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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5373_Библиотеки_им_академика_М_И_Перельмана.pdf
3 Topographic Properties ofPEGylated Nanocarriers
97
Fig. 3.19 AFM images of PEG7%-g-PLA rhodamine B-loaded nanocarriers before (a) and after
(b) lyophilization; surface morphology (left, S) and phase image (right, P). (Figure adapted with
permission from Essa et al. (2011) Copyright © 2011 Elsevier)
addition to particle size distribution, SEM can also be used to investigate the aggregation and dispersion of colloidal nanocarriers.
SEM requires specic sample preparation protocols based on the application.
For topographic and morphological investigation, nonconductive samples need to
be coated with a metal like platinum or gold. This not only improves imaging quality by enhancing secondary electron signal but also protects samples from radiation
damage. For any type of characterization, the samples should also be dried, and a
completely dry environment must be ensured in the vacuum chamber.
Most limitations of SEM arise due to its tedious sample preparation requirements. Dehydration of the sample may cause damage in the form of shrinking or
aggregation of particles. Even if solvent removal is not detrimental to a given
98
A. G. Prasad et al.
sample, dehydration will prevent characterization in the native aqueous environment for colloidal suspensions. Similarly, metal coating can obscure the original
topographic features of the specimen. In the context of polymeric nanoparticles like
PEGylated nanocarriers, vacuum conditions can collapse the grafted PEG chains
thereby distorting the topography. Though it is a very valuable tool to characterize
the surface of the nanocarrier, SEM is not suitable for studying the internal structure. In addition to these technical challenges, SEM is also an expensive instrument
to install and maintain.
3.12.1 Application inPEGylated Nanocarriers
Ebbesen and colleagues synthesized PEGylated PLGA nanocarriers using two
methods—surface grafting of PEG chains and as a copolymer. SEM was used to
characterize the size and morphology of nanoparticles. The hydrodynamic diameter
was measured to be in the range of 500–1500nm, and these results were corroborated by dynamic light scattering (DLS) measurements. Figure3.20 shows the SEM
micrographs for (a) non-PEGylated nanocarriers; PEGylated nanocarriers with (b)
30% nonanchoring PEG, (c) 6% PLGA-b-PEG, and (d) 30% PLGA-b-PEG. All
nanocarriers were observed to be polydisperse with a spherical morphology and
smooth topography. Interestingly, PLGA-b-PEG nanocarriers with 6% PEG showed
signicantly larger particles, whereas other PEGylated nanocarriers did not show
any apparent difference in comparison to the blank (0% PEG) nanocarriers (Ebbesen
etal. 2013). In this report, the authors demonstrated the utility of SEM to investigate
the shape, size distribution, and morphology of polymeric nanoparticles.
In another report, Yoncheva etal. developed poly(methyl vinyl ether-co-maleic
anhydride) (PVM/MA) based PEGylated nanocarriers. SEM was used to determine
the shape and morphology of the nanoparticles. As seen in the SEM image in
Fig.3.21, nanoparticles displayed a size of 280–300nm with a spherical morphology. These results agreed with the photon correlation spectroscopy analysis. Based
on this, the authors concluded that grafting the surface with PEG chains had no
signicant inuence on the size of the PEGylated nanocarriers (Yoncheva
etal. 2005).
3.13 Transmission Electron Microscopy
Transmission electron microscopy (TEM) works on a similar principle to that of
SEM.The main difference between the two techniques is the higher voltages of
80–300keV used in TEM.A tungsten or lanthanum hexaboride electric gun emits
high-energy electrons. The electrons are focused through an electromagnetic lens
and the ne beam passes through the specimen. Scattered and transmitted electrons
are detected and the difference in electron densities produces 2-D images with contrast on a phosphorescent screen. The brightness of the image is directly related to
the fraction of transmitted electrons.
3 Topographic Properties ofPEGylated Nanocarriers
99
Fig. 3.20 SEM images of nanocarriers: (a) 0% (blank), (b) 30% nonanchoring PEG, (c) 6%
PLGA-b-PEG, and (d) 30% PLGA-b-PEG. (Figure adapted with permission from Ebbesen et al.
(2013) Copyright © 2013, Springer Nature)
TEM can be used to determine the size distribution, morphology, and atomic
structure of the sample at ultra-high resolution. Elemental analysis is also possible
if emitted X-rays are detected or when combined with spectroscopic techniques. In
addition to the topography of the sample, TEM is also routinely used to characterize
interactions of nanoparticles with biological milieu shedding light on aspects such
as uptake and localization in cells. Cryo-TEM is particularly apt for such applications wherein aqueous dispersion of particles can be observed to analyze them in
their native environment.
As is the case with SEM, sample preparation for TEM is cumbersome. Polymeric
and other nanoparticles are drop-casted before immobilizing the dried specimen
onto a copper grid coated with a carbon lm. This is followed by the negative staining with phosphotungstic acid, uranyl acetate, or ammonium molybdate solution.
Negative staining is important to ensure an opaque background that enables the
translucent specimen to be viewed easily. TEM requires the sample to be thin for the
100
Fig. 3.21 SEM images of
PVM/MA-based
PEGylated nanocarriers
(Figure adapted with
permission from Yoncheva
et al. (2005) Copyright ©
2004 Elsevier).
A. G. Prasad et al.
beam to get transmitted through it. If the sample is larger than ~500nm, ultramicrotomy or a focused ion beam is necessary.
Though it is the most preferred electron microscopy technique, TEM suffers
from several limitations as follows. The instrumentation and maintenance of equipment are not only expensive but it also requires highly trained users. All drawbacks
of SEM apply to TEM as well. As it is operated in vacuum conditions, the samples
must be completely dry. Drying protocols can result in sample damage or distort the
topography and produce artifacts at best. This is a major challenge, particularly for
the characterization of polymeric nanocarriers. This can be addressed by spiking the
staining solution with a small concentration of glucose. Interaction with high-energy
scattered electrons may also damage the sample. This can be prevented by preparing
samples that are thinner than 500nm.
3.13.1 Application inPEGylated Nanocarriers
Ana Vila etal. fabricated PEGylated poly (lactic acid)-poly (ethylene glycol) (PLA–
PEG) nanoparticles and studied the impact of PEG coating density on transport
across the mucosal membrane. PLA–PEG copolymers of different molecular
weights were used to form the nanocarriers through nanoprecipitation and multiple
emulsion techniques. The authors used TEM to investigate the topography and morphology of nanocarriers. TEM images (Fig.3.22) showed that particles synthesized
with different molecular weights of PEG displayed no signicant differences in
appearance for a given preparation technique. However, different preparation techniques inuenced changes in the size and morphology of nanocarriers. As seen in
3 Topographic Properties ofPEGylated Nanocarriers
101
Fig. 3.22 TEM images of nanocarriers prepared with PLA–PEG copolymers of 37kDa weight,
using different preparation techniques. (Figure adapted with permission from Vila et al. (2004)
Copyright © 2004 Published by Elsevier)
the image, the emulsion technique formed particles that were more spherical than
those prepared using the nanoprecipitation technique (Vila etal. 2004). This report
demonstrates how TEM can be used to not only study the morphology of PEGylated
nanocarriers but also compare different preparation techniques.
Carl Walkey and colleagues prepared gold nanoparticles of different sizes and
studied the impact of size and PEG coating on serum protein adsorption and macrophage uptake. The authors used TEM to prove that PEGylated nanocarriers localized in the endosomes are independent of PEG grafting density. Figure3.23 shows
that non-PEGylated carriers formed dense aggregates, whereas PEGylated carriers
were well dispersed (Walkey etal. 2012). TEM can therefore be used to analyze
biological samples and study interactions of nanocarriers with cells and serum
proteins.
102
Fig. 3.23 TEM images of nanocarriers with PEG at 0, 0.48, and 0.96PEG/nm2. (Figure adapted
with permission from Walkey et al. (2012) Copyright © 2011 American Chemical Society)
A. G. Prasad et al.
3.14 Conclusion
Surface characterization is a very essential tool to probe the surface topography and
morphology of nanomaterials. As interfacial parameters dictate the stability and
properties of PEGylated nanocarriers, there is a need to determine the critical attributes accurately and precisely. Several spectroscopic techniques have been routinely applied to investigate the surface topography of nanocarriers. Modications
to the existing methods can provide useful information about the conformation and
arrangement of PEG chains. Advanced electron microscopic techniques and AFM
can achieve high-resolution topographic images of the core of the nanocarriers,
whereas other methods like thermogravimetric and calorimetric analysis provide
vital complementary information about the tethered PEG chains. As all techniques
suffer from specic limitations with respect to sample preparation and method
applicability, combining two or three tools would be the most effective approach to
characterize the topographic features of PEGylated nanocarriers.
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