CN106146761A - The nano-micelle of a kind of amphipathic multifunctional polymer and preparation thereof and application - Google Patents
The nano-micelle of a kind of amphipathic multifunctional polymer and preparation thereof and application Download PDFInfo
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- CN106146761A CN106146761A CN201610385359.4A CN201610385359A CN106146761A CN 106146761 A CN106146761 A CN 106146761A CN 201610385359 A CN201610385359 A CN 201610385359A CN 106146761 A CN106146761 A CN 106146761A
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- amphipathic
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- multifunctional polymer
- polymer
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Classifications
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F290/00—Macromolecular compounds obtained by polymerising monomers on to polymers modified by introduction of aliphatic unsaturated end or side groups
- C08F290/02—Macromolecular compounds obtained by polymerising monomers on to polymers modified by introduction of aliphatic unsaturated end or side groups on to polymers modified by introduction of unsaturated end groups
- C08F290/06—Polymers provided for in subclass C08G
- C08F290/062—Polyethers
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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
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
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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
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
- A61K49/0039—Coumarin dyes
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
- A61K49/0054—Macromolecular compounds, i.e. oligomers, polymers, dendrimers
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
- A61K49/0069—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form
- A61K49/0076—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form dispersion, suspension, e.g. particles in a liquid, colloid, emulsion
- A61K49/0082—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form dispersion, suspension, e.g. particles in a liquid, colloid, emulsion micelle, e.g. phospholipidic micelle and polymeric micelle
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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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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F8/00—Chemical modification by after-treatment
- C08F8/30—Introducing nitrogen atoms or nitrogen-containing groups
- C08F8/32—Introducing nitrogen atoms or nitrogen-containing groups by reaction with amines
Abstract
The invention discloses nano-micelle and the application of a kind of amphipathic multifunctional polymer PMNCF and preparation thereof, as shown in the formula (I), wherein, x is the positive integer of 10～1000 to its general structure, and y is the positive integer of 5～500, and z is the positive integer of 0～500；In x, y, z, x mole percent is 40 ~ 75%, and y is 10 ~ 30%, and z is 0～35%.The PMNCF of preparation is assembled into medicament-carried nano micelle with medicine easily by hydrophobe effect in aqueous, the imitating cell outer-layer film hydrophilic shell being formed can reduce immune system identification and scavenging action in vivo, dramatically increase Nano medication circulation timei in blood and the chance being enriched in tumor locus, easily with the receptor binding of cancer cell surfaces by targeting of taking the initiative in Nano medication introducing cancer cell, existing chemotherapeutics can be upgraded to " stealthy biological missile ", significantly reduce chemical therapy toxic side effect.
Description
Technical field
The present invention relates to nano-micelle and the application of a kind of amphipathic multifunctional polymer and preparation thereof, particularly to one
Side chain contains extracellular tunic amphion Phosphorylcholine respectively, the amphipathic multifunctional polymer of cholesterol and folic acid and receiving
The preparation method and application of rice carrier micelle, belongs to Chemistry and Physics of Polymers and biology medical material technical field.
Background technology
With the fast development of nanometer medicine, nano-medicament carrier receives much concern in recent decades.Nano medication transmits body
System (DDS) is using nano particle as cancer therapy drug and genophore, and the bags such as medicine are loaded in nano particle space or are adsorbed
On its surface, by sanguimotor mode, enhanced infiltration and delay (EPR) effect is utilized to realize that nano particle physics targets
To cancerous tissue and taken in intracellular by cancer cell, it is achieved safely and effectively targeted drug conveying and gene therapy.
However as research deepen continuously, discovery when nano particle by intravenous injection or oral by way of follow at blood
During ring, can experience before reaching its diseased region and be identified as allogenic material and pass through reticuloendothelial system (RES)
High phagocytic mononuclear phagocyte system (MPS) is absorbed and is left the circulatory system.The Nano medication of this picked-up is accumulated at other
Normal organ tissue produces toxic and side effect, and has a strong impact on the efficiency of medicine conveying.Additionally, nano drug-carrying carrier enters internal
Being affected by dilution for many times and other factors afterwards, carrier dissociation causes medicine to discharge the clinical practice affecting carrier in advance.Further,
Although folic acid has been used for the targeting component research of different anticarcinogen, reduce the injury of medicine normal tissue cell, increase
Add to tumor cytotoxicity effect, but the more difficult performance of targeting (Lee J. H., the et of many research report folic acid components
al. J. Controlled Release, 2015, 209, 219-228;Qiu L. Y., et al. International
Journal of Pharmaceutics, 2013,456,315-324).Therefore, nano particle is carried out from bionical angle
The design of carrier or surface biomimetic modification are possible to improve its stability in vivo, outer surface carry extracellular tunic both sexes from
Subbase group makes the nano particle of carrying medicament be difficult to by vivo immuning system identification and removing, it is easier to realize blood long circulating
Energy and high selective targeting.
Content of the invention
The present invention with improve nano medicament carrying system to the targeting of cancer cell, extend in blood circulation timei for breaking through
Mouthful, from cellular membrane biomimetic angle, devise a kind of group folic acid containing targeting, the amphion hydrophilic group of imitative membrane structure
Group and the amphipathic randomcopolymer of multifunctional bionic of strong-hydrophobicity cholesterol side chain.
Another object of the present invention is to provide the preparation method of the amphipathic randomcopolymer micella of a kind of imitative membrane structure,
Reach folic acid group by controlling preparation condition as much as possible in the distribution of micella outer surface, in order to reach more effectively thin with cancer
Cellular surface folacin receptor protein combination enters the purpose of cancer cell, it is thus achieved that a kind of both had good micella stability, internal hidden
Shape, hypotoxicity, again there is passive and active targeting effect Multifunction carrier micelle.
It is a still further object of the present invention to provide the internal stealth with imitative membrane structure and tumor-targeting nanometer carries
The concrete application of medicine micella.It can as the red blood cell of people long circulating in blood, be difficult to be identified by human immune system,
Cancer site efficiently concentrating, reaches to treat the purpose of cancer.
The present invention realizes that process is as follows:
Amphipathic multifunctional polymer shown in general structure (I), referred to as PMNCF, wherein R is hydrogen or methyl；
Wherein, x is the positive integer of 10～1000, and y is the positive integer of 5～500, and z is the positive integer of 0～500；In x, y, z, x
Mole percent is 40 ~ 75%, and y is 10 ~ 30%, and z is 0～35%；Being preferably x mole percent is 40 ~ 75%, and y is 10 ~ 30%, z
It is 10～35%；
n1It is the positive integer of 1 ~ 3, R1For the amphion hydrophilic radical simultaneous with positive and negative charge
n2It is the positive integer of 5 ~ 50, R2For the hydrophobic group containing cholesterol
n3It is the positive integer of 5 ~ 50, R3For the tumour folacin receptor targeting group containing folic acid
The preparation method of above-mentioned amphipathic multifunctional polymer: PMNCF is by two kinds of polymerizables containing amphion and active ester
Monomer causes random copolymerization first to obtain by free radical in the solution, and amphion unit molar fraction x is 40% ~ 75% two
Unit randomcopolymer PMN(Gong Y. K., et al. Macrom. Biosci., 2012,12,979-985), then use
Cholesterol containing amino and the folate molecule containing amino carry out amidatioon graft reaction to the active ester units in polymer,
Active ester units 1-x=60% ~ 25%, obtaining cholesterol unit molar fraction y is 10% ~ 30%, and folic acid unit molar fraction z is
The multifunctional polymer of 0% ~ 35%.
Above-mentioned polymerisation is carried out in anhydrous organic solvent dimethyl sulfoxide.
The method that amphipathic multifunctional polymer prepared by said method prepares nano drug-carrying micella: molten with dimethyl sulfoxide (DMSO)
Solve PMNCF polymer and hydrophobic drug, the drug solution of polymer quality percentage 10% ~ 50% will be accounted for and polymer solution adds
The aqueous solution entering quick stirring automatically forms the carrier micelle of diameter 30 ~ 150 nanometer, forms stable nano drug-carrying micella
The aqueous solution.
In above-mentioned preparation process, the pH of the regulation aqueous solution is 4 ~ 6 very crucial, is more than 6 or is less than 4, will cause micella shakiness
Fixed, targeting group exposes insufficient, targeting difference.
Described have hydrophobic drug selected from adriamycin, taxol, epirubicin, HCPT, THP, Duo Xi
His match, vinorelbine, oxaliplatin.
Above-mentioned Mobyneb amphipathic nature polyalcohol can be self-assembly of micella in aqueous, can be as hydrophobic anticancer medicine
The carrier of thing, in preparation process, is separated to organic solvent and free drug by ultrafiltration centrifugal method, purifies and concentrate nanometer
Carrier micelle.
Specifically, multi-functional amphiphilic micella takes following method to prepare: by amphipathic nature polyalcohol
(PMNCF) it is dissolved in DMSO with medicine such as adriamycin, is added drop-wise in the water that pH is 4 ~ 6, utilize amphipathic nature polyalcohol self assembly real
The load of existing medicine.Then by way of dialysis or ultrafiltration are centrifugal, remove organic solvent and free adriamycin obtains medicine carrying
Micella.The carrier micelle of formation is gulped down by measuring its particle diameter, Zeta potential, storage stability, cytotoxicity experiment and cell
Bite experiment etc., the performance of assessment carrier micelle.
Advantages of the present invention and positive effect: (1) present invention is by introducing extracellular on amphipathic nature polyalcohol side chain
Tunic Phosphorylcholine strong hydrophilicity group, the strong hydrophobic grouping of cholesterol and folate-targeted group, self assembly in slightly acidic water solution
Easily formed stable can the imitative membrane structure micella of carrying medicament, give " stealthy " function in glue bundle body；(2) present invention exists
Assembling the dissolving adding folic acid group in slightly acidic water solution, having more folic acid group to be distributed in micella outer surface can maximum limit
Degree targeting of taking the initiative；(3) Inventive polymers each monomeric groups molar content can control conjunction according to the actual requirements
One-tenth, the micella particle size range therefore being formed, targeting group content etc. are controlled, can carry out the optimization sieve of long circulating and Targeting Performance
Choosing；(4) the nano drug-carrying micella hydrophilic layer being formed is externally connected with the folic acid group of design content, with breast cancer cell, KB cell
And HeLa cell has the high selective targeting of 2 ~ 4 times；Nano drug-carrying micella has imitating cell outer-layer film hydrophilic layer structure,
Internal anti-immune system identification and scavenging action are excellent；(5) preparation method of the present invention is simple, it is possible to decrease application cost.With generally
The synthetically prepared of amphipathic nature block polymer using is compared, and prepares the condition of amphipathic randomcopolymer and technical process very
Simple and easy to do, easy popularization and application.
Brief description
Fig. 1 is polymer P MNCF-231H NMR spectra；
Fig. 2 is series drug accumulation release rate (a) pH=5.0 in different dissolution medium for the medicine carrying PMNCF micella; (b) pH
=7.4；
Fig. 3 variable concentrations carries the PMNCF micella of adriamycin and MADB-106 cell trains the cancer cell survival rate after 48 h support；
Fig. 4 is that Turnover of Mouse Peritoneal Macrophages is to the fluorescent microscopy images after different polymer micelle picked-up 2 h；
Fig. 5 is that the micella modified without FA and FA molar percentage is different is taken the photograph by rat breast cancer cell MADB-106 different time
Fluorescent microscopy images after taking.
Detailed description of the invention
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention do not limit
In this.
The preparation of embodiment 1 amination cholesterol
Join the anhydrous methylene chloride of 50 mL and the anhydrous ethylenediamine of 37.5 mL in 250 mL three neck round bottoms, be placed in
Cryosel bath make reaction temperature regulate to 0 DEG C.Then weigh 5.02 g(0.0112 mol) cholesteryl chloroformate, it is molten
Solution, in the anhydrous methylene chloride of 60 mL, after dissolving fully is transferred to solution in constant pressure funnel.At high-speed stirred shape
Being added drop-wise in aforementioned three neck round bottom by this solution under state, reaction structure formula is as follows:
After dropping finishes, under room temperature condition, lucifuge is reacted overnight.Reactant liquor is extracted three times by isopyknic distilled water, removes second
Diamine hydrochloride and ethylenediamine, be dried organic phase with a large amount of anhydrous magnesium sulfates, overnight remove a small amount of water.By dried organic
The suction filtration that reduces pressure mutually removes magnesium sulfate solid, and the rotation of the clear liquid obtaining lucifuge at ambient temperature is steamed then in 30 DEG C of vacuum drying, institute
Obtain Off-white solid and be Chol-NH2。
The preparation of embodiment 2 amination folic acid
The first step: mono amino protects ethylenediamine (BOC-NH2) synthesis:
By 4.40 g(0.0732 mol) ethylenediamine be dissolved in 110 mL containing 10% triethylamine methanol solution in, be stirred vigorously
Under, the 3.63 g(0.0172 mol being dissolved in 10 mL methyl alcohol) two dimethyl dicarbonate butyl esters are slowly added drop-wise to above-mentioned solution
In, reaction is stirred at room temperature overnight.After completion of the reaction, room temperature rotation is evaporated off methyl alcohol, obtains oil product, is dissolved in 100 mL
In dichloromethane, removing ethylenediamine with 10% aqueous sodium carbonate washing, organic phase anhydrous magnesium sulfate is dried overnight, and filters, very
Sky obtains the product (BOC-NH of mono amino protection after being dried2).
Second step: folic acid active ester synthesizes with the reacting ethylenediamine of mono amino protection:
By 1.00 g(0.0023 mol) folic acid (FA) joins in the 40 anhydrous DMSO of mL, adds 0.5 mL triethylamine, lucifuge
It is stirred overnight.Add 0.50 g(0.0023 mol) DCC and 0.52 g(0.0046 mol) NHS, lucifuge continues stirring 24 h pair
The γ position carboxyl of folic acid activates.After being filtered to remove insoluble matter, in filtrate, add 0.39 g(0.0025 mol) BOC-NH2,
Room temperature lucifuge is reacted overnight.It after being filtered to remove insoluble matter, is slowly added drop-wise in the 400 mL absolute ethers that are stirred vigorously and acetone
(1:1, V/V), then ice-water bath 0 DEG C cooling, stand and obtain yellow mercury oxide.Then the centrifugal yellow mercury oxide that obtains washs three with ether
The secondary DMSO removing residual, the yellow powder normal-temperature vacuum obtaining is drying for one day, obtains the product (FA-of mono amino protection
NHBOC).
3rd step: FA-BOC deprotection:
By 0.46 g(0.0008 mol) product is dissolved in 4 mL trifluoroacetic acids, after after 2 h are stirred at room temperature, rotary evaporation concentrates
By 30 mL absolute ether precipitations.Precipitate after dissolving with 5 mL DMF again with 25 mL ether sedimentations, after abandoning supernatant, use ether
Washing three times, 4 000 rpm/min are centrifugal obtains yellow mercury oxide.Last room temperature in vacuo is drying for one day, obtains product FA-NH2。
The preparation of embodiment 3 polymerizable activity alicyclic monomer (NPEM)
P-nitrophenyl oxygen formyl polyethylene glycol methacrylate (NPEM) containing the easy leaving group of p-nitrophenyl oxygen formoxyl
Preparation to make process as follows: in 250 mL three neck round bottoms, add 17.05 g (0.0324 mol) methacrylic acid to gather
Glycol ester, 4.80 g(0.0475 mol) triethylamine and 60 mL chloroforms make it dissolve.Weigh 9.62 g(0.0531 mol) right
Nitrobenzene oxygen formyl chloride is dissolved in 100 mL chloroformic solutions and drops to mechanical agitation with constant pressure funnel with the speed of 3 s/d
Under the conditions of in the three-neck flask of-20 DEG C.Dropping keeps this temperature to continue reaction 2 h after finishing, then recover room temperature reaction overnight.
After completion of the reaction, with Rotary Evaporators, reactant liquor is concentrated, add the cold ether sedimentation of equal-volume 50 mL and filter triethylamine salt
Hydrochlorate.Room temperature rotation is evaporated off ether, adds isopyknic chloroform, obtains flaxen liquid.With isopyknic pH=3.0 ~ 4.0
Phosphate buffer solution extraction with remove remnants triethylamine salt and triethylamine, after being repeated 3 times by collect organic phase at 30 DEG C
Rotation is steamed to not having drop to occur, and the pale yellow oil obtaining is active ester monomer p-nitrophenyl oxygen formyl polyethylene glycol
Methacrylate (NPEM).
Embodiment 4 synthesizes the binary polymer (PMN) of phosphorous phatidylcholine group and active ester group
Under nitrogen protection, in 250 mL three neck round bottoms, add 10 mL absolute ethyl alcohols, weigh 0.11 g azo two isobutyl
Nitrile (AIBN) is dissolved in 6 mL THF, and 1/4th volumes taking this solution until completely dissolved are transferred in above-mentioned three-necked bottle, rises
Temperature, to 70 DEG C of constant temperature, magnetic agitation, keeps this state 30 min.Add in 100 mL constant pressure funnels 60 mL dissolved with
NPEM 3.57 g(0.0068 mol) and 2.00 g(0.0068 mol) ethanol solution of Phosphorylcholine (MPC), Yi Jiyu
Lower 3/4ths volumes are dissolved with the THF solution of AIBN.It after being sufficiently mixed, is slowly added dropwise this solution to aforementioned three-necked bottle
Interior reaction 24 h, obtain flaxen clear solution.After reaction completes, after vacuum rotary steam removes 2/3rds solvents, retaining
Molecular weight is the interior dialysis of bag filter of 3 500 Da.First dialysed 3 times by its good solvent absolute ethyl alcohol, then with pH=3.0 ~ 4.0
Phosphate buffer is dialysed 6 times, 3 ~ 8 h every time.-50 DEG C of freeze-dryings afterwards, the White Flocculus obtaining is copolymer p MN
。1The molar content that H-NMR records NPEM component in copolymer p MN is 39%.Same method synthesizes a series of NPEM
The molar content of component is respectively the copolymer of the 27%th, 36% and 42%.
Embodiment 5 imitates the synthesis of the amphipathic multi-functional co-polymer PMNCF of membrane structure
Utilize active ester group that amidized folic acid and cholesterol are grafted under weak basic condition polymer P MN strand
On, weighing 0.50 gram of PMN-39, transfer them in 250 mL three neck round bottoms, the DMSO measuring 20 mL is transferred to it
In, adding 200 μ L triethylamines, temperature regulation makes it fully dissolve to 80 DEG C of constant temperature stirring 4 h.Weigh amination cholesterol
0.15 g(0.0003 mol), be dissolved in the DMSO of 30 mL, to be dissolved fully, solution is transferred to separatory funnel and drips
Enter isothermal reaction 3 h in flask.Then in this reaction flask, dropping contains 0.59 g(0.0012 mol) FA-NH230 mL
DMSO solution, 80 DEG C of constant temperature lucifuge reaction 20 h.Reaction terminate after by dialysis that reactant liquor molecular cut off is 3 500 Da
Bag dialysis.First with DMSO after 45 DEG C of dialysis 3 times, then dialysed 8 times by distilled water lucifuge, 3 ~ 8 h every time.After dialysis finishes, will
Product freeze-drying at-50 DEG C, obtains polymer P MNCF.With1H-NMR determines that the Mole percent of polymer and each component contains
Amount, the molar content recording each component in copolymer p MNCF is MPC/Chol/FA=61/16/23, uses PMNCF-23
Represent (Fig. 1).
Prepared by embodiment 6 PMNCF-9 micella
The DMSO solution of 2 mL 5 mg/mL PMNCF-9 polymer is slowly dropped into 10 mL pH's 5.0 of quick stirring
In deionized water, after dropping finishes, continue stirring 6 h.Mixed solution is transferred to retain relative molecular mass be 3 500 saturating
In analysis bag, dialyse in 20 mL ultra-pure waters 48 h, changes fresh ultra-pure water every 3 ~ 4 h therebetween.After dialysis completes, removal is thoroughly
Solution in analysis bag, centrifuges 20 min with 6 000 rpm/min and removes large granular impurity.Pipette supernatant dynamic light scattering
(DLS) Zeta potential of micella dispersion liquid and particle size and distribution thereof are measured respectively.
Prepared by embodiment 7 PMNCF-31 micella
50 mg PMNCF-31 polymer are dissolved in 10 mL DMSO with stirring, then this solution are slowly dropped into 50mL
In the deionized water of the lower pH=5.0 of quick stirring, dropping finishes follow-up continuous stirring 6 h.Micellar solution is transferred to retain and relatively divides
Protonatomic mass is in the ultra-filtration centrifuge tube of 3 000, with 4 000 rpm/min centrifugal ultrafiltration 20 min, is repeated 3 times removing DMSO.Dense
Contracting liquid ultra-pure water constant volume obtains the micellar solution of finite concentration (1 mg/mL), dynamic light scattering obtain Zeta potential-13.9 ±
0.8 mV, number is averagely hydrated particle diameter 56.0 ± 7.5 nm, PDI=0.219.
Embodiment 8 carries adriamycin PMNCF micella
The PMNCF-23 polymer weighing 30 mg is dissolved in 6 mL DMSO.11.4 mg ADMhs are dissolved in 2 mL DMSO
Rear addition 6.1 mg triethylamines stirring 4 h.It is added drop-wise to quickly to stir with 3 s/d after mixing with above-mentioned PMNCF-23 solution
In 30 mL deionized waters (pH=5.12), after being added dropwise to complete, continue stirring 6 h.Again mixed solution is transferred to retain average molecular
Quality is in the bag filter of 3 500 Da, and dialyse in 500 mL ultra-pure waters 48 h, changes fresh ultrapure every 3 ~ 8 h therebetween
Water.After dialysis completes, take out the solution in bag filter, centrifuge 20 min with 6 000 rpm/min, remove large granular impurity.On
Clear liquid molecular cut off be the ultra-filtration centrifuge tube of 3 000 Da at 4 000 rpm/min centrifugal ultrafiltration 20 min, finally use
0.45 μm of membrane filtration, dilution constant volume obtains 6 mg/mL and the micellar solution of 1 mg/mL.Pipette the carrier micelle of 1 mg/mL
Solution dynamic light scattering records Zeta potential-8.7 ± 1.7 mV, and number is averagely hydrated particle diameter 87.1 ± 4.9 nm, PDI=
0.072。
Embodiment 9 carrier micelle controls release characteristic curve
Take 4.0 mL medicine carrying adriamycin micella (1 mg/mL) solution additions and retain the bag filter that relative molecular mass is 3 500 Da
In, and bag filter is placed in 50 mL spiral cover centrifuge tubes, the dissolution medium (pH=7.4 or pH=5.0) of 20 mL is measured with graduated cylinder
Cushioning liquid.Carrying out extracorporeal releasing experiment on constant temperature 37 DEG C, 100 rpm/min shaking tables, separated in time takes dialysis and is situated between
Matter 3 mL analyzes release concentration, adds 3 mL fresh medium dislysates simultaneously and continues drug release process.The adriamycin release obtaining is bent
Line is as shown in Figure 2.Compared with the release profiles of free adriamycin, carrier micelle has obvious slow release effect.
Embodiment 10 carries the cytotoxicity of adriamycin PMNCF micella
Detect cell survival rate by Tetrazolium salt colorimetric assay (mtt assay), thus judge cytotoxicity, the blank micella of assessment, medicine carrying
Micella and free adriamycin are to cytotoxicity, concentration dependent and carrier micelle slow release effect.This experiment rat breast cancer
(MADB-106) cell is evaluated.
MADB-106 cell after adhere-wall culture 24 h, is replaced by the RPMI RPMI-1640 of micella containing PMNCF in 96 orifice plates
Change cell culture fluid, cultivate 24 h, 48 h.Sucking-off adds aseptic PBS to wash away dead cell, drip washing after cultivating the nutrient solution in plate hole
3 times.With the impact on cellular morphology for the polymer micelle of observation variable concentrations under inverted microscope.Then 20 μ are added to every hole
L concentration is that the RPMI RPMI-1640 of 5 mg/mL MTT continues at 37 DEG C, 5% CO24 h are hatched in incubator.Carefully sop up
After supernatant, every hole adds DMSO 200 μ L, is placed on the shaking table of constant temperature 37 DEG C and vibrates 10 min and make its gray crystals fully molten
Solve, at 490 nm wavelength, survey the OD value in each hole with ELIASA, be calculated each sample cell relative survival (Fig. 3).
Embodiment 11 carries the preparation of fluorescence probe Coumarin-6 micella
Weighing 25 mg PMNCF polymer to be dissolved in 5 mL dimethyl sulfoxide (DMSO)s, stirring 6 h makes it dissolve fully.Add 50 μ L dense
Degree is, after the chloroform soln of 0.5 mg/mL Coumarin-6 mixes, to be added drop-wise to 25 mL deionized waters with vigorous stirring
In (pH=5.12), after being added dropwise to complete continue stirring 6 h.Then being transferred to micellar solution retain relative molecular mass is 3 500
In the bag filter of Da, dialyse in 500 mL distilled water 48 h, changes first water every 3 ~ 8 h therebetween.Take out after dialysis
Solution in bag filter, centrifuges 20 min with 6 000 rpm/min in centrifuge, removes large granular impurity.Take supernatant (10
ML) in centrifuge, 20 mins are centrifuged with 4 000 rpm/min with the ultra-filtration centrifuge tube that molecular cut off is 3 000 Da, then
Add ultra-pure water constant volume again to 10 mL, centrifuge supernatant for several times in this approach again and reach to concentrate and remove free tonka-bean further
The purpose of element-6.It is 5 mg/mL or the load fluorescence probe of 1 mg/mL that concentrate constant volume to 5 or 25 mL finally obtain concentration
Micellar solution.
Embodiment 12 cell in vitro uptake ratio measures
Measure the uptake ratio to different micellas or nano particle by Turnover of Mouse Peritoneal Macrophages (MPM).
The first step: with the RPMI RPMI-1640 cell culture medium compound concentration 1.0 × 10 containing 10% calf serum6Individual/
The MPM cell suspension dispensing of mL in 24 well culture plates, every hole 1 000 μ L, in the constant temperature cell culture incubator of 37 DEG C train
Supporting 5 h makes cell attachment.
Second step: after discarding former cultivation, the load Coumarin-6 that experimental group addition RPMI RPMI-1640 has diluted
PMNCF micella nutrient solution (0.20 mg/mL) 1 000 μ L, blank group adds cell culture medium 1 000 μ L, and control group adds 0.20
The polylactic acid nano particle 1 000 μ L of the load equivalent Coumarin-6 of mg/mL, often group sets 4 multiple holes；Insert 37 DEG C, 5% CO2、
2 h are cultivated respectively in the constant incubator of saturated humidity.
3rd step: remove after 2 h and reclaim containing the Coumarin-6 nutrient solution not being ingested in centrifuge tube, 1 500
Centrifuging 10 min under the conditions of rpm/min, supernatant sepectrophotofluorometer excites at 430 nm, excites slit and launches narrow
Seam width is 5 nm, sweep speed 1200 nm/min, excitation voltage 700 V, and the fluorescence of scanning 445 ~ 630 nm wave-length coverages is sent out
Penetrate spectrum, read the intensity of fluorescence emission peak at 500 nm.
With the load Coumarin-6 micella dispersion liquid fluorescence intensity of addition for comparison, calculate macrophage pair according to below equation
The uptake ratio of polymer micelle.
WhereinI 0The fluorescence intensity of the load fluorescence probe polymer micelle dispersion liquid adding
I After being absorbed by macrophage, carry the fluorescence intensity of fluorescence probe polymer micelle dispersion liquid supernatant.
4th step: it is infirm at cell surface that the 200 L PBS drip washing of each hole of Tissue Culture Plate wash away absorption for three times
In conjunction with polymer micelle or nano particle, then under inverted fluorescence microscope pass through light field and fluorescence field observation of cell pattern
And have or not fluorescence phenomenon, qualitatively judge the phagocytosis situation to micella for the macrophage.Sepectrophotofluorometer is utilized to calculate uptake ratio
Result basically identical with fluorescence inverted microscope qualitative observation light field and fluorescence field macrophage phagocytic situation.Comparative illustration contains
PC radical polymerisation thing micella greatly reduces by the probability of macrophage phagocytic.Phagocytic rate result of calculation is as shown in Figure 4.
The targeting of embodiment 13 folacin receptor targeting micella measures
With rat breast cancer cell MADB-106 cell as model cell, investigate the situation of tumour cell picked-up micella, evaluate body
Outer cancer cell Targeting Performance.Specific experiment process is as follows:
MADB-106 cell is 5 × 10 with density in containing 10% FBS, containing dual anti-and RPMI-1640 nutrient solution without folic acid4
The density kind of individual cells/well is in 24 well culture plates.Removing nutrient solution after cultivating 24 h, the one of every hole addition 0.20 mg/mL is
Row load Coumarin-6 micella dispersion liquid 1.0 mL, 37 DEG C, under saturated humidity, 5% CO2Incubator is hatched different time (2
H, 6 h, 12 h), investigate, by light field and fluorescence field, the situation that micella is absorbed by different incubation time tumour cell qualitatively.Training
After supporting different time, collect containing the micella nutrient solution not being ingested, survey the picked-up effect of fluorescence intensity rational judgment tumour cell
Rate.With PBS drip washing orifice plate inner cell three times repeatedly, wash away the micella adsorbing in cell surface not strong bonded.Glimmering being inverted
Viewed under light microscopy light field and fluorescence field, qualitatively judge tumour cell picked-up situation.As Fig. 5 result shows, PMNCF-31's
Micella uptake ratio is 3 ~ 5 times without modified with folic acid micella, illustrates that micella can significantly improve cancer cell by folacin receptor mediated
Picked-up ability to micella, thus realize the cell targeted of carrier micelle.
Claims (9)
1. the amphipathic multifunctional polymer shown in general structure (I), referred to as PMNCF, wherein R is hydrogen or methyl；
Wherein, x is the positive integer of 10～1000, and y is the positive integer of 5～500, and z is the positive integer of 0～500；In x, y, z, x
Mole percent is 40 ~ 75%, and y is 10 ~ 30%, and z is 0～35%；
n1It is the positive integer of 1 ~ 3, R1For the amphion hydrophilic radical simultaneous with positive and negative charge
n2It is the positive integer of 5 ~ 50, R2For the hydrophobic group containing cholesterol
n3It is the positive integer of 5 ~ 50, R3For the tumour folacin receptor targeting group containing folic acid
。
2. amphipathic multifunctional polymer according to claim 1, it is characterised in that: in x, y, z, x mole percent is
40 ~ 75%, y are 10 ~ 30%, and z is 10～35%.
3. the preparation method of amphipathic multifunctional polymer described in claim 1, it is characterised in that: PMNCF by containing both sexes from
Two kinds of polymerisable monomers of son and active ester cause random copolymerization first to obtain amphion unit by free radical in the solution
Molar fraction x is the binary randomcopolymer of 40% ~ 75%, then the folate molecule with the cholesterol containing amino and containing amino
Carry out amidatioon graft reaction, active ester units 1-x=60% ~ 25% to the active ester units in polymer, obtain cholesterol list
Unit's molar fraction y is 10% ~ 30%, and folic acid unit molar fraction z is the multifunctional polymer of 0% ~ 35%.
4. the preparation method of amphipathic multifunctional polymer according to claim 3, it is characterised in that: above-mentioned polymerisation exists
Anhydrous organic solvent dimethyl sulfoxide is carried out.
5. the method that amphipathic multifunctional polymer described in claim 1 prepares nano drug-carrying micella, it is characterised in that use diformazan
Base sulfoxide dissolves PMNCF polymer and hydrophobic drug, will account for the drug solution of polymer quality percentage 10% ~ 50% and be polymerized
Thing solution adds the carrier micelle automatically forming diameter 30 ~ 150 nanometer in the quick aqueous solution stirring, and forms stable nanometer
The carrier micelle aqueous solution.
6. the preparation method of multifunctional polymer nano drug-carrying micella according to claim 5, it is characterised in that prepared
Cheng Zhong, the pH of the regulation aqueous solution is 4 ~ 6.
7. the preparation method of multifunctional polymer nano drug-carrying micella according to claim 5, it is characterised in that have and hate
Aqueous pharmaceutical is selected from adriamycin, taxol, epirubicin, HCPT, THP, docetaxel, vinorelbine, Austria
Husky profit platinum.
8. the preparation method of multifunctional polymer nano drug-carrying micella according to claim 5, it is characterised in that use ultrafiltration
Organic solvent and free drug are separated by centrifugal method, purify and concentrate nano drug-carrying micella.
9. the amphipathic multifunctional polymer described in claim 1 is as the application of nano drug-carrying carrier.
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