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Although interest and funding in nanotechnology for oncological applications is thriving, translating these novel therapeutics through the earliest stages of preclinical assessment remains challenging. Upon intravenous administration, nanomaterials interact with constituents of the blood inducing a wide range of associated immunotoxic effects. The literature on the immunological interactions of nanomaterials is vast and complicated. A small change in a particular characteristic of a nanomaterial (e.g., size, shape, or charge) can have a significant effect on its immunological profile in vivo, and poor selection of specific assays for establishing these undesirable effects can overlook this issue until the latest stages of preclinical assessment. This work describes the current literature on unintentional immunological effects associated with promising cancer nanomaterials (liposomes, dendrimers, mesoporous silica, iron oxide, gold, and quantum dots) and puts focus on what is missing in current preclinical evaluations. Opportunities for avoiding or limiting immunotoxicity through efficient preclinical assessment are discussed, with an emphasis placed on current regulatory views and requirements. Careful consideration of these issues will ensure a more efficient preclinical assessment of cancer nanomedicines, enabling a smoother clinical translation with less failures in the future. Here, the immunotoxic effects that occur after intravenous administration of promising cancer nanomedicines are described. The large number of preclinical failures associated with these nanomedicines is mainly due to unintentional immunological affects and methods to overcome these are detailed with reference to current regulatory requirements.

We present here a perspective detailing the current state-of-the-art technologies for the characterisation of nanoparticles (NPs) in liquid suspension. We detail the technologies involved and assess their applications in the determination of NP size and concentration. We also investigate the parameters that can influence the results and put forward a cause and effect analysis of the principle factors influencing the measurement of NP size and concentration by NP tracking analysis and dynamic light scattering, to identify areas where uncertainties in the measurement can arise. Also included are technologies capable of characterising NPs in solution, whose measurements are not based on light scattering. It is hoped that the manuscript, with its detailed description of the methodologies involved, will assist scientists in selecting the appropriate technology for characterising their materials and enabling them to comply with regulatory agencies' demands for accurate and reliable NP size and concentration data.

When assessing the risk and hazard of a non-pharmaceutical compound, the first step is determining acute toxicity, including toxicity following inhalation. Inhalation is a major exposure route for humans, and the respiratory epithelium is the first tissue that inhaled substances directly interact with. Acute inhalation toxicity testing for regulatory purposes is currently performed only in rats and/or mice according to OECD TG403, TG436, and TG433 test guidelines. Such tests are biased by the differences in the respiratory tract architecture and function across species, making it difficult to draw conclusions on the potential hazard of inhaled compounds in humans. Research efforts have been therefore focused on developing alternative, human-relevant models, with emphasis on the creation of advanced In vitro models. To date, there is no In vitro model that has been accepted by regulatory agencies as a stand-alone replacement for inhalation toxicity testing in animals. Here, we provide a brief introduction to current OECD test guidelines for acute inhalation toxicity, the interspecies differences affecting the predictive value of such tests, and the current regulatory efforts to advance alternative approaches to animal-based inhalation toxicity studies. We then list the steps that should allow overcoming the current challenges in validating In vitro alternatives for the successful replacement of animal-based inhalation toxicity studies. These steps are inclusive and descriptive, and should be detailed when adopting in house-produced 3D cell models for inhalation tests. Hence, we provide a checklist of key parameters that should be reported in any future scientific publications for reproducibility and transparency.When assessing the risk and hazard of a non-pharmaceutical compound, the first step is determining acute toxicity, including toxicity following inhalation. Inhalation is a major exposure route for humans, and the respiratory epithelium is the first tissue that inhaled substances directly interact with. Acute inhalation toxicity testing for regulatory purposes is currently performed only in rats and/or mice according to OECD TG403, TG436, and TG433 test guidelines. Such tests are biased by the differences in the respiratory tract architecture and function across species, making it difficult to draw conclusions on the potential hazard of inhaled compounds in humans. Research efforts have been therefore focused on developing alternative, human-relevant models, with emphasis on the creation of advanced In vitro models. To date, there is no In vitro model that has been accepted by regulatory agencies as a stand-alone replacement for inhalation toxicity testing in animals. Here, we provide a brief introduction to current OECD test guidelines for acute inhalation toxicity, the interspecies differences affecting the predictive value of such tests, and the current regulatory efforts to advance alternative approaches to animal-based inhalation toxicity studies. We then list the steps that should allow overcoming the current challenges in validating In vitro alternatives for the successful replacement of animal-based inhalation toxicity studies. These steps are inclusive and descriptive, and should be detailed when adopting in house-produced 3D cell models for inhalation tests. Hence, we provide a checklist of key parameters that should be reported in any future scientific publications for reproducibility and transparency.

Carbon-based nanomaterials (C-BNM) have recently attracted an increased attention as the materials with potential applications in industry and medicine. Bioresistance and proinflammatory potential of C-BNM is the main obstacle for their medicinal application which was documented in vivo and in vitro. However, there are still limited data especially on graphene derivatives such as graphene platelets (GP). In this work, we compared multi-walled carbon nanotubes (MWCNT) and two different types of pristine GP in their potential to activate inflammasome NLRP3 (The nod-like receptor family pyrin domain containing 3) in vitro. Our study is focused on exposure of THP-1/THP1-null cells and peripheral blood monocytes to C-BNM as representative models of canonical and alternative pathways, respectively. Although all nanomaterials were extensively accumulated in the cytoplasm, increasing doses of all C-BNM did not lead to cell death. We observed direct activation of NLRP3 via destabilization of lysosomes and release of cathepsin B into cytoplasm only in the case of MWCNTs. Direct activation of NLRP3 by both GP was statistically insignificant but could be induced by synergic action with muramyl dipeptide (MDP), as a representative molecule of the family of pathogen-associated molecular patterns (PAMPs). This study demonstrates a possible proinflammatory potential of GP and MWCNT acting through NLRP3 activation.

A comprehensive physicochemical characterization of heterogeneous nanoplastic (NPL) samples remains an analytical challenge requiring a combination of orthogonal measurement techniques to improve the accuracy and robustness of the results. Here, batch methods, including dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), tunable resistive pulse sensing (TRPS), transmission electron microscopy (TEM), and scanning electron microscopy (SEM), as well as separation/fractionation methods such as centrifugal liquid sedimentation (CLS) and field-flow fractionation (FFF)–multi-angle light scattering (MALS) combined with pyrolysis gas chromatography mass spectrometry (pyGC–MS) or Raman microspectroscopy (RM) were evaluated for NPL size, shape, and chemical composition measurements and for quantification. A set of representative/test particles of different chemical natures, including (i) polydisperse polyethylene (PE), (ii) (doped) polystyrene (PS) NPLs, (iii) titanium dioxide, and (iv) iron oxide nanoparticles (spherical and elongated), was used to assess the applicability and limitations of the selected methodologies. Particle sizes and number-based concentrations obtained by orthogonal batch methods (DLS, NTA, TRPS) were comparable for monodisperse spherical samples, while higher deviations were observed for polydisperse, agglomerated samples and for non-spherical particles, especially for light scattering methods. CLS and TRPS offer further insight with increased size resolution, while detailed morphological information can be derived by electron microscopy (EM)–based approaches. Combined techniques such as FFF coupled to MALS and RM can provide complementary information on physical and chemical properties by online measurements, while pyGC–MS analysis of FFF fractions can be used for the identification of polymer particles (vs. inorganic particles) and for their offline (semi)quantification. However, NPL analysis in complex samples will continue to present a serious challenge for the evaluated techniques without significant improvements in sample preparation.

Respiratory diseases constitute a huge burden in our society, and the global respiratory drug market currently grows at an annual rate between 4% and 6%. Inhalation is the preferred administration method for treating respiratory diseases, as it: (i) delivers the drug directly at the site of action, resulting in a rapid onset; (ii) is painless, thus improving patients’ compliance; and (iii) avoids first-pass metabolism reducing systemic side effects. Inhalation occurs through the mouth, with the drug generally exerting its therapeutic action in the lungs. In the most recent years, orally inhaled drugs (OIDs) have found application also in the treatment of systemic diseases. OIDs development, however, currently suffers of an overall attrition rate of around 70%, meaning that seven out of 10 new drug candidates fail to reach the clinic. Our commentary focuses on the reasons behind the poor OIDs translation into clinical products for the treatment of respiratory and systemic diseases, with particular emphasis on the parameters affecting the predictive value of animal preclinical tests. We then review the current advances in overcoming the limitation of animal animal-based studies through the development and adoption of in vitro, cell-based new approach methodologies (NAMs).

Background Lung cancer is the leading cause of cancer-related deaths worldwide. This study focuses on its most common form, Non-Small-Cell Lung Cancer (NSCLC). No cure exists for advanced NSCLC, and patient prognosis is extremely poor. Efforts are currently being made to develop effective inhaled NSCLC therapies. However, at present, reliable preclinical models to support the development of inhaled anti-cancer drugs do not exist. This is due to the oversimplified nature of currently available in vitro models, and the significant interspecies differences between animals and humans. Methods We have recently established 3D Multilayered Cell Cultures (MCCs) of human NSCLC (A549) cells grown at the Air-Liquid Interface (ALI) as the first in vitro tool for screening the efficacy of inhaled anti-cancer drugs. Here, we present an improved in vitro model formed by growing A549 cells and human fibroblasts (MRC-5 cell line) as an ALI multilayered co-culture. The model was characterized over 14-day growth and tested for its response to four benchmarking chemotherapeutics. Results ALI multilayered co-cultures showed an increased resistance to the four drugs tested as compared to ALI multilayered mono-cultures. The signalling pathways involved in the culture MultiDrug Resistance (MDR) were influenced by the cancer cell-fibroblast cross-talk, which was mediated through TGF-β1 release and subsequent activation of the PI3K/AKT/mTOR pathway. As per in vivo conditions, when inhibiting mTOR phosphorylation, MDR was triggered by activation of the MEK/ERK pathway activation and up-regulation in cIAP-1/2 expression. Conclusions Our study opens new research avenues for the development of alternatives to animal-based inhalation studies, impacting the development of anti-NSCLC drugs.

Evidence supports the advantages of inhalation over other drug-administration routes in the treatment of lung diseases, including cancer. Although data obtained from animal models and conventional in vitro cultures are informative, testing the efficacy of inhaled chemotherapeutic agents requires human-relevant preclinical tools. Such tools are currently unavailable. Here, we developed and characterized in vitro models for the efficacy testing of inhaled chemotherapeutic agents against non-small-cell lung cancer (NSCLC). These models recapitulated key elements of both the lung epithelium and the tumour tissue, namely the direct contact with the gas phase and the three-dimensional (3D) architecture. Our in vitro models were formed by growing, for the first time, human adenocarcinoma (A549) cells as multilayered mono-cultures at the Air-Liquid Interface (ALI). The in vitro models were tested for their response to four benchmarking chemotherapeutics, currently in use in clinics, demonstrating an increased resistance to these drugs as compared to sub-confluent monolayered 2D cell cultures. Chemoresistance was comparable to that detected in 3D hypoxic tumour spheroids. Being cultured in ALI conditions, the multilayered monocultures demonstrated to be compatible with testing drugs administered as a liquid aerosol by a clinical nebulizer, offering an advantage over 3D tumour spheroids. In conclusion, we demonstrated that our in vitro models provide new human-relevant tools allowing for the efficacy screening of inhaled anti-cancer drugs.

The use of nanotechnology in medical products has been demonstrated at laboratory scale, and many resulting nanomedicines are in the translational phase toward clinical applications, with global market trends indicating strong growth of the sector in the coming years. The translation of nanomedicines toward the clinic and subsequent commercialization may require the development of new or adaptation of existing standards to ensure the quality, safety and efficacy of such products. This work addresses some identified needs, and illustrates the shortcomings of currently used standardized methods when applied to medical-nanoparticles to assess particle size, drug loading, drug release and safety. Alternative physicochemical, and toxicology methods, with the potential to qualify as future standards supporting the evaluation of nanomedicine are provided.

One of the key challenges in the field of nanoparticle (NP) analysis is in producing reliable and reproducible characterisation data for nanomaterials. This study looks at the reproducibility using a relatively new, but rapidly adopted, technique, Nanoparticle Tracking Analysis (NTA) on a range of particle sizes and materials in several different media. It describes the protocol development and presents both the data and analysis of results obtained from 12 laboratories, mostly based in Europe, who are primarily QualityNano members. QualityNano is an EU FP7 funded Research Infrastructure that integrates 28 European analytical and experimental facilities in nanotechnology, medicine and natural sciences with the goal of developing and implementing best practice and quality in all aspects of nanosafety assessment. This study looks at both the development of the protocol and how this leads to highly reproducible results amongst participants. In this study, the parameter being measured is the modal particle size.