Nanotechnology management for a safer work environment

Why should there be a point of concern for nanomaterials?

Useful as they may seem, nanomaterials are a point of concern for many laboratories worldwide. Because of their very small size (as seen in Fig. 1), they can enter the body through a variety of routes and effectively have unknown effects in living organisms. Several laboratories worldwide have recently adopted a more specialized safety policy in handling nanoparticles.

Fig. 1

Examples of synthesized gold nanomaterials.

Pulmonary studies in the past suggest that smaller particles are able to pass the normal macrophage defense of the pulmonary system, and that these particles tend to deposit on the lungs and eventually obstruct normal breathing [1, 6]. Other nanomaterials such as carbon nanotubes, graphene, and metal nanoparticles have different sets of hazards and dangers that should be taken seriously for proper precautions.

In this contribution, we aim to provide an overview of techniques that can be used to synthesize nanoparticles and assess their nanotoxicity. Further, management protocols on laboratory safety and handling of nanomaterials will be discussed.

Surface modifications for safer nanoparticles

Biological systems interact with nanoparticles through the surface; this is also the very reason why many nanoparticles have become good drug carriers as well as carriers of other bioactive compounds. Recently, they have been used to selectively deliver chemotherapeutic drugs to cancer cells. In catalysis, they are functionalized with catalytic functional groups to help address mass transfer issues in heterogeneous systems due to their large surface areas and tendency to aggregate/coalescence to stabilize [13]. In this section, we will discuss the different possible methods by which NPs can be functionalized with safer functional groups.

Graphene

Graphene is a carbon allotrope comprising sp² hybridized carbon atoms arranged as a monolayer and in a honeycomb lattice. Due to its interesting electronic properties, it has attracted a remarkable deal of attention in recent years. Graphene is usually prepared via exfoliation using a mineral acid, and then subsequent reduction. The imperfect reduction usually yields C=O (or COOH) in the surface which makes the material an oxide (graphene oxide, GO), with a type of surface relevant for biomedical applications [14] (Fig. 3a, b). However, GO has been shown to have various toxicities in many in-vivo testing which includes platelet aggregation in mice. To address this, a promising approach developed by Singh et al. [15] involves the use of an amine-functionalized graphene to substitute the highly carboxylated GO. This work demonstrates that via functional group modification, the interaction of the material with living systems can be minimized.

Fig. 3

TEM images of literature reported graphene [23], graphene oxide [24], and iron nanoparticles [25] respectively. Reprinted with permission from Refs. [23] (Copyright © 2007 Nature Publishing), [24] (Copyright © 2008 Elsevier Publishing), and [25] (Copyright © 2006 American Chemical Society).

Mechanism-based predictive toxicology approach

Predictive testing essentially describes a useful tool that factors in chemical composition, structure, crystal structure, shape, state of agglomeration as well as other surface properties of a nanomaterial to determine its response with high throughput screening, hazard ranking, and bioactivity [22]. Using a library of engineered nanomaterials, scientists can also predict potential molecular interactions between nanomaterials and cell surfaces. In order to start up a data set library, a comprehensive database of surface properties of materials including TiO₂, CeO2, and ZnO has to be compiled and then tested for biological assay (oxygen-radical generation and pro-inflammatory responses in cells) [23]. A second important requirement relates to the development of a good high-throughput screening method, which in this case was performed in a 384-well plate. Healthy cell lines were exposed to specific dosages of the nanoparticles and then subsequently analysed for mitochondrial membrane potential, intracellular calcium influx as well as increased membrane permeability in dying cells [24]. Last, but not the least, studies and knowledge on the mechanism injury pathway taken by the cells in the presence of the nanoparticles are required essential information to complete the predictive model, with a data processing framework that can perform calculations based on the physico-chemical properties and HTS results. Using advanced mathematical computational methods, structure-activity relationships can be established and subsequently validated using a more systematic animal model study. Also, this predictive approach can be used to obtain pulmonary and environmental hazard assessments.

Biological assays

The use of different cell lines to determine nanomaterials cytotoxicity remains one of the most convenient and powerful methods of analysis to determine safety issues of nanoentities. Although biological assays are currently slowly moving towards the direction of high-throughput screening using large micro-well plates and more advanced imaging instruments, data gathered using traditional techniques are still of practical importance to toxicologists. Nanoparticles testing can often mask their actual toxicities because of many factors related to NP-assay component interactions [25], but data obtained in traditional assays serves as starting point for further in vivo animal testing (usually using mice).

In a comprehensive cytotoxicity screening conducted by Kroll et al. [26], 23 engineered nanomaterials (titania, aluminum, zirconium and their composites) were tested against ten different human cell lines, with results showing that most of the particles cause oxidative stress to the cells (Fig. 4). All but one of the nanomaterials did not reduce cellular metabolic activity. A similar study specifically utilized cancel cell lines such as osteocytic and HeLa to determine the toxicity of AuNP, AgNP and single-walled carbon nanotubes (SWCNT). SWCNT was found to be most toxic among the three nanomaterials. The observed toxicity of SWCNT to cancer cells certainly poses restrictions in its possible use as a safe carrier of chemotherapeutic agents [27].

Fig. 4

HTS screening and data processing factoring in the surface properties of particles in order to predict the biological effects of nanoparticles. (Reprinted with permission from Ref. [26], Copyright © 2012 American Chemical Society.)

Laboratory practices for safer nanomaterials research

In terms of useful and safe practises to handle nanomaterials in research, laboratories can use three different control approaches in order to ensure a safety working environment: 1) engineering control (Fig. 5, highest panel), 2) administrative control, and 3) personal protective equipment (Fig. 5, two lower panels).

Fig. 5

The first panel shows the engineering controls used in most laboratories. The two lower panels, on the other hand, show the personal protective equipment that should be used to ensure complete safety. Reproduced with permission from http://www.uvm.edu/safety/lab/ppe-gloves-lab-coats-respirator

Engineering control including the installation of ventilation, the preferential use of less toxic materials and the use of designated storage cabinets remained to be the most important aspect of laboratory safety. Administrative control, on the other hand, refers to the use of proper warning notices and appropriate chemical hazard labels. Although this could be effective, they could have the tendency to be overseen by the researchers involved. Lastly, personal protective equipment includes (the use of) appropriate gloves, lab coat, and face protection. Although third in the hierarchy of laboratory safety controls, personal protective equipment is considered every researcher’s first line of defences against danger [1, 28].

Conti et al. [29] recently surveyed the environmental and health safety practices of many laboratories worldwide towards engineered nanomaterials. Including key laboratories from Asia, North America, Australia, and Europe, the study showed that most laboratories strictly employ engineering controls to ensure safety, while most laboratories reported standard non-high efficiency particulate filters (non-HEPA). Some responded that they use more extensive air filter apparatus as well as wet scrubbers for water-soluble organic pollutants. For personal protective equipment (PPE), most institutions recommend nano-specific PPE depending on the type of research and development.

In a later survey conducted by Balas et al. [30] some appalling results were found as to how researchers handle nanomaterials in the laboratory by direct contact with researchers working on the field. By contacting almost 3000 researchers (of which 10 % responded) gathered from the Web of Knowledge database of publication, these authors were able to get a semi-randomized sample of the practices of many research laboratories worldwide. Roughly 90 % of the respondents were not aware of local and national regulations for nanosafety. More disturbingly, most respondents do not protect themselves even with the knowledge that handled NP could be airborne.

Although engineering controls remain to be the most important safety precaution in every laboratory, researchers should always take into account potential materials toxicity and conduct scientific procedures in the safest possible ways. Ideally, research laboratories should adopt a detailed laboratory safety protocol for nanosafety, with detailed knowledge between both researchers and supervisors on the toxicity and hazards of investigated nanomaterials. Secondly, handling procedures should be properly documented and reported by the researchers and kept in a file so that everyone in the lab can possibly access all safety protocols.

Many safety aspects of nanotechnology are already in place in many laboratories. Importantly, decision-making tools in handling nanomaterials have been proposed by experienced scientists in the field. The importance of risk assessments cannot be overlooked in every laboratory experiment. This does not only give the researchers an idea of the overall experimental flow, but also allows for the preconception of safety back-up plans in case a hazard occurs. A good risk assessment provides two important parts that are essential to ensure full safety: 1) hazard assessment and 2) exposure assessment (Fig. 6). A detailed risk assessment, risk characterization and risk management can be obtained when these two elements are put together [31, 32].

Fig. 6

a) A good risk assessment prior to any experiment involves the evaluation of the chemical hazards and an estimate of the level of exposure. b) The creation of a risk assessment is the first step in producing a detailed risk management procedure involving prevention, mitigation, and communication. (Reprinted with permission from Ref. [26], Copyright © 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A separate assessment is proposed by Meili et al. [33] divides nanomaterials into low, intermediate and high priority depending on their qualities and where they fit in a developed map for such purposes (Fig. 7). It is also important to determine the set of protocols that fit within each assessment level: What safety precautions should at least be present if the risk is considered a low, intermediate or high priority? These questions should be answered in detail and kept in file accessible to all researchers in the lab.

Fig. 7

Risk assessment can be divided into a question-and-answer portion type of evaluation in order to determine whether the nanoparticle in question is considered a low priority, intermediate priority, and high priority. (Reprinted with permission from Ref. [31], Copyright © 2012 American Chemical Society.)