CN107684628B

CN107684628B - Preparation method and application of fluorine-19 magnetic resonance contrast probe for sulfydryl detection

Info

Publication number: CN107684628B
Application number: CN201711121575.9A
Authority: CN
Inventors: 黄平升; 王伟伟; 秦怡博; 孔德领
Original assignee: Institute of Biomedical Engineering of CAMS and PUMC
Current assignee: Institute of Biomedical Engineering of CAMS and PUMC
Priority date: 2017-11-14
Filing date: 2017-11-14
Publication date: 2020-05-22
Anticipated expiration: 2037-11-14
Also published as: CN107684628A

Abstract

The invention relates to a preparation method and application of a fluorine-19 magnetic resonance contrast probe for sulfydryl detection. The structure and the composition of the material are as follows, wherein x is an integer of 5-50, and y is an integer of 5-50. The invention relates to a novel oxidation-reduction response activated amine protonation¹⁹The nanometer probe for magnetic resonance imaging is used for detecting sulfydryl in organisms. The nanometer probe has high selectivity to sulfhydryl compounds, and detection signals are easy to analyze and process. Meanwhile, the nano probe has the characteristics of high sensitivity, simplicity and convenience in operation, small tissue damage, high resolution and the like, and provides an important means for real-time in-situ visual monitoring of the biological sulfhydryl compound.

Description

Preparation method and application of fluorine-19 magnetic resonance contrast probe for sulfydryl detection

Technical Field

The invention relates to a preparation method and application of a fluorine-19 magnetic resonance imaging probe for sulfydryl detection, in particular to a polymer nano probe suitable for high-sensitivity and high-selectivity sulfydryl detection, which utilizes fluorine-19 magnetic resonance imaging (¹⁹F MRI) to detect the horizontal sulfydryl of cells or living bodies, belonging to the nano probe in the technical field of biological analysis.

Background

With the rapid development of life science and medical science in recent years, the detection of active substances in cells gradually draws attention. Active sulfhydryl micromolecules in organisms such as cysteine (Cys), homocysteine (Hcy), Glutathione (GSH) and the like play an important role in physiological and pathological processes of human bodies. Despite their very similar structures, biological functions are completely different. Cys is the only naturally essential amino acid containing a thiol group (-SH), and is an important component of synthetic proteins. Hcy is usually derived from the metabolism of methionine in vivo, and maintains the change of the levels of sulfur amino acids contained in the organism. GSH is a non-protein sulfhydryl compound with the highest intracellular content (1-10 mmol/L), and participates in a plurality of physiological functions of an organism, such as maintaining the oxidation-reduction dynamic balance of the organism, removing exogenous toxins, participating in signal transduction, completing gene regulation and the like. Abnormal levels of these sulfhydryl compounds in the body often induce a variety of diseases. Studies show that the abnormal Cys level in organisms can cause various diseases, such as slow growth, edema, liver injury, skin injury and the like. Too high Hcy level can also induce various diseases of human body, such as cardiovascular disease, senile dementia, vitamin B deficiency, etc. Modern medicine suggests that abnormalities in GSH are directly associated with many diseases and cancers. The understanding of the generation, distribution and level fluctuation of the sulfhydryl compounds has important scientific significance for the research of cell activity, organism physiology, pathology and the like. Therefore, quantitative detection of the change of the content of the sulfhydryl micromolecule in the organism becomes a hotspot of current research in the fields of chemistry, medicine, life science and the like.

At present, many methods for detecting sulfhydryl compounds have been reported, such as capillary electrophoresis, high performance liquid chromatography, liquid-mass spectrometry, microfluidic chip method, radioactive enzyme assay, fluorescence method, and the like. The fluorescent probe based on the chemical recognition function has high selectivity on sulfhydryl compounds, and detection signals are easy to analyze and process. Meanwhile, the fluorescence method has the characteristics of high sensitivity, simple and convenient operation, small tissue damage, no need of expensive and complex instruments and the like, is widely used for analyzing biological samples, and provides an important means for real-time and in-situ visual monitoring of the biological sulfhydryl compound. However, the application of the bio-fluorescence analysis at the living body level still has some problems, and the low resolution is the most critical problem limiting the detection application.

MRI is a technique of obtaining detailed three-dimensional images of the physiology and pathology of the body without radioactive damage by using the principle of nuclear magnetic resonance. The imaging technology can observe the internal structure of the human body by a non-invasive means and has revolutionary significance in the field of medical diagnostics. Now, MRI is not only a common clinical disease diagnostic tool, but has been increasingly applied to the field of basic life science research to clarify the mechanism of disease development, assist in drug development and visualization of disease treatment. Currently, the clinical application is mainly hydrogen-nuclear-based magnetic resonance imaging (¹H MRI), theoretically¹H MRI can examine various parts of the whole body of a human body and provide a three-dimensional spatial image. But clinicallyPractice shows that the wide distribution of water molecules in the body causes strong background interference, and the obtained imaging effect is very poor, thereby influencing the judgment of the diagnosis result. To achieve better imaging, a variety of imaging agents have been investigated, and it is desirable to enhance the contrast of the detected signals at different tissue sites and to improve the resolution of the imaging of different soft tissues by the presence of an accumulation of the imaging agent at the target site. Fortunately, it is a long-standing need,¹⁹f MRI is very good at overcoming¹Defects of H MRI: first, with respect to¹H MRI is limited by strong background interference caused by water molecules present in the body, which are not detectable in the body¹⁹F MRI signal (no detectable solid state fluorine compounds in bone)¹⁹F MRI signal) thus¹⁹F MRI is completely free of background interference; secondly, the first step is to carry out the first,¹⁹f NMR sensitivity is second only to¹Highly sensitive spin nuclei for H NMR; again, the process of the present invention,¹⁹f is a stable isotope with a natural abundance of 100%, and is therefore useful as a stabilizer for a variety of different types of medical devices¹⁹F MRI does not need isotope enrichment before use and does not need radioactive protection during use; finally, the process is carried out in a batch,¹⁹f MRI signals have a chemical shift range of approximately 400 ppm, and¹the H MRI signal is only about 20 ppm and is very sensitive to the change of chemical bonds and microenvironment, so that the H MRI signal is very sensitive to the change of chemical bonds and microenvironment¹⁹F MRI is particularly suitable for providing in vivo drug existing forms and microenvironment (such as pH value, oxygen concentration, viscosity and the like) of lesion areas. These pathophysiological information are crucial for the diagnosis and treatment of diseases. In any case, it is preferable that,¹⁹f MRI has advantages of non-traumatic, non-endogenous background interference and high spatial resolution, and is concerned more and more in fields such as biological imaging and disease diagnosis.

Disclosure of Invention

The invention aims to provide a preparation method and application of a fluorine-19 magnetic resonance contrast probe for sulfydryl detection. Polyethylene glycol monomethyl ether methyl methacrylate (mPEGMA) and 2- ((2,4 dinitro-N- (3,3, 3-trifluoropropyl) phenylsulfonamide) ethyl methacrylate (AMA-DNBSF) are used as monomers, and a random copolymer is obtained by reversible addition chain transfer radical polymerization, and the two monomers can be adjustedThe proportion of the hydrophilic and hydrophobic chain segments of the polymer is controlled by the feed ratio of the nano probe, and the assembly of the nano probe is further regulated and controlled. The invention relates to a novel oxidation-reduction response activated amine protonation¹⁹The polymer nano probe for the F magnetic resonance imaging can be used for detecting sulfydryl in a living body. The nanometer probe has high selectivity to sulfhydryl compounds, and detection signals are easy to analyze and process. Meanwhile, the nano probe has the characteristics of high sensitivity, simplicity and convenience in operation, small tissue damage, high resolution and the like, and provides an important means for real-time in-situ visual monitoring of the biological sulfhydryl compound.

The fluorine-19 magnetic resonance contrast probe for sulfydryl detection provided by the invention is polyethylene glycol (PEG) -poly 2- ((2,4 dinitro-N- (3,3, 3-trifluoropropyl) phenylsulfonamide) ethyl methacrylate (AMA-DNBSF) (PEDF) amphiphilic polymer, and the structure and the composition of the amphiphilic polymer are as follows:

wherein x is an integer of 5 to 50, and y is an integer of 5 to 50.

The probe is self-assembled in an aqueous solution to obtain a core-shell structure, the outer layer is a polyethylene glycol (PEG) hydrophilic layer, and the inner core is poly (2- ((2, 4-dinitro-N- (3,3, 3-trifluoropropyl) phenylsulfonamide) ethyl methacrylate.

The preparation method of the fluorine-19 magnetic resonance contrast probe for sulfydryl detection provided by the invention comprises the following steps:

1) using dimethylformamide as a solvent, and reacting phenyl- (2-hydroxyethyl) thiocarbonate, polyethylene glycol monomethyl ether methyl methacrylate and ethyl 2- ((2, 4-dinitro-N- (3,3, 3-trifluoropropyl) phenylsulfonamide) methacrylate at 68-72 ℃ for 20-24 h in the presence of azodiisobutyronitrile serving as an initiator; after the reaction is finished, adding DMF for dissolving, filling the solution into a dialysis bag, dialyzing the solution for 72 hours by using deionized water, replacing dialyzate every 12 hours, freezing and drying the product to obtain PEDF amphiphilic polymer, and characterizing the polymer by using nuclear magnetic hydrogen spectrum and fluorine spectrum; the molar ratio of the phenyl- (2-hydroxyethyl) thiocarbonate to the polyethylene glycol monomethyl ether methyl methacrylate to the 2- ((2, 4-dinitro-N- (3,3, 3-trifluoropropyl) phenylsulfonamide) ethyl methacrylate is 1:5:5-1:50: 50.

2) The obtained amphiphilic polymer is directly dispersed in 0.01M phosphate buffer solution with pH =7.4 under the action of ultrasound, so that a polymer nano probe is obtained, and the particle size and the form of the nanoparticles are detected by using a laser particle sizer and a transmission electron microscope.

The particle size of the nanoparticles is 20-200 nm.

The fluorine atom concentration in the dispersion liquid of the nano probe can be regulated and controlled by regulating and controlling the polymer proportion, and the concentration is 0.05-0.5 mol/L.

The fluorine-19 magnetic resonance contrast probe for detecting sulfydryl takes methyl polyethylene glycol monomethyl ether methacrylate (mPEGMA) and ethyl 2- ((2,4 dinitro-N- (3,3, 3-trifluoropropyl) phenylsulfonamide) methacrylate (AMA-DNBSF) as monomers, obtains a random copolymer through reversible addition chain transfer free radical polymerization, can control the hydrophilic-hydrophobic chain segment proportion of the polymer by adjusting the charge ratio of the two monomers, and further regulates the assembly of a nano probe, and particularly, the nano probe generates a secondary amino group through the rapid nucleophilic substitution reaction between sulfydryl and the hydrophobic section 2, 4-dinitro-phenylsulfonamide of the nano probe under the condition of pH 7.4, so that the spin-spin relaxation of the fluorine-containing chain segment is severely disturbed, make it¹⁹The F MRI signal goes from "off" to "on".¹⁹The F chain segment is frozen in the inner core of the nano probe, so that the F chain segment is frozen in the absence of sulfydryl¹⁹F magnetic resonance (¹⁹F NMR) signal is completely masked.

A strong electron group 2, 4-dinitro-benzenesulfonamide structure exists in a nano probe poly 2- ((2, 4-dinitro-N- (3,3, 3-trifluoropropyl) phenylsulfonamide) ethyl methacrylate chain segment, a nucleophilic substitution reaction can be rapidly carried out on the strong electron group 2, 4-dinitro-benzenesulfonamide structure and a mercapto group to generate a secondary amine group, and the secondary amine group is completely protonated under the condition of pH 7.4 to cause the nano probe to be assembled and disassembled, so that the nano probe is detected to be disassembled¹⁹F NMR signal.

The invention provides polyethylene glycol (PEG) -poly 2- ((2, 4-dinitro-N- (3)3, 3-trifluoropropyl) phenylsulfonamide) ethyl methacrylate (AMA-DNBSF) (PEDF) amphiphilic polymer for mercapto detection has fast response and can be used in the detection of mercapto,¹⁹the F MRI signal increases rapidly.

The invention provides a fluorine-19 magnetic resonance contrast probe for detecting sulfydryl, which is a novel oxidation-reduction response activated amine protonation¹⁹The polymer nano probe for the F magnetic resonance imaging can be used for detecting sulfydryl in a living body. The responsiveness to sulfydryl is high selectivity, and the detection signal is easy to analyze and process. Only responds to sulfydryl existing in biological environment, and is chemically inert to amino acid without sulfydryl such as lysine, active oxide (tert-butyl peroxide, hydrogen peroxide, sodium hypochlorite), reducing agent (ascorbic acid) and the like. The nano probe can detect glutathione in tumor tissues at the living body level, thereby realizing the purpose of tumor¹⁹And F, MRI detection. The method has the characteristics of high sensitivity, simple and convenient operation, small tissue damage, high resolution and the like, and provides an important means for real-time in-situ visual monitoring of the biological sulfhydryl compound.

Drawings

FIG. 1 Polymer P4 nuclear magnetic hydrogen and fluorine spectra. (A) Nuclear magnetic hydrogen spectrum, (B) nuclear magnetic fluorine spectrum characterization.

FIG. 2 nanoprobe N4 particle size distribution and microscopic morphology.

FIG. 3 of the nanoprobe N4¹⁹The F NMR signal varied with reaction time.

FIG. 4 thiol-selective characterization of nanoprobe N4.

FIG. 5 nanoprobe N4 detects glutathione in tumor tissue.

Detailed Description

The present invention will be described in further detail with reference to the following examples. The experimental methods in the examples, in which specific conditions are not specified, are generally performed under the conditions described in the manual and the conventional conditions, or under the conditions recommended by the manufacturer; general equipment, materials, reagents and the like used are commercially available unless otherwise specified.

Example 1 Synthesis of Polymer P4

Adding reversible addition-fragmentation chain transfer polymerization (RAFT) chain transfer agents phenyl- (2-hydroxyethyl) thiocarbonate (12.2 mg, 0.1 mM), polyethylene glycol monomethyl ether methyl methacrylate (mPEG MA) (500 mg, 1 mM) and 2- ((2,4 dinitro-N- (3,3, 3-trifluoropropyl) phenylsulfonamide) ethyl methacrylate (AMA-DNBSF) (455 mg, 1 mM), An Initiator (AIBN) (1.64 mg, 0.01 mM) and 3 mL solvent Dimethylformamide (DMF) into a shlenk reaction tube in sequence, sealing the reaction tube after three cycles of vacuumizing and introducing argon, reacting for 24 hours in an oil bath at 68-72 ℃, adding DMF for dissolving, filling into a dialysis bag (molecular weight cut-off 3500 Da), dialyzing for 72 hours by deionized water, the dialysate was changed every 12 h. Thereafter, the PEDF polymer was obtained by freeze-drying. The P4 polymer was characterized by nuclear magnetic hydrogen and fluorine spectra, and the results are shown in FIG. 1.

According to the method in example 1, polymers of other composition ratios can be obtained.

TABLE 1 composition of the Polymer and particle size and distribution of the corresponding nanoprobes

EXAMPLE 2 preparation of nanoprobe N4

20 mg of PEDF polymer is weighed and directly dispersed in 10 mL of 0.01M phosphate buffer solution with pH =7.4 under the action of ultrasound (25 ℃, 50Hz, 5 min), so that N4 nanoparticles are obtained, wherein the concentration of the nanoparticles is 2 mg/mL. The particle size and morphology of the nanoparticles were examined by a laser particle sizer and a transmission electron microscope, and the examination results are shown in fig. 2, in which the particle size of the nanoparticles prepared in this example was 45 nm, the particle size distribution was 0.12, and an obvious core-shell structure was present.

Nanoparticles of other polymer compositions can be prepared according to the preparation method in example 2.

EXAMPLE 3 preparation of nanoprobe N4¹⁹F NMR Signal Change with reaction time

5 mL of 2 mg/mL N4 nanoprobe solution was taken and kept under argonAdding cysteine (Cys) mother liquor under the protection condition to enable the final concentration of Cys to be 5 mg/mL, taking 300 mu L at a set time point, adding 100 mu L of heavy water for field locking, and utilizing nuclear magnetic resonance to carry out treatment on the nanoparticle solution¹⁹F NMR signals were detected. As shown in figure 3, the nanoprobe can generate a rapid response to sulfydryl, and the rapid response is within 30 min under the condition of existence of cysteine¹⁹The F NMR signal can be significantly enhanced.

EXAMPLE 4 thiol Selective characterization of nanoprobe N4

Numbering 5 mL of N4 nanoprobe solution of 2 mg/mL in sequence, respectively adding mother liquor of cysteine (Cys), homocysteine, reduced glutathione, lysine, tert-butyl peroxide, hydrogen peroxide, sodium hypochlorite, reducing agent ascorbic acid and the like under the condition of argon protection to enable the final concentration of a substance to be detected to be 5 mg/mL, taking 300 mu L after 30 min, adding 100 mu L of heavy water for field locking, and utilizing nuclear magnetic resonance to carry out nuclear magnetic resonance on the nanoparticle solution¹⁹F NMR signals were detected. As shown in fig. 4, the nanoprobe can only generate a fast response to thiol, and is chemically inert to amino acids without thiol group such as lysine, active oxides (t-butyl peroxide, hydrogen peroxide, sodium hypochlorite), reducing agents (ascorbic acid), and the like.

Example 5 Nanoprobe N4 detection of glutathione in tumor tissue

Taking a healthy Bab/c mouse, a female and a body weight of 19 +/-2 g, quickly inoculating the cultured HepG2 cells under the aseptic condition, and injecting 0.2 mL of cytoma liquid into the right hind leg of the mouse subcutaneously with the cell concentration of 5.0 multiplied by 10⁶A/only. After 2 weeks of inoculation, tumor volumes of 250 mm were selected³(V =1/2(a × b 2)). Before imaging experiments were performed, 100 μ L of N4 nanoprobe at a concentration of 100 mg/mL was injected into the tumor of the mice. And then, when the MRI test is carried out on the living mouse, 100 muL and 7% chloral hydrate are injected into the abdominal cavity of the mouse to serve as anesthetic, so that the normal operation of the test is ensured. The test was performed with a 7.0T Bruker BioSpec 70/20 USR.¹The H MRI test uses RARE-T2 pulse sequence, and the specific parameters are as follows: scan field of view (FOV) 50 mm,slice thickness 1 mm, excitation times 2, repetition time (Tr) 300ms, echo time (Te) 50 ms, matrix size 256 × 256.¹⁹The F MRI test uses a FLASH method, and the specific parameters are as follows: scan field of view (FOV) 50 mm x 50 mm, slice thickness 10 mm, excitation times 64, repetition time (Tr) 300ms, echo time (Te) 4.2 ms, matrix size 90 x 90. The test results are shown in fig. 5, the tumor position is clearly visible, and the imaging has a high signal-to-noise ratio.

Claims

1. A fluorine-19 magnetic resonance contrast probe for sulfydryl detection is characterized by being polyethylene glycol (PEG) -poly 2- ((2,4 dinitro-N- (3,3, 3-trifluoropropyl) phenylsulfonamide) ethyl methacrylate (AMA-DNBSF) (PEDF) amphiphilic polymer, and the structure and the composition of the polymer are as follows:

wherein x is an integer of 5 to 50, and y is an integer of 5 to 50.

2. The probe as claimed in claim 1, which is self-assembled in an aqueous solution to obtain a core-shell structure, the outer layer is a polyethylene glycol (PEG) hydrophilic layer, and the inner core is poly (2- ((2, 4-dinitro-N- (3,3, 3-trifluoropropyl) phenylsulfonamide) ethyl methacrylate.

3. The method for preparing a fluorine-19 magnetic resonance imaging probe for mercapto group detection as set forth in claim 1, characterized by the steps of:

1) using dimethylformamide as a solvent, and reacting phenyl- (2-hydroxyethyl) thiocarbonate, polyethylene glycol monomethyl ether methyl methacrylate and ethyl 2- ((2, 4-dinitro-N- (3,3, 3-trifluoropropyl) phenylsulfonamide) methacrylate at 68-72 ℃ for 20-24 h in the presence of azodiisobutyronitrile serving as an initiator; after the reaction is finished, adding dimethylformamide for dissolving, putting the solution into a dialysis bag, dialyzing the solution for 72 hours by using deionized water, replacing dialyzate every 12 hours, freezing and drying a product to obtain an amphiphilic Polymer (PEDF), and characterizing the polymer by using a nuclear magnetic hydrogen spectrum and a fluorine spectrum;

2) the obtained amphiphilic polymer is directly dispersed in 0.01M phosphate buffer solution with pH =7.4 under the action of ultrasound, thus obtaining amphiphilic polymer nanoparticle dispersion liquid, and the particle size and the morphology of the nanoparticles are detected by utilizing a laser particle sizer and a transmission electron microscope.

4. The process according to claim 3, wherein the mass or molar ratio of the phenyl- (2-hydroxyethyl) thiocarbonate, polyethylene glycol monomethyl ether methyl methacrylate and ethyl 2- ((2, 4-dinitro-N- (3,3, 3-trifluoropropyl) phenylsulfonamide) methacrylate is 1:5:5 to 1:50: 50.

5. The method according to claim 3, wherein the nanoparticles have a particle size of 20 to 200 nm.

6. The method according to claim 3, wherein the fluorine atom concentration in the dispersion of the nanoprobe is controlled by controlling the polymer ratio to a concentration of 0.05 to 0.5 mol/L.