Zhongdang Xiao

Visualizing of the cellular uptake and intracellular trafficking of exosomes by live-cell microscopy.

Cells release exosomes to transfer various molecules to other cells. Exosomes are involved in a number of physiological and pathological processes. They are emerging great potential utility for diseases diagnosis and treatment recently. However, the internalization and intracellular trafficking of exosomes have not been described clearly. In this work, exosomes were isolated from the culture medium of PC12 cells, labeled by lipophilic dye and amino‐reactive fluorophore, incubated with resting PC12 cells. The results of live‐cell microscopy indicated that exosomes were internalized through endocytosis pathway, trapped in vesicles, and transported to perinuclear region. Particle tracking fluorescent vesicles suggested that the active transport of exosomes may be mediated by cytoskeleton. The proteins on exosome membrane were found to be released from exosomes and trapped in lysosome. The inverted transport of lipophilic dye from perinuclear region to cell peripheries was revealed, possibly caused by recycling of the exosome lipids. This study provides new sight into the mechanisms of exosome uptake and intracellular fate. J. Cell. Biochem. 111: 488–496, 2010.

Exosome Uptake through Clathrin-mediated Endocytosis and Macropinocytosis and Mediating miR-21 Delivery

Background: Exosomes can transfer information between cells and facilitate tumor development. Results: PC12 cell-derived exosomes enter into BMSCs through clathrin-mediated endocytosis and macropinocytosis, and decrease the expression of TGFβRII and TPM1 through miR-21. Conclusion: The results dissect the pathway of exosome internalization and demonstrate their regulation ability. Significance: These findings enhanced our knowledge of the internalization and function of tumor exosomes. Exosomes are nanoscale membrane vesicles secreted from many types of cells. Carrying functional molecules, exosomes transfer information between cells and mediate many physiological and pathological processes. In this report, utilizing selective inhibitors, molecular tools, and specific endocytosis markers, the cellular uptake of PC12 cell-derived exosomes was imaged by high-throughput microscopy and statistically analyzed. It was found that the uptake was through clathrin-mediated endocytosis and macropinocytosis. Furthermore, PC12 cell-derived exosomes can enter and deliver microRNAs (miRNAs) into bone marrow-derived mesenchymal stromal cells (BMSCs), and decrease the expression level of transforming growth factor β receptor II (TGFβRII) and tropomyosin-1 (TPM1) through miR-21. These results show the pathway of exosome internalization and demonstrate that tumor cell-derived exosomes regulate target gene expression in normal cells.

Dynamics of exosome internalization and trafficking

Cells release exosomes into extracellular medium. Although the important roles of exosomes in many physiological and pathological processes are being revealed, the mechanism of exosome–cell interaction remains unclear. In this article, employing real‐time fluorescence microscopy, the motion of exosomes on the plasma membrane or in the cytoplasm of recipient PC12 cells was observed directly. In addition, several motion modes of exosomes were revealed by single particle tracking (SPT). The changes between motion modes were also detected, presenting the dynamic courses of exosome attachment onto plasma membrane and exosome uptake. Octadecyl rhodamine B chloride (R18) was found to be useful to distinguish endocytosis from fusion during exosome uptake. Colocalization with organelle markers showed exosomes were sorted to acidic vesicles after internalization. The results provide new sight into the exosome–cell interaction mode and the intercellular trafficking of exosomes. This study will help to understand the roles of exosomes at cell level. J. Cell. Physiol. 228: 1487–1495, 2013.

Effects of hydroxyapatite-containing composite nanofibers on osteogenesis of mesenchymal stem cells in vitro and bone regeneration in vivo.

Among a variety of polymers, poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a microbial polyester, with biodegradable, nonantigenic, and biocompatible properties, is attracting more and more attention in tissue engineering. Hydroxyapatite (HA), similar to the mineral component of natural bone, is known to be osteoconductive, nontoxic, and noninflammatory. In this study, aligned and random-oriented PHBV nanofibrous scaffolds loaded with HA nanoparticles were fabricated through electrospinning technique. Mesenchymal stem cells (MSCs) derived from rat bone marrow were used to investigate the effects of HA and orientation of fibers on cell proliferation and differentiation in vitro. Cell proliferation tested with CCK-8 assay indicated that the MSCs attached and proliferated more favorably on random-oriented PHBV nanofibrous meshes without HA. After one, two and four weeks of cell seeding, osteogenic markers including alkaline phosphate (ALP), osteocalcin (OCN), and mineralized matrix deposits were detected, respectively. The results indicated that the introduction of HA could induce MSCs to differentiate into osteoblasts. Moreover, 3D PHBV/HA scaffolds made from aligned and random-oriented nanofibers were implanted into critical-sized rabbit radius defects and exhibited significant effects on the repair of critical bone defects, implying their promising applications in bone tissue engineering.

Introducing RGD peptides on PHBV films through PEG-containing cross-linkers to improve the biocompatibility.

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a biodegradable polyester, has been a good candidate of biomaterial employed in tissue engineering. However, the PHBV film is hydrophobic and has no recognition sites for cell attachment. In this study, PHBV films are activated by ammonia plasma treatment to produce amino groups on the surface, followed by sequential reactions with a heterobifunctional cross-linker containing a segment of poly(ethylene glycol) (PEG) and further with RGD-containing peptides. XPS analyses of modified surfaces after each reaction step reveal that the RGD-containing peptides have been covalently grafted onto PHBV films. The result of cell viability assay indicates that the RGD-modified PHBV films exhibit a distinctly improved cellular compatibility. Moreover, according to the results of serum adsorption tests by optical waveguide lightmode spectroscopy (OWLS) and fibrinogen adsorption tests by enzyme-linked immunosorbent assay (ELISA) on unmodified and modified PHBV surfaces, the introduced PEG chains can significantly decrease the nonspecific adsorption of proteins from serum and fibrinogen from plasma, thus decreasing the risk of thrombus formation and improving the blood compatibility of implanted materials.

Assessing the survival of exogenous plant microRNA in mice

MicroRNAs (miRNAs), a class of small RNAs, are important molecules that influence several developmental processes and regulate RNA interference (RNAi), and are abundant in animals, plants, and plant tissues that are traditionally consumed in the diet. The survival of plant small RNAs from the diet in animals, however, remains unclear, and the persistence of miRNAs from dietary plants in the animal gastrointestinal (GI) tract is still under debate. In this study, ICR mice were fed plant total RNAs in quantities of 10–50 μg, extracted from Brassica oleracea. Serum, feces, and various tissues were collected from the mice after RNA consumption and analyzed for several miRNAs. Exogenous plant miRNAs were present in the sera, feces, and tissues of animals and these exogenous plant miRNAs were primarily acquired orally. MiR-172, the most highly enriched exogenous plant miRNA in B. oleracea, was found in the stomach, intestine, serum, and feces of mice that were fed plant RNA extracts including miR-172. The amount of miR-172 that survived passage through the GI tract varied among individuals, with a maximum of 4.5% recovered at the stomach of one individual, and had a range of 0.05–4.5% in different organs. Furthermore, miR-172 was detected in the blood, spleen, liver, and kidney of mice.

MicroRNAs as participants in cytotoxicity of CdTe quantum dots in NIH/3T3 cells

Epigenetic aspects of the cytotoxicity of CdTe quantum dots (QDs) recently have attracted more attention for their ability to reprogram gene expression after initial signals have been removed. And the involvement of epigenetic mechanisms in microRNA (miRNA) biogenesis suggests that miRNAs act as participants in the cytotoxicity of CdTe QDs. According to the results of SOLiD sequencing, the expression patterns of miRNAs are widely affected after CdTe QD exposure, resulting in the apoptosis-like cell death. Compared with 86 miRNAs with down-regulated expression, the expression levels of 121 miRNAs are up-regulated by CdTe QD treatment. The Z-test is used to find out miRNAs with significantly regulated expression, and the results indicate that the expression levels of 16 and 35 miRNAs are down- and up-regulated, respectively. And the expression levels of some significantly regulated miRNAs have time- and dose-dependent tendencies, which are similar to cell survival ratios affected by CdTe QDs. The fluctuations of miRNA expression start from the transcription of pri-miRNA, and are strengthened by the processing of pri-miRNA to pre-miRNA. As a regulator in miRNA biogenesis, p53 is involved in the transcription and processing of pri-miRNA. With no significant changes in the mRNA levels of p53, the increase in overall p53 protein levels and its post-translational modification by phosphorylation at Ser-15 are induced by CdTe QD treatment. Therefore, the differential expression of miRNAs are induced by CdTe QDs at the processing of miRNA biogenesis, which is an adaptive process of cells to external stimuli.

Assessment of nanomaterial cytotoxicity with SOLiD sequencing-based microRNA expression profiling.

The cytotoxicity of nanomaterials has become a major concern in the field of nanotechnology. The key challenge is the lack of reliable methods to examine the overall cellular effects of nanomaterials. Here, a new method is developed to assess the cytological effects of nanomaterial basing on miRNA expression profiling. The SOLiD sequencing is used to acquire the miRNAs expression profiling in NIH/3T3 cells after exposure to Fe(2)O(3) NPs, CdTe QDs and MW-CNTs, respectively. The systematic analysis of miRNAs expression profiling is established by taking account of all miRNAs into their regulatory networks. By affecting the output of targeted mRNAs, miRNAs widely regulated the KEGG pathways and GO biological processes in nanomaterial treated cells. Therefore, the miRNA expression profiling can well reflect the characteristic of nanomaterials, and the method not only provide more evidences to assess biocompatibility of nanomaterials and but also clues to discover new biological effects of nanomaterials.

Electrochemical biosensor based on CdS nanostructure surfaces

Well-defined hexangularly faced CdS nanorod arrays have been grown directly on a conductive ITO glass via a facile one-step and non-template hydrothermal approach. Gold nanoparticles were decorated onto the nanorods to enhance the electron transfer process of electrode. Glucose oxidase (GOD) was then immobilized on the CdS through crosslinking with chitosan (CS), which resulted in a glucose biosensor with high enzyme loading and excellent sensitivity. Such a chitosan-encapsulated GOD-based biosensor revealed a relatively rapid response time of less than 50s, and an approximate linear detection range of glucose concentration, from 50 to 500 μmol L(-1) with a detection limit of 38 μmol L(-1) and an electrode sensitivity of 5.9 μA mM(-1).

The effects of PHBV electrospun fibers with different diameters and orientations on growth behavior of bone-marrow-derived mesenchymal stem cells

Microenvironments in which cells live play an important role in the attachment, growth and interactions of cells. To mimic the natural structure of extracellular matrices, electrospinning was applied to fabricate biomaterials into ultrafine fibers. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a biocompatible and biodegradable polyester, has been shown to be an excellent biomaterial candidate for tissue engineering. In this study, five types of PHBV fibrous scaffolds with different diameters and orientations were obtained by changing solvents, concentration of electrospun solution and collector. Three kinds of scaffolds with good continuity and suitable mechanical properties, selected according to the morphology and mechanical properties of the scaffolds, were used for studying the influence of fiber diameter and orientation on growth behavior of bone-marrow-derived mesenchymal stem cells (MSCs). The results indicated that the random-oriented nanofibrous scaffold is most favorable for cell growth compared to other scaffolds, while the microfibrous scaffold resulted in the lowest viability of MSCs. The orientation of nanofibers showed a distinct effect on cell morphology by guiding cell skeleton extension. Both the random-oriented and aligned PHBV nanofibrous scaffolds showed to be good candidates for applications in tissue engineering.