CN110423487A - A kind of Rhodol derivative dye and its application - Google Patents

Abstract

The present invention relates to a kind of Rhodol derivative dye and a kind of photoacoustic imaging PA probe more particularly to Rhodol derivative dye and a kind of preparations and its application of photoacoustic imaging PA probe.The present invention designs the new Rhodol derivative dye Rhodol-NIR of synthesis, with big molar extinction coefficient, the novel Rhodol-NIR chromophore of excellent light stability and ideal quantum yield, shows excellent photophysical property when designing high contrast activation type PA probe.The Rhodol-PA probe that the present invention designs synthesis is proved to absorb by PA in vitro and fluorescence measurement is shown to the high sensitivity of hNQO1 and highly selective detection.The present invention is domestic and international first time to develop high contrast using spirolactone open loop switching strategy to activate PA probe.Switching strategy of the invention is a kind of blanket design, PA probe can be activated to provide new platform for exploitation high contrast.

Description

A kind of Rhodol derivative dye and its application

technical field

The invention relates to a Rhodol derivative dye and a photoacoustic imaging PA probe, in particular to the preparation and application of the Rhodol derivative dye and a photoacoustic imaging PA probe.

Background technique

Photoacoustic (PA) imaging is a powerful biomedical imaging modality that enables the noninvasive visualization of biological processes at the molecular and cellular levels in deep tissues with high spatial resolution. Utilizing a near-infrared (NIR) operating window, the PA can provide a penetration depth of several centimeters with a resolution of about 100 μm. Due to its advantages, it provides a useful tool for clinical imaging of various diseases including cancer diagnosis, metastasis assessment and treatment monitoring.

Molecular probes are essential in PA imaging because they can confer molecular or cellular specificity and enhance imaging contrast. PA probes typically rely on designs that allow selective accumulation of photon absorbers in diseased regions through passive or active targeting strategies. Activating PA probes, which can generate enhanced PA signals upon interaction with specific molecular or cellular events, are very promising. These probes have the advantages of low background and high sensitivity, which help to achieve real-time imaging with higher depth and spatial resolution. Current activated PA probes are designed based on intramolecular charge transfer (ICT) and contact quenching mechanisms. These two mechanisms have been well-documented for activating PA probes and have been applied to explore several target molecules such as metal ions, reactive oxygen species, nitric oxide, anaerobes, and enzymes. However, most probes have low imaging contrast due to the small shifts in absorption spectra or quantum yields induced by targeting molecules. However, the rational design of activated PA probes with excellent photophysical properties, such as large absorption shifts, strong near-infrared absorption, low quantum yield, and high photostability, and finally probes that provide high-contrast PA signals is still a challenge. Difficult to achieve.

The common spirocyclic anthracene dye, Rhodol, because it has a large extinction coefficient, ideal photostability and unique analyte-responsive ring-opening characteristics, after hydroxyl acetylation, it forms a spironolactone structure and is in a closed-loop state. It is often used as an activation Formula fluorescent reporter group, but its maximum absorption wavelength is only 550nm, and its maximum fluorescence emission is only 580nm, which limits its application range. Moreover, Rhodol only has an absorption band in the visible region and has a relatively large quantum yield, which is not suitable for in vivo PA imaging, let alone PA/NIRF dual-mode imaging. Therefore, it becomes extremely necessary to design a Rhodol variant with near-infrared (NIR) absorption, lower quantum yield, and Rhodol-like spironolactone ring-opening reaction.

Contents of the invention

Aiming at the deficiencies of the prior art, one of the purposes of the present invention is to develop a novel dye with near-infrared (NIR) absorption, capable of activating a high signal ratio PA response indicating enzyme activity.

The second purpose of the present invention is to develop a new high-contrast activated near-infrared PA probe using the unique spironolactone ring-opening reaction of the new dye, and to determine its performance in PA/NIRF dual-mode imaging of hNQO1 overexpressed tumors Applications.

In order to solve the problems of the technologies described above, the technical solution of the present invention is as follows:

A Rhodol derivative dye characterized in that the dye produces a significantly enhanced absorption band in the NIR region from 620 nm to 700 nm.

A Rhodol derivative dye is characterized in that its structural formula is as shown in formula (I):

Wherein, R1 is taken from H, F, Cl.

The preparation method of the dye comprises the following steps: mixing cyclohexanone, concentrated H ₂ SO ₄ , 4-diethylamino ketoacid (1a), and perchloric acid, filtering, and washing with water to obtain 9-(2-carboxy Phenyl)-6-(diethylamino)-1,2,3,4-tetrahydrooxyalkyl (2a), and then 3,5-difluoro-4-hydroxybenzaldehyde or 3,5- Dichloro-4-hydroxybenzaldehyde or hydroxybenzaldehyde react to give the dye.

Spiroanthracene dyes, such as fluorescein and Rhodol, have an equilibrium between the "open" form of the π-conjugated system and the "closed" spironolactone structural form. Furthermore, the "opened" form with a large π-conjugated system exhibits a large red-shift of the absorption spectrum and enhanced absorption bands compared with the "closed" spironolactone structure. To our knowledge, this property of spiroanthracene dyes has not been exploited in the design of activated PA probes.

When its phenolic hydroxyl group is blocked by esterification, the chromophore exists in the form of spironolactone, which shows negligible near-infrared absorption due to the breaking of the conjugated system. The analyte releases the phenolic hydroxyl moiety with a "ring-opening" reaction that restores its conjugated structure with strong NIR absorption and produces a high-contrast PA signal.

To design an activated PA probe using the spironolactone ring-opening reaction, we chose a common spiroanthracene dye, Rhodol, because of its large extinction coefficient, ideal photostability, and unique analyte-responsive ring-opening characteristic. However, Rhodol only has an absorption band in the visible region and has a relatively large quantum yield, which is not suitable for in vivo PA imaging. Therefore, we set out to design a variant of Rhodol with NIR absorption, lower quantum yield, and Rhodol-like spironolactone ring-opening reaction.

The present invention synthesizes a new Rhodol derivative (Rhodol-NIR) by combining the structure of the Rhodol core with the structure of difluoro-4-hydroxybenzaldehyde. This new Rhodol derivative not only has a large π-conjugated system, but also the fluoride ion can fine-tune the protonation constant of the phenolic hydroxyl moiety, showing excellent photophysical properties in the design of high-contrast activated PA probes. nature.

Compared with the existing Rhodol structure, we extend the conjugated system of the dye structure by coupling p-hydroxybenzaldehyde and its derivatives so that it has near-infrared absorption and emission.

The present invention also provides an application of the dye, and realizes the design of the PA probe for photoacoustic imaging by utilizing the spironolactone ring-closing-ring-opening control of the dye.

The present invention also provides a photoacoustic imaging PA probe, the structural formula of which is shown in formula (II):

Wherein, R ₂ is selected from dimethylmethylene quinone propionate, phosphate, galactosyl, trifluoromethanesulfonate, phenol.

The present invention also provides a method for preparing a photoacoustic imaging PA probe. The preparation method comprises mixing Rhodol derivative dye with trimethyl-locked quinonepropionic acid, phosphoric acid, galactoside, trifluoromethanesulfonate and The corresponding probes were prepared by reacting p-bromophenol.

The probe prepared by reacting the Rhodol derivative dye with trimethyl-locked quinonepropionic acid can be used to detect the overexpressed biomarker enzyme hydroquinone reductase in tumor cells;

The probe prepared by reacting the Rhodol derivative dye with phosphoric acid can be used to detect the overexpressed biomarker enzyme alkaline phosphatase in tumor cells;

The probe prepared by reacting the Rhodol derivative dye with galactoside can be used to detect the overexpressed biomarker enzyme galactosidase in tumor cells;

The probe prepared by reacting the Rhodol derivative dye with trifluoromethanesulfonate can be used to detect superoxide anion in inflammation;

The probe prepared by reacting the Rhodol derivative dye with p-bromophenol can be used to detect hydroxyl free radicals in inflammation.

Preferably, a photoacoustic imaging PA probe has a structural formula as shown in formula (III):

The preparation method of the photoacoustic imaging PA probe is characterized in that it includes synthesizing trimethyl-locked quinonepropionic acid, and self-assembling after mixing Rhodol derivative dyes with the synthesized trimethyl-locked quinonepropionic acid.

The preparation method of the described photoacoustic imaging PA probe comprises the following steps:

A. Synthesis of trimethyl-locked quinonepropionic acid: after mixing 3,3-dimethacrylate, 2,3,5-trimethyl-1,4-benzenediol and methanesulfonic acid, extraction, Wash, concentrate in vacuo, and recrystallize to obtain 6-hydroxy-3,3,5,7,8-pentamethylcyclolactone-2-one, add N-bromosuccinimide to the compound 1 solution, and stir , remove the solvent, extract, concentrate in vacuo, and purify to obtain 3-methyl-3-(2,4,5-trimethyl-3,6-dioxane-1,4-dien-1-yl) butyric acid.

B. Preparation of PA probe: Rhodol derivative dye, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 3-methyl-3-(2,4 , 5-trimethyl-3,6-dioxan-1,4-dien-1-yl)butanoic acid, 4-dimethylaminopyridine mixed reaction, washing, vacuum concentration, purification, to obtain light Acoustic Imaging PA Probe.

The present invention also provides the 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 overexpressed in tumor cells, as the case study. A PA probe for hNQO1 detection was designed by blocking the hydroxyl group with a trimethyl-locked quinonepropionic acid moiety ( _Q3 ). The esterification of the hydroxyl group promotes the formation of a "closed" spironolactone structure of the PA probe, which shows negligible absorption in the visible-near infrared region. When _hNQO1 catalyzes the reduction and eliminates the Q moiety in the PA probe, the resulting Rhodol-NIR dye undergoes a spontaneous ring-opening process and recovers its large π-conjugated system. Therefore, this elimination reaction is able to activate a high signal ratio PA signal and indicate enzyme activity.

In addition to the PA response, the Rhodol-NIR dye also provides an ideal fluorescent signal to achieve dual-mode detection of PA/NIRF. Furthermore, we demonstrate that the PA probe has very low NIR absorption background and high specificity for hNQO1, while the Rhodol-NIR dye produced after the enzymatic reaction exhibits a large molar extinction coefficient, excellent photostability and ideal quantum yield Rate. These excellent photophysical properties make PA probes useful for high-contrast PA/NIRF dual-modal imaging of living cells and animals. This probe also enables the determination of hNQO1 differential expression in live cells and tumors, offering great diagnostic potential for diseases associated with hNQO1 overexpression. To the best of our knowledge, this is the first activatable near-infrared PA probe developed based on the spironolactone ring-opening reaction. It is expected that our protocol can provide a new platform for studying high-contrast imaging-activated PA probes.

Compared with the prior art, the beneficial effects of the present invention are as follows:

1. The new Rhodol derivative dye Rhodol-NIR designed and synthesized by the present invention not only has a large π-conjugated system, but also the fluorine ion can finely adjust the protonation constant of the phenolic hydroxyl group, and develops a dye with a large molar extinction coefficient. , a novel Rhodol-NIR chromophore with excellent photostability and ideal quantum yield, exhibits excellent photophysical properties in the design of high-contrast activated PA probes.

2. The high-contrast activatable PA probe designed and synthesized by the present invention is based on the spironolactone ring-opening strategy of the unique analyte-induced spirocyclanthracene dye. Due to its "closed" spironolactone structure, the probe exhibited no NIR absorption, and its reaction with hNQO1 generated a Rhodol-NIR chromophore through a spironolactone ring-opening transition, allowing high-contrast PA/NIRF dual-mode detection and hNQO1 imaging.

3. The Rhodol-PA probe designed and synthesized by the present invention has been proved to have high sensitivity and high selectivity for hNQO1 detection by PA and fluorescence measurement in vitro.

4. The present invention is the first time at home and abroad to use spironolactone ring-opening transformation strategy to develop high-contrast activatable PA probes. The present switching strategy is a generally applicable design that may provide a new platform for the development of high-contrast activatable PA probes.

Description of drawings

Fig. 1 is the pKa value of Rhodol-NIR of the present invention;

Fig. 2 is the photostability working curve figure of Rhodol-NIR of the present invention;

Fig. 3 is the in vitro response spectrogram of Rhodol-PA of the present invention and control probe to hNQO1;

Fig. 4 is the high performance liquid phase chromatogram of Rhodol-PA of the present invention, Rhodol-NIR, Rhodol-PA and hNQO1 reaction mixture;

Fig. 5 is the high-resolution mass spectrogram of the reaction mixture of Rhodol-PA and hNQO1 of the present invention;

Fig. 6 is the fluorescence spectrogram of hNQO1 detected in vitro by the Rhodol-PA probe of the present invention;

Fig. 7 is the ultraviolet absorption spectrogram of the Rhodol-PA probe of the present invention detecting hNQO1 interference experiment in vitro;

Fig. 8 is the PA response diagram of the Rhodol-PA probe of the present invention to different concentrations of hNQO1;

Fig. 9 is a linear working curve of the photoacoustic signal intensity of the Rhodol-PA probe of the present invention at 680nm and different concentrations of hNQO1 fitting;

Figure 10 is the multispectral optical tomography (MSOT) imaging system imaging of hNQO1 by the Rhodol-PA probe explored in two cell lines HT-29 cells and MDA-MB-231 cells;

Fig. 11 is the result figure of the cytotoxicity experiment of Rhodol-PA of the present invention to HT-29 and MDA-MB-231 cells;

Figure 12 is the MSOT and fluorescence imaging images of mice with HT-29 and MDA-MB-231 tumors administered by tail vein injection of Rhodol-PA probe in vivo experiments;

Figure 13 is the MSOT and fluorescence imaging images of mice with HT-29 and MDA-MB-231 tumors administered by tail vein injection of control mice in vivo experiments.

Detailed ways

The present invention will be described in detail below in conjunction with examples. It should be noted that, in the case of no conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other.

Example 1

Design and Synthesis of Rhodol Derivative Dyes--Rhodol-NIR.

Freshly distilled cyclohexanone (3.3 mL, 31.9 mmol) was added dropwise to concentrated _H2SO4 (35 mL) and the mixture was cooled to ₀ °C. Then, 4-diethylaminoketoacid (16 mmol) was added portionwise with stirring. After reacting at 90°C for 1.5 hours, the mixture was poured into ice (150 g). Perchloric acid (70%, 3.5 mL) was then added, and the resulting precipitate was filtered and washed with cold water (50 mL) to give 9-(2-carboxyphenyl)-6-(diethylamino)-1,2,3, 4-Tetrahydrooxyalkyl (2a), using 9-(2-carboxyphenyl)-6-(diethylamino)-1,2,3,4-tetrahydrooxyalkyl (237.6mg, 0.5mmol ) and 3,5-difluoro-4-hydroxybenzaldehyde (118.5mg, 0.75mmol), refluxed in acetic acid at a high temperature of 110°C, and spin-dried through a silica gel column to obtain Rhodol-NIR as a blue solid (89.2mg, yield 29%), the specific synthesis process is as follows:

The structural formula of Rhodol-NIR is shown in formula (Ⅳ):

In a preliminary study, it was found that the π-conjugated system extension reaction is not only easy to synthesize, but also has an ideal non-rigid structure with low quantum yield, which is beneficial to enhance the PA signal.

Example 2

Photophysical Properties of Rhodol-NIR

Steps:

In order to determine the pKa value of Rhodol-NIR, use NaOH and hydrochloric acid to adjust the pH, prepare phosphate buffer solution containing 0.5% DMSO as a co-solvent and have different pH values, mix Rhodol-NIR (15 μ M) with different buffer systems, use The absorption spectrum of Rhodol-NIR in different pH buffer systems was measured by UV-1800 spectrophotometer, and the fluorescence spectrum of Rhodol-NIR in different pH buffer systems was measured by FS5 fluorometer, and the excitation wavelength was 620nm. The pH curve was then plotted using absorbance at 650 nm and 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 Figure 1.

Rhodol-NIR has an absorption maximum at 630nm, but it produces a significantly enhanced absorption band in the broad NIR region from 620nm to 700nm. It can be seen from Fig. 1 that the pKa value of Rhodol-NIR is 6.1, which proves our design that the introduction of electron-withdrawing substituents is beneficial to the reduction of pKa and enhanced NIR absorption of Rhodol derivatives. Taken together, the results demonstrate that Rhodol-NIR provides an ideal chromophore for the development of activatable PA probes.

We found that Rhodol-NIR exhibits a large molar extinction coefficient (ε=2.67×10 ⁵ M ^-1 ·cm ^-1 ) at 630 nm, which is comparable to existing small molecule chromophores for PA imaging. advantageous.

The quantum yield of Rhodol-NIR was measured in PBS (pH 7.4), using cresyl violet (Φs = 0.54 in MeOH) as a reference, and the quantum yield was calculated according to Equation 1:

Φ _X ＝Φ _S (A _S F _X /AXF _S )(n _X /n) ² (1)

Φ: Quantum yield; A: Absorbance at excitation wavelength; F: Integrated area of fluorescence spectrum at the same excitation wavelength; n: Refractive index of solvent; S and X represent reference standard sample and unknown sample, respectively.

The quantum yield Φ _F of Rhodol-NIR in pH=7.4 phosphate buffer solution was calculated to be 1.0%. This quantum yield demonstrates the high efficiency of Rhodol-NIR in light-to-heat conversion for PA imaging, considering that it is not substituted with heavy atoms to generate other competing photoconversion pathways. This quantum yield can also be used for fluorescence imaging studies with suitable sensitivity.

Rhodol-NIR also has good photostability, as shown in Figure 2.

The specific steps are as follows: In order to determine the photostability of Rhodol-NIR, the prepared Rhodol-NIR (15 μM) sample was placed on a microscope and irradiated with a mercury lamp (100 W). The samples were taken out every about 5 minutes, and the fluorescence spectrum was measured with a FS5 fluorometer. 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 showed a negligible decrease in fluorescence after 70 min of continuous irradiation, while the fluorescence of the commercial NIR dye indocyanine green (ICG) decreased by 90%. The high molar extinction coefficient in the NIR region, ideally low quantum yield, and high photostability suggest that Rhodol-NIR provides an ideal contrast agent for PA imaging.

Example 3

Design and Synthesis of PA Probe Based on Rhodol-NIR

The specific synthesis process is:

B. Synthesis of trimethyl-locked quinonepropionic acid: 3,3-dimethacrylate (1.60mL, 12mmol) was added to 2,3,5-trimethyl-1,4-benzenediol (1.52 g, 10 mmol) and methanesulfonic acid (15 mL), the mixture was stirred at 70°C for 2 hours. After cooling to room temperature, the reaction mixture was diluted with water to 150 mL and extracted three times with 70 mL of dichloromethane. The extract was washed with saturated NaHCO ₃ solution and NaCl solution, dried over anhydrous Na ₂ SO ₄ and concentrated in vacuo. Recrystallization from petroleum ether with 30% _CHCl3 gave 6-hydroxy-3,3,5,7,8-pentamethylcyclolactone-2-one as a white solid (1.85 g, 79.2% yield) .

To a solution of 6-hydroxy-3,3,5,7,8-pentamethylcyclic lactone-2-one (1.17 g, 5 mmol) in acetonitrile (60 mL) and water (25 mL) was added NBS (0.98 g, mmol). The reaction mixture was then stirred at room temperature for 1 hour. After removing most of the organic solvent, the residue was extracted three times with 30 mL of dichloromethane. _The organic layer was dried over anhydrous _Na2SO4 and concentrated under vacuum. The crude product was purified by column chromatography using PE/EtOAc 2:1 (v/v) as eluent to afford trimethylquinonepropionic acid (0.92 g, 73.5% yield).

C, preparation of PA probe: Rhodol-NIR (234.0mg, 0.38mmol) was slowly added to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (72.8mg, 0.38mmol), a solution of trimethyl-locked quinonepropionic acid (167.3mg, 0.58mmol), at 0°C, in CH ₂ Cl ₂ (30mL) was added 4-dimethylaminopyridine (46.4mg, 0.38 mmol). After reacting for 12 hours, the mixture was washed with HCl (1M). The organic layer was dried over _MgSO4 , filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography using _{CH2Cl2 -} MeOH (100:1 to 20:1) as eluent to obtain Rhodol-PA probe as purple solid (164.7 mg, 58% yield).

The specific route is shown below:

Example 4

Response properties of the Rhodol-PA probe.

The operation steps are as follows: In order to test the in vitro response performance of Rhodol-PA to hNQO1, first use PBS buffer (10 mM, pH=7.4) to prepare a 15 μM Rhodol-PA solution containing 0.5% (v/v) dimethyl sulfoxide and 100 μM NADH, and then Rhodol-PA and hNQO1 (3.0 μg/mL) were incubated at 37° C. for 1.5 h, and the ultraviolet absorption spectrum was measured 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 exists mainly in the form of a "closed ring" spironolactone structure with a broken π-conjugated structure.

After incubation of the probe with hNQO1 for 90 min, we observed a sharp increase in the NIR absorption band at 630 nm, indicating the production of Rhodol-NIR (Fig. 3). This finding suggested a two-step mechanism of the hNQO1-catalyzed reaction, in which the spironolactone ring-opening reaction spontaneously followed after the blocking group _Q3 in Rhodol-PA was reduced-eliminated. It was found that Rhodol-NIR can specifically respond to hNQO1, and has a large red shift (△λ>230nm) and high absorption signal ratio (>57 times at 630nm,>110 times at 680nm). This desirable performance is attributed to our spironolactone ring-opening design, in which the probe exhibits a "ring-closed" spironolactone structure with a broken π-conjugated system, while the product has an extended "ring-opened" large π-conjugated system form.

Example 5

Validating the Necessity of Spiral Opening Designs

We synthesized a control chromophore using a variant of Rhodol without the spiro moiety. A control probe was also prepared by esterifying the control emitter with the recognition substrate _Q3 of hNQO1.

The preparation process of the control probe is: the control chromophore (234.0mg, 0.38mmol) is slowly added to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (72.8mg , 0.38mmol), 3-methyl-3-(2,4,5-trimethyl-3,6-dioxane-1,4-dien-1-yl)butanoic acid (167.3mg, 0.58 mmol) was added 4-dimethylaminopyridine (46.4 mg, 0.38 mmol) in _{CH2Cl2 (} 30 mL) at 0 °C. After reacting for 12 hours, the mixture was washed with HCl (1M). The organic layer was dried over _MgSO4 , filtered and concentrated in vacuo. The residue was purified by column chromatography on silica gel using _{CH2Cl2 -} MeOH (100:1 to 20:1) as eluent to obtain the control probe as a purple solid (164.7 mg, 58% yield).

The specific route is as follows:

Wherein, Control probe refers to the control probe. Similarly, the present invention tests the in vitro response performance of the control probe to hNQO1, first configures a 15 μM Control probe solution containing 0.5% (v/v) dimethyl sulfoxide and 100 μM NADH with PBS buffer (10 mM, pH=7.4) , and then mixed with hNQO1 (3.0μg/mL), reacted at 37°C for 1.5h, and measured the ultraviolet absorption spectrum with a UV-1800 spectrophotometer.

Surprisingly, the control probe did exhibit a distinct vis-NIR absorption band (Fig. 3). Notably, the control probe has the same parental structure as the Rhodol-PA probe. Therefore, the different absorption spectra of the control probe and Rhodol-PA provided direct evidence for the broken π-conjugated system in Rhodol-PA, verifying the "closed ring" spironolactone structure in Rhodol-PA.

In addition, according to Figure 3, after the control probe was reacted with hNQO1, there was a slight increase in the absorption peak, and the maximum value was only enhanced by 3 times. This slight increase in absorption was attributed to enhanced intramolecular charge transfer effects due to the formation of free phenolic hydroxyl groups in the enzymatic reaction. Based on these results, we infer that the high-contrast response of Rhodol-PA is attributed to the spironolactone ring-opening mechanism, validating the potential of this new strategy to develop high-contrast activatable PA probes.

Example 6

Research on the Reaction Mechanism of Rhodol-PA Probe

As shown in the above formula, when the ring-closed probe Rhodol-PA was incubated with hNQO1, hNQO1 reacted with the recognition site to release the ring-opened dye Rhodol-NIR, generating photoacoustic and fluorescent signals.

In order to verify the reaction mechanism of the Rhodol-PA probe, we first carried out the HPLC analysis of Rhodol-PA, Rhodol-NIR and the reaction products of Rhodol-PA and hNQO1. With methanol/water (0.2% CF ₃ COOH=90/10 (v/v), flow rate 1ml/min, detection wavelength 550nm C18 column (250nm × 4.6mm, 5μm) as chromatographic conditions, in order to further verify the reaction mechanism, use The high-resolution mass spectrometry method analyzed the reaction product of Rhodol-PA and hNQO1.In the PBS buffer solution (10mM, pH=7.4) containing 0.5% (v/v) DMSO and 100μM NADH, at 37 ℃ with hNQO1 (1.5 μg/ml) was incubated for 1.5 hours. Then 1 mL of MeCN was added dropwise to the reaction mixture and centrifuged at 10,000 rpm for 15 minutes. The supernatant was analyzed by mass spectrometry in positive ion mode.

For the product from the reaction between the probe and hNQO1, two elution peaks were obtained at 8.8 min and 5.0 min, respectively, corresponding to the Rhodol-NIR chromophore and the unreacted probe (Figure 4). In addition, the reaction solution showed two new peaks, one corresponding to Rhodol-NIR (calculated [M+H] ⁺ m/z 516.1981, found 516.1987), and the other as a reduction by-product (calculated as [M+H] ⁺ ) m/z 235.1289, found 235.0541), as shown in Figure 5. This result suggested that the Rhodol-PA probe was converted into Rhodol-NIR after reacting with hNQO1.

Example 7

In vitro performance of PA probes on hNQO1

Using the synthesized Rhodol-PA probe, we then investigated its performance as a probe to detect hNQO1 in vitro.

The operation steps are as follows: In order to test the in vitro response performance of Rhodol-PA to hNQO1, first use PBS buffer (10 mM, pH=7.4) to prepare a 15 μM Rhodol-PA solution containing 0.5% (v/v) dimethyl sulfoxide and 100 μM NADH, then Rhodol-PA and hNQO1 (3.0 μg/mL) were reacted at 37°C for 1.5 h, the photoacoustic spectrum signals were collected by the inVision256-TF multi-wavelength photoacoustic imaging system (MSOT), and the fluorescence spectra were measured by the FS5 fluorometer.

As expected, the Rhodol-PA probe exhibited a very low PA signal in the range of 680nm to 800nm (Figure 6). After the probe was reacted with hNQO1, a strong PA response signal was obtained, with ~11-fold increase in 680nm double ratio. Similarly, the Rhodol-PA probe has only very low fluorescence, while its fluorescence signal is significantly enhanced after reacting with hNQO1, with ~59-fold signal ratio at 720 nm. The results hint at the potential of Rhodol-PA for high-contrast PA/NIRF dual-modal imaging of hNQO1.

To test its specificity, control experiments were performed in which the probe was incubated with various biologically relevant species with no uptake response (Fig. 7) demonstrating the high selectivity of the Rhodol-PA probe for hNQO1.

Further evidence was obtained from assays of two lysates of HT-29 cells, which are known to overexpress hNQO1. After incubating the probe with HT-29 cell lysates, we obtained an uptake signal (Fig. 7). In contrast, negligible uptake signals were observed in assays of HT-29 cell lysates pretreated with excess dicoumarin, a specific hNQO1 inhibitor. Additionally, lysates of MDA-MB-231 cells, which were reported to have very low expression of hNQO1, were probed to show very little response in UV absorbance (Fig. 7). Taken together, these results clearly demonstrate the high specificity of the Rhodol-PA probe for hNQO1.

Example 8

Determination of the Quantitative Capacity of Rhodol-PA Probes

The test method is as follows: In order to detect the quantitative ability of Rhodol-PA to hNQO1, different concentrations of hNQO1 (0-3.0 μg/mL) were used to incubate 15 μM Rhodol-PA at 37°C for 1.5 h, and the inVision256-TF multi-wavelength photoacoustic imaging The system collects photoacoustic signals, and then uses the photoacoustic signal intensity at 680nm to fit a linear working curve with different concentrations of hNQO1.

The PA signal at 680nm was found to exhibit a dynamic response to hNQO1 in the concentration range of 0.25-3.0 μg/mL (Figure 8). The response signal-to-fold ratio was ~11, indicating high contrast in the PA detection of hNQO1. A linear correlation was obtained over the range of 0.25 to 1.1 μg/mL with an estimated limit of detection (LOD) of 0.08 μg/mL (Figure 9).

Example 9

Ability of Rhodol-PA probe to image hNQO1 in living cells

Motivated by the promising in vitro results, we then explored the ability of the Rhodol-PA probe to image hNQO1 with PA/NIRF in living cells using two cell lines, HT-29 cells and MDA-MB-231 cells.

Specific operation steps: incubate HT-29 or MDA-MB-231 cells (approximately 7×10 ⁶ cells in a 75 cm ² cell culture flask) with Rhodol-PA (30 μM) at 37°C for 2 h, wash with 10 mL PBS for 3 The cells were digested with 0.25% trypsin and counted with TC20 ^TM automatic cell counter. Collect the cell suspension with a centrifuge microtube (200 μL). Insert the tube with the cell pellet into the MSOT imaging system holder and image it with the MSOT imaging system. When conducting inhibitor research, the cells were pretreated with dicoumarin (100 μM) inhibitor for 1 h before the cells were incubated with Rhodol-PA (30 μM), and the subsequent steps were the same as those without adding dicoumarin inhibitor. same.

Cells (7×10 ⁶ ) were centrifuged to obtain cell pellets, which were imaged with a multispectral optical tomography (MSOT) imaging system ( FIG. 10 ). As expected, the PA signal at 680 nm was significantly increased in HT-29 cells incubated with the Rhodol-PA probe (Fig. 10) when cells were pretreated with the hNQO1 inhibitor dicoumarin and then incubated with the probe , a 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 hNQO1 levels. This result demonstrates that the Rhodol-PA probe provides a molecular tool for specific differentiation activity, as well as detection of hNQO1 activity in living cells using MSOT imaging.

In addition, we also carried out the cytotoxicity test of Rhodol-PA to HT-29 and MDA-MB-231 cells.

Cytotoxicity experiment operation: The cytotoxicity of Rhodol-PA to HT-29 and MDA-MB-231 cells was studied by standard MTT method. HT-29 and MDA-MB-231 cells were seeded in 96-well plates with 5×10 ³ cells/well, and the culture medium was 100 μL. The cells were cultured at 37°C for 24 hours, then cultured with fresh medium containing different concentrations of Rhodol-PA (0-300 μM) 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 μL, 5 mg/ml) at 37° C. for 4 h. Then DMSO (100 μL/well) was added for 10 min to dissolve the precipitated formazan crystals. Absorbance at 570 nm was measured using an ELX800 ^™ microplate reader.

Furthermore, cytotoxicity assays showed that Rhodol-PA probe concentrations above 300 μM had little effect (>90%) on the cell viability (>90%) of the two cell lines HT-29 and MDA-MB-231 (Fig. Potential of low cytotoxic Rhodol-PA probes for biological applications.

Example 10

Tail vein administration demonstrates the ability of the Rhodol-PA probe to detect hNOQ1 activity in PA in living animals

Specific experimental procedure: For MSOT imaging, 200 μL of Rhodol-PA (300 μM) was injected via tail vein into HT-29 tumor or MDA-MB-231 tumor mice. The control group was injected with sterilized PBS (200 μL). 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. Placed in a water bath at 34°C, from 680nm to 800nm, collect photoacoustic signals at different wavelengths every 10nm, and use the photoacoustic intensity at 850nm as the background. For tumor sites, data were collected every 0.3 mm. MSOT images were collected at different time points (0, 0.5h, 1h, 3h, 5h, 7h and 12h) before Rhodol-PA injection (0 min) and after injection. Use the independent component spectral analysis technology of the photoacoustic instrument to analyze the collected signals, separate the signal of the probe after the response from the signal of other background light absorbers (such as hemoglobin) in the tissue, and obtain the photoacoustic imaging map. And calculate the average photoacoustic signal intensity of different parts of the tumor at the same time point, subtract the average photoacoustic signal intensity of different parts of the tumor before injection, and obtain the enhanced photoacoustic signal intensity.

In this study, Rhodol-PA probe (2.56 mg/kg) or PBS were administered to HT-29 and MDA-MB-231 tumor mice by tail injection, respectively. MSOT and fluorescence imaging images were acquired at different intervals (0, 0.5, 1, 3, 5, 7, 12 h) after injection. We observed a clear PA signal 1 hour after injection of the probe, and the strongest signal was obtained 5 hours after injection in the tumor area of HT-29 cell xenografted mice (Fig. 12a1). The PA signal after injection of the PBS control did not show a significant increase throughout the experiment, demonstrating that the activation of the PA signal comes from the response of the probe in the tumor area (Fig. 13). No significant increase in PA signal was found in the tumor area of MDA-MB-231 cell xenografted mice after injection of the probe (Fig. 12a1). Fluorescent images showed similar responses in tumor regions of HT-29 and MDA-MB-231 cell xenografted mice (Fig. 12a1). Data collected from six HT-29 tumor mice in repeated assays showed that the mean PA signal in the tumor area showed a rapid increase at 1 h after injection and reached a maximum at 5 h and then gradually decreased to 12 h ( Figure 12a2). In contrast, MDA-MB-231 tumor-implanted mice showed only a slight increase in PA signal in the tumor area after injection. The average signal ratio of HT-29 tumors to MDA-MB-231 tumors was approximately 7-fold, confirming that high-contrast PA imaging of hNQO1-overexpressing tumors was achieved using the Rhodol-PA probe. Furthermore, we found that in vivo PA spectra of tumor regions of HT-29 tumor-implanted mice obtained 5 h after injection showed a maximum PA signal at 680 nm, with a spectral distribution similar to that of in vitro spectra (Fig. 12a3). This result further confirmed that PA signaling in HT-29 tumors was attributed to hNQO1-mediated specific activation of Rhodol-PA probes in HT-29 cells. In vivo PA spectra from MDA-MB-231 tumor-bearing mice also showed similar but smaller features, suggesting low expression of hNQO1 in these cells.

In conclusion, the live mouse imaging results demonstrate that our probe can be selectively activated in hNQO1-upregulated tumors in living animals, enabling PA/NIRF dual-modal imaging of hNQO1-overexpressing tumors, implying that it has tumors in vivo. Potential for diagnostic and therapeutic evaluation.

The above-mentioned embodiments should be understood that these embodiments are only used to illustrate the present invention more clearly, and are not intended to limit the scope of the present invention. After reading the present invention, those skilled in the art will understand the various equivalent forms of the present invention All modifications fall within the scope defined by the appended claims of this application.

Claims (10)

1. A Rhodol derivative dye, characterized in that the dye produces an absorption band in the NIR region of 620nm to 700nm. 2. a Rhodol derivative dye is characterized in that its structural formula is as shown in formula (I): Wherein, R1 is taken from H, F, Cl. 3. a preparation method of dyestuff as claimed in claim 2, is characterized in that, comprises the following steps: cyclohexanone, concentrated H ₂ SO ₄ , 4-diethylamino keto acid, perchloric acid are mixed, filter, After washing with water, 9-(2-carboxyphenyl)-6-(diethylamino)-1,2,3,4-tetrahydrooxyalkyl was obtained, followed by 3,5-difluoro-4-hydroxybenzaldehyde Or 3,5-dichloro-4-hydroxybenzaldehyde or hydroxybenzaldehyde reaction to obtain dyes. 4. An application of designing and synthesizing a photoacoustic imaging PA probe utilizing the spironolactone ring-closing-ring-opening regulation of the dye as claimed in claim 1 or 2. 5. A photoacoustic imaging PA probe, characterized in that its structural formula is as shown in formula (II): Wherein, R ₂ is selected from dimethylmethylene quinone propionate, phosphate, galactosyl, trifluoromethanesulfonate, phenol. 6. The photoacoustic imaging PA probe according to claim 5, wherein its structural formula is as shown in formula (III): 7. a kind of preparation method of photoacoustic imaging PA probe as claimed in claim 5, is characterized in that, described preparation method is the quinone propionic acid, phosphoric acid, galactoside of Rhodol derivative dye and trimethyl lock , trifluoromethanesulfonate or p-bromophenol in any reaction preparation. 8. a kind of preparation method of photoacoustic imaging PA probe as claimed in claim 6, is characterized in that, described preparation method comprises the quinone propionic acid of synthesizing trimethyl lock, with the trimethyl of Rhodol derivative dye and synthesis Preparation of base-locked quinonepropionic acid mixtures. 9. The preparation method of photoacoustic imaging PA probe according to claim 8, is characterized in that, comprises the following steps: A. Synthesis of trimethyl-locked quinonepropionic acid: after mixing 3,3-dimethacrylate, 2,3,5-trimethyl-1,4-benzenediol and methanesulfonic acid, extraction, Wash, concentrate in vacuo, and recrystallize to obtain 6-hydroxy-3,3,5,7,8-pentamethylcyclolactone-2-one, add N-bromosuccinimide to the compound 1 solution, and stir , remove the solvent, extract, concentrate in vacuo, and purify to obtain 3-methyl-3-(2,4,5-trimethyl-3,6-dioxane-1,4-dien-1-yl) butyric acid. B, preparation of PA probe: Rhodol derivative dye, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 3-methyl-3-(2,4 , 5-trimethyl-3,6-dioxan-1,4-dien-1-yl)butanoic acid, 4-dimethylaminopyridine mixed reaction, washing, vacuum concentration, purification, to obtain light Acoustic Imaging PA Probe. 10. An application of the photoacoustic imaging PA probe according to claim 5 or 7 in high-contrast PA/NIRF dual-mode imaging of living cells and animals.