Diffusion of 2,2,6,6-tetramethylpiperidine 1-oxyl derivatives of variable hydrophobicity in tropocollagen I solution Chi Kin Liu, Judith Valle, Katarzyna Slowinska ⁎ Department of Chemistry and Biochemistry, California State University Long Beach, Long Beach, CA 90840, United States abstract article info Article history: Received 26 November 2007 Received in revised form 29 July 2008 Accepted 6 August 2008 Available online 19 August 2008 Keywords: Collagen Cross-linking Diffusion Hydrophobicity Electrochemical time-of-flight was used to measure the diffusion coefficients of 2,2,6,6-tetramethylpiperidine 1-oxyl derivatives, C n TPA, (3 to 7 CH 2 groups), in tropocollagen I solution, as a function of the chain length and the cross-linking with glutaraldehyde. The values of the diffusion coefficient of C n TPA in pure aqueous electrolyte follow the Stokes–Einstein law, i.e. the diffusion coefficient is inversely proportional to the size of the redox probe. Different behavior is observed in 0.5% (w/v) tropocollagen solution where the molecules with longer alkyl chains show larger diffusion coefficients than the smaller molecules. This behavior is explained in terms of electrostatic interactions between tropocollagen chains and the C n TPA molecules. The measurements of the diffusion coefficients of C n TPA in 0.5% tropocollagen cross-linked with glutaraldehyde indicate that while the C 7 TPA and C 5 TPA probes exhibit lower diffusion coefficients upon addition of 0.05% GA and 0.1% (v/v) GA respectively, the other C n TPA molecules exhibit either unchanged or increased diffusion coefficients under the same conditions thus indicating the presence of hydrophobic pockets selectively interacting with C n TPAs. These results demonstrate the utility of electrochemical time-of-flight in measurements of diffusion coeffi- cients in complex biopolymeric media. © 2008 Elsevier B.V. All rights reserved. 1. Introduction Collagen is a main component of connective tissue [1]. It is also one of the most promising biomaterials for targeted drug delivery systems, scaffolding, and mechanical parts fabrication [2–4]. The tropocollagen consists of rod-like polypeptide chains about 300 nm in length and 1.4 nm in diameter. The conformational stabilization of the collagen triple helix is often explained by its imino- rich structure [5,6]. Since the imino residues are incapable of hydrogen bonding and are not uniformly distributed, they create local hydrophobic “pockets” within the collagen structure where the sequence shows multiple repeats of proline and hydroxyproline in the X and Y position (general sequence for helical peptides can be expressed as Gly-X-Y). In fact, the NMR study by Charlton et al. has shown that the association between the polyphenol and proline rich peptides is of a hydrophobic nature even though the collagen matrix is in general hydrophilic [7]. Collagen used in drug delivery systems is rapidly biodegradable in vivo without chemical modifications. In order to stabilize its structure, intramolecular and intermolecular cross-linking is employed [8]. The commonly used cross-linking agent, glutaraldehyde (GA), forms a bridge by reacting with side chain ɛ-amino groups [1,9,10]. The addi- tion of GA to the solution of tropocollagen leads to the formation of both intramolecular as well as intermolecular cross-linking resulting in a polymeric gel-like network. The cross-linking with GA not only changes the structure of collagen, but it also potentially increases the hydrophobic character of the matrix. In fact, partial hydrophobicity of collagen cross-linked with glutaraldehyde or modified by methylation of the matrix was previously reported via water swelling experiments [11,12]. Hydrophobic interactions between collagen and small molecules are important in designing new drug delivery systems. The mechan- ism of association of small molecules with collagen is also important to understand the process of the collagen fibril formation [3,13,14]. Therefore there is a need to systematically study the problem of hydrophobic interactions between small molecules and collagen matrix both in the native and cross-linked form. The electrochemical time-of-flight (ETOF) technique is particularly useful in measurements involving polymeric systems because it does not require prior knowledge of the redox probe concentration, thus the possible adsorption of matrix molecules on the electrodes does not complicate the interpretation of experimental data. In addition, in polymeric systems the exact concentration of the redox probe could be difficult to determine, so the value of ETOF in studying this type of systems cannot be underestimated [16]. In this report we describe the measurements of diffusion coeffi- cients of several (2,2,6,6-tetramethylpiperidine 1-oxyl (Tempo) amide derivatives (C n TPA, n =3–7) in a tropocollagen I solution both in the native form and cross-linked via reaction with glutaraldehyde. The C n TPA molecules are modified with alkyl chains of various lengths (Fig. 1) and thus exhibit different hydrophobicities. Our goal is to Bioelectrochemistry 74 (2008) 195–200 ⁎ Corresponding author. E-mail address: kuslowin@csulb.edu (K. Slowinska). 1567-5394/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bioelechem.2008.08.002 Contents lists available at ScienceDirect Bioelectrochemistry journal homepage: www.elsevier.com/locate/bioelechem