[show abstract][hide abstract] ABSTRACT: BACKGROUND: With the prompt developments of regenerative medicine, the potential clinical applications of human embryonic stem cells have attracted intense attention. However, the labor-intensive and complex manual cell selection processes required during embryonic stem cell culturing have seriously limited large-scale production and broad applications. Thus, availability of a label-free, non-invasive platform to replace the current cumbersome manual selection has become a critical need. RESULTS: A non-invasive, label-free, and time-efficient optical platform for determining the quality of human embryonic stem cell colonies was developed by analyzing the scattering signals from those stem cell colonies. Additionally, confocal microscopy revealed that the cell colony morphology and surface structures were correlated with the resulting characteristic light scattering patterns. Standard immunostaining assay (Oct-4) was also utilized to validate the quality-determination from this light scattering protocol. The platform developed here can therefore provide identification accuracy of up to 87% for colony determination. CONCLUSIONS: Our study here demonstrated that light scattering patterns can serve as a feasible alternative approach to replace conventional manual selection for human embryonic stem cell cultures.
[show abstract][hide abstract] ABSTRACT: Quantum dots (QDs) are engineered nanoparticles (ENs) that have found increasing applications and shown great potential in drug delivery, biological imaging and industrial products. Knowledge of their stability, fate and transport in the aquatic environment is still lacking, including details of how these nanomaterials interact with marine phytoplankton. Here, we examined the toxicity of functionalized CdSe/ZnS QDs (amine- and carboxyl-) by exposing them for five days to Thalassiosira pseudonana (marine diatom) grown under different nutrient-conditions (enriched versus nitrogen-limited media). The released polysaccharides and proteins, the major components of extracellular polymeric substances (EPS), were measured to assess their potential effects on the interactions between QDs and T. pseudonana. The partitioning of QDs was analyzed by monitoring the concentration of Cd in different size fractions of the cultures (i.e., filtrate, <0.22μm and permeate, <3kDa). We found that the Cd release of QDs in the T. pseudonana culture was dependent on the nutrient conditions and nature of QDs' surface coating. Both amine- and carboxyl-functionalized QDs exhibited higher rates of Cd release in N-limited cultures than in nutrient enriched cultures. The results also showed that amine-functionalized QDs aggregate with minimal Cd release, independent of nutrient conditions. Laser scanning confocal microscopy images confirmed that aggregates are composed of QDs and the culture matrix (EPS). In addition, both types of QDs showed limited toxicity to T. pseudonana. The increasing production of proteins induced by QDs suggests that extracellular proteins might be involved in the detoxification of QDs to T. pseudonana via the Cd release of QDs. Our results here demonstrated that EPS can play an ameliorating role in QD toxicity, fate and transport in the aquatic environment.
[show abstract][hide abstract] ABSTRACT: There is an increasing concern that a considerable fraction of engineered nanoparticles (ENs), including quantum dots (QDs), will eventually find their way into the marine environment and have negative impacts on plankton. As ENs enter the ocean, they will encounter extracellular polymeric substances (EPS) from microbial sources before directly interacting with plankton cells. In this study, EPS harvested from four phytoplankton species, Amphora sp., Dunaliella tertiolecta, Phaeocystis globosa, and Thalassiosira pseudonana, were examined for potential interactions with CdSe nonfunctionalized and functionalized (carboxyl- and amine-) QDs in artificial seawater. Our results show that EPS do not reduce the solubility of QDs but rather decrease their stability. The degradation rate of QDs was positively correlated to the protein composition of EPS (defined by the ratio of protein/carbohydrate). Two approaches showed significant inhibition to the degradation of carboxyl-functionalized QDs: (1) the presence of an antioxidant, such as N-acetyl cysteine, and (2) absence of light. Owing to the complexity in evaluating integrated effects of QDs intrinsic properties and the external environmental factors that control the stability of QDs, conclusions must be based on a careful consideration of all these factors when attempting to evaluate the bioavailability of QDs and other ENs in the marine environments.
[show abstract][hide abstract] ABSTRACT: Transplantation of biomaterial scaffolds encasing human embryonic stem cells (hESCs) has been proposed as a clinical therapy for various neurological lesions and disorders. In light of recent developments, artificially synthesized carbon-based biomaterials such as carbon nanotubes and graphene have demonstrated feasibility in supporting stem cell attachment and differentiation. However, the applicability is significantly hampered by evidence of nanotoxic effects on multiple cell types. Thus, an emergent drive for an innovative carbonaceous biomaterial calls for a safer platform with comparable advantageous characteristics. Here, we showed for the first time, a natural coal-based activated charcoal (AC) composite biosubstrate can support and promote neuronal differentiation in hESCs. The bio-friendly AC composite biomatrices resulted in more matured neuron-like cells. Both of axonal length and density were at least twice as long and abundant, respectively, when compared with control groups. A functional assay demonstrated that the derived neuron-like cells responded to depolarization-dependent synaptic recycling and may contain active synapses. In addition, the AC composite substrate can serve to concentrate growth factors and cell adhesion proteins, further encouraging attachment and hESC differentiation. Moreover, the AC composite biomaterial can potentially be economically manufactured as implantable three-dimensional bioscaffolds, facilitating the regeneration of damaged neural and other tissues.
Journal of Biomedical Materials Research Part A 05/2012; 100(8):2006-17. · 2.83 Impact Factor
[show abstract][hide abstract] ABSTRACT: Water molecules play critical roles in many biological functions, such as protein dynamics, enzymatic activities, and cellular responses. Previous nuclear magnetic resonance and neutron scattering studies have shown that water molecules bind to specific sites on surfaces and form localized clusters. However, most current experimental techniques cannot measure dynamic behaviors of ordered water molecules on cell-size (10 μm) scale. Recently, the long-distance effect of structured water has been demonstrated by Pollack and his colleagues. Namely, there is a structured water layer near the hydrophilic surface that can exclude solutes (Zheng et al, Adv Colloid Interface Sci 127:19-27, 2006; Pollack 2006, Adv Colloid Interface Sci 103:173-196, 2003). The repelling forces of water clusters inside this exclusion region are investigated in this study. With a laser tweezers system, we found the existence of an unexpected force fields inside the solute-free exclusion zone near a Nafion surface. Our results suggest that the water clusters could transduce mechanical signals on the micrometer range within the exclusion zone. This unexpected inhomogeneous force field near the hydrophilic surface would provide a new insight into cellular activities, leading to a potential new physical chemistry mechanism for cell biology.
Journal of Biological Physics 01/2012; 38(1):113-20. · 0.95 Impact Factor
[show abstract][hide abstract] ABSTRACT: Luminal accumulation of viscous, poorly hydrated, and less transportable
mucus has been associated with altered mucus rheology and reduced
mucociliary clearance. These symptoms are some of the cardinal clinical
manifestations found throughout major respiratory diseases as well as
gastrointestinal and digestive disorders. Applications of current
mucolytics may yield short-term improvements but are continuously
challenged by undesirable side-effects. While nanoparticles (NPs) can
interact with mucin polymers, whether functionalized NPs can rectify
mucus rheology is unknown. Herein, we report that
carboxyl-functionalized NPs (24 nm and 120 nm) dramatically reduced
mucin gel size and accelerated mucin matrix hydration rate
(diffusivity). Our results suggest that carboxyl-functionalized NPs
disperse mucin gels possibly by enhancing network hydration. This report
highlights the prospective usages of carboxyl-functionalized NPs as a
novel mucus dispersant or mucolytic agent in adjusting mucus rheological
properties and improving mucociliary transport to relieve clinical
symptoms of patients suffering from relevant diseases.
[show abstract][hide abstract] ABSTRACT: Histamine released from mast cells, through complex interactions involving the binding of IgE to FcεRI receptors and the subsequent intracellular Ca²⁺ signaling, can mediate many allergic/inflammatory responses. The possibility of titanium dioxide nanoparticles (TiO₂ NPs), a nanomaterial pervasively used in nanotechnology and pharmaceutical industries, to directly induce histamine secretion without prior allergen sensitization has remained uncertain.
TiO₂ NP exposure increased both histamine secretion and cytosolic Ca²⁺ concentration ([Ca²⁺]C) in a dose dependent manner in rat RBL-2H3 mast cells. The increase in intracellular Ca²⁺ levels resulted primarily from an extracellular Ca²⁺ influx via membrane L-type Ca²⁺ channels. Unspecific Ca²⁺ entry via TiO₂ NP-instigated membrane disruption was demonstrated with the intracellular leakage of a fluorescent calcein dye. Oxidative stress induced by TiO₂ NPs also contributed to cytosolic Ca²⁺ signaling. The PLC-IP₃-IP₃ receptor pathways and endoplasmic reticulum (ER) were responsible for the sustained elevation of [Ca²⁺]C and histamine secretion.
Our data suggests that systemic circulation of NPs may prompt histamine release at different locales causing abnormal inflammatory diseases. This study provides a novel mechanistic link between environmental TiO₂ NP exposure and allergen-independent histamine release that can exacerbate manifestations of multiple allergic responses.
Particle and Fibre Toxicology 01/2012; 9:2. · 9.18 Impact Factor
[show abstract][hide abstract] ABSTRACT: Human embryonic stem cells [hESCs] are able to differentiate into specific lineages corresponding to regulated spatial and temporal signals. This unique attribute holds great promise for regenerative medicine and cell-based therapy for many human diseases such as spinal cord injury [SCI] and multiple sclerosis [MS]. Carbon nanotubes [CNTs] have been successfully used to promote neuronal differentiation, and silk has been widely applied in tissue engineering. This study aims to build silk-CNT composite scaffolds for improved neuron differentiation efficiency from hESCs.Two neuronal markers (β-III tubulin and nestin) were utilized to determine the hESC neuronal lineage differentiation. In addition, axonal lengths were measured to evaluate the progress of neuronal development. The results demonstrated that cells on silk-CNT scaffolds have a higher β-III tubulin and nestin expression, suggesting augmented neuronal differentiation. In addition, longer axons with higher density were found to associate with silk-CNT scaffolds.Our silk-CNT-based composite scaffolds can promote neuronal differentiation of hESCs. The silk-CNT composite scaffolds developed here can serve as efficient supporting matrices for stem cell-derived neuronal transplants, offering a promising opportunity for nerve repair treatments for SCI and MS patients.
Nanoscale Research Letters 01/2012; 7(1):126. · 2.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: Most cell culture systems grow and spread as contact-inhibited monolayers on flat culture dishes, but the embryonic stem cell (ESC) is one of the cell phenotypes that prefer to self-organize as tightly packed three-dimensional (3D) colonies. ESC also readily form 3D cell aggregates, called embryoid bodies (EB) that partially mimic the spatial and temporal processes of the developing embryo. Here, the rationale for ESC aggregatation, rather than "spreading" on gelatin-coated or mouse embryonic fibroblast (MEF)-coated dishes, is examined through the quantification of the expression levels of adhesion molecules on ESC and the calculation of the adhesive forces on ESC. Modeling each ESC as a dodecahedron, the adhesive force for each ESC-ESC binding was found to be 9.1 x 10(5) pN, whereas, the adhesive force for ESC-MEF binding was found to be an order of magnitude smaller at 7.9 x 10(4) pN. We also show that E-cadherin is the dominating molecule in the ESC-ESC adhesion and blocking E-cadherin leads to a significant reduction in colony formation. Here, we mathematically describe the preference for ESC to self-assemble into ESC-ESC aggregates and 3D colonies, rather than to bind and spread on gelatin or MEF-coated dishes, and have shown that these interactions are predominantly due to E-cadherin expression on ESC.
[show abstract][hide abstract] ABSTRACT: The unique properties of engineered nanoparticles (ENs) that make their industrial applications so attractive simultaneously raise questions regarding their environmental safety. ENs exhibit behaviors different from bulk materials with identical chemical compositions. Though the nanotoxicity of ENs has been studied intensively, their unintended environmental impacts remain largely unknown. Herein we report experimental results of EN interactions with exopolymeric substances (EPS) from three marine phytoplankton species: Amphora sp., Ankistrodesmus angustus and Phaeodactylum tricornutum. EPS are polysaccharide-rich anionic colloid polymers released by various microorganisms that can assemble into microgels, possibly by means of hydrophobic and ionic mechanisms. Polystyrene nanoparticles (23 nm) were used in our study as model ENs. The effects of ENs on EPS assembly were monitored with dynamic laser scattering (DLS). We found that ENs can induce significant acceleration in Amphora sp. EPS assembly; after 72 hours EN-EPS aggregation reached equilibrium, forming microscopic gels of ∼4-6 µm in size. In contrast, ENs only cause moderate assembly kinetic acceleration for A. angustus and P. tricornutum EPS samples. Our results indicate that the effects of ENs on EPS assembly kinetics mainly depend on the hydrophobic interactions of ENs with EPS polymers. The cycling mechanism of EPS is complex. Nonetheless, the change of EPS assembly kinetics induced by ENs can be considered as one potential disturbance to the marine carbon cycle.
PLoS ONE 01/2011; 6(7):e21865. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Nanoparticle (NP) exposure has been closely associated with the exacerbation and pathophysiology of many respiratory diseases such as Chronic Obstructive Pulmonary Disease (COPD) and asthma. Mucus hypersecretion and accumulation in the airway are major clinical manifestations commonly found in these diseases. Among a broad spectrum of NPs, titanium dioxide (TiO(2)), one of the PM10 components, is widely utilized in the nanoindustry for manufacturing and processing of various commercial products. Although TiO(2) NPs have been shown to induce cellular nanotoxicity and emphysema-like symptoms, whether TiO(2) NPs can directly induce mucus secretion from airway cells is currently unknown. Herein, we showed that TiO(2) NPs (<75 nm) can directly stimulate mucin secretion from human bronchial ChaGo-K1 epithelial cells via a Ca(2+) signaling mediated pathway. The amount of mucin secreted was quantified with enzyme-linked lectin assay (ELLA). The corresponding changes in cytosolic Ca(2+) concentration were monitored with Rhod-2, a fluorescent Ca(2+) dye. We found that TiO(2) NP-evoked mucin secretion was a function of increasing intracellular Ca(2+) concentration resulting from an extracellular Ca(2+) influx via membrane Ca(2+) channels and cytosolic ER Ca(2+) release. The calcium-induced calcium release (CICR) mechanism played a major role in further amplifying the intracellular Ca(2+) signal and in sustaining a cytosolic Ca(2+) increase. This study provides a potential mechanistic link between airborne NPs and the pathoetiology of pulmonary diseases involving mucus hypersecretion.
PLoS ONE 01/2011; 6(1):e16198. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: It is now widely recognized that dissolution plays an important role in metallic nanoparticle toxicity, but to what extent remains unclear. In the present study, it was found that ZnO-engineered nanoparticle (ZnO-EN) toxicity to the marine diatom Thalassiosira pseudonana could be solely explained by zinc ion (Zn(2+) ) release. This is based on comparable inhibitive effects from ZnO-EN addition media, with or without the ultrafiltration through a 3-kD membrane, and from the media in which only Zn(2+) was added. Considering the importance of dissolution in ZnO-EN toxicity, Zn(2+) release kinetics was systematically examined under different conditions for the first time. It was found to be mainly influenced by pH as well as the specific surface area of the nanoparticles. In contrast, natural organic compounds either enhance or reduce Zn(2+) release, depending on their chemical composition and concentration. Compared with deionized water, ZnO-EN dissolution rates were accelerated in seawater, whereas ZnO-EN concentration itself only had a very small effect on Zn(2+) release. Therefore, dissolution as affected by several physicochemical factors should not be neglected in the effects, behavior, and fate of ENs in the environment.
Environmental Toxicology and Chemistry 09/2010; 29(12):2814-22. · 2.62 Impact Factor
[show abstract][hide abstract] ABSTRACT: The behavior and toxicity of silver engineered nanoparticles (Ag-ENs) to the mixotrophic freshwater alga Ochromonas danica were examined in the present study to determine whether any other mechanisms are involved in their algal toxicity besides Ag(+) liberation outside the cells. Despite their good dispersability, the Ag-ENs were found to continuously aggregate and dissolve rapidly. When the initial nanoparticle concentration was lower than 10 µM, the total dissolved Ag(+) concentration ([Ag(+)](T)) in the suspending media reached its maximum after 1 d and then decreased suggesting that Ag(+) release might be limited by the nanoparticle surface area under these conditions. Furthermore, Ag-EN dissolution extent remarkably increased in the presence of glutathione. In the Ag-EN toxicity experiment, glutathione was also used to eliminate the indirect effects of Ag(+) that was released. However, remarkable toxicity was still observed although the free Ag(+) concentration in the media was orders of magnitude lower than the non-observed effect concentration of Ag(+) itself. Such inhibitive effects were mitigated when more glutathione was added, but could never be completely eliminated. Most importantly, we demonstrate, for the first time, that Ag-ENs can be taken in and accumulated inside the algal cells, where they exerted their toxic effects. Therefore, nanoparticle internalization may be an alternative pathway through which algal growth can be influenced.
PLoS ONE 01/2010; 5(12):e15196. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Multi-functionalized nanoparticles (NPs) have been extensively investigated for their potential in household and commercial products, and biomedical applications. Previous reports have confirmed the cellular nanotoxicity and adverse inflammatory effects on pulmonary systems induced by NPs. However, possible health hazards resulting from mucus rheological disturbances induced by NPs are underexplored. Accumulation of viscous, poorly dispersed, and less transportable mucus leading to improper mucus rheology and dysfunctional mucociliary clearance are typically found to associate with many respiratory diseases such as asthma, cystic fibrosis (CF), and COPD (Chronic Obstructive Pulmonary Disease). Whether functionalized NPs can alter mucus rheology and its operational mechanisms have not been resolved. Herein, we report that positively charged functionalized NPs can hinder mucin gel hydration and effectively induce mucin aggregation. The positively charged NPs can significantly reduce the rate of mucin matrix swelling by a maximum of 7.5 folds. These NPs significantly increase the size of aggregated mucin by approximately 30 times within 24 hrs. EGTA chelation of indigenous mucin crosslinkers (Ca(2+) ions) was unable to effectively disperse NP-induced aggregated mucins. Our results have demonstrated that positively charged functionalized NPs can impede mucin gel swelling by crosslinking the matrix. This report also highlights the unexpected health risk of NP-induced change in mucus rheological properties resulting in possible mucociliary transport impairment on epithelial mucosa and related health problems. In addition, our data can serve as a prospective guideline for designing nanocarriers for airway drug delivery applications.
PLoS ONE 01/2010; 5(11):e15434. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Human embryonic stem cells (hESCs) hold great promise for regenerative medicine and transplantation therapy due to their self-renewal and pluripotent properties. We report that 2D thin film scaffolds composed of biocompatible polymer grafted carbon nanotubes (CNTs), can selectively differentiate human embryonic stem cells into neuron cells while maintaining excellent cell viability. According to fluorescence image analysis, neuron differentiation efficiency of poly(acrylic acid) grafted CNT thin films is significant greater than that on poly(acrylic acid) thin films. When compared with the conventional poly-L-ornithine surfaces, a standard substratum commonly used for neuron culture, this new type thin film scaffold shows enhanced neuron differentiation. No noticeable cytotoxic effect difference has been detected between these two surfaces. The surface analysis and cell adhesion study have suggested that CNT-based surfaces can enhance protein adsorption and cell attachment. This finding indicates that CNT-based materials are excellent candidates for hESCs' neuron differentiation.
Biochemical and Biophysical Research Communications 06/2009; 384(4):426-30. · 2.41 Impact Factor
[show abstract][hide abstract] ABSTRACT: We present a novel approach for the ultra-rapid direct patterning of complex three-dimensional, stacked polystyrene (PS) microfluidic chips. By leveraging the inherent shrinkage properties of biaxially pre-stressed thermoplastic sheets, microfluidic channels become thinner and deeper upon heating. Design conception to fully functional chips can thus be completed within minutes.
Lab on a Chip 05/2008; 8(4):622-4. · 5.70 Impact Factor
[show abstract][hide abstract] ABSTRACT: Embryoid bodies (EB) are aggregates of embryonic stem cells. The most common way of creating these aggregates is the hanging drop method, a laborious approach of pipetting an arbitrary number of cells into well plates. The interactions between the stem cells forced into close proximity of one another promotes the generation of the EBs. Because the media in each of the wells has to be manually exchanged every day, this approach is manually intensive. Moreover, because environmental parameters including cell-cell, cell-soluble factor interactions, pH, and oxygen availability can be functions of EB size, cell populations obtained from traditional hanging drops can vary dramatically even when cultured under identical conditions. Recent studies have indeed shown that the initial number of cells forming the aggregate can have significant effects on stem cell differentiation. We have developed a simple, rapid, and scalable culture method to load pre-defined numbers of cells into microfabricated wells and maintain them for embryoid body development. Finally, these cells are easily accessible for further analysis and experimentation. This method is amenable to any lab and requires no dedicated equipment. We demonstrate this method by creating embryoid bodies using a red fluorescent mouse cell line (129S6B6-F1).
[show abstract][hide abstract] ABSTRACT: Rapid and effective mixing of macromolecular solutions remains a persistent challenge when studying biochemical reactions. We show here that rapid and enhanced micromixing can be achieved in an easily fabricated (requiring no lithography), topologically simple 3D microvortex mixer at low Reynolds numbers. Experiments indicate dramatically improved mixing performance when compared with the traditional 2D serpentine design. Direct numerical simulation is used to examine vortex formation and to offer mechanistic understanding of our experimental data.