Transient Dynamic Operation of G-Quadruplex-Gated Glucose Oxidase-Loaded ZIF-90 Metal-Organic Framework Nanoparticle Bioreactors.

Glucose oxidase-loaded ZIF-90 metal-organic framework nanoparticles conjugated to hemin-G-quadruplexes act as functional bioreactor hybrids operating transient dissipative biocatalytic cascaded transformations consisting of the glucose-driven H2O2-mediated oxidation of Amplex-Red to resorufin or the glucose-driven generation of chemiluminescence by the H2O2-mediated oxidation of luminol. One system involves the fueled activation of a reaction module leading to the temporal formation and depletion of the bioreactor conjugate operating the nickase-guided transient biocatalytic cascades. The second system demonstrates the fueled activation of a reaction module yielding a bioreactor conjugate operating the exonuclease III-dictated transient operation of the two biocatalytic cascades. The temporal operations of the bioreactor circuits are accompanied by kinetic models and computational simulations enabling us to predict the dynamic behavior of the systems subjected to different auxiliary conditions.

Visualization of Molecular Permeation into a Multi-compartment Phospholipid Vesicle.

Passive permeation of small molecules into vesicles with multiple compartments is a critical event in many chemical and biological processes. We consider the translocation of the peptide NAF-144-67 labeled with a fluorescent fluorescein dye across membranes of rhodamine-labeled 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) into liposomes with internal vesicles. Time-resolved microscopy revealed a sequential absorbance of the peptide in both the outer and inner micrometer vesicles that developed over a time period of minutes to hours, illustrating the spatial and temporal progress of the permeation. There is minimal perturbation of the membrane structure and no evidence for pore formation. On the basis of molecular dynamics simulations of NAF-144-67, we extended a local defect model to migration processes that include multiple compartments. The model captures the long residence time of the peptide within the membrane and the rate of permeation through the liposome and its internal compartments. Imaging experiments confirm the semi-quantitative description of the permeation of the model by activated diffusion and open the way for studies of more complex systems.

Aptamer-Functionalized Ce4+-Ion-Modified C-Dots: Peroxidase Mimicking Aptananozymes for the Oxidation of Dopamine and Cytotoxic Effects toward Cancer Cells.

Aptamer-functionalized Ce4+-ion-modified C-dots act as catalytic hybrid systems, aptananozymes, catalyzing the H2O2 oxidation of dopamine. A series of aptananozymes functionalized with different configurations of the dopamine binding aptamer, DBA, are introduced. All aptananozymes reveal substantially enhanced catalytic activities as compared to the separated Ce4+-ion-modified C-dots and aptamer constituents, and structure-catalytic functions between the structure and binding modes of the aptamers linked to the C-dots are demonstrated. The enhanced catalytic functions of the aptananozymes are attributed to the aptamer-induced concentration of the reaction substrates in spatial proximity to the Ce4+-ion-modified C-dots catalytic sites. The oxidation processes driven by the Ce4+-ion-modified C-dots involve the formation of reactive oxygen species (•OH radicals). Accordingly, Ce4+-ion-modified C-dots with the AS1411 aptamer or MUC1 aptamer, recognizing specific biomarkers associated with cancer cells, are employed as targeted catalytic agents for chemodynamic treatment of cancer cells. Treatment of MDA-MB-231 breast cancer cells and MCF-10A epithelial breast cells, as control, with the AS1411 aptamer- or MUC1 aptamer-modified Ce4+-ion-modified C-dots reveals selective cytotoxicity toward the cancer cells. In vivo experiments reveal that the aptamer-functionalized nanoparticles inhibit MDA-MB-231 tumor growth.

DNA-Tetrahedra Corona-Modified Hydrogel Microcapsules: "Smart" ATP- or microRNA-Responsive Drug Carriers.

The assembly of adenosine triphosphate (ATP)-responsive and miRNA-responsive DNA tetrahedra-functionalized carboxymethyl cellulose hydrogel microcapsules is presented. The microcapsules are loaded with the doxorubicin-dextran drug or with CdSe/ZnS quantum dots as a drug model. Selective unlocking of the respective microcapsules and the release of the loads in the presence of ATP or miRNA-141 are demonstrated. Functionalization of the hydrogel microcapsules a with corona of DNA tetrahedra nanostructures yields microcarriers that revealed superior permeation into cells. This is demonstrated by the effective permeation of the DNA tetrahedra-functionalized microcapsules into MDA-MB-231 breast cancer cells, as compared to epithelial MCF-10A nonmalignant breast cells. The superior permeation of the tetrahedra-functionalized microcapsules into MDA-MB-231 breast cancer cells, as compared to analog control hydrogel microcapsules modified with a corona of nucleic acid duplexes. The effective permeation of the stimuli-responsive, drug-loaded, DNA tetrahedra-modified microcapsules yields drug carriers of superior and selective cytotoxicity toward cancer cells.

Aptamer-Modified Au Nanoparticles: Functional Nanozyme Bioreactors for Cascaded Catalysis and Catalysts for Chemodynamic Treatment of Cancer Cells.

Polyadenine-stabilized Au nanoparticles (pA-AuNPs) reveal dual nanozyme catalytic activities toward the H2O2-mediated oxidation of dopamine to aminochrome and toward the aerobic oxidation of glucose to gluconic acid and H2O2. The conjugation of a dopamine-binding aptamer (DBA) to the pA-AuNPs yields aptananozyme structures catalyzing simultaneously the H2O2-mediated oxidation of dopamine to aminochrome through the aerobic oxidation of glucose. A set of aptananozymes consisting of DBA conjugated through the 5'- or 3'-end directly or spacer bridges to pA-AuNPs were synthesized. The set of aptananozymes revealed enhanced catalytic activities toward the H2O2-catalyzed oxidation of dopamine to dopachrome, as compared to the separated pA-AuNPs and DBA constituents, and structure-function relationships within the series of aptananozymes were demonstrated. The enhanced catalytic function of the aptananozymes was attributed to the concentration of the dopamine at the catalytic interfaces by means of aptamer-dopamine complexes. The dual catalytic activities of aptananozymes were further applied to design bioreactors catalyzing the effective aerobic oxidation of dopamine in the presence of glucose. Mechanistic studies demonstrated that the aptananozymes generate reactive oxygen species. Accordingly, the AS1411 aptamer, recognizing the nucleolin receptor associated with cancer cells, was conjugated to the pA-AuNPs, yielding a nanozyme for the chemodynamic treatment of cancer cells. The AS1411 aptamer targets the aptananozyme to the cancer cells and facilitates the selective permeation of the nanozyme into the cells. Selective cytotoxicity toward MDA-MB-231 breast cancer cells (ca. 70% cell death) as compared to MCF-10A epithelial cells (ca. 2% cell death) is demonstrated.

Topologically switchable and gated transcription machinery.

Topological barriers control in nature the transcription machinery, thereby perturbing gene expression. Here we introduce synthetically designed DNA templates that include built-in topological barriers for switchable, triggered-controlled transcription of RNA aptamers. This is exemplified with the design of transcription templates that include reversible and switchable topological barriers consisting of a Sr2+-ion-stabilized G-quadruplex and its separation by kryptofix [2.2.2], KP, for the switchable transcription of the malachite green (MG) RNA aptamer, the T-A·T triplex barrier being separated by a fuel-strand for the cyclic triggered transcription of the 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI)-binding aptamer, and the use of a photoactivated cis/trans azobenzene-modified nucleic acid barrier for the switchable "ON"/"OFF" transcription of the MG RNA aptamer. By applying a mixture of topologically triggered templates consisting of the photoresponsive barrier and the T-A·T triplex barrier, the gated transcription of the MG aptamer or the DFHBI-binding aptamer is demonstrated. In addition, a Sr2+-ion/KP topologically triggered DNA tetrahedra promoter-transcription scaffold, for the replication of the MG RNA aptamer, and T7 RNA polymerase are integrated into DNA-based carboxymethyl cellulose hydrogel microcapsules acting as cell-like assemblies. The switchable, reversible transcription of the MG RNA aptamer in a cell-like containment is introduced.

Biocatalytic cascades and intercommunicated biocatalytic cascades in microcapsule systems.

Biomolecule-loaded nucleic acid-functionalized carboxymethyl cellulose hydrogel-stabilized microcapsules (diameter ca. 2 µm) are introduced as cell-like containments. The microcapsules are loaded with two DNA tetrahedra, T1 and T2, functionalized with guanosine-rich G-quadruplex subunits, and/or with native enzymes (glucose oxidase, GOx, and/or ß-galactosidase, ß-gal). In the presence of K+-ions and hemin, the T1/T2 tetrahedra constituents, loaded in the microcapsules, assemble into a hemin/G-quadruplex bridged tetrahedra dimer DNAzyme catalyzing the oxidation of Amplex Red to Resorufin by generating H2O2. In the presence of co-loaded GOx or GOx/ß-gal, the GOx//T1/T2 hemin/G-quadruplex cascade catalyzing the glucose-mediated oxidation of Amplex Red to Resorufin, and the three-biocatalysts cascade consisting of ß-gal//GOx//hemin/G-quadruplex bridged T1/T2 catalyzing the lactose-driven oxidation of Amplex Red to Resorufin proceed in the microcapsules. Enhanced biocatalytic transformations in the microcapsules, as compared to the performance of the reactions in a homogeneous phase, are observed, due to the proximity of the biocatalysts in a confined volume. As the synthetic methodology to prepare the microcapsules yields boundaries functionalized with complementary nucleic acid tethers, the dynamic association of different microcapsules, loaded selectively with biomolecular catalysts, proceeds. The dynamic dimerization of GOx-loaded microcapsules and hemin/G-quadruplex bridged T1/T2 DNAzyme-loaded microcapsules yields effective intercommunicated microcapsules driving the GOx//hemin/G-quadruplex bridged T1/T2 DNAzyme cascade. In addition, the dynamic dimerization of GOx-loaded microcapsules with ß-gal//hemin/G-quadruplex bridged T1/T2-loaded microcapsules enables the bi-directional intercommunicated operation of the lactose-stimulated three catalysts ß-gal//GOx//hemin/G-quadruplex bridged T1/T2 DNAzyme cascade. The guided separation and formation of dynamic supramolecular dimer microcapsular containments, and the dictated switchable operation of intercommunicated biocatalytic cascades are demonstrated.

A peptide-derived strategy for specifically targeting the mitochondria and ER of cancer cells: a new approach in fighting cancer.

An effective anti-cancer therapy should exclusively target cancer cells and trigger in them a broad spectrum of cell death pathways that will prevent avoidance. Here, we present a new approach in cancer therapy that specifically targets the mitochondria and ER of cancer cells. We developed a peptide derived from the flexible and transmembrane domains of the human protein NAF-1/CISD2. This peptide (NAF-144-67) specifically permeates through the plasma membranes of human epithelial breast cancer cells, abolishes their mitochondria and ER, and triggers cell death with characteristics of apoptosis, ferroptosis and necroptosis. In vivo analysis revealed that the peptide significantly decreases tumor growth in mice carrying xenograft human tumors. Computational simulations of cancer vs. normal cell membranes reveal that the specificity of the peptide to cancer cells is due to its selective recognition of their membrane composition. NAF-144-67 represents a promising anti-cancer lead compound that acts via a unique mechanism.

A VDAC1-mediated NEET protein chain transfers [2Fe-2S] clusters between the mitochondria and the cytosol and impacts mitochondrial dynamics.

Mitochondrial inner NEET (MiNT) and the outer mitochondrial membrane (OMM) mitoNEET (mNT) proteins belong to the NEET protein family. This family plays a key role in mitochondrial labile iron and reactive oxygen species (ROS) homeostasis. NEET proteins contain labile [2Fe-2S] clusters which can be transferred to apo-acceptor proteins. In eukaryotes, the biogenesis of [2Fe-2S] clusters occurs within the mitochondria by the iron-sulfur cluster (ISC) system; the clusters are then transferred to [2Fe-2S] proteins within the mitochondria or exported to cytosolic proteins and the cytosolic iron-sulfur cluster assembly (CIA) system. The last step of export of the [2Fe-2S] is not yet fully characterized. Here we show that MiNT interacts with voltage-dependent anion channel 1 (VDAC1), a major OMM protein that connects the intermembrane space with the cytosol and participates in regulating the levels of different ions including mitochondrial labile iron (mLI). We further show that VDAC1 is mediating the interaction between MiNT and mNT, in which MiNT transfers its [2Fe-2S] clusters from inside the mitochondria to mNT that is facing the cytosol. This MiNT-VDAC1-mNT interaction is shown both experimentally and by computational calculations. Additionally, we show that modifying MiNT expression in breast cancer cells affects the dynamics of mitochondrial structure and morphology, mitochondrial function, and breast cancer tumor growth. Our findings reveal a pathway for the transfer of [2Fe-2S] clusters, which are assembled inside the mitochondria, to the cytosol.

miRNA-Guided Imaging and Photodynamic Therapy Treatment of Cancer Cells Using Zn(II)-Protoporphyrin IX-Loaded Metal-Organic Framework Nanoparticles.

An analytical platform for the selective miRNA-21-guided imaging of breast cancer cells and miRNA-221-guided imaging of ovarian cancer cells and the selective photodynamic therapy (PDT) of these cancer cells is introduced. The method is based on Zn(II)-protoporphyrin IX, Zn(II)-PPIX-loaded UiO-66 metal-organic framework nanoparticles, NMOFs, gated by two hairpins Hi/Hj through ligation of their phosphate residues to the vacant Zr4+-ions associated with the NMOFs. The hairpins are engineered to include the miRNA recognition sequence in the stem domain of Hi, and in the Hi and Hj, partial locked stem regions of G-quadruplex subunits. Intracellular phosphate-ions displace the hairpins, resulting in the release of the Zn(II)-PPIX and intracellular miRNAs open Hi, and this triggers the autonomous cross-opening of Hi and Hj. This activates the interhairpin hybridization chain reaction and leads to the assembly of highly fluorescent Zn(II)-PPIX-loaded G-quadruplex chains. The miRNA-guided fluorescent chains allow selective imaging of cancer cells. Moreover, PDT with visible light selectively kills cancer cells and tumor cells through the formation of toxic reactive oxygen species.

Aptamer-modified DNA tetrahedra-gated metal-organic framework nanoparticle carriers for enhanced chemotherapy or photodynamic therapy.

UiO-66 metal-organic framework nanoparticles (NMOFs) gated by aptamer-functionalized DNA tetrahedra provide superior biomarker-responsive hybrid nano-carriers for biomedical applications. Hybrid nano-carriers consisting of ATP-aptamer or VEGF-aptamer functionalized tetrahedra-gated NMOFs are loaded with the chemotherapeutic drug, doxorubicin (DOX). In the presence of ATP or VEGF, both abundant in cancer cells, the tetrahedra-gated NMOFs are unlocked to release the drug. Enhanced and selective permeation of the DOX-loaded ATP/VEGF-responsive tetrahedra-gated NMOFs into MDA-MB-231 breast cancer cells as compared to the reference ATP/VEGF-responsive duplex-gated NMOFs or non-malignant MCF-10A epithelial breast cells is observed. This results in enhanced and selective cytotoxicity of the tetrahedra-gated DOX-loaded NMOFs toward the malignant cells. Additional nano-carriers, consisting of photosensitizer Zn(ii) protoporphyrin IX (Zn(ii)-PPIX)-loaded VEGF-responsive tetrahedra-gated NMOFs, are introduced. The VEGF-triggered unlocking of the NMOFs yields separated G-quadruplex-VEGF aptamer complexes conjugated to the tetrahedra, resulting in the release of loaded Zn(ii)-PPIX. Association of the released Zn(ii)-PPIX to the G-quadruplex structures generates highly fluorescent supramolecular Zn(ii)-PPIX/G-quadruplex VEGF aptamer-tetrahedra structures. The efficient and selective generation of the highly fluorescent Zn(ii)-PPIX/G-quadruplex VEGF aptamer-tetrahedra nanostructures in malignant cells allows the light-induced photosensitized generation of reactive oxygen species (ROS), leading to high-efficacy PDT treatment of the malignant cells.

Disrupting CISD2 function in cancer cells primarily impacts mitochondrial labile iron levels and triggers TXNIP expression.

The CISD2 (NAF-1) protein plays a key role in regulating cellular homeostasis, aging, cancer and neurodegenerative diseases. It was found to control different calcium, reactive oxygen species (ROS), and iron signaling mechanisms. However, since most studies of CISD2 to date were conducted with cells that constitutively lack, overexpress, or contain mutations in CISD2, the relationships between these different signaling processes are unclear. To address the hierarchy of signaling events occurring in cells upon CISD2 disruption, we developed an inducible system to express CISD2, or the dominant-negative H114C inhibitor of CISD2, in human breast cancer cells. Here, we report that inducible disruption of CISD2 function causes an immediate disruption in mitochondrial labile iron (mLI), and that this disruption results in enhanced mitochondrial ROS (mROS) levels. We further show that alterations in cytosolic and ER calcium levels occur only after the changes in mLI and mROS levels happen and are unrelated to them. Interestingly, disrupting CISD2 function resulted in the enhanced expression of the tumor suppressor thioredoxin-interacting protein (TXNIP) that was dependent on the accumulation of mLI and associated with ferroptosis activation. CISD2 could therefore regulate the expression of TXNIP in cancer cells, and this regulation is dependent on alterations in mLI levels.

A Combined Drug Treatment That Reduces Mitochondrial Iron and Reactive Oxygen Levels Recovers Insulin Secretion in NAF-1-Deficient Pancreatic Cells.

Decreased insulin secretion, associated with pancreatic ß-cell failure, plays a critical role in many human diseases including diabetes, obesity, and cancer. While numerous studies linked ß-cell failure with enhanced levels of reactive oxygen species (ROS), the development of diabetes associated with hereditary conditions that result in iron overload, e.g., hemochromatosis, Friedreich's ataxia, and Wolfram syndrome type 2 (WFS-T2; a mutation in CISD2, encoding the [2Fe-2S] protein NAF-1), underscores an additional link between iron metabolism and ß-cell failure. Here, using NAF-1-repressed INS-1E pancreatic cells, we observed that NAF-1 repression inhibited insulin secretion, as well as impaired mitochondrial and ER structure and function. Importantly, we found that a combined treatment with the cell permeant iron chelator deferiprone and the glutathione precursor N-acetyl cysteine promoted the structural repair of mitochondria and ER, decreased mitochondrial labile iron and ROS levels, and restored glucose-stimulated insulin secretion. Additionally, treatment with the ferroptosis inhibitor ferrostatin-1 decreased cellular ROS formation and improved cellular growth of NAF-1 repressed pancreatic cells. Our findings reveal that suppressed expression of NAF-1 is associated with the development of ferroptosis-like features in pancreatic cells, and that reducing the levels of mitochondrial iron and ROS levels could be used as a therapeutic avenue for WFS-T2 patients.

pH- and miRNA-Responsive DNA-Tetrahedra/Metal-Organic Framework Conjugates: Functional Sense-and-Treat Carriers.

The synthesis of stimuli-responsive hybrid structures composed of drug-loaded UiO-66 metal-organic framework nanoparticles, NMOFs, locked by DNA tetrahedra gates is presented. The hybrid systems combine the high loading capacity of drugs in the porous NMOFs and the effective cell permeation properties of the DNA tetrahedra. The nucleic acid-functionalized UiO-66 NMOFs are loaded with drugs (doxorubicin, DOX, or camptothecin, CPT) or with dyes as drug models (Rhodamine 6G or fluorescein) and used to prepare stimuli-responsive carriers. In this study, two different stimuli-responsive NMOFs are presented. One system introduces the drug-loaded NMOFs locked by pH-responsive DNA tetrahedra. At acidic pH values, the gating tetrahedra are dissociated from the NMOFs through the formation of i-motif structures, resulting in the unlocking of the NMOFs and the release of the drugs. In addition, the tetrahedra gates are modified with AS1411 aptamer tethers, and these target the drug-loaded NMOFs to nucleolin receptors overexpressed in certain malignant cells. A second system involves the preparation of NMOFs loaded with drugs/dyes and gated by the microRNA (miRNA)-responsive tetrahedra (miRNA-21 or miRNA-155). In the presence of miRNAs, the dissociation of miRNA-responsive tetrahedra from the NMOFs leads to the unlocking of the NMOFs and the release of the loads. Further developments of the miRNA-responsive tetrahedra-gated hybrid carriers include the following. (i) By appropriate engineering of the miRNA gating units, the exonuclease III (Exo III)-amplified unlocking of the carriers, through the regeneration of the miRNA triggers, and the enhanced release of the loaded drugs are demonstrated. (ii) By applying mixtures of miRNA-21-responsive DNA tetrahedra-gated DOX-loaded NMOFs and miRNA-155-responsive DNA tetrahedra-gated CPT-loaded NMOFs, the multiplexed miRNA-21/miRNA-155-dictated release of the drugs is demonstrated. As compared to the analog DNA duplex-modified NMOFs, DNA tetrahedra-gated, drug-loaded NMOFs permeation into malignant MDA-MB-231 breast cancer cells presents more effective cell permeation. Effective and selective cytotoxicity toward the malignant cells, as compared to nonmalignant epithelial MCF-10A breast cells, is demonstrated due to the acidic pH, present in cancer cells, or the miRNA-21, present in MDA-MB-231 malignant cells.

Triggered Dimerization and Trimerization of DNA Tetrahedra for Multiplexed miRNA Detection and Imaging of Cancer Cells.

The reversible and switchable triggered reconfiguration of tetrahedra nanostructures from monomer tetrahedra structures into dimer or trimer structures is introduced. The triggered bridging of monomer tetrahedra by K+ -ion-stabilized G-quadruplexes or T-A•T triplexes leads to dimer or trimer tetrahedra structures that are separated by crown ether or basic pH conditions, respectively. The signal-triggered dimerization/trimerization of DNA tetrahedra structures is used to develop multiplexed miRNA-sensing platforms, and the tetrahedra mixture is used for intracellular sensing and imaging of miRNAs.

Chemical targeting of NEET proteins reveals their function in mitochondrial morphodynamics.

Several human pathologies including neurological, cardiac, infectious, cancerous, and metabolic diseases have been associated with altered mitochondria morphodynamics. Here, we identify a small organic molecule, which we named Mito-C. Mito-C is targeted to mitochondria and rapidly provokes mitochondrial network fragmentation. Biochemical analyses reveal that Mito-C is a member of a new class of heterocyclic compounds that target the NEET protein family, previously reported to regulate mitochondrial iron and ROS homeostasis. One of the NEET proteins, NAF-1, is identified as an important regulator of mitochondria morphodynamics that facilitates recruitment of DRP1 to the ER-mitochondria interface. Consistent with the observation that certain viruses modulate mitochondrial morphogenesis as a necessary part of their replication cycle, Mito-C counteracts dengue virus-induced mitochondrial network hyperfusion and represses viral replication. The newly identified chemical class including Mito-C is of therapeutic relevance for pathologies where altered mitochondria dynamics is part of disease etiology and NEET proteins are highlighted as important therapeutic targets in anti-viral research.

The balancing act of NEET proteins: Iron, ROS, calcium and metabolism.

NEET proteins belong to a highly conserved group of [2Fe-2S] proteins found across all kingdoms of life. Due to their unique [2Fe2S] cluster structure, they play a key role in the regulation of many different redox and oxidation processes. In eukaryotes, NEET proteins are localized to the mitochondria, endoplasmic reticulum (ER) and the mitochondrial-associated membranes connecting these organelles (MAM), and are involved in the control of multiple processes, ranging from autophagy and apoptosis to ferroptosis, oxidative stress, cell proliferation, redox control and iron and iron­sulfur homeostasis. Through their different functions and interactions with key proteins such as VDAC and Bcl-2, NEET proteins coordinate different mitochondrial, MAM, ER and cytosolic processes and functions and regulate major signaling molecules such as calcium and reactive oxygen species. Owing to their central role in cells, NEET proteins are associated with numerous human maladies including cancer, metabolic diseases, diabetes, obesity, and neurodegenerative diseases. In recent years, a new and exciting role for NEET proteins was uncovered, i.e., the regulation of mitochondrial dynamics and morphology. This new role places NEET proteins at the forefront of studies into cancer and different metabolic diseases, both associated with the regulation of mitochondrial dynamics. Here we review recent studies focused on the evolution, biological role, and structure of NEET proteins, as well as discuss different studies conducted on NEET proteins function using transgenic organisms. We further discuss the different strategies used in the development of drugs that target NEET proteins, and link these with the different roles of NEET proteins in cells.

DNA Tetrahedra Modules as Versatile Optical Sensing Platforms for Multiplexed Analysis of miRNAs, Endonucleases, and Aptamer-Ligand Complexes.

The sensing modules for analyzing miRNAs or the endonucleases consist of tetrahedra functionalized with three different fluorophore-quencher pairs in spatially quenched configurations and hairpin units acting as recognition elements for the analytes. Three different miRNAs (miRNA-21, miRNA-221, and miRNA-155) or three different endonucleases (Nt.BbvCI, EcoRI, and HindIII) uncage the respective hairpins, leading to the switched-on fluorescence of the respective fluorophores and to the multiplex detection of the respective analytes. In addition, a tetrahedron module for the multiplexed analysis of aptamer ligand complexes (ligands = ATP, thrombin, VEGF) is introduced. The module includes edges modified with three spatially separated fluorophore-quencher pairs that were stretched by the respective aptamer strands to yield a switched-on fluorescent state. Formation of the respective aptamer ligands reconfigures the edges into fluorophore-quenched caged-hairpin structures that enable the multiplexed analysis of the aptamer-ligand complexes. The facile permeation of the tetrahedra structures into cells is used for the imaging of MCF-7 and HepG2 cancer cells and their discrimination from normal epithelial MCF-10A breast cells.

Thermoplasmonic-Triggered Release of Loads from DNA-Modified Hydrogel Microcapsules Functionalized with Au Nanoparticles or Au Nanorods.

Microcapsules consisting of hydrogel shells cross-linked by glucosamine-boronate ester complexes and duplex nucleic acids, loaded with dyes or drugs and functionalized with Au nanoparticles (Au NPs) or Au nanorods (Au NRs), are developed. Irradiation of Au NPs or Au NRs results in the thermoplasmonic heating of the microcapsules, and the dissociation of the nucleic acid cross-linkers. The separation of duplex nucleic acid cross-linkers leads to low-stiffness hydrogel shells, allowing the release of loads. Switching off the light-induced plasmonic heating results in the regeneration of stiff hydrogel shells protecting the microcapsules, leading to the blockage of release processes. The thermoplasmonic release of tetramethylrhodamine-dextran, Texas Red-dextran, doxorubicin-dextran (DOX-D), or camptothecin-carboxymethylcellulose (CPT-CMC) from the microcapsules is introduced. By loading the microcapsules with two different drugs (DOX-D and CPT-CMC), the light-controlled dose release is demonstrated. Cellular experiments show efficient permeation of Au NPs/DOX-D or Au NRs/DOX-D microcapsules into MDA-MB-231 cancer cells and inefficient uptake by MCF-10A epithelial breast cells. Cytotoxicity experiments reveal selective thermoplasmon-induced cytotoxicity of the microcapsules toward MDA-MB-231 cancer cells as compared to MCF-10A cells. Also, selective cytotoxicity towards MDA-MB-231 cancer cells upon irradiation of the Au NPs- and Au NRs-functionalized microcapsules at λ = 532 or 910 nm is demonstrated.

Triggered Release of Loads from Microcapsule-in-Microcapsule Hydrogel Microcarriers: En-Route to an "Artificial Pancreas".

A method to assemble stimuli-responsive nucleic acid-based hydrogel-stabilized microcapsule-in-microcapsule systems is introduced. An inner aqueous compartment stabilized by a stimuli-responsive hydrogel-layer (∼150 nm) provides the inner microcapsule (diameter ∼2.5 µm). The inner microcapsule is separated from an outer aqueous compartment stabilized by an outer stimuli-responsive hydrogel layer (thickness of ∼150 nm) that yields the microcapsule-in-microcapsule system. Different loads, e.g., tetramethyl rhodamine-dextran (TMR-D) and CdSe/ZnS quantum dots (QDs), are loaded in the inner and outer aqueous compartments. The hydrogel layers exist in a higher stiffness state that prevents inter-reservoir or leakage of the loads from the respective aqueous compartments. Subjecting the inner hydrogel layer to Zn2+-ions and/or the outer hydrogel layer to acidic pH or crown ether leads to the triggered separation of the bridging units associated with the respective hydrogel layers. This results in the hydrogel layers of lower stiffness allowing either the mixing of the loads occupying the two aqueous compartments, the guided release of the load from the outer aqueous compartment, or the release of the loads from the two aqueous compartments. In addition, a pH-responsive microcapsule-in-microcapsule system is loaded with glucose oxidase (GOx) in the inner aqueous compartment and insulin in the outer aqueous compartment. Glucose permeates across the two hydrogel layers resulting in the GOx catalyzed aerobic oxidation of glucose to gluconic acid. The acidification of the microcapsule-in-microcapsule system leads to the triggered unlocking of the outer, pH-responsive hydrogel layer and to the release of insulin. The pH-stimulated release of insulin is controlled by the concentration of glucose. While at normal glucose levels, the release of insulin is practically prohibited, the dose-controlled release of insulin in the entire diabetic range  is demonstrated. Also, switchable ON/OFF release of insulin is achieved highlighting an autonomous glucose-responsive microdevice operating as an "artificial pancreas" for the release of insulin.