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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.