December 1, 2005 (Vol. 25, No. 21)
New Applications for Drug Discovery, Delivery, and Diagnostics
Nanotechnology is a “disruptive technology,” which will drive a new generation of cancer diagnostic and therapeutic products and result in dramatically improved cancer outcomes, according to Piotr Grodzinski, Ph.D., program director for cancer nanotechnology at the NCI.
His presentation was given at the recent Strategic Research Institute “Nanomedicine: Commercializing Drug Discovery, Delivery, and Diagnostics”, held in Cambridge, MA. Dr. Grodzinski painted a grim picture of the current status of the cancer. This year 570,000 Americans will die of cancer, and the costs of treatment will run in the neighborhood of $200 billion. Despite this vast expenditure of funds, the death rate has declined slowly in the last decade.
Meanwhile the number of individuals living with cancer has increased dramatically over the last 35 years, from 3 million to 10 million. On one hand this is welcome news, as cancer victims find themselves alive and their diseases managed effectively. On the other hand, it raises the disturbing question of the staggering cost of new treatments, especially biological molecules, including proteins.
To change this bleak situation, an armory of nanoparticle technologies is being mobilized, including dendrimers, nanoparticles, quantum dots, and fullerenes. All of these technologies offer unique opportunities for biotech companies to develop entirely new strategies for the treatment of malignancies.
Applications of nanoparticles include tissue targeting, sensing and imaging, localized therapy, and the use of much lower doses.
Nanotechnology’s benefits are especially relevant to cancer since the potential sensitivity of these platforms could allow the early detection of tumors before the cancer metastasizes. Technologies under development could allow DNA and protein markers to be detected in the same sample simultaneously.
Nanostructures lend themselves to loading with either drugs or tags, including fluors, so the tumor can be targeted, identified, and treated using much lower levels of the therapeutic agents.
Because nanotechnology draws on diverse disciplines, including engineering, physics, and biology, it requires an unprecedented level of collaboration and mutual support. For this reason, the NCI has created the Alliance for Nanotechnology in Cancer, working over the past six years to integrate nanotechnology into biomedical research.
“We know that melding nanotech plus cancer research produces near-term, clinically relevant advances,” said Dr. Grodzinski. The Alliance embarked on an $144 million initiative in 2004. This massive program encompasses the public and private sectors, emphasizing cross-disciplinary collaborations to ignite nano-product development and commercialization.
Private sector participation will help to ensure development and create a commercialization pathway to accelerate these nanotechnology applications in the clinic. The Nanotechnology Characterization Laboratory (NCL), in particular, is consolidating standards through assembly of its assay cascade and prequalification of new materials to drive product development.
Encouraging such collaborations and leveraging existing federal resources reduce the risk of new product investment to commercial entities, as illustrated by the range of activities in the field of nanomedicine presented at the conference.
Dendrimers are a major architectural class of nanoscale chemical polymers, according to Donald A. Tomalia, Ph.D., president of Dendritic Nanotechnologies (Mount Pleasant, MI). The term, coined by Dr. Tomalia in 1984, describes a large, synthetically produced precise polymer in which the atoms are arranged in many branches and sub-branches radiate out from a central core.
Dendrimers are built from a starting atom, such as nitrogen, to which carbon and other substances are added by a repeating series of chemical reactions that produce a spherical branching structure. As the process repeats, successive layers are added, and the sphere can be expanded to the size required by the investigator.
The result is a spherical macromolecular structure whose size is similar to albumin and hemoglobin, but smaller than such multimers as the gigantic IgM antibody complex. By manipulating the chemistry of the dendrimer, the geometry and properties of the structure can be altered to perform a vast array of functions, including magnetic resonance imaging contrast agents.
Dr. Tomalia and his colleagues developed a new class of such agents with large proton relaxation enhancements and high molecular relaxivities. The reagents are built from the polyamidoamine form of Starburst dendrimers in which free amines have been conjugated to the chelator 2-(4-isothiocyanatobenzyl)-6-methyl-diethylenetriaminepentaacetic acid.
By altering the size of the dendrimers, their properties of elimination through excretion can be profoundly changed. Consequently, a range of patterns of localization in kidneys, lymphatics, liver, and blood pool can be specified.
This new class of contrast agents has the potential for extensive applications in MR imaging. They are currently being evaluated by the NCI. This assessment, expected to take 1215 months, will include physical characterization, in vitro studies, and in vivo studies to determine their absorption, distribution, metabolism, excretion, and toxicity.
These investigations will generate data in support of an investigative new drug filing with the FDA. The development of dendrimers-based MRI contrast agents has the potential to yield sensitive, non-invasive intravascular compounds.
By the same rationale, dendrimers can be engineered for nano-based drug delivery. Dendrimers can be designed to be the appropriate size and perfectly encapsulate the drug, allowing optimum delivery. The degree of encapsulation can dictate the rate of release in a controlled manner. Also, the nanodimensions of the dendrimers can be specified so as to fit a specific receptor, further targeting delivery of the drug.
Whereas traditional drug delivery is monovalent, that is, a single drug molecule binds to a single cellular receptor, dendrimers can be engineered to carry a large number of drug molecules on their spherical exteriors, in such a fashion that interaction with the receptor-studded cellular membrane mimics the natural binding of a large viral entity to the target cell.
This approach has been the basis for a successful Phase I anti-HIV drug trial in 2004 by Starpharma (Melborne, Australia). Still other applications of dendrimers include Superfect gene vector transport systems for possible gene therapy (Qiagen), and dendrimer-linked antibodies (developed by Dade Behring for the detection of heart damage in their Stratus CS systems).
Nanoparticles in Diagnostics
William Moffitt, president and CEO of Nanosphere (Chicago, IL), discussed his firm’s applications of nanotechnology to diagnostics. Originating from basic research by Chad Mirkin, Ph.D., and others at Northwestern University, the company constructed a nanoparticle probe platform capable of direct genomic analysis without recourse to PCR. The company is also designing sensitive methods of protein detection, orders of magnitude more sensitive than conventional ELISA assays.
PCR proved to be one of the most significant research tools of the 20th century. However, it has not been widely adopted for diagnostic procedures due to its complexity and cost, even though a pressing need exists for sensitive methods for detection of DNA in genetic typing and infectious disease diagnosis.
Other requirements in the diagnostic field include sensitivity in protein detection. Various conditions, including cancer, cardiovascular disease, Alzheimer’s disease, and mad cow disease, produce signature proteins whose presence is indicative of the particular disorder. Yet these proteins are frequently produced in such infinitesimal amounts that their recognition requires higher levels of sensitivity than that provided by conventional ELISA-based assays.
Nanosphere bases much of its technology on the use of gold nanoparticles coupled to DNA or protein probes. Starting from a known sequence of DNA, Nanosphere scientists create a DNA probe whose nucleotide sequence is complementary to a specific region of a DNA target.
If the sequence is present in the sample being tested, the DNA probe binds to the target. The subsequent signal from the DNA probe establishes the presence of the target.
The unique properties of the gold nanoparticle probes allow detection through optical, electrical, or magnetic processes. Frequently the gold nanoparticle serves as a seed to which silver metal will bind, amplifying the original material to a detectable level. A similar approach can be performed for protein detection, with antibodies replacing the DNA probes bound to the nanoparticles.
Moffitt related studies that his company has carried for the assessment of a proprietary ovarian cancer marker in the circulation of patients. There is a desperate need for a serum-based assay with enough sensitivity to detect a handful of molecules per mL of blood.
Another area of great medical concern is the field of neurodegenerative dementia, especially Alzheimer’s disease. Using nanoparticle detection systems and an antibody specific for the amyloid beta-derived diffusible ligands, the nanoparticle technology could clearly separate a group of Alzheimer’s patients from matched controls in a post mortem analysis. Samples of cerebrospinal fluid were obtained, and the cases were confirmed through histological examination of neural tissue.
The advantages of the nanosphere technology over conventional PCR systems are notable, according to the company. The technology is simpler and more user-friendly than PCR, and so it follows that it is less costly and does not have the requirements for highly trained lab personnel. Furthermore, these nanotechnologies promise decentralization since their simplicity allows them to be adapted by small community hospitals and eventually configured as home diagnostic kits.
“Our platform for ultrasensitive detection is the Verigene System,” said Moffitt. “This device allows high count multiplexing for clinically relevant panels of nucleic acids and proteins.” The Verigene System is actually two instruments: the Auto Processing System (APS) and the Verigene ID, which permits analysis of a variety of assays on nucleic acids and proteins with the simplicity of a sandwich assay.
The device is engineered for nucleic acid and protein detection in one instrument, making it applicable throughout the life sciences industry. The company currently has feasibility assessments and development efforts ongoing for key market opportunities.
“Quantum dots are nanoscale semiconductor crystals that perform as fluorescing beacons in many life science applications,” stated Clinton Ballinger, Ph.D., CEO of Evident Technologies (Troy NY). These colloidal single crystals are a few nanometers in diameter, and can be precisely constructed with attention to size and shape, yielding qdots with composition and size-dependent absorption and emission. Because of their size, energy levels are quantized, and energy emission of a photon is in a narrow range, whereas absorption is in a broad band range.
Because of their adherence to quantum behavior, qdots have properties that make them superior to older, organic fluorescent molecules, which bleach rapidly and excite only in a narrow absorption range. They can emit at long wavelengths in the infrared, which allows them to be used as in vivo tags for localizing tumors. This allows the use of a single excitation source thus every color of quantum dot can be excited by the same source, as long as the wavelength light source is shorter than the emission.
Since quantum dot emission frequency is size-dependent, EviTags can come in any color. Evident’s current offering ranges from lake placid blue (490 nm) to maple red-orange (620 nm), with high signal intensity and minimal background interference.
Traditionally, quantum dots contained heavy metals, including lead and cadmium, but Evident developed a new panel of products in which toxic heavy metals are eliminated in order to reduce toxicity and environmental hazards. “We believe quantum dots are the ideal materials for imaging, tracking and diagnostic use in vivo,” Ballinger said.
“Our goal for the next decade is to apply nanotherapeutics to a range of human ills, including cancer, cardiovascular disease, genetic disorders, and trauma,” said Chiming Wei, M.D., Ph.D. Dr. Wei, who is head of cardiovascular/renal molecular research at Johns Hopkins University Medical School, discussed the nanotools and approaches that his team is exploiting.
Dr. Wei characterizes the long-term goal of nanomedicine as the control and manipulation of supramolecular assemblies in living cells in order to improve the quality of human health.
To reach these long-term objectives, Dr. Wei spelled out a set of five-year goals for his program, including: (1) development of smart biosensors that employ fluorescence resonance energy transfer or other molecular activation techniques; (2) optimization of performance of quantum dots and other nanoparticles; and (3) movement through the clinic to FDA approval of this new family of imaging agents.
Dr. Wei’s focus on nanomedicine is in the area of nanotherapeutics, at present under development at the Johns Hopkins Institute. Three main areas of study include drug therapy, with particular concern for size reductions of the drug-transporting particles; gene therapy, taking advantage of an endovascular model; and immunotherapy, targeting the mucosal tissues with local injections of nanoparticles.
Dr. Wei voiced particular concerns for environmental issues, an area referred to as nanotoxicology. While no adverse consequences of the application of nanotechnology to living creatures have yet been detected, there is apprehension within the industry over this issue. Many nanotechnology products are composed of heavy metals and other potential toxins, such as carbon, titanium, cadmium and gallium.
Because of their extremely small size, nanoparticles have a large surface area per unit of mass, thus enabling absorption and recruitment of exogenous toxins. Since they were explicitly designed to cross the surface membranes, organ exposure could potentially be enhanced. Thus the intrinsic qualities of nanoparticles, which endow them with so much potential, could have negative and dangerous outcomes.
A Future of Growth for Nanomedicine
Raj Bawa, Ph.D., president of Bawa Biotechnology and the conference chair, discussed the outlook for nanomedicine in the coming years. According to Dr. Bawa, the U.S. government is currently investing $1 billion a year in funding for nanotechnology research, while the global total is $4 billion per annum. Venture capital’s contribution has been much less, with $900 million expended in the past four years, and half of that going to fund nanobiotech projects.
The fact that the public contribution is so much larger than the private outlay clearly reflects a long-term optimism on the part of the U.S. and other governments that the venture capital sector hasn’t yet picked up on. This may be due to the fact that while the technology seems to have unlimited potential, there are few products that have moved through clinical trials and received approval by American or foreign regulatory bodies.
While the market for nanobiotechnology products is very new, it is expected to grow rapidly, reaching over $3 billion by 2008, for an annual growth rate of 28%. If this projected expansion comes to pass, nanobiotechnology will rapidly overwhelm conventional drug development and other traditional approaches.