Evaluation of the Transport and Binding of Dopamine-Loaded PLGA Nanoparticles for the Treatment of Parkinson's Disease Using In Vitro Model Systems.

The treatment of Parkinson's disease has been moving into the focus of pharmaceutical development. Yet, the necessity for reliable model systems in the development phase has made research challenging and in vivo models necessary. We have established reliable, reproducible in vitro model systems to evaluate the binding and transport of dopamine-loaded PLGA nanoparticles for the treatment of Parkinson's disease and put the results in context with comparable in vivo results. The in vitro models have provided similar results concerning the usability of the investigated nanoparticles as the previously used in vivo models and thus provide a good alternative in line with the 3R principles in pharmaceutical research.

Distribution of SiO2 nanoparticles in 3D liver microtissues.

INTRODUCTION: Nanoparticles (NPs) are used in numerous products in technical fields and biomedicine; their potential adverse effects have to be considered in order to achieve safe applications. Besides their distribution in tissues, organs, and cellular localization, their impact and penetration during the process of tissue formation occurring in vivo during liver regeneration are critical steps for establishment of safe nanomaterials. MATERIALS AND METHODS: In this study, 3D cell culture of human hepatocarcinoma cells (HepG2) was used to generate cellular spheroids, serving as in vitro liver microtissues. In order to determine their differential distribution and penetration depth in HepG2 spheroids, SiO2 NPs were applied either during or after spheroid formation. The NP penetration was comprehensively studied using confocal laser scanning microscopy and scanning electron microscopy. RESULTS: Spheroids were exposed to 100 µg mL-1 SiO2 NPs either at the beginning of spheroid formation, or during or after formation of spheroids. Microscopy analyses revealed that NP penetration into the spheroid is limited. During and after spheroid formation, SiO2 NPs penetrated about 20 µm into the spheroids, corresponding to about three cell layers. In contrast, because of the addition of SiO2 NPs simultaneously to cell seeding, NP agglomerates were located also in the spheroid center. Application of SiO2 NPs during the process of spheroid formation had no impact on final spheroid size. CONCLUSION: Understanding the distribution of NPs in tissues is essential for biomedical applications. The obtained results indicate that NPs show only limited penetration into already formed tissue, which is probably caused by the alteration of the tissue structure and cell packing density during the process of spheroid formation.

Targeted T1 Magnetic Resonance Imaging Contrast Enhancement with Extraordinarily Small CoFe2O4 Nanoparticles.

Extraordinarily small (2.4 nm) cobalt ferrite nanoparticles (ESCIoNs) were synthesized by a one-pot thermal decomposition approach to study their potential as magnetic resonance imaging (MRI) contrast agents. Fine size control was achieved using oleylamine alone, and annular dark-field scanning transmission electron microscopy revealed highly crystalline cubic spinel particles with atomic resolution. Ligand exchange with dimercaptosuccinic acid rendered the particles stable in physiological conditions with a hydrodynamic diameter of 12 nm. The particles displayed superparamagnetic properties and a low r2/ r1 ratio suitable for a T1 contrast agent. The particles were functionalized with bile acid, which improved biocompatibility by significant reduction of reactive oxygen species generation and is a first step toward liver-targeted T1 MRI. Our study demonstrates the potential of ESCIoNs as T1 MRI contrast agents.

Kinetic and spectroscopic responses of pH-sensitive nanoparticles: influence of the silica matrix.

Intracellular pH sensing with fluorescent nanoparticles is an emerging topic as pH plays several roles in physiology and pathologic processes. Here, nanoparticle-sized pH sensors (diameter far below 50 nm) for fluorescence imaging have been described. Consequently, a fluorescent derivative of pH-sensitive hydroxypyrene with pK a = 6.1 was synthesized and subsequently embedded in core and core-shell silica nanoparticles via a modified Stöber process. The detailed fluorescence spectroscopic characterization of the produced nanoparticles was carried out for retrieving information about the environment within the nanoparticle core. Several steady-state and time-resolved fluorescence spectroscopic methods hint to the screening of the probe molecule from the solvent, but it sustained interactions with hydrogen bonds similar to that of water. The incorporation of the indicator dye in the water-rich silica matrix neither changes the acidity constant nor dramatically slows down the protonation kinetics. However, cladding by another SiO2 shell leads to the partial substitution of water and decelerating the response of the probe molecule toward pH. The sensor is capable of monitoring pH changes in a physiological range by using ratiometric fluorescence excitation with λ ex = 405 nm and λ ex = 488 nm, as confirmed by the confocal fluorescence imaging of intracellular nanoparticle uptake.

Silica Nanoparticles for Intracellular Protein Delivery: a Novel Synthesis Approach Using Green Fluorescent Protein.

In this study, a novel approach for preparation of green fluorescent protein (GFP)-doped silica nanoparticles with a narrow size distribution is presented. GFP was chosen as a model protein due to its autofluorescence. Protein-doped nanoparticles have a high application potential in the field of intracellular protein delivery. In addition, fluorescently labelled particles can be used for bioimaging. The size of these protein-doped nanoparticles was adjusted from 15 to 35 nm using a multistep synthesis process, comprising the particle core synthesis followed by shell regrowth steps. GFP was selectively incorporated into the silica matrix of either the core or the shell or both by a one-pot reaction. The obtained nanoparticles were characterised by determination of particle size, hydrodynamic diameter, ζ-potential, fluorescence and quantum yield. The measurements showed that the fluorescence of GFP was maintained during particle synthesis. Cellular uptake experiments demonstrated that the GFP-doped nanoparticles can be used as stable and effective fluorescent probes. The study reveals the potential of the chosen approach for incorporation of functional biological macromolecules into silica nanoparticles, which opens novel application fields like intracellular protein delivery.

Different phase behavior and packing of ceramides with long (C16) and very long (C24) acyls in model membranes: infrared spectroscopy using deuterated lipids.

Ceramides (Cer) are the central molecules in sphingolipid metabolism that participate in cellular signaling and also prevent excessive water loss by the skin. Previous studies showed that sphingosine-based Cer with a long 16C chain (CerNS16) and very long 24C-chain ceramides (CerNS24) differ in their biological actions. Increased levels of long CerNS16 at the expense of the very long CerNS24 have been found in atopic dermatitis patients, and this change correlated with the skin barrier properties. To probe the membrane behavior of the long CerNS16 and the very long chain CerNS24, we studied their interactions with fatty acids and cholesterol in model stratum corneum membranes using infrared spectroscopy. Using Cer with deuterated acyls and/or deuterated fatty acids, we showed differences in lipid mixing, packing, and thermotropic phase behavior between long and very long Cer. These differences were observed in the presence of lignoceric acid or a heterogeneous fatty acid mixture (C16-C24), in the presence or absence of cholesterol sulfate, and at 5-95% humidity. In these membranes, very long CerNS24 prefers an extended (splayed-chain) conformation in which the fatty acid is associated with the very long Cer chain. In contrast, the shorter CerNS16 and fatty acids are mostly phase separated.

Interaction of serine proteases from polymorphonuclear leucocytes with the cell surface and heparin.

Polymorphonuclear leucocytes (PMNs) accumulate at inflammatory sites and contribute to host defence, regulation of the inflammatory process, and also to tissue injury. Upon activation, these cells release the serine proteases elastase, cathepsin G, and proteinase 3 that are involved in multiple processes such as microbicidal activity, penetration of PMNs through endothelium and adjacent connective tissue to inflammatory sites, and processing of various cytokines. Here, we compared the three serine proteases for their release from PMNs and their ability to interact with resting PMNs and the highly sulphated glycosaminoglycan heparin. Unlike elastase, proteinase 3 and cathepsin G were released from resting PMNs as evidenced by flow cytometry, confocal fluorescence microscopy, and activity measurements. While proteinase 3 binds heavily to surface targets on vital PMNs, cathepsin G and elastase interact preferentially with sulphated glycosaminoglycans. These data revealed a differentiated picture about the individual functions of the PMN serine proteases during inflammatory response.