ABSTRACT
Objective To construct 131 I labeled anti?EGFR immunoliposome nanoparticle ( 131 I?Cetuaximab ( C225)?BSA?PCL) , and investigate its inhibitory effect on EGFR?overexpressing cancer cells in vitro. Methods Anti?EGFR liposome nanoparticle C225?BSA?PCL and non?targeted liposomes BSA?PCL were constructed. The products were observed with transmission electron microscopy and dynamic light scat?tering. The EGFR?targeted binding and cellular uptake in EGFR?overexpressing cancer cells were observed with flow cytometry and confocal microscopy. Anti?EGFR and non?targeted liposomes were labeled with 131 I using the chloramine?T method. The targeted cell killing effects of 131 I labeled liposomes were analyzed using MTT assay. The time?dependent cellular uptake analysis was used to evaluate the slow?release effects of the 131 I labeled liposomes. The independent?samples t test was used for data analysis. Results The EG?FR?targeted liposome C225?BSA?PCL and non?targeted liposome BSA?PCL were successfully constructed, and the effective diameters were approximately 130-180 nm. Flow cytometry and confocal microscopy re?vealed significant uptake of C225?BSA?PCL in EGFR?overexpressing tumor cells. BSA?PCL could also bind to cells with minimal and weak tumor retention. The EGFR?targeted radioactive liposome 131I?C225?BSA?PCL showed greater targeted cell killing effect than non?targeted liposome 131I?BSA?PCL,the IC50 values of 131I?C225?BSA?PCL and 131 I?BSA?PCL were 0. 03-1. 32 and 0. 25-12. 19, respectively. The uptakes of 131 I?C225?BSA?PCL was higher than that of 131 I?BSA?PCL ( t=3.03-16.86, all P<0.05) and reached the maxi?mal level at 4 h after incubation. Conclusions The EGFR?targeted liposome C225?BSA?PCL demonstrated superior cellular binding and uptake on EGFR?overexpressing cancer cells compared with BSA?PCL. The EGFR?targeted radioactive liposome 131 I?C225?BSA?PCL had favorable intracellular retention and excellent targeted cell killing effect, and could effectively suppress the growth of EGFR?overexpressing cancer cells.
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Objective To prepare and evaluate 99Tcm radiolabeled Cetuximab (C225) for imaging of EGFR specific binding.Methods Cetuximab antibody was reduced by 2-iminothiophene (2-IT).The radiolabeling of IT-Cetuximab with 99Tcm(99Tcm-IT-Cetuximab) was analyzed by HPLC,and was tested for in vitro stability and molecular integrity.The human lung cancer line (A549)-bearing nude mouse model was prepared for biodistribution and tumor targeted study.Tumor uptake was also measured by in vivo γ imaging.Results The labeling efficiency was 93.15％.The radiochemical purity was 96.46％ after purification.The in vitro stability was good in 5％ HSA,in which the radiochemical purity maintained above 80％ at 4 ℃ for 24 h.99Tcm-IT-Cetuximab showed good specific binding to tumor with peak uptake of (3.417±0.769) ％ID/g after 4 h.The T/NT ratio of blood increased to 1.454±0.174 at 24 h.γ imaging of A549-bearing nude mice xenografts also showed high T/NT ratio.Conclusions 99Tcm-IT-Cetuximab molecular probe could be prepared with high radiolabeling yield and radiochemical purity.It has excellent in vitro and in vivo stability,and shows specific uptake in A549 tumor.
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Objective To evaluate the in vitro effect on tumor cell uptake,tumor imaging and in vivo biodistribution of 99Tcm-epidermal growth factor receptor (EGFR) mRNA antisense PNA probe mediated by cationic liposome.Methods The oligonucleotide with sequence complementary to part of the EGFR mRNA antisense PNA was hybridized in an anti-parallel orientation targeted PNA.PNA hybridization complexes were labeled with 99Tcm by ligand exchange.The assembly of lipofectamine and 99Tcm-labeled heteroduplex was achieved by electrostatic interactions,and the radiolabeled purity was determined by reversedphase HPLC (RP-HPLC).The disparities of cell uptake in SKOV3 cells and the differences of biodistribution and molecular imaging in BALB/c nude mice bearing SKOV3 xenografts between lipofectanine-mediated 99Tcm-EGFR mRNA antisense PNA (group 1) and 99Tcm-EGFR mRNA antisense PNA (group 2) were analyzed.Two-sample t (or t') test and Wilcoxon rank sum test were used for statistical analysis.Results The labeling rates of both group 1 and group 2 were more than 95％ within 6 h.The cell uptake at 1,2,4,6,12,24 h after injection was (28.90±1.12)％,(32.76±1.20)％,(38.20±3.11)％,(41.23±1.60)％,(46.63±1.55)％ and (46.78±2.14)％ in group 1,and was (3.51±0.39)％,(3.90±0.40)％,(4.69±0.18)％,(5.91±0.26)％,(5.30±0.22)％ and (5.39±0.17)％ in group 2 respectively (t'=47.11-58.67,Z=2.80,all P＜0.05).The retention ratios showed significant difference between the two groups (t'=7.25-11.55,Z=2.80,all P＜0.05).The SKOV3 tumor could be visualized in both groups at 1 h post injection but much better visualized in group 1.The T/NT ratios were higher in group 1 at all time points (t =3.96,t'=12.65-14.69,Z=2.83-5.29,all P＜0.05).The T/NT ratios at uptake peak were 5.02 and 3.95,respectively.The probe accumulated mainly in tumor,kidneys and liver.Tumor uptake increased with time ((1.49±0.09) ％ID/g and (2.15±0.21) ％ID/g at 1 h,(3.90±0.65) ％ID/g and (5.00±0.10) ％ID/g at 6 h) after lipofectamine treatment.The ratios of tumor to contralateral muscle were also higher in group 1 (t =11.24,t' =3.96-11.94,all P＜0.05).Conclusions Lipofectamine-mediation can significantly improve the intracellular delivery of radionuclide molecular probe.Lipofectamine-mediated 99Tcm-EGFR mRNA antisense PNA can greatly improve imaging contrast and visualization of EGFR-over-expressing tumors.
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Objective To label the modified human epidermal growth factor receptor 2 (HER2) affibody with 68Ga at cysteine position of carboxyl-terminal Gly-Gly-Gly-Cys (GGGC) sequence,and to image the mice model bearing HER2-expressed breast cancer with microPET.Methods The HER2 affibody with carboxyl-terminal cysteine was produced by gene engineering.The 1,4,7,10-tetraazacyclododecane-1,4,7-tris-aceticacid (DOTA) was conjugated to the thiol (SH)-group of cysteine by DOTA-10-maleimidoethylacetamide (MMA-DOTA).The newly eluted 68Ga was labeled to DOTA-HER2 affibody at 90 ℃.The labeled mixture was purified by C18 cartridge and eluted with anhydrous alcohol.The purified 68Ga-labeled affibody was prepared for cell binding analysis and microPET imaging.The HER2-expressed breast cancer cells MBA-MD-361 were used to analyze cell binding and establish cancer model with BALB/c nude mice.68Ga-labeled affibody (3.7 MBq) was administered to 4 nude mice with breast cancer via tail vein for microPET imaging at 20 and 60 min post injection.The mice were sacrificed immediately after imaging in unconscious state and their tissue/organs (tumor,muscle,bone and heart) were removed for weighing and radioactivity counting by γ counter.The tumor to normal organ ratios of radioactivity (tumor to liver,muscle,bone and heart)were calculated.Results The purity of HER2 affibody was more than 95％.The radiochemical purity of 68Ga-labeled HER2 affibody was 97％.The KD value (affinity) of 68Ga-labeled HER2 affibody was 1.5 nmol/L in HER2positive cells.MicroPET imaging showed that 68Ga-labeled HER2 affibody could bind HER2-positive cancer rapidly and excreted mainly through urinary system.The radioactivity ratios of tumor to liver,muscle,bone and heart were 17.4±1.0,35.1±10.9,20.7±6.2 and 20.9±4.0,respectively.Conclusions 68Ga could be labeled to HER2 affibody.The 68Ga-labeled HER2 affibody may be useful for PET imaging of HER2-positive tumor.