Endocytotic uptake of iron oxide nanoparticles by cultured brain microglial cells

Microglia are the phagocytotic cells of the brain that respond rapidly to alterations in brain homeostasis. Since iron oxide nanoparticles (IONPs) are used for diagnostic and therapeutic applications in the brain, the consequences of an exposure of microglial cells to IONPs are of particular interest. To address this topic we have synthesized and characterized fluorescent BODIPY®-labelled IONPs (BP-IONPs). The average hydrodynamic diameter and the ζ-potential of BP-IONPs in water were ～65 nm and ?49 mV, respectively. Both values increased after dispersion of the particles in serum containing incubation medium to ～130 nm and ?8 mV. Exposure of cultured rat microglial cells with BP-IONPs caused a time-, concentration- and temperature-dependent uptake of the particles, as demonstrated by strong increases in cellular iron contents and cellular fluorescence. Incubation for 3 h with 150 and 450 μM iron as BP-IONPs increased the cellular iron content from a low basal level of ～50 nmol iron mg^?1 to 219 ± 52 and 481 ± 28 nmol iron (mg protein)^?1, respectively. These conditions did not affect cell viability, but exposure to higher concentrations of BP-IONPs or for longer incubation periods severely compromised cell viability. The BP-IONP fluorescence in viable microglial cells was co-localized with lysosomes. In addition, BP-IONP accumulation was lowered by 60% in the presence of the endocytosis inhibitors 5-(N-ethyl-N-isopropyl)amiloride, tyrphostin 23 and chlorpromazin. These results suggest that the rapid accumulation of BP-IONPs by microglial cells is predominantly mediated by macropinocytosis and clathrin-mediated endocytosis, which direct the accumulated particles into the lysosomal compartment.     

Size effect of amphiphilic poly(γ-glutamic acid) nanoparticles on cellular uptake and maturation of dendritic cells in vivo

We prepared size-regulated nanoparticles (NPs) composed of amphiphilic poly(γ-glutamic acid) (γ-PGA). In this study, 40, 100 and 200 nm γ-PGA-graft-l-phenylalanine ethylester (γ-PGA-Phe) NPs were employed. The size of NPs significantly influenced the uptake and activation behaviors of antigen-presenting cells (APCs). When 40 nm γ-PGA-Phe NPs were applied to these cells in vitro, they were highly activated compared with 100 and 200 nm NPs, while cellular uptake was size dependent. The size of the γ-PGA-Phe NPs also significantly affected their migration to the lymph nodes and uptake behavior of NPs by dendritic cells (DCs) in vivo. The 40 nm γ-PGA-Phe NPs migrated more rapidly to the lymph nodes and were taken up by a greater number of DCs compared with 100 and 200 nm NPs. On the other hand, when the amount of γ-PGA-Phe NPs taken up per DC was evaluated, it was higher for 100 and 200 nm NPs than for 40 nm NPs, which suggests that the larger γ-PGA-Phe NPs can deliver a large amount of antigen to a single DC compared with smaller NPs. Furthermore, when examined the maturation of DCs in lymph nodes, 40 nm γ-PGA-Phe NPs efficiently stimulated DCs. These results suggest that the activation, uptake behavior by APCs, migration to lymph nodes, and DC maturation can be controlled by the size of γ-PGA-Phe NPs.     

Poly(ethylene glycol) analogs grafted with low molecular weight poly(ethylene imine) as non-viral gene vectors

A novel class of non-viral gene vectors consisting of low molecular weight poly(ethylene imine) (PEI) (molecular weight 800 Da) grafted onto degradable linear poly(ethylene glycol) (PEG) analogs was synthesized. First, a Michael addition reaction between poly(ethylene glycol) diacrylates (PEGDA) (molecular weight 258 Da) and d,l-dithiothreitol (DTT) was carried out to generate a linear polymer (PEG–DTT) having a terminal thiol, methacrylate and pendant hydroxyl functional groups. Five PEG–DTT analogs were synthesized by varying the molar ratio of diacrylates to thiols from 1.2:1 to 1:1.2. Then PEI (800 Da) was grafted onto the main chain of the PEG–DTTs using 1,1′-carbonyldiimidazole as the linker. The above reaction gave rise to a new class of non-viral gene vectors, (PEG–DTT)–g-PEI copolymers, which can effectively complex DNA to form nanoparticles. The molecular weights and structures of the copolymers were characterized by gel permeation chromatography, ¹H nuclear magnetic resonance and Fourier transform infrared spectroscopy. The size of the nanoparticles was <200 nm and the surface charge of the nanoparticles, expressed as the zeta potential, was between +20 and +40 mV. Cytotoxicity assays showed that the copolymers exhibited much lower cytotoxicities than high molecular weight PEI (25 kDa). Transfection was performed in cultured HeLa, HepG2, MCF-7 and COS-7 cells. The copolymers showed higher transfection efficiencies than PEI (25 kDa) tested in four cell lines. The presence of serum (up to 30%) had no inhibitory effect on the transfection efficiency. These results indicate that this new class of non-viral gene vectors may be a promising gene carrier that is worth further investigation.     

The behavior of endothelial cells on polyurethane nanocomposites and the associated signaling pathways

A series of nanocomposites from polyurethane (PU) incorporated with various low concentrations (17.4–174 ppm) of gold nanoparticles (approximately 5 nm) (denoted “PU–Au”) were used as a model system to study the mechanisms that influenced endothelial cell (EC) migration on biomaterial surfaces. The migration rate of ECs on the PU–Au nanocomposites was determined by a real-time image system. It was found that ECs had the highest migration rate on the nanocomposite containing 43.5 ppm of gold (“PU–Au 43.5 ppm”). The high EC migration rate was associated with increased levels of endothelial nitric oxide synthase (eNOS) and phosphorylated-Akt (p-Akt) expressed by ECs cultured on PU–Au. The inductions of both eNOS and p-Akt on PU–Au were abolished by the addition of LY294002 (PI3K inhibitor), suggesting that these cellular events may be regulated through the PI3K signaling pathway. Using a biotinylated VEGF-165 that recognizes VEGF receptors and by FACS analysis, slightly higher expression of VEGF receptors for ECs on PU–Au was also demonstrated. Phalloidin staining showed that actin appeared as a circumferential band surrounding each cell on tissue culture polystyrene, whereas on PU–Au, especially on PU–Au 43.5 ppm, the cells had their margin spread out and extend processes with stress fibers in the protruding lamellipodia. Moreover, the higher EC migration rate on PU–Au 43.5 ppm was suppressed by LY294002. The higher protein expression of focal adhesion kinase (FAK) on PU–Au 43.5 ppm was observed in FAK-GFP transfected ECs. It was concluded that PU–Au nanocomposites activated FAK and the PI3K/Akt signaling pathway in ECs, leading to proliferation and migration of ECs on these surfaces.     

Short-chain PEG molecules strongly bound to magnetic nanoparticle for MRI long circulating agents

This study developed an approach for the synthesis of magnetic nanoparticles coated with three different polyethylene glycol (PEG)-derived molecules. The influence of the coating on different properties of the nanoparticles was studied. Magnetite nanoparticles (7 and 12 nm in diameter) were obtained via thermal decomposition of a coordination complex as an iron precursor to ensure nanoparticle homogeneity in size and shape. Particles were first coated with meso-2,3-dimercaptosuccinic acid by a ligand exchange process to remove oleic acid, followed by modification with three distinct short-chain PEG polymers, which were covalently bound to the nanoparticle surface via 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride activation of the carboxylic acids. In all cases, colloidal suspensions had hydrodynamic sizes <100 nm and low surface charge, demonstrating the effect of PEG coating on the aggregation properties and steric stabilization of the magnetic nanoparticles. The internalization and biocompatibility of these materials in the HeLa human cervical carcinoma cell line were tested. Cells preincubated with PEG-coated iron nanoparticles were visualized outside the cells, and their biocompatibility at high Fe concentrations was demonstrated using a standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Finally, relaxivity parameters (r₁ and r₂) were used to evaluate the efficiency of suspensions as magnetic resonance imaging contrast agents; the r₂ value was similar to that for Resovist and up to four times higher than that for Sinerem, probably due to the larger nanoparticle size. The time of residence in blood of the nanoparticles measured from the relaxivity values, and the Fe content in blood was doubled for rats and rabbits due to the PEG on the nanoparticle surface. The results suggest that this PEGylation strategy for large magnetic nanoparticles (>10 nm) holds promise for biomedical applications.     

Covalent attachment of Mn-porphyrin onto doxorubicin-loaded poly(lactic acid) nanoparticles for potential magnetic resonance imaging and pH-sensitive drug delivery

In this paper, theranostic nanoparticles (MnP-DOX NPs) were fabricated by conjugating Mn-porphyrin onto the surface of doxorubicin (DOX)-loaded poly(lactic acid) (PLA) nanoparticles (DOX NPs) for potential T₁ magnetic resonance imaging and pH-sensitive drug delivery. An in vitro drug release study showed that the release rate of DOX from MnP-DOX NPs was slow at neutral pH but accelerated significantly in acidic conditions. It was found that MnP-DOX NPs could be easily internalized by HeLa cells and effectively suppressed the growth of HeLa cells and HT-29 cells due to the accelerated drug release in acidic lysosomal compartments. Magnetic resonance imaging (MRI) scanning analysis demonstrated that MnP-DOX NPs had much higher longitudinal relaxivity in water (r₁ value of 27.8 mM^?1 s^?1 of Mn³⁺) than Mn-porphyrin (Mn(III)TPPS3NH₂; r₁ value of 6.70 mM^?1 s^?1 of Mn³⁺), behaving as an excellent contrast agent for T₁-weighted MRI both in vitro and in vivo. In summary, such a smart and promising nanoplatform integrates multiple capabilities for effective cancer diagnosis and therapy.     

PEGylated PEI-based biodegradable polymers as non-viral gene vectors

Novel functional biodegradable gene vectors, poly(l-succinimide)-g-polyethylenimines-g-poly(ethylene glycol) (PSI-g-PEI-g-PEGs) were synthesized by conjugating methoxy poly(ethylene glycol) (mPEG, M_w = 750 Da) to PEI segments (M_w = 800 Da) of PSI-g-PEI. The physicochemical properties of PSI-g-PEI-g-PEGs, including buffering capability, pDNA binding ability, cytotoxicity, zeta potential and the particle size of polymer/pDNA complexes, were explored. The influence of PEGylation was discussed based on a comparative study of PSI-g-PEI-g-PEGs, PSI-g-PEI and PEI25k (M_w = 25 kDa). SEM images revealed that PSI-g-PEI-g-PEG/pDNA particles have a regular shape with the diameter ranging from 70 to 170 nm. PEGylation could suppress the aggregation occurrence between complexes, resulting in a reduction of the polymer/pDNA complex size. PSI-g-PEI-g-PEGs exhibited remarkably lower cytotoxicity compared to PSI-g-PEI and PEI25k. In 293T and HeLa cells, the obtained PSI-g-PEI-g-PEGs showed very high transfection efficiency compared to PEI25k. Fluorescent confocal microscopy demonstrated that PSI-g-PEI-g-PEGs could effectively transport pGL-3 plasmids into the nuclei of HeLa cells. Taking into account the continued high transfection efficacy and decreased toxicity after PEG modification, PSI-g-PEI-g-PEGs show great potential as the non-viral vectors for gene transfection.     

Factors influencing the transfection efficiency of ultra low molecular weight chitosan/hyaluronic acid nanoparticles

The present work describes nanoparticles made of ultra low molecular weight chitosan (ULMWCh)/hyaluronic acid (HA) as novel potential carriers for gene delivery. Small and monodispersed nanoparticles with high in vitro transfection capabilities have been obtained by the complexation of these two polyelectrolytes. ULMWCh (<10 kDa) presents more advantageous characteristics over the higher molecular weight chitosan for clinical applications, namely increased solubility at physiological pH and improved DNA release. The ULMWCh:HA ratio and the HA molecular weights were varied with the aim of obtaining particles in the 100 nm range. Using chitosan (Ch) with a molecular weight of 5 kDa, HA with a molecular weight of 64 kDa, and a weight ratio of 4:1, nanoparticles with a Z-average size of 146 ± 1 nm and narrow size distribution (polydispersity index: 0.073 ± 0.030) were obtained. Nanoparticle images taken in dry conditions by SEM and AFM showed spherical particles. The optimal pH for transfection ranged from 6.4 to 6.8 for 0.25 μg of EGFP plasmid per well, with an incubation time of 4 h. Using these optimized parameters, DNA/ULMWCh:HA nanoparticles successfully transfected 25 ± 1% of the 293T cells with pEGFP, compared to 0.7% obtained for DNA/ULMWCh under the same conditions. This high transfection efficiency of our non-viral gene delivery system could be attributed to the synergic effect of ULMWCh and low charge density of the HA chain for easy release of DNA which makes the system suitable for targeted gene delivery.     

Evaluation of the activity of a novel metabotropic glutamate receptor antagonist (±)-2-amino-2-(3-cis and trans-carboxycyclobutyl-3-(9-thioxanthyl)propionic acid) in the in vitro neonatal spinal cord and in an in vivo pain model

The cyclobutylglycine (±)-2-amino-2-(3-cis and trans-carboxycyclobutyl-3-(9-thioxanthyl)propionic acid) (LY393053) has been identified as a functionally potent metabotropic glutamate receptor antagonist. It is most potent on the two group I metabotropic glutamate receptors, 1α and 5α, with IC₅₀ values of 1.0±0.4 μM and 1.6±1.4 μM, respectively. In this study, LY393053 has also been evaluated electrophysiologically on native group I metabotropic glutamate receptors in an in vitro spinal cord preparation as well as behaviourally, in a mouse model of visceral pain. LY393053 dose-dependently antagonised group I agonist, (RS)-3, 5-dihydroxyphenylglycine, or a broad-spectrum agonist (1S,3R)-amino-1,3-cyclopentanedicarboxylic acid-induced depolarisation of spinal motoneurons. The apparent K_d values were estimated to be 0.3 μM against (RS)-3, 5-dihydroxyphenylglycine-induced depolarisation and 0.5 μM against (1S,3R)-amino-1,3-cyclopentanedicarboxylic acid-induced depolarisation, respectively. On the other hand, the dorsal root–ventral root potential elicited at 8× threshold was depressed by LY393053 with IC₅₀ values of 9.0±0.7 μM and 12.7±1.7 μM on monosynaptic and polysynaptic responses, respectively. When investigated using the mouse acetic acid writhing test, LY393053 showed significant analgesic effects at doses of 1–10 mg/kg intraperitoneally. An ED₅₀ value of 6.0 mg/kg was obtained in this test.By revealing a potent effect of LY393053 in antagonising the native group I metabotropic receptor-mediated responses in the spinal cord in rodents, and an antinociceptive efficacy in a mouse visceral pain model, these results, therefore, provide additional evidence in support of the analgesic potential of metabotropic glutamate receptor antagonists.     

Administration of a polyvalent mechanical bacterial lysate to elderly patients with COPD: Effects on circulating T,B and NK cells

The modifications of the subsets of circulating lymphocytes were evaluated in a group of patients with COPD undergoing treatment with a polyvalent mechanical bacterial lysate (PMBL), a drug that is able to significantly modify the natural history of these patients. Using multicolor immune-florescence and flow cytometry, T, B subsets and NK cells were extensively studied both in the group of treated patients and in a disease and age matched controls. Despite the age, in treated patients, T and NK cells were significantly increased in numbers of circulating cells, but not in percentages, while B cells remained unmodified. CD3 + 4+ T cells were increased in treated patients, while CD3 + CD8 T cells were unmodified by the treatment. Activated T cells were increased but Treg, resulted reduced both in percentage than in absolute numbers. Transitional B cells resulted increased (in percentage and in absolute numbers) in their late maturation step (T3), while only early Naïve B cells were increased by the treatment, while other naïve subpopulations were unmodified. Memory B cells were reduced in percentage (but remained unmodified as absolute numbers), while the most immature form of memory B cells was significantly increased. Finally, both switch memory B cells and plasma cells resulted unmodified by the PMBL treatment. These results clearly indicated that the administration of the PMBL, even in elderly patients with COPD, was able to induce a significant immune-stimulation and these results, at cellular level, clearly support the evidence that the mechanism of action of PMBL is strictly related to a direct effect on immune-competent cells.     

A microfluidic device for continuous manipulation of biological cells using dielectrophoresis

The present study demonstrates the design, simulation, fabrication and testing of a label-free continuous manipulation and separation micro-device of particles/biological cells suspended on medium based on conventional dielectrophoresis. The current dielectrophoretic device uses three planner electrodes to generate non-uniform electric field and induces both p-DEP and n-DEP force simultaneously depending on the dielectric properties of the particles and thus influencing at least two types of particles at a time. Numerical simulations were performed to predict the distribution of non-uniform electric field, DEP force and particle trajectories. The device is fabricated utilizing the advantage of bonding between PDMS and SU8 polymer. The p-DEP particles move away from the center of the streamline, while the n-DEP particles will follow the central streamline along the channel length. Dielectrophoretic effects were initially tested using polystyrene beads followed by manipulation of HeLa cells. In the experiment, it was observed that polystyrene beads in DI water always response as n-DEP up to 1 MHz frequency, whereas HeLa cells in PBS medium response as n-DEP up to 400 kHz frequency and then it experiences p-DEP up to 1 MHz. Further, the microscopic observations of DEP responses of HeLa cells were verified by performing trapping experiment at static condition.     

Silica–collagen bionanocomposites as three-dimensional scaffolds for fibroblast immobilization

Silica–collagen bionanocomposite hydrogels were obtained by addition of silica nanoparticles to a protein suspension followed by neutralization. Electron microscopy studies indicated that larger silica nanoparticles (80 nm) do not interact strongly with collagen, whereas smaller ones (12 nm) form rosaries along the protein fibers. However, the composite network structurally evolved with time due to the contraction of the cells and the dissolution of the silica nanoparticles. When compared to classical collagen hydrogels, these bionanocomposite materials showed lower surface contraction in the short term (1 week) and higher viability of entrapped cells in the long term (3 weeks). A low level of gelatinase MMP2 enzyme expression was also found after this period. Several proteins involved in the catabolic and anabolic activity of the cells could also be observed by immunodetection techniques. All these data suggest that the bionanocomposite matrices constitute a suitable environment for fibroblast adhesion, proliferation and biological activity and therefore constitute an original three-dimensional environment for in vitro cell culture and in vivo applications, in particular as biological dressings.     

Synergistic effects of electrospun PLLA fiber dimension and pattern on neonatal mouse cerebellum C17.2 stem cells

Topographical features, including fiber dimensions and pattern, are important aspects in developing fibrous scaffolds for tissue engineering. In this study aligned poly(l-lactide) (PLLA) fibers with diameters of 307 ± 47, 500 ± 53, 679 ± 72 and 917 ± 84 nm and random fibers with diameters of 327 ± 40, 545 ± 54, 746 ± 82 and 1150 ± 109 nm were obtained by optimizing the electrospinning parameters. We cultured neonatal mouse cerebellum C17.2 cells on the PLLA fibers. These neural stem cells (NSCs) exhibited significantly different growth and differentiation depending upon fiber dimension and pattern. On aligned fibers cell viability and proliferation was best on 500 nm fibers, and reduced on smaller or larger fibers. However, on random fibers cell viability and proliferation was best with the smallest (350 nm) and largest (1150 nm) diameter fibers. Polarized and elongated cells were orientated along the fiber direction on the aligned fibers, with focal contacts bridging the cell body and aligned fibers. Cells of spindle and polygonal morphologies were randomly distributed on the random fibers, with no focal contacts observed. Moreover, longer neurites were obtained on the aligned fibers than random fibers within the same diameter range. Thus, the surface topographic morphologies of fibrous scaffolds, including fiber pattern, dimensions and mesh size, play roles in regulating the viability, proliferation and neurite outgrowth of NSCs. Nevertheless, our results indicated that aligned 500 nm fiber are most promising for fine tuning the design of a nerve scaffold.     

Cell micropatterning on superhydrophobic diamond nanowires

Cell micropatterning was achieved in a spatially controlled manner based on heterogeneously wetted superhydrophilic/superhydrophobic diamond nanowire (NW) surfaces. Diamond NWs were synthesized on boron-doped diamond substrates using reactive ion etching and functionalized with octadecyltrichlorosilane to achieve superhydrophobicity. Superhydrophilic motifs of 400 × 400 μm² and 10 × 10 μm² single cell-sized motifs, surrounded by superhydrophobic regions, were then generated by selectively exposing the substrates to UV light. This design allowed successful patterning of single HeLa and MCF-10A cells within the superhydrophilic regions without additional surface modification. To add a further level of complexity, micropatterned co-cultures were obtained using bovine serum albumin to promote cell adhesion. This method is simple and does not require any complicated processing steps such as mask deposition or template removal. Potential applications are in the development of cell-based biological assays in well-controlled and biologically relevant environments.     

The role of nanoparticle concentration-dependent induction of cellular stress in the internalization of non-toxic cationic magnetoliposomes

Magnetoliposomes (MLs), built up of ultrasmall iron oxide cores each individually surrounded by a lipid bilayer, have emerged as highly biocompatible nanoparticles and promising tools in many biomedical applications. To improve cell uptake, cationic amphiphiles are inserted into the ML coat, but this often induces cytotoxic effects. In the present work, we synthesized and tested a cationic peptide–lipid conjugate (dipalmitoylphosphatidylethanolamine-succinyl-tetralysine [DPPE-succ-(Lys)₄]) which is entirely composed of biodegradable moieties and specifically designed to exert minimal cytotoxic effects. Uptake studies with both murine 3T3 fibroblasts and C17.2 neural progenitor cells shows 95.63 ± 5.83 pg Fe and 87.46 ± 5.62 pg Fe per cell after 24 h, respectively, for 16.66% DPPE-succ-(Lys)₄-containing MLs, with no effect on cell viability. However, these high intracellular nanoparticle concentrations transiently affect actin cytoskeleton architecture, formation of focal adhesion complexes and cell proliferation, returning to control levels after approximately 7 days post ML-incubation in both cell types. This study points out the great need for thorough characterization of cell–nanoparticle interactions as subtle time-dependent effects are hard to monitor and commonly used viability and functionality assays are not sufficient to address the broad spectrum of possible interferences of the nanoparticle with normal cell functioning.     

Methotrexate,combined with cyclophosphamide attenuates murine collagen induced arthritis by modulating the expression level of Breg and DCs

To explore the mechanism of methotrexate (MTX) and its combination with cyclophosphamide (CTX) in collagen-induced arthritis (CIA), we investigated the levels of several immune cells and cytokines in mice with different treatments. CIA was induced in DBA/1 mice at the age of 7 weeks by primary immunization with 100 μl emulsion containing 2 mg/ml bovine type II collagen which was mixed with complete Freund's adjuvant (CFA). The booster immunization was performed with 50–100 μl emulsion containing 2 mg/ml bovine type II collagen (CII) mixed with incomplete Freund's adjuvant (IFA). MTX, CTX or both were administrated after the booster immunization. Therapeutic effect was evaluated by arthritic scores, X-rays and assessment of histopathological joint destruction. The expression of TNF-α, IL-6, IL-23, IL-10 were also measured. The frequencies of different immune cell subsets in the lymph node, spleen and bone marrow were determined by flow cytometry analysis. Our results showed that CTX and MTX treatment attenuated the severity of arthritis of CIA mice and reduced the levels of several cytokines. CTX and MTX treated mice showed a lower frequency of B cells in bone marrow. Also, when treated the CIA mice with MTX, alone or together with CTX, the lymph nodes and spleen exhibited a decrease in regulatory B cells (Breg) and dendritic cells (DCs). Notably, the combination of MTX and CTX had a more pronounced effect. By measuring the levels of different immune cells those participated in the development of rheumatoid arthritis (RA), our experiment may help to evaluate the therapeutic effects and prognosis of arthritic diseases.     

Protective effect of quercitrin against hydrogen peroxide-induced dysfunction in osteoblastic MC3T3-E1 cells

The protective effect of quercitrin on the response of osteoblastic MC3T3-E1 cells to oxidative stress was evaluated. Osteoblasts were incubated with H₂O₂ and/or quercitrin, and markers of osteoblast function and oxidative damage were examined. Quercitrin treatment reversed the cytotoxic effect of H₂O₂ significantly (P < 0.05). This effect was blocked by ICI182780 and LY294002, suggesting that quercitrin's effect might be involved in estrogen action and results from PI3K mediated signaling pathway. Pretreatment of quercitrin increased collagen content, alkaline phosphatase (ALP) activity, and calcium deposition of osteoblasts compared with H₂O₂ treated cells and these effects were blocked by ERKs and p38 mitogen-activated protein kinases (MAPKs) inhibitors such as PD98059 and SB203580, respectively. These suggest that quercitrin-induced protective effect against osteoblast dysfunction by oxidative stress is associated with increased activation of ERKs and p38 MAPK. Pretreatment with quercitrin also reduced the increase in bone-resorbing factor, receptor activator of nuclear factor-kB ligand (RANKL) and oxidative damage markers (malondialdehyde, protein carbonyl, and nitrotyrosine) induced by H₂O₂. These results suggest that quercitrin may be protective against H₂O₂-induced dysfunction in osteoblasts.     

Association of high HLA-E expression during acute cellular rejection and numbers of HLA class I leader peptide mismatches with reduced renal allograft survival

Non-classical Human Leukocyte Antigen (HLA)-E preferentially presents leader peptides derived from classical HLA-class I molecules. HLA-E can trigger opposed immune responses by interacting with inhibitory NKG2A or by activating NKG2C receptors on NK and T-cells. We studied the impact of HLA-E on renal allograft survival during acute cellular rejection. HLA-E expression was up-regulated in acute cellular rejection (ACR) biopsies (n = 12) compared to biopsies from 13 renal allografts with no rejection-signs. HLA-E up-regulation was correlated with numbers of HLA-class I leader peptide mismatches (p = 0.04). CD8+ and CD56+ infiltrating cells correlated with HLA-E expression (p < 0.0001 and p = 0.0009, respectively). Activating NKG2C receptor dominated on effector cells in biopsies and peripheral blood during ACR potentially allowing HLA-E-mediated immune activation. Moreover, HLA-E expression correlated with deterioration in renal allograft function (p < 0.008) and reduced allograft survival (p = 0.002). Our findings provide evidence that during renal allograft rejection HLA-E along with high numbers of mismatched HLA-class I leader peptides might represent additional targets for immune-activating responses.     

The penetration of fresh undiluted sputum expectorated by cystic fibrosis patients by non-adhesive polymer nanoparticles

Highly viscoelastic and adhesive sputum has precluded efficient nanoparticle-based drug and gene delivery to the lungs of patients with cystic fibrosis (CF). We sought to determine whether nanoparticles coated with non-mucoadhesive polymers could penetrate CF sputum, and to use these “muco-inert particles” (MIPs) as non-destructive nanoprobes to characterize the sputum microstructure. Particles as large as 200 nm in diameter that were densely coated with low MW polyethylene glycol (PEG) moved through undiluted CF sputum with average speeds up to 90-fold faster than similarly-sized uncoated particles. On the other hand, the transport of both coated and uncoated 500 nm particles was strongly hindered. The local viscosity of sputum, encountered by the fastest 10% of 200 nm MIPs, was only 5-fold higher than that of water, whereas the bulk viscosity was 10,000-fold higher at low shear rates. Using measured transport rates of various sized MIPs combined with an obstruction-scaling model, we determined that the average 3D mesh spacing of CF sputum is ～140 ± 50 nm (range: 60–300 nm). Taken together, these results demonstrate that nanoparticles up to 200 nm in diameter that do not adhere to CF sputum can move rapidly through this critical barrier by accessing pores that are filled with a low viscosity fluid. The results also offer hope that desperately needed sputum-penetrating drug- and gene-carrier nanoparticles can be developed for CF.     

Manganese ferrite nanoparticle micellar nanocomposites as MRI contrast agent for liver imaging

Iron oxide nanoparticles are effective contrast agents for enhancement of magnetic resonance imaging at tissue, cellular or even molecular levels. In this study, manganese doped superparamagnetic iron oxide (Mn-SPIO) nanoparticles were used to form ultrasensitive MRI contrast agents for liver imaging. Hydrophobic Mn-SPIO nanoparticles are synthesized in organic phase and then transferred into water with the help of block copolymer mPEG-b-PCL. These Mn-SPIO nanoparticles are self-assembled into small clusters (mean diameter ≈ 80 nm) inside micelles as revealed by transmission electron microscopy. Mn-SPIO nanoparticles inside micelles decrease PCL crystallization temperatures, as verified from differential scanning calorimetry and Fourier transform infrared spectroscopy. The Mn-SPIO based nanocomposites are superparamagnetic at room temperature. At the magnetic field of 1.5 T, Mn-SPIO nanoparticle clustering micelles have a T₂ relaxivity of 270 (Mn + Fe) mM^?1 s^?1, which is much higher than single Mn-SPIO nanoparticle containing lipid–PEG micelles. This clustered nanocomposite has brought significant liver contrast with signal intensity changes of ?80% at 5 min after intravenous administration. The time window for enhanced-MRI can last about 36 h with obvious contrast on liver images. This sensitive MRI contrast agent may find applications in identification of small liver lesions, evaluation of the degree of liver cirrhosis, and differential diagnosis of other liver diseases.