Analytical and toxicological aspects of nanomaterials in different product groups: Challenges and opportunities

The widespread integration of engineered nanomaterials into consumer and industrial products creates new challenges and requires innovative approaches in terms of design, testing, reliability, and safety of nanotechnology. The aim of this review article is to give an overview of different product groups in which nanomaterials are present and outline their safety aspects for consumers. Here, release of nanomaterials and related analytical challenges and solutions as well as toxicological considerations, such as dose-metrics, are discussed. Additionally, the utilization of engineered nanomaterials as pharmaceuticals or nutraceuticals to deliver and release cargo molecules is covered. Furthermore, critical pathways for human exposure to nanomaterials, namely inhalation and ingestion, are discussed in the context of risk assessment. Analysis of NMs in food, innovative medicine or food contact materials is discussed. Specific focus is on the presence and release of nanomaterials, including whether nanomaterials can migrate from polymer nanocomposites used in food contact materials. With regard to the toxicology and toxicokinetics of nanomaterials, aspects of dose metrics of inhalation toxicity as well as ingestion toxicology and comparison between in vitro and in vivo conclusions are considered. The definition of dose descriptors to be applied in toxicological testing is emphasized. In relation to potential exposure from different products, opportunities arising from the use of advanced analytical techniques in more unique scenarios such as release of nanomaterials from medical devices such as orthopedic implants are addressed. Alongside higher product performance and complexity, further challenges regarding material characterization and safety, as well as acceptance by the general public are expected

On the reproducibility of extrusion-based bioprinting: round robin study on standardization in the field

The outcome of three-dimensional (3D) bioprinting heavily depends, amongst others, on the interaction between the developed bioink, the printing process, and the printing equipment. However, if this interplay is ensured, bioprinting promises unmatched possibilities in the health care area. To pave the way for comparing newly developed biomaterials, clinical studies, and medical applications (i.e. printed organs, patient-specific tissues), there is a great need for standardization of manufacturing methods in order to enable technology transfers. Despite the importance of such standardization, there is currently a tremendous lack of empirical data that examines the reproducibility and robustness of production in more than one location at a time. In this work, we present data derived from a round robin test for extrusion-based 3D printing performance comprising 12 different academic laboratories throughout Germany and analyze the respective prints using automated image analysis (IA) in three independent academic groups. The fabrication of objects from polymer solutions was standardized as much as currently possible to allow studying the comparability of results from different laboratories. This study has led to the conclusion that current standardization conditions still leave room for the intervention of operators due to missing automation of the equipment. This affects significantly the reproducibility and comparability of bioprinting experiments in multiple laboratories. Nevertheless, automated IA proved to be a suitable methodology for quality assurance as three independently developed workflows achieved similar results. Moreover, the extracted data describing geometric features showed how the function of printers affects the quality of the printed object. A significant step toward standardization of the process was made as an infrastructure for distribution of material and methods, as well as for data transfer and storage was successfully established

On the reproducibility of extrusion-based bioprinting: round robin study on standardization in the field

The outcome of three-dimensional (3D) bioprinting heavily depends, amongst others, on the interaction between the developed bioink, the printing process, and the printing equipment. However, if this interplay is ensured, bioprinting promises unmatched possibilities in the health care area. To pave the way for comparing newly developed biomaterials, clinical studies, and medical applications (i.e. printed organs, patient-specific tissues), there is a great need for standardization of manufacturing methods in order to enable technology transfers. Despite the importance of such standardization, there is currently a tremendous lack of empirical data that examines the reproducibility and robustness of production in more than one location at a time. In this work, we present data derived from a round robin test for extrusion-based 3D printing performance comprising 12 different academic laboratories throughout Germany and analyze the respective prints using automated image analysis (IA) in three independent academic groups. The fabrication of objects from polymer solutions was standardized as much as currently possible to allow studying the comparability of results from different laboratories. This study has led to the conclusion that current standardization conditions still leave room for the intervention of operators due to missing automation of the equipment. This affects significantly the reproducibility and comparability of bioprinting experiments in multiple laboratories. Nevertheless, automated IA proved to be a suitable methodology for quality assurance as three independently developed workflows achieved similar results. Moreover, the extracted data describing geometric features showed how the function of printers affects the quality of the printed object. A significant step toward standardization of the process was made as an infrastructure for distribution of material and methods, as well as for data transfer and storage was successfully established

Controlled Release of Vascular Endothelial Growth Factor from Heparin-Functionalized Gelatin Type A and Albumin Hydrogels

Bio-based release systems for pro-angiogenic growth factors are of interest, to overcome insufficient vascularization and bio-integration of implants. In this study, we investigated heparin-functionalized hydrogels based on gelatin type A or albumin as storage and release systems for vascular endothelial growth factor (VEGF). The hydrogels were crosslinked using carbodiimide chemistry in presence of heparin. Heparin-functionalization of the hydrogels was monitored by critical electrolyte concentration (CEC) staining. The hydrogels were characterized in terms of swelling in buffer solution and VEGF-containing solutions, and their loading with and release of VEGF was monitored. The equilibrium degree of swelling (EDS) was lower for albumin-based gels compared to gelatin-based gels. EDS was adjustable with the used carbodiimide concentration for both biopolymers. Furthermore, VEGF-loading and release were dependent on the carbodiimide concentration and loading conditions for both biopolymers. Loading of albumin-based gels was higher compared to gelatin-based gels, and its burst release was lower. Finally, elevated cumulative VEGF release after 21 days was determined for albumin-based hydrogels compared to gelatin A-based hydrogels. We consider the characteristic net charges of the proteins and degradation of albumin during release time as reasons for the observed effects. Both heparin-functionalized biomaterial systems, chemically crosslinked gelatin type A or albumin, had tunable physicochemical properties, and can be considered for controlled delivery of the pro-angiogenic growth factor VEGF

Extrusion-based 3D printing of poly(ethylene glycol) diacrylate hydrogels containing positively and negatively charged groups

Hydrogels are an interesting class of materials used in extrusion-based 3D printing, e.g., for drug delivery or tissue engineering. However, new hydrogel formulations for 3D printing as well as a detailed understanding of crucial formulation properties for 3D printing are needed. In this contribution, hydrogels based on poly(ethylene glycol) diacrylate (PEG-DA) and the charged monomers 3-sulfopropyl acrylate and [2-(acryloyloxy)ethyl]trimethylammonium chloride are formulated for 3D printing, together with Poloxamer 407 (P407). Chemical curing of formulations with PEG-DA and up to 5% (w/w) of the charged monomers was possible without difficulty. Through careful examination of the rheological properties of the non-cured formulations, it was found that flow properties of formulations with a high P407 concentration of 22.5% (w/w) possessed yield stresses well above 100 Pa together with pronounced shear thinning behavior. Thus, those formulations could be processed by 3D printing, as demonstrated by the generation of pyramidal objects. Modelling of the flow profile during 3D printing suggests that a plug-like laminar flow is prevalent inside the printer capillary. Under such circumstances, fast recovery of a high vicosity after material deposition might not be necessary to guarantee shape fidelity because the majority of the 3D printed volume does not face any relevant shear stress during printing

Physical interactions strengthen chemical gelatin methacryloyl gels

Chemically cross-linkable gelatin methacryloyl (GM) derivatives are getting increasing attention regarding biomedical applications. Thus, thorough investigations are needed to achieve full understanding and control of the physico-chemical behavior of these promising biomaterials. We previously introduced gelatin methacryloyl acetyl (GMA) derivatives, which can be used to control physical network formation (solution viscosity, sol-gel transition) independently from chemical cross-linking by variation of the methacryloyl-to-acetyl ratio. It is known that temperature dependent physical network formation significantly influences the mechanical properties of chemically cross-linked GM hydrogels. We investigated the temperature sensitivity of GM derivatives with different degrees of modification (GM2, GM10), or similar degrees of modification but different methacryloyl contents (GM10, GM2A8). Rheological analysis showed that the low modified GM2 forms strong physical gels upon cooling while GM10 and GM2A8 form soft or no gels. Yet, compression testing revealed that all photo cross-linked GM(A) hydrogels were stronger if cooling was applied during hydrogel preparation. We suggest that the hydrophobic methacryloyl and acetyl residues disturb triple helix formation with increasing degree of modification, but additionally form hydrophobic structures, which facilitate chemical cross-linking

Extrusion-Based 3D Printing of Poly(ethylene glycol) Diacrylate Hydrogels Containing Positively and Negatively Charged Groups

Hydrogels are an interesting class of materials used in extrusion-based 3D printing, e.g., for drug delivery or tissue engineering. However, new hydrogel formulations for 3D printing as well as a detailed understanding of crucial formulation properties for 3D printing are needed. In this contribution, hydrogels based on poly(ethylene glycol) diacrylate (PEG-DA) and the charged monomers 3-sulfopropyl acrylate and [2-(acryloyloxy)ethyl]trimethylammonium chloride are formulated for 3D printing, together with Poloxamer 407 (P407). Chemical curing of formulations with PEG-DA and up to 5% (w/w) of the charged monomers was possible without difficulty. Through careful examination of the rheological properties of the non-cured formulations, it was found that flow properties of formulations with a high P407 concentration of 22.5% (w/w) possessed yield stresses well above 100 Pa together with pronounced shear thinning behavior. Thus, those formulations could be processed by 3D printing, as demonstrated by the generation of pyramidal objects. Modelling of the flow profile during 3D printing suggests that a plug-like laminar flow is prevalent inside the printer capillary. Under such circumstances, fast recovery of a high vicosity after material deposition might not be necessary to guarantee shape fidelity because the majority of the 3D printed volume does not face any relevant shear stress during printing

Active ester containing surfmer for one-stage polymer nanoparticle surface functionalization in mini-emulsion polymerization

Functional surface active monomers (surfmers) are molecules that combine the functionalities of surface activity, polymerizability and reactive groups. This study presents an improved pathway for the synthesis of the active ester containing surfmer p-(11-acrylamido)undecanoyloxyphenyl dimethylsulfonium methyl sulfate (AUPDS). Further, the preparation of poly(methyl methacrylate) and polystyrene nanoparticles (NPs) by miniemulsion polymerization using AUPDS is investigated, leading to NPs with active ester groups on their surface. By systematically varying reaction parameters and reagent concentrations, it was found that AUPDS feed concentrations between 2-4 mol-% yielded narrowly distributed and stable spherical particles with average sizes between 83 nm and 134 nm for non-cross-linked NPs, and up to 163 nm for cross-linked NPs. By basic hydrolysis of the active ester groups in aqueous dispersion, the positive ζ-potential (ZP) was converted into a negative ZP and charge quantities determined by polyelectrolyte titrations before and after hydrolysis were in the same range, indicating that the active ester groups were indeed accessible in aqueous suspension. Increasing cross-linker amounts over 10 mol-% also led to a decrease of ZP of NPs, probably due to internalization of the AUPDS during polymerization. In conclusion, by using optimized reaction conditions, it is possible to prepare active ester functionalized NPs in one stage using AUPDS as a surfmer in miniemulsion polymerization

Covalent incorporation of tobacco mosaic virus increases the stiffness of poly(ethylene glycol) diacrylate hydrogels

Hydrogels are versatile materials, finding applications as adsorbers, supports for biosensors and biocatalysts or as scaffolds for tissue engineering. A frequently used building block for chemically cross-linked hydrogels is poly(ethylene glycol) diacrylate (PEG-DA). However, after curing, PEG-DA hydrogels cannot be functionalized easily. In this contribution, the stiff, rod-like tobacco mosaic virus (TMV) is investigated as a functional additive to PEG-DA hydrogels. TMV consists of more than 2000 identical coat proteins and can therefore present more than 2000 functional sites per TMV available for coupling, and thus has been used as a template or building block for nano-scaled hybrid materials for many years. Here, PEG-DA (Mn = 700 g/mol) hydrogels are combined with a thiol-group presenting TMV mutant (TMVCys). By covalent coupling of TMVCys into the hydrogel matrix via the thiol-Michael reaction, the storage modulus of the hydrogels is increased compared to pure PEG-DA hydrogels and to hydrogels containing wildtype TMV (wt-TMV) which is not coupled covalently into the hydrogel matrix. In contrast, the swelling behaviour of the hydrogels is not altered by TMVCys or wt-TMV. Transmission electron microscopy reveals that the TMV particles are well dispersed in the hydrogels without any large aggregates. These findings give rise to the conclusion that well-defined hydrogels were obtained which offer the possibility to use the incorporated TMV as multivalent carrier templates e.g. for enzymes in future studies