Abstract
The polyplexes of polyethylene glycol (PEG) grafted polyethylenimine (PEI) and conjugated with gene can be used as a gene carrier in gene therapy for its low toxicity. However, the polyplexes displayed lower transfection capability in vitro and in vivo in the past studies. Modification of the polyplexes by binding a targeting ligand may improve the transfection capability. Integrin αvβ3 plays a critical role in tumor angiogenesis and becomes a promising diagnostic and therapeutic target for various solid tumors. It is well known that Arg-Gly-Asp (RGD) peptide has high affinity with αvβ3 integrin. The studies of targeting ability of a gene delivery vehicle usually rely on a design by expression of reporter gene in vivo. In this study, we have attempted to label a targeting gene delivery vehicle with a gamma emitter for evaluating its biodistribution and targeting ability by single photon emission computed tomography/computed tomography (SPECT/CT).The scope of this study was to prepare targeting polymeric gene carriers based on dimeric cyclic RGD binding PEG-g-PEI (abbreviated as E[c(RGDyK)]2- PEG-g-PEI) and labeling with 99mTc for in vivo study. We have prepared the PEG-g-PEI products with PEG to PEI ratios of 1 to 1, 3 to 1, and 10 to 1, abbreviated as PP1, PP3 and PP10, respectively. The PEG to PEI ratios in the products were confirmed by 1H-NMR. Furthermore, the PEG-g-PEI/DNA polyplexes with different polymer/DNA weight ratios (abbreviated as P/D) were prepared and their surface charges and zeta potentials and formation abilities with DNA were measured. The particles sizes of the PEG-g-PEI/DNA polyplexes were measured to be 90-135 nm. The zeta potential of the polyplexes was measured to be 40-50 mV. The polyplexes had high formation ability with DNA in P/D ratio at 1:1.We compared the cytotoxicities of PEI/DNA and PEG-g-PEI/DNA fabricated at different PEG/PEI ratios and different P/D ratios. The polyplex of PEG-g-PEI/DNA fabricated with PEG/PEI ratio at 10: 1 and P/D=1, i.e., PP10/D, showed lowest cytotoxicity whereas PEI/DNA showed highest cytotoxicity. In the transfection studies, PP10/D with PP10 concentration at 0.025 mg/ml displayed a highest transfection efficiency in comparison with the other two polyplexes. The RPP10/D polyplex demonstrated significantly higher binding affinity and transfection efficiency than non-targeting PP10/D. Both PP10/D and RPP10/D had high radiolabeling efficiency at greater than 95% and borne with radiochemical stability above 80% either in saline and in rat plasma when stored for 24 h. In in vivo SPECT/CT studies, it was found from the images that RPP10/D presented higher uptake in the tumor than PP10/D through all the postinjection times studied. From the analyses on the regions of interest (ROI) by the SPECT/CT imaging, RPP10/D also showed higher tumor to liver and tumor to blood ratios than PP10/D. The conventional biodistribution study presented corresponding results and confirmed the targeting effect of RPP10/D. The plasmid EGFP (enhanced green fluorescent protein) of RPP10/D for gene transfection was also observed to successfully transfect into the tumor site by fluorescence microscope in this study.In conclusion, E[c(RGDyK)]2 bound PEGylated PEI complex is a potential gene delivery vehicle as a targeting and imaging agent for gene therapy as evidenced by the study with the Hela tumor bearing mice model.