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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
Fig. 3.12 Elemental analysis of SSNs and PEG-SSNs by EDX. (a) EDX spectrum of SSNs, (b)
weight and atomic percentage of SSNs, (c) EDX spectrum of PEG-SSNs, and (d) weight and
atomic percentage of PEG-SSNs. (Figure adapted with permission from Karim and Chowdhury
(2022) under the terms and conditions of the Creative Commons Attribution (CC BY) license
(https://creativecommons.org/licenses/by/4.0/))
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3.8 Mass Spectroscopy (MS)
Mass spectroscopy (MS) can be used for the quantitative analysis of the structure of
nanocarriers. When a sample is ionized, it degrades into fragments which can be
analyzed based on the differences in atomic masses. As discussed below, there are
several common mass spectroscopic techniques that can be used to probe critical
topographic information of nanocarriers.
Secondary ion mass spectroscopy (SIMS) is a technique that can provide information about the polymer surface structure and composition. Due to its smaller
sampling depth, SIMS provides high spectral resolution. The sample is bombarded
by an ion beam at a low current density, and secondary ions are sputtered from the
surface in response. Low beam density is important to ensure the topography of the
carriers is not damaged or altered. The sputtered ions are analyzed by a mass analyzer and the distribution of the molecules on the surface can be ascertained.
In time-of-ight secondary ion mass spectroscopy (TOF–SIMS), the mass of the
ions can be determined using time-of-ight. TOF–SIMS is a highly sensitive chemical analysis technique that can be used to investigate the topmost layer of the polymeric surface (~5 nm). As the resulting mass spectrum is of a high resolution,
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detailed information at the molecular level can be acquired at detection limits as low
as ppb–ppm. These benets make TOF–SIMS a more attractive technique compared to other spectroscopic methods like XPS and AES. Matrix-assisted laser
desorption/ionization (MALDI) is a method employed in mass spectrometry
wherein a high-energy laser is used for sample desorption and ionization. MALDITOF MS provides relatively better estimates of dispersity. In addition to topographical properties, MALDI can also be used to determine the size of the nanocarrier.
Sample preparation methods vary based on the specic mass spectroscopic technique and application. Typically, colloidal dispersions or lyophilized powder can be
used with minimal processing. As the mass analyzer tends to be very sensitive,
samples should be devoid of all contaminants. To achieve this, high-purity solvents
are required, and wearing silicone-free gloves is recommended while handling the
samples and operating the instrument.
Though mass spectroscopy enables topographic characterization at a high resolution, it suffers from a few limitations. The main drawback of SIMS is that it is not
amenable to quantitative analysis. TOF–SIMS requires the sample to be dry and the
analysis can only be performed in high vacuum. Before drying, care should also be
taken to ensure the solvent is free of any salts. Its high sensitivity can also be a
shortcoming if the sample surface has contaminants. Samples should therefore be
handled and processed very carefully. As with SIMS, quantitative analysis may be
challenging due to the matrix effect (dependence of secondary ion emission on the
sample matrix). Even though high-depth resolution is possible, the lateral resolution
is relatively low (~50nm). If the ion beam is at a high current density, the sample
surface will be damaged resulting in poor data interpretation. Due to these reasons,
advanced mass spectroscopic techniques like TOF–SIMS and MALDI need highly
trained users.
3.8.1 Application inPEGylated Nanocarriers
Hinterwirth and coworkers synthesized PEGylated gold nanoparticles with different
PEG chain lengths. PEGylation was achieved through thiol-based conjugation and
surface coverage by PEG was determined using inductively coupled plasma mass
spectrometry (ICP-MS). The results were also compared with a lipophilic thiol
ligand. The authors reported a novel gold-to-sulfur ratio measurement that could not
only estimate the surface PEG density but also predicted the impact of chain length
and nanoparticle size on the stability induced by Van der Waals interactions
(Hinterwirth etal. 2013).
In a similar report, Sebby etal. modied 10 nm and 30 nm gold nanoparticles
with uorescently labeled PEG.The authors determined PEG surface coverage for
nanoparticles conjugated through thiolate as well as lipoic acid-based linkers. While
thermogravimetric analysis was the primary technique used for analysis, MALDITOF- MS was utilized for the determination of molecular weights of the PEG derivatives. Figure 3.13 shows the Gaussian curve ts for lipoic acid-PEG-amine
(LA-PEG-NH3) and Thiol-PEG-FITC (HS-PEG-FITC). The estimates obtained
3 Topographic Properties ofPEGylated Nanocarriers
Fig. 3.13 MALDI-TOF-MS data (solid, black lines) with Gaussian ts (red, dashed lines) for (a)
HS-PEG-FITC and (b) LA-PEG-NH3. (Figure adapted with permission from Sebby and Manseld
(2015) Copyright © 2015, Springer Nature)
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Fig. 3.14 MALDI-TOF-MS data for the naked aptamer and PEGylated aptamer. Figure adapted
with permission from Ara et al. (2014) Copyright © 2014 Elsevier Ltd.)
from the Gaussian ts were 3900g/mol and 3850g/mol, respectively, and were in
close agreement with the manufacturer’s specications of 3400g/mol (Sebby and
Manseld 2015). This work illustrates how mass spectroscopy can complement
other surface analysis techniques to deduce critical topographical information about
nanocarriers.
MALDI-TOF-MS can also be used to monitor the PEG conjugation process. Ara
and colleagues developed PEGylated liposomes that were also modied with an
aptamer to endow the nanocarrier with a targeting functionality. As shown in
Fig.3.14, MALDI-TOF-MS was employed to conrm the PEGylation of nanocarriers (Ara et al. 2014). Hence, mass spectroscopy can detect small changes in
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molecular weights enabling the detection and monitoring of the PEG conjugation
process.
3.9 Thermogravimetric Analysis
Thermogravimetric analysis (TGA) is based on the principle of loss in the mass of
the sample upon heating it to controlled elevated temperatures. The components of
the nanocarrier degrade and vaporize at different temperatures. A thermally isolated, sensitive weighing balance and temperature-tunable furnace are part of the
apparatus. The relationship between mass and temperature is plotted as a decomposition curve. Oxidation temperature and loss of mass are detected and the residual
mass, which includes inorganic components of the nanocarriers, residual metals,
and other sample impurities, can be obtained. An IR spectrometer can be combined
with TGA to identify and analyze gases produced during thermal decomposition.
The main advantages of TGA include the precise quantication in a wide detection range, fast, and reliable analysis. In the context of PEGylated nanocarriers,
TGA can be used to characterize the surface and determine the purity. TGA can also
be used to determine the PEG surface density of nanoparticles by calculating the
degradation of PEG in the temperature range of 300–450°C.The mass fraction of
the organic components can be obtained at a precision as high as 10−4 (Corbierre
etal. 2004).
TGA analysis does not involve cumbersome sample preparation except for drying. However, the sample requirement can be relatively high, in the order of several
milligrams. This poses a serious limitation when the sample quantity is limited. It is
therefore recommended to combine TGA with other analytical tools to characterize
the topography. To improve the sensitivity of thermogravimetric analysis for smaller
sample amounts, an improved microscale method has been developed that provides
comparable results at sample amounts three orders of magnitude less than the
requirement for the conventional TGA method (Manseld etal. 2014).
3.9.1 Application inPEGylated Nanocarriers
Manseld etal. used a micro thermogravimetric analysis (μ-TGA) method to characterize PEGylated silica nanoparticles. The authors were able to conrm the presence of PEG and quantify surface-bound PEG content. The effectiveness of the
developed microscale technique was veried by comparing it with the conventional
TGA method. Figure3.15 shows the thermogram for SiO2–PEG nanoparticles. The
decline in mass above 100°C is due to the decomposition of surface PEG chains.
Above 500°C, mass increases due to the oxidation of silica nanoparticles. This
method quantied a mass change of 31.79% (blue line) compared to 34.1% by conventional TGA measurement (red line) (Manseld etal. 2014).
Maccarini etal. used several analytical techniques including TGA to characterize
gold nanoparticles derivatized with thiolated PEG.The authors were able to detect
3 Topographic Properties ofPEGylated Nanocarriers
Fig. 3.15 Mass % versus temperature thermogram of SiO2−PEG (n=25) nanoparticle samples
evaluated using μ-TGA (red) as compared to a conventional TGA measurement (blue). (Figure
adapted with permission from Manseld et al. (2014) Copyright © 2014, American Chemical
Society)
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PEG chain terminations at the water interface and obtain information about the
hydrated structure of PEGylated nanocarriers. TGA could precisely determine the
gold to polymer ratio in desiccated samples (Maccarini etal. 2010).
3.10 Differential Scanning Calorimetry
Differential scanning calorimetry (DSC) is a thermal analysis method that measures
heat ows associated with transitions in materials. The transitions can include physical or chemical changes that result in a change in the heat capacity of the material.
Sample temperature may change due to several events like solvent loss, phase transition, or thermal decomposition. To monitor such processes, DSC allows for a very
wide temperature range from −60°C to greater than 1500°C.
DSC can be run on two modes—power compensation and heat ux. In the power
compensation mode, the sample and an inert reference material are maintained at
the same temperature. The instrument records the amount of heat needed to increase
the temperature of each as a function of temperature. The heat capacity of the sample relative to the reference is equivalent to the energy required to maintain both at
an equal temperature. Here, enthalpy change is expressed as energy per unit time
and is plotted against time. In the heat ux mode, the analyte and reference are
assembled in a single furnace system. Changes in enthalpy of the sample result in a
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temperature difference between the sample and reference. The subsequent ow of
heat that brings the system to a thermal equilibrium can be correlated with temperature changes. This relationship is established through a calibration curve prior to
sample analysis.
DSC can be used to determine the phase transition points for melting and crystallization. Latent heat capacity and the heat of fusion may also be ascertained. For
polymeric nanoparticles, DSC may be used to investigate the molecular interactions
between the drug and PEG chains upon PEGylation. Differences in the glass transition temperature (Tg) can point to changes in the topography of the nanocarriers as
well as the crystalline state of the encapsulated drug. The main advantage of DSC is
its very low sample requirement (a few milligrams), and the determination of weak
phase transitions can be fairly accurate.
Solid and liquid samples can be analyzed by DSC.To improve the peak sharpness and resolution of the thermogram, the contact surface between the sample and
the crucible should be maximized. This ensures low resistance to heat ow through
the system. For solid samples, a thin lm or ne powder spread at the bottom of the
crucible is ideal. It is important to note that there are no interactions between the
sample and the crucible. Even though the measurement requires a very small amount
of sample, DSC is a destructive analysis technique. There are also limitations with
respect to volatile samples which pose difculties due to evaporation during sample
preparation. Increased resolution may be achieved at the cost of sensitivity and vice
versa. As a topographical characterization tool, DSC does not provide direct structural information. However, thermal transitions may be indicative of structural
changes.
3.10.1 Application inPEGylated Nanocarriers
Essa etal. developed poly(, -lactide) (PLA) based polymeric nanocarriers that
were PEGylated in two ways—grafted on the surface as 7%PEG-g-PLA and as a
multiblock copolymer (PLA–PEG–PLA)n. Both formulations had a similar PEG
insertion ratio and chain lengths. The effect of PEGylation on the thermal properties
of both types of nanocarriers was evaluated using DSC.Pure PLA was measured to
have a Tg of 46.4 °C. As seen in Fig. 3.16, PEG-grafted formulation showed an
increase in Tg of ~7°C (a) that was attributed to increased chain rigidity. On the
other hand, the multiblock copolymer formulation showed a lower Tg value of 39°C
(b). This could be due to the higher PEG content of 8.9mol% and comparatively
lower molecular weight. The authors inferred this result to also be indicative of a
fraction of PEG chains being present inside the PLA core of the nanoparticles.
Furthermore, the interaction between the loaded drug and polymeric matrix was
also investigated from the DSC thermograms. As shown in Fig.3.16a, pure ibuprofen has an endothermic peak at 78°C, whereas the melting endotherm of the drug
as a physical mixture with PEG-grafted polymer dropped to 74°C. This shift in
peak could be due to weak interactions between ibuprofen and the polymers. The
3 Topographic Properties ofPEGylated Nanocarriers
Fig. 3.16 DSC curves of ibuprofen (solid line), (a) physical mixture of PEG7%-g-PLA with
ibuprofen (dashed pink line), PEG7%-g-PLA polymer (dotted line), and ibuprofen-loaded NPs
(dash-dotted line); (b) physical mixture of (PLA–PEG–PLA)n with ibuprofen (dashed pink line),
(PLA–PEG–PLA)n polymer (dotted line), and ibuprofen-loaded NPs (dash-dotted line). (Figure
adapted with permission from Essa et al. (2010) Copyright © 2010 Elsevier)
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authors also used DSC to demonstrate that the drug exists in a crystalline state in
both formulations (Essa etal. 2010).
As discussed previously, Rabanel et al. synthesized PEGylated polymeric
nanoparticles in the “brush” conformation. The authors found that the Tg of PEG-gPLA polymers had a strong dependence on the concentration of PEG.Compared to
the bulk polymer, the lyophilized nanocarriers showed a very distinct thermogram.
In PEG-g-PLA polymers, the presence of PEG was conrmed by a PEG fusion
endotherm and a reduction in Tg. However, with an increase in the PEG content,
there was no further change in the glass transition temperature. These results were
also corroborated by NMR and XPS analyses (Rabanel etal. 2015).
3.11 Atomic Force Microscopy
Atomic force microscopy (AFM) is a robust scanning probe microscopy technique
that is based on the determination of interaction forces between the sample and a
probe. The ne probe of the AFM is coupled to a cantilever that can retrace the
topography of the sample. As the probe traverses the sample surface, there is a
repulsion between the tip and the sample. A constant force is maintained between
the sample and the tip which causes deection of the cantilever. The movements of
the cantilever are supported by a piezoelectric scanner. A laser beam captures the
degree of bending of the cantilever which correlates with the variation in the
reected beam. This variation is detected by a photodiode, and along with the
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rigidity of the cantilever, the interaction force and height of the surface can be
quantied.
AFM can be operated on three modes based on contact between sample and
probe: contact, noncontact, and tapping modes. Contact mode is accompanied by
strong lateral forces and this can potentially distort the sample surface and may also
damage the probe. The intermittent contact (tapping) mode, most commonly used
for nanoparticle characterization, is sensitive to the amplitude of the oscillating tip
and is hence also referred to as the oscillating mode. In the tapping mode, oscillation amplitudes can be typically close to 200nm compared to ~10nm in the noncontact mode. For sensitive samples that are susceptible to contact by the pointed
probe, noncontact mode is ideal.
AFM can be used to generate magnied 3-D images of the surface topography at
a very high resolution (~nm). High resolution is possible due to its wide surface
scanning range, the sensitivity of the piezoelectric crystal, and the nanometric size
of the probe. The resolution is not only similar to that obtained with SEM and TEM,
but AFM is also a less-expensive technique that can be easily installed while requiring a low footprint in the laboratory. Surface morphology, including the shape and
size of nanoparticles, can be determined precisely. AFM is frequently combined
with other spectroscopic techniques like FTIR and TOF–SIMS to improve spatial
resolution and enable the detection of elemental composition, respectively. AFM is
also one of a few available techniques that can be used to determine the surface
texture and roughness of nanomaterials. AFM can also be used to investigate thermal and magnetic interactions if the probe is functionalized based on the application.
Samples can be imaged directly without any surface modication, and measurement is possible in any environment—gas, liquid, or vacuum. However, the sample
must be stably immobilized on an inert substrate to enable accurate determination
of the morphology. The concentration of colloidal dispersions should also be optimized to prevent the formation of aggregates which adversely affect the topographical analysis. Factors like tip curvature radius and surface properties of the material
affect the measured topological properties. Particle characterization results may be
difcult to interpret if tip artifacts are not dealt with appropriately.
Even though AFM is a powerful topographical characterization tool, it suffers
from several limitations. Its high-resolution images require signicantly longer scan
times compared to electron microscopic techniques. If multiple samples need to be
tested, the low throughput of this method causes further delays in measurement.
Properties of the probe tip like shape and curvature can complicate measurements
and interpretation of results.
3.11.1 Application inPEGylated Nanocarriers
Backmann and coworkers reported a microcantilever-based sensor that was used to
characterize the conformation of PEG chains grafted on a gold surface. The authors
investigated two solvent conditions—PBS and 20% 2-propanol, by AFM imaging
and force–distance curve measurements. Figure3.17 shows that the PEG chains are
3 Topographic Properties ofPEGylated Nanocarriers
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Fig. 3.17 Representative force curves obtained by approaching the AFM tip to the Au surface
grafted with 20 kDa mPEG–SH immersed in PBS (black squares) and 20% 2-propanol (gray
squares). (Figure adapted with permission from Backmann et al. (2010) under the terms of the
Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0))
in a “brush” conguration when immersed in PBS (black squares). When the tip–
sample distance (D)=0, we are in the region of “hardwall repulsion.” As D increases
to ~25nm, the long-range repulsive force decays exponentially indicating the characteristic repulsive forces of a polymer brush. However, when the solvent was
switched to 20% 2-propanol (gray squares), the long-range repulsion disappeared
and a weak repulsion was observed at ~5nm. Here, the PEG monolayer was in a
“collapsed” state. The authors further investigated the solvent effect by acquiring
images at varying contact imaging forces. Their observations corroborated the
results of the force measurements and the authors concluded that grafted PEG
monolayers reversibly change their conformation when exposed to “good” and
“bad” solvents (Backmann etal. 2010).
Shilpa Sant and colleagues developed multiple types of PEGylated nanoparticles
with PEG grafted on the surface or as a constituent of a copolymer matrix. All nanocarriers were characterized for their surface morphology using AFM.The authors
found that the PEG content was comparatively lower for the multiblock copolymer.
This was attributed to some chains that may have interpenetrated inside the nanoparticle core. Figure 3.18 shows AFM phase images (lower panel) and topography
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Fig. 3.18 Tapping mode AFM images of NPs: topography (upper panel) and phase images (lower
panel). PLA (a), PEG1%-g-PLA (b), PEG5%-g-PLA (c), and PLA–PEG–PLA)n (d). (Figure
adapted with permission from Sant et al. (2008) Copyright © 2008 Wiley Periodicals, Inc.)
A. G. Prasad et al.
(upper panel) for both types of PEGylated nanocarriers. As seen in the lower panel,
sharp phase contrast was observed on all nanocarriers that were PEGylated. The
authors suggested this could be due to PVA coating on PLA on non-PEGylated
particles. However, PVA did not adsorb on hydrophilic PEGylated particles thereby
leading to a sharper contrast (Sant etal. 2008).
Essa etal. studied the effect of PEG surface PEG density of PEG-g-PLA nanoparticles on their physicochemical and biological properties including protein binding
and macrophage uptake. AFM was used to study the morphology of nanocarriers
pre- and post-lyophilization. Figure3.19 shows AFM images (surface morphology
on the left and phase image on the right) of these nanocarriers encapsulated with
rhodamine B.Before lyophilization, nanoparticles were spherical in shape, whereas
aggregates were observed after lyophilization. The authors also determined the surface chemistry of the particles and conrmed the presence of PEG monolayers at
the surface of nanoparticles (Essa etal. 2011).
3.12 Scanning Electron Microscopy
Scanning electron microscopy (SEM) is an electron microscopy technique wherein
the sample surface is scanned by a focused electron beam to produce a 3-D topographic image. The primary electron beam, collimated through a probe, hits the
sample to generate X-rays and secondary electrons which are collected by detectors.
Although it is run at low voltages of 500V to 30kV, SEM can produce images of
10–20nm particles at a high resolution. SEM is a very useful tool to characterize the
morphology and structure of nanoparticles. Combining it with spectroscopic techniques like EDX can provide the elemental composition of the sample surface. In