Biopharma

The analysis of nanoparticles is becoming increasinglyimportant in a broad range of sectors – including thepharmaceutical industry, where nanoparticles are beingused in a variety of applications. Nanoparticle drugdelivery systems can be used to protect drugs from beingmetabolised by enzymes and to help control their rate ofrelease – leading to fewer doses being necessary andthereby reducing the occurrence of adverse side effectsand toxicity. This is starting to enable delicate biologicaldrugs to be delivered accurately and opens up thepotential for using siRNA (small interfering RNA) astherapeutic agents.

Liposomes are one of the best-known examples ofnanoparticle delivery systems; new research using single-walled carbon nanotubes and other ‘smart’ nanoparticles isshowing great potential. The size of such particles isbecoming increasingly recognised as an important factorin determining the efficacy of a treatment as a particle’ssize can affect its circulation and residence-time in theblood, as well as the rate of absorption into cells.

As the industrial use of nanoparticles increases,investigation of their toxicity is becoming increasinglyimportant. Particle size can play a key role in biologicalactivity, with size being critical to a particle’s ability toinfiltrate cells. This has led to an increased need to fullycharacterise particle size distributions – rather than just theaverage size data that some techniques provide. Two of themost widely used nanoparticle sizing methods are photoncorrelation spectroscopy (PCS), also referred to as dynamiclight scattering (DLS), and electron microscopy (EM),usually known as scanning electron microscopy (SEM).

Photon correlation spectroscopy examines the lightscattered from particles detecting the rate of change ofinterference resulting from particle Brownian motion.Rather than giving the particle size based on a particle-by-particle calculation, it produces an average figure. It is agood technique for looking at samples with a very narrowrange of particle sizes (mono-dispersed), but has problemswith samples that contain a range of particle sizes (poly-dispersed) with larger particles biasing the average sizevalue produced.

Electron microscopy is a very exact method of measuringdry particles but may often require significant samplepreparation prior to inspection and measurement.

A NEW TECHNIQUEA new nanoparticle sizing method – nanoparticle trackinganalysis (NTA, NanoSight) – offers a unique way ofvisualising and analysing particles in liquids in that itrelates a particle’s Brownian motion to its size. Particlemovement rate is related to the size of the particle and theviscosity of the liquid through which it is moving, as wellas the temperature. Interestingly, particle density does notinfluence the rate of movement. Compared with otherlight scattering techniques, NTA enables higher resolutionparticle size distribution profiles to be obtained.

Individual particles from 10 to 1,000nm can be sized togive particle-by-particle size distribution data, rather thanthe average size data generated by PCS. The upper sizelimit is restricted by Brownian motion limitations. Largeparticles of one micron in size move relatively slowly andreduce the accuracy of the technique. The viscosity of the

Nanoparticle Tracking AnalysisA novel nanoparticle sizing method offers a unique way of visualising and analysing particles in liquids by correlating size with Brownian motion.

By Bob Carr, Andrew Malloy and Jeremy Warren at NanoSight Limited

Dr Bob Carr, Chief Technical Officer and Founder of NanoSight, previously worked for 20 years at a leading governmentresearch establishment in Wiltshire, UK before founding NanoSight in 2002. His background is in biodetectiontechniques employing laser optics and microsystems leading to more than 100 publications and patents. Currentinterests include nanoparticle detection techniques for application in the biotech and industrial chemical sectors.

Andrew Malloy was educated at the University of Liverpool where he completed a Masters degree in BiomedicalEngineering. Andrew has worked for NanoSight for three years where he has an active role in many areas of researchrelating to product development. He also has an active sales role within the company.

With a degree in Chemical Engineering from the University of Birmingham, UK, Jeremy Warren worked for Unilever inproduction management where he qualified as a chartered engineer. He then set up a successful chemicals manufacturingbusiness in Belgium, before completing an MBA at INSEAD in France. After a period in strategy consultancy with Booz Allen,Warren began a series of CEO roles in SMEs across a wide range of industries. This experience has focused on developingtechnology businesses with private equity finance. Jeremy Warren became CEO of NanoSight in 2005.

38 Innovations in Pharmaceutical Technology

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solvent used also plays a role in determining the upper sizelimit as it influences the movement of the particles. Thelower size limit is dictated by a particle’s ability to scattersufficient light to be detectable, with analysis of 10nmparticles only being possible with materials having highrefractive indices such as gold and silver.

The technique requires minimal sample preparation andinvolves simply ensuring that the particle concentration isin the range 10-5 to 10-10 particles per ml. Ideally, a samplewould have a concentration in the range of 10-6 to 10-9

particles per ml, which equates to less than 1 wt%. Carehas to be taken when diluting a sample as this maysometimes lead to particle aggregation.

Accurate and reproducible analyses are obtained fromrecording short video clips lasting only a few seconds toproduce particle number and concentration data. Giventhe close to real-time nature of the technique, particle-particle interaction information is accessible, as isinformation about sample aggregation and dissolution. Alltypes of particle can be measured and in any solvent typeas long as they do not have the same refractive index.

The NTA technique is robust and low cost. Uniquely, it allows the user a simple and direct qualitative view ofthe sample under analysis (perhaps to validate dataobtained from other techniques such as PCS); from this, an independent quantitative estimation of samplesize, size distribution and concentration can beimmediately obtained.

HARDWARE REQUIREMENTSNTA employs a specially designed flow cell that ismounted on a conventional optical microscope equippedwith a CCD camera capable of operating at 30 framesper second (see Figure 1). A suitably prepared sample ofnanoparticles in solution is injected into the cell (seeFigure 2), and the beam from a laser diode is then passedthrough the liquid sample cell.

Particles in the laser beam produce light scattering whichis viewed with a suitable optical microscope and CCDcamera against a proprietary metallised surface (see Figure3). The nanoparticles move randomly under solventbombardment (Brownian motion) in the beam of the laserat a speed related to their size, with smaller particlesmoving faster and further than larger particles.

The distance moved by each particle is measured and theaverage is determined before the Particle DiffusionCoefficient is calculated; this relates to a sphere equivalenthydrodynamic diameter of the Stokes-Einstein equation.

Nanoparticles are too small to beimaged directly by a microscope;instead, they are observed as ‘pointscatterers’ moving under Brownianmotion. Larger particles scattersignificantly more light than smallerparticles, while the speed of theparticles varies strongly according totheir size.

The process of collection through toanalysis is illustrated by the threeimages in Figure 4 (page 40). Figure 4ashows a frame from the video captureprocess that tracks the particlemovement under Brownian motion.The second image (see Figure 4b)shows the trajectory of each particle asderived from the series of CCD imagescaptured over a few seconds. The finalparticle size distribution is shown inFigure 4c; this is calculated from all theseparate measurements. The scalesshown are linear with the horizontalscale running from 1 to 1,000nm,whilst the vertical scale is a directnumber count of particles, each onederived from an individual measurement.

COMPARISON WITH PCS DATAWhen measuring a narrow range of particle sizes (forexample, a mono-dispersion), both PCS and NTA willdeliver equivalent results. However, NTA comes to thefore when studying poly-dispersed samples where a rangeof sizes is present. If a sample containing two differentsizes of particles is studied by PCS and by NTA, thenclearly different results are produced.

This is illustrated in a study of a solution containingparticles of 204nm and 384nm. While the NTA data

39Innovations in Pharmaceutical Technology

Metallised surface

Liquid

Glass

Microscope

Particles scatter laser beam

Particles to be viewed aresuspended in liquid

Laser beam (approximately50µm wide)

Figure 3: Schematic showing the light-scattering principle

Figure 2: Injection of a nanoparticle solution into the sample cell

Figure 1: Image showing the NanoSight sample cell in action

IPT 26 2008 28/8/08 10:19 Page 39

shown in Figure 5 clearly indicates the twosizes (a), the results from PCS only reportthe larger size particle (b). Thedistribution curves clearly show how PCSstruggles to detect the smaller particles astheir signals are overwhelmed by thesignals from the bigger particles. Thisbiasing of the results occurs because PCSmeasures light intensity, and largerparticles scatter much more intensely thansmaller ones.

APPLICATIONSThe breadth of samples that may bestudied using NTA is extremely wide,answering the particle sizing needs offields as diverse as toxicology and materialsscience. In liposomal drug delivery, thesize of a liposome is increasingly beingrecognised as an important factor in theability to deliver drugs effectively as it canaffect circulation and residence-time in theblood, the efficacy of the targeting, therate of cell absorption (or endocytosis)and, ultimately, the successful release ofthe payload. Such considerations are alsohugely important in the case of nanoscalepolymer-encapsulated drug deliverysystems. Accurate size measurement of allof the particles being administered istherefore imperative so that formulationchemists can rationally design optimaldelivery systems.

Driven by the ever-increasing use ofnanoparticles in industry, a new area of research knownas nanotoxicology has started to emerge. Recent work byIker Montes-Burgos, a graduate student in ProfessorKenneth Dawson’s group at University College Dublin,has shown the utility of the NTA approach tocharacterise silica nanoparticles before studying how theyenter cells and decrease cell viability. Mono-modal

particle sizes obtained using NTA broadly agreed withsizes obtained using PCS (with NTA typically giving asmaller particle size, as expected, due to the intensityweighting of PCS compared with particle-by-particlesizing of NTA). The group is currently trying to correlatehow the size and charge of the particles correlates with areduction in cell viability.

The need to determine the toxicity of a material inboth its bulk and nano forms stems from the fact thatwhen particles enter a biological fluid they becomecoated with proteins that may transmit undesiredbiological effects. This means that while a material maynot be toxic in its own right, when in a nanoparticulateform it may have the ability to present proteins in altered conformations that transmit unwantednegative effects.

Montes-Burgos has also found that it is possible tomeasure the increase in particle size when goldnanoparticles adsorb proteins from complex solutionssuch as minimum essential medium (MEM); particlediameters were shown to increase from 30nm to 40nmon protein adsorption. The work continues with thestudy of adsorption equilibrium kinetics in differentprotein solutions including blood plasma. Thesemeasurements may not be possible using PCS as thetechnique struggles to differentiate poly-dispersedparticles. Thus while both PCS and NTA are validtechniques for nanoparticle dispersion analysis, when itcomes to poly-dispersed samples, the NTA systemdelivers more realistic results.

CONCLUSIONNanoparticle size and distribution analysis is becomingincreasingly important in many industries, with scientistsneeding fast and accurate methods of analysis to assistthem in their research. The examples reported hereclearly show the strength of the nanoparticle trackinganalysis (NTA) technique. It is ideal for analysing poly-dispersed nanoparticles in liquids and offers significantbenefits over the established techniques of PCS

and EM. This, together with the ease of samplepreparation, means that NTAis rapidly gaining acceptanceas a critical instrumentationtechnique for groups studyingand applying nanoparticles intheir work.

The authors can be contacted atjeremy.warren@nanosight.co.uk

40 Innovations in Pharmaceutical Technology

Figure 5a:NTA data showing poly-dispersed sample

Figure 5b: The PCSresult appearing to show a mono-dispersed samplewith a higher and narrowerdistribution of particles

Figure 4a: A real-time video clip showing individual scattering of particles

Figure 4b: The individual particles aretracked as shown here

Figure 4c: The particle size distributioncalculation is shown

100 200 300 400 500 600 700 800 900 1,000 nm

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