CN117164845B - Preparation method and application of polymethine nanocluster - Google Patents

Preparation method and application of polymethine nanocluster Download PDF

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CN117164845B
CN117164845B CN202311446255.6A CN202311446255A CN117164845B CN 117164845 B CN117164845 B CN 117164845B CN 202311446255 A CN202311446255 A CN 202311446255A CN 117164845 B CN117164845 B CN 117164845B
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flavp
dspe
polymethine
ipa
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CN117164845A (en
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朱守俊
党泽韬
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First Hospital Jinlin University
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Abstract

The invention is suitable for the technical field of molecular probes, and provides a preparation method of a polymethine nanocluster and application thereof, wherein the polymethine nanocluster prepared by the preparation method of the polymethine nanocluster is applied to blood vessel imaging, and is directly used for imaging by tail vein injection when the blood vessel imaging is applied; the prepared polymethine nanoclusters are applied to nerve imaging, and are directly injected into the sciatic nerve in situ for imaging when the nerve imaging is carried out. Compared with the defect that the traditional encapsulation method cannot effectively inhibit aggregation-induced quenching, the polymethine nanocluster prepared by the method can completely release the brightness potential of polymethine dye and has excellent biological imaging performance.

Description

Preparation method and application of polymethine nanocluster
Technical Field
The invention belongs to the technical field of molecular probes, and particularly relates to a preparation method and application of a polymethine nanocluster.
Background
Fluorescence-based in vivo imaging is a rapidly evolving field due to its broad biomedical application prospect and potential for clinical transformations. Organic fluorophores are the most suitable contrast agents from the standpoint of biocompatibility and biosafety, because of their relatively low toxicity. A number of NIR-II organic fluorophores with excellent properties have been developed based on molecular engineering strategies, including polymethine scaffolds. The adjustable photophysical property, higher molar extinction coefficient and strong fluorescence emission make the polymethine scaffold an excellent candidate for near infrared developer. However, hydrophobic polymethine dyes require a critical hydrophilization process prior to use in vivo imaging. However, the significant solvent-borne color shifting behavior and aggregation-induced quenching effects in aqueous solutions severely limit the polymethine dye from exerting its brightness potential.
In order to address these challenges in developing efficient hydrophilization strategies, a completely new and efficient approach is needed. Most of the existing near infrared two-region polymethine dyes for in vivo imaging are prepared by surfactant (e.g., DSPE-PEG 2000 I.e., distearoyl phosphatidylethanolamine-polyethylene glycol 2000). However, most of the dyes in the formed micelles remain in an aggregation-induced quenched state, which limits their imaging in a longer wavelength windowIs not limited by the potential of (a). Here, we designed and synthesized a series of new polymethine dyes, with the fly backbone selected for its remarkable fluorescent brightness and high quantum yield. The Flav is then modified by a two-step hydrophilic process to yield a pegylated Flav (i.e., flavP) 2000 ). Using FlavP 2000 And DSPE-PEG 2000 Self-adaptive co-assembly strategy between the two, we prepared efficient luminescent DSPE-PEG 2000 @FlavP 2000 (abbreviated as DSPE@FlavP) 2000 ) And (3) nanoparticles. When FlavP 2000 With DSPE@FlavP 2000 FlavP when co-assembled or co-assembled in situ with proteins in a living organism 2000 Has the ability to autonomously adjust its conformation. And use of DSPE-PEG 2000 Compared with the traditional encapsulation method for directly encapsulating completely hydrophobic dye, DSPE@FlavP 2000 Shows significantly improved photophysical properties, including sharp absorption in aqueous solution and higher fluorescence quantum yield.
Disclosure of Invention
The invention aims to provide a preparation method and application of a polymethine nanocluster, and aims to solve the problems in the background technology.
In order to achieve the above purpose, the present invention provides the following technical solutions:
a method for preparing a polymethine nanocluster, comprising the steps of:
step S1, synthesizing polymethine dye with the following structure:
；
；
；
or dyes corresponding to the general structural formula:
；
step S2, modifying the Flav through a two-step hydrophilic process to obtain a pegylated Flav:
；
in the above formula, n=12 is FlavP 550 The method comprises the steps of carrying out a first treatment on the surface of the Flavp when n=44 2000 ；
Step S3, synthesis of IPA grafted IPA-FlavP 2000 ：
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Step S4, flavP 2000 And DSPE-PEG 2000 Inter-or FlavP 550 And DSPE-PEG 2000 And then the components are assembled together to prepare DSPE@FlavP 2000 Or DSPE@FlavP 550。
Further, the DSPE@FlavP 2000 Or DSPE@FlavP 550 The preparation method of the co-assembled nanocluster specifically comprises the following steps:
a. DSPE-PEG 2000 Dissolving in DMSO to form a mother solution;
b. 50 microliters of mother liquor and 5 microliters of FlavP 2000 Or FlavP 550 Mixing by vortex to obtain a mixture;
c. adding the mixture into PBS to form 10 ml solution, performing ultrasonic treatment on the solution at 0-4 ℃ for 30 min, and then incubating the solution in an oven at 40-60 ℃ for 1h, and then stabilizing the solution at room temperature in the absence of light for 1 h;
d. concentrating the solution obtained in the step c to 1 ml by an ultrafiltration centrifuge tube to obtain DSPE@FlavP 2000 Or DSPE@FlavP 550 The mother liquor is stored at 0-4deg.C.
Further, the DSPE-PEG 2000 With FlavP 2000 Molar ratio of (C) or DSPE-PEG 2000 With FlavP 550 The molar ratio of (2) was 50:1.
Further, the FlavP 2000 Is prepared through the steps of self-assembled nano-cluster preparation,the method specifically comprises the following steps:
flavp of 2-20 mM 2000 Dissolving the mother liquor in PBS 1 ml to obtain FlavP 2000 Self-assembled nanoclusters are stored at 0-4 ℃.
Further, the FlavP is subjected to 2000 Replacement with IPA-FlavP 2000 Obtaining IPA-FlavP 2000 Self-assembling nanoclusters.
Further, the IPA-FlavP 2000 Or FlavP 2000 In situ co-assembly with proteins in serum or blood occurs in an in vivo or in vitro environment.
The polymethine nanoclusters prepared by the preparation method are provided.
Use of a polymethine nanocluster as described above for vascular imaging, in which case the polymethine nanocluster is directly used for imaging by tail vein injection.
Use of a polymethine nanocluster as described above for neuroimaging, wherein imaging is performed by direct injection of sciatic nerve in situ.
Compared with the prior art, the invention has the beneficial effects that:
compared with the traditional method of directly packaging by the surfactant, the method has the advantages of simple and efficient preparation, high luminous efficiency and the like. In vivo bioimaging applications, DSPE@FlavP 2000 Can image the cerebral vessels and hind limb vessels of mice in the near infrared region above 1500 nm with high signal to noise ratio. FlavP 2000 And IPA-FlavP 2000 The brightness activation phenomenon caused by disassembly can occur in specific environments in vivo and in vitro, and the brand new phenomenon is first discovered. At the same time, IPA-FlavP 2000 Has a significantly prolonged blood circulation time, which makes it of great potential in clinical transformations.
Drawings
In FIG. 1, (a) is the absorption spectra of Flav, thFlav, and PhFlav; (b) fluorescence emission spectra for Flav, thFlav, and PhFlav; (c) Fluorescence intensity values for Flav, thFlav, and PhFlav at 980 nm excitation.
FIG. 2 is a schematic diagram of a process for preparing polymethine nanoclusters in accordance with the present invention.
In FIG. 3, (a) is FlavP 2000 Or FlavP 550 A luminance optimization graph of (2); (b) For DSPE@FlavP 2000 、DSPE@FlavP 550 And an absorption spectrum of dspe@leave; (c) For DSPE@FlavP 2000 、DSPE@FlavP 550 And fluorescent emission of dspe@leave (ratio after brightness optimization); (d) For DSPE@FlavP 2000 A dynamic light scattering particle size distribution histogram; (e) For DSPE@FlavP 550 Dynamic light scattering particle size distribution histogram.
In FIG. 4, (a) is DSPE@FlavP 2000 And dspe@flash for hindlimb vascular imaging in mice; (b) For DSPE@FlavP 2000 And dspe@leave for fluorescence emission of hindlimb vascular imaging of mice; (c) For DSPE@FlavP 2000 And dspe@leave for cerebrovascular imaging in mice; (d) For DSPE@FlavP 2000 And dspe@leave for fluorescence emission for cerebral vascular imaging of mice.
In FIG. 5, (a) is FlavP 2000 And IPA-FlavP 2000 Imaging images in centrifuge tubes containing different liquids; (b) Is a statistical measurement of fluorescence intensity in fig. 5 (a).
In FIG. 6, (a) is FlavP 2000 And IPA-FlavP 2000 After the administration, a blood vessel imaging diagram of the hind limb of the mice is obtained; (b) Statistical measurements of fluorescence intensity of hind limb blood vessels for four mice per group. Wherein FlavP 2000 The fluorescence intensity value at the time point of 1 min after administration was set to 100%; while IPA-FlavP 2000 The fluorescence intensity at 15 min before the administration was in an unstable growth phase, and thus the fluorescence intensity at 15 min after the administration was set to 100%.
In fig. 7, (a) is a schematic diagram of in situ injection of sciatic nerve in rats; (b) For FlavP 2000 Imaging sciatic nerves of the rats at selected time points after administration to both groups of rats; (c) Is IPA-FlavP 2000 Imaging sciatic nerves of the rats at selected time points after administration to both groups of rats; (d) For FlavP 2000 After administration, the sciatic nerve of the rat at the selected time point was imaged in the figureNormalized fluorescence intensity values at the cross section are shown (dash-dot lines); (e) Is IPA-FlavP 2000 After dosing, the rat sciatic nerve at the selected time point was imaged and normalized fluorescence intensity values at the cross section shown (dash-dot line).
FIG. 8 is a nuclear magnetic hydrogen spectrum of intermediate 2a of the present invention.
FIG. 9 is a nuclear magnetic hydrogen spectrum of intermediate 2b of the present invention.
FIG. 10 is a nuclear magnetic hydrogen spectrum of intermediate 3a of the present invention.
FIG. 11 shows nuclear magnetic resonance hydrogen spectra of intermediate 3b of the present invention.
FIG. 12 is a nuclear magnetic hydrogen spectrum of intermediate 3c of the present invention.
FIG. 13 is a nuclear magnetic resonance spectrum of intermediate 4a of the present invention.
FIG. 14 is a nuclear magnetic resonance spectrum of intermediate 4b of the present invention.
FIG. 15 is a nuclear magnetic resonance spectrum of intermediate 4c of the present invention.
FIG. 16 is a nuclear magnetic resonance spectrum of the polymethine dye Flav of the present invention.
FIG. 17 is a nuclear magnetic resonance spectrum of the polymethine dye PhFlav of the present invention.
FIG. 18 is a nuclear magnetic resonance spectrum of the polymethine dye ThFlav of the present invention.
FIG. 19 is a nuclear magnetic hydrogen spectrum of the first step intermediate 6a of the present invention for Flav hydrophilization.
FIG. 20 shows a modified FlavP after hydrophilization according to the invention 550 Nuclear magnetic hydrogen spectrum of (2).
FIG. 21 is a chart showing FlavP after hydrophilization modification according to the present invention 2000 Nuclear magnetic hydrogen spectrum of (2).
FIG. 22 is a nuclear magnetic hydrogen spectrum of an IPA graft-introduced intermediate 8a of the present invention.
FIG. 23 shows IPA-FlavP after hydrophilization modification according to the invention 2000 Nuclear magnetic hydrogen spectrum of (2).
Detailed Description
The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Specific implementations of the invention are described in detail below in connection with specific embodiments.
The preparation method of the polymethine nanocluster provided by the embodiment of the invention comprises the following steps:
a polymethine dye of the following structure was synthesized:
；
；
；
flav is a polymethine dye with emission wavelengths in the near infrared two region. It is mainly a large conjugated hydrophobic structure composed of an oxygen-containing heterocycle with various substituents and a multiolefin centered on chlorocyclohexene. As shown by the general formula above, both PhFlav and ThFlav are variants of the Flav structure, represented by R 1 Or R is 2 The substitution results in a change in photophysical properties, such as absorption and emission wavelengths, of both PhFlav and ThFlav compared to Flav.
The preparation method of polymethine dyes Flav, phFlav and ThFlav comprises the following synthetic route:
；
；
；
；
in the synthesis process, raw materials 1a and 1b respectively carry out aldol condensation and intramolecular cyclization reaction with aromatic ketone with a structure shown above an arrow in a route a, namely 2a and 2b molecules are obtained through a one-pot method. The reaction of the 2a and 2b molecules with diethylamine or diphenylamine, respectively, as shown in scheme b, under the action of a catalyst yields 3a, 3b and 3c molecules. The 3a, 3b and 3c molecules were reacted with methyl grignard reagents shown in scheme c, respectively, followed by quenching with tetrafluoroboric acid to give 4a, 4b and 4c molecules. The molecules 4a, 4b and 4c react with the structure shown above the arrow in scheme d in different solvents under weak base catalysis to give heptamethine dyes Flav (5 a), phFlav (5 b) and ThFlav (5 c), respectively.
In the above scheme, the method is a preferred embodiment, and any one or a combination of several of methanol, ethanol, acetonitrile, n-butanol, toluene, xylene, acetic acid or acetic anhydride can be selected as a reaction solvent in the preparation process; any one or a combination of a plurality of triethylamine, N-diisopropylethylamine, 2, 6-di-tert-butyl tetramethylpyridine, ammonium acetate, potassium acetate and sodium acetate is selected as a weak base catalyst, and a certain temperature from room temperature to reflux temperature of a solvent is selected as a reaction temperature, including but not limited to the combination.
In the preferred embodiment of the present invention, as shown in fig. 1, the absorption spectra and fluorescence emission spectra of Flav, phFlav and ThFlav are respectively given, and the fluorescence intensities of the three under excitation of 980 nm are compared, and finally, flav is selected as the preferred embodiment of the next step.
As a preferred embodiment of the present invention, the modification of Flav by a two-step hydrophilic process results in a pegylated Flav (i.e., flavP 550 Or FlavP 2000 Wherein n=12 is FlavP 550 The method comprises the steps of carrying out a first treatment on the surface of the Flavp when n=44 2000 ）。
As a preferred embodiment of the present invention, the DSPE@FlavP 2000 Or DSPE@FlavP 550 The preparation method of the co-assembled nanocluster specifically comprises the following steps:
a. DSPE-PEG 2000 Dissolving in DMSO to form mother liquor (10-100 mM);
b. 50 microliters of mother liquor and 5 microliters of FlavP 2000 Or FlavP 550 (concentration 2-20 in DMSO mM) by vortexing to give a mixture; the model of the vortex equipment is QL-902;
c. adding the mixture into PBS to form 10 ml solution, performing ultrasonic treatment on the solution at 0-4 ℃ for 30 min, and then incubating the solution in an oven at 40-60 ℃ for 1h, and then stabilizing the solution at room temperature in the absence of light for 1 h; wherein the model of the ultrasonic instrument is KQ5200DE;
d. concentrating the solution obtained in step c to 1 ml by ultrafiltration centrifuge tube (8000 rpm;30000 M.w.) to obtain DSPE@FlavP 2000 Or DSPE@FlavP 550 The mother liquor is stored at 0-4 ℃ for standby.
As a preferred embodiment of the present invention, the DSPE-PEG 2000 With FlavP 2000 Molar ratio of (C) or DSPE-PEG 2000 With FlavP 550 The molar ratio of (2) was 50:1.
In the embodiment of the invention, preferably, as shown in fig. 3, a polymethine nanocluster based on a brand-new self-adaptive co-assembly strategy is prepared, and brightness optimization and characterization are performed on the polymethine nanocluster. Specifically, referring to FIG. a, by optimizing DSPE-PEG 2000 With FlavP 2000 Or DSPE-PEG 2000 With FlavP 550 The final selected molar ratio was 50:1, at which time DSPE@FlavP 2000 Or DSPE@FlavP 550 Has higher brightness. DSPE@FlavP was then determined 2000 And DSPE@FlavP 550 With respect to the absorption and fluorescence emission spectra of (a) and (b) and (c), compared with the DSPE@Flav (DSPE-PEG) of the conventional encapsulation method 2000 DSPE@FlavP) compared to Flav also at a 50:1 molar ratio) 2000 And DSPE@FlavP 550 Has a sharp absorption peak that is significantly improved at high wavelengths, and significantly enhanced fluorescence emission. Finally, the invention characterizes DSPE@FlavP by dynamic light scattering 2000 And DSPE@FlavP 550 See figures d and e for particle size distribution characteristics.
As shown in FIG. 4, DSPE@FlavP 2000 Was used for imaging of the hind limb blood vessels and cerebral blood vessels in mice, and dspe@leave was used as a control. In particular, in hindlimb vascular imagingBefore starting, we first injected two groups of mice with an equal dose of DSPE@FlavP by tail vein respectively 2000 And DSPE@Flav (doses of 500. Mu. Mol, 200. Mu.l each). Imaging was then performed around 1 min after dosing. Imaging conditions were 980 nm excitation and 1500 nm long pass filters were used. As shown in FIG. a and FIG. b, DSPE@FlavP 2000 Clear imaging of the blood vessels of the hind limb of the mice was possible with a signal-to-noise ratio as high as 44.8, whereas dspe@leave no significant signal due to aggregation-induced quenching in the region above 1500 nm. Cerebral vessel imaging experimental procedure and imaging conditions are consistent with hindlimb vessel imaging, as shown in figure c and figure d, DSPE@FlavP 2000 The mouse cerebral vessels can be imaged clearly with a signal-to-noise ratio as high as 44.8, whereas dspe@leave has no apparent signal due to aggregation-induced quenching in the region above 1500 nm.
As a preferred embodiment of the present invention, IPA-FlavP is synthesized 2000 Molecular IPA grafting can extend the circulation time of grafted molecules in the blood of an organism through dynamic intermolecular interactions with albumin. IPA-FlavP 2000 The structure of the molecule is as follows:
；
as a preferred embodiment of the present invention, the FlavP 2000 The preparation steps of the self-assembled nanoclusters comprise:
flavp of 2-20 mM 2000 Dissolving the mother liquor in PBS 1 ml to obtain FlavP 2000 Self-assembled nanoclusters are stored at 0-4 ℃ for later use.
As a preferred embodiment of the present invention, the FlavP 2000 Replacement with IPA-FlavP 2000 Obtaining IPA-FlavP 2000 Self-assembling nanoclusters.
As a preferred embodiment of the present invention, flavP 2000 With IPA-FlavP 2000 Can generate the brightness activation phenomenon (Brightness activation induced by disassembly, namely BAD phenomenon) caused by disassembly under the specific environment in vitro and generate the in situ phenomenon with proteinAnd (5) co-assembling.
In an embodiment of the present invention, preferably, referring to FIG. 5, IPA-FlavP is aggregation-induced quenched in PBS 2000 And FlavP 2000 Self-assembled nanoclusters capable of significant fluorescence intensity in mouse whole blood (micblood), mouse serum (micser), fetal Bovine Serum (FBS), FBS (FBS after ultrafiltration) after filtration through ultrafiltration centrifuge tubes (see FIG. a), demonstrating IPA-FlavP 2000 And FlavP 2000 Self-assembled nanoclusters undergo a fluorescence activation phenomenon induced by disassembly in the above medium, the statistics of which are summarized in graph b. Notably, this was not observed in BSA (50 mg/ml).
We consider that this fluorescence activation phenomenon induced by disassembly is essentially consistent with polymethine nanoclusters based on an entirely new adaptive co-assembly strategy. The difference is that when the former occurs, the proteins in serum or blood act as "surfactants" and are compatible with IPA-FlavP 2000 Or FlavP 2000 In situ co-assembly occurs and thus fluorescence activation phenomena induced by such disassembly should also be divided into the core content of the present invention.
As shown in fig. 6, we verified that this fluorescence activation phenomenon induced by disassembly can occur in mice. Specifically, we performed long-term mice hind limb vascular imaging monitoring experiments. Before imaging begins we first injected tail vein in equal doses of FlavP into two groups of mice, respectively 2000 And IPA-FlavP 2000 (the doses were 500. Mu. Mol, 200. Mu. L each). Imaging was then performed at selected time points after dosing (panel a). A curve of the statistical fluorescence intensity values over time is then given (panel b). It can be seen that first the original aggregation-induced quenched IPA-FlavP 2000 And FlavP 2000 Self-assembled nanoclusters are capable of illuminating the blood vessels of mice after tail vein injection. IPA-FlavP can be seen additionally 2000 Fluorescent intensity with a blood circulation half-life of up to 10 h is FlavP 2000 (1 h) 10 times.
As shown in fig. 7, weIn situ injection and imaging experiments of the sciatic nerve of the rats were then performed (figure a). See panels b and c, flavP 2000 And IPA-FlavP 2000 After each of the two groups of rats was administered (the dose was 500. Mu. Moles, 2. Mu.l, and a microinjector was used in practice), the sciatic nerve of the rat was imaged at the selected time point. See panels d and e, flavP 2000 And IPA-FlavP 2000 After each administration, the rat sciatic nerve at the selected time point was imaged and normalized fluorescence intensity values at the cross section shown (dash-dot line). We can see that this de-assembly-induced fluorescence activation phenomenon can also occur in the nerves of rats. FlavP 2000 And IPA-FlavP 2000 The fluorescence intensity was very low at 0.1. 0.1 s after administration, and then increased gradually, each peaking at about 5.5 min after administration.
Example 1, preparation of intermediates 2a, 2b, 3a, 3b, 3c, 4a, 4b, 4c, flav (5 a), phFlav (5 b), thFlav (5 c);
2a, 2b, 3a, 3b, 3c, 4a, 4b, 4c, fly (5 a), phfly (5 b), thfly (5 c) structural formula and characterization data are as follows:
；
intermediate 2a: 4-bromo-2-hydroxyacetophenone (1 mmol) and 2, 6-dimethoxybenzaldehyde (1 mmol) were dissolved in dimethyl sulfoxide (10 ml) together with pyridine (0.5 mmol), one equivalent of elemental iodine was added, and the mixture solution was heated under nitrogen atmosphere at 150℃under reflux with stirring for 24: 24 h. After completion of the reaction, the reaction mixture was extracted three times with ethyl acetate and washed with water several times, the organic layer was sufficiently dried over anhydrous magnesium sulfate and filtered, and the organic solvent was removed by rotary evaporation to give a crude product, which was isolated and purified by column chromatography to give pale yellow solid 2a (yield 67%). 1 H NMR (400 MHz, CDCl3) δ 8.11 (d, J = 8.5 Hz, 1H), 7.66 (d, J = 1.8 Hz, 1H), 7.52 (dd, J = 8.5, 1.8 Hz, 1H), 7.42 (t, J = 8.4 Hz, 1H), 6.64 (d, J = 8.5 Hz, 2H), 6.42 (s, 1H), 3.80 (s, 6H). LC-HRMS (ESI-TOF): calcd. for C 17 H 13 BrO 4 [M + H] + 360.9997; found 361.0082；
Intermediate 2b:2b and 2a, and the raw material equivalent ratio is substantially identical. The purified product was a yellow solid which was darker than the purified 2a (63% yield). 1 H NMR (400 MHz, Chloroform-d) δ 8.07 (d, J = 8.5 Hz, 1H), 7.78 – 7.70 (m, 2H), 7.61 (dd, J = 5.0, 1.2 Hz, 1H), 7.54 (dd, J = 8.5, 1.7 Hz, 1H), 7.20 (dd, J = 5.0, 3.8 Hz, 1H), 6.72 (s, 1H). LC-HRMS (ESI-TOF): calcd. for C 13 H 7 BrO 2 S [M + H] + 306.9350; found 306.9439；
Intermediate 3a: 2a (1 mmol), sphos-Pd-G3 (0.1 mmol), sphos (0.1 mmol) and cesium carbonate (1.5 mmol) were dissolved together in absolute dry toluene (0.175. 0.175M), then diethylamine (3 mmol) was added to the mixture solution, and the mixture solution was subjected to "warm-freeze" pumping operation three times to provide an anhydrous and anaerobic system environment. The solution was refluxed with stirring at 110℃and the solution gradually turned reddish brown. 24 After h the reaction was complete and monitored by TLC. The mixture solution was extracted three times with methylene chloride, then dried sufficiently with anhydrous magnesium sulfate and filtered, and the organic solvent was removed by rotary evaporation to give a crude product, which was purified by column chromatography to give the product as a yellow oil (yield 81%). 1 H NMR (400 MHz, DMSO-d6) δ 7.80 (d, J = 9.1 Hz, 1H), 7.47 (t, J = 8.5 Hz, 1H), 6.84 (dd, J = 9.1, 2.4 Hz, 1H), 6.78 (d, J = 8.5 Hz, 2H), 6.54 (d, J = 2.4 Hz, 1H), 6.00 (s, 1H), 3.76 (s, 6H), 3.44 (q, J = 7.0 Hz, 4H), 1.11 (t, J = 7.0 Hz, 6H). LC-HRMS (ESI-TOF): calcd. for C 21 H 23 NO 4 [M + H] + 353.1627; found 354.1680；
Intermediate 3b: the synthesis procedure of 3b and the equivalent ratio of the starting materials and catalyst remained the same as the synthesis of 3a (yield 70%). 1 H NMR (400 MHz, DMSO-d6) δ 7.87 (d, J = 8.9 Hz, 1H), 7.53 – 7.18 (m, 11H), 6.89 (dd, J = 8.9, 2.3 Hz, 1H), 6.76 (d, J = 8.5 Hz, 2H), 6.55 (d, J = 2.2 Hz, 1H), 6.13 (s, 1H), 3.74 (s, 6H). LC-HRMS (ESI-TOF): calcd. for C 29 H 23 NO 4 [M + H] + 449.1627; found 450.1696；
Intermediate 3c: the equivalent ratio between the synthetic steps of 3c and 3a and the reaction raw materials and the catalyst remained the same (yield 68%). 1 H NMR (400 MHz, DMSO-d6) δ 7.94 (ddd, J = 15.6, 4.4, 1.2 Hz, 2H), 7.77 (d, J = 9.0 Hz, 1H), 7.27 (dd, J = 5.0, 3.7 Hz, 1H), 6.84 (dd, J = 9.1, 2.4 Hz, 1H), 6.67 (s, 1H), 6.33 (d, J = 2.4 Hz, 1H), 3.47 (q, J = 7.0 Hz, 4H), 1.15 (t, J = 7.0 Hz, 6H). LC-HRMS (ESI-TOF): calcd. for C 17 H 17 NO 2 S [M + H] + 299.0980; found 300.1062；
Intermediate 4a: 3a (1 mmol) was dissolved in absolute dry tetrahydrofuran (0.1M), the temperature of the solution was reduced to below 0 ℃ by ice water bath, methylmagnesium chloride (dissolved in tetrahydrofuran, 1.0 m,2.4 mmol) stored at 0 ℃ was slowly added dropwise to the above solution under ice water bath, the ice water bath was removed, and the reaction system was gradually returned to room temperature. The reaction was carried out overnight. After completion of the reaction, the reaction mixture was quenched by TLC with ethyl acetate/n-hexane (1:10) mixed solvent system using tetrafluoroboric acid (aqueous solution, 5%) coupling, then extracted with dichloromethane/water system, dried thoroughly over anhydrous magnesium sulfate, filtered and the organic solvent was removed by rotary evaporation. The crude product was purified by precipitation in ethyl acetate solution to give the product as a dark red solid (85% yield). 1 H NMR (400 MHz, DMSO-d6) δ 8.23 (d, J = 9.7 Hz, 1H), 7.63 (t, J = 8.5 Hz, 1H), 7.59 – 7.57 (m, 2H), 7.18 (d, J = 2.2 Hz, 1H), 6.92 (d, J = 8.5 Hz, 1H), 3.82 (s, 6H), 3.76 – 3.67 (m, 4H), 2.89 (s, 1H), 1.23 (t, J = 7.0 Hz, 6H). LC-HRMS (ESI-TOF): calcd. for C 22 H 26 NO 3 + [M] + 352.1907; found 352.1914；
Intermediate 4b: the equivalent ratio of the synthetic steps and the reaction raw materials of 4b are consistent with that of the synthetic steps of 4a (yield 80%). 1 H NMR (400 MHz, CDCl 3 ) δ 8.20 (d, J = 9.5 Hz, 1H) 7.59 (s, 1H), 7.53 – 7.47 (m, 5H), 7.41 – 7.36 (m, 3H), 7.33-7.30 (m, 4H), 6.97 (s, 1H), 6.66 (d, J = 8.5 Hz, 2H), 3.84 (s, 6H), 3.02 (s, 3H). LC-HRMS (ESI-TOF): calcd. for C 30 H 26 NO 3 + [M] + 448.1907; found 448.1910；
Intermediate 4c: the equivalent ratio of the synthetic steps of 4c and 4a to the starting materials remained the same (78% yield). 1 H NMR (400 MHz, DMSO-d6) δ 8.39 (dd, J = 4.0, 1.3 Hz, 1H), 8.31 (dd, J = 4.9, 1.2 Hz, 1H), 8.13 (d, J = 9.6 Hz, 1H), 8.02 (s, 1H), 7.51 – 7.40 (m, 2H), 7.20 (d, J = 2.5 Hz, 1H), 3.71 (q, J = 7.1 Hz, 4H), 2.82 (s, 3H), 1.24 (t, J= 7.0 Hz, 6H). LC-HRMS (ESI-TOF): calcd. for C 18 H 20 NO + [M] + 298.1260; found 298.1267；
Flav (5 a): 4a (1 mmol), N- [ (3- (anilinomethylene) -2-chloro-1-cyclohexen-1-yl) methylene]The aniline hydrochloride (0.45 mmol) and 2, 6-di-tert-butyl-4-methylpyridine (3 mmol) are all dissolved in a mixed solvent of n-butyl alcohol (0.165, M) and toluene (0.35, M), the operation of freezing and pumping heating is carried out for three times, then the reaction system is heated to 100 ℃, the reaction is stirred for 10 min to obtain a crude product, and then the crude product is purified by column chromatography to obtain a dark purple black solid (the yield is 40%). 1 H NMR (400 MHz, DMSO-d6) δ 8.10 – 7.72 (m, 4H), 7.37 (t, J = 8.8 Hz, 2H), 7.00 – 6.56 (m, 10H), 6.62 (s, 2H), 3.66 (s, 12H), 3.48 – 3.35 (m, 8H), 2.76 – 2.6 (m, 4H), 1.91 – 1.76 (m, 2H), 1.10 (t, J = 6.4 Hz, 12H). LC-HRMS (ESI-TOF): calcd. for C 52 H 56 ClN 2 O 6 + [M] + 839.3821; found 839.3788；
Phflash (5 b): the synthetic route for Ph-Flav was essentially identical to Flav, with the first step being identical, except that diethylamine, one of the starting materials in the second step, was replaced with diphenylamine (37% yield). 1 H NMR (400 MHz, CD 3 CN) δ 8.29 – 8.18 (m, 2H), 8.02 (d, J = 9.2 Hz, 2H), 7.59 – 7.36 (m, 16H), 7.34 – 7.23 (m, 8H), 7.17 (s, 2H), 7.04 – 6.93 (m, 2H), 6.89 (s, 2H), 6.69 (d, J = 8.3 Hz, 4H), 3.85 (s, 12H), 2.93 – 2.84 (m, 4H), 2.16 – 2.05 (m, 2H). MALDI-TOF-MS: calcd. for C 68 H 56 ClN 2 O 6 + [M] + 1031.382; found 1031.907；
Thfly (5 c): 4c (1 mmol), N- [ (3- (anilinomethylene) -2-chloro-1-cyclohexen-1-yl) methylene]Aniline hydrochloride (0.45 mmol) and sodium acetate (3 mmol) were added together into an absolute dry flask and dissolved in absolute ethanol (0.5M) and subjected to three "freeze-pump warm" operations, then the reaction system was heated to 80 ℃, refluxed and stirred for 10 min to give the crude product, which was then purified by column chromatography to give the final product as a dark purple solid (yield 40%). 1 H NMR (400 MHz, CD 3 CN) δ 8.19 – 7.93 (m, 8H), 7.52 – 7.29 (m, 4H), 7.07 – 6.79 (m, 4H), 6.70 – 6.55 (m, 2H), 3.59 – 3.45 (m, 8H), 2.86 – 2.76 (m, 4H), 1.95 – 1.84 (m, 2H), 1.18 (t, J = 7.6 Hz, 12H). LC-HRMS (ESI-TOF): calcd. for C 44 H 44 ClN 2 O 2 S 2 + [M] + 731.2527; found 731.2471。
Example 2 hydrophilized FlavP 2000 And FlavP 550 Is prepared by the steps of (1);
the structural formula and the preparation process are as follows:
；
；
in the above synthesis, flav (5 a) is first nucleophilic substituted with mercaptopropionic acid under weak base conditions to give 6a molecule, and then 6a molecule is reacted with two methoxy-terminated aminopolyethylene glycols of different molecular weights to give FlavP 550 Or FlavP 2000 。
Intermediate 6a: 3-mercaptopropionic acid (3 mmol) and N, N-diisopropylethylamine (3 mmol) were added to a dry flask and dissolved in anhydrous DMSO (0.1M)The mixture was stirred at room temperature for 30 min. Then, flav (5 a) (1 mmol) was added to the mixture with stirring, and the mixture was heated to 70℃under a protective gas atmosphere and reacted for 1h. The reaction was monitored by TLC. The crude product was purified by flash column chromatography on silica gel to give 6a as a purplish black solid (70% yield). 1 H NMR (400 MHz, CDCl 3 ) δ 8.60 – 8.46 (m, 2H), 7.91 (d, J = 8.6 Hz, 2H), 7.40 (t, J = 8.4 Hz, 2H), 7.02 (s, 2H), 6.93 – 6.78 (m, 4H), 6.66 (d, J = 8.4 Hz, 4H), 6.50 (s, 2H), 3.83 (s, 12H), 3.55 – 3.35 (m, 8H), 3.00 – 2.89 (m, 2H), 2.75 – 2.89 (m, 4H), 1.96 – 1.82 (m, 2H), 1.53 – 1.50 (m, 2H), 1.30 – 1.18 (m, 12H). MALDI-TOF-MS: calcd. for C 55 H 61 N 2 O 8 S + [M] + 909.414; found 909.026；
Product FlavP 2000 And FlavP 550 : 6a (1 mmol) and 2- (7-azabenzotriazol-1-yl) -N, N, N ', N' -tetramethylurea hexafluorophosphate (1 mmol) were added to a dry flask and dissolved in anhydrous THF (0.1M), and the mixture was stirred at room temperature for 30 min. Subsequently adding mPEG to the mixture 2000 - NH 2 / mPEG 550 - NH 2 (1.1 mmol) and N, N-diisopropylethylamine (2 mmol), followed by stirring under a protective gas atmosphere for 12. 12 h. The reaction was monitored by TLC. The crude product was purified by flash column chromatography on silica gel to give a black solid (67% yield). 1 H NMR of FlavP 2000 (400 MHz, CDCl 3 ) δ 8.83 – 7.96 (m, 4H), 7.60 – 7.35 (m, 6H), 7.02 – 6.64 (m, 8H), 3.85 (s, 12H), 3.65 (s, 201H), 3.57 – 3.52 (m, 8H), 3.39 (s, 3H), 2.83 – 2.71 (m, 2H), 2.60 – 2.52 (m, 4H), 2.27 – 2.17 (m, 2H), 2.06 – 1.96 (m, 2H), 1.66 – 1.57 (m, 2H), 1.31 (t, J = 7.3 Hz, 12H). FlavP 2000 was confirmed using MALDI-TOF-MS. Expected M.W. 1441, Measured M.W. 1449; 1 H NMR of FlavP 550 (400 MHz, CDCl 3 ) δ 8.79 – 7.95 (m, 4H), 7.63 – 7.39 (m, 6H), 7.00 – 6.89 (m, 4H), 6.68 (d, J = 8.8 Hz, 4H), 3.85 (s, 12H), 3.65 (s, 56H), 3.57 – 3.53 (m, 8H), 3.38 (s, 3H), 2.84 – 2.74 (m, 2H), 2.61 – 2.49 (m, 4H), 2.22 (t, J = 7.4 Hz, 2H), 1.95 – 1.91 (m, 2H), 1.64 – 1.59 (m, 5H), 1.34 – 1.31 (m, 12H). FlavP 550 was confirmed using MALDI-TOF-MS. Expected M.W. 2891, Measured M.W. 2901。
Example 3 IPA-FlavP 2000 Is prepared by the steps of (1);
the structural formula and the preparation process are as follows:
；
intermediate 8a: 6a (1 mmol) and 2- (7-azabenzotriazol-1-yl) -N, N, N ', N' -tetramethylurea hexafluorophosphate (1 mmol) were added to a dry flask and dissolved in anhydrous DMF (0.1M) and the mixture was stirred at room temperature for 30 min. Subsequently, raw material 7a (1.1 mmol) and N, N-diisopropylethylamine (2 mmol) were added to the mixture, followed by stirring under a protective gas atmosphere 12. 12 h. The reaction was monitored by TLC. The crude product was purified by flash column chromatography on silica gel to give IPA-flash (8 a) as a black solid (70% yield). 1 H NMR (500 MHz, CDCl 3 ) δ 8.54 (s, 2H), 7.93 (d, J = 9.3 Hz, 2H), 7.50 (d, J = 8.2 Hz, 2H), 7.42 (t, J = 8.4 Hz, 2H), 7.08 – 6.91 (m, 4H), 6.89 (d, J = 7.9 Hz, 4H), 6.67 (d, J = 8.5 Hz, 4H), 6.57 (d, J = 7.1 Hz, 1H), 6.54 – 6.46 (m, 2H), 6.19 (t, J = 6.0 Hz, 1H), 6.19 (q, J = 6.2 Hz, 1H), 3.83 (s, 12H), 3.58 (s, 3H), 3.54 – 3.41 (m, 8H), 3.09 (q, J = 6.2 Hz, 2H), 3.02 – 2.87 (m, 2H), 2.79 – 2.60 (m, 4H), 2.50 (t, J = 7.9 Hz, 2H), 2.44 (t, J = 7.2 Hz, 2H), 2.16 (t, J = 7.4 Hz, 2H), 1.98 – 1.87 (m, 2H), 1.85 – 1.78 (m, 2H), 1.63 – 1.57 (m, 4H), 1.34 (q, J = 6.7 Hz, 2H), 1.25 (t, J = 7.1 Hz, 12H). MALDI-TOF-MS: calcd. for C 72 H 84 IN 4 O 10 S + [M] + 1323.4947; found 1322.860；
Product IPA-FlavP 2000 : 8a (1 mmol) and 2- (7-azabenzotriazol-1-yl) -N, N, N ', N' -tetramethylurea hexafluorophosphate (1 mmol) were added to a dry flask and dissolved in anhydrous tetrahydrofuran (0.1M), and the mixture was stirred at room temperature for 30 min. Subsequently adding mPEG to the mixture 2000 - NH 2 (1.1 mmol) and N, N-diisopropylethylamine (2 mmol), followed by stirring under a protective gas atmosphere for 12. 12 h. The reaction was monitored by TLC. The crude product was purified by flash column chromatography on silica gel to give a black solid (41% yield). 1 H NMR (400 MHz, CDCl 3 ) δ 8.55 (d, J = 13.5 Hz, 2H), 7.92 (d, J = 9.2 Hz, 2H), 7.52 (d, J = 7.4 Hz, 2H), 7.42 (t, J = 8.4 Hz, 2H), 7.05 (s, 2H), 6.96 – 6.79 (m, 6H), 6.68 (d, J = 8.4 Hz, 4H), 6.51 (s, 2H), 3.83 (s, 12H), 3.65 (s, 194H), 3.50 – 3.45 (m, 8H), 3.39 (s, 3H), 3.05 – 2.92 (m, 2H), 2.84 – 2.69 (m, 4H), 2.61 – 2.50 (m, 4H), 2.33 – 2.21 (m, 4H), 2.00 – 1.84 (m, 6H), 1.63 – 1.59 (m, 2H), 1.30 – 1.19 (m, 12H). IPA-FlavP 2000 was confirmed using MALDI-TOF-MS. Expected M.W. 3291, Measured M.W. 3303。
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that modifications and improvements can be made by those skilled in the art without departing from the spirit of the present invention, and these should also be considered as the scope of the present invention, which does not affect the effect of the implementation of the present invention and the utility of the patent.

Claims (7)

1. The preparation method of the polymethine nanocluster is characterized by comprising the following steps of:
step S1, synthesizing polymethine dye with the following structure:
；
step S2, modifying the Flav through a two-step hydrophilic process to obtain a pegylated Flav:
；
in the above formula, n=12 is FlavP 550 The method comprises the steps of carrying out a first treatment on the surface of the Flavp when n=44 2000 ；
Step S3, flavP 2000 And DSPE-PEG 2000 Inter-or FlavP 550 And DSPE-PEG 2000 And then the components are assembled together to prepare DSPE@FlavP 2000 Or DSPE@FlavP 550 。
2. The method for preparing polymethine nanoclusters according to claim 1, wherein the dspe@flavpp 2000 Or DSPE@FlavP 550 The preparation method of the co-assembled nanocluster specifically comprises the following steps:
a. DSPE-PEG 2000 Dissolving in DMSO to form a mother solution;
b. 50 microliters of mother liquor and 5 microliters of FlavP 2000 Or FlavP 550 Mixing by vortex to obtain a mixture;
c. adding the mixture into PBS to form 10 ml solution, performing ultrasonic treatment on the solution at 0-4 ℃ for 30 min, and then incubating the solution in an oven at 40-60 ℃ for 1h, and then stabilizing the solution at room temperature in the absence of light for 1 h;
d. concentrating the solution obtained in the step c to 1 ml by an ultrafiltration centrifuge tube to obtain DSPE@FlavP 2000 Or DSPE@FlavP 550 The mother liquor is stored at 0-4deg.C.
3. The method for preparing polymethine nanoclusters according to claim 2, characterized in that the DSPE-PEG 2000 With FlavP 2000 Molar ratio of (C) or DSPE-PEG 2000 With FlavP 550 The molar ratio of (2) was 50:1.
4. A method for preparing a polymethine nanocluster, comprising:
preparation of FlavP 2000 Self-assembled nanoclusters or preparation of IPA-FlavP 2000 Self-assembling nanoclusters;
preparation of FlavP 2000 Specific steps of self-assembling nanoclustersComprising the following steps:
flavp of 2-20 mM 2000 Dissolving the mother liquor in PBS 1 ml to obtain FlavP 2000 Self-assembling nano clusters, and storing at 0-4 ℃;
preparation of IPA-FlavP 2000 The specific steps of self-assembling nanoclusters include:
synthesis of IPA grafted IPA-FlavP 2000 The method comprises the steps of carrying out a first treatment on the surface of the IPA-FlavP of 2-20 mM 2000 Dissolving the mother liquor in PBS 1 ml to obtain IPA-FlavP 2000 Self-assembling nano clusters, and storing at 0-4 ℃;
wherein, in the following formula, n=44 is FlavP 2000 ；
；
The structural formula of the IPA molecule is as follows:
；
IPA-FlavP 2000 the structural formula is as follows:
。
5. a polymethine nanocluster produced according to the production method of any one of claims 1 to 4.
6. Use of a polymethine nanocluster according to claim 5 for the preparation of a reagent for vascular imaging.
7. Use of a polymethine nanocluster according to claim 5 for the preparation of a reagent for neuroimaging.
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