CN104650307A - PH-sensitive pentablock linear polymer based on PDEAEMA and micelle - Google Patents
PH-sensitive pentablock linear polymer based on PDEAEMA and micelle Download PDFInfo
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Abstract
The invention belongs to the technical field of high polymer materials for biological medicines and discloses a pH-sensitive pentablock linear polymer based on PDEAEMA, a preparation method of the polymer and a micelle system obtained based on the polymer and the application of the micelle system. The polymer has the structure shown in a formula described in the specification, wherein x is equal to 45, y is equal to 19-37, and z is equal to 15-19. A symmetrical pentablock linear polymer is designed and synthesized, the drug encapsulation amount of the polymer can be obviously improved, the pH response range of the self-assembly micelle can be well regulated by virtue of the intermediate layer PDEAEMA so that the drug loading micelle can rapidly respond the change of the pH value in a tumor environment, the drug release can be effectively controlled, and burst release of the drug is avoided. A thick pH responsive intermediate layer is formed in the micelle formed by the polymer, the encapsulation capacity is improved, the drug release performance is improved, the particle size of the micelle after drug release is reduced, the drug discharge is facilitated, and the polymer can be widely applied to the field of medicines.
Description
Technical field
The invention belongs to biological medicine technical field of polymer materials, particularly responsive five block linear polymers of a kind of pH based on PDEAEMA and preparation method thereof and based on its micellar system obtained and application.
Background technology
Cancer (malignant tumour) is owing to controlling the not normal and disease that causes of growth and proliferation of cell mechanism, but cancer cells except grow out of control except, also locally can invade the healthy tissues of human body, and other positions can be transferred to very easily.According to up-to-date " report of world's cancer " display, in newly-increased 1,400 ten thousand cases of cancers in the whole world, have 3,070,000 to be Chinese, account for 21.8%, newly-increased cases of cancer occupies first of the whole world.Death toll ratio accounts for 26.9% of global death toll especially.Visible China is for the treatment still comparatively backwardness of cancer, and the research thus for cancer therapy is also especially aobvious important.Treatment mainly operative treatment, radiotherapy and chemotherapy three kinds of methods of current cancer, wherein chemical medicinal treatment remains and uses maximum methods.But still there are some problems needing to solve in chemical medicinal treatment, the utilization ratio of such as dewatering medicament is low, genomic medicine is easy to degraded, problem that the toxic side effect of medicine normal tissue cell is large etc.
In order to solve the problem, in the last few years, novel intelligent response (pH response, temperature response, enzyme response) drug delivery nano-carrier (polymer nanoparticle, liposomes, hydrogel, polymer micelle) arouses great concern.These nano anti-cancer pharmaceutical carriers, by changing the bio distribution of cancer therapy drug, improve the efficiency of killing cancer cells and the toxic side effect reduced human normal cell.These anti-cancer medicament carriers can also prolong drug cycling time in blood, improves the meltage of dewatering medicament in human body, and then decrease the dosage of administration, thus improve the utilization ratio of medicine and reduce toxic side effect.
Desirable anti-cancer medicament carrier can in normal tissue and blood the stablizing of holding structure, substantially do not discharge medicine, then can locate release medicine fast and effectively at tumor tissues position.And should have preparation method simply, higher drug loading, size be suitable for, the character such as degradation property is in vivo excellent.
Block polymer refers to a kind of particular polymer be prepared from that to be connected together by the polymer segment of two or more different in kind.The character of multiple polymers can be combined, obtain the polymer materials with multiple high-performance.These functional polymer materials can be widely used in the every field such as biological medicine, chemical industry, building according to its different properties, are that theoretical investigation or practical application all have a very big significance.
PH responsive polymer micella mainly refers to that polymer micelle is comparatively responsive to the change of the pH value of surrounding environment, and corresponding change can occur the physico-chemical property of micella, is a kind of newtype drug delivery vehicles material having application prospect.But this material is also still faced with series of problems, such as micella is easily disintegrated by dilution after entering blood, the particle diameter of micella is excessive to be easy to again reticuloendothelial system in body (RES) and to engulf or by tissue resorption such as liver spleens, and the size distribution of micella is wider etc.The self-assembly of polymer micelle is a Thermodynamically stable process, namely only its concentration higher than micella CMC value in micellar structure keep stable, the current concentration that is diluted to easily is disintegrated lower than below the CMC of micella, thus there is prominent releasing in the medicine in carrier micelle, so just limits the application of polymer micelle in human body.The polymer micelle that five block polymers are formed by self-assembly, owing to having longer hydrophobic block, make polymer micelle have less CMC value to maintain the stable of micella, there is compacter hydrophobic inner core simultaneously and make the particle diameter of micella not too large and the application prospect that has influence on it.
Amino containing functional group uncle in PDEAEMA, its pKb value is 6.9.The amino easy trap proton in sour environment of uncle and positively charged, apparent is hydrophilic radical, and micellar structure fluffs loose, and the medicine be embedded in micella is discharged in environment fast; In neutral or basic conditions, hardly protonation occurs, apparent is hydrophobic grouping, keeps the compact form of micella, also can effectively stop drug release in micella in environment while the pharmaceutical pack carrying capacity improving micella.GU [Journal of Polymer Science:PartA:Polymer Chemistry, 47 (2009): 3142-3153] the pH sensitive property of PDEAEMA is utilized, synthesize bi-block copolymer PPEGMEA-g-PDEAEMA, under different pH value, micella is prepared by self-assembly, and have studied pH response self-assembly behavior, the particle diameter of micella, the transmittance of micellar solution are all along with there is significantly change in the change of pH.The particle diameter of micella can obviously increase in the basic conditions, and the transmittance of solution significantly declines, and its change procedure is subject to the impact of protonation.Yang [ActaBiomaterials, 9 (2013): 7679-7690] 4/6 equal arm star polymkeric substance has been synthesized, its arm is the PCL-b-PDEAEMA-b-PPEGMA of three blocks, and for the preparation of amphipathic nano-micelle, carry hydrophobic anticancer drugs, doxorubicin (DOX) for bag, carrier micelle has lower micelle-forming concentration and sensitive pH sensitive property.Under pH7.4, drug release is slow, and when pH value drops to 5.0, the rate of release of medicine has had obvious quickening, and it is substantially nontoxic to demonstrate material by cytotoxicity experiment.Determan [Polymer, 46 (2005): 6933-6946] method of ATRP is adopted, five block polymers that various block length is controlled have been synthesized centered by Pluronic F127, reaction conditions is gentle, productive rate is high, and polymers exhibit has gone out good pH sensitivity and temperature sensitive performance.
Patent application CN200910024899.X discloses a kind of method adopting ATRP method block polymer synthesis, and the length of block is easy to regulation and control, and the mechanism of reaction is ripe, and various reaction conditions gentleness is feasible.Patent application CN20131014153.1 and CN201310141556.8 all discloses bag and carries the preparation method of hydrophobic anticancer drug target polymer micella, due to the difference of tumor tissue environment and healthy tissues environmental pH, realizes the set point control release of medicine.
Summary of the invention
In order to overcome above-mentioned prior art micella easily disintegrate and the shortcoming that is easily trapped with not enough, primary and foremost purpose of the present invention is to provide the responsive five block linear polymers of a kind of pH based on PDEAEMA.The structure of this polymkeric substance comprises: hydrophilic block polyoxyethylene glycol, and pH responds block methacrylic acid N, N-lignocaine ethyl ester, hydrophobic block methyl methacrylate, and combination obtains the responsive five block linear polymers of pH.
Another object of the present invention is to provide a kind of above-mentioned pH based on PDEAEMA preparation method of responsive five block linear polymers.The method, by adopting acyl bromination reaction, with 2-bromine isobutyl acylbromide for bromide reagent, is carried out acyl bromination reaction with macromole polyoxyethylene glycol, is prepared macromole evocating agent (Br-PEG-Br); Adopt transfer transport activating and regenerating Transfer Radical Polymerization (ARGET ATRP) again, with bromination polyoxyethylene glycol (Br-PEG-Br) for macromole evocating agent, cause pH successively and respond monomer methacrylic acid N, N-lignocaine ethyl ester (DEAEMA) and methyl methacrylate (MMA) obtain responsive five block linear polymers (polyoxyethylene glycol-[b-polymethyl acrylic acid N, the N-lignocaine ethyl ester-b-polymethylmethacrylate] of pH
2).
Still a further object of the present invention is to provide responsive five application of block linear polymer in field of medicaments of the above-mentioned pH based on PDEAEMA.
Still a further object of the present invention is to provide a kind of micellar system based on the responsive five block linear polymers of the above-mentioned pH based on PDEAEMA.Adopting dialysis method to prepare internal layer is hydrophobic block, and middle layer is that pH responds block, and shell is the nano grade polymer micella of hydrophilic block, realizes carrying the bag of hydrophobic drug.Under tumor tissues solutions of weak acidity (pH 5 ~ 6), the pH of this micellar system responds group generation protonation and shows as wetting ability, and micella dissociates, thus realizes by the quick controllable release of bag medicine carrying thing.The micella of the responsive five block linear polymer structures of pH of the present invention under the prerequisite keeping micella stable, can improve bag carrying capacity and the pH response sensitivity of medicine, more effective Drug controlled release, improves therapeutic efficiency, reduce side effect.
Still a further object of the present invention is to provide the application of above-mentioned micellar system in field of medicaments.
Object of the present invention is realized by following proposal:
The responsive five block linear polymers of pH based on PDEAEMA, have structure shown in following formula:
Wherein, x=45, y=19 ~ 37, z=15 ~ 19.
The structural formula of the responsive five block linear polymers of the pH based on PDEAEMA of the present invention is PEG-[b-PDEAEMA-b-PMMA]
2, number-average molecular weight is 10131 ~ 14536g/mol.
Present invention also offers the preparation method of the responsive five block linear polymers of a kind of above-mentioned pH based on PDEAEMA, comprise following concrete steps:
(1) prepare macromole evocating agent: catalyzer, polyoxyethylene glycol (PEG) are dissolved in solvent, then add acid binding agent and stir, dropwise add bromizating agent, react under room temperature, obtain macromole evocating agent (Br-PEG-Br);
(2) the responsive five block linear polymers of pH are prepared: the macromole evocating agent (Br-PEG-Br) step (1) prepared, pH respond monomer methacrylic acid N, N-lignocaine ethyl ester (DEAEMA), catalyzer, ligand 1,1,4,7,10,10-hexamethyl triethylene tetramine (HMTETA) is dissolved in solvent, add reductive agent again, after reacting by heating, add monomers methyl methacrylate (MMA) again, isothermal reaction, obtain the responsive five block linear polymers (PEG-[b-PDEAEMA-b-PMMA] of pH
2).
In step (1), the molfraction formula of reactant is as follows:
In step (2), the molfraction formula of reactant is as follows:
The room temperature reaction time described in step (1) is preferably 12 ~ 36h.
Bromizating agent described in step (1) preferably drips in 1h, more preferably drips in ice-water bath.
Solvent described in step (1) is preferably methylene dichloride.
Catalyzer described in step (1) is preferably DMAP (DMAP).
Acid binding agent described in step (1) can be the conventional any acid binding agent in this area, is preferably triethylamine (TEA).
Bromizating agent described in step (1) can be the conventional any bromizating agent in this area, is preferably 2-bromine isobutyl acylbromide (BMPB).
Catalyzer described in step (2) is preferably CuBr
2.
Reductive agent described in step (2) is preferably Sn (Oct)
2.
After having reacted in step (1), preferably use 5wt%NaHSO respectively
4, Na
2sO
4, deionized water extractive reaction liquid 3 times, obtain the macromole evocating agent after purifying.More preferably filter after dried overnight in anhydrous magnesium sulfate after having extracted, the most of solvent in rotary evaporation removing filtrate, then uses 10 times of volume cold diethyl ether solution precipitations, centrifugal, and with cold ether three times, then vacuum-drying.By dried sample dissolution in dehydrated alcohol, spend the night recrystallization, more namely obtain product B r-PEG-Br macromole evocating agent white solid after vacuum-drying.
The condition optimization of the reacting by heating described in step (2), for being heated to 60 ~ 90 DEG C, reacts 5 ~ 10h.The time of described isothermal reaction is preferably 5 ~ 10h.
Solvent described in step (2) is preferably toluene.
Preferably, after step (2) described isothermal reaction completes, by reaction system cooling, purifying, drying, obtain the product after purifying.After described purifying refers to that reaction product is dissolved in tetrahydrofuran (THF), cross neutral alumina chromatography column, tetrahydrofuran (THF) wash-out, removing catalyzer, then by elutriant concentrated by rotary evaporation, the 0 DEG C of normal hexane adding 10 times of volumes precipitates, and filters and obtains product after purification.
Preferably, above-mentioned reaction is all preferred carries out under atmosphere of inert gases, anhydrous condition.
The responsive five block linear polymers of pH based on PDEAEMA of the present invention can be applicable in field of medicaments, are specially adapted to bag and carry in hydrophobic drug.
Based on a micellar system for the responsive five block linear polymers of the above-mentioned pH based on PDEAEMA, obtain by responsive for the above-mentioned pH based on PDEAEMA five block linear polymers are dissolved in solvent.
Present invention also offers the application of above-mentioned micellar system in field of medicaments, be specially adapted to the Bao Zaizhong of hydrophobic drug, specifically comprise the following steps: the responsive five block linear polymers of pH based on PDEAEMA are dissolved in organic solvent and obtain micellar system, hydrophobic drug is dissolved in identical organic solvent, then mixes with above-mentioned micellar system, stir, dialysis, filter, dry, obtain bag and carry hydrophobic drug micellar system.
Described stirring is preferably 4 ~ 6h.
Described dialysis preferably to be dialysed 48h with deionized water, more preferably, within first 12 hours in dialysis procedure every 2 hours, changes a water, within 12 ~ 24 hours every 4 hours, changes a water, within 24 ~ 48 hours every 6 hours, change a water.
Described hydrophobic drug refers to that solubleness is less than or equal to the medicine of 1g in 1L water.
Described organic solvent is preferably the wherein one in dimethyl sulfoxide (DMSO) and dimethyl formamide.
The bag that the present invention prepares carries hydrophobic drug micellar system can control the medicine of loading at healthy tissues (pH 7.4) place's slow releasing, realize quick, controllable release at tumour cell solutions of weak acidity (pH 5 ~ 6) place, can be widely used in field of medicaments.
The responsive five block linear polymers of pH based on PDEAEMA of the present invention, adopting dialysis method to prepare internal layer is hydrophobic block, and middle layer is that pH responds block, and shell is the nano grade polymer micella of hydrophilic block, realizes carrying the bag of hydrophobic drug.Under tumor tissues solutions of weak acidity (pH 5 ~ 6), pH responds group generation protonation and shows as wetting ability, and micella dissociates, thus realizes by the quick controllable release of bag medicine carrying thing.The micella that this pH responds five block linear polymer structures under the prerequisite keeping micella stable, can improve the bag carrying capacity of medicine, pH response sensitivity, more effective Drug controlled release, improves therapeutic efficiency, reduce side effect.
Mechanism of the present invention is:
The present invention utilizes hydrophilic block polyoxyethylene glycol, pH to respond block methacrylic acid N, N-lignocaine ethyl ester and the combination of hydrophobic block methyl methacrylate obtain amphipathic pH and respond five block linear polymers, be dissolved in solvent, obtaining internal layer is hydrophobic block, middle layer is that pH responds block, shell is the nano grade polymer micella of hydrophilic block, the PEG of micella outer layer hydrophilic has nontoxic, the advantage such as non-immunogenicity and no antigen, while increase micella stability, extend micella cycling time in blood; Hydrophobic PMMA kernel can strengthen and carries performance to the bag of insoluble medicine; The PDEAEMA in middle layer shows as hydrophobicity when pH 7.4, and can jointly form the hydrophobic inner core of micella with cholesterol, this not only can prevent burst drug release, can strengthen the stability of micelle inner core simultaneously; Under tumor tissues slightly acidic (pH 6.0) condition, in PDEAEMA block side chain tertiary amine groups generation protonation and show as wetting ability, it is swelling that micella starts to occur to a certain degree; If carrier micelle enters in the endosome and lysosome with lower pH, PDEAEMA will be completely protonated, and now micella degree of swelling becomes large, and micella starts to occur assembling behavior of even dissociating, thus by the drug release of package-contained in tumour cell.By the ratio of block each in telomerized polymer, can the rate of release of regulating medicine, meet the release request of different pharmaceutical.
The present invention, relative to prior art, has following advantage and beneficial effect:
(1) design and synthesis of the present invention obtains symmetry five block linear polymer structure, the pharmaceutical pack carrying capacity of polymkeric substance can be improved significantly, the pH responding range of self-assembled micelle can better be regulated by middle layer PDEAEMA, allowing carrier micelle reach can under tumor environment, the change of quick response pH value, effectively can control the release of medicine, avoid the prominent of medicine to release.
(2) the micelle formation thicker pH that Inventive polymers is formed responds middle layer, while improving bag loading capability, improves medicine-releasing performance, reduces the particle diameter of micella after drug release, is conducive to the discharge of carrier.
(3) the responsive five block linear polymers of the pH based on PDEAEMA for preparing of the present invention, the length of each block is all easy to regulation and control, can carry different medicines according to applied environment different choice bag.
Accompanying drawing explanation
Fig. 1 is the GPC elution curve of the Br-PEG-Br in embodiment 1, and moving phase is tetrahydrofuran (THF).
Fig. 2 is Br-PEG-Br's in embodiment 1
1h NMR composes, and solvent is deuterochloroform.
Fig. 3 is the PEG-[b-PDEAEMA-b-PMMA] in embodiment 2
2gPC elution curve, moving phase is tetrahydrofuran (THF).
Fig. 4 is the PEG-[b-PDEAEMA-b-PMMA] in embodiment 2
2's
1h NMR composes, and solvent is deuterochloroform.
Fig. 5 is the PEG-[b-PDEAEMA-b-PMMA] in embodiment 5
2micelle-forming concentration test curve.
Fig. 6 is the PEG-[b-PDEAEMA-b-PMMA] in embodiment 6
2the potentiometric titration curve of self-assembled micelle.
Fig. 7 is the PEG-[b-PDEAEMA-b-PMMA] in embodiment 7
2the relation of the particle diameter of self-assembled micelle, zeta current potential and pH.
Fig. 8 is the PEG-[b-PDEAEMA-b-PMMA] in embodiment 8
2the transmission electron microscope picture of micella.
Fig. 9 is the PEG-[b-PDEAEMA-b-PMMA] in embodiment 9
2carry the In-vitro release curves of Zorubicin micella.
Figure 10 is PEG-[b-PDEAEMA-b-PMMA] in embodiment 10
2the vitro cytotoxicity of blank micella.
Figure 11 is PEG-[b-PDEAEMA-b-PMMA] in embodiment 10
2carry the vitro cytotoxicity of Zorubicin micella.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.
Embodiment 1: the synthesis of macromole evocating agent Br-PEG-Br
Building-up reactions formula is as follows:
The synthesis of macromole evocating agent Br-PEG-Br: load magnetic stir bar in 250mL round bottom reaction flask, with anti-mouth soft rubber ball sealing, vacuumizes-Tong argon gas three times, is placed in frozen water.0.4g (3.28mmol) DMAP (Shanghai covalent chemical) and 2.0mL (14.0mmol) TEA is dissolved in 20mL methylene dichloride, solution is joined in reaction flask by syringe.6.6g (3.30mmol) PEG (Sigma-Aldrich) is dissolved in 80mL methylene dichloride, by syringe, PEG solution is injected reaction flask.In reaction flask, dropwise drip 1.6mL (6.60mmol) BMPB (J & K, lark prestige) with syringe, added in one hour, react 2 hours at keeping 0 DEG C, remove ice bath, continue at room temperature to react 24 hours.React rear and used 5wt%NaHSO respectively
4, Na
2sO
4extract 3 times, and then with distilled water wash 3 times, be placed in anhydrous magnesium sulfate dried overnight.Then remove most solvent by rotary evaporation, then precipitate in cold diethyl ether concentrated solution slowly being instilled 10 times of volumes, centrifugal, the centrifugal solid obtained 35 DEG C, vacuum-drying 48h obtains Br-PEG-Br white solid under 35mbar.Utilize GPC to measure its molecular weight, and carry out nmr analysis, see Fig. 1 and Fig. 2.Productive rate is 85%, M
n=2073, M
w/ M
n=1.19.
Embodiment 2: the responsive five block linear polymer PEG-[b-PDEAEMA-b-PMMA] of pH
2synthesis
Building-up reactions formula is as follows:
Magnetic stir bar, 26.8mg (0.12mmol) cupric bromide and 2.2g (1mmol) macromole evocating agent Br-PEG-Br is loaded in the dry eggplant-shape bottle of 50mL, with anti-mouth soft rubber ball sealing, vacuumize-Tong argon gas three times, inject toluene 20mL, monomer DEAEMA (4.02mL with syringe successively, 20mmol) (J & K, lark prestige), part HMTETA (0.31ml, 1.2mmol) (J & K, lark prestige) add in bottle, stir and catalyst complexes was formed in 10 minutes.Subsequently by 0.486g (1.2mmol) reductive agent Sn (Oct)
2be dissolved in 2mL toluene and add in reaction flask.Then after reaction flask being transferred to 60 DEG C of reaction 7h, add 1.86mL (17.5mmol) monomer M MA (J & K, lark prestige), continue reaction 12h.Reaction solution is cooled to room temperature, add 50mL THF and stir make it dissolve, then use neutral alumina pillar Filtration of catalyst CuBr
2(making eluent with THF).Slowly join in the cold normal hexane of ten times amount (volume ratio) after the elutriant obtained is concentrated and precipitate.Precipitated product is at 35 DEG C, vacuum-drying 48h under 35mbar.Utilize GPC to measure its molecular weight, and carry out nmr analysis, see Fig. 3 and Fig. 4.Productive rate is 95%, Mn=10089, Mw/Mn=1.33.
The responsive five block linear polymer PEG-[b-PDEAEMA-b-PMMA] of embodiment 3:pH
2synthesis
Magnetic stir bar, 17.9mg (0.08mmol) cupric bromide and 1.76g (0.8mmol) macromole evocating agent Br-PEG-Br is loaded in the dry eggplant-shape bottle of 50mL, with anti-mouth soft rubber ball sealing, vacuumize-Tong argon gas three times, inject toluene 20mL, monomer DEAEMA (5.02mL with syringe successively, 25mmol), part HMTETA (0.21ml, 0.8mmol) add in bottle, stir and catalyst complexes was formed in 10 minutes.Subsequently by 0.324g (0.8mmol) reductive agent Sn (Oct)
2be dissolved in 2mL toluene and add in reaction flask.Then after reaction flask being transferred to 60 DEG C of reaction 7h, add 2.12mL (20mmol) monomer M MA, continue reaction 12h.Reaction solution is cooled to room temperature, add 50mL THF and stir make it dissolve, then use neutral alumina pillar Filtration of catalyst CuBr
2(making eluent with THF).Slowly join in the cold normal hexane of ten times amount (volume ratio) after the elutriant obtained is concentrated and precipitate.Precipitated product is at 35 DEG C, vacuum-drying 48h under 35mbar, and productive rate is 95%, Mn=12153, Mw/Mn=1.33.
Embodiment 4: based on the responsive five block linear polymer PEG-[b-PDEAEMA-b-PMMA] of pH
2synthesis
Magnetic stir bar, 22.3mg (0.10mmol) cupric bromide and 2.64g (1.2mmol) macromole evocating agent Br-PEG-Br is loaded in the dry eggplant-shape bottle of 50mL, with anti-mouth soft rubber ball sealing, vacuumize-Tong argon gas three times, inject toluene 20mL, monomer DEAEMA (8.04mL with syringe successively, 40mmol), part HMTETA (0.26ml, 1.0mmol) add in bottle, stir and catalyst complexes was formed in 10 minutes.Subsequently by 0.405g (1.0mmol) reductive agent Sn (Oct)
2be dissolved in 2mL toluene and add in reaction flask.Then after reaction flask being transferred to 60 DEG C of reaction 7h, add 1.59mL (15mmol) monomer M MA, continue reaction 12h.Reaction solution is cooled to room temperature, add 50mL THF and stir make it dissolve, then use neutral alumina pillar Filtration of catalyst CuBr
2(making eluent with THF).Slowly join in the cold normal hexane of ten times amount (volume ratio) after the elutriant obtained is concentrated and precipitate.Precipitated product is at 35 DEG C, vacuum-drying 48h under 35mbar, and productive rate is 95%, Mn=14536, Mw/Mn=1.39.
Embodiment 5: measure PEG-[b-PDEAEMA-b-PMMA]
2cMC value.
The pH utilizing fluorescence probe method testing example 2 to prepare responds five block linear polymer PEG-[b-PDEAEMA-b-PMMA]
2micelle-forming concentration.
(1) configuration of pyrene solution: use acetone solution pyrene, configuration concentration is 12 × 10
-5the pyrene solution of M.
(2) configuration of sample solution: take 8mg PEG-[b-PDEAEMA-b-PMMA]
2be dissolved in 10mL acetone, solution joined in 80mL deionized water, stir 24h to vapor away acetone, obtain the polymer mother liquor that concentration is 0.1mg/mL, be diluted to a series of concentration (concentration range is 0.0001 ~ 0.1mg/mL).Get 20 10mL volumetric flasks, add the pyrene solution that 0.1mL step (1) configures respectively, then add the copolymer solution constant volume of above-mentioned different concns respectively, shake up, obtain sample solution.In sample solution, the concentration of pyrene is 12 × 10
-7m.
(3) fluorescence spectrum test: take 373nm as emission wavelength, at the fluorescence excitation spectrum of 300 ~ 350nm scanning samples solution.Get the intensity rate (I that wavelength is 339nm and 336nm
339/ I
336) to the mapping of polymer concentration logarithm, as shown in Figure 5, the X-coordinate corresponding to curve catastrophe point is lg (CMC).Record the PEG-[b-PDEAEMA-b-PMMA] that embodiment 2 prepares
2micelle-forming concentration be 2.80mg/L.
Embodiment 6: potentiometric determination PEG-[b-PDEAEMA-b-PMMA]
2pKb interval.
PEG-[b-PDEAEMA-b-PMMA] prepared by 30mg embodiment 2
2fully being dissolved in 30mL deionized water, making polymer dissolution be the polymers soln of concentration 1mg/mL by adding a little HCl solution (0.1M) and ultrasonic method.Regulate the pH value of micellar solution to be about 3 with NaOH or HCl solution (0.1M), stir balance for some time and stablize to pH, select NaOH (0.1M) as titrating solution, dropwise add in polymers soln, read each pH value, as shown in Figure 6.The pKb interval of polymers soln is 6.5 ~ 7.4.
Embodiment 7:PEG-[b-PDEAEMA-b-PMMA]
2the pH of micellar system responds self-assembly behavior
PEG-[b-PDEAEMA-b-PMMA] prepared by 5mg embodiment 2
2fully be dissolved in 20mL acetone, join in 50mL deionized water under fast stirring fast, stirred at ambient temperature 24h, to vapor away acetone, obtains the polymer micelle solution that ultimate density is 0.1mg/mL.Regulate the pH value of micellar solution with NaOH or HCl solution (0.1M), stir balance for some time and stablize to pH, read pH value now.Adopt DLS to test particle diameter and the zeta current potential of micella under different pH value, as shown in Figure 7 simultaneously.From Fig. 7 (a), along with pH reduces, particle diameter reduces gradually, and is about 7 reach minimum in pH value, is that 7 to 5 particle diameters slightly increase subsequently, can reduces to some extent when pH value is less than 5 in pH value.From Fig. 7 (b), along with the reduction of pH value, zeta current potential increases gradually, tends towards stability when pH value is 7, and pH value continues to be decreased to when being less than 5, and zeta current potential has reduction slightly.
Embodiment 8:PEG-[b-PDEAEMA-b-PMMA]
2the morphology characterization of carrier micelle and blank micella
PEG-[b-PDEAEMA-b-PMMA] prepared by preparation embodiment 2
2the blank micella of polymkeric substance and carrier micelle, and characterize pattern.Dialysis method is adopted to prepare carrier micelle.Accurately take 10mg Zorubicin (DOX) to be dissolved in 20mL DMSO, add the TEA 20 μ L of 2 times of molar weights, stirring is spent the night.Take 20mg PEG-[b-PDEAEMA-b-PMMA] simultaneously
2be dissolved in 20mL DMSO.4 ~ 6h will be stirred under both mixed room temperatures, then (within first 12 hours every 2 hours, a water is changed with the deionized water 48h that dialyses, within 12 ~ 24 hours every 4 hours, change a water, within 24 ~ 48 hours every 6 hours, change a water), filter with 0.45 μm of filtering head again, lyophilize, obtains DOX carrier micelle powder.Take 40mgPEG-[b-PDEAEMA-b-PMMA]
2be dissolved in 40mL DMSO.Stirred at ambient temperature 4 ~ 6h, then to dialyse 48h (change a water in first 12 hours every 2 hours, change a water in 12 ~ 24 hours every 4 hours, change a water in 24 ~ 48 hours every 6 hours) with deionized water, obtains PEG-[b-PDEAEMA-b-PMMA]
2blank micella, adopts its pattern of tem observation to be spherical, sees Fig. 8.
Embodiment 9:PEG-[b-PDEAEMA-b-PMMA]
2the release in vitro of carrier micelle
The responsive five block polymer PEG-[b-PDEAEMA-b-PMMA] of pH based on PDEAEMA
2the release in vitro performance of carrier micelle adopts medicament dissolution instrument and purple instrument to measure.Concrete steps are as follows: accurately take 5mg DOX carrier micelle (preparing in embodiment 8) respectively and be dispersed in 5mL damping fluid (pH 7.4, pH 5.0), then dialysis tubing is placed in, proceed in the damping fluid of 45mL same pH and be placed in medicament dissolution instrument at 37 DEG C, release in vitro is carried out under 110rpm rotating speed, the timing damping fluid got outside 4mL bag is analyzed, and adds 4mL fresh buffer.By DOX concentration in determined by ultraviolet spectrophotometry different time release liquid, draw its In-vitro release curves, as shown in Figure 9.
As shown in Figure 9, DOX carrier micelle is under healthy tissues environment (pH 7.4), and slowly, the cumulative release amount of 24 hours is less than 20%, and rate of release subsequently also tends to be steady, and after 96 hours, cumulative release amount is less than 40% for the rate of release of DOX.And under the faint acidic conditions of tumour cell (pH 5.0), the rate of release of DOX obviously accelerates, the cumulative release amount of 24 hours reaches 60%, and after 96 hours cumulative release amount more than 80%.
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(preparing in embodiment 8) vitro cytotoxicity of carrier micelle and blank micella.Get 96 hole flat bottomed tissue culture plates, 200 μ L cell culture mediums (DMEM) will be added in surrounding orifice plate respectively as blank group.Middle Zhong Mei hole, 60 holes is with 1 × 10
4cell concn inoculation Hela cell (being purchased from ATCC, USA) of cells/well (200 μ L), wherein 96 orifice plates in contrast, are placed into 37 DEG C, saturated humidity, 5%CO by the 2nd row
224h is cultivated in incubator.Subsequently the blank of free Zorubicin, freeze-drying or carrier micelle cell culture medium are diluted to different polymer concentrations (blank micella 1 ~ 400mg/L) or drug level (free Zorubicin or year Zorubicin micella, 0.1 ~ 20mg/L).Remove in 96 orifice plates from the 2nd row to the 11st row institute porose cell culture medium after, the 2nd arrange in add fresh developing medium, in contrast.From the 3rd row to the 10th row, in all holes, add the sample solution of 200 μ L respectively, the sample of each concentration joins in 6 holes and carries out repetition.
After the cultivation of 24h or 48h, siphon away all containing the supernatant liquor in the hole of cell, add the PBS rinse cell of 200 μ L, then siphon away PBS.From the 2nd row to the 11st row, respectively to the developing medium of the MTT solution and 180 μ L that add 20 μ L in each hole, then 96 orifice plates are positioned in incubator and cultivate 4h.Siphon away unreduced MTT solution and developing medium subsequently.Each hole PBS of 200 μ L washes one time, and siphons away PBS.The DMSO adding 200 μ L in each hole dissolves MTT crystallization.Whole 96 orifice plates are placed in 37 DEG C of shaking tables the 10min that vibrates, and then utilize microplate reader to measure the absorbancy in each hole, 490nm place, and then calculate cell survival rate.
The results are shown in Figure 10 and Figure 11, Figure 10 is the toxicity data of blank micella, and from figure, blank micella is substantially nontoxic to Hela cell, and under the ultrahigh concentration of 400mg/L, the survival rate of cell still can reach close to 90%.Figure 11 is the toxicity data of carrier micelle, as can be seen from the figure, when concentration is 20mg/L, the survival rate of cell is only about 60%, and and free Zorubicin have cytotoxicity closely, illustrate that Zorubicin is through solid support material Bao Zaihou, its antitumour activity does not significantly weaken.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (10)
1., based on the responsive five block linear polymers of pH of PDEAEMA, it is characterized in that having structure shown in following formula:
Wherein, x=45, y=19 ~ 37, z=15 ~ 19.
2. a preparation method for the responsive five block linear polymers of the pH based on PDEAEMA according to claim 1, is characterized in that comprising following concrete steps:
(1) prepare macromole evocating agent: catalyzer, polyoxyethylene glycol are dissolved in solvent, then add acid binding agent and stir, dropwise add bromizating agent, react under room temperature, obtain macromole evocating agent;
(2) the responsive five block linear polymers of pH are prepared: macromole evocating agent step (1) prepared, pH respond monomer methacrylic acid N, N-lignocaine ethyl ester, catalyzer, ligand 1, Isosorbide-5-Nitrae, 7,10,10-hexamethyl triethylene tetramine is dissolved in solvent, then adds reductive agent, after reacting by heating, add monomers methyl methacrylate again, isothermal reaction, obtain the responsive five block linear polymers of pH.
3. the preparation method of the responsive five block linear polymers of the pH based on PDEAEMA according to claim 2, is characterized in that:
In step (1), the molfraction formula of reactant is as follows:
In step (2), the molfraction formula of reactant is as follows:
4. the preparation method of the responsive five block linear polymers of the pH based on PDEAEMA according to claim 2, is characterized in that: the room temperature reaction time described in step (1) is 12 ~ 36h; The condition of the reacting by heating described in step (2), for being heated to 60 ~ 90 DEG C, reacts 5 ~ 10h; The time of the isothermal reaction described in step (2) is 5 ~ 10h.
5. the preparation method of the responsive five block linear polymers of the pH based on PDEAEMA according to claim 2, is characterized in that: the catalyzer described in step (1) is DMAP; Acid binding agent described in step (1) is triethylamine; Bromizating agent described in step (1) is 2-bromine isobutyl acylbromide; Catalyzer described in step (2) is CuBr
2; Reductive agent described in step (2) is Sn (Oct)
2.
6. responsive five application of block linear polymer in field of medicaments of the pH based on PDEAEMA according to claim 1.
7. a micellar system, is characterized in that obtaining based on the responsive five block linear polymers of the pH based on PDEAEMA according to claim 1.
8. micellar system according to claim 7, is characterized in that obtaining by being dissolved in solvent by responsive for the pH based on PDEAEMA according to claim 1 five block linear polymers.
9. the application of the micellar system according to any one of claim 7 ~ 8 in field of medicaments.
10. the micellar system according to any one of claim 7 ~ 8 is in the application of the Bao Zaizhong of hydrophobic drug, it is characterized in that specifically comprising the following steps: responsive for the pH based on PDEAEMA according to claim 1 five block linear polymers are dissolved in organic solvent and obtain micellar system, hydrophobic drug is dissolved in identical organic solvent, mix with above-mentioned micellar system again, stir, dialysis, filter, drying, obtains bag and carries hydrophobic drug micellar system.
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