Beyond cleaning

Researchers are taking surfactants and emulsions, the ingredients of liquid soaps and face creams, and using them to tackle some of the world’s most challenging infectious diseases. Fiona Case finds out more

It was an unexpected result. James Baker and his group at the University of Michigan Medical Center, US, were hoping to use nanoemulsions to deliver molecules through biological membranes - through the lipid bilayers that protect and contain the contents of living cells. ’We were using bacteria as model systems,’ he recalls, ’and we noticed that the nanoemulsions were killing our models.’

They were using formulas similar to those used in high-end cosmetics and personal care products. The ingredients were unremarkable - oil, water, and detergents (surfactants). But, as the medical group continued to investigate their surprising results - and found that their nanoemulsions were not only active against a wide range of bacteria but also against enveloped viruses and fungi - they knew they had stumbled upon something important.

’Most medicines for the treatment of infections of the skin or mucous membranes contain antibiotics or chemotherapeutics that inhibit specific biochemical pathways,’ Baker comments. ’Indiscriminate use results in the emergence of resistant pathogens.’ Materials that act in a nonspecific manner - such as aldehydes and phenols used as surface disinfectants - are often too toxic and aggressive to the skin for use as topical treatment.

The nanoemulsion system is nonspecific, and it is a pleasure to use (it is the sensorial properties; rapid penetration and pleasant texture, that motivate the use of nanoemulsions in cosmetics and face creams).

Baker took his results to the Defense Advanced Research Project Agency (DARPA) at the US Department of Defense - which was particularly interested in new approaches to kill potential biological weapons such as anthrax. DARPA was impressed, and encouraged him to form a company, NanoBio, to commercialise his technology.

Forming a company has, Baker comments, been ’a strange journey’, but one that he has enjoyed. NanoBio received money from DARPA, and also benefited from its location in Michigan. The Michigan Economic Development Corporation, which promotes its Life Science Corridor with the slogan ’And you thought all we made in Michigan was cars,’ claims that the state leads the US as one of the fastest growing life sciences areas. It provided funding and advice in NanoBio’s critical start-up phase. The young biopharmaceutical company now has a commercial product pipeline of six antimicrobial treatments based on its nanoemulsion technology.

Even washing with soap and water has value in preventing infection and the spread of disease - provided the surfactants stay in contact with your hands for at least 15 or 20 seconds. (The US Centers for Disease Control and Prevention recommends that you sing the ’happy birthday’ song twice to time your wash). This is partly the result of cleaning. But, the surfactants do kill bacteria and viruses. The proposed mechanism is that the surfactants disrupt the biological lipid membranes.

Kyle Vanderlick, professor of chemical engineering at Princeton University, US, describes this as an attack ’below the belt’. ’Biological membranes are amazing packaging materials,’ she comments. ’They are essential to cell viability, but they can’t be perfect barriers: all organisms rely on finely tuned mechanisms for transferring specific materials across this membrane, in and out of their cells.’ This makes them susceptible to attack. For example, antimicrobial peptides, an integral weapon of our bodies’ natural defence systems, are designed to disrupt the membranes of invading bacteria. They appear to mediate the formation of a pore in the bacterial membrane, breaching the defences, allowing vital cell contents to escape (or allowing harmful external material to enter), hence killing the cell.

Vanderlick and her group are working to characterise the material properties of lipid membranes. They want to understand their interaction with various perturbing agents such as surfactants. They study the mechanical properties of model membranes in giant vesicles, which they probe with a micropipette providing a microscopic scale stress-strain experiment. They also look at the rate at which fluorescent probe molecules leak from their model systems.

’We are interested in identifying which surfactants, or surfactant mixtures, will be effective against particular classes of disease,’ she explains. ’For example, bacterial membranes tend to be negatively charged, making them more susceptible to attack by positively charged perturbers.’ Her research has also revealed a useful consequence of a fundamental difference between the membranes that surround bacterial cells, and those of eukaryotic cells (such as animal cells).

’Eukaryotic cell membranes contain cholesterol. The bacterial membrane does not.’ She explains: ’our research has shown that this makes the bacteria more susceptible to surfactant attack (regardless of charge effects). This can be used to advantage in targeting microbiological applications.’

Vanderlick is also working on developing polymer-based perturbing agents that will be used to target selected membranes (such as the HIV envelope membrane).

Baker has developed an incendiary system for antimicrobial warfare. The nanoemulsions provide an effective vehicle for delivering high local concentrations of surfactant to the microbial membrane, and they bring a second agent (the oil) into the disruptive mechanism.

Baker has noticed the effects of the emulsion fusing with the membrane, bringing this second method of attack into the fray. ’The bacteria actually change shape as soya-bean oil is delivered to their interior,’ he says.

The molecular scale mechanism by which lipid membranes are breached, allowing uncontrolled loss of cell contents, or delivery of external material into a cell, is still unclear - although computer modelling studies are offering considerable insight.

’Mesoscale simulation methods such as dissipative particle dynamics provide one of the few means for predicting the behaviour of biological membranes on realistic length and time scales,’ comments Julian Shillcock, from the Max Planck Institute of Colloids and Interfaces in Potsdam, Germany. ’The general shape and chemical character of the molecules is retained, so the molecular scale mechanism can be predicted, but without the atomistic scale details that limit the practical size of molecular mechanics models.’

In a modelling equivalent of Vanderlick’s stress-strain experiments Shillcock has been studying the effect of tension on the vulnerability of membranes, particularly to being breached by fusion with vesicles. ’When the bilayer is under tension, fusion and delivery of the vesicle contents through the bilayer is much more likely to occur,’ he observes. ’This may explain the increased susceptibility of cells to attack by surfactant-based systems during cell division.’

Other modelling studies have shown the importance of a locally high concentration of the membrane-perturbing agent. For example, a group of antimicrobial peptides are proposed to bridge the membrane in the so-called ’barrel stave’ mechanism, creating the pore through which material can be delivered or lost (down the middle of the ’barrel’).

These fundamental studies are providing guidance for designing drug delivery systems. This is in addition to systems designed to kill cells such as Vanderlick’s perturbing agents, or Baker’s nanoemulsions.

The future looks good for NanoBio and for its new form of nanoemulsion antimicrobial treatment. It has been an awarded a US Environmental Protection Agency contract to develop an anthrax decontamination product. It has identified a partner for scale-up, and the nanoemulsions appear to be doing well in phase II clinical trials for treating cold sores (herpes labialis). ’We have treated almost 300 patients with no adverse events,’ comments Baker. ’In fact they were pleased with therapy. By the end of June 2005 we should be able to confirm the efficacy.’ A treatment for nail fungus (onychomycosis) is also in clinical trails and treatments for genital herpes, shingles and vaginal infection are in varying stages of pre-clinical development.

Fiona Case is a freelance science writer based in Vermont, US