Therapeutic Micro-Nano Technology BioMEMs - Tejlal Desai & Sangeeta Bhatia

11.1. INTRODUCTION

Diseases that affect the lungs are among the most frequently-encountered and deadly we face as a population. During the period from 1982 to 1992 (the most recent decade for which data are available), the overall annual age-adjusted prevalence rate of self-reported asthma increased to 49.4 per 1000, and we have seen an age adjusted mortality due to asthma increase to nearly 5 in 1 million in the United States [90, 91]. Lung cancer is the deadliest of the solid tumors, leading to the deaths of more individuals than the combination of the three next most lethal cancers [78]. Emphysema and other chronic obstructive pulmonary diseases affect an estimated 2 million Americans [88]. Venous thrombosis becomes symptomatic in approximately 100 per 100,000 Americans per year, with thirty to forty percent of those resulting in pulmonary embolism [3, 17, 40, 59, 83]. Cystic fibrosis, a genetic disease that causes abnormally thick lung secretions with subsequent recurrent infections and extensive scarring of lung tissues, affects one in four thousand Caucasian births. The lungs are the delicate casualty of many inflammatory insults, from sepsis to environmental antigen exposure. These represent only the most common of diseases that manifest primarily in the lungs.

Clinical medicine has seen tremendous improvements that have allowed individuals to survive many acute illnesses to which they would have succumbed in the past. The body’s efforts to heal, however, often lead paradoxically to further injury, abnormality, and disability. Physicians are frustrated in their inability to monitor the body’s injury and repair processes as they occur on a sub-clinical basis in the lungs and elsewhere, prior to the time at which they may currently be practically detected by means of screening, onset of symptoms, or acute deteriorations. Doctors are often incapable of supervising directly or intervening specifically and locally as injuries occur and afterward—able instead only to make gross assessments of global dysfunctions and either stand by as damage accumulates or to offer therapeutics that may not address specific pathogenic events in a timely manner.

On the other hand, the worldwide scientific community has achieved great advances toward understanding many of the basic mechanisms behind lung and other diseases. It is increasingly apparent that most disease states come about not as a result of merely a single insult or abnormality, but as complex matrices of events and responses that evolve over time. As scientists gain specific knowledge of isolated relationships that define the complexities of our inner biology [47], it becomes ever less satisfying to approach problems by blanket assumptions, offering interventions that may or may not have beneficial effects for a specific individual at a particular site and point in time, and that in fact could even be detrimental in the given situation.

It is clear, then, that we must develop methods to thoughtfully unite our varied and burgeoning pieces of medical knowledge with practical means to precisely apply it clinically. We must span the gaps between our needs, knowledge, and expectations and the medical reality. Medical nanotechnology promises just such a bridge. By working on a nanometer, molecular scale, there is potential to simultaneously assess and intervene with accuracy in space, time, and activity that we have never before enjoyed.

The public imagination has been piqued by works such as Asimov’s Fantastic Voyage and Michael Crichton’s Prey. Despite this interest, however, the entry of nanotechnology into the medical realm has seemed to advance at an our-world pace that reflects the dimensions of its products. Well, there is no teeny Raquel Welch in a skin-tight inner-space suit, but

many physicians are quietly incorporating advances made through nanotechnology into their practices even now, and even if they might not be aware of the origins or nomenclature. We will explore such current applications in this chapter, particularly in relation to diseases of the lungs, after discussing a few of the challenges faced in introducing new technology into the general medical environment, discussing particularly dangers that must be anticipated in interventions that target the lungs. The bulk of this chapter will encompass a few of the theoretical applications of nanotechnology to diseases of the lungs and conclude with a call to continued cooperation and communication between physicians who identify the unsolved clinical frustrations and failures at the bedside and the biologists, chemists, engineers, and other visionaries who may provide the tools to solve them.

11.1.1. Today’s Medical Environment

While often frustrated with current limitations in diagnostic and therapeutic options, the majority of physicians do not have sufficient free time to dream of, pursue, or develop new technologies for many reasons. Medical practice generally has little room for creativity, no potential downtime to re-think and re-configure. Much of the literature on nanotechnology traditionally has not appeared in medical journals, but instead in those of chemistry, engineering, and physics and in the newer nanotechnology and microtechnology literature. Thus, information regarding new technology is not readily accessible nor comprehensible to physician clinicians, even were they capable of determining which of the developments may hold potential usefulness in their practices, for the diseases they treat. The technology then often lacks advocates for its rapid advancement and direction toward meaningful medical applications.

Also, emphasis on existing management strategies and treatments, coming from outcomes research and consensus-derived practice guidelines that go on to dictate current patient care protocols, builds the perception that there is no room for revolutionary improvements in existing practices. The legal and third party payer environments inflict similar constraints on straying from routines of diagnosis and disease management. Most changes creep slowly into medical practice, relative to the general explosion of information and innovation outside of medicine, and inch into common use based upon awareness of the medical community, availability, proven safety record, improved efficacy over existing treatments, demand of the public and clinicians, and affordability. Further, regulatory bodies have also slowed the progress of advancement and implementation of nanotechnology in medicine, in part because they have not known how to proceed. Experts are few and the potential dangers have not all been explored. Caution is clearly and understandably the prevailing attitude. Predicting future developments with some degree of realism is difficult. No one can guarantee what is possible or likely, thus complicating the planning process. Still, there is exponential growth in our knowledge and capabilities that should continue to produce exciting medical applications.

11.1.2. Challenges for Pulmonary Disease-Directed Nanotechnology Devices

The respiratory system is open to the environment, and thus is armed with means to protect against infections and other environmental threats. When considering inhaled access, the pattern of laminar flow of air within the upper airways and lungs; branching of

FIGURE 11.1. Relationship between particle size and location of deposition in the respiratory tract. [25]

the airways; and moisture, mucous, and ciliation of the epithelial lining make it difficult for larger particles (greater than approximately 5 microns, depending upon shape, composition, charge, etc.) (Figure 11.1) to travel as far as and remain in the lower reaches (alveolar spaces) of the lungs. The lungs are rich in macrophages that are readily capable of engulfing and neutralizing foreign material (including intentionally-introduced medical devices) and initiating an aggressive acute inflammatory response. While this capability may have allowed our forebears the opportunity to survive infectious onslaughts that might otherwise have been fatal, it may impair the access of devices that are to employ in the lungs and could also present a threat to the entire organism in the potential triggering of overwhelming systemic inflammatory responses. This is of special concern were there unintentional contamination of inhalable devices with infectious material, lipopolysaccharide, or other toxins. The intricacies of the immune system response are not fully understood, and concerns that inhalable interventions may either de-sensitize to true pathogens or “overwhelm” the first-line defense mechanisms have still not been completely allayed. The lungs are rich in lymphatic vessels and lymph tissue, assuring that subsequent exposures to materials the body perceives as “undesirable” will be met with even more immediate and vigorous humoral protection. The lung is the initiating site of many allergic responses as well—with unpleasant effects running the gamut from runny nose through rash to severe, life-threatening bronchospasm and anaphylaxis. Devices that remain in the lungs, accessing via airways, vasculature, or otherwise, are potentially subject to local healing/walling-off granulomatous or fibrosis responses that may of themselves be potential threats (as might occur with occlusion of the pulmonary vasculature leading to pulmonary hypertension and consequent right heart failure) or may impede the device function. Also, materials and devices that are sufficiently small to achieve the alveoli and traverse the extensive alveolar-capillary membrane can enter the circulation in a very rapid manner, as the surface area over which diffusion may take place is large and such diffusion is generally quite efficient, with the diffusion gradient maintained by the rapid movement of the entirety of the cardiac output through the lungs. Materials may also migrate in an unpredictable manner through the tissues of the lungs and into the entire body, where local response may cause secondary and unintended effects [9]. Implantable drug delivery devices have the potential for toxic effects, secondary to the

device itself, its carrier, or the contents. Special care must be taken to assure that any toxic contents will not leak unexpectedly and that the device is impervious to potential traumas. Also, any material that is foreign to the body serves as a potential focus for infection, so care must be taken to minimize this risk.

11.2. CURRENT APPLICATIONS OF MEDICAL TECHNOLOGY IN THE LUNGS

11.2.1. Molecularly-derived Therapeutics

The lungs, directly accessible via the airways to the outside environment, were the target of one of the first molecularly-based medical therapies. Dornase alpha recombinant human deoxyribonuclease I (rhDNase), launched in 1994, is a proteinaceous enzyme that selectively cleaves DNA [43]. Cystic fibrosis patients use rhDNAase, administered by inhalation of an aerosol mist, to cleave the sticky free DNA that has been released from destroyed inflammatory cells and that comprises a significant portion of mucous. This lysis of the DNA facilitates expectoration. DNAase therapy has contributed to improvements in the multicomponent management of an autosomal recessive inherited disease that had previously resulted in near uniform mortality by the early teens to result in a life expectancy for these patients that may reach now into the mid-thirties and even beyond [81].

After having been originally applied to treat acute coronary syndromes in the late 1980’s, tissue plasminogen activators have come to use in the lungs as treatment for large and life-threatening pulmonary emboli. Their value in this setting, though, continues to be questioned, plagued by complications related to inability to target their site of action, resulting in the potential for bleeding complications, and the sheer volume of clot that is often seen in acute thromboembolic disease. Thus, the role of tissue plasminogen activators in this setting is still under exploration [43].

11.2.2. Liposomes

Liposomes in many sizes and compositions are in fairly common use in cosmetic and medical applications and are continuing to undergo further refinement and testing for additional medical uses [49]. The liposome refinements attempt to overcome early problems related to variability in size, amount of medication incorporation into the particles, integrity of payload encapsulation, and targeting to diseased tissue and organs. Encapsulation of drugs in liposomes has often resulted in improved therapeutic efficacy over that of the drug alone following administration by multiple routes, including topical, injection, and inhalation. Physicians are accustomed especially to intravenous use of liposomal formulations of toxic azole antifungal medications [39].

Additional non-cosmetic liposomal formulations are in various stages of testing. Lung deposition of the antibiotics gentamicin, amikacin, and tobramycin markedly improves when these antibiotics are administered in inhalable liposomal forms, compared to nonliposomal inhalation [18, 26, 35, 36, 41, 66, 71]. A liposomal form of ketotifen fumarate, an experimental asthma bronchodilator, is in human trials [55], as is liposomal cyclosporine. Cyclosporin is currently most often utilized as an anti-rejection medication for transplant patients in oral or intravenous form, where its potential for system-wide side effects is significant. When given via liposomal inhalation for lung transplants, the peak serum level

of cyclosporin is on the order of 110 nanograms per milliliter, a fraction of that necessary if the drug is administered by other routes, thus reducing its toxic potential [89]. Interleukin 2 has been in use for many years to combat various cancers. Its administration, usually intravenously or subcutaneously, is often complicated by uncomfortable and occasionally life-threatening infection-like symptoms. Liposome-encapsulated Interleukin 2 via inhalation has been tested with promising results in animal models of several cancers that metastasize to the lungs [60]. Clodronate disodium encapsulated within liposomes administered via the airway in a murine model of Pseudomonas bacterial pneumonia and sepsis results in depletion of alveolar macrophages, thus reducing the initial inflammatory response and bringing about improvements in mortality [46]. Evaluation of gene therapies with the genetic material introduced via liposomal inhalation is underway. Liposomal formulations of the gene for α-tocopherol, a naturally-occurring anti-oxidant, have been administered in rats in anticipation of severe hypoxia (low oxygen levels). After hypoxia, the treated rats show downregulation of caspases, resulting in a limitation of apoptosis in the lungs. Their mortality is reduced from 60 to 30 percent [67, 86]. This approach also may be useful to protect the lungs during time-limited risks such as radiation therapy through the expression of manganese superoxide dismutase plasmids [28]. These are but a few of the many liposomal delivery systems that are under current use and study.

11.2.3. Devices with Nanometer-scale Features

Filters with pores of 15–40 nanometers have seen increased use in the manufacture of plasma-derived biopharmaceuticals as a means of size-excluding viral and other potentially infectious elements from therapeutic protein mixtures [13, 93]. Microarrays of many types and varieties are under development to aide in the identification of disease states or monitoring of disease activity. Many of the emerging systems utilize micron and nanometer scale manufactured features. Though these are still primarily a research tool, as the results become more reliable and the array results are correlated with particular disease states, we can anticipate more common use in the patient population.

11.3. POTENTIAL USES OF NANOTECHNOLOGY IN PULMONARY DISEASES

It is truly impossible for us to conceive or predict how all of the achievements in science may eventually channel into advances in medicine. It is, though, a useful pleasure to make the attempt. We can anticipate that particular tasks will continue to need to be accomplished. We will frame this discussion using those basic medical tasks. These immutable tasks are: 1) diagnosis and 2) therapeutics or management of disease. Therapeutics may be divided further into the administration of therapeutic agents versus provision of mechanical or structural improvements. We will begin with a description of the needs and potentials within each of these areas, then elaborate based upon available technology or technology in near development, as applied in specific examples to common pulmonary diseases. There are thousands of potential medical applications and nearly as many individual technology platforms. It is impossible to achieve complete comprehensiveness, but we will attempt to be as inclusive as space and reason will allow. We will not mention anything along the lines of “bioreactors,” or implantation or transplantation of cell colonies or tissues designed or programmed to act in a particular biologic fashion. We will make minimal mention of

nanotechnologic approaches to systemic, body-wide, delivery via the inhalational route, which could encompass a chapter in itself.

11.3.1. Diagnostics

The first broad category of need in pulmonary medicine, as in all of medicine, is in the area of diagnostics. This topic is huge, and the potential for improvement in existing practice is significant. Diagnosis encompasses identification of indicators, or markers, of disease states; various forms of imaging; localization, staging, and quantification of disease; prognostics; and monitoring of the progression of disease. It is conceivable that many techniques that may be used to localize and identify a disease may also be applied toward targeting for therapeutic intervention.

11.3.1.1.Disease Markers and Localization A disease state may be recognized or localized by the identification of markers of disease. These may include any of several potential biomolecular clues. Proteins may manifest disease in the form of either completely abnormal proteins / peptides, chemical or electrical changes that are encountered in places and at times where they should not normally be located, or proteins that are processed abnormally. Similarly, a disease state (or predisposition to it) may be predicted by the presence of particular genes within an individual’s total genome. In fact, nanotechnology provides a bridging step toward “personal medicine,” where an individual’s response to a planned therapeutic intervention may be predicted by the presence of particular genetic polymorphisms, as may their risks for certain disorders. Also, abnormalities in the structure of genetic material may come about as a result of local injury or other insult (structural genetic aberrations), or abnormal patterns of gene expression for the given conditions (upor down-regulations). These changes may be associated with particular diseases or may predispose to disease states such as cancer.

Disease markers may be identifiable in exceedingly small amounts or found only in relatively inaccessible anatomic locales. Nanotechnology may permit less invasive, less uncomfortable means of making diagnoses. Additionally, nanotechnology may allow for improvement in the lower limits of detection of disease, and thus improve the chances for early diagnosis as well as providing more accurate monitoring of therapeutic response or progression of disease. This can be accomplished by several means. Local chemical, electrical or physical property-changes in cells or tissues may be monitored by means of nanometer scale tubes and wires in the form of field effect transistors [23, 56]. Concentration of markers that are present only in small amounts, either by attraction of markers of interest to a stationary binding site or by the binding of biomarkers remotely onto particulates, with subsequent harvesting of bound particulates for query may be accomplished. Alternatively, identification of biomarkers found in low concentrations may be improved through amplification in cascade reactions or by tagging known biomarkers with indicators that may be more easily detected. Imaging, or localization of abnormalities, may take advantage of the presence of markers, the function or behavior of organs or tissues, or the architectural structure of the tissue or organ.

11.3.1.2.Imaging There are many means of imaging that may be used to detect local abnormalities. Positron emission tomography (PET) scanning demonstrates differences in metabolism between diseased tissues from that of normal tissue. Nanotechnology should

FIGURE 11.2. Transmission electron micrograph (TEM) showing the size and morphology of folate-coated gadolinium nanoparticles [75].

permit increased sensitivity through amplification of these detected chemical changes or by increasing their specificity, allowing improved differentiation between “normal” areas of increased metabolic activity and areas of true disease, or even potentially permitting discrimination among tumor types and of biologic behavior in the case of cancer. Contrast agents have been used for many years to improve the diagnostic capabilities in magnetic resonance imaging (MRI), with more nanometer-scale contrast agents in development. Electron Paramagnetic Resonance (EPR) is an evolving imaging technique that may allow an even greater understanding of the regional metabolic activity within diseased tissues, also enhanced through administration of nanometer-scale contrasts [74] (Figure 11.2). Nanotechnology may allow improvement in the sensitivity of detection or even allow differentiation between similar disease states (i.e. distinguish between different types of infectious pathogens) using traditional radiographic techniques such as computerized tomography (CT) or plain radiographs along with labeling of organisms or features of organisms. Mechanical differences between normal and diseased tissue may be identified through the employment of specialized precision ultrastructural ultrasound techniques [64], and these characteristics further enhanced by employing nanoscale contrast agents. Disease-related abnormalities that may not be otherwise directly visually identifiable can be made manifest directly

FIGURE 11.5. Commonly used silastic and expandable wire stents for airway obstruction. (Kaplan P)

Possibly one day in the future we will be able to utilize nanotechnologic self-assembling components to support airways and provide infrastructure for damaged alveolar lung tissue so that the patient may then be able to complete the healing process with her own cells and tissues. Such an intervention would extend tremendous hope for those who suffer from emphysema, whose only option at this point may be lung transplantation.

One of the major difficulties of patients who suffer from many lung diseases is that of cough. Damaged or inflamed airway lining cells and glands respond in part through increased mucous and other secretion production. Cilia, normally responsible for sweeping out the material lining the inner airways, are sometimes damaged by exposure to toxins or may be genetically pre-destined to malfunction. The nanotechnology of our children and grandchildren could provide direct help with the breakdown and clearance of secretions. One might envision in the distant future the inhalation of single-task nano-robots designated to break apart mucous and carry it out along the oxygen gradient. Damaged or disordered cilia components could assemble in-situ, within pulmonary epithelial lining cells, to assist in mucociliary clearance.

11.3.3.Evolving Nanotechnology in Pulmonary Diseases

11.3.3.1.Lung Cancer With lung cancer as most cancers, one of the primary goals is to determine earlier, at a time when intervention may be more effective, that an individual may have cancer. We would like to identify pre-cancerous lesions to avert the risk of malignancy. We want to use more accurate means to identify metastatic spread or recurrence of previously-treated cancer so that it may be addressed. We want to provide more effective means of targeting therapeutics to diseased tissues and cells. Nanotechnology offers many potential tools to achieve these goals [32], first in the form of nanoparticulates. There are numerous targets that may be utilized to identify and localize cancerous lesions with nanoparticulates. Detection of abnormal areas of endothelial growth factors

may be associated with (and provide a target against) pulmonary metastasis [54]. Methylation of p16INK4a, DAPK, MGMT, and FHIT is seen in lung epithelial tissue at risk for