Finely tuned ionizable lipid nanoparticles for CRISPR/Cas9 ribonucleoprotein delivery and gene editing.

Nonviral delivery of the CRISPR/Cas9 system provides great benefits for in vivo gene therapy due to the low risk of side effects. However, in vivo gene editing by delivering the Cas9 ribonucleoprotein (RNP) is challenging due to the poor delivery into target tissues and cells. Here, we introduce an effective delivery method for the CRISPR/Cas9 RNPs by finely tuning the formulation of ionizable lipid nanoparticles. The LNPs delivering CRISPR/Cas9 RNPs (CrLNPs) are demonstrated to induce gene editing with high efficiencies in various cancer cell lines in vitro. Furthermore, we show that CrLNPs can be delivered into tumor tissues with high efficiency, as well as induce significant gene editing in vivo. The current study presents an effective platform for nonviral delivery of the CRISPR/Cas9 system that can be applied as an in vivo gene editing therapeutic for treating various diseases such as cancer and genetic disorders.

Ratiometric Strategy Based on Intramolecular Internal Standard for Reproducible and Simultaneous Fingerprint Recognition of Diols via 19F NMR Spectroscopy.

19F NMR spectroscopy has been widely used as a convenient and noninvasive analytical technique for understanding complex natural phenomena at the atomic level. However, current NMR referencing techniques are most optimized for 1H NMR, which causes some limitations while referencing heteronuclear NMR. Despite its promising advantages, 19F NMR spectroscopy often exhibits large variations in experimental results and lacks consistency compared with 1H NMR. Herein, we propose a new strategy to improve the consistency of 19F NMR referencing using an internal standard method. As a proof-of-concept, BA-Py-TFP was applied as a sensor for diols via 19F NMR spectroscopy. This strategy proved to be a robust and reproducible referencing method with acceptable deviation (ΔδF = 43-58 ppb) across diverse NMR spectrometers at different institutions. In particular, this new strategy allows reliable fingerprint recognition for analytes and enables qualitative and quantitative analyses of mixtures of multiple analytes simultaneously. The high recovery rates for d-glucose in the human serum matrix suggest its potential suitability for a diverse range of applications, such as in diabetes-related diagnostics.

Inhalation Delivery of Interferon-λ-Loaded Pulmonary Surfactant Nanoparticles Induces Rapid Antiviral Immune Responses in the Lung.

The interferon-λ (IFN-λ)-regulated innate immune responses in the airway expand our understanding toward antiviral strategies against influenza A virus (IAV). The application of IFN-λ as mucosal antiviral therapeutic is still challenging, and advanced research will be necessary to achieve more efficient delivery of recombinant IFN-λs to the damaged respiratory mucosa. In this study, we examine the capability of IFN-λ to stimulate the innate immune response, promoting the swift elimination of IAV in the lungs. Additionally, we develop IFN-λ-loaded nanoparticles incorporated into pulmonary surfactant for inhalation therapy aimed at treating lung infections caused by IAV. We found that inhaled delivery of IFNλ-PSNPs significantly restricted IAV replication in the lungs from 3 days after infection (dpi), and IAV-caused lung histopathologic findings were completely improved in response to IFNλ-PSNPs. More significant and rapid attenuation of viral RNA was observed in the lung of mice with inhaled delivery of IFNλ-PSNPs compared to mice with recombinant IFN-λs. Inhalation treatment of IFNλ-PSNPs to IAV-infected mice can result in the increase of monocyte frequency in concert with restoration of T and B cells composition. Furthermore, the transcriptional profiles of monocytes shifted toward heightened IFN responses following IFNλ-PSNP treatment. These results imply that IFN-λ could serve as a robust inducer of innate immunity in the lungs against IAV infection, and inhalation of IFN-λs encapsulated in PSNPs effectively resolves lung infections caused by IAV through rapid viral clearance. PSNPs facilitated improved delivery of IFN-λs to the lungs, triggering potent antiviral immune responses upon IAV infection onset.

A pH-responsive sensor based on intramolecular internal standard for reproducible detection of strong acids and bases via 19F NMR spectroscopy.

Numerous methods, including pH meters and optical sensors, have been developed for the detection of pH, which is an important indicator in various fields. However, those methods are susceptible to errors in strongly acidic and basic ranges and inaccurate pH measurement due to sample turbidity, hindering their application such as photographic industries and wastewater treatment facilities. Eco-friendly and non-invasive 19F NMR spectroscopy is a promising technique for measurement of strong acids and bases owing to its high sensitivity and little interference; nevertheless, inconsistencies in reproducibility impede its widespread adoption. Herein, we developed a19F NMR-based pH sensor by introducing an intramolecular internal standard strategy into a pH-responsive fluorinated material. Based on the acceptable deviation (ΔδF = 17-19 ppb) in the evaluation of the internal standard signal, this pH-sensing platform enabled reproducible pH measurements in strongly acidic and basic environments. Moreover, its 19F NMR response showed reversibility and high stability to potential interfering factors, and the low absolute difference (0.026-0.086 in pH) for real samples such as diet Coke suggests its potential suitability for various acidic beverages.

A fluorous-tag-assisted fluorescent probe for simple and selective detection of hydrogen sulfide: application for turbid dyeing solutions.

Accurate hydrogen sulfide (H2S) detection has attracted much attention because its toxicity may affect aquatic environments and human health. However, recognizing H2S levels by conventional fluorescent probes in turbid wastewater has been challenging because the opaque environment interferes with their photophysical properties. To overcome this limitation, a fluorous-tagging strategy can be used for the development of fluorescent sensors to detect H2S in turbid solutions. The use of fluorescent probe assisted with fluorous-tag allowed for easy isolation of the probe using polytetrafluoroethylene (PTFE) material, while disturbing species were eliminated through a simple aqueous wash. This approach enabled the fluorescent probe to effectively quantify H2S, even in opaque solutions containing organic dyes that could interfere with fluorescence emission.