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Langmuir 2008, 24, 2064-2071
Core-Shell Nanoparticles: Characterization and Study of Their Use for the Encapsulation of Hydrophobic Fluorescent Dyes Jorge L. Cha´vez, Jeffrey L. Wong, and Randolph S. Duran* Butler Polymer Research Laboratories, Department of Chemistry, UniVersity of Florida, P.O. Box 117200, GainesVille, Florida 32611 ReceiVed July 23, 2007. In Final Form: October 18, 2007 Core-shell nanocapsules intended to be used as drug scavengers were prepared using a surfactant mixture containing octadecyltrimethoxysilane (OTMS) as a reactive amphiphile, to form spherical templates. A siloxane shell was grown on the surface of the templates by reacting tetramethoxysilane (TMOS) with the silanol groups obtained after the hydrolysis and condensation of OTMS. Dynamic light scattering (DLS) showed that particles with diameters in the range of 100-200 nm were obtained, with core and shell sizes controlled by varying component compositions. Atomic force microscopy (AFM) was used to study the effect of the silica coating of the templates on their robustness after deposition on a substrate. Subsequently, we present studies on the encapsulation of two hydrophobic fluorescent dyes, which are sensors of polarity and rigidity. Steady-state fluorescence spectroscopy was used to examine the fluorescence response of the dyes before and after shell growth. Changes in the emission of the encapsulated dyes were related to changes in the polarity and rigidity of the microenvironment where the dyes were located and correlated to the AFM results. Finally, dye-free core-shell particles were used to sequester the dyes from aqueous suspensions. Fluorescence of the sequestered species was compared to the dye-loaded particles to determine the final fate of the fluorophores in the nanoparticles.
Introduction In the past few years, different authors have emphasized the importance of compartmentalized sub-micrometer particles, especially when different microenvironments inside these architectures are offered for guest molecules.1-6 One of the most promising alternatives in nanoparticle research is the synthesis of particles that have a polar surface, allowing their use in aqueous systems. This offers potential application in the biomedical field,4,7 where particles with diameters smaller than 100 nm are generally used.7,8 When the interior of these particles is hydrophobic, they have potential applications as carriers for drugs,8-10 fluorescent labels,11-13 and reactive molecules,14 which allow their use in different applications ranging from drug delivery15-19 to nano* Corresponding author. Tel.: (352) 392-2011; fax: (352) 392-9741; e-mail:
[email protected]. (1) Kataoka, K.; Harada, A.; Nagasaki, Y. AdV. Drug. DeliVery ReV. 2001, 47, 113-131. (2) Couvreur, P.; Barratt, G.; Fattal, E.; Legrand, P.; Vauthier, C. Crit. ReV. Therap. Drug Carrier Syst. 2002, 19, 99-134. (3) Gittins, P.; Twyman, L. Supramol. Chem. 2003, 15, 5-23. (4) Saviæ, R.; Luo, L.; Eisenberg, A.; Maysinger, D. Science (Washington, DC, U.S.) 2003, 300, 625-627. (5) Cha´vez, J. L.; Wong, J. L.; Jovanovic, A. V.; Sinner, E. K.; Duran, R. S. IEE Proc.-Nanobiotechnol. 2005, 152, 73-84. (6) Caruso, F. AdV. Mater. 2001, 13, 11-22. (7) Li, Z. F.; Ruckenstein, E. Nano Lett. 2004, 4, 1463-1467. (8) Rangel-Yagui, C.; Pessoa, A.; Costa Tavares, L. J. Pharm. Pharmaceut. Sci. 2005, 8, 147-163. (9) Haag, R. Angew. Chem., Int. Ed. 2004, 43, 278-282. (10) Torchilin, V. P. Cell. Mol. Life Sci. 2004, 61, 2549-2559. (11) Koh, K.; Ohno, K.; Tsuji, Y.; Fukuda, T. Angew. Chem., Int. Ed. 2003, 42, 4194-4197. (12) Chen, G.; Guan, Z. J. Am. Chem. Soc. 2004, 126, 2662-2663. (13) Turner, J.; Wooley, K. Nano Lett. 2004, 4, 683-688. (14) Schappacher, M.; Putaux, J.; Lefebvre, C.; Deffieux, A. J. Am. Chem. Soc. 2005, 127, 2990-2998. (15) Nasongkla, N.; Shuai, X.; Ai, H.; Weinberg, B.; Pink, J.; Boothman, D.; Gao, J. Angew. Chem., Int. Ed. 2004, 43, 6323-6327. (16) Miller, D.; Kabanov, A. Colloids Surf., B 1999, 16, 321-330. (17) Allen, C.; Maysinger, D.; Eisenberg, A. Colloids Surf., B 1999, 16, 3-27. (18) Soppimath, K.; Aminabhavi, T.; Kulkarni, A.; Rudzinski, W. J. Controlled Release 2001, 70, 1-20. (19) Bae, Y.; Kukushima, S.; Harada, A.; Kataoka, K. Angew. Chem., Int. Ed. 2003, 42, 4640-4643.
reactors.20,21 A number of different ways to prepare core-shell particles has been proposed; one of the most common approaches is based on the synthesis of block copolymers,22-24 which form unimolecular micelles in solution if the blocks have complementary polarities. To improve the stability of the aggregates formed, cross-linking of the core or the shell has been proposed.25,26 In another typical approach, polymeric cores are synthesized inside microemulsion droplets, followed by the synthesis of a shell on the surface; hollow particles can be obtained by removal of the core by different means.27-32 Similar architectures are obtained by the layer-by-layer deposition of oppositely charged macromolecules on the surface of charged particles.33 Our group has reported the use of a mixed surfactant system as a template for the formation of spherical particles for use as drug scavengers,5,34,35 as shown in Scheme 1. The first step (20) Vriezema, E.; Aragone`s, M.; Elemans, J.; Cornelissen, J.; Rowan, A.; Nolte, L. Chem. ReV. 2005, 105, 1445-1490. (21) Lee, M.; Jang, C.; Ryu, J. J. Am. Chem. Soc. 2004, 126, 8082-8083. (22) Webber, G.; Wanless, E.; Armes, S.; Tang, Y.; Li, Y.; Biggs, S. AdV. Mater. 2004, 16, 1794-1798. (23) Webber, S. E. J. Phys. Chem. B 1998, 102, 2618-2626. (24) Kang, N.; Perron, M.; Prud’homme, R.; Zhang, Y.; Gaucher, G.; Leroux, J. Nano Lett. 2005, 5, 315-319. (25) Ro¨sler, A.; Wandermeulen, G.; Klok, H. AdV. Drug. DeliVery ReV. 2001, 53, 95-108. (26) Huang, H.; Kowalewski, T.; Wooley, K. L. J. Polym. Sci., Part A: Polym. Chem. 2003, 41, 1659-1668. (27) Jungmann, N.; Schmidt, M.; Ebenhoch, J.; Weis, J.; Maskos, M. Angew. Chem., Int. Ed. 2003, 42, 1714-1717. (28) Ni, K. F.; Shan, G. R.; Weng, Z. X.; Sheibat-Othman, N.; Fevotte, G.; Lefebvre, F.; Bourgeat-Lami, E. Macromolecules 2005, 38, 7321-7329. (29) Kobayashi, Y.; Misawa, K.; Kobayashi, M.; Takeda, M.; Konno, M.; Satake, M.; Kawazoe, Y.; Ohuchi, N.; Kasuya, A. Colloids Surf., A 2004, 242, 47-52. (30) Zoldesi, C. I.; van Walree, C. A.; Imhof, A. Langmuir 2006, 22, 43434352. (31) Ha, J.-W.; Park, I. J.; Lee, S.-B.; Kim, D.-K. Macromolecules 2002, 35, 6811-6818. (32) Wang, H.; Chen, P.; Zheng, X. J. Mater. Chem. 2004, 14, 1648-1652. (33) Caruso, F.; Caruso, R. A.; Mo¨hwald, H. Science (Washington, DC, U.S.) 1998, 282, 1111-1114. (34) Underhill, R. S.; Jovanovic, A. V.; Carino, S. R.; Varshney, M.; Shah, D. O.; Dennis, D. O.; Morey, T. E.; Duran, R. S. Chem. Mater. 2002, 14, 49194925.
10.1021/la702227d CCC: $40.75 © 2008 American Chemical Society Published on Web 01/26/2008
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Scheme 1. Overall Process for Formation of Core-Shell Particles
Chart 1. Chemical Structures of Surfactants and Dyes Used in This Study
Chart 2. List of Abbreviations Used in This Study
consists of the mixing of poly(oxyethylene)[20]-sorbitan monooleate (T80) and octadecyltrimethoxysilane with a small organic molecule, such as a fluorescent dye, or a small ester, see Chart 1 for chemical structures and Chart 2 for abbreviations. The methoxy groups of OTMS are then hydrolyzed and condensed to fix the geometry of the structures obtained, producing what we call templates. Finally, a siloxane network is synthesized on (35) Jovanovic, A. V.; Underhill, R. S.; Bucholz, T. L.; Duran, R. S. Chem. Mater. 2005, 17, 3375-3383.
the surface of the templates, using the free silanol groups as anchoring points. In the first part of this paper, we report on the characterization of the system by three techniquessdynamic light scattering, AFM, and transmission electron microscopy (TEM). The information obtained by the AFM analysis was found to be especially useful to study the effects of the molecules encapsulated and the effect of silica coating on the flexibility and robustness of the particles. We believe that these particles have potential applications as drug scavengers in biomedical applications. Hence, we focused our attention on studying the sequestration of hydrophobic molecules to see if the particles can internalize active probes. To study the sequestration of species from solution into particles, UV active species are commonly used.36-38 Typically, a volume of a known concentration of the absorbent is mixed with a suspension of the particles. After a stabilization period, the particles are removed, and the UV absorption of the species left on the absorbent phase is analyzed to obtain the amount of molecules that were sequestered. Although this is an effective and simple way to quantify the uptake capacities of the particulate systems used, this technique gives no information on the sequestration process, as it is unable to differentiate if the UV active species are adsorbed on the surface or transported into the interior of the particles. In the second part of this article, we report on the use of steady-state fluorescence spectroscopy to study the encapsulation of hydrophobic dyes in the core-shell nanoparticles. Contrary to the case explained previously, we studied the fluorescence response of the dyes captured by the particles. The emission profiles of the two fluorophores used are sensitive to polarity and rigidity changes in the environment where they are placed. On the basis of this, dye-doped particles were prepared in a simple way, by including coumarin 153 (C153) or pyrene in the synthesis of the nanoparticle. The emission maxima of the dyes in the templates and the core-shell particles were analyzed and compared, providing a way to identify as to whether the dyes were located near the surface, in contact with the solvent, or protected from it after shell growth. Finally, we used dye-free core-shell particles to sequester the dyes from aqueous suspensions. On the basis of the information obtained with the dye-loaded particles, we used the emission maximum of the dyes to identify the final fate of the sequestered dyes in the nanoparticles. Experimental Procedures Materials. Poly(oxyethylene)[20]-sorbitan monooleate, sodium octanoate (SO), 1-dodecene (1-d), C153, pyrene, and ethyl butyrate (EB) were purchased from Aldrich. OTMS and tetramethoxysilane (36) Meier, M. A. R.; Gohy, J.-F.; Fustin, C.-A.; Schubert, U. S. J. Am. Chem. Soc. 2004, 11517-11521. (37) Aathimanikandan, S. V.; Savariar, E. N.; Thayumanavan, S. J. Am. Chem. Soc. 2005, 127, 14922-14929. (38) Tamano, K.; Imae, T.; Yusa, S.; Shimada, Y. J. Phys. Chem. B 2005, 1226-1230.
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2066 Langmuir, Vol. 24, No. 5, 2008 Table 1. Composition of Formulations and Size Data for T80/SO/OTMS Combinationsa T80 (g) SO (g) T80/SO (mol)
OTMS (mg) T80/SO/OTMS (mol) d(DLS) (nm) d(TEM) (nm)
A
B
0.7 0.7 0.13
0.4 1.0 0.05
shell particles, see Supporting Information Figure S1). The shell thickness (ST) was calculated as follows: ST ) (d2 - d1)/2
A1
A2
A3
B1
B2
B3
10 20:156:1 47 ND
22.4 9:70:1 54 62
43.3 5:35:1 64 69
7 13:316:1 63 ND
22.2 5:100:1 75 68
45.4 3:50:1 88 84
a All DLS measurements were performed at 20 °C and 90°. TEM results were obtained by sizing the particles from micrographs similar to the ones shown in Figure 1. ND: not determined.
were purchased from Gelest Inc. OTMS was distilled at 0.05 mmHg prior to use and stored under Ar. All other reagents were used without further purification. Preparation of Templates. T80 and SO were mixed with 13.4 mL of saline (9 g of NaCl in 1 L of deionized water) at room temperature (RT) in an orbital shaker (J-Kem Scientific, model BTS 1500) at 300 rpm (see Table 1 for quantities). After 4 h, the samples were divided in three equal volumes, and different amounts of OTMS were added (Table 1). The mixtures were placed in an orbital shaker at 300 rpm and heated to 75 ( 0.5 °C overnight to promote the inclusion of OTMS in the system. After cooling down to RT, the pH of the solution was increased to approximately 10 using a 0.5 M NaOH aqueous solution. After 30 min, the pH was decreased to 7.4 using a 0.5 M HCl aqueous solution. Prior to any characterization or fluorescence studies, the templates were purified through 25 nm pore sized membranes (Millipore). Finally, the concentrated suspensions were diluted to their original volume, sonicated for 15 min, and filtered with 450 nm pore sized filters (Fisherbrand). Synthesis of Doped Core-Shell Nanoparticles. The core-shell particle synthesis started with the template formation as explained previously. For dye-loaded particles, the dye, pyrene for NPY or C153 for NCU, was mixed with the surfactants at room temperature in the first step of the synthesis. For the NEB system, EB and 1-dodecene were mixed with the surfactants at 75 ( 0.5 °C for 4 h. This was followed by OTMS addition as explained previously. Once the templates were obtained, a 5-fold dilution of the original template suspension was necessary before adding TMOS, to avoid cross-linking. TMOS was added in small portions (approximately 20 mg every 24 h), and the solution was allowed to stir until the size reached the desired range, as observed by DLS. Unlike the templates, the core-shell particles could be centrifugated. On the basis of this, the nanocapsules were centrifuged in a Sorvall RC 5B centrifuge (GMI, Inc.) at 2990g for 80 min to remove excess reagents. The nanocapsules were recovered as a pellet and resuspended by sonication for approximately 1 h. This process was repeated twice. After resuspension, the samples were filtered with 450 nm pore sized filters. Loading of Nanocapsules with Dye. The nanocapsule suspension (10 mL, purified and resuspended to its original volume) was mixed with the proper volume of a stock solution of C153 (system NCUSL) or pyrene (system NPYSL) dissolved in methanol, to obtain the desired final dye concentration as reported in Table 4. The samples were vortexed and then stirred for 1 h. Size Analysis. Size analysis was performed at two different stages: after purification of the templates and after centrifugation of the core-shell particles. This allowed determination of the core diameter and shell thickness for each nanocapsule suspension. Measurements were performed using a Precision Detectors PDDLS/ CoolBatch+90T instrument. The data were analyzed with the Precision Deconvolve32 Program. The measurements were taken at 20 °C and a 90° scattering angle, using a 683 nm laser source. Final sizes were obtained from the average of five trials (for a comparison of the correlation function obtained for the templates and core-
where d1 is the template diameter and d2 the core-shell particle diameter. TEM. One drop of a diluted template/nanocapsule suspension was placed on top of a Formvar carbon coated nickel grid, and the solvent was evaporated at RT. Samples for the NEB system were stained with OsO4 vapors for 15 min and for 90 s with a methanolic solution of uranyl acetate (UA, 2 wt %) immediately before measurement. Dye-loaded particles were stained with UA only. TEM images were taken with a Hitachi H-7000 microscope at 75 kV. Sizing was performed with the image J software; approximately 50 particles were used in each case. AFM. Samples for AFM analysis were prepared by depositing a few drops of a diluted suspension of nanocapsules on a piece of freshly cleaved mica substrate. The samples were stored at RT until use. AFM analysis was performed by scanning in tapping mode with a Nanoscope III AFM instrument (Digital Instruments, Inc.), using silicon probes (Nanosensor dimensions: T ) 3.8-4.5 µm, W ) 26-27 µm, and L ) 128 µm). The z-calibration was performed with a silicon grating (TGZ01, Mikromash), with a step height of 20 nm (accuracy 1 nm). Images were analyzed with the Nanoscope III software, using 150 particles in each case. Fluorescence Images. A drop of the nanocapsule suspension was placed on a clean glass slide (Corning Inc.) and dried at room temperature. Imaging was performed on a fluorescence microscope (Olympus, model IX70) with an excitation maximum at 360 nm ((20 nm) and emission maximum at 525 nm ((25 nm). Fluorescence Spectroscopy. Steady-state fluorescence spectroscopy measurements were carried out in a FluorologMax-3 (Horiba Jobin Yvon) with the following setup: 2 mm slits and 1 s integration time. The dye-loaded particles were dialyzed until no more dye could be removed, as observed by UV. After this, they were stored at room temperature. Before the measurements, the suspensions were diluted 5 times to reduce the scattering of light due to the size of the particles and were stirred for 1 h. It was not possible to avoid the presence of the scattering peaks, as observed in the emission spectra of encapsulated C153, but these appeared far from the maximum. As a consequence, no complications in the determination of the maximum in the emission of the dye were encountered. The dye-free particles were diluted and homogenized similarly to the doped particles. After addition of the dye, the samples were vortexed and stirred for 1 h before measurement.
Results and Discussion Previous reports from our group have demonstrated the possibility of using mixed surfactant systems to form templates for the synthesis of core-shell particles, as shown in Scheme 1.5,34,35 In this report, we included in the synthesis of the particles another surfactant, sodium octanoate, to increase the stability of the templates obtained due to the presence of negative charges at a neutral pH. Different T80/SO/OTMS mixtures were used to prepare templates, and their sizes were obtained by DLS and TEM, as shown in Table 1. Size analysis by DLS was performed for the T80/SO combinations before the addition of OTMS to the systems, showing a low intensity of the scattered light and diameter values between 6 and 8 nm in all cases, similar to T80 micelles.35,39 After OTMS addition, the pH of the suspension was changed to promote its hydrolysis and condensation, which produced templates with sizes between 50 and 90 nm. The formulations containing more OTMS produced more templates, observed as a dominant signal for the larger scatterers in the (39) Heydenreich, A. V.; Westmeier, R.; Pedersen, N.; Poulsen, H. S.; Kristensen, H. G. Int. J. Pharmaceut. 2003, 83-87.
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Figure 1. TEM and AFM height images of NEB templates (left) and core-shell particles (right), including the section analysis obtained by AFM (bottom). Scale bars in all images is 500 nm.
DLS results. TEM images shown in Figures 1-3 confirmed that the templates were spherical. Having in mind that one of our goals is the use of these particles as drug detoxification agents, we selected a formulation similar to samples A2 and A3, with template sizes around 60 nm, which is optimal for the production of particles for biomedical applications.4,7 Nanoparticles NCU and NPY were prepared including the hydrophobic dyes, C153 and pyrene, respectively, to study their fluorescence when placed in the interior of the particles, as will be discussed later. The system NEB was prepared with a combination of two small hydrophobic molecules to study the distribution of a dopant in the particles with the electron microscope and how it affects the robustness of the particles with the atomic microscope, as explained next. The results for the size characterization of the particles obtained are shown in Table 2. An increment in the diameter obtained after the addition of TMOS was observed. The shell thickness was obtained by the difference in size between the templates and the core-shell particles, assuming that one template produced one particle, as explained in the Experimental Procedures. The results obtained show that the templates were successfully coated with a siloxane shell.
A number of authors has used TEM for the characterization of core-shell particles, especially in cases where the shell was prepared with silicate precursors.11,29,40 In our case, a difficulty for imaging arises from the lack of high electron density in the different parts of the capsules. The NEB system was prepared with a mixture of dopants containing 1-dodecene, to react OsO4 with its unsaturations.35 This will fix the metal atom in the interior of the particles, increasing the contrast. Before imaging by TEM, the particles were stained with uranyl acetate to facilitate the visualization of their periphery.26 Figures 1-3 show TEM images of the NEB, NCU, and NPY systems, respectively. In all cases, spherical particles were observed, with sizes corresponding to the values obtained with DLS (Table 2). The images of the NEB templates showed particles uniformly stained, which indicates a homogeneous distribution of the filler molecules in their interior, as observed in Figure 1. In our case, the nonreactive surfactants are expected to be removed during purification due to their water solubility and the fact that they are not chemically connected to the templates. It was expected that only a thin skin that collapses (40) Gonza´les-Pe´rez, A.; Ruso, J. M.; Prieto, G.; Sarmiento, F. Langmuir 2004, 20, 2512-2514.
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Figure 2. TEM and AFM height images of NCU templates (left) and core-shell particles (right), including the section analysis obtained by AFM (bottom). Scale bars in all images is 300 nm.
after deposition on the substrate would be seen with the electron microscope, if the condensation of OTMS happened only on the surface of the particles. The TEM images suggested that we actually obtained templates that can maintain their geometry (at least in two dimensions) after the solvent has evaporated, suggesting that a more robust network has formed. This was further studied by AFM, as discussed next. AFM was found to be a valuable tool to characterize the templates and core-shell particles, as shown in Figure 1 for the NEB system. The diameter and height values for the particles were obtained from the section analysis (bottom image in Figure 1). The NEB templates showed a larger diameter than the other systems due to the presence of filler molecules in the core and a very low height value that indicated that they flattened but not collapsed on the substrate after solvent evaporation, suggesting a non-rigid interior. These images confirmed the TEM results, indicating that the templates are made of a robust material that let them maintain their shape. We used the diameter/height ratio (DHR) as a comparison of the rigidity of the templates and
particles, with a large DHR value as an indication of greater flexibility.32 The NEB templates showed hollow-like structures, with a DHR of 15.3, indicating a very flexible system (Table 3). After the shell growth, it is possible to observe changes on the surface of the particles due to the silica coating, as well as a decrease in the DHR value to 9.3. On the basis of these results, we believe that the shell built on the periphery prevents the particles from spreading as much as the templates on the substrate, producing a more robust system. This is confirmed by the results obtained from Figures 2 and 3 for the NCU and NPY systems. In both cases, the same diameter and height values were obtained, 63 and 5 nm, respectively, with a DHR value of 12.6. This value is lower than the one for NEB templates since these are smaller and more rigid templates with just a small amount of dye in their interior. After the shell growth, both the diameter and the height increased considerably for both dye-doped systems. Surprisingly, despite using similar amounts of the siloxane shell precursor, NPY grew a thicker shell than NCU, observed as a different diameter by DLS (200 and 120 nm, respectively). These values
Core-Shell Nanoparticles: Characterization and Study
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Figure 3. TEM and AFM height images of NPY templates (left) and core-shell particles (right), including the section analysis obtained by AFM (bottom). Scale bars in all images is 500 nm. Table 2. Formulations Used in Synthesis of Core-Shell Particles and DLS Characterization Resultsa SO T80 EB 1-d OTMS NEB 125 125 29 NCU 125 125 NPY 125 125 -
10 -
19 12 13
Py
C153 TMOS
0.70 0.45 -
42 15 14
d1
d2
ST
175 203 14 80 120 20 74 200 63
a All quantities given in milligrams. d1: template diameter; d2: coreshell nanocapsule diameter; and ST: shell thickness; all these values are in nanometers. Py: pyrene.
Table 3. AFM Characterization Results for Templates (1) and Core-Shell Particles (2) Prepared as Indicated in Table 2a NEB NCU NPY a
d1
h1
DHR1
d2
h2
DHR2
183 63 63
12 5 5
15.3 12.6 12.6
195 87 141
21 21 42
9.3 4.1 3.4
d: diameter and h: height; values are in nanometers.
correlate to the diameters obtained in the AFM analysis (141 and 87 nm, respectively) and, more importantly, to the height values, which increased dramatically to 41 and 21 nm, resulting in a decrease in the DHR values to 3.4 for NPY and 4.1 for NCU. This is a more dramatic change than the NEB templates to particles since the dye-doped particles grew a thicker shell.
We performed preliminary studies on the robustness of the templates and core-shell particles. They were calcinated by being heated slowly to 600 °C at a speed of 1 °C/min. The species left after this process were imaged by TEM as shown in Supporting Information Figures S2 and S3. In the case of the templates, for the most part, only a fiber-like material was observed with a few species resembling the original spherical templates. In the case of the core-shell particles, spherical particles with small holes in their interior were observed. This seems to indicate that the templates tend to break apart upon heating and removal of the organic moieties in their interior, while the core-shell particles seemed to be more resistant to this process. These are preliminary data that are being studied in more detail. Encapsulation of dyes in different systems, especially for labeling and imaging purposes, has been an important topic of research in the past few years.41-44 The characterization studies discussed previously suggest that our particles consist of a porous (41) Rigler, P.; Meier, W. J. Am. Chem. Soc. 2006, 128, 367-373. (42) Len, Y.; Tsai, P.; Huang, H.; Kuo, C.; Hung, Y.; Huang, D.; Chen, Y.; Mou, C. Chem. Mater. 2005, 17, 4570-4573. (43) Sun, H.; Scharff-Poulsen, A. M.; Gu, H.; Almdal, K. Chem. Mater. 2006, 18, 3381-3384. (44) Kalyankar, N. D.; Sharma, N. K.; Vaidya, S. V.; Calhoun, D.; Maldarelli, C.; Couzis, A.; Gilchrist, L. Langmuir 2006, 22, 5403-5411.
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2070 Langmuir, Vol. 24, No. 5, 2008 Table 4. Steady-State Fluorescence Data for Dye-Doped Templates and Core-Shell Particlesa NCU λmaxc a
NCUSL
Templates
np
533
525
0.26
µMb
543
NPY 2.5 µM
b
541
I3/I1d I1/Iexc
NPYSL
templates
np
0.12 µMb
2.1 µMb
0.94 4.44
0.94 12.1
1.38 -
0.84 1.90
np: core-shell particles. b Final dye concentration in the nanoparticle suspension. c Excitation wavelength 420 nm. d Excitation wavelength 339
nm.
and flexible core, protected from the exterior by the silica shell. We believe that these particles are a promising alternative for the encapsulation of nonpolar or harmful dyes, which need to be isolated from the aqueous media to be useful in bioapplications. To prove this, we decided to encapsulate hydrophobic dyes whose fluorescence responds to changes in the environment where they are placed. We used these fluorophores as sensors of polarity and rigidity, by identifying the maximum in their emission and comparison with values reported in the literature in different solvents and matrices. First, we studied the emission profile of the dyes encapsulated by the templates and the core-shell particles to investigate if the coating process isolates the core of the particles from the solvent. We start our discussion with the NCU system. C153 is a highly hydrophobic, fluorescent dye that has been used extensively in different systems as a probe for polarity and rigidity due to its large change in the dipole moment after its excitation.45-47 This dye can be excited between 400 and 450 nm and emits above 500 nm, a profile similar to dyes commonly used in protein labeling. A fluorescence microscopy image of the NCU core-shell particles is presented in the Supporting Information (Figure S4). The size of the particles was below the resolution limits of the microscope, so it was necessary to perform the imaging after the solvent had evaporated to promote particle aggregation. The emission is observed to be confined to spots with diameters in the range of a few micrometers, which corresponds to the aggregates observed in the AFM images. Table 4 shows the emission maximum of C153 in the templates and in the core-shell nanocapsules (for a comparison of the emission of the dye in the templates and core-shell particles, see Figure S5 in the Supporting Information). The maximum shifted from 533 to 525 nm after the shell growth, indicating a decrease in the polarity of the environment where the dye is placed, similar to a change in the emission maximum of the dye when dissolved in ethanol (530 nm) and butanol (525 nm).45-47 We interpret this decrease in the apparent polarity of the environment surrounding the dye as a consequence of the shell acting as a barrier to isolate the interior of the particles, where hydrophobic molecules tend to reside, from the polar solvent. To investigate if these results were a general trend for encapsulated species in our particles, we chose to study the fluorescence of pyrene in a similar manner due to its well-known emission dependence on solvent polarity.48 The ratio of the third and first peak intensities of its emission (I3/I1) has been qualitatively related to changes in polarity in the environment where the dye is placed. This is a consequence of the perturbations of the intensities of the vibrational fine structures in its emission spectrum, due to the extent of interactions between the solvent dipoles and the excited singlet states of pyrene. In the presence of polar solvents, there is an enhancement of the 0-0 band at (45) Grant, C. D.; DeRitter, M. R.; Steege, K. E.; Fadeeva, T. A.; Castner, E. W., Jr. Langmuir 2005, 21, 1745-1752. (46) Kumbhakar, M.; Nath, S.; Mukherjee, T.; Pal, H. J. Chem. Phys. 2004, 121, 6026-6033. (47) Stathatos, E.; Lianos, P.; Stangar, U. L.; Orel, B. Chem. Phys. Lett. 2001, 345, 381-385. (48) Kalyanasundaram, K.; Thomas, J. K. J. Am. Chem. Soc. 1977, 99, 20392044.
the expense of the others. This efficient dipolar coupling, with solvents such as acetonitrile or dimethyl sulfoxide, causes a drop in the peak ratio to as low as 0.5. However, when there is minimal coupling, the ratio is 2.0 (as in perfluoromethylcyclohexane).49,50 In our case, the fluorescence of the encapsulated dye was dominated by the excimer emission, observed as a broad peak centered at 475 nm, in both the templates and the core-shell particles, which prevented us from using I3/I1 to investigate the polarity of the interior of the particles. Excimers are formed if the photoexcited molecule, during its lifetime, approaches an unexcited dye molecule to form a collisional complex. Since this process is dominated by translational diffusion, it has been related to the viscosity of the media where the dye resides.51 Hence, the ratio of the intensities of the emission of the monomer and excimer (I1/Iexc) is related to the ability of the dye molecules to form dimers and can be used as an indicator of changes in the viscosity of the media.50 Despite that no change in the dye concentration is expected during the particle synthesis, it was observed that the I1/Iexc ratio increased from 4.41 to 12.1. We believe that this is a consequence of the increased difficulty that the excited monomers encounter to form excimers with ground-state monomers in the more rigid interior of the core-shell particles52 (see Figure S6 for a comparison of the emission spectra of the dye in the templates and core-shell particles). The results of the steady-state fluorescence studies for both dyes correlated with the AFM observations, showing a decrease in the flexibility of the core-shell particles as compared to the templates. In our earlier reports, we showed that these core-shell particles can be used as model systems for drug detoxification studies. It was observed that they can sequester quinoline from saline solutions, but it was not clear if the molecules were adsorbed on the surface or effectively internalized by the particles.35 On the basis of the fluorescence results explained in the previous sections, we decided to use the polarity/rigidity sensitivity of the dyes to study their uptake from aqueous suspensions and to identify their final location in the nanoparticles. To do this, we prepared dye-free nanoparticles similarly to the systems studied previously (see Supporting Information for quantities) and mixed them with a volume of methanolic solution of C153 and pyrene, samples NCUSL and NPYSL, respectively. Because of the low concentrations of the dyes used and their hydrophobicity, we expected them to reside preferentially in the particles. The maximum in the C153 emission showed a value shifted to the red, as compared to the NCU system, which we interpreted as a more polar microenvironment sensed by the probe. This value shifted slightly to the blue from 543 to 541 nm with a 10-fold increment in the dye concentration. The polarity sensed by the probe is higher than when the dye is dissolved in methanol (536 nm) and similar to the values obtained by Stathatos et al. in silica/poly(propylene oxide) composites.47 The emission maximum indicates that the dye molecules did not penetrate the shell of the particles but (49) Wilhelm, M.; Zhao, C.-L.; Wang, Y.; Renliang X. a.; Winnik, M. A. Macromolecules 1991, 24, 1033-1040. (50) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 2nd ed.; Kluwer Academic/Plenum Publishers: New York, 1999. (51) Vanderkooi, J. M.; Callis, J. B. Biochemistry 1974, 13, 4000-4006. (52) Jang, J.; Oh, J. H. AdV. Mater. 2003, 15, 977-980.
Core-Shell Nanoparticles: Characterization and Study
stayed close to the surface. This is probably due to the interaction of the carbonyl and amino groups with the silanol groups on the shell, similarly to what was observed by our group with an electroactive probe.53 Pyrene was used in a similar manner, at a low concentration, when no excimers were formed, and we obtained a I3/I1 value of 1.38. This value indicates a polarity sensed by the probe in between that of isopropyl alcohol and n-hexane (1.1 and 1.65, respectively), lower than the one obtained with C153, indicating effective protection from the solvent. The difference in the polarity experienced by the dye may be due to the lack of functional groups in pyrene that prevents interaction of the probe with the silanol groups in the shell. An increment in the dye concentration to 2 µM promoted excimer formation, similarly to what has been observed by Lee et al. with poly(acrylonitrile) nanotubes.54 Interestingly, the I1/Iexc ratio in this case is 1.9, which is lower than the value obtained from the particles prepared including the dye in their synthesis. We do not believe that this is an indication that it is easier to form dimers after uptake in the interior of the particles, but rather that at higher concentrations, pyrene is being internalized as aggregates due to its low solubility in water.
Conclusion A mixed surfactant system was used to form templates for the synthesis of nanoparticles. A silica shell was grown on the surface of the templates, resulting in core-shell particles. DLS, TEM, and AFM showed the effectiveness of the templating process. It was observed by AFM that the rigidity of the particles increased after the shell growth, producing a more robust system. (53) Joncheray, T. J.; Audebert, P.; Schwartz, E.; Jovanovic, A. V.; Ishaq, O.; Cha´vez, J. L.; Pansu, R.; Duran, R. S. Langmuir 2006, 22, 8684-8689. (54) Lee, K. J.; Oh, J. H.; Kim, J.; Jang, J. Chem. Mater. 2006, 18, 5002-5008.
Langmuir, Vol. 24, No. 5, 2008 2071
We prepared fluorescent core-shell particles whose emission can be tuned by selecting a different dye to encapsulate. Steadystate fluorescence spectroscopy was used to study the encapsulation of these hydrophobic dyes in the templates and core-shell nanoparticles. C153 and pyrene fluorescence was used to characterize the microenvironment of the dyes before and after the silica coating. It was observed that both dyes were protected from the polar solvent in the templates. C153 results showed a decrease in the polarity after the shell growth, while pyrene fluorescence indicated an increase in the viscosity of the interior of the particles, which was observed with the AFM studies as well. Finally, we studied the uptake of these dyes with dye-free core-shell particles from aqueous suspensions, observing that the dyes can be removed from the aqueous media. C153 was adsorbed on the surface of the core-shell particles, probably due to hydrogen bonding interactions; pyrene, on the other hand, could be internalized in the particles. Acknowledgment. Dr. Valeria Kleiman is thanked for valuable discussions, and Karen Kelley and Lynda Schneider from the Electron Microscopy Core Laboratory at the University of Florida are thanked for technical assistance in the electron microscopy studies. Supporting Information Available: Comparison of correlation function obtained by DLS for NPY templates and core-shell particles; TEM images of calcinated templates and core-shell particles; comparison of emission spectra for C153 in NCU templates, core-shell particles, and NCUSL; and comparison of emission spectra for pyrene in NPY templates, core-shell particles, and NPYSL. This material is available free of charge via the Internet at http://pubs.acs.org. LA702227D
