Nile red fluorescence labeled polyglycolic acid and application thereof
By mixing Nile red with a specific solvent and combining with polyglycolic acid, the problems of staining stability and fluorescence preservation in in vivo tracing research of polyglycolic acid were solved, and effective observation of its distribution and metabolism in vivo was achieved. , laying the foundation for its safety in the body and drug delivery research.
Patent Information
- Application Number
- CN202411279566.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-12
- Publication Date
- 2024-12-13
AI Technical Summary
The prior art is difficult to avoid the staining of cells or tissues by Nile red dye while maintaining fluorescence ability, and then be used for traceability of polyglycolic acid in vivo.
Nile red fluorescent labeled polyglycolic acid dispersion mixed with water and dimethyl sulfoxide was prepared by mixing Nile red with anhydrous ethanol and acetone into a staining solution, and after filtering and washing, Nile red fluorescently labeled polyglycolic acid was prepared. , and the dyeing conditions combined with heating and cooling cycle are used to ensure dyeing stability and fluorescence intensity.
The stable staining of nano-level polyglycolic acid was successfully achieved, avoiding the staining of cells or tissues in the body, while maintaining good fluorescence ability. It is used to observe its distribution and metabolic status in the body, and for the study of the safety and targeted delivery of biological vectors. Provides a basis.
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Figure CN119119697A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology, and particularly relates to a method for labeling polyglycolic acid with Nile red fluorescence, polyglycolic acid labeled with Nile red fluorescence and application thereof, and a tracer preparation. Background Art
[0002] Polyglycolic acid (PGA), also known as polyhydroxyacetic acid, is an aliphatic polyester polymer material with the least carbon number per unit, a completely decomposable ester structure, and the fastest degradation rate. Unlike traditional polymer materials with stable performance such as plastics and rubber, PGA as a material gradually degrades after a certain period of use. It is one of the polymer materials with the best known degradation performance and one of the few polymer materials that degrade rapidly in the marine environment. Its degradation products are water and CO2, which are harmless to the environment and have great significance for solving the current severe white pollution problem. At the same time, due to its good biodegradability and biocompatibility, PGA is also a biodegradable polymer widely used in the medical field. It is widely used in medical devices, drug delivery systems, tissue engineering and repair, and sutures. Due to its wide application in medicine, studying its metabolic distribution process in the body is particularly important for its targeting and safety as a drug carrier. However, due to its high crystallinity, high density and high degradation rate, PGA is more difficult to be labeled and stained than conventional polymer materials.
[0003] Nile Red (NR) is a chemical dye that belongs to the benzocyclopentadiene structure. It is soluble in organic solvents, but has low solubility in water. Nile Red has high brightness and fluorescence stability under a fluorescence microscope, so it is widely used in the fields of life sciences and materials science. By injecting Nile Red solution into the animal body, it is absorbed and stained by tissues and cells in the blood circulation, and the animal can be marked and visualized. At the same time, in the field of materials, NR can also stain ordinary micron-level polymer materials and use them for quantitative analysis. However, there has been no report on the use of NR to stain nano-level materials, especially PGA, and then transfer it into the body to avoid staining of cells or tissues in the body while maintaining good luminescence ability, and then it can be used for PGA in vivo tracing. Summary of the invention
[0004] In view of this, the object of the present invention is to provide a method for labeling polyglycolic acid with Nile red fluorescence, which can obtain stably stained polyglycolic acid. After the polyglycolic acid is transferred into the body, it can avoid staining of cells or tissues in the body while maintaining good luminescence ability, and can thus be used for PGA tracing in vivo.
[0005] The present invention provides a method for labeling polyglycolic acid with Nile red fluorescence, comprising the following steps:
[0006] Mixing Nile red with a first solvent to obtain a dye solution; the first solvent consists of anhydrous ethanol and acetone;
[0007] Mixing polyglycolic acid with a second solvent to obtain a polyglycolic acid dispersion; the second solvent consists of water and dimethyl sulfoxide;
[0008] The polyglycolic acid dispersion liquid is mixed with the dyeing liquid for dyeing, and then filtered and washed to obtain polyglycolic acid labeled with Nile red fluorescence.
[0009] In some embodiments, the volume ratio of anhydrous ethanol to acetone is (1:2) to (2:1).
[0010] In some embodiments, the volume ratio of water to dimethyl sulfoxide is (1:2) to (2:1).
[0011] In some embodiments, the particle size of the polyglycolic acid is 472.5 nm to 1000 nm.
[0012] In some embodiments, the concentration of Nile red in the staining solution is 0.5 mg / mL to 2 mg / mL.
[0013] In some embodiments, the concentration of polyglycolic acid in the polyglycolic acid dispersion is 1 mg / mL to 3 mg / mL.
[0014] In some embodiments, the volume ratio of the dye solution to the polyglycolic acid dispersion is (1:2) to (2:1).
[0015] In some embodiments, the dyeing conditions are heating at 30-42°C for 2-4 hours, cooling to 22-28°C, cycling the heating and cooling, and then soaking at room temperature. Preferably, the heating and cooling are performed once as a cycle, and the number of cycles is 2-4 times. Preferably, the soaking time at room temperature is 8-12 hours.
[0016] The present invention also provides polyglycolic acid labeled with Nile red fluorescence, and the polyglycolic acid labeled with Nile red fluorescence is prepared by the method for labeling polyglycolic acid with Nile red fluorescence.
[0017] The present invention also provides the use of the polyglycolic acid labeled with Nile red fluorescence in the preparation of a tracer preparation. Preferably, the tracer preparation is an in vivo tracer preparation.
[0018] In one embodiment, the in vivo includes in vitro / ex vivo cells, in vitro / ex vivo tissues, or in vivo in humans or animals.
[0019] The present invention also provides a tracer preparation, which comprises polyglycolic acid labeled with Nile red fluorescence prepared by the method for labeling polyglycolic acid with Nile red fluorescence.
[0020] Preferably, the tracer preparation is an in vivo tracer preparation.
[0021] More preferably, the in vivo includes in vitro / ex vivo cells, in vitro / ex vivo tissues or in vivo of humans or animals.
[0022] Beneficial effects of the present invention:
[0023] The present invention first mixes Nile red with a first solvent to obtain a dye solution, mixes polyglycolic acid with a second solvent to obtain a polyglycolic acid dispersion, then mixes the polyglycolic acid dispersion with the dye solution for dyeing, and obtains fluorescently labeled polyglycolic acid after filtering and washing. The present invention successfully dyes nano-scale polyglycolic acid by selecting a suitable solvent and an appropriate raw material ratio, and the obtained fluorescently labeled polyglycolic acid is stable in dyeing. After entering the tissue or cell, not only can its fluorescence intensity be maintained, but also the tissue and cell can be prevented from being stained. The state of polyglycolic acid being taken up by the tissue or cell and the distribution and metabolism of polyglycolic acid in the tissue or cell at different times can be observed, which plays a good tracing role in the distribution and metabolism of polyglycolic acid in the body, and lays a foundation for the safety research of polyglycolic acid as a biological carrier in the body and the research on targeted delivery of drugs. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 The graphs show the changes of THP-1 cells after co-incubation with Nile red-labeled PGA under confocal microscopy; wherein, a is the observation graph of THP-1 cells incubated with PGA for 0 h; b is the observation graph of THP-1 cells incubated with PGA for 1 h; c is the observation graph of a single THP-1 cell incubated with PGA for 0 h; d is the observation graph of a single THP-1 cell incubated with PGA for 1 h; from left to right in each row of graphs are bright field images for observing cell morphology; Hoechst staining observation graphs for observing the cell nuclei of co-incubated THP-1 cells; Nile red staining observation graphs for observing the distribution of Nile red-stained PGA in THP-1 cells; and cell fusion graphs for observing the distribution and relative positions of cell nuclei and PGA in THP-1 cells.
[0025] Figure 2 This is a diagram showing the distribution of PGA labeled with Nile red after being ingested into mice, where Heart represents heart, Liver represents liver, Spleen represents spleen, Lung represents lung, Kidney represents kidney, Stomach represents stomach, and Intestine represents intestine. DETAILED DESCRIPTION
[0026] Typical embodiments that embody the features and advantages of the present invention will be described in detail in the following description. It should be understood that the present invention can have various changes in different embodiments without departing from the scope of the present invention, and the descriptions therein are essentially used as illustrations rather than to limit the present invention.
[0027] In one aspect, the present invention provides a method for labeling polyglycolic acid with Nile red fluorescence, comprising the following steps:
[0028] Mixing Nile red with a first solvent to obtain a dye solution; the first solvent consists of anhydrous ethanol and acetone;
[0029] Mixing polyglycolic acid with a second solvent to obtain a polyglycolic acid dispersion; the second solvent consists of water and dimethyl sulfoxide;
[0030] The polyglycolic acid dispersion liquid is mixed with the dyeing liquid for dyeing, and then filtered and washed to obtain polyglycolic acid labeled with Nile red fluorescence.
[0031] The present invention has no particular limitation on the sources of Nile red, polyglycolic acid, anhydrous ethanol, acetone, water or dimethyl sulfoxide, and conventional commercial products in the art may be used.
[0032] In the present invention, the particle size of the polyglycolic acid is 472.5 nm to 1000 nm. In a specific embodiment, the average particle size of the polyglycolic acid is 687.5 nm, and the polymer dispersion index (PDI) is 0.182.
[0033] In the present invention, the first solvent is composed of anhydrous ethanol and acetone, and the volume ratio of the anhydrous ethanol to acetone is (1:2) to (2:1), preferably (1:1.5) to (1.5:1), and more preferably 1:1.
[0034] In the present invention, the concentration of Nile red in the dyeing solution is preferably 0.5 mg / mL to 2 mg / mL, more preferably 0.8 mg / mL to 1.5 mg / mL, and further preferably 1 mg / mL. The use of the first solvent of the present invention to dissolve Nile red can better dye PGA, and the effect is significantly better than a single solvent or other solvent ratios.
[0035] In the present invention, the second solvent is composed of water and dimethyl sulfoxide, and the volume ratio of water to dimethyl sulfoxide is (1:2) to (2:1), preferably (1:1.5) to (1.5:1), and more preferably 1:1.
[0036] In the present invention, the concentration of polyglycolic acid in the polyglycolic acid dispersion is preferably 1 mg / mL to 3 mg / mL, more preferably 1.5 mg / mL to 2.5 mg / mL, and further preferably 2 mg / mL. The second solvent of the present invention can solve the problem that PGA floats on the water surface and cannot be dyed, thereby obtaining a better dyeing effect.
[0037] In the present invention, when the polyglycolic acid dispersion and the dyeing solution are mixed for dyeing, the volume ratio of the dyeing solution to the polyglycolic acid dispersion is preferably (1:2) to (2:1), more preferably (1:1.5) to (1.5:1), and most preferably 1:1.
[0038] As an embodiment, the dyeing condition is preferably heated at 30-42°C for 2-4h, then cooled to 22-28°C, the heating and cooling are circulated, and then soaked at room temperature. The heating temperature is more preferably 38-41°C, and more preferably 40°C. The heating time is more preferably 2.5-3.5h, and more preferably 3h. The temperature reached by the cooling is more preferably 23-26°C, and more preferably 25°C. In the present invention, the heating and cooling are performed once as a cycle, and the number of cycles is preferably 2-4 times, and more preferably 3 times. The soaking time at room temperature is preferably 8-12h, more preferably 9-11h, and more preferably 10h, and the room temperature refers to 20-25°C, such as 20°C, 21°C, 22°C, 23°C, 24°C, and 25°C. The present invention adopts a heating and cooling cycle combined with a room temperature soaking method for dyeing, which can achieve a better dyeing effect while avoiding PGA degradation, so that the dyed PGA can still stably emit light after entering the body and avoid tissues and cells from being stained. The present invention has no particular limitation on the heating method, and heating may be performed in a water bath, for example.
[0039] As an embodiment, after dyeing, filtration is performed to remove impurities, and washing is performed to remove adsorbed dyes to obtain polyglycolic acid labeled with Nile red fluorescence. The present invention has no particular limitation on the filtering and washing methods, and conventional methods in the art can be used.
[0040] In another aspect, the present invention further provides polyglycolic acid labeled with Nile red fluorescence, wherein the polyglycolic acid labeled with Nile red fluorescence is prepared by the above method for labeling polyglycolic acid with Nile red fluorescence.
[0041] In another aspect, the present invention also provides the use of the polyglycolic acid labeled with Nile red fluorescence in the preparation of body tracer preparations.
[0042] Preferably, the tracer preparation may be an in vivo tracer preparation.
[0043] After the polyglycolic acid labeled with Nile red fluorescence of the present invention enters tissues or cells, it can not only maintain its fluorescence intensity, but also prevent the tissues and cells from being stained. It is possible to observe the uptake of polyglycolic acid by tissues or cells and the distribution and metabolism of polyglycolic acid in tissues or cells at different times, and play a good tracing role in the distribution and metabolism of polyglycolic acid in the body.
[0044] In the present invention, the in vivo preferably includes in vitro / ex vivo cells, in vitro / ex vivo tissues or in vivo of humans or animals.
[0045] In another aspect, the present invention further provides a tracer preparation, wherein the tracer preparation comprises polyglycolic acid labeled with Nile red fluorescence prepared by the above method for labeling polyglycolic acid with Nile red fluorescence.
[0046] Preferably, the tracer preparation is an in vivo tracer preparation.
[0047] More preferably, the in vivo includes in vitro / ex vivo cells, in vitro / ex vivo tissues or in vivo of humans or animals.
[0048] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.
[0049] In the following embodiments, unless otherwise specified, all of them are conventional methods.
[0050] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.
[0051] Example 1
[0052] a. Prepare a staining solution: Mix 25 mL of anhydrous ethanol and 25 mL of acetone to obtain a first solvent, and mix Nile red with the first solvent to obtain a staining solution having a Nile red concentration of 1 mg / mL.
[0053] b. Preparation of PGA dispersion: 25 mL of water and 25 mL of dimethyl sulfoxide were mixed to obtain a second solvent, and PGA having an average particle size of 687.5 nm was mixed with the second solvent to obtain a PGA dispersion having a PGA concentration of 2 mg / mL.
[0054] c. Mix the PGA dispersion and the dye solution, heat in a 40°C water bath for 3 hours, and cool to 25°C. The above water bath heating and cooling are performed once as one cycle, and the cycle is repeated 3 times, and then soak at room temperature for 10 hours.
[0055] d. The dyed solution is filtered to obtain PGA that retains fluorescence, and washed with deionized water to remove the adsorbed dye, obtaining PGA that retains fluorescence, showing red or purple.
[0056] Example 2
[0057] a. Prepare a staining solution: Mix 15 mL of anhydrous ethanol and 30 mL of acetone to obtain a first solvent, and mix Nile red with the first solvent to obtain a staining solution having a Nile red concentration of 0.5 mg / mL.
[0058] b. Preparation of PGA dispersion: 60 mL of water and 30 mL of dimethyl sulfoxide were mixed to obtain a second solvent, and PGA having an average particle size of 687.5 nm was mixed with the second solvent to obtain a PGA dispersion having a PGA concentration of 1 mg / mL.
[0059] c. Mix the PGA dispersion and the dye solution, heat in a 42°C water bath for 2 hours, and cool to 28°C. The above water bath heating and cooling are performed once as one cycle, and the cycle is repeated twice, and then soak at room temperature for 12 hours.
[0060] d. The dyed solution is filtered to obtain PGA that retains fluorescence, and washed with deionized water to remove the adsorbed dye, obtaining PGA that retains fluorescence, showing red or purple.
[0061] Example 3
[0062] a. Prepare a staining solution: Mix 60 mL of anhydrous ethanol and 30 mL of acetone to obtain a first solvent, and mix Nile red with the first solvent to obtain a staining solution having a Nile red concentration of 2 mg / mL.
[0063] b. Preparation of PGA dispersion: 15 mL of water and 30 mL of dimethyl sulfoxide were mixed to obtain a second solvent, and PGA having an average particle size of 687.5 nm was mixed with the second solvent to obtain a PGA dispersion having a PGA concentration of 3 mg / mL.
[0064] c. Mix the PGA dispersion and the dye solution, heat in a 30°C water bath for 4 hours, and cool to 22°C. The above water bath heating and cooling are performed once as one cycle, and the cycle is repeated 4 times, and then soak at room temperature for 8 hours.
[0065] d. The dyed solution is filtered to obtain PGA that retains fluorescence, and washed with deionized water to remove the adsorbed dye, obtaining PGA that retains fluorescence, showing red or purple.
[0066] Comparative Example 1
[0067] The only difference from Example 1 is that the first solvent is replaced with 50 mL of anhydrous ethanol, and the remaining steps are the same as Example 1. Results The prepared PGA has only weak fluorescence and cannot be stably maintained.
[0068] Comparative Example 2
[0069] The only difference from Example 1 is that the first solvent is replaced with 50 mL of acetone, and the remaining steps are the same as Example 1. Results The prepared PGA has only weak fluorescence and cannot be stably maintained.
[0070] Comparative Example 3
[0071] The only difference from Example 1 is that the second solvent is replaced with 50 mL of dimethyl sulfoxide, and the remaining steps are the same as Example 1. Results The prepared PGA has only weak fluorescence and cannot be stably maintained.
[0072] Experimental Example 1 Distribution of PGA after ingestion into THP-1 cells
[0073] THP-1 cells are a commonly used acute monocytic leukemia cell line in laboratories and are an ideal tool for studying immunity and inflammation. The Nile red-labeled PGA prepared in Example 1 was co-incubated with THP-1 cells, and the THP-1 cell nuclei were stained with Hoechst to observe the intracellular distribution of PGA labeled with Nile red at 0 h and 1 h after co-incubation with cells. The specific steps are as follows: THP-1 cells were incubated with 10 5 The density of cells / plate was inoculated in a confocal microscopy dish, and the cells were treated with 10 μg / mL Hoechst dye for 5 min and then washed with PBS. The cells were incubated with PGA labeled with Nile red at a concentration of 1 μg / mL at 37°C, 5% CO2 for 0 h and 1 h. After incubation, the cells were washed with PBS and operated under dark conditions. Image acquisition and analysis were performed using a Leica TCS SP8 X confocal microscope (Leica, Germany).
[0074] The results are as follows Figure 1 As shown. From the confocal photography results, it can be seen that PGA can be well stained by Nile red dye and can be phagocytosed by THP-1 cells. The distribution of PGA in cells is different at different incubation times. At 0h, the red fluorescence is mainly distributed on the cell membrane and endocytic vesicles, indicating that the cells are transporting and absorbing PGA across the membrane at this time. At 1h, the red fluorescence is widely distributed in the cells, and the cells have already taken up a large amount of PGA. It can be seen that PGA labeled with Nile red fluorescence can be used to observe the uptake of PGA by cells and the distribution of PGA in cells at different times.
[0075] Experimental Example 2 In vivo imaging study of the distribution of PGA after ingestion
[0076] The Nile red fluorescently labeled PGA prepared in Example 1 was dissolved in PBS solution to prepare mixed PBS solutions with fluorescently labeled PGA concentrations of 1 mg / L and 100 mg / L, respectively. The mice were divided into two groups: a low-dose group (corresponding to Figure 2The mice were gavaged with 1 mg / L mixed PBS solution for environmental concentration exposure. The high-dose group (corresponding to Figure 2 The mice were gavaged with a 100 mg / L mixed PBS solution for high concentration exposure. The gavage dose was 100 μL. After gavage, the mice were imaged in vivo to observe the distribution of PGA in the mice after 0.5h, 2h and 4h of ingestion. By observing the fluorescence distribution of PGA in the mice at different times after Nile red staining, the distribution of PGA in the mice at different times after ingestion was explored.
[0077] The results are as follows Figure 2 As shown in the figure, after PGA enters the body, it is almost not distributed in the heart, lungs and spleen, but mainly distributed in the liver, kidneys, stomach and intestines, and the distribution amount gradually decreases with the extension of time. Especially 4 hours after PGA enters the body, the distribution amount of PGA in the liver and intestines is significantly reduced, and there is almost no distribution in the kidneys. In the stomach, PGA still maintains a high distribution amount 4 hours after entering the body. At the same time, it can be seen from the figure that there is no significant difference in the distribution of PGA in the body between the low-dose group and the high-dose group. It can be seen that PGA labeled with Nile red fluorescence can be used for in vivo imaging research, and the distribution and metabolism of PGA at different times after being ingested into the body can be observed, which plays a good tracing role. At the same time, the distribution and transfer of PGA in different organs also show that the fluorescence color of Nile red will decrease with the transfer of PGA into different organs and the metabolism of PGA, indicating that PGA can be stably labeled by Nile red, and after entering the body, Nile red will not fall off from PGA and stain cells and tissues.
[0078] Experimental Example 3: Uptake of PGA in Caco-2 cells after Nile red staining
[0079] Caco-2 cells were used instead of intestinal epithelial cells to investigate the uptake of PGA in the intestine.
[0080] The Nile red fluorescent labeled PGA prepared in Example 1 was co-cultured with Caco-2 cells for different time periods, and the cell nuclei were stained with Hoechst dye to observe the location of the cells, and the extent of PGA uptake by Caco-2 cells at different times and the distribution of PGA in the cells were observed. 5The density of cells / plate was inoculated in a confocal microscopy dish, and the cells were treated with 10 μg / mL Hoechst dye for 5 min and then washed with PBS. The cells were incubated with PGA labeled with Nile red at a concentration of 1 μg / mL at 37°C, 5% CO2 for 0 h and 1 h. After incubation, the cells were washed with PBS and operated under dark conditions. Image acquisition and analysis were performed using a Leica TCS SP8 X confocal microscope (Leica, Germany).
[0081] Results: Caco-2 cells took up PGA to a certain extent, and co-incubation with PGA and THP-1 cells stained with Nile red showed similar results.
[0082] Experimental Example 4: Uptake of PGA in LO2 cells after Nile red staining
[0083] LO2 cells were used to investigate the uptake of PGA in liver cells.
[0084] The Nile red fluorescently labeled PGA prepared in Example 1 was co-cultured with LO2 cells for different time periods, and the cell nuclei were stained with Hoechst dye to observe the location of the cells, the extent of PGA uptake by LO2 cells at different times, and the distribution of PGA in the cells. 5 The density of cells / plate was inoculated in a confocal microscopy dish, and the cells were treated with 10 μg / mL Hoechst dye for 5 min and then washed with PBS. The cells were incubated with PGA labeled with Nile red at a concentration of 1 μg / mL at 37°C, 5% CO2 for 0 h and 1 h. After incubation, the cells were washed with PBS and operated under dark conditions. Image acquisition and analysis were performed using a Leica TCS SP8 X confocal microscope (Leica, Germany).
[0085] Results LO2 cells took up PGA to a certain extent, and co-incubated with PGA and THP-1 cells stained with Nile red showed similar results.
[0086] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.
Claims
1. A method for labeling polyglycolic acid with Nile red fluorescence, characterized in that: The following steps are involved: Mixing Nile red with a first solvent to obtain a dye solution; the first solvent consists of anhydrous ethanol and acetone; Mixing polyglycolic acid with a second solvent to obtain a polyglycolic acid dispersion; the second solvent consists of water and dimethyl sulfoxide; The polyglycolic acid dispersion liquid is mixed with the dyeing liquid for dyeing, and then filtered and washed to obtain polyglycolic acid labeled with Nile red fluorescence.
2. The method according to claim 1, characterized in that The volume ratio of anhydrous ethanol to acetone is (1:2) to (2:1); and / or The volume ratio of water to dimethyl sulfoxide is (1:2) to (2:1).
3. The method according to claim 1, characterized in that The particle size of the polyglycolic acid is 472.5 nm to 1000 nm.
4. The method according to claim 1, characterized in that: The concentration of Nile red in the staining solution is 0.5 mg / mL to 2 mg / mL; The concentration of polyglycolic acid in the polyglycolic acid dispersion is 1 mg / mL to 3 mg / mL; and / or The volume ratio of the dyeing liquid to the polyglycolic acid dispersion liquid is (1:2) to (2:1).
5. The method according to claim 1, characterized in that The dyeing conditions are heating at 30-42° C. for 2-4 hours, cooling to 22-28° C., cyclically performing the heating and cooling, and then soaking at room temperature; Preferably, the heating and cooling are performed once as one cycle, and the number of cycles is 2 to 4 times; and / or the time for immersing at room temperature is 8 to 12 hours.
6. A polyglycolic acid labeled with Nile red fluorescence, characterized in that: The Nile red fluorescently labeled polyglycolic acid is prepared by the method according to any one of claims 1 to 5.
7. Use of the polyglycolic acid labeled with Nile red fluorescence according to claim 6 in the preparation of a tracer preparation.
8. The use according to claim 7, characterized in that: The tracer preparation is an in vivo tracer preparation.
9. The use according to claim 8, characterized in that: The in vivo includes in vitro / ex vivo cells, in vitro / ex vivo tissues or in vivo of humans or animals.
10. A tracer preparation, characterized in that A polyglycolic acid comprising the Nile Red fluorescent label according to claim 6; Preferably, the tracer preparation is an in vivo tracer preparation; More preferably, the in vivo includes in vitro / ex vivo cells, in vitro / ex vivo tissues or in vivo of humans or animals.