¹³¹I Labeled Tyr-GX1Peptide Homing To Tumor Vessels For Molecular Imaging And Radiotherapeutic Study In Vitro

【Background】Gastrointestinal cancer is most common carcinoma which threating human health, thedisease situation of the most people is in aggressive-phase or advanced stage when theyvisited doctors, because of lacking effective therapeutics, the recovery rate is very low.Scientific researchers and doctors are trying to find the effective therapeutics to curetumors. In1971, professor J. Folkman hypothesized that tumor growth was dependent onangiogenesis, which represented a new territory in oncotherapy, then we screened andprepared many monoclonal antibodies or peptides, but now we still lack an effectivetargeted molecule for tumors diagnosis and therapy.Due to the development of molecule imaging technique in recent years, radiolabelledsmall molecule peptides or monoclonal antibodies are more attractive candidates in tumortargeting imaging or therapy, especially in the radiotherapy. GX1is a specific cyclicpeptide （CGNSNPKSC） screened by Min Zhi et al. from nude mice bearing human gastric cancers with a phage-displayed peptide library technology in vivo in2004. Previousresearches results confirmed that GX1could target to tumor-associated vascularendothelial cells, but the previous researches were focused on identification of theproperties, PEGylation,⁹⁹Tc^mlabeling and so on. But there is no research about GX1labeled by¹³¹I for imaging and radiotherapy at present, So in this study we designed andmodified tumor vasculature homing peptide GX1with tyrosine （Tyr）, and to evaluate thepossibility of¹³¹I-Tyr-GX1peptide as tumor targeted radiotracer by analysing the ECTimaging in nude mice bearing human gastrointestinal tumors and radiotherapy in vitro ofCo-HUVEC.【Objectives】1. To chemosynthesis the Tyr-GX1peptide and identify the qualities of chemicalproperties, combinability and targeting, then evaluate them with GX1.2. Use Na¹³¹I labeling Tyr-GX1peptide to prepare for the isotope probe of¹³¹I-Tyr-GX1and identify its labeling rate，stability and radiochemical purity，then theSPECT imaging and Cerenkov optical imaging were performed to evaluate the specificityof tumor vessels in vivo.3. To evaluate the radiotherapeutic effect of¹³¹I-Tyr-GX1to Co-HUVEC in vitro.【Methods】1. The Tyr-GX1peptide was synthesized according to the amino acid sequence, thenthe mass spectrometry and the identification of chemical properties were conducted; Toprepare biotin labeling Tyr-GX1and observe its combinability in HUVEC/Co-HUVEC byimmunofluorescence for evaluating its specificity; Tyr-GX1/GX1peptide was labeled by⁹⁹Tc^mand then was injected into nude mice bearing tumors via a vein of mouse tail, thenthe SPECT imaging of different times （4h,8h,12h,18h,24h） were observed and thetumor/non-tumor （T/NT） was calculated with ROI technology to demonstrate the targetingto tumor vessels in vivo.2. Tyr-GX1peptide was labeled with Na¹³¹I by Iodogen method under the optimumlabeling conditions, then the labeling efficiency, radiochemical purity and the stabilitywere detected in vivo and in vitro. Nude mice bearing tumor xenografts of human gastric carcinoma were intravenously injected with¹³¹I-Tyr-GX1by caudal vain and then SPECTimaging, biodistribution and Cerenkov optical imaging were performed for confirming itsspecificity.3. The radiotherapeutic effect of¹³¹I-Tyr-GX1was detected with FCM and MTT indifferent dose or concentration in vitro.【Results】1. Tyr-GX1peptide was confirmed by mass spectrometry. Immunofluorescenceshowed that Tyr-GX1could bind to Co-HUVEC/HUVEC comparing to SGC7901/GEScells and it had no obvious diversity in binding to Co-HUVEC with GX1comparing toURP.⁹⁹Tc^m-GX1/⁹⁹Tc^m-Tyr-GX1/⁹⁹Tc^m-URP was injected into nude mice bearing tumorxenografts of human colon cancer, respectively.24h SPECT imaging showed that thetumor were seen clearly in12h or18h. The radioactive uptake of⁹⁹Tc^m-Tyr-GX1wasobvious higher than⁹⁹Tc^m–URP.24h biodistribution showed that⁹⁹Tc^m-GX1/⁹⁹Tc^m-Tyr-GX1were mainly detained in kidney, liver and tumor which were higher thanother organs. It also illustrated that they were metabolized by kidney. Selectedtumor/organ （muscle, brain, blood, liver, kidney） ratios showed that radioactivityaccumulation of tumor were higher than non-tumorous organs （muscle, brain et al.）. In aword, the properties of Tyr-GX1were conformable with GX1.2. Paper chromatography showed that the labeling efficiency and the radiochemicalpurity of¹³¹I-Tyr-GX1peptide were above90%. The stability of¹³¹I-Tyr-GX1in differentsolutions（human serum, mouse serum, PBS, EDTA solution） were maintained about90%in24h, illustrating that¹³¹I-Tyr-GX1was stable in vivo or in vitro. Then the¹³¹I-Tyr-GX1was injected into nude mice bearing gastric cancer by caudal vein for dual-modalityimaging.24h SPECT showed increased¹³¹I-labelled GX1peptide was uptaked by thetumor from4h to24h gradually after injection, especially in12-18h. It was similar with¹³¹I-Tyr-RGD in imaging situation. In biodistribution, high radioactivity was found inkidney, then in the liver, tumor, and lower radioactivity were found in muscle, brain andbone. The Cerenkov optical signals were collected in abdomen and tumor tissue of nudemice bearing tumor, which was identical with the results of SPECT. 3. Na¹³¹I, Tyr-GX1and¹³¹I-Tyr-GX1were demonstrated to have an inhibitive effecton Co-HUVEC in different concentration in vitro and it had a dependence relationshipwith concentration. what was more,¹³¹I-Tyr-GX1could cause apoptosis and radiationdamage to Co-HUVEC in certain does.【Conclusions】1. Tyrosine modified GX1peptide was designed and synthesized successfully,Tyr-GX1had the same properties as GX1in affinity and targeting ability.2. Tyr-GX1peptide could be labeled with Na¹³¹I by Iodogen method easily andeffectively. We obtained a radioactive probe with high labeling efficiency, radiochemicalpurity and stability. SPECT and Cerenkov optical imaging showed that¹³¹I-Tyr-GX1had agood tumor-targeting efficacy in vivo.3.¹³¹I-Tyr-GX1was demonstrated that it had inhibitive effect or radiation damage toCo-HUVEC in certain does in vitro and had a dependence relationship with concentration.It also provided references for internal research, indicating it may be developed for a newpromising gastrointestinal tumor targeted radiotracer.