«COLLOIDAL GOLD NANOPARTICLES FOR CANCER THERAPY: EFFECTS OF PARTICLE SIZE ON TREATMENT EFFICACY, TOXICOLOGY, AND BIODISTRIBUTION A Dissertation ...»
This visible cue also corresponds to the ICP-MS analysis of lymph nodes (Figure 5.3 (a)) where concentration of gold decreased over time in the lymph nodes. Interestingly, larger 60nm gold system did not display any pigmentation within the lymph node that it looked similar in hue compared to the Control lymph nodes. This is also evidenced in the TEM images of the lymph nodes (Figure 5.7) where only fewer number of particles were seen in the 60nm gold system images compared to 5nm gold system. We believe that large size of 60nm limited the lymphatic clearance of the gold system and resulted in decreased appearance in the lymph nodes. Also, TEM images demonstrate number of particles within the lymph nodes decreases over time for the 5nm gold system, whereas there seems to be an increase in the number of particles in the lymph nodes for 60nm gold system over time. Again, this is also consistent with the ICP-MS results in Figure 5.3 (b) where the concentration of 60nm gold system in the lymph nodes did not decrease over time in contrast to 5nm gold system. Accumulation of gold nanoparticle after intravenous injection has been reported in previous literature that high concentrations of 4, 13, and 100 nm gold nanoparticles were detected in the mesenteric lymph nodes .
Lymphatic Clearance of Gold Nanoparticle (a) Schematic drawing of general lymph node locations (b) Pictures of extracted raw lymph nodes for Control versus 5nm versus 60nm gold nanoparticle [Mouse drawing modified from http://www.informatics.
jax.org/greenbook/figures/figure13-4.shtml; D#=day#] Detection of gold nanoparticles in the lymph nodes could be due to translocation of gold nanoparticles from the lungs. It has been reported that lymphatic drainage plays an important role in the uptake of particulates in the respiratory system [222, 223]. The findings indicate that lung could serve as a reservoir for nanoparticles after acute exposure, and there is significant translocation of nanoparticles out of the lungs through lymphatic vessels and/or the bloodstream to other organs with time. Balasubramanian et al. reported that gold nanoparticle concentration in the lungs increased dramatically after an injection then there was a continual decrease of nanoparticle in the lungs with time after an initial peak . Thus, this could account for the higher accumulation of gold nanoparticles in the lymph nodes at earlier times. As the amount of gold in the lung decreases over time, the according gold nanoparticle being translocated from lung to lymph nodes also decrease over time.
In a typical lymphatic clearance, molecules leak out of the blood vessels into the interstitial space and are cleared via the lymphatic system [224, 225]. Thus, small-sized 5nm gold nanoparticles could have easily extravasated from the blood vessels and into the interstitial space to result in lymphatic clearance. Indeed, dark circles seen in the lymph node pictures (Figure 5.4) indicate high accumulation of extravasated 5nm gold nanoparticles. Compared to the 5nm, 60nm lymph nodes did not display any discoloration in the lymph nodes (Figure 5.4), even though same concentration of gold was injected for both 5nm and 60nm. This suggests that larger 60nm gold nanoparticle was unable to extravasate from the blood vessel as easily as the 5nm gold nanoparticles that less of them end up in the lymph node. Another reason for gold detection in the lymph nodes could be accounted from general lymphatic clearance from the interstitial spaces of various organs, including the RES.
(a) 5nm Lymph
TEM Images of (a) 5nm Versus (b) 60nm Gold Nanoparticles in lymph nodes
5.5 CONCLUSION Here, we showed the size-dependent biodistribution and clearance of colloidal gold nanoparticles that (1) increased circulation time for 5nm gold system (due to size and PEG) resulted in biodistribution of gold in various organs compared to 60nm gold system, (2) 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, and (3) size dependent clearance was observed where 5nm gold system gets cleared out via renal (urine) and hepatobiliary (feces) 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 60nm or larger nanoparticles.
6.1 ABSTRACT In this chapter, we summarize the important findings from previous chapters and significance of our work in cancer nanotechnology. We then examine the future applications of gold nanoparticles in drug delivery applications, specifically focusing on the size and its impact on biological applications.
6.2 SUMMARY This dissertation focused on the design and development of various sizes of colloidal gold nanoparticle drug delivery systems for cancer nanotherapy. In particular, two representative sizes of gold nanoparticles, 5nm and 60nm, were investigated for the size effect on therapeutic efficacy, toxicity, and biodistribution in cancer nanotherapy. In the first chapter, we described the general background on cancer and emphasized the role of nanotechnology, particularly focusing on the size effect for successful detection, diagnosis, and treatment of cancer. The complexity and heterogeneity nature of cancer makes it difficult to successfully diagnose and treat cancer, and nanotechnology plays a critical role in overcoming the obstacles in cancer biology. It is the size-scale of nanotechnology that provides a powerful tool to easily manipulate the complex cancer environment by distinctively size-tuning the nanomaterial to interact with biological molecules in tumor.
In the second chapter, we closely looked at one class of nanoparticles, namely colloidal gold nanoparticle, and its unique physical and chemical properties that are attractive for applications in cancer nanotechnology. Gold nanoparticles confer several advantages such as biocompatibility, size-tunability, and easy surface modification methods. Furthermore, due to its unique optical properties, multiple analytical chemistry methods such as UV-vis spectrophotometry, SERS, TEM, ICP-MS, darkfield microscopy, fluorescence can be used. Previously, gold nanoparticles have been mainly used for chemical sensing, photothermal therapy, and diagnostic purposes. The idea of using gold nanopartcle as a carrier for drug delivery is recent that further attention and study is required.
In the third chapter, we start off our drug delivery study with larger 60nm colloidal gold nanoparticle system. Here, we report development and characterization of multifunctional drug delivery system for simultaneously treatment and SERS spectroscopic detection of tumor. Doxorubicin, serving a dual function of chemotherapeutic agent and SERS reporter molecule, was chemically conjugated to gold nanoparticle via pH-sensitive hydrazone linker then PEG was added to develop multifunctional delivery system. Doxorubicin occupied maximum of 20% of total surface area of gold nanoparticle to result in colloidal stability. The multifunctional delivery system demonstrated pH-dependent drug release profile, therapeutic effect on tumor cells, along with in vitro spectroscopic detection based on SERS. SERS spectra were detected for non-aggregated gold system at near-infrared wavelength. Thus, the development of multifunctional drug delivery system raises exciting opportunities for simultaneous spectroscopic detection and therapy for tumors.
In the fourth chapter, we switched our focus to smaller-sized 5nm gold nanoparticle for drug delivery applications. Despite successful treatment and SERS spectroscopic detection in tumor, 60nm gold nanoparticle system resulted in a limitation of low drug-loading efficiency (0.1 wt-%). In order to test in vivo, our calculation results showed that high concentration of gold nanoparticles were needed for 60nm gold nanoparticle system (calculation not shown). Thus, in order to increase the drug loading efficiency and minimize the amount of gold injected in vivo, 5nm gold nanoparticle was selected for the study. Similar to 60nm gold system, 5nm gold nanoparticles were coated with doxorubicin which was modified with pH-sensitive hydrazone linker. Then the resulting gold system was coated with PEG to give colloidal stability and biocompatibility. 5nm gold nanoparticle drug delivery system resulted in a higher drug loading efficiency of 5.5 wt-%. However, as a trade off for having a higher drug loading efficiency, 5nm gold system no longer displayed SERS like the 60nm gold system due to its size. When tested in a tumor mouse model, 5nm gold drug delivery system resulted in therapeutic efficacy against tumor with no apparent systemic toxicity. In contrast, pure doxorubicin resulted in kidney, heart, and lung toxicity, along with insignificant therapeutic efficacy compared to other groups tested. The success of 5nm gold system resulted from (1) “high” accumulation at the tumor site compared to other non-tumor sites via EPR effect, (2) ideal spatial distribution and successful penetration at tumor site, and (3) slow, controlled release of drug via pH-sensitive linker to result in no apparent systemic toxicity. All of these factors owe to the small size scale of our 5nm gold system.
Finally, in the fifth chapter, we closely looked at the 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) that led to size-dependent biodistribution of gold nanoparticles. We showed that 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. We also demonstrated size dependent clearance where 5nm gold system was excreted via urine and feces, whereas 60nm gold was mostly retained in the spleen and liver after 6 months. Thus, 5nm gold system is a potential candidate for drug delivery applications where 5nm gold core displays inherently different biodistribution and clearance characteristics than 60nm or larger nanoparticles.
In summary, 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 biodistribution in the system.
6.3 FUTURE DIRECTIONS The work presented in this thesis mainly focuses on the two different systems to represent application of colloidal gold nanoparticle in cancer nanotherapy. While the drug delivery application and the in vivo fate of gold nanoparticles have been demonstrated conclusively, there is still a large amount of further developments needs to be made for optimization. In the following section, we suggest specific studies and future directions to be undertaken for futher improvement of our study.
Improvement of Drug Loading Efficiency Maximum drug loading is desired for high therapeutic efficacy for drug delivery system. Currently, drug loading is limited to the “surface” of the gold nanoparticle, thus somewhat limiting the drug loading efficiency. Drug loading can be further enhanced by increasing the surface area for drug-hydrazone linker conjugation site by adopting a different type of polymer coatings such dendrimers. Dendrimeric polymer provides multiple conjugation sites per polymer by branching characteristics. However, further study on toxicity of dendrimeric system, along with drug release profile has to be investigated.
In this study, 60nm gold nanoparticle system had a low drug loading efficiency, thus limiting its use for in vivo applications. Also, as seen from the biodistribution and clearance studies, larger 60nm gold system was retained in the RES and was not cleared over time. Thus, the 60nm gold system could be more optimal for ex vivo diagnostic purposes in a long run. The SERS feature of 60nm gold system makes it an attractive candidate for cancer diagnostics where antibody-targeted 60nm gold nanoparticle with SERS tag could be used to capture cancer cells from the blood drawn from patients.
Improvement of Therapeutic Efficacy Hydrazone bond is highly sensitive to pH changes that it is cleaved under mild acidic conditions of less than pH 5.5. The slow, delayed release of doxorubicin from gold nanoparticle-anticancer agent-PEG system is due to (1) the diffusion barrier created by PEG coating and (2) interactions between the polymer and the drug. To ensure rapid release of the bound drug, drug needs to be placed closer to the outer most layer of the system to decrease any diffusional barrier and accessibility of drug release triggering molecules. However, placing the drug near the outer most layer will limit the drug loading efficiency and change the property of the system compared to the current system.
On the other hand, the PEG layer can be decreased to smaller size (or lower molecular weight) to minimize any diffusional barrior. However, decreasing the protective PEG layer will compromise the colloidal stability of the gold nanoparticle. Alternatively, different linker system such as glutathione-activated prodrug system could be adopted.
Alternatively, assuming that the drug loading percentage is fixed, doxorubicin (our model drug for the study) can be replaced with more potent drug to increase the therapeutic efficacy of the system.
Finally, targeting ligand can be adopted to increase the therapeutic efficacy of the system. It has been reported that targeting ligands increase the internalization of drug carrier by the cancer cells at tumor site. This requires testing the optimal concentration of targeting ligands to be used, pharmacokinetics of the carrier, and toxicity exerted by the targeting ligand itself.