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Control of drug accessibility on functional polyelectrolyte multilayer films
ARTICLE IN PRESS
Biomaterials 27 (2006) 4149–4156 www.elsevier.com/locate/biomaterials
Control of drug accessibility on functional polyelectrolyte multilayer films Constant Vodouheˆa,b,, Erell Le Guena,b, Juan Mendez Garzaa,b, Gregory Franciusa,b, Christophe De´jugnatc, Joe¨lle Ogiera,b, Pierre Schaafd, Jean-Claude Voegela,b, Philippe Lavallea,b, a INSERM Unite´ 595, 11 rue Humann, F-67085 Strasbourg Cedex, France Faculte´ de Chirurgie Dentaire, Universite´ Louis Pasteur, Strasbourg, F-67085, France c Max Planck Institute of Colloids and Interfaces, Golm/Potsdam, Germany d CNRS, UPR22, Institut Charles Sadron, Strasbourg, F-67083, France
b
Received 1 February 2006; accepted 16 March 2006 Available online 4 April 2006
Abstract A surface coating based on polylysine/hyaluronic acid multilayers was designed and acted as a reservoir for an antiproliferative agent, paclitaxel (Taxol). Absolutely no chemical modification of polyelectrolytes or of the drug was needed and the final architecture was obtained in an extremely simple way using the layer-by-layer method. The paclitaxel dose available for human colonic adenocarcinoma cells HT29 seeded on the films could be finely tuned. Moreover, the accessibility of the drugs was controlled by adding on the top of the drug reservoir a capping made of synthetic polyelectrolyte multilayers. This capping was also required to allow adhesion of HT29 cells. Paclitaxel activity was maintained after embedding in the polyelectrolyte multilayers and cellular viability could be reduced by about 80% 96 h after seeding. The strategy described in this paper could be valuable for various other drug/cell systems. r 2006 Elsevier Ltd. All rights reserved. Keywords: Paclitaxel; HT29 cells; Polyelectrolyte multilayers; Bioactive films
1. Introduction In the last two decades, much effort has been put forth to provide biomaterial surfaces bearing sophisticated properties [1]. In particular, protein adsorption control and cellular adhesion properties were deeply investigated [2,3]. With this goal in mind, surfaces have been equipped with specific peptides or proteins by direct grafting on the substrate while controlling the density and/or spatial orientation [4,5]. More recently, polymeric matrices have been designed as substrate’s coatings to include biological factors [6,7]. The final aim of such approaches consists of controlling the rate and selectivity of cellular adhesion. Corresponding authors. INSERM Unite´ 595, 11 rue Humann, F-67085 Strasbourg Cedex, France. Tel.: +33 3 90243378; fax: +33 3 90243379. E-mail addresses: [email protected] (C. Vodouheˆ), [email protected] (P. Lavalle).
0142-9612/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2006.03.024
Mediation of the cellular responses and subsequent processes such as inflammation and tissue regeneration around the biomaterial have been controlled using these methods [8,9]. To build up polymeric matrices to surfaces in a controlled manner, an approach was developed 15 years ago based on alternated deposition of polyanion and polycation layers leading to polyelectrolyte multilayer films also called layer-by-layer films (PEM or LbL films, respectively). This method has emerged as a new and simplified strategy to modify surfaces [10–15]. For conventional polyelectrolyte multilayer systems, the driving force of the build-up comes from the alternate overcompensation of the surface charges appearing after each new oppositely charged polyelectrolyte deposition. To confer specific biological activities to multilayered films one can incorporate peptides, proteins, hormones, growth factors or drugs while maintaining their native
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structures and their activities [8,16–20]. Following this scheme, films with effective anti-inflammatory [9,21] or anti-microbial [22,23] properties were recently constructed. Paclitaxel (Taxol) is a microtubule-stabilizing agent with efficient antiproliferative activity through antimitotic properties. This drug is one of the most potent chemotherapeutic molecule in particular in the treatment of breast and ovarian cancers [24]. Many studies have focused on the local delivery of paclitaxel from polymeric microspheres [25–28]. Paclitaxel has also been used in the treatment and prevention of restenosis after percutaneous coronary intervention. In particular, this drug leads to a drastic reduction of restenosis when applied to the stent surface [29–31]. Systems able to release antiproliferative agents from the stent itself are under development, the rapamycincoated stents having already entered the world market 3 years ago [32]. Luo and Prestwich [33,34] developed a prodrug consisting in paclitaxel bioconjugates with hyaluronic acid as a drug carrier. More recently, Thierry et al. [35] described a chemical modification of hyaluronic acid by grafting paclitaxel through a labile succinate ester linkage. They developed films with chitosan and hyaluronic acid-paclitaxel using the layer-by-layer method. To incorporate specific molecules into polyelectrolyte multilayers and in particular to design films with antithrombotic properties, other approach used precomplexation methods of the drug with the polycations or the polyanions involved in the multilayers [36]. Following this strategy an efficient entrapment is then obtained especially in the case of hydrophobic molecules [37]. In this study, we propose to design a surface coating which acts as a reservoir for drugs like paclitaxel based on polylysine/hyaluronic acid (PLL/HA) multilayers. Absolutely no chemical modification of polyelectrolytes or of the drug is needed and the final architecture is constructed in a simple way. The paclitaxel dose available for human colonic adenocarcinoma cells HT29 can be finely tuned. Furthermore, the accessibility of the drugs can be controlled by adding a capping made of synthetic polyelectrolyte multilayers on top of the drug reservoir. Activity of paclitaxel against these cells was maintained after embedding in the multilayers. This capping is also required to allow adhesion of the HT29 cells. Finally, the strategy described in this paper could be valuable for various other drug/cell systems. 2. Experimental 2.1. Polyelectrolytes and drug solutions Poly(L-lysine) (PLL, Mw ¼ 58 900 Da), PLLFITC (Fluorescein isothiocyanate labeled poly(L-lysine) Mw ¼ 50 000 Da), poly(sodium 4 styrene sulfonate) (PSS, Mw ¼ 70 000 Da) and poly(allylamine hydrochloride) (PAH, Mw ¼ 70 000 Da) were purchased from Sigma (St. Quentin Fallavier, France). Hyaluronic acid (HA, Mw ¼ 411500 Da) was obtained from BioIberica (Barcelona, Spain). Rhodamine B (Invitrogen, Cergy Pontoise, France) was coupled to PAH as previously described [38]. An
appropriate amount of PAH (1 mg mL1) was dissolved in a Na2CO3 solution (0.1 M, pH ¼ 8.5). Rhodamine (Rho) at 2 mg mL1 was dissolved in dimethylsulfoxyde (DMSO). We used a ratio of 1 mg of rhodamine for 50 mg of PAH. The solutions were gently mixed at room temperature for 2 h. PAHRho was purified by dialysis against water during 3 days and then the absence of free rhodamine in the solution was verified by UV spectroscopy. Rhodamine-labelled PSS (PSSMRho) was synthesized adopting the procedure described elsewhere [39]. Typically, 0.516 g (2.5 mmol) of SS (4styrene sulfonate, sodium salt/Sigma) was first dissolved in 7.5 mL pure water. A solution containing 8.3 mg (0.0125 mmol, 0.5% mol with respect to SS) of MRho (methacryloxyethyl thiocarbamoyl rhodamine B/ Polysciences, Eppelheim, Germany) in 2 mL methanol was prepared and added to the previous SS aqueous solution. This mixture was flushed with nitrogen while stirring for one hour. Then 250 mL of a 0.1 M (1% mol) TMEDA (tetramethylethylenediamine/Sigma) aqueous solution and 250 mL of a 0.05 M (0.5% mol) (NH4)2S2O8 (ammonium peroxodisulfate/ Sigma) aqueous solution were added to the previous PSS-MRho mixture under nitrogen to initiate the polymerization reaction. The solution was heated to 40 1C for 4 h under nitrogen, and then stirred at room temperature for further 12 h. The final crude solution was dialyzed against water for 1 week and finally freeze-dried, resulting in a dark red solid. Polyelectrolyte solutions (1 mg mL1) were prepared by dissolving adequate amounts of polyelectrolytes in 0.15 M NaCl solution, pH ¼ 6.5. All solutions were prepared using ultrapure water (Milli Q-plus system, Millipore) with a resistivity of 18.2 MO cm. Paclitaxel, commercially known as Taxol (gift from Hoˆpitaux Universitaires de Strasbourg, France) was adsorbed overnight at adequate concentration on polyelectrolyte mutlilayer films. Films were then rinsed three times in NaCl 0.15 M (pH 6.5) followed by an optional deposition of PSS/PAH layers. Paclitaxel Oregon Green 488 labeled (paclitaxelGreen 488, Molecular Probes, Oregon, USA) was previously dissolved in ethanol/cremophor solution (v/v at a concentration of 500 mg mL1) and then adjusted at 5, 10, 50 and 200 mg mL1 with NaCl 0.15 M (pH ¼ 6.5).
2.2. Build-up of polyelectrolyte multilayer films The multilayered films were deposited using an automated dipping robot (Riegler & Kirstein GmbH, Berlin, Germany) to glass coverslips. The slides were first dipped for 10 min in a polycation solution. Then, a rinsing step was achieved by dipping the substrates for an equal period in a 0.15 M NaCl (pH ¼ 6.5) solution. The polyanion was deposited in a similar way and the build-up process was continued until the deposition of the required pair of layers. After the deposition of n poly(L-lysine)/hyaluronic acid layer pairs [denoted (PLL/HA)n] the build-up was pursued in some cases by a further deposition of PSS/PAH bilayers in the same conditions as for PLL/HA depositions. Typically a film constituting 30 PLL/HA layer pairs and capped by one PSS/PAH bilayer will be denoted as (PLL/ HA)30/PSS/PAH.
2.3. Confocal laser scanning microscopy Confocal laser scanning microscopy (CLSM) observations were carried out with a Zeiss LSM 510 microscope using a 40/1.4 oil immersion objective and with 0.4 mm z-section intervals. Green 488 fluorescence was detected after excitation at 488 nm with a cutoff dichroic mirror 488 nm and an emission band-pass filter 505–530 nm (green). Rhodamine fluorescence was detected after excitation at 543 nm, dichroic mirror 543 nm, and emission long pass filter 585 nm (red). An average of four images in the same location were acquired at 512 512 pixels. Virtual film section images were performed in liquid conditions (NaCl 0.15 M, pH ¼ 6.5).
2.4. HT 29 cell culture A human colonic adenocarcinoma cell line HT29 was kindly provided by Dr. M. Kedinger, (INSERM UMR-S 682, Strasbourg, France).
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12 µm
(A)
12 µm
They were routinely cultured in 25 cm2 culture flasks, in Glutamax I DMEM 25 mM glucose (Invitrogen, France), without sodium pyruvate, and supplemented with 10% v/v heat-inactivated fetal calf serum and penicillin streptomycin 100 U mL1 and 0.1 mg mL1 (Invitrogen, France). Cultures were incubated at 37 1C in a humidified atmosphere of air (95%)CO2 (5%). The medium was changed every 2 days. For subcultures, cells were incubated in 0.05% trypsin-0.53 mM EDTA solution and then harvested. The polyelectrolyte multilayer films were sterilized for 10 min by exposure to UV light (254 nm, 30 W, illumunation distance 20 cm). Cells were seeded at a density of 8000 cells mL1 in 24 well plates, containing precoated glass coverslips with polyelectrolyte multilayer films.
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2.5. Cell viability measurement (B)
12 µm
The measurement of viability by the acid phosphatase method allows to quantify the number of living cells with greater sensitivity than the XTT test. It consists in the titration of the enzyme whose activity increases proportionally with the number of viable cells. In our study, the culture medium was eliminated and each well washed with 300 mL PBS. The buffer used and added to each well contains 0.1 M sodium acetate (pH 5.5), 0.1% of X-100 Triton and 10 mM of p-nitrophenyl phosphate [pNPP] (Sigma). The plates were placed in an incubator for 3 h at 37 1C with the air (95%)–CO2 (5%) suitably humidified. The reaction was stopped by the addition of 1 N sodium hydroxide and the absorbance of the yellow colored solution was measured at 405 nm using a spectrophotometer (Labsystems, iEMS Reader MF, Gibco). Cell viability was measured at 24 and 96 h. Statistical analyses were performed using Sigma-Stat software.
(C) 25
10 µg mL-1 50 µg mL-1 200 µg mL-1
20
Build-up of multilayer films made of polysaccharides has been recently investigated, in particularly for the polylysine/alginate, [40,41] polylysine/chondroitin, [20] chitosan/hyaluronic acid [42,43] and polylysine/hyaluronic acid (PLL/HA) [44–47] systems. It happens that these films increase exponential in thickness according to the number of deposited layers. This exponentially increase is related to the diffusivity through the film section of at least one of the polyelectrolytes involved in the polycation/polyanion multilayer. In the PLL/HA system, PLL chains constitute the diffusing species. Typically in the presence of 0.15 M NaCl, several mm thick films were obtained after the deposition of 30 PLL/HA layer pairs. It was recently suggested that PLL/HA multilayers act as reservoirs for PLL chains [48,49]. In this study, we investigated first the paclitaxel uptake by PLL/HA films. Paclitaxel molecules fluorescently labelled with Oregon Green 488 molecules were used for confocal microscopy studies. It was previously shown that this molecule despite the labelling with the dye maintains its biological activity [50]. Fig. 1 reveals the results for various fluorescently green-labelled paclitaxelGreen 488 concentrations varying from 10 to 200 mg mL1. First of all, one observes that the paclitaxelGreen 488 molecules diffuse through the whole (PLL/HA)60 film section. In particular, for paclitaxelGreen 488 adsorbed from solutions at 50 and 200 mg mL1, the fluorescence is homogeneously distributed over the whole film thickness. The total thickness of the (PLL/HA)60 is approximately 12 mm as expected from previous studies. Increasing the paclitaxelGreen 488 concentration of the solution during deposition leads to a higher
Z distance (µm)
3. Results and discussion 15
10
5
0 0
(D)
50
100
150
200
250
Fluorescence intensity (u.a.)
Fig. 1. Confocal laser scanning (CLSM) images of (PLL/HA)60/ paclitaxelGreen 488 film sections. Paclitaxel Green 488 was adsorbed from solutions at 10 mg mL1 (A), 50 mg mL1 (B) and 200 mg mL1 (C). Image sizes are 76.8 23.9 mm2. From these images, distance Z as a function of the fluorescence intensity was plotted for the three paclitaxelGreen 488 concentrations. All CLSM settings are identical for the three images. Z ¼ 0 corresponds to the location of the glass coverslips on which the film was deposited.
amount of drug taken up as materialized by the increase in green intensity (Fig. 1(D)). We determined the paclitaxel concentration in the PLL/HA films from fluorescence intensities. For this purpose, a calibration curve was first established by imaging paclitaxelGreen 488 solutions at predetermined concentrations (see Fig. S1 in Supplementary materials section). When paclitaxel was deposited at 10 mg mL1, the concentration in the films was about 500 mg mL1, i.e. 50 times more than the concentration in solution whereas for the paclitaxel solutions at 50 and 100 mg mL1, the concentration in the films were of about
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1200 and 1900 mg mL1, respectively, thus about 20 times more concentrated in the film than in solution. From these data it is possible to plot the adsorption isotherm of the drug in the (PLL/HA)60 film (see Fig. S2 in Supplementary material section). It appears that the paclitaxel concentration within the films depends on the initial concentrations of paclitaxel in the solutions during deposition. However, for high solution concentrations (between 50 and 100 mg mL1) a saturation process should take place and films would probably not contain paclitaxel concentrations up to 2000–2200 mg mL1. Finally, the drug content in PLL/HA films can be finely tuned in a large range concentration. These behaviors would probably not be observed for linear growing multilayer films like PSS/PAH. Actually, such films exhibit very different properties compared to PLL/HA, in particular they are very dense, thin and the embedding of a biological factors often leads to a loss of their activity [17]. We also checked the evolution of fluorescence intensity with time and found that it remained constant after being stored for four days at ambient temperature in NaCl 0.15 M, pH ¼ 6.5. This indicates that no or very low passive release occurs in a short time span. To control the efficiency of the paclitaxel embedded into polyelectrolyte multilayers, viability of HT29 cell line cultured on films with various architectures was tested. For all of the biological tests presented below, paclitaxel solutions were maintained at 5 mg mL1 during adsorption and the PLL/HA films were constituted of 30 bilayers. Further investigations are underway to study the efficiency of films containing various paclitaxel concentration on cell viability. From the adsorption isotherm of paclitaxel in PLL/HA films (see Fig. S2 in Supplementary material section), and if we consider that the relationship between bulk concentration and film concentration is linear for the low bulk concentration studied, it was possible to estimate that the amount of paclitaxel in the PLL/HA film was of about 250 mg mL1. The first viability tests demonstrated that HT29 cells cultured on (PLL/HA)30 films were not viable probably due to weak adhesion on such films. This could be attributed to the mechanical properties of the PLL/HA multilayers as previously described in the case of adhesion on such films of chondrosarcoma cells [51] or osteoblasts [52]. These films exhibit very low stiffness properties due to the high water content and as a consequence cells could not develop focal spots and thus did not adhere to the films [53,54]. In the present study, we wish to use polyelectrolyte multilayers as a kind of trap against HT29 cells: cells should first adhere on top of the film and then they should internalize paclitaxel to become apoptotic. We tried to modify the film surface by the addition of a PSS layer or PSS/PAH multilayers on top of PLL/HA films (Fig. 2). As described previously by Kidambi et al. [55] in the case of hepatocytes, sulfonate groups of PSS chains adsorbed on the surface promote cellular adhesion. This was also recently confirmed for motoneuron cells which
Fig. 2. Confocal laser scanning (CLSM) images of (A), (PLL/HA)29/ PLLFITC/HA/PSSMRho, (B), (PLL/HA)29/PLLFITC/HA/PSSMRho/PAHRho/ PSSMRho, (C), (PLL/HA)29/PLLFITC/HA/(PSSMRho/PAHRho)2/PSSMRho film sections. Image sizes are 13.4 29.4 mm2.
preferentially spread and adhere to PSS ending multilayer films [19]. With this in mind, we investigated the influence on cell viability of PSS chains deposited on a non-adhesive PLL/HA film. Despite the negative charge of the PSS chains and the negative surface charge of the (PLL/HA)30/PLLFITC/HA films [44], we observed the deposition of a PSS layer on top of this film (Fig. 2(A)). The whole film section is green due to PLLFITC (PLL-fluorescein isothiocyanate) diffusion [45] and a thin red line corresponding to PSSMRho (methacryloxyethyl thiocarbamoyl rhodamine B) is seen on top of the film. Some exchange mechanisms between the HA chains adsorbed on top of the film and the new incoming PSS chains probably take place. However the PSS chains didn’t diffuse into the PLL/HA film and seem to be adsorbed only on the upper part of the film. Adding PAHRho/PSSMRho or (PAHRho/PSSMRho)2 to the (PLL/HA)30/PLLFITC/HA/ PSSMRho film leads to similar observations with the confocal microscope (Figs. 2(B) and (C), respectively). Adhesion of HT29 cells became significant once the top of the PLL/HA film was capped by a PSS layer (Fig. 3) as
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AP activity (%)
100
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(PLL/HA)30 ~ at 24h (PLL/HA)30 ~ at 96h ***
80
** ***
60
***
40 ** *** 20
/P SS PA H )2 (P SS / ~
~
PS S/
PA H
~
PS
/P SS
S
0
Fig. 3. Acid phosphatase (AP) activity for HT29 cells cultured on (PLL/ HA)30/PSS, (PLL/HA)30/PSS/PAH/PSS, (PLL/HA)30/(PSS/PAH)2/PSS films determined at 24 h (dark bars) and at 96 h (gray bars). Data is divided into three groups: (PLL/HA)30 multilayers capped by PSS, by PSS/PAH/PSS or by (PSS/PAH)2/PSS. Results are expressed as an average of four independent measurements. The error bars represent the standard deviation. The value of 100% has been arbitrarily attributed to AP activity of HT29 cells on (PSS/(PAH)2/PSS ending films after 96 h of seeding. No activity was determined for (PLL/HA)30 films without any capping because HT29 cells are not viable on these films. * po0.05, ** po0.01, *** po0.001 vs (PSS/PAH)2/PSS ending films.
suggested by the acid phosphatase (AP) activity determined at 24 and 96 h. The adhesion was more pronounced for the PLL/HA film capped by PSS/PAH/PSS, in particularly after 96 h of culture. Adding (PSS/PAH)2/PSS onto (PLL/ HA)30 films again resulted in a significant increase in AP activity at 96 h. This effect could be attributed to a change in chemical composition of the top of the multilayer film (PSS instead of HA chains) and/or to a change in the mechanical properties of the PLL/HA film that possibly stiffens after capping with PSS/PAH multilayers. Further investigations are underway to study long-term activity of the paclitaxel-embedded films on HT29 cells. We determined that deposition of PSS, PSS/PAH/PSS or (PSS/PAH)2/PSS on (PLL/HA)30/paclitaxel multilayers doesn’t allow the release of paclitaxel contained in the films. Fig. 4(A) shows an example of a green labelled (PLL/ HA)30/paclitaxelGreen 488 film capped with a red labelled PSSMRho/PAHRho/PSSMRho multilayer. PSS and PAH multilayers are well deposited on films containing paclitaxel which is maintained in the PLL/HA film. Same conclusions can be drawn for cappings made of only PSSMRho, or of (PSSMRho/PAHRho)2/PSSMRho (data not shown). Next we evaluated if the paclitaxel embedded in (PLL/ HA)30 film still remains active and can be sensed by HT29 cells through the PSS, PSS/PAH or (PSS/PAH)2/PSS cappings. The AP activities induced at 24 and 96 h are
Fig. 4. Confocal laser scanning (CLSM) images of (A), a (PLL/HA)30/ paclitaxelGreen 488/PSSMRho/PAHMRho/PSSMRho film section, (B), HT29 cell section 24 h after seeding on a (PLL/HA)30/paclitaxelGreen 488/ PSSMRho/PAHMRho/PSSMRho film, and (C), top view (x,y) of the same cell. Image sizes are 29.4 13.9 mm2 (A), 46.0 22.6 mm2 (B), and 22 22 mm2 (C). Paclitaxel is deposited on the (PLL/HA)30 film at 100 mg mL1.
shown in Fig. 5 (normalized values). It becomes clear that the effect of the paclitaxel embedded in the (PLL/HA)30 film capped with a PSS layer induces a 45% AP decrease in activity at 24 h and thus strongly affects cell viability. Paclitaxel is probably in an active and free conformation when embedded in the PLL/HA multilayers. This effect is more pronounced at 96 h where only 20% of the initial activity is maintained. If the (PLL/HA)30/paclitaxel film is capped with PSS/PAH/PSS multilayers instead of one PSS layer, the AP activity of HT29 cells is less strongly affected by paclitaxel at 24 and at 96 h (respective decreases of about 30% and 70%). When (PSS/PAH)2/PSS constitutes the capping, the efficiency of paclitaxel on the reduction of AP activity is only 20% at 24 h and 35% at 96 h. Clearly, the number of PSS and PAH layers constituting the capping strongly influences the cell accessibility for the underlying paclitaxel. We also confirm by confocal microscopy that cells will sense the paclitaxel -embedded PLL/HA film, even through a PSS/PAH multilayer (Fig. 4(B)). Cells grown on a (PLL/HA)29/PLLFITC/HA/ PSSMRho/PAHMRho/PSSMRho film internalized the green labelled paclitaxelGreen 488. However, no red labelled PSSMRho neither PAHRho were visualized in the cells. PaclitaxelGreen 488 appears strongly concentrated in the cytoplasm and in the nucleolus while the nucleus is less dyed (Fig. 4(C)). Identical fluorescence patterns were also observed with lung cancer cells H460 cells dyed with various fluorescent paclitaxel [56]. PSS and PAH being synthetic polymers, cells are probably not able to degrade the PSS/PAH multilayers [48]. However they could very likely form pseudopodes through a PSS/PAH bilayer to reach the paclitaxel molecules located in the PLL/HA film as has been previously observed with monocytic cells [8,9]. However such pseudopodes could not be clearly visualized in
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(PLL/HA)30 ~ as control (24h or 96h) (PLL/HA) /paclitaxel ~ at 24h 30
(PLL/HA)30 /paclitaxel ~ at 96h
***
***
100
AP activity (%)
*** 80
in cellular adhesion. These PSS/PAH multilayers also permit tuning the accessibility of HT29 cells to the paclitaxel in terms of delay and/or kinetic. The structures designed in the present study constitutes a new route to control and to adjust the accessibility of HT29 cells to paclitaxel. Furthermore, this approach could present attractive versatility in the future for other cell/drug systems.
** ***
Acknowledgements
60
40
*** 20
/P SS S/ PS ~(
~P
SS
/P
PA
A
H
)2
H
~
/P SS
PS S
0
Fig. 5. Acid phosphatase (AP) activity of HT29 cells cultured on (PLL/ HA)30/PSS, (PLL/HA)30/paclitaxel/PSS, (PLL/HA)30/PSS/PAH/PSS, (PLL/ HA)30/paclitaxel/PSS/PAH/PSS, (PLL/HA)30/paclitaxel/(PSS/(PAH)2/PSS and determined at 24 h (gray bars) and 96 h (dark gray bars). Data is divided into three groups: (PLL/HA)30 multilayers with or without paclitaxel capped with PSS, PSS/PAH/PSS or (PSS/PAH)2/PSS. For each group, cell activity on functionalized films with paclitaxel is normalized by the values on the same films without paclitaxel at 24 or 96 h (black bars, activity of 100%). Paclitaxel is deposited on the (PLL/HA)30 film at 5 mg/mL. Results are expressed as an average of four independent measurements. The error bars corresponds to the standard deviation. * po0.05, ** po0.01, *** po0.001 vs control (multilayers without paclitaxel).
Fig. 4(B) because PSS/PAH layers are extremely thin in regards to CLSM resolution. Time required to obtain such contact between cells and paclitaxel should depend on the number of layers constituting the capping. It is also known that deposition of at least five bilayers of PSS/PAH are required to fully covered a surface [57]. Thus only cells in the proximity of some uncoated PSS/PAH areas reach paclitaxel molecules. When the number of deposited PSS/PAH layers increases, the uncoated PSS/PAH areas rarefied and the probability for cells to contact the PLL/HA film containing paclitaxel will then be reduced. 4. Conclusion In the present paper, we design thick polyelectrolyte multilayers containing an active drug, paclitaxel. We demonstrate that the film can act as a reservoir for paclitaxel and that the amount of drug embedded in PLL/HA films can be finely tuned. HT29 cells seeded on this film adhere poorly but deposition of PSS/PAH multilayers on top of PLL/HA films results in an increase
This work was supported by the ACI ‘‘Nanosciences 2004’’ from the Ministe`re de la Recherche et des Nouvelles Technologies (Project NR204). We thank Dr. M. Ke´dinger (INSERM UMR-S 682, Strasbourg) for kindly giving us HT29 cells and J. Mutterer (Institut de Biologie Mole´culaire des Plantes, CNRS/ULP, Strasbourg, France) for his assistance with the CLSM. We also thank D. Affleck for critical reading of the manuscript. The CLSM platform used in this study was co-financed by the Re´gion Alsace, the Universite´ Louis Pasteur, and the Association pour la Recherche sur le Cancer. Appendix A. Supplementary materials Supplementary data associated with this article can be found in the online version at doi:10.1016/j.biomaterials.2006.03.024
References [1] Ratner BD, Hoffman A, Schoen FJ, Lemons JE. Biomaterials science: an introduction to materials in medecine. New York: Academic Press; 2004. [2] Jones FH. Teeth and bones: applications of surface science to dental materials and related biomaterials. Surf Sci Rep 2001;42:75–205. [3] Kasemo B. Biological surface science. Surf Sci 2002;500:656–77. [4] Ito Y, Zheng J, Imanishi Y, Yonezawa K, Kasuga M. Protein-free cell culture on an artificial substrate with covalently immobilized insulin. Proc Natl Acad Sci USA 1996;93:3598–601. [5] Ito Y, Chen GP, Imanishi Y. Micropatterned immobilization of epidermal growth factor to regulate cell function. Bioconjugate Chem 1998;9:277–82. [6] Murphy WL, Peters MC, Kohn DH, Mooney DJ. Sustained release of vascular endothelial growth factor from mineralized poly(lactideco-glycolide) scaffolds for tissue engineering. Biomaterials 2000;21: 2521–7. [7] Grayson ACR, Choi IS, Tyler BM, Wang PP, Brem H, Cima MJ, et al. Multi-pulse drug delivery from a resorbable polymeric microchip device. Nat Mater 2003;2:767–72. [8] Jessel N, Atalar F, Lavalle P, Mutterer J, Decher G, Schaaf P, et al. Bioactive coatings based on a polyelectrolyte multilayer architecture functionalized by embedded proteins. Adv Mater 2003;15:692–5. [9] Benkirane-Jessel N, Lavalle P, Meyer F, Audouin F, Frisch B, Schaaf P, et al. Control of monocyte morphology on and response to model surfaces for implants equipped with anti-inflammatory agents. Adv Mater 2004;16:1507–11. [10] Decher G, Hong JD, Schmitt J. Buildup of ultrathin multilayer films by a self-assembly process. Consecutively alternating adsorption of anionic and cationic polyeletrolytes on charges surface. Thin Solid Films 1992;210:831–5.
ARTICLE IN PRESS C. Vodouheˆ et al. / Biomaterials 27 (2006) 4149–4156 [11] Decher G. Fuzzy nanoassemblies: Toward layered polymeric multicomposites. Science 1997;277:1232–7. [12] Ladam G, Schaad P, Voegel J- C, Schaaf P, Decher G, Cuisinier F. In situ determination of the structural properties of initially deposited polyelectrolyte multilayers. Langmuir 2000;16:1249–55. [13] Zhu YB, Gao CY, He T, Liu XY, Shen JC. Layer-by-layer assembly to modify poly(L-lactic acid) surface toward improving its cytocompatibility to human endothelial cells. Biomacromolecules 2003;4: 446–52. [14] Decher G, Schlenoff JB, editors. Multilayer thin films—sequential assembly of nanocomposite materials. Weinheim: Wiley–VCH; 2003. [15] Sukhishvili SA. Responsive polymer films and capsules via layer-bylayer assembly. Curr Opin Coll Int Sci 2005;10:37–44. [16] Caruso F, Niikura K, Furlong DN, Okahata Y. 2 Assembly of alternating polyelectrolyte and protein multilayer films for immunosensing. Langmuir 1997;13:3427–33. [17] Chluba J, Voegel J-C, Decher G, Erbacher P, Schaaf P, Ogier J. Peptide hormone covalently bound to polyelectrolytes and embedded into multilayer architectures conserving full biological activity. Biomacromolecules 2001;2:800–5. [18] Caruso F, Schuler C. Enzyme multilayers on colloid particles: assembly, stability, and enzymatic activity. Langmuir 2000;16:9595–603. [19] Vodouhe C, Schmittbuhl M, Boulmedais F, Bagnard D, Vautier D, Schaaf P, et al. Effect of functionalization of multilayered polyelectrolyte films on motoneuron growth. Biomaterials 2005;26:545–54. [20] Tezcaner A, Hicks D, Boulmedais F, Sahel J, Schaaf P, Voegel J-C, et al. Polyelectrolyte multilayer films as substrates for photoreceptor cells. Biomacromolecules 2006;7:86–94. [21] Benkirane-Jessel N, Schwinte´ P, Falvey P, Darcy R, Haı¨ kel Y, Schaaf P, et al. Build-up of polypeptide multilayer coatings with antiinflammatory properties based on the embedding of piroxicamcyclodextrin complexes. Adv Funct Mater 2004;14:174–82. [22] Boulmedais F, Frisch B, Etienne O, Lavalle P, Picart C, Ogier J, et al. Polyelectrolyte multilayer films with pegylated polypeptides as a new type of antimicrobial protection for biomaterials. Biomaterials 2004; 25:2003–11. [23] Etienne O, Picart C, Taddei C, Haikel Y, Dimarcq JL, Schaaf P, et al. Multilayer polyelectrolyte films functionalized by insertion of defensin: a new approach to protection of implants from bacterial colonization. Antimicrob Agents Chemother 2004;48:3662–9. [24] Gibbs JB. Mechanism-based target identification and drug discovery in cancer research. Science 2000;287:1969–73. [25] Attawia MA, Borden MD, Herbert KM, Katti DS, Asrari F, Uhrich KE, et al. Regional drug delivery with radiation for the treatment of Ewing’s sarcoma: In vitro development of a taxol release system. J Control Release 2001;71:193–202. [26] Fonseca C, Simoes S, Gaspar RE. Paclitaxel-loaded PLGA nanoparticles: preparation, physicochemical characterization and in vitro anti-tumoral activity. J Control Release 2002;83:273–86. [27] Ruan G, Feng SS. Preparation and characterization of poly(lactic acid)-poly(ethylene glycol)-poly(lactic acid) (PLA-PEG-PLA) microspheres for controlled release of paclitaxel. Biomaterials 2003;24: 5037–44. [28] Soga O, van Nostrum CF, Fens M, Rijcken CJF, Schiffelers RM, Storm G, et al. Thermosensitive and biodegradable polymeric micelles for paclitaxel delivery. J Control Release 2005;103:341–53. [29] Heldman AW, Cheng L, Jenkins GM, Heller PF, Kim DW, Ware M, et al. Paclitaxel stent coating inhibits neointimal hyperplasia at 4 weeks in a porcine model of coronary restenosis. Circulation 2001; 103:2289–95. [30] Park SJ, Shim WH, Ho DS, Raizner AE, Park SW, Hong MK, et al. A paclitaxel-eluting stent for the prevention of coronary restenosis. New Engl J Med 2003;348:1537–45. [31] Liistro F, Stankovic G, Di Mario C, Takagi T, Chieffo A, Moshiri S, et al. First clinical experience with a paclitaxel derivate-eluting polymer stent system implantation for in-stent restenosis—immediate and long-term clinical and angiographic outcome. Circulation 2002; 105:1883–6.
4155
[32] Morice M, Serruys PW, Sousa JE, Fajadet J, Hayashi EB, Perin M, et al. A randomized comparison of a sirolimus-eluting stent with a standard stent for coronary revascularization. New Engl J Med 2002; 346:1773–80. [33] Luo Y, Prestwich GD. Synthesis and selective cytotoxicity of a hyaluronic acid-antitumor bioconjugate. Bioconjugate Chem 1999;10:755–63. [34] Luo Y, Ziebell MR, Prestwich GD. A hyaluronic acid-taxol antitumor bioconjugate targeted to cancer cells. Biomacromolecules 2000;1:208–18. [35] Thierry B, Kujawa P, Tkaczyk C, Winnik FM, Bilodeau L, Tabrizian M. Delivery platform for hydrophobic drugs: prodrug approach combined with self-assembled multilayers. J Am Chem Soc 2005;127:1626–7. [36] Thierry B, Winnik FM, Merhi Y, Silver J, Tabrizian M. Bioactive coatings of endovascular stents based on polyelectrolyte multilayers. Biomacromolecules 2003;4:1564–71. [37] Guyomard A, Nysten B, Muller G, Glinel K. Loading and release of small hydrophobic molecules in multilayer films based on amphiphilic polysaccharides. Langmuir 2006;22:2281–7. [38] Hermanson GT. In: Hermanson GT, editor. Bioconjugate techniques. San Diego: Academic Press; 1996. p. 169–76. [39] Dahne L, Leporatti S, Donath E, Mohwald H. Fabrication of micro reaction cages with tailored properties. J Am Chem Soc 2001;123: 5431–6. [40] Elbert DL, Herbert CB, Hubbell JA. Thin polymer layers formed by polyelectrolyte multilayer techniques on biological surfaces. Langmuir 1999;15:5355–62. [41] Schuler C, Caruso F. Decomposable hollow biopolymer-based capsules. Biomacromolecules 2001;2:921–6. [42] Thierry B, Winnik FM, Merhi Y, Tabrizian M. Nanocoatings onto arteries via layer-by-layer deposition: toward the in vivo repair of damaged blood vessels. J Am Chem Soc 2003;125:7494–5. [43] Richert L, Lavalle P, Payan E, Zheng XS, Prestwich GD, Stoltz JF, et al. Layer by layer buildup of polysaccharide films: physical chemistry and cellular adhesion aspects. Langmuir 2004;20: 448–58. [44] Picart C, Lavalle P, Hubert P, Cuisinier FJG, Decher G, Schaaf P, et al. Buildup mechanism for poly(L-lysine)/hyaluronic acid films onto a solid surface. Langmuir 2001;17:7414–24. [45] Picart C, Mutterer J, Richert L, Luo Y, Prestwich GD, Schaaf P, et al. Molecular basis for the explanation of the exponential growth of polyelectrolyte multilayers. Proc Natl Acad Sci USA 2002;99: 12531–5. [46] Khademhosseini A, Suh KY, Yang JM, Eng G, Yeh J, Levenberg S, et al. Layer-by-layer deposition of hyaluronic acid and poly L-lysine for patterned cell co-cultures. Biomaterials 2004;25:3583–92. [47] Kujawa P, Moraille P, Sanchez J, Badia A, Winnik FM. Effect of molecular weight on the exponential growth and morphology of hyaluronan/chitosan multilayers: a surface plasmon resonance spectroscopy and atomic force microscopy investigation. J Am Chem Soc 2005;127:9224–34. [48] Garza JM, Schaaf P, Muller S, Ball V, Stoltz JF, Voegel J-C, et al. Multicompartment films made of alternate polyelectrolyte multilayers of exponential and linear growth. Langmuir 2004;20:7298–302. [49] Garza JM, Jessel N, Ladam G, Dupray V, Muller S, Stoltz J-F, et al. Polyelectrolyte multilayers and degradable polymer layers as multicompartment films. Langmuir 2005;21:12372–7. [50] Krouglova T, Vercammen J, Engelborghs Y. Correct diffusion coefficients of proteins in fluorescence correlation spectroscopy. Application to tubulin oligomers induced by Mg2+ and paclitaxel. Biophys J 2004;87:2635–46. [51] Richert L, Boulmedais F, Lavalle P, Mutterer J, Ferreux E, Decher G, et al. Improvement of stability and cell adhesion properties of polyelectrolyte multilayer films by chemical cross-linking. Biomacromolecules 2004;5:284–94. [52] Picart C, Elkaim R, Richert L, Audoin T, Arntz Y, Cardoso MD, et al. Primary cell adhesion on RGD-functionalized and covalently crosslinked thin polyelectrolyte multilayer films. Adv Funct Mater 2005;15:83–94.
ARTICLE IN PRESS 4156
C. Vodouheˆ et al. / Biomaterials 27 (2006) 4149–4156
[53] Mendelsohn JD, Yang SY, Hiller J, Hochbaum AI, Rubner MF. Rational design of cytophilic and cytophobic polyelectrolyte multilayer thin films. Biomacromolecules 2003;4:96–106. [54] Richert L, Engler AJ, Discher DE, Picart C. Elasticity of native and cross-linked polyelectrolyte multilayer films. Biomacromolecules 2004;5:1908–16. [55] Kidambi S, Lee I, Chan C. Controlling primary hepatocyte adhesion and spreading on protein-free polyelectrolyte multilayer films. J Am Chem Soc 2004;126:16286–7.
[56] Rao CS, Chu JJ, Liu RS, Lai YK. Synthesis and evaluation of [C-14]-labelled and fluorescent-tagged paclitaxel derivatives as new biological probes. Bioorg Med Chem 1998;6:2 193–204. [57] Lavalle P, Gergely C, Cuisinier FJG, Decher G, Schaaf P, Voegel J-C, et al. Comparison of the structure of polyelectrolytes multilayer films exhibiting a linear and an exponential growth regime: an in situ atomic force microscopy study. Macromolecules 2002;35: 4458–65.
Biomaterials 27 (2006) 4149–4156 www.elsevier.com/locate/biomaterials
Control of drug accessibility on functional polyelectrolyte multilayer films Constant Vodouheˆa,b,, Erell Le Guena,b, Juan Mendez Garzaa,b, Gregory Franciusa,b, Christophe De´jugnatc, Joe¨lle Ogiera,b, Pierre Schaafd, Jean-Claude Voegela,b, Philippe Lavallea,b, a INSERM Unite´ 595, 11 rue Humann, F-67085 Strasbourg Cedex, France Faculte´ de Chirurgie Dentaire, Universite´ Louis Pasteur, Strasbourg, F-67085, France c Max Planck Institute of Colloids and Interfaces, Golm/Potsdam, Germany d CNRS, UPR22, Institut Charles Sadron, Strasbourg, F-67083, France
b
Received 1 February 2006; accepted 16 March 2006 Available online 4 April 2006
Abstract A surface coating based on polylysine/hyaluronic acid multilayers was designed and acted as a reservoir for an antiproliferative agent, paclitaxel (Taxol). Absolutely no chemical modification of polyelectrolytes or of the drug was needed and the final architecture was obtained in an extremely simple way using the layer-by-layer method. The paclitaxel dose available for human colonic adenocarcinoma cells HT29 seeded on the films could be finely tuned. Moreover, the accessibility of the drugs was controlled by adding on the top of the drug reservoir a capping made of synthetic polyelectrolyte multilayers. This capping was also required to allow adhesion of HT29 cells. Paclitaxel activity was maintained after embedding in the polyelectrolyte multilayers and cellular viability could be reduced by about 80% 96 h after seeding. The strategy described in this paper could be valuable for various other drug/cell systems. r 2006 Elsevier Ltd. All rights reserved. Keywords: Paclitaxel; HT29 cells; Polyelectrolyte multilayers; Bioactive films
1. Introduction In the last two decades, much effort has been put forth to provide biomaterial surfaces bearing sophisticated properties [1]. In particular, protein adsorption control and cellular adhesion properties were deeply investigated [2,3]. With this goal in mind, surfaces have been equipped with specific peptides or proteins by direct grafting on the substrate while controlling the density and/or spatial orientation [4,5]. More recently, polymeric matrices have been designed as substrate’s coatings to include biological factors [6,7]. The final aim of such approaches consists of controlling the rate and selectivity of cellular adhesion. Corresponding authors. INSERM Unite´ 595, 11 rue Humann, F-67085 Strasbourg Cedex, France. Tel.: +33 3 90243378; fax: +33 3 90243379. E-mail addresses: [email protected] (C. Vodouheˆ), [email protected] (P. Lavalle).
0142-9612/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2006.03.024
Mediation of the cellular responses and subsequent processes such as inflammation and tissue regeneration around the biomaterial have been controlled using these methods [8,9]. To build up polymeric matrices to surfaces in a controlled manner, an approach was developed 15 years ago based on alternated deposition of polyanion and polycation layers leading to polyelectrolyte multilayer films also called layer-by-layer films (PEM or LbL films, respectively). This method has emerged as a new and simplified strategy to modify surfaces [10–15]. For conventional polyelectrolyte multilayer systems, the driving force of the build-up comes from the alternate overcompensation of the surface charges appearing after each new oppositely charged polyelectrolyte deposition. To confer specific biological activities to multilayered films one can incorporate peptides, proteins, hormones, growth factors or drugs while maintaining their native
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structures and their activities [8,16–20]. Following this scheme, films with effective anti-inflammatory [9,21] or anti-microbial [22,23] properties were recently constructed. Paclitaxel (Taxol) is a microtubule-stabilizing agent with efficient antiproliferative activity through antimitotic properties. This drug is one of the most potent chemotherapeutic molecule in particular in the treatment of breast and ovarian cancers [24]. Many studies have focused on the local delivery of paclitaxel from polymeric microspheres [25–28]. Paclitaxel has also been used in the treatment and prevention of restenosis after percutaneous coronary intervention. In particular, this drug leads to a drastic reduction of restenosis when applied to the stent surface [29–31]. Systems able to release antiproliferative agents from the stent itself are under development, the rapamycincoated stents having already entered the world market 3 years ago [32]. Luo and Prestwich [33,34] developed a prodrug consisting in paclitaxel bioconjugates with hyaluronic acid as a drug carrier. More recently, Thierry et al. [35] described a chemical modification of hyaluronic acid by grafting paclitaxel through a labile succinate ester linkage. They developed films with chitosan and hyaluronic acid-paclitaxel using the layer-by-layer method. To incorporate specific molecules into polyelectrolyte multilayers and in particular to design films with antithrombotic properties, other approach used precomplexation methods of the drug with the polycations or the polyanions involved in the multilayers [36]. Following this strategy an efficient entrapment is then obtained especially in the case of hydrophobic molecules [37]. In this study, we propose to design a surface coating which acts as a reservoir for drugs like paclitaxel based on polylysine/hyaluronic acid (PLL/HA) multilayers. Absolutely no chemical modification of polyelectrolytes or of the drug is needed and the final architecture is constructed in a simple way. The paclitaxel dose available for human colonic adenocarcinoma cells HT29 can be finely tuned. Furthermore, the accessibility of the drugs can be controlled by adding a capping made of synthetic polyelectrolyte multilayers on top of the drug reservoir. Activity of paclitaxel against these cells was maintained after embedding in the multilayers. This capping is also required to allow adhesion of the HT29 cells. Finally, the strategy described in this paper could be valuable for various other drug/cell systems. 2. Experimental 2.1. Polyelectrolytes and drug solutions Poly(L-lysine) (PLL, Mw ¼ 58 900 Da), PLLFITC (Fluorescein isothiocyanate labeled poly(L-lysine) Mw ¼ 50 000 Da), poly(sodium 4 styrene sulfonate) (PSS, Mw ¼ 70 000 Da) and poly(allylamine hydrochloride) (PAH, Mw ¼ 70 000 Da) were purchased from Sigma (St. Quentin Fallavier, France). Hyaluronic acid (HA, Mw ¼ 411500 Da) was obtained from BioIberica (Barcelona, Spain). Rhodamine B (Invitrogen, Cergy Pontoise, France) was coupled to PAH as previously described [38]. An
appropriate amount of PAH (1 mg mL1) was dissolved in a Na2CO3 solution (0.1 M, pH ¼ 8.5). Rhodamine (Rho) at 2 mg mL1 was dissolved in dimethylsulfoxyde (DMSO). We used a ratio of 1 mg of rhodamine for 50 mg of PAH. The solutions were gently mixed at room temperature for 2 h. PAHRho was purified by dialysis against water during 3 days and then the absence of free rhodamine in the solution was verified by UV spectroscopy. Rhodamine-labelled PSS (PSSMRho) was synthesized adopting the procedure described elsewhere [39]. Typically, 0.516 g (2.5 mmol) of SS (4styrene sulfonate, sodium salt/Sigma) was first dissolved in 7.5 mL pure water. A solution containing 8.3 mg (0.0125 mmol, 0.5% mol with respect to SS) of MRho (methacryloxyethyl thiocarbamoyl rhodamine B/ Polysciences, Eppelheim, Germany) in 2 mL methanol was prepared and added to the previous SS aqueous solution. This mixture was flushed with nitrogen while stirring for one hour. Then 250 mL of a 0.1 M (1% mol) TMEDA (tetramethylethylenediamine/Sigma) aqueous solution and 250 mL of a 0.05 M (0.5% mol) (NH4)2S2O8 (ammonium peroxodisulfate/ Sigma) aqueous solution were added to the previous PSS-MRho mixture under nitrogen to initiate the polymerization reaction. The solution was heated to 40 1C for 4 h under nitrogen, and then stirred at room temperature for further 12 h. The final crude solution was dialyzed against water for 1 week and finally freeze-dried, resulting in a dark red solid. Polyelectrolyte solutions (1 mg mL1) were prepared by dissolving adequate amounts of polyelectrolytes in 0.15 M NaCl solution, pH ¼ 6.5. All solutions were prepared using ultrapure water (Milli Q-plus system, Millipore) with a resistivity of 18.2 MO cm. Paclitaxel, commercially known as Taxol (gift from Hoˆpitaux Universitaires de Strasbourg, France) was adsorbed overnight at adequate concentration on polyelectrolyte mutlilayer films. Films were then rinsed three times in NaCl 0.15 M (pH 6.5) followed by an optional deposition of PSS/PAH layers. Paclitaxel Oregon Green 488 labeled (paclitaxelGreen 488, Molecular Probes, Oregon, USA) was previously dissolved in ethanol/cremophor solution (v/v at a concentration of 500 mg mL1) and then adjusted at 5, 10, 50 and 200 mg mL1 with NaCl 0.15 M (pH ¼ 6.5).
2.2. Build-up of polyelectrolyte multilayer films The multilayered films were deposited using an automated dipping robot (Riegler & Kirstein GmbH, Berlin, Germany) to glass coverslips. The slides were first dipped for 10 min in a polycation solution. Then, a rinsing step was achieved by dipping the substrates for an equal period in a 0.15 M NaCl (pH ¼ 6.5) solution. The polyanion was deposited in a similar way and the build-up process was continued until the deposition of the required pair of layers. After the deposition of n poly(L-lysine)/hyaluronic acid layer pairs [denoted (PLL/HA)n] the build-up was pursued in some cases by a further deposition of PSS/PAH bilayers in the same conditions as for PLL/HA depositions. Typically a film constituting 30 PLL/HA layer pairs and capped by one PSS/PAH bilayer will be denoted as (PLL/ HA)30/PSS/PAH.
2.3. Confocal laser scanning microscopy Confocal laser scanning microscopy (CLSM) observations were carried out with a Zeiss LSM 510 microscope using a 40/1.4 oil immersion objective and with 0.4 mm z-section intervals. Green 488 fluorescence was detected after excitation at 488 nm with a cutoff dichroic mirror 488 nm and an emission band-pass filter 505–530 nm (green). Rhodamine fluorescence was detected after excitation at 543 nm, dichroic mirror 543 nm, and emission long pass filter 585 nm (red). An average of four images in the same location were acquired at 512 512 pixels. Virtual film section images were performed in liquid conditions (NaCl 0.15 M, pH ¼ 6.5).
2.4. HT 29 cell culture A human colonic adenocarcinoma cell line HT29 was kindly provided by Dr. M. Kedinger, (INSERM UMR-S 682, Strasbourg, France).
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12 µm
(A)
12 µm
They were routinely cultured in 25 cm2 culture flasks, in Glutamax I DMEM 25 mM glucose (Invitrogen, France), without sodium pyruvate, and supplemented with 10% v/v heat-inactivated fetal calf serum and penicillin streptomycin 100 U mL1 and 0.1 mg mL1 (Invitrogen, France). Cultures were incubated at 37 1C in a humidified atmosphere of air (95%)CO2 (5%). The medium was changed every 2 days. For subcultures, cells were incubated in 0.05% trypsin-0.53 mM EDTA solution and then harvested. The polyelectrolyte multilayer films were sterilized for 10 min by exposure to UV light (254 nm, 30 W, illumunation distance 20 cm). Cells were seeded at a density of 8000 cells mL1 in 24 well plates, containing precoated glass coverslips with polyelectrolyte multilayer films.
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2.5. Cell viability measurement (B)
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The measurement of viability by the acid phosphatase method allows to quantify the number of living cells with greater sensitivity than the XTT test. It consists in the titration of the enzyme whose activity increases proportionally with the number of viable cells. In our study, the culture medium was eliminated and each well washed with 300 mL PBS. The buffer used and added to each well contains 0.1 M sodium acetate (pH 5.5), 0.1% of X-100 Triton and 10 mM of p-nitrophenyl phosphate [pNPP] (Sigma). The plates were placed in an incubator for 3 h at 37 1C with the air (95%)–CO2 (5%) suitably humidified. The reaction was stopped by the addition of 1 N sodium hydroxide and the absorbance of the yellow colored solution was measured at 405 nm using a spectrophotometer (Labsystems, iEMS Reader MF, Gibco). Cell viability was measured at 24 and 96 h. Statistical analyses were performed using Sigma-Stat software.
(C) 25
10 µg mL-1 50 µg mL-1 200 µg mL-1
20
Build-up of multilayer films made of polysaccharides has been recently investigated, in particularly for the polylysine/alginate, [40,41] polylysine/chondroitin, [20] chitosan/hyaluronic acid [42,43] and polylysine/hyaluronic acid (PLL/HA) [44–47] systems. It happens that these films increase exponential in thickness according to the number of deposited layers. This exponentially increase is related to the diffusivity through the film section of at least one of the polyelectrolytes involved in the polycation/polyanion multilayer. In the PLL/HA system, PLL chains constitute the diffusing species. Typically in the presence of 0.15 M NaCl, several mm thick films were obtained after the deposition of 30 PLL/HA layer pairs. It was recently suggested that PLL/HA multilayers act as reservoirs for PLL chains [48,49]. In this study, we investigated first the paclitaxel uptake by PLL/HA films. Paclitaxel molecules fluorescently labelled with Oregon Green 488 molecules were used for confocal microscopy studies. It was previously shown that this molecule despite the labelling with the dye maintains its biological activity [50]. Fig. 1 reveals the results for various fluorescently green-labelled paclitaxelGreen 488 concentrations varying from 10 to 200 mg mL1. First of all, one observes that the paclitaxelGreen 488 molecules diffuse through the whole (PLL/HA)60 film section. In particular, for paclitaxelGreen 488 adsorbed from solutions at 50 and 200 mg mL1, the fluorescence is homogeneously distributed over the whole film thickness. The total thickness of the (PLL/HA)60 is approximately 12 mm as expected from previous studies. Increasing the paclitaxelGreen 488 concentration of the solution during deposition leads to a higher
Z distance (µm)
3. Results and discussion 15
10
5
0 0
(D)
50
100
150
200
250
Fluorescence intensity (u.a.)
Fig. 1. Confocal laser scanning (CLSM) images of (PLL/HA)60/ paclitaxelGreen 488 film sections. Paclitaxel Green 488 was adsorbed from solutions at 10 mg mL1 (A), 50 mg mL1 (B) and 200 mg mL1 (C). Image sizes are 76.8 23.9 mm2. From these images, distance Z as a function of the fluorescence intensity was plotted for the three paclitaxelGreen 488 concentrations. All CLSM settings are identical for the three images. Z ¼ 0 corresponds to the location of the glass coverslips on which the film was deposited.
amount of drug taken up as materialized by the increase in green intensity (Fig. 1(D)). We determined the paclitaxel concentration in the PLL/HA films from fluorescence intensities. For this purpose, a calibration curve was first established by imaging paclitaxelGreen 488 solutions at predetermined concentrations (see Fig. S1 in Supplementary materials section). When paclitaxel was deposited at 10 mg mL1, the concentration in the films was about 500 mg mL1, i.e. 50 times more than the concentration in solution whereas for the paclitaxel solutions at 50 and 100 mg mL1, the concentration in the films were of about
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1200 and 1900 mg mL1, respectively, thus about 20 times more concentrated in the film than in solution. From these data it is possible to plot the adsorption isotherm of the drug in the (PLL/HA)60 film (see Fig. S2 in Supplementary material section). It appears that the paclitaxel concentration within the films depends on the initial concentrations of paclitaxel in the solutions during deposition. However, for high solution concentrations (between 50 and 100 mg mL1) a saturation process should take place and films would probably not contain paclitaxel concentrations up to 2000–2200 mg mL1. Finally, the drug content in PLL/HA films can be finely tuned in a large range concentration. These behaviors would probably not be observed for linear growing multilayer films like PSS/PAH. Actually, such films exhibit very different properties compared to PLL/HA, in particular they are very dense, thin and the embedding of a biological factors often leads to a loss of their activity [17]. We also checked the evolution of fluorescence intensity with time and found that it remained constant after being stored for four days at ambient temperature in NaCl 0.15 M, pH ¼ 6.5. This indicates that no or very low passive release occurs in a short time span. To control the efficiency of the paclitaxel embedded into polyelectrolyte multilayers, viability of HT29 cell line cultured on films with various architectures was tested. For all of the biological tests presented below, paclitaxel solutions were maintained at 5 mg mL1 during adsorption and the PLL/HA films were constituted of 30 bilayers. Further investigations are underway to study the efficiency of films containing various paclitaxel concentration on cell viability. From the adsorption isotherm of paclitaxel in PLL/HA films (see Fig. S2 in Supplementary material section), and if we consider that the relationship between bulk concentration and film concentration is linear for the low bulk concentration studied, it was possible to estimate that the amount of paclitaxel in the PLL/HA film was of about 250 mg mL1. The first viability tests demonstrated that HT29 cells cultured on (PLL/HA)30 films were not viable probably due to weak adhesion on such films. This could be attributed to the mechanical properties of the PLL/HA multilayers as previously described in the case of adhesion on such films of chondrosarcoma cells [51] or osteoblasts [52]. These films exhibit very low stiffness properties due to the high water content and as a consequence cells could not develop focal spots and thus did not adhere to the films [53,54]. In the present study, we wish to use polyelectrolyte multilayers as a kind of trap against HT29 cells: cells should first adhere on top of the film and then they should internalize paclitaxel to become apoptotic. We tried to modify the film surface by the addition of a PSS layer or PSS/PAH multilayers on top of PLL/HA films (Fig. 2). As described previously by Kidambi et al. [55] in the case of hepatocytes, sulfonate groups of PSS chains adsorbed on the surface promote cellular adhesion. This was also recently confirmed for motoneuron cells which
Fig. 2. Confocal laser scanning (CLSM) images of (A), (PLL/HA)29/ PLLFITC/HA/PSSMRho, (B), (PLL/HA)29/PLLFITC/HA/PSSMRho/PAHRho/ PSSMRho, (C), (PLL/HA)29/PLLFITC/HA/(PSSMRho/PAHRho)2/PSSMRho film sections. Image sizes are 13.4 29.4 mm2.
preferentially spread and adhere to PSS ending multilayer films [19]. With this in mind, we investigated the influence on cell viability of PSS chains deposited on a non-adhesive PLL/HA film. Despite the negative charge of the PSS chains and the negative surface charge of the (PLL/HA)30/PLLFITC/HA films [44], we observed the deposition of a PSS layer on top of this film (Fig. 2(A)). The whole film section is green due to PLLFITC (PLL-fluorescein isothiocyanate) diffusion [45] and a thin red line corresponding to PSSMRho (methacryloxyethyl thiocarbamoyl rhodamine B) is seen on top of the film. Some exchange mechanisms between the HA chains adsorbed on top of the film and the new incoming PSS chains probably take place. However the PSS chains didn’t diffuse into the PLL/HA film and seem to be adsorbed only on the upper part of the film. Adding PAHRho/PSSMRho or (PAHRho/PSSMRho)2 to the (PLL/HA)30/PLLFITC/HA/ PSSMRho film leads to similar observations with the confocal microscope (Figs. 2(B) and (C), respectively). Adhesion of HT29 cells became significant once the top of the PLL/HA film was capped by a PSS layer (Fig. 3) as
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AP activity (%)
100
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(PLL/HA)30 ~ at 24h (PLL/HA)30 ~ at 96h ***
80
** ***
60
***
40 ** *** 20
/P SS PA H )2 (P SS / ~
~
PS S/
PA H
~
PS
/P SS
S
0
Fig. 3. Acid phosphatase (AP) activity for HT29 cells cultured on (PLL/ HA)30/PSS, (PLL/HA)30/PSS/PAH/PSS, (PLL/HA)30/(PSS/PAH)2/PSS films determined at 24 h (dark bars) and at 96 h (gray bars). Data is divided into three groups: (PLL/HA)30 multilayers capped by PSS, by PSS/PAH/PSS or by (PSS/PAH)2/PSS. Results are expressed as an average of four independent measurements. The error bars represent the standard deviation. The value of 100% has been arbitrarily attributed to AP activity of HT29 cells on (PSS/(PAH)2/PSS ending films after 96 h of seeding. No activity was determined for (PLL/HA)30 films without any capping because HT29 cells are not viable on these films. * po0.05, ** po0.01, *** po0.001 vs (PSS/PAH)2/PSS ending films.
suggested by the acid phosphatase (AP) activity determined at 24 and 96 h. The adhesion was more pronounced for the PLL/HA film capped by PSS/PAH/PSS, in particularly after 96 h of culture. Adding (PSS/PAH)2/PSS onto (PLL/ HA)30 films again resulted in a significant increase in AP activity at 96 h. This effect could be attributed to a change in chemical composition of the top of the multilayer film (PSS instead of HA chains) and/or to a change in the mechanical properties of the PLL/HA film that possibly stiffens after capping with PSS/PAH multilayers. Further investigations are underway to study long-term activity of the paclitaxel-embedded films on HT29 cells. We determined that deposition of PSS, PSS/PAH/PSS or (PSS/PAH)2/PSS on (PLL/HA)30/paclitaxel multilayers doesn’t allow the release of paclitaxel contained in the films. Fig. 4(A) shows an example of a green labelled (PLL/ HA)30/paclitaxelGreen 488 film capped with a red labelled PSSMRho/PAHRho/PSSMRho multilayer. PSS and PAH multilayers are well deposited on films containing paclitaxel which is maintained in the PLL/HA film. Same conclusions can be drawn for cappings made of only PSSMRho, or of (PSSMRho/PAHRho)2/PSSMRho (data not shown). Next we evaluated if the paclitaxel embedded in (PLL/ HA)30 film still remains active and can be sensed by HT29 cells through the PSS, PSS/PAH or (PSS/PAH)2/PSS cappings. The AP activities induced at 24 and 96 h are
Fig. 4. Confocal laser scanning (CLSM) images of (A), a (PLL/HA)30/ paclitaxelGreen 488/PSSMRho/PAHMRho/PSSMRho film section, (B), HT29 cell section 24 h after seeding on a (PLL/HA)30/paclitaxelGreen 488/ PSSMRho/PAHMRho/PSSMRho film, and (C), top view (x,y) of the same cell. Image sizes are 29.4 13.9 mm2 (A), 46.0 22.6 mm2 (B), and 22 22 mm2 (C). Paclitaxel is deposited on the (PLL/HA)30 film at 100 mg mL1.
shown in Fig. 5 (normalized values). It becomes clear that the effect of the paclitaxel embedded in the (PLL/HA)30 film capped with a PSS layer induces a 45% AP decrease in activity at 24 h and thus strongly affects cell viability. Paclitaxel is probably in an active and free conformation when embedded in the PLL/HA multilayers. This effect is more pronounced at 96 h where only 20% of the initial activity is maintained. If the (PLL/HA)30/paclitaxel film is capped with PSS/PAH/PSS multilayers instead of one PSS layer, the AP activity of HT29 cells is less strongly affected by paclitaxel at 24 and at 96 h (respective decreases of about 30% and 70%). When (PSS/PAH)2/PSS constitutes the capping, the efficiency of paclitaxel on the reduction of AP activity is only 20% at 24 h and 35% at 96 h. Clearly, the number of PSS and PAH layers constituting the capping strongly influences the cell accessibility for the underlying paclitaxel. We also confirm by confocal microscopy that cells will sense the paclitaxel -embedded PLL/HA film, even through a PSS/PAH multilayer (Fig. 4(B)). Cells grown on a (PLL/HA)29/PLLFITC/HA/ PSSMRho/PAHMRho/PSSMRho film internalized the green labelled paclitaxelGreen 488. However, no red labelled PSSMRho neither PAHRho were visualized in the cells. PaclitaxelGreen 488 appears strongly concentrated in the cytoplasm and in the nucleolus while the nucleus is less dyed (Fig. 4(C)). Identical fluorescence patterns were also observed with lung cancer cells H460 cells dyed with various fluorescent paclitaxel [56]. PSS and PAH being synthetic polymers, cells are probably not able to degrade the PSS/PAH multilayers [48]. However they could very likely form pseudopodes through a PSS/PAH bilayer to reach the paclitaxel molecules located in the PLL/HA film as has been previously observed with monocytic cells [8,9]. However such pseudopodes could not be clearly visualized in
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(PLL/HA)30 ~ as control (24h or 96h) (PLL/HA) /paclitaxel ~ at 24h 30
(PLL/HA)30 /paclitaxel ~ at 96h
***
***
100
AP activity (%)
*** 80
in cellular adhesion. These PSS/PAH multilayers also permit tuning the accessibility of HT29 cells to the paclitaxel in terms of delay and/or kinetic. The structures designed in the present study constitutes a new route to control and to adjust the accessibility of HT29 cells to paclitaxel. Furthermore, this approach could present attractive versatility in the future for other cell/drug systems.
** ***
Acknowledgements
60
40
*** 20
/P SS S/ PS ~(
~P
SS
/P
PA
A
H
)2
H
~
/P SS
PS S
0
Fig. 5. Acid phosphatase (AP) activity of HT29 cells cultured on (PLL/ HA)30/PSS, (PLL/HA)30/paclitaxel/PSS, (PLL/HA)30/PSS/PAH/PSS, (PLL/ HA)30/paclitaxel/PSS/PAH/PSS, (PLL/HA)30/paclitaxel/(PSS/(PAH)2/PSS and determined at 24 h (gray bars) and 96 h (dark gray bars). Data is divided into three groups: (PLL/HA)30 multilayers with or without paclitaxel capped with PSS, PSS/PAH/PSS or (PSS/PAH)2/PSS. For each group, cell activity on functionalized films with paclitaxel is normalized by the values on the same films without paclitaxel at 24 or 96 h (black bars, activity of 100%). Paclitaxel is deposited on the (PLL/HA)30 film at 5 mg/mL. Results are expressed as an average of four independent measurements. The error bars corresponds to the standard deviation. * po0.05, ** po0.01, *** po0.001 vs control (multilayers without paclitaxel).
Fig. 4(B) because PSS/PAH layers are extremely thin in regards to CLSM resolution. Time required to obtain such contact between cells and paclitaxel should depend on the number of layers constituting the capping. It is also known that deposition of at least five bilayers of PSS/PAH are required to fully covered a surface [57]. Thus only cells in the proximity of some uncoated PSS/PAH areas reach paclitaxel molecules. When the number of deposited PSS/PAH layers increases, the uncoated PSS/PAH areas rarefied and the probability for cells to contact the PLL/HA film containing paclitaxel will then be reduced. 4. Conclusion In the present paper, we design thick polyelectrolyte multilayers containing an active drug, paclitaxel. We demonstrate that the film can act as a reservoir for paclitaxel and that the amount of drug embedded in PLL/HA films can be finely tuned. HT29 cells seeded on this film adhere poorly but deposition of PSS/PAH multilayers on top of PLL/HA films results in an increase
This work was supported by the ACI ‘‘Nanosciences 2004’’ from the Ministe`re de la Recherche et des Nouvelles Technologies (Project NR204). We thank Dr. M. Ke´dinger (INSERM UMR-S 682, Strasbourg) for kindly giving us HT29 cells and J. Mutterer (Institut de Biologie Mole´culaire des Plantes, CNRS/ULP, Strasbourg, France) for his assistance with the CLSM. We also thank D. Affleck for critical reading of the manuscript. The CLSM platform used in this study was co-financed by the Re´gion Alsace, the Universite´ Louis Pasteur, and the Association pour la Recherche sur le Cancer. Appendix A. Supplementary materials Supplementary data associated with this article can be found in the online version at doi:10.1016/j.biomaterials.2006.03.024
References [1] Ratner BD, Hoffman A, Schoen FJ, Lemons JE. Biomaterials science: an introduction to materials in medecine. New York: Academic Press; 2004. [2] Jones FH. Teeth and bones: applications of surface science to dental materials and related biomaterials. Surf Sci Rep 2001;42:75–205. [3] Kasemo B. Biological surface science. Surf Sci 2002;500:656–77. [4] Ito Y, Zheng J, Imanishi Y, Yonezawa K, Kasuga M. Protein-free cell culture on an artificial substrate with covalently immobilized insulin. Proc Natl Acad Sci USA 1996;93:3598–601. [5] Ito Y, Chen GP, Imanishi Y. Micropatterned immobilization of epidermal growth factor to regulate cell function. Bioconjugate Chem 1998;9:277–82. [6] Murphy WL, Peters MC, Kohn DH, Mooney DJ. Sustained release of vascular endothelial growth factor from mineralized poly(lactideco-glycolide) scaffolds for tissue engineering. Biomaterials 2000;21: 2521–7. [7] Grayson ACR, Choi IS, Tyler BM, Wang PP, Brem H, Cima MJ, et al. Multi-pulse drug delivery from a resorbable polymeric microchip device. Nat Mater 2003;2:767–72. [8] Jessel N, Atalar F, Lavalle P, Mutterer J, Decher G, Schaaf P, et al. Bioactive coatings based on a polyelectrolyte multilayer architecture functionalized by embedded proteins. Adv Mater 2003;15:692–5. [9] Benkirane-Jessel N, Lavalle P, Meyer F, Audouin F, Frisch B, Schaaf P, et al. Control of monocyte morphology on and response to model surfaces for implants equipped with anti-inflammatory agents. Adv Mater 2004;16:1507–11. [10] Decher G, Hong JD, Schmitt J. Buildup of ultrathin multilayer films by a self-assembly process. Consecutively alternating adsorption of anionic and cationic polyeletrolytes on charges surface. Thin Solid Films 1992;210:831–5.
ARTICLE IN PRESS C. Vodouheˆ et al. / Biomaterials 27 (2006) 4149–4156 [11] Decher G. Fuzzy nanoassemblies: Toward layered polymeric multicomposites. Science 1997;277:1232–7. [12] Ladam G, Schaad P, Voegel J- C, Schaaf P, Decher G, Cuisinier F. In situ determination of the structural properties of initially deposited polyelectrolyte multilayers. Langmuir 2000;16:1249–55. [13] Zhu YB, Gao CY, He T, Liu XY, Shen JC. Layer-by-layer assembly to modify poly(L-lactic acid) surface toward improving its cytocompatibility to human endothelial cells. Biomacromolecules 2003;4: 446–52. [14] Decher G, Schlenoff JB, editors. Multilayer thin films—sequential assembly of nanocomposite materials. Weinheim: Wiley–VCH; 2003. [15] Sukhishvili SA. Responsive polymer films and capsules via layer-bylayer assembly. Curr Opin Coll Int Sci 2005;10:37–44. [16] Caruso F, Niikura K, Furlong DN, Okahata Y. 2 Assembly of alternating polyelectrolyte and protein multilayer films for immunosensing. Langmuir 1997;13:3427–33. [17] Chluba J, Voegel J-C, Decher G, Erbacher P, Schaaf P, Ogier J. Peptide hormone covalently bound to polyelectrolytes and embedded into multilayer architectures conserving full biological activity. Biomacromolecules 2001;2:800–5. [18] Caruso F, Schuler C. Enzyme multilayers on colloid particles: assembly, stability, and enzymatic activity. Langmuir 2000;16:9595–603. [19] Vodouhe C, Schmittbuhl M, Boulmedais F, Bagnard D, Vautier D, Schaaf P, et al. Effect of functionalization of multilayered polyelectrolyte films on motoneuron growth. Biomaterials 2005;26:545–54. [20] Tezcaner A, Hicks D, Boulmedais F, Sahel J, Schaaf P, Voegel J-C, et al. Polyelectrolyte multilayer films as substrates for photoreceptor cells. Biomacromolecules 2006;7:86–94. [21] Benkirane-Jessel N, Schwinte´ P, Falvey P, Darcy R, Haı¨ kel Y, Schaaf P, et al. Build-up of polypeptide multilayer coatings with antiinflammatory properties based on the embedding of piroxicamcyclodextrin complexes. Adv Funct Mater 2004;14:174–82. [22] Boulmedais F, Frisch B, Etienne O, Lavalle P, Picart C, Ogier J, et al. Polyelectrolyte multilayer films with pegylated polypeptides as a new type of antimicrobial protection for biomaterials. Biomaterials 2004; 25:2003–11. [23] Etienne O, Picart C, Taddei C, Haikel Y, Dimarcq JL, Schaaf P, et al. Multilayer polyelectrolyte films functionalized by insertion of defensin: a new approach to protection of implants from bacterial colonization. Antimicrob Agents Chemother 2004;48:3662–9. [24] Gibbs JB. Mechanism-based target identification and drug discovery in cancer research. Science 2000;287:1969–73. [25] Attawia MA, Borden MD, Herbert KM, Katti DS, Asrari F, Uhrich KE, et al. Regional drug delivery with radiation for the treatment of Ewing’s sarcoma: In vitro development of a taxol release system. J Control Release 2001;71:193–202. [26] Fonseca C, Simoes S, Gaspar RE. Paclitaxel-loaded PLGA nanoparticles: preparation, physicochemical characterization and in vitro anti-tumoral activity. J Control Release 2002;83:273–86. [27] Ruan G, Feng SS. Preparation and characterization of poly(lactic acid)-poly(ethylene glycol)-poly(lactic acid) (PLA-PEG-PLA) microspheres for controlled release of paclitaxel. Biomaterials 2003;24: 5037–44. [28] Soga O, van Nostrum CF, Fens M, Rijcken CJF, Schiffelers RM, Storm G, et al. Thermosensitive and biodegradable polymeric micelles for paclitaxel delivery. J Control Release 2005;103:341–53. [29] Heldman AW, Cheng L, Jenkins GM, Heller PF, Kim DW, Ware M, et al. Paclitaxel stent coating inhibits neointimal hyperplasia at 4 weeks in a porcine model of coronary restenosis. Circulation 2001; 103:2289–95. [30] Park SJ, Shim WH, Ho DS, Raizner AE, Park SW, Hong MK, et al. A paclitaxel-eluting stent for the prevention of coronary restenosis. New Engl J Med 2003;348:1537–45. [31] Liistro F, Stankovic G, Di Mario C, Takagi T, Chieffo A, Moshiri S, et al. First clinical experience with a paclitaxel derivate-eluting polymer stent system implantation for in-stent restenosis—immediate and long-term clinical and angiographic outcome. Circulation 2002; 105:1883–6.
4155
[32] Morice M, Serruys PW, Sousa JE, Fajadet J, Hayashi EB, Perin M, et al. A randomized comparison of a sirolimus-eluting stent with a standard stent for coronary revascularization. New Engl J Med 2002; 346:1773–80. [33] Luo Y, Prestwich GD. Synthesis and selective cytotoxicity of a hyaluronic acid-antitumor bioconjugate. Bioconjugate Chem 1999;10:755–63. [34] Luo Y, Ziebell MR, Prestwich GD. A hyaluronic acid-taxol antitumor bioconjugate targeted to cancer cells. Biomacromolecules 2000;1:208–18. [35] Thierry B, Kujawa P, Tkaczyk C, Winnik FM, Bilodeau L, Tabrizian M. Delivery platform for hydrophobic drugs: prodrug approach combined with self-assembled multilayers. J Am Chem Soc 2005;127:1626–7. [36] Thierry B, Winnik FM, Merhi Y, Silver J, Tabrizian M. Bioactive coatings of endovascular stents based on polyelectrolyte multilayers. Biomacromolecules 2003;4:1564–71. [37] Guyomard A, Nysten B, Muller G, Glinel K. Loading and release of small hydrophobic molecules in multilayer films based on amphiphilic polysaccharides. Langmuir 2006;22:2281–7. [38] Hermanson GT. In: Hermanson GT, editor. Bioconjugate techniques. San Diego: Academic Press; 1996. p. 169–76. [39] Dahne L, Leporatti S, Donath E, Mohwald H. Fabrication of micro reaction cages with tailored properties. J Am Chem Soc 2001;123: 5431–6. [40] Elbert DL, Herbert CB, Hubbell JA. Thin polymer layers formed by polyelectrolyte multilayer techniques on biological surfaces. Langmuir 1999;15:5355–62. [41] Schuler C, Caruso F. Decomposable hollow biopolymer-based capsules. Biomacromolecules 2001;2:921–6. [42] Thierry B, Winnik FM, Merhi Y, Tabrizian M. Nanocoatings onto arteries via layer-by-layer deposition: toward the in vivo repair of damaged blood vessels. J Am Chem Soc 2003;125:7494–5. [43] Richert L, Lavalle P, Payan E, Zheng XS, Prestwich GD, Stoltz JF, et al. Layer by layer buildup of polysaccharide films: physical chemistry and cellular adhesion aspects. Langmuir 2004;20: 448–58. [44] Picart C, Lavalle P, Hubert P, Cuisinier FJG, Decher G, Schaaf P, et al. Buildup mechanism for poly(L-lysine)/hyaluronic acid films onto a solid surface. Langmuir 2001;17:7414–24. [45] Picart C, Mutterer J, Richert L, Luo Y, Prestwich GD, Schaaf P, et al. Molecular basis for the explanation of the exponential growth of polyelectrolyte multilayers. Proc Natl Acad Sci USA 2002;99: 12531–5. [46] Khademhosseini A, Suh KY, Yang JM, Eng G, Yeh J, Levenberg S, et al. Layer-by-layer deposition of hyaluronic acid and poly L-lysine for patterned cell co-cultures. Biomaterials 2004;25:3583–92. [47] Kujawa P, Moraille P, Sanchez J, Badia A, Winnik FM. Effect of molecular weight on the exponential growth and morphology of hyaluronan/chitosan multilayers: a surface plasmon resonance spectroscopy and atomic force microscopy investigation. J Am Chem Soc 2005;127:9224–34. [48] Garza JM, Schaaf P, Muller S, Ball V, Stoltz JF, Voegel J-C, et al. Multicompartment films made of alternate polyelectrolyte multilayers of exponential and linear growth. Langmuir 2004;20:7298–302. [49] Garza JM, Jessel N, Ladam G, Dupray V, Muller S, Stoltz J-F, et al. Polyelectrolyte multilayers and degradable polymer layers as multicompartment films. Langmuir 2005;21:12372–7. [50] Krouglova T, Vercammen J, Engelborghs Y. Correct diffusion coefficients of proteins in fluorescence correlation spectroscopy. Application to tubulin oligomers induced by Mg2+ and paclitaxel. Biophys J 2004;87:2635–46. [51] Richert L, Boulmedais F, Lavalle P, Mutterer J, Ferreux E, Decher G, et al. Improvement of stability and cell adhesion properties of polyelectrolyte multilayer films by chemical cross-linking. Biomacromolecules 2004;5:284–94. [52] Picart C, Elkaim R, Richert L, Audoin T, Arntz Y, Cardoso MD, et al. Primary cell adhesion on RGD-functionalized and covalently crosslinked thin polyelectrolyte multilayer films. Adv Funct Mater 2005;15:83–94.
ARTICLE IN PRESS 4156
C. Vodouheˆ et al. / Biomaterials 27 (2006) 4149–4156
[53] Mendelsohn JD, Yang SY, Hiller J, Hochbaum AI, Rubner MF. Rational design of cytophilic and cytophobic polyelectrolyte multilayer thin films. Biomacromolecules 2003;4:96–106. [54] Richert L, Engler AJ, Discher DE, Picart C. Elasticity of native and cross-linked polyelectrolyte multilayer films. Biomacromolecules 2004;5:1908–16. [55] Kidambi S, Lee I, Chan C. Controlling primary hepatocyte adhesion and spreading on protein-free polyelectrolyte multilayer films. J Am Chem Soc 2004;126:16286–7.
[56] Rao CS, Chu JJ, Liu RS, Lai YK. Synthesis and evaluation of [C-14]-labelled and fluorescent-tagged paclitaxel derivatives as new biological probes. Bioorg Med Chem 1998;6:2 193–204. [57] Lavalle P, Gergely C, Cuisinier FJG, Decher G, Schaaf P, Voegel J-C, et al. Comparison of the structure of polyelectrolytes multilayer films exhibiting a linear and an exponential growth regime: an in situ atomic force microscopy study. Macromolecules 2002;35: 4458–65.