Uptake and cytotoxicity of citrate-coated gold nanospheres : comparative studies on human endothelial and epithelial cells

The use of gold nanoparticles (AuNPs) for diagnostic applications and for drug and gene-delivery is currently under intensive investigation. For such applications, biocompatibility and the absence of cytotoxicity of AuNPs is essential. Although generally considered as highly biocompatible, previous in vitro studies have shown that cytotoxicity of AuNPs in certain human epithelial cells was observed. In particular, the degree of purification of AuNPs (presence of sodium citrate residues on the particles) was shown to affect the proliferation and induce cytotoxicity in these cells. To expand these studies, we have examined if the effects are related to nanoparticle size (10, 11 nm, 25 nm), to the presence of sodium citrate on the particles' surface or they are due to a varying degree of internalization of the AuNPs. Since two cell types are present in the major barriers to the outside in the human body, we have also included endothelial cells from the vasculature and blood brain barrier. Results Transmission electron microscopy demonstrates that the internalized gold nanoparticles are located within vesicles. Increased cytotoxicity was observed after exposure to AuNPs and was found to be concentration-dependent. In addition, cell viability and the proliferation of both endothelial cells decreased after exposure to gold nanoparticles, especially at high concentrations. Moreover, in contrast to the size of the particles (10 nm, 11 nm, 25 nm), the presence of sodium citrate on the nanoparticle surface appeared to enhance these effects. The effects on microvascular endothelial cells from blood vessels were slightly enhanced compared to the effects on brain-derived endothelial cells. A quantification of AuNPs within cells by ICP-AES showed that epithelial cells internalized a higher quantity of AuNPs compared to endothelial cells and that the quantity of uptake is not correlated with the amount of sodium citrate on the nanoparticles’ surface. Conclusions In conclusion the higher amount of citrate on the particle surface resulted in a higher impairment of cell viability, but did not enhance or reduce the uptake behavior in endothelial or epithelial cells. In addition, epithelial and endothelial cells exhibited different uptake behaviors for citrate-stabilized gold nanoparticles, which might be related to different interactions occurring at the nanoparticle-cell-surface interface. The different uptake in epithelial cells might explain the higher reduction of proliferation of these cells after exposure to AuNPs treatment although more detailed investigations are necessary to determine subcellular events. Nevertheless an extrinsic effect of sodium-citrate stabilized particles could not be excluded. Thus, the amount of sodium citrate should be reduced to a level on which the stability of the particles and the safety for biomedical applications are guaranteed

A novel blood-brain barrier co-culture System for drug targeting of Alzheimer’s disease : establishment by using acitretin as a model drug

In the pathogenesis of Alzheimer’s disease (AD) the homeostasis of amyloid precursor protein (APP) processing in the brain is impaired. The expression of the competing proteases ADAM10 (a disintegrin and metalloproteinase 10) and BACE-1 (beta site APP cleaving enzyme 1) is shifted in favor of the A-beta generating enzyme BACE-1. Acitretin–a synthetic retinoid–e.g., has been shown to increase ADAM10 gene expression, resulting in a decreased level of A-beta peptides within the brain of AD model mice and thus is of possible value for AD therapy. A striking challenge in evaluating novel therapeutically applicable drugs is the analysis of their potential to overcome the blood-brain barrier (BBB) for central nervous system targeting. In this study, we established a novel cell-based bio-assay model to test ADAM10-inducing drugs for their ability to cross the BBB. We therefore used primary porcine brain endothelial cells (PBECs) and human neuroblastoma cells (SH-SY5Y) transfected with an ADAM10-promoter luciferase reporter vector in an indirect co-culture system. Acitretin served as a model substance that crosses the BBB and induces ADAM10 expression. We ensured that ADAM10-dependent constitutive APP metabolism in the neuronal cells was unaffected under co-cultivation conditions. Barrier properties established by PBECs were augmented by co-cultivation with SH-SY5Y cells and they remained stable during the treatment with acitretin as demonstrated by electrical resistance measurement and permeability-coefficient determination. As a consequence of transcellular acitretin transport measured by HPLC, the activity of the ADAM10-promoter reporter gene was significantly increased in co-cultured neuronal cells as compared to vehicle-treated controls. In the present study, we provide a new bio-assay system relevant for the study of dru

Crosstalk between osteoblasts and endothelial cells co-cultured on a polycaprolactone-starch scaffold and the in vitro development of vascularization

The reconstruction of bone defects based on cell-seeded constructs requires a functional microvasculature that meets the metabolic demands of the engineered tissue. Therefore, strategies that augment neovascularization need to be identified. We propose an in vitro strategy consisting of the simultaneous culture of osteoblasts and endothelial cells on a starch-based scaffold for the formation of pre-vascular structures, with the final aim of accelerating the establishment of a vascular bed in the implanted construct. Human dermal microvascular endothelial cells (HDMECs) were co-cultured with human osteoblasts (hOBs) on a 3D starch-based scaffold and after 21 days of culture HDMEC aligned and organized into microcapillary-like structures. These vascular-like structures evolved from a cord-like configuration to a more complex branched morphology, had a lumen and stained in the perivascular region for type IV collagen. Genetic profiling of 84 osteogenesis-related genes was performed on coculture vs. monoculture. Osteoblasts in co-culture showed a significant up-regulation of type I collagen and immunohistochemistry revealed that the scaffold was filled with a dense matrix stained for type I collagen. In direct contact with HDMEC hOBs secreted higher amounts of VEGF in relation to monoculture and the highest peak in the release profile correlated with the formation of microcapillary-like structures. The heterotypic communication between the two cell types was also assured by direct cell– cell contact as shown by the expression of the gap junction connexin 43. In summary, by making use of heterotypic cellular crosstalk this co-culture system is a strategy to form vascular-like structures in vitro on a 3D scaffold.M.I. Santos would like to acknowledge the Portuguese Foundation for Science and Technology (FCT) for her PhD scholarship (SFRH/BD/13428/2003). This work was partially supported by FCT through funds from POCTI and/or FEDER programs and by the European Union funded STREP Project HIPPOCRATES (NMP3-CT-2003-505758). This work was carried out under the scope of the European NOE EXPERTISSUES (NMP3-CT-2004-500283)

In vitro investigation of silica nanoparticle uptake into human endothelial cells under physiological cyclic stretch

BACKGROUND In general the prediction of the toxicity and therapeutic efficacy of engineered nanoparticles in humans is initially determined using in vitro static cell culture assays. However, such test systems may not be sufficient for testing nanoparticles intended for intravenous application. Once injected, these nanoparticles are caught up in the blood stream in vivo and are therefore in continuous movement. Physical forces such as shear stress and cyclic stretch caused by the pulsatile blood flow are known to change the phenotype of endothelial cells which line the luminal side of the vasculature and thus may be able to affect cell-nanoparticle interactions. METHODS In this study we investigated the uptake of amorphous silica nanoparticles in primary endothelial cells (HUVEC) cultured under physiological cyclic stretch conditions (1 Hz, 5% stretch) and compared this to cells in a standard static cell culture system. The toxicity of varying concentrations was assessed using cell viability and cytotoxicity studies. Nanoparticles were also characterized for the induction of an inflammatory response. Changes to cell morphology was evaluated in cells by examining actin and PECAM staining patterns and the amounts of nanoparticles taken up under the different culture conditions by evaluation of intracellular fluorescence. The expression profile of 26 stress-related was determined by microarray analysis. RESULTS The results show that cytotoxicity to endothelial cells caused by silica nanoparticles is not significantly altered under stretch compared to static culture conditions. Nevertheless, cells cultured under stretch internalize fewer nanoparticles. The data indicate that the decrease of nanoparticle content in stretched cells was not due to the induction of cell stress, inflammation processes or an enhanced exocytosis but rather a result of decreased endocytosis. CONCLUSIONS In conclusion, this study shows that while the toxic impact of silica nanoparticles is not altered by stretch this dynamic model demonstrates altered cellular uptake of nanoparticles under physiologically relevant in vitro cell culture models. In particular for the development of nanoparticles for biomedical applications such improved in vitro cell culture models may play a pivotal role in the reduction of animal experiments and development costs

Effect of endothelial cell heterogeneity on nanoparticle uptake

Endothelial cells exhibit distinct properties in morphology and functions in different organs that can be exploited for nanomedicine targeting. In this work, endothelial cells from different organs, i.e. brain, lung, liver, and kidney, were exposed to plain, carboxylated, and amino-modified silica. As expected, different protein coronas were formed on the different nanoparticle types and these changed when foetal bovine serum (FBS) or human serum were used. Uptake efficiencies differed strongly in the different endothelia, confirming that the cells retained some of their organ-specific differences. However, all endothelia showed higher uptake for the amino modified silica in FBS, but, interestingly, this changed to the carboxylated silica when human serum was used, confirming that differences in the protein corona affect uptake preferences by cells. Thus, uptake rates of fluid phase markers and transferrin were determined in liver and brain endothelium to compare their endocytic activity. Overall, our results showed that endothelial cells of different organs have very different nanoparticle uptake efficiency, likely due to differences in receptor expression, affinity, and activity. A thorough characterization of phenotypic differences in the endothelia lining different organs is key to the development of targeted nanomedicine

Response of micro- and macrovascular endothelial cells to starch-based fiber meshes for bone tissue engineering

The establishment of a functional vasculature is as yet an unrealized milestone in bone reconstruction therapy. For this study, fibermesh scaffolds obtained from a blend of starch and poly(caprolactone) (SPCL), that have previously been shown to be an excellent material for the proliferation and differentiation of bone marrow cells and thereby represent great potential as constructs for bone regeneration, were examined for endothelial cell (EC) compatibility. To be successfully applied in vivo, this tissue engineered construct should also be able to support the growth of ECs in order to facilitate vascularization and therefore assure the viability of the construct upon implantation. The main goal of this study was to examine the interactions between ECs and SPCL fiber meshes. Primary cultures of HUVEC cells were selected as a model of macrovascular cells and the cell line HPMEC-ST1.6R as a model for microvascular ECs. Both macro- and microvascular ECs adhered to SPCL fiber-mesh scaffolds and grew to cover much of the available surface area of the scaffold. In addition, ECs growing on the SPCL fibers exhibited a typical morphology, maintained important functional properties, such as the expression of the intercellular junction proteins, PECAM-1 and VE-cadherin, the expression of the most typical endothelial marker vWF and sensitivity to pro-inflammatory stimuli, as shown by induction of the expression of cell adhesion molecules (CAMs) by lipopolysaccharide (LPS). These data indicate that ECs growing on SPCL fiber-mesh scaffolds maintain a normal expression of ECspecific genes/proteins, indicating a cell compatibility and potential suitability of these scaffolds for the vascularization process in bone tissue engineering in vivo

Gold nanoparticles induce cytotoxicity in the alveolar type-II cell lines A549 and NCIH441.

International audienceBackground: During the last years engineered nanoparticles (NPs) have been extensively used in different technologies and consequently many questions have arisen about the risk and the impact on human health following exposure to nanoparticles. Nevertheless, at present knowledge about the cytotoxicity induced by NPs is still largely incomplete. In this context, we have investigated the cytotoxicity induced by gold nanoparticles (AuNPs), which differed in size and purification grade (presence or absence of sodium citrate residues on the particle surface) in vitro, in the human alveolar type-II (ATII)-like cell lines A549 and NCIH441

Inflammatory and cytotoxic responses of an alveolar-capillary coculture model to silica nanoparticles: Comparison with conventional monocultures

<p>Abstract</p> <p>Background</p> <p>To date silica nanoparticles (SNPs) play an important role in modern technology and nanomedicine. SNPs are present in various materials (tyres, electrical and thermal insulation material, photovoltaic facilities). They are also used in products that are directly exposed to humans such as cosmetics or toothpaste. For that reason it is of great concern to evaluate the possible hazards of these engineered particles for human health. Attention should primarily be focussed on SNP effects on biological barriers. Accidentally released SNP could, for example, encounter the alveolar-capillary barrier by inhalation. In this study we examined the inflammatory and cytotoxic responses of monodisperse amorphous silica nanoparticles (aSNPs) of 30 nm in size on an <it>in vitro </it>coculture model mimicking the alveolar-capillary barrier and compared these to conventional monocultures.</p> <p>Methods</p> <p>Thus, the epithelial cell line, H441, and the endothelial cell line, ISO-HAS-1, were used in monoculture and in coculture on opposite sides of a filter membrane. Cytotoxicity was evaluated by the MTS assay, detection of membrane integrity (LDH release), and TER (Transepithelial Electrical Resistance) measurement. Additionally, parameters of inflammation (sICAM-1, IL-6 and IL-8 release) and apoptosis markers were investigated.</p> <p>Results</p> <p>Regarding toxic effects (viability, membrane integrity, TER) the coculture model was less sensitive to apical aSNP exposure than the conventional monocultures of the appropriate cells. On the other hand, the <it>in vitro </it>coculture model responded with the release of inflammatory markers in a much more sensitive fashion than the conventional monoculture. At concentrations that were 10-100fold less than the toxic concentrations the apically exposed coculture showed a release of IL-6 and IL-8 to the basolateral side. This may mimic the early inflammatory events that take place in the pulmonary alveoli after aSNP inhalation. Furthermore, a number of apoptosis markers belonging to the intrinsic pathway were upregulated in the coculture following aSNP treatment. Analysis of the individual markers indicated that the cells suffered from DNA damage, hypoxia and ER-stress.</p> <p>Conclusion</p> <p>We present evidence that our <it>in vitro </it>coculture model of the alveolar-capillary barrier is clearly advantageous compared to conventional monocultures in evaluating the extent of damage caused by hazardous material encountering the principle biological barrier in the lower respiratory tract.</p

The impact of intercellular communication for the generation of complex multicellular prevascularized tissue equivalents

In reconstructive surgery the use of prevascularized soft tissue equivalents is a promising approach for wound coverage of defects after tumor resection or trauma. However, in previous studies to generate soft tissue equivalents on collagen membranes, microcapillaries were restricted to superficial areas. In this study, to understand which factors were involved in the formation of these microcapillaries, the levels of the angiogenic factors vascular endothelial growth factor (VEGF), Interleukin-8 (IL-8), and basic fibroblast growth factor (bFGF) in the supernatants of the tissue equivalents were examined at various time points and conditions. Additionally, the influence of these factors on viability, proliferation, migration, and tube formation in monocultures compared to cocultures of fibroblast and endothelial cells was examined. The results showed that VEGF production was decreased in cocultures compared to fibroblast monocultures and the lowest VEGF levels were observed in endothelial cell monocultures. Additionally, the highest levels of IL-8 were observed in cocultures compared to monocultures. Similar results were observed for bFGF with lowest levels seen within the first 24 hr and highest levels in cocultures. VEGF and IL-8 were shown to promote endothelial cell viability, proliferation and migration and angiogenic parameters such as tube density, total tube length, and number of tube branches. Addition of VEGF and IL-8 to cocultures resulted in accelerated and denser formation of capillary-like structures. The results indicate that VEGF, IL-8, and bFGF strongly influence cellular behavior of endothelial cells and this information should be useful in promoting the formation of microcapillary-like structures in complex tissue equivalents

Surface-modified 3D starch-based scaffold for improved endothelialization for bone tissue engineering

Providing adequate vascularization is one of the main hurdles to the widespread clinical application of bone tissue engineering approaches. Due to their unique role in blood vessel formation, endothelial cells (EC) play a key role in the establishment of successful vascularization strategies. However, currently available polymeric materials do not generally support EC growth without coating with adhesive proteins. In this work we present argon plasma treatment as a suitable method to render the surface of a 3D starch-based scaffold compatible for ECs, this way obviating the need for protein precoating. To this end we studied the effect of plasma modification on surface properties, protein adsorption and ultimately on several aspects regarding EC behaviour. Characterization of surface properties revealed increased surface roughness and change in topography, while at the chemical level a higher oxygen content was demonstrated. The increased surface roughness of the material, together with the changed surface chemistry modulated protein adsorption as indicated by the different adsorption profile observed for vitronectin. In vitro studies showed that human umbilical vein ECs (HUVECs) seeded on plasma-modified scaffolds adhered, remained viable, proliferated, and maintained the typical cobblestone morphology, as observed for positive controls (scaffold pre-coated with adhesive proteins). Furthermore, genotypic expression of endothelial markers was maintained and neighbouring cells expressed PECAM-1 at the single-cell-level. These results indicate that Ar plasma modification is an effective methodology with potential to be incorporated in biomaterial strategies to promote the formation of vascularized engineered bone