Generation of Bioactive Materials with Rapid Self-Assembling Resorcinarene-Peptides

The aim of this work was to immobilize short melanocyte-stimulating hormone (MSH) anti-inflammatory peptide sequences onto a model surface using resorcinarenes, which are known to attach to a wide variety of hydrophilic materials. The first stage comprised the biological evaluation of a synthetic MSH peptide attached to different linking tethers (poly(ethylene glycol), PEG 350, octanoic acid or cholesterol) for the ability to inhibit inflammatory signaling. Findings showed that a glycinelysine-proline-D-valine sequence inhibited inflammatory signaling most effectively when attached to a PEG 350 tether. This molecule was selected for a second stage which comprised of synthesizing the MSH peptide attached to resorcinarene groups via a PEG tether and immobilizing it onto glass coverslips. X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF–SIMS) indicated the presence of a surface attached peptide. The ability of the immobilized peptide to inhibit inflammatory signaling was then determined by culturing RN22 Schwann neuronal cells and human dermal fibroblast cells on functional surfaces and measuring NF-kB/p65 inflammatory transcription factor activation. Significant inhibition of inflammatory signaling was observed in cells cultured on functional surfaces. In conclusion, we therefore describe the development of a new approach enabling the rapid immobilization of short biologically active peptides. This work has potential for generating a ‘‘dip-and-dry’’ approach for altering the surface properties of biomaterial and medical devices. Many implantable medical devices can initiate an acute inflammatory response which in turn is detrimental to the function of the device. This can be triggered either by the initial implantation injury or by the biomaterial itself. Examples of this include coronary restenosis after stent implantation, the ‘stress cracking’ of pacemakers and the failure of peripheral nerve guides. Inflammation can be treated by steroids or non-steroidal anti-inflammatories (NSAIDs). However, these compounds frequently lack sensitivity or a specific targeted site of action, leading to side effects. Therefore the development of more specific and localized forms of treatment is important for controlling inflammation associated with biomaterials. Previous work has investigated modifying the material surface chemistry or immobilizing non-bio-fouling molecules, such as PEG. However, these techniques are not always appropriate because they do not specifically target inflammatory signaling. An alternative approach is through the exploitation of naturally occurring peptides to reduce inflammation. One potential peptide is a-MSH which has multiple roles in the body including the control of inflammation. An analogue of a-MSH, NleDPhe-a-MSH, has already been shown to increase allograft survival when injected into rats. Previous research has shown that the minimal sequence required for anti-inflammatory biology is a tripeptide at the carboxyl terminal consisting of lysine-proline-valine (KPV). This tripeptide mimics the anti-inflammatory effect of the full-length peptide, but is more specific as it does not exhibit melanotropic activity or stimulate dopa oxidase activity. This peptide has the ability to inhibit nuclear factor-kB (NF-kB) activation when stimulated by a wide range of proinflammatory agents, reducing the production of proinflammatory cytokines under its control. KPV can stimulate the direct production of the anti-inflammatory cytokine interleukin-10 (IL-10) and inhibit inflammation in vivo. Additionally, the peptide has been demonstrated to possess antimicrobial properties in vitro. The short tripeptide sequence makes it amenable for straightforward laboratory synthesis and this, in combination with its pharmacological potency, makes it an attractive candidate for immobilization on to medical device surfaces for creating a localized anti-inflammatory effect. a-MSH and its analogues have previously been immobilized using polyelectrolyte films and also as an analogue of the tripeptide (GKP-D-V), where it was attached to polystyrene beads. Both techniques demonstrated that the peptide retained anti-inflammatory properties when immobilized in vitro. Prostheses of titanium beads coated with polyelectrolyte multilayer films functionalized with a-MSH also retained the ability to inhibit inflammation when implanted into rats. The D-Val substitution in KP-D-V leads to greater anti-inflammatory properties than the KPV peptide and the parent molecule, a-MSH, probably due to reduced degradation. The present work describes a new methodology for immobilization of the tripeptide analogue GKP-D-V using resorcinarene group molecules. Resorcinarenes can be immobilized through hydrogen bonding of the hydroxylated bowl to hydrophilic materials through simple immersion of the material into a solution of the resorcinarene compound. This forms a new surface on that material, the properties of which then depend on the nature of the group covalently attached to the resorcinarene. Previous research has investigated the use of attaching alkyl chains and