CN104231155B - Cholesterol modifies amphipathic pH response brush copolymer and preparation and micella thereof - Google Patents
Cholesterol modifies amphipathic pH response brush copolymer and preparation and micella thereof Download PDFInfo
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- CN104231155B CN104231155B CN201410410038.6A CN201410410038A CN104231155B CN 104231155 B CN104231155 B CN 104231155B CN 201410410038 A CN201410410038 A CN 201410410038A CN 104231155 B CN104231155 B CN 104231155B
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Abstract
The invention belongs to biological medical polymer polymeric material preparing technical field, disclose a kind of cholesterol and modify amphipathic pH response brush copolymer and preparation method and the micellar system prepared based on it.This copolymer has a structure as shown in following formula I:
Description
Technical field
The invention belongs to biological medical polymer polymeric material preparing technical field, solid particularly to a kind of courage
Alcohol modifies amphipathic pH response brush copolymer and preparation method and the micellar system prepared based on it.
Background technology
Cancer is the disease of a kind of serious threat human health, in current existing cancer treatment method, and effect
Best surely belongs to chemical medicinal treatment.But chemotherapeutics is also a double-edged sword, and on the one hand it is controlled in cancer
Having irreplaceable effect in treatment, on the other hand it is while killing cancer cell, also can be to normal cell
Produce the biggest toxic and side effect.There is poorly water-soluble, biological utilisation in existing anticancer class chemicals major part
Spend low, in body the defect such as non-specific distribution.
In order to overcome drawbacks described above, while making cancer therapy drug reach effective dose, reduce normal tissue
Toxic and side effect, novel delivery system (Drug Delivery System, DDS) gradually grows up.
Since the sixties in 20th century finds that liposome can be used in medicine conveying, medicine is carried by nanometer technology
System creates tremendous influence, and the organic/inorganic nano material as pharmaceutical carrier gushes like the mushrooms after rain
Reveal to come.Nano-medicament carrier based on polymer is a study hotspot of delivery system.This type of medicine
Thing carrier and simple medicine can effectively extend medicine circulation timei in blood compared with carrying, and increase
The water solubility of dewatering medicament, reduces bad immune response, thus reaches to reduce dosage and reduce poison secondary
The purpose of effect.A kind of preferably nano-medicament carrier should be provided simultaneously with following character: simply prepares and carries
Pure method, higher drugloading rate and envelop rate, excellent biological degradability, low toxicity or nontoxic, particle diameter is suitable
And circulation time in vivo is longer etc..
Copolymer is to be polymerized, by two or more monomers, the polymer formed.The structure of copolymer has to
Few two kinds of construction units, connect with chemical bond between construction unit, and this and two kinds of polymer are with mechanically or physically
The blend being mixed to form is different.There is the copolymer of difference in functionality in chemical industry, biology, medicine, building etc.
Field suffers from being widely applied.
PH responsive polymer micella, as the newtype drug delivery vehicles of a kind of great development prospect, has all
The most excellent performance characteristics, if realize the target administration of cancer therapy drug, strengthen bioavilability, reduce right
The toxic and side effect etc. of normal structure.But this novel intelligent nano-medicament carrier is also faced with a series of difficulty
Topic, in terms of carrier material technology of preparing: how to obtain the carrier micella of size tunable and narrowly distributing；How
Improve the drug encapsulation amount etc. of carrier；Carrier micelle controlled release properties aspect: how to ensure that carrier micelle is in difference
Structural stability under environmental condition, the most precisely responds small pH change and realizes the position control of medicine and release
Put.
Biomolecule cholesterol is due to the hydrophobicity sterol backbone of its height, good biological degradability and biology
Compatibility so that one of its optimal selection becoming many water-soluble polymer hydrophobically modifieds.Cholesterol is hydrophobic
The self-assembly of modified amphipathic nature polyalcohol ties up to the application aspect of medicine delivery the most also to be come by more
The most concerns.Prior art has carried out more research for an above-mentioned difficult problem, carries out multiple polymers monomer
Combination copolymerization is to obtain multi-functional copolymer, but still is difficult to obtain preferable effect.
Summary of the invention
In order to overcome the shortcoming of above-mentioned prior art with not enough, the primary and foremost purpose of the present invention is to provide a kind of courage
Sterol-modified amphipathic pH responds brush copolymer.The structure of this copolymer is: hydrophilic block metering system
Acid hydroxyl ethyl ester, hydrophobic cholesterol and pH respond block methacrylic acid N, and N-lignocaine ethyl ester is randomly altogether
Poly-, and then obtain amphipathic pH response brush copolymerization with the combination of hydrophilic block polyethylene glycol methacrylate-styrene polymer
Thing.
Another object of the present invention is that providing a kind of above-mentioned cholesterol to modify amphipathic pH responds brush copolymer
Preparation method.The method is by using electro transfer activating and regenerating Transfer Radical Polymerization
(ARGET ATRP), is little initiator molecule with 2-isobutyl ethyl bromide (EBriB), causes first successively
Base hydroxy-ethyl acrylate (HEMA), pH respond monomer methacrylic acid N, N-lignocaine ethyl ester (DEA)
And polyethylene glycol methacrylate-styrene polymer (PEGMA) carries out radical polymerization, obtain poly-(hydroxyethyl methacrylate
Ethyl ester-co-methacrylic acid N, N-lignocaine ethyl ester) the poly-polyethylene glycol methacrylate-styrene polymer of-b-
(P(HEMA-co-DEA)-b-PPEGMA).Then, it is polymerized with this with cholesterol formyl chloride (Chol)
Thing carries out alcoholysis reaction and prepares the cholesterol poly-(hydroxyethyl methacrylate-co-methacrylic acid of modification
N, N-lignocaine ethyl ester) the poly-polyethylene glycol methacrylate-styrene polymer of-b-
(Chol-P(HEMA-co-DEA)-b-PPEGMA)。
Still a further object of the present invention is that providing a kind of modifies amphipathic pH response brush altogether based on above-mentioned cholesterol
The micellar system of polymers.Modify amphipathic pH based on above-mentioned cholesterol and respond brush copolymer, use dialysis
Method prepare internal layer be cholesterol modify hydrophobic segment, intermediate layer be pH response segment, shell for parent
The nano grade polymer micella of water segment, it is achieved the loading to slightly water-soluble cancer therapy drug.Weak at tumor tissues
Under acid condition (pH 5～6), pH responds group generation protonation and shows as hydrophily, micella
Dissociate, thus realize being loaded quick, the controlled release of medicine.This cholesterol is modified amphipathic
PH response brush copolymer structure on the premise of maintaining high drug load, can improve the pH response of micella
Sensitivity, more effective Drug controlled release, improves the therapeutic efficiency of medicine.
It is still another object of the present invention to provide above-mentioned cholesterol modification amphipathic pH response brush copolymer to exist
Load the application in poorly water soluble drugs.
It is still another object of the present invention to provide the application in loading poorly water soluble drugs of the above-mentioned micellar system.
The purpose of the present invention is realized by following proposal:
A kind of cholesterol is modified amphipathic pH and is responded brush copolymer, has a structure as shown in following formula I:
Wherein, x=10～36, y=15～40, z=8～30.
The cholesterol of the present invention modifies amphipathic pH response brush copolymer
Chol-P(HEMA-co-DEA)-b-PPEGMA。
Preferably, the number-average molecular weight of the cholesterol modification amphipathic pH response brush copolymer of the present invention is
11891～40460g/mol.
The invention provides a kind of described cholesterol and modify the preparation side of amphipathic pH response brush copolymer
Method, including step in detail below:
(1) by catalyst CuBr2, monomers hydroxy ethyl ester (HEMA) and methacrylic acid
N, N-lignocaine ethyl ester (DEA), ligand 1,1,4,7,10,10-hexamethyl triethylene tetramine (HMTETA)
It is dissolved in solvent, adds reducing agent Sn (Oct)2, after stirring, it is added dropwise over initiator 2-isobutyl bromide
Ethyl ester (EBriB), after adding thermal response, adds monomer polyethylene glycol methacrylate-styrene polymer (PEGMA), continues
Continuous isothermal reaction, obtains amphipathic pH response brush copolymer (P (HEMA-co-DEA)-b-PPEGMA)；
(2) prepare cholesterol and modify amphipathic pH response brush copolymer: obtained by step (1)
Amphipathic pH response brush copolymer is dissolved in anhydrous methylene chloride (DCM), is sequentially added into solubilized under ice bath
Solution, in the triethylamine (TEA) of anhydrous methylene chloride and cholesteryl chloroformate, reacts 2～4h, and room temperature continues
Continuous reaction 24～48h, obtains cholesterol and modifies amphipathic pH response brush copolymer.
In described step (1), the molfraction formula of reactant is as follows:
In described step (2), the molfraction formula of reactant is as follows:
P (HEMA-co-DEA)-b-PPEGMA 1 part
Chol 9～15 parts
TEA 9～15 parts
The thermal response that adds described in step (1) refers to react at 60～90 DEG C 5～7h.Described isothermal reaction
Time be preferably 24～48h.
Preferably, after step (1) has been reacted, reaction system cooled down, purify, be dried, obtain pure
Product after change.After described purifying refers to product is dissolved in oxolane, cross neutral alumina chromatography
Post, oxolane elutes, and removes catalyst, then by eluent concentrated by rotary evaporation, adds 0 DEG C of 10 times of volumes
N-hexane precipitates, and is filtrated to get product after purification.
Solvent in step (1) is preferably toluene.
After step (2) has been reacted, preferably application system filtered, precipitate, be dried, obtain after purification
Product.Described filtration is used for removing insoluble triethylamine hydrochloride.Described precipitation refers to after filtration
Filtrate rotation is steamed, and the 0 DEG C of n-hexane adding 10 times of volumes precipitates.
Preferably, above-mentioned reaction is all carried out under inert gas shielding and anhydrous condition.
Its structural formula of described HEMA and DEA is as follows:
The above-mentioned cholesterol of the present invention is modified amphipathic pH response brush copolymer and be can be applicable to load slightly water-soluble
In medicine.
A kind of carrier micelle system modifying amphipathic pH response brush copolymer based on above-mentioned cholesterol, logical
Cross and cholesterol is modified amphipathic pH response brush copolymer be dissolved in dicyandiamide solution and obtaining.
Above-mentioned micellar system can be applicable to load in poorly water soluble drugs.
Above-mentioned micellar system is applied to load in poorly water soluble drugs and comprises the following steps: by poorly water soluble drugs
It is dissolved in organic solvent and overnight processing, cholesterol is modified amphipathic pH response brush copolymer simultaneously and is dissolved in
In same organic solvent, thing to be polymerized obtains micellar system after being completely dissolved, by this micellar system and shipwreck
Soluble drug solution mixes, stirring 4～6h under room temperature, deionized water dialysis 24h, filtration, freeze-drying,
Obtain loading poorly water soluble drugs micellar system.
Described poorly water soluble drugs refers to the solubility medicine less than or equal to 1g in 1L water.
Described organic solvent is preferably the one of which in dimethyl sulfoxide (DMSO) and dimethylformamide.
The medicine that described loading poorly water soluble drugs micellar system can control to load is at normal structure (pH
7.4) place slowly discharges, and realizes quick, controlled release at tumour cell solutions of weak acidity (pH 5～6) place.
Above-mentioned based on cholesterol of the present invention modification amphipathic pH response brush copolymer, use dialysis to prepare
Obtain internal layer be the hydrophobic segment modified of cholesterol, intermediate layer be pH response segment, shell be hydrophilic segment
Nano grade polymer micella, it is achieved the loading to slightly water-soluble cancer therapy drug.At tumor tissues faintly acid bar
Under part (pH 5～6), pH responds group generation protonation and shows as hydrophily, and micella solves
From, thus realize being loaded quick, the controlled release of medicine.This cholesterol modifies amphipathic pH response
Brush copolymer structure on the premise of maintaining high drug load, can improve the pH response sensitivity of micella,
More effectively Drug controlled release, improves the therapeutic efficiency of medicine.
The mechanism of the present invention is:
The present invention utilizes hydrophilic block hydroxyethyl methacrylate, hydrophobic cholesterol and pH to respond block methyl
Acrylic acid N, N-lignocaine ethyl ester random copolymerization, so with hydrophilic block polyethylene glycol methacrylate-styrene polymer
Combination obtains amphipathic pH and responds brush copolymer, is dissolved in solvent, and obtaining internal layer is that cholesterol is repaiied
The hydrophobic segment of decorations, intermediate layer be pH response segment, shell be the nano grade polymer micella of hydrophilic segment,
The PPEGMA of micella outer layer hydrophilic has the advantages such as nontoxic, non-immunogenicity and no antigen, is increasing
While adding micella stability, extend micella circulation timei in blood；Hydrophobic cholesterol kernel can increase
By force the bag of insoluble medicine is carried performance；The PDEA in intermediate layer shows as hydrophobicity when pH 7.4, can be with
Cholesterol collectively constitutes the hydrophobic inner core of micella, and this is possible not only to prevent burst drug release, can strengthen glue simultaneously
The stability of bundle kernel；Under the conditions of tumor tissues faintly acid (pH 6.0), side chain in PDEA block
Tertiary amine groups generation protonation and show as hydrophily, micella starts to occur a certain degree of swelling；Medicine carrying
If micella enters in endosome and the lysosome with lower pH, PDEA will protonate completely, now glue
Bundle degree of swelling becomes big, and micella starts appearance and assembles behavior of even dissociating, thus by the insoluble drug release of package-contained
In tumour cell.By the ratio of each block in regulation polymer, can with the rate of release of regulating medicine,
Meet the release request of different pharmaceutical.
The present invention, relative to prior art, has such advantages as and beneficial effect:
(1) hydrophobic grouping and pH are responded group and be designed to random copolymerization form, both can maintain higher
Drugloading rate, can effectively regulate again the pH response range of its self-assembled micelle, makes micella not only can respond rapidly to
The change of environmental pH, and can effectively alleviate prominent releasing, Drug controlled release.
(2) the bag loading capability of poorly water soluble drugs is strengthened by the copolymer after hydrophobic cholesterol is modified.
(3) cholesterol that the present invention prepares is modified amphipathic pH and is responded brush copolymer, it is easy to adjust
Control the ratio of each block, be applied to preparation and load poorly water soluble drugs micellar system, different pharmaceutical can be met
Release request.
Accompanying drawing explanation
Fig. 1 is the GPC elution curve of P (HEMA-co-DEA)-b-PPEGMA in embodiment 1.
Fig. 2 is the cholesterol modifying polymer front and back of embodiment 11H H NMR spectroscopy, wherein, (A) is
P (HEMA-co-DEA)-b-PPEGMA's1H H NMR spectroscopy, solvent is d-CDCl3；(B) it is
Chol-P (HEMA-co-DEA)-b-PPEGMA's1H H NMR spectroscopy, solvent is d-CDCl3。
Fig. 3 is the GPC elution curve of Chol-P (HEMA-co-DEA)-b-PPEGMA in embodiment 1.
Fig. 4 is that the critical micelle concentration of Chol-P (HEMA-co-DEA)-b-PPEGMA in embodiment 5 is surveyed
Examination curve.
Fig. 5 is DOX/DMSO calibration curve in embodiment 6.
Fig. 6 is the current potential of Chol-P (HEMA-co-DEA)-b-PPEGMA self-assembled micelle in embodiment 7
Titration curve.
Fig. 7 be the particle diameter of Chol-P (HEMA-co-DEA)-b-PPEGMA self-assembled micelle in embodiment 7,
Zeta current potential and the relation of pH.
Fig. 8 is the In-vitro release curves carrying adriamycin micella in embodiment 8.
Detailed description of the invention
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but the embodiment party of the present invention
Formula is not limited to this.
Embodiment 1: cholesterol is modified amphipathic pH and responded brush copolymer
The preparation of Chol-P (HEMA-co-DEA)-b-PPEGMA
(1) preparation of amphipathic pH response brush copolymer P (HEMA-co-DEA)-b-PPEGMA:
Stirrer and copper bromide (22.3mg, 1.0mmol) is loaded, with anti-mouth rubber in 50mL is dried eggplant-shape bottle
Leather plug is sealed, and vacuumizes-lead to argon gas three times.Successively with syringe by solvent toluene (20mL), monomer HEMA
(1.30g, 10mmol), DEA (6.48g, 35mmol), part HMTETA (0.31mL, 1.2
Mmol) adding in bottle, stirring 10min makes catalyst complexes be formed.Subsequently by reducing agent Sn (Oct)2
(0.40g, 1.0mmol) is dissolved in 2mL toluene and adds in reaction bulb.Syringe is used after stirring 5min
Quickly it is added dropwise over initiator EBriB (0.15mL, 1.08mmol), 60 DEG C of oil baths are stirred reaction 6
After h, add monomer PEGMA (4.75g, 10mmol), continue reaction 30h.Reactant liquor is cooled to
Room temperature, adds 50mL THF and stirring makes it dissolve, and is then filtered to remove with neutral alumina pillar and urges
Agent CuBr2(making eluant, eluent with THF).The eluent obtained is slowly added into ten times amount (bodies after concentrating
Long-pending than) cold n-hexane in precipitate.Precipitated product 35 DEG C, be vacuum dried 48h under 35mbar.Synthesis
Reaction equation is shown in formula (1).Utilize GPC to measure its molecular weight, and carry out nmr analysis, see Fig. 1 and Tu
(A) in 2.Productivity is 98%, Mn=12481, Mw/Mn=1.60.
(2) cholesterol is modified amphipathic pH and is responded brush copolymer Chol-P (HEMA-
Co-DEA) preparation of-b-PPEGMA: step (1) is prepared
P (HEMA-co-DEA)-b-PPEGMA (5.68g, 0.25mmol) is placed in 100mL eggplant shaped reaction device
In and with anti-mouth rubber stopper seal, vacuumize-lead to argon gas three times, holding reactor in be argon gas atmosphere.Add
Enter 20mL anhydrous methylene chloride, reactant is fully dissolved.Respectively by triethylamine (2.1mL, 15
Mmol), Chol (3.86g, 10mmol) be dissolved in 5mL anhydrous methylene chloride, condition of ice bath
Lower syringe is added sequentially in reactor, reacts 2h.Transfer room temperature condition to, continue reaction 30h.
After having reacted, it is filtered to remove insoluble triethylamine hydrochloride, precipitates three times with cold n-hexane, precipitation
Vacuum drying (35mbar, 35 DEG C) 48h, obtains light yellow product, and synthetic reaction formula is shown in formula (2).
Utilize GPC to measure its molecular weight, and carry out nmr analysis, see (B) in Fig. 2 and Fig. 3.Mn=16341,
Mw/Mn=1.54.
Embodiment 2: cholesterol is modified amphipathic pH and responded brush copolymer
The preparation of Chol-P (HEMA-co-DEA)-b-PPEGMA
(1) preparation of amphipathic pH response brush copolymer P (HEMA-co-DEA)-b-PPEGMA:
In 50mL is dried eggplant-shape bottle, loads stirrer and copper bromide (26.8mg, 0.8mmol), uses anti-mouth
Rubber stopper seals, and vacuumizes-lead to argon gas three times.Successively with syringe by solvent toluene (20mL), list
Body HEMA (1.30g, 10mmol), DEA (6.475g, 40mmol), part HMTETA
(0.31mL, 1.2mmol) adds in bottle, and stirring 10min makes catalyst complexes be formed.Subsequently will
Reducing agent Sn (Oct)2(0.486g, 1.2mmol) is dissolved in 2mL toluene and adds in reaction bulb.Stirring
Initiator EBriB (0.17mL, 1.2mmol) quickly it is added dropwise over syringe, at 70 DEG C after 5min
In oil bath after stirring reaction 5h, add monomer PEGMA (14.25g, 30mmol), continue reaction
24h.Reactant liquor is cooled to room temperature, adds 50mL THF and stirring makes it dissolve, then by neutrality
Aluminum oxide pillar Filtration of catalyst CuBr2(making eluant, eluent with THF).The reactant liquor obtained concentrates
After be slowly added in the cold n-hexane of ten times amount (volume ratio) precipitation.Product is at 35 DEG C, 35mbar
Lower vacuum drying 48h.Productivity is 89%, Mn=20634, Mw/Mn=1.51.
(2) cholesterol is modified amphipathic pH and is responded brush copolymer Chol-P (HEMA-co-
DEA) preparation of-b-PPEGMA: P (HEMA-co-DEA)-b-PPEGMA prepared by step (1)
(5.27g, 0.25mmol) is placed in 100mL eggplant shaped reaction device and seals with anti-mouth rubber stopper, takes out true
Empty-logical argon gas three times, keeping in reactor is argon gas atmosphere.Add 20mL anhydrous methylene chloride, will be anti-
Thing is answered fully to dissolve.Respectively by triethylamine (1.25mL, 9mmol), Chol (5.79g, 15mmol)
It is dissolved in 5mL anhydrous methylene chloride, is added sequentially in reactor, instead with syringe under condition of ice bath
Answer 2h.Transfer room temperature condition to, continue reaction 24h.After having reacted, it is filtered to remove insoluble three second
Amine hydrochlorate, precipitates three times with cold n-hexane, is vacuum dried (35mbar, 35 DEG C) 48h, obtains light
Yellow product.Mn=24494, Mw/Mn=1.47.
Embodiment 3: cholesterol is modified amphipathic pH and responded brush copolymer
The preparation of Chol-P (HEMA-co-DEA)-b-PPEGMA
(1) preparation of amphipathic pH response brush copolymer P (HEMA-co-DEA)-b-PPEGMA:
Stirrer and copper bromide (26.8mg, 0.12mmol) is loaded, with anti-in 50mL is dried eggplant-shape bottle
Mouth rubber stopper sealing, vacuumizes-leads to argon gas three times.Successively with syringe by solvent toluene (20mL),
Monomer HEMA (4.68g, 36mmol), DEA (2.775g, 15mmol), part HMTETA
(0.31mL, 1.2mmol) adds in bottle, and stirring 10min makes catalyst complexes be formed.Subsequently will
Reducing agent Sn (Oct)2(0.486g, 1.2mmol) is dissolved in 2mL toluene and adds in reaction bulb.Stirring
Initiator EBriB (0.17mL, 1.2mmol) quickly it is added dropwise over syringe, at 90 DEG C after 5min
In oil bath after stirring reaction 7h, add monomer PEGMA (14.25g, 30mmol), continue reaction
48h.Reactant liquor is cooled to room temperature, adds 50mL THF and stirring makes it dissolve, then by neutrality
Aluminum oxide pillar Filtration of catalyst CuBr2(making eluant, eluent with THF).The reactant liquor obtained concentrates
After be slowly added in the cold n-hexane of ten times amount (volume ratio) precipitation.Product is at 35 DEG C, 35mbar
Lower vacuum drying 48h.Productivity is 96%, Mn=21061, Mw/Mn=1.48.
(2) cholesterol is modified amphipathic pH and is responded brush copolymer Chol-P (HEMA-co-
DEA) preparation of-b-PPEGMA: P (HEMA-co-DEA)-b-PPEGMA prepared by step (1)
(4.3g, 0.25mmol) is placed in 100mL eggplant shaped reaction device and seals with anti-mouth rubber stopper, takes out true
Empty-logical argon gas three times, keeping in reactor is argon gas atmosphere.Add 20mL anhydrous methylene chloride, will
Reactant fully dissolves.Respectively by triethylamine (2.1mL, 15mmol), Chol (3.47g, 9mmol)
It is dissolved in 5mL anhydrous methylene chloride, is added sequentially in reactor, instead with syringe under condition of ice bath
Answer 4h.Transfer room temperature condition to, continue reaction 48h.After having reacted, it is filtered to remove insoluble three second
Amine hydrochlorate, precipitates three times with cold n-hexane, is vacuum dried (35mbar, 35 DEG C) 48h, obtains light
Yellow product.Mn=28781, Mw/Mn=1.46.
Embodiment 4: cholesterol is modified amphipathic pH and responded brush copolymer
The preparation of Chol-P (HEMA-co-DEA)-b-PPEGMA
(1) preparation of amphipathic pH response brush copolymer P (HEMA-co-DEA)-b-PPEGMA:
Stirrer and copper bromide (17.84mg, 0.08mmol) is loaded, with anti-in 50mL is dried eggplant-shape bottle
Mouth rubber stopper sealing, vacuumizes-leads to argon gas three times.Successively with syringe by solvent toluene (20mL),
Monomer HEMA (1.3g, 10mmol), DEA (2.775g, 15mmol), part HMTETA
(0.2mL, 0.8mmol) adds in bottle, and stirring 10min makes catalyst complexes be formed.Subsequently will
Reducing agent Sn (Oct)2(0.324g, 10.8mmol) is dissolved in 2mL toluene and adds in reaction bulb.Stir
Quickly it is added dropwise over initiator EBriB (0.12mL, 0.8mmol) with syringe after mixing 5min,
In 80 DEG C of oil baths after stirring reaction 6h, add monomer PEGMA (14.25g, 30mmol), continue
Reaction 36h.Reactant liquor is cooled to room temperature, adds 50mL THF and stirring makes it dissolve, then use
Neutral alumina pillar Filtration of catalyst CuBr2(making eluant, eluent with THF).The reactant liquor obtained
Precipitation it is slowly added in the cold n-hexane of ten times amount (volume ratio) after concentration.Product is at 35 DEG C, 35mbar
Lower vacuum drying 48h.Productivity is 96%, Mn=17187, Mw/Mn=1.43.
(2) cholesterol is modified amphipathic pH and is responded brush copolymer Chol-P (HEMA-co-
DEA) preparation of-b-PPEGMA: P (HEMA-co-DEA)-b-PPEGMA prepared by step (1)
(4.3g, 0.25mmol) is placed in 100mL eggplant shaped reaction device and seals with anti-mouth rubber stopper, takes out true
Empty-logical argon gas three times, keeping in reactor is argon gas atmosphere.Add 20mL anhydrous methylene chloride, will be anti-
Thing is answered fully to dissolve.Respectively by triethylamine (1.25mL, 9mmol), Chol (3.47g, 9mmol)
It is dissolved in 5mL anhydrous methylene chloride, is added sequentially in reactor, instead with syringe under condition of ice bath
Answer 3h.Transfer room temperature condition to, continue reaction 36h.After having reacted, it is filtered to remove insoluble three second
Amine hydrochlorate, precipitates three times with cold n-hexane, is vacuum dried (35mbar, 35 DEG C) 48h, obtains light
Yellow product.Mn=21047, Mw/Mn=1.52.
Embodiment 5: cholesterol modifies critical micelle concentration CMC of amphipathic pH response brush copolymer
Value
The cholesterol utilizing fluorescence probe method testing example 1 to prepare modifies amphipathic pH response brush
The critical micelle concentration of shape copolymer Chol-P (HEMA-co-DEA)-b-PPEGMA.
(1) configuration of pyrene solution: with acetone solution pyrene (Sigma-Aldrich), configuration concentration is 12
×10-5The pyrene solution of M.
(2) configuration of sample solution: weigh 5mg Chol-P (HEMA-co-DEA)-b-PPEGMA molten
In 10mL acetone, quickly being joined by solution in 50mL deionized water, 24h is to vapor away in stirring
Acetone, obtains the polymer mother liquor that concentration is 0.1mg/mL, is diluted to a series of concentration (concentration range
It is 0.0001～0.1mg/mL).Take 20 10mL volumetric flasks, be separately added into 0.1mL step (1)
The pyrene solution of configuration, is then respectively adding the copolymer solution constant volume of above-mentioned variable concentrations, shakes up, obtain
Sample solution.In sample solution, the concentration of pyrene is 12 × 10-7M。
(3) fluorescence spectrum test: with 373nm for launching wavelength, scan sample 300～350nm
The fluorescence excitation spectrum of solution.Take the intensity rate (I that wavelength is 339nm and 336nm339/I336) right
Polymer concentration logarithm is mapped, as it is shown in figure 5, the abscissa corresponding to curve catastrophe point is lg (CMC).
The critical micell recording Chol-P (the HEMA-co-DEA)-b-PPEGMA that embodiment 1 prepares is dense
Degree is 6.92mg/L.
Embodiment 6: cholesterol modifies the preparation of amphipathic pH response brush copolymer carrier micelle
Using dialysis preparation to carry the polymer micelle of adriamycin (DOX), concrete grammar is as follows: weigh
10mg DOX HCl is dissolved in 20mL DMSO, adds the TEA of 2 times of molar equivalents, is stirred overnight
Obtain DOX alkali.Take what 40mg embodiment 1 prepared
Chol-P (HEMA-co-DEA)-b-PPEGMA is dissolved in 20mL DMSO, and thing to be polymerized is the most molten
After solution obtains micellar system, in the DOX solution prepared before micellar system is joined, continue
After stirring 6h, proceeding to bag filter and dialyse, every 2h changes a deionized water, every 6h after 12h
Changing once, dialyse 24h altogether.After having dialysed, dislysate is filtered with 0.45 μm filter membrane,
After filtered fluid freeze-drying, obtain red powder solid and be DOX carrier micelle.
The preparation method of blank micella is identical with this.
Use the drugloading rate LC and envelop rate EE of ultraviolet visible spectrometry detection DOX.Concrete steps are such as
Under: accurately weigh 1mg and be lyophilized carrier micelle and be substantially soluble in 10mL DMSO, survey it at 480nm
The UV absorption intensity at place, by DOX/DMSO calibration curve, is shown in Fig. 5, is calculated DOX dense
Degree, calculates drugloading rate and envelop rate respectively.Calculated drugloading rate is 10.8%, and envelop rate is 48.5%.
The method for drafting of DOX/DMSO calibration curve is as follows: prepare the DOX/DMSO of a series of concentration
Standard liquid.With DMSO as blank, the DOX/DMSO solution of prepared series concentration is existed
Trap is measured at 480nm wavelength.After the absorbance of the blank DMSO solution of deduction, with the suction recorded
Luminosity A to concentration C mapping go forward side by side line linearity return, obtain if the DOX of Fig. 5 is in DMSO solution
Concentration and the canonical plotting of absorbance.
Dynamic light scattering method (DLS) is used to measure blank micella and the particle diameter of carrier micelle, distribution and zeta
Current potential.The particle diameter D of blank micellahBeing 0.29 for 155.6nm, PDI, zeta current potential is 21.1mV.
The particle diameter D of carrier micellehBeing 0.31 for 178.5nm, PDI, zeta current potential is 19.7mV.
Embodiment 7: cholesterol is modified the pH of amphipathic pH response brush copolymer micella and responded self assembly
Behavioral study
The pK of PDEA block in embodiment 1 polymer is determined initially with potentiometric titrationbValue, specifically
Step is as follows: Chol-P (the HEMA-co-DEA)-b-PPEGMA of 50mg embodiment 1 preparation is abundant
It is dissolved in 20mL acetone, is added rapidly under fast stirring in 50mL deionized water, under room temperature
24h is to vapor away acetone in stirring, obtains the polymer micelle solution that ultimate density is 1mg/mL.With
NaOH or the pH value of HCl solution (0.1M) regulation micellar solution, stirring balance a period of time to pH
Stable, read each pH value, as shown in Figure 6.Use micella under the different pH value of DLS test simultaneously
Particle diameter and zeta current potential, as shown in Figure 7.It will be appreciated from fig. 6 that the pK of polymer micellebValue is 7.2.By
Fig. 7 understands, and along with pH reduces, particle diameter and zeta current potential are gradually increased, and finally tend to definite value.
Embodiment 8: the release in vitro of carrier micelle
DOX In-vitro release curves under different pH uses medicament dissolution instrument test to obtain, concrete steps
As follows: to use method similarly to Example 5, prepare DOX medicine carrying glue with the polymer of embodiment 2
Bundle, represents with PMs-1.The DOX carrier micelle of embodiment 5 preparation represents with PMs-2.Claim respectively
Take 5mg DOX carrier micelle PMs-1 and PMs-2 to be scattered in 5mL PBS, buffer solution
PH value is respectively 6.0 and 7.4.Above-mentioned solution is placed in bag filter, proceeds to 40mL same pH
In buffer solution, it is placed in medicament dissolution instrument, at 37 DEG C, under 110rpm rotating speed, carries out release in vitro.Regularly
Sampling 4mL carries out ultra-violet analysis, and adds 4mL fresh buffer simultaneously.Use ultraviolet spectrophotometry
Measure DOX concentration in different time release liquid, draw In-vitro release curves, as shown in Figure 8.
As shown in Figure 8, DOX carrier micelle PMs-1 with PMs-2 shows consistent release in vitro row
For.Under normal structure environment (pH 7.4), the rate of release of DOX is very slow, and the accumulation of 24h is released
High-volume less than 35%, rate of release subsequently tends to be steady substantially.And in the subacidity of tumour cell
Under the conditions of (pH 6.0), the rate of release of DOX is substantially accelerated, and the cumulative release amount of 24h reaches 80%
Above.And, the PMs-1 drug release rate than PMs-2 is the most faster.
Above-described embodiment is the present invention preferably embodiment, but embodiments of the present invention are not by above-mentioned reality
Execute the restriction of example, the change made under other any Spirit Essence without departing from the present invention and principle, modification,
Substitute, combine, simplify, all should be the substitute mode of equivalence, within being included in protection scope of the present invention.
Claims (9)
1. a cholesterol is modified amphipathic pH and is responded brush copolymer, it is characterised in that have such as following formula
Structure shown in I:
Wherein, x=10～36, y=15～40, z=8～30.
Cholesterol the most according to claim 1 is modified amphipathic pH and is responded brush copolymer, and it is special
Levy and be that its number-average molecular weight is 11891～40460g/mol.
3. the cholesterol described in an any one of claim 1～2 is modified amphipathic pH and is responded brush copolymerization
The preparation method of thing, it is characterised in that include step in detail below:
(1) prepare amphipathic pH and respond brush copolymer: by catalyst CuBr2, monomer methacrylic acid
Hydroxyl ethyl ester and methacrylic acid N, N-lignocaine ethyl ester, ligand 1,1,4,7,10,10-hexamethyl triethylene tetramine
It is dissolved in solvent, adds reducing agent Sn (Oct)2, after stirring, it is added dropwise over initiator 2-bromine isobutyl
Acetoacetic ester, adds thermal response, adds monomer polyethylene glycol methacrylate-styrene polymer, continues isothermal reaction, has reacted
Cheng Hou, obtains amphipathic pH and responds brush copolymer；
(2) prepare cholesterol and modify amphipathic pH response brush copolymer: obtained by step (1)
Amphipathic pH response brush copolymer be dissolved under anhydrous methylene chloride, ice bath be sequentially added into be dissolved in anhydrous
The triethylamine of dichloromethane and cholesteryl chloroformate, react 2～4h, and room temperature continues reaction 24～48h,
Obtain cholesterol and modify amphipathic pH response brush copolymer.
Cholesterol the most according to claim 3 modifies the preparation of amphipathic pH response brush copolymer
Method, it is characterised in that:
In step (1), the molfraction formula of each reactant is as follows:
In step (2), the molfraction formula of each reactant is as follows:
Amphipathic pH responds brush copolymer 1 part
Cholesteryl chloroformate 9～15 parts
Triethylamine 9～15 parts.
Cholesterol the most according to claim 3 modifies the preparation of amphipathic pH response brush copolymer
Method, it is characterised in that: the thermal response that adds described in step (1) refers to react at 60～90 DEG C 5～7h；
The time of described isothermal reaction is 24～48h.
6. a micellar system, it is characterised in that repair based on the cholesterol described in any one of claim 1～2
Adorn amphipathic pH response brush copolymer to prepare.
The micellar system the most according to claim 6 application in loading poorly water soluble drugs.
The micellar system the most according to claim 7 application in loading poorly water soluble drugs, it is special
Levy and be to comprise the following steps: poorly water soluble drugs is dissolved in organic solvent and overnight processes, cholesterol is repaiied
Adoring amphipathic pH response brush copolymer to be dissolved in same organic solvent, thing to be polymerized obtains after being completely dissolved
To micellar system, this micellar system is mixed with poorly water soluble drugs solution, stir 4～6h under room temperature, go
Ionized water dialysis 24h, filtration, freeze-drying, obtain loading poorly water soluble drugs micellar system.
9. modify amphipathic pH according to the cholesterol described in any one of claim 1～2 and respond brush copolymerization
Thing application in loading poorly water soluble drugs.
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| CN104856952B (en) * | 2015-05-13 | 2017-08-04 | 江南大学 | A kind of preparation of poly- (L glutamic acid) b polyethylene glycol medicament-carried nano micelles |
| CN106565962B (en) * | 2016-10-24 | 2020-05-19 | 东莞理工学院 | Cholesterol grafted pH-responsive triblock amphiphilic copolymer and preparation method and application thereof |
| CN106883360B (en) * | 2017-02-14 | 2020-01-17 | 上海大学 | Functionalized stimulus-responsive polymer and preparation method thereof |
| CN106957401B (en) * | 2017-04-01 | 2020-03-17 | 苏州大学 | Cholesterol group anchored poly (ethylene glycol) methacrylate polymer and synthesis method and application method thereof |
| CN110041475B (en) * | 2019-04-11 | 2021-02-12 | 中国科学技术大学 | Amphiphilic block copolymer, shell-crosslinked micelle thereof, preparation method and application |
| CN110229295A (en) * | 2019-05-30 | 2019-09-13 | 湘潭大学 | A kind of copolymer and preparation method thereof with amphipathic block |
| CN112851854B (en) * | 2021-01-11 | 2022-06-28 | 上海市第十人民医院 | Random copolymer based on natural cholesterol, preparation method and application thereof |
| CN113698556A (en) * | 2021-08-18 | 2021-11-26 | 新乡医学院 | Preparation method of redox-sensitive targeted drug-loaded polymer |
| CN113881002B (en) * | 2021-11-17 | 2023-08-22 | 湘潭大学 | Amphiphilic block copolymer and preparation thereof, and preparation and application of nano micelle |
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