NANOPARTICLE TRACKING ANALYSIS (NTA)

CHARACTERISATION OF POLYMER COLLOIDS Agnieszka Siupa1, Pauline Carnell1, Dr. Bob Carr1, Edoardo Scarpa2

, Dr. Maria Molina Soler2

1NanoSight Ltd., a Malvern Company, Minton Park, London Road, Amesbury, Wiltshire, SP4 7RT, UK 2The Bone & Joint Research Group at the Institute of De1velopmental Sciences. Southampton University, Faculty of Medicine Southampton General Hospital, Tremona Road, Southampton, SO16 6YD, UK

2Organic and Macromolecular Chemistry, Institut für Chemie und Biochemie, Freie Universität Berlin, Takustrasse 3, 14195 Berlin, Germany

Nanoparticle Tracking Analysis (NTA) is a relatively new technique for characterization of nanomaterial

that allows detecting, tracking, sizing and concentration of polymer nanoparticles in a liquid suspension.

The colloidal suspension is illuminated by a specially configured laser assembly with the scattering signal

from individual particles passing through the specific optics which is collected by a high sensitivity

camera. Each individual particle is simultaneously tracked and analysed utilising its Brownian motion to

accurately determine the particle size. This technique provides a model-free number-based particle size

distribution profile. An additional benefit of this particle-by-particle approach is the ability to determine

total particle concentration as well as providing concentration data for each size class within the

distribution.

The technique was sucessfully used to characterise a broad range of polymer particles and polymer

suspensions for various applications including thermo responsive polymers, mix size polymer

microspheres as well as drug delivery vesicles.

Figure 1. Schematic of NTA equipment

Nanoparticle Tracking Analysis

A laser diode (642nm, 532nm, 488nm or 405nm) is used to pass a

finely focussed beam through a sample chamber containing nano-

material in liquid

The light scattered by individual particles is collected using optical

microscopy components, allowing a direct visualisation of them mov-

ing under Brownian motion

A video file of this movement is captured and the NTA software

tracks the movement of each particle on an individual basis.

NTA calculates Diffusion coefficient and by using Stokes—Einstein

equation particle hydrodynamic diameter is estimated

Particles can be tracked under both light scatter and fluorescence

mode

Introduction

Technique

NTA—High resolution of particle size

Drug delivery

Polymerosomes— multi parameter

characterisation under both light scatter and

fluorescence modes

References

[1] Hole P, Sillence K, Hannell C, et al. 2013. Interlaboratory comparison of size measurements on nanoparti-

cles using nanoparticle tracking analysis (NTA). Journal of Nanoparticle Research 12: 1-12

[2] Beverly S. Packard, David E. Wolf “Fluorescence lifetimes of carbocyanine lipid analogs in phospholipid

bilayers

NTA technique was used to characterise one of the polymeric drug delivery vesicles, polymersomes

(PMs) made of a block copolymer PEG (polyethylene glycol)-b-PCL (polycaprolactone).

Polymersomes are a versatile type of nanoparticle as they can be loaded with both hydrophilic and hy-

drophobic compounds, and they can also be engineered with the conjugation of antibodies on their sur-

face. The aim was to load PMs with an agonist of a signalling pathway (Wnt) in order to target specifi-

cally mesenchymal stem cells (MSC) in the bone marrow. The activation of the Wnt pathway is known

to determine the differentiation of MSC towards osteoblasts phenotype (bone forming cells), but its ac-

tivation can have stimulatory or inhibitory effects depending on the time of delivery. Thus, hypothesis

is that PMs conjugated with specific antibodies can deliver Wnt agonists specifically to populations of

MSC to induce bone regeneration.

Polymersomes were loaded with 0.1mg/ml DiI*, a hydrophobic fluorescent dye for a “proof of con-

cept” study that polymersomes can be loaded with hydrophobic cargos. Next sample was analysed un-

der both light scatter and fluorescence modes.

Figure 4. Polymerosomes loaded with Dil under both light scatter and fluorescence modes

The group from Freie University use NTA as a complimentary technique to DLS to characterise their

polymeric materials of PNIPAM (Poly(N-isopropylacrylamide)) and PPG (Polypropylene glycol). NTA

forms an innovative and robust tool in characterising thermoresponsive material behaviour in a range of

temperature providing detailed information on size distribution and especially on concentration.

Responsive dendritic systems and nanogels are used as smart polymeric systems for the improvement of

the therapeutic index of drugs. One potential application area is cancer therapy by targeted drug release

in thermally stimulated areas.

2 samples : PG and PNIPAM were measured at two different temperatures : 250C and 400C with Na-

noSight LM10HSB system with a temperature equilibrium time of 5 minutes.

Conclusion

Figure 7. Sample dilution against NTA measured concentration for 100nm standard particles

NTA represents a rapid multi-parameter characterisation method allowing the user to obtain particle

concentration and size distributions of polydisperse nanoscale systems, while the use of fluorescent

labels enables actual size distribution analysis of a specific sample sub-population.

NTA has been successfully used to characterize polymeric drug delivery carriers. Moreover the use of

fluorescence to detect labelled particles allows for better understanding of a drug localization, in-

creasing specificity of the detection.

Changes in size and concentration can be easily monitored at the temperature ranges from 15-50 0C

for thermo responsive polymers. Visualization in a real time allows these effects to be watched and

recorded.

NTA—Concentration measurement

Thermoresponsive polymers

Direct visualization,

Size and concentration measurement

NTA also generates a number-based concentration measurement directly, a crucial parameter in the as-

sessment of dosimetry for nanoparticles where weight-based concentration measurements are less rele-

vant .

The particle-by-particle approach of NTA allows accurate measurement of particle concentration within a

liquid suspension (particles per mL).

*Dil belongs to a family of dialkylcarbocyanine dyes that can be characterised by extremely high extinction coeffi-

cients, moderate fluorescence quantum yields and short excited state lifetimes in lipid environments. They are insolu-

ble in water but their fluorescence is readily detected when incorporated into membranes[2]. The excitation maxi-

mum for Dil is 550nm, approximately, and it emits bright red-orange, emission maximum at 570nm, approximately.

To excite the fluorescent molecules of Dil, NanoSight LM10HSGF system equipped with 532nm laser was used. For

the fluorescence signal detection 565nm long pass filter was applied. To prevent stain photobleaching sample was

analysed under precise and constant flow controlled by syringe pump.

0.0E+0

2.0E+7

4.0E+7

6.0E+7

8.0E+7

1.0E+8

1.2E+8

1.4E+8

0 50 100 150 200 250 300 350 400

Co

nce

ntr

atio

n [

par

ticl

es/

ml]

size [nm]

Polypropylene glycol measured at 250C and 40 0C

25 degress 40 degress

0.0E+0

2.0E+7

4.0E+7

6.0E+7

8.0E+7

1.0E+8

1.2E+8

1.4E+8

1.6E+8

0 50 100 150 200 250 300 350 400

Co

nce

ntr

atio

n [

par

ticl

es/

ml]

Size [nm]

Poly(N-isopropylacrylamide) measured at 250C and 400C

25 degress 40 degress

Total particle concentration of PNIPAM:

250C: 1.0*10

13particles/ml

400C: 1.1*10

13 particles/ml

Total particle concentration of PG:

250C: 6.6*10

12particles/ml

400C: 7.2*10

12 particles/ml

Together with size and size distribution NTA delivers the concentration of both stained PMs

(fluorescence) and total sample nanoparticles (light scatter) which is crucial for any drug delivery

system. Fluorescence is an important characterisation tool of the system and is the quickest way to

detect the efficiency of loading and delivery.

0.0E+00

2.0E+12

4.0E+12

6.0E+12

8.0E+12

0 50 100 150 200 250 300 350 400 450 500

Co

nce

ntr

atio

n [p

art

icle

s/m

l]

size [nm]

Polymerosomes loaded with Dil

scatter light mode fluorescence mode

NanoSight instruments provide a unique ability to directly visualise and size nanoparticles in a

liquid suspension. The visualisation of the Brownian motion allows each particle to be simultane-

ously and individually sized, overcoming inherent problems associated with techniques such as

Photon Correlation Spectroscopy (PCS, or Dynamic Light Scattering). Speed of the particle is re-

lated to the diffusion coefficient and reflects the particle size. As NTA tracks trajectories of an in-

dividual particle, a high resolution size distribution profile is generated. Furthermore different

particle populations can be easily separated by a the software and imaged on a 3D plot.

Figure 2. Visualization of 100nm, 200nm and 400nm PS particles

Figure 3. 3D plot of 100nm, 200nm and 400nm PS particles

Mixture of 100nm, 200nm and 400nm polystyrene particles (NIST standards) were measured

with NanoSight LM10HSB instrument.

Figure 8. Polypropylene glycol measured at 250C and 400C

Figure 9. Poly(N-isopropylacrylamide) measured at 250C and 400C

Figure 5. PMs loaded with Dil, light scatter

mode

Figure 6. PMs loaded with Dil, fluorescence

mode

Total particle concentration :

Light scatter: 4.7*1013

particles/ml

Fluorescence 1.6*1013

particles/ml

34% PMs particles loaded with Dil

In both cases with lower temperatures, faster moving, smaller particles were detected. In a higher

temperatures the size distribution shifted towards larger sizes. However the sample concentration

remained the same at both temperatures.