Synthesis And Preclinical Evaluation Of The Fibrin Binding Activatable Cell-penetrating Peptide ¹⁸F-iCREKA

Objectives:Our previous study has constructed a cyclic activatable cell-penetrating peptide(ACPP)called iCREKA which was consisted of CREKA,cell-penetrating peptides Tat(49?57)(RKKRRQRRR)and an enzyme cutting site PLGLAG.The florescence assays revealed that it could enter into tumor cells by the enzyme digestion of matrix metalloproteinases(MMP)-2/9.However,the florescence imaging cannot meet the clinical requirements.Some clinical imaging methods should be explored for the use of iCREKA.PET can be used as a method for real-time visualization of pharmacological action on the molecular level.['8F]fluorine is an optimal radionuclide in clinical practice because of the moderate radioactive half-life and the high-quality images it provides on PET/CT.Fluorine-18 labeled iCREKA was preliminarily studied in our previous study.But the[18F]SFB method in our previous study or the[18F]NFP method to obtain fluorine-18 labeled peptides was proved to be time-consuming with low yield.It was reported that the A118F labeling method was convenient and rapid and the labeling yield of this method was relatively high.Thus,we synthesized the fluorine-18 labeled peptides by A118F labeling method in this study.We evaluated the[18F]NOTA-iCREKA in vitro and in vivo with comparison of its contrasted linear peptide(LP)which was consisted of the same three components to iCREKA and the fluorine-18 labeled CREKA alone.MethodsAll chemicals obtained commercially were used without further purification.No-carrier-added fluorine-18 was obtained from GE PETtrace cyclotron(GE Healthcare,USA).Unmodified precursor peptides and FITC conjunct peptides were bought from ChinaPeptides CO.,LTD(China,Shanghai).NOTA conjunct peptides were bought from ChinesePeptides CO.,LTD(China,Hangzhou).MS spectrograms of the peptides were obtained from those two companies.The BALB/c nude mice(4-6 weeks old,18-22 g)were purchased from the Experimental Animal Center of Southern Medical University.All animal studies were conducted in accordance with the protocols approved by the Experimental Animal Center of Southern Medical University.Reversed-phase high-performance liquid chromatography(HPLC)analyses of the peptides were performed on a SHIMAZU system,including an SPD-M20A UV detector,a flow counts radiation detector(Bioscan),and a Shimadzu LC-10AD pump.HPLC solvents were consisted of pure water containing 0.1%trifluoroacetic acid(solvent A)and acetonitrile containing 0.1%trifluoroacetic acid(solvent B).A ZORBAX Eclipse XDB-C18 column(5 ?m,150 × 4.6 mm)was used with a flow rate of 1 ml/min.The HPLC gradient system began with an initial solvent composition of 95%solvent A and 5%solvent B for 2 min,followed by a linear gradient to 20%solvent A and 80%solvent B in 25 min,after which the column was re-equilibrated.Radiosynthesis of fluorine-18 labeled peptidesManual labeling:No-carrier-added aqueous[18F]fluoride was produced from the 18018F reaction.40ul deionized water,6ul 0.01mmol/l AlCl3,5ul glacial acetic acid,324ul acetonitrile and 0.2mg NOTA conjunct peptide were mixed well in a 1.5ml EP tube by an ultrasonic oscillator.555MBq(15mCi)[18F]fluoride(volume less than 100ul)was added into the EP tube.Then the tube was sealed and placed in a thermostatic oscillator(Allsheng Instrument Co.,Ltd,China).The reaction was last for lOmin with a temperature of 100 ?.HPLC analysis of the mixture was then performed to assess the radiochemical yield.The mixture was then passed a reversed-phase C-18 Sep-Pak plus extraction cartridge(Waters Corporation,USA)which was pre-conditioned with ethanol and water before use.The cartridge was washed by 10 mL of PBS and 20 mL of water.Then 0.8 ml of ethanol containing 10 mM of hydrochloric acid was used to elute the product.Semi-automatic labeling:This procedure was performed in a home-made multifunctional synthesis module(PET CO.,LTD.,China)within a lead shielded hot cell.100ul deionized water,15ul 0.01mmol/l AlCl3,15ul glacial acetic acid,780ul dimethyl formamide(DMF)and 0.5mg NOTA conjunct peptide were mixed well and transferred into the reaction tube in the module.Then[18F]fluoride(14.8-44.4 GBq/0.4-1.2 Ci)was transferred to the tube and the radiosynthesis was proceeded with the temperature of 100 0C for 10min.The mixture was then transferred to a semi-preparative HPLC to make separation.Water partition coefficientThe water partition coefficient was reflected by the LogP value of the fluorine-18 labeled peptides.500?l of octanol and 500?l of pure water were added into an EP tube.185KBq(5?Ci)of fluorine-18 labeled peptides were added into the solution and well mixed.After centrifuged for 5min at a speed of 1000r/min,300?l of supernatant liquid and 300?l of lower liquid were carefully pipetted into two y counter tubes respectively.The logarithm of the radioactivity ratio of'octanol to water was the LogP value.The test was repeated four times for each fluorine-18 labeledpeptide.Confocal microscope analysisFITC-LP or FITC-iCREKA(0.2 mg)was sterilized by 2ml DMEM medium and filtrated by an MF-MilliporeTM Membrane Filter(Merkemillipore,USA).Subsequently,1 ml of the solution was added to U87MG cells and Caov3 cells which were adhere to glass bottom cell culture dishes or microscope cover glasses.After incubating at 37 C for 60 min,tumor cells were washed 5 times carefully with PBS and immobilized by 75%ethanol.The nucleuses were stained by 4',6-diamidino-2-phenylindole(DAPI).The fluorescence distribution in tumor cells was examined on a ZEISS LSM 880(ZEISS,Germany)after final washing by PBS for 3 times.Fluorescence grayscale analyses were operated by ZEN 2 software(ZEISS,Germany)to make semiquantitative analyses.U87MG and Caov3 Nude Xenograft ModelsThe human primary glioblastoma cell line U87MG and the human ovarian adenocarcinoma cell line Caov3 were purchased from the Guangzhou Jennio Biotech Co.,Ltd.These two cells were both grown in Dulbecco's Modified Eagle's Medium(DMEM)(ThermoFisher Scientific,USA)containing 10%fetal bovine serum(PAN biotech,Germany).Cells were passaged twice per week,at 80%confluence,and incubated in 5%C02 at 37?.Animal procedures were performed according to the guideline of the Animal Ethics Committee of the Southern Medical University.Approximately 10×106 U87MG cells and Caov3 cells were suspended in PBS and subcutaneously implanted in the right armpit of female BALB/c nude mice.Tumors were allowed to grow to a size of 0.5-1.5 cm in diameter.Fluorine-18 labeled peptide binding affinity to fibrin clotsAs previously reported,to synthesize fibrin clots,75 ?l of fibrinogen solution(2 mg/ml)in 0.9%NaCl was added to four 1.5ml EP tubes.30?l of a 2.5 U/ml thrombin solution in 0.9%NaCl was subsequently added to these tubes.The tubes were placed in the thermostatic oscillator at 37? for four hours and gels representative of fibrin clots were formed in the tubes.370KBq(10?Ci)fluorine-18 labeled peptide was diluted in 1ml PBS.100ul of the solution was added into each tube respectively and added into four empty tubes for reference.All tubes were incubated at 37? for one hour.After that,the supernatant liquid was carefully pipetted.PBS was added and pipetted thrice to remove any CREKA or peptides not bond to the fibrin clots.Finally,all tubes were measured by a CAPRAC(?)-R radioactive counter(Imaxeon)to obtain the bond-to-gross ratio.Fluorine-18 labeled peptide binding affinity to U87MG and Caov3 cellsU87MG cells and Caov3 cells were counted and uniformly inoculated into 24-well plates.The 24-well plates were marked and cells were incubated for 24 hours in complete DMEM with 10%fetal bovine serum.Radio-medium was prepared by adding 888KBq(24?Ci)fluorine-18 labeled peptide into 15ml DMEM without any serum.After incubation,the complete DMEM were poured and the radio-medium were added in with 500?l/well.The remainder medium were collected into 4 y counter tubes with 500?l/tube to be representative of radioactivity gross.When the time arrived at 2,5,15,30,60 and 120min,radio-medium were carefully pipetted out and the wells were carefully rinsed by 1ml of PBS for 3 times.4 wells were divided into a group corresponding to one time point.After the rinsing,SDS-NaOH solutions were added into the wells to dissociate the cells.The solutions were collected into ?counter tubes to measure the radioactivity.The binding affinity was reflected by the radioactivity percentage,which was recorded as ID%,of the wells and the gross.The final results were recorded as time-radioactivity curve including the six time points.Fluorine-18 labeled peptides binding affinities after activation were also measured by this protocol while the radio-medium were mixed with activated MMP-2(Abeam).In Vitro StabilityAbout 3.7MBq(0.1mCi)fluorine-18 labeled peptide was respectively added to a 1.5ml EP tube which contained 1 ml phosphate buffer(PBS)and another tube which contained 1ml mouse serum.These tubes were incubated at 37? for 2 hours.Then the PBS sample(50 ?l)and filtered serum sample were injected into the HPLC column to analyze the stability of fluorine-18 labeled peptide.In Vivo StabilityA nude with U87MG Xenograft was injected with a dose of approximately 11.1 MBq(0.3mCi)of 18F-peptide via the tail vein.The mouse was euthanized at 30 min after injection,the tumor and liver were carefully excised,and the blood was collected.The tumor and liver were cut into pieces and made into homogenates by mixing 200ul normal saline.The mixture was centrifuged for 10min at a speed of 12000r/min.The supernatant liquid was carefully pipetted and transferred into an Amicon(?)Ultra centrifugal filter device for further centrifugation.The filtrate was injected into an HPLC column to analyze the in vivo stability of fluorine-18 labeled peptide.Small-Animal PET/CTSmall-animal PET/CTs were performed at the PET center of Southern Medical University Nanfang Hospital using a PET/CT scanner(Simens Inveon Micro PET/CT).Tumor-bearing mice(n=3/group)were intravenously injected with 5.55 MBq of[18F]NOTA-LP or[18F]NOTA-iCREKA under isoflurane anesthesia.A ten-minute static PET scan for each animal was acquired at 30,60,and 120 min after the injection.120 min dynamic scans were also obtained for[18F]NOTA-LP and[18F]NOTA-iCREKA(n=2/group).3-Dimensional ordered subset expectation maximization(3D-OSEM)algorithm was used for the PET reconstruction,and CT was applied for attenuation correction.Inveon Research Workplace(IRW)3.0 software(Siemens,Germany)was used to measure the regions of interest(ROIs).The radioactivity concentrations were computed automatically and the results were reflected by the unit of percent injected dose per gram(%ID/g).The maximum%ID/g of the ROIs were used to statistical analyses.Tumor-to-muscle(T/M)uptake ratios were calculated by dividing the radioactivity uptake in the tumor by the contralateral shoulder muscle.BiodistributionThree U87MG nude xenografts were injected with 1.85MBq(0.05mCi)[18F]NOTA-LP and other three U87MG nude xenografts were injected with 1.85MBq[18F]NOTA-iCREKA.These mice were euthanized at 1 h after injection.The tumor and organ samples were excised and transferred to pre-weighed y counting tubes for weighing and gamma counting.The radioactivity associated with each sample tube was measured on the radioactive counter.Four empty tubes were measured as the background.Four tubes containing 37KBq(1?Ci)[18F]NOTA-iCREKA in each tube were measured as the reference for conversion The radioactivity counts(counts per minute)were corrected by the background and radioactive decaying.The%ID/g of each tissue and organ were calculated.The radioactivities in the stomach and intestines were including the contents in them.Statistical AnalysisAll results are expressed as the mean ±SD.To determine the statistical significance of differences between the 2 groups,comparisons were made with the 2-tailed Student t test for paired data.A P value of less than 0.05 was considered to be statistically significant.ResultsChemistryThe mass spectrometry(MS)revealed that the molecular weights of our peptides were consistent with the theoretical molecular weight.The retention time of the CREKA,LP and iCREKA were 8.413,9.539 and 11.580 min,respectively.The LogP values of[18F]NOTA-CREKA,[18F]NOTA-LP and[18F]NOTA-iCREKA were-2.47±0.05,-2.57±0.02 and-2.51±0.01The radiochemical yields(decay-corrected)of the three peptides labeled by manual methods were 18.73?33.41%,13.28?28.05%and 8.78?25.39%for[18F]NOTA-CREKA,[18F]NOTA-LP and[18F]NOTA-iCREKA,respectively,after 10min's reaction time.Most of the[18F]NOTA-CREKA could not be adsorbed by the C-18 cartridge.[18F]NOTA-LP and[18F]NOTA-iCREKA could not be completely adsorbed by the C-18 cartridge and most of them could not be eluted from the cartridge.So the specific activity was calculated using the product of semi-automatic labeling method.The specific activity of[18F]NOTA-CREKA was above 349.55 MBq/?mol and the specific activity of[18F]NOTA-LP and[18F]NOTA-iCREKA were above 466.33 MBq/?mol and 179.53MBq/?mol,respectively.The retention time of the[18F]NOTA-CREKA,[18F]NOTA-LP and[18F]NOTA-iCREKA were 8.80mim,11.78min and 11.92 min,respectively.In vitro stabilityNo decomposition was observed for these three peptides.However,defluorinations were discovered for the[18F]NOTA-CREKA and[18F]NOTA-LP.The[18F]NOTA-iCREKA was consistently stable in PBS and serum during the 2 hours'incubation.In vitro stabilityOnly the[18F]NOTA-iCREKA kept part of the primary form stable in the blood and tumor at 30min after vein injection of radiolabeled peptides.The other two peptides,especially the[18F]NOTA-CREKA,decomposed obviously in the blood and tumor.In vitro binding affinityThe fibrin-binding percentages of[18F]NOTA-CREKA,[18F]NOTA-LP and[18F]NOTA-iCREKA to fibrin clots were 69.31±6.82%,67.09±6.20%and 67.92±6.71%.There was no statistical difference among the fibrin-binding percentages of these three peptides to fibrin clots(P=0.95)The binding percentages of[18F]NOTA-iCREKA to U87MG cells were high and could reach 23.90 percent injected dose(%ID)as the peak at 60min.As for the binding of[18F]NOTA-LP to U87MG cells,by contrast,the binding percentages reached 16.05(L/%ID as the peak at 30min.For[18F]NOTA-CREKA,nearly no radioactivity was revealed in U87MG cells.After activated by MMP-2,the binding affinity of[18F]NOTA-LP and[18F]NOTA-iCREKA to U87MG cells were both increased while still no radioactivity was revealed in U87MG cells for[18F]NOTA-CREKAFor Caov3 cells,there was still nearly no radioactivity in tumor cells,no matter with or without the activation of MMP-2.The binding affinity of[18F]NOTA-LP to Caov3 cells was high and it became slightly higher by the activation of MMP-2.As a contrast,the binding affinity of[18F]NOTA-iCREKA to Caov3 cells was low and became obviously increased by the activation of MMP-2Confocal microscope analysesThe uptake of FITC-LP in U87MG and Caov3 cells were both high.By contrast,the uptake of FITC-iCREKA in U87MG cells was high while the uptake of FITC-iCREKA in Caov3 cells was relatively low.In U87MG cells,there was no significant difference between the uptake of FITC-iCREKA and FITC-LP(t=0.20,P=0.85).In Caov3 cells,the uptake of FITC-LP was obviously higher than the uptake of FITC-iCREKA(t=27.24,P<0.01)ImmunohistochemistryThe IHC results of MMP-2 revealed that U87MG tumor expressed MMP-2 abundantly while the Caov3 tumor nearly expressed no MMP-2In vivo PET imagingIn U87MG xenograft-bearing mice,[18F]NOTA-LP and[18F]NOTA-iCREKA exhibited the highest uptake in the livers and kidneys.The U87MG tumor revealed moderate uptake of[18F]NOTA-iCREKA.By contrast,the uptake of[18F]NOTA-LP by U87MG tumor was relatively low.At 60min after radiotracer injection,the U87MG tumor revealed higher uptake of[18F]NOTA-iCREKA than[18F]NOTA-LP(t=6.90,P=0.01).The T/M ratio for[18F]NOTA-iCREKAwas higher than the T/M ratio for[18F]NOTA-LP(t=15.275,P<0.01).In Caov3 xenograft-bearing mice,[18F]NOTA-LP and[18F]NOTA-iCREKA exhibited a biodistribution similar to U87MG xenograft-bearing mice in most organs,with the highest uptake in the kidneys.The Caov3 tumor revealed nearly no uptake of radiotracer,no matter[18F]NOTA-LP or[18F]NOTA-iCREKA.Radioactivities could be observed in bone for two U87MG mice and two Caov3 xenograft-bearing mice.Besides,the dynamic time-radioactivity curve could demonstrate the change of radioactivity in tumors and organs in real-time.At the 65min,the T/M ratio of[18F]NOTA-iCREKA in U87MG xenograft-bearing mice reached the maximum.For[18F]NOTA-LP,the T/M ratio reached the maximum at 95min.BiodistributionThe biodistribution showed similar results to Micro-PET imaging.The radioactivities of[18F]NOTA-iCREKA were higher than the radioactivities of[18F]NOTA-LP in U87MG xenografts(t=8.47,P<0.01).T/M ratio for[18F]NOTA-LP was 3.01±0.37 while T/M ratio for[18F]NOTA-iCREKA was 4.31±0.22,and there were statistic differences between them(t=6.71,P=0.04).The%ID/g of the spleen,which was difficult to be delineated on Micro-PET imaging,was relatively high(2.74±1.97).ConclusionIn this study,we successfully synthesized fluorine-18 labeled cyclic ACPPs named iCREKA via the Al18F chelation reaction and further evaluated it with a comparison of its linear status peptide(LP).[18F]NOTA-iCREKA was stable in vitro and could get high uptake in MMP-2/9 highly expressed U87MG tumor cells while the uptake in MMP-2/9 lowly expressed Caov3 cells was extremely low.Micro-PET imaging and biodistribution showed similar results.The[18F]NOTA-iCREKA was more stable and could get higher uptake and higher T/M ratio than the[18F]NOTA-LP.The iCREKA could be a potential carrier which could transfer targeted drugs or therapeutic radiopharmaceuticals into tumor cells.