CN110804046B - Zinc ion proportional photoacoustic probe with mitochondrial targeting function and application thereof - Google Patents
Zinc ion proportional photoacoustic probe with mitochondrial targeting function and application thereof Download PDFInfo
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- C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings
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Abstract
本发明公开了一类具有线粒体靶向功能的锌离子比例光声探针，具有如下结构式：或者
本发明所述探针能够被可见光激发，具有红光发射，可减弱激发光源对生物样品的损伤，且具有较深的组织穿透深度。体外和体内光声成像实验表明本发明所述探针对Zn2+有较好的光声成像性能，可应用于生物成像，线粒体标记，Zn2+光声成像，具有简单的化学机构和良好的生物相容性，具有很好的应用前景。The invention discloses a zinc ion proportional photoacoustic probe with mitochondrial targeting function, which has the following structural formula: 
or The probe of the invention can be excited by visible light, has red light emission, can reduce the damage of the excitation light source to the biological sample, and has a deep tissue penetration depth. In vitro and in vivo photoacoustic imaging experiments show that the probe of the present invention has good photoacoustic imaging performance for Zn 2+ , and can be applied to biological imaging, mitochondrial labeling, Zn 2+ photoacoustic imaging, and has a simple chemical mechanism and good performance. The biocompatibility has good application prospects.
Description
Technical Field
The invention belongs to the technical field of biosensing. In particular to a xanthene derivative which can be used as Zn with a mitochondrion targeting function2+Proportional photoacoustic probes are used in the fields of cell imaging, mitochondrial marking, and photoacoustic imaging.
Background
In recent years, photoacoustic imaging has become a focus of attention as a new imaging mode. The photoacoustic imaging combines the advantages of optical imaging and ultrasonic imaging, on one hand, the photoacoustic imaging penetrates deeper than pure optical imaging (can break through high-resolution optical imaging depth 'soft' limit (1mm) such as laser confocal microscopy (LCSM), two-photon excitation microscopy (TPEF) and optical weak coherence tomography (OCT)), on the other hand, the photoacoustic imaging has higher resolution than traditional MRI and PET imaging, the image resolution can reach submicron and micron order, and high-resolution molecular imaging can be realized, on the other hand, due to the narrow line width of laser, the photoacoustic imaging can realize selective excitation of high-specificity spectral tissues by utilizing the high spectral selective absorption difference of biological tissues, not only can reflect the structural characteristics of tissues, but also can realize functional imaging, and a new imaging method and technical means different from the traditional medical imaging technology are created, the advantages of optical imaging and the acoustic imaging are combined, photoacoustic imaging is a background-free and non-invasive imaging technique that is very important for in vivo imaging.
Most of the current reports on Probes for detecting ions in organisms by using a Photoacoustic Imaging mode are used for detecting cell microenvironment or other biological species, such as Jefferson Chan project group, an example of aza-fluoro-borane probe APC-1 is designed to realize proportion Photoacoustic Imaging on Copper ions, but is irreversible due to reactive response and difficult to be used for real-time tracking and monitoring fluctuation of metal ions (see Li Hao, Zhang Pamela, Smaga Lukas P., Hoffman Ryan A., Chan Jefferson, Photoacustic Probes for Ratiometric Imaging of Copper (II), J.Am.chem.Soc. 2015,50, 15628-); one near-infrared proportional photoacoustic probe ER-P was designed in Tangbo project group for detecting cell polarity and realizing imaging of liver tissue of diabetic mice (see Xiao Haibin, Wu Chuanchhen, Li Ping, Gao Wen, Zhang Wen, Zhang Wei, Tong Lili, Tang Bo, Ratiometric photo-acoustic imaging of end plastic particulate in injected liquid tissues of diabetes, chem. Sci.,2017,10, 7025-; gil G. Westmeyer topic group synthesized an example of a Calcium-ion Photoacoustic Imaging probe CaSPA based on the partial cyanine structure design and achieved dynamic monitoring of Calcium ions in heart organs and zebra fish brains (see Roberts Sheryl, Seeger Markus, Jiang Yuanyuun, Mishra Angrag, Sigmuld Felix, Stelzl Anja, Lauri Antonella, Symvulidis Panagitis, Rolbeski Hannes, Preller Mathias, De n-Ben X. Lu ions, Razansky Daniel, Orschmann Tanja, Desboures Sabrina C., tscher Vea Paul, Bacth Thoron, Ntzeristoris Valis, Westmeyer G., Seecor G, Seoac J, phosphor J.2718, Checker J.2718, Souchi Osaka J.2718).
Intracellular metal ions play a vital role in cells and provide an important environment for the normal operation of cells. And zinc ion (Zn)2+) The second most abundant transition metal ions in the human body are mainly distributed in nerve cells (Zn in brain tissue)2+The content of the active component reaches 0.1-0.5 mM). Zn2+Is an indispensable element for normal physiological development and metabolism of human bodies. About 90% Zn in living body2+Tightly combined with metalloproteins and various enzymes, playing an important role in physiological function regulation and cell construction, Zn2+Transport protein and metallothionein strictly control Zn in vivo2+Dynamic balancing of (2). A great deal of research shows that Zn in the life body2+The deficiency of (b) will lead to a series of diseases: zn2+Promoting the growth and the tissue regeneration of organisms and Zn in the bodies of male teenagers2+The absence will result in short stature and incomplete development of sex characteristics; furthermore Zn2+The zinc has important physiological effects on brain development and functions, zinc participates in the process of transmitting signals to a central nervous system, memory and thinking skills are increased, and the brain development and maturation lag is caused by zinc deficiency. Zn2+Metabolic disorders play a major role in the development and progression of many neurodegenerative diseases such as Alzheimer's Disease (AD), and the like. Zn2+Are associated with many diseases, but Zn is involved in2+The roles played by these physiological processes are not fully understood, and thus the understanding of Zn in the body of a living body is well understood2+The dynamic process and spatial-temporal distribution of (2) appear to be very urgent.
Disclosure of Invention
The invention discloses a Zn used for Zn for the first time2+A probe for proportional photoacoustic imaging. The pyridine dimethylamine is connected to a xanthene fluorophore through nucleophilic addition with aldehyde group and then through hydroboration reduction, and forms a four-coordination structure with ortho-hydroxyl to identify Zn2+Meanwhile, the probe contains positive charges and can target mitochondria. By confocal imaging, the mitochondrial targeting ability of the probe can be determined.
The specific technical scheme of the invention is as follows:
a xanthene derivative has the following structural formula:
said xanthene derivatives being characterised by being capable of binding anions, such as Cl-、Br-，I-，NO3 -Or PF4 -。
Another object of the present invention is to provide the xanthene derivatives of the present invention in Zn2+Application in photoacoustic imaging. Further, the xanthene derivative is proportional Zn2+Photoacoustic probe, useful for in vitro or in vivo Zn2+Proportional photoacoustic imaging can also be used for marking mitochondria of living cells.
The xanthene derivative can be applied to the fields of cell imaging, biomarkers, photoacoustic imaging and sensing.
The invention connects the pyridine dimethylamine with xanthene fluorophore through nucleophilic addition with aldehyde group and then through hydroboration reduction, and forms a four-coordination structure with ortho-position hydroxyl group to identify zinc ion, and meanwhile, the probe contains positive charge and can target mitochondria. By confocal imaging, the mitochondrial targeting ability of the probe can be determined. Explanation of probe pair Zn by in vitro and in vivo photoacoustic imaging experiments2+Has better photoacoustic imaging performance.
The synthetic route of the xanthene fluorescent probe is as follows:
specifically, after adding sodium triacetoxyborohydride to 2, 4-dihydroxybenzaldehyde and dimethylpyridine amine, carrying out nucleophilic addition with aldehyde groups, carrying out a secondary hydroboration reduction reaction to obtain a compound 1, and carrying out a retro-Knoevenagel reaction, a ring formation dehydration reaction on the compound 1 and a compound 2 or 3 to obtain the xanthene derivative.
Has the advantages that: the photoacoustic probe provided by the invention can be used for detecting Zn2+The small-molecule photoacoustic imaging probe is simple in preparation method.
Zn of the invention2+The photoacoustic probe is capable of being excited by visible light and has red light emission, thereby attenuating damage to the biological sample from the excitation light source and having a deep tissue penetration depth.
In vitro and in vivo photoacoustic imaging experiment tableThe probe pair of the invention is Zn2+Has better photoacoustic imaging performance, can be applied to biological imaging, mitochondrial marking and Zn2+The photoacoustic imaging has a simple chemical mechanism, good biocompatibility and good application prospect.
Drawings
FIG. 1 shows an emission spectrum of HD-Zn.
FIG. 2 shows an absorption spectrum of HD-Zn.
FIG. 3.HD-Zn2+Ultraviolet absorption intensity ratio at concentration (A)680/A750) Fitted curve with concentration.
Figure 4. variation of the ratio of the absorption intensity of HD-Zn for different metal ions.
Figure 5. variation of the ratio of HD-Zn absorption intensity for different pH.
FIG. 6.HD-Zn chelated Zn2+Is detected.
Figure 7 in vitro photoacoustic imaging experimental results of HD-Zn.
FIG. 8 shows the result of cytotoxicity test of HD-Zn.
Figure 9 shows the results of experiments on HD-Zn targeting to live cell mitochondria.
FIG. 10 shows in vivo photoacoustic imaging experimental results for HD-Zn.
FIG. 11 addition of 1eq Zn to HD-Zn2+ESI-MS graph of (A).
FIG. 12 shows different equivalent of Zn added to HD-Zn2+Nuclear magnetic titration of (c).
Detailed Description
The following is a detailed description of the embodiments of the present invention, which is implemented on the premise of the technical solution of the present invention, and detailed implementation manners and specific operation procedures are given, but the scope of the present invention is not limited to the following examples.
Example 1: preparation of Compound 1
2, 4-dihydroxybenzaldehyde (10mmol), dimethylpyridine amine (10mmol) and triacetoxyborohydrideSodium chloride (20mmol) was dissolved in 100mL Dichloroethane (DCE), reacted for 24 hours under Ar atmosphere in ice bath, and then 100mL of NaHCO was added3The reaction was quenched, extracted 3 times with 50mL portions of dichloromethane, the organic phase collected, Na2SO4Drying, spin-drying the solvent to obtain a yellow solid, performing column chromatography, and performing methanol: dichloromethane (CH)2Cl2) After the crude product is obtained by 50:1(v/v), CH is added2Cl2And methanol, the final white product compound 1, yield: 31 percent.1H NMR(400 MHz,DMSO-d6)δ10.38(s,1H),9.13(s,1H),8.51(ddd,J＝4.9,1.8,0.9Hz,2H),7.85–7.70(m, 2H),7.42(dt,J＝7.9,1.1Hz,2H),7.26(ddd,J＝7.6,4.8,1.1Hz,2H),6.94(d,J＝8.1Hz,1H), 6.21(d,J＝2.4Hz,1H),6.19-6.14(m,J＝8.1Hz,1H),3.75(s,4H),3.57(s,2H).13C NMR(101 MHz,DMSO-d6)δ159.00,158.27,158.21,149.17,137.28,131.23,123.15,122.70,114.42,106.30, 103.31,58.72,54.53,40.68,40.62,40.47,40.42,40.21,40.00,39.79,39.58,39.37。
Example 2: preparation of photoacoustic probe HD-Zn
In a dry round-bottom flask, compound 1(1mmol), K was added2CO3(1mmol) and anhydrous DMF (3ml) and the mixture was stirred at room temperature for 30min under nitrogen blanket and then starting compound 2(0.5 mmol dissolved in 5ml anhydrous DMF) was injected with syringe. Heating the reaction mixture to 70 deg.C under nitrogen protection for 3 hr, evaporating solvent under reduced pressure, and purifying the residue by silica gel column Chromatography (CH)2Cl2Methanol 50:1, v/v) gave the desired product HD-Zn in a bluish-green color with a yield of 52.6%.1H NMR(400MHz,Methanol-d4)δ8.61(d,J＝14.7Hz,1H),8.40(d, J＝4.8Hz,2H),7.67(td,J＝7.7,1.7Hz,2H),7.55(d,J＝7.3Hz,1H),7.48–7.28(m,7H),7.19 (dd,J＝7.4,5.0Hz,2H),6.78(s,1H),6.34(d,J＝14.7Hz,1H),4.20(t,J＝7.3Hz,2H),3.80(d,J ＝18.7Hz,6H),2.72-2.55(m,2H),1.83(t,J＝7.1Hz,4H),1.71(s,6H),0.98(t,J＝7.3Hz, 3H).13C NMR(101MHz,Methanol-d4)δ177.05,162.35,162.20,157.86,154.53,148.02,145.08, 141.82,141.77,137.48,137.42,134.82,129.55,128.74,126.59,126.18,123.24,122.57,122.37, 11453,114.25,112.24,102.49,102.42,58.38,55.08,50.34,48.25,48.11,48.04,47.90,47.82, 47.68,47.61,47.40,47.19,46.97,46.01,28.59,27.10,23.74,20.77,20.33,10.25 High Resolution Mass Spectrometry (HRMS): Calc.623.3381, found.623.3378.
Example 3: emission Spectrum test of HD-Zn
The concentration of the spectroscopic test used in the present invention was 5. mu.M, and the test solvent was HEPES solution (50mM,100 mM KNO) mixed with 1% DMSO3pH7.4), and the excitation wavelength is 633nm when the emission spectrum is measured. Zn2+The solution being ZnCl2Dissolving in water.
The emission spectrum of HD-Zn is shown in FIG. 1. When 1 equivalent of Zn is added2+Then, the fluorescence is enhanced by about 1/3 times, and the wavelength is blue-shifted; shows that the probe HD-Zn is used for Zn in the fluorescence spectrum2+The response of (a) is not significant.
Example 3: HD-Zn absorption spectrum test and detection limit calculation
The absorption spectrum used in the present invention was measured at a concentration of 5. mu.M in HEPES solution (50mM,50mM KNO) mixed with 1% DMSO as a test solvent3pH7.4) as shown in FIG. 2, when Zn is added2+The absorption at 680nm is gradually increased, the absorption at 750nm is gradually decreased, the ratio of the absorption at 680nm to the absorption at 750nm is plotted against the concentration, as shown in FIG. 3, it can be found that the absorption ratio has a better linear relationship with the concentration, and 1 equivalent of Zn is added2+The rear absorption ratio is enhanced by about 10 times. The wavelength of the current commercial photoacoustic detector is required to be between 680nm and 950nm, and the molar absorption coefficient is required to be large, and the molar absorption coefficient of the probe is 3.62 multiplied by 10 calculated by Labert's law A ∈ bc4L·mol-1·cm-1And the molar absorption coefficient increased to 5.88X 10 after adding 1 equivalent4L·mol-1·cm-1Therefore, the absorption wavelength of the probe meets the requirement of a commercial photoacoustic instrument and can be used for photoacoustic imaging. The detection limit can be calculated according to the formula 3s/slope, where s represents the background noise during detection and slope represents the slope of the titration curve, and the formula defines the concentration corresponding to a signal that exceeds background noise by a factor of 3 as the detection limit. According to 0-5. mu.M Zn2+Data on UV titration at concentrationThe slope of the line can be determined by linear fitting (FIG. 3), and the detection limit of the probe is calculated to be about 24.2nM, which indicates that the probe has a lower detection limit and high sensitivity.
Example 3: selectivity test for HD-Zn
To 3mL of HEPES buffer solution (50mM, pH 7.40, containing 1% DMSO, v/v) of a fluorescent probe (5. mu.M), 100. mu.M Na was added, respectively+、K+、Mg2+、Ca2+、10μM Fe2+、Fe3+、Cu2+、Cu+、Ba2+、Hg2+、Cr3+、Ni2+、Cd2+And Zn2+And (5) carrying out absorption spectrum test after uniformly mixing.
As can be seen from FIG. 4, the addition of other cations to the probe solution caused little change in the ratio of the absorption intensities of the solution when 10. mu.M Zn was added2+Ratio of absorption intensity of rear solution (A)680/A750) The change is obvious, and the enhancement is about 10 times. These experimental results show that the probe HD-Zn is coupled with Zn2+Has good selectivity and Zn resistance2+The proportional metering response capability is not disturbed by other cations.
Example 4: influence of pH on the Probe HD-Zn
1 equivalent of Zn was added to 3mL of the probe solution (5. mu.M) and the probe solution (5. mu.M)2+Adding a certain volume of HCl aqueous solution or NaOH aqueous solution into the HEPES solution, adjusting the solution to different pH values, and performing fluorescence spectrum test after the solution is stable.
As shown in FIG. 5, the ratio of the absorption intensity of the probe HD-Zn at physiological pH 4.5-8 remained substantially constant. The experiment shows that the probe has small change of the absorption intensity ratio in the physiological pH range and is suitable for Zn in a life system2+And (4) detecting and imaging.
Example 4: reversibility test of HD-Zn
The chelate fluorescent probe has the advantages that the reversible detection of metal ions can be realized, and the embodiment detects Zn for the probe HD-Zn2+Was investigated for reversibility. According to previous studies, when 5. mu.M Zn was added to a 5. mu.M probe solution2+Then, A680/A750It will be enhanced. As can be seen from FIG. 6, Zn was added to the resulting solution by means of chelate competition2+Chelating agent TPEN (5. mu.M), all of which can convert Zn2+Deprivation of blood, A680/A750Returning to near original level, adding 5 μ M Zn2+Then, A680/A750The probe can be enhanced and repeated for three times, and the probe has better reversibility. The experimental result proves that HD-Zn can be applied to Zn2+Reversible ratiometric assays were performed.
Example 5: in vitro photoacoustic imaging experiment of HD-Zn
Considering the high molar extinction coefficient of HD-Zn probe and the absorption spectrum for Zn2+According to the absorption spectrum, 680nm and 750nm lasers are selected to be used in HEPES buffer solution (50mM,100 mM KNO) of 5 MuM HD-Zn3Ph7.4, 2% DMSO) for photoacoustic signal acquisition. It was found that the probe solution had a strong photoacoustic signal at 750nm laser, while the signal was weak at 680 nm. With Zn2+The increase of the concentration of the photoacoustic signal PA680 begins to increase, the photoacoustic signal PA750 begins to decrease, and the variation trend of the photoacoustic signal is more consistent with the absorption spectrum result (FIG. 7).
Example 6: cytotoxicity assay of HD-Zn
The cells adopted by the invention are all human breast cancer cells (MCF-7 cells). The digested cells were seeded in 96-well plates at a density of 10 per well4One/well at 37 ℃ 5% CO2The culture was continued for 24 hours. After the removal of stale culture medium, cells were cultured for 12 hours in cell culture medium with different concentrations of HD-Zn (1, 5, 10, 20. mu.M). The culture was terminated by adding 10. mu.L of MTT (5mg/mL) per well and continuing the culture for 4 hours. The culture medium was aspirated, 150. mu.L of DMSO was added to each well, and OD570 was measured using a microplate reader after shaking for 10 minutes on a shaker.
MTT cytotoxicity test results are shown in FIG. 8, and when the concentration of the complex is 0-20 mu M, the cell survival rate after 12-hour culture is more than 80%, which proves that the probe has low cytotoxicity and can be used for cell imaging.
Example 7: experiment for co-staining experiment of HD-Zn and commercial mitochondrial dye Mito-Tracker Green on living cell mitochondria
The digested MCF-7 cells were seeded in petri dishes at 37 ℃ with 5% CO2The culture was continued for 24 hours under the conditions of (1) to allow adhesion. After washing the cells with PBS solution and incubating the cells with HD-Zn (5. mu.M) cell culture for 10 minutes, the cells were incubated with Mito-Tracker Green (500nM) cell culture for 30 minutes, washed three times with PBS solution and imaged.
FIG. 9 shows the cell co-staining images of HD-Zn and the commercial mitochondrial dye Mito-Tracker Green, (a) the co-staining image collected by the Mito-Tracker Green channel with an excitation wavelength of 488nm and an emission wavelength range of 490-540nm, (b) the co-staining image collected by the HD-Zn channel with an excitation wavelength of 633nm and an emission wavelength range of 640-750nm, and (c) the superimposed images of the images a and b; (d) MCF-7 cell brightfield; (e) graphs (a-c) fluorescence intensity of probes HD-Zn and Mito-Tracker Green on the white line; (f) correlation graph of probe HD-Zn and Mito-Tracker Green fluorescence intensity. The emitting region of mitochondrial dye Mito-Tracker Green and the emitting region of HD-Zn are overlapped, the contact ratio of the emitting region and the emitting region of HD-Zn is found to be high, and the co-staining coefficient can be calculated to be as high as 0.92, so that the HD-Zn disclosed by the invention can target living cell mitochondria and can be used for marking the living cell mitochondria.
Example 8: in vivo photoacoustic imaging experiment of HD-Zn
Photoacoustic experiments of living bodies were performed on nude mice, and photoacoustic signals were collected under two laser light sources of 680nm and 750 nm. Before injecting HD-Zn, the hind limb of the mouse is firstly subjected to photoacoustic imaging, and the photoacoustic signal of the subcutaneous part of the hind limb of the mouse is found to be very weak. Then 50. mu.L of 20. mu.M HD-Zn HEPES buffer (50mM,100 mM KNO) was injected into the left and right hind limbs, respectively3Ph7.4, 2% DMSO) signal acquisition, it was found that the mouse subcutaneous injection region had a strong photoacoustic signal PA680 at 680nm, while the photoacoustic signal PA750 at 750nm of the same region was weak, which is consistent with photoacoustic imaging in solution (fig. 10 a). The ratio PA680/PA750 of the photoacoustic signals of the left and right hind limbs is 1.85 and 2.36 respectively. Thereafter, 100. mu.L of ZnCl was injected into the right limb of the mouse2Aqueous solution (1mM) to the left of mouseThe hind limb was injected with the same volume of saline and signal acquisition was performed after 10 minutes. Both PA680 and PA750 signals were found to be reduced in the left hind limb, probably because the signal of the probe HD-Zn solution was weakened as a result of dilution of the subcutaneous solution or diffusion under the skin. It is noted that when the ratio of the signals measured in the area is plotted, it can be found that the ratio PA680/PA750 of the left hind limb is 2.07, the value is slightly increased compared with the value before injection, and the ratio PA680/PA750 of the photoacoustic signals of the right hind limb is 3.54 (FIG. 10b), and the experimental result shows that the HD-Zn can realize subcutaneous Zn2+The photoacoustic detection of (2). Compared with the intensity-variable photoacoustic imaging probe, the proportional metering photoacoustic imaging probe has the characteristic that the detection is not influenced by the probe concentration. HD-Zn thus achieves subcutaneous Zn in mice2+The ratio of (a) to (b) measures photoacoustic imaging detection.
Example 9: HD-Zn combined with Zn2+Mechanism of (2)
The invention combines HD-Zn and Zn2+The coordination of zinc ions with 3N and ortho-O of the pyridyldimethylamino is presumed in response to the absorption spectrum data and the mass spectrum data, and a mass spectrum peak with m/z of 343.33 appears in the mass spectrum (figure 11) and is assigned as [ HD-Zn + Zn ] & lt & gt]2+The molecular weight of (2) can also be found by NMR titration (FIG. 12) along with Zn2+The concentration is increased, and the proton of the pyridine dimethylamino group is shifted, thereby indicating Zn2+The coordination to N and the shift of the proton on the xanthene ring indicate that the O conjugated to it is also coordinated. Thus, with Zn2+The coordination structure is as shown in FIG. 11.
Claims (10)
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