Microtechniques Focus Nanoparticle Characterisation for Drug Delivery Systems “
The use of nanoparticles within drug delivery is a growing area of research with wide ranging implications and is one of the major focuses of Professor Wim Jiskoot’s group at Leiden University. To successfully use nanoparticles is a challenge in that their characterisation is not straightforward. If all the particles are of the same size, known as ‘monodisperse’, there are several techniques available. However, where there are mixes of particles sizes and ratios of different sized particles, it is more difficult to make measurements on these ‘polydisperse’ systems. Jiskoot’s group has recently completed a review of a new technique called nanoparticle tracking analysis (NanoSight, Amesbury, UK) in comparison with other techniques.
The Centre for Drug Research, Leiden University is the home for Wim Jiskoot and his team of research scientists. Professor Jiskoot’s research is focused on the formulation and delivery of biopharmaceuticals. Biopharmaceuticals are different from conventional drugs because they are based on large, complex molecules (mostly proteins), which are difficult to produce, stabilise, and administer to the patient. He has two lines of work.
The first is devoted to the study of unwanted immunogenicity of therapeutic proteins. Although highly pure and (nearly) identical to endogenous proteins, most therapeutic proteins elicit antibodies in patients. Improved fundamental insight into the causative factors of antibody formation will enable the design of better (for example, more effective and safer) protein drugs. The second research line is vaccine delivery, with the intent to make (for example, bacterial or viral) proteins as immunogenic as possible.
When formulated as a vaccine, these proteins should induce immunity, preferably life-long after a single administration. The aim is to identify the immunogenicity- limiting steps after a vaccine is administered to the patient and thus optimising the performance of the vaccine.
The beam is caused to refract at the interface between the liquid sample and the optical element through which it is passed such that it describes a path close to parallel to the glass-sample interface.
The vaccine delivery group aims to develop innovative delivery systems, such as polymeric nanoparticles and liposomes, for the delivery of different types of vaccines through the conventional (injection) or needle-free administration routes (such as transcutaneous or intranasal delivery). It is very important to know the size of the delivery systems as the size can influence the uptake by the cells of the immune system, the diffusion through the skin, the release of vaccine components, and thus the immune response. The protein characterisation group seeks to understand the causes of unwanted immunogenicity of therapeutic proteins and develop transgenic mouse models capable of predicting immunogenicity of human/humanised proteins in a preclinical setting.
For the protein group, a good size characterisation of protein aggregates is essential to better understand which size class is responsible for triggering unwanted immunogenicity of therapeutic proteins which is believed to be related to the presence of aggregates in the protein formulations. The group aims to stress and thoroughly characterise protein formulations to then test which ones are more immunogenic after their injection in the mouse models.
Prior to using NanoSight’s LM-20 system, the Leiden group used a variety of established particle characterisation techniques such as Dynamic Light Scattering (DLS), Light Obscuration Particle Counting (LOPC) and Electron Microscopy (EM). However, each has deficiencies in terms of parameters such as sample preparation and speed of use. The work reported here evaluates the nanoparticle tracking analysis (NTA) technique, compares it with dynamic light scattering (DLS) and tests its performance in characterizing drug delivery nanoparticles and protein aggregates.
DYNAMIC LIGHT SCATTERING Author Details:
Andrew Malloy, Head of Applications Science & Jeremy Warren, CEO, NanoSight Limited, Minton Park, London Road, Amesbury, Wiltshire, SP4 7RT Email:
andrew.malloy@
nanosight.com
While Dynamic Light Scattering (DLS) methods (also known as Photon Correlation Spectroscopy - PCS) is an industry standard technique that is used routinely and very successfully for the analysis of monodisperse and homogenous sample types. It is however well recognised that DLS can become unreliable when presented with heterogeneous samples which contain a wide range of particle sizes, and that the mean particle size (z-average) will be intensity weighted towards the larger brighter particles within the sample.
Furthermore, successful analysis of the correlation function by classical deconvolution algorithms to extract, for instance, multimodal distributions are realistically limited to sample types containing only two (or exceptionally three) monodisperse particle sizes, each needing to differ from each other by a size factor of, in practice, >3:1. DLS is also limited in its ability to allow the user to recognise when the sample is unsuitable for analysis by that method and that the data (for example, the particle size distribution profile) obtained should accordingly be treated with some suspicion.
NANOPARTICLE TRACKING ANALYSIS
An alternative light scattering method for nanoparticle analysis is Nanoparticle Tracking Analysis (NTA). It is being increasingly used for determining nanoparticle size through simultaneously but individually tracking and analysing the trajectories described nanoparticles undergoing Brownian motion in a fluid.
HOW DOES NTA WORK?
The technique is centred on a sample analysis module (Figure 1), which comprises a small metal housing containing a solid-state, single-mode laser diode (<35mW, 638nm) configured to launch a finely focused beam through the sample of liquid containing a dilute suspension of nanoparticles placed directly above a specially designed optical flat. The sample chamber is approximately 250µl in volume and 500µm deep. Samples are introduced by syringe via a Luer port and allowed to thermally equilibrate for 20 seconds prior to analysis.
The beam is caused to refract at the interface between the liquid sample and the optical element through which it is passed such that it describes a path close to parallel to the glass-sample interface.
Figure 1. Picture of laser module showing beam passing through sample and viewed from above via microscope objective.
Particles in the beam (which is approximately 100µm wide by 15µm deep) are visualised by a conventional optical microscope aligned normally to the beam axis to collect light scattered from each and every particle in the field of view.
Given NTA is not an imaging technique per se; the total magnification of the system is quite modest (x100 via a x20 0.4 NA long working distance microscope objective). The particles are seen down the microscope as small points of light moving rapidly under Brownian motion (Figure 2a).
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