Synthesis and Characterization of Dually Labeled Pickering-Type

Jun 12, 2012 - Dual fluorescently labeled polymer particles were prepared in a downscaled Pickering-type miniemulsion system. Stable dispersions were ...
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Synthesis and Characterization of Dually Labeled Pickering-Type Stabilized Polymer Nanoparticles in a Downscaled Miniemulsion System Biao Kang,†,‡ Anika Schrade,† Yang Xu,§ Yinthai Chan,§ and Ulrich Ziener*,† †

Institute of Organic Chemistry III − Macromolecular Chemistry and Organic Materials, University of Ulm, Albert-Einstein-Allee 11, 89081 Ulm, Germany ‡ Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany § Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543 S Supporting Information *

ABSTRACT: Dual fluorescently labeled polymer particles were prepared in a downscaled Pickering-type miniemulsion system. Stable dispersions were obtained and the size of the hybrid particles could be varied between ca. 180 and 430 nm. Silica nanoparticles were employed as sole emulsifier, which were labeled by a fluorescein dye (FITC) or (encapsulated) quantum dots, and the polymer core was labeled by a perylene derivative. Downscaling of the Pickering-type miniemulsion system is intriguing by itself as it allows the use of precious nanoparticles as emulsifiers. Here, silica particles with a fluorescent core and an overall diameter between 20 and 40 nm were prepared and employed as stabilizer. The dual excitation and emission of both dyes was tested by fluorescence measurements and confocal laser scanning microscopy (cLSM).

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INTRODUCTION Nanotechnology based on particulate structures has received more and more interest in both fundamental research and industrial applications due to the extremely small size and unique electrical, magnetic, mechanical, and optical properties of nanoparticles.1 There is a variety of different approaches to (polymeric) nanoparticles; among them, miniemulsion polymerization is a versatile technique with excellent control over size, size distribution, and functionality of the particles.2 In spite of all the advantages, the surfactant used as an emulsifier during preparation of the latex is often cytotoxic, which is a large hindrance for e.g. biomedical applications.3 Although it can be removed by dialysis, the process is time-consuming and usually results in insufficient removal of surfactant or instability of the latexes in the end. In order to circumvent this problem, surfactant-free latex particles stabilized by negatively or positively charged silica particles can be prepared without the help of any surfactant.4 However, the introduction of silica particles makes the latex−cell interaction mechanism in biomedical applications more complicated.5 If the particles are used e.g. for diagnostics, localization by specific reporters is an important issue. While the uptake and final position of the hybrid particles within a living cell can be easily traced by encapsulating a fluorescent dye in it, the tracing of bare silica nanoparticles is difficult. The independent retrieval of both the polymer cores and the stabilizing silica particles is essential to keep full control over the cell−particle interaction and to © 2012 American Chemical Society

guarantee that the inorganic particles are not detached from the polymer core. To fulfill this purpose, magnetically or fluorescently labeled inorganic nanoparticles could be used as emulsifier to stabilize the hybrid particles. Hui-Ying Wen et al. have prepared magnetite-coated polystyrene hybrid microspheres by miniemulsion polymerization.6 Yingda Luo et al. have investigated the nucleation mechanism and morphology of polystyrene/Fe3O4 hybrid particles.7 In these works, a small amount of surfactant is always used in order to prepare stable hybrid particles; hence, it is less suitable for application in cell experiments. In another work, Shulai Lu et al. have prepared self-stabilized magnetic polymeric nanoparticles by utilizing sodium p-styrenesulfonate as monomer.8 However, to the best of our knowledge, there is no report on inorganic/organic hybrid particles using fluorescently labeled particulate emulsifiers. The synthesis of monodisperse silica particles is described in the literature by Stöber,9 while the detailed mechanism is discussed by Bogush et al.10 It is the aim of the present work to prepare Pickering-type stabilized polymer particles with independently traceable inorganic nanoparticles as emulsifier for cellular uptake experiments. Furthermore, a downscaling procedure was developed enabling one to prepare small-scale Pickering-type Received: March 30, 2012 Revised: May 3, 2012 Published: June 12, 2012 9347

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Table 1. Composition of the Normal (Runs NS1, NSL2) and Micro Systems (Runs MS3−MSL9)

a

run

styrene (g)

4-vinylpyridine (mg)

HD (mg)

AIBN (mg)

perylene dye (mg)

water (g)

silicaa (g)

pH

NS1 NSL2 MS3 MS4 MS5 MS6 MS7 MS8 MSL9

2.50 0.50 0.13 0.13 0.13 0.13 0.13 0.13 0.018

500 100 25 25 25 25 25 25 3.6

120 24 6 6 6 6 6 6 1.2

70 18 3 3 3 3 3 3 1.0

1.26 0.55 0.48 0.55 0.68 0.54 0.52 0.60 0.13

16.8 16.8 0.84 0.84 0.84 0.84 0.84 0.84 0.84

3.2 0.64 0.17 0.17 0.17 0.17 0.17 0.17 0.04

10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0

The amount of silica stands for the mass of silica dispersion with a solid content of 34 wt %. 25 mL bottle as water phase. Then, the water phase is added to the oil phase and an aqueous solution of ammonia is added to adjust the pH of the system. The whole mixture is magnetically stirred at 1000 rpm for 1 h. Then the system is transferred into a 50 mL beaker and homogenized by direct ultrasonication for 120 s at 0 °C, using a Branson 450 W digital sonifier at 90% amplitude. Finally, the miniemulsion is transferred into a 25 mL glass bottle and polymerized at 72 °C for 6 h. The detailed conditions of preparation and the composition of the different runs are given in Table 1. For the micro systems, different components are either added separately, like in the normal system, or first prepared as standard stock solutions of water and oil phase independently and then mixed to form the final system. In the latter case, the standard oil phase is always prepared correspondingly to the normal systems, while the theoretical solid content of the standard water phase is 1.2% for MSL10 and MSL11 and 0.6% for MSL12, MSL13, and MSL14 (Table

dispersions. The cellular uptake of nanoparticles has been shown to be a complicated process, influenced by various factors such as the nanoparticle size, charge, composition, morphology, etc. As there is only less known about the cellular uptake rate and route of rough nanoparticles, silica armored hybrid nanoparticles were prepared as a kind of model system. To get additional information what happens to the silica on the polymeric core during cellular internalization or metabolism, two independent fluorescent dyesone to label the polymeric core and the other for tracing the silicaseparately were introduced. With the dually labeled hybrid particles additional information about the fate of the silica after cellular internalization in a time-dependent manner shall be obtained in future cellular uptake experiments to address questions like the following: are the silica particles wiped off or do they stay on the polymeric core during uptake/metabolism, how about the stability of the hybrids in the cellular environment over time, and where does the silica/polymeric core end up in the cells?

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Table 2. Composition of the Micro Systems Homogenized by Indirect Sonication

EXPERIMENTAL SECTION

Materials. Styrene (Merck, 99%) was purified by running through an alumina column and stored at −20 °C. 4-Vinylpyridine (Aldrich, 95%) was purified by vacuum distillation at 50 °C and stored at −20 °C. Tetraethoxysilane (TEOS, VWR, 99%) was purified by vacuum distillation at 40 °C and protected against light. Ludox TMA colloidal silica (Aldrich, 34 wt %), α,α′-azoisobutyronitrile (AIBN, Merck, 98%), hexadecane (HD, Merck, 99%), perylene dye (Lumogen F Rot, Kremer), (3-aminopropyl)triethoxysilane (APS, Aldrich, 98%), fluorescein isothiocyanate (FITC, Aldrich, 90%), absolute ethanol (Merck, 99.9%), ammonia solution (Aldrich, 28 wt % and Merck, 25 wt %, respectively), cadmium acetylacetonate (Cd(acac)2, Aldrich, 99.9%), diisooctylphosphinic acid (DIPA, Fluka, 90%), 1,2-hexadecanediol (HDDO, Aldrich, 90%), 1-hexadecylamine (HDA, Aldrich, 90%), Igepal CO520 (Aldrich), 1-octadecene (ODE, Aldrich, 90%), sulfur (S, Aldrich, reagent grade), selenium pellet (Se, Aldrich, 99.99%), tetraethoxysilane (TEOS, Alfa Aesar, 99+%), trioctylphosphine (TOP, Strem, 97%), trioctylphosphine oxide (TOPO, Aldrich, 90%), and zinc acetylacetonate (Zn(acac)2, Aldrich, 99%) were used as received without further purification. AR grade ethanol, methanol, butanol, cyclohexane, and hexane and demineralized water with MilliQ grade (resistivity 18 MΩ) were employed. Synthesis. Synthesis of Pickering-Type Stabilized Polymer Particles. In the following, the Pickering-type systems with a water phase of ca. 18 mL are termed as NS (normal system), while those with ca. 1 mL are termed as MS (micro system). The NS and MS having a low solid content are termed correspondingly as NSL and MSL. For NS, the oil phase is prepared by mixing a certain amount of perylene dye (if added), AIBN, HD, styrene, and 4-vinylpyridine and magnetic stirring for 6 min in a 25 mL glass bottle. Certain amounts of Ludox silica dispersion and water are magnetically stirred in another

runa

water phase (mg)

oil phase (mg)

pH

MSL10 MSL11 MSL12 MSL13 MSL14

875 875 805 800 800

22 16 10 9 9

10.0 10.0 10.2 10.1 9.7

a

Fluorescent silica (MSL10−12) and encapsulated quantum dots (MSL13, 14) as stabilizer, respectively.

2). In either case, the system is prepared in a 1.5 mL glass vial, and all the subsequent procedures are carried out in this glass vial. In the end, the system is homogenized by indirect sonication, at 0 °C, 90% amplitude, for 2 min. Other conditions for the preparation of the micro systems are the same as for the normal system. The detailed preparation conditions and the compositions are given in Tables 1 and 2. Synthesis of Fluorescently Labeled Silica. Fluorescent silica nanoparticles with different sizes are prepared according to a modified protocol from the literature of van Blaaderen et al.11 For coupling APS with FITC, a mixture of 2.8 mg of APS and 1.6 mg of FITC is added to 0.6 g of absolute ethanol in a 1.5 mL glass vial. The vial is protected against light by aluminum foil and put into a shaker (HTM, 130 LP), which is set at room temperature and 40% shaking amplitude for 24 h. For preparing the fluorescent silica core, 6.8 g of ammonia is added to 200 mL of absolute ethanol in a 250 mL flask in an oil bath at 40 °C. The flask is protected against light by aluminum foil. The mixture is magnetically stirred at 300 rpm for 10 min. Then the coupling reaction system from above is transferred droplet wise into the flask, followed by the droplet wise addition (0.17 g min−1) of 0.888 g of TEOS. The whole system is kept at 40 °C for 48 h. 9348

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Transmission Electron Microscopy. The diluted dispersions, which are used for the DLS measurement (see above), are employed for the preparation of TEM samples. 2 μL of the diluted dispersion is taken and dropped onto a hydrophilyzed (by O2 plasma) carbon-coated copper grid. The samples are left at room temperature overnight to evaporate the water. TEM measurements were performed on a Zeiss EM 10 microscope with an acceleration voltage of 80 kV. Fluorescence Measurements and Microscopy. A multimode microplate reader (TECAN, Infinite M1000) was employed for fluorescence measurements. The samples were diluted with water (pH = 10) by 10 times prior to measurement and analyzed in 96-well plates (Greiner). Confocal laser scanning microscopy (cLSM, Zeiss LSM 710, 40× oil lense) was used to image the fluorescing particles. Images were taken by using the ZEN2010 software. The samples were diluted with water (pH = 10) by 10 times prior to measurement and analyzed in 8-well microcopy chambers (μ-Slide, Ibidi).

For growing a shell around the preformed core, 1.202 g of TEOS is added into 15 g of absolute ethanol, and the mixture is transferred into a 20 mL syringe and added to the silica core dispersion by a syringe pump (43.40 μL min−1). After the whole mixture is added, the system is kept at constant temperature and constant stirring speed for 48 h to guarantee complete conversion. Then, the majority of the ethanol is removed by rotary evaporation, until the volume of the dispersion is reduced to 9 mL. Ultracentrifugation (Beckman L8-M Ultracentrifuge, 26 000 rpm for 43 min) is used to precipitate the silica from the dispersion. After redispersion of the silica in 5 g of water with the help of ultrasonication, the system is again ultracentrifuged at 15 000 rpm for 20 min in order to remove all the bigger coagulates. Synthesis of Core−Shell CdSe/ZnS Quantum Dots. Synthesis of CdSe quantum dots (QDs) proceeded via a previously reported procedure12 with slight modifications. A bath of 9 g of TOPO, 6 g of HDA, and 0.25 mL of DIPA was degassed at 100 °C for 1.5 h. A precursor solution comprising a mixture of 317 mg of Cd(acac)2 and 567 mg of HDDO in 6 mL of ODE was degassed at 120 °C for 1.5 h, after which 4 mL of 1.5 M trioctylphosphine selenide (TOPSe) was added at room temperature. This precursor solution was then rapidly injected into the bath at 360 °C and allowed to cool to 80 °C immediately. The resulting CdSe QDs were subsequently processed via three cycles of precipitation and redispersion in butanol/methanol and hexane, respectively, before finally dispersing in hexane for further use. The processed CdSe QDs were overcoated with a ZnS shell via modifications of a previously reported procedure.13 Typically, a zinc stock solution was prepared by degassing a desired amount of zinc(acac)2 and HDDO in 6 mL of ODE at 130 °C for 1 h. The sulfur stock solution was prepared by degassing elemental sulfur in 6 mL of ODE at 160 °C for 1 h. A bath of 9 g of TOPO and 6 g of HDA was degassed at 120 °C for 1 h. A known amount of CdSe in minimal hexane was added, and the hexane was removed under vacuum at 80 °C for 0.5 h. 1 mL of the zinc stock solution was then injected under N2 before the temperature was increased to 230 °C where the same amount of S stock solution was added dropwise. The remaining zinc and sulfur stock solutions were successively injected dropwise when the temperature reached 240 °C. The solution was left to anneal for 20 min at 240 °C before cooling down to 80 °C. The QDs obtained were subsequently processed via three cycles of precipitation and redispersion in butanol/methanol and hexane, respectively, before finally dispersing in cyclohexane for further use. Silica Encapsulation. Uniform silica encapsulated CdSe/ZnS quantum dots (QDs) were synthesized through a previously reported reverse microemulsion method.14 Typically, 2 mL of Igepal was mixed with 15 mL of cyclohexane and stirred for 30 min. To this mixture, 500 μL of 25 wt % NH4OH was then added and stirred for another 30 min. Approximately 1 mL of 20 μM CdSe/ZnS QDs in cyclohexane was then introduced to the mixture to form stable reverse microemulsions and stirred for an additional 20 min. Finally, 200 μL of TEOS was added and stirred for 24 h to produce uniform SiO2coated QDs. The SiO2-coated QDs were subsequently precipitated out from the reaction mixture by addition of 10 mL of ethanol and centrifugation (3900 rpm) for 10 min. The product was then washed with ethanol three times to remove excess surfactant and unreacted precursor and finally dispersed in water. Characterization. Dynamic Light Scattering. To measure the particle size and size distribution, 2−10 μL of the hybrid particle systems or silica dispersions, respectively, are added to 1 mL of Milli-Q water, and the measurement was carried out on a Nano-Zetasizer (Malvern Instruments) at 25 °C in a standard polystyrene cuvette (Sarstedt, cuvette, 55 mm, PS). Z-average particle sizes in nm and PDIs are given as the average of four measurements. The PDI is a measure of the particle size distribution, and the PDI is a dimensionless number that describes the heterogeneity of the sample; it can range from 0 (monodisperse) to 1 (polydisperse). Zeta-Potential. 2−10 μL of the dispersion is diluted with 1 mL of aqueous KCl (10−3 M), and the measurement was performed with a Nano-Zetasizer (Malvern Instruments) at 25 °C in the zeta potential mode.

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RESULTS AND DISCUSSION The results of the Pickering-type dispersions prepared by miniemulsion with respect to colloidal stability, average size, and size distribution are given in Table 3. Table 3. Colloidal Characterization of the Hybrid Particles content of the dispersion run

stabilitya

hybrid particles

NS1 NSL2 MS3 MS4 MS5 MS6 MS7 MS8 MSL9 MSL10 MSL11 MSL12 MSL13 MSL14

Y N N Y Y Y Y Y Y Y Y Y N Y

Y N Y Y Y Y Y Y N Y Y Y Y Y

size

free silica

DLS (nm)

PDI

TEMb (nm)

Y N N Y Y Y Y Y Y N N N Y Y

226 >10000c 1724 320 287 431 268 220 157 186 227 183 1071 236

0.044 0.543 0.642 0.191 0.091 0.121 0.101 0.047 0.091 0.085 0.062 0.011 0.751 0.103

189 123 175 266 221 401 217 171 138 118 141 146 181 105

a

Determined visually by observing the coagulation. bAverage size from at least 50 particles. cOut of the meaningful range of DLS measurement.

Preparation of Stable Hybrid Particles with Normal Volume and Normal Solid Content. In the first step, Ludox silica is used, and a Pickering-type stabilized system with normal volume (ca. 22 mL) and normal solid content (ca. 18 wt %) is prepared (run NS1, Table 1) as reference sample. The recipe for this system is based on the pioneer work of Tiarks et al.15 The average size measured by DLS is 226 nm with a PDI below 0.1, indicating a narrow size distribution as confirmed by TEM (Figure 1a,b). The size determined by TEM is significantly smaller (189 nm), which is in line with previous findings and attributed to the water layer surrounding the particles (hydrodynamic diameter) and eventual swelling in dispersion. The ζ-potential of run NS1 of around −60 mV indicates the adsorption of negatively charged silica on the surface of the polymer particles, which is also expressed by the raspberry-like morphology seen in the TEM image (Figure 1a,b). The interaction between negatively charged silica and monomer droplets formed during ultrasonication is presumably based on hydrogen bonding between the nitrogen atom of 49349

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Figure 1. TEM images of a Pickering-type stabilized system in two magnifications (runNS1, Table 1).

order to prepare dispersions of hybrid particles with both a reduced volume (0.85 mL) and a reduced solid content (3.6 wt %). Attempts to prepare such microsystems analogously to the normal system by direct ultrasonication resulted in the formation of hardly any hybrid particles (MSL9, Table 1). We attribute this finding to partial oxidation of the monomers by air in the open reaction vessel owing to the small amount of monomer. In order to avoid oxidation, we applied indirect ultrasonication in a sealed container for further experiments. In run MSL10 the oil-to-water ratio is adjusted corresponding to the normal system with reduced solid content, and the reaction conditions of the system with a reduced volume are applied. The system is degassed before it is well sealed; then indirect ultrasonication is carried out, followed by polymerization. The size of the system measured by DLS is 186 nm, with a PDI of 0.085. As can be seen in the TEM image (Figure S1c,d), monodisperse hybrid particles are obtained. As discussed in the previous section, the size is smaller than found for the particles prepared with normal solid content. Besides, there is only little free silica left in the aqueous dispersion, which we attribute to the adsorption of free silica to the wall of the glass container. The solid content could be even further reduced by a factor of 2, resulting in quite monodisperse hybrid particles with a diameter of 183 nm with a PDI of 0.011 by DLS (run MSL12, Table 3). The average size measured by TEM is 146 nm (Figure S1e,f). Preparation and Characterization of Fluorescent Silica. Fluorescent silica with a core shell structure has been prepared by van Blaaderen et al.11 However, the diameters of the fluorescent particles they prepared are always larger than 100 nm.11,18 It is claimed that the size of the silica particles obtained by the Stöber process is determined by the stability of so-called subparticles,10 and the concentrations of all the reaction components need to be reduced, in order to prepare silica particles in the lower nanometer range. It is further reported that by increasing temperature, silica particles with a smaller diameter and narrower size distribution will be obtained.10 In the present contribution, the protocol from van Blaaderen et al.11 is followed in principle, while the concentration of water and ammonia in absolute ethanol is reduced. Silica particles with a fluorescent silica core doped with fluorescein isothiocyanate (FITC) and a pure silica shell are prepared. The fluorescein derivative is chosen because its excitation and emission maxima are well separated from the maxima of the

vinylpyridine and the silanol groups (−Si−OH) on the silica surface.4b,16 Effect of Small Solid Content on the Size of Final Polymer Particles. The downscaling of the Pickering-type system is meaningful in terms of materials’ savings so that precious nanoparticles can be used as stabilizer, and the properties of corresponding Pickering-type systems can be studied. Hence, in the second step, a corresponding system to run NS1 with normal volume of water phase (17 mL) but reduced solid content (3.6 wt %) is prepared. For system NSL2, the amount of monomer is reduced 5-fold together with the amount of Ludox silica dispersion. The system is not stable after the polymerization reaction. Phase separation occurs shortly after stirring is stopped. The DLS measurement gives a size above 10 μm, with a PDI larger than 0.5. In the TEM image (Figure S1a,b), it is obvious that hybrid particles are formed beside larger aggregates. However, these hybrid particles coagulate, and large clusters exist in the system as reflected by the DLS measurement. Besides, TEM reveals that the average size of the hybrid particles is 123 nm, which is much smaller compared to NS1. Additionally, there are always some free silica particles in NS1 present that do not bind to the surface of hybrid particles. In contrast to that, no free silica is found in NSL2. We attribute the decrease of size to the reduction of the oil-to-water ratio. This leads to a reduced rate of droplet fusion shifting the fusion−fission equilibrium to a reduced size of the particles.17 The formation of clusters of hybrid particles and absence of free silica in NSL2 could be also explained by the decreased size. While hybrid particles with a smaller size are produced, more surface area is generated at the same time; hence, there is a demand for silica to stabilize these interfaces. However, in run NSL2 the monomer-to-silica ratio is kept the same as in run NS1. This will result in an insufficient amount of silica in the system. In the end, all hybrid particles are partially coated with silica, and the steric and electrostatic repulsion is not sufficient to provide full colloidal stability. In order to prepare a stable system, the ratio between monomer and silica needs to be reduced. Microsystems. After reducing the volume of the system, it can be effectively homogenized by indirect ultrasonication, which has the advantage over direct sonication that closed reaction vessels can be easily employed, and thus oxidation by air can be avoided. This makes it possible to use oxidationsensitive nanoparticles as stabilizers for Pickering-type systems as well. The results of the previous section are combined in 9350

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Figure 2. TEM images of (a) FITC labeled silica, (b) quantum dots encapsulated in silica, and in two magnifications micro systems stabilized by (c, d) fluorescent silica (run MSL11, Table 2) and (e, f) quantum dots (run MSL14, Table 2).

70 free FITC molecules. Furthermore, the encapsulation efficiency of dye can be calculated to be 41% with respect to employed dye for the coupling with APS at the beginning of the synthesis. In contrast to the work of Boiko Cohen et al.,19 we did not expect a strong enhancement of the relative fluorescence efficiency of FITC dye after encapsulation, since the dye used in their report (DY-630-MI)19 has a more flexible structure compared to FITC and opens up more possibilities for cis−trans transformations, intermolecular interactions, and interactions with the solvent. Hence, after limiting these interactions by encapsulation, the fluorescence efficiency is strongly enhanced.19 In contrast, our FITC molecule has a relatively rigid structure by nature, and encapsulation should not have a significant effect on the fluorescence efficiency. Preparation and Characterization of Dually Labeled Hybrid Particles. Fluorescent Silica-Stabilized Polymer Particles. In run MSL11 (Table 2), hybrid polymer particles stabilized by fluorescent silica are prepared accordingly to the recipe for the systems with the nonfluorescing silica. The

perylene dye in the polymer core. Additionally, it can be easily covalently bound to silica and encapsulated in the silica matrix by coupling with APS. By the employed protocol particles with an overall diameter of around 26 nm (by TEM, see Figure 2a) could be obtained which perfectly match the commercial nonfluorescing Ludox silica (diameter 26 nm). The excitation and emission spectra of the fluorescent silica are displayed in Figure S2 showing maxima at λex = 490 nm and λem = 520 nm, respectively. The values are in accordance with literature data18 of the free dye (λex = 492 nm and λem = 518 nm), confirming that the dye molecules were incorporated into the silica particles without damage. Assuming that the optical properties of the fluorescent dye molecules are not altered by encapsulation, the number of encapsulated dye molecules can be calculated from the solid content (1.3 wt %, average diameter 25.9 nm, density 1.9 g cm−3), the fluorescence intensity of the silica dispersion, and a calibration curve of molecularly dissolved dye. The fluorescence intensity of every single silica particle is corresponding to about 9351

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Furthermore, the QD particles display a broad absorption below 550 nm, while the emission spectrum shows a narrow profile with a maximum at around 570 nm (Figure S3). Thus, they show similar optical and surface properties as the FITC labeled silica particles and should be analogously applicable for fluorescently labeled Pickering-type stabilization. However, QD stabilized hybrid particles prepared under the same conditions as MSL12 showed poor stability and resulted in an average size of 1071 nm (PDI 0.751) by DLS (run MSL13, Table 3). Probably, the interactions between the silica and the polymer surface are too weak which might be caused by the presence of polymeric stabilizers employed during the synthesis of the encapsulated QDs. As the pH plays a significant role in the stabilization mechanism,4b in a second run (MSL14, Table 3) the pH was reduced to 9.7 while keeping the other conditions constant. After polymerization, a stable dispersion was obtained with an average particle size of 236 nm (PDI 0.103) by DLS. TEM measurements revealed a size of 105 nm (Figure 2e,f), and the resulting morphology of the particles is similar to that stabilized by bare or fluorescent silica. At lower pH values, the interaction between silica and monomer droplets is indeed enhanced, and basically all the silica particles are fixed on the surface of the polymer particles. Further experiments have to be carried out to enhance the stability of the hybrid silica encapsulated QD stabilized particles. In Figure 4, the emission spectra of the dually labeled hybrid particles (λex = 360 and 561 nm, respectively) are shown. After

average size measured by DLS is 227 nm with a PDI of 0.062. TEM shows an average size of 141 nm (see Figure 2c,d). The fluorescence properties of the hybrid particles were measured as shown in Figure 3 with λex = 488 and 561 nm.

Figure 3. Emission spectra of the dispersion of hybrid particles (run MSL11, Table 2) with λex = 488 nm (FITC) and 561 nm (perylene dye) separately, with a concentration of hybrid particles of 100 μg mL−1. Note the different scale on the left and right y-axis.

After being excited at 488 nm, the fluorescence intensity mainly comes from FITC, while at 561 nm from the perylene dye. The two emission spectra differ from each other apparently. In order to further confirm the separate excitation and detection, measurements with the confocal laser scanning microscope (cLSM) were performed (Figure 5A−C). The spots in the green channel (FITC, Figure 5A) correspond well to those in the red channel (perylene dye, Figure 5B) and by overlaying the two channels an image with mainly yellow spots is generated (Figure 5C). There are some large spots, where intensity from the red channel is much stronger compared to that of the green channel. This could be explained by the overlapping of the excitation wavelength of perylene dye with the emission wavelength of FITC. When a large cluster of hybrid particles is formed in the cLSM sample, the emission of FITC in the center of the cluster will be absorbed by the perylene dye, which will emit in the red channel. This will lead to a higher intensity of the red emission although the emission intensity of FITC in nonclustered samples is much stronger than the intensity from the perylene dye (see Figure 3). Despite the different color intensities in Figure 5, one can exclude leakage of dye molecules from the particles within the accuracy of the detection method as only spots with overlaid colors are detected. Polymer Particles Stabilized by Silica Encapsulated Core− Shell CdSe/ZnS Quantum Dots. Organic dyes often display problems in optical applications like, e.g., photobleaching. Therefore, core−shell CdSe/ZnS based quantum dots encapsulated in silica were employed for stabilizing Pickeringtype polymer particles in analogy to the previous systems. The synthesis of the silica encapsulated quantum dots is detailed in the Experimental Section.14 The particles possess a silica shell resulting in a total average diameter of 29 nm via TEM analysis (Figure 2b) and a ζ-potential of −29 mV, which is quite similar to that of the Ludox silica (−27 mV, diameter 26 nm) and the fluorescent silica (−32 mV, diameter 26 nm).

Figure 4. Emission spectra of the dispersion of hybrid particles stabilized by QDs encapsulated in silica (run MSL14, Table 2) after excitation at λex = 360 and 561 nm.

being excited at 360 nm, the fluorescence intensity mainly originates from the QDs, while at 561 nm from the perylene dye. The two emission spectra differ from each other distinctly. In order to further confirm the separate excitation and detection, cLSM measurements are performed (see Figure 5D−F). The results are similar to that of fluorescent silica stabilized polymer particles (see above) and again demonstrate the possibility to excite and detect separately two different fluorescent signals from the same particles.

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CONCLUSION In this work, Pickering-type stabilized hybrid particles with a diameter of around 150 nm are prepared. These poly(styreneco-4-vinylpyridine) particles are monodisperse and stabilized by 9352

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Figure 5. (A, B, C) cLSM micrographs of run MSL11 (see Table 2) with (A) excitation at λex = 488 nm, recording the emission between 500 and 530 nm, (B) excitation at λex = 561 nm, recording the emission between 580 and 630 nm, and (C) overlay of both channels from (A) and (B); the contrast and brightness of the images are adjusted, so that the coherence of the red and green signals is apparent. (D, E, F) cLSM micrographs of run MSL14 (see Table 2) with (D) excitation at λex = 488 nm, recording the emission between 520 and 550 nm, (E) excitation at λex = 561 nm, recording the emission between 580 and 630 nm, and (F) overlay of both channels from (D) and (E).

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negatively charged silica particles with a diameter of around 26 nm, which is the commercially available Ludox silica. Successful downscaling of the original system by 200 times can be achieved. In this procedure, oxidation of monomer during preparation is minimized. The final systems possess an extremely low solid content (0.6%) and low volume (around 0.85 mL). This opens up possibilities to investigate Pickeringtype stabilized miniemulsion systems with precious stabilizers, for example, dendrimers, proteins, nanodiamonds, and so on. Fluorescent silica particles with core−shell structures and a diameter below 30 nm are also prepared. The fluorescent core has a diameter ranging from 15 to 22 nm, while a silica shell with a thickness of about 4 nm is deposited, which serves as protection layer of the fluorescent core. The fluorescent signal released from every single silica particle is strong and can be easily detected by spectroscopic and microscopic methods. The average size and surface charge of the fluorescent silica particles are close to the values of the commercially available Ludox silica. In the end, monodisperse dual fluorescently labeled hybrid particles stabilized either by fluorescent silica particles or quantum dots encapsulated in silica particles are prepared. Separate excitation and detection of both fluorescence signals for each system are demonstrated by the emission spectra of the dually labeled particles and further confirmed by confocal laser scanning microscopy (cLSM) investigations. This offers the possibility to trace the silica particles separately from the polymer particles in a time-dependent manner e.g. inside of cells in biomedical applications.

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AUTHOR INFORMATION

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to Prof. Katharina Landfester for fruitful discussions.

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REFERENCES

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