Keeping tabs on nanoparticles

As the importance of nanoparticles increases in both industry and academia, measuring and characterising them is becoming vital. With legislative issues also becoming a concern, a technique is needed which allows easy characterisation of nanoparticles. Could NTA be that technique?

In less than eight years, nearly 1,000 peer-reviewed papers have been published citing the nanoparticle characterisation technology known as NTA, Nanoparticle Tracking Analysis. It is many years since a new technique has generated such an exponential output of papers with perhaps the method of SPR, surface plasmon resonance, being the most recent. Third party papers are a clear validation that the technology has been accepted by both industry and academia researching into the quantification and behaviour of nanoparticles in liquids.

Legislation is increasingly a driving force in the world of nanotechnology. There have been several instances of government and industry bodies looking into ways to first define a nanoparticle and then to come up with methods of characterisation. Techniques such as scanning electron microscopy are well established and accepted for quantitative measurements though issues of sample preparation and time taken for a single analysis mean that this is not likely to become a routine or quality control procedure in a manufacturing process. Looking at testing in laboratories around the world, it is clear that cumulative measuring techniques such as Dynamic Light Scattering (DLS) are very widely used. However, when studying nanoparticles, particularly when complex dispersions (also known as polydisperse systems) are present, cumulative measurements have been shown to skew the results to higher size values than the actual dispersions present with reduced resolution between different particle populations.

NTA is a particle by particle method and thus by measuring and reporting individual particles, the final data is proven to be more insightful and reliable. This has particular benefit in the study of systems that may be prone to aggregation such as proteins where monomers can aggregate with respect to time. In this review, we seek to inform the reader about NTA – how it works; to illustrate the technique with a selection of examples of cutting edge nanotechnology research; and finally, to look at the state of legislation today reporting on how different countries and industries are responding to the growing challenges of better quantifying and understanding the performance of nanoparticles in real life scenarios.

NTA visualises measures and characterises virtually all nanoparticles (10-2000nm) in liquids. Particle size, concentration, zeta potential and aggregation can all be analysed while a fluorescence mode provides speciation of suitably-labelled particles. NTA provides real time monitoring of the subtle changes in the characteristics of particle populations with all of these analyses uniquely confirmed by visual validation.

From loading the sample into the cell to getting results can take as little as 2-3 minutes, with the ability to run batches of samples under the same conditions and directly compare results.

NTA is a process for direct and real-time visualisation and analysis of nanoparticles in liquids. Based on a laser-illuminated microscopy technique, Brownian motion of nanoparticles is analysed in real-time by a CCD or CMOS camera, particles are simultaneously but separately visualised and tracked by a dedicated particle image analysis program. The NTA program simultaneously identifies and tracks the centre of each particle on a frame-by-frame basis throughout the length of the video – typically 30 seconds. The average distance each particle moves in the image is automatically calculated. From this value the particle diffusion coefficient can be obtained and knowing the sample temperature and solvent viscosity the particle hydrodynamic diameter is identified. Because each particle is visualised and analysed separately the resulting particle size measurement and size distribution does not suffer from the limitations of being the intensity weighted, z-average distribution from DLS. The ability of NTA to simultaneously measure particle size and particle scatter intensity allows heterogeneous particle mixtures to be resolved, and particle concentration can be measured directly; the particle size distribution profile obtained by NTA being a direct number/frequency distribution. Because this is an absolute method, no user calibration is required.

More than 600 instruments are employed worldwide for a broad range of applications. While the initial applications tended to be found in the world of materials science, e.g. metals, ceramics and the like, significant growth is now being seen in the life sciences. Sectors include drug discovery and delivery, protein manufacture and aggregation, viral vaccine production and early-stage disease detection where particles known as exosomes are being used in the role of a labelled biomarker to provide a rapid and direct means to identify the presence of a variety of cancers. Aligned to these areas are researchers who are concerned with the effects of nanoparticles on the environment and their environmental fate. NTA also finds applications in assessing the purity of river water.

Exosomes and microvesicles The detection and analysis of exosomes and microvesicles can be extremely challenging as many have a diameter of less than 100nm, have a low refractive index, are highly heterogeneous and are sensitive to collection and handling conditions. Given these issues detection by conventional methodologies such as Flow Cytometry is difficult and sometimes impossible. NTA allows both size and concentration measurements to be taken simultaneously, whilst visual validation gives added confidence in the results obtained. The addition of the fluorescent module allows detection of particular biomarkers allowing phenotyping and measurement in biologically relevant media, overcoming many of the limitations cited above.

Protein aggregation

[caption id="attachment_36160" align="alignright" width="200"] Real time observation of proteins aggregating with time[/caption]

Over recent years interest in therapeutic proteins has dramatically increased. The global therapeutic proteins market is forecast to reach an estimated $142 billion in 2017. To be efficacious for a patient, and to prevent possible immunogenic side effects, the therapeutic needs to be available in its monomeric form. However, proteins have a well-deserved reputation for forming aggregates, and this aggregation can be caused by various stresses that the protein encounters during its production, purification, packaging transport and storage. Although current legislation requires data on particles 10µm or greater, typically being millions of monomer units, aggregates of this size are formed from smaller aggregates. When working with proteins at any stage of research, development and production it is therefore necessary to understand and control the profile of smaller aggregates before these larger ones appear.

NTA can provide high resolution particle size versus number distributions of aggregated proteins in the 30nm - 1.5 micron region, and when combined with DLS can also include the protein monomer in the size distribution profile.

Viruses and viral vaccines Viral vaccines are usually analysed using plaque assays or cell based bioassays, these methods give a direct count of only the infective particles present in the sample and any aggregates will only produce a count of one. So the total number of viral particles and the degree of aggregation is not reported. These additional parameters can give vital insight into product shelf life and purity.

NTA technology can measure total viral titre in minutes. The total viral titre is termed as the number of infectious and non-infectious virus particles within a preparation. It is often critical to be able to measure the total number of viruses and compare this to the infectious titre as measured by infectivity assays for example. The ratio of infectious:non-infectious particles varies widely and by monitoring this ratio, it provides the manufacturer the ability to optimise the purification process.

Drug delivery As the use of nanoscale materials in the field of medicine and drug delivery continues to grow, so does the need for methodology to effectively characterise these materials. At this scale, small changes in characteristics such as size, zeta potential, aggregation state and concentration of materials can have a large impact on the effectiveness, bioavailability and even potential toxicology of a product.

NTA represents a rapid multi-parameter characterisation method allowing the user to obtain particle count and size distributions of polydisperse nanoparticulate systems, while the use of fluorescent labels enables actual size distribution analysis in biologically relevant media. Consequently NTA has been rapidly adopted to characterise commonly defined nanoparticle vectors including micelles, liposomes, solid lipid nanoparticles and polymeric nanoparticles when studying the delivery of drugs, genes and vaccines into cells.

It is appropriate that governments are taking notice of the burgeoning use of nanoparticles and are driving legislators to look in depth at the issues.

Research has clearly taken the lead here with multiple groups in universities and industrial blue sky research organisations already applying nanoparticles in ways never before considered practical. The progress has also driven the development of new measurement technologies such as NTA to assist in their characterisation and use. There is a long way to go before legislation becomes effective worldwide but the development of appropriate tools progresses rapidly.

Nanoparticle characterisation and legislation Growing Nanomaterials legislation now impacts on sectors including biocides, cosmetics and food labelling. The  most recent implementation (11 July 2013) is  European Cosmetics Regulation 1223/2009 requiring cosmetics containing nanomaterials to be thoroughly assessed for safety and notified to the relevant authorities with the particle size and percentage of nanomaterial required for compliance of material data. Further discussions are underway to include the same approach to general chemicals regulation (REACH). NTA is cited in the EU’s 2012 report on candidate techniques to address the characterisation difficulties which followed the publication of their definition. Although the EU Definition is tailored to sectors it retains number count at its core and NTA is unique in providing this data.

The American Society of Testing Materials published a new standard in 2012, ASTM E2834 - Standard Guide for Measurement of Particle Size Distribution of Nanomaterials in Suspension by Nanoparticle Tracking Analysis (NTA). This document details use of NTA to measurement of particle size distributions for suspended particles from ~10nm to the onset of sedimentation.

Particle Technology Labs is the leading particle characterisation research and advisory company in the United States, providing assistance to a wide variety of industries. Recently, NTA has been adopted for both research-based projects and as a quality control tool for regulated industries. PTL’s NanoSight LM10-HSB instrument is certified cGMP as of 4^th June 2013.  The term cGMP refers to the Current Good Manufacturing Practice regulations enforced by the US Food and Drug Administration (FDA). With cGMP compliance established, NTA via PTL is now fully available to the pharmaceutical industry. This is a significant breakthrough in the commercial/industrial acceptance for NTA.

Authors: Sarah Newell & Jeremy Warren of NanoSight Limited