CN116510043A - Molecular image nano probe for glioma boundary judgment and preparation method thereof - Google Patents
Molecular image nano probe for glioma boundary judgment and preparation method thereof Download PDFInfo
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- CN116510043A CN116510043A CN202211130455.6A CN202211130455A CN116510043A CN 116510043 A CN116510043 A CN 116510043A CN 202211130455 A CN202211130455 A CN 202211130455A CN 116510043 A CN116510043 A CN 116510043A
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- glioma
- dendritic polymer
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Classifications
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- 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
- A61K49/1857—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 otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. PLGA
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- 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
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Abstract
The invention discloses a molecular image nano probe for glioma boundary judgment and a preparation method thereof. The nanoprobe comprises: the tumor targeting substance comprises a dendritic polymer, a tumor targeting substance and gadolinium, wherein the tumor targeting substance and gadolinium are respectively connected to the surface of the dendritic polymer; the tumor targeting substance is an inhibitor for targeting acetyl transferase GCN 5; the tumor targeting substance is connected with the dendritic polymer through PEG; the gadolinium is connected with the dendritic polymer through a chelating agent; the dendritic polymer is polyamide-amine type dendritic molecule or polylysine dendritic molecule. The nano probe provided by the invention can specifically target glioma, has good safety to organisms and in vivo stability, provides a novel imaging method for judging glioma boundaries, evaluating curative effects and grading in living bodies, brings a novel approach for glioma accurate diagnosis and treatment, and has great scientific significance and potential application value.
Description
Technical Field
The invention relates to the field of medical diagnosis, in particular to a molecular image nano probe for glioma boundary judgment and a preparation method thereof.
Background
Gliomas are the most common malignant tumors of the central nervous system, have special positions, have the characteristics of rapid growth, abundant blood vessels and the like, and have biological behaviors of highly invading and infiltrating to surrounding healthy brain tissues along with the progress of the tumors, so that the boundary between the gliomas and the surrounding brain tissues is unclear.
The search for effective diagnostic and therapeutic methods must be based on a deep understanding of the pathophysiological molecular mechanisms underlying glioma development. A large number of clinical practices show that the early diagnosis and treatment of glioma complement each other, and the accurate early diagnosis is beneficial to the patients to obtain long-term survival. If the glioma focus area can be found and successfully sketched in early stage of the disease, accurate examination and diagnosis can be facilitated, and targeted treatment can be further applied, so that tumor tissues can be removed as much as possible, and damage to surrounding brain tissues can be reduced. Therefore, how to effectively distinguish the biological boundary between glioma tissues and normal brain tissues, judge the tumor grade, truly display the glioma infiltration range, and still be a key problem of glioma diagnosis and treatment.
At present, imaging examination (such as CT and MRI) is a main means for clinically finding glioma, but glioma usually lacks obvious clinical signs in early stage, so that the imaging examination is difficult to accurately determine and the tumor limit cannot be clearly shown, and the glioma cells infiltrated at the boundary of tumor and normal brain tissue are difficult to clear by surgical operation or radiotherapy. In recent years, molecular imaging technology has been rapidly developed, and new molecular imaging markers are continuously emerging. These markers are typically genes or proteins highly associated with the tumor. Under the condition of good imaging technology means, the expression condition of the molecular markers can judge the tumor grade, effectively distinguish tumor and paraneoplastic tissue, accurately determine tumor boundaries, guide operation and radiotherapy and predict tumor prognosis. In the aspect of early diagnosis targets, a large number of basic researches screen out a plurality of mutant genes and protein targets to form a target library, particularly thousands of tumor targets in the age of post-genome and proteomics, and transformation medical researches are hopeful to screen out molecular imaging targets or multi-combination targets which can be used for early diagnosis from the target library. In the aspect of molecular probe construction, a radiolabelling method based on small molecules, macromolecules and nano particles is established, and a series of intelligent response molecular probes, double-targeting (fusion peptide) probes and multi-mode probes are obtained.
Despite the rapid development of molecular imaging techniques, progress in the display of glioma biological boundaries has remained unsatisfactory. Because of the existence of brain blood brain barrier, many molecular image contrast agents are difficult to cross the blood brain barrier and enter glioma growth sites, and are difficult to monitor glioma biological boundaries.
Therefore, development of molecular imaging probes capable of effectively penetrating the blood brain barrier of glioma patients and having glioma targeting is still urgent for glioma treatment.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, an object of the present invention is to provide a molecular imaging nanoprobe for glioma boundary determination, and a preparation method and application thereof. The invention discovers that the key driving gene acetyl transferase GCN5 for revealing occurrence and development of glioma is subjected to imaging tracing, so that early diagnosis, boundary sketching and imaging dynamic monitoring of the development of glioma are realized, a novel target of glioma is utilized to construct a specific image platform of a nano probe, specific imaging of glioma is realized, positioning and boundary sketching of glioma are improved, and subsequent curative effect evaluation is carried out.
In order to achieve the above object, the present invention provides a molecular imaging nanoprobe for glioma boundary determination, the nanoprobe comprising: the tumor targeting substance comprises a dendritic polymer, a tumor targeting substance and gadolinium, wherein the tumor targeting substance and gadolinium are respectively connected to the surface of the dendritic polymer;
the tumor targeting substance is an inhibitor for targeting acetyl transferase GCN 5;
the tumor targeting substance is connected with the dendritic polymer through PEG;
the gadolinium is connected with the dendritic polymer through a chelating agent;
the dendritic polymer is polyamide-amine type dendritic molecule or polylysine dendritic molecule.
Optionally, the tumor targeting substance is 9- (4-hydroxy-3-methoxyphenyl) -3,4,6,7,9, 10-hexahydroacridine-1, 8 (2 h,5 h) -dione (DC-G16) and derivatives thereof, and the structural formula of the DC-G16 and derivatives thereof comprises the following components:
optionally, the tumor targeting agent is DC-G16-11. The structural formula of the DC-G16-11 is as follows:
optionally, one end of the tumor targeting substance is connected with PEG, and the other end of the PEG is connected with maleimide.
Optionally, the number of amino groups contained in the dendrimer is 20 to 80 amino groups.
Optionally, the particle size of the dendrimer is 5-30 nm. After the tumor targeting substance and gadolinium are loaded on the surface of the dendritic polymer, the particle size is almost unchanged, and the particle size of the dendritic polymer is approximately equal to that of the nano probe.
Optionally, the molecular weight of the PEG is 1000-40000, preferably, the molecular weight is 1000-3000; more preferably, the molecular weight is 2000.
Optionally, the chelating agent is a metal ion chelating agent having a macrocyclic ligand. Preferably, the chelating agent is any one of 1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid (DOTA), NOTA and DTPA. More preferably, the chelator has maleimide attached.
Optionally, the gadolinium loading is 106-110 g/mg based on the total mass of the nano probe.
The invention also provides application of the molecular image nano probe for glioma boundary judgment in preparing a product for diagnosing or screening tumors, wherein the acetyl transferase GCN5 in the tumors is expressed in a high way.
Optionally, the tumor is selected from breast cancer, pancreatic cancer, liver cancer, lung cancer, colon cancer, glioma or leukemia; preferably glioma.
Optionally, the product is a contrast agent or an imaging agent.
The invention also provides a preparation method of the molecular image nanoprobe for glioma boundary judgment, which specifically comprises the following steps:
s1, connecting gadolinium to the surface of a dendritic polymer through a chelating agent to obtain an intermediate product;
s2, mixing and reacting the intermediate product with a tumor targeting substance in a solvent to obtain the nano probe.
Optionally, step S1 includes:
s1.1, chelating gadolinium ions and a chelating agent to form a chelating product;
s1.2, mixing and reacting the chelate product with a dendritic polymer to enable gadolinium to be connected to the surface of the dendritic polymer to serve as an intermediate product;
or alternatively, the first and second heat exchangers may be,
s'1.1, mixing and reacting the dendritic polymer with a chelating agent to enable the chelating agent to be connected to the dendritic polymer;
s'1.2, and chelating gadolinium ions, so that gadolinium is connected to the surface of the dendritic polymer and is used as an intermediate product.
Optionally, any one of the following technical features is included:
the mass ratio of gadolinium to the chelating agent is 2: (4-10);
the mass ratio of the chelating agent to the dendrimer is 1: (8-15);
s1.1, wherein the reaction temperature is 40-80 ℃;
s1.1, wherein the pH value is 4-7;
s1.2, wherein the reaction temperature is 10-40 ℃;
s1.2, wherein the pH value is 7-10;
s'1.1, wherein the reaction temperature is 10-40 ℃;
s'1.1, wherein the pH value is 7-10;
s'1.2, wherein the reaction temperature is 40-80 ℃;
s'1.2, the pH value is 4-6.
In S2, the mass ratio of the intermediate product to the tumor targeting substance is (15-30): 1, a step of;
s2, the reaction temperature is 10-40 ℃;
in S2, the pH value is 7-10.
Compared with the prior art, the invention has the following beneficial effects:
1) The molecular image characteristics of the molecular image nano probe for glioma boundary judgment in glioma cell lines, blood brain barrier bionic chips and mouse in-situ transplantation tumor models show that the molecular image nano probe can judge malignant evolution visualization and biological boundaries of glioma high-field MRI, can realize visualization of glioma evolution in vivo, can accurately define the biological boundaries of glioma, is beneficial to grading glioma by imaging, and evaluates the curative effect of subsequent treatment.
2) The nano probe provided by the invention has good in vivo stability and high targeting property, provides a new imaging method for judging glioma boundaries, evaluating curative effects and grading in vivo, and simultaneously brings a new way for glioma accurate diagnosis and treatment, and has great scientific significance and potential application value.
Drawings
FIG. 1 is a schematic structural diagram of a molecular image nanoprobe for glioma boundary determination according to the present invention.
FIG. 2 is a flow chart showing a method for preparing molecular image nanoprobes for glioma boundary determination according to the present invention.
Fig. 3 is a transmission electron microscope image of molecular image nanoprobe Den-Gd-DC-G16 for glioma boundary determination obtained in example 1 of the present invention.
Fig. 4 shows a distribution diagram of Gd element in molecular image nanoprobe de-Gd-DC-G16 for glioma boundary determination obtained in example 1 of the present invention, wherein each green dot is energy spectrum data of Gd, which shows gadolinium element in the field of view alone.
FIG. 5 shows a hydrated particle size distribution diagram of Den-Gd, an intermediate product obtained in example 1 of the present invention.
FIG. 6 shows a hydrated particle size distribution diagram of molecular imaging nanoprobe Den-Gd-DC-G16 for glioma boundary determination obtained in example 1 of the present invention.
FIG. 7 shows Zeta potential diagrams of dendrimer Den, intermediate Den-Gd and nanoprobe Den-Gd-DC-G16 in example 1 of the present invention.
FIG. 8 shows a relaxation rate graph of nanoprobe Den-Gd-DC-G16 in example 6 of the present invention.
FIG. 9 is a graph showing the survival rate of U251 cells treated with nanoprobe Den-Gd-DC-G16 at various time points in example 7 of the present invention.
FIG. 10 shows T1-weighted MR diagrams of different time points after injection of nanoprobe Den-Gd-DC-G16 into tumor-bearing nude mice in example 8 of the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made apparent and fully in view of the accompanying drawings, in which some, but not all embodiments of the invention are shown. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The invention discovers that the key driving gene of the occurrence and development of glioma, namely, acetyl transferase GCN5, is highly expressed at the glioma site. For this reason, the present invention has found small molecule inhibitors capable of specifically binding to GNC5, structural DC-G16 and its derivatives. Wherein the functional targeting group is 9- (4-hydroxy-3-methoxyphenyl) -3,4,6,7,9, 10-hexahydroacridine-1, 8 (2H, 5H) -dione, and the derivative thereof improves the binding efficiency of the functional targeting group and GCN5 by further modifying the molecular structure so as to improve the in-vivo distribution efficiency of the probe.
The performance requirements of molecular imaging nanoprobes for glioma boundary determination at least include: 1) A blood brain barrier crossing ability; 2) Has the capability of specifically combining with GCN 5; 3) Biosafety. The invention designs a probe which can cross a blood brain barrier and gather at a glioma part, and can precisely delineate the boundary of the glioma by radiography.
The invention selects dendritic polymer as carrier, which comprises the following selection criteria: 1) The particle size is controllable, the uniformity is good, and the particle size of the nano probe can be controlled to be lower than 50nm. The dendritic polymer carries the DC-G16 nano probe and has certain lipid solubility, so that the requirements of crossing blood brain barriers can be met. 2) Specific binding to GCN5 requires that the material from which the probe is made be capable of readily modifying the inhibitors (DC-G16 and its derivatives) that are capable of binding specifically to GCN 5. Therefore, the invention selects the polyamide-amine type dendritic molecule or polylysine dendritic molecule as a carrier, is rich in amino groups, is easy to modify, and has good biological safety.
Fig. 1 is a schematic structural diagram of a molecular image nanoprobe for glioma boundary determination according to the present invention. Wherein Den represents a dendrimer, and DC-G16 is an inhibitor of targeted acetyltransferase GCN 5. The nano probe comprises a dendritic polymer (Den), an inhibitor (DC-G16) connected to the surface of the dendritic polymer and gadolinium (Gd) connected to the surface of the dendritic polymer; the DC-G16 is connected with the Den through PEG; the Gd is linked to the Den via a chelating agent (e.g., DOTA).
FIG. 2 is a flow chart showing a method for preparing the molecular image nanoprobe for glioma boundary determination according to the present invention. Den, mal-DOTA and gadolinium trichloride react to obtain an intermediate product Den-Gd, and the intermediate product Den-Gd reacts with DC-G16 in HEPES (as buffer solution, water or water contained) to obtain the nano probe DEN-Gd-DC-G16. As shown in fig. 2, the method specifically comprises the following steps:
s1, connecting gadolinium to the surface of a dendritic polymer through a chelating agent to obtain an intermediate product; the mass ratio of gadolinium to the chelating agent is 2: (4-10). The mass ratio of gadolinium to the chelating agent can be 2: (4 to 6) may be 2: (5-8), may be 2: (7-10). In a preferred embodiment, the mass ratio of gadolinium to chelating agent is 2:5. the mass ratio of the chelating agent to the dendrimer is 1: (8-15). In some embodiments, the mass ratio of the chelating agent to the dendrimer may be 1: (8-10), may be 1: (9-12), can also be 1: (11-13), may also be 1: (12-15). In a preferred embodiment, 1:10.
s2, mixing and reacting the intermediate product with a tumor targeting substance in a solvent to obtain the nano probe. The reaction temperature is 10-40 ℃. The mass ratio of the intermediate product to the tumor targeting substance is (15-30): 1. preferably, the mass ratio of the intermediate product to the tumor targeting substance is (15-25): 1, may be (20 to 30): 1. in a preferred embodiment, 20:1. the reaction temperature may be 10 to 25 ℃, 20 to 35 ℃, or 30 to 40 ℃. In a preferred embodiment, 25 ℃. The pH value is 7-10. Preferably, the pH value can be 7-9 or 8-10. In a preferred embodiment, 8.4. The reaction time is 0.5-3 h. Preferably, the reaction time may be 0.5 to 1.5 hours, 1 to 2 hours, or 2 to 3 hours. In a preferred embodiment, 1h.
In some embodiments, step S1 comprises:
s1.1, chelating gadolinium ions and a chelating agent to form a chelating product; the reaction temperature can be 40-80 ℃; preferably, the temperature may be 40 to 60 ℃, 50 to 70 ℃, or 60 to 80 ℃. In a preferred embodiment, 60 ℃; the pH value is 4-7; further, the pH value is 4 to 6. Preferably, the pH value can be 4-5, also can be 4.5-5.5, also can be 5-6. In a preferred embodiment, the pH is 6. The reaction time is 10-60 min. Preferably, the time is 10 to 30 minutes, 20 to 50 minutes or 40 to 60 minutes. In a preferred embodiment, 30 minutes.
S1.2, the chelating product is mixed and reacted with the dendritic polymer, so that gadolinium is connected to the surface of the dendritic polymer and is used as an intermediate product. The reaction temperature is 10-40 ℃. Preferably, the temperature may be 10 to 20 ℃, 15 to 30 ℃, or 25 to 40 ℃. In a preferred embodiment, 25 ℃. The pH value is 7-10. Preferably, the pH value may be 7 to 8, 7.5 to 9, or 8.5 to 10. In a preferred embodiment, the pH is 8.4. The reaction time is 1-4 h. Preferably, the time can be 1-3 h or 2-4 h. In a preferred embodiment, 2h.
In some embodiments, step S1 comprises:
s'1.1, mixing and reacting the dendritic polymer with a chelating agent to enable the chelating agent to be connected to the dendritic polymer; the reaction temperature is 10-40 ℃. Preferably, the temperature may be 10 to 20 ℃, 15 to 30 ℃, or 25 to 40 ℃. In a preferred embodiment, 25 ℃. The pH value is 7-10. Preferably, the pH value may be 7 to 8, 7.5 to 9, or 8.5 to 10. In a preferred embodiment, the pH is 8.4. The reaction time is 1-4 h. Preferably, the time can be 1-3 h or 2-4 h. In a preferred embodiment, 2h.
S'1.2, and chelating gadolinium ions, so that gadolinium is connected to the surface of the dendritic polymer and is used as an intermediate product. The reaction temperature is 40-80 ℃. Preferably, the temperature may be 40 to 60 ℃, 50 to 70 ℃, or 60 to 80 ℃. In a preferred embodiment, 60 ℃. The pH value is 4-6. Preferably, the pH value can be 4-5, also can be 4.5-5.5, also can be 5-6. In a preferred embodiment, the pH is 6. The reaction time is 10-60 min. Preferably, the time can be 10-30 min, 20-50 min or 40-60 min. In a preferred embodiment, 30 minutes.
DOTA in the present invention means 1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetracarboxylic acid (DOTA); HEPES refers to 4-hydroxyethylpiperazine ethanesulfonic acid.
Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.
Before the embodiments of the invention are explained in further detail, it is to be understood that the invention is not limited in its scope to the particular embodiments described below; it is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention.
Where numerical ranges are provided in the examples, it is understood that unless otherwise stated herein, both endpoints of each numerical range and any number between the two endpoints are significant both in the numerical range. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention may be used to practice the present invention according to the knowledge of one skilled in the art and the description of the present invention.
In the following examples of the present application: dendrimers (Den) dendritic polyamidoamines purchased from WiHai Chen Chemie New Material Co., ltd.) have the following molecular structure:
maleimide-DOTA, available from Hangzhou New Joe BioCo., ltd, labeled Mal-DOTA.
In some embodiments, the inhibitor targeting acetyltransferase GCN5 is selected from DC-G16-11, modified at one end with PEG and modified at the other end with maleimide, designated Mal-PEG-DC-G16-11. Maleimide-polyethylene glycol-DC-G16-11 entrusted to Shanghai blaze Biotechnology Co., ltd, the structure is as follows:
example 1
In this example, a nanoprobe is prepared comprising the steps of:
dissolving gadolinium trichloride in 0.25M hydrochloric acid to form a hydrochloric acid solution with the final concentration of 50mg/mL gadolinium trichloride, mixing 10 mu L of the hydrochloric acid solution of gadolinium trichloride with 1mg of Mal-DOTA (molecular structure shown in the specification), regulating the pH value to 6 by using 0.25M sodium acetate solution, heating to 60 ℃, carrying out chelation reaction for 30min, and chelating gadolinium by DOTA to obtain Mal-DOTA-Gd.
Then, 20mg/mL of 1mL of Den HEPES buffer solution was added to the chelate product Mal-DOTA-Gd, the pH of the HEPES buffer solution was 8.4, the mixture was stirred at room temperature (25 ℃) and reacted for 2 hours, the Mal end of Mal-DOTA was bonded to the amino group of Den by Michael addition reaction, and the mixture was centrifuged to obtain an intermediate Den-Gd.
20mg of Den-Gd obtained above was mixed with 1mg of Mal-PEG-DC-G16-11 in HEPES buffer solution having pH=8.4, and the mixture was stirred at room temperature (25 ℃) for 1 hour, DC-G16 was bonded to the amino group of Den by Mal-terminal Michael addition reaction, and the mixture was centrifuged to obtain a nanoprobe labeled Den-Gd-DC-G16. Transmission electron microscope observation, particle diameter and Zeta potential measurement were performed.
Particle size measurement: the sample was purified with a filter having a pore size of 0.45 μm and diluted to 100g/mL with 1 XPBS. At the same time, the apparatus was calibrated with a standard solution of 2.0mg/mL bovine serum albumin. Particle size distribution was determined by dynamic light scattering. In determining the surface charge of the nanoprobe, the nanoprobe solution was filtered with a 0.45 μm frit and diluted into 10mM NaCl solution.
Zeta potential measurement: the surface potentials of Den, den-Gd and Den-Gd-DC-G16 in example 1 were measured using Malvern Zetasizer.
FIG. 3 is a transmission electron microscope image of the nanoprobe DEN-Gd-DC-G16 in the present embodiment. Wherein A is a transmission electron microscope image of a nano probe Den-Gd-DC-G16 at 100 nm; b is a transmission electron microscope image of the nano probe Den-Gd-DC-G16 at 50nm. As can be seen from FIG. 3, the nano probe Den-Gd-DC-G16 obtained by the invention has uniform morphology, about 20 nm.
Fig. 4 is an EDS diagram (Mapping diagram of TEM spectroscopy) of the nanoprobe Den-Gd-DC-G16 in this example. As can be seen from fig. 4, the surface of the nanoprobe obtained in the present invention successfully chelates Gd.
FIG. 5 is a graph showing the hydrated particle size distribution of Den-Gd, an intermediate product of this example. As can be seen from FIG. 5, the intermediate product Den-Gd has a particle size distribution around 20 nm.
FIG. 6 is a graph showing the particle size distribution of the nanoparticle probe Den-Gd-DC-G16 in this example. As can be seen from FIG. 6, the particle size distribution of the nanoprobe Den-Gd-DC-G16 was around 20 nm.
FIG. 7 is a Zeta potential diagram of dendrimer Den, intermediate Den-Gd and nanoprobe Den-Gd-DC-G16 in this example. As can be seen from fig. 7, the surface potential of the dendrimer Den is 31MV; after chelating Gd by chelating agent Mal-DOTA, the surface potential of the obtained intermediate product Den-Gd is 8.6mV; after further connection with PEG-DC-G16, the surface potential of the obtained nano probe Den-Gd-DC-G16 is-25.3 mV, and the surface potential of the probe is obviously changed, which proves that the connection between Den and Gd and the PEG-DC-G16 is successful.
Example 2
In this example, a nanoprobe is prepared comprising the steps of:
20mg of Den was mixed with 1mg of Mal-DOTA chelator in HEPES buffer at pH=8.4, reacted at room temperature (25 ℃) for 2 hours with stirring, and the solid was collected by centrifugation to obtain Den-DOTA.
Mixing 10 mu L of hydrochloric acid solution of 50mg/mL gadolinium trichloride with 20mg of Den-DOTA, regulating the pH value to 6 by using 0.25M sodium acetate solution, heating to 60 ℃, reacting for 30min, and centrifuging to obtain an intermediate product Den-Gd.
20mg of Den-Gd and 1mg of Mal-PEG-DC-G16-11 are mixed in HEPES buffer solution with pH=8.4, stirred at room temperature (25 ℃) for reaction for 1h, and centrifugally separated, so as to prepare the nano probe Den-Gd-DC-G16.
Example 3
In this example, a nanoprobe is prepared comprising the steps of:
dissolving gadolinium trichloride in 0.25M hydrochloric acid to form a hydrochloric acid solution with the final concentration of 50mg/mL gadolinium trichloride, taking 10 mu L of the hydrochloric acid solution of the gadolinium trichloride and carrying out chelation reaction with 0.8mg of maleimide-DOTA chelating agent (Mal-DOTA), regulating the pH value to 4 by using 0.25M sodium acetate solution, heating to 40 ℃, and reacting for 50min to obtain Mal-DOTA-Gd. And adding 20mg/mL of 1mL of Den HEPES buffer solution into the Mal-DOTA-Gd solution, stirring at room temperature for reaction for 1h, and centrifuging to obtain an intermediate product Den-Gd, wherein the pH value of the HEPES buffer solution is 10.
Mixing 20mg of Den-Gd obtained in the above with 1mg of Mal-PEG-DC-G16-11 in HEPES buffer solution with pH=10, stirring at 10 ℃ for reaction for 2 hours, and centrifugally separating to prepare the nano probe Den-Gd-DC-G16.
Example 4
In this example, a nanoprobe is prepared comprising the steps of:
gadolinium trichloride is dissolved in 0.25M hydrochloric acid to form a hydrochloric acid solution with the final concentration of 50mg/mL gadolinium trichloride, 10 mu L of the hydrochloric acid solution of gadolinium trichloride is taken to carry out chelation reaction with 2mg of maleimide-DOTA (Mal-DOTA) chelating agent, the pH value is regulated to 7 by 0.25M sodium acetate solution, the temperature is raised to 80 ℃ and the reaction is carried out for 20min. Then, 20mg/mL of 1mL of Den HEPES buffer solution was added thereto, the pH of the HEPES buffer solution was 7, and the reaction was stirred at room temperature for 3 hours, followed by centrifugation to obtain Den-Gd as an intermediate.
Mixing 20mg of Den-Gd obtained in the above with 1mg of Mal-PEG-DC-G16-11 in HEPES buffer solution with pH=10, stirring at 40 ℃ for reaction for 0.5h, and centrifugally separating to prepare the nano probe Den-Gd-DC-G16.
Measurement of Gd loading in nanoprobes
The loading of Gd in the nano probe obtained in the example 1 is measured, and the specific method comprises the following steps:
the nano-probe with unit mass (10 mg) is dissolved by hydrochloric acid, the solution is centrifuged, the supernatant is taken and quantitatively diluted to 10mL, and the mass of Gd element in the solution is measured by an atomic absorption spectrophotometer.
The final measured labelling rate of gadolinium was 108.+ -.2. Mu.g of Gd loading per mg of nanoprobe.
Relaxation rate determination of nanoprobes
The relaxation rate of the nanoprobe obtained in example 1 was measured, comprising the following:
the nano probe Den-Gd-DC-G16 prepared in example 1 was dispersed in 25. Mu.L of water to prepare aqueous solutions of Den-Gd-DC-G16 having final Gd concentrations of 0.5, 1.0 and 1.5mM, respectively, while water was used as a control group. Then placing the mixture in a relaxation tube, and measuring the T of the aqueous solution of Den-Gd-DC-G16 with different concentrations in a Bruker MQ60 relaxation analyzer 1 Time.
At 1/T 1 The time is the ordinate, and the atomic absorption spectrophotometer is used for measuring Gd 3+ The concentrations are on the abscissa and a linear fit is performed to obtain a relaxation rate curve as shown in fig. 8. As can be seen from fig. 8, y=10.65x+0.14 (R 2 =0.997), transverse relaxation rate r 1 ＝10.65mM -1 S -1 . The nano probe has magnetism and can be used for magnetic resonance imaging.
In vitro toxicity study of nanoprobes on cells
The in vitro toxicity test of cells was performed on the nanoprobe prepared in example 1, including the following:
experimental group: u251 cells were seeded into 96-well plates, the density was controlled to 10000 cells per well, and incubated at 37 ℃ for 24h to allow cell attachment. Den-Gd-DC-G16 was obtained in example 1 at a final concentration of 1mM, co-cultured at 37℃for 12 hours, 24 hours and 48 hours, and then the supernatant was aspirated, 100. Mu.L of CCK-8 medium was added, and the average absorbance of each well was measured at 425nm using a microplate reader.
Control group: the nanoprobe Den-Gd-DC-G16 in the experimental group is replaced by double distilled water with the same volume.
The toxicity of the nanoprobe Den-Gd-DC-G16 to cells was evaluated according to the ratio of the average absorbance of the experimental group and the control group.
FIG. 9 is a graph showing the survival rate of U251 cells treated with the nanoprobe Den-Gd-DC-G16 at various time points in this example. As can be seen from FIG. 9, the 1mM nanoprobe Den-Gd-DC-G16 treated U251 cells for 48 hours, which was found to have little effect on cell activity and be safe to human body.
Magnetic resonance imaging study of nanoprobes in vivo
The magnetic resonance imaging study of the nanoprobe prepared in example 1 in vivo was performed, and included as follows:
will be 1X 10 7 The individual U251 cells were suspended in PBS solution and subcutaneously injected to the right hindlimb side of 4-6 week old female mice to construct a U251 tumor xenograft model. Raising mice until tumor volume reaches 100-120mm 3 Obtaining the tumor-bearing mice.
The nanoprobe Den-Gd-DC-G16 obtained in example 1 was dissolved in physiological saline at a probe concentration of 10mg/ml and injected into tumor-bearing mice via tail vein, and 300ul of the probe at the above concentration was injected per mouse (i.e., the amount of the injected probe was 3mg per mouse). And respectively carrying out magnetic resonance T1 imaging at 0.5h, 3h and 6h, and observing the T1 imaging effect of the tumor part.
FIG. 10 is a T1-weighted MR plot of the present example at different time points after injection of the nanoprobe Den Gd-DC-G16 solution into tumor-bearing nude mice. Wherein baseline represents prior to injection. As can be seen from fig. 10, the tumor tissue was not significantly delimited from the surrounding muscle tissue prior to injection; after injection, the tumor site becomes bright, the MR signal is significantly enhanced, the tumor region magnetic resonance signal is uniformly raised, clearly distinguished from surrounding tissues, and the signal becomes strong as time increases.
The MR image proves that the nano probe can obviously improve the contrast effect of the tumor part, which proves that the nano probe has excellent targeting property and can obviously enhance the magnetic resonance imaging effect.
The molecular image nano probe for glioma boundary judgment provided by the invention can be used for preparing a product for tumor diagnosis or tumor screening, wherein the acetyl transferase GCN5 in the tumor is expressed in a high way. In some embodiments, the product is a contrast agent or an imaging agent.
The tumor is selected from breast cancer, pancreatic cancer, liver cancer, lung cancer, colon cancer, glioma or leukemia; preferably glioma.
In summary, the targeting nanomolecular probe Den-Gd-DC-G16 of glioma is synthesized by using Dendrimer (Dendrimer, den) and combining with small molecule inhibitor DC-G16 of GCN5 and gadolinium (Gd) respectively. The nano probe is used for positioning glioma in a human body by magnetic resonance and accurately defining the boundary of the glioma, is beneficial to grading glioma by imaging, and evaluates the curative effect of subsequent treatment.
While the present invention has been described in detail through the foregoing description of the preferred embodiment, it should be understood that the foregoing description is not to be considered as limiting the invention. Many modifications and substitutions of the present invention will become apparent to those of ordinary skill in the art upon reading the foregoing. Accordingly, the scope of the invention should be limited only by the attached claims.
Claims (10)
1. A molecular imaging nanoprobe for glioma boundary determination, said nanoprobe comprising: the tumor targeting substance comprises a dendritic polymer, a tumor targeting substance and gadolinium, wherein the tumor targeting substance and gadolinium are respectively connected to the surface of the dendritic polymer;
the tumor targeting substance is an inhibitor for targeting acetyl transferase GCN 5;
the tumor targeting substance is connected with the dendritic polymer through PEG;
the gadolinium is connected with the dendritic polymer through a chelating agent;
the dendritic polymer is polyamide-amine type dendritic molecule or polylysine dendritic molecule.
2. The molecular imaging nanoprobe for glioma boundary determination according to claim 1 wherein the tumor targeting substance is 9- (4-hydroxy-3-methoxyphenyl) -3,4,6,7,9, 10-hexahydroacridine-1, 8 (2 h,5 h) -dione (DC-G16) and its derivative, the structural formula comprises:
3. the molecular imaging nanoprobe for glioma boundary determination according to claim 1, comprising at least one of the following technical features:
one end of the tumor targeting substance is connected with PEG, and the other end of the PEG is connected with maleimide;
the grain diameter of the dendritic polymer is 5-30 nm;
the molecular weight of the PEG is 1000-40000;
the chelating agent is a metal ion chelating agent with a macrocyclic ligand;
the chelating agent is connected with maleimide;
the loading amount of gadolinium is 106-110 g/mg based on the total mass of the nano probe.
4. A molecular imaging nanoprobe for glioma boundary determination according to any one of claims 1 to 3 comprising at least one of the following technical features:
the number of amino groups contained in the dendritic polymer is 20-80;
the chelating agent is any one of 1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid (DOTA), NOTA and DTPA.
5. Use of a molecular imaging nanoprobe for glioma boundary determination according to any one of claims 1 to 4 for the preparation of a product for tumor diagnosis or screening of tumors, characterized in that acetyltransferase GCN5 is highly expressed in said tumors.
6. The use of claim 5, wherein the tumor comprises any one of glioma, breast cancer, pancreatic cancer, liver cancer, lung cancer, colon cancer, or leukemia.
7. The use according to claim 5, wherein the product is a contrast agent or an imaging agent.
8. A method for preparing a molecular imaging nanoprobe for glioma boundary determination according to any one of claims 1 to 4, comprising the steps of:
s1, connecting gadolinium to the surface of a dendritic polymer through a chelating agent to obtain an intermediate product;
s2, mixing and reacting the intermediate product with a tumor targeting substance in a solvent to obtain the nano probe.
9. The method for preparing a molecular imaging nanoprobe for glioma boundary determination according to claim 8, wherein step S1 comprises:
s1.1, chelating gadolinium ions and a chelating agent to form a chelating product;
s1.2, mixing and reacting the chelate product with a dendritic polymer to enable gadolinium to be connected to the surface of the dendritic polymer to serve as an intermediate product;
or alternatively, the first and second heat exchangers may be,
s'1.1, mixing and reacting the dendritic polymer with a chelating agent to enable the chelating agent to be connected to the dendritic polymer;
s'1.2, and chelating gadolinium ions, so that gadolinium is connected to the surface of the dendritic polymer and is used as an intermediate product.
10. The method for preparing the molecular imaging nanoprobe for glioma boundary determination according to claim 9, which is characterized by comprising any one of the following technical characteristics:
the mass ratio of gadolinium to the chelating agent is 2: (4-10);
the mass ratio of the chelating agent to the dendrimer is 1: (8-15);
s1.1, wherein the reaction temperature is 40-80 ℃;
s1.1, wherein the pH value is 4-7;
s1.2, wherein the reaction temperature is 10-40 ℃;
s1.2, wherein the pH value is 7-10;
s'1.1, wherein the reaction temperature is 10-40 ℃;
s'1.1, wherein the pH value is 7-10;
s'1.2, wherein the reaction temperature is 40-80 ℃;
s'1.2, the pH value is 4-6.
In S2, the mass ratio of the intermediate product to the tumor targeting substance is (15-30): 1, a step of;
s2, the reaction temperature is 10-40 ℃;
in S2, the pH value is 7-10.
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