Patent Application
20240165272
This application claims priority benefit of Chinese Patent Application No. 202211349415.0, filed on Oct. 31, 2022, which is hereby incorporated by reference in its entirety.
In general, the invention relates to in vivo visualization of the concentration and distribution of superoxide (O2·−) with a small molecular fluorescent/photoacoustic dual-modal probe, and in particular to a hemi-cyanine fluorophore based probe for accurately detecting endogenous O2·−, which belongs to the field of fluorescence/photoacoustic dual-modal bioimaging.
Liver is the main metabolic organ of the human body. The incidence of hepatic inflammation is gradually increasing, and the higher the risk of exposure to adverse conditions such as overdose of drugs, intemperance, allergies, viruses and intestinal microbial metabolites, the greater the incidence of hepatic inflammation, with varying degrees of hypohepatia. At present, the incidence of hepatic inflammation is gradually increasing. If left untreated, hepatic inflammation may continue to progress and eventually develop into severe complications. Therefore, accurate diagnosis of early hepatic inflammation may provide the opportunity for timely intervention to reduce the risk of disease progression. Currently, the clinical diagnosis of hepatic inflammation depends on liver function tests, which mainly check the abnormal levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum. Unfortunately, hepatic inflammation is easy to miss in its early stages because the symptoms are atypical and the abnormal blood markers detected are associated with many diseases throughout the body. Therefore, the early clinical detection of hepatic inflammation is a challenge, and there is an urgent need to develop new strategies capable of detection and accurate pathological analysis of early hepatic inflammation in real time.
Intracellular reactive oxygen species (ROS), as the key participants in oxidative stress, fluctuate dynamically with pathological processes. It is worth noting that O2·− is the primary ROS in the event of hepatocellular oxidative stress, and is the main source of other ROS and reactive nitrogen species (RNS). Therefore, O2·− can be considered as a pivotal early parameter of liver dysfunction, which in turn is involved in hepatic inflammation and ultimately hepatocyte necrosis. Therefore, it can be used as a key analyte for the early diagnosis of hepatic inflammation. Due to the high reactivity and short half-life of O2·−, real-time detection of O2·− in vivo is difficult. Therefore, it is necessary to develop alternative methods to monitor the fluctuation of hepatic O2·− in situ in order to promote the early diagnosis of hepatic inflammation.
NIR fluorescence probes are a promising diagnostic tool with strong imaging sensitivity for non-invasive detection of analytes of interest in vivo. In recent years, more attention has been paid to the development of fluorescent probes with high sensitivity and specificity for early markers of hepatic inflammation, which are expected to be used for in situ detection of hepatic inflammation. However, the tissue penetration ability of single NIR imaging is not enough to realize high spatial resolution visualization of deep tissue pathology information. Notably, photoacoustic (PA) imaging combines the imageological superiorities of fluorescence and ultrasound imaging to further facilitate real-time monitoring of internal physiological and pathological conditions in deep tissues. Because the imaging mechanism of NIRF imaging is similar to that of PA imaging, it makes sense to integrate the two imaging modalities with a probe. Through the two-in-one strategy, NIRF/PA dual-modal imaging possess both high sensitivity of fluorescence imaging and high penetration depth and high spatial resolution of PA, and can capture more comprehensive pathological information, becoming a promising strategy for precision medicine.
Although dual-modal probes provide novel strategies for precision medicine, small molecule dual-modal probes for in vivo bioimaging still face several challenges, such as low signal-to-background ratio (SBR) caused by their repaid clearance and unpredictable biodistribution in the lesion area. High dose probes can enhance SBR to some extent, but may cause potential metabolic stress. Enhancing active targeting capability is one of the most common strategies to improve the imaging performance of molecular probes. For example, based on the abundant galactose receptor protein on the surface of hepatocytes, galactose decoration can effectively promote probe accumulation in the liver. Therefore, in order to accurately diagnose liver diseases, more alternative targets that can improve liver targeting and facilitate probe design need to be developed. Cholic acid (CA) is a high affinity substrate for several hepatocyte-surface receptor and can be efficiently internalized into the liver, which can be used as a targeting group to achieve efficient enrichment of probes in the liver.
In order to overcome the poor accuracy of liver inflammation detection, liver-targeted fluorescence/photoacoustic dual-modal probes for the specific response to early markers of liver inflammation should be developed. We suppose that the active targeting strategy combined with the activatable dual-modal imaging synergistically improve in situ imaging performance and allow for early and accurate diagnosis of hepatic inflammation.
The invention provides a liver-targeted fluorescent/photoacoustic dual-modal imaging probe (hCy-Tf-CA) for real-time imaging of in situ hepatic inflammation, and its preparation method. The probe has the advantages of liver targeting ability and fluorescence/photoacoustic dual-modal imaging. With these advantages, the probe can in situ visualize O2·− in early hepatic inflammation with high selectivity and sensitivity, providing an effective method to facilitate precise imaging of hepatic inflammation.
In order to solve the above technical problems, the technical solutions are as follows:
The invention provides a liver-targeted fluorescent/photoacoustic dual-modal probe for real-time imaging of in situ hepatic inflammation, and the chemical makeup is as follows:
The invention provides a method for preparing a liver-targeted fluorescent/photoacoustic bimodal probe. The detailed synthesis is as follow:
Trifluoromethanesulfonic anhydride was dropwise added to a mixture of hCy-NHBoc and triethylamine in anhydrous CH2Cl2 in an ice bath under nitrogen atmosphere. When the reaction is complete, it was quenched by ice water. The organic layer was carefully separated and directly purified by column chromatography to afford the intermediate hCy-Tf-NHBoc as a purple solid. Subsequently, the intermediate hCy-Tf-NHBoc was added anhydrous CH2Cl2 containing 25% trifluoroacetic acid. After the reaction is complete, the reaction solution was evaporated under reduced pressure and the resulting residue was washed with diethyl ether to afford crude intermediate hCy-Tf-NH2. Then hCy-Tf-NH2, cholic acid, HBTU, HOBT and DIPEA were dissolved in DMF. After the reaction is complete, the solvent was removed by excess petroleum ether, and the resulting residue was purified by column chromatography to afford the liver-targeted fluorescence/photoacoustic dual-modal probe hCy-Tf-CA as a purple solid.
The synthesis route of hCy-Tf-CA is as follow:
In addition, the invention aims to provide a fluorescent probe for the detection of endogenic O2·− in cells. More importantly, the invention aims to in situ visualization of pathological O2·− in early hepatic inflammation by fluorescence/photoacoustic dual-modal imaging, providing an effective method to facilitate precise imaging of hepatic inflammation.
The invention has the following advantages:
The liver-targeted fluorescence/photoacoustic dual-modal probe provided by the invention can specifically react with O2·−, and its fluorescence intensity is significantly increased by 17 times. At the same time the probe shows good chemical stability and biocompatibility.
The probe of the invention has high selectivity and anti-interference. It barely responds to various potential interferents (Na+, K+, Ca2+, Mg2+, Zn2+, Cu2+, HS−, HClO, H2O2, ·OH, ONOO−, glucose, glutathione, cysteine, vitamin C, nitroreductase, alkaline phosphatase, β-galactosidase and leucine aminopeptidase), just shows significant response to O2·− with rapidly increased fluorescence.
The probe of the invention shows good biocompatibility, and can visual monitor O2·− fluctuation in cells under different drug stimulation.
By fluorescence/photoacoustic dual-mode imaging, the invention provides a reliable method for in situ real-time monitoring hepatic O2·− and early hepatic inflammation in a noninvasive manner, which shows a broad application prospect in bioanalysis field.
The present disclosure will be further described below with the preferred embodiment, but the present invention is not limited to the following examples.
Trifluoromethanesulfonic anhydride was dropwise added to a mixture of hCy-NHBoc and triethylamine in anhydrous dichloromethane in an ice bath under nitrogen atmosphere. After stirring for 5 minutes, it was quenched by ice water. The organic layer was carefully separated and directly purified by column chromatography to afford the intermediate hCy-Tf-NHBoc as a purple solid. Subsequently, the intermediate hCy-Tf-NHBoc was added anhydrous dichloromethane containing 25% trifluoroacetic acid. After stirring for 5 minutes, the reaction solution was evaporated under reduced pressure and the resulting residue was washed with diethyl ether to afford crude intermediate hCy-Tf-NH2. Then hCy-Tf-NH2, cholic acid, o-Benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium hexafluorophosphate, 1-Hydroxybenzotriazole and N,N-Diisopropylethylamine were dissolved in N,N-Dimethylformamide. After the reaction is complete, the solvent was removed by excess petroleum ether, and the resulting residue was purified by column chromatography to afford the liver-targeted fluorescence/photoacoustic dual-modal probe hCy-Tf-CA as a purple solid.
The molar ratio of hCy-NHBoc, trifluoromethanesulfonic anhydride and triethylamine was 1:1.2:1.2.
The volume ratio of methanol to dichloromethane was 1:50 in the first silica gel column chromatography.
The molar ratio of hCy-Tf-NHBoc to trifluoroacetic acid was 1:536.
The molar ratio of hCy-Tf-NH2, cholic acid, o-Benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium hexafluorophosphate, 1-Hydroxybenzotriazole and N,N-Diisopropylethylamine is 1:1.2:1.2:1.2:2.
The volume ratio of methanol to dichloromethane was 1:10 in the second silica gel column chromatography.
Nuclear magnetic resonance CH NMR and 13C NMR) confirmed the structures of the intermediate hCy-Tf-NHBoc and the final probe hCy-Tf-CA (
Absorption Spectra of hCy-Tf-CA Before and After the Reaction With O2·−
10 μL of probe stock solution (1 mM) was added to 1 mL of ethanol-phosphate buffer solution (ethanol/PBS=1/1, v/v, pH=7.4). To characterize the responsiveness of hCy-Tf-CA, 40 μL O2·− stock solution (2.5 mM) was added to above solution.
Fluorescence Spectra of hCy-Tf-CA Before and After the Reaction With O2·−
The reaction system was prepared under the same experimental conditions.
Photoacoustic Spectra of hCy-Tf-CA Before and After the Reaction With O2·−
The reaction system was prepared under the same experimental conditions.
Fluorescence Response of hCy-Tf-CA to Different Concentration of O2·−
Under the same experimental conditions, 10 μL of probe stock solution (1 mM) was added to 1 mL of ethanol-phosphate buffer solution, the O2·− titration experiment was then carried out. The fluorescence intensity at 730 nm were tested 10 minutes later.
Photoacoustic Response of hCy-Tf-CA to Different Concentration of O2·−
Under the same experimental conditions, 10 μL of probe stock solution (1 mM) was added to 1 mL of ethanol-phosphate buffer solution, the O2·− titration experiment then was carried out and the photoacoustic intensity at 710 nm were tested 10 minutes later.
Selective Response of hCy-Tf-CA to O2·−
The specificity of hCy-Tf-CA was determined against various potential interferents (Na+, K+, Ca2+, Mg2+, Zn2+, Cu2+, HS−, HClO, H2O2, ·OH, ONOO−, glucose, glutathione, cysteine, vitamin C, nitroreductase, alkaline phosphatase, β-galactosidase and leucine aminopeptidase). Under the same experimental conditions, 10 μL of probe stock solution (1 mM) was added to 1 mL of ethanol-phosphate buffer solution. Then stock solutions of various potential interferents were respectively added to above solution, then the fluorescence intensity at 730 nm was tested. The probe hCy-Tf-CA barely respond to various potential interferents (analytes: 100 μM, enzyme: 0.1 U·mL−1), but show significant response to O2·− with rapidly increased fluorescence (
In summary, the liver-targeted fluorescent/photoacoustic dual-modal probe hCy-Tf-CA prepared by the invention has high sensitivity when applied for O2·− detection in vitro, and can satisfy to selective detection of O2·− in dynamic and complex organism.
Inspired by the excellent responsiveness of the probe hCy-Tf-CA to O2·−, its biocompatibility and potential for further applications in living cells were assessed. HepG2 cells were plated in a 96-well plates at 6×105 cells/well and incubated overnight in the cell incubator. Then the cells were washed with culture medium and incubated with various concentrations of hCy-Tf-CA (1, 2, 4, 8, 10 μM) at 37° C. for 24 h. After removing the above solution, CCK-8 reagent diluted with culture medium was added to each well and incubated at 37° C. for 60 min. Finally, the absorbance was finally measured at 450 nm by a microplate reader. Cell viability was calculated using the following formula:
Calculation of cell viability: Cell viability (%)=(Aexperimental group−Ablank group)/(Acontrol group−Ablank group)×100%.
Standard CCK-8 assay showed that HepG2 cells were still more than 90% viable after 24 h exposure to 10 μM hCy-Tf-CA, which proved that the cytotoxicity of hCy-Tf-CA was negligible (
Fluorescence Imaging of Endogenous O2·− in HepG2 Cells
To evaluate the capacity of hCy-Tf-CA to image O2·− fluctuation in living cells, HepG2 cells were plated in a glass-bottom dish for 24 h to 80% density before the experiment. Cell fluorescence imaging of hCy-Tf-CA was divided into five groups. In the first group, hCy-Tf-CA (10 μM) was incubated with cells for 30 min. In the second group, cells were pretreated with lipopolysaccharide (LPS, 1 μg/mL) and interferon-gamma (IFN-γ, 50 ng/mL) for 12 h, then washed twice with PBS buffer before incubating with hCy-Tf-CA (10 μM) for 30 min. In the third group, cells were pretreated with LPS (1 μg/mL), IFN-γ (50 ng/mL) and 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO, 300 μM) for 12 h, then washed twice with PBS buffer before incubating with hCy-Tf-CA (10 μM) for 30 min. In the fourth and fifth groups, cells were pretreated with H2O2 or Na2SO3 (100 μM) for 30 min, then washed twice with PBS buffer before incubating with hCy-Tf-CA (10 μM) for 30 min. The cells were washed 3 times with PBS buffer before cell imaging. Fluorescence images of the cells were captured by a Nikon AX R microscope with DAPI channel (λex'404 nm, λem=425-475 nm) and Cy5.5 channel (λex=639 nm, λem=663-738).
As shown in
In order to visually detect hepatic O2·− concentration and drug-induced hepatic inflammation by fluorescence/photoacoustic dual-modal imaging, Balb/c female mice were intraperitoneally injected with overdose of APAP (300 mg/Kg) to cause drug-induced hepatic inflammation, and PBS injected mice served as the control group. The probe hCy-Tf-CA (50 μM, 100 μL) were administrated intravenously for in vivo fluorescence/photoacoustic dual-modal imaging. Images at predetermined time points were recorded on IVIS imaging system (Perkin Elmer, λex=675 nm, λem=760 nm) and InVision 256-TF imaging system (iThera Medical, λex=710 nm).
As shown in
In order to visually detect autoimmune hepatitis by fluorescence/photoacoustic dual-modal imaging, Balb/c female mice were intravenously injected with a single dose Con A (20 mg/Kg, dissolved in 100 μL PBS) for 2 h to induce early autoimmune hepatitis, and the remaining mice were treated with PBS solution as controls. hCy-Tf-CA was then intravenously injected into mice for in vivo imaging, and representative NIRF/PA dual-modal images of different treated mice were recorded at chosen times. Images at predetermined time points were recorded on IVIS imaging system (Perkin Elmer, λex=675 nm, λem=760 nm) and InVision 256-TF imaging system (iThera Medical, λex=710 nm).
As shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 202211349415.0 | Oct 2022 | CN | national |
