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DOI: 10.1039/C4RA05864J (Paper) RSC Adv., 2014, 4, 46536-46540

Microfluidic fabrication of microparticles with multiple structures from a biodegradable polymer blend†

Sang Woo Kim, Kyung-Hee Hwangbo, Joo Hyuk Lee and Kuk Young Cho*
Division of Advanced Materials Engineering, Kongju National University, 1223-24, Cheonan-daero, Seobuk-gu, Cheonan, Chungnam 331-717, Republic of Korea. E-mail: kycho@kongju.ac.kr

Received 17th June 2014 , Accepted 16th September 2014

First published on 19th September 2014

Abstract

We demonstrate control over the morphology and shape of microparticles. Two different types of microparticles (core–shell or acorn shaped) were produced by the varying the blend ratio of two immiscible biodegradable polymers in a tube-type microfluidic device. Subsequent use of selective solvent extraction resulted in the formation of hollow-core, crescent-shaped and dimple surfaced microparticles. A selective solvent, tetraglycol, which has been applied for intravenous injection in the human body, is used instead of organic solvents which can be toxic if remaining in microparticles.

Introduction

Anisotropic microparticles, including patch, asymmetrical, and Janus particles, are widely observed in nature (e.g. pollen grains, virus, biological cells) and the anisotropic character is strongly related with their ability for a specific role.1–3 Inspired by the impact of physical parameters (shape and morphology) on the performances, complex forms of synthesized microparticles are gaining recognition for their importance in various applications, such as building blocks,4,5 two-phase stabilizers,6,7 optical sensors,8 and electronic devices. Exploration of the use of complex-shaped microparticles in biomedical applications revealed their potential for improving performance compared to that of conventional smooth round-shaped microparticles,2,9 which is general feature of the particles fabricated in the aqueous media. Because biodegradable and biocompatible polymer is favourable for biomedical applications, it is a continuous challenge to provide tailor-made anisotropic microparticles using materials suitable for biomedical applications.10

Simulations of composite latex particles with two immiscible phases has been performed, and showed the formation of a variety of structures including core–shell, hollow particle, moon-like, and salami-like structures.11 Subsequent research has focused on the fabrication of structures predicted by this simulation study and the formation of Janus-like microparticles from blends of poly(methyl methacrylate) and polystyrene via internal phase separation has been reported.12 Although this approach provides a facile fabrication route for anisotropic microparticles from blend materials, there are few studies that used biodegradable polymer blends. Spherical microparticles fabricated from blends of biomedical polymers is reported to alter degradation and drug delivery characteristics.13 Addition of anisotropic features can be advantageous in obtaining tailor-made microparticles for precise control of performance of microparticles such as cell internalization, drug release behaviour for biomedical applications.2b,c

The microfluidic approach to fabrication of microparticles has the advantage of producing microparticles of a uniform-size. Because organic droplets continuously move with the laminar flow, there is difficulty in fabrication of anisotropic microparticles compared to a stagnant process such as micromolding. This limitation has been overcome by adopting specific techniques, such as photopolymerization or photolithographic polymerization.14–16 However, the materials used in these methods are based on UV-curable materials. The use of biodegradable polymers which are generally non-curable aliphatic polyester has difficulty in anisotropic microparticle fabrication via the microfluidic approach. Recently, we succeeded in the fabrication of golf ball-shaped microparticles, which possess dimple structures on the surface, from various biodegradable polymers. A tube-type microfluidic device incorporating an inert organic phase change material, was responsible for the formation of new structure.17 Inspired by the success of morphology-specific microparticle fabrication via microfluidic approach using addition of organic phase change material, mixtures of immiscible biodegradable polymer blends have attracted our attention for possibility of uniform-sized anisotropic microparticle formation from microfluidic device.

The blend of PLGA (poly(D,L-lactide-co-glycolide)) and PCL (poly(ε-caprolactone)) is one of the most attractive biodegradable polymer blends owing to its availability in biomedical applications. Tang et al., reported an enhancement of adhesion and growth of osteoblast cells using PLGA/PCL blend film, which is resulted from the change in surface morphology with the variation in composition.18,19 This blend has also been studied in vascular tissue engineering applications in the form of hollow fibre membranes.20 The degradation profile of microparticles prepared from a blend of PLGA/PCL (1[thin space (1/6-em)]:[thin space (1/6-em)]1, w/w) was intermediate between those of PCL and PLGA, indicating a tailoring of the degradation rate.21

Herein, we report the fabrication of various forms of uniform microparticles by variation of the PLGA/PCL blend composition. Furthermore, coupling this fabrication technique with the selective solvent extraction of PLGA by tetraglycol allowing the production of various shapes of PCL microparticles is discussed.

Experimental

Materials and fabrication of microparticles

Poly(ε-caprolactone) (PCL, Mw = 43[thin space (1/6-em)]300) and poly(D,L-lactide-co-glycolide) (PLGA, lactide[thin space (1/6-em)]:[thin space (1/6-em)]glycolide = 65[thin space (1/6-em)]:[thin space (1/6-em)]35, η = 0.55–0.75 dl g−1 in hexafluoroisopropanol, Lactel) were dissolved in dichloromethane (DCM), which served as an organic phase. The ratio of DCM to the mixture of PLGA and PCL was fixed to 9[thin space (1/6-em)]:[thin space (1/6-em)]1 by weight. The amount of PLGA and PCL was varied. The ratios of PLGA to PCL were 10[thin space (1/6-em)]:[thin space (1/6-em)]0, 8[thin space (1/6-em)]:[thin space (1/6-em)]2, 7[thin space (1/6-em)]:[thin space (1/6-em)]3, 6.5[thin space (1/6-em)]:[thin space (1/6-em)]3.5, 6[thin space (1/6-em)]:[thin space (1/6-em)]4, 5.5[thin space (1/6-em)]:[thin space (1/6-em)]4.5, 5[thin space (1/6-em)]:[thin space (1/6-em)]5, 4[thin space (1/6-em)]:[thin space (1/6-em)]6, 3[thin space (1/6-em)]:[thin space (1/6-em)]7, 2[thin space (1/6-em)]:[thin space (1/6-em)]8, and 0[thin space (1/6-em)]:[thin space (1/6-em)]10 by weight. After complete dissolution of PLGA and PCL, the organic phase was placed in a syringe, which was equipped with a 30G needle. Poly(vinyl alcohol) (PVA, 87–89% hydrolyzed, Mw of 13[thin space (1/6-em)]000 to 23[thin space (1/6-em)]000, Aldrich) was used as a stabilizer in continuous flow at concentration of 1 wt% in aqueous solution. Discontinuous and continuous phases were introduced by syringe pumps (KDS 100 and KDS Legato 200, KS Scientific) with independently adjusted flow rates. The microparticles are fabricated using a simple microfluidic device that uses a co-flow capillary system (continuous and discontinuous flow are in same direction). The rate of discontinuous flow (Qd) was 0.1 mL h−1. The rate of continuous flow (Qc) was determined as the condition at which droplets pinch off near the capillary tip (Qc = 200 mL h−1). The inner and outer diameter of the tubing (E-3603, Tygon) were 0.79 mm and 2.38 mm, respectively. DCM in the oil phase was removed by evaporation at ambient temperature for 3 h. Microparticles were washed with distilled water three times. The final product was first air dried in a hood for 24 h and then vacuum dried for 24 h. Rhodamine 6G (Aldrich) was used as a hydrophobic dye to support facile visualization of the organic microparticles (0.5 wt% based on PCL weight). Tetraglycol for selective extraction of PLGA was purchased from Sigma. To extract PLGA from the blended microparticles, microparticles were placed in tetraglycol (5 wt% microparticles in tetraglycol) and maintained for 48 h for complete extraction of PLGA. Microparticles were washed with distilled water three times. Selectively extracted product was first air dried in a hood for 24 h and then vacuum dried for 24 h.

Measurements

Thermal analysis was carried out using differential scanning calorimetry (DSC, Q20, TA Instruments). Heating thermograms were obtained using a heating rate of 5 °C min−1 in the temperature range from −120 °C to 200 °C. Field emission electron scanning microscopy (FE-SEM, S-4800, Hitachi) was used to investigate the morphology of the microparticles. Rhodamine 6G within the microparticles was observed using a confocal fluorescence microscope (LSM510-Meta NLO, Carl Zeiss) equipped with a 1 mW helium–neon laser and a Plan Neofluar 20× objective (NA 0.5). Red fluorescence was observed with a long-pass 560 nm emission filter under 543 nm laser illumination, and variation of the focus enable separate observation of the distribution of the dye in the core and the shell. The water contact angles of the single layers of uniform microparticles are measured using a contact angle analyzer (Phoenix 300 Plus, SEO).

Thermogravimetric analysis (TGA) was conducted to investigate existence of remaining PLGA after selective extraction using a TA instruments TGA Q50. The specimen was heated to 800 °C at a rate of 10 °C min−1 under nitrogen atmosphere.

Results and discussion

In our previous work, we found that monodisperse microparticles could be prepared with a tube-type microfluidic device when the length of tube exceeded 50 cm.17 It seems that some extents of DCM should evaporate from organic droplets, sufficient for maintaining their shape integrity, before they reach the Petri-dish for further solidification. In the present study, microfluidic channels with a length of 1 m were used to prepare various shapes of microparticles from the PLGA/PCL blend as shown in Fig. 1.
image file: c4ra05864j-f1.tif
Fig. 1 Fabrication of various shapes of microparticles from blends of immiscible biocompatible polymers using a tube-type microfluidic device.

PLGA (65/35) is an amorphous biodegradable polymer and PCL is semi-crystalline biodegradable polymer; the blends of these two polymers exhibit immiscibility (DSC thermograms of the PLGA/PCL blend is shown in Fig. S1†). Phase separation is governed kinetically by the evaporation of solvent in oily droplets. Thus, slow evaporation can induce complete phase separation. Unlike conventional methods, such as the dropping method in which microparticles are prepared by formation of droplets in an air atmosphere and subsequent introduction of the droplets into the aqueous media, the microfluidic approach enables droplet formation directly in the immiscible (oily droplet into aqueous media) liquid media. As a result, different shapes of microparticles could be obtained by compositional changes of the blend (Fig. 2).


image file: c4ra05864j-f2.tif
Fig. 2 SEM images of the microparticles with blend ratios (PLGA/PCL) of (a) 7[thin space (1/6-em)]:[thin space (1/6-em)]3, (b) 6.5[thin space (1/6-em)]:[thin space (1/6-em)]3.5, (c) 6[thin space (1/6-em)]:[thin space (1/6-em)]4, and (d) 2[thin space (1/6-em)]:[thin space (1/6-em)]8.

Spherical core–shell type microparticles, where one component exceeds 70% and acorn-shaped microparticles were obtained with the composition variation. Similar shapes of microparticles can be obtained from immiscible non-degradable blends, depending on the value of interfacial energy between the three phases.22 The formation of core–shell, acorn-type, or separated single component microparticles was controlled, in this case, by varying the spread coefficient based on interfacial energy of the three phases. However, our current work differs in that the determination of the shape is governed only by the composition of the blend under the condition of fixed materials.

Interestingly, small dots composed of PLGA are observed on the surface of the microparticle at the PCL region. It was reported that increasing the proportion of PLGA in the range of 10–30% resulted in PLGA filling the voids in the spherical aggregates of PCL region in films prepared from PLGA/PCL blends (1[thin space (1/6-em)]:[thin space (1/6-em)]9, 2[thin space (1/6-em)]:[thin space (1/6-em)]8, and 3[thin space (1/6-em)]:[thin space (1/6-em)]7).18 Similarly, microparticles with low amounts of PLGA (Fig. 2d) did not exhibit crump surface of microparticles of pure PCL,19 but a rather smooth surface.

To investigate the effect of changes in the blend composition on the hydrophobicity of the microparticles, the water contact angle was measured for single layers of spherical microparticles (Fig. 3 and S2†). It is well known that PCL exhibits a more hydrophobic character compared with PLGA. The water contact angles measured for the films composed of microparticles show an increase in hydrophobicity corresponding to the increase in the proportion of PCL.


image file: c4ra05864j-f3.tif
Fig. 3 The relationship between the water contact angles with the microparticle films and the various blend ratios.

The fluorescent dye, rhodamine 6G, was used to investigate how the distribution of PLGA and PCL in the microparticles changes with the blend ratios. Because rhodamine 6G is hydrophobic, it tends to be located in regions containing PCL, which is more hydrophobic than PLGA. The rhodamine 6G distributions in the microparticles were observed using confocal laser scanning microscopy (CLSM).23 As clearly indicated in the Fig. 4, uniform-sized microparticles (Fig. S3, S4 and Table S1†), with different distributions of rhodamine 6G, were observed. The distribution depends on the phase separation of the two biodegradable polymers with different hydrophobicity. As double walled and differentially degradable microparticles have been reported to show potential as a multi-drug release system, where the release of the active agent should differ during the release period,24 it is expected that changing the blend composition in this system can be applied to multi-drug release microparticles.


image file: c4ra05864j-f4.tif
Fig. 4 Confocal fluorescence microscopy images of microparticles with different PLGA/PCL blend ratios, (a) 7[thin space (1/6-em)]:[thin space (1/6-em)]3, (c) 6[thin space (1/6-em)]:[thin space (1/6-em)]4, and (e) 3[thin space (1/6-em)]:[thin space (1/6-em)]7, and the corresponding plots of rhodamine 6G distribution with surface intensity plot, (b), (d) and (f), respectively.

Tetraglycol, which is used as a solvent in parenteral products for intravenous or intramuscular injections (dosage limit of 0.07 mL kg−1 d−1)25 can dissolve PLGA and is miscible with water.26 This solvent has been used as an injectable, in situ forming gel for PLGA, and can be safer than using toxic organic solvents, such as DMSO.27 Selective solvent extraction can be used to prepare Janus particles. Generally an organic solvent that acts as a good solvent for one polymer and a poor solvent for another polymer is used. Because any remaining solvent may influence the toxicity of the prepared particles, the choice of the selective solvent is extremely important. The various forms of microparticles fabricated from PLGA/PCL blends were treated with tetraglycol to selectively remove PLGA. Complete removal of PLGA from the blended microparticles using tetraglycol was confirmed by the disappearance of specific degradation temperature of PLGA in TGA analysis (Fig. S5 and Table S2†).

As a result, different shapes of microparticles were obtained (Fig. 5). When the composition of PLGA/PCL changes to 7[thin space (1/6-em)]:[thin space (1/6-em)]3, 6.5[thin space (1/6-em)]:[thin space (1/6-em)]3.5, 6[thin space (1/6-em)]:[thin space (1/6-em)]4, and 2[thin space (1/6-em)]:[thin space (1/6-em)]8, final microparticle showed dimpled surface, crescent-like, and hollow-core microparticle composed of PCL were obtained (Fig. S6†). This result exactly matches with the image when PLGA region is removed from CLSM investigations. Closer investigation of the microparticles at the surface after selective leaching showed dimpled surface and these are originated from the PLGA dots that are spread over the PCL region in the microparticles from PLGA/PCL blend (Fig. 6). The size and the number of PLGA dots decreased corresponding to the amount of PLGA used in blend microparticles, this feature also reflected to the size and numbers of dimples.


image file: c4ra05864j-f5.tif
Fig. 5 SEM images of the Janus particles obtained from the selective extraction of PLGA. Blend ratio of PLGA/PCL were (a) 7[thin space (1/6-em)]:[thin space (1/6-em)]3, (b) 6.5[thin space (1/6-em)]:[thin space (1/6-em)]3.5, (c) 6[thin space (1/6-em)]:[thin space (1/6-em)]4, and (d) 2[thin space (1/6-em)]:[thin space (1/6-em)]8.

image file: c4ra05864j-f6.tif
Fig. 6 SEM images showing surface morphologies of the microparticles before (left) and after selective extraction of PLGA. (a) and (e) PLGA/PCL = 6[thin space (1/6-em)]:[thin space (1/6-em)]4, (b) and (f) PLGA/PCL = 5[thin space (1/6-em)]:[thin space (1/6-em)]5, (c) and (g) PLGA/PCL = 4[thin space (1/6-em)]:[thin space (1/6-em)]6, and (d) and (h) PLGA/PCL = 2[thin space (1/6-em)]:[thin space (1/6-em)]8.

Conclusion

Various complex-shaped microparticles were fabricated using a microfluidic approach and a biodegradable polymer blend (PLGA/PCL). Phase separation of the two immiscible polymers resulted in different shapes with the change of blend ratio. Further treatment consisting of selective extracting of PLGA using tetraglycol provides a less toxic approach to expanding the possible shapes of microparticles (Fig. 7).
image file: c4ra05864j-f7.tif
Fig. 7 Various particle shapes obtained from blending PLGA (red) and PCL (blue) and the subsequent selective leaching of PLGA from the microparticle.

It is anticipated that the various architectural features obtained by the combination of blending immiscible biodegradable polymer and selective extraction can be applied to various biomedical applications where biodegradable and anisotropic particles are required.

Acknowledgements

This work was supported by the research grant of the Kongju National University in 2013.

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Footnote

† Electronic supplementary information (ESI) available: DSC thermograms of blends and additional images of microparticles are available. See DOI: 10.1039/c4ra05864j
This journal is © The Royal Society of Chemistry 2014
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