18892
J. Phys. Chem. C 2007, 111, 18892-18898
Synthesis and Characterization of Organosilica Nanoparticles Prepared from 3-Mercaptopropyltrimethoxysilane as the Single Silica Source 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: July 23, 2007; In Final Form: October 10, 2007
Novel organosilica nanoparticles made of 3-mercaptopropyltrimethoxysilane as the single silica source (MPS NPs) have been synthesized successfully by using a Sto¨ber method. The MPS NPs were well dispersed in solution and have unique surface properties such as thiol residues on the surface and reduced zeta potential compared with the nanoparticles made of tetraethoxysilane. The MPS NPs with fluorescent dye were synthesized by a method that deposited fluorescent dye on the silica network. The resulting MPS NPs with fluorescence were bright, nonaggregated in solution, and photostable. The fluorescence intensity and photostability of fluorescent MPS NPs were sufficient for detection as a single fluorescent particle using flow cytometry and fluorescence microscopy. Protein-modified MPS NPs were prepared easily by absorption and by maleimide coupling. In this paper, we demonstrate the usefulness of MPS NPs and their surface properties and discuss their advantages for biological applications.
Introduction Nanoparticle-based technologies have been developed for bioanalysis and biomedical applications, specifically in ultrahigh-throughput and diagnostic screening, chip-based technology, multitarget detection systems, and imaging in vitro and in vivo.1-16 Nanoparticles have been conjugated with a variety of biomolecules including proteins, enzymes, peptides, and DNA and have been used as fluorescent biomarkers to monitor biological events or to identify and detect biological targets.17-20 Fluorescent nanoparticles, including quantum dots, have found widespread application; among these, silica nanoparticles have desirable qualities such as their high quantum yield, photostability, and water dispersibility. Preparation techniques allow the size, fluorescence intensity, and other photophysical characteristics of these silica nanoparticles to be tailored to specific needs for biological applications. Surface functionalization of silica nanoparticles with various reactive groups for subsequent bioconjugation using silica chemistry is a particularly desirable characteristic, but problems of efficacy and aggregation remain to be solved. Mesoporous organic-inorganic hybrid materials using silica compounds have been developed for various applications. The functionalization of silica-based mesoporous organic-inorganic hybrid materials can be achieved in three ways.21 These include post-attachment of organic components onto a pure silica matrix (grafting), simultaneous reaction of condensable inorganic silica species and silylated organic compounds (co-condensation on the particle surface), and the use of bis-silylated organic precursors that lead to periodic mesoporous organosilicas (PMOs). PMOs have been synthesized by reaction of dipodal alkoxysilanes [(RO)3Si-R′-Si(OR)3] such as 1,2-bis (trimethoxysilyl)ethane27 and 1,2-bis (triethoxysilyl)ethane28,29 in the * To whom correspondence should be addressed. Phone: +81-88-633-9220. Fax: +81-88-633-9426. E-mail:
[email protected]. tokushima-u.ac.jp.
presence of ionic structure-directing agents such as octadecyltrimethylammonium bromide and chloride salts. Silanizations of silica nanoparticles with 3-mercaptopropyltrimethoxysilane (MPS) and with N1-[3-(trimethoxysilyl)propyl]diethylenetriamine have been developed and used for immobilization of oligonucleotides22 and proteins.17 These methods correspond to grafting of silica-based mesoporous organic-inorganic hybrid materials onto the particle surface. Recently, it has been reported that silica nanoparticles were prepared and subsequently surface-modified via cohydrolysis of tetraethyl orthosilicate (TEOS) with various organosilane reagents,23 which corresponds to co-condensation, as defined above. Methods for surface functionalization of PMO nanoparticles have not yet been reported. Traditionally, silica nanoparticles have been prepared from TEOS by using Sto¨ber’s method24 or the reverse microemulsion method.25 There are no reports of other compounds that could make silica nanoparticles from a single silica source using a one-pot synthesis. In this paper, we describe novel silica nanoparticles made of MPS alone (MPSNPs) and displaying with thiol groups on the surface. We discuss the synthetic procedure, the properties of these novel fluorescent nanoparticles produced thereby, and the possibility for biological applications that make use of their modifiable surface. Experimental Section Materials. (3-Aminopropyl) trimethoxysilane (APS), 3-mercaptopropyltrimethoxysilane (MPS), and tetraethoxysilane (TEOS) were purchased from Sigma-Aldrich Chemical Co., (St. Louis, MO). Ethyl alcohol and 30% NH4OH were from Wako Fine Chemicals Inc. (Osaka, Japan). 5(6)-Carboxy-fluorecein-Nhydroxy succinimide ester was from Roche Molecular Biochemicals (Tokyo, Japan); rhodamine red-N-hydroxy succinimide ester, rhodamine red C2-maleimide, fluorescein-5maleimide, and Q-dot 605 were from Invitrogen (Carlsbad, CA). EZ-link maleimide activated neutravidin protein was from Pierce
10.1021/jp075798o CCC: $37.00 © 2007 American Chemical Society Published on Web 12/05/2007
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Figure 1. Transmission electron microscopy images of fluorescent silica nanoparticles as a function of time. TEOS NPs (a-c) and MPS NPs (d-f) were observed after 9 h (a and d), 1 day (b and e), and 2 days (c and f). Scale bars are 500 nm.
TABLE 1: Comparison of Fluorescence Intensity of Rhodamine Red-Containing Silica Nanoparticles Prepared from MPS (MPS NPs-R) and from TEOS (TEOS NPs-R) and Quantum Dots 605 (Q-dot 605) under the Optimum Conditions for Rhodamine Red and Q-dot 605 concentration diameter (Ave.) (nm) particle counts (counts/mL)a measurement λ(Ex/Em) (nm) intensity intensity/particleb ratio specific intensityc ratio
MPS NPs-R
TEOS NPs-R
Q-dot 605
Q-dot 605
0.10mg/mL 490 7.1 × 109 570/590 14.7 6.8 × 10-8 1 1.5 × 102 1
0.11 mg/mL 200 2.0 × 109 570/590 13.7 6.8 × 10-9 0.1 1.2× 102 0.8
40 nM 20 2.4 × 1013 570/590 17.3 7.2× 10-13 1.1 × 10-5 2.2 × 101 0.15
40 nM 20 2.4× 1013 350/605 505.4 2.1 × 10-11 3.1 × 10-4 6.3 × 102 4.2
a Concentration divided by weight of one particle. The weight of one particle was calculated from the volume of one particle: 4π(0.000290/2)3/3 (mm3) × 2.3 (specific gravity). b Intensity divided by the particle count. c Intensity divided by the particle count and then by the volume of one particle.
(Rockford, IL). Phycoerythrin-conjugated streptavidin (PCS) was from eBioscience (San Diego, CA). Green fluorescent protein (GFP) was from Upstate (Lake Placid, NY). Cy3conjugated anti-goat IgG wasfrom Jackson Immunoreserch (West Grove, PA). Preparation of Organosilica Nanoparticles and FluorescentContaining Organosilica Nanoparticles. TEOS (21 µL) or 19 µL of MPS, 325 µL of ethyl alcohol, 36 µL of NH4OH, and 68 µL of distilled water were combined using Sto¨ber’s method24 and incubated for 2 days. Fluorescent silica nanoparticles were prepared as described previously.26 APS-fluorescent dye conjugates or MPS-fluorescent dye conjugates were prepared by gently stirring a mixture of 10 mM of APS and 10 mM of 5(6)-carboxyfluorescein-N-hydroxysuccinimide ester or 10 mM MPS and 10 mM rhodamine red C2-maleimide in DMSO for 1 h. The Silica-fluorescent dye conjugates were mixed with a mixture of 21 µL of TEOS or 19 µL of MPS, 325 µL of ethyl alcohol, 36 µL of NH4OH, and 68 µL of distilled water using Sto¨ber’s method38 and incubated with gentle mixing for 2 days.
After incubation, the reaction mixture were applied to centrifugation to remove the remaining reagents. The particles were washed extensively with 70% ethyl alcohol and water. Electron Microscopy of Fluorescent Silica Nanoparticles. The nanoparticles were fixed on a 400-mesh copper grid coated with nitrocellulose, and transmission electron microscopy images were obtained with a Hitachi H800 electron microscope (Hitachi, Tokyo, Japan) or a JEOL JEM-1200EXII electron microscope (JEOL Ltd., Tokyo, Japan). Absorbance and Fluorescence Studies. Absorbance and fluorescence spectra of about 0.10 mg/mL solutions of the fluorescent nanoparticles and 40 nM Q-dot 605 were obtained with a U-3000 spectrophotometer (Hitachi, Tokyo, Japan) and an F-2500 fluorescence spectrophotometer (Hitachi, Tokyo, Japan), respectively. Preparation of Surface-Modified Nanoparticles. To prepare surface-modified nanoparticles using maleimide reaction, we reacted 95 µL of solutions of MPS NPs or TEOS NPs with 5 µL of 10 mM rhodamine red C2-maleimide, 5 µL of various
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Figure 2. Fluorescence microscopy of silica nanoparticles modified with the maleimide-rhodamine red conjugate on the surface. MPS NPs (a and b) and TEOS NPs (c and d) modified with the maleimiderhodamine red conjugate on the surface. Observation was made under bright field (a and c) or with excitation at 540/12 nm (b and d).
concentrations (from 0.25 to 2 mM) of fluorescein-5-maleimide, or 5 µL of 2 mg/mL EZ-link maleimide activated neutravidin protein for 2 h at room temperature. After the reaction was complete, the reaction mixture was centrifuged to remove remaining reagents. The particles were washed extensively with water or phosphate-buffered saline. Fluorescence and Light Microscopic Analysis of Fluorescent Silica Nanoparticles. Aliquots of the solutions containing the nanoparticles reacted with rhodamine red C2-maleimide were placed on glass slides and allowed to dry at room temperature. We analyzed the nanoparticles attached to the glass slides by using a fluorescence and light microscopy system consisting of an inverted fluorescence microscope (TE 2000, Nikon, Kanagawa, Japan) equipped with a 100-W mercury lamp as a light source and a CCD camera (Digital Sight DS-L1, Nikon, Kanagawa, Japan). To evaluate the photostability of the fluorescence intensity, we viewed a fixed area of a single fluorescent silica nanoparticle or Q-dot 605 particle attached to a glass slide through a CCD camera (Rolera-XR Mono Fast 1394 Cooled, Qimaging, Burnaby, BC, Canada) with continuous excitation and analyzed it with Image-Pro Plus software (MediaCybernetics, Silver Spring, MD). MPS NPs modified with GFP were prepared by mixing 1 µL of MPS NPs solution and 1 µL of 10 µg/mL of GFP, placed on glass slide, and observed before the drying of the mixture. Flow Cytometry. Flow cytometry analysis was performed on FACSCalibur flow cytometers (Becton Dickinson, San Jose, CA) with 488- and 635-nm excitation lasers. Green fluorescence was detected on the FL1 channel (530/30-nm band-pass filter), and red fluorescence was detected on the FL4 channel (661/ 16-nm band-pass filter). Surface-modified nanoparticles with various concentrations of fluorescein-5-maleimide as described above were diluted and analyzed. Surface-modified nanoparticles with proteins using absorption were prepared by simple mixing. Five microliters of the solutions of MPS NPs or TEOS NPs were mixed with an equal volume of 10 µg/mL of GFP or 10 µg/mL PCS, incubated for 5 min, diluted with 500 µL of water, and analyzed. All data were obtained without compensation. Zeta Potential Analysis. The zeta potential of nanoparticles was determined with a NICOMP submicron particle sizer, model 380/ZLS (Nicomp Particle Sizing Systems, Santa Barbara, CA) at room temperature using the zeta supplement. Electrodes were dipped directly in the solution containing freshly prepared silica nanoparticles. On the basis of the principles of electrophoretic
Nakamura and Ishimura
Figure 3. Flow cytometry analysis of surface fluorescence-tuned MPS NPs. MPS NPs modified with various concentrations of maleimidefluorescein conjugate on the surface were analyzed under the same conditions.
Figure 4. Fluorescence microscopy was used to compare the photostability of TEOS NPs (open boxes) and MPS NPs (solid circles), both incorporating fluorescent dye, and MPS NPs surface-modified with rhodamine red (solid triangles).
light scattering, we performed quantitative measures of the charge on colloidal particles in liquid suspension. Dot Blotting using Nanoparticles. The solution containing TEOS NPs or MPS NPs (0.2 µL) was spotted on glass slides and then dried. Two microliters of 7.5 µg/mL Cy3-conjugated anti-goat IgG was added to spots of TEOS NPs and MPS NPs and on the glass slide as a control and incubated at room temperature for 5 min in a moist chamber. After several washings, we analyzed the glass slides using a fluorescence image analyzer, FM BIO II Multi-View with FM BIO Analysis version 8.0 (Takara, Tokyo, Japan). Results and Discussion Preparation of Organosilica Nanoparticles. We synthesized nanoparticles successfully using MPS as the sole silica source, without other silicates such as TEOS and sodium silicate, by using a Sto¨ber method. The resulting MPS NPs were welldispersed. The rate of formation of MPS NPs was compared with that of TEOS NPs under the same conditions using transmission electron microscopy (TEM). TEOS NPs are clearly observable 9 h after the start of the reaction (Figure 1a-c). In the TEM studies, TEOS NPs did not change significantly after 1 and up to 2 days; thus, TEOS NPs are completely formed within the first 9 h of reaction time. Formation of MPS particles proceeded differently. After 9 h, the margins of the products were unclear; some of them were fused with each other, and others were separate (Figure 1d). We then washed the products in 70% ethyl alcohol/water, and followed up with centrifugation, upon which no nanoparticles were recovered. After day 2,
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TABLE 2: Zeta Potential of Silica Nanoparticles nanoparticles
zeta potential (mV)
TEOS NPs TEOS NPs containing rhodamine red MPS NPs MPS NPs containing rhodamine red MPS NPs displaying rhodamine red MPS NPs displaying NeutrAvidin
-38.7 -36.0 -52.2 -52.1 -32.2 -19.2
distinct MPS NPs were observed (Figure 1e and f). These results indicated that MPS NP formation was slower than TEOS NP formation under the same conditions. The particle growth process for MPS may differ from that for TEOS. The formation of TEOS NPs by the Sto¨ber method begins with hydrolysis of a silica precursor by ammonium hydroxide, followed by self-polymerization, formation of silica matrices, and precipitation of TEOS NPs. The mechanism by which MPS NPs form is not clear. The MPS reaction mixture became cloudy within a few hours, but no MPS particles could be recovered after the washing procedure. One hypothesis is that MPS micelles were formed first, followed by hydrolysis and polymerization of MPS by ammonium hydroxide in the micelles, resulting in the formation of nanoparticles. As compared with TEOS nanoparticles, MPS nanoparticles tend to have a wide distribution of sizes dependent on the concentration of MPS in the reaction mixture (data not shown). The size distributions of the TEOS and MPS NPs differ. For example, on day 2, the size of TEOS NPs ranged from 250 to 570 nm (Figure 1c), and that of MPS NPs from 350 to 1200 nm (Figure 1f). MPS NPs with a narrow size distribution are required for several applications, and methods for preparing them are under investigation by us. We hypothesized that MPS could make dipodal alkoxysilanes via formation of disulfide bonds between the thiol residues of MPS and the resulting dipodal alkoxysilane could produce POM nanoparticles. The other silica compounds that could produce nanoparticles like MPS are being sought and will be investigated by us. Preparation and Charaterization of Fluorescent-Containing Organosilica Nanoparticles. We synthesized MPS NPs and TEOS NPs containing fluorescent dyes as described in our previous report.26 In the present study, we characterized these particles and compared them with quantum dots (Table 1). The fluorescence intensity of Q-dot 605 was evaluated under two sets of conditions: The first set of conditions was the same as that used to evaluate the fluorescence intensity of the fluorescent silica nanoparticles containing rhodamine red (excitation and emission wavelengths were 570 and 590 nm, respectively) (Table 1 third column); the second set employed the optimum conditions for Q-dot 605 (excitation and emission wavelengths were 350 and 605 nm, respectively) (Table 1 fourth column). As summarized in Table 1, the fluorescence intensity divided by the particle count of the MPS NPs containing rhodamine was higher than that of quantum dots, perhaps because of the larger size of the silica nanoparticles and the amount of dye incorporated. Next we calculated the specific fluorescence intensity, which is the total fluorescence intensity divided by the particle volume (Table 1). The specific fluorescence intensity of the MPS nanoparticles measured with excitation at 570 nm and emission at 590 nm was 7 times that of the quantum dots measured under the same conditions and 1/4 that of quantum dots measured under the optimum conditions for Q-dot 605. In our previous work,26 TEOS NPs containing fluorescence were prepared using the same method and were compared with Q-dot 525. The
Figure 5. Dot-blot analysis of the ability of the silica nanoparticles to bind proteins. Nanoparticles dotted on glass slides were reacted with Cy3-conjugated anti-goat IgG, analyzed with a fluorescence image analyzer (a), and intensities were plotted (b). The fluorescence intensity of MPS NPs was higher than that of TEOS NPs.
specific intensity of TEOS NPs containing fluorescence was 1/3 that of the quantum dots with under the same conditions and 1/48 that of under the optimum conditions for Q-dot 525. In the case of the silica nanoparticle containing rhodamine red, the relative intensity to the quantum dot was improved as compared with the silica nanoparticle containing fluorescence. These results indicated that the selection of a suitable dye to incorporate into a silica network could improve the fluorescence intensity. The specific intensity of TEOS NPs and MPS NPs was similar; thus, the efficacy of rhodamine dye impregnation onto the silica network did not differ significantly between the two. In addition, fluorescent MPS NPs could be prepared as fluorescent-tuned silica nanoparticles and multiple fluorescent silica nanoparticles containing two kinds of fluorescent dyes like that of TEOS-NPs as reported previously (data not shown).26 Fluorescent nanoparticles, including MPS NPs and TEOS NPs, were of high fluorescence intensity; the fluorescence intensity of silica nanoparticles may have been enhanced relative to that of quantum dots by improvement of the synthetic method and the selection of fluorescent dyes. Preparation and Charaterization of Organosilica Nanoparticles with Fluorescent Attached to the Surface. MPS NPs have yet another advantage over conventional TEOS NPs: the surface of MPS NPs can be modified easily. The reactivity of the MPS NPs is a result of the thiol residue on the surface, which allows modification using, for example, maleimideconjugated fluorescent dyes. MPS NPs were reacted with dyes conjugated with maleimide and then characterized and compared with TEOS NPs by using fluorescence microscopy and flow cytometry. MPS NPs were reacted with a rhodamine red conjugated with maleimide, and the resulting particles were fluorescent. After the reaction, washing, and centrifugation, the pellets of MPS-NPs changed color from white to red. MPS NPs clearly fluoresced as was clear in fluorescence microscopy (Figure 2a and b). As a control experiment, TEOS NPs were also reacted with the rhodamine red-maleimide conjugate. The pellets of TEOS-NPs stayed white after washing, and no fluorescence from these NPs was observed by fluorescence microscopy (Figure 2c and d) under the same conditions used for MPS NPs. These findings indicated that surface attachment with the rhodamine red-maleimide conjugate was specific to MPS NPs, indicating that MPS NPs displayed on their surface abundant thiol residue that reacted with dye.
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Figure 6. Flow cytometry analysis of silica nanoparticles surface-modified with proteins. MPS NPs (a and c) and TEOS NPs (b and d) modified with GFP (a and b) or phycoerythrin-conjugated streptavidin (PCS) (c and d) were analyzed. Red lines and green lines indicate before and after reaction, respectively.
We next examined MPS NPs with fluorescein attached to the surface, using flow cytometry analysis to evaluate the intensity of fluorescence. MPS NPs (average diameter, about 450 nm; distribution of diameter from 200 to 600 nm) were reacted with various concentrations of the fluorescein-maleimide conjugate and then analyzed by flow cytometry. These MPS NPs fluoresced with various intensities (Figure 3). These results suggest that surface attachment of fluorescent dye on MPS NPs via thiol residues is effective for flow cytometric analysis of nanoparticles and that the fluorescence intensity is tunable. We have shown that MPS NPs can be made amenable to flow cytometric analysis and microscopic observation after surface modification with fluorescent dyes. Using fluorescence microscopy, we compared the photostabilities of TEOS and MPS NPs incorporating rhodamine red within the particles and MPS NPs with rhodamine red attached to the surface. The normalized curves of the single-particle fluorescence intensity of TEOS and MPS NPs, both with rhodamine inside the particles, showed good photostability; the intensities of both remained above 50% of their initial values after 250 s under continuous excitation, and their stabilities were about the same (Figure 4). The photostability of MPS NPs with rhodamine red on the surface was only about 40% after 250 s. The single-particle fluorescence intensities of TEOS containing rhodamine red, MPS NPs containing rhodamine red, and MPS NPs with rhodamine red on the surface at time 0 were 175 889, 120 582, and 403 338 a.u. and those after 250 s were 111 351, 66 549, and 147 294 a.u., respectively, and the fluorescence of them remained visible. The fluorescence intensity of surfacemodified MPS NPs was the highest among all of the NPs tested, but the photostability was no better than that for the others. In the case of MPS NPs with rhodamine red on the surface, abundant fluorescent dye was attached to the surface and the surface layer was accessible to the solvent and the oxygen it contained. In contrast, in NPs incorporating fluorescent dye inside, the dye was somewhat isolated by the particle from the outside environment and less exposed to oxygen molecules. This
difference might account for the observed differences in photostability. Zeta Potential Analyses of Organosilica Nanoparticles. Zeta potential analyses of TEOS NPs and MPS NPs were performed to characterize the surface charge of the NPs and to confirm surface functionalization. As shown in Table 2, the zeta potentials of MPS NPs were more negative than those of TEOS NPs. The zeta potentials of MPS NPs and TEOS NPs incorporating rhodamine red in their interior were not markedly different from those without dye. Alternatively, the zeta potential of MPS NPs surface-treated with rhodamine red-maleimide or NeutrAvidin maleimide conjugate were substantially lower. These results indicated that NPs incorporating rhodamine red inside the particle did not show the presence of fluorescent dyes on the surface; consequently, the zeta potentials were not affected. As will be discussed later in this paper, conjugation of protein to the surface of MPS NPs via the thiol-maleimide reaction was also effective and can change the zeta potential of NPs significantly. Surface Modifications of Organosilica Nanoparticles. MPS NPs are unique because of the thiol residues on the surface as prepared, without requiring any additional procedure. They also have more negative zeta potentials as compared with TEOS NPs. We examined the surface modification property of MPS NPs by using flow cytometry, fluorescence image analysis, and fluorescence microscopy. To compare the surface properties of MPS and TEOS NPs, we performed dot-blotting using these NPs. The dot-blotting could evaluate the ability of nanoparticles to absorb proteins on glass slides. The same amount of each NP was deposited on glass slides, dried, then reacted with a solution containing Cy3-conjugated anti-goat IgG, and examined with a fluorescence image analyzer. The spots of MPS NPs showed markedly higher intensity derived from Cy3-conjugated anti-goat IgG as compared with that of TEOS NPs (Figure 5a). The fluorescence intensity from MPS NPs was about 3 times that of TEOS NPs (Figure 5b). These results indicated that MPS NPs on glass slides could absorb a greater amount of protein
Organosilica Nanoparticles
Figure 7. Fluorescence microscopy of MPS NPs modified with GFP. MPS NPs modified with GFP can be detected under fluorescence microscopy. The particles were fluorescent and remained dispersed.
than TEOS NPs. In addition, glass slides with modified MPS NPs could improve protein absorption significantly, making them potentially useful for chip-based technology. We next performed flow cytometry to compare the surface properties of MPS and TEOS NPs in solution. The NP solutions were mixed with protein solutions containing either green fluorescent protein (GFP) or phycoerythrin-conjugated streptavidin. After mixing with GFP or phycoerythrin-conjugated streptavidin, the flow cytometry peaks for MPS NPs were markedly shifted to right due to fluorescence from GFP as compared with those of TEOS NPs (Figure 6). These findings indicated that MPS NPs absorb protein more effectively than TEOS NPs in solution. Microscopic observations were performed to evaluate the dispersion of MPS NPs modified with protein on the surface. The MPS NPs solution mixed with a solution containing GFP showed well-dispersed NPs with distinct fluorescence (Figure 7). These findings indicated that MPS NPs could be modified with GFP very effectively while still retaining good dispersion as compared with that of TEOS NPs. MPS NPs modified with GFP could be detected and observed by flow cytometry and fluorescence microscopy. The potential usefulness of MPS NPs for biological investigations, including bead assay using flow cytometry with MPS NPs, are under investigation. Previous studies reported modification of TEOS NPs by silanizations of silica nanoparticles with 3-mercaptopropyltrimethoxysilane or with N1-[3-(trimethoxysilyl)-propyl]diethylenetriamine. The silanized silica NPs were conjugated with disulfide-modified oligonucleotides via the thiol/disulfide exchange reaction,22 and with enzymes and antibodies via crosslinking between amine using glutaraldehyde.17 More recently, it has been reported that silica nanoparticles were prepared and subsequently surface-modified via cohydrolysis with TEOS and various organosilane reagents.23 This method can reduce the aggregation of amine-modified silica NPs. In the present study, MPS NPs were synthesized via a one-pot synthesis resulting in thiol residue on the surface without any additional procedure; they were well-dispersed and had a more negative zeta potential than TEOS NPs. MPS NPs hold promise for various biological applications because of their potential for surface modification; this aspect including bioassays and drug delivery systems is currently under investigation. Thiol residues on NPs have various advantages for modification and functionalization of nanoparticles. Thiol residues could
J. Phys. Chem. C, Vol. 111, No. 51, 2007 18897 react with various chemical couplings such as alkyl halide and maleimide, and could form covalent bonds with other molecules easily. Recently, various maleimide-conjugated molecules, including fluorescent dyes, streptavidin, polyethylene glycol, and others, have become commercially available. As described in this paper, reaction procedures are simple and conjugation efficiencies are high. In addition, a thiol/disulfide exchange reaction is also useful for conjugating thiol residues between oligonucleotides and silica nanoparticles,22 and between one protein and another.30 In this paper, we have demonstrated a better way to formulate MSP NPs that could be utilized in a variety of applications. Using thiol residues on the surface, novel nanoparticle-based drug delivery and release systems have been proposed and, in some cases, demonstrated.34,35 Glutathione (GSH) is the most abundant thiol species in cytoplasm, and it could be the major reducing agent. Using the difference in concentration between the intracellular GSH (1-10 mM) and extracellular GSH (2 µM in plasma),33-35 selective intracellular releases of substances were reported. Nanoparticles coated with plasmid DNA via disulfide bonds could release plasmid DNA via dissociation by intracellular disulfide reduction.34 Gold nanoparticles with layers containing thiolated Bodipy dyes have been prepared, and the thiolated Bodipy dyes were released in living cells successfully via a GSH-mediated release system.35 Our studies of MPS NPs modified with surface thiol residues suggest the possibility of adapting them as a carrier for nanoparticle-based drug delivery and release systems that use GSH to realize selective intracellular release of drugs and biomolecules. Conclusions Novel organosilica nanoparticles, MPS NPs, were synthesized by using one-pot synthesis based on the Sto¨ber method. MPS NPs have the important potential for surface modification as a result of the thiol residue on their surface. MPS NPs and MPS NPs modified with various molecules, including fluorescent dyes and proteins, have potential for use in various applications, such as biomedical analysis, chip-based technology, multi-target detection systems, imaging in vitro and in vivo, and drug delivery systems. 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). References and Notes (1) Salata, O. V. J. Nanobiotechnol. 2004, 2, 1-6. (2) Gao, X.; Cui, Y.; Levenson, R. M.; Chung, L. W. K.; Nie, S. Nat. Biotechnol. 2004, 22, 969-976. (3) 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. (4) Wang, L.; Yang, C. Y.; Tan, W. H. Nano Lett. 2005, 5, 17-43. (5) Wang, L.; Tan, W. H. Nano Lett. 2006, 6, 84-88. (6) 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. (7) Santra, S.; Zhang, P.; Wang, K.; Tapec, R.; Tan, W. Anal. Chem. 2001, 73, 4988-4993. (8) Tan, W.; Wang, K.; He, X.; Zhao, J.; Drake, T.; Wang, L.; Bagwe, R. P. Med. Res. ReV. 2004, 24, 621-638. (9) Zhao, X. J.; Bagwe, R. P.; Tan, W. H. AdV. Mater. 2004, 16, 173176. (10) Zhao, X.; Dytioco, R.; Tan, W. J. Am. Chem. Soc. 2003, 125, 11474-11475.
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