研究目的
To design a near-infrared (NIR) light–responsive nanocarrier of CRISPR-Cas9 for cancer therapeutics based on upconversion nanoparticles (UCNPs) for spatiotemporal manipulation of CRISPR-Cas9 delivery.
研究成果
The UCNPs-Cas9@PEI platform can be effectively internalized by cells via endocytosis pathways, followed by endosomal escape and cytosolic releases of the loaded UCNPs-Cas9 in the cytoplasm. When exposed to NIR light, UCNPs can emit local UV light and trigger the cleavage of the photocaged linkers, releasing Cas9 for gene editing. This strategy underlines the remote-operated nature of the noninvasive NIR light governing CRISPR-Cas9 release, enhancing the appeal of CRISPR-Cas9 technology and encouraging the developed smart delivery in nanomedicine.
研究不足
Owing to the lack of property of targeting tumors, a relatively small number of the nanovehicles were observed in the tumor tissues via intravenous administration; thus, not enough Cas9 accumulated in tumor to trigger gene editing by NIR. The proof of concept is done in such a way that UCNPS-cas9@PEI allows gene editing, but only after local administration to accessible tumors for which surgery may, however, be preferred.
1：Experimental Design and Method Selection:
Designed a NIR light–responsive nanocarrier of CRISPR-Cas9 based on UCNPs.
2：Sample Selection and Data Sources:
Used A549 cells (a human lung adenocarcinoma cell line) and KB cells (human oral epidermoid carcinoma) for in vitro studies and xenograft nude mice model bearing A549 cells for in vivo studies.
3：List of Experimental Equipment and Materials:
UCNPs, photosensitive molecules (ONA), polyethylenimine (PEI), Cas9 proteins, sgRNA, etc.
4：Experimental Procedures and Operational Workflow:
Synthesis of UCNPs, preparation of UCNPs-Cas9@PEI, NIR light–controlled release of CRISPR-Cas9, evaluation of cell viability, imaging of cellular uptake, EGFP gene disruption assay, Western blot analysis, SURVEYOR assay, tumor xenografts, and detection of tumor suppression in vivo.
5：Data Analysis Methods:
Confocal laser scanning microscopy (CLSM), flow cytometry (FCM), Western blot, SURVEYOR assay, etc.
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Microplate photometer
Multiskan FC
Thermo Fisher Scientific Inc.
Acquire UV-visible spectra and evaluate cell viability.
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Transmission electron microscope
JEM-200CX
JEOL Ltd.
Detect the morphology of nanoparticles.
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Confocal laser scanning microscope
LSM 710
Zeiss
Observe the process of cellular uptake and gene editing.
ZEISS LSM 990 Spectral Multiplex
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UCNPs
Serve as 'nanotransducers' that can convert NIR light (980 nm) into local ultraviolet light for the cleavage of photosensitive molecules, thereby resulting in on-demand release of CRISPR-Cas9.
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CRISPR-Cas9
Mediate genome modification with respect to versatility and high precision.
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PEI
Assist endosomal escape.
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ONA
Photosensitive molecules for the cleavage of ester bond under UV light.
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Flow cytometer
BD FACSVerse
BD Biosciences
Analyze EGFP disruption in cells and cell apoptosis.
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