Stimuli-Responsive Hydrogel Microcapsules Harnessing the COVID-19 Immune Response for Cancer Therapeutics.

The combination of gene therapy and immunotherapy concepts, along recent advances in DNA nanotechnology, have the potential to provide important tools for cancer therapies. We present the development of stimuli-responsive microcapsules, loaded with a viral immunogenetic agent, harnessing the immune response against the Coronavirus Disease 2019, COVID-19, to selectively attack liver cancer cells (hepatoma) or recognize breast cancer or hepatoma, by expression of green fluorescence protein, GFP. The pH-responsive microcapsules, modified with DNA-tetrahedra nanostructures, increased hepatoma permeation by 50 %. Incorporation of a GFP-encoding lentivirus vector inside the tumor-targeting pH-stimulated miRNA-triggered and Alpha-fetoprotein-dictated microcapsules enables the demonstration of neoplasm selectivity, with approximately 5,000-, 8,000- and 50,000-fold more expression in the cancerous cells, respectively. The incorporation of the SARS-CoV-2 spike protein in the gene vector promotes specific recognition of the immune-evading hepatoma by the COVID-19-analogous immune response, which leads to cytotoxic and inflammatory activity, mediated by serum components taken from vaccinated or recovered COVID-19 patients, resulting in effective elimination of the hepatoma (>85 % yield).

Electro-metabolic coupling in multi-chambered vascularized human cardiac organoids.

The study of cardiac physiology is hindered by physiological differences between humans and small-animal models. Here we report the generation of multi-chambered self-paced vascularized human cardiac organoids formed under anisotropic stress and their applicability to the study of cardiac arrhythmia. Sensors embedded in the cardiac organoids enabled the simultaneous measurement of oxygen uptake, extracellular field potentials and cardiac contraction at resolutions higher than 10 Hz. This microphysiological system revealed 1 Hz cardiac respiratory cycles that are coupled to the electrical rather than the mechanical activity of cardiomyocytes. This electro-mitochondrial coupling was driven by mitochondrial calcium oscillations driving respiration cycles. Pharmaceutical or genetic inhibition of this coupling results in arrhythmogenic behaviour. We show that the chemotherapeutic mitoxantrone induces arrhythmia through disruption of this pathway, a process that can be partially reversed by the co-administration of metformin. Our microphysiological cardiac systems may further facilitate the study of the mitochondrial dynamics of cardiac rhythms and advance our understanding of human cardiac physiology.

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.

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.

Aminoglycoside-induced lipotoxicity and its reversal in kidney on chip.

Aminoglycosides are an important class of antibiotics that play a critical role in the treatment of life-threatening infections, but their use is limited by their toxicity. In fact, gentamicin causes severe nephrotoxicity in 17% of hospitalized patients. The kidney proximal tubule is particularly vulnerable to drug-induced nephrotoxicity due to its role in drug transport. In this work, we developed a perfused vascularized model of human kidney tubuloids integrated with tissue-embedded microsensors that track the metabolic dynamics of aminoglycoside-induced renal toxicity in real time. Our model shows that gentamicin disrupts proximal tubule polarity at concentrations 20-fold below its TC50, leading to a 3.2-fold increase in glucose uptake, and reverse TCA cycle flux culminating in a 40-fold increase in lipid accumulation. Blocking glucose reabsorption using the SGLT2 inhibitor empagliflozin significantly reduced gentamicin toxicity by 10-fold. These results demonstrate the utility of sensor-integrated kidney-on-chip platforms to rapidly identify new metabolic mechanisms that may underly adverse drug reactions. The results should improve our ability to modulate the toxicity of novel aminoglycosides.

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.

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.

Constitutional Dynamic Networks-Guided Synthesis of Programmed "Genes", Transcription of mRNAs, and Translation of Proteins.

Inspired by nature, where dynamic networks control the levels of gene expression and the activities of transcribed/translated proteins, we introduce nucleic acid-based constitutional dynamic networks (CDNs) as functional modules mimicking native circuits by demonstrating CDNs-guided programmed synthesis of genes, controlled transcription of RNAs, and dictated transcription/translation synthesis of proteins. An auxiliary CDN consisting of four dynamically equilibrated constituents AA', AB', BA', and BB' is orthogonally triggered by two different inputs yielding two different compositionally reconfigured CDNs. Subjecting the parent auxiliary CDN to two hairpins, HA and HB, and two templates TA and TB and a nicking/replication machinery leads to the cleavage of the hairpins and to the activation of the nicking/replication machineries that synthesize two "genes", e.g., the histidine-dependent DNAzyme g1 and the Zn2+-ion-dependent DNAzyme g2. The triggered orthogonal reconfiguration of the parent CDN to the respective CDNs leads to the programmed preferred CDN-guided synthesis of g1 or g2. Similarly, the triggered reconfigured CDNs are subjected to two hairpins HC and HD, the templates I'/I and J'/J, and the RNA polymerase (RNAp)/NTPs machinery. While the cleavage of the hairpins by the constituents associated with the parent CDN leads to the transcription of the broccoli aptamer recognizing the DFHBI ligand and of the aptamer recognizing the malachite green (MG) ligand, the orthogonally triggered CDNs lead to the CDNs-guided enhanced transcription of either the DFHBI aptamer or the MG aptamer. In addition, subjecting the triggered reconfigured CDNs to predesigned hairpins HE and HF, the templates M'/M and N'/N, the RNAp/NTPs machinery, and the cell-free ribosome t-RNA machinery leads to the CDNs-guided transcription/translation of the green fluorescence protein (GFP) or red fluorescence protein (RFP).

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.

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.

Biocatalytic reversible control of the stiffness of DNA-modified responsive hydrogels: applications in shape-memory, self-healing and autonomous controlled release of insulin.

The enzymes glucose oxidase (GOx), acetylcholine esterase (AchE) and urease that drive biocatalytic transformations to alter pH, are integrated into pH-responsive DNA-based hydrogels. A two-enzyme-loaded hydrogel composed of GOx/urease or AchE/urease and a three-enzyme-loaded hydrogel composed of GOx/AchE/urease are presented. The biocatalytic transformations within the hydrogels lead to the dictated reconfiguration of nucleic acid bridges and the switchable control over the stiffness of the respective hydrogels. The switchable stiffness features are used to develop biocatalytically guided shape-memory and self-healing matrices. In addition, loading of GOx/insulin in a pH-responsive DNA-based hydrogel yields a glucose-triggered matrix for the controlled release of insulin, acting as an artificial pancreas. The release of insulin is controlled by the concentrations of glucose, hence, the biocatalytic insulin-loaded hydrogel provides an interesting sense-and-treat carrier for controlling diabetes.