Becker, Christian F. W.

Bialas, Friedrich

Bräuer, Birgit

Del Favero, Giorgia

Echalier, Cécile

Gerthsen, Dagmar

Grabher, Stephanie

Kamalov, Meder

Marko, Doris

Wittig, Anja

KITopen

This journal is©The Royal Society of Chemistry 2019 Chem. Commun., 2019, 55, 9649--9652 | 9649Cite this:Chem. Commun., 2019,55, 9649Silica particles with a quercetin–R5 peptideconjugate are taken up into HT-29 cells andtranslocate into the nucleus†Giorgia Del Favero, ‡a Friedrich Bialas,‡b Stephanie Grabher,a Anja Wittig,aBirgit Bra¨uer,b Dagmar Gerthsen,c Ce´cile Echalier, d Meder Kamalov, bDoris Marko *a and Christian F. W. Becker *bIntracellular delivery of bioactive polyphenols is currently evaluatedas a protective strategy for cells under pharmaceutical stress. To thisend, the 20mer R5 peptide from the marine diatom C. fusiformis wasN-terminally modified with a quercetin derivative. This polyphenol–peptide conjugate was used to generate homogeneous silica parti-cles under biomimetic conditions that are efficiently taken up byeukaryotic cells without being cytotoxic. However, not only wasaccumulation in the cytoplasm of living cells observed via electronand fluorescencemicroscopy but also translocation into the nucleus.The latter was only seen when the quercetin–peptide conjugate waspresent within the silica particles and provides a novel targetingoption for silica particles to nuclei.Delivering biological cargo into cells is a complex process thatis diﬃcult to control and recent studies have taken advantageof mesoporous silica nanoparticles as a suitable delivery tool.1,2Here we focused on a biomimetic strategy to generate silicaparticles that is based on the naturally occurring peptide R5found in the marine diatom C. fusiformis.3 This 20mer peptidegenerates, under biomimetic conditions, homogeneous sphe-rical silica particles (SiPs). Alterations to the peptide structureand sequence, e.g. by adding posttranslational modifications orby shuﬄing the amino acid sequence, can lead to diﬀerentparticle morphologies.4,5 We have previously demonstrated thatpeptide and protein cargo can be eﬃciently encapsulated intothe resulting silica particles without aﬀecting their function.6,7Together with our current findings that hydrophobic N-terminalmodifications of the R5 sequence support self-assembly of thepeptide prior to silica precipitation,8 this has led us to generate acovalent conjugate of a bioactive polyphenol, namely quercetin,with the R5 peptide (Fig. 1A–C). Quercetin is a common dietarypolyphenol, also widely used as food supplement.9 Even if well-known and characterized, thanks to its strong biological activity,quercetin is of continuously rising scientific interest.10–12For introducing a carboxylic acid linker into quercetin with-out aﬀecting its function we chose to address the 7-hydroxygroup. This was achieved by first completely acetylating quer-cetin with an excess of acetic anhydride in pyridine at 70 1C. Itwas then regioselectively deprotected with imidazole and thio-phenol in NMP as described by Kim et al. to give the 7-O-monodeacetylated product.13 Subsequently, the carboxylic acidlinker was introduced by addition of ethyl iodoacetate. Theintermediate 3 was then deprotected using hydrochloric acid inrefluxing acetone as described by Mattarei et al. (11% yield over4 steps, Fig. 1A).14 Coupling to the 20mer R5 peptide wasperformed on resin prior to cleavage and purification (Fig. 1B).The cleaved conjugate was then purified by RP-HPLC (Fig. 1C).The resulting quercetin–R5 conjugate was dissolved at 1 mgml1concentration in phosphate buffer at pH 7 and freshly generatedsilicic acid was added to give highly homogeneous silica particleswith a peptide loading of 495%. Particles were separated bycentrifugation, washed and imaged by scanning electron micro-scopy (SEM, Fig. 1D). The obtained particles were spherical witha diameter ofB400 nm. Release of the conjugate from the silicaparticles in 50 mM potassium phosphate buffer at neutral pHand pH 4 was followed by UV-VIS spectroscopy, based on theabsorbance of quercetin at 375 nm. Here, a pH-dependentrelease could be observed. At pH 4, 45% of the quercetin–R5conjugate was found in the supernatant after 5 h, whereas atneutral pH only 10% were released after 5 h and no furtherincrease in released conjugate was observed (Fig. 1F).Therefore, quercetin–R5 containing SiPs were incubated inaqueous buffer at pH 7.4 before incubation with cells for 5 h toremove loosely associated quercetin–R5. To investigate thea Institute of Food Chemistry and Toxicology, Faculty of Chemistry, University ofVienna, Waehringer Strasse 38, 1090 Vienna, Austria.E-mail: doris.marko@univie.ac.atb Institute of Biological Chemistry, Faculty of Chemistry, University of Vienna,Waehringer Strasse 38, 1090 Vienna, Austria. E-mail: christian.becker@unvie.ac.atc Laboratory for Electron Microscopy, Karlsruhe Institute of Technology (KIT),Engesserstrasse 7, 76131 Karlsruhe, Germanyd Institut des Biomole´cules Max Mousseron (IBMM), UMR5247 CNRS, ENSCM,Universite´ de Montpellier, 15 Avenue Charmes Flahault, 34093 Montpellier Cedex05, France† Electronic supplementary information (ESI) available: Experimental proce-dures, supplementary figures and tables. See DOI: 10.1039/c9cc02215e‡ Authors contributed equally.Received 20th March 2019,Accepted 16th July 2019DOI: 10.1039/c9cc02215ersc.li/chemcommChemCommCOMMUNICATIONOpen Access Article. Published on 17 July 2019. Downloaded on 9/10/2019 11:32:52 AM.  This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article OnlineView Journal  | View Issue9650 | Chem. Commun., 2019, 55, 9649--9652 This journal is©The Royal Society of Chemistry 2019morphology of the particles after release of a large fraction of theconjugate, a sample of the particles was incubated for 4.5 h in50 mM phosphate buffer at pH 4. The particles were thenimaged by SEM showing that they retained their spherical shape(Fig. 1E). The supernatant of the particles after 10 h incubationat pH 4 was also analysed by LC-MS and clearly indicated thatthe released conjugate was fully intact (Fig. S1, ESI†).To test uptake of R5-based silica particles, human colonadenocarcinoma HT-29 cells were cultivated and incubated withR5-silica particles not containing quercetin. High-angle annulardark-field scanning transmission electron microscopy (HAADF-STEM) experiments on ultrathin cell sections were performed toanalyse intact cells and locate SiPs. Biomimetic SiPs show strongbright contrast in HAADF-STEM images and appear to accumu-late at the cellular membrane already after 1 h of incubation withHT-29 cells (Fig. 2A). Accordingly, after 24 h of incubationmassive accumulation of SiPs in the intracellular compartmentbut not in the nucleus was observed (Fig. 2B). Once we verifiedthat biomimetic R5-SiPs can enter the intracellular compart-ment, a fluorescent variant was used in combination with livecell imaging to follow the kinetic of uptake at the cellular level.The fluorescein-labeled silane triethoxysilyl fluorescein wasincorporated into SiPs at a ratio of 1 to 20 to the R5 peptide.15Similarly, a progressive uptake was observed that led to accu-mulation of the fluorescent SiPs in the cytoplasm of HT-29 cellsafter 3 h (Fig. 2C and D). Cytotoxicity measurement revealed notoxicity of biomimetic SiPs in HT-29 cells (Fig. S2, ESI†).16After demonstrating uptake of R5-SiPs into HT-29 cellswithout detectable eﬀects on cell viability, we moved on toquercetin–R5-based SiPs. These SiPs behaved similarly to theR5– and fluorescein–R5-SiPs described above. Interestingly, wenoticed that the intrinsic fluorescence of the quercetin–R5peptide, even when incorporated in SiPs, was sufficient for livecell imaging (Fig. S3, ESI†). Concentration-dependent quercetinfluorescence was measured in HT-29 cells to determine theoptimal concentration for live cell imaging (Fig. S4, ESI†).Based on these results, in combination with cytotoxicity datafor R5 SiP, uptake experiments were performed at concentra-tions of 70 mM quercetin or quercetin–R5 SiP, respectively.The intrinsic fluorescence of the quercetin–R5 conjugateallowed us to study uptake without any additional labelling,which also excludes undesired effects of labels on the uptakeand targeting mechanisms. Here, uptake into HT-29 cells wasfollowed by live cell imaging and proved that already after 3 h amajor fraction of quercetin–R5 SiPs was found in cells (Fig. 3A–D).However, uptake of quercetin–R5 SiPs starts immediately afterthe application but it takes at least 90 min to reach the nucleus/peri-nuclear region (Fig. S5, ESI†). In order to verify if the uptakeof quercetin–R5-SiPs was an active process or a consequence ofan alteration of the permeability of the cell membrane, respec-tive live cell imaging experiments were performed (Fig. S6, ESI†).Confocal live imaging microscopy allowed us to follow the uptakeof quercetin–R5 SiPs and subsequent interaction with the plasmamembrane (Fig. S6, ESI†). Uptake of the quercetin–R5 SiPsFig. 1 Quercetin–R5 conjugate. (A) Synthesis of modified quercetin (11% overall yield). Conditions: (I) Ac2O, pyridine, 70 1C; (II) imidazole, PhSH, NMP,0 1C – rt; (III) DMF, Cs2CO3, ethyl iodoacetate, 0 1C rt; (IV) acetone, HCl, reflux; (B) quercetin modified R5 peptide; (C) analytical data of quercetin–R5conjugate (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 mm.(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).Communication ChemCommOpen Access Article. Published on 17 July 2019. Downloaded on 9/10/2019 11:32:52 AM.  This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.View Article OnlineThis journal is©The Royal Society of Chemistry 2019 Chem. Commun., 2019, 55, 9649--9652 | 9651occurred through active engulfment of the particles followed byrecovery of the membrane surface to its original integrity, thussuggesting the SiPs uptake to be an active, energy-dependentprocess and not a secondary effect of the particles sustained by aloss of membrane integrity. These findings are supported byuptake experiments at 4 1C and in the presence of wortmannin asan inhibitor of phagocytosis/micropinocytosis17 and after treat-ment with methyl-b-cyclodextrin (mbCD) to deplete cholesteroland induce alterations of membrane organization and fluidity(Fig. S7, ESI†).18 The uptake of quercetin–R5 SiPs was severelyreduced at 4 1C and SiPs are mainly found on the cell surfaceafter 3 h. However, as the wortmannin and mbCD treatment didnot lead to significant changes in uptake of quercetin–R5 SiPs,we can currently only exclude the related uptake mechanisms ofphagocytosis/micropinocytosis and caveolin-mediated endocyto-sis for HT-29 cells. Moreover, the quercetin-modified SiPs accu-mulated inside the nucleus (Fig. 3E and F and cross sections ofthe cell surface/central section), a behaviour not observed for anyother SiPs investigated here and rarely for silica (nano-) particlestested in other studies.19,20To control if this targeting eﬀect was accompanied by abiological eﬀect, comet assay (single cell gel electrophoresis)experiments were performed with the quercetin–R5 SiPs incomparison to the corresponding concentration of quercetin(70 mM), quercetin–R5 and R5 SiPs. Interestingly, binding ofquercetin to SiPs abolished the genotoxic effect of the uncon-jugated compound (Fig. S8, ESI†).Overall, we describe a new strategy to target biomimetic silicaparticles to the nucleus of eukaryotic cells without any detect-able eﬀects on cell viability. Covalent conjugation of the bioac-tive food constituent quercetin to a similarly safe silaﬃn-basedpeptide that is used to generate biomimetic silica, provides analmost ideal cellular shuttle. To attach cargo to this shuttle thecysteine residue oﬀers itself as an attachment point that couldbe used to release cargo via redox-mediated processes on its waythrough the cytoplasm or via more stable linkages within thenucleus.We gratefully acknowledge help from Carolin Lechner, HelgeGehrke, Georg Aichinger, Svenja Hankele and Holger Blank withsample preparation and data collection. This project has receivedfunding from the European Union’s Horizon 2020 research andinnovation programme under the Marie Sklodowska-Curie grantagreement no. 675007.Conflicts of interestThere are no conflicts to declare.Notes and references1 J. Lee, D. G. Jo, D. Park, H. Y. Chung and M. P. Mattson, Pharmacol.Rev., 2014, 66, 815–868.Fig. 2 High-angle annular dark-field scanning transmission electronmicroscopy (HAADF-STEM) images of ultrathin sections of HT-29 cellsafter 1 h (A) and 24 h (B) incubation with R5-peptide containing SiPs. SiPsshow strong bright contrast in HAADF-STEM images. Uptake was alsovisualized by live cell fluorescence measurements with fluorescein-labeledR5 SiPs at t = 0 (C) and t = 180 min. (D) The bright lines in the upper part of(A) are artifacts from ultramicrotomy used for thin-section preparation.Fig. 3 Analysis of quercetin–R5 SiP (green) uptake into HT-29 cells by livecell fluorescence imaging (cell membrane is depicted in white). Pictures areshowing 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 sectionsSURFACE level and central section of HT-29 cells (CENTER) 1 min after theapplication of SiPs (E) and at the end of the incubation (180 min (F)).ChemComm CommunicationOpen Access Article. Published on 17 July 2019. Downloaded on 9/10/2019 11:32:52 AM.  This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.View Article Online9652 | Chem. Commun., 2019, 55, 9649--9652 This journal is©The Royal Society of Chemistry 20192 A. Watermann and J. Brieger, Nanomaterials, 2017, 7, 189–205.3 R. D. Nils Kro¨ger and M. Sumper, Science, 1999, 286, 1129–1132.4 C. C. Lechner and C. F. W. Becker, Chem. Sci., 2012, 3, 3500.5 C. C. Lechner and C. F. Becker, J. Pept. Sci., 2014, 20, 152–158.6 C. C. Lechner and C. F. Becker, Bioorg. Med. Chem., 2013, 21, 3533–3541.7 C. C. Lechner and C. F. Becker, Biomater. Sci., 2015, 3, 288–297.8 M. Kamalov, A. Hajradini, C. Rentenberger and C. F. W. Becker,Mater.Lett., 2018, 212, 114–117.9 C. Chalet, J. Rubbens, J. Tack, G. S. Duchateau and P. Augustijns,J. Pharm. Pharmacol., 2018, 70, 1002–1008.10 Q. Wu, P. A. Kroon, H. Shao, P. W. Needs and X. Yang, J. Agric. FoodChem., 2018, 66, 7181–7189.11 Y. Jin, Z.-l. Huang, L. Li, Y. Yang, C.-h. Wang, Z.-t. Wang and L.-l. Ji,Acta Pharmacol. Sin., 2018, 40, 75–85.12 F. Aghapour, A. A. Moghadamnia, A. Nicolini, S. N. M. Kani,L. Barari, P. Morakabati, L. Rezazadeh and S. Kazemi, Biochem.Biophys. Res. Commun., 2018, 500, 860–865.13 M. K. Kim, H. Choo and Y. Chong, J. Med. Chem., 2014, 57, 7216–7233.14 A. Mattarei, L. Biasutto, F. Rastrelli, S. Garbisa, E. Marotta, M. Zorattiand C. Paradisi, Molecules, 2010, 15, 4722–4736.15 J. Ciccione, T. Jia, J.-L. Coll, K. Parra, M. Amblard, S. Jebors,J. Martinez, A. Mehdi and G. Subra, Chem. Mater., 2016, 28, 885–889.16 A. Wittig, H. Gehrke, G. Del Favero, E. M. Fritz, M. Al-Rawi,S. Diabate, C. Weiss, H. Sami, M. Ogris and D. 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Silica particles with a quercetin–R5 peptide conjugate are taken up into HT-29 cells and translocate into the nucleus

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

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