One-Pot Synthesis and Characterization of Three Kinds of Thiol

Mar 27, 2008 - Using a one-pot synthesis, thiol−organosilica nanoparticles (NPs) made from (3-mercaptopropyl)trimethoxysilane, (3-mercaptopropyl)tri...
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Langmuir 2008, 24, 5099-5108

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One-Pot Synthesis and Characterization of Three Kinds of Thiol-Organosilica Nanoparticles Michihiro Nakamura* and Kazunori Ishimura Department of Anatomy and Cell Biology, Medical Informatics, Institute of Health Biosciences, The UniVersity of Tokushima Graduate School, 3-18-15 Kuramoto, Tokushima 770-8503, Japan ReceiVed October 31, 2007. In Final Form: February 8, 2008 Using a one-pot synthesis, thiol-organosilica nanoparticles (NPs) made from (3-mercaptopropyl)trimethoxysilane, (3-mercaptopropyl)triethoxysilane, and (3-mercaptopropyl)methyldimethoxysilane have been successfully prepared. We compared the synthesis processes of thiol-organosilica NPs made of these three kinds of organosilicates, as well as particles made from tetraethoxysilicate (TEOS), at concentrations varying between 6.25 and 200 mM. We examined three types of synthetic conditions: the Sto¨ber method, in which particles are prepared in 65% ethanol, and two entirely aqueous solvent syntheses, containing either 2% or 27% ammonium hydroxide. The synthetic mixtures were examined using transmission electron microscopy (TEM) to evaluate the as-prepared NPs. The formation trends and rates for these organosilica NPs vary with differing organosilicate precursors, concentrations, and synthetic conditions. The Sto¨ber method is not suitable for formation of thiol-organosilica NPs as compared with the case of TEOS, but the conditions without ethanol and with 27% ammonium hydroxide are suitable for the formation of thiol-organosilica NPs. The size distributions of the formed NPs were evaluated using TEM and dynamic light scattering. The mean diameters of NPs increase with increasing concentrations of silicate, but the size distributions of NPs prepared under various conditions also differ between silicate sources. Thiol-organosilica NPs internally functionalized with fluorescent dye were also prepared using a one-pot synthesis and were characterized using fluorescence microscopy. The thiolorganosilica NPs retain fluorescent dye maleimide very well. In addition, rhodamine B-doped thiol-organosilica NPs show higher fluorescence than thiol-organosilica NPs prepared with rhodamine red maleimide. The surface of thiolorganosilica NPs contains exposed thiol residues, allowing the covalent attachment of fluorescent dye maleimide and protein maleimide. This is the first report describing the synthesis of thiol-organosilica NPs made of three kinds of thiol-organosilicates, differences in nanoparticle formation due to the kinds and concentrations of thiol-organosilicate and due to synthetic conditions, and the advantages of thiol-organosilica NPs due to the existence of both interior and exterior thiol residues.

Introduction Nanoparticles (NPs) are becoming an important component of biomedical applications including bioanalysis, multitarget detectionsystems,invitroandinvivoimaging,andnanomedicine.1-16 Nanoparticles for these technologies must be able to provide strong analytical signals, bind targets with high affinity and * To whom correspondence should be addressed. Phone: +81-88-6339220. Fax: +81-88-633-9426. E-mail: [email protected]. (1) Gao, X.; Cui, Y.; Levenson, R. M.; Chung, L. W. K.; Nie, S. Nat. Biotechnol. 2004, 22, 969-976. (2) Roy, I.; Ohulchanksy, T. Y.; Bharali, D. J.; Pudavar, H. E.; Mistretta, R. A.; Kaur, N.; Prasad, P. N. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 279-284. (3) Wang, L.; Yang, C. Y.; Tan, W. H. Nano Lett. 2005, 5, 17-43. (4) Wang, L.; Tan, W. H. Nano Lett. 2006, 6, 84-88. (5) Zhao, X. J.; Hilliard, L. R.; Mechery, S. J.; Wang, Y.; Bagwe, R.; Jin, S.; Tan, W. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 15027-15032. (6) Santra, S.; Zhang, P.; Wang, K.; Tapec, R.; Tan, W. Anal. Chem. 2001, 73, 4988-4993. (7) Tan, W.; Wang, K.; He, X.; Zhao, J.; Drake, T.; Wang, L.; Bagwe, R. P. Med. Res. ReV. 2004, 24, 621-638. (8) Zhao, X. J.; Bagwe, R. P.; Tan, W. H. AdV. Mater. 2004, 16, 173-176. (9) Zhao, X.; Dytioco, R.; Tan, W. J. Am. Chem. Soc. 2003, 125, 1147411475. (10) Song, H.-T.; Choi, J.; Huh, Y.-M.; Kim, S.; Jun, Y.-W.; Suh, J.-S.; Cheon, J. J. Am. Chem. Soc. 2005, 127, 9992-9993. (11) He, X. X.; Wang, K. M.; Tan, W.H.; Liu, B.; Lin, X.; He, C. M.; Li, D.; Huang, S. S.; Li, J. J. Am. Chem. Soc. 2003, 125, 7168-7169. (12) Quellec, P.; Gref, R.; Perrin, L.; Dellacherie, E.; Sommer, F.; Verbavatz, J. M.; Alonso, M. J. J. Biomed. Mater. Res. 1998, 42, 45-54. (13) Schroedter, A.; Weller, H. Angew. Chem., Int. Ed. 2002, 41, 3218-3221. (14) Santra, S.; Bagwe, R. P.; Dutta, D.; Stanley, J. T.; Walters, G. A.; Tan, W. H.; Moudgil, B. J.; Mericle, R. A. AdV. Mater. 2005, 17, 2165-2169. (15) Wang, L.; Wang, K. M.; Santra, S.; Zhao, X. J.; Hilliard, L. R.; Smith, J. E.; Wu, J. R.; Tan, W. H. Anal. Chem. 2006, 78, 646-654. (16) Liu. Y; Miyoshi, H., Nakamura, M. Int. J. Cancer 2007, 120 (12), 25272537.

specificity, and be adapted to controlled release systems. To this end, silica NPs are often used as core particles for biomedical applications because they are easy to prepare and separate, their surfaces may be modified or labeled, they are highly hydrophilic, and they are safe. Functionalization of both the interior and exterior (surfaces) of silica NPs is very important for various applications. The internal functionalization of silica NPs with signal molecules, such as fluorescent dyes, has been well investigated. Various kinds of fluorescent silica NPs have been prepared by doping the reaction solution with fluorescent dye during particle formation,7,8,17-21 imposing the dye on the silica network via the formation of bonds between the dye and a silane coupling reagent22-26 or other methods.27 Fluorescent silica NPs possess several advantages compared with other fluorescent NPs such as quantum dots and latex beads, including high fluorescence intensity, good photostability due to the exclusion of oxygen by (17) Vanzo, E. U.S. Patent 4,077,804, 1978. (18) Peterson, J. I. U.S. Patent 4,194,877, 1980. (19) Yabuuchi, N.; Otsuka, C.; Kashihara, A. U.S. Patent 5,367,039, 1994. (20) Lian, W.; Litherland, S. A.; Badrane, H.; Tan, W.; Wu, D.; Baker, H. V.; Gulig, P. A.; Lim, D. V.; Jin, S. Anal. Biochem. 2004, 334, 135-144. (21) Rossi, L. M.; Shi, L; Quina, F. H.; Rosenzweig, Z. Langmuir 2005, 21 (10), 4277-4280. (22) Van Blaaderen, A.; Vrij, A. Langmuir 1992, 8, 2921-2931. (23) Verhaegh, N. A. M.; van Blaaderen, A. Langmuir 1994, 10, 1427-1438. (24) Imhof, A.; Megens, M.; Engelberts, J. J.; de Lang, D. T. N.; Sprik, R.; Vos, W. L. J. Phys. Chem. B 1999, 103, 1408-1415. (25) Rossi, L. M.; Shi, L; Quina, F. H.; Rosenzweig, Z. Langmuir 2005, 21 (10), 4277-4280. (26) Nakamura, M.; Shono, M.; Ishimura, K. Anal. Chem. 2007, 79, 65076514. (27) Ow, H.; Larson, D. R.; Srivastava, M.; Baird, B. A.; Webb, W. W.; Wiesner, U. Nano Lett. 2005, 5, 113-117.

10.1021/la703395w CCC: $40.75 © 2008 American Chemical Society Published on Web 03/27/2008

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tionalization of NPs made of the three thiol-organosilicates. Thiol-organosilica NPs are internally functionalized with fluorescent dye using a one-pot synthesis, demonstrating the NPs’ unique property of internal functionalization. The surface of thiol-organosilica NPs is modified with fluorescent dye and proteins, also demonstrating the unique property of surface functionalization. Figure 1. Molecular structures of TEOS, MPMS, MPES, and MPDMS.

silica encapsulation, good potential for surface modification with various biomolecules, and toxicity. Surface functionalization of silica NPs with various biomolecules such as proteins, enzymes, peptides, and DNA is a key technology for various applications including bioassays, imaging, and drug delivery systems. The types and extent of possible surface functionalizations are largely controlled by the surface properties of the as-prepared NPs. Traditionally, silica NPs have been prepared from tetraethoxysilicate (TEOS) using Sto¨ber’s method28 or the reverse microemulsion method.29 As prepared, TEOS NPs display surface silanol groups, which require further modification prior to surface functionalization. Silanizations of TEOS NPs have been developed by grafting organosilicate compounds such as (3-mercaptopropyl)trimethoxysilane (MPMS) to the silica matrix.30,31 Recently, silica NPs were prepared and subsequently surface-modified via cohydrolysis of TEOS with various organosilane reagents.32 Various methods to modify the surface of TEOS NPs with organic functional groups capable of covalent bonding, such as amines, are also under development for use in various applications.7 Recently, a one-pot synthesis of organosilica NPs comprising a single organosilicate was developed and reported.33 These thiolorganosilica NPs had surface thiol residues as prepared, allowing for surface functionalization through adsorption, thiol-exchange reactions, and maleimide-coupled covalent bonding. Therefore, additional procedures such as organosilica grafting and cohydrolysis with TEOS and organosilicates were not required to functionalize the NPs’ surfaces. These organosilica NPs allow for facile surface functionalization, offering new opportunities for applications in various fields. However, silica NPs made of a single organosilicate have not been investigated and reported extensively, and more information is needed regarding the synthetic procedures of these particles, as well as the characteristics of their formation. In this paper we report the one-pot synthesis of thiolorganosilica NPs using MPMS, (3-mercaptopropyl)triethoxysilane (MPES), and (3-mercaptopropyl)methyldimethoxysilane (MPDMS) as single silica sources. As shown in Figure 1, all three thiol-organosilicates have a mercaptopropyl chain as a thiol residue, but otherwise they vary in structure. We describe and examine three kinds of synthetic conditions to prepare NPs: the Sto¨ber method,28 which uses ethanol as a solvent, and two syntheses in aqueous solvent, using 2% and 27% ammonium hydroxide. Nanoparticles formed from these three synthetic procedures are compared with each other, as well as with TEOS NPs. In addition, we evaluate the internal and surface func(28) Sto¨ber, W.; Fink, A.; Bohn, E. J. Colloid Interface Sci. 1968, 26, 62-69. (29) Yanagi, M.; Asano, Y.; Kandori, K.; Kon-no, K. Abstracts of the 39th Symposium of the DiVision of Colloid Interface Chemistry; Chemical Society of Japan: Tokyo, 1986; p 386. (30) Qhobosheane, M.; Santra, S.; Zhang, P.; Tan, W. Analyst 2001, 126, 1274-1278. (31) Hilliard, L.; Zhao, X.; Tan, W. Anal. Chim. Acta 2002, 470, 51-56. (32) Bagwe, R. P.: Hilliard, L. R.; Tan, W. Langmuir 2006, 22, 4357-4362. (33) Nakamura, M; Ishimura, K. J. Phys. Chem. C 2007, 111, 18892-18898.

Experimental Section Materials. MPMS, MPES, MPDMS, TEOS, rhodamine B, fluorescein, and tris dichlororuthenium (II) hexahydrate (Ru(bpy)) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). Rhodamine red C2-maleimide and fluorescein 5-maleimide were from Invitrogen (Carlsbad, CA). Green fluorescent protein (GFP) was from Upstate (Lake Placid, NY). Ethyl alcohol and 30% NH4OH were from Wako Fine Chemicals Inc. (Osaka, Japan). Preparation of Organosilica Nanoparticles and Internally Functionalized Fluorescent Organosilica Nanoparticles Using One-Pot Synthesis. Nanoparticles were prepared using three kinds of synthetic conditions, referred to herein as A, B, and C. Under condition A, various concentrations of TEOS or thiol-organosilicates (MPMS, MPES, or MPDMS), as listed in Tables 1 and 2, were mixed with 325 µL of ethyl alcohol and 36 µL of 28% NH4OH and diluted to 500 µL with distilled water. Under condition B, various concentration of TEOS or thiol-organosilicate were mixed with 36 µL of 28% NH4OH and diluted to 500 µL with distilled water. Under condition C, various concentrations of TEOS or thiolorganosilicate were diluted to 500 µL with 28% NH4OH. All synthesis mixtures were incubated at room temperature for 1 day or 3 days with occasional mixing. Internally functionalized fluorescent organosilica NPs were also prepared using a one-pot synthesis. Various concentration solutions of rhodamine red C2-maleimide, fluorescein 5-maleimide, rhodamine B, or fluorescein were mixed with a condition C solution containing 50 mM thiol-organosilicates (MPMS, MPES, or MPDMS) or with a condition A solution containing 50 mM TEOS, and the resulting mixture was incubated for 3 days. After incubation, the reaction mixture was centrifuged to remove unbound reagents. The particles were washed extensively with 70% ethyl alcohol and water. Preparation of Surface-Modified Organosilica Nanoparticles. To modify the surface of the silica NPs with fluorescent dye, 135 µL of thiol-organosilica NP solution made with 50 mM silicate (TEOS under condition A; MPMS, MPES, or MPDMS under condition C) was reacted with 15 µL of 1 mM rhodamine red C2maleimide for 9 h at room temperature. After the reaction was complete, the reaction mixture was centrifuged at 10000g for 5 min to remove unbound reagents, and the pellets were dispersed by sonication. The particles were washed extensively with water and stored in water. To modify the surface of the silica NPs with proteins, 10 µL of thiol-organosilica NPs made using condition A (200 mM TEOS) or condition C (200 mM MPMS or MPES or 50 mM MPDMS) was reacted with 10 µL of 10 µg/mL GFP in phosphate-buffered saline (pH 7.4) for a few minutes. After the reaction was complete, the reaction mixture was centrifuged at 10000g for 5 min to remove unbound reagents, and the pellets were dispersed by sonication. The particles were washed extensively with water and stored in water. Electron, Fluorescence, and Light Microscopic Analysis of Silica Nanoparticles. The NPs were observed with a Hitachi H500 or H7650 electron microscope (Hitachi, Tokyo, Japan) or a JEOL JEM-1200EXII electron microscope (JEOL Ltd., Tokyo, Japan) and with an inverted fluorescence microscope (TE 2000, Nikon, Kanagawa, Japan) equipped with a 100 W mercury lamp as a light source and a CCD camera as reported previously.33 Dynamic Light Scattering and ζ Potential Analysis. Dynamic light scattering to analyze the size distributions and ζ potential of NPs were determined with a NICOMP submicrometer particle sizer, model 380/ZLS (Nicomp Particle Sizing Systems, Santa Barbara, CA) at room temperature. For ζ potential analysis electrodes were dipped directly into the solution containing freshly prepared silica

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Table 1. Formation of Silica NPs under Conditions A, B, and C with Incubation Periods of 1 day (A) and 3 days (B)a concn (mM) TEOS

MPMS

MPES

MPDMS

TEOS

MPMS

MPES

MPDMS

condition A

condition B

condition C

200 100 50 25 12.5 6.25 200 100 50 25 12.5 6.25 200 100 50 25 12.5 6.25 200 100 50 25 12.5 6.25

(A) 1 day O O O O O O O X X X X X X X X X X X X X X X X X

X X X X X X O O O X X X O O O X X X X X X X X X

O X X X X X X O O O O O X X X O O O X X X O O O

200 100 50 25 12.5 6.25 200 100 50 25 12.5 6.25 200 100 50 25 12.5 6.25 200 100 50 25 12.5 6.25

(B) 3 days O O O O O O O O O O O O X X X O O O X X X X X X

X X X X X X O O O O O O O O O O O O X X X X X X

O O O X X X X O O O O O X X O O O O X X O O O O

a Formations and incomplete formations of NPs are indicated as O and X, respectively.

NPs. On the basis of the principles of electrophoretic light scattering, quantitative measures of the charge on colloidal particles in a liquid suspension were performed. Flow Cytometry. Flow cytometry analysis was performed on FACSCalibur flow cytometers (Becton Dickinson, San Jose, CA) with 488 and 635 nm excitation lasers. Fluorescence was detected on the FL2 channel (585/30 nm band-pass filter) and on the FL3 channel (670 nm long-pass filter). The fluorescent-tuned MPMS NP fluorescent dye was diluted and analyzed. All data were obtained without compensation.

Results and Discussion Synthesis of Three Kinds of Thiol-Organosilica NPs. Varying concentrations of thiol-organosilicates (MPMS, MPES, and MPDMS) and TEOS were used in three kinds of synthetic

Table 2. Size Evaluation of Silica NPs Prepared Using Conditions A, B, and C silica

condition

concn (mM)

av diam (nm)

CV (%)

TEOS TEOS TEOS TEOS TEOS TEOS TEOS TEOS TEOS MPMS MPMS MPMS MPMS MPMS MPMS MPMS MPMS MPMS MPMS MPMS MPMS MPMS MPMS MPMS MPMS MPMS MPES MPES MPES MPES MPES MPES MPES MPES MPES MPES MPES MPES MPES MPDMS MPDMS MPDMS MPDMS

A A A A A A C C C A A A A A A B B B B B B C C C C C A A A B B B B B B C C C C C C C C

200 100 50 25 12.5 6.25 200 100 50 200 100 50 25 12.5 6.25 200 100 50 25 12.5 6.25 100 50 25 12.5 6.25 25 12.5 6.25 200 100 50 25 12.5 6.25 50 25 12.5 6.25 50 25 12.5 6.25

447 381 314 192 117 62 177 80 40 705 484 380 333 233 154 740 677 502 295 234 147 882 407 248 125 66 388 329 313 736 680 412 295 186 114 378 264 134 81 1266 620 281 155

44.7 27.8 29.0 35.9 59.8 72.5 34.5 40.0 40.0 51.9 49.2 39.2 33.6 51.1 43.5 50.8 42.4 24.9 25.4 35.0 31.3 43.3 50.1 29.8 21.6 40.9 24.2 39.8 25.3 72.8 45.4 45.6 24.4 27.4 19.8 61.1 36.0 29.9 34.0 53.4 28.9 23.5 14.8

conditions to prepare NPs from a single silicate source. The first condition, condition A, followed traditional Sto¨ber methods28 (65% ethanol, 33% water, and 2% ammonium hydroxide). The second condition, condition B, contained 98% water and 2% ammonium hydroxide but did not contain ethanol. The third condition, condition C, contained a higher concentration of ammonium hydroxide (73% water and 27% ammonium hydroxide). Conditions B and C were varied only slightly from the Sto¨ber method in efforts to determine the minimum solution components necessary to form thiol-organosilica NPs. The synthesized NPs were evaluated by transmission electron microscopy (TEM), as shown in Figures 2-5. Nanoparticles were labeled as being completely or incompletely formed by analyzing these TEM images, as summarized in Table 1. The formation of a given batch of NPs was deemed complete if unaggregated NPs of >10 nm diameter with clearly visible edges were observed in the TEM images. 1.1. TEOS. As shown in Figure 2A, synthetic mixtures containing varying concentrations of TEOS produce defined particles after incubation for 1 day under condition A. Under condition B (Figure 2B), no NPs were observed, even after the solutions were incubated for 3 days. Under condition C (Figure 2C), NPs are observed using the highest concentration of TEOS

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Figure 2. TEM images of organosilica NPs prepared from TEOS as a function of time and TEOS concentration. TEOS NPs prepared under condition A (A), condition B (B), and condition C (C) were observed after incubation periods of 1 day ((A) and upper row in (B) and (C)) and 3 days (lower row in (B) and (C)). Scale bars: (A) 500 nm, (B) 5000 nm, (C) 200 nm.

(200 mM) after 1 day, and 50 and 100 mM TEOS synthetic mixtures produce NPs after 3 days. These findings indicate that formation of TEOS NPs requires ethanol when less than 2% ammonium hydroxide is used in the synthesis. Mixtures containing higher TEOS concentrations (from 50 to 200 mM) can form NPs in the presence of 27% ammonium hydroxide without ethanol. However, the rate of nanoparticle formation using this synthetic method (condition C) is slower than that observed for condition A, as a 3 day incubation period is required to form NPs from 50 and 100 mM TEOS under condition C compared with 1 day under condition A. 1.2. MPMS. As shown in Figure 3A, synthetic mixtures with the highest MPMS concentration (200 mM) form particles after 1 day under condition A, but mixtures of lower MPMS concentrations do not form particles. After 3 days under condition A, synthetic mixtures of all MPMS concentrations show clearly formed NPs. Under condition B, synthetic mixtures with higher MPMS concentrations (from 50 to 200 mM) form particles, but lower MPMS concentrations do not produce NPs after 1 day (Figure 3B). After 3 days under condition B, synthetic mixtures of all MPMS concentrations show clearly formed NPs. Under condition C, NPs are observed with all concentrations of MPMS except 200 mM after 1 day (Figure 3C). After 3 days under condition C, synthetic mixtures containing 200 mM MPMS show NPs, but they appear immature and aggregated. Compared with the formation of TEOS NPs, the formation of MPMS NPs under condition A is slower. Under conditions B and C, the formation of MPMS NPs significantly differs from that of TEOS NPs. Under condition B, MPMS NPs are formed from all MPMS concentrations after 3 days, with higher concentrations of MPMS (50-200 mM) forming particles after only 1 day. By contrast, TEOS particles are not formed from any concentration of TEOS under condition B. Under condition C, NPs are formed from all MPMS concentrations except for the highest concentration after 1 day, while only the highest concentration of TEOS forms NPs under this condition. These results indicate that the conditions without ethanol are suitable for MPMS nanoparticle formation, but Sto¨ber methods are not suitable. Nearly opposite results are obtained using TEOS. Compared with condition B of MPMS, condition C forms NPs at MPMS concentrations lower than 200 mM within 1 day. These

findings indicate that 27% ammonium hydroxide may accelerate the formation of MPMS NPs from lower concentrations of MPMS, and the formation rate of MPMS NPs is faster than those under conditions A and B. 1.3. MPES. As shown in Figure 4A, no NPs are observed after 1 day under condition A. After 3 days under condition A, MPES concentrations between 6.25 and 25 mM show clearly formed NPs, but higher concentrations do not. Under condition B (Figure 4B), synthetic mixtures with higher MPES concentrations (from 50 to 200 mM) form particles after 1 day, but lower MPES concentrations do not form particles. After 3 days under condition B, synthetic mixtures containing 6.25 and 25 mM MPES do not show clear NPs. Under condition C (Figure 4C), clearly formed NPs are observed using MPES concentrations between 6.25 and 25 mM after 1 day. After 3 days under condition C, synthetic mixtures containing 100 and 200 mM MPES show NPs, but they appear immature and aggregated. The formation trends of MPES NPs are similar to those of MPMS, but the range of MPES concentrations that form NPs is narrower, and MPES formation rates are slower than the formation rates of MPMS NPs. As with MPMS NPs, MPES NPs exhibit opposite formation trends compared to TEOS NPs. 1.4. MPDMS. As shown in Figure 5A,B, no MPDMS NPs are observed at any MPDMS concentration using either condition A or condition B, even after 3 days. Under condition C (Figure 5C), clearly formed NPs are observed with lower and middle MPDMS concentrations (from 6.25 to 25 mM) after 1 day. After 3 days under condition C, we observed NPs from 50 mM MPDMS, but not from higher MPDMS concentrations (100 and 200 mM). The formation trends of MPDMS NPs are different from not only those of TEOS but also those of the other thiol-organosilica sources, MPMS and MPES. The synthetic conditions using low ammonium hydroxide (conditions A and B) do not form MPDMS NPs. Only condition C, with 27% ammonium hydroxide, forms MPDMS NPs successfully. However, the range of MPDMS concentrations that form NPs is narrower, and formation rates are slower compared with the formation rates of MPMS and MPES. The formation trends of MPDMS NPs give important insight into the MPDMS nanoparticle formation mechanism. As shown in Figure 1, MPDMS has a mercaptopropyl chain, a methyl chain,

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Figure 3. TEM images of organosilica NPs prepared from MPMS as a function of time and MPMS concentration. MPMS NPs prepared under condition A (A), condition B (B), and condition C (C) were observed after incubation periods of 1 day (upper row) and 3 days (lower row). Scale bars: 1000 nm.

and two methoxy chains. The two methoxy chains might form two silanols by hydrolysis, and then two siloxane bridges might condense from one MPDMS molecule. Nevertheless, the formation of three-dimensional networks and particles from only two siloxane bridges of one MPDMS molecule is not likely. In fact, the formation of MPDMS NPs suggests that another chain, such as the mercaptopropyl chain, contributes to the formation of particles. The exact formation mechanism and molecular structure of the organosilica NPs are not known. Additional studies, including structural analysis, are required to understand them. 1.5. Synthetic Conditions. As shown by the TEM images in Figures 2-5 and the data in Table 1, the formations of TEOS and thiol-organosilica NPs differ appreciably from each other; they also vary between the three kinds of synthetic conditions used for each silica source. Condition A, the traditional Sto¨ber method, is very suitable for TEOS nanoparticle formation (Table 1), as NPs are formed from all TEOS concentrations within a short incubation time (1 day). By contrast, condition A is not suitable for thiol-organosilica nanoparticle formation. Clearly defined NPs are formed under condition A from only the highest

concentration of MPMS after 1 day. After 3 days, MPMS NPs and MPES NPs of almost all concentrations are formed, but MPDMS NPs still do not form. Condition B contains the same concentration of ammonium hydroxide as condition A but does not contain ethanol. TEOS solutions do not form any NPs under condition B. These results indicate that ethanol is essential for the formation of NPs from TEOS when 2% ammonium hydroxide is used. In the cases of MPMS and MPES, ethanol is not essential for the formation of particles under condition B when higher concentrations (50200 mM) of thiol-organosilicate are used, as NPs are formed from these solutions after 1 day (Table 1A). After 3 days, MPMS NPs and MPES NPs of all concentrations are formed, but MPDMS NPs still do not form. Condition C contains a higher concentration of ammonium hydroxide than conditions A and B. This condition allows the formation of TEOS NPs without ethanol from mixtures containing high concentrations of TEOS (from 50 to 200 mM) after 3 days. Under condition C, both MPMS and MPES NPs are formed from low concentrations of thiol-organosilicates (6.25-100 mM for MPMS and 6.25-25 mM for MPES) after 1 day. After 3

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Figure 4. TEM images of organosilica NPs prepared from MPES as a function of time and MPES concentration. MPES NPs prepared under condition A (A), condition B (B), and condition C (C) were observed after incubation periods of 1 day (upper row) and 3 days (lower row). Scale bars: 1000 nm.

days, higher concentrations of MPMS and MPES (200 mM for MPMS and 100-200 mM for MPES) form NPs, but they are aggregated. Remarkably, MPDMS NPs are formed successfully only under condition C. MPDMS NPs are formed from low concentrations of MPDMS (from 6.25 to 25 mM) after 1 day, similar to the formation trends of MPMS and MPES under condition C. After 3 days, a higher concentration of MPDMS (50 mM) forms NPs. The incorporation of high ammonium hydroxide concentrations promotes nanoparticle formation from lower concentrations of thiol-organosilicates and higher concentrations of TEOS. Ammonium hydroxide is a catalyst for the hydrolysis and condensation of silicates. An increasing amount of ammonium hydroxide could increase the rates of silicate hydrolysis and condensation. In our study, a high concentration of ammonium hydroxide in the absence of ethanol contributes to the formation of TEOS NPs from higher concentrations of TEOS after 3 days. In the cases of MPMS and MPES, formation trends differ from those in the case of TEOS: condition B, containing only 2% ammonium hydroxide, forms NPs from high concentrations of MPMS and MPES after 1 day, while condition C, containing

27% ammonium hydroxide, forms particles from lower concentrations of MPMS and MPES after 1 day, but does not promote particle formation from higher concentrations of MPMS and MPES. These results indicate that the effect of ammonium hydroxide on the formation of NPs depends on both the concentration and type of silicate used in the synthesis. A higher concentration of ammonium hydroxide could accelerate hydrolysis and condensation of silicate, causing a quicker formation of MPMS and MPES NPs than that observed using 2% ammonium hydroxide (conditions A and B). Under condition B, 2% ammonium hydroxide fails in forming NPs from low concentrations of MPMS and MPES after 1 day. Previously we proposed a hypothesis about formation of thiolorganosilica NPs: During nanoparticle formation, micelles of thiol-organosilicate are first formed, which are then hydrolyzed and condensed by ammonium hydroxide, resulting in the formation of NPs.33 Lower concentrations of thiol-organosilicates form smaller micelles, and low concentrations of ammonium hydroxide might reduce the contacts with these smaller thiolorganosilcate micelles (less than 300 nm diameter). Smaller NPs could form when the concentration of ammonium hydroxide

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Figure 5. TEM images of organosilica NPs prepared from MPDMS as a function of time and MPDMS concentration. MPDMS NPs prepared under condition A (A), condition B (B), and condition C (C) were observed after incubation periods of 1 day (upper row) and 3 days (lower row). Scale bars: (A) 200 nm, (B) 500 nm, (C) 1000 nm.

like in condition C is sufficient to contact the surface of the thiol-organosilicate micelles. High concentrations of ammonium hydroxide fail in forming clearly defined NPs from high concentrations of thiol-organosilicates. The MPMS and MPES particles formed from solutions of 200 mM thiol-organosilicate show aggregations of NPs. By contrast, low concentrations of ammonium hydroxide form particles from higher concentrations of MPMS and MPES. Should an imbalance exist between the formation of micelles and hydrolysis and condensation, incomplete formations of particles might occur. In the case of higher concentrations of MPMS and MPES with high concentrations of ammonium hydroxide, the hydrolysis and condensation preceded the formation of micelles, and then incomplete micelles became aggregates. Therefore, a combination of the concentration of ammonium hydroxide, the formation and condition of the thiol-organosilicate micelles, and the reaction kinetics of hydrolysis and condensation might be very important to consider in the formation of thiolorganosilica NPs. Characterization of Three Kinds of Thiol-Organosilica Nanoparticles. Size Distribution. Size distributions of the three kinds of thiol-organosilica NPs, as well as the TEOS NPs, formed under the three synthetic conditions were evaluated using dynamic light scattering analysis and electron microscopy. These results are summarized in Table 2, and mean particle diameters are plotted in Figure 6. Under condition A, the mean diameter of TEOS NPs was smaller than those of MPMS and MPES NPs at the same concentration (Figure 6A). The sizes of TEOS NPs were dependent on the TEOS concentration, but a decline in the dependency was observed above 100 mM TEOS. The sizes of MPMS NPs were also dependent on concentration, but a decline in the dependency was observed above 50 mM MPMS. The MPES NPs were bigger than the others and dependent on the concentration for all MPES preparations. Sto¨ber et al.28 reported

no effect of the TEOS concentration on the size of TEOS particles. Among other investigations,34-36 some discrepancies exist concerning the relationship between the TEOS concentration and the particle size: increasing TEOS concentrations have been shown to both increase and decrease particle sizes. In the present study, increasing the concentration of silicates increases the size of the formed NPs under all three kinds of conditions. Also increasing ratios of size are dependent on the type and concentration of the silicate, as well as the synthetic conditions, as described later. Under condition B, the size distributions of MPMS and MPES are similar to each other and are concentration-dependent (Figure 6B). The decline in the concentration dependency is observed above 50 mM MPMS and above 100 mM MPES. It has been reported previously that ethanol could affect the monodispersity and size of TEOS NPs: at lower ethanol concentrations, between 4 and 6 M, the particle size decreases with decreasing ethanol concentration.36 In the present study, TEOS NPs are not formed when ethanol is absent from the synthetic mixture. In the case of MPMS NPs, the absence of ethanol does not affect the size distribution of the formed NPs substantially in comparison with condition A: The average sizes of MPES NPs are only slightly smaller under condition B than those of particles prepared under condition A. Under condition C, the mean diameters of MPMS and MPES are also similar; for a given silicate concentration, mean diameters decrease in the order of MPDMS, MPMS and MPES, and then TEOS (Figure 6C). TEOS NPs are one-fifth the diameter of MPMS and MPES NPs, and the diameters of MPDMS NPs are (34) Van Helden, A. K.; Jansen, J. W.; Vrij, A. J. Colloid Interface Sci. 1981, 81, 354-368. (35) Bogush, G. H.; Tracy, M. A.; Zukoski, C. F. J. Non-Cryst. Solids 1988, 104, 95-106. (36) Rao, K. S.; El-Hami, K.; Kodaki, T.; Matsushige, K.; Makino, K. J. Colloid Interface Sci. 2005, 289, 125-1.

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Figure 6. Mean particle diameter as a function of silicate concentration for TEOS (black circles), MPMS (blue squares), MPES (red tilted squares), and MPDMS (light green triangles) under condition A (A), condition B (B), and condition C (C).

about double those of the corresponding MPMS or MPES particles. The diameters of TEOS NPs prepared under condition C are substantially smaller than those prepared under condition A. However, the sizes of MPMS and MPES NPs do not vary much between conditions. Remarkably, a decline in dependency is not observed among any of the four kinds of formed NPs prepared under condition C. We synthesized three kinds of thiol-organosilica NPs under three kinds of conditions, but these synthetic conditions do not form NPs with narrow size distributions, i.e., less than 20% coefficient of variation (Table 2). Thiol-organosilica NPs with a narrow size distribution are required for several applications, and we are currently investigating methods for preparing them. Internal Functionalization. We prepared thiol-organosilica NPs containing fluorescent dyes by modifying a previously reported synthesis.26 In the present study, we tried a one-pot

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synthesis of fluorescent NPs to determine whether thiolorganosilica NPs contain internal thiol residues that are capable of binding fluorescent dye maleimide. Thiol-organosilica NPs were synthesized with rhodamine red maleimide or fluorescein maleimide by adding each conjugate at the beginning of the reaction. As shown in Figure 7, all three kinds of thiolorganosilica NPs show fluorescence of rhodamine or fluorescein. As a control experiment, TEOS NPs were also prepared with fluorescent dye maleimide, but no fluorescence from these NPs is observed (Figure 7). These results indicate that fluorescent silica NPs may be prepared successfully with fluorescent dye maleimide using a one-pot synthesis. Fluorescent dye-doped thiol-organosilica NPs were also prepared using a one-pot synthesis. Thiol-organosilica NPs were synthesized with rhodamine B or fluorescein by adding each dye at the beginning of the reaction. As a control experiment, TEOS NPs were also prepared with rhodamine B or fluorescein, but no fluorescence from these NPs is observed. As shown in Figure 7A (lower panels), rhodamine B-doped thiol-organosilica NPs show high fluorescence. By contrast, fluorescein-doped thiolorganosilica NPs do not show clear fluorescence (Figure 7B, lower panels). These results indicate that fluorescent dye doping of thiol-organosilica NPs differs from the fluorescent dye doping of TEOS NPs. TEOS NPs cannot retain rhodamine B, but thiolorganosilica NPs retain rhodamine B very well. Fluorescent-tuned MPMS NPs were analyzed by means of flow cytometry. MPMS NPs prepared with rhodamine red maleimide were detected as three peaks with different fluorescence intensities depending on the concentration (between 6.25 and 25 µM) of rhodamine red maleimide used to prepare the particles (emission at 585/42 nm, FL-2; Figure 7C). Rhodamine B-doped thiol-organosilica NPs were detected as six peaks (Figure 7D), and the peak at a concentration of 200 mM was the highest as compared with that of NPs prepared with rhodamine red maleimide. This is the first report demonstrating that fluorescent dye-doped silica NPs can possess fluorescence intensities higher than those of fluorescent silica NPs prepared using chemical cross-linking reagents, such as maleimides and succinimidyl esters. In addition to rhodamine B, Ru(bpy) was also doped into thiol-organosilica NPs very well. As shown in Figure 7E, Ru(bpy)-doped thiol-organosilica NPs were also detected as six peaks (emission above 670 nm, FL-3). The cost to prepare fluorescent silica NPs thus may be reduced by omitting crosslinking reagents and by using a one-pot synthesis. Surface Characterization and Functionalization. ζ potential analyses of thiol-organosilica NPs were performed to characterize the surface charge of the NPs. Each type of thiol-organosilica NP was prepared using 50 mM thiol-organosilica solution under condition C for 3 days. The ζ potentials of MPMS NPs, MPES NPs, and MPDMS NPs are -50.2, -49.4, and -50.6 mV, respectively; their values are almost the same. As reported previously,33 the ζ potentials of thiol-organosilica NPs are more negative than those of TEOS NPs. We also examined the surface functionalization of all three types of thiol-organosilica NPs prepared in this study by modifying them with fluorescent dyes, as facile modification of the surface thiol residues of MPMS NPs has been reported previously.33 Thiol-organosilica NPs were reacted with rhodamine red maleimide and then characterized and compared with TEOS NPs using fluorescence microscopy. The surface-modified thiolorganosilica NPs show prominent fluorescence, as seen in Figure 8. As a control experiment, TEOS NPs were also reacted with rhodamine red maleimide, but no fluorescence from these NPs is observed. These findings indicate that surface modification

Synthesis of Thiol-Organosilica Nanoparticles

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Figure 7. Fluorescence microscopy of internally functionalized fluorescent thiol-organosilica and TEOS NPs. (A) Nanoparticles were internally functionalized with rhodamine red maleimide (upper panels) and with rhodamine B (lower panels). (B) Nanoparticles were internally functionalized with fluorescein maleimide (upper panels) and with fluorescein (lower panels). Flow cytometry analysis of fluorescent-tuned MPMS NPs prepared with rhodamine red maleimide (C) and rhodamine B-doped MPMS NPs containing rhodamine B (D) and Ru(bpy) (E). Fluorescent-tuned NPs were prepared with 200 µM (green lines), 100 µM (red lines), 50 µM (light blue lines), 25 µM (orange lines), 12.5 µM (blue lines), and 6.25 µM (yellow lines) rhodamine red maleimide, rhodmaine B, or Ru(bpy).

Finally, we examined the surface modification of thiolorganosilica NPs with proteins. The NP solutions were mixed with protein solutions containing GFP. After the solutions were mixed with GFP, the surface modification and the dispersion of thiol-organosilica NPs modified with protein on the surface were evaluated using fluorescence microscopy. The thiolorganosilica NP solution mixed with GFP solution shows welldispersed NPs with distinct fluorescence (Figure 8). These findings indicate that thiol-organosilica NPs may be modified with GFP very effectively while still retaining good dispersion, as opposed to TEOS NPs showing no clear fluorescence.

Conclusion Figure 8. Fluorescence microscopy of surface-modified thiolorganosilica and TEOS NPs. Nanoparticles were mixed with rhodamine red maleimide (upper panels) and with GFP (lower panels).

with rhodamine red maleimide is specific to thiol-organosilica NPs, indicating that all three kinds of thiol-organosilica NPs display abundant thiol residues on their surfaces that may react with the dye molecules.

Thiol-organosilica NPs were created from three kinds of thiol-organosilicates by using three kinds of one-pot syntheses, including the Sto¨ber method. TEOS NPs were also prepared for comparison with the thiol-organosilica particles. The formation of thiol-organosilica NPs differed between different starting materials and was dependent on the type and concentration of organosilicate, as well as the synthetic conditions. Under condition A, the Sto¨ber method containing ethanol, synthetic mixtures of all tested concentrations of TEOS could form particles after an

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incubation period of 1 day. By contrast, only the synthetic mixtures with high concentrations of MPMS and low concentrations of MPES formed particles after 1 day, but after 3 days nearly all concentrations of MPMS and MPES formed NPs. However, MPDMS solutions did not produce NPs, even after 3 days. Under condition B, containing 2% ammonia and no ethanol, TEOS solutions could not form particles after 3 days. The synthetic mixtures with high concentrations of MPMS and MPES formed particles after 1 day. After 3 days, all concentrations of MPMS and nearly all concentrations of MPES formed particles. Again, no MPDMS particles were formed under condition B, even after 3 days. Under condition C, containing 27% ammonia and no ethanol, high concentrations of TEOS formed particles after 1 day, but low concentrations of TEOS could not form particles, even after 3 days. The synthetic mixtures containing low concentrations of MPMS, MPES, and MPDMS could form particles after 1 day, but those with high concentrations of MPMS and MPDMS failed to form particles after 3 days. The size distributions of the organosilica NPs were also dependent on the type and concentration of organosilicate, as well as the synthetic conditions. The sizes of the formed thiol-organosilica NPs were

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dependent on the concentrations of the thiol-organosilicates, but the ratios of increasing size varied with varying synthetic conditions and thiol-organosilicate concentrations. Importantly, thiol-organosilica NPs may be internally modified due to the presence of interior thiol residues, permitting the preparation of fluorescent silica NPs using a one-pot synthesis. The as-prepared particles also contain surface thiol residues, permitting surface modification with various molecules including fluorescent dyes and proteins. These thiol-organosilica NPs have a high potential for use in various applications, such as biomedical analysis, chip-based technology, multitarget detection systems, in vitro and in vivo imaging, and nanomedicine. Acknowledgment. This work was supported in part by a Grant-in-Aid for Younger Scientists (to M.N.), by a Grant for Practical Application of University R&D Results under the Matching Fund Method (to M.N.) from the New Energy and Industrial Technology Development Organization (NEDO) of Japan, and by a Grant-in-Aid for Scientific Research (C) (to M.N.). The work is under patent pending (PCT/JP2007/61587). LA703395W