Huntingtin Co-Isolates with Small Extracellular Vesicles from Blood Plasma of TgHD and KI-HD Pig Models of Huntington's Disease and Human Blood Plasma.

(1) Background: Huntington's disease (HD) is rare incurable hereditary neurodegenerative disorder caused by CAG repeat expansion in the gene coding for the protein huntingtin (HTT). Mutated huntingtin (mHTT) undergoes fragmentation and accumulation, affecting cellular functions and leading to neuronal cell death. Porcine models of HD are used in preclinical testing of currently emerging disease modifying therapies. Such therapies are aimed at reducing mHTT expression, postpone the disease onset, slow down the progression, and point out the need of biomarkers to monitor disease development and therapy efficacy. Recently, extracellular vesicles (EVs), particularly exosomes, gained attention as possible carriers of disease biomarkers. We aimed to characterize HTT and mHTT forms/fragments in blood plasma derived EVs in transgenic (TgHD) and knock-in (KI-HD) porcine models, as well as in HD patients' plasma. (2) Methods: Small EVs were isolated by ultracentrifugation and HTT forms were visualized by western blotting. (3) Results: The full length 360 kDa HTT co-isolated with EVs from both the pig model and HD patient plasma. In addition, a ~70 kDa mutant HTT fragment was specific for TgHD pigs. Elevated total huntingtin levels in EVs from plasma of HD groups compared to controls were observed in both pig models and HD patients, however only in TgHD were they significant (p = 0.02). (4) Conclusions: Our study represents a valuable initial step towards the characterization of EV content in the search for HD biomarkers.

Anti-inflammatory potential of composites of yeast glucan particles and geranylated flavonoid diplacone.

Geranylated flavanone diplacone is a flavanone iso- lated from Paulownia tomentosa (Thunb.) Steud. (Paulowniaceae) with anti-inflammatory and antioxidant properties, nevertheless showing high lipophilicity and low solubility in water. Diplacone was therefore used as a model molecule for incorporation into glucan particles (GPs). GPs are prepared by intensive washing of yeast (Saccharomyces cerevisiae) leading to hollow shells consisting of β-(13)/β-(16) glucan mainly. The aim of this study was to compare anti-inflammatory potential of GPs-diplacone composites with the compound itself, GPs themselves and the physical mixture of GPs and diplacone. The cell line THP1-XBlueTM-MD2-CD14 derived from human leukemic monocytes was stimulated with lipopolysaccharide (LPS) from Escherichia coli to trigger inflammatory reaction. The composites of GPs with diplacone significantly decreased the activity of pro-inflammatory transcription factors nuclear factor κB (NF-κB) and activator protein 1 (AP-1).

Remotely controlled diffusion from magnetic liposome microgels.

The reversible, temperature-dependent change in the permeability of a phospholipid bilayer has been used for controlling the diffusion rate of encapsulated molecular payload from liposomes. Liposomes were preloaded with a fluorescent dye and immobilized in calcium alginate hydrogel microparticles that also contained iron oxide nanoparticles. The composite microparticles were produced by a drop-on-demand inkjet method. The ability of iron oxide nanoparticles to locally dissipate heat upon exposure to a radio-frequency (RF) alternating magnetic field was used to control the local temperature and therefore diffusion from the liposomes in a contactless way using an RF coil. Several different release patterns were realized, including repeated on-demand release. The internal structure of the composite alginate-liposome-magnetite microparticles was investigated, and the influence of microparticle concentration on the heating rate was determined. In order to achieve a temperature rise required for the liposome membrane melting, the concentration of alginate beads should be at least 25% of their maximum packing density for the nanoparticle concentration and specific absorption rate used.

Modified activities of macrophages' deubiquitinating enzymes after Francisella infection.

Francisella tularensis influences several host molecular/signaling pathways during infection. Ubiquitination and deubiquitination are among the most important regulatory mechanisms and respectively occur through attachment or removal of the ubiquitin molecule. The process is necessary not only to mark molecules for degradation, but also, for example, to the activation of signaling pathways leading to pro-inflammatory host response. Many intracellular pathogens, including Francisella tularensis, have evolved mechanisms of modifying such host immune responses to escape degradation. Here, we describe that F. tularensis interferes with the host's ubiquitination system. We show increased total activity of deubiquitinating enzymes (DUBs) in human macrophages after infection, while confirm reduced enzymatic activities of two specific DUBs (USP10 and UCH-L5), and demonstrate increased activity of USP25. We further reveal the enrichment of these three enzymes in exosomes derived from F. tularensis-infected cells. The obtained results show the regulatory effect on ubiquitination mechanism in macrophages during F. tularensis infection.

Surprising efficacy twist of two established cytostatics revealed by a-la-carte 3D cell spheroid preparation protocol.

Three-dimensional cell culture systems are increasingly used for biological and anticancer drug screening as they mimic the structure and microenvironment of tumors more closely than conventional two-dimensional cell models. In this study, the growth kinetics of colon adenocarcinoma-derived spheroids (HT-29 cell line) cultivated in liquid marble micro-bioreactors and nonadherent PDMS-coated well plates was investigated in detail and enabled precise control of the spheroid size by the seed cell density and cultivation time. The therapeutic effect of 5-fluorouracil and irinotecan hydrochloride in 2D monolayer cell culture and 3D tumor spheroids revealed an unexpected twist in their efficacy due to different ability to penetrate through 3D microtissue. For 5-fluorouracil, the inhibitory concentration IC50 after 48 h exposure increased from 11.3 µM for a 2D cell culture to 707.7 µM for a 3D spheroid. In the case of irinotecan, IC50 increased from 24.9 µM to 77.8 µM. Despite its higher molar weight, irinotecan appeared to penetrate the 3D spheroid structure more efficiently than 5-fluorouracil. While 5-fluorouracil mainly caused a suppression of spheroid growth from the outside, irinotecan affected the entire spheroid and caused its originally compact structure to disintegrate. The acquired results highlight the need to screen cancer chemotherapeutics on 3D tumor models, as contrasting results can be obtained compared to standard 2D cell cultures.

The effect of curcumin encapsulation into yeast glucan particles on antioxidant enzyme expression in vitro.

Glucan particles (GPs) from Saccharomyces cerevisiae consist mainly of ß-1,3-d-glucan. Curcumin is a phenolic compound of plant origin. A 24 h incubation with a mixture of GPs and curcumin increased the expression of the Nrf2 protein and increased the activation of the Nrf2-ARE system significantly.

Evaluation of ß-glucan particles as dual-function carriers for poorly soluble drugs.

Yeast glucan particles are porous polysaccharide cell walls extracted from Saccharomyces cerevisiae. Being mildly immunogenic, they are efficiently phagocytosed and have therefore been proposed as possible vehicles for drug delivery. Using curcumin as a model poorly water-soluble drug, a systematic comparison of three different physical loading methods - incipient wetness impregnation, slurry evaporation, and spray drying - was carried out and their influence on the particle morphology, encapsulation efficiency, amorphous drug content and release kinetics was evaluated. It was found that yeast glucan particles can contain up to 30% wt. of curcumin in the amorphous form when prepared by slurry evaporation. The dissolution of curcumin from glucan particles lead to a supersaturated solution in asimilar way as amorphous solid dispersions do, despite the fact that glucan particles themselves do not dissolve. Bi-phasic dissolution tests revealed up to 4-fold acceleration of curcumin dissolution rate from amorphous glucan particles compared to its crystalline form. Crucially, glucan particles were shown to retain the ability to be recognised and phagocytosed even after drug encapsulation.

Incorporating natural anti-inflammatory compounds into yeast glucan particles increases their bioactivity in vitro.

Yeast glucan particles (GPs) are promising agents for the delivery of biologically active compounds as drugs. GPs possess their own biological activities and can act synergistically with their cargo. This study aimed to determine how incorporating artemisinin, ellagic acid, (-)-epigallocatechin gallate, morusin, or trans-resveratrol into GPs affects their anti-inflammatory and antioxidant potential in vitro. Two different methods - slurry evaporation and spray drying - were used to prepare composites (GPs + bioactive compound) and the anti-inflammatory and antioxidative properties of the resultant products were compared. Several of the natural compounds showed the beneficial effects of being combined with GPs. The materials prepared by spray drying showed greater activity than those made using a rotary evaporator. Natural compounds incorporated into yeast GPs showed greater anti-inflammatory potential in vitro than simple suspensions of these compounds as demonstrated by their inhibition of the activity of transcription factors NF-κB/AP-1 and the secretion of the pro-inflammatory cytokine TNF-α.

Composites of yeast glucan particles and curcumin lead to improvement of dextran sulfate sodium-induced acute bowel inflammation in rats.

The goal of this work was to assess the usability of yeast glucan particles (GPs) as carriers for curcumin and determine the beneficial effect of a pharmacological composite of curcumin in GPs on dextran sulfate sodium induced colitis in rats. The assessment of the anti-inflammatory effect of particular substances was evaluated on the basis of the calculated disease activity index and by assessment of cytokines and enzymes from the gut tissue - tumor necrosis factor α (TNF-α), transforming growth factor ß1, interleukin (IL)-1ß, IL-6, IL-10, IL-17, catalase, superoxide dismutase 2, myeloperoxidase (MPO), and matrix metalloproteinase 9. Composites of GPs with incorporated curcumin showed promising results with the capability to lower symptoms of colitis and significantly decrease the production of pro-inflammatory cytokines TNF-α, IL-1ß, IL-6, and the activity of MPO, as well. The anti-inflammatory effect of the composites was greater than those of pure GPs or curcumin.

Encapsulation of poorly soluble drugs in yeast glucan particles by spray drying improves dispersion and dissolution properties.

In this work, novel amorphous solid dispersions based on yeast glucan particles were produced. Yeast glucan particles are hollow and porous, and they are mainly composed of amorphous polysaccharides. We hypothesized that these particles are suitable candidates for the amorphization of drugs with low water solubility. Model drugs ibuprofen and curcumin were successfully encapsulated in glucan particles by spray drying. Different spray-drying parameters were tested to evaluate the influence of atomizing droplet size and initial solid content on encapsulation efficiency. It was shown that higher solid content and, more significantly, larger droplet sizes lead to higher encapsulation efficiencies. The encapsulation efficiency of ibuprofen (10 wt%) into glucan particles was considerably improved from 41.3 ± 0.5% to 64.3 ± 0.2% by increasing initial solid content and droplet size with the two-fluid nozzle. The spray drying process was further optimized by using the ultrasonic nozzle and it was possible to achieve complete encapsulation of ibuprofen and curcumin without any precipitation of the active compound outside of the glucan particles. Overall, it was possible to produce completely amorphous composites with outstanding wettability and dispersion properties, and with significantly faster dissolution rates when compared to the micronized crude drug.

Glucan particles as suitable carriers for the natural anti-inflammatory compounds curcumin and diplacone - Evaluation in an ex vivo model.

Natural compounds offer a wide spectrum of potential active substances, but often they have a poor bioavailability. To increase the bioavailability and bioactivity of the natural anti-inflammatory molecules curcumin and diplacone, we used glucan particles (GPs), hollow shells from Saccharomyces cerevisiae composed mainly of ß-1,3-d-glucan. Their indigestibility and relative stability in the gut combined with their immunomodulatory effects makes them promising carriers for such compounds. This study aimed to determine how curcumin and diplacone, either alone or incorporated in GPs, affect the immunomodulatory activity of the latter by assessing the respiratory burst response and the secretion of pro-inflammatory cytokines by primary porcine innate immune cells. Incorporating curcumin and diplacone into GPs by controlled evaporation of the organic solvent substantially reduced the respiratory burst response mediated by GPs. Incorporated curcumin in GPs also reduced GPs mediated secretion of IL-1ß and TNF-α by innate immune cells. The obtained results indicate a potentially beneficial effect of the incorporation of curcumin or diplacone into GPs against inflammation.

Curcumin encapsulation in yeast glucan particles promotes its anti-inflammatory potential in vitro.

Glucan particles (GPs) from Saccharomyces cerevisiae are hollow shells that are composed mainly of ß-1,3-d-glucan, which has demonstrated immunomodulatory and anti-inflammatory potential both in vitro and in vivo. Curcumin is a natural hydrophobic phenolic compound, which possesses a significant anti-inflammatory effect and is used as supportive therapy in the treatment of many inflammatory diseases. The aim of this study is to evaluate the possible synergic effect and other benefits of the co-application of GPs and curcumin in the form of pharmaceutical composites. GP/curcumin composites were prepared using controlled evaporation of the organic solvent and their anti-oxidative effect and anti-inflammatory potential were tested on THP1­XBlue™­MD2­CD14 human monocytes cell line. The anti-oxidative effect was measured on pyocyanin-stimulated cells in vitro and the NF-κB/AP-1 signaling pathway on lipopolysaccharide pre-treated monocytes was chosen for anti-inflammatory assays. The secretion of pro-inflammatory cytokines TNF-α and IL-1ß was evaluated as well. Results mostly showed a pro-oxidative activity of empty GPs, however, pharmaceutical composites demonstrated an anti-oxidative effect. The activity of NF-κB/AP-1 was substantially decreased by the tested GP/curcumin composites, which also caused the attenuation of cytokines secretion. The obtained results indicate a beneficial effect of the incorporation of curcumin into GPs.

Magnetically Controlled Liposome Aggregates for On-Demand Release of Reactive Payloads.

A colloidal system able to act as a miniature reactor for on-demand release of reactive payloads has been demonstrated. The system is based on submicrometer aggregates consisting of anionic liposomes that act as storage reservoirs for the reactants, superparamagnetic iron oxide nanoparticles (SPIONs) that enable magnetic positioning in space and controlled release of reactants from the liposomes by radiofrequency stimulation, and an oppositely charged polyelectrolyte (poly-l-lysine) that keeps the constituent elements within the aggregates at a defined ratio. The kinetics of liposome-PLL-SPION heteroaggregation was systematically mapped and a suitable composition of the liposome bilayer was found such that the system exhibits stability at ambient conditions and radiofrequency triggered release at physiological temperature. The functionality of the system was demonstrated using a reaction between resazurin and ascorbic acid. The ability to release the reactants on-demand at defined time points was demonstrated. The system opens up opportunities for the controlled local delivery of unstable of highly bioactive molecules produced in situ and on demand from stable precursors.

Effect of lipid nanoparticle formulations on skin delivery of a lipophilic substance.

The aim of this study was to follow the skin penetration of a model lipophilic compound (Nile red) delivered by nanoparticulate carriers, the so-called lipid nanocapsules. The nanocapsules consisting of an oil core stabilized by amixture of surfactants were prepared by the phase inversion temperature method. Varying the particle composition (the oil/surfactant ratio) nanoparticles of different size were prepared and characterized. The penetration profile of Nile red delivered into the porcine skin by the nanoparticles compared to non-particulate samples was determined using fluorescence microscopy combined with a novel, statistically robust quantitative image analysis method. This study demonstrated that lipid nanoparticles promoted the skin penetration of encapsulated Nile red in comparison with all the non-particulate samples. Nile red delivered by the lipid-based nanoparticles was able to diffuse across the stratum corneum and partition itself uniformly in the epidermis. No relationship between Nile red penetration into the skin and the particle size was found. Moreover, the presence of sodium chloride in the water phase had a negative impact on the Nile red penetration into the skin. The results indicate that the physico-chemical circumstances of the nanoparticulate formulation play the major role in the penetration of lipophilic substances into the skin.

Non-invasive insight into the release mechanisms of a poorly soluble drug from amorphous solid dispersions by confocal Raman microscopy.

In this study, we investigated the release mechanism of the poorly water soluble drug aprepitant from different amorphous solid dispersions using confocal Raman microscopy (CRM). Solid dispersions were fabricated based on either Soluplus®, as an amphiphilic copolymer and solubilizer, or on polyvinylpyrrolidone, as a hydrophilic polymer, in order to elucidate the influence of the polymer characteristics on the drug form and dissolution mechanisms. Aprepitant exhibited its amorphous form in both solid dispersions. However, the release differed depending on the polymer. The high complexation effect of Soluplus was shown to be a crucial factor for stabilization of the amorphous drug, resulting in continuous release without any recrystallization of aprepitant. In contrast, solid dispersions based on polyvinylpyrrolidone showed a different mechanism of dissolution; due to the good affinity of PVP and water, the polymer is dissolving fast, leading to phase separation and local recrystallization of the drug. The study highlights the complexity of release processes from solid dispersions and elucidates the influence of the polymer on drug release kinetics.

Nanocrystals for dermal penetration enhancement - Effect of concentration and underlying mechanisms using curcumin as model.

Nanocrystals have received considerable attention in dermal application due to their ability to enhance delivery to the skin and overcome bioavailability issues caused by poor water and oil drug solubility. The objective of this study was to investigate the effect of nanocrystals on the mechanism of penetration behavior of curcumin as a model drug. Curcumin nanocrystals were produced by the smartCrystals® process, i.e. bead milling followed by high pressure homogenization. The mean particle size of the curcumin crystals was about 200nm. Stabilization was performed with alkyl polyglycoside surfactants. The distribution of curcumin within the skin was determined in vitro on cross-sections of porcine skin and visualized by fluorescent microscopy. The skin penetration profile was analyzed for the curcumin nanosuspension with decreasing concentrations (2%, 0.2%, 0.02% and 0.002% by weight) and compared to nanocrystals in a viscous hydroxypropylcellulose (HPC) gel. This study demonstrated there was minor difference between low viscous nanosuspension and the gel, but low viscosity seemed to favor skin penetration. Localization of curcumin was observed in the hair follicles, also contributing to skin uptake. Looking at the penetration of curcumin from formulations with decreasing nanocrystal concentration, formulations with 2%, 0.2% and 0.02% showed a similar penetration profile, whereas a significantly weaker fluorescence was observed in the case of a formulation containing 0.002% of curcumin nanocrystals. In this study we have shown that curcumin nanocrystals prepared by the smartCrystal® process are promising carriers in dermal application and furthermore, we identified the ideal concentration of 0.02% nanocrystals in dermal formulations. The comprehensive study of decreasing curcumin concentration in formulations revealed that the saturation solubility (Cs) is not the only determining factor for the penetration. A new mechanism based also on the concentration of the nanocrystals on skin surface was proposed.

Encapsulation stability and temperature-dependent release kinetics from hydrogel-immobilised liposomes.

Composite microparticles consisting of a calcium alginate gel matrix with embedded liposomes made from cholesterol:DPPC (dipalmitoylphosphatidylcholine) mixtures were considered. Factors affecting the encapsulation stability of liposomes during the gel formation by ionic cross-linking--namely temperature and the cholesterol:DPPC ratio--were systematically investigated. The liposomes were found to be tolerant to Ca(2+) ions during cross-linking of the gel and stable in the hydrogel matrix for extended periods of time when cholesterol was present in the phospholipid bilayer and temperature was kept sufficiently below the phase transition. The temperature-controlled release rate of encapsulated fluorescent dye was quantified. It is shown that a defined quantity of encapsulated substance can be repeatedly released from the embedded liposomes "on-demand" by short temperature pulses of suitably chosen duration and amplitude. This makes the hydrogel-liposome composites potential candidates for applications such as controlled drug delivery or study of reaction-diffusion phenomena in compartmentalised systems.

Biologically triggered liberation of sub-micron particles from alginate microcapsules.

A new method for triggering the burst liberation of encapsulated sub-micron particles from carrier particles using embedded microorganisms has been developed. Triggering mechanisms such as those based on chemical, light, thermal, or magnetic stimuli are known, but man-made particles are not yet able to replicate the concept of "dormancy" found in biological systems in the form of spores or seeds that survive in an inactive state and start to grow only once favourable environmental conditions are encountered. An engineered particle system that mimics this property by embedding viable yeast cells into synthetically made alginate microcapsules is reported in the present work. Cell growth and division is used as a triggering mechanism for stimuli-responsive release of the encapsulated content. The hybrid living/artificial capsules were formed by an inkjet printing process and the mechanism of biologically triggered release was shown using fluorescently labelled liposomes.

Reversible buckling and diffusion properties of silica-coated hydrogel particles.

The structure and diffusion properties of composite particles consisting of a calcium alginate hydrogel core and a thin SiO(2) surface layer have been investigated. The composite particles were formed by depositing a silica layer onto calcium alginate cores using a sol-gel process starting from alkoxysilane precursors. The composite particles were found to have a remarkable ability to reversibly rehydrate and return to their original size and shape after partial drying. The organo-silica skin was able to sustain large local deformations (such as complete folding) without the formation of cracks or defects. Such mechanical properties are uncharacteristic of pure silica and they can be attributed to the specific microstructure of the alginate-silica composite. The structure and composition of the alginate-silica particles were characterised by SEM, X-ray micro-tomography, Laser Scanning Confocal Microscopy and Thermo-gravimetry. In order to quantify the effect of the organo-silica layer on the diffusional transport into and out of the alginate particles, the uptake and release rates of several test molecules with increasing molecular weight were measured for both un-coated and silica-coated particles. While the diffusion rate of small and medium-size molecules (water, vitamin B12) was essentially unaffected by the presence of the silica layer, the diffusion rate of a larger biomolecule (lysozyme) was found to be slowed down by the presence of the surface layer. The flexibility of the organo-silica layer combined with the ability of even large biomolecules to diffuse through it indicate that the silica layer is macroporous, formed by individual SiO(2) nanoparticles dispersed and immobilised in the surface layer of the alginate hydrogel.

Investigation of internal microstructure and thermo-responsive properties of composite PNIPAM/silica microcapsules.

Composite microcapsules consisting of a thermo-responsive hydrogel poly-N-isopropylacrylamide (PNIPAM) and coated by silica (SiO(2)) nanoparticles have been synthesized by the inverse Pickering emulsion polymerization method. The composite capsules, whose mean diameter is in the 25-86 microm range in the expanded state, were characterized by static light scattering, atomic force microscopy (AFM), scanning electron microscopy (SEM), and laser scanning confocal microscopy (LSCM). It is reported that the hydrogel surface is uniformly covered by a monolayer of silica nanoparticles and that depending on the capsule size and degree of polymer cross-linking, either hollow-core or partially-filled hydrogel-core microcapsules can be created. Equilibrium thermo-responsive behavior of the composite microcapsules is investigated and it is found that after heating the particles above the lower critical solution temperature (LCST) of PNIPAM, the shrinkage ratio V/V(max) varies from 0.8 to 0.4 for a cross-linking ratio from 0.6% to 9% on a mass basis. Dynamic temperature cycling studies reveal no hysteresis in the shrinking and recovery phases, but a small measurable dependence of the asymptotic shrinkage ratio V/V(max) on the rate of temperature change exists. The composite capsules are stable under long-term storage in both dried and hydrated states and easily re-dispersible in water.