Imagine bandages that could kill bacteria or drinking glasses that sanitize themselves. Last year, a research group reported a treatment for glass surfaces that killed a range of bacteria on contact. Since then, they've extended the system to polymer surfaces.

Now, another group has independently developed an antibacterial treatment that's applicable to porous materials that are carbohydrate based, such as clothing and paper. And a third group has developed a family of compounds that could potentially lead to other antibacterial surface treatments. None of the approaches is expected to exacerbate the problem of growing bacterial resistance to antibiotics.

In the study reported last year, researchers demonstrated that covalent attachment of N-alkylated poly(4-vinylpyridine) groups (or N-alkyl PVP groups) to glass surfaces made those surfaces le-thal on contact to both gram-positive and gram-negative bacteria, the two major classes of bacteria [Proc. Natl. Acad. Sci. USA, 98, 5981 (2001) and C&EN, May 28, 2001, page 13]. That study was carried out by postdoc Joerg C. Tiller, visiting scientist Chun-Jen (Jason) Liao, and professor of chemistry and bioengineering Alexander M. Klibanov of Massachusetts Institute of Technology, in collaboration with biology professor Kim Lewis of Northeastern University. The most effective agents they identified were PVPs with N-alkyl chains three to eight carbon units in length.

Klibanov, Lewis, and MIT postdocs Tiller, Jian Lin, and Sang Beom Lee have since found that N-hexyl PVP treatments can be used to create surfaces that kill wild-type and antibiotic-resistant bacteria, not only on glass but also on commercial polyethylene, polypropylene, nylon, and poly(ethylene terephthalate) plastics [Biotechnol. Lett., 24, 801 (2002) and Biotechnol. Bioeng., 79, 466 (2002)]. And Klibanov, Lewis, Lin, and MIT visiting scientist Shuyi Qiu also recently discovered that alkylated polyethyleneimines can be used to create bactericidal coatings on glass surfaces and even on magnetic iron oxide nanoparticles [Biotechnol. Prog., submitted]. The MIT/Northeastern group has applied for broad patent protection on these bactericidal technologies.

Last year, the group's initial antibacterial coating study received extensive coverage in major newspapers and magazines. But another antibacterial surface treatment that's just as promising has received virtually no media attention. In a Division of Carbohydrate Chemistry poster session at the American Chemical Society national meeting this April in Orlando, Fla., a New York City-based team revealed a surface treatment that could do for clothing and paper what the MIT/Northeastern treatment does for glass, plastics, and nanoparticles.

"It's difficult at this time to imagine bacteria mutating to avoid this type of action--although bacteria can be sharp little devils."

THE NEW TREATMENT was conceived and developed by assistant professor JaimeLee Iolani Cohen and undergraduates Tanya Abel and Maya Filshtinskaya in the department of chemistry and physical sciences at Pace University, research assistant Alice Melkonian and assistant professor Karin Melkonian in the department of biology at the C. W. Post Campus of Long Island University, and professor Robert Engel of the department of chemistry and biochemistry at Queens College of the City University of New York.

The carbohydrate-based materials the group surface-treated were cotton cloth, gauze, wood, paper, and bulk cellulose. The researchers activated the materials' carbohydrate surface groups by tosylation and then displaced the tosylate groups by reaction with an amine reagent--a 1,4-diazabicyclo[2.2.2]octane (DABCO) group with an attached lipophilic alkyl chain. The inspiration for the reagent was a set of 1993 studies--carried out by associate professor of polymer chemistry Akihiko Kanazawa and professors of polymer chemistry Tomiki Ikeda and Takeshi Endo of Tokyo Institute of Technology, Yokohama, Japan--in which phosphonium and ammonium polymers were found to exhibit antibacterial activity.

After derivatizing the surfaces of the five carbohydrate-based materials with the DABCO-alkyl reagents, Cohen, Karin Melkonian, Engel, and coworkers exposed the materials to three types of gram-positive bacteria (including staph) and four types of gram-negative bacteria (including Escherichia coli). Treatments with reagents containing 10-, 12-, and 18-carbon alkyl groups were active against gram-positive bacteria but not against the gram-negative strains.

The agent containing a 16-carbon lipophilic chain, however, was effective against both gram-positive and gram-negative bacteria. A number of gram-negative bacteria are found in the gastrointestinal tract and are responsible for diseases such as sepsis, so it's particularly important that a proposed antibacterial surface treatment not leave them out.

The DABCO-hexadecane treatment kills a wide range of bacteria on simple contact. It is useful both for clothing and wound dressings, it doesn't require additional agents, and it is not removed from surfaces on washing. "The agent is chemically bonded to the surface such that it cannot be washed out under normal conditions and is not modified upon interaction with the bacteria," Cohen notes. "Thereby, it remains capable of continually acting against the bacteria."

INDEED, SURFACES derivatized with the DABCO-hexadecane reagent prevented growth of all seven bacterial strains tested, even after repeated washing and reapplication of bacteria to the surfaces. Engel says that the agent also kills yeast.

Cohen, Melkonian, Engel, and coworkers have since extended the applicability of the antibacterial effect from the five types of surfaces they initially tested to wool and silk. In fact, the researchers believe the approach will be applicable to any carbohydrate-based material their antibacterial reagent can react with.

They have applied for patent protection on synthesis of the surface-active agent and on a wide range of potential applications, including antibacterial and antifungal effects. And they have submitted a paper on the work to a journal.

A major difference between the antibacterial treatments devised by the MIT/Northeastern and the New York groups is that, so far, the former has been shown to be effective only on nonporous surfaces while the latter has been demonstrat-ed to be applicable only to carbohydrate-based porous surfaces. The mechanism of both techniques most likely involves disruption of bacterial cell walls.

"The recent findings of the Klibanov and Lewis collaboration and the Engel group have demonstrated that very simple chemical functionalization of surfaces is sufficient to endow materials with strong antimicrobial properties," comments Jonathan S. Dordick, department head and professor of chemical engineering at Rensselaer Polytechnic Institute. "In both cases, the researchers have functionalized surfaces with polyalkyl cation derivatives, which are known to disrupt bacterial cell walls and/or cell membranes, leading to cell death. The alkyl chain length strongly influences the antimicrobial properties of the surface coatings," says Dordick, a specialist in enzyme engineering.

The techniques involved are not synthetically demanding, and the studies don't provide "deep mechanistic insight into the antimicrobial properties of these materials," he adds. But the work is nevertheless "elegant in its simplicity and certainly successful in preventing the attachment and growth of bacteria on treated surfaces. Moreover, the ability to prevent colonization of bacteria from water and air (in the case of the Lewis and Klibanov study) or water (in the case of the Engel study) may have significant potential in preventing the unwanted transfer of microbial contaminants and in alleviating the eventual formation of biofilms on materials in contact with microbial solutions," he says.

Meanwhile, research by a third group suggests that further surface treatments may be on the way. The team has synthesized a novel family of arylamide polymers that are antibacterial and could eventually be used to treat surfaces [Proc. Natl. Acad. Sci. USA, 99, 5110 (2002)]. The work was carried out by University of Pennsylvania chemistry professor Michael L. Klein; biochemistry and biophysics professor William F. DeGrado; postdocs Gregory N. Tew (now assistant professor of polymer science at the University of Massachusetts, Amherst), Dahui Liu, Bin Chen, and Robert J. Doerksen; graduate student Justin Kaplan; and chemistry department X-ray facility director Patrick J. Carroll.

"THE POTENTIAL ability to keep surfaces and materials permanently antiseptic has significant implications and is very exciting," Tew says. The researchers have applied for patent protection on the technology.

According to Tew, the polymers they identified "mimic the complex structures and remarkable biological properties of proteins that fight bacteria"--specifically, endogenous defense peptides, such as the magainins. Defense peptides often have facially amphiphilic conformations, in which positively charged hydrophilic and uncharged hydrophobic groups segregate onto opposite faces of the structures. The peptides' facial amphilicity is believed to be responsible for their ability to kill bacteria and other cells by disrupting their phospholipid membranes.

Earlier, -peptides that mimic the properties of defense peptides were synthesized by DeGrado's group and a group led by chemistry professor Samuel H. Gellman of the University of Wisconsin, Madison. And self-assembling cyclic peptides that mimic defense peptides were designed and synthesized last year by chemistry professor M. Reza Ghadiri of Scripps Research Institute and coworkers. The arylamide polymers synthesized by Tew, Klein, DeGrado, and coworkers now extend the range of defense-peptide mimics still further. The polymers attach to cell membrane surfaces and punch holes in the membranes, causing cell death--just like defense peptides do.

"Such surface-active polymers could be used for a variety of purposes, such as antimicrobial materials and surfaces," the researchers note. They hope in the future to refine the cell-type selectivity of the polymers and to possibly test them as drugs.

"The potential ability to keep surfaces and materials permanently antiseptic has significant implications and is very exciting."

THE THREE research teams believe their antibacterial treatments will not worsen the growing problem of bacterial resistance to antibiotics.

The MIT/Northeastern bactericidal surfaces are unlikely to cause antibiotic resistance "because their putative mechanism of action is the disruption of bacterial membranes, as opposed to a specific biochemical pathway," Klibanov says. Bacteria with severely damaged membranes can't easily remain viable and produce mutated offspring resistant to the antibacterial agents, he notes.

Engel likewise says that because his group's treatment is "seemingly not acting on a metabolic process of the bacteria but rather seems to attack the cell wall directly," it probably won't evoke antibiotic resistance. "It's difficult at this time to imagine bacteria mutating to avoid this type of action--although bacteria can be sharp little devils," Engel concedes. However, "we don't have the data yet to provide a complete picture of the mode of action," Engel says, "and one of the things we want to look at is the possibility of development of resistance."

The only known mechanism of antibiotic resistance to antibacterial cationic monomers like the common antiseptics benzalkonium chloride and chlorhexidine, Lewis points out, is extrusion of the compounds by a bacterium's multi-drug-resistance pump. In the group's Biotechnology Letters paper, Lewis, Klibanov, and colleagues found that cells engineered to overexpress multidrug pumps exhibited considerable resistance to such conventional antimicrobial agents but were nevertheless killed by the group's N-hexyl PVP. "Apparently, a multidrug pump can extrude one cationic molecule at a time but not a polymer thread covered with cationic antimicrobials," Lewis says. "The designed polymer does not have a direct analog in nature, suggesting that resistance will not be easily developed."

As for the antibacterial polymers developed by Tew, Klein, DeGrado, and coworkers, "the multi-drug-resistance pumps that typically protect cells against small amphiphilic compounds are likely to be ineffective against our polymers due to their size," Tew says.

The applicability of antimicrobial coating techniques may eventually be extended to a broader range of microbes, Dordick notes. "Will it be possible to generate polymer-containing materials with different functionalities and/or alkyl compositions and chain lengths that target specific microbes, and will it be possible to extend this approach to nonbacterial contaminants, such as fungal and parasitic solutions or even viruses?" he asks rhetorically. "One may envision a range of polymers coated on a surface to prevent a wide range of microbial colonization," Dordick says.

Alternatively, "it may be possible to generate arrays of coatings that can be used to identify specific microbes, based on the sensitivity of given organisms to different surface treatments," he continues. "You can put a number of different chemistries there that might be able to target various types of cells." For example, specific biowarfare agents might be detected by incorporating enzymes or other biologically active compounds into surfaces and then analyzing the way unknown microorganisms respond to or interact with those surfaces. "This is something that we are now working on in our lab," he says.

MIMICKERS Tew (at right) and (from left) Liu, Chen, DeGrado, Doerksen, and Klein identified antibacterial arylamide polymers based on repeating unit shown.

PHOTO BY FELICE MACERA

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