DOI: 10.1002/chem.201404382
Communication
& Mesoporous Materials
Cathepsin-B Induced Controlled Release from Peptide-Capped Mesoporous Silica Nanoparticles Cristina de la Torre,[a, b, c] Laura Mondragn,[d] Carmen Coll,[a, b, c] Flix Sancenn,[a, b, c] Mara D. Marcos,[a, b, c] Ramn Martnez-MÇez,*[a, b, c] Pedro Amors,[e] Enrique Prez-Pay†,[f] and Mar Orzez*[f] Abstract: New capped silica mesoporous nanoparticles for intracellular controlled cargo release within cathepsin B expressing cells are described. Nanometric mesoporous MCM-41 supports loaded with safranin O (S1-P) or doxorubicin (S2-P) containing a molecular gate based on a cathepsin B target peptidic sequence were synthesized. Solids were designed to show “zero delivery” and to display cargo release in the presence of cathepsin B enzyme, which selectively hydrolyzed in vitro the capping peptide sequence. Controlled delivery in HeLa, MEFs WT, and MEFs lacking cathepsin B cell lines were also tested. Release of safranin O and doxorubicin in these cells took place when cathepsin B was active or present. Cells treated with S2-P showed a fall in cell viability due to nanoparticles internalization, cathepsin B hydrolysis of the capping peptide, and cytotoxic agent delivery, proving the possible use of these nanodevices as new therapeutic tools for cancer treatment.
In the treatment of cancer, chemotherapy constitutes the main therapeutic approach. Traditional chemotherapeutic agents target cells that divide rapidly, one of the main characteristics of cancer cells. However, this also means that cells belonging to healthy tissues that divide quickly are also targeted and destroyed constituting one of the main drawbacks of chemotherapy. To diminish these side effects, several strategies have been developed, among them: 1) the development of chemotherapeutic derivatives and analogues with less toxic effects
[a] C. de la Torre,+ C. Coll, F. Sancenn, M. D. Marcos, R. Martnez-MÇez Centro de Reconocimiento Molecular y Desarrollo Tecnolgico (IDM) Unidad Mixta Universidad Politcnica de ValenciaUniversidad de Valencia (Spain) [b] C. de la Torre,+ C. Coll, F. Sancenn, M. D. Marcos, R. Martnez-MÇez Departamento de Qumica, Universidad Politcnica de Valencia. Camino de Vera s/n, 46022, Valenicia (Spain) E-mail: [email protected] [c] C. de la Torre,+ C. Coll, F. Sancenn, M. D. Marcos, R. Martnez-MÇez CIBER de Bioingenieria, Biomateriales y Nanomedicina (CIBER-BBN) (Spain)
and better pharmacological properties or 2) the design of new drug delivery systems for better targeting of cancer cells. The arrival of nanotechnology has fuelled the latter approach and has brought about new innovative concepts to drug-delivery therapies.[1] Specifically attention has been put on drug-delivery systems capable of releasing active molecules to certain cells in a controlled fashion.[2] Among the different drug-delivery materials developed till present, silica mesoporous supports (SMPS) have undergone an exponential growth as carriers for drug storage and delivery in recent years, thanks to their unique properties, such as large loading capacity, low toxicity, and easy functionalization.[3] Notwithstanding, the most appealing feature of SMPS as carriers is, perhaps, the possibility of functionalizing them with molecular/supramolecular ensembles in their external surface to develop gated-SMPS, which show “zero delivery” and can release their cargo on-command in response to specifically designed external stimuli.[4] Since the first example of gated SMPS developed by Fujiwara’s laboratory,[5] a certain number of capped supports have been created responding to a wide variety of stimuli, such as light,[6] redox reactions,[7] pH,[8] changes in polarity,[9] temperature[10] etc. However, the use of biomolecules[11] as triggers provided the ultimate proof that SMPS may applied to more biological and realistic settings. In this context, the possibility of using enzymes as “biological-keys” has opened a wide range of new perspectives for the development of biocompatible gated SMPS carriers and this approach is envisioned to have a large potential to provide exquisite selectivity in the design of advanced gate-opening devices.[12] This development is reinforced by the cellular internalization mechanism of nanoparti[e] P. Amors Institut de Cincia dels Materials (ICMUV), Universitat de Valncia P.O. Box 2085, 46071 Valencia (Spain) [f] E. Prez-Pay, M. Orzez Laboratorio Pptidos y Protenas. Centro de Investigacin Prncipe Felipe., C/Eduardo Primo Yfflfera 3 (junto Oceanogr fic), 46012 Valencia (Spain) Fax: (+ 34) 96-328-97-01 E-mail: [email protected] [+] These authors contributed equally to this work. [†] Deceased.
[d] L. Mondragn+ Centre Mditerranen de Mdecine Molculaire (C3 M) quipe “contrle mtabolique des morts cellulaires” Institut national de la sant et de la recherche mdicale (Inserm) U1065, BP 2 3194, 06204, Nice (France) Chem. Eur. J. 2014, 20, 15309 – 15314
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201404382.
15309
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Communication cles, dependent on endocytosis and the subsequent arrival to lysosomes, for which the existent lysosomal enzymes can degrade the capping molecules in SMPS allowing the release of the cargo that will reach its target and provide the therapeutic benefit expected. Seminal examples of capped SMPS containing peptides were able to be opened by enzymes present in the lysosomes with a broad spectra of action, such as esterases,[13] amylases,[14, 15] amidases[16] or proteases.[17] However, a more advanced approach could be envisioned by the use of gated ensembles that could be opened by lysosome enzymes that are overexpressed in certain target diseases. In this context some of us have recently described the first example of cell-specific cargo release based on a nanoparticle able to display a selective and controlled cargo delivery in senescent cells.[15] Despite these interesting results, there are not, as far as we know, other examples that, through the use of gated SMPS, are able to deliver the cargo selectively in particular cells overexpressing a target enzyme that is related with a certain disease. In this context it is well-known that cathepsins are highly associated to cancer development and metastasis.[18] Cathepsins are a family of fifteen enzymes distinguished by their structure, catalytic mechanism, and the proteins they cleave. Inside this family there are 11 cystein proteases (B, C, F, H, L, K, O, S, V, W, X), two aspartic proteases (D and E), and two serine carboxiproteases (A and G). Most of them are only active thanks to the low pH of the lysosomes and they are able to cleave specific substrates. Cysteine cathepsins exhibit broad specificity, cleaving their substrates preferentially after basic or hydrophobic residues.[18c] Cathepsin B is probably one of the most wellknown cathepsin enzymes and many studies have shown that cathepsin B overexpression is correlated with invasive and metastatic phenotypes in cancers.[18c] In this context some strategies developed till present are based on the development of cathepsin B inhibitors[19] or in the use of cathepsin B target sequences for the release of therapeutic agents.[20] These examples have been based on polymer-therapeutics or small peptides bound to cytotoxic agents. However, capped SMPS that are able to be preferentially opened by cathepsin B have not been described so far. Among sequences targeted by cathepsin B, Silva et al.[21] demonstrated recently that the peptide Abz-GIVRAK(Dnp)-OH, was able to be hydrolyzed highly specifically by cathepsin B and proved that this peptide is highly selective for this enzyme among lysosomal cysteine proteases. Taking into account the above cited facts, the aim of this study was to design capped mesoporous materials capable of selectively delivering their cargo in the presence cathepsin B enzyme that is overexpressed in certain types of cancers. To achieve this goal, MCM41 silica mesoporous nanoparticles were selected as inorganic scaffolds[22] and were loaded with safranin O dye (S1). Then, based on the study of Silva et al.[21] the outer surface was functionalized, through a “click chemistry” reaction,[23] with the peptide alkynyl-GIVRAKEAEGIVRAK-OH (P) that contains the cathepsin B target sequence. This procedure yielded the final material, S1-P (see Figure 1A). The specific cargo release of the solid synthesized was studied by employing several cell lines Chem. Eur. J. 2014, 20, 15309 – 15314
www.chemeurj.org
Figure 1. Synthesis and characterization of S1 nanoparticles: A) Representation of the gated material S1-P functionalized with 3-(azidopropyl)triethoxysilane and capped with the peptide P and the selective delivery mechanism in the presence of cathepsin B. B) Power X-ray patterns of MCM-41 as synthesized, calcined MCM-41, S1 and final S1-P. C) TEM images of calcined MCM-41 and solid S1-P showing the typical porosity of the MCM-41 mesoporous matrix. D) Release profiles of safranin O in the absence (&) and in the presence of lysosomal extracts from HeLa cells (^) at 37 8C and pH 5.4.
expressing or not cathepsin B enzyme and using specific cathepsin B inhibitors. Once synthesized, solids were characterized by using standard procedures. The mesoporous structure of S1-P was confirmed by XRD and TEM studies (see Figure 1B and 1C). The nanoparticles obtained presented a spherical structure with an average diameter of 106 nm (confirmed by dynamic light-scattering measurements, see the Supporting Information) and an average pore diameter of 3.23 nm. N2 adsorption–desorption isotherms of S1 were typical of mesoporous systems with capped mesopores, and a marked reductions in the N2 volume adsorbed and surface area (90.7 m2g 1) were observed when compared with the initial MCM-41-based MSN (1028.5 m2g 1). Finally, the content of grafted peptide and cargo in solid S1-P was determined by thermogravimetric and elemental analysis
15310
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Communication
Figure 2. Cellular uptake and lysosomal cathepsin B cargo release of S1-P studies. A) HeLa and MEFs WT were incubated 24 h with 100 mg mL 1 of S1-P. Then, nanoparticle internalization was monitored by measuring safranin O associated fluorescence by flow cytometry. Histograms corresponding to HeLa and MEFs WT treated (red) or not with the nanoparticle are depicted. Quantification of safranin O positive cells is shown in the graphic. B) Once the internalization is confirmed, HeLa, MEFs WT, and MEFs CatB / were incubated for 12 h in the presence of S1-P 100 mg mL 1. The cellular uptake of S1-P was followed by safranin O associated fluorescence (red) in the presence of DNA marker Hoechst 33342 (blue) and plasma membrane marker WGA-Alexa-Fluor 647 (green). Cytoplasmic safranin O associated fluorescence was observed except in cells lacking cathepsin B enzyme (MEFs Cat B / ). To determine the specific role of cathepsin B in cargo release, cells were also pretreated for 4 h with the cathepsin B inhibitor 4 mm Ac-LVK-CHO (CatB Inh) previous to the addition of S1-P 100 mg mL 1 for 12 h. A reduction or disappearance of safranin O cytoplasmic signal was clearly observed in all cell lines. C) Quantification of the safranin O associated fluorescence intensity of the different cell samples was carried out. Comparison among the non (black bars) and cathepsin B inhibitor treated (white bars) cells was done. Three independent experiments were developed obtaining similar results. Data are expressed as mean s. Asterisc indicates significant differences *** (p < 0.001) when paired t Student tests were applied, while n.s. indicates nonsignificant differences statistically.
Chem. Eur. J. 2014, 20, 15309 – 15314
www.chemeurj.org
15311
and amounted to 0.084 and 0.185 g g 1 SiO2, respectively (see Supporting Information for further details). As a final step in the characterization of the nanoparticles, in vitro studies of the delivery of the safranin O from S1-P solid were performed (see Figure 1D). For that purpose, lysosomal extracts from HeLa cells were prepared and the presence of cathepsine B in the extract was confirmed by using the Z-RR-AMC fluorogenic substrate of this enzyme (see the Supporting Information).[24] In a typical experiment S1-P was suspended at 37 8C and pH 5.4 in the presence of lysosomal extract and dye release was tracked by following the emission of the safranin O in the solution as a function of lem = time (lex = 520 nm, 585 nm). Moreover, as a control, the same amount of solid S1-P was suspended in aqueous media at the same pH and a mixture of proteins with the same concentration as that measured for the lysosomal extract was added (see the Supporting Information). In the absence of the lysosomal extract, a poor release was found, which indicated that the safranin O cargo remained in the nanoparticles without delivery. In contrast, in the presence of the lysosomal extracts delivery of the safranin O was found as an increase of the dye fluorescence as a function of time. In additional studies we also found that the presence of recombinant cathepsin B enzyme was able to induce cargo delivery, whereas enzymes, such as amylases or ureases were unable to open the gate in S1-P (see the Supporting Information). Cargo release in these experiments was attributed to the specific cathepsin B cleavage of the capping peptide, P. This cleavage was expected to occur in the amide bond from positively charged arginine (R) and alanine (A) amino
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Communication acids in the target GIVRAK sequence which resulted in a reduction of the size of the attached peptides finally allowing delivery of the entrapped cargo.[21] Moreover note that although it is known that many proteins could be adsorbed onto the nanoparticle surface and formed a ‘corona’, a selective delivery in a complex medium such as a lysosomal extract was observed.[25] After demonstrating the effective safranin O release from S1P in the presence of cathepsin B in vitro, our next goal was to prove the possible use of the peptide-capped materials as a specific delivery system in cell models. HeLa cells and primary culture cells from wild-type mouse embryonic fibroblasts (WT MEFs) were employed for the experiments. In a first step, the internalization of S1-P nanoparticles was confirmed by flow cytometry analysis. MEFS and HeLa cells were treated for 24 h with S1-P and safranin O associated fluorescence was monitored (Figure 2A). Almost all the cell population incorporated the nanoparticle. In a further analysis cell lines were treated with S1-P for 12 h and safranin O associated fluorescence was monitored by confocal microscopy (see Figure 2B). Cytoplasmic safranin O associated fluorescence was observed in both treated cells, which indicated the proper internalization and release of the content of S1-P. Once cellular uptake was confirmed, evidence of the cathepsin B dependent aperture of the nanodevice was studied. For that purpose, HeLa and WT MEFs were treated with the inhibitor of cathepsin B enzyme Ac-LVK-CHO[26] 4 h prior the addition of S1-P. A remarkable reduction in the presence of cytoplasmic safranin O was perceived when compared to cells treated only in the presence of S1-P, thus indicating the reduction in the degradation of P when cathepsin B is not active. To further confirm this fact, MEFs primary cells deficient for cathepsin B were also employed (MEFs CatB / ).[27] These cells lack the enzyme cathepsin B and therefore they were not able to induce the degradation of P. When MEFs CatB / were treated with S1P no cytoplasmic safranin O was detected (see Figure 2B). Furthermore, pretreatment of MEFs CatB / with Ac-LVK-CHO before the addition of S1-P did not induce changes in the cytoplasmic safranin O associated fluorescence, proving the specific role of cathepsin B in the peptide degradation. Subsequent quantification of the cytoplasmic fluorescence (see Figure 2C) associated to nanoparticles among the different treatments indicated very significant differences in HeLa and WT MEFs when cathepsin B is inhibited, whereas no significant delivery was observed in case of MEFs CatB / cells. Moreover WST-1 cell viability assays with HeLa cells demonstrated that S1-P nanoparticles were not toxic at concentrations up to 200 mg mL 1. To extend the potential applicability of the peptide-capped nanoparticles, new SMPS containing peptide P and loaded with doxorubicin were prepared (S2-P). When characterized, S2-P presented a similar cargo delivery profile to S1-P in the presence of lysosomal extract at 37 8C. Moreover, the content of grafted peptide and cargo in solid S2-P amounted to 0.068 and 0.085 g g 1 SiO2, respectively (see the Supporting Information for further details). Doxorubicin is commonly employed in the treatment of various types of cancer. However, the nonspeChem. Eur. J. 2014, 20, 15309 – 15314
www.chemeurj.org
cific cellular internalization of this drug is associated with major secondary effects, such as cardio- and nephrotoxicity.[28] Confining doxorubicin in nanoparticles for more specific cellular release has been reported to be an interesting strategy to decrease the unwanted secondary effects, thereby increasing the efficiency of this drug. To prove the possible therapeutic application of this new nanodevice, confocal microscopy studies were developed to test the internalization of S2-P nanoparticles by HeLa cells. Cells treated with S2-P presented a cell death associated phenotype with cell detaching from the plate and a reduction in cellular density compared to control samples (see Figure 3A). Subsequent quantification of cell viability employing WST-1 assays showed a decrease of 35 % in cell viability of HeLa cells treated with S2-P when compared to control cells (see Figure 3B). Moreover HeLa cells treated with S2-P in the presence of cathepsin B inhibitor showed nonaltered cell viability due to the lack of capping-peptide degradation by cathepsin B. Similar results were obtained for MEFs WT; in this case a 50 % reduction in cell viability was obtained when cathepsin B was present. Finally, no significant cell death was detected in MEFs CatB / cells (lacking cathepsin B) treated with S2-P (see Figure 3B). In summary these results supported the cathepsin B hydrolysis of the anchored peptide in S2-P as a responsible mechanism for doxorubicin release and the subsequent reduction in cell viability.
Figure 3. Cellular uptake of S2-P and cell viability studies: A) HeLa cells were treated with S2-P for 48 h in the absence or in the presence of cathepsin B inhibitor Ac-LVK-CHO. Cellular uptake was determined by confocal microscopy. Transmitted light images and doxorubicin-associated fluorescence (green) images were taken. Control cells showed no fluorescence (a). Doxorubicin-associated fluorescence was located in the cytoplasm of the cells when they were treated with S2-P (b). Cells previously treated with cathepsin B inhibitor did not show doxorubicin cytoplasmic associated fluorescence (c). B) WST-1 viability assays in HeLa, MEFs WT, and MEFs CatB / cells treated with 100 mg mL 1 of S2-P. In case of HeLa cells studies were performed in the absence or the presence of the cathepsin B inhibitor Ac-LVKCHO. Three independent experiments containing triplicates were carried out. Data are expressed as mean s.e. Asterisc indicates significant differences ** (p < 0.01) when paired t Student tests were applied.
15312
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Communication In summary, we have reported here the synthesis and characterization of two hybrid organic–inorganic-capped nanoparticles S1-P and S2-P. Both solids consisted of a MCM-41 nanoparticulated mesoporous scaffold loaded with the dye safranin O (S1-P) or with the cytotoxic drug doxorubicin (S2-P) and capped with a peptidic sequence designed to be selectively hydrolyzed by cathepsin B enzyme overexpressed in several cancer cells. We demonstrated how this cathepsin B target peptidic sequence was only opened in the presence of cathepsin B in in vitro and in cellular assays. These results validate the potential use of silica mesoporous supports as therapeutic tools for the treatment of cathepsin B overexpressing cancer cells thanks to the specific enzyme-dependent cargo release. Besides, the data obtained in this study opens a wide range of possibilities in the design of advanced nanodevices for enzyme-controlled delivery applications, and a number of new advances in this area are anticipated.
[7]
[8]
[9] [10]
Acknowledgements [11]
We thank the Spanish Government (Project MAT2012-38429C04 and SAF2010-15512) and the Generalitat Valenciana (Project PROMETEO/2009/016 and PROMETEOII/2014/061) for support. C.T. is grateful to the Spanish Ministry of Science and Innovation for her PhD fellowship. L.M. thanks the Generalitat Valenciana and Nice city council for their postdoctoral contracts VALI + D and “Aides Individuelles aux Jeunes Chercheurs 2011”. C.C. thanks the Generalitat Valenciana for their postdoctoral contract VALI + D. M.O. thanks the CIPF for her postdoctoral fellowship. We thank the confocal microscopy service, Alberto Hernndez and Eva Mara La Fuente from CIPF for their technical support.
[13]
Keywords: Cathepsin B · controlled release mesoporous materials · nanoparticles · peptides
[14]
·
gated
[1] a) Z. Ge, S. Liu, Chem. Soc. Rev. 2013, 42, 7289 – 7325; b) O. C. Farokhzad, R. Langer, ACS Nano 2009, 3, 16 – 20; c) S. Wang, Microporous Mesoporous Mater. 2009, 117, 1 – 9; d) X. X. Zhang, H. S. Eden, X. Chen, J. Controlled Release 2012, 159, 2 – 13; e) Q. He, J. Shi, Adv. Mater. 2014, 26, 391 – 411. [2] a) K. M. L. Taylor-Pashow, J. Della Rocca, R. C. Huxford, W. Lin, Chem. Commun. 2010, 46, 5832 – 5849; b) P. Yang, S. Gai, J. Lin, Chem. Soc. Rev. 2012, 41, 3679 – 3698; c) Z. Li, J. Barnes, A. Bosoy, J. F. Stoddart, J. I. Zink, Chem. Soc. Rev. 2012, 41, 2590 – 2605; d) M. Colilla, B. Gonzlez, M. ValletReg, Biomater. Sci. 2013, 1, 114 – 134; e) Q. He, J. Shi, J. Mater. Chem. 2011, 21, 5845 – 5855. [3] a) J. S. Beck, J. C. Vartuli, W. J. Roth, M. E. Leonowicz, C. T. Kresge, K. D. Schmitt, C. T. W. Chu, D. H. Olson, E. W. Sheppard, S. B. McCullen, J. B. Hoggins, J. L. Schlenker, J. Am. Chem. Soc. 1992, 114, 10834 – 10843; b) A. P. Wight, M. E. Davis, Chem. Rev. 2002, 102, 3589 – 3614; c) G. Kickelbick, Angew. Chem. 2004, 116, 3164 – 3166; Angew. Chem. Int. Ed. 2004, 43, 3102 – 3104. [4] K. Cot, M. E. Belowich, M. Liong, M. W. Ambrogio, Y. A. Lau, H. A. Khatib, J. I. Zink, N. M. Khashab, J. F. Stoddart, Nanoscale 2009, 1, 16 – 39. [5] N. K. Mal, M. Fujiwara, Y. Tanaka, Nature 2003, 421, 350 – 353. [6] a) J. Lai, X. Mu, Y. Xu, X. Wu, C. Wu, C. Li, J. Chen, Y. Zhao, Chem. Commun. 2010, 46, 7370 – 7372; b) A. Agostini, F. Sancenn, R. Martnez-MÇez, M. D. Marcos, J. Soto, P. Amors, Chem. Eur. J. 2012, 18, 12218 – 12221; c) J. Lee, J. Park, K. Singha, W. J. Kim, Chem. Commun. Chem. Eur. J. 2014, 20, 15309 – 15314
www.chemeurj.org
[12]
[15]
[16]
[17]
[18]
[19]
15313
2013, 49, 1545 – 1547; d) T. M. Guardado-Alvrez, L. S. Devi, M. M. Russell, B. J. Schwartz, J. I. Zink, J. Am. Chem. Soc. 2013, 135, 14000 – 14003. a) R. Hernandez, H. R. Tseng, J. W. Wong, J. F. Stoddart, J. I. Zink, J. Am. Chem. Soc. 2004, 126, 3370 – 3371; b) R. Mortera, J. Vivero-Escoto, I. I. Slowing, E. Garrone, B. Onida, V. S.-Y. Lin, Chem. Commun. 2009, 3219 – 3221; c) L. Wang, M. Kim, Q. Fang, J. Min, W. I. Jeon, S. Y. Lee, S. J. Son, S. W. Joo, S. B. Lee, Chem. Commun. 2013, 49, 3194 – 3196; d) C. Wang, Z. Li, D. Cao, Y. L. Zhao, J. W. Gaines, O. A. Bozdemir, M. W. Ambrogio, M. Frasconi, Y. Y. Botros, J. I. Zink, J. F. Stoddart, Angew. Chem. 2012, 124, 5556 – 5561; Angew. Chem. Int. Ed. 2012, 51, 5460 – 5465. a) H. Meng, M. Xue, T. Xia, Y. L. Zhao, F. Tamanoi, J. F. Stoddart, J. I. Zink, E. A. Nel, J. Am. Chem. Soc. 2010, 132, 12690 – 12697; b) R. Guillet-Nicolas, A. Popat, J. L. Bridot, G. Monteith, S. Z. Qiao, F. Kleitz, Angew. Chem. 2013, 125, 2374 – 2378; Angew. Chem. Int. Ed. 2013, 52, 2318 – 2322; c) M. Xue, J. I. Zink, J. Am. Chem. Soc. 2013, 135, 17659 – 17662. C. Coll, R. Casasffls, E. Aznar, M. D. Marcos, R. Martnez-MÇez, F. Sancenn, J. Soto, P. Amors, Chem. Commun. 2007, 1957 – 1959. a) E. Aznar, L. Mondragn, J. V. Ros-Lis, F. Sancenn, M. D. Marcos, R. Martnez-MÇez, J. Soto, E. Prez-Pay, P. Amors, Angew. Chem. 2011, 123, 11368 – 11371; Angew. Chem. Int. Ed. 2011, 50, 11172 – 11175; b) C. de La Torre C, A. Agostini, L. Mondragn, M. Orzez, F. Sancenn, R. Martnez-MÇez, M. D. Marcos, P. Amors, E. Prez-Pay, Chem. Commun. 2014, 50, 3184 – 3186; c) A. Schlossbauer, S. Warncke, P. M. E. Gramlich, J. Kecht, A. Manetto, T. Carell, T. Bein, Angew. Chem. 2010, 122, 4842 – 4845; Angew. Chem. Int. Ed. 2010, 49, 4734 – 4737. a) P. Zhang, F. Cheng, R. Zhou, J. Cao, J. Li, C. Burda, Q. Min, J. J. Zhu, Angew. Chem. Int. Ed. 2014, 53, 2371 – 2375; b) E. Climent, R. MartnezMÇez, F. Sancenn, M. D. Marcos, J. Soto, A. Maquieira, P. Amors, Angew. Chem. 2010, 122, 7439 – 7441; Angew. Chem. Int. Ed. 2010, 49, 7281 – 7283; c) M. Oroval, E. Climent, C. Coll, R. Eritja, A. AviÇ, M. D. Marcos, F. Sancenn, R. Martnez-MÇez, P. Amors, Chem. Commun. 2013, 49, 5480 – 5482; d) E. Climent, L. Mondragn, R. Martnez-MÇez, F. Sancenn, M.-D. Marcos, J. R. Murgua, P. Amors, K. Rurack, Angew. Chem. 2013, 125, 9106 – 9110; Angew. Chem. Int. Ed. 2013, 52, 8938 – 8942; e) F. J. Hernandez, L. I. Hernandez, A. Pinto, T. Schfer, V. C. zalp, Chem. Commun. 2013, 49, 1285 – 1287; f) E. Climent, R. Martnez-MÇez, A. Maquieira, F. Sancenn, M. D. Marcos, E. M. Brun, J. Soto, P. Amors, ChemistryOpen 2012, 1, 251 – 259. C. Coll, A. Bernardos, R. Martnez-MÇez, F. Sancenn, Acc. Chem. Res. 2013, 46, 339 – 349. a) Y. L. Sun, Y. Zhou, Q. L. Li, Y. W. Yang, Chem. Commun. 2013, 49, 9033 – 9035; b) A. Agostini, L. Mondragn, L. Pascual, E. Aznar, C. Coll, R. Martnez-MÇez, F. Sancenn, J. Soto, M. D. Marcos, P. Amors, A. M. Costero, M. Parra, M. S. Gil, Langmuir 2012, 28, 41, 14766. a) Z. Chen, Z. Li, Y. Lin, M. Yin, J. Ren, X. Qu, Chem. Eur. J. 2013, 19, 1778 – 1783; b) A. Bernardos, L. Mondragn, E. Aznar, M. D. Marcos, R. Martnez-MÇez, F. Sancenn, J. Soto, J. M. Barat, E. Prez-Pay, C. Guillem, P. Amors, ACS Nano 2010, 4, 6353 – 6368; c) A. Bernardos, E. Aznar, M. D. Marcos, R. Martnez-MÇez, F. Sancenn, J. Soto, J. M. Barat, P. Amors, Angew. Chem. Int. Ed. 2009, 48, 32, 5884 – 5887; d) C. Park, H. Kim, S. Kim, C. Kim, J. Am. Chem. Soc. 2009, 131, 16614 – 16615. A. Agostini, L. Mondragn, A. Bernardos, R. Martnez-MÇez, M. D. Marcos, F. Sancenn, J. Soto, A. Costero, C. Manguan-Garca, R. Perona, M. Moreno-Torres, R. Aparicio-Sanchis, J. R. Murgua, Angew. Chem. Int. Ed. 2012, 51, 10556 – 10560. a) A. Agostini, L. Mondragn, C. Coll, E. Aznar, M. D. Marcos, R. MartnezMÇez, F. Sancenn, J. Soto, E. Prez-Pay, P. Amors, ChemistryOpen 2012, 1, 17 – 20; b) I. Candel, E. Aznar, L. Mondragn, C. de La Torre, R. Martnez-MÇez, F. Sancenn, M. D. Marcos, P. Amors, C. Guillem, E. Prez-Pay, A. M. Costero, S. Gil, M. Parra, Nanoscale 2012, 4, 7237 – 7245. a) L. Mondragn, N. Mas, V. Ferragud, C. De La Torre, A. Agostini, R. Martnez-MÇez, F. Sancenn, P. Amors, E. Prez-Pay, M. Orzez, Chem. Eur. J. 2014, 20, 5271 – 5281; b) C. Coll, L. Mondragn, R. MartnezMÇez, F. Sancenn, M. D. Marcos, J. Soto, P. Amors, E. Prez-Pay, Angew. Chem. Int. Ed. 2011, 50, 2138 – 2140. a) G. J. Tan, Z. K. Peng, J. P. Lu, F. Q. Tang, World J. Biol. Chem. 2013, 4, 91 – 101; b) C. S. Gondi, J. S. Rao, Expert Opin. Ther. Targets 2013, 17, 281 – 291; c) V. Turk, V. Stoka. O. Vasiljeva, M. Renko, T. Sun, B. Turk, D. Turk, Biochim. Biophys. Acta. 2012, 1824, 68 – 88. S. Gobec, R. Frlan, Curr. Med. Chem. 2006, 13, 2309 – 2327. 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Communication [20] M. Caldern, R. Graeser, F. Kratz, R. Haag, Bioorg. Med. Chem. Lett. 2009, 19, 3725 – 3728; b) R. Haag, F. Kratz, Angew. Chem. 2006, 118, 1218 – 1237; Angew. Chem. Int. Ed. 2006, 45, 1198 – 1215; c) R. Duncan, Nat. Rev. Cancer 2006, 6, 688 – 701; d) K. L. Kiick, Science 2007, 317, 1182 – 1183; e) Y. J. Zhong, L. H. Shao, Y. Li, Int. J. Oncol. 2013, 42, 373 – 383. [21] S. Silva Cotrin, L. Puzer, W. A. de Souza Judice, L. Juliano, A. K. Carmona, M. A. Juliano, Anal. Biochem. 2004, 335, 244 – 252. [22] a) C. Argyo, V. Weiss, C. Bruchle, T. Bein, Chem. Mater. 2014, 26, 435 – 451; b) J. El Haskouri, D. Ortiz de Zarate, C. Guillem, J. Latorre, M. Calds, A. Beltrn, D. Beltrn, A. B. Descalzo, G. Rodrguez-Lpez, R. MartnezMÇez, M. D. Marcos, P. Amors, Chem. Commun. 2002, 330 – 331. [23] a) V. V. Rostovtsev, L. G. Green, V. V. Fokin, K. B. Sharpless, Angew. Chem. 2002, 114, 2708 – 2711; Angew. Chem. Int. Ed. 2002, 41, 2596 – 2599; b) C. W. Tornoe, C. Christensen, M. Meldal, J. Org. Chem. 2002, 67, 3057 – 3064; c) H. C. Kolb, M. G. Finn, K. B. Sharpless, Angew. Chem. 2001, 113, 2056 – 2075; Angew. Chem. Int. Ed. 2001, 40, 2004 – 2021.
Chem. Eur. J. 2014, 20, 15309 – 15314
www.chemeurj.org
[24] L. Foghsgaard, D. Wissing, D. Mauch, U. Lademann, L. Bastholm, M. Boes, F. Elling, M. Leist, M. Jttel, J. Cell. Biochem. 2001, 153, 999 – 1010. [25] a) I. Lynch, K. A. Dawson, Nano Today 2008, 3, 40 – 47; b) M. Lundqvist, J. Stigler, G. Elia, I. Lynch, T. Cedervall, K. A. Dawson, Proc. Natl. Acad. Sci. USA 2008, 105, 14265 – 14270. [26] R. M. McConnell, J. L. York, D. Frizzell, C. Ezell, J. Med. Chem. 1993, 36, 1084 – 1089. [27] J. Deussing, W. Roth, P. Saftig, C. Peters, H. L. Ploegh, J. A. Villadangos, Proc. Natl. Acad. Sci. USA 1998, 95, 4516 – 4521. [28] F. A. Formari, J. K. Randolph, J. C. Yalowich, M. K. Ritke, D. A. Gewirtz, Mol. Pharmacol. 1994, 45, 649 – 656.
Received: July 13, 2014 Published online on October 10, 2014
15314
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Communication
& Mesoporous Materials
Cathepsin-B Induced Controlled Release from Peptide-Capped Mesoporous Silica Nanoparticles Cristina de la Torre,[a, b, c] Laura Mondragn,[d] Carmen Coll,[a, b, c] Flix Sancenn,[a, b, c] Mara D. Marcos,[a, b, c] Ramn Martnez-MÇez,*[a, b, c] Pedro Amors,[e] Enrique Prez-Pay†,[f] and Mar Orzez*[f] Abstract: New capped silica mesoporous nanoparticles for intracellular controlled cargo release within cathepsin B expressing cells are described. Nanometric mesoporous MCM-41 supports loaded with safranin O (S1-P) or doxorubicin (S2-P) containing a molecular gate based on a cathepsin B target peptidic sequence were synthesized. Solids were designed to show “zero delivery” and to display cargo release in the presence of cathepsin B enzyme, which selectively hydrolyzed in vitro the capping peptide sequence. Controlled delivery in HeLa, MEFs WT, and MEFs lacking cathepsin B cell lines were also tested. Release of safranin O and doxorubicin in these cells took place when cathepsin B was active or present. Cells treated with S2-P showed a fall in cell viability due to nanoparticles internalization, cathepsin B hydrolysis of the capping peptide, and cytotoxic agent delivery, proving the possible use of these nanodevices as new therapeutic tools for cancer treatment.
In the treatment of cancer, chemotherapy constitutes the main therapeutic approach. Traditional chemotherapeutic agents target cells that divide rapidly, one of the main characteristics of cancer cells. However, this also means that cells belonging to healthy tissues that divide quickly are also targeted and destroyed constituting one of the main drawbacks of chemotherapy. To diminish these side effects, several strategies have been developed, among them: 1) the development of chemotherapeutic derivatives and analogues with less toxic effects
[a] C. de la Torre,+ C. Coll, F. Sancenn, M. D. Marcos, R. Martnez-MÇez Centro de Reconocimiento Molecular y Desarrollo Tecnolgico (IDM) Unidad Mixta Universidad Politcnica de ValenciaUniversidad de Valencia (Spain) [b] C. de la Torre,+ C. Coll, F. Sancenn, M. D. Marcos, R. Martnez-MÇez Departamento de Qumica, Universidad Politcnica de Valencia. Camino de Vera s/n, 46022, Valenicia (Spain) E-mail: [email protected] [c] C. de la Torre,+ C. Coll, F. Sancenn, M. D. Marcos, R. Martnez-MÇez CIBER de Bioingenieria, Biomateriales y Nanomedicina (CIBER-BBN) (Spain)
and better pharmacological properties or 2) the design of new drug delivery systems for better targeting of cancer cells. The arrival of nanotechnology has fuelled the latter approach and has brought about new innovative concepts to drug-delivery therapies.[1] Specifically attention has been put on drug-delivery systems capable of releasing active molecules to certain cells in a controlled fashion.[2] Among the different drug-delivery materials developed till present, silica mesoporous supports (SMPS) have undergone an exponential growth as carriers for drug storage and delivery in recent years, thanks to their unique properties, such as large loading capacity, low toxicity, and easy functionalization.[3] Notwithstanding, the most appealing feature of SMPS as carriers is, perhaps, the possibility of functionalizing them with molecular/supramolecular ensembles in their external surface to develop gated-SMPS, which show “zero delivery” and can release their cargo on-command in response to specifically designed external stimuli.[4] Since the first example of gated SMPS developed by Fujiwara’s laboratory,[5] a certain number of capped supports have been created responding to a wide variety of stimuli, such as light,[6] redox reactions,[7] pH,[8] changes in polarity,[9] temperature[10] etc. However, the use of biomolecules[11] as triggers provided the ultimate proof that SMPS may applied to more biological and realistic settings. In this context, the possibility of using enzymes as “biological-keys” has opened a wide range of new perspectives for the development of biocompatible gated SMPS carriers and this approach is envisioned to have a large potential to provide exquisite selectivity in the design of advanced gate-opening devices.[12] This development is reinforced by the cellular internalization mechanism of nanoparti[e] P. Amors Institut de Cincia dels Materials (ICMUV), Universitat de Valncia P.O. Box 2085, 46071 Valencia (Spain) [f] E. Prez-Pay, M. Orzez Laboratorio Pptidos y Protenas. Centro de Investigacin Prncipe Felipe., C/Eduardo Primo Yfflfera 3 (junto Oceanogr fic), 46012 Valencia (Spain) Fax: (+ 34) 96-328-97-01 E-mail: [email protected] [+] These authors contributed equally to this work. [†] Deceased.
[d] L. Mondragn+ Centre Mditerranen de Mdecine Molculaire (C3 M) quipe “contrle mtabolique des morts cellulaires” Institut national de la sant et de la recherche mdicale (Inserm) U1065, BP 2 3194, 06204, Nice (France) Chem. Eur. J. 2014, 20, 15309 – 15314
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201404382.
15309
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Communication cles, dependent on endocytosis and the subsequent arrival to lysosomes, for which the existent lysosomal enzymes can degrade the capping molecules in SMPS allowing the release of the cargo that will reach its target and provide the therapeutic benefit expected. Seminal examples of capped SMPS containing peptides were able to be opened by enzymes present in the lysosomes with a broad spectra of action, such as esterases,[13] amylases,[14, 15] amidases[16] or proteases.[17] However, a more advanced approach could be envisioned by the use of gated ensembles that could be opened by lysosome enzymes that are overexpressed in certain target diseases. In this context some of us have recently described the first example of cell-specific cargo release based on a nanoparticle able to display a selective and controlled cargo delivery in senescent cells.[15] Despite these interesting results, there are not, as far as we know, other examples that, through the use of gated SMPS, are able to deliver the cargo selectively in particular cells overexpressing a target enzyme that is related with a certain disease. In this context it is well-known that cathepsins are highly associated to cancer development and metastasis.[18] Cathepsins are a family of fifteen enzymes distinguished by their structure, catalytic mechanism, and the proteins they cleave. Inside this family there are 11 cystein proteases (B, C, F, H, L, K, O, S, V, W, X), two aspartic proteases (D and E), and two serine carboxiproteases (A and G). Most of them are only active thanks to the low pH of the lysosomes and they are able to cleave specific substrates. Cysteine cathepsins exhibit broad specificity, cleaving their substrates preferentially after basic or hydrophobic residues.[18c] Cathepsin B is probably one of the most wellknown cathepsin enzymes and many studies have shown that cathepsin B overexpression is correlated with invasive and metastatic phenotypes in cancers.[18c] In this context some strategies developed till present are based on the development of cathepsin B inhibitors[19] or in the use of cathepsin B target sequences for the release of therapeutic agents.[20] These examples have been based on polymer-therapeutics or small peptides bound to cytotoxic agents. However, capped SMPS that are able to be preferentially opened by cathepsin B have not been described so far. Among sequences targeted by cathepsin B, Silva et al.[21] demonstrated recently that the peptide Abz-GIVRAK(Dnp)-OH, was able to be hydrolyzed highly specifically by cathepsin B and proved that this peptide is highly selective for this enzyme among lysosomal cysteine proteases. Taking into account the above cited facts, the aim of this study was to design capped mesoporous materials capable of selectively delivering their cargo in the presence cathepsin B enzyme that is overexpressed in certain types of cancers. To achieve this goal, MCM41 silica mesoporous nanoparticles were selected as inorganic scaffolds[22] and were loaded with safranin O dye (S1). Then, based on the study of Silva et al.[21] the outer surface was functionalized, through a “click chemistry” reaction,[23] with the peptide alkynyl-GIVRAKEAEGIVRAK-OH (P) that contains the cathepsin B target sequence. This procedure yielded the final material, S1-P (see Figure 1A). The specific cargo release of the solid synthesized was studied by employing several cell lines Chem. Eur. J. 2014, 20, 15309 – 15314
www.chemeurj.org
Figure 1. Synthesis and characterization of S1 nanoparticles: A) Representation of the gated material S1-P functionalized with 3-(azidopropyl)triethoxysilane and capped with the peptide P and the selective delivery mechanism in the presence of cathepsin B. B) Power X-ray patterns of MCM-41 as synthesized, calcined MCM-41, S1 and final S1-P. C) TEM images of calcined MCM-41 and solid S1-P showing the typical porosity of the MCM-41 mesoporous matrix. D) Release profiles of safranin O in the absence (&) and in the presence of lysosomal extracts from HeLa cells (^) at 37 8C and pH 5.4.
expressing or not cathepsin B enzyme and using specific cathepsin B inhibitors. Once synthesized, solids were characterized by using standard procedures. The mesoporous structure of S1-P was confirmed by XRD and TEM studies (see Figure 1B and 1C). The nanoparticles obtained presented a spherical structure with an average diameter of 106 nm (confirmed by dynamic light-scattering measurements, see the Supporting Information) and an average pore diameter of 3.23 nm. N2 adsorption–desorption isotherms of S1 were typical of mesoporous systems with capped mesopores, and a marked reductions in the N2 volume adsorbed and surface area (90.7 m2g 1) were observed when compared with the initial MCM-41-based MSN (1028.5 m2g 1). Finally, the content of grafted peptide and cargo in solid S1-P was determined by thermogravimetric and elemental analysis
15310
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Communication
Figure 2. Cellular uptake and lysosomal cathepsin B cargo release of S1-P studies. A) HeLa and MEFs WT were incubated 24 h with 100 mg mL 1 of S1-P. Then, nanoparticle internalization was monitored by measuring safranin O associated fluorescence by flow cytometry. Histograms corresponding to HeLa and MEFs WT treated (red) or not with the nanoparticle are depicted. Quantification of safranin O positive cells is shown in the graphic. B) Once the internalization is confirmed, HeLa, MEFs WT, and MEFs CatB / were incubated for 12 h in the presence of S1-P 100 mg mL 1. The cellular uptake of S1-P was followed by safranin O associated fluorescence (red) in the presence of DNA marker Hoechst 33342 (blue) and plasma membrane marker WGA-Alexa-Fluor 647 (green). Cytoplasmic safranin O associated fluorescence was observed except in cells lacking cathepsin B enzyme (MEFs Cat B / ). To determine the specific role of cathepsin B in cargo release, cells were also pretreated for 4 h with the cathepsin B inhibitor 4 mm Ac-LVK-CHO (CatB Inh) previous to the addition of S1-P 100 mg mL 1 for 12 h. A reduction or disappearance of safranin O cytoplasmic signal was clearly observed in all cell lines. C) Quantification of the safranin O associated fluorescence intensity of the different cell samples was carried out. Comparison among the non (black bars) and cathepsin B inhibitor treated (white bars) cells was done. Three independent experiments were developed obtaining similar results. Data are expressed as mean s. Asterisc indicates significant differences *** (p < 0.001) when paired t Student tests were applied, while n.s. indicates nonsignificant differences statistically.
Chem. Eur. J. 2014, 20, 15309 – 15314
www.chemeurj.org
15311
and amounted to 0.084 and 0.185 g g 1 SiO2, respectively (see Supporting Information for further details). As a final step in the characterization of the nanoparticles, in vitro studies of the delivery of the safranin O from S1-P solid were performed (see Figure 1D). For that purpose, lysosomal extracts from HeLa cells were prepared and the presence of cathepsine B in the extract was confirmed by using the Z-RR-AMC fluorogenic substrate of this enzyme (see the Supporting Information).[24] In a typical experiment S1-P was suspended at 37 8C and pH 5.4 in the presence of lysosomal extract and dye release was tracked by following the emission of the safranin O in the solution as a function of lem = time (lex = 520 nm, 585 nm). Moreover, as a control, the same amount of solid S1-P was suspended in aqueous media at the same pH and a mixture of proteins with the same concentration as that measured for the lysosomal extract was added (see the Supporting Information). In the absence of the lysosomal extract, a poor release was found, which indicated that the safranin O cargo remained in the nanoparticles without delivery. In contrast, in the presence of the lysosomal extracts delivery of the safranin O was found as an increase of the dye fluorescence as a function of time. In additional studies we also found that the presence of recombinant cathepsin B enzyme was able to induce cargo delivery, whereas enzymes, such as amylases or ureases were unable to open the gate in S1-P (see the Supporting Information). Cargo release in these experiments was attributed to the specific cathepsin B cleavage of the capping peptide, P. This cleavage was expected to occur in the amide bond from positively charged arginine (R) and alanine (A) amino
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Communication acids in the target GIVRAK sequence which resulted in a reduction of the size of the attached peptides finally allowing delivery of the entrapped cargo.[21] Moreover note that although it is known that many proteins could be adsorbed onto the nanoparticle surface and formed a ‘corona’, a selective delivery in a complex medium such as a lysosomal extract was observed.[25] After demonstrating the effective safranin O release from S1P in the presence of cathepsin B in vitro, our next goal was to prove the possible use of the peptide-capped materials as a specific delivery system in cell models. HeLa cells and primary culture cells from wild-type mouse embryonic fibroblasts (WT MEFs) were employed for the experiments. In a first step, the internalization of S1-P nanoparticles was confirmed by flow cytometry analysis. MEFS and HeLa cells were treated for 24 h with S1-P and safranin O associated fluorescence was monitored (Figure 2A). Almost all the cell population incorporated the nanoparticle. In a further analysis cell lines were treated with S1-P for 12 h and safranin O associated fluorescence was monitored by confocal microscopy (see Figure 2B). Cytoplasmic safranin O associated fluorescence was observed in both treated cells, which indicated the proper internalization and release of the content of S1-P. Once cellular uptake was confirmed, evidence of the cathepsin B dependent aperture of the nanodevice was studied. For that purpose, HeLa and WT MEFs were treated with the inhibitor of cathepsin B enzyme Ac-LVK-CHO[26] 4 h prior the addition of S1-P. A remarkable reduction in the presence of cytoplasmic safranin O was perceived when compared to cells treated only in the presence of S1-P, thus indicating the reduction in the degradation of P when cathepsin B is not active. To further confirm this fact, MEFs primary cells deficient for cathepsin B were also employed (MEFs CatB / ).[27] These cells lack the enzyme cathepsin B and therefore they were not able to induce the degradation of P. When MEFs CatB / were treated with S1P no cytoplasmic safranin O was detected (see Figure 2B). Furthermore, pretreatment of MEFs CatB / with Ac-LVK-CHO before the addition of S1-P did not induce changes in the cytoplasmic safranin O associated fluorescence, proving the specific role of cathepsin B in the peptide degradation. Subsequent quantification of the cytoplasmic fluorescence (see Figure 2C) associated to nanoparticles among the different treatments indicated very significant differences in HeLa and WT MEFs when cathepsin B is inhibited, whereas no significant delivery was observed in case of MEFs CatB / cells. Moreover WST-1 cell viability assays with HeLa cells demonstrated that S1-P nanoparticles were not toxic at concentrations up to 200 mg mL 1. To extend the potential applicability of the peptide-capped nanoparticles, new SMPS containing peptide P and loaded with doxorubicin were prepared (S2-P). When characterized, S2-P presented a similar cargo delivery profile to S1-P in the presence of lysosomal extract at 37 8C. Moreover, the content of grafted peptide and cargo in solid S2-P amounted to 0.068 and 0.085 g g 1 SiO2, respectively (see the Supporting Information for further details). Doxorubicin is commonly employed in the treatment of various types of cancer. However, the nonspeChem. Eur. J. 2014, 20, 15309 – 15314
www.chemeurj.org
cific cellular internalization of this drug is associated with major secondary effects, such as cardio- and nephrotoxicity.[28] Confining doxorubicin in nanoparticles for more specific cellular release has been reported to be an interesting strategy to decrease the unwanted secondary effects, thereby increasing the efficiency of this drug. To prove the possible therapeutic application of this new nanodevice, confocal microscopy studies were developed to test the internalization of S2-P nanoparticles by HeLa cells. Cells treated with S2-P presented a cell death associated phenotype with cell detaching from the plate and a reduction in cellular density compared to control samples (see Figure 3A). Subsequent quantification of cell viability employing WST-1 assays showed a decrease of 35 % in cell viability of HeLa cells treated with S2-P when compared to control cells (see Figure 3B). Moreover HeLa cells treated with S2-P in the presence of cathepsin B inhibitor showed nonaltered cell viability due to the lack of capping-peptide degradation by cathepsin B. Similar results were obtained for MEFs WT; in this case a 50 % reduction in cell viability was obtained when cathepsin B was present. Finally, no significant cell death was detected in MEFs CatB / cells (lacking cathepsin B) treated with S2-P (see Figure 3B). In summary these results supported the cathepsin B hydrolysis of the anchored peptide in S2-P as a responsible mechanism for doxorubicin release and the subsequent reduction in cell viability.
Figure 3. Cellular uptake of S2-P and cell viability studies: A) HeLa cells were treated with S2-P for 48 h in the absence or in the presence of cathepsin B inhibitor Ac-LVK-CHO. Cellular uptake was determined by confocal microscopy. Transmitted light images and doxorubicin-associated fluorescence (green) images were taken. Control cells showed no fluorescence (a). Doxorubicin-associated fluorescence was located in the cytoplasm of the cells when they were treated with S2-P (b). Cells previously treated with cathepsin B inhibitor did not show doxorubicin cytoplasmic associated fluorescence (c). B) WST-1 viability assays in HeLa, MEFs WT, and MEFs CatB / cells treated with 100 mg mL 1 of S2-P. In case of HeLa cells studies were performed in the absence or the presence of the cathepsin B inhibitor Ac-LVKCHO. Three independent experiments containing triplicates were carried out. Data are expressed as mean s.e. Asterisc indicates significant differences ** (p < 0.01) when paired t Student tests were applied.
15312
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Communication In summary, we have reported here the synthesis and characterization of two hybrid organic–inorganic-capped nanoparticles S1-P and S2-P. Both solids consisted of a MCM-41 nanoparticulated mesoporous scaffold loaded with the dye safranin O (S1-P) or with the cytotoxic drug doxorubicin (S2-P) and capped with a peptidic sequence designed to be selectively hydrolyzed by cathepsin B enzyme overexpressed in several cancer cells. We demonstrated how this cathepsin B target peptidic sequence was only opened in the presence of cathepsin B in in vitro and in cellular assays. These results validate the potential use of silica mesoporous supports as therapeutic tools for the treatment of cathepsin B overexpressing cancer cells thanks to the specific enzyme-dependent cargo release. Besides, the data obtained in this study opens a wide range of possibilities in the design of advanced nanodevices for enzyme-controlled delivery applications, and a number of new advances in this area are anticipated.
[7]
[8]
[9] [10]
Acknowledgements [11]
We thank the Spanish Government (Project MAT2012-38429C04 and SAF2010-15512) and the Generalitat Valenciana (Project PROMETEO/2009/016 and PROMETEOII/2014/061) for support. C.T. is grateful to the Spanish Ministry of Science and Innovation for her PhD fellowship. L.M. thanks the Generalitat Valenciana and Nice city council for their postdoctoral contracts VALI + D and “Aides Individuelles aux Jeunes Chercheurs 2011”. C.C. thanks the Generalitat Valenciana for their postdoctoral contract VALI + D. M.O. thanks the CIPF for her postdoctoral fellowship. We thank the confocal microscopy service, Alberto Hernndez and Eva Mara La Fuente from CIPF for their technical support.
[13]
Keywords: Cathepsin B · controlled release mesoporous materials · nanoparticles · peptides
[14]
·
gated
[1] a) Z. Ge, S. Liu, Chem. Soc. Rev. 2013, 42, 7289 – 7325; b) O. C. Farokhzad, R. Langer, ACS Nano 2009, 3, 16 – 20; c) S. Wang, Microporous Mesoporous Mater. 2009, 117, 1 – 9; d) X. X. Zhang, H. S. Eden, X. Chen, J. Controlled Release 2012, 159, 2 – 13; e) Q. He, J. Shi, Adv. Mater. 2014, 26, 391 – 411. [2] a) K. M. L. Taylor-Pashow, J. Della Rocca, R. C. Huxford, W. Lin, Chem. Commun. 2010, 46, 5832 – 5849; b) P. Yang, S. Gai, J. Lin, Chem. Soc. Rev. 2012, 41, 3679 – 3698; c) Z. Li, J. Barnes, A. Bosoy, J. F. Stoddart, J. I. Zink, Chem. Soc. Rev. 2012, 41, 2590 – 2605; d) M. Colilla, B. Gonzlez, M. ValletReg, Biomater. Sci. 2013, 1, 114 – 134; e) Q. He, J. Shi, J. Mater. Chem. 2011, 21, 5845 – 5855. [3] a) J. S. Beck, J. C. Vartuli, W. J. Roth, M. E. Leonowicz, C. T. Kresge, K. D. Schmitt, C. T. W. Chu, D. H. Olson, E. W. Sheppard, S. B. McCullen, J. B. Hoggins, J. L. Schlenker, J. Am. Chem. Soc. 1992, 114, 10834 – 10843; b) A. P. Wight, M. E. Davis, Chem. Rev. 2002, 102, 3589 – 3614; c) G. Kickelbick, Angew. Chem. 2004, 116, 3164 – 3166; Angew. Chem. Int. Ed. 2004, 43, 3102 – 3104. [4] K. Cot, M. E. Belowich, M. Liong, M. W. Ambrogio, Y. A. Lau, H. A. Khatib, J. I. Zink, N. M. Khashab, J. F. Stoddart, Nanoscale 2009, 1, 16 – 39. [5] N. K. Mal, M. Fujiwara, Y. Tanaka, Nature 2003, 421, 350 – 353. [6] a) J. Lai, X. Mu, Y. Xu, X. Wu, C. Wu, C. Li, J. Chen, Y. Zhao, Chem. Commun. 2010, 46, 7370 – 7372; b) A. Agostini, F. Sancenn, R. Martnez-MÇez, M. D. Marcos, J. Soto, P. Amors, Chem. Eur. J. 2012, 18, 12218 – 12221; c) J. Lee, J. Park, K. Singha, W. J. Kim, Chem. Commun. Chem. Eur. J. 2014, 20, 15309 – 15314
www.chemeurj.org
[12]
[15]
[16]
[17]
[18]
[19]
15313
2013, 49, 1545 – 1547; d) T. M. Guardado-Alvrez, L. S. Devi, M. M. Russell, B. J. Schwartz, J. I. Zink, J. Am. Chem. Soc. 2013, 135, 14000 – 14003. a) R. Hernandez, H. R. Tseng, J. W. Wong, J. F. Stoddart, J. I. Zink, J. Am. Chem. Soc. 2004, 126, 3370 – 3371; b) R. Mortera, J. Vivero-Escoto, I. I. Slowing, E. Garrone, B. Onida, V. S.-Y. Lin, Chem. Commun. 2009, 3219 – 3221; c) L. Wang, M. Kim, Q. Fang, J. Min, W. I. Jeon, S. Y. Lee, S. J. Son, S. W. Joo, S. B. Lee, Chem. Commun. 2013, 49, 3194 – 3196; d) C. Wang, Z. Li, D. Cao, Y. L. Zhao, J. W. Gaines, O. A. Bozdemir, M. W. Ambrogio, M. Frasconi, Y. Y. Botros, J. I. Zink, J. F. Stoddart, Angew. Chem. 2012, 124, 5556 – 5561; Angew. Chem. Int. Ed. 2012, 51, 5460 – 5465. a) H. Meng, M. Xue, T. Xia, Y. L. Zhao, F. Tamanoi, J. F. Stoddart, J. I. Zink, E. A. Nel, J. Am. Chem. Soc. 2010, 132, 12690 – 12697; b) R. Guillet-Nicolas, A. Popat, J. L. Bridot, G. Monteith, S. Z. Qiao, F. Kleitz, Angew. Chem. 2013, 125, 2374 – 2378; Angew. Chem. Int. Ed. 2013, 52, 2318 – 2322; c) M. Xue, J. I. Zink, J. Am. Chem. Soc. 2013, 135, 17659 – 17662. C. Coll, R. Casasffls, E. Aznar, M. D. Marcos, R. Martnez-MÇez, F. Sancenn, J. Soto, P. Amors, Chem. Commun. 2007, 1957 – 1959. a) E. Aznar, L. Mondragn, J. V. Ros-Lis, F. Sancenn, M. D. Marcos, R. Martnez-MÇez, J. Soto, E. Prez-Pay, P. Amors, Angew. Chem. 2011, 123, 11368 – 11371; Angew. Chem. Int. Ed. 2011, 50, 11172 – 11175; b) C. de La Torre C, A. Agostini, L. Mondragn, M. Orzez, F. Sancenn, R. Martnez-MÇez, M. D. Marcos, P. Amors, E. Prez-Pay, Chem. Commun. 2014, 50, 3184 – 3186; c) A. Schlossbauer, S. Warncke, P. M. E. Gramlich, J. Kecht, A. Manetto, T. Carell, T. Bein, Angew. Chem. 2010, 122, 4842 – 4845; Angew. Chem. Int. Ed. 2010, 49, 4734 – 4737. a) P. Zhang, F. Cheng, R. Zhou, J. Cao, J. Li, C. Burda, Q. Min, J. J. Zhu, Angew. Chem. Int. Ed. 2014, 53, 2371 – 2375; b) E. Climent, R. MartnezMÇez, F. Sancenn, M. D. Marcos, J. Soto, A. Maquieira, P. Amors, Angew. Chem. 2010, 122, 7439 – 7441; Angew. Chem. Int. Ed. 2010, 49, 7281 – 7283; c) M. Oroval, E. Climent, C. Coll, R. Eritja, A. AviÇ, M. D. Marcos, F. Sancenn, R. Martnez-MÇez, P. Amors, Chem. Commun. 2013, 49, 5480 – 5482; d) E. Climent, L. Mondragn, R. Martnez-MÇez, F. Sancenn, M.-D. Marcos, J. R. Murgua, P. Amors, K. Rurack, Angew. Chem. 2013, 125, 9106 – 9110; Angew. Chem. Int. Ed. 2013, 52, 8938 – 8942; e) F. J. Hernandez, L. I. Hernandez, A. Pinto, T. Schfer, V. C. zalp, Chem. Commun. 2013, 49, 1285 – 1287; f) E. Climent, R. Martnez-MÇez, A. Maquieira, F. Sancenn, M. D. Marcos, E. M. Brun, J. Soto, P. Amors, ChemistryOpen 2012, 1, 251 – 259. C. Coll, A. Bernardos, R. Martnez-MÇez, F. Sancenn, Acc. Chem. Res. 2013, 46, 339 – 349. a) Y. L. Sun, Y. Zhou, Q. L. Li, Y. W. Yang, Chem. Commun. 2013, 49, 9033 – 9035; b) A. Agostini, L. Mondragn, L. Pascual, E. Aznar, C. Coll, R. Martnez-MÇez, F. Sancenn, J. Soto, M. D. Marcos, P. Amors, A. M. Costero, M. Parra, M. S. Gil, Langmuir 2012, 28, 41, 14766. a) Z. Chen, Z. Li, Y. Lin, M. Yin, J. Ren, X. Qu, Chem. Eur. J. 2013, 19, 1778 – 1783; b) A. Bernardos, L. Mondragn, E. Aznar, M. D. Marcos, R. Martnez-MÇez, F. Sancenn, J. Soto, J. M. Barat, E. Prez-Pay, C. Guillem, P. Amors, ACS Nano 2010, 4, 6353 – 6368; c) A. Bernardos, E. Aznar, M. D. Marcos, R. Martnez-MÇez, F. Sancenn, J. Soto, J. M. Barat, P. Amors, Angew. Chem. Int. Ed. 2009, 48, 32, 5884 – 5887; d) C. Park, H. Kim, S. Kim, C. Kim, J. Am. Chem. Soc. 2009, 131, 16614 – 16615. A. Agostini, L. Mondragn, A. Bernardos, R. Martnez-MÇez, M. D. Marcos, F. Sancenn, J. Soto, A. Costero, C. Manguan-Garca, R. Perona, M. Moreno-Torres, R. Aparicio-Sanchis, J. R. Murgua, Angew. Chem. Int. Ed. 2012, 51, 10556 – 10560. a) A. Agostini, L. Mondragn, C. Coll, E. Aznar, M. D. Marcos, R. MartnezMÇez, F. Sancenn, J. Soto, E. Prez-Pay, P. Amors, ChemistryOpen 2012, 1, 17 – 20; b) I. Candel, E. Aznar, L. Mondragn, C. de La Torre, R. Martnez-MÇez, F. Sancenn, M. D. Marcos, P. Amors, C. Guillem, E. Prez-Pay, A. M. Costero, S. Gil, M. Parra, Nanoscale 2012, 4, 7237 – 7245. a) L. Mondragn, N. Mas, V. Ferragud, C. De La Torre, A. Agostini, R. Martnez-MÇez, F. Sancenn, P. Amors, E. Prez-Pay, M. Orzez, Chem. Eur. J. 2014, 20, 5271 – 5281; b) C. Coll, L. Mondragn, R. MartnezMÇez, F. Sancenn, M. D. Marcos, J. Soto, P. Amors, E. Prez-Pay, Angew. Chem. Int. Ed. 2011, 50, 2138 – 2140. a) G. J. Tan, Z. K. Peng, J. P. Lu, F. Q. Tang, World J. Biol. Chem. 2013, 4, 91 – 101; b) C. S. Gondi, J. S. Rao, Expert Opin. Ther. Targets 2013, 17, 281 – 291; c) V. Turk, V. Stoka. O. Vasiljeva, M. Renko, T. Sun, B. Turk, D. Turk, Biochim. Biophys. Acta. 2012, 1824, 68 – 88. S. Gobec, R. Frlan, Curr. Med. Chem. 2006, 13, 2309 – 2327. 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Communication [20] M. Caldern, R. Graeser, F. Kratz, R. Haag, Bioorg. Med. Chem. Lett. 2009, 19, 3725 – 3728; b) R. Haag, F. Kratz, Angew. Chem. 2006, 118, 1218 – 1237; Angew. Chem. Int. Ed. 2006, 45, 1198 – 1215; c) R. Duncan, Nat. Rev. Cancer 2006, 6, 688 – 701; d) K. L. Kiick, Science 2007, 317, 1182 – 1183; e) Y. J. Zhong, L. H. Shao, Y. Li, Int. J. Oncol. 2013, 42, 373 – 383. [21] S. Silva Cotrin, L. Puzer, W. A. de Souza Judice, L. Juliano, A. K. Carmona, M. A. Juliano, Anal. Biochem. 2004, 335, 244 – 252. [22] a) C. Argyo, V. Weiss, C. Bruchle, T. Bein, Chem. Mater. 2014, 26, 435 – 451; b) J. El Haskouri, D. Ortiz de Zarate, C. Guillem, J. Latorre, M. Calds, A. Beltrn, D. Beltrn, A. B. Descalzo, G. Rodrguez-Lpez, R. MartnezMÇez, M. D. Marcos, P. Amors, Chem. Commun. 2002, 330 – 331. [23] a) V. V. Rostovtsev, L. G. Green, V. V. Fokin, K. B. Sharpless, Angew. Chem. 2002, 114, 2708 – 2711; Angew. Chem. Int. Ed. 2002, 41, 2596 – 2599; b) C. W. Tornoe, C. Christensen, M. Meldal, J. Org. Chem. 2002, 67, 3057 – 3064; c) H. C. Kolb, M. G. Finn, K. B. Sharpless, Angew. Chem. 2001, 113, 2056 – 2075; Angew. Chem. Int. Ed. 2001, 40, 2004 – 2021.
Chem. Eur. J. 2014, 20, 15309 – 15314
www.chemeurj.org
[24] L. Foghsgaard, D. Wissing, D. Mauch, U. Lademann, L. Bastholm, M. Boes, F. Elling, M. Leist, M. Jttel, J. Cell. Biochem. 2001, 153, 999 – 1010. [25] a) I. Lynch, K. A. Dawson, Nano Today 2008, 3, 40 – 47; b) M. Lundqvist, J. Stigler, G. Elia, I. Lynch, T. Cedervall, K. A. Dawson, Proc. Natl. Acad. Sci. USA 2008, 105, 14265 – 14270. [26] R. M. McConnell, J. L. York, D. Frizzell, C. Ezell, J. Med. Chem. 1993, 36, 1084 – 1089. [27] J. Deussing, W. Roth, P. Saftig, C. Peters, H. L. Ploegh, J. A. Villadangos, Proc. Natl. Acad. Sci. USA 1998, 95, 4516 – 4521. [28] F. A. Formari, J. K. Randolph, J. C. Yalowich, M. K. Ritke, D. A. Gewirtz, Mol. Pharmacol. 1994, 45, 649 – 656.
Received: July 13, 2014 Published online on October 10, 2014
15314
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
