CN106913882B - Polyethylene glycol-gambogic acid liposome, preparation method and application thereof in treating malignant tumor - Google Patents
Polyethylene glycol-gambogic acid liposome, preparation method and application thereof in treating malignant tumor Download PDFInfo
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Abstract
The invention discloses a polyethylene glycol-gambogic acid liposome, which consists of a polyethylene glycol-gambogic acid conjugate, polyethylene glycol, vitamin E, cholesterol and phospholipid, wherein the weight ratio of the polyethylene glycol-gambogic acid conjugate to the polyethylene glycol to the vitamin E to the cholesterol to the phospholipid is 1-10:0.5-2:0.05-0.2:1-4: 10-30. According to the invention, an amphiphilic polymer PEG and an antitumor drug gambogic acid are connected into conjugate amphiphilic PEG-GA by ester bonds, and the PEG-GA is inserted into a liposome bilayer to construct a nano targeted drug delivery system, so that the water solubility of the drug can be improved, the stability and the antitumor effect of the drug can be enhanced, the accumulation of the drug in normal tissues can be reduced, the survival time of tumor-bearing mice can be prolonged, the treatment effect of the tumor-bearing mice can be improved, and the toxic and side effects can be reduced. The invention also provides a preparation method and application of the liposome, and the liposome can be applied to treating lung cancer, ovarian cancer, breast cancer, colorectal cancer, melanoma, head and neck cancer, lymphoma or cerebroma.
Description
Technical Field
The invention relates to the field of pharmaceutical preparations, in particular to a polyethylene glycol-gambogic acid liposome, a preparation method and application thereof in treating malignant tumors.
Background
Garcinia cambogia (gamboge), a natural product and derivatives thereof, play an increasingly important role in human health and in the development of medicinegamboge): is Garcinia cambogia of GuttiferaeGarcinia hanbaryi Hook. fSecreted dry resin. It is mainly produced in Cambodia, India, Thailand and Vietnam, and is cultivated in Guangdong province and Hainan province in China. The gamboge has long been recorded in traditional Chinese medicine, is cold in nature, sour, pungent, astringent and toxic in taste, has the effects of breaking blood and resolving masses, detoxifying, stopping bleeding and killing parasites, and is used for treating chronic diseases such as scrofula, carbuncle and jaundice, furuncle and the like since ancient times. Garcinia has been used abroad as a diuretic for the treatment of edema and elevated blood pressure in cerebral hemorrhage and is now recorded in the United states Pharmacopeia, 10 th edition. Gambogic acid (A), (B)Gambogic Acid, GA): the gambogic acid is an effective component extracted from the traditional Chinese medicine gamboge, is a broad-spectrum anti-tumor medicine, has an inhibiting effect on most tumor cells, and has a research showing that gambogic acid has an obvious dose-dependent inhibiting effect on the proliferation of human liver cancer cell strains SMMC-7721 and QGY-7701 and has a relatively weak effect on a normal human liver tissue cell strain L-02. Gambogic acid can also obviously inhibit proliferation of human gastric adenocarcinoma cell strain SGC-7901. The antineoplastic action mechanism of gambogic acid is multifaceted, including induction of tumor cell apoptosis, inhibition of cell cycle, influence of oncogene and expression of cancer-inhibiting gene and its related protein, etc., and in addition, gambogic acid also has the function of inhibiting tumor cell metastasis, which can not be simultaneously possessed by the existing chemotherapeutic drugs.
Poor solubility of gambogic acid, low bioavailability and short half-life (T in vivo1/2< 1h, in vivo T in rats1/2Less than 20 min), and simultaneously, the free gambogic acid has certain toxicity such as vascular stimulation, digestive tract side effect and the like, is easy to cause phlebitis at the administration part during clinical trial, also shows certain lethal toxicity or causes the animal to have hair erection, weight loss and the like in animal experiments, greatly limits the application of the gambogic acid, and limits the more excellent treatment effect of the gambogic acid to a certain extent. Therefore, it is necessary to develop a structural modification or a new formulation of gambogic acid. The gambogic acid used clinically at present is mainly an injection, and because the water solubility of the gambogic acid is poor, the gambogic acid raw material reported at present is mainly prepared by dissolving borax solution, and the method has poor stability and unsafe clinical use. In order to enhance the feasibility of developing various formulations such as tablets, capsules, oral liquids, granules and injections of gambogic acid, solutions are neededThe solubility of gambogic acid is poor. The existing research shows that the gambogic acid solution prepared by adding cosolvents such as L-arginine, meglumine and lysine or solubilizers such as polyoxyethylene castor oil and polysorbate into the gambogic acid bulk drug can obviously improve the solubility and stability of the gambogic acid solution, and is suitable for the development of various dosage forms; the L-arginine can be directly added or combined with L-arginine, meglumine, lysine and the like as cosolvent to prepare the gambogic acid freeze-dried preparation, which is very important for research on gambogic acid injection. However, long-term use of these cosolvents or solubilizers may cause a series of adverse reactions such as allergy, cardiovascular toxicity, renal toxicity, neurotoxicity, etc.
The prodrug plays an important role in the development process of the drug, and mainly aims to solve the water solubility problem of a parent drug, prolong the half-life period, reduce the toxicity of the drug and improve the drug effect. One of the important means for preparing the prodrug is to connect the compound with water-soluble polyethylene glycol (PEG), so that the water solubility of the compound can be greatly increased, and the change of the drug in the aspects of physicochemical property, pharmacokinetics and pharmacodynamics is caused. The products of the prodrug developed by the PEG reaction at present comprise PEG-paclitaxel, PEG-camptothecin and the like, and the PEG reaction prodrug has greatly increased water solubility, prolonged half-life period, reduced toxicity and increased activity. In recent years, various nano-carriers including liposome, nanoparticles, micelle and the like can improve the solubility of chemotherapeutic drugs, prolong the half-life of the chemotherapeutic drugs in vivo, reduce the absorption of normal cells to the drugs and increase the accumulation of the drugs in tumor cells, thereby achieving the maximum antitumor curative effect and the minimum toxic and side effect. The liposome has high biocompatibility and low biological toxicity due to the phospholipid as the main component, and can load hydrophilic and hydrophobic drugs. In addition, one of the powerful advantages of liposomes is their particular use, such as long circulation or targeting functions, which can be achieved by modifying the lipid composition or adding functional components. Over the past few decades, liposomal formulations of chemotherapeutic drugs have met with significant success in vitro experiments. The goal of the patent is to prepare GA into PEG-GA to achieve the purpose of solubilization, prolong the half-life of the drug and enhance the anti-tumor effect, and then combine PEG-GA with phospholipid molecules and the like to prepare the drug-loaded liposome by using the nanotechnology, thereby achieving the purposes of enhancing drug solubility, enhancing drug efficacy and reducing drug toxic and side effects.
Disclosure of Invention
The invention aims to overcome the defects of poor antitumor activity, short blood circulation time, poor preparation stability, large toxic and side effects and the like in the existing gambogic acid dosage form, and provides a polyethylene glycol-gambogic acid liposome which has excellent water solubility, can enhance the stability and antitumor effect of a medicament, greatly reduces the toxic and side effects of the medicament, and prolongs the life cycle of a tumor-bearing mouse.
In order to achieve the above purpose, the invention provides the following technical scheme:
a polyethylene glycol-gambogic acid liposome is composed of polyethylene glycol-gambogic acid conjugate, polyethylene glycol, vitamin E, cholesterol and phospholipid, wherein the weight ratio of the polyethylene glycol-gambogic acid conjugate to the polyethylene glycol to the vitamin E to the cholesterol to the phospholipid is 1-10:0.5-2:0.05-0.2:1-4: 10-30.
Through a large number of researches and experiments, the applicant finds that polyethylene glycol (PEG) and antitumor drug gambogic acid are combined into an amphiphilic conjugate, namely PEG-GA, and the PEG-GA is inserted into a liposome bilayer to construct a nano targeted drug delivery system, so that the water solubility of the drug can be improved, and the stability and the antitumor effect of the drug can be enhanced. The results of in vivo anti-tumor tests of melanoma and colon cancer transplantation tumor model mice show that the polyethylene glycol-gambogic acid liposome has obvious tumor growth inhibition effect, and the tumor inhibition rate is higher than that of the gambogic acid injection group; in the toxicity evaluation experiment process, the model mice can tolerate the polyethylene glycol-gambogic acid liposome with the preset dose, and do not show obvious abnormal hair, behavior, feeding and the like, and the weight of the mice normally increases during the treatment period, so that the polyethylene glycol-gambogic acid liposome is proved to reduce the toxicity of the medicine. In the treatment process of the B16 tumor-inhibiting model and the C26 model, the survival time experimental data show that after the treatment by using the polyethylene glycol-gambogic acid liposome, the survival time of the model mouse with 100 percent survival is obviously prolonged compared with other groups. And the conditions of the polyethylene glycol-gambogic acid liposome group are the same as those of the normal saline group, and the tail vein and tail tissues of the injection part are not abnormal all the time. Therefore, the polyethylene glycol-gambogic acid liposome further improves the anticancer activity of the medicament and reduces the toxic and side effects of the medicament.
English name of gambogic acid:Gambogic acidthe chemical name is 1, 5-methylene-1H, 3H, 11H-furo (3, 4-g) pyrano (3, 2-b) xanthene-1-crotonic acid, 3a, 4,5, 7-tetrahydro-8-hydroxy- α,3, 3, 11-tetramethyl-13- (3-methyl-2-butenyl) -11- (4-methyl-3-pentenyl) -7, 15-dioxo, the molecular weight is 628.7512, the molecular formula is C38H44O8
Wherein the structure of the polyethylene glycol-gambogic acid conjugate (PEG-GA) is shown as the following formula:
preferably, the weight ratio of the polyethylene glycol-gambogic acid conjugate, the polyethylene glycol, the vitamin E, the cholesterol and the phospholipid is 2:1:0.2:4: 15.
The water solubility, the stability and the antitumor activity of the polyethylene glycol-gambogic acid liposome are further optimized by further optimizing the compatibility proportion of liposome raw materials.
Preferably, the polyethylene glycol-gambogic acid conjugate is formed by ester bond connection of hydroxyl at one end of methoxy polyethylene glycol and carboxyl at C-30 position of gambogic acid.
Preferably, the methoxy polyethylene glycol comprises one or more of methoxy polyethylene glycol 1000, methoxy polyethylene glycol 2000, methoxy polyethylene glycol 4000, methoxy polyethylene glycol 6000 and methoxy polyethylene glycol 8000.
Preferably, the phospholipid comprises one or more of soybean lecithin, dilauroyl lecithin, dimyristoyl lecithin, dipalmitoyl lecithin, distearoyl lecithin, egg yolk lecithin, hydrogenated soybean lecithin, dioleoyl lecithin, dilauroyl phosphatidylglycerol, dipalmitoyl glycerol, distearoyl phosphatidylglycerol, dioleoyl phosphatidylglycerol and dimyristoyl phosphatidic acid.
Another object of the present invention is to provide the above method for preparing polyethylene glycol-gambogic acid liposome, a method for preparing polyethylene glycol-gambogic acid liposome, comprising the steps of:
s1 dissolving polyethylene glycol-gambogic acid conjugate, polyethylene glycol, vitamin E, cholesterol and phospholipid in ethanol to form organic phase, and heating to 40-60 deg.C;
s2, injecting the organic phase into the aqueous phase through a needle tube pump to form an alcohol-containing liposome solution, and removing ethanol from the alcohol-containing liposome solution at 40-60 ℃ under reduced pressure by using a rotary evaporator to obtain a liposome primary solution;
s3, granulating the liposome primary solution by using a high-pressure homogenizer; then, extruding and filtering the liposome primary solution by using an extruder and a filter membrane with the aperture of 0.1-0.45 mu m to obtain a liposome solution;
s4 adding freeze-drying protective agent into the liposome solution, and then carrying out vacuum freeze drying to obtain the polyethylene glycol-gambogic acid liposome.
Preferably, the volume ratio of the organic phase to the aqueous phase in step S2 is 1:1 to 1: 5.
Preferably, the whole-grain process in step S3 comprises feeding the liposome primary solution into a high-pressure homogenizer twice, wherein the first pressure is 100-300bar, and the second pressure is 700-900 bar.
Preferably, the lyoprotectant in step S4 is one or more of trehalose, lactose, sucrose, maltose, mannitol, proline and glycine.
Preferably, the prefreezing temperature and the sublimation drying temperature of the vacuum freeze drying process in the step S4 are lower than-30 ℃, and the desorption drying end point temperature is 20-30 ℃.
Preferably, the liposome solution is extrusion filtered in step S3 by using a filter with a pore size of 0.45 μm for the first time and a filter with a pore size of 0.22 μm for the second time.
Still another object of the present invention is to provide the use of the above polyethylene glycol-gambogic acid liposome, wherein the liposome can be used for treating lung cancer, ovarian cancer, breast cancer, colorectal cancer, melanoma, head and neck cancer, lymphoma or cerebroma.
Compared with the prior art, the invention has the beneficial effects that:
1. according to the invention, hydrophilic polyethylene glycol PEG and hydrophobic antitumor drug gambogic acid are combined into an amphiphilic polymer, and then the amphiphilic polymer and phospholipid are combined into a liposome drug delivery system, so that the water solubility of gambogic acid is improved, the drug is more suitable for intravenous injection, and the bioavailability of the drug is improved.
2. According to the invention, hydrophilic polyethylene glycol PEG and hydrophobic antitumor drug gambogic acid are combined into an amphiphilic polymer, and then the amphiphilic polymer and phospholipid are combined into a liposome drug delivery system, so that the release of the drug is delayed, the stability of the drug is enhanced, and the acute toxicity of the drug is expected to be reduced.
3. The invention combines hydrophilic polyethylene glycol PEG and hydrophobic antitumor drug gambogic acid into amphiphilic polymer, carries a liposome drug delivery system, has high biocompatibility of liposome and passive targeting property to tumor, is expected to improve the drug concentration at tumor parts and reduce drug accumulation in normal tissues, thereby prolonging the life cycle of tumor patients, improving the treatment effect and reducing toxic and side effects.
Drawings
FIG. 1 is an appearance diagram, a particle size distribution diagram and a transmission electron microscope diagram of a PEG-GA liposome preparation prepared by the embodiment of the invention.
FIG. 2 is a graph showing the effect of PEG-GA liposome preparation, PEG-GA group, GA group and blank liposome group prepared in the examples of the present invention on the body weight of mice and the survival time of mice.
FIG. 3 shows the B16-F10 and C26 tumor growth inhibition curves and tumor inhibition rates of the PEG-GA liposome group, the PEG-GA group, the GA group and the blank liposome group prepared in the example of the present invention in tumor-bearing mice.
FIG. 4 shows the stimulation of tail vein injection in mice with PEG-GA liposome preparation, PEG-GA group, GA group and blank liposome group prepared in the examples of the present invention.
FIG. 5 is a graph showing in vitro release rate profiles of the PEG-GA liposome preparation group, PEG-GA group and GA group prepared in the examples of the present invention.
Detailed Description
The present invention will be described in further detail with reference to test examples and specific embodiments. It should be understood that the scope of the above-described subject matter is not limited to the following examples, and any techniques implemented based on the disclosure of the present invention are within the scope of the present invention.
Example 1A polyethylene glycol-gambogic acid liposome comprising a polyethylene glycol-gambogic acid conjugate, polyethylene glycol, vitamin E, cholesterol and egg yolk lecithin in a weight ratio of 2:1:0.2:4:15
First, a polyethylene glycol-gambogic acid conjugate is prepared by first preparing methoxy polyethylene glycol 2000 (mPEG)2000) End group oxidation of (2), synthesis of mPEG2000-COOH; second step of reacting mPEG2000The hydroxyl at one end of-COOH and carboxyl at C-30 position of gambogic acid are subjected to esterification reaction under the action of EDA and DMAP to generate polyethylene glycol-gambogic acid conjugate PEG2000GA for use.
Secondly, preparing the polyethylene glycol-gambogic acid liposome, which comprises the following steps:
s1 dissolving PEG-GA, polyethylene glycol, vitamin E, cholesterol and egg yolk lecithin in ethanol to form an organic phase, and heating to 45 deg.C;
s2, injecting the organic phase into the water phase through a syringe pump to form an alcohol-containing liposome solution, wherein the weight ratio of the organic phase to the water phase is 1:3, removing ethanol from the alcoholic liposome solution by a rotary evaporator under reduced pressure at 45 ℃ to obtain a liposome primary solution;
s3, granulating the liposome primary solution in a high-pressure homogenizer twice, wherein the first pressure is 200bar, and the second pressure is 800 bar; then, the liposome primary solution is extruded and filtered, a filter membrane with the aperture of 0.45 mu m is used for the first time, and a filter membrane with the aperture of 0.22 mu m is used for the second time, so that a liposome solution is obtained;
s4 adding 10% mannitol into the liposome solution, and vacuum freeze drying at-40 deg.C, sublimation drying at-35 deg.C, and resolution drying at 25 deg.C to obtain polyethylene glycol-gambogic acid liposome.
0.4ml of the polyethylene glycol-gambogic acid liposome prepared in example 1 was taken out and put into a vial, and 1.6ml of deionized water was added to dilute five times to obtain a clear solution with blue opalescence, and the particle size was measured by a Malvern laser scattering particle size analyzer, and the laser particle size analysis result showed that the mean particle size was 70 ± 10nm and the particle size distribution of the liposome was narrow. The particle size distribution is shown in figure 1.
And (3) dropwise adding the liposome solution onto a silicon wafer with a proper size, and drying at room temperature. Plating gold on the surface of a silicon wafer sample to enhance the conductivity, attaching a conductive adhesive, placing the silicon wafer sample in a sample chamber, selecting an accelerating voltage of 3.0 kV, and observing the surface morphology of the liposome by adopting a scanning electron microscope. The SEM observation result is shown in figure 1, and the polyethylene glycol-gambogic acid liposome is approximately spherical, uniform in size and smooth in surface.
Example 2A PEG-gambogic acid liposome comprising a PEG-gambogic acid conjugate, PEG, vitamin E, cholesterol and soy phospholipids in a weight ratio of 1:2:0.2:4:10
First, a polyethylene glycol-gambogic acid conjugate is prepared by first preparing methoxypolyethylene glycol 1000 (mPEG)1000) End group oxidation of (2), synthesis of mPEG1000-COOH; second step of reacting mPEG1000The hydroxyl at one end of-COOH and carboxyl at C-30 position of gambogic acid are subjected to esterification reaction under the action of EDA and DMAP to generate polyethylene glycol-gambogic acid conjugate PEG1000-GA is reserved.
The preparation method of the polyethylene glycol-gambogic acid liposome comprises the following steps:
s1 formula amount of GA-mPEG1000Dissolving polyethylene glycol, vitamin E, cholesterol and phospholipid in ethanol to form an organic phase, and heating to 40 ℃;
s2, injecting the organic phase into the aqueous phase through a syringe pump to form an alcohol-containing liposome solution, wherein the weight ratio of the organic phase to the aqueous phase is 1:1, and removing ethanol from the alcohol-containing liposome solution at 50 ℃ under reduced pressure by using a rotary evaporator to obtain a primary liposome solution;
s3, granulating the liposome primary solution in a high-pressure homogenizer twice, wherein the first pressure is 100bar, and the second pressure is 900 bar; then, the liposome primary solution is extruded and filtered, a filter membrane with the aperture of 0.45 mu m is used for the first time, and a filter membrane with the aperture of 0.22 mu m is used for the second time, so that a liposome solution is obtained;
s4, finally, adding trehalose with the mass fraction of 5% into the liposome solution, and then carrying out vacuum freeze drying, wherein the prefreezing temperature is-38 ℃, the sublimation drying temperature is-32 ℃, and the final temperature of desorption drying is 24 ℃, so as to obtain the polyethylene glycol-gambogic acid liposome.
Example 3A polyethylene glycol-gambogic acid liposome comprising a polyethylene glycol-gambogic acid conjugate, polyethylene glycol, vitamin E, cholesterol and distearoyl phosphatidyl glycerol at a weight ratio of 10:0.5:0.05:1:30
First, a polyethylene glycol-gambogic acid conjugate is prepared, which comprises methoxy polyethylene glycol 8000 (mPEG)8000) End group oxidation of (2), synthesis of mPEG8000-COOH; second step of reacting mPEG8000The hydroxyl at one end of-COOH and carboxyl at C-30 position of gambogic acid are subjected to esterification reaction under the action of EDA and DMAP to generate polyethylene glycol-gambogic acid conjugate PEG8000-GA。
Secondly, preparing the polyethylene glycol-gambogic acid liposome, which comprises the following steps:
s1 formula amount of PEG-GA8000Dissolving polyethylene glycol, vitamin E, cholesterol and distearoyl phosphatidyl glycerol in ethanol to form an organic phase, and heating to 50 ℃;
s2, injecting the organic phase into the aqueous phase through a needle tube pump to form an alcohol-containing liposome solution, wherein the weight ratio of the organic phase to the aqueous phase is 1:5, and removing ethanol from the alcohol-containing liposome solution at 40 ℃ under reduced pressure by using a rotary evaporator to obtain a primary liposome solution;
s3, granulating the liposome primary solution in a high-pressure homogenizer twice, wherein the first pressure is 300bar, and the second pressure is 700 bar; then the liposome primary solution is extruded and filtered, a filter membrane with the aperture of 0.45 mu m is used for the first time, and a filter membrane with the aperture of 0.22 mu m is used for the second time, so that the liposome solution is obtained.
S4, finally, adding maltose with the mass fraction of 15% into the liposome solution, and then carrying out vacuum freeze drying, wherein the prefreezing temperature is-37 ℃, the sublimation drying temperature is-31 ℃, and the final temperature of desorption drying is 22 ℃, so as to obtain the polyethylene glycol-gambogic acid liposome.
Test example 1 in vitro cytotoxicity test of polyethylene glycol-gambogic acid liposomes
In the experimental example, the in vitro anti-tumor activity of the polyethylene glycol-gambogic acid liposome is detected by adopting an MTT method, and the experimental tumor cell strains comprise mouse skin melanoma cells B16-F10, mouse colon cancer cells C26 and human umbilical vein endothelial cells HUVEC. The procedures and methods of the MTT method are described by taking B16-F10 cell line as an example.
The B16-F10 cell suspension was inoculated into a 96-well culture plate at 100. mu.L per well and 4500 cells, and cultured overnight under conventional conditions, after which 100. mu.L per well of the cells were administered with different concentrations of the formulated PEG-GA liposome preparation, PEG-GA solution or GA injections corresponding to different concentrations, and the control group was supplemented with the same amount of the medium as a control. The culture was carried out for 48 hours, and 20. mu.L of MTT was added 4 hours before the end of the culture, and the culture was continued for 4 hours. Finally the culture supernatant was discarded thoroughly and 150. mu.L of DMSO was added to each well. Shaking at 37 deg.C for 10min to dissolve the crystals. On a microplate reader, setting the wavelength of 570nm to detect the absorbance A of each hole cell570The cell survival rate was calculated by the following formula, cell survival rate (%). test group A570Value/control group A570Value X100%. The dose-response curve obtained by plotting different concentrations of the same sample on the inhibition rate of tumor cell growth is shown in FIG. 3, from which the half-killing concentration IC of the sample is determined50The results are shown in table 1 below:
Table 1 The IC50（µM）of equivalent GA on different cells afterincubation with different fumulations for 48h
the results of in vivo anti-tumor tests of melanoma and colon cancer transplantation tumor model mice show that the polyethylene glycol-gambogic acid liposome has obvious tumor growth inhibition effect, and the tumor inhibition rate is higher than that of the gambogic acid injection group.
Test example 2 examples effects of PEG-GA Liposome preparation, PEG-GA group, GA group and blank liposome group on body weight and survival time of mice
Using ICR mice (body weight about 20 g) 6-8 weeks old as animal models, 24 rats were randomly selected and set up into 3 groups of 8 rats each with half male and female: the tail of the blank control group is injected with physiological saline intravenously, and the tail of the test group is injected with PEG-GA liposome preparation, PEG-GA group, GA injection and blank liposome group, and the dosage is 7.5 mg/kg. And observing for 7 days, wherein the main observation indexes comprise: weight, diet, behavioral activities, presence or absence of secretions, excretions, death and toxic reactions, presence or absence of normality of secretions and excretions, symptoms, severity, onset time, duration of toxic reactions, reversibility or absence of death of mice, anatomical observation of dying or dead animals, etc. The stimulation after tail vein injection is shown in FIG. 4, and the weight change and survival of the mice are shown in FIG. 2.
It can be seen that in the toxicity evaluation experiment process, the model mice can tolerate the polyethylene glycol-gambogic acid liposome with the preset dosage, and do not show obvious hair, behavior, eating abnormality and the like, and the weight of the mice normally increases during the treatment period, which proves that the polyethylene glycol-gambogic acid liposome reduces the toxicity of the medicament. In the treatment process of the B16 tumor-inhibiting model and the C26 model, the survival time experimental data show that after the treatment by using the polyethylene glycol-gambogic acid liposome, the survival time of the model mouse with 100 percent survival is obviously prolonged compared with other groups. And the conditions of the polyethylene glycol-gambogic acid liposome group are the same as those of the normal saline group, and the tail vein and tail tissues of the injection part are not abnormal all the time. Therefore, the polyethylene glycol-gambogic acid liposome further improves the anticancer activity of the medicament and reduces the toxic and side effects of the medicament.
Experimental example 3 PEG-GA liposome preparation group prepared in example, in vitro Release Rate test of PEG-GA and GA groups
The in vitro release of the liposome is studied by dialysis with NaHCO3Washing dialysis bag with EDTA solution (relative molecular mass cut-off 12000 ~ 14000)2mL of the PEG-GA liposome suspension prepared in example 1 was put in a dialysis bag, and the release medium was 500 mL of phosphate buffered saline (PBS, pH 7.4) containing 5% bovine serum, and the mixture was kept at 37 ℃ and stirred at a rate of 300 r/min. At fixed time points, 1mL of dialysis solution was aspirated and an equal amount of fresh release medium was replenished into the buffer solution.
Preparing a liposome solution: ultrapure water was added to PEG-GA liposome having a mass concentration of 1mg/mL to prepare a sample solution, and the release rate was measured under the above conditions. Preparation of a reference solution: the control sample was prepared as a 1mg/ml gambogic acid solution using ultrapure water containing L-arginine as a cosolvent, and the release rate was measured under the same conditions as the liposomes.
The release of the polyethylene glycol-gambogic acid liposome and the gambogic acid bulk drug in the PBS is shown in figure 5, wherein the 48h cumulative release rate of the polyethylene glycol-gambogic acid liposome is more than 50%, the 4h cumulative release rate is about 30%, and the release is more gradual compared with the bulk drug.
Claims (8)
1. The polyethylene glycol-gambogic acid liposome is characterized by consisting of a polyethylene glycol-gambogic acid conjugate, polyethylene glycol, vitamin E, cholesterol and phospholipid, wherein the weight ratio of the polyethylene glycol-gambogic acid conjugate to the polyethylene glycol to the vitamin E to the cholesterol to the phospholipid is 1-10:0.5-2:0.05-0.2:1-4: 10-30;
the polyethylene glycol-gambogic acid conjugate is formed by connecting hydroxyl at one end of methoxy polyethylene glycol and carboxyl at C-30 position of gambogic acid through ester bond;
the methoxy polyethylene glycol comprises one or more of methoxy polyethylene glycol 1000, methoxy polyethylene glycol 2000, methoxy polyethylene glycol 4000, methoxy polyethylene glycol 6000 and methoxy polyethylene glycol 8000.
2. The polyethylene glycol-gambogic acid liposome of claim 1, wherein the weight ratio of the polyethylene glycol-gambogic acid conjugate, polyethylene glycol, vitamin E, cholesterol and phospholipid is 2:1:0.2:4: 15.
3. The liposome of polyethylene glycol-gambogic acid of claim 1, wherein said phospholipid comprises one or more of soybean phospholipid, dilauroyl lecithin, dimyristoyl lecithin, dipalmitoyl lecithin, distearoyl lecithin, egg yolk lecithin, hydrogenated soybean lecithin, dioleoyl lecithin, dilauroyl phosphatidyl glycerol, dipalmitoyl glycerol, distearoyl phosphatidyl glycerol, dioleoyl phosphatidyl glycerol, and dimyristoyl phosphatidic acid.
4. A method for preparing the polyethylene glycol-gambogic acid liposome of claims 1-3, comprising the steps of:
s1 dissolving polyethylene glycol-gambogic acid conjugate, polyethylene glycol, vitamin E, cholesterol and phospholipid in ethanol to form organic phase, and heating to 40-60 deg.C;
s2, injecting the organic phase into the aqueous phase through a needle tube pump to form an alcohol-containing liposome solution, and removing ethanol from the alcohol-containing liposome solution at 40-60 ℃ under reduced pressure by using a rotary evaporator to obtain a liposome primary solution;
s3, granulating the liposome primary solution by using a high-pressure homogenizer; then, extruding and filtering the liposome primary solution by using an extruder and a filter membrane with the aperture of 0.1-0.45 mu m to obtain a liposome solution;
s4 adding freeze-drying protective agent into the liposome solution, and then carrying out vacuum freeze drying to obtain the polyethylene glycol-gambogic acid liposome.
5. The method for preparing the liposome of polyethylene glycol-gambogic acid according to claim 4, wherein the volume ratio of the organic phase to the aqueous phase in step S2 is 1:1-1: 5.
6. The method of claim 4, wherein the step S3 comprises subjecting the liposome primary solution to two high pressure homogenizer treatment steps, the first pressure is 100-300bar, and the second pressure is 700-900 bar.
7. The method for preparing the liposome of polyethylene glycol-gambogic acid according to claim 4, wherein the lyoprotectant in step S4 is one or more of trehalose, lactose, sucrose, maltose, mannitol, proline and glycine.
8. The use of the liposome of polyethylene glycol-gambogic acid according to claim 1 in the preparation of a medicament for the treatment of a tumor, wherein said tumor is lung cancer, ovarian cancer, breast cancer, melanoma, head and neck cancer, lymphoma or brain tumor.
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| CN1840201A (en) * | 2006-01-20 | 2006-10-04 | 中国药科大学 | Polyethylene glycol prodrug of gambogicacid, its preparation method, formulation and use |
| CN101028250A (en) * | 2007-04-03 | 2007-09-05 | 中国药科大学 | Garcinolic acid liposome and freezing-drying powdery preparation and its making method |
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| CN1840201A (en) * | 2006-01-20 | 2006-10-04 | 中国药科大学 | Polyethylene glycol prodrug of gambogicacid, its preparation method, formulation and use |
| CN101028250A (en) * | 2007-04-03 | 2007-09-05 | 中国药科大学 | Garcinolic acid liposome and freezing-drying powdery preparation and its making method |
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