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Synthesis of Biofunctional Janus Particles Binghui Li, Man Wang, Kui Chen, Zhifeng Cheng, Gaojian Chen,* Zexin Zhang* Janus particles with anisotropic biofunctionalities are perfect models to mimic anisotropic architectures and directional interactions that occur in nature. It is therefore highly desirable to develop reliable and efficient methods to synthesize biofunctional Janus particles. Herein, a facile method combining seeded-emulsion polymerization and thiol-click chemistry has been developed to synthesize Janus particles with glucose moieties on one side. These biofunctional Janus particles show region-selective binding of protein, which represents a big step toward biomimicry, and demonstrates the potential of the bioJanus particles for targeted drug delivery and binding.

1. Introduction Symmetrical shapes but anisotropic architectures are widely observed in nature ranging from complex living organisms to simple bacterium, such as left-side location of human heart and compartmentalization of components in cells.[1] Synthetic particles with anisotropic presentation of functionalities, therefore, form perfect models to mimic many of nature’s anisotropic architectures. They enable the study of the anisotropic interaction between the synthetic particles and the organisms, and hold the key to understanding the intriguing anisotropic behavior in all living organisms. One of the promising candidates for such synthetic models is Janus particle. Janus particles, which have different properties on two sides, are anisotropic materials of growing interest from both scientific and technological points of view.[2] B. Li, M. Wang, K. Chen, Z. Cheng, Prof. G. Chen, Prof. Z. Zhang Center for Soft Condensed Matter Physics and Interdisciplinary Research & Collaborative Innovation Center of Suzhou Nano Science and Technology and College of Physics, Optoelectronics and Energy Soochow University Suzhou 215006, China E-mail: [email protected]; [email protected]

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Biofunctional Janus particles, a new member to the family of Janus particles, add important biological features to the already unique Janus properties. For example, encoded with directional information, biofunctional Janus particles are able to bind biomolecules region-selectively, and can orientate themselves during cellular attachment and internalization processes.[3] Possible applications of biofunctional Janus particles, such as bioanalysis, cell detection, and biomedical imaging, have been envisioned.[4] As a result, there is emerging interest in the synthesis of biofunctional Janus particles. However, plain Janus particles without any biofunctionalities are already difficult to synthesize as surface tension typically produce homogenous particles. The common methods to produce Janus particles fall in three categories, namely fluidics methods,[5] selective chemical modifications,[6] and phase separation in confinement.[7] The fluidics methods such as microfluidics polymerizations can only make relatively large, nonBrownian particles, while the selective chemical modification and confined phase separation approaches, such as evaporation of metal layer and emulsion templating, suffer from low yields or polydispersity in shape and Janus balance.[2] Compared with plain Janus particles, biofunctional Janus particles are even more challenging to prepare, mainly due to the fact that an

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DOI: 10.1002/marc.201500063

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2. Experimental Section

Cross-linker

(a)

2.1. Materials

(b)

All chemicals (typical purity ≥98%) were purchased from SigmaAldrich and Acros Organics, and used as received.

Styrene monomer mixture

PS dimple particle

Dimple particle with liquid protrusion

2.2. Synthesis of Polystyrene (PS) Dimple Seed Particles (c)

(d)

Bio-functional Janus particle

Cl Cl

Cl Cl Cl Cl Cl Cl

PS/PVBC Janus particle

Figure 1. Schematic of the synthesis by SEP and click chemistry. a) Polymerization of styrene with programmed feeding of cross-liker to form dimple particles; b) Swelling of dimple particles by VBC, and then heated to form VBC liquid protrusion; c) Polymerization of the liquid VBC to form solid Janus particle; and d) Bio-functionalization of the Janus particle with glucose using thiol-chloride, “click” chemistry.

effective and efficient way to render particle bioactivity is still rare. Reports on the synthesis of biofunctional Janus particles are limited. Only recently, electrohydrodynamic cojetting has been demonstrated to produce biohybrid particles with spatially-controlled affinity to cell.[4e,f] It is therefore highly desirable to develop reliable and efficient methods to synthesize biofunctional Janus particles. An alternative chemical strategy towards the synthesis of Janus particles is seeded-emulsion polymerization (SEP). The SEP has great advantages over other methods as it can be easily scaled up to make large quantity of particles, which is vital for practical applications. Recently, we have reported a SEP approach to synthesize shape-anisotropic but chemically isotropic particles.[8] To the best of our knowledge, SEP has not been applied to synthesize biofunctional Janus particles. Herein, we report a facile approach to synthesize biofunctional Janus particles combining the well-documented SEP and the efficient thiol-based “click” chemistry.[9] Briefly, polystyrene (PS) dimple particles were first synthesized and used as seeds. Then vinylbenzyl chloride (VBC), was selected as the swelling monomer for the seed particles, for its similar molecular structure to styrene, and for its ability to be further chemically modified via the robust thiol-chloride chemistry.[10] Spherical Janus particle of PS/PVBC are formed after SEP. Finally, the PVBC part of the Janus particles are rendered biofunctionality through click chemistry using carbohydrates, a class of vital compounds found in many bioprocesses such as human metabolism, and often recognized as the “face” of cells,[9,10] see Figure 1 for a schematic of the synthesis.

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A mixture of styrene (St) with polyvinyl pyrrolidone (PVP) in ethanol was deoxygenated and then polymerized at 73 °C for 1 h using 2,2′-azobis(2-methylpropionitrile) (AIBN) as initiator. In the meantime, another batch of styrene mixture with cross-linker, divinylbenzene (DVB) in ethanol was prepared and deoxygenated. To obtain the dimple particle, we followed a programmed DVB-feeding methods reported in our previous studies.[8] DVB feeding time (5 h in total) was divided into fast and slow stages. Specifically, the feeding speed was set at 0.25 mL min−1 in the first hour, then reduced to 0.1 mL min−1 in the last 4 h. After finishing the DVB feeding, the reaction was left to last for 19 h with continuously bubbling of nitrogen gas.

2.3. Synthesis of Polystyrene/Poly(vinylbenzyl chloride) (PS/PVBC) Janus Particles by Seeded Emulsion Polymerization PS dimple seed particles dispersed in 1% polyvinyl alcohol (PVA) solution, monomer emulsion consisting of swelling monomer vinylbenzyl chloride (VBC, 5 wt%), cross-linker (DVB, 0.8 wt%), and initiator (AIBN, 0.5 wt%) were prepared and mixed. The mixture was tumbled at the speed of 40 rpm at 25 °C for 10 h to allow dimple particles to swell. Polymerization of the VBC was carried out at 72 °C for 8 h. After the polymerization, the particles were cleaned by repeated centrifugations and washed with ethanol. The morphology of the particles was examined by scanning electron microscope (SEM) and optical microscope.

2.4. Biofunctionalization of the PS/PVBC Particles Through Thiol-Click Chemistry The PS/PVBC Janus particles were mixed with glucothiose (1-thio-β-D-glucose sodium salt) and reacted at 40 °C for 110 h using the thiol-click chemistry of chloride groups from PVBC and thiol groups from the glucothiose.[10] The modifications were confirmed using a Bruker Vertex 80 Fourier transform-infrared (FT-IR) spectrometer in the attenuated total reflectance (ATR) mode, and elemental analysis with a Vario MICRO cube Elemental Analyzer.

2.5. Binding of Lectin to BioJanus Particles A solution of 0.5 mg mL−1 fluorescein isothiocyanate-modified Concanavalin A (FITC-ConA) in Phosphate Buffered Saline (PBS, PH 7.4), was prepared and mixed with a dilute (0.1% by weight) suspension of glucose-functionalized PS/PVBC Janus particles. The mixtures were tumbled slowly at 25 °C for 30 min to allow the specific carbohydrate-lectin binding. The particles were then washed with PBS several times to remove the excess FITC-ConA,

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and redispersed in deionized water for microscopy characterization. Bright field and fluorescence microscopy images were recorded on a Zeiss Axio Observer A1 microscope, to investigate the distribution of fluorescent FITC-ConA over the particles. The amount of fluorescent lectin, FITC-Con A, bound on the particles was determined by quantitative fluorescence spectroscopy technique using a Horiba FluoroMax 4 spectrometer.

3. Results and Discussion The simplest form of biofunctional Janus particles is sphere with biochemistry anisotropy but not shape anisotropy. Hence, we started with dimple seed particles, which can be swollen and polymerized to produce Janus spheres. After the seeded emulsion polymerization, most of the dimple particles were converted into spherical Janus particles, with a yield of 96.1% ± 1.5% (average and standard deviation). As shown in the SEM and optical microscope images (Figure 2a–c), the average diameter of the Janus particles, evaluated by image analysis of the micrographs, is 3.05 ± 0.09 μm. The PS and PVBC Janus lobes are not distinguishable in the micrographs, possibly due to the similar chemical and hence similar electron and optical contrast properties of the two polymers. To better visualize the PS and PVBC lobes, we enhance the optical contrast of the two polymers by solvent-swelling the particles. Dimethylformamide

(DMF) is a better solvent for PVBC than for PS. As a result, the PVBC lobe swells more and an enhanced contrast of the two Janus parts is achieved. It is clearly demonstrated in the bright-field micrograph that the Janus particle consists of two parts: one is the PS dimple particles, and the other is the PVBC in the shape of new moon in the 2D view (Figure 2d). By brief ultrasonication, the PVBC part detaches from the PS dimple particles (Figure 2e). Thus, uniform PVBC bowl-shaped particles can be obtained after removing the PS dimple particles by centrifugation (Figure 2f). This simple treatment, as an added benefit, uncovers a facile method to produce moon/bowl-shaped particles (Figure 2g). Possible applications of these particles can be expected such as directional assemblies and microreactors.[11] In addition, the successful incorporation of PVBC was confirmed by FT-IR spectrometer. Compared with the spectrum of PS dimple particles, a new, sharp peak at 1260 cm−1 was observed for the PS/PVBC Janus particles, which corresponds to the CH2–Cl vibration from the PVBC[12] (Figure 3). The chloride groups on the PVBC lobe of the Janus particle can be easily converted to other functionalities, providing a versatile chemical switch for further surface modifications on one of the well-defined Janus parts of the particles. To demonstrate the proof of concept of making bio-functional Janus particles, thiol-glucose was chosen to render the particle bioactivity using the straightforward thiol-click chemistry of thiol and chloride

Figure 2. Micrographs of the seed and Janus particles. SEM images of a) PS dimple seed particles; and b) PS/PVBC Janus particles. Brightfield optical microscope images of c) Janus particles; d) Janus particles swollen by dimethylformamide (DMF); e) swollen Janus particles after brief sonication; and f) PVBC bowl-shaped particles, after centrifugation to remove the PS lobe. g) Schematic of the process to separate the two Janus parts. The scale bars are 2 μm.

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*

3300 cm -1

*

1050 cm -1

PS/PVBCThio-glucose

* 1260 cm

-1

PS/PVBC

PS

4000

3500

3000

2500

2000

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Wave number [cm-1]

Figure 3. FT-IR spectra for PS dimple particles (bottom), PS/ PVBC Janus particles (middle), and PS/PVBC Janus particles after reaction with 1-thio-β-D-glucose sodium salt (top). The asterisks highlight the new peaks formed after SEP (≈1260 cm−1), and after thiol-click reaction (≈1050 and ≈3300 cm−1), respectively.

groups. After the click reaction and being washed, the Janus particles were recovered for chemical analysis using FT-IR spectrometer. Compared with the IR spectrum of bare Janus particles, the sharp peak at 1260 cm−1 due to CH2–Cl vibration disappears in the IR spectrum of thio-glucose functionalized Janus particles, and two new peaks show up at 1050 and 3300 cm−1, which correspond to the C–O and OH groups, respectively (top curve, Figure 3). This offers direct evidence that the thiol groups from 1-thio-β-D-glucose sodium salt successfully reacted with the chloride groups, and the thio-glucose is attached onto the Janus particles. The successful conjugation of glucothiose was also confirmed via elemental analysis of sulfur. Assuming glucothiose only react with vinyl groups on the particle surface, we found a surface coverage of 8.2 molecules nm–2 (see Supporting Information for calculation details). Note there are possibilities that some glucothiose molecules penetrate into the swollen Janus particles and react with the vinyl groups inside. To assess the biofunctionality of the Janus particle, lectin binding abilities of the glucose-functionalized Janus particles were investigated by using Concanavalin A (Con A), a lectin protein specific for binding carbohydrates such as glucose and mannose.[12,13] The amount of FITC-Con A bound on the particles were determined by quantitative fluorescence spectroscopy, giving a surface coverage of 0.083 molecules nm–2 (≈352 ng cm–2), which is consisted with the specific Con A adsorption ability of a bioactive surface reported recently.[14] The distribution of FITC-ConA over the particles are evaluated by fluorescent microscopy. The particles showed strong fluorescence at only the bioactive lobe (PVBC part) of the Janus particles, indicating the specific adsorption properties of the

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Figure 4. Binding biomolecules by biofunctional Janus particle. a) Schematic showing the nonfluorescent PS lobe and the fluorescent PVBC lobe after specific bio-binding of FITC-Con A. b) brightfield microscopy image; c) fluoresce microscopy image; and d) overlay of the images in (b) and (c). The scale bars are 2 μm.

Janus particles to the florescent biomolecules, FITC-Con A (Figure 4). The anisotropic presentation of carbohydrates and proteins are believed to guarantee further bioapplications of the bioJanus particles.

4. Conclusion In summary, we provided a facile and robust method to synthesize bioactive Janus particles by SEP and thiolclick chemistry. Our approach is distinguished from other methods, and has several advantages. First, thanks to emulsion polymerization, the synthesis can be scaled up to synthesize large quantities of biofunctional Janus particles. Second, equipped with click-chemistry, our method is general and can be extended to synthesize a myriad of biofunctional Janus particle by using vast selection of available bio-molecules. Last but not least, the synthesized Janus particles are highly bioactive and demonstrate strong region-selective affinity to specific biomolecules. We believe the method will benefit researchers in chemistry and material science with bioapplications and beyond.

Supporting Information Supporting Information is available from the Wiley Online Library or from the author.

Acknowledgements: B.L. and M.W. contributed equally to this work. This work was partially supported by the National

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Basic Research Program of China (Grant No. 2012CB821500), the National Natural Science Foundation of China (Grant Nos. 21174101, 910270401, and 21374069), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) and the Project for Jiangsu Scientific and Technological Innovation Team (2013).

[5]

[6] Received: February 2, 2015; Revised: March 10, 2015; Published online: April 9, 2015; DOI: 10.1002/marc.201500063 Keywords: colloids; biobinding; emulsion glucose; Janus particles; thoil-click chemistry

polymerization;

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Synthesis of Biofunctional Janus Particles.

Janus particles with anisotropic biofunctionalities are perfect models to mimic anisotropic architectures and directional interactions that occur in n...
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