Multifunctional poly(organosiloxane) nanoparticles as a model system for biomedical applications

Polymere Nanomaterialien finden bereits ein sehr breites Anwendungsspektrum in der Biomedizin, speziell in Bereichen des Wirkstofftransports und der Krebsforschung. Dennoch ist die Synthese geeigneter Modellsysteme für ein besseres Verständnis der Wechselwirkungen von Nanopartikeln mit biologischen Systemen notwendig. Diese Studie berichtet über die Synthese und Charakterisierung multifunktioneller, fluoreszenzmarkierter Poly(organosiloxan)-Kern-Schale-Nanopartikel mit veränderbaren Oberflächeneigenschaften. Die Kern-Schale-Architektur erlaubt eine unabhängige Funktionalisierung unterschiedlicher Partikelkompartimente. So wurde der Kern der Partikel während der Polykondensation mit RhodaminB-Monomer markiert. Durch Einführung geladener Gruppen in der Partikelschale wurden elektrostatisch stabilisierte Partikel erhalten. Alternativ führt das Aufpfropfen von Polymeren (thermoresponsives Poly(2-isopropyl-2- oxazolin) oder Poly(ethylenglykol)) auf die Oberfläche zu sterisch stabilisierten, biokompatiblen Nanopartikeln. Schließlich wurde Biotin an die Partikeloberfläche gekuppelt, um die Immobilisierung weiterer Liganden wie Antikörper oder Enzyme auf der Partikeloberfläche zu ermöglichen. Die Nanopartikel wurden mittels Elektronenmikroskopie, winkelabhängiger dynamischer Lichtstreuung (DLS), asymmetrischer Fluss Feld-Fluss Fraktionierung (AF-FFF) und ζ-Potential-Messungen charakterisiert. Die spektroskopischen Eigenschaften wurden mit der Fluoreszenzspektroskopie untersucht, einschließlich der Bestimmung der absoluten Fluoreszenzquantenausbeuten. Auf Grund ihrer modularen Struktur und Vielfalt an möglichen Funktionalisierungen eignet sich das Poly(organosiloxan)-System für diverse biomedizinische Anwendungen, wie z.B. Abbildungsmethoden, spezifisches Targeting und Wirkstofftransport. Die zelluläre Aufnahme der Nanopartikel in vivo wird oft durch die Bildung einer Proteinkorona beeinflusst, deshalb wurde weiterhin das Verhalten unterschiedlich funktionalisierter Partikel unter physiologischen Bedingungen und in Anwesenheit von Serumproteinen mittels DLS und AF-FFF untersucht. Neben der Synthese und physikochemischen Charakterisierung wurden auch Zellaufnahmeexperimente durchgeführt, in denen die Wechselwirkung der Nanopartikel mit Lungengewebemodellen erforscht wurde. Diese Versuche zeigen Unterschiede in der zellulären Aufnahme der elektrostatisch und der sterisch stabilisierten Partikel.Polymeric nanomaterials offer a wide range of biomedical applications especially in the fields of drug delivery and cancer research. However, suitable model systems are still required to gain deeper knowledge of interactions of nanoparticles with biological systems. This study reports on the synthesis and characterization of multifunctional, fluorescent poly(organosiloxane) core-shell nanoparticles with tunable surface properties. The core-shell approach allows the independent functionalization of different compartments. Thus, rhodamine b-labeled monomer was efficiently incorporated into the core during polycondensation. By introduction of charged groups on the surface, electrostatic stabilization of the particles was achieved. Alternatively, grafting of polymers (e.g. thermo-responsive poly(2-isopropyl-2-oxazoline) or poly(ethylene glycol)) leads to sterically stabilized, biocompatible nanoparticles. Finally biotin was introduced on the surface allowing the immobilization of further ligands such as antibodies or enzymes on the particle’s surface. The nanoparticles were characterized by electron microscopy, multi-angle dynamic light scattering (DLS), asymmetrical flow field-flow fractionation (AF-FFF) and zeta potential measurements. The spectroscopic properties were studied by fluorescence spectroscopy, including the determination of absolute fluorescence quantum yields. Due to their modular structure and the variety of possible functionalizations, the poly(organosiloxane) system is suitable for diverse biomedical applications, e.g. imaging methods, specific targeting and drug delivery. The cellular uptake of nanoparticles in vivo is often determined by the formation of a protein corona. Therefore, the behavior of nanoparticles with different surface characteristics was compared under physiological conditions and in presence of serum proteins using DLS and AF-FFF. Besides the synthesis and the physico-chemical characterization, cell experiments are performed where nanoparticles are applied to lung tissue models. These experiments demonstrate differences in the uptake behavior of electrostatically and sterically stabilized nanoparticles

Real-time ³¹P NMR reveals different gradient strengths in polyphosphoester copolymers as potential MRI-traceable nanomaterials

Polyphosphoesters (PPEs) are used in tissue engineering and drug delivery, as polyelectrolytes, and flame-retardants. Mostly polyphosphates have been investigated but copolymers involving different PPE subclasses have been rarely explored and the reactivity ratios of different cyclic phospholanes have not been reported. We synthesized binary and ternary PPE copolymers using cyclic comonomers, including side-chain phosphonates, phosphates, thiophosphate, and in-chain phosphonates, through organocatalyzed ring-opening copolymerization. Reactivity ratios were determined for all cases, including ternary PPE copolymers, using different nonterminal models. By combining different comonomers and organocatalysts, we created gradient copolymers with adjustable amphiphilicity and microstructure. Reactivity ratios ranging from 0.02 to 44 were observed for different comonomer sets. Statistical ring-opening copolymerization enabled the synthesis of amphiphilic gradient copolymers in a one-pot procedure, exhibiting tunable interfacial and magnetic resonance imaging (MRI) properties. These copolymers self-assembled in aqueous solutions, 31 P MRI imaging confirmed their potential as MRI-traceable nanostructures. This systematic study expands the possibilities of PPE-copolymers for drug delivery and theranostics.</p

Vitamin D₃ Modulates NF-kB/p65, 17β-Estradiol, and Vitamin D Receptors Expression at Estrogen Deficiency

The aim of the present study was to focus on the effects of Vitamin D3 (VD3) supplementation (5.0 mg/kg, s.c.) on the NF-kB/p65, 17β-estradiol (17β-E2)/VD3 receptors expression in the hippocampus in the long-term ovariectomized (OVX) rats treated with low dose of 17β-E2 (0.5 μg/rat, s.c.) submitted for the chronic unpredictable mild stress (CUMS) for 28 days. Sucrose preference (SPT), forced swimming (FST), and open-field (OFT) tests were conducted to estimate the anhedonia-/depression-like states. NF-kB/p65, 17β-E2/VD3 receptors levels in the hippocampus were evaluated by ELISA and Western blot assays. The findings demonstrated that VD3 at high dose (5.0 mg/kg, s.c.) in a combination with low dose of 17β-E2 decreased anhedonia in the SPT and depression-like behavior in the FST of the long-term OVX rats submitted to CUMS. VD3 (5.0 mg/kg) resulted in significant decreased levels of hippocampal NF-kB/p65 protein expression, as well as to the normalization of hippocampal 17β-E2/VD3 receptors levels in long-term OVX rats treated with 17β-E2 exposed to CUMS. In conclusion, VD3 (5.0 mg/kg, s.c.) in a combination with low dose of 17β-E2 had a synergic antianhedonic- and antidepressant-like effects in the adult female rats following long-term ovariectomy submitted to CUMS

Abstract P-24: Microscopic Analyses of Liquid-Liquid Phase Separation Induced by Linker Histone H1.0

Background: Liquid-liquid phase separation (LLPS) that leads to the formation of temporary functional domains in cells plays an important role in the processes of chromatin condensation and gene regulation. Earlier, it was demonstrated that histone H1.4 can form LLPS droplets with DNA. In the present work, LLPS was studied for histone H1.0, which is mainly expressed in differentiated and non-dividing cells. H1.0 is involved in cancer development: its amount decreases with the progression of tumor cells to malignancy. Methods: LSM710 confocal microscope (Zeiss) equipped with the 40x/1.2W objective was used to image mixtures of H1.0 with Cy3/Cy5 labeled DNA or nucleosomes in fluorescent and transmitted-light channels at the excitation of 514 nm. The formation of condensates as a result of LLPS was confirmed by salt-jump and FRAP/FLIP experiments. Results: Condensates were not observed when the ratio of negative to positive charges (N/P) in the samples was >1. At N/P~0.7, optically homogeneous droplet-like condensates were found. The appearance of condensates, their size and shape depended on concentrations of H1.0 and DNA. LLPS condensates but not aggregates disappeared by salt-jump to 650 mM NaCl. FRAP/FLIP experiments revealed a moderate rate of fluorescence recovery (τ½22s) indicating moderate DNA mobility of the H1.0-mediated condensates. The appearance of condensates was also observed in the mixtures of H1.0, DNA and Cy3/Cy5-labeled nucleosomes. Nucleosomes were involved in the condensate formation and found to be 2-fold more mobile (τ½10 s) than DNA. Conclusion: LLPS-related properties of H1.0 were studied for DNA and nucleosomes in vitro. Comparison with H1.4 shows that H1.0 forms liquid condensates of approximately the same size. Our result also may indicate that chromatin retains pronounced dynamic properties in H1.0-induced droplets despite the fact that H1.0 induces the formation of more compact chromatin

The internal structure of gadolinium and perfluorocarbon-loaded polymer nanoparticles affects ¹⁹F MRI relaxation times

19F magnetic resonance imaging (19F MRI) is an emerging technique for quantitative imaging in novel therapies, such as cellular therapies and theranostic nanocarriers. Nanocarriers loaded with liquid perfluorocarbon (PFC) typically have a (single) core-shell structure with PFC in the core due to the poor miscibility of PFC with organic and inorganic solvents. Paramagnetic relaxation enhancement acts only at a distance of a few angstroms. Thus, efficient modulation of the 19F signal is possible only with fluorophilic PFC-soluble chelates. However, these chelates cannot interact with the surrounding environment and they might result in image artifacts. Conversely, chelates bound to the nanoparticle shell typically have a minimal effect on the 19F signal and a strong impact on the aqueous environment. We show that the confinement of PFC in biodegradable polymeric nanoparticles (NPs) with a multicore structure enables the modulation of longitudinal (T1) and transverse (T2) 19F relaxation, as well as proton (1H) signals, using non-fluorophilic paramagnetic chelates. We compared multicore NPs versus a conventional single core structure, where the PFC is encapsulated in the core(s) and the chelate in the surrounding polymeric matrix. This modulated relaxation also makes multicore NPs sensitive to various acidic pH environments, while preserving their stability. This effect was not observed with single core nanocapsules (NCs). Importantly, paramagnetic chelates affected both T1 and T219F relaxation in multicore NPs, but not in single core NCs. Both relaxation times of the 19F nucleus were enhanced with an increasing concentration of the paramagnetic chelate. Moreover, as the polymeric matrix remained water permeable, proton enhancement additionally was observed in MRI.</p

Selective footprints and genes relevant to cold adaptation and other phenotypic traits are unscrambled in the genomes of divergently selected chicken breeds

Background: The genomes of worldwide poultry breeds divergently selected for performance and other phenotypic traits may also be affected by, and formed due to, past and current admixture events. Adaptation to diverse environments, including acclimation to harsh climatic conditions, has also left selection footprints in breed genomes. Results: Using the Chicken 50K_CobbCons SNP chip, we genotyped four divergently selected breeds: two aboriginal, cold tolerant Ushanka and Orloff Mille Fleur, one egg-type Russian White subjected to artificial selection for cold tolerance, and one meat-type White Cornish. Signals of selective sweeps were determined in the studied breeds using three methods: (1) assessment of runs of homozygosity islands, (2) FST based population differential analysis, and (3) haplotype differentiation analysis. Genomic regions of true selection signatures were identified by two or more methods or in two or more breeds. In these regions, we detected 540 prioritized candidate genes supplemented them with those that occurred in one breed using one statistic and were suggested in other studies. Amongst them, SOX5, ME3, ZNF536, WWP1, RIPK2, OSGIN2, DECR1, TPO, PPARGC1A, BDNF, MSTN, and beta-keratin genes can be especially mentioned as candidates for cold adaptation. Epigenetic factors may be involved in regulating some of these important genes (e.g., TPO and BDNF). Conclusion: Based on a genome-wide scan, our findings can help dissect the genetic architecture underlying various phenotypic traits in chicken breeds. These include genes representing the sine qua non for adaptation to harsh environments. Cold tolerance in acclimated chicken breeds may be developed following one of few specific gene expression mechanisms or more than one overlapping response known in cold-exposed individuals, and this warrants further investigation

Multicore liquid perfluorocarbon-loaded multimodal nanoparticles for stable ultrasound and ¹⁹ F MRI applied to in vivo cell tracking

Ultrasound is the most commonly used clinical imaging modality. However, in applications requiring cell-labeling, the large size and short active lifetime of ultrasound contrast agents limit their longitudinal use. Here, 100 nm radius, clinically applicable, polymeric nanoparticles containing a liquid perfluorocarbon, which enhance ultrasound contrast during repeated ultrasound imaging over the course of at least 48 h, are described. The perfluorocarbon enables monitoring the nanoparticles with quantitative 19 F magnetic resonance imaging, making these particles effective multimodal imaging agents. Unlike typical core–shell perfluorocarbon-based ultrasound contrast agents, these nanoparticles have an atypical fractal internal structure. The nonvaporizing highly hydrophobic perfluorocarbon forms multiple cores within the polymeric matrix and is, surprisingly, hydrated with water, as determined from small-angle neutron scattering and nuclear magnetic resonance spectroscopy. Finally, the nanoparticles are used to image therapeutic dendritic cells with ultrasound in vivo, as well as with 19 F MRI and fluorescence imaging, demonstrating their potential for long-term in vivo multimodal imaging. </p

INVESTIGATION OF MAGNETIZATION REVERSAL PROCESSES IN TWO-LAYER FeNiCo/Ta/CoW AND THREE-LAYER FeNiCo/FeNiCo FILMS

The aim is to study the nature of interacting multilayer film structures. It has been discovered that the heterophase character of one layer can lead to the asymmetry of other layer behaviour in the external magnetic field. The qualitative analysis of the hysteresis loop shift field value coincides with relation of the shift field for two-layer FeNiCo/CoW film. The experimental model was a base of the patent &quot;Procedure of Making Magnetostatic-Connected Films&quot;. In three-layer FeNiCo/Ta/FeNiCo the difference in the motion rate relations of the domen boundaries in the different layers has been explained from conceptions about dependence of the effective bond field of the layers upon the external magnetic field. The observed domen transfers from layer into layer have been explained by a combined action of the scattering fields from domen tops and external magnetic field. The investigation results are used at development of the aids in the magnetoelectronicsAvailable from VNTIC / VNTIC - Scientific & Technical Information Centre of RussiaSIGLERURussian Federatio