CN110563719A - Synthesis method of small-molecule hypoxic probe - Google Patents

Abstract

The invention discloses a synthesis method of a micromolecular hypoxic probe, which comprises the steps of firstly adding IRDye800CW NHS into 100ul of dimethylformamide for dissolving, adding equimolar micromolecular targeting agent into a dye solution, and mixing the components according to the weight ratio of 1: adding triethylamine as a reaction catalyst into an equivalent of 6, placing the mixture on a magnetic stirrer, rotating at the speed of 300rmp, finishing the reaction for about 2 hours, adding ultrapure water into the product to 5ml, placing the mixture in a-80-DEG refrigerator overnight for the second day, freeze-drying the mixture in a freeze dryer, stopping freeze-drying after 12 hours, and collecting the product.

Description

synthesis method of small-molecule hypoxic probe

Technical Field

the invention relates to the field of biomedicine, in particular to a method for synthesizing a small-molecule hypoxic probe.

Background

In recent years, with the breakthrough of molecular imaging, these new imaging technologies have realized intuitive imaging at the molecular level in vivo by introducing molecular imaging probes capable of binding to specific targets in vivo, using one or more imaging technologies, such as radionuclide imaging, magnetic resonance imaging, optical imaging, photoacoustic imaging, and even multi-modal fusion imaging. The novel imaging technology converts the traditional invasive in vitro assay into image-based noninvasive living body detection, and can acquire accurate and quantitative three-dimensional spatial information based on cell and molecular level, so that noninvasive real-time three-dimensional quantitative tumor hypoxia becomes possible.

Disclosure of Invention

The invention aims to provide a method for synthesizing a small-molecule hypoxic probe.

A method for synthesizing micromolecular hypoxic probe, which is characterized in that the marked fluorescent dye is operated in the whole course of keeping out of the sun at room temperature, firstly, IRDye800CW NHS is added into 100ul of dimethylformamide for dissolution, equimolar micromolecular targeting agent is added into dye solution, and the ratio of the molecular weight of IRDye800CW NHS to the total molecular weight of the micromolecular targeting agent is calculated according to the proportion of 1: adding triethylamine serving as a reaction catalyst into an equivalent of 6, placing the mixture on a magnetic stirrer at the rotating speed of 300rmp, finishing the reaction for about 2 hours, adding ultrapure water into the product to 5ml, placing the mixture in a refrigerator with the temperature of-80 ℃ overnight, freeze-drying the mixture in a freeze dryer the next day, stopping freeze-drying after 12 hours, and collecting the product.

As a further scheme of the invention: the targeted uptake method of the small molecule hypoxic probe specifically comprises the following steps:

1) And (3) probe incubation: diluting the probe concentration to 50nM with fresh complete medium solution; discarding the original culture medium in the confocal culture dish, adding a probe solution, placing the probe solution in a cell incubator for incubation for 4 hours, discarding the solution in the dish, and washing the solution for 3 times by using a PBS (phosphate buffer solution);

2) fixing paraformaldehyde: a1 ml pipette sucks 500. mu.L of 4% paraformaldehyde, covers the cell surface for cell fixation, and places in a cell incubator for 15 minutes. Removing the paraformaldehyde solution, and carefully washing the cell surface 3 times with PBS solution;

3) Triton X-100 membrane rupture: 200. mu.L of 0.1% Triton X-100/PBS was added thereto, and the mixture was allowed to stand at room temperature for 3 to 5 minutes. After removing the solution, adding PBS to wash for 3 times;

4) Bovine Serum Albumin (BSA) blocking: adding 500 mu L of 0.01g/ml BSA, incubating with the cells for 30 min, and washing again with PBS 3 times;

5) staining cytoskeleton: adding 200 mu L of 100nM rhodamine-phalloidin working solution to stain cytoskeleton, and standing for half an hour; washing with PBS for 3 times;

6) And (3) cell nucleus staining: 15ug/ml of 4', 6-diamidino-2-phenylindole (DAPI) were added in an amount of 500. mu.L. Standing for 5 minutes, discarding the liquid in the dish, and washing with PBS for 3 times;

7) Refrigerating at 4 degrees for standby: adding 100 μ L PBS solution to keep the cells in the confocal culture dish moist, wrapping the confocal culture dish with tinfoil, and refrigerating at 4 deg.C in a refrigerator;

8) The whole process is finished under the condition of keeping out of the sun; the control experiment adopts FITC with the same concentration to incubate with cells, and the experimental method is consistent;

9) laser confocal imaging: by observation under a Zeiss LSM710 laser confocal microscope and analysis processing of Zen software, green represents FITC fluorescence, red represents rhodamine-phalloidin fluorescence, and blue represents DAPI fluorescence.

the invention has the beneficial effects that: the invention is from the in vitro cell level to the in vivo subcutaneous tumor model, from the living body near infrared optical 2D imaging to the photoacoustic 3D real-time imaging, and from the macroscopic imaging to the microscopic imaging, the CAIX-800 can be efficiently and specifically combined with a CAIX receptor so as to target hypoxic 5-8F tumor, and powerful guarantee can be provided for realizing the subsequent hypoxic imaging research of in situ and transfer models.

additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

drawings

in order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.

FIG. 1 shows the chemical reaction scheme for the synthesis of CAIX-800.

Detailed Description

Example 1

1 biological materials and test reagents

1) Chemical reagents: small molecule targeting agents for CAIX were synthesized at the medical college at hopkins university. The materials used in the chemical synthesis in this chapter: n, N-diethylethylamine (triethylamine), N-Dimethylformamide (DMF), etc., were purchased from Sigma-Aldrich. The fluorescent dyes IRDye800CW NHS (LI-COR) and FITC NHS (Thermo Scientific) were purchased from Shanghai Chu peptide biol.

2) Cell lines: the human nasopharyngeal carcinoma cell line 5-8F, C666-1 is provided by southern medical university.

3) Experimental animals: male BALB/C4-week-old nude mice were provided by Experimental animals technology, Inc., Vitongli, Beijing.

4) The relevant reagents in the experimental process of this chapter are listed in Table 1

TABLE 1 test reagents

2 instruments and equipments used in the experimental process are listed in Table 2

TABLE 2 Experimental Equipment

3 Experimental methods

3.1 Synthesis of CAIX-800 and CAIX-FITC

1) The labeled fluorescent dye is operated in the dark at room temperature. Firstly, IRDye800CW NHS was dissolved in 100ul of dimethylformamide, equimolar amounts of small molecule targeting agent were added to the dye solution, and the ratio of 1: 6, adding triethylamine as a reaction catalyst, placing the mixture on a magnetic stirrer at the rotating speed of 300rmp, and finishing the reaction for about 2 hours. The product was added to 5ml of ultrapure water, placed in a-80 ℃ freezer overnight for the next day to be lyophilized in a lyophilizer, and after 12h the lyophilization was stopped and the product was collected. The method for synthesizing CAIX-FITC for in vitro experiments is the same as that of CAIX-800.

3.2 characterization and in vitro Performance analysis of CAIX-800 and CAIX-FITC

1) The collected product was subjected to structure detection by Shanghai Chu peptide BioLimited. Mass spectrometry and High Performance Liquid Chromatography (HPLC) purity analysis were performed on both products.

2) And detecting the ultraviolet absorption wavelength of the solution by using an ultraviolet absorption spectrum measuring instrument. Deionized water is selected as a solvent to prepare samples to be detected with different concentrations. And (3) taking a quartz cuvette as a sample pool, transferring 5ml of solution to be detected by using a pipette with the measuring range of 1ml, and placing the sample to be detected in a sample detection groove. Before measurement, deionized water is used as a reference solution, a measurement baseline is zeroed, the initial wavelength of detection is 400nm and the end wavelength of detection is 1200nm, and the peak value of a spectrum is analyzed after the detection is finished.

3.3 Targeted uptake assay of nasopharyngeal carcinoma cells to Probe

(1) Cell recovery: quickly taking out the freezing tube from the liquid nitrogen tank, putting the freezing tube into a constant-temperature water bath kettle, setting the temperature to be 37 ℃, clamping the freezing tube by using forceps, and repeatedly shaking the freezing tube in the water bath kettle to quickly melt the cell freezing liquid in the freezing tube for about 40 s; after the cryopreservation tube is disinfected by alcohol, transferring the cryopreservation liquid into a culture dish containing a complete culture medium in a biological safety cabinet, slightly shaking the culture dish, putting the culture dish into a cell culture box for overnight, after cells adhere to the wall, removing a supernatant, slightly washing the cells for 3 times by using PBS (phosphate buffer solution) pre-warming buffer solution, adding a fresh culture medium, and continuing culturing.

(2) Cell passage: after the cells are in the logarithmic growth phase (the fusion degree is about 80%), discarding the old culture medium, washing the adherent cells for 3 times by using pre-warmed PBS (phosphate buffer solution), adding about 0.5mL of 0.25% pancreatin, putting the cells into a cell culture box for incubation for about 0.5min, and adding 2mL of complete culture medium to terminate the digestion process when observing the cell shape under a microscope and starting to become round; gently blow and beat adherent cells by using a 1mL pipette gun, when the cells completely fall off, transferring the cells into a 15mL centrifuge tube, centrifuging for 5min at the rotating speed of 800rpm, discarding the supernatant after the cells are precipitated, adding a fresh complete culture medium to gently blow and beat the cells uniformly, and then according to the following steps of 1: 3 to a new dish and continued culturing.

(3) Cell plating: and (3) placing a 35mm glass bottom dish (a special laser confocal culture dish) under the ultraviolet light of an ultra-clean bench for irradiation for 30 minutes for sterilization. When the growth of 5-8F and C666-1 cells reached 80%, the medium in the dish was discarded, the cell surface was carefully washed 3 times with pre-warmed PBS solution, 0.5ml of 0.25% trypsinized cells were added for 30s, and the cells were collected and centrifuged (800rpm,5 min). After centrifugation, the supernatant was discarded and 1mL of complete medium was added for resuspension. After the mixture is blown to be even, 200 mu L of cell suspension is absorbed by a liquid transfer gun with the dosage range of 200 mu L, and the cell suspension is slowly dripped into and covers the small hole in the confocal culture dish. The cells were placed in a cell incubator and incubated for 4 hours to allow the cells to adhere. Subsequently, 1ml of complete medium was slowly added to the edge of the confocal dish and left in the cell culture chamber overnight.

probe targeted uptake experiments:

1) And (3) probe incubation: the probe concentration was diluted to 50nM with fresh complete medium solution. And (3) discarding the original culture medium in the confocal culture dish, adding the probe solution, placing the confocal culture dish in a cell incubator for incubation for 4 hours, discarding the solution in the dish, and washing the confocal culture dish for 3 times by using a PBS (phosphate buffer solution).

2) fixing paraformaldehyde: a1 ml pipette sucks 500. mu.L of 4% paraformaldehyde, covers the cell surface for cell fixation, and places in a cell incubator for 15 minutes. The paraformaldehyde solution was removed and the cell surface was carefully washed 3 times with PBS solution.

3) Triton X-100 membrane rupture: 200. mu.L of 0.1% Triton X-100/PBS was added thereto, and the mixture was allowed to stand at room temperature for 3 to 5 minutes. After removal of the solution, PBS was added and washed 3 times.

4) Bovine Serum Albumin (BSA) blocking: 500. mu.L of 0.01g/ml BSA was added, incubated with the cells for 30 minutes, and washed again 3 times with PBS.

5) Staining cytoskeleton: adding 100nM rhodamine-phalloidin working solution 200. mu.L to stain cytoskeleton, and standing for half an hour. PBS was washed 3 times.

6) and (3) cell nucleus staining: 15ug/ml of 4', 6-diamidino-2-phenylindole (DAPI) were added in an amount of 500. mu.L. And standing for 5 minutes, discarding the liquid in the dish, and washing with PBS 3 times.

7) Refrigerating at 4 degrees for standby: adding 100 μ L PBS solution to keep the cells in the confocal culture dish moist, wrapping the confocal culture dish with tinfoil, and refrigerating at 4 deg.C in a refrigerator.

8) The whole process is finished under the condition of keeping out of the sun. In the control experiment, the cells were incubated with FITC at the same concentration, and the experimental method was consistent.

9) laser confocal imaging: by observation under a Zeiss LSM710 laser confocal microscope and analysis processing of Zen software, green represents FITC fluorescence, red represents rhodamine-phalloidin fluorescence, and blue represents DAPI fluorescence.

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Claims (2)

1. A method for synthesizing micromolecular hypoxic probe is characterized in that labeled fluorescent dye is operated in a dark way at room temperature in the whole process, firstly, IRDye800CW NHS is added into 100ul dimethylformamide for dissolution, equimolar micromolecular targeting agent is added into dye solution, and the ratio of the molecular weight of IRDye800CW NHS to the total molecular weight of the micromolecular targeting agent is calculated according to the proportion of 1: adding triethylamine serving as a reaction catalyst into an equivalent of 6, placing the mixture on a magnetic stirrer at the rotating speed of 300rmp, finishing the reaction for about 2 hours, adding ultrapure water into the product to 5ml, placing the mixture in a refrigerator with the temperature of-80 ℃ overnight, freeze-drying the mixture in a freeze dryer the next day, stopping freeze-drying after 12 hours, and collecting the product.

2. The targeted uptake method of the small molecule hypoxic probe according to claim 1, characterized by comprising the following steps:

1) And (3) probe incubation: diluting the probe concentration to 50nM with fresh complete medium solution; discarding the original culture medium in the confocal culture dish, adding a probe solution, placing the probe solution in a cell incubator for incubation for 4 hours, discarding the solution in the dish, and washing the solution for 3 times by using a PBS (phosphate buffer solution);

2) Fixing paraformaldehyde: a1 ml pipette sucks 500. mu.L of 4% paraformaldehyde, covers the cell surface for cell fixation, and places in a cell incubator for 15 minutes. Removing the paraformaldehyde solution, and carefully washing the cell surface 3 times with PBS solution;

3) Triton X-100 membrane rupture: 200. mu.L of 0.1% Triton X-100/PBS was added thereto, and the mixture was allowed to stand at room temperature for 3 to 5 minutes. After removing the solution, adding PBS to wash for 3 times;

4) bovine Serum Albumin (BSA) blocking: adding 500 μ L of 0.01g/ml BSA, incubating with the cells for 30 min, and washing again with PBS 3 times;

5) Staining cytoskeleton: adding 200 mu L of 100nM rhodamine-phalloidin working solution to stain cytoskeleton, and standing for half an hour; washing with PBS for 3 times;

6) And (3) cell nucleus staining: 15ug/ml of 4', 6-diamidino-2-phenylindole (DAPI) were added in an amount of 500. mu.L. Standing for 5 minutes, discarding the liquid in the dish, and washing with PBS for 3 times;

7) Refrigerating at 4 degrees for standby: adding 100 μ L PBS solution to keep the cells in the confocal culture dish moist, wrapping the confocal culture dish with tinfoil, and refrigerating at 4 deg.C in a refrigerator;

8) The whole process is finished under the condition of keeping out of the sun; the control experiment adopts FITC with the same concentration to incubate with cells, and the experimental method is consistent;

9) laser confocal imaging: by observation under a Zeiss LSM710 laser confocal microscope and analysis processing of Zen software, green represents FITC fluorescence, red represents rhodamine-phalloidin fluorescence, and blue represents DAPI fluorescence.