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Directing nanoparticles to the nucleus by attachment of nuclear localization sequences (NLS) is an aim in many applications. Gold nanoparticles modified with two different NLS were studied while crossing barriers of intact cells, including uptake, endosomal escape, and nuclear translocation. By imaging of the nanoparticles and by characterization of their molecular interactions with surface-enhanced Raman scattering (SERS), it is shown that nuclear translocation strongly depends on the particular incubation conditions. After an 1 h of incubation followed by a 24 h chase time, 14 nm gold particles carrying an adenoviral NLS are localized in endosomes, in the cytoplasm, and in the nucleus of fibroblast cells. In contrast, the cells display no nanoparticles in the cytoplasm or nucleus when continuously incubated with the nanoparticles for 24 h. The ultrastructural and spectroscopic data indicate different processing of NLS-functionalized particles in endosomes compared to unmodified particles. NLS-functionalized nanoparticles form larger intraendosomal aggregates than unmodified gold nanoparticles. SERS spectra of cells with NLS-functionalized gold nanoparticles contain bands assigned to DNA and were clearly different from those with unmodified gold nanoparticles. The different processing in the presence of an NLS is influenced by a continuous exposure of the cells to nanoparticles and an ongoing nanoparticle uptake. This is supported by mass-spectrometry-based quantification that indicates enhanced uptake of NLS-functionalized nanoparticles compared to unmodified particles under the same conditions. The results contribute to the optimization of nanoparticle analysis in cells in a variety of applications, e.g., in theranostics, biotechnology, and bioanalytics.

The penetration of topically applied tacrolimus formulated in micelles into murine skin is reported, measured by X-ray microscopy. Tacrolimus and micelles are probed for the first time by this high spatial resolution technique by element-selective excitation in the C 1s- and O 1s-regimes. This method allows selective detection of the distribution and penetration depth of drugs and carrier molecules into biologic tissues. It is observed that small, but distinct quantities of the drug and micelles, acting as a drug carrier, penetrate the stratum corneum. A comparison is made with the paraffin-based commercial tacrolimus ointment Protopic®, where local drug concentrations show to be low. A slight increase in local drug concentration in the stratum corneum is observed, if tacrolimus is formulated in micelles, as compared to Protopic®. This underscores the importance of the drug formulations for effective drug delivery. Time-resolved penetration shows presence of drug in the stratum corneum 100 min after formulation application, with penetration to deeper skin layers at 1000 min. High resolution micrographs give indications for a penetration pathway along the lipid membranes between corneocytes, but also suggest that the compound may penetrate corneocytes.

Loading of antigen on particles as well as the choice of skin as target organ for vaccination were independently described as effective dose-sparing strategies for vaccination. Combining these two strategies, sufficient antigen recognition may be achievable via the transcutaneous route even with minimal-invasive tools. Here, we investigated the skin penetration and cellular uptake of topically administered virus-like particles (VLPs), composed of the HIV-1 precursor protein Pr55gag, as well as the migratory activity of skin antigen-presenting cells (APCs). We compared VLP administration on ex vivo human skin pre-treated with cyanoacrylate tape stripping (CSSS, minimal-invasive) to administration by skin pricking and intradermal injection (invasive). CSSS as well as pricking treatments resulted in penetration of VLPs in the viable skin layers. Electron microscopy confirmed that at least part of VLPs remained intact during the penetration process. Flow cytometry of epidermal, dermal, and HLA-DR+ APCs harvested from culture media of skin explants cultivated at air-liquid interface revealed that a number of cells had taken-up VLPs. Similar results were found between invasive and minimal-invasive VLP application methods. CSSS pre-treatment was associated with significantly increased levels of IL-1α levels in cell culture media as compared to untreated and pricked skin. Our findings provide first evidence for effective cellular uptake of VLPs after dermal application and indicate that even mild physical barrier disruption, as induced by CSSS, provides stimulatory signals that enable the activation of APCs and uptake of large antigenic material.

The penetration of dexamethasone into human skin ex vivo is reported. X-ray microscopy is used for label-free probing of the drug and quantification of the local drug concentration with a spatial resolution reaching 70±5nm. This is accomplished by selective probing the dexamethasone by X-ray absorption. Varying the penetration time between 10min and 1000min provides detailed information on the penetration process. In addition, the stratum corneum has been damaged by tape-stripping in order to determine the importance of this barrier regarding temporally resolved drug penetration profiles. Dexamethasone concentrations distinctly vary, especially close to the border of the stratum corneum and the viable epidermis, where a local minimum in drug concentration is observed. Furthermore, near the basal membrane the drug concentration strongly drops. High spatial resolution studies along with a de-convolution procedure reveal the spatial distribution of dexamethasone in the interspaces between the corneocytes consisting of stratum corneum lipids. These results on local drug concentrations are interpreted in terms of barriers affecting the drug penetration in human skin.

Drugs that influence enzymes of lipid metabolism can cause pathological accumulation of lipids in animal cells. Here, gold nanoparticles, acting as nanosensors that deliver surface-enhanced Raman scattering (SERS) spectra from living cells provide molecular evidence of lipid accumulation in lysosomes after treatment of cultured cells with the three tricyclic antidepressants (TCA) desipramine, amitryptiline, and imipramine. The vibrational spectra elucidate to great detail and with very high sensitivity the composition of the drug-induced lipid accumulations, also observed in fixed samples by electron microscopy and X-ray nanotomography. The nanoprobes show that mostly sphingomyelin is accumulated in the lysosomes but also other lipids, in particular, cholesterol. The observation of sphingomyelin accumulation supports the impairment of the enzyme acid sphingomyelinase. The SERS data were analyzed by random forest based approaches, in particular, by minimal depth variable selection and surrogate minimal depth (SMD), shown here to be particularly useful machine learning tools for the analysis of the lipid signals that contribute only weakly to SERS spectra of cells. SMD is used for the identification of molecular colocalization and interactions of the drug molecules with lipid membranes and for discriminating between the biochemical effects of the three different TCA molecules, in agreement with their different activity. The spectra also indicate that the protein composition is significantly changed in cells treated with the drugs.

Label-free detection of core-multishell (CMS) nanocarriers and the anti-inflammatory drug dexamethasone is reported. Selective excitation by tunable soft X-rays in the O 1s-regime is used for probing either the CMS nanocarrier or the drug. Furthermore, the drug loading efficiency into CMS nanocarriers is determined by X-ray spectroscopy. The drug-loaded nanocarriers were topically applied to human skin explants providing insights into the penetration and drug release processes. It is shown that the core-multishell nanocarriers remain in the stratum corneum when applied for 100 min to 1000 min. Dexamethasone, if applied topically to human ex vivo skin explants using different formulations, shows a vehicle-dependent penetration behavior. Highest local drug concentrations are found in the stratum corneum as well as in the viable epidermis. If the drug is loaded to core-multishell nanocarriers, the concentration of the free drug is low in the stratum corneum and is enhanced in the viable epidermis as compared to other drug formulations. The present results provide insights into the penetration of drug nanocarriers as well as the mechanisms of controlled drug release from CMS nanocarriers in human skin. They are also compared to related work using dye-labeled nanocarriers and dyes that were used as model drugs.

The hepatic differentiation of human induced pluripotent stem cells (hiPSC) holds great potential for application in regenerative medicine, pharmacological drug screening, and toxicity testing. However, full maturation of hiPSC into functional hepatocytes has not yet been achieved. In this study, we investigated the potential of a dynamic three-dimensional (3D) hollow fiber membrane bioreactor technology to improve the hepatic differentiation of hiPSC in comparison to static two-dimensional (2D) cultures. A total of 100 × 106 hiPSC were seeded into each 3D bioreactor (n = 3). Differentiation into definitive endoderm (DE) was induced by adding activin A, Wnt3a, and sodium butyrate to the culture medium. For further maturation, hepatocyte growth factor and oncostatin M were added. The same differentiation protocol was applied to hiPSC maintained in 2D cultures. Secretion of alpha-fetoprotein (AFP), a marker for DE, was significantly (p

Loading of antigen on particles as well as the choice of skin as target organ for vaccination were independently described as effective dose-sparing strategies for vaccination. Combining these two strategies, sufficient antigen recognition may be achievable via the transcutaneous route even with minimal-invasive tools. Here, we investigated the skin penetration and cellular uptake of topically administered virus-like particles (VLPs), composed of the HIV-1 precursor protein Pr55

In this paper we present a comprehensive study for the ability of thermoresponsive nanogels (tNG) to act as cutaneous penetration enhancers. Given the unique properties of such molecular architectures with regard to their chemical composition and thermoresponsive properties, we propose a particular mode of penetration enhancement mechanism, i.e. hydration of the stratum corneum. Different tNG were fabricated using dendritic polyglycerol as a multifunctional crosslinker and three different kinds of thermoresponsive polymers as linear counterpart: poly(N-isopropylacrylamide) (pNIPAM), p(di(ethylene glycol) methyl ether methacrylate - co - oligo ethylene glycol methacrylate) (DEGMA-co-OEGMA475), and poly(glycidyl methyl ether - co - ethyl glycidyl ether) (tPG). Excised human skin was investigated by means of fluorescence microscopy, which enabled the detection of significant increment in the penetration of tNG as well as the encapsulated fluorescein. The morphology of the treated skin samples was thoroughly investigated by transmission electron microscopy and stimulated Raman spectromicroscopy. We found that tNG can perturbate the organization of both proteins and lipids in the skin barrier, which was attributed to tNG hydration effects. Interestingly, different drug delivery properties were detected and the ability of each investigated tNG to enhance skin penetration correlated well with the degree of induced stratum corneum hydration. The differences in the penetration enhancements could be attributed to the chemical structures of the nanogels used in this study. The most effective stratum corneum hydration was detected for nanogels having additional or more exposed polyether structure in their chemical composition.

The assumption that mesenchymal stromal cell (MSC)based-therapies are capable of augmenting physiological regeneration processes has fostered intensive basic and clinical research activities. However, to achieve sustained therapeutic success in vivo, not only the biological, but also the mechanical microenvironment of MSCs during these regeneration processes needs to be taken into account. This is especially important for e.g., bone fracture repair, since MSCs present at the fracture site undergo signifi cant biomechanical stimulation. This study has therefore investigated cellular characteristics and the functional behaviour of MSCs in response to mechanical loading. Our results demonstrated a reduced expression of MSC surface markers CD73 (ecto-5’-nucleotidase) and CD29 (integrin β1) after loading. On the functional level, loading led to a reduced migration of MSCs. Both effects persisted for a week after the removal of the loading stimulus. Specifi c inhibition of CD73/CD29 demonstrated their substrate dependent involvement in MSC migration after loading. These results were supported by scanning electron microscopy images and phalloidin staining of actin fi laments displaying less cell spreading, lamellipodia formation and actin accumulations. Moreover, focal adhesion kinase and Src-family kinases were identifi ed as candidate downstream targets of CD73/CD29 that might contribute to the mechanically induced decrease in MSC migration. These results suggest that MSC migration is controlled by CD73/CD29, which in turn are regulated by mechanical stimulation of cells. We therefore speculate that MSCs migrate into the fracture site, become mechanically entrapped, and thereby accumulate to fulfi l their regenerative functions.

Cultivation of primary human liver cells in innovative 3D perfusion multi-compartment capillary membrane bioreactors using decentralized mass exchange and integral oxygenation provides in vitro conditions close to the physiologic environment in vivo. While a few scale-up bioreactors were used clinically, inoculated liver progenitors in these bioreactors were not investigated. Therefore, we characterized regenerative processes and expression patterns of auto- and paracrine mediators involved in liver regeneration in bioreactors after patient treatment. Primary human liver cells containing parenchymal and non-parenchymal cells co-cultivated in bioreactors were used for clinical extra-corporeal liver support to bridge to liver transplantation. 3D tissue re-structuring in bioreactors was studied; expression of proteins and genes related to regenerative processes and hepatic progenitors was analyzed. Formation of multiple bile ductular networks and colonies of putative progenitors were observed within parenchymal cell aggregates. HGF was detected in scattered cells located close to vascular-like structures, expression of HGFA and c-Met was assigned to biliary cells and hepatocytes. Increased expression of genes associated to hepatic progenitors was detected following clinical application. The results confirm auto- and paracrine interactions between co-cultured cells in the bioreactor. The 3D bioreactor provides a valuable tool to study mechanisms of progenitor activation and hepatic regeneration ex vivo under patient plasma treatment.

Sustained proteinuria and tubulointerstitial damage have been closely linked with progressive renal failure. Upon excess protein endocytosis, tubular epithelial cells are thought to produce mediators that promote inflammation, tubular degeneration, and fibrosis. This concept was tested in a transgenic mouse model with megalin deficiency. Application of an anti-glomerular basement membrane serum to transgenic megalin-deficient mice [Cre(+)/GN] and megalin-positive littermates [Cre(-)/GN] produced the typical glomerulonephritis (GN) with heavy proteinuria in both groups. Tubulointerstitial damages correlated closely with glomerular damages in pooled Cre(+)/GN and Cre(-)/GN mice. Owing to a mosaic pattern of megalin expression in the mutant mice, Cre(+)/GN kidneys permitted side-by-side analysis of megalin-deficient and megalin-positive tubules in the same kidney. Protein endocytosis was found only in megalin-positive cells. TGF-beta, intercellular adhesion molecule, vascular cellular adhesion molecule, endothelin-1, and cell proliferation were high in megalin-positive cells, whereas apoptosis, heat-shock protein 25, and osteopontin were enhanced in megalin-deficient cells. No fibrotic changes were associated with either phenotype. Tubular degeneration with interstitial inflammation was found only in nephrons with extensive crescentic lesions at the glomerulotubular junction. In sum, enhanced protein endocytosis indeed led to an upregulation of profibrotic mediators in a megalin-dependent way; however, there was no evidence that endocytosis played a pathogenetic role in the development of the tubulointerstitial disease.

Selective probing of dexamethasone in excised human skin using soft X-ray spectromicroscopy provides quantitative concentration profiles as well as two-dimensional drug distribution maps. Element- and site-selective excitation of dexamethasone at the oxygen K-edge with the lateral step width adjusted to 1 μm provides detailed information on the location of the drug in the different skin layers. The key of this work is to probe dexamethasone selectively at the carbonyl site (C3) by the O 1s → π* transition, providing also a most efficient way to quantify the drug concentration as a function of penetration depth in correlation with structural properties of the skin containing carboxyl and amide oxygen sites occurring at higher transition energy than dexamethasone. Following drug exposure for 4 h, the glucocorticoide is located in about equal amounts in the stratum corneum, the outermost horny layer of skin, and in the viable epidermis, whereas in the dermis no dexamethasone is detected. In the stratum corneum, most of the lipophilic drug is found in regions between corneocytes, where epidermal lipids are dominating.

The accumulation of mitochondria containing mutated genomes was proposed to be an important factor involved in aging. Although the level of mutated mtDNA has shown to increase over time, it is currently not possible to directly measure the mtDNA mutation rate within living cells. The combination of mathematical modeling and controlled experiments is an alternative approach to obtain an estimate for the mutation rate in a well-defined system. In order to judge the relevance of mitochondrial mutations for the aging process, we used a mouse model to study age-related alterations of the mitochondrial proteins. Based on these experimental data we constructed a mathematical model of the mitochondrial population dynamics to estimate mtDNA mutation rates. Mitochondria were isolated from mouse brain and liver at six different ages (newborn to 24-months). A large-gel 2D-electrophoresis-based proteomics approach was used to analyze the mitochondrial proteins. The expression of two respiratory chain complex I subunits and one complex IV subunit decreased significantly with age. One subunit of complex III and one subunit of complex V increased in expression during aging. Together, these data indicate that complex I and IV deficiency in aged tissues might be accompanied by feedback regulation of other protein complexes in the respiratory chain. When we fitted our experimental data to the mathematical model, mtDNA mutation rate was estimated to be 2.7x10(-8) per mtDNA per day for brain and 3.2x10(-9) per mtDNA per day for liver. According to our model and in agreement with the mitochondrial theory of aging, mtDNA mutations could cause the detrimental changes seen in mitochondrial populations during the normal lifespan of mice, while at the same time ensure that the mitochondrial population remains functional during the developmental and reproductive period of mice.

Introduction. The development of a bioreactor providing a three-dimensional network of interwoven capillary membranes with integrated oxygenation and decentralized mass exchange enables the culture of primary human liver cells from discarded donor organs for extracorporeal liver support. Methods. Primary liver cells were isolated from 54 discarded organs (donor age 56.7±13.2 years). Between 2.8×10 10 and 6.4×10 10 parenchymal cells (PC) were cocultured with nonparenchymal cells (NPC) of the same organ in bioreactors (n=36). The metabolic activity of the cells was regularly determined during culture. The cell morphology and ultrastructure were investigated after culture periods of 1 to 5 weeks. Results. Cell metabolism was maintained over at least 3 weeks after a phase of adaptation lasting 2 to 3 days. Through the use of transmission electron microscopy and immunohistochemistry, it was demonstrated that PC and NPC spontaneously formed tissue-like structures. Vascular cavities (CD 31 immunoreactivity [IR]) and bile duct-like channels (CK 19 IR), both exhibiting proliferation activity (Ki-67 IR), were regularly distributed. Some of the bile duct-like channels showed similarities to the Canals of Hering found in the natural liver. Cells expressing morphologic and antigenic characteristics of adult liver stem cells (CD 34 IR and c-kit IR) and areas with cells that showed both hepatocyte and biliary characteristics were detected. Conclusion. The results show that primary human liver cells obtained from discarded donor organs recover and can be maintained in bioreactors for clinical liver support therapy. In addition, initial observations on adult liver stem-cell culture in bioreactors are presented.

The cellular response to nanoparticle exposure is essential in various contexts, especially in nanotoxicity and nanomedicine. Here, 14-nm gold nanoparticles in 3T3 fibroblast cells are investigated in a series of pulse-chase experiments with a 30-min incubation pulse and chase times ranging from 15 min to 48 h. The gold nanoparticles and their aggregates are quantified inside the cellular ultrastructure by laser ablation inductively coupled plasma mass spectrometry micromapping and evaluated regarding the surface-enhanced Raman scattering (SERS) signals. In this way, both information about their localization at the micrometre scale and their molecular nanoenvironment, respectively, is obtained and can be related. Thus, the nanoparticle pathway from endocytotic uptake, intracellular processing, to cell division can be followed. It is shown that the ability of the intracellular nanoparticles and their accumulations and aggregates to support high SERS signals is neither directly related to nanoparticle amount nor to high local nanoparticle densities. The SERS data indicate that aggregate geometry and interparticle distances in the cell must change in the course of endosomal maturation and play a critical role for a specific gold nanoparticle type in order to act as efficient SERS nanoprobe. This finding is supported by TEM images, showing only a minor portion of aggregates that present small interparticle spacing. The SERS spectra obtained after different chase times show a changing composition and/or structure of the biomolecule corona of the gold nanoparticles as a consequence of endosomal processing.