Confocal Laser Scanning Fluorescence Microscopy Research Articles

Multifunctional silica-coated mixed polymeric micelles for integrin-targeted therapy of pediatric patient-derived glioblastoma

Glioblastoma multiforme (GBM) remains a major cause of mortality because treatments are precluded by to the limited transport and penetration of chemotherapeutics across the blood–brain barrier. Pitavastatin (PTV) is a hydrophobic Food and Drug Administration (FDA)-approved anticholesterolemic agent with reported anti-GBM activity. In the present study, we encapsulate PTV in silica-coated polymeric micelles (SiO2 PMs) surface-modified with the cyclic peptide Arg-Gly-Asp-Phe-Val (cRGDfV) that actively targets the αvβ3 integrin overexpressed in the BBB endothelium and GBM. A central composite design is utilized to optimize the preparation process and improve the drug encapsulation ratio from 131 to 780 μg/mL. The silica shell provides full colloidal stability upon extreme dilution and enables a better control of the release kinetics in vitro with 28% of the cargo released after 12 h. Furthermore, SiO2 PMs show excellent compatibility and are internalized by human BBB endothelial cells, astrocytes and pericytes, as shown by confocal laser scanning fluorescence microscopy and flow cytometry. Finally, the anticancer efficacy is assessed in a pediatric patient-derived glioma cell line expressing high levels of the integrin subunits αv, β3 and β5. This PTV-loaded nanocarrier triggers apoptosis by reducing the mRNA level of anti-apoptotic genes NF-kβ, IL-6, BIRC1 and BIRC5 by 89%, 33%, 81% and 63%, respectively, and the cell viability by >60%. Overall, our results suggest the potential of these hybrid nanocarriers for the targeted therapy of GBM and other tumors overexpressing integrin receptors.

Combined fluorescence imaging and LA-ICP-MS trace element mapping of stalagmites: Microfabric identification and interpretation

Here, we map a series of stalagmites from Asturias, Spain, by laser ablation inductively-coupled-plasma mass spectrometry and confocal laser scanning (fluorescence) microscopy and discuss the origins of trace element and fluorescence variations. Seasonal banding is evident with both methods and may be attributed to lignins/humic acids based on fluorescence absorption and emission characteristics. Some lateral variations in fluorescence present as saw-tooth fluorescent “spires” and demonstrate disruptions of seasonal banding, corroborated by trace element variations (most prominently Mg and Na). Such features likely reflect the differential partitioning of trace elements by sectoral zoning as a result of low supersaturation and/or high organic matter load, combined with the effects of dissolved organic matter on the calcite growth surface and the association of each element with colloidal organic matter. The lateral variability of trace elements demonstrates the pitfalls of obtaining trace element information from one-dimensional transects without prior reconnaissance mapping. It is, however, possible that traditional drilling with ~1 mm holes homogenizes these features and provides reliable trace element estimates.

Unraveling Depth-Specific Ionic Conduction and Stiffness Behavior across Ionomer Thin Films and Bulk Membranes

Interfacial behavior of submicron thick polymer films critically controls the performance of electrochemical devices. We developed a robust, everyday-accessible, fluorescence confocal laser scanning microscopy (CLSM)-based strategy that can probe the distribution of mobility, ion conduction, and other properties across ionomer samples. When fluorescent photoacid probe 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS) was incorporated into <1 μm thick Nafion films on substrates, the depth-profile images showed thickness- and interface-dependent proton conduction behavior. In these films, proton conduction was weak over a region next to substrate interface, then gradually increased until air interface at 88% RH. Conversely, consistently high proton conduction with no interface dependence was observed across 35-50 μm thick bulk, free-standing Nafion membranes. A hump-like mobility/stiffness distribution was observed across Nafion films containing mobility-sensitive probe (9-(2-carboxy-2-cyanovinyl)julolidine) (CCVJ). The proton conduction and mobility distribution were rationalized as a combinatorial effect of interfacial interaction, ionomer chain orientation, chain density, and ionic domain characteristics.

Phase Aggregation Suppression of Homogeneous Perovskites Processed in Ambient Condition toward Efficient Light‐Emitting Diodes

AbstractPerovskite light‐emitting diodes (PeLEDs) have attracted attention because of their high efficiencies. However, due to the sensitivity of perovskites to ambient condition, perovskite emitter layers are generally fabricated under an inert gas environment (e.g., dry N2), which increases processing complexity and cost. Here, air‐prepared quasi‐2D perovskites are reported for efficient PeLEDs. It is found that the phase aggregation is the major obstacle deteriorating the characteristics of air‐prepared perovskites. Through antisolvent engineering to modulate the nucleation and growth characteristics of perovskite films from precursor solution, phase aggregations are restrained. Confocal laser scanning fluorescence microscopy results demonstrate homogeneous perovskite films with uniform photoluminescence distributions. Traps at grain boundaries are passivated, and exciton transfer among perovskite phases becomes effective. Finally, efficient green PeLEDs based on air‐prepared perovskites are realized with an external quantum efficiency of 15.4%. This work provides a promising strategy to fabricate cost‐effective perovskite devices in ambient air condition.

Intrapulpal Concentration of Hydrogen Peroxide of Teeth Restored With Bulk Fill and Conventional Bioactive Composites.

This study evaluated intrapulpal concentration and hydrogen peroxide (HP) penetration at the interface of teeth restored with bioactive composites, using conventional or bulk-fill composites. Cylindrical cavities were prepared on the buccal surface of bovine incisor crowns (n=20) and restored with: resin modified glass-ionomer (RMGI, Riva Light Cure, SDI), non-bioactive bulk-fill composite (FB, Filtek Bulk, 3M Oral Care), non-bioactive conventional composite (FZ, Filtek Z350, 3M Oral Care), bioactive bulk-fill composite (AC, Activa BioActive, Pulpedent), and bioactive conventional composite (BII, Beautifil II, Shofu). After 5,000 thermal cycles, restorations (n=10) were exposed to high (35% HP [4 applications of 8 min/session-4 sessions]) or low (9.5% HP [30 min/day-14 days]) concentration bleaching protocols. After the last bleaching application, the HP intrapulpal concentration was determined. Additional teeth were restored, pigmented with rhodamine B solution, and HP penetration around the interface was observed under laser scanning confocal fluorescence microscopy (LSCFM, n=3). The presence of gaps at the interface was observed on replicas of the cross-sectioned samples under scanning electron microscopy (SEM, n=5). Data were submitted to one-way (gap analysis) and twoway analysis of variance (ANOVA; HP intrapulpal concentration) and Tukey test (α=0.05). The LSCFM images were qualitatively analyzed. The restored teeth submitted to 35% HP presented higher HP intrapulpal concentration than teeth submitted to 9.5% HP (p<0.05). No differences in HP intrapulpal concentration was observed among groups (p>0.05) when exposed to 9.5% HP. Lower HP intrapulpal concentration was observed for teeth restored with RMGI exposed to HP 35%, when compared with teeth restored with nonbioactive conventional (FZ; p=0.004) and bulk-fill composites (FB; p=0.01). No gap formation was observed at the outer enamel adhesive interface for all restorative materials. LSCFM images showed that 35% HP promoted greater degradation of rhodamine B at the enamel, except for RMGI. In this context, RMGI promoted lower HP intrapulpal concentration than non-bioactive conventional and bulk-fill composites.

Ventricular Dysfunction and Calcium Handling Derangements in Isolated Hearts From Duchenne Muscular Dystrophy (DMD mdx‐4CV ) Mice Following Sustained Ventricular Preload

Duchenne muscular dystrophy (DMD) is the most common muscular dystrophy and is caused by mutations in the dystrophin gene. Subclinical signs of cardiac disease present early and usually progress to dilated cardiomyopathy in late stage DMD patients. Dystrophin deficiency associates with stretch-induced myocyte calcium entry, adverse calcium overload, and cardiomyocyte damage. In this investigation we test the hypothesis that dystrophin deficiency leads to ventricular dysfunction and calcium handling abnormalities following sustained ventricular preload challenge. Hearts of 3-10 month male Control and Dmdmdx-4CV dystrophic mice were isolated, cannulated via both the aorta and left atrium, and perfused with physiological saline solution in working heart mode. Following elevation in preload to 20 mmHg, hearts of Control and Dmdmdx-4CV mice exhibited similar systolic maximum pressure development (PMax: 105±13 mmHg Control versus 102±7 mmHg Dmdmdx-4CV). However, while hearts of Control mice maintained systolic performance following the 30 minute pre-load challenge (PMax, 30 minutes - PMax, peak response: -6±3 mmHg, N=5), hearts of Dmdmdx-4CV mice exhibited a marked decline in systolic performance (PMax,30 minutes - PMax, peak response: -29±6 mmHg, N=7, P<0.01 vs. Control). The Dmdmdx-4CV hearts also had visible signs of damage following preload challenge. Using Control and Dmdmdx-4CV mice with transgenic, cardiac-specific expression of the GCaMP6f calcium indicator (MerCreMer-GCaMP6f), we examined incidence of calcium overload behavior following preload challenge. Isolated working hearts were subjected to 20 mmHg preload in working heart mode for 30 minutes, switched to Langendorff mode with physiological saline containing 40 uM blebbistatin, and imaged using laser-scanning confocal fluorescence microscopy. Calcium overload behavior was observed in 21% of cardiomyocytes of Dmdmdx-4CV hearts (n=30/140 cells equally sampled from N=4 hearts) compared to 1% of cardiomyocytes of Control hearts (n=2/140 cells from N=4 hearts). Taken together, these data indicate susceptibility to stretch-induced calcium overload, ventricular damage, and ventricular dysfunction in male Dmdmdx-4CV hearts.

Back to the Basics: Two Approaches for the Identification and Extraction of Lipid Droplets from Malassezia pachydermatis CBS1879 and Malassezia globosa CBS7966.

Malassezia spp. are lipid-dependent yeasts that have been related to skin mycobiota and dermatological and systemic diseases. Study of lipid droplets (LDs) is relevant to elucidate the unknown role of these organelles in Malassezia and to gain a broader overview of lipid metabolism in Malassezia. Here, we standardized two protocols for the analysis of LDs in M. pachydermatis and M. globosa. The first describes co-staining for confocal laser-scanning fluorescence microscopy, and the second details extraction and purification of LDs. The double stain is achieved with three different neutral lipid fluorophores, namely Nile Red, BODIPY™ 493/503, and HCS LipidTOX™ Deep Red Neutral, in combination with Calcofluor White. For LD extraction, cell wall rupture is conducted using Trichoderma harzianum enzymes and cycles of vortexing with zirconium beads. LD purification is performed in a three-step ultracentrifugation process. These standardizations will contribute to the study of the dynamics, morphology, and composition of LDs in Malassezia. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Lipid droplet fluorescence staining Basic Protocol 2: Lipid droplet extraction and purification Support Protocol: Malassezia spp. culture conditions.

Building Block Symmetry Relegation Induces Mesopore and Abundant Open-Metal Sites in Metal–Organic Frameworks for Cancer Therapy

Building Block Symmetry Relegation Induces Mesopore and Abundant Open-Metal Sites in Metal–Organic Frameworks for Cancer Therapy

Heterocellular spheroids of the neurovascular blood-brain barrier as a platform for personalized nanoneuromedicine

SummaryNanoneuromedicine investigates nanotechnology to target the brain and treat neurological diseases. In this work, we biofabricated heterocellular spheroids comprising human brain microvascular endothelial cells, brain vascular pericytes and astrocytes combined with primary cortical neurons and microglia isolated from neonate rats. The structure and function are characterized by confocal laser scanning and light sheet fluorescence microscopy, electron microscopy, western blotting, and RNA sequencing. The spheroid bulk is formed by neural cells and microglia and the surface by endothelial cells and they upregulate key structural and functional proteins of the blood-brain barrier. These cellular constructs are utilized to preliminary screen the permeability of polymeric, metallic, and ceramic nanoparticles (NPs). Findings reveal that penetration and distribution patterns depend on the NP type and that microglia would play a key role in this pathway, highlighting the promise of this platform to investigate the interaction of different nanomaterials with the central nervous system in nanomedicine, nanosafety and nanotoxicology.

3D Imaging of Whole-Mount Optically Cleared Organs as a Tool to Estimate Pathogen Distribution in the Lungs

Daily, people inhale corpuscular pathogens of different origin and sizes. Among them are 3 um conidia of Aspergillus fumigatus fungi. Precise location of the pathogen in the airways can be helpful in indicating the potential cells that provide sufficient antifungal defense.The aim of the study was to build a 3D model of the mouse lung using fluorescence confocal laser scanning microscopy. Using the model we traced the location of A. fumigatus conidia in the different airway generations of immunocompetent mice and mice with induced immunosuppression. A. fumigatus conidia were labeled with Alexa594-NHS and applied to mice via the oropharyngeal route. At different time points mouse lungs were fixed, airways were labeled with streptavidin-Alexa488. Specimens were delipidated and optically cleared as whole-mounts. Specimens were subjected to confocal microscopy.Based on the fluorescence signal, the binary mask for the airways in the entire lung was built. We identified A. fumigatus conidia inside of bronchial branches and outside of them – in the alveolar space. At 6 hours after A. fumigatus conidia application, the percentage of conidia in the alveolar space of mice with immunosuppression was significantly decreased compared to immunocompetent mice, while in the bronchial branches the percentage of conidia was elevated in mice with immunosuppression. Thus using 3D imaging of whole-mount optically cleared lungs we showed the difference in A. fumigatus conidia distribution in bronchial branches and alveolar space in mice with induced immunosuppression compared to immunocompetent mice. The work was supported by RSF No 19-75-00082.

Poly(lactide-co-glycolide) Nanoparticles Mediate Sustained Gene Silencing and Improved Biocompatibility of siRNA Delivery Systems in Mouse Lungs after Pulmonary Administration.

Pulmonary delivery of small interfering RNA (siRNA)-based drugs is promising in treating severe lung disorders characterized by the upregulated expression of disease-causing genes. Previous studies have shown that the sustained siRNA release in vitro can be achieved from polymeric matrix nanoparticles based on poly(lactide-co-glycolide) (PLGA) loaded with lipoplexes (LPXs) composed of cationic lipid and anionic siRNA (lipid-polymer hybrid nanoparticles, LPNs). Yet, the in vivo efficacy, potential for prolonging the pharmacological effect, disposition, and safety of LPNs after pulmonary administration have not been investigated. In this study, siRNA against enhanced green fluorescent protein (EGFP-siRNA) was either assembled with 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) to form LPX or co-entrapped with DOTAP in PLGA nanoparticles to form LPNs. The disposition and clearance of LPXs and LPNs in mouse lungs were studied after intratracheal administration by using single-photon emission computed tomography/computed tomography (SPECT/CT) and gamma counting. Fluorescence spectroscopy, Western blot, and confocal laser scanning microscopy were used to evaluate the silencing of the EGFP expression mediated by the LPXs and LPNs after intratracheal administration to transgenic mice expressing the EGFP gene. The in vivo biocompatibility of LPXs and LPNs was investigated by measuring the cytokine level, total cell counts in bronchoalveolar lavage fluid, and observing the lung tissue histology section. The results showed that the silencing of the EGFP expression mediated by LPNs after pulmonary administration was both prolonged and enhanced as compared to LPXs. This may be attributed to the sustained release characteristics of PLGA, and the prolonged retention in the lung tissue of the colloidally more stable LPNs in comparison to LPXs, as indicated by SPECT/CT. The presence of PLGA effectively alleviated the acute inflammatory effect of cationic lipids to the lungs. This study suggests that PLGA-based LPNs may present an effective formulation strategy to mediate sustained gene silencing effects in the lung via pulmonary administration.

Revisiting the Polyol Synthesis and Plasmonic Properties of Silver Nanocubes

Background: Noble-metal nanocrystals have been extensively studied over the past decades because of their unique optical properties. The polyol process is considered an effective method for silver (Ag) nanocrystals’ synthesis in solution even though the reproducibility of its shape controlling is still a challenge. Here, Ag nanowires and nanocubes were synthesized by the polyol process, in which the Ag+ ions are directly reduced by ethylene glycol with a certain amount of Cl− ions added. We present the relationship between the final morphology of the Ag nanostructures with the parameters of reaction, including temperature, growth time, injection rate, and the amount of sodium chloride. The as-synthesized nanowires and nanocubes were characterized by scanning electron microscopy, transmission electron microscopy and X-ray diffraction. The uniformly distributed nanocubes with a mean edge length of 140 nm were obtained. The localized surface plasmon resonance of Ag nanocubes was characterized by laser scanning fluorescence confocal microscopy. The photoluminescence enhancement was observed on the perovskite film coupled with Ag nanocubes. Objective: We aimed to synthesize uniform and controllable silver nanocubes and nanowires through the polyol process and explore the interaction between CsPbBr3 perovskite film and Ag nanocubes antennas. Methods: We synthesized silver nanocubes and nanowires through the polyol process where the silver nitrate (AgNO3) was reduced by Ethylene Glycol (EG) in the presence of a blocking agent polyvinylpyrrolidone (PVP). Results: We successfully synthesized Ag nanocubes with an average edge length of 140 nm and Ag nanowires with a uniform distribution in terms of both shape and size through a polyol process with sodium chloride (NaCl) as the additive. In addition, the local photoluminescence (PL) enhancement was observed in a perovskite film by combining Ag nanocubes, which is attributed to the antennas plasmonic effect of the Ag nanocubes. Conclusion: In summary we studied the parameters in the polyol process such as reaction temperature, growth time, injection rate, kind of halide ion and NaCl amount for the synthesis of Ag nanowires and nanocubes. Our results suggest that the concentration of Cl- and the growth time have the main influence on Ag nanowires and nanocubes formation. The optimum growth time was found to be 60 min and 120 min for the formation of Ag nanowires and nanocubes, respectively. In addition, we revealed that the opportune reaction temperature of Ag nanowires was 140 °C. The injection rate of precursors was also found to play an important role in the final morphology of Ag nanowires and nanocubes. In addition, for the generation of Ag nanocubes, the presence of Cl− ion in the reaction is critical, which can eliminate most of the byproducts. We obtained the Ag nanowires with a uniform distribution in terms of both shape and size, and nanocubes with average lengths of 140 nm by the polyol process with the optimal parameters. Plasmon-coupled emission induced by noble-metal nanocrystals has attracted more attention in recent years. In this work, the PL of a perovskite film was enhanced by the coupling of Ag nanocubes due to the surface plasmonic effect.

Application of quantitative cell imaging using label-free optical diffraction tomography.

The cell is three-dimensionally and dynamically organized into cellular compartments, including the endoplasmic reticulum, mitochondria, vesicles, and nucleus, which have high relative molecular density. The structure and functions of these compartments and organelles may be deduced from the diffusion and interaction of related biomolecules. Among these cellular components, various protein molecules can freely access the nucleolus or mitotic chromosome through Brownian diffusion, even though they have a densely packed structure. However, physicochemical properties of the nucleolus and chromosomes, such as molecular density and volume, are not yet fully understood under changing cellular conditions. Many studies have been conducted based on high-resolution imaging and analysis techniques using fluorescence. However, there are limitations in imaging only fluorescently labeled molecules, and cytotoxicity occurs during three-dimensional imaging. Alternatively, the recently developed label-free three-dimensional optical diffraction tomography (ODT) imaging technique can divide various organelles in cells into volumes and analyze them by refractive index, although specific molecules cannot be observed. A previous study established an analytical method that provides comprehensive insights into the physical properties of the nucleolus and mitotic chromosome by utilizing the advantages of ODT and fluorescence techniques, such as fluorescence correlation spectroscopy and confocal laser scanning microscopy. This review article summarizes a recent study and discusses the future aspects of the ODT for cellular compartments.

Chronic Systemic Infection of Mice with Leishmania infantum Leads to Increased Bone Mass.

Vector-borne infections of humans with the protozoan parasite Leishmania (L.) infantum can cause a systemic and potentially lethal disease termed visceral leishmaniasis. In the corresponding mouse model, an intravenous infection with L. infantum leads to the persistence of parasites in various organs, including bone marrow (BM). Considering the anatomical proximity between the BM and the cortical bone, we investigated whether a chronic infection with L. infantum affected bone homeostasis. Unexpectedly, chronic infection with L. infantum caused an increase in bone mass in mice. In vivo, an increased number of osteoblasts and osteocytes and a decreased maturation of osteoclasts characterized the phenotype. Confocal laser scanning fluorescence microscopy confirmed the infection of BM macrophages but also revealed the presence of parasites in osteoclasts. In vitro, mature osteoclasts took up L. infantum parasites. However, infection of osteoclast progenitors abolished their differentiation and function. In addition, secretory products of infected BM-derived macrophages inhibited the maturation of osteoclasts. Both in vitro and in vivo, infected macrophages and osteoclasts showed an enhanced expression of the anti-osteoclastogenic chemokine CCL5 (RANTES). Neutralization of CCL5 prevented the inhibition of osteoclast generation seen in the presence of culture supernatants from L. infantum-infected macrophages. Altogether, our study shows that chronic infection with Leishmania increases bone mass by inducing bone formation and impairing osteoclast differentiation and function. © 2022 American Society for Bone and Mineral Research (ASBMR).

Slippery liquid‐infused porous surfaces: The effect of oil on the water repellence of hydrophobic and superhydrophobic soils

AbstractSoil wettability is important for understanding a wide range of earth system processes, from agricultural productivity to debris flows and sediment fan formation. However, there is limited research considering how soil–water interactions, where the soil grains are naturally hydrophobic, might change in the presence of oil from natural hydrocarbon leakage or oil spills. Here we show how slippery liquid‐infused porous surfaces (SLIPS) apply to hydrophobic soils, by physical modelling of surfaces of different grain sizes and examining their interactions with water before and after impregnation with silicone oil. Using contact and sliding angle measurements and laser scanning fluorescence confocal microscopy, we demonstrate that soil SLIPS can be created with thick oil layers and thin conformal oil layers on median grain sizes of 231 μm and 32 μm, respectively. Until now, SLIPS have only been observed in human‐made materials and biological surfaces. The mechanisms reported here demonstrate that SLIPS can occur in natural granular materials, providing a new mechanism for water‐shedding in soil and sediment systems. Furthermore, the water‐shedding properties may be long lasting as conformal oil layers are stabilized by capillary forces. These results have important implications for understanding soil physics and mechanics where oil is present in a soil, and for agricultural hydrophobicity on shallow slopes.Highlights We model oil contamination on a hydrophobic model soil as a mechanism for creating SLIPS. Soil SLIPS have implications for water‐shedding, oil spill remediation and earth processes. Our model soils exhibit extreme water‐shedding, illustrated by low water droplet sliding angles. This is the first physical modelling observation of SLIPS arising from hydrophobic soil.

Luminescent amphiphilic nanogels by terpyridine-Zn(II) complexation of polymeric micelles

In this work, we investigated terpyridine (tpy)/Zn(II) complexation for the crosslinking of polymeric micelles of the branched poly(ethylene oxide)–poly(propylene oxide) block copolymer Tetronic® 1107 (T1107) in water and produce physically stable amphiphilic luminescent nanogels. Nanoparticles displayed a size of 235 ± 25 and 318 ± 57 nm before and after Zn(II) crosslinking, respectively, as measured by dynamic light scattering. High-resolution scanning electron microscopy analysis revealed the multimicellar nature of the crosslinked nanoparticles. In addition, Zn(II) complexation prevented nanoparticle disassembly after extreme dilution below the critical micellar concentration and reduced the minimum concentration required for the reverse thermal gelation of concentrated aqueous T1107 systems. The cell compatibility and uptake were initially assessed in the murine macrophage cell line RAW 264.7. Results showed that complexation increases the cell compatibility of the nanoparticles with respect to the non-complexed counterparts. In addition, non-crosslinked nanoparticles accumulated in the cell membrane, while the complexed ones were internalized, as observed by confocal laser scanning fluorescence microscopy. Then, the antiproliferative activity of the crosslinked nanoparticles was confirmed in the rhabdomyosarcoma cell line Rh30; their inhibitory concentration 50 (IC50) being 101 μg/mL (6.7 μM). Finally, the encapsulation and release of the hydrophobic antiretroviral efavirenz was characterized in vitro. Complexation slightly reduced the release kinetics with respect to the pristine nanoparticles. Overall results demonstrate the promise of this simple modification strategy to produce amphiphilic nanogels with a set of advantageous physicochemical, optical, and biological properties.

Magnetically gradient-distributed microcapsule/epoxy composites: Low capsule load and highly targeted self-healing performance

One-component self-healing epoxy-matrix composites are needed urgently in the electronics industry because of their ability to repair surface damage autonomously. However, their practical applications are limited by unsuitable external stimuli and heavy microcapsule loads. Here, we report a magnetic-field and ultraviolet dual-responsive microcapsule to endow epoxy composites with a highly targeted self-healing capability under low microcapsule loads. Specifically, 5 wt% Fe3O4@SiO2 nanoparticles that were incorporated into the core material served as magnetic targets and helped the microcapsules to navigate to the near-surface region of a high-failure probability, to form magnetically gradient-distributed microcapsule/epoxy composites. This resulted in a more than fourfold increase in the local microcapsule concentration. The successful targeted self-healing with a microcapsule doping percentage as low as 4 wt% was confirmed by laser-scanning fluorescence confocal microscopy and scanning electron microscopy observations, and electrochemical testing. In addition, a kinetic model based on isothermal photo-differential scanning calorimetry measurements and a molecular-dynamics simulation model were both established to analyze the self-healing mechanism. Because of the facility and universality of this magnetic control method, these magnetic microcapsules could be guided to concentrate in vulnerable parts of other electrical insulators as desired, which highlights the good potential of this targeted self-healing method in energy fields.

Nanomicellar Lenalidomide-Fenretinide Combination Suppresses Tumor Growth in an MYCN Amplified Neuroblastoma Tumor.

PurposeIn a previous study, we demonstrated that the combination of fenretinide with lenalidomide, administered by a novel nanomicellar formulation (FLM), provided a strong antitumor effect in a neuroblastoma TrkB-expressing tumor. In this study, we tested the nanomicellar combination in an MYCN amplified neuroblastoma xenograft to assess its efficacy in different tumor genotypes and evaluate the interactions of the nanomicelles with the tumor cells.Experimental DesignFLM was administered to mice bearing human NLF xenografts to evaluate its efficacy in comparison with the nanomicelles containing fenretinide alone (FM). Confocal laser-scanning fluorescence microscopy images of the NLF cells treated with FLM and FM allowed us to estimate the nanomicelle ability to transport the encapsulated drugs inside the tumor cells. Flow cytometric analysis of the cells from treated tumors was performed to assess the effect of treatment on GD2 expression and NK cell infiltration.ResultsFLM and FM decreased the growth of NLF xenografts at comparable extents during the treatment period. Afterwards, FLM induced a progressive tumor regression without regrowth, while FM treatment was followed by regrowth within 15–20 days after the end of treatment. Both FLM and FM were able to penetrate the tumor cells transporting the encapsulated drugs. FLM transported higher amount of fenretinide inside the cells. Also, FLM treatment strongly increased GD2 expression in treated tumors and slightly decreased the NK infiltration compared to FM.ConclusionFLM treatment induced a superior antitumor response than FM in NLF xenografts, presumably due to the combined effects of fenretinide cytotoxicity and lenalidomide antiangiogenic activity. The ability of FLM to penetrate tumor cells, transporting the encapsulated drugs, substantially improved the therapeutic efficiency of this system. Moreover, the enhancement of GD2 expression in FLM treated tumors offers the possibility to further increase the antitumor effect by the use of anti-GD2 CAR-T cells and anti-GD2 antibodies in combination with FLM in multimodal therapies.

Amphiphilic Polymeric Nanoparticles Modified with a Retro-Enantio Peptide Shuttle Target the Brain of Mice

The blood–brain barrier (BBB) is a challenge for the treatment of diseases of the central nervous system (CNS) from the systemic circulation. The design of novel strategies to increase drug bioavailability in the CNS is called for. In this work, we synthesized amphiphilic polymeric nanoparticles by the self-assembly of graft copolymers of chitosan (CS, hydrophilic copolymer backbone) hydrophobized in the side-chain with poly­(methyl methacrylate) (PMMA)/poly­(acrylic acid) (PAAc) blocks and surface-decorated with a biologically stable retro-enantio peptide shuttle that improves brain transport. Nanoparticles showed one size population in the 190–210 nm range (intensity distribution) and a relatively small polydispersity index, as measured by dynamic light scattering. The surface charge estimated by the zeta-potential decreased from +35 mV in the unmodified nanoparticles to +14 mV in the modified ones, confirming the exposure of the peptide shuttle at the nanoparticle surface. The cell compatibility and uptake were assayed in hCMEC/D3 cells, a model of BBB endothelium, by a metabolic assay, confocal laser scanning fluorescence microscopy, and imaging flow cytometry in the absence and the presence of endocytosis inhibitors. Results indicated that the peptide shuttle modification improves their cell compatibility and that they are internalized by a clathrin-mediated pathway. In vitro permeability studies conducted in hCMEC/D3 cell monolayers showed that peptide shuttle-modified nanoparticles increase the apparent permeability with respect to the unmodified ones by 3.4 times. Finally, the brain accumulation was investigated upon i.v. administration to Hsd:ICR mice by using fluorescently labeled nanoparticles in an in vivo imaging system and light sheet fluorescence microscopy. Unmodified nanoparticles could be hardly detected in the brain blood vessels and parenchyma. Conversely, nanoparticles modified with the peptide shuttle could be detected after 10 min, with a maximum accumulation at 30 min and a slow concentration decline later on. Calculation of the area under the curve confirmed a 4-fold statistically significant increase in the accumulation of the modified nanoparticles with respect to the unmodified counterparts. These findings demonstrate the promise of this strategy to improve the delivery of nanoencapsulated cargos to the CNS.

Immunohistochemical Detection of 5-Hydroxymethylcytosine and 5-Carboxylcytosine in Sections of Zebrafish Embryos.

5-methylcytosine (5mC) is an epigenetic modification to DNA which modulates transcription. 5mC can be sequentially oxidized to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Collectively, these marks are referred to as the oxidized derivatives of 5mC (i.e., oxi-mCs). Their formation is catalyzed by the ten-eleven translocation methylcytosine dioxygenases (TETs 1, 2 and 3). Various techniques have been developed for the detection of oxi-mCs. The following chapter describes an immunochemical protocol for the simultaneous detection of 5hmC and 5caC in embryonic zebrafish tissue sections. The embryos are fixed, permeabilized and embedded in paraffin blocks. The blocks are cut into sections that are mounted onto slides. Depurination of the DNA is performed to allow immunodetection of the oxi-mCs. The 5hmC is detected with the help of a mouse anti-5hmC monoclonal primary antibody and a goat anti-mouse Alexa Fluor 633-conjugated secondary antibody. The weak 5caC signal requires enzymatic amplification. Its detection involves a rabbit anti-5caC polyclonal primary antibody and a goat anti-rabbit secondary antibody that is conjugated to horseradish peroxidase (HRP). HRP amplifies the 5caC signal by catalyzing the deposition of large quantities of fluorescein-labeled tyramide. Sections immunostained for 5hmC and 5caC are analyzed by fluorescent light or confocal laser scanning microscopy. This immunochemical method allows for highly sensitive detection of 5hmC and 5caC in zebrafish tissues.