Molecular Imaging and Preclinical Studies of Radiolabeled Long-Term RGD Peptides in U-87 MG Tumor-Bearing Mice

The Arg–Gly–Asp (RGD) peptide shows a high affinity for αvβ3 integrin, which is overexpressed in new tumor blood vessels and many types of tumor cells. The radiolabeled RGD peptide has been studied for cancer imaging and radionuclide therapy. We have developed a long-term tumor-targeting peptide DOTA-EB-cRGDfK, which combines a DOTA chelator, a truncated Evans blue dye (EB), a modified linker, and cRGDfK peptide. The aim of this study was to evaluate the potential of indium-111(111In) radiolabeled DOTA-EB-cRGDfK in αvβ3 integrin-expressing tumors. The human glioblastoma cell line U-87 MG was used to determine the in vitro binding affinity of the radiolabeled peptide. The in vivo distribution of radiolabeled peptides in U-87 MG xenografts was investigated by biodistribution, nanoSPECT/CT, pharmacokinetic and excretion studies. The in vitro competition assay showed that 111In-DOTA-EB-cRGDfK had a significant binding affinity to U-87 MG cancer cells (IC50 = 71.7 nM). NanoSPECT/CT imaging showed 111In-DOTA-EB-cRGDfK has higher tumor uptake than control peptides (111In-DOTA-cRGDfK and 111In-DOTA-EB), and there is still a clear signal until 72 h after injection. The biodistribution results showed significant tumor accumulation (27.1 ± 2.7% ID/g) and the tumor to non-tumor ratio was 22.85 at 24 h after injection. In addition, the pharmacokinetics results indicated that the 111In-DOTA-EB-cRGDfK peptide has a long-term half-life (T1/2λz = 77.3 h) and that the calculated absorbed dose was safe for humans. We demonstrated that radiolabeled DOTA-EB-cRGDfK may be a promising agent for glioblastoma tumor imaging and has the potential as a theranostic radiopharmaceutical.


Introduction
Cancer is the second leading cause of death in the world. According to statistics from the World Health Organization (WHO), about 9.6 million people die from cancer each year, and on average, 1 in 6 people die from cancer. In recent years, integrin-mediated biological activity targeted against cancer has been improving. Mammals have 24 different integrins, consisting of 18 α-subunits and 8 β-subunits, which are cell adhesion receptors connected to the extracellular matrix (ECM) and other cells [1]. Integrins participate in signal transduction pathways to regulate cell growth, migration, motility, proliferation and survival [2,3]. The α v β 3 integrin is overexpressed in new tumor blood vessels and various tumor cells including neuroblastoma, osteosarcoma, melanoma, glioblastoma, breast cancer and lung cancer [4,5]. Furthermore, integrin plays important roles in cancer proliferation, invasion, survival, metastasis and angiogenesis [6][7][8]. According to the above characteristics, integrin is developed as a suitable target for cancer diagnosis and therapy [9][10][11].

Radiolabeling with Indium-111
The structure of targeting peptide DOTA-EB-cRGDfK (A) and the two control peptides DOTA-cRGDfK (B) and DOTA-EB (C) are shown in Figure 1. After radiolabeling with 111 In, the labeling efficiency of these peptides was higher than 95% determined by radio-TLC analysis (Figure 2A). The R f of 111 In-DOTA-EB-cRGDfK, 111 In-DOTA-cRGDfK, and 111 In-DOTA-EB were close to 0, and the R f of free 111 In was approximately 1. In addition, HPLC analysis showed the radiochemical purity of the 111 In-DOTA-EB-cRGDfK, 111 In-DOTA-cRGDfK, and 111 In-DOTA-EB was greater than 95% ( Figure 2B). The retention time of free 111 In, 111 In-DOTA-EB-cRGDfK, 111 In-DOTA-cRGDfK, and 111 In-DOTA-EB was 4.8, 7.1, 5.9 and 8.2 min, respectively. Because the labeling efficiency of 111 In-DOTA-EB-cRGDfK was over 95% (96.3 ± 1.4%, n = 3), no further purification was performed for the subsequent studies.

In Vitro Competitive Binding Assay
Competitive binding assay with U-87 MG cells revealed specific cell uptake of 111 In-DOTA-EB-cRGDfK. As the concentration of unlabeled DOTA-EB-cRGDfK or DOTA-cRGDfK increased, the binding ratio of 111 In-DOTA-EB-cRGDfK decreased, indicating the presence of specific binding affinity ( Figure 2C). The half maximal inhibitory concentration (IC 50 ) values of DOTA-EB-cRGDfK or DOTA-cRGDfK were obtained from curve fitting, which were 71.7 nM and 35.2 nM, respectively.

Biodistribution Studies
Biodistribution of 111 In-DOTA-EB-cRGDfK at 2, 24, 48, 72, and 96 h after intraveno injection is listed in Table 2. The results showed that the top three organs for drug acc mulation were the tumor, kidney, and spleen. The radioactivity level at the tumor si reached a peak at 24 h (27.1 ± 2.7% ID/g) and was steadily maintained until 96 h aft administration (18.6 ± 2.0% ID/g). The Tumor/Muscle (T/M) ratio of 111 In-DOTA-E cRGDfK was 8.9 at 2 h and reached to 22.85 at 24 h. The highest uptake in the kidney an spleen was 14.4 ± 0.4% ID/g at 24 h and 12.9 ± 2.8% ID/g at 48 h, respectively. Album binding ability of EB may cause accumulation in the spleen, and the intense radioactivi seen in the kidneys and bladder suggests 111 In-DOTA-EB-cRGDfK was cleared by ren excretion. In addition, low levels of radioactivity were observed in the musculoskelet systems and other organs.

Biodistribution Studies
Biodistribution of 111 In-DOTA-EB-cRGDfK at 2, 24, 48, 72, and 96 h after intravenous injection is listed in Table 2. The results showed that the top three organs for drug accumulation were the tumor, kidney, and spleen. The radioactivity level at the tumor site reached a peak at 24 h (27.1 ± 2.7% ID/g) and was steadily maintained until 96 h after administration (18.6 ± 2.0% ID/g). The Tumor/Muscle (T/M) ratio of 111 In-DOTA-EB-cRGDfK was 8.9 at 2 h and reached to 22.85 at 24 h. The highest uptake in the kidney and spleen was 14.4 ± 0.4% ID/g at 24 h and 12.9 ± 2.8% ID/g at 48 h, respectively. Albumin binding ability of EB may cause accumulation in the spleen, and the intense radioactivity seen in the kidneys and bladder suggests 111 In-DOTA-EB-cRGDfK was cleared by renal excretion. In addition, low levels of radioactivity were observed in the musculoskeletal systems and other organs. Values were presented as %ID/g, mean ± SD, (n = 4-5 at each time point). T/M, tumor/muscle; T/B, tumor/blood.

Pharmacokinetic and Excretion Studies
The pharmacokinetic parameters were estimated with the WinNonlin program and summarized in Table 3  The excretion data of 111 In-DOTA-EB-cRGDfK from U-87 MG tumor-bearing mice are presented in Figure 4. The cumulative radioactivity excreted via urine and feces were 38.7 ± 7.0% ID and 21.6 ± 5.6% ID up to Day 8 post-injection, respectively. After 8 days of the administration, 111 In-DOTA-EB-cRGDfK was excreted from the body by 60%, and the radioactivity was primarily cleared through the urine, accounting for 64% of the total excretion.

Dosimetry
According to the determination of the residence time in mice, the radiation absorbed dose prediction of 111 In-DOTA-EB-cRGDfK administered to humans is shown in Table 4. Urinary excretion was assumed to be 37%, biologic half-time was 17.6 h, and intestinal excretion was assumed to be 25%. The radiation absorbed dose was low, of which the first three absorbed doses were lower large intestine (0.622 mSv/MBq), upper large intestine (0.379 mSv/MBq), and kidneys (0.214 mSv/MBq). In other treatment-limiting tissues, such as heart, liver, lungs, ovaries, red marrow and spleen, the absorption values were 0.102 mSv/MBq, 0.187 mSv/MBq, 0.096 mSv/MBq, 0.202 mSv/MBq, 0.08 mSv/MBq, 0.186 mSv/MBq, respectively. The effective dose was 0.201 mSv/MBq.

Dosimetry
According to the determination of the residence time in mice, the radiation absorbed dose prediction of 111 In-DOTA-EB-cRGDfK administered to humans is shown in Table 4. Urinary excretion was assumed to be 37%, biologic half-time was 17.6 h, and intestinal excretion was assumed to be 25%. The radiation absorbed dose was low, of which the first three absorbed doses were lower large intestine (0.622 mSv/MBq), upper large intestine (0.379 mSv/MBq), and kidneys (0.214 mSv/MBq). In other treatment-limiting tissues, such as heart, liver, lungs, ovaries, red marrow and spleen, the absorption values were 0.102 mSv/MBq, 0.187 mSv/MBq, 0.096 mSv/MBq, 0.202 mSv/MBq, 0.08 mSv/MBq, 0.186 mSv/MBq, respectively. The effective dose was 0.201 mSv/MBq.

Discussion
Radiolabeled peptides that bind to cancer cells with high affinity and specificity may have great potential for both diagnostic imaging and targeted radionuclide therapy. Such peptide molecules are cleared from circulation very quickly. Therefore, radiotherapy requiring high doses and frequent clinical administration results in higher clinical costs and systemic toxicity [27,28]. In this study, we present the preclinical evaluation of a novel 111 In-DOTA-EB-cRGDfK peptide. It consists of a tumor-targeting peptide cRGDfK, an albumin-binding agent (EB), a modified linker, and a DOTA chelator for radionuclide labeling. The cRGDfK peptide shows specificity towards α v β 3 integrin receptors that are most commonly over-expressed in tumor cells, which can guide peptides to tumor sites [29]. Binding to plasma protein is an effective strategy to increase the pharmacokinetic properties of short-lived molecules [30]. The EB has an affinity to albumin and can improve the pharmacokinetic properties of the peptide.
Because 111 In is a long-lived radioisotope and can be used to prepare tracers for diagnostic imaging, the radiolabeling of peptides with 111 In has been used for tumor detection [31,32]. We radiolabeled DOTA-EB-cRGDfK with gamma-emitting radionuclide 111 In and obtained high radiochemical purity (>95%) without further purification. Additionally, it showed high stability, as the radiochemical purity of 111 In-DOTA-EB-cRGDfK remained above 94% for at least 96 h. The competitive binding assay showed either DOTA-EB-cRGDfK or DOTA-cRGDfK had specificity to U-87 MG human glioblastoma cell. The IC 50 of DOTA-cRGDfK (35.2 nM) was almost identical to the previous report [24,33]. Although the results showed that the conjugate of RGD peptide to EB molecule slightly reduced the affinity (IC 50 = 71.7 nM), it was still at the nM level with good binding specificity. The binding properties of EB to albumin can be used to retain drugs in the blood and be supported by the in vivo results.
NanoSPECT/CT images of 111 In-DOTA-EB-cRGDfK showed significant tumor signal within 0.5 h, and gradually increased with time, and maintained to 72 h overtime. On the contrary, the signal of 111 In-DOTA-cRGDfK was not obvious under the same scale bar and was quickly eliminated from the body. Another control group, 111 In-DOTA-EB, also had significant tumor accumulation and long half-life, but obvious radiation signals could be observed in non-tumor organs within 4 h after administration. According to image quantification results, the highest tumor uptake of 111 In-DOTA-EB-cRGDfK, 111 In-DOTA-cRGDfK, and 111 In-DOTA-EB was 25.5 ± 3.9% ID/g at 48 h, 2.0 ± 0.5% ID/g at 0.5 h, and 10.3 ± 4.4% ID/g at 24 h, respectively. The tumor uptake of 111 In-DOTA-cRGDfK was consistent with the previous study [24,33]. The accumulation of 111 In-DOTA-EB in tumors is mainly due to the enhanced permeability and retention (EPR) effect. EB may bind strongly to albumin, extravasates, and remains for a prolonged time in the extravascular space due to the EPR effect of tumors [34]. The tumor accumulation of 111 In-DOTA-EB-cRGDfK is greater than the sum of 111 In-DOTA-cRGDfK and 111 In-DOTA-EB. When the RGD peptide prolongs its retention time in the body, it may increase the chance of tumor targeting, leading to increased tumor uptake; then, competing with excess DOTA-EB-cRGDfK, the inhibitory effect gradually increased with time and reached 71% in 24 h. This indicated 111 In-DOTA-cRGDfK has specific binding toward U-87 MG solid tumor.
Biodistribution results were consistent with the drug properties shown in the bioimaging results. The maximum accumulation of the drug at the tumor site can reach 27.12 ± 2.70% ID/g, and it remains at 18.55 ± 2.01% ID/g after 96 h administration; then, the maximum tumor to muscle ratio and tumor to blood ratio was 22.85 and 23.61, respectively. These indicate that DOTA-EB-cRGDfK has a good specific binding ability to tumors. The results show that the high non-specific intake in the spleen is high due to the albumin binding affinity of the EB portion. In addition, the radioactivity was seen in the kidneys and urinary bladder suggested renal excretion, which is consistent with the results of the excretion test. The excretion data of 111 In-DOTA-EB-cRGDfK showed that urinary excretion accounted for 64% of the total excretion, and 60% injection dose was excreted from the body 8 days after administration.
Plasma protein binding can be an effective strategy for improving the pharmacokinetic properties of short-lived molecules [30]. For the pharmacokinetics, the experimental results demonstrated a pharmacologic advantage of 111 In-DOTA-EB-cRGDfK. The C max of 111 In-DOTA-EB-cRGDfK in the body is 3.5 times that of 111 In-DOTA-cRGDfK, and the half-life is 4.5 times longer. Moreover, 111 In-DOTA-EB-cRGDfK showed slow blood clearance, slow mean residence time, and prolonged retention. The biodistribution and pharmacokinetic results demonstrated the longer retention time in the blood, long-term tumor localization, and high concentration of DOTA-EB-cRGDfK.
In terms of drug toxicity, the first-in-human study of 64 Cu-NOTA-EB-RGD demonstrated its safety, favorable pharmacokinetic, and dosimetry profile [35]. The DOTA-EB-cRGDfK also showed safe dosimetric calculations through the OLINDA/EXM code, providing estimates of the absorbed doses from animal species to humans. In order to evaluate the potential toxicity of 111 In-DOTA-EB-cRGDfK in clinical use, we took the recommended intravenous dose (222 MBq) for SPECT imaging of an FDA-approved 111 In-labeled peptide radiotracer, 111 In-pentetreotide, as reference. The radiation doses of 11 In-DOTA-EB-cRGDfK from such an administration for the lower large intestine, kidney, and liver were 0.138 Gy, 0.048 Gy, and 0.042 Gy, respectively. Compare to the tolerance of normal tissues to radiation doses are 25 Gy, 20 Gy, and 8 Gy, respectively [36], we found that the radiation absorbed dose was low and does not affect the human body. This shows the safety of the drug and is also conducive to future related trials of labeling for therapeutic radionuclides.
The novel DOTA-EB-cRGDfK peptide presented several advantages in U-87 MG human glioblastoma. First, it shows a high tumor uptake in vivo biodistribution (27.12 ± 2.70% ID/g), and it is higher than dimeric, trimeric, and tetramer c(RGD) radioligands [37][38][39], and the tumor imaging quantification of 111 In-DOTA-EB-cRGDfK is 50% higher than 64 Cu-NMEB-RGD (25.09 ± 4.76% ID/g and 16.64 ± 1.99% ID/g at 24 h after injection) [24]. In addition, the tumor uptake of 18 F-BPA is 1.44 ± 0.44% ID/g, which is usually used in boron neutron capture therapy (BNCT) [40]. The tumor absorption of 111 In-DOTA-EB-cRGDfK is more than 20 times that of 18 F-BPA. Second, 111 In-DOTA-EB-cRGDfK shows favorable pharmacokinetics. The radiolabeled peptides usually showed fast blood clearance, but the T 1/2λz of 111 In-DOTA-EB-cRGDfK was 77.3 h. The extended half-life may increase the fully useful dosage. Third, DOTA is a good chelator for labeling different radionuclides, such as 111 In for SPECT imaging and 177 Lu for treatment. DOTA-EB-cRGDfK shows the possibility of being a theranostic radiopharmaceuitcal. On the other hand, blood-brain barrier (BBB) may be considered in the future application. Radiolabeled peptides may pass the barrier because BBB breakdown is common in high-grade gliomas and brain metastases [41,42]. Hematotoxicity may be concerned due to albumin binding ability, and further investigations with kidneys and intestine toxicities of administration are warranted. In addition, we have other experiments with DOTA-EB-cRGDfK in different cell types in progress. Experiments therapeutic radionucleotide 177 Lu labeling are also ongoing.

Cell Culture and Tumor Model
The human glioblastoma cell line, U-87 MG (stock number: BCRC 60360), was obtained from Bioresource Collection and Research Center. Cells were grown in Minimum Essential Medium (MEM; Gibco, Grand Island, NY, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco, Gaithersburg, MD, USA) and 1% antibiotic antimycotic solution (Sigma-Aldrich, St. Louis, MO, USA) at 37 • C under 5% CO 2 incubation. Female non-obese diabetic, severe combined immunodeficiency (NOD/SCID) mice were obtained from BioLASCO Taiwan Co., Ltd. The 6-week-old mice were housed in a 12 h light cycle at 22 • C, with food and water provided ad libitum. 2 × 10 6 U-87 MG cells resuspended in 100 µL of phosphate-buffered saline (PBS; Gibco, Gaithersburg, MD, USA) were injected subcutaneously into each nude mouse. The tumor model was studied when the tumor volume reached 300-500 mm 3 . Animal protocols were approved by the Institutional Animal Care and Use Committee at the Institute of Nuclear Energy Research, Taoyuan, Taiwan (approval ID: 107169; approval date:13 February 2018).

Radiolabeling with Indium-111
The targeting peptide DOTA-EB-cRGDfK, DOTA-cRGDfK, and DOTA-EB were purchased from Ontores Biotechnologies (Zhejiang, China). Indium [ 111 InCl 3 ] chloride (gamma emitter) was generated from INER (Lung-Tan, Taiwan). For indium-111 labeling, 30 µg targeting peptide and 222 MBq 111 InCl 3 were mixed in 1 M sodium acetate (pH 6.0) to a final volume of 300 µL. After incubation for 15 min at 95 • C and 500 rpm shaking in a thermal controller, the radiolabeling product was obtained without further purification. Product quality was analyzed by instant thin layer chromatography (iTLC) and high-performance liquid chromatography (HPLC).
The iTLC method was used on the glass microfiber chromatography paper impregnated with a silica gel (Agilent Technologies, Santa Clara, CA, USA), whereas the mobile phase was used as 0.1 M citric acid/0.1 M sodium citrate (v/v = 2.1/7.9) buffer. Then, the sheets were measured using a radioactive scanner (AR-2000 radio-TLC Imaging Scanner, Bioscan, France), and the relative front (Rf) value of radiochemical was calculated. Rf value is defined as the ratio of distance traveled by the component to the distance traveled by the solvent front from the sample spot. HPLC-analysis was performed using UV detection at 220 nm and a radio detector with a Zorbax SB-C18 column (Agilent Technologies, Santa Clara, CA, USA). The flow rate was 0.8 mL/min with the gradient mobile phase going from 80% A buffer (0.1% TFA in water) and 20% B buffer (0.1% TFA in acetonitrile) to 60% B buffer within 10 min.

In Vitro Competitive Binding Assays
U-87 MG cells were seeded into 24-well plates at a density of 1 × 10 5 cells per well and incubated overnight. Different concentrations ranging from 0.01 nM to 5000 nM of DOTA-EB-cRGDfK or DOTA-cRGDfK were added to the wells. U-87 MG cells were then incubated with 10 nM 111 In-DOTA-EB-cRGDfK for 4 h at 37 • C. Cells were washed once with 0.5 mL PBS and were removed from each well by 0.25% trypsin-EDTA (Gibco, Gaithersburg, MD, USA). The cell suspensions were collected and measured by using a PerkinElmer 2480 Automatic Gamma Counter (PerkinElmer, Waltham, MA, USA). Binding results were expressed as a percent of total counts, and IC 50 values were calculated using SigmaPlot 12.5 (Systat Software, Inc., San Jose, CA, USA).

In Vitro Stability Study
In vitro stability of 111 In-DOTA-EB-cRGDfK was evaluated by incubation with normal saline (in 1:1 vol ratio) or rat plasma (in 1:19 vol ratio) at room temperature. The radiochemical purity was determined by iTLC analysis as previously described [43]. At desired times (0, 2, 4, 24, 48, 72, and 96 h), 1 µL was using for iTLC on the glass microfiber chromatography paper impregnated with a silica gel (Agilent Technologies, Santa Clara, CA, USA), whereas the mobile phase was used as 0.1 M citric acid/0.1 M sodium citrate (v/v = 2.1/7.9) buffer. Then, the sheets were measured using a radioactive scanner (AR-2000 radio-TLC Imaging Scanner, Bioscan, France).

NanoSPECT/CT Imagings
The procedure for nanoSPECT/CT imaging has been previously described [43]. Each mouse was tail-vein injected with about 18.5 MBq and 2.5 µg (50-100 µL) of radiolabeled 111 In-DOTA-EB-cRGDfK, 111 In-DOTA-cRGDfK, or 111 In-DOTA-EB. For the blocking study, animals were pre-treated with a 120-fold molar excess (300 µg) DOTA-EB-cRGDfK by tail vein injection 10 min ago. SPECT images and X-ray CT images were acquired using a nanoSPECT/CT ® plus scanner system (Mediso Medical Imaging Systems; Budapest, Hungary). The mice were anesthetized with 1-2% isoflurane during the imaging acquisition. The imaging acquisition was accomplished at 60 s per frame. The energy window was set at 171 and 245 KeV ± 10%, the image size was set at 256 × 256, and the field of view of 60 mm × 100 mm. For image reconstruction, the HiSPECT and Nucline software were used for the SPECT and CT images, respectively. The InVivoScope software was used for the fusion of SPECT and CT images. The image quantification of the region of interest (ROI) was acquired by PMOD v. 3.3 (Zürich, Switzerland). The SPECT images were presented on a scale of 2.5% ID/g to 25% ID/g.

Biodistribution, Pharmacokinetic and Excretion Studies
Thirty U-87 MG tumor-bearing mice (five mice per group) received an intravenous injection of about 1.85 MBq and 0.25 µg of 111 In-DOTA-EB-cRGDfK for biodistribution study. They were sacrificed by CO 2 asphyxiation at 2, 4, 24, 48, 72, and 96 h. At each time point, the organs of interest were sampled and whole organs collected where possible. The samples were rinsed in saline, blotted dry, weighed, and then counted using a PerkinElmer 2480 gamma counter (PerkinElmer, Waltham, MA, USA). Data were expressed as the percentage injected dose per gram of tissue (% ID/g).
For the pharmacokinetic study, five U-87 MG tumor-bearing mice received an intravenous injection of about 1.85 MBq and 0.25 µg of 111 In-DOTA-EB-cRGDfK or 111 In-DOTA-cRGDfK. Blood samples (10 µL) were collected by heart puncture under 2% isoflurane anesthesia at 0.083, 0.5, 2, 4, 24, 48, 72, 96, and 168 h. The radioactivity of blood samples was measured by PerkinElmer 2480 gamma counter and expressed as the percentage injected dose per gram (% ID/g). Data was used to determine the pharmacokinetic parameters by noncompartmental analysis (NCA) and the analysis software was WinNonlin 7.0 (Pharsight Corporation, Mountain View, CA, USA). Parameters, including terminal half-life (T 1/2λz ), maximum concentration (Cmax), total body clearance (Cl), area under curve (AUC), and mean residence time (MRT) were determined. Pharmacokinetic parameters associated with the terminal phase were calculated using the best-fit method to estimate the terminal half-life.
For excretion studies, five U-87 MG tumor-bearing mice received an intravenous injection of about 1.85 MBq and 0.25 µg of 111 In-DOTA-EB-cRGDfK. They were placed in metabolic cages individually for 8 days. Food and water were adequately provided. Urine and feces were continuously collected in each metabolic cage, weighed daily, and counted for radioactivity on a gamma counter. The %ID excreted at each time was thus determined. Cumulative excretion curves (urine and feces) were fitted with nonlinear regression by SigmaPlot 12.5 (Systat Software, Inc., San Jose, CA, USA).

Dosimetry
Dosimetry analysis of 111 In-DOTA-EB-cRGDfK was based on biodistribution and excretion results and was performed as previously described [44,45]. The uptake and doses in various tissues/organs were derived from the radioactivity concentration in tissues and organs of interest, assuming a homogeneous distribution within each source region. The calculated mean value of % ID/g for the organs in mice was extrapolated to uptake in the organs of a 70 Kg adult. The extrapolated values (% IA) in the human organs at 2, 24, 48,72 and 96 h were fitted with exponential functions and integrated to obtain the number of disintegrations in the source organs; This information was entered into the OLINDA/EXM computer program. The International Commission on Radiological Protection (ICRP) 30 Gastrointestinal (GI) model and voiding urinary bladder model in OLINDA/EXM were used to estimate the number of disintegrations occurring in the excretory organs. this information was input into the OLINDA/EXM computer program. The integrals (MBqh/MBq administered) for organs were calculated and used for the dosimetry estimation.

Statistical Analysis
The results are expressed as mean and standard deviation (mean ± SD). The unpaired t-test was used for group comparisons. Data fitting and statistical analyses were computed using the SigmaPlot 12.5 (Systat Software, Inc., San Jose, CA, USA). Values of p < 0.05 were considered significant.

Conclusions
This study proposes a novel integrin-targeted molecule, DOTA-EB-cRGDfK, with long-term tumor retention ability. The results demonstrated that 111 In-DOTA-EB-cRGDfK had higher accumulation in xenograft integrin-expressing U-87 MG tumor and that low imaging dose appears to be effective in treating tumors with high 111 In-DOTA-EB-cRGDfK uptakes. It shows the possibility of being a therapeutic drug and is suitable for labeling diagnostic and therapeutic radionuclides. For future theranostic clinical use, 111 In-DOTA-EB-cRGDfK/ 177 Lu-DOTA-EB-cRGDfK has the potential to improve treatment efficacy using significantly lower quantities of 177 Lu, and is a promising candidate for clinical translation to treat human brain cancer at the administered low dose. Further investigations with hematotoxicity, kidneys and intestine toxicities of administration are warranted.