CN106913882A - A kind of polyethylene glycol gambogicacid liposome and preparation method and its application in malignant tumour is treated - Google Patents
A kind of polyethylene glycol gambogicacid liposome and preparation method and its application in malignant tumour is treated Download PDFInfo
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
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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
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
Abstract
The invention discloses a kind of polyethylene glycol gambogicacid liposome, the liposome is made up of polyethylene glycol gambogicacid conjugate, polyethylene glycol, vitamin E, cholesterol and phosphatide, and the weight ratio of the polyethylene glycol gambogicacid conjugate, polyethylene glycol, vitamin E, cholesterol and phosphatide is 1 10:0.5‑2:0.05‑0.2:1‑4:10‑30.Amphipathic nature polyalcohol PEG and antineoplastic gambogicacid ester bond are connected into the amphipathic PEG GA of conjugate by the present invention, liposome bilayers are inserted by by PEG GA, build nano target medicine delivery system, the water solubility of medicine can not only be improved, the stability and antitumor action of medicine can be strengthened simultaneously, accumulation of the medicine in normal structure is reduced, extends the time-to-live of tumor-bearing mice, improved its therapeutic efficiency and reduce toxic and side effect.Invention also provides the preparation method and applications of the liposome, the liposome can be applied to treatment lung cancer, oophoroma, breast cancer, colorectal cancer, melanoma, incidence cancer, lymthoma or brain tumor.
Description
Technical field
The present invention relates to field of pharmaceutical preparations, more particularly to a kind of polyethylene glycol-gambogicacid liposome and preparation method and
Its application in malignant tumour is treated.
Background technology
Natural products and its derivative play more and more important effect, gamboge in terms of human health with medical development
（gamboge）：It is Garcinia maingayii gamboge treeGarcinia hanbaryi Hook. fThe dry resin secreted.Its main product
In Cambodia, India, Thailand and Vietnam, there is cultivation in Guangdong Province of China and Hainan Province.Motherland's medical science is to its early on the books, gamboge
It is cold in nature, it is sour, pungent, puckery, it is poisonous, the effect of tool blood-breaking dissipating bind, removing toxic substances, hemostasis, desinsection, treatment scrofula is just used for since ancient times
The persistent ailments such as scrofula, carbuncle subcutaneous ulcer, furuncle.Gamboge is often used as diuretics abroad, and blood pressure during treatment oedema and cerebral hemorrhage is raised,
Record in《American Pharmacopeia》10th edition.Gambogicacid（Gambogic Acid, GA）：It is the active ingredient of extraction in Chinese medicine gamboge,
It is broad-spectrum anti-cancer drug, it is all inhibited for most tumors cell, there are some researches show gambogicacid is to human liver cancer cell
The propagation of strain SMMC-7721 and QGY-7701 has obvious dose-dependent inhibition to act on, and to the strain of normal person's hepatic tissue cell
L-02 effects are relatively weak.Gambogicacid can also substantially suppress the propagation of BGC823 cell line SGC-7901.Gambogicacid is antitumor
Mechanism of action be it is many, including inducing apoptosis of tumour cell, suppress the cell cycle, influence oncogene and tumor suppressor gene and its
Expression of GAP-associated protein GAP etc., also with the function of suppressing Nasopharyngeal neoplasms, these are current chemotherapeutics institutes to gambogicacid in addition
Can not be provided simultaneously with.
Gambogicacid poor solubility, bioavilability is low, half-life short（T in dog body1/2＜ 1h, T in rat body1/2＜
20min）, while free gambogicacid has the toxicity such as certain vascular stimulation, digestive tract side effects, it is easy in clinic trial
Trigger the phlebitis of medicine-feeding part, certain lethal toxicity is also demonstrated by zoopery or animal perpendicular hair, body weight is occurred
Mitigate etc., the application of gambogicacid is greatly limit, the outstanding therapeutic effect of gambogicacid is also limit to a certain extent.Therefore have
The necessary research for carrying out structural modification or novel form to gambogicacid.The gambogicacid of current Clinical practice is normal based on injection, by
Very poor in gambogicacid water solubility, the gamboge acid starting material of existing report is mainly what is prepared using borax soln dissolving, and the method is steady
Qualitative difference, Clinical practice is dangerous.It is various to strengthen the tablet of gambogicacid, capsule, oral liquid, granule and injection etc.
The feasibility of formulation development is, it is necessary to solve gambogicacid poor solubility this problem.There are some researches show in gambogicacid bulk drug
The solubilizer such as cosolvent or Emulsifier EL-60, polysorbate such as L-arginine, meglumine, lysine are added to prepare rattan
Yellow acid solution can significantly improve its solubility and stability, it is adaptable to the exploitation of various formulations；And pass through directly to add L- essence ammonia
Acid is used in combination L-arginine, meglumine, lysine etc. the lyophilized formulations of gambogicacid can be prepared as cosolvent, this
Injection research for gambogicacid is particularly important.But these cosolvents of long-term use or solubilizer, may cause a series of
Adverse reaction, such as allergy, Cardiovascular Toxicity, renal toxicity, neurotoxicity.
Prodrug plays important role in the development process of medicine, and its main purpose is the water-soluble of solution parent drug
Sex chromosome mosaicism, extension half-life period, reduction drug toxicity and raising effect of drugs.It is by chemical combination to prepare one of important means of prodrug
Thing and water-soluble polyethylene glycol（PEG）Connection, the water solubility of compound can be greatly increased, so as to cause medicine physicochemical property,
Change in terms of pharmacokinetics and pharmacodynamics.At present using PEGylation successfully develop prodrug product have PEG- taxols,
PEG- camptothecines etc., the preceding aqueous solubility after PEGylation is greatly increased, the reduction of Increased Plasma Half-life, toxicity, and activity also increased.
Various nano-carriers include liposome in recent years, and nanoparticle and micella etc. can improve the dissolubility of chemotherapeutics, extend its
Internal half-life period, absorption of the normal cell to medicine is reduced, increase accumulation of the medicine in tumour cell, so as to reach most
Big antitumor curative effect and the toxic and side effect of minimum.Liposome is phosphatide, thus the biofacies with height due to main component
Capacitive and relatively low bio-toxicity, can load hydrophily and hydrophobic medicine.Additionally, one of powerful advantage of liposome is
Its special-purpose, such as can realize long circulating or targeting work(by changing lipid components or the functional composition of increase
Energy.In the past few decades, the Lipidosome of chemotherapeutics achieves significant success in testing in vitro.This patent
Target be mainly GA is first prepared into PEG-GA reach solubilising purpose, extend medicine half-life period and enhancing antitumous effect,
PEG-GA and phospholipid molecule etc. are combined again, drug-loaded liposome is prepared using nanometer technology, reach enhancing drug solubility, enhancing
The purpose of curative effect of medication and reduction poisonous side effect of medicine.
The content of the invention
It is an object of the invention to overcome, the antitumor activity in the presence of existing gambogicacid formulation is poor, blood circulation time
The above-mentioned deficiency such as short, preparation stability is poor and toxic and side effect is big, there is provided a kind of polyethylene glycol-gambogicacid liposome, the liposome
With excellent water solubility, the stability and antitumor action of medicine can be strengthened, the toxic and side effect of medicine is greatly reduced, extended
The life cycle of tumor-bearing mice.
In order to realize foregoing invention purpose, the invention provides following technical scheme：
A kind of polyethylene glycol-gambogicacid liposome, the liposome is by polyethylene glycol-gambogicacid conjugate, polyethylene glycol, vitamin
E, cholesterol and phosphatide composition, the polyethylene glycol-gambogicacid conjugate, polyethylene glycol, vitamin E, cholesterol and phosphatide
Weight ratio is 1-10:0.5-2:0.05-0.2:1-4:10-30.
Applicant has found through numerous studies and experiment, by polyethylene glycol（PEG）Two are combined into antineoplastic gambogicacid
Parent's property conjugate is PEG-GA, and liposome bilayers are inserted by by PEG-GA, builds nano target medicine delivery system,
The water solubility of medicine can not only be improved, while the stability and antitumor action of medicine can be strengthened.Melanoma and colon cancer are moved
Plant antitumor result of the test in knurl model mice body and show that polyethylene glycol-gambogicacid liposome has obvious Tumor growth inhibition
Effect, its tumour inhibiting rate is higher than gamboge acid injection group；In toxicity assessment experimentation, model mice is resistant to predetermined close
Polyethylene glycol-gambogicacid liposome, does not show obvious hair, behavior, feed exception etc., and Mouse Weight is normal during treatment
Increase, it was demonstrated that polyethylene glycol-gambogicacid liposome reduces the toxicity of medicine.Suppress the treatment of knurl model and C26 models in B16
During, life cycle experimental data shows, after being treated using polyethylene glycol-gambogicacid liposome, model mice and other
Group is obviously prolonged compared to the life cycle of 100% survival.And polyethylene glycol-gambogicacid liposome group is as physiological saline group situation,
The tail vein and tail tissue of injection site all no abnormality seen always.Therefore polyethylene glycol-gambogicacid lipid is further illustrated
Body improves the active anticancer of medicine, while reducing the toxic and side effect of medicine.
The English name of gambogicacid：Gambogic acid；Chemical name：1,5- methylene -1H, 3H, 11H- furans is simultaneously（3,
4-g）Pyrans is simultaneously（3,2-b）Xanthene -1- crotonic acids, 3a, 4,5,7- tetrahydrochysene -8- hydroxyl-alphas, 3,3,11- tetramethyl -13-（3- first
Base -2- cyclobutenyls）-11-（4- methyl-3-pentenyls）- 7,15- dioxo, molecular weight：628.7512, molecular formula：C38H44O8
Wherein polyethylene glycol-gambogicacid conjugate（PEG-GA）Structure be shown below：
Preferably, the weight ratio of polyethylene glycol-gambogicacid conjugate, polyethylene glycol, vitamin E, cholesterol and phosphatide is 2:1:
0.2:4:15。
By the proportion compatibility of further preferred liposome raw material, polyethylene glycol of the invention-gambogicacid liposome is water-soluble
Property, stability and antitumor activity are further optimized.
Preferably, polyethylene glycol-gambogicacid conjugate is by the hydroxyl of methoxy poly (ethylene glycol) one end and gambogicacid C-30
Carboxyl connects to be formed by ester bond.
Preferably, methoxy poly (ethylene glycol) includes methoxy poly (ethylene glycol) 1000, methoxy poly (ethylene glycol) 2000, methoxyl group
One or more in Macrogol 4000, methoxy poly (ethylene glycol) 6000 and methoxy poly (ethylene glycol) 8000.
Preferably, phosphatide includes soybean lecithin, DLPC, two myristoyl lecithin, two palmityl lecithins
Fat, distearoylphosphatidylcholine, distearoylphosphatidylcholine, egg yolk lecithin, hydrogenated soybean lecithin, DOPC, two bays
Acyl phosphatidyl glycerol, two palm phosphatidyl glycerols, DSPG, DOPG and two myristoyl phosphorus
One or more in resin acid.
Preparation method another object of the present invention is to provide above-mentioned polyethylene glycol-gambogicacid liposome, a kind of poly- second
The preparation method of glycol-gambogicacid liposome, comprises the following steps：
Polyethylene glycol-gambogicacid conjugate, polyethylene glycol, vitamin E, cholesterol and phosphatide is dissolved in ethanol and is formed with by S1
Machine phase, is heated to 40-60 DEG C；
S2 forms containing alcohol liposome solutions during above-mentioned organic phase is entered into water phase by needle tubing infusion, and the containing alcohol liposome solutions are used
Rotary Evaporators are removed under reduced pressure ethanol at 40-60 DEG C, obtain liposome just solution；
By above-mentioned liposome, just solution carries out whole grain to S3 with high pressure homogenizer；Then use extruder and the 0.1-0.45 μm of filter in aperture
To liposome, just solution carries out extrusion filtering to film, obtains liposome solutions；
Then S4 carries out vacuum freeze drying to freeze drying protectant is added in above-mentioned liposome solutions, obtains final product polyethylene glycol-rattan
Yellow acid liposome.
Preferably, organic phase and the volume ratio of water phase are 1 in step S2:1-1:5.
Preferably, whole grain process includes that just solution enters high pressure homogenizer treatment, first at twice by liposome in step S3
Secondary pressure is 100-300bar, and second pressure is 700-900 bar.
Preferably, freeze drying protectant is trehalose, lactose, sucrose, maltose, mannitol, proline and sweet in step S4
One or more in propylhomoserin.
Preferably, the pre-freezing temperature and lyophilization temperature of vacuum freeze drying process are less than -30 DEG C, parsing in step S4
Outlet temperature is dried for 20-30 DEG C.
Preferably, when liposome solutions being carried out into extrusion filtering in step S3, for the first time using 0.45 μm of filter membrane in aperture,
Use 0.22 μm of filter membrane in aperture second.
It is still another object of the present invention to provide the application of above-mentioned polyethylene glycol-gambogicacid liposome, the liposome can
It is applied to treatment lung cancer, oophoroma, breast cancer, colorectal cancer, melanoma, incidence cancer, lymthoma or brain tumor.
Compared with prior art, beneficial effects of the present invention：
1st, hydrophilic polyethylene glycol PEG is combined into amphipathic nature polyalcohol by the present invention with hydrophobic antineoplastic gambogicacid,
Liposomal drug delivery system is combined into phosphatide etc. again, the water solubility of gambogicacid is improve, medicine is noted more suitable for vein
Penetrate, improve the bioavilability of medicine.
2nd, hydrophilic polyethylene glycol PEG is combined into amphipathic polymerization by the present invention with hydrophobic antineoplastic gambogicacid
Thing, then liposomal drug delivery system is combined into phosphatide etc., delay the release of medicine, enhance the stability of medicine and have
Hope the acute toxicity for reducing medicine.
3rd, hydrophilic polyethylene glycol PEG is combined into amphipathic polymerization by the present invention with hydrophobic antineoplastic gambogicacid
Thing, carries liposome administration system, is the describing property of biology and the passive targeting to tumour of liposome height, is expected to improve
In the drug concentration of tumor locus, the drug accumulation in normal structure is reduced, so as to extend the life cycle of tumor patient, improvement is controlled
Treat effect and reduce toxic and side effect.
Brief description of the drawings
Fig. 1 is PEG-GA Liposomal formulations outside drawing, grain size distribution and transmission electron microscope picture prepared by the embodiment of the present invention.
Fig. 2 is PEG-GA Liposomal formulations, PEG-GA groups, GA groups and blank liposome group pair prepared by the embodiment of the present invention
The influence of Mouse Weight and the influence to survival time of mice.
Fig. 3 is PEG-GA liposomes group, PEG-GA groups, GA groups and the blank liposome group of embodiment of the present invention preparation to lotus
B16-F10 and C26 Tumor growth inhibitions curve and tumour inhibiting rate in knurl Mice Body.
Fig. 4 is PEG-GA Liposomal formulations, PEG-GA groups, GA groups and blank liposome group pair prepared by the embodiment of the present invention
The stimulation situation of mouse tail intravenous injection.
Fig. 5 is the release in vitro rate of PEG-GA Liposomal formulation groups prepared by the embodiment of the present invention, PEG-GA groups and GA groups
Curve.
Specific embodiment
With reference to test example and specific embodiment, the present invention is described in further detail.But this should not be understood
For the scope of above-mentioned theme of the invention is only limitted to following embodiment, all technologies realized based on present invention belong to this
The scope of invention.
A kind of polyethylene glycol of embodiment 1-gambogicacid liposome, is 2 by weight ratio:1:0.2:4:15 polyethylene glycol-rattan
Yellow acid conjugate, polyethylene glycol, vitamin E, cholesterol and egg yolk lecithin composition
Polyethylene glycol-gambogicacid conjugate is prepared first, including first by methoxy poly (ethylene glycol) 2000（mPEG2000）End group oxygen
Change, synthesize mPEG2000-COOH；Second step makes mPEG2000The hydroxyl of-COOH one end and gambogicacid C-30 carboxyl in EDA and
There is esterification in the presence of DMAP, generate polyethylene glycol-gambogicacid conjugate PEG2000- GA is standby.
Next prepares polyethylene glycol-gambogicacid liposome, comprises the following steps：
In being dissolved in ethanol be formed with for the PEG-GA of formula ratio, polyethylene glycol, vitamin E, cholesterol and egg yolk lecithin by S1
Machine phase, is heated to 45 DEG C；
S2 enters above-mentioned organic phase by needle tubing infusion to form containing alcohol liposome solutions in water phase, wherein the weight of organic phase and water phase
Amount is than being 1：3, the containing alcohol liposome solutions Rotary Evaporators are removed under reduced pressure ethanol at 45 DEG C, obtain liposome just molten
Liquid；
By above-mentioned liposome, just solution enters the treatment of high pressure homogenizer whole grain to S3 at twice, and first time pressure is 200bar, second
Pressure is 800bar；Then to liposome, just solution carries out extrusion filtering again, and 0.45 μm of filter membrane in aperture, second are used for the first time
It is secondary to use 0.22 μm of filter membrane in aperture, obtain liposome solutions；
S4 is last, and 10% mannitol is added in above-mentioned liposome solutions, then carries out vacuum freeze drying, wherein pre-freezing temperature
It it is -40 DEG C, lyophilization drying temperature is -35 DEG C, and parsing-desiccation outlet temperature is 25 DEG C, obtains final product polyethylene glycol-gambogicacid fat
Plastid.
Take 0.4ml above-described embodiments 1 preparation polyethylene glycol-gambogicacid liposome in cillin bottle, add 1.6ml go from
Sub- water dilutes five times, obtains the settled solution of blue-opalescent, with Malvern laser diffraction particle size analysis-e/or determining particle diameters, swashs
Light results of grain size analysis shows its average grain diameter for 70 ± 10nm, liposomal particle size narrow distribution.Particle diameter distribution such as Fig. 1 institutes
Show.
Liposome solutions are added drop-wise on the silicon chip being of moderate size, drying at room temperature.Led in the enhancing of silicon chip sample surface gold-plating
Electrically, sample room is placed in after sticking conducting resinl, selection accelerating potential is 3.0 kV, using the surface of scanning electron microscopic observation liposome
Form.SEM observes result as shown in Fig. 1, and polyethylene glycol-gambogicacid liposome is similar to spherical, and size is homogeneous, surface light
It is sliding.
A kind of polyethylene glycol of embodiment 2-gambogicacid liposome, is 1 by weight ratio:2:0.2:4:10 polyethylene glycol-rattan
Yellow acid conjugate, polyethylene glycol, vitamin E, cholesterol and soybean lecithin composition
Polyethylene glycol-gambogicacid conjugate is prepared first, including first by methoxy poly (ethylene glycol) 1000（mPEG1000）End group oxygen
Change, synthesize mPEG1000-COOH；Second step makes mPEG1000The hydroxyl of-COOH one end and gambogicacid C-30 carboxyl in EDA and
There is esterification in the presence of DMAP, generate polyethylene glycol-gambogicacid conjugate PEG1000-GA is standby.
The preparation method of polyethylene glycol-gambogicacid liposome, comprises the following steps：
S1 is by the GA- mPEG of formula ratio1000, polyethylene glycol, vitamin E, cholesterol and phosphatide form organic in being dissolved in ethanol
Phase, is heated to 40 DEG C；
S2 enters above-mentioned organic phase by needle tubing infusion to form containing alcohol liposome solutions in water phase, wherein the weight of organic phase and water phase
Amount compares 1:1, the containing alcohol liposome solutions Rotary Evaporators are removed under reduced pressure ethanol at 50 DEG C, obtain liposome just solution；
By above-mentioned liposome, just solution enters the treatment of high pressure homogenizer whole grain to S3 at twice, and first time pressure is 100bar, second
Pressure is 900bar；Then to liposome, just solution carries out extrusion filtering again, and 0.45 μm of filter membrane in aperture, second are used for the first time
It is secondary to use 0.22 μm of filter membrane in aperture, obtain liposome solutions；
S4 is last, and the trehalose of 5% mass fraction is added in above-mentioned liposome solutions, then carries out vacuum freeze drying, its
Middle pre-freezing temperature is -38 DEG C, and lyophilization drying temperature is -32 DEG C, and parsing-desiccation outlet temperature is 24 DEG C, obtains final product poly- second two
Alcohol-gambogicacid liposome.
A kind of polyethylene glycol of embodiment 3-gambogicacid liposome, is 10 by weight ratio:0.5:0.05:1:30 poly- second two
Alcohol-gambogicacid conjugate, polyethylene glycol, vitamin E, cholesterol and DSPG composition
Polyethylene glycol-gambogicacid conjugate is prepared first, including first by methoxy poly (ethylene glycol) 8000（mPEG8000）End group oxygen
Change, synthesize mPEG8000-COOH；Second step makes mPEG8000The hydroxyl of-COOH one end and gambogicacid C-30 carboxyl in EDA and
There is esterification in the presence of DMAP, generate polyethylene glycol-gambogicacid conjugate PEG8000-GA。
Next prepares polyethylene glycol-gambogicacid liposome, comprises the following steps：
S1 is by the PEG-GA of formula ratio8000, polyethylene glycol, vitamin E, cholesterol and DSPG are dissolved in second
Organic phase is formed in alcohol, 50 DEG C are heated to；
S2 enters above-mentioned organic phase by needle tubing infusion to form containing alcohol liposome solutions in water phase, wherein the weight of organic phase and water phase
Amount is than being 1:5, the containing alcohol liposome solutions Rotary Evaporators are removed under reduced pressure ethanol at 40 DEG C, obtain liposome just molten
Liquid；
By above-mentioned liposome, just solution enters the treatment of high pressure homogenizer whole grain to S3 at twice, and first time pressure is 300bar, second
Pressure is 700bar；Then to liposome, just solution carries out extrusion filtering again, and 0.45 μm of filter membrane in aperture, second are used for the first time
It is secondary to use 0.22 μm of filter membrane in aperture, obtain liposome solutions.
S4 is last, and the maltose of 15% mass fraction is added in above-mentioned liposome solutions, then carries out vacuum refrigeration and does
Dry, wherein pre-freezing temperature is -37 DEG C, and lyophilization drying temperature is -31 DEG C, and parsing-desiccation outlet temperature is 22 DEG C, is obtained final product poly-
Ethylene glycol-gambogicacid liposome.
The vitro cytotoxicity test of 1 polyethylene glycol of test example-gambogicacid liposome
This test example detects the anti tumor activity in vitro of polyethylene glycol-gambogicacid liposome using mtt assay, tests tumour cell
Strain includes mouse skin MC B16-F10, mouse colonic cell C26 and Human umbilical vein endothelial cells HUVEC.With
The flow and method of mtt assay are introduced as a example by B16-F10 cell lines.
By B16-F10 cell suspension inoculations in 96 well culture plates, per the μ L of hole 100,4500 cells, trained under normal condition
Support overnight, hereafter, give the PEG-GA Liposomal formulations of the various concentrations that μ L are prepared of every hole cell 100, PEG-GA solution or
The GA injections of correspondence various concentrations, control group adds the culture medium of equivalent as control.Culture 48h, the 4h before culture is terminated, plus
Enter the μ L of MTT 20, continue to cultivate 4h.Culture supernatant is thoroughly finally discarded, 150 μ L DMSO are added per hole.Shaking table is low at 37 DEG C
Speed rocks 10min, crystal is fully dissolved.On ELIASA, absorbance of the detection per hole cell under setting 570nm wavelength
A570Value, cell survival rate is calculated by following equation:Cell survival rate (%)=experimental group A570Value/control group A570Value ×
100%.The dose-effect curve such as institute of accompanying drawing 3 can obtain to the mapping of growth of tumour cell inhibiting rate with the various concentrations of same sample
Show, therefrom obtain the half casualty-producing concentrations IC of sample50, it is as a result as shown in table 1 below：
Table 1 The IC50（µM）of equivalent GA on different cells after incubation
with different fumulations for 48h
Antitumor result of the test shows in melanoma and colon cancer Transplanted tumor model Mice Body, polyethylene glycol-gambogicacid lipid
Body has obvious Tumor growth inhibition to act on, and its tumour inhibiting rate is higher than gamboge acid injection group.
The embodiment PEG-GA Liposomal formulations of test example 2, PEG-GA groups, GA groups and blank liposome group are to Mouse Weight
Influence and the influence to survival time of mice
With 6-8 week old（Body weight about 20g）ICR small white mouses be animal model, take 24 rats at random, set 3 groups, every group 8
Only, male and female half and half：Blank control group tail vein injections physiological saline, test group afterbody injection PEG-GA Liposomal formulations,
PEG-GA groups, GA injections and blank liposome group, dosage is 7.5mg/kg.Observation 7 days, MAIN OUTCOME MEASURES includes：Body weight,
Diet, behavioral activity, if having secretion, excreta, death condition and toxic reaction, whether secretion is normal with excreta,
It is the symptom of toxic reaction, the order of severity, initial time, the duration, whether reversible, if dead mouse occur, for dying
Or the anatomic observation of dead animal etc..Stimulation situation wherein after tail vein injections as shown in figure 4, Mouse Weight change and
Mouse survival situation is as shown in Figure 2.
It can be seen that in toxicity assessment experimentation, model mice is resistant to the polyethylene glycol-gambogicacid of predetermined close
Liposome, does not show obvious hair, behavior, feed exception etc., and Mouse Weight normally increases during treatment, it was demonstrated that poly- second two
Alcohol-gambogicacid liposome reduces the toxicity of medicine.In the therapeutic process that B16 suppresses knurl model and C26 models, life cycle reality
Data display is tested, after being treated using polyethylene glycol-gambogicacid liposome, model mice 100% survival compared with other groups
Life cycle is obviously prolonged.And polyethylene glycol-gambogicacid liposome group is as physiological saline group situation, the tail vein of injection site
With tail tissue all no abnormality seen always.Therefore further illustrate polyethylene glycol-gambogicacid liposome and improve the anti-of medicine
Cancer activity, while reducing the toxic and side effect of medicine.
PEG-GA Liposomal formulation groups prepared by the embodiment of test example 3, the release in vitro rate experiment of PEG-GA and GA groups
Using the outer release conditions of dialysis research Via Liposomes, NaHCO is used3Bag filter is washed away with EDTA solution（Average molecular
Quality interception 12000~14000）The glycerine and impurity of upper residual, the PEG-GA liposomes for taking the preparation of 2mL embodiments 1 suspend
Liquid is placed in bag filter, and dissolution medium is the phosphate buffer of 5% cow's serum（PBS, pH 7.4）500 mL, 37 DEG C of constant temperature,
Mixing speed 300r/min.Point draws 1mL dialysis solutions at a fixed time, and the fresh dissolution medium of equivalent amount is added to
In cushioning liquid.
It is prepared by liposome solutions：Ultra-pure water is added to be configured to sample in mass concentration is for the PEG-GA liposomes of 1mg/mL
Solution, determines its release rate under these conditions.It is prepared by reference substance solution：Control sample is used added with cosolvent L-arginine
Ultra-pure water be configured to 1mg/ml gamboge acid solutions, and determine its release rate under with liposome the same terms.
Polyethylene glycol-gambogicacid liposome discharges as shown in Fig. 5 with gambogicacid bulk drug in PBS, polyethylene glycol-rattan
Yellow acid liposome 48h preparations are about 30% more than 50%, 4h preparations, discharge gentler compared with bulk drug.
Claims (10)
1. a kind of polyethylene glycol-gambogicacid liposome, it is characterised in that the liposome is by polyethylene glycol-gambogicacid conjugate, poly-
Ethylene glycol, vitamin E, cholesterol and phosphatide composition, the polyethylene glycol-gambogicacid conjugate, polyethylene glycol, vitamin E, courage
The weight ratio of sterol and phosphatide is 1-10:0.5-2:0.05-0.2:1-4:10-30.
2. polyethylene glycol according to claim 1-gambogicacid liposome, it is characterised in that the polyethylene glycol-gambogicacid
The weight ratio of conjugate, polyethylene glycol, vitamin E, cholesterol and phosphatide is 2:1:0.2:4:15.
3. polyethylene glycol according to claim 1 and 2-gambogicacid liposome, it is characterised in that the polyethylene glycol-rattan
Yellow acid conjugate is connected by ester bond and formed by the hydroxyl and gambogicacid C-30 carboxyl of methoxy poly (ethylene glycol) one end.
4. polyethylene glycol according to claim 3-gambogicacid liposome, it is characterised in that the methoxy poly (ethylene glycol)
Including methoxy poly (ethylene glycol) 1000, methoxy poly (ethylene glycol) 2000, methoxy poly (ethylene glycol) 4000, methoxy poly (ethylene glycol)
6000 and methoxy poly (ethylene glycol) 8000 in one or more.
5. polyethylene glycol according to claim 1-gambogicacid liposome, it is characterised in that the phosphatide includes soybean phosphorus
Fat, DLPC, two myristoyl lecithin, DPPC, distearoylphosphatidylcholine, distearyl lecithin
Fat, egg yolk lecithin, hydrogenated soybean lecithin, DOPC, PE, two palm phosphatidyl glycerols, two
One or more in stearyl phosphatidyl glycerine, DOPG and two myristoyl phosphatidic acids.
6. a kind of method of the polyethylene glycol-gambogicacid liposome prepared described in claim 1-5, it is characterised in that including with
Lower step：
Polyethylene glycol-gambogicacid conjugate, polyethylene glycol, vitamin E, cholesterol and phosphatide is dissolved in ethanol and is formed with by S1
Machine phase, is heated to 40-60 DEG C；
S2 forms containing alcohol liposome solutions during above-mentioned organic phase is entered into water phase by needle tubing infusion, and the containing alcohol liposome solutions are used
Rotary Evaporators are removed under reduced pressure ethanol at 40-60 DEG C, obtain liposome just solution；
By above-mentioned liposome, just solution carries out whole grain to S3 with high pressure homogenizer；Then use extruder and the 0.1-0.45 μm of filter in aperture
To liposome, just solution carries out extrusion filtering to film, obtains liposome solutions；
Then S4 carries out vacuum freeze drying to freeze drying protectant is added in above-mentioned liposome solutions, obtains final product polyethylene glycol-rattan
Yellow acid liposome.
7. the preparation method of polyethylene glycol according to claim 6-gambogicacid liposome, it is characterised in that the step
Organic phase and the volume ratio of water phase are 1 in S2:1-1:5.
8. the preparation method of polyethylene glycol according to claim 6-gambogicacid liposome, it is characterised in that the step
Whole grain process includes that just solution enters high pressure homogenizer treatment at twice by liposome in S3, and first time pressure is 100-300bar,
Second pressure is 700-900 bar.
9. the preparation method of polyethylene glycol according to claim 6-gambogicacid liposome, it is characterised in that the step
Freeze drying protectant is one or more in trehalose, lactose, sucrose, maltose, mannitol, proline and glycine in S4.
10. the application of polyethylene glycol according to claim 1-gambogicacid liposome, it is characterised in that the liposome can
It is applied to treatment lung cancer, oophoroma, breast cancer, colorectal cancer, melanoma, incidence cancer, lymthoma or brain tumor.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111617264A (en) * | 2020-07-09 | 2020-09-04 | 西安交通大学医学院第二附属医院 | Preparation method of lipid nano microbubble ultrasound contrast agent carrying dabrafenib |
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| CN114767620A (en) * | 2022-03-16 | 2022-07-22 | 四川省医学科学院·四川省人民医院 | Multistage response injectable hydrogel loaded with gambogic acid and application thereof |
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