Synthesis of Collagen Nanotubes with Highly Regular Dimensions

Apr 16, 2009 - On the basis of the ProtParam tool available on the ExPASy molecular biology server, a value of iep close to 9 was calculated.20 This m...
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May 2009

Published by the American Chemical Society

Volume 10, Number 5

 Copyright 2009 by the American Chemical Society

Communications Synthesis of Collagen Nanotubes with Highly Regular Dimensions through Membrane-Templated Layer-by-Layer Assembly Jessem Landoulsi,†,‡ Ce´cile J. Roy,† Christine Dupont-Gillain,‡ and Sophie Demoustier-Champagne*,† Unite´ de Physique et de Chimie des Hauts Polyme`res (POLY), Universite´ catholique de Louvain, Place Croix du Sud 1, B-1348 Louvain-la-Neuve, Belgium, and Unite´ de Chimie des Interfaces (CIFA), Universite´ catholique de Louvain, Place Croix du Sud 2/18, B-1348, Louvain-la-Neuve, Belgium Received February 26, 2009; Revised Manuscript Received April 7, 2009

Nanotubes made from a fibrillar protein, namely, collagen, were fabricated by a template-based method combined with layer-by-layer (LbL) deposition. The ability to incorporate collagen in LbL multilayered film was first demonstrated by in situ quartz crystal microbalance and ex situ ellipsometry on a flat model substrate, using poly(styrene sulfonate) (PSS) as polyanion. Collagen-based nanotubes were then fabricated by alternately immersing a polycarbonate membrane, used as template, in PSS and collagen aqueous solutions. Direct evidence for nanotube formation was obtained by dissolving the membrane and imaging the liberated (PSS/collagen)n nanostructures by scanning electron microscopy and by transmission electron microscopy. The proposed strategy constitutes a practical alternative to electrospinning as it allows a very good control over the dimensions (outside and inside diameters and length) of the resulting nanotubes. Besides their fundamental interest, collagen-based nanotubes are useful nano-objects for the creation of new nanostructured biomaterials with numerous potential applications in the biomedical field.

Introduction The creation of environments mimicking the extracellular matrix (ECM) has become one of the challenging aims in biomaterials science. This is due to the pivotal role of the ECM, which offers physical and biochemical cues for the regulation of cell behavior. ECM also promotes intercellular interactions, as well as intracellular responses.1 Artificial nanofibers, whose dimensions, strength, high surface area/volume ratio, and biocompatibility resemble those of natural ECM fibers are, thus, highly desired.2 Currently, electrospinning is the mostly used technique to produce nanofibers made of polymers or proteins.3 However, though recent advances have been reported on the electrospinning process, improvements are still required regarding the control of the nanofibers dimensions.4 Moreover, * To whom correspondence should be addressed. Tel.: +32-10-472702. Fax: +32-10-451593. E-mail: [email protected]. † Unite´ de Physique et de Chimie des Hauts Polyme`res (POLY). ‡ Unite´ de Chimie des Interfaces (CIFA).

electrospinning of natural molecules is still challenging because they do not behave like classical polymers, lacking the viscoelastic properties required for stable electrospinning.5 In this paper, we describe a simple and versatile approach for synthesizing collagen-based nanotubes with controlled and tunable dimensions. Collagen is the main structural protein found in ECM and is broadly used as a substrate or scaffold for cell attachment, proliferation, and differentiation.6 Hence, the use of this protein may have a beneficial role regarding cell-nanotube interactions. The strategy is based on the nanotube template synthesis through layer-by-layer (LbL) assembly. An important feature of the template synthesis method, using track-etched membranes as templates, is the ability to control the dimensions of the resulting nanotubes over a wide range. The outside diameter of the nanotubes, determined by the diameter of the template pores, can indeed be varied from 30 nm up to a few µm, and the length of the nanotubes, determined by the thickness of the template, can range from 5 to 50 µm.7 The LbL technique, based on the alternate adsorption of oppositely charged species,

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has attracted much interest for biomaterial applications as it is a versatile technique, allowing the control of the multilayers properties (composition, thickness, and function).8,9 Combining this LbL process with the template method allows the wall thickness to be controlled, and correspondingly, the inside diameter of template-synthesized nanotubes, by adjusting the number of layers of the material deposited along the pore walls. LbL deposition of polyelectrolytes was demonstrated to occur in nanopores by several groups.10-13 However, up to now, only few papers described the fabrication of nanotubes made from common globular proteins, such as glucose oxidase, hemoglobin, and cytochrome c,14-17 and to our knowledge, the assembly of collagen in LbL multilayered nanotubes has not yet been reported. The use of collagen to build polyelectrolyte multilayers is not trivial. Indeed, owing to its fibrillar structure, collagen has a particular charge distribution. Accordingly, contrary to globular proteins, the isoelectric point (iep) of collagen is not well-defined and is still under debate. Experimental values of 6.0 and 9.3 have been reported in the literature.18,19 A theoretical iep can also be computed using the amino acid composition of collagen. On the basis of the ProtParam tool available on the ExPASy molecular biology server, a value of iep close to 9 was calculated.20 This may explain why in spite of the great interest of collagen in many biomedical applications, up to now, very few papers reported on its incorporation in LbL assembly.21,22 In the present contribution, the ability to incorporate collagen in LbL multilayered film was therefore first investigated on a flat model substrate. Then, conditions allowing collagen diffusion and assembly into template pores were identified, leading to the successful fabrication of collagen-based nanotubes.

Experimental Section Materials. Sodium poly(styrene sulfonate) (PSS, Mw ∼ 70 kDa) and poly(allylamine hydrochloride) (PAH, Mw ∼ 15 kDa) were purchased from Sigma-Aldrich. Type I collagen from calf skin (4 mg/ mL at pH 3.0) was purchased from AutogenBioclear (U.K.). Polyelectrolytes solutions were prepared in 100 mM acetate buffer (pH ) 4.7) at concentration of 1 mg/mL. Collagen was also prepared by dissolution in acetate buffer solution at concentrations values of 10, 100, and 500 mg/mL. All solutions were freshly prepared before use. Quartz Crystal Microbalance (QCM). The build up of PAH/(PSS/ collagen)n multilayers on flat substrate was monitored in situ by quartz crystal microbalance. Measurements were performed with a Q-Sense E4 System (Gothenborg, Sweden) at a temperature of 22.0 ( 0.1 °C. The crystal used is a thin AT-cut quartz coated with a thin SiO2 film (thickness ∼ 50 nm). Oscillations of the crystal at the resonant frequency (5 MHz) or at one of its overtones (15, 25, 35, 45, 55, 65 MHz) were obtained when applying ac voltage. The variation of the resonance frequency (∆f) was monitored upon adsorption of the polyelectrolytes. Solutions were injected into the measurement cell using a peristaltic pump (Ismatec IPC-N 4) at a flow rate of 50 mL/s. Prior to the multilayers build up, acetate buffer solution was injected to establish the baseline. The construction of the PAH/(PSS/collagen)n multilayers was performed as follows: first, PAH soluion was brought into the measurement cell, during 15 min, for allowing the establishment of the adsorption equilibrium at the crystal surface. Subsequently, rinsing was performed for 10 min using acetate buffer solution. PSS and collagen were then alternately injected according to the same procedure, except that the adsorption time for collagen was 30 min. After the build up of 3.5 bilayers, the response of the sensor could no longer be monitored for all harmonics, probably because the film became too thick. Nanotubes Synthesis. The process of nanotube synthesis, depicted in Scheme 1, consists of the build up of PAH/(PSS/collagen)n

Communications Scheme 1. Nanotube Fabrication via the Membrane-Templated Layer-by-Layer Assembly

multilayers onto the pore walls of a nanoporous template. The templates used in this study are 21 µm thick polycarbonate track-etched membranes with pore size of 200 and 500 nm (provided by It4ip, Seneffe, Belgium). After the adsorption of PAH (anchoring layer), the template was alternately immersed in PSS and collagen solutions. The dipping time was fixed at 1 h for polyelectrolyte solutions and varied from 1 to 3 h for collagen. Electronic Microscopies. After the LbL assembly, nanotubes were extracted for observations by dissolving the template in dichloromethane without subsequent sonication (Scheme 1). The LbL thin films deposited on the top and bottom surfaces of the template easily came off during the membrane dissolution process and remained insoluble. For transmission electron microscopy (TEM), nanotubes were collected on carbon grids and imaged with a LEO 922 TEM operating at 200 kV. For scanning electron microscopy analyses, the templates were deposited on a silver membrane (SPI supplies, U.S.A.) and then dissolved and rinsed with dichloromethane to entrap the liberated nanotubes. The samples were then imaged using a field-effect gun digital scanning electron microscope (FE-SEM, DSM 982 Gemini from LEO) operating at 1 kV.

Results and Discussion The LbL assembly of (PSS/collagen)n multilayers on flat model substrate (where n represents the number of adsorbed bilayers) was monitored in situ using QCM as shown in Figure 1. Prior to the multilayers build up, PAH was adsorbed on the negatively charged surface of the SiO2-coated quartz crystal to form an anchoring layer. A shift of the resonant frequency was recorded for each added layer corresponding to the adsorption of PSS and collagen. The frequency shifts and, thereby, the amounts of adsorbed collagen are appreciably higher than those observed for PSS. These results evidenced the build up of PAH/ (PSS/collagen)n multilayers, suggesting that, in the chosen experimental conditions, collagen may be involved as a polycation in LbL assembly. Furthermore, ex situ ellipsometry measurements have shown an increase of the film thickness with the number n (data not shown) to reach approximately 30 nm after the adsorption of 3 bilayers. Based on the results obtained on flat substrate, collagen-based nanotubes were fabricated. One key issue for the LbL assembly of collagen in nanopores, as a result of its high molecular dimensions, was to find the right experimental conditions

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the obtained collagen-based nanostructures and confirmed that nanotubes have an outside diameter equivalent to the diameter of the template nanopores. Finally, the potential influence of dichloromethane (the solvent used for removing the polycarbonate template) on the structure of adsorbed collagen layers was examined by X-ray photoelectron spectroscopy (XPS) and by atomic force microscopy (AFM). This study reveals that CH2Cl2 treatment has no significant effect on the nanoscale organization (Supporting Information, Figure S1) and, on the chemical surface composition (Supporting Information, Figure S2 and Table S1) of adsorbed collagen layers. Though, in this study we used PSS, a strong polyanion, as model system for investigating the LbL assembly of collagen into nanopores, the method described here can, of course, be exploited for fabricating nanotubes using collagen and a variety of other polyanions of biological interest, such as polysaccharides. Figure 1. Build up of PAH/(PSS/collagen)n multilayers monitored in situ by quartz crystal microbalance.

Conclusion We present here the first successful fabrication of nanotubes made from a fibrillar protein, namely collagen, by a templatebased method combined with LbL deposition. The proposed strategy constitutes a practical alternative to electrospinning as it allows a very good and simple control over the dimensions (outside and inside diameters and length) of the resulting nanotubes. Besides their fundamental interest, nanotubes made of fibrillar proteins forming the natural ECM are useful nanoobjects for the creation of new nanostructured biomaterials with numerous potential applications in the biomedical field, especially in regenerative medicine. Moreover, the formation of nanotubes, instead of nanofibers, presents another substantial advantage, as the nanotubular structure allows the easy incorporation of biologically active molecules, such as DNA or growth factors that could be delivered further, for instance during cell culture. These developments are currently under investigation.

Figure 2. SEM (A, B) and TEM (C, D) images of (PSS/collagen)6 nanotubes synthesized by LbL in template with nanopore size of 200 (A, C) and 500 nm (B, D). The concentration of polyelectrolyte solutions used for the LbL process was [PSS] ) 1 mg/mL and [collagen] ) 100 mg/mL.

allowing the diffusion of the fibrillar protein into the nanopores and its deposition onto the pore walls. Our current results show that the formation of nanotubes depends mainly on the collagen solution concentration and the diffusion time of collagen solution into the nanopores. Accordingly, no nanotubes were observed at collagen concentration and diffusion time lower than 100 µg/ mL and 1 h and 30 min, respectively. SEM images show that PAH/(PSS/collagen)6 nanotubes were successfully synthesized using templates with pore size of either 200 or 500 nm (Figure 2A and B). The nanotubes present a smooth surface, a narrow size distribution and, in both cases, an outside diameter corresponding to the diameter of the template nanopores. Furthermore, the nanotubes length corresponds to the thickness of the template. It should be noted that mechanically stable nanotubes were only obtained for at least six deposited PSS/collagen bilayers. More direct evidence for nanotube formation can be obtained by dissolving the membrane and imaging the liberated (PSS/ collagen)n nanostructures using TEM. As shown in Figure 2C and D, TEM images clearly revealed the hollow structure of

Acknowledgment. The authors thank Etienne Ferain and it4ip company for supplying polycarbonate membranes. This work was financially supported by BELSPO in the frame of network IAP 6/27. S.D.-C. thanks the F.R.S.-FNRS for her Senior Research Associate position. C.R. acknowledges financial support from FRIA. Supporting Information Available. XPS spectra and AFM images on adsorbed collagen layers before and after immersion into dichloromethane (the solvent used for removing the polycarbonate template) are given. This material is available free of charge via the Internet at http://pubs.acs.org.

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