Aggregation-induced emission near-infrared thermal molecule as well as preparation method and application thereof

Abstract

The invention discloses an aggregation-induced emission near-infrared photothermal molecule as well as a preparation method and application of the aggregation-induced emission near-infrared photothermal molecule, and belongs to the technical field of biomedicine. The invention provides the preparation method of the aggregation-induced emission near-infrared photothermal molecule, the synthesis process is simple and efficient, and the obtained novel near-infrared photothermal molecule has better luminescence performance or photothermal performance of a near-infrared second region. Meanwhile, the near-infrared aggregation-induced emission protein nanoparticles provided by the invention are simple in preparation process and excellent in performance, and can effectively cross nasal mucosa and target an A beta plaque part, so that AD visualization and photo-thermal / hypoxia relief combined treatment are realized.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical technology, and particularly relates to a near-infrared photothermal molecule for aggregation-induced emission and a preparation method and application thereof. BACKGROUND

[0002] Alzheimer's disease (AD) is a central nervous system degenerative disease that is becoming increasingly severe. Its complex pathological mechanism makes single drug treatment ineffective, and there is an urgent need to develop new strategies for multi-modal collaborative treatment. In recent years, near-infrared photothermal diagnosis and treatment technology has shown great potential in the field of AD treatment due to its unique advantages: in terms of diagnosis, functionalized near-infrared fluorescent nanoprobes can specifically recognize Aβ plaques and tau proteins, and combined with multi-modal imaging technology, early and accurate diagnosis of AD can be achieved; in terms of treatment, precise photothermal regulation can not only promote the disaggregation of Aβ plaques and enhance the clearance function of microglia, but also significantly improve the brain microcirculation and neuroinflammatory microenvironment.

[0003] Notably, the chronic hypoxic state commonly present in the brains of AD patients can further exacerbate Aβ deposition and tau protein pathology. In view of this key pathological link, perfluorocarbon (PFCs) materials have become a breakthrough treatment option due to their excellent oxygen delivery capacity. PFCs not only serve as efficient oxygen carriers to directly improve brain tissue hypoxia, but also can be used in conjunction with near-infrared photothermal therapy or ultrasound therapy to enhance local oxygen metabolism efficiency through sonodynamic / photothermal effects, while simultaneously regulating the HIF-1α signaling pathway, achieving the dual therapeutic effects of reducing Aβ neurotoxicity and inhibiting neuroinflammation. This combined treatment strategy based on hypoxia improvement and photothermal therapy provides a new research direction and treatment approach for clinical intervention of AD.

[0004] The breakthrough progress in nanomedicine has opened up new avenues for the development of multifunctional targeted delivery systems. Albumin-based drug carrier systems have become an ideal platform for achieving AD combination therapy due to their excellent biocompatibility, controllable drug release characteristics, and surface modifiability. By rationally designing albumin nanocarriers, near-infrared photothermal drugs and perfluorocarbon (PFCs) oxygen carriers can be simultaneously encapsulated, and dual-functional ligands of GM1 (targeting Aβ plaques) and TAT peptides (promoting blood-brain barrier penetration) can be precisely modified on the surface of the carriers. This innovative design not only enables precise brain delivery of drugs, but also synergistically achieves the dual therapeutic effects of photothermal therapy (promoting Aβ clearance) and hypoxia improvement (relieving neuroinflammation), providing a new solution for the synergistic treatment of AD.

[0005] Traditional near-infrared photothermal drugs (such as indocyanine green ICG) have significant limitations. After binding with proteins, they exhibit decreased imaging performance due to the aggregation-induced quenching (ACQ) effect, and have insufficient photothermal conversion efficiency and poor stability, which severely affects their application effectiveness in the diagnosis and treatment of brain diseases.

[0006] In addition, the traditional nanomedicine for AD treatment also has many defects, such as: (1) The traditional AD nanomedicine adopts polymers (such as PEG-DSPE) and inorganic substances (such as silicon dioxide) for drug delivery, and such materials have poor biological safety and low biocompatibility; some nanomedicines adopt biological extracts (such as liposomes), but the preparation of such materials is complex, difficult to prepare, and expensive; (2) When combined with photothermal therapy, the traditional nanomedicine for AD treatment selects inorganic materials (such as carbon dots, quantum dots and Prussian blue) and metals (such as gold) as the photothermal material, and such materials have problems of high toxicity, poor biocompatibility, large dosage and single function (only photothermal effect); (3) The traditional nanomedicine has a single treatment mode for AD, which causes limited intervention effect. SUMMARY

[0007] In view of the above problems, the application provides a near-infrared photothermal molecule with aggregation-induced emission and a preparation method and application thereof, provides a preparation method of a near-infrared photothermal molecule using electron-donating atom localization effect, synthesizes a new near-infrared photothermal molecule with aggregation-induced emission characteristics, improves the molecular thermal conversion efficiency and imaging effect, and integrates the near-infrared photothermal molecule into a protein nanoparticle for AD diagnosis and treatment, which is expected to break through the bottleneck of the prior art and open up a new way for the application of nanomedicine in the field of neurodegenerative diseases.

[0008] One of the technical solutions provided by the application is:

[0009] A near-infrared photothermal molecule with aggregation-induced emission, the chemical structure of the near-infrared photothermal molecule is as follows:

[0010]

[0011] wherein R1 and R2 are independently selected from groups containing alkyl chains;

[0012] R3, R4, R5 and R6 are substituents at ortho, meta and para positions of a benzene ring, and are independently selected from groups containing electron-donating atoms;

[0013] The group containing an alkyl chain is a C1-C20 straight chain, a C1-C20 branched chain or a C1-C20 cyclic alkyl chain; an alkyl chain substituted by one or more of oxygen atoms, alkenyl groups, alkynyl groups, aryl groups, carbonyl groups, hydroxyl groups, amino groups, carboxyl groups, cyano groups, nitro groups and ester groups, and an alkyl chain substituted by one or more of hydrogen atoms, fluorine atoms, chlorine atoms, bromine atoms and iodine atoms;

[0014] The group containing an electron-donating atom is a methoxy group, a methylthio group or a dimethylamino group.

[0015] Further, the near-infrared photothermal molecule is selected from the following structures:

[0016]

[0017] Further, the near-infrared photothermal molecule is selected from the following structure:

[0018]

[0019] The second technical solution of the present application provides:

[0020] A preparation method of the above-mentioned aggregation-induced emission near-infrared photothermal molecule, comprising the following steps:

[0021] Under the protection of nitrogen, NDA-PBr, diphenylamine compound, sodium tert-butoxide, 2-dicyclohexylphosphine-2', 4', 6'-triisopropyl biphenyl and tris(dibenzylideneacetone) dipalladium (0) are dissolved in an organic solvent to carry out a reflux reaction; after the reaction is completed, cooling, extraction, washing, drying, purification are carried out to prepare the aggregation-induced emission near-infrared photothermal molecule;

[0022] The diphenylamine compound is selected from bis(4-methoxyphenyl)amine, bis(3-methoxyphenyl)amine or bis(2-methoxyphenyl)amine, bis(4-methylthiophenyl)amine, bis(3-methylthiophenyl)amine, bis(2-methylthiophenyl)amine, bis(4-dimethylaminophenyl)amine, bis(3-dimethylaminophenyl)amine, bis(2-dimethylaminophenyl)amine, bis(4-trimethylphosphinophenyl)amine, bis(3-trimethylphosphinophenyl)amine, bis(2-trimethylphosphinophenyl)amine, bis(4-selenophenyl)amine, bis(3-selenophenyl)amine, bis(2-selenophenyl)amine, bis(4-tellurophenyl)amine, bis(3-tellurophenyl)amine, bis(2-tellurophenyl)amine, bis(4-arsino phenyl)amine, bis(3-arsino phenyl)amine or bis(2-arsino phenyl)amine;

[0023] The structure of the NDA-PBr is as follows:

[0024] The present application provides an innovative molecular design strategy for regulating the fluorescence performance and photothermal performance of DAD type near-infrared photothermal molecules. The present application synthesizes a series of molecules by introducing methoxy groups at the para, meta and ortho positions of triphenylamine, including NDA-TpMT, NDA-TmMT or NDA-ToMT. By introducing methoxy groups at the ortho and meta positions of triphenylamine, the methoxy groups have a high limiting effect to inhibit the intramolecular motion ability. In the aggregate state, the ortho methoxy group inhibition is particularly obvious, and the quantum yield is significantly improved, which is beneficial to the improvement of fluorescence imaging effect; by introducing methoxy groups at the para position of triphenylamine, the para methoxy group only provides electron effect through lone pair electrons, but does not have any limiting effect, so that more efficient photothermal performance is obtained. The difference in electron-donating atom limiting effect of different substitution positions can precisely regulate the fluorescence and heat production performance of D-A type NIR-II dyes, providing material support for subsequent construction of combined diagnosis and treatment integrated near-infrared aggregation-induced emission protein nanoparticles.

[0025] Further, the molar ratio of the NDA-PBr, diphenylamine compound, sodium tert-butoxide, 2-dicyclohexylphosphino-2',4',6'-triisopropyl biphenyl and tris(dibenzylideneacetone)dipalladium(0) is 7:21:28:2:1.

[0026] Further, the temperature of the reflux reaction is 120 DEG C.

[0027] The third technical solution provided by the present application is:

[0028] The application of the above-mentioned aggregation-induced emission near-infrared photothermal molecule in the preparation of near-infrared aggregation-induced emission protein nanoparticles.

[0029] The fourth technical solution provided by the present application is:

[0030] The present application first prepares an aggregation-induced emission near-infrared photothermal molecule; selects serum albumin nanomaterial as the support material of the motor; uses PFC to load oxygen and jointly encapsulate in the protein nanoparticle, and finally performs AD targeting modification and transmembrane peptide modification.

[0031] A preparation method of a near-infrared aggregation-induced emission protein nanoparticle, specifically comprising the following steps: mixing the above-mentioned near-infrared photothermal molecule solution, PFC solution, serum albumin aqueous solution and GM1 solution, performing a self-assembly reaction, adding a crosslinking agent, obtaining a primary nanoparticle after reaction, and sequentially performing activation, surface modification and targeting modification on the primary nanoparticle, stirring in an oxygen environment to complete oxygen loading, and preparing the near-infrared aggregation-induced emission protein nanoparticle.

[0032] Further, the volume ratio of the near-infrared photothermal molecule solution, the PFC solution, the serum albumin aqueous solution and the GM1 solution is (100-500):(1-3):(200-250):(0.5-1).

[0033] Further, the serum albumin is one or several of human serum albumin, bovine serum albumin and serum albumin modified protein.

[0034] Further, the concentration of the near-infrared photothermal molecule solution is 1-5 mg / mL; the concentration of the PFC solution is 80-100 mg / mL; the concentration of the serum albumin aqueous solution is 10-20 mg / mL; and the concentration of the GM1 solution is 100 mg / mL.

[0035] The serum albumin has the advantages of safety, non-toxicity, non-immunogenicity, biodegradability and good biocompatibility. The amino acids in the albumin are connected by peptide bonds and twisted into a group shape with a reticular gap, which creates favorable conditions for inlaid drug carrying. The albumin itself also contains amino and carboxyl groups which can be used as modification sites for targeting peptides. After being combined with drugs, the release of the drugs at the injection site can be delayed, and the therapeutic effect of the drugs can be improved and the toxic side effects can be reduced. The hydrophobic cavity formed during preparation overcomes the defects of hydrophobic drugs. Compared with liposomes and emulsions, the in-vivo and storage stability of the albumin nanoparticles is better, and the albumin nanoparticles have good hydrophilicity. Therefore, the serum albumin can effectively load various drugs and be modified as a potential nanocarrier. The near-infrared photothermal molecule designed in the application has the characteristics of near-infrared two-region luminescence, heat production and good light stability. The serum albumin is used as a biological functional carrier to finally construct a combined diagnosis and treatment integrated near-infrared aggregation-induced emission protein nanoparticle which integrates targeting, fluorescence / photothermal imaging, photothermal / alleviation of hypoxia treatment, and has important research and clinical application prospects in the fields of biotechnology and medical technology.

[0036] The fifth technical solution provided by the application is as follows:

[0037] The near-infrared aggregation-induced emission protein nanoparticle prepared by the preparation method has a spherical shape and a particle size of 50-100 nm.

[0038] The near-infrared aggregation-induced emission protein nanoparticle with a particle size of less than 100 nm can more effectively cross the nasal mucosa, enter the brain through the trigeminal nerve-olfactory bulb after nasal instillation, and improve the targeting effect on the Aβ lesion.

[0039] Compared with the prior art, the application has the following advantages and technical effects:

[0040] This invention utilizes the para-, meta-, and ortho-conjugation and steric repulsion interactions of the donor-acceptor type near-infrared molecule triphenylamine to propose a method for preparing aggregation-induced emission near-infrared photothermal molecules. This synthetic process is simple and efficient, and the resulting novel near-infrared photothermal molecules possess superior luminescent or photothermal properties in the near-infrared II region. Simultaneously, the near-infrared aggregation-induced emission protein nanoparticles provided by this invention have a simple preparation process, excellent performance, and can effectively cross the nasal mucosa and target Aβ plaque sites, enabling visualization of AD and combined photothermal / hypoxia relief therapy. Attached Figure Description

[0041] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0042] Figure 1 The 1H NMR spectrum of the near-infrared photothermal molecule NDA-DmMT;

[0043] Figure 2 The carbon spectrum of the near-infrared photothermal molecule NDA-DmMT;

[0044] Figure 3 The 1H NMR spectrum of the near-infrared photothermal molecule NDA-DoMT;

[0045] Figure 4 The carbon spectrum of the near-infrared photothermal molecule NDA-DoMT;

[0046] Figure 5 The emission spectra of the near-infrared photothermal molecules NDA-DpMT, NDA-DmMT, and NDA-DoMT are homogenized.

[0047] Figure 6 For NDA-DpMT NPs, NDA-DmMT NPs and NDA-DoMT NPs at 0.8W cm -2 Photothermal curves under 808nm excitation;

[0048] Figure 7 Electron microscopy images of the morphology of the combined diagnostic and therapeutic near-infrared aggregation-induced emission protein nanoparticles prepared in Examples 5-7;

[0049] Figure 8 The oxygen loading capacity test diagrams are for the near-infrared aggregation-induced emission protein nanoparticles for combined diagnosis and treatment prepared in Examples 5-7.

[0050] Figure 9a is the TEM picture of Aβ fibers; b, c and d are the TEM pictures of Aβ fibers after the photothermal destruction of near-infrared aggregation-induced emission protein nanoparticles prepared in Example 5, Example 6 and Example 7, respectively;

[0051] Figure 10 a is the ThT fluorescence picture of Aβ fibers; b, c and d are the ThT fluorescence pictures of Aβ fibers after the photothermal destruction of near-infrared aggregation-induced emission protein nanoparticles prepared in Example 5, Example 6 and Example 7, respectively;

[0052] Figure 11 a is the fluorescence picture of microglia cells stained by hypoxia probe; b, c and d are the fluorescence pictures of microglia cells treated by near-infrared aggregation-induced emission protein nanoparticles prepared in Example 5 (TGAP NPs), Example 6 (TGMP NPs) and Example 7 (TGOP NPs) and laser;

[0053] Figure 12 a is the fluorescence picture of microglia cells stained by hypoxia probe; b, c and d are the fluorescence pictures of microglia cells treated by near-infrared aggregation-induced emission protein nanoparticles prepared in Example 5 (TGAP NPs), Example 6 (TGMP NPs) and Example 7 (TGOP NPs) and laser; DETAILED DESCRIPTION

[0054] The following detailed description is provided to understand the various example embodiments of the present application, which should not be considered limiting, but rather as a description of certain aspects, features and embodiments of the present application.

[0055] It should be understood that the terms used in the present application merely describe particular embodiments, and are not intended to limit the present application. In addition, for numerical ranges in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is specifically disclosed. Each smaller range between any stated value or intervening value in any stated range and any other stated value or intervening value in any stated range is also encompassed within the application. The upper and lower limits of these smaller ranges can independently be included or excluded in the range.

[0056] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, preferred methods and materials are described. All publications mentioned in this specification are herein incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any reference in this specification is not intended as an admission that the reference is prior art, but rather that the reference is part of the technical literature that is relevant to the disclosure.

[0057] Many modifications and variations of the present application described in the specification are possible without departing from the scope or spirit of the application. Other implementations of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples are illustrative only.

[0058] As used herein, the terms "comprise", "comprising", "include", "including", "have" and "having" and the like are open-ended and do not exclude additional elements or steps.

[0059] The room temperature of the present application refers to 25±2℃.

[0060] The raw materials in the embodiments of the present application are obtained by market purchase.

[0061] In view of the problems that the traditional nanomedicine adopts polymers and inorganic substances for drug delivery, and such materials have poor biological safety and low biocompatibility, the present application adopts serum albumin as a carrier, which is simple, cheap and has high biocompatibility, thereby avoiding the poor biocompatibility of polymers and the like, and avoiding the defects of complex preparation and high cost of plasmids. Meanwhile, the serum albumin has low immunogenicity, is biodegradable, and contains rich modifiable groups on the surface, so the serum albumin is a new ideal carrier material for AD nanotherapy.

[0062] In view of the problems of high toxicity, large dosage and single function of inorganic and metal materials selected as the light-thermal material if the traditional nano drug for AD treatment is combined with photothermal therapy, the organic near-infrared light diagnosis and treatment molecules are used as the light-thermal material (which also has the near-infrared fluorescence imaging capability) in the present application. Compared with inorganic materials, the organic near-infrared light diagnosis and treatment molecules have the advantages of low toxicity, simple synthesis and controllable and adjustable performance. However, the traditional near-infrared light diagnosis and treatment molecules (such as FDA approved drug ICG) have the defects of poor light / thermal stability, aggregation-induced fluorescence quenching (ACQ) after being combined with proteins, and low signal-to-noise ratio. Therefore, the donor-acceptor-donor (DAD) type near-infrared light-thermal molecules (new structure) are designed by using the aggregation-induced emission (AIE) technology concept in the present application. The new type of near-infrared light-thermal molecules prepared in the present application contain a highly rigid conjugated structure, a stable covalent bond structure and good light-thermal stability. In addition, the classic AIE unit triphenylamine is introduced into the new molecule as the D unit, and the rotation between D and A is allowed at the same time, which prevents strong p-p stacking of the new molecule in the aggregated state and avoids the occurrence of ACQ, thereby ensuring that excellent light-thermal and fluorescence effects can still be achieved after being combined with proteins. Compared with the existing aggregation-induced emission (AIE) light-thermal material, the DAD type near-infrared light-thermal molecule provided in the present application is a completely new structure, and a completely new strategy (near-infrared light diagnosis and treatment molecule with electron-donating atom positioning effect) is used. By introducing electron-donating atoms (such as oxygen atoms, sulfur atoms and nitrogen atoms) and adjusting the position (ortho, meta and para) of the electron-donating atoms in the D structure, a new design strategy for regulating the fluorescence and light-thermal performance of the DAD type near-infrared light-thermal molecule is found.

[0063] In view of the problems of single treatment mode and limited intervention effect of the traditional nano material, the present application provides a new type of nano drug for AD treatment by nasal administration, which can bypass the blood-brain barrier and directly reach the brain. The transmembrane peptide is modified to improve the efficiency of the particles crossing the nasal mucosa. The Ab targeting preparation GM1 is introduced to improve the targeting effect of the nano drug on the lesion. The light-thermal material (photothermal therapy) and perfluorocarbon (oxygen release therapy) are combined to ensure efficient AD treatment and reduce damage to normal brain tissue.

[0064] The purpose of the present application is to develop a near-infrared light diagnosis and treatment molecule design method using electron-donating atom positioning effect, to design and synthesize near-infrared AIE light-thermal molecules with higher light-thermal efficiency, to solve the problems of poor imaging and insufficient light-thermal conversion efficiency of the existing materials, and to explore the synthesis of light-thermal and oxygen relief combined diagnosis and treatment integrated near-infrared aggregation-induced emission protein nanoparticles and their application in AD diagnosis and treatment.

[0065] The PFC (perfluorocarbon) used in the embodiment of the present application is perfluorooctyl bromide; the serum albumin is human serum albumin; and the sequence of the TAT (cell penetrating peptide) is YGRKKRRQRRR.

[0066] The raw materials required in the embodiment of the present application are all commercially available. The concentration of the glutaraldehyde crosslinking agent aqueous solution used in the embodiment of the present application is 2.5 wt.%.

[0067] Example 1

[0068] The synthesis route of NDA-PBr is as follows:

[0069]

[0070] The synthesis method of NDA-PBr comprises the following steps: under nitrogen protection, sodium hydride (210 mg, 5.24 mmol) is added into 10 mL of redistilled DMF and stirred under ice bath condition; then 2-(4-bromophenyl)acetonitrile (513 mg, 2.62 mmol) is added and reacted for 30 minutes, carbon disulfide (300 mg, 3.93 mmol) is added, the ice bath is removed and the reaction is continued at room temperature for 2 hours, and the reaction solution changes from colorless to light green and gradually to brown. Then 4,5,9,10-tetrabromo-2,7-bis(2-octyldodecyl)benzo[lmn][3,8]phenanthrolin-1,3,6,8(2H,7H)-tetraone (300 mg, 0.262 mmol) is added at one time, and the reaction solution is black purple after stirring at room temperature for 1 hour. The reaction is quenched with 20 mL of normal saline, extracted with ethyl acetate, and the organic phase is dried over anhydrous sodium sulfate and then rotary evaporated. The residue is separated by column chromatography with dichloromethane / petroleum ether (2:1), and finally the ink green solid product is obtained (yield 71%).

[0071] NMR data 1 H NMR, 500 MHz, CDCl3, δ / ppm): 7.67 (d, J = 8.0 Hz, 4H), 7.56 (d, J = 7.9 Hz, 4H), 4.22-4.08 (m, 4H), 1.99 (s, 2H), 1.27 (t, J = 35.6 Hz, 64H), 0.85 (d, J = 5.6 Hz, 12H).

[0072] Example 2

[0073] The synthesis route of the near-infrared photothermal molecule NDA-DpMT is as follows:

[0074]

[0075] NDA-PBr (100 mg, 0.07 mmol), bis(3-methoxyphenyl)amine (50 mg, 0.21 mmol), sodium tert-butoxide (26.8 mg, 0.28 mmol), 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (X-phos, 11.5 mg, 0.02 mmol) and tris(dibenzylideneacetone)dipalladium(0) (Pd2(dba)3, 9.2 mg, 0.01 mmol) were dissolved in toluene under nitrogen and refluxed at 120 °C for 12 hours. After the reaction was completed, it was cooled, extracted with dichloromethane, and washed with saturated brine three times. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: dichloromethane / petroleum ether = 2:1) to obtain the product as a green powder (yield: 53%).

[0076] NMR spectrum of hydrogen 1 H NMR, 500 MHz, CDC13, δ / ppm): 7.54-7.39 (d, 4H), 6.86-6.69 (d, 4H), 4.23-4.10 (d, 4H), 3.99 (s, 2H), 2.00 (s, 2H), 1.27 (m, 64H), 0.85 (dt, 12H).

[0077] Example 3

[0078] The synthetic route of the near-infrared photothermal molecule NDA-DmMT is as follows:

[0079]

[0080] NDA-PBr (100 mg, 0.07 mmol), bis(3-methoxyphenyl)amine (50 mg, 0.21 mmol), sodium tert-butoxide (26.8 mg, 0.28 mmol), 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (X-phos, 11.5 mg, 0.02 mmol) and tris(dibenzylideneacetone)dipalladium(0) (Pd2(dba)3, 9.2 mg, 0.01 mmol) were dissolved in toluene under nitrogen and refluxed at 120 °C for 12 hours. After the reaction was completed, it was cooled, extracted with dichloromethane, and washed with saturated brine three times. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: dichloromethane / petroleum ether = 2:1) to obtain the product as a green powder (yield: 53%).

[0081] NMR spectrum of hydrogen 1H NMR (500 MHz, CDC13, δ / ppm): 7.49-7.44 (d, 4H), 7.18-7.12 (d, 8H), 6.99-6.94 (d, 4H), 6.94-6.86 (d, 8H), 4.24-4.16 (d, 4H), 4.00-3.96 (m, 8H), 2.05 (s, 2H), 1.27 (m, 64H), 0.85 (dt, 12H).

[0082] 13 C NMR (126 MHz, CDC13, δ / ppm): 162.19, 156.44, 149.39, 139.32, 129.46, 127.95, 127.68, 127.34, 118.37, 115.52, 115.31, 114.99, 77.32, 77.07, 76.82, 68.55, 68.30, 31.96, 31.94, 31.67, 31.51, 31.40, 30.20, 29.75, 29.69, 29.40, 26.46, 25.83, 25.79, 22.73, 22.68, 22.66, 14.16, 14.09.

[0083] Mass (MALDI-TOF): calculated for C 120 H 158 N6O8S4): [M] + 1940.1058 found: 1940.1410

[0084] Figure 1 H NMR of near-infrared photothermal molecule NDA-DmMT.

[0085] Figure 2 C NMR of near-infrared photothermal molecule NDA-DmMT.

[0086] Example 4

[0087] Preparation method of near-infrared photothermal molecule NDA-DoMT, the synthetic route is as follows:

[0088]

[0089] NDA-PBr (100 mg, 0.07 mmol), bis(2-methoxyphenyl)amine (50 mg, 0.21 mmol), sodium tert-butoxide (26.8 mg, 0.28 mmol), 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (X-phos, 11.5 mg, 0.02 mmol) and tris(dibenzylideneacetone)dipalladium(0) (Pd2(dba)3, 9.2 mg, 0.01 mmol) were dissolved in toluene under nitrogen and refluxed at 120 °C for 12 h. After cooling, the reaction was extracted with dichloromethane and washed with saturated brine three times. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: dichloromethane / petroleum ether = 2:1) to obtain the product as a green powder (yield: 40%).

[0090] NMR of the near-infrared photothermal molecule NDA-DoMT. 1 H NMR, 500 MHz, CDC13, δ / ppm) 7.49-7.44 (d, 4H), 7.18-7.12 (d, 8H), 6.99-6.94 (d, 4H), 6.94-6.86 (d, 8H), 4.24-4.16 (d, 4H), 4.00-3.96 (m, 8H), 2.05 (s, 2H), 1.27 (m, 192H), 0.85 (dt, 24H).

[0091] NMR of the near-infrared photothermal molecule NDA-DoMT. 13 C NMR, 126 MHz, CDC13, δ / ppm) 162.33, 156.40, 149.46, 139.37, 133.98, 128.05, 127.61, 124.01, 118.49, 115.50, 115.19, 115.10, 77.29, 77.04, 76.78 (solvent peak of CDC13), 68.45, 68.31, 34.89, 31.96, 31.90, 31.46, 30.21, 30.13, 29.74, 29.69, 29.66, 29.50, 29.40, 29.35, 29.00, 26.44, 26.15, 25.95, 22.72, 14.15. Mass (MALDI-TOF) calculated for C 168 H 254 N6O8S4): [M] + 2613.8603 found: 2613.7075

[0092] Figure 3 NMR of the near-infrared photothermal molecule NDA-DoMT.

[0093] Figure 4 NMR of the near-infrared photothermal molecule NDA-DoMT.

[0094] Figure 5 Normalized emission spectra of near-infrared photothermal molecules NDA-DpMT, NDA-DmMT and NDA-DoMT.

[0095] Example 5

[0096] A preparation method of near-infrared aggregation-induced emission protein nanoparticles, comprising the following steps:

[0097] A clean small glass bottle is taken, 2 mL of serum albumin (HSA) aqueous solution with a concentration of 10 mg / mL is measured into the glass bottle, 1 mL of the near-infrared photothermal molecule NDA-DpMT solution prepared in Example 2 is added, the concentration of which is 1 mg / mL and the volume of which is 1 mL, 20 uL of PFC solution with a concentration of 100 mg / mL is added, and 10 uL of GM1 (monosialotetrahexosyl ganglioside) solution with a concentration of 100 mg / mL is added; after magnetic stirring at 800 rpm / min and under the condition of 25°C for 3 min, 100 uL of glutaraldehyde crosslinking agent aqueous solution is added, and finally the reaction is continuously stirred under the condition of 25°C; after the reaction is completed, the reaction system is filtered with a 220 nm water filter membrane; the filtrate is centrifuged at 5000 rpm / min for 10 min, and is washed with deionized water for 3 times to obtain primary nanoparticles; EDC / NHS (the molar ratio of EDC and NHS is 1:1, and the molar ratio of EDC, NHS and the carboxyl content in serum albumin is 4.8:4.8:1.6) is added to the primary nanoparticles, and the obtained solution is stirred at 400 rpm / min in a 25 mL round-bottom flask at room temperature for 15 min; Sulfo-SMCC (sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate) is added to the obtained solution, and the molar ratio of Sulfo-SMCC and the carboxyl content in serum albumin is 1:4; the obtained solution is stirred at 800 rpm / min in a 25 mL round-bottom flask at room temperature for 30 min; finally, TAT aqueous solution is added to the above-mentioned aqueous solution, the molar mass ratio of HSA and TAT is 1:4, and the obtained solution is stirred at 800 rpm / min in a 25 mL round-bottom flask at room temperature for 12 h; after the reaction is terminated, the reaction system is filtered with a 220 nm water filter membrane; the filtrate is centrifuged at 5000 rpm / min for 10 min, and is washed with deionized water for 3 times; the obtained solution is stirred in an oxygen environment for 15 min to obtain oxygen-loaded near-infrared aggregation-induced emission protein nanoparticles (TGAPNPs).

[0098] Example 6

[0099] The same as Example 5, except that the near-infrared photothermal molecule solution prepared in Example 1 is replaced by the near-infrared photothermal molecule NDA-DmMT prepared in Example 3, that is, TGMPNPs.

[0100] Example 7

[0101] The same as example 5, except that the near-infrared photothermal molecule solution prepared in example 1 is replaced by the near-infrared photothermal molecule NDA-DoMT prepared in example 4, i.e. TGOP NPs.

[0102] Example 8

[0103] A clean small glass bottle is taken, and 2 mL of serum albumin (HSA) aqueous solution with a concentration of 10 mg / mL is measured into the bottle; 1 mL of the near-infrared photothermal molecule NDA-DpMT solution prepared in example 2 with a concentration of 1 mg / mL is added, which is stirred at 800 rpm and 25°C for 3 min, then 100 μL of glutaraldehyde crosslinking agent aqueous solution is added, and finally the reaction is continued to be stirred at 25°C; after the reaction is completed, the reaction solution is filtered with a 220 nm water filter membrane; the filtrate is centrifuged at 5000 rpm for 10 min, and washed with deionized water for 3 times to obtain NDA-DpMT NPs.

[0104] The above NDA-DpMT solution is replaced by the near-infrared photothermal molecule NDA-DmMT solution (1 mg / mL) prepared in example 3 in an equal volume to prepare NDA-DmMT NPs.

[0105] The above NDA-DpMT solution is replaced by the near-infrared photothermal molecule NDA-DoMT solution (1 mg / mL) prepared in example 4 in an equal volume to prepare NDA-DoMT NPs.

[0106] The above NDA-DpMT NPs, NDA-DmMT NPs and NDA-DoMT NPs are respectively added to water to prepare a 100 μM solution, and then irradiated under 0.8 W cm -2 808 nm laser for 5 min, and the temperature change of each solution is recorded.

[0107] Figure 6 The photothermal curves of NDA-DpMT NPs, NDA-DmMT NPs and NDA-DoMT NPs under 0.8 W cm -2 808 nm excitation. The results verify that the methoxy para-substitution has a greater steric repulsion interaction, and the large ortho-methoxy group in the limited space of the electron donor promotes the formation of a highly twisted molecular structure, thereby improving the photothermal effect of the ortho-substituted molecule.

[0108] Figure 7 The morphology electron microscope pictures of the near-infrared aggregation-induced emission protein nanoparticles prepared in examples 5-7 are shown, and it can be observed that the protein particles are spherical particles with a particle size of less than 100 nm.

[0109] Figure 8 Figure. Oxygen loading capacity test of NIR-AIE protein nanoparticles prepared in Example 5-Example 7.

[0110] Performance test 1

[0111] In vitro photothermal therapy test of NIR-AIE protein nanoparticles prepared in Example 5-Example 7:

[0112] Take 10 uL of NIR-AIE protein nanoparticle solution (10 mg / mL) prepared in Example 5-Example 7 respectively, add 50 uL of 50 uM Aβ fiber solution respectively, and irradiate with 808 nm laser for 15 minutes. Take 10 uL and drop it on the TEM copper grid, observe the Aβ fiber structure by electron microscopy, and at the same time use ThT staining to observe the fiber fluorescence change under the fluorescence microscope.

[0113] Figure 9 In a, it is the TEM electron micrograph of Aβ fiber; in b, c and d, it is the TEM electron micrograph of Aβ fiber after photothermal destruction by NIR-AIE protein nanoparticles prepared in Example 5, Example 6 and Example 7 respectively. From Figure 9 It can be observed from the above that the obvious fiber structure is destroyed, which proves that the NIR-AIE protein nanoparticles can achieve photothermal destruction of Aβ fiber.

[0114] Figure 10 In a, it is the ThT fluorescence picture of Aβ fiber; in b, c and d, it is the ThT fluorescence picture of Aβ fiber after photothermal destruction by NIR-AIE protein nanoparticles prepared in Example 5, Example 6 and Example 7 respectively. ThT can recognize the fiber structure of Aβ, and the obvious decrease of ThT fluorescence also confirms the destruction effect of NIR-AIE protein nanoparticles on Aβ.

[0115] Performance test 2

[0116] In vitro hypoxia relief therapy of NIR-AIE protein nanoparticles prepared in Example 5-Example 7:

[0117] Microglial cells were seeded in a 24-well plate at a density of 5x10 4 After 24 hours of culture (37℃, 5% CO2), the cells were transferred to a hypoxic incubator for 24 hours (4 groups were prepared in parallel). NIR-AIE protein nanoparticles prepared in Example 5, Example 6 and Example 7 (10 mg / mL, 50 uL) were added to the culture medium of three groups of cells respectively, and the other group of cells was not added with NIR-AIE protein nanoparticles as a control group. After 12 hours of incubation, the cells were stained with a hypoxia probe and observed under a fluorescence microscope.

[0118] Figure 11 Fluorescence pictures of microglial cells stained by hypoxia probe, wherein a is hypoxia control group, b, c and d are near-infrared aggregation-induced emission protein nanoparticles prepared by example 5 (TGAP NPs), example 6 (TGMP NPs) and example 7 (TGOP NPs) respectively, and laser treatment group; the results prove that the near-infrared aggregation-induced emission protein nanoparticles can effectively alleviate the hypoxia state of cells, and the feasibility of treating AD brain hypoxia is verified.

[0119] Performance test 3

[0120] Brain targeting imaging test of near-infrared aggregation-induced emission protein nanoparticles prepared in examples 5-7:

[0121] The near-infrared aggregation-induced emission protein nanoparticles (50 mg / mL, 20 μL) aqueous solution prepared in examples 5, 6 and 7 were respectively administered to the mouse nose, and the NIR-II fluorescence images of the brain were taken at different time points.

[0122] Figure 12 Fluorescence imaging pictures of near-infrared aggregation-induced emission protein nanoparticles prepared in examples 5 (TGAP NPs), example 6 (TGMP NPs) and example 7 (TGOP NPs) in the brain, proving that the near-infrared aggregation-induced emission protein nanoparticles can effectively cross the nasal mucosa and target the Aβ-rich location in the brain, providing the possibility for subsequent precise treatment of AD, and reducing the damage to normal tissues in the brain.

[0123] The above, only for the preferred specific embodiments of the present application, but the protection scope of the present application is not limited to this, any person skilled in the art in the technical field disclosed in the present application, can easily think of the changes or replacement, should be covered in the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. An aggregation-induced emission near-infrared photothermal molecule, characterized in that, A chemical structure general formula of the near-infrared photothermal molecule is as follows: The R1 and R2 are independently selected from groups containing alkyl chains; The R3, R4, R5 and R6 are substituents at ortho, meta and para positions of a benzene ring, and are independently selected from groups containing electron-donating atoms; The group containing alkyl chains is a C1-C20 straight chain, a C1-C20 branched chain or a C1-C20 cyclic alkyl; The group containing electron-donating atoms is a methoxy group, a methylthio group or a dimethylamino group. 2.The aggregation-induced emission near-infrared photothermal molecule of claim 1, wherein, The near-infrared photothermal molecule is selected from the following chemical structures:

3. A method for preparing the aggregation-induced emission near-infrared photothermal molecule according to claim 1 or 2, characterized in that, The method comprises the following steps: The NDA-PBr, the diphenylamine compound, the sodium tert-butoxide, the 2-dicyclohexylphosphino-2', 4', 6'-triisopropyl biphenyl and the tris(dibenzylideneacetone)dipalladium(0) are dissolved in an organic solvent to perform a reflux reaction under nitrogen protection; after the reaction is completed, cooling, extraction, washing, drying, purification are performed to prepare the aggregation-induced emission near-infrared photothermal molecule. The diphenylamine compound is selected from bis(4-methoxyphenyl)amine, bis(3-methoxyphenyl)amine, bis(2-methoxyphenyl)amine, bis(4-methylthiophenyl)amine, bis(3-methylthiophenyl)amine, bis(2-methylthiophenyl)amine, bis(4-dimethylaminophenyl)amine, bis(3-dimethylaminophenyl)amine, bis(2-dimethylaminophenyl)amine, bis(4-trimethylphosphinophenyl)amine, bis(3-trimethylphosphinophenyl)amine, bis(2-trimethylphosphinophenyl)amine, bis(4-methylselenophenyl)amine, bis(3-methylselenophenyl)amine, bis(2-methylselenophenyl)amine, bis(4-methyltellurophenyl)amine, bis(3-methyltellurophenyl)amine, bis(2-methyltellurophenyl)amine, bis(4-methylarsenophenyl)amine, bis(3-methylarsenophenyl)amine or bis(2-methylarsenophenyl)amine. The NDA-PBr structural formula is as follows:

4. The production method according to claim 3, characterized by, The molar ratio of the NDA-PBr, the diphenylamine compound, the sodium tert-butoxide, the 2-dicyclohexylphosphino-2', 4', 6'-triisopropyl biphenyl and the tris(dibenzylideneacetone)dipalladium(0) is 7:21:28:2:

1.

5. The preparation method according to claim 3, characterized in that, The temperature of the reflux reaction is 120 DEG C.

6. Use of the aggregation-induced emission near-infrared photothermal molecule of claim 1 or 2 in the preparation of a near-infrared aggregation-induced emission protein nanoparticle.

7. A method of preparing near-infrared aggregation-induced emission protein nanoparticles, characterized in that, The method comprises the following steps: mixing a near-infrared photothermal molecule solution of claim 1 or 2, a PFC solution, a serum albumin aqueous solution and a monosialotetrahexosyl ganglioside solution to perform a self-assembly reaction, adding a crosslinking agent, obtaining a primary nanoparticle after the reaction, and sequentially performing activation, modification and targeting modification on the primary nanoparticle, stirring in an oxygen environment to complete the loading of oxygen, and preparing the near-infrared aggregation-induced emission protein nanoparticle.

8. The preparation method according to claim 7, characterized in that, The volume ratio of the near-infrared photothermal molecule solution, the PFC solution, the serum albumin aqueous solution and the GM1 solution is (100-500):(1-3):(200-250):(0.5-1).

9. The production method according to claim 8, characterized by, The concentration of the near-infrared photothermal molecule solution is 1-5 mg / mL; the concentration of the PFC solution is 80-100 mg / mL, the concentration of the serum albumin aqueous solution is 10-20 mg / mL, and the concentration of the monosialotetrahexosyl ganglioside solution is 100 mg / mL.

10. Near-infrared aggregation-induced emission proteinic nanoparticles prepared by the method of any one of claims 7 to 9, characterized in that, The near-infrared aggregation-induced emission protein nanoparticle is spherical and has a particle size of 50-100 nm.

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