Controllable Preparation of Monodisperse Microspheres Using

*E-mail: [email protected] (C.-S.L.), [email protected] (J.L.); Telephone: .... of thermosensitive gelatin hydrogel microspheres in a microfluidic sy...
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Controllable Preparation of Monodisperse Microspheres Using Geometrically Mediated Droplet Formation in a Single Mold Chang-Hyung Choi,† Jongmin Kim,† Sung-Min Kang,† Jinkee Lee,*,‡ and Chang-Soo Lee*,† †

Department of Chemical Engineering, Chungnam National University, Yuseong-gu, Daejeon, 305-764, South Korea School of Mechanical Engineering, Sungkyunkwan University, Suwon, Gyeong Gi-Do 440-746, South Korea

‡

S Supporting Information *

ABSTRACT: We present a surfactant-free fabrication method for simultaneous generation of monodisperse microspheres with controllable size manner. Droplets that become microspheres by solidification processes are made in a two-step process: capillary rising-induced fluid division and wetting of immiscible fluid in a micromold. Design of the mold geometry and the monomer concentration primarily determines the microsphere size and the size distribution. Furthermore, the synergistic effect of two parameters is able to efficiently manipulate the microsphere sizes from submicrometers to a few hundred micrometers.

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polymerization,11 seeded polymerization,12 and suspension polymerization.14 However, there is no method available to control the size of particles within a broad range from submicrometers to a few hundred micrometers. In addition, there is a limited ability to select materials, use complicated formulations, and use elegant procedures to control the nucleation and growth processes.15 Recently, microfluidics has been developed for the preparation of monodisperse particles over a broad size range through T-junction,16 coflowing,17 flow focusing,18 and stopflow lithography.19 Although microfluidic systems can be used to control size, shape, porosity, internal structure, and composition, the controllable ranges of particles are tens of micrometers in diameter because they strongly depend on the channel geometry, interfacial properties between immiscible fluids, and flow rates.20 Recently, Tabeling et al. presented a nanofluidic approach including submicrometric channels that generates droplets, particles, Janus droplets, and microcapsules with sizes from 1 to 3 μm.21 However, this technique is not suitable for the preparation of larger microspheres with sizes over 3 μm. In addition, surfactants are commonly added to stabilize individual droplets and reduce the droplet size in nano/microfluidic systems.22 In industry, however, extra separation processes that have a high associated cost are required to remove the surfactants because it causes acute toxicity such as respiratory distress, metabolic acidosis, tachycardia, renal failure, and electrolyte imbalances.23 Thus, the widespread use of microfluidics is limited in biomedical

INTRODUCTION Microspheres play critical roles in many applications, such as drug delivery systems,1 optical materials,2 cosmetics,3 and chemical and biological diagnostics.4 The ability to confer a variety of functions through physical and chemical modification can be beneficial for many applications.5 The particles decorated with multifunctionality require narrowly controlled chemical composition and precisely engineered physical properties.6 Hence, many researchers are increasingly devoted to designing particles by considering physical factors such as size, shape, mechanical property, roughness, and compartmentalization without modifying the particles’ chemical properties.7,8 In particular, the size of microspheres has been much studied in the context of developing biomaterials for drug delivery. First, simply controlling the size of microspheres can provide a significant potential for smart drug carriers due to their various release profiles. By mixing microspheres of different sizes, it is possible to obtain another degree of control over the release. For example, zero-order kinetics (i.e., a linear release profile) can be obtained by combining the proper formulation of microsphere sizes.9 Second, several important in vivo functions of drug-carrying particles depend on their size: these include circulation times, extravasation, targeting, immunogenicity, internalization, intracellular trafficking, degradation, flow properties, clearance, and uptake mechanisms.5 Thus, there is a significant need for a method that can be used to synthesize monodisperse particles with tunable sizes. Previously, several methods have been presented to synthesize monodisperse microspheres.10−14 In general, emulsion polymerization can produce polymeric spheres with sizes ranging from 0.1 to 1 μm.14 Larger spheres with sizes ranging from 1 to 10 μm can be produced by dispersion © XXXX American Chemical Society

Received: April 16, 2013 Revised: June 21, 2013

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solution (poly(ethylene glycol) diacrylate (PEG-DA)) diluted with volatile solvent is introduced into the polydimethylsiloxane (PDMS) mold, and the excess solution is removed by suction with pipetting (Figure 1A, step 1). Second, the volatile solvent starts to evaporate at room temperature. The evaporation of the volatile solvent results in a volume reduction of the solution in the mold. The remaining photocurable solution is divided by capillary rising at the edge of each arm of the cross-shaped mold (step 2). Third, wetting fluid (nhexadecane) is added to the PDMS mold and wets the exposed PDMS surface. Eventually, the photocurable solution is fully surrounded by the wetting fluid, which results in the formation of spherical droplets for energy minimization. Sequential images of each step are shown in Figure 1B. Importantly, the symmetry given by the cross-shaped mold allows the photocurable solution to be divided into four arms having identical volume, thereby forming monodisperse droplets as a result of the wetting process. To confirm the principle of the formation of droplets in Figure 1A, we have solidified the photocurable solution confined in the mold in the intermediate step. Scanning electron microscopy (SEM) images show the resulting structure of the cured photocurable solution, and its curvature is well matched with our expectation for each arm (Figure 1C). This result proves that the photocurable solution is equally divided by the capillary force driven by the mold geometry and equivalent evaporation of the photocurable solution at each arm. We expect that the interfacial tension plays a pivot role in droplet formation. First, as the interfacial tension between PEG-DA and PDMS is increasing, the binding strength between the PDMS surface and the PEG-DA solution is stronger. Thus, the summation of gravitational force and the binding force between PDMS and PEG-DA hinders the generation of the spherical droplet. Second, when the interfacial tension between PEG-DA and wetting fluid is higher than the summation of gravitational force and the binding force, PEGDA adheres to PDMS surface. In the case of reverse conditions, PEG-DA solution is detached from the PDMS surface and becomes a spherical droplet. We have previously demonstrated the micromolding method based on Laplace pressure-induced flow for the synthesis of monodisperse microspheres. However, the method should use different size of micromolds when we want to synthesize different size of microspheres. Therefore, there is a great need for an even more simple and robust technique to generate highly controllable multiple-size microspheres in a single mold. To improve the micromolding technique, we used a photocurable solution with different monomer concentrations (10−100% of PEG-DA in ethanol) to precisely control the dimension of microspheres over a wide range. First, in the range of formation B (60−100% monomer), a single droplet is formed by Laplace pressure-induced flow through the wetting fluid, which was reported in our previous study (Figure 2).25 Although volume of the monomer dramatically decreases by 60%, it still fully covers the bottom of the PDMS (fluid division does not occur), resulting in a single droplet in a mold. The diameter of the droplet is determined by volume of the monomer after the solvent evaporation of the photocurable solution. Thus, the diameter (dB) of a single droplet formed in a cross-shaped mold is given by dB = (6Vmonomer/π)1/3 (Figure 2B). In contrast, in the range of formation A (10−40%), fluid division occurs, and the divided monomer volume is

applications. Desimone et al. have presented a pioneering alternative micromolding technique, particle replication in nonwetting templates (PRINT), to produce monodisperse particles with various sizes and shapes.24 This technique involves simple and reproducible systems and can generate highly uniform polymer particles; however, it has limitations for fabricating three-dimensioonal (3D)-shaped particles such as spheres and concave/convex particles. As an improvement to the approach, we have recently presented a novel micromolding technique to produce monodisperse microspheres25 and anisotropic microparticles26 using surface-tension-induced fluid flows in micromolds. However, it is still challenging to precisely control particle size range and its distribution of size through a greener way. This study reports a surfactant-free method for simultaneous generation of monodisperse microspheres with multiple sizes in a single mold. The geometrically mediated droplet formation results from two-step process that involves fluid division driven by solvent evaporation and the wetting of immiscible fluid. Importantly, mold geometry and concentration of monomer primarily determines the size and distribution. Therefore, we can efficiently produce microspheres with sizes from submicrometers to a few hundred micrometers in a simple manner.

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RESULTS AND DISCUSSION The basic principle of the formation of droplets in a crossshaped micromold is depicted in Figure 1. First, a photocurable

Figure 1. Droplet formation in a mold. (A) Schematics of three major steps involved in the generation of a droplet in a micromold. Each step sequentially represents the addition of solvent-diluted photocurable solution into a mold (step 1), fluid division driven by solvent evaporation and capillary rise (step 2), and droplet formation resulting from the wetting process (step 3). (B) Sequential images of the resulting formation from each step, which is clearly visualized by incorporating rhodamine B into the photocurable monomer. (C) SEM images of the resulting formation from step 2 showing the contact angle of the monomer. 30% PEG-DA (diluted with ethanol) is used as a photocurable solution. The calculated interfacial tension between PEG-DA and PDMS is about 3.5 mN/m. B

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Figure 2. Ability to control the size of microspheres using a single mold. (A) Distinctive formation of the remaining monomer is observed after solvent evaporation of the photocurable solution with different concentrations of monomer; while fluid division occurs for conditions involving low concentrations of monomer (10−40% PEGDA, Formation A), no division occurs at conditions involving relatively high concentrations of monomer (60−100% PEG-DA, Formation B). Upon the addition of wetting fluid, the monomer confined in the mold becomes a spherical droplet due to the principle of energy minimization. A cross-shaped mold including four arms results in the formation of four droplets. (B) A plot of the diameter of the microspheres versus the concentration of monomer. The transition state is defined as the coexistence of droplets formed from both formations (data not shown). (C) SEM image of PEG microspheres. (D) Size distribution of PEG microsphere. The polydispersity values are below 2% for the microspheres.

Figure 3. Ability to control the size of microspheres with diameters ranging from 700 nm to 2 μm using a star-shaped mold with ten arms. (A) SEM image of the star-shaped mold used for synthesizing colloidal microspheres. (B−C) SEM images of colloidal microspheres formed from 2% PEG-DA solution. (D−F) SEM image of colloidal microspheres formed when the concentration of PEG-DA solution was varied (0.1−1%). (G) Size distribution of PEG microspheres formed from 0.5% PEG-DA solution. The polydispersity is approximately 5.4%. (H) A plot of the diameter of the colloidal microsphere versus the concentration of the monomer. The height of the micromold is 10 μm.

The next challenge for this technique is to generate multiplesized microspheres in a single mold. Here, we have used simple branched molds consisting of different numbers of arms with distinct lengths, providing precision control of the microsphere size. The different length of the arms results in a capillary force gradient through solvent evaporation, making it possible to control the volume of monomer. For example, a symmetric micromold (S 1 = S 2 = S 3) allows for generation of monodisperse microspheres, whereas an asymmetric micromold (S 1 < S 2 < S 3) allows for generation of microspheres of different sizes by dividing the volume of monomer fluid proportionally to the length of the arms (Figure 4). The corresponding SEM images show the “particle cocktail” containing various sizes of microspheres. The polydispersity of the microspheres formed in each arm is less than 3%. On the basis of image analysis, microsphere size increases proportionally to approximately S 1/3 (Figure S1). The approximate relationship between the length of the arms and the size of the microspheres is shown in Figure S2. Although the capillary force is determined by the perimeter of each arm, it can be controlled by changing the length of the arms when the width and height of the arm is constant. The result indicated that particle mixtures with diverse size distribution combinations can be prepared by simply designing the mold geometry. This method produces several types of microspheres such as polymer microspheres, silica microspheres, and colloid crystals through combination of various solidification methods including photopolymerization, hydrolysis/polycondensation, and evaporation-based self-assembly, respectively.25 In addition, we can further produce monodisperse microgels by physical or chemical solidification. We synthesize physical microgels

determined by the concentration of the photocurable solution. This phenomenon is attributed to the interplay between solvent evaporation and capillary rise; thus, the bottom surface of PDMS at the center of the micromold is exposed, and fluid division occurs. The diameter (dA) of the droplets formed in the mold is given by dA = (6Vmonomer/π)1/3/N, where N is the number of arms of the mold (Figure 2B) when the fluid division occurs. For instance, N is four for the cross-shaped mold. Herein, we have obtained uniform microspheres after solidifying these droplets via photopolymerization (Figure 2C). In the case of the cross-shaped mold having an identical volume for each arm, the synthesized microspheres are highly monodisperse (CV = 2%) (Figure 2D). Although we used the same mold repeatedly, size control can be achieved by simply changing the concentration of the monomer. Furthermore, we can expect that the mold geometry offers an additional advantage for manipulating the retention volume and thus extending the controllable range of microsphere sizes down to a few hundred nanometers. To confirm the capability, we introduced a photocurable solution with a concentration of PEG-DA ranging from 0.1 to 2% into a star-shaped micromold having ten arms. As the solvent evaporates, the volume of droplets is significantly decreased due to the mold shape or dimension. Upon addition of wetting fluid, droplet formation occurs and becomes colloidal particles by photopolymerization. SEM images of the resulting colloidal particles in micromolds are shown in Figure 3A−F. Thus, the combination of monomer concentration and mold geometry obviously has a synergistic effect for efficiently controlling individual droplet volume, allowing monodisperse colloidal particles ranging from 700 nm to 2 μm to be produced (Figure 3G,H). C

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economical problem caused by separation and purification processes in the conventional methods. (3) This method is the first to demonstrate the capability to produce microspheres with sizes ranging from submicrometers to a few hundred micrometers. We envision that our proposed eco-friendly method could be used as a surfactant-free process to produce highly monodisperse microspheres with many potential biomedical applications such as drug-delivery or medical imaging.5

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ASSOCIATED CONTENT

S Supporting Information *

Details of experimental methods, size controllability of microspheres by modulation of the length of the mold arms, and theoretical estimation of the droplet formation. This material is available free of charge via the Internet at http:// pubs.acs.org.

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Figure 4. Ability to produce multiple-sized microspheres through the design of the mold geometry. Representations of the mold geometry used are shown: (A) symmetric branched mold with three arms, (B) asymmetric branched mold with three arms, (C) four arms, and (D) five arms. The length of the basic unit (square) is S = 100 μm, and the height = 25 μm. Monodisperse microspheres are formed with a symmetric mold (S 1 = S 2 = S 3), while an asymmetric mold (S 1 < S 2 < S 3) causes capillary force differences across the micromold, which allowing the fluid to be divided into different amounts of monomer, thus generating multiple-sized microspheres. The multiple-sized microspheres generated from 30% PEG-DA solution. The polydispersity values are below 3% for the microspheres.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (C.-S.L.), [email protected] (J.L.); Telephone: +82-42-821-5896. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by a National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2011-0017322) and by the National Space Laboratory (NSL) program through the National Research Foundation of Korea funded by the Ministry of Education, Science, and Technology (20100026155). We also thank Professor Hyunmin Yi at Tuft University and Professor Eunji Lee at Chungnam National University for fruitful discussion.

through temperature-induced gelation.27 Gellan solution (2% gellan gum in water) is heated to 80 °C to dissolve and hydrate the microgel. After the solution is introduced into the micromold, the addition of wetting fluid leads to the generation of droplets and subsequently cooling of the gellan droplets below the gelling temperature (40−45 °C), thereby making a spherical gel network (Figure S3-A). In a chemical manner, we produce Na-alginate droplets containing CaCO3 nanoparticles (3 wt %), and then wetting fluid containing acetic acid (5 wt %) is loaded.28 Upon external diffusion of the acetic acid, the pH of the droplet is rapidly decreased, which allows for the release of Ca2+ ions from the CaCO3 nanoparticles. Finally, alginate microgels are formed through a chemical chelation method. In addition, this approach can be extended to the encapsulation of active agents. We successfully encapsulate fluorescent-labeled bovine serum albumin (FITC-BSA) as an active drug model into alginate microgels (Figure S3−B). Overall, the proposed method can produce microspheres while offering the flexibility to choose components and the corresponding functionality.

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REFERENCES

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CONCLUSIONS We have demonstrated a novel method for simultaneous generation of monodisperse microspheres with multiple sizes in a single mold. The geometrically mediated droplet formation results from two steps involving fluid division and wetting. Importantly, simply changing the monomer concentration and the mold geometry can precisely control the size of the microspheres. In addition, we can also produce spherical microgels through droplet template synthesis using physical and chemical routes. The proposed method has remarkable advantages: (1) monodisperse microspheres with multiple sizes can be generated by simply varying the mold geometry without delicate flow control. (2) surfactant-free method can solve an D

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