CN112898578A - Preparation method of indocyanine green derivative and application of indocyanine green derivative in detection of oxidized low-density lipoprotein - Google Patents
Preparation method of indocyanine green derivative and application of indocyanine green derivative in detection of oxidized low-density lipoprotein Download PDFInfo
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- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
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- A61K49/0032—Methine dyes, e.g. cyanine dyes
- A61K49/0034—Indocyanine green, i.e. ICG, cardiogreen
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Abstract
The invention discloses a preparation method of an indocyanine green derivative, which comprises the following steps of (1) preparing an indocyanine green derivative ICG-CO-NHS modified by-CO-NHS; (2) PEG-NH 2-modified indocyanine green derivative ICG-PEG500 was prepared. The invention also discloses application of the indocyanine green derivative ICG-PEG500 in detection of oxidized low-density lipoprotein. The ICG metabolism time was 0.06421h and the ICG-PEG500 metabolism time was 4.2261h, suggesting that the PEG-modified ICG derivatives have significantly extended residence time in plasma. The half-life of the blood plasma is prolonged, the trouble that the retention time of the blood plasma needs to be prolonged by multiple times or continuous injection in fluorescence radiography can be avoided, and the ICG derivative and the ICG have the fluorescence and spectral properties close to each other, have good stability and have good application prospect.
Description
Technical Field
The invention belongs to the technical field of indocyanine green modification, and particularly relates to a preparation method of an indocyanine green derivative. The invention also relates to application of the indocyanine green derivative in detection of oxidized low-density lipoprotein.
Background
Indocyanine Green (ICG) is a cyanine dye with a molecular weight of 751.4Da used for medical diagnosis, and has been used in the medical field for measuring liver blood flow, ophthalmic angiography, detecting atherosclerotic plaques, and the like. With the development of imaging technologies such as MRI, CT, ultrasound and the like, in-vivo imaging of atherosclerotic plaques is not limited to traditional vascular structure imaging, but also is expanded to accurate detection of plaques in blood vessels, so that imaging monitoring and analysis of key molecular events of plaque development, erosion and rupture become possible. Indocyanine green is a tricarbocyanine infrared photosensitive dye and needs to be stored away from light, because imine positive ions in a five-membered ring are unstable, can be broken under long-time illumination, and has short plasma half-life and fast in-vivo metabolism. With this feature, there have been studies to detect the vulnerability of atherosclerotic plaques in animal models using ICG. ICG has a maximum absorption spectrum around 800nm, a half-life of 150s to 180s, is tightly bound with hemoglobin in blood, can be rapidly distributed in systemic blood vessels, and is completely excreted by liver bile. A recent study showed that in an atherosclerotic animal rabbit model, atherosclerotic plaque signals were detectable by ICG injection for 20min, and the detectable ICG fluorescence showed plaques with higher target background ratios.
However, the previous experiments have several problems, namely 1) the ICG solution is unstable to light and easy to gather in aqueous solution, the half-life of the ICG in plasma is very short, and the clinical administration needs to adopt an injection or slow intravenous drip mode to keep the imaging time, thereby limiting the application of the ICG as a near infrared fluorescence imaging material. 2) ICG solutions are currently only suitable for detecting plaque density and do not provide much information on the distribution of plaque and the actually harmful ox-LDL.
Cell-mediated oxidative modification of LDL (ox-LDL) can be marked by the generation of thiobarbituric acid-reactive substituents (TBARS), while Malondialdehyde (MDA) can be reacted with thiobarbituric acid (TBA) to produce reddish brown 3, 5, 5-trimethyloxazole-2, 4-dione, the TBARS content is calculated, and the degree of oxidative modification of LDL is expressed as the TBARS content per gram of LDL cholesterol. ox-LDL produced early in plaque formation is actually a danger signal of abnormal cell lipid loading, and the detection of ox-LDL is helpful to provide information for abnormal lipid loading of vascular endothelium early in plaque formation.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a preparation method of indocyanine green derivatives for distribution of plaques and research of actually harmful ox-LDL.
The invention solves the technical problems through the following technical scheme,
a preparation method of indocyanine green derivatives comprises the following steps,
(1) preparation of-CO-NHS-modified indocyanine green derivative ICG-CO-NHS:
a commercial poly (lactic-co-glycolic acid) copolymer, PLGA, was reacted with a commercial indocyanine green imide derivative, ICG-NHS, PLGA: ICG-NHS is 1: 5-10, stirring and reacting for 24 hours at room temperature in a weakly alkaline environment, wherein the modified indocyanine green ICG is a-CO-NHS modified indocyanine green derivative ICG-CO-NHS;
(2) preparation of PEG-NH2-modified indocyanine green derivative ICG-PEG 500:
taking the indocyanine green derivative ICG-CO-NHS modified by-CO-NHS and m-PEG-NH prepared in the step (1)2-in a molar ratio of 1: 10 to 15, reacting in a weak alkaline environment at room temperature in a shaking table for 12 hours, wherein m-PEG-NH2-carrying out ester substitution reaction with-CO-NHS modified indocyanine green derivative ICG-CO-NHS, removing a molecule of N-hydroxysuccinimide, stopping the reaction, concentrating under reduced pressure, and drying with anhydrous sodium sulfate to obtain PEG-NH2-a modified indocyanine green ICG derivative ICG-PEG 500.
In order to obtain better technical effect, in the step (1), the pH value is 7.35-7.44 in a weak alkaline environment;
in order to obtain better technical effect, in the step (2), a phosphate buffered saline solution with the pH value of 7.0-8.0 is used in a weak alkaline environment;
in order to obtain better technical effect, in the step (2), m is monomethyl ether in m-PEG-NH 2-;
in order to obtain better technical effect, in the step (2), the indocyanine green derivative ICG-CO-NHS modified by-CO-NHS is mixed with PEG-NH2-in a molar ratio of 1: 12, and mixing.
The invention also provides an application of indocyanine green derivative ICG-PEG500 in detection of oxidized low-density lipoprotein.
The invention solves the technical problems through the following technical scheme,
an application of indocyanine green derivative ICG-PEG500 in detecting oxidized low-density lipoprotein, which comprises the following steps,
(1) will be PEG-NH2-the modified indocyanine green derivative ICG-PEG500 dissolved in physiological saline to a concentration of 20. mu.g.ml-1The mixed solution of (1 mg/kg)-1Measuring tail vein and injecting into mouse;
(2) carrying out near-infrared II-zone fluorescence imaging and photographing on the mice at the time points of 3min, 6min, 30min, 120min, 240min, 480min and 600min after tail vein injection respectively;
(3) taking a picture by using a SWIR near-infrared camera, taking a picture under a long-wave pass filter with the excitation spectrum of 808nm and the excitation spectrum of 900nm, observing fluorescence signal areas of an aortic arch area and a leg skin area of the mouse, taking images by using a Zeiss Axio Scope A1 fluorescence microscope, and analyzing the fluorescence areas in ImageJ software;
(4) the picture was converted into an 8bit gray scale image using ImageJ software, and then the picture was subjected to black-and-white inversion, the optical density was corrected, and pixels were selected as a unit of measurement, the Area of the fluorescence region was measured, and data was measured and recorded.
Another application of indocyanine green derivative ICG-PEG500 in detecting oxidized low-density lipoprotein comprises the following steps,
(1) ICG or ICG-PEG500 is dissolved in high-sugar DMEM medium, and ICG or ICG-PEG500 with the final concentration of 10 mu M is administered to endothelial cells;
(2) performing near-infrared II-zone fluorescence imaging on endothelial cells at time points of 1min, 5min, 15min, 30min and 60min respectively, and taking a picture;
(3) taking a picture by using a SWIR near-infrared camera, taking a picture under a long-wave pass filter with the excitation spectrum of 808nm and the excitation spectrum of 900nm, taking a cell fluorescence image by using a Zeiss Axio Scope A1 fluorescence microscope, and analyzing a fluorescence area in ImageJ software;
(4) the picture was converted into an 8bit gray scale image using ImageJ software, and then the picture was subjected to black-and-white inversion, the optical density was corrected, and pixels were selected as a unit of measurement, the Area of the fluorescence region was measured, and data was measured and recorded.
The ICG metabolism time was 0.06421h and the ICG-PEG500 metabolism time was 4.2261h, suggesting that the PEG-modified ICG derivatives have significantly extended residence time in plasma. The half-life of the blood plasma is prolonged, the trouble that the retention time of the blood plasma needs to be prolonged by multiple times or continuous injection in fluorescence radiography can be avoided, and the ICG derivative and the ICG have the fluorescence and spectral properties close to each other, have good stability and have good application prospect.
Drawings
FIG. 1 is a chart of HPLC absorption peaks according to an embodiment of the present invention;
FIG. 2 is an absorption spectrum of an embodiment of the present invention;
FIG. 3 is a graph showing the near infrared region I fluorescence intensity of the example of the present invention;
FIG. 4 is a graph of near infrared region II fluorescence intensity for the examples of the present invention;
FIG. 5 is a graph of stability testing in a neutral solution in accordance with an embodiment of the present invention;
FIG. 6 is a graph of stability in vitro serum test according to an embodiment of the present invention;
FIG. 7 is a comparison graph of the concentration of high fluorescence signals in the aortic arch region after ICG or ICG-PEG500 injection in accordance with an embodiment of the present invention;
FIG. 8 is a graph comparing the metabolism of fluorescence signals on the medial side of the leg after ICG or ICG-PEG500 injection in accordance with an embodiment of the present invention;
FIG. 9 is a graph showing the accumulation of high fluorescence signals in endothelial cells after ICG or ICG-PEG500 injection in accordance with an embodiment of the present invention;
wherein in FIGS. 2-6, the continuous line represents unmodified ICG and the broken line represents ICG-PEG 500.
Detailed Description
The invention is explained in further detail below with reference to the figures and examples.
Example 1 PEG-NH-modified Indolocyanine Green derivatives
A preparation method of indocyanine green derivatives comprises the following steps,
(1) preparation of-CO-NHS-modified indocyanine green derivative ICG-CO-NHS:
a commercially available polylactic-co-glycolic acid copolymer PLGA (polylactic-co-glycolic acid) was reacted with a commercially available indocyanine green imide derivative ICG-NHS, PLGA: ICG-NHS molar ratio of 1: 5-10, in this example, PLGA: ICG-NHS molar ratio of 1: stirring and reacting for 24 hours at room temperature under the condition that the pH value of the alkalescent environment is 7.35-7.44, wherein the modified indocyanine green ICG is a-CO-NHS modified indocyanine green derivative ICG-CO-NHS;
polylactic-co-glycolic acid PLGA:
indocyanine green ICG-NHS:
indocyanine green derivative ICG-CO-NHS modified by-CO-NHS:
reaction equation for synthesizing-CO-NHS modified indocyanine green derivative ICG-CO-NHS:
(2) preparation of PEG-NH2-modified indocyanine green derivative ICG-PEG 500:
taking the indocyanine green derivative ICG-CO-NHS modified by-CO-NHS and m-PEG-NH prepared in the step (1)2-in a molar ratio of 1: 10 to 15, in this example ICG-CO-NHS and m-PEG-NH2-in a molar ratio of 1: 12, reacting in phosphate buffer solution with pH of 7.0-8.0 at room temperature in a shaking table for 12h, and reacting with m-PEG-NH2-reacting with ICG-CO-NHS for ester substitution, removing one molecule of N-hydroxysuccinimide, stopping reaction, concentrating under reduced pressure, and drying with anhydrous sodium sulfate to obtain PEG-NH2Modified indocyanine green ICG derivative ICG-PEG500, in this example, m-PEG-NH 2-is monomethyl ether, m-PEG is polyethylene glycol monomethyl ether (Methoxypolyethylene glycols), and the reaction process is shown as:
m-PEG-NH2-：
via PEG-NH2-modified indocyanine green ICG derivative ICG-CO-NHS:
the reaction process is as follows:
PEG-NH2- + Indolocyanine green derivative ICG-CO-NHS-modified with-CO-NHS-PEG-NH2-a modified indocyanine green ICG derivative ICG-PEG500,
example 2 detection by PEG-NH2-modified indolesCyanine green derivative ICG-PEG500
The PEG-NH-modified indocyanine green derivative ICG-PEG500 prepared in example 1 is detected to judge whether PEG-NH is successfully prepared2-a modified indocyanine green derivative ICG-PEG 500.
1) And (3) carrying out high performance liquid chromatography analysis and detection: an absorption peak was detected under a 780nm channel by High Performance Liquid Chromatography (HPLC), and an unmodified ICG and the PEG-NH-modified indocyanine green derivative (ICG-PEG 500 for short) prepared in example 1 were selected, and a separation Column (C4Column, 300A, 3.5 μm, 4.6 mm. times.250 mm) was selected.
TABLE 1 HPLC conditions
In FIG. 1, A is the HPLC absorption peak pattern of ICG-NHS, and B is the HPLC absorption peak pattern of ICG-PEG 500. ICG-NHS is small in polarity, poor in lipophilicity and water solubility, and the peak time is 22.4943min, while ICG-PEG500 is 20.2263min, which is earlier than that of ICG-NHS.
The PEG is successfully connected with the ICG, the polarity is increased, the water solubility is improved, the retention time in the chromatographic column is shortened, and the peak-appearing time is shortened.
2) And (3) performing absorption spectrum analysis and detection: taking out the concentration of 20 mu g/ml-1Unmodified ICG and ICG-PEG500 were subjected to absorption spectroscopy.
2.1) spectral property testing: the UV-Vis-NIR spectrometer cary 5000 detects the spectral absorption intensity of ICG-PEG500 compared with that of unmodified ICG and of ICG-PEG500 in the spectral range from 500nm to 1000 nm. The emission spectrum refers to a spectrum emitted by a light source, and provides energy for atoms or molecules at a higher energy level, so that the atoms or molecules at the high energy level generate radiation when transiting to a lower energy level, and emits redundant energy to form a spectrum, and the light of the light source with the continuous spectrum is measured by a spectrometer to obtain an absorption spectrum.
The continuous line in FIG. 2 represents unmodified ICG and the broken line represents ICG-PEG500, showing that ICG-PEG500 has a spectral absorbance capacity substantially identical to that of unmodified ICG.
2.2) near-infrared I-region fluorescence intensity: unmodified ICG and ICG-PEG500 in the near infrared I region, using a spectrophotometer Spectronic 200, set the excitation light 808nm, wavelength 650 nm-900 nm relative fluorescence intensity, respectively test the unmodified ICG and ICG-PEG500 relative fluorescence intensity.
The continuous line in FIG. 3 represents unmodified ICG and the broken line represents ICG-PEG 500.
In the near infrared I region, ICG-PEG500 has good fluorescence property equivalent to that of unmodified ICG, and ICG-PEG500 has no influence on the fluorescence property of ICG after modification of PEG.
2.3) near-infrared II-zone fluorescence intensity: the relative fluorescence intensity of unmodified ICG and ICG-PEG500 in the near infrared II region at the wavelength of 900-1200 nm is detected by using spectrographs NIRQUEST512 and CVH100/M and setting the exciting light at 808 nm.
The continuous line in FIG. 4 represents unmodified ICG and the broken line represents ICG-PEG 500.
It is shown that ICG-PEG500 has good fluorescence properties comparable to unmodified ICG in the near infrared region II.
Example 3 stability testing
Unmodified ICG and ICG-PEG500 were taken for stability testing in neutral solution and serum stability testing in vitro, respectively.
3.1) stability test in neutral solution: preparation of 20. mu.g.ml-1Unmodified ICG and ICG-PEG500 solutions, unmodified ICG and ICG-PEG500 were dissolved in PBS phosphate buffered saline (phosphate buffer saline) at pH 7.4, respectively.
The PBS buffer was configured to weigh NaCl: 8g, KCl: 0.2g, Na2HPO4：12g，H2O：3.63g， NaH2PO4: dissolving 0.24g in 900mL double distilled water, adjusting pH to 7.4 with hydrochloric acid, adding water to constant volume of 1L, and storing at normal temperature for use.
Standing in a constant temperature oven in dark place, standing for 3h, 6h, 9h, 12h, 15h, 18h, 21h, and 24h, measuring fluorescence intensity of the sample, setting exciting light 808nm with spectrophotometer Spectronic 200, and calculating the reduction rate.
The continuous line in FIG. 5 represents unmodified ICG and the broken line represents ICG-PEG 500.
As shown in the figure, the fluorescence intensity of the unmodified ICG is reduced along with the time, the fluorescence intensity of the unmodified ICG is reduced rapidly along with the time within 1-12h, and the fluorescence intensity of the ICG-PEG500 is in a relatively stable state along with the time change, which indicates that the ICG-PEG500 has better stability in a neutral environment in a neutral solution.
3.2) in vitro serum stability test: preparation of 20. mu.g.ml-1Unmodified ICG and ICG-PEG500 solutions, the unmodified ICG and ICG-PEG500 are respectively dissolved in FBS (fetal bovine serum), then the mixture is kept still in an incubator, the fluorescence intensity of a sample is measured after the mixture is kept still for 3 hours, 6 hours, 9 hours, 12 hours, 15 hours, 18 hours, 21 hours and 24 hours, a spectrophotometer Spectronic 200 is used for testing, the exciting light nm is set, and the reduction rate is calculated to 808 nm.
The continuous line in FIG. 6 represents unmodified ICG and the broken line represents ICG-PEG 500. As shown, the fluorescence intensity of both unmodified ICG and ICG-PEG500 was in a steady state over time.
Example 4 application of indocyanine green derivative ICG-PEG500
Experimental groups: an application of indocyanine green derivative ICG-PEG500 in detecting oxidized low-density lipoprotein, which comprises the following steps,
(1) ICG-PEG500 was dissolved in physiological saline to a final concentration of 20. mu.g/ml-1The amount of ICG-PEG500 is 1 mg/kg-1The tail vein is measured and injected into the body of a mouse
(2) After tail vein injection, near infrared II region fluorescence imaging and photographing are carried out on the aortic arch region of the mouse at the time points of 3min, 6min, 30min, 120min, 240min, 480min and 600min respectively.
(3) Taking a picture with a SWIR near-infrared camera with an excitation spectrum of 808nm and a long-wave pass filter of 900nm, observing the main concentration region of the fluorescence signal, and taking an image with a Zeiss Axio Scope a1 fluorescence microscope and analyzing the fluorescence region in ImageJ software;
(4) the image was converted into an 8-bit gray scale image using ImageJ software, and then the image was subjected to black-and-white inversion and corrected to an appropriate optical density, pixels were selected as measurement units, the Area of the fluorescence region was measured, and data was measured and recorded.
Control group: the same procedure and procedure were followed for the experimental group, using only ICG instead of ICG-PEG 500.
In this example, 6 healthy mice (each weighing about 20 g) were divided into 2 groups including an experimental group and a control group, and 3 mice were each group.
Wherein the amount of ICG-PEG500 in the experimental group of mice is 1mg kg-1The injection contains ICG-PEG500 with a concentration of 20 μ g/ml-1ICG-PEG500 was injected into the tail vein.
The amount of ICG in control group mice is 1 mg/kg-1The injection contains ICG with concentration of 20 μ g/ml-1ICG was injected tail vein.
The experimental group and the control group were subjected to fluorescence area concentrated region detection after injecting indocyanine green derivatives as described in this example.
Example 5 fluorescence area concentrated region detection
Acute cardiovascular and cerebrovascular events (including cerebrovascular diseases, acute coronary syndromes and the like) are mainly caused by unstable plaques, the existing imaging means such as angiography, intravascular ultrasound, CT or MRI angiography and the like have certain diagnostic value on atherosclerosis, morphological changes caused by atherosclerotic lesions, such as the stenosis degree of an arterial lumen, the thickness of a blood vessel wall or the shape and the size of plaques, and the application of the imaging means is limited due to invasive, high-price and other factors. In recent years, fluorescence and near infrared emission spectra provide a feasible method for early detection of AS plaque and vulnerable plaque. In-vivo imaging of atherosclerotic plaques is not limited to traditional vascular structure imaging, but also extends to accurate detection of intravascular plaques, enabling imaging monitoring analysis of key molecular events of plaque progression, erosion and rupture. ICG solutions are currently only suitable for detecting plaque density and do not provide much information on the distribution of plaque and the actually harmful ox-LDL. ox-LDL produced early in plaque formation is actually a danger signal of abnormal cell lipid loading, and the detection of ox-LDL is helpful to provide information for abnormal lipid loading of vascular endothelium early in plaque formation. During the oxidative modification of LDL to ox-LDL, cell-mediated oxidative modification of LDL can be marked by the generation of thiobarbituric acid reactant (TBARS), while Malondialdehyde (MDA) can react with thiobarbituric acid (TBA) to produce reddish brown 3, 5, 5-trimethyloxazole-2, 4-dione, the TBARS content is calculated, and the degree of oxidative modification of LDL is expressed as the TBARS content per gram of LDL cholesterol.
The experimental group and the control group in example 4 were photographed by a SWIR near-infrared camera with an excitation spectrum of 808nm and a 900nm long-wave pass filter.
After tail vein injection, near infrared II region fluorescence imaging and photographing are carried out on the aortic arch region of the mouse at the time points of 3min, 6min, 30min, 120min, 240min, 480min and 600min respectively, and the figure 8 shows.
The high fluorescence signal is mainly selectively gathered at the aortic root, aortic arch, descending aorta initiation, abdominal aorta renal artery bifurcation and other parts, and the areas are common AS plaque forming parts.
The high fluorescence intensity region of the aortic arch was analyzed, the calculated background signal was subtracted, and the entire artery was outlined.
The region of interest (ROI) was manually delineated from the aortic arch, the beginning of descending aorta and abdominal aorta, and the percentage of the ROI area of the high fluorescence emission region of each experimental group in the entire aortic arch area was calculated.
In FIG. 7, the fluorescence of the aortic arch region began to appear 3min after ICG-PEG500 injection in mice, and the fluorescence signal of the aortic arch region was very clear at 30min, which is clearer than the image of ICG injection in mice 3 min. The imaging after 240min for ICG-PEG500 injected mice was close to 3min for ICG injected mice, suggesting that ICG-PEG500 may be enriched in the aortic arch region of mice and persist for a relatively long time.
Example 6 plasma half-life test
Plasma half-life tests were performed after injection of indocyanine green derivatives as in example 5.
The experimental group and the control group in example 4 were photographed by a SWIR near-infrared camera with an excitation spectrum of 808nm and a 900nm long-wave pass filter.
After tail vein injection, near infrared II region fluorescence imaging is carried out on the femoral vein of the mouse at the time points of 3min, 6min, 30min, 120min, 240min, 480min and 600min respectively, pictures are taken, the fluorescence intensity of the aortic arch region in the image picture is compared with the fluorescence intensity of the skin part of legs, the plasma half-life period is calculated by the ratio, and the blood clearance rates of ICG and ICG-PEG500 are compared.
In FIG. 8, the respective metabolic times of ICG and ICG-PEG500 in plasma were calculated based on the ratio of fluorescence intensities. The ICG metabolism time was 0.06421h and the ICG-PEG500 metabolism time was 4.2261h, suggesting that the PEG-modified ICG derivatives have significantly extended residence time in plasma.
The half-life of the plasma is prolonged, the trouble that the plasma retention time needs to be prolonged by multiple times or continuous injection in fluorescence radiography can be avoided, and the ICG-PEG500 and the ICG have the fluorescence and spectrum properties close to each other, have good stability and have good application prospect.
Example 7 Another application of the indocyanine Green derivative ICG-PEG500
Experimental groups: another application of indocyanine green derivative ICG-PEG500 in detecting oxidized low-density lipoprotein comprises the following steps,
(1) ICG or ICG-PEG500 is dissolved in high-sugar DMEM medium, and ICG or ICG-PEG500 with the final concentration of 10 mu M is administered to endothelial cells;
(2) performing near-infrared II-zone fluorescence imaging on endothelial cells at time points of 1min, 5min, 15min, 30min and 60min respectively, and taking a picture;
(3) taking a picture by using a SWIR near-infrared camera, taking a picture under a long-wave pass filter with the excitation spectrum of 808nm and the excitation spectrum of 900nm, taking a cell fluorescence image by using a Zeiss Axio Scope A1 fluorescence microscope, and analyzing a fluorescence area in ImageJ software;
(4) the image was converted into an 8-bit gray scale image using ImageJ software, and then the image was subjected to black-and-white inversion and corrected to an appropriate optical density, pixels were selected as measurement units, the Area of the fluorescence region was measured, and data was measured and recorded.
Control group: the same procedure and procedure were followed for the experimental group, using only ICG instead of ICG-PEG 500.
In this example, mouse b.End3 endothelial cells were divided into 2 groups of 9 dishes, including experimental and control groups.
Wherein the experimental group was administered 10 μ M ICG-PEG500 to endothelial cells.
Control endothelial cells were given 10 μ M ICG.
The experimental group and the control group were subjected to fluorescence area concentrated region detection after injecting indocyanine green derivatives as described in this example.
Example 8 endothelial cell fluorescence detection assay
Endothelial cell fluorescence detection was performed after injection of indocyanine green derivatives as in example 7.
The experimental group and the control group in example 7 were photographed by a SWIR near-infrared camera with an excitation spectrum of 808nm and a 900nm long-wave pass filter.
After the recovery of the endothelial cells of the mouse b.End3 cells, the cells were cultured in a DMEM medium containing 10% fetal bovine serum. After 80% of cells in the culture bottle grow full, removing the culture solution, adding 2ml of PBS buffer solution for gentle washing, removing the washing solution, adding 2ml of 0.125% pancreatin solution, digesting the cells for 50s, removing the digestion solution, adding DMEM culture medium containing 10% fetal calf serum to stop digestion, adding 4ml of 10% DMEM culture medium, gently blowing and beating to form single cell suspension, passaging in a new culture bottle, and carrying out 5% CO treatment at 37 ℃ to obtain the single cell suspension2Culturing in an incubator.
End3 cells were grown to confluency in flasks, digested with 0.125% trypsin solution, diluted to 1.0X 10 in high-glucose DMEM medium containing 10% fetal bovine serum5Each.ml-1Inoculating into 96-well culture plate with 100 μ l/well, and culturing at 37 deg.C with 5% CO2Culturing for 24h under the condition to obtain a fusion state, and synchronously culturing for 1h by changing a serum-free high-sugar DMEM culture medium before an experiment.
Mouse b.End3 endothelial cells were divided into 2 groups of 9 dishes, each group of endothelial cells were given 10. mu.M ICG or 10. mu.M ICG-PEG 500. After each group of cells was treated, the cells were washed 2 times with PBS buffer, and 0.5 mg/ml of MTT working solution was added thereto-1At 37 ℃ and 5% CO2After 3 hours of incubation under the conditions, the culture medium was removed, crystals were dissolved by adding 150. mu.l of DMSO to each well, and the absorbance (measurement wavelength 570nm, reference wavelength 650nm) was measured to calculate the cell viability, which was ═ total number of cells-dead cells/total number of cells × 100%. The images were photographed by SWIR near infrared camera with excitation spectrum of 808nm and 900nm long-wave pass filter at 1min, 5min, 15min, 30min and 60 min. Endothelial cells were subjected to near infrared zone ii fluorescence imaging and photographed. The high fluorescence signal is mainly selectively concentrated on the endothelial cell membrane, and these regions are the main penetration and enrichment sites of ox-LDL. The red high fluorescence intensity area of endothelial cells was analyzed, the calculated background signal was subtracted, and the vascular endothelial intensity of the high fluorescence emission of each group of endothelial cells was calculated as a percentage of the total endothelial cell membrane.
In FIG. 9, after 5min, fluorescence began to appear on the endothelial cell membrane after administration of ICG-PEG500, and the fluorescence signal was very clear on the endothelial cell membrane after 15-30min, which was more clear than the image formed by the endothelial cell injected with ICG for 15 min. Imaging of ICG-PEG500 administered endothelial cells 15min later, which was close to 5min later, suggests that ICG-PEG500 can be enriched on the endothelial cell membrane and persist for a relatively long time.
Claims (7)
1. A preparation method of indocyanine green derivatives comprises the following steps,
(1) preparation of-CO-NHS-modified indocyanine green derivative ICG-CO-NHS:
a commercial poly (lactic-co-glycolic acid) copolymer, PLGA, was reacted with a commercial indocyanine green imide derivative, ICG-NHS, PLGA: ICG-NHS molar ratio of 1: 5-10, stirring and reacting for 24 hours at room temperature in a weakly alkaline environment, wherein the modified indocyanine green ICG is a-CO-NHS modified indocyanine green derivative ICG-CO-NHS;
(2) preparation of PEG-NH2-modified indocyanine green derivative ICG-PEG 500:
taking the indocyanine green derivative ICG-CO-NHS modified by-CO-NHS and m-PEG-NH prepared in the step (1)2-in a molar ratio of 1: 10 to 15, reacting in a weak alkaline environment at room temperature in a shaking table for 12 hours, wherein m-PEG-NH2-carrying out ester substitution reaction with-CO-NHS modified indocyanine green derivative ICG-CO-NHS, removing a molecule of N-hydroxysuccinimide, stopping the reaction, concentrating under reduced pressure, and drying with anhydrous sodium sulfate to obtain PEG-NH2-a modified indocyanine green ICG derivative ICG-PEG 500.
2. The method for preparing indocyanine green derivative according to claim 1, wherein in step (1), the pH in the weakly alkaline environment is 7.35 to 7.44.
3. The method for preparing indocyanine green derivative according to claim 1, wherein in step (2), the weakly alkaline environment is phosphate buffered saline solution having a pH of 7.0 to 8.0.
4. The method for preparing an indocyanine green derivative according to claim 1, wherein in step (2), m is monomethyl ether in m-PEG-NH 2-.
5. The method for preparing indocyanine green derivative according to claim 1, wherein in step (2), the indocyanine green derivative ICG-CO-NHS modified by-CO-NHS is reacted with PEG-NH2-in a molar ratio of 1: 12, and mixing.
6. Use of the indocyanine green derivative ICG-PEG500 according to any one of claims 1 to 5 for the detection of oxidized low-density lipoproteins, which comprises the steps of,
(1) will be PEG-NH2-the modified indocyanine green derivative ICG-PEG500 dissolved in physiological saline to a concentration of 20. mu.g.ml-1The mixed solution of (1 mg/kg)-1Measuring tail vein and injecting into mouse;
(2) carrying out near-infrared II-zone fluorescence imaging and photographing on the mice at the time points of 3min, 6min, 30min, 120min, 240min, 480min and 600min after tail vein injection respectively;
(3) taking a picture by using a SWIR near-infrared camera, taking a picture under a long-wave pass filter with the excitation spectrum of 808nm and the excitation spectrum of 900nm, observing fluorescence signal areas of an aortic arch area and a leg skin area of the mouse, taking images by using a Zeiss Axio Scope A1 fluorescence microscope, and analyzing the fluorescence areas in ImageJ software;
(4) the picture was converted into an 8bit gray scale image using ImageJ software, and then the picture was subjected to black-and-white inversion, the optical density was corrected, and pixels were selected as a unit of measurement, the Area of the fluorescence region was measured, and data was measured and recorded.
7. Use of the indocyanine green derivative ICG-PEG500 according to any one of claims 1 to 5 for the detection of oxidized low-density lipoproteins, which comprises the steps of,
(1) ICG or ICG-PEG500 is dissolved in high-sugar DMEM medium, and ICG or ICG-PEG500 with the final concentration of 10 mu M is administered to endothelial cells;
(2) performing near-infrared II-zone fluorescence imaging on endothelial cells at time points of 1min, 5min, 15min, 30min and 60min respectively, and taking a picture;
(3) taking a picture by using a SWIR near-infrared camera, taking a picture under a long-wave pass filter with the excitation spectrum of 808nm and the excitation spectrum of 900nm, taking a cell fluorescence image by using a Zeiss Axio Scope A1 fluorescence microscope, and analyzing a fluorescence area in ImageJ software;
(4) the picture was converted into an 8bit gray scale image using ImageJ software, and then the picture was subjected to black-and-white inversion, the optical density was corrected, and pixels were selected as a unit of measurement, the Area of the fluorescence region was measured, and data was measured and recorded.
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