«COLLOIDAL GOLD NANOPARTICLES FOR CANCER THERAPY: EFFECTS OF PARTICLE SIZE ON TREATMENT EFFICACY, TOXICOLOGY, AND BIODISTRIBUTION A Dissertation ...»
death. Cancer is caused by both external factors (tobacco, infectious organisms, chemicals, and radiation) and internal factors (inherited mutations, hormones, immune conditions, and mutations that occur from
Cancer has been the second leading cause of death worldwide, following heart diseases . Cases of cancer doubled globally between 1975 and 2000, will double again by 2020, and will nearly triple by 2030, according to the World Cancer Report by World Health Organization. There were an estimated 12 million new cancer diagnoses and more than 7 million deaths worldwide in 2009. The projected numbers for 2030 are 20 to 26 million new diagnoses and 13 to 17 million deaths. Looking at the United States alone, approximately 1.5 million new cancer cases are expected to be diagnosed in 2010 .
Over the past 50 years, the death rate due to top mortality causes (heart diseases, cerebrovascular diseases, and pneumonia/influenza) has declined dramatically in the US . However, despite the vast knowledge and efforts made over the past decade to fight cancer, relatively little progress has been made to reduce the cancer death rate. Thus, the rapid increase in the global cancer burden represents a real challenge for health systems worldwide.
Leading Causes of Death Worldwide in 2001 (thousands)  Figure 1.1. Comparison of Death Rates for Top Four Leading Causes of Mortality in the United States  As seen in Figure 1.1, compared to the peak rate of 215.1 per 100,000 in 1991, the cancer death rate decreased 17% to 178.4 in 2007. Rates for other major chronic diseases decreased substantially during this period.
The complexity and heterogeneity nature of cancer makes it difficult to successfully diagnose and treat cancer. Advances in cancer research have been focused on studying molecular level of the disease, and “nanotechnology” plays a critical role in overcoming the obstacles in cancer biology. Nanotechnology is an interdisciplinary research field that combines chemistry, engineering, biology, and medicine that allows early detection, accurate diagnosis, and personalized treatment of cancer .
Nanotechnology adopts a size scale that is equivalent to biological molecules (i.e protein, DNA, etc.) of 5-100 nm in diameter. Due to its small size, nanoparticle improves the availability of particular agent by increasing its interactions with biomolecules both inside and outside surface of the cells. The most well-studied nanoparticles include quantum dots [4-7], iron oxide [8-11], polymer-based nanoparticles [12-14], carbon nanotube , gold nanoparticle [16-19], and many others .
One of the most extensively studied subjects in nanotechnology is “size”. In particular, the relationship between size and various aspects such as tumor accumulation/ targeting, cellular uptake, toxicity, biodistribution, and clearance has been the major focus for successful application of nanoparticles in detection, diagnosis, and treatment of cancer. In this chapter, we will closely look at the general characteristics of cancer and the role of nanotechnology in cancer biology, especially focusing on the “size effect”.
1.3 CHARACTERISTICS OF CANCER Research over the past decades have revealed that tumorigenesis in humans is a multi-step process that results in dynamic changes in the genome. The transformation of normal cells to malignant cells is an extremely complicated process that brings heterogeneity in cancer cells. To make the matter more complex, more than 100 distinctive types of human cancer have been identified, and multiple subtypes of tumors are found within a specific type of cancer. However, it has been reported that most and possibly all types of human cancer share common traits that are acquired during tumor development: 1) self-sufficiency in growth signals, 2) insensitivity to anti-growth signals,
3) evasion of programmed cell death or apoptosis, 4) limitless replicative potential, 5) sustained angiogenesis, 6) tissue invasion and metastasis .
Normal cells heavily depend on growth factors and signals from its environment to control proliferation. However, tumor cells provide their own growth factors and proliferate independently from external growth signals. Furthermore, tumor cells display abnormal cell growth by disrupting the anti-growth signal pathway to result in uncontrollable growth.
Six Acquired Traits of Human Cancer  Apoptosis is body’s defense mechanism where diseased cells are removed from the system through programmed cell death. Tumor cells avoid apoptosis by altering the cellular pathway for programmed cell death, which allows them to grow without any restriction or confinement. As the tumor cells proliferate, they provide their own nutrients via creating their own network of blood vessels to access oxygen and other required nutrients. Finally, tumor cells invade adjacent tissues and travel distant sites where nutrients and space is not limited initially. It is this metastasis of tumor cells that results in 90% of human cancer deaths.
1.4 ENHANCED PERMEABILITY AND RETENTION (EPR) EFFECTTumor vasculature is characterized as “leaky” due to its irregular-shaped, dilated, disorganized, and poorly-aligned endothelial cells [22-24]. Additionally, poor lymphatic drainage results in leakage of plasma components from the circulation into the interstitial space of the tumor. This phenomenon, originally described by Matsumura and Maeda, is called enhanced permeability and retention (EPR) effect .
As seen in Figure 1.3, normal, healthy vasculature displays continuous morphology where pores are 2-6 nm in size . Tumor vasculature has larger pores than the normal vessels that size ranges from 100 to 2000 nm [27-29]. Increased cutoff pore size for tumor vasculature allows increased permeability of plasma proteins for tumor and lack of functional lymphatic vessels within tumor decreases the rate of clearance.
The EPR effect now has become the “gold standard” in anticancer drug delivery that takes advantage of the unique anatomical-pathophysiological nature of the tumor blood vessels. The EPR effect is a molecular weight dependent phenomenon that only occurs in the tumor tissue. Particles larger than 40 kDa selectively leak out from the tumor vessel to accumulate in the tumor tissue. The increased accumulation of 40 kDa are due to prolonged circulation time and decreased clearance rate from the body. In attempt to prolong the drug residence time and selectively trap the nanoparticle in the tumor cells, biocompatible poly (ethylene glycol) is commonly used to prevent rapid clearance of the nanoparticle by the reticuloendothelial system.
a) b) Figure 1.3. Normal and Tumor Vasculature. a) Normal vessels are aligned in parallel to one another (A), whereas tumor vessels have chaotic morphology with uneven diameters ; b) SEM images of normal blood vessels of pancreas (A), colon (B), and liver (C) compared to tumor vessels of liver (D, metastatic tumor nodule marked as “T” and normal liver tissue marked as “N”), tumor nodule (E), and empty void in tumor vascular bed (F) 
1.5 SIZE EFFECT Various nanoparticles such as semiconductor quantum dots, iron oxide, polymerbased nanoparticles, and gold nanoparticles are present for in vitro and in vivo applications in nano-diagnostics and nano-therapy. Quantum dots are approximately 2-9 nm in size and are widely used in cell-tracking, solution-based detection, and in vivo imaging in whole animals [4, 7, 30]. Quantum dots display unique physical and chemical properties of narrow emission peaks, high photostability and brightness, broad absorption peaks, and size-tunable emission wavelengths throughout visible and infrared spectrum . Paramagnetic iron oxides are widely used in vivo MRI contrast imaging agent [8, 9, 11, 31] and is also used as a carrier for targeted delivery of anticancer agent .
Polymer-based nanoparticles are commonly used for drug delivery applications and can be synthesized in various shapes and sizes that can carry multiple agents to the diseased cells [12-14]. Some of the polymer-based nanoparticles, such as PLGA-PEG, are biodegradable that can be cleared out from the body after the application over time .
Finally, gold nanoparticles are widely used for chemical sensing such as surfaceenhanced Raman scattering (SERS), photo-thermal therapy, dark-field optical microscopy, and drug delivery applications due to its unique optical properties .
All of the above quantum dots, iron oxide, polymer-based nanoparticle, and gold nanoparticles can be synthesized in various sizes for biological applications. Not only each type of nanoparticle has unique chemical and physical characteristics but different sizes within each type of nanoparticle also results in unique property. Thus, size becomes an important factor for successful applications of these nanoparticles in cancer nanotechnology.
Size Effect on Tumor Targeting and Accumulation Solid tumors are characterized by defective vascular architecture and impaired lymphatic drainage/ recovery system that lead to enhanced permeability and retention (EPR) effect . The key mechanism to this phenomenon is the retention of the macromolecules (MW 40KDa, which is renal clearance cutoff) in solid tumors, where low-molecular weight particles are returned (instead of getting retained) back into the blood circulation by diffusion . Many nanoparticle systems take advantage of EPR effect to increase the bioavailability of the delivered agent for successful cancer detection and treatment.
Recently, Perrault and coworkers have tested wide size ranges of gold nanoparticles (10-100nm) and found that accumulation of 40−100 nm particles is exclusively dependent on blood half-life, whereas the accumulation of particles in the 20 nm range depends on size and half-life . They concluded that particles with hydrodynamic diameter of 60-100 nm with PEGylation (5 or 10kDa) would provide excellent candidate to utilized EPR effect for increased tumor accumulation. However, Perrault et al. found that larger particles (60-100nm) permeated less into the tumor and localized in the perivascular region. For 20nm particles, they permeated far from the vessel centers and may have cleared into the surrounding tissues that led to lower degree of accumulation.
Similarly, Dreher and coworkers used dextrans to study the tumor vascular permeability and accumulation of drug carriers . They found that tumor accumulation increased with larger molecular weight dextrans (40−70 kDa or 11.2−14.6 nm). They accounted poor accumulation of low molecular weight dextrans (3.3-10kDa) is due to the increased permeation of rate and clearance of dextrans into interstitial space.
Thus, EPR effect is very sensitive to size and appropriate size needs to be selected for the successful accumulation and permeation of nanoparticle in solid tumor.
Size Effect on Cellular Uptake Many studies in the past have demonstrated that size plays a critical role in cellular uptake of various nanoparticles such as liposomes, polymer nanoparticles, DNAcoated glycocluster nanoparticles, and inorganic nanostructures [38-42]. Compared to other sizes (range of 14-100 nm), Chithrani et al. found that nanoparticles with a diameter of approximately 50nm were taken up by cells at a higher concentration with a faster rate.
Particularly, Osaki et al. showed that 50nm quantum dot nanoparticles entered the cell more efficiently than smaller nanoparticles via receptor-mediated endocytosis.
Within the same size, additional factors such as surface charge can complicate the cellular uptake process of nanoparticles [38, 41-44]. Generally, cationic particles tend to bind to the cell surface more efficiently than the anionic particles due to electrostatic attraction to the negatively charged cellular membrane. Surface charge of the nanoparticle can be easily modified with various coatings. However, surface coatings with thiolated PEG (MW 1500) can increase the nanoparticle diameter up to ~6nm , which affects the overall size of the nanoparticle for cellular uptake.
Size Effect on Nanoparticle Toxicity A concern for nanomaterial toxicity arises as the physical and chemical properties of the material changes dramatically at a nanoscopic scale. Various sizes of nanoparticles, especially gold nanoparticles, have been frequently been in the focus of interest for testing nanotoxicity. It has been reported that gold nanoparticles or clusters as small as
1.4nm in diameter lead to unusual cytotoxicity, where 1.4nm particles strongly interact with the major grooves of the DNA . Similarly, Pan et al. found that 1.4 nm gold particles predominantly cause necrosis while 1.2nm gold particles mainly cause apoptosis in connective tissue fibroblast, epithelial cells, macrophage, and melanoma cells . For a given concentration, Pan et al. reported that 15nm gold nanoparticle did not show toxicity compared to 1.4 nm gold particles.
Connor et al. reports gold nanoparticles with 4nm, 12nm, and 18nm in diameters did not display toxicity towards human leukemia cell lines . Furthermore, Shukla et al. reported lysine and poly-lysine capped 35 ± 7 Å gold nanoparticle lacked toxicity towards macrophages but reactive oxygen and nitride species were observed .
Finally, 50nm gold nanoparticles were uptaken by HeLa cells without any toxicity .
Thus, for a given nanomaterial (gold in this case), size plays a critical role in exerting toxicity to cells.
Size Effect on Nanoparticle Biodistibution The unique physico-chemical properties of nanoscale particles results in an increased reactivity with the biological systems that it renders different effects in the system compared to the larger, bulk materials. It is important to know the distribution and the effects of absorbed nanoparticle in various organs after an exposure. Generally, nanoparticles with size less than 10nm get distributed throughout the system, whereas larger particles like ~60nm is mostly confined to the liver and spleen after intravenous injection . Furthermore, in detailed studies on various nanoparticle size and its distribution confirm that majority of nanoparticles accumulated in the “liver” and “spleen” regardless of size (1.9nm~250nm), shape (sphere or rod), type (carbon nanotube, quantum dots, iron oxide, gold nanoparticle), and dose of exposure (0.01~2700 mg/kg) after intravenous injection [18, 49-62].