CN104974745A - Amphiphilic luminescent substance with aggregation-induced luminescent properties and its application - Google Patents

Abstract

The invention relates to an amphiphilic luminescent substance with aggregation-induced emission characteristic and application thereof. The luminescent material is prepared by connecting a hydrophilic unit on a hydrophobic unit with typical aggregation-induced emission characteristics (AIE), can be used for a fluorescence chemical sensor and a fluorescent stain for preparing fluorescent stains for imaging living cells and animals, is particularly suitable for fluorescent labeling of biopolymers, and can be used as an AIE activated biocompatible probe, so that the luminescent material can be applied to the aspects of clinical cancer imaging, diagnosis and treatment.

Description

Amphiphilic luminescent substance with aggregation-induced luminescent properties and its application

technical field

The invention relates to the technical field of fluorescent materials, in particular to an amphiphilic luminescent substance with aggregation-induced luminescent properties and an application thereof.

Background technique

In recent years, it has been found that a series of helical molecules do not emit light in solution, but are induced to emit strong light after being formed by aggregation. The aggregation fluorescence quenching effect is exactly the opposite. Using this new effect, AIE luminescent materials can be applied in many high-tech fields, such as chemical sensors, biological probes, immunolabeling, stimuli-responsive materials and solid-state emitters, etc. [Chem.Commun.2001,18,1740; J.Mater. Chem., 2001, 11, 2974; Chem. Soc. Rev., 2011, 40, 5361; Adv. Funct. Mater., 2012, 22, 771; Chem. Commun., 2011, 47, 7323; , 2,500; Acc.Chem.Res., 2013,46,2441; Chem.Commun.2013,49,11335; Biomaterials 2008,29,1345; US 8,029,767; US 2013/0029325; CN103175768]. Tetraphenylethylene (tetraphenylethene, TPE) and hexaphenylsilacyclopentadiene (hexaphenylsiole, HPS) are prototype AIE molecules, which have easy synthesis, high quantum yield in solid state, high chemical stability and photostability, etc. Advantages, but its hydrophobic nature greatly limits its application in the biological field. In order to reduce its hydrophobicity or improve its hydrophilicity, charged functional groups are introduced into its structure. For example, TPE with two or four ammonium groups emits weakly in aqueous solution, but becomes a strong emitter when added with negatively charged biomolecules, such as calf thymus DNA and bovine serum albumin (BSA)[ Chem. Commun., 2006, 3705; Chem.–Eur. J., 2008, 14, 6428.]. The anionic TPE derivative brings the sulfonic acid group into the hydrophobic cavity of the main folding structure of BSA, and the luminescence is enhanced due to the restriction of intramolecular rotation (RIR). When BSA is unfolded by introducing a surfactant (such as sodium dodecyl sulfate SDS), it no longer emits light [J. Phys. Chem. B, 2007, 111, 11817]. Tang and his co-workers reported poly-N-isopropylacrylamide (PNIPAM) polymers containing TPE ligands [Chem. Can be used to track temperature-induced conformational changes in polymers. On the other hand, there are few studies and reports on amphiphilic AIE molecules. Structurally, amphiphilic molecules are surfactants and organic compounds that generally contain both hydrophobic and hydrophilic structural units. The hydrophobic tails of most amphiphilic molecules are branched, linear or aromatic hydrocarbon chains, while the hydrophilic heads are uncharged, negatively charged and positively charged, sometimes referred to as nonionic surfactants, anionic surfactants and cationic surfactant. Thus, an amphiphilic molecule is composed of a water-insoluble ligand and a water-soluble ligand. In aqueous solution, amphiphilic molecules form polymers, such as colloids and micelles, in which the hydrophobic tails gather to form a core, and the hydrophilic heads are concentrated on the surface and in contact with the surrounding aqueous environment. The shape and size of polymers mainly depend on the structure of amphiphilic molecules and the balance between hydrophilicity and hydrophobicity of molecules. Amphiphilic molecules are widely found in our daily life items, including detergents, fabric softeners, emulsions, paints, inks and cosmetics, as well as in biological utilization, such as protein extraction, cell plasma membrane, drug delivery and so on. The combination of amphiphilic and AIE properties can generate a series of new fluorescent molecules for chemical and biological applications.

Contents of the invention

The purpose of the present invention is to provide an amphiphilic luminescent substance with aggregation-induced luminescent properties and its application, which solves the problem that the luminescent substance with aggregation-induced luminescent characteristics in the prior art is hydrophobic, which leads to limited applications. question.

The technical solution adopted by the present invention to solve the technical problem is: an amphiphilic luminescent substance with aggregation-induced luminescent properties, comprising a hydrophobic unit with aggregation-induced/enhanced luminescent properties, and a hydrophobic A hydrophilic unit is attached to the unit, and the structural formula of the luminescent substance is selected from any of the following I, II, III, IV, V and VI;

in, Represents a hydrophobic unit with aggregation-inducing/enhanced luminescence properties;

represents a hydrophilic unit;

In the amphiphilic luminescent substance with aggregation-induced luminescent properties of the present invention, the luminescent substance is soluble in water, and micelles can be formed when the concentration of the aqueous solution formed by the luminescent substance is greater than or equal to the critical micelle concentration.

In the amphiphilic luminescent substance with aggregation-induced luminescent properties of the present invention, the hydrophobic unit with aggregation-induced/enhanced luminescent properties includes at least one first host structure, and the host structure is selected from any of the following: A group of structures:

Wherein, R and R(X) respectively represent a hydrophilic unit connected to the hydrophobic unit with aggregation-inducing/enhancing luminescence properties.

In the amphiphilic luminescent substance with aggregation-induced luminescent properties of the present invention, the hydrophilic units are selected from the group consisting of OH, ammonium salt, amino group, mercaptan, ethyl glycol, sulfonate, phosphate and at least one of carboxylate groups.

In the amphiphilic luminescent substance with aggregation-induced luminescent properties of the present invention, the hydrophobic unit with aggregation-induced/enhanced luminescent properties contains a structure of The group, the hydrophilic unit contains an ethyl ethylene glycol group; specifically, the luminescent substance contains a group of any one of the following structural formulas P1/6, P2/6 and P3/6 : where n, m, o and p represent natural numbers from 2 to 3000 respectively,

In the amphiphilic luminescent substance with aggregation-induced luminescent properties of the present invention, the application of the luminescent substance in tracking cells, in the preparation of cell trackers, and in the preparation of probes for tracking cells Or in monitoring the application of intracellular drug release process.

In the amphiphilic luminescent substance with aggregation-induced luminescent properties of the present invention, the hydrophilic unit includes at least one second host structure, and the second host structure is selected from groups including any of the following structures :

and

Wherein R ₁ , R ₂ , R ₃ , R ₄ and R ₅ are respectively selected from the group consisting of H, alkyl, unsaturated alkyl, isoalkyl, cycloalkyl, heterocycloalkyl, aromatic hydrocarbon group, heteroaryl hydrocarbon group , and C _n H _2n+1 , C ₁₀ H ₇ , C ₁₂ H ₉ , OC ₆ H ₅ , OC ₁₀ H ₇ , OC ₁₂ H ₉ , C _n H _2n COOH, C _n H _2n NCS, C _n H _2n A group of at least one of N ₃ , C _n H _2n NH ₂ , C _n H _2n SH, C _n H _2n Cl, C _n H _2n Br, and C _n H _2n I, n is a natural number;

X ^- is a counter ion, and X ^- is selected from I ^- , Cl ^- , Br ^- , PF ₆ ^- , ClO ₄ ^- , BF ₄ ^- , BPh ₄ ^- , CH ₃ PhSO ₃ ^- .

In the amphiphilic luminescent substance with aggregation-induced luminescent properties of the present invention, the hydrophobic unit with aggregation-induced/enhanced luminescent properties contains a structure of The group, the hydrophilic unit contains a structure of group, wherein R ₁ is a propane group, X ^- is Br ^- ; specifically, the luminescent substance contains a group with the following structural formula TPE-MEM:

In the amphiphilic luminescent substance with aggregation-induced luminescent properties of the present invention, the application of the luminescent substance in the staining of specific selection cell membrane, the application of the preparation of specific selection cell membrane staining agent, the preparation of specific selection cell membrane Use in dyed probes, use in the preparation of photosensitizers and use in the preparation of phototherapy medicaments or in the preparation of phototherapy medicaments for the treatment of cancer.

In the amphiphilic luminescent substance with aggregation-induced luminescent properties of the present invention, the luminescent substance can be complexed with metal ions selected from La ³⁺ , Pr ³⁺ , Nd ³⁺ , Pm ³⁺ , At least one of Sm ³⁺ , Eu ³⁺ , Gd ³⁺ , Tb ³⁺ , Ce ³⁺ , Dy ³⁺ , Ho ³⁺ , Er ³⁺ , Tm ³⁺ , Yb ³⁺ and Lu ³⁺ .

In the amphiphilic luminescent substance with aggregation-induced luminescent properties of the present invention, the metal ion is Gd ³⁺ , and the hydrophobic unit with aggregation-induced/enhanced luminescent properties contains a structure of The group, the hydrophilic unit contains an ethyl glycol group; specifically, the luminescent substance contains a group of the following structural formula TPE-2Gd:

In the amphiphilic luminescent substance with aggregation-induced luminescent properties of the present invention, the application of the luminescent substance in the specific selection of cytoplasmic region staining , for use in the preparation of stains for the specific selection of staining in cytoplasmic regions, in cell imaging, in tracking tumor cells, in the preparation of tumor cell trackers for tracking tumor cells, or in the preparation of nuclear magnetic resonance imaging Application of contrast agents.

The present invention also specifies the application of the above-mentioned amphiphilic luminescent substances with aggregation-induced luminescent properties in tracking cells, the application in preparing cell trackers, the application in preparing probes for tracking cells, and the application in monitoring cells. Application in the process of intracellular drug release, application in specific selection of cell membrane staining, application in preparation of specific selection cell membrane staining, application in preparation of probes for specific selection of cell membrane staining, application in preparation of photosensitizers application in the preparation of phototherapy drugs or in the preparation of phototherapy drugs for the treatment of cancer, the application in the staining of specific selective cytoplasmic regions, the use in the preparation of staining agents for specific selective cytoplasmic region staining applications, applications in cell imaging, applications in tracking tumor cells, applications in the preparation of tumor cell trackers for tracking tumor cells, or applications in the preparation of MRI contrast agents.

The implementation of the amphiphilic luminescent substance with aggregation-induced luminescent properties and its application of the present invention has the following beneficial effects: the luminescent substance of the present invention is connected with a hydrophilic unit on a typical aggregation-induced luminescent characteristic (AIE) hydrophobic unit. The luminescent substance can be used for fluorescent chemical sensors and fluorescent dyes for imaging fluorescent dyes in living cells and animals. These amphiphilic dyes are especially suitable for fluorescent labeling of biopolymers and can be used as The biocompatible probe activated by AIE makes it applicable in clinical cancer imaging, diagnosis and therapy. Specifically, (1) the present invention prepares non-ionic water-soluble polyethylene glycol (PEG) modified tetraphenylethylene (TPEs) luminescent material, which is prepared by azide alkyne cycloaddition reaction, and its aggregation Induced luminescence, micellization, thermosensitivity, and the fluorescent visualization agent that can be used as intracellular imaging have all been studied; (2) the present invention also prepared a cationic tetrastyryl pyridinium salt luminescent substance, which has an AIE characteristics, can selectively dye the cell membrane, and the luminescent substance can also generate reactive oxygen species (ROS) under room light irradiation, which makes it possible to observe the effects of cell necrosis and phototherapy under mild conditions; (3) the present invention also Successfully synthesized a novel dual-mode magnetic resonance imaging (MRI) contrast agent (TPE-2Gd), which contains hydrophobic tetraphenylethylene units and hydrophilic diethylenetriaminepentaacetic acid-gadolinium complexes, is magnetic and can fluoresce Imaging, experimental results show that it is an ideal MRI contrast agent with a long cycle life for diagnosis and a short cycle life short enough for body clearance.

Description of drawings

Figure 1 is a synthetic route diagram of P1/6, P2/6 and P3/6 luminescent substances in Example 1 of the present invention;

Fig. 2 is a synthetic route diagram of TPE-MEM luminescent substance in Example 2 of the present invention;

Figure 3 is a synthetic route diagram of TPE-2Gd luminescent material in Example 3 of the present invention;

4A is a photo of P1/6 luminescent substance (0.25mg/mL) irradiated by ultraviolet light with a wavelength of 365nm in THF/H ₂ O mixtures with different water contents;

Figure 4B is a photo of P1/6 luminescent material (0.25mg/mL) irradiated by ultraviolet light with a wavelength of 365nm in THF/hexane mixtures with different hexane contents;

Figure 4C shows the correlation between the relative intensities of P1/6, P2/6 and P3/6 luminescent substances in THF/H ₂ O mixtures of different compositions and the water content in THF/H ₂ O mixtures of different compositions Graph; wherein, the concentration of the luminescent substance is 0.25 mg/mL, the excitation wavelength (nm): 320 (P1/6), 335 (P2/6) and 350 (P3/6);

Figure 4D is the correlation curve between the relative intensities of P1/6, P2/6 and P3/6 luminescent substances in THF/hexane mixtures of different compositions and the hexane content in THF/hexane mixtures of different compositions Figure; wherein, the concentration of the luminescent substance is 0.25 mg/mL, the excitation wavelength (nm): 320 (P1/6), 335 (P2/6) and 350 (P3/6);

Fig. 5A is a photo of aqueous solutions (0.002-2mg/mL) of P1/6 luminescent substances with different concentrations irradiated by ultraviolet light with a wavelength of 365nm;

Figure 5B is a PL spectrum diagram of aqueous solutions of P1/6 luminescent substances with different concentrations;

Figure 5C is a correlation graph between the I/I ₀ values of P1/6, P2/6 and P3/6 luminescent substances and the corresponding concentrations in water;

Figure 6 is a correlation diagram of analyzing the 24-hour cytotoxicity of P1/6 luminescent substance to HepG2 cells by CCK8 experiment;

Figure 7 is an imaging diagram of different concentrations of P1/6 luminescent substances staining live HepG2 cells for 12 hours;

Figure 8 is an imaging image of 200 μg/mL concentrations of P2/6 and P3/6 luminescent substances staining live HepG2 cells for 24 hours;

Figure 9 is a long-term tracking image of HepG2 cells from the first to fifth passages stained with 150 μg/mL P1/6 luminescent material for 24 hours;

Figure 10 is the UV and PL spectrum of TPE-MEM (40μM) in water, the excitation wavelength is 405nm;

Figure 11A is the PL spectrum of TPE-MEM in THF/DMSO mixed solvents containing different contents of THF (f _THF ), wherein the concentration of TPE-MEM is 25 μM, and the excitation wavelength is 405 nm;

Figure 11B is the correlation curve between the PL intensity of TPE-MEM at 625nm and f _THF in THF/DMSO mixed solvent, where the concentration of TPE-MEM is 25 μM; the inner inset is TPE-MEM in DMSO solvent and THF/DMSO Photographs irradiated by ultraviolet light with a wavelength of 365nm in DMSO mixed solvent ( _fTHF =99%);

Figure 12 is the correlation curve between the PL intensity and concentration of TPE-MEM at 600nm; the inner illustration is a photo of different concentrations of TPE-MEM irradiated by ultraviolet light with a wavelength of 365nm;

Figure 13A is a photo of the particle size of TPE-MEM (100 μM) in an aqueous solution measured by a transmission electron microscope (TEM);

Fig. 13B is a particle size curve diagram of TPE-MEM with different concentrations measured by a zeta potential particle size analyzer; the inner illustration is a particle size distribution diagram of TPE-MEM (100 μM) in an aqueous solution measured by a zeta potential particle size analyzer;

Figure 14 is an analysis diagram of the influence of TPE-MEM measured by MTT method on the proliferation of HeLa cells; cells were exposed to different concentrations of TPE-MEM for 6 hours;

Figure 15A is a laser scanning confocal microscope image of HeLa cells stained by TPE-MEM (5 μM, λ _ex =405nm and λ _em =550±70nm);

Figure 15B is a laser scanning confocal microscope image of HeLa cells stained by CellMask ^TM deep red cell membrane staining agent (C10046, 5 μg/mL, λ _ex =633nm and λ _em =685±55nm);

Fig. 15C is a bright field imaging diagram corresponding to Fig. 15B;

Fig. 15D is the merged imaging image of Fig. 15A-15C, and the overlapping coefficient of Fig. 15A and Fig. 15B is calculated to be 72%;

Fig. 16 is the change curve diagram of the signal loss (%) of TPE-MEM (solid line circle) fluorescence emission during the increase of scanning time; the inner illustrations are before and after the continuous scanning of 325.7s respectively by TPE -Fluorescence imaging of live HeLa cells stained with MEM (5 μM), λ _ex =405nm and λ _em =550±50nm;

Figure 17A is the PL spectrum of H2DCFDA (1 μM) in the presence of TPE-MEM (10 μM) under the illumination of room light, and the excitation wavelength is 488 nm;

17B is a graph showing the influence of irradiation time on the PL intensity at 535 nm of a solution containing TPE-MEM, a solution containing H2DCFDA, or a solution containing both TPE-MEM and H2DCFDA;

Photos AD in Figure 18 are confocal imaging images of HeLa cells stained with TPE-MEM and PI before irradiation; photos EH are confocal imaging images of HeLa cells stained with TPE-MEM and PI after irradiation; photos IL are confocal imaging images of HeLa cells stained with TPE-MEM and PI after irradiation; Confocal images of HeLa cells stained with PI only after irradiation; photos C, G, and K are bright field images corresponding to A, E, and I; D, H, and L are images of A/B/C, respectively Combined imaging image, combined imaging image of E/F/G and combined imaging image of I/J/K; [TPE-MEM]=5 μM; [PI]=3 μM; Channel I: λ _ex ＝405nm, λ _em ＝550 ±50nm; Channel II: λ _ex ＝560nm, λ _em ＝620±65nm;

Figure 19 is a comparison chart of the effects of room light irradiation on TPE-MEM and non-irradiation of TPE-MEM on the proliferation of HeLa cells measured by the MTT method;

Figure 20A is a photo of TPE-2Gd irradiated by 365nm ultraviolet light in THF/H2O mixed solutions with different water contents (f _w );

Figure 20B is the emission spectrum of TPE-2Gd in THF/H2O mixed solutions with different water contents (f _w ), the concentration is 100 μM, and the excitation wavelength is 330 nm;

Figure 20C is a graph showing the correlation between the relative PL intensity (I/I ₀ ) of different concentrations of TPE-2Gd (1 μM and 100 μM) and the water content in the THF/H ₂ O mixed solution;

Fig. 21A is the photograph of the TPE-2Gd aqueous solution of different concentrations under ultraviolet light irradiation;

Figure 21B is an emission spectrum diagram of TPE-2Gd aqueous solutions with different concentrations, and the excitation wavelength is 330nm;

Figure 21C is a correlation curve between the PL intensity of TPE-2Gd and the concentration in water, and the critical micelle concentration (CMC) of TPE-2Gd is 70 μM;

Figure 22A is a transmission electron microscope image of TPE-2Gd (100 μM) in water;

Figure 22B is a particle size analysis diagram of TPE-2Gd (100 μM) in water;

Figure 23A is a fluorescence imaging image of HeLa cells stained with TPE-2Gd, [TPE-2Gd]=30 μM;

Figure 23B is a fluorescence imaging image of PI-stained HeLa cells after TPE-2Gd staining, [TPE-2Gd]=30 μM;

Figure 23C is the merged image of Figure 23A and Figure 23B, [TPE-2Gd]=30μM;

Figure 23D is a bright field image of HeLa cells, [TPE-2Gd]=30 μM;

Figure 24 is an analysis diagram of TPE-2Gd on the cell proliferation of HeLa cells measured by the MTT method. The cells were exposed to different concentrations of TPE-2Gd for 4 hours, and continued to culture for 24 hours after replacing the fresh medium;

Figure 25 is TPE-2Gd and commercial reference substances with different Gd ³⁺ concentrations The T ₁ -weighted nuclear magnetic resonance spectrum (MR), the sample is diluted by normal saline;

Figure 26 is TPE-2Gd and commercial reference substances with different Gd ³⁺ concentrations The graph of the water proton longitudinal relaxation rate (1/T ₁ ), according to the equation: 1/T ₁ =1/T _1,0 +R ₁ ×[C _Gd ], the relaxation rate R _1,TPE-2Gd = 3.36±0.10mM ⁻¹ ·s ⁻¹ ; R ₁ , magnevist＝3.70±0.02mM ⁻¹ ·s ⁻¹ ;

Figure 27 shows the intravenous injection of TPE- ^2Gd and Post coronal T ₁ -weighted magnetic resonance (MR) imaging image;

Figure 28A is a quantitative analysis graph of T1 _- weighted imaging of the heart;

Figure 28B is a quantitative analysis graph of T ₁ -weighted imaging of the liver;

Figure 29 shows the intrahepatic intravenous injection of TPE- ^2Gd and Post-axial T ₁ -weighted magnetic resonance (MR) image.

Detailed ways

The amphiphilic luminescent substance with aggregation-induced luminescent properties and its applications of the present invention will be further described in conjunction with the accompanying drawings and examples:

The aggregation-induced eimission (AIE) phenomenon was discovered by Tang in 2001. At present, the AIE phenomenon is due to its potential applications in organic light-emitting diodes (OLEDs), chemical sensors, biosensors, and biological imaging agents in vivo and in vitro. It has become one of the hottest research fields in the world, but most AIE molecules are composed of aromatic rings, so AIE molecules are hydrophobic and insoluble in aqueous media. On the other hand, there are few reports about water-soluble AIE molecules or amphiphilic AIE molecules. Considering this aspect, the present invention designs and synthesizes some water-soluble and amphiphilic AIE molecules, and develops their potential biological applications.

The following examples are merely illustrative examples of the present invention, not restrictive.

Example 1: Synthesis of non-ionic amphiphilic luminescent substances P1/6, P2/6 and P3/6 and experimental research on their application

(1) Synthesis (n, m, o and p respectively represent natural numbers from 2 to 3000)

As shown in Figure 1, the supply ratio of the reagents used in the synthesis of luminescent substance P1/6 is as follows, the concentration ratio between the sixth compound 6, the first compound 1, CuBr and PMDETA [6]/[1]/[CuBr]/ [PMDETA] is 1/4/4/4, the supply ratio of the reagents for synthesizing luminescent substances P2/6 and P3/6 is different from that of luminescent substance P1/6 in that the sixth compound 6 is different from the second compound The concentration ratio of 2 and the third compound 3 is [6]/[2]/[3]=1/1/0.5. At room temperature, the sixth compound 6 (1.05 mg, 0.3 mmol) and the first compound 1 (430 mg, 1.2 mmol) was carried out chain reaction, after stirring for 36 hours, the reaction mixture was diluted with water (300mL), the aqueous solution was extracted four times with dichloromethane, all the organic phases were mixed together, further washed six times by concentrated brine, and passed _Na2SO4 dry _. After solvent evaporation, the residue was concentrated to ~20 mL, precipitated three times in diethyl ether (300 mL), the precipitate was filtered and rinsed with excess diethyl ether to give the corresponding product.

Luminescent substance P1/6, yield 82%, M _w 3200; M _w /M _n 1.03; IR (KBr), ν (cm ^-1 ): 3435, 2885, 1641, 1466, 1346, 1281, 1244, 1111, 953,841,756,700,623,581,523; ¹ HNMR (400MHz, DMSO-d ₆ ), δ (ppm): 8.46 (2H, s, benzotriazole triazole-H), 7.62 (4H, d, J = 8.0Hz, benzotriazole triazole-Ar- H), 7.17-6.99 (34H, m, Ar-H), 4.56 (4H, t, J = 4.8Hz, CH ₂ -triazole benzotriazole), 3.85 (4H, t, J = 4.8Hz, CH ₂ CH ₂ -triazole (benzotriazole), 3.53–3.46 (300H, m, OCH ₂ ); ¹³ C NMR (100MHz, DMSO-d ₆ ), δ (ppm): 145.86, 143.15, 143.02, 142.63, 140.77, 140.19, 131.20 ,130.70,130.65,128.97,127.86,127.82,126.62,124.50,121.72,72.36,69.90,69.73,69.64,68.67,60.23,49.58.

Luminescent substance P2/6, yield 81%, M _w 34700; M _w /M _n 1.39; IR (KBr), ν (cm ^-1 ): 3416, 2880, 1639, 1466, 1352, 1281, 1250, 1113, 951,845,770,704,675,527; ¹ HNMR (400MHz, DMSO-d ₆ ), δ (ppm): 8.41 (2H, s, triazole-H), 7.57 (4H, t, J=8.0Hz, benzotriazole triazole-Ar-H), 7.14-6.99 (14H, m, Ar-H), 4.50 (4H, m, CH ₂ -triazole), 3.80 (4H, t, J=4.8Hz, OCH ₂ CH ₂ -triazole), 3.60–3.39(300H,m,OCH ₂ ); ¹³ C NMR(100MHz,DMSO-d ₆ ),δ(ppm):145.87,145.86,143.06,142.65,140.42,131.26,130.77,129.03,127.93,126.72,124.5 ,121.76,72.37,69.92,69.74,69.53,68.69.60.25,49.60.

Luminescent substance P3/6, yield 88%, M _w 27,600; M _w /M _n 1.39; IR (KBr), ν (cm ^-1 ): 3439, 3136, 2873, 1639, 1463, 1350, 1286, 1249, 1109,954,840,663,530; ¹ H NMR(400MHz,DMSO-d ₆ ),δ(ppm):8.41(4H,s,triazole-H),7.61(8H,d,J＝8.0Hz,triazole -Ar-H), 7.07 (8H, d, J=8.0Hz, Ar-H), 4.49 (8H, s, CH ₂ -triazole benzotriazole), 3.79 (8H, s, OCH ₂ CH ₂ -triazole benzene Triazole), 3.64–3.40 (600H, m, OCH ₂ ); ¹³ C NMR (100MHz, DMSO-d ₆ ), δ (ppm): 145.85, 142.53, 140.21, 131.42, 129.24, 124.66, 121.79, 72.36, 69.68 , 68.68, 60.23, 49.59.

(2) Applied research:

Synthesis of nonionic, water-soluble tetraphenylethylene (TPE)-functionalized polyethylene glycol (PEG) luminophores with different chain numbers of polyethylene glycol (PEG) via azide-alkyne cycloaddition ( P1/6, P2/6 and P3/6), to change the hydrophobic TPE into a hydrophilic molecule. These luminescent materials show good thermal stability, see Table 1, where the T _d of P1/6, P2/6 and P3/6 are 351.6°C, 342.2°C and 352.1°C, respectively. As shown in Figures 4A–4D, these as-synthesized polymers are water-soluble and amphiphilic, and exhibit AIE characteristics in both THF/water solvent systems and THF/hexane solvent systems. In bulk aqueous solutions, these polymers form micellar polymers above the critical micelle concentration (CMC). As shown in Figures 5A, 5B and 5C, the CMCs of P1/6, P2/6 and P3/6 were 0.09mM, 0.12mM and 0.20mM respectively by utilizing the properties of autofluorescence in the aggregation state of these polymers. about 100nm, respectively. All the results indicated that the micelles of these polymers consisted of non-polar TPE heads on the inside and hydrophilic PEG chains directed towards the aqueous medium. Below the CMC, these polymers are soluble in the medium alone and do not emit light. When the concentration is increased above the CMC, these polymers begin to form micelles, and the TPE part gathers in the hydrophobic interior, showing strong fluorescence. These polymers thus become luminescent due to the effect of restriction of intramolecular rotation (RIR) on micellization.

To investigate the effect of P1/6 on living cells, a cytotoxicity assay was performed using CCK8. WST-8([2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazoliun, monosodium salt], [2-(2-methyl Oxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, glutamate sodium salt] through intracellular The dehydrogenase is converted into formazan, which is proportional to the number of living cells. As shown in Figure 6, the cytotoxicity of P1/6 was dose-dependent after P1/6 was exposed to HepG2 cells for 24h. Concentrations were 50, 75 , 100, and 125 μg/mL corresponded to 99.93%, 98.1%, 95.82% and 92.94% cell viability respectively, and when the concentration reached 150 μg/mL, the cell survival rate still remained above 86%. All data show that P1/6 has Good biocompatibility for live cell imaging.

Table 1: Molecular weight and thermal properties of P1/6, P2/6 and P3/6

For clinical applications as a drug carrier, PEG is biocompatible and suitable for delivering organic stains into cells. Pure stain TPE was unable to penetrate into intracellular regions due to its high hydrophobicity (data not shown). TPE-functionalized PEG polymers were used to stain adherent HepG2 cells as bioprobes due to better optical properties with AIE properties. Amphiphilic polymers tend to form nanopolymers in aqueous solution, which can facilitate cellular uptake through endocytosis. As shown in Figure 7, when HepG2 cells were stained by P1/6 for 12h, there was light from the cytoplasmic region. The fluorescence became stronger as the concentration of P1/6 increased, indicating that the efficiency of endocytosis depends on the dose of P1/6 in the medium. As shown in Figure 8, although the cells were incubated with high concentrations of P2/6 and P3/6 for a longer period of time, the luminescence was much weaker compared to P1/6. This difference is attributed to the ratio between TPE and PEG.

In order to monitor cell morphology over a long period of time, manufacturers have tried to improve the loading time after staining. For example, commercial cell trackers, CellTracker ^TM blue CMAC (7-Amino-4-Chloromethylcoumarin, 7-amino-4-chloromethylcoumarin) and blue CMHC (4-chloromethyl-7-hydroxycoumarin, 4-chloro methyl-7-hydroxycoumarin) functionalized with chloromethyl functional groups. When the tracer's probes are inside the cells, the probes can react with thiols on proteins and peptides, but according to the protocol the stains only illuminate the cells for about 24 hours after staining. As shown in Figure 9, as the first passage, live HepG2 cells were exposed to P1/6 polymer for 24 hours, emitting strong light. Because the biological macromolecule PEG will lock the fluorophore in the cell, the P1/6 in the cell is delivered to the daughter cell. The probe was able to trace to the fifth generation, although the fluorescence became weaker due to the increase of the proliferative generation. Therefore, P1/6 can be used as a long-term live cell tracker, which is a potential alternative. Therefore, this fluorescent polymer can be applied to monitor intracellular drug release.

Example 2: Synthesis of cationic amphiphilic luminescent material TPE-MEM and experimental research on its application

(1) synthesis

As shown in Figure 2, under a nitrogen atmosphere, 1-(3-trimethylaminopropyl)-4-methylpyridine dibromide (1-(3-trimethylammoniopropyl)-4-methylpyridinium dibromide) (0.5g , 1.4mmol) and the third compound 3 (1.5016g, 2.8mmol) were refluxed in absolute ethanol, and three drops of piperidine were added for catalysis. After cooling to room temperature, the solvent was evaporated under reduced pressure and passed through a silica gel column. The residue was purified by chromatography using a mixed solvent of dichloromethane and methanol (2:1 v/v) as eluent to give a yellow product TPE-MEM (0.72 g, 59%). ¹ H NMR (400MHz, methanol-d ₄ ,δ): 8.948 (d, 2H, J = 6.8Hz), 8.213-8.190 (m, 2H), 7.946 (6, 1H, J = 16Hz), 7.737-7.717 ( m,2H),7.576(d,2H,J＝8Hz),7.430-7.331(m,3H),7.061-6.843(m,14H),6.605-6.570(m,2H),4.698(t,2H,J =15.2Hz), 3.797(t, 2H, J=14.8Hz), 3.644(t, 2H, J=16.4Hz), 3.240(s, 9H), 2.663-2.581(m, 2H), 1.693-1.619(m ,2H),1.421-1.262(m,6H),0.871(t,3H,J＝14.4Hz); ¹³ C NMR(100MHz,CDCl ₃ ,δ):157.362,157.260,153.875,143.411,143.306,143.250,143.227 ,143.198,141.993,140.990,140.597,138.760,136.680,135.055,133.295,131.619,131.584,131.140,130.482,128.135,126.902,126.855,126.798,126.683,126.281,125.676,125.507,125.457,125.216,125.115,123.440,121.585 , 112.874, 112.700, 66.856, 61.787, 55.965, 52.066, 30.728, 30.716, 28.311, 24.791, 24.216, 21.625 ^{, 12.399} ; Value, 791.3570[M-Br ^- ] ⁺ .

(2) Applied research

The cell membrane is the cell's protective barrier and is essential for cell integrity, growth, and death. Luminescents that image cell membranes in living and dying cells are urgently needed. In this example, a novel amphiphilic tetraphenylethene-based pyridinium salt (tetraphenylethene-basedpyridinium salt, TPE-MEM) with remarkable aggregation-induced emission (AIE) properties and used for selective cell membrane staining is provided . The high-yield luminescent fluorescent probe (TPE-MEM) was synthesized by the following reactions, including asymmetric McMurry reaction, Suzuki coupling reaction and doubly charged pyridinium salt and hexyloxytetraphenylethene benzaldehyde (hexyloxytetraphenylethene benzaldehyde) condensation reaction between them. Due to the double charge characteristics, TPE-MEM has poor solubility in non-polar solvents, such as in THF and DCM non-polar solvents, but soluble in polar solvents, such as in water, DMF, DMSO and methanol Soluble in.

Figure 10 shows the ultraviolet (UV) and photoluminescence (PL) spectra of TPE-MEM in aqueous solution (40 μΜ). The absorption maximum wavelength of TPE-MEM falls at 395nm. For the convenience of biological applications, PL measurements were performed using 405 nm as the excitation wavelength. Photoexcitation of the aqueous solution induced yellow emission at 590 nm, indicating a large shift at 185 nm, caused by extended conjugation and intramolecular charge transfer from the electron donor TPE ligand to the electron acceptor pyridine unit (intramolecular charge transfer, ICT).

As shown in Figures 11A and 11B, TPE-MEM exhibited clearly opposite AIE properties due to its strongly polar nature. The DMSO solution of TPE-MEM (25μM) emits weakly, but in 99% THF, the luminescence becomes stronger at 625nm. Considering the molecular structure of TPE-MEM, it is speculated that the amphiphilic TPE-MEM molecules can form micelles at high concentrations. Thanks to the properties of AIE, the critical micelle concentration (CMC) of TPE-MEM can be determined using its own fluorescence intensity. As shown in Figure 12, when the concentration of TPE-MEM is below the CMC, the molecule dissolves, so no fluorescence is produced; above 0.01mM, the PL intensity increases sharply; the correlation curve between the PL intensity and the concentration of the dye produces two lines, The intersection of the two lines determines the CMC to be 0.02 mM. As shown in Figures 13A and 13B, the formation of nanopolymers and CMC values at high dye concentrations were also confirmed by transmission electron microscopy and zeta potential particle size analyzer. The effective diameter of the aqueous micelles is 77.4nm, and the effective diameter shrinks to 40nm with dehydration. When TPE-MEM is molecularly dissolved below the CMC, no microparticles can be detected; when above the CMC (0.02mM), microparticles can be observed. The inflection point determined by PL is the same as the CMC value. In addition, the particle size of nanopolymers is suitable for both in vitro cellular uptake and in vivo circulation, as well as biodistribution.

Cell viability and cytotoxicity of TPE-MEM can be measured in HeLa cells using MTT colorimetry prior to cell imaging. At 37°C, cells were exposed to different concentrations (0, 2.5 μM, 5 μM, 10 μM, 20 μM) of TPE-MEM for 6 hours in a dark CO2 incubator, and then cultured in fresh medium in the incubator for 18 hours. hours for cell proliferation. As shown in Fig. 14, the results show that TPE-MEM concentrations up to 20 μM are generally not cytotoxic in the dark. TPE-MEM was then evaluated for selective membrane staining, as expected. As shown in Figures 15A-15D, co-staining experiments using CellMask ^™ Deep Red cell membrane stain (C10046), which is a A commercially available cell membrane imaging agent. By using software for a confocal microscope (CLSM LSM7; Carl Zeiss, Germany), the overlap factor between Figure 15A and Figure 15B was determined to be 72%. The overlap factor is quite high due to the thin cell membrane structure and competitive inhibition between the two stains. Compared to C10046, TPE-MEM showed less internalization and imaged cell membranes with better imaging resolution. What's more, cell microvilli can be clearly seen through TPE-MEM. In addition to compound staining experiments, Z-stack CLSM scanning was also performed.

A cell membrane is an organelle showing a large negative potential at the interface of the membrane, and the phospholipid bilayer on this organelle is mainly composed of a thin amphipathic phospholipid bilayer. Therefore, cell membrane target biostains generally need to be both amphipathic and cationic. Coordinating and balancing the hydrophobicity and hydrophilicity of molecules to meet the amphipathic nature of cell membranes is crucial for the design of new biostains for cell membranes. TPE-MEM is amphiphilic and positively charged, so it can be used as an excellent biological stain to specifically stain the cell membrane of living cells. The amphiphilic TPE-MEM is spontaneously arranged in the phospholipid bilayer such that the hydrophobic tail region is electrostatically attracted to the negatively charged phosphate within the bilayer and the hydrophilic head region is isolated from the surrounding polar liquid. Forces such as electrostatic forces and van der Waals interactions (van der Waals interactions) lead to specific target staining.

For cell imaging stains, photostability is one of the most important criteria. Some AIE stains used for specific cell staining are highly photostable. The helical molecular structure and its polymerization formation process can prevent the diffusion of oxygen into the AIE particles, and the oxygen entering into the AIE particles will oxidize the fluorophore and make its PL emission white. As shown in Figure 16, similar photostability results were obtained in living cells. Normalize the fluorescence intensity based on the initial fluorescence intensity. As shown in Figure 16, the signal loss of TPE-MEM was less than 40% with a total irradiation time of 5 minutes (30 scans). From 0 s to 325.7 s, there is a slight drop in signal loss and imaging brightness due to diffusion of the stain and movement of the cells.

As shown in Figure 16, bubbles can be seen in the series of scans over the CLSM time course. Apparently, the cell villi shrank and disappeared, and then based on laser scanning, the cells swelled and leaked, showing that the plasma membrane of the cells became discontinuous and leaky. This phenomenon strongly suggested that the cell death was caused by laser scanning, which is why the follow-up research was motivated. Phenothiazinium-based molecules with positive charges in π-conjugated systems are widely used in reactive oxygen species (ROS) generation and phototherapy. Compared with phenothiazine, TPE-MEM also has a positive charge in the π-conjugated system, which may allow light-induced ROS generation, resulting in cell death.

To confirm the above hypothesis, ROS detection was performed using a commercial ROS fluorescent probe H2DCFDA. When H2DCFDA is oxidized by pre-existing ROS, strong fluorescence emission at 535 nm (λ _ex =488 nm) can be detected. Surprisingly, normal white room light (LED bulb, 3W) was sufficient to generate ROS when irradiated on the TPE-MEM solution. The PL spectra of the PBS solution containing H2DCFDA, the PBS solution containing TPE-MEM, and the PBS solution containing both H2DCFDA and TPE-MEM were obtained in the same room illuminated by light. The PL spectra of the samples at different irradiation times were recorded ( FIG. 17A , λ _ex =488nm) and the correlation curve between the peak intensity at 535nm and the irradiation time ( FIG. 17B ). In FIG. 17A , when H2DCFDA and TPE-MEM exist in the solution simultaneously, the characteristic peak of oxidized H2DCFDA appears at 535 nm and increases with light irradiation. The measurement results showed that the PL intensity continued to increase even when the room light was irradiated for more than 120 min, while the UV light whitened the fluorescence within only a few minutes (data not shown). The PL intensity of H2DCFDA solution alone and TPE-MEM solution alone changed slightly after being irradiated by room light. The observations showed that it was actually ROS generated when the light irradiated the TPE-MEM, causing cell damage and death.

However, the pathology of cell death remains unexplained. Cell membrane integrity is one of the most important morphological features used to differentiate apoptosis from necrosis. During necrosis, the cell swells, the cell membrane becomes porous and splits, and eventually the cell exchanges material with its surroundings. Propidium iodide (PI) is impermeable through cell membranes and is generally excluded from living cells. PI is often used to stain the nuclei of dead cells when the cell membrane is porous. Thus, PI opens up for the identification of dead cells in large numbers of cells and as a counterstain in multicolor fluorescence techniques. In this example, as shown in Figure 18, TPE-MEM was added to the culture medium with living cells for labeling, and then PI was introduced into the observation base. As shown in A-D of Figure 18, only yellow light from TPE-MEM (channel I) on the cell membrane could be detected before light irradiation. When the cells were irradiated for about 5 minutes (30scans), and then irradiated for another 5 minutes for PI uptake, as shown in E-H in Figure 18, changes in cell morphology could be observed in the cytoplasm and nucleus inside the cells and from Red light emitted by PI (channel II). The red light emitted by PI in the nucleus is due to the insertion and binding of PI to DNA, which causes PI to emit red light, while the red signal in the cytoplasm may be caused by the cleavage of the nucleus into the cytoplasm. In the control experiment without TPE-MEM, no red signal (channel II) was detected before irradiation (data not shown) and after irradiation (I–L in Fig. 18). All observations showed that the cell membrane became porous, indicating that light exposure in the presence of TPE-MEM caused cell necrosis.

All the results indicated that TPE-MEM promoted ROS generation under room light irradiation. In addition to the membrane selectivity and excellent photostability of TPE-MEM, TPE-MEM can be potentially used as a phototherapy drug for cancer treatment. In order to evaluate the effect of light therapy on the proliferation of HeLa cancer cells by using normal room light to irradiate TPE-MEM, as shown in Figure 19, by the MTT method, two conditions containing different concentrations of Cell viability of TPE-MEM. A standard curve was prepared from the relationship between the measured MTT value and the measured MTT value of the sample not containing TPE-MEM and not illuminated, and then the cell viability was calculated.

The results in Figure 19 show that the unirradiated cells have a survival rate and the cell survival rate from 0 μM-10 μM is about 90%, and the sample without TPE-MEM shows no toxicity after being irradiated by room light. However, TPE-MEM irradiated with room light produced ROS, and the cell viability dropped to 47% in the TPE-MEM containing 10 μM. In the case of containing TPE-MEM, a relatively large difference in cell viability was obtained between samples irradiated with room light and samples not irradiated with room light. White room lighting is mild, readily available, and cheap, which, combined with high yields of ROS, makes phototherapy non-hazardous, photostable, and low in dark toxicity. All the advantages make TPE-MEM a better photosensitizer (also called photosensitizer).

Although it is difficult to identify ROS substances and explain the mechanism of ROS generation, ROS generation can be observed. ROS have been shown to enhance the proliferation of cancer cells, but excessive ROS levels can lead to apoptosis and necrosis of cancer cells. In this example, cell membrane biostain (TPE-MEM) generates excess ROS by room light irradiation, which can visualize the process of necrosis of cancer cells in situ in real time.

In conclusion, the synthesized asymmetric amphiphilic tetraphenylethene-based pyridinium salt (TPE-MEM) with strong AIE properties can be used for cell membrane staining. Due to the cationic and amphiphilic properties, TPE-MEM has high specificity for cell membranes and excellent photostability in living cells. Surprisingly, TPE-MEM can effectively induce ROS and lead to cell necrosis only by ordinary room light irradiation. These unique features allow real-time observation of the process of necrosis and phototherapy in situ. Therefore, the result of generating ROS and phototherapy makes the luminescent material of this embodiment can be used to prepare new AIE phototherapy drugs for treating cancer.

Example 3: Synthesis of anionic amphiphilic luminescent material TPE-2Gd and experimental research on its application

(1) Synthesis of TPE-2+

As shown in Figure 3, under nitrogen atmosphere, 1-(3-trimethylaminopropyl)-4-methylpyridine dibromide (1-(3-trimethylammoniopropyl)-4-methylpyridinium dibromide) (0.5g , 1.4mmol) and 4-(1,2,2-triphenylvinyl)benzaldehyde (4-(1,2,2-triphenylvinyl)benzaldehyde) (1.01g, 2.8mmol) in anhydrous methanol Reflux reaction, add three drops of piperidine for catalysis, after cooling to room temperature, evaporate the solvent under reduced pressure, and purify the residue by silica gel column chromatography, using a mixture of dichloromethane and methanol (2:1v/v) The solvent was used as eluent to give the yellow product TPE-2+ (0.56 g, 57%). ¹ H NMR (400MHz, methanol-d ₄ ,δ): 8.913 (d, 2H, J = 6.8Hz), 8.177 (d, 2H, J = 6.8Hz), 7.875 (d, 1H, J = 16.0Hz), 7.501(d, 2H, J=8.4Hz), 7.352(d, 1H, J=16.4Hz), 7.120-6.985(m, 17H), 4.678(t, 2H, J=12.4Hz), 3.611(t, 2H , J=16.8Hz), 3.224(s,9H), 2.600(m,2H); ¹³ C NMR (100MHz, methanol-d ₄ ,δ): 153.985, 146.064, 143.359, 142.763, 142.666, 142.535, 141.710, 141.079 ,139.581,132.603,131.121,130.342,130.261,126.910,126.741,125.901,125.794,123.366,121.556,61.776,55.939,52.015,24.136；HRMS(MALDI-TOF)m/z:计算值,535.3102[M-HBr- Br ^- ] ⁺ ; measured value, 537.3263[M-HBr-Br ^- ] ⁺ .

(2) Synthesize the fifth compound 5, and synthesize TPE-2Gd through the fifth compound 5

As shown in Figure 3, the third compound 3 (163.3mg, 0.2mmol), the fourth compound 4 (214.4mg, 0.6mmol), DCC (136.2mg, 0.66mmol) and DMAP (80.6mg, 0.66mmol) were completely dissolved After adding 3 mL of triethylamine to 30 mL of DMF, the above mixture was stirred for 48 hours under nitrogen atmosphere at room temperature, and 10 mL of trifluoroacetic acid was added to the stirred mixture to acidify for 30 minutes, then filtered, and dissolved in hexane Precipitation in 3 times, the corresponding required fifth compound 5 was obtained with a yield of 92%. ¹ H NMR (400MHz, DMSO-d ₆ ,δ): 8.45(m,2H,H oftriazole), 8.05(m,2H,H of amide), 7.60(m,4H; H-Ar-triazole Benzenetriazole), 7.12-7.01(m, 14H; H-Ar), 4.53(s, 4H; CH ₂ -triazole benzotriazole), 3.83(m, 4H; CH ₂ C-triazole benzotriazole), 3.57 -3.13 (m, 20H; OCH ₂ ), 3.02 (s, 20H; CH ₂ C=O), 2.90 (s, 20H; NCH ₂ ); ¹³ C NMR (100MHz, DMSO-d ₆ , δ): 172.6, 169.2, 145.9, 143.0, 142.6, 140.4, 131.3, 130.7, 129.0, 127.9, 126.7, 124.6, 121.8, 69.6, 68.6, 64.9, 55.1, 51.7, 49.6; , 1460.1, 1396.4, 1226.7, 1089.8, 974.1, 908.5, 700.2cm ^-1 ; HRMS (MALDI-TOF) m/z: calculated value, 1670.5896[M-4H+3Na+K]; measured value, 1669.1871[M-5H +3Na+K] ⁺ ; calculated value, 1698.5795[M-6H+6Na]; measured value, 1697.1949[M-7H+6Na] ⁺ ; calculated value, 1724.8194; [M-5H+2Na+3K]; measured value, 1724.2223[M-6H+2Na+3K] ⁺ .

As shown in Figure 3, then the fifth compound 5 (235.0mg, 0.15mmol), GdAc ₃ (107.0mg, 0.32mmol) and NaAc (131.2mg, 1.6mmol) were dissolved in 20mL of DMF, at 70°C After stirring overnight, the resulting mixture was filtered and precipitated three times in hexane to give the corresponding desired compound TPE-2Gd in 95% yield. IR (KBr): ν = 3439.1, 1595.1, 1409.9, 1330.9, 1273.0, 1220.6, 1095.6, 933.5, 707.9, 653.9 cm ^-1 ; HRMS (MALDI-TOF) m/z: Calculated value, 1984.5525 [M+6H ₂ O ]; measured value, 1984.4323 [M+6H ₂ O].

(3) Applied research

In order to achieve dual functions, a TPE derivative (TPE-2Gd) containing GD-diethylenetriaminepentaacetic acid (Gd-diethylenetriamine pentaacetic acid, DTPA) chelate was synthesized in this example, and then its photophysical properties were analyzed. detection. UV-vis spectroscopy showed that TPE-2Gd had an absorption maximum at 330 nm, similar to that of the parental fluorescein-TPE (data not shown). As shown in Figures 20A-20C, when the water content (f _w ) in THF/water solution is less than 50% when excited at 330 nm, the fluorescence emission of TPE-2Gd is weak. With the increase of f _w , the fluorescence of the solution gradually becomes stronger, and becomes high-intensity luminescence in pure aqueous solution, showing an obvious AIE effect. The photographs in Fig. 20A clearly show that the fluorescence of TPE-2Gd increases with increasing f _w in THF/water solution.

It can be noticed from Fig. 20C that the fluorescence intensity of TPE-2Gd in pure water doubled when the dye concentration was increased from 1 μM to 100 μM. It is speculated that the amphiphilic TPE-2Gd molecules can form micelles at high concentrations. Due to the AIE properties, the critical micelle concentration (CMC) of TPE-2Gd can be estimated using the fluorescence intensity. When the concentration is lower than the CMC, the dye molecules can be better dissolved in the solution and therefore do not emit luminescence. When the dye concentration is higher than 10 μM, the solution emits light. As shown in Figures 21A-21C, the correlation curve between the fluorescence intensity and the concentration of the dye forms two lines, and the intersection of the two lines gives a CMC of 70 μM, which is higher than the value of sodium dodecyl sulfate (CMC=8.2 mM) is much lower, mainly due to the strong hydrophobicity of the TPE ligand. Such a low CMC ensures the formation of nanopolymers in vivo even under blood dilution. As shown in Figures 22A and 22B, the formation of nanopolymers at high dye concentrations was confirmed by transmission electron microscopy and zeta potential particle size analyzer. The effective diameter of the aqueous micelles is 164.9nm, which shrinks to 70nm after dehydration. The particle size of the nanopolymer is suitable for in vitro cellular uptake and in vivo circulation and biodistribution.

A commercial Gd-based MR (Gd-based MR) contrast agent approved by the FDA It is a typical extracellular fluid reagent, which can circulate rapidly in the extracellular area or interstitial space. On the other hand, TPE-2Gd can enter tumor cells in the form of nanopolymers, which has been demonstrated by live cell imaging. HeLa cells were cultured with 30 μM TPE-2Gd for 4 hours, and TPE-2Gd entered the cells and illuminated the cytoplasmic region through blue fluorescence. To further confirm the staining area of TPE-2Gd, PI was used as a counterstain. PI is a nuclear stain that stains the nucleus of fixed cells. As shown in Figures 23A-23D, the blue fluorescence around the nucleus clearly indicates that TPE-2Gd selectively stains only the cytoplasmic region. The nanopolymers of TPE-2Gd are internalized into living cells through the pathway of endocytosis, so that TPE-2Gd can track tumors at the cellular level, making up for the Tracking tumor defects at the tissue level.

The toxicity of TPE-2Gd in HeLa cells was determined by MTT assay. Cells were exposed to different concentrations of TPE-2Gd (0, 15 μM, 30 μM, 45 μM, and 60 μM) for 4 h, and then cultured in fresh medium for 24 h to evaluate the internalization effect of nanopolymers on cell proliferation. As shown in Figure 24, when the concentration of TPE-2Gd was lower than 30 μM, it was generally not toxic, and about 98.8% of the cells were viable after treatment with the stain. Even when the concentration was increased to 60 μM, which is 2 times higher than the working concentration of the cell imaging experiment, the cell viability was still about 87.8%. This result indicates that TPE-2Gd is biocompatible for cell imaging and holds promise for further in vivo studies.

On the other hand, in order to detect whether TPE-2Gd is an effective MRI contrast agent, the longitudinal relaxation time or spin of TPE- ^2Gd in aqueous solution was determined according to the Gd concentration at room temperature using a 3.0T nuclear magnetic resonance imaging (MRI) instrument. Lattice (T ₁ ). As shown in Figure 25, as the concentration of TPE-2Gd in the buffer solution increases, the signal intensity (brightness) of the mixture becomes stronger, which is comparable to that of the same Gd ³⁺ concentration resemblance. As shown in Figure 26, the measured relaxation performance of TPE-2Gd is 3.36±0.10mM ^-1 ·s ^-1 , The relaxation efficiency of is 3.70±0.02mM ^-1 ·s ^-1 , and the relaxation efficiency refers to the paramagnetic component of the spin lattice in the contrast agent per unit concentration. and In contrast, TPE-2Gd has a high relaxation rate, indicating that TPE-2Gd can enhance the relaxation rate of its neighboring water protons, which in turn leads to enhanced signal intensity. As shown in Figure 27, Figure 28A, Figure 28B and Figure 29, as nanoparticles, TPE-2GD has a longer half-life in blood, and can be used to extend the time window of liver and cardiac MRI imaging.

It should be understood that those skilled in the art may make improvements or changes based on the above description, and all such improvements or changes shall fall within the protection scope of the appended claims of the present invention.

Claims (13)

1. An amphiphilic luminescent substance with aggregation-induced emission characteristics, comprising a hydrophobic unit with aggregation-induced/enhanced emission characteristics, wherein the hydrophobic unit with aggregation-induced/enhanced emission characteristics is connected with a hydrophilic unit, and the structural formula of the luminescent substance is selected from any one of the following I, II, III, IV, V and VI;

wherein,indicates that there is aggregation induction/augmentationA hydrophobic unit having strong light-emitting characteristics;

represents a hydrophilic unit;

2. the amphiphilic luminescent substance with aggregation-induced emission characteristics according to claim 1, wherein the luminescent substance is soluble in water, and the concentration of the aqueous solution formed by the luminescent substance is greater than or equal to the critical micelle concentration, so that micelles can be formed.

3. The amphiphilic luminescent substance with aggregation-induced emission properties according to claim 1, wherein the hydrophobic unit with aggregation-induced/enhanced emission properties comprises at least a first host structure selected from the group consisting of:

wherein R and R (X) each represent a hydrophilic unit linked to the hydrophobic unit having aggregation-inducing/luminescent-enhancing properties.

4. The amphiphilic luminescent substance having aggregation-induced emission properties according to claim 3, wherein the hydrophilic units are each selected from the group consisting of OH, ammonium salt, amino group, thiol, ethylglycol, sulfonate, phosphate, and carboxylate.

5. The amphiphilic luminescent substance with aggregation-induced emission properties according to claim 4, wherein said luminescent substance with aggregation-inducing/increasing propertiesThe hydrophobic unit with strong light-emitting property comprises the structureThe hydrophilic unit comprises an ethyl glycol group; specifically, the luminescent material comprises a group of any one of the following structural formulas P1/6, P2/6 and P3/6: wherein n, m, o and p each represent a natural number of 2 to 3000,

6. the amphiphilic luminescent substance with aggregation-induced emission properties according to claim 5, wherein the luminescent substance is used for tracking cells, for preparing a cell tracker, for preparing a probe for tracking cells, or for monitoring the release of a drug in cells.

7. The amphiphilic luminescent substance with aggregation-induced emission properties of claim 3, wherein the hydrophilic unit comprises at least one second host structure selected from the group consisting of:

wherein R is₁、R₂、R₃、R₄And R₅Are respectively selected from the group consisting of H, alkyl, unsaturated alkyl, isoalkyl, cycloalkyl, isocycloalkyl, aromatic, isoaromatic, and C_nH_2n+1、C₁₀H₇、C₁₂H₉、OC₆H₅、OC₁₀H₇、OC₁₂H₉、C_nH_2nCOOH、C_nH_2nNCS、C_nH_2nN₃、C_nH_2nNH₂、C_nH_2nSH、C_nH_2nCl、C_nH_2nBr、C_nH_2nAt least one group of I, n is a natural number;

X^-is a counter ion, X^-Is selected from I^-、Cl^-、Br^-、PF₆ ^-、ClO₄ ^-、BF₄ ^-、BPh₄ ^-、CH₃PhSO₃ ^-。

8. The amphiphilic luminescent substance with aggregation-induced emission properties of claim 7, wherein the hydrophobic unit with aggregation-induced/enhanced emission properties comprises a structure ofThe hydrophilic unit comprises a group of the structureWherein R is₁Is a propyl group, X^-Is Br^-(ii) a Specifically, the phosphor comprises a group of the following structural formula TPE-MEM:

9. the amphiphilic luminescent substance with aggregation-induced emission properties according to claim 8, wherein the luminescent substance is used for specific selective cell membrane staining, for preparing a probe for specific selective cell membrane staining, for preparing a photosensitizer, for preparing a phototherapeutic agent or for preparing a phototherapeutic agent for treating cancer.

10. The amphiphilic luminescent substance with aggregation-induced emission properties according to claim 1, wherein the luminescent substance is capable of complexing with metal ions selected from the group consisting of La³⁺、Pr³⁺、Nd³⁺、Pm³⁺、Sm³⁺、Eu³⁺、Gd³⁺、Tb³⁺、Ce³⁺、Dy³⁺、Ho³⁺、Er³⁺、Tm³⁺、Yb³⁺And Lu³⁺At least one of (1).

11. The amphipathic emitter of claim 10, wherein said metal ion is Gd³⁺The hydrophobic unit with aggregation-induced/enhanced luminescence property comprises the structureThe hydrophilic unit comprises an ethyl glycol group; specifically, the luminescent material comprises a group with the following structural formula of TPE-2 Gd:

12. the amphiphilic luminescent substance with aggregation-induced emission properties according to claim 11, wherein the luminescent substance is used for staining specifically selective for cytoplasmic domains, for preparing staining agents specifically selective for cytoplasmic domains, for cell imaging, for tracking tumor cells, for preparing tumor cell trackers for tracking tumor cells, or for preparing mri contrast agents.

13. An amphiphilic luminescent substance with aggregation-induced emission characteristics for use in tracking cells, for use in the preparation of cell trackers, for use in the preparation of probes for tracking cells, for monitoring drug release in cells, for use in specific selective cell membrane staining, for use in the preparation of probes for specific selective cell membrane staining, for use in the preparation of photosensitizers, for use in the preparation of phototherapeutic agents or for the preparation of phototherapeutic agents for the treatment of cancer, for use in specific selective cytoplasmic zone staining, the application in preparing a staining agent for specifically selecting cytoplasmic zone staining, the application in cell imaging, the application in tracking tumor cells, the application in preparing a tumor cell tracker for tracking tumor cells or the application in preparing a nuclear magnetic resonance imaging contrast agent.