CN113755160B - Stress mitochondria localization imaging probe composition and application thereof - Google Patents
Stress mitochondria localization imaging probe composition and application thereof Download PDFInfo
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Abstract
The invention discloses a stress mitochondria positioning imaging probe composition and application thereof, wherein the composition comprises a first probe and a second probe which can be gathered in a line granule body under the drive of transmembrane potential delta psi m of mitochondria, and the first probe and the second probe are connected in the mitochondria through biological orthogonal reaction to form a combined probe, so that when the transmembrane potential delta psi m is reduced or lost due to the stress reaction of the mitochondria, the combined probe still remains in the mitochondria, thereby realizing stable positioning imaging of the stress mitochondria.
Description
Technical Field
The invention belongs to the technical field of cell biology, and particularly relates to a stress mitochondria positioning imaging probe composition and application thereof.
Background
Mitochondria are ubiquitous organelles in mammals, and their transmembrane potential (Δψm) is critical for various organelle functions, from ATP production to cell signaling. The decrease in mitochondrial membrane potential Δψm is associated with a variety of cellular activities including oxidative stress, inflammation, and mitochondrial autophagy. Mitochondrial autophagy disorders are also associated with a variety of diseases such as parkinson.
The mitochondrial imaging probe in the prior art mainly consists of a cationic fluorophore and a dye combined with a lipophilic cation, is enriched in mitochondria under the drive of mitochondrial transmembrane potential delta psim, and has the defects that: when Δψm is reduced or lost, the probe can leak out of the mitochondria. Conventional mitochondrial imaging probes are difficult to use for stressed mitochondrial analysis because stressed mitochondria are often accompanied by loss of mitochondrial transmembrane potential.
Disclosure of Invention
The present invention aims to overcome the defects of the prior art and provide a composition for stimulating mitochondrial localization imaging probes.
It is another object of the present invention to provide a method of imaging the localization of the excited mitochondria.
It is a further object of the present invention to provide the use of the stress mitochondrial localization imaging probe composition described above.
The technical scheme of the invention is as follows:
a stress mitochondrial localization imaging probe composition comprising a first probe and a second probe capable of aggregating in vivo under the drive of mitochondrial transmembrane potential Δψm, wherein
A first probe having a first indicator group and a first targeting group that targets mitochondria;
a second probe having a second indicator group and a second targeting group for targeting mitochondria;
the first probe and the second probe are connected through bioorthogonal reaction in the mitochondria to form a combined probe, so that when the transmembrane potential delta psi m is reduced or lost due to the stress reaction of the mitochondria, the combined probe still remains in the mitochondria, thereby realizing stable positioning imaging of the stress mitochondria.
The other technical scheme of the invention is as follows:
a method of stress mitochondrial localization imaging comprising: the method comprises the steps that a first probe and a second probe which can be gathered in a line granule body under the drive of transmembrane potential delta psi m of mitochondria are incubated with living cells in sequence, so that the first probe and the second probe are gathered into appointed mitochondria, and are connected through bioorthogonal reaction in the mitochondria to form a combined probe, when the transmembrane potential delta psi m of the mitochondria is reduced or lost due to the stress reaction, the combined probe is still remained in the mitochondria, and therefore stable positioning imaging of the stress mitochondria is realized;
the first probe has a first indicator group and a first targeting group that targets mitochondria;
the second probe has a second indicator group and a second targeting group that targets mitochondria.
Still another technical scheme of the invention is as follows:
the application of the stress mitochondrial localization imaging probe composition in mitochondrial autophagy imaging analysis.
Still another technical solution of the present invention is as follows:
the application of the stress mitochondrial localization imaging probe composition in the assessment of the mitochondrial autophagy inducer.
The beneficial effects of the invention are as follows:
1. the first probe and the second probe can generate conjugation reaction in the line granule body, and the obtained combined probe is stably kept in the mitochondria, so that the stress mitochondrial marking is realized.
2. The first probe and the second probe in the invention have simple synthesis process and are easy to popularize.
3. The invention can be accurately positioned on the mitochondria of living cells, and has high signal to noise ratio;
4. the invention can also be used for monitoring specific parameters of stress mitochondria including pH through regulating and controlling structures.
Drawings
Fig. 1 is a schematic diagram of the operation of embodiment 1 of the present invention.
FIG. 2 shows a fluorescent probe according to example 1 of the present invention Az Red-ΔΨm、Red-ΔΨm、 DBCO Mitochondrial localization map of Blue Δψm in cells.
FIG. 3 shows a fluorescent probe according to example 1 of the present invention Az Red-ΔΨm、Red-ΔΨm、Red-ΔΨm/ DBCO Blue-ΔΨm、 Az Red-ΔΨm/ DBCO Retention profile of Blue Δψm after complete loss of mitochondrial potential.
FIG. 4 shows the fluorescent probe Red- Δψm +.1 of example 1 of the present invention DBCO Imaging of Blue Δψm remaining in mitochondria after complete loss of mitochondrial potential.
FIG. 5 shows a fluorescent probe according to example 1 of the present invention Az Emission spectrum of the response of ProRed- Δψm to pH.
FIG. 6 shows a fluorescent probe according to example 2 of the present invention Az ProRed-ΔΨm/ DBCO Blue- Δψm was used for cell mitochondrial autophagy imaging analysis under starvation induction conditions.
FIG. 7 shows a fluorescent probe according to example 3 of the present invention Az ProRed-ΔΨm/ DBCO Evaluation results of Blue- Δψm for mitochondrial autophagy inducers rapamycin, etoposide, rotenone and oligomycin.
Detailed Description
The technical scheme of the invention is further illustrated and described below by the specific embodiments in combination with the accompanying drawings.
In a preferred embodiment of the invention, the first targeting group comprises triphenylphosphine and positively charged rhodamine; the second targeting group includes triphenylphosphine and a positively charged rhodamine.
In a preferred embodiment of the invention, the first indicator group comprises coumarin and a pH-sensitive indicator group; the second indicator group includes coumarin and a pH-sensitive indicator group.
In a preferred embodiment of the invention, the first probe has an azide group and the second probe has an alkyne group, and the bioorthogonal reaction comprises an alkyne-azide click reaction.
Example 1
The specific principle of this embodiment is shown in fig. 1, and is specifically as follows:
(1) Preparation of fluorescent Probe at 10mM concentration Az Standard solution of Red- Δψm: weigh 7.3mg Az Red-Deltapsim is dissolved in 1mL of dimethyl sulfoxide to obtain 10mmol/L (10 mM) fluorescent probe Az Standard solution of Red- Δψm.
(2) Preparation of fluorescent Probe at 10mM concentration DBCO Standard solution of Blue- Δψm: weigh 9.6mg DBCO Blue-Deltapsim is dissolved in 1mL of dimethyl sulfoxide to obtain 10mmol/L (10 mM) fluorescent probe DBCO Standard solution of Blue- Δψm.
(3) Preparation of fluorescent Probe at 10mM concentration Az Standard solution of proRed- Δψm: weigh 11.4mg Az ProRed-Deltaψm was dissolved in 1mL of dimethyl sulfoxide to obtain 10mmol/L (10 mM) of fluorescent probe Az Standard solution of ProRed- Δψm.
Above-mentioned DBCO Blue-ΔΨm、 Az Red- Δψm, and Az ProRed- Δψm has the following structural formula:
(4) Taking the above Az Standard of ProRed- Δψm1. Mu.L of the solution was diluted to 1000. Mu.L (PBS, 10mM,30%DMSO) to obtain 10. Mu.M of a fluorescent probe Az ProRed- Δψm solution. 400 mu L of the solution is taken and fluorescent probes are detected Az ProRed- Δψm (10 μM) has a fluorescence emission spectrum with excitation wavelength of 580nm at different pH (4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5 and 9.0). The experimental results are shown in FIG. 5, fluorescent probe Az ProRed- Δψm is pH responsive.
(5) HeLa-Tom20-GFP + Cells (a genetically edited HeLa cell, which expresses Tom20-GFP protein, which is widely used to indicate the location of mitochondria in cells) were seeded in different 35mm glass bottom dishes. After the next day of cell attachment, these HeLa-Tom20-GFP cells were then isolated + Cells were incubated at 0.3. Mu.M Az Red- Δψm60min,0.3 μMRed- Δψm60min or 2 μM DBCO Blue- Δψm90min followed by 3 washes with fresh cell culture broth. Finally, 3 untreated cells were washed 3 times with PBS for confocal imaging. The results are shown in fig. 2, where all probes are well localized in mitochondria.
(6) HeLa cells in four 35mm glass bottom dishes were designated a, b, c, d, respectively. HeLa cells of a and b discs were treated with 0.3. Mu.M Red-. DELTA.ψm for 60min, heLa cells of c and d discs were treated with 0.3. Mu.M Az Red- Δψm was treated for 60min. The b-and d-disk cells were then treated with 2. Mu.M DBCO Blue- Δψm was incubated for 90min, and the a and c plates were untreated as controls. Four discs of cells were treated with 25 μm CCCP (all known in english as carboyl cyanide 3-chlorophenylhydrozon, a drug that depolarizes the mitochondria and deprives them of electrical potential) for one hour and confocal imaging was performed in the presence of CCCP. The experimental results are shown in FIG. 3, with only sequential additions Az Red- Δψm and DBCO the experimental group d of Blue Δψm had a signal retention after potential loss.
(7)HeLa-Tom20-GFP + Cells were seeded on 35mm glass bottom dishes and the next day will contain 0.3. Mu.M Az After incubating the cells in cell culture broth of Red- Δψm for 60min, the cells were washed three times with PBS. Then incubate 2. Mu.M in the same manner DBCO Blue- Δψm90 min. After three washes with PBS, 25. Mu.M CCCP was added and treated for 1h in the presence of CCCPConfocal imaging was performed. The experimental results are shown in FIG. 4, where the probe signal was completely co-localized with Tom20 GFP, demonstrating that the present invention is able to retain the fluorescent signal of the probe in the mitochondria that lose potential.
Example 2
Referring to step (7) of example 1, heLa-Lamp2-GFP was used + (Gene-edited HeLa cell, expressed Lamp2-GFP protein, lamp2 is a lysosomal membrane protein widely used to indicate the position of mitochondria in cells) were used sequentially Az ProRed- Δψm (2 μM,60 min) and DBCO blue-. DELTA.ψm (3. Mu.M, 90 min) was treated. After three washes with PBS, HBSS (Hank's Balanced Salt Solution) was added and incubated for 6 hours to starve the cells, and the control group was incubated with normal whole serum cell culture medium for the same 6 hours. Confocal imaging was performed after the time point. The results are shown in fig. 6, where the red light signal of starved HeLa cell lysosomes is greatly enhanced, demonstrating that the present invention can be used to indicate the extent of mitochondrial autophagy.
Example 3
Referring to step (7) of example 1, heLa-Lamp2-GFP was used + Sequentially use Az ProRed- Δψm (2 μM,60 min) and DBCO blue-. DELTA.ψm (3. Mu.M, 90 min) was treated and then washed three times with PBS. The cells were incubated with HBSS containing 15. Mu.M rapamycin, HBSS containing 15. Mu.M Rotenone, HBSS containing 15. Mu.M Etoposide, HBSS containing 15. Mu.M Oligomycin for 1 hour and confocal imaging. The experimental results are shown in fig. 7, and in this case, the red light of the experimental group containing the Rapmycin or Rotenone is enhanced, which indicates that the Rapmycin and the Rotenone can enhance autophagy under starvation conditions, and further proves that the invention has the potential of drug screening.
The foregoing description is only illustrative of the preferred embodiments of the present invention and is not to be construed as limiting the scope of the invention, i.e., the invention is not to be limited to the details of the invention.
Claims (4)
1. A stress mitochondrial localization imaging probe composition characterized by: comprising a first probe and a second probe capable of being aggregated in vivo under the drive of mitochondrial transmembrane potential Δψm, wherein
A first probe of Az Red- Δψm or Az ProRed-ΔΨm， Az Red- Δψm has the structural formula of
，R=N 3 ， Az ProRed- Δψm is of the formula +.>
；
A second probe of DBCO Blue-Deltaψm of the structural formula
；
The first probe and the second probe are connected in the mitochondria through alkynyl-azido click reaction to form a combined probe, so that when the transmembrane potential delta psi m is reduced or lost due to mitochondrial stress reaction, the combined probe still remains in the mitochondria, thereby realizing stable positioning imaging of the stress mitochondria.
2. The stress mitochondria positioning imaging method is characterized in that: comprising the following steps: the method comprises the steps that a first probe and a second probe which can be gathered in a line particle body under the drive of transmembrane potential delta psi m of mitochondria are incubated with living cells in sequence, so that the first probe and the second probe are gathered into appointed mitochondria, and are connected through alkynyl-azido click reaction in the mitochondria to form a combined probe, so that when the transmembrane potential delta psi m is reduced or lost due to mitochondrial stress reaction, the combined probe still remains in the mitochondria, and stable positioning imaging of the stress mitochondria is realized;
the first probe is Az Red- Δψm or Az ProRed-ΔΨm， Az Red- Δψm has the structural formula of
，R=N 3 ， Az ProRed- Δψm is of the formula +.>
；
The second probe is DBCO Blue-Deltaψm of the structural formula
。
3. Use of the stressed mitochondrial localization imaging probe composition of claim 1 in a mitochondrial autophagy imaging assay.
4. Use of the stress mitochondrial localization imaging probe composition of claim 1 in the assessment of mitochondrial autophagy inducers.
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| CN107417714A (en) * | 2017-07-06 | 2017-12-01 | 南开大学 | A kind of highly sensitive fluorescence probe and its synthetic method and application based on BODIPY |
| CN109810138A (en) * | 2018-12-26 | 2019-05-28 | 浙江工业大学 | A kind of targetted mitochondria Small-molecule probe and its preparation method and application |
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| CN107417714A (en) * | 2017-07-06 | 2017-12-01 | 南开大学 | A kind of highly sensitive fluorescence probe and its synthetic method and application based on BODIPY |
| CN109810138A (en) * | 2018-12-26 | 2019-05-28 | 浙江工业大学 | A kind of targetted mitochondria Small-molecule probe and its preparation method and application |
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