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August 26, 2002
Volume 80, Number 34
CENEAR 80 34 pp. 39-47
ISSN 0009-2347

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THE LACK OF experience with drug delivery systems composed of dendrimers is a disadvantage to people who are developing them now, according to Frechet. "You are starting a whole new ball game," he says. "You have no history of their application."

Frechet collaborates with Francis C. Szoka Jr. of the departments of biopharmaceutical sciences and pharmaceutical chemistry at the University of California, San Francisco, to deliver anticancer agents such as doxorubicin with 2,2-bis(hydroxymethyl) propanoic acid dendrimers [Bioconjugate Chem., 13, 443 and 453 (2002)].

One of the molecules Frechet is working on is actually a hybrid between a linear polymer and a dendrimer, consisting of a three-arm poly(ethylene oxide) star attached to dendritic moieties. "Essentially, we are using the dendrimer to provide us with multiple sites of attachment, and we are using the linear polymer to provide us with water solubility," Frechet says.

The doxorubicin is attached to the dendrimer through a hydrazone linker, which can be cleaved simply by changing the pH. "It's a fairly simple bond to break. It's a linkage between the amino group of a hydrazine and keto group of the drug. Doxorubicin has a ketone that can be used for that purpose," Frechet says. "Most people have chosen to attach doxorubicin through its amino group, but that gives an amide. Amide linkages are very hard to break, so we have avoided that."

It's necessary to adapt dendrimers to different drugs, Frechet says, although his group is interested in making as generic a system as possible. They are simplifying the system by breaking it into two parts: a drug carrier and a portion that would confer solubility and the ability to circulate in the system.

"We are looking at making them such that they will self-assemble. You have two components, you mix them, and bang, you get one," he says. "To the outside world, it would look just like the solubilizing component. It will essentially surround, engulf the other one, and help in its transport and delivery." So far, Frechet only has chemical data about this system, but biological studies with Szoka will start later this year, Frechet says.

"You provide a quantifiable benefit by attaching a drug--a very toxic drug used in chemotherapy--to a dendrimer," Frechet says. "You may reduce or eliminate its toxicity. If you can target it properly, you eliminate the side effects." Frechet describes their initial mouse in vivo work as "pretty good."

AT THE CRS MEETING, Alexander T. Florence, dean of the University of London School of Pharmacy, described his group's work in forming particles with dendrimers to serve as drug carriers.

Dendrimers can aggregate to form larger structures that Florence called "dendrisomes" and "dendriplexes." Dendrisomes are formed by combining dendrons (dendrimer segments) with cholesterol to form a vesicle. Dendrisomes are similar to liposomes, which have long been the workhorses of drug delivery.

One of the ways that Florence is using dendrimers is to form complexes with DNA. The resulting particles are then used to orally administer DNA. For example, mice that were fed particles containing DNA coding for b-galactosidase did indeed express the protein.

In addition, Florence's group is interested in using targeting ligands to direct where the particles go. For example, they have used internalin, a protein from the bacterium Listeria monocytogenes, as a targeting ligand. Internalin is a ligand for the receptor E-cadherin, which is expressed in the intestine. So far, the research "has not gone as far as hoped," Florence said.

Despite much talk about targeting ligands, it has proven to be more difficult than anticipated to develop active targeting strategies. "It's easy to put up a cartoon," Florence said, "but difficult to actually do."

Duncan believes that people were "a bit naive" about active targeting of drugs. Targeting cells in a dish is one thing, but the physiological system is much more complex. On the issue of targeting in cancer applications, Duncan tells C&EN: "Imagine an intravenous injection, with all the complexity of blood proteins, the cells lining the vessels, the endothelial cells of the vessel wall, and other cells which interface with the blood in the liver. You have to make sure that you're not getting any interaction with all those normal cells before you arrive at the tumor site."

Another focus for drug delivery researchers is DNA delivery, in which DNA is treated as a macromolecular drug (C&EN, Nov. 26, 2001, page 35). Nonviral gene delivery has several advantages over gene delivery with viral vectors, according to Sung Wan Kim, a professor of pharmaceutics and pharmaceutical chemistry at the University of Utah. These advantages include versatility, no integration into the host chromosome, and fewer problems with immunogenicity, he told the audience for his plenary lecture at the CRS meeting. 

POLYMERS SERVE TO condense DNA and protect it from degradation before it can express the desired protein. But because what works in vitro doesn't always work in vivo and vice versa, coming up with a good gene carrier can be tough. "In vivo, you have to go through more barriers than in cell culture," says Kam W. Leong, professor of biomedical engineering at Johns Hopkins University.

There are three stages in delivering the DNA to the nucleus. First, the particle has to be taken up by the cell through endocytosis. After it's in the cell, the polymer must be able to escape the endosome (a type of vesicle) by disrupting the membrane. Then the polymer has to transport the DNA to the cell nucleus. "We can control the specific design of polymers for each function," Kim says.

The polymer requirements for gene delivery depend on how the DNA will be administered, according to Kim. "If we want to deliver the gene systemically, I think we have to use a water-soluble, biodegradable, cationic polymer," he told C&EN. For local delivery, Kim conjugates cholesterol with polymers to promote interaction with cells in the vascular wall and enhance uptake. In addition, targeting ligands can direct the DNA to a specific location. For example, Kim has used galactose to target the liver and various antibodies to home in on leukemia cells, myocardial cells, and angiogenic tissue.

Leong has used both naturally occurring polymers, such as chitosan, and synthetic polymers for DNA delivery. Designing polymers for DNA delivery involves a "tricky balance," he says. "The DNA has to be free before it can work, but part of the function of the gene carrier is to protect the DNA from enzymatic degradation before it can reach the nucleus of the cell. If the nanoparticle is breaking down too fast, then it will not work well."

Leong and Hai-Quan Mao developed new polyphosphoesters as DNA carriers at the Johns Hopkins Singapore Biomedical Center. The researchers chose the polyphosphoesters because of their structural versatility, as they can be varied at either the backbone or the side chain. At the CRS meeting, Leong described research in which they systematically evaluated different poly(phosphoroamidates), which consist of a phosphoester backbone with amine side chains.

Another application in which drug delivery and materials development play a role is in tissue engineering. At first the relationship between the two areas seems tenuous, but for some types of tissue engineering, it's not a stretch at all.

For example, Jeffrey A. Hubbell, professor of biomedical engineering and director of the Institute for Biomedical Engineering at the University of Zurich and the Swiss Federal Institute of Technology, Zurich, performs tissue engineering by delivering growth factors to the desired site.

"Doing tissue engineering with factors to stimulate cells in the body is really just fancy drug delivery," Hubbell says. "One is delivering a drug--like a protein, a morphogenic factor--that stimulates cellular responses at a site with the goal of ending up with some overall tissue reconstruction or regeneration at that site." Hubbell is using drug delivery for such tissue engineering applications as angiogenesis, bone repair, and nerve regeneration.

 
8034cov1.Right1 8034cov1.Right
ANCHORED When the angiogenic growth factor VEGF is incorporated into a fibrin matrix but allowed to diffuse freely, angiogenesis occurs both in the matrix and elsewhere on the chorioallantoic membrane of a fertile chicken egg (left). When an engineered form of VEGF is covalently coupled to the fibrin matrix, cells migrate into the matrix, cleave the coupled growth factor, and release it on demand, resulting in a much more localized response (right).
COURTESY OF JEFFREY HUBBELL

THIS APPLICATION OF drug delivery presents challenges for materials development. Hubbell is using naturally occurring molecules that are normally involved in development. These molecules "operate in a particularly complex environment in development. Cells are evolved to respond to them in that complex environment," Hubbell says.

"The naive approach from drug delivery was just to take materials that were developed to deliver steroids--utterly different molecules--and to use those same sorts of materials," Hubbell says. Such an approach has been ineffective, he notes, because the growth factors "have been presented completely out of context, completely in a different environment than nature intended them to be presented." One of the main needs in drug delivery for tissue engineering, therefore, is the development of materials that will present the growth factors in the way that they would be presented during development.

One challenge, Hubbell believes, is to use biochemical approaches in materials development. That means designing materials that degrade by processes other than hydrolysis. "Building in biological character to synthetic materials or to natural materials that have been engineered--I think that's a route that is likely to meet with success." One example of this biological character is making "the material degrade in a way that's triggered by the healing response."

Hubbell and his group mimic the natural system by chemically coupling the growth factors to a gel matrix or by building affinity sites into the material. "The growth factors are coupled to the matrix by a linker that cells can cleave," Hubbell says. The matrix "can release the factor when the cells are ready for it."

Hubbell has used natural materials such as fibrin and synthetic materials as matrices for delivering factors. He thinks that synthetic materials will ultimately be the way to go. "At the end of the day, you have much more control over the characteristics of the material," he says. "You should be able to design everything, select everything, instead of just modifying what nature gave you.

 
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FULL SIZE - CLICK IMAGE
TWEAKING Replacing aromatic side chains with acyl side chains improves the interaction between the block copolymer and the drug amphotericin B.

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Copyright © 2002 American Chemical Society



 
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Gene Delivery--Witout Viruses
[C&EN, Nov. 26, 2001]

Robert Langer's Engineering Magic
[C&EN, Feb. 18, 2002]

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