History

Selenium (Se) is an essential trace element for humans and all other animals. The paradox of selenium, however, is that it is both essential and also toxic (Spallholz, 1994). Selenium deficiency is responsible for disorders such as "white muscle disease" in several animal species and Keshan disease, a cardiomyopathy affecting primarily young women and children in China (Oldfield, 1987;Yang et al., 1983). Toxic levels of selenium in animals are responsible for "blind staggers", a CNS disorder in livestock. Since the discovery of selenium in 1817 it was understood only as a toxic element until 1957, when Klaus Schwarz revealed the ability of selenium to protect against dietary liver necrosis. It is now known that an average adult human male requires approximately 40 ug Se/day to make the 25 known human selenium proteins. In 1989 the USDA established a recommended daily allowance for selenium of 70 ug Se/day for adult males, 55 ug Se/day for adult females, and many individuals may ingest up to 600 ug Se/day or more as nutritional supplements. The UTL, upper tolerable limit, for selenium is greater than 800 ug Se/day, and some studies have reported patients taking up to 3.2 mgs of selenium per day for a year with no serious toxicity. In fulfilling the nutritional requirement, dietary selenium from the inorganic salts, selenate and selenite, and the organic selenium compounds, mainly the primary protein amino acid selenomethionine, are metabolized by cells into the selenocysteine found in all selenium enzymes and proteins (Hawkes et al., 1985).

Redox Chemistry

Very recently, an understanding of selenium as a catalytic generator of superoxide (O2-.) from the oxidation of thiols has emerged. The catalytic attribute of selenium has been known for nearly five decades, but the pro-oxidative characteristics of several selenium compounds has only recently been elucidated. In general, selenite and selenium dioxide are catalytic by their oxidation of thiols, such as glutathione (GSH), forming the glutathione selenopersufide anion, GSSe- that oxidizes GSH producing superoxide. Diselenides, such as selenocystine, are reduced by thiols forming the selenide anion, RSe-, which is also catalytic. The selenide anion is the catalytic species, catalyzing the oxidation of thiols producing superoxide, hydrogen peroxide (H2O2) and a putative thiyl radical. These and likely other reactive oxygen species appear to account for selenium’s toxicity to cells. The selenide anion in these small organoselenium compounds, which produces O2-, is also the catalytic moiety of the selenocysteine residue in all known selenium enzymes (Spallholz, 1994). This selenium free radical generating chemistry is shown below:

R-Se- + 2 GSH + O2 —› R-Se- + GSSG + O2-.

Covalent Linkage

Unlike other biocidal agents like copper and silver ions which can also generates reactive oxygen species, selenium can be covalently attached to various materials with no loss of its catalytic activity. Selenium can be covalently substituted for sulfur, oxygen, and nitrogen in most organic molecules. It may also be covalently attached to any terminal carbon atom of any molecule forming a catalytic selenide anion. Using standard and variations of typical protein and carbohydrate attachment chemistries, carboxyl and amino containing selenides may be routinely attached to many polymers, peptides, antibodies, steroids and drugs. Polymers and other molecules with attached selenides generate superoxide in a dose dependent manner in biological solutions, in cells or attached to insoluble matrixes such as silicones.

Because of the benefits of selenium we are applying the SeLECT technology to a wide range of applications ranging from armed therapeutics to coatings for medical devices, industry, and consumer products.

Therapeutics

Selenium compounds may be used as an arming agent for antibodies, peptides, small molecules, and nucleotides (e.g. aptamers). The selenium technology attached to a targeting agent delivers a lethal payload to tumor cells, pathogenic microbes, or other specific cells. Because of selenium’s unique mode of action, selenium-armed agents have been shown to kill bacteria at 4o C, indicating that there need not be metabolic activity for selenium to be effective. Selenium-armed molecules have even been shown to directly oxidize virus particles. Due to the limited localization of the free radical production, bystander cells are not damaged and the selenium is metabolized without adverse effects. In fact, a therapeutic dose of selenium is half of the recommended daily allowance and several-fold less than the amount taken by many as a
nutritional supplement.

Most cancer therapies, such as chemotherapy and radiation, are intrinsically toxic and can be lethal to both healthy and diseased cells. While antibodies are very specific in the cells they target, they also have several drawbacks. If an antibody does not directly block signal transmission, then its effectiveness depends on activating other components of the immune system, delivering toxic drugs which must be internalized by the cell, or delivering a dose of radiation ( See Chart ). SeLECT armed antibodies overcome most of the potency obstacles pack the punch of a radiolabeled antibody without the hepatic or renal toxicity. Internalization of the antibody is not required since the free radical reaction is lethal to the targeted cells even at the cell surface and specific binding is not required. If the SeLECT antibody binds, the cell will be killed. Free radicals are extremely reactive and typically only travel a very short distance before finding something to react with. This extreme localization of the toxic effects means that SeLECT antibodies are very safe for nontargeted bystander cells.

Lethal Specific Side Effects Profile Cytotoxic Drug A D F Blocking Ab C A A Immune recruiting Ab C A A Drug armed Ab B- A B Isotope armed Ab A B C SeLECT Ab A A A

In vitro and in vivo results have demonstrated the effectiveness of selenium with various targeting agents. Of particular note are results achieved in a nude mouse colorectal tumor model. All mice received treatment with an antibody targeting the colorectal tumor cells. One group, however, received this antibody armed with selenium; the other group received unarmed antibody. The figure shows the dramatic effect of utilizing selenium as an arming agent with this antibody.

Colorectal tumor model: the effect of arming with selenium.

	Antibody With Selenium	Antibody Without Selenium

Coatings

Because selenides can be covalently attached without loss of catalytic activity, it is particularly well suited for applications requiring a biocidal surface coating agent.We are investigating and developing use of selenium coatings for applications including medical devices, textiles, paints and sealants, consumer products (e.g. sponges), and other industrial/consumer uses and products. Materials to which selenium has been already successfully attached include cellulose sponges, silicone hydrogel lenses, polymethymethacrylate (PMMA, using a CO2 plasma generator), epoxy, as well as biological molecules such as peptides and antibodies.

	1) Surface is treated with Selenium	2) When bacteria attempt to colonize the coated surface….
	3) Selenium kills the bacteria on contact	4) Bacteria can’t live on the Selenium coated surface

As a demonstration of the ability of selenium-coated materials in inhibiting bacterial cell growth, selenium-coated and control sponges were tested against the ability of the pathogenic bacteria Staphylococcus aureus to colonize and form a biofilm. Selenium-coated and control sponges were immersed in L-broth and inoculated with Staphylococcus aureus which was allowed to proliferate in the broth for 4 days. At this time sponges were fixed in gluteraldehyde and visually examined using a scanning electron microscope following gold sputtering. The figures below show electron micrographs (EM) from the selenium-coated sponge and untreated control sponge at 700 X magnification. The inset photo on the right shows the untreated sponge at 3000 X magnification. In the EM of the selenium-coated sponge one can easily see the cellulose fibers of the sponge. In the untreated control sponge the entire sponge is obscured by a Staphylococcus biofilm. In the inset photo one can see the bacterial cells in the untreated sponge.

Cellulose sponges: antibacterial effect of coating with selenium.

	Selenium-treated Sponge	Untreated Control