Mitochondrial targeting type viscosity fluorescent probe for detecting Alzheimer's disease as well as synthesis method and application of mitochondrial targeting type viscosity fluorescent probe

Abstract

The invention discloses a mitochondrial targeting type viscosity fluorescent probe as well as a synthesis method and application thereof, and belongs to the technical field of biological materials. The chemical structure of the probe is shown as a formula I. The mitochondrial targeting viscosity fluorescent probe provided by the invention targets a mitochondrial inner membrane, structurally comprises a twisted intramolecular charge transfer (TICT) fluorescent rotor, and can emit a fluorescence signal with adjustable intensity along with the change of microenvironment viscosity. Compared with the traditional mitochondrial dye, the probe does not depend on mitochondrial membrane potential, and has excellent light stability and ultrahigh resolution imaging capability. Cell experiments show that the probe can be used for Alzheimer's disease (AD) imaging, and can indicate mitochondrial inner membrane viscosity change in real time and reveal a dynamic process of interaction between mitochondria and lysosome; pathological characteristics such as mitochondrial viscosity increase, ridge structure disorder, network fragmentation and lysosomal dyskinesia are observed in an AD model.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, specifically to a mitochondrial-targeted viscosity fluorescent probe for detecting Alzheimer's disease, its synthesis method, and its application. Background Technology

[0002] Alzheimer's disease (AD) is the most common neurodegenerative disease, characterized by the deposition of β-amyloid (Aβ) plaques, Tau protein neurofibrillary tangles, and synaptic dysfunction in the brain, leading to a gradual decline in cognitive abilities. Recent studies have shown that mitochondrial dysfunction plays a crucial role in the pathogenesis of AD, including disruptions in mitochondrial dynamics (fusion, division) and homeostasis (mitochondrial autophagy), which result in impaired cellular energy metabolism, increased oxidative stress, and neuronal death. Mitochondrial morphological abnormalities and impaired quality control mechanisms are commonly observed in cellular and animal models of AD, but the biophysical changes involved in these defects (such as changes in mitochondrial viscosity) remain insufficiently understood.

[0003] In mitochondrial research, commonly used fluorescent probes such as Rhodamine 123 and MitoTracker Red have played a significant role. However, these traditional probes are highly dependent on mitochondrial membrane potential. Under pathological conditions, mitochondrial membrane potential may be lost, rendering these dyes ineffective in indicating mitochondrial function. Furthermore, existing probes have inherent limitations, such as insufficient photostability and limited spatial resolution, preventing the resolution of mitochondrial inner membrane structures at the nanoscale. For example, previous studies have used mitochondrial-targeting probes to observe mitochondrial-lysosome interactions during apoptosis, developed the near-infrared mitochondrial probe HCy-1 to detect perchlorate (HClO) in the brains of AD model mice, and used aggregation-induced emission (AIE)-based probes to identify Aβ amyloid protein to aid in early diagnosis. However, imaging tools targeting AD-related changes in mitochondrial microenvironment viscosity remain lacking. Therefore, a novel fluorescent molecular probe is urgently needed to overcome these shortcomings and achieve real-time, high-resolution imaging of mitochondrial viscosity changes and mitochondrial-lysosome interactions. Summary of the Invention

[0004] The purpose of this invention is to provide a mitochondrial-targeted viscosity fluorescent probe for detecting Alzheimer's disease, its synthesis method, and its application. This probe can specifically target the inner mitochondrial membrane and generate a fluorescent response to changes in microenvironment viscosity. Through a unique molecular structure design, it achieves highly sensitive imaging of abnormal changes in mitochondria in Alzheimer's disease, thereby solving the technical problem of the lack of mitochondrial viscosity imaging methods in the prior art.

[0005] This invention is achieved through the following technical solution:

[0006] This invention provides a mitochondrial-targeting viscosity fluorescent probe, the chemical structure of which is shown in Formula I:

[0007]

[0008] Further specifying, in the mitochondrial-targeted viscosity fluorescent probe, the probe molecule has a donor-π-bridge-acceptor molecular structure, with a diphenylamine group as an electron donor, a thiophene ring and a double bond as a π-conjugated bridge, and a pyridinium cation as an electron acceptor and a mitochondrial-targeting group; wherein the pyridinium cation is substituted with 4-(ethoxycarbonyl)pentyl to form a quaternary ammonium salt structure.

[0009] More specifically, in the mitochondrial-targeted viscosity fluorescent probe, the probe molecule comprises a twisted intramolecular charge transfer fluorescent rotor structure.

[0010] More specifically, in the mitochondrial-targeted viscosity fluorescent probe, the fluorescence emission wavelength of the probe is located in the visible red light region, with a wavelength range of 610 nm to 630 nm.

[0011] More specifically, in the mitochondrial-targeted viscosity fluorescent probe, the probe is localized independently of the mitochondrial membrane potential.

[0012] This invention also provides a method for synthesizing the above-mentioned mitochondrial-targeted viscosity fluorescent probe, wherein the synthetic route of the probe is as follows:

[0013]

[0014] More specifically, the method for synthesizing the mitochondrial-targeted viscosity fluorescent probe includes the following steps:

[0015] 5-Halothiophen-2-carboxaldehyde was reacted with diphenylamine in the presence of a palladium catalyst to obtain an intermediate of 5-(diphenylamino)thiophen-2-carboxaldehyde.

[0016] The intermediate was condensed with 4-methylpyridine in Knoevenagel to form a 2-thienylvinyl-substituted pyridine compound;

[0017] The pyridine compound is reacted with ethyl 5-halopentanoate in an N-alkylation quaternization reaction to introduce a positive charge into the pyridine, thereby obtaining the halide salt of the fluorescent probe;

[0018] The obtained halide salt was subjected to anion exchange to obtain the fluorescent probe.

[0019] The present invention also provides the application of the above-mentioned mitochondrial-targeted viscosity fluorescent probe in mitochondrial imaging reagents.

[0020] The present invention also provides the application of the above-mentioned mitochondrial-targeted viscosity fluorescent probe in multiplex co-staining imaging reagents for mitochondria and organelles.

[0021] The present invention also provides the application of the above-mentioned mitochondrial-targeted viscosity fluorescent probe in the preparation of imaging reagents for the diagnosis of Alzheimer's disease.

[0022] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0023] This invention provides a mitochondrial-targeted viscosity fluorescent probe that targets the inner mitochondrial membrane. Structurally, it incorporates a twisted intramolecular charge transfer (TICT) fluorescent rotor, emitting a fluorescent signal with adjustable intensity in response to changes in microenvironment viscosity. Compared to traditional mitochondrial dyes, this probe is independent of mitochondrial membrane potential, exhibiting excellent photostability (fluorescence intensity remains above 95% after 200 consecutive excitations) and ultra-high resolution imaging capability (resolving mitochondrial substructures of approximately 90 nm). Cellular experiments demonstrate that this probe can be used for Alzheimer's disease (AD) imaging, providing real-time indication of changes in mitochondrial inner membrane viscosity and revealing the dynamic processes of mitochondrial-lysosome interactions. Pathological features such as increased mitochondrial viscosity, cristae structure disorder, network fragmentation, and lysosomal motility impairment were observed in AD models. In the brain tissue of AD transgenic mice, the probe's fluorescence signal was approximately 2.3 times stronger than that of normal controls, directly indicating an abnormal increase in mitochondrial viscosity during the AD disease process. The fluorescent probe provided by this invention offers an innovative tool for subcellular microenvironment viscosity imaging and can be used for AD pathological research. Attached Figure Description

[0024] To more clearly illustrate the technical solutions of the exemplary embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of the present invention and should not be considered as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort. In the drawings:

[0025] Figure 1 The proton spectrum (DMSO-d6, 400MHz) of the MTDTP probe provided by this invention.

[0026] Figure 2 The carbon spectrum (DMSO-d6, 100MHz) of the MTDTP probe provided for this invention;

[0027] Figure 3 High-resolution mass spectrum of the MTDTP probe provided by this invention;

[0028] Figure 4The following are the spectral and viscosity response characteristics of the MTDTP probe provided by this invention: (a) shows the UV-Vis absorption spectrum of the MTDTP probe in different solvents; (b) shows the fluorescence emission spectrum of the probe in different polar environments, indicating that the emission peak at approximately 620 nm remains largely unchanged with solvent polarity; (c) shows the fluorescence intensity variation curve of the probe at different viscosities (PBS and glycerol mixed solution); and (d) shows the linear relationship between fluorescence intensity and viscosity after taking the logarithm of viscosity, indicating that the probe fluorescence is highly correlated with viscosity.

[0029] Figure 5 The images show the imaging results of the MTDTP probe provided by this invention applied to live cells; (a) a schematic diagram of MTDTP and PKDMR co-labeling of the mitochondrial inner membrane; (b) a SIM super-resolution (SR) image of HeLa cells after co-labeling with MTDTP and PKDMR; (c) intensity profile analysis based on the yellow line in the magnified view of Figure b; (d) a representative SIM image of MTDTP-labeled HeLa cells and intensity profile analysis of the yellow line; (e) evaluation of the photostability of live HeLa cells labeled with MTDTP and two commercial mitochondrial dyes (MTG and MTDR). Each dye was scanned 200 times individually using a confocal microscope, and the experiment was repeated three times to ensure the reliability of the analysis; (f) time-lapse SIM imaging of live HeLa cells after co-labeling with MTDTP and Sir-tubulin.

[0030] Figure 6 Imaging results of the MTDTP probe provided by this invention applied to an AD cell model; wherein, (a) a schematic flowchart of constructing an AD model cell in vitro using SH-SY5Y cells, and performing combined staining with MTDTP and LyDR. (bd) SIM images of SH-SY5Y cells after 24 or 48 hours of Aβo pretreatment, stained with combined MTDTP and LyDR. Quantitative statistics of mitochondrial surface area (e), mitochondrial length (f), and proportion of different types of mitochondria (g) of SH-SY5Y cells after 24 or 48 hours of Aβo pretreatment. Quantitative statistics of total lysosomal migration distance (h), lysosomal trajectory displacement (i), and average lysosomal trajectory velocity (j) of SH-SY5Y cells after 24 or 48 hours of Aβo pretreatment.

[0031] Figure 7The image shows the imaging results of the MTDTP probe provided by this invention in the brain tissue of AD model mice; (a1) is a co-localization image of mitochondria (green) labeled by the MTDTP probe and the mitochondrial outer membrane protein TOMM20 immunostaining (red) in fresh brain slices, showing that MTDTP is mainly stained in the mitochondrial structure; (a2) is the intensity profile analysis of the selected line segment in (a1), which proves that the MTDTP signal and mitochondrial markers highly overlap. (b) Images of MTDTP staining (green) and Aβ antibody staining (red) in the brain tissue of normal mice and AD model mice show a significant increase in MTDTP fluorescence signal in the brain of AD mice. (c) and (d) are quantitative statistical bar charts of the average fluorescence intensity of MTDTP in the cortex and hippocampus, respectively. AD mice show an intensity increase of more than 2 times compared with normal mice, reflecting a significant increase in mitochondrial viscosity. (e) and (f) are quantitative analysis diagrams of mitochondrial damage accumulation, such as the area of ​​co-localization of mitochondria and lysosomes or the proportion of damaged mitochondria. The results support the conclusion that a large number of damaged mitochondria accumulate in the brain of AD mice. (g) is a three-dimensional reconstruction diagram of the process of mitochondrial autophagy in brain tissue. It can be seen that LAMP1-positive lysosomes (red) attempt to engulf MTDTP-labeled mitochondria (green), indicating that mitochondrial clearance is blocked but autophagy attempts are active in the AD model. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments and accompanying drawings. The illustrative embodiments and descriptions of this invention are for explanation only and are not intended to limit the invention. Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0033] This invention provides a mitochondrial-targeting viscosity fluorescent probe, the chemical structure of which is shown in Formula I:

[0034]

[0035] The chemical name of this probe is 4-[2-(5-(diphenylamino)-2-thienyl)vinyl]-1-(4-(ethoxycarbonyl)butyl)pyridine hexafluorophosphate. The molecular structure shows the donor-π-bridge-acceptor structure of the probe molecule, as well as components such as the diphenylamine group, thienyl ring, vinylpyridinium acceptor, and 4-(ethoxycarbonyl)butyl side chain. This fluorescent probe is used for mitochondrial viscosity imaging in Alzheimer's disease, selectively localizing to the inner mitochondrial membrane and generating fluorescent signals in response to changes in microenvironment viscosity.

[0036] Further specified, the probe molecule has a donor-π bridge-acceptor molecular structure, with a diphenylamine group as an electron donor, a thiophene ring and a double bond as a π conjugated bridge, and a pyridinium cation as an electron acceptor and a mitochondrial targeting group; wherein the pyridinium cation is substituted with 4-(ethoxycarbonyl)pentyl to form a quaternary ammonium salt structure.

[0037] The probe molecule provided by this invention adopts a donor-π-bridge-acceptor (D-π-A) structural architecture, which includes the following key structural units: (1) a diphenylamine group as a strong electron donor, providing the chromophore core; (2) a thiophene ring as an electron-rich aromatic π-bridge, enhancing conjugation and improving the lipophilicity of the molecule; (3) a pyridinium cationic group as an electron acceptor and mitochondrial targeting group, introducing a positive charge to utilize the electrostatic attraction of the mitochondrial membrane potential for aggregation (even when the membrane potential is reduced, there is still a certain enrichment effect); (4) the donor and acceptor ends are connected by conjugated vinyl groups. (CH=CH) bonds are linked to form a D-π-A conjugated system to promote charge transfer; (5) a single bond connection configuration exists between the diphenylamine-substituted thiophene unit and the vinylpyridinium unit, giving intramolecular torsional freedom, thereby utilizing the twisted intramolecular charge transfer (TICT) mechanism to generate a rotation-sensitive fluorescence response to changes in microenvironment viscosity; (6) a flexible 4-(ethoxycarbonyl)butyl chain (CH3CH2CH2CH2COOEt) is bonded to the pyridine nitrogen atom, improving molecular hydrophobicity and membrane permeability, ensuring that the probe enters living cells efficiently. The above structural units work synergistically to enable the probe of the present invention to significantly improve the sensitivity to changes in microenvironment viscosity, photostability, and mitochondrial imaging specificity while maintaining long-wavelength red emission.

[0038] More specifically, the probe molecule comprises a twisted intramolecular charge transfer (TICT) fluorescent rotor structure. In low-viscosity environments, its intramolecular rotation is free, leading to fluorescence quenching; in high-viscosity environments, its intramolecular rotation is restricted, resulting in significantly enhanced fluorescence intensity, achieving a reversible response to environmental viscosity.

[0039] More specifically, the fluorescence emission wavelength of the probe is located in the visible red light region, with a wavelength range of 610 nm to 630 nm. This is suitable for imaging biological samples, and the fluorescence intensity increases at least 50-fold when the solution viscosity increases from 1.0 cp to 1150 cp.

[0040] More specifically, the probe is not dependent on mitochondrial membrane potential for localization. Even in the event of loss of mitochondrial membrane potential, it can still specifically anchor to the inner mitochondrial membrane, thereby maintaining reliable mitochondrial staining performance under pathological conditions.

[0041] This invention also provides a method for synthesizing the above-mentioned mitochondrial-targeted viscosity fluorescent probe, wherein the synthetic route of the probe is as follows:

[0042]

[0043] The synthetic route demonstrates the synthetic method of the probe molecule, including the preparation of intermediates, the coupling reaction to form a conjugated structure, and the process of quaternization to introduce a positive charge into pyridine and generate hexafluorophosphate.

[0044] More specifically, the method for synthesizing the mitochondrial-targeted viscosity fluorescent probe includes the following steps:

[0045] 5-Halothiophen-2-carboxaldehyde was reacted with diphenylamine in the presence of a palladium catalyst to obtain an intermediate of 5-(diphenylamino)thiophen-2-carboxaldehyde.

[0046] The intermediate was condensed with 4-methylpyridine in Knoevenagel to form a 2-thienylvinyl-substituted pyridine compound;

[0047] The pyridine compound is reacted with ethyl 5-halopentanoate in an N-alkylation quaternization reaction to introduce a positive charge into the pyridine, thereby obtaining the halide salt of the fluorescent probe;

[0048] The obtained halide salt was subjected to anion exchange to obtain the fluorescent probe.

[0049] The working mechanism of the probe provided by this invention lies in TICT viscosity response: in a low-viscosity environment, the rotational groups within the probe molecule can freely twist, and the excited-state energy is dissipated through non-radiative pathways, leading to fluorescence quenching; while in a high-viscosity environment, intramolecular rotation is restricted, and the excited-state energy is released through radiative transitions, resulting in a significant enhancement in fluorescence intensity. Experimental measurements show that when the solution viscosity increases from approximately 1.0 cP to 1150 cP, the fluorescence intensity of this probe can increase by approximately 59 times, and the absolute quantum yield increases from 0.016 to 0.388, exhibiting excellent viscosity-sensitive characteristics and a linear response relationship. This highly quantifiable fluorescence enhancement enables it to accurately report the magnitude of changes in the viscosity of the mitochondrial inner membrane microenvironment.

[0050] The innovation of the probe provided by this invention lies in its superior imaging performance and functional applications targeting Alzheimer's disease (AD) pathology. On one hand, the probe molecules achieve highly specific staining without relying on mitochondrial membrane potential, and can still target the inner mitochondrial membrane even when mitochondria are depolarized or functionally impaired. This characteristic overcomes the limitation of traditional membrane potential-dependent dyes failing under pathological conditions. On the other hand, the probe exhibits extremely high photostability; after 200 repeated scans with a confocal laser, the fluorescence signal retains more than 95% of its initial intensity, far superior to commercial mitochondrial dyes, meeting the requirements for long-term live-cell imaging. Furthermore, the probe emits at a wavelength of approximately 620 nm, located in the red light region, which is beneficial for multiple staining and tissue penetration; it is also compatible with super-resolution imaging technology, achieving a spatial resolution of approximately 90 nm under structured illumination microscopy (SIM), clearly revealing subcellular structures such as mitochondrial cristae. The probe provided by this invention can also be used in conjunction with other fluorescent labels (such as microtubule dyes and lysosomal dyes) to achieve multicolor high-resolution imaging of the interactions between mitochondria and various cellular components such as the cytoskeleton and lysosomes, providing a powerful tool for studying the dynamic behavior of mitochondria within cells.

[0051] Through the above technical solutions, the probe of this invention has achieved significant application results in Alzheimer's disease models. At the cellular level, this probe revealed the staged pathological changes in mitochondria under AD pathological conditions, including mitochondrial cristae structural disorder, mitochondrial network fragmentation, and impaired lysosomal motility. These changes collectively exacerbate the decline in mitophagy function. At the animal level, staining fresh brain tissue sections from AD transgenic mice with this probe revealed a significant increase in mitochondrial inner membrane viscosity in the brains of AD mice, manifested as an approximately 2.3-fold increase in probe fluorescence intensity compared to normal mice. Simultaneously, a large number of damaged mitochondria were observed to accumulate in the brains of AD mice, with lysosomes attempting to engulf these probe-labeled mitochondria, indicating impaired mitochondrial clearance. This demonstrates that the probe of this invention can not only be used for the visualization of AD-related mitochondrial lesions but also establishes "abnormal mitochondrial viscosity" as a new marker for AD pathogenesis, providing a new biomarker and imaging strategy for the diagnosis and treatment evaluation of AD.

[0052] In summary, the mitochondrial viscosity imaging fluorescent probe provided by this invention effectively overcomes the limitations of existing technologies and has the following beneficial effects:

[0053] (1) Membrane potential independent: This probe achieves mitochondrial targeting through the positive charge of pyridine, which is independent of membrane potential. It can still effectively stain the inner mitochondrial membrane under pathological conditions where the mitochondrial membrane potential is reduced or even disappeared.

[0054] (2) High viscosity sensitivity: The probe contains TICT rotation group, which enhances the fluorescence response to changes in environmental viscosity by tens of times. It can quantitatively monitor subtle changes in mitochondrial viscosity and provide a quantitative means for studying the biophysical state of mitochondria.

[0055] (3) Super photostability: During live cell imaging, the probe molecules have excellent photostability and the fluorescence signal hardly decays after repeated excitation over a long period of time (maintaining >95% intensity after 200 scans), making it suitable for long-term, super-resolution dynamic imaging observation.

[0056] (4) Super-resolution imaging capability: This probe can resolve substructures such as mitochondrial cristae at a scale of about 90 nm under super-resolution imaging conditions such as SIM, which is significantly better than the conventional optical microscopy limit, and realizes the imaging observation of mitochondrial ultrastructure.

[0057] (5) Inner membrane-specific localization: The probe achieves selective anchoring to the mitochondrial inner membrane through a D-π-A structure design. Double staining experiments with mitochondrial inner membrane-specific dyes showed a colocalization coefficient (PCC) as high as 0.93, proving that this probe can accurately indicate the condition of the mitochondrial inner membrane region.

[0058] (5) Multifunctional multicolor imaging: This probe emits red light and can be combined with other fluorescent probes such as green light and near-infrared light to achieve multi-channel imaging. For example, dual labeling with microtubule dyes can reveal the migration and interaction of mitochondria along the cytoskeleton, and dual labeling with lysosome dyes can capture the dynamic processes of mitochondrial-lysosome contact, fusion / phagocytosis in real time, providing new means for studying organelle interactions.

[0059] (6) Pathological diagnostic value in Alzheimer's disease: In Alzheimer's cell and animal models, this probe successfully revealed the key pathological phenomenon of increased mitochondrial viscosity and observed pathological features such as mitochondrial debris accumulation and impaired lysosomal clearance. Therefore, this probe is expected to serve as an imaging tool for the diagnosis of Alzheimer's disease, used for early detection of mitochondrial dysfunction, and for evaluating the impact of interventions on mitochondrial health.

[0060] The technical solution provided by this invention overcomes the shortcomings of existing fluorescent probes in AD-related research, links the mitochondrial biophysical microenvironment with the pathology of neurodegenerative diseases, and has important prospects for scientific research and clinical application.

[0061] This invention also provides the application of the aforementioned mitochondrial-targeting viscosity fluorescent probe in mitochondrial imaging reagents. This probe exhibits extremely high photostability; after 200 consecutive excitations in confocal microscopy, its fluorescence intensity remains above 90% of its initial value, making it suitable for long-term live-cell imaging and super-resolution imaging. This probe possesses super-resolution imaging capabilities; using the probe in conjunction with structured illumination microscopy, sub-microstructures such as mitochondrial cristae at the approximately 90-nanometer scale can be resolved, achieving ultrastructural imaging of mitochondria.

[0062] This invention also provides the application of the above-mentioned mitochondrial-targeted viscosity fluorescent probe in multiplex co-staining imaging reagents for mitochondria and organelles. It can monitor the dynamic interactions between mitochondria and lysosomes, microtubules, etc.

[0063] This invention also provides the application of the aforementioned mitochondrial-targeted viscosity fluorescent probe for detecting Alzheimer's disease in the preparation of imaging reagents for Alzheimer's disease diagnosis. The intensity of the probe's fluorescence signal can be used to assess mitochondrial viscosity status, thereby detecting early mitochondrial functional abnormalities in Alzheimer's disease or evaluating the effectiveness of therapeutic interventions.

[0064] To further illustrate the present invention, the following description, in conjunction with embodiments, illustrates the mitochondrial-targeted viscosity fluorescent probe, its synthesis method, and its applications provided by the present invention. However, it should be understood that these embodiments are implemented under the premise of the technical solution of the present invention, providing detailed implementation methods and specific operating procedures. They are only for further illustrating the features and advantages of the present invention and are not intended to limit the scope of the claims of the present invention. The scope of protection of the present invention is not limited to the following embodiments.

[0065] The synthetic route of the fluorescent probe provided by this invention is shown below:

[0066]

[0067] 5-(diphenylamino)-2-halothiophene is designated as M, the pyridine compound as R2, and the fluorescent probe of this invention as the MTDTP probe.

[0068] Example 1: Synthesis of the Fluorescent Probe (MTDTP) of the Present Invention

[0069] This embodiment provides a method for preparing a fluorescent probe (MTDTP), the specific steps of which are as follows:

[0070] (1) Synthesis of compound M: Under nitrogen protection, 100 mL of anhydrous dimethyl sulfoxide was added to a 250 mL three-necked flask. Then, 0.45 g (2.3 mmol) of 1,10-phenanthroline was added, and the mixture was stirred for 30 minutes until completely dissolved. Next, 0.46 g (2.4 mmol) of cuprous iodide (I) was added, maintaining a molar ratio of 1,10-phenanthroline to cuprous iodide (I) of 1:1.04. Stirring was continued for 20 minutes to ensure the reaction of 1,10-phenanthroline with Cu... +The ions were fully coordinated, forming a stable catalytically active species. Then, 5.00 g (36 mmol) of anhydrous potassium carbonate was added, and the mixture was stirred for 20 minutes. Subsequently, 2.80 g (11 mmol) of diphenylamine, 1.90 g (10 mmol) of 5-bromothiophene-2-carboxaldehyde, 0.04 g (0.15 mmol) of 18-crown-6, and 2 drops (approximately 0.02 g, 0.05 mmol) of Aliquat-336 were added sequentially. 18-crown-6 and Aliquat-336 constitute a biphase transfer catalytic system, synergistically promoting the efficiency of the heterogeneous reaction. The reaction mixture was refluxed at 95 °C for 48 hours, cooled to room temperature, and filtered under reduced pressure to remove insoluble matter. The filtrate was slowly poured into 800 mL of deionized water (in a 1000 mL beaker), and stirred at room temperature for 24 hours to precipitate a dark gray solid.

[0071] After the solid was filtered under reduced pressure, the crude product was purified by column chromatography (petroleum ether / ethyl acetate = 30:1) to give 1.25 g of yellow crystals, with a yield of 45%.

[0072] The detection data for compound M is: Mp = 97℃. 1 H-NMR (d6-acetone, 400MHz, ppm): δ = 6.39 (d, J = 4.4Hz, 1H), 7.29 (t, J = 10.0Hz, 2H), 7 .35(d,J=8.0Hz,4H),7.46(q,J=8.0&10.0Hz,4H),7.66(d,J=4.4Hz,1H),9.64(s,1H). 13 C-NMR (d6-DMSO, 150MHz): δ = 125.59, 126.58, 127.94, 129.46, 130.03, 139.53, 145.46, 163.29, 182.19. IR (KBr, cm -1 ):3087(w),3039(w),2805(m),2760(m),1889(w),1663(vs),1580m),1524(m ),1485(s),1424(s),1386(s),1349(s),1220(m),755(m),691(m).Calcd.for C 17 H 13 NOS(279.36):C,73.09;H,4.69;N,5.01%.Found:C,73.13;H,4.71;N,5.03%.MALDI-TOF:m / z,cal:279.07,found:279.26[M + ].

[0073] (2) Synthesis of compound R2: 9.32 g (0.1 mol) of 4-methylpyridine and 26.77 g (0.12 mol) of ethyl 5-bromopentanoate were added to a 250 mL round-bottom flask and refluxed at 120 °C for 10 hours. Then, 20 g (0.12 mol) of ammonium hexafluorophosphate dissolved in acetonitrile was added to displace the anion, and the reaction was allowed to proceed for 2 hours. After filtration through a Buchner funnel, the solvent was evaporated to dryness and filtered again to obtain 16 g of a yellow oily substance.

[0074] Yield of compound R2: 72%.

[0075] The detection data for compound R2 are: MALDI-TOF: m / z, calcd for C 13 H 20 NO2 + =222.15,found222.15.

[0076] (3) Synthesis of probe MTDTP: Compound M and compound R2 (containing PF6) - A mixture of pyridine salt derivatives (1:1, 0.01 mol) was dissolved in ethanol, and 8 drops of piperidine were added and reacted for 24 hours. After evaporating the solvent, the crude product was ultrasonically dispersed in cyclohexane and then recrystallized from ethanol to give 2 g of red solid. Yield: 31.81%.

[0077] The detection data for the MTDTP probe are: mp 154-156℃. MS (ESI): m / z, calcd for C 30 H 31 N2O2S + =483.21,found:483.21. 1 H NMR (400MHz, DMSO-d6) δ8.69(d,J=6.6Hz,2H),7.96(d,J=6.6Hz,2H),7.43–7.35(m,4H),7.22(dd,J=9.0,7.2Hz,8H),6.69(d,J=15.7Hz,1H),6.40(d, J=4.1Hz,1H),4.36(t,J=7.1Hz,2H),4.00(q,J=7.1Hz,2H),2.31(t,J=7.4H z, 2H), 1.84 (p, J = 7.3Hz, 2H), 1.46 (p, J = 7.5Hz, 2H), 1.13 (t, J = 7.1Hz, 3H). 13C NMR(100MHz,DMSO-d6)δ173.03,158.03,153.60,146.61,144.00,135.56,134.81,130.51,126 .18,125.29,122.87,118.13,114.97,60.38,59.33,33.19,30.28,21.39,14.63.FT-IR(KBr,cm -1 ):ν C-H (sp 3 ),3086(s),2938(s);ν C＝O ,1720(s);ν C-O-C ,1175(s);ν C＝C ,1423(m),1585(m);ν C-N ,1300(m),ν C-S, 963(s).

[0078] Example 3: Photophysical Properties and Viscosity Response Test

[0079] In this embodiment, the spectral properties and viscosity response behavior of the MTDTP probe were tested. A UV-265 spectrophotometer was used to measure the UV-Vis absorption spectrum. Five solvents—acetonitrile, dimethyl sulfoxide, ethanol, water, and tetrahydrofuran—were selected, and the effect of solvent polarity on the UV-Vis absorption spectrum of the probe was tested. The wavelength range measured was 300–700 nm, and the sample concentration was c = 1.0 × 10⁻⁶. -5 The concentration was mol / L, and a quartz cuvette with dimensions of 1 cm × 1 cm was used as the sample cell. The test temperature was 25℃. The single-photon emission spectrum was measured on a Hitachi F-7000 fluorescence spectrometer. The sample concentration was c = 1.0 × 10⁻⁶ mol / L. -5 The concentration was mol / L, the sample container was a 1cm×1cm transparent quartz cuvette, the test temperature was 25℃, the data acquisition step size was 1nm, the width of the incident and exit slits was 10.0nm, and the excitation voltage was 500mV.

[0080] The UV-Vis absorption spectra of MTDTP were measured in solvents of different polarities, and the results were... Figure 4The probe exhibits two significant absorption bands in the 300–700 nm range: approximately 300 nm corresponds to the π-π* transition in the diphenylamine-thiophene aromatic system, and approximately 500 nm corresponds to the intramolecular charge transfer (ICT) transition. The positions of these absorption peaks remain almost unchanged when the solvent polarity changes, indicating that solvent polarity has little effect on the probe's ground state energy level, and the intramolecular charge transfer state is stable. Fluorescence emission tests show that the emission peak of MTDTP is stable at around 615–630 nm in various solvents, and the emission intensity is insensitive to solvent polarity. This demonstrates that the fluorescence performance of this probe is mainly controlled by its internal molecular structure and is less affected by external polar environments, which is beneficial for maintaining signal stability in complex biological environments.

[0081] The response characteristics of MTDTP to microenvironment viscosity were tested in detail: solutions of different viscosities (range 1.0–1150 cP) were prepared by mixing glycerol-phosphate buffer (PBS), and the fluorescence intensity of the probe was measured as a function of viscosity. Solution systems of different viscosities were prepared by mixing PBS and glycerol in different volume ratios, and the fluorescence intensity of MTDTP in solutions of different viscosities was detected after adding the probe.

[0082] The results are as follows Figure 5 As shown in Figure c, the fluorescence intensity of MTDTP increased significantly with increasing solution viscosity. At a viscosity of 1150 cP (99% glycerol), the fluorescence enhancement was approximately 59-fold compared to the initial concentration (pure PBS, 1.0 cP), and the fluorescence intensity showed a good linear correlation with the logarithm of viscosity (R²≈0.97). Simultaneously, the absolute fluorescence quantum yield of the probe gradually increased from 1.6% at low viscosity to 38.8% at high viscosity. These results demonstrate that MTDTP is extremely sensitive to environmental viscosity and can amplify microviscosity changes into observable fluorescence signal changes through the TICT mechanism. Further selectivity experiments showed that MTDTP has a specific response to membrane lipid environments: when liposomes simulating the mitochondrial membrane were added, the probe fluorescence intensity increased by approximately 16-fold, while the addition of common mitochondrial macromolecules such as DNA, RNA, ATP, or thiol-containing amino acids resulted in virtually no significant change in fluorescence. Therefore, MTDTP is primarily sensitive to hydrophobic viscous membrane environments, consistent with its design intent (targeting the inner mitochondrial membrane and sensing membrane viscosity). In summary, the probe of this invention meets the requirements of biological imaging in terms of photophysical properties, and achieves highly sensitive detection of microenvironment viscosity through the TICT intramolecular rotation mechanism.

[0083] Example 4: Live Cell Imaging and Functional Verification

[0084] In this embodiment, the MTDTP probe was applied to live-cell experiments. The conditions for incubating live cells with this probe were: a 2 μM concentration at 37°C for 15 min, followed by washing the cells three times with sterile PBS. This was to verify its mitochondrial targeting, imaging performance, and role in mitochondrial-lysosome interaction studies (see [link to study]). Figure 5 and Figure 6 ).

[0085] First, the cell membrane permeability and biocompatibility of the probe were tested: MTDTP at a concentration of 2 μM was added to the culture medium of various cell types (HeLa cervical cancer cells, 4T1 breast cancer cells, HepG2 liver cancer cells, QSG normal liver cells, etc.) and incubated for 15 minutes, and the intracellular fluorescence distribution was observed. The results showed that mitochondrial-like fluorescent signals appeared in all cell types, located in the cytoplasmic region, and pre-fixed cells could also be stained, indicating that this probe has good universal applicability. The MTT cell viability assay results showed that incubation at the working concentration (2 μM) for 24 hours had no significant effect on cell viability (cell viability was above 90%), indicating that the probe has no obvious toxicity to cells and is suitable for live-cell imaging experiments.

[0086] Furthermore, the mitochondrial targeting and specificity of MTDTP were verified through co-localization experiments with commercial mitochondrial dyes. HeLa cells were simultaneously incubated with the probe (green fluorescence, 2 μM for 15 min) and MitoTracker Deep Red. The results showed a high degree of overlap in the fluorescence signals of the two dyes in the cells, with a Pearson correlation coefficient (PCC) of approximately 0.93. This demonstrates that MTDTP is primarily enriched in the mitochondrial region, and its localization is consistent with that of classic mitochondrial dyes. Simultaneously, co-staining results with other organelle-specific dyes (such as endoplasmic reticulum and lysosomes) showed no significant overlap, indicating that the probe has high selectivity for mitochondria and will not non-specifically stain other cellular structures.

[0087] Notably, the MTDTP probe selectively anchors to the inner mitochondrial membrane. To confirm this, we co-stained it with a commercially available fluorescent dye (PKMTDR) that specifically indicates the inner mitochondrial membrane. Super-resolution microscopy (SIM) results are shown. Figure 5 (b, c) shows that MTDTP and PKMTDR are highly congruent in their location on the inner mitochondrial membrane, and their fluorescence signals completely overlap. Furthermore, at higher resolution, MTDTP clearly depicts the fine ladder-like structure of the mitochondrial cristae. Figure 5d) This further proves that MTDTP is indeed located on the inner mitochondrial membrane and can resolve its ultrastructure. Correspondingly, the pyridinium cation targeting group used in MTDTP is more likely to accumulate near the inner membrane (due to the higher negative potential on the inner side of the inner membrane), and its lipophilic side chain can insert into the membrane, thereby achieving selective staining of the inner membrane.

[0088] The photostability of the probe of this invention is outstanding in live-cell imaging. MTDTP-labeled live cells were placed under a confocal laser scanning microscope, and the same field of view was repeatedly scanned 200 times with a laser. The fluorescence intensity change curve over time was recorded. Figure 5 e). The results showed that the fluorescence intensity of MTDTP hardly decayed under continuous excitation, maintaining more than 95% of its initial intensity. In contrast, the fluorescence of control commercial mitochondrial dyes (such as MitoTracker Green and MitoTracker Deep Red) decayed much faster than that of MTDTP. The excellent photobleaching resistance of MTDTP ensures its suitability for long-duration dynamic imaging and super-resolution imaging experiments requiring multiple excitations and acquisitions.

[0089] Subsequently, we used the MTDTP probe combined with high-resolution imaging to observe the interaction between mitochondria and other cellular structures.

[0090] (1) Mitochondrial-microtubule interaction: MTDTP-labeled mitochondria were co-incubated with SiR-tubulin in live cells, and dual-channel SIM live-cell imaging was performed. The results showed that mitochondrial movement and positioning were finely spatially coordinated with the microtubule network: mitochondria moved and remained along microtubule tracks, exhibiting a morphology that matched the microtubule skeleton. Figure 5 f). Time-series images further reveal that microtubules act as "tracks" during mitochondrial transport, with the location and direction of mitochondrial movement clearly guided by microtubule distribution. This observation suggests that MTDTP can be used to study the interaction between mitochondria and the cytoskeleton and the mechanisms of mitochondrial migration.

[0091] (2) Mitochondrial-lysosome interactions: HeLa cells were co-incubated using MTDTP and the lysosomal tracking dye LysoTracker DeepRed (LyDR) to observe mitochondrial-lysosome interactions (MLIs) in real time. MTDTP revealed the fine structure of the mitochondrial cristae with a high signal-to-noise ratio and recorded several key mitochondrial dynamics processes, including clear mitochondrial fusion and fission events. Through analysis of a large number of images, we classified the observed mitochondrial-lysosome interactions into four types: dissociation (lysosomes and mitochondria are independent and do not contact each other), contact (lysosomes and mitochondria are in close contact but not encapsulated), encapsulation (lysosomes are partially embedded in the mitochondrial network), and fusion (lysosomes and mitochondria fuse, and signals overlap). These interaction types reflect the characteristics of different stages in mitophagy: from initial contact, membrane fusion to the final lysosomal engulfment of damaged mitochondria. In the real-time images, we indeed captured the process of damaged mitochondria being encapsulated and engulfed by lysosomes, which is of great significance for understanding how cells clear defective mitochondria. Therefore, the superior imaging performance of the MTDTP probe enables us to visualize the dynamic interaction between mitochondria and lysosomes like never before, providing an effective means to elucidate the mitochondrial quality control mechanism.

[0092] Example 5: Application Study in AD Cell Model

[0093] This embodiment uses the MTDTP probe in an in vitro AD cell model to reveal mitochondrial dysfunction associated with Alzheimer's disease. The method for establishing the AD cell model is as follows: Human neuroblastoma cells (SH-SY5Y) are selected and Aβ1-42 oligomers are added to a final concentration of 10 μM, followed by incubation for 24 and 48 hours to induce pathological changes at different stages of AD progression. Cells without Aβ treatment serve as a control. Subsequently, mitochondria and lysosomes are stained with MTDTP and LysoTracker, respectively, and observed using SIM super-resolution microscopy (e.g., mitochondria and lysosomes). Figure 6 (As shown).

[0094] Imaging results showed that, compared with the untreated control group, the morphology of mitochondria in Aβ-treated cells changed significantly: mitochondria changed from long filamentous or branched to more short rod-shaped and granular shapes. After 48 hours of Aβ treatment, mitochondria in the cells showed a tendency to swell and rupture, with many mitochondria shortening to less than 1 μm in length, forming dot-like / elliptical shapes (defined as "round mitochondria"), while the number of long rod-shaped mitochondria longer than 3 μm was significantly reduced (defined as a significant reduction in "elongated mitochondria"). Statistical classification showed that approximately 12% of mitochondria in untreated cells were round (<1 μm), 25% were medium rod-shaped (1–3 μm), and 63% were elongated (>3 μm); after 48 hours of Aβ treatment, the proportion of round mitochondria increased to 29% (a 2.4-fold increase), while the proportion of elongated mitochondria decreased to 22%. Simultaneously, the mitochondrial surface area also decreased significantly (average surface area reduction of approximately 56%). Figure 6 e) The average length is shortened (reduced by approximately 39.5%) Figure 6 These results indicate that Aβ-induced mitochondrial structural damage and network fragmentation occur extensively.

[0095] In addition to changes in the mitochondria themselves, we also assessed the kinetic behavior of lysosomes under Aβ treatment. By tracking lysosomal movement trajectories and combining this with image analysis, we found that lysosomal motility was significantly slowed down after Aβ treatment. Figure 6 f). Specifically, the average distance traveled per unit time in the 48h treatment group was reduced by approximately 78% compared to the control group, the overall trajectory length was shortened, and the average speed decreased (f). Figure 6 Our analysis reveals that Aβ disrupts mitochondrial-lysosomal interactions (MLIs) through dual organelle damage, including mitochondrial structural collapse and lysosomal motility impairment, synergistically exacerbating mitophagy dysfunction and thus worsening the course of Alzheimer's disease. These findings provide the first nanoscale visualization of the mechanism of MLI dysregulation in an Alzheimer's disease model, suggesting that restoring lysosomal motility can block this pathogenic cascade, offering a new avenue for treatment.

[0096] Example 6: Validation by brain tissue imaging in AD model mice

[0097] This embodiment uses the MTDTP probe for brain tissue imaging in transgenic Alzheimer's disease mice to verify the probe's performance in live samples and to investigate changes in mitochondrial viscosity and dynamics under AD pathological conditions. Three APP / PS1 transgenic AD model mice (12 months old, exhibiting obvious AD-like pathology) and three wild-type control mice of the same age were used in the experiment. Brains were immediately harvested after euthanasia, and fresh brain slices (approximately 10 μm thick) were cut on ice. Immunofluorescence staining was performed, including an antibody against amyloid Aβ (to label AD plaques) and an antibody against the mitochondrial outer membrane protein TOMM20 (to label mitochondrial locations). The slices were then incubated with 10 μM MTDTP for 30 minutes to allow the probe to penetrate the tissue and stain the mitochondria. Free probes were washed away with PBS, and cell nuclei were stained with DAPI. Multichannel images were acquired using confocal microscopy, and three-dimensional spatial reconstruction analysis was performed (see [link to relevant documentation]). Figure 7 ).

[0098] Imaging results showed that MTDTP successfully stained mitochondrial structures in brain tissue. In wild-type mouse brain slices, MTDTP primarily labeled the mitochondrial network, which highly overlapped with the TOMM20 immune signal. Figure 7 a) This verified that the probe still retained mitochondrial specificity in tissue samples. In the brain tissue of AD model mice, the overall fluorescence signal of MTDTP was significantly enhanced, especially in the cerebral cortex and hippocampus, where it was far stronger than the control. Figure 7 b). Quantitative analysis showed that, compared with wild-type mice, the mean fluorescence intensity of MTDTP in the cortical region of AD mice increased by approximately 3.6 times, and in the hippocampus by approximately 2.5 times. Figure 7 The differences (c, d) were statistically significant (P < 0.01). This result directly indicates that the mitochondrial viscosity in the brains of AD mice was significantly higher than normal, supporting "high mitochondrial viscosity" as one of the important pathological features of AD.

[0099] In addition to changes in overall fluorescence intensity, MTDTP also revealed abnormalities in mitochondrial quality control in the AD brain. Numerous clusters of MTDTP bright spots were observed in brain slices of AD mice. Comparison with Aβ antibody and LAMP1 (lysosomal membrane protein) immunosignals revealed that these strong fluorescent clusters corresponded to the aggregation of damaged mitochondria and increased co-localization with lysosomes. Three-dimensional reconstructed images showed that in the neurons of AD mice, many LAMP1-positive lysosomes closely adhered to or even enveloped MTDTP-labeled mitochondria. Figure 7g) The morphology shows lysosomes attempting to engulf mitochondria. Further quantitative tissue analysis revealed that the proportion of mitochondria with lysosomal co-localization in the brains of AD mice was significantly lower than that in normal mice, suggesting impaired mitophagy. These findings are consistent with the known pathology of AD: impaired mitochondrial function and degradation disorders coexist, leading to the gradual accumulation of defective mitochondria within neurons. This is also one of the reasons for chronic neuronal stress and death during the course of AD. Through MTDTP imaging, we have, for the first time, directly verified this hypothesis at the tissue level.

[0100] In summary, in AD model mice, the probe of this invention successfully indicated pathological changes such as abnormally increased mitochondrial viscosity and impaired mitochondrial clearance in brain tissue. This not only demonstrates the effectiveness and specificity of MTDTP in complex biological tissues but also suggests its potential application in the early diagnosis of AD (by detecting abnormal mitochondrial viscosity) and the evaluation of subsequent treatment interventions. For example, MTDTP could be considered for in vivo brain imaging or the detection of mitochondrial components in cerebrospinal fluid, thereby developing it into a molecular imaging reagent for the auxiliary diagnosis of AD.

[0101] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A mitochondrial-targeted viscosity fluorescent probe, characterized in that, Its chemical structure is shown in Formula I:

2. The mitochondrial-targeted viscosity fluorescent probe according to claim 1, characterized in that, The probe molecule has a donor-π bridge-acceptor molecular structure, with a diphenylamine group as an electron donor, a thiophene ring and a double bond as a π-conjugated bridge, and a pyridinium cation as an electron acceptor and a mitochondrial targeting group; wherein the pyridinium cation is substituted with 4-(ethoxycarbonyl)pentyl to form a quaternary ammonium salt structure.

3. The mitochondrial-targeted viscosity fluorescent probe according to claim 1, characterized in that, The probe molecule contains a twisted intramolecular charge-transfer fluorescent rotor structure.

4. The mitochondrial-targeted viscosity fluorescent probe according to claim 1, characterized in that, The fluorescence emission wavelength of the probe is located in the visible red light region, with a wavelength range of 610 nm to 630 nm.

5. The mitochondrial-targeted viscosity fluorescent probe according to claim 1, characterized in that, The probe is localized without relying on mitochondrial membrane potential.

6. A method for synthesizing a mitochondrial-targeting viscosity fluorescent probe according to any one of claims 1-5, characterized in that, The synthesis route of the probe is as follows:

7. The method for synthesizing the mitochondrial-targeted viscosity fluorescent probe according to claim 6, characterized in that, Includes the following steps: 5-Halothiophen-2-carboxaldehyde was reacted with diphenylamine in the presence of a palladium catalyst to obtain an intermediate of 5-(diphenylamino)thiophen-2-carboxaldehyde. The intermediate was condensed with 4-methylpyridine in Knoevenagel to form a 2-thienylvinyl-substituted pyridine compound; The pyridine compound is reacted with ethyl 5-halopentanoate in an N-alkylation quaternization reaction to introduce a positive charge into the pyridine, thereby obtaining the halide salt of the fluorescent probe; The obtained halide salt was subjected to anion exchange to obtain the fluorescent probe.

8. The application of a mitochondrial-targeting viscosity fluorescent probe as described in any one of claims 1-5 in mitochondrial imaging reagents.

9. The application of a mitochondrial-targeting viscosity fluorescent probe as described in any one of claims 1-5 in a multiplex co-staining imaging reagent for mitochondria and organelles.

10. The use of a mitochondrial-targeting viscosity fluorescent probe as described in any one of claims 1-5 in the preparation of imaging reagents for the diagnosis of Alzheimer's disease.

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