Preparation Of A New Molecular Probe ^99mTc-HYNIC-ACPP Targeting MMP-2/9 And Experimental Study On The Imaging Of A549 Lung Cancer Mice

Objective The activated cell penetrating peptide?ACPP?is composed of three parts which include a cationic peptide segment with the cell membrane penetration ability,an anionic peptide segment playing the role of neutralization and substrate polypeptide connecting two segments of the charged peptide which can be specifically hydrolyzed by enzymes.When ACPP is not activated,the whole is neutral and has no ability to penetrate the cell membrane.While entering the microenvironment which the activated matrix metalloproteinase-2/9?MMP-2/9?exists in,the substrate is hydrolyzed and those two electrified peptides are separated.Subsequently,the cationic peptide recovers the ability to penetrate membranes and takes the connecting signal molecule(e.g.,^99mTc)into the cell.The connecting signal molecules accumulate in the cell and finally the purpose of target imaging is achieved.MMP-2/9 can promote the occurrence,invasion and metastasis of tumor by degrading the extracellular matrix,promoting tumor angiogenesis,activating a variety of growth factors and other pathophysiological processes,which is considered as an important target for the diagnosis and treatment of lung cancer.In this study,a novel molecular probe ^99mTc-HYNIC-ACPP was prepared by using a bifunctional chelating agent HYNIC to carry out the radionuclide ^99mTc labeling of ACPP.The biological distribution and SPECT imaging of the probe in nude mice model of lung cancer A549 cell were studied to explore the value of ^99mTc-HYNIC-ACPP in targeting MMP-2/9 imaging in the nude mice bearing A549 lung cancer.Method Human non-small cell lung cancer cell line A549 cells were cultured in vitro,and A549 lung cancer mouse model was constructed.PO17061301 polypeptide?HYNIC-ACPP?was marked with Tricine freeze-dried kit by two steps.The labeled products were separated and purified by PD-10 desalting column and the radioactivity map of the eluent was drawn.The radiochemical purity of ^99mTc-HYNIC-ACPP in the separation products was measured by Thin-layer chromatography.The tumor bearing mice were randomly divided into five groups,and the ^99mTc-HYNIC-ACPP probe was injected respectively.After 30 min,1 h,2 h,3 h and6 h,we carried out the biological distribution,calculated the ratio of tumor/muscle?T/M?and plot the radioactivity map.Another Five groups of tumor bearing mice were given SPECT imaging after administration of 30 min,1 h,2 h,3 h and 6 h.Result Human non-small cell lung cancer cell line A549 cells were cultured and A549 lung cancer mouse model was constructed successfully.The molecular probe ^99mTc-HYNIC-ACPP was successfully prepared.After separation and purification by PD-10 desalting column,the radioactivity map of the eluent showed an obvious high peak?polypeptide peak?and a low peak?salt peak?.The radiochemical purity was 92.3%by Thin-layer chromatography.The results of biological distribution study showed that the distribution of radiopharmaceuticals was higher in the kidney,liver and spleen at first.The radioactivity of the liver and spleen gradually decreased with time while the kidney gradually concentrated.Meanwhile,T/M were2.0±0.3,2.9±0.4,1.9±0.2,1.5±0.3 and 0.5±0.1 respectively after injection of 30 min,1 h,2 h,3 h and 6 h.The SPECT imaging displayed that radioactivity concentration began to appear in the tumor site after injection of 30 min,the most obvious concentration of radioactivity occurred after 1 h.Then it gradually decreased and almost invisible after 6 h.However,the very high concentration of radioactive concentration could be seen in the liver,kidney,spleen and bladder all the time.Conclusion The radioactive technetium labeled ^99mTc-HYNIC-ACPP can be prepared simply and has a high radiochemical purity.It can be concentrated in the tumor tissue and the tumor development image is clearly visible,which suggests that ^99mTc-HYNIC-ACPP SPECT imaging can detect MMP-2/9 in vivo.This experiment provides an experimental basis for non-small cell lung cancer specific diagnosis and target therapy clinically,and lays a foundation for targeted MMP-2/9 imaging of other types of tumors.