Fluorescent tracer system and method suitable for studying liposome distribution in vivo and in vitro
A fluorescent tracer and liposome technology, applied in liposome delivery, preparations for in vivo tests, fluorescence/phosphorescence, etc., can solve problems such as difficulties in studying the distribution of liposomes in vivo and in vitro, and avoid intra-group differences , fast response, easy to operate
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Embodiment 1
[0081] Example 1 Preparation and characterization of trans-cyclooctene-labeled liposomes
[0082] (1) Trans-cyclooctene active ester (TCO-NHS) and amino lipid (in this example, DSPE-PEG2000-NH 2 For example) were dissolved in organic solvents (such as dichloromethane, chloroform, absolute ethanol, etc.), so that the final concentration of the solution was 10 mg / mL; the trans-cyclooctene active ester solution was added to the amino lipid solution. , so that the number of moles of trans-cyclooctene active ester is 2 to 5 times that of amino lipids, add 0.3% to 0.5% of triethylamine by volume, shake gently at room temperature, react for 8h, use ethyl acetate The unreacted trans-cyclooctene active ester was removed by washing with a mixed solution of diethyl ether to obtain trans-cyclooctene lipid (DSPE-PEG2000-TCO). The organic solvent in the trans-cyclooctene lipid solution was removed, and an appropriate amount of deionized water was added to make the concentration higher than...
Embodiment 2
[0089] Example 2 Study on the stability of trans-cyclooctene liposome preparations
[0090] The trans-cyclooctene liposomes prepared in Example 1 (the molar ratio of trans-cyclooctene to lipids were 1%, 3%, and 5%) were redispersed in deionized water, PBS, HEPES, 1640 culture medium and saline. The particle size, polydispersity coefficient (PDI) of particle size and zeta potential were measured at different time points using dynamic light scattering nanoparticle size analyzer (DLS) to investigate its stability.
[0091] The drug-loaded trans-cyclooctene liposomes (the molar ratio of trans-cyclooctene was 1%, 3%, and 5%) were detected by ultraviolet absorption (UV) with a microplate reader after dialysis to analyze the leakage of drugs at different time points. In some cases, doxorubicin is used as the model drug.
[0092] like Figures 5a-5dAs shown, the particle size of trans-cyclooctene liposomes (1%) did not change significantly in deionized water, PBS, HEPES, 1640 mediu...
Embodiment 3 4
[0094] Example 3 Synthesis of Tetrazine Fluorescent Probes
[0095] Dissolve the tetrazine active ester (Tetrazine-PEG5-NHS Ester) and the near-infrared fluorescent dye (Cy5.5 in this example) in an organic solvent, respectively, and mix the two so that the number of moles of the tetrazine active ester is near-infrared. 3 to 5 times the fluorescent dye, add triethylamine with a volume ratio of 0.3% to 0.5%, and react at room temperature in the dark and shake for 12 hours. After the reaction is completed, wash with ether and ethyl acetate to separate and obtain blue color. The solid is freeze-dried to obtain a dark blue powder, which is a tetrazine near-infrared fluorescent probe (Tz-Cy5.5). When ready to use, it can be redissolved in buffers such as PBS, physiological saline, and serum-free medium.
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