Direct cytosolic delivery of large biomolecules that bypass the endocytic pathways is a promising strategy for therapeutic applications. Recent works have shown that small-molecule, nanoparticle, and polymer-based carriers can be designed for direct cytosolic delivery. It has been shown that the specific surface chemistry of the carrier, nanoscale assembly between the carrier and cargo molecule, good colloidal stability, and low surface charge of the nano-assembly are critical for non-endocytic uptake processes. Here we report a guanidinium-terminated polyaspartic acid micelle for direct cytosolic delivery of protein and DNA. The polymer delivers the protein/DNA directly to the cytosol by forming a nano-assembly, and it is observed that <200 nm size of colloidal assembly with near-zero surface charge is critical for efficient cytosolic delivery. This work shows the importance of size and colloidal property of the nano-assembly for carrier-based cytosolic delivery of large biomolecules.

lncRNA genes can be genic or "intergenic". "Genic" RNAs can be further divided into six biotypes. Through genomewide analysis of a publicly available data set on corticogenesis, we found that the divergent lncRNA (XH) biotype, comprising the lncRNA and the coding gene being in opposite directions in a head-to-head manner, was most prominent during neural commitment. Within this biotype, a coding gene/divergent RNA pair of the BASP1 gene and the uncharacterized RNA loc285696 (hitherto referred as BASP1-AS1) formed a major HUB gene during neuronal differentiation. Experimental validation during the in vitro differentiation of human neural progenitor cells (hNPCs) showed that BASP1-AS1 regulates the expression of its adjacent coding gene, BASP1. Both transcripts increased sharply on the first day of neuronal differentiation of hNPCs, to fall steadily thereafter, reaching very low levels in differentiated neurons. BASP1-AS1 RNA and the BASP1 gene formed a molecular complex that also included the transcription factor TCF12. TCF12 is coded by the DYX1 locus, associated with inherited dyslexia and neurodevelopmental defects. Knockdown of BASP1-AS1, BASP1, or TCF12 impaired the neuronal differentiation of hNPCs, as seen by reduction in DCX and TUJ1-positive cells and by reduced neurite length. There was also increased cell proliferation. A common set of critical genes was affected by the three molecules in the complex. Our study thus identified the role of the XH biotype and a novel mediator of neuronal differentiation-the complex of BASP1-AS1, BASP1, and TCF12. It also linked a neuronal differentiation pathway to inherited dyslexia.

Although subcellular targeting can enhance the therapeutic performance of most drugs, such targeting requires appropriate carrier-based delivery that can bypass endosomal/lysosomal trafficking. Recent works show that nanocarriers can be designed for direct cell membrane translocation and nonendocytic uptake, bypassing the usual endocytosis processes. Here we show that this approach can be adapted for the rapid cell nucleus delivery of molecular drugs. In particular, a guanidinium-terminated nanocarrier is used to create a weak interaction-based carrier-drug nanoassembly for direct membrane translocation into the cytosol. The rapid and extensive entry of a drug-loaded nanocarrier into the cell without any vesicular coating and affinity of the drug to the nucleus allows their nucleus labeling. Compared to endocytotic uptake that requires more than hours for cell uptake followed by predominant lysosomal entrapment, this nonendocytic uptake labels the nucleus within a few minutes without any lysosomal trafficking. This approach may be utilized for nanocarrier-based subcellular targeting of drugs for more effective therapy.

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