CN101348570A - Preparation of star copolymer self-assembled nano micelle - Google Patents
Preparation of star copolymer self-assembled nano micelle Download PDFInfo
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Abstract
A preparation method for a radial copolymer self-assembly nanometer micelle comprises the following steps of: preparing carboxyl terminated polymethyl methacrylate; preparing a tri-arm polymer (NIPAAm-co-DMAEMA); taking the redox system formed by cerium-alcohol as an initiator, and carrying out the free radical copolymerization of the monomers of N-isopropyl acrylamide and N, N-dimethyl amine ethyl methyl metharcrylate; preparing a radical copolymer, dissolving the obtained radical amphiphilic copolymer into a filter bag, and putting the filter bag in distilled water for dialysis to obtain the copolymer micelle. The radical copolymer micelle which is used as a medicine carrier is capable of reaching the targeted release of the medicine in a pathological area through making use of external or in-vivo intrinsic environmental changes, thereby improving the curing efficiency of the medicine.
Description
Background technology
In the past ten years, amphipathic copolymer is not only in the basic science field, and at biomedicine field, particularly in the medicine carrier systems, attracted the numerous concern of people.In selective solvent, amphiphilic block copolymer can self-assembly forms the nanometer aggregate of variform, and especially the spherical polymer micella of being made up of hydrophobic kernel and hydrophilic shell has important use in medicament carrier system.Intrafascicular at the core-shell type polymer latex that is used for medicament carrier system; hydrophobic kernel can be used as a nano container and is used for loading hydrophobic medicine; hydrophilic shell then plays effective space protection and prevents intermicellar gathering, and also may modify hydrophilic outer shell in addition increases target or other functional group.The micellar phase that forms with traditional tensio-active agent relatively, polymer micelle has much higher thermodynamic stability.The low micelle-forming concentration (CMC) of polymer micelle is than traditional tensio-active agent glue (10
-3-10
-4Mol/L) bundle is low about 1000 times.Same, polymer micelle also has many advantages: simple and easy to do as the preparation method; Need not modify drug loading and can obtain higher entrapment; The risk of chronic accumulation is less and be easy to realize control to the drug release behavior etc. in vivo.Compare with the other medicines carrier that size of particles is bigger, since the size of polymer micelle in the aqueous solution less (＜200nm), not only can reduce RE and engulf the non-specific absorption of system (RES) it, can also utilize non-selective retention effect (EPR effect), thereby realize selectivity transfer function cancer therapy drug.As mentioned above, at present, polymer micelle is considered to one of extremely promising drug delivery system (DDSs).
The main purpose of pharmaceutical carrier is in order to control and regulate medicine distribution in vivo.Since in many pathology zone of different tissues or organ (as inflammation, tumour and wound etc.) temperature that all can show the zone raises (2-5 ℃) or pH descend (a 1-2.5 pH unit), and the technology that positions heating in the noumenal tumour zone is existing to be used, and the application of the pharmaceutical carrier that stimuli responsive (as temperature or environment pH etc.) excites will be the effective way that strengthens pathology zone pharmaceutical activity.Can prepare stimulation sensitive polymers micella at an easy rate by chemical design.Usually, this base polymer is that the segment by temperature or pH sensitivity is constituted, as PNIPAM (PNIPAAm), polyacrylic acid (PAA), polymethyl acrylic acid (PMAA), polymethyl acrylic acid dimethyl amido ethyl ester (PDMAEMA) and their multipolymer etc.
In the past few years, the investigator studies the temperature responsive polymer micella based on PNIPAAm.Yokoyama etc. have reported by the Poly of temperature response (NIPAAm-co-DMAAm) segment as hydrophilic block, biodegradable poly(lactic acid) (poly (D, L-lactide)), (poly (D, L-lactide-co-ε-caprolactone)) is as the copolymer micelle of hydrophobic segment for the multipolymer of polycaprolactone (poly (ε-caprolactone)) or lactic acid and caprolactone.These copolymer micelles can load anticarcinogen Zorubicin (doxorubicin), and have shown the drug release behavior of temperature response.Same, based on the amphiphilic block polymer PSt-b-PNIPAAm of PNIPAAm and PBMA-b-PNIPAAm synthetic bibliographical information is arranged also.This analog copolymer micella has demonstrated reversible dispersion/gathering behavior corresponding to the multiple temperature variation in the aqueous solution.The investigator wishes that these copolymer micelles not only can utilize the EPR effect selectivity of display space in vivo, and can strengthen the hold facility of polymer micelle in target area by the method for location heating.Yang etc. have synthesized poly (NIPAAm-co-DMAAm)-b-poly (D, the L-lactide-co-glycolide) segmented copolymer, and studied the temperature sensitivity of copolymer micelle and to the release behavior of Zorubicin of different compositions.Zhang etc. have reported that several have the self-assembling polymers micella based on PNIPAAm of unique texture, comprise a kind of star-like segmented copolymer
[27]The multipolymer of " Y " type.Because they have unique micellar structure, these polymkeric substance have all demonstrated higher entrapment efficiency and unique temperature response drug release behavior.Their research also shows, can effectively control the drug release kinetics characteristic of corresponding medicine carrier systems by selecting the suitable copolymers structure, as the encapsulation efficiency of medicine and release rate of drugs etc.
On the other hand, also be used for loading and sustained release based on the polymer micelle of the responsive segmented copolymer of the pH of PEG and butyl methacrylate, isobutyl acrylate, propyl methacrylate or butyl acrylate to medicine candesartan cilexetil (CDN).The group of Leroux has reported that these multipolymers can self-assembly form micella, and demonstrates the high carrying drug ratio of CDN and the drug release pattern of pH dependent form.The work group of Yang has reported the copolymer micelle based on di-block copolymer poly (2-ethyl-2-oxazoline)-b-poly (L-lactide).Their experimental result shows, the structure of (pH 7.4) polymer micelle can keep dewatering medicament DOX under common physiological condition, experience when being similar to intracellular pH and changing (being about pH 4 ~ 5 in endosome and the lysosome) when polymer micelle, can optionally discharge DOX.The research group of Bae has developed a kind of tumor-localizing polymer micelle of pH sensitivity, and this micella is formed by multipolymer poly (L-lacticacid)-b-poly (ethylene glycol)-b-poly (L-histidine) self-assembly.They infer that it is hydrophobic inner core that this polymer micelle can form with PLA and polyHis segment in the aqueous solution, be " flower " shape (flower-like) micella of shell with the PEG segment.The pH susceptibility of polymer micelle derives from when the slight acidifying of environment, polyHis segment generation ionization, the dissociating of caused micellar hydrophobic inner core.This mechanism just provides possibility for the small pH of polymer micelle experience changes (pH 7.2-6.5) trigger polymers micella release drug loading.
Recently, in order to improve the target station-keeping ability and the therapeutic efficiency of pharmaceutical carrier, people have prepared many pharmaceutical carriers with two stimuli responsives.People have synthesized a kind of cholesterol grafted multipolymer poly (NIPAAm-co-DMAAm-co-UA), and are used to seal the cancer therapy drug of water-soluble extreme difference, taxol (paclitaxel).Carrier micelle based on this cholesterol graftomer has demonstrated a kind of extremely useful pH relevant temperature response characteristic, and be significantly higher than under the environment of pH 7.4 at its drug release rate under the environment of pH 5.0, help cancer therapy drug and discharge in the location of tumor region.The research group of Zhang has developed based on the temperature of undecylenic acid and NIPAAm, pH double-bang firecracker and has answered multipolymer poly (UA-b-NIPAAm), and as pharmaceutical carrier dewatering medicament prednisone (prednisone acetate) is sealed.This medicament carrier system has shown sustained release behavior and unique pH response medicine release behavior of significant temperature response.The research group of Jiang has reported a kind of nanometer self-assembly particle based on copolymer p (NIPAAm-co-AA)-b-PCL.This particle has shown answers characteristic to the double-bang firecracker of temperature and pH sensitivity, and the scope of its response temperature and pH variation is fit to be applied to the carrier system of targeted anticancer medicine very much.Yet we notice, all have been to use the monomer (AA, MAor UA) that contains carbonyl as the pH sensitive group mostly, usually all in pH is lower than 7.4 environment, shows higher drug release rate, and are applied to the target location of tumour.In order to expand the range of application of pharmaceutical carrier in biological medicine that double-bang firecracker is answered, it is localized that exploitation can be carried out target to other pathology zone with different microenvironments, and novel double-bang firecracker is answered pharmaceutical carrier, also seems very important.
Summary of the invention
Purpose of the present invention is to provide a kind of preparation method of star copolymer self-assembled nano micelle, can form nanometer polymer micelle in the aqueous solution.
The preparation method of a kind of radial copolymer self-assembled nano micelle of the present invention comprises the steps:
A. prepare the polymethylmethacrylate that end group is a carboxyl: be chain-transfer agent with the thiohydracrylic acid, use the initiator Diisopropyl azodicarboxylate in organic solvent monomers methyl methacrylate, under the certain temperature, carry out Raolical polymerizable;
B. prepare three arm polymkeric substance tri-arm P (NIPAAm-co-DMAEMA): the redox system of forming with cerium-alcohol is an initiator, and with monomer N-N-isopropylacrylamide and N, N-dimethyl aminoethyl methyl methacrylate carries out radical copolymerization;
C. prepare radial copolymer: two kinds of polymkeric substance of above preparation are added N-hydroxy-succinamide and dicyclohexylcarbodiimide in solvent, the reaction certain hour, product precipitates in ether, filters final vacuum and is dried to constant weight;
D. the above-mentioned star amphipathic copolymer that makes is dissolved in the dialysis tubing, inserts in the distilled water and dialyse, obtain copolymer micelle.
The preparation method of above-mentioned radial copolymer self-assembled nano micelle, the described temperature of step a are between 50 ℃-80 ℃, and the reaction times is 5 hours-48 hours, and described organic solvent is a dioxane, methane amide, methylene dichloride, benzene, toluene any.
The preparation method of above-mentioned radial copolymer self-assembled nano micelle, among the step b, described initiator system is made up of Pehanorm and cerous nitrate, their mass ratio is 1: 5-1: between 100, the mass ratio of initiator system and polymerization single polymerization monomer is controlled at 1: 100-1: between 10000, temperature of reaction is 2-12 hour, and temperature of reaction is between 50 ℃-80 ℃.
The preparation method of above-mentioned radial copolymer self-assembled nano micelle, among the step c, described organic solvent be dioxane, methane amide, methylene dichloride, trichloromethane, benzene, toluene any, the reaction times is 10-72 hour, temperature is controlled between 0 ℃-80 ℃.
The present invention has adopted a kind of easy synthetic method, has prepared a kind of amphipathic copolymer of novel star, and can form nanometer polymer micelle in the aqueous solution.This radial copolymer by hydrophobic polymethyl acrylic acid (PMAA) the PMMA segment of a weak point and three hydrophilic multipolymer poly Poly (NIPAAm-co-DMAEMA) segment constitute.In hydrophilic segment, NIPAAm and DMAEMA are respectively as the temperature sensitive and the pH sensitive group of polymkeric substance.As shown in Figure 1, star polymer can self-assembly in the aqueous solution forms micella, and can response temperature and the variation of pH.Under different pH conditions, the size of lower critical solution temperature of polymkeric substance (LCST) and self-assembled micelle all changes.Thereby temperature and pH can both regulate and control the structure of polymer micelle.As a kind of effective stimulation responsive type drug delivery system, this polymer nano-particle has shown the drug release behavior of temperature and pH response to dewatering medicament methopterin-A (MTX).What is more important, the drug release behavior of acceleration can be easily regulation and control by temperature or pH reach.Therefore, this star-type polymer micella is as a kind of pharmaceutical carrier, and the drug targeting that can reach in the pathology zone by the variation that utilizes inherent environment in outside or the body discharges, and improves pharmacological agent efficient.
The present invention has synthesized the novel amphipathic star block copolymer of being made up of hydrophilic three arm tri-arm P (NIPAAm-co-DMAEMA) segments and hydrophobic PMMA segment.In the aqueous solution, this radial copolymer can self-assembly form the nano-micelle particle with nucleocapsid structure.Because ionization and the deionization effect of PDMAEMA segment under condition of different pH in the hydrophilic segment, copolymer micelle shows different LCST under different pH conditions.When the pH of environment is respectively 5,7.4 and at 9 o'clock, the corresponding LCST of copolymer micelle is respectively 32 ℃, 39.5 ℃ of 36.6 ℃ of and, and also subtle change also takes place in the particle diameter of copolymer micelle along with the variation of pH.Because temperature and pH double-bang firecracker that hydrophilic segment has are answered characteristic, the self-assembly copolymer micelle of medicine carrying has also demonstrated the drug release behavior of temperature and pH double-response.In the vitro drug release experiment, the medicine carrying copolymer micelle shows the drug release rate hastening phenomenon of temperature triggered under the condition of pH 7.4, and shows the drug release rate hastening phenomenon of pH division under 37 ℃ condition.Therefore, our synthetic new copolymer nano-micelle particle is that a kind of intellectual drug with very big potential using value transmits carrier, it can be by the minor alteration of envrionment temperature or pH, be implemented in the drug release and the accumulation in target pathology zone, to improve the therapeutic efficiency of medicine, reduce side effect.
Spend a step below a description is done in the formation of copolymer micelle of the present invention, as the carrier of the very little medicine of solubleness in water, hydrophobic kernel is the prerequisite of its drug loading.In addition, to have hydrophilic surface also be its assurance with long blood circulation time to carrier.Therefore, we wish that the synthetic polymer micelle can have the structure of hydrophobic core/hydrophilic shell in the aqueous solution.For our synthetic star amphiphilic block copolymer, by relatively it just can determine whether to form in the aqueous solution and have hydrophobic core/polymer micelle of hydrophilic shell structure respectively at CDCl3 and D2O solvent.Fig. 3 and Fig. 4 are the multipolymer 1H NMR collection of illustrative plates in CDCl3 and D2O solvent respectively, and the ownership of resonance peak.We find, as Fig. 3, all composition segmental resonance peaks in the multipolymer occurred in the collection of illustrative plates of CDCl3 solvent, yet, in the collection of illustrative plates such as Fig. 4 of D2O solvent, PNIPAAm and PDMAEMA segmental resonance peak have only appearred, do not detect PMMA segmental resonance peak.This shows that in the aqueous solution, the red hydrophobic PMMA segment of multipolymer oneself is wrapped up by hydrophilic segment, has formed to have hydrophobic core/copolymer micelle of hydrophilic shell structure.
Definite method that the present invention hangs down micelle-forming concentration (CMC) is as follows:
CMC is that research is micelle formation, and estimates one of important parameter of micella stability.Usually, in amphipathic copolymer, the increase of hydrophobic segment ratio can cause the CMC of multipolymer to reduce, and means that also thermodynamic stability increases to some extent.Thereby we wish that amphiphilic polymer can have lower CMC usually.In this chapter, we select the fluorescent probe of pyrene as external source.The CMC of multipolymer is changed to determine in aqueous phase and the caused fluorescent emission of ratio of distributing in the micella hydrophobic core or excitation spectrum by pyrene.Fig. 3-the 4th, in different pH environment, the graphic representation that the logarithm of copolymer concentration (lgc) is done the intensity rate I391/I374 in the pyrene emmission spectrum in the polymers soln.Along with the increase of polymer concentration, ratio is also increasing.This be since since polymer formation micella, more pyrene molecule has entered in the hydrophobic interior nuclear environment.The tangent line point of crossing of these curve break both sides promptly is defined as the CMC value of multipolymer.We can see that under condition of different pH I391/I374 is to the graphic representation of logC in the fluorescence emission spectrum from Fig. 5.λ ex=342nm, [pyrene]=6 * 10-7M. multipolymer is in different pH environment, and the CMC value has certain variation.When the pH value of solution of polymkeric substance is respectively 5,7.4 and at 9 o'clock, pairing CMC is respectively 7.5mg/L, 8.7mg/L and 11.2mg/L.Obviously, the CMC value of radial copolymer under different pH conditions is all smaller, shows in the scope of pH from 5 to 9, and it can both form stable self-assembling polymers micella in the aqueous solution.Owing to have lower CMC value, this copolymer micelle is expected in extremely rare aqueous solution, as the human body fluid environment, uses as pharmaceutical carrier.Simultaneously, we notice that also the CMC value of this copolymer micelle has shown significant pH dependency.This is because under different pH conditions, the DMAEMA group has taken place protonated or deprotonation in the hydrophilic segment of multipolymer, has changed the hydrophilic-hydrophobic matter of hydrophilic segment, thereby has influenced the CMC of multipolymer, has shown the pH correlation properties of CMC.
The copolymer micelle structural changes of temperature of the present invention and pH sensitivity is as follows:
PNIPAAm is the polymkeric substance with lower critical solution temperature (LCST) that a kind of people know, and its transformation temperature is approximately 32 ℃.Be lower than this temperature, PNIPAAm is a water-soluble polymers, and polymer molecular chain exists with the form of extended chain.Be higher than this temperature, reversible of PNIPAAm experience changes mutually, changes water-insoluble aggregate into from water-soluble polymers.In addition, hydrophilic or hydrophobic monomer carries out copolymerization by other of NIPAAm monomer and different ratios, can regulate the LCST value of PNIPAAm at an easy rate within the specific limits.Owing to have a large amount of tertiary amine groups, PDMAEMA shows as a kind of pH sensitive polymers in the aqueous solution.Under different pH conditions, PDMAEMA can show the state of ionization or deionization.Therefore, the water-soluble of PDMAEMA is relevant with the pH of solution.In the tart environment, the PDMAEMA ionization has positive charge, shows extremely strong water-soluble.In alkaline environment, the PDMAEMA deionization, electric charge reduces, and becomes the water insoluble polymer.We not only pay close attention to the variation of copolymer micelle structure under differing temps, also pay close attention in the different pH environment change of temperature sensitive characteristic simultaneously.Expect as us, Fig. 6 has provided the LCST value of multipolymer under the condition of different pH, Fig. 6 under condition of different pH the aqueous copolymers solution transmitance with the variation of temperature curve, show the rising along with temperature, the hydrophilic chain structure of copolymer micelle is changed into the hydrophobic aggregation body of contraction by the hydrophilic chain that stretches.Along with variation of temperature, the change of hydrophilic outer shell chain structure has also destroyed the stable polymer micella.What is more important, we notice that polymer micelle has demonstrated pH relevant temperature response characteristic.Corresponding to pH 5, pH 7.4 and pH 9, the multipolymer LCST value that records is respectively 32 ℃, 36.6 ℃ and 39.5 ℃.The temperature that copolymer sheet reveals in order to explain, pH Lazer sense characteristic, we can be divided into the PDMAEMA segment in the radial copolymer two parts: the hydrophilic segment that the hydrophobic segment that a part is made up of the DMAEMA monomer of deionization, another part then are made up of Ionized DMAEMA monomer.Obviously, this two-part ratio is to regulate by the variation of pH.Therefore, in tri-arm P (NIPAAm-co-DMAEMA) segment, hydrophilic/hydrophobic segmental ratio changes along with the variation of pH, raises thereby influenced the LCST value of multipolymer: pH, and the hydrophobic segment ratio increases, and LCST descends, and vice versa.As the carrier of medicine, the structural response temperature of copolymer micelle, the variation of pH and change will inevitably cause its variation to the release behavior of medicine, and this will discussed with the lower section.
The pH dependency of the form of nano-micelle particle of the present invention and size thereof is as follows:
In solvent optionally, amphiphilic block copolymer can self-assembly forms the aggregate of variform, and is bar-shaped as sphere, vesica, lamelliform, tubulose etc.As everyone knows, diameter is the optimal selection of antitumor drug pharmaceutical carrier less than the spherical micelle particle of 200nm].This is because it can make full use of selectivity retention effect (EPR effect) and gather at tumor region, and can avoid RE to engulf the removal effect of system.Fig. 3-the 6th, under pH 7.4 conditions, the empty polymer micelle (Fig. 7) and the Electronic Speculum picture of drug-carrying polymer micelle (Fig. 7).Picture shows, no matter is the micella behind polymer micelle or the medicine carrying, can both disperse to become the isolated spheric nano-micelle particle that has in the aqueous solution.And we notice that more greatly, after this showed the copolymer micelle medicine carrying, the micellar size slightly increased the micella particle (80-120nm) behind the medicine carrying than empty micella particle (60-100nm).In addition, the measurement result of particle nano-particles size (Fig. 8) shows that the particle diameter of polymer micelle has small increase after being not only medicine carrying, and along with the variation of pH value of solution, the micellar particle diameter also has small variation.Under the condition of pH 5, the diameter of polymer latex bundle of particle is about 113nm, and along with pH is increased to 9, the particle diameter of polymer micelle is decreased to about 85nm, and the polymer micelle behind the medicine carrying also has similar Changing Pattern.What this polymer micelle particle diameter changed along with the pH variation also is because in different pH environment, the degree of ionization difference of the tertiary amine groups in the PDMAEMA segment (pKa-7.4) is caused.Under lower pH condition, PDMAEMA segmental degree of ionization is higher, has more positive charge, therefore, the hydrophilic shell of polymer micelle has demonstrated stronger Coulomb repulsion effect, has stretched the hydrophilic segment of shell, has caused bigger copolymer micelle size.Yet, under higher pH condition, the deionization of PDMAEMA segment, the positive charge that has reduces, and hydrophilic outer shell shrinks, and has caused reducing of copolymer micelle particle.
The vitro drug release behavior of temperature of the present invention and pH response is as follows:
Intelligent pharmaceutical carrier during stable circulation, does not discharge contained medicine in vivo as far as possible, and when the experience external stimulus, during as the variation of temperature or pH, then can quicken release rate of drugs in the pathology zone.From as can be seen at experimental section, the entrapment efficiency of copolymer micelle (encapsulation efficiency, EE) be 31.5%, show the hydrophobic core that nearly 31.5% (2.45mg) entered polymer micelle in the model drug MTX (8.0mg) that uses.More traditional polymer micelle, this copolymer micelle have demonstrated higher medicine carrying ability, and this may be to have had special star structure [39,40] owing to form the micellar multipolymer, has strengthened the interaction ability of hydrophobic inner core and medicine.
Fig. 9 be the medicine carrying copolymer micelle in the environment of pH 7.4, respectively at 25 ℃, the accumulation drug release graphic representation when being lower than lower critical solution temperature (LCST) and 40 ℃ (being higher than LCST).When the drug release envrionment temperature was lower than the LCST (36.6 ℃, pH 7.4) of multipolymer, multipolymer existed with stable hud typed micellar form, and medicine is encapsulated in the hydrophobic kernel, so drug release rate is less.When temperature was higher than the LCST of multipolymer, the shell of polymer micelle became the hydrophobic chain of contraction from the hydrophilic chain that stretches, and the stable nucleus shell structure of copolymer micelle is destroyed, and drug release rate is accelerated.Based on the drug release behavior of the temperature triggered of this kind under approximate physiological environment, this copolymer nano micella particle will be a kind of very promising location pharmaceutical carrier.It can utilize regional heating technique or pathology zone inherent temperature to raise and realize the triggering of drug release.
From Figure 10, we can observe, and under given pH condition, at 25 ℃, the rate of release of MTX from the copolymer micelle particle and accumulative total burst size (being lower than 35%) all are lower.In addition, although we it is further noted that under different pH conditions, total the accumulative total release amount of medicine substantially the same, but in the starting stage of drug release, slightly higher when the drug release rate when pH is 5 will be 7.4 and 9 than pH.In this experiment, release temperature is 25 ℃, is lower than the multipolymer LCST value under the given pH condition.Therefore, in whole drug release process, the copolymer micelle particle all is the nucleocapsid structure that is keeping stable, under different pH conditions, does not show the characteristic of pH response.Under different pH conditions, the difference of the initial drug rate of release of medicine carrying copolymer micelle particle, show under higher pH condition, the contraction of micella particle shell hydrophilic segment also might hinder medicine in the hydrophobic core to the diffusion of release medium, thereby reduced drug release rate, but to the but not influence basically of total release amount of medicine.
37 ℃ of release temperature maintenances are constant, and the medicine carrying copolymer micelle shows significantly different drug release behavior such as Figure 11 under the different pH conditions, has shown significant pH response medicine release characteristics.When pH=5, release rate of drugs is less, and the accumulative total release amount of medicine is less than 35%.And when pH=7.4 or 9, drug release rate significantly strengthens, and time of releasing, the accumulative total release amount of medicine reached 70-80% when being 40h.Different pharmaceutical release behavior under condition of different pH can be by under the condition of different pH, and the difference of the LCST of multipolymer makes an explanation.When pH=5, the LCST of multipolymer is defined as about 39.5 ℃, is higher than the release temperature of experiment, at this moment, the medicine carrying copolymer micelle is to discharge under the temperature of the LCST that is lower than multipolymer, and copolymer micelle is stable nucleocapsid structure, therefore rate of release is less, and the accumulative total burst size is also lower.When pH changes into 7.4 and 9, the LCST of multipolymer is respectively 36.6 ℃ and 32 ℃, be lower than the release temperature of experiment, at this moment, the medicine carrying copolymer micelle is to discharge under the temperature of the LCST that is higher than multipolymer, and the rock steady structure of multipolymer is destroyed, copolymer micelle is transformed into precipitation, the ability drop of entrapped drug, thus medicine to enter the speed of release medium bigger, the accumulative total burst size is also higher.Based on the pH response medicine release characteristics of this copolymer micelle under the normal physiological body temperature condition, this novel nano micella particle is expected to become a kind of oral pharmaceutical carrier with huge potential using value.It can be under tart pH condition (as in the stomach environment) keep the medicine that loaded, and (in environment in intestines) quickens to discharge the medicine that is loaded under the condition near normal physiological pH.
In addition, for temperature, the pH double-bang firecracker of confirming the medicine carrying copolymer micelle are further answered the drug release behavior, when we have investigated in drug release process the temperature that changes release medium or pH, the variation of drug release behavior.Figure 12 has shown that under the condition of pH 7.4 medicine of temperature triggered quickens release behavior.This is that the medicine carrying copolymer micelle is changed into the cause of insoluble aggregate (37 ℃) by stable hud typed micella (25 ℃) because temperature raises.Similarly, Figure 13 has shown that under 37 ℃ condition the medicine that pH triggers quickens release behavior.The result shows, discharges in the medicine process at the medicine carrying copolymer micelle near the Human Physiology environment, and the change of temperature or pH all can cause the variation of drug release behavior.Therefore, we think that this nanometer copolymer particle with temperature and pH double-response can be as the location pharmaceutical carrier of temperature and pH triggering, it can pass through the temperature of target area or the variation of pH, thereby strengthen medicine improves medicine in the accumulation in target pathology zone therapeutic efficiency.
Description of drawings
Fig. 1 is the synoptic diagram of the structural changes of star copolymer self-assembly and response temperature and pH
Fig. 2 is the synthetic route of star copolymer
Fig. 3 is that (a) three arm P (NIPAAm-co-DMAEMA) multipolymers are at CDCl
3In and (b) star copolymer at CDCl
3In
1H NMR spectrogram
Fig. 4 is that star copolymer is at D
2Among the O
1H NMR spectrogram
Fig. 5 is I in the fluorescence emission spectrum under condition of different pH
391/ I
374Graphic representation to logC.
Fig. 6 be under condition of different pH the aqueous copolymers solution transmitance with the variation of temperature curve
Fig. 7 is the nano-micelle particle behind empty nano-micelle particle (b) medicine carrying of transmission electron microscope picture (a)
Fig. 8 is under condition of different pH, the change of size figure of micella particle
Fig. 9 is a temperature sensitive drug release curve in the PBS of pH 7.4 buffered soln
Figure 10 is the pH sensitive medicaments release profiles under 25 ℃
Figure 11 is the drug release curve 37 ℃ of following pH sensitivities
Figure 12 be the temperature triggered medicine under pH 7.4 conditions quicken release graphics and
Figure 13 quickens releasing curve diagram at the medicine that 37 ℃ of following pH trigger.
Specific embodiment
The N that the specific embodiment of the invention is used, N-dimethyl aminoethyl methyl methacrylate (DMAEMA, 98%) is purchased the company in Arcos, purifies through underpressure distillation before using.N-N-isopropylacrylamide (NIPAAm) is purchased the company in Arcos, purifies through toluene/normal hexane recrystallization before using.Pehanorm and ceric ammonium nitrate (IV) are Shanghai chemical reagent factory products, unprocessed direct use.3-thiohydracrylic acid (MPA), N-hydroxy-succinamide (NHS) and dicylohexyl-carbodiimide (DCC) are Acros company product, unprocessed direct use.Diisopropyl azodicarboxylate (AIBN) is purchased in the Shanghai laboratory, purifies through recrystallizing methanol before using.Dioxane and dimethyl formamide are Shanghai chemical reagent factory product, handle according to standard method before using.Bromination-3-(4,5-dimethylthiazole base-2)-2,5-phenylbenzene tetrazolium (MTT) is a Sigma company product.Vero cell (African green monkey kidney cell) is provided by China typical thing preservation center (CCTCC).Improved Eagle substratum (MEM) and phosphate buffer solution (PBS) are purchased the company in Gibco.All the other reagent and solution are commercially available, unprocessed direct use.
The specific embodiment of the invention is finished by following steps:
1, the preparation of semiremote pawl polymer P MMA-COOH
By being that to have prepared end be the polymethylmethacrylate (PMMA-COOH) of carboxyl for the radical polymerization of chain-transfer agent with MPA.With MMA (9 * 10
-2Mol), MPA (2.2 * 10
-3And AIBN (4.5 * 10 mol))
-4Mol) be dissolved among the 40mL DMF.Solution feeds nitrogen bubble deoxygenation 30min (processing parameter please be given a numerical value interval, and to enlarge scope of patent protection, what relate to later also will handle like this).System is warming up to 60 ℃, stirs polymerization 8h.After reaction is finished, in reaction solution impouring water, separate out white precipitate, washing filtering.The white solid that obtains is dissolved among the DMF, precipitates in water, three times repeatedly, product vacuum-drying is to constant weight.
2, the preparation of three arm polymkeric substance tri-arm P (NIPAAm-co-DMAEMA)
The copolymerization of monomer NIPAAm and DMAEMA, the redox system of forming with cerium-alcohol is an initiator, carries out in the there-necked flask with whipping appts, reflux condensing tube and nitrogen protection device.With NIPAAm 3.4g (3 * 10
-2Mol), DMAEMA 0.94g (6 * 10
-3And Pehanorm 0.18g (1.5 * 10 mol),
-3Mol) and 70% salpeter solution 1mL be dissolved in the 70mL deionized water, then mixing solutions is fed nitrogen gas stream deoxygenation 30min.Be heated to 55 ℃, stir, insulation 1h (will be with the word of standard, H change into hour).Be dissolved with 3.278g (6 * 10 with the syringe adding
-3Mol) the 0.5M salpeter solution 3mL initiated polymerization of ceric ammonium nitrate (IV).Under the protection of nitrogen gas stream, insulated and stirred reaction 8 hours is cooled to room temperature then.Reaction solution moves in the dialysis tubing (molecular weight cut-off is 8,000-10,000g/mol, Shanghai chemical reagents corporation), immerses in the distilled water and dialyses 4 days, and to remove unreacted monomer, lyophilize obtains product.
3, the preparation of star amphipathic copolymer
With tri-arm P (NIPAAm-co-DMAEMA) the copolymer 1 .0g of above preparation, PMMA-COOH0.5g, and N-hydroxy-succinamide (NHS) 6.0 * 10
-4Mol is dissolved in the 10mL dioxane.In nitrogen protection and stir down, dropwise adding contains DCC (6.0 * 10 in the above-mentioned solution
-4Mol) dioxane 5mL.After dropwising, stirring reaction 48h under the room temperature.Product precipitates the PMMA-COOH that does not participate in reaction to remove in a large amount of ether.Product is dried to constant weight in a vacuum.
4, star amphipathic copolymer micellar forms
This chapter adopts dialysis method to prepare copolymer micelle.Concrete grammar is as follows: star amphipathic copolymer 2mg is dissolved in the dialysis tubing (molecular weight cut-off 8000-10,000g/mol, Shanghai chemical reagents corporation), inserts and dialysed in the 500mL distilled water 48 hours, lyophilize obtains copolymer micelle.
The mensuration of the invention described above embodiment polymericular weight
Resulting polymers molecular weight (number-average molecular weight M
nAnd weight-average molecular weight M
w) mensuration use and to be equipped with the gel permeation chromatography (GPC) of Waters 2690D separation module and Waters 2410 refractive index detectors to carry out.DMF is an elutriant, flow velocity 0.3mL/min.Polystyrene with narrow molecular weight distributions is a standard, and Waters millennium module software is used to calculate molecular weight.
The mensuration of H NMR of the present invention
H NMR spectrogram with Varian VX-300 Mercury (300MHz) nmr determination (in be designated as TMS, solvent is respectively CDCl
3And D
2O).
The foregoing description optical transmittance is measured
Radial copolymer concentration under the condition of different pH is that the optical transmittance of the phosphate buffer solution (0.1M) of 4mg/mL is measured UV-8500 ultraviolet spectrometer (China) mensuration that use is furnished with the circulator bath chuck.The mensuration wavelength is 500nm, and temperature rise rate is 0.5 ℃/min.It is 50% pairing temperature that the LCST of polymers soln is defined as optical transmittance.
The observation of the foregoing description transmission electron microscope
In order to observe size and the form of polymer micelle in solution, specimen preparation is as follows: the aqueous solution of a multipolymer places on the copper mesh that is covered with the polyvinyl formal film, and is dry down in room temperature (being lower than the LCST of polymkeric substance).Observe acceleration voltage 80kV with JEM-100CX II transmission electron microscope (Japan).
The mensuration of the foregoing description copolymer micelle particle dia
The mensuration of copolymer micelle particle dia is used Malvern Zetasizer Zs90, and (Britain Malvern) carries out.During mensuration, temperature is 25 ℃, is lower than the LCST value of multipolymer, and solution is the phosphate buffer solution (0.1M) that contains the different pH of 0.01M NaCl.
The mensuration of the above embodiment of the present invention aqueous copolymers solution fluorescence
With the described unanimity of work before us, static fluorescence spectrum is measured with Shimadzu RF-5301PC fluorescence spectrophotometer (Japan).Pyrene is as hydrophobic fluorescent probe.The acetone soln (6 * 10 of 1mL pyrene
-6M) insert in the test tube, acetone spontaneous evaporation in air is removed.A series of different concns, the aqueous solutions of polymers of 10mL adds respectively in the above-mentioned test tube that contains remaining pyrene.Has identical pyrene concentration (6 * 10 in all polymers soln samples to be measured
-7M).Polymers soln at room temperature leaves standstill 24 hours, so that the dissolving of pyrene in solution reaches balance.Excitation wavelength is 340nm, emmission spectrum record 350 to 600nm scopes.Excite and launch slit width and be 5nm.In the emmission spectrum of pyrene, the 3rd emission band (391nm, I
3) and first emission band (374nm, I
1) the ratio (I of emissive porwer (emission peak height)
3/ I
1) concentration of polymkeric substance is mapped.The tangent line point of crossing of these curve break both sides promptly is defined as the CMC value of polymkeric substance.
The loading of the above embodiment of the present invention medicine
8mg radial copolymer and 8mg methotrexate (MTX) are dissolved in the 4mL diformamide.(000g/mol), dialysis is 24 hours in 1000mL distilled water for Shanghai chemical reagents corporation product, molecular weight cut-off: 8000-10 in the solution immigration dialysis tubing.In order to measure the drug loading of polymkeric substance, drug-carrying polymer solution is filtered, remove insolubles, lyophilize then obtains the drug-carrying polymer powder.The drug-carrying polymer powder of certainweight is dissolved in the diformamide, measures the amount of medicine, absorbing wavelength 371nm with the UV spectrum calibration curve method.Methotrexate/diformamide typical curve is obtained by experiment.Experiment records, and the carrying drug ratio of this multipolymer is about 31.5wt.%.
The mensuration of the above embodiment of the present invention vitro drug release behavior
After the dialysis drug loading, dialysis tubing is directly immersed 400mL have in the release medium of different pH or temperature, stir.At regular intervals, from distilled water, pipette 3mL solution, measure drug level.After having got sample, add the release medium of 3mL, to keep liquor capacity constant at every turn.Measure absorbing wavelength 371nm with ultraviolet absorption spectroscopy from the intrafascicular drug level that discharges of polymer latex.
Synthetic and the structure of the above embodiment of the present invention amphiphilic radial copolymer
Star block copolymer is that the PMMA segment of carboxyl and tri-armP (NIPAAm-co-DMAEMA) the polymer segment coupling of three arms prepare by end.The synthetic route of multipolymer as shown in Figure 2.
The first step is to adopt radical polymerization, is that chain-transfer agent has synthesized the PMMA with carboxy blocking with MPA, as the hydrophobic block of subject polymer.Second step was the hydrophilic segment tri-armP (NIPAAm-co-DMAEMA) of synthetic multi-arm.The cerium salt of high price with go back original reagent, as the common initiator system of forming such as alcohol, aldehyde, ketone, the polyreaction that causes vinyl monomer has bibliographical information.Among this chapter, the redox system that the copolymerization of NIPAAm and DMAEMA is made up of ceric ammonium nitrate and Pehanorm causes.In the Pehanorm molecule, the carbon atom that links to each other with hydroxyl generates free radical under the effect of cerium (IV), thus the monomeric polymerization of initiated polymerization.Shown in 3-1, at Ce
+ 4Oxygenizement under, in the Pehanorm-CH
2The OH group has produced free radical.In polymerization, the free radical of formation is from-CH
2The OH group is to NIPAAm and DMAEMA monomer transfer, thereby realized the growth of polymeric chain.Owing in a Pehanorm molecule, three-CH is arranged
2The OH group can form three and cause free radical, therefore, has obtained having the NIPAAm of three arms and the multipolymer of DMAEMA.At last, terminal for the PMMA segment of carboxyl and hydrophilic three arm tri-arm P (NIPAAm-co-DMAEMA) multipolymers carry out coupling under the effect of NHS and DCC, obtained the amphipathic star block copolymer.The molecular weight polymkeric substance that obtains in each step and subject polymer and molecular weight distribution result are shown in table 3-1.The molecular weight result of polymkeric substance shows, in the 3rd step synthetic in, the increase of the molecular weight of star polymer is suitable with the molecular weight of PMMA-COOH, has proved the coupling between two corresponding segments.
Table 1PMMA, the molecular weight of tri-arms P (NIPPAm-co-DMAEMA) and radial copolymer
In addition, by D and star block copolymer at CCl
3Among the D
1H NMR Fig. 3 as seen, star block copolymer
1H NMR has not only demonstrated the characteristic peak of PNIPAAm, PDMAEMA, the characteristic peak that tangible PMMA also occurred, and the PMMA-COOH that does not participate in reaction has removed by precipitating in ether, thereby has confirmed that this multipolymer is to be made of PMMA segment and NIPAAm, the coupling of DMAEMA cosegment.Based on multipolymer
1The intensity (3.97) that H NMR figure, the multipolymer of tri-arm P (NIPAAm-co-DMAEMA) form resonance peak that can be by belonging to NIPPAm is calculated with resonance peak (2.25) intensity that belongs to DMAEMA and is obtained.The result shows that PNIPAAm is consistent with ratio and the feed ratio of PDMAEMA in the multipolymer.This that is to say, the preparation of our success a kind of novel amphiphilic star block copolymer.This multipolymer is made up of a relatively short hydrophobic PMMA segment and the hydrophilic segment with three arms of temperature and pH responding ability.
Claims (4)
1, a kind of preparation method of radial copolymer self-assembled nano micelle is characterized in that, comprises the steps:
E. prepare the polymethylmethacrylate that end group is a carboxyl: be chain-transfer agent with the thiohydracrylic acid, use the initiator Diisopropyl azodicarboxylate in organic solvent monomers methyl methacrylate, under the certain temperature, carry out Raolical polymerizable;
F. prepare three arm polymkeric substance tri-arm P (NIPAAm-co-DMAEMA): the redox system of forming with cerium-alcohol is an initiator, and with monomer N-N-isopropylacrylamide and N, N-dimethyl aminoethyl methyl methacrylate carries out radical copolymerization;
G. prepare radial copolymer: two kinds of polymkeric substance of above preparation are added N-hydroxy-succinamide and dicyclohexylcarbodiimide in solvent, the reaction certain hour, product precipitates in ether, filters final vacuum and is dried to constant weight;
H. the above-mentioned star amphipathic copolymer that makes is dissolved in the dialysis tubing, inserts in the distilled water and dialyse, obtain copolymer micelle.
2, the preparation method of radial copolymer self-assembled nano micelle as claimed in claim 1 is characterized in that, among the step a, described temperature is between 50 ℃-80 ℃, reaction times is 5 hours-48 hours, and described organic solvent is a dioxane, methane amide, methylene dichloride, benzene, toluene any.
3, the preparation method of radial copolymer self-assembled nano micelle as claimed in claim 1, it is characterized in that, among the step b, described initiator system is made up of Pehanorm and cerous nitrate, their mass ratio is 1: 5-1: between 100, the mass ratio of initiator system and polymerization single polymerization monomer is controlled at 1: 100-1: between 10000, temperature of reaction is 2-12 hour, and temperature of reaction is between 50 ℃-80 ℃.
4, the preparation method of radial copolymer self-assembled nano micelle as claimed in claim 1, it is characterized in that, among the step c, described organic solvent be dioxane, methane amide, methylene dichloride, trichloromethane, benzene, toluene any, reaction times is 10-72 hour, and temperature is controlled between 0 ℃-80 ℃.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2013150796A (en) * | 2011-12-28 | 2013-08-08 | National Cerebral & Cardiovascular Center | Temperature-sensitive material and hemostatic agent |
| CN103772714A (en) * | 2014-01-24 | 2014-05-07 | 同济大学 | Starlike segmented copolymer and preparation method thereof |
| CN103992451A (en) * | 2014-05-13 | 2014-08-20 | 同济大学 | Diblock copolymer with sugar, temperature and pH triple sensitivities, and preparing method thereof |
| CN114797512A (en) * | 2021-01-29 | 2022-07-29 | 深圳市碳源生物科技有限公司 | Star-poly (dimethylaminoethyl acrylate) polymer modified PVDF (polyvinylidene fluoride) membrane and application thereof |
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2008
- 2008-09-03 CN CN2008101070542A patent/CN101348570B/en not_active Expired - Fee Related
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2013150796A (en) * | 2011-12-28 | 2013-08-08 | National Cerebral & Cardiovascular Center | Temperature-sensitive material and hemostatic agent |
| CN103772714A (en) * | 2014-01-24 | 2014-05-07 | 同济大学 | Starlike segmented copolymer and preparation method thereof |
| CN103772714B (en) * | 2014-01-24 | 2016-04-06 | 同济大学 | A kind of star block copolymer and preparation method thereof |
| CN103992451A (en) * | 2014-05-13 | 2014-08-20 | 同济大学 | Diblock copolymer with sugar, temperature and pH triple sensitivities, and preparing method thereof |
| CN103992451B (en) * | 2014-05-13 | 2016-06-15 | 同济大学 | A kind of diblock copolymer possessing sugar, temperature, the triple sensitivity of pH and preparation method thereof |
| CN114797512A (en) * | 2021-01-29 | 2022-07-29 | 深圳市碳源生物科技有限公司 | Star-poly (dimethylaminoethyl acrylate) polymer modified PVDF (polyvinylidene fluoride) membrane and application thereof |
| CN114797512B (en) * | 2021-01-29 | 2023-02-03 | 深圳市碳源生物科技有限公司 | Star-poly (dimethylaminoethyl acrylate) polymer modified PVDF (polyvinylidene fluoride) membrane and application thereof |
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