Surface-Modification of Polyelectrolyte Multilayer-Coated Particles for

Max Planck Institute of Colloids and Interfaces, D-14424 Potsdam, Germany, and ... the modification of the PSS/4G PAMAM multilayer coatings on the par...
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Langmuir 2003, 19, 6219-6225

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Surface-Modification of Polyelectrolyte Multilayer-Coated Particles for Biological Applications Ajay J. Khopade† and Frank Caruso*,‡ Max Planck Institute of Colloids and Interfaces, D-14424 Potsdam, Germany, and Department of Chemical and Biomolecular Engineering, The University of Melbourne, Victoria 3010, Australia Received January 21, 2003. In Final Form: April 24, 2003 Layer-by-layer multilayer films of poly(styrenesulfonate) (PSS) and 4th generation poly(amidoamine) dendrimer (4G PAMAM) assembled on particle supports were modified by a stepwise process involving (i) cross-linking of free amino groups of PSS/4G PAMAM multilayers using a bifunctional cross-linking agent, gluteraldehyde, which simultaneously chemically activates the surface, (ii) covalently linking the free aldehyde groups made available from the cross-linker with octadecylamine (fatty-acylation), and (iii) adsorbing a poly(ethylene glycol) (molecular weight ∼2000) derivative of distearylphosphatidylethanolamine (PEG2000-DSPE) on the fatty-acylated surface (PEGylation). Microelectrophoresis, confocal laser scanning microscopy, transmission electron microscopy, and differential scanning calorimetry were used to follow the modification of the PSS/4G PAMAM multilayer coatings on the particles. Analogous experiments were performed on planar supports (silicon, glass, and gold) to additionally quantify the process by Fourier transform infrared spectroscopy, atomic force microscopy, contact-angle, ellipsometry, and quartz crystal microbalance techniques. The experimental data from the colloid and planar supports were in mutual agreement, providing evidence for the stepwise surface modification of the multilayer films. Adsorption experiments involving biological cells or proteins and the surface-modified PSS/4G PAMAM multilayers were conducted to establish the surface biocompatibility of the modified films. Compared with the respective unmodified films, the surface-modified PSS/4G PAMAM multilayer-coated colloid particles showed reduced adhesion to the biological cell surfaces (macrophage cell line TPH-1), while the adsorbed amount of human serum albumin decreased upon exposure to the surface-modified PSS/4G PAMAM multilayer-coated planar substrates. This demonstrates the biocompatibility of the surface-modified multilayer films.

Introduction A number of promising carrier systems have been proposed for drug delivery, but the major hindrance to their clinical acceptance is their rejection by the body, which recognizes them as foreign entities. However, it is interesting that the endogenous carrier systems (biovectors) of the body, like lipoproteins1,2 (cholesterol and fat carriers), are not immediately recognized and, hence, are not rejected for days. The differences in the recognition processes are mainly dependent on the surface characteristics of these systems. The technological challenge, therefore, is to modify the surface properties of the carriers to mimic biovectors by blocking the recognition process. The supramolecular biovector3,4 (SMBV) is a drug delivery system that is engineered to imitate nanometer-sized lipoproteins. Lipoproteins consist of a fatty core stabilized by a phospholipid monolayer on the outer surface, which has embedded apoproteins, thus giving them a high circulation half-life in the body for the delivery of fat, other fatty precursors, or loaded bioactives to various sites.1,2 Along similar lines, SMBVs are prepared by grafting fatty * To whom correspondence should be addressed. Fax: +61 3 8344 4153. E-mail: [email protected]. † Max Planck Institute of Colloids and Interfaces. ‡ The University of Melbourne. (1) Biochemistry, 2nd ed.; Voet, D., Voet, J. G., Eds.; John Wiley and Sons: New York, 1995; p 316-322. (2) Lestavel-Delattre, S.; Matrin-Nizard, F.; Clavery, V.; Testard, P.; Favre, G.; Doualin, G.; Sqallihoussaini, H.; Bard, J. M.; Duriez, P.; Delbart, C.; Soula, G.; Lesieur, D.; Lesisur, I.; Cazin, J. C.; Cazin, M.; Fruchart, J. C. Cancer Res. 1992, 52, 3629. (3) Peyrot, M.; Sautereau, A. M.; Rabanel, J. M.; Nguyen, F.; Tocanne, J. F.; Samain, D. Int. J. Pharm. 1994, 102, 25. (4) (a) Nagaich, S.; Khopade, A. J.; Jain, N. K. Pharm. Acta Helv. 1999, 73, 227. (b) Shelly, U.; Khopade, A. J.; Jain, N. K. Pharm. Acta Helv. 1999, 73, 275.

acids on hydrophilic synthetic nanoparticle cores and further coating with a monolayer of phospholipids or poly(ethylene glycol) (PEG) phospholipid derivatives to mimic lipoprotein surfaces.3,4 One promising route to the preparation of carrier systems with engineered properties is via the layer-bylayer5 (LbL) templating of colloid particles.6 Over the past few years, the LbL assembly of oppositely charged species, including polyelectrolytes, nanoparticles, biomolecules, and metallosupramolecules, on colloid templates has been a major focus of our research.6 Our recent work has focused on the physicochemical characteristics of sequentially assembled multilayers of the linear polyelectrolyte, poly(sodium 4-styrenesulfonate) (PSS), and the hierarchical polyelectrolyte, 4th generation poly(amidoamine) dendrimer (4G PAMAM).7-9 These films were prepared with a view to develop novel drug delivery systems. We demonstrated that LbL assembled PSS/4G PAMAM multilayers could be used as nanoreservoirs for the loading and release of drugs when assembled onto planar and spherical supports.7 Additionally, they are suitable building blocks to form hollow capsules that can be subsequently loaded with low-molecular-weight drug compounds.7,9 The loading and release of the drugs could be tuned depending (5) (a) Decher, G. Science 1997, 277, 1232 and references therein. (b) Decher, G.; Hong, J. D. Macromol. Chem. Macromol. Symp. 1991, 46, 321. (6) (a) Caruso, F.; Caruso, R. A.; Mo¨hwald, H. Science 1998, 282, 1111. (b) Donath, E.; Sukhorukov, G. B.; Caruso, F.; Davis, S. A.; Mo¨hwald, H. Angew. Chem., Int. Ed. 1998, 37, 2202. (c) Caruso, F. In Nano-Surface Chemistry; Rosoff, M., Ed.; Marcel Dekker: New York, 2001; p 505. (d) Caruso, F. Adv. Mater. 2001, 13, 11. (e) Caruso, F. Chem. Eur. J. 2000, 6, 413. (7) Khopade, A. J.; Caruso, F. Nano Lett. 2002, 2, 415. (8) Khopade, A. J.; Caruso, F. Langmuir 2002, 18, 7669. (9) Khopade, A. J.; Caruso, F. Biomacromolecules 2002, 3, 1154.

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on a number of parameters, highlighting the potential of the capsules as drug delivery systems.9 Thus far, the surfaces of these systems have not been specifically and systematically modified with the view for their use in bioapplications. The application of the SMBV surface modification strategy to PSS/4G PAMAM multilayer films would be an important extension because of the interesting physiological consequences of these systems. For example, their interaction with biological surfaces, such as the corneal membrane, nasal, vaginal, and oral mucosa,10 could be controlled to increase the retention time and to improve subsequent delivery of the encapsulated core11 material on these surfaces. Thus, the carriers adhering to the biological surfaces are interesting for mucosal immunization when the core is comprised of an antigen and for ophthalmic and nasal pathologies when it is a bioactive drug. The surface modification of carriers with PEG (PEGylation) makes them less palatable to the phagocytes responsible for their clearance from the body12 and also less adhering to proteins and bacterial cells responsible for granuloma formation and infection, respectively, at an implant site.13 Thus, PEGylation of PSS/4G PAMAM LbL films on spherical and planar surfaces is useful for designing both StealthTM carrier systems12 and biocompatible implants,13 respectively. Building on our previous work,7-9 in this paper we investigate the surface modification of PSS/4G PAMAM multilayers assembled onto both spherical (particle) and planar supports through gluteraldehyde cross-linking, fatty acid grafting, and adsorption of a phospholipid derivative of PEG. We combine a range of techniques to follow the surface modification of the films step-by-step with surface characterization methods to examine their morphology. Further, we explore the influence of the surface modification of the PSS/4G PAMAM multilayers on their adhesion properties toward biological cells and model proteins. Experimental Section Materials. PSS, molecular weight (MW) ∼70 000, and poly(ethylenimine), MW ∼15 000, were obtained from Fluka. The 4G PAMAM, gluteraldehyde, octadecylamine (ODA), and dilauryl carboxyfluorescein were purchased from Sigma-Aldrich and were used without further purification. Human serum albumin (HSA) and other biochemicals and reagents for cell culture were also purchased from Sigma-Aldrich. The PEG (MW ∼2000) derivative of distearylphosphatidylethanolamine (PEG2000DSPE) was obtained from Lipoid GmbH. The melamine formaldehyde (MF) particles were purchased from Microparticles GmbH. Ultrapure water (Millipore) with a specific resistance of more than 18 MΩ cm was used in all experiments. Experiments were carried out at room temperature (25 °C). Formation and Surface Modification of Polyelectrolyte Multilayers. (a) Particles. The bare, positively charged MF particles were alternately incubated with polyelectrolyte solutions of PSS and 4G PAMAM (1 mg mL-1 containing 0.154 M NaCl) (10) Du, H.; Chandaroy, P.; Hui, S. W. Biochim. Biophys. Actass Biomembranes 1997, 1326, 236. (11) Other small-molecular-weight materials and enzyme crystals have been encapsulated by using the LbL strategy. (a) Caruso, F.; Trau, D.; Mo¨hwald, H.; Renneberg, R. Langmuir 2000, 16, 1485. (b) Caruso, F.; Yang, W.; Trau, D.; Renneberg, R. Langmuir 2000, 16, 8932. (12) (a) Bazile, D.; Homme, C. P.; Bassoullet, M. T.; Marlard, M.; Spentehauer, G.; Veillard, M. J. Pharm. Sci. 1995, 84, 493. (b) Illum, L.; Jacobson, L. O.; Mu¨ller, R. H.; Mak, E.; Davis, S. S. Biomaterials 1987, 8, 113. (13) (a) Uster, P. S.; Allen, T. M.; Daniel, B. E.; Mendez, C. J.; Newman, M. S.; Zhu, G. Z. FEBS Lett. 1996, 386, 243. (b) Laverman, P.; Brouwers, A. H.; Dams, E. T. M.; Oyen, W. J. G.; Storm, G.; Van Rooijen, N.; Corstens, F. H. M.; Boerman, O. C. J. Pharmacol. Exp. Ther. 2000, 293, 996. (c) Bradley, A. J.; Murad, K. L.; Regan, K. L.; Scott, M. D. Biochim. Biophys. ActasBiomembranes 2002, 1561, 147.

Khopade and Caruso Scheme 1. Schematic Illustration of the Preparation and Surface Modification of PSS/4G PAMAM Multilayer-Coated Colloid Particlesa

a (1) PSS/4G PAMAM multilayer deposition by the sequential adsorption of PSS and 4G PAMAM, with intermittent washing cycles. (2) Inter- and intramolecular crosslinking of amino groups within the multilayers using gluteraldehyde, which simultaneously activates the surface (due to the presence of unreacted aldehyde groups). (3) ODA grafting onto the crosslinked multilayers. (4) PEG2000-DSPE adsorption on the ODA anchor that facilitates adsorption through hydrophobic interactions. The colloid particle support shown can be replaced by a planar substrate (e.g., quartz, silicon wafer, or gold) to prepare the multilayer films (see text for details).

for 20 min, with three intermittent centrifugation/washing cycles with water to remove excess polyelectrolyte after each adsorption step. This resulted in the formation of multilayer-coated MF particles. Details may be found elsewhere.7,9 The particles were surface-modified according to the sequence illustrated in Scheme 1. First, the multilayer-coated particles [(PSS/4G PAMAM)5] were cross-linked by incubating the particle suspension (1 mL of ca. 0.5 wt %) in a 0.1% w/v gluteraldehyde solution for 6 h (step 2). Following cross-linking, the particles were washed with water. A total of 1 mL of an ODA solution (10 mg mL-1) in chloroform was then added to the suspension and shaken for ∼6 h, followed by three washing steps with pure chloroform (step 3). The particles were finally dispersed in water, and the dispersion was heated at 50-70 °C for another 1-2 h to remove excess chloroform. After cooling, the particles were dispersed in a PEG2000-DSPE solution (1 mg mL-1) and washed twice with pure water (step 4). Confocal micrographs were taken with a Leica (model TCS SP), equipped with a 100× oil immersion objective. The particles were labeled with fluorescent dyes by exposing them to a dye solution, followed by subsequent washing steps to remove the excess dye molecules. Transmission electron microscopy (TEM; Zeiss EM 912 Omega microscope operated at 120 kV) was used to examine the surface morphology of the particles before and after coating. The electrophoretic mobilities of the coated particles were measured using a Malvern Zetasizer 4.6 All measurements were performed in air-equilibrated pure water without added electrolyte. An average of five measurements at the stationary level were taken. The thermal properties of the fatty-acyl grafted PSS/4G PAMAM multilayer films were studied by differential scanning calorimetry (DSC) (Microcal Inc.). The phase transition behavior was monitored from 25 to 70 °C with a heating rate of 1 °C min-1. DSC of solid samples was performed by using a Netsch DSC 200 cell with a TASC 414/3 controller. The thermal transition behavior was recorded during the first heating. (b) Planar Supports. According to the previously reported method,7,8 multilayers were deposited by immersing substrates

Polyelectrolyte Multilayer-Coated Particles (appropriately treated gold or silicon to make them hydrophilic) alternately in 1 mg mL-1 polyelectrolyte solutions containing 0.154 M NaCl, followed by rinsing with water and drying with a gentle nitrogen gas stream after the deposition of each layer. The 10-layer film [(PSS/4G PAMAM)5], with an outermost 4G PAMAM layer, was dipped in a 0.1% w/v gluteraldehyde solution for 6 h, followed by washing and air-drying. The film was then dipped in an ODA solution in chloroform for another 6 h, followed by washing with chloroform. All the steps were performed at room temperature. The ODA-grafted multilayer film was then dipped in the solution of the amphipathic PEG2000-DSPE (0.1 mg mL-1, 20 mL) at 50-70 °C for 1-2 h to assist adsorption by hydrophobic interaction (PEGylation).14 The cross-linking and ODA grafting steps of the multilayer films on gold supports were monitored by Fourier transform infrared external reflection spectroscopy (FTIR-ERS) (Equinox 55/S, Bruker). The static contact angles between the sessile drop of water and the PSS/4G PAMAM films on a silicon wafer were measured by using a computer image analysis goniometer (Kreuss, Germany). The average of three measurements on five spots on the samples was taken. The quartz crystal microbalance (QCM) electrodes (9-MHz resonance frequency) were used to monitor in-air frequency changes after each adsorption and grafting step. The frequency difference was then used to determine the mass of material deposited after each step.15 Ellipsometry was performed using an optical null ellipsometer (Multiskop Ellipsometer, Optrel GmbH) with a He-Ne laser at 632.8 nm at an angle of 70°. The film thickness was calculated using a computer program, assuming a refractive index value of 1.47 for polyelectrolyte multilayers.8 Atomic force microscopy (AFM) images were recorded in air at room temperature (20-25 °C) using a NanoScope III Multimode AFM (Digital Instruments, Inc., Santa Barbara, CA) in tapping mode at a scan rate of 0.5-1 Hz. The images were processed using NanoScope software and Image PC software. Adhesion, Phagocytosis, and Protein Adsorption. A human monoblastoid cell line (TPH-1) suspension culture in a RPMI 1640 medium supplemented with 10 wt % fetal calf serum and 0.1 mg mL-1 of glutamine, mercaptoethanol, and gentamycin was used for studying adhesion and phagocytosis of the surfacemodified colloid particles labeled with dilaurylfluorescein. The cell lines were passaged three times per week and differentiated by exposure to 16 nM Phorbol Myristate Acetate for 18 h. Cells were pelleted by centrifugation and counted, and their viability was determined. The cell suspension (1 mL, 1 × 106 cells) was transferred to a 24-well culture plate (Corning, NY), and 50 µL (1 × 109 mL-1) of the dispersion of the PSS/4G PAMAM multilayer-coated and multilayer-modified particles was added to the cell suspension. The culture plates were incubated at 37 °C in an incubator at 95% relative humidity and 5% CO2 atmosphere. At hourly intervals, the cells were observed under a confocal microscope for colloid adhesion and phagocytosis. At least 100 cells were counted to determine the number of particles adhered to them by using Neubaur’s chamber9 (a hemocytometer used for red blood cell counting). Adhering particles could not be removed by dilution with a medium or by applying mild shear to the coverslip, whereas the nonadhering particles separated from the cell surface under these conditions. The adsorption of a model protein HSA was carried out by dipping unmodified and surface-modified PSS/4G PAMAM multilayer-coated QCM electrodes (gold-coated) in aqueous solutions of HSA of various concentrations (0.1-0.5 mg mL-1 in 0.154 M NaCl) for 30 min.

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Figure 1. DSC thermograms of pure ODA (dotted line) and ODA grafted onto PSS/4G PAMAM-coated particles (solid line).

Surface-Modification of PSS/4G PAMAM Films. In all cases, the multilayer films in this work were comprised of five pairs of PSS/4G PAMAM, that is, a [(PSS/ 4G PAMAM)5] film. The steps for surface modification of the PSS/4G PAMAM multilayer films consisted of glu-

teraldehyde cross-linking, octadecyl grafting, and PEG2000-DSPE adsorption (see Scheme 1). Treatment of PSS/4G PAMAM multilayers using a bifunctional crosslinking agent such as gluteraldehyde imparts two favorable properties: (i) stability of the PSS/4G PAMAM multilayer film due to inter- and intramolecular cross-linking16 and (ii) chemical reactivity to the surface due to the presence of unreacted aldehyde groups,17 which can then be utilized for additional chemistry (e.g., octadecyl grafting). The PEG2000-DSPE was adsorbed on the fatty-acyl surfaces produced via hydrophobic interactions between the phospholipid alkyl chains and the surface-grafted octadecyl chains to impart biocompatibility.12,13 It is wellknown that assembling a layer of closely packed hydrocarbon chains followed by exposure to either a dilute solution of emulsified lipids or vesicles facilitates the formation of surface-coupled bilayers.14 The amine groups of 4G PAMAM in the PSS/4G PAMAM multilayers (4G PAMAM outermost layer) were crosslinked to each other by gluteraldehyde. The covalent attachment of the amine groups of ODA with the aldehydeactivated PSS/4G PAMAM multilayer-coated particles was performed in water by the thorough shaking of the particles with ODA in chloroform. Qualitative observation of the interfacial behavior of the colloids revealed that the multilayer-coated particles initially accumulate at the airwater interface after cross-linking and then at the waterchloroform interface during reaction and in the washing steps with chloroform. Microelectrophoresis examination of the particles showed a decrease from 45 ( 3 to 19 ( 4 mV after cross-linking, to 12 ( 9 mV after ODA attachment. This clearly indicates surface modification of the colloid particles. Confocal laser scanning micrcoscopy (CLSM) experiments of the ODA-grafted particles, exposed to the hydrophobic dye dilaurylfluorescein, showed a fluorescent ring (not shown), suggesting the presence of hydrophobic alkyl groups on the surface of the colloid particles. The unmodified particle surface (control) did not show any fluorescent ring, indicating no significant dilaurylfluorescein adsorption. Additional studies by DSC on the ODA-grafted particles showed two endothermic peaks at 33.5 and 55.6 °C (Figure 1). These peaks may be

(14) (a) Liu, H.; Faucher, K. M.; Sun, X. L.; Feng, J.; Johnson, T. L.; Orban, J. M.; Apkarian, R. P.; Dluhy, R. A.; Chaikof, E. L. Langmuir 2002, 18, 1332. (b) Winger, T. M.; Ludovice, P. J.; Chaikof, E. L. Langmuir 1999, 15, 3866. (15) Caruso, F.; Niikura, K.; Furlong, D. N.; Okahata, Y. Langmuir 1997, 13, 3422.

(16) (a) Pastoriza-Santos, I.; Scho¨ler, B.; Caruso, F. Adv. Funct. Mater. 2001, 11, 122. (b) Dai, J. H.; Jensen, A. W.; Mohanty, D. K.; Erndt, J.; Bruening, M. L. Langmuir 2001, 17, 931. (17) Immobilized Affinity Ligand Techniques; Hermanson, G. T., Mallia, A. K., Smith, P. K., Eds.; Academic Press: London, 1992; p 69-79.

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attributed to the phase transition of ODA grafted to the polymer and the melting temperature of the ODA present as a result of intercalation in the grafted ODA, respectively. A sharp endothermic peak for pure ODA was obtained at 50.7 °C (Figure 1), corresponding to its melting temperature. The higher transition temperatures obtained for the ODA-grafted particles may be due to increased inter- and intramolecular interactions between the octadecyl chains in the presence of water.18 The DSC thermograms indicate the presence of a stable octadecyl phase after grafting. These results are consistent with those reported for other octadecyl-grafted comb polymers in an aqueous solution.19 Complementary qualitative and quantitative data on surface modification of PSS/4G PAMAM multilayer films were obtained by employing planar substrates. Contactangle measurements showed hydrophobization of the 4G PAMAM/PSS films as a result of the cross-linking and grafting steps.20 The contact angle increased from 27 ( 2° for the PSS/4G PAMAM multilayers to 55 ( 4° for the gluteraldehyde cross-linked films and to 94 ( 3° after ODA grafting. The contact angle is less than the 114° reported for an ODA monolayer.21 The QCM measurements revealed a surface coverage of the multilayer film by ODA (at saturation) of 58 ( 4%, calculated assuming an area per molecule of 0.24 nm2 for ODA. The ellipsometric data showed an increase in thickness of 1.6 nm, which is about 64% of the thickness of an ODA monolayer (ca. 2.5 nm). The contact-angle, QCM, and ellipsometry data are in mutual agreement, suggesting that for films prepared on planar supports there are ungrafted regions. Changes in the composition of the PSS/4G PAMAM films on a planar surface due to surface modification were additionally confirmed by FTIR-ERS. The bands observed at 1640 and 1550 cm-1 (amide I and amide II bands; Figure 2a, dotted spectra) and the broad band between 3100 and 3500 cm-1, due to primary amines and secondary amides within the dendrimer (Figure 2b, dotted spectra) for the non-cross-linked films, weakened after chemical crosslinking (Figure 2a,b, dashed spectra). The appearance of an imide band at 1725 cm-1 (Figure 2a, solid spectra) and -CH stretching bands at 2800-2900 cm-1 (Figure 2b, solid spectra) confirmed the attachment of ODA. The morphology of the surface-modified multilayercoated particles was examined by TEM. Figure 3 shows a TEM image of MF particles coated with cross-linked PSS/4G PAMAM multilayers. Compared with non-crosslinked multilayer-coated or uncoated particles, these particles display a higher surface roughness. This aspect was investigated in detail on PSS/4G PAMAM multilayer films formed on planar surfaces by AFM because AFM allows the observation of changes in the surface topography at the nanometer level. In agreement with our earlier reports,7,8 AFM revealed an average root-meansquare (rms) roughness value (1 × 1 µm2) of 0.7 nm for the PSS/4G PAMAM multilayer films. When the films were cross-linked at 50-70 °C for 2 h, the formation of distinct pores was observed and the rms value increased to 1.5 nm (Figure 4a). The formation of pores was most likely a result of a combination of both cross-linking of the dendrimer molecules and two-dimensional contraction due to improved hydrophobic interactions between the phenyl (18) Lee, J. W.; Park, J. K.; Lee, J. H. J. Polymer Sci., Part B: Polym. Phys. 2000, 38, 823. (19) (a) Park, J. H.; Lee, K. B.; Kwon, I. C.; Bae, Y. H. J. Biomater. Sci., Polym. Ed. 2001, 12, 629. (b) Inomata, K.; Sakamaki, Y.; Nose, T.; Sasaki, S. Polym. J. 1996, 28, 986. (20) Chen, J. Y.; Luo, G. B.; Cao, W. X. J. Colloid Interface Sci. 2001, 238, 62. (21) Lee, Y. L. Langmuir 1999, 15, 1796.

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Figure 2. FTIR-ERS spectra of PSS/4G PAMAM multilayer films on gold planar substrates at different stages of surface modification: dotted spectra, unmodified film; dashed spectra, gluteraldehyde cross-linked film; solid spectra, ODA-grafted film. Part a shows the spectra in the spectral range 1200-2000 cm-1, and part b shows the spectra in the range 2600-3600 cm-1. It is noted that complex reactions occurring during dendrimer cross-linking with polymers previously observed22 may be responsible for the apparent absence of the peak at 1640 cm-1 (part a, dashed spectrum).

groups of PSS in chloroform. An increase in the rms of the films (1.3 nm) and the formation of pores were also observed when the PSS/4G PAMAM films were heated at 70 °C for 2 h, with no cross-linking agent added. This points toward the heating being largely responsible for the roughness changes. It is also noted that heating leads to PAMAM dendrimer cross-linking as a result of retroMichael addition reactions.22 The increase in surface roughness of the coated colloids with cross-linked multilayer coatings is in qualitative agreement with the data obtained for the films on planar surfaces. Following ODA grafting onto the PSS/4G PAMAM multilayer-coated particles, the aqueous colloidal dispersion appeared curdy because the particles tended to aggregate as a result of hydrophobic interactions between the particles. However, this aggregation and fast sedimentation could be prevented by the presence of PEG2000(22) (a) Zhao, M.; Liu, Y.; Crooks, R. M.; Bergbreiter, D. E. J. Am. Chem. Soc. 1999, 121, 923. (b) Liu, Y.; Bruening, M. L.; Bergbreiter, D. E.; Crooks, R. M. Angew. Chem., Int. Ed. Engl. 1997, 36, 2114.

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Figure 3. TEM image of a gluteraldehyde cross-linked [PSS/ 4G PAMAM]5 film coated on MF particles. The surface roughness seen is attributable to the surface modification of the coating (see text for details).

DSPE in the aqueous phase, which suggested the formation of a PEG2000-DSPE coating over the fatty-acylated surface. The δ-potential for the particles decreased from 12 ( 2 to 5 ( 2 mV after PEG2000-DSPE coating. The existence of a small overall positive charge even after adsorption of neutral PEG2000-DSPE suggests that the polyelectrolyte underlayer contributes to the measured ζ-potential. On planar surfaces, PEG2000-DSPE adsorption led to a change of contact angle from 94 ( 3 to 37 ( 4°, suggesting that the hydrophilic PEG chains lie on the surface. A contact angle of 25 ( 2° has been reported earlier for a densely grafted hydrophilic PEG brush on a silicon surface.23 The higher contact-angle value obtained in our experiments suggests a nonbrush (i.e., pancake or mushroom) conformation24 of the PEG chains. The ellipsometry data showed a thickness increase of about 3.6 ( 0.2 nm after PEG2000-DSPE adsorption (air-dried films). A thickness in the range of about 2.2-6.5 nm (ellipsometry) has been reported for a PEG2000-DSPE monolayer deposited onto silicon substrates by the LangmuirBlodgett (LB) technique, depending upon the different surface pressures (PEG conformation) used for the monolayer deposition and the water content of the PEG chains.24 The thickness of a fully hydrated PEG layer adsorbed on a silane-modified silicon and quartz substrate from PEG2000-DSPE/distearoylphosphatidylamine mixtures, prepared by the LB method, is in the range 4.5-7.5 nm (measured by neutron reflectivity).25 The thickness depends on the amount of PEG2000-DSPE in the mixture, which is also related to the conformation of the extending PEG chains from the silanized surface. The thickness value obtained in our case indicates a noninteracting mushroom conformation of the PEG chains24,25 and correlates well with the contact-angle measurements. AFM examination revealed that the film becomes smoother upon PEG2000DSPE adsorption, with a rms value of 0.5 ( 0.1 nm (Figure (23) Alcantar, N. A.; Aydil, E. S.; Israelachvili, J. N. J. Biomed. Mater. Res. 2000, 51, 343. (24) Baekmark, T. R.; Elender, G.; Lasic, D. D.; Sackmann, E. Langmuir 1995, 11, 3975. (25) (a) Kuhl, T. L.; Majewski, J.; Wong, J. Y.; Steinberg, S.; Leckband, D. E.; Israelachvili, J. N.; Smith, G. S. Biophys. J. 1998, 75, 2352. (b) Majewski, J.; Kuhl, T. L.; Gerstenberg, M. C.; Israelachvili, J. N.; Smith, G. S. J. Phys. Chem. B 1997, 101, 3122.

Figure 4. AFM image of (a) a gluteraldehyde cross-linked [PSS/4G PAMAM]5 film on a silicon wafer, showing distinct pores, and (b) an ODA-grafted and PEG2000-DSPE-coated [PSS/ 4G PAMAM]5 film, showing a significant change in surface morphology as a result of further modification.

4b; versus the 1.5 nm for the non-PEGylated films), indicating a rather uniform distribution of PEG2000DSPE on the surface. A decreasing rms value (in the range of ∼1.0-0.2 nm for a 1 × 1 µm2-area) has been reported for a Langmuir mixed monolayer film of PEG-grafted phospholipid and pure phospholipid on a planar substrate in a dried state with an increasing concentration of PEGgrafted phospholipid in the mixed monolayer.26 The rms value of ∼0.5 nm, obtained for 5 mol % PEG-grafted phospholipid in the mixed monolayer,26 corresponds to a weakly interacting mushroom conformation (based on neutron reflectivity and ellipsometry studies with 4.5 mol % PEG-grafted phospholipid) of the PEG chains on the surface.25 Thus, the AFM data from our studies can be interpreted as suggesting a uniformly distributed mushroom conformation of the PEG chains. The contact-angle, ellipsometry, and AFM data obtained are in mutual agreement. Biocompatibility Studies of PEGylated PSS/4G PAMAM Multilayer Films. We explored the biocompatibility of the PSS/4G PAMAM multilayer-coated colloids as a function of cell adhesion using a macrophage (26) Kim, K.; Kim, C.; Byun, Y. J. Biomed. Mater. Res. 2000, 52, 836.

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Khopade and Caruso

Figure 6. Adsorption behavior of a model protein, HSA, on unmodified and surface-modified PSS/4G PAMAM films prepared on planar substrates (gold-coated QCM electrodes) as a function of the HSA concentration. The square, circle, and triangle data points represent unmodified, ODA-grafted, and PEG2000-DSPE-coated PSS/4G PAMAM films, respectively. The lines drawn are to guide the eye.

Figure 5. CLSM image of biological cells with adhered [PSS/ 4G PAMAM]5-coated MF particles in (a) transmission and (b) fluorescence modes. The arrows indicate fluorescence (in part b) due to the cell-adhered labeled particles. Cells could not be observed in this mode. The cell adhesion studies were performed using a human monoblastoid cell line (TPH-1) suspension culture in a RPMI 1640 medium. The image was taken after an incubation time of 2 h. It is noted that there were more than 15 particles adhered to each cell but only a few particles are seen in the image shown because it was captured at a particular focal plane.

data indicate that there was a decrease in adhesion of the PEG2000-DSPE-modified coated particles to the cell membrane compared with that of the control particles. This further supports the PEGylation of the cross-linked and activated multilayer-coated particles. To further demonstrate the compatibility of PEG2000DSPE-modified multilayer surfaces with biological systems, adsorption studies with the model protein, HSA, were conducted on PSS/4G PAMAM films that were crosslinked, alkylated, and PEG2000-DSPE-coated on planar supports. The adsorption amount initially increased with an increasing concentration of HSA and at higher HSA concentrations reached a saturation value (Figure 6). The adsorption of HSA at saturation decreased from 2.0 mg m-2 for the unmodified polyelectrolyte multilayer film to about 0.5 mg m-2 for the PEG2000-DSPE coated surface. This is due to the steric repulsion of proteins by the hydrophilic PEG chains present on the surface, which is in agreement with the earlier contact-angle measurements and earlier reports on the binding of proteins onto PEGcoated surfaces.27 It can be concluded that the surface modification of PSS/4G PAMAM multilayers yields a protein-retarding surface that would help combat undesired biological processes, such as the phagocytosis of colloidal carriers and granuloma formation or infection at an implant site, demonstrating the biological compatibility of these surface-modified multilayer films. Conclusions

cell line TPH-1. The unmodified multilayer-coated particles (control) adhered to cell lines (biological cells are negatively charged) almost immediately, probably as a result of their positive charge (Figure 5). Almost all of the particles (>99%) adhered to the cell lines after 2 h of incubation. Each cell was coated with more than 15 particles. However, there was no indication of phagocytosis (i.e., engulfment of particles by the macrophages), probably as a result of the large particle size. After 4 h, some of the cells (