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.
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.
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.
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