X-Message-Number: 32494
Date: Fri, 19 Mar 2010 19:53:11 -0800 (PST)
From: 
Subject: New Technique Turns Proteins Into Glass


[Could this technique turn organs into glass? At the very least, dehydration in 
decanol, would reduce ice formation without having to load additional toxic 
cryoprotectants. It is interesting, that although decanol will not dissolve in 
water, water can still dissolve in decanol.]

New Technique Turns Proteins Into Glass

ScienceDaily (Mar. 17, 2010) - Duke University researchers have devised a method
to dry and preserve proteins in a glassified form that seems to retain the 
molecules' properties as workhorses of biology.


They are exploring whether their glassification technique could bring about 
protein-based drugs that are cheaper to make and easier to deliver than current 
techniques which render proteins into freeze dried powders to preserve them.

Duke engineer and chemist David Needham describes this glassification process as
"molecular water surgery" because it removes virtually all the water from 
around a dissolved protein by almost magically pulling the water into a second 
solvent.

"It's like a sponge sucking water off a counter," said Needham, a professor of 
mechanical engineering and materials science at Duke's Pratt School of 
Engineering, who has formed a company called Biogyali ("gyali" means glass in 
Greek) to develop the innovation. That firm has also applied to patent the idea 
of turning proteins into tiny glass beads at room temperature for drug delivery 
systems.

A report by Needham, graduate student Deborah Rickard and former graduate 
student P. Brent Duncan online in the Biophysical Journal describes how his team
carefully controlled water removal during glassification by releasing single 
tiny droplets of water-dissolved protein into the organic solvent decanol with a
micropipette.

Preliminary evaluations by his senior scientist David Gaul and a team of 
undergraduate students showed that four test proteins undergoing such procedures
retained all or most of their original activity when water was restored. His 
group has received about $1 million from the National Institutes of Health 
grants for the research.

Having devised a way to turn proteins into glassy microbeads measuring only 
about 26 millionths of a meter in diameter, Needham hopes those can be directly 
injected into the body for use as "biologic" drugs.

His group's early research shows high concentrations of such tiny beadlets would
not be as viscous as proteins dehydrated into the normal powder form, which 
tend to clog up syringes, he said.

These microbeads might also be packaged for slow time-release by surrounding 
them with a polymer that would biodegrade over time, though how to do that has 
not been resolved yet, he added.

In collaborations with Duke's Brain Tumor Center and Comprehensive Cancer 
Center, the researchers are seeking additional funding to do initial evaluations
on glassified forms of three molecules with drug potential.

One, known as O6-AMBG, can help the cancer drug Temozolomide work better when 
infused into brain tumors. A second, Lapatinib, is designed to knock out other 
molecules that help cancer cells grow in the breast and elsewhere. The third, 
shepherdin, also targets breast cancers.

Their discovery of protein glassification grew out of a basic exploration of a 
general question: What can dissolve in what?

Needham's research group found, for example, that air and the organic liquid 
chloroform will both dissolve in water at about the same rate. It also found 
that water will dissolve in decanol, a substance it cannot even mix with in 
large quantities.

These experiments, and the theory underlying them, are described in a second 
report led by Needhams's graduate student Jonathan Su, now published online in 
the Journal of Chemical Physics ( http://link.aip.org/link/?JCP/132/044506 ).

"Mixing" and "dissolving" are not the same thing, Needham said. "A good example 
of a suspended mixture is salad dressing, where oil and water are mixed but oil 
does not appreciably dissolve in water, nor water in oil."

They next tried a more complex variation of a familiar high school experiment 
which dissolves so much salt in water that some begins coming back out of the 
solution as a crystal.

In this case, after dissolving the salt in water, Needham's group then inserted 
a microbubble of that solution into immiscible decanol in a microscopic chamber.
The water itself then dissolved into the decanol and left behind the salt, 
which also crystallized.

According to his group's Biophysical Journal report, while decanol has 
practically no tendency to dissolve in water, water has a high probability of 
dissolving in decanol, allowing the latter to be used as a "drying" agent to 
remove the former.

"So then we asked: what if we did the same thing with the protein albumin?" 
Needham said. "I expected to maybe get crystallized albumin," Needham recalled. 
"But, in just a few minutes, we instead formed a glassified microbead of protein
on the tip of a micropipette, at a high density just a bit more dense than 
water itself. That protein glass is not a crystal. It's really a solid liquid."

Many proteins can be coaxed into forming crystals, solids created by repeating 
three dimensional patterns of atoms as surrounding water is removed. On the 
other hand, Needham said he was not really surprised that his protein samples 
instead formed into glasses, which are more unorganized assemblage of molecules 
that can still "flow" over very long time scales.

The water loss in his process is apparently too rapid for the molecules of big 
and irregular proteins to reorganize into a crystal form in such a short time, 
he explained.

Careful studies by his graduate student Rickard found that the decanol removed 
all the water that is not bound up in the proteins' molecular structures. And 
the remaining "bound" water was insufficient to support the growth of bacteria 
and fungi. Storing proteins as microbeads could thus preserve them.

Proteins are currently dried into clumpy, irregular powders by several 
industrial processes -- usually freeze-drying -- to protect them from such 
microbe damage. Drying also avoids the chemical breakdowns that can also occur 
when proteins are kept in solution. "But in the freeze-drying process itself, 
some very sensitive biologic drugs can also get damaged," Needham said.

Freeze-drying proteins into solids is also slower and more expensive than 
glassifying them, he added. And the resulting "flaky" powder is harder to handle
than glassified beads.

Glassification "is a fast process," said Gaul, a senior research scientist in 
Needham's lab. Unlike freeze-drying, "we can dry particles within minutes, if 
not seconds, and don't need any specialized equipment."
Email or share this story:
| More
________________________________________
Story Source:

Adapted from materials provided by Duke University, via EurekAlert!, a service 
of AAAS.
________________________________________
Journal Reference:

Deborah L. Rickard, P. Brent Duncan, and David Needham. Hydration Potential of 
Lysozyme: Protein Dehydration Using a Single Microparticle Technique. 
Biophysical Journal, 2010; 98 (6): 1075-1084 DOI: 10.1016/j.bpj.2009.11.043
Abstract

For biological molecules in aqueous solution, the hydration pressure as a 
function of distance from the molecular surface represents a very short-range 
repulsive pressure that limits atom-atom contact, opposing the attractive van 
der Waals pressure. Whereas the separation distance for molecules that easily 
arrange into ordered arrays (e.g., lipids, DNA, collagen fibers) can be 
determined from x-ray diffraction, many globular proteins are not as easily 
structured. Using a new micropipette technique, spherical, glassified protein 
microbeads can be made that allow determination of protein hydration as a 
function of the water activity (aw) in a surrounding medium (decanol). By 
adjusting aw of the dehydration medium, the final protein concentration of the 
solid microbead is controlled, and ranges from 700 to 1150 mg/mL. By controlling
aw (and thus the osmotic pressure) around lysozyme, the repulsive pressure was 
determined as a function of distance between each globular, ellipsoid protein. 
For separation distances, d, between 2.5 and 9 A, the repulsive decay length was
1.7 A and the pressure extrapolated to d = 0 was 2.2  108 N/m2, indicating that
the hydration pressure for lysozyme is similar to other biological interfaces 
such as phospholipid bilayers.

Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=32494