Nuclear-targeted CRISPR/Cas delivery by using aptamer-modified polymer lipid nanoparticles

Efficient nuclear delivery of CRISPR/Cas ribonucleoproteins (RNP) remains a significant hurdle for non-viral systems. To address this, we developed a polymer-lipid hybrid nanoparticle functionalized with the AS1411 aptamer, targeting nucleolin to facilitate nucleus-directed delivery. Confocal imaging confirmed the accumulation of these aptamer-modified nanoparticles within the cell nuclei. For precise quantification, we utilized an AI-assisted segmentation approach based on deep convolutional neural networks (CNN) to analyse nanoparticle and DAPI colocalization. We further evaluated in vitro gene knockout efficiency of Cas9/sgRNA by using this nucleus-targeted system. Aptamer-functionalised nanoparticles reduced GFP-positive cells to 30.0%, compared with 40.8% for untargeted nanoparticles. Further evaluation targeting the Lcn2 gene demonstrated higher knockout efficacy and a more potent inhibition of breast cancer cell proliferation. These findings indicate that aptamer-mediated nuclear targeting enhances CRISPR/Cas9 editing efficacy and may offer the potential to advance the performance of non-viral gene therapies. This graphical abstract illustrates the design and mechanism of an AS1411 aptamer-functionalized polymer-lipid hybrid nanoparticle, engineered for nucleus-targeted CRISPR/Cas9 delivery. The process begins with the nanoparticles binding on cell-surface nucleolin, followed by cellular internalization and nucleolin-mediated trafficking, which significantly enhances the nuclear accumulation of Cas9/sgRNA complexes. By integrating aptamer-mediated targeting with a non-viral nanocarrier, this strategy provides an effective platform for enhancing both the precision of nuclear delivery and overall gene knockout efficiency in cancer cells. • AS1411-functionalized hybrids enhance nucleolin-mediated nuclear CRISPR/Cas9 delivery. • Targeted nanoparticles increase nucleus colocalization in MDA-MB-231 and HCT-116. • U-Net CNN provides objective, reproducible nuclear colocalization quantification. • Aptamer targeting improves GFP and Lcn2 editing, reducing proliferation and migration.