«COLLOIDAL GOLD NANOPARTICLES FOR CANCER THERAPY: EFFECTS OF PARTICLE SIZE ON TREATMENT EFFICACY, TOXICOLOGY, AND BIODISTRIBUTION A Dissertation ...»
Similarly, kidney and heart toxicities seen in Doxorubicin group are further complemented with the serum protein profile results. Kidney toxicity can be measured through total bilirubicin, creatinine, and LDH levels, whereas heart toxicty can be measured via creatine kinase and LDH. Creatinine (~0.2 mg/dl) and total bilirubicin (~0.15 mg/dl) levels for all experimental groups were similar, but increased LDH level in Doxorubicin group suggests kidney toxicity in doxorubicin treated mice. Moreover, increased creatine kinase and LDH levels in Doxorubicin group also suggests cardiotoxicity in doxorubicin-treated mice, as evidenced in histology sections.
We believe that the pharmacokinetics of the encapsulated doxorubicin influences the toxicity profile of such formulation of Au-DOX-PEG. It is the competition between the tumor accumulation rate and the drug release rate that it is preferable for the drug to be released after significant amount of drug carrier has accumulated at the tumor site to exert its therapeutic effect and minimize any side effects to normal, healthy cells.
No apparent toxicity from Au-DOX-PEG system comes from change in pharmacokinetics and biodistribution of doxorubicin that was linked via pH-sensitive linker to gold nanoparticle. One hypothesis is that for our Au-DOX-PEG system, the slow, controlled release of the drug (Figure 4.3) resulted in no apparent toxicity in vital organs. If you manipulate the drug release rate, you can reduce the toxicity at healthy organs and not interfere with therapeutic activity of Au-DOX-PEG. Other hypothesis is that due to increased vascular permeability at the tumor site , Au-DOX-PEG accumulates more rapidly with higher concentration at the tumor site compared to nontumor sites. Indeed, as seen in the ICP-MS results from Figure 4.8 (a), majority of gold was concentrated at the tumor. Less than 16% of total single injected gold went to spleen and liver, whereas less than 1.5% of the total single injected gold went to kidney, lung, and heart. Addition to the small amount of total gold accumulating in the vital organs, the ultra-structural differences between tumor and vital organs have resulted in no apparent toxicities at non-tumor sites. As mentioned earlier, increased permeability and retention effect at the tumor site resulted in a toxicity or therapeutic efficacy toward cancer cells for Au-DOX-PEG. Due to their leaky vasculatures, Au-DOX-PEG is easily extravasated to access cancer cells. In contrast, normal, healthy cells have tightly regulated blood vessels (smaller pore size cutoff of 2-6nm) that transport of nanoparticles are restricted and Au-DOX-PEG have less chance of accessing the healthy, normal cells .
Furthermore, combined with clearance of Au-DOX-PEG within the normal tissues due to functional lymphatic drainage (unlike tumor lymphatics) might have reduced the exposure of Au-DOX-PEG to the vital organs. Also seen in the TEM images (Figure 4.8 (d)), most of the Au-DOX-PEG in vital organs are contained within the blood vessels or macrophages outside the cells, unlike the tumor.
Finally, there also has been a controversy for in vivo toxicity exerted by differently sized gold nanoparticles themselves. Indeed, it is hard to conclude what is the exact size of the gold nanoparticle to result in toxicity. Toxicity not only depends on the size of the nanoparticle but also depends on the surface-ligand chemistry, charge, shape, chemical composition of the particle itself, and the route of administration. The in vivo study done by Chen, Y et al. indicates no inherent toxicity exerted by the naked 5nm gold nanoparticles . On the other hand, 13nm PEGlyated gold nanoparticle induced acute inflammation and toxicity to liver . Similarly, Terentyuk G. et al. also reports toxicity exerted by 15nm PEGylated gold nanoparticle in rabbit organs after intravenous injection . They found that 15nm PEGylated gold nanoparticle resulted in hemodynamic disorders (congestion of the blood) in liver and spleen, along with thickening of kidney basal membrane. Niidome et al. have shown that the toxic potential is triggered by the surface modification of the gold nanoparticles. In fact, bromidestabilized gold nanorods induced severe cytotoxicity in HeLa cells, whereas PEGmodified gold particles, which displayed a neutral surface, could only induce moderate toxicity . Nevertheless, Connor et al. demonstrated that neither the surface characteristics nor the size of gold nanoparticles seemed to play a role in inducing cytotoxicity in the human leukemic cell line K562 . Connor et al. stated that 4, 12, 18nm gold nanoparticles with various surface modifiers were not inherently toxic to human cells, despite being taken up by the cells. Similarly, Shukla et al. found that 3.5nm gold nanoparticles lacked toxicity towards macrophages but reactive oxygen and nitride species were observed . For extremely small-sized gold nanoparticles or gold clusters (less than 2nm in diameter), Pan et al. and Tsoli et al. stated that they are toxic to cells [45, 46]. As far as we are concerned, our 5nm core Au-DOX-PEG gold nanoparticle did not display any apparent toxicity to the vital organs when administered in high dosage via tail-vein. This is evident in the MTT assay, serum anlaysis, and histology results in Figure 4.4 and Figure 4.8.
4.5 CONCLUSION We took a “tumor activated prodrug therapy” approach, where drug bound to the gold surface remains inactive until it reaches the acidic tumor site or intracellular environment, where change in pH triggers the release of doxorubicin via cleavage of hydrazone bond. We took doxorubicin as a model drug to test the feasibility of using small-sized 5nm gold nanoparticle for drug delivery applications. The water-soluble AuDOX-PEG resulted in similar toxicity to cancer cells as pure doxorubicin in vitro.
However, when tested in vivo, high concentration of Au-DOX-PEG accumulated at the tumor site via EPR effect to result in therapeutic efficacy. Unlike the pure doxorubicin, Au-DOX-PEG did not result in any apparent toxicity to vital organs.
The success of Au-DOX-PEG system resulted from (1) “high” accumulation at the tumor site compared to other non-tumor sites, (2) ideal spatial distribution and successful penetration at tumor site (i.e. Au-DOX-PEG were present both inside and outside the cancer cells), and (3) slow, controlled release of drug via pH-sensitive linker (i.e. state of the drug), all owing to the small size scale of the system. The small size of the system, along with PEGylation, gave prolonged blood circulation time to result in high accumulation of at the tumor site. Also, the small size scale allowed Au-DOX-PEG to easily extravasate into the tumor environment to result in therapeutic efficacy. The slow, controlled release of drug and high accumulation at the tumor site resulted in no apparent toxicity at vital organs, whereas pure doxorubicin displayed heart, kidney, and lung toxicity. Thus, our results demonstrated that functionalized 5nm gold nanoparticlebased drug delivery system represents a highly attractive candidate as a potential drug delivery carrier for cancer nanotherapy.
5.1 ABSTRACT Here, we closely looked at the size-dependent biodistribution and clearance of both 5nm and 60nm gold nanoparticle systems. In addition to therapeutic efficacy of colloidal gold system, it is important to study the long-term clearance and the fate of the delivered colloidal gold system for in vivo applications. Compare to the short blood circulation half time (9 hours) for 60nm gold system, 5nm gold system resulted in a longer circulation half time (1.6 days). Larger 60nm gold nanoparticles were mostly uptaken in the liver and the spleen, whereas smaller sized 5nm gold nanoparticle was visible in the various organs in the system, especially resulting in pigmentation in the skin and the lymph nodes. Size dependent clearance was observed that 5nm gold system was excreted via renal and hepatobiliary pathways, whereas 60nm gold was mostly retained in the spleen and liver after 6 months. Thus, 5nm gold system is a potential candidate for biomedical applications, where 5nm gold core displays inherently different biodistribution and clearance characteristics than larger nanoparticles.
5.2 INTRODUCTION 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. Moreover, it is important for the particle to be delivered to the desired, targeted site, such as tumor for drug delivery applications, and “size” plays a critical role.
Generally, when the system is injected with the nanoparticle, it is uptaken by the reticulo-endothelial System (RES) such as spleen or liver . The surface modification of the nanoparticle can change the dynamics of nanoparticle circulation time that various coating techniques can be adopted to prolong the circulation time of the nanoparticle in the blood. In particular, coating the surface of the nanoparticle with hydrophilic polymer such as poly(ethylene glycol) or PEGylation can render “stealth” characteristics to the nanoparticles, thus resulting prolonged circulation time [169, 201]. Moreover, the circulation time of this the nanoparticle not only depends on the coating but also depends on the core size of the nanoparticle. Consequently, the circulation time of the nanoparticle will affect the distribution and efficacy of the nanoparticle within the system, especially for intravenously injected nanoparticles.
Currently, several studies have been reported on the size-dependent biodistribution and clearance of gold nanoparticles. There is a correlation between size and biodistribution of nanoparticles. Generally, nanoparticles with size less than 10nm gets 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, 49-51, 53, 56, 57, 59-62, 199, 202-204]. Thus, if all the particles accumulate and distribute in a similar manner despite of size, shape, and dose, then “clearance” plays a critical role that determines the success of the nanoparticle for in vivo applications.
After delivering the therapeutic or imaging agent with a nanocarrier to the target site, it is desirable to see the delivery vehicle to clear out from the body, minimizing any harm to the healthy, normal cells. It has been reported that larger particles such as 20nm gold nanoparticle is minimally excreted through feces and urine that there is significant and persistent accumulation of gold nanoparticle in the liver and spleen through intravenous exposure . Similarly, metal-based 13nm quantum dot showed accumulation in kidney but there was no urinary excretion up to 28 days after the injection in mice . Also, 40nm gold nanoparticle was removed from the circulation primarily by Kupffer cells in the liver and remained as clusters even after six months . In contrast, particles with less than ~6nm in diameter displayed clearance from the system. For example, 77% of the injected 1.9nm gold nanoparticle was rapidly cleared through the kidney and excreted 5 hours after the intravenous injection in the mouse .
Similarly, Choi et al showed rapid clearance of zwitterionic coated quantum dots (4.36nm) through kidney and urinary excretion within 4 hours after the intravenous injection. Based on these findings, we believe that nanoparticle “size” plays a critical role for not only delivering the drug delivery system to the target site but also determining the in vivo behavior such as clearance and distribution of the nanoparticle throughout the body . However, there is no golden standard for the optimal size to be used in cancer nanotechnology and it is still debatable.
In this study, we look closely at the special properties inherent to 5nm gold nanoparticle, compared to the larger 60nm gold nanoparticle. It was found that 5nm gold nanoparticle distributed throughout the system, slowly clearing out of the system with time via urine and feces. Furthermore, it was found that lymphatic system was involved in the clearance of 5nm gold nanoparticle. In contrast, the larger 60nm gold nanoparticle system steadily accumulated in the spleen and liver at a greater amount than the 5nm system over time and showed decreased clearance from the system in long term. Thus, we hypothesize that it is the “nano-size scale”, under the renal and urinary excretion threshold, of the polymer-gold system that is responsible for clearance of polymer-gold system over time. This study will provide insight on specific size of nanoparticle that could be applicable for other nanoparticle delivery systems.
5.3 MATERIALS AND METHODS Materials Chemical Reagents Citrate-stabilized gold colloids, 5 nm and 60nm in size, were obtained from Ted Pella, Inc. (Redding, CA). Poly(ethylene glycol) (CH3O-PEG-SH) of molecular weight 5000 was purchased from Rapp Polymere (Germany). Mili-Q deionized water (Millipore, 18.2 MΩ cm-1) was used throughout the experiments. All of the products were used without modification or purification unless as noted.
Instrumentation Nanoparticle surface charge (zeta potential) and size were measured by ZetaSizer Nano-ZS90 (Malvern Instrument). Adsorption spectra were obtained through ultravioletvisible spectrophotometer (Beckman Coulter DU530). Gold content was analyzed by ICP-MS (HP 4500, Agilent Technologies). TEM were taken by using Hitachi H7500 high-magnification electron microscope.
Mouse Model 5-6 weeks old CrTac: NCr-Foxn1nu male nude mice were obtained from a commercial vendor (Taconic). The protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Emory University.
Statistical Analysis Statistical analysis was performed using one-way ANOVA followed by multiple comparison Bonferroni’s test. Data were collected from at least three different animals and P0.05 was considered statistically significant.