A Journal of Accepted Article Title: Mesoporous Silica Nanocarriers with Cyclic Peptide Gatekeeper: Specific Targeting of Aminopeptidase N and Triggered Drug Release by Stimuli-Responsive Conformational Transformation Authors: Jeonghun Lee, Eun-Taex Oh, Yeji Han, Ha Gyeong Kim, Heon Joo Park, and Chulhee Kim This manuscript has been accepted after peer review and appears as an Accepted Article online prior to editing, proofing, and formal publication of the final Version of Record (VoR). This work is currently citable by using the Digital Object Identifier (DOI) given below. The VoR will be published online in Early View as soon as possible and may be different to this Accepted Article as a result of editing. Readers should obtain the VoR from the journal website shown below when it is published to ensure accuracy of information. The authors are responsible for the content of this Accepted Article. To be cited as: Chem. Eur. J. 10.1002/chem.201704309 Link to VoR: http://dx.doi.org/10.1002/chem.201704309 Supported by 10.1002/chem.201704309 Chemistry - A European Journal COMMUNICATION Mesoporous Silica Nanocarriers with Cyclic Peptide Gatekeeper: Specific Targeting of Aminopeptidase N and Triggered Drug Release by Stimuli-Responsive Conformational Transformation Abstract: Utilizing stimuli-responsive conformational transformation of a cyclic peptide as a gatekeeper for mesoporous nanocarriers has several advantages such as facile introduction of targeting capabilities, low enzymatic degradation during blood circulation and enhanced specific binding to selected cells. In this report, a NGRcontaining dual functional cyclic peptide gatekeeper on the surface of mesoporous nanocarrier is prepared not only for active targeting of the aminopeptidase N (APN) expressed on cancer cells but also stimuli-responsive intracellular drug release triggered by a GSHinduced conformational transformation of the peptide gatekeeper. The peptide gatekeeper on the surface of nanocarriers exhibits onoff gatekeeping via conformational transformation triggered by intracellular glutathione of the cancer cells. H1299 cells (high APN expression) showed greater uptake of the nanocarrier by endocytosis and higher apoptosis than A549 cells (low APN expression). Reducing the side effects of anticancer chemotherapy can increase its therapeutic efficacy. Among many approaches in such reduction, use of smart delivery carriers containing anticancer drug is of great interest due to its fascinating approach such as targeted delivery and on-site drug release.[1-3] Many delivery carriers have been investigated, including liposomes, polymer micelles, dendrimers, proteins, and inorganic nanoparticles.[1, 4-7] In particular, mesoporous silica nanocarriers (MSNs) have attracted considerable attention because of their benefits, which include ease of surface modification, chemical stability, adjustable size, low cytotoxicity and high surface area and pore volume for drug loading.[8-14] Moreover, MSNs with gatekeepers such as cyclodextrins, cucurbiturils, polymers, dendrimers, inorganic nanoparticles and peptides could achieve the zero release characteristics during blood circulation, thereby reducing undesirable side effects at off-target sites.[15-16] Furthermore, proper design of the [a] [b] [c] [+] Dr. J. Lee, Y. Han, Prof. Dr. C. Kim Department of Polymer Science and Engineering Inha University Yonghyun-dong, Nam-gu, Incheon 22212, Korea E-mail: firstname.lastname@example.org Dr. E.-T. Oh Department of Biomedical Sciences, School of Medicine Inha University Yonghyun-dong, Nam-gu, Incheon 22212, Korea H. G. Kim, Prof. Dr. H. J. Park Department of Microbiology, Hypoxia-related Disease Research Center, College of Medicine Inha University Yonghyun-dong, Nam-gu, Incheon 22212, Korea E-mail: email@example.com These Authors contributed equally to this work. Supporting information for this article is given via a link at the end of the document. gatekeeper structure enables the release of drugs entrapped in the mesopores of MSNs in response to stimuli such as pH, light, enzymes and redox potential.[11, 17-28] Notably, using peptides as gatekeepers of MSNs has several advantages, including high biocompatibility, specific active targeting of the desired cells, enhanced endosomal escape and/or the use of enzymatic degradation as a specific stimulus for the triggered release of entrapped drugs. [11, 24-27, 29] Recently, we developed a cyclic peptide gatekeeper with the capability of triggered drug release by stimuli-responsive conformational conversion.[26-27, 30] Based on the difference of the gatekeeping capability of the peptide with a turn and a random conformation on the surface of MSNs, we developed a stimuliresponsive peptide gatekeeper with an intramolecular disulfide bond that caused the peptide with a random structure to take on a turn structure. MSNs with this peptide gatekeeper (FmocCGGC-SS-Si) retained drug molecules in their mesopores. Upon addition of glutathione (GSH), a biological reductase upregulated in various cancer cells,[31-33] the intramolecular disulfide bond of the peptide gatekeeper was reduced, resulting in the conformational conversion of the peptide from a turn to a random structure and releasing the entrapped guests. Then, we reported the MSN (PEG-WCGKC-SS-Si) with optimized sequence of the peptide gatekeeper for enhanced biocompatibility. In response to intracellular GSH-induced conformational transformation, PEG-WCGKC-SS-Si selectively released entrapped drugs in A549 human lung cancer cells (which express high GSH level) in a controlled manner but not in CCD normal lung cells (which express low GSH level). Utilizing stimuli-responsive conformational transformation of a cyclic peptide as a gatekeeper for MSNs has several advantages. First, targeting capabilities could be easily introduced into an on-off cyclic gatekeeper by inserting appropriate peptide sequences, such as RGD or NGR, between the two cysteine units. These sequences can act as ligands that selectively target certain cells that contain receptors that bind these ligands maintaining the capability of stimuli-responsive intracellular drug release. Second, because many enzymes have a lower ability to degrade cyclic than linear peptides, nanocarriers with cyclic peptide gatekeepers can circulate for longer periods in the blood, while retaining their targeting capability.[34-35] Third, in several cases such as RGD and NGR, cyclic structure of the peptide targeting ligand enhances the specific binding to selected cells via their specific receptors than linear one.[34, 36-38] Therefore, as a proof of concept, we here demonstrate the dual function of MSN (PEG-WKCNGRC-SS-Si) with the NGR between the two cysteine units of the cyclic peptide gatekeeper not only for active targeting of the cancer cells but also stimuliresponsive intracellular drug release triggered by the GSH- This article is protected by copyright. All rights reserved. Accepted Manuscript Jeonghun Lee+,[a] Eun-Taex Oh+,[b] Yeji Han,[a] Ha Gyeong Kim,[c] Heon Joo Park,*[c] and Chulhee Kim*[a] 10.1002/chem.201704309 Chemistry - A European Journal induced conformational transformation of the peptide gatekeeper, as shown in Figure 1. The NGR peptide can specifically bind to the aminopeptidase N (APN, also known as CD13), a receptor overexpressed in tumour endothelial cells and in various tumour cell lines.[37-40] Cyclic NGR has shown stronger affinity and higher specificity towards APN receptors than linear NGR.[37-38] Furthermore, cyclic peptides generally exhibit greater resistance to enzymatic degradation than linear peptides,[34-35] Therefore, the introduction of the cyclic CNGRC peptide as a gatekeeper onto the surface of mesoporous nanocarriers would result in an effective drug carrier with greater targeting capacity and therapeutic efficacy via increased intratumoral uptake.[34-36, 41] reaction, generating DOX-loaded MSNs with a peptide gatekeeper (WKCNGRC-SS-Si). The peptide with azide functionality (WKCNGRC-N3) was prepared by a solid-phase peptide synthesis method using Fmoc-chemistry (see Figure S3 and the experimental section for details). The intramolecular disulfide bond was introduced by oxygen bubbling into a solution of WKCNGRC-N3 in acetonitrile/water (50:50 v/v), forming the cyclic peptide (WKCNGRC-SS-N3). The presence of the peptide gatekeeper on the surface of MSNs (WKCNGRC-SS-Si) was confirmed by FT-IR (amide II near 1554 cm−1), as shown in Figure S2. Based on the 5,5’-dithio-bis-(2-nitrobenzoic acid) (DTNB) titration of thiol units after reduction of the disulfide bond on the surface of WKCNGRC-SS-Si using GSH, the weight percentage of the peptide units on the surface of MSN was 1.8 wt%. Figure 2. Release profiles of DOX from WKCNGRC-SS-Si (a) and CNGRCSS-Si (b) in PBS buffer (pH = 7.4, 30 mM). The final concentration of GSH was 0.1 mM and the concentration of the nanocarrier was 0.1 mg/mL. Figure 1. Schematic representation of the MSNs with peptide gatekeepers for targeting APN and drug release in response to GSH-induced conformational transformation. Conditions: i) 3-aminopropyltriethoxysilane; ii) propargyl bromide; iii) surfactant removal, DOX loading, WKCNGRC-SS-N3, sodium ascorbate, copper (II) sulfate; iv) PEG-NCO. To investigate the gatekeeping characteristics and targeting capacity of the MSN (PEG-WKCNGRC-SS-Si) with this peptide gatekeeper, we prepared MCM-41-type MSNs with a diameter of 80 nm and hexagonally ordered pores as described previously.[26-27] The Barrett-Joyner-Halenda (BJH) pore size distribution analysis (Figure S1) showed that the average diameter of the mesopore was 2.5 nm. Next, we modified the surface of MSNs with 3-aminopropyltriethoxysilane by a sol–gel reaction to introduce amino groups (Si-NH2), which was confirmed by Fourier transform infrared (FT-IR) spectroscopy (N–H bend absorption at 1490 cm−1, as shown in Figure S2) and a positive zeta potential (+11.54 mV). Then, alkyne groups were introduced onto the surface of MSNs (Si-alkyne) by reacting the Si-NH2 with propargyl bromide. The introduction of the alkyne group was confirmed by FT-IR (alkyne stretching band at 2133 Figure S2). After removing the surfactant, cm−1, cetyltrimethylammonium bromide, from the mesopores of Sialkyne using ammonium nitrate in ethanol, we loaded the guest anticancer drug (doxorubicin, DOX) by immersing the nanoparticles in a DMF solution of DOX. Finally, the cyclic peptide with an azide group at its C-terminal (WKCNGRC-SSN3) was conjugated onto the surface of Si-alkyne by a click The gatekeeping abilities of the peptide gatekeepers with linear (WKCNGRC) and cyclic (WKCNGRC-SS) structures were investigated by monitoring the change in the photoluminescence (PL) intensity of DOX entrapped in the mesopores of MSNs in phosphate-buffered saline (PBS, pH 7.4). In the absence of any stimulus, DOX in WKCNGRC-SS-Si was retained within the mesopores of MSNs for more than 700 min (Figure 2a). Upon the addition of GSH, a reducing agent expressed at high levels in various cancer cells, the DOX entrapped in WKCNGRC-SS-Si was released over time. These results indicate that GSH triggered the structural transformation of the peptide gatekeeper, from a cyclic structure (WKCNGRC-SS) with gatekeeping ability to a linear structure (WKCNGRC) lacking gatekeeping ability. Therefore, the peptide gatekeeper (WKCNGRC-SS) could be utilized as a stimuli-responsive gatekeeper on the surface of MSNs. To investigate the effect of the tryptophan moiety at the end of the peptide gatekeeper, the CNGRC-SS-N3 peptide was synthesized and MSNs with the peptide (CNGRC-SS-Si) were prepared as described in the experimental section of the Supporting Information. The entrapped DOX in CNGRC-SS-Si was released in the absence of any external stimulus (Figure 2b). This result indicates that the cyclic CNGRC sequence itself does not have gatekeeping ability, and that the tryptophan unit of WKCNGRC-SS-Si is important, along with the conformational transformation, for on-off gatekeeping. Because the terminal tryptophan unit has a bulky indole ring, the steric hindrance induced by tryptophan units would interfere the transport of the drug molecules from the mesopore to the aqueous media. This article is protected by copyright. All rights reserved. Accepted Manuscript COMMUNICATION 10.1002/chem.201704309 Chemistry - A European Journal COMMUNICATION results demonstrate that the NGR sequence on the surface of MSNs increased the cellular uptake of MSNs by targeting high levels of cell surface APN in cancer cells. Figure 4. APN-mediated cellular uptake of silica nanoparticles (PEGWKCNGRC-SS-Si) and intracellular release of DOX from MSNs in APNexpressing cancer cells. CLSM images were taken showing the time course of DOX fluorescence intensity in H1299 and A549 cells treated with PEGWKCNGRC-SS-Si for 12 hr. Figure 3. a) TEM images of PEG-WKCNGRC-SS-Si. b) Release profile of DOX from PEG-WKCNGRC-SS-Si in PBS buffer. The final concentration of GSH was 0.1 mM and the concentration of the nanocarrier was 0.1 mg/mL. H1299 and A549 cells have been shown to express high and low levels of APN, respectively. Similarly, we found that APN was overexpressed in H1299, but not in A549 cells (Figure S6). We therefore investigated APN-mediated cellular uptake by PEG-WKCNGRC-SS-Si and intracellular GSH-induced release of DOX from PEG-WKCNGRC-SS-Si in H1299 and A549 cells. The nucleus is the primary site of cytotoxic action of DOX, where it intercalates into DNA, forming DNA adducts and inhibiting topoisomerase II. Therefore, the release of DOX from PEGWKCNGRC-SS-Si in the cells was determined by confocal laser scanning microscopy (CLSM). CLSM images showed that the fluorescence intensity of DOX increased in H1299 and A549 cells incubated for 12 h with PEG-WKCNGRC-SS-Si loaded with 3 μM DOX (Figure 4). Marked accumulation of DOX was observed in the nuclei of H1299, but not of A549 cells. To confirm the intracellular release of DOX from PEG-WKCNGRCSS-Si and the accumulation of DOX in the nuclei, the nucleic acids of these cells were stained with DAPI (4’,6’-diamidino-2phenyl-indole). DOX and DAPI fluorescence overlapped in H1299 cells, but not in A549 cells (Figure 5). Collectively, these Next, we investigated the ability of PEG-WKCNGRC-SS-Si to kill target cancer cells expressing high levels of surface APN. Treatment of both H1299 and A549 cells with free DOX for 4 hr decreased their clonogenic survival (Figure 6). Incubation with PEG-WKCNGRC-SS-Si loaded with 3 μM DOX for 4 hr reduced the surviving fractions of H1299 and A549 cells to 1.9 × 10-4 and 4.7 × 10-3, respectively (Figure 6). To confirm these results, we investigated the ability of PEG-WKCNGRC-SS-Si loaded with 3 μM DOX to induce apoptosis using TUNEL (terminal deoxytransferase-mediated dUTP-biotin nick-end labeling) assays. PEG-WKCNGRC-SS-Si loaded with 3 μM DOX induced significant apoptosis in H1299 cells, whereas it induced negligible apoptosis in A549 cells (Figures 7 and 8). In these results, PEG-WKCNGRC-SS-Si loaded with 3 μM DOX showed significant clonogenic cell death (Figure 6) and acute cell death (Figure 7 and 8) in HT1299 cells expressing high APN. In A549 cells expressing low APN, it showed significant clonogenic cell death compared to the control (Figure 6). However, it showed negligible acute apoptosis in TUNEL assay. A previous report demonstrated that clonogenic survival assay is a realistic model to determine the effect of drugs on proliferating cancer cells. This assay is time-consuming to set up and analyse. Another report described that a high concentration (~μM) of doxorubicin causes an apoptotic cancer cell death after 1 day of doxorubicin This article is protected by copyright. All rights reserved. Accepted Manuscript To investigate the in vitro stimuli-responsive drug-release and targeting properties of the nanocarrier, we enhanced its biocompatibility and dispersion stability by modifying the amine group of DOX-loaded WKCNGRC-SS-Si using monomethoxy poly(ethylene glycol) isocyanate (PEG-NCO; MW 2000) (see experimental section for details). After PEGylation, the zeta potential value was changed from +17.05 to -22.54 mV, and the hydrodynamic radius was changed from 235 nm to 107 nm as shown in Figure S4. TEM images showed that the mesoporous nature and diameter of PEG-WKCNGRC-SS-Si were preserved during surface modification (Figure 3a and Figure S5). In addition, gatekeeping ability was maintained after PEGylation (Figure 3b). In the absence of an external stimulus, DOX in PEG-WKCNGRC-SS-Si was not released over 700 min in PBS buffer. Upon addition of GSH, DOX in the pores was released due to structural transformation of the peptide from a cyclic to a linear conformation. These results indicate that the stimuliresponsive gatekeeping capacity of WKCNGRC-SS was maintained even after functionalization of the peptide gatekeeper with PEG. Based on the total released amounts of DOX for the MSN, the loading percent of DOX in PEGWKCNGRC-SS-Si was 2.5 wt%. 10.1002/chem.201704309 Chemistry - A European Journal COMMUNICATION Figure 6. Clonogenic survival of H1299 and A549 cells treated for 4 hr with 3 μM DOX or PEG-WKCNGRC-SS-Si alone or loaded with 3 μM DOX. The cells were subsequently washed three times with PBS and cultured for an additional 14 days, and the proportions of surviving cells were calculated. *P < 0.05. Figure 5. APN-mediated cellular uptake of PEG-WKCNGRC-SS-Si and intracellular release of DOX from MSNs in APN-expressing cancer cells. H1299 and A549 cells were treated with PEG-WKCNGRC-SS-Si for 4 hr, were fixed with 4% PFA, washed three times with PBS, and stained with DAPI. The fluorescence intensities of DOX and DAPI-stained nuclei were examined using a TE2000E laser-scanning confocal microscope. Figure 7. Representative photomicrographs of apoptotic cells (i.e., TUNELpositive cells). H1299 and A549 cells were treated with 3 μM DOX or PEGWKCNGRC-SS-Si, alone or loaded with 3 μM DOX, for 12 hr. TUNEL-positive cells were detected by confocal microscopy. This article is protected by copyright. All rights reserved. Accepted Manuscript treatment, while a low concentration (~nM) of doxorubicin causes a senescence like cancer cell death after 6 days of doxorubicin treatment. Therefore, little intracellular uptake of PEG-WKCNGRC-SS-Si loaded with 3 μM DOX by endocytosis caused significant clonogenic cell death in A549 cells expressing low APN in clonogenic survival assay (Figure 6), while it did not cause acute DNA damage in TUNEL assay (Figure 7 and 8). These results indicate that the APN-targeting ligand of the peptide gatekeeper plays a central role in the cellular uptake of MSNs in cancer cells expressing high surface levels of APN and their resultant DOX-induced cell death. 10.1002/chem.201704309 Chemistry - A European Journal COMMUNICATION  J. A. Barreto, W. O’Malley, M. Kubeil, B. Graham, H. Stephan, L. Spiccia, Adv. Mater. 2011, 23, H18-H40.  P. G. Corrie, Medicine (Baltimore). 2008, 36, 24-28.  R. A. Petros, J. M. DeSimone, Nat. Rev. Drug Discov. 2010, 9, 615-627.  V. Bagalkot, L. Zhang, E. Levy-Nissenbaum, S. Jon, P. W. Kantoff, R. Langer, O. C. Farokhzad, Nano Lett. 2007, 7, 3065-3070.  C. Vauthier, K. Bouchemal, Pharm. Res. 2008, 26, 1025-1058.  N. Nasongkla, E. Bey, J. Ren, H. Ai, C. Khemtong, J. S. Guthi, S.-F. Chin, A. D. Sherry, D. A. Boothman, J. Gao, Nano Lett. 2006, 6, 2427-2430.  Khatib, J. I. Zink, N. M. Khashab, J. F. Stoddart, Nanoscale 2009, 1, 16-39.  C. T. Kresge, M. E. Leonowicz, W. J. Roth, J. C. Vartuli, J. S. Beck, Nature 1992, 359, 710-712.  E. Aznar, M. Oroval, L. Pascual, J. R. n. Murguía, R. n. Martínez-Máñez, F. l. Sancenón, Chem. Rev. 2016, 116, 561-718. In conclusion, we prepared a dual functional MSN (PEGWKCNGRC-SS-Si) with a cyclic peptide gatekeeper containing the NGR sequence not only for active targeting of the APN expressed on cancer cells but also stimuli-responsive intracellular drug release triggered by a GSH-induced conformational transformation of the peptide gatekeeper. In the absence of any stimuli, WKCNGRC-SS-Si showed zero-release property over 700 min. Upon addition of GSH, a bioreductase upregulated in various cancer cells, the intramolecular disulfide bond of the gatekeeper was reduced to two thiol units, resulting in the conformational transformation of the peptide gatekeeper from a cyclic to a linear conformation, and the release of DOX in WKCNGRC-SS-Si. After PEGylation to for enhanced dispersion stability and biocompatibility of the nanocarrier (PEGWKCNGRC-SS-Si), the stimuli-responsive conformational transformation of the peptide gatekeeper and the resulting triggered release was preserved. H1299 cells, with high APN expression, showed greater uptake of the nanocarrier by endocytosis than A549 cells, with low APN expression. Moreover, the therapeutic efficacy of DOX-loaded nanocarriers, as shown by cell apoptosis and cleavage of caspase 3 and PARP, was greater in H1299 than in A549 cells, due to the ability of the cyclic NGR sequence in the peptide gatekeeper to target APN. Therefore, our strategy using dual functional stimuliresponsive peptide gatekeepers would be valuable to prepare mesoporous nanocarriers with enhanced therapeutic efficacy by targeting cancer cells.  S. Mura, J. Nicolas, P. Couvreur, Nat. Mater. 2013, 12, 991-1003.  C. Coll, A. Bernardos, R. Martínez-Máñez, F. Sancenón, Acc. Chem. Res. 2013,  I. I. Slowing, J. L. Vivero-Escoto, C.-W. Wu, V. S. Y. Lin, Adv. Drug Del. Rev. 46, 339-349. 2008, 60, 1278-1288.  J. L. Vivero-Escoto, I. I. Slowing, B. G. Trewyn, V. S. Y. Lin, Small 2010, 6, 1952-1967.  F. Tang, L. Li, D. Chen, Adv. Mater. 2012, 24, 1504-1534.  Y.-W. Yang, Med. Chem. Commun. 2011, 2, 1033-1049.  M. Liong, J. Lu, M. Kovochich, T. Xia, S. G. Ruehm, A. E. Nel, F. Tamanoi, J. I. Zink, ACS Nano 2008, 2, 889-896.  Y.-Z. You, K. K. Kalebaila, S. L. Brock, Chem. Mater. 2008, 20, 3354-3359.  J. L. Vivero-Escoto, I. I. Slowing, C.-W. Wu, V. S. Y. Lin, J. Am. Chem. Soc. 2009, 131, 3462-3463.  C. Park, H. Kim, S. Kim, C. Kim, J. Am. Chem. Soc. 2009, 131, 16614-16615.  H. Kim, S. Kim, C. Park, H. Lee, H. J. Park, C. Kim, Adv. Mater. 2010, 22, 4280-4283.  J. Lee, H. Kim, S. Kim, H. Lee, J. Kim, N. Kim, H. J. Park, E. K. Choi, J. S. Lee, C. Kim, J. Mater. Chem. 2012, 22, 14061-14067.  J. Zhang, Z.-F. Yuan, Y. Wang, W.-H. Chen, G.-F. Luo, S.-X. Cheng, R.-X. Zhuo, X.-Z. Zhang, J. Am. Chem. Soc. 2013, 135, 5068-5073.  D. M. Copolovici, K. Langel, E. Eriste, Ü. Langel, ACS Nano 2014, 8, 19721994.  P. D. Thornton, A. Heise, J. Am. Chem. Soc. 2010, 132, 2024-2028.  C. Coll, L. Mondragón, R. Martínez-Máñez, F. Sancenón, M. D. Marcos, J. Soto, P. Amorós, E. Pérez-Payá, Angew. Chem. Int. Ed. 2011, 50, 2138-2140.  J. Lee, H. Kim, S. Han, E. Hong, K.-H. Lee, C. Kim, J. Am. Chem. Soc. 2014, 136, 12880-12883.  Acknowledgements J. Lee, E.-T. Oh, H. Yoon, H. Kim, H. J. Park, C. Kim, Nanoscale 2016, 8, 8070-8077. C. K. thanks the National Research Foundation of Korea (NRF) (NRF-2015M2B2B1068625 and NRF-2016R1D1A1B03930953) for support. H.J.P. also thanks NRF (NRF-2014R1A5A2009392 and NRF-2015M2B2B1068623 (NRF-2015M2B2B1068599)) for support. Keywords: peptides • gatekeepers • conformational transformation • stimuli-responsiveness • mesoporous silica nanocarriers  J. Lee, H. Kim, C. Kim, Macromol. Res. 2016, 24, 478-481.  C. d. l. Torre, A. Agostini, L. Mondragón, M. Orzáez, F. Sancenón, R. Martínez-Máñez, M. D. Marcos, P. Amorós, E. Pérez-Payá, Chem. Commun. 2014, 50, 3184-3186.  J. Lee, S. Han, J. Lee, M. Choi, C. Kim, New J. Chem. 2017, 41, 6969-6972.  G. K. Balendiran, R. Dabur, D. Fraser, Cell Biochem. Funct. 2004, 22, 343-352.  J. M. Estrela, A. Ortega, E. Obrador, Crit. Rev. Clin. Lab. Sci. 2006, 43, 143181.  H. Liu, H. Wang, S. Shenvi, T. M. Hagen, R.-M. Liu, Ann. N.Y. Acad. Sci. 2004, 1019, 346-349. This article is protected by copyright. All rights reserved. Accepted Manuscript Figure 8. Ratio of TUNEL-positive to DAPI-stained H1299 and A549 cells treated with 3 μM DOX or PEG-WKCNGRC-SS-Si, alone or loaded with 3 μM DOX, for 12 hr. Data are expressed as the mean ± standard deviation of three independent experiments. Columns represent the compiled data derived from five independent experiments. ****P < 0.0001 K. K. Cotí, M. E. Belowich, M. Liong, M. W. Ambrogio, Y. A. Lau, H. A. 10.1002/chem.201704309 Chemistry - A European Journal COMMUNICATION  F. Danhier, A. L. Breton, V. Préat, Mol. Pharm. 2012, 9, 2961-2973.  K. Temming, R. M. Schiffelers, G. Molema, R. J. Kok, Drug Resist. Updates  R. A. Ashmun, L. H. Shapiro, W. Arap, E. Ruoslahti, Cancer Res. 2000, 60, 722-727. 2005, 8, 381-402.   S. Verrier, S. Pallu, R. Bareille, A. Jonczyk, J. Meyer, M. Dard, J. Amédée,  S. Liu, Mol. Pharm. 2006, 3, 472-487. Biomaterials 2002, 23, 585-596.  S. Akita, N. Hattori, T. Masuda, Y. Horimasu, T. Nakashima, H. Iwamoto, K. D. Majhen, J. Gabrilovac, M. Eloit, J. Richardson, A. Ambriović-Ristov, Biochem. Biophys. Res. Commun. 2006, 348, 278-287.  Fujitaka, M. Miyake, N. Kohno, Cancer Sci. 2015, 106, 921-928.  K. M. Laginha, S. Verwoert, G. J. R. Charrois, T. M. Allen, Clin. Cancer Res. 2005, 11, 6944-6949. G. Colombo, F. Curnis, G. M. S. De Mori, A. Gasparri, C. Longoni, A. Sacchi, R. Longhi, A. Corti, J. Biol. Chem. 2002, 277, 47891-47897.  R. M. Hoffman, J. Clin. Lab. Anal. 1991, 5, 133-143. M. Wickström, R. Larsson, P. Nygren, J. Gullbo, Cancer Sci. 2011, 102, 501-  Y.-W. Eom, M. A. Kim, S. S. Park, M. J. Goo, H. J. Kwon, S. Sohn, W.-H. 508. Kim, G. Yoon, K. S. Choi, Oncogene 2005, 24, 4765-4777. Accepted Manuscript  R. Pasqualini, E. Koivunen, R. Kain, J. Lahdenranta, M. Sakamoto, A. Stryhn, This article is protected by copyright. All rights reserved. 10.1002/chem.201704309 Chemistry - A European Journal COMMUNICATION Entry for the Table of Contents COMMUNICATION J. Lee, E.-T. Oh, Y. Han, H. G. Kim, H. J. Park*, C. Kim* Page No. – Page No. Mesoporous Silica Nanocarriers with Cyclic Peptide Gatekeeper: Specific Targeting of Aminopeptidase N and Triggered Drug Release by StimuliResponsive Conformational Transformation This article is protected by copyright. All rights reserved. Accepted Manuscript A NGR-containing dual functional cyclic peptide gatekeeper on the surface of mesoporous nanocarrier is prepared not only for active targeting of the aminopeptidase N expressed on cancer cells but also stimuliresponsive intracellular drug release triggered by a glutathione-induced conformational transformation of the peptide gatekeeper.