CN111518546A - Hypoxic microenvironment response fluorescent probe and preparation method and application thereof - Google Patents
Hypoxic microenvironment response fluorescent probe and preparation method and application thereof Download PDFInfo
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- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
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- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
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- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
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Abstract
A fluorescent probe responding to a hypoxic microenvironment and a preparation method and application thereof are disclosed. Cell experiments prove that the fluorescent probe provided by the invention can react with azo reductase overexpressed by cells under the hypoxic condition to generate obvious fluorescence enhancement, thereby realizing the fluorescence response of a hypoxic microenvironment. Meanwhile, the fluorescent probe provided by the invention also has the advantages of low background fluorescent signal, good biocompatibility, simple structure, definite composition, easiness in preparation and purification and the like, so that the fluorescent probe has good application prospect in the aspects of early diagnosis of tumors, preoperative evaluation, intraoperative navigation and the like.
Description
Technical Field
The invention relates to a hypoxic microenvironment response fluorescent probe and a preparation method and application thereof, belonging to the technical field of biochemical analysis.
Background
When the tumor grows rapidly, the oxygen demand of the tumor cells is increased, and the internal blood supply of the tumor is insufficient, so that the oxygen demand for proliferation and metabolism of the tumor cells is difficult to meetEventually, it leads to a significant hypoxic state (3-0.1% O) in some of the intratumoral microenvironments2). Hypoxia is a common feature of all solid tumors, and most tumors have a certain internal hypoxic microenvironment during growth. At present, the hypoxic state has become an important index for clinically assessing the degree of tumor deterioration. Therefore, development of a hypoxic microenvironment detection and even imaging technology which is convenient, sensitive and good in specificity is helpful for clinicians to diagnose tumors as soon as possible, determine the positions of patients and make appropriate treatment strategies, so that the treatment effect is improved. O is2Needle electrode method as clinical detection of local O2Concentration of gold standard by inserting a fine needle electrode into the tumor site, local O can be accurately measured2The concentration varies in different pathological states, but this method is not efficient and invasive to measure. In contrast, fluorescence imaging diagnostic methods with high spatial and temporal resolution, non-invasive, etc., represent a significant advantage in this respect. Research shows that the expression level of azoreductase in cells is greatly increased in the anaerobic state, so that the development of a specific azoreductase fluorescent probe can effectively evaluate the hypoxic degree of tumor tissues and cells, is further applied to the fluorescent imaging diagnosis of tumors, and has important significance for early diagnosis, preoperative evaluation and intraoperative navigation of tumors.
Disclosure of Invention
The invention aims to solve the technical problem of developing a hypoxic microenvironment detection and even imaging technology which is convenient, sensitive and good in specificity, improving the early diagnosis of a tumor by a clinician, determining the position of a patient and formulating a proper treatment strategy, thereby being beneficial to improving the treatment effect.
In order to solve the technical problems, the invention adopts the following technical scheme:
a fluorescent probe responding to a hypoxic microenvironment is shown as a formula I:
the synthetic method of the hypoxic microenvironment responsive fluorescent probe I comprises the following steps:
the method comprises the following steps: refluxing 3-hydroxy-N, N-diethylaniline and phthalic anhydride in toluene solution to react to obtain an intermediate 1;
step two: carrying out reflux reaction on the intermediate 1 and 6-amino-1, 2,3, 4-tetrahydro-1-naphthalenone in concentrated sulfuric acid to generate an intermediate 2;
step three: the intermediate 2 and N, N-diethylaniline are reacted with sodium nitrite to obtain the fluorescent probe shown in the formula I; see the following reaction scheme:
the fluorescent probe I responding to the hypoxic microenvironment is applied to imaging diagnosis.
The imaging diagnosis is a fluorescence imaging diagnosis in molecular imaging.
The beneficial effect of adopting above-mentioned technical scheme is:
1. the probe of the invention has low self-fluorescence background signal, and can improve the sensitivity of imaging detection;
2. the probe molecule of the invention has positive charge and has targeting effect on tumor tissue cell mitochondria with higher membrane potential than normal tissue cell mitochondria;
3. the invention can give fluorescence turn-on response to azo reductase in tumor tissue hypoxia microenvironment, thereby realizing the fluorescence imaging diagnosis of tumor;
4. the invention has low cytotoxicity and good biocompatibility;
5. the invention has simple structure, definite composition, easy preparation and purification, mass production and easy storage.
Drawings
FIG. 1 shows a process for producing an intermediate 1 of the present invention1H NMR Spectrum (400MHz, DMSO-d)6)。
FIG. 2 shows a process for producing intermediate 1 of the present invention13C NMR spectrum (100MHz, DMSO-d)6)。
FIG. 3 shows a process for preparing intermediate 2 of the present invention1H NMR Spectrum (400MHz, DMSO-d)6)。
FIG. 4 shows a process for producing intermediate 2 of the present invention13C NMR spectra (100MHz, CD)3OD)。
FIG. 5 is a HR-ESI-MS spectrum of intermediate 2 according to the present invention.
FIG. 6 shows a schematic diagram of the fluorescent probe of formula I1H NMR Spectrum (400MHz, DMSO-d)6)。
FIG. 7 shows a schematic representation of the fluorescent probe of formula I according to the invention13C NMR spectrum (100MHz, DMSO-d)6)。
FIG. 8 is a HR-ESI-MS spectrum of the fluorescent probe of formula I according to the present invention.
FIG. 9 is a graph showing the absorption spectrum of the fluorescent probe of formula I (20. mu.M) of the present invention in methanol.
FIG. 10 shows fluorescence spectra of a fluorescent probe of formula I of the present invention (10. mu.M) before and after reaction with sodium dithionite in PBS.
FIG. 11 shows the fluorescence spectra of the fluorescent probe of formula I of the present invention after reduction reaction with sodium dithionite of different concentrations.
FIG. 12 shows the fluorescence intensity of the fluorescent probe of formula I of the present invention after reaction with Sodium Dithionite (SDT) and other common bioreductive agents.
FIG. 13 shows fluorescence spectra of the fluorescent probe of formula I of the present invention after reaction with sodium dithionite and other different analytes.
FIG. 14 shows the activity of several common cells of the present invention after culturing with different concentrations of the fluorescent probe of formula I.
FIG. 15 shows fluorescence imaging of several common cells with different hypoxic time by the fluorescent probe of formula I of the present invention.
FIG. 16 is a graph showing the fluorescence imaging of the fluorescent probe of formula I under the effect of the azoreductase inhibitor rotenone on different cells in the anaerobic state.
Detailed Description
Exemplary embodiments of the present disclosure are described in more detail below with reference to the accompanying drawings. It should be noted that the following embodiments illustrate rather than limit the invention. These embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.
Example 1:
preparation of fluorescent probes of formula I:
this example provides a method for preparing a fluorescent probe represented by formula I, which comprises the following synthetic route:
the method comprises the following specific steps:
the method comprises the following steps: taking 1g of 3-hydroxy-N, N-diethylaniline and 0.9g of phthalic anhydride in 30mL of toluene solution, stirring and refluxing for 8h at 110 ℃, filtering to obtain mauve solid, dissolving in ethanol and recrystallizing to obtain the mauve solid, namely the intermediate 1, wherein the mauve solid is obtained1H NMR and13the C NMR spectrum is shown in the attached figures 1 and 2.
Step two: putting 40mL of concentrated sulfuric acid into a 125mL reaction bottle, placing the reaction bottle in a ice salt bath for 5min, weighing 0.6411g of 6-amino-1, 2,3, 4-tetrahydro-1-naphthalenone, slowly adding the weighed materials into sulfuric acid, stirring the mixture for 5min, then weighing 1.3924g of intermediate 1, adding the mixture into the system, stirring the mixture for 3min, carrying out reflux reaction at 90 ℃, and tracking the reaction process by using a thin layer chromatography. After the reaction is finished, pouring the reaction liquid into 150g of ice, adding 2mL of perchloric acid, stirring overnight, and filtering to obtain a purple black solid, namely an intermediate 21H NMR、13C NMR and HR-ESI-MS are shown in FIGS. 3 to 5.
Step three: 0.439g of intermediate 2 was added to 20mL of acetonitrile/water mixture (volume ratio 1:1), the reaction was stirred at 0 ℃ under nitrogen protection, 75mg of sodium nitrite was added, stirring was carried out for 10min, 115mg of trifluoroacetic acid was added and stirring was carried out for 30min, then 300mg of N, N-diethylaniline was dissolved in 5mL of acetonitrile and added to the above system, stirring was continued at 0 ℃ for 4h, finally 100mL of purified water was added and extraction was carried out with dichloromethane, the organic phase was dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. Separating the crude product by silica gel column chromatography, eluting with methanol and dichloromethane (v/v,1:50-1:20) to obtain blue solid, which is the fluorescent probe shown in formula I1H NMR、13C NMR and HR-ESI-MS are shown in FIGS. 6 to 8.
Example 2:
spectral properties of the fluorescent probe of formula I:
dissolving the fluorescent probe in methanol to prepare a 20 mu M solution, and measuring the absorption spectrum of the solution in an ultraviolet-visible spectrophotometer to obtain the result shown in figure 9, wherein the maximum absorption wavelength is 509 nm; in addition, the fluorescent probe (10 μ M) of the invention is reacted with a reducing agent Sodium Dithionite (SDT) in PBS, fluorescence emission spectra before and after the reaction are measured on a fluorescence spectrophotometer, the result is shown in figure 10, the background fluorescence signal of the probe is low, and after the reaction with the sodium dithionite, the fluorescence enhancement is obviously generated at 615 nm.
Example 3:
the fluorescent response of the fluorescent probe to SDT of formula I:
taking 50 mu L of methanol solution (1mmol/L) of the fluorescent probe shown in the formula I, placing the methanol solution into a 5mL colorimetric tube with a plug, adding a proper amount of PBS solution, respectively adding SDT solutions (50mmol/L and dissolved by double distilled water) with different volumes, fixing the volume to 5mL by the PBS solution, uniformly mixing to ensure that the concentration of the probe is 10 mu M, the concentration of the SDT is 0, 0.5, 1, 1.5 and 2 mu M respectively, and measuring the fluorescence emission spectrum after uniformly mixing. As a result, in FIG. 11, the fluorescence at 615nm gradually increased with the increase in the concentration of SDT.
Example 4:
selectivity of the fluorescent probe of formula I:
the fluorescent probe (10 μ M) shown in formula I reacts with SDT (25 μ M) and common biological reducing agents (Cys, GSH, Vc and NADH, the concentration is 100 μ M) in PBS respectively, and then the fluorescence emission spectrum is measured. The results are shown in FIG. 12, and only SDT can cause the fluorescence enhancement of the probe, which shows that biological reducing agents such as Cys and the like do not interfere with the fluorescence imaging of the probe. The fluorescent probe (10. mu.M) of formula I was separately mixed with SDT (25. mu.M) and common inorganic salt ions (including Co) in PBS2+、Cu2+、Fe2+、K+、Li+、Mg2 +、Ni2+、Pd2+、Zn2+And an anion: AC-、Br-、Cl-、CO3 2-、H2PO4 -、HPO4 2-、I-、NO2-、PO4 3-、S2 -、S2O3 2-And the concentration was 100. mu.M), and the fluorescence emission spectrum was measured. The results are shown in FIG. 13, which also shows that these inorganic salt ions do not interfere with fluorescence imaging of the probe.
Example 5:
cytotoxicity of the fluorescent probe of formula I:
several cells (16HBE, L02, 4T1, A549, HeLa) in logarithmic growth phase were taken according to 1 × 104The cells were seeded in 96-well plates at a density of one mL/mL and 5% CO at 37 ℃2And (3) incubating for 24 h. The probe was added after being dissolved in cell-grade DMSO so that the final concentration of the compound was 0, 5, 10, 20, 30, 40, 60, 80, 120. mu.M, and DMSO was not more than 0.2%, and after further incubation for 24 hours, 20. mu.L (5mg/L) of MTT solution (PBS dissolved) was added, reaction was carried out for 4 hours, all the culture medium was discarded, 150. mu.L of DMSO was added, shaking was performed sufficiently, and the absorbance value OD at 490nm was measured using a SpectraMax Plus 384 microplate reader. The survival rate of the cells is calculated,
survival rate ═ OD(sample)-OD(blank)]/[OD(No sample)-OD(blank)]×100％
Wherein OD(sample)Absorbance, OD, of fluorescent probe set at different concentrations(No sample)Absorbance, OD, of a probe set without addition of fluorescence(blank)Absorbance of blank wells. The results are shown in FIG. 14, and the fluorescent probe has no obvious toxicity to the cells, which indicates that the probe has good biocompatibility.
Example 6:
hypoxic cell fluorescence imaging of a fluorescent probe of formula I:
cells in logarithmic growth phase (16HBE, L02, 4T1, A549, HeLa) were taken according to 1 × 106The cells were seeded in 6-well plates at 37 ℃ in 5% CO2After 24h incubation under the conditions of (1). The probes were added after lysis with cell-grade DMSO, at a final concentration of 10. mu.M per well, DMSO notOver 0.2%, incubate for an additional 4h, after which time the medium is aspirated, washed 3 times with PBS, and 1mL of fresh medium is added to each well. Normal oxygen control wells were placed on the plate, and a clean coverslip was added to each of the remaining wells to create a hypoxic environment, allowing azoreductase to be overexpressed, timed out for 2, 4, 6, 8 hours respectively, and fluorescence pictures taken under an inverted fluorescence microscope. The results are shown in figure 15, and the fluorescence intensity of the probe in each cell is gradually enhanced along with the prolonging of the hypoxic time, which indicates that the probe can be used for the fluorescent imaging diagnosis of the hypoxic microenvironment of the cells.
Example 7:
and (3) performing fluorescence imaging on the hypoxic cells by using the fluorescent probe shown in the formula I under the action of rotenone:
after several cells (16HBE, L02, 4T1, a549, HeLa) were loaded with probes as described in example 6, normoxic group, hypoxic group, and hypoxic + rotenone (azo reductase inhibitor) group were set, wherein rotenone was added after dissolving in sterile water to a final concentration of 0.2 μ M each. Except for the normoxic group, the other two groups of cell holes are covered by a clean cover glass to block oxygen, a hypoxic environment is manufactured to ensure that the azoreductase is over-expressed, the cells are put into a carbon dioxide incubator to be continuously cultured for 8 hours and taken out, and a fluorescence picture is taken under an inverted fluorescence microscope. The results are shown in figure 16, the cells of the hypoxic group all show obvious fluorescent signals, and the rotenone can obviously inhibit the fluorescent signals of the hypoxic cells, which indicates that the hypoxic fluorescent response of the probe to the cells is caused by azoreductase.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.
Claims (7)
1. A fluorescent probe responding to hypoxic microenvironment, which is characterized in that: the structural formula is shown as formula I:
2. the method for synthesizing the hypoxic microenvironment-responsive fluorescent probe I as claimed in claim 1, comprising the steps of:
the method comprises the following steps: refluxing 3-hydroxy-N, N-diethylaniline and phthalic anhydride in toluene solution to react to obtain an intermediate 1;
step two: carrying out reflux reaction on the intermediate 1 and 6-amino-1, 2,3, 4-tetrahydro-1-naphthalenone in concentrated sulfuric acid to generate an intermediate 2;
step three: the intermediate 2 and N, N-diethylaniline are reacted with sodium nitrite to obtain the fluorescent probe shown in the formula I; see the following reaction scheme:
3. the method for synthesizing the hypoxic microenvironment-responsive fluorescent probe I as claimed in claim 2, wherein the method comprises the following steps: generating the intermediate 1 in the step one, wherein the generating of the intermediate 1 specifically comprises the following steps:
taking 1g of 3-hydroxy-N, N-diethylaniline and 0.9g of phthalic anhydride in 30mL of toluene solution, stirring and refluxing for reaction for 8h at 110 ℃, filtering to obtain mauve solid, dissolving with ethanol and recrystallizing to obtain the mauve solid, namely the intermediate 1.
4. The method for synthesizing the hypoxic microenvironment-responsive fluorescent probe I as claimed in claim 2, wherein the method comprises the following steps: generating the intermediate 2 in the step one, wherein the generating of the intermediate 2 specifically comprises the following steps:
putting 40mL of concentrated sulfuric acid into a 125mL reaction bottle, placing the reaction bottle in a ice salt bath for 5min, weighing 0.6411g of 6-amino-1, 2,3, 4-tetrahydro-1-naphthalenone, slowly adding the weighed materials into sulfuric acid, stirring the mixture for 5min, then weighing 1.3924g of intermediate 1, adding the mixture into the system, stirring the mixture for 3min, carrying out reflux reaction at 90 ℃, and tracking the reaction process by using a thin layer chromatography. After the reaction is finished, pouring the reaction solution into 150g of ice, adding 2mL of perchloric acid, stirring overnight, and filtering to obtain a purple black solid, namely the intermediate 2.
5. The synthesis method of the tumor double-targeting diagnosis and treatment combined photosensitizer I according to claim 2, is characterized in that: the generation of the fluorescent probe shown in the formula I in the step I specifically comprises the following steps:
0.439g of intermediate 2 was added to 20mL of acetonitrile/water mixture (volume ratio 1:1), the reaction was stirred at 0 ℃ under nitrogen protection, 75mg of sodium nitrite was added, stirring was carried out for 10min, 115mg of trifluoroacetic acid was added and stirring was carried out for 30min, then 300mg of N, N-diethylaniline was dissolved in 5mL of acetonitrile and added to the above system, stirring was continued at 0 ℃ for 4h, finally 100mL of purified water was added and extraction was carried out with dichloromethane, the organic phase was dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. And (3) carrying out column chromatography separation on the crude product by adopting a silica gel column, and taking methanol and dichloromethane as eluent (v/v,1:50-1:20) to obtain a blue solid, namely the fluorescent probe shown in the formula I.
6. The hypoxic microenvironment-responsive fluorescent probe of claim 1, wherein: the fluorescent probe I responding to the hypoxic microenvironment is applied to imaging diagnosis.
7. The hypoxic microenvironment-responsive fluorescent probe of claim 1, wherein: the imaging diagnosis is a fluorescence imaging diagnosis in molecular imaging.
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