Silica Ouzo Effect: Amphiphilic Drugs Facilitate Nanoprecipitation of

Dec 16, 2015 - Silica Ouzo Effect: Amphiphilic Drugs Facilitate Nanoprecipitation of Polycondensed Mercaptosilanes. Shih-Jiuan Chiu†, Chien-Yu Linâ€...
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Silica Ouzo Effect: Amphiphilic Drugs Facilitate Nanoprecipitation of Polycondensed Mercaptosilanes Shih-Jiuan Chiu,† Chien-Yu Lin,†,‡ Hung-Chang Chou,‡ and Teh-Min Hu*,‡ †

College of Pharmacy, Taipei Medical University, Taipei 11031, Taiwan, ROC School of Pharmacy, National Defense Medical Center, Taipei 11490, Taiwan, ROC

‡

S Supporting Information *

ABSTRACT: Amphiphilic drugs are therapeutic agents whose molecular structures contain both hydrophobic and hydrophilic portions. Here we report a systematic study on how amphiphilic drugs can assist in silica nanoprecipitation. 3-Mercaptopropyltrimethoxysilane (MPTMS) was used as the sole silica material and 12 amphiphilic drugs spanning a wide spectrum of therapeutic categories were included. MPTMS polycondensation was conducted in a DMSO-based organic phase. After a sufficient time, particle formation was induced by injecting a small amount of the organic phase into a water solution containing various amphiphiles. The results show that all amphiphilic drugs studied exerted concentration-dependent facilitating effect on nanoparticle formation. Under certain preparation conditions, the particle solution showed physical stability over a long period and the formed particles could be as small as 100 nm. By systematically varying drug concentrations and injection volumes, the ability of each amphiphile to promote nanoprecipitation can be quantified and compared, based on two novel indices: the area under the critical volume-concentration curve (AUC) and the critical stabilization concentration (CSC). We demonstrate that both ability indices significantly correlated with the drug’s log P and critical micelle concentrations (CMC). Furthermore, we have optimized the aging and particle purification condition and extensively characterized our system through comprehensive TEM and zeta-potential measurements, as well as determinations for drug entrapment and release. In conclusion, we have established a quantitative structure−activity relationship for amphiphilic small-molecular drugs in their ability to interact with poly(mercaptopropyl)silsesquioxane species and form nanoparticles via solvent shifting. We speculate that both hydrophobic and electrostatic interactions play important roles in the formation and stabilization of nanoparticles.

1. INTRODUCTION Amphiphilic drugs are pharmacologically active compounds whose molecular structures contain both hydrophobic and hydrophilic portions. They can be found in a broad range of therapeutic categories, e.g. antihistamines, beta-blockers, local anesthetics, phenothiazines, and tricyclic antidepressants (TCAs), etc.1 Given their amphiphilic nature, these drugs possess surface activity and behave similarly to classical detergents. In aqueous media, amphiphilic drugs tend to selfassociate and form organized structures (e.g., micelles) at critical concentrations. In addition, analogous to the action of detergent, the drugs can interact with cell membranes.1 Many of these surface active drugs contain one or more aromatic ring systems which contribute to their hydrophobicity. For example, the antipsychotic phenothiazines and TCAs are two families of structurally similar compounds whose self-association properties have been well studied.2−7 These drugs are alike in that they bear a planar tricyclic ring system with a short alkyl side chain carrying a positively charged nitrogen atom. Nanoprecipitation method (solvent displacement or solvent shifting) is a popular and straightforward method for the preparation of nanoparticles. 8−14 The method can be © XXXX American Chemical Society

conducted in one step, using less toxic solvents, and without the need of prior emulsification (compared with emulsionbased methods). To perform nanoprecipitation, the hydrophobic solute (either polymers or low-water-solubility drugs) is first dissolved in a water-miscible polar solvent, e.g. alcohols, acetone, THF, and DMSO, which constitutes the organic phase (or solvent phase). Then, the particles of the hydrophobic solute are formed when the organic phase is added to a larger quantity of a nonsolvent (usually water)−i.e., the water phase. Because the solute has low solubility in water, under appropriate conditions the solvent shifting (displacement) process can produce submicrometer particles. Recently, this technical procedure has adopted a lively namethe Ouzo effectwhich was originally coined by Vitale and Katz in describing the formation of liquid dispersions of small waterinsoluble molecules upon solvent displacement.15 Ouzo is the Greek alcoholic beverage (Pastis in France) made from the extract of anise seed. Since 2005, after the seminal review of Ganachaud and Katz,16 the Ouzo effect has gradually gained Received: November 3, 2015

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Figure 1. Effect of thioridazine (TDZ) concentrations and injection volumes (organic phase) on particle size and the corresponding appearance of the colloidal dispersion after 24-h standing. Red asterisks indicate unstable dispersion. The resulting colloid dispersion was left aging for 2 h at 60 °C (unless otherwise indicated) and then was centrifuged at 5000 rpm (2810g) for 30 min at 4 °C (Sorvall Super T21, Wilmington, DE, USA). After removing the supernatant, the particle pellet was washed and recentrifuged with 10 mL of water. The final particle pellet was redispersed in water by repeat pipetting. 2.3. Characterization of Particles. The hydrodynamic diameters of particles were determined by dynamic-light-scattering (DLS) measurements (LB-500, Horiba instruments, Inc.). The turbidity of colloid solutions was determined by monitoring the absorbance at 800 nm (Shimadzu UV-2450, Kyoto, Japan). The particle surface charges were measured using a zeta-potential analyzer (ZetaPlus, Brookhaven Instruments Co.). TEM images of nanoparticles were acquired on a Hitachi H-600 transmission electron microscope operated at 75 kV. The entrapment ratio of thioridazine (TDZ) in silica nanoparticles was determined using UV spectrophotometry (Shimadzu UV-2450, Kyoto, Japan). The concentration of TDZ in the supernatant (collected from centrifuging 1 mL of particle dispersion at 14 000 rpm (15 996g, for 20 min) was determined at 313 nm. The entrapment ratio was calculated according to the following equation: entrapment (%) = 100 × (total TDZ concn − supernatant TDZ concn)/total TDZ concn. Direct determination of entrapped TDZ was conducted by using DMSO extraction and sonication (10 min), followed by fluorescence measurements (excitation/emission at 260 nm/460 nm). The thiol concentration in particle solution was determined using a modified colorimetric method.30 Briefly, 0.5 mL of diluted particle solution was mixed with 0.5 mL of a DTNB (5,5-dithiol (2nitrobenzoic acid)) solution (0.2 mM in Tris buffer, pH 8). Then, the reaction mixture was incubated for 15 min at room temperature, followed by centrifugation at 14 000 rpm (15 996g) for 10 min. The absorbance of the resulting supernatant was measured at 412 nm. The thiol concentrations were estimated using standard solutions of cysteine HCl. The drug release study was conducted for thioridazine-loaded nanoparticles using a dialysis method. A sealed dialysis bag (MW cutoff 12−14 kDa) containing 1 mL of particle solution (particle dispersed in PBS containing 0.2% bovine serum albumin) was immersed in 225 mL of PBS-based release medium (pH 7.4 or 5.7, 37 °C) with constant stirring at 300 rpm. At each sampling time, 25 mL of the medium was removed and then replenished with 25 mL of fresh medium. The concentration of thioridazine was determined using a microplate fluorometer (excitation/emission at 260 nm/460 nm). The cumulative percentage of drug release as a function of time was reported. 2.4. Data Analysis. The hydrodynamic size data were mean of three determinations. Data were collected and analyzed using

popularity in the fields of polymers and drug delivery.9,10,17−21 Also, solvent displacement has been used for the formation of droplets at solid−liquid interfaces.22−25 We have recently developed a facile procedure to synthesize silica nanoparticles with great flexibility. Using the method, nitric oxide (NO)-delivery silica nanoparticles can be directly prepared from a single organosilane source, 3-mercaptopropyltrimethoxysilane (MPTMS); the conjugation of the NO moiety and particle formation can be achieved in one pot.26−28 Moreover, thiol and amine-cofunctionalized silica nanoparticles can be conveniently prepared by simply combining two organosilane precursors in the procedure.29 In this present study, the versatility of the “silica Ouzo effect” is further revealed by the novel finding that the amphiphilic drugs can facilitate silica nanoprecipitation. We demonstrate that a series of amphiphilic drugs interacts, both hydrophobically and electrostatically, with MPTMS-derived, polycondensed silica species during the solvent shifting process. Remarkably, the comprehesive study allows us to deduce a quantitative relationship between molecular structure and a drug’s ability in promoting nanoprecipitation. Our systematic approach offers insights into the role of the amphiphilicity of drugs in forming supramolecular complexes with polymers, which has potential implication in developing novel drug delivery systems.

2. EXPERIMENTAL SECTION 2.1. Materials. Organosilanes, amphiphilic drugs, phenothiazine, pyrene, and sodium 1-pentanesulfonate were purchased from SigmaAldrich (St. Louis, MO, USA). All chemicals and solvents were of analytical-reagent grade and used as received. Deionized water was used throughout the study (18.2 MΩ·cm; Millipore Milli-Q gradient A-10, Bedford, MA, USA). 2.2. Preparation and Purification of Nanoparticles. In a typical synthesis, MPTMS (3-mercaptopropyltrimethoxysilane) and an amphiphilic drug were dissolved in 8.8 mL of DMSO and mixed with 1 mL of 5 M HCl to form the organic phase (cooled on ice for 10 min). The MPTMS concentration was 100 mM unless otherwise indicated. The concentrations of amphiphilic drugs were an important variable, ranging from 2.5 to 40 mM in the organic phase. The organic phase was allowed to stand at ambient temperature for 24 h (in the absence of light). Then, an aliquot (0.25−2 mL) of the organic phase was aspirated and injected rapidly (∼10 s, 1-mL needle, 27 gauge) into 10 mL of water under constant stirring (300 rpm) at room temperature. B

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Langmuir Microsoft Excel and SigmaPlot. Correlation analysis was conducted by using the Correlation option in Data Analysis Tools of Microsoft Excel. Student’s t test was used to compare two independent samples. P < 0.05 was considered statistically significant.

24 h) were shown in Figure S5. As can be seen, the drugs tested demonstrate similarities and differences, i.e., all can facilitate silica nanoprecipitation, yet the exact parametric space for the formation of stable nanoparticles varies. Specifically, some drugs have a narrower parametric region (enclosed by concentration and volume) for the generation of small and stable particles; these drugs include chlorcyclizine and diphenhydramine (both are antihistamines), dibucaine (a local anesthetic), and propranolol (an antihypertensive betablocker). In contrast, drugs belonging to tricyclic antidepressants (TCAs) and phenothiazines facilitate silica nanoprecipitation with a wider parameter range. 3.4. Area under the Critical Volume−Concentration Curve (AUC) and Quantitative Structure−Function Relationship. To provide a quantitative understanding of the structure−activity relationship, we propose a method to quantify the parametric region for generation of stable nanoparticles. At each drug concentration, we define the critical volume as the injected organic-phase volume beyond which the formed particles had a measured size of >1000 nm (Figure S4) and/or exhibited significant sedimentation after 24 h (visible from Figure S5). The critical volume itself can be used to compare the ability of various drugs in facilitating nanoprecipitation. Therefore, drugs with higher ability are those that allow for injection of larger amounts of organic phase into the water phase without generating large unstable particles. By plotting critical volumes versus drug concentrations, a curve can be obtained (as exemplified in Figure 2), which divides the

3. RESULTS AND DISCUSSION 3.1. Baseline. The present study uses the nanoprecipitation approach described previously for the synthesis of SiNPs.26,29 3Mercaptopropyltrimethoxysilane (MPTMS) was used as the sole silica source for synthesis. Briefly, the mercaptosilane monomer was subjected to acid-catalyzed hydrolytic condensation in an organic phase consisting of a water-miscible solvent (e.g., DMSO). After 24 h, a small quantity of the organic phase (still clear solution) was rapidly injected into water to trigger silica precipitation. Supporting Information Figure S1 shows that, when a wide concentration range of MPTMS was tested, particle formation occurred only at MPTMS ≥ 10 mM. Notably, once the silica species were precipitated, their sizes were all greater than 2 μm, regardless of which concentrations and injection volumes were used. Therefore, nanoscale particles cannot be formed without the structural modification of MPTMS (such as S-nitrosation26) or addition of other silanes,29 as shown in our previous studies. 3.2. The Case of Thioridazine (TDZ). Thioridazine (TDZ) is a phenothiazine antipsychotic drug which is considered amphiphilic because its chemical structure contains both hydrophobic (the phenothiazine ring) and hydrophilic (the aminoalkyl side chain) portions (Table S1). To investigate how amphiphilic drugs would interact with silica species in the context of nanoprecipitation, the MPTMS-containing organic phase was supplemented with various concentrations (2.5−40 mM) of TDZ, and the volume of the organic phase injected into the water phase was varied (0.25−2 mL). Surprisingly, particles with the hydrodynamic sizes ranging from 100−1000 nm can be produced after mixing the organic and water phases. Figure 1 shows that the particle size is a function of both TDZ concentrations and injection volumes: At a fixed injection volume, higher TDZ concentrations resulted in the formation of smaller particles; however, at a fixed TDZ concentration, larger particles were generated with increasing injection volumes. Photographs of the formed particle dispersions after 24-h standing were taken, and it can be judged from the appearance that certain dispersions were unstable with significant particle sedimentation. The unstable dispersions correspond to the conditions which initially produce large particles (i.e., low [TDZ] and high volumes). Nevertheless, when the added amount of TDZ is sufficient, the particles generated were small enough and maintain a stable colloid system. The stability of the system can be further realized by monitoring the turbidity change over time: the kinetic turbidity traces were almost flattened for high [TDZ] and low solvent/ water volume ratios (Figure S2), suggesting that particle coalescence was inhibited by TDZ. Figure S3 shows that the system can be stable for at least five weeks under an adequate preparation condition. 3.3. Extention to 12 Therapeutic Drugs. The finding of the TDZ experiment encourages us to conduct a systematic investigation over 12 amphiphilic drugs covering a wide range of therapeutic categories (Table S1). For each drug, a 3-D plot was constructed to show the dependence of measured particle sizes on drug concentrations and injection volumes; and all the 3-D plots are shown and compared in Figure S4. The corresponding photographs of the resulting dispersions (after

Figure 2. Representation of the critical volume vs drug concentration curve. The critical volume is defined as the injected organic-phase volume beyond which the formed particles had a measured size of >1000 nm (Figure S4) and/or exhibited significant sedimentation after 24 h (Figure S5). AUC was calculated by the trapezoidal rule.

concentration−volume parametric space into two regions (stable vs unstable). Therefore, the area under the curve (AUC) can be used as an index that characterizes the inherent ability of amphiphilic drugs in promoting stable nanoparticle formation: the larger the AUC is, the more effective is the drug to enhance silica nanoprecipitation. The critical volume curves for all 12 drugs are shown in Figure S6 and the AUC for each curve was estimated accordingly using a trapezoidal rule. Then, the relationships between AUC and the physicochemical properties, such as log P and critical micelle concentrations (CMC), of amphiphilic drugs (Table S2) were examined. Remarkably, a strong positive correlation (p < 0.001) between AUC and log P was observed for drugs with log P C

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hydrophobic, tricyclic ring system and an extended side chain with an amine functionality. The result indicates that for structurally similar amphiphilic drugs, the drug’s capability to promote silica nanoprecipitation can be predicted by its hydrophobicity: more hydrophobic means more powerful. In contrast, AUC is negatively correlated with CMC (Figure S7), suggesting that the nanoenhancing activity is also related to the ability of amphiphilic drugs to self-assemble. 3.5. Critical Stabilization Concentration (CSC): Its Relation with log P and Critical Micelle Concentration. The finding that TCAs and phenothiazines antipsychotics act similarly in the system is of particular interest. Indeed, the following analysis further epitomizes the homogeneity of the two drug classes. By a closer look of the particle size data as a function of drug concentrations, one immediately finds that the size of formed silica particles decreased with increasing concentrations of amphiphilic drugs in the final particle dispersions. There is clearly a transition from larger particles to smaller particles at a critical concentration, which is here referred to as a critical stabilization concentration (CSC) for an amphiphilic drug (Figure 4A). The use of the term “stabilization” is to reflect the fact that the original MPTMS

Figure 3. Tendency of amphiphilic drugs to promote silica nanoprecipitation (the AUC index) as a function of log P of the drugs. The dashed line is the regression line for the data points of phenothiazines antipsychotics and TCAs combined (AUC = 10.9*log P − 8.0; r = 0.935, p < 0.001).

Figure 4. Determination of the critical stabilization concentration (CSC) and the quantitative structure−activity relationship for phenothiazines antipsychotics and TCAs combined. (A) Hydrodynamic sizes as a function of drug concentrations in the final solvent/water binary mixture. The CSC was determined by extrapolating the linear regression line to obtain the intercept on the X-axis as exemplified (inset). The injection volume was fixed at 1 mL. (B) Chemical structures of phenothiazine antipsychotics. (C) Chemical structures of tricyclic antidepressants (TCAs). (D) Relationship between CSC and log P. (E) Relationship between CSC and CMC. D

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ion-pair reagent, 1-pentanesulfonate (PS). Figure 6 shows that particle sizes increased with increasing amounts of PS added.

system in the absence of amphiphilic drugs only produces large unstable particles; the effect of added drugs is to facilitate the formation of stable silica nanoparticles, possibly by direct interactions between amphiphilic drugs and polycondensed silica species. When CSC was plotted against log P of drugs, the two drug classes (TCAs and phenothiazines, Figure 4B and 4C) can be unified in a straight line with only a slight deviation (r = 0.95, p = 0.0003; Figure 4D). Such a relationship suggests that drugs with higher hydrophobicity exert a greater effect on silica precipitation and stabilization. Thus, thioridazine is the most effective silica-nanoprecipitation agent, because it has the highest log P among all drugs tested in the present study. Figure 4E shows that CSC is positively correlated with CMC; remarkably, CSC is much lower than CMC (in average, the CSC/CMC ratio is about 0.025). 3.6. Role of Amino Side Chains. The above quantitative analysis indicates that hydrophobic interaction plays an important role in controlling the particle formation. However, is hydrophobicity sufficient to bring amphiphilic drugs together with silica species? To answer this question, two hydrophobic compounds (underivatized phenothiazine and pyrene, see Figure 5) were included in the nanoprecipitation experiment.

Figure 6. Effect of sodium 1-pentanesulfonate and sodium chloride on thioridazine (TDZ)-mediated silica nanoprecipitation. Experimental conditions: MPTMS concn, 100 mM; TDZ concn, 20 mM; organic solvent, DMSO; reaction time, 24 h; injection volume, 1 mL; water phase (10 mL) containing sodium 1-pentanesulfonate or sodium chloride (0, 5, 10, 15, 20, 30, 40, 60, and 80 mM).

Besides, the turbidity of the system increased rapidly over time in the presence of high PS concentrations (Figure S8). The data suggest that PS blocks the effect of TDZ on silica nanoprecipitation by reducing the binding of TDZ to MPTMSderived silica polymeric species. Second, sodium choride was used as an electrostatic shielding agent and its effect on particle formation was also examined. As can be seen in Figure 6, the size of particles produced from solvent displacement increased exponentially with [NaCl], suggesting that the electrostatic interaction between amphiphilic drugs and silica species had been disrupted. Third, the pH titration study shows that low pHs favor the formation of stable nanoparticles (Figure S9). Taken together, it is very likely that the amino group and the associated electrostatic attraction is the driving force for the initial drug−silica interaction. It has been shown that nanoparticles prepared from nanoprecipitation (Ouzo effect) can be stabilized by the presence of amphiphilic polymers.9,31,32 Moreorver, the particle stabilization mechanism has been attributed to the surface adsorption of polymers.33 Therefore, it is likely that in our system the amphiphilic drugs may also exert a stabilization effect via particle surface adsorption. To test this hypothesis, a systematic experiment was performed in which silica nanoparticles were prepared in the presence of various concentrations of TDZ. The zeta potentials of the final purified nanoparticles were then measured. The result shows that the nanoparticles exhibited positive surface potentials with magnitudes sufficient to render particles stable (i.e., > +30 mV, pH ∼ 3.4, Figure S10.A). Furthermore, zeta potentials and drug entrapment were increased with increasing amounts of TDZ used for preparation (Figure S10.B). These findings, along with the observation that particle sizes negatively correlated with zeta potentials (Figure 7), indicate that significant surface adsorption of TDZ may stabilize particles by creating a repulsive, postively charged surface with amine groups. This is further supported by the fact that, like many postively charged particles, the TDZ-stabilized nanoparticles were unstable in a buffer solution; however, the physical stability can be maintained in the presence of protein molecules (Figure S11).

Figure 5. Effect of hydrophobic compounds lack of an amino side chain (e.g., plain phenothiazine and pyrene) on silica precipitation, compared with that of thioridazine. Particle sizes were measured immediately after precipitation. Note that unstable particles were produced for underivatized phenothiazine and pyrene and undergo significant sedimentation after 24 h. Experimental conditions: MPTMS, 100 mM; drug concentration in the organic phase, 20 mM; water phase, 10 mL; injection volume (organic phase), 0.5 and 1 mL.

The result shows that both compounds were unable to produce stable nanoparticles (Figure 5), suggesting that the hydrophobic structure alone is not sufficient to render effective drug−silica interaction. As both underivatized phenothiazine and pyrene do not contain an aminoalkyl side chain as TDZ does, they may lack the amphiphilicity required for the interaction. Accordingly, the amine side chain becomes indispensable: it may facilitate the silica−drug interaction by providing positive charges that bind to negatively charged silanolate groups (i.e., the electrostatic attraction). Further experiments were conducted to dissect the important roles of the electrostatic interaction. First, TDZ-mediated silica nanoprecipitation was performed in the presence of an anionic E

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after solvent displacement, suggesting that the mercaptopropyl group in MPTMS is unique. 3.9. Facilitated Ouzo Effect: Optimal Reaction Conditions. The most important finding of the present study is to demonstrate that amphiphilic drugs facilitate the unstable MPTMS system into a metastable nanoscale system, in which MPTMS-derived silica species form nanoparticles in a controllable and predictable way. The phenomenon therefore can be viewed as “facilitated Ouzo effect of silica”. Analogous to the common Ouzo effect, the system is modifiable by altering the solvent/nonsolvent ratio and solvent property, etc.9,10 However, some unique features of the silica Ouzo effect are emphasized here. First, unlike the common polymeric Ouzo effect where precipitated polymers are preformed and welldefined, the precipitable silica species was formed in situ from a single silane. Therefore, sufficient reaction time in the organic phase was needed to ensure substantial silica polymerization. In fact, Figure S14.A shows that TDZ facilitated the formation of stable nanoparticles only after an extended reaction period. Second, the effect of solvent types on nanoprecipitation is more complicated in the silica Ouzo effect, because, not only affecting the phase-mixing property, the solvent type may also affect the sol−gel reaction in the organic phase. Indeed, it has been reported that the rate of sol−gel polymerization of organosilanes varies in different organic solvents.36 In the present study, nanoprecipitation occurred more easily in DMSO than in other solvents (Figure S14.B), which is consistent with our previous findings.26,29 Third, signifiant amounts of the amphiphilic drug (e.g., TDZ) was entrapped in the particle formation process, and the entrapment efficiency can be increased by using higher organosilane concentrations; for example, at MPTMS = 400 mM, the entrapment efficiency reached 65% (Figure S15). 3.10. Purification and Collection of Particles, and Transmission Electron Microscopy Studies. A centrifugation procedure was used to separate nanoparticles from the reaction solution and to collect purified particles. Previously, we have shown that an appropriate aging condition is required for obtaining particles with good dispersion.29 In the present study, the optimal aging condition was also tested by varying aging time and temperature. Figure S16 shows the TEM micrographs of TDZ-stabilized silica nanoparticles with different aging parameters (1 h, 2 h, 24 h at room temperature, and 2 h at 60 °C, respectively). The TEM images show that particles collected with short aging times (e.g., 1 or 2 h) at room temperature tended to aggregate, whereas better dispersion quality can be achieved by prolonging the aging time or elevating the aging temperature. To further demonstrate the temperature effect, the aging time was fixed at 2 h and the effect of aging on particle collection at room temperature was compared with that at 60 °C. As indicated in Figure S17.A, particle sizes were barely changed right after aging and before washing/centrifugation at room temperature (around 100 nm); however, after the final washing step, the size was increased by 4-fold (i.e., from 100 to 400 nm). In contrast, when aged at 60 °C, the size was initially increased to 170 nm after aging (before washing) and the final size (340 nm) were smaller than that for aging at room temperature. Remarkably, the fundamental difference between the two aging conditions is that many more pellets can be obtained at 60 °C than at room temperature after the first centrifugation following aging (Figure S17.B). It was speculated that, for TDZ-stabilized particles, increasing the aging temperature may favor the formation of more solidified,

Figure 7. Correlation between hydrodynamic sizes and zeta potentials for particles prepared in the presence of TDZ with various concentrations.

3.7. Drug in the Organic Phase vs Drug in the Water Phase. As experiments unfolded, we learned that hydrophobic and electrostatic interactions may dictate amphiphilic drugmediated silica nanoprecipitation. Moreover, although up to now the experiments have been conducted such that amphiphilic drugs were initially added in the organic phase, we consider that the major interactions may occur mainly at the mixing of the organic phase with the water phase, during which amphiphilic drugs bind to polycondensed silica species. Therefore, it was assumed that if an amphiphilic drug was instead added in the water phase, it would also produce similar nanoprecipitation effect. The following experiment was performed to test this hypothesis. TDZ was added either in the organic phase or water phase, and after the nanoprecipitation step the particles obtained from the two approaches were compared as shown in Table S3. The result clearly shows that regardless of where TDZ was initially located, the system produced comparable colloidal dispersions. Furthermore, the particles formed by either approach had similar spherical morphology (Figure S12). 3.8. Silane Structural Effect. The Ouzo effect has been studied for small hydrophobic molecules and drugs, as well as polymers. It operates when a water-miscible solvent phase containing a dissolved hydrophobic solute mixes with a water phase, leading to spontaneous generation of colloidal dispersions. The solvent/water intermixing and low water solubility of the solute drive instantaneous particle formation in water. Indeed, it has been shown that smaller and more stable nanoparticles can be formed with more hydrophobic solutes.34,35 In the present study, MPTMS was the starting monomeric material which was subjected to acid-catalyzed silica condensation reaction and formed polycondensed mercaptopropyl functionalized silica species. The in situ formed silica oligomeric species has low solubility in water; but apparently, the solubility is not low enough to induce fast nucleation and form small nanoparticles; instead, large unstable particles were produced. However, our major finding indicates that the presence of small-molecular amphiphiles facilitates the formation of nanoparticles in such a system. To investigate the silane structural effect, we conducted similar experiments for various silanes, including tetramethylorthosilicate (TMOS), (3-aminopropyl)trimethoxysilane (APTMS), and two hydrophobic trimethoxysilanes (Figure S13) whose log P values are even higher than that of MPTMS (log P = 1.09). We found that all silanes tested, regardless of their hydrophobicity, did not mediate particle formation and only clear solution was formed F

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relationship. However, to gain better understanding of the system, we further conducted a systematic comparison among different drugs for various properties in the particle preparation and purification process. Figure 9 shows that the particle sizes

bigger particles that can be more easily brought down by centrifugation, but less prone to agglomeration. One possible mechanism for increasing particle growth is via accelerated Ostwald ripening at an elevated temperature, in which small and unstable particles first dissociate and then reassemble with larger particles. In our system, the process may be associated with thiol−disulfide chemistry: i.e., conjugation of dissolved species to existing particles via the formation of disulfide bonds. To test this premise, we measured thiol contents before and after aging, and the result shows significant thiol loss after aging at 60 °C, but not at room temperature (Figure S18). Apparently, high temperature has resulted in enhanced thiol oxidation, which in turn favors particle growth. The optimized aging condition was applied for purifying and collecting nanoparticles prepared using various concentrations of TDZ, and the TEM images of the resultant particles were systematically acquired. Figure 8 shows clear TEM images with

Figure 9. Correlation between hydrodynamic sizes and critical stabilization concentrations for particles prepared in the presence of various phenothiazine antipsychotics and tricyclic antidepressants.

of formed nanoparticles significantly correlated with the critical stabilization concentrations (CSC) of drugs. It can be seen that smaller particles were produced for drugs with lower CSC, such as thioridazine and chlorpromazine. Apparently, drugs with higher ability in assisting nanoprecipitation (i.e., those with lower CSC, lower CMC, and higher log P values, as shown in Figure 4) tend to mediate the formation of smaller nanoparticles. Furthermore, after nanoprecipitation, the effect of the particle purification procedure (i.e., aging/washing/centrifugation) on nanoparticle collection was extensively studied and compared for various drugs. Interestingly, when the newly synthesized particle solution was aged at 60 °C for 2 h, six of eight drugs exhibited particle coalescence (Figure S20); only thioridazine and chlorpromazine can be purified under the high-intensity aging condition. When a low-intensity aging condition (room temperature, 1 h) was used, the aging-induced particle aggregation became less severe; however, particles produced from the six drugs cannot withstand subsequent washing and centrifugation steps, as evidenced by TEM images (Figure S21). Taken together, the observed instability supports the premise that less hydrophobic and amphiphilic drugs have lower tendency to interact with silica species. Because these drugs have much higher CMC and CSC values than thioridazine and chlorpromazine, much higher concentrations may be needed to produce more stable nanoparticles. 3.12. Drug Release Study. The drug release characteristics of TDZ-loaded nanoparticles was studied. Figure 10 shows the release profile of TDZ in albumin-containing phosphatebuffered saline at 37 °C. It is apparent that slow release occurred at pH 7.4; and about 25% of drug can be released over a period of 6 days. Decreasing the pH of the release medium, however, resulted in burst release. This pH-sensitive release phenomenon further suggests that the binding and release of TDZ involves electrostatic interaction between the negatively charged silica species and positively charged TDZ. 3.13. Proposed Mechanisms for Nanoparticle Formation and Stabilization. Scheme 1 depicts the potential mechanism underlying the effect of amphiphilic drugs on promoting silica nanoprecipitation. In this model, mercaptopropyl polysilsesquioxane is formed via acid-catalyzed poly-

Figure 8. TEM micrographs and size distributions of TDZ-stabilized nanoparticles prepared at TDZ concentration (organic phase) of (A) 5 mM, (B) 10 mM, (C) 20 mM, and (D) 40 mM.

distinct, well-distributed spherical particles whose sizes are decreased with increasing TDZ concentrations. The quantitative data shown in Figure S19 further confirm the concentration-dependent effect of TDZ observed by DLS measurements. 3.11. Closer Look at Similarities and Dissimilarities among Drugs. In the present study, we demonstrate the ability of amphiphilic drugs in facilitating silica nanoprecipitation by including a wide range of drugs. Particularly, the inclusion of several structurally related compounds belonging to phenothiazines and TCAs allows us to obtain a quantitative G

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binding would induce the formation of drug−silica complex that is more hydrophobic than the original species, thereby reaching a higher supersaturation state in water. Moreover, the binding may decrease the surface tension of the particle/water interface. According to classical nucleation theory, either increasing supersaturation or lowering surface tension would result in an increased nucleation rate with the formation of a large quantity of small nuclei, thus favoring nanoprecipitation.9 Indeed, we have shown that hydrophobic compounds without the amino side chain did not facilitate the formation of nanoparticles (Figure 5), and that charge shielding agents disrupted thioridazine-mediated silica nanoprecipitation (Figure 6). Second, the hydrophobic π system of amphiphilic drugs plays an important role in governing nanoprecipitation, given that more hydrophobic drugs produced smaller and more stable nanoparticles. One can understand the importance of the hydrophobic force by drawing an analogy with the systems that involve the interaction between polyelectrolytes and oppositely charged surfactants, such as mixing DNA with cationic lipids in aqueous solution.37−40 In such systems, in addition to electrostatic interaction, cooperative surfactant−polymer binding may be induced by surfactant−polymer and surfactant− surfactant binding involving hydrophobic aliphatic tails. Accordingly, the system often has a characteristic concentration (e.g., critical aggregation concentration) much lower than the critical micelle concentration (CMC).41,42 In our study, we argue that the aromatic system resembles the aliphatic tails of surfactants and its tendency to self-assemble may mediate cooperative drug−silica aggregation and help nanoprecipitation. Indeed, we show that the characteristic concentration in our system (i.e., critical stabilization concentration, CSC) is also much lower than the CMC of amphiphilic drugs. This leads to an intriguing, untested question as to whether commonly used cationic surfactants can also exhibit similar nanoprecipitation effect. In fact, we found that CTAB (cetyltrimethylammonium bromide), a cationic surfactant, did promote silica nanoprecipitation with a much lower CSC value (0.025 mM) than its CMC (0.9 mM,43 Figure S22.A). Remarkably, the CSC/ CMC ratio for CTAB is 0.028, which is close to the average value for amphiphilic drugs (0.024, Figure 4E). However, for comparison, Tween 20 (a nonionic surfactant) can only facilitate nanoparticle formation at a CSC value much higher than its CMC (i.e., CSC = 2.02 mM (Figure S22.B) vs CMC = 0.04 mM;44 CSC/CMC ∼ 50). Finally, it was suggested that the mechanism of stabilization and drug entrapment may be further realized by studying spatial distribution/localization of drugs inside the particles. Although an advanced method such as electron energy loss spectroscopy (EELS) may be used to provide semiquantitative information on spatial drug distributions,45 here we address the issue based on an alternative method. In Figure S23, we show that a certain amount of entrapped drug molecules cannot be extracted by consecutive stepwise water extraction steps; however, this nonwater-extractable portion accounted for about 30% of total drug loaded, and can be recovered by subsequent addition of DMSO, which disassembled the particles. The result suggests that a small number of amphiphiles were entrapped within particles during nanoprecipitation, whereas the majority of amphiphiles were adsorbed on the surface of particles.

Figure 10. Release profile of TDZ-loaded nanoparticles and free TDZ in albumin-containing phosphate-buffer saline solutions. n = 3.

Scheme 1. Proposed Mechanisms for Amphiphilic DrugsMediated Nanoprecipitation of Polycondensed Mercaptopropyl Silica Species upon Solvent/Water Mixinga

a

(A) In the absence of amphiphilic drugs, unstable microparticles were formed via a nucleation/growth mechanism. (B) In the presence of amphiphilic drugs, electrostatic, hydrophobic and π-stacking interactions may facilitate the nucleation process and contribute to the stabilization of formed nanoparticles.

condensation of MPTMS monomer in the organic phase. The polycondensed species contains sufficient hydrophilic, dissociable silanol groups, given that under similar conditions about 40−50% T2 silicon structures are present.26,29 Apparently, despite the presence of the mercaptopropyl group, the polymeric structure is not hydrophobic enough to render a supersaturated state for nanoparticle formation. Instead, microparticles were formed in the control experiments (Scheme 1A). Here we report, for the first time, that amphiphilic drugs can facilitate the formation of drugentrapping polysilsesquioxane nanoparticles (Scheme 1B). Many of the amphiphilic drugs studied possess planar hydrophobic aromatic ring systems and hydrophilic side chains with positively charged amino nitrogen. Thus, the drugs can be viewed as surface active agents capable of interacting with oppositely charged, polycondensed organosilane species. Specifically, two supramolecular driving forces are proposed to control the formation of nanoparticles: i.e., electrostatic and hydrophobic π-stacking interactions (Scheme 1B). First, upon solvent/water intermixing (pH = 2), amphiphilic drugs may bind to the polysiloxane structure via electrostatic interaction between the protonated amine and the anionic SiO−. The initial H

DOI: 10.1021/acs.langmuir.5b04048 Langmuir XXXX, XXX, XXX−XXX

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(4) Attwood, D.; Mosquera, V.; Garcia, M.; Suarez, M. J.; Sarmiento, F. Comparison of the Micellar Properties of Structurally Related Antidepressant Drugs. J. Colloid Interface Sci. 1995, 175 (1), 201−206. (5) Sarmiento, F.; Lopez-Fontan, J. L.; Prieto, G.; Attwood, D.; Mosquera, V. Mixed micelles of structurally related antidepressant drugs. Colloid Polym. Sci. 1997, 275 (12), 1144−1147. (6) Kabir-ud-Din; Yaseen, Z. Formulation of amphiphilic drug amitriptyline hydrochloride by polyoxyethylene sorbitan esters in aqueous electrolytic solution. Colloids Surf., B 2012, 93, 208−214. (7) Alam, M. S.; Ghosh, G.; Mandal, A. B.; Kabir-ud-Din. Aggregation behavior and interaction of an amphiphilic drug imipramine hydrochloride with cationic surfactant cetyltrimethylammonium bromide: Light scattering studies. Colloids Surf., B 2011, 88 (2), 779−784. (8) Yan, X. B.; Delgado, M.; Fu, A.; Alcouffe, P.; Gouin, S. G.; Fleury, E.; Katz, J. L.; Ganachaud, F.; Bernard, J. Simple but Precise Engineering of Functional Nanocapsules through Nanoprecipitation. Angew. Chem., Int. Ed. 2014, 53 (27), 6910−6913. (9) Lepeltier, E.; Bourgaux, C.; Couvreur, P. Nanoprecipitation and the ″Ouzo effect″: Application to drug delivery devices. Adv. Drug Delivery Rev. 2014, 71, 86−97. (10) Beck-Broichsitter, M.; Nicolas, J.; Couvreur, P. Solvent selection causes remarkable shifts of the “Ouzo region” for poly(lactide-coglycolide) nanoparticles prepared by nanoprecipitation. Nanoscale 2015, 7 (20), 9215−9221. (11) Ford, J.; Chambon, P.; North, J.; Hatton, F. L.; Giardiello, M.; Owen, A.; Rannard, S. P. Multiple and Co-Nanoprecipitation Studies of Branched Hydrophobic Copolymers and A-B Amphiphilic Block Copolymers, Allowing Rapid Formation of Sterically Stabilized Nanoparticles in Aqueous Media. Macromolecules 2015, 48 (6), 1883−1893. (12) D’Addio, S. M.; Prud’homme, R. K. Controlling drug nanoparticle formation by rapid precipitation. Adv. Drug Delivery Rev. 2011, 63 (6), 417−26. (13) Yu, Y.; Chen, C. K.; Law, W. C.; Weinheimer, E.; Sengupta, S.; Prasad, P. N.; Cheng, C. Polylactide-graft-doxorubicin Nanoparticles with Precisely Controlled Drug Loading for pH-Triggered Drug Delivery. Biomacromolecules 2014, 15 (2), 524−532. (14) Tang, C.; Amin, D.; Messersmith, P. B.; Anthony, J. E.; Prud’homme, R. K. Polymer Directed Self-Assembly of pH-Responsive Antioxidant Nanoparticles. Langmuir 2015, 31 (12), 3612−3620. (15) Vitale, S. A.; Katz, J. L. Liquid droplet dispersions formed by homogeneous liquid-liquid nucleation: ″the ouzo effect″. Langmuir 2003, 19, 4105−10. (16) Ganachaud, F.; Katz, J. L. Nanoparticles and nanocapsules created using the Ouzo effect: spontaneous emulisification as an alternative to ultrasonic and high-shear devices. ChemPhysChem 2005, 6 (2), 209−16. (17) Beck-Broichsitter, M.; Rytting, E.; Lebhardt, T.; Wang, X.; Kissel, T. Preparation of nanoparticles by solvent displacement for drug delivery: a shift in the ″ouzo region″ upon drug loading. Eur. J. Pharm. Sci. 2010, 41 (2), 244−53. (18) Aschenbrenner, E.; Bley, K.; Koynov, K.; Makowski, M.; Kappl, M.; Landfester, K.; Weiss, C. K. Using the polymeric ouzo effect for the preparation of polysaccharide-based nanoparticles. Langmuir 2013, 29 (28), 8845−55. (19) Aubry, J.; Ganachaud, F.; Cohen Addad, J. P.; Cabane, B. Nanoprecipitation of polymethylmethacrylate by solvent shifting: 1. Boundaries. Langmuir 2009, 25 (4), 1970−9. (20) Botet, R. The “ouzo effect”, recent developments and application to therapeutic drug carrying. J. Phys. Conf Ser. 2012, 352, 012047. (21) Shah, M.; Agrawal, Y. Development of Ciprofloxacin HCl-Based Solid Lipid Nanoparticles Using Ouzo Effect: An Experimental Optimization and Comparative Study. J. Dispersion Sci. Technol. 2013, 34 (1), 37−46. (22) Peng, S. H.; Xu, C. L.; Hughes, T. C.; Zhang, X. H. From Nanodroplets by the Ouzo Effect to Interfacial Nanolenses. Langmuir 2014, 30 (41), 12270−12277.

4. CONCLUSIONS Silica particles have wide applications, particularly in the field of drug delivery. Many methods lead to the formation of bare silica nanoparticles without drug loading. For example, the most well-known method to prepare plain silica nanoparticles is perhaps the Stöber approach, which takes a simple sol−gel condensation step.46 For silica particles to carry theranostics, making the plain particles is just the first step. It normally takes extra steps to get the drugs attached or entrapped in the preformed silica structure. Here we report a novel method in which silica nanoparticles can be prepared by one-step nanoprecipitation facilitated by the presence of amphiphilic drugs, with simultaneous drug entrapment. We demonstrate that, by including a wide range of amphiphilic drugs, a quantitative structure−activity relationship can be obtained: i.e., the nanoprecipitation capability of an amphiphilic drug positively and negatively correlated with drug’s log P and CMC, respectively. The finding suggests that hydrophobicity and surface activity of amphiphilic drugs govern the cooperative aggregation between drugs and polycondensed silane species. Moreover, electrostatic interaction plays an important role in enhancing nanoparticle formation and stabilization. In recent drug-delivery studies, focuses have been on the amphiphilicity of polymeric carrier materials, such as the use of amphiphilic block copolymers.47−49 Our study, however, manifests that the amphiphilicity of the theranostic payload may be another important parameter to consider in preparing a nanoscale drugdelivery system.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.5b04048. Supplementary tables (Tables S1−S3) and figures (Figures S1−S23) (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the Ministry of Science and Technology (MOST) of Taiwan (NSC 102-2320-B-016-003MY3, TM Hu). We thank Ms Huei-Min Chen and the Core Facility Center, Office of Research and Development (Taipei Medical University) for the technical support of TEM.

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REFERENCES

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