CN106692053A - Amphipathic segmented copolymer micelle compound drug loading and delivery system and preparation method of brain-targeted water-soluble micelle - Google Patents
Amphipathic segmented copolymer micelle compound drug loading and delivery system and preparation method of brain-targeted water-soluble micelle Download PDFInfo
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- CN106692053A CN106692053A CN201710029453.0A CN201710029453A CN106692053A CN 106692053 A CN106692053 A CN 106692053A CN 201710029453 A CN201710029453 A CN 201710029453A CN 106692053 A CN106692053 A CN 106692053A
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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
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
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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
- 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 provides a brain-targeted water-soluble micelle. The brain-targeted water-soluble micelle is characterized in that a compound nano micelle loaded with paclitaxel and lapatinib is connected with a short peptide molecule Angiopep-2 of a cerebral endothelial cell. In the brain-targeted water-soluble micelle, the drug loading micelle having a brain-targeted function is prepared on the basis by preparing the compound water-soluble drug loading nano micelle. The molecule is connected with the targeted molecule Angiopep-2 by utilizing amidogen and carboxyl bonds, and the nano drug loading micelle having the brain-targeted function is prepared.
Description
Technical field
The present invention relates to Brain targeting formulation art, in particular it relates to a kind of Amphiphilic Block Copolymer Micelles compound carries medicine
The preparation method of delivery system and Brain targeting water solubility micella.
Background technology
Taxane family (taxol, Docetaxel) is to treat one of mainstay of metastatic breast cancer at present, to the greatest extent
Pipe Taxane family is widely used, but it has many unavoidable toxic and side effects, including：Alopecia, tired, courbature, marrow
Suppression, Nervous toxicity, allergic reaction etc..
Meanwhile, Taxane family is highly insoluble in water, and its solubility in water is 0.5 μ g/ml, thus is needed in administration
With Emulsifier EL-60 and absolute ethyl alcohol (1:1, V/V) it is cosolvent, and Emulsifier EL-60 may induce patient's product
The fatal allergic reaction of life, thus, it is prevention allergic reaction, it is necessary to heavy dose of antihistamine drug and glucocorticoid to patient
Pre-processed.Many undesirable elements can then reduce the compliance of patient above, and then limit Japanese yew class to a certain extent
The application of medicine.
Lapatinib is a kind of oral small molecule epidermal growth factor (EGFR：ErbB-1, ErbB-2) EGFR-TK
Inhibitor.Clinic is mainly used in the Her-2 positives and previously received to include anthracycline, taxol, Herceptin (Trastuzumab)
The late period for the treatment of or metastatic breast cancer.
Based on a series of result of clinical tests, Lapatinib is for relapse and metastasis, the HER2 positives to Trastuzumab resistance
Patient with breast cancer, there is good clinical effectiveness, therefore Lapatinib can exist as single medicine or with chemotherapy drugs in combination at present
Clinically apply.
Lapatinib is late or in the treatment of the positive breast cancer of metastatic Her-2, with irreplaceable advantage.But
Be, Lapatinib it is water-soluble very poor, the solubility in water is only 0.007mg/ml, it is difficult to prepare Aqueous injection agent, only
Can be administered in the way of oral tablet.Moreover, the bioavilability of Lapatinib also very low (being less than 20%) is, it is necessary to increase
Dosage (unit dosage form reaches 1.25g) and dosage rate.Because the drug price is expensive, therefore current drawing handkerchief of taking is replaced
Buddhist nun has been significantly greatly increased toxic and side effect and the financial burden of tumor patient, and reduces the compliance of patient to a certain extent.
For advanced breast cancer patient, the side effect of said medicine significantly reduces compliance so that curative effect is big
Give a discount, it is often more important that, because taxol, Lapatinib are difficult to reach effective dose in brain metastes focus, these are difficult to
The pharmaceutical dosage form stood is substantially invalid to breast cancer patients with brain metastatic tumor.
Therefore, taxol, Lapatinib compound Aqueous injection delivery system are researched and developed and medicine is passed with Brain targeting function
System, is expected to improve bioavilability and antitumous effect, reduces toxic and side effect, Pharmacoeconomic benefit is improved, with good
Researching value and clinical meaning.
The content of invention
It is contemplated that overcoming drawbacks described above, taxol, Lapatinib are made the water-soluble injection of compound and with brain target
To the delivery system of function, as the short peptide molecules Angiopep-2 of close brain endothelial cell of delivery system connection on this so that
Carrier micelle has Brain targeting function, across blood-brain barrier and can reach brain metastes lesions position, plays curative effect.
Additionally, in the present invention, curative effect is played with different anticancer mechanisms, lacking for the easy resistance of single medicine can not only be overcome
Point, and due to target administration, ill-effect of the chemotherapeutics to whole body can be reduced, it is expected to further improve bioavilability
And antitumous effect, toxic and side effect is reduced, improve pharmacoeconomics benefit.
The invention provides a kind of Brain targeting water solubility micella, it is characterised in that：Answering for taxol and Lapatinib will be carried
Square nano-micelle is connected with the short peptide molecules Angiopep-2 of close brain endothelial cell.
Further, a kind of preparation method of Brain targeting water solubility micella that the present invention is provided, it is characterised in that specific system
Make operation as follows：
Step one, activator is put into the compound nano micellar solution for carrying taxol and Lapatinib, outside activation micella
Carboxyl key in one polymer；
Step 2, in the amphipathic block nano micellar solution of the load Lapatinib for having activated Angiopep-2 is put into,
Hatch at a temperature of 10-40 DEG C, the carboxyl key of micella is connected with the amino linkage of small peptide, form the glue with target function
Beam；
Step 3, except deactivator, obtain the pure Brain targeting nano-micelle aqueous solution.
Further, the present invention provide a kind of Brain targeting water solubility micella preparation method, also with it is such the characteristics of：
I.e., the addition of above-mentioned activator is 1-10 times of Lapatinib consumption；Preferably 3-6 times.
The consumption of above-mentioned Angiopep-2 is 0.1-5 times of Lapatinib consumption；Preferably 0.3-0.7 times.Should
The consumption of Angiopep-2 is generally 0.08-0.5 times of activator addition.Above-mentioned consumption is mass ratio.
Wherein, during above-mentioned Lapatinib refers to the amphipathic block nano micellar solution that Lapatinib is carried for preparing
The amount of the Lapatinib for being used.
Further, the present invention provide a kind of Brain targeting water solubility micella preparation method, also with it is such the characteristics of：
I.e., in above-mentioned steps one, activator be selected from can activated carboxyl any reagent, most preferably with N-hydroxy-succinamide and
1- ethyls-(3- dimethylaminopropyls) carbodiimide hydrochloride.
Further, the present invention provide a kind of Brain targeting water solubility micella preparation method, also with it is such the characteristics of：
I.e., the soak time of above-mentioned steps one is 0.5-2 hours, and activation temperature is 10-80 DEG C；
The brooding time of above-mentioned steps two is 4-12 hours.
Additionally, the present invention provide a kind of Brain targeting water solubility micella, also with it is such the characteristics of：I.e., above-mentioned load Japanese yew
The compound nano-micelle of alcohol and Lapatinib is prepared by film aquation method by Lapatinib, taxol and polymer and formed；
Wherein, above-mentioned polymer is selected from polyethylene glycol, polyethylene glycol maleimide or methoxy poly (ethylene glycol) and poly- breast
In acid, PLLA-glycolide, poly-epsilon-caprolactone, PLGA, PLLA, PLLA
The copolymerization product of one or more；Formed such as：PEG-PLA, mPEG-PLA, malePEG-PLA (polyethylene glycol maleimide
Amine) etc. isostructural polymer；
And/or
Above-mentioned polymer be selected from polyethylene glycol or methoxy poly (ethylene glycol) and PLA, PLLA-glycolide, poly- ε-
The end of one or more in caprolactone, PLGA, PLLA, PLLA carries out carboxyl
The copolymerization product of end-blocking.Formed such as：The isostructural polymer such as HOOC-PEG-PLA, HOOC-mPEG-PLA.
Wherein, in above-mentioned copolymer, polyethylene glycol or methoxy poly (ethylene glycol) are selected from the poly- second that molecular weight is 500-50000
Glycol or methoxy poly (ethylene glycol)；It is the polyethylene glycol or the poly- second two of methoxyl group in the range of 1000-10000 preferably to be selected from molecular weight
Alcohol；Most preferably from polyethylene glycol or methoxy poly (ethylene glycol) that molecular weight is 1000,2000,3000,4000,5000.
In above-mentioned copolymer, PLA, PLLA-glycolide, poly-epsilon-caprolactone, poly- (DL- lactides-second friendship
Ester), to be selected from molecular weight be the above-claimed cpd of 100-50000 for PLLA or PLLA；Preferably being selected from molecular weight is
It is one or more in above-claimed cpd in the range of 200-20000, embedding with what polyethylene glycol or methoxy poly (ethylene glycol) were formed
The product of Duan Juhe.
Additionally, the present invention provide a kind of Brain targeting water solubility micella, also with it is such the characteristics of：I.e., above-mentioned load Japanese yew
The specific preparation method of the compound nano-micelle of alcohol and Lapatinib is as follows：
Step one, by Lapatinib, taxol and polymer co-dissolve in organic solvent；
Step 2, at a temperature of 10-60 DEG C, remove organic solvent, obtain film；
Step 3, under the conditions of 10-60 DEG C of water bath with thermostatic control, film is fully dissolved in water, obtain carrier micelle liquid
Body；The consumption of water according to being added the need for subsequent reactions, preferably 0.5-5 times of consumption of organic solvent.
Step 4, by filtering, obtain pure carrier micelle liquid.
Additionally, the present invention provide a kind of Brain targeting water solubility micella, also with it is such the characteristics of：I.e., above-mentioned drawing handkerchief is replaced
The mass ratio of Buddhist nun, taxol and polymer is 1:0.2-1：5-20.Preferably 1:0.3-0.6：8-14.Aforementioned proportion refers both to quality
Than.
Additionally, the present invention provide a kind of Brain targeting water solubility micella, also with it is such the characteristics of：I.e., it is above-mentioned organic molten
It is lower boiling easy that agent is selected from, preferably alcohols solvent, most preferably methyl alcohol；
Above-mentioned polymer is selected from copolymerization product and end and carries out the copolymerization product of carboxy blocking；Both polymer temperatures are mixed
Compound；
Wherein, the mass ratio that above-mentioned copolymerization product and end carry out the copolymerization product of carboxy blocking is 10:0.1-5.
PEG-PLA and COOH-PEG-PLA=10 is selected from most preferably from the polymer:The mixing of 0.8-2 (mass ratio)
Thing, or malePEG-PLA and COOH-PEG-PLA=10:The mixture of 0.8-2 (mass ratio).
Additionally, present invention also offers a kind of application of above-mentioned Brain targeting water solubility micella, it is characterised in that：With double
Weight Brain targeting function, is applied to treat the illness of breast cancer patients with brain transfer.
Effect of the invention and effect：
In the present invention, brain is connected with by preparing the water-soluble medicament-carried nano micelle of compound, and prepare on this basis
The carrier micelle of target function.Micella is connected with targeted molecular Angiopep-2 using amino and carboxyl key, being prepared into has
The nano drug-carrying micella of dual Brain targeting function.Understood through the capability study experiment of the saturating in vitro blood-brain barrier of target drug-carrying micella,
The micella for being connected to targeted molecular of the present invention has the good function of penetrating blood-brain barrier.
Additionally, the preparation method of micella is mainly dialysis, self-assembly method, electrostatic interaction method, chemical bonding processes at present, but
It is that these methods all have various shortcomings, such as：Dialysis electrostatic interaction method, is not suitable for large-scale production；Self assembly
Method, only relies on the connection of the non-covalent bonds such as Van der Waals force, and gained micella is unstable, and requirement of the chemical bonding processes to material is very high.
In order to overcome drawbacks described above, in the present invention, prepared using film aquation method and carry answering for taxol and Lapatinib
Side's water solubility micella, using the biological nature of polymer, forms hydrophobic inner casing packaging medicine taxol and Lapatinib, hydrophilic outer
Shell provides good water solubility, can improve the water-soluble energy of the medicine of taxol and Lapatinib this two class water solubility extreme difference,
So as to extend the application of such medicine.Additionally, after testing the film aquation method prepare compound micella particle diameter and electron microscope
It was found that, its drugloading rate envelop rate is entirely capable of meeting clinical demand.
Additionally, being found by studying, such compound carrier micelle that the present invention is provided is smaller than the particle diameter of blank micella, energy
Make the medicine during application, smoothly reach target position, therefore, substantially increase its application clinically.
Thereafter, the water miscible micella is formed with amino carboxyl key covalent bond connection micella and target short peptide molecule, make
Obtaining micella has close brain endothelial cell function.And the Brain targeting function that carrier micelle has, can across blood-brain barrier and
Brain metastes lesions position is reached, curative effect is played.
As can be seen here, the Brain targeting nano-micelle for being obtained using the method for the present invention, that is, disclosure satisfy that water solubility can be noted by force
The requirement penetrated, improves bioavilability and reduces unit dosage form, reduces patient economy burden, improves patient compliance.
And, the Brain targeting water solubility micella that the present invention is provided, also with Brain targeting function, can be used for clinical breast cancer brain
The treatment of transfer, improves the survival rate and quality of life of brain metastes patient.
Importantly, in the present invention, there is provided a kind of water-soluble carrier micelle, played with different anticancer mechanism and treated
Effect, can not only overcome the shortcoming of the easy resistance of single medicine, and due to target administration, can reduce chemotherapeutics to the bad of whole body
Effect, is expected to further improve bioavilability and antitumous effect, reduces toxic and side effect, improves pharmacoeconomics benefit.
As can be seen here, product of the invention, possesses good stability and biocompatibility, can both solve taxol and
The practical problem of Lapatinib slightly solubility, and a kind of delivery system for being capable of active targeting brain metastes focus, contribution can be provided
A kind of brand-new form of administration, solves breast cancer patients with brain and shifts patient without the available predicament of medicine.
Brief description of the drawings
The system of the amphipathic compound block nano-micelle 1# for carrying taxol and Lapatinib that accompanying drawing 1, embodiment one are related to
Standby flow chart；
The grain of the amphipathic compound block nano-micelle 1# for carrying taxol and Lapatinib that accompanying drawing 2, embodiment one are related to
Footpath；
The electricity of the amphipathic compound block nano-micelle 1# for carrying taxol and Lapatinib that accompanying drawing 3, embodiment one are related to
Mirror figure；
The amphipathic compound block nano-micelle 2# and brain that carry taxol and Lapatinib that accompanying drawing 4, embodiment two are related to
The preparation flow figure of targeted nano micella.
The grain of the amphipathic compound block nano-micelle 2# for carrying taxol and Lapatinib that accompanying drawing 5, embodiment two are related to
Footpath；
The amphipathic compound block nano-micelle 2#'s for carrying taxol and Lapatinib that accompanying drawing 6, embodiment two are related to
Zeta potential figure；
The capability study figure of accompanying drawing 7, the saturating in vitro blood-brain barrier of target drug-carrying micella, i.e., set up in vitro blood-brain barrier model
Afterwards, two kinds of micellas penetrate BBB models in same time and compare figure into the qualitative of Skbr-3 cells, wherein, a is load cou-
6 hungry area beam in intracytoplasmic distribution, b be DAPI in intracytoplasmic distribution, c is a, the superposition of b pictures.
Specific embodiment
Embodiment one, the amphipathic compound block nano-micelle 1# for carrying taxol and Lapatinib
A. preparatory phase：The amphipathic compound block nano-micelle for carrying taxol and Lapatinib that the present embodiment one is related to
1# is prepared using preparation flow as shown in Figure 1.
In the present embodiment, using film aquation method, using methyl alcohol as solvent, by polymer malePEG-PLA and drawing
Handkerchief dissolves altogether for Buddhist nun, taxol, and rotary evaporation eliminates organic solvent and using distilled water that film is soluble in water, formation load medicine glue
Beam, is filtrated to get pure water miscible load Lapatinib nano-micelle.
Concrete technology step is as follows：
Step one, by 4mg Lapatinibs, 2mg taxols and 40mg polymer malePEG-PLA co-dissolves in 6ml's
In methyl alcohol (find under study for action, Lapatinib can not reach the solubility needed for testing in multi-solvents, and in methyl alcohol
Dissolved state is good)；
Step 2, mixed solution pipettor is transferred in 250ml eggplant-shape bottles, in 40 DEG C of water bath with thermostatic control condition backspin
Turn evaporation and eliminate organic solvent, obtain being attached to the faint yellow film in eggplant-shape bottle.
Step 3, eggplant-shape bottle is removed, the distilled water of 4ml is drawn with liquid-transfering gun, made under the conditions of 40 DEG C of water bath with thermostatic control thin
Film is fully dissolved in water, obtains the carrier micelle liquid of pale yellow transparent.
Step 4, the micellar liquid filtering by gained, remove free drug, obtain pure carrier micelle liquid.
B. micella physicochemical property is determined
B-1. micella particle diameter potential measurement
I () adds distilled water that carrier micelle LPTN+PTX-MIC is diluted into debita spissitudo, surveyed using granularity/Zeta potential
Determine instrument and determine particle size values.
As shown in Fig. 2 the particle diameter for carrying the amphipathic compound block nano-micelle 1# of taxol and Lapatinib is 16.3nm.
As comparison, film aquation method is also used in the present embodiment, appropriate malePEG2000-PLA200 is dissolved in
After methyl alcohol, the blank micella being prepared into, its particle diameter is 20nm or so>The particle diameter of carrier micelle.
It is possible thereby to, it will be seen that, after taxol and Lapatinib is loaded into, micella particle diameter is reduced on the contrary.
(ii) it is added dropwise after dilutes micellar solution on the copper mesh of covering carbon film, with phosphotungstic acid (pH 7.0,2%, w/v)
Negative staining, is placed in observation micella formalness under transmission electron microscope, as shown in figure 3, its dispersiveness is good after drying at room temperature.
B-2. the measure of drugloading rate envelop rate
The chromatographic condition of (i) taxols HPLC analyses
Chromatographic column：Dikma Diamonsil C18 (4.6 × 250mm, 5 μm)
Mobile phase：Acetonitrile: distilled water (50: 50)
Flow velocity：1.0mL/min
Detector：UV-detector
Detection wavelength：228nm
Column temperature：35℃
Sample size：20μL
(ii) chromatographic condition of Lapatinibs HPLC analyses
Chromatographic column：Dikma Diamonsil C18 (4.6 × 250mm, 5 μm)
Mobile phase：Acetonitrile: 0.01MKH2PO4 solution (PH=6.2) (65: 35)
Flow velocity：1.0ml/min
Detector：UV-detector
Detection wavelength：253nm
Column temperature：35℃
Sample size：20μL
Precision draws the LPTN+PTX-MIC solution after filtering, adds quantitative methanol dilution and discharges Lapatinib, surpasses
It is complete that sound 5min dissolves polymer, 0.45 μm of filtering with microporous membrane, takes solution sample introduction measure.Computational envelope is distinguished according to the following formula
Rate (Entrapping Efficiency, EE) and drugloading rate (Drug Loading Capacity, DLC)：
The drugloading rate DLC of taxol is 2%, and envelop rate EE is 79%,
The drugloading rate DLC of Lapatinib is 5%, and envelop rate EE is 81%.
Embodiment two, the amphipathic compound block nano-micelle 2# and Brain targeting nano-micelle that carry taxol and Lapatinib
A. preparatory phase：The amphipathic compound block nano-micelle for carrying taxol and Lapatinib that the present embodiment two is related to
2# and Brain targeting nano-micelle are prepared using preparation flow as shown in Figure 4.
It is same to use film aquation method in the present embodiment two, using methyl alcohol as solvent, by polymer malePEG-
PLA, COOH-PEG-PLA, taxol and Lapatinib proportionally co-dissolve in methyl alcohol, rotary evaporation eliminates organic molten
Agent is simultaneously soluble in water by film using distilled water, forms carrier micelle, and utilize the covalent bond that amino and carboxyl key are formed by glue
Beam is connected with targeted molecular Angiopep-2, is prepared into the nano drug-carrying micella with Brain targeting function.
Concrete technology step is as follows：
Step one, by 4mg Lapatinibs, 2mg taxols and 40mg polymer malePEG-PLA, 4mg polymer COOH-
(the dissolving needed for experiment can not be reached in multi-solvents because of Lapatinib in the methyl alcohol of 6ml of PEG-PLA co-dissolves
Degree, and dissolved state is good in methyl alcohol)
Step 2, mixed solution pipettor is transferred in 250ml eggplant-shape bottles, the rotary evaporation under the conditions of water bath with thermostatic control
Organic solvent is eliminated, obtains being attached to the faint yellow film in eggplant-shape bottle.
Step 3, eggplant-shape bottle is removed, 4ml distilled water is drawn with liquid-transfering gun, make film all molten under the conditions of water bath with thermostatic control
In Xie Yushui, the carrier micelle liquid of pale yellow transparent is obtained.
Step 4, the micellar liquid filtering by gained, remove free drug, obtain pure carrier micelle liquid.
Step 5, EDC (1- (3- the dimethylamino-propyls) -3- ethyl carbon for putting into micellar solution excessive 14.2mg
Diimmonium salt hydrochlorate) and 4mg NHS (N-hydroxy-succinamide), incubation at room temperature 0.5 hour, for activating the outer strata of micella
Carboxyl key (COOH-) on compound.
Step 6, the Angiopep-2 for putting into the micellar solution for having activated 2mg, incubation at room temperature 6 hours makes micella
Carboxyl key is connected with the amino linkage of small peptide, forms the micella with target function.
Step 7, ultrafiltration, except deactivator EDC, NHS, obtain pure target drug-carrying micellar aqueous solution.
B. micella physicochemical property is determined
B-1. the particle diameter potential measurement of target drug-carrying micella
I () adds distilled water that target drug-carrying micella ANG-LPTN+PTX-MIC is diluted into debita spissitudo, using granularity/
Zeta potential analyzer determines particle size values.
As shown in figure 5, the particle diameter for carrying the amphipathic block nano-micelle 2# of Lapatinib is 19.9nm.
It is seen from the above data that the amphipathic block nano-micelle 2# of compound manufactured by the method for the present embodiment two
Particle diameter of the particle diameter again smaller than blank micella.
(ii) it is added dropwise after dilutes micellar solution on the copper mesh of covering carbon film, with phosphotungstic acid (pH 7.0,2%, w/v)
Negative staining, is placed in observation micella formalness under transmission electron microscope, as shown in fig. 6, it is uniformly dispersed after drying at room temperature.
B-2. the capability study of the saturating in vitro blood-brain barrier of target drug-carrying micella
In vitro blood-brain barrier model is built according to document utilization Transwell Tissue Culture Plates, is prepared according to the method described above
Two kinds of micellas of fluorescent coumarin 6 are carried, one kind does not connect targeted molecular, and one kind carries targeted molecular.
Can enter cytoplasm using coumarin 6 but nucleus can not be entered, and the spy of the main staining cell cores of dyestuff DAPI
Property, two kinds of micellas of observation pass through the amount of blood-brain barrier model in same time.
As shown in fig. 7, after setting up in vitro blood-brain barrier model, two kinds of micellas penetrate BBB models in same time and go forward side by side
Enter the qualitative comparing of Skbr-3 cells.
A be the hungry area beam for carrying cou-6 in intracytoplasmic distribution, b be DAPI in intracytoplasmic distribution, c is a, b pictures
Superposition, can be visually observed that figure below, that is, be connected to penetration capacity of the micella of targeted molecular to BBB in same time obvious
It is better than the micella for not connecing targeted molecular.
As can be seen here, the compound carrier micelle that the present embodiment is provided possesses good physical property, prepares on this basis
Brain targeting compound carrier micelle, with the good function of penetrating blood-brain barrier.
There is the medicine of different antitumor mechanisms using two kinds, and have confirmed the medicine with synergy, system
It is standby to disclosure satisfy that reduction toxic and side effect reduces dosage into compound intelligent drug delivery system, treat the demand of brain metastes focus.
In above-described embodiment one and two, the malePEG-PLA of selection is malePEG2000-PLA200, and researcher is also led to
Cross using different polymer to synthesize nano-micelle, and find after measured, such nano-micelle possesses good stability
And biocompatibility, the practical problem of taxol and Lapatinib slightly solubility can be both solved, and can be actively there is provided one kind
Realize the delivery system of dual-target brain metastes focus.
Specific example is as follows：
Embodiment three, the amphipathic block nano-micelle 3# for carrying Lapatinib
It is in the present embodiment three, 4mg Lapatinibs, 2mg taxols and 40mg polymer mPEG2000-PLA200 is common
It is dissolved in the methyl alcohol of 6ml；Mixed solution pipettor is transferred in 250ml eggplant-shape bottles, in 40 DEG C of water bath with thermostatic control condition
Lower rotary evaporation eliminates organic solvent, obtains being attached to the faint yellow film in eggplant-shape bottle；Eggplant-shape bottle is removed, is drawn with liquid-transfering gun
The distilled water of 4ml, in film is fully dissolved in water under the conditions of 40 DEG C of water bath with thermostatic control, obtains the load medicine glue of pale yellow transparent
Beam liquid；The micellar liquid of gained is filtered, free drug is removed, pure carrier micelle liquid is obtained.
Example IV, the amphipathic block nano-micelle 4# for carrying Lapatinib
In the present embodiment four, by 4mg Lapatinibs, 1mg taxols and 20mg polymer Ps EG (2000)-b-PLGA
(10000) co-dissolve is in the methyl alcohol of 5ml；Mixed solution pipettor is transferred in 250ml eggplant-shape bottles, in 40 DEG C of perseverance
Rotary evaporation eliminates organic solvent under the conditions of tepidarium, obtains being attached to the film in eggplant-shape bottle；Eggplant-shape bottle is removed, liquid-transfering gun is used
The distilled water of 5ml is drawn, in film is fully dissolved in water under the conditions of 40 DEG C of water bath with thermostatic control, transparent carrier micelle is obtained
Liquid；The micellar liquid of gained is filtered, free drug is removed, pure carrier micelle liquid is obtained.
Embodiment five, the amphipathic block nano-micelle 5# for carrying Lapatinib
In the present embodiment five, by 4mg Lapatinibs, 4mg taxols and 80mg polymer Ps EG (5000)-b-PCL
(10000) co-dissolve is in the methyl alcohol of 10ml；Mixed solution pipettor is transferred in 250ml eggplant-shape bottles, at 40 DEG C
Rotary evaporation eliminates organic solvent under the conditions of water bath with thermostatic control, obtains being attached to the film in eggplant-shape bottle；Eggplant-shape bottle is removed, liquid relief is used
Rifle draws the distilled water of 8ml, in film is fully dissolved in water under the conditions of 60 DEG C of water bath with thermostatic control, obtains transparent load medicine glue
Beam liquid；The micellar liquid of gained is filtered, free drug is removed, pure carrier micelle liquid is obtained.
The particle diameter of the amphipathic block nano-micelle 3#-5# of above-mentioned load Lapatinib is respectively less than 20nm.
The drugloading rate DLC of taxol is 2-4%, and envelop rate EE is 77-80%,
The drugloading rate DLC of Lapatinib is 5-7%, and envelop rate EE is 79-83%.
Embodiment six, the amphipathic block nano-micelle 6# and its Brain targeting nano-micelle that carry Lapatinib
In the present embodiment six, by 4mg Lapatinibs, 2mg taxols and 40mg polymer mPEG2000-PLA200,4mg
Mixed solution pipettor is transferred to 250ml by polymer COOH-mPEG2000-PLA200 co-dissolves in the methyl alcohol of 6ml
In eggplant-shape bottle, rotary evaporation eliminates organic solvent under the conditions of water bath with thermostatic control, obtains being attached to the faint yellow film in eggplant-shape bottle.
Eggplant-shape bottle is removed, 4ml distilled water is drawn with liquid-transfering gun, in film is fully dissolved in water under the conditions of water bath with thermostatic control, obtain light
The carrier micelle liquid of yellow transparent.The micellar liquid of gained is filtered, free drug is removed, pure carrier micelle liquid is obtained
Body 6#.The NHS of the EDC and 4mg of excessive 14.2mg, incubation at room temperature 0.5 hour are put into micellar solution.In the glue for having activated
The Angiopep-2 of 2mg is put into beam solution, incubation at room temperature 6 hours, ultrafiltration removes deactivator EDC, NHS, obtains pure target
To carrier micelle aqueous solution 6#.
Embodiment seven, the amphipathic block nano-micelle 7# and its Brain targeting nano-micelle that carry Lapatinib
In the present embodiment seven, by 4mg Lapatinibs, 3mg taxols and 21mg polymer Ps EG (2000)-b-PLGA
(10000), 5mg polymer COOH-PEG (2000)-b-PLGA (10000) co-dissolves are in the methyl alcohol of 7ml, by mixed solution
It is transferred in 250ml eggplant-shape bottles with pipettor, rotary evaporation eliminates organic solvent under the conditions of water bath with thermostatic control, obtains being attached to eggplant
Film in shape bottle.Eggplant-shape bottle is removed, 6ml distilled water is drawn with liquid-transfering gun, film is all dissolved under the conditions of water bath with thermostatic control
Yu Shuizhong, obtains transparent carrier micelle liquid.The micellar liquid of gained is filtered, free drug is removed, pure load is obtained
Medicine micellar liquid 7#.The NHS of the EDC and 4mg of excessive 4mg, incubation at room temperature 1 hour are put into micellar solution.Activating
Micellar solution in put into 4mg Angiopep-2, incubation at room temperature 8 hours, ultrafiltration remove deactivator EDC, NHS, obtain pure
Target drug-carrying micellar aqueous solution 7#.
Embodiment eight, the amphipathic block nano-micelle 8# and its Brain targeting nano-micelle that carry Lapatinib
In the present embodiment seven, by 4mg Lapatinibs, 4mg taxols and 55mg polymer Ps EG (5000)-b-PCL
(10000), 15mg polymer COOH-PEG (5000)-b-PCL (10000) co-dissolves are in the methyl alcohol of 6ml, by mixed solution
It is transferred in 250ml eggplant-shape bottles with pipettor, rotary evaporation eliminates organic solvent under the conditions of water bath with thermostatic control, obtains being attached to eggplant
Film in shape bottle.Eggplant-shape bottle is removed, 8ml distilled water is drawn with liquid-transfering gun, film is all dissolved under the conditions of water bath with thermostatic control
Yu Shuizhong, obtains transparent carrier micelle liquid.The micellar liquid of gained is filtered, free drug is removed, pure load is obtained
Medicine micellar liquid 8#.The NHS of the EDC and 8mg of excessive 25mg, incubation at room temperature 2 hours are put into micellar solution.Activating
Micellar solution in put into 1mg Angiopep-2, incubation at room temperature 12 hours, ultrafiltration remove deactivator EDC, NHS, obtain pure
Target drug-carrying micellar aqueous solution 8#.
The particle diameter of the amphipathic block nano-micelle 6#-8# of above-mentioned load Lapatinib is respectively less than 20nm.
The drugloading rate DLC of taxol is 1-4%, and envelop rate EE is 76-82%,
The drugloading rate DLC of Lapatinib is 4-9%, and envelop rate EE is 78-85%.
By above-mentioned experiment it can be found that the above is passed for a kind of new compound water solubility carrier micelle that this patent is provided
Medicine system, possesses good stability and biocompatibility, can both solve the reality of taxol and Lapatinib slightly solubility
Problem, additionally using the antitumor principle that two kinds of medicines are different, the lethal effect to tumour is strengthened by acting synergistically, and
And because being connected to targeted molecular, blood-brain barrier can be actively penetrated, brain tumor tissue site is gathered in, by dual-target
Effect plays a role to breast cancer patients with brain transfer patient, even if previously using taxol, has produced the patient of resistance, also can be from
Middle benefit.Thus, a kind of brand-new form of administration is contribute to, breast cancer patients with brain is solved and is shifted patient without the available predicament of medicine.
Claims (10)
1. a kind of Brain targeting water solubility micella, it is characterised in that：
The compound nano-micelle of taxol and Lapatinib and the short peptide molecules Angiopep-2 phases of close brain endothelial cell will be carried
Connection.
2. a kind of preparation method of Brain targeting water solubility micella as claimed in claim 1, it is characterised in that specific manufacturing process
It is as follows：
Step one, activator is put into the compound nano micellar solution for carrying taxol and Lapatinib, the outer strata of activation micella
Carboxyl key on compound；
Step 2, in the amphipathic block nano micellar solution of the load Lapatinib for having activated Angiopep-2 is put into, in 10-
Hatch at a temperature of 40 DEG C, the carboxyl key of micella is connected with the amino linkage of small peptide, form the micella with target function；
Step 3, except deactivator, obtain the pure Brain targeting nano-micelle aqueous solution.
3. the preparation method of a kind of Brain targeting water solubility micella as claimed in claim 2, it is characterised in that：
The addition of the activator is 1-10 times of Lapatinib consumption；
The consumption of the Angiopep-2 is 0.1-5 times of Lapatinib consumption；
Wherein, the Lapatinib is made during referring to the amphipathic block nano micellar solution that Lapatinib is carried for preparing
The amount of Lapatinib.
4. the preparation method of a kind of Brain targeting water solubility micella as claimed in claim 2, it is characterised in that：
In the step one, activator is selected from N-hydroxy-succinamide and 1- ethyls-(3- dimethylaminopropyls) carbon two is sub-
Amine hydrochlorate.
5. the preparation method of a kind of Brain targeting water solubility micella as claimed in claim 2, it is characterised in that：
The soak time of the step one is 0.5-2 hours, and activation temperature is 10-80 DEG C；
The brooding time of the step 2 is 4-12 hours.
6. a kind of Brain targeting water solubility micella as claimed in claim 1, it is characterised in that：
It is described to carry the compound nano-micelle of taxol and Lapatinib by Lapatinib, taxol and polymer by film aquation
Method is prepared from；
Wherein, the polymer is selected from polyethylene glycol, polyethylene glycol maleimide or methoxy poly (ethylene glycol) and PLA, gathers
One kind in L- lactide coglycolides, poly-epsilon-caprolactone, PLGA, PLLA, PLLA
Or several copolymerization products；
And/or
The polymer be selected from polyethylene glycol or methoxy poly (ethylene glycol) and PLA, PLLA-glycolide, poly- ε-oneself in
The end of one or more in ester, PLGA, PLLA, PLLA carries out carboxy blocking
Copolymerization product.
7. a kind of Brain targeting water solubility micella as claimed in claim 6, it is characterised in that：
The specific preparation method of the compound nano-micelle for carrying taxol and Lapatinib is as follows：
Step one, by Lapatinib, taxol and polymer co-dissolve in organic solvent；
Step 2, at a temperature of 10-60 DEG C, remove organic solvent, obtain film；
Step 3, under the conditions of 10-60 DEG C of water bath with thermostatic control, film is fully dissolved in water, obtain carrier micelle liquid；
Step 4, by filtering, obtain pure carrier micelle liquid.
8. a kind of Brain targeting water solubility micella as claimed in claim 6, it is characterised in that：
The mass ratio of the Lapatinib, taxol and polymer is 1:0.2-1：5-20.
9. a kind of Brain targeting water solubility micella as claimed in claim 6, it is characterised in that：
The organic solvent is selected from alcohols solvent；
The polymer is selected from copolymerization product and end and carries out the copolymerization product of carboxy blocking；
Wherein, the mass ratio that the copolymerization product and end carry out the copolymerization product of carboxy blocking is 10:0.1-5.
10. a kind of water-soluble micella of Brain targeting as described in claim 1-9 is any, it is characterised in that：
With dual Brain targeting function, it is applied to treat the illness of breast cancer patients with brain transfer.
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