Targeting oncogenic KRAS with molecular brush-conjugated antisense oligonucleotides.

The mutant form of the guanosine triphosphatase (GTPase) KRAS is a key driver in human tumors but remains a challenging therapeutic target, making KRASMUT cancers a highly unmet clinical need. Here, we report a class of bottlebrush polyethylene glycol (PEG)-conjugated antisense oligonucleotides (ASOs) for potent in vivo KRAS depletion. Owing to their highly branched architecture, these molecular nanoconstructs suppress nearly all side effects associated with DNA-protein interactions and substantially enhance the pharmacological properties of the ASO, such as plasma pharmacokinetics and tumor uptake. Systemic delivery to mice bearing human non-small-cell lung carcinoma xenografts results in a significant reduction in both KRAS levels and tumor growth, and the antitumor performance well exceeds that of current popular ASO paradigms, such as chemically modified oligonucleotides and PEGylation using linear or slightly branched PEG. Importantly, these conjugates relax the requirement on the ASO chemistry, allowing unmodified, natural phosphodiester ASOs to achieve efficacy comparable to that of chemically modified ones. Both the bottlebrush polymer and its ASO conjugates appear to be safe and well tolerated in mice. Together, these data indicate that the molecular brush-ASO conjugate is a promising therapeutic platform for the treatment of KRAS-driven human cancers and warrant further preclinical and clinical development.

Thermal and mechanical THz modulation of flexible all-dielectric metamaterial.

The implementation of Terahertz (THz) modulation is critical for applications in high-speed wireless communications, security screening and so on. Therefore, it is particularly significant to obtain THz wave modulation devices with stable and flexible performance, easy manipulation of the modulation method, and multi-functionality. Here, we propose a flexible all-dielectric metamaterial by embedding zirconia (ZrO2) microspheres into a vanadium dioxide/polydimethylsiloxane (VO2/PDMS) composite, which can achieve thermal and mechanical tuning of THz wave transmission. When the temperature of the ZrO2/VO2/PDMS metamaterial increases, VO2 changes from the insulating phase to the metallic phase, and the 1st (at 0.304 THz) and 2nd (at 0.414 THz) order magnetic resonances exhibit the tunability of 20 GHz and 15 GHz, respectively. When stretched, the 1st and 2nd order magnetic resonances show the tunability of 12 GHz and 10 GHz, respectively. In the meantime, there are accompanying changes in transmittance at the resonances. The ZrO2/VO2/PDMS all-dielectric metamaterial presented in this work provides an alternative strategy for developing actively tunable, flexible, and versatile THz devices. In addition, it has the merits of simple preparation and low cost, promising large-area and rapid preparation of meta-arrays.

Terahertz switchable VO2-Au hybrid active metasurface holographic encryption.

The combination of metasurface and holographic technology is the most cutting-edge development, but most of the proposed designs are static and do not allow active changes through external stimulation after fabrication, which takes only a limited part of the advantage provided by metasurface. Here, we propose and demonstrate a switchable hybrid active metasurface hologram in the terahertz (THz) regime composed of dynamic pixels (VO2-CSRR) and static pixels (Au-CSRR) based on an intelligent algorithm, which can display some/all information in different temperature ranges. In particular, such performance shows excellent potential in the field of optical communication security, making it a promising candidate. To prove this possibility, we propose a scheme for optical information encryption/decryption and transmission, which takes metasurfaces as carriers of encrypted information and state/polarization/positions as the secret key components. Only when the two matches correctly can we get the hidden real information. The security of our proposed scheme has reached an unprecedented level, providing a new road for communication security.

Doxorubicin and CpG loaded liposomal spherical nucleic acid for enhanced Cancer treatment.

Chemotherapeutics that can trigger immunogenic cell death (ICD) and release tumor-specific antigens are effective on treating a variety of cancers. The codelivery of chemotherapeutics with adjuvants is a promising strategy to achieve synergistic therapeutic effect. However, low drug loading and complicated preparation of current delivery systems lead to carrier-associated toxicity and immunogenicity. Herein, we developed a facile approach to construct liposomal spherical nucleic acids (SNA) by the self-assembly of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)-doxorubicin conjugate and DOPE-matrix metalloproteinases-9 (MMP-9) responsive peptide-CpG conjugate (DOPE-MMP-CpG). Liposomal SNAs efficiently co-delivered DOX and CpG into tumors and released the two drugs upon biological stimuli of MMP-9 enzyme in tumor microenvironment (TME) and high concentration of endogenous glutathione in tumor cells. We demonstrated that liposomal SNA enhanced activation of dendritic cells (DCs), promoted expansion of CD8+ and CD4+ T cells in both tumors and spleen, inhibited tumor growth, and extended animal survival. This work provided a simple strategy of delivering chemotherapeutics and adjuvants to tumors with synergistic therapeutic effect and reduced side effect.

Mild Acidosis-Directed Signal Amplification in Tumor Microenvironment via Spatioselective Recruitment of DNA Amplifiers.

DNA biotechnology offers intriguing opportunities for amplification-based sensitive detection. However, spatiotemporally-controlled manipulation of signal amplification for in situ imaging of the tumor microenvironment remains an outstanding challenge. Here, we demonstrate a DNA-based strategy that can spatial-selectively amplify the acidic signal in the extracellular milieu of the tumor to achieve specific imaging with improved sensitivity. The strategy, termed mild acidosis-targeted amplification (MAT-amp), leverages the specific acidic microenvironment to engineer tumor cells with artificial DNA receptors through a pH (low) insertion peptide, which permits controlled recruitment of fluorescent amplifiers via a hybridization chain reaction. The acidosis-responsive amplification cascade enables significant fluorescence enhancement in tumors with a reduced background signal in normal tissues, leading to improved signal-to-background ratio. These results highlight the utility of MAT-amp for in situ imaging of the microenvironment characterized by pH disequilibrium.

Spherical Nucleic Acids for Topical Treatment of Hyperpigmentation.

Oligonucleotide-based materials such as spherical nucleic acid (SNA) have been reported to exhibit improved penetration through the epidermis and the dermis of the skin upon topical application. Herein, we report a self-assembled, skin-depigmenting SNA structure, which is based upon a bifunctional oligonucleotide amphiphile containing an antisense oligonucleotide and a tyrosinase inhibitor prodrug. The two components work synergistically to increase oligonucleotide cellular uptake, enhance drug solubility, and promote skin penetration. The particles were shown to reduce melanin content in B16F10 melanoma cells and exhibited a potent antimelanogenic effect in an ultraviolet B-induced hyperpigmentation mouse model.

Molecular spherical nucleic acids.

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.

DNA-Mediated Step-Growth Polymerization of Bottlebrush Macromonomers.

Herein, we report the DNA-mediated self-assembly of bivalent bottlebrush polymers, a process akin to the step-growth polymerization of small molecule monomers. In these "condensation reactions", the polymer serves as a steric guide to limit DNA hybridization in a fixed direction, while the DNA serves as a functional group equivalent, connecting complementary brushes to form well-defined, one-dimensional nanostructures. The polymerization was studied using spectroscopy, microscopy, and scattering techniques and was modeled numerically. The model made predictions of the degree of polymerization and size distribution of the assembled products, and suggested the potential for branching at hybridization junctions, all of which were confirmed experimentally. This study serves as a theoretical basis for the polymer-assembly approach which has the potential to open up new possibilities for suprapolymers with controlled architecture, macromonomer sequence, and end-group functionalities.

Improving the Enzymatic Stability and the Pharmacokinetics of Oligonucleotides via DNA-Backboned Bottlebrush Polymers.

Herein, we design and synthesize site-specifically PEGylated oligonucleotide hairpins and demonstrate that their ability to undergo hybridization chain reaction is nearly unaffected by the PEGylation. The resulting DNA-backboned bottlebrush polymers with PEG side chains exhibit increased resistance against nucleolytic degradation, enhanced thermal stabilities, and elevated blood retention times in vivo, which collectively pave the way for more therapeutically focused DNA nanostructure designs.

Depth-Profiling the Nuclease Stability and the Gene Silencing Efficacy of Brush-Architectured Poly(ethylene glycol)-DNA Conjugates.

PEGylation of an oligonucleotide using a brush polymer can improve its biopharmaceutical characteristics, including enzymatic stability and biodistribution. Herein, we quantitatively explore the nuclease accessibility of the nucleic acid as a function of "depth" toward the backbone of the brush polymer. It is found that protein accessibility decreases as the nucleotide is located closer to the backbone. Thus, by moving the conjugation point from the terminus of the nucleic acid strand to an internal position, much smaller brushes can be used to achieve the same level of steric shielding. This finding also makes it possible to assess antisense gene regulation efficiency of these brush-DNA conjugates as a function of their nuclease stability.

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.

Human Y-Family DNA Polymerase κ Is More Tolerant to Changes in Its Active Site Loop than Its Ortholog Escherichia coli DinB.

DNA damage is a constant threat and can be bypassed in a process called translesion synthesis, which is typically carried out by Y-family DNA polymerases. Y-family DNA polymerases are conserved in all domains of life and tend to have specificity for certain types of DNA damage. Escherichia coli DinB and its human ortholog pol κ can bypass specific minor groove deoxyguanine adducts efficiently and are inhibited by major groove adducts, as Y-family DNA polymerases make contacts with the minor groove side of the DNA substrate and lack contacts with the major groove at the nascent base pair. DinB is inhibited by major groove adducts more than pol κ, and they each have active site loops of different lengths, with four additional amino acids in the DinB loop. We previously showed that the R35A active site loop mutation in DinB allows for bypass of the major groove adduct N6-furfuryl-dA. These observations led us to investigate the different active site loops by creating loop swap chimeras of DinB with a pol κ loop and vice versa by changing the loop residues in a stepwise fashion. We then determined their activity with undamaged DNA or DNA containing N2-furfuryl-dG or N6-furfuryl-dA. The DinB proteins with the pol kappa loop have low activity on all templates but have decreased misincorporation compared to either wild-type protein. The kappa proteins with the DinB loop retain activity on all templates and have decreased misincorporation compared to either wild-type protein. We assessed the thermal stability of the proteins and observed an increase in stability in the presence of all DNA templates and additional increases generally only in the presence of the undamaged and N2-furfuryl-dG adduct and dCTP, which correlates with activity. Overall we find that pol κ is more tolerant to changes in the active site loop than DinB.

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.

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.

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.

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.

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.

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.

A terahertz meta-sensor array for 2D strain mapping.

Large-scale stretchable strain sensor arrays capable of mapping two-dimensional strain distributions have gained interest for applications as wearable devices and relating to the Internet of Things. However, existing strain sensor arrays are usually unable to achieve accurate directional recognition and experience a trade-off between high sensing resolution and large area detection. Here, based on classical Mie resonance, we report a flexible meta-sensor array that can detect the in-plane direction and magnitude of preloaded strains by referencing a dynamically transmitted terahertz (THz) signal. By building a one-to-one correspondence between the intrinsic electrical/magnetic dipole resonance frequency and the horizontal/perpendicular tension level, arbitrary strain information across the meta-sensor array is accurately detected and quantified using a THz scanning setup. Particularly, with a simple preparation process of micro template-assisted assembly, this meta-sensor array offers ultrahigh sensor density (~11.1 cm-2) and has been seamlessly extended to a record-breaking size (110 × 130 mm2), demonstrating its promise in real-life applications.

Tunable C4-Symmetry-Broken Metasurfaces Based on Phase Transition of Vanadium Dioxide (VO2).

Coupling is a ubiquitous phenomenon observed in various systems, which profoundly alters the original oscillation state of resonant systems and leads to the unique optical properties of metasurfaces. In this study, we introduce a terahertz (THz) tunable coupling metasurface characterized by a four-fold rotation (C4) symmetry-breaking structural array achieved through the incorporation of vanadium dioxide (VO2). This disruption of the C4 symmetry results in dynamically controlled electromagnetic interactions and couplings between excitation modes. The coupling between new resonant modes modifies the peak of electromagnetic-induced transparency (EIT) within the C4 symmetric metasurfaces, simulating the mutual interference process between modes. Additionally, breaking the C4 symmetry enhances the mirror asymmetry, and imparts distinct chiral properties in the far-field during the experimental process. This research demonstrates promising applications in diverse fields, including biological monitoring, light modulation, sensing, and nonlinear enhancement.