Molecular probe for imaging coronavirus infected living body and preparation method thereof
A coronavirus and molecular probe technology, applied in the field of biomedical applications, to achieve enhanced stability, good application prospects, and improved uptake
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example 1
[0030] Example 1 Molecular probe 64 Preparation of Cu-NOTA-HR-609
[0031] (1) Synthesis of NOTA-HR-609
[0032] (2) Radioactive labeling reaction
[0033] Dissolve 30-500 μg of labeled precursor NOTA-HR-609 in 100 μL of sodium acetate buffer (0.1-0.5 M, pH = 5.5) (peptides that are not easily soluble can be solubilized by ultrasound), and add 65-85 MBq of [ 64 Cu]CuCl 2 (usually as CuCl 2 The chemical form exists in 0.01 M HCl), and the mixture is reacted at room temperature for 30-60 min (the time is generally controlled within 60 min) to complete the labeling. The labeling rate can be determined by HPLC or TLC. If the labeling rate is greater than 95%, it can be used directly without purification. If the labeling rate is less than 90%, Sep-pak C18 column separation and purification to obtain molecular probes 64 Cu-NOTA-HR-609.
[0034] (3) Radiochemical purity and labeling rate
[0035] Determination of Molecular Probes by Thin Layer Chromatography Radiation Scanner...
example 2
[0036] Example 2 Molecular probe Cy5-HR-609 in vitro cell experiment
[0037] Put HEK293 / SARS-CoV-2 cells stably expressing S protein into flow tubes, 100 μL per tube, 10 5 cells were divided into: a. Negative control, no antibody; b. Positive control, first add anti-S2 antibody, incubate for 30 min, then add AlexaFluor®488-AffiniPure Goat Anti-Human IgG and incubate for 30 min; c. Add Cy5- HR-609, incubate for 1 hour; d. First add anti-S2 antibody and Cy5-HR-609, incubate for 30 minutes, then add Alexa Fluor®488-AffiniPureGoat Anti-Human IgG and incubate for 30 minutes. Analysis was performed using a flow cytometer (CytoFLEX, BECKMAN COULTER). For specific results, see image 3 . In vitro cell experiments show that Cy5-HR-609 has a very strong ability to target target proteins at the cellular level.
example 3
[0038] Example 3 Molecular probe Cy5-HR-609 in vivo animal experiment
[0039] Approved by the Ethics Committee of the Fifth Affiliated Hospital of Sun Yat-sen University, SCID mice were purchased from Guangdong Experimental Animal Center under a suitable environment (21°C, humidity 60%, 15 air circulation / h, 12h light / d, 2 cleanings / d) Eat and drink freely for 2 days, and carry out environmental pre-adaptation. Afterwards, the animals were anesthetized by inhalation of 2.5% isoflurane. HEK293 cells highly expressing or not expressing S protein suspended in PBS were subcutaneously injected into the right axilla for tumor formation in animals. Fluorescent imaging was performed when the subcutaneous tumor reached a diameter of 0.8-1.0 cm. Cy5-HR-609 dissolved in 40% cyclodextrin solution was injected into the tail vein at a dose of 4 mg / Kg, and the image acquisition time was 0 h, 1 h, 2 h, 4 h, 12 h, 24 h, 36 h, 48 h h. A region of interest (Region of Interest, ROI) was draw...
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