CN114736938A - A hypoxia-responsive alkyne-amine click polymerization method for intracellular polymerization and its application - Google Patents

Abstract

The invention provides an intracellular polymerization method of hypoxia-responsive alkyne-amine click polymerization and application thereof, belonging to the technical field of biochemistry. The intracellular polymerization method of hypoxia-responsive alkyne-amine click polymerization provided by the present invention includes the following steps: (1) culturing cells in a binary nitro compound solution under hypoxia; The cells after the oxygen culture are transferred to the binary alkynyl compound solution for normoxia culture; (3) step (2) the cells after the normoxia culture are transferred to the medium for normoxia culture, that is, the hypoxia-responsive alkynylamine click is realized Aggregate intracellular aggregates. The dibasic nitro compound in the present invention is reduced to a dibasic amine compound by the intracellular nitroreductase under hypoxic conditions, and the reduced dibasic amine compound and the dibasic alkynyl compound are polymerized in the cell, In this way, the anchoring of fluorescent small molecules is realized, resulting in the "lighting up" of the fluorescent signal, and the fluorescent imaging of cells is realized.

Description

A hypoxia-responsive alkyne-amine click polymerization method for intracellular polymerization and its application

technical field

The invention relates to the technical field of biochemistry, in particular to an intracellular polymerization method of hypoxia-responsive alkyne-amine click polymerization and application thereof.

Background technique

In 2014, in the process of expanding the click polymerization process based on alkyne monomers, the project of Academician Tang Benzhong established a spontaneous thiol-alkyne click polymerization reaction without catalyst. The reaction can be carried out under very mild conditions without the use of external catalysts. High molecular weight polymers were obtained in as little as 2 hours with yields as high as 97% (Macromalecules, 2014, 47: 1325-1333). However, when alkyne-amine click polymerization is constructed in tissue cells, carbonyl-activated alkyne monomers and diamines of tetraphenylethylene (TPE) can be taken up by normal cells and tumor cells at the same time, and spontaneously undergo intracellular polymerization. To achieve "light-on" imaging, it is difficult to distinguish between normal cells and tumor cells.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide an intracellular polymerization method of hypoxia-responsive alkyne-amine click polymerization and its application, so as to achieve precise localization of tumor cells.

In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

The invention provides an intracellular polymerization method of hypoxia-responsive alkyne-amine click polymerization, comprising the following steps:

(1) Hypoxic culture of cells in binary nitro compound solution;

(2) transferring the cells after the hypoxic culture in step (1) to a binary alkynyl compound solution for normoxia culture;

(3) In step (2), the cells after the normoxia culture are transferred to the culture medium for normoxia culture, that is, the intracellular polymerization of the hypoxia-responsive alkyne-amine click polymerization is realized;

The oxygen concentration of the hypoxic culture in step (1) is 1-20%.

Preferably, the structural formula of the binary nitro compound is

The structural formula of the binary alkynyl compound is

In formulas (I) to (III), R ₁ and R ₃ are selected from any one of (1) to (18); R ₂ and R ₄ are selected from hydrogen atoms or any of (19) to (22). One or more of:

Wherein, m, h and k are integers from 1 to 100; X is selected from N, P, O, S or Si elements; * represents a substitution position.

Preferably, the concentration of the binary nitro compound solution is 0.1 μmol/L˜150 mmol/L.

Preferably, the concentration of the cells is 1×10 ⁵ to 2×10 ⁵ cells/mL.

Preferably, the cells are tumor cells.

Preferably, the concentration of the binary alkynyl compound solution is 1 μmol/L˜100 mmol/L.

Preferably, the solvent of the binary nitro compound solution, the solvent of the binary alkynyl compound solution and the medium in step (3) are independently PBS phosphate buffered saline, HEPES buffer, DMEM cell culture medium and One of the 1640 cell culture media.

Preferably, the temperature of the hypoxic culture in step (1) and the normoxic culture in steps (2) and (3) are independently 4-37° C., and the time is independently 1-600 min.

The invention also provides an application of the hypoxia-responsive intracellular polymerization method of alkyne-amine click polymerization to realize cell fluorescence imaging.

The hypoxia-responsive intracellular polymerization method of alkyne-amine click polymerization provided by the invention utilizes the advantages of high reaction efficiency and mild conditions of alkyne-amine click polymerization, and can realize intracellular fluorescence imaging. The binary nitro compound in the present invention does not emit light or emits very weak light due to the nitro quenching effect, but the binary nitro compound is reduced by the intracellular nitroreductase under hypoxic conditions to After the diamine-based compound, the diamine-based compound emits stronger light. Then the diamine-based compound and the dibasic alkynyl compound are polymerized in the cell, so as to realize the anchoring of the fluorescent small molecule, resulting in the "lighting" of the fluorescent signal, and realizing the fluorescence imaging of the cell. Further, because nitroreductase is highly expressed in tumor cells compared with normal tissue cells, the concentration of diamine-based compounds in normal cells is relatively low, and the fluorescence imaging is relatively weak, which is compared with those with strong fluorescence. Image the tumor cells to differentiate. The above research results of the present invention can be used for hypoxia analysis research of in vitro cells.

Description of drawings

Fig. 1 is the fluorescence spectrogram of compound 1 and compound M1 in embodiment 2;

Fig. 2 is a confocal laser microscope image of hypoxia-responsive intracellular aggregation pre-incubated in 21% O ₂ , 5% O ₂ and 1% O ₂ and 5% O ₂ + dicoumarin in Example 3;

Fig. 3 is the laser confocal fluorescence image of normal cells (L929) and cancer cells (4T1, A549 and HeLa) treated by hypoxia-responsive intracellular polymerization in Example 4;

Figure 4 shows the effect of compound M2 on hypoxia-responsive intracellular aggregation in Example 5, wherein (A) is the fluorescence imaging of compound 1 without compound M2 after incubation, (B) is compound 1 added after incubation Fluorescence imaging of M2;

Fig. 5 is the toxicity test result of compound 1 to HaLe cell in embodiment 6;

Fig. 6 is the toxicity test result of compound M2 to HaLe cell in embodiment 6;

Fig. 7 is the toxicity test result of hypoxia-responsive intracellular polymerization to HaLe cells in Example 6;

8 is a scanning electron microscope characterization diagram of HeLa cells in the blank group and hypoxia-responsive intracellular aggregation (experimental group) in Example 6;

FIG. 9 is a cryo-electron microscope characterization diagram of HeLa cells in the blank group and hypoxia-responsive intracellular aggregation (experimental group) in Example 6;

10 is a confocal laser microscope image of HeLa cells co-stained with calreticulin in the blank group and hypoxia-responsive intracellular aggregation (experimental group) in Example 7;

Figure 11 is a confocal laser microscope image of HeLa cells co-stained with Annexin V and PI in the blank group and hypoxia-responsive intracellular aggregation (experimental group) in Example 8;

Figure 12 is the tumor volume change of mice in Example 9;

FIG. 13 shows changes in body weight of mice in Example 9. FIG.

Detailed ways

The invention provides an intracellular polymerization method of hypoxia-responsive alkyne-amine click polymerization, comprising the following steps:

(1) Hypoxic culture of cells in binary nitro compound solution;

(2) transferring the cells after the hypoxic culture in step (1) to a binary alkynyl compound solution for normoxia culture;

(3) In step (2), the cells after the normoxia culture are transferred to the culture medium for normoxia culture, that is, the intracellular polymerization of the hypoxia-responsive alkyne-amine click polymerization is realized;

The oxygen concentration of the hypoxic culture in step (1) is 1-20%.

In the present invention, the cells are mixed with the binary nitro compound solution to carry out hypoxic culture.

In the present invention, the oxygen concentration of the hypoxic culture in step (1) is 1-20%, preferably 3-10%, more preferably 5%.

所述二元炔基化合物的结构式优选为

In the present invention, the structural formula of the binary nitro compound is preferably

The structural formula of the binary alkynyl compound is preferably

In formulas (I) to (III), R ₁ and R ₃ are selected from any one of (1) to (18); R ₂ and R ₄ are selected from hydrogen atoms or any of (19) to (22). One or more of:

Wherein, m, h and k are integers from 1 to 100; X is selected from N, P, O, S or Si elements; * represents a substitution position.

In the present invention, the cells are preferably tumor cells, and the tumor cells are more preferably HeLa cells.

In the present invention, the concentration of the cells is preferably 1×10 ⁵ to 2×10 ⁵ cells/mL, and more preferably 1.5×10 ⁵ cells/mL.

In the present invention, the concentration of the binary nitro compound solution is preferably 0.1 μmol/L to 150 mmol/L, more preferably 1 μmol/L to 1500 μmol/L, and still more preferably 150 μmol/L.

In the present invention, the solvent of the binary nitro compound solution is preferably one or more of PBS phosphate buffered saline solution, HEPES buffer, DMEM cell culture medium and 1640 cell culture medium, more preferably PBS phosphate buffered saline Salt solution or DMEM cell culture medium, still more preferably DMEM cell culture medium containing 10% fetal bovine serum.

In the present invention, the temperature of the hypoxic culture is preferably 4 to 37°C, more preferably 37°C.

In the present invention, the time of the hypoxic culture is preferably 1-600 min, more preferably 60-180 min, and still more preferably 120 min.

In the present invention, after the cells are cultured in the binary nitro compound solution, the culture medium is discarded, and the cells are preferably washed with PBS phosphate buffered saline.

In the present invention, the dosage of the PBS phosphate buffered saline solution is preferably 0.5-1.5 ml, more preferably 1 ml.

In the present invention, the number of times of washing with the PBS phosphate buffered saline solution is preferably 2 to 4 times, more preferably 3 times.

Then, the washed cells were mixed with a binary alkynyl compound solution to perform normoxia culture.

In the present invention, the concentration of the binary alkynyl compound solution is preferably 1 μmol/L to 100 mmol/L, more preferably 5 μmol/L to 1000 μmol/L, and still more preferably 10 μmol/L.

In the present invention, the solute of the binary alkynyl compound solution is preferably one or more of PBS phosphate buffered saline solution, HEPES buffer, DMEM cell culture medium and 1640 cell culture medium, more preferably PBS phosphate buffer Saline solution or DMEM cell culture medium, still more preferably PBS phosphate buffered saline.

In the present invention, the temperature of the normoxia culture is preferably 4°C to 37°C, more preferably 37°C.

In the present invention, the time of the normoxia culture is preferably 1-600 min, more preferably 5-30 min, and still more preferably 10 min.

Finally, after the cells are cultured in the binary alkynyl compound solution, the culture medium is discarded, the culture medium is added, and the normoxia culture is continued to realize the intracellular polymerization of the hypoxia-responsive alkyne-amine click polymerization.

In the present invention, after the culture medium is discarded, the cells are preferably washed with PBS phosphate buffered saline.

In the present invention, the dosage of the PBS phosphate buffered saline solution is preferably 0.5-1.5 ml, more preferably 1 ml.

In the present invention, the number of times of washing with the PBS phosphate buffered saline solution is preferably 2 to 4 times, more preferably 3 times.

In the present invention, the culture medium is preferably one or more of PBS phosphate buffered saline, HEPES buffer, DMEM cell culture medium and 1640 cell culture medium, more preferably PBS phosphate buffered saline or DMEM cell culture The base is further preferably DMEM cell culture medium containing 10% fetal bovine serum.

In the present invention, the temperature of the normoxia culture is preferably 4 to 37°C, more preferably 37°C.

In the present invention, the time of the normoxia culture is preferably 1-600 min, more preferably 60-180 min, and still more preferably 120 min.

The invention also provides an application of the hypoxia-responsive intracellular polymerization method of alkyne-amine click polymerization to realize cell fluorescence imaging.

The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the protection scope of the present invention.

Example 1

HeLa cells up to 80% cell density were digested, resuspended in fresh DMEM (10% fetal bovine serum) medium, and diluted to ^1.5 x 105 cells/mL, inoculated in each confocal dish 1mL, after culturing until the cells adhered, add 150μM aromatic binary nitro compound 1 solution (the solvent is DMEM medium containing 10% fetal bovine serum), and place it at 37°C and 5% O ₂ for hypoxia culture 2h; after the hypoxic incubation, the culture medium was discarded at room temperature (20°C), and the cells were carefully washed 3 times with 1 ml of PBS phosphate buffered saline solution, and then the washed cells were added with a concentration of 10 μM binary alkynyl compounds M2 solution (the solvent is PBS), placed in normoxia (21% O ₂ ) for 10 min at 37°C; after the end of normoxia, the culture medium was discarded at room temperature (20°C), and 1 ml of PBS phosphate buffered saline was used for The cells were carefully washed 3 times, and then fresh 10% fetal bovine serum DMEM medium was added to the washed cells to continue culturing in normoxia at 37° C. for 2 h to complete the polymerization.

(即式Ⅰ中R₃为16所示的结构式、R₄为氧原子时的化合物)，二元炔基化合物M2为

(即式Ⅲ中R₁为8所示的结构式、R₂为氢原子时的化合物)。Wherein, the aromatic binary nitro compound 1 is:

(that is, in formula I, R ₃ is a compound of the structural formula represented by 16, and R ₄ is an oxygen atom), and the binary alkynyl compound M2 is

(that is, the compound in the formula III where R ₁ is the structural formula represented by 8 and R ₂ is a hydrogen atom).

The hypoxia response process is realized by the reduction of the nitro group to the amine group by the action of nitroreductase (NTR) and coenzyme (NAD(P)H) (to obtain an aromatic binary nitro compound M1), and the response is The process is as formula (1):

The acetylenic monomer (M2) and the diamine (M1) are subjected to the click hydrogen amination polymerization reaction to obtain the monomer P1, and the reaction equation is as formula (2):

Among them, the synthesis method of compound M1 can be synthesized according to the synthesis method in the published literature (Macromolecules 1997, 30:3547-3552); the synthesis method of compound M2 can be synthesized according to the synthesis method in the published literature (Macromolecules 2020, 53:2516-2525) .

Example 2

The luminescence of the aromatic dibasic nitro compound 1 and the aromatic dibasic amine compound M1 in Example 1 were detected, and the results are shown in FIG. 1 . It can be seen from Figure 1 that compound M1 has strong fluorescence, while compound 1 hardly emits light. It shows that the "lighting" of the fluorescent signal cannot occur before the binary nitro compound is converted into the binary amine compound.

Example 3

Add 1 mL of HeLa cells with a concentration of 1.5 × 10 ⁵ cells/mL to a laser confocal culture dish, and culture until the cells adhere to the wall. % fetal bovine serum in DMEM medium) at 37°C in normoxia (21% O ₂ ), 5% O ₂ , 1% O ₂ and 5% O ₂ + dicoumarin (nitroreductase inhibitory After incubation, the culture medium was discarded at room temperature (20°C), and the cells were carefully washed 3 times with 1 ml of PBS phosphate buffered saline solution, and then the washed cells were added with a concentration of 10 μM binary Alkynyl compound M2 solution (the solvent is PBS), and then both were incubated in normoxia at 37°C. After the normoxic incubation, the culture medium was discarded at room temperature (20°C), and the cells were carefully plated with 1 ml of PBS phosphate buffered saline. After washing 3 times, fresh DMEM medium containing 10% fetal bovine serum was added to the washed cells, and the cells were incubated under normoxia at 37°C to complete the polymerization. Fluorescence imaging of cells in each group was detected. The results are shown in Figure 2.

It can be seen from Figure 2 that under normoxic conditions (21% O ₂ ) HeLa cells treated with compound 1 could not detect the fluorescence signal, but only under hypoxic conditions (5% O ₂ , 1% O ₂ ) Fluorescent signal was detected. After nitroreductase was inhibited (TPE-2NO ₂ -dicoumarin group), the fluorescence was also significantly inhibited. It shows that compound 1 can be reduced to compound M1 by nitroreductase only in hypoxic environment, and realizes cell fluorescence imaging.

Example 4

The fluorescence imaging of normal cells L929 and cancer cells 4T1, A549 and HeLa treated with compound 1 under hypoxic conditions was determined (the specific experimental steps of each treatment are the same as those in Example 1). The results are shown in Figure 3.

In Figure 3, under the same conditions (37°C, 5% O ₂ ), cancer cells 4T1, A549, and HeLa all have obvious fluorescence imaging, indicating that this response process can also occur in cancer cells 4T1 and A549, and has a general effect on cancer cells. suitability. Meanwhile, the fluorescence of normal cell L929 is weak, indicating that the intracellular hypoxia-responsive polymerization reaction of the present invention has no obvious effect on normal cells.

Example 5

The effect of compound M2 on hypoxia-responsive intracellular aggregation was observed.

Treatment without compound M2: Add 1 mL of HeLa cells with a concentration of 1.5×10 ⁵ cells/mL to a laser confocal culture dish, and after the cells adhere to the wall, add a solution of aromatic binary nitro compound 1 with a concentration of 150 μM (The solvent is DMEM medium containing 10% fetal bovine serum), placed at 37°C and 5% O ₂ for 2h hypoxia culture. After the hypoxic culture, the culture medium was discarded at room temperature (20°C), and 1ml of The cells were carefully washed three times with PBS phosphate buffered saline, and then fresh DMEM medium containing 10% fetal bovine serum was added to the washed cells for 2 h at 37°C for normoxia. Confocal cytofluorescence imaging was then performed.

Treatment with compound M2: Add 1 mL of HeLa cells with a concentration of 1.5 × 10 ⁵ cells/mL to a laser confocal culture dish, and cultivate until the cells adhere to the wall. The concentration is 150 μM aromatic binary nitro compound 1 solution ( The solvent is DMEM medium containing 10% fetal bovine serum), placed at 37°C and 5% O ₂ under hypoxia for 2h. After the hypoxia culture, the culture medium was discarded at room temperature (20°C), and 1ml PBS was used. The cells were carefully washed three times with phosphate-buffered saline, and then the washed cells were added to a solution of 10 μM dibasic alkynyl compound M2 (the solvent was PBS), and incubated at 37 °C for 10 min under normoxia; After the end, the culture medium was discarded at room temperature (20°C), and the cells were carefully washed 3 times with 1 ml of PBS phosphate buffered saline, and then fresh DMEM medium containing 10% fetal bovine serum was added to the washed cells to continue. Incubate in normoxia at 37°C for 2 h to complete the polymerization. Confocal cytofluorescence imaging was then performed for comparison. The results are shown in Figure 4. Figure 4 (A) is the fluorescence imaging without compound M2, and Figure 4 (B) is the fluorescence imaging with compound M2 added.

It can be seen that in (A) of Figure 4, due to the easy expulsion of small molecules, the fluorescence after the reduction of the nitro group to the amine group quickly disappeared; while in (B), the intracellular fluorescence was fixed after the addition of compound M2. Thanks to the high efficiency of alkyne-amine polymerization, the introduction of compound M2 can achieve hypoxia-responsive intracellular polymerization with compound M1 generated in situ in cells to generate monomer P1, thereby immobilizing small fluorescent molecules (ie compound M1) to achieve Intracellular Stable Fluorescence Imaging.

Example 6

Cell viability was tested by MTT method. The detection principle is that succinate dehydrogenase (located in mitochondria) in well-grown cells is reduced by MTT reagent to purple crystals that are insoluble in water but soluble in DMSO, and the cell survival is reflected by detecting its absorption value at 570nm. state.

Add 1×10 ⁵ cells/ml of HeLa cell suspension to a 96-well plate, add 100 μl to each well, that is, 10,000 cells per well, and place the 96-well plate confluent with cells into an incubator (37°C) overnight. The next day, after checking that the cells adhere well, set aside.

Toxicity test of TPE-2NO ₂ (compound 1) under hypoxia: take a well-adhered 96-well cell plate, discard the culture medium, and add compound 1 solutions of different concentrations (50, 100, 150, 200, 250 μM) respectively ( The solvent is DMEM medium containing 10% fetal bovine serum), 100 μl/well, incubate at 37°C, 5% O ₂ for 2 h, then discard the culture medium, add DMEM (10% fetal bovine serum) medium, 1 ml / well, and continue to incubate for 22 h at 37 °C under normoxia. Then discard the culture medium, add 10 μg/ml MTT solution, 100 μl/well, and continue to incubate for 4 h at 37°C under normoxia. At this time, obvious purple crystals are formed at the bottom of the dish, then discard the culture medium, and add 100 μl to no holes. DMSO was dissolved, and then placed in a microplate reader to test its absorption value at 570 nm and the measured data were processed.

Toxicity test of activated alkyne (compound M2): take a well-adhered 96-well cell plate, discard the culture medium and add compound M2 solution (solvent is PBS) of different concentrations (1, 2, 4, 8, 16 μM), 100 μl/ Well, incubate for 10 min at 37°C under 20% O ₂ conditions, then discard the culture medium, add DMEM (10% fetal bovine serum) medium, 1ml/well, and continue to incubate at 37°C under normoxia for 24h. Then discard the culture medium, add 10 μg/ml MTT solution, 100 μl/well, and continue to incubate at 37°C for 4 hours under normoxia. At this time, obvious purple crystals are formed at the bottom of the dish, then discard the culture medium and add 100 μl of DMSO was dissolved, and then placed in a microplate reader to test its absorption value at 570 nm and the measured data were processed.

Intracellular aggregation toxicity test: take a well-adhered 96-well cell plate, discard the culture medium, add compound 1 (100 μl/well) at a concentration of 150 μM, incubate at 37°C, 5% O ₂ for 2 h, then discard the culture The solution was carefully washed three times with PBS, the culture medium was discarded, and different concentrations (1, 2, 4, 8, 16 μM) of compound M2 solution (solvent in PBS) were added, 100 μl/well, and incubated at 37 °C under normoxia for 10 min. Then the culture medium was discarded, and the incubation was continued at 37°C under normoxia for 22h. Then discard the culture medium, add 10 μg/ml MTT solution, 100 μl/well, continue to incubate at 37°C for 4 h in normoxia, at this time, obvious purple crystals are formed at the bottom of the dish, then discard the culture medium, and add 100 μl of DMSO to every hole. Dissolved, and then placed in a microplate reader to test its absorption value at 570nm and processed the measured data.

The cell viability ratio (VR) was estimated according to the following equation:

where A ₀ is the absorbance of cells without any drug, and A is the absorbance of cells incubated with the sample to be tested.

The results are shown in FIGS. 5 to 7 . Figure 5 shows the toxicity test results of compound 1 on HaLe cells, Figure 6 shows the toxicity test results of compound M2 on HaLe cells, and Figure 7 shows the toxicity test results of hypoxia-responsive intracellular polymerization on HaLe cells.

The results showed that the alkyne-amine click polymerization of TPE-2NH ₂ generated in situ after the reduction of TPE-2NO ₂ by intracellular nitroreductase and carbonyl-activated terminal alkynes had a strong toxic effect on HaLe cells, resulting in cell viability. reduce. However, compound 1 and compound M2 alone had no toxic effect on HaLe cells, and the cell survival rate was higher.

Taking the cells in the intracellular aggregation toxicity test as the control group, and taking the cells without any drug as the blank group, electron microscope scanning was performed, and the results are shown in Fig. 8 . It can be seen from Figure 8 that compared with the cells of the blank group, the cell structure of the experimental group was severely damaged.

Taking the cells in the intracellular aggregation toxicity test as the control group, and taking the cells without any drug as the blank group, cryo-electron microscopy was performed, and the results are shown in Figure 9. The severe disruption of the endoplasmic reticulum structure in the experimental group can be seen from Figure 9.

Example 7

488二次抗体进一步孵育2h。最后，在PBS中与Hoechst(10μg/ml)一起孵育5min。然后使用CLSM对细胞进行成像。对于Alexa

488，激发波长为488nm，发射滤光片为500～540nm；对于Hoechst，激发波长为405nm，发射滤光片为430～478nm。HeLa cells were grown adherently in confocal imaging dishes. At 37°C and 5% O ₂ , HeLa cells were first incubated with 150 μM compound 1 solution (the solvent was DMEM medium containing 10% fetal bovine serum) for 2 h, the culture medium was discarded, and 1 ml of room temperature (20° C.) The cells were carefully washed 3 times with PBS, and then a 10 μM solution of Compound 2 (solvent in PBS) was added. Incubate in normoxia at 37°C for 10 min, discard the culture medium, then add fresh DMEM (10% FBS) and incubate at 37°C for 24 h under normoxia, then fix the cells with 4% paraformaldehyde at room temperature 15min, permeabilized with 0.2% Triton X-100 for 15min at room temperature (20°C), then with 5% nonfat milk in 0.2% PBS-T (0.5g nonfat dry milk + 20μl of Tween20 were dissolved in 10ml of PBS buffer together solution prepared) and blocked for 1 h. Cells were incubated with anti-CRT antibody overnight at 4°C, then with Alexa at room temperature

488 secondary antibody was further incubated for 2h. Finally, incubated with Hoechst (10 μg/ml) in PBS for 5 min. The cells were then imaged using CLSM. for Alexa

488, excitation wavelength is 488nm, emission filter is 500-540nm; for Hoechst, excitation wavelength is 405nm, emission filter is 430-478nm.

Confocal fluorescence imaging experiments were performed on the above-treated cells, and at the same time, the above-mentioned treatments were also performed on HeLa cells without drug treatment, as a control group. The results are shown in Fig. 10. It can be seen from Fig. 10 that fluorescence appeared in the experimental group, indicating that its calreticulin had an eversion phenomenon.

Example 8

HeLa cells were grown adherently in confocal imaging dishes. At 37°C and 5% O ₂ , HeLa cells were first incubated with 150 μM compound 1 solution (the solvent was DMEM medium containing 10% fetal bovine serum) for 2 h, the culture medium was discarded, and 1 ml of room temperature (20° C.) The cells were carefully washed 3 times with PBS, and then a 10 μM solution of Compound 2 (solvent in PBS) was added. Incubate in normoxia at 37°C for 10 min, followed by incubation with fresh DMEM (10% FBS) medium for 120 min at 37°C in normoxia, followed by Annexin V-FITC (1/200 dilution) and 5 μg/mL PI. After incubation for 30 min, confocal fluorescence imaging was performed. The excitation wavelength of Annexin V-FITC is 488 nm, and the emission filter wavelength is 490-557 nm; the excitation wavelength of PI is 543 nm, and the emission filter is 557-680 nm.

Confocal fluorescence imaging experiments were performed on the above-treated cells, and at the same time, the above-mentioned treatments were also performed on HeLa cells without drug treatment, as a control group. The results are shown in Figure 11, the cells in the experimental group undergo apoptosis.

Example 9

BALB/C mice (6-8 weeks old) used in the experiments were purchased from the Guangdong Animal Center, and the experiments were carried out in the Laboratory Animal Center of South China Agricultural University. The mice entered the animal center for one week to complete routine medical examinations and fully adapt to the new environment before starting the experiment.

1. Divide the mice into five groups, with 4 mice in each group, divided into the experimental group and the control group.

2. Use 5% chloral hydrate as an anesthetic, inject 100 μL of anesthetic into each mouse intraperitoneally, and wait for coma;

3. Depilate, wash and dry respectively, and inoculate 100 μL (1×10 ⁷ ) 4T1 tumor cells in each group;

4. Use when the tumor grows to the desired size of ^50mm3 ;

5. In situ injection of drugs into subcutaneous tumor sites, among which, compound 1 and compound M2 were injected in experimental group 1, and 25 μL/50 mm ³ of compound 1 (concentration of 10 mM/PBS) was injected first, and after 6h interval, Inject 25μL/ ^50mm3 of compound M2 (concentration of 1mM/PBS); experimental groups 2, 3, and 4 were injected with 25μL/50mm3 ^of compound 1 (concentration of 10mM/PBS), 25μL/50mm3 ^of compound M2 (concentration of 1mM/PBS) PBS), 25 μL/50 mm ³ of compound M1 (at a concentration of 1 mM/PBS); the control group was injected with 25 μL/50 mm ³ of PBS.

6. Every other day, measure the body weight and tumor volume of the mice, and record the data for 15 days. The results are shown in Figures 12 and 13.

As can be seen from Figure 12, the tumor volume in each group increased to varying degrees and was finally higher than the initial tumor volume. Compared with PBS treatment, the inhibitory effect of Compound 1 and Compound M1 treatment on tumor growth was negligible, and M2 treatment also had a negligible effect on tumor growth. Similar to the tumor growth curve of PBS, it is worth noting that the anti-tumor effect of the compound 1/M2 treatment group (experimental group 1) was the most significant, and the tumor growth rate was very slow.

As can be seen from Figure 13, during the treatment process, the volume of mice in each group did not decrease, but showed a slight upward trend. The slowly rising body weight of mice indicated that various treatment methods had good biocompatibility.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made. It should be regarded as the protection scope of the present invention.

Claims (9)

1. A hypoxia response type alkyne-amine click polymerization intracellular polymerization method is characterized by comprising the following steps:

(1) carrying out hypoxic culture on cells in a binary nitro compound solution;

(2) transferring the cells cultured by the hypoxia in the step (1) into a binary alkynyl compound solution for normoxic culture;

(3) transferring the cells subjected to the normoxic culture in the step (2) to a culture medium for normoxic culture, namely realizing the intracellular polymerization of hypoxia response type alkyne-amine click polymerization;

the oxygen concentration of the hypoxic culture in the step (1) is 1-20%.

2. The hypoxic responsive alkyne-amine click polymerization of claim 1The intracellular polymerization method is characterized in that the structural formula of the binary nitro compound is shown in the specification

The structural formula of the binary alkynyl compound is

In the formulae (I) to (III), R₁，R₃Any one selected from (1) to (18); r₂，R₄Selected from hydrogen atoms or any one or more of (19) to (22):

wherein m, h and k are integers of 1-100; x is selected from N, P, O, S or Si element; indicates the substitution position.

3. The method for the intracellular polymerization of hypoxia-responsive alkyne-amine click polymerization of claim 2, wherein the concentration of the solution of the dinitrogen compound is 0.1 μmol/L to 150 mmol/L.

4. The method for the intracellular polymerization of hypoxia-responsive alkyne-amine click polymerization of claim 3, wherein the concentration of the cells is 1 x 10⁵～2×10⁵one/mL.

5. The hypoxia-responsive alkyne-amine click polymerized intracellular polymerization method of claim 4, wherein the cell is a tumor cell.

6. The hypoxia-responsive alkyne-amine click polymerization intracellular polymerization method of claim 5, wherein the concentration of the dibasic alkynyl compound solution is 1 μmol/L to 100 mmol/L.

7. The hypoxia-responsive alkyne-amine click polymerization intracellular polymerization method according to claim 6, wherein the solvent of the binary nitro compound solution, the solvent of the binary alkynyl compound solution and the medium in the step (3) are independently one of PBS phosphate buffered saline, HEPES buffer, DMEM cell medium and 1640 cell medium.

8. The method for intracellular polymerization of hypoxia-responsive alkyne-amine click polymerization according to any one of claims 1 to 7, wherein the temperature of the hypoxic culture in the step (1) and the temperature of the normoxic culture in the steps (2) and (3) are independently 4 to 37 ℃ and independently 1 to 600 min.

9. The application of the hypoxia-responsive alkyne-amine click polymerization intracellular polymerization method disclosed by any one of claims 1-8 in cell fluorescence imaging.

Images