Bioactive Galactose-Branched Polyelectrolyte Multilayers and

Sep 2, 2006 - The experimental protocols were first optimized on flat, smooth silica substrates using UV−vis, contact angle, and atomic force micros...
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Langmuir 2006, 22, 8458-8464

Bioactive Galactose-Branched Polyelectrolyte Multilayers and Microcapsules: Self-Assembly, Characterization, and Biospecific Lectin Adsorption Fu Zhang, Qi Wu, Zhi-Chun Chen, Xia Li, Xiu-Ming Jiang, and Xian-Fu Lin* Department of Chemistry, Zhejiang UniVersity, Hangzhou 310027, People’s Republic of China ReceiVed March 29, 2006. In Final Form: July 27, 2006 We describe the fabrication of multilayers and microcapsules with biologically designed targeting activity using chemoenzymatic synthesized carbohydrate-branched polyelectrolytes. A novel cationic D-galactose-branched copolymer [poly(vinyl galactose ester-co-methacryloxyethyl trimethylammonium chloride), PGEDMC] is alternated with poly(styrene sulfonate) (PSS) to form thin multifilms by the layer-by-layer (LbL) technique on such different solid surfaces as quartz slides, poly(ethylene terephthalate) (PET) films, silicon wafers, and polystyrene (PS) microparticles. The experimental protocols were first optimized on flat, smooth silica substrates using UV-vis, contact angle, and atomic force microscopy (AFM) measurements. The film properties of PGEDMC/PSS multilayers are modified by varying polyelectrolyte concentration, ionic strength, and counteranion types. Hollow capsules were formed after the removal of colloidal templates; transmission (TEM) and scanning (SEM) electron microscopy were used to verify the LbL process integrity. PGEDMC/PSS planar films and capsules carrying β-galactose as recognition signals have specific recognition abilities with peanut agglutinin (PNA) lectin rather than concanavalin A (Con A) lectin observed by fluorescence spectroscopy.

Introduction The versatile strategies based on layer-by-layer (LbL) selfassembly have been widely applied for preparing ultrathin functional films.1 This technology has been extended for fabricating hollow capsules with well-defined size.2 Poly(styrene sulfonate) (PSS) and poly(allylamine hydrochloride) (PAH), which was the most thoroughly studied polyelectrolyte (PE) pair in the past decade, have been successfully used to produce multilayer films and microcapsules.3 Recently some novel polyelectrolytes have been successfully constructed in order to obtain particles and capsules with specifically desired properties, which endow responsiveness to temperature, light, magnetic field, chemical or biological stimuli, and specific analyte molecules in solution, as well as capsule manipulation and targeting.4 von Klitzing5 and Sukhorukov6 and coworkers had reported the self-assembly of polyelectrolytes containing a thermosensitive poly(N-isoproprylacrylamide) (PNIPAM) block for fabrication of thermosensitive hollow capsules. Textor7 and co-workers demonstrated the fabrication of multilayers and microcapsules, which are able to resist protein adsorption and opsonization by macrophages, through the LbL assembly of PAH/PSS and a final monomolecular coating of the polycationic graft copolymer poly(L-lysine)-graft-poly(ethylene glycol). In consideration of drug-delivery applications of multilayers and microcapsules, there is a particularly important need for tailored carriers in order to efficiently target specific types of * Corresponding author: e-mail [email protected]. (1) (a) Decher, G. Science 1997, 277, 1232. (b) Chung, A. J.; Rubner, M. F. Langmuir 2002, 18, 1176. (c) Hiller, J.; Mendelsohn, J. D.; Rubner, M. F. Nat. Mater. 2002, 1, 59. (2) (a) Donath, E.; Sukhorukov, G. B.; Caruso, F.; Davis, S.; Mo¨hwald, H. Angew. Chem. 1998, 110, 2323. (b) Caruso, F.; Lichtenfeld, H.; Giersig, M.; Mo¨hwald, H. J. Am. Chem. Soc. 1998, 120, 8523. (3) (a) Chen, W.; McCarthy, T. J. Macromolecules 1997, 30, 8453. (b) Hammond, P. T. Curr. Opin. Colloid Interface Sci. 2000, 4, 430. (c) Tong, W. J.; Gao, C. Y.; Mo¨hwald, H. Chem. Mater. 2005, 17, 4610. (4) Sukhishvili, S. A. Curr. Opin. Colloid Interface Sci. 2005, 10, 37. (5) Steitz, R.; Leiner, V.; Tauer, K.; Khrenov, V.; von Klitzing, R. Appl. Phys. A- 2002, 74, 519. (6) Glinel, K.; Sukhorukov, G. B.; Mo¨hwald, H.; Khrenov, V.; Tauer, K. Macromol. Chem. Phys. 2003, 204, 1784. (7) Heuberger, R.; Sukhorukov, G.; Vo¨ro¨s, J.; Textor, M.; Mo¨hwald, H. AdV. Funct. Mater. 2005, 15, 357.

cells, tissues, or organs. Among the many approaches used to manipulate delivery of drugs to a targeted tissue, the recognition processes based on carbohydrate-protein interaction are expected to be one of the most promising routes in cellular-specific drug targeting;8 thus a variety of glycopolymers have been synthesized.9 Incorporating specific carbohydrate into multilayers by the LbL method is a facile and competitive procedure to construct drugdelivery systems for specific targeting of cells and tissues.10 In this context, the preparation of carbohydrate-branched PEs and the subsequent fabrication of multilayers and microcapsules with biologically designed targeting surface properties are particularly attractive. D-Galactose is a well-known targeting molecule directing to the hepatic cells through strongly binding with large numbers of asialoglycoprotein receptors, which are exclusively expressed by liver parenchymal cells.11 It can also be specifically recognized by some lectins such as peanut agglutinin (PNA), which binds preferentially to a commonly occurring structure, β-D-galactose or galactosyl-(β-1,3)-N-acetylgalactosamine. It is reported that the glycopolymers showed stronger interactions with lectins than those in aqueous solution due to the glycocluster effects of the polymers.10 Therefore many polymers modified with D-galactose were synthesized and successfully applied in hepatic cellularspecific drug targeting.11 The chemoenzymatic synthesis of glycoconjugates has been well documented in the literature.12 In our previous paper, a series of carbohydrate-branched polymers have been prepared via enzymatic synthesis and subsequent redox polymerization.13 The aim of this work was to produce multilayers and microcapsules with biologically designed targeting activity by (8) (a) Tayler, M. E.; Kurt, D. Introduction to Glycobiology; Oxford University Press: New York, 2003. (b) Lee, Y. C. Carbohydr. Res. 1978, 67, 509. (9) (a) Narain, R.; Armes, S. P. Biomacromolecules 2003, 4, 1746. (b) Dong, C. M.; Chaikof, E. L. Colloid Polym. Sci. 2005, 283, 1366. (c) Uzawa, H.; Ito, H.; Izumi, M.; Tokuhisa, H.; Taguchi, K.; Minoura, N. Tetrahedron 2005, 61, 5895. (d) Zhu, J. M.; Marchant, R. E. Biomacromolecules 2006, 7, 1036. (10) Miura, Y.; Sato, H.; Ikeda, T.; Sugimura, H.; Takai, O.; Kobayashi, K. Biomacromolecules 2004, 5, 1708. (11) Kim, I. S.; Kim S. H. Int. J. Pharm. 2002, 245, 67. (12) (a) Chen, X.; Martin, B. D.; Neubauer, T. K.; Linhardt, R. J.; Dordick, J. S.; Rethwisch, D. G. Carbohydr. Polym. 1995, 28, 15. (b) Miura, Y.; Ikeda, T.; Kobayashi, K. Biomacromolecules 2003, 4, 410.

10.1021/la060847u CCC: $33.50 © 2006 American Chemical Society Published on Web 09/02/2006

Polyelectrolyte Multilayers and Microcapsules

Figure 1. Molecular structure of poly(vinyl galactose ester-comethacryloxyethyl trimethylammonium chloride) (PGEDMC).

use of chemoenzymatic synthesized cationic D-galactose-branched polyelectrolytes, which were alternated with PSS by the LbL technique. The self-assembly nature of the novel polycation PGEDMC was investigated in detail by varying PE concentration, ionic strength, and counteranion type. The assembly process and structure of multilayers were characterized by UV-vis, contact angle, and atomic force microscopy (AFM). Hollow capsules on PS microparticles were formed after the removal of colloidal templates to entrap drugs and other molecules. Furthermore, the specific interaction of PNA lectin with PGEDMC/PSS planar film and capsule was studied with fluorescence spectroscopy. The core-shell system has the advantage as drug carriers in controlled drug delivery with potential targeting of hepatic cells. Experimental Section Materials. Poly(sodium 4-styrenesulfonate) (PSS, MW 70 000) (Sigma-Aldrich), poly(allylamine hydrochloride) (PAH, MW 70 000) (Sigma-Aldrich), fluorescein-labeled peanut agglutinin (FLPNA, β-galactose-binding lectin) (Vector), and fluorescein-labeled concanavalin A (FL-Con A, R-glucose-binding lectin) (Vector) were used without further purification. The polystyrene (PS) particles (about 2 µm diameter) were synthesized according to the literature.14 Poly(vinyl galactose ester-co-methacryloxyethyl trimethylammonium chloride) (PGEDMC, Mw 54 200, Mw/Mn ) 2.4; data from gelpermeation chromatography, GPC) was prepared according to the literature procedure.13a The content of D-galactose in the copolymers, defined as the number of d-galactose monomers per copolymer chain, was calculated from their 1H NMR spectra. Figure 1 shows the molecular structure of PGEDMC. Multilayer Assembly on Planar Substrates. The quartz slides or one-side-polished silicon (100) wafers cut into squares 1 cm by 1 cm were soaked in “piranha” solution (30% H2O2/concentrated H2SO4, 1:3 v/v) at 80 °C for 30 min, then rinsed with pure water with ultrasonication assistance, and finally dried with nitrogen. Poly(ethylene terephthalate) (PET) films (1.5 cm × 8 cm) were first rinsed in methanol for 30 min and then washed with water. Then they were soaked in 15% (m/v) NaOH solution for about 4 h, followed by a rinse in diluted hydrochloric acid and water, and dried with a stream of nitrogen. The alternating assembly of the planar substrate was done as follows: The substrate was first soaked in a polycationic PGEDMC solution with a certain salt concentration for 10 min and then rinsed with water three times. Then it was dipped in the polyanionic PSS solution for another 10 min and washed with water three times. The assembly procedure described above was repeated until the bilayers needed were obtained. After each bilayer was prepared, UV-vis measurement was performed to follow the multilayer growth; here the outermost layer was always the PSS. A water contact angle was also measured to follow the LbL procedure after each monolayer was adsorbed. Typical AFM images were recorded on PET samples when 8 bilayers had been deposited, and (13) (a) Wang, X.; Wu, Q.; Wang, N.; Lin, X. F. Carbohydr. Polym. 2005, 60, 357. (b) Wu, Q.; Chen, Z. C.; Lu, D. S.; Lin, X. F. Macromol. Biosci. 2006, 6, 78. (14) Poptoshev, E.; Rutland, M. W.; Claesson, P. M. Langmuir 1999, 15, 7789.

Langmuir, Vol. 22, No. 20, 2006 8459 the bilayer film thickness was measured on silicon substrates by AFM as well. Multilayer Assembly on Colloidal Particles and Preparation of Hollow Capsules. The PGEDMC/PSS multilayer-coated PS particles were prepared as follows: 1 mL of a 2 mg/mL aqueous PSS solution at pH 7.0 (containing 0.5 M NaCl) was added to 10 mg of PS particles in a centrifuge tube. The PSS was allowed to adsorb on the particles for 10 min with shaking (200 rpm at 20 °C). Then the excess polyelectrolyte was removed by two repeated cycles of centrifugation (7300 rpm, 8 min)/washing/redispersion by shaking and gentle ultrasonication. The following PAH layer was deposited by the same procedure with 1 mL of PAH solution (2 mg/mL, containing 0.5 M NaCl at pH 7.0). After the precursor bilayer of (PSS/PAH) was deposited, the positively charged PGEDMC was alternately with oppositely charged PSS by the same method until the desired number of (PSS/PGEDMC) layers was obtained and the outermost layer was PGEDMC. For dissolution of PS particles, 100 µL of the coated PS particles was exposed to 1 mL of tetrahydrofuran (THF). After 5 h at 20 °C the supernatant was replaced with a new portion of THF for 12 h. Then the capsules were centrifuged at 7200 rpm for 8 min and rinsed with THF and water three times each. Lectin Binding on Galactose-Branched Multilayers and Microcapsules. The binding of the galactose copolymer PGEDMC with PNA lectin was conducted on both quartz slide substrates and microcapsules. The (PGEDMC/PSS)4PGEDMC multilayer-coated quartz slide was incubated in 200 µg/mL FL-PNA in 10 mM phosphate-buffered saline (PBS, pH 7.8, containing 0.15 M NaCl) at 25 °C for 2 h and then washed with PBS to remove weakly bounded lectin. For colloidal particles, 100 µL of (PSS/PGEDMC)4coated PS microparticles was dropped into 0.5 mL of FL-PNA or FL-Con A (200 µg/mL in 10 mM PBS buffer) at 25 °C for 2 h under gentle shaking. Finally, the capsules were rinsed with PBS four times and the supernatant was removed. The fluorescence images were observed with a fluorescence microscope. UV-vis Measurement. An Analytikjena Specord 200 UV-vis spectrophotometer was used to monitor the absorbance increment of the films on quartz slides. PSS has a maximum absorption peak at 226 nm, while PGEDMC shows no absorption in the UV-vis region. Data were evaluated after the spectrum of the “piranha”treated blank quartz sample was subtracted from each of the measured spectra. AFM Measurement. The morphology of multilayer-deposited PET films and bilayer thickness on silicon substrates were determined by atomic force microscopy (AFM) on a Multimode Nanoscope IIIA (Veeco, Santa Barbara, CA) apparatus. All the AFM images were obtained by the tapping mode in ambient air, and the silicon cantilever used was 125 µm in length with a resonance frequency of 325 kHz. Deflection and height mode images were scanned simultaneously at a fixed scan rate of 1 Hz. The scan sizes varied from 10 × 10 µm to 15 × 15 µm. The film thickness was determined by scratching a furrow on the film with a razor blade and analyzing the cross sections of the scanned area. Contact Angle Measurement. An OCA20 video-based contact angle measuring device (Data Physics, Germany) was used to measure the static contact angles of water on the prepared multilayer surfaces at 25 °C. Typically, 1 µL of water was dropped on PET films, and then the drop shape was recorded and analyzed by the system within 1 s. Transmission Electron Microscopy. The capsules were loaded on a carbon-coated 200 mesh copper grid and observed under a transmission electron microscope (TEM, JEM 200CX) at 100 kV electron beam accelerating voltage. Typically, 25 µL of suspension was dropped on the grid, and the extra solution was then allowed to air-dry. Scanning Electron Microscopy. Samples were prepared by loading 20 µL of the capsule suspension onto a glass slide. After drying overnight, the samples were sputtered with gold and measured by scanning electron microscopy on a Sirion-100 instrument (FEI) at an operation voltage of 5.0 keV.

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Figure 2. UV-Vis spectra of (PGEDMC/PSS)n on quartz slide as a function of bilayer number. (Inset) Typical increment of absorbance at 226 nm depending on assembly cycles. The multilayer was deposited in 1 mg/mL polyelectrolyte solution with 0.5 M NaCl.

Figure 3. PGEDMC/PSS film thickness determined with AFM. (Inset) Typical dependence of bilayers thickness on deposition cycles. The multilayer was deposited in 1 mg/mL polyelectrolyte solution with 0.5 M NaCl. Confocal Laser Scanning Microscopy. The fluorescence images were taken by a Zeiss LSM 510 scanning device (Zeiss, Germany) mounted on a Zeiss Axiovert 100 inverted microscope equipped with external argon laser (for excitation at 488 nm). Observations were taken by using a 60× water immersion objective with a numerical aperture of 1.4 for capsules and 20× water for slides. Typically, 30 µL of lectin-adsorbed capsule suspension was dropped on a glass slide and then observed. The FL-PNA-deposited quartz slide was placed in the holder of the confocal microscope and observed.

Results and Discussion PGEDMC/PSS Multilayer Formation: Nature of the Polycation. PGEDMC polymer was assembled in alternation with the strong polyanion of PSS. Exponential-like growth is observed in Figure 2 for the PGEDMC/PSS films. We found that less PSS molecules were adsorbed in the first few PGEDMC/ PSS multilayers than the following ones. Film thickness was also increased after deposition of another bilayer, as shown in Figure 3. AFM data indicate that the thicknesses of inner deposited layers are somewhat thinner than the outer ones: ca. 1.7 nm for the first bilayer, 5.7 nm for interbilayer, and 8.7 nm for outer bilayer; the average thickness of 10 PGEDMC/PSS bilayers is ca. 6 nm. Such behavior may be caused by the roughness of the film increasing with the number of deposited layers.15 The polymers adsorbed in the initial layers adopt a flat conformation because of the strong interactions with the substrate surface; however, the influence of the substrate decreases with increasing (15) McAloney, R. A.; Sinyor, M.; Dudnik, V.; Goh, C. Langmuir 2001, 17, 6655.

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Figure 4. Relationship of contact angle and number of layers (PGEDMC/PSS system). Number of layers: 0, bare PET; 1, PGEDMC; 2, PGEDMC/PSS; 3, PGEDMC/PSS/PGEDMC, and so on.

number of deposited cycles, and the polyelectrolyte chains start to adsorb in denser coil form, which results in an increasing surface area. Besides, the bulky chains grafted on the PGEDMC backbone would make a stiff polymer conformation and free polyelectrolytes with slower diffusion coefficients could no longer fully diffuse out of the formed multilayer after diffusing into the existing polyion network during a new adsorption,16 resulting in nonlinear multilayer growth. Porcel et al.17 suggested that in the hyaluronic acid/poly(L-lysine) (HA/PLL) system after a given number of deposition steps the film thickness evolution changed from exponential to linear, due to a film restructuring that progressively forbids the diffusion of one of the polyelectrolytes constituting the film over part of the film. The stable tendency of the film to grow with deposition of each layer was also confirmed by contact angle measurement. The hydrophobic/hydrophilic properties of coated PET films were expected to vary as compared with the original hydrophobic PET film. Figure 4 shows the change of water contact angle of coated PET surfaces with the number of coating layers. For the initial two layers, a gradual decrease of the water contact angle was observed while the coating layer number was increased. After the first bilayer was deposited, the uniformity of anionized PET surface was decreased. From the 10-layer coating of (PGEDMC/PSS)5, the contact angle began to jump alternatively between 73° and 10°, relying on the outermost layer component. Upon further increasing the deposition cycles, the contact angle values of the same outermost layer component remained almost constant. This alternative change in contact angle was indicating that fully covered and layered coatings were well developed on PET films after several deposition cycles of (PGEDMC/PSS). The wettability of fully coated PET films is dominantly controlled by the outmost coating component. Polyelectrolyte Concentration. To observe the influence of the PE concentration on the growth behavior of this newly copolymerized polyelectrolyte and PSS, a series of concentration gradients were adjusted as follows: 0.1, 0.5, 1.0, 2.0, and 5.0 mg/mL, with the same ionic strength of 0.5 M NaCl for each solution. UV-vis spectrometry was performed to monitor evolution of the PSS absorption as a function of deposited layer number. Figure 5 shows the multilayer absorbance as a function of PE concentration for eight-bilayer films. The data shown are (16) (a) Lavalle, P.; Vivet, V.; Jessel, N.; Decher, G.; Voegel, J. C.; Messini, P. J.; Schaaf, P. Macromolecules 2004, 37, 1159. (b) Hu¨bsch, E.; Ball, V.; Senger, B.; Decher, G.; Voegel, J. C.; Schaaf, P. Langmuir 2004, 20, 1980. (17) Porcel, C.; Lavalle, P.; Ball, V.; Decher, G.; Senger, B.; Voegel, J. C.; Schaaf, P. Langmuir 2006, 22, 4376.

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Figure 5. Influence of PE concentration on (PGEDMC/PSS)8 multilayer formation investigated with UV-vis. Absorbance at 226 nm are plotted as a function of the PE concentration. In all of the adsorption solutions, 0.5 M NaCl was used.

Figure 7. (PGEDMC/PSS)8 multilayer absorbance at λ 226 nm determined with UV-vis. The multilayer was deposited in 0.5 M sodium salt of the corresponding counteranions. Figure 6. Influence of ionic strength (NaCl concentration) on (PGEDMC/PSS) multilayer formation investigated with UV-vis. The inset data (from bottom to top) are total absorbance at 226 nm of 1-8 bilayers vs salt concentration.

for films obtained not by drying after each layer so as to remain the original conformation during PE assembly. The absorbance increased rapidly with the increased PE concentration, and when the PE solution gradually grows dense, the increment of absorbance becomes small. Here we did not observe an absorption saturation, which usually appears as a plateau in other polyelectrolyte systems in this concentration range.14 This possibly resulted from the positive charge insufficiency of the copolymerized polycation, which prolonged the occurrence of saturation range. Moreover, although increasing solution concentration tends to produce a thicker assembled film due to the adsorption driving forces such as viscous and electrostatic forces, the excess polyelectrolytes adsorbed with weaker binding sites are readily eliminated by the desorption driving forces such as air shear forces.18 Ionic Strength. The experimental variable that greatly influences the film thickness is the ionic strength of the solution.19 The thickness of films composed of consecutively alternating layers of PGEDMC and PSS on quartz can be fine-tuned by changing the ionic strength of the solution from which the PEs are adsorbed. Figure 6 depicts the dependence of UV absorbance at 226 nm wavelength on salt concentration in the range from 0.1 to 2 M NaCl in 1 mg/mL PE solution. It can be found that the dependence on salt concentration is almost linear for the first four bilayers, and after more bilayers had been deposited, it followed an exponential relation (see the inset in Figure 6). In (18) Cho, J. H.; Lee, S. H. Polymer 2003, 44, 5455. (19) Dubas, S. T.; Schlenoff, J. B. Macromolecules 1999, 32, 8153.

all cases a higher NaCl concentration yields a thicker film. This is presumably due to screening of charges on the polymer by salt ions, which causes the polyelectrolyte to adopt a more compact conformation. Coupled with this is a decrease in the electrostatic repulsion between oppositely charged surfaces and PE, also due to screening. A lower degree of interaction classically leads to adsorption with more loops in solution and fewer surface trains. In low ionic strength solutions, highly charged polyelectrolytes adopt extended conformations and are fairly inflexible due to the strong repulsion between charged monomers.20 Counteranions. The amount of flexible polyelectrolyte deposited onto a surface depends to a great extent on the ionic strength of the polyelectrolyte solution. The electrolyte species is also important.19 Saloma¨ki et al.21 had reported the influence of a variety of counteranions on the properties of PSS/PDADMA multilayers and illustrated that the thickness follows reasonably well the position of the counteranion in the Hofmeister series. In this paper, sodium salts of ClO4-, F-, Cl-, NO3-, Br-, I-, SO42-, CO32-, PO43-, CH3COO-, tartrate2-, and citrate3- were selected for PGEDMC/PSS multilayer formation, including common inorganic salts (monovalent and multivalent) and two organic salts (sodium potassium tartrate and sodium citrate). However, NaClO4 was found to precipitate PGEDMC under the same condition. The deposition of eight-bilayer PGEDMC/PSS system was carried out in the presence of different counteranions with a concentration of 0.5 M, and the layers were followed step by step via UV-vis (Figure 7). The UV absorbance of multilayers (20) (a) Lo¨sche, M.; Schmitt, J.; Decher, G.; Bouwman, W. G.; Kjaer, K. Macromolecules 1998, 31, 8893. (b) Ullner, M.; Woodward, C. E. Macromolecules 2002, 35, 1437. (21) Saloma¨ki, M.; Tervasma¨ki, P.; Areva, S.; Kankare, J. Langmuir 2004, 20, 3679.

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Figure 9. (a) TEM image of [PAH/PSS-(PGEDMC/PSS)4]-coated PS particles. The PS particle templates have an average diameter of 1.85 µm (inset). (b) TEM image of hollow [PAH/PSS-(PGEDMC/ PSS)4].

Figure 8. AFM images of the eight-layer PGEDMC/PSS copolymer films prepared on PET substrates from 0.5 M counteranion adsorption solution. Films with (a) no salt, (b) Cl-, (c) NO3-, and (d) Br- are shown. The image area is 4 µm2. The vertical scale bar for all of the images is 80 nm. (e) RMS roughness of AFM measurement of eight-bilayer PGEDMC/PSS films deposited in 0.5 M sodium salt of the corresponding anion, the average values from left to right are as follows: 7.637, 10.882, 21.978, and 25.273 nm.

ranked by the depositing anion, beginning from the maximum, is the following: I- . Br- > NO3- > Cl- > CH3COO- > Ffor monovalent ions and SO42- > tartrate2- ≈ citrate3- . CO32-, PO43- for multivalent ions. The effect of I- was the largest, while CO32- and PO43- hardly stimulated the multilayer growth, which was identical to salt-free fabrication. Small ions are strongly hydrated, with small or negative entropies of hydration, creating local order and higher local density. In the case of the halide series (I-, Br-, Cl-, and F-), the surface charge density increases with the decrease of the radius of hydrated anion. A large singly charged ion such as I- possesses low surface charge density and would have weaker hydration acting like hydrophobic molecules, which may additionally be pushed on by strong water-water interactions. The less hydrated anion produces a lower viscosity polymer solution, which shrinks the polyelectrolyte. Moreover, it would have a stronger interaction with oppositely charged groups that screen strongly the polymer charges, resulting in a loopy conformation of multilayers and a thick film.21 Anions screen less charges and thus induce smooth thin layers. For characterization of the surfaces of the multilayer films, AFM measurements were performed after eight-bilayer deposition. The root-mean-square roughness (RMS) of four typical types of eight-bilayer PGEDMC/PSS films on PET substrate is shown in Figure 8 with AFM data. Clear differences in the surface

morphology of the four samples with different counteranions can be observed. The assembly without any salt led to a small RMS value, obviously being indistinguishable from the roughness of PET substrate (corresponding to growth halting after one bilayer had been fabricated by UV-vis). For films with Cl-, NO3-, and Br-, not only the surface roughness but also the grain size increases, which correlates with the increasing PGEDMC/PSS film thickness. This is in accordance with UV-vis data. It is indicated that the adsorbed amounts and morphology properties of galactose-branched polymer multilayers can be controlled by adding various counterions into the polyelectrolyte solution, and the significant differences of these properties are of interest for permeability and mechanics studies and offer possible applications in membrane technology or drug delivery and targeting. Microcapsules on the Basis of PGEDMC. Direct visualization of (PGEDMC/PSS)4-coated PS particles and hollow capsules were provided by TEM measurements. Figure 9a shows the TEM micrograph of an uncoated PS particle (average diameter ) 1.85 µm, inset) and [PSS/PAH-(PSS/PGEDMC)4]-coated PS particles. The coated particles showed increased surface roughness compared to the PS particle. With increasing coated bilayers, the diameter of microparticles also increased. The average diameter of [PSS/PAH-(PSS/PGEDMC)4]-coated PS particles was 1.95 µm (data from measuring the diameters of >15 capsules), and the double-wall thickness of the capsules is about 100 nm. For a (PAH/PSS)5 capsule, the double-wall thickness is approximately 44 nm,3c corresponding to a bilayer thickness of 4.4 nm. The average film thickness for a PSS/PGEDMC bilayer is ca. 11.4 nm. This is probably due to a lower charge density of PGEDMC backbone as well as the bulky galactose side groups. A TEM image of (PSS/PAH-(PSS/PGEDMC)4) hollow capsules is shown in Figure 9b. Typical hollow capsules were observed with characterizations of folds, creases, and flattening, which were due to the drying procedure. From the image, it can be seen that hollow microspheres were prepared by exposing the PGEDMC/PSS-coated particles to THF to remove the PS core via permeation through the PGEDMC/PSS multilayer walls. The hollow microspheres kept the original shape of the PS templates. SEM investigation also proved that the creases and folds of the continuous polyelectrolyte films had an average template diameter

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Figure 10. SEM image of [PAH/PSS-(PGEDMC/PSS)4] hollow capsule with a PS template of about 4 µm.

Figure 11. CLSM images of (a) (PGEDMC/PSS)4-PGEDMC multilayers and (b) PNA binding quartz slides coated with (PGEDMC/ PSS)4-PGEDMC multilayers. The arrow indicates the edge of the quartz slide.

Figure 12. CLSM images of (a) PNA binding microcapsules coated with [PSS/PAH-(PSS/PGEDMC)4], (b) single PNA binding microcapsules, and (c) Con A binding microcapsules coated with [PSS/ PAH-(PSS/PGEDMC)4].

of 4 µm (Figure 10), which were induced by the collapse of the films after evaporation of the aqueous content. PNA Lectin Recognition of Galactose-Branched Multilayers and Microcapsules. Lectin recognition of the deposited carbohydrates on quartz slides and microcapsules was visualized by confocal laser scanning microscopy (CLSM) using FL-labeled PNA lectin. Peanut agglutinin is a 110 000 molecular weight protein composed of four identical subunits, each of which can specifically combine with one galactose molecule. Fluorescence images were observed along the whole surface of the coated slides (Figure 11b), which showed the specific precipitation of lectin through the affinity with carbohydrate ligands along the polymer chain, compared to a black background of (PGEDMC/ PSS)4-PGEDMC before lectin binding (Figure 11a). Strong and specific response of PNA to the grafted galactose was also observed on the PGEDMC-assembled microcapsules. Figure 12 shows high fluorescent intensity at the surface of (PSS/ PGEDMC)4-PNA particles due to the adsorption of FL-PNA on the galactose residue-modified multilayers. In PBS buffer solution, the capsules appeared in most of the cases as a bright green ring with weaker fluorescence inside (Figure 12a,b). Compared with a good monodispersion of capsules before recognition, there was an obvious aggregation of particles after

addition of PNA. Single capsules were “cross-linked” by specific lectin affinity with galactose and gathered as some particle aggregations. The free PNA lectin was not observed, indicating that the free PNA molecules had been removed through rinsing and lectin molecules were specifically and firmly bonded to the microcapsules. In the control experiment of FL-Con A lectin recognition of (PSS/PGEDMC)4 capsules (Figure 12c), a very weak fluorescence signal was observed by CLSM under the same experimental conditions, resulting from much less FL-Con A lectin absorption. It was proved that the galactose groups of PGEDMC retained their biological ability during LbL assembly with polyanions. Combining the preparation of hollow capsules with the specific recognition of β-D-galactose residues for the asialoglycoprotein receptor, these multilayers had the potential for further studies of hepatocyte-targeting drug controlled delivery systems.

Conclusions This paper has demonstrated that a new bioactive polyelectrolyte containing galactose residues can be well assembled with anionic polymers on both planar substrates and colloid particles by LbL techniques. The nonreducing terminal D-galactose linking to the backbone of the polymer was specifically and strongly

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recognized with PNA lectin rather than nonspecific lectin of Con A observed by fluorescence spectroscopy, which can be used as an important recognition molecule for targeting drugcontrolled delivery systems. The film thickness and roughness of (PGEDMC/PSS) multilayers could be tailored by varying PE concentration, salt concentration, and counteranion types. Hollow capsules were successfully prepared by alternating deposition of PGEDMC and PSS on PS particles followed by template removal with THF. The formation of multilayers, especially on colloidal

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particles, is of great interest for encapsulation and release of a variety of substances such as organic dyes, drugs, and biomolecules. Studies are in progress concerning the specific interaction between the capsules containing β-D-galactose residues and HepG2 cells mediated by asialoglycoprotein receptor ASGP-R. Acknowledgment. Financial support from the National Natural Science Foundation of China is gratefully acknowledged. LA060847U