Nanomedicine:
Should NAPE Be Interested?

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By Kattesh V. Katti and Raghuraman Kannan,
Departments of Radiology and Physics, 
Missouri University Research Reactor,
University of Missouri-Columbia, MO

Introduction

Nanotechnology is projected to be a major key to 21st century technological and medical progress, and its success will depend on continued collaborative innovative efforts among the natural sciences, medicine, engineering and allied disciplines. Although it may be too early to gauge, its economic and societal impact is estimated by the National Science Foundation report “Societal Implications of Nanoscience and Nanotechnology” (March 2001) at one trillion dollars by 2015 [1]. This new field, referred to as “Disruptive Science,” is inherently interdisciplinary and has the power to break boundaries between traditional areas of science, agriculture, medicine and engineering. It opens new opportunities to miniaturize today’s products, to provide new materials with exceptional performance properties and to enrich our understanding of nature and life itself. This interdisciplinary field is being discussed in terms of its potential to deliver a Second Industrial Revolution, radically transforming manufacturing processes. In medicine it is poised to bring about a paradigm shift in the way diseases are diagnosed and treated.

Nanomedicine is an emerging medical area that utilizes nanoparticles for the detection and treatment of various diseases and disorders. It relies on nanoparticles which are tiny fragments of metals (or non-metals) that are 100,000 times smaller than the width of human hair. Why is this tiny size so important in medical applications? Size is important; but, it is not size alone that matters. It also is the collateral properties that emanate when materials, especially metals, are reduced to nanometers. As shown in Figure 1, nanoparticles within the size domains of 1-50 nanometers can be related to the sizes of various biological entities including cells, viruses, proteins and antibodies.

Man-made nanoparticles, with sizes in the range of living cells, are the focus of current medical research. Nanoparticles produced using nanoscience and nanotechnology principles exhibit properties unique to nanometer size. For example: (i) gold nanoparticles can release a certain amount of heat when placed within oscillating magnetic fields for potential applications to control or eradicate specific tumors; (ii) nanoparticles display photo absorbance or emission characteristics that can be used in imaging for the diagnosis of various diseases; (iii) selective absorption of x-rays by metallic nanoparticles can lead to measurable contrasts for use in computer tomographic (CT) imaging of diseases/disorders. Release of absorbed x-ray energy onto tumors can suppress or eradicate them, thus utilizing nanoparticles as mediators for dual imaging and therapy applications to diagnose, stage and treat various diseases. These and a host of useful diagnostic/therapeutic properties are attainable only when metallic (or non-metallic) substances are reduced to nanometer sizes, thus making the interplay of size and properties of nanoparticles the essence of an emerging medical modality referred to as nanomedicine (Figure 2) [2].

A variety of such nanoparticulate vectors and nanoscale devices have demonstrated efficacy in their utility as in vivo tumor imaging agents in animal models and in human clinical trials. The ubiquitous place of gold in nanomedicine stems from its chemical ability to serve in an unoxidized state at the nano size when most surfaces of less noble metals oxidize to a depth of several nanometers or more, often obliterating their nanoscale properties. The high reactivity of gold nanoparticles (AuNPs) juxtaposed with their biocompatibility has spawned great interest in their utility for in vivo imaging and therapy. Recent work is centered around development of hybrid AuNPs starting from nascent metal nanoparticles. Hybrid nanoparticles are produced by coating AuNPs with tumor cell specific biomolecules, including monoclonal antibodies, aptamers, peptides and various receptor specific substrates. Receptor specific hybrid nanoparticles are used mainly for targeting three different markers that are over-expressed on cancer cells. They include: matrix metalloproteases (MMPs), epidermal growth factor receptor (EGFR), and oncoproteins that are associated with human papillomavirus (HPV) infection. 

Synthesis of Biocompatible Gold Nanoparticles

Biocompatibility is an important prerequisite in utilizing AuNPs for in vivo imaging and therapy applications. It requires the stabilizing of nanoparticles in a biologically benign medium. Currently available techniques utilize harsh conditions, such as the application of sodium borohydride to reduce AuCl4- in order to produce AuNPs. While these methods work efficiently, they are unsuitable because sodium borohydride will reduce the chemical functionality present on peptide backbones, either reducing or eliminating the biospecificity of the biomolecules. The sodium borohydride reduction method also uses thiols to stabilize AuNPs from agglomeration. Although this protocol leads to enhanced stability of AuNPs, such AuNPs cannot be readily anchored onto peptides or other biomolecules because of the strong interaction of gold metal with thiol groups. This means that thiol-stabilized AuNPs have limited applicability in the development of AuNP tagged biomolecules for use in target-specific nanoscale imaging or therapeutic agents. Other methods described in the literature have similar drawbacks.

For sustained research aimed at the design and development of AuNP-based imaging or therapeutic agents, we felt it imperative to develop new and efficient methods that lead to the production of biocompatible AuNPs under physiologically benign conditions, using biologically benign chemical molecules such as carbohydrates (starch, glucose, agarose) and proteins (e.g., glyco proteins). As part of our long standing interest in the design and development of gold and other metal-based cancer imaging and therapeutic agents and sensor devices [3-10], we performed extensive studies on the chemical, structural, photophysical and biological aspects of a wide spectrum of gold compounds. We recently discovered that a new trimeric alanine phosphine conjugate (THPAL, P(CH2NHCH3COOH)3), interacting with commercially available NaAuCl4 in water at 25 0C at physiological pH, results in the production of biocompatible gold nanoparticles in excellent yields with complete retention of nanoparticulate properties [3,4]. This AuNP production process allows systematic variation in nanoparticulate size via changes in the stabilizer and stoichiometry of NaAuCl4 and THPAL (Figure 3).

Extensive studies of the interrelationship of reaction conditions and nanoparticulate sizes have provided optimized conditions to produce AuNPs of well-defined sizes. Transmission electron microscopic (TEM) analysis has been used to view gold nanoparticulate sizes that are 100,000 times smaller than the width of human hair. Sizes and shapes of gold nanoparticles are depicted in Figure 4. We have used various biocompatible matrices including, starch, agarose, glucose, and Gum Arabic to stabilize gold nanoparticles. The sizes of resulting AuNPs are: Starch stabilized: 20 nm, Agarose stabilized: 13 nm, Glucose stabilized: 22 nm, and Gum Arabic stabilized: 10 nm. These nanoparticle size variations are ideally suited for biomedical applications because 15-30 nm sizes allows direct attachment of gold nanoparticles to specific cells for diagnostic imaging and therapy applications.

Detection and Therapy of Early Stage Diseases

Nanoparticles, because of their sizes within the cellular domain, can be effectively used to target individual cells for early stage detection and therapy of cancer and other diseases. Almost all types of mammalian cancers over-express receptors for specific peptides (and proteins). Therefore, cancer specific peptides can be anchored on the surface of nanoparticles to give them the “sense of direction” to seek and home in selectively onto tumor cells and cancer tissue. Because of the large surface area of nanoparticles, each one can carry more than one peptide or more than one type of peptide (Figure 5). This multifunctionality not only leads to significantly higher diagnostic/therapeutic efficacy, the incorporation of peptides with multiple receptor targeting capabilities produces multiple antigen-binding nanoparticles for targeting multiple cancer receptors in patients. Schematic sketches, as shown in Figure 6, outline new approaches being developed in our laboratory for the creation of prostate tumor-specific hybrid gold nanoparticles for use in imaging and therapy.

Impairment of vision is a common manifestation in various ophthalmic diseases and disorders, including age related macular degeneration and pseudoxanthoma elasticum. Potential treatment approaches based on gold nanoparticles are on the horizon for these problems thanks to the biocompatibility of gold nanoparticles and their ability to deliver diagnostic/therapeutic probes without harmful side effects.

Gold Nanoparticles In Therapy

Another attractive approach for the application of nanotechnology to nanomedicine is the utility of nanoparticles that display inherent therapeutic properties. For example, radioactive AuNPs present attractive prospects in cancer therapy and other diseases. The radioactive properties of Au-198 (bmax = 0.96, MeV; t½= 2.7 d) and Au-199 (bmax = 0.46 MeV; t½= 3.14 d) make them ideal candidates for use in radiotherapeutic applications. Such gold isotopes have imageable gamma emissions for dosimetry and pharmacokinetic studies. Gold nanoparticles can be delivered directly into cells and cellular components with a high concentration (dose) of radioactivity to cancerous tumor cells. Nanoparticulate therapeutic agents derived from radioactive AuNPs provide higher therapeutic payload to tumor sites as each gold nanoparticle contains hundreds/thousands of atoms of gold. This unique advantage of achieving a substantial increase in therapeutic dose to tumor site coupled with the feasibility of tagging nanoparticles of Au-198 with oligonucleotides and peptides that are selective for receptors over-expressed by diseased tissue, presents a remarkable new potential for the treatment of cancer. Recent studies in our laboratories have provided “Proof of Principle” for the production and stabilization of biocompatible radioactive gold nanoparticles for potential applications in therapeutic nanomedicine (Figure 7) [3].

Readily Injectable Nontoxic Gold Nanoparticles for Treating Cancer and Other Disorders

Cancer and other disorders often require repeated administration of medication, making the development of nontoxic pharmaceuticals of major significance in modern treatment protocols. From the above discussions, it is clear that gold nanoparticulate vectors can play a significant role in the advancement of clinically useful diagnostic and therapeutic medical products. A serious drawback in this effort is the rarity of nontoxic gold nanoparticulate constructs and formulations that can be administered as described. The ability of plants to absorb and assimilate metals provides the potential to utilize plant extracts as nontoxic vehicles to stabilize and deliver nanoparticles for in vivo nanomedicinal applications. In this context, we have recently discovered the application of Gum Arabic (Acasia Gum) as a plant-derived construct for stabilizing gold nanoparticles. This natural gum is exuded by various species of Acacia commonly grown in the sub-Saharan regions of Africa, Australia, India and South America. Gum Arabic is a widely accepted nontoxic ingredient in both food and pharmaceutical products. Emulsification, acid stability, low viscosity at high temperatures, adhesive and binding properties and good mouth feel are among the reasons for its wide acceptance as an additive in confectionaries, beverages, bakery products, brewing, and in pharmaceutical formulations. In addition, Gum Arabic has unique structural features that attracted our attention. It has a highly branched polysaccharide structure consisting of a complex mixture of potassium, calcium and magnesium salts derived from arabic acid with galactose, rhamose, glucuronic acid, 4-O-methyl glucuronic acid and arabinos residues. Its molecular structure is comprised of three main components: the dominant being arabinogalactan (90%) which has a low protein content (5%), a high protein content (10%) segment with 10% arabinogalactan and the third component (<1%) contains glycoproteins with over 50% protein content. Our recent studies have produced a new class of injectable, in vivo stable hybrid nanoparticles derived from the tagging of Gum Arabic glycoprotein matrix with gold nanoparticles (Figure 8). Preliminary results of in vivo pharmacokinetics studies of GA-AuNP in pigs have demonstrated that these nanoparticulate phyto-constructs are nontoxic and thus may be utilized for human imaging and therapy applications [4]. 

Societal Impact and “The Big Nanomedicine Picture”

The unique abilities of nanoparticles to serve as diagnostic/therapeutic probes and also as carriers of drug molecules for delivery at specific sites provide exciting opportunities in treating various diseases. Nanoparticles present realistic prospects to serve as platforms for carrying both diagnostic and therapeutic vectors within the same entity, and thus may provide future pharmaceuticals with both capabilities built within the same pill. Indeed, it has been estimated that over 80% of all future drugs will utilize some form of nanotechnology. “Readily Injectable” gold nanoparticles that are stable in vivo and nontoxic at therapeutic doses will play pivotal roles for site-specific in vivo delivery, as in vivo sensors, semiconducting slow bleaching photo-active agents for optical imaging, as carriers of very high diagnostic or therapeutic loads to tumor/disease sites, in photodynamic therapy by carrying a plethora of free radical-generating chemicals to tumor sites, as contrast enhancers in CT imaging and as x-ray absorbers at tumor sites for x-ray based therapy. Conjugation of nontoxic gold nanoparticulate vectors to stem cells will provide major advances in stem cell based imaging and therapy of cancer and other disorders. Despite the “super spectrum” of current and realistic future applications offered by hybrid gold nanoparticles, there is still a severe paucity of in vivo studies demonstrating low toxicity of gold nanoparticulate constructs and formulations. Therefore, the development of readily injectable, in vivo stable nontoxic gold nanoparticulate vectors, especially created from commonly accepted human food ingredients, are needed for major advances in nanomedicine. Our results demonstrating the ability of Gum Arabic to provide in vitro and in vivo stability to maintain the nanoparticulate properties of gold nanoparticles intact for several months in aqueous/saline/phosphate buffered solutions, as well as in the solid state, represent a significant advance in nanoscience with realistic implications for safe delivery of nanoparticles for a variety of diagnostic and therapeutic applications. The ready availability and adaptability of Gum Arabic within the human food chain make our approach a viable strategy for storage, shipment and in vivo delivery of gold nanoparticle-based nanomedicine products world wide.

Although there is no question of the scientific power and the positive impact of nanoscience and nanotechnology in transforming medical diagnosis and therapy, the potential toxic side effects of nanoparticles administered via intravenous or oral pathways cannot be discounted. Concerted effort must be invested in gaining new insights concerning near and long term pharmacology and toxicology of a wide spectrum of nanoparticles that are being considered for medical use.

Acknowledgment: This work has been generously supported by the National Institutes of Health/National Cancer Institute under the Cancer Nanotechnology Platform program (grant number: 1R01CA119412-01).

References:
1. http://www.wtec.org/loyola/nano/NSET.Societal.Implications/
2. P. Grodzinski, M. Silver, L. K. Molnar, Expert Review of Molecular Diagnostics 2006, 6, 307.
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