Fei Jia

Light-Triggered, Self-Immolative Nucleic Acid-Drug Nanostructures

The simultaneous intracellular delivery of multiple types of payloads, such as hydrophobic drugs and nucleic acids, typically requires complex carrier systems. Herein, we demonstrate a self-deliverable form of nucleic acid-drug nanostructure that is composed almost entirely of payload molecules. Upon light activation, the nanostructure sheds the nucleic acid shell, while the core, which consists of prodrug molecules, disintegrates via an irreversible self-immolative process, releasing free drug molecules and small molecule fragments. We demonstrate that the nanostructures exhibit enhanced stability against DNase I compared with free DNA, and that the model drug (camptothecin) released exhibits similar efficacy as free, unmodified drugs toward cancer cells.

Blurring the Role of Oligonucleotides: Spherical Nucleic Acids as a Drug Delivery Vehicle

Nucleic acids are generally regarded as the payload in gene therapy, often requiring a carrier for intracellular delivery. With the recent discovery that spherical nucleic acids enter cells rapidly, we demonstrate that nucleic acids also have the potential to act as a delivery vehicle. Herein, we report an amphiphilic DNA-paclitaxel conjugate, which forms stable micellar nanoparticles in solution. The nucleic acid component acts as both a therapeutic payload for intracellular gene regulation and the delivery vehicle for the drug component. A bioreductively activated, self-immolative disulfide linker is used to tether the drug, allowing free drug to be released upon cell uptake. We found that the DNA-paclitaxel nanostructures enter cells ∼100 times faster than free DNA, exhibit increased stability against nuclease, and show nearly identical cytotoxicity as free drug. These nanostructures allow one to access a gene target and a drug target using only the payloads themselves, bypassing the need for a cocarrier system.

Temperature-activated nucleic acid nanostructures

DNA and poly(N-isopropylacrylamide) are co-assembled onto gold nanoparticles. The DNA sequences can be reversibly exposed or hidden from the polymer surface in response to temperature cues, thereby translating the temperature trigger to the on-off switching of the surface chemistry and function. When exposed by heating (∼30 °C), the DNA rapidly hybridizes to complementary strands, and chain-end biotin groups become readily accessible, while at lower temperatures these activities are largely blocked.

Polycondensation of Polymer Brushes via DNA Hybridization

Triblock copolymer brushes were functionalized with nucleic acid sequences, which allowed the polymers to connect head-to-tail and form supramolecular nanostructures. Two approaches were designed and implemented, using either a palindromic DNA attached to both ends of the polymer or two different DNA sequences attached regiospecifically. Given appropriate conditions, the DNA-brush conjugates self-assembled to form either nanoworms with length up to several microns or cross-linked networks. This process is analogous to the step-growth polymerization of small molecule monomers.

Providing Oligonucleotides with Steric Selectivity by Brush-Polymer-Assisted Compaction

Difficult biopharmaceutical characteristics of oligonucleotides, such as poor enzymatic stability, rapid clearance by reticuloendothelial organs, immunostimulation, and coagulopathies, limit their application as therapeutics. Many of these side effects are initiated via sequence-specific or nonsequence-specific interactions with proteins. Herein, we report a novel form of brush-polymer/DNA conjugate that provides the DNA with nanoscale steric selectivity: Hybridization kinetics with complementary DNA remains nearly unaffected, but interactions with proteins are significantly retarded. The relative lengths of the brush side chain and the DNA strand are found to play a critical role in the degree of selectivity. Being able to evade protein adhesion also improves in vivo biodistribution, thus making these molecular nanostructures promising materials for oligonucleotide-based therapies.

Effective Antisense Gene Regulation via Noncationic, Polyethylene Glycol Brushes

Negatively charged nucleic acids are often complexed with polycationic transfection agents before delivery. Herein, we demonstrate that a noncationic, biocompatible polymer, polyethylene glycol, can be used as a transfection vector by forming a brush polymer-DNA conjugate. The brush architecture provides embedded DNA strands with enhanced nuclease stability and improved cell uptake. Because of the biologically benign nature of the polymer component, no cytotoxicity was observed. This approach has the potential to address several long-lasting challenges in oligonucleotide therapeutics.

Effect of PEG Architecture on the Hybridization Thermodynamics and Protein Accessibility of PEGylated Oligonucleotides

PEGylation is an attractive approach to modifying oligonucleotides intended for therapeutic purposes. PEG conjugation reduces protein interactions with the oligonucleotide, and helps to overcome their intrinsic biopharmaceutical shortcomings, such as poor enzymatic stability, rapid body clearance, and unwanted immunostimulation. However, the effect of PEG architecture and the manner in which the PEG component interferes with the hybridization of the oligonucleotide remain poorly understood. In this study, we systematically compare the hybridization thermodynamics and protein accessibility of several DNA conjugates involving linear, Y-shaped, and brush-type PEG. It is found that PEGylated DNA experiences two opposing effects: local excluded volume effect and chemical interactions, the strengths of which are architecture-dependent. Notably, the brush architecture is able to offer significantly greater protein shielding capacity than its linear or Y-shaped counterparts, while maintaining nearly identical free energy for DNA hybridization compared with free DNA.

Modulating the Cellular Immune Response of Oligonucleotides by Brush Polymer-Assisted Compaction

Unwanted stimulation of the innate immune system by foreign nucleic acids has been one of the major barriers preventing bioactive sequences from reaching market. Foreign nucleic acids can be recognized by multiple pattern recognition receptors (PRRs), which trigger a signaling cascade to activate host defense systems, leading to a range of side effects. This study demonstrates that polyethylene glycol (PEG)-modified DNA strands can greatly reduce the activation of the innate immune system, and the extent of reduction is dependent upon polymer architecture. Highly branched brushes with long PEG side chains achieve the best suppression by blocking PRR interactions via a local steric effect. Interestingly, the brush polymer creates little barrier toward DNA-DNA interaction. Quantification of inflammatory cytokines in both mRNA and protein levels as well as the extent of cellular uptake shows a direct correlation between steric congestion and reduction of cellular immune response. These results suggest that the brush architecture offers unique advantages for PEGylating oligonucleotides in the context of minimizing unwanted immune system activation.

Precision Tuning of DNA- and Poly(ethylene glycol)-based Nanoparticles via Co-assembly for Effective Antisense Gene Regulation

Development of safe and effective delivery vectors remains a critical challenge for oligonucleotides-based biopharmaceuticals. In this work, we incorporate antisense oligonucleotides into micellar nanoparticles via co-assembly of two block copolymers, DNA-b-poly(ɛ-caprolactone) and poly(ethylene glycol)-b-poly(ɛ-caprolactone), and the hydrophobic homopolymer, poly(ɛ-caprolactone). By fine-tuning the spacing of particle surface moieties through adding a diluent homopolymer to the micellar core, we conduct a systematic investigation of structure-property correlations among hybridization kinetics, nuclease resistance, cellular uptake, and antisense efficacy. It is found that, when an intermediate density is achieved, nuclease access of the DNA is greatly hindered, but formation of double-stranded DNA is nearly unaffected. Remarkably, micelles showing the highest selectivity (good nuclease resistance while still maintaining hybridization kinetics) also exhibit the highest antisense gene silencing efficacies. ...

Molecular spherical nucleic acids

Significance Spherical nucleic acids (SNAs) made thus far are inherently polydisperse due to variations in surface nucleic acid density, particle size, or both. In this article, we describe the synthesis and characterization of two types of molecular SNAs with precise numbers of surface DNA strands using T8 polyoctahedral silsesquioxane and buckminsterfullerene C60 scaffolds. The surface DNA densities for these molecular structures fall inside the range of typical SNAs, which allows them to exhibit SNA-related properties, including enhanced cellular uptake and the ability to function as a gene regulation agent. With a route to molecularly pure SNAs opened, it becomes possible to use them to unveil the molecular details of SNA interactions with complementary ligands and living systems. Herein, we report a class of molecular spherical nucleic acid (SNA) nanostructures. These nano-sized single molecules are synthesized from T8 polyoctahedral silsesquioxane and buckminsterfullerene C60 scaffolds, modified with 8 and 12 pendant DNA strands, respectively. These conjugates have different DNA surface densities and thus exhibit different levels of nuclease resistance, cellular uptake, and gene regulation capabilities; the properties displayed by the C60 SNA conjugate are closer to those of conventional and prototypical gold nanoparticle SNAs. Importantly, the C60 SNA can serve as a single entity (no transfection agent required) antisense agent to efficiently regulate gene expression. The realization of molecularly pure forms of SNAs will open the door for studying the interactions of such structures with ligands and living cells with a much greater degree of control than the conventional polydisperse forms of SNAs.