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Flowing through Gastrointestinal Barriers with Model Nanoparticles:

From Complex Fluids to Model Human Intestinal Epithelium
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Iris Renata Sousa Ribeiro, Raquel Frenedoso da Silva, Renata Santos Rabelo, Talita Miguel Marin,
Jefferson Bettini, and Mateus Borba Cardoso*
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ABSTRACT: Most nanomaterial-based medicines are intravenously

applied since oral administration comprises challenging-related bio-
logical obstacles, such as interactions with distinct digestive fluids and
their transport through the intestinal barrier. Moreover, there is a lack of
nanoparticle-based studies that faithfully consider the above-cited
obstacles and boost oral-administered nanomedicines’ rational design.
In this study, the physicochemical stability of fluorescent model silica
nanoparticles (f-SiO2NPs) passing through all simulated gastrointestinal
fluids (salivary, gastric, and intestinal) and their absorption and
transport across a model human intestinal epithelium barrier are
investigated. An aggregation/disaggregation f-SiO2NPs process is
identified, although these particles remain chemically and physically
stable after exposure to digestive fluids. Further, fine imaging of f-
SiO2NPs through the absorption and transport across the human intestinal epithelium indicates that nanoparticle transport is time-
dependent. The above-presented protocol shows tremendous potential for deciphering fundamental gastrointestinal nanoparticles’
evolution and can contribute to rational oral administration-based nanomedicine design.
KEYWORDS: nanoparticles, simulated gastrointestinal fluids, intestinal epithelium model, stability, transport

■ INTRODUCTION
Nanomedicine has significantly evolved over the last 20
years,1−4 and nanoparticle (NP)-based formulations have been
successfully used for diagnosis and disease treatments.5−11
While the oral route of nanomedicine administration provides
benefits such as reduced infection risks, low cost, patient
comfort, and easy use, intravenous injection has been the most
employed route. This is mainly due to obstacles related to
orally administered nanomedicines which undergo aggregation
and degradation induced by the acidic pH in the stomach and
digestive enzymes.2,12−20 Consequently, these structural
changes can affect the absorption of NPs by the small intestine
and their ultimate fate.14,17,21,22 Therefore, the rational
development of nanomaterial-based drugs that can successfully
pass through the gastrointestinal fluids and be sufficiently
absorbed and transported across the intestinal epithelium is of
paramount importance.14,15,23−25
One of the limiting factors facing the development of
sustainable NP-based medicine for oral administration relies
on the lack of protocols that simultaneously deal with the NPs’
stability and degradation in complex digestive fluids and their
transport and absorption through optimized human epithelial
models. The INFOGEST community recently proposed a
static in vitro digestion model based on conditions physiolog-
ically relevant to the human gastrointestinal tract.26,27 The
method comprises the three digestion steps (oral, gastric, and
intestinal), considering pH, duration, ionic strength, compo-
sition, and enzyme concentration. In parallel, in vitro models
have been developed to mimic small intestine enterocytes,
allowing absorption and transport analysis with high
accuracy.14,28−36 Optimized intestinal models involve the co-
cultivation of human colon carcinoma epithelial (Caco-2, non-
mucus secretion) and human colorectal adenocarcinoma (HT-
29, high mucus secretion) cells in a 9:1 ratio.29,32,37,38
Nevertheless, most reports have scrutinized the transport of
nanomedicines across nonideal intestine monoculture models,
which cannot mimic the human intestinal epithelium
accordingly.36,39,40
Received: May 16, 2023
Accepted: July 6, 2023
Published: July 19, 2023

© 2023 American Chemical Society https://doi.org/10.1021/acsami.3c07048

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ACS Applied Materials & Interfaces
Figure 1. Representation of the protocol to evaluate the gastrointestinal evolution of orally administered NPs. Step 1: NPs’ incubation with
simulated salivary, gastric, and intestinal fluids. Fluids’ preparation followed the INFOGEST model. Step 2: Schematic representation of NPs’
absorption and transport across the intestinal barrier (not to scale). Optimized epithelial models were prepared by co-cultivation of Caco-2 and
HT-29 cells (9:1 ratio) in a polycarbonate cell culture insert. Mucus is represented in blue. The porosity of the polycarbonate membrane was not
represented in the drawing to enhance the visualization of the intestinal epithelium.
Most NP-driven studies have only focused on the
permeation through the intestinal epithelium, while studies
on complex digestive system-simulating fluids are still lacking
in the literature.33,36,38,41−44 These studies in complex media
are compulsory to draw a widespread understanding of their
absorption and transport in conditions that mimic real-world
events. Recent efforts to evaluate the impact of simulated
digestive fluids on the absorption and transport of NPs
through the intestinal model have faced physical and chemical
stability issues.45 Consequently, robust studies that simulta-
neously probe the stability and degradation of NPs in complex
digestive fluids and their transport and absorption through
optimized human epithelium models are still lacking in the
literature.
Herein, we present a new protocol to investigate the stability
and degradation of fluorescent model silica nanoparticles (f-
SiO2NPs) in simulated salivary, gastric, and intestinal fluids
simultaneously while in-depth probing their absorption and
transport through an optimized intestinal epithelium model
(Caco-2:HT-29) (Figure 1). The first innovative strategy
combines scattering and microscopy techniques to follow NPs’
aggregation state, surface degradation, and organic material
adsorption while passing through salivary, gastric, and
intestinal fluids (step 1�Figure 1). The second meaningful
advance is based on visualizing the intimate interaction of f-
SiO2NPs with the epithelial barrier model during absorption
and transport processes (step 2�Figure 1). This protocol
overcomes drawbacks found in previous research studies,
where the obstacles of the gastrointestinal tract are not
analyzed in their entirety but addressed separately and,
generally, under nonoptimized conditions.
■ RESULTS AND DISCUSSION
Characterization of f-SiO2NPs. f-SiO2NPs were chosen
due to their promising nanomedicine-related properties, such
as high area/volume ratio, physicochemical stability, easy
detection by incorporating a fluorescent dye, biocompatibility,
and low toxicity.46,47 f-SiO2NPs were synthesized (Figure S1)
using a modified Stöber method where a fluorescent dye
(rhodamine B isothiocyanate) was covalently conjugated to a
silica precursor and subsequently condensed, forming a
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fluorescent core. Afterward, an additional raw silica shell was
formed around the fluorescent core to prevent dye-biological
environment unspecific interactions, and a set of comple-
mentary techniques thoroughly characterized the synthesized
system. Scanning electron microscope (SEM) images showed a
typical quasi-spherical morphology and indicated the average
particle size of 86.4 nm and a half-width at half-maximum of
12.0 nm (Figure S2). Furthermore, these structures presented
an average hydrodynamic diameter of 106.8 ± 1.6 nm with a
low polydispersity index (PDI, 0.06) and a ζ-potential of −49.1
± 1.8 mV. Also, f-SiO2NPs showed red fluorescence with
maximum emission at 580 nm, characteristic of the rhodamine
B isothiocyanate presence (Figure S3).
In Vitro Digestion Experiments. f-SiO2NPs were
submitted to gastrointestinal digestion procedures,26,27 which
included the oral, gastric, and intestinal phases since they
mimic in vivo conditions, taking into account the presence of
digestive enzymes and their concentrations, pH, digestion
time, and salt concentrations, among other factors (Table S1).
First, f-SiO2NPs (final concentration of 0.5 mg mL−1) were
incubated with simulated salivary fluid (SSF) for 2 min,
followed by 2 h incubations with simulated gastric fluid (SGF)
and with simulated intestinal fluid (SIF), respectively. It is
essential to highlight that the transit time of the small intestine
is relatively constant (2−4 h).23 INFOGEST static in vitro
model considers 2 h of incubation not only because of the
accurate transit times in the gastrointestinal tract but also due
the process is a static method, so no eventual products are
removed, which may impair the functioning of the enzymes for
prolonged periods.48 The conductivity measurements indi-
cated values of 3.60 mS cm−1 for SSF, 16.30 mS cm−1 for SGF,
and 11.80 mS cm−1 for SIF, and the magnitude of these
numbers correlates with the concentration of electrolytes in
solution. According to dynamic light scattering (DLS, Figure
2a), f-SiO2NPs in the presence of SSF (pH 7.4) revealed to be
stable, with an average hydrodynamic diameter of 115.5 ± 0.3
nm and low PDI (0.08). However, NPs underwent an
aggregated state in SGF (pH 3.0), likely associated with the
low pH and high concentration of electrolytes. Silica
nanoparticles reach their isoelectric point at pH ∼2, resulting
in a decrease of the electric field formed in the double electric
layer and consequent aggregation (Figure S4).49,50 Moreover,
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Figure 2. NPs’ incubation with gastrointestinal fluids. (a) Size distribution curves obtained using a Zetasizer Nano ZS instrument and (b)
transmission electron microscopy (TEM) images of f-SiO2NPs in water (control, before incubation) and after undergoing interactions with all of
the simulated fluids used in the digestion experiments (after incubation). The samples were centrifuged and redispersed in water for analysis. After
incubation, organic material can be found around the NP as well as salts (black dots). The salt was evidenced by the diffraction pattern highlighted
in the top right inset in (b). Scale bar: 50 nm (TEM images) and 20 nm−1 (diffraction pattern). (c) Schematic representation of the salts around f-
SiO2NPs after incubation in digestive fluids. (d) EFTEM micrographs of f-SiO2NPs before and after SSF, SGF, and SIF incubation. Silicon is
represented in red, oxygen in blue, and carbon in green. As observed, f-SiO2NPs passing through the digestive process have a large amount of
carbon, indicating the presence of organic material. Scale bar: 50 nm. (e) Statistical analysis of height data obtained by atomic force microscopy
(AFM). The analyzes were performed for f-SiO2NPs in water (control) and after incubation in SSF, SGF, and SIF. Results are displayed as mean ±
SD. The one-way analysis of variance (ANOVA) and Tukey’s test were used for data analysis (n = 12). Differences were considered significant
when p < 0.05. Common symbols on top of the bars indicate no statistical difference between them. (f) Representation of f-SiO2NPs surface
roughness at different length scales L (40, 25, and 10 nm) characterized by AFM. (g) Root-mean-square (RMS) roughness as a function of L
obtained from AFM measurements carried out for f-SiO2NPs in water (control) as well as for f-SiO2NPs after incubation in SSF, SGF, and SIF.
Results are displayed as mean ± SD. Two-way ANOVA and Tukey’s test were used for data analysis. Differences were considered significant when
p < 0.05. Common symbols on top of the bars indicate no statistical difference between them.

the effects of ions also play a prominent role and likely induce
unspecific interaction between particles as SGF contains high
concentrations of electrolytes. Meanwhile, certain disaggrega-
tion was observed after incubating f-SiO2NPs in SIF (pH 7.4),
which presented an average hydrodynamic diameter of 187.1 ±
13.2 nm. Silica nanoparticles are negatively charged at neutral
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pH due to the deprotonated hydroxyl groups on their surface,
inducing an increase in the electric field formed in the electric
double layer and consequent repulsion among NPs (Figure
S4).49 It is worth mentioning that SSF, SGF, and SIF present
distinct ionic strengths, and the presence of different enzymes
can ultimately play an essential role in the NPs’ stability. The
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Figure 3. NPs’ stability and absorption and transport across the intestinal barrier. (a) Size distribution curves obtained using a Zetasizer Nano ZS
instrument of f-SiO2NPs (0.5 mg mL−1) after incubation in culture medium DMEM supplemented with 10% FBS. The incubation times were 0, 1,
2, 3, and 24 h at 37 °C. (b) Representation of the intestinal barrier (green) formed by Caco-2 and HT-29 cells in culture inserts. The DMEM
culture medium and f-SiO2NPs are represented by pink and pink spheres, respectively. (c) Percentage of absorption and transport of f-SiO2NPs
(0.5 mg mL−1) through the intestinal epithelium, where the upper compartment is represented in green, the bottom compartment in orange, and
the intestinal epithelium in pink. The concentration of f-SiO2NPs in samples was measured directly for fluorescence considering the excitation
parameter fixed at 524 nm, the gain value at 120, and the maximum emission at 580 nm. Results are displayed as mean ± SD. The ANOVA and
Tukey’s test were used for data analysis (n = 10). Differences were considered significant when p < 0.05. Common symbols on top of the bars
indicate no statistical difference between them. (d) Confocal microscopy images (intermediate slices) for the intestinal epithelium after incubation
for 4 and 24 h with f-SiO2NPs. (e, f) Magnified images of the region with SiO2NPs in (d) referring to the incubation times of 4 and 24 h,
respectively. The cells were stained with DAPI and phalloidin 488 and observed under a confocal microscope using lasers at 405 nm for DAPI
(nuclei), at 490 nm for phalloidin 488 (actin filaments), and at 561 nm for f-SiO2NPs. Scale bar for (d, e, f): 25 μm.

aggregation/disaggregation pattern has already been demon-
strated for nano-sized silica during in vitro digestion of foods
containing silica as a food additive.49 Nonetheless, little has
been evaluated regarding the effect of digestive fluids on the
resulting NPs’ surface. Understanding possible NP surface-
induced degradation is paramount for developing oral
nanomedicines since it may trigger a premature drug release
process and induce surface-functionalized nanocarriers mis-
targeting. Therefore, an in-depth morphological investigation
was carried out, considering the evolution of f-SiO2NPs
through the gastrointestinal fluids.
Transmission electron microscopy (TEM) (Figure S5) and
atomic force microscopy (AFM) (Figure S6) images
corroborated the dynamic light scattering (DLS) results
indicating that f-SiO2NPs underwent aggregation in the
stomach with subsequent disaggregation in intestinal con-
ditions. Moreover, TEM analysis also showed no significant
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difference in particle size and form when pristine f-SiO2NPs
were compared with those passed through all gastrointestinal
fluids (Figure S7). It is also worth mentioning that some TEM
images revealed the presence of small structures around f-
SiO2NPs (Figure 2b,c), attributed to the fluids’ salts and
organic material content. The diffraction pattern confirmed the
presence of the salts (Figures 2b,c and S8), and the organic
material by energy-filtered transmission electron microscopy
(EFTEM) images. EFTEM images indicated the deposition of
organic moieties, especially in the carbon channel, around the
f-SiO2NPs that passed through all gastrointestinal fluids
(Figure 2d).51 These residues hampered the effective particle
height acquisition by AFM, so a purification column was
employed to remove these impurities (Figure S9).
AFM images revealed a statistically significant height
reduction after impurities removal for f-SiO2NPs exposed to
SGF and SIF, which may indicate NP leaching (Figures 2e and
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Figure 4. NPs’ absorption and transport across the intestinal barrier. (a, b) Scanning electron micrographs of intestinal epithelium after incubation
for 24 h with f-SiO2NPs (0.5 mg mL−1). (c) Schematic representation of the intestinal epithelium (green), showing f-SiO2NPs (pink) on the
microvilli. (d, e) Transmission electron micrographs of resin-embedded intestinal epithelium before incubation with f-SiO2NPs. Abbreviations: DE,
desmosome; TJ, tight junction. (f) Representation of junctions formed between adjacent cell membranes and of some internal cell structures, where
1: zonule of occlusion, 2: zonule of adhesion, 3: desmosome, 4: gap junction, 5: hemidesmosome, 6: mucus granules, and 7: lipid drops. (g, h)
Transmission electron micrographs of resin-embedded intestinal epithelium after incubation for 24 h with f-SiO2NPs. (i) Schematic representation
of f-SiO2NPs located in the vicinity of the microvilli and crossing the intestinal epithelium and reaching the polycarbonate membrane. (j) EDS
analysis of resin-embedded intestinal epithelium after incubation with f-SiO2NPs for 4 h. Maps of the constituent elements of f-SiO2NPs, silicon
(pink) and oxygen (blue) are shown in panels (k) and (l), respectively.

S8). In contrast, this height reduction was not observed by in the samples’ roughness were observed at different
TEM analysis (Figure S6). In addition, no significant changes magnifications (Figure 2f,g), indicating that the f-SiO2NPs
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are likely chemically stable to the digestion simulated process.
Thus, we suggest that AFM height reduction is an artifact
attributed to the NPs’ passage through the purification column.
Larger structures elute first during the desalination procedure,
and the analyzed aliquots were likely rich in smaller NPs.
Stability of f-SiO2NPs in Culture Medium and Cell
Viability Assays. Following in vitro digestion experiments, we
attempted to evaluate the absorption and transport of f-
SiO2NPs through the model intestinal epithelium since this is
one of the critical barriers before NPs are distributed
throughout the organism. For this purpose, we first tested
the f-SiO2NPs colloidal stability in the epithelium culture
medium (Dulbecco’s modified Eagle’s medium, (DMEM))
supplemented with 10% of fetal bovine serum (FBS) and
evaluated the toxicity of these particles against the constituent
cells of the intestinal barrier (Caco-2 and HT-29). The average
hydrodynamic diameter of f-SiO2NPs (0.5 mg mL−1) remained
at ∼160 nm, and PDI at 0.14 after 24 h of incubation suggests
the absence of massive aggregation (Figure 3a). Particularly, a
subtle difference is observed in the 24 h curve in relation to the
other incubation times, which can be likely attributed to the
sedimentation of the few aggregates formed at the beginning of
the incubation process, slightly shifting the curve to smaller
diameter values. This maintenance in the colloidal stability of
NPs is a favorable condition for the cellular internalization
process. Also, f-SiO2NPs presented a negligible influence on
Caco-2 and HT-29-cells survival rates (Figures S10 and S11).
The viability always remained above 80% irrespective of the
tested NP concentration, indicating that f-SiO2NPs are
nontoxic to these cell lines.52 The high stability of f-SiO2NPs
associated with their nontoxicity suggests that these particles
can translocate through the intestinal epithelial model without
causing cell damage.
Transport of f-SiO2NPs across the Intestinal Epithe-
lium. After 21 days of culture, Caco-2/HT-29 intestinal
epithelia were exposed to f-SiO2NPs for 4, 15, and 24 h
(Figure 3b). Fluorescence measurements were used to
quantitatively estimate the transport of f-SiO2NPs across the
intestinal barrier. The incubation time of 4 h corresponds to
regular intestine transit time in humans and is an indirect
strategy to probe NPs-epithelium interaction reliably. After 4 h,
70 ± 12% of f-SiO2NPs remained in the upper compartment,
while a low transport to the bottom region was observed
(Figure 3c). The remaining 30 ± 11% of f-SiO2NPs were
either deposited on the intestinal epithelium surface or
absorbed by the epithelium. The discrimination between
both scenarios was done qualitatively by imaging the f-
SiO2NPs-treated intestines with a confocal microscope (Figure
3d,e). Using such images for quantitative assessment of f-
SiO2NPs absorption by the epithelium is inaccurate, as they do
not statistically represent the entire specimen. Intermediate
slice images of the epithelium indicated the presence of f-
SiO2NPs, most likely around the nucleus, which is a common
intracellular destination for particles similar to the ones used
here (Figure 3d,e).53,54 A recent report assessed the passage of
SiO2NPs through a nonoptimized intestinal barrier after 6 h of
incubation. Low NP absorption was observed, likely due to the
use of a mucus-free Caco-2 monoculture since HT-29 cells
were not considered in the experimental model.36 Afterward,
we hypothesized that increasing the incubation time would
allow some intracellular f-SiO2NPs to be transported through
the intestines. As expected, the percentage of f-SiO2NPs that
passed through the intestine was significantly higher with
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increasing incubation times (Figure 3c). The results showed
that around 50% f-SiO2NPs could be found in the intestinal
epithelium after 15 and 24 h of incubation, either deposited on
its surface or absorbed by the cells, as shown above.
Electron microscopy imaging was further employed to
unriddle the intimate interaction between f-SiO2NPs and the
intestinal epithelium. Regardless of the incubation time, SEM
images confirmed the presence of f-SiO2NPs outside the cells,
mainly associated with microvilli (Figures 4a,b, and S12). A
schematic epithelium representation is presented in Figure 4c,
where the interaction between f-SiO2NPs and the microvilli
becomes evident. TEM images allowed us to identify particular
structures of the intestinal epithelium, such as microvilli, cell
junctions (e.g., desmosomes [DE] and tight junctions [TJ]),
and the lipid drops produced by HT-29 cells (Figures 4d,e, and
S13). These structures in the intestinal epithelia validate our
co-culture model as a reliable strategy to mimic a lifelike
epithelial barrier. A drawing containing the most relevant
epithelium structures is presented in Figure 4f and agrees well
with the TEM images described above. The presence of f-
SiO2NPs in TEM images was ascribed to high electron density
structures within the same size range as the NPs synthesized in
this work (Figures 4g,h, and S13). f-SiO2NPs are more
electronically dense than those present in the cytosol or within
the lysosomal compartments in cells (lower mass atoms), so
these NPs can be easily distinguished from cellular compart-
ments. 55 According to TEM images, f-SiO 2NPs were
simultaneously seen in the vicinity of microvilli and crossing
the intestinal epithelium before reaching the polycarbonate
membrane, as depicted in Figure 4i. Energy-dispersive
spectroscopy (EDS) images (Figure 4j−l) confirmed that the
high electron density structures seen by TEM, on the surface
and inside the intestinal epithelium correspond to f-SiO2NPs.
After 4 h of incubation, the elements silicon (Si) and oxygen
(O) were identified as components of these nanometric
structures, confirming that these NPs can be internalized and
transported across the cells and finally reach the bottom
compartment (Figure 4j−l). Osmium (Os) and uranium (U)
were typically seen in the regions of resin-embedded
epithelium, and their presence is inherent in the sample
preparation. Similar EDS results were seen when f-SiO2NPs
incubation time is increased to 24 h (Figure S14).
The results mentioned above reveal that f-SiO2NPs can be
absorbed and transported through the intestinal epithelium,
which is critical for oral drug administration. However,
improvements in the particle are needed to increase transport
efficiency across the intestinal epithelium. Using NPs surface
functional groups is a relevant strategy that can be used to
enhance cell interaction and ultimately facilitate absorption
and transport.
Finally, although f-SiO2NPs were used as model nano-
particles, this entire protocol (in vitro digestion and transport
across intestinal epithelium) can be extended for an in-depth
understanding of the behavior of other types of nanoparticles
(e.g., metallic and polymeric nanoparticles) aimed at oral
administration.
■ CONCLUSIONS
In summary, we presented a successful protocol that
simultaneously evaluates the NPs’ stability and degradation
in digestive fluids (salivary, stomach, and intestinal fluids) as
well as their absorption and transport through a model human
intestinal epithelial barrier (co-culture of Caco-2/HT-29 cells).
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f-SiO2NPs exhibited no irreversible aggregation and degrada-
tion when exposed to simulated gastrointestinal fluids, and
their absorption and permeation through the epithelial cells
were time-dependent. The in vitro protocol described here
provides a comprehensive picture of the behavior of NPs when
exposed to the obstacles of oral administration. We suggest
that this protocol can be employed to evaluate the effect of
NPs bioaccumulation on the intestinal barrier over time as well
as to deeply investigate the ultimate behavior of degradable
and functional NPs in the gastrointestinal tract. The above-
described strategy can easily be expanded to other nano-
structured systems and might be employed as a quality control
platform that supports complex oral methods and in vivo
assays.
■ MATERIALS AND METHODS
Materials. Ammonium hydroxide (28−30%) and tetraethyl
orthosilicate (TEOS) were obtained from Sigma-Aldrich, and ethanol
(absolute) was purchased from Merck. All reagents and chemicals
were used as received without further purification. Water used in
described procedures was obtained from the Water purification
system (Purelab from ELGA, resistivity of 18.2 MΩ·cm−1). Cell
culture supplies were obtained from Gibco (Thermo Fisher
Scientific).
f-SiO2NPs Synthesis. The dye precursor was synthesized by
conjugation of the isothiocyanate group of rhodamine B isothiocya-
nate to (3-aminopropyl)triethoxysilane (APTES) at a molar ratio of
50:1 (dye:APTES) in absolute ethanol under nitrogen flow. The
resulting solution was continuously stirred for 24 h. The conjugate
was then hydrolyzed in a basic ethanolic solution with TEOS and
catalyzed by ammonia.56 In summary, 0.02 mL of dye precursor and
0.51 mL of TEOS were added to 30 mL of ethanol under continuous
stirring for 30 min. Then, 1.50 mL of ammonia solution 28% was
added and the resulting mixture was stirred for 3 h. Subsequently,
0.02 mL of conjugate and 0.51 mL of TEOS were added once again
and the reaction was stirred overnight. A silica shell was synthesized
around the preformed fluorescent core by the addition of ethanol (60
mL), ammonia solution (1.40 mL), and TEOS (1.70 mL). TEOS was
added at a flow rate of 1.00 mL min−1 using a syringe pump (New Era
Pump Systems, NE-8000). The resulting suspension was continually
stirred for 24 h and, then the f-SiO2NPs were centrifuged (10,640g for
15 min) and, then washed once with ethanol and four times with
deionized water. f-SiO2NPs were dispersed in aqueous solution with
the final concentration adjusted to 5.70 g L−1.
f-SiO2NPs Characterization. The particle size (Z-average), PDI,
and ζ-potential measurement of the dispersions were determined
using a Zetasizer Nano ZS instrument (Malvern Instruments Ltd.).
The size and PDI measurements were taken at 25 °C at an angle of
173°, and the data were calculated as the mean of three successive
analyses (100 s) for at least three independent samples. The
dispersions were diluted with water to adjust the signal level. ζ-
Potential measurements were used to evaluate the residual charge of
the f-SiO2NPs. The measurements were taken using a He−Ne (633
nm) laser. A dispersion of nanoparticles (1 mg mL−1) was prepared in
10 mM phosphate buffer (pH 7.4) for the analysis. Three
measurements (each consisting of at least 10 scans) were taken for
each sample.
Electron microscopy was performed using a SEM-FEM HR (FEI
Inspect) operating at an accelerating voltage of 10 kV. Images were
acquired in scanning mode. The samples were deposited onto 400
mesh holey carbon-coated copper grids.
Fluorescence measurements were performed using a TECAN
spectrophotometer (Infinite 2000). The analyses were performed on a
96-well plate. The excitation parameter was fixed at 524 nm, and
emission spectra were generated at 560−700 nm. The gain value was
kept constant at 120, and three measurements were taken for each
sample.
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In Vitro Digestion Experiments. The prepared fluids (Table S1,
Supporting Information) were designed as SSF, SGF, and SIF. The
conductivity of each fluid was measured using a pH/Cond 3320
conductivity meter (WTW). f-SiO2NPs were added to the respective
fluids at the final concentration of 0.5 mg mL−1. Between each studied
phase, NPs were centrifuged (23,940g for 5 min) and suspended in
the subsequent medium. In addition, a sample of f-SiO2NPs in water,
without incubation in the simulated fluids, was analyzed as a control.
The particle size was evaluated after the incubation time of the f-
SiO2NPs in each medium using a Zetasizer Nano ZS instrument.
Besides, TEM, EFTEM, and AFM images were obtained to assess
changes in the particles’ surface along the digestion process in
simulated fluids.
TEM images of the f-SiO2NPs were obtained using TALOS F200C
(Thermo Fisher Scientific) microscope operating at 200 kV
(conventional mode, room temperature) with FEI Ceta 16 M Pixel
CMOS 4 K × 4 K camera (Thermo Fisher Scientific). The samples
were centrifuged (23,940g for 5 min) and redispersed in water for
analysis. Drop casting was used to previously deposit the sample on
the holey carbon-coated copper grid.
Core-loss EFTEM analyses were carried out at JEOL JEM2100F
(200 kV). After incubation of f-SiO2NPs (0.5 mg mL−1) in SSF, SGF,
and SIF fluids, the samples were centrifuged (23,940g for 5 min) and
redispersed in water for analysis. In addition, a sample of f-SiO2NPs in
water, without incubation in the simulated fluids, was analyzed as a
control. Drop casting was used to previously deposit the sample on
the holey carbon-coated copper grid. For EFTEM analysis, the
elemental maps of silicon, oxygen, and carbon were acquired.
High-resolution AFM images were obtained using a Bruker
MultiMode 8 instrument with Peak Force Tapping mode. The
analyses were performed with a ScanAsyst-Air cantilever, charac-
terized by a nominal spring constant of 0.4 N m−1 and resonance
frequency of 70 kHz. The f-SiO2NPs, after incubation in SSF, SGF,
and SIF, were centrifugated and resuspended in ultrapure water for
AFM characterization or submitted to the illustra NAP-25 column for
desalting and buffer exchange of NPs dispersions. Then, the samples
were deposited in a mica substrate and overnight dried under a gentle
nitrogen flow. The heights and the surface roughness of the f-
SiO2NPs as the root-mean-square (RMS) values Rq of the distribution
of heights in the AFM high-resolution topographical images were
evaluated with open-source software available at http://gwyddion.
net/.
Stability of f-SiO2NPs in Biological Medium. The stability of f-
SiO2NPs (0.5 mg mL−1) in the biological medium was verified
considering 0, 1, 2, 3, and 24 h of incubation at 37 °C. The medium
used was DMEM supplemented with 10% of FBS, and the
measurements were performed using a Zetasizer Nano ZS instrument.
Three measurements (each consisting of at least 10 scans) were taken
for each sample.
Cell Viability Assays. Alamar Blue. Caco-2 and HT-29 cells were
seeded in 96-well microplates at a density of 5 × 105 cells mL−1 and
incubated for 24 h in a humidified atmosphere at 37 °C and 5% CO2
to obtain a subconfluent monolayer. Cells were exposed to f-SiO2NPs
at concentrations of 0.05, 0.10, 0.50, and 1.00 mg mL−1. After 24 h
incubation, the cells were washed three times with phosphate-buffered
saline (PBS), Alamar Blue (Invitrogen, 10% in DMEM) was added to
each well, and samples were incubated for 3 h at 37 °C. The
supernatant was posteriorly collected from the wells, put in a fresh 96-
well plate, and analyzed with a spectrophotometer (1420 Victor,
PerkinElmer) at 560 nm excitation wavelength and 590 nm emission
wavelength. Results are displayed as mean ± standard deviation (SD).
LIVE/DEAD. Caco-2 and HT-29 cells were incubated for 24 h under
standard conditions. Culture medium was removed, and the viability
was assessed by LIVE/DEAD Viability/Cytotoxicity Kit assays
(Invitrogen). LIVE/DEAD kit quickly discriminates live from dead
cells by simultaneously staining with green fluorescent calcein-AM to
indicate intracellular esterase activity and red-fluorescent ethidium
homodimer-1 to indicate loss of plasma membrane integrity.
However, to carry out these experiments, ethidium was replaced by
DAPI, since f-SiO2NPs, like ethidium homodimer-1, show fluo-
https://doi.org/10.1021/acsami.3c07048
ACS Appl. Mater. Interfaces 2023, 15, 36025−36035
ACS Applied Materials & Interfaces
rescence in the red region, which could interfere with the result. The
samples were incubated with calcein-AM (1.8 μM) and DAPI (1.8
μM) for 20 min. The reading of fluorescence was carried out in a
macro-confocal microscope (Leica TCS LSI, Leica).
Small Intestine Model Production. Caco-2 and HT-29 cells
were cultivated in high-glucose DMEM, supplemented with 10% FBS,
100 units per mL penicillin, 100 μg mL−1 streptomycin, and 1%
nonessential amino acids. Cell cultures were incubated at 37 °C in a
fully humid, 5% CO2 environment. The medium was renewed every
two or three days. Polycarbonate cell culture inserts were used to
produce a functional intestinal cell barrier model (intestine
equivalent), with a 0.40 μm pore size and an area of 0.60 cm2
(Millicell, Millipore, Merck KGaA). The inserts were integrated in a
24-well cell culture plate (Sarstedt) with 800 μL of DMEM per well in
the bottom compartment (which represents the human intestinal
bloodstream). To produce the intestine equivalent, 2.50 × 105 cells in
200 μL of DMEM57,58 were seeded in the insert’s upper compartment
(which represents the human intestinal lumen), considering the 9:1
ratio of Caco-2:HT-29, corresponding to 2.25 × 105 Caco-2 and 2.5 ×
104 HT-29 per insert. About 21 days are required for Caco-2 cells to
fully differentiate and form confluent and tight monolayers in cell
culture inserts.59 The media were changed every two or three days for
cell differentiation and barrier formation during the culture period.
Trans-epithelial electrical resistance (TEER) was used to further
confirm barrier formation and to test the cell monolayer health and
confluence, besides the absence of leaks before. The measurements
were performed every three days with the Millicell ERS-2 meter
(Millipore, Merck KGaA).60 Intestinal models with a TEER value of
150−300 Ω·cm2 were chosen for nanoparticle transport assays.61
Transport of f-SiO2NPs across the Intestinal Epithelium. For
transport studies, 200 μL nanoparticles (0.5 mg mL−1) in DMEM
containing 10% FBS were added to the upper compartment of the
insert and 800 μL of the medium was added to its bottom
compartment. The analyses were performed at the incubation times
of 4, 15, and 24 h. The concentration of f-SiO2NPs in samples was
measured directly for fluorescence using a Tecan Infinite M200 Pro
multimode reader (Tecan US, Inc, microplate reader). The results
were obtained from 5 batches of intestines produced on different
days, and for each batch, duplicates were made for the incubation
times of 4, 15, and 24 h. Results are displayed as mean ± standard
deviation (SD). It is noteworthy that TEER measurements were
performed to test the cell monolayer health after incubation with f-
SiO2NPs.
Confocal Microscopy. After 4, 15, and 24 h of incubation with f-
SiO2NPs, the upper and bottom compartments of the insert were
washed three times with PBS 1× and 500 μL of paraformaldehyde
(4.0% w/v) was added on each side for fixing the cells. After 1 h of
fixation, the inserts were again washed with PBS 1× and 500 μL of
permeabilization and blocking solution (0.1% (v/v) Triton X-100 and
1.0% (w/v) bovine serum albumin) was added on each side for 1 h.
Following incubation and washing inserts, the membrane with the
cells was cut out, stained with DAPI (2.5 μM, 10 min, Thermo Fisher
Scientific) to mark nucleic acids and phalloidin 488 (0.2 μM, 1 h 30
min, Thermo Fisher Scientific) to bind actin filaments, and then
washed with PBS 1×. The cells were observed under a confocal
microscope Leica TCS SP8 from the Brazilian Biosciences National
Laboratory - LNBio/CNPEM using lasers at 405 nm for DAPI, at 490
nm for phalloidin 488 and 561 nm for f-SiO2NPs.
SEM. At the end of incubation times, f-SiO2NPs were fixed for 30
min with 2.5% glutaraldehyde in cacodylate buffer (0.1 M, pH 7.2−
7.4) with calcium chloride (3 mM) and rinsed three times with wash
buffer for 5 min in an ice bath. Then, the samples were dehydrated
through a series of increasing concentrations (15, 30, 50, 70, 90, and
100%) of ethanol and dried in a desiccator. They were then sputtered
with a 5 nm thick gold coat and analyzed in an FEI Inspect
microscope (FEI Company) operated at 10−20 kV.
TEM. After incubation with f-SiO2NPs, the intestinal epithelium
samples were fixed for 30 min with 2.5% glutaraldehyde in cacodylate
buffer (0.1 M, pH 7.2−7.4) with calcium chloride (3 mM) and rinsed
three times with wash buffer for 5 min in an ice bath. Subsequently,
36032
www.acsami.org Research Article
the samples were post-fixed with 1% osmium tetroxide, 0.8%
potassium ferricyanide, 3 mM calcium chloride, and 0.1 M sodium
cacodylate buffer for 30 min on ice. After post-fixation, the sample was
washed with ultrapure water and stained with 2% uranyl acetate
overnight at 4 °C. The dehydration process was performed in 15−
100% ethanol ascending series. Then, the samples were gradually
infiltrated in EMbed812 and polymerized at 60 °C for 72 h. Finally,
the samples were cut into ultrathin sections with an ultramicrotome
and imaged in a TEM 1400-JEOL (Jeol) operating at 120 kV. The
microscope is equipped with a OneView CMOS 4 k × 4 k camera
(GATAN) for digital image acquisition.
EDS. The samples prepared for TEM imaging were also evaluated
for their chemical composition using EDS. The images were obtained
in a double-beam electron microscope Helios Nanolab 660 (Thermo
Scientific), in which an Oxford Instruments X-Maxn EDS detector is
installed. For the analysis, an acceleration voltage of 10 kV, a current
of 1.6, and a working distance of approx. 4 mm were used.
Statistical Analysis. The Shapiro−Wilk test evaluated the
normality of data. The Student t-test was used to make comparisons
between two sets of data with a normal distribution (parametric data),
while the Mann−Whitney test was used to make comparisons
between two nonparametric data sets. One-way and two-way analysis
of variance (ANOVA) and Tukey’s test were used to compare three
or more parametric data. Differences were considered significant
when p < 0.05. All statistical calculations were carried out using the R
program version 3.6.2 (R Development Core Team, 2019).
■
*
ASSOCIATED CONTENT
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsami.3c07048.
Representation of nanoparticles core−shell synthesis;
nanoparticle characterization in water (By SEM and
fluorescence measurements); description of the prep-
aration of the simulated fluids used in the in vitro
digestion experiments; nanoparticle characterization
submitted to in vitro digestion assays (by TEM and
AFM analyses); viability of Caco-2 and HT-29 cells
using Alamar Blue and LIVE/DEAD assays; and
absorption and transport of NPs across the intestinal
epithelium (by SEM, TEM, and EDS analyses) (PDF)
■ AUTHOR INFORMATION
Corresponding Author
Mateus Borba Cardoso − Institute of Chemistry (IQ),
University of Campinas (UNICAMP), Campinas, SP 13083-
970, Brazil; Brazilian Synchrotron Light Laboratory
(LNLS), Brazilian Center for Research in Energy and
Materials (CNPEM), Campinas 13083-970, Brazil;
orcid.org/0000-0003-2102-1225; Email: cardosomb@
lnls.br
Authors
Iris Renata Sousa Ribeiro − Institute of Chemistry (IQ),
University of Campinas (UNICAMP), Campinas, SP 13083-
970, Brazil; Brazilian Synchrotron Light Laboratory
(LNLS), Brazilian Center for Research in Energy and
Materials (CNPEM), Campinas 13083-970, Brazil
Raquel Frenedoso da Silva − Brazilian Synchrotron Light
Laboratory (LNLS), Brazilian Center for Research in Energy
and Materials (CNPEM), Campinas 13083-970, Brazil
Renata Santos Rabelo − Brazilian Synchrotron Light
Laboratory (LNLS), Brazilian Center for Research in Energy
and Materials (CNPEM), Campinas 13083-970, Brazil
https://doi.org/10.1021/acsami.3c07048
ACS Appl. Mater. Interfaces 2023, 15, 36025−36035
ACS Applied Materials & Interfaces
Talita Miguel Marin − Brazilian Biosciences National
Laboratory (LNBio), Brazilian Center for Research in
Energy and Materials (CNPEM), Campinas 13083-970,
Brazil; Department of Biochemistry and Tissue Biology,
Institute of Biology, University of Campinas (UNICAMP),
Campinas, SP 13083-970, Brazil
Jefferson Bettini − Brazilian Nanotechnology National
Laboratory (LNNano), Brazilian Center for Research in
Energy and Materials (CNPEM), Campinas 13083-970,
Brazil
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsami.3c07048
Author Contributions
M.B.C. directed the research. M.B.C., I.R.S.R., and R.F.S.
designed the experiments. I.R.S.R. carried out all of the
nanoparticle synthesis and characterization tests. I.R.S.R. and
R.F.S. performed all of the biological tests. T.M.M.
collaborated with the preparation of the intestinal epithelium
model. R.S.R. processed the samples for analysis by TEM,
SEM, and AFM. J.B. performed the TEM and EFTEM analyses
and processed the images. I.R.S.R., R.F.S., R.S.R., and M.B.C.
analyzed the data and wrote the manuscript with contributions
from all authors. All authors have given approval to the final
version of the manuscript.
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
This work was supported by Fundação de Amparo à Pesquisa
do Estado de São Paulo (processes 2015/25406-5, 2017/
21318-0, and 2021/12071-6). The authors acknowledge ME
laboratory of Brazilian Nanotechnology National Laboratory
(LNNano) for the use of electron microscopy facility
(proposal TEM-C2-27225, TEM-FT-28036, and SEM-FT-
28100). LNBio and Brazilian Biorenewables National Labo-
ratory (LNBr) are acknowledged for laboratory infrastructure
for biological experiments and for fluorescence measurements
(MAC-27630). The authors thank Nathalia de Carvalho
Indolfo for assistance in the preparation of the intestinal
model and Tiago M. dos Santos for trimming and slicing
samples embedded in resin to acquire TEM images. They
acknowledge Carla Pollo and Florian Meneau for ptychog-
raphy tests, Raul Freitas for nano-IR imaging, and Francesco
Lena and Helio Tolentino for fluorescent experiments done,
respectively, at Cateretê, Imbuia, and Carnaúba beamlines at
Sirius-LNLS.
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