CN110041475B - Amphiphilic block copolymer, shell-crosslinked micelle thereof, preparation method and application - Google Patents
Amphiphilic block copolymer, shell-crosslinked micelle thereof, preparation method and application Download PDFInfo
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Abstract
本发明提供一种两亲性嵌段共聚物、由其组成的聚合物纳米壳交联胶束以及它们的制备方法和应用。所述两亲性嵌段共聚物由亲水链段和在中性以及碱性环境中呈现疏水特性的疏水链段通过可逆加成‑断裂链转移(RAFT)聚合获得，其中所述亲水链段是寡聚乙二醇甲基丙烯酸酯与带有用于壳交联的醛基功能团的甲基丙烯酸酯类化合物共聚得到，其分子量为5000～10000Da。本发明实现了聚合物纳米壳交联胶束载体负载的疏水药物的可控释放，同时两亲性嵌段共聚物中共价键结合的具有红光发射的光敏剂以及用于壳交联的醛基功能团配合在中性以及碱性环境中呈现疏水特性的疏水链段，能够在弱酸的微环境中以及红光照射的激发下准确地实现药物的可控释放。 The invention provides an amphiphilic block copolymer, a polymer nanoshell cross-linked micelle composed thereof, and a preparation method and application thereof. The amphiphilic block copolymer is obtained by reversible addition-fragmentation chain transfer (RAFT) polymerization of hydrophilic segments and hydrophobic segments exhibiting hydrophobic properties in neutral and alkaline environments, wherein the hydrophilic chains The segment is obtained by copolymerizing oligoethylene glycol methacrylate and a methacrylate compound with an aldehyde functional group for shell crosslinking, and its molecular weight is 5000-10000 Da. The invention realizes the controllable release of the hydrophobic drug loaded by the polymer nanoshell cross-linked micelle carrier, and at the same time, the amphiphilic block copolymer is covalently bonded to a photosensitizer with red light emission and an aldehyde for shell cross-linking. The functional group is matched with a hydrophobic segment that exhibits hydrophobic properties in neutral and alkaline environments, and can accurately achieve controlled release of drugs in a weak acid microenvironment and under the excitation of red light irradiation. 
Description
Technical Field
The invention belongs to the field of high polymer materials and preparation and application thereof. In particular to an amphiphilic block copolymer with stimulus responsiveness, a polymer nano micelle composed of the amphiphilic block copolymer, and a preparation method and application thereof.
Background
The high bionics of various structural functions realized by simulating a cell structure by using a supermolecular assembly is always concerned by people, and particularly, the nano-assembly of the amphiphilic block copolymer is used for realizing the drug delivery of the focus part of a human body. However, although the block copolymer formed assemblies exhibit better thermodynamic stability compared to small molecule surfactants due to their lower Critical Micellization Concentration (CMC), there are some uncontrollable factors that may lead to the destruction and dissociation of the block copolymer assemblies, such as temperature, concentration, and solvent conditions (Elsabayy M, Heo G S, LimSM, Chemical revlews, 2015, 115 (19): 10967-11011). For drug delivery systems using block copolymer assemblies as carriers, stability of the assemblies is particularly important in avoiding premature release of the drug, prolonging blood circulation time, and increasing the therapeutic effect of the drug (Talelli M, Barz M, Rijcken C JF, Nano today, 2015, 10 (1): 93-117). The focus of the research in recent years is also directed to increasing the accuracy of drug delivery and improving the biological activity of drugs. Responsive polymers to physiologically relevant signals have also been a major research hotspot. The low pH environment exhibited by human tumor sites compared to normal tissue cells is an important reference point. There is also a wealth of research into pH sensitive polymer systems.
How to design a shell cross-linked nano micelle by utilizing a pH and illumination dual stimulus responsive polymer system so as to effectively improve the stability of a nano assembly and achieve the aim of controlling the release of a medicament, which is a research subject explored by scientists on cumin.
Disclosure of Invention
The invention aims to construct a polymer nano micelle which can stably exist under the pH condition of a normal physiological environment and responds to stimulus under a weak acid environment and red light irradiation, and provides a stimulus-responsive amphiphilic block copolymer, a polymer nano shell cross-linked micelle composed of the amphiphilic block copolymer, a preparation method and application thereof.
To this end, the present invention provides the following aspects of the invention:
an amphiphilic block copolymer, comprising a hydrophilic chain segment and a hydrophobic chain segment, wherein the hydrophobic chain segment is a hydrophobic chain segment shown as the following formula A.
Formula A:
wherein m is 0.95-0.99, n is 0.05-0.01, and a is 52-58.
The hydrophilic chain segment is obtained by copolymerizing oligoethylene glycol methacrylate and a methacrylate compound with an aldehyde functional group for shell crosslinking, and the molecular weight of the hydrophilic chain segment is 5000-10000 Da.
<2> the amphiphilic block copolymer according to <1>, wherein the hydrophobic segment is a hydrophobic segment obtained by covalently bonding the hydrophobic segment of formula a with a photosensitizer having red light emission.
<3> the amphiphilic block copolymer according to <2>, wherein the photosensitizer having red light emission is a photosensitizer PpIX having the following formula, and the resulting hydrophobic segment represented by the following formula B,
PpIX：
formula B:
the amphiphilic block copolymer according to any one of <1> - <3>, wherein the amphiphilic block copolymer has a structure of the following formula I
Formula I:
wherein m is 0.95-0.99, n is 0.05-0.01, x is 0.6-0.8, y is 0.4-0.2, a is 52-58, and b is 15-20.
A method for preparing the amphiphilic block copolymer of any one of <1> to <4>, the method comprising the steps of:
a) polymerizing a micromolecular chain transfer agent with reactants shown in a formula II and a formula III to obtain a hydrophilic macromolecular chain transfer agent;
b) continuously reacting the obtained hydrophilic macromolecular chain transfer agent with reactants shown in formulas IV and V to obtain an amphiphilic block copolymer;
c) optionally, reacting the amphiphilic block copolymer obtained in b) with a photosensitizer having red light emission, preferably a photosensitizer PpIX represented by formula C below, to obtain the amphiphilic block copolymer,
formula II:
formula III:
formula IV:
formula V:
formula C:
[ 6 ] A polymer nanoshell crosslinked micelle obtained by shell crosslinking of the micelle using a shell crosslinking agent of formula D after assembly of the amphiphilic block copolymer of any one of <1> to <4> in an aqueous system having a pH of 7.3 to 7.4, the polymer nanoshell crosslinked micelle having a size of 50 to 80nm and having pH and red light responsivity,
formula D:
the amphiphilic block copolymer nanoshell crosslinked micelle of <6>, wherein the polymer nanoshell crosslinked micelle further comprises a drug to be released.
A method for controlling the dissociation of the amphiphilic block copolymer nanoshell crosslinked micelles of any one of <6> to <7>, the method comprising the steps of:
a) preparing the amphiphilic segmented copolymer nanoshell crosslinked micelle into an aqueous dispersion;
b) slowly acidifying the pH value of the obtained aqueous dispersion from the range of 7.3-7.4 to 5.0-5.5 to convert the hydrophobic chain segment in the amphiphilic block copolymer into hydrophilic;
c) irradiating with red light to dissociate the connecting part of formula D in the amphiphilic block copolymer nano-shell cross-linked micelle, and finally disintegrating the nano-micelle assembly.
The application of the amphiphilic block copolymer nanoshell crosslinked micelle as a drug carrier according to any one of <6> to <7>.
Use of the amphiphilic block copolymer nanoshell crosslinked micelle of any one of <6> to <7> for achieving controlled release of a hydrophobic drug under excitation of a weak acid microenvironment and red light irradiation.
Drawings
FIG. 1 shows NMR spectra of a hydrophilic macromolecular chain transfer agent and an amphiphilic block copolymer according to one embodiment of the present invention, used to characterize the chemical structure and chain structure of the block copolymer. Wherein, the curve a shows the nuclear magnetic resonance hydrogen spectrum of the hydrophilic macromolecular chain transfer agent, and the curve b shows the nuclear magnetic resonance hydrogen spectrum of the amphiphilic block copolymer.
Fig. 2 shows a gel permeation chromatography spectrum of a hydrophilic macromolecular chain transfer agent and an amphiphilic block copolymer according to one embodiment of the present invention, for characterization of the molecular weight and polydispersity of the block copolymer. Wherein the curve a shows the gel permeation chromatogram of the hydrophilic macromolecular chain transfer agent, and the curve b shows the gel permeation chromatogram of the amphiphilic block copolymer.
Fig. 3 shows dynamic light scattering and electron microscope photographs of an amphiphilic block copolymer nano-micelle under different pH environments and in a shell cross-linking state according to an embodiment of the present invention, where fig. a shows particle size results of a dynamic light scattering test of the amphiphilic block copolymer nano-micelle under different pH environments and in a shell cross-linking state, fig. b shows electron microscope photographs of the amphiphilic block copolymer nano-micelle under a pH of 7.3 to 7.4 and in a shell cross-linking state, and fig. c shows electron microscope photographs of the amphiphilic block copolymer nano-micelle under a pH of 5.0 to 5.5 and in a shell cross-linking state.
Fig. 4 shows the change of the photosensitizer fluorescence intensity of the amphiphilic block copolymer nanoshell crosslinked micelle in different pH environments and the corresponding fluorescence intensity normalization curve, wherein fig. a shows that the photosensitizer PpIX fluorescence emission (625nm) intensity of the nanoshell crosslinked micelle increases with the decrease of the pH value of the aqueous system, indicating the restoration of the photodynamic ability of the photosensitizer; the graph b shows the trend of the intensity of the fluorescence emission (625nm) of the photosensitizer in the shell cross-linked micelle showing corresponding changes along with the change of the pH value of the water system.
Fig. 5 shows the change of fluorescence intensity of a shell cross-linking agent of micelle particles (also called micelles) in An environment with a pH of 5 of amphiphilic block copolymer nanoshell cross-linked micelles according to An embodiment of the present invention, wherein fig. a shows the shell cross-linking agent 9, 10-aminoethoxyanthracene (An-2 NH) of the amphiphilic nanoshell cross-linked micelles under the continuous irradiation of red light (wavelength range of 570nm to 590nm) in An environment with a pH of 5.0 to 5.52) The intensity of fluorescence emission (430nm) of the micelle is continuously reduced along with the intensity of fluorescence emission, and the shell cross-linking agent is broken and dissociated from the shell layer of the micelle particle; fig. b shows the change of fluorescence emission (430nm) intensity of the shell cross-linking agent of the shell cross-linked micelle with illumination time under different pH environments, which also shows that the shell cross-linking agent can be more effectively cleaved and dissociated by continuous illumination stimulation when the shell cross-linked micelle is under the environment of pH 5.0 to 5.5.
FIG. 6 shows the pair of the change of fluorescence intensity of the cross-linking agent of the shell of the micelle particle and the change of the particle size of the micelle particle in different environments of pH 5.0-5.5 and pH 7.3-7.4 of the amphiphilic block copolymer nanoshell cross-linked micelle according to one embodiment of the present inventionThe figure shows that when the pH value is 5.0-5.5, the shell cross-linking agent is 9, 10-aminoethoxy anthracene (An-2 NH)2) The intensity of fluorescence emission (430nm) of the polymer is continuously reduced along with the increase of illumination time, the particle size of the polymer is slightly enlarged due to the initial swelling effect, then the assembly is dissociated along with the breakage of the connecting structure of the shell cross-linking agent, and finally the particle size is reduced until the state of the particle size of the single chain of the polymer is almost reached; however, when the pH value is 7.3-7.4, the fluorescence intensity of the shell cross-linking agent and the particle size of the assembly are not obviously changed, which shows good stability.
Fig. 7 shows the cumulative release curve of the hydrophobic drug Camptothecin (CPT) in the amphiphilic block copolymer nanoshell cross-linked micelles according to one embodiment of the present invention over time, which shows that at a pH of 7.3 to 7.4, the cumulative release of the drug begins to equilibrate after 17 hours in both the illuminated and non-illuminated conditions, the cumulative release of the CPT is 30% in the illuminated condition, and only 25% of the drug is released in the non-illuminated condition. And under the environment of pH 5.0-5.5, the cumulative release amount of the CPT technical can reach 80% after being irradiated by red light for 40 hours. The result shows that the shell crosslinked micelle can effectively release the loaded hydrophobic drug under the stimulation of weak acid environment and red light (the wavelength range is 570 nm-590 nm) illumination.
Detailed Description
The technical solution of the present invention is further elaborated below. It should be noted that the various embodiments of the invention may be combined in any manner as desired.
Polymeric nanomicelles are typically self-assembled from amphiphilic block copolymers, which contain an oleophilic (also called hydrophobic) core and a hydrophilic shell. Therefore, a hydrophobic substance, such as a hydrophobic drug, can be loaded into the inner core of the nanomicelle, and then the controlled release of the hydrophobic drug can be realized by designing the responsive polymer nanoparticles. However, most block copolymer nano-assemblies have stability problems, and dissociation can occur in the blood circulation process, so that the drugs are released in advance, and the circulation time of the assemblies and the drugs in vivo is shortened. Therefore, not only is a certain influence on normal cells caused, but also an ideal effect on the release of the medicine at the focus part cannot be achieved.
In the present invention, we propose a novel strategy to solve the stability problem of polymer nano-assemblies. Specifically, we have designed a block copolymer that contains a hydrophobic segment that is pH sensitive. The chain segment presents hydrophobic characteristics in neutral and alkaline environments (pH is more than 7), and the hydrophobic characteristics are changed into hydrophilic characteristics in weak acid (pH is 5-6). Photosensitizers such as protoporphyrin ix (PpIX) photosensitizers covalently bound to a hydrophobic segment aggregate together due to assembly of polymeric nanomicelles, at which time the fluorescence emission and photodynamic ability of PpIX is impaired by the photoinduced electron transfer effect (PET) of the amino groups of the micelle core layer and the self-quenching effect. When the hydrophobic chain segment of the polymer nano assembly is changed into the hydrophilic chain segment to swell, the fluorescence emission and the photodynamic capacity of PpIX are recovered, and then the external red light is used for illumination stimulation to generate singlet oxygen, so that the connection structure of the assembly shell cross-linking agent is broken, and finally the hydrophobic drug loaded in the assembly is released along with the dissociation of micelle particles.
In the present invention, a photosensitizer suitable for use is a photosensitizer having red light emission. The photosensitizer is preferably at least one selected from protoporphyrin ix (ppix) photosensitizer, eosin Y photosensitizer, porphyrin-based photosensitizer, nile blue photosensitizer, phthalocyanine photosensitizer, and the like, and most preferably protoporphyrin ix (ppix) photosensitizer.
In general, in one aspect, the invention provides an amphiphilic block copolymer derived from a hydrophilic segment and a hydrophobic segment by polymerization, such as reversible addition-fragmentation chain transfer (RAFT) polymerization. The hydrophilic chain segment of the amphiphilic block copolymer is obtained by copolymerizing oligo-polyethylene glycol methacrylate and methacrylate (such as 4-formyl phenyl oxyethyl methacrylate) with an aldehyde group functional group for shell crosslinking, and the molecular weight of the hydrophilic chain segment is 5000-10000 daltons (Da), preferably 6000-9000 Da, more preferably 6000-8000 Da, and most preferably 7000-8000 Da. The hydrophobic segment has a chemical structure represented by formula a or formula B below.
Formula A:
formula B:
wherein m is 0.95-0.99, preferably 0.96-0.98, more preferably 0.97-0.98, n is 0.05-0.01, preferably 0.04-0.02, more preferably 0.03-0.02, a is 52-58, preferably 53-57, more preferably 54-56.
The term "oligoethylene glycol methacrylate" as used herein refers to methacrylate compounds containing an oligoethylene glycol structure with a degree of polymerization in the range of 8 to 9 in the ester side group.
The term "methacrylate having an aldehyde functional group for shell crosslinking" as used herein refers to a methacrylate-based compound carrying an aldehyde functional group capable of undergoing a crosslinking reaction with the shell crosslinking agent of the present invention, and a specific example thereof is 4-formylphenoxyethyl methacrylate and the like.
In a preferred embodiment, the amphiphilic block copolymer has a chemical structure of formula I below, and a hydrophilic segment obtained by reversible addition-fragmentation chain transfer (RAFT) polymerization of oligo (ethylene glycol) methacrylate of formula II and 4-formylphenoxyethyl methacrylate of formula III, and a hydrophobic segment obtained by reversible addition-fragmentation chain transfer (RAFT) polymerization of N, N' -diisopropylaminoethyl methacrylate of formula IV and hydroxyethyl methacrylate of formula V. Finally, the amphiphilic block copolymer with the red light emitting photosensitizer (PpIX) is obtained by esterification reaction of post-modification and the formula C.
Formula I:
formula II:
formula III:
formula IV:
formula V:
formula C:
wherein m is 0.95-0.99, preferably 0.96-0.98, more preferably 0.97-0.98; n is 0.05 to 0.01, preferably 0.04 to 0.02, and more preferably 0.03 to 0.02; x is 0.6 to 0.8, preferably 0.65 to 0.8, and more preferably 0.7 to 0.8; y is 0.4 to 0.2, preferably 0.35 to 0.2, more preferably 0.3 to 0.2; a is 52-58, preferably 53-57, more preferably 54-56; b is 15 to 20, preferably 16 to 19, and more preferably 17 to 19.
In the present invention, the term "reversible addition-fragmentation chain transfer (RAFT)" has a general meaning in the field of polymer synthesis, and specifically means that a disulfide or a trithioester derivative is used as a chain transfer agent to form a dormant intermediate with radicals during polymerization, thereby limiting irreversible biradical termination side reactions between extended chain radicals and effectively controlling the polymerization reaction. Meanwhile, the dormant intermediate can be cracked by itself, new active free radicals are released from corresponding sulfur atoms, and a lengthening chain is formed by combining monomers. Since the rate of addition or cleavage is much faster than the rate of chain growth, the dithioester and trithioester derivatives rapidly migrate between the living and dormant radicals, narrowing the molecular weight distribution, and allowing the polymerization to exhibit controlled/"living" characteristics. This is a very efficient method for achieving a controlled activity of radical polymerization and has been used since 1998.
In another aspect, the invention provides a polymer nanoshell crosslinked micelle composed of the amphiphilic block copolymer, wherein the polymer nanoshell crosslinked micelle is uniformly dispersed and has a size of 50-80 nm. In a preferred embodiment, the nanomicelle size is about 50nm or so. In a further preferred embodiment, the nanomicelle has pH and red light responsiveness.
In another aspect of the present invention, there is provided a method for controlling dissociation of polymer nanoshell crosslinked micelles, the method comprising the steps of: assembling the amphiphilic segmented copolymer into an aqueous dispersion of the polymer nanoshell crosslinked micelle under the environment of pH 7.3-7.4; and then, slowly acidifying the pH value of the assembly water system from 7.3-7.4 to 5.0-5.5, stirring for a period of time, and then carrying out red light (the wavelength range is 570-590 nm) illumination stimulation from the outside, so that the cross-linked connecting part of the nano micelle shell is dissociated, and finally the nano micelle assembly is disintegrated.
In a preferred embodiment, the aqueous polymer nanomicelle dispersion is formed as follows: dissolving 5mg of the amphiphilic block copolymer in an organic solvent serving as a cosolvent, dropwise adding 9mL of deionized water with the pH value of 10-10.1 at a certain temperature under stirring, continuously stirring and volatilizing after the deionized water is dropwise added, and diluting with a phosphate buffer solution (with the pH value of 7.3-7.4) to remove the organic solvent. The shell crosslinker and sodium cyanoborohydride are then added to the micellar dispersion and after an overnight reaction, the unreacted impurities are removed by dialysis in pure water. And finally diluting the mixture to a total volume of 10mL by using a phosphate buffer solution (with the pH value of 7.3-7.4) again to obtain the 0.5mg/mL aqueous polymer nanoparticle dispersion.
In a preferred embodiment, the reaction occurs as follows: assembling, crosslinking and dispersing the amphiphilic block copolymer in an environment with the pH value of 7.3-7.4 to obtain an aqueous dispersion of the polymer nanoshell crosslinked micelle; and then, slowly acidifying the pH value of the assembly water system from the range of 7.3-7.4 to 5.0-5.5, stirring for a period of time, converting the original hydrophobic chain segment in the assembly into a hydrophilic chain segment under an acidic condition, swelling the nano-micelle, recovering the photodynamic capacity of the red light emitting photosensitizer (PpIX) covalently bonded on the hydrophobic chain segment, generating singlet oxygen under the illumination stimulation of external red light (the wavelength range is 570-590 nm), further dissociating the connecting part of the nano-micelle shell cross-linking, and finally disintegrating the nano-micelle assembly.
In another aspect, the invention provides an application of the amphiphilic block copolymer nanoshell crosslinked micelle as a drug carrier, for example, a carrier of a hydrophobic anticancer drug Camptothecin (CPT) and the like.
In another aspect, the invention provides the amphiphilic block copolymer nanoshell crosslinked micelle, which can realize the controlled release of the hydrophobic drug in a weak acid microenvironment.
In another aspect, the invention provides the use of the amphiphilic block copolymer nanoshell crosslinked micelle for achieving controlled release of a hydrophobic drug under the excitation of a weak acid microenvironment and red light irradiation.
The purpose of the invention can be realized by the following technical scheme:
first, a monomer with An aldehyde functional group for shell crosslinking is prepared by a chemical reaction in combination with organic chemistry and is named ABEMA, which is reacted with a shell crosslinking agent such as 9, 10-aminoethoxyanthracene dihydrochloride (An-2 NH)2) And carrying out chemical combination reaction to cross-link the polymer micelle particle shell. The monomer ABEMA and a shell crosslinking agent 9, 10-aminoethoxy anthracene (An-2 NH)2) Has the following structure:
then, oligoethylene glycol methacrylate (e.g., 475Da molecular weight) and ABEMA are polymerized, preferably by reversible addition-fragmentation chain transfer (RAFT) polymerization, followed by polymerization, preferably by reversible addition-fragmentation chain transfer (RAFT) polymerization, with N, N' -diisopropylaminoethyl methacrylate and hydroxyethyl methacrylate, using a small molecule chain transfer agent such as CEP (2-cyano-2-propyltrithiocarbonate). This gives an amphiphilic block copolymer which is finally esterified by a post-modification with a photosensitizer (e.g. PpIX) to give the final amphiphilic block copolymer with a red-emitting photosensitizer (e.g. PpIX). The micromolecule chain transfer agent CEP and the photosensitizer have the following structures:
CEP：
pIX：
eosin Y:
porphyrin:
nile blue:
phthalocyanine:
preferably, the resulting amphiphilic block copolymers of suitable hydrophilic and hydrophobic segment lengths are self-assembled using a co-solvent-water addition process. The specific process is as follows: fully dissolving a polymer in tetrahydrofuran, slowly adding deionized water with the pH value of 10-10.1 under the action of stirring at a certain temperature, continuously stirring and volatilizing after assembly to remove an organic solvent, and diluting with a phosphate buffer solution (with the pH value of 7.3-7.4). A shell crosslinking agent such as 9, 10-aminoethoxyanthracene dihydrochloride (An-2 NH) is then added to the micellar dispersion2) And sodium cyanoborohydride, and after reacting overnight, unreacted impurities were removed by dialysis in pure water. And finally diluting with phosphate buffer solution (pH 7.3-7.4) again to obtain nano particle aqueous dispersion with fifty to eighty more nano size.
By using the obtained nano-shell crosslinked micelle, the hydrophilic and hydrophobic properties of the micelle core can be changed by changing the pH value of an external water system, the nano-micelle swells after a hydrophobic chain segment is converted into a hydrophilic chain segment, then the photodynamic ability of a red light emitting photosensitizer (such as PpIX) which is covalently bonded on the hydrophobic chain segment is recovered, singlet oxygen is generated under the stimulation of external red light illumination, then the connecting structure of the nano-micelle shell crosslinking is dissociated, and finally the nano-micelle assembly is disintegrated, so that the dissociative release of the hydrophobic drug is realized.
The invention will be further illustrated by the following examples, which are intended only for the purpose of a better understanding of the invention and do not limit the scope of the invention.
Examples
Preparation example 1
Step one, preparing ABEMA containing aldehyde functional groups:
it is characterized in that: the monomer can be well copolymerized with hydrophilic monomer oligo-polyethylene glycol methacrylate to obtain a hydrophilic chain segment, and meanwhile, the side group of the monomer contains an aldehyde functional group, and the monomer can perform crosslinking reaction with a shell crosslinking agent 9, 10-aminoethoxy anthracene to finally obtain a shell crosslinked nano micelle assembly.
The preparation method comprises the following steps:
2.44g of 4-hydroxybenzaldehyde (guo-drug) was dissolved in 50mL of acetone, and then potassium carbonate (4.146g, 30mmol, guo-drug) and bromoethanol (3.75g, 30mmol, guo-drug) were added to the system, and refluxed overnight. The mixture was then filtered to remove inorganic salts and acetone by rotary evaporation, then extracted after dissolution in dichloromethane, the organic phase was retained, dichloromethane was removed by rotary evaporation, and finally purified by column chromatography on silica gel to give intermediate 1(1.89g, 57%). The structural formula is as follows:
thereafter, 1.66g of intermediate 1 was dissolved in 40mL of anhydrous dichloromethane, 2mL of triethylamine (TEA, national drug) was added in an ice bath, and 5mL of anhydrous dichloromethane in which methacryloyl chloride (1.568g, 15mmol, national drug) was dissolved was added dropwise while maintaining the ice bath. After the addition was complete, the mixture was stirred at 0 ℃ for 1h and then at room temperature for 4 h. Then washed with saturated sodium bicarbonate water, extracted three times to retain the organic phase, and dried over anhydrous sodium sulfate overnight. Finally, the monomer was isolated and purified by silica gel column chromatography to give ABEMA (1.05g, 45%).
Secondly, an amphiphilic block copolymer is further prepared by using a RAFT polymerization method, and the reaction formula of the amphiphilic block copolymer is shown as follows:
it is characterized in that: the copolymer chain segment of the oligo-polyethylene glycol methacrylate and the ABEMA is used as a hydrophilic chain segment, the polymerization degree of the chain segment is 15-20, wherein x is 0.6-0.8, and y is 0.4-0.2. The copolymer chain segment of the N, N' -diisopropylaminoethyl methacrylate and hydroxyethyl methacrylate is a hydrophobic chain segment, the polymerization degree of the chain segment is 52-58, wherein m is 0.95-0.99, and N is 0.05-0.01.
The preparation method comprises the following steps: to a 50mL glass sealed tube were added the chain transfer agents trithioester CEP (0.32g, 1.52mmol), oligo-ethylene glycol methacrylate OEGMA (21.66g, 45.6mmol, Aldrich's reagent), monomer ABEMA (3.56g, 15.2mmol), azobisisobutyronitrile AIBN (1.28g, 7.6mmol, national medicine) and 25mL dioxane and a stirring magneton. The whole system is degassed by three times of freezing-degassing-unfreezing circulation in a sealed tube, then the tube is sealed under the conditions of vacuum and freezing, and the polymerization reaction is carried out for 8 hours under the condition of stirring at 70 ℃. Stopping the polymerization reaction by using liquid nitrogen after the reaction is finished, opening a sealing tube to expose the system in the air, diluting the reaction mixture to room temperature, dropwise adding the diluted reaction mixture into excessive ethyl glacial ether, collecting precipitates, dissolving the precipitates by using a small amount of dichloromethane, and then precipitating in the excessive ethyl glacial ether; after this dissolution-precipitation process was repeated three times, the resulting precipitated product was dried overnight in a vacuum oven to give a hydrophilic macromolecular chain transfer agent (17.21g, 67%).
Then a 50mL glass sealed tube was charged with hydrophilic macromolecular chain transfer agent (2.28g, 0.3mmol), N, N '-diisopropylaminoethyl methacrylate (3.42g, 30mmol, Aldrich's reagent), hydroxyethyl methacrylate (1.95g, 15mmol, Shanghai SulBiotech), azobisisobutyronitrile (1.28g, 7.6mmol, Guardian) and 25mL dioxane and stirring magnetons. The whole system is degassed by three times of freezing-degassing-unfreezing circulation in a sealed tube, then the tube is sealed under the conditions of vacuum and freezing, and the polymerization reaction is carried out for 8 hours under the condition of stirring at 70 ℃. Stopping the polymerization reaction by using liquid nitrogen, opening a sealing pipe to expose the system to the air, dropwise adding the reaction mixture into excessive petroleum ether after the system is recovered to the room temperature, collecting precipitates, dissolving the precipitates by using a small amount of dichloromethane, and then precipitating in the excessive petroleum ether; after repeating the dissolution-precipitation process three times, the finally obtained precipitated product was dried overnight in a vacuum oven to obtain a yellow solid. Next, the yellow solid described above was transferred with excess azobisisobutyronitrile and DMSO solvent into a glass sealed tube and stirring magnetons were added. After the whole system is degassed by three times of freezing-degassing-unfreezing cycles in a sealed tube, the tube is sealed under the conditions of vacuum and freezing, and the polymerization reaction is carried out for 2 hours under the stirring condition at 70 ℃. Stopping the polymerization reaction by using liquid nitrogen after the reaction is finished, opening a sealing pipe to expose the system in the air, diluting the reaction mixture to room temperature, dripping the diluted reaction mixture into excessive petroleum ether, collecting precipitates, dissolving the precipitates by using a small amount of dichloromethane, and then precipitating in the excessive petroleum ether; after repeating the dissolution-precipitation process three times, the finally obtained precipitated product was dried overnight in a vacuum oven to finally obtain 4.32g (56%) of a white solid polymer.
The chemical structures and chain structures of the hydrophilic macromolecular chain transfer agent and the amphiphilic block copolymer, and the molecular weight and molecular weight distribution thereof are respectively obtained by nuclear magnetic resonance hydrogen spectrum and gel permeation chromatography tests, and the results are respectively shown in fig. 1 and fig. 2.
Thirdly, the final amphiphilic block copolymer is subjected to esterification reaction with a photosensitizer with carboxylic acid groups through post-modification to obtain the final amphiphilic block copolymer with the red light emitting photosensitizer (PpIX), and the esterification reaction and the structural formula of the photosensitizer are shown as follows:
the preparation method comprises the following steps: 4.3g of the polymer obtained in the second step, 4-dimethylaminopyridine (DMAP, 0.03g, Chinese medicine), dicyclohexylcarbodiimide (DCC, 0.1g, Chinese medicine), and a photosensitizer protoporphyrin IX (PpIX, 0.15g, Aldrich reagent) were dissolved in anhydrous dichloromethane. And stirred at room temperature overnight. The unmodified photosensitizer protoporphyrin IX was then removed by basic alumina column chromatography. The product was finally spin dried to give 3.92g (88%) of a polymer as a dark red solid powder.
The fourth step: self-assembly of an amphiphilic block copolymer loaded with hydrophobic drug Camptothecin (CPT) is carried out to prepare the nano-shell cross-linked micelle particle. The method comprises the following specific steps:
first 5mg of polymer and 1mg of camptothecin were weighed out and dissolved in THF (1 mL). Then, 9mL of deionized water (pH 10-10.1) was added at a rate of 9mL/h at room temperature. The organic solvent is then evaporated under agitation and the undissolved hydrophobic anticancer drug is removed by filtration through a water filtration membrane. Finally, the assembly is dissolved by deionized waterAfter diluting to 9mL, the solution was diluted with phosphate buffer (PBS, 0.2mol/L, pH 7.3-7.4, Chinese medicine) to a total volume of 10 mL. Then, 2mL of the micelle dispersion (0.5g/L, PBS, 0.02mol/L, pH 7.3-7.4) was added with 9, 10-aminoethoxyanthracene dihydrochloride (An-2 NH)2) And stirred at room temperature for 4 h. Then NaBH is added3CN (0.1mg, Chinese medicine) is used for reducing imine bonds to obtain the final shell crosslinked nano micelle particles. After the reaction overnight, unreacted impurities were removed by dialysis against pure water. And finally, diluting the mixture by using phosphate buffer solution (PBS, 0.2mol/L, pH 7.3-7.4, Chinese medicine) to the total volume of 2mL to obtain the polymer nanoshell crosslinked micelle particle loaded with the hydrophobic medicine camptothecin.
The nanoparticles obtained as described above are characterized as follows:
1) the nano micelle particle is characterized in that a hydrophilic shell layer is formed by a copolymerization chain segment of oligo-polyethylene glycol methacrylate and ABEMA, and a hydrophobic core is formed by a copolymerization chain segment of N, N' -diisopropylaminoethyl methacrylate and a methacrylate monomer connected with a photosensitizer. The particle size is about 50nm, and the size distribution is uniform. Figure 3 shows dynamic light scattering data and a TEM photograph.
2) The polymer nanoshell crosslinked micelle particles can stably exist in neutral and alkaline environments, and can effectively dissociate and release the internally coated hydrophobic drug in a weakly acidic environment and under red light illumination. Fig. 4 shows that the shell-crosslinked micelle particles effectively respond to a weak acid environment. FIG. 5 shows that the shell-crosslinked micelle particle assembly dissociates to release the hydrophobic drug only after illumination with red light in a weak acid environment.
The fifth step: a method for controlling the dissociation of amphiphilic block copolymer nanoshell crosslinked micelle particles. The method comprises the following specific steps:
and (2) under the conditions that the pH value of the aqueous dispersion of the polymer nanoshell crosslinked micelle particles is 7.3-7.4 and the temperature is 37 ℃, the pH value of the obtained aqueous dispersion of the polymer nanoshell crosslinked micelle particles is slowly adjusted from 7.3-7.4 to 5.0-5.5. The original hydrophobic chain segment is changed into hydrophilicity, and the shell crosslinked micelle particle assembly swells. The system is then illuminated with a red light source. At this time, the photosensitizer located in the micelle core layer recovers its photodynamic ability due to the swelling of the assembly, and it can efficiently generate singlet oxygen under illumination of red light wavelength. Then the shell layer cross-linked structure is broken under the action of singlet oxygen. Finally, the assembly is dissociated to release the hydrophobic drug. FIG. 6 shows that the assembly is relatively stable at pH 7.3-7.4, and the assembly can be effectively dissociated at pH 5.0-5.5 under red (580nm) light.
Application example 1: loading and controllable release of amphiphilic block copolymer nanoshell crosslinked micelle particle on hydrophobic drug
In the prepared polymer nanoshell crosslinked micelle particle loaded with the hydrophobic drug camptothecin, a fluorescence spectrum is utilized to track the release process of Camptothecin (CPT) technical from a hydrophobic region of the micelle nanoparticle under the condition of weakly acidic pH value and under the condition of red light irradiation. First, 0.3mL of drug-loaded shell cross-linked micelle with a mass concentration of 0.5mg/mL was added to each of the four dialysis dishes. The pH value of two systems is 5.0-5.5, and the pH value of the other two systems is 7.3-7.4. One set of the systems with different pH values was illuminated with red light (580nm) at room temperature, and the other two sets were placed in a dark room. Measuring ultraviolet absorption of camptothecin in the dialysate at 228nm at intervals, and periodically updating the dialysate to replace the dialysate with phosphate buffer solution with corresponding volume and pH value. The cumulative release of CPT was calculated from the previously measured standard UV absorption curve of CPT at 228 nm. The results show that the hydrophobic drug can exist in the assembly stably in relative terms under the environment with the pH value of 7.3-7.4. And when the pH value is 5.0-5.5, the hydrophobic drug can be effectively released, and especially under the condition of combining with red light (580nm), the cumulative release amount can reach as much as 80%. Figure 7 shows the cumulative release profile of camptothecin encapsulated in a hydrophobic core in various pH environments and in the absence of light.
Industrial applicability
The invention realizes the controllable release of the hydrophobic drug loaded by the polymer nano-shell cross-linked micelle carrier, simultaneously, the photosensitizer with red light emission combined by covalent bonds in the amphiphilic segmented copolymer and the hydrophobic chain segment with hydrophobic property in neutral and alkaline environments are matched by the aldehyde functional group for shell cross-linking, and the controllable release of the drug can be accurately realized in the microenvironment of weak acid and under the excitation of red light irradiation, so the invention has great potential industrial application in the aspect of being used as a drug carrier or the controllable release of the drug.
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