Abstract
Nanoparticles, labeled with a signaling moiety for in vivo imaging, and one or more ligands for molecularly targeted specificity, hold considerable promise in oncology. Nanoparticles can serve as modular platforms, from which a wide variety of highly sensitive and specific imaging agents can be created. For example, many hundreds or thousands of atoms that provide imaging signals, such as radioisotopes, lanthanides, or fluorophores, can be attached to each nanoparticle, to form imaging agents that would provide higher sensitivity that can be obtained from agents based on small molecules. Similarly, many copies of targeted ligands can be attached to nanoparticles to markedly inrease specific binding. Drugs or therapeutic isotopes can be added to create multifunctional nanoparticles. Appropriately labeled and targeted nanoparticles could lead to a paradigm change in which cancer detection, diagnosis, and therapy are combined in a single molecular complex.
What is Nanotechnology?
Nanotechnology is the creation and use of materials or devices at the level of atoms, molecules, and supramolecular structures, and the exploitation of the unique properties and phenomena of matter at the nanoscale (1–100 nm). Advances in physics, chemistry, materials sciences, and engineering now allow us to fabricate new molecular complexes by combining atoms or molecules one at a time, and in arrangements that do not occur in nature. Such new macromolecules or materials may have heretofore unknown properties, completely different from any naturally occurring molecular entities. Nanoscience and nanomedicine refer to the use of nanotechnology principles, materials, or devices in scientific and medical pursuits, respectively. Nanoscale devices or macromolecules are similar in size to large biological molecules, such as enzymes and receptors. Therefore, nanoscale devices or particles can readily interact with biological molecules either on the surface or inside of cells. The potential offered by such engineered interactions is the basis for the excitement surrounding the possibilities inherent in nanomedicine.
In the world of in vivo imaging, nanoparticles represent the nanotechnology area of most interest. Nanoparticles essentially are large macromolecules that serve as a “platform” to which a variety of signaling and targeting molecules can be attached. From the signaling perspective, the potential advantage of nanoparticles is that hundreds, thousands, or even tens of thousands of signaling molecules, or combinations of signaling molecules for different imaging modalities, can be attached to a single nanoparticle leading to a dramatic increase in signal sensitivity. Similarly from the targeting perspective, multiple molecules of ligand, or combinations of ligands, can be attached to a single nanoparticle, leading potentially to increased specificity.
Why Nanotechnology in Oncology?
Two of the most pressing needs in oncology are to develop new ways of detecting primary cancers as early as possible, and methods to match the appropriate molecular therapy to each patient's tumor. Early detection is, and will continue to be, the single best way to significantly improve therapeutic outcomes, and personalized, targeted therapies represent the “holy grail” that the revolution in molecular medicine promises. One expectation of the cancer field is that noninvasive imaging technologies, such as MRI, optical imaging, PET, and others, will play a critical role in the early detection of cancer. However, this is likely to require the development of new imaging (contrast) agents that will improve the sensitivity of these technologies and enable them to spot the early molecular changes associated with cancer rather than the gross structural abnormalities that mark the disease's more advanced stages. For molecularly targeted therapies to work optimally, we need noninvasive or minimally invasive methods such as imaging to identify whether the tumor phenotype or molecular signature is responding to the selected targeted therapy. Nanotechnology is rapidly making a mark among a small but growing group of cancer researchers as a potential tool for creating such imaging agents. Indeed, at least one nanotechnology-based imaging product has demonstrated success in detecting micrometastases in humans and is awaiting FDA approval, and several other nanoparticulate imaging agents are either in or are close to human clinical trials.
In the materials sciences world, nanotechnology, referring to the interactions of cellular and molecular components and engineered materials with dimensions that are typically smaller than 100 nm, has achieved the status as one of the critical research endeavors of the early 21st century. As researchers make ever smaller and more powerful electronic devices, chemical catalysts, and innovative materials by harnessing the quantum-related and other unique properties of molecular assemblages built at the nanometer-scale, it is clear that nanoscale research will have an impact on society that we can as yet only imagine. Indeed, for those of us in the cancer field, nanotechnology affords the opportunity to radically change the way we detect, treat, and prevent both cancer and its neoplastic predecessors.
By working with molecular-scale objects, nanotechnology can serve as a core technological platform that will enable us to both interrogate and perturb the molecular pathways of cancer and thereby fundamentally alter how we think about the clinical management of neoplasia. Nanotechnology will help blur the boundaries between detection, treatment, and prevention in much the same way that colonoscopy has combined detection, treatment, and prevention of colon cancer in one procedure [1].
However, scale is not the main reason for nanotechnology's potential in imaging. Although small enough to reach almost anywhere in the body, nanoscale devices are versatile and may bring fundamentally new properties to bear on the problem. They can be easily derivatized with a variety of ligands that might distinguish between tumor and healthy tissue. Yet unlike monoclonal antibodies, which can also target tumors, nanoscale devices can be loaded with or constructed from thousands of molecules of contrast agent—iron oxide or gadolinium, for example—providing a huge boost in signal intensity for diverse imaging modalities. The result can be a nanoscale device that yields simultaneous improvements in specificity and sensitivity. Nanotechnology can thus serve as a technology enabler that can leverage the nearly continuous discovery of new tumor markers into novel, more powerful imaging agents.
With only limited penetration in the biomedical research arena, nanotechnology is nonetheless already serving as a synergistic enabler of progress throughout oncology, bringing about an integration of the now largely separate paths of diagnosing malignancy, treating it, and ultimately preventing or reversing the genetic and biochemical transformations that lead to cancer. It is likely that with concerted application of nanotechnology across the field of oncology, we will dramatically increase our chances of detecting cancer and treating at its earliest stages, a paradigm change that must occur in order to radically improve the outlook for cancer patients.
In Vivo Tumor Imaging
A variety of nanoscale particles have already demonstrated utility in imaging tumors and the tumor microenvironment in both animal models and human clinical trials. The most advanced work in this area utilized dextran-coated ultra-small superparamagnetic iron oxide (USPIO) nanoparticles to image lymph nodes containing micrometastases in patients with prostate cancer [2], whereas other studies have used paramagnetic gadolinium-labeled nanoparticulate dendrimers to image lymphatic micrometastases in a mouse breast cancer model [3]. The operative principle here is that normal lymph nodes, namely, those with no micrometastases, rapidly accumulate these particles, leading to a significant decrease in MRI signal intensity resulting from high local concentration of superparamagnetic material. Tumor cells do not take up these particles, so tumor deposits create a “signal void.” When imaged using a standard 1.5 T MRI instrument, normal lymph nodes appear black, whereas those loaded with metastatic cells, or portions of lymph nodes with micrometastases, appear white.
In a clinical trial, researchers administered USPIO nanoparticles, using a dose of 2.6 mg of iron per kilogram of body weight, to 88 patients scheduled to undergo surgical resection of prostate tumor and/or pelvic lymph nodes. Although 71% of the nodes containing metastases did not meet the usual CT imaging criteria for malignancy, MRI with the superparamagnetic nanoparticles correctly identified every patient with metastatic nodes. In addition, suspicious nodes were identified with the superparamagnetic nanoparticles in 9 patients, who then underwent more extensive exploratory surgery that confirmed the presence of distant metastases. An unexpected finding was that the nanoparticles were able to occasionally identify micrometastases less than 2 mm in diameter in normal-sized nodes, well below the typical limit of detection of positron emission tomography, which is considered the most sensitive imaging technique in several clinical scenarios.
Gold nanoparticles, coated with cancer cell-specific ligands, can act as molecularly targeted contrast agents for optical imaging. Using a variety of ligands, including monoclonal antibodies, receptor substrates, and aptamers, these particles have been targeted to three different markers overexpressed on cancer cells: epidermal growth factor receptor (EGFR), matrix metalloproteases (MMPs), and oncoproteins associated with human papillomavirus (HPV) infection, the primary cause of cervical cancer [4].
Although optical imaging does have limitations due to tissue absorption and reflection of light, the inherent low cost of optical imaging methods compared to more complex imaging techniques such as MRI and PET has attracted increasing interest from cancer researchers [5], particularly with the development of inexpensive fiber-optic confocal imaging systems capable of being fed through ducts and capillaries [6].
Near-infrared (NIR) imaging of targeted gold-coated nanoparticles containing a dielectric silicon core is showing promise as a means of detecting molecular signatures of cancer. By varying the thickness of gold coating and the diameter of the dielectric core, it is possible to tune the optical absorption and emission spectra of these particles from the near-UV to the mid-IR portion of the spectra. This is an example of one of the appealing characteristics of nanotechnology (i.e., that certain physical properties can be engineered into the nanodevice). These particles can also be modified with polyethylene glycol to increase circulation time and with sulfide-based linkers to add targeting ligands. Animal experiments have shown that gold/silicon nanoparticles coated with the breast carcinoma marker Her-2 successfully detect microscopic tumors in breast. Once the particles were localized to tumor tissue, increasing the power of the NIR beam for 4 min increased the particle's temperature by an average of 34.7°C, enough to induce irreversible heat damage in the carcinoma cells [7]. Subsequent experiments demonstrated that tumor-bearing mice subjected to this combination of detection and treatment experienced a marked increase in lifespan compared to animals treated with NIR without prior administration of the gold-coated nanoshells [8].
Indirect detection of tumors is also possible using nanoparticles to image changes that occur in the tumor microenvironment [9], including angiogenesis [10,11] and lymphocyte infiltration [12]. In one study, for example, gadolinium–perfluorocarbon nanoparticles were labeled with a ligand that binds to αvβ3, an epitope that is highly expressed on activated neovascular endothelial cells characteristic of angiogenic vessels and absent on quiescent endothelial cells. The particles were then administered to rabbits with implanted, established tumor, and used to image new vasculature using standard 1.5 T MRI [13]. The MR signal increased 56% in vessels surrounding tumors compared to areas distant from tumors. Control animals who received untargeted paramagnetic nanoparticles or targeted by nanoparticles with a nonspecific ligand showed no change in MR signal intensity. Similar results have been obtained using targeted nanoparticles containing ultrasound contrast agents and ultrasound imaging [14].
Informing Therapeutic Decisions
Merging nanotechnology and imaging technology also has the potential for changing the way we approach cancer therapy and clinical trial design by providing the means for detecting in real time whether a given therapeutic agent is actually killing malignant cells. Currently, an Achilles heel of nearly any form of cancer therapy, proven or experimental, is that the patient must often wait months to find out if therapy has been successful. In many instances, this delay means that should the initial therapy fail, subsequent treatments may have a reduced chance of success. This lag also adversely impacts how new therapies undergo clinical testing, as it leaves regulatory agencies reluctant to allow new cancer therapies to be tested on anyone but those patients who have exhausted all other therapeutic possibilities. Unfortunately, this set of patients is far less likely to respond to any therapy, particularly to those molecularly targeted therapies that aim to stop cancer early in its progression, an approach that virtually all of our knowledge says is the best approach for treating cancer.
Given the fact that most effective therapeutic agents induce apoptosis, it is not surprising that much of the work in the area of nanoscale “efficacy detectors” has so far concentrated on imaging large numbers of cells undergoing apoptosis. The molecule annexin V, which binds to phosphatidylserine exposed to a cell's environment during apoptosis [15], is a likely target for detecting apoptosis. Indeed, nanoparticles labeled with annexin V and containing a paramagnetic iron oxide [16] have successfully identified apoptosis using MRI following a positive response to an anticancer agent. Moreover, these nanoscale contrast agents did not indicate that apoptosis was occurring following administration of a nontoxic agent. Further development of this type of system could provide clinicians with a way of determining therapeutic efficacy in a matter of days after treatment.
Other systems could be designed to detect when the p53 system is reactivated [17,18] or when a therapeutic agent turns on or off the biochemical system that it targets in a cancer cell, such as angiogenesis. One approach to measuring the latter is to use a nanoscale ultrasound contrast agent to measure blood around tumors before and after anti-angiogenic therapy [19]. Another is to measure changes in the expression of surface markers of early angiogenesis [20].
Nanotechnology and imaging can also combine to provide surgeons with the means of detecting tumor margins prior to or even during resection, which would be particularly critical for the successful treatment of brain malignancies [21]. Although several methods have been developed using standard optical and MRI contrast agents [22,23], the creative use of nanoparticles offers the opportunity to combine imaging modalities into one agent that can provide preoperative tumor localization using MRI and interoperative imaging of tumor margins using optical fluorescence. One such multimodal nanoparticle has been developed for imaging gliomas [24].
As the examples cited above suggest, most current clinical applications of nanoparticle-based imaging agents are for MRI. Nanoparticles are also likely to be very useful as the basis for optical imaging agents, but clinically useful applications of in vivo optical imaging are predominantly in the development phase. Ultrasound microbubbles, with or without targeting moieties attached, are another class of nanoparticle-based imaging agents. Nanoparticles can easily be labeled with radioisotopes to form radiopharmaceuticals, and nanoparticle-based contrast agents for computed tomography (CT) are in development. Thus, nanoparticle imaging agents can potentially be used with all current modalities for in vivo imaging. It should also be noted that nanoparticle imaging agents will not replace, but are likely to be complementary to, conventional (e.g., small molecule) imaging agents for any modality. Many small molecule imaging agents are currently in development.
Nanoparticle imaging agents have some limitations. Because of their large size they are mostly applicable to targeting vascular, extracellular, or cell surface receptors. Getting nanoparticle into cells or nuclei is a challenge, which might require, for example, adding some molecule (such as the TAT protein) that will assist in transferring the nanoparticle through the cell membrane. Another issue that needs more study is the potential toxicology of nanoparticles. Some nanoparticles may contain elements or molecules that would be toxic if they become dissociated from the nanoparticle. In other cases, the nanoparticle may contain only elements that are known to be safe in their natural forms, but may have toxicity due to their unique properties in the nanoengineered form.
The NCI Alliance for Nanotechnology in Cancer
To help meet the NCI Director's Challenge Goal of eliminating death and suffering from cancer by 2015, the National Cancer Institute (NCI) is engaged in a concerted effort to harness the power of nanotechnology to radically change the way cancer is diagnosed, treated, and prevented. Over the past 6 years, the NCI has taken a lead role in integrating nanotechnology into biomedical research through a variety of programs. The results of these initial funding efforts have demonstrated clearly that melding nanotechnology and cancer research and development efforts will have a profound, disruptive effect on oncology.
To ensure that we capitalize on this opportunity to make dramatic progress today, the NCI has created the NCI Alliance for Nanotechnology in Cancer, a comprehensive, systematized initiative encompassing the public and private sectors and designed to accelerate the application of the best capabilities of nanotechnology to cancer. A major focus of the Alliance, which was formally approved by the NCI Board of Scientific Advisors this past July, is to fund efforts to develop imaging agents to detect cancer in its earliest, most easily treatable, presymptomatic stage and to provide real-time assessments of therapeutic and surgical efficacy. To meet the Alliance's goals in a timely manner, the NCI will issue Program Announcements, Requests For Applications and Request For Proposals, and will use a variety of program management and funding mechanisms that have been shown successful in prior technology development programs, to fund focused nanotechnology development initiatives. Many of these will be milestone driven and product oriented, with an emphasis on commercialization through small-business and larger private-sector project team members.
To accomplish its goals, the Alliance will rely on three strategies to support nanotechnology based platforms. First, the NCI will establish approximately four Centers of Cancer Nanotechnology Excellence (CCNEs) to integrate nanotechnology development into the basic and applied cancer research that is necessary to rapidly facilitate the application of this science to clinical research. The critical requirements for each CCNE will be:
Integration with a Comprehensive Cancer Center/SPORE program
Affiliation with university or research centers of engineering and physical sciences (e.g., mathematics, chemistry, physics, and material sciences)
Advanced biocomputing capabilities
Required existing not-for-profit/private technology development partnerships
One CCNE will serve as a coordinating center for all the CCNEs, to facilitate data and technology transfer across centers, interconnecting and leveraging the strengths and advances of each.
The NCI Alliance recognizes that there is a critical need to train a cadre of researchers who are skilled in applying the tools of nanotechnology to critical problems in cancer research and clinical oncology. And given the complex nature of this endeavor, building multidisciplinary teams will be essential to realizing this vision [25]. Thus, the NCI will provide the necessary funds and opportunities for the cross-disciplinary training and collaboration that will be needed to maximize the impact that nanotechnology can have on the development of powerful new imaging agents and methodologies. Initially, the Alliance will rely on existing training and career development mechanisms to direct talent to this area as quickly as possible. The NCI recognizes, however, that new mechanisms for developing multidisciplinary teams may be needed. The NCI will also encourage programs to be developed with interfaces to the training programs of other Federal agencies, including the FDA, the National Institute of Standards and Technology (NIST), and the Defense Advanced Research Projects Agency (DARPA).
In addition, the NCI has established the Nanotechnology Characterization Laboratory (NCL) at the NCI-Frederick campus to work in concert with NIST and FDA to perform and standardize the preclinical characterization of nanoscale devices in a way that will facilitate the accelerated regulatory review and translation of these devices into the clinical realm. A key activity of the NCL will be to work together with FDA scientists to develop an assay cascade that can serve as the standard protocol for preclinical toxicology, pharmacology, and efficacy testing of nanoscale devices. This assay cascade will characterize a nanoscale device's physical attributes, its in vitro biological properties, and its in vivo compatibility.
The NCL will also address the lack of knowledge concerning the health and safety of nanomaterials that could become an obstacle to the rapid implementation of nanotechnology. Although industry has long manufactured fine and ultra-fine particles for use in a variety of applications, the effects of those particles on human health has been studied only for a small number of materials and applications. The assay cascades developed by the NCL to characterize the effect of nanomaterials and platforms in in vitro and in vivo tests can also provide standardized measures of the effect of these materials, devices, and waste products on human safety. This additional NCL service will require close collaboration with nanotechnology research institutions and product developers and manufacturers to develop the appropriate standard assays and protocols in response to this public need.
Ultimately, the Alliance is not just an initiative for the NCI, but a call to action for the cancer research and technology communities. It emphasizes the process of building partnerships between the private and public sector with the goal of creating teams best equipped to translate today's knowledge about cancer biology and nanotechnology into clinically useful products, including imaging agents.
Conclusion
Advances in nanotechnology and cancer research have created an opportunity to develop novel imaging agents that stand to change the way cancer is detected and treated. Although the application of nanotechnology to the field of cancer imaging is still in its infancy, work done to date clearly shows the promise of this nascent multidisciplinary enterprise, enough so that the NCI believes the time is ripe to undertake a concerted effort to develop nanoscale imaging agents that will impact clinical practice in the near-term. Given the enormous impact that cancer has both in terms of morbidity and cost, the successful application of nanotechnology to cancer imaging could have profound societal implications.