Nanotoxicity in Cancer Research: Technical Protocols and Considerations for the Use of 3D Tumour Spheroids

The poor clinical translation of oncological nanomedicine products is one of the greatest challenges faced by research today. The use of reductionist in vitro models of human cancer and non-predictive animal models is generally considered as one of the main causes of such very low translation rate. The integration of three-dimensional (3D) tumour spheroids in the early stages of the preclinical screening pipeline could significantly facilitate the translation of nanomedicine candidates into clinical practice, by allowing for a more reliable prediction of their efficacy and safety in humans. To lead a successful integration of 3D spheroids, protocols that satisfy issues of ease-of-use, reproducibility and compatibility with conventional and high-throughput assays, without losing the advantages offered by two-dimensional (2D) cell systems, are still needed. To address such need, protocols for the formation and characterisation of scaffold-free 3D tumour spheroids of human adenocarcinoma cells were developed and optimised in this study for their application in nanomedicine safety testing. The protocols reported in this chapter provide the ground on how 3D tumour spheroids could be implemented to design nanomedicine products and speed up experimental cancer research, eliminating those candidates that are likely to be ineffective or unsafe in human at early development stages

Graphene toxicity as a double-edged sword of risks and exploitable opportunities: A critical analysis of the most recent trends and developments

Increased production volumes and a broadening application spectrum of graphene have raised concerns about its potential adverse effects on human health. Numerous reports demonstrate that graphene irrespective of its particular form exerts its effects on a widest range of living organisms, including prokaryotic bacteria and viruses, plants, micro-and macro-invertebrates, mammalian and human cells and whole animals in vivo. However, the available experimental data is frequently a matter of significant divergence and even controversy. Therefore, we provide here a critical analysis of the most recent (2015-2016) reports accumulated in the graphene-related materials biocompatibility and toxicology field in order to elucidate the cutting edge achievements, emerging trends and future opportunities in the area. Experimental findings from the diverse in vitro and in vivo model systems are analysed in the context of the most likely graphene exposure scenarios, such as respiratory inhalation, ingestion route, parenteral administration and topical exposure through the skin. Key factors influencing the toxicity of graphene and its complex derivatives as well as potential risk mitigation approaches exploiting graphene physicochemical properties, surface modifications and possible degradation pathways are also discussed along with its emerging applications for healthcare, diagnostics and innovative therapeutic approaches

Editorial: Use of 3D Models in Drug Development and Precision Medicine - Advances and Outlook

Three-dimensional (3D) in vitro models in the drug development pipeline can help selecting the most promising and safe drug candidates at the pre-clinical stage, prior to clinical trials, reducing and sometimes even replacing animal studies in accordance with the “3Rs (Reduction, Refinement and Replacement) principle”

Towards the Identification of an In Vitro Tool for Assessing the Biological Behavior of Aerosol Supplied Nanomaterials

Nanoparticles (NP)-based inhalation systems for drug delivery can be administered in liquid form, by nebulization or using pressurized metered dose inhalers, and in solid form by means of dry powder inhalers. However, NP delivery to the lungs has many challenges including the formulation instability due to particle-particle interactions and subsequent aggregation, causing poor deposition in the small distal airways and subsequent alveolar macrophages activity, which could lead to inflammation. This work aims at providing an in vitro experimental design for investigating the correlation between the physico-chemical properties of NP, and their biological behavior, when they are used as NP-based inhalation treatments, comparing two different exposure systems. By means of an aerosol drug delivery nebulizer, human lung cells cultured at air-liquid interface (ALI) were exposed to two titanium dioxide NP (NM-100 and NM-101), obtained from the JRC repository. In parallel, ALI cultures were exposed to NP suspension by direct inoculation, i.e., by adding the NP suspensions on the apical side of the cell cultures with a pipette. The formulation stability of NP, measured as hydrodynamic size distributions, the cell viability, cell monolayer integrity, cell morphology and pro-inflammatory cytokines secretion were investigated. Our results demonstrated that the formulation stability of NM-100 and NM-101 was strongly dependent on the aggregation phenomena that occur in the conditions adopted for the biological experiments. Interestingly, comparable biological data between the two exposure methods used were observed, suggesting that the conventional exposure coupled to ALI culturing conditions offers a relevant in vitro tool for assessing the correlation between the physico-chemical properties of NP and their biological behavior, when NP are used as drug delivery systems

Multifactorial determinants that govern nanoparticle uptake by human endothelial cells under flow

Vascular endothelium is a potential target for therapeutic intervention in diverse pathological processes, including inflammation, atherosclerosis, and thrombosis. By virtue of their intravascular topography, endothelial cells are exposed to dynamically changing mechanical forces that are generated by blood flow. In the present study, we investigated the interactions of negatively charged 2.7 nm and 4.7 nm CdTe quantum dots and 50 nm silica particles with cultured endothelial cells under regulated shear stress (SS) conditions. Cultured cells within the engineered microfluidic channels were exposed to nanoparticles under static condition or under low, medium, and high SS rates (0.05, 0.1, and 0.5 Pa, respectively). Vascular inflammation and associated endothelial damage were simulated by treatment with tumor necrosis factor-α (TNF-α) or by compromising the cell membrane with the use of low Triton X-100 concentration. Our results demonstrate that SS is critical for nanoparticle uptake by endothelial cells. Maximal uptake was registered at the SS rate of 0.05 Pa. By contrast, endothelial exposure to mild detergents or TNF-α treatment had no significant effect on nanoparticle uptake. Atomic force microscopy demonstrated the increased formation of actin-based cytoskeletal structures, including stress fibers and membrane ruffles, which have been associated with nanoparticle endocytosis. In conclusion, the combinatorial effects of SS rates, vascular endothelial conditions, and nanoparticle physical and chemical properties must be taken into account for the successful design of nanoparticle–drug conjugates intended for parenteral delivery

Characterization of interaction of magnetic nanoparticles with breast cancer cells

Background: Different superparamagnetic iron oxide nanoparticles have been tested for their potential use in cancer treatment, as they enter into cells with high effectiveness, do not induce cytotoxicity, and are retained for relatively long periods of time inside the cells. We have analyzed the interaction, internalization and biocompatibility of dimercaptosuccinic acid-coated superparamagnetic iron oxide nanoparticles with an average diameter of 15 nm and negative surface charge in MCF-7 breast cancer cells. Results: Cells were incubated with dimercaptosuccinic acid-coated superparamagnetic iron oxide nanoparticles for different time intervals, ranging from 0.5 to 72 h. These nanoparticles showed efficient internalization and relatively slow clearance. Time-dependent uptake studies demonstrated the maximum accumulation of dimercaptosuccinic acid-coated superparamagnetic iron oxide nanoparticles after 24 h of incubation, and afterwards they were slowly removed from cells. Superparamagnetic iron oxide nanoparticles were internalized by energy dependent endocytosis and localized in endosomes. Transmission electron microscopy studies showed macropinocytosis uptake and clathrin-mediated internalization depending on the nanoparticles aggregate size. MCF-7 cells accumulated these nanoparticles without any significant effect on cell morphology, cytoskeleton organization, cell cycle distribution, reactive oxygen species generation and cell viability, showing a similar behavior to untreated control cells. Conclusions: All these findings indicate that dimercaptosuccinic acid-coated superparamagnetic iron oxide nanoparticles have excellent properties in terms of efficiency and biocompatibility for application to target breast cancer cellsThe research leading to these results have received partial funding from the European Seventh Framework Programme (FP7/2007-2013) under the project MULTIFUN grant agreement no. 262943, and the project Nanofrontmag-CM (S2013/MIT-2850) from the Comunidad de Madrid. Additional grants were obtained from BFU 2011–29038 and CTQ2013-48767-C3-3-R from the Ministerio de Economia y Competitividad and S2009/Mat 1507 from the Comunidad de Madrid (to JLC), from EU FP7 project NAMDIATREAM (ref 246479) and from “la Caixa” / CNB International PhD Programme Fellowship