CN104398501A - Polymeric micelle composition for treating recrudescent cancer and preparation method thereof - Google Patents
Polymeric micelle composition for treating recrudescent cancer and preparation method thereof Download PDFInfo
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- CN104398501A CN104398501A CN201410715412.3A CN201410715412A CN104398501A CN 104398501 A CN104398501 A CN 104398501A CN 201410715412 A CN201410715412 A CN 201410715412A CN 104398501 A CN104398501 A CN 104398501A
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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
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
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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
- A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
- A61K31/05—Phenols
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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
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
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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
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
Abstract
The invention discloses a polymeric micelle composition for treating recrudescent cancer and a preparation method thereof. The composition comprises two amphiphilic copolymers and active components, wherein the active components are taxane and resveratrol as a promoter for inducing tumor cells apoptosis. The polymeric micelle composition for treating the recrudescent cancer can be accumulated in a tumor tissue; according to the polymeric micelle composition, tumor cells and tumor stem cells are synchronously killed, the treatment effect of the tumors is improved, and the transfer and the recurrence probabilities of the tumors are reduced.
Description
Technical field
The present invention relates to polymer micelle composition being used for the treatment of easy relapsed cancer and preparation method thereof.
Background technology
Malignant tumor is commonly encountered diseases and the frequently-occurring disease of serious threat human life health, has become in world wide the primary disease causing human death.With the antitumor drug that paclitaxel and Docetaxel are representative, it is the treatment being widely used in the malignant tumor such as breast carcinoma, ovarian cancer, nonsmall-cell lung cancer.But taxane anticancer agents is as very low in paclitaxel, Docetaxel dissolubility in water, non-ionic surface active agent is usually adopted to carry out solubilising.Paclitaxel injection taxol (Taxol) is dissolved in by medicine in the mixed solvent of polyoxyethylene castor oil and dehydrated alcohol (1:1).Docetaxel injection taxotere (Taxotere) be by medicine dissolution in surface active agent tween-80 (40g/L), then subpackage, before Clinical practice again with 13% ethanol water dilution and 5% glucose solution dilute.Discharge histamine during surfactant polyoxyethylene Oleum Ricini degradation in vivo, can cause the side effect such as severe allergic reaction and bone marrow depression, surface active agent tween-80 has hemolytic and stickiness is large, also can produce obvious anaphylaxis after injection.
Although taxane has become the first-line drug of the various malignant tumor of clinical treatment, its antitumous effect have also been obtained general accreditation.But as everyone knows, the cure rate of chemotherapy of tumors is extremely low, one of them most important reason is exactly the tumor recurrence after chemotherapy.Once there is tumor recurrence, current clinical treatment means can not produce any therapeutic effect substantially.Nearest research shows, the cell in tumor tissues exists heterogeneous, comprises Common tumors cell and a small amount of tumor stem cell.The conventional chemotherapeutic drugs comprising taxane is merely able to kill and wound common tumor cell, and does not have kill capability to tumor stem cell.After end of chemotherapy, due to the existence of tumor stem cell, result in recurrence and the transfer of tumor, therefore, tumor stem cell is considered to the main cause that tumor produces propagation, recurrence and shifts.Because tumor stem cell does not respond treatment meanss such as common chemotherapy, radiotherapies, lack the therapeutic strategy for tumor stem cell at present clinically.
Resveratrol is a kind of polyphenols extracted from plant, and research shows, it can induced tumor stem cell apoptosis, and resveratrol can by activation P53 gene, and the cytokine causing proteasome to rely on is degraded, induced tumor stem cell generation apoptosis.But the dissolubility of resveratrol in water is extremely low, not easily makes intravenous injection, needs to select suitable adjuvant to its solubilising, meet clinical demand.
On the other hand, conbined usage resveratrol and taxane have synergism, can reduce the dosage of taxane, reduce the generation of toxic and side effects, increase the therapeutic effect of tumor.
Also comprise taxane and tumor stem cell apoptosis promoter resveratrol without any a kind of polymer micelle composition at present simultaneously, also do not occur the micelle composition utilizing two kinds of easy relapsed cancer of Drug therapy.
Summary of the invention
Technical problem
The present invention aims to provide a kind of polymer micelle composition for the treatment of easy relapsed cancer, said composition comprises Amphipathilic block polymer and active component, wherein, described active component is taxane and tumor stem cell apoptosis promoter, and described tumor stem cell apoptosis promoter is resveratrol.
The present invention also aims to provide a kind of preparation method for the treatment of the polymer micelle composition of easy relapse cancer.
The present invention is achieved through the following technical solutions:
The invention provides a kind of polymer micelle composition for the treatment of easy relapsed cancer, said composition comprises Amphipathilic block polymer and active component, wherein, described active component is taxane and tumor stem cell apoptosis promoter, and described tumor stem cell apoptosis promoter is resveratrol.
Polymer micelle composition of the present invention comprises:
(1) be encapsulated in the mixture of taxane in a micelle and resveratrol simultaneously;
(2) can also be encapsulating taxane polymer micelle and the mixture encapsulating resveratrol polymer micelle;
It can also be the mixture of as above (1) and (2).
The mass ratio of its active component taxane and resveratrol is: 0.1-10:1, preferred 0.5-2:1, and the summation of taxane and resveratrol accounts for the 0.1%-45wt% of micelle composition.
Polymer micelle composition of the present invention by weight percentage, comprises following composition:
Taxane 0.2-30%;
Resveratrol 0.2-20%;
Amphipathic nature block polymer 50-99.6%.
Wherein said taxane can be paclitaxel, DTX, 7-acetyl paclitaxel, t-acetyl paclitaxel, 10-deacetylate paclitaxel, 10-deacetylate-7-Epitaxol, 7-xyloside paclitaxel, 10-deacetylate-7-glutarylpaclitaxel, 7-N, N-dimethylglycinamidyl paclitaxel, 7-L-alanyl paclitaxel or above every mixture.Especially, can be Docetaxel or paclitaxel.Described taxane can be noncrystalline or crystallization.Can be dehydrate or hydrate.
Described amphipathic nature block polymer is for comprising the polymer of hydrophilic block (A) and hydrophobic block (B), and wherein, hydrophilic block is selected from one or more blocks of Polyethylene Glycol and methoxy poly (ethylene glycol); Hydrophobic block is selected from one or more blocks of the following: poly-newborn sour ﹑ gathers and hands over the acid of ester ﹑ polylactic acid second alcohol acid ﹑ PVOH to hand over ester ﹑ to gather flat Fructus Persicae acid ﹑ to gather own acid lactone ﹑ and gather and gather ammonia base acid ﹑ to dioxa Ketohexamethylene ﹑ and gather former sour ester ﹑ polyanhydride and above every copolymer.
Described hydrophilic block number-average molecular weight is 500-20,000 dalton, and described hydrophobic block number-average molecular weight is 500-20,000 dalton, and the content of hydrophilic block shared by described block copolymer gross mass is 20-80wt%.
Described resveratrol is used for induced tumor stem cell apoptosis, can be cis or transconfiguration.Resveratrol is a kind of natural polyphenol compounds, extracts the earliest from white hellebore, and be present in Shu ﹑ Hua Sheng ﹑ Fructus Vitis viniferae various plants, resveratrol has cis and trans two kinds of isomers.
In one embodiment of the invention, the polymer micelle composition being used for the treatment of easy relapsed cancer comprises the amphipathic nature block polymer that Zi Shan Wan ﹑ resveratrol and two kinds of poorly water soluble drugs carry out solubilising.
In another embodiment, described polymer micelle is nano-particle.Especially, the particle diameter of described micellar nanoparticles is 10-200nm.
Term " easy relapsed cancer " refers to due to anticarcinogen as taxane etc. can not kill tumor stem cell at this, and after causing using anticarcinogen chemotherapy, tumor stem cell continues differentiation and becomes tumor cell, causes the tumor recurrence or transfer that obtain controlling.Described cancer comprises all types of cancer, such as: breast carcinoma, ovarian cancer, pulmonary carcinoma, carcinoma of prostate, etc.; Without any restriction.
Term " nano-particle " herein means the granule with nano-scale particle size, comprises the nano-particle of micellar structure.
For describing " composite micelle ", " " the statement language " compound " of composite micelle and nanoparticle ” ﹑ or this type of micelle refers to so a kind of state to composite nano-granule ” ﹑: wherein, and taxane is encapsulated in a micelle or nano-particle together with resveratrol.Composite micelle or nano-particle can by obtaining two kinds of drug encapsulation in micelle or nano-particle.
Polymer micelle composition according to invention comprises the mixed micelle and composite micelle and the mixture of the two that are encapsulated with taxane and resveratrol.In one embodiment, the micelle composition being used for the treatment of easy relapsed cancer comprises and is encapsulated in active component taxane in micelle and resveratrol, and described micelle is made up of amphipathic nature block polymer.Described compositions can be composite micelle form or mixed form especially, if composite micelle form, taxane is encapsulated in the same micelle particle of amphipathic nature block polymer formation together with resveratrol, and if mixed form, taxane and resveratrol are encapsulated in different micelle particles and mix.In another embodiment, described compositions can be the mixture of following material: the compositions containing the taxane be encapsulated in together in same micelle particle and resveratrol; And the compositions of encapsulating taxane polymer micelle or encapsulating resveratrol polymer micelle mixture.
According in the anti-cancer agent composition of the embodiment of the present invention, the mass ratio (taxane quality: the quality of resveratrol) of taxane and resveratrol can be 0.1-10:1.Preferred 0.5-2:1, in this quality than in scope, taxane is optimum as the effect of anti-cancer agent therapy and prophylaxis of tumours recurrence.
In another embodiment, based on the gross mass of compositions, the total content of taxane and resveratrol can be 0.1-45wt%.Due to taxane and resveratrol, to be encapsulated in polymer latex intrafascicular, and the content that therefore can be encapsulated in taxane in polymer micelle core and resveratrol is restricted.In one embodiment of the invention, based on the gross mass of compositions, the content of taxane is 0.2-30wt%, preferred 0.2-20%; And the content of resveratrol is 0.2-15wt%, preferred 0.2-10%.
In one embodiment, amphipathic nature block polymer can be the di-block copolymer of A-B type, comprises hydrophilic A and hydrophobic B.Described hydrophilic block A can be Polyethylene Glycol, and hydrophobic B block can be polylactic acid or derivatives thereof.
Polyethylene Glycol as hydrophilic block A can select one or more of Polyethylene Glycol or methoxy poly (ethylene glycol), but is also not limited to these Polyethylene Glycol.Especially, preferred methoxy poly (ethylene glycol).
Polylactic acid or derivatives thereof as hydrophobic block B can be selected from one or more every materials: poly-newborn sour ﹑ gathers and hands over ester ﹑ to gather the acid of second alcohol ﹑ PVOH to hand over ester ﹑ to gather flat Fructus Persicae acid ﹑ to gather own acid lactone ﹑ and gather and gather ammonia base acid ﹑ to dioxa Ketohexamethylene ﹑ and gather former sour ester ﹑ polyanhydride and above every copolymer.Preferably, described polylactic acid or derivatives thereof can be polylactic acid, the acid of polyactide, PVOH hand over ester ﹑ to gather flat Fructus Persicae acid ﹑ to gather own acid lactone ﹑ poly-to dioxanone.
In one embodiment, the number-average molecular weight of described hydrophilic block is 500-20,000 dalton, preferably 1,000-10,000 dalton.The number-average molecular weight of hydrophobic block B is 500-10,000 dalton.In another embodiment, based on the gross weight of described diblock copolymer, the content of hydrophilic block A can be 20-80wt%, preferred 40%-70wt%.
The mass percentage content of described amphipathic nature block polymer shared by compositions gross mass can be 50-99.5%, preferred 50-90%.
The granularity of compound amphipathic nature block polymer compositions (taxane is encapsulated in micelle together with resveratrol) in water is 10-180nm, can be solid-state after lyophilizing.
Present invention also offers a kind of preparation method being used for the treatment of the micelle composition of easy relapsed cancer.According to one embodiment of present invention, be used for the treatment of easy relapsed cancer, can comprise the following steps containing as the preparation method of the taxane of active component and the polymer micelle composition of resveratrol: taxane, resveratrol and amphipathic nature block polymer dissolve in organic solvent by (a); B organic solvent mixes with aqueous medium by (); (c) by solution separating device glass container, lyophilizing and get final product.
Taxane and resveratrol can also respectively from described dissolution of polymer in different organic solvents, thus obtained containing taxane polymer micelle with containing resveratrol polymer micelle, then by these two kinds of micelles mixing, obtained mixed micelle compositions.
Can be compound according to micelle composition of the present invention; also can be mixed type; if compound; then taxane is encapsulated in polymer micelle or nano-particle together with resveratrol; and if mixed type, then the polymer micelle containing taxane or nano-particle are together with the polymer micelle containing resveratrol or mixture of nanoparticles.Preferably, the compositions of above-mentioned two types can be prepared by the following method.
A preparation method for compound anticarcinogen polymer micelle composition, wherein, taxane is encapsulated in described micelle composition as active component together with resveratrol, and described method can comprise the following steps:
A taxane, resveratrol and amphipathic nature block polymer dissolve in organic solvent by (), described organic solvent is selected from: ethanol, methanol, propanol, butanols, amylalcohol, hexanol, acetone, acetonitrile, dichloromethane, chloroform, methyl ethyl ketone and ethyl acetate.
B (), by organic solvent and aqueous solution, obtains mixture, in the mixture, taxane, resveratrol and polymer uniform mix;
C solution is sub-packed in glass container by (), lyophilizing and get final product.
After described preparation method step (b), also can comprise the following steps:
D () carries out sterilizing and depyrogenation to the micelle composition obtained;
In step (d), by solution being filtered sterilizing filter, described aqueous solution is sterilized.
In one embodiment, the aqueous solution used in step (b) can be the aqueous solution of distilled water, normal saline or lyophilizing adjuvant.Lyophilizing adjuvant can be selected from one or more materials in mannitol, sorbitol, lactose, trehalose and sucrose, preferred lactose and trehalose.
A preparation method for mixed type anticarcinogen polymer micelle composition, described micelle composition comprises containing the polymer micelle of taxane or nano-particle and containing the polymer micelle of resveratrol or the mixture of nano-particle, said method comprising the steps of:
A taxane, resveratrol dissolve in organic solvent with amphipathic nature block polymer by () respectively, described organic solvent is selected from: ethanol, methanol, propanol butanols, amylalcohol, hexanol, acetone, acetonitrile, dichloromethane, chloroform, methyl ethyl ketone and ethyl acetate;
B (), by the organic solution that obtains respectively and aqueous solution, different micelle, containing taxane polymer micelle with containing resveratrol polymer micelle, mixes by preparation, preparation mixed type micelle composition.
C solution is sub-packed in glass container by (), lyophilizing and get final product.
Described preparation method also can comprise other step after being positioned at step (c):
D () carries out sterilizing and depyrogenation to the micelle composition obtained.
In step (b), the aqueous solution of use can be the aqueous solution of distilled water, normal saline or lyophilizing adjuvant.Lyophilizing adjuvant can be selected from one or more materials in mannitol, sorbitol, lactose, trehalose and sucrose, preferred lactose and trehalose.
In step (b) and (C), also can will comprise organic solution and the aqueous solution of two kinds of medicines respectively, carry out lyophilizing separately, before use micelle is mixed.
Polymer micelle composition of the present invention can be prepared into acceptable preparation clinically with pharmaceutically acceptable carrier.As the preparation for parenteral, administration can be carried out in the following manner: sublingual administration administration, rectally, percutaneous dosing, intravenous administration, administered intramuscular, intraperitoneal injection etc.A typical example is for intravenous isotonic aqueous solution or suspension.In one embodiment of the present of invention, the micelle composition being used for the treatment of easy relapsed cancer can be made into lyophilized formulations, and after can redissolving in water for injection, 5% glucose solution, normal saline or analog, by the mode administration of vein.
Because taxane and resveratrol are all insoluble in water, therefore need to adopt specific technology and compositions to carry out solubilising, to carry out intravenous administration.Common technological means carries out solubilising with organic solvent (ethanol) or surfactant (polyoxyethylene castor oil or polysorbate) to this type of medicine.But due to when intravenous administration, above-mentioned two kinds of surfactants can cause serious anaphylaxis, therefore need careful use.The invention provides the polymer composition that one does not have apparent side effect (as haemolysis and anaphylaxis), also do not have apparent side effect to produce even if carry out administration in intravenous injection mode.
The polymer micelle composition being used for the treatment of easy relapsed cancer according to the present invention comprises amphipathic nature block polymer, due to permeability and anelasticity (the enhanced permeability and retention of enhancing, EPR) effect, described polymer micelle composition can being deposited on tumor tissues of high concentration.And owing to containing taxane and tumor stem cell apoptosis promoter in described compositions, because it shows outstanding anticancer function to killing and wounding of tumor stem cell.In addition, compositions of the present invention can not produce the haemolysis and anaphylaxis that produce owing to using polyoxyethylene castor oil.The using dosage of taxane can be reduced by conbined usage resveratrol, reduce the generation of toxic reaction, strengthen the therapeutic effect of tumor.
Accompanying drawing explanation
Fig. 1 is the particle size distribution result of simultaneously wrapping the polymer micelle being loaded with Docetaxel and resveratrol, and the described polymer micelle analyzed with dynamic light scattering method is at the grain size distribution of aqueous solution;
Fig. 2 wraps the polymer micelle stability measurement result being loaded with Docetaxel and resveratrol simultaneously, and wherein, Docetaxel and resveratrol use high performance liquid chromatography (HPLC) to measure;
Fig. 3 is that Docetaxel and resveratrol carrier micelle are to the cytotoxicity of MCF-7 cell;
Fig. 4 is that Docetaxel and resveratrol carrier micelle are to the cytotoxicity of MCF-7 tumor stem cell;
Fig. 5 is that Docetaxel and resveratrol carrier micelle are to the drug action of MCF-7 tumor-bearing mice.
Detailed description of the invention
The preparation of [embodiment 1] compound anticarcinogen micelle composition containing amphipathic nature block polymer, wherein Docetaxel is encapsulated in described micelle composition together with resveratrol.
For the preparation of the compound anti-cancer agent composition of the easy relapsed cancer for the treatment of, this anti-cancer agent composition comprises mPEG-PLA (mPEG-PDLLA), PEG-PASP (PEG-PASP), PEG-PDLLA Acetic acid, hydroxy-, bimol. cyclic ester (mPEG-PLGA) amphipathic nature block polymer, and taxane is encapsulated in polymer latex bundle of particle together with resveratrol.
By the substance dissolves in table 1 in the propanol of 15.0ml, then mix with water, in this mixed liquor, Docetaxel, resveratrol and mPEG-PDLLA polymer (mono methoxy polyethylene glycol weight average molecular weight 2000, polyactide weight average molecular weight 2000, mPEG2000-PLA2000) Homogeneous phase mixing.Take lactose 500mg and be dissolved in 10ml water for injection.By the third alcohol and water mix homogeneously under agitation, solution, through 0.22 micron membrane filter aseptic filtration, is sub-packed in after sterilizing in clean cillin bottle.Cillin bottle is placed in, freezing under-30 DEG C of-50 DEG C of conditions, after lyophilizing, through filling nitrogen, jumping a queue, rolling lid, to obtain final product.
[table 1]
[embodiment 2] is encapsulated with the preparation of the composite polymer nano-particle composition of Docetaxel and resveratrol simultaneously.
By the substance dissolves in table 2 in 20ml amylalcohol, then mix with water, in this mixed liquor, Docetaxel, resveratrol and mPEG-PDLLA polymer (mono methoxy polyethylene glycol weight average molecular weight 2000, polyactide weight average molecular weight 6000, MPEG2000-PLA6000) Homogeneous phase mixing.Take trehalose 500mg and be dissolved in 10ml water for injection.By the third alcohol and water mix homogeneously under agitation, solution, through 0.22 micron membrane filter aseptic filtration, is sub-packed in after sterilizing in clean cillin bottle.Cillin bottle is placed in, freezing under-30 DEG C of-50 DEG C of conditions, after lyophilizing, through filling nitrogen, jumping a queue, rolling lid, to obtain final product.
[table 2]
The preparation of [embodiment 3] mixed type anticarcinogen micelle composition, described micelle composition comprises encapsulating Docetaxel polymer micelle and encapsulating contains resveratrol polymer micelle.After preparation is encapsulated with the polymer micelle composition of Docetaxel and resveratrol respectively, by these two kinds of micelle composition mixing, with the micelle composition for the preparation of the easy relapsed cancer for the treatment of.
A) preparation of the Amphiphilic Block Copolymer Micelles of resveratrol is encapsulated
Take resveratrol 10.0mg and mPEG-PDLLA polymer (mono methoxy polyethylene glycol weight average molecular weight 2000, polyactide weight average molecular weight 2000, mPEG2000-PLA2000) 100mg, be dissolved in 20ml butanols, then mix with water, in this mixed liquor, resveratrol and mPEG-PDLLA polymer (mono methoxy polyethylene glycol weight average molecular weight 2000, polyactide weight average molecular weight 2000, mPEG2000-PLA2000) Homogeneous phase mixing, decompression removing organic solvent.
B) preparation of the amphipathic nature block polymer of Docetaxel is encapsulated
Take Docetaxel 10.0mg and mPEG-PDLLA polymer (mono methoxy polyethylene glycol weight average molecular weight 2000, polyactide weight average molecular weight 2000, MPEG2000-PLA2000) 100mg, be dissolved in 20ml butanols, then mix with water, in this mixed liquor, Docetaxel and mPEG-PDLLA polymer (mono methoxy polyethylene glycol weight average molecular weight 2000, polyactide weight average molecular weight 2000, MPEG2000-PLA2000) Homogeneous phase mixing, decompression removing organic solvent.
C) preparation of mixed type polymer micelle composition
By above-mentioned two solution mixing.Taking trehalose 500mg is dissolved in solution, and solution, through 0.22 micron membrane filter aseptic filtration, is sub-packed in after sterilizing in clean cillin bottle.Cillin bottle is placed in, freezing under-30 DEG C of-50 DEG C of conditions, after lyophilizing, through filling nitrogen, jumping a queue, rolling lid, to obtain final product.
D) preparation of mixed type polymer micelle composition
Adopt step a) and step and step b) respectively preparation bag carry the lyophilized products of resveratrol polymer micelle composition and a lyophilized products for Docetaxel polymer micelle composition, lyophilized products is redissolved in an aqueous medium, then mixes.
Measure obtained compositions particle diameter.Result is as shown in table 4
[table 4]
[test case 1] is encapsulated with the granularity of the compound micelle of Docetaxel and resveratrol simultaneously.
The compositions of preparation in embodiment 1 and 2 is redissolved in normal saline, obtains the sample for granulometry.Diluted composition solution granularity is measured with Particle Size Analyzer.The results are shown in Table 5.Typical grain size distribution as shown in Figure 1.As seen from the figure, the particle size distribution range of carrier micelle is narrower, and polydispersity index is less than 0.1.
| Sample | Particle diameter (nm) |
| Compositions 1 | 20-30 |
| Compositions 2 | 17-28 |
| Compositions 3 | 16-30 |
| Compositions 4 | 22-34 |
| Compositions 5 | 20-30 |
| Compositions 6 | 22-34 |
| Compositions 7 | 20-30 |
[measurement example 2] encapsulates the stability of Docetaxel and resveratrol micelle simultaneously
Compositions 4 diluted in test case 1 is at room temperature placed 24 hours, to assess the stability of solution.In testing time section, measure the envelop rate of Docetaxel and resveratrol with high performance liquid chromatograph (HPLC).As shown in Figure 2, concrete HPLC condition is as follows for result:
As shown in Figure 2, the concentration of paclitaxel and resveratrol remains on more than 98%, and this shows that the leakage rate of two kinds of medicines is within 2%.
[test case 3] associating Docetaxel and resveratrol are to the cytotoxicity of MCF-7.
Precision takes Docetaxel and resveratrol, is dissolved in a small amount of DMSO respectively, by DMEM culture fluid dilution Docetaxel to 20.0 μ g/mL concentration; Dilution resveratrol to 10.0,20.0,40.0,80.0 μ g/mL series concentration.By Docetaxel and the mixing of resveratrol equal-volume, be mixed with the medicine series mixed solution that concentration ratio is respectively 2:1 ﹑ 1:1 ﹑ 1:2,1:4.(survey the concentration of medicine should in as above concentration range) be got MCF-7 cell single cell suspension and is inoculated in 96 porocyte culture plates (every hole 7 × 10 with every hole 200 μ L
3individual cell).Cultivate 24h and treat cell attachment, exhaust culture fluid, add variety classes variable concentrations need testing solution, and establish zeroing group, matched group (cell culture fluid), often group establishes 5 multiple holes.After cultivating 48h respectively, every hole adds 5mgmL
-1the PBS solution 20 μ L of MTT, continues to cultivate 4h, discards culture fluid, every hole adds 150 μ L dimethyl sulfoxide, shake about 10min to dissolve completely to precipitation, measure each component, each ratio makes in MCF-7 cell 48 hours optical density value at 490nm place (OD value) with combining separately.
As CDI < 1, show as synergism, show as obvious synergism as CDI < 0.7; Show as summation action as CDI=1, as CDI > 1, then show as antagonism.The a of Fig. 3 is the inhibitory action that Docetaxel and resveratrol combine to MCF-7, the b of Fig. 3 is the synergy index of two medicine different proportions, as can be seen from the figure within the scope of 1:0.1-1:10, shows obvious synergism at the concentration ratio of Docetaxel and resveratrol.
[test case 4] associating Docetaxel and resveratrol are to the cytotoxicity of MCF-7 stem cell
The cultivation of MCF-7 stem cell
After the enzymic digestion of MCF-7 cell tryptase, after the piping and druming of DMEM-F12 culture fluid, with 30,000/mL density is placed in culture bottle and hatches.Under this condition of culture, MCF-7 cell can be dead gradually, and MCF-7 tumor stem cell can be survived, and non-adherent is produced into sphaerocyst bunch, is called mammary gland ball.The centrifugal 5min of 1000rpm can collect mammary gland ball, and this cultural method is called Serium-free Culture.
Add MCF-7 stem cell 10,000/hole, then add the resveratrol solution of variable concentrations, and establish zeroing group, matched group (cell culture fluid), 5 multiple holes are established in every hole.Then cultivate after 48 hours, add CellTiter96Aqueous reagent, measure OD value at 490nm place by microplate reader, calculate cell survival rate.
As can be seen from Figure 4, resveratrol has obvious lethal effect to tumor stem cell, and along with the increase of concentration, apoptosis-induced effect is more obvious.Make veratryl alcohol clear and can induce MCF-7 apoptosis of tumor cells.
[test case 5] Docetaxel and resveratrol carrier micelle are to the drug action of MCF-7 tumor-bearing mice
Detect the active anticancer of 8-10 compositions in embodiment
Cultivated by the DMEM culture fluid of the MCF-7 cell be stored in liquid nitrogen containing 10%FBS, before use, with trypsinization, the centrifugal 4min of 1000rpm, takes out supernatant, is resuspended in by cell in the normal saline of 0.9%, adjustment cell concentration to 5 × 10
6cell/200 μ L, is then seeded to nude mice on the right side of oxter by cell suspension according to every nude mice 200 μ L liquid volume with 1mL syringe.When tumor grows to 50mm
3time start administration, and administration is designated as 0 day that day, nude mice is divided into 5 groups, at the 0th day, 3rd day and the 6th day, respectively with the dosage of 35mg/kg Docetaxel, 35mg/kg resveratrol, undertaken giving matched group normal saline, compositions 8 (resveratrol micelle composition), compositions 9 (Docetaxel micelle composition) and compositions 10 (resveratrol/Docetaxel polymer micelle composition) by tail vein.
As time goes on, gross tumor volume is measured.Gross tumor volume is calculated by formula 1.
Formula 1
Gross tumor volume (TV)=(W
2× L)/2 (wherein, L is major axis, and W is minor axis)
In order to the effectiveness of validation test, each treatment uses at least 4 nude mices, and each group uses at least 4 tumors.In the starting point for the treatment of, the size of tumor is: volume is 50mm
3.If last administration 2 weeks interior animals die, then think to be poisoned to death, need to reject from assessment.There is the group dead more than in 3 animals, or comprise the group that weight loss more than 15% and thoroughly recovers, be considered to there is no active anticancer.Result as shown in Figure 5.
As can be seen from Figure 5, give resveratrol micelle composition (compositions 8) compared with matched group, there is certain inhibitory action to cancer, but be not so good as the micelle group (compositions 9) obvious (P < 0.05) of Docetaxel.Especially, by mixed type polymer micelle composition (compositions 10) treatment group with compared with the group for the treatment of containing paclitaxel polymer micelle composition group (compositions 9) (P < 0.05), the former shows the inhibit activities better to growth of cancer cells.
Claims (10)
1. be used for the treatment of a polymer micelle composition for easy relapsed cancer, comprise Amphipathilic block polymer and active component, it is characterized in that, described active component is taxane and tumor stem cell apoptosis promoter resveratrol.
2. according to the polymer micelle composition being used for the treatment of easy relapsed cancer described in claim 1, it is characterized in that: described compositions comprises and is encapsulated in taxane in micelle and resveratrol simultaneously, or the mixture of encapsulating taxane polymer micelle and encapsulating resveratrol polymer micelle, or the compositions of the taxane be encapsulated in a micelle and resveratrol and encapsulating taxane polymer micelle and the mixture encapsulating resveratrol polymer micelle.
3. the polymer micelle composition being used for the treatment of easy relapsed cancer according to claim 1 or 2; it is characterized in that: described taxane is selected from the every mixture of paclitaxel, Docetaxel, 7-acetyl paclitaxel, t-acetyl paclitaxel, 10-deacetylate paclitaxel, 10-deacetylate-7-Epitaxol, 7-xyloside paclitaxel, 10-deacetylate-7-glutarylpaclitaxel, 7-N, N-dimethylglycinamidyl paclitaxel, 7-L-alanyl paclitaxel or more.
4. according to the polymer micelle composition that be used for the treatment of easy relapsed cancer of claim 1-3 described in any one, it is characterized in that: described amphipathic nature block polymer is the polymer comprising hydrophilic block and hydrophobic block, wherein, hydrophilic block is selected from one or more blocks of Polyethylene Glycol and methoxy poly (ethylene glycol); Hydrophobic block is selected from one or more blocks of the following: poly-newborn sour ﹑ gathers and hands over the acid of ester ﹑ polylactic acid second alcohol acid ﹑ PVOH to hand over ester ﹑ to gather flat Fructus Persicae acid ﹑ to gather own acid lactone ﹑ and gather and gather ammonia base acid ﹑ to dioxa Ketohexamethylene ﹑ and gather former sour ester ﹑ polyanhydride and above every copolymer.
5. the polymer micelle composition being used for the treatment of easy relapsed cancer according to claim 4, its feature is being: described hydrophilic block number-average molecular weight is 500-20,000 dalton, described hydrophobic block number-average molecular weight is 500-20,000 dalton, and the content of hydrophilic block shared by described block copolymer gross mass is 20-80wt%.
6. the polymer micelle composition being used for the treatment of easy relapsed cancer according to claim 1 and 2, is characterized in that: the mass ratio of taxane and resveratrol is 0.1-10:1.
7. the polymer micelle composition being used for the treatment of easy relapsed cancer according to claim 1 or 2, is characterized in that: it is 0.1-45wt% that taxane and resveratrol summation account for described micelle composition gross mass.
8. the polymer micelle composition being used for the treatment of easy relapsed cancer according to claim 1 and 2, is characterized in that, described micelle composition by weight percentage, comprising:
The taxane of 0.2-30wt%
The resveratrol of 0.2-20wt%
The amphipathic nature block polymer of 50-99.6wt%.
9., according to the preparation method being used for the treatment of the polymer micelle composition of easy relapsed cancer described in claim 1, it is characterized in that, comprise the following steps:
(1) taxane, resveratrol and amphipathic nature block polymer dissolve in organic solvent;
(2) organic solvent is mixed with water, be uniformly mixed rear lyophilizing;
Or
(1) taxane, resveratrol dissolve in organic solvent with amphipathic nature block polymer respectively;
(2) organic solvent containing taxane obtained and the organic solvent containing resveratrol are mixed with water respectively, be uniformly mixed rear lyophilizing.
10. the preparation method being used for the treatment of easy relapsed cancer polymer micelle composition according to claim 9, it is characterized in that, described organic solvent is selected from: at least one of methanol, ethanol, propanol, butanols, amylalcohol, hexanol, acetone, acetonitrile, dichloromethane, chloroform, methyl ethyl ketone or ethyl acetate.
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| CN105232459A (en) * | 2015-09-24 | 2016-01-13 | 沈阳药科大学 | Redissolving self-assembly type poorly-water-soluble drug-polymer micelle composition and preparation method thereof |
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