Characterisation of particles in solution - a perspective on light scattering and comparative technologies.

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.

Cellular uptake and biocompatibility of bismuth ferrite harmonic advanced nanoparticles.

Bismuth Ferrite (BFO) nanoparticles (BFO-NP) display interesting optical (nonlinear response) and magnetic properties which make them amenable for bio-oriented diagnostic applications as intra- and extra membrane contrast agents. Due to the relatively recent availability of this material in well dispersed nanometric form, its biocompatibility was not known to date. In this study, we present a thorough assessment of the effects of in vitro exposure of human adenocarcinoma (A549), lung squamous carcinoma (NCI-H520), and acute monocytic leukemia (THP-1) cell lines to uncoated and poly(ethylene glycol)-coated BFO-NP in the form of cytotoxicity, haemolytic response and biocompatibility. Our results support the attractiveness of the functional-BFO towards biomedical applications focused on advanced diagnostic imaging. FROM THE CLINICAL EDITOR: Bismuth Ferrite nanoparticles (BFO-NP) have been recently successfully introduced as photodynamic tools and imaging probes. However, how these nanoparticles interact with various cells at the cellular level remains poorly understood. In this study, the authors performed in vitro experiments to assess the effects of uncoated and PEG-coated BFO-NP in the form of cytotoxicity, haemolytic response and biocompatibility.

Heparin conjugated quantum dots for in vitro imaging applications.

In this work heparin-gelatine multi-layered cadmium telluride quantum dots (QDgel/hep) were synthesised using a novel 'one-pot' method. The QDs produced were characterised using various spectroscopic and physiochemical techniques. Suitable QDs were then selected and compared to thioglycolic acid stabilised quantum dots (QDTGA) and gelatine coated quantum dots (QDgel) for utilisation in in vitro imaging experiments on live and fixed permeabilised THP-1, A549 and Caco-2 cell lines. Exposure of live THP-1 cells to QDgel/hep resulted in localisation of the QDs to the nucleus of the cells. QDgel/hep show affinity for the nuclear compartment of fixed permeabilised THP-1 and A549 cells but remain confined to cytoplasm of fixed permeabilised Caco-2 cells. It is postulated that heparin binding to the CD11b receptor facilitates the internalisation of the QDs into the nucleus of THP-1 cells. In addition, the heparin layer may reduce the unfavourable thrombogenic nature of quantum dots observed in vivo. FROM THE CLINICAL EDITOR: In this study, heparin conjugated quantum dots were found to have superior imaging properties compared to its native counterparts. The authors postulate that heparin binding to the CD11b receptor facilitates QD internalization to the nucleus, and the heparin layer may reduce the in vivo thrombogenic properties of quantum dots.

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.

Are existing standard methods suitable for the evaluation of nanomedicines: some case studies.

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 in vitro safety. Alternative physicochemical, and in vitro toxicology methods, with the potential to qualify as future standards supporting the evaluation of nanomedicine are provided.

Targeted polyethylene glycol gold nanoparticles for the treatment of pancreatic cancer: from synthesis to proof-of-concept in vitro studies.

The main objective of this study was to optimize and characterize a drug delivery carrier for doxorubicin, intended to be intravenously administered, capable of improving the therapeutic index of the chemotherapeutic agent itself, and aimed at the treatment of pancreatic cancer. In light of this goal, we report a robust one-step method for the synthesis of dicarboxylic acid-terminated polyethylene glycol (PEG)-gold nanoparticles (AuNPs) and doxorubicin-loaded PEG-AuNPs, and their further antibody targeting (anti-Kv11.1 polyclonal antibody [pAb]). In in vitro proof-of-concept studies, we evaluated the influence of the nanocarrier and of the active targeting functionality on the anti-tumor efficacy of doxorubicin, with respect to its half-maximal effective concentration (EC50) and drug-triggered changes in the cell cycle. Our results demonstrated that the therapeutic efficacy of doxorubicin was positively influenced not only by the active targeting exploited through anti-Kv11.1-pAb but also by the drug coupling with a nanometer-sized delivery system, which indeed resulted in a 30-fold decrease of doxorubicin EC50, cell cycle blockage, and drug localization in the cell nuclei. The cell internalization pathway was strongly influenced by the active targeting of the Kv11.1 subunit of the human Ether-à-go-go related gene 1 (hERG1) channel aberrantly expressed on the membrane of pancreatic cancer cells. Targeted PEG-AuNPs were translocated into the lysosomes and were associated to an increased lysosomal function in PANC-1 cells. Additionally, doxorubicin release into an aqueous environment was almost negligible after 7 days, suggesting that drug release from PEG-AuNPs was triggered by enzymatic activity. Although preliminary, data gathered from this study have considerable potential in the application of safe-by-design nano-enabled drug-delivery systems (ie, nanomedicines) for the treatment of pancreatic cancer, a disease with a poor prognosis and one of the main current burdens of today's health care bill of industrialized countries.

A safe-by-design approach to the development of gold nanoboxes as carriers for internalization into cancer cells.

Gold nanomaterials are currently raising a significant interest for human welfare in the field of clinical diagnosis, therapeutics for chronic pathologies, as well as of many other biomedical applications. In particular, gold nanomaterials are becoming a promising technology for developing novel approaches and treatments against widespread societal diseases such as cancer. In this study, we investigated the potential of proprietary gold nanoboxes (AuNBs) as carriers for their perspective translation into multifunctional, pre-clinical nano-enabled systems for personalized medicine approaches against lung cancer. A safe-by-design, tiered approach, with systematic tests conducted in the early phases on uncoated AuNBs and more focused testing on the coated, drug-loaded nanomaterial toward the end, was adopted. Our results showed that uncoated AuNBs could effectively penetrate into human lung adenocarcinoma (A549) cells when in simple (mono-cultures) or complex (co- and three-dimensional-cultures) in vitro microenvironments mimicking the alveolar region of human lungs. Uncoated AuNBs were biologically inert in A549 cells and demonstrated signs of biodegradability. Concurrently, preliminary data revealed that coated, drug-loaded AuNBs could efficiently deliver a chemotherapeutic agent to A549 cells, corroborating the hypothesis that AuNBs could be used in the future for the development of personalized nano-enabled systems for lung cancer treatment.

Interlaboratory comparison of size measurements on nanoparticles using nanoparticle tracking analysis (NTA).

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.