CN114288425A - A protein-cyanine dye composite fluorophore and its preparation method and application - Google Patents

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. self-assembled complexes. Compared with most existing near-infrared second-region probes, the complex of the present invention has good biocompatibility and rapid renal metabolism in vivo, and has outstanding quantum yield in near-infrared first/second region and luminescence capability, which can be applied to in vivo fluorescence imaging and molecular imaging. The invention also provides a preparation method of the compound and its application as a fluorescent imaging imaging agent in the near-infrared first/second region, such as blood pool development, first-level lymph node surgical navigation, lymphedema, visualization of proliferating organisms, and the like.

Description

A protein-cyanine dye composite fluorophore and its preparation method and application

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 technique

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 method to understand the tissue structure of organisms and clarify various physiological functions of organisms. Among them, fluorescence imaging stands out in the research of disease observation, medicine and biology in vivo due to its advantages of low experimental cost, simple imaging process, fast imaging speed, high sensitivity, and no ionizing radiation to living organisms. Compared to imaging in the visible (400-750 nm) and conventional near-infrared (NIR-I, 750-900 nm) regions, near-infrared (NIR-II, 1000-1700 nm) fluorescence imaging can be extremely It greatly reduces the absorption, scattering and autofluorescence of light by biological tissues, which can significantly improve the imaging depth and imaging effect, which has led to a series of preclinical imaging studies. However, most currently available NIR-II fluorescent probes, such as inorganic probes (usually containing heavy atoms in their backbones) and organic dyes (containing large π-conjugated groups), exhibit low levels of biosafety and Poor fluorescence quantum yield and/or uncontrollable pharmacokinetic properties, long-term retention and accumulation in vivo after imaging limit its clinical translation potential.

So far, only a handful of NIR-II fluorophores with good biocompatibility and renal excretion have been successfully reported. Successful examples include small molecules CH1055-PEG, IR-E1 and IR-BGP6. However, these fluorescent molecules exhibit relatively low fluorescence quantum yields in aqueous solutions, limiting their practical applications to some extent. At the same time, in recent years, researchers have successfully combined the fluorescent emission tail (>1000 nm) of commercial/clinically approved cyanine dyes in the near-infrared region with albumin to obtain a series of biologically compatible Capacitive and NIR-II fluorescent probes with bright luminescence in the near-infrared second region, but no mention of renal excretion. Therefore, there is an urgent need to develop NIR-II fluorophores with high quantum efficiency, good biosafety and effective renal clearance, which will effectively promote the clinical application of NIR-II fluorescence imaging technology.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a protein-cyanine dye composite fluorophore and its preparation method and application, so as to solve the problems in the background technology.

To achieve the above object, the present invention provides the following technical solutions:

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 above technical solutions, the present invention also provides the following optional technical solutions:

In an alternative: the protein is β-lactoglobulin in the lipocalin family.

In an optional solution: the cyanine dye molecule is one of IR-780, IR-783 and IR-808, it is worth noting that this method can be extended to other cyanine dyes with chlorine atoms .

In an optional solution: the complex is β-lactoglobulin according to the reaction of its specific site with the chlorine atom of the six-membered ring in the middle of the cyanine dye molecule. Binding complex; the outer layer of the covalently binding complex is β-lactoglobulin, and the interior of the β-lactoglobulin is wrapped with cyanine dye molecules.

A method for preparing a protein-cyanine dye composite fluorophore as described above, characterized by comprising the following steps: Step S1: Mixing solution A containing β-lactoglobulin and solution B containing cyanine dye molecules A reaction solution is obtained, wherein the molar ratios of cyanine dye molecules and β-lactoglobulin in the reaction solution are 0.5:1, 1:1, 2:1, 3:1, 4:1, and 5:1, respectively. The concentration of β-lactoglobulin in the reaction solution is 0-400 μM; step S2: heating and co-incubating for 0-5 hours at a temperature of 30-90° C. to obtain a complex.

In an optional solution: in step S1, the molar ratio of cyanine dye molecules to β-lactoglobulin is 1:1; in step S1, the concentration of β-lactoglobulin in the reaction solution is 10 μM; in step S2 The heating co-incubation temperature is 70-80° C.; the mixing incubation time in step S2 is 2 hours.

Application of the protein-cyanine dye composite fluorophore described in claim 1 as a fluorescent imaging imaging agent in the near-infrared first/second region window.

In an optional solution: the fluorescent imaging imaging agent is used for imaging in various cells, tissues or other living organisms.

In an optional solution: the application of the composite fluorophore for near-infrared first/second zone imaging in primary lymph node surgical navigation, lymphedema and hyperplasia assessment.

Compared with the prior art, the beneficial effects of the present invention are as follows:

The covalently bound composite fluorophore of the present invention has good biocompatibility and rapid renal clearance in vivo, exhibits high quantum yield and bright luminescence in the near-infrared first/second region, and is used in fluorescence imaging and molecular Excellent imaging effect in imaging applications;

Inspired by the albumin coating technology, the present invention utilizes β-lactoglobulin to externally modify the cyanine dye molecules to obtain a near-infrared first/second region molecular probe with bright luminescence, which effectively reduces the concentration of the cyanine dye molecules in the Aggregation quenching in aqueous solution limits the spatial structure of cyanine dye molecules, reduces vibrational relaxation, promotes charge transfer in twisted molecules, and greatly increases the quantum yield in the near-infrared first and second regions. In addition, β-lactoglobulin also greatly improved the biocompatibility of the probe and effectively changed the metabolic pathway of the probe;

The invention realizes that while increasing the depth and brightness of the probe to detect the organism, the compound probe can be given rapid renal clearance capability, thereby effectively promoting the clinical application of the near-infrared first/second region fluorescence imaging technology.

At the same time, the present invention also successfully verifies the feasibility of the composite and magnetic resonance (MRI) imaging for near-infrared first/second area/magnetic resonance dual-modal imaging application, which is expected to greatly improve clinical detection and surgical navigation. accuracy.

Description of drawings

1 is a schematic diagram of the combination of β-lactoglobulin and cyanine dye according to Example 1 of the present invention;

Fig. 2 is the mass spectrometry characterization of Example 1 of the present invention before and after the compounding of β-lactoglobulin and cyanine dye;

Fig. 3 is the first-area/second-area fluorescence spectrum characterization before and after the compounding of β-lactoglobulin and cyanine dye in Example 1 of the present invention;

Fig. 4 is the quantum yield of Example 1 of the present invention to β-lactoglobulin@cyanine dye composite fluorescent probe and the comparison chart with clinical ICG;

Fig. 5 is the optimization of reaction temperature and its gel electrophoresis analysis diagram of embodiment 1 of the present invention;

Fig. 6 is embodiment 1 of the present invention to the optimization of reaction feed ratio and its gel electrophoresis analysis diagram;

Fig. 7 is the optimization of reaction time, gel electrophoresis analysis diagram of embodiment 1 of the present invention;

Fig. 8 is the optimization of reactant concentration and its gel electrophoresis analysis diagram according to Embodiment 1 of the present invention;

Fig. 9 is the cyanine dye molecule of β-lactoglobulin and the chlorine atom in the middle of the molecule in Example 1 of the present invention, including IR-780, IR-783, IR-808, and the cyanine dye molecule without the middle of the molecule The complex situation of ICG of chlorine atom and its gel electrophoresis analysis chart;

Figure 10 is the blood safety evaluation of the β-lactoglobulin@IR-780 composite fluorophore in Example 2 of the present invention;

Figure 11 is the biocompatibility evaluation of Example 2 of the present invention to β-lactoglobulin@IR-780 composite fluorophore;

Fig. 12 is the in vivo metabolic behavior exploration of β-lactoglobulin@IR-780 composite fluorophore in Example 2 of the present invention;

Fig. 13 is the head blood vessel imaging of the β-lactoglobulin@IR-780 composite fluorophore in the near-infrared region 1 and region 2 windows in Example 2 of the present invention;

Fig. 14 is the imaging of leg blood vessels in the near-infrared region one and region two windows of β-lactoglobulin@IR-780 composite fluorophore in Example 2 of the present invention;

Figure 15 is the lymphatic imaging of the β-lactoglobulin@IR-780 composite fluorophore in the near-infrared region one and region two windows in Example 2 of the present invention;

Figure 16 is the application exploration of β-lactoglobulin@IR-780 composite fluorescent probe in first-level lymph node surgical navigation in Example 3 of the present invention;

Fig. 17 is the application exploration of the β-lactoglobulin@IR-780 composite fluorescent probe in the evaluation of lymphedema and hyperplasia in Example 4 of the present invention;

Figure 18 is the magnetic resonance imaging of β-lactoglobulin after chelation with DOTA/Gd in Example 3 of the present invention.

Detailed ways

In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. The embodiments listed in the present invention are only used to illustrate the present invention, but not to limit the scope of the present invention. Any obvious modifications or changes made to the present invention do not depart from the spirit and scope of the present invention.

Please refer to FIG. 1-FIG. 8, the embodiments of the present invention are described in detail;

Example 1

This embodiment provides a protein-cyanine dye composite fluorescent probe, the probe is composed of β-lactoglobulin through its specific site and the chlorine atom ring carried by the six-membered ring in the middle of the cyanine dye molecule. Atoms react to obtain a covalently bound complex, the outer layer of the covalently bound complex is β-lactoglobulin, the interior of β-lactoglobulin is wrapped with cyanine dye molecules, and the cyanine dye molecules are surrounded by β-lactoglobulin itself. The hydrophobic cavity is coated to form a stable and firm fluorescent probe.

The preparation method of the above-mentioned protein-cyanine dye complex comprises the following steps:

S1: mixing solution A containing β-lactoglobulin and solution B containing cyanine dye molecules to obtain a reaction solution;

The molar ratio of cyanine dye molecules to β-lactoglobulin in the reaction solution is 0.5:1, and the concentration of β-lactoglobulin in the reaction solution is 400 μM;

S2: then heat and co-incubate at 30°C for 2 hours to obtain a covalently bound complex, and the cyanine dye molecule is IR-780;

Example 2

This embodiment provides a protein-cyanine dye composite fluorescent probe, the probe is composed of β-lactoglobulin through its specific site and the chlorine atom ring carried by the six-membered ring in the middle of the cyanine dye molecule. Atoms react to obtain a covalently bound complex, the outer layer of the covalently bound complex is β-lactoglobulin, the interior of β-lactoglobulin is wrapped with cyanine dye molecules, and the cyanine dye molecules are surrounded by β-lactoglobulin itself. The hydrophobic cavity is coated to form a stable and firm fluorescent probe.

The preparation method of the above-mentioned protein-cyanine dye complex comprises the following steps:

S1: mixing solution A containing β-lactoglobulin and solution B containing cyanine dye molecules to obtain a reaction solution;

The molar ratio of cyanine dye molecules to β-lactoglobulin in the reaction solution is 1:1, and the concentration of β-lactoglobulin in the reaction solution is 100 μM;

S2: then heat and co-incubate at 90°C for 2 hours to obtain a covalently bound complex, and the cyanine dye molecule is IR-783;

Example 3

This embodiment provides a novel protein-cyanine dye composite fluorescent probe, which is composed of β-lactoglobulin through its specific site and the chlorine atom ring carried by the six-membered ring in the middle of the cyanine dye molecule. The chlorine atom reacts to obtain a covalently bound complex, the outer layer of the covalently bound complex is β-lactoglobulin, the interior of β-lactoglobulin is wrapped with cyanine dye molecules, and the cyanine dye molecules are surrounded by β-lactoglobulin. It is coated with its own hydrophobic cavity to form a stable and firm fluorescent probe.

The preparation method of the above-mentioned protein-cyanine dye complex comprises the following steps:

S1: mixing solution A containing β-lactoglobulin and solution B containing cyanine dye molecules to obtain a reaction solution, wherein the molar ratio of cyanine dye molecules and β-lactoglobulin in the reaction solution is 2:1, respectively, The concentration of β-lactoglobulin in the reaction solution is 20 μM;

S2: then heating and co-incubating at 70°C for 1 hour to obtain a covalently bound complex, and the cyanine dye molecule is IR-808;

Example 4

This embodiment provides a novel protein-cyanine dye composite fluorescent probe, which is composed of β-lactoglobulin through its specific site and the chlorine atom ring carried by the six-membered ring in the middle of the cyanine dye molecule. The chlorine atom reacts to obtain a covalently bound complex, the outer layer of the covalently bound complex is β-lactoglobulin, the interior of β-lactoglobulin is wrapped with cyanine dye molecules, and the cyanine dye molecules are surrounded by β-lactoglobulin. It is coated with its own hydrophobic cavity to form a stable and firm fluorescent probe.

The preparation method of the above-mentioned protein-cyanine dye complex comprises the following steps:

S1: mixing solution A containing β-lactoglobulin and solution B containing cyanine dye molecules to obtain a reaction solution, wherein the molar ratio of cyanine dye molecules and β-lactoglobulin in the reaction solution is 3:1, respectively, The concentration of β-lactoglobulin in the reaction solution is 200 μM;

S2: then heat and co-incubate at 80°C for 5 hours to obtain a covalently bound complex, and the cyanine dye molecule is ICG;

Example 5

This embodiment provides a novel protein-cyanine dye composite fluorescent probe, which is composed of β-lactoglobulin through its specific site and the chlorine atom ring carried by the six-membered ring in the middle of the cyanine dye molecule. The chlorine atom reacts to obtain a covalently bound complex, the outer layer of the covalently bound complex is β-lactoglobulin, the interior of β-lactoglobulin is wrapped with cyanine dye molecules, and the cyanine dye molecules are surrounded by β-lactoglobulin. It is coated with its own hydrophobic cavity to form a stable and firm fluorescent probe.

The preparation method of the above-mentioned protein-cyanine dye complex comprises the following steps:

S1: mixing solution A containing β-lactoglobulin and solution B containing cyanine dye molecules to obtain a reaction solution, wherein the molar ratio of cyanine dye molecules and β-lactoglobulin in the reaction solution is 4:1, respectively, The concentration of β-lactoglobulin in the reaction solution is 10 μM;

S2: then heat and co-incubate at 80°C for 5 hours to obtain a covalently bound complex, and the cyanine dye molecule includes IR-780;

Example 6

This embodiment provides a novel protein-cyanine dye composite fluorescent probe, which is composed of β-lactoglobulin through its specific site and the chlorine atom ring carried by the six-membered ring in the middle of the cyanine dye molecule. The chlorine atom reacts to obtain a covalently bound complex, the outer layer of the covalently bound complex is β-lactoglobulin, the interior of β-lactoglobulin is wrapped with cyanine dye molecules, and the cyanine dye molecules are surrounded by β-lactoglobulin. It is coated with its own hydrophobic cavity to form a stable and firm fluorescent probe.

The preparation method of the above-mentioned protein-cyanine dye complex comprises the following steps:

S1: Mix solution A containing β-lactoglobulin and solution B containing cyanine dye molecules to obtain a reaction solution, and the molar ratio of cyanine dye molecules to β-lactoglobulin in the reaction solution is 5:1, so The concentration of β-lactoglobulin in the reaction solution was 1 μM;

S2: then heating and co-incubating at 90° C. for 0.5 hours to obtain a covalently bound complex, and the cyanine dye molecule is IR-783.

Experiments have verified that when the concentration of β-lactoglobulin in the reaction solution in the above step S1 is 20 μM, the brightness is the best, but the composite fluorophore has been partially quenched at this concentration, which does not conform to the linear increasing trend, so β-lactoglobulin is selected. The concentration of lactoglobulin was 10 μM as the optimal reaction condition.

In the above step S2, the most preferred heating and co-incubation temperature is 70°C.

In addition, as shown in Figure 9, by comparing the fluorescence intensity before and after the complexation of β-lactoglobulin with cyanine dyes and the running data, it can be found that β-lactoglobulin has no effect on chlorine-containing/chlorine-free cyanine dyes. Both molecules have fluorescence enhancement effect, but the enhancement effect is more obvious for chlorine-containing cyanine dye molecules (IR-780 > IR-808 > IR-783), and an effective covalent bond is formed between the two.

In vivo imaging application of β-lactoglobulin@cyanine dye composite fluorescent probe

Taking β-lactoglobulin@IR-780 as an example, the biosafety of the composite probe was first explored. As shown in Figures 10 and 11, the complex exhibited good blood safety and biocompatibility, with negligible cytotoxicity against LO2 and 4T1 cells.

On the basis of the above-mentioned good biological safety, the in vivo metabolic behavior of the β-lactoglobulin@cyanine dye composite fluorescent probe was also explored. The composite fluorescent probe was injected into Balb/c mice through tail vein injection and monitored by a near-infrared second-zone imaging device. As shown in Figure 12, the composite probe showed controllable and rapid metabolic behavior - Renal metabolism with almost complete excretion and negligible dermal absorption 120 hours after injection. This excellent renal clearance property further indicates the good biosafety of the complex;

test

Based on the above characterization, the in vivo imaging effect of the composite prepared in Example 1 under the near-infrared first and second region imaging windows was evaluated.

The composite fluorescent probe was injected into C57 mice through tail vein injection, and the head and leg blood vessels were monitored by near-infrared second-zone imaging equipment;

As shown in Fig. 13 and Fig. 14, the composite fluorescent probe exhibits excellent blood vessel imaging capability in the near-infrared second region, and with the red-shift of the imaging window, a clearer blood vessel image is obtained in the wavelength band above 1200 or 1300 nm. like ability. At the same time, the imaging ability of the lymphatic system of the composite fluorescent probe was explored by injection into the sole of the foot. As shown in Fig. 15, the composite probe also showed excellent imaging ability for the lymphatic system, which could clearly illuminate the popliteal lymph nodes and sacral lymph nodes, and even showed excellent imaging ability for lymphatic vessels.

In conclusion, the complex has excellent imaging ability of blood vessels/lymphatic system in the near-infrared first/second area window, and is expected to be applied to the follow-up vascular/lymphatic system disease research and surgical navigation, and accelerate the near-infrared first/second area fluorescence imaging. The process of realizing clinical application of technology.

To explore the application of β-lactoglobulin@cyanine dye composite fluorescent probe in the assessment of lymphedema and hyperplasia

Kunming mice were selected for modeling lymphedema. First, the soles of the feet were injected with patent blue to visualize the lymphatic vessels of the lower limbs of the mice, and then one side of the blue-stained lymphatic vessels was ligated.

Three days after modeling, β-lactoglobulin@cyanine dye composite fluorescent probe was injected into the sole of the foot, and the hindlimb lymphedema was observed under the near-infrared camera.

As shown in Figure 17, it can be clearly observed that the lymphatic drainage system of the mice has changed, and multiple lymph nodes have been successfully illuminated. At the same time, massive lymphatic hyperplasia after surgery can also be clearly observed. In conclusion, the above results successfully verified the advantages of β-lactoglobulin@cyanine dye composite fluorescent probe in near-infrared lymphography, which can provide a comprehensive understanding of the lymphatic drainage map, and is expected to be quantitatively evaluated by in vivo imaging. Lymphatic transport and function.

In order to accelerate the clinical transformation process of the composite fluorescent probe;

The β-lactoglobulin was further covalently grafted with the DOTA chelating ring and chelated with Gd to realize the dual imaging probe of near-infrared first/second zone/magnetic resonance (MRI).

The specific preparation method includes the following steps:

S1: Weigh a certain amount of DOTA-NHS-ester and dissolve it in DMSO to prepare a 20mM solution. According to the molar ratio of DOTA-NHS-ester and β-lactoglobulin 2:1, mix the prepared DOTA-NHS-ester Add to 100 µM of β-lactoglobulin in water. Then, the mixed solution was stirred at room temperature for 12 h. At the end of the reaction, the mixed solution was subjected to repeated ultrafiltration three times with a 10k ultrafiltration centrifuge tube to remove unreacted DOTA-NHS-ester.

S2: Weigh a certain amount of gadolinium chloride and dissolve it in DMSO to prepare a 20 mM solution. According to the molar ratio of gadolinium chloride and DOTA-NHS-ester 2:1, slowly add the prepared gadolinium chloride to the above-mentioned ultrafiltration solution. After β-lactoglobulin-DOTA mixed solution. Then, the mixed solution was further stirred for 12 h at room temperature. At the end of the reaction, the mixed solution was repeatedly ultrafiltered three times with a 10k ultrafiltration centrifuge tube to remove unreacted Gd3+, thereby obtaining a β-lactoglobulin-DOTA complex with magnetic resonance imaging capability.

Next, the magnetic resonance imaging capability of the prepared β-lactoglobulin-DOTA complex was investigated.

As shown in Figure 18, the complex exhibited potent magnetic resonance imaging capabilities. It can be seen that the successful preparation of the complex will lay an effective foundation for the realization of the preparation of near-infrared first/second zone/magnetic resonance (MRI) dual-modality imaging probes, which is also expected to greatly improve clinical detection and Accuracy of Surgical Navigation.

The above are only specific embodiments of the present disclosure, but the protection scope of the present disclosure is not limited to this. should be included within the scope of protection of the present disclosure. Therefore, the protection scope of the present disclosure should 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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