Temporal Control of Efficient In Vivo Bioconjugation Using a Genetically Encoded Tetrazine-Mediated Inverse-Electron-Demand Diels-Alder Reaction.

An inverse-electron-demand Diels-Alder (IEDDA) reaction using genetically encoded tetrazine variants enables rapid bioconjugation for diverse applications in vitro and in cellulo. However, in vivo bioconjugation using genetically encoded tetrazine variants is challenging, because the IEDDA coupling reaction competes with rapid elimination of reaction partners in vivo. Here, we tested the hypothesis that a genetically encoded phenylalanine analogue containing a hydrogen-substituted tetrazine (frTet) would increase the IEDDA reaction rate, thereby allowing for successful bioconjugation in vivo. We found that the in vitro IEDDA reaction rate of superfolder green fluorescent protein (sfGFP) containing frTet (sfGFP-frTet) was 12-fold greater than that of sfGFP containing methyl-substituted tetrazine (sfGFP-Tet_v2.0). Additionally, sfGFP variants encapsulated with chitosan-modified, pluronic-based nanocarriers were delivered into nude mice or tumor-bearing mice for in vivo imaging. The in vivo-delivered sfGFP-frTet exhibited almost complete fluorescence recovery upon addition of trans-cyclooctene via the IEDDA reaction within 2 h, whereas sfGFP-Tet_v2.0 did not show substantial fluorescence recovery. These results demonstrated that the genetically encoded frTet allows an almost complete IEDDA reaction in vivo upon addition of trans-cyclooctene, enabling temporal control of in vivo bioconjugation in a very high yield.

Giant Decrease in Interfacial Energy of Liquid Metals by Native Oxides.

Native oxides form on the surface of many metals. Here, using gallium-based liquid metal alloys, Johnson-Kendall-Roberts (JKR) measurements are employed to show that native oxide dramatically lower the tension of the metal interface from 724 to 10 mN m-1. Like conventional surfactants, the oxide has asymmetry between the composition of its internal and external interfaces. Yet, in comparison to conventional surfactants, oxides are an order of magnitude more effective at lowering tension and do not need to be added externally to the liquid (i.e., oxides form naturally on metals). This surfactant-like asymmetry explains the adhesion of oxide-coated metals to surfaces. The resulting low interfacial energy between the metal and the interior of the oxide helps stabilize non-spherical liquid metal structures. In addition, at small enough macroscopic contact angles, the finite tension of the liquid within the oxide can drive fluid instabilities that are useful for separating the oxide from the metal to form oxide-encased bubbles or deposit thin oxide films (1-5 nm) on surfaces. Since oxides form on many metals, this work can have implications for a wide range of metals and metal oxides in addition to explaining the physical behavior of liquid metal.

Self-assembled hemin-conjugated heparin with dual-enzymatic cascade reaction activities for acute kidney injury.

Nanozymes have prominent catalytic activities with high stability as a substitute for unstable and expensive natural enzymes. However, most nanozymes are metal/inorganic nanomaterials, facing difficulty in clinical translation due to their unproven biosafety and limited biodegradability issues. Hemin, an organometallic porphyrin, was newly found to possess superoxide dismutase (SOD) mimetic activity along with previously known catalase (CAT) mimetic activity. However, hemin has poor bioavailability due to its low water solubility. Therefore, a highly biocompatible and biodegradable organic-based nanozyme system with SOD/CAT mimetic cascade reaction activity was developed by conjugating hemin to heparin (HepH) or chitosan (CS-H). Between them, Hep-H formed a smaller (<50 nm) and more stable self-assembled nanostructure and even possessed much higher and more stable SOD and CAT activities as well as the cascade reaction activity compared to CS-H and free hemin. Hep-H also showed a better cell protection effect against reactive oxygen species (ROS) compared to CS-H and hemin in vitro. Furthermore, Hep-H was selectively delivered to the injured kidney upon intravenous administration at the analysis time point (24 h) and exhibited excellent therapeutic effects on an acute kidney injury model by efficiently removing ROS, reducing inflammation, and minimizing structural and functional damage to the kidney.

Nanoreactor for cascade reaction between SOD and CAT and its tissue regeneration effect.

Nanoreactors for scavenging reactive oxygen species (ROS), a major factor in inflammatory diseases, can reduce overproduced ROS, and thus can prevent further progress of the diseases or facilitate the regeneration of damaged inflamed tissues. Herein, we designed a pluronic-based nanocarrier loaded with dual antioxidant enzymes present in vivo (superoxide dismutase (SOD) and catalase (CAT)) as a nanoreactor system for the regeneration of inflammatory tissue. The catalytic activity of each enzyme was enhanced by loading it into the nanocarrier. More importantly, the nanocarrier could enhance the cascade reaction between SOD and CAT, which converts the superoxide anion to oxygen. The synergistic anti-inflammatory effect of the nanoreactor based on the cascade reaction was verified in vitro. Furthermore, in an inflammatory bowel disease (IBD) mouse model, the dual enzyme (SOD/CAT)-loaded nanocarrier could result in significantly enhanced tissue regeneration and notably alleviated inflammation activities upon intravenous administration of them compared to other control groups, including single enzyme (SOD or CAT)-loaded nanocarrier and the free mixture of both enzymes without the nanocarrier. Thus, the efficacy of the nanoreactor for the cascade reaction on tissue regeneration in vivo was proved. Accordingly, the nanoreactor could be applied for tissue regeneration therapy against various inflammatory diseases.

Solvent-Assisted Filling of Liquid Metal and Its Selective Dewetting for the Multilayered 3D Interconnect in Stretchable Electronics.

As stretchable electronics are rapidly developing and becoming complex, the requirement for stretchable, multilayered, and large-area printed circuit boards (PCBs) is emerging. This demands a stretchable electrode and its vertical interconnect access (via) for 3-dimensional (3D) connectivity between layers. Here, we demonstrate solvent-assisted liquid metal (LM) filling into the submicrometer channel (∼400 nm), including via-hole filling and selective dewetting of LM. We provide the theoretical background of solvent-assisted LM filling and selective dewetting and reveal the osmotic pressure arising from anomalous mass transport phenomena, case II diffusion, which drives negative pressure, the spontaneous pulling of LM into the open channel. Also, we suggest design criteria for the geometry and dimension of LM interconnects to obtain structural stability without dewetting, based on the theoretical and computational background. We demonstrate a simple stretchable near-field communication (NFC) device including transferred micrometer-size light-emitting diodes (LEDs) with only 230 µm to the stretchable liquid metal PCB, without any soldering process. The device operates stably under repetitive stretching and releasing (∼50% uniaxial strain) due to the stable connection through the LM via between the upper and lower layers. Finally, we propose a concept for modular-type stretchable electronics, based on the cohesive liquid nature of LM. As a building block, the functional module can be easily removed from a mainframe, and replaced by another functional module, to suit user demand.

Enhanced Transport and Permeation of a Polymeric Nanocarrier across the Retina by Mixing with ATP upon Intravitreal Injection.

The outer part of the retina pigment epithelium (RPE) in the retina is the main site of neovascularization associated with retinal diseases. However, various obstacles interrupt the delivery of medicines across the RPE, mainly due to the well-developed tight junctions in the RPE. Currently, there is no practical formulation to overcome this issue. In this study, we demonstrated that simple mixing with adenosine tetraphosphate (ATP) has the potential to greatly enhance the transport and permeation of a polymeric nanocarrier across the retina via intravitreal administration. Chitosan-functionalized, pluronic-based nanocarrier (NC), which can deliver various biomolecules efficiently, was used as a polymeric nanocarrier. Mixing with ATP facilitated the diffusion of the nanocarrier in the vitreous humor by reducing the electrostatic interaction between NC and negatively charged glycosaminoglycans (GAGs) in the vitreous humor. Mixing with ATP also allowed the penetration of NC across the whole retina, and it resulted in a great increase (approximately nine times) in the transport of NC across the retina, as well as spreading it throughout the whole retina upon intravitreal administration in a mouse model. This enhanced permeation across the retina was specific to ATP but not to GTP, suggesting the possibility of P2Y receptor-mediated tight junction disruption by ATP.

Soft and elastic hollow microcapsules embedded silicone elastomer films with enhanced water uptake and permeability for mechanical stimuli responsive drug delivery applications.

Polydimethylsiloxane (PDMS) film with significantly enhanced water permeability and uptake was prepared by incorporating spherical elastic hollow microcapsules (eHMCs) in it. eHMCs were prepared through O/W/O emulsification method. Water permeability and uptake of the film increased significantly in proportion to the amount of embedded eHMCs while minimizing the changes in elastic characteristics and transparency of PDMS. The release rate of loaded water soluble model drug from the eHMC-embedded PDMS film could be controlled by the magnitude of uniaxial mechanical stimulus applied over the film and initial drug loading amount, with negligible release of drug from the film in the absence of external stimulation. Thus, these biocompatible and elastic composite PDMS films are potentially useful, including as an easily accessible and instantly effective way of controlling hydrophilic drug release using the mechanical stimulus as well as a soft elastomer with enhanced water uptake and permeability.

Pluronic-Based Nanocarrier Platform Encapsulating Two Enzymes for Cascade Reactions.

Enzyme immobilization is very important for diverse enzyme applications. Particularly, there is a growing need for coimmobilization of multiple enzymes for biosensing and synthetic applications. However, it is still challenging to coimmobilize two enzymes with desirable features, including high immobilization yield, retention of enzymatic activity, and low leaching. In this study, we demonstrated that a pluronic-based nanocarrier (PNC) can be an encapsulation platform for immobilization of various single enzymes. Since the PNC is temperature-sensitive, a simple temperature change from 4 to 37 °C led to a substantial size reduction and enzyme encapsulation. All six enzymes tested were encapsulated by the PNC in high yield (∼90%) with the retained enzymatic activity (>95%). The leaching of encapsulated enzymes was very minimal (<0.13% for 2 weeks). Then, we demonstrated that the PNC can efficiently coencapsulate two enzymes, formate dehydrogenase (FDH) and mannitol dehydrogenase (MDH), for a cascade reaction producing d-mannitol. Coencapsulation of FDH and MDH resulted in an over 10-fold increase in d-mannitol production compared to the free mix of FDH and MDH, likely due to the enhanced local concentrations of FDH and MDH inside the PNC.

Co-delivery of therapeutic protein and catalase-mimic nanoparticle using a biocompatible nanocarrier for enhanced therapeutic effect.

Therapeutic proteins are indispensable in the treatment of various human diseases. Despite the many benefits of therapeutic proteins, they also exhibit diverse side effects. Therefore, reducing unwanted side effects of therapeutic proteins as well as enhancing their therapeutic efficacy are very important in developing therapeutic proteins. Urate oxidase (UOX) is a therapeutic enzyme that catalyzes the conversion of uric acid (UA) into a soluble metabolite, and it is used clinically for the treatment of hyperuricemia. Since UA degradation by UOX generates H2O2 (a cytotoxic side product), UOX was co-delivered with catalase-mimic nanoparticles (AuNPs) using biocompatible pluronic-based nanocarriers (NCs) to effectively reduce H2O2-associated toxicity in cultured cells and to enhance UA degradation efficiency in vivo. Simple temperature-dependent size changes of NCs allowed co-encapsulation of both UOX and AuNPs at a high loading efficiency without compromising critical properties, resulting in efficient modulation of a mixing ratio of UOX and AuNPs encapsulated in NCs. Co-localizing UOX and AuNPs in the NCs led to enhanced UA degradation and H2O2 removal in vitro, leading to a great reduction in H2O2-associated cytotoxicity compared with UOX alone or a free mixture of UOX and AuNPs. Furthermore, we demonstrated that co-delivery of UOX and AuNPs using NCs significantly improves in vivo UA degradation compared to simple co-injection of free UOX and AuNPs. More broadly, we showed that biocompatible pluronic-based nanocarriers can be used to deliver a target therapeutic protein along with its toxicity-eliminating agent in order to reduce side effects and enhance efficacy.

Photosensitizer-mediated mitochondria-targeting nanosized drug carriers: Subcellular targeting, therapeutic, and imaging potentials.

Mitochondria-targeting drug carriers have considerable potential because of the presence of many molecular drug targets in the mitochondria and their pivotal roles in cellular viability, metabolism, maintenance, and death. To compare the mitochondria-targeting abilities of triphenylphosphonium (TPP) and pheophorbide a (PhA) in nanoparticles (NPs), this study prepared mitochondria-targeting NPs using mixtures of methoxy poly(ethylene glycol)-(SS-PhA)2 [mPEG-(SS-PhA)2 or PPA] and TPP-b-poly(ε-caprolactone)-b-TPP [TPP-b-PCL-b-TPP or TPCL], which were designated PPAn-TPCL4-n (0≤n≤4) NPs. With increasing TPCL content, the formed PPAn-TPCL4-n NPs decreased in size from 33nm to 18nm and increased in terms of positive zeta-potentials from -12mV to 33mV. Although the increased TPCL content caused some dark toxicity of the PPAn-TPCL4-n NPs due to the intrinsic positive character of TPCL, the NPs showed strong light-induced killing effects in tumor cells. In addition, the mitochondrial distribution of the PPAn-TPCL4-n NPs was analyzed and imaged by flow cytometry and confocal microscopy, respectively. Thus, the PhA-containing NPs specifically targeted the mitochondria, and light stimulation caused PhA-mediated therapeutic effects and imaging functions. Expanding the capabilities of these nanocarriers by incorporating other drugs should enable multiple potential applications (e.g., targeting, therapy, and imaging) for combination and synergistic treatments.