:

[0138] Table 8. Prescription Composition and Characterization

[0139]

[0140] a) Dissolve the prescribed amount of phospholipid (PC: 80-85%) and ascorbate 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, which is the mixed membrane material.

[0141] b) The obtained mixed membrane material is dissolved in dichloromethane with paclitaxel and medium-chain oil to obtain a homogeneous solution. The dichloromethane is removed by vacuum rotary evaporation in a water bath at 30-40°C to obtain the lipid membrane complex.

[0142] 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 approximately 2.5% glycerol was slowly added with stirring to obtain a crude emulsion. The crude emulsion was homogenized under high pressure to obtain a paclitaxel lipid core with a negatively charged surface.

[0143] (2) Preparation method of nanocapsules covering the first layer of capsule wall: Take an appropriate amount of paclitaxel lipid core solution with negative surface charge, and slowly add it dropwise into an appropriate amount of chitosan solution of a certain concentration (0.005mol / L hydrochloric acid as solvent) under stirring. After self-assembly, chitosan is adsorbed on the surface of lipid core, thereby obtaining nanocapsules with positive surface charge.

[0144] (3) Prescription design and effect index of the star point design-response surface optimization method:

[0145] Using the central composite (CCD) method of the star-response surface methodology, a two-factor, five-level formulation design was employed. Chitosan concentration (X1) and the volume ratio of chitosan solution to lipid core solution (Vchitosan:Vlipid core) (X2) were the two factors investigated. Five levels were selected for each factor, resulting in a total of 13 experiments, including 8 non-centropoints and 5 centropoints.

[0146] The factor level table is as follows:

[0147] Table 9. Star Design Factor Level Table

[0148]

[0149] Effect index and analysis method: The total normalized value (OD) obtained by mathematical conversion 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.

[0150] Calculation of the overall normalized value (OD): First, the Hassan method is used to calculate the d-value for effects where smaller values ​​are better and larger values ​​are better, respectively. Smaller particle size and PDI values ​​are better, while larger surface potential values ​​are better. The overall normalized value (OD) is the geometric mean of all effects.

[0151] d 粒径 =(y max -y i ) / (y max -y min )

[0152] d PDI =(y max -y i ) / (y max -y min )

[0153] d 电位 =(y i -y min ) / (y max -ymin )

[0154] Among them, y max For the maximum value of this effect, y min For the minimum value of this effect, y i Current measurement value

[0155] Total score normalized value (OD) = geometric mean (d 粒径 d PDI d 电位 )

[0156] (4) Results:

[0157] Table 10. Formulations for self-assembly of chitosan-coated nanocapsules

[0158]

[0159] The results showed that the higher the chitosan concentration and the greater the volume ratio of chitosan solution to lipid core solution in the formulation, the smaller the particle size and the higher the positive surface charge of the resulting nanocapsules. Therefore, the amount of chitosan can be varied according to requirements, including changing the chitosan concentration and solution volume, to obtain nanocapsules with the desired particle size and potential coating the first layer of the capsule wall.

[0160] The results from the 13 experiments were fitted with a quadratic polynomial, and the actual equation is as follows:

[0161] OD=-6.601+32.072X1+3.241X2-7.001X1X2-33.994X1 2 -0.328X2 2 .

[0162] The coefficient evaluation model (Coded Equation) is as follows:

[0163] OD=0.826+0.224X1+0.266X2-0.175X1X2-0.085X1 2 -0.082X2 2 .

[0164] The regression coefficients R of the quadratic polynomial fitting 2 =0.9848, r=0.9924, F-test p-value less than 0.01, indicating a good fit model. Contour plots of X1 and X2 with the same OD drawn using Design-Expert 8.0 software are attached. Figure 3 .

[0165] Based on comprehensive experimental measurements and contour plots, the preferred range is: chitosan concentration of 0.15–0.3 mg / mL, and the volume ratio of chitosan solution to lipid core solution of 1.5–3.0. A more preferred range is: chitosan concentration of 0.2–0.3 mg / mL, and the volume ratio of chitosan solution to lipid core solution of 2.0–3.0.

[0166] After conversion, the preferred range based on mass ratio is: chitosan relative to each gram of phospholipid is 0.015 to 0.045 grams, more preferably 0.01875 to 0.03125 grams.

[0167] Example 10: Dosage of Low Molecular Weight Heparin

[0168] The effects of low molecular weight heparin dosage, including different low molecular weight heparin concentrations and solution volumes, on the preparation of composite lipid nanocapsules coating the second layer of the capsule wall were investigated.

[0169] (1) Preparation method of nanocapsules covering the first layer of the capsule wall:

[0170] Table 11 Prescription Composition and Characterization

[0171]

[0172] a) Dissolve the prescribed amount of phospholipid (PC: 80-85%) and ascorbate 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, which is the mixed membrane material.

[0173] b) The obtained mixed membrane material is dissolved in dichloromethane with paclitaxel and medium-chain oil to obtain a homogeneous solution. The dichloromethane is removed by vacuum rotary evaporation in a water bath at 30-40°C to obtain the lipid membrane complex.

[0174] 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 approximately 2.5% glycerol was slowly added with stirring to obtain a crude emulsion. The crude emulsion was homogenized under high pressure to obtain a paclitaxel lipid core with a negatively charged surface.

[0175] d) Take 1 part of paclitaxel lipid core solution and slowly add it dropwise to 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 the surface of lipid core, so that the lipid core is coated with the first layer of capsule wall, thereby obtaining nanocapsules (CS-NCs) with positively charged surface coated with the first layer of capsule wall.

[0176] (2) Preparation method of composite lipid nanocapsules with a second layer of capsule wall: Take an appropriate amount of CS-NCs solution with positive charge on the surface and slowly add it dropwise into an appropriate amount of low molecular weight heparin solution (pH 7.5) with a certain concentration under stirring. After self-assembly, the low molecular weight heparin is adsorbed on the surface of the nanocapsule, so that the nanocapsule is coated with a second layer of capsule wall, thereby obtaining paclitaxel composite lipid nanocapsules.

[0177] (3) Prescription design and effect index of the star point design-response surface optimization method:

[0178] Using the central composite (CCD) method of the star-point design-response surface optimization approach as a model, a two-factor, five-level formulation design was conducted. The low molecular weight heparin concentration and the volume ratio (V0.05) of the low molecular weight heparin solution to the CS-NCs nanocapsule solution were used as the basis for formulation design. 低分子肝素 V CS-NCs The two factors were investigated, with five levels selected for each factor, resulting in a total of 13 experiments, including 8 non-centrocenter points and 5 centrocenter points. The particle size, PDI, and potential of the prepared composite lipid nanocapsules were used as effect indicators.

[0179] The factor level table is as follows:

[0180] Table 12 Star Design Factor Level Table

[0181]

[0182] (4) Results of the star-point design-response surface optimization method

[0183] Table 13 Self-assembled formulations of composite lipid nanocapsules coated with low molecular weight heparin

[0184]

[0185] The results showed that different low molecular weight heparin concentrations and different volume ratios of low molecular weight heparin solution to CS-NCs nanocapsule solution in the star-shaped design formulations could all produce composite lipid nanocapsules with small particle size and high surface negative charge, with no significant differences between the formulations. Therefore, the amount of low molecular weight heparin can be changed according to requirements, including changing the concentration of low molecular weight heparin and the solution volume, to obtain the desired composite lipid nanocapsules with a second layer of capsule wall.

[0186] The concentration of low molecular weight heparin investigated was 0.05–0.35 mg / mL, and the volume ratio of low molecular weight heparin solution to chitosan-coated nanocapsule solution was 1.25–2.75.

[0187] (5) Continue to change the concentration and solution volume of low molecular weight heparin:

[0188] Table 14. Self-assembled formulation of composite lipid nanocapsules coated with low molecular weight heparin.

[0189]

[0190] 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, composite lipid nanocapsules with small particle size and high surface negative charge could still be obtained.

[0191] Based on the experimental results of (4) and (5), the concentration of low molecular weight heparin investigated was 0.05–0.35 mg / mL, and the volume ratio of low molecular weight heparin solution to chitosan-coated nanocapsule solution was 1.00–2.75. After conversion, the preferred range based on mass ratio is: the amount of low molecular weight heparin relative to each gram of chitosan is 0.42–5.25 grams.

[0192] Example 11 Preparation and characterization of paclitaxel-based lipid nanocapsules

[0193] Table 15 Formulation composition of composite lipid nanocapsules

[0194]

[0195] (I) Preparation method:

[0196] (1) Dissolve the prescribed amount of phospholipid (PC: 80-85%) and ascorbic acid 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 precipitate, which is the mixed membrane material.

[0197] (2) Dissolve the mixed membrane material, paclitaxel and medium-chain oil obtained in step 1 in dichloromethane to obtain a homogeneous solution. Remove the dichloromethane by vacuum rotary evaporation in a water bath at 30-40°C to obtain the lipid membrane complex.

[0198] (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 with stirring to obtain a crude emulsion. The crude emulsion was homogenized under high pressure to obtain a paclitaxel lipid core with a negatively charged surface.

[0199] (4) Take 1 part of paclitaxel lipid core solution and slowly add it dropwise to 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 the surface of lipid core, so that the lipid core is coated with the first layer of capsule wall, thereby obtaining nanocapsules (CS-NCs) with positive surface charge.

[0200] (5) Take one part of the positively charged CS-NCs solution 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, the low molecular weight heparin is adsorbed on the surface of the nanocapsule, so that the nanocapsule is coated with a second capsule wall, thereby obtaining the paclitaxel composite lipid nanocapsule (LH-NCs).

[0201] (II) Encapsulation efficiency determination method:

[0202] The drug encapsulation efficiency of nanocapsules (CS-NCs) and composite lipid nanocapsules (LH-NCs) coated with the first layer of the capsule wall was determined by low-speed centrifugation, as follows:

[0203] (1) HPLC chromatographic conditions: ZORBAX Eclipse XDB-C18 (4.6mm×150mm, 3.5μm) column; mobile phase: methanol-water-acetonitrile (23:41:36); detection wavelength: 227nm; column temperature: 35℃; flow rate: 1.2mL / min; injection volume: 50μL.

[0204] (2) Separation of encapsulated and free drugs by low-speed centrifugation: Take 0.5 mL of CS-NCs solution or paclitaxel-based lipid nanocapsule solution, dilute with water to 10 mL, shake well, take about 8 mL, and centrifuge at 1000 rpm. -1 Centrifuge for 10 minutes to precipitate unencapsulated paclitaxel crystals. Transfer 2 mL of the supernatant to a 25 mL volumetric flask, add 12 mL of methanol-glacial acetic acid (200:1) to dissolve completely, then dilute to the mark with the mobile phase and mix well. Determine the paclitaxel content using HPLC, which is taken as the amount of encapsulated drug, E1. Take another 2 mL of the solution before centrifugation and repeat the same procedure, determining the paclitaxel content by HPLC, which is taken as the total amount of paclitaxel in the nanocapsule solution before centrifugation, E0. Calculate the encapsulation efficiency using the following formula.

[0205] Encapsulation rate % = E1 / E0 × 100%

[0206] (III) Results:

[0207] Table 16 Characterization of composite lipid nanocapsules

[0208]

[0209] The results showed that during the preparation of the composite lipid nanocapsules, the particle size slightly increased with the increase of the number of lipid core layers covering the capsule wall, and the surface charge of the particles exhibited an alternating pattern of positive and negative charges. The drug encapsulation efficiency was greater than 90%, indicating that paclitaxel composite lipid nanocapsules could be successfully prepared through electrostatic adsorption of positive and negative charges. The particle size distribution diagram and transmission electron microscopy image of the composite lipid nanocapsules are attached. Figure 4 and attached Figure 5 .

[0210] Example 12 Preparation and characterization of paclitaxel-based lipid nanocapsule lyophilized powder

[0211] Preparation method: Take the paclitaxel composite lipid nanocapsule solution (LH-NCs) prepared by the method of Example 11 of this invention, add 6g of trehalose as a lyophilization support agent per 100mL, dissolve completely, shake well, and freeze dry to obtain lyophilized powder of composite lipid nanocapsules.

[0212] The freeze-drying process is shown in the table below.

[0213] Table 17 Freeze-drying process

[0214]

[0215] Characterization method: Take an appropriate amount of paclitaxel-based lipid nanocapsule lyophilized powder, reconstitute it with physiological saline, and then determine its composition.

[0216] Results: The average particle size was 159.2 ± 15.6 nm, the polydispersity index (PDI) was 0.273 ± 0.013, the zeta potential was -53.96 ± 5.67 mV, and the drug encapsulation efficiency was 99.47 ± 1.04%. Transmission electron microscopy images are attached. Figure 6 .

[0217] Example 13 Release rate of composite lipid nanocapsules

[0218] The drug release characteristics of the paclitaxel-based lipid nanocapsule lyophilized powder prepared using the method of Example 12 of this invention were investigated.

[0219] A solution of paclitaxel-based lipid nanocapsules with a concentration of approximately 0.12 mg / mL was prepared by oscillation dialysis. An appropriate amount of lyophilized paclitaxel-based lipid nanocapsule powder was reconstituted with physiological saline and shaken well. 1 mL of this solution was placed in a dialysis bag and sealed. The dialysis bag was placed in 50 mL of release medium at 37°C, with an oscillation rate of 100 times per minute. 1 mL of release medium was collected at 0.5, 1, 2, 4, 8, 12, 24, 36, and 48 hours, and 1 mL of fresh medium was added simultaneously. The drug content in the collected release medium was determined using the HPLC method described in Example 11 of this invention, and the cumulative drug release rate was calculated. The release curves of the lipid nanocapsules in two release media were investigated: pH 5.0 phosphate buffer (containing 0.5% Tween 80) and pH 7.4 phosphate buffer (containing 0.5% Tween 80).

[0220] Cumulative drug release rate = (cumulative drug release / total drug release) × 100%

[0221] The results showed that the cumulative drug release rate of paclitaxel-based lipid nanocapsules was 91.5% after 48 hours in pH 5.0 medium and 82.2% after 48 hours in pH 7.4 medium. The release curves are attached. Figure 7 .

[0222] The release curves were fitted with a mathematical model using DDSolver 1.0 software. The release curves at pH 5.0 and pH 7.4 best fit the classic Higuchi diffusion equation. The results are shown in the table below.

[0223] Table 18 Fitting of Release Curves

[0224]

[0225] The results showed that the drug release from paclitaxel-based lipid nanocapsules in both media was mainly affected by its own diffusion behavior, which belongs to the diffusion-controlled release mechanism.

[0226] Example 14: Tumor-targeted distribution in tumor-bearing mice using composite lipid nanocapsules

[0227] Composite lipid nanocapsules were prepared using the fluorescent dye DiR, and the in vivo tumor-targeting distribution of the composite lipid nanocapsules was investigated using small animal in vivo imaging technology.

[0228] (I) Preparation of DiR composite lipid nanocapsules

[0229] Table 19 Formulation composition of DiR composite lipid nanocapsules

[0230]

[0231] (1) Dissolve the prescribed amount of phospholipid (PC≥98%) and ascorbic acid palmitate in methanol to obtain a clear and transparent solution. Remove the methanol by vacuum rotary evaporation in a water bath at 30-40℃ and collect the precipitate, which is the mixed membrane material.

[0232] (2) Dissolve the mixed membrane material, DiR and medium chain oil obtained in step 1 in dichloromethane to obtain a homogeneous solution. Remove the dichloromethane by vacuum rotary evaporation in a water bath at 30-40°C to obtain the lipid membrane complex.

[0233] (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 with 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 charge, i.e., the lipid core.

[0234] (4) Take 1 part of the lipid core solution and slowly add it dropwise to 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 the surface of the lipid core, so that the lipid core is coated with the first layer of capsule wall, thereby obtaining nanocapsules (DiR-CS-NCs) with positive surface charge.

[0235] (5) Take one part of the positively charged DiR-CS-NCs solution 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, the low molecular weight heparin is adsorbed on the surface of the nanocapsule, so that the nanocapsule is coated with a second layer of capsule wall, thereby obtaining the negatively charged DiR composite lipid nanocapsule (DiR-LH-NCs).

[0236] Characterization results:

[0237] Table 20 Characterization of DiR composite lipid nanocapsules

[0238]

[0239] (II) Tumor-targeted distribution in tumor-bearing mice

[0240] Establishment of the 4T1 tumor-bearing mouse model: Balb / c mice, female, weighing 17±2g. Each mouse was in situ seeded with 1×104 4T1 breast cancer cells into the fourth mammary fat pad. 6 Each animal was fed normally until the tumor grew to approximately 800 mm in size. 3 Used for experiments.

[0241] Experimental group: 4T1 model mice were randomly divided into 3 groups (n=1 per group): DiR solution group (2% DMSO-water as solvent), DiR lipid core group, and DiR composite lipid nanocapsule group. Each group was administered DiR via tail vein injection at a dose of 0.1 mg / kg. In vivo imaging (IVIS) was performed at 2, 4, 8, 12, and 24 hours after administration. Tumor tissue was rapidly removed 24 hours after the experiment for luminescence detection, and the tumor luminescence intensity of each group was recorded.

[0242] The results showed that the fluorescence intensity of tumor tissues from the DiR composite lipid nanocapsule group and the lipid core group was significantly higher than that from the solution group (see attached figure). Figure 8 Tumor tissue was removed after the 24-hour test; see attached image. Figure 9 The luminescence values ​​of each group were as follows: DiR solution group (8.314 × 10⁻⁶) 8 p / s), DiR lipid core group (8.764×10 9 p / s), DiR composite lipid nanocapsules (1.073×10 10The p / s ratio showed that the composite lipid nanocapsules and lipid core had good tumor-targeting distribution.

Claims

1. A composite lipid nanocapsule composition, the composition comprising the following components: lipids, phospholipids, ascorbate palmitate, antitumor compounds, chitosan, and low molecular weight heparin; the composite lipid nanocapsule composition is formed by a negatively charged lipid core, a positively charged first capsule wall, and a negatively charged second capsule wall, which are bonded together by electrostatic adsorption between positively and negatively charged groups; the negatively charged lipid core comprises the following components: lipids, phospholipids, ascorbate palmitate, and antitumor compounds; the positively charged first capsule wall comprises chitosan; the negatively charged second capsule wall comprises low molecular weight heparin; and the composite lipid nanocapsules have a particle size ranging from 10 to 1000 nm. The amount of ascorbate palmitate relative to each gram of phospholipid is 0.08–0.12 g; the antitumor compound is paclitaxel, and the amount of paclitaxel relative to each gram of phospholipid is 0.02–0.03 g. The method for preparing the negatively charged lipid core includes the following preparation steps: (1) Dissolve phospholipids and ascorbate palmitate in organic solvent A to obtain a homogeneous solution, remove organic solvent A to obtain a mixed membrane material; (2) Dissolve the mixed membrane material, oil and antitumor compound obtained in step 1 in organic solvent B to obtain a homogeneous solution, remove organic solvent B to obtain lipid membrane complex; (3) The lipid membrane complex obtained in step 2 is dispersed in an aqueous phase, emulsified and homogenized to obtain lipid nanoparticles with negative surface charge, namely the lipid core with negative surface charge.

2. The composite lipid nanocapsule composition according to claim 1, characterized in that, The negatively charged lipid core contains cholesterol.

3. The composite lipid nanocapsule composition according to claim 1, characterized in that, The amount of oil used is 0.001 to 100 ml per gram of phospholipid; the amount of chitosan used is 0.001 to 100 g per gram of phospholipid; and the amount of low molecular weight heparin used is 0.001 to 100 g per gram of chitosan.

4. The composite lipid nanocapsule composition according to claim 1, characterized in that, 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 class of heparin and its salts prepared by depolymerization.

5. The method for preparing the composite lipid nanocapsules according to any one of claims 1-4, characterized in that, The preparation steps for a composite lipid nanocapsule consisting of a negatively charged lipid core, a positively charged first capsule wall, and a negatively charged second capsule wall, which are bonded together by electrostatic adsorption between positively and negatively charged groups, are as follows: (1) The negatively charged lipid core is dispersed in an aqueous phase containing chitosan. After self-assembly, the chitosan is adsorbed onto the surface of the lipid core, so that the lipid core is coated with the first layer of capsule wall, thereby obtaining a positively charged nanocapsule. (2) The positively charged nanocapsules obtained in step 1 are dispersed in an aqueous phase containing low molecular weight heparin. After self-assembly, the low molecular weight heparin is adsorbed onto the surface of the nanocapsules, so that the nanocapsules are coated with a second layer of capsule wall, thereby obtaining the composite lipid nanocapsules.

6. The composite lipid nanocapsule composition according to claim 1, characterized in that, The organic solvent A includes one or more of methanol, ethanol, and tetrahydrofuran; the organic solvent B includes one or more of chloroform, dichloromethane, tetrahydrofuran, n-hexane, cyclohexane, ethyl acetate, petroleum ether, methanol, and ethanol.

7. The use of the composite lipid nanocapsule composition according to any one of claims 1-4 in the preparation of antitumor drugs.

Images