**Biomimetic Materials as Potential Medical Adhesives – Composition and Adhesive Properties of the Material Coating the Cuvierian Tubules Expelled by** *Holothuria dofleinii*

 1Yong Y. Peng1, Veronica Glattauer1, Timothy D. Skewes2, Jacinta F. White1, Kate M. Nairn1, Andrew N. McDevitt3, Christopher M. Elvin3, Jerome A. Werkmeister1, Lloyd D. Graham4 and John A.M. Ramshaw1

### **1. Introduction**

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Novel, distinct adhesive systems have been described for a wide range of marine species (Kamino, 2008). These highly effective, natural materials provide a link between biological science and material science, and can serve as models on which new, bioinspired synthetic materials could be based. These various adhesive systems have developed independently, on many occasions, and provide a wide range of opportunities for the development of new, biologically-inspired adhesives. The natural adhesives include, for example, the marine mussel (*Mytilus sp.*) (Lin et al., 2007), barnacle (Nakano et al., 2007) and stickleback (Jones et al., 2001) adhesives, which are protein-based, as well as sponge, certain algal and marine bacterial adhesives (Mancuso-Nichols et al., 2009) that are polysaccharide-based.

In the present paper, we examine the adhesive system found associated with the Cuvierian tubules of a holothurian species (sea cucumber), *Holothuria dofleinii*. This is an example of the particularly rapid marine adhesive that is found on the surface of Cuvierian tubules when they are expelled (DeMoor et al., 2003; Müller et al., 1972; VandenSpiegel & Jangoux, 1987). The unique nature of this natural adhesive system, especially its rapid action under water, has suggested that if the mechanism can be understood, then it may prove to be possible to mimic the adhesive through biotechnology and/or synthetic chemistry. An adhesive that functions readily in an aqueous environment would be particularly valuable, especially in medical applications, as the majority of existing adhesives bind to dry surfaces more strongly than the same surfaces when wet.

Cuvierian tubules provide a host defence mechanism for certain species of holothurians (Lawrence, 2001; VandenSpiegel & Jangoux, 1987). It has long been known that, on expulsion, the Cuvierian tubules fill with liquid and lengthen, become sticky and rapidly

*<sup>1</sup>CSIRO Materials Science and Engineering, Bayview Avenue, Clayton, VIC 3169, Australia 2CSIRO Marine and Atmospheric Research, Middle Street, Cleveland, QLD 4163, Australia 3CSIRO Livestock Industries, Carmody Road, St Lucia, QLD 4067, Australia* 

*<sup>4</sup>CSIRO Food and Nutritional Sciences, Julius Ave, North Ryde, NSW 2113 ,Australia1*

Biomimetic Materials as Potential Medical Adhesives – Composition and Adhesive

euthanised by freezing at minus 20 °C.

pass filters on an Olympus BX61 fluorescence microscope.

XL30 FESEM microscope at an accelerating voltage of 2 kV.

**2.4 Gel electrophoresis analysis**

**2.5 Amino acid analysis**

glass slides. After air drying, the tubules were examined by microscopy.

insoluble material, although the yield of adhesive proteins was less.

**2.3 Microscopy**

Properties of the Material Coating the Cuvierian Tubules Expelled by *Holothuria dofleinii* 247

to drain briefly (<20 sec), but not by squeezing as had been proposed by others (Zahn et al., 1973). Intact tubules prior to expulsion were obtained by dissection of animals that had been

To look for the presence of glycoprotein on the surface of expelled tubules, samples were treated with fluorescently-labelled lectins; fluorescein isothiocyanate (FITC)-labelled concanavilin A (ConA), FITC-labelled *Datura stramonium* agglutinin (DSA), and FITClabelled *Lycopersicon esculentum* agglutinin (LEA) (all from Sigma, St Louis). All FITClabelled lectins were applied as 20 μg/mL solutions in Tris-buffered saline (TBS) for 60 min, followed by 3 × 5 min washes in TBS. Samples were examined using appropriate narrow

To examine the distribution of adhesiveness on tubules, individual freshly expelled tubules after draining (see above) were transferred to a wash solution in a plastic trough which contained a suspension of 0.5% w/v Bio-Gel P2 (45-90 m particle size) in 3.5% w/v NaCl, 10 mM sodium phosphate, pH 7.6. After 5 sec immersion, the tubules were washed 3 times in 3.5% NaCl, 10 mM sodium phosphate, pH 7.6 and were then drained and placed onto

For scanning electron microscopy (SEM) expelled tubules were examined using a Philips

Freshly expelled and drained tubules were allowed to adhere to a glass plate and were air dried. The tubules on glass plates were removed by peeling, leaving the layer of adhesive, and potentially other components of the tubule wall as a print on the glass (DeMoor et al., 2003). This material was collected by removal with a sharp razor blade and was then extracted in electrophoresis sample buffer, containing 2-mercaptoethanol. SDSpolyacrylamide gel electrophoresis (SDS-PAGE) was based on the method of Laemmli (1970) using Invitrogen NuPAGE Novex 4-14% Bis-Tris Gel with MES running gel buffer, at 180V for 60 min. Molecular weights were determined by comparison to globular protein standards (BioRad) using BioRad Quantity One v.4.4.0 software. For protein identification, gels were stained by Coomassie Blue R-250. Samples that had not been dried completely, but only sufficient to remove excess liquid, appeared to give samples that contained less

Protein extracts were separated by SDS-PAGE, followed by transfer of the protein bands to PVDF membrane using Invitrogen NuPAGE Transfer buffer (NP0006-1). Amino acid analysis of PVDF membrane pieces used vapour-phase hydrolysis (5.8 M HCl at 108 °C for 18 h), followed by precolumn derivatisation with 6-aminoquinolyl-*N*-hydroxysuccinimidyl carbamate (Cohen & DeAntonis, 1994). Derivatives were separated and quantified by reversed phase (C18 Waters AccQTag) HPLC at 37 C (Cohen, 2001) (Australian Proteome Analysis Facility), using a Waters Alliance 2695 Separation Module, a Waters 474

Fluorescence Detector and a Waters 2487 Dual Absorbance Detector in series.

immobilise most organisms with which they come into contact (VandenSpiegel & Jangoux, 1987). The tubules, once expelled, are immediately adhesive on contact with a solid surface (VandenSpiegel & Jangoux, 1987), such as the exoskeleton or skin of a predator. Crabs, molluscs and sea stars can stimulate tubule expulsion, and the tubules stick to these species. This adhesion happens entirely under water, and does not need the mixed environment of the intertidal zone where many of the other potential adhesives are sourced.

Sticky tubules are found only within the family Holothuridae within the order Aspidochirotida, and mostly in the genus Bohadschia and the genus Holothuria. Various authors have described the ultrastructure of the tubules, especially for *H. forskåli* (Lawrence, 2001; VandenSpiegel & Jangoux, 1987; VandenSpiegel et al., 2000), as well as their expulsion and release (Flammang et al., 2002) and the timeframe of regeneration (Flammang et al., 2002; VandenSpiegel et al., 2000).

Flammang and Jangoux (2004) suggested, from the differences in the surface (adhesive) protein types and compositions in *H. forskåli* and *H. maculosa*, that the adhesion proteins and mechanism may differ between species. Other studies showed that adhesive strengths varied between species, with the adhesion in *H. leucospilota* being several times greater than for six other species (Flammang et al., 2002). A limited number of studies have probed the mechanism of adhesion, focusing on *H. forskåli* and *H. leucospilota* (De Moor et al., 2003; Müller et al., 1972; Zahn et al., 1973). These studies have shown that best adhesion is found at temperatures, salinity and pH similar to those found in the marine environment in which the organism flourishes, and is most effective with hydrophilic surfaces (Flammang et al., 2002; Müller et al., 1972; Zahn et al., 1973). Increasing concentrations of urea led to a loss of adhesion, suggesting that native protein structure(s) or interctions(s) may be required for effective bonding (Müller et al., 1972). Later biochemical studies have also suggested that the adhesive mechanism involves protein components (DeMoor et al., 2003).

In the present study, we have extended the information on Cuvierian tubule adhesion. In this study we examined the tubules of a different species, *H. dofleinii* Augustin, 1908. We have examined the distribution of the adhesive substance on the surface of expelled tubules, along with the molecular weights and amino acid compositions of its main protein components. We have estimated the strength of adhesion of *H. dofleinii* tubules to different substrata, and examined the effects of salinity, pH, ionic strength and denaturants on the adhesive properties.
