Development of complex-shaped liver multicellular spheroids as a human-based model for nanoparticle toxicity assessment in vitro.

The emergence of human-based models is incontestably required for the study of complex physiological pathways and validation of reliable in vitro methods as alternative for in vivo studies in experimental animals for toxicity assessment. With this objective, we have developed and tested three dimensional environments for cells using different types of hydrogels including transglutaminase-cross-linked gelatin, collagen type I, and growth-factor depleted Matrigel. Cells grown in Matrigel exhibited the greatest cell proliferation and spheroid diameter. Moreover, analysis of urea and albumin biosynthesis revealed that the created system allowed the immortalized liver cell line HepG2 to re-establish normal hepatocyte-like properties which were not observed under the conditions of conventional cell cultures. This study presents a scalable technology for production of complex-shaped liver multicellular spheroids as a system which improves the predictive value of cell-based assays for safety and risk assessment. The time- and dose-dependent toxicity of nanoparticles demonstrates a higher cytotoxic effect when HepG2 cells grown as monolayer than embedded in hydrogels. The experimental setup provided evidence that the cell environment has significant influence on cell sensitivity and that liver spheroid is a useful and novel tool to examine nanoparticle dosing effect even at the level of in vitro studies. Therefore, this system can be applied to a wide variety of potentially hostile compounds in basic screening to provide initial warning of adverse effects and trigger subsequent analysis and remedial actions.

Differential Repair Protein Recruitment at Sites of Clustered and Isolated DNA Double-Strand Breaks Produced by High-Energy Heavy Ions.

DNA double-strand break (DSB) repair is crucial to maintain genomic stability. The fidelity of the repair depends on the complexity of the lesion, with clustered DSBs being more difficult to repair than isolated breaks. Using live cell imaging of heavy ion tracks produced at a high-energy particle accelerator we visualised simultaneously the recruitment of different proteins at individual sites of complex and simple DSBs in human cells. NBS1 and 53BP1 were recruited in a few seconds to complex DSBs, but in 40% of the isolated DSBs the recruitment was delayed approximately 5 min. Using base excision repair (BER) inhibitors we demonstrate that some simple DSBs are generated by enzymatic processing of base damage, while BER did not affect the complex DSBs. The results show that DSB processing and repair kinetics are dependent on the complexity of the breaks and can be different even for the same clastogenic agent.

A cell-based biosensor for nanomaterials cytotoxicity assessment in three dimensional cell culture.

Nanoparticles (NPs) are widely used in consumer and medicinal products. The high prevalence of nanoparticles in the environment raises concerns regarding their effects on human health, but there is limited knowledge about how NPs interact with cells or tissues. Because the European Union has called for a substantial reduction of animal experiments for scientific purposes (Directive 2010/63), increased efforts are required to develop in vitro models to evaluate potentially hazardous agents. Here, we describe a new cell-based biosensor for the evaluation of NPs cytotoxicity. The new biosensor is based on transgenic human hepatoblastoma cells (HepG2) that express a secreted form of alkaline phosphatase (SEAP) as a reporter protein whose expression is induced upon activation of a stress response pathway controlled by the transcription regulator nuclear factor-κB (NF-κB). The NF-κB_HepG2 sensor cells were cultured in a Matrigel-based three dimensional environment to simulate the in vivo situation. The new biosensor cells offer the advantage of generating fast and reproducible readout at lower concentrations and shorter incubation time than conventional viability assays, avoid possible interaction between nanomaterials and assay compounds, therefore, minimize generation of false positive or negative results and indicate mechanism of toxicity through NF-κB signaling.

Sensitive and rapid detection of aflatoxin M1 in milk utilizing enhanced SPR and p(HEMA) brushes.

The rapid and sensitive detection of aflatoxin M1 (AFM1) in milk by using surface plasmon resonance (SPR) biosensor is reported. This low molecular weight mycotoxin is analyzed using an indirect competitive immunoassay that is amplified by secondary antibodies conjugated with Au nanoparticles. In order to prevent fouling on the sensor surface by the constituents present in analyzed milk samples, an interface with poly(2-hydroxyethyl methacrylate) p(HEMA) brush was employed. The study presents a comparison of performance characteristics of p(HEMA)-based sensor with a regularly used polyethylene glycol-based architecture relying on mixed thiol self-assembled monolayer. Both sensors are characterized in terms of surface mass density of immobilized AFM1 conjugate as well as affinity bound primary and secondary antibodies. The efficiency of the amplification strategy based on Au nanoparticle is discussed. The biosensor allowed for highly sensitive detection of AFM1 in milk with a limit of detection (LOD) as low as 18pgmL(-1) with the analysis time of 55min.

Fast and sensitive detection of ochratoxin A in red wine by nanoparticle-enhanced SPR.

Herein, we present a fast and sensitive biosensor for detection of Ochratoxin A (OTA) in a red wine that utilizes gold nanoparticle-enhanced surface plasmon resonance (SPR). By combining an indirect competitive inhibition immunoassay and signal enhancement by secondary antibodies conjugated with gold nanoparticles (AuNPs), highly sensitive detection of low molecular weight compounds (such as OTA) was achieved. The reported biosensor allowed for OTA detection at concentrations as low as 0.75 ng mL(-1) and its limit of detection was improved by more than one order of magnitude to 0.068 ng mL(-1) by applying AuNPs as a signal enhancer. The study investigates the interplay of size of AuNPs and affinity of recognition elements affecting the efficiency of the signal amplification strategy based on AuNP. Furthermore, we observed that the presence of polyphenolic compounds in wine samples strongly interferes with the affinity binding on the surface. To overcome this limitation, a simple pre-treatment of the wine sample with the binding agent poly(vinylpyrrolidone) (PVP) was successfully applied.