PEGylation Of GX1Homing To Tumor Vasculature And Application For Molecular Imaging

【Background】In1971, J. Folkman hypothesized that tumor growth was dependent onangiogenesis, which represented a new perspective on the role of blood vesselsin tumor growth. Angiogenesis occurs in the microenvironment of many solidtumors, including colon cancer. The anti-angiogenesis strategy has beendeveloped through extensive and intensive research. A key aspect of thisstrategy is to identify the molecules that are able to deliver effectivelytherapeutic agents to the endothelial cells of the tumor vessels.GX1is a cyclic9-mer peptide CGNSNPKSC that targets tumor-derivedvascular endothelial cells. GX1was identified by Min Zhi et al. in2004byscreening a phage-displayed peptide library in vivo, and was appraised andfurther studied by Xiaoli Hui et al. Previous results indicated that GX1was ableto bind specifically to the tumor vasculature and could be applied toanti-angiogenesis iatreusis of cancer, together with anti-neoplastic agents, as anovel tumor vascular marker. However, GX1is not able to bind to tumor vessels perfectly and has a short half-life in vivo. The development of a clinicalapplication for the use of GX1requires additional research.A key goal of anti-cancer drug design is to increase the specific targeting ofthe drug to tumor tissues to decrease unwanted side effects. According toprevious research, polyvalence and chemical modifications are effectivemethods for increasing the receptor binding affinity, in vivo pharmacokineticsand tumor retention of molecular therapies. Bivalent GX1was synthesized withdicarboxylic monomethoxy polyethylene glycol5000（mPEG, molecular weight5000）, which is referred to as the PEGylated GX1dimer or PEG-（GX1）₂. Aseries of tests were conducted to estimate the physiological properties of theresultant probe.【Objectives】1. To PEGylate GX1and screen the ideal peptide according to SPECTimaging.₂. To identify and evaluate PEGylated GX1in vivo and in vitro.【Methods】1. Polyvalent GX1s were synthesized using dicarboxylic mPEG5000. SPECTimagings were performed to screen the idealest peptide.₂. The synthetic peptide was conjugated with biotin forimmunocytofluorescence experiments. The peptide was labeled with theisotope^99mTc for SPECT imaging of tumor vasculature utilizing the LoVo orSGC7901tumor xenograft nude mice models and other studies. The invitro and in vivo binding affinity, specificity and targeting efficacy ofPEG-（GX1）₂was investigated to determine if the new probe is moreeffective than the homologous monomer.3. Biodistribution was performed using null mice with tumor xenografts of human gastrointestinal carcinoma. The radioactivity in various tissuesamples was measured with a γ-counter and was decay-corrected as to thetime of injection, and the percentage of injected dose per gram （%ID/g）for each organ or tissue was calculated. Each value represented the meanand SD of the three animals. The tumor/non-tumor ratios （T/NT ratios）were detected at the seven time points.【Results】1. Tetravalent and bivalent GX1s were synthesized and the idealest peptidewas screened by SPECT imaging—PEG-（GX1）₂.₂.^99mTc-PEG-（GX1）₂was found to have high labeling efficiency and high invitro stability. Immunofluorescence staining, the in vitro receptorcompetitive binding inhibition assay and the multidrop saturating receptorbinding assay demonstrated that PEG-（GX1）₂and^99mTc-PEG-（GX1）₂bound specifically to Co-HUVEC with a high affinity. SPECT imagingresults showed that^99mTc-PEG-（GX1）₂targeted the tumor tissue with higherradioactivity accumulation than did^99mTc-GX1.3. Biodistribution obtained the same results as SPECT imaging. All resultsrevealed that PEG-（GX1）₂had higher affinity with receptor, stronger tumortargeting.【Conclusions】PEG-（GX1）₂displayed higher affinity and targeting ability than did GX1.^99mTc-PEG-（GX1）₂is a promising radiotracer for tumor angiogenesis imagingand internal radiotherapy of gastrointestinal cancers.