«COLLOIDAL GOLD NANOPARTICLES FOR CANCER THERAPY: EFFECTS OF PARTICLE SIZE ON TREATMENT EFFICACY, TOXICOLOGY, AND BIODISTRIBUTION A Dissertation ...»
Improvement of Biodistribution, Toxicity, and Clearance Studies As seen with our studies, smaller-sized nanoparticles accumulate at various organs in the system. Thus, wide and even spread of smaller-sized gold nanoparticle in the sytem requires further investigations in other parts (rather than focusing only on the vital organs) of the system. Also, to strenghthen the toxicity studies, changes in gene expressions in various organs can be studied, along with in depth serum protein analysis for organ toxicity.
Different sizes, rather than 5nm and 60nm, of gold nanoparticles needs to be tested for further generalization of the biodistribution and clearance of nanoparticles.
Even smaller particles (like 2-3nm) and particles in between 5nm and 60nm can be used to study the different patterns in biodistribution and clearance. Additionally, different coating can be adopted to further strengthen the study. Different coatings on gold surface render different charge, shape, colloidal stability, and size. Thus, wide ranges of coatings such as multidentate polymer can be tested. Furthermore, different dosages of gold system can be used to test the therapeutic efficacy, biodistribution, and clearance.
For clearance study, urine and feces needs to be collected cumulatively. Due to limited resources, our study was conducted by collecting urine and feces at each specific time point. By collecting urine and feces cumulatively, we can accurately calculate the amount excreted after the intravenous injection.
Finally, alternative methods other than ICP-MS can be used to further verify the gold quantification results. Analysis of collected organs, feces, urine, and blood through ICP-MS is an extremely costly and time-consuming process that it requires extensive drying and acid digestion of the samples. Other analytical chemistry methods such as atomic emission spectroscopy could be utilized.
Future of Gold Nanoparticles in Drug Delivery Applications Nanotechnology plays a critical role in treatment of cancer. Currently, there are many ongoing clinical trials for various types of nanoparticles for cancer treatment. In particular, Doxil® and Abraxane® are one of few FDA approved nanoparticles for cancer therapy.
Doxil®, PEGylated liposomal doxorubicin, is widely used to treat ovarian cancer or multiple myeloma . Doxil® has been approved in the United States since 1995, and there are over 1219 clinical trials related to Doxil® alone [227, 228]. Doxil® is formulated by encapsulating anthracycline (i.e. doxorubicin hydrochloride) drug in liposomes with surface-bound methoxy-polyethylene glycol to result in mean size of 80nm and doxorubicin concentration of 2 mg/mL . Due to its unique pharmacokinetic properties such as long circulation time (i.e. elimination half-life of 20hours), restricted volume of distribution (i.e. close to blood volume), and stable retention of anthracycline inside the liposome during circulation, Doxil® has shown 6fold enhancement in antitumor activity compared to free doxorubicin with reduced cardiac toxicity . However, Doxil® induces dose-limiting toxicities such as asthenia, fatigue, fever, anorexia, nausea, vomiting, stomatitis (i.e. painful sores in mouth), diarrhea, constipation, hand-foot syndrome, rash, neutropenia (i.e. low white blood cell count), thrombocytopenia, and anemia . In particular, skin represents the major liposome accumulating site for long-circulating Doxil® that results in bothersome redness, flaking, swelling, and burning sensation on palms of hands or soles of feet.
Abraxane® was also approved by FDA in 2005, which overcomes the limitations of paclitaxel by formulating albumin-bound form of paclitaxel with a mean particle size of ~130nm . In general, paclitaxel is highly hydrophobic and conventionally delivered via Cremphor EL® and ethanol, which leads to hypersensitivity reactions, severe neuropoenia, and peripheral neuropathy in patients during therapy . When injected, this 130nm albumin-paclitaxel complex quickly dissolves into smaller endogenous albumin-sized (~10nm) complexes for effective accumulation at tumor sites.
Several clinical trials show that Abraxane® improves the solubility of paclitaxel and also improves the toxicity profile of conventional paclitaxel therapy with Cremphor EL® with greater anti-tumor activity . A Phase III clinical trial with metastatic breast cancer patients resulted in higher response rate, significantly lower rate of grade 4 neutropenia, but higher rates of grade 3 sensory neuropathy .
As seen above, despite advances made in toxic effects and therapeutic efficacy for cancer therapy, cancer patients still suffer from long-term adverse effects of the drug and cancer mortality rate is still unacceptably high. Nanotechnology still faces challenges in the drug delivery area for further improvement and solutions to resolve these issues. To pinpoint, the biggest problems in current drug delivery are 1) presence of systemic toxicity imposed by nanoparticles due to changed pharmacokinetics and 2) drug stabilization and increased accumulation at target site for improved drug efficacy.
New formulations introduced for particular chemotherapeutic agent can reduce or eliminate the toxicity native to that drug, but it can also introduce unexpected, new toxicities to the system. For example, prolonged circulation time of Doxil® resulted in a high accumulation of liposomes in the skin. Due to small size, pure doxorubicin gets cleared out from the system within few minutes. However, change in pharmacokinetics of the doxorubicin by increasing the delivery vehicle size and PEGylation allowed retention of the drug in the blood for longer periods of time and exposed other organs, such as skin, to result in toxicity. Longer circulation time does lead to increased accumulation of the drug at the target site, but at the same time there is a risk of exposing other, unrelated organs for potential toxicity.
In addition to high accumulation of drug at the target site, efficacy of the drug is determined by the stability of drug during circulation and appropriate release/delivery of drug at the tumor site. In other words, drug needs to be stable during circulation and get properly released at the desired target site. For both Doxil® and Abraxane®, active drug is loosely bound, not covalently linked, to the delivery vehicle that there is a chance for leakage of parent drug during circulation. Leakage of drug not only reduces the efficacy of the drug delivery system but also increases the risk for potential toxicity in the system.
Nanoparticle needs to be appropriately formulated so that drug is retained until it reaches its target site, and gets released in a controlled manner. Thus, there should be a balance between systemic toxicity and efficacy of the drug to optimally treat the disease.
Similarly, our gold nanoparticle drug delivery system also faced the same issues dealt above. Due to its stealth PEG layer and the size, our gold nanoparticle drug delivery system circulated in the blood for long period of time (~1.6 days), which led to wide distribution of the drug delivery system in non-tumor organs. Similar to Doxil®, we observed increased accumulation at the skin but no skin toxicity. Surprisingly, we observed non-toxic skin pigmentation, which faded with time. Unlike Doxil® and Abraxane®, our parent drug doxorubicin was covalently bound to the gold nanoparticle surface for stability during blood circulation. Due to smart, pH-activated drug release mechanism, delivery system was observed to release drug in acidic environment such as tumor stroma. Despite the accumulation at non-tumor sites, no apparent toxicity was observed for the given experimental period. Extremely high concentration of gold drug delivery system accumulation and retention at tumor site, relative to non-tumor site, outweighs the potential toxicity exerted on non-tumor sites. Furthermore, gold drug delivery system seems to be present mostly outside the cells in non-tumor sites. No apparent toxicity present in non-tumor site is mostly due to minimal or slow release of hydrazone-bound drug from the gold surface in non-acidic environment.
Overall, our gold drug delivery system provided new insight on 5nm gold system that led to “increased” tumor accumulation due to changed pharmacokinetics (i.e.
covalently bound doxorubicin was well-camouflaged or protected by PEG layer that stayed intact throughout the half-life circulation time of ~1.6 days) for “improved” therapeutic efficacy and overall “reduction” of side effects. Furthermore, small-sized gold drug system was slowly “cleared” out from the body over time after cessation of treatment. We believe that 5nm gold drug delivery system is an ideal candidate for future drug delivery application only when intricate balance amongst nanoparticle formulation, dosage, dosing schedule, and nanoparticle pharmacokinetics are met, as seen in our study.
Due to high surface-to-volume ratio, 5nm core allows increased drug loading efficiency.
Furthermore, 5nm core is right below the renal clearance threshold and above 2nm, which has been reported to result in cellular toxicity. The only issue with 5nm drug delivery was skin pigmentation, which seems to be mostly cosmetic issue. This can be resolved by adopting different dosage, dosing schedule, or different types of potent drugs.
Nanotechnology allows different delivery strategies in cancer therapy that balance between toxicity and efficacy of the drug system is crucial. Furthermore, combining effective dosing schedule for improved efficacy of the drug and safety profile is essential.
Nanotechnology offers new opportunities for currently available chemotherapeutic agents by 1) increasing solubility of drug, 2) increasing maximum allowed dosage, 3) decreasing toxicity of the native drug (but not complete elimination of toxicity related to the original drug and sometimes occurrence of unexpected, new toxicity), 4) allowing various delivery strategies by various formulation methods (i.e. biodegradable materials, liquidto-gel transitioning material, thermal ablation, etc.), 5) allowing controlled release of the drug, 6) allowing different methods of metabolism to bypass certain metabolic pathway, and 7) allowing different methods of administration (i.e. intravenous, intraperitoneal, local tumor administration).
Thus, future opportunities with gold nanoparticles in drug delivery, particularly with 5nm, are positive and limitless. As an ideal drug delivery vehicle, gold nanoparticle selectively delivers various kinds of drugs in a discrete quantity at specific time intervals, while minimizing systemic toxicity.
6.4 CONCLUSION In conclusion, it is the “size” that affects the behavior and fate of the nanoparticle in biological system. By choosing the appropriate size for the system, we were able to successfully demonstrate the use of gold nanoparticles in drug delivery applications, along with desirable clearance from the biological system.
This work is significant by providing an insight on a potential ideal candidate for gold nanoparticle-based drug delivery system that uses small (5nm) gold nanoparticle to study therapeutic efficacy on solid tumor and in vivo clearance and biodistribution. To our knowledge, we are the first team to investigate in detail for 5nm gold nanoparticle drug delivery system in vivo and its complete behavior for better understanding of the gold nanoparticle-based drug delivery. The findings from this study will have implications in the chemical design of nanostructures for biomedical applications.
UV-vis spectroscopy can be used to characterize the coatings on the gold nanoparticle. For 5nm gold nanoparticle, it displays maximum absorption peak around 514nm for raw, non-aggregated 5nm particle. To ensure that bound doxorubicin or PEG on gold surface does not affect the colloidal stability, absorption spectra were measured for gold-drug (Au-DOX) and gold-drug-PEG (Au-DOX-PEG) in water (Figure A.1).
Furthermore, to prevent formation of aggregates in various biological mediums such as salt, PEGylated drug-gold nanoparticle (Au-DOX-PEG) was incubated in 0.5M salt solution (reflecting the biological salt concentration). As seen in Figure A.1, Au-DOXPEG was stable in salt solutions when tested up to 1 week, as indicated by smooth tail in the red wavelength region of the spectrum measured by UV-vis spectroscopy. In contrast, when raw 5nm gold nanoparticle was incubated in 0.5M salt solution, broadening of spectra was observed, along with the red shift in the maximum absorption peak. This indicates formation of aggregates of gold nanoparticles, as black precipitates were seen in the solution.
Additionally, when Au-DOX only was incubated in 10% serum solution (not shown here), dark precipitates (gold nanoparticle aggregates) were seen within few hours, whereas Au-DOX-PEG was stable (no precipitates seen) in 10% serum solution when observed up to 4 days at 37°C.
Figure A.1. Characterizaion of 5nm Gold Drug Delivery System (Au-DOX-PEG) with UV-vis Spectrometer (spectra were scaled for better visualization).
NANOPARTICLES IN VARIOUS ORGANS AFTER TREATMENTDue to its unique optical property, colloidal gold nanoparticles can be imaged via darkfield microscopy. After mouse was treated with Au-DOX-PEG, various organs were harvested and embedded in paraffin for thin sections. The images obtained in Figure B.1 are unstained that orange dots spotted in various organs are solely from the deposited gold itself.
(a) (b) (c) (d) (e) Figure B.1. Darkfield Imaging of Liver, Spleen, Kidney, Lung, and Heart after 16 Days of 5nm Gold Nanoparticle Drug Delivery System Au-DOX-PEG Treatment (red arrows indicate dark grey spots which represent gold deposited within the tissue)
1. Lopez AD, Mathers CD, Ezzati M, Jamison DT, Murray CJ. Global and regional burden of disease and risk factors, 2001: systematic analysis of population health data. Lancet 2006 May 27;367(9524):1747-1757.
2. Cancer Statistics 2010. American Cancer Society, Inc.
3. Cai W, Chen X. Nanoplatforms for Targeted Molecular Imaging in Living Subjects. Small 2007;3(11):1840-1854.
4. Gao XH, Cui YY, Levenson RM, Chung LWK, Nie SM. In vivo cancer targeting and imaging with semiconductor quantum dots. Nature Biotechnology 2004 Aug;22(8):969-976.