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Chih-Hui Yang1 Chih-Yu Wang2 Alexandru Mihai Grumezescu3 Andrew H.-J. Wang1,4 Ching-Ju Hsiao1,5 Zu-Yu Chen1,5 Keng-Shiang Huang5 1 Department
of Biological Science and Technology, I-Shou University, Kaohsiung, Taiwan 2 Department of Biomedical Engineering, I-Shou University, Kaohsiung, Taiwan 3 Department of Science and Engineering of Oxide Materials and Nanomaterials, University Politehnica of Bucharest, Faculty of Applied Chemistry and Materials Science, Bucharest, Romania 4 Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan 5 The School of Chinese Medicine for Post-Baccalaureate, I-Shou University, Kaohsiung, Taiwan
Received April 20, 2014 Revised May 22, 2014 Accepted May 29, 2014
Research Article
Core-shell structure microcapsules with dual pH-responsive drug release function We report dual pH-responsive microcapsules manufactured by combining electrostatic droplets (ESD) and microfluidic droplets (MFD) techniques to produce monodisperse core (alginate)-shell (chitosan) structure with dual pH-responsive drug release function. The fabricated core-shell microcapsules were size controllable by tuning the synthesis parameters of the ESD and MFD systems, and were responsive in both acidic and alkaline environment, We used two model drugs (ampicillin loaded in the chitosan shell and diclofenac loaded in the alginate core) for drug delivery study. The results show that coreshell structure microcapsules have better drug release efficiency than respective core or shell particles. A biocompatibility test showed that the core-shell structure microcapsules presented positive cell viability (above 80%) when evaluated by the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The results indicate that the synthesized core-shell microcapsules were a potential candidate of dual-drug carriers. Keywords: Core-shell / Drug carriers / Drug release / Dual pH-responsive DOI 10.1002/elps.201400210
Additional supporting information may be found in the online version of this article at the publisher’s web-site
1 Introduction The researches on pH-responsive drug carriers were emerging due to their wide applications in recent years [1–3]. With pH-sensitivity, drug delivery would be controllable according to acidic or alkaline environment. For example, the pH-value in microenvironment of tumor tissue is often lower than healthy tissue about 0.5–1.0 units (i.e., mildly acidic than normal tissues) [4], therefore the development of pH-sensitive polymer-based drug carriers for cancer chemotherapy has attracted tremendous attention. In literature, the time and rate of microcapsule contents release can be controlled with trigger pH-value by using pH-responsive polymers and tuned their thickness or ingredient [5]. Cationic polymers (such as chitosan) can swell or degrade to release drugs in the stomach due to the acid gastric juice, while anionic polymers (such as alginate) is able to prevent gastric degradation but is responsive to alkaline pancreatic juice and thus can release weak ba-
Correspondence: Professor Keng-Shiang Huang, The School of Chinese Medicine for Post-Baccalaureate, I-Shou University, Kaohsiung, Taiwan E-mail: [email protected]
Abbreviations: ESD, electrostatic droplet; MFD, microfluidic droplet; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
sic drugs in intestinal and colon to achieve bioavailability [6]. In literature, chitosan coated alginate spheres has been used as a controlled pH/temperature sensitive drug delivery system, and embolization or chemoembolization [7, 8]. Alginate coated chitosan micro/nano- particles were applied for oral delivery drug or vaccine delivery [6,9]. Dual pH-responsive of oral delivery drug can offers effective solution to non-invasive patient administration. Multiple emulsions, in which emulsions enclose smaller emulsions being suspended in a continuous liquid phase, are known as “emulsions of emulsions” [10]. Multiple emulsions have been studied since they were first described in 1925 by Seifriz [11]. They possess extremely valuable therapeutic and biotechnological applications in the food, agricultural, pharmaceutical, and cosmetic industries, in which they facilitate the sustained release and transport of active material [12–14]. However, multiple emulsions are thermodynamically unstable because of the excess free energy associated with the surface of the emulsion droplets [15]. To support the stabilization of these multi-layered dispersions, surfactants are typically incorporated into the intermediate or external phase [16]. Multiple emulsions are facilely manufactured through a 2-step bulk emulsification process [17, 18]. However, this technique cannot easily control the size distribution of the resulting microspheres, and phase inversion techniques have Colour Online: See the article online to view Figs. 1–3 in colour.
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similar limitations [19]. Fabricating multiple emulsions with controlled sizes and internal structures has been a long-term challenge [12,20,21]. Several methods have recently achieved monodispersed single emulsions, which has enabled the fabrication of multiple emulsions with desired sizes, structures, and compositions [22]. These methods include membrane emulsification [23], microchannel emulsification [24], 2D microfluidic emulsification [25], 3D coaxial microcapillary emulsification [26], and electrostatic droplets [27], and others. Shirasu porous glass, for example, is a well-known permeable membrane for membrane emulsification. The coefficient of variation of the droplet diameters is approximately 10%. Two-step membrane emulsification has been proposed for generating multiple emulsions [28]. Microchannel emulsification is an alternative method for generating multiple emulsions, which combines microchannel array devices or the straight-through device with a 2-step emulsification process [29]. Microfluidic droplets (MFD) technology, one of the most widely used methods for constructing size-controlled spheres with a narrow size distribution, have been used to form particles, double emulsions, hollow capsules, and bubbles [12, 30–33]. Various channel designs have been developed, such as T-junction configuration, cross-junction configuration (flow-focusing configuration), microcapillary configuration, and others [34–42]. In fact, the advances of MFD technology help the formation of core-shell microspheres and multi-cored microcapsules [43–45]. Electrostatic droplets technology (ESD) was another approach that can be used to fabricate core-sell particles. By using the Taylor cone and applying voltages, ESD is used in the electrified jetting technique for producing core-shell droplets [46–49]. These methodologies can flexibly construct droplet size, internal structure, and the compositions of multiple emulsions. Each methodology has its own specific advantages, and combining various technologies provided alternative approaches for multiple emulsion synthesis. The objective of present work was to develop chitosan coated alginate core-shell microcapsules with dual pH-responsibility. The combination of the ESD and MFD techniques can generate uniform-size alginate-chitosan coreshell microcapsules. The microcapsules were characterized by morphology, dispersity, and pH-responsibility. We also discuss the influences of operation conditions (e.g., flow rate and apply voltage parameters). Drug loading and triggered release properties were also addressed.
2 Materials and methods 2.1 Experimental setup of the ESD system Supporting Information Figure S1 shows the schematic diagram of the experimental equipment for preparing the alginate particles. We used a syringe pump (KD Scientific KDS230, programmed by a PC) to drive the alginate solution into a needle that was connected to the positive electrode of a direct current power supply (the supplying voltage ranges C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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from 0 to 5 kV). A Petri-dish filled with solidification solution (CaCl2 , 25% w/v water solution) was placed on a metallic plate that was connected to the negative electrode of the power supply. The distance between the tip and the Petri-dish was about 10 cm. The applied high voltage induced a strong electrostatic field between the tip and the Petri-dish on the metallic plate. Positively charged droplets were dragged by the strong electrostatic field, dropped into the solidification solution and formed uniform-sized particles. The system was encased in an insulated acrylic cabinet to prevent accidental contact with any high-voltage sources during the experiment.
2.2 Experimental setup of the MFD system We constructed a cross-junction channel microfluidic chip from a conventional PMMA substrate using a CO2 laser machine (LaserPro Venus, GCC, Taiwan). The fabrication processes are similar to our previous report [50]. The proposed microfluidic chip was laid out on a conventional PMMA substrate (depth: 2 mm) using a CO2 laser machine (M300, Universal Laser System, USA). Fabricating a microchannel on a PMMA substrate by means of a laser machine is similar to R data using a laser printer to print a document. The CAD was sent from a PC to the laser machine and the design was then carried out on the PMMA substrate by the CO2 laser machine [51]. The structure and design are shown in Fig. 1B. We injected the dispersed phase at the center channel, and the continuous phase at the side channel. We simultaneously injected these fluids into the microfluidic chip using syringe pumps (KDS230, KD Scientific).
2.3 Characterization of microcapsules We characterized the collected solidified microcapsules under the optical microscopy system (TE2000U, Nikon, USA). We obtained the diameter of the microcapsules, expressed as mean ± SD, from the photomicrographs and counted an excess of 100 particles to ensure a statistical representation.
2.4 Activity measurement of core-shell microcapsules in various pH conditions We observed the degradation of the core-shell microcapsules using an optical imaging system in pancreatic juice (pH 7.7) and gastric juice (pH 1.3) at room temperature for 9 h and used the pure chitosan, pure alginate, and alginate-chitosan core-shell structure in this study. We recorded the images using image processing software.
2.5 Biocompatibility test We employed human glioblastoma multiforme (GBM) cell lines for a biocompatibility study. We evaluated the viability www.electrophoresis-journal.com
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Figure 1. Illustration for the microfluidic system. (A) Schematic illustrations for the working principle of the synthesis of core-shell droplets using microfluidic chip. (B) Design of the microfluidic chip. (a) A photo image of the microfluidic chip. Scale bar is 1.86 cm. (b) Design of the microfluidic chip. (c) Enlargement of cross-junction channel. The dispersed phase is 1% chitosan in acetic acid add various amount of size and concentrations alginate particles. Continuous phase is sunflower oil.
of the control and treated cells using a 3- (4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay with cells (1 × 104 /well) seeded in 96-well microtitre plates containing a 100 L culture medium (RPMI1640, DMEM), permitted to adhere for 24 h, and washed with PBS. After 24 h treatment, the cells were incubated at 37°C in 200 L MTT solution (1 mg/mL) for 4 h. After removing the medium and the MTT, 100 L DMSO was added to each well, and the assay plate was read at 595 nm using a microplate reader (Thermo Electron, Multiskan Accent). The absorbance of the untreated cells was considered to be 100%.
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3 Results and discussion 3.1 Preparation of the core (alginate)-shell (chitosan) microcapsules After loading in the syringe, sodium-alginate solution (dispersed phase) was driven out from the needle tip with a constant speed. We applied high voltage (0.15, 0.45, 0.6, 0.75, and 1.35 kV, respectively) to the liquid droplets to charge the liquid and split the pendant stream at the tip of the needle using electrostatic repulsion to form a series of isolatable Na-alginate droplets. The Petri-dish was filled with divalent cations (Ca2+ )
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in 25% w/v water solution and subjected to continuous gentle stirring. Sodium-alginate droplets were dropped into the Petri-dish and gelled to form Ca-alginate particles in situ by immersing Ca2+ ions for several minutes, with a shrinking rate from 44 to 59%. We fabricated the core-shell microcapsules using the MFD technique in a cross-junction channel. The dispersed phase, composed of both chitosan solution (1.5% w/v) and alginate particles with various sizes and concentrations in acetic acid, were injected in the central inlet (Fig. 1). Sunflower seed oil (continuous phase) was injected from the side inlet of both sides. The fluids entering through the Teflon tubes were then simultaneously injected into the microfluidic chip using syringe pumps (KDS230, KD Scientific). The flow rate of the dispersed phase and the continuous phase were from 0.03 to 0.06 mL/min and from 0.4 to 0.8 mL/min, respectively. We obtained uniform-sized droplets based on flowfocusing. The cores (alginate particles) contained in the chitosan droplets were then dropped into a solidification solution (20 wt% NaOH) to obtain core-shell structure microcapsules. In our previous studies, we successfully encapsulated several drugs (i.e., insulin, diclofenac, and ampicillin) in alginate or chitosan particles with similar preparation conditions to this study [35, 46, 49] indicating the potential of core-shell microcapsules as drug carriers. However, core-shell microcapsules might be unsuitable for encapsulating biological materials (such as enzymes) because of an acidic or alkaline environment during preparation.
Electrophoresis 2014, 35, 2673–2680 Table 1. The effects of the flow rate of disperse phase and the intensity of electric field on the particle size
Flow rate (mL/h)
Applied voltage (kV)
Particle diameter (m)
RSDa) (%)
Shrinking rateb) (%)
3
0.15 0.45 0.6 0.75 1.35 0.15 0.45 0.6 0.75 1.35 0.15 0.45 0.6 0.75 1.35
1732.5 691.7 441.1 215.8 177.8 1665.2 222.7 206.8 164.1 151.2 1565.3 216.7 210.3 199.1 175.9
0.2 1.5 0.7 1.3 7.4 0.1 1.3 1.9 5.5 1.7 0.5 2.4 6.7 1.3 2.4
59 56 57 57 51 50 48 48 45 45 46 44 47 42 45
5
7
a) The relative standard deviation. b) Particles obtained after solidification. Table 2. The relationships among the diameter of the microcapsules, the flow rate of dispersed phase and the flow rate of continuous phase
Flow rate of disperse phase (mL/min)
Flow rate of continuous phase (mL/min)
Particle diameter (m)
RSD (%)
0.03
0.4 0.5 0.6 0.7 0.8 0.4 0.5 0.6 0.7 0.8 0.4 0.5 0.6 0.7 0.8 0.4 0.5 0.6 0.7 0.8
669 566 530 489 472 703 639 581 528 489 713 627 592 542 534 740 670 626 586 552
0.4 0.9 0.6 0.8 0.2 0.9 0.9 0.7 1.5 1.2 0.7 0.6 1.5 0.9 1.7 0.9 1.1 1 0.9 1.1
3.2 Influences of synthesis parameters on the morphology of core-shell structures 0.04
The applied voltage intensity of the ESD system can be used to control the core size (alginate particles). For applying high voltage varying from 0.15 kV to 1.35 kV, the particle average diameters can be controlled from 1732 to 176 m, with a RSD less than 7.5%, which meets the typical monodispersity criterion (Table 1) [52]. As shown in Supporting Information Fig. S2, the particle diameter shrinks when the disperse flowrate decreases. The diameter of alginate particles dramatically decreases when the electric field changes from 0.15 to 0.45 kV, but the size decreasing became slower from 0.45 to 1.35 kV. We suppose that the larger particles (i.e. made from 0.15 to 0.45 kV) may have lower density and thus were more easily minified than smaller particles (i.e. made from 0.45 to 1.35 kV). Generally speaking, decreasing the applied voltage of the ESD system increases the alginate particle diameter. This trend is similar to previous studies [46–49]. The shear force in the MFD system can be adjusted by the flow-rate ratio between the continuous phase and the dispersed phase [29–42, 46]; therefore, the diameter of the fabricated particles can be tuned by altering the flow rate of the two phases [25]. We observed that the size of the coreshell droplets enlarged with increased flow rate of the dispersed phase, but decreased flow rate of the continuous phase C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
0.05
0.06
(Table 2). We obtained Eqs. (1)–(4) under various flow rates of the continuous and dispersed phase, respectively. D = 1143 − 1287F + 714F 2 , (R2 = 0.996)(dispersed phase 0.06 mL/ min)
(1)
D = 1167 − 1469F + 857F 2 , (R2 = 0.998)(dispersed phase 0.05 mL/ min)
(2)
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Figure 2. Optical microscope images of the chitosan encapsulating alginate microspheres with (A) single particle, (B) dual particles, (C) multiple particles, respectively. The faster of the flow rate of dispersed phase (0.04 0.06 mL/min), the more cores were encapsulated. The continuous phase was kept in 0.5 mL/min.
D = 1060 − 1045F + 443F 2 , (R2 = 0.998)(dispersed phase 0.04 mL/min)
(3)
D = 904 − 874F + 429F 2 , (R2 = 0.998)(dispersed phase 0.03 mL/min)
(4)
D = chitosan-shell diameter (m); F = flow rate of continuous phase (mL/min) In this study, both the ESD technique and the MFD technique both can fabricate uniform-sized particles with controllable diameters by changing the manufacturing conditions. The throughput can be estimated by dividing the flow rate of the dispersed phase into the average volume of the fabricated particles. Based on the calculation in Table 2, the throughput varied from 15 particles/h (0.03 mL/min of the dispersed phase and 0.4 mL/min of the continuous phase) to 55 particles/h (0.06 mL/min of the dispersed phase and 0.8 mL/min of the continuous phase). The throughput can be improved by raising the flow rate of the dispersed phase.
3.3 Various core-shell structures Figure 2 shows numerous alginate cores encapsulated in the chitosan shell. The conditions for fabricating the core-shell structure were described as following. The alginate cores, with average particle diameters of 178 ± 13 m, were fabricated by using a high voltage of 1.35 kV and flow rate of 3 mL/h. The core-shell structure with various encapsulated cores was achieved by changing the flow rate of the continuous and the dispersed phase; increasing the flow rate of the dispersed phase from 0.04 mL/min, through 0.05 mL/min C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 3. The chitosan encapsulating yellow color alginate particles and/or red color alginate particles.
to 0.06 mL/min, encapsulated 1, 2, and more cores (alginate particles) in the chitosan shells (Figs. 2A, B and C). Figure 3 shows different types of cores (i.e., alginate particles dyed with yellow or red color) simultaneously encapsulated in one chitosan shell, suggesting that cores with various functions can be concurrently encapsulated in the chitosan shell. To obtain desired ratio of different color alginate cores encapsulated in chitosan shell, we mixed proper proportions of various color alginate cores in chitosan solution and then synthesize the core-shell structure microcapsules via MFD systems. This approach would be also suitable for encapsulating alginate cores loaded with various types of drugs. Loading droplets with a controllable number of particles or cells is useful for chemical or biological assays in microfluidic devices. However, an uneven number of particles or cells may be encapsulated in the droplets according to Poisson www.electrophoresis-journal.com
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Cumulative release (%)
100
80
60
40
20
0
0
2
4
6
8
10
Time (hour) Figure 5. The drug release patterns of the core-shell structure with various model drugs. •: Ampicillin loaded in chitosan particles. ◦: Diclofenac loaded in alginate particles. : Ampicillin loaded in the chitosan shell of the core-shell particles. :Diclofenac loaded in alginate core of the core-shell particles.
120
Figure 4. Results of the in vitro dissolution of test capsules at (A) pancreatic juice (pH 7.7). (B) Gastric juice (pH 1.32). The scale bars are 500 m.
statistics [53] and channel clogging may cause an uncontrollable number of encapsulated particles [53, 54]. Abate et. al. employed compliant particles with close-packed ordering to solve these problems [53]. Cao et al. applied a simple onelayer microfluidic device, in which droplets with encapsulated particles flowed through a narrow interrogation channel and could be detected with fluorescence [54]. In our study, the relationship between the flow rate of the dispersed phase and the number of encapsulated alginate cores required further study. The structure of various cores with multiple functions provides a versatile and promising platform for encapsulating incompatible actives or chemicals. However, co-encapsulating a specified number and size of multicomponent cores in one shell is not easy to achieve. Wang et al. suggested a hierarchical and scalable microfluidic device constructed by combining three building blocks to generate multi-component multiple emulsions [55]. However, encapsulating multiple functionalized solid cores remains a challenge. Our work provides alternative methods to address this problem.
3.4 Activities of the core-shell particles in various pH environments Figure 4A shows the degradations of chitosan particles (first row), alginate particles (second row), and alginate-chitosan C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Cell Viability (%)
100 80 60 40
20 0
control
67.5
125
250
500
1000
Conc. (µg/mL) Figure 6. Analysis of cell viability.
core-shell particles (third row) immersed in pancreatic juice (pH 7.7) to mimic the conditions in the small intestine. The results show that chitosan particles did not degrade in a weak alkali environment for at least 9 h (first row), whereas the alginate particles quickly degraded in half an hour (second row). The third row shows that the alginate core is well protected by the chitosan shell in an alkaline environment. Figure 4B shows the degradations of chitosan particles (first row), alginate particles (second row), and alginate-chitosan core-shell particles (third row) immersed in gastric juice (pH 1.32) to mimic conditions in the stomach. The chitosan particles rapidly degraded in half an hour (first row), whereas the alginate particles were well preserved in the acid environment (second row). The third row shows that the core-shell particles could not last for more than half an hour in an acidic environment. Chitosan and alginates are 2 types of promising polymers that can be used as drug carriers, but suffer from hydrolysis of their glycosidic bonds in gastrointestinal tract fluids. It is necessary to prevent them from destruction before www.electrophoresis-journal.com
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achieving their destination. The pH-sensitive properties of the core-shell structure can be used to control the drug release process when the carriers pass through the gastrointestinal tract. Rao et al. proposed a chitosan-based pH sensitive micro-network for the controlled release of 5-fluorouracil [56]. Wang et al. presented pH-sensitive magnetic alginate-chitosan beads for albendazole delivery [57]. We synthesized pH-responsive core-shell particles as a positive drug carrier for controlled delivery.
3.5 Drug delivery and cell viability The efficiency of drug delivery for core-shell particles is shown in Fig. 5. We used Ampicillin (loaded in chitosan) and Diclofenac (loaded in alginate) as model drugs in this study and observed that the core-shell particle presented enhanced drug delivery efficiency for Ampicillin loaded in chitosan (Fig. 5). In the first 3 h, the core-shell particles loaded with both the Ampicillin in chitosan shell () or Diclofenac in aliginate core () had better efficiency than that for Diclofenac loaded in only alginate particles (•) or Ampicillin loaded in only chitosan particles (◦). (Fig. 5). The results were similar to some previous studies which have shown that alginatechitosan core-shell particles are efficient for drug delivery and gene transfection [7,9,58]. However, further researches would be required to clarify the exact mechanism of the presented results. We estimated the cytotoxicity using cell viability, using incubating doses (0–1,000 g/mL) of the prepared core-shell microparticles (with an outer diameter of 500 m and an inner diameter of 180 m). The results show positive viability (above 80%) with alginate-chitosan particles below 1000 g/mL (Fig. 6), indicating that the composite microcapsules are a potential candidate for a drug delivery system.
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5 References [1] Jiang, L., Gao, Z.-M., Ye, L., Zhang, A.-Y., Feng, Z.-G., Biomater. Sci. 2013, 1, 1282–1291. [2] Yoshida, T., Lai, T. C., Kwon, G. S., Sako, K., Expert Opin. Drug Deliv. 2013, 10, 1497–1513. [3] Li, Y., Gao, G. H., Lee, D. S., J. Polym. Sci. A: Polym. Chem. 2013, 51, 4175–4182. [4] Pasut, G., Veronese, F. M., Prog. Polym. Sci. 2007, 32, 933–961. [5] Abbaspourrad, A., Datta, S. S., Weitz, D. A., Langmuir 2013, 29, 12697–12702. [6] Bagre, A. P., Jain, K., Jain, N. K., Int. J. Pharm. 2013, 456, 31–40. [7] Shi, J., Alves, N. M., Mano, J. F., J. Biomed. Mater. Res. B, Appl. Biomater. 2008, 84, 595–603. [8] Eroglu, M., Kursaklioglu, H., Misirli, Y., Iyisoy, A., Acar, A., Isin Dogan, A., Denkbas, E. B., J. Microencapsul. 2006, 23, 367–376. [9] Li, X., Kong, X., Shi, S., Zheng, X., Guo, G., Wei, Y., Qian, Z., BMC Biotechnol. 2008, 8, 89. [10] McClements, D. J., Curr. Opin. Colloid Interface Sci. 2012, 17, 235–245. [11] William, S., J. Phys. Chem. 1925, 29, 738–749. [12] Utada, A. S., Lorenceau, E., Link, D. R., Kaplan, P. D., Stone, H. A., Weitz, D. A., Science 2005, 308, 537–541. [13] Gresham, P. A., Barnett, M., Smith, S. V., Schneider, R., Nature 1971, 234, 149–150. [14] Tahara, Y., Kaneko, T., Toita, R., Yoshiyama, C., Kitaoka, T., Niidome, T., Katayama, Y., Kamiya, N., Goto, M., J Control. Release 2012, 161, 713–721. [15] Aserin, A., Multiple Emulsions: Technology and Applications, Wiley-Interscience, Hoboken, NJ 2008. [16] Nisisako, T., Chem. Eng. Technol. 2008, 31, 1091–1098. [17] Matsumoto, S., Kita, Y., Yonezawa, D., J. Colloid. Interface Sci. 1976, 57, 353–361. [18] Pradhan, M., Rousseau, D., J. Colloid. Interface Sci. 2012, 386, 398–404.
4 Concluding remarks
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We proposed core-shell structure microcapsules with dual pH-responsive drug delivery function by combining ESD and MFD technologies. This two-step procedure allowed monodisperse particles in both shells and cores. The fabricated core-shell microcapsules have pH-controlled drug delivery function according to acidic and alkaline environment, and present positive biocompatibility, indicating their potential use in biological and biomedical applications, such as pHresponsive drug-delivery systems, scaffolding for bone tissues, an oral drug-delivery vehicle, and others.
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This work was financially supported by a grant from the National Science Council of Taiwan. The authors would like to thank Mr. Chien-Ming Pan (Department of Biomedical Engineering, IShou University) for his support in experiments. The authors have declared no conflict of interest. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Electrophoresis 2014, 35, 2673–2680
Chih-Hui Yang1 Chih-Yu Wang2 Alexandru Mihai Grumezescu3 Andrew H.-J. Wang1,4 Ching-Ju Hsiao1,5 Zu-Yu Chen1,5 Keng-Shiang Huang5 1 Department
of Biological Science and Technology, I-Shou University, Kaohsiung, Taiwan 2 Department of Biomedical Engineering, I-Shou University, Kaohsiung, Taiwan 3 Department of Science and Engineering of Oxide Materials and Nanomaterials, University Politehnica of Bucharest, Faculty of Applied Chemistry and Materials Science, Bucharest, Romania 4 Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan 5 The School of Chinese Medicine for Post-Baccalaureate, I-Shou University, Kaohsiung, Taiwan
Received April 20, 2014 Revised May 22, 2014 Accepted May 29, 2014
Research Article
Core-shell structure microcapsules with dual pH-responsive drug release function We report dual pH-responsive microcapsules manufactured by combining electrostatic droplets (ESD) and microfluidic droplets (MFD) techniques to produce monodisperse core (alginate)-shell (chitosan) structure with dual pH-responsive drug release function. The fabricated core-shell microcapsules were size controllable by tuning the synthesis parameters of the ESD and MFD systems, and were responsive in both acidic and alkaline environment, We used two model drugs (ampicillin loaded in the chitosan shell and diclofenac loaded in the alginate core) for drug delivery study. The results show that coreshell structure microcapsules have better drug release efficiency than respective core or shell particles. A biocompatibility test showed that the core-shell structure microcapsules presented positive cell viability (above 80%) when evaluated by the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The results indicate that the synthesized core-shell microcapsules were a potential candidate of dual-drug carriers. Keywords: Core-shell / Drug carriers / Drug release / Dual pH-responsive DOI 10.1002/elps.201400210
Additional supporting information may be found in the online version of this article at the publisher’s web-site
1 Introduction The researches on pH-responsive drug carriers were emerging due to their wide applications in recent years [1–3]. With pH-sensitivity, drug delivery would be controllable according to acidic or alkaline environment. For example, the pH-value in microenvironment of tumor tissue is often lower than healthy tissue about 0.5–1.0 units (i.e., mildly acidic than normal tissues) [4], therefore the development of pH-sensitive polymer-based drug carriers for cancer chemotherapy has attracted tremendous attention. In literature, the time and rate of microcapsule contents release can be controlled with trigger pH-value by using pH-responsive polymers and tuned their thickness or ingredient [5]. Cationic polymers (such as chitosan) can swell or degrade to release drugs in the stomach due to the acid gastric juice, while anionic polymers (such as alginate) is able to prevent gastric degradation but is responsive to alkaline pancreatic juice and thus can release weak ba-
Correspondence: Professor Keng-Shiang Huang, The School of Chinese Medicine for Post-Baccalaureate, I-Shou University, Kaohsiung, Taiwan E-mail: [email protected]
Abbreviations: ESD, electrostatic droplet; MFD, microfluidic droplet; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
sic drugs in intestinal and colon to achieve bioavailability [6]. In literature, chitosan coated alginate spheres has been used as a controlled pH/temperature sensitive drug delivery system, and embolization or chemoembolization [7, 8]. Alginate coated chitosan micro/nano- particles were applied for oral delivery drug or vaccine delivery [6,9]. Dual pH-responsive of oral delivery drug can offers effective solution to non-invasive patient administration. Multiple emulsions, in which emulsions enclose smaller emulsions being suspended in a continuous liquid phase, are known as “emulsions of emulsions” [10]. Multiple emulsions have been studied since they were first described in 1925 by Seifriz [11]. They possess extremely valuable therapeutic and biotechnological applications in the food, agricultural, pharmaceutical, and cosmetic industries, in which they facilitate the sustained release and transport of active material [12–14]. However, multiple emulsions are thermodynamically unstable because of the excess free energy associated with the surface of the emulsion droplets [15]. To support the stabilization of these multi-layered dispersions, surfactants are typically incorporated into the intermediate or external phase [16]. Multiple emulsions are facilely manufactured through a 2-step bulk emulsification process [17, 18]. However, this technique cannot easily control the size distribution of the resulting microspheres, and phase inversion techniques have Colour Online: See the article online to view Figs. 1–3 in colour.
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similar limitations [19]. Fabricating multiple emulsions with controlled sizes and internal structures has been a long-term challenge [12,20,21]. Several methods have recently achieved monodispersed single emulsions, which has enabled the fabrication of multiple emulsions with desired sizes, structures, and compositions [22]. These methods include membrane emulsification [23], microchannel emulsification [24], 2D microfluidic emulsification [25], 3D coaxial microcapillary emulsification [26], and electrostatic droplets [27], and others. Shirasu porous glass, for example, is a well-known permeable membrane for membrane emulsification. The coefficient of variation of the droplet diameters is approximately 10%. Two-step membrane emulsification has been proposed for generating multiple emulsions [28]. Microchannel emulsification is an alternative method for generating multiple emulsions, which combines microchannel array devices or the straight-through device with a 2-step emulsification process [29]. Microfluidic droplets (MFD) technology, one of the most widely used methods for constructing size-controlled spheres with a narrow size distribution, have been used to form particles, double emulsions, hollow capsules, and bubbles [12, 30–33]. Various channel designs have been developed, such as T-junction configuration, cross-junction configuration (flow-focusing configuration), microcapillary configuration, and others [34–42]. In fact, the advances of MFD technology help the formation of core-shell microspheres and multi-cored microcapsules [43–45]. Electrostatic droplets technology (ESD) was another approach that can be used to fabricate core-sell particles. By using the Taylor cone and applying voltages, ESD is used in the electrified jetting technique for producing core-shell droplets [46–49]. These methodologies can flexibly construct droplet size, internal structure, and the compositions of multiple emulsions. Each methodology has its own specific advantages, and combining various technologies provided alternative approaches for multiple emulsion synthesis. The objective of present work was to develop chitosan coated alginate core-shell microcapsules with dual pH-responsibility. The combination of the ESD and MFD techniques can generate uniform-size alginate-chitosan coreshell microcapsules. The microcapsules were characterized by morphology, dispersity, and pH-responsibility. We also discuss the influences of operation conditions (e.g., flow rate and apply voltage parameters). Drug loading and triggered release properties were also addressed.
2 Materials and methods 2.1 Experimental setup of the ESD system Supporting Information Figure S1 shows the schematic diagram of the experimental equipment for preparing the alginate particles. We used a syringe pump (KD Scientific KDS230, programmed by a PC) to drive the alginate solution into a needle that was connected to the positive electrode of a direct current power supply (the supplying voltage ranges C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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from 0 to 5 kV). A Petri-dish filled with solidification solution (CaCl2 , 25% w/v water solution) was placed on a metallic plate that was connected to the negative electrode of the power supply. The distance between the tip and the Petri-dish was about 10 cm. The applied high voltage induced a strong electrostatic field between the tip and the Petri-dish on the metallic plate. Positively charged droplets were dragged by the strong electrostatic field, dropped into the solidification solution and formed uniform-sized particles. The system was encased in an insulated acrylic cabinet to prevent accidental contact with any high-voltage sources during the experiment.
2.2 Experimental setup of the MFD system We constructed a cross-junction channel microfluidic chip from a conventional PMMA substrate using a CO2 laser machine (LaserPro Venus, GCC, Taiwan). The fabrication processes are similar to our previous report [50]. The proposed microfluidic chip was laid out on a conventional PMMA substrate (depth: 2 mm) using a CO2 laser machine (M300, Universal Laser System, USA). Fabricating a microchannel on a PMMA substrate by means of a laser machine is similar to R data using a laser printer to print a document. The CAD was sent from a PC to the laser machine and the design was then carried out on the PMMA substrate by the CO2 laser machine [51]. The structure and design are shown in Fig. 1B. We injected the dispersed phase at the center channel, and the continuous phase at the side channel. We simultaneously injected these fluids into the microfluidic chip using syringe pumps (KDS230, KD Scientific).
2.3 Characterization of microcapsules We characterized the collected solidified microcapsules under the optical microscopy system (TE2000U, Nikon, USA). We obtained the diameter of the microcapsules, expressed as mean ± SD, from the photomicrographs and counted an excess of 100 particles to ensure a statistical representation.
2.4 Activity measurement of core-shell microcapsules in various pH conditions We observed the degradation of the core-shell microcapsules using an optical imaging system in pancreatic juice (pH 7.7) and gastric juice (pH 1.3) at room temperature for 9 h and used the pure chitosan, pure alginate, and alginate-chitosan core-shell structure in this study. We recorded the images using image processing software.
2.5 Biocompatibility test We employed human glioblastoma multiforme (GBM) cell lines for a biocompatibility study. We evaluated the viability www.electrophoresis-journal.com
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Figure 1. Illustration for the microfluidic system. (A) Schematic illustrations for the working principle of the synthesis of core-shell droplets using microfluidic chip. (B) Design of the microfluidic chip. (a) A photo image of the microfluidic chip. Scale bar is 1.86 cm. (b) Design of the microfluidic chip. (c) Enlargement of cross-junction channel. The dispersed phase is 1% chitosan in acetic acid add various amount of size and concentrations alginate particles. Continuous phase is sunflower oil.
of the control and treated cells using a 3- (4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay with cells (1 × 104 /well) seeded in 96-well microtitre plates containing a 100 L culture medium (RPMI1640, DMEM), permitted to adhere for 24 h, and washed with PBS. After 24 h treatment, the cells were incubated at 37°C in 200 L MTT solution (1 mg/mL) for 4 h. After removing the medium and the MTT, 100 L DMSO was added to each well, and the assay plate was read at 595 nm using a microplate reader (Thermo Electron, Multiskan Accent). The absorbance of the untreated cells was considered to be 100%.
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3 Results and discussion 3.1 Preparation of the core (alginate)-shell (chitosan) microcapsules After loading in the syringe, sodium-alginate solution (dispersed phase) was driven out from the needle tip with a constant speed. We applied high voltage (0.15, 0.45, 0.6, 0.75, and 1.35 kV, respectively) to the liquid droplets to charge the liquid and split the pendant stream at the tip of the needle using electrostatic repulsion to form a series of isolatable Na-alginate droplets. The Petri-dish was filled with divalent cations (Ca2+ )
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in 25% w/v water solution and subjected to continuous gentle stirring. Sodium-alginate droplets were dropped into the Petri-dish and gelled to form Ca-alginate particles in situ by immersing Ca2+ ions for several minutes, with a shrinking rate from 44 to 59%. We fabricated the core-shell microcapsules using the MFD technique in a cross-junction channel. The dispersed phase, composed of both chitosan solution (1.5% w/v) and alginate particles with various sizes and concentrations in acetic acid, were injected in the central inlet (Fig. 1). Sunflower seed oil (continuous phase) was injected from the side inlet of both sides. The fluids entering through the Teflon tubes were then simultaneously injected into the microfluidic chip using syringe pumps (KDS230, KD Scientific). The flow rate of the dispersed phase and the continuous phase were from 0.03 to 0.06 mL/min and from 0.4 to 0.8 mL/min, respectively. We obtained uniform-sized droplets based on flowfocusing. The cores (alginate particles) contained in the chitosan droplets were then dropped into a solidification solution (20 wt% NaOH) to obtain core-shell structure microcapsules. In our previous studies, we successfully encapsulated several drugs (i.e., insulin, diclofenac, and ampicillin) in alginate or chitosan particles with similar preparation conditions to this study [35, 46, 49] indicating the potential of core-shell microcapsules as drug carriers. However, core-shell microcapsules might be unsuitable for encapsulating biological materials (such as enzymes) because of an acidic or alkaline environment during preparation.
Electrophoresis 2014, 35, 2673–2680 Table 1. The effects of the flow rate of disperse phase and the intensity of electric field on the particle size
Flow rate (mL/h)
Applied voltage (kV)
Particle diameter (m)
RSDa) (%)
Shrinking rateb) (%)
3
0.15 0.45 0.6 0.75 1.35 0.15 0.45 0.6 0.75 1.35 0.15 0.45 0.6 0.75 1.35
1732.5 691.7 441.1 215.8 177.8 1665.2 222.7 206.8 164.1 151.2 1565.3 216.7 210.3 199.1 175.9
0.2 1.5 0.7 1.3 7.4 0.1 1.3 1.9 5.5 1.7 0.5 2.4 6.7 1.3 2.4
59 56 57 57 51 50 48 48 45 45 46 44 47 42 45
5
7
a) The relative standard deviation. b) Particles obtained after solidification. Table 2. The relationships among the diameter of the microcapsules, the flow rate of dispersed phase and the flow rate of continuous phase
Flow rate of disperse phase (mL/min)
Flow rate of continuous phase (mL/min)
Particle diameter (m)
RSD (%)
0.03
0.4 0.5 0.6 0.7 0.8 0.4 0.5 0.6 0.7 0.8 0.4 0.5 0.6 0.7 0.8 0.4 0.5 0.6 0.7 0.8
669 566 530 489 472 703 639 581 528 489 713 627 592 542 534 740 670 626 586 552
0.4 0.9 0.6 0.8 0.2 0.9 0.9 0.7 1.5 1.2 0.7 0.6 1.5 0.9 1.7 0.9 1.1 1 0.9 1.1
3.2 Influences of synthesis parameters on the morphology of core-shell structures 0.04
The applied voltage intensity of the ESD system can be used to control the core size (alginate particles). For applying high voltage varying from 0.15 kV to 1.35 kV, the particle average diameters can be controlled from 1732 to 176 m, with a RSD less than 7.5%, which meets the typical monodispersity criterion (Table 1) [52]. As shown in Supporting Information Fig. S2, the particle diameter shrinks when the disperse flowrate decreases. The diameter of alginate particles dramatically decreases when the electric field changes from 0.15 to 0.45 kV, but the size decreasing became slower from 0.45 to 1.35 kV. We suppose that the larger particles (i.e. made from 0.15 to 0.45 kV) may have lower density and thus were more easily minified than smaller particles (i.e. made from 0.45 to 1.35 kV). Generally speaking, decreasing the applied voltage of the ESD system increases the alginate particle diameter. This trend is similar to previous studies [46–49]. The shear force in the MFD system can be adjusted by the flow-rate ratio between the continuous phase and the dispersed phase [29–42, 46]; therefore, the diameter of the fabricated particles can be tuned by altering the flow rate of the two phases [25]. We observed that the size of the coreshell droplets enlarged with increased flow rate of the dispersed phase, but decreased flow rate of the continuous phase C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
0.05
0.06
(Table 2). We obtained Eqs. (1)–(4) under various flow rates of the continuous and dispersed phase, respectively. D = 1143 − 1287F + 714F 2 , (R2 = 0.996)(dispersed phase 0.06 mL/ min)
(1)
D = 1167 − 1469F + 857F 2 , (R2 = 0.998)(dispersed phase 0.05 mL/ min)
(2)
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Figure 2. Optical microscope images of the chitosan encapsulating alginate microspheres with (A) single particle, (B) dual particles, (C) multiple particles, respectively. The faster of the flow rate of dispersed phase (0.04 0.06 mL/min), the more cores were encapsulated. The continuous phase was kept in 0.5 mL/min.
D = 1060 − 1045F + 443F 2 , (R2 = 0.998)(dispersed phase 0.04 mL/min)
(3)
D = 904 − 874F + 429F 2 , (R2 = 0.998)(dispersed phase 0.03 mL/min)
(4)
D = chitosan-shell diameter (m); F = flow rate of continuous phase (mL/min) In this study, both the ESD technique and the MFD technique both can fabricate uniform-sized particles with controllable diameters by changing the manufacturing conditions. The throughput can be estimated by dividing the flow rate of the dispersed phase into the average volume of the fabricated particles. Based on the calculation in Table 2, the throughput varied from 15 particles/h (0.03 mL/min of the dispersed phase and 0.4 mL/min of the continuous phase) to 55 particles/h (0.06 mL/min of the dispersed phase and 0.8 mL/min of the continuous phase). The throughput can be improved by raising the flow rate of the dispersed phase.
3.3 Various core-shell structures Figure 2 shows numerous alginate cores encapsulated in the chitosan shell. The conditions for fabricating the core-shell structure were described as following. The alginate cores, with average particle diameters of 178 ± 13 m, were fabricated by using a high voltage of 1.35 kV and flow rate of 3 mL/h. The core-shell structure with various encapsulated cores was achieved by changing the flow rate of the continuous and the dispersed phase; increasing the flow rate of the dispersed phase from 0.04 mL/min, through 0.05 mL/min C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 3. The chitosan encapsulating yellow color alginate particles and/or red color alginate particles.
to 0.06 mL/min, encapsulated 1, 2, and more cores (alginate particles) in the chitosan shells (Figs. 2A, B and C). Figure 3 shows different types of cores (i.e., alginate particles dyed with yellow or red color) simultaneously encapsulated in one chitosan shell, suggesting that cores with various functions can be concurrently encapsulated in the chitosan shell. To obtain desired ratio of different color alginate cores encapsulated in chitosan shell, we mixed proper proportions of various color alginate cores in chitosan solution and then synthesize the core-shell structure microcapsules via MFD systems. This approach would be also suitable for encapsulating alginate cores loaded with various types of drugs. Loading droplets with a controllable number of particles or cells is useful for chemical or biological assays in microfluidic devices. However, an uneven number of particles or cells may be encapsulated in the droplets according to Poisson www.electrophoresis-journal.com
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Cumulative release (%)
100
80
60
40
20
0
0
2
4
6
8
10
Time (hour) Figure 5. The drug release patterns of the core-shell structure with various model drugs. •: Ampicillin loaded in chitosan particles. ◦: Diclofenac loaded in alginate particles. : Ampicillin loaded in the chitosan shell of the core-shell particles. :Diclofenac loaded in alginate core of the core-shell particles.
120
Figure 4. Results of the in vitro dissolution of test capsules at (A) pancreatic juice (pH 7.7). (B) Gastric juice (pH 1.32). The scale bars are 500 m.
statistics [53] and channel clogging may cause an uncontrollable number of encapsulated particles [53, 54]. Abate et. al. employed compliant particles with close-packed ordering to solve these problems [53]. Cao et al. applied a simple onelayer microfluidic device, in which droplets with encapsulated particles flowed through a narrow interrogation channel and could be detected with fluorescence [54]. In our study, the relationship between the flow rate of the dispersed phase and the number of encapsulated alginate cores required further study. The structure of various cores with multiple functions provides a versatile and promising platform for encapsulating incompatible actives or chemicals. However, co-encapsulating a specified number and size of multicomponent cores in one shell is not easy to achieve. Wang et al. suggested a hierarchical and scalable microfluidic device constructed by combining three building blocks to generate multi-component multiple emulsions [55]. However, encapsulating multiple functionalized solid cores remains a challenge. Our work provides alternative methods to address this problem.
3.4 Activities of the core-shell particles in various pH environments Figure 4A shows the degradations of chitosan particles (first row), alginate particles (second row), and alginate-chitosan C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Cell Viability (%)
100 80 60 40
20 0
control
67.5
125
250
500
1000
Conc. (µg/mL) Figure 6. Analysis of cell viability.
core-shell particles (third row) immersed in pancreatic juice (pH 7.7) to mimic the conditions in the small intestine. The results show that chitosan particles did not degrade in a weak alkali environment for at least 9 h (first row), whereas the alginate particles quickly degraded in half an hour (second row). The third row shows that the alginate core is well protected by the chitosan shell in an alkaline environment. Figure 4B shows the degradations of chitosan particles (first row), alginate particles (second row), and alginate-chitosan core-shell particles (third row) immersed in gastric juice (pH 1.32) to mimic conditions in the stomach. The chitosan particles rapidly degraded in half an hour (first row), whereas the alginate particles were well preserved in the acid environment (second row). The third row shows that the core-shell particles could not last for more than half an hour in an acidic environment. Chitosan and alginates are 2 types of promising polymers that can be used as drug carriers, but suffer from hydrolysis of their glycosidic bonds in gastrointestinal tract fluids. It is necessary to prevent them from destruction before www.electrophoresis-journal.com
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achieving their destination. The pH-sensitive properties of the core-shell structure can be used to control the drug release process when the carriers pass through the gastrointestinal tract. Rao et al. proposed a chitosan-based pH sensitive micro-network for the controlled release of 5-fluorouracil [56]. Wang et al. presented pH-sensitive magnetic alginate-chitosan beads for albendazole delivery [57]. We synthesized pH-responsive core-shell particles as a positive drug carrier for controlled delivery.
3.5 Drug delivery and cell viability The efficiency of drug delivery for core-shell particles is shown in Fig. 5. We used Ampicillin (loaded in chitosan) and Diclofenac (loaded in alginate) as model drugs in this study and observed that the core-shell particle presented enhanced drug delivery efficiency for Ampicillin loaded in chitosan (Fig. 5). In the first 3 h, the core-shell particles loaded with both the Ampicillin in chitosan shell () or Diclofenac in aliginate core () had better efficiency than that for Diclofenac loaded in only alginate particles (•) or Ampicillin loaded in only chitosan particles (◦). (Fig. 5). The results were similar to some previous studies which have shown that alginatechitosan core-shell particles are efficient for drug delivery and gene transfection [7,9,58]. However, further researches would be required to clarify the exact mechanism of the presented results. We estimated the cytotoxicity using cell viability, using incubating doses (0–1,000 g/mL) of the prepared core-shell microparticles (with an outer diameter of 500 m and an inner diameter of 180 m). The results show positive viability (above 80%) with alginate-chitosan particles below 1000 g/mL (Fig. 6), indicating that the composite microcapsules are a potential candidate for a drug delivery system.
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4 Concluding remarks
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We proposed core-shell structure microcapsules with dual pH-responsive drug delivery function by combining ESD and MFD technologies. This two-step procedure allowed monodisperse particles in both shells and cores. The fabricated core-shell microcapsules have pH-controlled drug delivery function according to acidic and alkaline environment, and present positive biocompatibility, indicating their potential use in biological and biomedical applications, such as pHresponsive drug-delivery systems, scaffolding for bone tissues, an oral drug-delivery vehicle, and others.
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This work was financially supported by a grant from the National Science Council of Taiwan. The authors would like to thank Mr. Chien-Ming Pan (Department of Biomedical Engineering, IShou University) for his support in experiments. The authors have declared no conflict of interest. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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