Click Chemistry-Enabled Conjugation Strategy for Producing Dibenzodiazepinone-Type Fluorescent Probes To Target M2 Acetylcholine Receptors.

The development of fluorescently labeled receptor-targeting compounds represents a powerful pharmacological tool to study and characterize ligand-receptor interactions. Despite significant advances in developing sub-type-specific antagonists for muscarinic acetylcholine receptors (mAChRs), reports on antagonists feasible for click chemistry are less common. Here, we designed and synthesized an antagonist suitable for probe attachment through click chemistry, namely, dibenzodiazepinone (DIBA)-alkyne, based on a previously reported DIBA scaffold with a high binding affinity to type-2 mAChR (M2R). To demonstrate the versatility of DIBA-alkyne as a building block for bioconjugates, we assembled DIBA-alkyne with Cyanine5 fluorophores (Cy5) and polyethylene glycol (PEG) biomolecules to obtain fluorescent DIBA antagonist (DIBA-Cy5) and fluorescent DIBA PEG derivatives. Flow cytometric analysis showed that DIBA-Cy5 possessed a high binding affinity to M2R (Kd = 1.80 nM), a two-order magnitude higher binding affinity than M1R. Fluorescent DIBA PEG derivatives maintained a potent binding to the M2R (Kd ≤ 4 nM), confirmed by confocal microscopic imaging. Additionally, DIBA-Cy5 can serve as a fluorescent ligand in the receptor-ligand competitive binding assay for other mAChR ligands, an attractive alternative to the traditional radioligand-based assay. The competitive binding mode between DIBA-Cy5 and orthosteric antagonist atropine/allosteric modulator LY2119620 indicated a dualsteric binding mode of the DIBA-type antagonist to M2R. Lastly, we demonstrated the direct staining of DIBA-Cy5 to M2R receptors in the sinoatrial node of a mouse heart. The adaptability of the clickable DIBA antagonist to a wide range of fluorophores and biomolecules can facilitate its use in various biomedical applications such as binding assays that screen compounds for M2R as the receptor target.

Magnetic-Responsive Surface-Enhanced Raman Scattering Platform with Tunable Hot Spot for Ultrasensitive Virus Nucleic Acid Detection.

Surface-enhanced Raman scattering (SERS)-based biosensors are promising tools for virus nucleic acid detection. However, it remains challenging for SERS-based biosensors using a sandwiching strategy to detect long-chain nucleic acids such as nucleocapsid (N) gene of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) because the extension of the coupling distance (CD) between the two tethered metallic nanostructures weakens electric field and SERS signals. Herein, we report a magnetic-responsive substrate consisting of heteoronanostructures that controls the CD for ultrasensitive and highly selective detection of the N gene of SARS-CoV-2. Significantly, our findings show that this platform reversibly shortens the CD and enhances SERS signals with a 10-fold increase in the detection limit from 1 fM to 100 aM, compared to those without magnetic modulation. The optical simulation that emulates the CD shortening process confirms the CD-dependent electric field strength and further supports the experimental results. Our study provides new insights into designing a stimuli-responsive SERS-based platform with tunable hot spots for long-chain nucleic acid detection.

Induced abundant oxygen vacancies in Sc2VO5-δ /g-C3N4 heterojunctions for enhanced photocatalytic degradation of levofloxacin.

Sc2VO5-δ /g-C3N4 heterojunctions (SVCs) with abundant oxygen vacancies (OVs) were synthesized by ultrasonic exfoliation combined with the thermal etching method. The structures, OVs and spatial separation of the photogenerated carriers were systematically characterized. The results manifested that the SVCs were successfully constructed via the strong interaction between g-C3N4 (CN) and Sc2VO5-δ (SV). The SVCs possessed a higher concentration of OVs than that of pristine CN and SV. The formation of the SVC heterostructures and the optimization of the OVs were the two major factors to accelerate the separation of the charge carriers and finally to improve the photocatalysis performance. The as-prepared 10%SVC (containing 10 wt% of SV) catalyst exhibited the highest OV concentration and the best photocatalytic performance. The levofloxacin (LVX) photodegradation activity showed a positive correlation with the OV concentration. The photocatalytic degradation efficiencies were 89.1, 98.8 and 99.0% on 10%SVC for LVX, methylene blue (MB) and rhodamine B (RhB), respectively. These photodegradation processes followed the pseudo first order kinetic equation. The apparent rate constant (k app) of LVX degradation on 10%SVC was 11.0 and 7.5 times that of CN and SV. The h+, ˙OH and ˙O2 - were the major reactive species in the photodegradation process.

Imaging in vivo acetylcholine release in the peripheral nervous system with a fluorescent nanosensor.

The ability to monitor the release of neurotransmitters during synaptic transmission would significantly impact the diagnosis and treatment of neurological diseases. Here, we present a DNA-based enzymatic nanosensor for quantitative detection of acetylcholine (ACh) in the peripheral nervous system of living mice. ACh nanosensors consist of DNA as a scaffold, acetylcholinesterase as a recognition component, pH-sensitive fluorophores as signal generators, and α-bungarotoxin as a targeting moiety. We demonstrate the utility of the nanosensors in the submandibular ganglia of living mice to sensitively detect ACh ranging from 0.228 to 358 µM. In addition, the sensor response upon electrical stimulation of the efferent nerve is dose dependent, reversible, and we observe a reduction of ∼76% in sensor signal upon pharmacological inhibition of ACh release. Equipped with an advanced imaging processing tool, we further spatially resolve ACh signal propagation on the tissue level. Our platform enables sensitive measurement and mapping of ACh transmission in the peripheral nervous system.

Specific Delivery of Oligonucleotides to the Cell Nucleus via Gentle Compression and Attachment of Polythymidine.

Nonviral delivery of nucleic acids to the cell nucleus typically requires chemical methods that do not guarantee specific delivery (e.g., transfection agent) or physical methods that may require extensive fabrication (e.g., microfluidics) or an elevated pressure (e.g., 105 Pa for microneedles). We report a method of delivering oligonucleotides to the nucleus with high specificity (relative to the cytosol) by synergistically combining chemical and physical approaches. Particularly, we demonstrate that DNA oligonucleotides appended with a polythymidine [poly(T)] segment (chemical) profusely accumulate inside the nucleus when the cells are under gentle compression imposed by the weight of a single glass coverslip (physical; ∼2.2 Pa). Our "compression-cum-poly(T)" delivery method is simple, can be generalizable to three "hard-to-transfect" cell types, and does not induce significant levels of cytotoxicity or long-term oxidative stress to the treated cells when provided the use of suitable compression times and oligonucleotide concentrations. In bEnd.3 endothelial cells, compression-aided intranuclear delivery of poly(T) is primarily mediated by importin ß and nucleoporin 62. Our method significantly enhances the intranuclear delivery of antisense oligonucleotides to bEnd.3 endothelioma cells and the inhibition of two target genes, including a reporter gene encoding the enhanced green fluorescent protein and an intranuclear lncRNA oncogene (metastasis-associated lung adenocarcinoma transcript 1), when compared with delivery without gentle compression or poly(T) attachment. Our data underscore the critical roles of pressure and nucleotide sequence on the intranuclear delivery of nucleic acids.

Promoting the Delivery of Nanoparticles to Atherosclerotic Plaques by DNA Coating.

Many nanoparticle-based carriers to atherosclerotic plaques contain peptides, lipoproteins, and sugars, yet the application of DNA-based nanostructures for targeting plaques remains infrequent. In this work, we demonstrate that DNA-coated superparamagnetic iron oxide nanoparticles (DNA-SPIONs), prepared by attaching DNA oligonucleotides to poly(ethylene glycol)-coated SPIONs (PEG-SPIONs), effectively accumulate in the macrophages of atherosclerotic plaques following an intravenous injection into apolipoprotein E knockout (ApoE-/-) mice. DNA-SPIONs enter RAW 264.7 macrophages faster and more abundantly than PEG-SPIONs. DNA-SPIONs mostly enter RAW 264.7 cells by engaging Class A scavenger receptors (SR-A) and lipid rafts and traffic inside the cell along the endolysosomal pathway. ABS-SPIONs, nanoparticles with a similarly polyanionic surface charge as DNA-SPIONs but bearing abasic oligonucleotides also effectively bind to SR-A and enter RAW 264.7 cells. Near-infrared fluorescence imaging reveals evident localization of DNA-SPIONs in the heart and aorta 30 min post-injection. Aortic iron content for DNA-SPIONs climbs to the peak (∼60% ID/g) 2 h post-injection (accompanied by profuse accumulation in the aortic root), but it takes 8 h for PEG-SPIONs to reach the peak aortic amount (∼44% ID/g). ABS-SPIONs do not appreciably accumulate in the aorta or aortic root, suggesting that the DNA coating (not the surface charge) dictates in vivo plaque accumulation. Flow cytometry analysis reveals more pronounced uptake of DNA-SPIONs by hepatic endothelial cells, splenic macrophages and dendritic cells, and aortic M2 macrophages (the cell type with the highest uptake in the aorta) than PEG-SPIONs. In summary, coating nanoparticles with DNA is an effective strategy of promoting their systemic delivery to atherosclerotic plaques.

Process and Results of Implementing Disease Management Program in Patients with First-time Ischemic Stroke.

BACKGROUND: This study aimed to examine the effect of disease management program (DMP) on the patients with first-time ischemic stroke (IS). METHODS: A DMP with 4 parts of performance indicators (PIs, including outpatient, emergency department, inpatient and follow-up treatment) was implemented in patients with stroke in 2 hospitals (Hospital T and R) in Shanghai China from 2007-2010. The effect of DMP on the outcome of IS patients was analyzed according to the criteria of the National Institute of Health Stroke Scale (NIHSS). Furthermore, the total effective rate of DMP, average length of stay, hospitalization cost, and cost-effectiveness ratio (CER) between DMP and non-DMP patients were calculated, followed by the cost-effectiveness analysis. RESULTS: The total effective rate of DMP (T: 69.9%; R: 76.6%) was significantly (P<0.05) higher than that of non-DMP (T: 60.8%; R: 62.7%) group in the same hospital. In addition, a significant (P<0.05) difference in effective rate was observed between DMP and non-DMP at the NIHSS score ≥ 7. Furthermore, the average length of stay and hospitalization cost of the patients in DMP group were significantly (P<0.05) lower than those in non-DMP group. A superior CER was also found in DMP group than non-DMP group. CONCLUSION: The implementation of DMP for IS can effectively improve the treatment outcome and reduce the average length of stay and hospitalization cost.

Promoting intracellular delivery of sub-25 nm nanoparticles via defined levels of compression.

Many investigations into the interactions between nanoparticles and mammalian cells entail the use of culture systems that do not account for the effect of extracellular mechanical cues, such as compression. In this work, we present an experimental set-up to systematically investigate the combined effects of nanoparticle size and compressive stress on the cellular uptake and intracellular localization of poly(ethylene glycol)-coated gold nanoparticles (Au-PEG NPs). Specifically, we employ an automated micromechanical system to apply defined levels of compressive strain to an agarose gel, which transmits defined amounts of unconfined, uniaxial compressive stress to a monolayer of C2C12 mouse myoblasts seeded underneath the gel without compromising cell viability. Notably, uptake of Au-PEG NPs smaller than 25 nm by compressed myoblasts is up to 5-fold higher than that by uncompressed cells. The optimal compressive stress for maximizing the cellular uptake of sub-25 nm NPs monotonically increases with NP size. With and without compression, the Au-PEG NPs enter C2C12 cells via energy-dependent uptake; they also enter compressed cells via clathrin-mediated endocytosis as the major pathway. Upon cellular entry, the Au-PEG NPs more readily reside in the late endosomes or lysosomes of compressed cells than uncompressed cells. Results from our experimental set-up yield mechanistic insights into the delivery of NPs to cell types under extracellular compression.

Recent Advances in Managing Atherosclerosis via Nanomedicine.

Atherosclerosis, driven by chronic inflammation of the arteries and lipid accumulation on the blood vessel wall, underpins many cardiovascular diseases with high mortality rates globally, such as stroke and ischemic heart disease. Engineered bio-nanomaterials are now under active investigation as carriers of therapeutic and/or imaging agents to atherosclerotic plaques. This Review summarizes the latest bio-nanomaterial-based strategies for managing atherosclerosis published over the past five years, a period marked by a rapid surge in preclinical applications of bio-nanomaterials for imaging and/or treating atherosclerosis. To start, the biomarkers exploited by emerging bio-nanomaterials for targeting various components of atherosclerotic plaques are outlined. In addition, recent efforts to rationally design and screen for bio-nanomaterials with the optimal physicochemical properties for targeting plaques are presented. Moreover, the latest preclinical applications of bio-nanomaterials as carriers of imaging, therapeutic, or theranostic agents to atherosclerotic plaques are discussed. Finally, a mechanistic understanding of the interactions between bio-nanomaterials and the plaque ("athero-nano" interactions) is suggested, the opportunities and challenges in the clinical translation of bio-nanomaterials for managing atherosclerosis are discussed, and recent clinical trials for atherosclerotic nanomedicines are introduced.

Polydopamine-based concentric nanoshells with programmable architectures and plasmonic properties.

Nanoshells, classically comprising gold as the metallic component and silica as the dielectric material, are important for fundamental studies in nanoplasmonics. They also empower a myriad of applications, including sensing, energy harvesting, and cancer therapy. Yet, laborious preparation precludes the development of next-generation nanoshells with structural complexity, compositional diversity, and tailorable plasmonic behaviors. This work presents an efficient approach to the bottom-up assembly of concentric nanoshells. By employing polydopamine as the dielectric material and exploiting its intrinsic adhesiveness and pH-tunable surface charge, the growth of each shell only takes 3-4 hours at room temperature. A series of polydopamine-based concentric nanoshells with programmable nanogap thickness, elemental composition (gold and silver), and geometrical configuration (number of layers) is prepared, followed by extensive structural characterization. Four of the silver-containing nanostructures are newly reported. Systematic investigations into the plasmonic properties of concentric nanoshells as a function of their structural parameters further reveal multiple Fano resonances and local-field "hot spots", infrequently reported plasmonic features for individual nanostructures fabricated using bottom-up wet chemistry. These results establish materials design rules for engineering complex plasmon-based systems originating from the integration of multiple plasmonic elements into defined locations within a compact nanostructure.

Mechanism for the Cellular Uptake of Targeted Gold Nanorods of Defined Aspect Ratios.

Biomedical applications of non-spherical nanoparticles such as photothermal therapy and molecular imaging require their efficient intracellular delivery, yet reported details on their interactions with the cell remain inconsistent. Here, the effects of nanoparticle geometry and receptor targeting on the cellular uptake and intracellular trafficking are systematically explored by using C166 (mouse endothelial) cells and gold nanoparticles of four different aspect ratios (ARs) from 1 to 7. When coated with poly(ethylene glycol) strands, the cellular uptake of untargeted nanoparticles monotonically decreases with AR. Next, gold nanoparticles are functionalized with DNA oligonucleotides to target Class A scavenger receptors expressed by C166 cells. Intriguingly, cellular uptake is maximized at a particular AR: shorter nanorods (AR = 2) enter C166 cells more than nanospheres (AR = 1) and longer nanorods (AR = 4 or 7). Strikingly, long targeted nanorods align to the cell membrane in a near-parallel manner followed by rotating by ≈90° to enter the cell via a caveolae-mediated pathway. Upon cellular entry, targeted nanorods of all ARs predominantly traffic to the late endosome without progressing to the lysosome. The studies yield important materials design rules for drug delivery carriers based on targeted, anisotropic nanoparticles.