Mimicking Horseradish Peroxidase and NADH Peroxidase by Heterogeneous Cu2+-Modified Graphene Oxide Nanoparticles.

Cu2+-ion-modified graphene oxide nanoparticles, Cu2+-GO NPs, act as a heterogeneous catalyst mimicking functions of horseradish peroxidase, HRP, and of NADH peroxidase. The Cu2+-GO NPs catalyze the oxidation of dopamine to aminochrome by H2O2 and catalyze the generation of chemiluminescence in the presence of luminol and H2O2. The Cu2+-GO NPs provide an active material for the chemiluminescence detection of H2O2 and allow the probing of the activity of H2O2-generating oxidases and the detection of their substrates. This is exemplified with detecting glucose by the aerobic oxidation of glucose by glucose oxidase and the Cu2+-GO NP-stimulated chemiluminescence intensity generated by the H2O2 product. Similarly, the Cu2+-GO NPs catalyze the H2O2 oxidation of NADH to the biologically active NAD+ cofactor. This catalytic system allows its conjugation to biocatalytic transformations involving NAD+-dependent enzyme, as exemplified for the alcohol dehydrogenase-catalyzed oxidation of benzyl alcohol to benzoic acid through the Cu2+-GO NPs-catalyzed regeneration of NAD+.

Mimicking Peroxidase Activities with Prussian Blue Nanoparticles and Their Cyanometalate Structural Analogues.

Nanoparticles composed of Prussian Blue, PB, and the cyanometalate structural analogues, CuFe, FeCoFe, and FeCo, are examined as inorganic clusters that mimic the functions of peroxidases. PB acts as a superior catalyst for the oxidation of dopamine to aminochrome by H2O2. The oxidation of dopamine by H2O2 in the presence of PB is 6-fold faster than in the presence of CuFe. The cluster FeCo does not catalyze the oxidation of dopamine to aminochrome. The most efficient catalyst for the generation of chemiluminescence by the oxidation of luminol by H2O2 is, however, FeCo, and PB lacks any catalytic activity toward the generation of chemiluminescence. The order of catalyzed chemiluminescence generation is FeCo ≫ CuFe > FeCoFe. The clusters PB, CuFe, FeCoFe, and FeCo mimic the functions of NADH peroxidase. The catalyzed oxidation of NADH by H2O2 to form NAD+ follows the order PB ≫ CuFe ∼ FeCoFe, FeCo. The efficient generation of chemiluminescence by the FeCo-catalyzed oxidation of luminol by H2O2 is used to develop a glucose sensor. The aerobic oxidation of glucose in the presence of glucose oxidase, GOx, yields gluconic acid and H2O2. The chemiluminescence intensities formed by the GOx-generated H2O2 relate to the concentration of glucose, thus providing a quantitative readout signal for the concentrations of glucose.

Nucleoapzymes: Hemin/G-Quadruplex DNAzyme-Aptamer Binding Site Conjugates with Superior Enzyme-like Catalytic Functions.

A novel concept to improve the catalytic functions of nucleic acids (DNAzymes) is introduced. The method involves the conjugation of a DNA recognition sequence (aptamer) to the catalytic DNAzyme, yielding a hybrid structure termed "nucleoapzyme". Concentrating the substrate within the "nucleoapzyme" leads to enhanced catalytic activity, displaying saturation kinetics. Different conjugation modes of the aptamer/DNAzyme units and the availability of different aptamer sequences for a substrate provide diverse means to design improved catalysts. This is exemplified with (i) The H2O2-mediated oxidation of dopamine to aminochrome using a series of hemin/G-quadruplex-dopamine aptamer nucleoapzymes. All nucleoapzymes reveal enhanced catalytic activities as compared to the separated DNAzyme/aptamer units, and the most active nucleoapzyme reveals a 20-fold enhanced activity. Molecular dynamics simulations provide rational assessment of the activity of the various nucleoapzymes. The hemin/G-quadruplex-aptamer nucleoapzyme also stimulates the chiroselective oxidation of L- vs D-DOPA by H2O2. (ii) The H2O2-mediated oxidation of N-hydroxy-L-arginine to L-citrulline by a series of hemin/G-quadruplex-arginine aptamer conjugated nucleoapzymes.

Light-Responsive and pH-Responsive DNA Microcapsules for Controlled Release of Loads.

A method to assemble light-responsive or pH-responsive microcapsules loaded with different loads (tetramethylrhodamine-modified dextran, TMR-D; microperoxidase-11, MP-11; CdSe/ZnS quantum dots; or doxorubicin-modified dextran, DOX-D) is described. The method is based on the layer-by-layer deposition of sequence-specific nucleic acids on poly(allylamine hydrochloride)-functionalized CaCO3 core microparticles, loaded with the different loads, that after the dissolution of the core particles with EDTA yields the stimuli-responsive microcapsules that include the respective loads. The light-responsive microcapsules are composed of photocleavable o-nitrobenzyl-phosphate-modified DNA shells, and the pH-responsive microcapsules are made of a cytosine-rich layer cross-linked by nucleic acid bridges. Irradiating the o-nitrobenzyl phosphate-functionalized microcapsules, λ = 365 nm, or subjecting the pH-responsive microcapsules to pH = 5.0, results in the cleavage of the microcapsule shells and the release of the loads. Preliminary studies address the cytotoxicity of the DOX-D-loaded microcapsules toward MDA-MB-231 breast cancer cells and normal MCF-10A breast epithelial cells. Selective cytotoxicity of the DOX-D-loaded microcapsules toward cancer cells is demonstrated.

Deterministic transfection drives efficient nonviral reprogramming and uncovers reprogramming barriers.

Safety concerns and/or the stochastic nature of current transduction approaches have hampered nuclear reprogramming's clinical translation. We report a novel non-viral nanotechnology-based platform permitting deterministic large-scale transfection with single-cell resolution. The superior capabilities of our technology are demonstrated by modification of the well-established direct neuronal reprogramming paradigm using overexpression of the transcription factors Brn2, Ascl1, and Myt1l (BAM). Reprogramming efficiencies were comparable to viral methodologies (up to ~9-12%) without the constraints of capsid size and with the ability to control plasmid dosage, in addition to showing superior performance relative to existing non-viral methods. Furthermore, increased neuronal complexity could be tailored by varying BAM ratio and by including additional proneural genes to the BAM cocktail. Furthermore, high-throughput NEP allowed easy interrogation of the reprogramming process. We discovered that BAM-mediated reprogramming is regulated by AsclI dosage, the S-phase cyclin CCNA2, and that some induced neurons passed through a nestin-positive cell stage. FROM THE CLINICAL EDITOR: In the field of regenerative medicine, the ability to direct cell fate by nuclear reprogramming is an important facet in terms of clinical application. In this article, the authors described their novel technique of cell reprogramming through overexpression of the transcription factors Brn2, Ascl1, and Myt1l (BAM) by in situ electroporation through nanochannels. This new technique could provide a platform for further future designs.

Magnetic tweezers-based 3D microchannel electroporation for high-throughput gene transfection in living cells.

A novel high-throughput magnetic tweezers-based 3D microchannel electroporation system capable of transfecting 40 000 cells/cm(2) on a single chip for gene therapy, regenerative medicine, and intracellular detection of target mRNA for screening cellular heterogeneity is reported. A single cell or an ordered array of individual cells are remotely guided by programmable magnetic fields to poration sites with high (>90%) cell alignment efficiency to enable various transfection reagents to be delivered simultaneously into the cells. The present technique, in contrast to the conventional vacuum-based approach, is significantly gentler on the cellular membrane yielding >90% cell viability and, moreover, allows transfected cells to be transported for further analysis. Illustrating the versatility of the system, the GATA2 molecular beacon is delivered into leukemia cells to detect the regulation level of the GATA2 gene that is associated with the initiation of leukemia. The uniform delivery and a sharp contrast of fluorescence intensity between GATA2 positive and negative cells demonstrate key aspects of the platform for gene transfer, screening and detection of targeted intracellular markers in living cells.

DNA-modified Prussian blue nanozymes for enhanced electrochemical biosensing.

Prussian blue nanoparticles exhibit the potential to be employed in bioanalytical applications due to their robust stability, peroxidase-like catalytic functionality, straightforward synthesis, and biocompatibility. An efficient approach is presented for the synthesis of nucleic acid-modified Prussian blue nanoparticles (DNA-PBNPs), utilizing nanoparticle porosity to adsorb nucleic acids (polyT). This strategic adsorption leads to the exposure of nucleic acid sequences on the particle surface while retaining catalytic activity. DNA-PBNPs further couple with functional nucleic acid sequences and aptamers through complementary base pairing to act as transducers in biosensors and amplify signal acquisition. Subsequently, we integrated a copper ion-dependent DNAzyme (Cu2+-DNAzyme) and a vascular endothelial growth factor aptamer (VEGF aptamer) onto screen-printed electrodes to serve as recognition elements for analytes. Significantly, our approach leverages DNA-PBNPs as a superior alternative to traditional enzyme-linked antibodies in electrochemical biosensors, thereby enhancing both the efficiency and adaptability of these devices. Our study conclusively demonstrates the application of DNA-PBNPs in two different biosensing paradigms: the sensitive detection of copper ions and vascular endothelial growth factor (VEGF). These results indicate the promising potential of DNA-modified Prussian blue nanoparticles in advancing bioanalytical sensing technologies.

Hybridization chain reaction-assisted enzyme cascade genosensor for the detection of Listeria monocytogenes.

Foodborne diseases caused by pathogens may threaten public health and the social economy. We demonstrated a method for identifying pathogenic Listeria monocytogenes using DNA logic operations. To achieve accurate species distinguishing, three specific sequences of Listeria monocytogenes genomic DNA were screened out and used as the feature sequences. Three complementary probes with tag modification were designed as sensing elements and exert affinity for magnetic beads, glucose oxidase (GOx), and horseradish peroxidase (HRP). To obtain a digital output (YES/NO answer) for rapid determination, a Boolean logic function was employed. Three sensing probes enabled the recognition of the target sequence (input) and the formation of a target DNA/probe hybrid. Through magnetic separation and affinity binding events, the target DNA/probes hybrid led to the construction of GOx/HRP enzyme cascade, which produced a visualized color signal (output) in the presence of substrates, glucose, and 3, 3', 5, 5'-tetramethylbenzidine (TMB). A hybridization chain reaction (HCR) was coupled with this sensing scaffold to increase the binding of the enzyme cascade and amplify the output signal. The logical functional biosensor showed high selectivity of Listeria monocytogenes over other Listeria species. This sensing platform provides a simple, sensitive, and highly specific method for detecting Listeria monocytogenes.

Ethylene Glycol-Manipulated Syntheses of Calcium Carbonate Particles and DNA Capsules toward Efficient ATP-Responsive Cargo Release.

Cargo molecule-encapsulated DNA capsules synthesized with a solid sacrificial template have elicited significant interest in the last decade and have been used for active materials in applications ranging from biosensors to drug delivery. However, the correlation between template properties and the subsequent assembly and triggered release behavior of the resultant carriers remain uninvestigated. In this study, ethylene glycol (EG) was added during the CaCO3 precipitation synthesis to yield particles of various sizes and surface properties, and the adenosine triphosphate (ATP)-responsive release characteristics of the fabricated DNA capsules affected by these particle properties were investigated. The geometry, crystallization, and surface morphology of the CaCO3 particles co-precipitated at various EG concentrations were characterized. We discuss the integrity of cross-linking hybridization, fluorescent molecule internalization, degree of leakage, and release efficiency of the resulting DNA capsules and their relevance brought by particle properties. To achieve efficient encapsulation and cargo release, the surface roughness of the CaCO3 particles was explored and was deemed a key determinant of the compactness of the DNA shell after template removal. This effect was particularly strong in CaCO3 particles in connection with high EG concentrations. The DNA capsules fabricated using 83% EG exhibited low leakage, high loading, and moderate release efficiencies as well as a greater apparent association constant with ATP due to their small particle size and the high-integrity DNA shells.

Engineered multivalent DNA capsules for multiplexed detection of genotoxicants via versatile controlled release mechanisms.

Assessing the risks associated with genotoxic compounds is challenging because of their complex genotoxicity and the difficulty in the dynamic monitoring of coexisting hazards. In this paper, DNA-assembly-based multistimulus responsive capsules that can detect multiple genotoxic agents simultaneously are presented. By exploiting the sequence- and reactivity-editable properties of DNA, DNA sequences in a DNA shell are designed to exhibit multivalent susceptibility against ultraviolet B radiation, aflatoxin B1, and styrene oxide. Upon exposure to genotoxicants, the developed DNA capsules dissociate because of the production of DNA adducts or aptamer-ligand complex-activated dehybridization, which results in the release of encapsulated fluorophores for a measure of the genotoxicant level. The fluorophore release kinetics for each genotoxicant is investigated. Moreover, the destruction behaviors of the developed capsules are evaluated in binary and ternary toxin mixtures. Multiple linear regression indicates the existence of a strong relationship between the fluorescent response and the genotoxicant level; the result highlights the significance of particular genotoxicant and the antagonistic effect of interacting genotoxic substances on capsule destruction. This DNA architecture allows the monitoring of human exposure to genotoxic agents, which enables the timely adoption of remedial measures, and benefits development of an endogenous genotoxin-responsive drug delivery system.

Stimuli-responsive hydrogel microcapsules for the amplified detection of microRNAs.

A method for the synthesis of DNA-based acrylamide hydrogel microcapsules loaded with quantum dots as a readout signal is introduced. The shell of DNA-acrylamide hydrogel microcapsules is encoded with microRNA-responsive functionalities, being capable of the detection of cancer-associated microRNA. The microRNA-141 (miR-141), a potential biomarker in prostate cancer, was employed as a model target in the microcapsular biosensor. The sensing principle of the microcapsular biosensor is based on the competitive sequence displacement of target miR-141 with the bridging DNA in the microcapsule's shell, leading to the unlocking of DNA-acrylamide hydrogel microcapsules and the release of the readout signal provided by fluorescent quantum dots. The readout signal is intensified as the concentration of miR-141 increases. While miR-141 was directly measured by DNA-acrylamide hydrogel microcapsules, the linear range for the detection of miR-141 is 2.5 to 50 µM and the limit of detection is 1.69 µM. To improve the sensitivity of the microcapsular biosensor for clinical needs, the isothermal strand displacement polymerization/nicking amplification machinery (SDP/NA) process was coupled to the DNA-acrylamide hydrogel microcapsule sensor for the microRNA detection. The linear range for the detection of miR-141 is improved to the range of 102 to 105 pM and the limit of detection is 44.9 pM. Compared to direct microcapsular biosensing, the detection limit for miR-141 by microcapsules coupled with strand-displacement amplification is enhanced by four orders of magnitude.

Conformation dependence of DNA electrophoretic mobility in a converging channel.

The electrophoresis of lambda-DNA is observed in a microscale converging channel where the center-of-masses trajectories of DNA molecules are tracked to measure instantaneous electrophoretic (EP) mobilities of DNA molecules of various stretch lengths and conformations. Contrary to the usual assumption that DNA mobility is a constant, independent of field and DNA length in free solution, we find DNA EP mobility varies along the axis in the contracting geometry. We correlate this mobility variation with the local stretch and conformational changes of the DNA, which are induced by the electric field gradient produced by the contraction. A "shish-kebab" model of a rigid polymer segment is developed, which consists of aligned spheres acting as charge and drag centers. The EP mobility of the shish-kebab is obtained by determining the electrohydrodynamic interactions of aligned spheres driven by the electric field. Multiple shish-kebabs are then connected end-to-end to form a freely jointed chain model for a flexible DNA chain. DNA EP mobility is finally obtained as an ensemble average over the shish-kebab orientations that are biased to match the overall stretch of the DNA chain. Using physically reasonable parameters, the model agrees well with experimental results for the dependence of EP mobility on stretch and conformation. We find that the magnitude of the EP mobility increases with DNA stretch, and that this increase is more pronounced for folded conformations.

Formation and finite element analysis of tethered bilayer lipid structures.

Rapid solvent exchange of an ethanolic solution of diphytanoyl phosphatidylcholine (DPhyPC) in the presence of a mixed self-assembled monolayer (SAM) [thiolipid/ß-mercaptoethanol (ßME) (3/7 mol/mol) on Au] shows a transition from densely packed tethered bilayer lipid membranes [(dp)tBLMs], to loosely packed tethered bilayer lipid membranes [(lp)tBLMs], and tethered bilayer liposome nanoparticles (tBLNs) with decreasing DPhyPC concentration. The tethered lipidic constructs in the aqueous medium were analyzed by atomic force microscopy (AFM) and electrochemical impedance spectroscopy (EIS). Finite element analysis (FEA) was applied to interpret spectral EIS features without referring to equivalent circuit modeling. Using structural data obtained earlier from neutron reflectometry and dielectric constants of lipid bilayers, we reproduced experimentally observed features of the electrochemical impedance (EI) spectra of complex surface constructs involving small pinhole defects, large membrane-free patches, and bound liposomes. We demonstrated by FEA that highly insulating (dp)tBLMs with low-defect density exhibit EI spectra in the shape of a perfect semicircle with or without low-frequency upward "tails" in the Cole-Cole representation. Such EI spectra were observed at DPhyPC concentrations of >5 × 10(-3) mol L(-1). While AFM was not able to visualize very small lateral defects in such films, EI spectra unambiguously signaled their presence by increased low frequency "tails". Using FEA we demonstrate that films with large diameter visible defects (>25 nm by AFM) produce EI spectral features consisting of two semicircles of comparable size. Such films were typically obtained at DPhyPC concentrations of <5 × 10(-3) mol L(-1). At DPhyPC concentrations of <1.0 × 10(-3) mol L(-1) the planar bilayer structures were replaced by ellipsoidal liposomes with diameters ranging from 50 to 500 nm as observed in AFM images. Despite the distinct surface morphology change, the EI curves exhibited two semicircle spectral features typical for the large size defects in planar tBLMs. FEA revealed that, to account for these EI features for bound liposome systems (50-500 nm diameter), one needs to assume much lower tBLM conductivities of the submembrane space, which separates the electrode surface and the phospholipid bilayer. Alternatively, FEA indicates that such features may also be observed on composite surfaces containing both bound liposomes and patches of planar bilayers. Triple semicircular features, observed in some of the experimental EI curves, were attributed to an increased complexity of the real tBLMs. The modeling demonstrated that such features are typical for heterogeneous tBLM surfaces containing large patches of different defectiveness levels. By integrating AFM, EIS, and FEA data, our work provides diagnostic criteria allowing the precise characterization of the properties and the morphology of surface supported bilayer systems.

Single and Bilayer Polyacrylamide Hydrogel-Based Microcapsules for the Triggered Release of Loads, Logic Gate Operations, and Intercommunication between Microcapsules.

A method to assemble loaded stimuli-responsive DNA-polyacrylamide hydrogel-stabilized microcapsules is presented. The method involves coating substrate-loaded CaCO3 microparticles, functionalized with nucleic acid promoter units, and cross-linking DNA-modified polyacrylamide chains on the microcapsules, using the hybridization chain reaction (HCR) to yield the DNA-cross-linked hydrogel coating. Dissolution of the CaCO3 particles generated the substrate-loaded hydrogel-protected microcapsules. The microcapsule-hydrogel shells include engineered stimuli-responsive oligonucleotide cross-linkers that control the stiffness of the hydrogel shells, allowing the triggered release of the loads. One approach includes the incorporation of cofactor-dependent DNAzyme units into the cross-linked hydrogel layers (cofactor = Mg2+ ions, Zn2+ ions, or histidine) as stimuli-responsive units. Cleavage of the cross-linking DNAzyme substrates by the respective cofactors yields hydrogel coatings with a reduced stiffness and higher porosity that allow the release of the loads. A further approach involved the application of the HCR process to assemble the bilayer hydrogel microcapsules that are unlocked by two cooperative triggers. Bilayer microcapsules consisting of a K+ ions-stabilized G-quadruplex/18-crown-6-ether (CE) responsive layer and a Mg2+ ion DNAzyme-responsive layers are presented. Unlocking and locking of the G-quadruplex cross-linked layer by 18-crown-6-ether and K+ ions, respectively, in the presence of Mg2+ ions allow the switchable controlled release of the load. In addition, the intercommunication of two kinds of stimuli-responsive bilayer hydrogel microcapsules carrying two different loads (tetramethylrhodamine-dextran, TMR-D, and CdSe/ZnS quantum dots) is demonstrated. The intercommunication process involves the stimuli-triggered generation of "information transfer" strands from one microcapsule to another that activate the release of the loads.

Attomole DNA electrochemical sensor for the detection of Escherichia coli O157.

Enterohemorrhagic Escherichia coli O157, a verocytotoxin (VT1/2)-producing pathogen, can be deadly because it can induce acute or chronic renal failure. To speed up the clinical diagnosis of related syndromes caused by E. coli O157, there is an urgent need for rapid, simple, and reliable analytical tools for its quantitation. In this study, we developed a novel electrochemical competitive genosensor, featuring gold-electrodeposited screen-printed electrodes (nanoAu/SPE) modified with a self-assembled monolayer of thiol-capped single-stranded DNA (capture probe), for the detection of the rfbE gene, which is specific to E. coli O157. This assay functions based on competition between the target gene (complementary to the capture probe DNA) and reporter DNA-tagged, hexaammineruthenium(III) chloride-encapsulated liposomes. The current signal of the released liposomal Ru(NH(3))(6)(3+) was measured using square wave voltammetry, yielding a sigmoidally shaped dose-response curve whose linear portion was over the range from 1 to 10(6) fmol. This liposomal competitive assay provides an amplification route for the detection of the rfbE gene at ultratrace levels; indeed, we could detect as little as 0.75 amol of the target rfbE DNA (equivalent to the amount present in 5 microL of a 0.15 pM solution).

Mimicking Horseradish Peroxidase Functions Using Cu2+-Modified Carbon Nitride Nanoparticles or Cu2+-Modified Carbon Dots as Heterogeneous Catalysts.

Cu2+-functionalized carbon nitride nanoparticles (Cu2+-g-C3N4 NPs), ∼200 nm, and Cu2+-carbon dots (Cu2+-C-dots), ∼8 nm, act as horseradish peroxidase-mimicking catalysts. The nanoparticles catalyze the generation of chemiluminescence in the presence of luminol/H2O2 and catalyze the oxidation of dopamine by H2O2 to form aminochrome. The Cu2+-g-C3N4-driven generation of chemiluminescence is used to develop a H2O2 sensor and is implemented to develop a glucose detection platform and a sensor for probing glucose oxidase. Also, the Cu2+-C-dots are functionalized with the ß-cyclodextrin (ß-CD) receptor units. The concentration of dopamine, at the Cu2+-C-dots' surface, by means of the ß-CD receptor sites, leads to a 4-fold enhancement in the oxidation of dopamine by H2O2 to yield aminochrome compared to that of the unmodified C-dots.

pH- and ligand-induced release of loads from DNA-acrylamide hydrogel microcapsules.

Herein, a method to construct stimuli-responsive DNA-acrylamide-based hydrogel microcapsules has been presented. This method involves the use of polyacrylamide chains modified with predesigned nucleic acid hairpin units and optionally single-strand tethers that provide the required hybridization and recognition functions to yield substrate-loaded stimuli-responsive hydrogel-based microcapsules. The synthesis of the microcapsules involves the loading of CaCO3 microparticles with the respective load substrates and the functionalization of the CaCO3 template particles with nucleic acid promoter units. In the presence of the hairpin-modified acrylamide chains, the promoter units induce the hybridization chain reaction (HCR), which leads to the formation of a hydrogel coating, which, after the dissociation of the CaCO3 cores, yields substrate-loaded stimuli-responsive hydrogel microcapsules. One of the microcapsule systems includes, in the hairpin-modified acrylamide constructs, and in the subsequent HCR-generated hydrogel shells, the caged sequences of anti-ATP or anti-cocaine aptamers. In the presence of ATP or cocaine, the duplex-caged aptamer sequences are separated via the formation of ATP- or cocaine-aptamer complexes, which results in the partial separation of the microcapsules and the release of the loads. The second type of microcapsule is cooperatively stabilized by bridges generated by HCR and pH-sensitive duplex units. Under acidic conditions, the pH-sensitive bridges dissociate via the formation of i-motif structures, which results in an increase in the fluidity of the microcapsule shells and the release of the loads. Preliminary studies indicate that ATP- or pH-responsive microcapsules loaded with the anticancer drug, doxorubicin, have a selective cytotoxic effect on MDA-MB-231 cancer cells.

Topical tissue nano-transfection mediates non-viral stroma reprogramming and rescue.

Although cellular therapies represent a promising strategy for a number of conditions, current approaches face major translational hurdles, including limited cell sources and the need for cumbersome pre-processing steps (for example, isolation, induced pluripotency). In vivo cell reprogramming has the potential to enable more-effective cell-based therapies by using readily available cell sources (for example, fibroblasts) and circumventing the need for ex vivo pre-processing. Existing reprogramming methodologies, however, are fraught with caveats, including a heavy reliance on viral transfection. Moreover, capsid size constraints and/or the stochastic nature of status quo approaches (viral and non-viral) pose additional limitations, thus highlighting the need for safer and more deterministic in vivo reprogramming methods. Here, we report a novel yet simple-to-implement non-viral approach to topically reprogram tissues through a nanochannelled device validated with well-established and newly developed reprogramming models of induced neurons and endothelium, respectively. We demonstrate the simplicity and utility of this approach by rescuing necrotizing tissues and whole limbs using two murine models of injury-induced ischaemia.

Programmed pH-Responsive Microcapsules for the Controlled Release of CdSe/ZnS Quantum Dots.

Two methods for the preparation of pH-responsive all-DNA microcapsules loaded with CdSe/ZnS quantum dots (QDs) are discussed. One approach involves the construction of DNA microcapsules composed of nucleic acid layers that include, at pH 7.2, "dormant" C-G·C(+) triplex sequences. The formation of the C-G·C(+) triplex structures at pH 5.0 leads to the cleavage of the microcapsules and to the release of the QDs. A second approach involves the synthesis of CdSe/ZnS QD-loaded DNA microcapsules, stabilized at pH 7.2 by T-A·T interlayer triplex bridges. The dissociation of the bridges at pH 9.0 separates the bridging triplex units, resulting in the degradation of the microcapsules and to the release of the QDs. The programmed pH-stimulated release of luminescent QDs, emitting at 620 and 560 nm, from the C-G·C(+) or T-A·T triplex-responsive microcapsules is demonstrated by subjecting the QD-loaded microcapsule mixtures to pH 5.0 or pH 9.0, respectively.

Adenosine Triphosphate-Triggered Release of Macromolecular and Nanoparticle Loads from Aptamer/DNA-Cross-Linked Microcapsules.

The synthesis of stimuli-responsive DNA microcapsules acting as carriers for different payloads, and being dissociated through the formation of aptamer-ligand complexes is described. Specifically, stimuli-responsive anti-adenosine triphosphate (ATP) aptamer-cross-linked DNA-stabilized microcapsules loaded with tetramethylrhodamine-modified dextran (TMR-D), CdSe/ZnS quantum dots (QDs), or microperoxidase-11 (MP-11) are presented. In the presence of ATP as trigger, the microcapsules are dissociated through the formation of aptamer-ATP complexes, resulting in the release of the respective loads. Selective unlocking of the capsules is demonstrated, and CTP, GTP, or TTP do not unlock the pores. The ATP-triggered release of MP-11 from the microcapsules enables the MP-11-catalyzed oxidation of Amplex UltraRed by H2O2 to the fluorescent product resorufin.