CN109481404B - Preparation method of pH-sensitive imidazole liposome - Google Patents
Preparation method of pH-sensitive imidazole liposome Download PDFInfo
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- CN109481404B CN109481404B CN201811554431.7A CN201811554431A CN109481404B CN 109481404 B CN109481404 B CN 109481404B CN 201811554431 A CN201811554431 A CN 201811554431A CN 109481404 B CN109481404 B CN 109481404B
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- 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
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
- A61K9/1277—Processes for preparing; Proliposomes
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- 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
- A61K31/12—Ketones
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
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- 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
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Abstract
The invention relates to a preparation method of pH sensitive imidazole liposome, belonging to the field of pharmacy, and the method comprises the following steps: firstly, weighing lecithin and cholesterol, placing the lecithin and the cholesterol in a glass container, and adding ethanol into the glass container to dissolve the lecithin and the cholesterol to obtain a mixed solution; secondly, adding a curcumin absolute ethyl alcohol solution into the mixed solution, uniformly mixing, and removing the organic solvent by reduced pressure evaporation until a uniform and transparent film is formed on the bottle wall; adding phosphate buffer salt solution containing imidazole to hydrate, elute and disperse the film, carrying out water bath at 35-45 ℃ and normal pressure rotary hydration for 20-40min, and carrying out ultrasonic treatment for 10-20min to fully mix to obtain the pH sensitive imidazole liposome. The method adopts liposome technology, adopts lecithin and cholesterol as materials, and adds curcumin and imidazole at appropriate stage during preparation process, so as to solve the problem that fat-soluble medicine is not easy to dissolve in water, and simultaneously reduce toxicity of encapsulated medicine.
Description
Technical Field
The invention belongs to the field of pharmacy, and particularly relates to a preparation method of a pH-sensitive imidazole liposome.
Background
Liposomes refer to the microvesicles formed by encapsulating a drug within a lipid bilayer. The targeting sustained-release tablet plays an important role in a drug delivery system because of the characteristics of targeting, stability, sustained release, low toxicity and the like. The tumor area has special microenvironment compared with normal tissues, such as low pH, high temperature (pH less than 6.0, about 39-42 ℃), partial enzyme over-expression and the like, so that the treatment medicine does not have difficulty in exerting effective curative effect on the focus. The stability of the lipid bilayer in the pH sensitive liposome structure changes with changes in the environmental pH and may also be referred to as an acid sensitive liposome. It can not only improve the targeting property of the liposome, but also promote the full play of the drug effect and improve the expression level of the gene.
Curcumin (CUR) is a phenolic compound extracted from Curcuma longa of Zingiberaceae, and is the main active ingredient of Curcuma longa. Research shows that the CUR is a safe and effective medicine with multiple functions of cancer resistance, inflammation resistance, oxidation resistance and the like, wherein the curcumin has the inhibiting effect on the generation, proliferation and metastasis of multiple tumor cells when being used for preventing and treating tumors. However, CUR is insoluble in water and aqueous solutions are unstable at neutral to alkaline pH, and their in vitro anticancer activity drops significantly when stored at 4 ℃ for three days. Therefore, how to prepare pH sensitive liposomes with excellent performance for the lipid-soluble therapeutic drugs is a key topic in the field of antitumor research.
Disclosure of Invention
In order to solve the defects in the prior art, the invention aims to provide a preparation method of a pH sensitive imidazole liposome, which adopts a liposome technology, takes lecithin and cholesterol as materials, and curcumin and imidazole are added at proper stages in the preparation process to prepare the pH sensitive imidazole liposome, thereby solving the problem that fat-soluble medicines are not easy to dissolve in water, improving the effective rate of administration, and simultaneously reducing the toxicity of the encapsulated medicines.
In order to achieve the purpose, the invention adopts the specific scheme that:
a preparation method of pH sensitive imidazole liposome is characterized in that: the method comprises the following steps:
weighing lecithin and cholesterol, placing the lecithin and cholesterol in a glass container, and adding ethanol into the glass container to dissolve the lecithin and cholesterol to obtain a mixed solution;
step two, adding a curcumin absolute ethyl alcohol solution into the mixed solution, uniformly mixing, and removing the organic solvent by reduced pressure evaporation until a uniform and transparent film is formed on the bottle wall; adding phosphate buffer salt solution containing imidazole to hydrate, elute and disperse the film, carrying out water bath at 35-45 ℃ and normal pressure rotary hydration for 20-40min, and carrying out ultrasonic treatment for 10-20min to fully mix to obtain the pH sensitive imidazole liposome.
As a further optimization of the scheme, the mass ratio of the lecithin to the cholesterol is 3: 1-8: 1; the curcumin content is 0.1-0.3 mg; the content of imidazole is 0.001-0.005 g; the addition amount of the phosphate buffer solution is 15-30 mL, and the concentration is 0.01 mol/L.
As a further optimization of the scheme, the mass of the lecithin is 0.08g, and the mass of the cholesterol is 0.01 g; the curcumin content is 0.1 mg; the imidazole content is 0.001 g; the amount of phosphate buffered saline added was 30 mL.
As a further optimization of the scheme, the frequency of the ultrasonic wave is 30KHZ, the temperature is 30 ℃, and the time is 15 min.
And as a further optimization of the scheme, the content of the curcumin in the anhydrous ethanol solution of the curcumin in the step two is 0.05-0.15 mg.
As a further optimization of the scheme, the absolute pressure of the reduced pressure evaporation in the step two is 0.
Has the advantages that:
the preparation method optimizes the addition ratio of lecithin and cholesterol, the addition amount of other elements and the like, and simultaneously adds imidazole in the film hydration process, because the tumor cell environment is slightly acidic and the imidazole is protonated in the acidic environment, the medicine can be promoted to be released, so the prepared pH sensitive liposome can be used as a carrier of the tumor treatment medicine. The invention has the advantages of simple prescription, good stability of process conditions, easy industrialization and great popularization value. The novel targeted liposome prepared by the method has uniform particle size distribution, good stability and strong pH sensitivity. In vitro toxicity tests of the liposome prove that the lecithin and cholesterol as materials for preparing the liposome and the imidazole as a pH sensitive material added have very low and basically negligible toxicity to cells, meet the requirement of low toxicity of a drug carrier, and can be used as the drug carrier. In addition, the imidazole hybrid liposome has pH sensitivity, can improve the targeting property of the liposome, can promote the full play of drug effect and improve the expression level of genes, and has good application value in a drug delivery system.
Drawings
FIG. 1 is a graph of particle size distribution for pH sensitive liposomes;
FIG. 2 is a transmission electron micrograph of pH sensitive liposomes;
FIG. 3 is a graph of in vitro drug release from several liposomes at different pH conditions;
FIG. 4 is a graph comparing the cytotoxicity of C6 cells against common liposomes and pH sensitive liposomes at different concentrations.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention.
Example 1
1. Experimental procedure
The liposome is prepared by a film dispersion method. Weighing a certain mass of lecithin and cholesterol, placing the lecithin and cholesterol in a 50mL eggplant-shaped bottle, dissolving the lecithin and cholesterol in 10mL absolute ethyl alcohol, shaking the bottle to dissolve the lecithin and cholesterol, adding a certain amount of curcumin ethanol solution, evaporating the curcumin ethanol solution in a constant-temperature water bath at 40 ℃ under reduced pressure to remove an organic solvent, forming a uniform and transparent film on the wall of the bottle, adding a certain amount of Phosphate Buffer Solution (PBS) for dissolving imidazole to hydrate, elute and disperse the film, carrying out normal-pressure rotary hydration in the water bath, and carrying out ultrasonic dissolution to obtain a sample, namely the pH-sensitive imidazole liposome solution.
The temperature of the water bath is 35-45 deg.C, and the time is 20-40 min; ultrasonic frequency is 30KHZ, temperature is 30 deg.C, and time is 10-20 min; the pressure of reduced pressure evaporation is: the relative pressure is-0.01 MPa, which is equivalent to the absolute pressure of 0, under the vacuum condition; the concentration of the phosphate buffer solution is 0.01 mol/L; the concentration of the ethanol solution of curcumin can be configured to be 0.05-0.15
mg/mL。
In the step, the mass ratio of the lecithin to the cholesterol is 3: 1-8: 1; the curcumin content is 0.1-0.3 mg; the content of imidazole is 0.001-0.005 g; the addition amount of the phosphate buffer solution is 15-30 mL.
2. Best raw material ratio for researching liposome preparation
An orthogonal test is adopted to search the optimal proportion, the encapsulation rate of the liposome is taken as the index for investigation, and the optimal prescription is finally determined. Reasonably arranging and analyzing a test method for a plurality of factors, and finally selecting three main factors for analysis, namely, the ratio A of lecithin to cholesterol, the curcumin dosage B and the PBS dosage C, wherein each factor determines three levels, and the number of the levels of the factor A is 3:1, 5:1 and 8:1 in sequence; the horizontal number of the factor B is 0.1, 0.2 and 0.3 in sequence; the horizontal number of the factor C is 15, 20 and 30 in sequence, and an L9(33) orthogonal design table is selected. The experimental design and results are shown in table 1 below.
Table 1: experimental design and results of orthogonal experimental design optimization liposome prescription and preparation process
And (3) measuring results: as the ratio of lecithin to cholesterol increased, the encapsulation efficiency increased; as the curcumin content increased, the encapsulation efficiency decreased; the encapsulation efficiency increased with increasing PBS usage. Analysis Table 1 gave the optimum level combination A3B1C3Namely the ratio of lecithin to cholesterol is 8:1, wherein 0.08g of lecithin and 0.01g of cholesterol are added; the curcumin content is 0.1 mg; PBS was used in an amount of 30 mL.
3. Prescription optimization
Under the optimized optimal conditions, different amounts of pH sensitive material imidazole are added to respectively prepare the pH sensitive liposome. The encapsulation efficiency is taken as a survey index, and the dosage of the better imidazole is finally determined to be 0.001-0.005g, and the optimal content is 0.001 g.
4. Measuring the shape and particle size distribution of the pH sensitive liposome
Dynamic Light Scattering (DLS) was used to detect the particle size distribution. 0.2mL of freshly prepared liposomal suspension was diluted with 2.5mL of double distilled water. Each assay was repeated 3 times. The results are shown in FIG. 2, and the measurement results are: the average particle diameter was 141nm (dispersion index PDI: 0.233). The transmission electron microscope shows that the liposome is spherical and has uniform size distribution.
5. In vitro drug release assay
After dialysis to remove free curcumin, the in vitro drug release of the liposomes was determined by dialysis. The pH-sensitive liposome containing imidazole and the ordinary liposome were placed in a dialysis bag, and dialyzed with PBS (simulated body fluid) having pH5.0(0.01M) and pH7.4(0.01M), respectively, as external fluids. Into 10mL of the buffer solution, dialysis bags containing 1mL of liposomes were placed, respectively. Drug release experiments were performed in a 37 + -1 deg.C constant temperature water bath. At fixed time intervals, the drug-releasing external liquid is taken out for drug content measurement, and the same amount of fresh buffer external liquid is added in time. The drug content in the drug-releasing external liquid is detected by fluorescence.
As shown in FIG. 3, the cumulative release of the liposome containing imidazole was larger than that of the ordinary liposome in the same period of time. In addition, curcumin released more rapidly at pH5.0 than at pH 7.4. This finding is due to the fact that imidazole has a pKa of about 6.0 and that protonation of imidazole in a meta-acid environment can disrupt the bilayer structure of liposomes, resulting in a decrease in binding at ph5.0 and a rapid release of drug.
6. Stability test
Several imidazole liposome suspensions with different compositions are kept standing and protected from light at 4 ℃, and the corresponding entrapment rates of the several liposome sample solutions are measured on days 0, 2, 5 and 7 respectively. The stability was evaluated by using the leakage rate as an index. The results are shown in table 2 below.
Table 2: stability of pH sensitive liposomes
As can be seen from the above table, the stability difference between the two is not large, and in general, the stability of the pH sensitive liposome is better, and the leakage rate of the blank liposome is larger.
7. In vitro toxicity testing of liposomes
The non-loaded liposomes were subjected to in vitro toxicity assay using MTT method with C6 cells. C6 cells were plated at 6000 cells/well in 96-well plates. After 24h incubation in DMEM (200. mu.L/well) containing 10% FBS, 200. mu.L of fresh medium containing liposomes at the specified concentration was added and incubation continued for 48 h. After replacing the well solution with 200. mu.L of fresh medium, 20. mu.L of MTT solution (5mg/mL) was added to each well and incubated for 4 h. The medium was removed and 150 μ L DMSO was added to each well. The absorbance value (OD) was measured at 570nm by a microplate reader (Bio-Rad, Model 550, USA). The relative cell activity was calculated by the following formula: cell activity (%) [ OD570 (sample)/OD 570 (control) ] × 100. Wherein OD570 (control) represents the measurement without liposomes, and OD570 (sample) represents the measurement with liposomes.
The determination result is shown in fig. 4, when the concentration of the liposome reaches 200 μ g/mL, the relative survival rate of the C6 cells is above 80%, which indicates that the lecithin and cholesterol as the materials for preparing the liposome and the imidazole as the added pH sensitive material have very little toxicity to the cells, are almost negligible, meet the requirement of low toxicity of the drug carrier, and can be used as the drug carrier.
Claims (5)
1. A preparation method of pH sensitive imidazole liposome is characterized in that: the method comprises the following steps:
weighing lecithin and cholesterol, placing the lecithin and cholesterol in a glass container, and adding ethanol into the glass container to dissolve the lecithin and cholesterol to obtain a mixed solution;
step two, adding a curcumin absolute ethyl alcohol solution into the mixed solution, uniformly mixing, and removing the organic solvent by reduced pressure evaporation until a uniform and transparent film is formed on the bottle wall; adding phosphate buffer salt solution containing imidazole to hydrate, elute and disperse the film, carrying out water bath at 35-45 ℃ and normal pressure rotary hydration for 20-40min, and carrying out ultrasonic treatment for 10-20min to fully mix to obtain the pH sensitive imidazole liposome;
the mass ratio of the lecithin to the cholesterol is 3: 1-8: 1; the curcumin content is 0.1-0.3 mg; the content of imidazole is 0.001-0.005 g; the addition amount of the phosphate buffer solution is 15-30 mL, and the concentration is 0.01 mol/L.
2. The method for preparing a pH-sensitive imidazole liposome according to claim 1, wherein: the mass of the lecithin is 0.08g, and the mass of the cholesterol is 0.01 g; the curcumin content is 0.1 mg; the imidazole content is 0.001 g; the amount of phosphate buffered saline added was 30 mL.
3. The method for preparing a pH-sensitive imidazole liposome according to claim 1, wherein: the ultrasonic frequency is 30KHZ, the temperature is 30 ℃, and the time is 15 min.
4. The method for preparing a pH-sensitive imidazole liposome according to claim 1, wherein: and secondly, the content of curcumin in the curcumin absolute ethanol solution is 0.05-0.15 mg.
5. The method for preparing a pH-sensitive imidazole liposome according to claim 1, wherein: and the absolute pressure of the reduced pressure evaporation in the step two is 0.
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