Fluorescent Features and Applicable Biosensing of a Core–Shell Ag Nanocluster Shielded by a DNA Tetrahedral Nanocage

The DNA frame structure as a natural shell to stably shield the sequence-templated Ag nanocluster core (csAgNC) is intriguing yet challenging for applicable fluorescence biosensing, for which the elaborate programming of a cluster scaffold inside a DNA-based cage to guide csAgNC nucleation might be crucial. Herein, we report the first design of a symmetric tetrahedral DNA nanocage (TDC) that was self-assembled in a one-pot process using a C-rich csAgNC template strand and four single strands. Inside the as-constructed soft TDC architecture, the template sequence was logically bridged from one side to another, not in the same face, thereby guiding the in situ synthesis of emissive csAgNC. Because of the strong electron-repulsive capability of the negatively charged TDC, the as-formed csAgNC displayed significantly improved fluorescence stability and superb spectral behavior. By incorporating the recognizable modules of targeted microRNAs (miRNAs) in one vertex of the TDC, an updated TDC (uTDC) biosensing platform was established via the photoinduced electron transfer effect between the emissive csAgNC reporter and hemin/G-quadruplex (hG4) conjugate. Because of the target-interrupted csAgNC switching in three states with the spatial proximity and separation to hG4, an “on–off–on” fluorescing signal response was executed, thus achieving a wide linear range to miRNAs and a limit of detection down to picomoles. Without complicated chemical modifications, this simpler and more cost-effective strategy offered accurate cell imaging of miRNAs, further suggesting possible therapeutic applications

Programming Intracellular Clustering of Spiky Nanoparticles via Liposome Encapsulation

The intracellular clustering of anisotropic nanoparticles is crucial to the improvement of the localized surface plasmon resonance (LSPR) for phototherapy applications. Herein, we programmed the intracellular clustering process of spiky nanoparticles (SNPs) by encapsulating them into an anionic liposome via a frame-guided self-assembly approach. The liposome-encapsulated SNPs (lipo-SNPs) exhibited distinct and enhanced lysosome-triggered aggregation behavior while maintaining excellent monodispersity, even in acidic or protein-rich environments. We explored the enhancement of the photothermal therapy performance for SNPs as a proof of concept. The photothermal conversion efficiency of lipo-SNPs clusters significantly increased 15 times compared to that of single lipo-SNPs. Upon accumulation in lysosomes with a 2.4-fold increase in clustering, lipo-SNPs resulted in an increase in cell-killing efficiency to 45% from 12% at 24 μg/mL. These findings indicated that liposome encapsulation provides a promising approach to programing nanoparticle clustering at the target site, which facilitates advances in the development of smart nanomedicine with programmable enhancement in LSPR

Programming Intracellular Clustering of Spiky Nanoparticles via Liposome Encapsulation

The intracellular clustering of anisotropic nanoparticles is crucial to the improvement of the localized surface plasmon resonance (LSPR) for phototherapy applications. Herein, we programmed the intracellular clustering process of spiky nanoparticles (SNPs) by encapsulating them into an anionic liposome via a frame-guided self-assembly approach. The liposome-encapsulated SNPs (lipo-SNPs) exhibited distinct and enhanced lysosome-triggered aggregation behavior while maintaining excellent monodispersity, even in acidic or protein-rich environments. We explored the enhancement of the photothermal therapy performance for SNPs as a proof of concept. The photothermal conversion efficiency of lipo-SNPs clusters significantly increased 15 times compared to that of single lipo-SNPs. Upon accumulation in lysosomes with a 2.4-fold increase in clustering, lipo-SNPs resulted in an increase in cell-killing efficiency to 45% from 12% at 24 μg/mL. These findings indicated that liposome encapsulation provides a promising approach to programing nanoparticle clustering at the target site, which facilitates advances in the development of smart nanomedicine with programmable enhancement in LSPR

Riociguat reduces hyperoxia-induced PH.

<p>(<b>A</b>) Right ventricular systolic pressure (RVSP) was significantly increased in the hyperoxia + placebo treated group as compared with normoxia group. Riociguat administration significantly decreased RVSP during hyperoxia. ***<i>P</i> < 0.001 compared with normoxia; ⁺<i>P</i> < 0.05 compared with hyperoxia + placebo (n = 6/group). (<b>B</b>) Right ventricular hypertrophy (RVH), also known as the Fulton’s index, was determined by the weight ratio of right ventricle (RV) to left ventricle + septum (LV + S). Hyperoxia exposed animals in the presence of placebo showed significant RVH as compared with normoxia group. Administration of riociguat decreased RVH in hyperoxia exposed lungs. ***<i>P</i> < 0.001 compared with normoxia; ⁺⁺<i>P</i> < 0.01 compared with hyperoxia + placebo (n = 6/group).</p

Riociguat prevents hyperoxia-impaired alveolarization.

<p>(<b>A</b>) H & E stained lung histology. Hyperoxia exposure in the presence of placebo decreased radial alveolar count (RAC) (<b>B</b>) and increased mean linear intercept (MLI) (<b>C</b>) as compared with normoxia. Administration of riociguat increased RAC and decreased MLI during hyperoxia. ***<i>P</i> < 0.001 compared with normoxia; ⁺⁺⁺<i>P</i> < 0.001 compared with hyperoxia + placebo (n = 6/group). Scale bar: 100 μm.</p

Riociguat increases lung tissue cGMP levels.

<p>Riociguat significantly increased cGMP concentration in hyperoxia-exposed rats as compared with placebo treated hyperoxic rats. *<i>P</i> < 0.05 compared with hyperoxia + placebo (n = 4/group).</p

Riociguat does not affect femur growth and structure.

<p>(<b>A</b>) Bone length. (<b>B</b>) Trabecular thickness (Tb.Th). (<b>C</b>) Trabecular number (Tb.N). (<b>D</b>) Bone volume fraction (BV/TV). (<b>E</b>) Structural model index (SMI). (<b>F</b>) Representative micro-CT images. n = 5/group.</p

Riociguat decreases hyperoxia-induced vascular remodeling.

<p>(<b>A</b>, <b>C</b>) Double immunofluorescence staining for vWF (green signal) and α-SMA (red signal) plus DAPI nuclear stain (blue signal). (<b>B</b>) Hyperoxia exposure in the presence of placebo increased muscularization of peripheral pulmonary vessels (<50 μm in diameter) as compared with normoxia group (red arrow). Administration of riociguat decreased muscularized vessels in hyperoxia exposed lungs. (<b>D</b>) Hyperoxia increased medial wall thickness (MWT) in presence of placebo as compared with normoxia group. Riociguat administration significantly decreased MWT in hyperoxia group. ***<i>P</i> < 0.001 compared with normoxia; ⁺⁺⁺<i>P</i> < 0.001 compared with hyperoxia + placebo (n = 6/group). Scale bar: 50 μm. (<b>E</b>) Double immunofluorescence staining with Ki67 (red arrow) and α-SMA (green signal) plus DAPI nuclear staining (blue signal). (<b>F</b>) Hyperoxia exposure in the presence of placebo increased vascular proliferation as compared with normoxia group. Administration of riociguat decreased vascular proliferation. ***<i>P</i> < 0.001 compared with normoxia; ⁺⁺⁺<i>P</i> < 0.001 compared with hyperoxia + placebo (n = 6/group). (<b>G</b>) CTGF gene expression was up-regulated by hyperoxia and it was down-regulated by riociguat. *<i>P</i> < 0.05 compared with normoxia; ⁺⁺⁺<i>P</i> < 0.001 compared with hyperoxia + placebo (n = 6/group). (<b>H</b>) Representative Western blots of CTGF and β-actin. (<b>I</b>). Expression of CTGF was increased by hyperoxia, while administration of riociguat decreased CTGF expression in hyperoxia exposed lungs. ***<i>P</i> < 0.001 compared with normoxia; ⁺<i>P</i> < 0.05 compared with hyperoxia + placebo (n = 6/group). RA: room air, normaxia; O₂: hyperoxia; PL: placebo; Rio: riociguat.</p

Riociguat reduces and alters hyperoxia-induced lung inflammation.

<p>(<b>A</b>) Immunostaining for Mac-3, a macrophage marker. (<b>B</b>) The alveolar airspace macrophage population was increased by hyperoxia exposure as compared to normoxia. Administration of riociguat decreased macrophage count during hyperoxia. ***<i>P</i> < 0.001 compared with normoxia; ⁺⁺<i>P</i> < 0.01 compared with hyperoxia + placebo (n = 6/group). (<b>C</b>) Immunostaining for the M1 marker, inducible nitric oxide synthase (iNOS), and M2 markers, chitinase 3-like 3 (Ym1) and resistin-like molecule alpha (RELM-α) showed that in hyperoxia plus placebo lungs, both M1 and M2 polarized macrophages were detected. But, treatment with riociguat decreased only M1 macrophages in hyperoxia-exposed lungs. (<b>D</b>) Representative Western blots for NLRP-1, active caspase-1 and active IL-1β. Administration of riociguat decreased hyperoxia-induced lung expression of (<b>E</b>) NLRP-1 (***<i>P</i> < 0.001 compared with normoxia; ⁺<i>P</i> < 0.05 compared with hyperoxia + placebo), (<b>F</b>) active caspase-1 (***<i>P</i> < 0.001 compared with normoxia; ⁺⁺<i>P</i> < 0.05 compared with hyperoxia + placebo), and (<b>G</b>) active IL-1β (*<i>P</i> < 0.05 compared with normoxia; ⁺⁺⁺<i>P</i> < 0.001 compared with hyperoxia + placebo). RA: room air, normoxia; O₂: hyperoxia; PL: placebo; Rio: riociguat.</p