CN114288425A - Protein-cyanine dye composite fluorophore and preparation method and application thereof - Google Patents
Protein-cyanine dye composite fluorophore and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a protein-cyanine dye composite fluorophore, a preparation method and application thereof, and relates to the technical field of near-infrared imaging probes. Compared with most of the existing near-infrared two-region probes, the compound has good biocompatibility and rapid renal metabolism capability in vivo, has outstanding quantum yield and luminous capability in a near-infrared one/two region, and can be applied to living body fluorescence imaging and molecular imaging. The invention also provides a preparation method of the compound and application of the compound as a fluorescence imaging developer in a near-infrared one/two region, such as blood pool development, first-level lymph node operation navigation, lymphedema, hyperplasia living body visualization and the like.
Description
Technical Field
The invention relates to the technical field of near-infrared imaging probes, in particular to a protein-cyanine dye composite fluorophore and a preparation method and application thereof.
Background
In recent years, with the development of optical imaging technology, especially the introduction of digital imaging technology and computer image analysis technology, biological imaging technology has become an important research means for understanding the tissue structure of living body and elucidating various physiological functions of living body. The fluorescence imaging is unique in the disease observation of living bodies and the research of medicines, biology and the like because of the advantages of low experimental cost, simple imaging process, high imaging speed, high sensitivity, no ionizing radiation to the living bodies and the like. Compared with the imaging in the visible light (400-. However, most of the currently available NIR-II fluorescent probes, such as inorganic probes (usually containing heavy atoms in their backbone) and organic dyes (containing large pi conjugated groups), exhibit low levels of biosafety and poor fluorescence quantum yields and/or uncontrollable pharmacokinetic properties, are retained and accumulated in vivo for long periods after imaging, rendering their clinical conversion potential limited.
NIR-II fluorophores with good biocompatibility and renal excretion ability have been reported successfully so far, and successful examples include small molecules CH1055-PEG, IR-E1, IR-BGP6, and the like. However, these fluorescent molecules exhibit relatively low fluorescence quantum yields in aqueous solutions, limiting their practical application to some extent. Meanwhile, in recent years, researchers successfully compound commercial/clinical approved near-infrared first-region cyanine dyes with fluorescence emission tails (> 1000 nm) and albumin to obtain a series of NIR-II fluorescent probes with good biocompatibility and near-infrared second-region bright luminous capacity, but no mention is made of renal excretion capacity. Therefore, there is an urgent need to develop NIR-II fluorophores that combine high quantum efficiency, good biosafety and enable efficient renal clearance, which will effectively advance the development of NIR-II fluorescence imaging technology for clinical applications.
Disclosure of Invention
The invention aims to provide a protein-cyanine dye composite fluorophore, a preparation method and application thereof, so as to solve the problems in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme:
a protein-cyanine dye complex fluorophore is a complex formed by controllable self-assembly of protein and cyanine dye molecules.
On the basis of the technical scheme, the invention also provides the following optional technical scheme:
in one alternative: the protein is beta-lactoglobulin in the lipocalin family.
In one alternative: the cyanine dye molecule is one of IR-780, IR-783 and IR-808, and the method can be extended to other cyanine dyes with chlorine atoms.
In one alternative: the compound is a covalent binding compound obtained by reacting beta-lactoglobulin with chlorine atoms contained in six-membered rings in the middle of cyanine dye molecules according to the specific position of the beta-lactoglobulin; the outer layer of the covalent bonding compound is beta-lactoglobulin, and cyanine dye molecules are wrapped inside the beta-lactoglobulin.
The preparation method of the protein-cyanine dye complex fluorophore is characterized by comprising the following steps: step S1: mixing a solution A containing beta-lactoglobulin with a solution B containing cyanine dye molecules to obtain a reaction solution, wherein the molar ratio of the cyanine dye molecules to the beta-lactoglobulin in the reaction solution is respectively 0.5: 1. 1: 1. 2:1, 3:1, 4:1, 5:1, the concentration of beta-lactoglobulin in the reaction solution is 0-400 mu M; step S2: heating and co-incubating at 30-90 deg.C for 0-5 hr to obtain complex.
In one alternative: the mole ratio of cyanine dye molecules to beta-lactoglobulin in step S1 was 1: 1; the concentration of beta-lactoglobulin in the reaction solution in the step S1 is 10 mu M; the heating co-incubation temperature in the step S2 is 70-80 ℃; the blending incubation time in step S2 was 2 hours.
Use of the protein-cyanine dye complex fluorophore of claim 1 as an imaging agent in a near-infrared one/two-region window.
In one alternative: the fluorescent imaging agents find use in imaging a variety of cells, tissues or other living organisms.
In one alternative: the composite fluorophore is applied to near-infrared one/two-region imaging in the aspects of primary lymph node surgical navigation, lymphedema and hyperplasia evaluation.
Compared with the prior art, the invention has the following beneficial effects:
the covalent bonding composite fluorophore has good biocompatibility and rapid renal clearance capability in vivo, shows high quantum yield and bright luminous capability in a near infrared region I/II, and has excellent imaging effect in fluorescence imaging and molecular imaging applications;
the invention is inspired by albumin coating technology, and utilizes beta-lactoglobulin to carry out external modification on cyanine dye molecules to obtain a near-infrared one/two zone molecular probe with bright luminous capability, thereby effectively reducing aggregation quenching of the cyanine dye molecules in aqueous solution, limiting the spatial structure of the cyanine dye molecules, reducing vibration relaxation, promoting charge transfer in twisted molecules and greatly increasing the quantum yield of the near-infrared one/two zones. In addition, the beta-lactoglobulin also greatly improves the biocompatibility of the probe and effectively changes the metabolic pathway of the probe;
the invention realizes that the depth and the brightness of the probe for detecting organisms are increased, and simultaneously, the composite probe is endowed with the capability of rapidly clearing the kidney, thereby effectively promoting the process of realizing the clinical application of the near-infrared one/two-region fluorescence imaging technology.
Meanwhile, the invention also successfully verifies the feasibility of the application of the compound and Magnetic Resonance Imaging (MRI) to near infrared one/two-region/MRI bimodal imaging, which is expected to greatly improve the accuracy of clinical detection and surgical navigation.
Drawings
FIG. 1 is a schematic representation of the binding of beta-lactoglobulin to cyanine dye according to example 1 of the present invention;
FIG. 2 is a mass spectrum characterization of beta-lactoglobulin before and after complexing with cyanine dye in example 1 of the present invention;
FIG. 3 is a graph showing the one-region/two-region fluorescence spectra before and after the complexation of beta-lactoglobulin and cyanine dye in example 1 of the present invention;
FIG. 4 is a graph showing the quantum yield of the beta-lactoglobulin @ cyanine dye composite fluorescent probe of example 1 of the present invention and comparison with clinical ICG;
FIG. 5 is a diagram showing the optimization of the reaction temperature and the gel electrophoresis analysis thereof in example 1 of the present invention;
FIG. 6 is a diagram of the optimization of the reaction charge ratio and the gel electrophoresis analysis thereof in example 1 of the present invention;
FIG. 7 is a graph of gel electrophoresis analysis with optimized reaction time in example 1 of the present invention;
FIG. 8 is a graph showing the optimization of reactant concentration and gel electrophoresis analysis thereof in example 1 of the present invention;
FIG. 9 is a gel electrophoresis analysis chart showing the complexation of beta-lactoglobulin with cyanine dye molecules having chlorine atoms in the middle of the molecules, including IR-780, IR-783 and IR-808, and ICG having no chlorine atoms in the middle of the molecules in example 1 of the present invention;
FIG. 10 is a blood safety assessment of example 2 of the present invention on the β -lactoglobulin @ IR-780 complex fluorophore;
FIG. 11 is a biocompatibility assessment of the β -lactoglobulin @ IR-780 complex fluorophore of example 2 of the present invention;
FIG. 12 is a study of the in vivo metabolic behavior of the β -lactoglobulin @ IR-780 complex fluorophore in example 2 of the present invention;
FIG. 13 is a diagram showing the head vessel imaging of the β -lactoglobulin @ IR-780 complex fluorophore in the near infrared first and second windows in example 2 of the present invention;
FIG. 14 is a leg vessel image of the β -lactoglobulin @ IR-780 complex fluorophore in the near infrared first and second zone windows in example 2 of this invention;
FIG. 15 is a lymph image of the β -lactoglobulin @ IR-780 complex fluorophore in the near infrared first and second windows in example 2 of this invention;
FIG. 16 is a study of the application of the beta-lactoglobulin @ IR-780 complex fluorescent probe in the surgical navigation of primary lymph nodes in example 3 of the present invention;
FIG. 17 is a study on the application of the beta-lactoglobulin @ IR-780 complex fluorescent probe in the evaluation of lymphedema and hyperplasia in example 4 of the present invention;
FIG. 18 is magnetic resonance imaging after chelation of beta-lactoglobulin with DOTA/Gd in example 3 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. The examples are given solely for the purpose of illustration and are not intended to limit the scope of the invention. Any obvious modifications or variations can be made to the present invention without departing from the spirit or scope of the present invention.
Please refer to fig. 1-8, which illustrate the embodiment of the present invention in detail;
example 1
The embodiment provides a protein-cyanine dye composite fluorescent probe, which is a covalent bonding compound obtained by reacting beta-lactoglobulin through a specific site thereof with chlorine atoms contained in a six-membered ring in the middle of a cyanine dye molecule, wherein the outer layer of the covalent bonding compound is beta-lactoglobulin, the inside of the beta-lactoglobulin is wrapped by the cyanine dye molecule, and the cyanine dye molecule is wrapped by a hydrophobic cavity of the beta-lactoglobulin itself to form a stable and firm fluorescent probe.
The preparation method of the protein-cyanine dye compound comprises the following steps:
s1: mixing a solution A containing beta-lactoglobulin and a solution B containing cyanine dye molecules to obtain a reaction solution;
the molar ratio of cyanine dye molecules to beta-lactoglobulin in the reaction solution is 0.5:1, and the concentration of the beta-lactoglobulin in the reaction solution is 400 mu M;
s2: then heating and co-incubating for 2 hours at the temperature of 30 ℃ to obtain a covalent binding complex, wherein the cyanine dye molecule is IR-780;
example 2
The embodiment provides a protein-cyanine dye composite fluorescent probe, which is a covalent bonding compound obtained by reacting beta-lactoglobulin through a specific site thereof with chlorine atoms contained in a six-membered ring in the middle of a cyanine dye molecule, wherein the outer layer of the covalent bonding compound is beta-lactoglobulin, the inside of the beta-lactoglobulin is wrapped by the cyanine dye molecule, and the cyanine dye molecule is wrapped by a hydrophobic cavity of the beta-lactoglobulin itself to form a stable and firm fluorescent probe.
The preparation method of the protein-cyanine dye compound comprises the following steps:
s1: mixing a solution A containing beta-lactoglobulin and a solution B containing cyanine dye molecules to obtain a reaction solution;
the molar ratio of cyanine dye molecules to beta-lactoglobulin in the reaction solution is 1:1, and the concentration of the beta-lactoglobulin in the reaction solution is 100 mu M;
s2: then heating and co-incubating for 2 hours at the temperature of 90 ℃ to obtain a covalent binding compound, wherein the cyanine dye molecule is IR-783;
example 3
The embodiment provides a novel protein-cyanine dye composite fluorescent probe, which is a covalent bonding compound obtained by reacting beta-lactoglobulin through a specific site with chlorine atoms contained in a six-membered ring in the middle of a cyanine dye molecule, wherein the outer layer of the covalent bonding compound is beta-lactoglobulin, the inside of the beta-lactoglobulin is wrapped by the cyanine dye molecule, and the cyanine dye molecule is wrapped by a hydrophobic cavity of the beta-lactoglobulin to form a stable and firm fluorescent probe.
The preparation method of the protein-cyanine dye compound comprises the following steps:
s1: mixing a solution A containing beta-lactoglobulin with a solution B containing cyanine dye molecules to obtain a reaction solution, wherein the molar ratio of the cyanine dye molecules to the beta-lactoglobulin in the reaction solution is 2:1 respectively, and the concentration of the beta-lactoglobulin in the reaction solution is 20 mu M;
s2: then heating and co-incubating for 1 hour at the temperature of 70 ℃ to obtain a covalent binding compound, wherein the cyanine dye molecule is IR-808;
example 4
The embodiment provides a novel protein-cyanine dye composite fluorescent probe, which is a covalent bonding compound obtained by reacting beta-lactoglobulin through a specific site with chlorine atoms contained in a six-membered ring in the middle of a cyanine dye molecule, wherein the outer layer of the covalent bonding compound is beta-lactoglobulin, the inside of the beta-lactoglobulin is wrapped by the cyanine dye molecule, and the cyanine dye molecule is wrapped by a hydrophobic cavity of the beta-lactoglobulin to form a stable and firm fluorescent probe.
The preparation method of the protein-cyanine dye compound comprises the following steps:
s1: mixing a solution A containing beta-lactoglobulin with a solution B containing cyanine dye molecules to obtain a reaction solution, wherein the molar ratio of the cyanine dye molecules to the beta-lactoglobulin in the reaction solution is 3:1 respectively, and the concentration of the beta-lactoglobulin in the reaction solution is 200 mu M;
s2: then heating and co-incubating for 5 hours at the temperature of 80 ℃ to obtain a covalent binding compound, wherein the cyanine dye molecule is ICG;
example 5
The embodiment provides a novel protein-cyanine dye composite fluorescent probe, which is a covalent bonding compound obtained by reacting beta-lactoglobulin through a specific site with chlorine atoms contained in a six-membered ring in the middle of a cyanine dye molecule, wherein the outer layer of the covalent bonding compound is beta-lactoglobulin, the inside of the beta-lactoglobulin is wrapped by the cyanine dye molecule, and the cyanine dye molecule is wrapped by a hydrophobic cavity of the beta-lactoglobulin to form a stable and firm fluorescent probe.
The preparation method of the protein-cyanine dye compound comprises the following steps:
s1: mixing a solution A containing beta-lactoglobulin with a solution B containing cyanine dye molecules to obtain a reaction solution, wherein the molar ratio of the cyanine dye molecules to the beta-lactoglobulin in the reaction solution is 4:1 respectively, and the concentration of the beta-lactoglobulin in the reaction solution is 10 mu M;
s2: then heating and co-incubating for 5 hours at the temperature of 80 ℃ to obtain a covalent binding complex, wherein the cyanine dye molecules comprise IR-780;
example 6
The embodiment provides a novel protein-cyanine dye composite fluorescent probe, which is a covalent bonding compound obtained by reacting beta-lactoglobulin through a specific site with chlorine atoms contained in a six-membered ring in the middle of a cyanine dye molecule, wherein the outer layer of the covalent bonding compound is beta-lactoglobulin, the inside of the beta-lactoglobulin is wrapped by the cyanine dye molecule, and the cyanine dye molecule is wrapped by a hydrophobic cavity of the beta-lactoglobulin to form a stable and firm fluorescent probe.
The preparation method of the protein-cyanine dye compound comprises the following steps:
s1: mixing a solution A containing beta-lactoglobulin with a solution B containing cyanine dye molecules to obtain a reaction solution, wherein the molar ratio of the cyanine dye molecules to the beta-lactoglobulin in the reaction solution is 5:1, and the concentration of the beta-lactoglobulin in the reaction solution is 1 mu M;
s2: and then heating and co-incubating for 0.5 hour at the temperature of 90 ℃ to obtain the covalent binding complex, wherein the cyanine dye molecule is IR-783.
Experiments prove that when the concentration of the beta-lactoglobulin in the reaction solution in the step S1 is 20 mu M, the brightness is the best, but the composite fluorophore has partial quenching phenomenon at the concentration and does not conform to the linear increasing trend, so that the concentration of the beta-lactoglobulin is 10 mu M to be the best reaction condition.
In the above step S2, the most preferable temperature for the heat co-incubation is 70 ℃.
In addition, as shown in fig. 9, by comparing the fluorescence intensity and running data before and after the beta-lactoglobulin is complexed with the cyanine dye, it can be found that the beta-lactoglobulin has fluorescence enhancement effect on both chlorine-containing and chlorine-free cyanine dye molecules, but has more significant enhancement effect on chlorine-containing cyanine dye molecules (IR-780 > IR-808 > IR-783), and effective covalent bond connection is formed between the two.
In-vivo imaging application of beta-lactoglobulin @ cyanine dye composite fluorescent probe
Taking beta-lactoglobulin @ IR-780 as an example, the biological safety of the composite probe was first investigated. As shown in fig. 10 and 11, the complex exhibited good blood safety and biocompatibility, exhibiting negligible cytotoxicity to LO2 and 4T1 cells.
On the basis of the good biological safety, the in vivo metabolic behavior of the beta-lactoglobulin @ cyanine dye composite fluorescent probe is also explored. The composite fluorescent probe was injected into Balb/c mice via tail vein injection and monitored by near infrared two-zone imaging equipment, as shown in FIG. 12, and exhibited a controlled and rapid metabolic behavior-renal metabolism-which was almost completely excreted out of the body 120 h after injection and exhibited negligible skin absorption. This excellent renal clearance property further indicates good biosafety of the complex;
test of
Based on the above characterization, the in vivo imaging effect of the compound prepared in example 1 under the near infrared first and second region imaging windows was evaluated.
Injecting the composite fluorescent probe into a C57 mouse body in a tail vein injection mode, and monitoring blood vessels of the head and the legs of the mouse body by a near-infrared two-region imaging device;
as shown in FIGS. 13 and 14, the composite fluorescent probe exhibits excellent near-infrared two-zone blood vessel imaging capability, and obtains clearer blood vessel imaging capability in a wave band above 1200 or 1300 nanometers along with the red shift of an imaging window. Meanwhile, the lymphatic system imaging capability of the composite fluorescent probe is explored in a sole injection mode. As shown in fig. 15, the composite probe also exhibited excellent imaging ability for the lymphatic system, was able to brighten the popliteal lymph node and sacral lymph node clearly, and exhibited excellent imaging ability even for the lymphatic vessels.
In conclusion, the compound has excellent imaging capability of a blood vessel/lymphatic system in a near-infrared first/second region window, is expected to be applied to subsequent research and surgical navigation of blood vessel/lymphatic system diseases, and accelerates the process of realizing clinical application of a near-infrared first/second region fluorescence imaging technology.
Application of beta-lactoglobulin @ cyanine dye composite fluorescent probe in lymphedema and hyperplasia evaluation
Kunming mice were selected for lymphedema model modeling. First, the mouse lower limb lymphatic vessels were visualized by injecting patent blue into the soles of the feet, and then one side of the blue-stained lymphatic vessels were ligated.
After three days of molding, the foot sole is injected with a beta-lactoglobulin @ cyanine dye composite fluorescent probe, and the hind limb lymphedema condition is observed under a near-infrared camera.
As shown in fig. 17, it was clearly observed that the mouse lymphatic drainage system was altered and that many lymph nodes were successfully lighted. At the same time, a large amount of lymphatic hyperplasia can be clearly observed after the operation. In conclusion, the above results successfully verify the advantages of the beta-lactoglobulin @ cyanine dye composite fluorescent probe in the near-infrared lymphography, can provide the capability of comprehensively understanding the lymphatic drainage pattern, and is expected to quantitatively evaluate lymphatic transport and function through in vivo imaging.
In order to accelerate the clinical transformation process of the composite fluorescent probe;
and further carrying out covalent grafting on the beta-lactoglobulin and a DOTA chelate ring, and chelating with Gd to realize a near infrared one/two region/Magnetic Resonance (MRI) double-imaging probe.
The preparation method comprises the following steps:
s1: weighing a certain amount of DOTA-NHS-ester, dissolving in DMSO to prepare a 20mM solution, and mixing the DOTA-NHS-ester with beta-lactoglobulin 2:1, adding the prepared DOTA-NHS-ester into 100 mu M beta-lactoglobulin aqueous solution. Then, the mixed solution was stirred at room temperature for 12 hours. At the end of the reaction, the mixed solution was repeatedly ultrafiltered three times with 10k ultrafiltration tubes to remove unreacted DOTA-NHS-ester.
S2: weighing a certain amount of gadolinium chloride, dissolving the gadolinium chloride in DMSO to prepare a 20mM solution, and mixing the gadolinium chloride with DOTA-NHS-ester 2:1, slowly adding the prepared gadolinium chloride into the beta-lactoglobulin-DOTA mixed solution after ultrafiltration. Then, the mixed solution was further stirred at room temperature for 12 hours. And after the reaction is finished, repeatedly carrying out ultrafiltration on the mixed solution three times by using a 10k ultrafiltration centrifugal tube to remove unreacted Gd3+, thereby obtaining the beta-lactoglobulin-DOTA compound with the magnetic resonance imaging capability.
Next, the magnetic resonance imaging ability of the prepared β -lactoglobulin-DOTA complex was investigated.
As shown in fig. 18, the composite exhibits effective magnetic resonance imaging capability. Therefore, the successful preparation of the compound lays an effective foundation for the preparation of a near infrared one/two region/Magnetic Resonance (MRI) bimodal imaging probe, and the measure is expected to greatly improve the accuracy of clinical detection and surgical navigation.
The above description is only for the specific embodiments of the present disclosure, but the scope of the present disclosure is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present disclosure, and all the changes or substitutions should be covered within the scope of the present disclosure. Therefore, the protection scope of the present disclosure shall be subject to the protection scope of the claims.
Claims (9)
1. A protein-cyanine dye composite fluorophore is characterized in that the fluorophore is a composite formed by controllable self-assembly of protein and cyanine dye molecules.
2. The protein-cyanine dye complex fluorophore of claim 1, wherein the protein is β -lactoglobulin in the lipocalin family.
3. The protein-cyanine dye complex fluorophore of claim 1, wherein the cyanine dye molecule is one of IR-780, IR-783 and IR-808.
4. The protein-cyanine dye complex fluorophore according to claim 1, wherein the complex is a covalently bound complex obtained by reacting β -lactoglobulin with a chlorine atom contained in a six-membered ring among cyanine dye molecules according to its own specific site; the outer layer of the covalent bonding compound is beta-lactoglobulin, and cyanine dye molecules are wrapped inside the beta-lactoglobulin.
5. A method for preparing a protein-cyanine dye complex fluorophore according to any one of claims 1-4, comprising the steps of: step S1: mixing a solution A containing beta-lactoglobulin with a solution B containing cyanine dye molecules to obtain a reaction solution, wherein the molar ratio of the cyanine dye molecules to the beta-lactoglobulin in the reaction solution is respectively 0.5: 1. 1: 1. 2:1, 3:1, 4:1, 5:1, the concentration of beta-lactoglobulin in the reaction solution is 0-400 mu M; step S2: heating and co-incubating at 30-90 deg.C for 0-5 hr to obtain complex.
6. The method for preparing a protein-cyanine dye complex fluorophore according to claim 1, wherein the molar ratio of cyanine dye molecules to β -lactoglobulin in step S1 is 1: 1; the concentration of beta-lactoglobulin in the reaction solution in the step S1 is 10 mu M; the heating co-incubation temperature in the step S2 is 70-80 ℃; the blending incubation time in step S2 was 2 hours.
7. Use of the protein-cyanine dye complex fluorophore of claim 1 as an imaging agent for fluorescence imaging in the near-infrared one/two-region window.
8. The use of the protein-cyanine dye complex fluorophore as a fluorescent imaging agent in the near-infrared one/two-region window according to claim 1, wherein the fluorescent imaging agent is used for imaging in various cells, tissues or other living organisms.
9. The use of the protein-cyanine dye complex fluorophore as a fluorescent imaging agent in the nir window of claim 7, wherein the complex fluorophore is used for nir imaging in primary lymph node surgical guidance, lymphedema and hyperplasia evaluation.
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