In this study, polyelectrolyte microcapsules have been fabricated by biocompatible ferrosoferric oxide nanoparticles (Fe3O4 NPs) and poly allyamine hydrochloride (PAH) using layer by layer assembly technique. The Fe3O4 NPs were prepared by chemical co-precipitation, and characterized by transmission electron microscopy (TEM) and infrared spectrum (IR). Quartz cell also was used as a substrate for building multilayer films to evaluate the capability of forming planar film. The result showed that Fe3O4 NPs were selectively deposited on the surface of quartz cell. Microcapsules containing Fe3O4 NPs were fabricated by Fe3O4 NPs and PAH alternately self-assembly on calcium carbonate microparticles firstly, then 0.2 molL(-1) EDTA was used to remove the calcium carbonate. Scanning electron microscopy (SEM), Zetasizer and vibrating sample magnetometer (VSM) were used to characterize the microcapsule's morphology, size and magnetic properties. The result revealed that Fe3O4 NPs and PAH were successfully deposited on the surface of CaCO3 microparticles, the microcapsule manifested superparamagnetism, size and saturation magnetization were 4.9 +/- 1.2 microm and 8.94 emu x g(-1), respectively. As a model drug, Rhodamin B isothiocyanate labeled bovine serum albumin (RBITC-BSA) was encapsulated in microcapsule depended on pH sensitive of the microcapsule film. When pH 5.0, drug add in was 2 mg, the encapsulation efficiency was (86.08 +/- 3.36) % and the drug loading was 8.01 +/- 0.30 mg x m(L-1).
Calcium Carbonate ; chemistry ; Capsules ; Chemical Precipitation ; Drug Carriers ; Drug Compounding ; methods ; Drug Delivery Systems ; Electrolytes ; chemistry ; Ferrosoferric Oxide ; chemistry ; Magnetite Nanoparticles ; Microscopy, Electron, Scanning ; Microscopy, Fluorescence ; Particle Size ; Rhodamines ; administration & dosage ; chemistry ; Serum Albumin, Bovine ; administration & dosage ; chemistry

Chronic granulomatous disease (CGD) is a rare genetic disease, which is caused by defects in the NADPH oxidase complex (gp91phox, p22phox, p40phox, p47phox, and p67phox) of phagocytes. This defect results in impaired production of superoxide anions and other reactive oxygen species (ROS), which are necessary for killing bacterial and fungal microorganisms and leads to recurrent, life-threatening bacterial and fungal infections and granulomatous inflammation. The dihydrorhodamine (DHR) flow cytometry assay is a useful diagnostic tool for CGD that can detect absent or reduced NADPH oxidase activity in stimulated phagocytes. We report a patient with X-linked CGD carrying a novel mutation of the CYBB gene whose chimerism status following hematopoietic stem cell transplantation (HSCT) has been rapidly determined using the DHR assay. The level of DHR activity correlates well with short tandem repeat PCR analysis. Considering the advantages of this simple, rapid, and cost-effective procedure, serial measurement of DHR assay would facilitate the rapid determination of a patient's engraftment status, as a supplementary monitoring tool of chimerism status following HSCT.
Base Sequence ; *Chimerism ; DNA Mutational Analysis ; Flow Cytometry ; Granulomatous Disease, Chronic/*diagnosis/*enzymology/genetics/surgery ; *Hematopoietic Stem Cell Transplantation ; Homozygote ; Humans ; Infant, Newborn ; Male ; Membrane Glycoproteins/chemistry/*genetics ; Mutation ; NADPH Oxidase/chemistry/*genetics ; Polymerase Chain Reaction ; Rhodamines/chemistry/metabolism

OBJECTIVE: To determine the feasibility of labeling human mesenchymal stem cells (hMSCs) with bifunctional nanoparticles and assessing their potential as imaging probes in the monitoring of hMSC transplantation. MATERIALS AND METHODS: The T1 and T2 relaxivities of the nanoparticles (MNP@SiO2[RITC]-PEG) were measured at 1.5T and 3T magnetic resonance scanner. Using hMSCs and the nanoparticles, labeling efficiency, toxicity, and proliferation were assessed. Confocal laser scanning microscopy and transmission electron microscopy were used to specify the intracellular localization of the endocytosed iron nanoparticles. We also observed in vitro and in vivo visualization of the labeled hMSCs with a 3T MR scanner and optical imaging. RESULTS: MNP@SiO2(RITC)-PEG showed both superparamagnetic and fluorescent properties. The r1 and r2 relaxivity values of the MNP@SiO2(RITC)-PEG were 0.33 and 398 mM-1 s-1 at 1.5T, respectively, and 0.29 and 453 mM-1 s-1 at 3T, respectively. The effective internalization of MNP@SiO2(RITC)-PEG into hMSCs was observed by confocal laser scanning fluorescence microscopy. The transmission electron microscopy images showed that MNP@SiO2(RITC)-PEG was internalized into the cells and mainly resided in the cytoplasm. The viability and proliferation of MNP@SiO2(RITC)-PEG-labeled hMSCs were not significantly different from the control cells. MNP@SiO2(RITC)-PEG-labeled hMSCs were observed in vitro and in vivo with optical and MR imaging. CONCLUSION: MNP@SiO2(RITC)-PEG can be a useful contrast agent for stem cell imaging, which is suitable for a bimodal detection by MRI and optical imaging.
Animals ; Biocompatible Materials ; Cells, Cultured ; Cobalt ; Feasibility Studies ; Ferric Compounds ; Humans ; Magnetic Resonance Imaging/*methods ; *Mesenchymal Stem Cells ; Mice ; Mice, Nude ; Microscopy, Confocal ; Microscopy, Electron ; Nanoparticles/*chemistry ; Phantoms, Imaging ; Polyethylene Glycols ; Rats ; Rhodamines ; Silicon Dioxide ; Staining and Labeling/methods

BACKGROUND: Fluorescent dye Rhodamine 6G (R6G) is a substrate of multidrug resistance pumps and its accumulation is reduced in some azole-resistant Candida isolates with the upregulation of multidrug efflux transporter genes. Despite reports on species-specific differences in azole susceptibility in various Candida species, only a few studies have been reported on the R6G accumulation among clinical isolates of Candida species. In this study, we compared R6G accumulation between six different Candida species. METHODS: The intracellular accumulation of R6G and minimal inhibitory concentrations (MICs) of three triazole agents were investigated in 48 strains of six Candida species (14 C. albicans, 9 C. tropicalis, 8 C. glabrata, 8 C. krusei, 7 C. parapsilosis, and 2 C. haemulonii). R6G accumulation was measured by using flow cytometry and the geometric mean of the fluorescence intensity (GMF) was used to compare the accumulation between the Candida isolates. RESULTS: The GMF values for the C. tropicalis, C. albicans, C. krusei, C. parapsilosis, and C. glabrata isolates were 167.3+/-18.5, 126.9+/-6.6, 88.5+/-18.5, 50.8+/-7.0, and 38.1+/-3.9, respectively. C. glabrata had a significantly lower mean GMF than all the other Candida species (P<0.05). While some Candida strains with trailing growth phenomenon and increased fluconazole MIC did not have a reduced GMF, three Candida strains with increased MICs to all three triazole agents had a reduced GMF. CONCLUSIONS: This study found species-specific differences in R6G accumulation in Candida. In addition, the intracellular R6G accumulation can be used to investigate the drug efflux mechanism in azole-resistant Candida strains.
Antifungal Agents/pharmacology ; Azoles/pharmacology ; Candida/chemistry/isolation & purification/*metabolism ; Candidiasis/drug therapy ; Drug Resistance, Fungal ; Flow Cytometry/*methods ; Fluconazole/pharmacology ; Fluorescent Dyes/*analysis ; Humans ; Microbial Sensitivity Tests ; Rhodamines/*analysis ; Species Specificity

OBJECTIVETo investigate the dose and time-dependent effects of lipopolysaccharide (LPS) on cytoskeletal F-acitn and G-actin reorganizations by visualizing their distribution and measuring their contents in human umbilical vein endothelial cell line ECV-304.

METHODSF-actin was labeled with rhodamine-phalloidin and G-actin with deoxyribonuclease I (DNase I)conjugated with fluorescein isothiocyanate (FITC). Contents of cytoskeletal proteins were obtained by flow cytometry.

RESULTSF-actin was mainly distributed peripherally in endothelial cells under normal conditions. LPS stimulation caused the formation of stress fibers and filopodia. G-actin was normally seen in perinuclear and nuclear areas in control ECV-304 cells. Under LPS stimulation, G-actin dots appeared in the cytoplasmic region. The actin disorganization was accompanied by the time- and dose- dependent decrease in F-actin pool and increase in G-actin pool.

CONCLUSIONSLPS can induce characteristic morphological alterations of actin cytoskeleton and formation of intercellular gap in endothelial cells, accompanied by changes in F-actin and G-actin pools.

Actins ; drug effects ; Analysis of Variance ; Cells, Cultured ; Deoxyribonuclease I ; Dose-Response Relationship, Drug ; Endothelial Cells ; chemistry ; Escherichia coli ; Fluorescein-5-isothiocyanate ; Fluorescent Dyes ; Humans ; Lipopolysaccharides ; pharmacology ; Phalloidine ; Rhodamines ; Umbilical Veins ; cytology

OBJECTIVETo synthesize a tumor-targeting cell-penetrating peptide (CPP) and evaluate its biological activity and cytotoxicity in vitro.

METHODSWith fluorenylmethyloxycarbonyl (Fmoc) as the protective group of α-amino acid, the tumor-targeting CPP were synthesized with stepwise amino acid extension using solid-phase synthesis method. 5-carboxytetramethylrhodamine was added for fluorescence labeling in the presence of the coupling agents HATU and DMF. The purity of the CPP was measured by high-performance liquid chromatography and its molecular weight measured by mass spectrometry. Fluorescence microscope was used to assess the cell-penetrating activity?of the CPP in hepatocellular carcinoma cell lines SMMC-7721 and normal hepatocellular cell lines LO2. The growth activity of CPP-treated SMMC-7721 cells was measured by MTT assay.

RESULTSWith a purity of 96.05% and a relative molecular mass of 3504.9, the synthesized CPP showed no translocation activity in normal hepatocellular cell lines LO2, but showed strong ability to translocate into SMMC-7721 cells without affecting the biological activity of the cells.

CONCLUSIONUsing Fmoc solid-phase synthesis method, we have successfully synthesized the CPP with tumor-targeting activity.

Cell Line, Tumor ; Cell-Penetrating Peptides ; chemical synthesis ; pharmacology ; Drug Delivery Systems ; Drug Design ; Humans ; Liver Neoplasms ; drug therapy ; metabolism ; pathology ; Matrix Metalloproteinase 2 ; metabolism ; Rhodamines ; chemistry ; Solid-Phase Synthesis Techniques

OBJECTIVE: This paper applied a transcriptomic approach to investigate the mechanisms of adriamycin (ADR) in treating proliferative vitreoretinopathy (PVR) using ARPE-19 cells. METHODS: The growth inhibitory effects of ADR on ARPE-19 cells were assessed by sulforhodamine B (SRB) assay and propidium iodide (PI) staining using flow cytometry. The differentially expressed genes between ADR-treated ARPE-19 cells and normal ARPE-19 cells and the signaling pathways involved were investigated by microarray analysis. Mitochondrial function was detected by JC-1 staining using flow cytometry and the Bcl-2/Bax protein family. The phosphorylated histone H2AX (γ-H2AX), phosphorylated checkpoint kinase 1 (p-CHK1), and phosphorylated checkpoint kinase 2 (p-CHK2) were assessed to detect DNA damage and repair. RESULTS: ADR could significantly inhibit ARPE-19 cell proliferation and induce caspase-dependent apoptosis in vitro. In total, 4479 differentially expressed genes were found, and gene ontology items and the p53 signaling pathway were enriched. A protein-protein interaction analysis indicated that the TP53 protein molecules regulated by ADR were related to DNA damage and oxidative stress. ADR reduced mitochondrial membrane potential and the Bcl-2/Bax ratio. p53-knockdown restored the activation of c-caspase-3 activity induced by ADR by regulating Bax expression, and it inhibited ADR-induced ARPE-19 cell apoptosis. Finally, the levels of the γ-H2AX, p-CHK1, and p-CHK2 proteins were up-regulated after ADR exposure. CONCLUSIONS The mechanism of ARPE-19 cell death induced by ADR may be caspase-dependent apoptosis, and it may be regulated by the p53-dependent mitochondrial dysfunction, activating the p53 signaling pathway through DNA damage.
Apoptosis ; Caspases/metabolism* ; Cell Proliferation ; Cell Survival/drug effects* ; Doxorubicin/pharmacology* ; Flow Cytometry ; Gene Expression Profiling ; Gene Expression Regulation ; Humans ; Membrane Potential, Mitochondrial ; Oligonucleotide Array Sequence Analysis ; Oxidative Stress/drug effects* ; Phosphorylation ; Propidium/chemistry* ; RNA, Small Interfering/metabolism* ; Retinal Pigment Epithelium/metabolism* ; Rhodamines/chemistry* ; Signal Transduction/drug effects* ; Transcriptome ; Tumor Suppressor Protein p53/metabolism* ; Vitreoretinopathy, Proliferative/drug therapy*