Hydroxyl radical near-infrared fluorescence-photoacoustic dual-mode molecular probe as well as preparation method and application thereof

Abstract

The invention relates to the technical field of fluorescence imaging, in particular to a hydroxyl radical near-infrared fluorescence-photoacoustic dual-mode molecular probe as well as a preparation method and application thereof. The hydroxyl radical near-infrared fluorescence-photoacoustic dual-mode molecular probe provided by the invention is obtained by reducing IR-775 dye, the preparation method is simple, the molecular structure is simple, near-infrared fluorescence is remarkably enhanced, an obvious photoacoustic signal is generated, and the hydroxyl radical near-infrared fluorescence-photoacoustic dual-mode molecular probe has the advantages of high detection sensitivity, high accuracy and good selectivity.

Description

Technical Field

[0001] This invention relates to the field of fluorescence imaging technology, and in particular to a hydroxyl radical near-infrared fluorescence-photoacoustic dual-mode molecular probe, its preparation method, and its application. Background Technology

[0002] Reactive oxygen species (ROS), a class of highly reactive substances derived from oxygen within cells, play a crucial role in various cellular signaling processes. Among them, hydroxyl radicals have attracted significant attention due to their extremely high oxidizing capacity. Hydroxyl radicals can induce DNA base oxidation and hydroxylation, lipid peroxidation, and protein oxidative damage. Their excessive production is closely linked to various pathophysiological processes related to oxidative stress, such as inflammation, cancer, and cardiovascular disease. Because hydroxyl radicals exist at extremely low concentrations (typically nanomolar to micromolar levels), have extremely short lifetimes (half-life ≤ 1 nanosecond), and exhibit extremely high reactivity under physiological and pathological conditions, their real-time and accurate detection in biological systems presents a significant challenge. Therefore, developing highly sensitive and selective hydroxyl radical detection tools is crucial for a deeper understanding of their biological functions and disease associations.

[0003] Currently, commonly used methods for detecting hydroxyl radicals are based on fluorescent probes excited by visible light (wavelength range typically 400-650 nm). The fluorescence signal generated by these probes after response is easily interfered with by the strong autofluorescence of endogenous substances widely present in the body (such as flavin, porphyrin, etc.), resulting in a significant reduction in the signal-to-noise ratio and insufficient selectivity and accuracy. In addition, the penetration depth of visible light in biological tissues is limited, which seriously restricts its application in deep tissues in vivo or in situ real-time detection.

[0004] Near-infrared (NIR) fluorescent molecular probes significantly improve accuracy and deep penetration compared to traditional probes. They utilize light in the 650-1700 nm wavelength range to excite probe molecules, causing them to transition to an excited state. Subsequently, they emit NIR fluorescence with a longer wavelength through radiative transitions. Because NIR light experiences less scattering and weak absorption in biological tissues, and tissue autofluorescence is extremely low, deeper tissue penetration, higher imaging signal-to-noise ratio, and lower photodamage are achieved. This makes them particularly suitable for high-contrast, high-sensitivity fluorescence imaging of deep tissues in vivo. Photoacoustic imaging, a physical phenomenon based on the photoacoustic effect, can be understood as the process by which matter absorbs light energy and generates sound waves (ultrasound). This effect cleverly combines the two physical signals of light and sound, possessing both the high contrast of optical imaging and the deep penetration advantages of ultrasound imaging. It is a core principle of the rapidly developing biomedical imaging technology in recent years. Combining near-infrared fluorescent probes with photoacoustic theory will be beneficial for improving the sensitivity, accuracy, and selectivity of deep detection.

[0005] The intensity of photoacoustic signals mainly depends on the probe's light absorption capacity, photothermal conversion efficiency, and thermal expansion characteristics. Near-infrared fluorescent molecular probes possess strong near-infrared absorption, providing a physical basis for photoacoustic conversion. However, such dual-mode probes are currently in the early stages of commercialization. Dual-mode probe molecules not only need to simultaneously achieve high fluorescence quantum yield and efficient photothermal conversion within the same molecule—these two energy release pathways are inherently competitive—but also need to ensure that the two signals respond stably and synchronously to the target analyte in complex biological environments, avoiding dual-signal decoupling due to differences in tissue microenvironment or asynchronous probe metabolic kinetics. Therefore, achieving high fluorescence and high photoacoustic signals usually relies on complex molecular structure design, resulting in lengthy synthetic routes and harsh reaction conditions, making large-scale preparation difficult. Furthermore, the response characteristics of different signal channels are easily affected by the biological environment, leading to signal asynchrony or unstable responses. Especially in complex in vivo environments, it is often difficult to simultaneously achieve probe structural stability, response consistency, and imaging signal-to-noise ratio, limiting their further applications. Summary of the Invention

[0006] This invention, based on the structurally simple IR-775 molecular framework, utilizes a concise reduction modification strategy to prepare a hydroxyl radical near-infrared fluorescence-photoacoustic dual-mode molecular probe. This probe exhibits selective responsiveness to hydroxyl radicals, achieving near-infrared fluorescence "off-on" response and synchronous photoacoustic signal enhancement within a single molecular structure. It combines high response sensitivity, imaging stability, and ease of preparation, reducing synthesis costs while improving the probe's practical application feasibility and overcoming multiple limitations in existing technologies. Furthermore, this hydroxyl radical near-infrared fluorescence-photoacoustic dual-mode molecular probe possesses advantages such as high detection sensitivity, high accuracy, and good selectivity.

[0007] The hydroxyl radical near-infrared fluorescence-photoacoustic dual-mode molecular probe provided by this invention has the structure shown in Formula I:

[0008]

[0009] In some embodiments, the preparation method of the hydroxyl radical near-infrared fluorescence-photoacoustic dual-mode molecular probe includes the following steps:

[0010] Under the protection of an inert gas, a reducing agent is added to the dye solution and mixed. The mixture is then stirred at 0-5°C for 1-3 hours to obtain a reduced reaction solution. The dye solution includes IR-775 dye and an organic solvent. The IR-775 dye has the structure shown in Formula II.

[0011] Glacial acetic acid was added to the reduced reaction solution and mixed to obtain a neutralized reaction solution; the pH of the neutralized reaction solution was 6.8-7.2.

[0012] Add a saturated aqueous solution of sodium chloride to the neutralized reaction solution and mix. Then add ethyl acetate for extraction and collect the organic phase.

[0013] The organic phase was dried and then concentrated to obtain the hydroxyl radical near-infrared fluorescence-photoacoustic dual-mode molecular probe.

[0014] In some embodiments, the organic solvent is a mixture of methanol and tetrahydrofuran; optionally, the volume fraction of methanol is 40-60%.

[0015] In some embodiments, the reducing agent includes at least one of sodium borohydride, sodium cyanoborohydride, and sodium triacetoxyborohydride.

[0016] In some embodiments, the molar ratio of the reducing agent to the IR-775 dye in the dye solution is 1.8-2.2:1.

[0017] In some embodiments, the concentration of IR-775 dye in the dye solution is 8-12 mmol / L.

[0018] In some embodiments, the step of adding a reducing agent to the dye solution and mixing it under the protection of an inert gas includes:

[0019] A reducing agent solution is prepared by dissolving the reducing agent in an alcohol solvent.

[0020] Under the protection of an inert gas and with stirring, the reducing agent solution is added dropwise to the dye solution and mixed.

[0021] In some embodiments, the concentration of the reducing agent in the reducing agent solution is 0.08-0.12 mol / L.

[0022] This invention also provides the application of the aforementioned hydroxyl radical near-infrared fluorescence-photoacoustic dual-mode molecular probe in the detection of hydroxyl radicals.

[0023] This invention also provides the application of the hydroxyl radical near-infrared fluorescence-photoacoustic dual-mode molecular probe described above in in vitro fluorescence response and cell imaging.

[0024] This invention relates to a hydroxyl radical near-infrared fluorescence-photoacoustic dual-mode molecular probe, obtained through the structural reduction of the heptamethrin indole anthocyanin dye IR-775. Initially, it is in an "off" state for both fluorescence and photoacoustic activity. When hydroxyl radicals are present in vivo or in vitro, IR-775-R is specifically oxidized, reconstructing the donor-acceptor electronic properties of its molecular structure, inducing intramolecular charge transfer, and thereby restoring and expanding its conjugated system. This results in a significant enhancement of near-infrared fluorescence and the simultaneous generation of a noticeable photoacoustic signal, achieving a synchronous "on" response for near-infrared fluorescence and photoacoustic signals. The near-infrared dual-mode detection characteristics of the fluorescence-photoacoustic molecular probe can improve detection depth and resolution. The excellent cell imaging results demonstrate the potential of this probe for clinical applications.

[0025] The hydroxyl radical near-infrared fluorescence-photoacoustic dual-mode molecular probe provided by this invention is based on the reduction-derived structure of the indole-based anthocyanin dye IR-775. After undergoing a specific oxidation reaction with hydroxyl radicals, it can reconstruct the intramolecular charge transfer process, significantly expand the conjugated system, and generate obvious near-infrared fluorescence and photoacoustic signals, achieving an "off-on" type dual-mode response. This probe integrates near-infrared fluorescence and photoacoustic dual imaging functions, possessing both high-sensitivity and high-contrast molecular imaging capabilities, as well as strong tissue penetration depth and spatial resolution, significantly improving the accuracy and application value of biological imaging. In addition, the probe is simple to synthesize under mild conditions, and the raw materials are readily available, showing good scalability and application prospects. Attached Figure Description

[0026] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0027] Figure 1 This invention relates to the mass spectrometry and proton NMR characterization (chloroform) of the hydroxyl radical near-infrared fluorescence-photoacoustic dual-mode molecular probe.

[0028] Figure 2 This is a graph showing the ultraviolet, fluorescence, and photoacoustic changes in the response of the hydroxyl radical near-infrared fluorescence-photoacoustic dual-mode molecular probe of this invention to hydroxyl radicals.

[0029] Figure 3 This is a selective experimental data graph of the hydroxyl radical near-infrared fluorescence-photoacoustic dual-mode molecular probe of the present invention.

[0030] Figure 4This is a graph showing the MTT experimental data of the hydroxyl radical near-infrared fluorescence-photoacoustic dual-mode molecular probe of this invention.

[0031] Figure 5 This is a confocal microscopy cell imaging image of the hydroxyl radical near-infrared fluorescence-photoacoustic dual-mode molecular probe of the present invention.

[0032] Figure 6 This is an intracellular photoacoustic imaging image of the hydroxyl radical near-infrared fluorescence-photoacoustic dual-mode molecular probe of the present invention. Detailed Implementation

[0033] The present invention will be further described below with reference to embodiments and examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the protection scope of the appended claims.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0035] The hydroxyl radical near-infrared fluorescence-photoacoustic dual-mode molecular probe provided by this invention has the structure shown in Formula I:

[0036]

[0037] The chemical formula of the compound with the structure shown in Formula I is C 32 H 37 ClN2, with a theoretical monoisotopic mass of 484.26, has a theoretical [M+H] content under positive ion electrospray mode. + The ion has an m / z of 485.26. Due to the instability of chlorine in electrospray mass spectrometry, especially when it is at a site that is easily dissociated in benzyl, allyl, or indole structures, selective desorption often occurs. In an ionized environment, chlorine atoms are easily removed in the form of neutral or negative ions (such as Cl- or HCl), thereby generating stable ion fragment structures without chlorine. Therefore, the mass spectrometry results of the compound with the structure shown in Formula I differ from the theoretical values.

[0038] In some embodiments, the mass spectrometry result of the compound with the structure shown in Formula I is m / z 483.26, corresponding to [M–Cl+H] after dechlorination and proton addition. + Or its equivalent structure, the detection result is not due to experimental abnormalities or operational deviations, but is determined by the characteristic peak of the main stable ion formed by the target molecule during ionization.

[0039] The compounds with the structure shown in Formula I have a certain structural correspondence. Some hydrogen atoms with the same chemical environment appear as the same set of shared signal peaks in nuclear magnetic resonance. Therefore, they are only counted as one signal in the integral calculation and are not reflected in the total integral value. As a result, the total number of integrals of the measured hydrogen spectrum is slightly lower than its theoretical number of hydrogen atoms.

[0040] The preparation method of this hydroxyl radical near-infrared fluorescence-photoacoustic dual-mode molecular probe includes the following steps:

[0041] Under the protection of an inert gas, a reducing agent is added to the dye solution and mixed. The mixture is then stirred at 0-5°C for 1-3 hours to obtain a reduced reaction solution. The dye solution includes IR-775 dye and an organic solvent. IR-775 dye has the structure shown in Formula II.

[0042] Glacial acetic acid was added to the reduced reaction solution and mixed to obtain a neutralized reaction solution; the pH of the neutralized reaction solution was 6.8-7.2.

[0043] Add a saturated aqueous solution of sodium chloride to the neutralized reaction solution and mix. Then add ethyl acetate for extraction and collect the organic phase.

[0044] After drying and concentration, the organic phase was used to obtain a hydroxyl radical near-infrared fluorescence-photoacoustic dual-mode molecular probe.

[0045] In some embodiments, the organic solvent is a mixture of methanol and tetrahydrofuran; optionally, the volume fraction of methanol is 40-60%, such as 40%, 45%, 50%, 55%, 60%, etc. Reaction in a suitable solvent is beneficial for forming a homogeneous and stable reaction system, thereby improving the conversion rate and yield of the reaction.

[0046] In some embodiments, the reducing agent includes at least one selected from sodium borohydride, sodium cyanoborohydride, and sodium triacetoxyborohydride. Preferably, sodium borohydride is used as the reducing agent, which can yield higher yields and purity.

[0047] In some embodiments, the molar ratio of the reducing agent to the IR-775 dye in the dye solution is 1.8-2.2:1, such as 1.8:1, 1.9:1, 2:1, 2.1:1, 2.2:1, etc. This molar ratio ensures that the cation of the IR-775 dye is reduced, while other easily reduced structures remain unaffected, resulting in a product with high purity.

[0048] In some embodiments, the concentration of IR-775 dye in the dye solution is 8-12 mmol / L, such as 8 mmol / L, 9 mmol / L, 10 mmol / L, 11 mmol / L, 12 mmol / L, etc.

[0049] In some embodiments, the step of adding a reducing agent to the dye solution and mixing it under the protection of an inert gas includes:

[0050] A reducing agent solution is prepared by dissolving the reducing agent in an alcohol solvent.

[0051] Under the protection of an inert gas and with stirring, a reducing agent solution is added dropwise to the dye solution and mixed.

[0052] Optionally, the alcohol solvent includes at least one of methanol and ethanol;

[0053] Optionally, in the step of adding the reducing agent solution to the dye solution, the temperature of the mixture is controlled at 0-5°C.

[0054] In some embodiments, the concentration of the reducing agent in the reducing agent solution is 0.08-0.12 mol / L, such as 0.08 mol / L, 0.09 mol / L, 0.1 mol / L, 0.11 mol / L, 0.12 mol / L, etc.

[0055] In some embodiments, in the step of adding the reducing agent solution to the dye solution, the dropping rate is 0.1-0.15 mL / min, which translates to approximately 0.1-0.15 mmol / min in moles. By controlling the dropping rate, the reaction rate can be controlled, which is beneficial for improving the conversion rate and yield of the reaction and obtaining a product with higher purity.

[0056] This invention also provides the application of the hydroxyl radical near-infrared fluorescence-photoacoustic dual-mode molecular probe described above in the detection of hydroxyl radicals.

[0057] This invention also provides the application of the hydroxyl radical near-infrared fluorescence-photoacoustic dual-mode molecular probe described above in in vitro fluorescence response and cell imaging.

[0058] The following are some specific examples.

[0059] For experimental parameters not specified in the following specific embodiments, please refer to the guidelines given in this invention document first, or refer to experimental manuals or other experimental methods known in the art, or refer to the experimental conditions recommended by the manufacturer.

[0060] The raw materials and reagents involved in the following specific embodiments can be obtained commercially or prepared by those skilled in the art using known methods.

[0061] IR-775 dye, CAS number 199444-11-6, has the following structure:

[0062] Example 1

[0063] This embodiment provides a method for preparing the hydroxyl radical near-infrared fluorescence-photoacoustic dual-mode molecular probe of the present invention, the steps of which are as follows:

[0064] (1) Under inert gas protection, weigh 71.1 mg of IR-775 dye and add it to a dry 50 mL round bottom flask. Add 10 mL of organic solvent (prepared by mixing methanol and tetrahydrofuran in a volume ratio of 1:1) and stir magnetically to fully dissolve the IR-775 dye to obtain the first solution.

[0065] (2) Cool the first solution to 0-5°C in an ice bath. While stirring, slowly add a 0.1 mol / L methanol solution of fresh sodium borohydride dropwise, maintaining the temperature of the mixture at 0-5°C during the addition. The molar amount of sodium borohydride added is twice that of the heptamethrin indole cyanine dye IR-775, and the addition rate is approximately 0.133 mL / min (corresponding to approximately 0.0133 mmol / min). After the addition is complete, continue stirring the resulting mixture at 0-5°C for 2 hours to obtain the reduced reaction solution.

[0066] (3) Slowly add 0.5 mL of glacial acetic acid to the reduced reaction solution to neutralize the residual sodium borohydride. Stop adding the acid when the pH of the reaction solution is adjusted to neutral (pH 6.8-7.2) to obtain the neutralized reaction solution.

[0067] (4) Add 10 mL of saturated sodium chloride aqueous solution to the neutralized reaction mixture, mix thoroughly, and perform liquid-liquid separation. Extract the lower aqueous phase with ethyl acetate three times, using 30 mL of ethyl acetate each time. Collect and combine the ethyl acetate phases, dry with anhydrous sodium sulfate, and then perform rotary evaporation under reduced pressure. Freeze-dry the resulting concentrate for 12 hours to obtain a yellow-green solid, 62.3 mg. Based on the raw material IR-775 dye, the yield is 87.6%, the purity is 96.3%, and the conversion rate is 84.4%. Mass spectrometry and 1H NMR spectroscopy are shown below. Figure 1 As shown, Figure 1 In Figure A, the mass spectrometry result of the probe is shown. Figure 1 B in the figure represents the 1H NMR spectrum of the probe. 1 H NMR (400MHz, CDCl3) δ = 8.18 (d, J = 13.2Hz, 2H), 7.18 (t, J = 7.3Hz, 4H), 6.90 (t, J = 7.4Hz, 2H), 6.68 (d, J = 7.9 Hz,2H),5.41(d,J=13.2Hz,2H),3.21(s,6H),2.62(t,J=6.2Hz,4H),1.87(p,J=6.4Hz,2H),1.68(s,12H).[M +[483.26] Structural characterization confirms the successful preparation of the hydroxyl radical near-infrared fluorescence-photoacoustic dual-mode molecular probe of the present invention.

[0068] Example 2

[0069] This embodiment tests the performance of the hydroxyl radical near-infrared fluorescence-photoacoustic dual-mode molecular probe of the present invention.

[0070] 1. Near-infrared fluorescence characteristics (1) Preparation of probe stock solution: Weigh 4.83 mg (10 μmol) of the probe prepared in Example 1, dissolve it in 1 mL of dimethyl sulfoxide (DMSO) to prepare a probe stock solution with a concentration of 10 mM for later use.

[0071] (2) Preparation of hydroxyl radical reaction solution: Weigh 27.8 mg FeSO4·7H2O and dissolve it in 1 mL of water (pH = 6) to prepare a 100 mM FeSO4·7H2O stock solution. The concentration ratio of Fenton's reagent FeSO4·7H2O to H2O2 is 1:6, and the concentration of FeSO4·7H2O represents the concentration of hydroxyl radicals.

[0072] (3) Sample preparation: Take 3 μL of the 10 mM probe stock solution and add it to a cuvette containing 3 mL of PBS buffer (pH 7.4). Mix well to make the final concentration 10 μM. Then add it to the Fenton reagent system according to the experimental design and incubate at 37 °C for 30 minutes in the dark.

[0073] (4) Fluorescence detection: After incubation, the fluorescence emission spectrum in the 750-900nm band was recorded using 630nm as the excitation wavelength, and the fluorescence intensity change at 770nm was read. The widths of both the excitation and emission slits were set to 5nm.

[0074] (5) Control group setup: A blank control group without ·OH was set up and fluorescence was measured under the same conditions as a background control.

[0075] The probe emits near-infrared fluorescence at a wavelength of 770 nm, has strong tissue penetration, and can effectively avoid interference from the autofluorescence of organisms when imaging in the near-infrared channel.

[0076] 2. In vitro UV response characteristics of hydroxyl radicals

[0077] Weigh 4.83 mg (10 μmol) of the probe prepared in Example 1 and dissolve it in 1 mL of DMSO to prepare a 10 mM probe stock solution. Weigh 27.8 mg of FeSO4·7H2O and dissolve it in 1 mL of water (pH = 6) to prepare a 100 mM FeSO4·7H2O stock solution. The concentration ratio of Fenton's reagent FeSO4·7H2O to H2O2 is 1:6, and the concentration of FeSO4·7H2O represents the concentration of hydroxyl radicals. Take 3 μL of the 10 mM probe stock solution and add it to a cuvette containing 3 mL of PBS buffer (pH = 7.4). At this point, the probe concentration is 10 μM. In the cuvette, add FeSO4·7H2O (final concentration 0-50 μM) for UV response experiments and add FeSO4·7H2O (final concentration 0-150 μM) for fluorescence response experiments (excitation wavelength 630 nm). The obtained data are processed using Origin software. Figure 2 ,like Figure 2 As shown in Figure A, the probe's maximum UV absorption before the addition of FeSO4·7H2O was at 430 nm, while the fluorescence emission at 770 nm was very low. After the addition of FeSO4·7H2O, the UV peak at 430 nm decreased, while the UV absorption peak at 790 nm gradually increased. Furthermore, as shown in Figure A... Figure 2 As shown in Figure B, the fluorescence emission of the probe at 770 nm is greatly enhanced, indicating that the probe has a good response to hydroxyl radicals. Figure 2 The linear results shown in Figure C indicate that the detection limit is 8.44 nM and the detection range is 0.01–50 μM. Meanwhile, as shown in Figure C... Figure 2 As shown in Figure D, the photoacoustic signal of IR-775-R at 720 nm is significantly increased in the presence of hydroxyl radicals.

[0078] Example 3

[0079] This embodiment demonstrates the selectivity of the hydroxyl radical near-infrared fluorescence-photoacoustic dual-mode molecular probe of the present invention.

[0080] The probe (1 mM) and substrate (100 mM) prepared in Example 1 were prepared using DMSO. 30 μL of each 100 mM substrate solution was added to 12 centrifuge tubes containing 2940 μL of PBS buffer (pH = 7.4) and mixed. Then, 30 μL of the 1 mM probe solution was added to each substrate solution. The substrates to be tested were FeSO4·7H2O+H2O2 and HROS (…). 1 O2, ONOO - NO, ClO - TBHP, H2O2, O2 ·- ), metal ions (Na) + K + Ca2+ Zn 2+ Cu 2+ CO 2+ Mg 2+ Fe 2+ Fe 3 + ), anion (NO3) - NO2 - CH3COO - S2O3 2- PO4 3- CO3 2- SO3 2- SO4 2- The study included biothiols (glutathione, vitamin C, lysine, methionine, serine, cysteine, homocysteine, arginine, and hydrogen sulfide). Except for glutathione and cysteine, which were at 5 mM, all other substances were at 100 μM. Fluorescence emission intensity was measured using a cuvette. The results are as follows: Figure 3 As shown, only the FeSO4·7H2O+H2O2 group exhibited a strong fluorescence signal, indicating that the probe of this invention has good selectivity for hydroxyl radicals.

[0081] Example 4

[0082] This embodiment tests the biocompatibility of the hydroxyl radical near-infrared fluorescence-photoacoustic dual-mode molecular probe of the present invention.

[0083] Add sterile PBS buffer (pH=7.4) around the outer edge of a 96-well plate, and fill the remaining inner wells with HK-2 cells. Incubate at 37°C for 24 hours. Weigh 4.83 mg of the probe prepared in Example 1 and dissolve it in 1 mL of biological DMSO solution to prepare a 10 mM biological probe stock solution. Prepare a 5 mg / mL MTT solution. In seven 5 mL sterile centrifuge tubes, add 3 mL of LDM / F12 medium to each tube. Add 0, 0.3, 0.6, 1.5, 3, 4.5, and 6 μL of biological probe stock solution to the centrifuge tubes sequentially. Pipette 190 μL of the solution from each of the seven centrifuge tubes and add it sequentially to five rows of wells at equal concentration. Incubate for 24 hours. Then, add 10 μL of LTT solution to each well and incubate at 37°C for another 4 hours. Discard the medium and add 100 μL of DMSO solution to each well. Shake for 10 minutes until fully dissolved, and then perform detection (detection wavelength: 550 nm). Data processing using Origin software Figure 4 ,like Figure 4 As shown, when the probe concentration reaches 20 μM (6 μL of biological probe stock solution), the cells still have a survival rate of over 90%, indicating that the probe has good biocompatibility and low cytotoxicity.

[0084] Example 5

[0085] This embodiment provides the application of the hydroxyl radical near-infrared fluorescence-photoacoustic dual-mode molecular probe of the present invention in cell imaging.

[0086] 1. Fluorescence signal response effect

[0087] HK-2 cells were seeded at appropriate concentrations in two confocal microscopy dishes and cultured until the cells reached optimal condition. The culture medium in one of the confocal microscopy dishes was then completely removed, and DMEM / F12 medium containing 100 μM gentamicin (GM) was added to induce excessive production of endogenous hydroxyl radicals in HK-2 cells. The cells were incubated at 37°C for 24 hours. The culture medium in the confocal microscopy dish was then completely removed, and 2 μL of the biological probe stock solution from Example 4 was added to 2 mL of DMEM / F12 medium and thoroughly mixed. 1 mL of this mixture was then added to both confocal microscopy dishes. After incubation at 37°C for 30 minutes, the culture medium was discarded, and Hoechst 33342 staining solution was added for staining for 30 minutes in each dish. The cells were then washed three times with sterile PBS buffer (pH 7.4). Images taken using the confocal microscope are shown below. Figure 5 ,like Figure 5 As shown, the near-infrared fluorescence signal was weak in the unstimulated group, while the drug-stimulated group showed a strong fluorescence signal due to the excessive generation of hydroxyl radicals, indicating that the probe can have a good cell imaging effect in cells.

[0088] 2. Photoacoustic imaging effect

[0089] A cell model was established following the steps outlined above. After incubation, the culture medium was discarded, and IR-775-R probe at a final concentration of 10 μM was added to the cells. The cells were then incubated at 37°C and 5% CO2 for 30 minutes to allow the probe to fully penetrate the cells and interact with intracellular hydroxyl radicals. Subsequently, the cell suspension was aspirated into PE-10 phantom tubes (0.28 × 0.61 mm in diameter) using a syringe. Photoacoustic imaging was performed using a Visualsonic Vevo 2100LAZER system to record changes in photoacoustic signal intensity in different treatment groups, thereby evaluating the probe's responsiveness to intracellular hydroxyl radicals. Intracellular photoacoustic imaging images are shown below. Figure 6 After co-incubating the modeled and drug-treated cells with the probe for 30 minutes, the photoacoustic signal of the HK-2 cell suspension treated with GM at 720 nm was observed to be higher than that of the HK-2 cell suspension co-treated with thiourea (hydroxyl radical scavenger) and GM. This indicates that the probe can have a good photoacoustic imaging effect in the cells.

[0090] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity, not all possible combinations of the technical features in the above-described embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0091] The embodiments described above merely illustrate several implementations of the present invention and should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Furthermore, it should be understood that after reading the above teachings, those skilled in the art can make various alterations or modifications to the present invention, and the equivalent forms obtained also fall within the protection scope of the present invention. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided by the present invention through logical analysis, reasoning, or limited experimentation are all within the protection scope of the appended claims. Therefore, the protection scope of this patent should be determined by the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. A hydroxyl radical near-infrared fluorescence-photoacoustic dual-mode molecular probe, characterized in that, It has a structure as shown in Equation I:

2. The method for preparing the hydroxyl radical near-infrared fluorescence-photoacoustic dual-mode molecular probe according to claim 1, characterized in that, Includes the following steps: Under the protection of an inert gas, a reducing agent is added to the dye solution and mixed. The mixture is then stirred at 0-5°C for 1-3 hours to obtain a reduced reaction solution. The dye solution includes IR-775 dye and an organic solvent. The IR-775 dye has the structure shown in Formula II. Glacial acetic acid was added to the reduced reaction solution and mixed to obtain a neutralized reaction solution; the pH of the neutralized reaction solution was 6.8-7.

2. Add a saturated aqueous solution of sodium chloride to the neutralized reaction solution and mix. Then add ethyl acetate for extraction and collect the organic phase. The organic phase was dried and then concentrated to obtain the hydroxyl radical near-infrared fluorescence-photoacoustic dual-mode molecular probe.

3. The preparation method according to claim 2, characterized in that, The organic solvent is a mixture of methanol and tetrahydrofuran; optionally, the volume fraction of methanol is 40-60%.

4. The preparation method according to claim 2, characterized in that, The reducing agent includes at least one of sodium borohydride, sodium cyanoborohydride, and sodium triacetoxyborohydride.

5. The preparation method according to claim 2, characterized in that, The molar ratio of the reducing agent to the IR-775 dye in the dye solution is 1.8-2.2:

1.

6. The preparation method according to claim 1, characterized in that, The concentration of IR-775 dye in the dye solution is 8-12 mmol / L.

7. The preparation method according to claim 1, characterized in that, The step of adding a reducing agent to a dye solution and mixing it under the protection of an inert gas includes: A reducing agent solution is prepared by dissolving the reducing agent in an alcohol solvent. Under the protection of an inert gas and with stirring, the reducing agent solution is added dropwise to the dye solution and mixed.

8. The preparation method according to claim 7, characterized in that, The concentration of the reducing agent in the reducing agent solution is 0.08-0.12 mol / L.

9. The application of the hydroxyl radical near-infrared fluorescence-photoacoustic dual-mode molecular probe according to claim 1 in the detection of hydroxyl radicals.

10. The application of the hydroxyl radical near-infrared fluorescence-photoacoustic dual-mode molecular probe of claim 1 in in vitro fluorescence response and cell imaging.

Images