Pitstop 2

Uptake Pathway of Apple-derived Nanoparticle by Intestinal
Cells to Deliver its Cargo
Mayumi Arai1 & Hisakazu Komori 1 & Daichi Fujita 1 & Ikumi Tamai1
Received: 11 January 2021 /Accepted: 17 February 2021
# The Author(s), under exclusive licence to Springer Science+Business Media, LLC part of Springer Nature 2021
Purpose Food-derived nanoparticles exert cytoprotective
effects on intestinal cells by delivering their cargo, which
includes macromolecules such as microRNAs and proteins,
as well as low-molecular weight compounds. We previously
reported that apple-derived nanoparticles (APNPs) downre￾gulate the expression of human intestinal transporter
OATP2B1/SLCO2B1 mRNA. To verify the involvement of
the cargo of APNPs in affecting the expression of transporters,
we characterized the uptake mechanism of APNPs in intesti￾nal cells.
Methods The uptake of fluorescent PKH26-labeled APNPs
(PKH-APNPs) into Caco-2, LS180, and HT-29MTX cells
was evaluated by confocal microscopy and flow cytometry.
Results The uptake of PKH-APNPs was prevented in the
presence of clathrin-dependent endocytosis inhibitors, chlor￾promazine and Pitstop2. Furthermore, PKH-APNPs were in￾corporated by the HT29-MTX cells, despite the disturbance
of the mucus layer. Additionally, the decrease in SLCO2B1
mRNA by APNPs was reversed by Pitstop 2 in Caco-2 cells,
indicating that APNPs decrease SLCO2B1 by being incorpo￾rated via clathrin-dependent endocytosis.
Conclusions We demonstrated that clathrin-dependent en￾docytosis was mainly involved in the uptake of APNPs by
intestinal cells, and that the cargo in the APNPs downregulate
the mRNA expression of SLCO2B1. Therefore, APNPs could
be a useful tool to deliver large molecules such as microRNAs
to intestinal cells.
KEY WORDS apple-derived nanoparticles .
clathrin-dependent endocytosis . food-drug interaction . intestinal
uptake . OATP2B1
APNPs Apple-derived nanoparticles
miRNA microRNA
NPs Nanoparticles
OATP2B1 Organic anion-transporting polypeptide 2B1
PKH-APNPs PKH26-labeled APNPs
Over the last few decades, food-drug interactions have been
reported to affect the intestinal absorption of clinically used
drugs (1,2). The clinically relevant interaction of dihydropyri￾midines, such as felodipine, with grapefruit juice on the intesti￾nal cytochrome P-450 enzyme is due to the inclusion of fura￾nocoumarins, which cause a mechanism-based inhibition (3,4).
The reduced bioavailability of fexofenadine via interaction
with fruit juices is explained by the inhibition of the organic
anion-transporting polypeptide 2B1 (OATP2B1) transporter
by naringin, hesperidin, and several other small molecules in
apple, grapefruit, and orange juices as inhibitors of OATP2B1
(5–7). Thus, it has been demonstrated that small molecules in
fruit juices could directly affect intestinal transporters and drug
metabolizing enzymes.
In recent years, food-derived nanoparticles (NPs) have
been discovered as food components that affect gastrointesti￾nal function. NPs are vesicle-like particles composed of a lipid
bilayer (8,9), with an average particle size of approximately
100 to 400 nm and a negative zeta potential (−49.2 to
1.52 mV) (10). Food-derived NPs contain macromolecules
* Ikumi Tamai
[email protected]
1 Faculty of Pharmaceutical Sciences, Institute of Medical, Pharmaceutical
and Health Sciences, Kanazawa University, Kakuma-machi,
Kanazawa 920-1192, Japan
Pharm Res (2021) 38:523–530
/Published online: 15 March 2021
such as proteins and nucleic acids, as well as low-molecular
weight compounds (11,12). It has also been reported that NPs
isolated or prepared from foods such as ginger, grape, grape￾fruit, lemon, and milk have biological functions in vitro and
in vivo (13–18). Thus, edible food can reach the intestinal
lumen and food-derived NPs may exert their biological effects
through macromolecules contained in NPs as cargo, since NPs
can protect the degradation of macromolecules in the intesti￾nal lumen and facilitate cellular uptake (10,15).
We recently reported that apple-derived nanoparticles
(APNPs) reduced the mRNA expression of intestinal trans￾porters, including OATP2B1/SLCO2B1, apical sodium￾dependent bile acid transporter (ASBT/SLC10A2), and carni￾tine transporter (OCTN2/SLC22A5) in the human intestinal
epithelial cell line Caco-2 (19). In this study, it was indicated
that a decrease in the expression of OATP2B1 was mediated
by microRNA (miRNA) derived from apples. This suggests
that apple miRNA is taken up by the cells via APNPs as cargo,
since naked miRNA is considered difficult to deliver into cells
due to their instability in the gut lumen and low membrane
permeability. However, the characteristics of the incorpora￾tion pathway of APNPs by Caco-2 cells have not yet been fully
demonstrated. Therefore, characterization of APNP uptake
by intestinal cells is required to clarify the postulated mecha￾nism by which APNPs regulate the expression of intestinal
transporters. Therefore, in the present study, we aimed to
characterize the mechanism of APNP uptake by intestinal
cells. In this study, the incorporation of APNPs into intestinal
cell lines was evaluated using the fluorescent dye PKH26,
which labels the lipid bilayer of APNPs (PKH-APNPs), to
clarify the endocytosis pathway by which the APNPs are in￾corporated into cells. Furthermore, the involvement of the
endocytic pathway in the downregulation of SLCO2B1
mRNA by APNPs was verified in Caco-2 cells.
Apples (Mallus pumila, Sun Fuji) were purchased from
Sawaguchi Farm (Morioka, Japan). The PKH26 labeling kit,
Pitstop 2, and amiloride were purchased from Sigma-Aldrich
(St. Louis, MO). Alexa-Fluor-488-conjugated WGA, Hoechst
33342, and FBS were obtained from Thermo Fisher Scientific
Inc. (Waltham, MA). Chlorpromazine and indomethacin
were purchased from Fuji Film Wako Pure Chemical
Corporation (Osaka, Japan). Filipin III and cyotochalasin D
were purchased from Cayman Chemical Company (Ann
Arbor, MI). RNAiso Plus and M-MLV reverse transcriptase
were purchased from Takara Bio Inc. (Shiga, Japan) and
Promega (Madison, WI), respectively. Brilliant III Ultra-Fast
SYBR Green QPCR Master Mix, Cellmatrix® collagen, and
Bradford protein assay reagents were purchased from Agilent
Technologies (Santa Clara, CA), Nitta Gelatin (Tokyo,
Japan), and Bio-Rad (Hercules, CA), respectively. All other
chemicals were of reagent grade or of the highest purity com￾mercially available.
Cell Culture
Caco-2 and LS180 cells were obtained from the RIKEN Cell
Bank (Tsukuba, Japan) and HT29-MTX E12 cells were
obtained from the Health Science Research Resources Bank
(Osaka, Japan). All cells were cultured in Dulbecco’s modified
Eagle’s medium (25 mM glucose) supplemented with 10%
FBS, 0.1 mM nonessential amino acids, benzylpenicillin
(100 U/mL), and streptomycin (100 μg/mL). Caco-2 cells
were used after a 3-day culture, while in some experiments,
they were cultured for 14 days on a collagen-coated plate.
When 14-day culture Caco-2 cells were used, they were indi￾cated in the results of each experiment. HT29-MTX E12 cells
were cultured on collagen-coated plates for two weeks to fa￾cilitate the secretion of mucus.
Isolation and Purification of APNPs
APNPs were prepared according to our previous reports (19).
Briefly, whole apples were washed with water for 10 min and
crushed with a grater. The supernatant obtained after centri￾fugation of the crushed apples at 2000×g at 4°C for 20 min
was then centrifuged at 13,000×g at 4°C for 70 min to exclude
debris. The final supernatant was then centrifuged at
120,000×g for 130 min, and the resulting pellet was used as
the NP fraction after resuspension in phosphate-buffered sa￾line (PBS). The concentration of APNPs was presented as a
protein concentration measured with a Bio-Rad protein
quantification assay kit, using bovine serum albumin (BSA)
as the standard.
Fluorescence Labeling of APNPs
The lipid membranes of the APNPs were labeled with PKH26
according to the manufacturer’s instruction (Sigma-Aldrich).
Briefly, APNPs were incubated with PKH26 at 37°C for
10 min. To terminate the labeling reaction, an equal volume
of 1% BSA in PBS was added. Unlabeled fluorescent dye was
removed by centrifugation at 120,000×g for 90 min, then the
labeled NP pellet was suspended in cell culture medium. The
concentration of the obtained PKH26-labeled APNPs (PKH￾APNPs) was determined by measuring the protein concentra￾tion with a Bio-Rad protein quantification assay kit, using
BSA as the standard.
524 Pharm Res (2021) 38:523–530
Confocal Microscopic Analysis of APNP Uptake
After incubation of the PKH-APNPs with cells at 37°C for
the indicated times, the Caco-2 and HT29-MTX cells were
washed with PBS. The cells were fixed in 4% paraformal￾dehyde solution at room temperature, washed with PBS,
and then incubated with 2 μg/mL Alexa-Fluor-488-
conjugated WGA to label the cell membrane markers.
Nuclei were counterstained with 2 μg/mL Hoechst
33342. Fluorescence images were captured with an
LSM710 confocal laser microscope (Carl Zeiss,
Oberkochen, Germany).
Flow Cytometry Analysis of APNP Uptake
Caco-2 cells and LS180 cells were incubated with PKH￾APNPs at 37°C for 12 or 24 h. To remove the mucus from
HT29-MTX E12 cells, 10 mg/mL N-acetylcysteine was
added and incubated with the cells at 37°C for 1 h.
Subsequently, the HT29-MTX E12 cells were incubated with
PKH-APNPs at 37°C for 12 h. The PKH-APNPs in the mu￾cus layer were then removed by shaking the plate at 700 rpm
for 10 min and the spent medium was replaced with fresh
medium. After three rounds of removal, the cells were washed
with PBS and harvested after incubation with 0.1% trypsin for
5 min. After washing with PBS, the cells were subjected to flow
cytometry. Analysis was performed using a BD FACSVerse™
flow cytometer (BD Bioscience, Franklin Lakes, MA). To
quantify the amount of PKH-APNPs taken up, the geometric
mean was used as the mean fluorescence intensity. Untreated
cells were used as a negative control and their fluorescence
was subtracted as the background value.
Evaluation of APNPs Uptake in the Mouse
Gastrointestinal Tract
This animal study was performed the requirement of the
Kanazawa University Institutional Animal Care and Use
Committee (permit number AP-163753). Slc; ICR mice
(8 weeks old, male) were purchased from Sankyo Labo
Service Corporation (Tokyo, Japan). Mice were orally ad￾ministered PKH-APNPs (1.0 mg/mouse). After 2 h, mice
were anesthetized and euthanized by cervical dislocation to
collect small intestine. The isolated tissues were washed
with PBS and frozen using OCT compound (Sakura
Finetek Japan, Tokyo, Japan). The frozen block was sliced
a thickness of 10 μm to prepare a tissue section. After drying
for 2 h, frozen sections washed with PBS and stained nuclei
with 2 μg/mL Hoechst 33342. Fluorescence images were
captured with an LSM710 confocal laser microscope (Carl
Quantitative Real-Time PCR
Total RNA was prepared from Caco-2 cells using RNAiso
Plus then reverse-transcribed to cDNA using M-MLV reverse
transcriptase (Promega, Madison, WI). Quantitative real-time
RT-PCR analysis of SLCO2B1 (Fw: 5’-TTCTTTGC
TCATTACA-3′) was performed in an Mx3000P QPCR sys￾tem (Agilent Technologies, Santa Clara, CA) using Brilliant
III Ultra-Fast SYBR Green QPCR Master Mix (Agilent
Technologies). The fold changes in these genes were normal￾ized to HPRT, and relative mRNA expression was analyzed
by the 2−ΔΔCT method.
Data Analysis
All data are expressed as means ± SEM unless otherwise noted,
and statistical analyses were performed using Student’s t test,
with p < 0.05 as the criterion of significance.
Uptake of APNPs by Intestinal Cell Lines
To evaluate the uptake of APNPs in human intestinal cells,
PKH-APNPs were added to the incubation medium of Caco-
2 cells cultured for 3 days and were detected using flow cytom￾etry. The fluorescence mean of PKH26 increased with the
incubation time (Fig. 1a) and concentration of PKH-APNPs
(Fig. 1b). Moreover, the uptake of 20 μg/mL PKH-APNPs
was visually evaluated by confocal microscopy after 24 h.
The fluorescence of PKH-APNPs was observed at the in￾tracellular locus and was found to increase with incubation
time (Fig. 1c). Thus, it was shown that APNPs were incor￾porated into Caco-2 cells. PKH-APNP uptake was also an￾alyzed in the human colon adenocarcinoma cell line LS180
(Fig. 1d). The mean fluorescence intensity increased pro￾portional to the treatment time of PKH-APNPs. This result
suggested that PKH-APNPs were also incorporated by
LS180 cells. Furthermore, uptake was measured in Caco-
2 cells cultured for 14 days, which are already differentiated
into cells like the intestinal epithelium. The uptake of PKH￾APNPs increased in a time-dependent manner, similar to
that observed in Fig. 1a. Furthermore, the incorporation of
PKH-APNPs was also observed in intestinal villi, when
mice were orally administrated PKH-APNPs (Fig. 1e).
Thus, it was demonstrated that APNPs were incorporated
into human intestinal epithelial cells.
Pharm Res (2021) 38:523–530 525
Effect of the Mucus Layer on the Uptake of APNPs
It was considered that orally administrated APNPs cross the
mucus layer before being incorporated by intestinal epithelial
cells in vivo. The mucus layer is composed of mucins pro￾duced from goblet cells and acts as a physical barrier by
covering the intestinal epithelium surface. Therefore, we ex￾amined whether APNPs were taken up even in the presence of
a mucus layer using HT29-MTX cells. These cells exhibit a
differentiated goblet cell-like phenotype, secreting low
amounts of MUC2 mucins predominantly expressed in the
small and large intestine (20). Quantitative analysis using flow
PKH-APNP exposure me (h)
Untreated 2 h 6 h 12 h 24 h
PKH-APNP concentraon (μg/mL)
PKH-APNP exposure me (h)
cMean fluorescent intensity
PKH-APNP exposure me (h)
Mean fluorescent intensity
Mean fluorescent intensity
300 μm
Mean fluorescent intensity
Fig. 1 Uptake of APNPs by
intestinal cell lines (a, c, d) Caco-2
cells (a), LS180 cells (c), and intes￾tinal epithelial-like differentiated
Caco-2 cells (d) were treated with
20 μg/mL PKH-APNPs and ana￾lyzed by flow cytometry. (a) PKH￾APNPs were treated for 6 h. Each
bar represents the mean ± SEM
(n = 3). (b) After 3 days culture,
Caco-2 cells were incubated with
20 μg/mL PKH-APNPs (red) and
PBS for 24 h at 37°C. The nuclei
and cell membranes were stained
with Hoechst 33342 (blue) and
Alexa-Fluor-488-conjugated WGA
(green), respectively. Lower panels
shows merged images. Upper
panels represent PKH26 shown in
white as a pseudo color. (e) Slc;ICR
mice were orally administrated
PKH-APNPs (1.0 mg/mouse). After
2 h, the small intestine was isolated
and frozen tissue section was
stained Nuclei with Hoechst
33342. Tissue section was ob￾served with a fluorescence micro￾scope. Arrowheads indicate the
PKH-APNPs accumulation.
526 Pharm Res (2021) 38:523–530
cytometry showed that the incorporation of PKH-APNPs was
increased in HT29-MTX cells in a time-dependent manner
(Fig. 2a). In addition, the fluorescence of PKH-APNPs was
observed in the intracellular compartment by confocal micros￾copy (Fig. 2b). Furthermore, to confirm the influence of the
mucus layer, APNP uptake was evaluated in HT29-MTX
cells in the presence or absence of mucus. When the mucus
layer was removed by pre-treatment with N-acetylcysteine, an
enhanced uptake of APNPs was observed (Fig. 2c). This result
indicates that the mucus layer functions as a barrier to APNP
uptake by intestinal epithelial cells. Taken together, APNPs
are taken up by intestinal epithelial cells despite the mucus
layer barrier.
Cell Incorporation of APNPs Via Endocytosis
To investigate the mechanism of uptake of APNPs by Caco-2
cells, the contribution of each endocytic pathway was exam￾ined. The uptake of PKH-APNPs was assessed in the presence
of chlorpromazine and Pitstop 2 as clathrin-dependent endo￾cytosis inhibitors, indomethacin and filipin III as caveolae￾mediated endocytosis inhibitors, and amiloride and cytocha￾lasin D as macropinocytosis inhibitors (13,15,21). It was con￾firmed that the positive controls for the endocytosis pathways
prevented the uptake of different molecules at the indicated
concentration of each inhibitor (Fig. 3a). The fluorescence
intensity of PKH-APNPs as measured by flow cytometry is
shown in Fig. 3. The mean fluorescence intensity was signifi￾cantly decreased after treatment with chlorpromazine and
Pitstop 2, while inhibitors of caveolae-mediated endocytosis
and macropinocytosis did not affect the incorporation of
APNPs into Caco-2 cells (Fig. 3b). Similarly, the uptake of
PKH-APNPs by LS180 cells (Fig. 3c) and Caco-2 cells cul￾tured for 14 days (Fig. 3d) were significantly reduced by treat￾ment with chlorpromazine or Pitstop 2. Thus, it was
demonstrated that APNPs were taken up by intestinal epithe￾lial cells via clathrin-dependent endocytosis.
Involvement of Clathrin-Mediated Endocytosis
in the Downregulation of SLCO2B1 by APNPs
We previously reported that APNPs suppressed the expression
of SLCO2B1 mRNA in Caco-2 cells (19). Therefore, we veri￾fied that such downregulation of SLCO2B1 was associated
with the incorporation of APNPs into Caco-2 cells via
clathrin-dependent endocytosis. Similar to our previous re￾port, the mRNA expression of SLCO2B1 was significantly de￾creased by treatment with APNPs for 48 h in the absence of
Pitstop 2 (Fig. 4a). When the cells were treated with 1 μM
Pitstop 2, which repressed the incorporation of APNPs by
Caco-2 cells (Fig. 4b), the mRNA expression of SLCO2B1
was significantly reduced (Fig. 4a). After treatment with
0.5 μM Pitstop 2, the uptake of APNPs and the expression
of SLCO2B1 mRNA were not significantly altered, although
they showed similar results to those obtained upon treatment
of 1 μM Pitstop 2 (Fig. 4a and b). Consequently, it was sug￾gested that the APNP-induced downregulation of SLCO2B1
mRNA was triggered by the incorporation of APNPs via
clathrin-dependent endocytosis.
Food-derived NPs have attracted attention as a tool for the oral
administration of macromolecular drugs. We suggest that
miRNAs contained in APNPs as cargo can regulate the expres￾sion of transporters in Caco-2 cells (19), which is a novel mech￾anism of drug-food interactions on intestinal transporters.
Here, we evaluated the incorporation of APNPs into several
intestinal cell lines to demonstrate the direct effect of food￾derived macromolecules through nanoparticles in food.
APNP Mean fluorescent intensity
Fig. 2 Effect of mucus on the incorporation of APNPs into intestinal cells (a) HT29-MTX cells were treated with the PKH-APNPs at a concentration of 20 μg/mL.
Uptake of PKH-APNPs was evaluated using flow cytometry. (b) HT29-MTX cells were incubated with 20 μg/mL PKH-APNPs and PBS for 12 h. The nuclei and
cell membranes were stained with Hoechst 33342 and Alexa-Fluor-488-conjugated WGA, respectively. (c) HT29-MTX cells were cultured with 20 μg/mL PKH￾APNPs for 24 h after the removal of mucus by N-acetylcysteine, and subsequently analyzed via flow cytometry. Each bar represents the mean ± SEM (n = 3).
Pharm Res (2021) 38:523–530 527
Our results showed that clathrin-dependent endocytosis
was involved in the incorporation of APNPs by intestinal ep￾ithelial cells (Fig. 3), while caveolin-dependent endocytosis or
macropinocytosis was not observed. It has been suggested that
several food-derived NPs are mainly taken up by cells via the
endocytic pathway (22). NPs isolated from grapefruit were
reported to be incorporated into RAW264 cells, mediated
by macropinocytosis and clathrin-dependent endocytosis
(15). However, in A549 cells, which are derived from human
alveolar epithelial adenocarcinoma, NPs prepared from
grapefruit were incorporated via phagocytosis and clathrin￾dependent endocytosis (21). Thus, the endocytosis pathway
in which food-derived NPs are taken up may differ depending
on the type of cells. Our results showed that in both Caco-2
and LS180 cells, which are intestinal cell lines, incorporated
APNPs through clathrin-dependent endocytosis. Therefore,
the clathrin-dependent endocytosis of APNPs in Caco-2 cells
and LS180 cells presented here may reflect the interaction
between intestinal epithelial cells and APNPs in vivo.
Generally, it is known that the endocytic pathway involved
depends on the particle size. Clathrin-dependent endocytosis
is regarded to occur in NPs with particle sizes below 300 nm
(23), while caveolae-mediated endocytosis is expected to hap￾pen to particles with sizes about 60–80 nm. Indeed, it has been
Fig. 3 Potential pathways utilized
by APNPs to enter intestinal cells.
Caco-2 cells (a and b), LS180 cells
(c), and differentiated Caco-2 cells
(d) were pre-incubated with chlor￾promazine (25 μM) and Pitstop 2
(20 μM), filipinIII (50 μM) and in￾domethacin (25 μM), and amiloride
(25 μM) and cytochalasin D
(10 μM), as clathrin-dependent en￾docytosis inhibitors, caveolae￾mediated endocytosis inhibitors,
and macropinocytosis inhibitors,
respectively. After 1 h, Fluorescently
labeled transferrin (10 μg/mL),
cholera toxin B subunit (0.5 μg/mL),
and dextran (0.5 mg/mL) were
used as positive controls taken up
via clathrin-dependent endocytosis,
caveolae-mediated endocytosis,
and micropinocytosis, respectively
(a). PKH-APNPs were treated at
37°C for 12 h (b and c) or 24 h (d).
Each bar represents the mean ±
SEM (n = 3).
p<0.05 p<0.05
Cytochalasin D
Mean fluorescent intensity
(% of untreated group)
Pitstop 2
p<0.05 p<0.05 Chlorpromazine Pitstop 2 DMSO
Mean fluorescent intensity
(% of untreated group)
Mean fluorescent intensity
(% of untreated group)
Pitstop 2
p = 0.06
Transferrin uptake
(% of untreated group)
Cholera toxin B subunit uptake
(% of untreated group)
Dextran uptake
(% of untreated group)
p < 0.05 120
Cytochalasin D
Pitstop 2
528 Pharm Res (2021) 38:523–530
reported that beads with a diameter < 200 nm tend to be
preferentially taken up via clathrin-dependent endocytosis in
B16 cells, which are mouse melanoma cells (24). On the other
hand, large particles are most likely to be engulfed via macro￾pinocytosis. Thus, such information is consistent with the fact
that the average particle size of APNPs is 170 nm, mainly in
the range of 100–200 nm (19). However, our preliminary
result showed that the average size of APNP was decreased
by approximately 20 nm after passing through the mimic
gastrointestinal fluid. Therefore, some of digested APNPs in
gastrointestinal fluids might be also incorporated into
caveolin-mediated endocytosis.
NPs endocytosed into cells are generally thought to mature
as early endosomes and are eventually degraded by lysosomes
via late endosomes (22). Therefore, in order for the apple
miRNAs in the APNPs that have been taken up to show any
biological effect, they need to escape from the endosomes to
the cytoplasm during endosome maturation. As shown in Fig.
3, since APNPs were incorporated into cells via clathrin￾dependent endocytosis whose cargo decreased the mRNA ex￾pression of SLCO2B1, it is possible that some miRNAs or other
macromolecules in the APNPs escaped the endosomes and
affected gene silencing. In clathrin-dependent endocytosis,
when the ligand binds to the receptor, the particles are cov￾ered with clathrin coat pits and taken up into cells (25), so
certain proteins on the membrane of APNPs may exist as
ligands. That ligand is likely to bind to specific receptors on
the cell surface membrane and cause clathrin-dependent en￾docytosis. If such a ligand located on the surface of APNPs and
its receptor on the cell membrane can be identified, APNPs
can then be used as a tool to deliver nucleic acids specifically
and efficiently to target cells.
In our previous study, it was shown that APNP treatment
reduced the mRNA expression of SLCO2B1 in Caco-2 cells,
and that the 3’-UTR of the SLCO2B1 gene was essential for
the reduction of SLCO2B1 mRNA expression by APNPs, sug￾gesting that miRNAs contained in the APNPs reduced the
mRNA expression of SLCO2B1 in Caco-2 cells (19).
Therefore, we clarified the relationship between the downre￾gulation of SLCO2B1 mRNA by APNPs and the incorpora￾tion of APNPs. As shown in Fig. 4, inhibition of clathrin￾dependent endocytosis reversed the decrease in mRNA ex￾pression of SLCO2B1 by APNPs. Thus, the decrease in
mRNA expression of SLCO2B1 after APNP treatment can
be explained by the clathrin-dependent endocytosis of
APNPs. In addition, we have previously shown that the trans￾port activity of the SLCO2B1 substrate estrone 3-sulfate was
reduced by APNPs treatment (19). It was also shown that the
APNPs fraction did not contain low molecular weight com￾pounds such as phloridzin and phloretin at concentrations
that inhibit SLCO2B1. Therefore, we concluded that the de￾crease of SLCO2B1 mRNA expression by APNPs treatment is
not due to the low molecular weight compounds contained in
the APNPs fraction. These results strongly support our hy￾pothesis that the miRNA contained in APNPs affects the ex￾pression of transporters in the intestine.
If orally administered APNPs are taken up by human
intestinal cells and miRNAs exert physiological functions,
they need to cross the mucus layer while maintaining the
shape of the particles in the gastrointestinal environment.
Despite the presence of a mucus layer, APNPs were taken
up by HT-29MTX cells, even though the incorporation of
APNPs was reduced by the mucus (Fig. 3). We previously
reported that APNPs were present in an intact form at
pH 1.0, and maintained a particle size distribution of about
200 nm (19). This suggests that orally administered APNPs
maintain their particle shape even in gastric juice. In fact,
our results showed that orally administrated APNPs were
taken up by intestinal epithelial cells in mice (Fig. 1e).
Therefore, it is expected that orally administrated APNPs
are incorporated into intestinal cells in vivo, and that
miRNAs in APNPs can affect intestinal function. In another
report, rice-derived miRNAs have been reported to be tak￾en up by intestinal tissues after ingestion of rice (26). In
Pitstop 2 (μM)
Relave SLCO2B1
mRNA expression
1.5 p<0.05
Pitstop 2 (μM) 0 0.5 1.0
Fig. 4 Effect of the inhibition of clathrin-dependent endocytosis on decreasing the mRNA expression of SLCO2B1 by APNPs. After pre-incubation with each
inhibitor for clathrin-dependent endocytosis for 1 h, APNPs (a) or PKH-APNP (b) were incubated at 37°C for 48 h. The mRNA expression of SLCO2B1 was
examined via qRT-PCR (a). Incorporated PKH-APNPs were evaluated using flow cytometry (b). Each bar represents the mean ± SEM (n = 3).
Pharm Res (2021) 38:523–530 529
addition, NPs isolated from edible plants such as grapefruit,
ginger, and carrot have been reported to be incorporated
into intestinal tissue (17). Thus, it was considered that var￾ious food-derived NPs were incorporated into the intestine
in vivo. In addition to other food-derived NPs, APNPs can
also be incorporated into intestinal cells in vivo.
The present study demonstrated that clathrin-dependent en￾docytosis was mainly involved in the uptake of APNPs by
intestinal epithelial cells such as Caco-2, and this uptake
mechanism contributed significantly to the downregulation
of the mRNA expression of intestinal transporters such as
OATP2B1. This study also provides novel knowledge that
large food-derived molecules can directly affect intestinal ep￾ithelial cellular function through food-derived NPs.
supported by a Grant-in-Aid for Scientific Research (B)
[16H05111] and a Grant-in-Aid for Challenging
Exploratory Research [20 K21474] from the Japan Society
for the Promotion of Science (JSPS). The authors declare no
conflicts of financial interest.
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