DOI: 10.1002/chem.201404296
Communication
& Nanoparticles
An Efficient Rose Bengal Based Nanoplatform for Photodynamic Therapy Enrica Gianotti,*[a] Bianca Martins Estev¼o,[a, b] Fabio Cucinotta,[a] Noboru Hioka,[b] Manuela Rizzi,[c] Filippo Ren,[c] and Leonardo Marchese[a] state (FT > 0.4) and long triplet-state lifetimes (tT > 1 ms), because the efficiency of the PS is dependent on the photophysical properties of its lowest excited triplet state, 5) a high yield of 1O2 generation, and 6) a high photostability and no dark toxicity.[3, 4] Halogen-xanthene dyes, such as rose bengal (RB), are wellknown molecules that exhibit intense absorption bands in the green area of the visible spectrum (480–550 nm) and produce singlet oxygen in high yield (RB shows F 1O2 = 0.75 under 540 nm light irradiation), an important factor for its use as a PS in PDT.[5–7] The minimally penetrating nature of such green light makes RB particularly useful in many cutaneous lesions and dermatological diseases.[8] However, as a hydrophilic photosensitizer, RB suffers from a poor intracellular uptake ability and, thus, cannot be used to treat solid tumors.[9, 10] Recently, the uptake efficiency of RB by cancer cells was enhanced by the use of nanoparticles as the vehicle to conjugate with RB.[11, 12] Among a variety of nanoparticles that can be used for therapy and diagnostics, gold nanoparticles[13–15] and mesoporous silica nanoparticles (MSNs) have attracted great attention in recent years as cell markers,[16, 17] drug- and gene-delivery platforms,[18, 19] and carriers of enzymes.[20] MSNs possess a large surface area, a high pore volume, a uniform pore size, and are known to be biocompatible systems.[21] In addition, the porous inorganic structure can also protect the loaded molecules from photochemical degradation.[22] Herein, we report on a simple surface-modification process that conjugates the photosensitizer RB with amine-functionalized MSNs through covalent bonding to yield RB-modified MSNs (RB-MSNs) for PDT studies. Anchoring into the mesoporous channels leads to site isolation of RB, avoiding the intermolecular quenching that takes place within aggregated PS. The embedded RB shows a highly enhanced photostability, which is relevant for practical applications. The release of 1O2 was evaluated by a chemical method using uric acid, which undergoes oxidation during PS illumination. In vitro tests revealed the effects of RB-MSNs on cell proliferation in the absence or presence of light activation in a melanoma cellular model (SK-MEL-28). Melanoma, according to the World Health Organization (WHO), is one of the most aggressive skin cancers and its occurrence is increasing faster than that of any other cancer type, becoming a major publichealth cancer in many countries.[23, 24] Moreover, this malignancy represents a great medical challenge, because it displays a high metastatic potential and a strong resistance to currently available clinical treatments.[25, 26] Light acts like a trigger to
Abstract: Organically modified mesoporous silica nanoparticles (MSNs) containing rose bengal (RB), a xanthene dye, were successfully synthesized. RB-modified MSNs have shown a relevant photostability and a high efficiency in the photoproduction and delivery of singlet oxygen (1O2), which is particularly promising for photodynamic therapy (PDT) applications. In vitro tests have evidenced that RB-MSNs are able to reduce cell proliferation in one of the most aggressive skin cancer types (SK-MEL-28) after green-light irradiation.
Photodynamic therapy (PDT) is an emerging therapeutic modality and a noninvasive treatment that involves the use of photosensitizers (PS), which, when irradiated with specific light sources, induce cell death. In fact, the PS in its excited state can interact with molecular oxygen to generate cytotoxic singlet oxygen (1O2), a highly reactive species that can produce necrosis and/or apoptosis of diseased tissues and further healing of local injuries. The advantage in using PDT is that the oxidative damage is limited to the immediate area surrounding the excited PS, thus reducing the toxic side effects that appear when chemotherapy or radiotherapy are applied.[1, 2] A good PDT photosensitizer should meet some important requirements that include: 1) a high absorption coefficient in the spectral region of the excitation light, 2) a high intersystem crossing (ISC) yield, 3) a triplet state of appropriate energy (ET > 95 kJ mol1) to allow for efficient energy transfer to ground-state oxygen, 4) a high quantum yield of the triplet
[a] Dr. E. Gianotti, B. Martins Estev¼o, Dr. F. Cucinotta, Prof. L. Marchese Dipartimento di Scienze e Innovazione Tecnologica and Centro Nano-SiSTeMI Universit del Piemonte Orientale “A. Avogadro” Viale T. Michel 11, 15121 Alessandria (Italy) E-mail: [email protected] [b] B. Martins Estev¼o, Prof. N. Hioka Nucleos Research of Photodynamic Therapy, Chemistry Department State University of Maring, Av. Colombo 5.790 87020-900, Maring, Paran (Brazil) [c] Dr. M. Rizzi, Prof. F. Ren Innovative Research Laboratory for Wound Healing Health Sciences Department Universit del Piemonte Orientale “A. Avogadro Via Solaroli 17, 28100 Novara (Italy) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201404296. Chem. Eur. J. 2014, 20, 1 – 6
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Communication specific surface area (SSA), pore diameter, and pore volume decreased when RB was covalently bound to MSNs, confirming that the RB molecules have been introduced into the mesoporous channels. To get more insights on the location of RB, RB molecules were covalently bound to MSNs without removing the CTAB surfactant; under these conditions, the mesopores were completely occluded and RB could only be attached to the external surface of the nanoparticles. In this latter case, we observed a RB leaching of 80 %, calculated from the UV/Vis specScheme 1. Schematic representation of the synthetic procedure used to obtain RB-modified MSNs (RB-MSNs). tra of the eluate by using the Lambert–Beer law. Conversely, when RB was attached after surfactant removal, ca. 85 % of the induce toxicity owing to 1O2 release, producing subsequent RB molecules were stabilized inside the pores (Figure S3 in the cell damage or even cell death. Supporting Information). The procedure for the synthesis of RB-MSNs is reported in The DR UV/Vis spectrum of the RB-MSNs solid is reported in Scheme 1. The amine-functionalized MSNs were obtained by Figure 2 together with the UV/Vis spectrum of RB in a solution a one-pot cetyltrimethylammonium bromide (CTAB) template of water. At physiological pH, the absorption maximum of free synthesis using 3-aminopropyltriethoxysilane (APTS) and tetRB is located at 547 nm, while the maximum of RB-MSNs is raethyl ortosilicate (TEOS), as described in literature.[27] The surred-shifted at 564 nm owing to the confinement effect and factant was removed by Soxhlet extraction in acidic conditions. a different chemical environment inside the mesopores. In adThe amine groups are necessary to conjugate the activated RB. dition, the weak shoulder at a lower wavelength observed in The covalent coupling between RB and NH2-MSNs was confirmed by FTIR spectral analysis (Figure S1 in the Supporting Information). UV/Vis absorption spectroscopy was used to evaluate the loading of RB on RB-MSMs (0.034 mmol per mg MSNs).The average number of RB molecules per nanoparticle was calculated to be about 4.20 104 by considering the density of the mesoporous silica nanoparticles[28] and the particle size. The RB-MSNs were characterized by XRD and HRTEM analyses to elucidate the structural modification and particle dimensions of the MSNs after the post-synthetic route to introduce the RB molecules inside the mesopores. Figure 1 A shows that both NH2-MSNs and RB-MSNs display the typical XRD pattern of an ordered hexagonal network of mesopores with well defined (100), (110), and (200) reflections,[29] indicating that the synthetic pathway adopted for anchoring RB on the silica surface did not affect the ordered assembly of the MSNs pores. The HRTEM images of RB-MSNs (Figure 1 B) reveal the presence of well-ordered nanostructured mesoporous particles with an Figure 1. A) XRD patterns of extracted NH2-MSNs and RB-MSNs. B) HRTEM average particle size in the 160–180 nm range, which was also images of RB-MSNs; scale bars: 20 nm (top), 50 nm (bottom). confirmed by DLS analysis (Table 1, see the Supporting Information). The textural properties of NH2-MSNs and RB-loaded MSNs Table 1. Textural properties of MSNs. were obtained by N2 adsorption–desorption isotherms at 77 K (Figure S2 in the Supporting Information) and are reported in DDFT [] V [cm3 g1] Samples SSABET [m2 g1] Table 1. The functionalized MSNs show type IV isotherms with NH2-MSNs 1120 37.5 1.3 H1-type hysteresis, which is clearly representative of standard RB-MSNs 885 36 0.9 M41S materials (Figure S2 in the Supporting Information). The &
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Figure 2. DR UV/Vis spectrum of RB-MSNs (curve a) and UV/Vis absorption spectrum of free RB in a solution of water (5 106 m, curve b).
both the solid sample (curve a) and the RB in solution (curve b) can be attributed to the presence of some RB aggregates[5] that, nonetheless, did not affect the performances of the RB-MSNs in the photoproduction and release of 1O2 (see below). The protection effect of the inorganic mesoporous nanostructure towards RB photodegradation was evidenced by the UV/Vis spectra of RB in solution and RB-MSNs samples upon irradiation with visible light for several days (Figure S4 in the Supporting Information). The decrease of the RB absorption band due to photodegradation is almost stopped when RB is embedded into MSNs nanoparticles, while photobleaching is rapid when RB is in solution. The singlet-oxygen generation was evaluated by a chemical method using uric acid (UA) as detector.[30–32] Under irradiation at neutral pH, UA is stable as a monoanion with a band centered at 292 nm. When singlet oxygen is released, as an effect of PS photoexcitation, UA is irreversibly oxidized and a decrease in the intensity of the band at 292 nm is observed. In our experiments, a solution of uric acid in water was irradiated at 540 nm in the presence of RB-MSNs solid nanoparticles, and the decreasing of the UA band at 292 nm was monitored for 4800 sec (Figure 3 A). The decay curves of UA absorption at 292 nm as a function of the irradiation time are reported in Figure 3 B in which the RB-MSNs decay is compared to the decay of MSNs without the photosensitizer (NH2-MSNs). Only in the presence of RB, a decay in the UA band due the 1O2 release was observed. The slope of the curve is roughly proportional to the efficiency of singlet-oxygen generation.[32–34] A direct comparison between the RB-MSNs and RB in solution by monitoring the UA photooxidation is not possible, because a water suspension of RB-MSNs is used for the photoproduction of 1O2 and this is affected by nanoparticle light scattering. Therefore, the efficiency of singlet-oxygen delivery of RB-MSNs (hRBMSNs) was calculated by using Equation (1)[35]:
hðRBMSNsÞ ¼ FRB 1 O2
tRB tðRBMSNsÞ
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Figure 3. A) Absorption spectra of UA in the presence of RB-MSNs in water after different irradiation times with 540 nm light. B) Decay curves of the UA absorption band at 292 nm as a function of the irradiation time in the presence of RB-MSNs and NH2-MSNs.
in which t(RB) is the UA decreasing time in the presence of RB in a solution of water, t(RB-MSNs) is the UA decreasing time in the presence of RB-MSNs in a suspension of water, both adjusted to a first-order exponential decay, and FRB is the singletoxygen quantum yield of free RB in a solution of water (F 1 O2 = 0.75 under 540 nm light irradiation). The h value obtained for RB-MSNs is 0.74, which is very close to the quantum yield of singlet-oxygen release of free RB; this means that the 1O2 produced by the excited RB immobilized on the silica surface diffuses efficiently through the MSNs mesopores. Owing to the high singlet-oxygen release achieved when RB is attached to the inner walls of the mesoporous silica nanostructure, in vitro tests were performed by using melanoma cells. The effects of rose bengal loaded silica nanoparticles on cell proliferation in the absence or presence of light activation was evaluated in a melanoma cellular model (SK-MEL-28).[23] The SK-MEL-28 cell line has been chosen for this test, because it undergoes cell death after RB high concentration (100– 200 mm) treatment, even in the absence of light.[36] As a further control, cells were also treated under the same conditions with unloaded silica nanoparticles. Cellular specimens were preincubated for 5 h in a serum-free medium before light stimulation (5 min under green light), and cell proliferation was evaluated after 18 h of incubation in a complete cell-culture medium. As shown in Figure 4, control cells proliferation was not affected
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Communication thylammoniumbromide (CTAB) as a structure-directing agent.[27] CTAB (1.9 mmol) was first dissolved in water (340 mL). Then, a solution of NaOH (2.0 m, 2.45 mL) was added to the solution of CTAB, and the temperature was adjusted to 80 8C. Finally, tetraethoxy silane (TEOS, 3.5 mL, 18.1 mmol) and 3-aminopropyltriethoxy silane (APTS, 0.43 mL, 2.04 mmol) were added simultaneously and dropwise to the solution over a period of 4 min. The mixture was stirred at 80 8C for 2 h to give rise to white precipitates. The solid product was filtered, washed with deionized water and ethanol, and dried in vacuo. The surfactant template (CTAB) was removed by Soxhlet extraction with 2-propanol/HCl for 96 h at 200 8C.
Covalent conjugation of Rose Bengal with NH2-MSNs Firstly, extracted NH2-MSNs were dispersed in DMF (15 mL) and then sonicated for 15 min. To covalently conjugate RB to NH2MSNs, a solution of DMF (15 mL) containing rose bengal (RB, 1 equiv), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5b]pyridinium 3-oxid hexafluorophosphate (HATU, 1 equiv), and N,Ndiisopropylethylamine (DIPEA, 2 equiv) was added to the NH2MSNs dispersion and stirred vigorously for 24 h at room temperature protected from light. The solid hybrid was then filtered and dried under vacuum. The as-synthesized RB-MSNs solids were washed with DMF several times to remove the unreacted RB molecules. After the washing procedure, the actual RB loading was calculated from the UV/Vis spectrum of the RB eluate by using the Lambert–Beer law (e566 = 95 780 m1 cm1 in DMF measured from the slope of a plot).
Figure 4. MSNs and RB-MSNs effects on SK-MEL-28 cell proliferation. Representative optical microscopy images (magnification 10 ) and quantification of MSNs and RB-MSNs effects on cell proliferation in the absence (white bars) or presence (grey bars) of light stimulation. Results are expressed as mean values standard deviation (SD). * p < 0.05.
Cellular toxicity
by light stimulation (83.93 25.62 cells/mm2 and 81.68 23.65 cells/mm2). In the absence of light stimulation, preincubation with RB-MSNs did not alter cell proliferation (87.36 26.48 cells/mm2), whereas after light activation it was able to reduce cell proliferation (54.61 11.54 cells/mm2, p < 0.05). As expected, unloaded silica nanoparticles did not significantly alter cellular proliferation, both in the absence or presence of light stimulation (64.10 8.79 cells/mm2 and 47.85 13.48 cells/ mm2, respectively). In summary, a simple surface-modification process was adopted to conjugate the photosensitizer RB with amine-functionalized MSNs through covalent bonding to yield a photostable RB-modified MSNs, that is, the porous inorganic structure protects the embedded molecules from photochemical degradation. The RB confined inside the mesopores of MSNs has been successfully photoexcited, and the singlet-oxygen generation has been monitored by a chemical method using uric acid (UA) as detector. The efficiency of singlet-oxygen delivery of RB-MSNs is high, meaning that 1O2 can easily diffuse through the MSNs mesopores into the UA solution. Promising results have been obtained by in vitro tests using an aggressive melanoma cellular model that remarks the potential application of RB-MSNs as a PDT agent.
2.5 104 cells were seeded in 24-wells culture plates and allowed to adhere overnight. Non adherent cells were then removed by a gentle wash in phosphate buffer (PBS, pH 7.4). Test silica nanoparticles aqueous suspensions (0–100 mg mL1) were added to the cells in a serum-free culture medium and incubated for 5 h before light irradiation. The cells were irradiated for 5 min with a green light in the absence of cell-culture medium. Immediately after each irradiation, cell-culture medium was added to the cells. In parallel, some samples were treated with silica nanoparticles but were not irradiated with light. After irradiation, the serum-free cell-culture medium was replaced by complete cell-culture medium in all samples. The cells were further incubated overnight and the cell viability was assessed by counting formalin-sucrose-fixed cells after crystal-violet staining. The counting procedure was performed by two different researchers blinded to experimental groups to assess the reproducibility of the analysis. Cell proliferation was evaluated by counting cells in each microscopic field in at least 3 fields for each experimental condition. The cell density was expressed as a mean number of cells/mm2 standard deviation (S.D.).[37]
Acknowledgements The authors thank the Masterlight project (Bando di Ateneo CSP 2012) for financial support. B.M.E. thanks CAPES (Coordenażo de AperfeiÅoamento de Pessoal de Nvel Superior) for financial support. The authors are grateful to Prof. Daniela Taverna from the Molecular Biotechnology Center, Department of Molecular Biotechnology and Health Sciences, University of Turin (Italy) for providing the highly metastatic human melanoma cell line SK-MEL-28 used in this work.
Experimental Section Synthesis of functionalized MSNs nanoparticles Functionalized mesoporous silica nanoparticles (NH2-MSNs) were prepared according to literature procedures by using cetyltrime-
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Communication [22] V. Ramamurthy, Photochemistry in Organized and Constrained Media, Wiley-VCH, Weinheim, 1991. [23] J. B. Jeong, S. C. Hong, J. S. Koo, H. J. Jeong, Food Nutr. Sci. 2011, 2, 128 – 132. [24] D. W. Mueller, A. K. Bosserhoff, Br. J. Cancer 2009, 101, 551 – 556. [25] J. Ma, H. Han, D. Liu, W. Ii, X. Feng, X. Xue, X. Wu, G. Niu, G. Zhang, Y. Zhao, C. Liu, H. Tao, B. Gao, Plos One 2013, DOI: 10.1371/journal.pone0073261. [26] E. Buommino, A. Baroni, N. Canozo, M. Petrazzullo, R. Nicoletti, A. Vozza, M. A. Tufano, Invest. New Drugs 2009, 27, 412 – 418. [27] D. R. Radu, C.-Y. Lai, K. Jeftinija, E. W. Rowe, S. Jeftinija, V. S.-Y. Lin, J. Am. Chem. Soc. 2004, 126, 13216 – 13217. [28] K. J. Edler, P. A. Reynolds, J. W. White, D. Cookson, J. Chem. Soc. Faraday Trans. 1997, 93, 199 – 202. [29] C. T. Kresge, M. E. Leonowicz, W. J. Roth, J. C. Vartuli, Nature 1992, 359, 710 – 712. [30] F. Fischer, G. Graschew, H. J. Sinn, W. Maier-Borst, W. J. Lorenz, P. M. Schlag, Clin. Chim. Acta 1998, 274, 89 – 104. [31] B. Ribeiro Rabello, A. Passarella Gerola, D. Silva Pellosi, A. Luiz Tessaro, J. Leandro Aparicio, W. Caetano, N. Hioka, J. Photochem. Photobiol. A 2012, 238, 53 – 62. [32] A. Passarella Gerola, J. Semensato, D. Silva Pellosi, V. Roberto Batistela, B. Ribeiro Rabello, N. Hioka, W. Caetano, J. Photochem. Photobiol. A 2012, 232, 14 – 21. [33] T. Y. Ohulchanskyy, L. Roy, L. N. Goswami, Y. Chen, J. Bergey, R. K. Pandey, A. R. Oseroff, P. N. Prasad, Nano Lett. 2007, 7, 2835 – 2842. [34] H.-L. Tu, Y.-S. Lin, H.-Y. Lin, Y. Hung, L.-W. Lo, Y.-F. Chen, C.-Y. Mou, Adv. Mater. 2009, 21, 172 – 177. [35] L. M. Rossi, P. R. Silva, L. L. R. Vono, A. U. Fernandes, D. B. Tada, M. S. Baptista, Langmuir 2008, 24, 12534 – 12538. [36] S. H. Mousavi, X. D. Zhang, A. M. Sharifi, P. Hersey, Iran. J. Basic Med. Sci. 2006, 9, 216 – 222. [37] C. Molinari, M. Rizzi, D. F. Squarzanti, P. Pittarella, G. Vacca, F. Ren, Cell. Physiol. Biochem. 2013, 31, 815 – 822.
Keywords: cytotoxicity · mesoporous silica nanoparticles · photodynamic therapy · Rose Bengal · singlet oxygen [1] J. P. Celli, B. Q. Spring, I. Rizvi, C. L. Evans, K. S. Samkoe, S. Verma, B. W. Pogue, T. Hasan, Chem. Rev. 2010, 110, 2795 – 2838. [2] C. Wang, L. Cheng, Z. Liu, Theranostic 2013, 3, 317 – 330. [3] M. C. DeRosa, R. J. Crutchley, Coord. Chem. Rev. 2002, 233 – 234, 351 – 371. [4] J. Jori, J. Photochem. Photobiol. B 1996, 36, 87 – 93. [5] D. C. Neckers, J. Photochem. Photobiol. A 1989, 47, 1 – 29. [6] E. Gandin, Y. Lion, A. Van der Vorst, Photochem. Photobiol. 1983, 37, 271 – 278. [7] R. W. Redmond, J. N. Gamlin, Photochem. Photobiol. 1999, 70, 391 – 475. [8] E. Panzarini, V. Inguscio, L. Dini, International Journal of Photoenergy 2011, DOI: 10.1155/2011/713726. [9] E. Fischer, F. Varga, Acta Physiol. Acad. Sci. Hung. 1979, 54, 89 – 94. [10] B. Wang, J.-H. Wang, Q. Liu, H. Huang, M. Chen, K. Li, C. Li, X.-F. Yu, P. K. Chu, Biomaterials 2014, 35, 1954 – 1966. [11] A. Uppal, B. Jain, PK. Gupta, K. Das, Photochem. Photobiol. 2011, 87, 1146 – 1151. [12] W. Lin, M. C. Garnett, S. S. Davis, E. Schacht, E. Ferruti, P. Illum, J. Controlled Release 2001, 71, 117 – 126. [13] Y. Cheng, A. C. Samia, J. D. Meyers, I. Panagopoulos, B. Fei, C. Burda, J. Am. Chem. Soc. 2008, 130, 10643 – 10647. [14] T. Doane, C. Burda, Adv. Drug Delivery Rev. 2013, 65, 607 – 621. [15] Y. Cheng, T. L. Doane, C.-H. Chuang, A. Ziady, C. Burda, Small 2014, 10, 1799 – 1804. [16] C.-P. Tsai, Y. Hung, Y.-H. Chou, D.-M. Huang, J.-K. Hisao, C. Chang, Y.-C. Chen, C.-Y. Mou, Small 2008, 4, 186 – 191. [17] S.-H. Wu, Y.-S. Lin, Y. Hung, Y.-H. Chou, Y.-H. Hsu, C. Chang, C.-Y. Mou, ChemBioChem 2008, 9, 53 – 57. [18] J. Lu, M. Liong, J. I. Zink, F. Tamanoi, Small 2007, 3, 1341 – 1346. [19] M. Colilla, B. Gonzalez, M. Vallet-Reg, Biomater.Sci. 2013, 1, 114 – 134. [20] I. I. Slowing, B. G. Trewyn, V. S.-Y. Lin, J. Am. Chem. Soc. 2007, 129, 8845 – 8849. [21] Z. Li, J. C. Barnes, A. Bosoy, J. Fraser Stoddart, J. I. Zink, Chem. Soc. Rev. 2012, 41, 2590 – 2605.
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Received: July 8, 2014 Published online on && &&, 0000
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COMMUNICATION & Nanoparticles
An efficient and photostable rose bengal (RB) nanoplatform for photodynamic therapy has been successfully synthesized. RB-modified mesoporous silica nanoparticles (MSNs) have shown a relevant photostability and a high efficiency in the photoproduction and delivery of singlet oxygen (1O2), and in vitro tests have evidenced that RB-MSNs are able to reduce cell proliferation in one of the most aggressive skin-cancer types (SK-MEL-28) after green-light irradiation.
E. Gianotti,* B. Martins Estev¼o, F. Cucinotta, N. Hioka, M. Rizzi, F. Ren, L. Marchese && – && An Efficient Rose Bengal Based Nanoplatform for Photodynamic Therapy
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An Efficient Rose Bengal Based Nanoplatform for Photodynamic Therapy Enrica Gianotti,*[a] Bianca Martins Estev¼o,[a, b] Fabio Cucinotta,[a] Noboru Hioka,[b] Manuela Rizzi,[c] Filippo Ren,[c] and Leonardo Marchese[a] state (FT > 0.4) and long triplet-state lifetimes (tT > 1 ms), because the efficiency of the PS is dependent on the photophysical properties of its lowest excited triplet state, 5) a high yield of 1O2 generation, and 6) a high photostability and no dark toxicity.[3, 4] Halogen-xanthene dyes, such as rose bengal (RB), are wellknown molecules that exhibit intense absorption bands in the green area of the visible spectrum (480–550 nm) and produce singlet oxygen in high yield (RB shows F 1O2 = 0.75 under 540 nm light irradiation), an important factor for its use as a PS in PDT.[5–7] The minimally penetrating nature of such green light makes RB particularly useful in many cutaneous lesions and dermatological diseases.[8] However, as a hydrophilic photosensitizer, RB suffers from a poor intracellular uptake ability and, thus, cannot be used to treat solid tumors.[9, 10] Recently, the uptake efficiency of RB by cancer cells was enhanced by the use of nanoparticles as the vehicle to conjugate with RB.[11, 12] Among a variety of nanoparticles that can be used for therapy and diagnostics, gold nanoparticles[13–15] and mesoporous silica nanoparticles (MSNs) have attracted great attention in recent years as cell markers,[16, 17] drug- and gene-delivery platforms,[18, 19] and carriers of enzymes.[20] MSNs possess a large surface area, a high pore volume, a uniform pore size, and are known to be biocompatible systems.[21] In addition, the porous inorganic structure can also protect the loaded molecules from photochemical degradation.[22] Herein, we report on a simple surface-modification process that conjugates the photosensitizer RB with amine-functionalized MSNs through covalent bonding to yield RB-modified MSNs (RB-MSNs) for PDT studies. Anchoring into the mesoporous channels leads to site isolation of RB, avoiding the intermolecular quenching that takes place within aggregated PS. The embedded RB shows a highly enhanced photostability, which is relevant for practical applications. The release of 1O2 was evaluated by a chemical method using uric acid, which undergoes oxidation during PS illumination. In vitro tests revealed the effects of RB-MSNs on cell proliferation in the absence or presence of light activation in a melanoma cellular model (SK-MEL-28). Melanoma, according to the World Health Organization (WHO), is one of the most aggressive skin cancers and its occurrence is increasing faster than that of any other cancer type, becoming a major publichealth cancer in many countries.[23, 24] Moreover, this malignancy represents a great medical challenge, because it displays a high metastatic potential and a strong resistance to currently available clinical treatments.[25, 26] Light acts like a trigger to
Abstract: Organically modified mesoporous silica nanoparticles (MSNs) containing rose bengal (RB), a xanthene dye, were successfully synthesized. RB-modified MSNs have shown a relevant photostability and a high efficiency in the photoproduction and delivery of singlet oxygen (1O2), which is particularly promising for photodynamic therapy (PDT) applications. In vitro tests have evidenced that RB-MSNs are able to reduce cell proliferation in one of the most aggressive skin cancer types (SK-MEL-28) after green-light irradiation.
Photodynamic therapy (PDT) is an emerging therapeutic modality and a noninvasive treatment that involves the use of photosensitizers (PS), which, when irradiated with specific light sources, induce cell death. In fact, the PS in its excited state can interact with molecular oxygen to generate cytotoxic singlet oxygen (1O2), a highly reactive species that can produce necrosis and/or apoptosis of diseased tissues and further healing of local injuries. The advantage in using PDT is that the oxidative damage is limited to the immediate area surrounding the excited PS, thus reducing the toxic side effects that appear when chemotherapy or radiotherapy are applied.[1, 2] A good PDT photosensitizer should meet some important requirements that include: 1) a high absorption coefficient in the spectral region of the excitation light, 2) a high intersystem crossing (ISC) yield, 3) a triplet state of appropriate energy (ET > 95 kJ mol1) to allow for efficient energy transfer to ground-state oxygen, 4) a high quantum yield of the triplet
[a] Dr. E. Gianotti, B. Martins Estev¼o, Dr. F. Cucinotta, Prof. L. Marchese Dipartimento di Scienze e Innovazione Tecnologica and Centro Nano-SiSTeMI Universit del Piemonte Orientale “A. Avogadro” Viale T. Michel 11, 15121 Alessandria (Italy) E-mail: [email protected] [b] B. Martins Estev¼o, Prof. N. Hioka Nucleos Research of Photodynamic Therapy, Chemistry Department State University of Maring, Av. Colombo 5.790 87020-900, Maring, Paran (Brazil) [c] Dr. M. Rizzi, Prof. F. Ren Innovative Research Laboratory for Wound Healing Health Sciences Department Universit del Piemonte Orientale “A. Avogadro Via Solaroli 17, 28100 Novara (Italy) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201404296. Chem. Eur. J. 2014, 20, 1 – 6
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Communication specific surface area (SSA), pore diameter, and pore volume decreased when RB was covalently bound to MSNs, confirming that the RB molecules have been introduced into the mesoporous channels. To get more insights on the location of RB, RB molecules were covalently bound to MSNs without removing the CTAB surfactant; under these conditions, the mesopores were completely occluded and RB could only be attached to the external surface of the nanoparticles. In this latter case, we observed a RB leaching of 80 %, calculated from the UV/Vis specScheme 1. Schematic representation of the synthetic procedure used to obtain RB-modified MSNs (RB-MSNs). tra of the eluate by using the Lambert–Beer law. Conversely, when RB was attached after surfactant removal, ca. 85 % of the induce toxicity owing to 1O2 release, producing subsequent RB molecules were stabilized inside the pores (Figure S3 in the cell damage or even cell death. Supporting Information). The procedure for the synthesis of RB-MSNs is reported in The DR UV/Vis spectrum of the RB-MSNs solid is reported in Scheme 1. The amine-functionalized MSNs were obtained by Figure 2 together with the UV/Vis spectrum of RB in a solution a one-pot cetyltrimethylammonium bromide (CTAB) template of water. At physiological pH, the absorption maximum of free synthesis using 3-aminopropyltriethoxysilane (APTS) and tetRB is located at 547 nm, while the maximum of RB-MSNs is raethyl ortosilicate (TEOS), as described in literature.[27] The surred-shifted at 564 nm owing to the confinement effect and factant was removed by Soxhlet extraction in acidic conditions. a different chemical environment inside the mesopores. In adThe amine groups are necessary to conjugate the activated RB. dition, the weak shoulder at a lower wavelength observed in The covalent coupling between RB and NH2-MSNs was confirmed by FTIR spectral analysis (Figure S1 in the Supporting Information). UV/Vis absorption spectroscopy was used to evaluate the loading of RB on RB-MSMs (0.034 mmol per mg MSNs).The average number of RB molecules per nanoparticle was calculated to be about 4.20 104 by considering the density of the mesoporous silica nanoparticles[28] and the particle size. The RB-MSNs were characterized by XRD and HRTEM analyses to elucidate the structural modification and particle dimensions of the MSNs after the post-synthetic route to introduce the RB molecules inside the mesopores. Figure 1 A shows that both NH2-MSNs and RB-MSNs display the typical XRD pattern of an ordered hexagonal network of mesopores with well defined (100), (110), and (200) reflections,[29] indicating that the synthetic pathway adopted for anchoring RB on the silica surface did not affect the ordered assembly of the MSNs pores. The HRTEM images of RB-MSNs (Figure 1 B) reveal the presence of well-ordered nanostructured mesoporous particles with an Figure 1. A) XRD patterns of extracted NH2-MSNs and RB-MSNs. B) HRTEM average particle size in the 160–180 nm range, which was also images of RB-MSNs; scale bars: 20 nm (top), 50 nm (bottom). confirmed by DLS analysis (Table 1, see the Supporting Information). The textural properties of NH2-MSNs and RB-loaded MSNs Table 1. Textural properties of MSNs. were obtained by N2 adsorption–desorption isotherms at 77 K (Figure S2 in the Supporting Information) and are reported in DDFT [] V [cm3 g1] Samples SSABET [m2 g1] Table 1. The functionalized MSNs show type IV isotherms with NH2-MSNs 1120 37.5 1.3 H1-type hysteresis, which is clearly representative of standard RB-MSNs 885 36 0.9 M41S materials (Figure S2 in the Supporting Information). The &
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Figure 2. DR UV/Vis spectrum of RB-MSNs (curve a) and UV/Vis absorption spectrum of free RB in a solution of water (5 106 m, curve b).
both the solid sample (curve a) and the RB in solution (curve b) can be attributed to the presence of some RB aggregates[5] that, nonetheless, did not affect the performances of the RB-MSNs in the photoproduction and release of 1O2 (see below). The protection effect of the inorganic mesoporous nanostructure towards RB photodegradation was evidenced by the UV/Vis spectra of RB in solution and RB-MSNs samples upon irradiation with visible light for several days (Figure S4 in the Supporting Information). The decrease of the RB absorption band due to photodegradation is almost stopped when RB is embedded into MSNs nanoparticles, while photobleaching is rapid when RB is in solution. The singlet-oxygen generation was evaluated by a chemical method using uric acid (UA) as detector.[30–32] Under irradiation at neutral pH, UA is stable as a monoanion with a band centered at 292 nm. When singlet oxygen is released, as an effect of PS photoexcitation, UA is irreversibly oxidized and a decrease in the intensity of the band at 292 nm is observed. In our experiments, a solution of uric acid in water was irradiated at 540 nm in the presence of RB-MSNs solid nanoparticles, and the decreasing of the UA band at 292 nm was monitored for 4800 sec (Figure 3 A). The decay curves of UA absorption at 292 nm as a function of the irradiation time are reported in Figure 3 B in which the RB-MSNs decay is compared to the decay of MSNs without the photosensitizer (NH2-MSNs). Only in the presence of RB, a decay in the UA band due the 1O2 release was observed. The slope of the curve is roughly proportional to the efficiency of singlet-oxygen generation.[32–34] A direct comparison between the RB-MSNs and RB in solution by monitoring the UA photooxidation is not possible, because a water suspension of RB-MSNs is used for the photoproduction of 1O2 and this is affected by nanoparticle light scattering. Therefore, the efficiency of singlet-oxygen delivery of RB-MSNs (hRBMSNs) was calculated by using Equation (1)[35]:
hðRBMSNsÞ ¼ FRB 1 O2
tRB tðRBMSNsÞ
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Figure 3. A) Absorption spectra of UA in the presence of RB-MSNs in water after different irradiation times with 540 nm light. B) Decay curves of the UA absorption band at 292 nm as a function of the irradiation time in the presence of RB-MSNs and NH2-MSNs.
in which t(RB) is the UA decreasing time in the presence of RB in a solution of water, t(RB-MSNs) is the UA decreasing time in the presence of RB-MSNs in a suspension of water, both adjusted to a first-order exponential decay, and FRB is the singletoxygen quantum yield of free RB in a solution of water (F 1 O2 = 0.75 under 540 nm light irradiation). The h value obtained for RB-MSNs is 0.74, which is very close to the quantum yield of singlet-oxygen release of free RB; this means that the 1O2 produced by the excited RB immobilized on the silica surface diffuses efficiently through the MSNs mesopores. Owing to the high singlet-oxygen release achieved when RB is attached to the inner walls of the mesoporous silica nanostructure, in vitro tests were performed by using melanoma cells. The effects of rose bengal loaded silica nanoparticles on cell proliferation in the absence or presence of light activation was evaluated in a melanoma cellular model (SK-MEL-28).[23] The SK-MEL-28 cell line has been chosen for this test, because it undergoes cell death after RB high concentration (100– 200 mm) treatment, even in the absence of light.[36] As a further control, cells were also treated under the same conditions with unloaded silica nanoparticles. Cellular specimens were preincubated for 5 h in a serum-free medium before light stimulation (5 min under green light), and cell proliferation was evaluated after 18 h of incubation in a complete cell-culture medium. As shown in Figure 4, control cells proliferation was not affected
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Communication thylammoniumbromide (CTAB) as a structure-directing agent.[27] CTAB (1.9 mmol) was first dissolved in water (340 mL). Then, a solution of NaOH (2.0 m, 2.45 mL) was added to the solution of CTAB, and the temperature was adjusted to 80 8C. Finally, tetraethoxy silane (TEOS, 3.5 mL, 18.1 mmol) and 3-aminopropyltriethoxy silane (APTS, 0.43 mL, 2.04 mmol) were added simultaneously and dropwise to the solution over a period of 4 min. The mixture was stirred at 80 8C for 2 h to give rise to white precipitates. The solid product was filtered, washed with deionized water and ethanol, and dried in vacuo. The surfactant template (CTAB) was removed by Soxhlet extraction with 2-propanol/HCl for 96 h at 200 8C.
Covalent conjugation of Rose Bengal with NH2-MSNs Firstly, extracted NH2-MSNs were dispersed in DMF (15 mL) and then sonicated for 15 min. To covalently conjugate RB to NH2MSNs, a solution of DMF (15 mL) containing rose bengal (RB, 1 equiv), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5b]pyridinium 3-oxid hexafluorophosphate (HATU, 1 equiv), and N,Ndiisopropylethylamine (DIPEA, 2 equiv) was added to the NH2MSNs dispersion and stirred vigorously for 24 h at room temperature protected from light. The solid hybrid was then filtered and dried under vacuum. The as-synthesized RB-MSNs solids were washed with DMF several times to remove the unreacted RB molecules. After the washing procedure, the actual RB loading was calculated from the UV/Vis spectrum of the RB eluate by using the Lambert–Beer law (e566 = 95 780 m1 cm1 in DMF measured from the slope of a plot).
Figure 4. MSNs and RB-MSNs effects on SK-MEL-28 cell proliferation. Representative optical microscopy images (magnification 10 ) and quantification of MSNs and RB-MSNs effects on cell proliferation in the absence (white bars) or presence (grey bars) of light stimulation. Results are expressed as mean values standard deviation (SD). * p < 0.05.
Cellular toxicity
by light stimulation (83.93 25.62 cells/mm2 and 81.68 23.65 cells/mm2). In the absence of light stimulation, preincubation with RB-MSNs did not alter cell proliferation (87.36 26.48 cells/mm2), whereas after light activation it was able to reduce cell proliferation (54.61 11.54 cells/mm2, p < 0.05). As expected, unloaded silica nanoparticles did not significantly alter cellular proliferation, both in the absence or presence of light stimulation (64.10 8.79 cells/mm2 and 47.85 13.48 cells/ mm2, respectively). In summary, a simple surface-modification process was adopted to conjugate the photosensitizer RB with amine-functionalized MSNs through covalent bonding to yield a photostable RB-modified MSNs, that is, the porous inorganic structure protects the embedded molecules from photochemical degradation. The RB confined inside the mesopores of MSNs has been successfully photoexcited, and the singlet-oxygen generation has been monitored by a chemical method using uric acid (UA) as detector. The efficiency of singlet-oxygen delivery of RB-MSNs is high, meaning that 1O2 can easily diffuse through the MSNs mesopores into the UA solution. Promising results have been obtained by in vitro tests using an aggressive melanoma cellular model that remarks the potential application of RB-MSNs as a PDT agent.
2.5 104 cells were seeded in 24-wells culture plates and allowed to adhere overnight. Non adherent cells were then removed by a gentle wash in phosphate buffer (PBS, pH 7.4). Test silica nanoparticles aqueous suspensions (0–100 mg mL1) were added to the cells in a serum-free culture medium and incubated for 5 h before light irradiation. The cells were irradiated for 5 min with a green light in the absence of cell-culture medium. Immediately after each irradiation, cell-culture medium was added to the cells. In parallel, some samples were treated with silica nanoparticles but were not irradiated with light. After irradiation, the serum-free cell-culture medium was replaced by complete cell-culture medium in all samples. The cells were further incubated overnight and the cell viability was assessed by counting formalin-sucrose-fixed cells after crystal-violet staining. The counting procedure was performed by two different researchers blinded to experimental groups to assess the reproducibility of the analysis. Cell proliferation was evaluated by counting cells in each microscopic field in at least 3 fields for each experimental condition. The cell density was expressed as a mean number of cells/mm2 standard deviation (S.D.).[37]
Acknowledgements The authors thank the Masterlight project (Bando di Ateneo CSP 2012) for financial support. B.M.E. thanks CAPES (Coordenażo de AperfeiÅoamento de Pessoal de Nvel Superior) for financial support. The authors are grateful to Prof. Daniela Taverna from the Molecular Biotechnology Center, Department of Molecular Biotechnology and Health Sciences, University of Turin (Italy) for providing the highly metastatic human melanoma cell line SK-MEL-28 used in this work.
Experimental Section Synthesis of functionalized MSNs nanoparticles Functionalized mesoporous silica nanoparticles (NH2-MSNs) were prepared according to literature procedures by using cetyltrime-
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Received: July 8, 2014 Published online on && &&, 0000
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COMMUNICATION & Nanoparticles
An efficient and photostable rose bengal (RB) nanoplatform for photodynamic therapy has been successfully synthesized. RB-modified mesoporous silica nanoparticles (MSNs) have shown a relevant photostability and a high efficiency in the photoproduction and delivery of singlet oxygen (1O2), and in vitro tests have evidenced that RB-MSNs are able to reduce cell proliferation in one of the most aggressive skin-cancer types (SK-MEL-28) after green-light irradiation.
E. Gianotti,* B. Martins Estev¼o, F. Cucinotta, N. Hioka, M. Rizzi, F. Ren, L. Marchese && – && An Efficient Rose Bengal Based Nanoplatform for Photodynamic Therapy
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