Self-Assembled DNA-Protein Hybrid Nanospheres: Biocompatible Nano-Drug-Carriers for Targeted Cancer Therapy.

We developed hybrid nanospheres comprised of two of the most important biomolecules in nature, DNA and proteins, which have excellent biocompatibility, high drug payload capacity, in vivo imaging ability, and in vitro/in vivo cancer targeting capability. The synthesis can be done in a facile one-pot assembly system that includes three steps: step-growth polymerization of two DNA oligomers, addition of streptavidin to assemble spherical hybrid nanostructures, and functionalization of hybrid nanospheres with biotinylated aptamers. To test the feasibility of cancer targeting and drug-loading capacity of the hybrid nanospheres, MUC1-specific aptamers (MA3) were conjugated to nanosphere surfaces (apt-nanospheres), and doxorubicin (Dox) was loaded into nanospheres by DNA intercalation. The successful construction of nanospheres and apt-nanospheres was confirmed by agarose gel electrophoresis and dynamic light scattering (DLS). Their uniform spherical morphology was confirmed by transmission electron microscopy (TEM). Fluorescence spectra of nanospheres demonstrated high Dox-loading capability and slow-release characteristics. In vitro MUC1-specific binding of the apt-nanospheres was confirmed by flow cytometry and confocal microscopy. Dox-loaded apt-nanospheres significantly increased cytotoxicity of the MUC1-positive cancer cells due to aptamer-mediated selective internalization, as shown via cell viability assays. Apt-nanospheres could also be imaged in vivo through the synthesis of hybrid nanospheres using fluorescent dye-conjugated DNA strands. Finally, in vivo specific targeting ability of apt-nanospheres was confirmed in a MUC1-positive 4T1 tumor-bearing mouse model, whereas apt-nanospheres did not cause any sign of systemic toxicity in normal mice. Taken together, our self-assembled DNA-streptavidin hybrid nanospheres show promise as a biocompatible cancer targeting material for contemporary nanomedical technology.

An efficient cell type specific conjugating method for incorporating various nanostructures to genetically encoded AviTag expressed optogenetic opsins.

Here, we report genetically encoded AviTag conjugating system for Channelrhodopsin-2(ChR2) in order to attach various nanostructures to the membrane protein in a cell type specific manner. First, AviTag peptide sequence is cloned to N-terminal site of ChR2 construct and expressed at the membrane of primary-cultured hippocampal neurons via lentiviral transduction. Second, with the help of BirA enzyme and ATP, biotin coated quantum dots (Qdots) and streptavidin (SAv) coated Qdots are successfully bound to AviTag sites at the membrane where ChR2 is located and confirmed by fluorescence imaging. Moreover, we synthesize biotinylated Traptavidin-DNA conjugate probes containing a desthio-biotin that has weaker affinity than a regular biotin, and successfully exchange them with pre-conjugated Biotin-AviTag-ChR2 site at the membrane of neuronal cells which can potentially solve the crosslinking issue of Avidin linked probes. Therefore, we expect the AviTag-ChR2 fusion platform to become a great tool for incorporating various nanostructures at the specific sites of a cellular membrane in order to overcome the limits of optogenetic opsins.

Multivalent Traptavidin-DNA Conjugates for the Programmable Assembly of Nanostructures.

Here, we explore the extended utility of two important functional biomolecules, DNA and protein, by hybridizing them through avidin-biotin conjugation. We report a simple yet scalable technique of successive magnetic separations to synthesize traptavidin-DNA conjugates with four distinct DNA binding sites that can be used as a supramolecular building block for programmable assembly of nanostructures. Using this nanoassembly platform, we fabricate several different plasmonic nanostructures with various metallic as well as semiconductor nanoparticles in predetermined ways. We also use the platform to construct dendrimer nanostructures using valency-controlled traptavidin-DNA conjugates in a programmable manner. These results suggest that our protein-DNA supramolecular building blocks would make a significant contribution to the assembly of multicomponent and complex nanostructures for numerous contemporary and future applications from molecular imaging to drug delivery.

Galactosylation of chitosan-graft-spermine as a gene carrier for hepatocyte targeting in vitro and in vivo.

Polyethyleneimine (PEI) has been described as a highly efficient gene carrier due to its efficient proton sponge effect within endosomes. However, many studies have demonstrated that PEI is toxic and associated with a lack of cell specificity despite high transfection efficiency. In order to minimize the toxicity of PEI, we prepared chitosan-graft-spermine (CHI-g-SPE) in a previous study. CHI-g-SPE showed low toxicity and high transfection efficiency. However, this compound also had limited target cell specificity. In the present study, we synthesized galactosylated CHI-g-SPE (GCS) because this modified GCS could be delivered specifically into the liver due to hepatocyte-specific galactose receptors. The DNA-binding properties of GCS at various copolymer/DNA weight ratios were evaluated by a gel retardation assay. The GCS copolymer exhibited significant DNA-binding ability and efficiently protected DNA from nuclease attack. Using energy-filtered transmission electron microscopy (EF-TEM), we observed dense spherical, nano-sized GCS/DNA complexes with a homogenous distribution. Most importantly, GCS was associated with remarkably low cytotoxicity compared to PEI in HepG2, HeLa, and A549 cells. Moreover, GCS carriers specifically delivered the gene-of-interest into hepatocytes in vitro as well as in vivo. Our results suggest that the novel GCS described here is a safe and highly efficient carrier for hepatocyte-targeted gene delivery.

Suppression of tumor growth in H-ras12V liver cancer mice by delivery of programmed cell death protein 4 using galactosylated poly(ethylene glycol)-chitosan-graft-spermine.

Non-viral gene delivery systems based on polyethyleneimine (PEI) are efficient due to their proton-sponge effect within endosomes, but they have poor physical characteristics such as slow dissociation, cytotoxicity, and non targeted gene delivery. To overcome many of the problems associated with PEI, we synthesized a galactosylated poly(ethylene glycol)-chitosan-graft-spermine (GPCS) copolymer with low cytotoxicity and optimal gene delivery to hepatocytes using an amide bond between galactosylated poly(ethylene glycol) and chitosan-graft-spermine. The GPCS copolymer formed complexes with plasmid DNA, and the GPCS/DNA complexes had well-formed spherical shapes. The GPCS/DNA complexes were nanoscale size with homogenous size distribution and a positive zeta potential by dynamic light scattering (DLS). The GPCS copolymer had lower cytotoxicity than that of PEI 25K in HepG2, HeLa, and A549 cell lines at various concentrations and showed good hepatocyte-targeting ability. Furthermore, GPCS/DNA complexes showed higher levels of GFP expression in the liver in model mice after intravenous injection than naked DNA and metoxy-poly(ethylene glycol)-chitosan-graft-spermine as controls without remarkable fibrosis, inflammation, lipidosis, or necrosis. In a tumor suppression study, an intravenous injection of the GPCS/Pdcd4 complexes significantly suppressed tumor growth, activated apoptosis, and suppressed proliferation and angiogenesis in liver tumor-bearing H-ras12V mice. Our results indicate that the GPCS copolymer has potential as a hepatocyte-targeting gene carrier.