Versatile Synthesis of Thiol- and Amine-Bifunctionalized Silica

Jun 13, 2014 - School of Pharmacy, National Defense Medical Center, Taipei 11490, Taiwan ... Department of Chemical Engineering, National Tsing Hua ...
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Versatile Synthesis of Thiol- and Amine-Bifunctionalized Silica Nanoparticles Based on the Ouzo Effect Shih-Jiuan Chiu,*,† Su-Yuan Wang,† Hung-Chang Chou,‡ Ying-Ling Liu,§ and Teh-Min Hu*,‡ †

School of Pharmacy, College of Pharmacy, Taipei Medical University, Taipei 11031, Taiwan ROC School of Pharmacy, National Defense Medical Center, Taipei 11490, Taiwan ROC § Department of Chemical Engineering, National Tsing Hua University, Hsinchu 30013, Taiwan ROC ‡

S Supporting Information *

ABSTRACT: In this article, we report a novel, nanoprecipitation-based method for preparing silica nanoparticles with thiol and amine cofunctionalization. (3Mercaptopropyl)trimethoxysilane (MPTMS) and 3-aminopropyltrimethoxysilane (APTMS) were used as the organosilane precursors, which were subjected to acidcatalyzed polycondensation in an organic phase containing a water-miscible solvent (e.g., dimethyl sulfoxide). A pale colloidal solution could be immediately formed when the preincubated organic phase was directly injected into water. The initial composition ratio between MPTMS and APTMS is an important factor governing the formation of nanoparticles. Specifically, large, unstable micrometer-sized particles were formed for preparation using MPTMS as the sole silane source. In contrast, when APTMS was used alone, no particles could be formed. By reducing the fraction of APTMS (or increasing that of MPTMS) in the initial mixture of organosilanes, the formation of nanometer-sized particles occurred at a critical fraction of APTMS (i.e., 25%). Remarkably, a tiny fraction (e.g., 1%) of APTMS was sufficient to produce stable nanoparticles with a hydrodynamic diameter of about 200 nm. Other factors that would also affect particle formation were determined. Moreover, an interesting temperature effect on particle formation was observed. The TEM micrographs show spherical nanospheres with mean sizes of 130−150 nm in diameter. The solid-state 29Si NMR spectra demonstrate that the hybrid silica materials contain fully and partially condensed silicon structures. The bifunctionalized silica nanoparticles have positive zeta potentials whose magnitudes are positively correlated with the amount of APTMS. The total thiol content, however, is negatively correlated with the amount of APTMS. The cationic nanoparticles can bind an antisense oligonucleotide in a composition-dependent manner.

1. INTRODUCTION The design, synthesis, and development of organic−inorganic hybrid materials have recently attracted considerable multidisciplinary interest.1,2 In particular, hybrid nanoscale silica materials have drawn much attention because of the following advantages: facile size- and shape-tunable synthesis, unique physicochemical properties, high stability, and versatile routes for surface modification.3 Silica nanoparticles have found applications in many fields, including biomedical and pharmaceutical applications.3−5 The surfaces of silica nanoparticles can be easily modulated via versatile silane chemistry. The postgrafting procedure is widely used, in which primary silica particles are first synthesized by the hydrolysis and condensation of tetraalkyl orthosilicates (e.g., by the Stöber method), followed by reacting (postgrafting) the surface silanol groups with organosilanes (usually functionalized trialkoxysilanes).2,6 Another commonly employed approach is the co-condensation of tetraalkyl orthosilicates and organosilanes, which generally requires the use of surfactants.2,7,8 While most literature reports describe monofunctionalized silica particles prepared by the methods mentioned above, it is, in theory, possible to modify the surfaces of particles with different kinds of functional groups by © 2014 American Chemical Society

simply combining two or more organosilane reagents in one pot during either the postgrafting or co-condensation process. For example, Bagwe et al.8 developed a microemulsion-based, postgrafting approach to preparing silica nanoparticles with dual surface functionalization. The authors demonstrated that additional surface modification of silica spheres can reduce particle aggregation and nonspecific binding. Functionalized silica particles can also be directly produced from a single source or mixtures of organosilanes, without the use of the orthosilicate silica substrate.9−16 A simple hydrolytic sol−gel process with or without surfactants is usually involved, and polysilsesquioxane structures are generally obtained.14,17,18 Using this approach, the surfaces of silica spheres have been modified with various reactive, monofunctional moieties including vinyl, thiol, and amine groups. There is, however, a paucity of reports on the synthesis of bifunctionalized hybrid silica spheres using only organosilanes. Nair and Pavithran19 described a novel method for preparing amine/vinyl group bifunctionalized silica spheres by a casting and drying Received: April 24, 2014 Revised: June 11, 2014 Published: June 13, 2014 7676

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preformed, well-defined organic polymers are mainly used in the nanoprecipitation process.30,35,39,40 We previously reported that the ouzo effect can be utilized in preparing nitric oxidecarrying silica nanoparticles starting from a single organosilane monomer precursor.41 Herein, we introduce a facile strategy for the preparation of amine- and thiol-bifunctionalized silica nanoparticles. For the first time, we demonstrate that the ouzo effect can be extended and applied to the synthesis of stable bifunctionalized silica nanospheres from a mixture of organosilanes of varying composition. The method is based on an intriguing observation that nanocolloidal dispersions were formed when APTMS (an amine-containing organosilane) and MPTMS (a thiol-containing organosilane) at certain composition ratios were first reacted in an acidic organic phase, followed by transferring an aliquot of the organic phase into the water phase. The resulting nanoparticles contain a polysilsesquioxane structure with variable amine and thiol functionalities, and because of their positive surface charges, they exhibit excellent colloid stability and the capability to bind a model oligonucleotide.

procedure. The method required the hydrolytic co-condensation of 3-aminopropyltriethoxysilane and vinyltriethoxysilane in an ethanol/water mixture for several days, followed by casting and solvent evaporation steps to facilitate the formation of particles.19 The hybrid silica particles were proposed to be formed from aggregates of both fully and incompletely condensed silsesquioxane structures.19 Thiols (SH) and amines (NH2) are two frequently used reactive functional groups for the bioconjugation reaction. In particular, nanoparticles can be made into a carrier for drugs, imaging agents, or biomolecules via various thiol or amine chemistries, such as the maleimide- and N-hydroxysuccinimide ester (NHS)-mediated conjugation, respectively.20 Given the recent endeavor to develop multifunctional nanoparticles,21 combining multiple reactive groups in the same nanoparticle would offer an attractive strategy for effectively conjugating different molecular/therapeutic entities or ligands to nanoparticles. While this is an area that remains to be further investigated, SH- and NH2-bifunctionalized silica materials have been prepared and have been shown to be potent adsorbents for inorganic and metal ions, which have been implicated in environmental protection.22,23 To achieve mixed surface functionalities, the previous studies usually adopted the hightemperature postgrafting scheme that requires multiple complex processing steps and the use of water-insoluble aromatic hydrocarbon solvents (e.g., toluene). Moreover, although the postgrafting approach has been proven to be useful for bulk silica gel, it would be challenging to apply the method to silica nanoparticles, given the potential of particle aggregation during synthesis. Recently, mesoporous silica nanoparticles (MSN) with mixed SH/NH2 surface groups have been synthesized in an attempt to investigate the effect of surface functionalization on drug loading and release.24 In this report,24 silica nanoparticles were synthesized by the basecatalyzed co-condensation of tetraethyl orthosilicate (TEOS) and organosilanes at an elevated reaction temperature. It should be mentioned that a surfactant template was needed because the goal was to obtain a mesoporous structure. Accordingly, a general surfactant-free and room-temperature-based approach to preparing bifunctional nonporous silica nanoparticles remains to be studied. Ouzo is an alcoholic beverage in Greece. This anise-flavored drink has different names in other countries (e.g., pastis in France25). Before drinking ouzo, one usually pours the clear liquor into a glass of water to turn immediately into an opaque, milky-white solution. This intriguing phenomenon has been attributed to the spontaneous aggregation (emulsification) of water-insoluble solute trans-anethol, upon mixing the alcoholic drink with water.26−28 The ouzo effect, originally coined by Vitale and Katz,29 has now been used to describe a general physical process that involves the spontaneous formation of metastable colloid dispersions via mixing water (nonsolvent) with a water-miscible solvent containing hydrophobic solutes.25,30−35 The process, widely known as nanoprecipitation, was first described by Fessi et al. in 1989 for the preparation of drug-carrying polymer nanoparticles.36 Recently, nanoprecipitation has become a popular approach to fabricating pharmaceutical nanocarriers possibly because it employs a simple and versatile procedure that generally requires no surfactants and minimal energy.30 Moreover, the method tends to produce drug nanoparticles with a high loading efficiency.36−38 The application of the method in drug delivery has recently been reviewed by Lepeltier et al.35 Notably,

2. EXPERIMENTAL SECTION 2.1. Materials. 3-Mercaptopropyltrimethoxysilane (MPTMS), 3aminopropyltrimethoxysilane (APTMS), and other aminotrimethoxysilanes were purchased from Sigma-Aldrich (St. Louis, MO). Dimethyl sulfoxide (DMSO), 5,5-dithiol (2-nitrobenzoic acid), L-cysteine hydrochloride, human serum albumin, and phosphate buffers were also from Sigma-Aldrich. Diethylenetriaminepentaacetic acid (DTPA) was obtained from TCI (Tokyo, Japan). Acetone and sodium chloride were obtained from J. T. Baker (Phillipsburg, NJ). Methanol, acetonitrile, and hydrochloric acid were purchased from Merck (Darmstadt, Germany). HEPES (sodium salt) was obtained from BioShop (Burlington, Canada). Cell culture medium RPMI was purchased from PAA Laboratories Pty Ltd (Morningside, Australia). Oligonucleotide G3139 (Genasense, 5′-TCT CCC AGC GTG CGC CAT-3′) with a phosphorothioate backbone was produced by Alpha DNA (Montreal, Canada). All chemicals and solvents were analytical reagent grade and used as received. Deionized water (18.2 MΩ·cm at 25 °C) was used throughout the study (Millipore Milli-Q gradient A10, Bedford, MA). 2.2. Synthesis of Bifunctional Silica Nanoparticles. The present study employed a nanoprecipitation procedure to prepare silica nanoparticles, which involves mixing an organic phase with a water phase. In a typical synthesis, 10 mL of the organic phase contains 8.13 mL of DMSO, a total of 0.37 mL of the organosilane precursors (with various molar ratios of MPTMS and APTMS), 0.5 mL of 10 mM DTPA (dissolved in deionized water to prevent thiol autoxidation), and 1 mL of 5 M HCl. The concentration of MPTMS and APTMS combined was generally kept at 200 mM. The organicphase materials were added to a 10 mL glass container immersed in an ice bath. After 10 min, the organic phase was left standing for 24 h at ambient temperature (to avoid light). At the end of the reaction, a 1 mL aliquot of the organic phase was aspirated using a 1 mL syringe (27 gauge) and injected rapidly (∼10 s) into 10 mL of water (the water phase) under constant stirring (e.g., 300 rpm) at room temperature. The resulting colloid dispersion was left to age for 2 h at 60 °C and then was centrifuged at 5500 rpm (3615g) for 30 min at 4 °C (Sorvall Super T21, DuPont Company, Wilmington, DE). After the supernatant was removed, the particle pellet was then subjected to repeat washing−centrifugation steps (10 mL of water × 2 cycles). Finally, the washed particles were redispersed in 10 mL of water by ultrasound. To optimize the preparation conditions, several experimental parameters were varied and tested, including the silane composition ratio, total silane concentration, reaction time and temperature, types of organic solvents, stirring rate, needle size, injection volume, ionic strength, and aging time and temperature. 7677

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2.3. Characterization of Bifunctional Silica Nanoparticles. The hydrodynamic sizes of particles were determined using a dynamiclight-scattering (DLS) instrument (LB-550, Horiba Instruments, Inc.). Generally, 1 mL of undiluted particle samples from three independent syntheses was taken for the measurement, and for each sample, the measurement was repeated three times. The polydispersity index (PDI) was calculated using the relationship PDI = (CV%/100)2, where CV% is the coefficient of variation (%) of the measured particle size distribution. The turbidity of colloid solutions was determined by measuring the optical density (O.D.) at 650 nm using a spectrophotometer (Shimadzu UV-2450, Kyoto, Japan). The surface charges of nanoparticles dispersed in water (1.5 mL) were characterized using a zeta-potential analyzer (ZetaPlus, Brookhaven Instruments Co.). For each sample, the zeta potential was measured 10 times at room temperature. TEM images of nanoparticles were acquired on a Hitachi H-600 transmission electron microscope operated at 75 kV. To prepare TEM samples, an aliquot (20 μL) of diluted aqueous particle dispersions was placed and air dried on carbon-coated nickel TEM grids (200 mesh; Electron Microscopy Sciences, Hatfield, PA). Particle sizes were analyzed using ImageJ software.42 Solid-state magic angle spinning (MAS) 29Si NMR spectroscopy was performed on a Bruker Avance III spectrometer equipped with a wide-bore 14.1 T magnet and a 4 mm double-resonance MAS probehead. The instrumental settings are as follows: sample spinning rate, 10 kHz; 29Si Larmor frequency, 119.24 MHz; spectra acquisition parameters, 2.5 μs pulse (π/4), 1H TPPM decoupling scheme (70 kHz rf); and recycle delay, 120 s. Elemental analysis was recorded on a Thermo Flash 2000 (for N, C, S, and H). The thiol contents on nanoparticle surfaces were determined using a colorimetric method.13 Briefly, 0.5 mL of diluted aqueous dispersions of silica nanoparticles was mixed with 0.2 mL of a 5,5-dithiol (2nitrobenzoic acid) solution (5 mM in 0.5 M phosphate buffer, pH 7.4), and the reaction mixture was incubated for 30 min at room temperature. Then, the reaction solution was centrifuged at 14 000 rpm (15 996g, Eppendorf Centrifuge 5402, Hamburg, Germany) for 10 min, and the absorbance of the resulting supernatant was measured at 412 nm using a UV−visible spectrophotometer (Shimadzu UV2450, Kyoto, Japan). The thiol concentrations were estimated from the standard curve of cysteine hydrochloride (7.8−250 μM). 2.4. Oligonucleotide Binding. The capacity of the prepared silica nanoparticles binding to a model antisense oligonucleotide, G3139, was measured by a depletion method. The silica nanoparticles prepared at various M/A ratios (5:1−99:1) were dispersed and diluted in water. The turbidity of the particle dispersions was adjusted and maintained at an optical density (650 nm) of 1 such that each sample contained comparable numbers of particles. To 1 mL of diluted particle dispersions, a 10 μL aliquot of a G3139 solution containing various amounts (2.5−20 μg) of G3139 in water was added, and the resulting reaction mixture was then incubated at room temperature for 30 min. After incubation, the solution was centrifuged (14 000 rpm, 10 min) to settle particles, and the amount of G3139 remaining in the supernatant was assayed by monitoring the absorbance at 260 nm and using a standard curve of G3139. The amount of oligonucleotide binding to silica nanoparticles was calculated by subtracting the amount of G3139 added from the amount of G3139 remaining in the supernatant.

Figure 1. Formation of colloidal dispersions via the nanoprecipitation procedure (the ouzo effect). (A) Hydrodynamic diameters and size distributions (inset) measured by DLS for particles formed at various MPTMS/APTMS (i.e., M/A) ratios. (Top inset) Photograph showing the appearance of the corresponding particle dispersions. (B) Hydrodynamic diameters and turbidity (mean ± SD, n = 3) as a function of the percentage of APTMS. (Note that the range of the corresponding M/A ratio was extended.)

the particles settled rapidly. Surprisingly, when MPTMS and APTMS were combined and left to react in the organic phase, colloid dispersions containing nanoscale particles could be formed after mixing the organic phase with the water phase for certain MPTMS/APTMS (M/A) molar ratios. It can be seen in Figure 1A that stable colloid dispersions were formed at a critical M/A ratio (e.g., 3:1). Below this critical ratio, the formed solutions were clear; above this ratio, however, the resulting solutions became increasingly turbid with increasing M/A ratios (see photographs in Figure 1A). There is no sign of particle settling or phase separation, suggesting that the colloid system is at least metastable and the ouzo effect is operative. Initially, we observed that stable particle dispersions can be produced at M/A molar ratios ranging from 3:1 to 19:1 (corresponding to 25−5% APTMS) and the hydrodynamic sizes of the particles formed were all around 200−250 nm with a similar size distribution (Figure 1A). We then conducted additional nanoprecipitation experiments for a wide range of organosilane ratios, including those that correspond to extremely low %APTMS. Strikingly, stable colloid dispersions can still be formed when the proportion of APTMS used is as low as 0.25% (i.e., M/A = 399:1), although the measured

3. RESULTS AND DISCUSSION 3.1. Silica Ouzo Effect and the Formation of Nanocolloidal Dispersions. The present study was initiated by an interesting finding shown in Figure 1A. When APTMS was used as the only organosilane and subjected to the acidcatalyzed precondensation and phase-mixing procedures as described in the Experimental Section, the final solution remained clear, indicating no particle formation. In contrast, when MPTMS was used alone, an opaque solution containing particles with sizes >3 μm was formed after phase mixing, and 7678

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keeping M/A at 5:1. After 24 h of incubation, 1 mL of the organic phase was injected into 10 mL of water. The result, unexpectedly, shows that nanoparticles with a mean diameter of 200−250 nm can be formed for all organosilane concentrations tested (Figure 2). The colloidal solutions were stable in terms

particle sizes are larger (Figure 1B). Furthermore, the use of smaller amounts of APTMS resulted in the formation of a more turbid colloid solution (turbidity measurement, Figure 1B). On the basis of comparable particle sizes, the result suggests that more particles were produced when smaller fractions of APTMS were used. This was confirmed by collecting the solid contents of the final dispersions and the result in Figure S1 of the Supporting Information showing that the solid recovery rate decreases from 89% at M/A = 99 (i.e., 1% APTMS) to 15% at M/A = 5 (i.e., 16.7% APTMS). To optimize the synthesis of nanoparticles, we investigated the effect of several experimental parameters on the formation of colloid dispersions. For most experiments, the M/A ratio was kept at 5:1, unless otherwise indicated. By varying the rate of magnetic stirring in the water phase, the result shows that particle sizes decreased with increasing stirring rates; however, wider size distributions (i.e., higher polydispersity index (PDI) values) were observed at higher stirring rates (Figure S2 in Supporting Information). Smaller particles can also be produced when smaller injection needles (e.g., 27 gauge) were used. Furthermore, the formation of stable nanocolloidal solutions was highly dependent on the volume of the organic phase injected. Our data show that in 10 mL of water, stable nanocolloidal solutions were obtained only when the volume of the injected organic phase was maintained below 2 mL. The above findings are consistent with the theory of rapid nanoprecipitation. Specifically, supersaturation is favored under conditions of intense micromixing and optimal mixing ratios of organic solvent to water.43 We also determined the effect of varying the type of organic solvents used on particle sizes. As depicted in Figure S2, DMSO was the best solvent because it produced the smallest particles with the most homogeneous size distribution. Although nanoparticles prepared in acetonitrile (the least water-miscible solvent tested) resulted in the largest particles, the apparent correlation between the water miscibility of the organic solvent and the size of nanoparticles does not seem to hold in our silica system.44,45 The fact that the ouzo process usually results in stable colloid dispersions is intriguing. In the present study, silica nanoparticles were immediately formed when mixing the organic phase with water by direct injection. Under conditions that led to the formation of sub-300-nm particles, we observed that the particle size remained unchanged in the final solvent−water mixture over an extended period (e.g., 24 h). Accordingly, there appears to be no particle growth or aggregation during the prolonged incubation. This apparent particle stability may be due to the high surface charges (zeta potentials > +40 mV) of nanoparticles, which can be modified by adding NaCl to the water phase (Figure S3 in Supporting Information). The presence of NaCl in solution may screen the surface charges of particles, thereby decreasing the repulsive electrostatic force between particles and promoting the aggregation of particles.46 The ouzo effect is believed to result from the delicate interaction among the triadsolute, solvent, and water. The formation of stable colloidal particles strongly depends on the physicochemical properties of the solute and solvent. Besides, the relative quantity of the solute and solvent in the system also plays an important role. To investigate the impact of varying the initial total silane concentrations on particle formation, we measured the particle size and turbidity of the colloidal solution formed. The total concentrations of the mixed organosilanes in the organic phase were varied from 100 to 800 mM while

Figure 2. Effect of varying the initial total silane concentration (MPTMS + APTMS) on the formation of the colloidal solution. The M/A ratio was kept at 5:1.

of the absence of particle aggregation and phase segregation. Most interestingly, the turbidity of the solution increased with increasing silane concentrations up to 300 mM and then decreased when the concentration was further increased (Figure 2). Given that particle sizes varied insignificantly with silane concentrations, the turbidity data suggest that the greatest number of particles was formed at 300−400 mM silane. Besides, the complexity of the system was further revealed by showing a peaked polydispersity profile (Figure S4 in Supporting Information). To the best of our knowledge, this bell-shaped relationship has not been previously reported. The formation of stable nanoparticles usually occurs in a tiny ouzo region on a phase diagram where the range of solute concentration is quite narrow. Increasing the solute concentration over the ouzo limit would induce particle aggregation and produce an unstable system.29,31,32 In our previous study,41 the range of solute concentrations within the ouzo region was estimated to be less than 3-fold. Nevertheless, in the present study, the presence of APTMS appears to extend the concentration range significantly for stable particle formation. Moreover, the data suggest that APTMS at high concentrations may inhibit particle formation. 3.2. Kinetic and Temperature Effects. In the initial experiments, the organosilane mixture was allowed to react in the organic phase at a fixed reaction time of 24 h before the nanoprecipitation procedure was performed. It was assumed that a suitable reaction time is required so that sufficient polycondensed silica species can be accumulated in the organic phase and later precipitated as nanospheres in the water phase. To further study the effect of varying the organic-phase reaction time on particle formation, various reaction times ranging from 1 to 24 h were tested. The results in Figure 3 show that substantial amounts of nanoparticles were produced only until t = 4 h (based on the turbidity data) and the average hydrodynamic size of the particles formed was 317 nm in diameter (PDI = 0.12). At t ≥ 8 h, however, smaller nanoparticles with almost constant mean particle sizes (ca. 230 nm) and similar size distributions (PDI ranging from 0.17 7679

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Figure 3. Formation of nanocolloidal solution as a function of reaction time in the organic phase (mean ± SD, n = 3). The organic phase (M/ A = 5:1) was incubated for various times before being mixed into water. After mixing, the particle size and turbidity were immediately measured by DLS and UV−visible spectrophotometry, respectively.

at 8 h to 0.111 at 24 h) were produced (Figure S5 in Supporting Information). Notably, the turbidity of the resulting colloid solution increased almost linearly with time (t ≥ 4 h, Figure 3), suggesting that the number of nanoparticles was constantly increased over the time interval studied. Therefore, the optimal reaction time is chosen to be 24 h because it results in a greater number of nanospheres with lower size variability and because it allows more convenient experimental setups. The findings of the kinetic study described above prompted us to further determine the effect of altering the reaction temperature on silica nanoprecipitation because the extent of polycondensation in the organic phase would be modified when the reaction temperature is changed. The results are shown in Figure 4. At M/A = 5:1, nanoscale silica particles can still be produced at a low temperature (2 °C), although the mean particle size is slightly larger (Figure 4A). Increasing the incubation temperature over room temperature, however, caused a drastic change in particle formation, which is epitomized by the formation of solutions with markedly reduced turbidity; at high temperatures (e.g., 30 and 37 °C), clear solutions were even formed (photographic inset in Figure 4A). Moreover, the size measurement reported values reaching the lowest detection limit of DLS, suggesting that particle formation was substantially inhibited at elevated temperatures. Interestingly, this apparent inhibitory effect was also observed when the organic-phase reaction was run at room temperature over a prolonged period (i.e., 36 or 48 h). Can this temperature dependency be reproduced when the M/A ratio is varied? To answer this question, similar experiments were conducted for M/A = 19:1 and 99:1. The results shown in Figure 4B, however, reveal remarkably different temperature effects larger particles were produced at both low and high temperatures (i.e., a U-shaped relationship). Thus, the hightemperature inhibition of particle formation appeared not to occur at higher M/A ratios. Moreover, while the U-shaped relationship is more prominent for the system of M/A = 99:1, particle formation is less temperature-dependent at M/A = 19:1. Furthermore, regardless of the M/A ratio, the smallest particles were produced at room temperature. Taken together, the dichotomous temperature effects highlight the role that APTMS plays in the formation of silica nanoparticles in the system studied. At higher initial APTMS concentrations, intensive polycondensation (at raised reaction temperature or

Figure 4. Effect of reaction temperature on nanoprecipitation. (A) M/ A = 5:1 and (B) M/A = 19:1 and 99:1. The organic phase containing a mixture of organosilanes was incubated at various temperatures for 24 h, and then 1 mL of the organic phase was injected into water. Data were presented as the mean ± SD from three separate experiments. Photographs showing the appearance of the final solution (M/A = 5:1) were inserted. For comparison, the photographs of the final solution obtained under a condition of prolonged incubation at room temperature were also inserted in (A).

prolonged reaction time) would result in the formation of condensed silica species that contain a greater number of amine groups, which would in turn render them too hydrophilic to form particles in the water phase. However, at significantly lower levels of APTMS, the hydrophilicity gained from the incorporated amine groups would reach a limit under intensive reaction conditions where unconjugated APTMS would be completely consumed. Accordingly, the ratio of incorporated SH to NH2 groups would be substantially increased at elevated reaction temperatures, which may explain why, instead of preventing particle formation, larger particles were produced at M/A = 19:1 and 99:1. Indeed, the premise is supported by the finding shown in Figure 1B that particles with increasing sizes were formed at room temperature when the percentage of APTMS is 2000 nm) were formed, regardless of which organosilane was used. The results presented therefore demonstrate that the hydrophobicity of the aminosilane is important in controlling the formation and thus the particle size of the hybrid silica nanospheres. 3.7. Discussion. The most important finding of the present study is that amine- and thiol-bifunctionalized silica nanoparticles can be conveniently and directly prepared from a mixture of mercaptosilane and aminosilane precursors, requiring no surfactants and high-shear energy and operating under mild ambient conditions. Our method uses only mixed trialkoxy organosilanes to form a silica structure and to simultaneously provide the functional groups of interest. The preparation involves two major steps. The first is the kinetically controlled step where acid-catalyzed hydrolysis and the

condensation of organosilanes formed oligomeric or polymeric silica species in the water-miscible (organic) solvent phase. Notably, the organic phase remained clear during the preincubation (reaction) phase, indicating that the silica oligomers/polymers formed were very soluble in the organic phase and particle formation was inhibited. In the second step, instantaneous particle formation was thermodynamically driven by rapid injection and mixing of a small aliquot of the clear organic phase in pure water. This phenomenon is analogous to that of pouring ouzo into a glass of water. We called this process the silica ouzo effect. Our approach, therefore, is fundamentally different from the conventional sol−gel process in which silica particles are generated during the extensive condensation process. Our method is remarkably simple and flexible in controlling the size and surface properties. Most importantly, it offers a promising general platform for fabricating multifunctional silica nanoparticles. The silica ouzo effect presented here and in our previous paper41 is simple in its procedure but by no means in its mechanism. Indeed, it may be far more complex than the ouzo effect involving a well-defined solute, such as anethol in ouzo or synthetic polymers for pharmaceutical preparations. The complexity of our system, especially in this study, lies in the fact that the silica oligomeric/polymeric structures responsible for self-assembly in the water phase were kinetically derived from two heterogeneous precursor monomers. The evolution and the identity of such structures would therefore be controlled by the rate of siloxane condensation and initial silane composition. Taking M/A = 5 as an example (Figure 3), our data suggest that a critical reaction time (4 h) was needed before a substantial quantity of hydrophobic silica species were produced in the organic phase. Self-assembly of the hydrophobic species was then triggered by the subsequent phasemixing procedure. According to the kinetic rate law, the initial condensation rate between the same M molecules (homogeneous condensation for MPTMS) would be much higher than that between M and A (heterogeneous condensation between MPTMS and APTMS) and that between the same A molecules (homogeneous condensation of APTMS) because the initial concentration of M is much higher than that of A. As the 7683

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(Figure 4B), since the temperature effects on the conjugated and isolated species were likely to be canceled out. Fourth, by further increasing the quantity of APTMS (e.g., M/A = 5), the number of amine species incorporated into some of the polymer structures would be just adequate for particle formation and stabilization (pathway IV). Finally, above a critical APTMS concentration, the formed silica polymers would contain a large fraction of charged amine species, thus rendering them too hydrophilic to generate particles in water (pathway V).

reaction propagates, we hypothesize that the silica polymeric structure would become much more dominated by M species, especially for a high initial M/A ratio, because of the kinetic screening effect. Thus, the fraction of M species in the polymeric structure would be much higher than that in the initial precursor mixture. To support this premise, we conducted an elemental analysis (for M/A = 19), and the result indicates that the molar ratio of sulfur/nitrogen (65.7) in the particles is 3-fold higher than the M/A ratio. Accordingly, the polymeric structures that can self-assemble in the water phase consist mainly of M species, as a result of preferential condensation among M species. This is particularly true when the initial composition of M is high (e.g., M/A = 99). As the fraction of APTMS increases (e.g., M/A = 5), the hydrophobicity of the structure may be significantly attenuated under conditions of elevated reaction temperature or prolonged reaction time (Figure 4A) because more hydrophilic A species would be incorporated into the structure. This may explain why the recovery of solid contents sharply decreased with increasing APTMS (Figure S1 in Supporting Information) and why particle formation is totally inhibited above a critical APTMS fraction (Figure1). The hydrophobic mercaptopropyl group contributed by MPTMS may govern the self-aggregation of the silica polymers in water. But what is the role of APTMS? How minuscule an amount of APTMS is sufficient to stabilize the hydrophobic structure, which would otherwise aggregate to form large particles in the absence of APTMS? Why would temperature exert a different effect on systems with high and low M/A ratios (Figure 4). To obtain the answers to these questions, a detailed mechanistic study is warranted. Here we attempt to offer some plausible explanations, which are schematically illustrated in Figure 9. First, in the absence of APTMS, the MPTMS polymers would nucleate and form small primary particles after being transferred into the water phase because of their low water solubility. Since the pH of the system is around the isoelectric point (IEP ≈ 1.2)53 of the silanol group, both silanol and the SH groups would be undissociated, leading to a low surface charge of the particles. It has been suggested that for small silica particles to be stable they should acquire high surface charges.54 Accordingly, the 100% MPTMS-based primary particles would immediately aggregate to form large particles (pathway I). Second, when a small amount of APTMS is added to the initial silane mixture (e.g., M/A ≥ 99 or APTMS ≤1%), the major constituent of the silica structure would still be MPTMS because the homogeneous condensation between M species would outcompete the heterogeneous condensation. There might be a small fraction of the co-condensed amine species, but they may not provide sufficient positive charges to stabilize the structure. Instead, we speculate that the remaining amine species in the bulk solution may act as surfactants (because of the amphiphilic nature of aminosilanes) and stabilize silica particles during nanoprecipitation (pathway II). This may explain why raising the reaction temperature for M/A = 99 would result in the formation of larger particles. In this case, although a more intensive reaction condition would increase the incorporation of the amine species into the dominant silica structure, their effect on reducing the bulk surfactant-like species would be more substantial. Third, as the amount of APTMS added is increased, both conjugated and bulk amine species would contribute to the stability of particles (pathway III). This would be exemplified by the synthesis at M/A = 19, in which particle formation was less sensitive to temperature perturbation

4. CONCLUSIONS Recently, there has been a trend toward developing multifunctional nanomaterials for delivering therapeutic cargo. An attractive approach to efficiently tether a therapeutic molecule to a nanocarrier is to employ various reactive thiol and amine chemistries. While a nanoparticle can be easily activated by attaching a single functional entity (e.g., SH or NH2) to its surface, it would become more challenging to incorporate multiple surface-functional moieties. Here, we present a facile, ambient-condition, surfactant-free, low-energy method to synthesize sub-150-nm silica-based nanoparticles that exhibit both surface thiol and amine functionalities. We demonstrate that the ouzo effect was involved in the formation of stable bifunctionalized silica nanospheres directly from a mixture of thiol- and amine-containing organosilane precursors (MPTMS and APTMS). Specifically, MPTMS and APTMS were first mixed and co-condensed in the organic phase under acidic conditions. A nanocolloidal dispersion can be immediately formed when an aliquot of the clear organic phase is injected into the water phase. We determined that the formation of stable colloidal dispersions depended mostly on the initial molar ratio of MPTMS and APTMS. Importantly, we show that APTMS played a crucial role in governing the formation of stable nanoparticles, and the result was generalizable to other aminosilanes. Moreover, the bifunctional silica particles have significant positive surface charges and thiol groups that can be easily tailored by varying the molar ratio of the organosilane precursors. In conclusion, stable silica nanoparticles with tunable surface thiol and amine bifunctional groups can be efficiently prepared by the nanoprecipitation approach proposed, which may facilitate the development of multifunctional silica-based nanomedicine.

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ASSOCIATED CONTENT

S Supporting Information *

Total solid content and the corresponding recovery rate of particles prepared at various MPTMS/APTMS ratios. Effect of varying preparation parameters on particle formation. Effect of adding NaCl to the water phase on particle formation during nanoprecipitation. Polydispersity of particles formed at various silane concentrations. Particle size distribution of particles formed after the nanoprecipitation process. Optimization of the aging condition for particle collection. Colloidal stability of the collected silica nanoparticles. Effect of the amine structure of aminoalkoxysilanes on particle formation. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail:[email protected]. *E-mail: [email protected]. 7684

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Ministry of Science and Technology of ROC (NSC 99-2320-B-038-003-MY3, NSC 101-2320-B-016-004, and NSC 102-2320-B-016-003-MY3). We thank Ms. Huei-Min Chen and the Core Facility Center, Office of Research and Development, Taipei Medical University for TEM technical support. We also thank the Instrumentation Center of the National Taiwan University for the solid-state 29Si NMR measurement and elemental analysis.

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