CN106902078A - One kind contains taxol micelle nano cluster and preparation method thereof - Google Patents
One kind contains taxol micelle nano cluster and preparation method thereof Download PDFInfo
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- CN106902078A CN106902078A CN201510964287.4A CN201510964287A CN106902078A CN 106902078 A CN106902078 A CN 106902078A CN 201510964287 A CN201510964287 A CN 201510964287A CN 106902078 A CN106902078 A CN 106902078A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
- A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
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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
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
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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
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
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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
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
Abstract
The invention belongs to biomedicine technical field, it is related to one kind to contain taxol micelle nano cluster and preparation method thereof.Taxol micella (the PTXPEG PLA micelles that the present invention will be prepared using emulsification ultrasonic method；PM taxol micelle nano cluster) is prepared by crosslinking agent, is eventually adding in the negatively charged small molecule of hydrophily and positive surface charge and is improved system hydrophily.Micelle nano cluster system of the present invention contains in the hydrophobic inner core of micella taxol, do not influenceing on basis of original micella compared with high drug load, effectively increase system particle diameter and improve system hydrophily, extension circulation time in vivo, increase tumour EPR effects, so as to improve the validity and security of the medicine clinical practice.
Description
Technical field
The invention belongs to biomedicine technical field, it is related to one kind to contain taxol micelle nano cluster and its preparation side
Method；It is described to contain taxol micelle nano cluster for a kind of extension polymer micelle circulation time in vivo and effectively play
The micelle nano cluster of tumour EPR effects, can improve oncotherapy effect.
Technical background
It is a line broad-spectrum anti-cancer drug prior art discloses taxol (PTX), by promoting spindle micro-
The polymerization of tubulin, suppresses depolymerization, keeps tubulin stabilization, so that suppress tumour cell mitosis process,
And then antitumor action is played, it is evident in efficacy；But its solubility in water is less than 1ug/m.The taxol
Listing preparationMainly using PEG-PLA micellar systems increases the dosage of taxol,
Amphipathic nature material can form the copolymer micelle with spherical core-shell structure in aqueous, in it
Core, reduces its toxic and side effect by taxol solubilising internally as the container of hydrophobic drug, and shell is to medicine
Shield, it is difficult by reticuloendothelial system phagocytic, improve the stability of medicine, it is to avoid traditional system
AgentThe use of middle solubilizer Emulsifier EL-60, so as to can obviously reduce the allergy of paclitaxel injection
Property, hematotoxicity, improve the security that uses of medicine.But practice display, it is describedCompared with
Do not increase the Relative drug accumulation of tumor locus, be slightly less than on the contraryAnd's
Circulation time in vivo compared withShorten, it is impossible to when reaching tumor locus to taxol and playing curative effect and provide enough
Between.
Research display, entity tumor has enhancing infiltration and retention effect (EPR), due to relatively strong capillary
Vascular permeability, nano-particle increases in the relative accumulation of tumor tissues；However, the performance of EPR effects for
The particle diameter of nano-particle has certain limitation.It is reported that the taxol micella of average grain diameter 10nm with it is free
Taxol is compared and is not almost shown any EPR effects；With average grain diameter more than 300nm or less than 50nm
Nano-particle compare, particle diameter shows accumulation higher for the particle of 100nm and 200nm；Average grain diameter
For the particle of 100nm shows longer body-internal-circulation and relatively low mononuclear macrophage intake.Though
So, the taxol PEG-PLA micellas have the distinctive advantage of the system, but due to most of polymer latex
The particle diameter of beam is smaller (10~50nm), it is impossible to effectively play tumour EPR effects, therefore, build a kind of new
Polymer micelle structure, i.e. polymer micelle nano-cluster, on the premise of maintaining original micellar structure constant,
Larger hydrophilic polymer backbones are formed by cross-linking reaction to carry micella so as to form micellar aggregates, both
The Drug loading capacity of the constant original micella of maintenance of micella original structure can be ensured, system particle diameter can be increased to again
100nm or so plays tumour EPR effects, so as to improve the validity and security of administering paclitaxel system.
The TPGS is a kind of nonionic surfactant, can be used as solubilizer, sorbefacient, emulsifying agent,
Precipitating inhibitor and the carrier as medicine etc. are widely used in preparation research.Currently have been reported that, TPGS
Taxol bioavilability can be improved, the therapeutic effect of taxol is improved, and reverse as p-gp inhibitor
Tumor multi-medicine drug-resistant.
The polyethyleneimine (PEI), is a kind of with functional water-soluble macromolecule, is broadly divided into two kinds
Structure type：Dendroid and linear；Relative molecular mass from hundreds of to hundreds of thousands, on its macromolecular chain
Possess substantial amounts of amino, only micella does not provide the barrier of stable existence, and system surface has been taken positive electricity
Lotus and functional groups, for the multi-functional modification in the surface of nano particle provides feasibility.
The content of the invention
Taxol micelle nano cluster is contained it is an object of the invention to provide one kind；It is described to contain taxol micelle nano
Cluster is a kind of new polymer micelle structure, i.e. polymer micelle nano-cluster；The nano-cluster is ensureing original poly-
On the constant basis of compound micellar structure, can effectively increase system particle diameter, extend circulation time in vivo, increase
Plus tumour EPR effects, so as to improve the validity and security of the medicine clinical practice.
It is of the present invention to contain taxol micelle nano cluster, hydrophilic cross-linking is built using TPGS and PEI netted
Skeleton, makes polymer micelle interspersed wherein so as to increase system particle diameter, and by IgG and positive surface charge simultaneously
Increase system hydrophily, is obtained and contains taxol micelle nano cluster；This contains the particle diameter of taxol micelle nano cluster
It is 100nm or so；In the present invention, take the micella after dilution and contain taxol micelle nano cluster solution in right amount,
It is added dropwise on the copper mesh of covering carbon film, 2.0% phosphotungstic acid negative staining, observation under transmission electron microscope is placed in after drying at room temperature,
Particle diameter is respectively 15nm, 100nm or so (respectively as shown in Figure 1 and Figure 2).
It is a further object of the present invention to provide the preparation method for containing taxol micelle nano cluster；
The preparation method for containing taxol micelle nano cluster of the present invention, prepares micella and receives using emulsification ultrasonic method
Rice cluster, comprises the following steps：
(1) taxol and carrier material are completely dissolved in a in organic solvent, and material and taxol are formed uniformly
Solution I；
(2) rotary evaporation of the solution I removes organic solvent a, and carrier and pharmaceutical film is obtained；
(3) by above-mentioned film aquation, polymer micelle solution II is obtained；
(4) PEI solution is added in the polymer micelle solution II, is added contain TPGS-CDI under agitation
Organic solvent b, formed white emulsion III；
(5) by the water bath sonicator of above-mentioned white emulsion III, rotary evaporation removes organic solvent b, obtains solution IV；
(6) solution IV adds IgG solution under agitation, 1h is stirred at room temperature and obtains micelle nano cluster solution
V, it is standby to be placed in 4 DEG C of refrigerators, is obtained and contains taxol micelle nano cluster.
In the present invention, the polymeric material of the polymer micelle is prepared for PEG2000-PLA, and through experiment
It has been shown that, the micella that other materials is formed is difficult to reach preferable drugloading rate；Wherein, it is described
The molecular weight of PEG2000-PLA, PEG is 2000, is (40 with the block ratio of PLA:60)~(60:40),
I.e. the molecular weight of PLA is 1333~3000, and total molecular weight is 3333~5000；
In preparation method of the present invention, the molecular weight of the PEI is in 600~25000Da；
In preparation method of the present invention, described TPGS is the esterification products of D-a-Vitamin E and PEG,
Wherein the molecular weight of PEG is 500~5000；In the present invention also can using molecular weight 500~5000 PEG or contain
Have molecular weight 500~5000 PEG macromolecular；
In preparation method of the present invention, the micellar carrier material taxol is with the mass ratio of PEG-PLA
(1:5)~(1:1000)；
In preparation method of the present invention, preferably emulsion is formed by emulsifying ultrasonic method；
In the present invention, the method for the micelle nano cluster is prepared for film dispersion method, but be not limited to the method,
Dialysis, emulsion process or desivac can also be used；
In preparation method of the present invention, the micellar carrier material PEG-PLA and micelle nano cluster skeleton material
The mass ratio of material is (5:1)~(1:1000)；
In preparation method of the present invention, the organic solvent a is selected from dichloromethane, chloroform, tetrahydrochysene furan
Mutter, acetonitrile, ether, acetone, methyl alcohol or ethanol any one；
In preparation method of the present invention, the organic solvent b is selected from any in dichloromethane or chloroform
Kind；
In preparation method of the present invention, the volume ratio of the reclaimed water phase of the white emulsion III and organic phase is (2:1)
~(20:1)；
In preparation method of the present invention, gained micella is with the particle size range of dynamic light scattering determination
10nm-30nm；
In preparation method of the present invention, gained micelle nano cluster is with the particle size range of dynamic light scattering determination
It is 50nm-200nm；
In preparation method of the present invention, (2) middle rotating evaporation temperature is 25 DEG C~60 DEG C to the step；
In preparation method of the present invention, the step (3) middle aqueous vehicles be selected from deionized water, physiological saline or
Any one of PBS；
In preparation method of the present invention, (5) middle water-bath ultrasonic time is 5~60min to the step；
In preparation method of the present invention, (5) middle rotating evaporation temperature is 25~60 DEG C to the step；
In preparation method of the present invention, the IgG is immunoglobulin G, is the main antibody component of serum,
It is a kind of hydrophilic small molecules, can also uses other electronegative hydrophilic small molecules.
The present invention has also carried out assessment and has contained taxol micelle nano cluster to A549 and RAW cellular uptake medicines
The experiment of influence：Result shows that the PTX concentration of micelle nano cluster is significantly stronger than after A549 cellular uptakes 1h
Common micella, and the PTX concentration of RAW cells is substantially less than common micella；The taxol PEG-PLA
IC of the micelle nano cluster to A549 cell lines50It is 2.39 ± 0.63ng/ml to be worth；Result shows that the A549 is thin
Born of the same parents are significantly more than common micella to the intake of micelle nano cluster, and the intake of RAW cells is substantially less than general
Logical micella；Confirm, the obtained micelle nano cluster can reduce macrophage phagocytic, increase it in tumour cell
Relative accumulation (as shown in Figure 3).
The present invention investigates empty vectors material to the toxicity of A549 tumour cells using mtt assay, as a result shows,
IC of the micelle nano cluster to A54950Value is more than 500ng/ml, shows that the carrier material of the micelle nano cluster is biological
Compatibility is preferably (as shown in Figure 4).
The present invention investigates the cell for carrying taxol micelle nano cluster to A549 tumour cells using mtt assay respectively
Toxic action, as a result shows, 3 kinds of IC of formulation for paclitaxel50Value is ordered as the common micella ﹤ trips of micelle nano cluster ﹤
From taxol, significant difference (P is respectively provided between any two<0.05)；Result shows, obtained micelle nano cluster
The cytotoxicity (as shown in Figure 5) of taxol can be improved.
The present invention with DiR fluorescence labelings micella and micelle nano cluster spike its distribution behavior in vivo, as a result
As shown in fig. 7, the extension micelle nano cluster after administration over time has in tumor locus significantly accumulate, and compared with
The accumulation of micella is more obvious；Result shows, because the EP R effects of particle diameter increase micelle nano cluster significantly increase
Plus.
The present invention carries out pharmacokinetic evaluation, as a result such as by being injected intravenously with healthy SD rat as model to it
Shown in Fig. 7, the medicine of rat intravenous injection is moved by the taxol PEG-PLA micelle nanos cluster obtained by the method
Process meets two compartment model, wherein t1/2It is 412.93 ± 120.21min, the scope of CL exists
Between 0.012 ± 0.003L/min/kg；Result shows that it meets two compartment model, and common micellar phase ratio, with band
Micelle nano cluster in the small molecule of negative electricity and afterwards can be obviously prolonged circulation time in vivo；
The present invention has the nude mice of A549 subcutaneous tumors as animal model with lotus, and common micella and glue are given by vein
Beam nano-cluster, carries out internal pharmacodynamic evaluation, and administration is put to death nude mice and separates subcutaneous tumors, as a result shows after 12 days
Show that the micelle nano cluster is substantially better than common micella (as shown in Figure 6) to the drug effect of A549 tumours.
It is of the invention to contain taxol micelle nano cluster, with commercially available common taxol micellar phase ratio, have into play
Advantage；
Can ensure on taxol PEG-PLA micellar systems high drug load basis, extend system body-internal-circulation
Time, strengthen EPR effects, so as to increase its relative accumulation in tumor locus, reduce the adverse reaction of medicine,
Improve antineoplastic therapeutic effect.
Brief description of the drawings
Fig. 1 is the grain size distribution of taxol PEG-PLA micellas.
Fig. 2 is the grain size distribution of taxol micelle nano cluster.
Fig. 3 is the transmission electron microscope photo of taxol PEG-PLA micellas, and the micella of preparation is evenly distributed, form circle
It is whole.
Fig. 4 is the transmission electron microscope photo of taxol micelle nano cluster, and the micelle nano cluster of preparation is uniformly dispersed, and
Soilless sticking phenomenon.
Fig. 5 is the dilution stability of taxol micelle nano cluster.Inspection target is micelle nano cluster particle diameter, one
In fixed dilution range, taxol micelle nano cluster has good stability.
Fig. 6 is RAW cells to taxol PEG-PLA micellas and the intake of taxol micelle nano cluster.
Fig. 7 is intake of the A549 tumour cells to taxol PEG-PLA micellas and taxol micelle nano cluster
Amount.
Fig. 8 is cell toxicant evaluation of the blank micella nanocluster material to A549 tumour cells.
Fig. 9 is that free paclitaxel (Free PTX), the common micella of taxol and taxol micelle nano cluster are right respectively
The CDCC of A549 cells.
Figure 10 is the pharmacokinetic curve (n=6) of healthy SD rat vein instillation micelle nano cluster (PNMC).
Figure 11 is common micella (the DiRPEG-PLA micelles of DiR spikes；) and micelle nano cluster (DiR DM
micelles nanocluster；DMNC) respectively different time lotus A549 subcutaneous tumors mouse internal distribution.
Figure 12 is that lotus subcutaneous tumors nude mice is injected intravenously the in vitro knurl ratio of common micella and micelle nano cluster after 12 days
Compared with.
Specific embodiment
The present invention is elaborated with reference to embodiment, but protection scope of the present invention is not limited to following realities
Apply example.
Embodiment 1：
50mg PEG-PLA, 10mg taxols are weighed, 4ml acetonitriles are added, ultrasound makes carrier material and medicine
Thing fully dissolves.Thoroughly be evaporated organic solvent by revolving 30min at the solution is placed in into 40 DEG C of Rotary Evaporators,
It is placed at room temperature in vacuum drying chamber overnight, removing a small amount of solvent of residual must dry transparent medicine film skeleton, then
10ml deionized waters constant speed stirring 30min under 500rpm rotating speeds is added, hydrating fluid is crossed into 0.22 μm of acetic acid
Cellulose ester membrane filter, obtains taxol PEG-PLA micellar solutions, in taxol under the stirring of 500rpm rotating speeds
The μ l of 0.5%PEI 200 are added in PEG-PLA micellar solutions, and is added dropwise 1ml contains 20mgTPGS-CDI two
Chloromethanes solution, ultrasound 30min is emulsified by gained emulsion, and organic solvent is evaporated off in 40 DEG C of backspins, plus
Enter 1mg IgG, be incubated at room temperature 1h, be obtained and contain taxol micelle nano cluster.
Embodiment 2：
70mg PEG-PLA, 10mg taxols are weighed, 4ml dichloromethane is added, ultrasound makes carrier material
Fully dissolved with medicine, revolving 30min is thorough by organic solvent at the solution is placed in into 40 DEG C of Rotary Evaporators
It is evaporated, is placed at room temperature in vacuum drying chamber overnight, removing a small amount of solvent of residual must dries transparent medicine film skeleton,
It is subsequently adding 10ml and removes physiological saline constant speed stirring 30min under 500rpm rotating speeds.Hydrating fluid is crossed 0.22 μm
Acetyl cellulose filter membrane, obtains taxol PEG-PLA micellar solutions, in Japanese yew under the stirring of 500rpm rotating speeds
The μ l of 0.5%PEI 200 are added in alcohol PEG-PLA micellar solutions, and 1ml is added dropwise and contain 20mgTPGS-CDI
Dichloromethane solution, gained emulsion is emulsified into ultrasound 30min, and organic solvent is evaporated off in 30 DEG C of backspins,
1mg IgG are added, 1h is incubated at room temperature, is obtained and is contained taxol micelle nano cluster.
Embodiment 3：
100mg PEG-PLA, 30mg taxols are weighed, 4ml dichloromethane is added, ultrasound makes carrier material
Fully dissolved with medicine, revolving 30min is thorough by organic solvent at the solution is placed in into 50 DEG C of Rotary Evaporators
It is evaporated, is placed at room temperature in vacuum drying chamber overnight, removing a small amount of solvent of residual must dries transparent medicine film skeleton,
It is subsequently adding 15ml physiological saline constant speed stirring 30min under 500rpm rotating speeds.Hydrating fluid is crossed 0.22 μm
Acetyl cellulose filter membrane, obtains taxol PEG-PLA micellar solutions, in Japanese yew under the stirring of 500rpm rotating speeds
The μ l of 0.5%PEI 300 are added in alcohol PEG-PLA micellar solutions, and 1ml is added dropwise and contain 20mgPEG-CDI's
Chloroform soln, ultrasound 30min is emulsified by gained emulsion, and organic solvent is evaporated off in 40 DEG C of backspins,
1.5mg IgG are added, 1h is incubated at room temperature, is obtained and is contained taxol micelle nano cluster.
Embodiment 4：
10mg PEG-PLA, 2mg taxols are weighed, 4ml acetonitriles are added, ultrasound fills carrier material and medicine
Divide dissolving.Thoroughly be evaporated organic solvent by revolving 30min at the solution is placed in into 30 DEG C of Rotary Evaporators, room temperature
Under be placed in vacuum drying chamber overnight, removing a small amount of solvent of residual must dry transparent medicine film skeleton.It is subsequently adding
15ml deionized waters constant speed stirring 30min under 500rpm rotating speeds.Hydrating fluid is crossed into 0.22 μm of acetyl cellulose
Filter membrane, obtains taxol PEG-PLA micellar solutions.In taxol PEG-PLA glue under the stirring of 500rpm rotating speeds
The μ l of 0.5%PEI 300 are added in beam solution, and the dichloromethane solution that 1.5ml contains 40mgPEG-CDI are added dropwise,
Gained emulsion is emulsified into ultrasound 30min, and organic solvent is evaporated off in 40 DEG C of backspins, add 1.5mg IgG,
Incubation at room temperature 1h, is obtained and contains taxol micelle nano cluster.
Embodiment 5：
50mg PEG-PLA, 2mg taxols are weighed, 4ml acetonitriles are added, ultrasound fills carrier material and medicine
Divide dissolving.Thoroughly be evaporated organic solvent by revolving 30min at the solution is placed in into 40 DEG C of Rotary Evaporators, room temperature
Under be placed in vacuum drying chamber overnight, removing a small amount of solvent of residual must dry transparent medicine film skeleton, be subsequently adding
10ml physiological saline constant speed stirring 30min under 500rpm rotating speeds.Hydrating fluid is crossed into 0.22 μm of acetyl cellulose
Filter membrane, obtains taxol PEG-PLA micellar solutions, in taxol PEG-PLA glue under the stirring of 500rpm rotating speeds
The μ l of 0.5%PEI 200 are added in beam solution, and the chloroform soln that 1ml contains 40mgTPGS-CDI are added dropwise,
Gained emulsion is emulsified into ultrasound 30min, and organic solvent is evaporated off in 50 DEG C of backspins, add 1mg IgG,
Incubation at room temperature 1h, is obtained and contains taxol micelle nano cluster.
Embodiment 6：
20mg PEG-PLA, 2mg taxols are weighed, 4ml acetonitriles are added, ultrasound fills carrier material and medicine
Divide dissolving, thoroughly be evaporated organic solvent by revolving 30min at the solution is placed in into 30 DEG C of Rotary Evaporators, room temperature
Under be placed in vacuum drying chamber overnight, removing a small amount of solvent of residual must dry transparent medicine film skeleton, be subsequently adding
20ml PBS (Ph7.4) constant speed stirring 30min under 500rpm rotating speeds, 0.22 μm of cellulose acetate is crossed by hydrating fluid
Ester filter membrane, obtains taxol PEG-PLA micellar solutions, in taxol PEG-PLA under the stirring of 500rpm rotating speeds
The μ l of 0.5%PEI 200 are added in micellar solution, and the chloroform soln that 1ml contains 40mgTPGS-CDI are added dropwise,
Gained emulsion is emulsified into ultrasound 30min, and organic solvent is evaporated off in 40 DEG C of backspins, add 1mg IgG,
Incubation at room temperature 1h, is obtained and contains taxol micelle nano cluster.
Embodiment 7：
20mg PEG-PLA, 2mg taxols are weighed, 4ml acetonitriles are added, ultrasound fills carrier material and medicine
Divide dissolving, thoroughly be evaporated organic solvent by revolving 30min at the solution is placed in into 30 DEG C of Rotary Evaporators, room temperature
Under be placed in vacuum drying chamber overnight, removing a small amount of solvent of residual must dry transparent medicine film skeleton, be subsequently adding
10ml physiological saline constant speed stirring 30min under 500rpm rotating speeds.Hydrating fluid is crossed into 0.22 μm of acetyl cellulose
Filter membrane, obtains taxol PEG-PLA micellar solutions, in taxol PEG-PLA glue under the stirring of 500rpm rotating speeds
The μ l of 0.5%PEI 200 are added in beam solution, and the chloroform soln that 1ml contains 40mgTPGS-CDI are added dropwise,
Gained emulsion is emulsified into ultrasound 30min, and organic solvent is evaporated off in 40 DEG C of backspins, add 1mg IgG,
Incubation at room temperature 1h, is obtained and contains taxol micelle nano cluster.
Embodiment 8：
20mg PEG-PLA, 2mg taxols are weighed, 4ml acetonitriles are added, ultrasound fills carrier material and medicine
Divide dissolving, thoroughly be evaporated organic solvent by revolving 30min at the solution is placed in into 30 DEG C of Rotary Evaporators, room temperature
Under be placed in vacuum drying chamber overnight, removing a small amount of solvent of residual must dry transparent medicine film skeleton, be subsequently adding
20ml PBS (pH7.4) constant speed stirring 30min under 500rpm rotating speeds.Hydrating fluid is crossed into 0.22 μm of cellulose acetate
Ester filter membrane, obtains taxol PEG-PLA micellar solutions, in taxol PEG-PLA under the stirring of 500rpm rotating speeds
The μ l of 0.5%PEI 200 are added in micellar solution, and the chloroform soln that 1ml contains 40mgTPGS-CDI are added dropwise,
Gained emulsion is emulsified into ultrasound 30min, and organic solvent is evaporated off in 40 DEG C of backspins, add 1mg IgG,
Incubation at room temperature 1h, is obtained and contains taxol micelle nano cluster.
Embodiment 9：
50mg PEG-PLA, 10mg taxols are weighed, 4ml acetonitriles are added, ultrasound makes carrier material and medicine
Thing fully dissolves, and thoroughly be evaporated organic solvent by revolving 30min at the solution is placed in into 40 DEG C of Rotary Evaporators,
It is placed at room temperature in vacuum drying chamber overnight, removing a small amount of solvent of residual must dry transparent medicine film skeleton, then
Add 10ml PBS (pH7.4) constant speed stirring 30min under 500rpm rotating speeds.Hydrating fluid is crossed into 0.22 μm of vinegar
Acid cellulose ester filter membrane, obtains taxol PEG-PLA micellar solutions.In taxol under the stirring of 500rpm rotating speeds
The μ l of 0.5%PEI 200 are added in PEG-PLA micellar solutions, and is added dropwise 2ml contains 20mgTPGS-CDI three
Chloromethanes solution, ultrasound 30min is emulsified by gained emulsion, and organic solvent is evaporated off in 40 DEG C of backspins, plus
Enter 1mg IgG, be incubated at room temperature 1h, be obtained and contain taxol micelle nano cluster.
Embodiment 10：
50mg PEG-PLA, 10mg taxols are weighed, 4ml acetonitriles are added, ultrasound makes carrier material and medicine
Fully dissolving, thoroughly be evaporated organic solvent by revolving 30min at the solution is placed in into 40 DEG C of Rotary Evaporators, room
It is placed under temperature in vacuum drying chamber overnight, removing a small amount of solvent of residual must dry transparent medicine film skeleton, Ran Houjia
Enter 20ml deionized waters constant speed stirring 30min under 500rpm rotating speeds.Hydrating fluid is crossed into 0.22 μm of acetate fiber
Plain ester filter membrane, obtains taxol PEG-PLA micellar solutions.In taxol PEG-PLA under the stirring of 500rpm rotating speeds
The μ l of 0.5%PEI 400 are added in micellar solution, and the chloroform soln that 5ml contains 20mgTPGS-CDI are added dropwise,
Gained emulsion is emulsified into ultrasound 30min, and organic solvent is evaporated off in 40 DEG C of backspins, add 4mg IgG,
Incubation at room temperature 1h, is obtained and contains taxol micelle nano cluster.
Embodiment 11：
50mg PEG-PLA, 12.5mg taxols are weighed, 4ml acetonitriles are added, ultrasound makes carrier material and medicine
Thing fully dissolves, and thoroughly be evaporated organic solvent by revolving 30min at the solution is placed in into 40 DEG C of Rotary Evaporators,
It is placed at room temperature in vacuum drying chamber overnight, removing a small amount of solvent of residual must dry transparent medicine film skeleton, then
10ml deionized waters constant speed stirring 30min under 500rpm rotating speeds is added, hydrating fluid is crossed into 0.22 μm of acetic acid fibre
The plain ester filter membrane of dimension, obtains taxol PEG-PLA micellar solutions.In taxol under the stirring of 500rpm rotating speeds
The μ l of 0.5%PEI 400 are added in PEG-PLA micellar solutions, and is added dropwise 0.5ml contains 40mgTPGS-CDI two
Chloromethanes solution, ultrasound 30min is emulsified by gained emulsion, and organic solvent is evaporated off in 50 DEG C of backspins, plus
Enter 1mg hyaluronic acids, be incubated at room temperature 1h, be obtained and contain taxol micelle nano cluster.
Embodiment 12：
Cellular uptake quantitative determination：Ratio according to carrier and medicine prepares the taxol of 5 μ g/ml respectively
The taxol micelle nano cluster of PEG-PLA micellas and 5 μ g/ml, and it is incubated 1h at 37 DEG C；
The A549 cells of exponential phase are inoculated in 6 orifice plates with the density of 1 × 105cells/pore, cell is treated
PBS washed cells individual layer is used after adherent 2 times, add the DMEM nutrient solutions without FBS, in 37 DEG C of incubations
30min；Incubation suctions out training liquid after terminating, embathed 3 times with the PBS of precooling, and taxol PEG-PLA is added per hole
Taxol each 1ml of micelle nano cluster of micella and 5 μ g/ml, 3 parts of operation repetitive is incubated 3h at 37 DEG C, is incubated knot
Shu Hou, rapidly removes medicine, with the PBS washed cells individual layer 3 times of precooling, adds the distilled water of 400 μ l, uses
Cell scraper scraping cell gently is transferred in EP pipes, takes 100 μ l cell samples, adds 200 μ l methyl alcohol
Probe Ultrasonic Searching extracts intracellular PTX, and 5min is centrifuged in 10000rpm, takes supernatant and determines thin using HPLC methods
Intracellular PTX contents, go the cell sample of remainder to determine intracellular protein content with BCA methods；Will be per hole
The content of PTX is used to eliminate the error that every hole cell quantity brings divided by the protein content of respective aperture.
Embodiment 13：
Taxol micelle nano cluster is diluted to containing the μ g/ml~10 μ g/ml's of taxol 0.001 with liquid is trained containing FBS
Series concentration, preparations. Control group is：A () (is dissolved in containing 1.0%DMSO's with the paclitaxel solution of concentration range
In PBS)；The taxol PEG-PLA micellas of (b) same concentration range；By Non-small cell lung carcinoma cell A549
With 5 × 103The density of cells/pore is inoculated into 96 well culture plates, after culture 24h, by above experimental program
Give containing drug solns, six parts of operation repetitive directly cultivates 96h under conventional culture conditions, and culture is used after terminating
Mtt assay calculates inhibitory rate of cell growth and seeks half-inhibition concentration IC50Value；Result shows, described to contain purple
The IC of China fir alcohol micelle nano cluster group50Significantly less than PEG-PLA micella groups, the micelle nano cluster really can be with
Increase particle diameter improves the cytotoxicity of taxol.
Embodiment 14：
Lotus A549 subcutaneous tumors nude mices are randomly divided into 4 groups, every group 6, physiological saline (saline) is set to
Control group, Taxol groups (PTX:10mg/kg), PEG-PLA micellas group (PTX:10mg/kg) and micelle nano
Cluster group (PTX:10mg/kg), it was administered in 0,4,8 days, is tested the 12nd day, put to death nude mice, is peeled off
Tumour, takes pictures and claims knurl weight, and Tumor growth inhibition percentage (IRT%) is calculated according to below equation：
Wherein WtreatedIt is the average knurl weight for the treatment of group, WcontrolIt is the average knurl weight of control group.
Result shows, the tumour tumour inhibiting rate for containing taxol micelle nano cluster group apparently higher than common micella,
The drug effect of good treatment tumour is produced in vivo.
Claims (19)
1. one kind contains taxol micelle nano cluster, it is characterised in that build hydrophilic sexual intercourse with TPGS and PEI
Connection mesh skeleton, makes polymer micelle interspersed wherein so as to increase system particle diameter, and by IgG and surface just
Electric charge simultaneously increases system hydrophily, is obtained and contains taxol micelle nano cluster；
The particle diameter for containing taxol micelle nano cluster is 100nm or so.
2. a kind of method for containing taxol micelle nano cluster prepared described in claim 1, it is characterised in that
Comprise the following steps：
(1) taxol and carrier material are completely dissolved in organic solvent a, material and taxol is formed uniformly
Solution I；
(2) the rotary evaporation of solution I is removed into organic solvent a, taxol and carrier material film is obtained；
(3), by above-mentioned film aquation, polymer micelle solution II is obtained；
(4) PEI solution is added in the polymer micelle solution II, and stirring is lower to add having containing TPGS-CDI
Machine solvent b, forms white emulsion III；
(5), by the water bath sonicator of above-mentioned white emulsion III, rotary evaporation removes organic solvent b, obtains solution IV；
(6) the solution IV adds IgG solution under agitation, 1h is stirred at room temperature and obtains micelle nano cluster solution V, puts
It is standby in 4 DEG C of refrigerators, it is obtained and contains taxol micelle nano cluster.
3. the preparation method as described in claim 2, it is characterised in that prepare the polymerization of the polymer micelle
Thing material is PEG2000-PLA, wherein, the molecular weight of described PEG2000-PLA, PEG is 2000, with
The block ratio of PLA is 40:60~60:The molecular weight of 40, i.e. PLA is 1333~3000, and total molecular weight is
3333~5000.
4. the preparation method as described in claim 2, it is characterised in that the molecular weight of the PEI is
600~25000Da.
5. the preparation method as described in claim 2, it is characterised in that described TPGS is D-a-Vitamin
The molecular weight of the esterification products of E and PEG, wherein PEG is 500~5000.
6. the preparation method as described in claim 2, it is characterised in that the micellar carrier material taxol with
The mass ratio of PEG-PLA is 1:5~1:1000.
7. the preparation method as described in claim 2, it is characterised in that super by emulsification in the preparation method
Sound method forms emulsion.
8. the preparation method as described in claim 2, it is characterised in that the method for preparing the micelle nano cluster
It is film dispersion method, dialysis, emulsion process or desivac.
9. the preparation method as described in claim 2, it is characterised in that the micellar carrier material PEG-PLA
It is 5 with the mass ratio of micelle nano cluster framework material:1~1:1000.
10. the preparation method as described in claim 2, it is characterised in that the organic solvent a is selected from dichloromethane
Alkane, chloroform, tetrahydrofuran, acetonitrile, ether, acetone, methyl alcohol or ethanol.
11. preparation method as described in claim 2, it is characterised in that the organic solvent b is selected from dichloromethane
Alkane or chloroform.
12. preparation method as described in claim 2, it is characterised in that the reclaimed water phase of the white emulsion III and
The volume ratio of organic phase is 2:1~20:1.
13. preparation method as described in claim 2, it is characterised in that gained micella is with dynamic light scattering method
The particle size range of measure is 10nm-30nm.
14. preparation method as described in claim 2, it is characterised in that gained micelle nano cluster is with dynamic optical
The particle size range of scattering method is 50nm-200nm.
15. preparation method as described in claim 2, it is characterised in that the step (2) middle rotary evaporation temperature
Spend is 25 DEG C~60 DEG C.
16. preparation method as described in claim 2, it is characterised in that the step (3) middle aqueous vehicles choosing
From deionized water, physiological saline or PBS.
17. preparation method as described in claim 2, it is characterised in that the step (5) middle water bath sonicator when
Between be 5~60min.
18. preparation method as described in claim 2, it is characterised in that the step (5) middle rotary evaporation temperature
Spend is 25~60 DEG C.
19. preparation method as described in claim 2, it is characterised in that the IgG is immunoglobulin G.
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