杏吧原创

MAKE a list of the things that are essential for life as we know it, and it鈥檚
a safe bet that oxygen and light will be up there near the top. But under the
right conditions, this combination can turn from a life-giver to a killer that
is merciless in its attack鈥攖earing molecules apart, shredding proteins and
bulldozing its way through cells.

Doctors have already harnessed this destructive power to deal with cancer
cells, in a technique known as photodynamic therapy (PDT). Blasting cells apart
with light and oxygen might seem like an extreme measure鈥攁 last resort
where there is no other effective treatment. But doctors are now directing this
big gun against a much more common problem. It has become the latest weapon in
the war against bacteria.

The hope is that PDT will be able to defeat some of the most troublesome
bacterial infections鈥攇iving us cheap and simple treatments for gum
disease, wound infections and stomach ulcers, for example. In contrast to
treatments that use antibiotics, the assault will be so violent that it is hard
to see how bacteria could ever develop immunity to it. And as well as treating
disease, PDT could one day take on a whole range of disinfection tasks, from
purifying water to creating bacteria-free fabrics, floors and walls for
hospitals.

Existing PDT cancer treatments involve two components: a light-sensitive
chemical, known as a photosensitiser, and bright light. The chemical is injected
into the body and collects in the tumour. Here it absorbs light energy, which it
then passes on to oxygen molecules. This transforms them into violent killers
that set off through the tumour cells on an orgy of destruction. For obvious
reasons, this treatment is most useful against cancers that are easily
accessible to the doctor鈥檚 light source, such as skin and mouth tumours. And
it鈥檚 very effective鈥擯DT can clear 90 per cent of skin tumours if they are
caught early enough.

The energy-rich form of oxygen that wields this destructive
power鈥攃alled singlet oxygen鈥攊s no ordinary molecule. The extra
energy pumped into it by the photosensitiser raises one of its electrons to a
high energy state, making the molecule reactive and unstable. 鈥淚t鈥檚 about as
reactive as chlorine,鈥� says Raymond Bonnett, a chemist at Queen Mary and
Westfield College in London who is one of the pioneers of the field.

Turning ordinary molecular oxygen into singlet oxygen is a two-stage affair.
First, light energy has to be captured by the photosensitiser, which is usually
some kind of dye. In the second step, an excited molecule of the photosensitiser
passes this energy directly to any oxygen molecule that it happens to meet,
turning it into singlet oxygen. Since almost all living cells are suffused with
oxygen, such encounters are not unusual.

Bonnett describes singlet oxygen formation as a relay race, with one molecule
passing a high-energy baton to another. But if this race is to generate singlet
oxygen every time, chemists must choose photosensitisers with care. When most
molecules absorb light, they don鈥檛 stay for long in an excited energy
state鈥攖hey lose excess energy as heat or re-emit it as light within
nanoseconds. An effective photosensitiser, on the other hand, must hang on to
this energy for much longer in order to meet an oxygen molecule.

Some of the best photosensitisers are porphyrins and
phthalocyanines鈥攎olecules with large ring-like structures which are
related to chlorophyll. Chemists have found that these molecules can trap their
energy for a millisecond or more, easily long enough to pass it on to an oxygen
molecule.

Singlet oxygen, once it has formed, will quickly spread havoc (see Diagram).
If antibiotics can be said to behave like a molecular scalpel, slicing precisely
through a cell鈥檚 metabolic pathways, singlet oxygen is more like a chain saw. It
hacks through almost everything it meets, oxidising proteins, sugars and
unsaturated lipids. That鈥檚 why it goes after the membranes that surround
bacterial cells, says Bonnett.

This sets the scene for a chain reaction. 鈥淭he singlet oxygen chews through
the cell membrane, making it more permeable, so that the photosensitiser can
then get inside the bacterium,鈥� says chemist Jackie Lacey from Imperial College,
London. Once inside, the photosensitiser absorbs more light and generates more
singlet oxygen, which chops into the bacterium鈥檚 structure. Amino acids,
including tryptophan, methionine and histidine, are sliced up, destroying or
damaging the bacterium鈥檚 proteins. And the excited photosensitiser can generate
reactive free radicals such as the superoxide ion O₂^鈥�路 and the
hydroxyl radical OH路, which drift through the cell, leaving their own
trails of destruction.

Chink in the armour

As drug designers know all too well, some bacteria are easier to kill than
others. 鈥淕ram-positive鈥� bacteria have thin cell membranes which are porous to
photosensitisers, so these organisms are easily killed with singlet oxygen. But
the rest of them, the 鈥淕ram negative鈥� type, are more of a challenge. They have a
complex outer structure that keeps many photosensitisers and antibiotics at bay.
Because of this extra armour, Gram negative bacteria such as Escherichia
coli and Salmonella can cause infections which won鈥檛 respond to
conventional treatments.

But photochemists have found a chink in this armour. Stan Brown from the
Centre for Photobiology and Photodynamic Therapy at Leeds University has found
that positively charged or 鈥渃ationic鈥� photosensitisers are more efficient
against cultures of Gram negative bacteria than their negatively charged
鈥渁nionic鈥� counterparts. Anionic photosensitisers can鈥檛 get into the bacteria
directly says Brown. But switch to cationic photosensitisers and it鈥檚 a
different story鈥攑hotosensitisation becomes deadly. 鈥淲e assume that there
is something about cationic photosensitisers that can disturb the cell wall,鈥�
says Brown. Since the outer membrane of Gram negative bacteria is negatively
charged, Lacey suspects that positively charged photosensitisers bind to the
membrane, disrupting it or damaging its proteins and lipids with singlet oxygen
or free radicals.

This is all very well for lab studies, but how do you apply the technique to
fight disease? One site of infection that appears ideally suited to singlet
oxygen treatment is the mouth. There are all kinds of harmful bacteria in there
(see 鈥淥pen wide, we鈥檙e going to explore鈥�, New 杏吧原创, 14 March, p
32). Treating them with antibiotics in toothpaste is pretty much a nonstarter,
because that would encourage the bacteria to develop resistance. But at the
Eastman Dental Institute in London, Mike Wilson and his team have found that a
combination of the cationic photosensitiser toluidine blue and 30 seconds of red
light from a low-power laser is all that they need to kill oral bacteria. Their
technique can wipe out Streptococcus mutans, which causes tooth decay,
and the Gram negative Porphyromonas gingivalis, which leads to gum
disease.

Ilya Eigenbrot and David Phillips at Imperial College have teamed up with
Wilson to develop a kind of 鈥渟mart toothpaste鈥�, which contains a photosensitiser
that is selectively absorbed by bacteria. Wilson says he can also get pinpoint
accuracy by attaching toluidine blue to antibodies directed against specific
bacteria. He is starting clinical trials soon, but admits the treatment is 鈥渁t a
very early stage鈥�.

The researchers have also designed a 鈥渓aser toothbrush鈥� that gets the light
to where it鈥檚 needed by making the bristles work as optical fibres (see
Technology, 23 November 1996, p 24). 鈥淎 tiny laser or bright diode fits inside
the handle, and the light is beamed onto the teeth through the bristles,鈥� says
Eigenbrot. The researchers say a healthcare company is already interested in
their device, but they are keeping its identity secret.

The use of optical fibres raises the possibility of directing light deep
inside the body to kill bacteria lurking there. Stomach and duodenal ulcers
might become one of the next targets for the technology: their main cause is a
bacterium called Helicobacter pylori. 鈥淚t鈥檚 staggering that a single
bacterium can be responsible for so much disease in the stomach and duodenum,鈥�
says Stephen Bown from the National Medical Laser Centre at University College
London. 鈥淚t affects almost 20 per cent of the world鈥檚 population.鈥� He has
already begun work to see if laser light delivered through optical fibres can
activate photosensitisers to combat this bacterium but, like Wilson, concedes
there is still a long way to go. Part of the problem is that the offending
bacteria have to be completely removed from the stomach, not just from the
ulcerated areas. 鈥淓radication in the stomach may be possible, but it is a huge
task,鈥� says Bown (left).

There could be other problems too. Once you have unleashed singlet oxygen,
how do you ensure that it kills only the cells that you want it to, and not
healthy tissue as well? 鈥淚t won鈥檛 discriminate between the baddies and the
goodies,鈥� says Bonnett. However, researchers have found that most bacteria are
more vulnerable to this treatment than healthy human tissue. 鈥淚t takes less
light to kill bacteria than healthy cells,鈥� says Bown. And if it does kill some
cells from healthy tissue, the damage usually heals quickly, he says.

One of the attractions of using oxygen to kill bacteria is that they are not
likely to develop the sort of resistance that is making some strains immune to
antibiotics. 鈥淚t is difficult to see how a cell could protect itself from this
sort of physical attack,鈥� says Eigenbrot. 鈥淚t鈥檚 a bit like asking whether you
could become immune to being run over by a bus.鈥� Not everyone is that confident.
鈥淭here are bacteria out there that can resist almost anything,鈥� says Stuart
Levy, director of the Center for Adaptation Genetics and Drug Resistance at
Tufts University in Boston, who points out that some bacteria can switch on
mechanisms to handle oxygen radicals. But Levy accepts that the sheer volume of
singlet oxygen generated by photosensitisers will probably overcome what
defences the bacteria may have. Toxicologist Bruce Demple of the Harvard School
of Public Health in Boston agrees that chemistry is on our side. 鈥淲e don鈥檛 know
enough yet to be sure,鈥� says Demple, 鈥渂ut you can probably overwhelm the
bacteria鈥檚 defences by upping the concentrations.鈥�

Brushing aside worries about resistance, Bonnett is taking the idea even
further, and testing it as an all-purpose disinfectant that kills bacteria
before they cause infections. He has incorporated a porphyrin photosensitiser
into polymers and fibres, which could be turned into films or woven into
clothing or bandages. He has already tested out the idea at home, with a cloth
impregnated with photosensitiser. After it had been in use for a week, he
compared the number of bacterial colonies it harboured with those on an
untreated one. 鈥淭here were about a hundred times less on the treated one,鈥� says
Bonnett. And he didn鈥檛 need a laser鈥� ordinary household lighting was
enough.

Useful discoveries

In a similar vein, Bonnett and his colleagues have fixed tetraphenyl
porphyrin photosensitisers into Cellophane films that could be used to wrap
food. Under tests lasting 24 hours, during which the films were exposed to
light, there was no growth of E. coli or Staphylococcus
aureus鈥攕ome strains of which are resistant to antibiotics and are
responsible for many infections in hospitals. Bonnet has also put the dye into
polystyrene films that could be used to make plastic tiles or wall coverings.
And cloth impregnated with photosensitiser could be used for antibacterial
clothing. 鈥淚t could be useful in kitchens and operating theatres,鈥� he says.

The chance discovery of unexpectedly low numbers of bacteria in the waste
water from a textile dyeing factory suggests another
application鈥攕ensitisers could be used to purify drinking water. The
photosensitiser could be bound to polymer beads or trapped inside a porous
material, and then mounted in reservoirs or holding tanks, while sunlight does
the rest. The sensitiser could generate a lethal dose of singlet oxygen, which
diffuses through the water to kill most waterborne bacteria. This could provide
a cheap way to purify water, especially in countries where there is plenty of
sun.

Best of all, its advocates say, singlet oxygen has none of the environmental
problems associated with other disinfecting agents such as chlorine, which can
react to form chlorinated hydrocarbons in the environment. Singlet oxygen only
remains active for microseconds. After that, says Bonnett, 鈥渋t just switches
back to gaseous oxygen鈥攖he oxygen of the air.鈥�

Parasitic fungi of the genus Cercospora have become experts in using chemical
warfare to break through their host鈥檚 defences. They synthesise cercosporin, a
singlet oxygen generator, which diffuses into the cell walls of banana palms or
coffee plants鈥攖he fungi鈥檚 main hosts. As light falls on the leaves,
singlet oxygen generated by the photosensitiser breaks down the fatty acids in
the cell鈥檚 membrane, allowing nutrients to leak out to the hungry fungus.

But securing its food source in this unorthodox manner is just one of
Cercospora鈥檚 tricks. Equally important is the way it protects itself from the
ravages of the singlet oxygen it produces. Plant pathologist Margaret Daub from
North Carolina State University has isolated two genes that are linked to this
resistance. Knocking out either one of these genes leaves the fungus sensitive
to its own attack. One gene is unique, but to Daub鈥檚 surprise, the other is
found in some organisms that are sensitive to singlet oxygen. At the moment she
has few clues to what either gene does.

There are danger signals here: the fact that some organisms contain genes for
resistance to singlet oxygen suggests that others could acquire similar
immunity. But Daub believes this is unlikely to happen: 鈥淲e have tried
mutagenesis and selection for resistance in tobacco cells, but we were never
able to pick up anything that was more resistant.鈥�

Cercospora fungi contain carotenoids鈥攎olecules that provide protection
against singlet oxygen by quenching light-activated forms of the
photosensitiser, as well as singlet oxygen itself. But Daub has found that
mutant strains of the fungus that do not make carotenoids are still just as
resistant. The mystery remains.