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MATERIAL MATTERS COLUMN

Oxides of nitrogen

Professor Williams retired from the University of Liverpool, after 40 years, at the end of 2007. He retains the position of Emeritus Professor there and now has a series of professorial appointments in North Carolina, USA; Sydney, Australia; Cape Town, South Africa; and in China. He offers consulting services from his company Morgan & Masterson, based in Brussels, Belgium. He is Editor-in-Chief of Biomaterials, the leading journal in the biomaterials field. He is Scientific Director of STEPS, the European Commission Framework VI Programme on a Systems Approach to Tissue Engineering Products and Processes.

Oxides of nitrogen tend to have a bad image in that they are often considered to be the major components of air pollution. We all know that nitrous oxide (NO) used to have some benefits because it was employed (as laughing gas) for anaesthesia, especially for dental procedures. However, these oxides in general are reactive chemicals and major constituents of automobile exhaust and play a significant role in the development of photochemical smogs that pollute the atmosphere of many major cities, which is obviously no laughing matter. Until recently, the monoxide NO received little attention apart from this focus on its detrimental effects and text books tended to ignore this obnoxious little molecule.

In the last couple of decades, the situation has changed. It has been realised that NO, as well as being highly reactive and labile, has considerable significance within biological systems. It is ubiquitous and plays a variety of roles in physiological processes, especially within immune and inflammatory processes. One of the consequences of this has been the attention given to the potential use of NO to control the foreign body response to biomaterials. This is not a trivial process because the molecule is one of those that can exert cytotoxic effects under some circumstances and control host defence mechanisms under others, especially within the vascular system.

The physiological functions of NO

NO is a gas and is produced by cells in the body through the enzymatic action of NO synthase (NOS) on the amino acid L-arginine. There are three forms of NOS, which partly explains the complexity of the activity of NO, and two of these are found in endothelial cells, which also partly explains the noticeable vascular effects. One form of NOS, called nNOS, where n signifies neural, serves as a transmitter in the brain and in the peripheral nervous system. The two endothelial forms: inducible NOS (iNOS) and constitutive NOS (cNOS), which is sometimes referred to as eNOS, affect blood flow and the action of smooth muscle cells in the endothelium. It is cNOS that is associated with the generation of NO under normal circumstances; the amount is dependent on shear stresses in the blood and on the availability of receptors on the cell surface However, under unusual circumstances it is iNOS that becomes active, for example, when there is bacterial infection or a cytokine-influenced inflammation such as in the presence of a stent, artificial artery or invasive catheter.

NO is known to relax the smooth muscle in the blood vessel walls. The endothelial walls under the influence of shear stress during systole release a burst of NO that allows the unimpeded flow of blood through the relaxed vessel. Reduced levels of NO production cause hypertension. The NO at the surface of the endothelium also resists platelet aggregation. Moreover, the NO produced by cNOS inhibits inflammation in the vessels and resists smooth muscle hyperplasia (associated with restenosis of intravascular stents). NO is also particularly effective in killing bacteria.

Although therapeutic use of NO offers the possibility of enhancing many physiological reactions, there is one serious issue that must be faced when considering this application. It has a half life of only a few seconds within biological systems, mainly because it quickly reacts with superoxide anions and with haemoglobin. Any successful therapy therefore has to involve the continuous generation of NO and the delivery of the NO by diffusion to the relevant cells and molecules. In this respect, the NO is also able to activate the enzyme guanylyl cyclise within the endothelium, and this will act as a secondary messenger to stimulate those reactions that lead to smooth muscle relaxation and other events. It therefore becomes of immense importance to be able to develop a system that is able to continuously generate NO.

NO generating systems

Several possibilities exist for the preparation of NO generating systems that could be applied to the surface of polymers to improve their biocompatibility. Most involve the use of certain types of chemical agent that can be doped into or grafted onto the surface of the polymer where they release NO within the body. There are two important compounds here, the diazeniumdiolates and the nitrosothiols. It is possible, for example, to take a diazeniumdiolate and incorporate it into a gel that can be applied to a silicone elastomer.¹ The release of NO is initiated through contact with aqueous solutions, where the water decomposes the diazeniumdiolate to yield a continuous flux of NO. The rate of release is controlled by the rate of diffusion into the surface and the rate of diffusion out again of the NO. Typically, much of the NO will be released during the first few hours, then prolonged but small fluxes are observed over several days. It is possible to tune the rate and extent of release to some degree by control of the thickness of the surface layer and its composition.

The value of this type of release profile will obviously depend on the required functionality of the NO in the particular application. In some situations, the most important events in the biocompatibility of a device take place in the first few hours and the type of profile mentioned above could be of considerable value. The protein adsorption that takes place on invasive biosensors (biofouling) and which limits their sensitivity has been shown to be reduced if those sensors have a NO releasing coating. It is also possible for this short term effect to be translated into longer term benefits through reduced platelet adhesion. It is not all good news, however, because it has been shown that as these substances react with water and leach into the body fluids, the NO can become oxidised, leading to the formation of nitrosamines, which may be carcinogenic. A great deal of work is being undertaken to control the formulation so that either noncarcinogenic residues are produced or the diazeniumdiolate is covalently linked to the polymer and is therefore incapable of leaching.

The second main group NO donor involves sulphur- (S) nitrosothiols. These are substances that occur naturally such as S-nitroso-serum albumin, which is found in circulating blood and where the bond between the S and the NO is cleaved through one of several catalytic mechanisms. S-nitrosothiols have been covalently linked to polymers that have been used experimentally to produce site-specific delivery of NO to, for example, arteries after balloon angioplasty.²

Limitations and possibilities

This concept of NO releasing polymers has been discussed in numerous publications during the past five to six years and several patents relating to devices have been filed Although there are a number of therapeutic possibilities, the primary target appears to be the development of thromboresistant coatings for intravascular devices such as extracorporeal circuits and intravascular oxygen sensors. As noted above, the principal methods investigated so far involve the incorporation of NO donor molecules into the polymer. The main limitations here are the small reservoir of NO releasing molecules, which tends to provide acute effects only, and the potentially harmful by-products. An alternative approach may involve the use of agents attached to a polymer surface that are able to release NO from endogenous NO donor molecules. This is the case with the circulating S-nitrosothiols mentioned above, which could in theory imply an unlimited supply of NO. These are attractive possibilities for future blood compatible medical devices.

David Williams Morgan & Masterson, Avenue de la Forêt 103, Brussels 1000, Belgium, tel. +32 4 7597 0556, e-mail: peggy@morgan-masterson.com, www.morgan-masterson.com