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Hydrophobic Nanobiological Probes

A nano-biological probe, hydrophobic technology, applied in the field of biomaterials and biomedicine, can solve problems such as cell death, long-term technical practice, labor, etc.

Active Publication Date: 2022-02-22
NANJING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Furthermore, even without increasing the diameter of the polymer coating, the size of the bare hydrophobic QDs approaches that of the protein, and intracellular vesicle capture remains an insurmountable hurdle: typically <1% of endocytosed proteins are estimated to (and other types of macromolecules) can escape intracellular vesicle capture and reach the cytoplasm
In addition, bypassing endocytosis by methods that physically disrupt cell membranes (such as microinjection and electroporation) often requires additional instrumentation, requiring prolonged technical practice and laborious manipulations, leading to significant cell death and producing irreversible duplicate results

Method used

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Examples

Experimental program
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Effect test

preparation example Construction

[0148] 2. Preparation of cS-bQDs-Tat

[0149] Surface covered with hydrophobic Hydrophobic QD of ligand tri-n-octylphosphine oxide [published in ACS Nano, 6(12), pp11066-11073 in 2012]. Hydrophobic QDs (in DMF) were incubated with cysteamine for 0.5 h to exchange a small portion of the surface ligands of the QDs such that the resulting QDs had -NH on a small portion of the QD surface 2 (The remaining hydrophobic surface coverage is estimated to be typically 90%). Binding of Tat peptides to -NH on the QD surface using EDC 2 group conjugation to form bQDs-Tat. Disperse the solution (DMF, or other organic solvents, such as acetone, ethanol, and DMSO) in water (or other aqueous environments, such as cell culture media, with a typical volume ratio of organic solvent to water of 1:99) to form cS-bQDs -TAT.

[0150] 3. Physicochemical characterization of cS-bQDs-Tat

[0151] The morphology of the nanoparticles was visualized by transmission electron microscopy (TEM, JEM-200CX, ...

Embodiment 1

[0184] Example 1: Preparation and physicochemical characterization of cS-bQDs-Tat

[0185] To prepare cS-bQDs-Tat, a small portion (eg, 10%) of the exposed hydrophobic QDs (bQDs; surface ligand trioctylphosphine, or is the surface of TOP) ( figure 2 a). Organic solvents tested included dimethylformamide (DMF), acetone, ethanol, and dimethylsulfoxide (DMSO). DMF has the best overall performance. The Tat peptide was then attached to the QD surface by conjugation with cysteamine via 1-semiethylamine-3-(3-dimethylaminopropyl)carbodiimide (EDC) chemistry to form bQDs-Tat( figure 2 a). Subsequently, bQDs-Tat stored in an organic solvent was diluted in water to form cS-bQDs-Tat ( figure 2 a). The surface modification was confirmed by zeta potential, Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) analysis. With the addition of cysteamine and Tat peptide, the surface charge positive potential becomes higher and higher, which is consi...

Embodiment 2

[0187] Example 2: Intracellular targeting of cS-bQDs-Tat in living cells

[0188] cS-bQDs-Tat exhibited striking intracellular targeting in living cells. In ~30 independent experiments (performed by 3 independent investigators), ~200 cells were studied by fluorescence imaging in each experiment, and in ~95% of the cells studied (approximately 5% of the cells studied had no internalized QDs), cS-bQDs-Tat reliably produced about 95% nuclear targeting specificity (defined as the percentage of the amount of QDs in the nucleus relative to the total amount of QDs in the cell) ( image 3 a includes more targeted confocal images). This result is consistent with that obtained by isolating nuclei from cell suspensions followed by fluorescence spectroscopic measurements. Within the nucleus, cS-bQDs-Tat was found to frequently accumulate in the nucleolus, a specific intranuclear region where ribosome biogenesis takes place. This result indicates that cS-bQD-Tat can also overcome the mo...

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Abstract

The present invention provides a novel nanobiological probe and its application in intracellular targeted delivery of biologically active molecules. Specifically, the present invention firstly develops a mixed solvent-bare hydrophobic QD-biomolecule (cS-bQD ‑BM, or 'SDot'). Using the nucleus as a model target, SDots display extraordinary intracellular targeting performance, including near-perfect specificity, excellent efficiency and reproducibility, high-throughput capability, minimal toxicity, ease of manipulation, and excellent optical properties and colloidal stability.

Description

technical field [0001] The invention relates to the fields of biological materials and biomedicine, in particular to a nanometer biological probe and its application in cells, especially in living cells, for targeted delivery of bioactive molecules. Background technique [0002] Fluorescent probes play a key role in live cell imaging as a visualized signal source. Compared with the other two main classes of fluorescent probes, small molecule fluorescent dyes and genetically encoded fluorescent proteins, quantum dots (QDs, nanoscale crystals of semiconductor materials) have several distinctly superior or unique properties, especially optical properties , including extraordinary fluorescence intensity and photostability, size- and composition-tunable fluorescence emission peaks with narrow peak widths, can be excited for multiple imaging by a single light source, and can be used as imaging probes for correlative optical and electron microscopy. However, unlike fluorescent dye...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K49/00A61K45/00
CPCA61K49/0056A61K49/0067A61K49/0017A61K45/00A61K49/00C12Q1/68C12N15/11G01N33/50
Inventor 阮刚王军谢金兵梅玲
Owner NANJING UNIV