br Determination of the in
3.5. Determination of the in vitro stability of 198AuNP-RGD under physio-logical conditions
In order to ascertain that 198Au does not leach out of the in-trinsically radiolabeled nanoparticles when administered in vivo, 198AuNP-RGD was incubated in excess volume of PBS and mouse serum and the in vitro stability was determined by radio-TLC analyses. The results of three independent studies are shown in Fig. 8. The results indicated that 198AuNP-RGD was highly stable (intact 198Au in nanoparticles was N96%) in both the media over the 7 d period of observation.
3.6. Cell binding assay
The binding of 198AuNP-RGD was analyzed in melanoma cell line (Fig. 9). The results indicated effective binding of the nanoparticles to the melanoma cells. The inhibition studies further established the spec-ificity of 198AuNP-RGD towards melanoma cell line.
3.7. Biodistribution studies
The high uptake and prolonged retention of the radiolabeled nanoparticles in liver and spleen is a cause of major concern and potentially compromises the safety of the therapeutic approach. Also, significant uptake of the radiolabeled nanoparticles was ob-served in the kidneys indicating that the nanoparticles cleared by both hepatobiliary as well as renal route. However, this behavior of radiolabeled nanoplatforms used in cancer targeting is quite common and similar biodistribution patterns were also observed in several studies reported earlier [11,30,31] Basically, the mono-nuclear phagocyte system (MPS), which consists of a system of phagocytic cells, mainly residing in the macrophages in the liver, spleen, and AR-13324 nodes, sequesters nanoparticles immediately after injection . This phenomenon results in high and rapid up-take in these organs. In fact, nonspecific uptake of radiolabeled
Large scale synthesis of 198AuNP-RGD using 198Au produced in the Dhruva reactor.
aAmount of Au foil irradiated (mg)
nanoparticles in these healthy organs is one of the major chal-lenges towards clinical translation of this class of agents for use in cancer imaging and therapy . When a high dose (equivalent to 20–25 mg/kg of body weight of mice) of c(RGDfK) was administered in mice along with 198AuNP-RGD, the uptake of radioactivity in the tumor was signif-icantly reduced to 2.9 ± 0.8%ID/g at 4 h p.i. (Fig. 11). Further, when non-targeted 198Au nanoparticles were administered, the uptake of radioactivity in the tumor was significantly low (3.0 ± 0.9%ID/g) at 4 h p.i. (Fig. 11). These studies established that the enhanced uptake of targeted 198AuNP-RGD was indeed receptor mediated.
3.8. Tumor regression studies
In order to elucidate the therapeutic efficacy of 198AuNP-RGD in effecting retardation of tumor growth, a group of melanoma tumor bearing C57BL/6 mice were intravenously administered with 3 different doses (18.5 MBq, 37.0 MBq and 55.5 MBq) of the radiolabeled agent and TGI and BWI were monitored over a period of 15 d (Fig. 12). Tumor growth was significantly retarded in treated mice and the deceleration in the growth was enhanced with the increasing dose of 198AuNP-RGD. One time injection of 18.5 MBq 198AuNP-RGD resulted in limited tumor growth delay as compared with mice injected with normal saline or non-radioactive AuNP-RGD as controls (Fig. 12A). For mice treated with 37.0 MBq or 55.5 MBq of 198AuNP-RGD, significant reduction in TGI was observed. Over this period of time, there was no signif-
Fig. 5. Representative γ-ray spectra of 198AuNP-RGD.
Overall, this study indicated that a single dose of 37.0 MBq of 198AuNP-RGD was most effective for targeted tumor therapy in melanoma tumor bearing mice.
The biodistribution studies described above showed high accumula-tion of radiolabeled nanoparticles occurred in healthy organs such as kidney, liver and spleen. Despite, high uptake of radiolabeled nanopar-ticles in these organs, no adverse effect (in terms of changes in normal behavior of animals and significant weight loss) was observed in the
Radioactivity eluted (%) 50
Volume of PBS (mL) Number of nanoparticles bound to cells (X 1015)
Binding in excess of c(RGDfK)
Fig. 7. Size exclusion chromatographic pattern of 198AuNP-RGD developed using a PD-10 column. Fig. 9. In vitro cell binding and inhibition studies with 198AuNP-RGD in melanoma cells.
group of animals treated with optimal radioactive dose (37.0 MBq) of 198AuNP-RGD over the 15 days period of study.