CN110423487B - Rhodol derivative dye and application thereof - Google Patents
Rhodol derivative dye and application thereof Download PDFInfo
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- CN110423487B CN110423487B CN201910706458.1A CN201910706458A CN110423487B CN 110423487 B CN110423487 B CN 110423487B CN 201910706458 A CN201910706458 A CN 201910706458A CN 110423487 B CN110423487 B CN 110423487B
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- rhodol
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- C09B23/14—Styryl dyes
- C09B23/145—Styryl dyes the ethylene chain carrying an heterocyclic residue, e.g. heterocycle-CH=CH-C6H5
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Abstract
The invention relates to a Rhodol derivative dye and a photoacoustic imaging PA probe, in particular to preparation and application of the Rhodol derivative dye and the photoacoustic imaging PA probe. The new Rhodol derivative dye Rhodol-NIR designed and synthesized by the invention has a large molar extinction coefficient, excellent photostability and ideal quantum yield, and shows excellent photophysical properties when designing a high-contrast activated PA probe. The Rhodol-PA probe designed and synthesized by the invention is proved to show high sensitivity and high selectivity detection on hNQO1 through PA absorption and fluorescence measurement in vitro. The invention develops the high-contrast activatable PA probe by utilizing the spironolactone ring-opening conversion strategy for the first time at home and abroad. The switching strategy of the present invention is a universally applicable design that can provide a new platform for developing high contrast activatable PA probes.
Description
Technical Field
The invention relates to a Rhodol derivative dye and a photoacoustic imaging PA probe, in particular to preparation and application of the Rhodol derivative dye and the photoacoustic imaging PA probe.
Background
Photoacoustic (PA) imaging is a powerful biomedical imaging modality that enables non-invasive visualization of biological processes at the molecular and cellular level of deep tissue at high spatial resolution. With a Near Infrared (NIR) operating window, the PA can provide a penetration depth of a few centimeters with a resolution of about 100 μm. Because of its advantages, it provides a useful tool for clinical imaging of various diseases, including diagnosis of cancer, metastasis assessment, and therapy monitoring.
Molecular probes are essential in PA imaging because they can confer molecular or cellular specificity and enhance imaging contrast. PA probes generally rely on designs that allow for selective accumulation of photon absorbers in the diseased region through passive or active targeting strategies. Activated PA probes, which produce enhanced PA signaling upon interaction with specific molecular or cellular events, are very promising. These probes have the advantage of low background and high sensitivity, facilitating real-time imaging at higher depth and spatial resolution. Currently activated PA probes are designed based on an Intramolecular Charge Transfer (ICT) and contact quenching mechanism. These two mechanisms have been well documented for activating PA probes and have been applied to the exploration of several target molecules, such as metal ions, reactive oxygen species, nitric oxide, anaerobes, and enzymes. However, most probes do not have high imaging contrast because the targeted molecules induce small changes in the absorption spectrum shift or quantum yield. However, rational design of activated PA probes with excellent photophysical properties, such as large absorption spectral shifts, strong near-infrared absorption, low quantum yield and high light stability, which ultimately provide high contrast PA signals, remains difficult to achieve.
The common spirocyclic anthracene dye, Rhodol, has a large extinction coefficient, ideal light stability and unique open-loop response characteristic of an analyte, and after hydroxyl group acetylation, a spirolactone structure is formed and is in a closed-loop state, and the spirolactone structure is often used as an activated fluorescent reporter group, but the maximum absorption wavelength is 550nm, and the maximum fluorescent emission is 580nm, so that the application range of the spirocyclic anthracene dye is limited. And Rhodol has an absorption band only in the visible region and has a relatively large quantum yield, which is not suitable for in vivo PA imaging, and is even more unsuitable for PA/NIRF dual-mode imaging. Therefore, it becomes necessary to design a Rhodol variant with Near Infrared (NIR) absorption, lower quantum yield and spirolactone ring opening reaction similar to Rhodol.
Disclosure of Invention
In response to the deficiencies of the prior art, it is an object of the present invention to develop a novel dye with Near Infrared (NIR) absorption capable of activating a high signal-to-multiple PA response indicative of enzymatic activity.
The invention also aims to develop a novel high-contrast activated near-infrared PA probe by utilizing the unique spirolactone ring-opening reaction of the novel dye and determine the application of the probe in PA/NIRF dual-mode imaging of tumors with hNQO1 over-expression.
In order to solve the technical problems, the technical scheme of the invention is as follows:
a Rhodol derivative dye, characterized in that it produces a significantly enhanced absorption band in the NIR region from 620nm to 700 nm.
A Rhodol derivative dye, which is characterized in that the structural formula is shown as the formula (I):
wherein R1 is taken from H, F, Cl.
The preparation method of the dye comprises the following steps: cyclohexanone, concentrated H2SO4Mixing 4-diethylamino keto acid (1a) and perchloric acid, filtering, washing with water to obtain 9- (2-carboxyphenyl) -6- (diethylamino) -1,2,3, 4-tetrahydroxyalkyl (2a), and reacting with 3, 5-difluoro-4-hydroxybenzaldehyde or 3, 5-dichloro-4-hydroxybenzaldehyde or hydroxybenzaldehyde to obtain the dye.
Spirocyclic anthracene dyes, such as fluorescein and Rhodol, have a balance between the "open-ring" form and the "closed" spirolactone structural form of the pi-conjugated system. Furthermore, the "open-ring" form with a large pi-conjugated system exhibits a large red-shift of the absorption spectrum and an enhanced absorption band compared to the "closed" spirolactone structure. To our knowledge, this property of the spirocyclic anthracene dye has not been exploited for the design of activated PA probes.
When its phenolic hydroxyl group is blocked by esterification, the chromophore exists as a spirolactone, exhibiting negligible near infrared absorption due to the breaking of the conjugated system. The analyte allows the phenolic hydroxyl moiety to be released with a "ring opening" reaction, restoring its conjugated structure with strong NIR absorption, and producing a high contrast PA signal.
To design an activated PA probe using spirolactone ring-opening reactions, we selected a common spirocyclic anthracene dye, Rhodol, because of its large extinction coefficient, desirable photostability, and unique analyte response ring-opening characteristics. However, Rhodol has an absorption band only in the visible region and has a relatively large quantum yield, which is not suitable for in vivo PA imaging. Therefore, we began to design a Rhodol variant with NIR absorption, lower quantum yield and spirolactone ring opening reaction similar to Rhodol.
The present invention synthesizes novel Rhodol derivatives (Rhodol-NIR) by combining the structure of Rhodol core with difluoro-4-hydroxybenzaldehyde structure. The new Rhodol derivative not only has a large pi-conjugated system, but also the fluorine ion can finely adjust the protonation constant of the phenolic hydroxyl part, and the Rhodol derivative shows excellent photophysical properties when designing a high-contrast activated PA probe.
Compared with the existing Rhodol structure, the conjugated system of the dye structure is prolonged by coupling p-hydroxybenzaldehyde and derivatives, so that the dye structure has near infrared absorption and emission.
The invention also provides application of the dye, and the design of the photoacoustic imaging PA probe is realized by utilizing the spirolactone closed-open loop regulation and control of the dye.
The invention also provides a photoacoustic imaging PA probe, which has a structural formula shown as a formula (II):
wherein R is2Is selected from dimethyl methylene quinone propionate, phosphate radical, galactosyl radical, trifluoro methane sulfonate radical and phenol radical.
The invention also provides a preparation method of the photoacoustic imaging PA probe, which comprises the step of reacting Rhodol derivative dye with trimethoprim quinonic acid, phosphoric acid, galactoside, trifluoromethanesulfonate and p-bromophenol to prepare the corresponding probe.
The probe prepared by the reaction of the Rhodol derivative dye and the trimethoprim quinoprotein can be used for detecting the over-expressed biomarker enzyme hydroquinone reductase in tumor cells;
the probe prepared by the reaction of the Rhodol derivative dye and phosphoric acid can be used for detecting over-expressed biomarker enzyme alkaline phosphatase in tumor cells;
the probe prepared by the reaction of the Rhodol derivative dye and galactoside can be used for detecting the over-expressed biomarker enzyme galactosidase in tumor cells;
the probe prepared by the reaction of the Rhodol derivative dye and trifluoromethanesulfonate can be used for detecting superoxide anions in inflammation;
the probe prepared by the reaction of the Rhodol derivative dye and p-bromophenol can be used for detecting hydroxyl radicals in inflammation.
Preferably, the structural formula of the photoacoustic imaging PA probe is shown as the formula (III):
the preparation method of the photoacoustic imaging PA probe is characterized by comprising the steps of synthesizing trimethyl-locked quinonic acid, and mixing the Rhodol derivative dye with the synthesized trimethyl-locked quinonic acid for self-assembly.
The preparation method of the photoacoustic imaging PA probe comprises the following steps:
A. synthesis of trimethyl locked quinone propionic acid: mixing 3, 3-dimethacrylate, 2,3, 5-trimethyl-1, 4-benzene diol and methanesulfonic acid, extracting, washing, vacuum concentrating and recrystallizing to obtain 6-hydroxy-3, 3,5,7, 8-pentamethyl cyclic lactone-2-ketone, adding N-bromosuccinimide into the solution of the compound 1, stirring, removing the solvent, extracting, vacuum concentrating and purifying to obtain the 3-methyl-3- (2,4, 5-trimethyl-3, 6-dioxan-1, 4-diene-1-yl) butyric acid.
B. Preparation of PA Probe: the photoacoustic imaging PA probe is obtained by mixing and reacting Rhodol derivative dye, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, 3-methyl-3- (2,4, 5-trimethyl-3, 6-dioxan-1, 4-diene-1-yl) butyric acid and 4-dimethylaminopyridine, and then washing, vacuum concentrating and purifying.
The invention also provides application of the photoacoustic imaging PA probe in high-contrast PA/NIRF dual-mode imaging of living cells and animals.
We chose human quinone oxidoreductase (hNQO1), a biomarker enzyme that is overexpressed in tumor cells, as the case for the study. PA probes for hNQO1 detection by use of a trimethlocked quinopropanoic acid moiety (Q)3) Blocking hydroxyl groups. Esterification of hydroxyl groups to promote PA probingThe needles form a "closed" spirolactone structure, exhibiting negligible absorption in the visible-near infrared region. When hNQO1 catalyzes the reduction and elimination of Q in PA probe3In part, the resulting Rhodol-NIR dye undergoes a spontaneous ring opening process and reverts to its large pi-conjugated system. Thus, this elimination reaction can activate a high signal-to-noise ratio of the PA signal and indicate enzymatic activity.
Besides the PA response, the Rhodol-NIR dye also provides ideal fluorescence signals, enabling dual mode detection of PA/NIRF. Furthermore, we demonstrate that PA probes have a very low NIR absorption background and high specificity for hNQO1, while the resulting Rhodol-NIR dye after the enzymatic reaction shows a large molar extinction coefficient, excellent photostability and ideal quantum yield. These excellent photophysical properties make the PA probe useful for high contrast PA/NIRF dual-mode imaging of living cells and animals. The probe can also determine the hNQO1 differential expression of living cells and tumors, and provides great diagnosis potential for diseases related to hNQO1 overexpression. To our knowledge, this was the first activatable near-infrared PA probe developed based on the spirolactone ring-opening reaction. Our approach is expected to provide a new platform for the study of high contrast imaging activated PA probes.
Compared with the prior art, the invention has the following beneficial effects:
1. the novel Rhodol derivative dye Rhodol-NIR designed and synthesized by the invention not only has a large pi-conjugated system, but also fluorine ions can finely adjust the protonation constant of a phenolic hydroxyl part, so that a novel Rhodol-NIR chromophore with a large molar extinction coefficient, excellent light stability and ideal quantum yield is developed, and excellent photophysical properties are shown when a high-contrast activated PA probe is designed.
2. The invention designs a synthesized high-contrast activatable PA probe based on a unique analyte-induced spirolactone ring-opening strategy of a spirocyclic anthracene dye. The probe does not exhibit NIR absorption due to its "closed" spirolactone structure, and its reaction with hNQO1 generates a Rhodol-NIR chromophore through spirolactone ring opening transition, allowing high contrast PA/NIRF dual mode detection and hNQO1 imaging.
3. The synthesized Rhodol-PA probe designed by the invention is proved to be high-sensitivity and high-selectivity detection on hNQO1 through PA and fluorescence measurement in vitro.
4. The invention develops the high-contrast activatable PA probe by utilizing the spironolactone ring-opening conversion strategy for the first time at home and abroad. The switching strategy of the present invention is a universally applicable design that can provide a new platform for developing high contrast activatable PA probes.
Drawings
FIG. 1 is the pKa value of Rhodol-NIR according to the invention;
FIG. 2 is a graph of the photostability operation of Rhodol-NIR according to the invention;
FIG. 3 is a graph of the in vitro response spectra of Rhodol-PA and control probes of the invention to hNQO 1;
FIG. 4 is a high performance liquid chromatogram of a reaction mixture of Rhodol-PA, Rhodol-NIR, Rhodol-PA and hNQO1 according to the invention;
FIG. 5 is a high resolution mass spectrum of the reaction mixture of Rhodol-PA and hNQO1 according to the present invention;
FIG. 6 is a diagram of the fluorescence spectrum of hNQO1 detected in vitro by the Rhodol-PA probe of the present invention;
FIG. 7 is a graph of the UV absorption spectrum of an in vitro interference test with hNQO1 using Rhodol-PA probe according to the present invention;
FIG. 8 is a graph of the PA response of the Rhodol-PA probe of the present invention to various concentrations of hNQO 1;
FIG. 9 is a graph of the photoacoustic signal intensity at 680nm of a Rhodol-PA probe of the present invention fitted with a linear working curve of hNQO1 at various concentrations;
FIG. 10 is a multi-spectral optical tomography (MSOT) imaging system of hNQO1 with Rhodol-PA probe explored on two cell lines HT-29 cells and MDA-MB-231 cells;
FIG. 11 is a graph showing the results of the cytotoxicity of Rhodol-PA against HT-29 and MDA-MB-231 cells according to the present invention;
FIG. 12 is a graph of MSOT and fluorescence images from HT-29 and MDA-MB-231 tumor mice administered by caudal intravenous injection of a Rhodol-PA probe in vivo;
FIG. 13 is a graph of MSOT and fluorescence images of experimental control tail vein administration HT-29 and MDA-MB-231 tumor mice in vivo.
Detailed Description
The present invention will be described in detail with reference to examples. It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.
Example 1
Design and synthesis of Rhodol derivative dye- -Rhodol-NIR.
Freshly distilled cyclohexanone (3.3mL, 31.9mmol) was added dropwise to concentrated H2SO4(35mL) and the mixture was cooled to 0 ℃. Then, 4-diethylaminoketoacid (16mmol) was added in portions with stirring. After 1.5 hours of reaction at 90 ℃, the mixture was poured into ice (150 g). Perchloric acid (70%, 3.5mL) was then added and the resulting precipitate was filtered and washed with cold water (50mL) to give 9- (2-carboxyphenyl) -6- (diethylamino) -1,2,3, 4-tetrahydroxyalkyl (2a), which was spun through a silica gel column to give Rhodol-NIR as a blue solid (89.2mg, 29% yield) using 9- (2-carboxyphenyl) -6- (diethylamino) -1,2,3, 4-tetrahydroxyalkyl (237.6mg, 0.5mmol) and 3, 5-difluoro-4-hydroxybenzaldehyde (118.5mg, 0.75mmol) at 110 ℃ under reflux in acetic acid:
the Rhodol-NIR structural formula is shown as formula (IV):
in preliminary study, the extension reaction of the pi-conjugated system is found to be not only easy to synthesize, but also have an ideal non-rigid structure with low quantum yield, which is beneficial to enhancing PA signals.
Example 2
Photophysical Properties of Rhodol-NIR
The method comprises the following operation steps:
to determine the pKa value of Rhodol-NIR, the pH was adjusted with NaOH and hydrochloric acid, phosphate buffer solutions containing 0.5% DMSO as a cosolvent and having different pH values were prepared, Rhodol-NIR (15. mu.M) was mixed with different buffer systems, the absorption spectra of Rhodol-NIR at different pH buffer systems were determined with a UV-1800 spectrophotometer and the fluorescence spectra of Rhodol-NIR at different pH buffer systems were determined with an FS5 fluorometer, the excitation wavelength being 620 nm. The pH curve was then plotted using the absorbance at 650nm and the fluorescence intensity at 720 nm. The pKa of the compound was calculated according to the henderson-hasselbach equation, and the specific results are shown in fig. 1.
Rhodol-NIR has a maximum absorption value of 630nm, but it produces a significantly enhanced absorption band in the broad NIR region from 620nm to 700 nm. As can be seen from FIG. 1, the pKa value of Rhodol-NIR is 6.1, demonstrating our design that the introduction of electron-withdrawing substituents facilitates the reduction of pKa and enhances NIR absorption by Rhodol derivatives. In summary, the results show that Rhodol-NIR provides an ideal chromophore for the development of activatable PA probes.
We found that Rhodol-NIR shows a large molar extinction coefficient at 630nm (. epsilon. times.2.67X 10)5M-1·cm-1) This compares favorably with existing small molecule chromophores used for PA imaging.
The quantum yield of Rhodol-NIR was determined in PBS (ph7.4) with reference to cresyl violet (Φ s in MeOH ═ 0.54) and calculated according to equation 1:
ΦX＝ΦS(ASFX/AXFS)(nX/n)2 (1)
phi: quantum yield; a: absorbance at the excitation wavelength; f: the integrated area of the fluorescence spectrum under the same excitation wavelength; n: the refractive index of the solvent; s and X represent a reference standard sample and an unknown sample, respectively.
Quantum yield PhiPhiO of Rhodol-NIR in pH7.4 phosphoric acid buffer solutionFThe calculation was 1.0%. This quantum yield indicates that Rhodol-NIR has high efficiency in the photo-thermal conversion of PA imaging given that it does not produce other competing photo-conversion pathways with heavy atom substitution. This quantum yield can also be used for the study of fluorescence imaging and has suitable sensitivityAnd (4) sensitivity.
Rhodol-NIR also has good photostability, as shown in FIG. 2.
The method comprises the following specific steps: to determine the photostability of Rhodol-NIR, samples of prepared Rhodol-NIR (15. mu.M) were placed on a microscope and irradiated with a mercury lamp (100W). Samples were removed every approximately 5min and the fluorescence spectra were measured using an FS5 fluorimeter. For comparison, the photostability of indocyanine green (ICG) under the same conditions was also investigated. Working curves were made with the maximum fluorescence intensity values of Rhodol-NIR and ICG as a function of time.
The results are shown in fig. 2 and show a negligible decrease in fluorescence after 70min of continuous irradiation, whereas the commercial NIR dye indocyanine green (ICG) decreased in fluorescence by 90%. The high molar extinction coefficient in the NIR region, desirably low quantum yield and high light stability indicate that Rhodol-NIR provides an ideal contrast agent for PA imaging.
Example 3
Rhodol-NIR-based PA probe design and synthesis
The specific synthetic process is as follows:
B. synthesis of trimethyl locked quinone propionic acid: 3, 3-dimethacrylate (1.60mL, 12mmol) was added to a mixture of 2,3, 5-trimethyl-1, 4-benzenediol (1.52g, 10mmol) and methanesulfonic acid (15mL) and the mixture was stirred at 70 ℃ for 2 hours. After cooling to room temperature, the reaction mixture was diluted to 150mL with water and extracted three times with 70mL dichloromethane. The extract is extracted with saturated NaHCO3Washing with NaCl solution and anhydrous Na2SO4Dried and concentrated in vacuo. With 30% CHCl3Recrystallization from petroleum ether gave 6-hydroxy-3, 3,5,7, 8-pentamethylcyclolacton-2-one as a white solid (1.85g, 79.2% yield).
To a solution of 6-hydroxy-3, 3,5,7, 8-pentamethylcyclolacton-2-one (1.17g, 5mmol) in acetonitrile (60mL) and water (25mL) was added NBS (0.98g, 5.5 mmol). The reaction mixture was then stirred at room temperature for 1 hour. After most of the organic solvent was removed, the residue was extracted three times with 30mL of dichloromethane. With anhydrous Na2SO4The organic layer was dried and concentrated under vacuum. Using PE/EtOAc 2:1 (v/v) as eluent, the crude product was purified by column chromatography to yield trimethyi locked quinopropanoic acid (0.92g, 73.5% yield).
C. Preparation of PA Probe: Rhodol-NIR (234.0mg, 0.38mmol) was slowly added to a solution of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (72.8mg, 0.38mmol), trimethlocked quinovonic acid (167.3mg, 0.58mmol) at 0 ℃ in CH2Cl2To (30mL) was added 4-dimethylaminopyridine (46.4mg, 0.38 mmol). After 12 hours of reaction, the mixture was washed with HCl (1M). With MgSO4The organic layer was dried, filtered and concentrated in vacuo. Purifying the residue by silica gel column chromatography using CH2Cl2MeOH (100:1 to 20:1) as eluent, affording Rhodol-PA probe as a purple solid (164.7mg, 58% yield).
The specific route is as follows:
example 4
The response performance of the Rhodol-PA probe.
The operation steps are as follows: to test the in vitro response of Rhodol-PA to hNQO1, a 15 μ M solution of Rhodol-PA containing 0.5% (v/v) dimethyl sulfoxide and 100 μ M NADH was first prepared in PBS buffer (10mM, pH7.4), then Rhodol-PA was incubated with hNQO1(3.0 μ g/mL) at 37 ℃ for 1.5h and the UV absorption spectrum was determined using a UV-1800 spectrophotometer.
As expected, the resulting Rhodol-PA probe showed no absorption band in the NIR region (FIG. 3). The disappearance of the NIR absorption means that the Rhodol-PA probe is predominantly present in the form of a "closed-loop" spirolactone structure with a broken pi-conjugated structure.
After incubation of the probe with hNQO1 for 90min, we observed a sharp increase in NIR absorption band at 630nm, indicating the generation of Rhodol-NIR (fig. 3). This finding suggests a two-step mechanism for hNQO1 catalyzed reactions, when blocking group Q in Rhodol-PA3After reduction-elimination, the spironolactone ring-opening reaction proceeds spontaneously. DiscoveryRhodol-NIR is able to respond specifically to hNQO1 and has a large red shift (Δ λ > 230nm) and a high absorption signal-to-magnification ratio (at 630 nm)>57 times at 680nm>110 times). This ideal performance is attributed to our spirolactone ring-opening design, where the probe exhibits a "closed-ring" spirolactone structure with a broken pi-conjugated system, while the product has an extended "open-ring" form of a large pi-conjugated system.
Example 5
Verification of necessity of design of spiro ring open loop
We synthesized the control chromophore using the Rhodol variant without the spiro moiety. Also by using hNQO1 recognition of substrate Q3Control probes were prepared by esterifying control projectiles.
The preparation process of the control probe comprises the following steps: the control chromophore (234.0mg, 0.38mmol) was added slowly to a solution of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (72.8mg, 0.38mmol), 3-methyl-3- (2,4, 5-trimethyl-3, 6-dioxan-1, 4-dien-1-yl) butyric acid (167.3mg, 0.58mmol) at 0 ℃ in CH2Cl2To (30mL) was added 4-dimethylaminopyridine (46.4mg, 0.38 mmol). After 12 hours of reaction, the mixture was washed with HCl (1M). With MgSO4The organic layer was dried, filtered and concentrated in vacuo. Purifying the residue by silica gel column chromatography using CH2Cl2MeOH (100:1 to 20:1) as eluent, obtained control probe as a purple solid (164.7mg, 58% yield).
The specific route is as follows:
wherein, the Control probe is a Control probe. Similarly, the present invention tested the in vitro response of Control probes to hNQO1 by first preparing a 15 μ M solution of Control probe containing 0.5% (v/v) dimethyl sulfoxide and 100 μ M NADH in PBS buffer (10mM, pH7.4), mixing with hNQO1(3.0 μ g/mL), reacting at 37 ℃ for 1.5h, and measuring the UV absorption spectrum with a UV-1800 spectrophotometer.
Surprisingly, the control probe did exhibit a significant vis-NIR absorption band (FIG. 3). Notably, the control probe had the same parent structure as the Rhodol-PA probe. Thus, the different absorption spectra of the control probe and Rhodol-PA provide direct evidence for a broken pi-conjugated system in Rhodol-PA, validating the "closed-loop" spirolactone structure in Rhodol-PA.
Furthermore, as shown in FIG. 3, a slight increase in the absorbance peak occurred after reaction of the control probe with hNQO1, with only a 3-fold increase in the maximum value. This slight increase in absorption is due to the enhanced intramolecular charge transfer effect due to the formation of free phenolic hydroxyl groups in the enzymatic reaction. Based on these results, we concluded that the high contrast response of Rhodol-PA is due to the spirolactone ring-opening mechanism, validating the potential of this new strategy to develop high contrast activatable PA probes.
Example 6
Reaction mechanism of Rhodol-PA probe
As shown in the above formula, when the closed-loop form of the probe Rhodol-PA is incubated with hNQO1, hNQO1 reacts with the recognition site to release the open-loop form of the dye Rhodol-NIR, resulting in photoacoustic and fluorescent signals.
To verify the reaction mechanism of Rhodol-PA probe, first, high performance liquid chromatography analysis was performed on Rhodol-PA, Rhodol-NIR and reaction products of Rhodol-PA and hNQO 1. With methanol/water (0.2% CF)3COOH (90/10 (v/v), flow rate 1ml/min, C18 column (250 nm. times.4.6 mm, 5 μm) with detection wavelength of 550nm as chromatographic conditions, and in order to further verify the reaction mechanism, the reaction product of Rhodol-PA and hNQO1 was analyzed using high resolution mass spectrometry. Incubation with hNQO1(1.5 μ g/ml) was performed in PBS buffer (10mM, pH7.4) containing 0.5% (v/v) dimethyl sulfoxide and 100 μ M NADH at 37 ℃ for 1.5 hours. Then 1mL of MeCN was added dropwise to the reaction mixture and centrifuged at 10000 rpm for 15 minutes. Taking supernatant to perform mass spectrometry in a positive ion mode.
For theThe product from the reaction between probe and hNQO1 obtained two elution peaks at 8.8min and 5.0min, respectively, corresponding to the Rhodol-NIR chromophore and unreacted probe (fig. 4). In addition, the reaction solution showed two new peaks, one corresponding to Rhodol-NIR (calculation of [ M + H ]]+M/z 516.1981, found 516.1987), and another as a reduction by-product (calculated as [ M + H ]]+) m/z 235.1289, found 235.0541) as shown in fig. 5. This result suggests that the Rhodol-PA probe was converted to Rhodol-NIR after reaction with hNQO 1.
Example 7
In vitro Performance of PA probes on hNQO1
Using the synthesized Rhodol-PA probe, we subsequently investigated its performance as a probe for detecting hNQO1 in vitro.
The operation steps are as follows: to test the in vitro response performance of Rhodol-PA to hNQO1, a 15 μ M solution of Rhodol-PA was first prepared with PBS buffer (10mM, pH7.4) containing 0.5% (v/v) dimethyl sulfoxide and 100 μ M NADH, and then Rhodol-PA was reacted with hNQO1(3.0 μ g/mL) at 37 ℃ for 1.5h, and photoacoustic spectra signals were collected using inVision256-TF multi-wavelength photoacoustic imaging system (MSOT), respectively, and fluorescence spectra were measured using FS5 fluorometer.
As expected, the Rhodol-PA probe showed very low PA signal in the 680nm to 800nm range (fig. 6) and after reaction of the probe with hNQO1, a strong PA response signal was obtained with a-11 fold ratio at 680 nm. Similarly, the Rhodol-PA probe has only very low fluorescence, while its fluorescence signal is significantly enhanced after reaction with hNQO1, with a-59 fold signal-to-fold ratio at 720 nm. The results suggest the potential for high contrast PA/NIRF dual mode imaging of Rhodol-PA to hNQO 1.
To test their specificity, control experiments were performed in which the probes incubated with various biologically relevant species did not respond to absorption (FIG. 7) demonstrating the high selectivity of Rhodol-PA probes for hNQO 1.
Further evidence was obtained from the assay of two lysates of HT-29 cells, which are known to overexpress hNQO 1. After incubation of the probes with HT-29 cell lysates, we obtained uptake signals (FIG. 7). In contrast, negligible uptake signal was observed in the assay of HT-29 cell lysates pretreated with excess dicoumarin, a specific hnqoo 1 inhibitor. In addition, lysates of MDA-MB-231 cells were assayed with a probe, which reportedly had very low hNQO1 expression, showing a very small response at UV absorption (FIG. 7). Taken together, these results clearly demonstrate the high specificity of the Rhodol-PA probe for hNQO 1.
Example 8
Determination of the quantitative Capacity of the Rhodol-PA Probe
The test method specifically comprises the following steps: in order to detect the quantitative capability of Rhodol-PA on hNQO1, 15 mu M Rhodol-PA was cultured with hNQO1(0-3.0 mu g/mL) of different concentrations for reaction at 37 ℃ for 1.5h, respectively, an INVision256-TF multi-wavelength photoacoustic imaging system was used to collect photoacoustic signals, and then a linear working curve was fitted with the photoacoustic signal intensity at 680nm and hNQO1 of different concentrations.
The PA signal at 680nm was found to exhibit a dynamic response to hNQO1 (FIG. 8) response signal ratio of 11 over the concentration range of 0.25-3.0 μ g/mL, indicating high contrast in the PA assay of hNQO 1. A linear correlation was obtained in the range of 0.25 to 1.1. mu.g/mL, with an estimated limit of detection (LOD) of 0.08. mu.g/mL (FIG. 9).
Example 9
The ability of Rhodol-PA probes to image hNQO1 in living cells
Driven by the ideal in vitro results, we subsequently explored the ability of the Rhodol-PA probe to image hNQO1 on PA/NIRF in living cells using two cell lines HT-29 cells and MDA-MB-231 cells.
The method comprises the following specific operation steps: HT-29 or MDA-MB-231 cells (about 7X 10)6The cells were placed at 75cm2In cell culture flasks) was incubated with Rhodol-PA (30. mu.M) at 37 ℃ for 2h, washed 3 times with 10mL PBS, then cells were collected by 0.25% trypsin digestion and TC20TMAn automated cell counter counts. The cell suspension was collected by centrifugation of a microtube (200. mu.L). The cuvette with the cell pellet was inserted into the MSOT imaging system holder and imaged with the MSOT imaging system. For inhibitor studies, cells were pretreated with dicoumarol (100 μ M) inhibitor for HT-29 fine cells before incubation with Rhodol-PA (30 μ M)Cell 1h, the subsequent steps were the same as without the addition of dicoumarin inhibitor.
Cells were centrifuged (7X 10)6) To obtain cell pellet, imaged with a multi-spectral optical tomography (MSOT) imaging system (fig. 10). As expected, HT-29 cells incubated with Rhodol-PA probe had a significant increase in PA signal at 680nm (FIG. 10) when cells were pretreated with hNQO1 inhibitor dicoumarin and then incubated with probe, much lower PA signal was observed. Furthermore, MDA-MB-231 cells incubated with the probe showed negligible PA signal. These results are consistent with literature reports that HT-29 cells overexpress hNQO1 levels and MDA-MB-231 cells have very low levels of hNQO 1. This result indicates that Rhodol-PA probes provide a specific differentiated active molecular tool, as well as the detection of hNQO1 activity in living cells using MSOT imaging.
Furthermore, we performed an experimental cytotoxicity assay of Rhodol-PA on HT-29 and MDA-MB-231 cells.
Cytotoxicity experimental procedure: the cytotoxicity of Rhodol-PA on HT-29 and MDA-MB-231 cells was investigated using the standard MTT method. HT-29 and MDA-MB-231 cells were seeded at 5X 10, respectively3Cells/well in 96-well plates, medium was 100. mu.L. The cells were incubated at 37 ℃ for 24 hours, then incubated with fresh medium containing different concentrations of Rhodol-PA (0-300. mu.M) for 12 hours, then the medium was removed and the cells were washed 3 times with cold PBS. Cells were incubated with MTT reagent (10. mu.L, 5mg/ml) for 4h at 37 ℃. DMSO (100. mu.L/well) was then added for 10min to dissolve the precipitated crystals of methylene chloride. Using ELX800TMThe microplate reader measures the absorbance at 570 nm.
Furthermore, cytotoxicity assay showed that concentrations of the Rhodol-PA probe above 300. mu.M had little effect (> 90%) on the cell viability of both cell lines HT-29 and MDA-MB-231 (FIG. 11), indicating the potential of using this low-cytotoxicity Rhodol-PA probe for biological applications.
Example 10
Tail vein administration demonstrated the ability of Rhodol-PA probe to detect hNOQ1 activity in a live animal by PA
The specific test steps are as follows: for MSOT imaging, 200. mu.L of Rhodol-PA (300. mu.M) was injected via the tail vein into HT-29 tumor or MDA-MB-231 tumor mice. Control groups were injected with sterile PBS (200. mu.L). First, mice were anesthetized in an anesthesia box containing 1% isoflurane-oxygen atmosphere. Then, the ultrasound gel was applied to the tumor site of the mouse, and the mouse was wrapped with a polyethylene film to allow coupling between the tissue and the aqueous medium. The photoacoustic signal was collected every 10nm at different wavelengths, placed in a 34 ℃ water bath ranging from 680nm to 800nm, and using a photoacoustic intensity of 850nm as background. Data were collected every 0.3 mm for tumor sites. MSOT images were collected before (0 min) and at different time points (0,0.5 h, 1h,3h,5h,7h and 12h) after injection for Rhodol-PA. And (3) carrying out data analysis on the acquired signals by utilizing an independent component spectrum analysis technology of the photoacoustic instrument, and separating the signals of the responded probe from the signals of other background light absorbers (such as hemoglobin) in the tissue to obtain a photoacoustic imaging graph. And calculating the average photoacoustic signal intensity of different parts of the tumor at the same time point, and deducting the average photoacoustic signal intensity of different parts of the tumor before injection to obtain the enhanced photoacoustic signal intensity.
In this study, HT-29 and MDA-MB-231 tumor mice were dosed via tail injection with Rhodol-PA probe (2.56mg/kg) or PBS, respectively. MSOT and fluorescence imaging images were obtained at different intervals (0,0.5,1,3,5, 7,12h) after injection. We observed a clear PA signal 1 hour after probe injection and the strongest signal was obtained 5 hours after injection in the tumor region of HT-29 cell xenograft mice (fig. 12a 1). The PA signal after injection of the PBS control showed no significant increase throughout the experiment, demonstrating that activation of the PA signal is from the response of the probe in the tumor area (fig. 13). After injection of the probe, no significant increase in PA signal was found in the tumor region of MDA-MB-231 cell xenografted mice (FIG. 12a 1). The fluorescence image showed similar responses in the tumor region of HT-29 and MDA-MB-231 cell xenografted mice (FIG. 12a 1). Data collected from six HT-29 tumor mice in duplicate assays showed that the mean PA signal in the tumor region showed a rapid increase 1 hour after injection and reached a maximum at 5 hours and then gradually decreased to 12 hours (fig. 12a 2). In contrast, mice implanted with MDA-MB-231 tumors showed only a slight increase in PA signal in the tumor area after injection. The mean signal ratio of HT-29 tumors to MDA-MB-231 tumors was about 7-fold, confirming that high contrast PA imaging of hNQO1 overexpressing tumors was achieved using the Rhodol-PA probe. Furthermore, we found that the in vivo PA spectra of the tumor region of HT-29 tumor-implanted mice obtained 5 hours after injection showed a maximal PA signal at 680nm with a spectral distribution similar to the in vitro spectrum (fig. 12a 3). This result further confirms that PA signaling in HT-29 tumors is due to specific activation of the Rhodol-PA probe mediated by hNQO1 overexpressed in HT-29 cells. The in vivo PA spectra from mice bearing MDA-MB-231 tumors also showed similar but minor features, indicating low expression of hNQO1 in these cells.
In summary, live mouse imaging results indicate that our probes can be selectively activated in tumors upregulated by hNQO1 in live animals, enabling PA/NIRF dual-mode imaging of hNQO1 overexpressed tumors, which means their potential for in vivo tumor diagnosis and therapy assessment.
The foregoing examples are set forth to illustrate the present invention more clearly and are not to be construed as limiting the scope of the invention, which is defined in the appended claims to which the invention pertains, as modified in all equivalent forms, by those skilled in the art after reading the present invention.
Claims (7)
1. A Rhodol derivative dye, wherein the dye produces an absorption band in the NIR region of 620nm to 700 nm; the structural formula of the dye is shown as the formula (I):
wherein R1 is F.
2. A process for the preparation of the dye according to claim 1, comprising the steps of: cyclohexanone, concentrated H2SO42-hydroxy groupMixing the 4-diethylamino-2' -carboxyl benzophenone and perchloric acid, filtering and washing to obtain 9- (2-carboxyphenyl) -6- (diethylamino) -1,2,3, 4-tetrahydroanthracene base, and then reacting with 3, 5-difluoro-4-hydroxybenzaldehyde or 3, 5-dichloro-4-hydroxybenzaldehyde to obtain the dye.
3. Use of spirolactone ring-opening closed-loop control design of a dye as claimed in claim 1, which dye generates an absorption band in the NIR region of 620 to 700nm, for the synthesis of photoacoustic imaging PA probes.
4. A photoacoustic imaging PA probe is characterized in that the structural formula is shown as a formula (III):
5. a method for preparing the photoacoustic imaging PA probe according to claim 4, wherein the method comprises synthesizing 3-methyl-3- (2,4, 5-trimethyl-3, 6-dioxan-1, 4-dien-1-yl) butyric acid, and mixing the Rhodol derivative dye according to claim 1 with the synthesized 3-methyl-3- (2,4, 5-trimethyl-3, 6-dioxan-1, 4-dien-1-yl) butyric acid.
6. The method for preparing a photoacoustic imaging PA probe according to claim 5, comprising the steps of:
A. synthesis of 3-methyl-3- (2,4, 5-trimethyl-3, 6-dioxa-1, 4-dien-1-yl) butanoic acid: mixing 3, 3-dimethacrylate, 2,3, 5-trimethyl-1, 4-benzenediol and methanesulfonic acid, extracting, washing, vacuum concentrating and recrystallizing to obtain 6-hydroxy-4, 4,5,7, 8-pentamethyl chroman-2-one, adding N-bromosuccinimide into acetonitrile and aqueous solution of the 6-hydroxy-4, 4,5,7, 8-pentamethyl chroman-2-one, stirring, removing a solvent, extracting, vacuum concentrating and purifying to obtain 3-methyl-3- (2,4, 5-trimethyl-3, 6-dioxan-1, 4-diene-1-yl) butyric acid;
B. preparation of PA Probe: mixing the Rhodol derivative dye, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, 3-methyl-3- (2,4, 5-trimethyl-3, 6-dioxa-1, 4-dien-1-yl) butyric acid and 4-dimethylaminopyridine for reaction, washing, vacuum concentrating and purifying to obtain the photoacoustic imaging PA probe.
7. Use of the photoacoustic imaging PA probe of claim 4 for high contrast PA/NIRF dual mode imaging of living cells and animals for non-diagnostic purposes.
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