CN111840579B - Hypoxic imaging agent and preparation method and application thereof - Google Patents
Hypoxic imaging agent and preparation method and application thereof Download PDFInfo
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- CN111840579B CN111840579B CN202010778883.4A CN202010778883A CN111840579B CN 111840579 B CN111840579 B CN 111840579B CN 202010778883 A CN202010778883 A CN 202010778883A CN 111840579 B CN111840579 B CN 111840579B
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/0002—General or multifunctional contrast agents, e.g. chelated agents
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
- A61K49/0069—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form
- A61K49/0089—Particulate, powder, adsorbate, bead, sphere
- A61K49/0091—Microparticle, microcapsule, microbubble, microsphere, microbead, i.e. having a size or diameter higher or equal to 1 micrometer
- A61K49/0093—Nanoparticle, nanocapsule, nanobubble, nanosphere, nanobead, i.e. having a size or diameter smaller than 1 micrometer, e.g. polymeric nanoparticle
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
- A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
- A61K49/10—Organic compounds
- A61K49/12—Macromolecular compounds
- A61K49/126—Linear polymers, e.g. dextran, inulin, PEG
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- A—HUMAN NECESSITIES
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- A61K49/00—Preparations for testing in vivo
- A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
- A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
- A61K49/1818—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles
- A61K49/1821—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles
- A61K49/1824—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles
- A61K49/1827—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle
- A61K49/1851—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle having a (super)(para)magnetic core coated or functionalised with an organic macromolecular compound, i.e. oligomeric, polymeric, dendrimeric organic molecule
- A61K49/1854—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle having a (super)(para)magnetic core coated or functionalised with an organic macromolecular compound, i.e. oligomeric, polymeric, dendrimeric organic molecule the organic macromolecular compound being obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. poly(meth)acrylate, polyacrylamide, polyvinylpyrrolidone, polyvinylalcohol
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Abstract
The invention provides an anaerobic imaging agent and a preparation method and application thereof, wherein the anaerobic imaging agent comprises UIO-Pimo nano particles, the UIO-Pimo nano particles comprise polyacrylic acid coated ferroferric oxide nano particles, probe molecules shown in a formula I and connected to polyacrylic acid segments through amido bonds, and molecules containing sulfydryl and connected to the polyacrylic acid segments through the amido bonds. In an ordinary oxygen environment, the imaging agent has small, uniform and stable particle size; in a hypoxic environment, under the coexistence of reductase and NADPH, the ultra-small particles can be assembled into aggregates with larger size, and fluorescence and nuclear magnetic resonance T2 imaging signals of the parts are enhanced, so that the sensitivity of hypoxic tumor detection is increased. In addition, the imaging agent has good penetrability, no obvious cytotoxicity and good biocompatibility; the preparation method is simple, mild in condition and low in cost.
Description
Technical Field
The invention belongs to the technical field of nano imaging agents, and relates to a hypoxic imaging agent, and a preparation method and application thereof.
Background
Hypoxia is a common phenomenon in most solid tumors, and is closely related to the occurrence, development, metastasis and drug resistance of tumors (J.Natl.cancer I.2007,99(19), 1441-1454.). The discoverer of the relevant factor (HIF-1 alpha) was awarded by Nobel physiology and medicine in 2019. Therefore, how to image hypoxic cells is very important for tumor diagnosis. However, the determination of the lesion site or the degree of lesion by a single mode imaging means has been very limited, and the possibility of collectively imaging the tumor hypoxic region by a plurality of means will become a trend in the future.
The sensitivity of fluorescence imaging is particularly high, but the tissue penetration capability is poor; the magnetic resonance imaging has strong penetrating power and high spatial resolution, the relationship between the imaging quality and the state of the object to be measured is large, and slight movement can cause motion artifacts, which affect the definition of the image and further affect the analysis of the disease condition, thus causing misjudgment (ACS appl. mater. interfaces 2016,8, 4424-4433.). If the two imaging modes can be combined and analyzed together, the analysis of the disease condition can be more accurate.
Due to the competition of oxygen deficiency, nitroimidazole and its derivatives can be reduced by nitroreductase in hypoxic cells to hydroxylamine imidazole, whose para-position can undergo irreversible addition reaction with thiol-containing molecules to form adducts (Gynecol. Oncol.1998,71(2), 270-. However, nitroimidazole and derivatives thereof are mostly small molecules, which are easy to metabolize, have short half-life period of blood and limited enrichment capacity at tumor sites, and the methods of slice immunostaining mediated by nitroimidazole and derivatives thereof require biopsy of pathological tissues of patients, and the staining method is complicated.
Therefore, it is desirable in the art to develop a hypoxic imaging agent that can self-assemble in tumor hypoxic microenvironments, enhancing fluorescence and mri signals in the area.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a hypoxic imaging agent, and a preparation method and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
on one hand, the invention provides a hypoxic imaging agent, which comprises UIO-Pimo nano particles, wherein the UIO-Pimo nano particles comprise polyacrylic acid coated ferroferric oxide nano particles, probe molecules shown in a formula I and connected to polyacrylic acid segments through amido bonds, and molecules containing sulfydryl and connected to the polyacrylic acid segments through the amido bonds;
in the present invention, the molecule of formula I to which the hypoxic imaging agent is attached provides the functions of the site of the hypoxic assembly of the particle (nitroimidazole) and the fluorescent molecule (NBD), the attached thiol-containing molecule is capable of providing the thiol group required for particle cross-linking, and the ultra-small iron oxide nanoparticles (UIO) are capable of T2-weighted imaging by nuclear magnetic resonance. When the nano particles reach the hypoxic region of the tumor, cross-linking occurs between the particles, the environment where the fluorescent molecules on the molecules of the formula I are located is changed, strong fluorescence is emitted, and the fluorescence detection sensitivity is improved; in addition, the size of the particles is increased after crosslinking, so that the signal intensity of nuclear magnetic resonance imaging can be greatly improved, the retention time of the nanoparticles at a tumor part can be prolonged, and the observation time window is prolonged.
In the present invention, the UIO-Pimo nanoparticle chinese meaning is a paramagnetic iron oxide nanoparticle in which an oxygen-poor ligand and a mercapto group-containing molecule are linked.
Preferably, the probe molecule shown in formula I is linked to the polyacrylic acid segment through an amide bond formed between a primary amino group on the structure of the probe molecule and a carboxyl group on the polyacrylic acid segment.
In the present invention, the polyacrylic acid fragment structure may be represented as:
wherein n is an integer of 7 to 50, for example, n can be 7, 8, 9, 10, 12, 15, 18, 20, 23, 25, 28, 30, 33, 35, 38, 40, 45, 48 or 50.
Preferably, the thiol-containing molecule is cysteine or penicillamine.
Preferably, the particle size of the UIO-Pimo nanoparticles is 5-20 nm, such as 5nm, 8nm, 10nm, 12nm, 14nm, 16nm, 18nm or 20 nm.
Preferably, the UIO-Pimo nanoparticles have a hydrated particle size of 10-100 nm, such as 10nm, 15nm, 20nm, 25nm, 30nm, 40nm, 50nm, 60nm, 70nm, 80nm, 90nm or 100 nm.
Preferably, the molar ratio of the iron element and the thiol-group-containing molecule to the probe molecule shown in formula I in the UIO-Pimo nanoparticle is (40-100): 1-40): 1, and may be, for example, 40:1:1, 42:1:1, 45:1:1, 50:1:1, 55:1:1, 60:1:1, 65:1:1, 68:1:1, 70:1:1, 75:1:1, 78:1:1, 80:1:1, 83:1:1, 90:1:1, 94:1:1, 98:1:1, 100:1:1, 55:3:1, 58:5:1, 60:8:1, 68:8:1, 70:10:1, 73:9:1, 75:13:1, 78:18:1, 80:20:1, 82:25:1, 85:28:1, 88:30:1, 92: 1, 35:1, 38:1, 98:1, 70:1, 9:1, 38:1, or 1 100:25:1, 100:30:1, 100:33:1, 100:40:1, etc.
The UIO-Pimo nano particle is self-assembled under the condition that reductase, NADPH and hypoxia exist together.
In a hypoxic environment, under the coexistence of reductase and NADPH, the ultra-small particles can be assembled into aggregates with larger size, and fluorescence and nuclear magnetic resonance T2 imaging signals of the parts are enhanced, so that the sensitivity of hypoxic tumor detection is increased.
In another aspect, the present invention provides a method for preparing the hypoxic imaging agent as described above, the method comprising the steps of:
(1) dispersing polyacrylic acid modified iron oxide nanoparticles (UIO) in a first reaction liquid, adding a condensing agent and an acylation activating agent, and carrying out an activation reaction;
(2) and dispersing the activated nano particles (UIO) in a second reaction solution, adding a molecule containing sulfydryl and a probe molecule shown in a formula I, and reacting to obtain the hypoxic imaging agent.
Preferably, the first reaction solution in step (1) is 2- (N-morpholino) ethanesulfonic acid buffer (MES) with pH5-6 (e.g., 5.1, 5.3, 5.5, 5.8, 5.9, etc.).
Preferably, the mass-to-volume ratio of the polyacrylic acid-modified iron oxide nanoparticles described in step (1) to the first reaction solution is (0.4-3.2) mg (4-6) mL, for example, 0.4mg:4mL, 0.6mg:4mL, 0.8mg:4mL, 1mg:4mL, 1.3mg:5mL, 1.5mg:6mL, 2mg:4mL, 2.5mg:4mL, 3mg:5mL, 3.2mg:6mL, 1.9mg:4mL, 2.4mg:4mL, 2.8mg:5mL, 2.1mg:5.5mL, 0.4mg:6mL, 0.8mg:6mL, 1.4mg:6mL, 2.3mg:6mL, 2.9mg:6mL, or the like, based on the mass of the iron element.
Preferably, the condensing agent of step (1) is a carbodiimide condensing agent, preferably 1-ethyl- (3-dimethylaminopropyl) carbodiimide.
Preferably, the acylation activator in step (1) is N-hydroxysuccinimide.
Preferably, the molar ratio of the condensing agent to the acylation activating agent in step (1) is 1 to 1.5:1, such as 1:1, 1.05:1, 1.1:1, 1.13:1, 1.15:1, 1.18:1, 1.2:1, 1.23:1, 1.25:1, 1.28:1, 1.3:1, 1.35:1, 1.38:1, 1.4:1, 1.45:1, 1.48:1 or 1.5: 1.
Preferably, the amount of the condensing agent is 40-80 μmol, such as 40 μmol, 43 μmol, 45 μmol, 48 μmol, 50 μmol, 52 μmol, 54 μmol, 58 μmol, 60 μmol, 65 μmol, 68 μmol, 70 μmol, 75 μmol, 78 μmol or 80 μmol, relative to the amount of iron element in the polyacrylic acid-modified iron oxide nanoparticles of step (1) of 1.6mg by mass.
Preferably, the activation reaction of step (1) is carried out at room temperature.
Preferably, the activation reaction of step (1) is carried out for a period of 2 to 4 hours, such as 2.2 hours, 2.5 hours, 2.8 hours, 3 hours, 3.3 hours, 3.5 hours, 3.8 hours or 4 hours.
Preferably, the activation reaction of step (1) is followed by purification by ultrafiltration to remove unreacted activating agent.
Preferably, the molecular weight cut-off of the ultrafiltration tube used for ultrafiltration purification after the activation reaction in step (1) is 1000-10,000 Da, such as 1000Da, 2000Da, 2500Da, 3000Da, 4000Da, 5000Da, 6000Da, 7000Da, 8000Da, 9000Da or 10,000 Da.
Preferably, the rotation speed of ultrafiltration purification after the activation reaction in step (1) is 4000-6000 rpm, such as 4000rpm, 4300rpm, 4500rpm, 4800rpm, 5000rpm, 5300rpm, 5500rpm, 5800rpm, or 6000 rpm.
Preferably, the time for ultrafiltration purification after the activation reaction in step (1) is 10-30 min, such as 10min, 15min, 18min, 20min, 25min, 28min or 30min, and the number of ultrafiltration times is 3-5.
Preferably, the second reaction solution in step (2) is Phosphate Buffered Saline (PBS) with pH 7.4.
Preferably, the molar ratio of the thiol-group-containing molecule in step (2) to the probe molecule represented by formula I is 1-80: 1, such as 1:1, 2:1, 5:1, 8:1, 10:1, 13:1, 15:1, 18:1, 20:1, 23:1, 25:1, 28:1, 30:1, 35:1, 38:1, 40:1, 45:1, 48:1, 50:1, 55:1, 58:1, 60:1, 65:1, 68:1, 70:1, 73:1, 75:1, 78:1, or 80: 1.
Preferably, the amount of the polyacrylic acid-modified iron oxide nanoparticles used in step (2) is 1.6mg in terms of the mass of iron element, and the thiol-group-containing molecule is 10 to 40. mu. mol, for example, 10. mu. mol, 15. mu. mol, 18. mu. mol, 20. mu. mol, 25. mu. mol, 28. mu. mol, 30. mu. mol, 35. mu. mol, 38. mu. mol, or 40. mu. mol.
Preferably, the temperature of the reaction in step (2) is 0 to 4 ℃, for example, 0 ℃,1 ℃,2 ℃, 2.5 ℃, 3 ℃, 3.5 ℃ or 4 ℃.
Preferably, the reaction time in step (2) is 8-24 hours, such as 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 17 hours, 18 hours, 20 hours, 22 hours, 24 hours, and the like.
Preferably, ultrafiltration purification is performed after the reaction in step (2) is completed.
Preferably, the molecular weight cut-off of the ultrafiltration tube used for ultrafiltration purification after the reaction in the step (2) is 1000-10,000 Da, such as 1000Da, 2000Da, 2500Da, 3000Da, 4000Da, 5000Da, 6000Da, 7000Da, 8000Da, 9000Da or 10,000 Da.
Preferably, the rotation speed of ultrafiltration purification after the reaction in step (2) is 4000-6000 rpm, such as 4000rpm, 4300rpm, 4500rpm, 4800rpm, 5000rpm, 5300rpm, 5500rpm, 5800rpm, or 6000 rpm.
Preferably, the time for performing ultrafiltration purification after the reaction in step (2) is 10-30 min, such as 10min, 15min, 18min, 20min, 25min, 28min or 30min, and the number of times of ultrafiltration is 3-5.
In the present invention, after the reaction in step (2) is completed, purification by ultrafiltration is performed, and then the filtrate is collected and the graft ratio is measured by an ultraviolet-visible spectrophotometer. The detection wavelength of the ultraviolet and visible spectrophotometer is 450-490 nm.
In the present invention, preferably, 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC) is used as a carbodiimide condensing agent, N-hydroxysuccinimide (NHS) is used as an acylation activating agent, cysteine is used as a small molecule containing a sulfhydryl group, and the reaction process for synthesizing UIO-Pimo is shown in FIG. 1. Wherein UIO is polyacrylic acid modified iron oxide nanoparticles.
In another aspect, the invention provides an application of the hypoxic imaging agent in preparation of a cell hypoxic detection material.
The hypoxic imaging agent of the invention can be assembled in a tumor hypoxic microenvironment and used to generate fluorescence and magnetic resonance imaging signals at the site.
Compared with the prior art, the invention has the following beneficial effects:
(1) the hypoxic imaging agent has small particle size, good penetrability, uniform particle size and easy purification, and the surface of the particles is negative charge; no obvious cytotoxicity and good biocompatibility; the long-circulation effect is better, and after the function is exerted, the blood can be metabolized by the organism;
(2) the imaging agent can be assembled in a tumor hypoxia microenvironment, the fluorescence and nuclear magnetic resonance imaging signal intensity of the part is enhanced, and the detection sensitivity is improved.
(3) The imaging agent of the invention has the advantages of simple preparation method, mild conditions, lower cost and easy popularization and application.
Drawings
FIG. 1 is a schematic diagram of an exemplary reaction process for synthesizing UIO-Pimo;
FIG. 2 is a transmission electron micrograph of UIO-Pimo nanoparticles of example 1 at 20 nm;
FIG. 3 is a plot of the particle size distribution of the UIO-Pimo nanoparticles of example 1;
FIG. 4 is a graph showing the results of measurement of hydrated particle size, polydispersity index (PDI) and zeta potential of UIO-Pimo nanoparticles in example 1;
FIG. 5 is a graph of the UV-VIS absorption spectra of UIO-Pimo and UIO nanoparticles of example 1;
FIG. 6 is the fluorescence emission spectra of UIO-Pimo and UIO nanoparticles of example 1 at an excitation wavelength of 480 nm;
FIG. 7 is a transmission electron micrograph of UIO-Pimo nanoparticles of example 1 before and after hypoxic aggregation;
FIG. 8 is a graph showing the results of measurement of hydrated particle size of UIO-Pimo nanoparticles of example 1 before and after hypoxic aggregation;
FIG. 9 is the UIO-Pimo nanoparticles of example 1T of particles before and after hypoxic aggregation 2 A graph of weighted nuclear magnetic signal measurements;
FIG. 10 is the fluorescence spectrum of UIO-Pimo nanoparticles of example 1 before and after hypoxic aggregation with an excitation wavelength of 480 nm;
FIG. 11 is an image of a confocal laser microscopy image after incubation of the UIO-Pimo nanoparticles of example 1 with cells cultured under different oxygen conditions;
FIG. 12 is a photograph of a fluorescent image of the UIO-Pimo nanoparticles of example 1 against hypoxic tumors in mice;
FIG. 13 is T at different time points of mouse tumor tissue after tail vein injection of UIO-Pimo nanoparticles of example 1 (20mg/kg) 2 A weighted nuclear magnetic resonance imaging map;
FIG. 14 is a graph showing the results of Prussian blue staining of mouse tumor tissues 3 hours after tail vein injection of UIO-Pimo nanoparticles (20mg/kg) of example 1, wherein the right graph is an enlarged view of the designated locations in the left graph.
Detailed Description
The technical solution of the present invention is further described below by way of specific embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
Example 1
This example prepares a tumor hypoxia multimodal imaging agent using the following steps:
(1) dispersing polyacrylic acid modified iron oxide nanoparticles (UIO, 1.6mg Fe) in MES buffer (4mL) with pH of about 6, and adding EDC/NHS (15.2mg/9.2 mg);
(2) after reacting for 2 hours at room temperature, performing ultrafiltration purification by using an ultrafiltration tube with the molecular weight cutoff of 10K, centrifuging at 6000rpm for 20 minutes, dispersing the upper concentrated solution by using deionized water, performing centrifugal cleaning again, and washing for three times;
(3) dispersing the activated UIOs in PBS buffer solution with pH of 7.4, adding L-cysteine (4.84mg, 40. mu. mol) and PLN (0.57mg, 1. mu. mol), and reacting at room temperature for 8 hours;
(4) the ultrafiltration method is the same as the step (2), filtrate is collected, the grafting rate is determined by measuring the absorption at 480nm through an ultraviolet-visible spectrophotometer, the upper layer concentrated solution of an ultrafiltration tube is UIO-Pimo solution, and the filtrate is collected and stored at 4 ℃.
The particle morphology was observed by a transmission electron microscope (FEI, Tecnai G220S-TWIN, 200kV) and, as a result, as shown in FIG. 2, the UIO-Pimo prepared in this example was spherical, and the hydrated particle size and the surface charge of the UIO-Pimo particles prepared in the example were measured by a dynamic light scattering instrument (Zetasizer NanoZS) and, as a result, as shown in FIGS. 3 and 4, it was found that the UIO-Pimo had an average particle size of 4.6 nm; the hydrated particle size is about 10nm, and the surface charge is-40.4.
The UV absorption of the UIO-Pimo prepared in this example was measured using a UV spectrophotometer (Perkin Elmer Lambda 850); the results are shown in FIG. 5, where the absorbance value of UIO-Pimo at 480nm was higher than that of UIO at the same iron concentration, indicating successful ligation of PLN; fluorescence spectroscopy was performed using a fluorescence spectrophotometer (F-4600, HITACHI, Japan) and the results are shown in FIG. 6, where when 480nm was used as excitation wavelength, UIO-Pimo showed a distinct absorption peak at 550nm compared to simple ultra-small iron oxide nanoparticles, further demonstrating the successful ligation of PLN.
The iron concentration in the UIO-Pimo prepared in this example was determined by inductively coupled spectroscopy (PE8000, Perkin Elmer, USA) and the molar ratio of iron element to PLN was determined by ICP-OES (inductively coupled spectroscopy) and uv spectrophotometer as: 21: 0.71.
example 2
This example prepared a tumor hypoxia multimodal imaging agent using the following steps:
(1) dispersing polyacrylic acid modified iron oxide nanoparticles (UIO, 0.8mg Fe) in MES buffer (4mL) with pH of about 6, and adding EDC/NHS (7.6mg/4.6 mg);
(2) after reacting for 2 hours at room temperature, performing ultrafiltration purification by using an ultrafiltration tube with the molecular weight cutoff of 10K, centrifuging at 6000rpm for 20 minutes, then dispersing upper-layer concentrated solution by using deionized water, performing centrifugal cleaning again, and washing for three times;
(3) the activated UIOs were dispersed in PBS buffer pH 7.4, and L-cysteine (4.84mg, 40. mu. mol) and PLN (0.29mg, 0.5. mu. mol) were added to react at room temperature overnight;
(4) the ultrafiltration method is the same as the step (2), filtrate is collected, the grafting rate is determined by measuring the absorption at 480nm through an ultraviolet-visible spectrophotometer, the upper layer concentrated solution of an ultrafiltration tube is UIO-Pimo solution, and the filtrate is collected and stored at 4 ℃.
Through transmission electron microscope observation, the UIO-Pimo prepared by the method is spherical, the hydration particle size is about 10nm, and the molar ratio of the iron element to the PLN is determined by ICP-OES and an ultraviolet spectrophotometer as follows: 21: 0.38.
example 3
This example prepared a tumor hypoxia multimodal imaging agent using the following steps:
(1) dispersing polyacrylic acid modified iron oxide nanoparticles (UIO, 1.6mg Fe) in MES buffer (4mL) with pH of about 6, and adding EDC/NHS (15.2mg/9.2 mg);
(2) after reacting for 2 hours at room temperature, performing ultrafiltration purification by using an ultrafiltration tube with the molecular weight cutoff of 10K, centrifuging at 6000rpm for 20 minutes, then dispersing upper-layer concentrated solution by using deionized water, performing centrifugal cleaning again, and washing for three times;
(3) dispersing the activated UIOs in PBS buffer solution with pH of 7.4, adding L-cysteine (2.42mg, 20. mu. mol) and PLN (0.57mg, 1. mu. mol), and reacting at room temperature overnight;
(4) and (3) performing the same ultrafiltration method as the step (2), collecting filtrate, determining the grafting rate by measuring the absorption at 480nm by using an ultraviolet-visible spectrophotometer, collecting and storing the concentrated solution on the upper layer of the ultrafiltration tube, wherein the concentrated solution is a UIO-Pimo solution.
Through transmission electron microscope observation, the UIO-Pimo prepared by the method is spherical, the hydrated particle size is about 10nm, and the molar ratio of the iron element to the PLN is determined by ICP-OES and an ultraviolet spectrophotometer as follows: 21: 0.67.
example 4
The method adopts the following steps to verify the hypoxic response sensitivity of UIO-Pimo under the coexistence of hypoxic oxygen, NADPH and reductase, and specifically comprises the following steps:
(1) to a 10mL EP tube was added PBS buffer (1mL, pH 7.4) followed by UIO-Pimo (25. mu.g Fe from example 1), and the EP tube was placed in a 37 ℃ water bath;
(2) placing the EP tube into a water bath kettle at 37 ℃, blowing argon for 10min, stopping argon, and adding NADPH (1mg/mL, 0.1mL) and cytochrome c reductase (1mg/mL, 0.1 mL);
(3) continuously blowing argon for 60min, taking out the liquid in the EP tube, and measuring the hydrated particle size, TEM and T 2 Weighting the magnetic resonance imaging signal and the fluorescence spectrum.
The transmission electron micrograph of the UIO-Pimo nanoparticles before and after the hypoxic aggregation is shown in FIG. 7, and it can be seen from FIG. 7 that the UIO-Pimo nanoparticles are uniformly dispersed in normal oxygen and aggregated into a larger-sized assembly under the hypoxic condition. The results of the measurement of the hydrated particle size of UIO-Pimo nanoparticles before and after hypoxic aggregation are shown in fig. 8, and fig. 8 shows that the hydrated particle size of the assembly is significantly increased in the hypoxic state.
The NMR imaging signals were measured using a multi-source NMR spectrometer (BioSpec70/20USR, Bruker). T of UIO-Pimo nanoparticles before and after hypoxic aggregation 2 The results of the weighted nuclear magnetic signal measurements are shown in FIG. 9, and the UIO-Pimo assembly significantly increases the T in the region 2 The relaxation value of the weighted nuclear magnetic resonance imaging, the fluorescence spectrogram of the UIO-Pimo nanoparticles before and after the hypoxic aggregation is shown in FIG. 10, and it can be seen that the fluorescence signal is improved in the anaerobic state.
Example 5
The following steps are adopted in the embodiment to verify the fluorescence imaging of the UIO-Pimo on the hypoxic cells and the tumor tissues, and the method specifically comprises the following steps:
(1) MDA-MB-231 cells were seeded at 8 million cells/well in a glass dish and after overnight adherence, the cells were placed under conditions of varying oxygen content (21% O) 2 ，2％O 2 ，0.1％O 2 ) After 12h incubation, UIO-Pimo (containing 20. mu.M PLN) was added and incubation continued for 3 h. Washing cells with PBS for 3 times, adding Hoechst3342 to stain cell nuclei for 10min, staining lysosome dye for 40min, washing the cells with PBS, observing the fluorescence condition of UIO-Pimo in the cells by using a laser confocal microscope, and recording and mapping.
(2) Female Bal/bc mice of 4-5 weeks are selected, 100 ten thousand 231 breast cancer cells are inoculated subcutaneously at the right hind leg part, and when the tumor grows to a proper size, UIO-Pimo (10mg/kg) is injected into the tail vein. After 4 hours, to verify that UIO-Pimo accumulates at the tumor hypoxic site, we dissected the mice to remove the tumor, embed the tumor tissue in OCT and section. Tumor sections were stained with DAPI and co-localized analysis of NBD green fluorescence and hypoxia inducible factor-1 alpha (HIF-1 alpha) red fluorescence was performed using confocal microscopy. While location of Fe in the tissue sections was confirmed with prussian blue staining. Quantitative analysis of all images was performed using Image Pro Plus (NIH).
FIG. 11 is a graph of the images of UIO-Pimo treated cells at different oxygen contents, as seen at 0.1% O 2 Next, UIO-Pimo exhibits a strong green fluorescence, and the green fluorescence coincides with the red fluorescence of lysosomes. At 2% O 2 Next, the green fluorescence was reduced to 21% O 2 Next, there was almost no green fluorescence, indicating that UIO-Pimo can aggregate into larger-sized assemblies under hypoxic conditions and emit green fluorescence of NBD.
FIG. 12 is an image of the fluorescence image of a tumor section, and it can be seen that the red fluorescence of HIF-1 α clearly coincides with the green fluorescence of UIO-Pimo. It is demonstrated that the UIO-Pimo of the present invention can effectively target hypoxic regions in vivo and aggregate to give green fluorescence.
Example 6
This example uses the following procedure to verify the T of UIO-Pimo on hypoxic tumor tissue 2 The method for weighting the nuclear magnetic resonance imaging signals specifically comprises the following steps:
female Bal/bc mice of 4-5 weeks were selected, 100 ten thousand 231 breast cancer cells were subcutaneously inoculated at the right hind leg, and when the tumor grew to an appropriate size, UIO-Pimo (20mg/kg) was injected into the caudal vein. Performing nuclear magnetic imaging before injection and after injection for 0.5h, 1h, 2h, 4h, 8h and 24h respectively, and collecting T 2 The image is weighted. Wherein images obtained before injection were used as controls. Magnetic imaging was performed on a 7.0T MRI scanner equipped with a mouse coil. Using T with the following parameters 2 Turbo RARE sequence acquisition image: TR 3000ms, TE 50ms, field of view 35 × 35mm, matrix size 256 × 256, slice number 20, slice thickness 1 mm, flip angle 90°，NEX＝5。
FIG. 13 is nuclear magnetic imaging of tumor T 2 The weighted graph shows that the tumor part gradually becomes black from 0.5h to 6h after the material injection, and gradually recovers to the level before the material injection from 8h and 24 h. It is shown that the UIO-Pimo of the present invention has a better in vivo T 2 The nature of the imaging is weighted and can be metabolized from the body. A distinct blue color appears in the prussian blue staining pattern of fig. 14, further illustrating that the imaging agent can be enriched at the tumor site.
The applicant states that the present invention is illustrated by the above examples to describe a hypoxic imaging agent of the invention, a method of making the same and applications thereof, but the present invention is not limited to the above examples, i.e. it is not meant that the invention must rely on the above examples to be practiced. It will be apparent to those skilled in the art that any modifications to the present invention, equivalent substitutions of selected materials and additions of auxiliary components, selection of specific forms, etc., are within the scope and disclosure of the present invention.
Claims (29)
1. The hypoxic imaging agent is characterized by comprising UIO-Pimo nanoparticles, wherein the UIO-Pimo nanoparticles comprise polyacrylic acid-coated ferroferric oxide nanoparticles, probe molecules shown in a formula I and molecules containing sulfydryl, wherein the probe molecules are connected to polyacrylic acid fragments through amide bonds;
the probe molecule shown in the formula I is connected to the polyacrylic acid segment through an amido bond formed by a primary amino group on the structure of the probe molecule and a carboxyl group on the polyacrylic acid segment;
the molecule containing a sulfhydryl group is cysteine.
2. The hypoxic imaging agent according to claim 1, wherein the polyacrylic acid fragment has the structure:
wherein n is an integer of 7 to 50.
3. The hypoxic imaging agent according to claim 1, wherein the UIO-Pimo nanoparticles have a particle size of 5 to 20 nm.
4. The hypoxic imaging agent according to claim 1, wherein the UIO-Pimo nanoparticles have a hydrated particle size of 10 to 100 nm.
5. The hypoxic imaging agent according to claim 1, wherein the molar ratio of the iron element, the thiol-containing molecule and the probe molecule shown in formula I in the UIO-Pimo nanoparticles is (40-100): 1-40): 1.
6. The method of preparing the hypoxic imaging agent of any one of claims 1-5, wherein the method of preparing comprises the steps of:
(1) dispersing polyacrylic acid modified iron oxide nanoparticles into a first reaction solution, adding a condensing agent and an acylation activating agent, and carrying out an activation reaction;
(2) and dispersing the activated nano particles in a second reaction solution, adding a molecule containing sulfydryl and a probe molecule shown in a formula I, and reacting to obtain the hypoxic imaging agent.
7. The method according to claim 6, wherein the first reaction solution in the step (1) is a 2- (N-morpholino) ethanesulfonic acid buffer solution having a pH of 5 to 6.
8. The preparation method according to claim 6, wherein the mass-to-volume ratio of the polyacrylic acid-modified iron oxide nanoparticles in the step (1) to the first reaction solution is (0.4-3.2) mg (4-6) mL based on the mass of the iron element.
9. The production method according to claim 6, characterized in that the condensing agent in step (1) is a carbodiimide condensing agent.
10. The production method according to claim 6, wherein the condensing agent in the step (1) is 1-ethyl- (3-dimethylaminopropyl) carbodiimide.
11. The process according to claim 6, wherein the acylation activator in the step (1) is N-hydroxysuccinimide.
12. The method according to claim 6, wherein the molar ratio of the condensing agent to the acylation activating agent in the step (1) is 1 to 1.5: 1.
13. The method according to claim 6, wherein the amount of the condensing agent is 40 to 80. mu. mol based on the mass of the iron element relative to the amount of the polyacrylic acid-modified iron oxide nanoparticles of step (1) which is 1.6mg based on the mass of the iron element.
14. The method according to claim 6, wherein the activation reaction in step (1) is carried out at room temperature.
15. The method according to claim 6, wherein the activation reaction of step (1) is carried out for 2 to 4 hours.
16. The method according to claim 6, wherein the activation reaction of step (1) is followed by purification by ultrafiltration to remove unreacted activating agent.
17. The method according to claim 16, wherein the molecular weight cut-off of the ultrafiltration tube used for the ultrafiltration purification after the activation reaction in step (1) is 1000 to 10,000 Da.
18. The preparation method according to claim 16, wherein the rotational speed of the ultrafiltration purification after the activation reaction in step (1) is 4000 to 6000 rpm.
19. The preparation method according to claim 16, wherein the time for ultrafiltration purification after the activation reaction in step (1) is 10-30 min, and the number of times of ultrafiltration is 3-5.
20. The method according to claim 6, wherein the second reaction solution in the step (2) is a phosphate buffer solution having a pH of 7.4.
21. The preparation method according to claim 6, wherein the molar ratio of the thiol-group-containing molecule in step (2) to the probe molecule represented by formula I is 1-80: 1.
22. The method according to claim 6, wherein the amount of the thiol-group-containing molecule is 10 to 40 μmol, relative to the amount of the polyacrylic acid-modified iron oxide nanoparticles of step (2), based on the mass of the iron element, which is 1.6 mg.
23. The method according to claim 6, wherein the temperature of the reaction in the step (2) is 0 to 4 ℃.
24. The method according to claim 6, wherein the reaction time in the step (2) is 8 to 24 hours.
25. The method according to claim 6, wherein the step (2) is followed by purification by ultrafiltration.
26. The preparation method of claim 25, wherein the molecular weight cut-off of the ultrafiltration tube used for ultrafiltration purification after the reaction in step (2) is 1000-10,000 Da.
27. The preparation method of claim 25, wherein the rotation speed of the ultrafiltration purification after the reaction in the step (2) is 4000-6000 rpm.
28. The preparation method of claim 25, wherein the time for ultrafiltration purification after the reaction in step (2) is completed is 10-30 min, and the number of times of ultrafiltration is 3-5.
29. Use of the hypoxic imaging agent according to any one of claims 1-5 in the preparation of a cell hypoxic detection material.
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