PEGylated Liposomes Can Be Modified in Myriad Ways
Gene Therapy with PEGylated Liposomes
Advanced applications of stealth technology
hold great promise for the future of medicine
By Will Block
In the last two months’ issues, we described a molecular “stealth technology” in some detail (“Better Supplementation with PEGylated Liposomes” and
“PEGylated Liposomes Increase Bioavailability”). This month we’ll look at the revolutionary potential of some advanced applications of this technology. Unless you’re already familiar with PEGylated liposomes, it would be helpful to read the previous articles first.
f you know guns, you can appreciate the difference between a rifle and a shotgun. The former delivers one bullet precisely to one tiny spot (not necessarily the spot you were aiming at, but that’s another story), whereas the latter blasts many pellets over a fairly large area (assuming the target is some distance away—the shot pattern expands with distance). In hunting, the main advantage of shotguns is that they greatly increase the hit probability on small moving targets (birds, mainly) that would be nearly impossible to hit with a rifle.
Taking drugs or supplements is somewhat analogous. The traditional approach, used for thousands of years, is to fire a shotgun blast, pharmacologically speaking, and let your circulation spread the medicinal molecules throughout your body, where they will be absorbed by any tissues that have an affinity for them. The molecules will have a therapeutic effect in some tissues, while in others they may produce unwanted side effects. In the shotgun analogy, this is like having one pellet kill a duck (OK, technically that’s not therapeutic for the duck, but let’s not quibble) while the rest go to waste and may contribute to lead contamination of the ground or water.
Duck Hunting in the Twenty-First Century
The modern alternative is the rifle approach: one target, one bullet, no waste, no side effects—perfect if the duck is a sitting duck. In medical terms, the target is a single type of tissue or organ, and the bullet is a minimal dosage (no waste) of a drug or supplement. It’s as easy as hitting a sitting duck—a duck sitting behind several rows of hedges, that is. And you’re firing a bullet that can easily be deflected by hedges. Oh, yes, and it’s pitch dark outside, so you can’t even see the hedges, let alone the duck. Good luck!
Believe it or not, it’s becoming increasingly possible to hit that “duck” hiding inside the human body, where the “hedges” are a variety of physical and chemical barriers that a drug or supplement molecule must traverse in order to reach the tissue or organ that needs therapy. Scientists have devised a number of ingenious methods for pulling off this trick. One of them involves an advanced application of PEGylated liposomes (called “stealth technology” in the pharmaceutical world). These microscopic, synthetic cells are designed to be sustained-action delivery vehicles for drugs or supplements in situations where low bioavailability or potential toxicity is a serious problem.
For the “duck trick,” the PEGylated liposomes are of a highly specialized type in which the PEG molecules have been chemically modified to enable them to recognize the target tissue or organ. The modification consists of attaching certain biologically active targeting molecules to the free ends of the PEGs (alternatively, these molecules can be attached to the liposomal cell membrane, but that doesn’t work nearly as well).
Duck sitting behind hedge
Immunoliposomes for Active Targeting
The targeting molecules are of various types, but most are antibodies of the human immune system. (Antibodies are immunoglobulins—Y-shaped glycoproteins that, like all other proteins, are very large and complex compared with most ordinary molecules.) PEGylated liposomes that have been modified in this manner are called immunoliposomes, and their special ability is active targeting. (See the sidebar “Active and Passive Targeting.”)
Active and Passive Targeting
The cell surface in every type of tissue or organ has a unique molecular “fingerprint,” just as, e.g., the skin of every type of fruit is distinctive in its appearance, texture, and aroma, enabling even a blind person to identify the fruit without having to bite into it. It is this chemical specificity of cell surfaces that scientists have exploited in developing immunoliposomes for active targeting. They use immunoliposomes incorporating antibodies that can “read” the molecular fingerprint of a certain type of cell and attach themselves to those cells, and no others.
By feeling their way blindly throughout the body as they go with the flow of the blood circulation, the immunoliposomes can, in effect, find the lone duck sitting behind the hedges (the accompanying article explains this analogy). From our perspective, it’s not so much like firing a bullet at the duck from a distance as it is blundering through the hedges in the dark, holding a bullet in our hand and touching everything with it until finally something feels like a duck.
Thus the immunoliposome bullet is not a “magic bullet” that seeks out its target tissue or organ, but one that finds its target by trial and error, relying on the specificity of its molecular interactions with the target. This is called active targeting because the target is preselected.
Passive targeting, by contrast, occurs with ordinary (nonimmunosomal) PEGylated liposomes when they’re preferentially absorbed by two types of tissues—tumors and inflamed tissues—that happen to be more accessible to them than normal tissues. Another form of passive targeting occurs when liposomes are scavenged by phagocytes: the payload molecules may benefit the phagocytes themselves, or, if they’re subsequently released unharmed by the phagocytes (a big if), they may benefit the liver and spleen, where phagocytes are found in particular abundance.
Aside from these examples of passive targeting, ordinary PEGylated liposomes have minimal interactions with tissues and organs of the body. For all practical purposes, they are inert carriers of drugs or supplements, and they remain in the bloodstream for long periods (hours to days), releasing their payloads slowly. The benefits are twofold: (1) because they’re administered parenterally (i.e., other than via the digestive tract), they can greatly increase the bioavailability of their payload molecules; and (2) because they produce only low (but sustained) blood levels of these molecules, they can minimize toxicity or unwanted side effects.
Active targeting with immunoliposomes is a new field of pharmacology that still faces tremendous obstacles in terms of the effective delivery of drugs to specified tissues or organs. Although the technology has been under development for several decades, its first clinical application using antibodies as the targeting molecules was a phase I trial published in 2004.
In this trial, the anticancer drug doxorubicin (which, like many such drugs, is highly toxic to various tissues) was administered intravenously to 23 patients with stomach cancer. The targeting molecules used were certain cancer-reactive human monoclonal antibodies (monoclonal means derived from a single cloned cell line). The results clearly demonstrated the technical feasibility of active targeting in clinical practice. The treatment itself, however, was unsuccessful, despite the known clinical efficacy of doxorubicin (much research still needs to be done on immunoliposomal delivery).
Gene Therapy Produces Predetermined Proteins
A revolutionary application of immunoliposomal technology is found in gene therapy. This is the treatment of certain disorders, especially those caused by genetic anomalies or deficiencies, by inserting specially engineered genes into targeted cells of the body. Thus the “drug” in this case is not a drug at all, but rather a DNA fragment, either natural or synthetic, consisting of genes whose purpose is to alter or replace defective genes, such as those involved in the hereditary disease cystic fibrosis.
The specific goal of gene therapy is the production, in the targeted cells, of therapeutic proteins that are encoded by the inserted genes. These may be proteins that the patient’s cells cannot produce; their purpose may be to restore normal function to the cells, to remove or render harmless some substance or some gene that is causing disease, or to make the cells (especially cancerous cells) more susceptible to attack by conventional drugs.
Cationic Liposomes—Pro and Con
What’s breathtaking about gene therapy is that it represents a direct medical intervention via the master molecule of life itself: DNA. Of the numerous techniques that have been developed to accomplish this stunning feat, DNA delivery by immunoliposomes appears to be one of the safest and potentially most versatile, even if it’s not as effective as some others.
Most of the immunoliposomes that have been used for gene therapy research in laboratory studies are further specialized through chemical modification of the liposomal membrane itself. They're called cationic liposomes because the surfaces, both exterior and interior, of their liposomal membranes are positively charged [most liposomal membrane surfaces are negatively charged (anionic) or neutral]. The cationic nature comes from the special phospholipids used in constructing the membranes, and the effect is to increase greatly the loading efficiency of their DNA payload, which is negatively charged.
Unfortunately, cationic liposomes are highly problematic in living organisms (they’re scavenged within minutes, for one thing), and progress in developing neutral immunoliposomes for DNA delivery has made it apparent that these have a much better chance of success in human gene therapy.
DNA Delivery Poses Huge Challenges
DNA cannot be taken orally, as it’s degraded by digestive juices (much of the food you eat contains the DNA of some plant or animal, and all of it is degraded). Nor can it be injected intravenously, as it’s quickly degraded in the blood. Even if it were not, however, one would still need a targeting vehicle for delivering it to those cells that needed it.
The daunting mission of immunoliposomes for DNA delivery is not just to find their target cells, but also, when they do find them, to interact with them chemically in such a way as to ensure that their payload will be transferred directly from the liposome into the cell. This may require, among other things, that the liposomal membrane be chemically modified in such a way that it will shed its coating of PEG molecules at the appropriate time, allowing the two membranes—the artificial liposomal one and the natural cell one—to fuse and open a channel through which the DNA can pass.
All this must be done safely and effectively, of course, i.e., with minimal toxicity to any tissues and with minimal waste of the immunoliposomes. Engineering such capabilities into the system requires great expertise in biochemistry and biophysics. Among the many legitimate concerns regarding gene therapy are that the immune system may attack cells that have been treated in this manner, that unwanted and potentially harmful mutations might occur, and that altered genes might be passed on to succeeding generations.
How to Penetrate the Blood-Brain Barrier
Although gene therapy is still in a rudimentary stage of development, its promise is enormous. Of the many tissues and organs that can be treated in this way, none is more important—or challenging—than the brain, which is largely isolated from foreign invaders, be they drug or supplement molecules, PEGylated liposomes, or other delivery vehicles. The primary obstacle is the blood-brain barrier, the name given to the epithelial cells that line the vast network of capillaries nourishing our brain cells. Because these epithelial cells are exceptionally tightly packed, it’s difficult for most molecules, and virtually impossible for liposomes, to squeeze between them to gain access to the brain cells.
Nature, however, has provided an ingenious solution to this problem: some chemical entities can diffuse right through the epithelial cells—in one side and out the other—in order to reach the brain’s inner sanctum. Ordinary PEGylated liposomes cannot do this, but some immunoliposomes can. The complex process, called transcytosis, enables drug or gene therapy of the brain to be carried out in ways that would otherwise be impossible.
A number of laboratory and rodent studies have shown that immunoliposomes using a monoclonal antibody called OX26 as the targeting molecule can penetrate the blood-brain barrier quite effectively via transcytosis. In one study in which the immunoliposomes carried a DNA payload, evidence of expression of the inserted gene was found throughout neurons of the central nervous system (brain and spinal cord), proving that the technique worked. In another gene-therapeutic study, positive pharmacological and physical effects were observed in rats with induced Parkinson’s disease.
Nanomedicine—In the Realm of the Very Large
Of the many buzzwords associated with modern science, one that has acquired a special mystique is nanotechnology. It has captured the imagination of scientists and laypeople alike, and new frontiers in this exciting domain are being explored all over the world. One of them is the emerging field of nanomedicine, which entails therapeutic entities and methods of novel kinds, characterized in part by the size range in which they occur.
The popular concept of nanotechnology is that it involves objects in the size range of nanometers (nm), or billionths of a meter.* Sizes within this domain, defined as 1 to 1000 nm, are very small by most standards, but they’re very large compared with those of most molecules, which are in the range of fractions of a nanometer to a few nanometers. The difference is particularly relevant in nanomedicine, which entails the development of drug-delivery vehicles, such as PEGylated liposomes, that can be hundreds or even thousands of times larger than most molecules.
Thus, although we think of nanotechnology as involving things that are infinitesimal, nanomedicines are enormous compared with the molecules that have constituted virtually all medicines known to man since time immemorial. This has advantages and disadvantages. Much more important than the sizes involved, however, are the molecular strategies employed. These depend largely on the chemistry of polymers, which are long molecular chains consisting of repeating units of smaller molecules.
See the tiny black dot? Imagine that it’s a sphere the size of a glucose molecule, about 0.7 nm in diameter. The green spot, drawn to scale, represents a rather small liposome of 70 nm in diameter, or 100 times wider than the glucose molecule. Its volume is therefore 1 million times greater, because volume increases as the cube of the radius. Get the picture?
The dot and the spot are shown here
about 560,000 times their actual sizes.
If you used 10 mg of phospholipids to
make unilamellar (single-walled)
liposomes of 70-nm diameter, you
would get about 150 trillion of them. Their combined total surface area would be huge, about 3 square meters, but their combined total “capture volume” would be minuscule, about 25
Some of the most important compounds in living organisms—notably proteins, nucleic acids (DNA and RNA), and polysaccharides—are polymers. It is not these polymers, however, with which nanomedicine is concerned, but rather a variety of synthetic polymers created in the laboratory, and the medicinal uses to which they can be adapted. One of these polymers is PEG (polyethylene glycol), the substance used to PEGylate liposomes (and other entities as well, including certain proteins and drug molecules). PEG can be used as itself or as any of a great variety of chemical derivatives, each one tailored by chemists to achieve a specific purpose in the body, such as the active targeting of preselected tissues or organs.
The field of nanomedicine is still in its infancy but is already bringing clinical benefits to thousands of patients worldwide. And it offers the prospect of further dramatic innovations and advances in therapeutics—some of which could be applied “over-the-counter” for nutritional supplements if they should fall into the public domain owing to unpatentability or patent expiration, or if the pharmaceutical companies were willing to license the technology at affordable cost.
- Duncan R. Nanomedicines in action. Pharmaceut J 2004;273:485-8.
Shooting for Future Capabilities
So what does all this have to do with nutritional supplements? Nothing at present—active targeting with immunoliposomes, whether for gene therapy or more conventional drug therapy, is far beyond the reach of most supplement manufacturers. As a logical outgrowth of ordinary PEGylated liposomal technology, however, it gives us a tantalizing glimpse of future capabilities in pharmacology—capabilities that we too may someday be able to aim for. With a rifle, of course.
- Janoff AS, ed. Liposomes: Rational Design. Marcel Dekker, New York, 1999.
- Svenson S, ed. Carrier-Based Drug Delivery. ACS Symposium Series 879, American Chemical Society, Washington, DC, 2004.
- Matsumura Y, Gotoh M, Muro K, Yamada Y, Shirao K, Shimada Y, et al. Phase I and pharmacokinetic study of MCC-465, a doxorubicin (DXR) encapsulated in PEG immunoliposome, in patients with metastatic stomach cancer. Ann Oncol 2004;15:517-25.
- Schnyder A, Huwyler J. Drug transport to brain with targeted liposomes. NeuroRx 2005;2(1):99-107
- Shi N, Pardridge WM. Noninvasive gene targeting to the brain. Proc Natl Acad Sci USA 2000;97:7567-72.
- Shi N, Zhang Y, Zhu C, Boado RJ, Pardridge WM. Brain-specific expression of an exogenous gene after i.v. administration. Proc Natl Acad Sci USA 2001;98:12754-9.
- Zhang Y, Calon F, Zhu C, Boado RJ, Pardridge WM. Intravenous nonviral gene therapy causes normalization of striatal tyrosine hydroxylase and reversal of motor impairment in experimental parkinsonism. Hum Gene Ther 2003;14:1-12.
Will Block is the publisher and editorial director of Life Enhancement magazine.