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«COLLOIDAL GOLD NANOPARTICLES FOR CANCER THERAPY: EFFECTS OF PARTICLE SIZE ON TREATMENT EFFICACY, TOXICOLOGY, AND BIODISTRIBUTION A Dissertation ...»

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PEGylation of colloidal gold Methoxy thiol-PEG (MW 5000) solution was added drop-wise to the colloidal gold solution. PEG solution was added accordingly to correspond to a complete coverage for PEG monolayer on gold particle surface. Simple geometric calculations showed that each thiol-PEG occupied a footprint area of 0.35 nm2 on the gold surface, consistent with the literature data reported for brush conformation of thiol-PEG. Excess of 10-20 fold PEG solution was added to the colloidal gold to ensure stability of gold colloids against aggregation under various in vivo conditions. The resulting 5nm and 60nm gold-PEG solutions were centrifuged to remove any unbound thiol-PEG. Then UV-vis spectrometer, DLS, and zeta potential were used to characterize the system.

Blood, urine, and feces collection Blood was drawn from the cheeks of the mice at different time points (1, 5, 10, 30 minutes; 1, 2, 4, 8, 20, 30 hours; 2, 3,4,5,6,7…25 days). Blood was weighed then further digested in nitric acid for ICP-MS analysis. Similarly, urine and feces were collected at different time points (30 minutes; 2.5, 4, 7.5 hours; 1,2,3,4….25 days). Note that urine and feces were collected at specific time points and was not cumulative. Collected urine and feces were weighed then further digested in nitric acid for ICP-MS analysis.

In vivo study of biodistribution and clearance Due to the nature of long term clearance and biodistribution study, non-tumor bearing nude mouse will be used with PEGylated 5nm and 60nm gold nanoparticles.

Mice were divided (n=4) randomly into three groups of control, 5nm, and 60nm groups.

Equal concentration of gold (0.04g/kg of body weight) was injected once for both 5nm and 60nm groups via tail vein. Mice were housed in sterile cages and any abnormal changes in body weight and behavior were observed every day. After the single injection, critical time points such as 1, 3, 10, 20, 35, 60, 90, and 180 day were chosen for harvesting organs (heart, kidney, liver, lung, brain, spleen, and skin) to quantify any gold concentration changes over time. Concurrently, pictures will be taken to document any skin and lymph node color changes over time. Presence of gold in various organs was verified by ICP-MS and TEM.

5.4 RESULTS AND DISCUSSION PEGylation of Gold Nanoparticle and Blood Circulation Time For biocompatibility and colloidal stability, 5nm and 60nm gold nanoparticles were coated with thiolated methoxy-PEG (MW 5000). The resulting gold nanoparticle had an average hydrodynamic size (diameter) of 18.2 ± 0.9 nm with a surface charge (zeta potential) of -5.04 ± 0.6 mV for 5nm gold nanoparticle system, whereas 60nm gold nanoparticle system had 78.5 ± 1.2 nm for average hydrodynamic diameter and -14.5 ±

1.4 mV for zeta potential. The resulting gold nanoparticle was injected once through the mouse tail vein to study the pharmacokinetics and its behavior.

After a single tail vein injection, blood samples were collected for 5nm gold nanoparticle system at various time points (between 0 and 25 days) from the mouse cheek, and ICP-MS was used to measure the gold concentration in blood. Using a simple mono-exponential decay model, the experimental data were fitted to result in a half decay time (t1/2) of ~1.6 days for the PEGylated gold nanoparticle. This is consistent with the previous literature reports where small-sized gold nanoparticles (1.4 nm and 15nm in diameter) resulted in high blood concentrations even after 24 hours after the intravenous injection [60, 205].

It has been reported that surface modification with long-chain PEG (with molecular weight of ≥2000 Da) significantly reduces protein adsorption on a surface [169], which in turn increases the circulation time of the nanoparticle in blood. The socalled “non-fouling” or protein resistant surface is controlled by two principles of 1) terminal hydrophilicity of the head group combined with 2) formation of a dense but disordered PEG brush with significant penetration of water into the PEG layer [171]. As seen in the TEM image from Figure 5.1, it is this thick and high surface density of PEG layer on the gold surface that resulted in a long blood circulation time. It has been reported that smaller gold nanoparticle (10nm) resulted in a higher surface density of the adsorbed single-stranded DNA compared to the larger gold nanoparticle (50nm) by 13 times [206]. Thus, it is the nature of the small 5nm gold nanoparticle core that resulted in dense “brush” configuration layer of PEG to minimize opsonization (adsorption of blood protein). Additionally, it is the non-targeted nature of the particle that resulted in a longer circulation time. It has been reported that targeted ligand exposed on the surface can accelerate the opsonization process [196, 207]. Finally, high concentration of small gold nanoparticle in the blood stream have possibly saturated the reticuloendothelial system (RES) and retarded the uptake by the RES. In comparison, larger gold nanoparticle of 60nm core that has been PEGylated (MW 5000) resulted in a half decay time of 9 hours (not shown). Thus, our 5nm core gold nanoparticle system resulted in ~4 times increase in half decay time compared to 60nm, having more advantage for the EPR effect for tumor accumulation.

Figure 5.1.

Blood Circulation Half Life of 5nm Gold Nanoparticle (~1.6 days) Biodistribution of Gold Nanoparticle in Skin and Pigmentation Skin is the largest organ in the body. Here, skin is used as a semi-quantitative assessment measure that is related to the gold concentration inside the body. It was found that internal gold concentration was related to the degree of visible skin pigmentation.





The skin pigmentation qualitatively measures and reflects the gold distribution and clearance within the system that has been injected with 5nm gold nanoparticle.

The deposition of cutaneous gold occurred in the reticular and papillary dermis in the absence of inflammatory change. Most of the gold nanoparticles were confined in the dermal macrophage, inside a lysosome, in an aggregated form as seen Figure 5.2.

The following skin pigmentation was due to presence of 5nm gold nanoparticle and was not a melanin-induced pigmentation. The mice used for this experiment were derived from an albino mouse (i.e. lacking melanin pigmentation) and further examination of epidermis under TEM confirmed the absence of melanin granules in the mouse skin. Thus, this reversible skin pigmentation was solely due to injection of 5nm gold nanoparticle. We believe that reversible characteristic (i.e skin darkening then lightening) is due to the clearance of small-sized 5nm gold nanoparticle over time (as seen in next section). Interestingly, the skin pigmentation was also size dependent. When equal amount of gold concentration of 60nm gold nanoparticle was injected, no skin pigmentation was present in the 60nm gold injected mouse (Figure 5.2).

For humans, it has been reported that high concentration of crystalline and amorphous gold “salt” (not nanoparticle) ingestion leads to an “irreversible” skin pigmentation called “chrysiasis”. Similar to the mouse skin pigmentation seen in our experiment, chrysiasis is characterized by the grayish-blue pigmentation of the skin but is “irreversible”. Also, unlike our mouse skin pigmentation, chrysiasis skin pigmentation preferentially occurs in the areas of sun exposure, where metal deposits stimulate melanin production [208].

To our knowledge, we are the first team to report the mouse skin pigmentation which qualitatively correlates to internal biodistribution and clearance of gold nanoparticle over time. Sonavane et al. reported that in vitro experiments revealed sizedependent gold nanoparticle accumulation in rat skin [209]. Similar to our results, Sonavane et al. reported that compared to the larger size gold nanoparticles, small-sized gold nanoparticle (15nm) displayed higher permeation and accumulation in the dermis and epidermis of the rat skin. Furthermore, Semmler-Behnke et al. briefly mentions high accumulation of 1.4 nm gold nanoparticle in the subcutaneous fatty tissue, which could not be explained. However, both papers mentioned gold nanoparticle accumulation in the skin, but not skin pigmentation [205].

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Figure 5.2.

Gold Nanoparticle Induced Skin Pigmentation and Qualitative Monitoring of Distribution of 5nm versus 60nm Gold Nanoparticle (a) Skin Color Change versus Time Picture (in order of Control vs. 5nm vs. 60nm) (b) 6 month multi-view of Control vs.

5nm vs. 60nm (c) TEM images of 5nm gold nanoparticle inside the skin around the head area for 3 day and 6 months (d) TEM images of 60nm gold nanoparticle inside the skin around the head area for 10 day and 6 months Despite same concentration of gold was injected for both 5nm and 60nm, the mouse injected with 60nm gold lack skin pigmentation due to its large size. As seen in Figure 5.2, the TEM images reveal that only few number of particles actually reach the dermis of the skin. Due to its large size, 60nm is not readily diffused to the dermis like the smallsized 5nm gold. Moreover, for both 5nm and 60nm, gold nanoparticles were only visible within the dermis layer, which is in proximity to the blood vessel. Based on the observation via TEM, deeper layers of dermis (closer to blood vessel and muscle layer), you see more gold. Gold nanoparticles were confined in an irregular-shaped cells, suspected to be a type of a macrophage, inside a lysosome.

Also, gold distributes in different patterns within skin over time for 5nm gold nanoparticle system. At the beginning, the gold spreads out evenly throughout the body, giving an even pigmentation in the skin. Then as the gold gets cleared out from the body, gold tend to concentrate more around the head and the buttocks area, and the dark pigmentation line appeared on the sides of the mice. The ICP-MS data in Figure 5.3 was measured by taking three points in the body (head, torso, and buttocks), then averaged the ICP-MS values for each time point.

Biodistribution of 5nm Gold Nanoparticle versus 60nm Gold Nanoparticle in Various Organs There was a size-dependent biodistribution of gold nanoparticles within the system (Figure 5.3). In general, smaller 5nm gold system resulted in a wide spread of gold in various organs with total gold mass being liver spleen kidney lung heart, skin, lymph brain at day 180. Although, the spleen exhibited higher capacities per gram of tissue (ng/g or ppb) for gold nanoparticle uptake, the liver took up the majority of the nanoparticle due to its larger mass. The amount of 5nm gold system in the liver did not decrease significantly until day 35. After day 35, significant amount of 5nm gold system was cleared out from the liver. The gold in the liver decreased from 263.66 ± 66.14 ng/g at day 35 to 142.43 ± 19.71 ng/g (P0.05) at day 90 and further decreased to 53.39 ng/g at day 180 (n=1 for day 180). For spleen, significant decrease in gold was also observed after day 35. The gold in the spleen decreased from 832.99 ± 138.57 ng/g at day 35 to

509.43 ± 27.57 ng/g (P0.05) at day 90 and further reduced to 291.8127 ng/g at day 180 (n=1 for day 180). Interestingly, the amount of total gold in the lymph also decreased significantly after day 35 that gold decreased from 494 ± 33.91 ng/g at day 35 to 228.36 ± 25.2 ng/g at day 90 (P0.05) and further reduced to 48.13 ng/g at day 180 (n=1 for day 180). For heart, kidney, lung, and skin, there was more gradual decrease in gold over time. Small amount of 5nm gold system was detected in the brain but it was less than 0.1% of the total injected dose. Approximately 44% of the total injected dose was uptaken by the liver and spleen at 3 day (36% in the liver and 8% in the spleen). After 180 days, approximately 15% of the total injected gold was detected in the liver and the spleen (10% in liver and 5% in spleen). Thus, the long-tern study in a naïve, non-tumor bearing mice study indicated that the majority of 5nm gold system accumulated in the liver and spleen, and it gradually cleared out over time at day 180. Unlike the 60nm gold system, the unexpected bell-shaped biodistribution (Figure 5.3 (a) histogram for lymph, spleen, liver, and skin) of 5nm gold system would most likely resulted from the high concentration (0.04g Au/kg of body weight) of gold injected to the mouse. As reported by literature, high concentrations (0.85mg/kg of body weight) of small-sized 4nm gold were detected in the blood up to7 days and continuted to detect gold in the blood up to one month [210]. Similarly, our 5nm gold system resulted in a prolonged blood half-time (1.6 days) that most likely the high concentrations of 5nm gold system did not get fully uptaken by the various organs up to ~35 days from circulation. The animals remained healthy for the entire duration of the study (up to 180 days) and no observable signs of weight loss, behavioral changes, and toxicity were detected.

(a)

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Figure 5.3.

Biodistribution of (a) 5nm versus (b) 60nm Gold Nanoparticles in Various Organs up to 180 days (dashline: P0.05) 60nm gold system also displayed similar biodistribution as 5nm gold system with highest uptake being the liver and the spleen. Overall, unlike the 5nm gold system, majority of gold was present in the liver and the spleen and much less so in other evaluated organs. Initially, approximately 60% of the total injected gold was uptaken by the liver and spleen (40% liver and 19 % spleen) at day 3. After 180 days of initial injection, there was no observable change in the amount of gold in the spleen (~23% of total injected gold). However, there was a slight decrease in the amount of 60nm gold system in the liver that the total injected gold decreased to ~37% after 180 days. The slight decrease in the gold concentration in the liver suggests hepatobiliary clearance of gold in the liver over time. Similar trend was seen for other organs (i.e. lymph, kidney, skin, heart, and lung) that there were no significant changes in the gold concentration over the span of 6 months. Consistent with the literature results, no gold was found in the brain for 60nm (but for 5nm gold system), which reflects the tight restriction of blood brain barrier for the passage of nanoparticles. It has been reported that particles smaller than 20nm in diameter results in translocation of nanoparticle into the brain, whereas the passage of larger particles (50nm) into the brain is restricted [49, 52].



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