Layer-by-Layer Polyelectrolyte–Polyester Hybrid Microcapsules for

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Layer-by-Layer Polyelectrolyte−Polyester Hybrid Microcapsules for Encapsulation and Delivery of Hydrophobic Drugs Rongcong Luo,† Subbu S. Venkatraman,*,† and Björn Neu‡ †

School of Materials Science and Engineering, Nanyang Technological University, 639798, Singapore School of Chemical and Biomedical Engineering, Nanyang Technological University, 639798, Singapore

‡

S Supporting Information *

ABSTRACT: A two-step process is developed to form layerby-layer (LbL) polyelectrolyte microcapsules, which are able to encapsulate and deliver hydrophobic drugs. Spherical porous calcium carbonate (CaCO3) microparticles were used as templates and coated with a poly(lactic acid-co-glycolic acid) (PLGA) layer containing hydrophobic compounds via an in situ precipitation gelling process. PLGA layers that precipitated from N-methyl-2-pyrrolidone (NMP) had a lower loading and smoother surface than those precipitated from acetone. The difference may be due to different viscosities and solvent exchange dynamics. In the second step, the successful coating of multilayer polyelectrolytes poly(allylamine hydrochloride) (PAH) and poly(styrene sulfonate) (PSS) onto the PLGA coated CaCO3 microparticles was confirmed with AFM and ζ-potential studies. The release of a model hydrophobic drug, ibuprofen, from these hybrid microcapsules with different numbers of PAH/PSS layers was investigated. It was found that the release of ibuprofen decreases with increasing layer numbers demonstrating the possibility to control the release of ibuprofen with these novel hybrid microcapsules. Besides loading of hydrophobic drugs, the interior of these microcapsules can also be loaded with hydrophilic compounds and functional nanoparticles as demonstrated by loading with Fe3O4 nanoparticles, forming magnetically responsive dual drug releasing carriers.

1. INTRODUCTION The encapsulation of therapeutic compounds in polymeric materials is an effective way to prevent their degradation and to control their release and thus not only to maximize the efficiency but also to reduce adverse side-effects.1−3 A simple yet highly versatile drug encapsulation technique is to form a polymeric multilayer microcapsule via the so-called layer-bylayer (LbL) assembly.4−7 These microcapsules are generated by sequential deposition of polymer layers onto a sacrificial template.4,5 Therapeutic compounds can be incorporated either in the microcapsule cavity or within the multilayer shells.6,7 Depending on the polymer nature, the LbL formation is facilitated via electrostatic interactions, hydrogen bonding or linkage via covalent bonds.8−10 LbL microcapsules have several unique advantages as compared to other microencapsulation techniques such as the simplicity of the assembly process, the possibility to easily control the size and shape of the capsules, the wall permeability, stability, and surface functionality of the capsules, and the possibility to fabricate multifunctional carrier systems.11,12 Despite the advantages of LbL polyelectrolyte microcapsules, the encapsulation of hydrophobic drugs into LbL polyelectrolyte microcapsules remains challenging, which is due to the fact that LbL microcapsules fabrication and loading is usually carried in aqueous solutions.13−15 Poorly water-soluble drugs or hydrophobic drugs are a heterogeneous group of molecules that exhibit poor water solubility but that are typically are soluble in various organic solvents.16 It was reported that more than 40% © 2013 American Chemical Society

of new pharmacologically active compounds identified through screening of combinatorial libraries have poor water solubility.17 Consequently, the encapsulation of hydrophobic substances continues to be an important topic for scientists and pharmaceuticals companies.18−20 To date, four main strategies have been proposed to incorporate and deliver hydrophobic drugs using LbL polyelectrolyte microcapsules. The first method is coating multilayer polyelectrolytes onto oil/water (O/W) emulsion microsized droplet where the hydrophobic drugs are dissolved.21,22 The second approach is embedding hydrophobic drugs loaded micelle or liposomes subunits into the LbL polyelectrolyte microcapsules interiors.23,24 The third technique is to infiltrate solution of hydrophobic drugs in volatile organic solvent into mesoporous silica microparticle. The organic solvents are then evaporated, and the drug-loaded mesoporous silica microparticles are subsequently coated with polyelectrolytes and decomposed to yield hydrophobic drugs loaded microcapsules.25 Finally, modified polyelectrolytes such as quaternized chitosan derivatives and decylaminohydrazide (C10 chain)-modified hyaluronic acid (HA), which can form stable complex with hydrophobic compounds were developed and used as a layer of the LbL polyelectrolyte microcapsules.26 However, there are some limitations associated with these Received: March 16, 2013 Revised: May 6, 2013 Published: May 21, 2013 2262

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entire treatment period. It has been proven that the codelivery system suppressed cancer growth more efficiently than the single drug system.35 Compared to other codelivery systems, these polyelectrolyte/PLGA hybrid microcapsule-based carriers offer some advantages, as they are simpler to fabricate, easier to modulate their size, shape, surface functionality/charge, compositions, and release behaviors, require no harsh reaction conditions and toxic chemicals, and exhibit ease of multifunctionality. They may find applications in synergetic drug delivery systems and as multifunctional carriers.

methods such as difficulty of handling soft liquified emulsion cores, which are easily broken, the necessity of an oil phase, the cytotoxicity of micelles, or the harsh solvents needed.21−26 On the other hand, copolymers of lactic acid and glycolic acid (PLGA) have been used widely as carrier systems for hydrophobic drugs.27−29 Such systems have a number of unique features such as biodegradability, biocompatibility, FDA (U.S. Food and Drug Administration) approval, commercial availability, and their hydrophobic nature, which allows carrying various hydrophobic drugs with a high loading capacity.27−29 Taking advantage of the above features of PLGA, it has been frequently used as a hydrophobic moiety to harbor hydrophobic drugs in some hydrophobic/hydrophilic dual drug codelivery systems. One typical example is the poly(ethylene glycol)-co-PLGA (PEG-PLGA) amphiphilic copolymer nanoparticles where the PLGA block acts as hydrophobic part to load paclitaxel.30 Besides acting as hydrophobic block in the amphiphilic copolymer, PLGA has also been designed as solid hydrophobic drugs loaded depots in various forms such as particulates, thin films, and hollow fibers.31 Among the encapsulation techniques of hydrophobic drug in PLGA, the in situ precipitating depot systems are attractive, since they can reduce the manufacturing costs and complexity as compared to conventional methods such as emulsion-based, melting-based or spraying drying techniques.32,33 In order to form a PLGA in situ depot delivery system, PLGA and the hydrophobic drugs are dissolved in water-miscible solvents such as N-methyl-2-pyrrolidone (NMP), dimethyl sulfoxide (DMSO), or acetone. Upon contact of the solution with water, the water-miscible solvents diffuse away from the polymer solution and the polymer precipitate to form a solid matrix in which the hydrophobic drugs are incorporated.32,33 In this study, PLGA/polyelectrolyte hybrid microcapsules where hydrophobic substances (e.g., ibuprofen (IBU), Nile Red, Coumarin-6) were loaded in PLGA matrix with further polyelectrolyte coating to impart them with controllable release ability were formed. Furthermore, a magnetically responsive, hydrophilic−hydrophobic dual-drug delivery vehicle can also be conveniently formed by filling the capsule interior cavity with hydrophilic substances and Fe3O4 nanoparticles via incorporating them into a CaCO3 template beforehand, and thus multifunctional microcapsules can be developed. The polyelectrolyte/PLGA hybrid microcapsules may find applications for hydrophilic/hydrophobic dual drug codelivery systems. Beneficial features of the dual drug codelivery system are its synergistic effects, ability to suppress drug resistance, and ability to tune the relative dosage of both drugs in a single integrated carrier. For example, Thakur and co-workers formed an electrospun mat loaded with an anesthetic, lidocaine hydrochloride (hydrophilic drug), and an antibiotic, mupirocin (hydrophobic drug) to treat wounds. They found that the dual drug codelivery system had synergistic effects where one is to relieve the patient’s pain and the other is to kill the bacteria.34 For many other clinical cases, especially for cancer treatments, combined therapy with hydrophilic/hydrophobic drugs having different therapeutic effects proves to be an effective way. One unique example is the codelivery of hydrophobic anticancer drugs and hydrophilic DNA using poly(ε-caprolactone)-graf t-polyethylenimine micelles. One important feature of this system is its ability to circumvent the multidrug resistance (MDR) problem. This is achieved through the simultaneously delivered DNA in the system which can keep targeted cells sensitive to the anticancer drug during the

2. EXPERIMENTAL SECTION 2.1. Materials. Poly(DL-lactide-co-glycolide) 53:47 (PLGA) (IV = 1.05, Mw/Mn = 1.88) was obtained from Purac Biochem (Gorinchem, Netherlands) and used as received. Poly(allylamine hydrochloride) (PAH) (average Mw ∼ 58 000), poly(styrene sulfonate) (PSS) (average Mw ∼ 70 000), IBU, Nile Red, Coumarin-6, and fluorescein isothiocyanate labeled dextran (FITC-dextran) (average Mw ∼4000), iron oxide (II, III) (Fe3O4) magnetic nanoparticles (5 nm average particles size) were purchased from Sigma-Aldrich. 2.2. Methods. 2.2.1. Coating CaCO 3 with PLGA Layer Containing Hydrophobic Compounds. Hydrophobic compounds such as Nile Red, Coumarin-6 and IBU are dissolved in NMP or acetone where PLGA is also dissolved. Two milliliters of PLGA/ hydrophobic compounds NMP or acetone solution is incubated with 10 mg of CaCO3 microparticles under stirring for ∼1 h. Subsequently, the CaCO3 microparticles are separated from the solution by centrifugation at 4000 × g for 1 min. The microparticles are then washed with NMP or acetone one time and separated by centrifugation at 4000 × g for 1 min. The obtained CaCO3 microparticles are immediately mixed with 1 mL 0.02% PVA aqueous solution by a vortex mixer for 1−2 min. Due to the diffusion of NMP or acetone to the aqueous solution, PLGA precipitates to form a solidified matrix containing the hydrophobic compounds coating onto the surface of CaCO3 microparticles. The PLGA coated CaCO3 microparticles are separated from the solution by centrifugation at 4000 × g for 1 min and then washed with water. The centrifugation and washing process are repeated two times. The obtained PLGAcoated CaCO3 microparticles are then used as a template for further PAH/PSS coating. In order to produce PLGA microcapsules, the CaCO3 microparticles templates can be removed by HCl. Briefly, the obtained PLGA-coated CaCO3 microparticles are mixed with 2 mL 0.1 M HCl for 2 min, HCl can dissolve the CaCO3 microparticles into CO2 and H2O. The PLGA microcapsules are separated from the supernatant by centrifugation at 6000 × g for 5 min and washed with water two times to remove the residual HCl. The ζ-potential measurement of bare and PLGA coated CaCO3 microparticles is carried out using a Malvern Zeta-sizer (Malvern Zetasizer Nano. UK). The bare or coated CaCO3 microparticles are suspended in 0.5 M NaCl (pH = 6.5) and then subjected to ζ-potential testing. The measured electrophoretic mobilities are converted into ζpotential using the Smoluchowski relationship. 2.2.2. Coating Polyelectrolytes (PAH/PSS) onto PLGA-Coated CaCO3 Microparticles. The above obtained CaCO3 microparticles covered with solidified PLGA layer containing hydrophobic compounds are further coated with multilayer polyelectrolytes. For the coating, the microparticles are added to PSS solution (1 mg/mL in 0.5 M NaCl) for 10 min at room temperature. Subsequently, the microparticles are separated by centrifugation and washed with 0.5 M NaCl twice. Following that, the microparticles are coated with PAH with the same conditions used for PSS coating. LbL consecutive adsorption of PAH and PSS is carried out by repeating these steps in sequence until the desired layer number is achieved. In order to produce the hollow polyelectrolyte/PLGA hybrid microcapsules, the CaCO3 microparticles templates can be removed by HCl. The washed polyelectrolyte/PLGA coated CaCO3 microparticles are mixed with 2 mL 0.1 M HCl for 2 min. The hybrid microcapsules are separated from the supernatant by centrifugation at 6000 × g for 5 min. The HCl dissolution and centrifugation steps are repeated two to three times. 2263

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Scheme 1. Schematic Illustration Showing the Preparation of Multifunctional Hybrid Microcapsules via Combining the PLGA in Situ Precipitation Technique and LbL Polyelectrolyte Depositiona

a

(I) NMP or acetone solution containing PLGA and hydrophobic compounds infiltrated onto CaCO3 template. (II) The loaded microparticles are exposed to H2O, which results in precipitation of PLGA containing hydrophobic compounds. (III) Further LbL polyelectrolyte coating to control the compounds release profiles. (IV) CaCO3 template cores were removed to produce hollow capsules. (V) Interior of the capsules can also be prefilled with hydrophilic compounds (FITC-dextran, denoted in green) and inorganic nanoparticles (Fe3O4 denoted by black dots) via encapsulating these substances into CaCO3 template core beforehand, thus producing multifunctional microcapsules. Finally, the obtained microcapsules are washed with water and separated by centrifugation. 2.2.3. Loading of FITC-Dextran and Fe3O4 into the Hybrid Microcapsules. FITC-dextran is preloaded into the CaCO3 microparticles templates using cocrystallization method during the formation process. Briefly, 10 mL 0.33 M CaCl2 dissolved in 4 mg FITC-dextran is quickly poured into 10 mL of 0.33 M Na2CO3 solution. The solutions are mixed and vigorously stirred using a magnetic stirrer for 2 min. White precipitation is immediately formed, indicating the formation of CaCO3 microparticles. The precipitates are then separated from the supernatant by centrifugation at 4000 × g for 1 min. The obtained microparticles are washed with water two times, ethanol one time, and then dried under vacuum. The washed particles are stored in dried conditions at 4 °C for further use. 5 mg of 5−10 nm Fe3O4 nanoparticles were suspended in 2 mL 0.01 mg/mL PSS solution and then incubated with 10 mg dried porous CaCO3 microparticles for 1h at room temperature. The mixture was then centrifuged at 1000 × g for 1 min to settle down the Fe3O4 attached CaCO3 microparticles. The CaCO3 microparticles were then washed with DI H2O two times and centrifuged to discard the supernatant. 2.2.4. Drug release from the hybrid microcapsules. The release behaviors of IBU from the hybrid microcapsules are carried out in 2 mL of phosphate buffered saline (PBS; pH 7.2) at 37 °C under constant shaking. After defined time intervals, the samples were centrifuged, and the supernatant was collected and replaced by fresh PBS. The drug release amounts are analyzed using UV spectroscopy. FITC-dextran release is determined by using fluorescence spectrometer (Infinite 200, Tecan, Switzerland) with 490 nm and 520 nm as the excitation and emission wavelength, respectively. Loading amounts of IBU are determined by the following. Briefly, the CaCO3 microparticles coated with IBU-loaded PLGA are removed using HCl as the above-mentioned method. The PLGA microcapsules are then collected and dissolved with 1 mL acetone to release the total IBU. The total IBU amounts are determined by UV spectroscopy. The

loading amount of FITC-dextran is determined by using 1 mL 0.1 M HCl to dissolve the FITC-dextran-loaded CaCO3 microparticles. 2.2.5. Microscopic Characterization. Confocal Laser Scanning Microscope (CLSM). A CLSM (Leica LAS Singapore) was used to characterize NMP or acetone microcapsule and PLGA−polyelectrolyte hybrid microcapsules. These microcapsules dyed with fluorescent agents were suspended in H2O, and the suspension was transferred to a chamber slides and viewed under a CLSM. Scanning Electron Microscope (SEM). SEM (JEOL JSM-6340F, JEOL Ltd., Japan) was used to study the morphology of NMP or acetone PLGA microcapsules. One drop of NMP or acetone PLGA microcapsules suspension was placed onto clean silicon wafers, dried, coated with Pt, and finally observed under a 5 keV accelerating voltage. Atomic force microscope (AFM). AFM was used to investigate the surface topography, capsule wall thickness, and capsule wall roughness. One drop of the capsule suspension was placed onto a clean silicon wafer and dried prior to imaging. Tapping-mode AFM was used to obtain the images. For surface roughness analysis, the arithmetic average roughness Ra was calculated according to the equipment attached software. For the capsule wall thickness determination, the capsule single-wall thickness was determined as half of the height of the collapsed flat regions on dried capsules according to the previous research.

3. RESULTS AND DISCUSSION In order to encapsulate hydrophobic drugs into LbL microcapsules, PLGA, which is a good candidate for hydrophobic drug encapsulation, is introduced here. By combining the PLGA in situ gelling technique and LbL polyelectrolytes deposition, hybrid LbL polyelectrolyte microcapsules that are capable of loading hydrophobic drugs were formed (see Scheme 1). 2264

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3.1. Formation of Porous CaCO3 Microparticles. Generally, the direct mixing of soluble salt of Ca2+ and CO32− results in an amorphous nanoprecipitate instantly, which will transform into aggregated CaCO3 microcrystals. The CaCO3 microcrystals have three morphologies: calcite, aragonite, and vaterite. Only vaterites are suitable templates for forming LbL polyelectrolyte microcapsules due to their positive surface charge and spherical shape.33 In this study, a simple and reproducible procedure for preparing spherical CaCO3 microparticles (vaterite) developed by Volodkin et al. was used.36 In this method, the amorphous nanoprecipitate (CaCO3 nanoparticles) forms instantly when mixing CaCl2 and Na2CO3 solution, which will immediately transform into spherical CaCO3 microparticles due to the nanoparticles aggregation. SEM observation indicates that CaCO3 microparticles have a size of about 3−5 μm (Figure S1a, Supporting Information), and that CaCO3 microparticles are formed by the assembly of CaCO3 nanoparticles (Figure S1b), resulting in a rough surface with interconnected channels and interspaces. 3.2. Coating Hydrophobic Compound-Loaded PLGA onto CaCO3 Microparticles. 3.2.1. Changes of Surface Potential. The bare CaCO3 microparticles have a positive surface potential of about +16 mV (Figure 1). It changes to −3

template removal (Figure 2a,b), demonstrating that CaCO3 microparticles were successfully coated with PLGA, which is either solidified from acetone or NMP solution. These welldispersed hollow PLGA microcapsules produced via in situ gelling of PLGA on the surface of CaCO3 microparticles provides an alternative way to manufacture hollow PLGA microcapsules. Traditionally, hollow PLGA microcapsules were produced by an emulsification/solvent evaporation method where a liquid oil core is necessary. 3.2.3. SEM and AFM Investigation. SEM was also used to investigate the surface morphology of these PLGA microcapsules in more detail (Figure 3). As can be seen, these PLGA microcapsules have less folds and creases than the traditional LbL polyelectrolyte microcapsules. The possible reason is that PLGA has stronger mechanical strength than polyelectrolyte, which can resist the collapse caused by the template removal. The enlarged view shows that PLGA microcapsules formed from NMP have a less porous surface (Figure 3a,b) while those solidified from acetone exhibit a spongy surface with pores (Figure 3c,d). The AFM images confirm these observations (Figure 4a,b). The mean roughness analysis by AFM implies that PLGA microcapsules solidified from acetone solution have a more porous surface (8−12 nm) than those formed from NMP solution (3−5 nm), indicating the porous nature of acetone PLGA microcapsules (Figure 4b). The morphology difference can be explained by phase inversion dynamics, which is largely affected by the solvent quality. NMP has a viscosity of 1.7 cP, while that for acetone is only 0.32 cP. Furthermore, it has been reported that the diffusion coefficient of water to NMP (Dwater‑NMP) is 18 cm2/s, which is much lower than that of acetone, having a Dwater‑acetone of up to 88.6 cm2/s. Consequently, PLGA NMP solution will induce slower water exchange than PLGA acetone solution.37 Upon contact with water, lower water influx rates and lower PLGA gelation rates can be expected for the PLGA NMP system, giving NMP PLGA microcapsules decreasing structure porosity (Figure 4a). 3.2.4. TGA Measurement. TGA was used to investigate the weight of PLGA coated onto CaCO3 microparticles after their solidification from NMP or acetone solution (Figure 5). Figure 5a shows the thermal degradation profiles of pure PLGA and pure CaCO3 microparticles. It was found that pure PLGA starts to degrade at about 250 °C and finish the degradation at 350 °C with no residues left (Figure 5a). By contrast, CaCO3 microparticles have a higher degradation temperature, where they start to degrade at about 650 °C. Consequently, the PLGA-coated CaCO3 microparticles should have a two-stage degradation profile under TGA analysis, where the first degradation temperature is for PLGA, while the second one is for CaCO3 microparticles. The TGA analysis again confirms the successful coating of PLGA onto CaCO3 either from acetone or NMP (Figure 5b 5c). A typical two-stage degradation profile was observed for both cases (Figure 5b,c). Through calculating the weight difference on the first stage of the TGA curve, the weight of PLGA coated onto CaCO3 after their solidification can be obtained. For CaCO3 microparticles coated with PLGA solidified from acetone solution, a 4.5% coating amount of PLGA can be obtained (Figure 5b). On the other hand, a 2.1% coating amount of PLGA was obtained for PLGA solidified from NMP solution (Figure 5c). The weight difference proves that more PLGA can be coated onto CaCO3 microparticles

Figure 1. ζ-Potential values for bare CaCO3 microparticles, PLGAcoated CaCO3 microparticles, and hollow PLGA microcapsules where CaCO3 microparticle template cores are dissolved.

mV (PLGA solidified from acetone solution) and −2 mV (PLGA solidified from NMP solution) after being covered by solidified PLGA film (Figure 1). Since PLGA is a neutral or slightly negatively charged polymer, the ζ-potential changes reflected here indicate the successful coating of a PLGA layer onto the surface of CaCO3 microparticles. Furthermore, the slightly negative ζ-potential values were also maintained even after the CaCO3 template cores are dissolved (Figure 1). These results suggest that the PLGA microcapsules remain integrated after the dissolution of CaCO3 template cores. 3.2.2. CLSM Observation. In order to further confirm the successful coating of PLGA onto the surface of CaCO3 and the integrality of the PLGA microcapsules after template removal, a CLSM was used to investigate the PLGA microcapsules suspension. To investigate the microcapsules via CLSM, a hydrophobic green fluorescent dye, Coumarin-6, was added into the PLGA acetone or NMP solution. It was found that spherical and integrated green microcapsules were observed after the CaCO3 2265

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Figure 2. CLSM observation of Coumarin-6-loaded PLGA microcapsules: (a) PLGA microcapsules solidified from acetone; (b) PLGA microcapsules solidified from NMP.

Figure 3. SEM observation of surface morphology for the PLGA microcapsules solidified from acetone or NMP solution: (a) PLGA microcapsules solidified from NMP solution; (b) enlarged view of single PLGA microcapsules solidified from NMP solution; (c) PLGA microcapsules solidified from acetone solution; (d) enlarged view of single PLGA microcapsules solidified from acetone solution.

within the PLGA layer. As illustrated in Scheme 1, an LbL coating of polyelectrolytes (PAH/PSS) was added onto the PLGA-coated CaCO3 templates in order to control the release behavior and to produce LbL microcapsules. ζ-Potential measurments were initially used to study the polyelectrolytes layering process (Figure 6). The PLGA coated CaCO3 microparticles have a slightly negative charge (−4 mV), possibly resulting from the ionization of −COOH groups in the PLGA layers.38 After depositing the first positive polyelectrolyte

when the PLGA are solidified from acetone solution. This is because of the different viscosity between PLGA acetone and NMP solution. PLGA NMP solution has a higher viscosity than PLGA acetone solution. The higher viscosity solution is more difficult to be infiltrated and immobilized into porous materials. 3.3. LbL Coating of Polyelectrolytes onto the PLGA Coated CaCO3 Microparticles. The above results fully confirm the coating of the PLGA layer onto CaCO 3 microparticles, where hydrophobic compounds can be loaded 2266

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Figure 4. AFM investigations of surface morphology and surface roughness for the PLGA microcapsules: (a) PLGA microcapsules solidified from NMP solution; (b) PLGA microcapsules solidified from acetone solution; (c) mean roughness of a PLGA microcapsule solidified from NMP and acetone solution, respectively. The data points represent mean ± standard deviation of date from three positions of the microcapsules wall.

the red capsule shell (Figure S2a,d). On the other hand, the FITC-PAH/PSS coating was observed being coated closely onto NMP or acetone PLGA microcapsules as the green capsule shell (Figure S2b,e). The overlay pictures demonstrated that the polyelectrolyte−PLGA hybrid microcapsules were successfully formed (Figure S2c,f). 3.4. IBU Release from the Hybrid Microcapsules. IBU was used as a model hydrophobic substance to investigate the release kinetics of these hybrid microcapsules (Figure 8). IBU is a nonsteroidal anti-inflammatory drug, which also exhibits antipyretic and analgesic properties. Due to its hydrophobic nature, which limits its further applications, microencapsulation of IBU is proposed to be an effective way to enhance their solubility, bioavailability, and maintain their blood level to be therapeutically active without overdose.39 A loading amount of 2.9% was achieved for acetone PLGA microcapsules and 2.2% for NMP PLGA microcapsules. The encapsulation efficiency for acetone PLGA microcapsules is 25.3%, while that for NMP PLGA microcapsules is 18.5%. Loading amounts and encapsulation efficiency in our system currently is relatively

layer (PAH), the negative surface potential reversed to +10 mV. The positive potential reversed again to −20 mV after the subsequent PSS coating (Figure 6). The ζ-potential studies suggest the successful coating of PAH/PSS onto the PLGAcoated CaCO3 templates. From the AFM observations in Figure 4, it is known that PLGA microcapsules solidified from acetone have a more porous surface than the ones from NMP solution (Figure 4). After coating eight layers of PAH/PSS, the pores on the surface of PLGA microcapsules disappear, and the surface topographies of these hybrid microcapsules are quite similar (Figure 7). The surface of these PLGA/polyelectrolyte hybrid microcapsules appears with tight, flat, and continuous morphology (Figure 7). CLSM observation was also used to confirm the PAH/PSS coating onto the PLGA layer (Figure S2). For CLSM observation, another hydrophobic red fluorescent dye, Nile Red, was loaded into the NMP or acetone PLGA layer coated onto the CaCO3 template. The coating of PAH/PSS was indicated by the FITC-PAH (FITC-PAH/PSS). NMP or acetone PLGA layers were successfully formed as implied from 2267

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Figure 5. Weight analysis of PLGA coated onto CaCO3 microparticles with TGA: (a) TGA degradation profiles for pure PLGA and pure CaCO3; (b) TGA degradation profile of CaCO3 coated with PLGA solidified from acetone solution; (c) TGA degradation profile of CaCO3 coated with PLGA solidified from NMP solution.

low due to the less efficient adsorption of PLGA solution onto the surface of CaCO3 microparticles, even when we increased the added amounts of IBU. We may enhance the loading through increasing the porosity of CaCO3 microparticles to absorb more PLGA solution, using smaller molecular weights of PLGA and application of pressure, which can facilitate the adsorption of PLGA onto the CaCO3 microparticles. For both uncoated NMP and acetone microcapsules, a burst release of about 90% IBU was observed within the first 5 h (Figure 8a,b). After coating two layers of PAH/PSS, the burst release was reduced to around 60%. With further coating of PAH/PSS to four and six layers, the burst release was further reduced, and a more sustainable release can be observed, indicating that the LbL PAH/PSS coatings have the barrier properties to reduce the transport of IBU to outside medium, and tunable release profiles were achieved by changing the layer numbers. Besides reducing the burst release, the subsequent linear release phase after about 20 h was also quite different with two, four, and six polyelectrolyte layer-coated NMP and acetone hybrid microcapsules as can be seen in the inset of the two graphs (Figure 8a,b). For uncoated NMP PLGA microcapsules, the IBU release from 20 h to 170 h remains unchanged. After

Figure 6. ζ-Potential studies for the PAH/PSS coating process onto the PLGA coated CaCO3 microparticles.

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Figure 7. AFM observation for the polyelectrolyte/PLGA hybrid microcapsules: (a) PAH/PSS coating onto the CaCO3 microparticles coated with PLGA solidified from NMP solution; (b) PAH/PSS coating onto the CaCO3 microparticles coated with PLGA solidified from acetone solution.

Figure 8. IBU release profiles from the hybrid microcapsules having different layer numbers: (a) release profiles of IBU loaded in PLGA solidified from NMP; (b) release profiles of IBU loaded in PLGA solidified from acetone solution. The insets in panels a and b indicate IBU release after 20 h. The data points represent mean ± standard deviation of data from three independent samples.

release is summarized in Figure 9f. Figure 9f shows that hydrophilic−hydrophobic dual drug release can be achieved in a controllable manner from the microcapsules. For either IBU or FITC-dextran, the release rate decreases with the increase of layer numbers. At the respective two, four, or six layers of PAH/PSS coating, IBU shows a faster release than FITCdextran, which might be due to the smaller size of IBU molecules. This multifunctional platform comprising inorganic nanoparticles entrapped inside showed the ability to deliver both hydrophobic and hydrophilic drugs in a temporal manner. Taking advantage of the simplicity and modular assembly of template-based LbL polyelectrolyte/PLGA hybrid microcapsules, cumulative desired functionalities such as targeting, magnetic, imaging, and dual drug codelivery system can be achieved using a minimum number of components in an integrated carrier.

coating two layers of PAH/PSS, a linear increase from 60% to 65% can be observed. For capsules coated with four layers and six layers, the linear increase is from 45%, 35% to 60%, 55%, respectively (Figure 8a). Similar trends are seen for acetone PLGA hybrid microcapsules (Figure 8b). These results imply that the multilayer polyelectrolyte coating onto the NMP or acetone microcapsules is able to control the release of the hydrophobic drug in a more sustainable manner over longer periods as compared to the uncoated microcapsules. 3.5. Formation of Magnetically Responsive, Hydrophilic/Hydrophobic Dual Drug Delivery Microcapsules. Beside hydrophobic drugs, other components such as hydrophilic drugs and inorganic nanoparticles can also be conveniently integrated into such hybrid microcapsules (Figure 9). Here, the interior of the microcapsules was filled in with hydrophilic substance (FITC-dextran, 0.4% loading and 30.3% encapsulation efficiency) and Fe3O4 nanoparticles according to previous procedures.40 Figure 9 suggests that a dual-drug containing magnetically responsive multifunctional microcapsule is successfully formed (Figure 9). The dual drug

4. CONCLUSION In conclusion, we introduced the PLGA in situ depot forming systems into the LbL polyelectrolyte microcapsules to produce 2269

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Figure 9. (a) Green channel of CLSM showing the aligned multifunctional microcapsules under magnetic force; the green color is from the hydrophilic model compound (FITC-dextran) prefilled in the interior of the microcapsules. (b) Enlarged view of capsules in the selected area of panel a. (c) Schematic illustration to show how the capsules are aligned. (d) Red channel of CLSM showing the aligned multifunctional microcapsules under magnetic force; the red color is from the hydrophobic model compound (Nile Red) incorporated in the acetone PLGA microcapsules. (e) Enlarged view of capsules in the selected area of panel d. (f) Dual-drug (IBU and FITC-dextran) release behaviors from the acetone PLGA hybrid microcapsules. The data points represent mean ± standard deviation of the data from three independent samples.

Notes

the novel hybrid microcapsules. Hydrophobic drugs can be incorporated into PLGA matrix while the further LbL polyelectrolyte coating serves as the release rate controlling membrane. Production of such hybrid microcapsules expands both the application fields of the PLGA delivery system and the LbL polyelectrolyte microcapsules. Meanwhile, this technique can be applied to encapsulate various aqueous soluble drugs. Hydrophilic drugs and other inorganic nanoparticles like silver, gold, and magnetic nanoparticles can also be incorporated into the hybrid microcapsules. This multifunctional platform simultaneously has dual-drug release and stimulation-responsive ability. It may find wide applications in a synergetic drug delivery system such as the gene and anticancer drug codelivery systems.

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from the Ministry of Education (Singapore) and the National Research Foundation (Singapore).

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ASSOCIATED CONTENT

S Supporting Information *

SEM observations of the surface and interior morphology of CaCO3 microparticles, and CLSM observation showing the PAH/PSS coating onto the coated PLGA layer. This information is available free of charge via the Internet at http://pubs.acs.org/.

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REFERENCES

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*Tel.: +65 6790 4259. E-mail address: [email protected]. 2270

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