Use of androst-4,6,8(9),13(14)-tetraen-3,11,16-trione as a fluorescent imaging agent

Abstract

This invention discloses a novel use of androstrol-4,6,8(9),13(14)-tetraen-3,11,16-trione, namely its application as a fluorescence imaging reagent. The androstrol-4,6,8(9),13(14)-tetraen-3,11,16-trione solution was co-incubated with human epidermal cells (HaCat) and acute lymphoblastic leukemia cells (BALL-1), respectively, achieving real-time, high-resolution fluorescence imaging of normal and tumor cells. This invention also achieved rapid, real-time in vivo fluorescence imaging in Balb / c nude mice. The compound of this invention is an androstrol natural product derived from *Smilax china*. Its application in cell fluorescence imaging, bioanalysis, and other fields has advantages such as rapid and sensitive cell staining, low cytotoxicity, strong fluorescence signal, and in vivo imaging capabilities, demonstrating practical application value.

Description

Technical Field

[0001] This invention belongs to the field of bioanalytical detection technology, specifically relating to the application of the natural compound androstenedin-4,6,8(9),13(14)-tetraene-3,11,16-trione in cell and animal in vivo fluorescence imaging. Background Technology

[0002] With the deepening of biomedical research, visualization-based bioimaging technology plays an increasingly important role in the life sciences and medicine. Compared with other bioimaging technologies, fluorescence imaging is characterized by low cost, rapid imaging, and sensitive single-molecule imaging at the molecular level, and can label and trace tumor growth. However, the current use of fluorescent dyes and quantum dots as fluorescent probes has some obvious drawbacks, such as low light penetration depth, potential damage to biological tissues, and the autofluorescence of biological samples, which limits its further application in the field of bioimaging. Currently, our understanding is still limited in the following aspects: first, suitable biomarkers for imaging; second, the selection of imaging targets and contrast enhancement materials; and third, the chemical methods for biochemically catalyzing imaging probes. In addition, many difficulties are encountered in the development of imaging reagents, including: poor transport of probes targeting tissues or tumors; high biotoxicity; poor probe stability; and low in vivo signal enhancement intensity. Therefore, it is very important to develop a fluorescent imaging compound with strong fluorescence signal, high stability, and low toxicity.

[0003] Epigynum auritum is a species of vine in the genus Epigynum of the family Apocynaceae, native to southern Yunnan, my country. Androst-4,6,8(9),13(14)-tetraen-3,11,16-trione is a natural small molecule compound derived from Epigynum auritum, with the molecular formula C. 19 H 18 O3, with a molecular weight of 294.13, belongs to the androsteroid class of compounds. Due to its small molecular weight and the presence of numerous double bonds in its structure, it is highly lipid-soluble and can easily cross the cell barrier. However, to date, no one has proposed its use in the field of fluorescence imaging. Summary of the Invention

[0004] To address the problems existing in the above-mentioned technologies, this invention provides a novel use for androstrol-4,6,8(9),13(14)-tetraen-3,11,16-trione, namely, its application as a fluorescence imaging reagent in the field of cell or biological in vivo fluorescence imaging. The chemical structural formula of androstrol-4,6,8(9),13(14)-tetraen-3,11,16-trione is as follows:

[0005]

[0006] The present invention used a fluorescence spectrophotometer to measure the optimal excitation wavelength of the compound as 450 nm and the optimal emission wavelength as 550 nm.

[0007] This invention validated the use of the above-mentioned compound through in vitro and in vivo experiments. In the in vitro experiments, human epidermal cell line HaCat, human acute lymphoblastic leukemia cell line BALL-1, and a certain concentration of androst-4,6,8(9),13(14)-tetraene-3,11,16-trione solution were mixed and incubated. A fluorescence signal of the compound was detected at a wavelength of 450 nm, and this concentration was non-toxic to normal cells. In vivo imaging experiments were conducted by intravenous injection of the compound into Balb / c nude mice. Dissection 7 days later showed no damage to the brain, heart, liver, spleen, lungs, kidneys, and testes of the Balb / c nude mice. Based on the above experimental results, it is indicated that the compound can be used in the fields of cell imaging and in vivo biological imaging.

[0008] Compared with the prior art, the present invention has the following advantages:

[0009] 1. This invention has discovered new biological applications for the monomer compound androstenedin-4,6,8(9),13(14)-tetraene-3,11,16-trione of Smilax china, opening up a new research field;

[0010] 2. Through experiments, this invention found that the monomer compound of *Syzygium sibiricum*, androst-4,6,8(9),13(14)-tetraene-3,11,16-trione, can be used to image normal cells and tumor cells at 60 μmol / L. At this concentration, no toxicity to normal cells was observed, but it had a good inhibitory effect on leukemia cells.

[0011] 3. The monomer compound of *Symplocos simaoensis*, androst-4,6,8(9),13(14)-tetraene-3,11,16-trione, is a natural product with high safety; moreover, the separation and purification process is simple and the cost is low, making it suitable for industrial production and market promotion. Attached Figure Description

[0012] Figure 1 The excitation spectrum (top) and emission spectrum (bottom) of androstened-4,6,8(9),13(14)-tetraene-3,11,16-trione are shown.

[0013] Figure 2 The inhibitory activity of androstenedione-4,6,8(9),13(14)-tetraen-3,11,16-trione on the proliferation of normal human epidermal cells (HaCat, top) and acute lymphoblastic leukemia cells (BALL-1, bottom); P<0.05(*), P<0.01(**), P<0.001(***) and P<0.0001(****) indicate significant differences compared with the negative control group (sample concentration of 0);

[0014] Figure 3 Fluorescence images of human epidermal cells HaCat and acute lymphoblastic leukemia cells BALL-1 after co-incubation with androst-4,6,8(9),13(14)-tetraene-3,11,16-trione for 24 h and 48 h.

[0015] Figure 4 In vivo imaging results of tail vein injection of androst-4,6,8(9),13(14)-tetraene-3,11,16-trione compound into Balb / c nude mice;

[0016] Figure 5 Seven days after injecting 50 μl of a 2 mg / mL androst-4,6,8(9),13(14)-tetraene-3,11,16-trione compound into the tail vein of Balb / c nude mice, organs were stained with hematoxylin and eosin (HE). Detailed Implementation

[0017] The androsterone-4,6,8(9),13(14)-tetraene-3,11,16-trione used in the embodiments of this invention was prepared using existing phytochemical separation and purification techniques. The invention is further illustrated below with reference to experimental data and accompanying drawings. These experimental examples are for illustrative purposes only and are not intended to limit the scope of application of this invention. After reading this invention, any equivalent modifications, alterations, and modifications made by those skilled in the art are within the scope defined by the claims of this invention. Unless otherwise specified, the reagents used in the embodiments are conventional commercially available products or reagents prepared using conventional methods. Unless otherwise specified, the methods used in the embodiments are conventional experimental methods.

[0018] In the examples, RPMI 1640 medium, DMEM medium, fetal bovine serum (FBS), phosphate-buffered saline (PBS), penicillin and streptomycin were all purchased from Gibco; DMSO was purchased from Beijing Solarbio Biotechnology Co., Ltd.; and the cell counting (CCK-8) assay kit was purchased from Shanghai Bioscient Biotechnology Co., Ltd.

[0019] Example 1: Fluorescence spectroscopy measurement of androstrol-4,6,8(9),13(14)-tetraen-3,11,16-trione

[0020] A precise amount of androstened 4,6,8(9),13(14)-tetraen-3,11,16-trione was dissolved in DMSO to prepare a sample solution with a concentration of 1 mg / mL. Approximately 2 / 3 of the sample solution was placed in a clean, four-sided transparent 10 mm quartz cuvette. The excitation and emission spectra of the solution were then measured using a HITACHI F-4600 fluorescence spectrophotometer. Excitation spectral detection conditions: emission wavelength Em was 900 nm, excitation wavelength Ex was 200-880 nm, Ex slit width was 10 nm, and Em slit width was 20 nm. Emission spectral detection conditions: excitation wavelength Ex was 430 nm, emission wavelength Em was 450-900 nm, Ex slit width was 10 nm, and Em slit width was 20 nm. The excitation and emission spectra of androstened 4,6,8(9),13(14)-tetraen-3,11,16-trione are shown in [reference needed]. Figure 1 ;

[0021] Depend on Figure 1 It is known that the optimal excitation wavelength of androstened-4,6,8(9),13(14)-tetraene-3,11,16-trione is about 450 nm and the optimal emission wavelength is about 550 nm.

[0022] Example 2: Toxicity experiment of androstenedione-4,6,8(9),13(14)-tetraene-3,11,16-trione on normal cells and tumor cells 1. Experimental materials

[0023] Solution preparation: Androst-4,6,8(9),13(14)-tetraene-3,11,16-trione was prepared into a 30 mmol / L stock solution using cell-grade DMSO under sterile conditions. The stock solution was sealed and stored in a -20℃ refrigerator protected from light for later use. In the experiment, the solution was diluted to different concentrations according to the final concentration requirements after drug addition.

[0024] Cell lines: Human acute lymphoblastic leukemia cells BALL-1 and human epidermal cells HaCat were purchased from the Kunming Cell Bank of the Chinese Academy of Sciences. These cell lines were cultured in RPMI 1640 and DMEM medium containing 10% fetal bovine serum + 1% penicillin and streptomycin, respectively, at 37°C in a 5% CO2 incubator for later use.

[0025] 2. Experimental Methods

[0026] Human epidermal cells (HaCat) in the exponential growth phase were prepared into a solution with a concentration of 2 × 10⁻⁶ using complete culture medium. 4Cell suspension was seeded into 96-well plates at 200 μL per well. The experiment was divided into a drug group and a negative control group, with 5 replicates per group (or per drug concentration). After 24 h of culture, the drug group was replaced with 200 μL of medium containing different concentrations of androstenedin-4,6,8(9),13(14)-tetraen-3,11,16-trione (final concentrations of 10 μmol / L, 20 μmol / L, 40 μmol / L, 80 μmol / L and 120 μmol / L), and the negative control group was replaced with 200 μL of fresh complete medium. After 22 h of culture, 20 μL of CCK-8 reagent was added to each well, and the plates were incubated for another 4 h. The absorbance (OD value) of each well was then measured at 490 nm using a microplate reader. The effect of different drug concentrations on cell viability was calculated using the formula: viability = (OD value of drug group / OD value of negative control group) × 100%. The results are shown in the figure. Figure 2 The image above;

[0027] BALL-1 acute lymphoblastic leukemia cells in the exponential growth phase were injected at a dose of 5 × 10⁻⁶. 4 The samples were seeded at a density of 200 μL in each well of a 96-well plate. The experiment consisted of a drug group and a negative control group. The final drug concentrations were 10 μM, 20 μM, 40 μM, 80 μM, and 120 μM. The negative control group received the same volume of culture medium. Five replicates were performed for each group (or each drug concentration). After 44 h of incubation, 20 μL of CCK-8 solution was added to each well, and incubation continued for another 4 h. The absorbance at 490 nm was measured using a microplate reader, and the survival rate was calculated as (OD value of drug group / OD value of negative control group) × 100%. Results are shown below. Figure 2 The image below;

[0028] like Figure 2 As shown, at a drug concentration of 120 μmol / L, the compound had no damaging effect on normal cells. Furthermore, androstenedione-4,6,8(9),13(14)-tetraen-3,11,16-trione significantly reduced tumor cell viability in a dose-dependent manner within 48 hours. In conclusion, it is worth emphasizing that androstenedione-4,6,8(9),13(14)-tetraen-3,11,16-trione is non-toxic to normal cells but has a growth-inhibiting effect on leukemia cells.

[0029] Experiment Example 3: Fluorescence Imaging Experiment of Androst-4,6,8(9),13(14)-Tetraene-3,11,16-Trione in Cells

[0030] Human epidermal cells HaCat in the exponential growth phase were subjected to 2×10 5Cells were seeded in copolymer dishes at a rate of 2 mL per dish. After 24 h of culture, the original culture medium was aspirated, and a 60 μmol / L solution of androstenedione-4,6,8(9),13(14)-tetraen-3,11,16-trione was added. The control group was treated with an equal volume of culture medium. Cells were cultured at 37 °C for 24 h and at 5% CO2 for 48 h. After culturing, the cells were washed twice with cold PBS. 1 mL of 4% paraformaldehyde fixative was added to each dish, and the cells were fixed at room temperature for 15 min. After fixing, the cells were washed twice with cold PBS. Then, the cells were stained with 1 mL of 0.5 μg / mL DAPI solution at room temperature in the dark for 15 min. After washing twice with cold PBS, 0.5 mL of PBS was added to infiltrate the cells and prevent them from drying out. The cells were then photographed under a laser confocal fluorescence microscope using excitation wavelengths of 405 nm and 450 nm to obtain fluorescence images.

[0031] The experimental results showed that blue fluorescence emitted by the nuclear dye DAPI was observed at an excitation wavelength of 405 nm, green fluorescence emitted by cells after sample treatment was observed at an excitation wavelength of 450 nm, and combined fluorescence images (Merged) under the two excitation light channels were observed. Furthermore, the experiment found that the fluorescence intensity gradually increased with the increase of sample incubation time.

[0032] BALL-1 acute lymphoblastic leukemia cells in the exponential growth phase were injected at a dose of 5 × 10⁻⁶. 5 Cells were seeded in 6-well plates at a concentration of 10 cells / well. 60 μmol / L androst-4,6,8(9),13(14)-tetraene-3,11,16-trione solution was added. An equal volume of culture medium was added to the control group. The total volume per well was 2 mL. The cells were cultured at 37°C for 24 h and in a 5% CO2 incubator for 48 h, respectively, before the culture was terminated. The cell suspension was gently blown to mix and then transferred to 2 mL centrifuge tubes. The cells were collected by centrifuging at 1000 rpm for 5 min. The cells were then washed twice with cold PBS. 1 mL of 4% paraformaldehyde fixative was added to each centrifuge tube. The cells were fixed at room temperature for 15 min and then centrifuged at 1000 rpm for 5 min to remove the fixative. The cell pellet was washed twice with cold PBS. Then, stain with 1 mL of 0.5 μg / mL DAPI solution at room temperature in the dark for 15 min, wash twice with cold PBS, add 0.1 mL of PBS to suspend the cells for cell slide preparation, and take pictures with a laser confocal fluorescence microscope. Excitation at wavelengths of 405 nm and 450 nm can be used to acquire cell fluorescence images.

[0033] See results Figure 3The experimental results showed that blue fluorescence emitted by the nuclear dye DAPI was observed at an excitation wavelength of 405 nm, green fluorescence emitted by cells after sample treatment was observed at an excitation wavelength of 450 nm, and combined fluorescence images (Merged) under the two excitation light channels were observed. Furthermore, the experiment found that the fluorescence intensity gradually increased with the increase of sample incubation time.

[0034] Experimental Example 4: Fluorescence Imaging Experiment of Androst-4,6,8(9),13(14)-Tetraene-3,11,16-Trione in Balb / c Nude Mice

[0035] Twelve 6-week-old male Balb / c nude mice (20±2g) were divided into two groups: a treatment group and a control group, with 6 mice in each group. After one week of acclimatization, the treatment group was injected with 50μL of androst-4,6,8(9),13(14)-tetraene-3,11,16-trione solution at a concentration of 2mg / mL via the tail vein, while the control group was injected with the same volume of solvent (cell-grade DMSO). After injection, the mice were anesthetized with isoflurane at different time points of 0.25 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, and 8 hours. The mice were then placed on the operating platform of a small animal in vivo imaging instrument. The excitation wavelength was set to 465nm and the emission wavelength was set to DsRed (580nm) to collect in vivo fluorescence distribution images.

[0036] See results Figure 4 As can be seen from the figure, compared with the control group (the nude mouse on the left in the picture), the nude mouse in the drug treatment group (the nude mouse on the right in the picture) was able to emit fluorescence in vivo at an excitation wavelength of 465nm and an emission wavelength of DsRed (580nm), and the fluorescence intensity first increased and then decreased over time.

[0037] Experimental Example 5: Organ HE staining experiment after 7 days of injection of the monomer compound androstenedinium-4,6,8(9),13(14)-tetraene-3,11,16-trione into Balb / c nude mice.

[0038] Twelve six-week-old male Balb / c nude mice (20±2g) were divided into two groups: a drug administration group and a control group, with six mice in each group. After one week of acclimatization, the drug administration group was injected with 50μL of androst-4,6,8(9),13(14)-tetraene-3,11,16-trione solution at a concentration of 2mg / mL via the tail vein, while the control group was injected with the same volume of solvent (cell-grade DMSO). Water and food were provided normally after drug administration, and weight changes were recorded daily. Seven days later, the brain, heart, liver, spleen, lungs, kidneys, and testes were dissected and subjected to HE staining for pathological observation to determine whether the drug caused damage to the animal organs.

[0039] See results Figure 5Compared with the control group, the treated group did not show any inflammation or necrotic cell accumulation, and therefore did not cause damage to the brain, heart, liver, spleen, lungs, kidneys and testes of Balb / c nude mice.

Claims

1. Use of androsta-4,6,8(9), 13(14)-tetraen-3, 11, 16-trione as a fluorescent imaging agent, characterized in that: For imaging human epidermal cells, acute lymphoblastic leukemia cells and mouse in vivo.

Images