Abstract: On average, only 1 out of 10,000-100,000 injected biomolecules reaches its therapeutic target. Biobarriers throughout the body present many obstacles for drugs to realize their therapeutic potentials. Nanovectors make it possible to achieve lesion site recognition and biobarrier avoidance.
Approximately one person a minute dies of cancer in the United States. Normalized per population size, this corresponds to a current mortality rate that is essentially identical to what it was in 1950 - a very counterintuitive finding, given the exceptional progress recorded in the fundamental scientific understanding of malignant disease in the last 50 years. Dominant among the reasons for the unsatisfactory progress in the treatment of cancer is our general inability to treat metastatic colonies, when surgical intervention and radiation therapy are no longer available options. Systemic injection with chemical and biological agents is then the choice, with the yet-unsolved problem of selectivity in the intervention on cancer cell population, or the ability to kill cancer without causing intolerable levels of unwanted collateral effects on the patient.
This treatment selectivity problem breaks down into three major, related components: the ability for the therapeutic substances to reach the cancer lesion, to recognize it as the target of its action, and to perform the therapeutic intervention solely at the site of the lesion. Many approaches have been developed to address these questions, and have met with different degrees of success. Particularly promising are the recent clinical advances recorded in the field of the so-called molecularly targeted therapies, which intervene in an exquisitely selective fashion on cancer-associated biological features, such as mutations in the receptor of epidermal growth factor in cancers of epithelial origin (Hynes, 2005), or the activation of the BCR-ABL tyrosine kinase pathway in chronic myelogenous leukemia.
Antibodies are directly employed as treatment agents, or are conjugated to chemo- or radio-therapeutic moieties to enhance treatment selectivity (Adams, 2005). However, the very nature of antibodies, and the relatively large sizes of their complexes with therapeutic agents make it very difficult for them to reach the intended targets, though their target-recognition capabilities are remarkable. The problem is in their ability to overcome the multiple, sequential, formidable biological barriers (”biobarriers”) that separate the target lesions from the site of injection of the therapy (Ferrari, 2005a). Typically, only one injected biomolecule per 10,000-100,000 reaches the intended destination. Thus, it may be recognized that lesion recognition and biobarrier-avoidance are largely decoupled problems, for all intended clinical purposes.
The development of cancer treatments has traditionally rested on the implicit assumption that it is possible to develop a single agent - biological or chemical - that can perform all three functions: reaching the target by bypassing all biobarriers, recognizing the lesion with substantial specificity, and performing the intended therapeutic action. This may appear to be too much to ask of a single, however complex, non-self molecule. The new perspective brought in by nanotechnology is that this elusive combination of different tasks can be successfully performed, by introducing nanometer-sized carrier particles (”nanovectors”) that have the primary function of transporting the active agent to the target site, performing the multiple biobarrier-avoidance tasks required along the way (Ferrari, 2005b). The nanovectors can be engineered to employ several, concurrent strategies to localize preferentially at the target lesion, and release its therapeutic payload. The active principles contained in the nanovector can be any conventional or novel drug, including those in the molecularly targeted generation, as well as combination of different agents: Nanovector technology is not about developing new therapeutic molecules, it is about their multi-functional transport capabilities. Still, nano-encapsulated treatments present just like any other injectable drug, from the viewpoint of clinical administration.
Nanotechnology, and Nano-in-Medicine
Nanotechnology pertains to man-made functional devices with dimensions or features on the order single to a few hundreds of nanometers. For perspective, the volume of a cancer cell of assumed spherical shape and 10-micron (10,000 nanometer) diameter could accommodate approximately 1 billion spherical nanovectors of 10-nanometer diameter. Still, each of these nanovectors would contain over a billion individual atoms. Thus, nanotechnology is the art and science of making, testing, and using supra-atomic and supra-molecular devices that are capable of performing functions that are only possible because of their size.
Nanotechnology is no stranger to medicine. Nano-featured components are crucial for the performance of laboratory functions, such as liquid chromatography. Photolithography, the very technological foundation of microarrays manufacturing, has now evolved to the point that it is possible to control features on the nanoscale (”nanoarrays”), yielding a million-fold increase in information density, and ushering in an era of unprecedented ability to detect and quantify, in real-time, a large number of molecular signatures from complex biological fluids. Nanoparticulate contrast agents are in clinical use as contrast agents for a variety of imaging modalities (Ferrari, 2005a). Nanotechnology-enabled therapeutics include liposomes, which are the archetypical nanovectors and, despite their relative simplicity, have engendered substantial progress in clinical oncology. Liposomes are currently used for the treatment of recurrent breast and ovarian cancer, and Kaposi’s sarcoma. The FDA has recently approved nano-sized particulates of paclitaxel, conjugated with albumin molecules, for the treatment of metastatic breast cancer.
It is estimated that several hundreds of different nanovectors are in varying stages on pre-clinical and clinical development toward regulatory approval in the USA and worldwide. Those derived from lipid chemistry include multiple generations of liposomes, solid-lipid nanoparticles, lipid-encapsulated perfluorocarbons, and “micro-bubbles.” Prominent among polymeric nano-drugs are dendrimers, polymer-drugs-conjugates, micelles, and biodegradable particles. Hybrid lipid-polymer constructs such as dendrisomes are under investigation. Inorganic nanovectors comprise gold-encapsulated oxide particles (”nanoshells”), silica- and silicon-based carriers, quantum dots, iron oxide-based nanoparticulates, and carbon-sixty molecules, such as fullerenes and nanotubes. Biological nano-delivery technology includes particles derived from nucleic acids and viruses (Ferrari, 2005b).
Each of these families of nanovectors presents advantages and limitations, in view of their application to cancer. A detailed review of their properties much exceeds the scope of this article - the point is simply that there are many, entirely different families of nanovectors that are poised to make an impact on cancer therapeutics. Each of these has very different properties, in terms of biodistribution, capacity to carry different drugs, release mechanisms and time profiles, conjugation with localizing agents, toxicity and biocompatibility in general. Any drug, whether currently in the clinic, under development, or shelved because of toxicity profile could be selectively delivered via one or more nanovector type, resulting, in principle, in an increase in therapeutic efficacy with a concurrent reduction of collateral damage. Success in bringing to the clinic even a modest number of nanovectors, each with the capabilities to carry different drugs and to be targeted to different cancers, could therefore yield an imposing positive impact on the pipeline of new therapeutic agents.
The Selective Localization of Nanovectors
The therapeutic index (TI) of a drug is the ratio between the benefit of the intervention for a given indication, and the undesired collateral damage. The TI generally increases with spatial localization of the active agent at the target cancer lesion site. Biological targeting, e.g., by way of recognition moieties such as antibodies, ligands, and aptamers yields localization advantages that increase with the specificity of the targeting moiety, and decrease with its inability to bypass biological barriers. The threshold level of TI gain that is sufficient to justify clinical development is not necessarily reached by biological targeting alone. Fortunately, it is possible to implement multiple, simultaneous targeting strategies on nanovectors, that allow for a cumulative gain in localization, and the potential to reach the desired TI threshold. In other words, the probabilities of selective lesion localization via different mechanisms are additive, and the fact that nanovectors can be designed to take advantage of several mechanisms at the same time makes them a potentially advantageous therapeutic strategy.
Nanoshells are a very promising example of multi-mechanism lesion localization. They are formed by a spherical, dielectric silicon-oxide core, of desired dimensions in the nanoscale range. The core is enveloped in a gold shell, the thickness of which is also a few nanometers, and can be exquisitely tailored by design to resonate with incident light radiation in the near-infrared spectral range. Light in this range is absolutely harmless, penetrates very deep into tissue, and causes the irradiated nanoshells to be selectively heated, causing thermal ablation of the tissue they contact. Nanoshells were injected in experimental animal models with cancer xenografts, and selectively concentrated at the tumor size since they were manufactured in sizes that allowed for their preferential permeation across leaky angiogenic tumor vasculature - a mechanism of passive targeting known as “enhanced permeation and retention” (EPR), which is also the main localization route for current liposomal treatment in the clinic. A further degree of therapeutic localization was granted by the fact that only the lesion-containing tissues and organs were irradiated. This resulted in the thermal ablation of all of the cancers, with complete and sustained remission (O’Neal, 2004). At no expense to these two localizing strategies, a third targeting mechanism can be added to the EPR and the remote activation through selective irradiation, by conjugating the nanoshells with biological recognition agents such as antibodies to cancer cell surface or vascular endothelial antigens. This illustrates the concept of multiple, concurrent localization strategies that can be exploited by nanovectors, with the potential to combine and yield gains in lesion targeting and ultimately in therapeutic index.
Nanovectors for the Bypassing of BioBarriers
In the history of drug development, the emphasis on targeting has much overshadowed the importance of overcoming biological barriers. While it is evident that lesion recognition capabilities are useless, unless the lesion to be recognized can be reached, the differentially inferior attention dedicated to biobarrier avoidance was perhaps the result of the foreboding complexity and diversity of these barriers.
Under the rubric of biobarriers we include those of endothelial and epithelial origins. Prime examples of the former are the tumor vascular endothelium, which is leaky in selected cases (Neri, 2005). Epithelial barriers to the penetration of active therapeutic agents, whether nanovectored or not, include the blood-brain barrier and the internal lining of the intestine. The attachment of albumin molecules to nanoparticles of paclitaxel in an FDA-approved treatment of breast cancer is specifically for the purpose of enhanced transport across an otherwise impermeable vascular endothelium. Synthetic carriers can be charged with a therapeutic moiety and permeation-enhancing molecules such as zonula-occludens toxins for the reversible opening of tight junctions in epithelial barriers (Ferrari, 2002). This provides the nanoparticle-enabled co-localization of the bio-barrier avoidances agent and the therapeutic molecule. Use of the former in areas broader than the intended target would expose the patient to intolerable risks for opportunistic pathogen penetration - therein resides the advantage of nano-vectored co-delivery.
Nanoparticles injected into the systemic circulation are generally cleared by the resident macrophages of the Reticulo-Endothelial System (RES), which form a third example of biobarriers. RES uptake has been bypassed by the use of polyethylene glycol (PEG) on the nanoparticle surface - a strategy first pioneered for application in “stealth liposomes.” An example of a biobarrier of physical nature is the increased hydrostatic pressure that develops internally to growing cancer lesions, and opposes drugs and nanovectors transport from the vascular compartment into the tumor, actually causing net flux in the opposite direction. The answers to this barrier, and to many others, are still not available. Progress in their identification will enable clinical practice-changing advances in the therapy of cancer and other pathologies.
Novel, yet more exciting therapeutic innovations might result from continued progress in nanovector-based delivery: once a therapeutic nanovector is validated, in combination with therapeutic and biobarrier-avoidance agents of choice, it might be envisioned that different, patient-specific biological moieties could be appended to its external surface, to provide patient- specific localization of the therapeutic action. The time evolution of the molecular expression of the cancer lesion and its microenvironment could be monitored, and the therapy localization strategies could then be tailored to meet the requirements present at the time of administration. Or, the nanovector itself could contain reporter mechanisms that release soluble signals in the bloodstream, to inform the health care provider of the efficacy or noxiousness of the treatment in real time.
These are strongly speculative scenarios, to be sure. However, they share a common foundation with the nanovector-based therapeutic agents that are currently entering clinical practice with increasing vigor: they are based on the notion of decoupling the therapeutic action, which is assigned to the vectored active principles, from the transport to the intended target site, and the avoidance of the biological barriers found along the way. These are functions that are optimally performed by multifunctional nanovectors.
References and Further Readings
Ferrari M, Grove C, Dehlinger P, Martin F. Particles for oral delivery of peptides and proteins, US Patent No. 6,355,270, March 12, 2002.
Ferrari M. Cancer nanotechnology: opportunities and challenges. Nature Reviews Cancer 5:161-171, 2005a.
Ferrari M. Nanovector Therapeutics. Current Opinion in Chemistry and Biology 9:343-346, 2005b.
Hynes NE, Lane HA. ERBB receptors and cancer: the complexity of targeted inhibitors. Nature Reviews Cancer 5:341-354, 2005.
Neri D, Bicknell R, Tumor vascular targeting. Nature Reviews Cancer 5:436-446, 2005.
O’Neal DP, Halas NJ, West JL. Photo-thermal tumor ablation in mice using near infrared-absorbing nanoparticles. Cancer Letter 209:171-176, 2004.
[Discovery Medicine, 5(28):363-366, 2005]