-SCIENCE & TECHNOLOGY PACIFICHEM
2000
ONESTEPTOBIOACTIVE POLYMERS Supercritical C 0 2 disperses inorganic materials, enzymes in biodegradable composites MAIRIN B. BRENNAN, C&EN WASHINGTON
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HASE DIAGRAMS AREN'T AN UN-
usual sight at scientific meetings. But the one Steven M. Howdle began his presentation with at a Pacifichem symposium on solvation structure and reactivity in supercritical fluids certainly was. Howdle, a professor of chemistry at the University of Nottingham in England, didn't show a phase diagram of a supercritical fluid—instead it depicted the interrelated phases of his research career, which evolved from spectroscopy to embrace microemulsions, polymers, biomaterials, and tissue engineering. Howdle described his group's most recent achievement: using supercritical C 0 2 to incorporate inorganic materials and enzymes into the biodegradable polymers poly(lactide-c0-glycolide), PLGA, andpoly(DL-lactide),PLA[Cfe#. Commum, 109 (2001)]. But first he reviewed the steps that led up to this accomplishment and influenced the focus of his research along the way including being inspired by a pro-
B I 0 C 0 M P 0 S I T E S Images of hydroxyapatite particles (top) and platelike catalase (bottom) dispersed in poly(lactide-co-glycolide) matrices were obtained by scanning electron microscopy. 34
C & E N / J A N U A R Y 2 9 , 2001
lar interactions in microemulsions could be tailored for polymers." AT THIS POINT, Howdle's research took a turn toward biomaterials. Surgeons at the university's medical school asked him if he could find a way to incorporate hydroxyapatite (calcium phosphate hydroxide) into a PLGA matrix as a first step toward creating artificial bone. Hydroxyapatite is the natural mineralizing agent in bone. PLGA is used in fashioning some drug-delivery devices and as scaffolding material in tissue engineering. Soon after, Howdle attended a conference where he heard a presentation by
cedure to make powdered coatings for paint applications. In 1996, Howdle collaborated with Keith P.Johnston, a professor of chemical engineering at the University of Texas, Austin, to make microemulsions of water in supercritical C0 2 , using a fluorinated surfactant to stabilize the water droplets. Experiments with infrared spectroscopy Poly(lactide-co-glycolide) (PLGA) proved that the microscopic droplets are stabilized by dual interactions: The surfactant's ionic head forms hydrogen bonds with the water while its fluorinated tail F-^CF — CF 2 — o4-CF — C02H confers solubility in C0 2 . 1 J I /i| CF3 Meanwhile, Joseph M. DiSimone, an CF, associate professor ofchemistry at the UniKrytox compound versity ofNorth Carolina, Chapel Hill, had n~U shown that free-radical polymerization of physical chemist Frederick methyl methacrylate can be m S. Mandel, project director carried out in supercritical 2 of supercritical fluids techC 0 2 in the presence of the | nology at Ferro Corp., Inderight stabilizing block co- > pendence, Ohio. Mandel polymer. The copolymer is g described a procedure to constructedfromaunit that's £ make powdered coatings for soluble in C0 2 and aunit that paint applications. Such attaches to the poly(methyl coatings are sprayed on cars, methacrylate) particles and refrigerators, and other stabilizes them as they begin metal surfaces. to grow The copolymer is a The standard approach "huge molecule," Howdle involves grinding the pigsaid. "I had an idea there ment into a fine powder might be another way of HOWDLE that's physically mixed into doing this." He wondered whether an analog of the surfactant mole- the plasticized polymer, a process "somecule he'd used for the water microemulsions what akin to mixing [talcum powder} in molasses," Howdle said. Both the grindmight be used as a stabilizer. Working with graduate student Paul ing and mixing are energy-intensive, highChristian at Nottingham and DerekJ. Irvine temperature processes that must be careat ICI Acrylics in Middlesborough, Eng- fully controlled to avoid cross-linking the land, Howdle chose as a putative stabilizer polymer, he noted. The paint industry was the commercially available compound Kry- looking to produce low-temperature-curtox 157FSL—a perfluoroether containing a ing paints for spraying applications. The approach that Mandel described is carboxylic acid head group. The researchers reasoned that the molecule's perfluoroether based on supercritical C0 2 . Polymer and tail would provide a strong interaction with powdered pigment are lumped together in C0 2 while the carboxylic acid head group an autoclave, which is filled with C 0 2 at interacted with the methyl methacrylate high pressure. The polymer is plasticized polymer {Macromolecules, 33, 237 (2000)}.by dissolved C0 2 , and then the pigment is 'We saw a beautiful polymer form," How- homogeneously dispersed through the dle said. "What we learned about molecu- polymer via a stirrer. Fast release of pres-
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sure forces a fine-powdered mixture of polymer and pigment through a nozzle for collection. The entire operation takes place at near ambient temperature. "I thought we could use the same mechanism to incorporate hydroxyapatite into PLGA," Howdle said. "We modified the technique by releasing the pressure slowly to make a spongelike apatite-containing foam." In collaboration with Mandel, pharmaceutical scientist Kevin M. Shakesheff and other colleagues at Nottingham, and physicist Vladimir K. Popov at the Russian Academy of Sciences' Institute on Laser & Information Technology in Troitsk, Howdle achieved this goal. "The nice thing is there's no other solvent involved, and the whole thing is carried out near room temperature," he said. The group now is working to grow bone tissue by introducing both bone-forming and bone-metabolizing cells into hydroxyapatite-loaded PLGA scaffolds. EARLIER WORK by other researchers had shown that supercritical C 0 2 can be used to create microcellular foams of amorphous polymers. It also showed that the rate of depressurization strongly influences pore size and number. With Howdle's approach, this control of pore size can be achieved at the same time the mixing process is carried out. The technique therefore should provide a one-step process for producing polymers for sustained release of bioactive compounds, Howdle claims. He and his colleagues have incorporated three different enzymes—catalase, ribonuclease A, and p-D-galactosidase—into either PLGA or PLA composites andhave shown that the enzymes retain full catalytic activity The inherently low solubility of proteins in supercritical C0 2 provides a key advantage in preserving protein structure and function, he noted. "The enzyme is packed into a crystal, it's held together by hydrogen bonds, and C 0 2 isn't going to interfere with that type of bonding." The group also has studied the release of ribonuclease A over 70 days and shown that, after an initial burst release ofless than 10% of the drug over thefirsttwo days, the enzyme is released steadily until its supply is exhausted. This is the type of response pharmaceuticalfirmslook for, Howdle said. The team believes their approach may have an impact on the design of tissue engineering scaffolds for generating cartilage, bone, and liver tissue. "We can make highly porous sponges that slowly release growth factors" that retain their activity in a threedimensional biodegradable complex, Howdle explained. • HTTP://PUBS.ACS.ORG/CEN
May 13-18, 2DD1 ID L.
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International Symposium on Applications of Enzymes in Chemical and Biological Defense 13-18 May, 2001 Sheraton World Resort Orlando, Florida • Presentations on the latest research on both medical and non-medical applications of enzymes • Emphasis on enzymatic reactions with biological and chemical warfare agents, carbamate and organophosphorus pesticides, and related materials • Topic areas: protection, detection, decontamination, prophylaxis and treatment Open to government agencies, academia and industry. Sponsored by: • U.S. Army Soldier and Biological Chemical Command (SBCCOM) • U.S. Army Research Office (ARO) • U.S. Army Medical Research and Material Command (MRMC) • Defense Advanced Research Projects Agency (DARPA) At this time, papers and posters are still being accepted. For an announcement on papers and posters or a registration packet, please contact: Debbie Bilotto Tel: 410.612.8247 Fax: 410.612.9968 E-mail: bilotto
[email protected] A block of rooms has been reserved under Enzyme Symposium at the host facility, Sheraton World Resort, 10100 International Drive, Orlando, Florida 32821. Reservations are to be made by calling 1-800-327-0363. Please identify yourself as attending the Enzyme Symposium when you call. Space is limited and will be distributed on a first-come, first-serve basis. All reservations should be made by 13 April, 2001. Room rate will be $86.00 per night.
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