A splice-switch oligonucleotide loaded self-cleavable DNA nanogel.

A self-cleavable DNA nanogel loaded with splice-switch oligonucleotide (SSO) has been developed. Under acidic conditions (pH 5.0), cleavage of the acid-labile chemical linker and generation of the i-motif structure led to the disintegration of the DNA nanogel and efficient release of SSO in its unaltered native state.

Self-Promoted Hydroxyl Radical Releasing Magnetic Zn@Fe Particles.

Semiconductor photocatalysts, such as TiO2 and ZnO, have garnered significant attention for their ability to generate hydroxyl radicals, offering various practical applications. However, the reliance on UV light to facilitate electron-hole separation for hydroxyl radical production poses limitations. In this study, a novel approach is presented utilizing Zn@Fe core/shell particles capable of generating hydroxyl radicals without external energy input. The generation process involves electron donation from Zn to O2, resulting in the formation of radical species .O2 -/H2O2, followed by Fe-catalyzed conversion of H2O2 into hydroxyl radicals through the Fenton reaction. The release of .OH imparts good antimicrobial and antiviral properties to the Zn@Fe particles. Furthermore, the inclusion of Fe confers magnetic properties to the material. This dual functionality holds promise for diverse potential applications for the Zn@Fe particles.

Redox Active Zn@MOFs as Spontaneous Reactive Oxygen Species Releasing Antimicrobials.

The rapid development of antimicrobial resistance (AMR) among infectious pathogens has become a major threat and challenge in healthcare systems globally. A strategy distinct from minimizing the overuse of antimicrobials involves the development of novel antimicrobials with a mode of action that prevents the development of AMR microbial strains. Reactive oxygen species (ROS) are formed as a natural byproduct of the cellular aerobic metabolism. However, it becomes pathological when ROS is produced at excessive levels. Exploiting this phenomenon, research on redox-active bactericides has been demonstrated to be beneficial. Materials that release ROS via photodynamic, thermodynamic, and photocatalytic interventions have been developed as nanomedicines and are used in various applications. However, these materials require external stimuli for ROS release to be effective as biocides. In this paper, we report novel zinc-based metal organic framework (Zn@MOF) particles that promote the spontaneous release of active ROS species. The synthesized Zn@MOF spontaneously releases superoxide anions and hydrogen peroxide, exhibiting a potent antimicrobial efficacy against various microbes. Zn@MOF-incorporated plastic films and coatings show excellent, long-lasting antimicrobial potency even under continuous microbial challenge and an aging process. These disinfecting surfaces maintain their antimicrobial properties even after 500× surface wipes. Zn@MOF is also biocompatible and safe on the skin, illustrating its broad potential applications in medical technology and consumer care applications.

A Self-Immolative DNA Nanogel Vaccine toward Cancer Immunotherapy.

The development of precisely engineered vehicles for intracellular delivery and the controlled release of payloads remains a challenge. DNA-based nanomaterials offer a promising solution based on the A-T-G-C alphabet-dictated predictable assembly and high programmability. Herein, we present a self-immolative DNA nanogel vaccine, which can be tracelessly released in the intracellular compartments and activate the immune response. Three building blocks with cytosine-rich overhang domains are designed to self-assemble into a DNA nanogel framework with a controlled size. Two oligo agonists and one antigen peptide are conjugated to the building blocks via an acid-labile chemical linker. Upon internalization into acidic endosomes, the formation of i-motif configurations leads to dissociation of the DNA nanogel vaccine. The acid-labile chemical linker is cleaved, releasing the agonists and antigen in their traceless original form to activate antigen-presenting cells and an immune response. This study presents a novel strategy for constructing delivery platforms for intracellularly stimuli-triggered traceless release of therapeutics.

Neural cell membrane-coated DNA nanogels as a potential target-specific drug delivery tool for the central nervous system.

A major bottleneck in drug/gene delivery to enhance tissue regeneration after injuries is to achieve targeted delivery to the cells of interest. Unfortunately, we have not been able to attain effective targeted drug delivery in tissues due to the lack of efficient delivery platforms. Since specific cell-cell interactions exist to impart the unique structure and functionality of tissues and organs, we hypothesize that such specific cellular interactions may also be harnessed for drug delivery applications in the form of cell membrane coatings. Here, we employed neural cell-derived membrane coating technique on DNA nanogels to improve target specificity. The efficacy of neural cell membrane-coated DNA nanogels (NCM-nanogels) was demonstrated by using four types of cell membranes derived from the central nervous system (CNS), namely, astrocytes, microglia, cortical neurons, and oligodendrocyte progenitor cells (OPCs). A successful coating of NCMs over DNA nanogels was confirmed by dynamic light scattering, zeta potential measurements and transmission electron microscopy. Subsequently, an overall improvement in cellular uptake of NCM-nanogels over uncoated DNA nanogels (p < 0.005) was seen. Additionally, we observed a selective uptake of OPC membrane-coated DNA nanogels (NCM-O mem) by oligodendrocytes over other cell types both in vitro and in vivo. Our quantitative polymerase chain reaction (qPCR) results also showed selective and effective gene knockdown capacity of NCM-O mem for OPC transfection. The findings in this work may be beneficial for future drug delivery applications targeted at the CNS.

Ultrasmall Coinage Metal Nanoclusters as Promising Theranostic Probes for Biomedical Applications.

Ultrasmall coinage metal nanoclusters (NCs, <3 nm) have emerged as a novel class of theranostic probes due to their atomically precise size and engineered physicochemical properties. The rapid advances in the design and applications of metal NC-based theranostic probes are made possible by the atomic-level engineering of metal NCs. This Perspective article examines (i) how the functions of metal NCs are engineered for theranostic applications, (ii) how a metal NC-based theranostic probe is designed and how its physicochemical properties affect the theranostic performance, and (iii) how metal NCs are used to diagnose and treat various diseases. We first summarize the tailored properties of metal NCs for theranostic applications in terms of biocompatibility and tumor targeting. We focus our discussion on the theranostic applications of metal NCs in bioimaging-directed disease diagnosis, photoinduced disease therapy, nanomedicine, drug delivery, and optical urinalysis. Lastly, an outlook on the challenges and opportunities in the future development of metal NCs for theranostic applications is provided.

Toxicity and efficacy of green tea catechin derivative-based micellar nanocomplexes for anticancer protein delivery.

Toxicity towards non-tumor cells during anticancer therapy can be reduced by using nanoscale systems for anticancer drug delivery. Usually only the loaded drug has anticancer activity. Recently, micellar nanocomplexes (MNCs) comprising green tea catechin derivatives for the delivery of the anticancer proteins, such as Herceptin, have been developed. Herceptin as well as the MNCs without the drug were effective against HER2/neu-overexpressing human tumor cells and had synergistic anticancer effects in vitro and in vivo. It remained unclear which kinds of negative effects the MNCs had on tumor cells exactly, and which of their components mediated them. Also, it was unclear if MNC has any toxicity effects on the normal cells of vital human organ systems. Herein we examined the effects of Herceptin-MNCs and their individual components on human breast cancer cells and on normal primary human endothelial and kidney proximal tubular cells. We applied a novel in vitro model that predicts nephrotoxicity in humans with high accuracy, as well as high-content screening and microfluidic mono- and co-culture models to thoroughly address effects on various cell types. The results showed that MNCs alone were profoundly toxic for breast cancer cells, and induced apoptosis regardless of HER2/neu expression levels. Apoptosis was induced by both green tea catechin derivatives contained within MNCs. In contrast, MNCs were not toxic for normal human cells, and the probability was low that MNCs would be nephrotoxic in humans. Together, the results supported the hypothesis that green tea catechin derivative-based MNCs could improve efficacy and safety of therapies with anticancer proteins.

Enhancing Protein Adsorption for Improved Lateral Flow Assay on Cellulose Paper by Depleting Inert Additive Films Using Reactive Plasma.

Paper-based platforms are ideal for on-site surveillance of infectious diseases in low-resource settings due to their simplicity, self-containment, and low cost. The two most popular materials used in paper-based platforms are nitrocellulose and cellulose. The nitrocellulose membrane has a high protein binding affinity, but its high price is an issue. Cellulose paper is inexpensive and allows intricate fluidic control for more sophisticated biochemical reactions, but it has a low protein binding affinity. By examining the microstructure of cellulose paper, we discover that cellulose fibers in the paper matrix are covered by thin films, which possibly result from the additives used in the paper-making process. Our finding suggests that the thin films are inert to protein adsorption. By selectively depleting the inert films with reactive plasma, we were able to enhance the protein adsorption to the cellulose paper and improve the performance of lateral flow assays. The performance of certain lateral flow assays on the plasma-treated cellulose paper is equivalent to or better than that on the nitrocellulose membrane. This leads us to believe that cellulose paper with a microstructure exclusively designed for protein binding, either by refined paper manufacturing process or by post-manufacture modification such as the plasma treatment presented herein, can potentially replace nitrocellulose as a less expensive paper substrate for point-of-care rapid test kits.

A Strong Acid-Induced DNA Hydrogel Based on pH-Reconfigurable A-Motif Duplex.

Under a pH value lower than the pKa of adenine (3.5), adenine-rich sequences (A-strand) form a unique parallel A-motif duplex due to the protonation of A-strand. At a pH above 3.5, deprotonation of adenines leads to the dissolution of A-motif duplex to A-strand single coil. This pH-reconfigurable A-motif duplex has been developed as a novel pH-responsive DNA hydrogel, termed A-hydrogel. The hydrogel state is achieved at pH 1.2 by the A-motif duplex bridging units, which are cross-linked by both reverse Hoogsteen interaction and electrostatic attraction. Hydrogel-to-solution transition is triggered by pH 4.3 due to the deprotonation-induced separation of A-motif duplex. The A-hydrogel system undergoes reversible hydrogel-solution transitions by subjecting the system to cyclic pH shifts between 1.2 and 4.3. An anti-inflammatory medicine, sulfasalazine (SSZ), which intercalates into A-motif duplex, is loaded into A-hydrogel. Its pH-controlled release from A-hydrogel is successfully demonstrated. The strong acid-induced A-hydrogel may fill the gap that other mild acid-responsive DNA hydrogels cannot do, such as protection of orally delivered drug in hostile stomach environment against strong acid (pH ~ 1.2) and digestive enzymes.

Directly interface microreaction tube and test strip for the detection of Salmonella in food with combined isothermal amplification and lateral flow assay.

Salmonella is a common foodborne bacterial pathogen that leads to severe illness or even death. The recommended method for Salmonella detection relies on the culture and has a long turnaround time of up to ∼1 week. In this study, we have developed a molecular assay that detects Salmonella in food by targeting the invA gene using loop-mediated isothermal amplification (LAMP) and lateral flow assay (LFA). The assay shortens the turnaround time considerably to ∼1 day, including pre-enrichment and DNA extraction. More importantly, we have developed a simple device that directly couples the LAMP microreaction tube to the LFA test strip, eliminating the need for manual liquid handling. The unique design greatly simplifies the device operation, rendering the device suitable for point-of-sampling applications. The method could be used as an initial screening tool, but would require confirmation testing to act on the result. Using the proposed device, we have successfully detected samples containing as little as 10 fg (3-4 copies) Salmonella genomic DNA. The detection limit of the device in terms of bacteria load is 4 colony forming units (CFUs). We have also correctly identified Salmonella at as little as 4-6 CFU bacteria per 25 g of food homogenate.

CRISPR-based systems for sensitive and rapid on-site COVID-19 diagnostics.

The COVID-19 pandemic has strained healthcare systems. Sensitive, specific, and timely COVID-19 diagnosis is crucial for effective medical intervention and transmission control. RT-PCR is the most sensitive/specific, but requires costly equipment and trained personnel in centralized laboratories, which are inaccessible to resource-limited areas. Antigen rapid tests enable point-of-care (POC) detection but are significantly less sensitive/specific. CRISPR-Cas systems are compatible with isothermal amplification and dipstick readout, enabling sensitive/specific on-site testing. However, improvements in sensitivity and workflow complexity are needed to spur clinical adoption. We outline the mechanisms/strategies of major CRISPR-Cas systems, evaluate their on-site diagnostic capabilities, and discuss future research directions.

Acid-Resistant and Physiological pH-Responsive DNA Hydrogel Composed of A-Motif and i-Motif toward Oral Insulin Delivery.

An acid-resistant DNA hydrogel that is stable in an extremely acidic environment with pH as low as 1.2 has not been reported before, largely due to the instability of DNA-hybridized structures. To achieve this, adenine (A)-rich and cytosine (C)-rich oligonucleotides are rationally designed and integrated to form copolymers with acrylamide monomers via free-radical polymerization. In an acidic environment (pH 1.2-6.0), the generated copolymers form a hydrogel state, which is cross-linked by parallel A-motif duplex configurations (pH 1.2-3.0) and quadruplex i-motif structures (pH 4.0-6.0) due to the protonation of A and C bases, respectively. Specifically, the protonated A-rich sequences under pH 1.2-3.0 form a stable parallel A-motif duplex cross-linking unit through reverse Hoogsteen interaction and electrostatic attraction. Hemi-protonated C bases under mildly acidic pH (4.0-6.0) form quadruplex i-motif cross-linking configuration via Hoogsteen interaction. Under physiological pH, both A and C bases deprotonated, resulting in the separation of A-motif and i-motif to A-rich and C-rich single strands, respectively, and thereby the dissociation of the DNA hydrogel into the solution state. The acid-resistant and physiological pH-responsive DNA hydrogel was further developed for oral drug delivery to the hostile acidic environment in the stomach (pH 1.2), duodenum (pH 5.0), and small intestine (pH 7.2), where the drug would be released and absorbed. As a proof of concept, insulin was encapsulated in the DNA hydrogel and orally administered to diabetic rats. In vitro and in vivo studies demonstrated the potential usage of the DNA hydrogel for oral drug delivery.

Engineered NS1 for Sensitive, Specific Zika Virus Diagnosis from Patient Serology.

Dengue virus (DENV) and Zika virus (ZIKV) belong to the Flaviviridae family of viruses spread by Aedes aegypti mosquitoes in tropical and subtropical areas. Accurate diagnostic tests to differentiate the 2 infections are necessary for patient management and disease control. Using characterized ZIKV and DENV patient plasma in a blind manner, we validated an ELISA and a rapid immunochromatographic test for ZIKV detection. We engineered the ZIKV nonstructural protein 1 (NS1) for sensitive serologic detection with low cross reactivity against dengue and developed monoclonal antibodies specific for the ZIKV NS1 antigen. As expected, the serologic assays performed better with convalescent than acute plasma samples; the sensitivity ranged from 71% to 88%, depending on the performance of individual tests (IgM/IgG/NS1). Although serologic tests were generally less sensitive with acute samples, our ZIKV NS1 antibodies were able to complement the serologic tests to achieve greater sensitivity for detecting early infections.

Evaluation of the ZnO Nanopillar Surface for Disinfection Applications.

Much attention has been devoted to the synthesis and antimicrobial studies of nanopatterned surfaces. However, factors contributing to their potential and eventual application, such as large-scale synthesis, material durability, and biocompatibility, are often neglected in such studies. In this paper, the ZnO nanopillar surface is found to be amenable to synthesis in large forms and stable upon exposure to highly accelerated lifetime tests (HALT) without any detrimental effect on its antimicrobial activity. Additionally, the material is effective against clinically isolated pathogens and biocompatible in vivo. These findings illustrate the broad applicability of ZnO nanopillar surfaces in the common equipment used in health-care and consumer industries.

Assessing Toxicity with Human Cell-Based In Vitro Methods.

In toxicology, there is a strong push towards replacing animal experiments with alternative methods, which include cell-based in vitro methods for the assessment of adverse health effects in humans. High-throughput methods are of central interest due to the large and steadily growing numbers of compounds that require assessment. Tremendous progress has been made during the last decade in developing and applying such methods. Innovative technologies for addressing complex biological interactions include induced pluripotent stem cell- and organoid-based approaches, organotypic coculture systems, and microfluidic 'multiorgan' chips. Combining in vitro methods with bioinformatics and in silico modeling generates new powerful tools for toxicity assessment, and the rapid progress in the field is expected to continue.

Miscible-Solvent-Assisted Two-Phase Synthesis of Monolayer-Ligand-Protected Metal Nanoclusters with Various Sizes.

Effective yet versatile synthetic strategies for size-tunable metal nanoclusters (NCs) are scarce. This has hampered the development of this unique class of nanomaterials. Here, a general protocol is reported for the synthesis of high-quality metal NCs protected by a variety of organic ligands (e.g., selenolate, thiolate, and phosphine) based on a miscible-solvent-assisted phase transfer between water and organic solution. This method is demonstrated to be facile, rapid (≤3 h), scalable (gram-scale), and versatile. The size of the selenolated and thiolated Au NCs can be tuned from Au10 to Au61 by simply varying the miscible solvent in proportions and types. The advantages of this method, such as quick phase separation and no need for purification treatment, enable real-time monitoring of metal NC growth within the NaBH4 reduction system. The results show that the size of Au NCs gradually increases with increasing valence electron count by a stepwise 2x e- hopping mechanism (x = 0-5), i.e., 0 e- → 2 e- → 4 e- → 8 e- → 18 e- → 22 e- → 32 e- .

Carrier-Enhanced Anticancer Efficacy of Sunitinib-Loaded Green Tea-Based Micellar Nanocomplex beyond Tumor-Targeted Delivery.

Although a few nanomedicines have been approved for clinical use in cancer treatment, that recognizes improved patient safety through targeted delivery, their improved efficacy over conventional drugs has remained marginal. One of the typical drawbacks of nanocarriers for cancer therapy is a low drug-loading capacity that leads to insufficient efficacy and requires an increase in dosage and/or frequency of administration, which in turn increases carrier toxicity. In contrast, elevating drug-loading would cause the risk of nanocarrier instability, resulting in low efficacy and off-target toxicity. This intractable drug-to-carrier ratio has imposed constraints on the design and development of nanocarriers. However, if the nanocarrier has intrinsic therapeutic effects, the efficacy would be synergistically augmented with less concern for the drug-to-carrier ratio. Sunitinib-loaded micellar nanocomplex (SU-MNC) was formed using poly(ethylene glycol)-conjugated epigallocatechin-3-O-gallate (PEG-EGCG) as such a carrier. SU-MNC specifically inhibited the vascular endothelial growth factor-induced proliferation of endothelial cells, exhibiting minimal cytotoxicity to normal renal cells. SU-MNC showed enhanced anticancer effects and less toxicity than SU administered orally/intravenously on human renal cell carcinoma-xenografted mice, demonstrating more efficient effects on anti-angiogenesis, apoptosis induction, and proliferation inhibition against tumors. In comparison, a conventional nanocarrier, SU-loaded polymeric micelle (SU-PM) comprised of PEG-b-poly(lactic acid) (PEG-PLA) copolymer, only reduced toxicity with no elevated efficacy, despite comparable drug-loading and tumor-targeting efficiency to SU-MNC. Improved efficacy of SU-MNC was ascribed to the carrier-drug synergies with the high-performance carrier of PEG-EGCG besides tumor-targeted delivery.

pH-Degradable imidazolium oligomers as antimicrobial materials with tuneable loss of activity.

Antimicrobial resistance (AMR) has become a global public health threat. One of the major causes of AMR development is the accumulation of low levels of antimicrobials in the environment. To tackle this problem, novel antimicrobial agents that do not leave active residues after treatment are needed. In this study, a strategy for synthesizing a series of main-chain imidazolium oligomers that incorporate carbonate, hemiaminal, ester and urea functional groups to serve as degradable linkers is presented. These oligomers exhibit excellent microbicidal activity and kill E. coli at low concentrations in a short time (99% killing efficiency in 2 min). Moreover, the oligomers are self-degradable and biocompatible. The degradation of these oligomers is studied in buffered solutions with different pH. Under basic conditions (pH = 8), carbonate-linked and ester-linked oligomers degrade to inactive and less toxic small molecules within weeks, making it less likely for these oligomers to induce antimicrobial resistance as compared to traditional antibiotics. The application of these oligomers for the in vivo treatment of S. aureus infected wounds is demonstrated in a mouse model. Notably, the oligomers demonstrate antibacterial efficacy and accelerated wound healing comparable to vancomycin, a first-line antibiotic for the treatment of complicated skin infections.

Molecular Swings as Highly Active Ion Transporters.

Ions are transported across membrane mostly via carrier or channel mechanisms. Herein, a unique class of molecular-machine-inspired membrane transporters, termed molecular swings is reported that utilize a previously unexplored swing mechanism for promoting ion transport in a highly efficient manner. In particular, the molecular swing, which carries a 15-crown-5 unit as the ion-binding and transporting unit, exhibits extremely high ion-transport activities with EC50 values of 46 nm (a channel:lipid molar ratio of 1:4800 or 0.021 mol % relative to lipid) and 110 nm for K+ and Na+ ions, respectively. Remarkably, such ion transport activities remain high in a cholesterol-rich environment, with EC50 values of 130 (0.045 mol % relative to lipid/cholesterol) and 326 nm for K+ and Na+ ions, respectively.

Sieve-through vertical flow platform for efficient liquid exchange in particle-based assays.

Particle-based assays are widely used in many biomedical applications. However, the performance of particle-based systems is often compromised by the carry-over contamination caused by the residual reagents during the liquid-exchange process. We have developed a sieve-through platform that utilizes a porous membrane to sieve out the particles, and an absorbent pad to remove the waste liquid by capillary force. The porous membrane is able to contain the liquid in the reaction chamber, and allows the waste liquid to flow through when it is brought into contact with the absorbent pad. The sieve-through platform is able to effectively remove the waste liquid, thereby achieving a more efficient liquid exchange as compared to the conventional process, and minimizing the carry-over contamination. In this study, we have determined the factors that affect the flow characteristics through the porous membrane on the sieve-through platform. We have shown that the sieve-through platform effectively reduces the carry-over contamination. In addition, we have shown particle-based ELISA on the sieve-through platform for the analysis of proteins and cells. We have further demonstrated the potential of the sieve-through platform for high-throughput analysis by presenting a sieve-array, which allows concurrent analysis of multiple samples in parallel. The sieve-through platform can significantly improve the performance of particle-based systems.