CN115006351A - PEG (polyethylene glycol) positively charged liposome and preparation method thereof - Google Patents
PEG (polyethylene glycol) positively charged liposome and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of medicines, and particularly relates to a PEG (polyethylene glycol) positively charged liposome and a preparation method thereof. The PEG-modified liposome with positive charge provided by the invention comprises a myelin basic protein compound of a compressed nucleic acid medicament, a liposome with positive charge wrapping the surface of the myelin basic protein compound of the compressed nucleic acid medicament, and polyethylene glycol modified on the surface of the liposome with positive charge; the positively charged liposome is DOTAP/DHA-DOPC/CTBBA liposome. The PEG-modified liposome with positive charge prepared by the invention has relatively small particle size and potential, good encapsulation efficiency and low toxicity; the liposome can be used for encapsulating more nucleic acid medicaments, can well encapsulate the nucleic acid medicaments, and has high stability within 30 days. In addition, the PEG positively charged liposome prepared by the invention has better capability of inhibiting the migration of B16-F1 cells.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a PEG (polyethylene glycol) positively charged liposome and a preparation method thereof.
Background
Liposome delivery systems are one of the most widely studied drug delivery systems today. Liposomes are closed vesicles composed of lipid bilayers consisting of phospholipids and cholesterol. Because the liposome kernel is a special structure of an aqueous phase, the liposome can be used for encapsulating hydrophilic drugs, such as DNA, protein, nucleic acid drugs and the like. The good liposome is used as a drug delivery carrier, can wrap a series of antigens and immunoadjuvants with different physicochemical properties, protects protein polypeptide antigens from being degraded, can promote phagocytosis and presentation of antigen presenting cells against the antigens, and improves the specific immunoreaction of an organism.
Liposome delivery systems, as a new class of drug delivery systems, are widely used in antineoplastic drugs, such as Doxil from Sequus, Myocet from Elan; antifungal agents, such as Vyxeos by Jaxx corporation; and Onpattro applied to the field of RNAi. The transportation of the drug of interest from the outside of the cell to the target and its expression requires a series of obstacles. Endocytosis is the main mechanism for liposome drug to enter cells, and in order to achieve effective delivery efficiency, liposome is used as a drug delivery system, and obstacles need to be overcome. Liposomes are of interest and benefit as drug delivery systems by effectively encapsulating a wide variety of water-soluble, differently sized molecules with different dissociation constants. The dosage is controlled to achieve the aim of reducing the administration dosage and reducing the side effect under the condition of meeting the curative effect. The targeting property of the liposome is improved by modifying the surface of the liposome and improving the function, and the circulation time and the action part are controlled. Liposomes have been developed as drug delivery systems that can now be loaded with chemotherapeutic drugs, antimicrobial and viral (SARS, HIV, AIV, RV, etc.) drugs, antiparasitic drugs, genetic material, vaccines, therapeutic proteins, anti-inflammatory drugs, hormones, natural drugs, and the like. The drug-loaded liposome has passive targeting property, active targeting property (surface modified liposome) and slow release property, and the drug is coated by the liposome to protect the drug, improve the stability of the drug, reduce the exposure of sensitive tissues to the drug with high toxicity and reduce the drug toxicity. The cationic liposome is used as one type of liposome delivery system, carries positive charges through the surface of the cationic liposome, is adsorbed to the surface of a negatively charged cell through electrostatic interaction, and enters the cell through endocytosis to form an inclusion body; the cationic lipid in the cationic liposome and the negatively charged lipid in the endosome generate electrostatic interaction, the negatively charged lipid is converted into the cavity from the outside of the endosome to form a neutral ion pair, and the drug is separated from the liposome, enters cytoplasm and finally plays a corresponding function.
Chinese patent application 201910847775.5 discloses a liposome, its preparation method, liposome assembly and carrying liposome complex, the liposome provided by the invention has long circulation, stimulation responsiveness, targeting property and other properties, and multiple modifiable sites, and can be used for preparing multifunctional biological agents, thereby providing more convenient choices for diagnosis and treatment of diseases. However, it does not consider the problems of encapsulation efficiency and stability of liposomes.
The study of liposomes has progressed from the early, common, ordinary liposomes to multifunctional liposomes and has shown its potential utility in many ways. However, the number of liposome preparations currently on the market is limited due to difficulties in ideal formulation, stability, etc. of liposomes. The existing liposome generally has the problems of low entrapment rate and low stability. Therefore, it is urgent to develop a liposome having not only a good encapsulation efficiency but also a high stability.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a PEGylated positively charged liposome and a preparation method thereof. The PEG-modified liposome with positive charge prepared by the invention has relatively small particle size and potential, good encapsulation efficiency and low toxicity; the liposome can be used for encapsulating more nucleic acid medicaments, can well encapsulate the nucleic acid medicaments, and has high stability within 30 days. In addition, the PEG positively charged liposome prepared by the invention has better capability of inhibiting the migration of B16-F1 cells.
The technical scheme of the invention is as follows:
a PEGylated positively charged liposome comprises a myelin basic protein complex of a compressed nucleic acid drug, a positively charged liposome wrapping the surface of the myelin basic protein complex of the compressed nucleic acid drug, and polyethylene glycol modified on the surface of the positively charged liposome; the positively charged liposome is DOTAP/DHA-DOPC/CTBBA liposome.
Further, the DOTAP is (2, 3-dioleoyl-propyl) -trimethylammonium-chloride salt.
Furthermore, the DHA-DOPC is neutral lipid and is a compound of docosahexaenoic acid and dioleoylphosphatidylcholine.
Further, the particle size of the PEGylated positively charged liposome is 110-130 nm.
Furthermore, the nucleic acid medicament is micromolecular RNA, including tumor RNA, the tumor RNA is encapsulated, and positive charge liposome encapsulating the tumor RNA can directly cause anti-tumor immune response.
Further, the polyethylene glycol is distearoylphosphatidylethanolamine-polyethylene glycol 2000(DSPE-PEG 2000).
The invention also provides a preparation method of the PEG positively charged liposome, which comprises the following steps:
(1) respectively dissolving DOTAP, DHA-DOPC and CTBBA in an organic solvent to obtain a DOTAP solution, a DHA-DOPC solution and a CTBBA solution, and dissolving polyethylene glycol in DEPC treated water to obtain a polyethylene glycol solution;
(2) preparing DOTAP/DHA-DOPC/CTBBA liposome by a thin film hydration method;
(3) mixing the nucleic acid drug and myelin basic protein to prepare a myelin basic protein compound of the compressed nucleic acid drug;
(4) mixing the DOTAP/DHA-DOPC/CTBBA liposome with the myelin basic protein complex of the compressed nucleic acid medicament to obtain the positively charged liposome of the myelin basic protein of the compressed nucleic acid medicament;
(5) adding polyethylene glycol solution into positive charged liposome of myelin basic protein of compressed nucleic acid medicine, placing the mixture at 40-50 deg.C for 1-2min, and then at 20-30 deg.C for 8-15 min.
Further, the preparation method of the DOTAP/DHA-DOPC/CTBBA liposome comprises the following steps:
(a) adding DOTAP solution, DHA-DOPC solution and CTBBA solution into a container, adding chloroform, and mixing to obtain a mixed solution;
(b) performing rotary evaporation on the mixed solution obtained in the step (a) for 40-50min under vacuum conditions and water bath conditions of 100rpm and 40-60 ℃ on a rotary evaporator to form a uniform lipid film on the wall of a bottle, and remaining concentrated solution in the bottle;
(c) cooling the bottle to 5 ℃, carrying out heat preservation and crystallization for 7-9h, filtering to obtain wet crystals, and drying the wet crystals at 60-70 ℃ until the drying weight loss is less than or equal to 1% to obtain a crude product;
(d) and (c) hydrating the crude product obtained in the step (c), carrying out ultrasonic treatment for 20-30min after full hydration, extruding through a 100nm polycarbonate film, and repeatedly extruding for 20 times to obtain the product.
Further, the volume ratio of the DOTAP solution, the DHA-DOPC solution and the CTBBA solution in the step (a) is 9-10:2.5-3.5: 12-14.
Further, the polyethylene glycol solution is added in the step (5) in an amount of 4-6% of the volume of the positively charged liposome of the myelin basic protein of the compressed nucleic acid drug.
Myelin basic protein is a strongly basic membrane protein synthesized by oligodendrocyte of central nervous system and Schwann cell of peripheral nervous system of vertebrate central nervous system, and contains multiple basic amino acids. Compared with the traditional liposome carrier constructed by complexing protamine with nucleic acid medicaments, myelin basic protein can replace protamine to be used as a part of the liposome carrier due to the strong basicity and the effect of easily permeating a blood brain barrier, and is applied to the entrapment of the nucleic acid medicaments.
The PEG liposome with positive charges prepared by the invention takes a compound of DHA and DOPC as neutral lipid, can better lead the liposome to pass through a blood brain barrier and stably release encapsulated drugs, increases the encapsulation of nucleic acid drugs by the complexation of myelin basic protein, can better improve the drug leakage problem of the liposome by using cholesterol derivative CTBBA to replace cholesterol in the traditional liposome, and can promote the blood circulation time of the liposome in vivo and improve the uptake efficiency of cells by embedding PEG on the surface of the liposome. The prepared PEG liposome with positive charge can be used for encapsulating more nucleic acid drugs by using less liposome, the drug encapsulation rate of the nucleic acid drugs is up to 77.45 percent, the prepared PEG liposome with positive charge finished product not only can be used for encapsulating more nucleic acid drugs under the condition of low phospholipid usage amount, but also has high stability and low toxicity, and the encapsulated RNA can be highly expressed in vivo.
Compared with the prior art, the invention has the following advantages:
(1) the PEG-based liposome with positive charge prepared by the invention has relatively small particle size and potential, good encapsulation efficiency and low toxicity;
(2) the PEG liposome with positive charge prepared by the invention can be used for encapsulating more nucleic acid medicaments by using less liposome, can well encapsulate the nucleic acid medicaments, and has high stability within 30 d;
(3) the PEG-modified positively charged liposome prepared by the invention has better capability of inhibiting migration of B16-F1 cells.
Drawings
FIG. 1 is a schematic of the synthesis of PEGylated positively charged liposomes of the present invention;
FIG. 2 is a graph of the particle size distribution of PEGylated positively charged liposomes prepared in example 3 of the present invention;
FIG. 3 is a graph of the potential distribution of PEGylated positively charged liposomes prepared in example 3 of the present invention;
FIG. 4 is a transmission electron microscope image of PEGylated positively charged liposomes prepared in example 3;
FIG. 5 is a graph of the effect of different concentrations of PEGylated positively charged liposome solutions on cell viability of B16-F1 cells.
Detailed Description
The present invention is further described in the following description of the specific embodiments, which is not intended to limit the invention, but various modifications and improvements can be made by those skilled in the art according to the basic idea of the invention, within the scope of the invention, as long as they do not depart from the basic idea of the invention.
The starting materials used in the present invention are all commercially available unless otherwise specified. For example, myelin basic protein is available from Shanghai Crystal wind Biotechnology, Inc.; siRNA is available from shanghai gimbals pharmaceutical technology ltd, cat #: A02025.
a schematic of the synthesis of PEGylated positively charged liposomes of the invention is shown in FIG. 1.
Example 1A PEGylated positively charged Liposome
The PEGylated positively charged liposome comprises a myelin basic protein complex of a compressed nucleic acid drug, a positively charged liposome wrapping the surface of the myelin basic protein complex of the compressed nucleic acid drug, and polyethylene glycol modified on the surface of the positively charged liposome; the positively charged liposome is a DOTAP/DHA-DOPC/CTBBA liposome; the DOTAP is (2, 3-dioleoyl-propyl) -trimethylammonium-chloride; the DHA-DOPC is neutral lipid and is a compound of docosahexaenoic acid and dioleoylphosphatidylcholine. The nucleic acid drug is siRNA; the polyethylene glycol is distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000(DSPE-PEG 2000).
The preparation method of the PEG positively charged liposome comprises the following steps:
(1) sucking a certain amount of chloroform, adding the chloroform into a standard subpackaged DOTAP (dot in ether) bottle of 100 mg/bottle, transferring the dissolved solution to a 15mL centrifuge tube, and adding the chloroform to 10mL scale marks to obtain a DOTAP solution; adding 2.5mL of the LDHA-DOPC suspension into a 15mL centrifuge tube, and adding chloroform to 10mL of a scale mark to obtain a DHA-DOPC solution; accurately weighing 50mg of CTBBA powder, adding the CTBBA powder into a 15mL centrifuge tube, and adding chloroform to 10mL scale marks; accurately weighing 30mg of DSPE-PEG2000 powder, adding 3mL of DEPC treated water, and performing ultrasonic dissolution in a water bath to obtain a polyethylene glycol solution;
(2) preparing DOTAP/DHA-DOPC/CTBBA liposome by a thin film hydration method;
(3) taking 13 mu L of myelin basic protein solution, 50 mu L of nucleic acid medicine solution and 35 mu L of DEPC (diethyl phthalate) treated water, mixing, whirling for 30s, and incubating at room temperature for 4min to obtain a myelin basic protein compound of compressed nucleic acid medicine; the preparation method of the myelin basic protein solution comprises the following steps: taking 18mg of myelin basic protein and mixing with 2mL of phosphate buffer solution with pH7.4 to obtain the compound; the preparation method of the nucleic acid medicine solution comprises the following steps: dissolving siRNA in sterile water to prepare the siRNA with the solubility of 0.5 mu g/mu L;
(4) mixing 65 mu L of DOTAP/DHA-DOPC/CTBBA liposome obtained in the step (2) with 10 mu L of the myelin basic protein complex of the compressed nucleic acid medicament obtained in the step (3), and obtaining positive charge liposome of the myelin basic protein of the compressed nucleic acid medicament at room temperature for 10 min;
(5) adding polyethylene glycol solution into positive charged liposome of myelin basic protein of compressed nucleic acid medicine, wherein the addition amount of polyethylene glycol solution is 4% of the positive charged liposome volume of myelin basic protein of compressed nucleic acid medicine, placing the mixture at 40 deg.C for 1min, and then at 20 deg.C for 8 min.
The structural formula of CTBBA is as follows:
the CTBBA synthetic route is as follows: prepared according to the prior literature. [1] Liu XM, Yang B, Wang YL, etc. Photo somerisable cholestol derivitives as photo-trigger of lipomes: Effect of lipid polarity, temperature, incorporation ratio, and cholestol, Biochim Biophys Acta,2005,1720(1-2),28-34.[2] Liu XM, Yang B, Wang YL, etc. A New nanoscopic pulse Drug Delivery System, Chem Mater,2005,17(11),2792-2795.
The preparation method of the DHA-DOPC suspension comprises the following steps:
precisely weighing 4mg of DOPC and 0.5mg of DHA0 in a glass tube respectively, adding 6mL of ethanol for dissolving, rotationally evaporating at the rotating speed of 100rpm at the temperature of 25 ℃ for 30min until the organic solvent is basically evaporated, observing the film forming effect after the rotational evaporation, and placing the film forming effect in a vacuum freeze dryer for freeze drying for 8h to remove residual ethanol. Taking out the sample with residual ethanol removed, adding 5mL ultrapure water, vortex-shaking for hydrating for 1min to form milky uniform lipid solution, transferring into an airtight syringe, and extruding 100nm polycarbonate membrane for 20 times to obtain the final product.
The preparation method of the DOTAP/DHA-DOPC/CTBBA liposome specifically comprises the following steps:
(a) adding a DOTAP solution, a DHA-DOPC solution and a CTBBA solution into a container, wherein the total volume of the DOTAP solution, the DHA-DOPC solution and the CTBBA solution is 15mL, the volume ratio of the DOTAP solution to the DHA-DOPC solution to the CTBBA solution is 9:3.5:14, adding 3mL of chloroform, and mixing to obtain a mixed solution;
(b) rotationally evaporating the mixed solution obtained in the step (a) for 40min under vacuum condition and water bath condition of 100rpm and 40 ℃ on a rotary evaporator to form a uniform lipid film on the bottle wall, and remaining the concentrated solution in the bottle;
(c) cooling the bottle to 5 ℃, carrying out heat preservation and crystallization for 7h, filtering to obtain wet crystals, and drying the wet crystals at 60 ℃ until the loss on drying is less than or equal to 1% to obtain a crude product;
(d) and (c) hydrating the crude product obtained in the step (c), carrying out ultrasonic treatment for 20min after full hydration, extruding the product through a 100nm polycarbonate film, and repeatedly extruding the product for 20 times to obtain the nano-crystalline polycarbonate.
The hydration treatment of step (d) is specifically operated as follows: and (c) adding 10mL of PBS buffer solution with the pH value of 7.4 into the crude product obtained in the step (c), and carrying out vortex oscillation for 2min to ensure that the lipid membrane is fully swelled until a milky uniform lipid solution is formed.
Example 2A PEGylated positively charged Liposome
The PEGylated positively charged liposome comprises a myelin basic protein complex of a compressed nucleic acid drug, a positively charged liposome wrapping the surface of the myelin basic protein complex of the compressed nucleic acid drug, and polyethylene glycol modified on the surface of the positively charged liposome; the positively charged liposome is a DOTAP/DHA-DOPC/CTBBA liposome; the DOTAP is (2, 3-dioleoyl-propyl) -trimethylammonium-chloride; the DHA-DOPC is neutral lipid and is a compound of docosahexaenoic acid and dioleoylphosphatidylcholine. The nucleic acid drug is siRNA; the polyethylene glycol is distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000(DSPE-PEG 2000).
The preparation method of the PEG positively charged liposome comprises the following steps:
(1) sucking a certain amount of chloroform, adding the chloroform into a standard subpackaged DOTAP (dot in ether) bottle of 100 mg/bottle, transferring the dissolved solution to a 15mL centrifuge tube, and adding the chloroform to 10mL scale marks to obtain a DOTAP solution; adding 2.5mL of the LDHA-DOPC suspension into a 15mL centrifuge tube, and adding chloroform to 10mL of a scale mark to obtain a DHA-DOPC solution; accurately weighing 50mg of CTBBA powder, adding the CTBBA powder into a 15mL centrifuge tube, and adding chloroform to 10mL scale marks; accurately weighing 30mg of DSPE-PEG2000 powder, adding 3mL of DEPC treated water, and performing ultrasonic dissolution in a water bath to obtain a polyethylene glycol solution;
(2) preparing DOTAP/DHA-DOPC/CTBBA liposome by a thin film hydration method;
(3) taking 13 mu L of myelin basic protein solution, 50 mu L of nucleic acid drug solution and 35 mu L of DEPC treated water, mixing, whirling for 30s, and incubating at room temperature for 7min to obtain myelin basic protein complex of compressed nucleic acid drug; the preparation method of the myelin basic protein solution comprises the following steps: taking 18mg of myelin basic protein and mixing with 2mL of phosphate buffer solution with pH7.4 to obtain the compound; the preparation method of the nucleic acid medicine solution comprises the following steps: dissolving siRNA in sterile water to prepare the siRNA with the solubility of 0.5 mu g/mu L;
(4) mixing 65 mu L of DOTAP/DHA-DOPC/CTBBA liposome obtained in the step (2) with 10 mu L of the myelin basic protein complex of the compressed nucleic acid medicament obtained in the step (3), and obtaining positive charge liposome of the myelin basic protein of the compressed nucleic acid medicament at room temperature for 20 min;
(5) adding polyethylene glycol solution into positive charged liposome of myelin basic protein of compressed nucleic acid medicine, wherein the addition amount of polyethylene glycol solution is 6% of the positive charged liposome volume of myelin basic protein of compressed nucleic acid medicine, placing the mixture at 50 deg.C for 2min, and then at 30 deg.C for 15 min.
The DHA-DOPC suspension was prepared in a similar manner to example 1.
The preparation method of the DOTAP/DHA-DOPC/CTBBA liposome specifically comprises the following steps:
(a) adding a DOTAP solution, a DHA-DOPC solution and a CTBBA solution into a container, wherein the total volume of the DOTAP solution, the DHA-DOPC solution and the CTBBA solution is 15mL, the volume ratio of the DOTAP solution to the DHA-DOPC solution to the CTBBA solution is 10:2.5:12, adding 4mL of chloroform, and mixing to obtain a mixed solution;
(b) rotationally evaporating the mixed solution obtained in the step (a) for 50min under vacuum condition and water bath condition of 100rpm and 60 ℃ on a rotary evaporator to form a uniform lipid film on the bottle wall, and remaining the concentrated solution in the bottle;
(c) cooling the bottle to 5 ℃, carrying out heat preservation and crystallization for 9 hours, filtering to obtain wet crystals, and drying the wet crystals at 70 ℃ until the drying weight loss is less than or equal to 1% to obtain a crude product;
(d) and (c) hydrating the crude product obtained in the step (c), carrying out ultrasonic treatment for 30min after full hydration, extruding the product through a 100nm polycarbonate film, and repeatedly extruding the product for 20 times to obtain the nano-crystalline polycarbonate.
Said step (d) of hydrating being specifically operative to: and (c) adding 10mL of PBS buffer solution with the pH value of 7.4 into the crude product obtained in the step (c), and carrying out vortex oscillation for 2min to ensure that the lipid membrane is fully swelled until a milky uniform lipid solution is formed.
Example 3A PEGylated positively charged Liposome
The PEGylated positively charged liposome comprises a myelin basic protein complex of a compressed nucleic acid drug, a positively charged liposome wrapping the surface of the myelin basic protein complex of the compressed nucleic acid drug, and polyethylene glycol modified on the surface of the positively charged liposome; the positively charged liposome is a DOTAP/DHA-DOPC/CTBBA liposome; the DOTAP is (2, 3-dioleoyl-propyl) -trimethylammonium-chloride; the DHA-DOPC is neutral lipid and is a compound of docosahexaenoic acid and dioleoylphosphatidylcholine. The nucleic acid drug is siRNA; the polyethylene glycol is distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000(DSPE-PEG 2000).
The preparation method of the PEG positively charged liposome comprises the following steps:
(1) sucking a certain amount of chloroform, adding the chloroform into a standard subpackaged DOTAP (dot in ether) bottle of 100 mg/bottle, transferring the dissolved solution to a 15mL centrifuge tube, and adding the chloroform to 10mL scale marks to obtain a DOTAP solution; adding 2.5mL of the LDHA-DOPC suspension into a 15mL centrifuge tube, and adding chloroform to 10mL of a scale mark to obtain a DHA-DOPC solution; accurately weighing 50mg of CTBBA powder, adding the CTBBA powder into a 15mL centrifuge tube, and adding chloroform to 10mL scale marks; accurately weighing 30mg of DSPE-PEG2000 powder, adding 3mL of DEPC treated water, and performing ultrasonic dissolution in a water bath to obtain a polyethylene glycol solution;
(2) preparing DOTAP/DHA-DOPC/CTBBA liposome by a film hydration method;
(3) taking 13 mu L of myelin basic protein solution, 50 mu L of nucleic acid drug solution and 35 mu L of DEPC treated water, mixing, whirling for 30s, and incubating at room temperature for 5min to obtain myelin basic protein complex of compressed nucleic acid drug; the preparation method of the myelin basic protein solution comprises the following steps: taking 18mg of myelin basic protein and mixing with 2mL of phosphate buffer solution with pH7.4 to obtain the compound; the preparation method of the nucleic acid medicine solution comprises the following steps: dissolving siRNA in sterile water to prepare the siRNA with the solubility of 0.5 mug/muL;
(4) mixing 65 mu L of DOTAP/DHA-DOPC/CTBBA liposome obtained in the step (2) with 10 mu L of the myelin basic protein complex of the compressed nucleic acid medicament obtained in the step (3) to obtain a positively charged liposome of the myelin basic protein of the compressed nucleic acid medicament;
(5) adding polyethylene glycol solution into positive charged liposome of myelin basic protein of compressed nucleic acid medicine, wherein the addition amount of polyethylene glycol solution is 4.8% of the positive charged liposome volume of myelin basic protein of compressed nucleic acid medicine, placing the mixture at 45 deg.C for 2min, and then at 25 deg.C for 12 min.
The DHA-DOPC suspension was prepared in a similar manner to example 1.
The preparation method of the DOTAP/DHA-DOPC/CTBBA liposome specifically comprises the following steps:
(a) adding a DOTAP solution, a DHA-DOPC solution and a CTBBA solution into a container, wherein the total volume of the DOTAP solution, the DHA-DOPC solution and the CTBBA solution is 15mL, the volume ratio of the DOTAP solution to the DHA-DOPC solution to the CTBBA solution is 9.4:3.1:13.2, adding 4mL of chloroform, and mixing to obtain a mixed solution;
(b) rotationally evaporating the mixed solution obtained in the step (a) for 45min under the conditions of vacuum and water bath at 50 ℃ and 100rpm on a rotary evaporator to form a uniform lipid film on the bottle wall, and remaining the concentrated solution in the bottle;
(c) cooling the bottle to 5 ℃, carrying out heat preservation crystallization for 8 hours, filtering to obtain wet crystals, and drying the wet crystals at 65 ℃ until the drying weight loss is less than or equal to 1% to obtain a crude product;
(d) and (c) hydrating the crude product obtained in the step (c), carrying out ultrasonic treatment for 25min after full hydration, extruding the product through a 100nm polycarbonate film, and repeatedly extruding the product for 20 times to obtain the product.
The hydration treatment of step (d) is specifically operated as follows: and (c) adding 10mL of PBS buffer solution with the pH value of 7.4 into the crude product obtained in the step (c), and carrying out vortex oscillation for 2min to ensure that the lipid membrane is fully swelled until a milky uniform lipid solution is formed.
The DHA-DOPC solution was prepared in a similar manner to example 1.
Comparative example 1A positively charged liposome
The composition and preparation of the positively charged liposomes are similar to those of example 3.
The difference from example 3 is that myelin basic protein is replaced by protamine.
Comparative example 2A positively charged Liposome
The composition and preparation of the positively charged liposomes are similar to those of example 3.
The difference from example 3 is that DOTAP/DHA-DOPC/CTBBA liposomes were replaced by DOTAP/DOPC/CTBBA liposomes.
Test example I measurement of particle size and potential of liposomes
The particle size and potential of the pegylated, positively charged liposomes prepared in example 3 of this invention were measured using a malvern particle sizer. The method specifically comprises the following steps: and (3) placing the prepared PEG-modified liposome with positive charge in a sample cell, sealing the sample cell, placing the sample cell with the sample in a sample injection groove, closing the cover of the sample cell area, and testing. The particle size distribution of the PEGylated positively charged liposomes prepared in example 3 of the present invention is shown in FIG. 2, and the potential distribution of the PEGylated positively charged liposomes prepared in example 3 of the present invention is shown in FIG. 3. As can be seen from FIG. 2, the particle size of the PEGylated positively charged liposomes prepared according to the present invention was 110.35 nm. As can be seen in FIG. 3, the potential of the PEGylated positively charged liposomes prepared according to the present invention was 57.32 mV.
Second test example, liposome morphology detection
The morphology of the PEGylated positively charged liposomes prepared in example 3 of the present invention was examined using a transmission electron microscope. The method specifically comprises the following steps: mu.L of PEGylated positively charged liposome solution prepared in example 3 was applied to a copper mesh with carbon membrane and dried at room temperature. Dropping 2% phosphotungstic acid solution on the copper net, and drying at room temperature. And observing under a transmission electron microscope. FIG. 4 shows a TEM image of PEGylated positively charged liposomes prepared in example 3 of the present invention. As can be seen from FIG. 4, the PEG-modified liposome with positive charges prepared by the invention has uniform distribution, regular shape and particle size of about 110nm, which is consistent with the particle size measured by a Malvern particle sizer.
Test example III measurement of encapsulation efficiency of liposomes
Respectively centrifuging the PEGylated positively charged liposomes prepared in example 3 of the present invention at 4 ℃ for 1h using an ultra high speed centrifuge, and collecting the supernatant in a volume of V The cleaning is carried out on the waste water, determination of RNA concentration C in supernatant Using Quant-iT RiboGreen RNA Assay Kit Cleaning the upper part (ii) a Dissolving the precipitate obtained by centrifugation in DMSO, taking out an appropriate amount, mixing with heparin sodium solution, standing at room temperature for 1h, and recording the volume V of displacement The precipitate is formed by the reaction of the raw materials, and the measured RNA concentration C The precipitate is formed by the precipitation of the mixture, the encapsulation efficiency and recovery were calculated according to the following formula: encapsulation efficiency (%) - (1- (C) Go to ×V Go to )/(C Cleaning the upper part ×V Cleaning the upper part +C Precipitation of ×V Precipitation of ) X 100%; recovery (%) ═ C Cleaning the upper part ×V Cleaning the upper part +C Precipitation of ×V Precipitation of )/C Feeding material ×V Feeding material X 100%. The encapsulation efficiency of the PEGylated positively charged liposome prepared in example 3 of the invention is 77.45% and the recovery rate is 31.5%.
Experimental example four, comparative study of particle size, potential and entrapment efficiency of different liposomes
The blank liposome is prepared by a thin film hydration method. The method comprises the following specific steps: precisely weighing 10mg of soybean lecithin and 2mg of cholesterol in an eggplant-shaped bottle, adding 2mL of chloroform for dissolving, rotationally evaporating at the rotating speed of 80rpm for 15min at the temperature of 25 ℃ until the chloroform is basically evaporated, forming a lipid film on the wall of the eggplant-shaped bottle, placing the eggplant-shaped bottle in a vacuum freeze dryer for freeze drying for 5h to remove residual chloroform, taking out the eggplant-shaped bottle, adding 1mL of phosphate buffer solution, continuously shaking until the eggplant-shaped bottle is fully hydrated, transferring the eggplant-shaped bottle to an airtight injector, and extruding a 100nm polycarbonate film for 20 times to obtain a blank liposome.
The common charged liposome is prepared by ethanol injection method. The method comprises the following specific steps: precisely weighing DOTAP70mg and cholesterol 10mg in a test tube, adding 30mL of absolute ethyl alcohol, fully dissolving at 37 ℃ to form an oil phase, weighing 2 μ L of nucleic acid drug (siRNA) aqueous solution, fully mixing in the test tube, weighing 10mL of PBS buffer solution in a round-bottom flask, slowly injecting the mixed solution in the test tube into the PBS buffer solution by using an airtight syringe under the magnetic stirring of 50rpm at the water bath temperature of 50 ℃, and continuously stirring for 30min to obtain the common charged liposome.
The particle size and potential of the liposomes prepared in the blank liposome, the ordinary charged liposome, examples 1 to 3, and comparative examples 1 to 2 were measured by a Malvern particle sizer, and the particle size distribution index (PDI) was calculated. The encapsulation efficiency of the liposomes prepared by the conventional charged liposome, example 1, example 2 and example 3, and comparative example 1 and comparative example 2 was measured by referring to the method of test example three. Triplicate determinations were made. The results of comparing the particle size, potential and encapsulation efficiency of different liposomes are shown in table 1.
Table 1: comparison results of particle size, potential and entrapment efficiency of different liposomes
| Sample (I) | Particle size/nm | PDI | Zeta potential | Encapsulation efficiency% |
| Blank liposomes | 94.72±0.22 | 0.211±0.023 | -35.63±0.34 | / |
| Common charged liposomes | 131.64±0.73 | 0.175±0.011 | 61.92±0.66 | 62.54±0.96 |
| Example 1 | 114.79±0.83 | 0.323±0.027 | 60.12±1.15 | 74.23±0.39 |
| Example 2 | 114.32±0.57 | 0.338±0.071 | 58.12±2.65 | 72.63±0.51 |
| Example 3 | 110.35±0.81 | 0.311±0.006 | 57.32±2.48 | 77.45±0.39 |
| Comparative example 1 | 121.27±0.23 | 0.354±0.018 | 61.42±4.31 | 68.32±0.22 |
| Comparative example 2 | 112.18±0.66 | 0.331±0.005 | 58.36±0.97 | 68.12±0.85 |
As can be seen from Table 1, the PEG-modified positively charged liposomes prepared in examples 1-3 of the present invention have good encapsulation efficiency while having relatively small particle size and potential, as shown by the comparison of blank liposomes, conventional charged liposomes, examples 1, 2,3, comparative examples 1 and 2 with different liposomes, i.e., the PEG-modified positively charged liposomes prepared in the present invention have improved encapsulation efficiency.
Test example five examination of liposome stability
The PEGylated positively charged liposomes prepared in example 3 of the present invention were placed in an environment of 4 ℃ respectively. Particle size, potential and encapsulation efficiency were measured at 0d, 10d, 20d and 30d using a Malvern particle sizer and a Quant-iT RiboGreen RNA Assay Kit, with reference to the methods of test example three and test example four, respectively. Triplicate determinations were made. The test results are shown in table 2.
Table 2: results of Liposome stability test
As can be seen from Table 2, the stability of PEGylated positively charged liposomes at different times was examined, and it was found that the PEGylated positively charged liposomes were able to entrap nucleic acid drugs well and had high stability within 30 days.
Test example six detection of Liposome cell proliferation toxicity
The PEGylated positively charged liposomes prepared in example 3 according to the present invention were examined for their cytotoxicity to increase cell proliferation. The specific method comprises the following steps: B16-F1 cells in logarithmic phase are taken, digested and blown into single cell suspension; centrifuging at 1000r/min for 10min, and removing supernatant; adjusting the cell density to 0.6X 10 7 Preparing 200 mu L of cell suspension in a 96-well plate, and placing the cell suspension in an incubator at 37 ℃ for 12 hours; taking out the 96-well plate, adding 20 mu L of PEG liposome solution with different concentrations and positive charges, and continuously incubating for 24 h; adding culture solution into the 96-well plate and continuing culturing for 4 h; the absorbance at 560nm was measured. The cell proliferation toxicity was measured by MTT method, the added culture medium was MTT solution, and OD value was measured by a microplate reader. Cell viability was 100% (OD-blank OD of experimental group)/(OD-blank OD of control group). The effect of different concentrations of PEGylated positively charged liposome solutions on cell viability of B16-F1 cells is shown in FIG. 5. As can be seen in FIG. 5, B16-F1 cells were found to have good growth potential at a PEGylated positively charged liposome concentration of 7. mu.L/mL.
Test example seven cell migration ability test
The cell migration capacity of the PEG-modified positively charged liposome prepared by the invention is detected. The specific method comprises the following steps: B16-F1 cells in logarithmic phase are taken, digested and blown into a single cell suspension; centrifuging at 1000r/min for 10min, and removing supernatant; inoculating cells onto 6-well culture plate, and adjusting cell density to 2 × 10 5 Per mL, 37 ℃, 5% CO 2 And incubating overnight; B16-F1 cell monolayers were plated to the well bottom and the plate bottom was then plated using a tipHorizontally scribing from one side to the other side, and measuring 1mL PBS in each hole; the ordinary positively charged liposome solution prepared by the method of reference example four, the PEGylated positively charged liposome solution prepared in examples 1-3 of the present invention, and the positively charged liposome prepared in comparative examples 1-2 were administered separately and left at 37 ℃ with 5% CO 2 The scratch was observed in the incubators 12h and 24h, and the scratch area was measured. The calculation formula of the scratch healing rate is as follows: scratch healing rate (mobility) ═ T 0 Time area-T t Area per hour)/T 0 Area x 100%. The rate of healing of the scratch is used to reflect the speed of migration. The test results are shown in table 3.
Table 3: results comparing the scratch healing rates of different liposomes to B16-F1 cells
| Sample (I) | |
24h |
| Common charged liposomes | (36.4±2.3)％ | (77.2±3.4)％ |
| Example 1 | (6.2±0.4)％ | (17.8±3.2)％ |
| Example 2 | (8.1±2.6)％ | (21.6±1.3)％ |
| Example 3 | (5.2±1.3)％ | (16.3±2.6)％ |
| Comparative example 1 | (11.3±2.4)％ | (24.4±1.7)％ |
| Comparative example 2 | (12.1±0.7)％ | (23.8±2.2)％ |
As can be seen from Table 3, the scratch healing rates of the conventional positively charged liposomes, the PEGylated positively charged liposomes prepared in examples 1 to 3, and the positively charged liposomes prepared in comparative examples 1 to 2 were (36.4. + -. 2.3)%, (6.2. + -. 0.4)%, (8.1. + -. 2.6)%, (5.2. + -. 1.3)%, (11.3. + -. 2.4)%, (12.1. + -. 0.7)%, and (77.2. + -. 3.4)%, (17.8. + -. 3.2)%, (21.6. + -. 1.3)%, (16.3. + -. 2.6)%, (24.4. + -. 1.7)%, and (23.8. + -. 2.2)%, respectively, for 12 hours, for B16-F1 cells. Therefore, the PEG-based positively charged liposome prepared by the invention has obviously better capability of inhibiting B16-F1 cell migration than that of the common positively charged liposome and the positively charged liposome prepared in the comparative examples 1-2. Comparing example 1, example 2 and example 3, the healing rate of the scratch of the liposome prepared in example 3 is lowest in 12h and 24h, so that the PEGylated positively charged liposome prepared in example 3 of the invention has better capability of inhibiting the migration of B16-F1 cells.
Claims (9)
1. A PEGylated positively charged liposome is characterized by comprising a myelin basic protein complex of a compressed nucleic acid drug, a positively charged liposome wrapping the surface of the myelin basic protein complex of the compressed nucleic acid drug, and polyethylene glycol modified on the surface of the positively charged liposome; the positively charged liposome is DOTAP/DHA-DOPC/CTBBA liposome.
2. The PEGylated positively charged liposome of claim 1, wherein said DOTAP is (2, 3-dioleoyl-propyl) -trimethylammonium-chloride.
3. The pegylated, positively charged liposome of claim 1, wherein said DHA-DOPC is a neutral lipid which is a complex of docosahexaenoic acid and dioleoylphosphatidylcholine.
4. The PEGylated positively charged liposome of claim 1, wherein the PEGylated positively charged liposome has a particle size of 110-130 nm.
5. The PEGylated positively charged liposome of claim 1, wherein said polyethylene glycol is distearoylphosphatidylethanolamine-polyethylene glycol 2000.
6. The method of preparing a PEGylated positively charged liposome of any one of claims 1 to 5, comprising the steps of:
(1) respectively dissolving DOTAP, DHA-DOPC and CTBBA in an organic solvent to obtain a DOTAP solution, a DHA-DOPC solution and a CTBBA solution, and dissolving polyethylene glycol in DEPC treated water to obtain a polyethylene glycol solution;
(2) preparing DOTAP/DHA-DOPC/CTBBA liposome by a thin film hydration method;
(3) mixing the nucleic acid drug and myelin basic protein to prepare a myelin basic protein compound of the compressed nucleic acid drug;
(4) mixing the DOTAP/DHA-DOPC/CTBBA liposome with the myelin basic protein complex of the compressed nucleic acid medicament to obtain the positively charged liposome of the myelin basic protein of the compressed nucleic acid medicament;
(5) adding polyethylene glycol solution into positive charged liposome of myelin basic protein of compressed nucleic acid medicine, placing the mixture at 40-50 deg.C for 1-2min, and then at 20-30 deg.C for 8-15 min.
7. The method of preparing PEGylated positively charged liposomes of claim 6, wherein said DOTAP/DHA-DOPC/CTBBA liposomes are prepared by:
(a) adding DOTAP solution, DHA-DOPC solution and CTBBA solution into a container, adding chloroform, and mixing to obtain a mixed solution;
(b) performing rotary evaporation on the mixed solution obtained in the step (a) for 40-50min under vacuum conditions and water bath conditions of 100rpm and 40-60 ℃ on a rotary evaporator to form a uniform lipid film on the wall of a bottle, and remaining concentrated solution in the bottle;
(c) cooling the bottle to 5 ℃, carrying out heat preservation and crystallization for 7-9h, filtering to obtain wet crystals, and drying the wet crystals at 60-70 ℃ until the drying weight loss is less than or equal to 1% to obtain a crude product;
(d) and (c) hydrating the crude product obtained in the step (c), carrying out ultrasonic treatment for 20-30min after full hydration, extruding through a 100nm polycarbonate film, and repeatedly extruding for 20 times to obtain the product.
8. The method of preparing PEGylated positively charged liposomes of claim 7 wherein the volume ratio of said DOTAP solution, DHA-DOPC solution and CTBBA solution in step (a) is from 9 to 10:2.5 to 3.5:12 to 14.
9. The method of claim 6, wherein the polyethylene glycol solution is added in an amount of 4-6% of the positively charged liposome volume of myelin basic protein of compressed nucleic acid drug in step (5).
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| JP2022075622A (en) * | 2020-11-04 | 2022-05-18 | ▲復▼旦大学 | Drug delivery carrier and pharmaceutical preparation for delivering many kinds of therapeutic agents in common using the same |
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