Synthesis and evaluation of the targeted binding of RGD-containing PEGylated-PEI/DNA polyplex micelles as radiotracers for a tumor-targeting imaging probe

Site-specific labeling of molecular imaging probes necessitates the topical administration of medications during the development of homogeneous tracers. Hence, receptor-mediated gene transfer is believed to be of enormous significance in the clinical translation of a promising gene delivery technique. Plasmid DNA (pEGFP) and polycations produce polyplexes, which can be proficient probes for molecular imaging when accompanied by a gamma emitter. Therefore, this study describes the physico-biological characterization of a radiotracer for tumor imaging in a HeLa tumor-bearing mouse model. Polyplex micelles were formed with pEGFP and Arg-Gly-Asp (RGD) peptide-modified poly(ethylene glycol)-grafted polyethylenimine (E[c(RGDyK)]2-PEG-g-PEI) and were labeled with 99mTc for the in vivo study. The various PEG-g-PEIs prepared by controlling the PEG-to-PEI ratios were confirmed by 1H-NMR. The sizes and zeta potentials of the PEG-g-PEI/DNA polyplexes were 90-135 nm and 40-50 mV, respectively. The biophysical characterization of pEGFP in polyplexes was evaluated via various methods, including determination of the condensation efficiency of the polymers and the biodistribution, in vitro stability, in vivo application, and kinetics of the radiolabeled polyplexes. These characteristics were studied as a function of time using 3D-SPECT/CT images and by end-point scintillation counting. The polyplex of PEG-g-PEI/DNA fabricated with a PEG/PEI ratio of 10 : 1 and N/P = 1, i.e., PP10/D, exhibited the lowest cytotoxicity and the highest transfection efficiency. The cyclic-RGD peptide-modified polyplex PEG-g-PEI/DNA (RPP10/D) had significantly higher binding affinity and transfection efficiency than the non-targeting PP10/D did. 99mTc radio-labeled PP10/D and RPP10/D were prepared with high radiolabeling efficiency of greater than 95% and radiochemical stability above 80%, both in saline and in rat plasma, when stored for 24 h and were evaluated for their tumor-targeting capability and biodistribution. Through in vivo SPECT/CT images, it was determined that RPP10/D-99mTc presented higher uptake in the tumor than PP10/D-99mTc at all of the post-injection times studied. We found that the two tracers of radioactive complexes mainly accumulated in the liver, spleen and kidneys at 24 h after intravenous injection in female BALB/c nude mice bearing subcutaneous HeLa tumors. The accumulation of the site-specifically labeled RPP10/D-99mTc was lower in liver, kidney and spleen compared w