Biomacromolecules 2005, 6, 3433-3439
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From Ultrathin Capsules to Biaqueous Vesicles Ajay J. Khopade,*,†,‡ N. Arulsudar,† Surekha A. Khopade,† Roy Knocke,‡ Ju¨rgen Hartmann,‡ and Helmuth Mo¨hwald‡ Sun Pharma Advanced Research Centre,Tandalja, Baroda 390 020, Gujarat, India, and Max Planck Institute of Colloids and Interfaces, D-14476, Am Mu¨hlenberg 1, Golm, Germany Received July 14, 2005; Revised Manuscript Received August 26, 2005
The layer-by-layer (LbL) adsorption of anionic polyelectrolytes (PE) and tobramycin sulfate (TbS) multilayers on zinc oxide core particles followed by the controlled core-removal process leads to the formation of ultrathin capsules, which gradually convert to biaqueous vesicles and emulsionlike systems depending on the hydrophilicity/hydrophobicity of the PE backbone, PE/TbS ratio, and Zn2+ concentration. The unique characteristics of the PE/TbS multilayer capsules result because of the formation of PE/TbS/H2O biphasic liquid systems unlike the other LbL capsular systems that form stiff PE coacervates when mixed together in water. This paper investigates the PE/TbS ultrathin capsule to biaqueous vesicle transition and its physicochemical properties. Introduction Layer-by-layer (LbL) assembly of oppositely charged polyelectrolytes (PE) on sacrificial core particles produces ultrathin, PE-multilayer microcapsules after the core removal.1 A variety of different materials such as dyes, oxometalates, inorganic particles, and dendrimers in combination with linear PEs have been used to prepare multilayer microcapsules so that their physicochemical properties can be customized for various applications.2a-e Recently, we have reported purely biocompatible, ultrathin, antibiotic-walled microcapsules for sustained drug delivery in the eye, which were prepared by LbL assembly of the anionic PEs and a polycationic aminoglycoside (AmG), tobramycin sulfate (TbS), on zinc oxide (ZnO) core particles and removal of the core with a weak organic acid.3 Incidentally, we discovered that these microcapsules slowly transformed into vesicles and emulsions as the Zn2+ ions from the capsule suspension were extracted. This paper attempts to investigate the ultrathin microcapsule to vesicle transition because such morphological changes are not reported for any other LbL microcapsules. Therefore, in the following paragraphs we discuss the properties of the polymer mixtures to understand the complexation behavior of the two types of PEs with TbS and its relevance to the system described herein. Aqueous polymer mixtures display various physicochemical characteristics such as synergistic viscosity enhancement and phase separation. The latter results either from the small repulsive or attractive interaction between unlike polymers or from a difference in the polymer-solvent interactions, or from a combination of both phenomena. The phase * Corresponding authors. (A.J.K.) Present address Sun Pharma Advanced Research Centre, Tandalja, Baroda 390 020, Gujarat, India. Tel +91 265 2350789; fax +91 265 2354897; e-mail
[email protected]. (H.M.) Tel +49 331 567 9201; fax +49 331 567 9202; e-mail Mohwald@ mpikg-golm.mpg.de. † Sun Pharma Advanced Research Centre. ‡ Max Planck Institute of Colloids and Interfaces.
separation, commonly known as “polymer incompatibility”, involves the formation of two liquid phases, where each phase is enriched in one of the polymer components, for example, mixtures of poly(ethylene oxide) (PEO) and dextran.5 The seggregative phase separation is also shown by similarly charged PE systems (both anionic), for example, poly(styrene sulfonate)(PSS)/poly(acrylic acid),5f though the literature on PE systems are relatively less. On the other hand, a preferential attraction between unlike polymers leads to an associative phase separation where a concentrated liquid phase, enriched in both polymers, is formed, commonly referred to as complex coacervation.6 The coacervates may range from stiff precipitatelike, highly viscous to less viscous, fluidlike materials depending upon the physicochemical properties of the PEs used, their mole ratio, pH and the temperature. The associative phase behavior is also observed with nonionic polymers in nonaqueous solvents.7 Reports on the associative fluidlike coacervate systems from oppositely charged PEs, relevant to the present context, are also largely limited to protein systems, such as bovine serum albumin/poly(dimethyldiallylammonium chloride)8a and PSS/ gelatin.8b To understand vesicle formation from the LbL microcapsules, various examples of nonionic polymer/ polymeric surfactant systems9 like mesophasic (lamellar)9a and micellar9b-c systems are interesting because they selfassemble and rely on the structuring properties of water for the formation of a heterogeneous multiphase system. The biocompatibility of the components and the predominance of water as a solvent make these polymeric phase systems suitable for use in bioseparation processes,10a designing solvent-free drug delivery systems,10b and biomineralization.9a In this paper, we have made an attempt to show and understand the transformation of the PE/TbS multilayer microcapsules, prepared from [PE/TbS]n@ZnO, to the vesicular structures when the TbS and Zn2+ ions are slowly removed from the capsule dispersion. On the basis of the
10.1021/bm0504936 CCC: $30.25 © 2005 American Chemical Society Published on Web 10/21/2005
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above-described behavior of polymer mixtures, it is likely that this phenomenon occurs because the components of the multilayer capsules, that is, anionic PEs and polycationic AmG, form a fluidlike coacervate, unlike the other previously reported PE components that form a stiff coacervated complex or a precipitatelike multilayer film. Besides showing that the PE/TbS mixture forms a fluid coacervate or an aqueous biphasic system (ABS), we also show that the PE/ TbS film displays order (depending on PE backbone chemistry) and fluidity. Hence, the PE/TbS vesicles, transformed from the PE/TbS multilayer capsules, are termed “biaqueous vesicles”. The foamlike biaqueous vesicular structures were also reported for coacervate systems prepared from gelatin/acacia, (Na+, K+) adenosine triphosphatase/ cholate, and the broth of pure amino acid mixture.11 However, these were formed spontaneously and not prepared and characterized as reported in the present paper. The common denominator in the reported vesicular systems of PEs is the presence of proteins and high molecular weight components, while this paper reports on the formation of biaqueous vesicles from a protein-free system and with a small PE molecule such as AmG antibiotic by LbL technique. Experimental Section Materials. The PEs, PSS (MW ∼70 000) and DxS (MW ∼100 000), were obtained from Sigma-Aldrich and used without further purification. The selected polycationic AmG component, TbS, was a kind gift from Sun Pharma, India. Other materials such as, solvents and reagents were of analytical reagent grade and used as such. Phase Diagram. Stock solutions of the PEs, TbS and DxS, were prepared in water and mixed in different proportions by weight in preweighed glass vials. Water was further added dropwise (microliter portions) until the turbidity was observed. The proportion at which turbidity was generated was considered as two-phase formation.6 The final weight of the vial was taken and the percentage weight of each component (PE, TbS, and water) was calculated on a weight/weight basis. The data were plotted on a triangular graph with PE, TbS, and water as three apexes representing 100% concentration. Preparation and Coating of ZnO Core Particles. Zinc oxide (ZnO) cores (0.3-0.9 µm) were synthesized in-house.3 Neutral to weakly charged, bare ZnO particles were incubated with PE solution for 10 min, followed by three centrifugation and wash cycles. PE-coated ZnO particles were then incubated with TbS solution for another 10 min and washed three times via a centrifugation cycle. The PE and TbS adsorption steps were repeated to build multilayers on the ZnO particles. Ultrathin capsules, vesicles, or emulsions were finally obtained by dissolving the ZnO core by the addition of 1 mL of 0.01 M acetic acid solution, followed by water washing via dialysis. This system is described in detail elsewhere.3 Labeling. Fluorescein- and rhodamine-labeled TbS were prepared by incubating fluorescein isothiocyanate (FITC) and rhodamine isothiocyanate (TRITC), respectively, with TbS
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(1:1 mol ratio) at pH 10.5 for 24 h. The formation of TbSFITC and TbS-TRITC was confirmed by the appearance of an amide bond at 1540-1560 cm-1 in the infrared spectrum. The labeled PE/TbS multilayer microcapsules and films were prepared with TbS-FITC and TbS-TRITC as required for characterization. Chemical Analysis. PSS and TbS quantities were determined by the ultraviolet spectroscopy and high-performance liquid chromatography (HPLC) methods, respectively. HPLC (Waters) was performed on a Hypersil BDS C-18, 250 × 4.6 mm, 5 µm column with a mixture of 1.0 g of Tris in 420 mL of water, 10 mL of 1 N H2SO4, and 570 mL of acetonitrile (total volume 1000 mL). The injection volume was 20 µL and the flow rate of the mobile phase was 1.5 mL min-1, with a total running time of 40 min. The eluted drug was detected at 365 nm by a UV detector. The working test and reference standard was a 20 µg mL-1 solution of TbS in a 80% aqueous dimethyl sulfoxide (containing 3 mg mL-1 Tris)/alcoholic dinitrofluorobenzene (10 mg mL-1)/ acetonitrile (1:1:3) mixture. The mixture was heated at 60 °C for 50-55 min to complete derivatization of TbS before injection into an HPLC. The test samples were also treated similarly. The analytical method has excellent specificity, sensitivity, and reproducibility. The samples consisting of PE/TbS complex (coacervate, multilayer capsules and vesicles) were dissolved in 0.01M NaOH before analysis. Instrumental Analysis. Small-angle X-ray scattering (SAXS) measurements were carried out with a Nonius rotating anode (U ) 40 kV, I ) 100mA, λ ) 0.154 nm) using image plates. With the image plates placed at a distance of 40 cm from the sample, a scattering vector range of s ) 0.07-1.6 nm-1 was available. Two-dimensional (2D) diffraction patterns were transformed into one-dimensional (1D) radial averages. A Nonius PDS120 powder diffractometer was used to perform wide-angle X-ray scattering (WAXS) measurements in transmission geometry. The source of Cu/K radiation (λ ) 0.154 nm) was a AFR590 generator. A monochromatic primary beam was obtained by use of a curved Ge crystal. Scattered radiation was measured with a Nonius CPS120 position-sensitive detector. The resolution of this detector in 2θ is 0.018°. Confocal laser scanning microscopy (CLSM) images, in both transmission and fluorescence modes, were taken with a Leica TCS SP microscope equipped with a 100× oil immersion objective. The excitation wavelength was set for FITC or TRITC, as required. The PE/AmG multilayer was bleached by switching on a high-intensity laser beam for 5 s. The fluorescence recovery was imaged at different time intervals. Raman spectra were recorded on a Raman microscope (WiTec, Germany). Transmission electron microscopy (TEM) images were taken on a Zeiss EM912 Omega microscope operated at 120 kV. Results and Discussion Phase Behavior of PE/TbS/H2O System. PSS and DxS are miscible with water over their whole composition range. If the ionic sulfate/sulfonate groups are disregarded, the backbones of PSS and DxS are water-insoluble (polystyrene)
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Figure 1. Chemical structures of: DxS, PSS and TbS.
Figure 3. Raman spectra of PSS (solid line), TbS (dotted line), and air-dried PSS/TbS complex (dashed line).
Figure 2. (a) Optical microscopic image showing phase separation (droplet budding phenomenon) occurring at a homogeneous PSS/ TbS aqueous solution and water interface. The budding is taking place into the PSS/TbS aqueous solution phase where the droplets are seen. (b) Triangular phase diagram for PSS/TbS/H2O ABS showing the enclosed area in solid dots as the two-phase region.
and water-soluble (dextran), respectively. The selected polycationic AmG component, TbS (Sun Pharma, India), is also soluble in water. The chemical structures of the selected PEs and AmG are given in Figure 1. When an equimolar or greater amount of TbS is added to the concentrated (5-10%) aqueous solutions of PSS or DxS, initially, a soluble complex is formed that separates into two liquid phases upon dilution with water (Figure 2a). Occurrence of liquid-liquid demixing might be explained by the exclusion of a PE/TbS complex from the aqueous solution associated with excess water, similar to what is reported for the gelatin/acacia ABS.11 Two-phase region formation in the PSS/TbS/H2O system at 25 °C is shown in a triangular phase diagram (Figure 2b, see Experimental Section). Figure 2b reveals that the biphasic system forms at low polymer fractions and is favored by the addition of excess water. The
phase diagram for DxS/TbS/H2O (not shown) was similar to that in Figure 2b, except that the two-phase area was smaller. The phase-separated portions are denser and form the bottom phase in the test tube. The bottom phase contains the two components (PE and TbS) in ∼1:0.96 mole ratio, demonstrating that it is the PE/TbS complex that phaseseparates. However, it does not form a stiff coacervate because the chemical characteristics of the two PEs and excess associated water complex render a liquid state. The phase formation was temperature- and ionic strengthdependent. The two-phase area was reduced with increasing amounts of NaCl (zero at ∼2.0 mM) and increasing temperatures (zero at ∼50 °C), which indicates the role of electrostatics in the (PE/TbS)/H2O ABS formation and water association with PE/TbS complex. The water-destructuring effect at high ionic strength and temperature of the PE/TbS complex leads to the disappearance of the ABS.10,12 Unlike DxS/TbS thin films, PSS/TbS complex films exhibit phenyl side-chain periodicity at the air-water interface. This creates terraces, appearing in the optical image as sharp fringes of the interference colors (each color change represents ∼30 nm step) and islands and holes under the atomic force microscope (AFM, not shown). A similar observation is made for the polymers that interact due to the covalently attached complementary hydrogen-bonding functionalities in a nonaqueous medium.13 PE/TbS Multilayer Capsule to Biaqueous Vesicle Transition. An ultrathin capsular system was prepared by LbL adsorption of the [PE/TbS]n multilayers on the ZnO core particles ([PE/TbS]n@ZnO) and subsequent removal of the ZnO core by acetic acid (see Experimental Section).3 Raman spectra of the air-dried [PSS/TbS]6 multilayer capsules showed all the peaks from the two components (PSS and TbS) confirming the multilayer wall as PSS/TbS complex (Figure 3). Depending on the type of PE backbone, the transformation of multilayer capsules to biaqueous vesicle or emulsion (ABS droplets) differs, which is observed by TEM. As shown in Figure 4a, ultrathin capsules were obtained from the [DxS/ TbS]6@ZnO system (TbS outer layer) at a concentration above 15 mM Zn2+ ions in the dispersion medium.3 As Zn2+ ions were further extracted by dialysis, the microcapsule collapsed into a droplet (Figure 4c) through a metastable, fluid-membrane capsular intermediate (Figure 4, panels b
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Figure 4. Transmission electron microscopic images of [DxS/TbS]6 LbL capsules (a), which convert to biaqueous emulsion droplets as Zn2+ ions are removed (c) through an intermediate fluid-capsular state showing spreading around the periphery (b). The fluid capsular state is termed as biaqueous vesicles. Biaqueous vesicles obtained from [DxS/TbS]6 (d) and [PSS/TbS]6 (e) multilayer capsules at a Zn2+ ion concentration corresponding to
