Anisotropically Functionalized Aptamer-DNA Nanostructures for Enhanced Cell Proliferation and Target-Specific Adhesion in 3D Cell Cultures.

The development of supramolecular hydrogel scaffolds for the precise positioning of biochemical cues is paramount for applications such as tissue engineering. Nucleic acid engineering allows fabrication of three-dimensional (3D) nanostructures with high variability and nanoscale precision. In this study, aptamers were anisotropically functionalized onto branched DNA nanostructures to control their cell adhesion capability, and their efficiency as biological signal inducers for 3D cell cultivation was investigated. Each arm of the X-shaped DNA nanostructure (X-DNA) was functionalized with photo-cross-linkable or cell adhesion moieties, and the steric hindrance of the 3D DNA nanostructures on a cell was optimized. X-DNA nanostructures with cell-positioning parameters were rapidly photopolymerized to form hybrid hydrogels, and their effects on cell behaviors and positions were investigated. We observed that aptamer-functionalized X-DNA nanostructures exhibited significantly enhanced cell proliferation and provided homogeneous distribution and target-specific adhesion of encapsulated cells within hydrogel matrices. Overall, the anisotropic functionalization of DNA nanostructures provides a controllable function for the advancement of conventional 3D culture platforms.

Functional polymeric DNA nanostructure-decorated cellulose nanocrystals for targeted and stimuli-responsive drug delivery.

Targeted and stimuli-responsive drug delivery enhances therapeutic efficacy and minimizes undesirable side effects of cancer treatment. Although cellulose nanocrystals (CNCs) are used as drug carriers because of their robustness, spindle shape, biocompatibility, renewability, and nontoxicity, the lack of programmability and functionality of CNCs-based platforms hampers their application. Thus, high adaptability and the capacity to form dynamic 3D nanostructures of DNA may be advantageous, as they can provide functionalities such as target-specific and stimuli-responsive drug release. Using DNA nanotechnology, the functional polymeric form of DNA nanostructures can be replicated using rolling circle amplification (RCA), and the biologically and physiologically stable DNA nanostructures may overcome the challenges of CNCs. In this study, multifunctional polymeric DNAs produced with RCA were strongly complexed with surface-modified CNCs via electrostatic interactions to form polymeric DNA-decorated CNCs (pDCs). Particle size, polydispersity, zeta potential, and biostability of the nanocomplexes were analyzed. As a proof of concept, the dynamic structural functionalities of DNA nanostructures were verified by observing cancer-targeted intracellular delivery and pH-responsive drug release. pDCs showed anticancer properties without side effects in vitro, owing to their aptamer and i-motif functionalities. In conclusion, pDCs exhibited multifunctional anticancer activities, demonstrating their potential as a promising hybrid nanocomplex platform for targeted cancer therapy.

Nanoengineered Polymeric RNA Nanoparticles for Controlled Biodistribution and Efficient Targeted Cancer Therapy.

RNA nanotechnology, including rolling circle transcription (RCT), has gained increasing interest as a fascinating siRNA delivery nanoplatform for biostable and tumor-targetable RNA-based therapies. However, due to the lack of fine-tuning technologies for RNA nanostructures, the relationship between physicochemical properties and siRNA efficacy of polymeric siRNA nanoparticles (PRNs) with different sizes has not yet been fully elucidated. Herein, we scrutinized the effects of size/surface chemistry-tuned PRNs on the biological and physiological interactions with tumors. PRNs with adjusted size and surface properties were prepared using sequential engineering processes: RCT, condensation, and nanolayer deposition of functional biopolymers. Through the RCT process, nanoparticles of three sizes with a diameter of 50-200 nm were fabricated and terminated with three types of biopolymers: poly-l-lysine (PLL), poly-l-glutamate (PLG), and hyaluronic acid (HA) for different surface properties. Among the PRNs, HA-layered nanoparticles with a diameter of ∼200 nm exhibited the most effective systemic delivery, resulting in superior anticancer effects in an orthotopic breast tumor model due to the CD44 receptor targeting and optimized nanosized structure. Depending on the type of PRNs, the in vivo siRNA delivery with protein expression inhibition differed by up to approximately 20-fold. These findings indicate that the types of layered biopolymers and the PRNs size mediate efficient polymeric siRNA delivery to the targeted tumors, resulting in high RNAi-induced therapeutic efficacy. This RNA-nanotechnology-based size/surface editing can overcome the limitations of siRNA therapeutics and represents a potent built-in module method to design RNA therapeutics tailored for targeted cancer therapy.

Mucoadhesive chitosan microcapsules for controlled gastrointestinal delivery and oral bioavailability enhancement of low molecular weight peptides.

A bioactive compound, collagen peptide (CP), is widely used for biological activities such as anti-photoaging and antioxidant effects, with increased oral bioavailability because of its low molecular weight and high hydrophilicity. However, controlling release time and increasing retention time in the digestive tract for a more convenient oral administration is still a challenge. We developed CP-loaded chitosan (CS) microcapsules via strong and rapid ionic gelation using a highly negative phytic acid (PA) crosslinker. The platform enhanced the oral bioavailability of CP with controlled gastrointestinal delivery by utilizing the mucoadhesiveness and tight junction-opening properties of CS. CS and CP concentrations varied from 1.5 to 3.5% and 0-30%, respectively, for optimal and stable microcapsule synthesis. The physicochemical properties, in vitro release profile with intestinal permeability, in vivo oral bioavailability, in vivo biodistribution, anti-photoaging effect, and antioxidant effect of optimized CS microcapsules were analyzed to investigate the impact of controlling parameters. The structure of CS microcapsules was tuned by PA diffused gradient ionic cross-linking degree, resulting in a controlled CP release region in the gastrointestinal tract. The optimized microcapsules increased Cmax, AUC, and tmax by 1.5-, 3.4-, and 8.0-fold, respectively. Furthermore, CP in microcapsules showed anti-photoaging effects by downregulating matrix metalloproteinases-1 via antioxidant effects. According to our knowledge, this is the first study to microencapsulate CP for oral bioavailability enhancement. The peptide delivery method employed is simple, economical, and can be applied to customize bioactive compound administration.

Crystallinity-tuned ultrasoft polymeric DNA networks for controlled release of anticancer drugs.

Despite the vast interest in utilizing rolling circle amplification (RCA)-based DNA networks for bioapplications, precise control of the mechanical and physicochemical properties is highly challenging. To address this concern, we aimed to develop ultrasoft self-supporting polymerized DNA networks (pDNets) of variable crystallinities to manipulate sequence-mediated drug release efficiency. A controlled ratio of the inorganic magnesium pyrophosphate (MgPPi) crystal to the organic polymeric DNA resulted in the synthesis of pDNets of various nanoporosities. The number of crystal microstructures influencing drug localization and release pattern and the tunable mechanical properties influencing injectability and structural stability under physiological conditions were investigated. The pDNets exhibited ultrasoft properties with Young's moduli of 0.06-0.54 Pa; approximately 9-fold differences in mechanical properties were obtained by varying the degree of crystallinity. With functional DNA sequences, the developed platforms showed pH stimuli-responsive drug release profiles of the dynamic DNA structures and aptamer-specific cell target adhesion efficiency. Analyses of controlled delivery of anticancer therapeutics in vitro and in vivo revealed crystallinity-dependent antitumor efficacy without side effects. This strategy provides an effective one-pot enzymatic polymerization methodology and a favorable microenvironment for a three-dimensional DNA network based on demand-localized drug delivery.

Silver Nanoclusters Serve as Fluorescent Rivets Linking Hoogsteen Triplex DNA and Hairpin-Loop DNA Structures.

Greater understanding of the mutual influence between DNA and the associated nanomaterial on the properties of each other can provide alternative strategies for designing and developing DNA nanomachines. DNA secondary structures are essential for encapsulating highly emissive silver nanoclusters (DNA/AgNCs). Likewise, AgNCs stabilize secondary DNA structures, such as hairpin DNA, duplex DNA, and parallel-motif DNA triplex. In this study, we found that the fluorescence of AgNCs encapsulated within a Hoogsteen triplex DNA structure can be turned on and off in response to pH changes. We also show that AgNCs can act as nanoscale rivets, linking two functionally distinctive DNA nanostructures. For instance, we found that a Hoogsteen triplex DNA structure with a seven-cytosine loop encapsulates red fluorescent AgNCs. The red fluorescence faded under alkaline conditions, whereas the fluorescence was restored in a near-neutral environment. Hairpin DNA and random DNA structures did not exhibit this pH-dependent AgNCs fluorescence. A fluorescence lifetime measurement and a small-angle X-ray scattering analysis showed that the triplex DNA-encapsulated AgNCs were photophysically convertible between bright and dark states. An in-gel electrophoresis analysis indicated that bright and dark convertibility depended on the AgNCs-riveted dimerization of the triplex DNAs. Moreover, we found that AgNCs rivet the triplex DNA and hairpin DNA to form a heterodimer, emitting orange fluorescence. Our findings suggest that AgNCs between two cytosine-rich loops can be used as nanorivets in designing noncanonical DNA origami beyond Watson-Crick base pairing.

Cellulose nanocrystals as support nanomaterials for dual droplet-based freeform 3D printing.

The selection of sacrificial support materials is important in the fabrication of complex freeform structures. In this study, a dual droplet-based, freeform 3D printing method for pseudoplastic alginate biomaterial inks was developed using Bingham plastic cellulose nanocrystals (CNCs) as support nanomaterials. CNCs-CaCl2 mixture compositions and alginate concentrations were varied to enhance printability with rheological properties of shape fidelity and structural stability. The mixtures supported the shape of alginate and allowed CaCl2 diffusion-based cross-linking during 3D printing. The hydrogels showed rheological and physicochemical properties similar to those of pure alginate hydrogel, as CNCs were removed during post-printing processing. BSA-loaded multi-layered spheres, freeform 3D-printed for oral protein drug delivery, protected BSA in the gastric environment and provided controlled and sustained release of BSA into the intestinal environment as layer width and alginate concentration increased. This method can facilitate freeform 3D printing of diverse pseudoplastic biomaterial inks for biomedical applications.

DNA-Assisted Smart Nanocarriers: Progress, Challenges, and Opportunities.

Due to powerful breakthroughs in nanotechnology, smart delivery mechanisms have rapidly emerged for use in diverse applications across biomedical research and therapeutic development. Recent efforts toward understanding stimuli-responsive strategies have led to substantial improvements in their conceptual application and in vitro efficiency. Because disease targets for therapy are often localized in specific cells, organs, or tissues, an enhanced permeability and retention (EPR)-based strategy remains inadequate for accurate drug delivery and release to target regions, resulting in an insufficient drug concentration reaching the target region and undesired side effects. To address these issues, more precise and remote-controlled stimuli-responsive systems, which recognize and react to changes in the pathophysiological microenvironment, were recently elucidated as feasible on-demand drug-delivery systems. In this Perspective, we focus on progress toward stimuli-responsive drug-delivery systems that utilize dynamic DNA molecules by exploiting DNA nanotechnology. DNA structures can be precisely reconfigured by external and internal stimuli to drive the release of a loaded drug in a target region with appropriate microenvironments. We describe the chemical, physical, and biological engineering principles and strategies for constructing DNA-assisted nanocarriers. We also provide a summary of smart nanocarrier systems, organized with respect to the structural changes in the DNA strand in the microenvironment, resulting from changes in pH and temperature and the presence of intracellular oligonucleotides. To do so, we highlight recent advances in related biomedical research and applications as well as discuss major challenges and opportunities for DNA-assisted nanocarriers to guide the development of future in vivo therapies and clinical translation strategies.

Cationic cellulose nanocrystals complexed with polymeric siRNA for efficient anticancer drug delivery.

Surface-modified cellulose nanocrystals (CNCs) were developed for efficient delivery of polymeric siRNA in cancer cells. Cationic CNCs were synthesized using the sequential process of hydrothermal desulfation and chemical modification following which, polymeric siRNA obtained using from a two-step process of rolling circle transcription and Mg2+ chelation was complexed with the modified CNCs by electrostatic interaction. The complexation efficiency was optimized for high drug loading and release in the cytoplasmic environment. The resultant nanocomplex showed significantly enhanced enzymatic stability, gene knockdown efficacy, and apoptosis-induced in vitro therapeutic effect. Our results suggest CNCs as a promising carbohydrate-based delivery platform which could be utilized for RNAi-mediated cancer therapeutics.

Dual-targeting RNA nanoparticles for efficient delivery of polymeric siRNA to cancer cells.

A new dual-targeting polymeric siRNA nanoparticle (Dual-PSNP) was developed via multiple processes: rolling circle transcription, condensation, electrostatic deposition, and click chemistry. The Dual-PSNP showed significantly improved cancer-specific intracellular delivery, gene knockdown efficacy, and apoptosis-mediated cytotoxicity through additive receptor-mediated interactions of the two ligands.

Noncanonical Head-to-Head Hairpin DNA Dimerization Is Essential for the Synthesis of Orange Emissive Silver Nanoclusters.

DNA secondary structures, such as dimers and hairpins, are important for the synthesis of DNA template-embedded silver nanoclusters (DNA/AgNCs). However, the arrangement of AgNCs within a given DNA template and how the AgNC influences the secondary structure of the DNA template are still unclear. Here, we introduce a noncanonical head-to-head hairpin DNA nanostructure that is driven by orange-emissive AgNCs. Through detailed in-gel analysis, sugar backbone switching, inductively coupled plasma mass spectrometry, small-angle X-ray scattering, and small angle neutron scattering, we show that the orange-emissive AgNCs mediate cytosine-Ag-cytosine bridging between two six-cytosine loop (6C-loop) hairpin DNA templates. Unlike green, red, or far-red emissive AgNCs, which are embedded inside a hairpin and duplex DNA template, the orange-emissive AgNCs are localized on the interface between the two 6C-loop hairpin DNA templates, thereby linking them. Moreover, we found that deoxyribose in the backbone of the 6C-loop at the third and fourth cytosines is crucial for the formation of the orange-emissive AgNCs and the head-to-head hairpin DNA structure. Taken together, we suggest that the specific wavelength of AgNCs fluorescence is determined by the mutual interaction between the secondary or tertiary structures of DNA- and AgNC-mediated intermolecular DNA cross-linking.

Size-controlled synthesis of polymerized DNA nanoparticles for targeted anticancer drug delivery.

We synthesized size-tunable polymerized DNA nanoparticles (PDNs) for cancer-targeted drug delivery via sequential processes of rolling circle amplification, condensation, and layer-by-layer assembly. The PDNs selectively delivered anti-sense oligonucleotides to target cancer cells and exhibited size-dependent gene regulation efficacy.

Synthesis of high-strength microcrystalline cellulose hydrogel by viscosity adjustment.

Developing hydrogels with enhanced mechanical strength is desirable for bio-related applications. For such applications, cellulose is a notable biopolymer for hydrogel synthesis due to its inherent strength and stiffness. Here, we report the viscosity-adjusted synthesis of a high-strength hydrogel through the physical entanglement of microcrystalline cellulose (MCC) in a solvent mixture of tetrabutylammonium fluoride/dimethyl sulfoxide (TBAF/DMSO). MCC was strategically dissolved with TBAF in DMSO at a controlled ratio to induce the formation of a liquid crystalline phase (LCP), which was closely related to the viscosity of the cellulose solution. The highest viscosity was obtained at 2.5% MCC and 3.5% TBAF, leading to the strongest high-strength MCC hydrogel (strongest HS-MCC hydrogel). The resulting hydrogel exhibited a high compressive strength of 0.38MPa and a densely packed structure. Consequently, a positive linear correlation was determined between the viscosity of the cellulose solution and the mechanical strength of the HS-MCC hydrogel.

Nanoparticle-Patterned Multicompartmental Chitosan Capsules for Oral Delivery of Oligonucleotides.

Orally administered antisense therapy has been introduced as an effective approach for treating cancer in the gastrointestinal tract. However, its practical application has been limited by the instability of oligonucleotides and their inefficient delivery. To overcome these problems, we synthesized size-dependent, oligonucleotide nanoparticle-patterned chitosan/phytic acid (ODN/CS/PA) capsules with protective shields via a three-step process of self-assembly, nanoparticle encapsulation, and shell formation. The multicompartmental capsule size and oligonucleotide nanoparticle-loading pattern were controlled by applying different potentials during the electrostatic extrusion process used for nanoparticle encapsulation. Over 95% of encapsulated oligonucleotides were protected from nuclease digestion (DNase I) and, depending on their size, showed 40-75% protection against simulated gastric fluid. Their controlled release from capsules correlated with the cellular delivery of released nanoparticles and the inhibition of protein expression in cancer cells. Specifically, large capsules showed approximately 32-fold greater delivery to cancer cells than nonencapsulated nanoparticles. We also confirmed delivery of oligonucleotide nanoparticles to the small intestine and colon of rats following oral administration. These findings demonstrate that the multicompartmental ODN/CS/PA capsules can facilitate efficient oral delivery of oligonucleotides for cancer treatment.