CRISPR/Cas9 system is a powerful toolbox for gene editing. However, the low delivery efficiency is still a big hurdle impeding its applications. Herein, we report a strategy to deliver Cas9-sgPlk-1 plasmids (CP) by a multifunctional vehicle for tumor therapy. We condensed CPs on TAT peptide-modified Au nanoparticles (AuNPs/CP, ACP) via electrostatic interactions, and coated lipids (DOTAP, DOPE, cholesterol, PEG2000-DSPE) on the ACP to form lipid-encapsulated, AuNPs-condensed CP (LACP). LACP can enter tumor cells and release CP into the cytosol by laser-triggered thermo-effects of the AuNPs; the CP can enter nuclei by TAT guidance, enabling effective knock-outs of target gene (Plk-1) of tumor (melanoma) and inhibition of the tumor both in vitro and in vivo. This AuNPs-condensed, lipid-encapsulated, and laser-controlled delivery system provides a versatile method for high efficiency CRISPR/Cas9 delivery and targeted gene editing for treatment of a wide spectrum of diseases.

No abstract

CRISPR/Cas9 system is apowerfultoolbox for gene editing. However,the low delivery efficiency is still abig hurdle impeding its applications.H erein, we report as trategy to deliver Cas9-sgPlk-1 plasmids (CP) by am ultifunctional vehicle for tumor therapy. We condensed CPs on TAT peptide-modified Au nanoparticles (AuNPs/CP,A CP) via electrostatic interactions,a nd coated lipids (DOTAP, DOPE, cholesterol, PEG2000-DSPE) on the ACPt of orm lipidencapsulated, AuNPs-condensed CP (LACP). LACP can enter tumor cells and release CP into the cytosol by lasertriggered thermo-effects of the AuNPs;the CP can enter nuclei by TATguidance,enabling effective knock-outs of target gene (Plk-1) of tumor (melanoma) and inhibition of the tumor both in vitro and in vivo.T his AuNPs-condensed, lipid-encapsulated, and laser-controlled delivery system provides aversatile method for high efficiency CRISPR/Cas9 delivery and targeted gene editing for treatment of aw ide spectrum of diseases.

BackgroundThe sentinel lymph node biopsy (SLNB) was developed as a new modality in the surgical diagnosis of lymph node metastases. Dye and radioisotope are major tracers for the detection of sentinel lymph nodes (SLN). Dye tends to excessively infiltrate into the interstitium due to their small size (less than several nanometers), resulting in difficulties in maintaining clear surgical fields. Radioisotopes are available in limited number of hospitals. Fluorescent nanoparticles are good candidates for SLN tracer to solve these problems, as we can choose suitable particle size and fluorescence wavelength of near-infrared. However, the use of nanoparticles faces safety issues, and many attempts have been performed by giving insulating coats on nanoparticles. In addition, the preparation of the uniform insulating layer is important to decrease variations in the quality as an SLN tracer.Methodology/Principal FindingsWe herein succeeded in coating fluorescent polystyrene nanoparticles of 40 nm with uniform silica layer of 13 nm by the modified Stöber method. The light stability of silica coated nanoparticles was 1.3-fold greater than noncoated nanoparticles. The popliteal lymph node could be visualized by the silica coated nanoparticles with injection in the rat feet.Conclusions/SignificanceThe silica coated nanoparticles in lymph nodes could be observed by transmission electron microscope, suggesting that our silica coating method is useful as a SLN tracer with highly precise distribution of nanoparticles in histological evaluation. We also demonstrated for the first time that a prolonged enhancement of SLN is caused by the phagocytosis of fluorescent nanoparticles by both macrophages and dendritic cells.

Contrast agents are often used to enhance the contrast of X-ray computed tomography (CT) imaging of tumors to improve diagnostic accuracy. However, because the iodine-based contrast agents currently used in hospitals are of low molecular weight, the agent is rapidly excreted from the kidney or moves to extravascular tissues through the capillary vessels, depending on its concentration gradient. This leads to nonspecific enhancement of contrast images for tissues. Here, we created gold (Au) nanoparticles as a new contrast agent to specifically image tumors with CT using an enhanced permeability and retention (EPR) effect. Au has a higher X-ray absorption coefficient than does iodine. Au nanoparticles were supported with polyethylene glycol (PEG) chains on their surface to increase the blood retention and were conjugated with a cancer-specific antibody via terminal PEG chains. The developed Au nanoparticles were injected into tumor-bearing mice, and the distribution of Au was examined with CT imaging, transmission electron microscopy, and elemental analysis using inductively coupled plasma optical emission spectrometry. The results show that specific localization of the developed Au nanoparticles in the tumor is affected by a slight difference in particle size and enhanced by the conjugation of a specific antibody against the tumor.