Study On Tumor Imaging Of Novel Tumor Targeting Anti-cancer Peptide

Objective:The newly developed tumor-targeted RKL and NKL were obtained by modifying RGD and NGR with anticancer peptide D-K₆L₉.The newly developed RKL,NKL and DKL were labeled by sulfo-Cy5.5-NHS ester.In order to test whether these three peptides could be used as probes for tumor molecular imaging,we verified and compared the binding affinity of Cy5.5-RKL,Cy5.5-NKL and Cy5.5-DKL to tumor in vitro and in vivo,through cell immunofluorescence test and near infrared fluorescence imaging of tumor-bearing mice.In order to benefit the treatment of glioblastoma,we chose the probe with the highest tumor binding affinity from those of the three,and verified its ability to locate tumor in orthotopic glioma model.We then tested its ability to delineate the boundary of glioblastoma which could contribute to the radical resection of glioma.In order to develop a probe which could be used in Positron emission computed tomography,DOTA-NKL was labeled with positron nuclide ⁶⁸Ga,and ⁶⁸Ga-DOTA-NKL was obtained.The imaging ability of ⁶⁸Ga-DOTA-NKL was verified by cell uptake test and PET/CT imaging of tumor model.Method:RKL,NKL and DKL were synthesized by company.Sulfo-Cy5.5-NHS esters dissolved in DMF were mixed with these three peptides with bicarbonate solution as buffer.Cy5.5-RKL,Cy5.5-NKL and Cy5.5-DKL were separated and obtained by high performance liquid chromatography and lyophilized for reserve.U87MG human glioma cells were incubated with Cy5.5-RKL,Cy5.5-NKL and Cy5.5-DKL,respectively for 1 h at 37℃,which was dissolved in phosphate buffer saline with a concentration of 50 nM.In the blocking group,U87MG human glioma cells were co-incubated with Cy5.5-RKL,Cy5.5-NKL and Cy5.5-DKL（50 nM）and their precursors（50μM）at 37℃ for 1 h.After staining the nucleus with DAPI,the binding specificity of these probes to tumor cells was verified by confocal laser microscopy.In order to verify their abilities to be used as cancer targeted probes in near infrared fluorescence imaging,U87MG human glioma-bearing mice was established.Cy5.5-RKL,Cy5.5-NKL and Cy5.5-DKL were injected into tumor-bearing mice through the tail vein at a dose of 1.5 nmol,respectively.Near infrared fluorescence imaging of tumor-bearing mice was continuously performed within 8 h after probes injection.In the blocking group,1.5 nmol Cy5.5 labeled peptides and their precursors（20 mg/kg）were co-injected into tumor-bearing mice.Near infrared fluorescence imaging was performed 3 hours after injection to test the specificity of the probes to U87MG glioma.The binding affinities of Cy5.5-RKL,Cy5.5-NKL and Cy5.5-DKL to tumor cells and tumor tissue were compared.The probe of these three with the highest tumor binding affinity was selected.U87MG orthotopic glioma models were injected with Cy5.5-RKL at a dose of 1.5 nmol via tail vein.Near infrared fluorescence imaging was performed within 8 h after probe injection.The imaging ability of Cy5.5-RKL to be used as a probe for near infrared fluorescence imaging for U87MG orthotopic glioma models was detected.And then verified its ability to be used for surgical navigation.DOTA-NKL was labeled with positron nuclide ⁶⁸Ga.The labeling rate of ⁶⁸Ga was verified by high performance liquid chromatography.In order to test its lipid-water partition coefficients,⁶⁸Ga-DOTA-NKL（370 kBq）was added into the mixture of n-octanol and phosphate buffer saline.The tube was then oscillated for 15 min and centrifuged for 5 min at 12,500 rpm.The radioactivities in both n-octanol and PBS were determined by gamma counter.⁶⁸Ga-DOTA-NKL was incubated in phosphate buffer saline and mouse serum respectively.The radiochemical purity of the probe was determined continuously by high performance liquid chromatography within 4 hours to verify the stability of the probe in vitro.The expression levels of CD13 in 22Rv1 human prostate cancer cell and HT-29 human colon cancer cell were detected by immunofluorescence assay.The uptake and efflux of ⁶⁸Ga-DOTA-NKL by 22Rv1 human prostate cancer cell and HT-29 human colon cancer cell was measured.The binding affinity of ⁶⁸Ga-DOTA-NKL to 22Rv1 human prostate cancer cell was determined.The IC₅₀ value of ⁶⁸Ga-DOTA-NKL to 22Rv1 human prostate cancer cell was determined.22Rv1 human prostate cancer and HT-29 human colon cancer tumor models were established respectively.⁶⁸Ga-DOTA-NKL（7.4 MBq）was injected into mice via tail vein,and PET/CT imaging of the tumor model was performed continuously within 3 hours.In the blocking group,⁶⁸Ga-DOTA-NKL（7.4 MBq）and NKL（20 mg/kg）were co-injected into mice.PET/CT imaging was performed 2 hours later to verify the specificity of the probe to tumor.2 h after the probe injection,the tumor-bearing mice were dissected to explore the biological distribution of the probe in tumor-bearing mice.Result:Cy5.5-RKL,Cy5.5-NKL and Cy5.5-DKL were successfully synthesis.The peak time of Cy5.5-RKL,Cy5.5-NKL and Cy5.5-DKL in high performance liquid chromatography were 19.39,19.44 and 19.40 min,respectively.The yields of Cy5.5-RKL,Cy5.5-NKL and Cy5.5-DKL were 84.95%,96.12%and 76.78%.The maximum absorption wavelengths of Cy5.5-RKL,Cy5.5-NKL and Cy5.5-DKL were 675,673 and 673 nm,and the maximum fluorescence emission wavelengths were 693,695 and 693 nm,respectively.Cy5.5-RKL had the highest binding ability to U87MG human glioma cells.Cy5.5-NKL had the moderate binding ability to U87MG human glioma cells.Cy5.5-DKL had the lowest binding ability.The binding affinities of these three probes were significantly reduced in the blocking test.Near infrared fluorescence imaging results showed that the maximum T/NT values of Cy5.5-RKL,Cy5.5-NKL,Cy5.5-DKL appeared at 7.5,7.5,3 h during the imaging process.The maximum T/NT values were 7.65,4.89 and 5.43,respectively.In the blocking group,the T/NT values of Cy5.5-RKL,Cy5.5-NKL and Cy5.5-DKL at 3 h during the imaging process were 0.73±0.06,1.07±0.04,1.40±1.23,respectively,which were much lower than the non-blocking group（7.65±0.72,5.43±0.72,4.89±0.26,（P<0.05））.Cy5.5-RKL with the highest binding affinity to tumor cells and tumor tissue was selected for orthotopic glioma imaging.The fluorescence intensity value of orthotopic glioma was 2.407×10¹⁰,which was significantly higher than that of normal brain tissue（4.178×10⁹）（P<0.05）.The ⁶⁸Ga labeling rate of DOTA-NKL was 94.51+0.49%.Lipid-water partition coefficient of ⁶⁸Ga-DOTA-NKL was 1.34+0.07.⁶⁸Ga-DOTA-NKL showed good stability in serum and phosphate buffer saline.The uptake of ⁶⁸Ga-DOTA-NKL by 22Rv1 human prostate cancer cells in 2 h was 3.15±0.04%.In HT-29 human colon cancer cells with negative CD13 expression,the uptake rate in 2 h was 1.03±0.003%（P<0.05）.The binding affinity test confirmed that the IC₅₀ value of DOTA-NKL to 22Rv1 human prostate cancer cells was 13.87 nM.⁶⁸Ga-DOTA-NKL was used as the probe for PET/CT imaging for 22Rv1 human prostate cancer models.The uptake of probe by 22Rv1 human prostate cancer reached 8.69±0.20,6.61±0.22,3.85±0.06 and 1.41±0.23%ID/g at 0.5,1,2 and 3 hours after injection.The uptake of the probe by HT-29 human colon cancer was only 0.59±0.04%ID/g at 1 h after injection.Conclusion:Newly developed probes,RKL,NKL and DKL,were successfully synthesized.The results of in vitro and in vivo experiments showed that these three probes can be used in near infrared fluorescence imaging.Among these three probes,RKL,which performed best in near infrared fluorescence imaging,was detected to determine its ability to be used as a probe for surgical navigation for glioma radical resection.The result of the intraoperative imaging was good.The boundary between glioma and normal brain tissue can be clearly delineated.⁶⁸Ga-DOTA-NKL has good physical and chemical properties,and has good targeting effect on CD13-positive tumors in vitro and in vivo.It is an ideal probe for PET/CT imaging.