Silica particles with a quercetin-R5 peptide conjugate are taken up into HT-29 cells and translocate into the nucleus

Article abstract of DOI:10.1039/c9cc02215e

Intracellular delivery of bioactive polyphenols is currently evaluated as a protective strategy for cells under pharmaceutical stress. To this end, the 20mer R5 peptide from the marine diatom C. fusiformis was N-terminally modified with a quercetin deriva

Full text of DOI:10.1039/c9cc02215e

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Silica particles with a Quercetin-R5 peptide conjugate are taken
up into HT-29 cells and translocate into the nucleus
Giorgia Del Favero#a, Friedrich Bialas#b, Stephanie Grabher,a Anja Wittiga, Birgit Bräuerb, Dagmar
iReceived 00th January 20xx,
Accepted 00th January 20xx
Gerthsenc, Cécile Echalierd, Meder Kamalovb, Doris Marko*a and Christian F.W. Becker*b
DOI: 10.1039/x0xx00000x
www.rsc.org/
7
Intracellular delivery of bioactive polyphenols is currently their function.6, Together with our current findings that
evaluated as a protective strategy for cells under pharmaceutical hydrophobic N-terminal modifications of the R5 sequence
stress. To this end, the 20mer R5 peptide from the marine diatom support self-assembly of the peptide prior to silica
C. fusiformis was N-terminally modified with a quercetin derivative. precipitation,8 this has led us to generate a covalent conjugate
This polyphenol-peptide conjugate was used to generate of a bioactive polyphenol, namely quercetin, with the R5
homogeneous silica particles under biomimetic conditions that are peptide (Figure 1 A-C). Quercetin is a common dietary
efficiently taken up by eukaryotic cells without being cytotoxic. polyphenol, also widely used as food supplement.9 Even if well-
However, not only accumulation in the cytoplasm of living cells was known and characterized, thanks to its strong biological activity,
observed via electron and fluorescence microscopy but also quercetin is of continuously rising scientific interest.10-12
translocation into the nucleus. The latter was only seen when the
quercetin-peptide conjugate was present within the silica particles For introducing a carboxylic acid linker into quercetin without
and provides a novel targeting option for silica particles to nuclei. affecting its function we chose to address the 7-hydroxy group.
Controls with quercetin- or peptide-only and particles carrying no This was achieved by first completely acetylating quercetin with
cargo only entered the cytoplasm with much lower efficiency.
an excess of acetic anhydride in pyridine at 70 °C. It was then
regioselectively deprotected with imidazole and thiophenol in
NMP as described by Kim et al. to give the 7-O-
monodeacetylated product.13 Subsequently, the carboxylic acid
linker was introduced by addition of ethyl iodoacetate. The
intermediate 3 was then deprotected using hydrochloric acid in
refluxing acetone as described by Mattarei et al. (11% yield over
4 steps, Figure 1A).14 Coupling to the 20mer R5 peptide was
performed on resin prior to cleavage and purification (Figure
1B). The cleaved conjugate was then purified by RP-HPLC
(Figure 1C). The resulting quercetin-R5 conjugate was dissolved
at 1 mg/ml concentration in phosphate buffer at pH 7 and
freshly generated silicic acid was added to give highly
homogeneous silica particles with a peptide loading of >95%.
Particles were separated by centrifugation, washed and imaged
by scanning electron microscopy (SEM, Figure 1D). The obtained
particles were spherical with a diameter of ~400 nm. Release of
the conjugate from the silica particles in 50 mM potassium
phosphate buffer at neutral pH and pH 4 was followed by UV-
VIS spectroscopy, based on the absorbance of quercetin at 375
nm. Here, a pH-dependent release could be observed. At pH 4,
45% of the quercetin-R5 conjugate was found in the
supernatant after 5 h, whereas at neutral pH only 10% were
released after 5 h and no further increase in released conjugate
was observed (Figure F).
Delivering biological cargo into cells is a complex process that is
difficult to control and recent studies have taken advantage of
mesoporous silica nanoparticles as a suitable delivery tool.
1,2
Here we focused on a biomimetic strategy to generate silica
particles that is based on the naturally occurring peptide R5
found in the marine diatom C. fusiformis.3 This 20mer peptide
generates, under biomimetic conditions, homogeneous
spherical silica particles (SiPs). Alterations to the peptide
structure and sequence, e.g. by adding posttranslational
modifications or by shuffling the amino acid sequence, can lead
5
to different particle morphologies.4, We have previously
demonstrated that peptide and protein cargo can be efficiently
encapsulated into the resulting silica particles without affecting
a. Institute of Food Chemistry and Toxicology, Faculty of Chemistry, University of
Vienna, Waehringer Strasse 38, 1090 Vienna, Austria
b. Institute of Biological Chemistry, Faculty of Chemistry, University of Vienna,
Waehringer Strasse 38, 1090 Vienna, Austria
c. Laboratory for Electron Microscopy, Karlsruhe Institute of Technology (KIT),
Engesserstrasse 7, 76131 Karlsruhe, Germany
d. Institut des Biomolécules Max Mousseron (IBMM), UMR5247 CNRS, ENSCM,
Université de Montpellier, 15 avenue Charmes Flahault, 34093 Montpellier Cedex
05, France
# Authors contributed equally
† Electronic Supplementary Information (ESI) available: Experimental procedures,
supplementary figures and tables. See DOI: 10.1039/x0xx00000x
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Figure 1. Quercetin-R5 conjugate. A) Synthesis of modified quercetin (11 % overall yield). Conditions: I) Ac2O, pyridine, 70 °C; II) imidazole, PhSH, NMP, 0 °C - rt; III)
DMF, Cs2CO3, ethyl iodoacetate, 0 °C rt; IV) acetone, HCl, reflux; B) Quercetin modified R5 peptide; C) Analytical data of quercetin-R5 conjugate (RP-HPLC, ESI-MS) and
SEM image of resulting silica particles before (D) and after (E) the release of the conjugate at pH 4, scale bars: 1 µm. F) Release of quercetin-R5 conjugate from silica
particles followed by absorbance of the supernatant at 375 nm at pH 7 (red) and at pH 4 (black).
Therefore, quercetin-R5 containing SiPs were incubated in observed that led to accumulation of the fluorescent SiPs in the
aqueous buffer at pH 7.4 before incubation with cells for 5 h to cytoplasm of HT-29 cells after 3 h (Figure 2 C and D). Cytotoxicity
remove loosely associated quercetin-R5. To investigate the measurement revealed no toxicity of biomimetic SiPs in HT-29
morphology of the particles after release of a large fraction of cells (Figure S2).16
the conjugate, a sample of the particles was incubated for 4.5 h After demonstrating uptake of R5-SiPs into HT-29 cells without
in 50 mM phosphate buffer at pH 4. The particles were then detectable effects on cell viability, we moved on to quercetin-
imaged by SEM showing that they retained their spherical shape R5-based SiPs. These SiPs behaved similarly to the R5- and
(Figure 1E). The supernatant of the particles after 10 h fluorescein-R5-SiPs described above. Interestingly, we noticed
incubation at pH 4 was also analysed by LC-MS and clearly that the intrinsic fluorescence of the quercetin-R5 peptide, even
indicated that the released conjugate was fully intact (Figure when incorporated in SiPs, was sufficient for live cell imaging
S1).
(Figure S3). Concentration-dependent quercetin fluorescence
To test uptake of R5-based silica particles, human colon was measured in HT-29 cells to determine the optimal
adenocarcinoma HT-29 cells were cultivated and incubated concentration for live cell imaging (Figure S4). Based on these
with R5-silica particles not containing quercetin. High-angle results, in combination with cytotoxicity data for R5 SiP, uptake
annular dark-field scanning transmission electron microscopy experiments were performed at concentrations of 70 µM
(HAADF-STEM) experiments on ultrathin cell sections were quercetin or quercetin-R5 SiP, respectively.
performed to analyse intact cells and locate SiPs. Biomimetica The intrinsic fluorescence of the quercetin-R5 conjugate
SiPs show strong bright contrast in HAADF-STEM images and allowed us to study uptake without any additional labelling,
appear to accumulate at the cellular membrane already after 1h which also excludes undesired effects of labels on the uptake
of incubation with HT-29 cells (Figure 2A). Accordingly, after 24h and targeting mechanisms. Here, uptake into HT-29 cells was
of incubation massive accumulation of SiPs in the intracellular followed by live cell imaging and proved that already after 3 h a
compartment but not in the nucleus was observed (Figure 2B). major fraction of quercetin-R5 SiPs was found in cells (Figure
Once we verified that biomimetic R5-SiPs can enter the 3A-D). However, uptake of quercetin-R5 SiPs starts immediately
intracellular compartment, a fluorescent variant was used in after the application but it takes at least 90 min to reach the
combination with live cell imaging to follow the kinetic of nucleus/peri-nuclear region (Figure S5). In order to verify if the
uptake at the cellular level. The fluorescein-labeled silane uptake of quercetin-R5-SiPs was an active process or a
triethoxysilyl fluorescein was incorporated into SiPs at a ratio of consequence of an alteration of the permeability of the cell
1 to 20 to the R5 peptide.15 Similarly, a progressive uptake was membrane, respective live cell imaging experiments were
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performed (Figure S6). Confocal live imaging microscopy quercetin to SiPs abolished the genotoxic effect of the
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DOI: 10.1039/C9CC02215E
allowed us to follow the uptake of quercetin-R5 SiPs and unconjugated compound (Figure S8).
subsequent interaction with the plasma membrane (Figure S6). Overall, we describe a new strategy to target biomimetic silica
Uptake of the quercetin-R5 SiPs occurred through active particles to the nucleus of eukaryotic cells without any
engulfment of the particles followed by recovery of the detectable effects on cell viability. Covalent conjugation of the
membrane surface to its original integrity, thus suggesting the bioactive food constituent quercetin to a similarly safe silaffin-
SiPs uptake to be an active, energy-dependent process and not based peptide that is used to generate biomimetic silica,
a secondary effect of the particles sustained by a loss of provides an almost ideal cellular shuttle. To attach cargo to this
membrane integrity. These findings are supported by uptake shuttle the cysteine residue offers itself as an attachment point
experiments at 4°C and in the presence of wortmannin as an that could be used to release cargo via redox-mediated
inhibitor of phagocytosis/ micropinocytosis17 and after processes on its way through the cytoplasm or via more stable
treatment with methyl-β-cyclodextrin (mβCD) to deplete linkages within the nucleus.
cholesterol and induce alterations of membrane organization
and fluidity (Figure S7).18 The uptake of quercetin-R5 SiPs was
severely reduced at 4°C and SiPs are mainly found on the cell
surface after 3 h. However, as the wortmannin and mβCD
treatment did not lead to significant changes in uptake of
quercetin-R5 SiPs, we can currently only exclude the related
uptake mechanisms of phagocytosis/ micropinocytosis and
caveolin-mediated endocytosis for HT-29 cells. Moreover, the
quercetin-modified SiPs accumulated inside the nucleus (Figure
3E, F and cross sections of the cell surface central section), a
behaviour not observed for any other SiPs investigated here and
rarely for silica (nano-) particles tested in other studies.19, 20
Figure 3: Analysis of quercetin-R5 SiP (green) uptake into HT-29 cells by live cell
fluorescence imaging (cell membrane is depicted in white). Pictures are showing
the time course of uptake (A) 1 min; (B) 60 min. (C) 120 min. (D) 180 min.
Appearance of the 3D reconstruction and of the cross sections SURFACE level and
central section of HT-29 cells (CENTER) 1 min after the application of SiPs (E) and
at the end of the incubation (180 min. F).
Conflicts of interest
There are no conflicts to declare.
Figure 2: High-angle annular dark-field scanning transmission electron microscopy
(HAADF-STEM) images of ultrathin sections of HT-29 cells after 1 h (A) and 24 h (B)
incubation with R5-peptide containing SiPs. SiPs show strong bright contrast in
HAADF-STEM images. Uptake was also visualized by life cell fluorescence
measurements with fluorescein-labeled R5 SiPs at t= 0 (C) and t = 180 min. (D).
Notes and references
We gratefully acknowledge help from Carolin Lechner, Helge
Gehrke, Georg Aichinger, Svenja Hankele and Holger Blank with
The bright lines in the upper part of (A) are artifacts from ultramicrotomy used for
thin-section preparation.
To control if this targeting effect was accompanied by a
biological effect, comet assay (single cell gel electrophoresis)
experiments were performed with the quercetin-R5 SiPs in
comparison to the corresponding concentration of quercetin
(70 μM), quercetin-R5 and R5 SiPs. Interestingly, binding of
sample preparation and data collection. This project has received
funding from the European Union’s Horizon 2020 research and
innovation programme under the Marie Sklodowska-Curie grant
agreement No 675007.
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TOC Figure
Particles generated by biomimetic silica precipitation with a quercetin-R5 peptide enter the
nucleus of HT-29 cells without inducing toxicity.