Bioactive multilayer thin films of charged N,N-disubstituted hydrazine phosphorus dendrimers fabricated by layer-by-layer self-assembly

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Thin Solid Films 516 (2008) 1256 – 1264 www.elsevier.com/locate/tsf

Bioactive multilayer thin films of charged N,N-disubstituted hydrazine phosphorus dendrimers fabricated by layer-by-layer self-assembly Jose-Luis Hernandez-Lopez a , Hwei Ling Khor a , Anne-Marie Caminade b , Jean-Pierre Majoral b , Silvia Mittler a,1 , Wolfgang Knoll a,⁎, Dong Ha Kim c,⁎ a Max-Planck-Institut für Polymerforschung, Ackermannweg 10, 55128 Mainz, Germany Laboratoire de Chimie de Coordination, CNRS UPR 8241, 205 Route de Narbonne, 31077 Toulouse Cedex 04, France Division of Nano Sciences and Department of Chemistry, Ewha Womans University, 11-1 Daehyun-Dong, Seodaemun-Gu, Seoul 120-750, Korea b

c

Received 27 May 2006; received in revised form 27 March 2007; accepted 18 May 2007 Available online 5 June 2007

Abstract Charged N,N-disubstituted hydrazine phosphorus-containing dendrimers are deposited either as alternate all-dendrimers multilayers or alternating with linear polymers on 3-mercaptopropionic acid or 3-aminopropyldimethylethoxysilane coated surfaces via electrostatic layer-bylayer self-assembly. The behavior of the film formation is investigated by surface plasmon resonance spectroscopy and ellipsometry. Fetal cortical rat neurons were cultured on the dendrimer films in order to investigate the influence of the surface charge of the outermost layer on their adhesion and maturation. It was found that neurons attached preferentially and matured slightly faster on film surfaces terminated with positively charged dendrimers than on negatively charged surfaces. © 2007 Elsevier B.V. All rights reserved. Keywords: Phosphorus dendrimer; Layer-by-layer self-assembly; Surface plasmon resonance; Cell adhesion

1. Introduction Dendrimers have attracted considerable attention as functional nanomaterials due to their perfectly branched architecture, monodisperse nature, and globular shape with high endgroup functionality [1–5]. This unique class of materials have served as valuable model compounds to study interdisciplinary research subjects in the field of encapsulation of guest molecules [6], optoelectronics [7,8], catalysis [9–11], biology [12– 14], photophysical processes [15–19], etc. Recently, there has been an increasing interest in using dendrimers as building blocks for the preparation of molecularly thin multilayer films on the basis of electrostatic layer-bylayer (LbL) self-assembly [20–32]. Most studies, however, have explored the preparation and structural investigation of ⁎ Corresponding authors. Knoll is to be contacted at Tel.: +49 6131 379 160; fax: +49 6130 379 360. Kim, Tel.: +82 2 3277 4517; fax: +82 2 3277 3419. E-mail addresses: [email protected] (W. Knoll), [email protected] (D.H. Kim). 1 Current address: Department of Physics and Astronomy, The University of Western Ontario, London, Ontario, Canada N6A 3K7. 0040-6090/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.tsf.2007.05.049

multilayers using poly(amidoamine) dendrimers combined with linear polyelectrolytes, biomolecules or inorganics, but less attention has been paid to their biomedical applications [26]. Within the last few years, polyelectrolyte multilayers (PEMs) have been exploited for the development of biofunctionalized coatings to enhance or resist cell adhesion by mimicking extra cellular matrix components. Rubner and coworkers have demonstrated that interactions of cells with PEMs based on linear polyelectrolytes could be modulated in correlation to the swelling properties of the films [33,34]. Similar issues have been addressed on poly(L-glutamic) acid containing PEMs by controlling the pH of the solutions or through crosslinking/functionalization of the film surfaces [35,36]. Control over the manner in which proteins and cells interact with surfaces is critical for the success of implanted biomedical devices. However, it has been shown that cellular behaviors depend on a complex combination of several parameters and a straightforward definition has not been made. Since the molecular architecture and chemical nature of dendritic polymers can be tailored with high precision in terms of rigidity, functionality, surface charge, surface free energy, roughness, and hydrophilicity, etc.,

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the development of dendrimer-based PEMs may facilitate the creation of versatile surface coatings by which the fundamental understanding of biomolecular recognition can be improved. Here, we report on multilayer thin films composed of hydrazine phosphorous-containing dendrimers or dendrimers/linear poly (styrene sulfonate) (PSS) fabricated by the LbL deposition strategy [37] and demonstrate the potential of such dendrimer PEMs as bioactive surfaces for the control of protein/cell adhesion. 2. Experimental details 2.1. Materials The synthesis of the N,N-disubstituted hydrazine phosphoruscontaining dendrimers of the 4th generation having 96 functional groups on the surface with either cationic (G4(NH+Et2Cl−)96) or anionic (G4(CH–COO− Na+ )96) character was described elsewhere [38–40]. The basic chemical structures of both dendrimers are shown in Fig. 1. Both materials were dissolved in ultra pure water at a concentration of 1.0 mg/ml or 2.9 × 10− 5 M. Dendrimer solutions of pH ∼ 5.5 were used for the studies of the film formation itself (Figs. 3–5). For cell adhesion experiments, the pH of the cationic and anionic dendrimer solutions was adjusted to ∼ 4.0 and 9.0, respectively (Figs. 6–8). Poly(styrenesulfonic acid sodium salt) (PSS) with a molecular weight of Mw = 77 400 g/mol was obtained from Fluka, Germany. Poly(allylamine hydrochloride) with a Mw of ca. 70 000 g/mol and 3-Mercaptopropionic acid (3-MPA) were obtained from Aldrich, Germany. Aminopropyldimethylethoxysilane (3-APDMES) was supplied by ABCR, Germany. Poly(diallyldimethylammonium chloride) (Mw ∼ 40 000–100 000 g/ mol) and poly-(ethylenesulfonic acid sodium salt) (Mw = 19 100 g/mol) were purchased from Mütek, Germany. Sodium chloride, sodium hydroxide, hydrochloric acid and ethanol (AR grade) were purchased from Riedel-deHaën, Germany. All chemicals were used without further purification. The ultra pure water used in this study was obtained by reversed osmosis

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(Milli-RO 35 TS; Millipore GmbH, Germany) followed by ionexchange and filtration steps (Milli-Q; Millipore GmbH, Germany). The specific resistance was better than 18.2 MΩ cm, and the total organic content was less than 10 ppb (according to the manufacturer). 2.2. Preparation of substrates Polished silicon wafers (one side polished, b100N orientation, doped with Boron to 1 Ω cm specific resistance) were purchased from CrysTec in Germany. Quartz slides were obtained from Heraeus (Jena, Germany). Prior to use they were cleaned according to the following procedures: immersion for 15 min each in a series of ultrasonically agitated solvents (acetone, ethanol, water) then for 15 min each in ultrasonically agitated a) 2% alkaline detergent solution (Hellmanex, Hellma, Germany; signification apparatus Super RK510, Sonorex, Germany), b) water and c) ethanol at room temperature. In between the sonication steps the samples were rinsed in ultrapure water. Finally the substrates were blown dry in a stream of nitrogen until further processing. In order to obtain a complete silanized surface, silicon and quartz substrates were dried at 100 °C in vacuum for 1 h and activated in a plasma reactor (Plasma System 200-G; Tepla, Kirchheim, Germany) under plasma normal conditions (90 Pa Ar and 10 Pa O2, 300 W) for the same time. This procedure rendered the surfaces hydrophilic, with a water–air contact angle of less than 10°. The substrates were placed in a closed glass vessel with 3-APDMES and heated to about 120–140 °C for 3 h. After reaction, the substrates were allowed to cool, ultrasonicated in ethanol for a few minutes, rinsed in ethanol and water and then stored for the adsorption experiments. This procedure rendered the surfaces hydrophilic, with an advancing water–air contact angle of 49 ± 2°. The self-assembled monolayers were made on thin-film gold (Au) surfaces prepared by thermal evaporation of Au (99.99%, Balzers) onto cleaned glass substrates (LaSFN9, Schott glass, Germany) in an evaporating chamber (Edwards, FL400) under ultra high vacuum at a pressure below 5 × 10− 4 Pa. After

Fig. 1. Structures of the N,N-disubstituted hydrazine phosphorus dendrimers: A) with the anionic end function (G4(CH–COO−Na+)96) and B) with the cationic end function (G4(NH+Et2Cl−)96).

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preparation, the Au-coated glass substrates were allowed to cool under vacuum for approximately 1 h and then used without delay for subsequent measurements. An Au-coated glass slide was placed on top of an o-ring in a poly(tetrafluoroethylene) flow cell forming a 500 μl chamber with two channels for injecting the sample or rinsing solution through the chamber. 2.3. Titration The amount of charged groups on the dendrimer as well as the isoelectric point was determined via polyelectrolyte titration. This method consists of the stoichiometric reaction of a polyanion with a polycation. The titration end point was determined by a particle charge detection apparatus of Mütek, Germany, whose operation mode is based on the electrokinetic occurrence of a flow potential [41]. It consists of a particle charge detector (Mütek PCD 03 pH) standard-equipped with a pH mini-electrode and a Metrohm Titrino 702 SM automatic titrator. The polymer solution to be titrated is placed in the device sample container. Some of the dendrimer molecules will stay in the bulk solution while a small fraction will adsorb on the container walls. An oscillating piston makes the dendrimer solution stream between the container wall and the piston itself. As a result of the solution movement and of the inertia of the molecules with respect to solvent, the diffuse part of the electrical double layer around the polymer molecules will be partly sheared off. This leads to a partial charge separation, which is measured as a potential between two Au electrodes on the wall of the sample container. Only the molecules adsorbed on the container wall will cause the potential measured by the electrodes. The titration end point was taken as the point of sign inversion of the streaming potential. In fact, if the polymer charge has been completely compensated, any excess amount of polyion added in the solution leads to a reversal of the measured streaming potential. 2.4. Surface plasmon resonance spectroscopy The behavior of film formation was characterized by surface plasmon resonance (SPR) spectroscopy on a home build spectrometer [42–44]. The Kretschmann configuration [45] is used with a 50 nm Au film evaporated onto a glass substrate, which is then optically matched (index-matching fluid with nD @ 25 °C = 1.700 from Cargille Laboratories) to the base of a 90 LaSFN9 glass prism (n = 1.850 at λ = 632.8 nm). Thus the surface plasmon is excited at the metal/dielectric interface, upon total internal reflection of the laser beam (HeNe, λ = 632.8 nm, power 5 mW) at the prism base. By varying the angles of incidence of the laser beam, a plot of reflected intensity as a function of the angle of incidence is obtained. The reflected intensity shows a sharp minimum at the resonance angle which depends upon the precise architecture of the metal/dielectric interface and is defined by the matching condition for energy and momentum between the evanescent photons and the surface plasmon. Adsorption processes occurring at the gold interface were followed in real time by selecting an appropriate angle of incidence and monitoring the reflected intensity as a function of

time. Knowledge of the form of the resonance curve allowed this intensity to be interpreted as a shift in the angle of resonance. From a Fresnel simulation to the resonance curve for bare Au surfaces, it is possible to obtain the dielectric constant and the thickness of the layer. Addition of a thin layer to the surface of the gold typically shifts the position of the resonance to a higher resonance angle, and fitting to this second curve determines the optical thickness, Δnd, of the layer. Although surface plasmon measurements allow for the determination of an average optical thickness of an adsorbed film, accurate conversion of this optical thickness to a geometrical thickness requires knowledge of the refractive index of the film, a parameter which depends on both the molecular composition of the film and the packing density. 2.5. Ellipsometry All ellipsometric measurements were performed with a polarizer–compensator–sample-analyser manual null ellipsometer [46] at a wavelength of λ = 632.8 nm in the dry state and an angle of incidence of 70°. Ellipsometry is a nondestructive and sensitive method to investigate interfaces and thin films based on the change of the polarization of a monochromatic light beam upon reflection off an interface. The change in the polarization is related to the optical and geometrical properties of the interface. In the case of a layer at the interface, the change in polarization is determined by the refractive indices of this layer. The change in polarization on a silicon wafer is measured in terms of two ellipsometric angles δΔ and δΨ. For very thin interfacial layers only the change in δΔ is significant. At least five different locations on each sample were measured and the average was calculated. 2.6. Contact angle measurement Advancing and receding contact angles of a water droplet on the films were measured using a contact angle microscope (Krüss G-10, Krüss GmbH, Hamburg, Germany) under ambient conditions, while the volume of the drop is increased or decreased at the minimum rate required for movement of the water/air/substrate triple line [47,48]. 2.7. Cell culture Glass coverslips were ultrasonically cleaned with 2% alkaline detergent solution (Hellmanex, Helma, Germany) for 15 min and rinsed thoroughly with Milli-Q water. Cleaned glass coverslips were then autoclaved and activated in a plasma reactor (Plasma System 200-G; Tepla, Kirchheim, Germany) under normal plasma conditions (90 Pa Ar and 10 Pa O2, 300 W) for 5 min prior to coating with sterile filtered (Minisart®, Sartorius AG, Germany) polyethyleneimine (PEI) and dendrimer solutions. An initial layer of positively charged linear polymer (PEI solution: 1 mg/ml) was coated on the glass coverslip before the build up of the alternate layers of anionic (G 4 (CH–COO − Na + ) 96 ) and cationic (G 4 (NH + Et 2 Cl − ) 96 )

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dendrimers (dendrimer solution: 1 mg/ml). The substrates were rinsed with Milli-Q water after each subsequent layer was adsorbed. For comparison, cleaned glass coverslips were also coated with linear-type poly-D-lysine (Sigma-Aldrich Vertriebs GmbH, Germany), a frequently used protein to coat surfaces for neuronal cultures, at a concentration of 0.1 mg/ml overnight. Cell culture was performed on 3 types of substrates, one group consisting of a final layer of the cationic functional group (8 dendrimer layers), the other group consisting of a final layer of anionic functional group (9 dendrimer layers) and a control group of poly-D-lysine coated substrates. Neurons were seeded on triplicates of each type of substrates for light and confocal laser microscopy studies.

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strate. 4 random areas of 240 μm × 200 μm from each image were chosen and analysed with ImageJ 1.37 h. Two morphological parameters were determined for the analysis. Neurons with dendrites longer than twice the length of the cell bodies were counted and expressed as a percentage of the total number of cells counted. The ratio of neuronal to nonneuronal cells on different substrates was also compared. The average neurite length per neuron was calculated by manually linearizing every segment of the neurite. Standard error was calculated and displayed in the bar chart. 3. Results and discussion 3.1. Fabrication and characterization of multilayers

2.8. Neuron isolation Cortical neurons were isolated from fetal CD rats after 18 or 19 days of gestation using methods modified from Brewer et al. [49]. Neurons were seeded on the glass coverslips coated with the corresponding dendrimer films at a density of 40 000 cells/ cm2. The cultures were maintained in Neurobasal™ medium (Gibco, Invitrogen GmbH, Germany) supplemented with B-27 supplement (Gibco, Invitrogen GmbH, Germany) and 0.5 mM L-glutamine (Gibco Invitrogen GmbH, Germany). Medium was changed every 2 days and the cultures were monitored up to 6 days by light microscopy (Olympus IX70, Olympus Optical Co., Germany). 2.9. Immunofluorescence Cultures were fixed with 4% paraformaldehyde with 3% sucrose (Sigma-Aldrich Vertriebs GmbH, Germany) in 0.1 M phosphate buffered solution (Gibco, Invitrogen GmbH, Germany) after 6 days and double immunostained against microtubule associated protein2 (MAP2) and Neurofilament 160 kD. Rabbit anti-microtubule-associated protein2 polyclonal antibody (Chemicon Europe Ltd, UK) and monoclonal antibody against Neurofilament 160 kD (Acris Antibodies GmbH, Germany) were both used at a dilution of 1:200 and incubated overnight at 4 °C. The cultures were then rinsed with phosphate buffered solution and incubated with donkey anti-rabbit antibodies conjugated to fluorescein (Chemicon Europe Ltd, UK) and anti-mouse conjugated to rhodamin (Chemicon Europe Ltd, UK) at a dilution of 1:100 overnight, counterstained with 4,6diamidino-2-phenylindole (Roche Diagnostics GmbH, Germany) and mounted on glass slides in fluorescent mounting medium (Dako Diagnostica GmbH, Germany). Samples were visualized using a confocal laser microscope (Carl Zeiss LSM 510, Carl Zeiss AG, Germany).

Titration measurements were performed in order to understand the effect of solution pH on the charge density for both types of dendrimers. The results from the respective titration experiments are shown in Fig. 2 as the percentage of surface charge versus pH of the solutions. As expected the surface charge decreases with increasing pH for cationic dendrimer, whereas an opposite behavior is observed for the anionic dendrimer. The crossing point of identical percentage of surface charges could be identified at a pH of ∼ 5.5 with ∼ 78% degree of charge. At first, the alternating deposition of the cationic dendrimer with an anionic linear polymer, poly(styrene sulfonate) (PSS), was performed. The in-situ kinetic mode SPR spectroscopy for the consecutive alternating deposition is shown in Fig. 3 starting with the surface functionalization of a gold (Au) layer with 3mercaptopropionic acid (3-MPA). The sharp increases in reflected intensity which is converted in Fig. 3A to the corresponding increase of the SPR resonance angle with the addition of the solutions indicate the starting points of the adsorption of each molecular layer. The broken and the open arrows indicate the injection of dendrimer and the PSS solution, respectively, whereas the thin arrows pointing upwards represent the rinsing protocol with Milli-Q water for removing any physisorbed

2.10. Morphological analysis of cell behavior Images of neurons fixed and stained for MAP2 and neurofilament 160 kD as described in the section above were used for morphological analysis. The images were first transformed to gray level images and converted into binary images for further analysis. Images were taken from 3–4 replicates of each sub-

Fig. 2. Titration curves for the cationic (○) and the anionic (□) dendrimer.

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as the SPR spectroscopy. The decrease in the ellipsometric angle, Δ, a measure for the increase in optical thickness of the deposited film, is steeper for the first two functional layers than for the later deposited ones. The effective optical thicknesses are calculated using the refractive index of 3-APDMES n ∼ 1.457. The change in film thickness as a function of the number of deposition steps is displayed in Fig. 4B. The film thickness was found to increase nearly linearly to the number of deposited layers beyond the third layer, and the increment in thickness per each bilayer was measured to be Δd ∼0.8 ± 0.2 nm. Next, the alternating deposition of positively and negatively charged dendrimers is also monitored by SPR spectroscopy, as shown in Fig. 5A. The open and broken arrows indicate the injection of positive and negative dendrimer solution, respectively, whereas the thin arrows pointing upwards show the rinsing step in between. In comparison with the behavior shown in Fig. 3, both materials contribute equally to the increase in optical thickness as evidenced by the regular increase in the accumulated intensity shift with the number of deposition steps (Figure not shown). The corresponding thickness values determined from the fitting are shown in Fig. 5B. The average thickness per bilayer, G4

Fig. 3. A) Time resolved alternate deposition of the cationic dendrimer and PSS measured by the kinetic mode surface plasmon resonance spectroscopy. B) Optical thickness versus the number of deposited layers (▴: 3-APDMES; □: PSS; ●: cationic dendrimer).

material leading to a small decrease in intensity. The adsorption and desorption behavior for both the dendrimer and the PSS are rather regular with the dendrimer layer leading to a higher increase in reflectivity than the PSS layer. The consecutive repetition of these alternating deposition steps leads to the formation of multilayers with a very regular signal increment, which could be more clearly seen when the accumulated intensity shift is plotted versus the number of deposited layers (data not shown). The corresponding thickness values can be estimated from Fresnel fitting to the data with the refractive indices of the dendrimer and PSS layers being n = 1.457 and n = 1.484, respectively, as summarized in Fig. 3B. The first bilayer, G4(NH+Et2Cl−)96/PSS, showed the biggest shift in the plasmon curve with a thickness of 14 Å. In order to evaluate the film thickness by other mean, ellipsometry measurement was carried out. To this end, the identical multilayers were fabricated onto oxide surfaces modified with 3-aminopropyldimethylethoxysilane (3-APDMES) onto which anionic PSS layer adsorbs first. The ellipsometric data obtained from these samples (Fig. 4) exhibit the same trend

Fig. 4. A) Ellipsometer angle θ versus the number of deposited layers of the cationic dendrimer and PSS. B) Ellipsometric thickness versus number of deposited layers (▴: 3-APDMES; ○: PSS; □: cationic dendrimer).

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crease because the dendrimers cannot smear out like a linear chain-polymer. The average thickness of the bilayer measured by different means was of the same order with a slight discrepancy which may be attributed to the different parameters involved in each method, e.g., underlying substrates, refractive index, dry/wet environment, etc. The contact angles of the surfaces of the films fabricated in this study were measured with Milli-Q water to provide supplementary information about the surface properties and the results are summarized in Table 1. The clean silicon wafer exhibits a very hydrophilic character with a contact angle smaller than 10°. The contact angle of a self-assembled monolayer of 3-APDMES increases, but still yields a hydrophilic surface. Both the monolayers of G4(NH+Et2Cl−)96 and G4(CH– COO−Na+)96 exhibit hydrophilic surfaces with contact angles of θ = 59° and θ = 67°, respectively. The hysteresis – a measure for the roughness of the surface – increases from the clean silicon wafer surface to the surface functionalized by 3APDMES and finally the dendrimer layers. 3.2. Cell adhesion Electrostatic LbL assembly allows us to prepare nanoscale coatings on heterogenous biological surfaces. Application of

Fig. 5. A) Time resolved alternate deposition of the cationic and the anionic dendrimer measured by the kinetic mode surface plasmon resonance spectroscopy. B) Optical thickness versus the number of deposited layers (▴: 3-MPA; ○: cationic dendrimer; ■: anionic dendrimer).

(NH+Et2Cl−)96 + G4(CH–COO−Na+)96, was evaluated to be d ∼17.5 Å. From the above results, it was found that the deposition of a charged linear polymer on the oppositely charged dendrimer led to a smaller increase in optical thickness. This can be interpreted in terms of the filling of the interstitial sites of the dendrimer layer by the linear polymers, whereas the pure dendrimer deposition of opposite charge leads to a higher thickness inTable 1 Contact angle of water on various surfaces Types of surfaces

Advancing Surface free Receding Contact angle angle [°] energy (σsv) angle [°] hysteresis [mJ/m2] (Δθ) [°]

Silicon wafer b10 Silicon wafer with 3- 49 ± 2 APDMES G4(NH+Et2Cl−)96 59 ± 1 67 ± 1 G4(CH–COO−Na+)96

– –

b10 37 ± 5

– 12

48.3 43.3

40 ± 3 49 ± 1

19 18

The surface free energy was calculated from the advancing contact angle using the Neumann's equation-of-state approach [46,47].

Fig. 6. Cortical neurons from fetal CD rats were cultured for 5 days on both positively (A) and negatively (B) charged surfaces. (A) Neurons were better attached and proliferated faster on the positively charged surface (A).

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Immunostaining performed on the positively charged substrates showed that neurons after 5 days in culture expressed MAP2, a microtubule associated protein found in developing dendrites (red) (Fig. 8). It was observed that 66% (3 samples) of the neurons on the positively charged surfaces (Fig. 8A) had

Fig. 7. Delicate linear neuronal extensions were observed branching out perpendicular to the main neuronal extension of neurons cultured on positively charged surface after 5 days.

such nanoscale coatings ranged from bioactive [50,51] to bioinert [52] functions with the latter giving rise to control of cell adhesion by patterning cell adhesion promoting regions [33,34]. Coatings with tunable cell adhesion properties for example by varying the pH during assembly can be created [35]. Richert et al. and other researchers have demonstrated that layers with high wettability increased cell resistivity [35,52]. Most of the LbL system tested in cell culture was based on linear polyelectrolytes. Initial in vitro studies demonstrated that higher initial cell attachment was observed on the substrates with G4(NH+Et2Cl−)96 as the outer most layer (net positive charged surface). More neurons were adhered to the surface with a net positive charge and proliferated at a faster rate than those cultured on the substrates with G4(CH–COO−Na+)96 as the outer most layer (net negative charged surface) (Fig. 6A and B). This observation agrees with what was observed with poly-lysine, a frequently used protein to improve adherence of neurons on surfaces. Poly-lysine also carries a net positive charge. Neurons preferentially attached to positively charged surface through means of electrostatic interaction between anionic sites located on lipids, proteins and/ or polysaccharides of plasma membranes and cationic sites of the surface. Higher connectivity of neuronal networks was thus, observed on positively charged surfaces compared to the negatively charged surfaces. An interesting feature was observed at higher magnification, thin linear neuronal extensions (arrows) were observed branching out perpendicular at regular intervals from the main neuronal extension (labeled as A in Fig. 7). These thin linear neuronal extensions were observed at isolated regions randomly distributed on both positively and negatively charged surfaces. The charged N,N-disubstituted hydrazine phosphoruscontaining dendrimers were rigidly built up as reported in the previous section when a more significant increase in optical thickness was observed with the deposition of only dendrimer layers. This results in an imperfect covering of the alternate layers creating topographical inhomogeneity to which the neurites align. It has been widely reported that cells were sensitive to topographical signals from the external environment [53–55].

Fig. 8. Immunofluorescence staining showed that neurons displayed normal dendritic (MAP2, red) extension and delicate neurofilament (Neurofilament 160 kD, green) on the positive dendrimer (A), negative dendrimer (B) and polyD-lysine coated (C) substrates after 5 days. Cell aggregates of more than 5 neurons were observed to form more frequently on the negatively charged surfaces. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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4. Conclusions

Fig. 9. A bar chart displaying the average neurite length per neuron on glass slides coated with final cationic layer (MJ+), final anionic layer (MJ-), poly-Dlysine (PDL).

dendrites two times longer than their cell bodies as compared to 62% (4 samples) on the negatively charged surfaces (Fig. 8B). There were also 9% more non-neuronal cells on the positively charged surface. The non-neuronal cells which should not overgrow the culture are a consequence of the primary cell culture and are necessary for the survival of neurons. A higher incident of non-neuronal cells could also enhance the maturity of neurons on the positively charged surfaces. LbL assembly of dendrimers increased the surface roughness with increased deposition as stated earlier resulting in an inhomogeneous exposure of oppositely charges. An overall slightly higher hydrophilicity of positively charged surfaces (59°) compared to that of negatively charged surfaces (67°) could have also enhanced cell adhesion and thus improved neuronal maturation. Cell aggregates of more than 5 cells were formed more frequently on negatively charged surface (Fig. 8B). Neurofilament (160 kD, green color in Fig. 8B) an intermediate filament found localized in axons during late postnatal development was also present as sinuous delicate short segments comparable with poly-lysine coated glass cover slips (Fig. 8C). It appeared that axonal development is still premature after 5 days of culture on both positively and negatively charged surfaces. The average neurite length per neuron on the positively charged surface after 2 and 5 days in vitro was comparable to that on poly-D-lysine (PDL) coated surfaces (Fig. 9). PDL is frequently used to coat the culture surface for the cultivation of neurons. On the other hand, the average neurite length per neuron on the negatively charged surface was drastically reduced after 2 days in vitro. After 5 days in vitro the average neurite length per neuron was not possible to be calculated as the neurons were mostly growing in aggregates such that individual neurons could not be made out clearly. The fetal cortical neurons preferentially adhered to the positively charged surface and matured at a slightly faster rate than those on the negatively charged surface.

We have demonstrated that multilayer thin films composed of pure cationic/anionic dendrimers and cationic dendrimer/ linear polyelectrolytes could be fabricated by layer-by-layer deposition using 10 N,N-disubstituted hydrazine phosphoruscontaining dendrimers as building blocks. The growth of multilayers of alternately charged dendrimers shows a regular linear increase in film thickness compared with the multilayers of linear polyelectrolyte and dendrimer, as evidenced by surface plasmon resonance spectroscopy and ellipsometry. Of the various issues that one can explore with the dendrimer containing multilayer films, biomolecular interaction was investigated using selected cells as a function of the number of bilayers and the surface charge. Fetal cortical neurons appeared to adhere and proliferate better and mature at a slightly faster rate on cationic dendrimer layer in our preliminary cell culture study. Further studies will be conducted with longer culture times to better understand the behavior of neuronal maturation and different types of cells using a wide range of model dendrimer systems. Acknowledgements This work was supported by the Seoul Research and Business Development Program (10816). Financial support from Deutsche Forschungsgemeinschaft (MU 334/22-2, and SFB 625) is gratefully acknowledged. Prof. J.-P. Majoral and Dr. A. M. Caminade thank CNRS (France) for financial support. The authors thank Melanie Jungblut at the Max Planck Institute for Polymer Research for the preparation of fetal cortical neurons. References [1] G.R. Newkome, F. Vögtle, C.N. Moorefield, Dendrimers and Dendrons, John Wiley and Sons, 2001. [2] J.M.J. Fréchet, D.A. Tomalia, Dendrimers and other Dendritic Polymers, John Wiley and Sons, 2001. [3] O.A. Matthews, A.N. Shipway, J.F. Stoddart, Prog. Polym. Sci. 23 (1998) 1. [4] F. Zeng, S.C. Zimmerman, Chem. Rev. 97 (1997) 1681. [5] J.-P. Majoral, A.M. Caminade, Chem. Rev. 99 (1999) 845. [6] J.F.G.A. Jansen, E.M.M. de Brabander-van der Berg, E.W. Meijer, Science 266 (1994) 1226. [7] V. Percec, M. Glodde, T.K. Bera, Y. Miura, I. Shiyanovskaya, K.D. Singer, V.S.K. Balagurusamy, P.A. Heiney, I. Schnell, A. Rapp, H.-W. Spiess, S.D. Hudson, H. Duan, Nature 419 (2002) 384. [8] J.L. Atwood, J.E.C. Davids, D.D. Macnicol, F. Vögtle, J.-M. Lehn, Comprehensive Supramolecular Chemistry, Pergamon Press, Oxford, 1996. [9] J.W.J. Knapen, A.W. van der Made, J.C. de Wilde, P.W.N.M. van Leeuwen, P. Wijkens, D.M. Grove, G. van Koten, Nature 372 (1994) 659. [10] D. Astruc, F. Chardac, Chem. Rev. 101 (2001) 2991. [11] A.M. Caminade, V. Maraval, R. Laurent, J.P. Majoral, Curr. Org. Chem. 6 (2002) 739. [12] S.-E. Stiriba, H. Frey, R. Haag, Angew. Chem., Int. Ed. Engl. 41 (2002) 1329. [13] J.D. Eichman, A.U. Bielinska, J.F. Kukowska-Latallo, B.W. Donovan, J.R. Baker Jr., in: J.M.J. Fréchet, D.A. Tomalia (Eds.), Dendrimers and other Dendritic Polymers, John Wiley and Sons, 2001, p. 441, Chaper 18. [14] P. Singh, in: J.M.J. Fréchet, D.A. Tomalia (Eds.), Dendrimers and other Dendritic Polymers, John Wiley and Sons, 2001, p. 463, Chapter 19.

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