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

DRUG DELIVERY
Materials scientists look for new materials and ways to manipulate existing ones in order to fulfill unmet needs
SCAFFOLD Highly branched dendrimers, depicted here among cells shown in green, may one day deliver drug molecules
CENTER FOR BIOLOGICAL NANOTECHNOLOGY, UNIVERSITY OF MICHIGAN

CELIA M. HENRY, C&EN WASHINGTON

In the context of drug delivery, the needs for materials can generally be broken into two categories, notes Robert S. Langer, a professor of chemical and biomedical engineering at Massachusetts Institute of Technology: the creation of new materials and better understanding of how to manipulate existing materials.

In both cases and in whatever route of administration, "you go to the unmet needs," Langer says. "The unmet needs lead you to where materials can do something." Current needs include reducing the toxicity of drugs, increasing their absorption, and improving their release profile.

In one fertile area of research, scientists are tailoring polymers to address those needs. They are using long-standing polymers like poly(ethylene glycol) (PEG) and newer types like dendrimers. And they are forming polymeric micelles and using polymer-drug conjugates as prodrugs, just to name a few.

Last month, scientists presented examples of research on materials for drug delivery at the annual meeting of the Controlled Release Society (CRS). More than a thousand scientists gathered in Seoul, South Korea, to hear talks on topics ranging from polymeric carriers for anticancer agents to particles for gene delivery to scaffolds for cell delivery and tissue engineering.

ONE AREA THAT researchers have particularly been focusing on is the delivery of anticancer agents. Polymers have already been shown to form effective delivery systems for localized treatment of cancer. But Ruth Duncan, director of the Center for Polymer Therapeutics at the Welsh School of Pharmacy at Cardiff University, wants to use polymer-drug conjugates to treat metastatic cancers as well, which are much more difficult to deal with.

"If we can give [the conjugates] by injection, then we have an opportunity to target the micrometastases that can be present throughout the whole organism," Duncan says.

Polymer carriers have several advantages over other delivery methods such as liposomes and antibodies, according to Duncan. Because liposomes--spherical vesicles made of phospholipids--are particles, they get taken up by macrophages. High levels can be found in the liver and spleen, even when the liposomes are given "stealth" characteristics by coating them with PEG. In addition, Duncan says, stealth liposomes have other side effects, such as extravasation, in which the liposome moves from the blood vessel into tissue where it's not wanted. Antibodies, meanwhile, have the disadvantage that most receptors on tumor cells are also present on normal cells, making it hard to find ones that are unique to cancer.

In contrast, water-soluble polymers allow Duncan to work with a single molecule rather than a large particle. "You can choose a material which doesn't go to the liver and the spleen and to which you can bind an anticancer agent using a linkage that's designed to be more specifically clipped at the tumor tissue," she says. "It's in effect a macromolecular prodrug."

To avoid the liver and spleen, Duncan works with uncharged hydrophilic polymers, such as PEG and N-(2-hydroxypropyl) methacrylamide. When these polymers are hydrated, they can circulate in the blood for periods of up to about 24 hours, according to Duncan.

Like many others, Duncan uses the fact that new blood vessels in tumors are "leaky" to passively target tumors. Because tumor blood vessels are more permeable than blood vessels in other tissue, drugs enter tumor tissue fairly easily. This effect, known as the enhanced permeability and retention effect, was first discovered by Hiroshi Maeda of the University of Kumamoto in Japan in 1986.

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IMPRINTS Recognition capabilities can be built into drug delivery using molecular imprinting. Monomers polymerize around a template molecule. The template is then removed, leaving a site that will interact selectively with the template. Such a site can be used to trigger drug delivery in response to the presence of a particular compound--for example, insulin in the presence of glucose.

ANOTHER ADVANTAGE of polymers is that the linkage can be designed to control where and when the drug is released. Duncan uses peptide linkages, which are cleaved after the polymer-drug conjugate is taken up into cells by the process of endocytosis. Within the resulting endosome, a family of enzymes called the lysosomal thiol-dependent proteases catalyzes the cleavage of the polymer-drug connection.

However, polymer carrier systems also have their disadvantages, Duncan points out. Compared to liposomes, which are basically empty vesicles that can be "stuffed full of drug," polymers have a low drug-carrying capacity. The payload that each polymer molecule can carry depends on the number of reactive groups where the drug can be attached. PEG, for example, can carry only two drug molecules, but other polymers can carry as much as 25 wt %, Duncan says.

At the CRS meeting, Glen S. Kwon, associate professor of pharmacy at the University of Wisconsin, Madison, described the development of polymeric micelles for drug delivery. The micelles are made of block copolymers of PEG and a poly(l-aspartamide) derivative. The copolymer forms a structure in which PEG serves as a hydrophilic shell and the amino acid forms the core. By fine-tuning the composition of the block copolymer, Kwon and his coworkers can control the structure of the resulting micelles, which are about 30 to 50 nm in diameter.

The side chains of the amino acid portion of the copolymer are crucial to the interactions with encapsulated drugs, so that portion is where Kwon and his group fine-tune the chemistry. The core-forming block needs to have a lower molecular weight than the PEG block or the micelles won't form.

Kwon started with an amino acid block with aromatic side chains. He wanted to know what would happen if those aromatic side chains were replaced with acyl chains. He chose hexyl, lauric, and stearic acyl chains.

Kwon and his group used the micelles to deliver the antifungal agent amphotericin B, which he said is called "amphoterrible" by AIDS patients who take it for life-threatening fungal infections. The drug is now formulated with liposomes, which reduce its toxicity but also diminish antifungal activity. In addition, the liposome formulations are expensive, costing approximately $1,000 a dose, Kwon said.

The longer side chains interact more strongly with amphotericin than the shorter side chains do, they found. The strong interaction with the stearic side chains means that the drug is released more slowly.

"We envision long-circulating nanoscopic drug depots that may produce controlled levels of free drug in blood over time and perhaps drug release solely at disease tissue," Kwon told C&EN.

Bioadhesive polymers to help improve the absorption of drugs are the focus of work by Edith Mathiowitz, an associate professor of medical science and engineering at Brown University. "You can find applications for bioadhesive polymers in almost any region that you have epithelial cells," she says, including oral, buccal (cheek), GI tract, rectal, or vaginal delivery. "The adhesive molecules bring the delivery system closer to the mucosa. If the particle is larger than 10 mm in diameter, it will stay for a prolonged time and deliver the drug. If the particle is small, the chances of being taken up are much higher. In the last case, the entire delivery system with the drug is delivered to the systemic circulation."

To accomplish this improved delivery, Mathiowitz designs polymers with a high amount of carboxylic acid, which hydrogen bonds with the carboxylic acids in epithelial cells. "You want light bonding," she says. "You don't want covalent bonding." Mathiowitz has started a company called Spherics to commercialize the polymeric drug delivery systems.

Relative newcomers to the collection of materials used for drug delivery are dendrimers, a type of highly branched macromolecule. One of the major advantages of dendrimers is their relatively small size, according to James R. Baker Jr., director of the Center for Biological Nanotechnology at the University of Michigan. "We can get a platform that we can target that's less than 5 nm in diameter. It provides a very nice scaffold and one that certainly can get through vascular pores and into tissue more efficiently than larger carriers," he says.

Another advantage of dendrimers is that their synthesis results in a single molecular weight rather than a distribution of sizes. "Although they're rather complicated, they can be synthesized so that you have a single molecular weight, a single species in the bottle," notes Duncan, who also works with dendrimers.

In addition, dendrimers have a high drug-carrying capacity because of their multivalency, according to Jean M. J. Frechet, a chemistry professor at the University of California, Berkeley. "You have many functional groups and can deliver a high payload," he says. "If you spend the effort for targeting, you are targeting a high payload as opposed to a single molecule."

Duncan has done work with platinate anticancer agents conjugated either to linear polymers or to dendrimers. The linear polymer could carry 10 wt % of the platinate, whereas the dendrimer could handle 25 wt %.

However, "the advantages of dendrimers are still being worked out," Duncan cautions. "The dendrimers seem to move out of the tumor tissue rather quickly," which can prevent the drug from concentrating in the tumor.

Baker and his colleagues use poly(amidoamine) dendrimers to deliver anticancer agents such as cisplatin and methotrexate. The drugs are conjugated to the dendrimers using photocleavable or labile linkers, which can be made to release the drug using light or through acid cleavage.

So far, dendrimers have not been used in people. "We haven't done large-scale human toxicity studies," Baker says, "but we've given up to 8 or 10 mg of this material in a single dose to a mouse without any toxicity. It's encouraging."

 
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Peppas Kwon
PHOTO BY LINDA FREI
Mathiowitz
BROWN MEDICINE PHOTO

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.

 
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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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TWEAKING Replacing aromatic side chains with acyl side chains improves the interaction between the block copolymer and the drug amphotericin B.

"THE REASON we started working with materials like fibrin and asking how to change them, how to fix them up as it were, was that evolution had done so much work already," Hubbell says. "Nature had already figured out how to make these materials from liquids into solids. Nature had already figured out how to make them remodel as cells migrate into them." However, he doesn't want "to be limited by the decisions that evolution made."

The synthetic materials that Hubbell's group uses are intended to be delivered to the body as a liquid, then to solidify to form gels. In the past, they have accomplished that by photopolymerizing acrylates or methacrylates in the body. Performing the polymerization in the body allows the precursors to be delivered through minimally invasive surgical procedures.

Now Hubbell is working with Michael-type additions involving thiols. One component has a Michael-type donor, and another has a Michael-type acceptor. A cross-linking reaction occurs in the body when the components are mixed.

One of the challenges is to make sure that no reaction causes heating. Hubbell accomplishes this by making sure that the number of moles of the reactive group is very low. "Even if the reaction is exothermic, you just don't have much of the reaction going on in order to get solidification," he says. "By using reactions that are not so exothermic and by using materials with macromonomers rather than low-molecular-weight monomers, you can get solidification without generating so much heat."

In addition, the reaction should be tolerant of both water and oxygen. "It's also pretty important to use precursors that don't easily cross cell membranes," Hubbell says. "If you keep this Michael-type reaction outside the cells, it is really very nontoxic, but if you allow it to happen inside the cell, it can be very toxic." The researchers keep the precursors out of the cells by making sure the precursors are large enough and hydrophilic enough not to cross the cell membrane.

In the area of angiogenesis, it's difficult to get normal blood vessels to form if the growth factors are free, Hubbell says. However, when they are complexed to a matrix, the resulting blood vessels are more normal.

Several things are necessary to move forward with this type of tissue engineering, according to Hubbell. First, materials must be tested in the clinic and be optimized. Second, it is necessary to learn how to manipulate the characteristics of the materials at different length scales. "Making hierarchically ordered materials is an important challenge to engineering their characteristics," Hubbell says. In addition, Hubbell would like to make materials that are conducive to one cell type but not another--for example, promoting healing but inhibiting scar formation. "Learning how to say no to some cell types or some biological responses is probably as important as saying yes to others."

Another type of material being developed for drug delivery is the so-called intelligent biomaterials. Such materials would combine molecular recognition with drug release. Nicholas A. Peppas, a chemical engineering professor at Purdue University, believes that such a strategy represents the future of drug delivery. Peppas will soon be moving to the chemical and biomedical engineering departments at the University of Texas, Austin, to head a new initiative in this field. In his vision, the recognition of a particular agent in the body--desirable or undesirable--would trigger the release of a therapeutic agent. Achieving this would result in a "new generation of drug delivery systems," he says.

Peppas and his group make polymers capable of recognizing certain compounds by using the technique of molecular imprinting, which is more often associated with chromatography. The molecule that the polymer will sense is used as a template around which the monomers are allowed to polymerize. The template molecule is then extracted from the polymer. 

"WHAT IS LEFT behind are nanopores or micropores that hopefully remember only the specific template," Peppas says. "For example, in the case of glucose, if I prepare a solution of glucose, sucrose, and galactose, this particular compound would recognize only the glucose. We've come close, but we're not at 100% recognition."

In the example of glucose-sensing molecularly imprinted nanoparticles, Peppas hopes that the detection of glucose would trigger the release of insulin from within the particle. "I'm describing to you something futuristic," Peppas tells C&EN, "something that is more or less like science fiction but is not 100% science fiction because we're already working on parts of it."

These are just a few examples of the work that materials scientists are doing to develop new polymers to effectively deliver drugs. As drugs become larger and less water soluble, the importance of new delivery systems will only increase.

GOOD CHOLESTEROL The cholesterol side chain in this water-soluble lipopolymer promotes interactions between the gene carrier and cells.

COMING UP

Meeting Targets Future Of Drug Delivery

ACS ProSpectives will be tackling the topic of drug delivery later this year in a conference titled "Future Directions of Drug Delivery Technologies: Molecular Design, Cellular Response, and Nanotechnology." The conference will include presentations on the design of new drug carriers, nanotechnology, protein delivery, gene delivery, medical applications, and tissue engineering. The meeting will be held Oct. 13–16 in Boston.

A goal of the meeting is to "bring together scientists with chief technical officers, chief executive officers, and other businesspeople who are interested in new technologies and new applications," says Nicholas A. Peppas, one of the meeting chairs and a professor of chemical and biomedical engineering at Purdue University. "It was important for us to concentrate on the future." The other meeting chairs are Robert S. Langer, professor of chemical and biomedical engineering at Massachusetts Institute of Technology, and Patrick Couvreur, pharmacy professor at Paris-Sud University.

"We want to have a really good scientific meeting, with leading speakers and cutting-edge topics from both academics and industry," Langer says. "Our goal is to have a forum for people to hear the latest cutting-edge science, with really good things and people they may not have seen all the time" at other drug delivery conferences.

ACS ProSpectives conferences are small meetings geared toward senior-level industry scientists on topics at chemistry's interdisciplinary frontiers. Other meetings this fall include one on combinatorial chemistry and another on proteomics. More information can be found on the Web at http://www.acsprospectives.org.



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