In Vitro Prostate Cancer Treatment via CRISPR-Cas9 Gene Editing Facilitated by Polyethyleneimine-Derived Graphene Quantum Dots.

CRISPR-Cas9 is a programmable gene editing tool with a promising potential for cancer gene therapy. This therapeutic function is enabled in the present work via the non-covalent delivery of CRISPR ribonucleic protein (RNP) by cationic glucosamine/PEI-derived graphene quantum dots (PEI-GQD) that aid in overcoming physiological barriers and tracking genes of interest. PEI-GQD/RNP complex targeting the TP53 mutation overexpressed in ~50% of cancers successfully produces its double-stranded breaks in solution and in PC3 prostate cancer cells. Restoring this cancer "suicide" gene can promote cellular repair pathways and lead to cancer cell apoptosis. Its repair to the healthy form performed by simultaneous PEI-GQD delivery of CRISPR RNP and a gene repair template leads to a successful therapeutic outcome: 40% apoptotic cancer cell death, while having no effect on non-cancerous HeK293 cells. The translocation of PEI-GQD/RNP complex into PC3 cell cytoplasm is tracked via GQD intrinsic fluorescence, while EGFP-tagged RNP is detected in the cell nucleus, showing the successful detachment of the gene editing tool upon internalization. Using GQDs as non-viral delivery and imaging agents for CRISPR-Cas9 RNP sets the stage for image-guided cancer-specific gene therapy.

Cancer Therapeutic siRNA Delivery and Imaging by Nitrogen- and Neodymium-Doped Graphene Quantum Dots.

While small interfering RNA (siRNA) technology has become a powerful tool that can enable cancer-specific gene therapy, its translation to the clinic is still hampered by the inability of the genes alone to cell transfection, poor siRNA stability in blood, and the lack of delivery tracking capabilities. Recently, graphene quantum dots (GQDs) have emerged as a novel platform allowing targeted drug delivery and fluorescence image tracking in visible and near-infrared regions. These capabilities can aid in overcoming primary obstacles to siRNA therapeutics. Here, for the first time, we utilize biocompatible nitrogen- and neodymium-doped graphene quantum dots (NGQDs and Nd-NGQDs, respectively) for the delivery of Kirsten rat sarcoma virus (KRAS) and epidermal growth factor receptor (EGFR) siRNA effective against a variety of cancer types. GQDs loaded with siRNA noncovalently facilitate successful siRNA transfection into HeLa cells, confirmed by confocal fluorescence microscopy at biocompatible GQD concentrations of 375 µg/mL. While the GQD platform provides visible fluorescence tracking, Nd doping enables deeper-tissue near-infrared fluorescence imaging suitable for both in vitro and in vivo applications. The therapeutic efficacy of the GQD/siRNA complex is verified by successful protein knockdown in HeLa cells at nanomolar siEGFR and siKRAS concentrations. A range of GQD/siRNA loading ratios and payloads are tested to ultimately provide substantial inhibition of protein expression down to 31-45%, comparable with conventional Lipofectamine-mediated delivery. This demonstrates the promising potential of GQDs for the nontoxic delivery of siRNA and genes in general, complemented by multiwavelength image tracking.