Aggregation-induced emission near-infrared photothermal molecule, preparation method and application thereof

Abstract

The application discloses an aggregation-induced emission near-infrared photothermal molecule and a preparation method and application thereof, and belongs to the technical field of biomedicine. The application utilizes conjugation and spatial repulsion interaction of a donor-acceptor type near-infrared molecule triphenylamine at para, meta and ortho positions, and proposes a 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 light emission performance or photothermal performance in the near-infrared two-zone. Meanwhile, the preparation process of the near-infrared aggregation-induced emission protein nanoparticle provided by the application is simple, and the performance is excellent. The near-infrared aggregation-induced emission protein nanoparticle can effectively cross the nasal mucosa and target the A beta plaque part, realizes visualization of AD and photothermal / hypoxia alleviation combined treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to an aggregation-induced emission near-infrared photothermal molecule, its preparation method, and its application. Background Technology

[0002] Alzheimer's disease (AD), an increasingly serious neurodegenerative disease, suffers from complex pathological mechanisms that render single-drug therapy ineffective, necessitating the development of novel multimodal synergistic treatment strategies. In recent years, near-infrared photothermal therapy has demonstrated significant potential in AD treatment due to its unique advantages: in diagnosis, functionalized near-infrared fluorescent nanoprobes can specifically identify Aβ plaques and tau protein, enabling early and accurate diagnosis of AD when combined with multimodal imaging technology; in treatment, precise photothermal modulation can not only promote Aβ plaque depolymerization and enhance microglial cell clearance function, but also significantly improve cerebral microcirculation and the neuroinflammatory microenvironment.

[0003] It is noteworthy that the prevalent chronic hypoxia in the brains of AD patients further exacerbates Aβ deposition and tau protein pathology. Targeting this key pathological aspect, perfluorocarbons (PFCs) have emerged as a breakthrough treatment option due to their superior oxygen delivery capabilities. PFCs not only directly improve brain tissue hypoxia as highly efficient oxygen carriers, but can also be synergistically applied with near-infrared photothermal therapy or ultrasound therapy. Through sonodynamic / photothermal effects, they enhance local oxygen metabolism efficiency while simultaneously modulating the HIF-1α signaling pathway, achieving a dual therapeutic effect 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 the clinical intervention of AD.

[0004] Breakthrough advancements in nanomedicine have opened new avenues for developing multifunctional targeted delivery systems. Albumin-based drug delivery systems, with their superior biocompatibility, tunable drug release characteristics, and surface modifiability, have become ideal platforms for combined treatment of Alzheimer's disease (AD). Through the rational design of albumin nanocarriers, near-infrared photothermal drugs and perfluorocarbon (PFC) oxygen carriers can be simultaneously encapsulated, with the carrier surface precisely modified with bifunctional ligands GM1 (targeting Aβ plaques) and TAT peptide (promoting blood-brain barrier penetration). This innovative design not only enables precise drug delivery to the brain but also synergistically exerts the dual therapeutic effects of photothermal therapy (promoting Aβ clearance) and improvement of cerebral hypoxia (alleviating 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 to proteins, their imaging performance is reduced due to aggregation-induced fluorescence quenching (ACQ) effect. Furthermore, their photothermal conversion efficiency is insufficient and their stability is poor, which seriously affects their application in the diagnosis and treatment of brain diseases.

[0006] In addition, traditional nanomedicines used for AD treatment have many defects, such as: (1) Traditional AD nanomedicines mostly use polymers (such as PEG-DSPE) and inorganic materials (such as silica) for drug delivery. These materials have poor biosafety and low biocompatibility. Some nanomedicines use biological extracts (such as liposomes), but these materials are complicated to prepare, difficult to manufacture, and expensive. (2) When combined with photothermal therapy, the photothermal materials selected by traditional nanomedicines used for AD treatment are mostly inorganic materials (such as carbon dots, quantum dots and Prussian blue) and metals (such as gold). These materials have problems such as high toxicity, poor biocompatibility, large dosage and single function (only photothermal effect). (3) Traditional nanomedicines have a single mode of AD treatment, resulting in limited intervention effect. Summary of the Invention

[0007] To address the aforementioned issues, this invention provides a near-infrared photothermal molecule with aggregation-induced emission, its preparation method, and its application. It offers a method for preparing near-infrared photothermal molecules utilizing the electron-donating atom confinement effect, synthesizing novel near-infrared photothermal molecules with aggregation-induced emission properties, thereby improving molecular thermal conversion efficiency and imaging effects. Furthermore, this method integrates these molecules into protein nanoparticles for AD diagnosis and treatment, potentially overcoming existing technological bottlenecks and opening new avenues for the application of nanomedicine in the field of neurodegenerative diseases.

[0008] One of the technical solutions provided by this invention:

[0009] An aggregation-induced luminescence near-infrared photothermal molecule, wherein the general chemical structural formula of the near-infrared photothermal molecule is:

[0010]

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

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

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

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

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

[0016]

[0017] Furthermore, the near-infrared photothermal molecule is selected from the following structures:

[0018]

[0019] The second technical solution provided by this invention:

[0020] A method for preparing the above-mentioned aggregation-induced emission near-infrared photothermal molecule includes the following steps:

[0021] Under nitrogen protection, NDA-PBr, diphenylamine compound, sodium tert-butoxide, 2-dicyclohexylphosphine-2',4',6'-triisopropylbiphenyl and tris(dibenzylacetone)dipalladium(O) were dissolved in an organic solvent and refluxed. After the reaction was completed, the mixture was cooled, extracted, washed, dried and purified 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-trimethylphosphophenyl)amine, bis(3-trimethylphosphophenyl)amine, bis(2-trimethylphosphophenyl)amine, bis(4-methylselenophenyl)amine, bis(3-methylselenophenyl)amine, bis(2-methylselenophenyl)amine, bis(4-methyltellurylphenyl)amine, bis(3-methyltellurylphenyl)amine, bis(2-methyltellurylphenyl)amine, bis(4-methylarsenylphenyl)amine, bis(3-methylarsenylphenyl)amine or bis(2-methylarsenylphenyl)amine;

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

[0024] This invention proposes an innovative molecular design strategy for regulating the fluorescence and photothermal properties of DAD-type near-infrared photothermal molecules. By introducing methoxy groups at the para, meta, and ortho positions of triphenylamine, a series of molecules, including NDA-TpMT, NDA-TmMT, and NDA-ToMT, were synthesized. Introducing methoxy groups at the ortho and meta positions of triphenylamine provides a highly efficient confinement effect, inhibiting intramolecular motion. In the aggregated state, the ortho-methoxy group exhibits particularly significant inhibition, resulting in a substantial increase in quantum yield and improved fluorescence imaging. Introducing a methoxy group at the para position of triphenylamine allows the para-methoxy group to donate electrons only through lone pairs without any confinement effect, thus achieving more efficient photothermal properties. This difference in electron-donating atom confinement effect at different substitution positions allows for precise regulation of the fluorescence and thermal properties of DA-type NIR-II dyes, providing material support for the subsequent construction of near-infrared aggregation-induced emission protein nanoparticles for integrated diagnosis and treatment.

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

[0026] Furthermore, the reflux reaction temperature is 120°C.

[0027] The third technical solution provided by this invention:

[0028] 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 this invention:

[0030] This invention first prepares aggregation-induced emission near-infrared photothermal molecules; selects serum albumin nanomaterials as the motor support material; utilizes PFC to load oxygen and co-encapsulates it in protein nanoparticles; and finally performs AD targeting modification and transmembrane peptide modification.

[0031] A method for preparing near-infrared aggregation-induced emission protein nanoparticles includes the following steps: mixing the above-mentioned near-infrared photothermal molecular solution, PFC solution, serum albumin aqueous solution and GM1 solution, carrying out a self-assembly reaction, adding a crosslinking agent, obtaining primary nanoparticles after the reaction, activating, surface modifying and targeting the primary nanoparticles in sequence, stirring in an oxygen environment to complete the oxygen loading, and preparing the near-infrared aggregation-induced emission protein nanoparticles.

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

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

[0034] Furthermore, the concentration of the near-infrared photothermal molecular 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] Serum albumin possesses advantages such as safety, non-toxicity, non-immunogenicity, biodegradability, and good biocompatibility. The amino acids in albumin are linked by peptide bonds and twisted into clusters with a network-like structure, creating favorable conditions for drug embedding and delivery. Albumin itself also contains amino and carboxyl groups, which can serve as modification sites for targeting peptides. When bound to drugs, it can delay drug release at the injection site, improve efficacy, and reduce toxic side effects. The preparation process forms hydrophobic cavities, overcoming the shortcomings of hydrophobic drugs. Compared with liposomes and emulsions, albumin nanoparticles exhibit better in vivo and storage stability and good hydrophilicity. Therefore, serum albumin, as a potential nanocarrier, can effectively encapsulate and modify various drugs. The near-infrared photothermal molecule designed in this invention features near-infrared II emission, heat generation, and good photostability. Using serum albumin as a biological functional carrier, a combined diagnostic and therapeutic near-infrared aggregation-induced emission protein nanoparticle integrating targeting, fluorescence / photothermal imaging, and photothermal / hypoxia relief therapy is ultimately constructed, showing significant research and clinical application prospects in the fields of biotechnology and pharmaceutical technology.

[0036] The fifth technical solution provided by this invention:

[0037] A near-infrared aggregation-induced emission protein nanoparticle prepared by the above preparation method, wherein the near-infrared aggregation-induced emission protein nanoparticle is spherical and has a particle size of 50-100 nm.

[0038] Near-infrared aggregation-induced luminescent protein nanoparticles smaller than 100 nm can more effectively cross the nasal mucosa and enter the brain via the trigeminal nerve-olfactory bulb after nasal instillation, thus improving the targeting effect on Aβ lesions.

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

[0040] This invention utilizes the para-, meta-, and ortho-conjugation interactions and steric repulsion 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 9In Figure a, the image is a TEM electron microscope image of Aβ fibers; b, c, and d are TEM electron microscope images of Aβ fibers after photothermal destruction of near-infrared aggregation-induced emission protein nanoparticles prepared in Examples 5, 6, and 7, respectively.

[0051] Figure 10 In the image, a is the ThT fluorescence image of Aβ fibers, and b, c and d are the ThT fluorescence images of Aβ fibers after photothermal destruction of near-infrared aggregation-induced luminescent protein nanoparticles prepared in Examples 5, 6 and 7, respectively.

[0052] Figure 11 Fluorescent images of microglia stained with hypoxia probes, where a is the hypoxia control group, and b, c and d are the near-infrared aggregation-induced luminescent protein nanoparticles plus laser treatment groups prepared in Example 5 (TGAP NPs), Example 6 (TGMP NPs) and Example 7 (TGOP NPs), respectively;

[0053] Figure 12 Fluorescence imaging images of near-infrared aggregation-induced luminescent protein nanoparticles prepared for Examples 5 (TGAP NPs), 6 (TGMP NPs), and 7 (TGOP NPs) in the brain. Detailed Implementation

[0054] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0055] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0056] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0057] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0058] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0059] The room temperature in this invention refers to 25±2℃.

[0060] The raw materials used in the embodiments of this invention were not purchased through commercial channels.

[0061] Traditional nanomedicines often use polymers and inorganic materials for drug delivery, which suffer from poor biosafety and low biocompatibility. This invention addresses these issues by using serum albumin, a simple, inexpensive, and highly biocompatible material, as a carrier. This avoids the poor biocompatibility of polymers and avoids the complex and costly preparation of plasmids. Furthermore, serum albumin exhibits low immunogenicity, is biodegradable, and contains abundant modifiable groups on its surface. Therefore, serum albumin is a novel and ideal carrier material for AD nanotherapy.

[0062] To address the issues of high toxicity, large quantities, and limited functionality in traditional nanomedicines used in conjunction with photothermal therapy for Alzheimer's disease (AD), this invention employs organic near-infrared photothermal molecules as photothermal materials (which also possess near-infrared fluorescence imaging capabilities). Compared to inorganic materials, organic near-infrared photothermal molecules offer advantages such as low toxicity, simple synthesis, and controllable and tunable performance. However, traditional near-infrared photothermal molecules (such as the FDA-approved drug ICG) suffer from poor photo / thermal stability, aggregation-induced fluorescence quenching (ACQ) upon protein binding, and low signal-to-noise ratio. Therefore, this invention utilizes aggregation-induced emission (AIE) technology to design a donor-acceptor-donor (DAD) type near-infrared photothermal molecule (a novel structure). The novel near-infrared photothermal molecule prepared by this invention contains a highly rigid conjugated structure, a stable covalent bond structure, and good photothermal stability. Furthermore, this invention introduces the classic AIE unit triphenylamine as the D unit in the new molecule, while allowing rotation between D and A units. This prevents strong p-p stacking in the aggregated state, avoids ACQ formation, and ensures excellent photothermal and fluorescence effects after protein binding. Compared to existing aggregation-induced emission (AIE) photothermal materials, the DAD-type near-infrared photothermal molecule provided by this invention has a novel structure and employs a novel strategy (near-infrared phototherapy molecule with electron-donating atom confinement effect). By introducing electron-donating atoms (such as oxygen, sulfur, and nitrogen atoms) and adjusting their positions (ortho, meta, and pair) in the D structure, this invention discovers a novel design strategy for regulating the fluorescence and photothermal properties of DAD-type near-infrared photothermal molecules.

[0063] To address the limitations of traditional nanomaterial treatments, which are often limited in their therapeutic efficacy, this invention provides a novel nasal drop delivery route for AD treatment, bypassing the blood-brain barrier to reach the brain directly. It modifies transmembrane peptides to improve the efficiency of particle crossing the nasal mucosa; introduces the Ab-targeting agent GM1 to enhance the nanomedicine's targeting effect on lesions; and combines photothermal materials (photothermal therapy) and perfluorinated carbon (oxygen release therapy) as dual treatment modes to ensure highly effective AD treatment while minimizing damage to normal brain tissue.

[0064] The purpose of this invention is to develop a near-infrared phototherapy molecular design method that utilizes the electron-donating atom confinement effect to design and synthesize near-infrared AIE photothermal molecules with higher photothermal efficiency, in order to solve the problems of poor imaging and insufficient photothermal conversion efficiency of existing materials. This invention also explores the synthesis of near-infrared aggregation-induced emission protein nanoparticles that integrate photothermal and hypoxia relief in diagnosis and treatment, and investigates their application in AD diagnosis and treatment.

[0065] In this embodiment of the invention, the PFC (perfluorocarbon) used is perfluorooctane; serum albumin is human serum albumin; and the sequence of TAT (cell-penetrating peptide) is YGRKKRRQRRR.

[0066] All raw materials required in the embodiments of this invention were purchased commercially. The concentration of the glutaraldehyde crosslinking agent aqueous solution used in the embodiments of this invention is 2.5 wt.%.

[0067] Example 1

[0068] The synthetic route for NDA-PBr is as follows:

[0069]

[0070] The synthesis of NDA-PBr includes the following steps: Under nitrogen protection, sodium hydride (210 mg, 5.24 mmol) was added to 10 mL of redistilled DMF, and the mixture was stirred in an ice bath. Then, 2-(4-bromophenyl)acetonitrile (513 mg, 2.62 mmol) was added and reacted for 30 minutes. Carbon disulfide (300 mg, 3.93 mmol) was then added, the ice bath was removed, and the mixture was allowed to rise to room temperature and continue reacting for 2 hours. The reaction solution changed from colorless to light green and then gradually turned brownish-red. Next, 4,5,9,10-tetrabromo-2,7-bis(2-octyldodecyl)benzo[lmn][3,8]phenanthroline-1,3,6,8(2H,7H)-tetraone (300 mg, 0.262 mmol) was added in a single batch. After stirring at room temperature for 1 hour, the reaction solution turned dark purple. The reaction was quenched with 20 mL of physiological saline, extracted with ethyl acetate, and the organic phase was dried over anhydrous sodium sulfate and then evaporated to dryness. The residue was separated by dichloromethane / petroleum ether (2:1) column chromatography, finally yielding a dark green solid product (71% yield).

[0071] NMR data ( 1 H NMR, 500MHz, CDCl3, δ / ppm): 7.67 (d, J=8.0Hz, 4H), 7.56 (d, J=7.9Hz, 4H), 4.2 2-4.08 (m, 4H), 1.99 (s, 2H), 1.27 (t, J=35.6Hz, 64H), 0.85 (d, J=5.6Hz, 12H).

[0072] Example 2

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

[0074]

[0075] Under nitrogen protection, NDA-PBr (100 mg, 0.07 mmol), bis(4-methoxyphenyl)amine (50 mg, 0.21 mmol), sodium tert-butoxide (26.8 mg, 0.28 mmol), 2-dicyclohexylphosphine-2',4',6'-triisopropylbiphenyl (X-phos, 11.5 mg, 0.02 mmol), and tris(dibenzylacetone)dipalladium(O) (Pd2(dba)3, 9.2 mg, 0.01 mmol) prepared in Example 1 were dissolved in toluene and refluxed at 120 °C for 12 hours. After the reaction was completed, the mixture was cooled, extracted with dichloromethane, and washed three times with saturated brine. 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 give a green powder product (yield: 60%).

[0076] 1H NMR (1H NMR) 1 H NMR, 500MHz, CDCl3, 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 for the near-infrared photothermal molecule NDA-DmMT is as follows:

[0079]

[0080] Under nitrogen protection, 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-dicyclohexylphosphine-2',4',6'-triisopropylbiphenyl (X-phos, 11.5 mg, 0.02 mmol), and tris(dibenzylacetone)dipalladium(0) (Pd2(dba)3, 9.2 mg, 0.01 mmol) were dissolved in toluene and refluxed at 120 °C for 12 hours. After the reaction was completed, the mixture was cooled, extracted with dichloromethane, and washed three times with saturated brine. 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 give a green powder product (yield: 53%).

[0081] 1H NMR spectrum 1H NMR (500MHz, CDCl3, 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 (126MHz, CDCl3, 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 spectrometry (MALDI-TOF): Calculated value (C 120 H 158 N6O8S4): [M] + Measured value: 1940.1058; actual value: 1940.1410

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

[0085] Figure 2 This is the carbon spectrum of the near-infrared photothermal molecule NDA-DmMT.

[0086] Example 4

[0087] The preparation method and synthetic route of the near-infrared photothermal molecule NDA-DoMT are as follows:

[0088]

[0089] Under nitrogen protection, 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-dicyclohexylphosphine-2',4',6'-triisopropylbiphenyl (X-phos, 11.5 mg, 0.02 mmol), and tris(dibenzylacetone)dipalladium(0) (Pd2(dba)3, 9.2 mg, 0.01 mmol) were dissolved in toluene and refluxed at 120 °C for 12 hours. After the reaction was completed, the mixture was cooled, extracted with dichloromethane, and washed three times with saturated brine. 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 give a green powder product (yield: 40%).

[0090] 1H NMR (1H NMR) 1 H NMR, 500MHz, CDCl3, 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] Carbon NMR (carbon nuclear magnetic resonance) 13 C NMR, 126MHz, CDCl3, 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 (CDCl3 solvent peak), 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. Calculated value of mass spectrometry (MALDI-TOF) (C 168 H 254 N6O8S4): [M] + Measured value: 2613.8603; Actual value: 2613.7075

[0092] Figure 3 The NMR spectrum of the near-infrared photothermal molecule NDA-DoMT is shown in 1H NMR.

[0093] Figure 4 This is the carbon spectrum of the near-infrared photothermal molecule NDA-DoMT.

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

[0095] Example 5

[0096] A method for preparing near-infrared aggregation-induced luminescent protein nanoparticles includes the following steps:

[0097] Take a clean small glass bottle and measure 2 mL of a 10 mg / mL serum albumin (HSA) aqueous solution into it; add 1 mL of a 1 mg / mL NDA-DpMT solution prepared in Example 2 using tetrahydrofuran, 20 μL of a 100 mg / mL PFC solution, and 100 mg / mL NDA-DpMT solution. 10 μL of GM1 (monosialotetrahexosylganglioside) solution was added; after magnetic stirring at 800 rpm and 25°C for 3 min, 100 μL of glutaraldehyde crosslinking agent aqueous solution was added, and the reaction was continued at 25°C. After the reaction was completed, the mixture was filtered through a 220 nm aqueous filter membrane; the filtrate was centrifuged at 5000 rpm for 10 min and washed three times with deionized water to obtain primary nanoparticles. EDC / NHS (molar ratio of EDC to NHS of 1:1, molar ratio of EDC, NHS to carboxyl groups in serum albumin of 4.8:4.8:1.6) was added to the above primary nanoparticles, and the mixture was activated at 400 rpm for 15 min in a 25 mL round-bottom flask at room temperature. Sulfo- SMCC (sulfosuccinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid ester, with a molar ratio of 1:4 to the carboxyl group content in serum albumin) was modified by stirring at 800 rpm / min for 30 min at room temperature in a 25 mL round-bottom flask. Finally, TAT aqueous solution was added to the above aqueous solution at a molar mass ratio of 1:4 (HSA:TAT), and the mixture was stirred at 800 rpm / min for 12 h at room temperature in a 25 mL round-bottom flask. After terminating the reaction, the reaction system was filtered through a 220 nm aqueous filter membrane. The filtrate was centrifuged at 5000 rpm for 10 min, washed three times with deionized water, and stirred for 15 min under oxygen to obtain oxygen-loaded near-infrared aggregation-induced luminescent protein nanoparticles (i.e., TGAPNs).

[0098] Example 6

[0099] Same as Example 5, except that the near-infrared photothermal molecular solution prepared in Example 1 is replaced by an equal mass of the near-infrared photothermal molecular NDA-DmMT, i.e., TGMPNPs, prepared in Example 3.

[0100] Example 7

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

[0102] Example 8

[0103] Take a clean small glass bottle and measure 2 mL of a 10 mg / mL serum albumin (HSA) aqueous solution into it; add 1 mL of a 1 mg / mL NDA-DpMT solution prepared in Example 2 using tetrahydrofuran, and magnetically stir at 800 rpm and 25°C for 3 min. Then add 100 μL of a glutaraldehyde crosslinking agent aqueous solution and continue stirring at 25°C. After the reaction is complete, filter the solution using a 220 nm aqueous filter membrane. Centrifuge the filtrate at 5000 rpm for 10 min and wash it three times with deionized water to obtain NDA-DpMT NPs.

[0104] The above NDA-DpMT solution was replaced with an equal volume of the near-infrared photothermal molecule NDA-DmMT solution (1 mg / mL) prepared in Example 3 to prepare NDA-DmMTNP.

[0105] The above NDA-DpMT solution was replaced with an equal volume of the near-infrared photothermal molecule NDA-DoMT solution (1 mg / mL) prepared in Example 4 to prepare NDA-DoMTNP.

[0106] After preparing 100 μM solutions by adding the above-mentioned NDA-DpMT NPs, NDA-DmMT NPs, and NDA-DoMT NPs to water, respectively, the solutions were then subjected to a 0.8 W cm⁻¹ solution. -2 Irradiate the solution with an 808nm laser for 5 minutes and record the temperature changes of each solution.

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

[0108] Figure 7 The images shown are electron micrographs of the near-infrared aggregation-induced emission protein nanoparticles prepared in Examples 5-7. It can be observed that the protein particles are spherical particles with a diameter of less than 100 nm.

[0109] Figure 8 The oxygen loading capacity test diagrams are for the near-infrared aggregation-induced emission protein nanoparticles prepared in Examples 5-7.

[0110] Performance Test 1

[0111] In vitro photothermal therapy tests of near-infrared aggregation-induced luminescent protein nanoparticles prepared in Examples 5-7:

[0112] Take 10 μL of the near-infrared aggregation-induced emission protein nanoparticle solution (10 mg / mL) prepared in Examples 5-7, and add it to 50 μL of 50 μM Aβ fiber solution, respectively. Irradiate with an 808 nm laser for 15 minutes. Take 10 μL and add it to a TEM copper grid. Observe the Aβ fiber structure by electron microscopy. At the same time, observe the changes in fiber fluorescence under a fluorescence microscope using ThT staining.

[0113] Figure 9 Image a is a TEM image of Aβ fibers; images b, c, and d are TEM images of Aβ fibers after photothermal destruction of near-infrared aggregation-induced emission protein nanoparticles prepared in Examples 5, 6, and 7, respectively. Figure 9 The obvious destruction of the fiber structure can be observed, proving that near-infrared aggregation-induced emission protein nanoparticles can achieve photothermal destruction of Aβ fibers.

[0114] Figure 10 Image a shows the ThT fluorescence image of Aβ fibers. Images b, c, and d show the ThT fluorescence images of Aβ fibers after photothermal destruction by near-infrared aggregation-induced emission protein nanoparticles prepared in Examples 5, 6, and 7, respectively. ThT can recognize the fiber structure of Aβ, and the significant reduction in ThT fluorescence confirms the destructive effect of near-infrared aggregation-induced emission protein nanoparticles on Aβ.

[0115] Performance Test 2

[0116] In vitro hypoxia relief using near-infrared aggregation-induced luminescent protein nanoparticles prepared in Examples 5-7:

[0117] Microglia were divided into 5x10 groups. 4 Cells were seeded at high density in 24-well plates and cultured for 24 hours (37°C, 5% CO2). After changing the culture medium, the cells were transferred to a hypoxic incubator and cultured for another 24 hours (four parallel preparations were made). Near-infrared aggregation-induced emission protein nanoparticles (10 mg / mL, 50 μL) prepared in Examples 5, 6, and 7 were added to the culture medium of three of the cell groups, respectively. The other group of cells did not contain near-infrared aggregation-induced emission protein nanoparticles and served as a control group. The cells were incubated for a total of 12 hours, stained with hypoxic probes, and observed under a fluorescence microscope.

[0118] Figure 11 The images show fluorescence images of microglia stained with hypoxia probes. In the images, a represents the hypoxia control group, while b, c, and d represent the near-infrared aggregation-induced emission protein nanoparticles (NIEPs) prepared in Examples 5 (TGAP NPs), 6 (TGMP NPs), and 7 (TGOP NPs), respectively, treated with laser therapy. The results demonstrate that NIEPs can effectively alleviate cellular hypoxia, validating the feasibility of treating hypoxia in the brain of Alzheimer's disease (AD).

[0119] Performance Test 3

[0120] Brain-targeting imaging experiments of near-infrared aggregation-induced luminescent protein nanoparticles prepared in Examples 5-7:

[0121] The near-infrared aggregation-induced luminescent protein nanoparticles (50 mg / mL, 20 μL) prepared in Examples 5, 6 and 7 were administered intranasally to mice, and NIR-II fluorescence images of the brain were captured at different time points.

[0122] Figure 12 Fluorescence imaging images of near-infrared aggregation-induced luminescent protein nanoparticles prepared in Examples 5 (TGAP NPs), 6 (TGMP NPs), and 7 (TGOP NPs) in the brain demonstrate that these near-infrared aggregation-induced luminescent protein nanoparticles can effectively cross the nasal mucosa and target Aβ-rich sites in the brain, providing a possibility for precise treatment of AD and reducing damage to normal brain tissue.

[0123] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for preparing near-infrared aggregation-induced luminescent protein nanoparticles, characterized in that, Includes the following steps: Near-infrared photothermal molecular solution, PFC solution, serum albumin aqueous solution and monosialotetrahexosylganglioside solution were mixed and subjected to self-assembly reaction. A cross-linking agent was added, and primary nanoparticles were obtained after the reaction. The primary nanoparticles were then activated, modified and targeted, and stirred in an oxygen environment to complete the oxygen loading, thus preparing the near-infrared aggregation-induced emission protein nanoparticles. The near-infrared photothermal molecules are selected from the following chemical structures: , or ; The preparation method of the near-infrared photothermal molecule includes the following steps: Under nitrogen protection, NDA-PBr, diphenylamine compound, sodium tert-butoxide, 2-dicyclohexylphosphine-2',4',6'-triisopropylbiphenyl and tris(dibenzylacetone)dipalladium(O) were dissolved in an organic solvent and refluxed. After the reaction was completed, the mixture was cooled, extracted, washed, dried and purified to prepare the aggregation-induced emission near-infrared photothermal molecule. 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-trimethylphosphophenyl)amine, bis(3-trimethylphosphophenyl)amine, bis(2-trimethylphosphophenyl)amine, bis(4-methylselenophenyl)amine, bis(3-methylselenophenyl)amine, bis(2-methylselenophenyl)amine, bis(4-methyltellurylphenyl)amine, bis(3-methyltellurylphenyl)amine, bis(2-methyltellurylphenyl)amine, bis(4-methylarsenylphenyl)amine, bis(3-methylarsenylphenyl)amine or bis(2-methylarsenylphenyl)amine; The NDA-PBr structure is as follows: ; The molar ratio of NDA-PBr, diphenylamine compound, sodium tert-butoxide, 2-dicyclohexylphosphine-2',4',6'-triisopropylbiphenyl and tris(dibenzylideneacetone)dipalladium(0) is 7:21:28:2:1; The reflux reaction temperature is 120°C.

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

3. The preparation method according to claim 2, characterized in that, The concentration of the near-infrared photothermal molecular 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 monosialotetrahexosylganglioside solution is 100 mg / mL.

4. Near-infrared aggregation-induced emission protein nanoparticles prepared by the preparation method according to any one of claims 1-3, characterized in that, The near-infrared aggregation-induced luminescent protein nanoparticles are spherical with a particle size of 50-100 nm.

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