CN111920782A - Composite lipid nanocapsule composition and preparation method and application thereof - Google Patents
Composite lipid nanocapsule composition and preparation method and application thereof Download PDFInfo
- Publication number
- CN111920782A CN111920782A CN201910392474.8A CN201910392474A CN111920782A CN 111920782 A CN111920782 A CN 111920782A CN 201910392474 A CN201910392474 A CN 201910392474A CN 111920782 A CN111920782 A CN 111920782A
- Authority
- CN
- China
- Prior art keywords
- lipid
- nanocapsule
- composite
- chitosan
- phospholipid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/513—Organic macromolecular compounds; Dendrimers
- A61K9/5161—Polysaccharides, e.g. alginate, chitosan, cellulose derivatives; Cyclodextrin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/5123—Organic compounds, e.g. fats, sugars
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Epidemiology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Optics & Photonics (AREA)
- Physics & Mathematics (AREA)
- Biomedical Technology (AREA)
- Nanotechnology (AREA)
- Dermatology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Medicinal Preparation (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a composite lipid nanocapsule composition, and a preparation method and application thereof, and belongs to the technical field of medicines. The composite lipid nano-capsule composition is formed by combining a lipid core with a negatively charged surface, a positively charged first layer of capsule wall and a negatively charged second layer of capsule wall through electrostatic adsorption between positive and negative charge groups, and can be used for intravenous injection. The lipid core is a storage for loading anti-tumor drugs, the chitosan has the effects of promoting transmembrane transport, controlling drug release and the like, and the low-molecular heparin coated outside the nanocapsule can act with heparinase, so that the nanocapsule is enriched and degraded on a malignant tumor part, and the function of targeting tumors is realized. In addition, the carrier material used by the composition has high biological safety, good tumor targeting property, high drug encapsulation rate, good stability, controllable release and simple preparation method, and has wide application prospect when being used for targeted delivery of antitumor drugs.
Description
Technical Field
The invention belongs to the technical field of medicines and nano-medicines, and relates to a composite lipid nanocapsule composition, and a preparation method and application thereof.
Background
Malignant tumors are one of the major diseases that threaten human health and cause death of patients. The chemotherapy is a conventional malignant tumor treatment means, but the chemotherapy generally has the defects of no treatment selectivity, large toxic and side effects, easy generation of drug resistance of cancer cells and the like, and poor tolerance of patients. In addition, many antitumor drugs have the defects of poor water solubility, poor stability and other physicochemical properties. Therefore, the physicochemical properties of the drug need to be improved by the preparation technology, and particularly, the tumor targeting property of the drug, the toxic and side effects of the organism and the treatment effect of the tumor need to be improved by the preparation technology according to the biological characteristics of the tumor and the microenvironment thereof.
Nanocapsules (Nanocapsules) are drug depot type nanoparticles formed by wrapping solid or liquid drugs as capsule cores, and are small-size nanometer drug carrier systems (10-1000 nm) formed by wrapping natural or synthetic high-molecular thin-layer polymer membranes and oily or aqueous cores. As a drug carrier, besides improving the conventional physicochemical properties of the drug such as solubility, dispersibility and the like, the drug carrier also has the following advantages: 1. the stability of the medicine is improved, and the influence of environmental factors on the medicine is reduced; 2. endows the drug with sustained and controlled release performance, and effectively controls the drug release; 3. the capsule material can be positioned in a specific tissue after surface modification, so that the purpose of active targeting is achieved; 4. the nanometer level particle and its small size may be used in intravenous injection without causing blood vessel embolism.
Based on the advantages, the nanocapsule has a wide prospect in antitumor drug delivery, but the nanocapsule preparation researched more at present still has some problems, such as poor biocompatibility of carrier materials, poor tumor targeting of the preparation, unsatisfactory drug release, poor controllability of preparation process, unstable quality and the like.
The prior art discloses a microcapsule, which is described in the Master academic paper "layer by layer self-assembled heparin/chitosan microcapsule preparation and performance research" (Liangyan, 2014, Jiangnan university, China). The microcapsule is prepared by adsorbing natural polysaccharide heparin with negative charge and natural polysaccharide chitosan with positive charge on core CaCO by layer-by-layer self-assembly technique3The surface of the template, then CaCO is removed3And (4) preparing a template. Research results show that the microcapsule has good biocompatibility and stability. However, the microcapsule prepared by the technology has the particle size of about 4-5 microns, is not suitable for intravenous injection administration, has insufficient drug targeted delivery capacity and limits the clinical application of the microcapsule.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a composite lipid nanocapsule composition, which has the following properties: the used polymer and functional material have good biocompatibility, safety and no toxicity; the particle size is nano (10-1000 nm), and the preparation can be used for intravenous injection; has good tumor targeting property; the medicine encapsulation rate is high, and the stability is good; the preparation method is simple and feasible.
Another technical problem solved by the present invention is to provide the use of the composite lipid nanocapsule composition.
The technical problem solved by the invention is realized by the following technical scheme:
in a first aspect of the technical scheme of the invention, a composition for compounding lipid nanocapsules is provided, which comprises the following components: grease, phospholipid, ascorbyl palmitate, an antitumor compound, chitosan and low molecular weight heparin. The particle size range of the composite lipid nano-capsule is 10-1000 nm, the preferable particle size is 10-500 nm, and the more preferable particle size is 10-200 nm. The particle size refers to the average light Intensity particle size (Intensity-Weighted Mean Diameter, abbreviated as particle size) measured by a laser particle size analyzer.
The complex lipid nanocapsule composition may also or may not comprise cholesterol.
The composite lipid nanocapsule is formed by combining a lipid core with a negatively charged surface, a positively charged first layer of capsule wall and a negatively charged second layer of capsule wall through electrostatic adsorption between positive and negative charge groups (shown in figure 1); the surface negatively charged lipid core comprises the following components: grease, phospholipid, ascorbyl palmitate and an antitumor compound; the positively charged first capsule wall comprises chitosan; the negatively charged second layer capsule wall comprises low molecular weight heparin; the surface negatively charged lipid core may or may not also contain cholesterol.
Furthermore, in the composite lipid nano-capsule composition, the dosage of the ascorbyl palmitate is 0.001-100 g, preferably 0.02-0.12 g, relative to each gram of phospholipid; the dosage of the grease relative to each gram of phospholipid is 0.001-100 ml, preferably 0.8-1.2 ml; the dosage of the chitosan relative to each gram of phospholipid is 0.001-100 g, preferably 0.015-0.045 g, and more preferably 0.01875-0.03125 g; the dosage of the low molecular weight heparin is 0.001-100 g, preferably 0.42-5.25 g per gram of chitosan.
Further, in the composite lipid nanocapsule composition, the oil is one or more oils of natural or synthetic origin, preferably including medium-chain triglycerides (abbreviated as medium-chain oils) or soybean oil; the phospholipid is one or more phospholipids from natural or synthetic sources, preferably comprises lecithin (mainly comprising phosphatidylcholine, and more preferably phosphatidylcholine content of 80% or more); the chitosan preferably comprises low molecular chitosan; the low molecular weight heparin is heparin with lower molecular weight prepared by depolymerization and salts thereof, preferably comprises low molecular weight heparin sodium, and more preferably comprises Enoxaparin sodium (Enoxaparin sodium).
Further, in the composite lipid nanocapsule composition, the anti-tumor compound comprises a taxane compound, and further, the taxane compound comprises paclitaxel; the dosage of the paclitaxel relative to each gram of the phospholipid is 0-100 g, preferably 0.02-0.03 g.
Further, the composite lipid nano-capsule composition and the preparation method thereof are described in the second aspect of the technical scheme of the invention. In a second aspect of the present invention, there is provided a method for preparing the complex lipid nanocapsule composition of the first aspect, wherein the lipid core having a negatively charged surface comprises the following steps:
(1) dissolving phospholipid and ascorbyl palmitate in an organic solvent A to obtain a uniform solution, and removing the organic solvent A to obtain a mixed membrane material;
(2) dissolving the mixed membrane material, the grease and the anti-tumor compound obtained in the step 1 in an organic solvent B to obtain a uniform solution, and removing the organic solvent B to obtain a lipid membrane compound;
(3) and (3) dispersing the lipid membrane compound obtained in the step (2) in a water phase, emulsifying and homogenizing to obtain the lipid nanoparticles with negative charges on the surface, namely the lipid core with negative charges on the surface.
In the components of the lipid core with the surface with negative charges, the dosage of the ascorbyl palmitate is 0.001-100 g, preferably 0.02-0.12 g, relative to each gram of phospholipid; the dosage of the grease is 0.001-100 ml, preferably 0.8-1.2 ml per gram of phospholipid.
Further, the anti-tumor compound comprises a taxane compound, and further, the taxane compound comprises paclitaxel; the dosage of the paclitaxel relative to each gram of the phospholipid is 0-100 g, preferably 0.02-0.03 g.
Further, the organic solvent a includes, but is not limited to, one or more of methanol, ethanol, and tetrahydrofuran, and 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, and dichloromethane is preferred.
Further, the aqueous phase for dispersing the lipid membrane complex may or may not contain glycerol.
Further, the methods for removing the organic solvent a and the organic solvent B include, but are not limited to, a reduced pressure drying method, a solvent evaporation method, a rotary evaporation method, a spray drying method, and a freeze drying method.
The preparation method of the composite lipid nanocapsule composition comprises the following preparation steps of:
(1) dispersing a lipid core with negative charges on the surface in a water phase containing chitosan, adsorbing the chitosan on the surface of the lipid core through self-assembly, and coating the first layer of capsule wall on the lipid core so as to obtain the nano capsule with positive charges on the surface.
(2) Dispersing the nano-capsules with positive charges on the surfaces obtained in the step (1) into a water phase containing low-molecular heparin, adsorbing the low-molecular heparin on the surfaces of the nano-capsules through self-assembly, and coating the nano-capsules with a second layer of capsule walls to obtain the composite lipid nano-capsules.
The chitosan concentration of the water phase containing chitosan is 0.001-100 mg/mL, preferably 0.15-0.3 mg/mL, more preferably 0.2-0.3 mg/mL, and further preferably 0.2 mg/mL; the chitosan solution is 0.001 to 10000 parts by volume per part of the lipid core solution, preferably 1.5 to 3.0 parts by volume, more preferably 2.0 to 3.0 parts by volume, and further preferably 2.5 parts by volume. The amount of chitosan 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 per gram of phospholipid.
The aqueous phase containing the low molecular heparin, wherein the concentration of the low molecular heparin is 0.001-100 mg/mL, preferably 0.05-0.35 mg/mL, and more preferably 0.08 mg/mL; the volume ratio of the low molecular heparin solution to each part of the chitosan-coated nanocapsule solution with positive charges on the surface is 0.001-10000 parts, preferably 1.00-2.75 parts, and more preferably 1.2 parts. The dosage of the low molecular weight heparin is 0.001-100 g, preferably 0.42-5.25 g, and more preferably 0.672 g per gram of chitosan according to the mass ratio.
Further, the aqueous phase containing chitosan, the medium for adjusting the acidity thereof, includes but is not limited to acetic acid, hydrochloric acid, phosphoric acid, preferably hydrochloric acid, and more preferably the concentration of hydrochloric acid in the aqueous phase is 0.005 mol/L.
The composite lipid nanocapsule composition can be prepared into various dosage forms containing the composite lipid nanocapsule, including but not limited to injection, freeze-dried powder and gel, and the medicines of the various dosage forms can be prepared according to a conventional method in the pharmaceutical field, and preferably are injection or freeze-dried powder.
The third aspect of the technical scheme of the invention provides an application of the composite lipid nanocapsule composition in preparing an anti-tumor medicament, wherein tumors comprise 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.
Advantageous technical effects
Compared with the prior art, the invention has the following beneficial technical effects:
the composite lipid nanocapsule composition provided by the invention has various properties, including improvement of drug stability, capability of being used for intravenous injection, sustained and controlled release performance, active targeting on tumor parts, reduction of toxic and side effects of normal tissues and improvement of tumor treatment effect. The advantages of the present invention are as follows,
1. the lipid composite nanocapsule prepared by the invention is small-size particles with the particle size of nanometer level, can improve the dispersibility, permeability and stability of the drug, can be used for intravenous injection, has the EPR effect (enhanced permeability and retentivity effect) of solid tumors, and is an excellent carrier for targeted delivery of antitumor drugs.
2. The lipid core with the surface with negative charges prepared by the invention is grease, can be loaded with hydrophobic drugs, and improves the solubility of the drugs.
3. The membrane material of the lipid core with the surface with negative charge prepared by the invention contains phospholipid. The main component of lecithin is phosphatidylcholine which is used alone as a membrane material, and the interface negative charge density of the prepared nanoparticles is small and can not be regulated, so that the preparation of the subsequent nanocapsules is not facilitated. The invention discovers that ascorbyl palmitate (AP, the chemical structural formula of which is shown in figure 2) has one side of lipophilic palmitate and the other side of hydrophilic ascorbic acid acidic polyhydroxy group, and the ascorbyl palmitate and phospholipid are made into a mixed membrane material after hydrolysis, so that the negative charge density of the surface of a lipid core can be obviously enhanced, the stability of the lipid core is improved, and the ascorbyl palmitate and the phospholipid mixed membrane material have an important function of coating a capsule wall with positive charges through electrostatic adsorption.
4. The composite lipid nanocapsule prepared by the invention has an active targeting tumor effect. Low-molecular-weight heparin (LMWH) is a general name of heparin with lower molecular weight prepared by depolymerization, has narrow average molecular weight distribution range, and has fewer side effects compared with non-depolymerized heparin. Heparanase (HPA, heparin enzyme for short) is endogenous endo-beta-D-glucuronic acid endonuclease and can specifically cut off heparin sulfate proteoglycan on extracellular matrix. A large number of researches find that the overexpression of the heparinase is closely related to the occurrence, development, invasion and metastasis of tumors, and is an important molecular target related to the tumors. Heparin and its lysate low molecular weight heparin as heparinase action substrate, can be combined with heparinase through enzyme-substrate interaction, inhibit endogenous heparan sulfate proteoglycan degradation, have antitumor metastasis, anti-inflammatory, anticoagulant and antithrombotic pharmacological effects. The invention coats the low molecular heparin on the outer side of the composite lipid nanocapsule, and utilizes the characteristics of the action of the low molecular heparin and the heparinase to enrich and degrade the nanocapsule on malignant tumor parts, thereby having active targeting effect on tumors and microenvironment thereof.
5. The composite lipid nanocapsule provided by the invention has a multifunctional synergistic effect. The composite lipid nanocapsule is formed by combining three parts: the lipid core is a reservoir for loading drugs, the chitosan has the effects of promoting transmembrane transport, controlling drug release and the like, and the low molecular heparin can actively target malignant tumor sites with high expression of heparinase. After entering systemic circulation, the composite lipid nanocapsule is easier to enrich and degrade at a tumor part compared with a normal tissue, and the chitosan-coated nanocapsule is exposed, so that the nanocapsule is easy to be absorbed by tumor cells, and a medicament is released intracellularly. The synergistic effect of multiple functional materials can enhance the tumor treatment effect of the medicine, reduce the toxic and side effects of the organism and improve the tolerance of patients.
6. The composite lipid nanocapsule provided by the invention has good drug encapsulation efficiency, stability and release controllability. The drug is loaded in the lipid core, is not interfered by the functional material of the capsule wall and the external environment, and improves the drug encapsulation efficiency and stability. The invention adopts the chitosan with strong positive charge, the chitosan can be tightly combined with the lipid core with negative charge on the surface and the low molecular heparin, and the stability of the nanocapsule in the body circulation can be improved; on the other hand, because the chitosan has strong positive charge and can interact with various drug molecules, the drug-loaded lipid nanoparticles (lipid core) are prepared firstly and then coated with the chitosan, the drug does not directly contact the chitosan, the interference on the drug is eliminated, the drug release rate is adjusted only by changing the amount of the chitosan adsorbed on the surface of the lipid core, and the drug release is more controllable.
7. The carrier material used by the composite lipid nanocapsule provided by the invention is mainly natural, has good biocompatibility, and is safe and nontoxic.
8. The preparation process of the composite lipid nanocapsule provided by the invention is simple and feasible, is beneficial to industrial production, and meets the requirement of clinical treatment.
Drawings
FIG. 1 schematic diagram of preparation method of composite lipid nanocapsule
FIG. 2 Structure of ascorbyl palmitate
FIG. 3 shows horizontal contour lines as factors of the star point design-effective surface optimization method in example 9 of the present invention.
FIG. 4 is a graph showing the distribution of particle size of the complex lipid nanocapsules of example 11 of the present invention
FIG. 5 shows a transmission electron micrograph of the complex lipid nanocapsule of example 11 of the present invention
FIG. 6 shows a transmission electron microscope image of the freeze-dried powder of the composite lipid nanocapsule of example 12 of the present invention
FIG. 7 shows the release profile of the composite lipid nanocapsule of example 13 of the present invention
Fig. 8 shows the in vivo tumor targeting distribution of the tumor-bearing mice injected with the complex lipid nanocapsules of example 14 of the present invention as a function of time, a: a common solution group; b: a lipid core group; c: composite lipid nanocapsule group
FIG. 9 shows the fluorescence intensity of tumor tissue of tumor-bearing mice injected with complex lipid nanocapsules for 24 hours in example 14 of the present invention
Detailed Description
The present invention is further described below with reference to specific examples, which are only illustrative and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and substitutions are within the scope of the invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. The instrument comprises the following steps:
experimental ultrahigh pressure homogenizer: nano DeBEE, BEE corporation, USA
Laser particle size potential meter: nicomp 380ZLS, PSS Corp USA
Reagent:
phospholipid: PC-98T, PC > 98%, Shanghai Avena pharmaceutical science and technology Limited
LIOID E80, PC 80-85%, Lipoid Gmbh in Germany
And (3) chitosan: CHITOSAN, Low molecular weight, Sigma-aldrich Sigma Aldrich
Low molecular weight heparin: enoxaparin sodium, Hebei dichroa febrifuga Biochemical pharmaceutical Co., Ltd
And (2) DiR: DiR iodide, cell membrane near-infrared fluorescent cyanine dye, AAT Bioquest, usa
EXAMPLE 1 Regulation of surface negative Charge effects of ascorbyl palmitate
Lipid cores containing different ratios of ascorbyl palmitate were prepared and compared with normal liposomes without ascorbyl palmitate.
TABLE 1 formulation and characterization of different ascorbyl palmitate ratios
1. Preparation method of formula A1 (common liposome):
(1) the prescribed amounts of phospholipids (PC: 80-85%) and cholesterol were dissolved in chloroform-methanol (88: 12) to give a clear and transparent solution, and the organic solvent was removed by rotary evaporation to give a lipid membrane.
(2) The lipid membrane obtained in step 1 was placed in a water bath at 40 to 60 ℃, and 25mL of a phosphate buffer (pH 7) containing about 0.2% tween 80 was slowly added with stirring to obtain a crude emulsion. Homogenizing the crude emulsion under high pressure to obtain liposome.
2. The preparation method of the formula A2-A5 comprises the following steps:
(1) dissolving the phospholipid (PC is more than or equal to 98%) and the ascorbyl palmitate in the formula amount in methanol to obtain a clear and transparent solution, removing the methanol in a water bath at the temperature of 30-40 ℃ by vacuum rotary evaporation, and collecting the obtained precipitate, namely the mixed membrane material.
(2) And (3) dissolving the mixed membrane material and the medium-chain oil obtained in the step (1) in dichloromethane to obtain a uniform solution, and removing the dichloromethane in a water bath at the temperature of 30-40 ℃ by vacuum rotary evaporation to obtain the lipid membrane compound.
(3) The lipid membrane complex obtained in step 2 was placed in a water bath at 40 to 60 ℃, and 50mL of a phosphate buffer (pH 7) containing about 2.5% glycerol was slowly added with stirring to obtain a crude emulsion. And homogenizing the coarse emulsion under high pressure to obtain blank nanoparticles with negative charges on the surface, namely blank lipid cores.
3. The characterization method comprises the following steps: the nano particles prepared from each part are diluted to proper concentration by water, and the average Diameter of light Intensity-Weighted Mean Diameter (particle Diameter for short), the polydispersity index (PDI) and the Zeta potential (potential for short) are measured by a laser particle size potential measuring instrument.
4. As a result: the potential absolute value of the negative charge on the surface of the common liposome without adding ascorbyl palmitate is lower, which is not beneficial to coating the capsule wall with positive charge subsequently. With the addition of ascorbyl palmitate, the negative charge density increased significantly. When the amount added reached a certain level (about 8% of the phospholipid), further increase in the amount of ascorbyl palmitate did not significantly change the negative charge density, presumably due to the fact that ascorbyl palmitate is close to saturation at the interfacial film.
Example 2 comparison of different Phospholipids
The effect of using phospholipids containing different proportions of phosphatidylcholine (PC: 80% to 100%) on the preparation of the lipid core was examined.
TABLE 2 prescription and characterization of different phospholipids
The preparation method comprises the following steps:
(1) dissolving the phospholipid and the ascorbyl palmitate in the prescription amount in methanol to obtain a clear and transparent solution, removing the methanol by vacuum rotary evaporation in a water bath at the temperature of 30-40 ℃, and collecting the obtained precipitate, namely the mixed membrane material.
(2) And (3) dissolving the mixed membrane material and the medium-chain oil obtained in the step (1) in dichloromethane to obtain a uniform solution, and removing the dichloromethane in a water bath at the temperature of 30-40 ℃ by vacuum rotary evaporation to obtain the lipid membrane compound.
(3) The lipid membrane complex obtained in step 2 was placed in a water bath at 40 to 60 ℃, and 50mL of a phosphate buffer (pH 7) containing about 2.5% glycerol was slowly added with stirring to obtain a crude emulsion. And homogenizing the coarse emulsion under high pressure to obtain blank nanoparticles with negative charges on the surface, namely blank lipid cores.
The results show that there is no significant difference in lipid cores made with phospholipids containing different proportions of phosphatidylcholine (PC: 80% to 100%).
Example 3 comparison of different greases
The effect of using different oils on the preparation of nanocapsule lipid cores was investigated.
TABLE 3 formulation and characterization of the different oil phases
The preparation method comprises the following steps:
(1) dissolving the phospholipid (PC is more than or equal to 98%) and the ascorbyl palmitate in the formula amount in methanol to obtain a clear and transparent solution, removing the methanol in a water bath at the temperature of 30-40 ℃ by vacuum rotary evaporation, and collecting the obtained precipitate, namely the mixed membrane material.
(2) And (3) dissolving the mixed membrane material obtained in the step (1) and the oil phase in the formula amount in dichloromethane to obtain a uniform solution, and removing the dichloromethane in a water bath at the temperature of 30-40 ℃ by vacuum rotary evaporation to obtain the lipid membrane compound.
(3) And (3) placing the lipid membrane complex obtained in the step (2) in a water bath at the temperature of 40-60 ℃, slowly adding 50mL of phosphate buffer solution (pH 7) containing about 2.5% of glycerol under stirring to obtain a crude emulsion, and homogenizing the crude emulsion at high pressure to obtain blank nanoparticles with negative charges on the surface, namely blank lipid cores.
The results show that lipid cores with small average particle size and rich surface negative charges can be prepared by using medium-chain oil or soybean oil as oil phase.
Example 4 preparation and characterization of paclitaxel lipid core
Preparing and characterizing a drug-loaded lipid core by taking an antitumor compound taxol as a model drug
TABLE 4 prescription composition and characterization
The preparation method comprises the following steps:
(1) dissolving phospholipid (80-85 percent of PC) and ascorbyl palmitate in the formula amount in methanol to obtain a clear and transparent solution, removing the methanol by vacuum rotary evaporation in a water bath at the temperature of 30-40 ℃, and collecting the obtained precipitate, namely the mixed membrane material.
(2) And (3) dissolving the mixed membrane material obtained in the step (1), paclitaxel and medium-chain oil in dichloromethane to obtain a uniform solution, and removing the dichloromethane in a water bath at the temperature of 30-40 ℃ by vacuum rotary evaporation to obtain the lipid membrane complex.
(3) The lipid membrane complex obtained in step 2 was placed in a water bath at 40 to 60 ℃, and 50mL of a phosphate buffer (pH 7) containing about 2.5% glycerol was slowly added with stirring to obtain a crude emulsion. Homogenizing the crude emulsion under high pressure to obtain paclitaxel lipid core with negative charges on the surface.
Example 5 encapsulation efficiency of paclitaxel lipid core
Method for measuring encapsulation efficiency of paclitaxel lipid core drug
(1) HPLC chromatographic conditions: ZORBAX Eclipse XDB-C18(4.6 mm. times.150 mm, 3.5 μm) as a column; the mobile phase was methanol-water-acetonitrile (23:41: 36): the detection wavelength was 227nm, the column temperature was 35 ℃, the flow rate was 1.2 mL/min, and the sample volume was 10. mu.L.
(2) The encapsulated and free drug was separated by microcolumn centrifugation: adding Sephadex G50 into 5mL syringe (filter paper at bottom), filling to 5mL scale, naturally flowing water, compacting to form uniform gel column without fracture, and rotating at 2000 rpm-1Centrifuging for 3 min to dehydrate the gel column to obtain gel microcolumn for use. 0.5mL of paclitaxel lipid core solution is uniformly dripped on the upper end of the microcolumn for 2000 r.m-1Centrifuging for 3 min, and collecting eluent. Washing the gel micro-column with water, 0.5mL each time, 2000 r.m.-1Centrifuging for 3 min, washing for 3 times, and collecting eluate. All eluates were combined, dissolved in methanol-glacial acetic acid (200: 1) and diluted to an appropriate concentration, and the paclitaxel content was determined by HPLC as the amount of the encapsulated drug (W1). Dissolving 0.5mL of paclitaxel lipid core solution in methanol-glacial acetic acid (200: 1), diluting to appropriate concentration, and determining paclitaxel content by HPLC method to obtain total amount of paclitaxel (Wtotal) in lipid core solution before column centrifugation
The encapsulation efficiency is W1/W total multiplied by 100%
(3) The drug encapsulation efficiency of the paclitaxel lipid core prepared by the method of example 4 was: 96.0 percent
EXAMPLE 6 Effect of cholesterol addition
The effect of adding different proportions of cholesterol on the preparation of the paclitaxel lipid core was examined.
TABLE 5 prescription and characterization of cholesterol at various ratios
The preparation method comprises the following steps:
(1) dissolving phospholipid (80-85 percent of PC) and ascorbyl palmitate in the formula amount in methanol to obtain a clear and transparent solution, removing the methanol by vacuum rotary evaporation in a water bath at the temperature of 30-40 ℃, and collecting the obtained precipitate, namely the mixed membrane material.
(2) And (3) dissolving the mixed membrane material obtained in the step (1), paclitaxel, medium-chain oil and cholesterol in a prescription amount in dichloromethane to obtain a uniform solution, and performing vacuum rotary evaporation in a water bath at the temperature of 30-40 ℃ to remove the dichloromethane to obtain the lipid membrane complex.
(3) The lipid membrane complex obtained in step 2 was placed in a water bath at 40 to 60 ℃, and 50mL of a phosphate buffer (pH 7) containing about 2.5% glycerol was slowly added with stirring to obtain a crude emulsion. Homogenizing the crude emulsion under high pressure to obtain paclitaxel lipid core with negative charges on the surface.
The results show that the paclitaxel lipid core with the drug encapsulation rate of more than 90 percent and rich negative charges can be prepared by adding the cholesterol with different proportions, which indicates that the addition of the cholesterol has no obvious influence on the preparation of the paclitaxel lipid core.
Example 7 comparison of different oil phase ratios
The effect of adding different proportions of oil on the preparation of the paclitaxel lipid core was examined.
TABLE 6 formulation and characterization of different oil phase ratios
The preparation method comprises the following steps:
(1) dissolving phospholipid (80-85 percent of PC) and ascorbyl palmitate in the formula amount in methanol to obtain a clear and transparent solution, removing the methanol by vacuum rotary evaporation in a water bath at the temperature of 30-40 ℃, and collecting the obtained precipitate, namely the mixed membrane material.
(2) And (3) dissolving the mixed membrane material obtained in the step (1) with the paclitaxel, the medium-chain oil and the cholesterol in the prescribed amount in dichloromethane to obtain a uniform solution, and removing the dichloromethane in a water bath at the temperature of 30-40 ℃ by vacuum rotary evaporation to obtain the lipid membrane compound.
(3) The lipid membrane complex obtained in step 2 was placed in a water bath at 40 to 60 ℃, and 50mL of a phosphate buffer (pH 7) containing about 2.5% glycerol was slowly added with stirring to obtain a crude emulsion. Homogenizing the crude emulsion under high pressure to obtain paclitaxel lipid core with negative charges on the surface.
The results show that the paclitaxel lipid core with the drug encapsulation rate of more than 90% and rich negative charges can be prepared by the oil phase formulas with different proportions.
Example 8 comparison of different drug loadings
And (3) investigating the influence of different paclitaxel drug-loading rates on the encapsulation efficiency of the lipid core drug.
TABLE 7 prescription for different drug loadings
The preparation method comprises the following steps:
(1) dissolving phospholipid (80-85 percent of PC) and ascorbyl palmitate in the formula amount in methanol to obtain a clear and transparent solution, removing the methanol by vacuum rotary evaporation in a water bath at the temperature of 30-40 ℃, and collecting the obtained precipitate, namely the mixed membrane material.
(2) And (3) dissolving the mixed membrane material obtained in the step (1) with paclitaxel and medium-chain oil in a prescription amount in dichloromethane to obtain a uniform solution, and removing the dichloromethane in a water bath at the temperature of 30-40 ℃ by vacuum rotary evaporation to obtain the lipid membrane compound.
(3) The lipid membrane complex obtained in step 2 was placed in a water bath at 40 to 60 ℃, and 50mL of a phosphate buffer (pH 7) containing about 2.5% glycerol was slowly added with stirring to obtain a crude emulsion. Homogenizing the crude emulsion under high pressure to obtain paclitaxel lipid core with negative charges on the surface.
The results showed that the paclitaxel lipid core of formula Z3 precipitated the drug after 24 hours of storage at room temperature. The paclitaxel lipid cores of the prescription Z1 and the prescription Z2 have good stability and higher encapsulation efficiency (more than 80%).
EXAMPLE 9 amount of Chitosan
And (3) observing the influence of the dosage of the chitosan, including different chitosan concentrations and solution volumes, on the preparation of the nanocapsule coated on the first layer of the capsule wall by adopting a star point design-effect surface optimization method. The results were analyzed with Design-Expert 8.0 software.
(1) The preparation method of the paclitaxel lipid core comprises the following steps:
TABLE 8 prescription composition and characterization
a) The prescribed amount of phospholipid (PC:80-85 percent) and ascorbyl palmitate are dissolved in methanol to obtain clear and transparent solution, the clear and transparent solution is subjected to vacuum rotary evaporation in water bath at the temperature of 30-40 ℃ to remove the methanol, and the obtained precipitate, namely the mixed membrane material, is collected.
b) And dissolving the obtained mixed membrane material, paclitaxel and medium-chain oil in dichloromethane to obtain a uniform solution, and removing the dichloromethane in a water bath at the temperature of 30-40 ℃ by vacuum rotary evaporation to obtain the lipid membrane complex.
c) The obtained lipid membrane complex was placed in a water bath at 40 to 60 ℃, and 50mL of a phosphate buffer (pH 7) containing about 2.5% glycerol was slowly added with stirring to obtain a crude emulsion. Homogenizing the crude emulsion under high pressure to obtain paclitaxel lipid core with negative charges on the surface.
(2) The preparation method of the nanocapsule coated with the first layer of the capsule wall comprises the following steps: slowly dripping a proper amount of paclitaxel lipid core solution with negative charges on the surface into a proper amount of chitosan solution (0.005mol/L hydrochloric acid is used as a solvent) with certain concentration under stirring, and adsorbing chitosan on the surface of a lipid core through self-assembly, thereby obtaining the nanocapsule with positive charges on the surface.
(3) The prescription design and effect index of the star point design-effect surface optimization method are as follows:
a Central combination method (CCD) of a star point design-effect surface optimization method is used as a model, and prescription design is carried out by adopting two factors and five levels. 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 two factors to be considered, selecting five levels for 13 experiments, wherein the number of non-central points is 8, the number of central points is 5,
the factor levels are tabulated below:
table 9 star point design factor horizontal table
Effect index and analysis method: and (3) analyzing the result by Design-Expert 8.0 software by taking the total evaluation normalization (OD) obtained by performing mathematical transformation on the particle size, PDI and surface potential of the prepared nanocapsule as an effect index.
Calculation of the overall score normalization value (OD): firstly, the effect that the smaller the value is, the better the effect is, and the effect that the larger the value is, the better the d value is calculated by adopting a Hassan method, wherein the smaller the particle size and the PDI value is, the better the effect is, and the larger the surface potential value is, the better the effect is. The overall normalized value (OD) is the geometric mean of the individual effects:
dparticle size＝(ymax-yi)/(ymax-ymin)
dPDI＝(ymax-yi)/(ymax-ymin)
dElectric potential＝(yi-ymin)/(ymax-ymin)
Wherein, ymaxIs the maximum value of the effect, yminIs the minimum value of the effect, yiIs the current measured value
Geometric mean (d) is the total normalized value (OD)Particle size，dPDI，dElectric potential)
(4) As a result:
TABLE 10 Chitosan-coated nanocapsule self-assembly formulation combination
The results show that the higher the chitosan concentration in the formulation, the larger the volume ratio of chitosan solution to lipid core solution, the smaller the particle size of the produced nanocapsule, and the higher the electropositivity of the surface charge. Therefore, the amount of chitosan used can be varied as desired, including varying the chitosan concentration and solution volume to produce nanocapsules of desired particle size and potential that coat the first capsule wall.
The results of the 13 trials were fitted by a quadratic polynomial fitting (Actual Equation) which was:
OD＝-6.601+32.072X1+3.241X2-7.001X1X2-33.994X1 2-0.328X2 2。
the coefficient evaluation model (Coded evaluation) is:
OD＝0.826+0.224X1+0.266X2-0.175X1X2-0.085X1 2-0.082X2 2。
regression coefficient R of quadratic polynomial fitting20.9848, r 0.9924, F test P value less than 0.01, is a better fit model. Drawing X by Design-Expert 8.0 software1、X2Contour plots of the same OD are shown in FIG. 3.
Combining experimental measurements and contour plots, the preferred ranges are: 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. More preferred ranges are: 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.
Through conversion, the preferable range according to the mass ratio is as follows: the dosage of the chitosan is 0.015-0.045 g, more preferably 0.01875-0.03125 g per gram of phospholipid.
Example 10 amount of Low molecular heparin
And (3) inspecting the influence of the dosage of the low molecular heparin, including the concentration and the solution volume of different low molecular heparins, on the preparation of the composite lipid nanocapsule coating the second layer of the capsule wall.
(1) The preparation method of the nanocapsule coated with the first layer of the capsule wall comprises the following steps:
TABLE 11 prescription composition and characterization
a) The prescribed amount of phospholipid (PC:80-85 percent) and ascorbyl palmitate are dissolved in methanol to obtain clear and transparent solution, the clear and transparent solution is subjected to vacuum rotary evaporation in water bath at the temperature of 30-40 ℃ to remove the methanol, and the obtained precipitate, namely the mixed membrane material, is collected.
b) And dissolving the obtained mixed membrane material, paclitaxel and medium-chain oil in dichloromethane to obtain a uniform solution, and removing the dichloromethane in a water bath at the temperature of 30-40 ℃ by vacuum rotary evaporation to obtain the lipid membrane complex.
c) The obtained lipid membrane complex was placed in a water bath at 40 to 60 ℃, and 50mL of a phosphate buffer (pH 7) containing about 2.5% glycerol was slowly added with stirring to obtain a crude emulsion. Homogenizing the crude emulsion under high pressure to obtain paclitaxel lipid core with negative charges on the surface.
d) Slowly dripping 1 part of paclitaxel lipid core solution into 2.5 parts of 0.20mg/mL chitosan solution (0.005mol/L hydrochloric acid is used as a solvent) under stirring, and performing self-assembly to make chitosan adsorbed on the surface of the lipid core so as to coat the lipid core with a first layer of capsule wall, thereby obtaining the nano-capsules (CS-NCs) with positive charges on the surface for coating the first layer of capsule wall.
(2) The preparation method of the composite lipid nanocapsule coated with the second layer of the capsule wall comprises the following steps: slowly dripping a proper amount of CS-NCs solution with positive charges on the surface into a proper amount of stirred low-molecular-weight heparin solution (pH7.5) with a certain concentration, and adsorbing the low-molecular-weight heparin on the surface of the nanocapsule through self-assembly so that the nanocapsule is coated with a second layer of capsule wall, thereby obtaining the paclitaxel composite lipid nanocapsule.
(3) The prescription design and effect index of the star point design-effect surface optimization method are as follows:
central combination method (Central C) of effect surface optimization method by star point designomposite, CCD) as a model, and a prescription design is performed using two factors, five levels. The volume ratio (V) of the low molecular heparin concentration to the low molecular heparin solution to the CS-NCs nanocapsule solution is usedLow molecular heparin：VCS-NCs) For two factors to be considered, five levels were selected for each factor, and 13 experiments were performed, wherein the number of non-central points was 8 and the number of central points was 5. The particle size, PDI and potential of the prepared composite lipid nanocapsule are taken as effect indexes for investigation.
The factor levels are tabulated below:
table 12 star point design factor horizontal table
(4) Investigation result of star point design-effect surface optimization method
TABLE 13 self-assembly formulation combination of composite lipid nanocapsules coated with low-molecular heparin
The results show that the composite lipid nanocapsules with small particle size and high surface negative charge can be prepared by different low-molecular heparin concentrations and the volume ratios of different low-molecular heparin solutions and CS-NCs nanocapsule solutions in the prescription combination designed by the asterisks, and the prescription has no obvious difference. Therefore, the amount of the low-molecular heparin can be changed according to requirements, including the concentration and the solution volume of the low-molecular heparin, so as to prepare the required composite lipid nanocapsule coating the second layer of the capsule wall.
The concentration of the low molecular heparin to be inspected is 0.05-0.35 mg/mL, and the volume ratio of the low molecular heparin solution to the chitosan-coated nanocapsule solution is 1.25-2.75.
(5) The concentration of low molecular weight heparin and the volume of the solution were continuously changed:
TABLE 14 self-assembly formulation combination of composite lipid nanocapsules coated with low molecular heparin
The result shows that when the concentration of the low molecular heparin is 0.06-0.08 mg/mL, and the volume ratio of the low molecular heparin solution to the chitosan-coated nano-capsule solution is 1.0-1.2, the composite lipid nano-capsule with smaller particle size and higher negative charges on the surface can still be obtained.
And (3) combining the experimental results of (4) and (5), wherein the concentration of the low molecular heparin to be investigated is 0.05-0.35 mg/mL, and the volume ratio of the low molecular heparin solution to the chitosan-coated nanocapsule solution is 1.00-2.75. Through conversion, the preferable range according to the mass ratio is as follows: the dosage of the low molecular weight heparin relative to each gram of chitosan is 0.42-5.25 g.
Example 11 preparation and characterization of paclitaxel Complex lipid nanocapsules
TABLE 15 formulation composition of composite lipid nanocapsules
The preparation method comprises the following steps:
(1) dissolving phospholipid (80-85 percent of PC) and ascorbyl palmitate in the formula amount in methanol to obtain a clear and transparent solution, removing the methanol by vacuum rotary evaporation in a water bath at the temperature of 30-40 ℃, and collecting the obtained precipitate, namely the mixed membrane material.
(2) And (3) dissolving the mixed membrane material obtained in the step (1), paclitaxel and medium-chain oil in dichloromethane to obtain a uniform solution, and removing the dichloromethane in a water bath at the temperature of 30-40 ℃ by vacuum rotary evaporation to obtain the lipid membrane complex.
(3) The lipid membrane complex obtained in step 2 was placed in a water bath at 40 to 60 ℃, and 50mL of a phosphate buffer (pH 7) containing about 2.5% glycerol was slowly added with stirring to obtain a crude emulsion. Homogenizing the crude emulsion under high pressure to obtain paclitaxel lipid core with negative charges on the surface.
(4) Slowly dripping 1 part of paclitaxel lipid core solution into 2.5 parts of 0.20mg/mL chitosan solution (0.005mol/L hydrochloric acid as solvent) under stirring, allowing chitosan to adsorb on lipid core surface by self-assembly, and coating the lipid core with first layer of capsule wall to obtain nanometer capsule (CS-NCs) with positive charges on surface
(5) Slowly dripping 1 part of CS-NCs solution with positive charges on the surface into 1.2 parts of 0.08mg/mL low molecular heparin solution (pH7.5) under stirring, and performing self-assembly to enable the low molecular heparin to be adsorbed on the surface of the nanocapsule so as to enable the nanocapsule to be coated with a second layer of capsule wall, thereby obtaining the paclitaxel composite lipid nanocapsule (LH-NCs).
(II) an encapsulation efficiency determination method:
and (2) determining the drug encapsulation efficiency of the nanocapsules (CS-NCs) and the composite lipid nanocapsules (LH-NCs) coating the first layer of the capsule wall by adopting a low-speed centrifugation method, wherein the method specifically comprises the following steps:
(1) HPLC chromatographic conditions: ZORBAX Eclipse XDB-C18(4.6 mm. times.150 mm, 3.5 μm) as a column; the mobile phase was methanol-water-acetonitrile (23:41: 36): the detection wavelength was 227nm, the column temperature was 35 ℃, the flow rate was 1.2 mL/min, and the sample volume was 50. mu.L.
(2) The encapsulated and free drug was separated using low speed centrifugation: taking 0.5mL of CS-NCs solution or paclitaxel composite lipid nanocapsule solution, diluting with water to 10mL, shaking up, taking about 8mL, rotating at 1000 r.m.-1Centrifugation is carried out for 10 minutes, and unencapsulated paclitaxel crystal aggregates are precipitated. Then 2mL to 25mL volumetric flask of the supernatant was taken, 12mL of methanol-glacial acetic acid (200: 1) was added to dissolve completely, and then diluted to the scale with mobile phase, shaken well, and the amount of paclitaxel was determined by HPLC method as the amount of the encapsulated drug E1. And taking 2mL of solution before centrifugation, and determining the content of the paclitaxel by using an HPLC method to obtain the total amount E0 of the paclitaxel in the nanocapsule solution before centrifugation. The encapsulation efficiency was calculated as follows.
Percent encapsulation rate is E1/E0 multiplied by 100 percent
(III) results:
TABLE 16 characterization of composite lipid nanocapsules
The results show that in the preparation process of the composite lipid nanocapsule, the particle size is slightly increased along with the increase of the number of layers of the capsule wall coated by the lipid core, the surface charge of the particles is in an alternating positive and negative charge form, the drug encapsulation rate is more than 90 percent, and the paclitaxel composite lipid nanocapsule can be successfully prepared through the electrostatic adsorption effect of the positive and negative charges. The particle size distribution diagram and transmission electron micrograph of the composite lipid nanocapsule are shown in figure 4 and figure 5 respectively.
Example 12 preparation and characterization of paclitaxel composite lipid nanocapsule lyophilized powder
The preparation method comprises the following steps: the paclitaxel composite lipid nanocapsule solution (LH-NCs) prepared by the method of the embodiment 11 of the invention is taken, 6g of trehalose serving as a freeze-drying propping agent is added into every 100mL of the solution to be completely dissolved, shaken up and freeze-dried to obtain freeze-dried powder of the composite lipid nanocapsule.
The freeze-drying procedure is as follows.
TABLE 17 Freeze drying procedure
The characterization method comprises the following steps: taking a proper amount of taxol complex lipid nanocapsule freeze-dried powder, and measuring after redissolving the taxol complex lipid nanocapsule freeze-dried powder by using normal saline.
As a result: the average particle diameter is 159.2 +/-15.6 nm, the polydispersity index (PDI) is 0.273 +/-0.013, the Zeta potential is-53.96 +/-5.67 mV, and the drug encapsulation efficiency is 99.47 +/-1.04%. The transmission electron micrograph is shown in figure 6.
Example 13 Release degree of Complex lipid nanocapsules
The paclitaxel composite lipid nanocapsule freeze-dried powder prepared by the method of the embodiment 12 is adopted to examine the drug release characteristics
By adopting an oscillation dialysis method, taking a proper amount of taxol composite lipid nanocapsule freeze-dried powder, re-dissolving with normal saline, shaking up to obtain a composite lipid nanocapsule solution with the taxol concentration of about 0.12mg/mL, putting 1mL into a dialysis bag, and sealing. The dialysis bag was placed in 50mL of release medium at 37 ℃ with shaking at 100 per minute. 1mL of release medium was removed at 0.5, 1, 2, 4, 8, 12, 24, 36, 48 hours, respectively, while 1mL of fresh medium was added. The amount of drug in the released release medium was measured by HPLC as described in example 11 of the present invention, and the cumulative release rate of the drug was calculated. Respectively investigating the release curves of the composite lipid nanocapsule in two release media, wherein the release media are respectively as follows: phosphate buffer (containing 0.5% tween 80) at ph5.0 and phosphate buffer (containing 0.5% tween 80) at ph 7.4.
The cumulative release rate of the drug is (cumulative drug release amount/total drug amount) multiplied by 100 percent
The result shows that the drug cumulative release rate of the paclitaxel complex lipid nano-capsule is 91.5% in pH5.0 medium at 48 hours, and 82.2% in pH7.4 medium at 48 hours, and the release curve is shown in figure 7.
The release curves were mathematically model-fitted using DDSolver 1.0 software, the release curves at pH5.0 and pH7.4 both best fit the classical Higuchi diffusion equation, the results are shown in the following table,
TABLE 18 fitting of Release curves
The result shows that the drug in the paclitaxel composite lipid nanocapsule is mainly influenced by the self-diffusion behavior in the drug release process of two mediums, and belongs to a diffusion controlled release mechanism.
Example 14 Targeted tumor distribution in tumor-bearing mice of Complex lipid nanocapsules
Preparing the composite lipid nanocapsule by using a fluorescent dye DiR, and inspecting the in-vivo tumor targeting distribution of the composite lipid nanocapsule by using a small animal living body imaging technology.
Preparation of (mono) DiR composite lipid nanocapsule
TABLE 19 formulation composition of DiR Complex lipid nanocapsules
(1) Dissolving the phospholipid (PC is more than or equal to 98%) and the ascorbyl palmitate in the prescription amount in methanol to obtain a clear and transparent solution, removing the methanol in a water bath at the temperature of 30-40 ℃ by vacuum rotary evaporation, and collecting the obtained precipitate, namely the mixed membrane material.
(2) And (3) dissolving the mixed membrane material obtained in the step (1), DiR and medium-chain oil in dichloromethane to obtain a uniform solution, and removing the dichloromethane in a water bath at the temperature of 30-40 ℃ by vacuum rotary evaporation to obtain the lipid membrane compound.
(3) The lipid membrane complex obtained in step 2 was placed in a water bath at 40 to 60 ℃, and 50mL of a phosphate buffer (pH 7) containing about 2.5% glycerol was slowly added with stirring to obtain a crude emulsion. And homogenizing the coarse emulsion under high pressure to obtain a DiR lipid nanoparticle solution with a negatively charged surface, namely a lipid core.
(4) Slowly dripping 1 part of lipid core solution into 2.5 parts of 0.20mg/mL chitosan solution (0.005mol/L hydrochloric acid is used as a solvent) under stirring, and performing self-assembly to enable the chitosan to be adsorbed on the surface of the lipid core so that the lipid core is coated with a first layer of capsule wall, thereby obtaining the nano-capsules (DiR-CS-NCs) with positive charges on the surface.
(5) Slowly dripping 1 part of DiR-CS-NCs solution with positive charges on the surface into 1.2 parts of 0.08mg/mL low molecular heparin solution (pH7.5) under stirring, and allowing the low molecular heparin to be adsorbed on the surface of the nanocapsule through self-assembly so as to coat the second layer of the capsule wall with the nanocapsule, thereby obtaining the DiR composite lipid nanocapsule (DiR-LH-NCs) with negative charges on the surface.
And (3) characterization results:
TABLE 20 characterization of DiR composite lipid nanocapsules
(II) Targeted tumor distribution in tumor-bearing mice
Establishment of 4T1 tumor-bearing mouse model: balb/c mice, female, body weight 17 + -2 g. Each mouse was inoculated with 1X 10 of 4T1 breast cancer cells in situ on the fourth mammary fat pad6Feeding normally until the tumor volume grows to about 800mm3For use in experiments.
Experimental groups: 4T1 model mice were randomly divided into 3 groups of 1 mouse each, namely a DiR solution group (2% DMSO-water as a solvent), a DiR lipid core group and a DiR complex lipid nanocapsule group. And (3) administering each group by tail vein injection with the dose of 0.1mg/kgDiR, carrying out IVIS imaging by using a small animal living body imaging system at 2, 4, 8, 12 and 24 hours after administration, rapidly taking out tumor tissues after the experiment is finished at 24 hours, carrying out luminescence detection, and recording the tumor luminescence intensity of each group.
The results show that the fluorescence intensity of tumor tissues of the DiR complex lipid nano-capsule group and the lipid core group is obviously higher than that of the solution group, and the results are shown in figure 8. After the 24-hour experiment, tumor tissues were taken out, and the luminescence values of the groups are shown in figure 9: DiR solution group (8.314X 10)8p/s), DiR lipid core group (8.764X 10)9p/s), DiR composite lipid nanocapsule group (1.073X 10)10p/s) shows good tumor targeting distribution effect of the composite lipid nanocapsule and the lipid core.
Claims (10)
1. A composite lipid nanocapsule composition, the composition comprising: grease, phospholipid, ascorbyl palmitate, an antitumor compound, chitosan and low molecular heparin; the particle size range of the composite lipid nanocapsule is 10-1000 nm.
2. The composite lipid nanocapsule composition of claim 1 wherein said composite lipid nanocapsule composition further comprises cholesterol.
3. The composite lipid nanocapsule composition of any one of claims 1-2, wherein said composite lipid nanocapsule composition is formed by combining a negatively charged lipid core, a positively charged first wall, and a negatively charged second wall, by electrostatic adsorption between the positively and negatively charged groups; the surface negatively charged lipid core comprises the following components: grease, phospholipid, ascorbyl palmitate and an antitumor compound; the positively charged first capsule wall comprises chitosan; the negatively charged second layer capsule wall comprises low molecular weight heparin; the surface negatively charged lipid core may also comprise cholesterol.
4. The complex lipid nanocapsule composition of claim 1, wherein the amount of ascorbyl palmitate is 0.001-100 g per g of phospholipid; the dosage of the grease relative to each gram of phospholipid is 0.001-100 ml; the dosage of the chitosan relative to each gram of phospholipid is 0.001-100 grams; the dosage of the low molecular weight heparin relative to each gram of chitosan is 0.001-100 g.
5. The composite lipid nanocapsule composition of claim 1 wherein the lipid is one or more lipids 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 heparin is heparin with lower molecular weight and salt thereof prepared by depolymerization; the anti-tumor compound comprises a taxane compound.
6. The composite lipid nanocapsule composition of claim 5, wherein the taxane compound comprises paclitaxel; the dosage of the paclitaxel relative to each gram of the phospholipid is 0-100 g.
7. The method of preparing the composite lipid nanocapsule composition of any one of claims 1-6, wherein the lipid core having a negatively charged surface comprises the steps of:
(1) dissolving phospholipid and ascorbyl palmitate in an organic solvent A to obtain a uniform solution, and removing the organic solvent A to obtain a mixed membrane material;
(2) dissolving the mixed membrane material, the grease and the anti-tumor compound obtained in the step 1 in an organic solvent B to obtain a uniform solution, and removing the organic solvent B to obtain a lipid membrane compound;
(3) and (3) dispersing the lipid membrane compound obtained in the step (2) in a water phase, emulsifying and homogenizing to obtain the lipid nanoparticles with negative charges on the surface, namely the lipid core with negative charges on the surface.
8. The method for preparing the composite lipid nanocapsule of any one of claims 1-6, wherein the composite lipid nanocapsule formed by combining the lipid core with a negatively charged surface, the first wall with a positive charge, and the second wall with a negative charge through electrostatic adsorption between the positively and negatively charged groups comprises the following steps:
(1) dispersing a lipid core with negative charges on the surface in a water phase containing chitosan, and adsorbing the chitosan on the surface of the lipid core through self-assembly so that the lipid core is coated with a first layer of capsule wall, thereby obtaining a nano capsule with positive charges on the surface;
(2) dispersing the nano-capsules with positive charges on the surfaces obtained in the step (1) into a water phase containing low-molecular heparin, adsorbing the low-molecular heparin on the surfaces of the nano-capsules through self-assembly, and coating the nano-capsules with a second layer of capsule walls to obtain the composite lipid nano-capsules.
9. The preparation method according to claim 7, wherein the organic solvent A includes but is not limited to one or more of methanol, ethanol, tetrahydrofuran; 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.
10. Use of the complex lipid nanocapsule composition of any one of claims 1-6 in the preparation of an anti-tumor medicament.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910392474.8A CN111920782A (en) | 2019-05-13 | 2019-05-13 | Composite lipid nanocapsule composition and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910392474.8A CN111920782A (en) | 2019-05-13 | 2019-05-13 | Composite lipid nanocapsule composition and preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111920782A true CN111920782A (en) | 2020-11-13 |
Family
ID=73282698
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910392474.8A Pending CN111920782A (en) | 2019-05-13 | 2019-05-13 | Composite lipid nanocapsule composition and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111920782A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114028623A (en) * | 2021-10-26 | 2022-02-11 | 江苏朴芃医疗科技有限公司 | Cardiac shunt |
| CN116236417A (en) * | 2023-03-07 | 2023-06-09 | 大连理工大学 | Polymer multilayer skin sun-screening agent coated carrier material and preparation method thereof |
| CN117368670A (en) * | 2023-11-07 | 2024-01-09 | 东莞市一丁精密模具组件有限公司 | Method and system for flexibly detecting discharge characteristic of mold |
Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1153474A (en) * | 1994-07-19 | 1997-07-02 | 海马金/全氟碳公司 | Stable oil-in-water emulsions incorporating taxine (taxol) and method of making same |
| CN1602854A (en) * | 2003-09-29 | 2005-04-06 | 北京华京五方实用纳米科技开发有限公司 | Taxel preparation for intravenous injection |
| CN101249063A (en) * | 2008-03-14 | 2008-08-27 | 褚红女 | Calciparine/sodium salt nano oral preparation and preparation technique thereof |
| CN101396343A (en) * | 2007-09-26 | 2009-04-01 | 中国医学科学院药物研究所 | Paclitaxel submicron emulsion using lipid composite as middle carrier |
| CN101601648A (en) * | 2009-06-09 | 2009-12-16 | 沈阳药科大学 | Sub-microemulsion used for intravenous injection of polyene yew alcohol phospholipid composite and preparation method thereof |
| CN102105134A (en) * | 2008-07-23 | 2011-06-22 | 印度血清及疫苗有限公司 | Stable injectable oil-in-water docetaxel nanoemulsion |
| US20120201862A1 (en) * | 2009-06-08 | 2012-08-09 | Cuesta Regueiro Ana Belen | Process for the preparation of colloidal systems for the delivery of active compounds |
| CN102847200A (en) * | 2011-06-28 | 2013-01-02 | 先健科技(深圳)有限公司 | A method for preparing electrostatically self-assembled drug carried layer |
| KR101255149B1 (en) * | 2011-10-14 | 2013-04-22 | 포항공과대학교 산학협력단 | Composition for nucleic acid delivery using metal nanoparticles and preparing method thereof |
| CN105769817A (en) * | 2016-03-09 | 2016-07-20 | 辽宁科技学院 | NLC (Nanostructured lipid carrier) with surface modified by TCS (thiolated chitosan) and preparation method of NLC |
| US20180200197A1 (en) * | 2015-07-17 | 2018-07-19 | Uniwersytet Jagiellonski | Nanocapsule for delivery of lipophilic compound and process of preparation thereof |
-
2019
- 2019-05-13 CN CN201910392474.8A patent/CN111920782A/en active Pending
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1153474A (en) * | 1994-07-19 | 1997-07-02 | 海马金/全氟碳公司 | Stable oil-in-water emulsions incorporating taxine (taxol) and method of making same |
| CN1602854A (en) * | 2003-09-29 | 2005-04-06 | 北京华京五方实用纳米科技开发有限公司 | Taxel preparation for intravenous injection |
| CN101396343A (en) * | 2007-09-26 | 2009-04-01 | 中国医学科学院药物研究所 | Paclitaxel submicron emulsion using lipid composite as middle carrier |
| CN101249063A (en) * | 2008-03-14 | 2008-08-27 | 褚红女 | Calciparine/sodium salt nano oral preparation and preparation technique thereof |
| CN102105134A (en) * | 2008-07-23 | 2011-06-22 | 印度血清及疫苗有限公司 | Stable injectable oil-in-water docetaxel nanoemulsion |
| US20120201862A1 (en) * | 2009-06-08 | 2012-08-09 | Cuesta Regueiro Ana Belen | Process for the preparation of colloidal systems for the delivery of active compounds |
| CN101601648A (en) * | 2009-06-09 | 2009-12-16 | 沈阳药科大学 | Sub-microemulsion used for intravenous injection of polyene yew alcohol phospholipid composite and preparation method thereof |
| CN102847200A (en) * | 2011-06-28 | 2013-01-02 | 先健科技(深圳)有限公司 | A method for preparing electrostatically self-assembled drug carried layer |
| KR101255149B1 (en) * | 2011-10-14 | 2013-04-22 | 포항공과대학교 산학협력단 | Composition for nucleic acid delivery using metal nanoparticles and preparing method thereof |
| US20180200197A1 (en) * | 2015-07-17 | 2018-07-19 | Uniwersytet Jagiellonski | Nanocapsule for delivery of lipophilic compound and process of preparation thereof |
| CN105769817A (en) * | 2016-03-09 | 2016-07-20 | 辽宁科技学院 | NLC (Nanostructured lipid carrier) with surface modified by TCS (thiolated chitosan) and preparation method of NLC |
Non-Patent Citations (6)
| Title |
|---|
| GIULIA IACCARINO ET AL: "Matrix metalloproteinase-cleavable nanocapsules for tumor-activated drug release", 《ACTA BIOMATERIALI》 * |
| R. VECCHIONE ET AL: "Tunable stability of monodisperse secondary O/W nano-emulsions", 《NANOSCALE》 * |
| SOON HONG YUK ET AL: "Enhancement of the Targeting Capabilities of the Paclitaxel-Loaded Pluronic Nanoparticles with a Glycol Chitosan/Heparin Composite", 《MOLECULAR PHARMACEUTICS》 * |
| 刘蜀宝 主编: "《药剂学》", 31 July 2007, 河南科学技术出版社 * |
| 梁艳: "层层自组装肝素/壳聚糖微胶囊的制备及其性能研究", 《中国优秀博硕士学位论文全文数据库(硕士)医药卫生科技辑》 * |
| 王沛 等: "层层自组装纳米药物载体在癌症治疗中的研究进展", 《科学通报》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114028623A (en) * | 2021-10-26 | 2022-02-11 | 江苏朴芃医疗科技有限公司 | Cardiac shunt |
| CN114028623B (en) * | 2021-10-26 | 2024-02-27 | 江苏朴芃医疗科技有限公司 | Cardiac shunt |
| CN116236417A (en) * | 2023-03-07 | 2023-06-09 | 大连理工大学 | Polymer multilayer skin sun-screening agent coated carrier material and preparation method thereof |
| CN117368670A (en) * | 2023-11-07 | 2024-01-09 | 东莞市一丁精密模具组件有限公司 | Method and system for flexibly detecting discharge characteristic of mold |
| CN117368670B (en) * | 2023-11-07 | 2024-03-26 | 东莞市一丁精密模具组件有限公司 | Method and system for flexibly detecting discharge characteristic of mold |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106177986B (en) | A kind of lipid-polymer medicine-carried nano particles and its preparation method and application | |
| CN102271659B (en) | Liposome of irinotecan or its hydrochloride and preparation method thereof | |
| Chi et al. | Docetaxel-loaded biomimetic nanoparticles for targeted lung cancer therapy in vivo | |
| CN111920782A (en) | Composite lipid nanocapsule composition and preparation method and application thereof | |
| CN103120645B (en) | Irinotecan or irinotecan hydrochloride lipidosome and preparation method thereof | |
| Song et al. | Surface-modified PLGA nanoparticles with PEG/LA-chitosan for targeted delivery of arsenic trioxide for liver cancer treatment: Inhibition effects enhanced and side effects reduced | |
| CN101396346B (en) | Paclitaxel lipid composite | |
| CN110408047B (en) | Nano coordination polymer and preparation method and application thereof | |
| CN113384530B (en) | Polysaccharide core Nanocells and preparation method and application thereof | |
| CN101708337B (en) | Preparation method of human serum albumin nano granules coated with oxaliplatin | |
| WO2008080369A1 (en) | Steady liposomal composition | |
| Karabasz et al. | Cytotoxic activity of paclitaxel incorporated into polyelectrolyte nanocapsules | |
| CN103655484B (en) | A kind ofly utilize self-assembling technique method preparing taxol slow release microballoons and products thereof | |
| CN110200921A (en) | A kind of anti-inflammatory liposome and its preparation and application with targeting | |
| CN112472686B (en) | Lipid nanoparticle of PEG-PLA-SN38 linker and preparation method thereof | |
| CN102805729A (en) | Vinflunine liposome preparation and preparation method of vinflunine liposome preparation | |
| KR20160117794A (en) | Drug delivery system comprising gelatine nano-particles slowly releasing hardly-water soluble substances and its preparation method | |
| CN105708800B (en) | A kind of Lovastatin silica nodule for targetting breast carcinoma stem cell | |
| CN100350912C (en) | Nanometer partical administration system of prostaglandin E1 coated with polyglycol derived phospholipid | |
| CN103263672A (en) | Preparation method and application of nanometer particles of taxane drugs | |
| CN107669637B (en) | Artemether liposome for injection and preparation method and application thereof | |
| CN111388449B (en) | Polyornithine/carboxymethyl lentinan layer-by-layer self-assembly drug carrier and preparation method thereof | |
| CN109700782B (en) | High-drug-loading-rate disulfiram nanoparticles and application thereof in tumor prevention and treatment | |
| CN104546718B (en) | A kind of long circulating Rabeprazole liposome composition and its preparation method and application | |
| CN106309411B (en) | A kind of Quercetin and taxol convey lung sucking nano target porous polymeric particle and preparation method thereof altogether |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |