the following description with reference to the accompanying drawings is provided to clearly and completely explain the exemplary embodiments of the disclosure . it is apparent that the following embodiments are merely some embodiments of the disclosure rather than all embodiments of the disclosure . according to the embodiments in the disclosure , all the other embodiments attainable by those skilled in the art without creative endeavor belong to the protection scope of the disclosure . 1 . materials and instruments : all the purified chemicals are obtained from sigma / aldrich in the us ( st . louis , mo .). trimeric cyclic peptide having four polyethylene glycol and three cyclic rgd ( 4p - rgd 3 , the chemical structure is shown in fig1 ) is obtained from peptides international , inc in the us ( louisville , ky .) ( as shown in annotation 6 ). hynic - osu ( succinimidyl 6 - hydrazinonicotinate ) is prepared based on the procedure cited in the reference ( as shown in annotation 7 ). 6 . peptides international , inc ; website : https :// www . pepnet . com / 7 . harris , t . d . ; sworin , m . ; williams , n . ; rajopadhye , m . ; damphousse , p . r . ; glowacka , d . ; poirier , m . j . ; yu , k . synthesis of stable hydrazones of a hydrazinonicatinyl - modifiedpeptide for the preparation of 99mtc - labeled radiopharmaceuticals . bioconjugate chem . 1998 , 10 , 808 - 814 . maldi ( matrix - assisted laser desorption ionization ) spectrum is obtained by applied biosystems voyager de pro mass spectrometer ( framingham , mass .). na 99m tco 4 is obtained from lantheus medical imaging or global medical solutions . 1 . method 1 : the instrument used for purification of semi - preparative hplc is a laballiance hplc system and a zorbax c18 semi - preparative column ( 9 . 4 nm × 250 mm , pore size : 100 å , agilent technologies , santa clara , calif .) including an uv detector ( wavelength = 254 nm ). the gradient elution conditions are : flow velocity : 2 . 5 ml / minute , the initial mobile phase is 90 % of solution a ( 0 . 1 % tfa aqueous solution ) and 10 % of solution b ( 0 . 1 % tfa acetonitrile solution ), the mobile phase is linearly changed to 80 % of solution a and 20 % of solution b after 5 minutes , as well as the mobile phase is linearly changed to 50 % of solution a and 50 % of solution b after 20 minutes . 2 . method 2 : laballiance hplc system is used for the purification of radio - hplc , which includes a ram in / usdetector ( tampa , fl ) and 1 zorbax c18 column ( 4 . 6 mm × 250 mm , pore size : 300 , agilent technologies , santa clara , calif .). the gradient elution conditions are : flow velocity : 1 ml / minute , the mobile phase is 90 % of solution a ( 25 mm ammonium acetate solution ) and 10 % of solution b ( acetonitrile solution ) between 0 - 5 minute , and solution b in the gradient mobile phase is linearly elevated to 60 % between 5 - 20 minutes . 13 . 5 mg hynic - osu ( 30 . 0 μmol ) and 9 . 0 mg 4p - rgd 3 ( 3 . 0 μmol ) are dissolved in 2 . 0 ml of dimethylformatnide ( dmf ). an excess amount of diisopropylethylamine ( diea ) ( 5 drops ) is dropped into the solution , and the mixture is stirred under room temperature until the reaction is completed ( about 24 hrs ). 2 . 0 ml of water is incorporated into the mixture , and the ph is adjusted to 3 - 4 by incorporating pure tfa . the product is purified by hplc ( hplc method 1 ), and the composition at 18 minute is collected . after freeze - drying the collected liquid , is 6 . 5 mg of white powder of hynic - 4p - rgd 3 is obtained as the target product ( about 50 %). the chemical structure is shown in fig1 , and maldi - ms : m / z mw of [ c 148 h 227 n 35 o 48 s ]= 3295 . 8240 . 5 mg of tppts , 6 . 5 mg of glycine , 40 mg of mannitol , 38 . 5 mg of disodium succinate hexahydrate , 12 . 7 mg of succinate acid solution and 1 . 0 - 1 . 5 ml of na 99m tco 4 solution ( a physiological saline with the activity of f1110 - 1850 mbq ) are injected into the lyophilized vial containing 20 - 25 μg of hynic - 4p - rgd 3 . the above vial is heated in boiling water bath for 10 - 20 minutes and then is standed for 5 minutes under room temperature , and a sample solution of 99m tc - 4p - rgd 3 is obtained ( the chemical structure is shown in fig2 ). the radiochemical purity is analyzed by radio - hplc ( hple method 2 ). according to the radio hplc spectrum of 99m tc - 4p - rgd - 3 ( fig3 ), it can be realized that 99m tc - 4p - rgd 3 has high purity of radiochemical purity ( the purity is greater than 90 %), such that it can be used in the research of biodistribution and tumor imaging . v . biodistribution and preparation of 99m tc - 4p - rgd 3 for tumor imaging : the preparation of 99m tc - 4p - rgd 3 for the research of biodistribution includes incorporating 99m tc - 4p - rgd 3 solution into physiological saline until the activity concentration is 10 - 30 mci / ml . the preparation of 99m tc - 4p - rgd 3 for tumor imaging includes incorporating 99m tc - 4p - rgd 3 solution into physiological saline until the activity concentration is about 10 mci / ml . during the experiments , each of the animal is injected with about 0 . 1 ml of the solution . biodistribution and tumor imaging are performed under the guidance of us national institutes of health animal experiments ( 1985 , nih 86 - 23 , laboratory animal care ). u87mg cells are purchased from us atcc ( manassas , va .). cell culture conditions : dmem medium ( non - essential amino acids sodium pyruvate ) containing 10 % of fetal bovine serum and 1 % of penicillin and streptomycin . the cells are cultured under 37 ° c . with 5 % of co 2 . when the cells are grown to 90 % confluence , they grow exponentially . the cells can be grown to a single layer , multiple layers or split layers . female athymic nu / nu nude mice aged 4 - 5 weeks are used for the experiments , 5 × 10 6 of tumor cells are incorporated into 0 . 1 ml of physiological saline and are planted to shoulder subcutaneous of the mice under sterile conditions . about 4 weeks after planting , the tumor cells grow to 0 . 1 - 0 . 5 g for the use of biodistribution and imaging . 7 random u87mg glioma nude mice ( 20 - 25 g ) are selected . each of the nude mice is injected at the tail vein with 3 μci of 99m tc - 4p - rgd 3 . after 60 minutes , a great amount of sodium pentobarbital is injected for executing the mice . blood samples are collected from the mouse hearts . tumor , normal organs ( brain , eyes , heart , spleen , lung , liver , kidney , muscle and intestine ) and tumor tissues are collected and washed by saline . then they are dried by absorbent tissue , weighed by perkin elmer wizard 1480 γ counter ( shelton , conn .). the organ intake is measured by the percentage of the injection dose per gram of the tissue (% id / g ). the following table compares the biodistribution in u87mg glioma nude mice 60 minutes after the injection between 99m tc - 4p - rgd3 and currently used medicine , 99m tc - galacto - rgd 2 and 99m tc - 3p - rgd 2 . viii . fig4 is the comparison of animal biodistributions in organs after 60 minutes between 99m tc - 4p - rgd 3 ( n = 7 ), 99m tc - 3p - rgd 2 ( n = 6 ) and 99m tc - galactose - rgd 2 ( n = 8 ) according to the embodiments of the disclosure . 60 minutes after the injection , the tumor intake of 99m tc - 4p - rgd 3 ( 7 . 34 ± 1 . 66 % id / g ) is highly similar to 99m tc - 3p - rgd 2 ( 7 . 24 ± 0 . 95 % id / g ) and 99m tc - galacto - rgd 2 ( 6 . 86 ± 1 . 33 % id / g ). however , the intake background in organs of the whole body , such as intestine , liver , lung , muscle , spleen and kidney , is significantly decreased . thus , it can be realized that 99m tc - 4p - rgd 3 can provide a better quality of tumor imaging for whole body as compared to 99m tc - 3p - rgd 2 and 99m tc - galacto - rgd 2 , and 99m tc - 4p - rgd 3 is more superior for the application of diagnosis of tumors in whole body . ix . fig5 is a 3d and sectional view of spect / ct image of us7mg glioma nude mice after injection of 99m tc - galactose - rgd 2 , 99m tc - 3p - rgd 2 and 99m tc - 4p - rgd 3 ( about 37 mbq ). the radioactive accumulation in animal abdomen is the lowest after the injection of 99m tc - 4p - rgd 3 ( especially in intestines ), and the tumor intake is about 7 . 34 id / g based on the spect quantization . the contrast ratio of the tumor and the background in the spect / ct image is great , such that the tumor can be clearly noticed . as compared with the images of 99m tc - 3p - rgd 2 and 99m tc - galacto - rgd 2 , it can be realized that the image of 99m tc - 4p - r . gd 3 does not have significant background intake of the tissues or organs . according to the spect / ct images , it can be clearly realized that is an excellent radioactive tumor imaging tracer for whole body . x . normal mice ( n = 2 ) are selected for the metabolism research of the stability of 99m mtc - 4p - rgd 3 in vivo , each animal is injected with about 10 mbq of 99m tc - 4p - rgd 3 . the bladders are squeezed manually after 30 minutes and 120 minutes , respectively . the urine samples are collected and are mixed with 50 % aqueous acetonitrile with the same volume . the mixture is centrifilged under 8 , 000 rpm . the supernatant is collected and is filtered by a 0 . 20 μm millex - lg filter . the filtrate is analyzed by the hplc . the fecal samples 120 minutes after the injection is collected , and is treated for homogenization by 20 % of aqueous acetonitrile . the obtained mixture is rotated for about 5 minutes and then is centrifuged under 8 , 000 rpm . the supernatant is collected and is filtered by a 0 . 20 μm . millex - lg filter . the filtrate is analyzed by the hplc . the radioactive recovery is greater than 95 % for the urine samples and the fecal samples ( counted by γ ). xi . fig6 indicates typical metabolisms of 99m tc - 4p - rgd 3 , and fig6 indicates radioactive hplc spectrums of the physiological saline ( a ) before the injection of 99m tc - 4p - rgd urine samples ( b ) 30 minutes after the injection as well as urine samples ( c ) and fecal samples ( d ) 120 minutes after the injection . according to the spectrums , during the research period , which is over 2 hours , no metabolites of 99m tc - 4p - rgd - 3 are detected in the urine samples and the fecal samples . thus , it indicates that 99m mtc - 4p - rgd ; can be excreted outside animals &# 39 ; bodies in the original form of 99m tc - 4p - rgd 3 after metabolism . note that the specifications relating to the above embodiments should be construed as exemplary rather than as limitative of the present disclosure . the equivalent variations and modifications on the structures or the process by reference to the specification and the drawings of the disclosure , or application to the other relevant technology fields directly or indirectly should be construed similarly as falling within the protection scope of the disclosure .
the disclosure is related to a chemical structure , a preparation and an imaging method of a tumor imaging tracer , especially to a spect radionuclide - labeled trimeric cyclic rgd peptide : 99m tc - 4p - rgd 3 , which functions as a ligand to be combined with integrin α v β 3 receptor in tumor for tumor imaging . the preparation includes linking hynic - osu with 4p - rgd 3 to form hynic - 4p - rgd 3 and then chelating hynic with 99m tc to create 99m tc - 4p - rgd 3 . animal biodistribution and imaging experiments reveal higher uptake in tumor and lower uptake in normal organs , therefore the disclosure can improve image quality , through animal metabolic experiments , the original form of 99m tc - 4p - rgd 3 can be still excreted without the evidence of further metabolites in urine and feces . according to the disclosure , the tumor imaging tracer 99m tc - 4p - rgd ; combined with the integrin α v β 3 solves the issue of high background uptake in normal organs , such that radio - labeled spect imaging tracers are more suitable for animal whole - body tumor evaluation .