Delivery of siRNA using cationic rosette nanotubes for gene silencing.

The quest for new therapeutic treatments for hereditary diseases has led to many advances in RNA interference (RNAi) and gene silencing. While this technique has the potential to address many problems, the key to its continued use is the development of effective delivery strategies that would reduce cellular toxicity and increase silencing efficiency. Rosette nanotubes (RNTs) are biomimetic supramolecular nanostructures formed through the self-assembly of hybrid guanine-cytosine (G∧C) DNA bases. Here, we used bioactive RNTs for siRNA delivery and gene silencing. Fifteen lysine-functionalized twin-G∧C motifs (KnT, n = 1 to 15) were synthesized using solid phase peptide synthesis to produce building blocks that self-assembled to produce cationic RNTs under physiological conditions. The intracellular uptake of siRNA delivered by the oligo-L-lysine RNTs was examined and it was found that the complexation of siRNA was affected by the cationic charges from the lysine residues and the length of RNTs formed, with the higher charged KnT RNTs delivering siRNA to the cells at a faster rate. In addition, by protecting siRNA from serum degradation, KnT RNTs were shown to deliver their cargo to the cells effectively via the endocytic pathway. A reduction in the expression (∼70%) of the target stat3 protein was observed during gene expression analysis in HCT116 and A549 cell lines.

Size Selective Corona Interactions from Self-Assembled Rosette and Single-Walled Carbon Nanotubes.

Nanoparticle corona phases, especially those surrounding anisotropic particles, are central to determining their catalytic, molecular recognition, and interfacial properties. It remains a longstanding challenge to chemically synthesize and control such phases at the nanoparticle surface. In this work, the supramolecular chemistry of rosette nanotubes (RNTs), well-defined hierarchically self-assembled nanostructures formed from heteroaromatic bicyclic bases, is used to create molecularly precise and continuous corona phases on single-walled carbon nanotubes (SWCNTs). These RNT-SWCNT (RS) complexes exhibit the lowest solvent-exposed surface area (147.8 ± 60 m-1 ) measured to date due to its regular structure. Through Raman spectroscopy, molecular-scale control of the free volume is also observed between the two annular structures and the effects of confined water. SWCNT photoluminescence (PL) within the RNT is also modulated considerably as a function of their diameter and chirality, especially for the (11, 1) species, where a PL increase compared to other species can be attributed to their chiral angle and the RNT's inward facing electron densities. In summary, RNT chemistry is extended to the problem of chemically defining both the exterior and interior corona interfaces of an encapsulated particle, thereby opening the door to precision control of core-shell nanoparticle interfaces.

Cellular Delivery of Plasmid DNA into Wheat Microspores Using Rosette Nanotubes.

Plant genetic engineering offers promising solutions to the increasing demand for efficient, sustainable, and high-yielding crop production as well as changing environmental conditions. The main challenge for gene delivery in plants is the presence of a cell wall that limits the transportation of genes within the cells. Microspores are plant cells that are, under the right conditions, capable of generating embryos, leading to the formation of haploid plants. Here, we designed cationic and fluorescent rosette nanotubes (RNTs) that penetrate the cell walls of viable wheat microspores under mild conditions and in the absence of an external force. These nanomaterials can capture plasmid DNA to form RNT-DNA complexes and transport their DNA cargo into live microspores. The nanomaterials and the complexes formed were nontoxic to the microspores.

Three-Dimensional Printing Biologically Inspired DNA-Based Gradient Scaffolds for Cartilage Tissue Regeneration.

Cartilage damage caused by aging, repeated overloading, trauma, and diseases can result in chronic pain, inflammation, stiffness, and even disability. Unlike other types of tissues (bone, skin, muscle, etc.), cartilage tissue has an extremely weak regenerative capacity. Currently, the gold standard surgical treatment for repairing cartilage damage includes autografts and allografts. However, these procedures are limited by insufficient donor sources and the potential for immunological rejection. After years of development, engineered tissue now provides a valuable artificial replacement for tissue regeneration purposes. Three-dimensional (3D) bioprinting technologies can print customizable hierarchical structures with cells. The objective of the current work was to prepare a 3D-printed three-layer gradient scaffold with lysine-functionalized rosette nanotubes (RNTK) for improving the chondrogenic differentiation of adipose-derived mesenchymal stem cells (ADSCs). Specifically, biologically inspired RNTKs were utilized in our work because they have unique surface chemistry and biomimetic nanostructure to improve cell adhesion and growth. Different ratios of gelatin methacrylate (GelMA) and poly(ethylene glycol) diacrylate (PEGDA) were printed into a three-layer GelMA-PEGDA gradient scaffold using a stereolithography-based printer, followed by coating with RNTKs. The pores and channels (∼500 µm) were observed in the scaffold. It was found that the population of ADSCs on the GelMA-PEGDA-RNTK scaffold increased by 34% compared to the GelMA-PEGDA scaffold (control). Moreover, after 3 weeks of chondrogenic differentiation, collagen II, glycosaminoglycan, and total collagen synthesis on the GelMA-PEGDA-RNTK scaffold significantly respectively increased by 59%, 71%, and 60%, as compared to the control scaffold. Gene expression of collagen II α1, SOX 9, and aggrecan in the ADSCs growing on the GelMA-PEGDA-RNTK scaffold increased by 79%, 52%, and 47% after 3 weeks, compared to the controls, respectively. These results indicated that RNTKs are a promising type of nanotubes for promoting chondrogenic differentiation, and the present 3D-printed three-layer gradient GelMA-PEGDA-RNTK scaffold shows considerable promise for future cartilage repair and regeneration.

Rosette Nanotube Porins as Ion Selective Transporters and Single-Molecule Sensors.

Rosette nanotubes (RNTs) are a class of materials formed by molecular self-assembly of a fused guanine-cytosine base (G∧C base). An important feature of these self-assembled nanotubes is their precise atomic structure, intriguing for rational design and optimization as synthetic transmembrane porins. Here, we present experimental observations of ion transport across 1.1 nm inner diameter RNT porins (RNTPs) of various lengths in the range 5-200 nm. In a typical experiment, custom lipophilic RNTPs were first inserted into lipid vesicles; the vesicles then spontaneously fused with a planar lipid bilayer, which produced stepwise increases of ion current across the bilayer. Our measurements in 1 M KCl solution indicate ion transport rates of ∼50 ions s-1 V-1 m, which for short channels amounts to conductance values of ∼1 nS, commensurate with naturally occurring toxin channels such as α-hemolysin. Measurements of interaction times of α-cyclodextrin with RNTPs reveal two distinct unbinding time scales, which suggest that interactions of either face of α-cyclodextrin with the RNTP face are differentiable, backed with all-atom molecular dynamics simulations. Our results highlight the potential of RNTPs as self-assembled nonproteinaceous single-molecule sensors and selective nanofilters with tunable functionality through chemistry.

Enhanced antibiotic activity of ampicillin conjugated to gold nanoparticles on PEGylated rosette nanotubes.

PURPOSE: This work presents the preparation of a nanocomposite of ampicillin-conjugated gold nanoparticles (AuNPs) and self-assembled rosette nanotubes (RNTs), and evaluates its antibacterial properties against two strains of drug-resistant bacteria (Staphylococcus aureus [S. aureus], methicillin-resistant S. aureus [MRSA]). MATERIALS AND METHODS: Small, nearly monodisperse AuNPs (1.43±0.5 nm in diameter) nucleated on the surface of polyethylene glycol-functionalized RNTs in a one-pot reaction. Upon conjugation with ampicillin, their diameter increased to 1.86±0.32 nm. The antibacterial activity of the nanocomposite against S. aureus and MRSA was tested using different concentrations of ampicillin. The cytocompatibility of the nanocomposite was also tested against human dermal fibroblasts. RESULTS: Based on bacterial inhibition studies, the nanocomposite demonstrated enhanced antibiotic activity against both bacterial strains. The minimum inhibitory concentration (MIC) of the nanocomposite against S. aureus was found to be 0.58 µg/mL, which was 18% lower than ampicillin alone. The nanocomposite also exhibited a 20 hrs MIC of 4 µg/mL against MRSA, approximately 10-20 times lower than previously reported values for ampicillin alone. In addition, at concentrations of 4 µg/mL of ampicillin (70 µg/mL of AuNPs), the nanocomposite showed negligible cytotoxic effects. CONCLUSION: Our findings offer a new approach for the treatment of drug-resistant bacteria by potentiating inhibitory effects of existing antibiotics, and delivering them using a non-toxic formulation.

A Barcoded Polymer-Based Cross-Reactive Spectroscopic Sensor Array for Organic Volatiles.

The development of cross-reactive sensor arrays for volatile organics (electronic noses, e-noses) is an active area of research. In this manuscript, we present a new format for barcoded polymer sensor arrays based on porous polymer beads. An array of nine self-encoded polymers was analyzed by Raman spectroscopy before and after exposure to a series of volatile organic compounds, and the changes in the vibrational fingerprints of their polymers was recorded before and after exposure. Our results show that the spectroscopic changes experienced by the porous spectroscopically encoded beads after exposure to an analyte can be used to identify and classify the target analytes. To expedite this analysis, analyte-specific changes induced in the sensor arrays were transformed into a response pattern using multivariate data analysis. These studies established the barcoded bead array format as a potentially effective sensing element in e-nose devices. Devices such as these have the potential to advance personalized medicine, providing a platform for non-invasive, real-time volatile metabolite detection.

Cross-Reactive, Self-Encoded Polymer Film Arrays for Sensor Applications.

The development of chemical sensors continues to be an active area of research, especially the development of a practical electronic nose. Here, we present a spectroscopic chemical sensor based on an array of 64 self-encoded polymer films deposited on a microfabricated silicon substrate. The polymer arrays were analyzed by FTIR and Raman spectroscopy before and after exposure to a series of organic volatiles to monitor changes in their vibrational fingerprints. We show here that the spectroscopic changes of self-encoded polymer films can be used to distinguish between volatile organic analytes. Changes induced in the sensor arrays by the analyte vapor were denoted by a spectroscopic response of the self-encoded polymer sensors and transformed into a response pattern by multivariate data analysis using partial least squares regression. The results indicated that the polymer sensors provide a unique and reproducible pattern for each analyte vapor and can potentially be used in the fabrication of a novel electronic nose device.

Computational Modeling for Biomimetic Sensors.

Computational modeling has become an important tool for scientists to both predict the properties of materials and systems and to gain a better understanding of the underlying mechanisms. This chapter is a brief yet holistic introduction to computational modeling, focusing on density functional theoretical (DFT) methods. The different types of computational modeling methods, including molecular mechanics, semiempirical, and ab initio methods, as well as the different software available for computational calculations are discussed. A step-by-step guide is presented using Gaussian16 software to introduce the basics of computational modeling based on our work with biomimetic polymer beads. However, the guide presented here is not limited to this particular system; it can be applied to any computational modeling case. The computational modeling methods for the building of the structures are described, and the calculation parameters, such as basis sets and exchange-correlation functionals, are explained. The output data and results are presented and discussed. Two simulation features were the focus of this work: (1) the simulation of the Raman spectra and (2) the different solvation environments. While some researchers in the field believe that computational simulation should be performed before the lab experiments, in fact they should be done simultaneously. This is so that the output of the experimental data can be used as the input of the computational parameters, as a form of semiempirical modeling, in order to achieve more accurate results for predicting the behavior of future experiments and understanding the atomic forces and mechanisms.

Zinc oxide end-capped Fe3O4@mSiO2 core-shell nanocarriers as targeted and responsive drug delivery system for chemo-/ions synergistic therapeutics.

Multifunctional core-shell nanocarriers based on zinc oxide (ZnO)-gated magnetic mesoporous silica nanoparticles (MMSN) were prepared for cancer treatment through magnetic targeting and pH-triggered controlled drug release. Under an external magnetic field, the MMSN could actively deliver chemotherapeutic agent, daunomycin (DNM), to the targeted sites. At neutral aqueous, the functionalized MMSN could stably accommodate the DNM molecules since the mesopores were capped by the ZnO gatekeepers. In contrast, at the acid intercellular environment, the gatekeepers would be removed to control the release of drugs due to the dissolution of ZnO. Meanwhile, ZnO quantum dots not only rapidly dissolve in an acidic condition of cancer cells but also enhance the anti-cancer effect of Zn2+. An in vitro controlled release proliferation indicated that the acid sensitive ZnO gatekeepers showed well response by the 'on-off' switch of the pores. Cellular experiments against cervical cancer cell (HeLa cells) further showed that functionalized MMSN significantly suppressed cancer cells growth through synergistic effects between the chemotherapy and Zn2+ ions with monitoring the treatment process. These results suggested that the ZnO-gated MMSN platform is a promising approach to serve as a pH-sensitive system for chemotherapies delivery and Zn2+ controlled release for further application in the treatment of various cancers by synergistic effects.

3D Printed scaffolds with hierarchical biomimetic structure for osteochondral regeneration.

Osteochondral defects resulting from trauma and/or pathologic disorders are critical clinical problems. The current approaches still do not yield satisfactory due to insufficient donor sources and potential immunological rejection of implanted tissues. 3D printing technology has shown great promise for fabricating customizable, biomimetic tissue matrices. The purpose of the present study is to investigate 3D printed scaffolds with biomimetic, biphasic structure for osteochondral regeneration. For this purpose, nano-hydroxyapatite and transforming growth factor beta 1 nanoparticles were synthesized and distributed separately into the lower and upper layers of the biphasic scaffold, which was fabricated using 3D stereolithography printer. Our results showed that this scaffold design successfully promoted osteogenic and chondrogenic differentiation of human bone marrow mesenchymal stem cells, as well as enhanced gene expression associated with both osteogenesis and chondrogenesis alike. The finding demonstrated that 3D printed osteochondral scaffolds with biomimetic, biphasic structure are excellent candidates for osteochondral repair and regeneration.

Stimuli-responsive hydrogels for manipulation of cell microenvironment: From chemistry to biofabrication technology.

Native tissues orchestrate their functions by complex interdependent cascades of biochemical and biophysical cues that vary spatially and temporally during cellular processes. Scaffolds with well-tuned structural, mechanical, and biochemical properties have been developed to guide cell behavior and provide insight on cell-matrix interaction. However, static scaffolds very often fail to mimic the dynamicity of native extracellular matrices. Stimuli-responsive scaffolds have emerged as powerful platforms that capture vital features of native tissues owing to their ability to change chemical and physical properties in response to cytocompatible stimuli, thus enabling on-demand manipulation of cell microenvironment. The vast expansion in biorthogonal chemistries and stimuli-responsive functionalities has fuelled further the development of new smart scaffolds that can permit multiple irreversible or reversible spatiotemporal modulation of cell-directing cues, thereby prompting in-depth studies to interpret the decisive elements that regulate cell behavior. Integration of stimuli-responsive hydrogels with current biofabrication technologies has allowed the development of dynamic scaffolds with organizational features and hierarchical architectures similar to native tissues. This review highlights the progress achieved using stimuli-responsive hydrogels in fundamental cell biology studies, with particular emphasis on the interplay between chemistry, biomaterials design, and biofabrication technologies for manipulation of cell microenvironment.

Synthesis of N-Bridged Pyrido[4,3-d]pyrimidines and Self-Assembly into Twin Rosette Cages and Nanotubes in Organic Media.

Two N-bridged pyrido[4,3-d]pyrimidine derivatives were synthesized toward realization of a self-assembled bis-rosette cage, in organic media. Starting from commercially available malononitrile dimer and dimethyl 5-aminoisophthalate, the target molecules were synthesized in 11 steps using a convergent approach. The final bridged compounds were characterized by nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry. The hierarchical self-assembly of the nanocages into rosette nanotubes and nanobundles was established by electron microscopy and molecular modelling studies.

Electroconductive Gelatin Methacryloyl-PEDOT:PSS Composite Hydrogels: Design, Synthesis, and Properties.

Electroconductive hydrogels are used in a wide range of biomedical applications, including electrodes for patient monitoring and electrotherapy, or as biosensors and electrochemical actuators. Approaches to design electroconductive hydrogels are often met with low biocompatibility and biodegradability, limiting their potential applications as biomaterials. In this study, composite hydrogels were prepared from a conducting polymer complex, poly(3,4-ethylenedioxythiophene):polystyrenesulfonate (PEDOT:PSS) dispersed within a photo-crosslinkable naturally derived hydrogel, gelatin methacryloyl (GelMA). To determine the impact of PEDOT:PSS loading on physical and microstructural properties and cellular responses, the electrical and mechanical properties, electrical properties, and biocompatibility of hydrogels loaded with 0-0.3% (w/v) PEDOT:PSS were evaluated and compared to GelMA control. Our results indicated that the properties of the hydrogels, such as mechanics, degradation, and swelling, could be tuned by changing the concentration of PEDOT:PSS. In particular, the impedance of hydrogels decreased from 449.0 kOhm for control GelMA to 281.2 and 261.0 kOhm for hydrogels containing 0.1% (w/v) and 0.3% (w/v) PEDOT:PSS at 1 Hz frequency, respectively. In addition, an ex vivo experiment demonstrated that the threshold voltage to stimulate contraction in explanted abdominal tissue connected by the composite hydrogels decreased from 9.3 ± 1.2 V for GelMA to 6.7 ± 1.5 V and 4.0 ± 1.0 V for hydrogels containing 0.1% (w/v) and 0.3% (w/v) PEDOT:PSS, respectively. In vitro studies showed that composite hydrogels containing 0.1% (w/v) PEDOT:PSS supported the viability and spreading of C2C12 myoblasts, comparable to GelMA controls. These results indicate the potential of our composite hydrogel as an electroconductive biomaterial.

Bioactive Organic Rosette Nanotubes Support Sensory Neurite Outgrowth.

Regardless of the intervention for peripheral nerve repair, slow rates of axonal regeneration often result in poor clinical outcomes. Thus, using new materials such as biologically inspired, biocompatible, organic rosette nanotubes (RNTs) could provide a tailorable scaffold to modulate neurite extension and attachment for improved nerve repair. RNTs are obtained through the spontaneous self-assembly of a synthetic DNA base analogue featuring the hydrogen bond triads of both guanine and cytosine, the G∧C base. Here, we investigated the potential of RNTs functionalized with lysine and Arg-Gly-Asp-Ser-Lys (RGDSK) peptide to support neural growth. We hypothesized that (a) due to their dimensions, the RNTs would support neuron attachment, and (b) their conjugation to the integrin-binding peptide RGDSK would further enhance neurite outgrowth compared to unfunctionalized RNT. Neurite extension was examined on a variety of RNT structures, including RNT with a lysine side chain (K1), a mixture of the K1 and a free RGDS peptide, RNT alone, an RGDSK-functionalized RNT, in addition to poly-d-lysine and laminin controls. Both whole dorsal root ganglion (DRG) and single dissociated DRG neurons were seeded onto RNT-coated substrates containing various ratios of peptides. Analysis of neuron morphometrics showed that RNT blends support DRG neuron attachment and neurite extension, with RGDS presentation increasing neurite outgrowth from whole DRG by up to 47% over a 7-day period compared to K1 alone (p < 0.013). In addition, while RNTs increased the sprouting of primary neurites extending from dissociated DRG neurons, the total neurite outgrowth per neuron remained the same. These results show that functionalized biomimetic RNTs provide a support for neurite growth and extension and have the ability to modulate neuronal morphology. These results also pave the way for the design of injectable RNT-based nanomaterials that support guided neural regeneration following traumatic injury.

Cutting Edge Methods for Non-Invasive Disease Diagnosis Using E-Tongue and E-Nose Devices.

Biomimetic cross-reactive sensor arrays (B-CRSAs) have been used to detect and diagnose a wide variety of diseases including metabolic disorders, mental health diseases, and cancer by analyzing both vapor and liquid patient samples. Technological advancements over the past decade have made these systems selective, sensitive, and affordable. To date, devices for non-invasive and accurate disease diagnosis have seen rapid improvement, suggesting a feasible alternative to current standards for medical diagnostics. This review provides an overview of the most recent B-CRSAs for diagnostics (also referred to electronic noses and tongues in the literature) and an outlook for future technological development.

Artificial Nose Technology: Status and Prospects in Diagnostics.

Biomimetic crossreactive sensor arrays have been used to detect and analyze a wide variety of vapor and liquid components in applications such as food science, public health and safety, and diagnostics. As technology has advanced over the past three decades, these systems have become selective, sensitive, and affordable. Currently, the need for noninvasive and accurate devices for early disease diagnosis remains a challenge. This Opinion article provides an overview of the various types of biomimetic crossreactive sensor arrays (also referred to as electronic noses or tongues in the literature), their current use and future directions, and an outlook for future technological development.

High Field Solid-State NMR Spectroscopy Investigation of (15)N-Labeled Rosette Nanotubes: Hydrogen Bond Network and Channel-Bound Water.

(15)N-labeled rosette nanotubes were synthesized and investigated using high-field solid-state NMR spectroscopy, X-ray diffraction, atomic force microscopy, and electron microscopy. The results established the H-bond network involved in the self-assembly of the nanostructure as well as bound water molecules in the nanotube's channel.

Rosette Nanotubes Alter IgE-Mediated Degranulation in the Rat Basophilic Leukemia (RBL)-2H3 Cell Line.

In this study, the effects of rosette nanotube (RNT) exposure on immune cell viability and function were investigated in vitro using the rat basophilic leukemia (RBL)-2H3 cell line. RBL-2H3 viability was decreased in a dose- and time-dependent manner after lysine-functionalized RNT (K-RNT) exposure. In addition, K-RNTs had a significant effect on RBL-2H3 degranulation. When K-RNT exposure was concurrent with IgE sensitization, 50 and 100 mg l(-1) K-RNTs elicited a heightened degranulatory response compared with IgE alone. Exposure to 50 and 100 mg l(-1) K-RNTs also caused degranulation in RBL-2H3 cells not sensitized with IgE (0 ng ml(-1) IgE). Furthermore, in cells preexposed to K-RNTs for 2 h and subsequently washed, sensitized, and stimulated with IgE, a potentiated degranulatory response was observed. Using confocal laser scanning microscopy and a fluorescein isothiocyanate (FITC)-functionalized RNT construct (termed FITC(1)/TBL(19)-RNT), we demonstrated a strong and direct affiliation between RNTs and RBL-2H3 cell membranes. We also demonstrated cellular internalization of RNTs after 2 h of exposure. Together, these data demonstrate that RNTs may affiliate with the cellular membrane of RBL-2H3 cells and can be internalized. These interactions can affect viability and alter the ability of these cells to elicit IgE-FcεR mediated degranulation.