Tumor angiogenesis imaging using cyclic RGD-PEGylated gold nanoparticles labeled with radioiodine

Abstract

Introduction: The biocompatibility of nanoparticles is important in biomedical field such as in vivo molecular imaging and drug delivery. To visualize the tumor angiogenesis, I developed 125I-labeled gold nanoparticles (AuNPs) conjugated with cyclic RGD-PEG peptides. Methods: I developed that cyclic RGD-PEGylated-AuNPs was labeled with 125I (125I-cRP-AuNPs) as molecular imaging probes for targeting integrin αvβ3. The stability of 125I-cRP-AuNPs was monitored by ITLC-SG with saline (pH 7) as the solvent for 16 hrs at 37oC in serum. For the competitive binding assay to the integrin αvβ3, 125I-echistatin was incubated with αvβ3-expressing cancer cell, U87MG and non-expressing cancer cell, MCF7 after pre-treating with cRP-AuNPs or cRGD peptides. Cellular uptakes of 125I were measured using gamma-counter. To confirm receptor specific internalization of cRP-AuNPs, TEM image was acquired in U87MG and MCF7 cell lines. The uptake and clearance of 125I-cRP-AuNPs in vivo was evaluated using serial SPECT/CT studies from 0 hr to 5 hrs after intravenous injection of 11.1 MBq 125I-cRP-AuNPs in a U87MG tumor bearing mice. To confirm specific integrin αvβ3 binding of 125I-cRP-AuNPs, the extracted tumor tissues were analyzed by TEM imaging technique and stained with αvβ3 integrin antibody. Renal clearance of 125I-cRP-AuNPs was confirmed with radio-TLC analysis of urine samples after injection. Results: The entire 125I labeling procedure to cyclic RGD-PEGylated gold nanoparticles was completed within 20 min including purification and concentration steps. Radio-chemical purity of 125I-labeled cRGD-AuNPs was very stable during the incubation in serum at 37oC. In competitive binding assay to U87MG cells, IC50 values for cRP-AuNPs and cRGD peptides were 0.33 nM and 51.34 nM, respectively. Remarkably, the 125I-cRP-AuNP probes showed ~150-fold higher αvβ3 avidity than the corresponding cRGD peptides. TEM images showed intracellular localization of the particles in U87MG cells only, not in MCF7 cells. SPECT/CT image showed uptakes of 125I-cRP-AuNPs in the U87MG xenografted tumor and blood pool until 1 hr after injection. In the extracted tumor analysis, the TEM images clearly showed the 125I-cRP-AuNPs are uptake by the tumor. And the tumor tissue was well correlated with αvβ3-PE staining regions and Au silver enhancement. These results demonstrate that the 125I-cRP-AuNPs entered the tumor via integrin αvβ3-receptor specific endocytosis. Radio-chemical yield of urine samples was observed over 50% of 125I-cRP-AuNPs, this result showed their efficient clearance from the body through renal and urinary excretion route. Conclusion: 125I-cRP-AuNPs defined αvβ3 expressing cancer cells both in vitro and in vivo. These functionalized 125I-cRP-AuNPs have a visualization in tumor and angiogenesis which will be useful for various diagnostic and therapeutic applications.