The Diagnostic Value of ACSL1, ACSL4, and ACSL5 and the Clinical Potential of an ACSL Inhibitor in Non-Small-Cell Lung Cancer.

Abnormal expression of ACSL members 1, 3, 4, 5, and 6 is frequently seen in human cancer; however, their clinical relevance is unclear. In this study, we analyzed the expression of ACSLs and investigated the effects of the ACSL inhibitor Triacsin C (TC) in lung cancer. We found that, compared to normal human bronchial epithelial (NHBE) cells, ACSL1, ACSL4, and ACSL6 were highly expressed, while ACSL3 and ACSL5 were lost in the majority of lung cancer cell lines. ACSL activity was associated with the expression levels of the ACSLs. In primary lung tumors, a higher expression of ACSL1, ACSL4, and ACSL5 was significantly correlated with adenocarcinoma (ADC). Moreover, ACSL5 was significantly reversely related to the proliferation marker Ki67 in low-grade tumors, while ACSL3 was positively associated with Ki67 in high-grade tumors. Combination therapy with TC and Gemcitabine enhanced the growth-inhibitory effect in EGFR wild-type cells, while TC combined with EGFR-TKIs sensitized the EGFR-mutant cells to EGFR-TKI treatment. Taken together, the data suggest that ACSL1 may be a biomarker for lung ADC, and ACSL1, ACSL4, and ACSL5 may be involved in lung cancer differentiation, and TC, in combination with chemotherapy or EGFR-TKIs, may help patients overcome drug resistance.

Long-Term Stability, Biocompatibility, and Magnetization of Suspensions of Isolated Bacterial Magnetosomes.

Magnetosomes are magnetic nanoparticles biosynthesized by magnetotactic bacteria. Due to a genetically strictly controlled biomineralization process, the ensuing magnetosomes have been envisioned as agents for biomedical and clinical applications. In the present work, different stability parameters of magnetosomes isolated from Magnetospirillum gryphiswaldense upon storage in suspension (HEPES buffer, 4 °C, nitrogen atmosphere) for one year in the absence of antibiotics are examined. The magnetic potency, measured by the saturation magnetization of the particle suspension, drops to one-third of its starting value within this year-about ten times slower than at ambient air and room temperature. The particle size distribution, the integrity of the surrounding magnetosome membrane, the colloidal stability, and the biocompatibility turn out to be not severely affected by long-term storage.

Histone demethylase KDM4C is a functional dependency in JAK2-mutated neoplasms.

Mutations of the JAK2 gene are frequent aberrations in the aging hematopoietic system and in myeloid neoplasms. While JAK-inhibitors efficiently reduce hyperinflammation induced by the constitutively active mutated JAK2 kinase, the malignant clone and abundance of mutated cells remains rather unaffected. Here, we sought to assess for genetic vulnerabilities of JAK2-mutated clones. We identified lysine-specific demethylase KDM4C as a selective genetic dependency that persists upon JAK-inhibitor treatment. Genetic inactivation of KDM4C in human and murine JAK2-mutated cells resulted in loss of cell competition and reduced proliferation. These findings led to reduced disease penetrance and improved survival in xenograft models of human JAK2-mutated cells. KDM4C deleted cells showed alterations in target histone residue methylation and target gene expression, resulting in induction of cellular senescence. In summary, these data establish KDM4C as a specific dependency and therapeutic target in JAK2-mutated cells that is essential for oncogenic signaling and prevents induction of senescence.

ITIH5 shows tumor suppressive properties in cervical cancer cells grown as multicellular tumor spheroids.

Cervical cancer (CC) arises from premalignant cervical intraepithelial neoplasia (CIN) induced by a persistent infection with human papillomaviruses. The multi-stepwise disease progression is driven by genetic and epigenetic alterations. Our previous studies demonstrated a clear downregulation of inter-α-trypsin-inhibitor-heavy chain 5 (ITIH5) at mRNA and protein levels in CC compared to CIN2/3 and normal cervical tissue. Initial in vitro functional analyses revealed a suppressive effect of ITIH5 on relevant mechanisms for cancer progression in conventional two dimensional (2D) cell culture model systems. Based on these studies, we aimed to investigate the functional relevance of ITIH5 in multicellular tumor spheroid (MCTS) models, which resemble in vivo tumors more closely. We successfully established CC cell line-derived MCTS using the hanging-drop technique. ITIH5 was ectopically overexpressed in HeLa and SiHa cells and its functional relevance was investigated under three dimensional (3D) culture conditions. We found that ITIH5 re-expression significantly suppressed tumor spheroid growth and spheroid invasiveness of both HeLa and SiHa spheroids. Immunohistochemical (IHC) analyses revealed a significant reduction in Ki-67 cell proliferation index and CAIX-positive areas indicative for hypoxia and acidification. Furthermore, we observed an increase in cPARP-positive cells suggesting a higher rate of apoptosis upon ITIH5 overexpression. An effect of ITIH5 expression on the susceptibility of cervical MCTS towards cytostatic drug treatment was not observed. Collectively, these data uncover pronounced anti-proliferative effects of ITIH5 under 3D cell culture conditions and provide further functional evidence that the downregulation of ITIH5 expression during cervical carcinogenesis could support cancer development.

Biocompatibility, uptake and subcellular localization of bacterial magnetosomes in mammalian cells.

Magnetosomes represent biogenic, magnetic nanoparticles biosynthesized by magnetotactic bacteria. Subtle biological control on each step of biomineralization generates core-shell nanoparticles of high crystallinity, strong magnetization and uniform shape and size. These features make magnetosomes a promising alternative to chemically synthesized nanoparticles for many applications in the biotechnological and biomedical field, such as their usage as biosensors in medical diagnostics, as drug-delivery agents, or as contrast agents for magnetic imaging techniques. Thereby, the particles are directly applied to mammalian cells or even injected into the body. In the present work, we provide a comprehensive characterization of isolated magnetosomes as potential cytotoxic effects and particle uptake have not been well studied so far. Different cell lines including cancer cells and primary cells are incubated with increasing particle amounts, and effects on cell viability are investigated. Obtained data suggest a concentration-dependent biocompatibility of isolated magnetosomes for all tested cell lines. Furthermore, magnetosome accumulation in endolysosomal structures around the nuclei is observed. Proliferation rates are affected in the presence of increasing particle amounts; however, viability is not affected and doubling times can be restored by reducing the magnetosome concentration. In addition, we evidence magnetosome-cell interactions that are strong enough to allow for magnetic cell sorting. Overall, our study not only assesses the biocompatibility of isolated magnetosomes, but also evaluates effects on cell proliferation and the fate of internalized magnetosomes, thereby providing prerequisites for their future in vivo application as biomedical agents.

Reactive Nanoparticles Derived from Polysaccharide Phenyl Carbonates.

Polysaccharide (PS) based nanoparticles (NP) are of great interest for biomedical applications. A key challenge in this regard is the functionalization of these nanomaterials. The aim of the present work was the development of reactive PS-NP that can be coupled with an amino group containing compounds under mild aqueous conditions. A series of cellulose phenyl carbonates (CPC) and xylan phenyl carbonates (XPC) with variable degrees of substitution (DS) was obtained by homogeneous synthesis. The preparation of PS-NP by self-assembling of these hydrophobic derivatives was studied comprehensively. While CPC mostly formed macroscopic aggregates, XPC formed well-defined spherical NP with diameters around 100 to 200 nm that showed a pronounced long-term stability in water against both particle aggregation as well as cleavage of phenyl carbonate moieties. Using an amino group functionalized dye it was demonstrated that the novel XPC-NP are reactive towards amines. A simple coupling procedure was established that enables direct functionalization of the reactive NP in an aqueous dispersion. Finally, it was demonstrated that dye functionalized XPC-NP are non-cytotoxic and can be employed in advanced biomedical applications.

Towards standardized purification of bacterial magnetic nanoparticles for future in vivo applications.

Bacterial magnetosomes (MS) are well-defined membrane-enveloped single-domain iron oxide (magnetite) nanoparticles, which are susceptible to genetic and chemical engineering. Additionally, the possibility to manipulate these particles by external magnetic fields facilitates their application in biomedicine and biotechnology, e.g. as magnetic resonance imaging probes or for drug delivery purposes. However, current purification protocols are poorly characterized, thereby hampering standardized and reproducible magnetosome production and thus, reliable testing for in vivo applications. In that context, the establishment of reproducible particle isolation procedures as well as the identification of high quality control parameters and the evaluation of potential cytotoxic effects of purified particles are of major importance. In this study, we characterize a multi-step purification protocol for MS with regard to purity, iron content, size and polydispersity of magnetite particles. In addition, we address potential cytotoxic effects of isolated MS when incubated with mammalian cells. Overall, we provide a detailed overview of the process-structure relationship during the isolation of MS and thus, identify prerequisites for high-yield MS production and their future application in the biomedical and biotechnological field. STATEMENT OF SIGNIFICANCE: Magnetic nanoparticles are of increasing interest for a variety of biomedical and biotechnological applications. Due to their unprecedented material characteristics, bacterial magnetosomes represent a promising alternative to chemically synthesized iron oxide nanoparticles. As applications require well-defined, highly purified and fully characterized nanoparticles, reliable protocols are necessary for efficient and reproducible magnetosome isolation. In our study, we evaluate an improved magnetosome extraction procedure and monitor quality parameters such as particle size distribution, membrane integrity and purity of the suspension by a combination of physicochemical and biochemical methods. Furthermore, the cytotoxicity of the isolated magnetosomes is assessed using different cell lines. In summary, our study helps to establish prerequisites for many real-world applications of magnetosomes in the field of biotechnology and biomedicine.

Polymethine Dye-Functionalized Nanoparticles for Targeting CML Stem Cells.

In chronic myelogenous leukemia (CML), treatment with tyrosine kinase inhibitors (TKI) is unable to eradicate leukemic stem cells (LSC). Polymethine dye-functionalized nanoparticles can be internalized by specific cell types using transmembrane carrier proteins. In this study we investigated the uptake behavior of various polymethine dyes on leukemia cell lines and searched for carrier proteins that guide dye transport using RNA interference. The results show that the uptake of DY-635 is dependent on organic anion transport protein 1B3 (OATP1B3) in CML cells and immature myeloid precursor cells of CML patients. In contrast to nonspecific poly(lactide-co-glycolic acid) (PLGA) nanoparticle constructs, DY-635-functionalization of nanoparticles led to an uptake in CML cells. Investigation of these nanoparticles on bone marrow of CML patients showed a preferred uptake in LSC. The transcription of OATP1B3 is known to be induced under hypoxic conditions via the hypoxia-inducing factor 1 alpha (HIF1α), thus also in the stem cells niche. Since these cells have the potential to repopulate the bone marrow after CML treatment discontinuation, eliminating them by means of drug-loaded DY-635-functionalized PLGA nanoparticles deployed as a selective delivery system to LSC is highly relevant to the ongoing search for curative treatment options for CML patients.

Biocompatible Magnetic Fluids of Co-Doped Iron Oxide Nanoparticles with Tunable Magnetic Properties.

Magnetite (Fe3O4) particles with a diameter around 10 nm have a very low coercivity (Hc) and relative remnant magnetization (Mr/Ms), which is unfavorable for magnetic fluid hyperthermia. In contrast, cobalt ferrite (CoFe2O4) particles of the same size have a very high Hc and Mr/Ms, which is magnetically too hard to obtain suitable specific heating power (SHP) in hyperthermia. For the optimization of the magnetic properties, the Fe2+ ions of magnetite were substituted by Co2+ step by step, which results in a Co doped iron oxide inverse spinel with an adjustable Fe2+ substitution degree in the full range of pure iron oxide up to pure cobalt ferrite. The obtained magnetic nanoparticles were characterized regarding their structural and magnetic properties as well as their cell toxicity. The pure iron oxide particles showed an average size of 8 nm, which increased up to 12 nm for the cobalt ferrite. For ferrofluids containing the prepared particles, only a limited dependence of Hc and Mr/Ms on the Co content in the particles was found, which confirms a stable dispersion of the particles within the ferrofluid. For dry particles, a strong correlation between the Co content and the resulting Hc and Mr/Ms was detected. For small substitution degrees, only a slight increase in Hc was found for the increasing Co content, whereas for a substitution of more than 10% of the Fe atoms by Co, a strong linear increase in Hc and Mr/Ms was obtained. Mössbauer spectroscopy revealed predominantly Fe3+ in all samples, while also verifying an ordered magnetic structure with a low to moderate surface spin canting. Relative spectral areas of Mössbauer subspectra indicated a mainly random distribution of Co2+ ions rather than the more pronounced octahedral site-preference of bulk CoFe2O4. Cell vitality studies confirmed no increased toxicity of the Co-doped iron oxide nanoparticles compared to the pure iron oxide ones. Magnetic heating performance was confirmed to be a function of coercivity as well. The here presented non-toxic magnetic nanoparticle system enables the tuning of the magnetic properties of the particles without a remarkable change in particles size. The found heating performance is suitable for magnetic hyperthermia application.

Inhibition of bone morphogenetic protein signaling reduces viability, growth and migratory potential of non-small cell lung carcinoma cells.

PURPOSE: BMP signaling has an oncogenic and tumor-suppressing activity in lung cancer that makes the prospective therapeutic utility of BMP signaling in lung cancer treatment complex. A more in-depth analysis of lung cancer subtypes is needed to identify BMP-related therapeutic targets. We sought to examine the influence of BMP signaling on the viability, growth and migration properties of the cell line LCLC-103H, which originates from a large cell lung carcinoma with giant cells and an extended aneuploidy. METHODS: We used BMP-4 and LDN-214117 as agonist/antagonist system for the BMP receptor type I signaling. Using flow cytometry, wound healing assay, trans-well assay and spheroid culture, we examined the influence of BMP signaling on cell viability, growth and migration. Molecular mechanisms underlying observed changes in cell migration were investigated via gene expression analysis of epithelial-mesenchymal transition (EMT) markers. RESULTS: BMP signaling inhibition resulted in LCLC-103H cell apoptosis and necrosis 72 h after LDN-214117 treatment. Cell growth and proliferation are markedly affected by BMP signaling inhibition. Chemotactic motility and migratory ability of LCLC-103H cells were clearly hampered by LDN-214117 treatment. Cell migration changes after BMP signaling inhibition were shown to be coupled with considerable down-regulation of transcription factors involved in EMT, especially Snail. CONCLUSIONS: BMP signaling inhibition in LCLC-103H cells leads to reduced growth and proliferation, hindered migration and accelerated cell death. The findings contribute to the pool of evidence on BMP signaling in lung cancer with a possibility of introducing BMP signaling inhibition as a novel therapeutic approach for the disease.

Protein corona formation and its constitutional changes on magnetic nanoparticles in serum featuring a polydehydroalanine coating: effects of charge and incubation conditions.

The inevitable formation of a protein corona upon contact of nanoparticles with different biological fluids is of great interest in the context of biomedical applications. It is well established that the surface chemistry of the respective nanomaterial has tremendous impact on protein adsorption, both in terms of the actual amount as well as the type of proteins adsorbed. In that regard, especially polyzwitterions are discussed as coating materials as they are known to partially inhibit protein adsorption. We herein present comparative incubation studies on iron oxide nanoparticles (either single core (SPION) or multicore nanoparticles (MCNP)) after coating with either polyanionic or polyzwitterionic polymeric shells based on polydehydroalanine (PDha). Apart from varying surface charge and chemistry, also the influence of incubation time and temperature on the formation and composition of a protein corona upon exposure to fetal calf serum was investigated. The amounts of adsorbed biomolecules were determined using thermogravimetric analysis. SDS-PAGE experiments revealed information on protein composition as major components of the biomolecule corona. Our results show that distinctly lower amounts of proteins are adsorbed onto polyzwitterionic hybrid nanoparticles in general, but also the corona composition varies as indicated by elevated relative ratios of medium molecular weight proteins (i.e. proteins 25-100 kDa) estimated by non-specific silver protein staining. In addition, increasing relative amounts of albumin (67 kDa) via specific Western blot assays on PDha-coated MCNP are detected.

Integrative genomic profiling of large-cell neuroendocrine carcinomas reveals distinct subtypes of high-grade neuroendocrine lung tumors.

Pulmonary large-cell neuroendocrine carcinomas (LCNECs) have similarities with other lung cancers, but their precise relationship has remained unclear. Here we perform a comprehensive genomic (n = 60) and transcriptomic (n = 69) analysis of 75 LCNECs and identify two molecular subgroups: "type I LCNECs" with bi-allelic TP53 and STK11/KEAP1 alterations (37%), and "type II LCNECs" enriched for bi-allelic inactivation of TP53 and RB1 (42%). Despite sharing genomic alterations with adenocarcinomas and squamous cell carcinomas, no transcriptional relationship was found; instead LCNECs form distinct transcriptional subgroups with closest similarity to SCLC. While type I LCNECs and SCLCs exhibit a neuroendocrine profile with ASCL1high/DLL3high/NOTCHlow, type II LCNECs bear TP53 and RB1 alterations and differ from most SCLC tumors with reduced neuroendocrine markers, a pattern of ASCL1low/DLL3low/NOTCHhigh, and an upregulation of immune-related pathways. In conclusion, LCNECs comprise two molecularly defined subgroups, and distinguishing them from SCLC may allow stratified targeted treatment of high-grade neuroendocrine lung tumors.

Magnetic Nanoparticles Interact and Pass an In Vitro Co-Culture Blood-Placenta Barrier Model.

Magnetic nanoparticles are interesting tools for biomedicine. Before application, critical prerequisites have to be fulfilled. An important issue is the contact and interaction with biological barriers such as the blood-placenta barrier. In order to study these processes in detail, suitable in vitro models are needed. For that purpose a blood-placenta barrier model based on the trophoblast-like cell line BeWo and primary placenta-derived pericytes was established. This model was characterized by molecular permeability, transepithelial electrical resistance and cell-cell-contact markers. Superparamagnetic iron oxide nanoparticles (SPIONs) with cationic, anionic or neutral surface charge were applied. The localization of the nanoparticles within the cells was illustrated by histochemistry. The time-dependent passage of the nanoparticles through the BeWo/pericyte barrier was measured by magnetic particle spectroscopy and atomic absorption spectroscopy. Cationically coated SPIONs exhibited the most extensive interaction with the BeWo cells and remained primarily in the BeWo/pericyte cell layer. In contrast, SPIONs with neutral and anionic surface charge were able to pass the cell layer to a higher extent and could be detected beyond the barrier after 24 h. This study showed that the mode of SPION interaction with and passage through the in vitro blood-placenta barrier model depends on the surface charge and the duration of treatment.

Influence of Sterilization and Preservation Procedures on the Integrity of Serum Protein-Coated Magnetic Nanoparticles.

Protein-coated magnetic nanoparticles are promising candidates for various medical applications. Prior to their application into a biological system, one has to guarantee that the particle dispersions are free from pathogens or any other microbiologic contamination. Furthermore, to find entrance into clinical routine, the nanoparticle dispersions have to be storable for several months. In this study, we tested several procedures for sterilization and preservation of nanoparticle containing liquids on their influence on the integrity of the protein coating on the surface of these particles. For this, samples were treated by freezing, autoclaving, lyophilization, and ultraviolet (UV) irradiation, and characterized by means of dynamic light scattering, determination of surface potential, and gel electrophoresis afterwards. We found that the UV sterilization followed by lyophilization under the addition of polyethylene glycol are the most promising procedures for the preparation of sterilized long-term durable protein-coated magnetic nanoparticles. Ongoing work is focused on the optimization of used protocols for UV sterilization and lyophilization for further improvement of the storage time.

RHEB1 insufficiency in aged male mice is associated with stress-induced seizures.

The mechanistic target of rapamycin (mTOR), a protein kinase, is a central regulator of mammalian metabolism and physiology. Protein mTOR complex 1 (mTORC1) functions as a major sensor for the nutrient, energy, and redox state of a cell and is activated by ras homolog enriched in brain (RHEB1), a GTP-binding protein. Increased activation of mTORC1 pathway has been associated with developmental abnormalities, certain form of epilepsy (tuberous sclerosis), and cancer. Clinically, those mTOR-related disorders are treated with the mTOR inhibitor rapamycin and its rapalogs. Because the effects of chronic interference with mTOR signaling in the aged brain are yet unknown, we used a genetic strategy to interfere with mTORC1 signaling selectively by introducing mutations of Rheb1 into the mouse. We created conventional knockout (Rheb1 +/- ) and gene trap (Rheb1 Δ/+ ) mutant mouse lines. Rheb1-insufficient mice with different combinations of mutant alleles were monitored over a time span of 2 years. The mice did not show any behavioral/neurological changes during the first 18 months of age. However, after aging (> 18 months of age), both the Rheb1 +/- and Rheb1 Δ /- hybrid males developed rare stress-induced seizures, whereas Rheb1 +/- and Rheb1 Δ /- females and Rheb1 Δ/+ and Rheb1 Δ/Δ mice of both genders did not show any abnormality. Our findings suggest that chronic intervention with mTORC1 signaling in the aged brain might be associated with major adverse events.

Human microRNA-299-3p decreases invasive behavior of cancer cells by downregulation of Oct4 expression and causes apoptosis.

PURPOSE: Oct4 was reported to be one of the most important pluripotency transcription factors in the biology of stem cells including cancer stem cells, and progressed malignant cells. Here we report the investigation of gene expression control of Oct4 by selected human microRNAs and the physiological effect of Oct4 silencing in invasive cancer cells. METHODS AND RESULTS: High throughput luciferase activity assay revealed the microRNA-299-3p to be the most effective in reducing gene expression of Oct4, which was confirmed by Western blot analysis and Oct4 promoter activity in a target luciferase assay. Furthermore, it could be demonstrated that downregulation of Oct4 by microRNAs-299-3p in breast cancer and fibrosarcoma cells lead to a decreased invasiveness in a microfluidic chip assay. Additionally, microRNA-299-3p causes apoptosis in cancer cells. Comparison with Oct4 specific siRNA transfection confirmed that this effect is primary due to the blockade of Oct4 expression. CONCLUSION: The results suggest that microRNA-299-3p is an interesting target for potential clinical use. It may be able to decrease invasive behaviour of carcinoma cells; or even kill these cells by causing apoptosis.

Comprehensive analysis of the in vitro and ex ovo hemocompatibility of surface engineered iron oxide nanoparticles for biomedical applications.

A set of biomedically relevant iron oxide nanoparticles with systematically modified polymer surfaces was investigated regarding their interaction with the first contact partners after systemic administration such as blood cells, blood proteins, and the endothelial blood vessels, to establish structure-activity relationships. All nanoparticles were intensively characterized regarding their physicochemical parameters. Cyto- and hemocompatibility tests showed that (1) the properties of the core material itself were not relevant in short-term incubation studies, and (2) toxicities increased with higher polymer mass, neutral = anionic < cationic surface charge and charge density, as well as agglomeration. Based on this, it was possible to classify the nanoparticles in three groups, to establish structure-activity relationships and to predict nanosafety. While the results between cyto- and hemotoxicity tests correlated well for the polymers, data were not fully transferable for the nanoparticles, especially in case of cationic low molar mass polymer coatings. To evaluate the prediction efficacy of the static in vitro models, the results were compared to those obtained in an ex ovo shell-less hen's egg test after microinjection under dynamic flow conditions. While the polymers demonstrated hemotoxicity profiles comparable to the in vitro tests, the size-dependent risks of nanoparticles could be more efficiently simulated in the more complex ex ovo environment, making the shell-less egg model an efficient alternative to animal studies according to the 3R concept.

Zwitterionic Iron Oxide (γ-Fe2 O3 ) Nanoparticles Based on P(2VP-grad-AA) Copolymers.

This study presents the synthesis and characterization of zwitterionic core-shell hybrid nanoparticles consisting of a core of iron oxide multicore nanoparticles (MCNPs, γ-Fe2 O3 ) and a shell of sultonated poly(2-vinylpyridine-grad-acrylic acid) copolymers. The gradient copolymers are prepared by reversible addition fragmentation chain transfer polymerization of 2-vinylpyridine (2VP), followed by the addition of tert-butyl acrylate and subsequent hydrolysis. Grafting of P(2VP-grad-AA) onto MCNP results in P(2VP-grad-AA)@MCNP, followed by quaternization using 1,3-propanesultone-leading to P(2VPS -grad-AA)@MCNP with a zwitterionic shell. The resulting particles are characterized by transmission electron microscopy, dynamic light scattering, and thermogravimetric analysis measurements, showing particle diameters of ≈70-90 nm and an overall content of the copolymer shell of ≈10%. Turbidity measurements indicate increased stability toward secondary aggregation after coating if compared to the pristine MCNP and additional cytotoxicity tests do not reveal any significant influence on cell viability.

Intentional formation of a protein corona on nanoparticles: Serum concentration affects protein corona mass, surface charge, and nanoparticle-cell interaction.

The protein corona, which immediately is formed after contact of nanoparticles and biological systems, plays a crucial role for the biological fate of nanoparticles. In the here presented study we describe a strategy to control the amount of corona proteins which bind on particle surface and the impact of such a protein corona on particle-cell interactions. For corona formation, polyethyleneimine (PEI) coated magnetic nanoparticles (MNP) were incubated in a medium consisting of fetal calf serum (FCS) and cell culture medium. To modulate the amount of proteins bind to particles, the composition of the incubation medium was varied with regard to the FCS content. The protein corona mass was estimated and the size distribution of the participating proteins was determined by means of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Additionally, the zeta potential of incubated particles was measured. Human blood-brain barrier-representing cell line HBMEC was used for in vitro incubation experiments. To investigate the consequences of the FCS dependent protein corona formation on the interaction of MNP and cells flow cytometry and laser scanning microscopy were used. Zeta potential as well as SDS-PAGE clearly reveal an increase in the amount of corona proteins on MNP with increasing amount of FCS in incubation medium. For MNP incubated with lower FCS concentrations especially medium-sized proteins of molecular weights between 30kDa and 100kDa could be found within the protein corona, whereas for MNP incubated within higher FCS concentrations the fraction of corona proteins of 30kDa and less increased. The presence of the protein corona reduces the interaction of PEI-coated MNP with HBMEC cells within a 30min-incubation.

RHEB1 expression in embryonic and postnatal mouse.

Ras homolog enriched in brain (RHEB1) is a member within the superfamily of GTP-binding proteins encoded by the RAS oncogenes. RHEB1 is located at the crossroad of several important pathways including the insulin-signaling pathways and thus plays an important role in different physiological processes. To understand better the physiological relevance of RHEB1 protein, the expression pattern of RHEB1 was analyzed in both embryonic (at E3.5-E16.5) and adult (1-month old) mice. RHEB1 immunostaining and X-gal staining were used for wild-type and Rheb1 gene trap mutant mice, respectively. These independent methods revealed similar RHEB1 expression patterns during both embryonic and postnatal developments. Ubiquitous uniform RHEB1/ß-gal and/or RHEB1 expression was seen in preimplantation embryos at E3.5 and postimplantation embryos up to E12.5. Between stages E13.5 and E16.5, RHEB1 expression levels became complex: In particular, strong expression was identified in neural tissues, including the neuroepithelial layer of the mesencephalon, telencephalon, and neural tube of CNS and dorsal root ganglia. In addition, strong expression was seen in certain peripheral tissues including heart, intestine, muscle, and urinary bladder. Postnatal mice have broad spatial RHEB1 expression in different regions of the cerebral cortex, subcortical regions (including hippocampus), olfactory bulb, medulla oblongata, and cerebellum (particularly in Purkinje cells). Significant RHEB1 expression was also viewed in internal organs including the heart, intestine, urinary bladder, and muscle. Moreover, adult animals have complex tissue- and organ-specific RHEB1 expression patterns with different intensities observed throughout postnatal development. Its expression level is in general comparable in CNS and other organs of mouse. Thus, the expression pattern of RHEB1 suggests that it likely plays a ubiquitous role in the development of the early embryo with more tissue-specific roles in later development.