Advances in Targeted Delivery and the Future of Bioweapons

Abstract

Biotechnology–and aerosol delivery specifically–is beginning to blur the line between solutions and threats. Could a cure for diabetes and a dangerous pathogen be delivered in the same way? Such a scenario isn't far off.

In the past 10 years there have been considerable advances in targeted delivery systems–the biological components that allow an activity to be directed to a particular site in the body–that are designed to fight serious diseases in a biomedical research and clinical application context. They are, of course, perfectly legitimate undertakings. Nevertheless, the question arises as to whether recent advances in targeted delivery technology increase the potential for biological agents to be misused. This is of particular concern since the possibilities for the use or misuse of traditional microbiological and toxin agents, biochemical bioregulators, and artificial agents depends in great part on the ability to deliver the biological agent to the target in a way that is effective. 1

There are several means of achieving a targeted delivery, but the two areas that have progressed most significantly and appear to be most relevant are advances in aerosol and viral vector delivery technology. By examining some of the developments in these areas, it is possible to come to basic conclusions about the feasibility of their being misused to disseminate biological agents.

Aerosol delivery of infectious microorganisms.

Aerosols are particles in the form of a liquid or powder that are suspended in air and can be inhaled and deposited on the mucous membranes lining the nasal passages and the respiratory tract. The size of the droplets determine to a great extent where they will be deposited in the airway after inhalation. Particles up to 5 micrometers in diameter can reach deep lung areas; larger particles will attach to more anterior parts of the respiratory tract. 2

Aerosols have long been considered the superior means of disseminating traditional biological agents (e.g., causative agents of anthrax, plague, tularemia, or smallpox) as biological weapons over large areas. 3 For instance, a 2003 study on the potential for aerosol dissemination of biological agents involved the use of the bacterium Bacillus thuringiensis to control insurgent populations of the European gypsy moth, which were posing a threat to the lumber industry in Victoria, British Columbia. 4 An aircraft sprayed the biological insecticide Foray 48B (a solution of B. thuringiensis endospores) over approximately 30,000 acres, including residential and rural areas. It resulted in more than 99 percent mortality in the gypsy moth population. Surprisingly, even though the equipment was designed to generate droplets of 110-130 micrometers in diameter, smaller droplets (2-7 micrometers) that could penetrate houses were also produced; these droplets were shown to contaminate the nasal passages of residents inside their homes. While exposure to B. thuringiensis has no detrimental effect on humans, this experiment shed light on how effective dissemination of microorganisms over a large area without using sophisticated technology can be.

Vaccine administration via aerosols.

Aerosols are known to be effective in vaccination with whole microorganisms, which is generally more successful than using components or parts of microorganisms. For example, field trials in Mexico established the effectiveness of mass immunization of children with the measles vaccine virus via aerosols. 5 The children breathed in the aerosol output of a classic jet nebulizer driven by an air compressor for 30 seconds by holding a conical paper mask over their mouths and noses. Subsequent tests showed that this type of vaccination compared favorably with conventional methods of administration and required only one-third of the dose normally needed. In the former Soviet Union, thousands of people were successfully vaccinated with aerosols of live, attenuated strains of anthrax, plague, tularemia, and smallpox agents using tent-exposure systems. 6 Although the aerosols were produced in an enclosed environment (i.e., a tent), these practical experiments demonstrated that vaccination against a wide array of bioweapons-relevant microorganisms could be achieved by inhaling aerosol clouds containing the agents.

Considering some recent advances that have been made in vaccine delivery technology via the aerosol route, the focus now has shifted toward improving delivery and uptake of antigens, the components of microorganisms that can elicit protective immune responses. In addition, aerosol delivery of vaccines consisting of DNA is being intensively investigated. The DNA encodes genes that will produce an antigen after the vaccine has been delivered and taken in. Subsequently, the antigen that is produced will trigger an immune response. Among the various mucosal sites, nasal delivery is especially attractive because of relatively high permeability, low activity of destructive enzymes, and the presence of a considerable number of immune response cells at this site. 7 In general, lipophilic substances–those having an affinity for fat–are readily absorbable over the nasal mucosa. On the other hand, hydrophilic compounds–those having an affinity for water, such as peptide and protein antigens or DNA vaccines–are taken up relatively poorly. Since many antigens of interest are hydrophilic in nature and are thus less readily taken up, researchers have been developing methods aimed at improving their permeability properties for nasal and respiratory tract uptake. 8

While exposure to B. thuringiensis has no detrimental effect on humans, this experiment shed light on how effective dissemination of microorganisms over a large area without using sophisticated technology can be.

In this context, the design of nanoparticles with specific properties is of particular interest. Nanoparticles, which cells take up more efficiently than larger particles, usually range in size between 1 nanometer (a billionth of a meter, or around 10 times the size of an atom) and 100 nanometers (the size of large molecules). 9 In addition to particle size, the physico-chemical properties of the nanoparticles are important for uptake. Accordingly, researchers have explored several strategies to enhance their affinity for mucosal surfaces. 10

Delivery of biochemical therapeutics via aerosols.

There is much interest in the potential of aerosols to deliver therapeutic drugs (e.g., insulin) for several reasons. For one, the surface area of the lung is between 80 and 140 square meters. Also, the alveolar (air sac) epithelium in most pulmonary regions is only about 0.1-0.2 micrometers thick, and the distance between the epithelial surface and the blood is much less than it is in the bronchial system, which should facilitate drug uptake. 11 There are, however, a number of absorption barriers in the human lung, including the mucus layer, alveolar lining fluid layer, and competing uptake pathways. The absorption of drugs over the mucosal surface can be increased by the aforementioned vaccine administration strategies, and the engulfment activity of macrophages can be reduced by packaging substances into porous particles. 12 Already some 15 proteins or peptides could feasibly be delivered via the lungs in order to treat various illnesses. 13

Two, the nasal route has the added potential of providing the drugs direct access to the brain by entering the olfactory bulb along nerve cells. However, this mechanism is slow and, therefore, not yet efficient for delivery. 14 The brain is normally protected from the potentially harmful effects of biologically active substances or cells in the blood by the blood-brain barrier, which prevents circulating substances of a particular size and chemical property (including cells) from entering the brain. 15 There has been progress with some enhanced substances that seem to be able to weaken this defense, therefore allowing them to permeate the blood-brain barrier. For example, the cancer drug doxorubicin was able to cross the intact blood-brain barrier when attached to nanoparticles coated with polysorbate, an absorption enhancer. 16

Several clinical applications and other studies have shown that the aerosol delivery of bioactive biochemicals–substances that regulate vital physiological systems, also commonly known as bioregulators–is feasible and has been put into practice. Inhaled insulin delivery has been explored for more than a decade, and one formulation called Exubera has been administered by a special inhaler in the form of a spray-dried powder that is deposited into the lungs. 17 Despite Exubera's success in treating diabetes, it has been withdrawn from the market, partly due to trial results showing an increase in lung cancer among patients receiving the drug. Other companies continue to develop inhaled insulin, one reporting that their studies in mice and rats have shown no indications of carcinogenicity. 18 The neuropeptide oxytocin, which was reported to increase trust behavior in humans when they are given a single dose by nasal spray, is another example of successful aerosol drug delivery. 19 Oxytocin has been marketed in nasal spray form by Vero Labs under the name Liquid Trust, which is advertised by the company to be “the world's first oxytocin product specially formulated to create a trusting atmosphere!” 20

The most prominent example of applied aerosol drug delivery is the Dubrovka Theater incident in which Russian military special forces tried to rescue hostages held at the Moscow theater by introducing an unidentified gaseous substance, which was supposed to have incapacitating effects, into the building's ventilation system. Of the 800 hostages held in the theater, 127 died and more than 650 of the survivors required hospitalization. 21 Many of the patients had classic signs of opioid intoxication, and the Russian health minister announced several days later that a derivative of the opioid fentanyl had been used. The precise derivative and its dosage were never revealed. Thus, the fact that theurapeutics can be delivered is not in doubt. The real question is how to deliver them properly.

Viral vector technology.

Advances in molecular biology, immunology, and tumor genetics have led to the design and implementation of novel viral vectors for use in vaccine, cancer, drug, and immune system therapies. 22 It is important to note, however, that these same delivery advances have dual-use implications–for example, arming viruses with a destructive or even deadly payload to be delivered to an unsuspecting population. In general, these viruses act as vehicles that carry and deliver foreign genes to the body. The theory is that infecting an organism with one of these novel viruses will lead to the expression of the foreign gene in the affected tissues' cells. In turn, the expression of the gene would initiate the synthesis of the active substance (the gene product), which would then exert its effect on the body. In effect, the virus, similar to a Trojan horse, is used to smuggle a foreign substance into the body and deliver it to a particular tissue where it will cause a reaction. The use of viral vectors is the subject of intense research and development.

The most prominent example of applied aerosol drug delivery is the Dubrovka Theater incident in which Russian military special forces tried to rescue hostages held at the Moscow theater by introducing an unidentified gaseous substance, which was supposed to have incapacitating effects, into the building's ventilation system.

Vaccinia virus, specifically, shows some promise as a viral vector in destroying certain cancerous tumors that are resistant to established methods of therapy. For such studies, the virus has been engineered to recognize and invade tumor cells and deliver substances that can boost anti-tumor immune responses. 23 For example, in clinical trials with metastatic melanoma patients, an engineered vaccinia virus could successfully deliver its package to selected tissues. 24 Vaccinia virus enhanced for tumor selectivity also has been armed with a pro-drug activation system that has been termed “suicide gene therapy.” 25 In this case, the virus delivers a nontoxic gene-encoding enzyme that is converted to its highly toxic form when the gene is expressed in tumor cells. This recombinant virus has shown promise in investigations of human and mouse ovarian tumor models. 26 In comparison, adenoviruses and adeno-as-sociated viruses–that is, those occurring in the respiratory tract–have been developed for therapy purposes, but the clinical benefits have been modest. 27 Besides showing generally poor gene transfer and expression, these two vectors induce potent immune responses that compromise their own efficacy. To solve this problem, new strategies that allow the virus to evade an immune response, including the generation of “immuno-stealth” proteins (invisible to the immune system) to coat the viruses, are actively being researched. 28

Much work also has been invested in the development of lentivirus (the subfamily of retroviruses to which the AIDS virus belongs) delivery systems, as these viruses are efficient in infecting non-dividing cells and elicit only a weak immune response. 29 Although lentiviruses have a very narrow host range, this can be broadened or altered by pseudotyping, which involves exchanging the surface proteins of particular strains of viruses during packaging of the virus's genetic material into its outer covering (a final step in the synthesis of the virus) before it is released from the invaded cell.

The great promise of lentiviral vector development for clinical use is dampened by the fact that they are retroviruses that insert their genetic material randomly into the genome of the host. This could lead to adverse mutations, depending upon where in the host genome this material is inserted. It was seen, for example, when two children developed leukemia following a bone marrow treatment with a retroviral vector. 30 Nevertheless, retroviral systems that correct genetic defects have been confirmed as efficient. In one trial, 17 of 18 patients treated in London and Milan thus far have gained a functionally reconstituted immune system. 31 For improved safety, lentivirus vectors are being designed to insert into specific host genome sites that will not lead to detrimental mutations. 32 There is also much interest in developing lentiviruses as vectors in combination with RNA interference (RNAi), since RNAi is emerging as one of the most potent, effective, and practical methods of interfering with, or silencing, the expression of a specific target gene. 33 Although lentiviral vectors have not yet been fully developed for clinical use, there has been some success, and improvements are inevitable. 34

Artificial viruses.

So-called artificial viruses (or non-viral vectors) for gene and cancer therapy are another area of advancement that is rapidly growing and must be monitored closely. These nanoparticle-sized, polymer-based complexes contain DNA and are being developed in an attempt to overcome the negative aspects of using viruses to deliver genes (e.g., safety and manufacturing problems, immunogenicity, limited targeting ability, or limited transport capacity). The main problem with non-viral vectors is that they have not yet consistently demonstrated gene transfer efficiency comparable to that of viruses, limiting their practical use. 35 Nevertheless, researchers have demonstrated a significant degree of effectiveness in gene delivery to airway cells in mice using a cationic (positively charged) non-viral vector administered through the nasal passages. 36 Because of this and other successes, there is great interest in developing these vectors further.

Feasibility of aerosol delivery of viral vectors.

Viral vectors have largely been administered by injection, in some cases using repeated application, which would not be practical for weapons delivery. However, some studies have indicated that administering a viral vector through inhalation is feasible. Treating cystic fibrosis patients by having them inhale an adeno-associated virus vector engineered with a gene to deliver the transmembrane conductance regulator resulted in “encouraging trends in improvement in pulmonary function.” 37 In some 20 clinical trials, the use of gene-transfer agents including adenovirus and adeno-associated virus has demonstrated “proof of principle for gene transfer to the airway.” But efficiency is still low. 38

Other studies have shown that lentiviral vectors pseudotyped with the glycoprotein from the Ebola Zaire virus could achieve gene transfer in the lungs of mice. 39 Although the mice were infected by direct instillation of a single dose of the vector, the potential for infection by inhalation at least was given by the investigation. This can be seen in another study with mice where a lentivirus vector was successfully administered by inhalation in a nose-only exposure chamber. The results showed that lentivirus-mediated delivery via an aerosol was effective to a significant degree. 40

Concerns about advances in science and technology leading to the creation of novel biological warfare agents are compounded by the recognition that new and improved ways of delivering them are already at hand and will be developed further at a rapid pace.

Many viruses (such as lentiviruses) are quite sensitive to environmental stress, reducing the feasibility of their dissemination via aerosols in the atmosphere. However, the development of methods for encapsulating or packaging sensitive substances for controlled drug delivery over the nasal and respiratory routes is an area of intense investigation, which could yield benefits for increasing the resistance of viruses and non-viral agents to environmental stress. 41

What aerosols tell us about bioweapons.

Concerns about advances in science and technology leading to the creation of novel biological warfare agents are compounded by the recognition that new and improved ways of delivering them are already at hand and will be developed further at a rapid pace. Indeed, great strides are being made in aerosol delivery techniques, particularly considering the interest in drug development and delivery. The production of defined nanoparticles and new methods for improving absorption of agents through the nasal passages and respiratory tract as well as across the blood-brain barrier create a potential for greatly improved aerosol delivery of bioactive agents.

Furthermore, when advances in aerosol delivery technology are combined with improvements in specific targeting, gene transfer, and gene expression efficacy of viral vectors, the potential synergy effects raise the dual-use risk aspect to a new level. It must be stressed, however, that the goals of using armed viruses for gene and cancer therapy are quite different from those of using armed viruses as weapons. In the latter case, an aggressor may not be bound by a high degree of efficacy in his delivery system, and the concerns about the safety of highly efficient retroviral vectors would presumably be of little concern for someone intent on delivering a biological weapon to a chosen target. The most sophisticated of these advances in technology are certainly not easy to put into practice, but require extensive expertise, well-equipped laboratories, and substantial funds. Yet, even quite demanding manipulations are continually being simplified, so that the considerable advances in targeted delivery technology could well make it easier for both state-supported actors and terrorists to disseminate biological agents as weapons in the not too distant future.