CN104650307B - PH five block linear polymers of sensitivity and micella based on PDEAEMA - Google Patents
PH five block linear polymers of sensitivity and micella based on PDEAEMA Download PDFInfo
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Abstract
The invention belongs to biological medicine technical field of polymer materials, a kind of pH five block linear polymers of sensitivity based on PDEAEMA and preparation method thereof and the micellar system that obtains based on it and application are disclosed.The polymer has structure shown in following formula：Wherein, x=45, y=19~37, z=15~19.Present invention design synthesis obtains the block linear polymer structure of symmetry five, the drug encapsulation amount of polymer can significantly be improved, the pH response ranges of self-assembled micelle can be preferably adjusted by intermediate layer PDEAEMA, allowing carrier micelle to reach can be under tumor environment, the change of quick response pH value, the release of medicine can effectively be controlled, it is to avoid the prominent of medicine is released.The thicker pH responses intermediate layer of micelle forma-tion that Inventive polymers are formed, raising improves medicine-releasing performance while contain ability, reduces the particle diameter of micella after insoluble drug release, is conducive to the discharge of carrier, in can be widely applied to field of medicaments.
Description
Technical field
The invention belongs to biological medicine technical field of polymer materials, more particularly to a kind of pH based on PDEAEMA is quick
Feel five block linear polymers and preparation method thereof and the micellar system that obtains based on it and application.
Background technology
Cancer (malignant tumour) is the disease caused because control growth and proliferation of cell mechanism is not normal, but cancer cell is removed
Outside growth is out of control, the normal structure of human body can be also locally invaded, and can easily be transferred to very much other positions.According to
Newest《World's cancer report》It has been shown that, in newly-increased 14,000,000 cases of cancers in the whole world, it is Chinese to have 3,070,000, account for 21.8%,
Newly-increased cases of cancer is occupied first of the whole world.Death toll ratio is even more and accounts for the 26.9% of global death toll.It can be seen that China is for cancer
The treatment of disease still more falls behind, and the research thus for treatment of cancer also shows important especially.The treatment of current cancer is mainly hand
Art treatment, three kinds of methods of radiotherapy and chemotherapy, wherein chemical medicinal treatment are still using most methods.But
It is that chemical medicinal treatment still has that some need to solve, such as the utilization rate of dewatering medicament is low, genomic medicine is easy to
Degraded, the toxic and side effect of medicine normal tissue cell is big the problems such as wait.
In order to be able to solve the above problems, in the last few years, novel intelligent response (pH responses, temperature-responsive, enzyme response) medicine
Transmission nano-carrier (polymer nanoparticle, liposomes, hydrogel, polymer micelle) is aroused great concern.These
Nano anti-cancer pharmaceutical carrier improves the efficiency of killing cancer cell and reduces to human body just by changing the bio distribution of cancer therapy drug
The toxic and side effect of normal cell.These anti-cancer medicament carriers can also extend medicine circulation time in blood, improve hydrophobic medicine
Meltage of the thing in human body, and then the dosage of administration is reduced, so as to improve the utilization rate of medicine and reduce malicious secondary work
With.
Preferable anti-cancer medicament carrier can not discharge drug substantially in normal tissue and the stabilization of holding structure in blood
Thing, and then can fast and effectively position release medicine at tumor tissues position.And should have preparation method simple, higher
Drugloading rate, particle size are suitable, the property such as degradation property in vivo is excellent.
Block polymer refers to that the different polymer segment of two or more property is connected together into what is be prepared from
A kind of particular polymer.The property of multiple polymers can be combined together, obtain the polymer with various superior functions
Material.These functional polymer materials can be widely used in biological medicine, chemical industry, building etc. according to its heterogeneity
Every field, either theoretical research or practical application all have a very big significance.
It is more sensitive that pH responsive polymer micellas are primarily referred to as change of the polymer micelle to the pH value of surrounding environment, micella
Physicochemical property corresponding change can occur, be a kind of newtype drug delivery vehicles material of great application prospect.But it is this
Material is also still faced with a series of problems, such as micella is into easily disintegration is diluted after blood, and the particle diameter of micella is excessive
It is easy to be swallowed or by tissue resorptions such as liver spleens by internal reticuloendothelial system (RES) again, the particle diameter distribution of micella is wider etc..It is poly-
The self assembly of compound micella is a Thermodynamically stable process, i.e., the micella only when CMC value of its concentration higher than micella
Structure remained stable, the current concentration that is diluted to easily is disintegrated less than below the CMC of micella, so that the medicine in carrier micelle occurs
It is prominent to release, so limit application of the polymer micelle in human body.Five block polymers pass through the polymer being self-assembly of
Micella, due to hydrophobic block more long so that polymer micelle has less CMC value to maintain the stabilization of micella, together
When there is greater compactness of hydrophobic inner core so that the particle diameter of micella is not too large and has influence on its application prospect.
Contain functional group's tertiary amino in PDEAEMA, its pKb value is 6.9.Tertiary amino easy trap proton in sour environment
And positively charged, apparent is hydrophilic radical, and micellar structure becomes loose, and the medicine being embedded in micella is quickly discharged into environment；
In neutral or basic conditions, hardly there is protonation, apparent is hydrophobic grouping, keep the cramped construction of micella,
Insoluble drug release in micella also can be effectively prevented while the drug encapsulation amount of micella is improved in environment.GU[Journal
of Polymer Science:Part A:Polymer Chemistry,47(2009):3142-3153] using PDEAEMA's
PH sensitive properties, have synthesized bi-block copolymer PPEGMEA-g-PDEAEMA, under different pH value, are prepared by self assembly
Micella, and pH response self assembly behaviors are have studied, the particle diameter of micella, the light transmittance of micellar solution are all as the change generation of pH is bright
Aobvious change.The particle diameter of micella can be significantly increased in the basic conditions, and the light transmittance of solution is remarkably decreased, and its change
Journey is influenceed by protonation.Yang[Acta Biomaterials,9(2013):7679-7690] synthesize 4/6 equal arm
Star-type polymer, its arm is the PCL-b-PDEAEMA-b-PPEGMA of three block, and for preparing amphipathic nano-micelle, is used
In containing hydrophobic anticancer drugs, doxorubicin (DOX), carrier micelle has relatively low critical micelle concentration and sensitive pH quick
Perceptual energy.Insoluble drug release is slow under pH7.4, and when pH value drops to 5.0, the rate of release of medicine has obvious quickening,
And it is substantially nontoxic by cytotoxicity experiment to demonstrate material.Determan[Polymer,46(2005):6933-6946] use
The method of ATRP, has synthesized five controllable block polymers of various block lengths, reaction condition centered on Pluronic F127
Gently, yield is high, and polymer shows preferable pH sensitivities and temperature sensitivity energy.
Patent application CN200910024899.X discloses a kind of method of use ATRP method block polymer synthesis, embedding
The length of section is easy to regulation and control, and the mechanism of reaction is ripe, and various reaction conditions are gently feasible.Patent application CN20131014153.1
The preparation method for containing hydrophobic anticancer drug target polymer micella is disclosed with CN201310141556.8, due to tumour
The difference of organizational environment and normal structure environmental pH, realizes the position control release of medicine.
The content of the invention
For the shortcoming and not enough, primary and foremost purpose of the invention that overcome above-mentioned prior art micella easily to disintegrate and be easily trapped
It is that a kind of pH five block linear polymers of sensitivity based on PDEAEMA are provided.The structure of the polymer is included：Hydrophilic block is gathered
Ethylene glycol, pH response block methacrylic acid N, N- lignocaine ethyl esters, hydrophobic block methyl methacrylate, combination obtains pH
Sensitive five block linear polymers.
Another object of the present invention is to provide a kind of system of the above-mentioned pH five block linear polymers of sensitivity based on PDEAEMA
Preparation Method.The method, with 2- bromine isobutyl acylbromides as bromide reagent, is carried out by using acyl bromination reaction with macromolecular polyethylene glycol
Acyl bromination reaction, prepares macromole evocating agent (Br-PEG-Br)；Gathered using electro transfer activating and regenerating atom transferred free radical again
Legal (ARGET ATRP), with bromination polyethylene glycol (Br-PEG-Br) as macromole evocating agent, triggers pH response monomer first successively
Base acrylic acid N, N- lignocaine ethyl ester (DEAEMA) and methyl methacrylate (MMA) obtain pH five block linear polymerizations of sensitivity
Thing (polyethylene glycol-[b- polymethylacrylic acid N, N- lignocaine ethyl ester-b- polymethyl methacrylates]2)。
Still a further object of the present invention is to provide the above-mentioned pH five block linear polymers of sensitivity based on PDEAEMA to be led in medicine
Application in domain.
Still a further object of the present invention is to provide a kind of based on the above-mentioned pH five block linear polymers of sensitivity based on PDEAEMA
Micellar system.It is hydrophobic block to use dialysis to prepare internal layer, and intermediate layer is that pH responds block, and shell is received for hydrophilic block
Meter level polymer micelle, realization is contained to hydrophobic drug.Under tumor tissues solutions of weak acidity (pH 5~6), the micella
There is protonation and show as hydrophily in the pH of system, micella dissociates, so as to realize being contained medicine in response to group
Quick controlled release.The micella of five block linear polymer structures of pH sensitivities of the invention can be before micella stabilization be kept
Put, improve the amount of containing and pH response sensitivities of medicine, more effective Drug controlled release improves therapeutic efficiency, reduces secondary
Effect.
Still a further object of the present invention is the application for providing above-mentioned micellar system in field of medicaments.
The purpose of the present invention is realized by following proposal：
A kind of pH five block linear polymers of sensitivity based on PDEAEMA, with structure shown in following formula：
Wherein, x=45, y=19~37, z=15~19.
The structural formula of the pH five block linear polymers of sensitivity based on PDEAEMA of the invention is PEG- [b-PDEAEMA-
b-PMMA]2, number-average molecular weight is 10131~14536g/mol.
Present invention also offers a kind of preparation method of the above-mentioned pH five block linear polymers of sensitivity based on PDEAEMA,
Including step in detail below：
(1) macromole evocating agent is prepared：Catalyst, polyethylene glycol (PEG) are dissolved in solvent, acid binding agent stirring is added
Uniformly, bromating agent is added dropwise over, is reacted at room temperature, obtain macromole evocating agent (Br-PEG-Br)；
(2) pH five block linear polymers of sensitivity are prepared：Macromole evocating agent (the Br-PEG- that step (1) is prepared
Br), pH responds monomer methacrylic acid N, N- lignocaine ethyl ester (DEAEMA), catalyst, ligand 1,1,4,7,10,10- six
Methyl triethylene tetramine (HMTETA) is dissolved in solvent, adds reducing agent, after heating response, adds monomer methacrylic acid
Methyl esters (MMA), isothermal reaction obtains pH five block linear polymers of sensitivity (PEG- [b-PDEAEMA-b-PMMA]2)。
The molfraction formula of reactant is as follows in step (1)：
The molfraction formula of reactant is as follows in step (2)：
The room temperature reaction time described in step (1) is preferably 12~36h.
Bromating agent described in step (1) is preferably dripped in 1h, is added dropwise more preferably in ice-water bath.
Solvent described in step (1) is preferably dichloromethane.
Catalyst described in step (1) is preferably DMAP (DMAP).
Acid binding agent described in step (1) can be any acid binding agent commonly used in the art, preferably triethylamine (TEA).
Bromating agent described in step (1) can be any bromating agent commonly used in the art, preferably 2- bromine isobutyl acylbromides
(BMPB)。
Catalyst described in step (2) is preferably CuBr2。
Reducing agent described in step (2) is preferably Sn (Oct)2。
In step (1) after the completion of reaction, 5wt%NaHSO is preferably used respectively4、Na2SO4, deionized water extractive reaction liquid 3
It is secondary, obtain macromole evocating agent after purification.Filtered after being dried overnight in anhydrous magnesium sulfate after more preferably having extracted, rotation is steamed
Hair removes the most of solvent in filtrate, then is precipitated with 10 times of volume cold diethyl ether solution, is centrifuged, and with cold ether three times,
Then it is vacuum dried.Dried sample is dissolved in absolute ethyl alcohol, is overnight recrystallized, then obtain final product product after being vacuum dried
Br-PEG-Br macromole evocating agent white solids.
The condition of the heating response described in step (2) is preferably and is heated to 60~90 DEG C, reacts 5~10h.Described perseverance
The time of temperature reaction is preferably 5~10h.
Solvent described in step (2) is preferably toluene.
Preferably, after the completion of step (2) described isothermal reaction, by reaction system cooling, purifying, dry, obtain after purification
Product.After described purifying refers to and for product to be dissolved in tetrahydrofuran, neutral alumina chromatographic column is crossed, tetrahydrofuran is washed
It is de-, catalyst is removed, then by eluent concentrated by rotary evaporation, add 10 times of the 0 of volume DEG C of n-hexanes to be precipitated, it is filtrated to get purifying
Product afterwards.
Preferably, above-mentioned reaction is preferably carried out under atmosphere of inert gases, anhydrous condition.
PH five block linear polymers of sensitivity based on PDEAEMA of the invention can be applied in field of medicaments, especially fit
In for containing hydrophobic drug.
A kind of micellar system based on the above-mentioned pH five block linear polymers of sensitivity based on PDEAEMA, by will be above-mentioned
PH five block linear polymers of sensitivity based on PDEAEMA are obtained in being dissolved in solvent.
Present invention also offers application of the above-mentioned micellar system in field of medicaments, the bag of hydrophobic drug is particularly well-suited to
In load, following steps are specifically included：PH five block linear polymers of sensitivity based on PDEAEMA are dissolved in organic solvent and are obtained
Micellar system, hydrophobic drug is dissolved in identical organic solvent, then is mixed with above-mentioned micellar system, stirring, dialysis, mistake
Filter, dries, and obtains containing hydrophobic drug micellar system.
Described stirring is preferably 4~6h.
Described dialysis preferably with deionized water dialyse 48h, it is highly preferred that first 12 hours in dialysis procedure are every 2 hours
A water is changed, a water is changed within every 4 hours 12~24 hours, a water is changed within every 6 hours 24~48 hours.
Described hydrophobic drug refers to medicine of the solubility less than or equal to 1g in 1L water.
Described organic solvent is preferably the one of which in dimethyl sulfoxide (DMSO) and dimethylformamide.
What the present invention was prepared contain hydrophobic drug micellar system can control the medicine of loading in normal structure (pH
7.4) place is slowly discharged, and quick, controlled release is realized at tumour cell solutions of weak acidity (pH 5~6) place, be can be widely applied to
In field of medicaments.
It is of the invention based on PDEAEMA pH sensitivity five block linear polymers, use dialysis prepare internal layer for
Hydrophobic block, intermediate layer is that pH responds block, and shell is the nano grade polymer micella of hydrophilic block, is realized to hydrophobic drug
Contain.Under tumor tissues solutions of weak acidity (pH 5~6), there is protonation and show as hydrophilic in pH in response to group
Property, micella dissociates, so as to realize being contained the quick controlled release of medicine.This pH responds five block linear polymer knots
The micella of structure can improve the amount of containing of medicine, more effectively pH response sensitivities, control on the premise of micella stabilization is kept
Insoluble drug release, improves therapeutic efficiency, reduces side effect.
Mechanism of the invention is：
The present invention is using hydrophilic block polyethylene glycol, pH response block methacrylic acid N, N- lignocaine ethyl esters and hydrophobic
The combination of block methyl methacrylate obtains amphipathic pH and responds five block linear polymers, is dissolved in solvent, obtains
Internal layer is hydrophobic block, and intermediate layer is that pH responds block, and shell is the nano grade polymer micella of hydrophilic block, micella outer layer parent
Aqueous PEG has the advantages that nontoxic, non-immunogenicity and no antigen, while micella stability is increased, extends micella
Circulation time in blood；Hydrophobic PMMA kernels can strengthen and contain performance to insoluble medicine；The PDEAEMA in intermediate layer exists
Hydrophobicity is shown as during pH 7.4, the hydrophobic inner core of micella can be collectively constituted with cholesterol, this can not only prevent burst drug release,
The stability of micelle inner core can be strengthened simultaneously；Under the conditions of tumor tissues faintly acid (pH 6.0), side chain in PDEAEMA blocks
Tertiary amine groups occur protonation and show as hydrophily, micella start occur it is a certain degree of swelling；If carrier micelle enters
Enter to have in the endosome and lysosome of lower pH, PDEAEMA will be protonated completely, now micella degree of swelling becomes big, micella
Start aggregation even dissociation behavior occur, so as to by the insoluble drug release of package-contained to tumour cell.In by adjusting polymer
The ratio of each block, can meet the release request of different pharmaceutical with the rate of release of regulating medicine.
The present invention has the following advantages and beneficial effect relative to prior art：
(1) present invention design synthesis obtains the block linear polymer structure of symmetry five, can significantly improve polymer
Drug encapsulation amount, the pH response ranges of self-assembled micelle can be preferably adjusted by intermediate layer PDEAEMA, allow carrier micelle to reach
To can be under tumor environment, the change of quick response pH value can effectively control the release of medicine, it is to avoid the prominent of medicine is released.
(2) the thicker pH responses intermediate layer of the micelle forma-tion of Inventive polymers formation, while raising contains ability, carries
Medicine-releasing performance high, reduces the particle diameter of micella after insoluble drug release, is conducive to the discharge of carrier.
(3) the five block linear polymers of sensitivity of the pH based on PDEAEMA that prepared by the present invention, the length of each block is all easy
In regulation and control, different medicines can be contained according to applied environment different choice.
Brief description of the drawings
Fig. 1 is the GPC elution curves of the Br-PEG-Br in embodiment 1, and mobile phase is tetrahydrofuran.
Fig. 2 is the Br-PEG-Br's in embodiment 11H H NMR spectroscopies, solvent is deuterochloroform.
Fig. 3 is the PEG- [b-PDEAEMA-b-PMMA] in embodiment 22GPC elution curves, mobile phase is tetrahydrofuran.
Fig. 4 is the PEG- [b-PDEAEMA-b-PMMA] in embodiment 22's1H H NMR spectroscopies, solvent is deuterochloroform.
Fig. 5 is the PEG- [b-PDEAEMA-b-PMMA] in embodiment 52Critical micelle concentration test curve.
Fig. 6 is the PEG- [b-PDEAEMA-b-PMMA] in embodiment 62The potentiometric titration curve of self-assembled micelle.
Fig. 7 is the PEG- [b-PDEAEMA-b-PMMA] in embodiment 72The particle diameter of self-assembled micelle, zeta current potentials and pH
Relation.
Fig. 8 is the PEG- [b-PDEAEMA-b-PMMA] in embodiment 82The transmission electron microscope picture of micella.
Fig. 9 is the PEG- [b-PDEAEMA-b-PMMA] in embodiment 92Carry the In-vitro release curves of adriamycin micella.
Figure 10 is PEG- [b-PDEAEMA-b-PMMA] in embodiment 102Blank micella vitro cytotoxicity.
Figure 11 is PEG- [b-PDEAEMA-b-PMMA] in embodiment 102Load adriamycin micella vitro cytotoxicity.
Specific embodiment
With reference to embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited
In this.
Embodiment 1：The synthesis of macromole evocating agent Br-PEG-Br
Synthetic reaction formula is as follows：
The synthesis of macromole evocating agent Br-PEG-Br：Load magnetic stir bar in 250mL round bottom reaction bulbs, use anti-mouth
Rubber stopper is sealed, and vacuumizes-lead to argon gas three times, is placed in frozen water.By 0.4g (3.28mmol) DMAP (Shanghai covalent chemical)
It is dissolved in 20mL dichloromethane with 2.0mL (14.0mmol) TEA, solution is added in reaction bulb by syringe.Will
6.6g (3.30mmol) PEG (Sigma-Aldrich) is dissolved in 80mL dichloromethane, is injected PEG solution by syringe
In reaction bulb.With syringe to 1.6mL (6.60mmol) BMPB (J＆K, lark prestige) is dropwise added dropwise in reaction bulb, in one hour
Add, reacted 2 hours at being kept for 0 DEG C, remove ice bath, continue to react 24 hours at room temperature.5wt% is used after the completion of reaction respectively
NaHSO4、Na2SO4Extraction 3 times, is then placed in anhydrous magnesium sulfate and is dried overnight with water washing is distilled 3 times again.Then by rotation
Turn evaporation and remove most solvent, then concentrate is slowly dropped into 10 times of cold diethyl ethers of volume precipitates, centrifugation, centrifugation is obtained
Solid 48h be vacuum dried under 35 DEG C, 35mbar obtain Br-PEG-Br white solids.Its molecular weight is determined using GPC, and
Nmr analysis are carried out, Fig. 1 and Fig. 2 is seen.Yield is 85%, Mn=2073, Mw/Mn=1.19.
Embodiment 2：PH sensitivity five block linear polymers PEG- [b-PDEAEMA-b-PMMA]2Synthesis
Synthetic reaction formula is as follows：
Load magnetic stir bar, 26.8mg (0.12mmol) copper bromides and 2.2g (1mmol) in 50mL dries eggplant-shape bottle
Macromole evocating agent Br-PEG-Br, is sealed with anti-mouth rubber stopper, vacuumizes-lead to argon gas three times, is injected first with syringe successively
Benzene 20mL, monomer DEAEMA (4.02mL, 20mmol) (J＆K, lark prestige), part HMTETA (0.31ml, 1.2mmol) (J＆K,
Lark prestige) add bottle in, stirring form catalyst complexes within 10 minutes.Then by 0.486g (1.2mmol) reducing agents Sn
(Oct)2In being dissolved in 2mL toluene and adding reaction bulb.Then after reaction bulb being transferred into 60 DEG C of reaction 7h, 1.86mL is added
(17.5mmol) monomer MMA (J＆K, lark prestige), continues to react 12h.Reaction solution is cooled to room temperature, 50mL THF is added and is stirred
Mixing dissolves it, then with neutral alumina pillar Filtration of catalyst CuBr2(eluant, eluent is made with THF).The wash-out for obtaining
Precipitation in ten times of cold n-hexanes of amount (volume ratio) is slowly added into after liquid concentration.Precipitated product vacuum under 35 DEG C, 35mbar
Dry 48h.Its molecular weight is determined using GPC, and carries out nmr analysis, see Fig. 3 and Fig. 4.Yield is 95%, Mn=10089,
Mw/Mn=1.33.
Embodiment 3：PH five block linear polymers PEG- [b-PDEAEMA-b-PMMA] of sensitivity2Synthesis
Load magnetic stir bar, 17.9mg (0.08mmol) copper bromides and 1.76g in 50mL dries eggplant-shape bottle
(0.8mmol) macromole evocating agent Br-PEG-Br, is sealed with anti-mouth rubber stopper, vacuumizes-lead to argon gas three times, successively with injection
Toluene 20mL, monomer DEAEMA (5.02mL, 25mmol), part HMTETA (0.21ml, 0.8mmol) are added bottle by device injection
In, stirring forms catalyst complexes in 10 minutes.Then by 0.324g (0.8mmol) reducing agents Sn (Oct)2It is dissolved in 2mL first
Benzo is added in reaction bulb.Then after reaction bulb being transferred into 60 DEG C of reaction 7h, 2.12mL (20mmol) monomer MMA is added, after
Continuous reaction 12h.Reaction solution is cooled to room temperature, 50mL THF is added and stirring is dissolved it, then use neutral alumina pillar
Filtration of catalyst CuBr2(eluant, eluent is made with THF).Ten times of amounts (volume ratio) are slowly added into after the eluent concentration for obtaining
Cold n-hexane in precipitate.Precipitated product is vacuum dried 48h under 35 DEG C, 35mbar, and yield is 95%, Mn=12153, Mw/
Mn=1.33.
Embodiment 4：Based on pH five block linear polymers PEG- [b-PDEAEMA-b-PMMA] of sensitivity2Synthesis
Load magnetic stir bar, 22.3mg (0.10mmol) copper bromides and 2.64g in 50mL dries eggplant-shape bottle
(1.2mmol) macromole evocating agent Br-PEG-Br, is sealed with anti-mouth rubber stopper, vacuumizes-lead to argon gas three times, successively with injection
Toluene 20mL, monomer DEAEMA (8.04mL, 40mmol), part HMTETA (0.26ml, 1.0mmol) are added bottle by device injection
In, stirring forms catalyst complexes in 10 minutes.Then by 0.405g (1.0mmol) reducing agents Sn (Oct)2It is dissolved in 2mL first
Benzo is added in reaction bulb.Then after reaction bulb being transferred into 60 DEG C of reaction 7h, 1.59mL (15mmol) monomer MMA is added, after
Continuous reaction 12h.Reaction solution is cooled to room temperature, 50mL THF is added and stirring is dissolved it, then use neutral alumina pillar
Filtration of catalyst CuBr2(eluant, eluent is made with THF).Ten times of amounts (volume ratio) are slowly added into after the eluent concentration for obtaining
Cold n-hexane in precipitate.Precipitated product is vacuum dried 48h under 35 DEG C, 35mbar, and yield is 95%, Mn=14536, Mw/
Mn=1.39.
Embodiment 5：Determine PEG- [b-PDEAEMA-b-PMMA]2CMC value.
The pH prepared using fluorescence probe method testing example 2 responds five block linear polymer PEG- [b-
PDEAEMA-b-PMMA]2Critical micelle concentration.
(1) configuration of pyrene solution：Acetone solution pyrene is used, configuration concentration is 12 × 10-5The pyrene solution of M.
(2) configuration of sample solution：Weigh 8mg PEG- [b-PDEAEMA-b-PMMA]2It is dissolved in 10mL acetone, will be molten
Liquid is added in 80mL deionized waters, and stirring 24h obtains the polymer mother liquor that concentration is 0.1mg/mL to vapor away acetone, dilute
It is interpreted into a series of concentration (concentration range is 0.0001~0.1mg/mL).20 10mL volumetric flasks are taken, 0.1mL steps are separately added into
(1) the pyrene solution of configuration, is then respectively adding the copolymer solution constant volume of above-mentioned various concentrations, shakes up, and obtains sample solution.Sample
The concentration of pyrene is 12 × 10 in product solution-7M。
(3) fluorescence spectrum test：With 373nm as launch wavelength, the fluorescence excitation of sample solution is scanned in 300~350nm
Spectrum.Take the intensity rate (I that wavelength is 339nm and 336nm339/I336) polymer concentration logarithm is mapped, as shown in figure 5, bent
Abscissa corresponding to line catastrophe point is lg (CMC).Measure the PEG- [b-PDEAEMA-b- that embodiment 2 is prepared
PMMA]2Critical micelle concentration be 2.80mg/L.
Embodiment 6：Potentiometric determination PEG- [b-PDEAEMA-b-PMMA]2PKb it is interval.
PEG- [b-PDEAEMA-b-PMMA] prepared by 30mg embodiments 22It is completely dissolved in 30mL deionized waters, leads to
Cross add a little HCl solution (0.1M) and ultrasound method polymer is dissolved as the polymer solution of concentration 1mg/mL.With
The pH value of NaOH or HCl solution (0.1M) regulation micellar solution is about 3, and stirring balance a period of time is stable to pH, from NaOH
(0.1M) is added dropwise over as titrating solution toward polymer solution, reads each pH value, as shown in Figure 6.The pKb of polymer solution
Interval is 6.5~7.4.
Embodiment 7：PEG-[b-PDEAEMA-b-PMMA]2The pH response self assembly behaviors of micellar system
PEG- [b-PDEAEMA-b-PMMA] prepared by 5mg embodiments 22It is completely dissolved in 20mL acetone, quick
It is added rapidly under stirring in 50mL deionized waters, 24h is stirred at room temperature to vapor away acetone, obtains ultimate density for 0.1mg/
The polymer micelle solution of mL.The pH value of micellar solution is adjusted with NaOH or HCl solution (0.1M), stirring balance a period of time is extremely
PH stabilizations, read pH value now.The particle diameter and zeta current potentials of micella under different pH value are tested using DLS simultaneously, such as Fig. 7 institutes
Show.From Fig. 7 (a), as pH reduces, particle diameter is gradually reduced, and is 7 or so to reach minimum in pH value, is then 7 in pH value
Slightly have increase to 5 particle diameters, can reduce when being less than 5 to pH value.From Fig. 7 (b), with the reduction of pH value, zeta current potentials
Gradually increase, to pH value be 7 when tend towards stability, when pH value continues to be decreased to less than 5, zeta current potentials have reduction slightly.
Embodiment 8：PEG-[b-PDEAEMA-b-PMMA]2The morphology characterization of carrier micelle and blank micella
Prepare PEG- [b-PDEAEMA-b-PMMA] prepared by embodiment 22The blank micella and carrier micelle of polymer, and
Characterize pattern.Carrier micelle is prepared using dialysis.Accurately weigh 10mg adriamycins (DOX) to be dissolved in 20mL DMSO, add 2
The μ L of TEA 20 of times mole, are stirred overnight.Weigh 20mg PEG- [b-PDEAEMA-b-PMMA] simultaneously2It is dissolved in 20mL DMSO
In.4~6h will be stirred under both mixed room temperatures, then with deionized water dialyse 48h (change a water within every 2 hours first 12 hours, 12
A water is changed within every 4 hours~24 hours, a water is changed within every 6 hours 24~48 hours), then filtered with 0.45 μm of filtering head, freezing
Dry, obtain DOX carrier micelle powder.Weigh 40mg PEG- [b-PDEAEMA-b-PMMA]2It is dissolved in 40mL DMSO.Room temperature
4~6h of lower stirring, then (is changed a water for every 2 hours first 12 hours, changed within every 4 hours 12~24 hours with deionized water dialysis 48h
Water, changes a water in every 6 hours 24~48 hours), obtain PEG- [b-PDEAEMA-b-PMMA]2Blank micella, using TEM
It is spherical to observe its pattern, sees Fig. 8.
Embodiment 9：PEG-[b-PDEAEMA-b-PMMA]2The release in vitro of carrier micelle
Five block polymer PEG- [b-PDEAEMA-b-PMMA] of pH sensitivities based on PDEAEMA2Carrier micelle it is external
Release performance is determined using medicament dissolution instrument and purple instrument.Comprise the following steps that：It is (real that 5mg DOX carrier micelles are accurately weighed respectively
Apply preparation in example 8) it is dispersed in 5mL buffer solutions (pH 7.4, pH 5.0), it is subsequently placed in bag filter, it is transferred to the identical pH of 45mL
Medicament dissolution instrument is placed in the buffer solution of value at 37 DEG C, release in vitro is carried out under 110rpm rotating speeds, regularly take the buffering outside 4mL bags
Liquid is analyzed, and adds 4mL fresh buffers.With DOX concentration in determined by ultraviolet spectrophotometry different time release liquid, paint
Its In-vitro release curves is made, as shown in Figure 9.
As shown in Figure 9, DOX carrier micelles are under normal structure environment (pH 7.4), the rate of release of DOX slowly,
Less than 20%, subsequent rate of release is also to tend to be steady to the cumulative release amount of 24 hours, and cumulative release amount is less than after 96 hours
40%.And under the faint acid condition of tumour cell (pH 5.0), the rate of release of DOX substantially accelerates, the accumulation of 24 hours
Burst size reaches 60%, and cumulative release amount is more than 80% after 96 hours.
Embodiment 10：PEG-[b-PDEAEMA-b-PMMA]2The in vitro toxicity evaluation of carrier micelle and blank micella
Test PEG- [b-PDEAEMA-b-PMMA]2(being prepared in embodiment 8) of carrier micelle and blank micella is external thin
Cellular toxicity.96 hole flat bottomed tissue culture plates are taken, 200 μ L cell culture mediums (DMEM) as blank will be separately added into surrounding orifice plate
Group.Per hole with 1 × 10 in middle 60 holes4Cells/well (200 μ L) cell concentration inoculation Hela cells (be purchased from ATCC,
USA), wherein 96 orifice plates are placed into 37 DEG C, saturated humidity, 5%CO by the 2nd row as control224h is cultivated in incubator.Then
Free adriamycin, lyophilized blank or carrier micelle cell culture medium are diluted to different polymer concentration (blank glue
1~400mg/L of beam) or drug concentration (free adriamycin carries adriamycin micella, 0.1~20mg/L).In 96 orifice plates are removed
After the 2nd row to the cell culture medium in the 11st all holes of row, fresh culture medium is added in being arranged the 2nd, as control.
From the 3rd row to the 10th row, to the sample solution that 200 μ L are separately added into all of hole, the sample of each concentration is added to 6 holes
In repeated.
By after the culture of 24h or 48h, siphoning away the supernatant in all holes containing cell, add 200 μ L's
PBS rinse cells, then siphon away PBS.From the 2nd row to the 11st row, respectively to the MTT solution and 180 μ that 20 μ L are added in each hole
, then be positioned over 96 orifice plates in incubator and cultivate 4h by the culture medium of L.Then siphon away unreduced MTT solution and culture is situated between
Matter.Each hole is washed one time with the PBS of 200 μ L, and siphons away PBS.Crystallized to adding the DMSO of 200 μ L to dissolve MTT in each hole.It is whole
Individual 96 orifice plate vibrates 10min in being placed on 37 DEG C of shaking tables, and the absorbance in each hole at 490nm is then determined using ELIASA, and then
Calculate cell survival rate.
Result is shown in that Figure 10 and Figure 11, Figure 10 are the toxicity data of blank micella, from figure, blank micella is thin to Hela
Born of the same parents are substantially nontoxic, and under the ultrahigh concentration of 400mg/L, the survival rate of cell can be still reached close to 90%.Figure 11 is load medicine
The toxicity data of micella, as can be seen from the figure when concentration is 20mg/L, the survival rate of cell is only 60% or so, and and
Free adriamycin has cytotoxicity closely, and after illustrating that adriamycin is contained through carrier material, its active anticancer does not have
It is obvious to weaken.
Above-described embodiment is the present invention preferably implementation method, but embodiments of the present invention are not by above-described embodiment
Limitation, it is other it is any without departing from Spirit Essence of the invention and the change, modification, replacement made under principle, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (10)
1. it is a kind of based on PDEAEMA pH sensitivity five block linear polymers, it is characterised in that with structure shown in following formula：
R is
Wherein, x=45, y=19~37, z=15~19.
2. it is a kind of it is according to claim 1 based on PDEAEMA pH sensitivity five block linear polymers preparation method, its
It is characterised by including step in detail below：
(1) macromole evocating agent is prepared：Catalyst, polyethylene glycol are dissolved in solvent, acid binding agent are added and is stirred, dropwise
Bromating agent is added, is reacted at room temperature, obtain macromole evocating agent；
(2) pH five block linear polymers of sensitivity are prepared：Macromole evocating agent, the pH response monomers that step (1) is prepared
Methacrylic acid N, N- lignocaine ethyl ester, catalyst, ligand 1,1,4,7,10,10- hexamethyl triethylene tetramines are dissolved in solvent
In, reducing agent is added, after heating response, monomers methyl methacrylate is added, isothermal reaction obtains pH five blocks of sensitivity
Linear polymer.
3. it is according to claim 2 based on PDEAEMA pH sensitivity five block linear polymers preparation method, its feature
It is：
The molfraction formula of reactant is as follows in step (1)：
The molfraction formula of reactant is as follows in step (2)：
4. it is according to claim 2 based on PDEAEMA pH sensitivity five block linear polymers preparation method, its feature
It is：The room temperature reaction time described in step (1) is 12~36h；The condition of the heating response described in step (2) is heating
To 60~90 DEG C, 5~10h is reacted；The time of the isothermal reaction described in step (2) is 5~10h.
5. it is according to claim 2 based on PDEAEMA pH sensitivity five block linear polymers preparation method, its feature
It is：Catalyst described in step (1) is DMAP；Acid binding agent described in step (1) is triethylamine；Step
(1) bromating agent described in is 2- bromine isobutyl acylbromides；Catalyst described in step (2) is CuBr2；Described in step (2)
Reducing agent is Sn (Oct)2。
6. it is according to claim 1 based on PDEAEMA pH sensitivity five block linear polymers in field of medicaments should
With.
7. a kind of micellar system, it is characterised in that linear based on five blocks of sensitivity of the pH based on PDEAEMA described in claim 1
Polymer is obtained.
8. micellar system according to claim 7, it is characterised in that by by described in claim 1 based on PDEAEMA
PH sensitivity five block linear polymers be dissolved in solvent in obtain.
9. application of the micellar system according to any one of claim 7~8 in field of medicaments.
10. application of the micellar system according to any one of claim 7~8 in the containing of hydrophobic drug, its feature
It is to specifically include following steps：The five block linear polymers of sensitivity of the pH based on PDEAEMA described in claim 1 are dissolved in
Micellar system is obtained in organic solvent, hydrophobic drug is dissolved in identical organic solvent, then mixed with above-mentioned micellar system,
Stirring, dialysis, filtering is dried, and obtains containing hydrophobic drug micellar system.
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| Pentablock copolymers of poly(ethylene glycol), poly((2-dimethyl amino) ethyl methacrylate) and poly(2-hydroxyethyl methacrylate) from consecutive atom transfer radical polymerizations for non-viral gene delivery;Fu-Jian Xu, et al.;《Biomaterials》;20080418;第29卷(第20期);第3023-3033页 * |
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