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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
77
Only moderately strong signals can be obtained due to the low cross section
available in conventional Raman scattering. To overcome this challenge and improve
the sensitivity and specicity of the technique, surface-enhanced Raman
Spectroscopy (SERS) uses electromagnetic enhancement and a chemical effect that
amplies the polarizability of the molecule due to charge-transfer. This is particularly useful in the case of metallic nanoparticles where enhancement can be exploited
to deduce information about surface binding and modications in functional groups
of molecules.
Tip-enhanced Raman Scattering (TERS) is analogous to SERS but also includes
a scanning probe microscopy such as AFM or scanning tunneling microscopy
(STM). The molecules on a metal nanoparticle directly under the sharp metal tip
show enhanced localized surface plasmon resonance (LSPR) thereby allowing for
single molecule detection (Bailo and Deckert 2008). The enhancement in the Raman
signal is due to the creation of a “hot spot” between the tip and the substrate. This
overcomes the limitations of spatial resolution and substrates in SERS.As the TERS
signal comes from the small gap between the apex of the tip and the substrate, a very
high resolution of 1–10nm can be obtained (Zhang etal. 2016). The major benet
of TERS is its applicability to characterize samples both in air and liquid. This is
particularly useful to study surfaces of modied nanocarriers in solution.
Even though Raman spectroscopy requires minimal to no sample preparation, it
is important to select the right substrate based on the sample and application. The
samples can be analyzed either as powders or resuspended in PBS to simulate physiologic conditions. Dried or colloidal suspensions of nanoparticles can be deposited
onto glass slides that are typically coated with poly(4-vinlypyridine) (PVP). To stably immobilize the nanoparticles on the glass support, the slides can also be functionalized with (3-aminopropyl) triethoxysilane.
As with IR spectroscopy, Raman spectroscopy may not be ideal for quantitative
analysis. The choice of substrate for particle immobilization may be limited to a
transparent substrate like glass. Though SERS and TERS have been successfully
used to study surfaces of metallic nanoparticles, whether they can also be applied to
nonmetallic nanoparticles is still under investigation.
3.4.1 Application inPEGylated Nanocarriers
Nuwanthi Katuwavila and co-workers described a sustained-release drug delivery
system for cephalexin (CEF) using PEGylated graphene oxide. Raman spectroscopy was used to conrm the successful conjugation of PEG.Raman spectra of
graphene oxide (GO) and PEGylated graphene oxide (GO-PEG) are shown in
Fig.3.4. The characteristic peaks corresponding to the D and G bands were located
for both nanocarriers at 1380/cm and 1580/cm. Intensity ratios of the G band to D
band were found to be very similar to GO and GO-PEG, at 0.840 and 0.844, respectively. Similarity in this ratio was used to conrm that conjugation with PEG did not
affect the aromatic structure of GO nanoparticles (Katuwavila etal. 2020). Therefore,
78
Fig. 3.4 Raman spectra of
(a) GO and (b) GO-PEG
where Go was
functionalized with PEG
by a simple esterication.
adapted with permission
from Katuwavila et al.
(2020) Copyright © 2020
Published by Elsevier)
Fig. 3.5 Raman spectra
for (a) TiO2, (b) PEG, and
(c) TiO2–PEG, recorded in
the zone 150–3200/cm.
(Figure adapted with
permission from León
etal. (2017) under Creative
Common CC BY license)
A. G. Prasad et al.
Raman spectroscopy can be used to assure the integrity of the core structure of
nanocarriers.
León and colleagues developed PEGylated TiO2 nanocarriers for targeted delivery of 2-methoxyestradiol (2ME). Raman spectroscopy was used to follow the PEG
conjugation process. In Fig.3.5, we see the Raman spectra for TiO2 (a), PEG (b),
and composite TiO2–PEG (c). The major characteristic peaks for PEG were observed
at 2938/cm, 2886/cm, and 2843/cm. As there is no appreciable shift in the bands in
TiO2–PEG compared to TiO2, it was concluded that modication with PEG did not
affect the crystalline structure of the TiO2 nanocarriers. Moreover, functionalization
with PEG was conrmed by locating broad peaks around 1000–1200/cm that correspond to C–C and C–O stretching vibrations. Sharp characteristic peaks around
3 Topographic Properties ofPEGylated Nanocarriers
79
2900/cm also prove that the nanocarriers were successfully PEGylated (León
etal. 2017).
Carly Levin etal. used surface-enhanced Raman spectroscopy to determine the
packing density of thiolated poly(ethylene glycol) adsorbates on gold nanoshells.
They developed a nondestructive assay to calculate the number of 2000MW and
5000 MW PEG molecules bound to gold nanoshell surfaces. p-Mercaptoaniline
(pMA) was used as the linker molecule and its Langmuir isotherm was interpolated
to deduce the packing density. Figure3.6 shows the SERS spectra for gold nanoshells
coated with (a) 5000 MW pMA-PEG-Fl conjugate, (b) 2000 MW pMA-PEG-Fl
conjugate, and (c) pMA only. The predominant bending and stretching modes arising due to the benzene moiety of pMA were observed on all spectra. This result
conrmed successful thiol-mediated conjugation of the uorescent PEG moiety.
Furthermore, the spectra for PEGylated nanoshells showed an additional peak at
1330/cm corresponding to the xanthene ring of the uorescent moiety. Interestingly,
the spectrum for the conjugate with 2000MW PEG showed a more dened Stokes
feature for uorescein. This was attributed to a higher number of PEG molecules on
the surface of the nanoshell compared to the 5000MW PEG conjugates that had a
larger molecular footprint. By comparing the estimated and theoretical PEG densities for PEGylated nanoshells, the authors were also able to determine the conformation of PEG chains. Their analysis on packing densities showed that both PEG
conjugates compactly bound to the interfaces covering surface areas that are consistent with the “brush” conformation, rather than the extended “mushroom” conguration (Levin etal. 2006). This report presented a quantitative analysis that could be
utilized to assess not only the number of PEG molecules bound to the surface but
also deduce the conformation of PEG chains.
Fig. 3.6 SERS spectra for
Au-silica nanoshells coated
with (a) pMAPEG5000- Fl, (b)
pMA-PEG2000-Fl, and (c)
pMA (offset for clarity).
(Figure adapted with
permission from Levin
etal. (2006) Copyright ©
2006 American Chemical
Society)
80
A. G. Prasad et al.
3.5 X-Ray Photoelectron Spectroscopy
X-ray photoelectron spectroscopy (XPS) is a powerful surface analysis technique
that can be used to characterize polymeric nanomaterials. As it provides valuable
chemical information at the surface, it is also known as electron spectroscopy for
chemical analysis (ESCA). This technique is based on the principle of photoelectric
effect wherein irradiation by a monochromatic X-ray beam results in the emission
of photoelectrons from the inner shell of atoms. The photoelectrons are collected by
detectors that identify and quantify their kinetic energy. The kinetic energy is equal
to the binding energy of the electron and this information is used to identify the element. With this method, the surface composition can be determined which can be
used to further evaluate the coverage density of the polymer.
The maximum sampling depth that corresponds to the upper layers of the sample
is typically less than 10nm. Unlike microscopic techniques, XPS provides elemental composition along the direction of the electron beam. Except hydrogen, XPS can
detect all other atoms on the surface. The kinetic energy of the photoelectrons can
be related to the electronic structure and oxidation states of the elements. Other
surface characteristics and properties relevant to ligand binding, attachment of functional moieties can be deduced through a high-resolution scan that can identify and
quantify chemical bonds. As XPS lacks spatial (lateral) resolution, individual
nanoparticles cannot be analyzed. However, collections of particles that form a single layer can be surveyed to obtain elemental compositions. In comparison to other
techniques like Auger electron spectroscopy (AES), XPS is less damaging to the
sample due to relatively lower energy of X-rays.
Most of the limitations of this technique arise due to the complexity of sample
handling. Operation of the XPS equipment also requires frequent maintenance and
highly trained users. XPS samples need to be prepared and handled carefully as
minute impurities can affect the analysis. To prevent contamination, the sample surface is thoroughly washed with volatile organic solvents and dried. The analysis is
performed on dehydrated samples in an ultra-high vacuum environment. For colloidal samples, the sample must be dried and immobilized on a at substrate only
after removing all salts from the solvent. As mentioned earlier, a major drawback of
this technique is its poor lateral resolution which complicates quantitative analysis
and interpretation of results. This can be aggravated by the presence of environmental carbon impurities.
Precision in surface PEG quantication in polymeric nanocarriers composed of
other ether compounds can be limited due to a similar proportion of carbon and
oxygen atoms. Additionally, sampling depths for C1s and O1s are ~10nm and
~8nm, respectively which can cause further discrepancies. This challenge can be
overcome by including heteroatoms such as nitrogen or sulfur in the PEG functional
group. Another drawback of XPS analysis for PEG measurements has to do with the
deleterious consequences of drying the samples. The sample in vacuum has a collapsed dehydrated PEG layer whose surface distribution and thickness can be markedly different from the native conformation in an aqueous solution.
3 Topographic Properties ofPEGylated Nanocarriers
81
3.5.1 Application inPEGylated Nanocarriers
Damodaran and coworkers used XPS to calculate PEG layer thickness and grafting
densities in amine functionalized poly(ethylene glycols) (PEGs). The amine conjugation method was used to graft 2000 and 4000Da PEGs on hydrophilic and hydrophobic derivatives. Elemental surface composition was evaluated by survey scans
and high-resolution C1s scans were used for detailed surface chemical analysis.
High-resolution C1s scans (Fig.3.7) show C–O peaks with increased intensity at
286.5eV.Increasing concentrations of PEG resulted in increasing intensities and
this conrmed successful PEG coupling. The authors also calculated the thickness
of the PEG layer followed by surface conjugation and density. The grafting densities were correlated with brush and mushroom conformations for the polystyrene
and Sephadex matrices, respectively (Damodaran etal. 2010).
Rabanel etal. synthesized PEGylated PLA polymeric nanocarriers with varying
PEG surface densities in the “brush” conformation. The orientation of PEG chains
at the surface was investigated by both XPS and nuclear magnetic resonance (NMR).
The two techniques showed exceptionally similar results conrming the segregation
of PEG chains at the surface. Minor discrepancies between XPS and NMR were
attributed to the depth resolution of XPS (~10nm) which may not be clearly distinguishable between PEG chains at and underneath the nanoparticle surface.
Figure3.8 shows high-resolution C1s (a) and O1s (b) scans for PEGylated nanoparticles. The authors used these scans to ascertain the constituent chemical bonds that
enable the identication of ether groups. As ether groups are specic to PEG, they
were able to successfully distinguish between PEG and PLA (Rabanel etal. 2015).
Fig. 3.7 XPS high-resolution C1s scans. (Figure adapted with permission from Damodaran et al.
(2010) Copyright © 2010 American Chemical Society)
82
Fig. 3.8 XPS high-resolution spectra with deconvoluted C1s (a) and O1s peaks (b) for PEG-g-
PLA NP with a 7.9% PEG content. (Figure adapted with permission from Rabanel et al. (2015)
Copyright © 2015 American Chemical Society)
A. G. Prasad et al.
3.6 Nuclear Magnetic Resonance
Nuclear magnetic resonance (NMR) is a quantitative analytical tool that exploits the
magnetic nature of nuclei of atoms of isotopes like 1H, 13C, 19F, and 31P to characterize the structure of materials at nanoscale. When a strong magnetic eld is applied,
the nuclei spin about the direction of the eld and at a frequency proportional to the
magnetic eld strength.
The “spin-up” and “spin-down” states of the nuclei correspond to their alignment
in the direction of the applied eld and its opposite direction, respectively. The
energy difference due to the transitions between these states can be investigated by
radiofrequency electromagnetic waves. The electromagnetic waves are irradiated at
right angles to the applied magnetic eld at a frequency close to the Larmor frequencies of the nuclei. When the frequency of nuclear precession and electromagnetic waves matches, magnetic resonance occurs which is the basis of this technique.
The resonance frequency depends both on the sample and the solvent. The energy
differences are measured, and the absorption energy is plotted versus chemical shift,
which is typically expressed in ppm. Chemical shift is the difference in absorption
frequencies for the sample and a standard normalized by the absorption frequency
of the standard. The integrated peak surface in the resulting plot is proportional to
the number of protons detected.
NMR enables the sensitive measurement of protein displacement and is also versatile to characterize particles in suspensions as well as gaseous environments.
NMR relies on the detection of spin coherences and can be used to investigate the
topographical properties of nanoparticles. Chemical shifts detected by NMR can be
used to obtain critical information about chemical bonds including bond lengths,
electron polarization, and dynamics. These properties help ascertain the chemical
identity of atoms at the surface and NMR; therefore, enabling monitoring and controlling the local structure. Though NMR is not the primary technique for topographical characterization of nanoparticles, chemical functionalization and the
enhanced surface area of nanoparticles make it amenable to investigate the chemical
3 Topographic Properties ofPEGylated Nanocarriers
83
structure at the surface. In PEGylated nanocarriers, NMR can be used to study
ligand binding interactions at the surface. In addition to the density and orientation
of ligands at the surface, it can also provide information on specic chemical bonds,
atomic composition, and molecular structure. PEG density at the surface can also be
estimated as exemplied later in this section.
Nanoparticles for analysis can be directly prepared in 0.5% D2O solution with a
surfactant like cholic acid sodium salt (CHA). Internal standards like
3-(trimethylsilyl)-1-propanesulfonic acid, sodium salt (DSS), and tetramethylsilane
(TMS) are required for 1H NMR analysis. Serial dilutions of PEG in D2O with the
internal standard are generated to obtain a calibration curve for the PEG signal. For
lyophilized samples, dissolution in deuterated chloroform (CDCl3) enables the
determination of the total PEG content.
A major limitation associated with NMR is that the nanoparticles must be dissolved in a deuterated solvent. However, with this resuspension step, the PEG chains
at the surface and the core can no longer be distinguished in the NMR spectrum. If
it can be guaranteed that the PEG is grafted exclusively on the surface, the NMR
signal can be considered to represent the surface-bound PEG.If lyophilized nanocarriers need to be characterized without dispersion in D2O, another challenge
arises. Particles typically aggregate upon freeze-drying and the concomitant
decrease in surface area can result in erroneous analysis that underestimates the
PEG surface area. Lyoprotectants may be added to prevent this, but this could alter
NMR signals due to interference with the internal standard of PEG.Though NMR
can be an effective tool to characterize diamagnetic and antiferromagnetic substances, it is not suitable for ferromagnetic and ferrimagnetic materials. Due to their
high saturation magnetization, local magnetic elds are affected, and signal frequencies are shifted. This results in broad signal peaks that cannot be interpreted for
accurate measurements.
3.6.1 Application inPEGylated Nanocarriers
Xu and colleagues synthesized poly(lactic-co-glycolic acid) (PLGA) based nanocarriers with varying surface densities of PEG to study the impact of PEG density
on interactions between nanoparticles and mucus. They used 1H NMR to determine
the PEG content phase separated at the surface and compared it to the total PEG
content, obtained by dissolving lyophilized nanoparticles in CDCl3. Figure 3.9
shows the 1H NMR spectra for (a) PLGA nanoparticles without PEG with DSS as
the internal standard, (b) PLGA nanoparticles with 10% PEG, dispersed in D2O
with DSS as the internal standard, and (c) lyophilized PLGA nanoparticles with
10% PEG, dissolved in CDCl3 with TMS as the internal standard. DSS shows its
characteristic peaks at 2.91, 1.76, 0.65, and 0ppm. Figure3.9b shows an additional
peak at 3.65 ppm conrming PEG functionalization at the nanoparticle surface.
This also allows for the quantitation of PEG density on the nanocarrier surface.
Figure3.9c shows the same peak at 3.65ppm demonstrating that all the PEG content is at the surface (Xu et al. 2015). In this study, the authors used 1H NMR
84
Fig. 3.9 Representative
1
H NMR spectra of (a)
PLGA (0% PEG)
nanoparticles with 1wt%
DSS as the internal
standard, (b) PLGAPEG10% nanoparticles
suspended in D2O with
1wt% DSS as the internal
standard, and (c)
lyophilized PLGAPEG10% nanoparticles
dissolved in CDCl3 with
TMS as the internal
standard. (Figure adapted
with permission from Xu
et al. (2015) Copyright ©
2015 American Chemical
Society)
A. G. Prasad et al.
spectroscopy to not only conrm the conjugation process but also quantitatively
prove that the method of synthesis enabled PEG to completely phase separate on the
surface of the nanocarriers.
Garcia-Fuentes etal. developed a novel drug nanocarrier made of tripalmitin,
lecithin, and PEG-stearate. They used NMR methods to characterize the structure,
composition, and architectural organization of the PEGylated nanocarriers.
Specically, PEG drafting on the surface of the nanoparticles was conrmed by
studying its interactions with the external aqueous phase. The authors generated a
nuclear Overhauser effect (NOE) spectrum (Vogeli 2014) (Fig.3.10) which demonstrates molecular interactions between the PEG chains and water. The 3.6ppm PEG
signal conrms the localization of PEG on the surface of nanocarriers (GarciaFuentes etal. 2004).
3 Topographic Properties ofPEGylated Nanocarriers
Fig. 3.10 NOE spectrum between H2O and the nanoparticle functional groups. (Figure adapted
with permission from Garcia-Fuentes et al. (2004) Copyright © 2004 American Chemical Society)
85
Fig. 3.11 Temporal evolution of the 1H NMR spectra and DOSY spectrum of the PEG signal
upon grafting to the surface of gold nanoparticles. (Figure adapted with permission from Lu et al.
(2019) under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence)
Lu etal. developed a nondestructive 1H NMR method to determine the grafting
density of PEG on gold nanoparticles. NMR signals from free and grafted PEG
were distinguished using a multi-Lorentzian-splitting algorithm. As seen in
Fig.3.11, the PEG grafting process was monitored with time, and the intensity of
the PEG peak at 3.70ppm decreased gradually. The authors inferred the broadening
of the bottom parts of the peak to correlate to the existence of two types of PEG
chains. This conclusion was also corroborated by diffusion-ordered spectroscopy
(DOSY) (Groves etal. 2004) which showed two peaks at 3.70ppm with diffusion
coefcients corresponding to free and grafted PEG chains. The authors were
86
A. G. Prasad et al.
therefore able to investigate the temporal evolution of the grafting process and analyze the grafting rates and surface densities of PEG by a unique NMR approach (Lu
etal. 2019).
3.7 Energy-Dispersive X-Ray Spectroscopy
Energy-dispersive X-ray spectroscopy (EDS/EDX) is primarily used to quantify
surface elemental compositions. It is sometimes combined with scanning electron
microscopy (SEM) wherein an electron beam is incident on the sample. As this
excites the surface atoms, electrons from the inner shells are ejected and valence
electrons occupy their place. This transition results in the emission of X-rays whose
wavelengths can be analyzed by a detector. Each element emits X-rays at a unique
wavelength, thereby enabling the determination of the surface compositions. EDX
is also compatible with other electron microscopic techniques like transmission
electron microscopy (TEM) and scanning transmission electron microscopy
(STEM). The spatial resolution of the data acquired is ~10nm and the enhanced
resolution of the morphology depends on the associated microscopic technique.
EDS samples used for TEM/STEM are required to be electron transparent.
Hence, samples are prepared on thin supporting foils, typically made from carbon.
The X-rays are emitted isotropically and the detector, usually placed at an elevation
angle of ~35°, can collect a very small fraction of the total X-rays emitted. It is also
important to note that X-ray photon energy may be signicantly attenuated due to
scattering and absorption before reaching the detector. The detector should therefore be sensitive and have a larger angular area to receive the photons. Extending
measurement times is another strategy to compensate for low signal-to-noise ratios
and overly attenuated photon energies. Although EDX is useful for surface compositional analysis, it does not provide accurate quantitative data. However, peak
intensities in the spectrogram can be used to estimate the abundance of each surface
element.
3.7.1 Application inPEGylated Nanocarriers
Karim etal. fabricated pH-responsive nanoparticles composed of strontium sulte
(SSNs) that were modied by biotinylated PEG to prevent rapid clearance from
systemic circulation. They used EDX to analyze the elemental compositions of the
nanocarriers. Figure3.12 shows a comparative analysis of nanoparticles before and
after PEGylation. Common elements such as C, O, Sr, and S were detected in both
the constructs although PEG-SSNs contained a signicantly higher percentage of C
and O.This demonstrated the presence of biotin-PEG in the PEGylated nanocarriers. Interestingly, Si and Pt were also detected, and the authors suggested this could
be due to the use of a glass holder and Pt sputtering during sample preparation
(Karim and Chowdhury 2022). This report underscores the utility of EDX to conrm PEGylation as well as its limitations in data interpretation that arise due to
surface contamination.