1. www.nanosight.com C NTA C haracterisation of Nanoparticles and Aggregates using N anoparticle T racking A nalysis (NTA)

2. LM10 Series NS300 NS500 The Instrument Range

3. Mai 13 As the schematic below shows, the NanoSight technology comprises: a metallised optical element illuminated by laser beam. Laser as seen at low magnification NanoSight LM20 device www.nanosight.com Hardware: laser illuminated sample chamber + optical microscope

4. Fast and easy to use system Loading of 200nm latex standard Mai 13 www.nanosight.com

5. Titanium Dioxide Mai 13 www.nanosight.com Polydisperse titanium dioxide particles as seen at standard magnification

6. Particles are Visualised, not imaged Particles are too small to be imaged by the microscope Particles seen as point scatterers moving under Brownian motion Larger particles scatter significantly more light Speed of particles varies strongly with particle size Microvesicles purified from serum by ultracentrifugation Mai 13 www.nanosight.com

7. Nanoparticles move under Brownian movement due the random movement of water molecules (red molecules in movie) surrounding them. Small particle move faster than larger particles. Diffusion Coefficient can be calculated by tracking the movement of each particle and then through application of the Stokes-Einstein equation particle size can be calculated. Principle of Measurement Mai 13 www.nanosight.com

8. Brownian motion of each particle is followed in real-time via video Tracking software establishes mean square displacement and diffusion coefficient (Dt) Then from Stokes Einstein can be obtained particle (sphere equivalent hydrodynamic) diameter, dh K B = Boltzmann Constant T = Temperature  = viscosity KBTKBT Stokes-Einstein equation D t = d h 3  d h Sizing is an absolute method Mai 13 www.nanosight.com

9. Nanoparticle Tracking Analysis (NTA) is the gathering of unique information and comes from assessment of individual particles, rather than averaging over a bulk sample. This provides a distinct advantage in determining particle size analysis tracking capture Nanoparticle Tracking Analysis Mai 13 www.nanosight.com

10. The NanoSight NTA 2.0 (nano-particle tracking) analysis suite allows for captured video footage to be simultaneously tracked and analysed… 100+200nm nanoparticles being tracked and analysed by NanoSight NTA 2.0 Particle Sizing in action - Software Analysis Mai 13 www.nanosight.com

11. Minimum size limit is related to: Material type Wavelength and power of illumination source Sensitivity of the camera Size Concentration Minimum concentration is related to: Poor statistics (Requiring longer analysis time) 10 – 40 nm 1 000 – 2 000 nm Maximum Size limit is related to: Limited Brownian motion Viscosity of solvent Approx 10 5-6 / mL Maximum concentration is related to: Inability to resolve neighboring particles Tracks too short before crossing occurs Approx 10 9 / mL NTA Detection Limits www.nanosight.com

12. True size distribution profile Mixture of 100nm and 200nm latex microspheres dispersed in water in 1:1 ratio Particle distribution displays a number count vs particle size. Mai 13 www.nanosight.com

13. Mai 13 www.nanosight.com True size distribution profile 164 On this experience, NTA accurately tracked 3 sub- populations of nanoparticles Mix of 3 populations in a real sample

14. Mai 13 Number count of particle in a determined volume : Absolute concentration measurement in 10 E 8 part/mL. Particles are Counted– By Number Count www.nanosight.com Global concentration of all populations Concentration of a population selected by user

15. Particles are Counted– By Number Count Mai 13 www.nanosight.com RPMI + FCS (2.54x10 E 8 part/mL) PBS (2.47x10 E 8 part / mL) On this experience, NTA accurately tracked the same population of particles but prepared in 2 different solvents Shift in size, but not in concentration

16. NanoSight has the unique ability to plot each particle’s size as a function of its scattered intensity 2D size v. number 2D size v. intensity 3D size v. Intensity v. concentration Relative intensity scattered Mai 13 www.nanosight.com

17. Relative intensity scattered

18. 100 PS 60nm Au 30nm Au In this mixture of 30 nm and 60 nm gold nanoparticles mixed with 100 nm polystyrene, the three particle types can be clearly seen in the 3D plot confirming indications of a tri-modal given in the normal particle size distribution plot. Despite their smaller size, the 60 nm Au can be seen to scatter more than the 100 nm PS. Resolving mixtures of different particle types and sizes Mai 13 www.nanosight.com

19. NanoSight systems can be fitted with Choice of long pass or band pass filters allow specific nanoparticles to be tracked in high backgrounds Applications in: Nanoparticle toxicity studies Nano-rheology Bio-diagnostics Phenotyping specific exosomes Laser diode capable of exciting fluorophores and quantum dots Fluorescent Mode available Mai 13 www.nanosight.com

20. Analysis of 100 nm Fluorescence standard particles suspended in FBS Mai 13 www.nanosight.com Modal particle size peaks: 103 nm Concentration: x10 8 particles/ml Concentration: 9.5 x10 8 particles/ml 100 nm fluorescently labelled particles suspended in 100% FBS Sample maintained at a constant temperature (37 o C) Sample viscosity – 1.33 cP Excitation wavelength - 532 nm Using a 565 nm Long pass optical filter No filter565 nm Long pass optical filter This allows selective visualising of fluorescent/fluorescently labelled particles

21. Dynamic Light Scattering (DLS or PCS) Mai 13 www.nanosight.com DLS (alternatively known as Photon Correlation Spectroscopy (PCS)) is deservedly an industry standard technique and widely used for 40 years... Also relies on Brownian motion and relates the random motion of particles in liquid suspension to a hydrodynamic radius. Technique relates the rate of change of the intensity of the speckle to particle size using the auto-correlation function. Very effective technique for measuring particle size for mono- dispersed samples from a few nm up to microns. Widely used, big installed base.

22. Comparison of different particle size distribution Particle by particle method Static light scattering Dynamic Light Scattering

23. DLS Analysis and NanoSight equivalent for monodisperse systems NTA vs DLS - Monodisperse Mai 13 www.nanosight.com DLS NTA

24. With NanoSight, the analysis shows both 100nm and 200nm peaks Polystyrene reference spheres in water (100nm and 200nm) NTA vs DLS – Bimodal Sample Mai 13 www.nanosight.com

25. 100nm particles not seen due to intensity bias Mixed Sample of 100nm+200nm particles at a ratio of 10:1 by weight ? Mai 13 NTA vs DLS – Bimodal Sample www.nanosight.com

26. Data reproduced from Filipe, Hawe and Jiskoot (2010) “Critical Evaluation of Nanoparticle Tracking Analysis (NTA) by NanoSight for the Measurement of Nanoparticles and Protein Aggregates”, Pharmaceutical Research, DOI: 10.1007/s11095-010-0073-2 NTA (red profiles) Mixtures of polystyrene of different sizes DLS (blue bars) Mai 13 www.nanosight.com NTA vs DLS in publications

27. NTA is not intensity-weighted towards larger particles, unlike DLS While DLS is an ensemble technique, NTA operates particle-by-particle NTA has much higher resolving power with respect to multimodal and polydisperse samples and heterogenous/mixed sample types NanoSight unique and direct view visually validates the particle size distribution data Number vs. Intensity vs. Size is provided for each particle size class NTA provides particle concentration information Fluorescence mode Zeta Potential, particle by particle NTA requires no information about collection angle, wavelength or solvent refractive index, unlike DLS NanoSight’s Advantages Vs. DLS/PCS Mai 13 www.nanosight.com

28. Comparing different Technologies Mai 13 www.nanosight.com TechniqueStrengthsWeaknesses Electronic microscope (TEM, SEM) -Higher magnification, greater resolution, compositional information, topography and morphology -High costs - Complex sample preparation procedure Analytical Ultra Centrifugation-High resolution-High cost, complex sample preparation & data evaluation, -Low sample throughput Coulter Counters (i.e IZON)-Very low cost. - Small size -Need calibration -Surface charge of particle influences result -Clogging of aperture is common Flow Cytometers-High Throughput -Sorting of different size of particle -Fluoresence -Not suitable for vesicles sizing due to no accurate refractive index to calibrate -Lower limit is about 300nm Dynamic Light Scattering-suited to mono-disperse -samples from a few nm to a few microns -Intensity-biased Plot -No Fluorescence and no counting ability

29. Mai 13 Application Example : Exosomes www.nanosight.com Microvesicles - typically 100nm to 1um range Nanovesicles or Exosomes - 30-100nm Originally thought to be cellular debris Now appreciated that these cell-derived particles are of significant importance and play an important role both in cellular communication and signalling Recently found to be present at significantly elevated levels in a number of diseases conditions Exosomes and Nanovesicles

30. Mai 13 Application Example : Exosomes www.nanosight.com Why use NanoSight in this application ? We COUNT particles – concentration is key to this application. Researchers can not get good concentration information any other way – this is KEY. Remember an increase in the number of exosomes could be related to the onset of disease. We can work in FLUORESCENCE. This means they can fluorescently label these particles and therefore SPECIATE – i.e. They know which exosomes they are looking at. High resolution SIZE. All of these parameters in a SINGLE MEASUREMENT – allows them to determine the size, concentration and type of exosome population.

31. Mai 13 Application Example : Exosomes www.nanosight.com Comparisons of different techniques Flow Cytometry: Limited lower limit of detection – can’t detect the smaller exosomes DLS: Samples are often not very homogenous – therefore not suited for measurement with DLS. DLS cannot count and also can’t phenotype the exosomes – meaning it has limited value. EM: Time consuming and expensive

32. Mai 13 Application Example : Exosomes (Fluoresence) www.nanosight.com

33. Mai 13 Application Example : Purified Influenza Virus www.nanosight.com Why Nanosight ? Manufacturer interested in the purity of their product – i.e. The size distribution of particles within a sample. Manufacturer interested in the dosage of the vaccine i.e. How many viruses are present? Manufacturers need to know the ratio of infective to non-infective viruses in their sample. Manufacturer interested in detecting specific viruses as the samples are purified – therefore fluorescence is of interest to them. COUNTING aspect is the most important feature as manufacturers often use long winded infectivity assays to characterise their samples.

34. Current methologies for counting such as plaque assay only count infectious particles which often represent a small component in attenuated vaccines i.e. perhaps only 1% of product remains infective. The ability to count viruses in liquid suspension is essential for those working in vaccine development. Particle aggregation and yield quality are factors which need to be understood when developing these viral vaccines. Mai 13 Application Example : Purified Influenza Virus www.nanosight.com

35. NanoSight technology has a unique application in the detection of early stage aggregation in protein therapeutics Protein monomer is too small to be individually resolved by this technique, but early stage aggregates are readily detected Protein monomer at high concentration causes high background noise in image, with the aggregate forming the resolvable particles Both size and number of aggregates can be calculated and studied, providing insight into product stability. Data reproduced from Filipe, Hawe and Jiskoot (2010) Pharmaceutical Research, DOI: 10.1007/s11095-010-0073-2 Mai 13 Application Example: Protein Aggregation at controlled temperature (15-55 o C) www.nanosight.com

36. DLS analysis of aggregated protein sample generally produces a bimodal analysis. The protein monomer and the very large aggregates get picked up through DLS as these are the regions which scatter most light. The monomer scatters a lot of light by virtue of its high concentration and the larger particles by virtue of their size. Clearly a protein sample cannot aggregate from monomer to large micron sized aggregates with nothing in between. Whilst NanoSight cannot measure the monomer it can provide valuable information in the 30nm and above range which is typically the region which is poorly served by alternative techniques. Mai 13 Application Example: Protein Aggregation: NTA vs DLS www.nanosight.com

37. Mai 13 Application Example: Drug Delivery system www.nanosight.com The size will affect its 1.Circulation and residence time in the blood, 2. the efficacy of the targeting, 3. the rate of cell absorption Advantage of using Iiposome as a drug deliver Drugs can be targeted to specific areas by attaching ligands to the liposome Readily absorbed by cells The rate of drug release can be controlled Allows potentially lower doses of drug to be used, reducing toxicity and side-effects.

38. Multi-walled Carbon nanotubes Cosmetics Foodstuffs Ink jet inks and pigment particles Ceramics Fuel additives Liposomes and other drug delivery vehicles Virus and VLP samples Exosomes and nanovesicles Nanobubbles Magnetic Nanoparticles Precursor chemical for wafer fabrication. Quantum dots Polymers and colloids CMP Slurries Mai 13 The NanoSight system is widely applicable www.nanosight.com

39. Size Number or concentration Polydispersity – true PSD “Relative Light Intensity” Fluorescence Surface Potential, Zeta Potential + + + + Parameters measured – simultaneously, ‘real time’, particle-by-particle... Mai 13 www.nanosight.com

40. BASF, Europe BP Castrol, Europe DuPont, USA Epson, Japan Exxon Mobile, USA GlaxoSmithKline, Europe Merck, USA Medimmune Nestle, Europe NIST, USA Selected Users Novartis, Europe Proctor & Gamble, USA Roche, Europe Smith & Nephew, Europe Solvay, Europe Toshiba, Japan Unilever, Europe US EPA US Forces Wyeth Biopharma, USA

41. NTA devices worldwilde mapping Mai 13 www.nanosight.com > 500 > 500 devices ≈ 800 ≈ 800 publications

42. Awards Mai 13 www.nanosight.com