CN113616815B - pH responsive T 1 /T 2 Switching type MRI contrast agent and preparation method and application thereof - Google Patents

pH responsive T 1 /T 2 Switching type MRI contrast agent and preparation method and application thereof Download PDF

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CN113616815B
CN113616815B CN202010381677.XA CN202010381677A CN113616815B CN 113616815 B CN113616815 B CN 113616815B CN 202010381677 A CN202010381677 A CN 202010381677A CN 113616815 B CN113616815 B CN 113616815B
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polyethylene glycol
glutamic acid
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裴仁军
贺祎霖
曹翼
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Suzhou Institute of Nano Tech and Nano Bionics of CAS
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Abstract

The invention discloses a pH responsive T 1 /T 2 A switching type MRI contrast agent, a preparation method and application thereof. The T is 1 /T 2 The switching type MRI contrast agent has a structure represented by the following formula:
Figure DDA0002482356440000011
wherein X is selected from
Figure DDA0002482356440000012
Or
Figure DDA0002482356440000013
m and n are both selected from 30 to 50; m is ferric oxide nano-particles. The MRI contrast agent provided by the invention can specifically respond in a weak acid microenvironment of a tumor, so that the surface charge of contrast agent particles is changed, and the aggregation of ferric oxide nanoparticles is realized through electrostatic interaction, thereby realizing T 1 Contrast agent to T 2 Switching and activating T of contrast agent 2 The contrast enhancement effect has the advantages of high sensitivity and selectivity, low toxicity, good biocompatibility and the like in tumor diagnosis.

Description

pH responsive T 1 /T 2 Switching type MRI contrast agent and preparation method and application thereof
Technical Field
The invention belongs to the technical field of medicament preparation, and particularly relates to pH-responsive T 1 /T 2 A switching MRI contrast agent (ESIONPs system) and its preparation method and application, such as application in preparing products with tumor detection function.
Background
Early diagnosis of cancer is of great importance in the treatment of cancer. Among many imaging techniques, magnetic Resonance Imaging (MRI) is widely used for clinical diagnosis of tumors due to its advantages of high spatial resolution, no radiation damage, and multi-aspect scanning imaging. Since MRI technology has the drawback of low sensitivity, and it is sometimes difficult to distinguish between diseased tissue and normal tissue in diagnosis, contrast agents are often introduced to address this problem. The contrast agent generally used in clinic is gadolinium-based small molecule contrast agent which can improve the contrast ratio of tumor and normal tissues but still causesThe risk of nephrogenic systemic fibrosis, and the defects of low relaxation rate and no specificity to tumor tissues of the small-molecule contrast agent exist. Therefore, with the growing awareness of tumor tissues, stimuli-responsive contrast agents are attracting increasing attention. The contrast agent responds to the endogenous stimulation of a tumor part, so that the signal is changed, the specificity to the tumor tissue and the MRI signal are enhanced, and the diagnosis accuracy is improved. For example, researchers have synthesized diblock copolymers through polyethylene glycol and polyhistidine, and connected small gadolinium chelates to the terminal of the copolymers and self-assembled to form pH-responsive polymer micelles, which can selectively enhance T in a tumor weak acid microenvironment by protonating imidazole groups in polyhistidine to cause micelle dispersion 1 A contrast signal. While such stimuli-responsive contrast agents may enhance MRI contrast at the tumor site, they may still produce signals in normal tissue that interfere with diagnosis. Thus, T 1 /T 2 Switching contrast agents are being developed to specifically enhance contrast by switching the contrast signal in response to endogenous stimuli at the tumor site, thereby reducing interference from background signals.
At present, T of this type 1 /T 2 Switching type contrast agents have also rarely been reported. Thus, new T is developed 1 /T 2 Switching type contrast agents are a problem to be solved.
Disclosure of Invention
The main purpose of the invention is to provide a pH-responsive T 1 /T 2 A switching type MRI contrast agent, a preparation method and an application thereof, which overcome the defects of the prior art.
In order to achieve the purpose, the technical scheme adopted by the invention comprises the following steps:
the embodiment of the invention provides a pH response T 1 /T 2 A switched MRI contrast agent having a structure according to formula (I):
Figure BDA0002482356420000021
wherein,x is selected from
Figure BDA0002482356420000022
m and n are both selected from 30 to 50; m is ferric oxide nano-particles.
The embodiment of the invention also provides a pH responsive T 1 /T 2 A method of preparing a switched MRI contrast agent, comprising:
at least leading a first uniform mixed reaction system containing benzyl glutamate cyclic anhydride and propargylamine to carry out ring-opening polymerization reaction to prepare poly gamma-benzyl-L-glutamic acid containing alkynyl;
at least enabling a second uniformly mixed reaction system containing the alkynyl-containing poly gamma-benzyl-L-glutamic acid and a hydrogen bromide acetic acid solution to react to prepare alkynyl-containing poly-L-glutamic acid;
at least enabling the poly gamma-benzyl-L-glutamic acid containing alkynyl to react with a third uniformly mixed reaction system of 2-hydroxypyridine and diethylenetriamine to prepare poly { N- [ N' - (2-aminoethyl) -2-aminoethyl ] glutamic acid } containing alkynyl;
reacting at least a fourth intimately mixed reaction system comprising said alkynyl-containing poly { N- [ N '- (2-aminoethyl) -2-aminoethyl ] glutamic acid } and acrylic acid under basic conditions to produce alkynyl-containing poly (N- { N' - [ N "- (2-carboxyethyl) -2-aminoethyl ] -2-aminoethyl } glutamine);
reacting at least a fifth uniformly mixed reaction system of polyethylene glycol with azide and amino end groups and ferric oxide nanoparticles with carboxyl activated ester modified on the surface to prepare ultra-small ferric oxide nanoparticles with azide partially modified by polyethylene glycol on the surface;
reacting at least a sixth uniformly mixed reaction system containing the ultra-small iron oxide nanoparticles with surfaces modified with azido groups by polyethylene glycol moieties, the alkynyl-containing poly-L-glutamic acid and a cuprous catalyst to prepare the ultra-small iron oxide nanoparticles with surfaces connected with the poly-L-glutamic acid by polyethylene glycol;
reacting at least a seventh uniformly mixed reaction system comprising ferric oxide nanoparticles with surfaces modified with azido groups by polyethylene glycol moieties, poly (N- { N '- [ N' - (2-carboxyethyl) -2-aminoethyl ] -2-aminoethyl } glutamine) containing alkynyl groups and a monovalent copper catalyst to prepare ferric oxide nanoparticles with surfaces connected with poly (N- { N '- [ N' - (2-carboxyethyl) -2-aminoethyl ] -2-aminoethyl } glutamine) by polyethylene glycol;
and attaching the surface to the ferric oxide nanoparticles of poly-L-glutamic acid by polyethylene glycol, and attaching the surface to poly (N- { N '- [ N' - (2-carboxyethyl) -2-aminoethyl) by polyethylene glycol]-2-aminoethyl } glutamine) with water to form a pH-responsive T 1 /T 2 Switching type MRI contrast agents.
The embodiment of the invention also provides pH-responsive T prepared by the method 1 /T 2 Switching type MRI contrast agents.
The embodiment of the invention also provides the pH response T 1 /T 2 Use of a switchable MRI contrast agent in the manufacture of a product having tumor detection capabilities.
The embodiment of the invention also provides a composition for radiography, which comprises the following components: t of the aforementioned pH response 1 /T 2 A switched-mode MRI contrast agent; and, a pharmaceutically acceptable adjuvant.
The embodiment of the invention also provides a medicinal composition, which comprises: the aforementioned pH responsive T 1 /T 2 A switchable MRI contrast agent, a drug, and a pharmaceutically acceptable carrier.
The embodiment of the invention also provides a non-medical purpose radiography method, which comprises the following steps: the aforementioned contrast agent or the aforementioned composition for contrast is administered to a subject to be contrasted, and contrast is performed.
Compared with the prior art, the invention has the beneficial effects that:
(1) The pH responsive T provided by the invention 1 /T 2 The switching type MRI contrast agent (ESIONPs system) takes ferric oxide nanoparticles as a carrier, can respond and initiate aggregation under the weak acid condition, has good biocompatibility, can specifically aggregate under the weak acid microenvironment of tumor and perform T 1 Contrast signal to T 2 The interference of background signals is reduced by switching the contrast signals, so that the sensitivity is effectively improved, the interference of the background signals is reduced, and the specificity and the selectivity are realized on the imaging of tumor parts. At the same time, the pH responds to T 1 /T 2 The switching type MRI contrast agent is rapidly aggregated in a tumor microenvironment to cause the particle size to be enlarged, so that the high permeability and retention Effect (EPR) can be effectively enhanced, and the enrichment of the contrast agent in a tumor part is improved;
(2) The pH responsive T provided by the invention 1 /T 2 The switching type MRI contrast agent consists of two iron sesquioxide nanoparticles, wherein the response of the particle with the zwitterionic polymer on the surface under the weak acid environment leads to the change from electric neutrality to positive surface charge, and the surface of the other particle with the carboxyl on the surface carries negative charge. The pH response T is realized through the electrostatic interaction between the positive and negative charges on the surfaces of two ferric oxide nanoparticles 1 /T 2 Switched MRI contrast agents (ESIONPs systems) aggregate, wherein the transition T is due to rapid progressive ionization of zwitterionic polymers 1 Contrast-like agents to T 2 Switching of the contrast-like agent will rapidly activate T at the tumor site 2 The contrast enhancement effect improves the imaging contrast of the tumor part.
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In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings needed to be used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments described in the present application, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of alkynyl-containing poly-gamma-benzyl-L-glutamic acid as a product in step (1) in example 1 of the present invention;
FIG. 2 is a nuclear magnetic resonance hydrogen spectrum of alkynyl group-containing poly-L-glutamic acid as a product in step (2) in example 1 of the present invention;
FIG. 3 is a NMR spectrum of an alkynyl-containing poly { N- [ N' - (2-aminoethyl) -2-aminoethyl ] glutamic acid } produced in step (3) of example 1 of the present invention;
FIG. 4 is a NMR spectrum of poly (N- { N' - [ N "- (2-carboxyethyl) -2-aminoethyl ] -2-aminoethyl } glutamine) of the product of alkynyl in step (4) of example 1 of the present invention;
FIG. 5 is an infrared spectrum of ferric oxide nanoparticles with azido groups modified by polyethylene glycol moieties on the surface of the product in step (5) of example 1 according to the present invention;
FIG. 6 is an infrared spectrum of iron trioxide nanoparticles whose surfaces are polyethylene glycol-linked to poly-L-glutamic acid and iron trioxide nanoparticles whose surfaces are polyethylene glycol-linked to poly (N- { N' - [ N "- (2-carboxyethyl) -2-aminoethyl ] -2-aminoethyl } glutamine) in steps (6) and (7) of example 1 of the present invention;
FIGS. 7 a-7 b show the longitudinal relaxivity (r) of the MRI contrast agent of example 1 under the conditions of physiological neutrality (pH 7.4) and weak acidity of the tumor microenvironment (pH 6.5) 1 ) Relaxation rate contrast maps and corresponding solution imaging;
FIGS. 8 a-8 b show the transverse relaxivity (r) of the MRI contrast agent of example 1 under the conditions of physiological neutrality (pH 7.4) and weak acidity of the tumor microenvironment (pH 6.5) 2 ) Relaxation rate contrast maps and corresponding solution imaging;
FIG. 9 is a cytotoxicity test chart of the MRI contrast agent in Human Umbilical Vein Endothelial Cells (HUVEC) and mouse breast cancer cells (4T 1) in example 1 of the present invention;
FIG. 10 is a graph of a tissue toxicology test in BALB/c mice with MRI contrast agent of example 1 of the present invention;
FIG. 11 is an in vivo imaging of MRI contrast agents in example 1 of the present invention on BALB/c mice transplanted with 4T1 cell tumors.
Detailed Description
In view of the defects of the prior art, the inventors of the present invention have made extensive studies and extensive practices to provide a technical solution of the present invention, which mainly uses the iron trioxide nanoparticles as carriers to design, and the iron trioxide nanoparticles show T due to their smaller magnetic moment and surface paramagnetic iron ions 1 Contrast effect, and T is shown by the non-uniformity of the surrounding magnetic field after particle aggregation 2 And (4) contrast effect. In addition, the ferric oxide nano-particles have low toxicity and good biocompatibility. The unique property and excellent biological safety make the ferric oxide nano-particles suitable as a carrier for constructing T 1 /T 2 Switching type contrast agents.
The technical solutions of the present invention will be described clearly and completely below, and it should be apparent that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
An aspect of an embodiment of the present invention provides a pH-responsive T 1 /T 2 A switched MRI contrast agent having a structure according to formula (I):
Figure BDA0002482356420000051
wherein X is selected from
Figure BDA0002482356420000052
m and n are both selected from 30 to 50; m is ferric oxide nano-particles.
In some more specific embodiments, the iron trioxide nanoparticles have a particle size of less than 5nm, and the iron trioxide nanoparticles are ultra-small iron trioxide nanoparticles.
The pH responsive T provided by the embodiments of the invention 1 /T 2 Switchable MRI contrast agents (ESIONPs systems) comprise iron trioxide nanoparticles that trigger aggregation in a weakly acidic tumor microenvironment, resulting in switching of the contrast effect, wherein the iron trioxide nanoparticles can act as a T due to their ultra-small magnetic moment and surface paramagnetic iron ion 1 The contrast agent is prepared by modifying polyethylene glycol on the surface of nanoparticles to obtain monodisperse ferric oxide nanoparticles in water. Subsequently, it will be under a weak acidThe zwitterionic polymer capable of being changed from neutral to positive charge and the polyglutamic acid (carboxyl is contained in a polymeric structure) which is electronegative under neutral and weak acid conditions are respectively modified on the surfaces of the nanoparticles through polyethylene glycol, the two nanoparticles are mixed at equal concentration to obtain the pH-responsive contrast agent which can generate electrostatic interaction and aggregate under the weak acid condition of a tumor microenvironment, and then T is used for controlling the pH value of the contrast agent to be higher than the pH value of the contrast agent 1 Contrast agent to T 2 Switching of contrast agent to selectively produce T at the tumor site 2 The contrast signal is enhanced. Meanwhile, as the size of the particles after aggregation is enlarged, the EPR effect (high permeability and retention effect) can be effectively enhanced, and the enrichment and residence time of the contrast agent in tumor tissues can be prolonged.
Experiments show that the pH response T provided by the embodiment of the invention 1 /T 2 Longitudinal relaxation rate r of switchable MRI contrast agents (ESIONPs systems) before and after response 1 From 5.7118mM -1 ·s -1 It was changed to 3.8804mM -1 ·s -1 Transverse relaxation rate r 2 From 9.1074mM -1 ·s -1 It was changed to 42.1950mM -1 ·s -1 R of which 2 /r 1 The ratio changed from 1.59 to 10.87, which also indicates that the contrast agent has a T value under weak acid conditions 1 Type is converted into T 2 Contrast agents of the type which selectively produce T at the tumour site 2 The contrast signal is enhanced, thereby reducing the interference of background signals and improving the specificity and selectivity of the contrast agent. In addition, the contrast agent has good biocompatibility and extremely low biotoxicity.
In another aspect of the embodiments of the present invention, there is also provided a pH responsive T 1 /T 2 A method of preparing a switched MRI contrast agent, comprising:
at least leading a first uniform mixed reaction system containing benzyl glutamate cyclic anhydride and propargylamine to carry out ring-opening polymerization reaction to prepare poly gamma-benzyl-L-glutamic acid containing alkynyl;
at least enabling a second uniformly mixed reaction system containing the alkynyl poly-gamma-benzyl-L-glutamic acid and a hydrogen bromide acetic acid solution to react to prepare alkynyl poly-L-glutamic acid;
at least enabling the alkynyl-containing poly gamma-benzyl-L-glutamic acid to react with a third uniformly mixed reaction system of 2-hydroxypyridine and diethylenetriamine to prepare alkynyl-containing poly { N- [ N' - (2-aminoethyl) -2-aminoethyl ] glutamic acid };
reacting at least a fourth uniformly mixed reaction system containing the alkynyl-containing poly { N- [ N '- (2-aminoethyl) -2-aminoethyl ] glutamic acid } and acrylic acid under basic conditions to obtain alkynyl-containing poly (N- { N' - [ N "- (2-carboxyethyl) -2-aminoethyl ] -2-aminoethyl } glutamine);
reacting at least a fifth uniform mixed reaction system of polyethylene glycol with terminal groups of azide and amino and polyethylene glycol with terminal groups of amino with the ultra-small ferric oxide nanoparticles with the surface modified with carboxyl activated ester to prepare ferric oxide nanoparticles with the surface partially modified with azide groups by polyethylene glycol;
reacting a sixth uniformly mixed reaction system which at least comprises the ferric oxide nanoparticles with surfaces modified with azido groups through polyethylene glycol moieties, the alkynyl-containing poly-L-glutamic acid and a cuprous catalyst to obtain the ferric oxide nanoparticles with surfaces connected with the poly-L-glutamic acid through polyethylene glycol;
reacting at least a seventh uniformly mixed reaction system comprising ferric oxide nanoparticles with surfaces modified with azide groups through polyethylene glycol moieties, poly (N- { N '- [ N' - (2-carboxyethyl) -2-aminoethyl ] -2-aminoethyl } glutamine) containing alkynyl groups and a monovalent copper catalyst to prepare the ferric oxide nanoparticles with surfaces connected with the poly (N- { N '- [ N' - (2-carboxyethyl) -2-aminoethyl ] -2-aminoethyl } glutamine) through polyethylene glycol;
and the surface is connected with the ferric oxide nano-particles of poly-L-glutamic acid through polyethylene glycol, and the surface is connected with the poly (N- { N '- [ N' - (2-carboxyethyl) -2-aminoethyl) through polyethylene glycol]-2-aminoethyl } glutamine) with water to form a pH-responsive T 1 /T 2 A switched MRI contrast agent.
In some more specific embodiments, the preparation method specifically comprises: under a protective atmosphere, dissolving glutamic acid benzyl ester cyclic internal anhydride in dichloromethane and dimethylformamide to form a solution, then adding propargylamine into the solution to form a first uniform mixed reaction system, and reacting at 20-25 ℃ for 24-48 h to prepare the alkynyl-containing poly gamma-benzyl-L-glutamic acid, wherein the volume of dichloromethane is larger than that of dimethylformamide.
Furthermore, the volume ratio of the dichloromethane to the dimethylformamide is 3-5:1.
Further, the molar ratio of the glutamic acid benzyl ester cyclic anhydride to propargylamine is greater than 40, and particularly preferably 60 to 40.
Further, the protective atmosphere includes a nitrogen atmosphere or an inert gas atmosphere.
In some more specific embodiments, the preparation method specifically comprises: dissolving the alkynyl-containing poly gamma-benzyl-L-glutamic acid in trifluoroacetic acid, adding 33 mass percent of hydrogen bromide acetic acid solution to form a second uniform mixed reaction system, reacting at 50-60 ℃ for 2-4 h, cooling the second uniform mixed reaction system to room temperature, and continuing to react for 12-24 h to obtain the alkynyl-containing poly-L-glutamic acid, wherein the hydrogen bromide acetic acid solution is a directly purchased reagent with CAS number of 37348-16-6.
In some more specific embodiments, the preparation method specifically comprises: dissolving the alkynyl-containing poly gamma-benzyl-L-glutamic acid and 2-hydroxypyridine in methyl pyrrolidone to form a solution, adding diethylenetriamine to form a third uniformly mixed reaction system, and reacting at 20-25 ℃ for 48-72 h to obtain the alkynyl-containing poly { N- [ N' - (2-aminoethyl) -2-aminoethyl ] glutamic acid }.
Furthermore, the mol ratio of benzyl and 2-hydroxypyridine in the alkynyl-containing poly gamma-benzyl-L-glutamic acid is 1:5-10.
Furthermore, the molar ratio of benzyl to diethylenetriamine in the alkynyl-containing poly gamma-benzyl-L-glutamic acid is 1.
In some more specific embodiments, the preparation method specifically comprises: dissolving the alkynyl-containing poly { N- [ N '- (2-aminoethyl) -2-aminoethyl ] glutamic acid } in an alkaline aqueous solution under a protective atmosphere, adding acrylic acid to form a fourth uniformly mixed reaction system, and reacting at 20-25 ℃ for 5-7 days to obtain the alkynyl-containing poly (N- { N' - [ N '- (2-carboxyethyl) -2-aminoethyl ] -2-aminoethyl } glutamine), wherein the molar ratio of the acrylic acid to the alkynyl-containing poly { N- [ N' - (2-aminoethyl) -2-aminoethyl ] glutamic acid } is 10-20.
Further, the basic aqueous solution includes an aqueous sodium bicarbonate solution, and is not limited thereto.
Further, the concentration of the sodium bicarbonate aqueous solution is 500mmol/L.
Further, the protective atmosphere includes a nitrogen atmosphere or an inert gas atmosphere.
In some more specific embodiments, the preparation method specifically comprises: mixing ferric oxide nanoparticles with surfaces modified with carboxyl activated ester, polyethylene glycol with end groups of azide and amino and polyethylene glycol with end groups of amino in chloroform, adding N, N-diisopropylethylamine to form a fifth uniform mixed reaction system, and reacting at 20-25 ℃ for 12-24 h to obtain the ferric oxide nanoparticles with surfaces partially modified with azide groups through polyethylene glycol; the mass ratio of the polyethylene glycol with the terminal groups of azide and amino to the polyethylene glycol with the terminal groups of amino is 1:1, and the mass ratio of the ferric oxide nanoparticles of the surface modified carboxyl activated ester to the polyethylene glycol with the terminal groups of azide and amino is 1:5-10.
Further, the iron trioxide nanoparticles of the surface-modified carboxyl-activated ester include, but are not limited to, iron trioxide nanoparticles of a surface-modified pentafluorophenol ester.
Furthermore, the molar ratio of the N, N-diisopropylethylamine to the polyethylene glycol with the amino end group is 100-200.
In some more specific embodiments, the preparation method specifically comprises: dissolving the ferric oxide nanoparticles with the surfaces modified with azido groups through polyethylene glycol in dimethylformamide, adding the alkynyl-containing poly-L-glutamic acid, then carrying out freezing deoxidization, adding sodium ascorbate and copper sulfate in a protective atmosphere to form a sixth uniform mixed reaction system, and reacting at 20-25 ℃ for 48-72 h to obtain the ferric oxide nanoparticles with the surfaces connected with poly-L-glutamic acid through polyethylene glycol.
In some more specific embodiments, the preparation method specifically comprises: dissolving the ferric oxide nanoparticles with the surfaces modified with azide groups through polyethylene glycol in dimethylformamide, adding the alkynyl-containing poly (N- { N '- [ N' - (2-carboxyethyl) -2-aminoethyl ] -2-aminoethyl } glutamine), carrying out freezing deoxidization, adding sodium ascorbate and copper sulfate in protective atmosphere to form a seventh uniform mixed reaction system, and reacting at 20-25 ℃ for 48-72 h to obtain the ferric oxide nanoparticles with the surfaces connected with poly (N- { N '- [ N' - (2-carboxyethyl) -2-aminoethyl ] -2-aminoethyl } glutamine) through polyethylene glycol.
In some more specific embodiments, the preparation method specifically comprises: connecting the ferric oxide nanoparticles with the surface connected with poly-L-glutamic acid through polyethylene glycol and connecting the poly (N- { N '- [ N' - (2-carboxyethyl) -2-aminoethyl) through polyethylene glycol]-2-aminoethyl } glutamine) in water, followed by mixing to form a pH-responsive T 1 /T 2 A switched MRI contrast agent.
Further, the molar ratio of the ferric oxide nanoparticles with the surface modified with azido groups by polyethylene glycol to the ferric oxide nanoparticles with the surface connected with poly (N- { N '- [ N' - (2-carboxyethyl) -2-aminoethyl ] -2-aminoethyl } glutamine) by polyethylene glycol is 1:1-3, preferably any one of 1:1, 1:2 and 1:3.
In some more specific embodiments, the preparation method further comprises: and (3) carrying out post-treatment steps such as purification and the like on the ferric oxide nanoparticles with the surfaces connected with the poly-L-glutamic acid through polyethylene glycol and the ferric oxide nanoparticles with the surfaces connected with the poly (N- { N '- [ N' - (2-carboxyethyl) -2-aminoethyl ] -2-aminoethyl } glutamine) through polyethylene glycol. For example, two kinds of iron sesquioxide nanoparticles, which are surface-linked with poly-L-glutamic acid and poly (N- { N' - [ N "- (2-carboxyethyl) -2-aminoethyl ] -2-aminoethyl } glutamine) respectively by polyethylene glycol, may be dispersed in water, dialyzed against a dialysis bag having a molecular weight cut-off of 30000 to 50000Da, and subjected to lyophilization treatment, particularly preferably 35000 to 50000Da.
In some more specific embodiments, the method of making can comprise:
(1) Preparation of alkynyl-containing poly gamma-benzyl-L-glutamic acid: dichloromethane was stirred overnight with calcium hydride and distilled to give a dried solution. Dimethylformamide was dried over calcium hydride and distilled under reduced pressure to obtain a dried solution. Dissolving benzyl glutamate cyclic anhydride in dichloromethane and dimethylformamide under a protective atmosphere, adding propargylamine, and reacting at room temperature for 24-48 h to obtain alkynyl-containing poly gamma-benzyl-L-glutamic acid. Wherein the volume of the dichloromethane is larger than that of the dimethylformamide. The molar ratio of the glutamic acid benzyl ester cyclic anhydride to the propargylamine is 60-40.
(2) Preparation of alkynyl-containing poly-L-glutamic acid: dissolving poly-gamma-benzyl-L-glutamic acid containing alkynyl in trifluoroacetic acid, placing in a 100mL round-bottom flask, adding 33wt% hydrogen bromide acetic acid solution, reacting at 50-60 ℃ for 2-4 h, cooling to room temperature, and reacting for 12-24 h to obtain poly-L-glutamic acid containing alkynyl.
(3) Preparation of alkynyl-containing poly { N- [ N' - (2-aminoethyl) -2-aminoethyl ] glutamic acid }: adding poly gamma-benzyl-L-glutamic acid containing alkynyl and 2-hydroxypyridine into a 100mL round-bottom flask according to the molar ratio of 1:5-10 of benzyl to 2-hydroxypyridine, dissolving in methylpyrrolidone under a protective atmosphere to form a solution, adding diethylenetriamine (the molar ratio of benzyl to diethylenetriamine is 1-50), and reacting at room temperature for 48-72 h to obtain poly { N- [ N' - (2-aminoethyl) -2-aminoethyl ] glutamic acid } containing alkynyl.
(4) Preparation of alkynyl-containing poly (N- { N' - [ N "- (2-carboxyethyl) -2-aminoethyl ] -2-aminoethyl } glutamine): dissolving alkynyl-containing poly { N- [ N '- (2-aminoethyl) -2-aminoethyl ] glutamic acid } in 400-600 mmol/L aqueous solution of sodium carbonate, dripping acrylic acid, and reacting at room temperature for 5-7 days under a protective atmosphere to obtain alkynyl-containing poly (N- { N' - [ N '- (2-carboxyethyl) -2-aminoethyl ] -2-aminoethyl } glutamine), wherein the using amount of the acrylic acid is far larger than that of alkynyl-containing poly { N- [ N' - (2-aminoethyl) -2-aminoethyl ] glutamic acid }.
(5) Preparing ferric oxide nanoparticles with surfaces modified by azido groups through polyethylene glycol moieties: mixing ferric oxide nanoparticles of which the surfaces are modified with pentafluorophenol ester, polyethylene glycol of which the end groups are azide and amino and polyethylene glycol of which the end group is amino with chloroform according to the mass ratio of 1. Wherein, the mol ratio of the N, N-diisopropylethylamine to the polyethylene glycol with the end group as amino is 100-200.
(6) Preparing ferric oxide nanoparticles with surfaces modified by polyethylene glycol parts, wherein the ferric oxide nanoparticles comprise poly-L-glutamic acid and are prepared by the following steps: dissolving the ferric oxide nanoparticles with the surfaces modified by the azido groups through the polyethylene glycol moieties and poly-L-glutamic acid containing the alkynyl groups in the dimethylformamide according to a mass ratio of 1:5-10, then freezing and deoxidizing for three times, quickly adding sodium ascorbate and copper sulfate pentahydrate into a solution of the ferric oxide nanoparticles with the surfaces modified by the azido groups through the polyethylene glycol moieties and the poly-L-glutamic acid containing the alkynyl groups in a protective atmosphere, and reacting for 48-72 h at room temperature in the protective atmosphere to obtain the ferric oxide nanoparticles with the surfaces modified by the polyethylene glycol moieties.
(7) Preparation of iron sesquioxide nanoparticles of poly (N- { N' - [ N "- (2-carboxyethyl) -2-aminoethyl ] -2-aminoethyl } glutamine) surface modified by polyethylene glycol moieties: dissolving the ferric oxide nanoparticles with the azido groups modified by the polyethylene glycol moieties on the surface and poly (N- { N ' - [ N ' - (2-carboxyethyl) -2-aminoethyl ] -2-aminoethyl } glutamine) containing alkynyl in dimethylformamide according to the mass ratio of 1:5-10, then freezing for deoxygenation for three times, quickly adding sodium ascorbate and copper sulfate pentahydrate into a solution of the ferric oxide nanoparticles with the azido groups modified by the polyethylene glycol moieties on the surface and poly (N- { N ' - [ N ' - (2-carboxyethyl) -2-aminoethyl ] -2-aminoethyl } glutamine) containing alkynyl in a protective atmosphere, and reacting for 48-72 hours at room temperature in the protective atmosphere to obtain the ferric oxide nanoparticles with the poly { N- [ N ' - (2-aminoethyl) -2-aminoethyl ] glutamic acid } modified by the polyethylene glycol moieties on the surface.
(8) pH responsive T 1 /T 2 Preparation of switchable MRI contrast agents (ESIONPs systems): ferric oxide nanoparticles with surfaces modified by polyethylene glycol part and poly (N- { N '- [ N' - (2-carboxyethyl) -2-aminoethyl) with surfaces modified by polyethylene glycol part]-2-aminoethyl } glutamine) to obtain pH-responsive T 1 /T 2 Switchable MRI contrast agents (ESIONPs systems).
In the preparation step of the alkynyl-containing poly-gamma-benzyl-L-glutamic acid, the alkynyl-containing poly-gamma-benzyl-L-glutamic acid is obtained through ring-opening polymerization, slowly dropped into glacial ethyl ether, filtered to collect solid, dissolved in dichloromethane, dropped into the glacial ethyl ether again for precipitation and purification, repeated for three times or more, and then subjected to rotary evaporation to remove the ethyl ether, and vacuum drying and purification.
In the preparation step of the alkynyl-containing poly-L-glutamic acid, the alkynyl-containing poly-L-glutamic acid can be dripped into excessive acetone to obtain a white solid, the white solid is collected by filtration and washed by acetone for a plurality of times, and then the diethyl ether is removed by rotary evaporation, and the white solid is dried and purified in vacuum.
Wherein, in the preparation step of the alkynyl-containing poly { N- [ N '- (2-aminoethyl) -2-aminoethyl ] glutamic acid }, the alkynyl-containing poly { N- [ N' - (2-aminoethyl) -2-aminoethyl ] glutamic acid } can be precipitated in a large amount of diethyl ether, dissolved in dichloromethane after centrifugation and then precipitated in a large amount of diethyl ether, the solid is collected by filtration, dissolved in dichloromethane and precipitated again in diethyl ether, repeated for 3 times or more, the precipitate is collected by centrifugation and dissolved in deionized water, the pH is adjusted to neutrality by hydrochloric acid, dialyzed in 10 to 20mmol/L HCl aqueous solution for two days by using a dialysis bag with molecular weight cut-off of 3500 to 5000Da, dialyzed in deionized water for three days, and freeze-dried.
Wherein in the preparation step of the alkynyl-containing poly (N- { N '- [ N' - (2-carboxyethyl) -2-aminoethyl ] -2-aminoethyl } glutamine), the alkynyl-containing poly (N- { N '- [ N' - (2-carboxyethyl) -2-aminoethyl ] -2-aminoethyl } glutamine) is obtained by Michael addition reaction, and is dialyzed for 12h in 10-20 mmol/L NaOH aqueous solution by using a dialysis bag with molecular weight cut-off of 3500-5000 Da, dialyzed for 12h in 10-20 mmol/L HCl aqueous solution, dialyzed for three days in deionized water, and freeze-dried.
Wherein, in the step of preparing the ferric oxide nanoparticles with the surfaces modified with azido groups through polyethylene glycol moieties, the ferric oxide nanoparticles with the surfaces modified with azido groups through polyethylene glycol moieties can be concentrated, dropped into a large amount of ether, centrifugally collected, dissolved in deionized water, dialyzed for three days by a dialysis bag with cut-off molecular weight of 35000-50000 Da, and freeze-dried.
Wherein, in the preparation step of the ferric oxide nanoparticles with the surfaces modified with the poly-L-glutamic acid through the polyethylene glycol part, the ferric oxide nanoparticles with the surfaces modified with the poly-L-glutamic acid through the polyethylene glycol part are obtained through click reaction, are directly dialyzed in deionized water for three days by a dialysis bag with the molecular weight cutoff of 35000-50000 Da, and are frozen and dried.
Wherein in the preparation step of the ferric oxide nanoparticles of the poly { N- [ N '- (2-aminoethyl) -2-aminoethyl ] glutamic acid } partially modified on the surface by polyethylene glycol, the ferric oxide nanoparticles of the poly { N- [ N' - (2-aminoethyl) -2-aminoethyl ] glutamic acid } partially modified on the surface by polyethylene glycol are obtained by click reaction, directly dialyzed in deionized water for three days by a dialysis bag with molecular weight cutoff of 35000-50000 Da, and freeze-dried.
The dichloromethane, chloroform and dimethylformamide employed in the above embodiments of the present invention may also be replaced by other suitable organic solvents.
In another aspect of embodiments of the invention there is also provided a pH responsive T prepared by the foregoing method 1 /T 2 A switched MRI contrast agent.
Hair brushAnother aspect of an illustrative embodiment also provides the foregoing pH responsive T 1 /T 2 Use of a switchable MRI contrast agent in the manufacture of a product having tumor detection capabilities.
Further, fe in the product 3+ The concentration is 30-50 mmol/L.
For example, another aspect of an embodiment of the present invention also provides a composition for contrast, including: the aforementioned pH responsive T 1 /T 2 A switched-mode MRI contrast agent; and, a pharmaceutically acceptable adjuvant.
Further, the pharmaceutically acceptable adjuvant includes a diluent, and is not limited thereto.
For example, another aspect of an embodiment of the present invention also provides a pharmaceutical composition comprising: the aforementioned pH responsive T 1 /T 2 A switchable MRI contrast agent, a drug, and a pharmaceutically acceptable carrier.
The term "carrier" as used herein has a meaning well known to those skilled in the art and can include any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, the like, and combinations thereof.
Another aspect of an embodiment of the present invention also provides a method for imaging for non-medical purposes, which includes: the aforementioned contrast agent or the aforementioned composition for contrast is administered to a subject to be contrasted, and contrast is performed.
The object to be imaged may be a living tissue of a living body, such as a tumor tissue, and the like, but is not limited thereto.
The pH responsive T provided by the above examples of the invention 1 /T 2 The switching type MRI contrast agent has high specificity and selectivity on tumor parts, good biocompatibility and low toxicity, and can be metabolized in vivo quickly, so that the contrast agent has excellent imaging contrast performance, can avoid the interference of background signals and is a tumor magnetic resonance imaging agentVibroseis imaging offers high sensitivity and tumor targeting specificity. Further, the above examples of the present invention provide a pH responsive T 1 /T 2 The size of the switching MRI contrast agent after aggregation becomes large such that its retention effect at tumor tissue is significant.
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention, and that experimental conditions and set parameters should not be construed as limiting the basic embodiments of the invention. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
Example 1
pH responsive T 1 /T 2 A method for preparing switchable MRI contrast agents (ESIONPs systems) comprises the steps of:
(1) Preparation of alkynyl-containing poly gamma-benzyl-L-glutamic acid: stirring dichloromethane through calcium hydride overnight and distilling to obtain a dried solution, drying dimethylformamide through calcium hydride and distilling under reduced pressure to obtain a dried solution, dissolving benzyl glutamate intra-cyclic anhydride (BLG-NCA) in dichloromethane and dimethylformamide (the volume ratio is 4:1) under a protective atmosphere, adding propargylamine, reacting at 25 ℃ for 24 hours, wherein the molar ratio of the benzyl glutamate intra-cyclic anhydride to the propargylamine is 40;
(2) Preparation of alkynyl-containing poly-L-glutamic acid: dissolving poly-gamma-benzyl-L-glutamic acid containing alkynyl in trifluoroacetic acid, placing the solution in a 100mL round-bottom flask, adding 33wt% of hydrogen bromide acetic acid solution, reacting at 50 ℃ for 2h, and cooling to 25 ℃ for 12h to obtain poly-L-glutamic acid containing alkynyl. And after the reaction is finished, dropwise adding excessive acetone into the solution to obtain a white solid, filtering and collecting, washing with acetone for multiple times, then performing rotary evaporation to remove diethyl ether, and performing vacuum drying and purification to obtain alkynyl-containing poly-L-glutamic acid (alkynyl-PGA). The synthetic routes of the steps (1) and (2) can be represented by the following chemical equations:
Figure BDA0002482356420000121
(3) Preparation of alkynyl-containing poly { N- [ N' - (2-aminoethyl) -2-aminoethyl ] glutamic acid }: adding poly gamma-benzyl-L-glutamic acid containing alkynyl and 2-hydroxypyridine into a 100mL round-bottom flask according to the molar ratio of 1:5 of benzyl to 2-hydroxypyridine, dissolving into methyl pyrrolidone to form a solution under a protective atmosphere, adding diethylenetriamine (the molar ratio of benzyl to diethylenetriamine is 1: 50), and reacting at 25 ℃ for 48h to obtain poly { N- [ N' - (2-aminoethyl) -2-aminoethyl ] glutamic acid } containing alkynyl; after the reaction is finished, precipitating the obtained solution in a large amount of diethyl ether, dissolving the solution by using dichloromethane after centrifugation, then precipitating the solution by using a large amount of diethyl ether, filtering, collecting a solid, dissolving the solid in dichloromethane, precipitating the solid by using the diethyl ether again, repeating the steps for 3 times, centrifugally collecting the precipitate, dissolving the precipitate in deionized water, adjusting the pH to be neutral by using hydrochloric acid, dialyzing the precipitate in a 10mmol/L HCl aqueous solution for two days by using a dialysis bag with the molecular weight cut-off of 3500Da, dialyzing the precipitate in the deionized water for three days, and then carrying out freeze drying treatment to obtain alkynyl-containing poly { N- [ N' - (2-aminoethyl) -2-aminoethyl ] glutamic acid } (alkynyl-PGlu (DET));
(4) Preparation of alkynyl-containing poly (N- { N' - [ N "- (2-carboxyethyl) -2-aminoethyl ] -2-aminoethyl } glutamine): dissolving alkynyl-containing poly { N- [ N ' - (2-aminoethyl) -2-aminoethyl ] glutamic acid } in 500mmol/L aqueous sodium carbonate solution, dropping acrylic acid, and reacting at 25 ℃ for 7 days under a protective atmosphere to obtain alkynyl-containing poly (N- { N ' - [ N "- (2-carboxyethyl) -2-aminoethyl ] -2-aminoethyl } glutamine), wherein the molar amount of acrylic acid is 100 times that of alkynyl-containing poly { N- [ N ' - (2-aminoethyl) -2-aminoethyl ] glutamic acid }. After the reaction is finished, dialyzing the mixture for 12h in 10mmol/L NaOH aqueous solution by using a dialysis bag with the molecular weight cut-off of 3500Da, dialyzing the mixture for 12h in 10mmol/L HCl aqueous solution, finally dialyzing the mixture for three days in deionized water, and performing freeze drying treatment to obtain alkynyl-containing poly (N- { N '- [ N' - (2-carboxyethyl) -2-aminoethyl ] -2-aminoethyl } glutamine) (alkinyl-PGlu (DET-Car)), wherein the synthetic routes of the steps (3) and (4) can be represented by the following chemical equations:
Figure BDA0002482356420000141
(5) Preparing ferric oxide nanoparticles with surfaces modified by azido groups through polyethylene glycol moieties: mixing ferric oxide nanoparticles of surface-modified pentafluorophenol ester, polyethylene glycol with end groups of azide and amino and polyethylene glycol with end group of amino in trichloromethane according to a mass ratio of 1 3 )；
(6) Preparing ferric oxide nanoparticles with surfaces modified by polyethylene glycol parts, wherein the ferric oxide nanoparticles comprise poly-L-glutamic acid and are prepared by the following steps: dissolving the ferric oxide nanoparticles with azido groups modified by polyethylene glycol moieties on the surface and poly-L-glutamic acid containing alkynyl in dimethylformamide according to a mass ratio of 1:5, then carrying out freezing deoxidization for three times, quickly adding sodium ascorbate and copper sulfate pentahydrate into a solution of the ferric oxide nanoparticles with azido groups modified by polyethylene glycol moieties on the surface and poly-L-glutamic acid containing alkynyl in a protective atmosphere, then carrying out reaction for 48 hours at 25 ℃ in the protective atmosphere to obtain the ferric oxide nanoparticles with poly-L-glutamic acid modified by polyethylene glycol moieties on the surface, directly dialyzing the ferric oxide nanoparticles with a dialysis bag with molecular weight cutoff of 35000Da in deionized water for three days after the reaction is finished, and carrying out freeze drying treatment to obtain the ferric oxide nanoparticles (ESIONPs-PEG-PGA) with poly-L-glutamic acid modified by polyethylene glycol moieties on the surface;
(7) Preparation of iron sesquioxide nanoparticles of poly (N- { N' - [ N "- (2-carboxyethyl) -2-aminoethyl ] -2-aminoethyl } glutamine) surface modified by polyethylene glycol moieties: dissolving the ferric oxide nanoparticles with the surface modified by the polyethylene glycol part and poly (N- { N '- [ N' - (2-carboxyethyl) -2-aminoethyl ] -2-aminoethyl } glutamine) containing alkynyl according to the mass ratio of 1:5 in dimethylformamide, then carrying out freeze deoxidization three times, quickly adding sodium ascorbate and copper sulfate pentahydrate into a solution of the ferric oxide nanoparticles with the surface modified by the polyethylene glycol part and poly (N- { N '- [ N' - (2-carboxyethyl) -2-aminoethyl ] -2-aminoethyl } glutamine) containing alkynyl in a protective atmosphere, then carrying out reaction for 48 hours at 25 ℃ in the protective atmosphere to obtain the ferric oxide nanoparticles with the surface modified by the polyethylene glycol part, namely poly { N- [ N '- (2-aminoethyl) -2-aminoethyl ] glutamic acid }, directly dialyzing the ferric oxide nanoparticles with a dialysis bag with the molecular weight of 35000Da in deionized water for three days, carrying out freeze drying treatment to obtain the ferric oxide nanoparticles with the surface modified by the polyethylene glycol part, namely poly (N- { N' - [ N '- (2-aminoethyl) -2-aminoethyl ] glutamic acid }, wherein the chemical formula of poly (N- { N' - [ 2-aminoethyl ] -2-aminoethyl } glutamine) can be synthesized by the following steps of PEG ONEqui:
Figure BDA0002482356420000151
(8) pH responsive T 1 /T 2 Preparation of switchable MRI contrast agents (ESIONPs systems): ferric oxide nanoparticles (ESIONPs-PEG-PGA for short) with surface modified with poly-L-glutamic acid through polyethylene glycol part and poly (N- { N '- [ N' - (2-carboxyethyl) -2-aminoethyl) with surface modified with polyethylene glycol part]-2-aminoethyl } glutamine) in water at equal concentration to obtain pH-responsive T 1 /T 2 Switched MRI contrast agents (esionpsystem).
Comparative example 1: this comparative example was substantially the same as example 1, except that steps (7) and (8) were not included, and finally iron oxide nanoparticles (ESIONPs-PEG-PGA) having poly-L-glutamic acid modified on the surface thereof by a polyethylene glycol moiety were obtained.
Comparative example 2: this control example was essentially identical to example 1, but without steps (6) and (8), and finally yielded iron trioxide nanoparticles (ESIONPs-PEG-PDC) surface-modified with polyethylene glycol moieties poly (N- { N '- [ N' - (2-carboxyethyl) -2-aminoethyl ] -2-aminoethyl } glutamine).
Example 2
pH responsive T 1 /T 2 A preparation method of the switching type MRI contrast agent comprises the following steps:
(1) Step (1) of example 1, the reaction time of benzyl glutamate cyclic anhydride (BLG-NCA) and propargylamine was adjusted to 22 ℃ for 36h, and the molar ratio of benzyl glutamate cyclic anhydride to propargylamine was 50;
(2) This step is similar to step (2) in example 1, except that: dissolving poly-gamma-benzyl-L-glutamic acid containing alkynyl in trifluoroacetic acid, placing the solution in a 100mL round-bottom flask, adding 33wt% of hydrogen bromide acetic acid solution, reacting at 55 ℃ for 3h, and cooling to room temperature for reacting for 18h to obtain poly-L-glutamic acid containing alkynyl;
(3) This step is similar to step (3) in example 1 except that the alkynyl group-containing poly-gamma-benzyl-L-glutamic acid and 2-hydroxypyridine were adjusted to 1:8 in terms of the molar ratio of benzyl group and 2-hydroxypyridine, and the molar ratio of benzyl group and diethylenetriamine was adjusted to 1;
(4) This step is similar to step (4) in example 1, except that: dissolving alkynyl-containing poly { N- [ N' - (2-aminoethyl) -2-aminoethyl ] glutamic acid } in 400mmol/L aqueous solution of sodium carbonate, dropping acrylic acid, and reacting at 22 deg.C for 5 days under protective atmosphere;
(5) This step is similar to step (5) in example 1, except that: the reaction time is adjusted to 24h at 22 ℃, and the molar ratio of the N, N-diisopropylethylamine to the polyethylene glycol with the end group being amino is adjusted to 100;
(6) This step is similar to step (6) in example 1, except that: adjusting the mass ratio of the ferric oxide nanoparticles with the surfaces modified with azido groups through polyethylene glycol to poly-L-glutamic acid containing alkynyl to 1:8, and adjusting the reaction time to 60h at 22 ℃;
(7) This step is similar to step (7) in example 1, except that: adjusting the mass ratio of the ferric oxide nanoparticles with the surfaces modified with the azido groups through polyethylene glycol moieties to poly (N- { N '- [ N' - (2-carboxyethyl) -2-aminoethyl ] -2-aminoethyl } glutamine) containing alkynyl to 1:8, and adjusting the reaction time to 60h at 22 ℃;
(8) Ferric oxide nanoparticles with surfaces modified by polyethylene glycol part and poly (N- { N '- [ N' - (2-carboxyethyl) -2-aminoethyl) with surfaces modified by polyethylene glycol part]-2-aminoethyl } glutamine) to obtain pH-responsive T 1 /T 2 Switching type MRI contrast agents.
Example 3
pH responsive T 1 /T 2 A preparation method of the switching type MRI contrast agent comprises the following steps:
(1) This procedure is similar to procedure (1) in example 1 except that the reaction time of benzyl glutamate cyclic anhydride (BLG-NCA) and propargylamine is adjusted to 20 ℃ for 48h, and the molar ratio of benzyl glutamate cyclic anhydride to propargylamine is adjusted to 60;
(2) This step is similar to step (2) in example 1, except that: dissolving poly-gamma-benzyl-L-glutamic acid containing alkynyl in trifluoroacetic acid, placing the solution in a 100mL round-bottom flask, adding 33wt% of hydrogen bromide acetic acid solution, reacting at 20 ℃ for 4h, and cooling to 20 ℃ for 24h to obtain poly-L-glutamic acid containing alkynyl;
(3) This step is similar to step (3) in example 1 except that poly-gamma-benzyl-L-glutamic acid containing an alkynyl group and 2-hydroxypyridine are adjusted to 1;
(4) This step is similar to step (4) in example 1, except that: dissolving alkynyl-containing poly { N- [ N' - (2-aminoethyl) -2-aminoethyl ] glutamic acid } in 600mmol/L sodium carbonate water solution, dripping acrylic acid, and reacting at 20 ℃ for 6 days under a protective atmosphere;
(5) This step is similar to step (5) in example 1, except that: adjusting the reaction time to 20h, wherein the molar ratio of the N, N-diisopropylethylamine to the polyethylene glycol with the end group as amino is adjusted to 200;
(6) This step is similar to step (6) in example 1, except that: adjusting the mass ratio of the ferric oxide nanoparticles with the surfaces modified with azido groups through polyethylene glycol moieties to poly-L-glutamic acid containing alkynyl to 1, adjusting the reaction time to 72h, directly dialyzing the product in deionized water by a dialysis bag with the molecular weight cutoff of 50000Da for three days after the reaction is finished, and performing freeze-drying treatment to obtain the ferric oxide nanoparticles (ESIONPs-PEG-PGA) with the surfaces modified with poly-L-glutamic acid through polyethylene glycol moieties;
(7) This step is similar to step (7) in example 1, except that: adjusting the mass ratio of the ferric oxide nanoparticles with the surfaces modified by the azido groups through polyethylene glycol moieties to poly (N- { N '- [ N' - (2-carboxyethyl) -2-aminoethyl ] -2-aminoethyl } glutamine) containing alkynyl to 1;
(8) Ferric oxide nano-particles of which the surfaces are partially modified by polyethylene glycol to poly-L-glutamic acid and poly (N- { N '- [ N' - (2-carboxyethyl) -2-aminoethyl group]-2-aminoethyl } glutamine) to obtain pH-responsive T 1 /T 2 Switching type MRI contrast agents.
Next, the iron oxide nanoparticles (ESIONPs-PEG-PGA) obtained in example 1, which were surface-modified with poly-L-glutamic acid by polyethylene glycol moiety, and poly (N- { N' - [ N "- (2-carboxyethyl) -2-aminoethyl) which was surface-modified with polyethylene glycol moiety, were demonstrated by several performance tests]-2-aminoethyl } glutamine) iron trioxide nanoparticles (esinops-PEG-PDC) and pH-responsive T 1 /T 2 The use of switched MRI contrast agents (esionpsystems) as contrast agents is advantageous.
Performance test one
The longitudinal relaxation times T of the contrast agent product obtained in example 1 of the invention were tested on a 0.5T MRI tester under physiologically neutral (pH 7.4) and weakly acidic (pH 6.5) tumor microenvironment 1 And T 1 Weighted imaging, the method of operation of which comprises:
respectively preparing the two samples with iron concentration of 2-0.125 mmol/L (mmol/L can be abbreviated as mM), after testing on a 0.5T MRI tester, carrying out linear fitting by taking the iron ion concentration as abscissa and the reciprocal of the longitudinal relaxation time as ordinate to obtain the contrast agent of the invention with longitudinal relaxation rates of 5.7118mM respectively under the conditions of pH 7.4 and 6.5 -1 ·s -1 And 3.8804mM -1 ·s -1 (see fig. 7 a-7 b), it can be seen that the longitudinal relaxation rate of the contrast agent of the present invention is reduced in a weak acid environment.
By T of both at different concentrations 1 Weighted imaging As can be seen, T of the contrast agent obtained in example 1 of the present invention 1 The imaging brightness of the contrast effect under the neutral condition is far higher than that under the weak acidic condition.
Performance test 2
The transverse relaxation time T of the contrast agent obtained in example 1 of the invention under the conditions of physiological neutrality (pH 7.4) and weak acidity of the tumor microenvironment (pH 6.5) was tested on a 0.5T MRI tester 2 And T 2 Weighted imaging, the method of operation of which comprises:
separately preparing the iron concentrationAfter the two samples with the concentration of 2-0.125 mM are tested on a 0.5T MRI tester, linear fitting is carried out by taking the concentration of iron ions as abscissa and the reciprocal of transverse relaxation time as ordinate to obtain the transverse relaxation rates of 9.1074mM of the contrast agent of the invention before and after incubation and DTT respectively -1 ·s -1 And 42.1950mM -1 ·s -1 (see FIGS. 8 a-8 b), it can be seen that the transverse relaxation rate of the contrast agent of the present invention is significantly increased in a weak acid environment. The ratio of transverse relaxation rate to longitudinal relaxation rate at pH 7.4 was 1.59. The ratio of transverse relaxation rate to longitudinal relaxation rate at pH 6.5 was 10.87, and it can be seen from the change in the ratio that the contrast agent obtained in this example can respond in a weak acid environment and is shown by T 1 Contrast agents of type T 2 Contrast agents of the type (I).
By T of both at different concentrations 2 Weighted imaging can show the T of the contrast agent obtained in example 1 2 The contrast effect is clearly darker under weakly acidic conditions than under neutral conditions.
Performance test three
The operation method of the contrast agent obtained by the contrast agent obtained in the embodiment 1 of the invention for detecting toxicity of normal non-cancer cells and cancer cells (4T 1) comprises the following steps:
the cytotoxicity of the contrast agent obtained in this example was measured in human umbilical vein endothelial cells (HUVEC cells) and mouse breast cancer cells (4T 1) by tetrazolium salt colorimetry (WST method).
HUVEC cells or 4T1 cells were seeded into 100. Mu.L of 96-well plates at a density of 5000 to 8000 cells per well, and the 96-well plates were placed in CO 2 The cells were cultured in an incubator at 37 ℃ for 24 hours. Dissolving the contrast agent in a complete culture medium, and filtering and sterilizing; then, the contrast agent is diluted into a plurality of groups of culture media with different concentrations of 0.10-4 mM by using complete culture media (culture media without the contrast agent).
The old medium in the 96-well plate was aspirated, and then medium at different concentrations was added to the 96-well plate at 100. Mu.L per well, and 100. Mu.L of complete medium was added to the control group, and incubation was continued for 24h. Finally all media was removed, 100. Mu.L of fresh complete media was added to each well, and then 10. Mu.L of WST-1 (which was one) was added to each wellMTT-like compounds, in the presence of electron-coupling agents, can be reduced by some dehydrogenases in the mitochondria to produce orange-yellow formazan. The more rapid the cell proliferation, the darker the color; the more cytotoxic, the lighter the color. All of them are called 2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulphophenyl) -2H-tetrazolium, monosodium salt,2- (4-iodobenzene) -3- (4-nitrobenzene) -5- (2,4 dithiobenzene) -2H-tetrazolium monosodium salt, cultured in incubator for 2H, and measured by microplate reader for absorbance OD at 450nm 450nm .4 replicates were made for each contrast agent concentration (referred to as experimental) and control. Relative viability of the cells was calculated from absorbance values. The blank group was complete medium without cells, and the control group was cells without medium.
Relative cell survival rate (%) =100 × (OD of experiment group OD-blank OD)/(OD of control group OD-blank OD)
As shown in FIG. 9, even if the iron concentration reached 4mM, the cell survival rate of ESIONPsystem group for HUVEC cells and 4T1 cells was still 95% or more, indicating that pH-responsive T cells were prepared 1 /T 2 The switching type contrast agents ESIONPs systems have little toxicity to normal cells and good biocompatibility.
Performance test four
The tissue toxicity detection method of the contrast agent obtained in the embodiment 1 of the invention comprises the following steps:
the tissue toxicity of the contrast agent obtained in this example in normal BALB/c mice was determined by hematoxylin-eosin staining (H & E staining).
Four week old normal BALB/c mice were divided into two groups:
the first group was given saline as a control group;
the second group of tail vein was injected with a physiological saline solution containing the contrast agent obtained in this example, in which the iron ion concentration was 0.1 mmol/kg;
after 2 days of feeding under normal conditions, the neck was sacrificed by dislocation, and the heart, liver, spleen, lung, and kidney were collected and stained by H & E section and observed by taking a photograph with a microscope.
As shown in fig. 10, the contrast agent obtained in example 1 had little damage to each organ tissue, and the high concentration sample did not significantly increase. In particular, hepatocytes in liver sections were relatively normal and did not show any signs of inflammatory responses. Pulmonary fibrosis was also not observed in lung sections. No tissue necrosis was observed in all other section samples. The contrast agent has no obvious pathological change or damage to important organs, and the contrast agent has very good biocompatibility and safety.
Performance test five
The operation method of the in vivo MRI imaging experiment of the contrast agent obtained in the embodiment 1 of the invention comprises the following steps:
mouse breast cancer cell (4T 1) BALB/c mouse models were constructed and divided into four groups. First, a control group treated with sodium bicarbonate was prepared by intratumorally injecting a group of mice with 25 μ L of sodium bicarbonate to neutralize the acidity at the tumor site. Then, four groups of tumor-bearing mice were intraperitoneally injected with a 20% urethane solution at a dose of 5mL/kg body weight. After four groups of mice are deeply anesthetized, a blank scan is performed before the injection of the contrast medium. Then, tumor-bearing mice of ESIONPsystem were used as an experimental group, tumor-bearing mice of ESIONPs-PEG-PGA were used as a control group A, tumor-bearing mice of ESIONPs-PEG-PDC were used as a control group B, tumor-bearing mice of ESIONPs system were used as a control group C, and the iron ion dose of each of the four groups was 0.1mmol/kg body weight. Then fixing the tumor-bearing mice, placing the tumor-bearing mice in a 1.5T miniature magnetic resonance imaging instrument, and respectively shooting T1 hour, 2 hours and 3 hours after injection 2 The magnetic resonance image is weighted.
As shown in fig. 11, after four groups of contrast agents were injected into tumor-bearing mice from the tail vein at an injection amount of 0.1mmol/kg, the four groups exhibited significantly different image enhancement effects. In order to make the images obtained by four sets of MRI scans mutually contrastive, the sequence parameters are uniformly set to TE =60.86ms and TR =3000ms. After ESIONPs system is injected into the tail vein of the mice of the first group of experimental groups for 1 hour, the MR images of the tumor parts are obviously darkened, the contrast is increased, and the images are lightened at the two subsequent time pointsThe degree has no obvious change, which indicates that the contrast agent has obvious T at the tumor part 2 A signal enhancement effect. The images of the tumor sites of the mice became significantly brighter after injection by the second control group aesiops-PEG-PGA and the third control group besiops-PEG-PDC, wherein the brightness of the images of the third group was higher, which may be due to the fact that esinops-PEG-PDC have positive charges on the surface in a weak acid environment and have higher cellular uptake rate. These two sets of images demonstrate that ESIONPs systems electrostatically aggregate in the acidic tumor environment resulting in T 2 Activation of the contrast signal. When the acidity of the tumor sites was neutralized, the tumor sites of the mice in the fourth control group C were not darkened compared to ESIONPs system, and T was 2 The MR signal is not activated, which verifies the T of ESIONPs systems 2 MR signals can only be activated in acidic environments. Therefore, according to the analysis of the results, ESIONPs system composed of ESIONPs-PEG-PGA and ESIONPs-PEG-PDC can generate electrostatic interaction to cause aggregation in the weak acidic environment of tumor, and T is the ratio of the total amount of ESIONPs-PEG-PGA and ESIONPs-PEG-PDC 1 Conversion of contrast agents to T 2 Contrast agent, thereby activating T 2 MR enhances the effect and achieves high selectivity and specificity at the tumor site.
In summary, the pH responsive T provided by the present invention 1 /T 2 The switching type MRI contrast agent has high specificity and selectivity on tumor parts, good biocompatibility and low toxicity, can be metabolized in vivo quickly, and can selectively activate T in a tumor weak acid microenvironment 2 MR signals, from T 1 Conversion of contrast agents to T 2 Contrast agent, producing T 2 The contrast enhancement effect is achieved, so that the imaging contrast performance is excellent, the interference of a background signal can be avoided, the EPR effect is enhanced, and T is activated 2 The contrast enhancement effect, high selectivity and specificity to tumor parts and good biocompatibility.
In addition, the inventors of the present invention conducted corresponding experiments using other materials listed above and other process conditions instead of the materials and process conditions described in examples 1 to 3, and the obtained MRI contrast agent is also excellent in biocompatibility, safety, relaxivity and imaging contrast.
It should be noted that the above-mentioned embodiments of the present invention do not limit the scope of the present invention. Any other corresponding changes and modifications made according to the technical idea of the present invention should be included in the protection scope of the claims of the present invention.
The aspects, embodiments, features and examples of the present invention should be considered illustrative in all respects and not restrictive, the scope of the invention being defined solely by the claims. Other embodiments, modifications, and uses will be apparent to those skilled in the art without departing from the spirit and scope of the claimed invention.
The use of headings and chapters in this disclosure is not meant to limit the disclosure; each section may apply to any aspect, embodiment, or feature of the invention.
Throughout this specification, where a composition is described as having, containing, or comprising specific components or where a process is described as having, containing, or comprising specific process steps, it is contemplated that the composition of the present teachings also consist essentially of, or consist of, the recited components, and the process of the present teachings also consist essentially of, or consist of, the recited process steps.
It should be understood that the order of steps or the order in which particular actions are performed is not critical, so long as the teachings of the invention remain operable. Further, two or more steps or actions may be performed simultaneously.
While the invention has been described with reference to illustrative embodiments, it will be understood by those skilled in the art that various other changes, omissions and/or additions may be made and substantial equivalents may be substituted for elements thereof without departing from the spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from its scope. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed for carrying out this invention, but that the invention will include all embodiments falling within the scope of the appended claims. Moreover, unless specifically stated any use of the terms first, second, etc. do not denote any order or importance, but rather the terms first, second, etc. are used to distinguish one element from another.

Claims (22)

1. pH responsive T 1 /T 2 A switchable MRI contrast agent characterized in that it has a structure represented by the following formula (I)
Figure FDA0003845649130000011
Wherein X is selected from
Figure FDA0003845649130000012
m and n are both selected from 30 to 50; m is ferric oxide nano-particles, and the particle size of the ferric oxide nano-particles is less than 5nm;
t of the pH response 1 /T 2 The switching type MRI contrast agent is prepared by connecting ferric oxide nanoparticles with poly-L-glutamic acid on the surface through polyethylene glycol and poly (N- { N '- [ N' - (2-carboxyethyl) -2-aminoethyl) on the surface through polyethylene glycol]-2-aminoethyl } glutamine) are dissolved in water and mixed.
2. pH responsive T 1 /T 2 A method for preparing a switchable MRI contrast agent, characterized in that it comprises:
at least leading a first uniform mixed reaction system containing benzyl glutamate cyclic anhydride and propargylamine to carry out ring-opening polymerization reaction to prepare poly gamma-benzyl-L-glutamic acid containing alkynyl;
at least enabling a second uniformly mixed reaction system containing the alkynyl-containing poly gamma-benzyl-L-glutamic acid and a hydrogen bromide acetic acid solution to react to prepare alkynyl-containing poly-L-glutamic acid;
at least enabling the poly gamma-benzyl-L-glutamic acid containing alkynyl to react with a third uniformly mixed reaction system of 2-hydroxypyridine and diethylenetriamine to prepare poly { N- [ N' - (2-aminoethyl) -2-aminoethyl ] glutamic acid } containing alkynyl;
reacting at least a fourth uniformly mixed reaction system containing the alkynyl-containing poly { N- [ N '- (2-aminoethyl) -2-aminoethyl ] glutamic acid } and acrylic acid under basic conditions to obtain alkynyl-containing poly (N- { N' - [ N "- (2-carboxyethyl) -2-aminoethyl ] -2-aminoethyl } glutamine);
reacting at least a fifth uniformly mixed reaction system comprising polyethylene glycol with azide and amino as end groups, polyethylene glycol with amino as end groups and ferric oxide nanoparticles with carboxyl activated ester modified on the surface to prepare ferric oxide nanoparticles with azide partially modified by polyethylene glycol on the surface; wherein the particle size of the ferric oxide nanoparticles in the surface modified carboxyl activated ester is less than 5nm;
reacting a sixth uniformly mixed reaction system which at least comprises ferric oxide nanoparticles with surfaces modified with azido groups by polyethylene glycol moieties, poly-L-glutamic acid containing alkynyl and a cuprous catalyst to obtain the ferric oxide nanoparticles with surfaces connected with the poly-L-glutamic acid by polyethylene glycol;
reacting at least a seventh uniformly mixed reaction system comprising ferric oxide nanoparticles with surfaces modified with azide groups through polyethylene glycol moieties, poly (N- { N '- [ N' - (2-carboxyethyl) -2-aminoethyl ] -2-aminoethyl } glutamine) containing alkynyl groups and a monovalent copper catalyst to prepare the ferric oxide nanoparticles with surfaces connected with the poly (N- { N '- [ N' - (2-carboxyethyl) -2-aminoethyl ] -2-aminoethyl } glutamine) through polyethylene glycol;
and attaching the surface to the ferric oxide nanoparticles of poly-L-glutamic acid by polyethylene glycol, and attaching the surface to poly (N- { N '- [ N' - (2-carboxyethyl) -2-aminoethyl) by polyethylene glycol]-2-aminoethyl } glutamine) with water to form a pH-responsive T 1 /T 2 A switched MRI contrast agent.
3. The method according to claim 2, comprising:
under a protective atmosphere, dissolving glutamic acid benzyl ester cyclic internal anhydride in dichloromethane and dimethylformamide to form a solution, then adding propargylamine into the solution to form a first uniform mixed reaction system, and reacting at 20-25 ℃ for 24-48 h to prepare the alkynyl-containing poly gamma-benzyl-L-glutamic acid, wherein the volume ratio of dichloromethane to dimethylformamide is 3-5: 1;
the molar ratio of the glutamic acid benzyl ester cyclic anhydride to the propargylamine is more than 40:1.
4. The production method according to claim 3, characterized in that: the molar ratio of the glutamic acid benzyl ester cyclic internal anhydride to the propargylamine is 60-40: 1.
5. The preparation method according to claim 2, characterized by specifically comprising:
dissolving the alkynyl-containing poly-gamma-benzyl-L-glutamic acid in trifluoroacetic acid, adding 33 mass percent of hydrogen bromide acetic acid solution to form a second uniform mixed reaction system, reacting for 2-4 h at 50-60 ℃, cooling the second uniform mixed reaction system to room temperature, and continuing to react for 12-24 h to obtain the alkynyl-containing poly-L-glutamic acid.
6. The preparation method according to claim 2, characterized by specifically comprising:
dissolving the alkynyl-containing poly gamma-benzyl-L-glutamic acid and 2-hydroxypyridine in methyl pyrrolidone to form a solution, adding diethylenetriamine to form a third uniformly mixed reaction system, and reacting at 20-25 ℃ for 48-72 h to obtain alkynyl-containing poly { N- [ N' - (2-aminoethyl) -2-aminoethyl ] glutamic acid };
the mol ratio of benzyl to 2-hydroxypyridine in the alkynyl-containing poly gamma-benzyl-L-glutamic acid is 1: 5-10;
the mol ratio of benzyl to diethylenetriamine in the poly-gamma-benzyl-L-glutamic acid containing alkynyl is 1: 50-100.
7. The method according to claim 2, comprising:
dissolving the alkynyl-containing poly { N- [ N ' - (2-aminoethyl) -2-aminoethyl ] glutamic acid } in an alkaline aqueous solution under a protective atmosphere, adding acrylic acid to form a fourth uniformly mixed reaction system, and reacting at 20-25 ℃ for 5-7 days to obtain the alkynyl-containing poly (N- { N ' - [ N "- (2-carboxyethyl) -2-aminoethyl ] -2-aminoethyl } glutamine), wherein the molar ratio of the acrylic acid to the alkynyl-containing poly { N- [ N ' - (2-aminoethyl) -2-aminoethyl ] glutamic acid } is 10-20: 1, the alkaline aqueous solution is selected from sodium bicarbonate aqueous solution.
8. The method according to claim 2, comprising:
mixing ferric oxide nanoparticles with carboxyl activated ester modified on the surface, polyethylene glycol with end groups of azide and amino and polyethylene glycol with end group of amino in chloroform, adding N, N-diisopropylethylamine to form a fifth uniform mixed reaction system, and reacting at 20-25 ℃ for 12-24 h to obtain the ferric oxide nanoparticles with azido partially modified by polyethylene glycol on the surface; wherein, the mass ratio of the polyethylene glycol with the end group of azide and amino to the polyethylene glycol with the end group of amino is 1:1, and meanwhile, the mass ratio of the ferric oxide nano particles of the surface modification carboxyl activated ester to the polyethylene glycol with the end group of azide and amino is 1: 5-10;
the ferric oxide nanoparticles of the surface modified carboxyl activated ester are selected from ferric oxide nanoparticles of surface modified pentafluorophenol ester;
the mol ratio of the N, N-diisopropylethylamine to the polyethylene glycol with the amino group as the end group is 100-200: 1.
9. The method according to claim 2, comprising:
dissolving the ferric oxide nanoparticles with azido groups modified on the surfaces by polyethylene glycol in dimethyl formamide, adding the poly-L-glutamic acid containing alkynyl, then carrying out freezing deoxidization treatment, adding sodium ascorbate and copper sulfate in a protective atmosphere to form a sixth uniform mixed reaction system, and reacting at 20-25 ℃ for 48-72 h to obtain the ferric oxide nanoparticles with surfaces connected with the poly-L-glutamic acid by polyethylene glycol.
10. The method of claim 9, further comprising: after the reaction of the sixth uniform mixing reaction system is finished, dialyzing and freeze-drying the obtained mixture; the cut-off molecular weight of the dialysis bag adopted in the dialysis treatment is 30000-50000 Da.
11. The method of manufacturing according to claim 10, wherein: the cut-off molecular weight of the dialysis bag adopted in the dialysis treatment is 35000-50000 Da.
12. The preparation method according to claim 2, characterized by specifically comprising:
dissolving the ferric oxide nanoparticles with the surfaces partially modified by azido through polyethylene glycol into dimethyl formamide, adding the alkynyl-containing poly (N- { N '- [ N' - (2-carboxyethyl) -2-aminoethyl ] -2-aminoethyl } glutamine), freezing for deoxygenation, adding sodium ascorbate and copper sulfate in a protective atmosphere to form a seventh uniform mixed reaction system, and reacting at 20-25 ℃ for 48-72 h to obtain the ferric oxide nanoparticles with the surfaces connected with poly (N- { N '- [ N' - (2-carboxyethyl) -2-aminoethyl ] -2-aminoethyl } glutamine) through polyethylene glycol.
13. The method of claim 12, further comprising: after the reaction of the seventh uniformly mixed reaction system is finished, dialyzing and freeze-drying the obtained mixture; the cut-off molecular weight of the dialysis bag adopted in the dialysis treatment is 30000-50000 Da.
14. The method of manufacturing according to claim 13, wherein: the cut-off molecular weight of the dialysis bag adopted in the dialysis treatment is 35000-50000 Da.
15. The method of claim 2, wherein: the molar ratio of the ferric oxide nanoparticles with the surfaces partially modified by the azido groups through the polyethylene glycol to the ferric oxide nanoparticles with the surfaces connected with the poly (N- { N '- [ N' - (2-carboxyethyl) -2-aminoethyl ] -2-aminoethyl } glutamine) through the polyethylene glycol is 1: 1-3.
16. The method of claim 15, wherein: the mole ratio of the ferric oxide nanoparticles with the surface modified with azido groups through polyethylene glycol to the ferric oxide nanoparticles with the surface connected with poly (N- { N '- [ N' - (2-carboxyethyl) -2-aminoethyl ] -2-aminoethyl } glutamine) through polyethylene glycol is 1:1, 1:2 or 1: 3.
17. A pH-responsive T prepared by the method of any one of claims 2 to 16 1 /T 2 Switching type MRI contrast agents.
18. The pH responsive T of claim 1 or 17 1 /T 2 Use of a switchable MRI contrast agent in the manufacture of a product having tumor detection capabilities.
19. Use according to claim 18, characterized in that: fe in said product 3+ The concentration is 30-50 mmol/L.
20. A composition for imaging comprising: the pH responsive T of claim 1 or 17 1 /T 2 A switched-mode MRI contrast agent; and, a pharmaceutically acceptable adjuvant.
21. The contrast composition according to claim 20, wherein the pharmaceutically acceptable adjuvant comprises a diluent.
22. A pharmaceutical composition characterized by comprising: the pH responsive T of claim 1 or 17 1 /T 2 A switchable MRI contrast agent, a drug and a pharmaceutically acceptable carrier.
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