Oligonucleotide/atom fine nanocluster compound capable of efficiently penetrating through blood brain barrier and preparation method and application thereof
A technology of oligonucleotide and nanocluster, which is applied in the field of oligonucleotide/atom fine nanocluster complex and its preparation, can solve the problems of lack of efficient materials for crossing the blood-brain barrier, and achieve good biological learning effect, improving crossing efficiency, and efficient crossing effect
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Embodiment 1
[0044] The atomically fine gold nanoclusters protected by oligonucleotides in this example include Au8 clusters with a specific structure, containing 40 thymines (T 40 ) single-stranded oligonucleotide, and dimethylformamide, an organic solvent miscible with water.
[0045] The preparation method of the atomically fine gold nanoclusters protected by the oligonucleotide is as follows:
[0046] 1) Dissolve Au8 in acetonitrile to a final concentration of 100 μM;
[0047] 2) Put T 40 Single-stranded oligonucleotides were dissolved in deionized water to a final concentration of 100 μM;
[0048] 3) Take 200 μL of the Au8 solution and add 100 μL of T 40 Add 100 μL of water-miscible dimethylformamide to the single-stranded oligonucleotide solution, mix well, shake at 400 rpm at room temperature for 12 hours overnight at 25°C.
[0049] 4) Add 3mL of deionized water to the mixed solution obtained in step 3), use a K ultrafiltration tube, 12000rpm, ultrafiltration three times for 15m...
Embodiment 2
[0053] Oligonucleotide-protected atomically fine gold nanoclusters cross the blood-brain barrier in mice as follows:
[0054] 1) The aqueous solution of atomically fine gold nanoclusters protected by oligonucleotides obtained in Example 1 was mixed with 10xPBS at a ratio of 9:1, and sonicated for 30 minutes.
[0055] 2) Use a 1mL syringe to inject into the mouse body through the tail vein, and observe the fluorescence of the mouse brain (1h, 6h, 12h, 36h) with a small animal in vivo imager. The results are as follows Figure 4 shown.
[0056] 3) After 12 hours of administration, the mice were dissected, and the mouse brain tissue was taken for frozen section observation. The thickness of the slice was 10 μm. DAPI was used to stain the nucleus of the mouse brain tissue, and the red color of the brain could be seen under fluorescent confocal microscope observation. Fluorescence is the atomically fine gold nanoclusters protected by oligonucleotides in the present invention, such...
Embodiment 3
[0059] 1) Culture human blood-brain barrier brain endothelial cells bEnd.3 to 1×10 5 About (100×20mm Petri dish, Corning, USA). The culture conditions are: containing growth factors, DMEM medium (Gibco LifeTechnologies Inc., UK), adding 10% (v / v) fetal bovine serum, penicillin and streptomycin (100U / mL, Sigma-Aldrich) and streptomycin (100 μg / mL, Sigma-Aldrich), 37 ° C, 5% CO 2 cultivated under conditions.
[0060] 2) Mix the atomically fine gold nanoclusters protected by the oligonucleotide obtained in Example 1 with the medium, and the final concentration of the gold clusters is 2 μM. After incubation with the cells at 37°C for 30 min, observed under a confocal fluorescence microscope, it can be seen that atomically fine gold nanoclusters protected by oligonucleotides can be effectively taken up by brain endothelial cells. Such as Figure 7 shown.
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