E technetium-99 m. doi:10.1371/journal.pone.0061043.ggroups showed that the biodistribution characteristics of 99m Tc-RRL were better than 131I-RRL. The fast blood clearance and high tumor uptake was similar to the results on cyclic RGD radiolabeled with 99mTc, which is integrin avb3-specific and widely used in integrin expression imaging [25,26]. CD13 also plays an important role in tumor angiogenesis, Pathuri G, etc. reported the radiosynthesis and biodistribution of a high affinity CD13 inhibitor in nude mice with human fibrosarcoma xenografts [27]. The tumor uptake value and tumor-to-muscle ratios of their work was 2.8860.64 ID/g and 5.3, respectively. Our results showed a better ID/g value and ratio of tumor-to-nontumor.Most small molecule radiolabeled with 99mTc was excreted by urinary system preclinically or clinically [28,29]. The data in this study also showed that the molecular probe predominantly accumulated in kidneys. It indicated that the tracer was cleared through the urinary system, whose molecular weight is below the threshold that can be filtered by the glomerular membrane (, 60 kDa) [30], and displayed a property of good target/nontarget result. Blocking group was designed for illustrating the specificity of 99m Tc-RRL targeting tumors. Compared with the experimental groups, the radioactivity of the blocking group showed only a little concentration in tumor, which was similar with the control group, but much in heart, liver, spleen, lung and stomach at the time ofA Novel99mTc-Labeled Molecular ProbeTable 2. Biodistribution ( ID/g) of99mTc-RRL in Mice Bearing HepG2 Xenografts.15 min Blood Heart Liver Spleen Lung Gracillin price Kidney Stomach Small Intestine Bladder Bone Skeletal Muscle Tumor 6.9760.56 2.6760.19 2.1660.32 1.7760.49 6.0060.58 22.7963.06 10.6062.65 3.1760.68 6.3363.17 4.0161.47 1.1460.22 4.1060.30 min 4.7160.57 2.5060.44 2.0760.46 1.7360.36 5.1360.79 16.8664.32 10.8261.66 3.1560.92 6.0661.74 3.5561.33 0.8060.09 5.2561.1h 4.0760.99 1.9760.28 2.1860.60 1.5060.28 3.8360.76 14.6563.80 12.9963.16 3.9861.32 5.4061.45 4.6561.77 0.7260.37 4.4361.2h 2.5960.44 1.5360.16 1.5560.23 0.9860.11 2.9260.46 10.8562.58 9.5461.69 2.9361.48 4.2262.55 4.0661.22 0.5560.11 3.1760.4h 2.0060.4 1.2560.15 1.6760.33 
1.0360.27 2.2260.37 8.3061.86 7.3161.52 1.7560.77 2.3161.33 3.6361.62 0.5560.16 3.9261.6h 2.0960.68 1.3360.30 1.9160.93 1.0560.35 2.0660.88 9.1661.90 8.3561.51 2.4160.92 2.3460.52 3.8260.89 0.6560.19 3.2660.Each value SMER-28 manufacturer represents average of 5 mice 6 SD and is expressed as ID radioactivity per gram organ or tissue. doi:10.1371/journal.pone.0061043.tFigure 6. Relationship between tumor size and tumor uptake. A linear relationship between tumor size and uptake was shown with R2 = 0.821. doi:10.1371/journal.pone.0061043.gA Novel99mTc-Labeled Molecular ProbeFigure 7. Planar imaging of tumor xenografts. Images of nude mice bearing HepG2 cells were displayed at 1 h (A), 2 h (B), 4 h(C) and 6 h (D) post injection of the 99mTc-RRL. Tumors on the front right upper extremities were shown clearly. doi:10.1371/journal.pone.0061043.g6 h (P,0.05). The ID/g value of tumor was not statistically significant between blocking and control group (P.0.05), but thatof experimental group was significant higher than them (P,0.05). The reason for high uptake in heart, spleen and lung and lower uptake in tumor in blocking group may illustrate the tumorspecific characteristics of 99mTc-RRL. These biodistribution results supported from pro and con that the radiola.E technetium-99 m. doi:10.1371/journal.pone.0061043.ggroups showed that the biodistribution characteristics of 99m Tc-RRL were better than 131I-RRL. The fast blood clearance and high tumor uptake was similar to the results on cyclic RGD radiolabeled with 99mTc, which is integrin avb3-specific and widely used in integrin expression imaging [25,26]. CD13 also plays an important role in tumor angiogenesis, Pathuri G, etc. reported the radiosynthesis and biodistribution of a high affinity CD13 inhibitor in nude mice with human fibrosarcoma xenografts [27]. The tumor uptake value and tumor-to-muscle ratios of their work was 2.8860.64 ID/g and 5.3, respectively. Our results showed a better ID/g value and ratio of tumor-to-nontumor.Most small molecule radiolabeled with 99mTc was excreted by urinary system preclinically or clinically [28,29]. The data in this study also showed that the molecular probe predominantly accumulated in kidneys. It indicated that the tracer was cleared through the urinary system, whose molecular weight is below the threshold that can be filtered by the glomerular membrane (, 60 kDa) [30], and displayed a property of good target/nontarget result. Blocking group was designed for illustrating the specificity of 99m Tc-RRL targeting tumors. Compared with the experimental groups, the radioactivity of the blocking group showed only a little concentration in tumor, which was similar with the control group, but much in heart, liver, spleen, lung and stomach at the time ofA Novel99mTc-Labeled Molecular ProbeTable 2. Biodistribution ( ID/g) of99mTc-RRL in Mice Bearing HepG2 Xenografts.15 min Blood Heart Liver Spleen Lung Kidney Stomach Small Intestine Bladder Bone Skeletal Muscle Tumor 6.9760.56 2.6760.19 2.1660.32 1.7760.49 6.0060.58 22.7963.06 10.6062.65 3.1760.68 6.3363.17 4.0161.47 1.1460.22 4.1060.30 min 4.7160.57 2.5060.44 2.0760.46 1.7360.36 5.1360.79 16.8664.32 10.8261.66 3.1560.92 6.0661.74 3.5561.33 0.8060.09 5.2561.1h 4.0760.99 1.9760.28 2.1860.60 1.5060.28 3.8360.76 14.6563.80 12.9963.16 3.9861.32 5.4061.45 4.6561.77 0.7260.37 4.4361.2h 2.5960.44 1.5360.16 1.5560.23 0.9860.11 2.9260.46 10.8562.58 9.5461.69 2.9361.48 4.2262.55 4.0661.22 0.5560.11 3.1760.4h 2.0060.4 1.2560.15 1.6760.33 1.0360.27 2.2260.37 8.3061.86 7.3161.52 1.7560.77 2.3161.33 3.6361.62 0.5560.16 3.9261.6h 2.0960.68 1.3360.30 1.9160.93 1.0560.35 2.0660.88 9.1661.90 8.3561.51 2.4160.92 2.3460.52 3.8260.89 0.6560.19 3.2660.Each value represents average of 5 mice 6 SD and is expressed 
as ID radioactivity per gram organ or tissue. doi:10.1371/journal.pone.0061043.tFigure 6. Relationship between tumor size and tumor uptake. A linear relationship between tumor size and uptake was shown with R2 = 0.821. doi:10.1371/journal.pone.0061043.gA Novel99mTc-Labeled Molecular ProbeFigure 7. Planar imaging of tumor xenografts. Images of nude mice bearing HepG2 cells were displayed at 1 h (A), 2 h (B), 4 h(C) and 6 h (D) post injection of the 99mTc-RRL. Tumors on the front right upper extremities were shown clearly. doi:10.1371/journal.pone.0061043.g6 h (P,0.05). The ID/g value of tumor was not statistically significant between blocking and control group (P.0.05), but thatof experimental group was significant higher than them (P,0.05). The reason for high uptake in heart, spleen and lung and lower uptake in tumor in blocking group may illustrate the tumorspecific characteristics of 99mTc-RRL. These biodistribution results supported from pro and con that the radiola.