Early evaluation of anti-angiogenic effects with gadolinium (III) labeled APN/CD13 specific binding peptides magnetic resonance imaging

Early evaluation of anti-angiogenic effects with gadolinium (III) labeled APN/CD13 specific binding peptides magnetic resonance imaging | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Early evaluation of anti-angiogenic effects with gadolinium (III) labeled APN/CD13 specific binding peptides magnetic resonance imaging Sheng Han, Song-Song Liu, Gong-Wei Jing, Pin-Qin Wang, Yan-Teng Zhang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5274314/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Jul, 2025 Read the published version in Scientific Reports → Version 1 posted 11 You are reading this latest preprint version Abstract Background Anti-angiogenesis has been recognized as a crucial strategy in anti-tumor therapy, and the early assessment of its efficacy is equally significant. In this study, we developed a magnetic resonance (MR) probe specifically targeting angiogenesis to facilitate targeted imaging for the early evaluation of anti-angiogenic effects. Methods We synthesized DOTA-G3CNGRC, conjugated it with gadolinium (III), and subsequently evaluated the labeled probe in vitro. Tumor-bearing mouse models of HT-29 (CD13-negative expression) and HT-1080 (CD13-positive expression) were established, followed by MR Imaging conducted via intraperitoneal injection of the labeled probe and Gd-DOTA at a dosage of 0.5 mg/kg/day before and after ubenimex treatment over a consecutive period of seven days. The average pixel ratio of the transplanted tumor (target tissue, T) to the left hind leg (non-target tissue, NT) was determined using the region of interest technique (ROI), while changes in tumor size were meticulously recorded. Additionally, APN/CD13 expression levels in transplanted tumors were assessed both prior to and following treatment. Results The labeling rate of probes was 88.99%. The IC50 of the probes was 61.66 nM. The T/NT ratio of HT-1080 was significantly higher than that of HT-29 ( P < 0.001, n = 5). After treatment, the T/NT value of HT-1080 transplanted tumors was significantly decreased ( P < 0.001, n = 5), accompanied by a significant decrease in CD13 expression and unapparent changes in tumor size ( P = 0.710, n = 5). Conclusion The Gd-DOTA-G3CNGRC probes appeared potential for targeted MR imaging in assessing the early efficacy of anti-APN/CD13 drugs. Biological sciences/Cancer Biological sciences/Molecular biology Health sciences/Biomarkers Health sciences/Nephrology Health sciences/Oncology APN/CD13 Angiogenesis Anti-angiogenesis Asn-Gly-Arg (NGR) peptide Gadolinium (III) Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The healing process of almost all chronic diseases and acute injuries is accompanied by endothelial cell activation and angiogenesis, and the process of angiogenesis has an important impact on the final prognosis of different diseases. For some diseases, providing conditions to promote angiogenesis is considered to be one of the measures to promote the improvement of these diseases, such as fractures and wound healing disorders. However, for other diseases, especially malignant tumors, anti-angiogenesis is considered one of the therapeutic means to achieve better outcomes for these diseases. Therefore, the search for and identification of new receptors selectively expressed by activated endothelial cells and the discovery of vascular function regulators or new ligands that can interact with them has become a hot spot in vascular target imaging and therapy research [ 1 – 5 ]. Aminopeptidase N (APN/CD13) (EC 3.4.11.2) is a type II exonuclide of approximately 150–240 kDa that is involved in the degradation of neutral or alkaline N-terminal residues of biologically active peptides. This protein is a member of the zinc metallopeptidase M1 family and consists of an enzyme-promoting extracellular domain, a transmembrane region and a short cytoplasmic domain involved in signal transduction [ 6 ]. It was found that the expression of this enzyme was up-regulated in angiogenic vessels caused by various etiologies, while it was little or no expression in normal vessels [ 7 – 8 ]. Studies have shown that APN/CD13 plays an important role in cancer development, progression and even treatment, especially the importance of APN expression and activities related to cancer characteristics, especially tumor angiogenesis and metastasis [ 9 , 10 ]. The Asn-Gly-Arg (NGR) peptide was originally discovered by selecting peptide-phage libraries in tumor mice [ 11 ]. A large number of previous studies have shown that the NGR peptide can bind specifically to aminopeptidase N (APN/CD13) in vivo and in vitro. Because of these properties, the NGR peptide has been used by many researchers as a ligand-directed vector for the delivery of various therapeutics and imaging agents to angiogenic vessels [ 12 – 15 ]. Most of the previous research reports focused on targeted therapeutic drugs or imaging probes based on NGR carriers, while relatively few reports have evaluated the efficacy of anti-angiogenic drugs based on NGR carrier probes[ 10 – 12 ]. Therefore, in this study, Gd-DOTA-G3CNGRC was prepared, and imaging was performed on tumor-bearing mouse models to provide a magnetic resonance molecular imaging method for the early efficacy evaluation of antiangiogenic drugs. 1. Experimental materials and instruments 1.1 Experimental materials 2-Chloro-triphenylmethyl chloride resin, Fmoc-AA-OH (N-[fluorent-methoxycarbonyl]-l-alanyl-L-alanine), 5 g of indanhydrone-100 ml of ethanol, pyridine, N-(9-fluorent-methoxycarbonyl)-S-triphenylmethyl-L-cysteine (Fmoc-CYS (TRT)-Oh), Fmoc-ARG (PBF)-OH (N- (9-fluorene methoxycarbonyl)-S-triphenylmethyl-L-cysteine), N-fluorenyl methoxyl-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine (FMOC-Arg(Pbf) OH), and DOTA-NHS (1,4,7,10-tetraazecyclododecane-1,4,7,10-tetraacetic acid) were of analytical grade and purchased from Hefei Bairdi Chemical Technology Co., LTD., China. Anhui). Analytical grade DCM (dichloromethane), DIEA (N,N-diisopropyl ethylamine), methanol, DMF (N,N-dimethylformamide), HOBT (1-hydroxybenzotriazole), and DIC (N,N'-diisopropyl carbodiimide) were purchased from Tianjin Concord Technology Co., LTD. (Tianjin, China). Bestatin (Ubenimex), GdCl 3 .6H 2 O, Gd-DOTA,and xylenol orange were purchased from Sigma‒Aldrich. The cell culture and immunohistochemical staining related reagents used in this study were purchased from Gibco, a brand owned by Thermo Fisher Scientific Inc. 1.2 Main experimental instruments Vacuum freeze-dryer/freeze-dryer (LGJ-30FD, Beijing Songyuan Huaxing Technology Development Co., LTD.), preparation-grade HPLC (P3000Ⅰ, Beijing Innovation Tongheng Chromatography Technology Co., LTD.), Shimadzu Liquid chromatocl-Mass Spectrometry Instrument (LCMS2020) and Shimadzu HPLC analysis instrument (LC-10AT 2010A) are the products of Jinna Instrument Equipment (Shanghai) Co., LTD., continuous electric deionization ultra-pure water machine (YL-EDI60, Shanghai Yilan Environmental Protection Technology Co., LTD.) magnetic resonance imaging instrument (SIEMENS, MAGNETOM, skyra3.0T). 2. Methods 2.1 Synthesis of DOTA-G3CNGRC Firstly, the 2-chloro-triphenylmethyl chloride resin (500 mg) was immersed in DCM for a duration of 30 minutes, followed by three washes with DMF and one wash with DCM. Then a mixture containing 0.15 mM of Fmoc-CYS (TRT) OH, 8 ml anhydrous DCM and 800 µL DIEA was added and reacted under nitrogen bubble condition for 1.5 hours. After washing with DMF for 3 times, a mixture containing 0.5 ml DIEA, 0.5 ml methanol and 2 ml DCM was added and the reaction continued for 20 minutes. Then draining the liquid in the reactor with a circulating water vacuum pump, add DMF about 3 times the volume of resin to the reactor, wash for 30 seconds, drain the liquid, and repeat the operation 4 times. Secondly, 20% of piperidine / DMF solution (with 3 times the volume of resin) was added to the reactor and bubble nitrogen for 20 minutes. Then wash 4 times according to the above washing method. Finally, take the above 10–20 pieces of resin, add two drops of detection reagent A and B, and heat for 2 minutes in a 100℃ incubator to observe the color of the resin. The coloring of the resin means that the Fmoc was successfully removed; if no color appears, the operation needs to be repeated again. Coupling: 0.5 mM of Fmoc-ARG (PBF)-OH and 0.5 mM of HOBT (75 mg) were dissolved in 1ml DMF, followed by 100 µL DIC, mixed for 1min, and then the mixture was added to the drained resin for a nitrogen bubble reaction of 1 hour. The resin was then tested and washed repeatedly. After the peptide synthesis, the Fmoc was removed and washed, and then DMF containing 0.3 nM DOTA-NHS 2 ml and 100 µL DIEA were added in sequence for 20 minutes to make the N-terminal of the polypeptide modified by DOTA. Finally, following the method described above, the resin is washed three times with methanol, followed by the removal of the liquid and vacuum drying until the resin appeared as a granular substance. Cut the polypeptide from the resin and purify the polypeptide: add the cut solution (100 ml of the cutting reagent contains 95 ml TFA + 1 ml water + 2 ml EDT + 2 ml Tis) to the dried resin, and then add methyl tert-butyl ether (pre-cooled at -20℃) equivalent to 10 times the volume of the cutting solution for sedimentation of the cut polypeptide. Finally, the peptide was purified using an 8 micron 30×250 mm diagesol column. Mobile phase: A: 0.1% TFA aqueous solution B: 0.1% TFA acetonitrile solution; the flow rate was 12 ml/min, the sample was loaded through pump A, and then 10% acetonitrile water was operated to balance the gradient for 5 min, and the sample peak for collection and detection was prepared. When the purity of the analysis was greater than 80%, the sample was freezable for use. 2.2 Labeling DOTA-G3CNGRC with Gd (III) Firstly, 1 mg (0.9µM) of DOTA-NGR was dissolved in 9 ml of acetic acid buffer with a pH of 7.4, followed by 10 ml of GdCl3 solution with a concentration of 10 µM/L, the molar ratio of Gd (III) to NGR was about 11:1, and the reaction was oscillated in a 38℃ water bath for 30 minutes. Secondly, the mixture was transferred into a 10 ml dialysis tube (Spectra/Por® Flot-A-Lyzer ®, MWCO: 500 − 100), then the tube was placed in a beaker containing 500 ml of deionized water and dialyzed with a magnetic stirrer for 1 hour. Finally, the marking rate was determined: 6 ml of the prepared xylenol orange solution (concentration 2 g/L) was added to a 12-well cell culture plate at 1 ml per well, GdCl3 solution at different concentrations (0.1 ml) was added, and the enzyme marker (Varioskan LUX, Thermo Fisher Scientific Inc.) was used. The OD value of each well was determined at 575 nm, and the standard curve and equation of Gd (III) content were established. After dialysis, the liquid in the beaker was added to the xylenol-containing orange hole at a rate of 0.1 ml per hole, the OD value of each hole was determined, the concentration of free Gd (III) was calculated, and the loading rate of Gd (III) was calculated according to the concentration. To further verify the successful labeling of the probe, we performed magnetic resonance imaging on the labeled probe. The sequence used for magnetic resonance imaging was T1-weighted TSE dark-fluid, and the collection conditions were as follows: TR: 2310.00 ms, TE: 13.00 ms, DFOV: 38.70 × 23.00 cm, Matrix: 256 × 216, slice thickness 3 mm. Ammonium acetate buffer (0.2 µM/ml) was set as the negative control, gadolinium (III) labeled DOTA-G3CNGRC (0.18 µM/ml) as the labeled probe concentration, and Gd-DTPA (2.5 µM/ml) as the positive control. The average pixels in the region above the transverse location were counted using the region of interest technique. 2.3 Cell binding experiment Cell binding experiments were carried out according to the methods reported in the references [ 16 ] and were summarized as follows: HT-1080 cells (1×10 6 cells/well) (purchased from the Cell Bank of the Chinese Academy of Sciences) were plated at uniform cell density and incubated overnight. The cells were washed 3 times with cold buffer (25 mM HEPES and 1% BSA) for 2 minutes. The cells were then mixed with 5 µL of 99m Tc-labelled G3CNGRC [the preparation conditions were as follows: freshly washed 99m TcO 4 − 20 mCi (740 MBq) and 10 µg of stannous chloride (SnCl 2 ) were added to 2 mg of DOTA G3CNGRC dissolved in deionized water, mixed evenly, and oscillated in a water bath at 38°C for 30 minutes. The reaction solution was transferred into a dialysis tube and oscillated at room temperature for 30 minutes (the dialysate was 500 ml of deionized water)], and different concentrations of Gd-DOTA − G3CNGRC were incubated for 1 hour. After washing with cold binding buffer 3 times, the cells were lysed with 200 µL lysis buffer. A gamma counter was then used to measure the radioactivity counts in each well. SPSS 29.0 software was used to perform nonlinear regression fitting on the data, and the 50% inhibition concentration (half-inhibition concentration, IC50) value of the best fit was calculated. 2.4 Establishment of a tumor-bearing animal model and magnetic resonance imaging 2.4.1 Culture of cell lines and establishment of the tumor model HT-1080 (human fibrosarcoma) (CD13 positive expression) and HT-29 (human colorectal adenocarcinoma) (CD13 negative expression) cells (purchased from Cell Bank of the Chinese Academy of Sciences) were cultured in a 75 ml flask with 10 ml complete DMEM (HT-1080)/RM1570 (HT-29)/medium with 1% penicillin and 1% streptomycin. The culture conditions were 37°C, 10% FBS, and 5% CO 2 . BALB/c nude mice were 8 weeks old (purchased from the Experimental Animal Center of Zunyi Medical University), with a total of 14 males and females randomly weighing approximately 25 g per mouse. The mice were kept in an environment free of special pathogens (SPF). HT-29 and HT-1080 cells were collected and inoculated into the right axilla of mice at 5×10 6 cells per mouse and continued to be fed, and magnetic resonance imaging was performed when the tumor grew up to approximately 1 cm. 2.4.2 Magnetic resonance imaging of the tumor model Gd-DOTA-G3CNGRC (200 µg per mouse) was injected intraperitoneally, mice were fixed on a flat board, and a 3.0T MRI T1WI scan was performed with a special coil for small animals. The sequence used was T1 weighted flash 2d (t1_fl2d) with the following imaging parameters: TR: 200 ms, TE: 2.46 ms, DFOV: 40.4×20.0 cm, Matrix: 320×272, 3 mm slice thickness. Image processing: the region of interest technique (ROI) was adopted, the tumor was selected as target tissue (T), and the muscle tissue of the left hind leg was selected as nontarget tissue (NT). The average pixels of the ROI region were counted and the T/NT ratio was calculated. After the first imaging, HT-1080 tumor-bearing mice were placed in SPF environment and given 0.5 mg/kg/ day intraperitoneal administration of ubenimex for 7 days, and magnetic resonance imaging was performed again on the 8th day to detect the changes in tumor T/NT values. To confirm the specificity of the probe, Gd-DOTA imaging of the transplanted tumor (by intraperitoneal injection of 1 mg/kg) was performed using the same sequence as Gd-DOTA-G3CNGRC. Scanning time points were set to 0.5 h, 1 h, 2 h and 4 h after injection, respectively. 2.5 Pathological examination After the first MRI scan, two HT-1080 and HT-29 tumor-bearing mice were sacrificed through cervical dislocation and tumor tissues were collected. The specimens were placed in formaldehyde solution for 48 hours and then embedded in paraffin blocks to prepare 5 µm thickness of sections, and then immunohistochemistry was performed to detect the expression of APN/CD13 in each sample. At the end of the second MRI scan, all mice were sacrificed to detect APN/CD13 expression in transplanted tumors by the same method. 2.6 Statistical analysis The statistical software used for all data analysis was SPSS 29.0. One-way analysis of variance was used to compare the means of multiple groups. P < 0.05 was set as a statistically significant difference, and measurement data were expressed as the mean ± standard deviation (mean ± std.). 3. Results 3.1 Synthesis of DOTA-G3CNGRC and Gd (III) labeling HPLC results showed that the peak retention time of DOTA-G3CNGRC was 15.136 minutes, and the purity was up to 96.39% (Supplementary Fig. 1). Mass spectrometry results showed that the actual molecular weight of DOTA-G3CNGRC was 1107.20, which was very close to the theoretical molecular weight of 1107.18 (Supplementary Fig. 2). According to the established GdCl 3 standard curve (Fig. 1 ), the labeling rate of Gd-DOTA-G3NGR was measured to be 88.99%. The average pixels of the labeled probe (Fig. 2 B) was significantly higher than that of the Ammonium acetate solution (Fig. 2 A ), but lower than that of the Gd-DTPA (Fig. 2 C) ( Table 1 ). Table 1 Signal intensity of the gadolinium labeled probe under magnetic resonance T1WI imaging(Mean ± std., n = 6) Group Concentration (µM/L) signal intensity (pixeles) F P P (LSD) Gd-DOTA 2.50 4054.33 ± 29.75 37345.10 .000 .000 * Gd-DOTA-G3CNGRC 0.18 463.67 ± 16.76 .000 ** Ammonium acetate 0.20 235.00 ± 32.33 .000 *** *, Gd-DOTA vs. Gd-DOTA-G3CNGRC; **, Gd-DOTA vs. Ammonium acetate; ***, Gd-DOTA-G3CNGRC vs. Ammonium acetate (SPSS 29.0, One-way analysis of variance, LSD test ) 3.2 Results of the cell binding experiment The CD13 receptor-binding affinity of DOTA − G3CNGRC was measured by a competitive cell binding assay using HT-1080 cells, and the 99m Tc-labelled DOTA − G3CNGRC peptide was used for competitive substitution as a CD13-specific radioligand. The IC50 of DOTA − G3CNGRC was 61.66 nM (95% CI: 57.92 nM to 65.81 nM, n = 5) (Fig. 3 ). 3.3 Results of magnetic resonance imaging in animal models When imaging was performed after intraperitoneal injection of Gd-DOTA, we found that the transplanted tumors of HT-1080 (Fig. 4 , mouse 1) and HT-29 (Fig. 4 , mouse 2) showed similar enhancement, but the peak time of enhancement was at 1 hour after injection (Fig. 5 ). HT-1080 grafts (Fig. 4 , mouse 3) showed high signals on T1WI, while HT-29 grafts showed equal signals (Fig. 4 , mouse 4). After 1 week of treatment with ubenimex, the signal intensity of HT-1080 grafts decreased significantly on T1WI (Fig. 4 , mouse 5). The highest signal intensity of HT-1080 and HT-29 grafts appeared at 2 hours after injection (Fig. 5 ). Statistical analysis results showed that the T / NT value of HT-1080 graft was significantly higher than HT-29 graft both before and after treatment (Table 2 ) at 2 hours post-injection; one hour after injection of Gd-DOTA, no difference of T/NT values was found among HT-1080 transplanted tumors (whether treated or not) and HT-29 (Table 2 ). To observe the changes in size of the transplanted tumors, the transverse diameter of the transplanted tumor were measured and recorded before and after treatment with ubenimex. The results showed that the transverse diameter of HT-1080 transplanted tumors did not change significantly before and after treatment (1.3 ± 0.1 cm vs.1.4 ± 0.1 cm, n = 5, P = 0.710), while the transverse diameter of HT-29 transplanted tumors was significantly larger after 1 week (1.5 ± 0.2 cm vs. 1.2 ± 0.2 cm, P = 0.009). Table 2 T/NT values of transplanted tumor at different time points after injection of different contrast agents(Mean ± std., n = 5) Group T/NT (Gd-DOTA) F 1 P 1 T/NT (Gd-DOTA-G3CNGRC) F 2 P 2 P (LSD) 0.5 hour 1 hour 2 hours 4 hours 0.5 hour 1 hour 2 hours 4 hours HT-1080 P 1.99 ± 0.10 2.54 ± 0.20 2.00 ± 0.24 1.92 ± 0.07 0.829 0.46 2.83 ± 0.98 3.20 ± 0.46 4.42 ± 0.38 2.69 ± 0.41 186.04 .000 .000 # HT-29 2.13 ± 0.20 2.53 ± 0.06 1.90 ± 0.14 1.81 ± 0.18 1.16 ± 0.12 1.12 ± 0.16 1.21 ± 0.18 1.13 ± 0.03 .000 ## HT-1080 A 1.97 ± 0.08 2.69 ± 0.34 2.03 ± 0.15 1.85 ± 0.17 1.51 ± 0.16 1.92 ± 0.19 1.89 ± 0.22 1.77 ± 0.15 .002 ### HT-1080 P , HT-1080 grafts were not treated with ubenimex; HT-1080 A , HT-1080 transplanted tumors were treated with ubenimex for 7 consecutive days; F 1 and P 1 , F and P were derived from 1 hour after injection of Gd-DOTA, respectively; F 2 and P 2 , F and P were derived from 2 hours after injection of Gd-DOTA-G3CNGRC, respectively; #, HT-1080 P VS. HT-1080 A ; ##, HT-1080 P vs. HT-29; ###, HT-1080 A vs. HT-29 (SPSS 29.0, One-way analysis of variance, LSD test ) 3.5 Pathology Immunohistochemical staining showed that APN/CD13 was not expressed in HT-29 transplanted tumors (Fig. 6 A, B), but was highly expressed in HT-1080 transplanted tumors (Fig. 6 C). The expression of APN/CD13 in HT-1080 grafts decreased significantly one week after treatment with ubenimex (Fig. 6 D). Discussion In addition to pathological examination methods, there are many imaging methods used to evaluate tumor angiogenesis status, and each evaluation method has unique advantages and application ranges. Nuclear medicine has the advantage of high sensitivity but low spatial resolution [ 16 , 17 ]. Magnetic resonance imaging equipment can provide a clearer anatomical structure, but magnetic resonance technology often cannot distinguish between normal (or mature) and new blood vessels [ 18 ]. The sensitivity of optical devices is high, and the imaging process is simple. However, due to the weak penetration ability of fluorescence photons and the characteristics of fluorescence quenching, the clinical application of this kind of imaging technology is currently restricted [ 19 , 20 ]. MR angiography and MR perfusion imaging are commo clinical methods used for tumor vascular evaluation [ 18 , 21 ], and the lack of specificity for neovascularization is a common defect of these methods. Whereas, magnetic resonance imaging with paramagnetic materials labeled neovasculotargeting peptides is expected to solve the problem of insufficient resolution in nuclear medicine imaging, and can obtain images with better tissue and spatial resolution, without the risk of ionizing radiation [ 22 – 24 ]. The intensity of the T1-weighted image signal of the magnetic resonance probe is related to the Gd (III) loading rate of the probe and the concentration of Gd (III) chelate [ 25 ]. The loading rate of the Gd-DOTA-G3CNGRC probes prepared in the study was similar to those of previous research reports [ 22 – 24 ]. However, there have been few previous studies on the use of magnetic resonance targeting probes to evaluate the efficacy of antiangiogenic drugs or antitumour therapy drugs. Our results showed that the T/NT ratio of the same group of tumour-bearing mice decreased significantly after 7 days of ubenimex treatment, but the transverse diameter of the tumor did not change significantly, suggesting that the CD13 activity expressed by the tumor was inhibited after treatment. In addition, on Gd-DOTA imaging, HT-1080 transplanted tumors (pre - and post-treatment) and HT-29 transplanted tumors showed significant enhancement. Moreover, the enhancement time was different from that of Gd-DOTA-G3CNGRC and there was no difference in T/NT values, which further confirmed the high specificity of Gd-DOTA-G3CNGRC in CD13-positive tumors. In the mouse tumor model tested in this experiment, HT-29 tumors with negative expression of APN/CD13 had little uptake of Gd-DOTA-G3CNGRC, while HT-1080 tumors with positive expression of CD13 obviously took up GD-DOTA-G3CNGRC, suggesting that during the synthesis and labeling of DOTA-G3CNGRC, the activity of G3CNGRC was not significantly affected. Compared with previous reports, our imaging results basically reached the same conclusion [ 15 , 22 – 24 ]. However, in contrast to these reports, our study mainly focused on the feasibility of early evaluation of the efficacy of antiangiogenic drugs, so DOTA-G3CNGRC was not used for blocking experiments in our study. Studies have shown that APN/CD13 is upregulated in the blood vessels of many solid tumors, and the expression of cancer cells, stroma and/or vasculature can be observed depending on the tumor type [ 12 , 13 ]. The formation of neovascularization is closely related to tumor growth, metastasis and invasion, so reducing the expression of APN/CD13 in tumor cells has a significant promoting effect on improving the therapeutic effect of antitumour drugs [ 26 – 28 ]. At present, a variety of antagonists targeting APN/CD13 have entered the clinical application or clinical trial stage, such as bestatin (ubenimex), amastatin, and probestin [ 13 , 29 , 30 ], among which ubenimex is the most commonly used in the clinic. Its mechanism of action may be through targeting the CD13/EMP3/FAK/NF-κB pathway, affecting autophagy and apoptosis, improving chemotherapy drug sensitivity, and thus reversing drug resistance [ 30 ]. Anti-tumour angiogenesis is currently a commonly used method, including many types of drugs, which have also been clinically applied [ 31 , 32 ]. Most patients may experience significant side effects for a period of time after usage, and resistance may develop subsequently [ 33 – 34 ]. Therefore, in addition to detecting serum tumor markers, imaging of a target should also be performed for direct evaluation, especially early assessment. Our findings introduced a new aspect to these approaches, but further research should be needed on various types of tumor models. Conclusion The Gd-DOTA-G3CNGRC probe prepared in this study showed a good affinity for CD13-positive tumor cells in vitro experiments, and a high T/NT ratio for APNCD13-positive graft tumors. After 1 week of anti-APN /CD13 treatment, although there was no significant change in the transverse diameter of the APN/ CD13-positive graft tumor, the uptake of the probe was significantly reduced. Therefore, we believed that Gd-DOTA-G3CNGRC magnetic resonance imaging can be used as an imaging method to evaluate the early efficacy of anti-angiogenesis studies. Declarations Competing Interests The authors declare no conflicts of interest. Funding This work supported by Science and Technology Department of Guizhou Province grant: No. [2019]1325. Author Contribution Authors in order:The author, Songsong Liu, participated in the research process, data collection, analysis and interpretation of data, drafting papers, statistical analysis;Sheng Han participated in the research process, data collection, analysis and interpretation of data, drafting papers, statistical analysis;Gongwei Jing participated in research process, data collection, analysis and interpretation of data, statistical analysis;Pinqin Wang participated in the research process, analysis and interpretation of MRI data, and supporting work;Yanteng Zhang participated in the research process, MRI data collection, statistical analysis,technical and material support;Ling Xiong participated in the research process, data collection, administration, technical and material support;Yingfang Zhang , participated in the research process, IC50 data collection, literature review and material support;Huasheng Qu participated in the research process, 99mTc-DOTA-G3CNGRCdata collection, literature review and material support;Bingxiu Ren participated in the design, implementation research, data collection, analysis and interpretation of data, drafting papers, critical review of the intellectual content of the manuscript, access to research funding. Acknowledgement Thanks to all the staff of the central laboratory of the Third Affiliated Hospital of Zunyi Medical University (Zunyi First People's Hospital) for their selfless help in this study. Thanks for the funding of this study from Guizhou Science and Technology Department project: [2019] 1325. Data Availability All data generated or analysed during this study are included in this published article [and its supplementary information files]. References Jiajie Kuai, Chenchen Han, Wei Wei. Potential Regulatory Roles of GRK2 in Endothelial Cell Activity and Pathological Angiogenesis. Frontiers in immunology 2021; 698424. Luca Crippa, Mimma Bianco, Barbara Colombo, et al. A new chromogranin A-dependent angiogenic switch activated by thrombin. Blood 2013; 121:392-402. Liu Chao, Castillo Alesha B. Targeting Osteogenesis-Angiogenesis Coupling for Bone Repair. Journal of the American Academy of Orthopaedic Surgeons 2018; 26 (7): e153-55. Arjan W. Griffioen and Andrew C. Dudley. 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Synthesis, characterization and relaxivity validations of Gd (III) complex of DOTA tetrahydrazide as MRI contrast agent. Journal of Molecular Structure 2022; 1255: 132474. Pereira Flavia E, Cronin Chunxia, Ghosh Mallika, et al. CD13 is essential for inflammatory trafficking and infarct healing following permanent coronary artery occlusion in mice. Cardiovascular Research 2013; 100 (1): 74-83. Juho J Miettinen, Romika Kumari, Gunnhildur Asta Traustadottir, et al. Aminopeptidase expression in multiple myeloma associates with disease progression and sensitivity to melflufen. Cancers 2021; 13 (7) : 1527. Ting Xiu, Qie Guo, Fanjing Jing, Yunyan Shi, Fanbo Jing. CD13 downregulation mediated by ubenimex inhibits autophagy to overcome 5-FU resistance by disturbing the EMP3/FAK/NF-κB pathway in gastric cancer cells. Transl Cancer Res 2022; 11 (8): 2487-2500. Yangyang Liu, Dongsheng Zhao, Chenghua Zhang, et al. Development of Hydroxamate Derivatives Containing a Pyrazoline Moiety as APN Inhibitors to Overcome Angiogenesis. Molecules 2022; 27: 8339. Jiangying Cao, Wei Zhao, Chunlong Zhao, et al. Development of a Bestatin-SAHA Hybrid with Dual Inhibitory Activity against APN and HDAC. Molecules 2020; 25: 4991. Samman Munir, Asad Ali Shah, Muhammad Shahid, et al. Anti-angiogenesis potential of phytochemicals for the therapeutic management of tumors. Current pharmaceutical design 2020; 26 (2): 265-78. Raluca Ioana Teleanu, Cristina Chircov, Alexandru Mihai Grumezescu, Daniel Mihai Teleanu. Tumor Angiogenesis and Anti-Angiogenic Strategies for Cancer Treatment. Journal of clinical medicine 2019; 9 (1): 84. Shinji Nakamichi, Kaoru Kubota,Kotone Matsuyama, et al. A PhaseⅡStudy of Ubenimex Combined With Pembrolizumab, Nab-Paclitaxel, and Carboplatin for Previously Untreated Advanced Squamous Non-Small-Cell Lung Cancer: TORG2241 (UBE-Q) 2023. Clinical Lung Cancer 2024; 25 (1): 85-90. Yunyan Shi, Qie Guo, Fanjing Jing, Xiuling Shang, Changkai Zhou, Fanbo Jing. Ubenimex suppresses glycolysis mediated by CD13/Hedgehog signaling to enhance the effect of cisplatin in liver cancer. Transl Cancer Res 2023;12 (10): 2823-36. Additional Declarations No competing interests reported. Supplementary Files Supportinginformation.doc Cite Share Download PDF Status: Published Journal Publication published 01 Jul, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 03 Feb, 2025 Reviews received at journal 03 Feb, 2025 Reviews received at journal 16 Jan, 2025 Reviewers agreed at journal 15 Jan, 2025 Reviewers agreed at journal 09 Jan, 2025 Reviewers agreed at journal 04 Jan, 2025 Reviewers invited by journal 13 Nov, 2024 Editor assigned by journal 07 Nov, 2024 Editor invited by journal 07 Nov, 2024 Submission checks completed at journal 04 Nov, 2024 First submitted to journal 16 Oct, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5274314","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":377781159,"identity":"9153ad93-1e10-4923-8f03-51c249b8ebcc","order_by":0,"name":"Sheng Han","email":"","orcid":"","institution":"The Third Affiliated Hospital of Zunyi Medical University, The First People's Hospital of Zunyi City)","correspondingAuthor":false,"prefix":"","firstName":"Sheng","middleName":"","lastName":"Han","suffix":""},{"id":377781160,"identity":"77537d88-1c3d-40be-b501-101f0cacfd97","order_by":1,"name":"Song-Song Liu","email":"","orcid":"","institution":"The Third Affiliated Hospital of Zunyi Medical University, The First People's Hospital of Zunyi City)","correspondingAuthor":false,"prefix":"","firstName":"Song-Song","middleName":"","lastName":"Liu","suffix":""},{"id":377781161,"identity":"47580de3-315f-4691-94e0-0cda04b684f2","order_by":2,"name":"Gong-Wei Jing","email":"","orcid":"","institution":"The Third Affiliated Hospital of Zunyi Medical University, The First People's Hospital of Zunyi City)","correspondingAuthor":false,"prefix":"","firstName":"Gong-Wei","middleName":"","lastName":"Jing","suffix":""},{"id":377781162,"identity":"25f109ac-dca5-4f0e-8976-800f7c534014","order_by":3,"name":"Pin-Qin Wang","email":"","orcid":"","institution":"The Third Affiliated Hospital of Zunyi Medical University, The First People's Hospital of Zunyi City)","correspondingAuthor":false,"prefix":"","firstName":"Pin-Qin","middleName":"","lastName":"Wang","suffix":""},{"id":377781163,"identity":"f1e0c2a3-a774-4118-8afb-bccb75636f82","order_by":4,"name":"Yan-Teng Zhang","email":"","orcid":"","institution":"The Third Affiliated Hospital of Zunyi Medical University, The First People's Hospital of Zunyi City)","correspondingAuthor":false,"prefix":"","firstName":"Yan-Teng","middleName":"","lastName":"Zhang","suffix":""},{"id":377781164,"identity":"4baad8de-6c26-4305-8f3c-6ec1efb590a2","order_by":5,"name":"Ling Xiong","email":"","orcid":"","institution":"The Third Affiliated Hospital of Zunyi Medical University, The First People's Hospital of Zunyi City)","correspondingAuthor":false,"prefix":"","firstName":"Ling","middleName":"","lastName":"Xiong","suffix":""},{"id":377781165,"identity":"07ced840-5120-45d0-8bb5-fdb0d26cd1e6","order_by":6,"name":"Ying-Fang Zhang","email":"","orcid":"","institution":"The Third Affiliated Hospital of Zunyi Medical University, The First People's Hospital of Zunyi City)","correspondingAuthor":false,"prefix":"","firstName":"Ying-Fang","middleName":"","lastName":"Zhang","suffix":""},{"id":377781166,"identity":"90ac0371-4432-46e1-b0f9-9cba38e577f6","order_by":7,"name":"Hua-Sheng Qu","email":"","orcid":"","institution":"The Third Affiliated Hospital of Zunyi Medical University, The First People's Hospital of Zunyi City)","correspondingAuthor":false,"prefix":"","firstName":"Hua-Sheng","middleName":"","lastName":"Qu","suffix":""},{"id":377781168,"identity":"242476a8-e84d-42b7-9f8a-3ac40e460252","order_by":8,"name":"Bing-Xiu Ren","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvUlEQVRIiWNgGAWjYFAC/o8PgKQcG3vzAaL1GBuACD6eYwlEazETABKJ8yRyFIhTb3C7IY25ouJOehtDDgPDj4ptRGi5c+DYwzNnnuW2MZw9wNhz5jYRWm4kths2th3ObWPsS2BmbCNKSzKbZOO/w+lszDwGxGpJA2ppOJzAxkasFskbOcyGDccOG7bxsCUcJMovfDdyGB821ByWl5//+OCDHxVEaFE4gMQ5gEMRKpBvIErZKBgFo2AUjGgAAOrIPvRGDkGdAAAAAElFTkSuQmCC","orcid":"","institution":"The Third Affiliated Hospital of Zunyi Medical University, The First People's Hospital of Zunyi City)","correspondingAuthor":true,"prefix":"","firstName":"Bing-Xiu","middleName":"","lastName":"Ren","suffix":""}],"badges":[],"createdAt":"2024-10-16 08:53:37","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5274314/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5274314/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-05905-1","type":"published","date":"2025-07-01T15:58:35+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":70385352,"identity":"d9942197-9dac-402e-a4ad-75bd1407a4b2","added_by":"auto","created_at":"2024-12-02 17:07:39","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":89947,"visible":true,"origin":"","legend":"\u003cp\u003eStandard curve of\u0026nbsp; Gd (III) concentration and OD value of xylenol orange mixture. After gadolinium (III) of different concentrations were mixed with xylenoline orange, the relationship between optical density (x) and gadolinium (III) concentration (y) at a wavelength of 570 nm was calculated by SPSS 29.0, and the relationship between the two can be expressed by the equation y=0.0649x+0.0439. The ‘R\u003csup\u003e2\u003c/sup\u003e =0.9033’ indicated a high correlation between the ‘x’ and ‘y’.\u003c/p\u003e","description":"","filename":"Figure1.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5274314/v1/a38aad72a922394a8a44c060.jpg"},{"id":70385355,"identity":"28c5752c-d010-42e3-b82b-e024f3093b74","added_by":"auto","created_at":"2024-12-02 17:07:43","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":63772,"visible":true,"origin":"","legend":"\u003cp\u003eMagnetic resonance imaging of Gd-DOTA-G3CNGRC. TheT1-weighted TSE dark-fluid was performed under the following condition :TR: 2130.00 ms, TE: 13.00 ms, DFOV: 38.70× 23.00 cm, Matrix: 256×216, slice thickness 3 mm. Ammonium acetate (A) showed a slightly low signal , gadolinium (III)-labeled DOTA-G3CNGRC (B) showed a high signal, and Gd-DTPA (C) showed a significantly high signal.\u003c/p\u003e","description":"","filename":"Figure2.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5274314/v1/4b6836be18660a188ff18c67.jpg"},{"id":70385364,"identity":"8f034048-c901-482f-93f1-43841c786911","added_by":"auto","created_at":"2024-12-02 17:08:02","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":318853,"visible":true,"origin":"","legend":"\u003cp\u003eCell binding experiment of DOTA-G3CNGRC. In HT-1080 cells, \u003csup\u003e99m\u003c/sup\u003eTc-DOTA-G3CNGRC binding to the CD13 receptor was inhibited by DOTA-G3-CNGRC in a dose-dependent manner. The concentration of DOTA-G3-CNGRC peptide was between 1.0~120 nM. The half-inhibition concentration (IC50) of DOTA-G3-CNGRC was 61.66 nM (95% CI: 57.92 nM to 65.81 nM, n = 5).\u003c/p\u003e","description":"","filename":"Figure3.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5274314/v1/10255c5f7926ebb22d9382d4.jpg"},{"id":70385431,"identity":"df63611b-5955-4dbb-bf78-c0a9799e01a0","added_by":"auto","created_at":"2024-12-02 17:10:21","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":858584,"visible":true,"origin":"","legend":"\u003cp\u003eMagnetic resonance imaging of tumor-bearing mouse model. After intraperitoneal injection of Gd-DOTA-G3CNGRC (200ug per mouse) and Gd-DOTA (1 mg/kg), T1-weighted images were acquired with the sequence of t1_fl2d and the following parameters, TR: 258 ms, TE: 2.46 ms, DFOV：40.4 × 20.0 cm, Matrix：320 × 272 , 3 mm slice thickness. The solid green circle is located in the target tissue (the transplanted tumor), and the dotted green circle is located in the left hind leg (the non-target tissue), and the area of the two coils is equal for each mouse.HT-1080 (mouse 1) and HT-29 (mouse 2) grafts in the Gd-DOTA group showed similar intensification trends and the highest signal intensity occurred within 1 hour after injection. After injection of Gd-DOTA-G3CNGRC, HT-1080 tumor graft (mouse 3) showed the highest signal intensity at 2 hours, while HT-29 tumor graft (mouse 4) only showed similar signal intensity to non-target tissue (left hind leg). After 1 week of intraperitoneal injection of 0.5 mg /kg/ day of ubenimex, HT-1080 tumor graft signal decreased significantly, but slightly higher signal intensity was still seen within 2 hours after injection (mouse 5).\u003c/p\u003e","description":"","filename":"figure4.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5274314/v1/300f2727b130ab78167f7f68.jpg"},{"id":70385407,"identity":"96cac787-6237-483f-818e-539b7bb97528","added_by":"auto","created_at":"2024-12-02 17:09:20","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":325319,"visible":true,"origin":"","legend":"\u003cp\u003eTarget (T) to non-target ratio (NT) statistics of HT-1080 and HT-29 grafts. After intraperitoneal injection of Gd-DOTA-G3CNGRC (200ug per mouse) and Gd-DOTA (1 mg/kg), region of interest technique (ROI) was adopted, tumor tissue was used as target tissue and left hind leg muscle was used as non-target tissue. Tissues with the same area were selected and average pixels in ROI regions were counted at different time points. In the Gd-DOTA-G3CNGRC group, the maximum T/NT value of HT-1080 grafted tumors appeared at 2 hours after injection, but decreased significantly at 1 week after treatment with ubenimex (4.22 ± 0.64 vs. 1.90± 0.22, n = 5, the \u003cem\u003eP\u003c/em\u003e-value was less than 0.001); The T/NT ratio of HT-29 transplanted tumors showed a peak value at 0.5 h after injection, but it was significantly lower than that of HT-1080 transplanted tumors before and after treatment ( 1.21 ± 0.18 vs. 4.22± 0.64, 1.21 ± 0.18 vs. 1.90 ± 0.22, n = 5, the \u003cem\u003eP\u003c/em\u003e-values were 0.001 and 0.002, respectively). In the Gd-DOTA group, the T/NT ratio of HT-1080 transplanted tumors appeared at 1 hour after injection whether before or after treatment, and the same situation also appeared in HT-29 transplanted tumors, and there was no significant difference in T/NT ratio among the three groups (the T/NT ratios of the three groups of transplanted tumors at 1 h after injection were HT-1080: 2.55 ± 0.20, HT-1080 after treatment: 2.70 ± 0.34, and HT-29: 2.53 ± 0.06, respectively，n = 5, all the P values were greater than 0.05).\u003c/p\u003e","description":"","filename":"Figure5.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5274314/v1/88049e453f722438baba0917.jpg"},{"id":70385448,"identity":"7d564678-2034-44e6-8470-6482f2f5bc87","added_by":"auto","created_at":"2024-12-02 17:10:54","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2098106,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemical staining of ANP/CD13 expression in transplanted tumors. HT-29 grafts (A,B) showed no significant expression of ANP/CD13, HT-1080 grafts showed high expression of ANP/CD13 before treatment with ubenimex (arrows), and significantly decreased expression of ANP/CD13 at 7 days after treatment with ubenimex (arrows) (20×).\u003c/p\u003e","description":"","filename":"Figure6.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5274314/v1/7654e008f9dfcbb14b7b5782.jpg"},{"id":86179226,"identity":"10b0509c-f7e6-4569-896d-873f0942fff9","added_by":"auto","created_at":"2025-07-07 16:17:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4321512,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5274314/v1/fd4c39ef-f9b1-4a0b-96f7-c11cefae593f.pdf"},{"id":70385372,"identity":"fc659315-2fc7-40ff-a321-04e2ff64937a","added_by":"auto","created_at":"2024-12-02 17:08:12","extension":"doc","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":536576,"visible":true,"origin":"","legend":"","description":"","filename":"Supportinginformation.doc","url":"https://assets-eu.researchsquare.com/files/rs-5274314/v1/f2da326fe53060a054c31d48.doc"}],"financialInterests":"No competing interests reported.","formattedTitle":"Early evaluation of anti-angiogenic effects with gadolinium (III) labeled APN/CD13 specific binding peptides magnetic resonance imaging","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe healing process of almost all chronic diseases and acute injuries is accompanied by endothelial cell activation and angiogenesis, and the process of angiogenesis has an important impact on the final prognosis of different diseases. For some diseases, providing conditions to promote angiogenesis is considered to be one of the measures to promote the improvement of these diseases, such as fractures and wound healing disorders. However, for other diseases, especially malignant tumors, anti-angiogenesis is considered one of the therapeutic means to achieve better outcomes for these diseases. Therefore, the search for and identification of new receptors selectively expressed by activated endothelial cells and the discovery of vascular function regulators or new ligands that can interact with them has become a hot spot in vascular target imaging and therapy research [\u003cspan additionalcitationids=\"CR2 CR3 CR4\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e–\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAminopeptidase N (APN/CD13) (EC 3.4.11.2) is a type II exonuclide of approximately 150–240 kDa that is involved in the degradation of neutral or alkaline N-terminal residues of biologically active peptides. This protein is a member of the zinc metallopeptidase M1 family and consists of an enzyme-promoting extracellular domain, a transmembrane region and a short cytoplasmic domain involved in signal transduction [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. It was found that the expression of this enzyme was up-regulated in angiogenic vessels caused by various etiologies, while it was little or no expression in normal vessels [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e–\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Studies have shown that APN/CD13 plays an important role in cancer development, progression and even treatment, especially the importance of APN expression and activities related to cancer characteristics, especially tumor angiogenesis and metastasis [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe Asn-Gly-Arg (NGR) peptide was originally discovered by selecting peptide-phage libraries in tumor mice [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. A large number of previous studies have shown that the NGR peptide can bind specifically to aminopeptidase N (APN/CD13) in vivo and in vitro. Because of these properties, the NGR peptide has been used by many researchers as a ligand-directed vector for the delivery of various therapeutics and imaging agents to angiogenic vessels [\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e–\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Most of the previous research reports focused on targeted therapeutic drugs or imaging probes based on NGR carriers, while relatively few reports have evaluated the efficacy of anti-angiogenic drugs based on NGR carrier probes[\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e–\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Therefore, in this study, Gd-DOTA-G3CNGRC was prepared, and imaging was performed on tumor-bearing mouse models to provide a magnetic resonance molecular imaging method for the early efficacy evaluation of antiangiogenic drugs.\u003c/p\u003e"},{"header":"1. Experimental materials and instruments","content":"\u003cp\u003e1.1 Experimental materials\u003c/p\u003e\u003cp\u003e2-Chloro-triphenylmethyl chloride resin, Fmoc-AA-OH (N-[fluorent-methoxycarbonyl]-l-alanyl-L-alanine), 5 g of indanhydrone-100 ml of ethanol, pyridine, N-(9-fluorent-methoxycarbonyl)-S-triphenylmethyl-L-cysteine (Fmoc-CYS (TRT)-Oh), Fmoc-ARG (PBF)-OH (N- (9-fluorene methoxycarbonyl)-S-triphenylmethyl-L-cysteine), N-fluorenyl methoxyl-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine (FMOC-Arg(Pbf) OH), and DOTA-NHS (1,4,7,10-tetraazecyclododecane-1,4,7,10-tetraacetic acid) were of analytical grade and purchased from Hefei Bairdi Chemical Technology Co., LTD., China. Anhui). Analytical grade DCM (dichloromethane), DIEA (N,N-diisopropyl ethylamine), methanol, DMF (N,N-dimethylformamide), HOBT (1-hydroxybenzotriazole), and DIC (N,N'-diisopropyl carbodiimide) were purchased from Tianjin Concord Technology Co., LTD. (Tianjin, China). Bestatin (Ubenimex), GdCl\u003csub\u003e3\u003c/sub\u003e.6H\u003csub\u003e2\u003c/sub\u003eO, Gd-DOTA,and xylenol orange were purchased from Sigma‒Aldrich. The cell culture and immunohistochemical staining related reagents used in this study were purchased from Gibco, a brand owned by Thermo Fisher Scientific Inc.\u003c/p\u003e\u003cp\u003e1.2 Main experimental instruments\u003c/p\u003e\u003cp\u003eVacuum freeze-dryer/freeze-dryer (LGJ-30FD, Beijing Songyuan Huaxing Technology Development Co., LTD.), preparation-grade HPLC (P3000Ⅰ, Beijing Innovation Tongheng Chromatography Technology Co., LTD.), Shimadzu Liquid chromatocl-Mass Spectrometry Instrument (LCMS2020) and Shimadzu HPLC analysis instrument (LC-10AT 2010A) are the products of Jinna Instrument Equipment (Shanghai) Co., LTD., continuous electric deionization ultra-pure water machine (YL-EDI60, Shanghai Yilan Environmental Protection Technology Co., LTD.) magnetic resonance imaging instrument (SIEMENS, MAGNETOM, skyra3.0T).\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cp\u003e2.1 Synthesis of DOTA-G3CNGRC\u003c/p\u003e\u003cp\u003eFirstly, the 2-chloro-triphenylmethyl chloride resin (500 mg) was immersed in DCM for a duration of 30 minutes, followed by three washes with DMF and one wash with DCM. Then a mixture containing 0.15 mM of Fmoc-CYS (TRT) OH, 8 ml anhydrous DCM and 800 µL DIEA was added and reacted under nitrogen bubble condition for 1.5 hours. After washing with DMF for 3 times, a mixture containing 0.5 ml DIEA, 0.5 ml methanol and 2 ml DCM was added and the reaction continued for 20 minutes. Then draining the liquid in the reactor with a circulating water vacuum pump, add DMF about 3 times the volume of resin to the reactor, wash for 30 seconds, drain the liquid, and repeat the operation 4 times. Secondly, 20% of piperidine / DMF solution (with 3 times the volume of resin) was added to the reactor and bubble nitrogen for 20 minutes. Then wash 4 times according to the above washing method. Finally, take the above 10–20 pieces of resin, add two drops of detection reagent A and B, and heat for 2 minutes in a 100℃ incubator to observe the color of the resin. The coloring of the resin means that the Fmoc was successfully removed; if no color appears, the operation needs to be repeated again. Coupling: 0.5 mM of Fmoc-ARG (PBF)-OH and 0.5 mM of HOBT (75 mg) were dissolved in 1ml DMF, followed by 100 µL DIC, mixed for 1min, and then the mixture was added to the drained resin for a nitrogen bubble reaction of 1 hour. The resin was then tested and washed repeatedly. After the peptide synthesis, the Fmoc was removed and washed, and then DMF containing 0.3 nM DOTA-NHS 2 ml and 100 µL DIEA were added in sequence for 20 minutes to make the N-terminal of the polypeptide modified by DOTA. Finally, following the method described above, the resin is washed three times with methanol, followed by the removal of the liquid and vacuum drying until the resin appeared as a granular substance. Cut the polypeptide from the resin and purify the polypeptide: add the cut solution (100 ml of the cutting reagent contains 95 ml TFA + 1 ml water + 2 ml EDT + 2 ml Tis) to the dried resin, and then add methyl tert-butyl ether (pre-cooled at -20℃) equivalent to 10 times the volume of the cutting solution for sedimentation of the cut polypeptide. Finally, the peptide was purified using an 8 micron 30×250 mm diagesol column. Mobile phase: A: 0.1% TFA aqueous solution B: 0.1% TFA acetonitrile solution; the flow rate was 12 ml/min, the sample was loaded through pump A, and then 10% acetonitrile water was operated to balance the gradient for 5 min, and the sample peak for collection and detection was prepared. When the purity of the analysis was greater than 80%, the sample was freezable for use.\u003c/p\u003e\u003cp\u003e2.2 Labeling DOTA-G3CNGRC with Gd (III)\u003c/p\u003e\u003cp\u003eFirstly, 1 mg (0.9µM) of DOTA-NGR was dissolved in 9 ml of acetic acid buffer with a pH of 7.4, followed by 10 ml of GdCl3 solution with a concentration of 10 µM/L, the molar ratio of Gd (III) to NGR was about 11:1, and the reaction was oscillated in a 38℃ water bath for 30 minutes. Secondly, the mixture was transferred into a 10 ml dialysis tube (Spectra/Por® Flot-A-Lyzer ®, MWCO: 500 − 100), then the tube was placed in a beaker containing 500 ml of deionized water and dialyzed with a magnetic stirrer for 1 hour. Finally, the marking rate was determined: 6 ml of the prepared xylenol orange solution (concentration 2 g/L) was added to a 12-well cell culture plate at 1 ml per well, GdCl3 solution at different concentrations (0.1 ml) was added, and the enzyme marker (Varioskan LUX, Thermo Fisher Scientific Inc.) was used. The OD value of each well was determined at 575 nm, and the standard curve and equation of Gd (III) content were established. After dialysis, the liquid in the beaker was added to the xylenol-containing orange hole at a rate of 0.1 ml per hole, the OD value of each hole was determined, the concentration of free Gd (III) was calculated, and the loading rate of Gd (III) was calculated according to the concentration. To further verify the successful labeling of the probe, we performed magnetic resonance imaging on the labeled probe. The sequence used for magnetic resonance imaging was T1-weighted TSE dark-fluid, and the collection conditions were as follows: TR: 2310.00 ms, TE: 13.00 ms, DFOV: 38.70 × 23.00 cm, Matrix: 256 × 216, slice thickness 3 mm. Ammonium acetate buffer (0.2 µM/ml) was set as the negative control, gadolinium (III) labeled DOTA-G3CNGRC (0.18 µM/ml) as the labeled probe concentration, and Gd-DTPA (2.5 µM/ml) as the positive control. The average pixels in the region above the transverse location were counted using the region of interest technique.\u003c/p\u003e\u003cp\u003e2.3 Cell binding experiment\u003c/p\u003e\u003cp\u003eCell binding experiments were carried out according to the methods reported in the references [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] and were summarized as follows: HT-1080 cells (1×10\u003csup\u003e6\u003c/sup\u003e cells/well) (purchased from the Cell Bank of the Chinese Academy of Sciences) were plated at uniform cell density and incubated overnight. The cells were washed 3 times with cold buffer (25 mM HEPES and 1% BSA) for 2 minutes. The cells were then mixed with 5 µL of \u003csup\u003e99m\u003c/sup\u003eTc-labelled G3CNGRC [the preparation conditions were as follows: freshly washed \u003csup\u003e99m\u003c/sup\u003eTcO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e−\u003c/sup\u003e 20 mCi (740 MBq) and 10 µg of stannous chloride (SnCl\u003csub\u003e2\u003c/sub\u003e) were added to 2 mg of DOTA G3CNGRC dissolved in deionized water, mixed evenly, and oscillated in a water bath at 38°C for 30 minutes. The reaction solution was transferred into a dialysis tube and oscillated at room temperature for 30 minutes (the dialysate was 500 ml of deionized water)], and different concentrations of Gd-DOTA − G3CNGRC were incubated for 1 hour. After washing with cold binding buffer 3 times, the cells were lysed with 200 µL lysis buffer. A gamma counter was then used to measure the radioactivity counts in each well. SPSS 29.0 software was used to perform nonlinear regression fitting on the data, and the 50% inhibition concentration (half-inhibition concentration, IC50) value of the best fit was calculated.\u003c/p\u003e\u003cp\u003e2.4 Establishment of a tumor-bearing animal model and magnetic resonance imaging\u003c/p\u003e\u003cp\u003e2.4.1 Culture of cell lines and establishment of the tumor model\u003c/p\u003e\u003cp\u003eHT-1080 (human fibrosarcoma) (CD13 positive expression) and HT-29 (human colorectal adenocarcinoma) (CD13 negative expression) cells (purchased from Cell Bank of the Chinese Academy of Sciences) were cultured in a 75 ml flask with 10 ml complete DMEM (HT-1080)/RM1570 (HT-29)/medium with 1% penicillin and 1% streptomycin. The culture conditions were 37°C, 10% FBS, and 5% CO\u003csub\u003e2\u003c/sub\u003e. BALB/c nude mice were 8 weeks old (purchased from the Experimental Animal Center of Zunyi Medical University), with a total of 14 males and females randomly weighing approximately 25 g per mouse. The mice were kept in an environment free of special pathogens (SPF). HT-29 and HT-1080 cells were collected and inoculated into the right axilla of mice at 5×10\u003csup\u003e6\u003c/sup\u003e cells per mouse and continued to be fed, and magnetic resonance imaging was performed when the tumor grew up to approximately 1 cm.\u003c/p\u003e\u003cp\u003e2.4.2 Magnetic resonance imaging of the tumor model\u003c/p\u003e\u003cp\u003eGd-DOTA-G3CNGRC (200 µg per mouse) was injected intraperitoneally, mice were fixed on a flat board, and a 3.0T MRI T1WI scan was performed with a special coil for small animals. The sequence used was T1 weighted flash 2d (t1_fl2d) with the following imaging parameters: TR: 200 ms, TE: 2.46 ms, DFOV: 40.4×20.0 cm, Matrix: 320×272, 3 mm slice thickness. Image processing: the region of interest technique (ROI) was adopted, the tumor was selected as target tissue (T), and the muscle tissue of the left hind leg was selected as nontarget tissue (NT). The average pixels of the ROI region were counted and the T/NT ratio was calculated. After the first imaging, HT-1080 tumor-bearing mice were placed in SPF environment and given 0.5 mg/kg/ day intraperitoneal administration of ubenimex for 7 days, and magnetic resonance imaging was performed again on the 8th day to detect the changes in tumor T/NT values. To confirm the specificity of the probe, Gd-DOTA imaging of the transplanted tumor (by intraperitoneal injection of 1 mg/kg) was performed using the same sequence as Gd-DOTA-G3CNGRC. Scanning time points were set to 0.5 h, 1 h, 2 h and 4 h after injection, respectively.\u003c/p\u003e\u003cp\u003e2.5 Pathological examination\u003c/p\u003e\u003cp\u003eAfter the first MRI scan, two HT-1080 and HT-29 tumor-bearing mice were sacrificed through cervical dislocation and tumor tissues were collected. The specimens were placed in formaldehyde solution for 48 hours and then embedded in paraffin blocks to prepare 5 µm thickness of sections, and then immunohistochemistry was performed to detect the expression of APN/CD13 in each sample. At the end of the second MRI scan, all mice were sacrificed to detect APN/CD13 expression in transplanted tumors by the same method.\u003c/p\u003e\u003cp\u003e2.6 Statistical analysis\u003c/p\u003e\u003cp\u003eThe statistical software used for all data analysis was SPSS 29.0. One-way analysis of variance was used to compare the means of multiple groups. \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 was set as a statistically significant difference, and measurement data were expressed as the mean ± standard deviation (mean ± std.).\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e3.1 Synthesis of DOTA-G3CNGRC and Gd (III) labeling\u003c/p\u003e\u003cp\u003eHPLC results showed that the peak retention time of DOTA-G3CNGRC was 15.136 minutes, and the purity was up to 96.39% (Supplementary Fig.\u0026nbsp;1). Mass spectrometry results showed that the actual molecular weight of DOTA-G3CNGRC was 1107.20, which was very close to the theoretical molecular weight of 1107.18 (Supplementary Fig.\u0026nbsp;2). According to the established GdCl\u003csub\u003e3\u003c/sub\u003e standard curve (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), the labeling rate of Gd-DOTA-G3NGR was measured to be 88.99%. The average pixels of the labeled probe (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) was significantly higher than that of the Ammonium acetate solution (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA ), but lower than that of the Gd-DTPA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) ( Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"±\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSignal intensity of the gadolinium labeled probe under magnetic resonance T1WI imaging(Mean ± std., n = 6)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eConcentration (µM/L)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003esignal intensity (pixeles)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e (LSD)\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGd-DOTA\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.50\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c3\"\u003e \u003cp\u003e4054.33 ± 29.75\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e37345.10\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e.000\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e.000\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGd-DOTA-G3CNGRC\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.18\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c3\"\u003e \u003cp\u003e463.67 ± 16.76\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e.000\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmmonium acetate\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.20\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c3\"\u003e \u003cp\u003e235.00 ± 32.33\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e.000\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003e*, Gd-DOTA vs. Gd-DOTA-G3CNGRC; **, Gd-DOTA vs. Ammonium acetate; ***, Gd-DOTA-G3CNGRC vs. Ammonium acetate (SPSS 29.0, One-way analysis of variance, LSD test )\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003cp\u003e3.2 Results of the cell binding experiment\u003c/p\u003e\u003cp\u003eThe CD13 receptor-binding affinity of DOTA − G3CNGRC was measured by a competitive cell binding assay using HT-1080 cells, and the \u003csup\u003e99m\u003c/sup\u003eTc-labelled DOTA − G3CNGRC peptide was used for competitive substitution as a CD13-specific radioligand. The IC50 of DOTA − G3CNGRC was 61.66 nM (95% CI: 57.92 nM to 65.81 nM, n = 5) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e3.3 Results of magnetic resonance imaging in animal models\u003c/p\u003e\u003cp\u003eWhen imaging was performed after intraperitoneal injection of Gd-DOTA, we found that the transplanted tumors of HT-1080 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, mouse 1) and HT-29 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, mouse 2) showed similar enhancement, but the peak time of enhancement was at 1 hour after injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). HT-1080 grafts (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, mouse 3) showed high signals on T1WI, while HT-29 grafts showed equal signals (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, mouse 4). After 1 week of treatment with ubenimex, the signal intensity of HT-1080 grafts decreased significantly on T1WI (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, mouse 5). The highest signal intensity of HT-1080 and HT-29 grafts appeared at 2 hours after injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Statistical analysis results showed that the T / NT value of HT-1080 graft was significantly higher than HT-29 graft both before and after treatment (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) at 2 hours post-injection; one hour after injection of Gd-DOTA, no difference of T/NT values was found among HT-1080 transplanted tumors (whether treated or not) and HT-29 (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). To observe the changes in size of the transplanted tumors, the transverse diameter of the transplanted tumor were measured and recorded before and after treatment with ubenimex. The results showed that the transverse diameter of HT-1080 transplanted tumors did not change significantly before and after treatment (1.3 ± 0.1 cm vs.1.4 ± 0.1 cm, n = 5, \u003cem\u003eP\u003c/em\u003e = 0.710), while the transverse diameter of HT-29 transplanted tumors was significantly larger after 1 week (1.5 ± 0.2 cm vs. 1.2 ± 0.2 cm, \u003cem\u003eP\u003c/em\u003e = 0.009).\u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c14\" colnum=\"14\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eT/NT values of transplanted tumor at different time points after injection of different contrast agents(Mean ± std., n = 5)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"14\"\u003e\u003c/colgroup\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003eT/NT (Gd-DOTA)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eF\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eP\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"4\" nameend=\"c11\" namest=\"c8\"\u003e \u003cp\u003eT/NT (Gd-DOTA-G3CNGRC)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003eF\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003eP\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e (LSD)\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5 hour\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 hour\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2 hours\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4 hours\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.5 hour\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1 hour\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e2 hours\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e4 hours\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHT-1080 \u003csup\u003eP\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.99 ± 0.10\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.54 ± 0.20\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.00 ± 0.24\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.92 ± 0.07\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e0.829\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e0.46\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.83 ± 0.98\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3.20 ± 0.46\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e4.42 ± 0.38\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e2.69 ± 0.41\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e186.04\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e.000\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e.000\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHT-29\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.13 ± 0.20\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.53 ± 0.06\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.90 ± 0.14\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.81 ± 0.18\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.16 ± 0.12\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1.12 ± 0.16\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e1.21 ± 0.18\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e1.13 ± 0.03\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e.000\u003csup\u003e##\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHT-1080\u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.97 ± 0.08\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.69 ± 0.34\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.03 ± 0.15\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.85 ± 0.17\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.51 ± 0.16\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1.92 ± 0.19\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e1.89 ± 0.22\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e1.77 ± 0.15\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e.002\u003csup\u003e###\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"14\"\u003eHT-1080 \u003csup\u003eP\u003c/sup\u003e, HT-1080 grafts were not treated with ubenimex; HT-1080\u003csup\u003eA\u003c/sup\u003e, HT-1080 transplanted tumors were treated with ubenimex for 7 consecutive days; F\u003csup\u003e1\u003c/sup\u003e and \u003cem\u003eP\u003c/em\u003e\u003csup\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sup\u003e, F and P were derived from 1 hour after injection of Gd-DOTA, respectively; F\u003csup\u003e2\u003c/sup\u003e and \u003cem\u003eP\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e, F and P were derived from 2 hours after injection of Gd-DOTA-G3CNGRC, respectively; #, HT-1080 \u003csup\u003eP\u003c/sup\u003e VS. HT-1080\u003csup\u003eA\u003c/sup\u003e ; ##, HT-1080 \u003csup\u003eP\u003c/sup\u003e vs. HT-29; ###, HT-1080\u003csup\u003eA\u003c/sup\u003e vs. HT-29 (SPSS 29.0, One-way analysis of variance, LSD test )\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e3.5 Pathology\u003c/p\u003e\u003cp\u003eImmunohistochemical staining showed that APN/CD13 was not expressed in HT-29 transplanted tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B), but was highly expressed in HT-1080 transplanted tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). The expression of APN/CD13 in HT-1080 grafts decreased significantly one week after treatment with ubenimex (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn addition to pathological examination methods, there are many imaging methods used to evaluate tumor angiogenesis status, and each evaluation method has unique advantages and application ranges. Nuclear medicine has the advantage of high sensitivity but low spatial resolution [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Magnetic resonance imaging equipment can provide a clearer anatomical structure, but magnetic resonance technology often cannot distinguish between normal (or mature) and new blood vessels [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The sensitivity of optical devices is high, and the imaging process is simple. However, due to the weak penetration ability of fluorescence photons and the characteristics of fluorescence quenching, the clinical application of this kind of imaging technology is currently restricted [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. MR angiography and MR perfusion imaging are commo clinical methods used for tumor vascular evaluation [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], and the lack of specificity for neovascularization is a common defect of these methods. Whereas, magnetic resonance imaging with paramagnetic materials labeled neovasculotargeting peptides is expected to solve the problem of insufficient resolution in nuclear medicine imaging, and can obtain images with better tissue and spatial resolution, without the risk of ionizing radiation [\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The intensity of the T1-weighted image signal of the magnetic resonance probe is related to the Gd (III) loading rate of the probe and the concentration of Gd (III) chelate [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The loading rate of the Gd-DOTA-G3CNGRC probes prepared in the study was similar to those of previous research reports [\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. However, there have been few previous studies on the use of magnetic resonance targeting probes to evaluate the efficacy of antiangiogenic drugs or antitumour therapy drugs. Our results showed that the T/NT ratio of the same group of tumour-bearing mice decreased significantly after 7 days of ubenimex treatment, but the transverse diameter of the tumor did not change significantly, suggesting that the CD13 activity expressed by the tumor was inhibited after treatment. In addition, on Gd-DOTA imaging, HT-1080 transplanted tumors (pre - and post-treatment) and HT-29 transplanted tumors showed significant enhancement. Moreover, the enhancement time was different from that of Gd-DOTA-G3CNGRC and there was no difference in T/NT values, which further confirmed the high specificity of Gd-DOTA-G3CNGRC in CD13-positive tumors. In the mouse tumor model tested in this experiment, HT-29 tumors with negative expression of APN/CD13 had little uptake of Gd-DOTA-G3CNGRC, while HT-1080 tumors with positive expression of CD13 obviously took up GD-DOTA-G3CNGRC, suggesting that during the synthesis and labeling of DOTA-G3CNGRC, the activity of G3CNGRC was not significantly affected. Compared with previous reports, our imaging results basically reached the same conclusion [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. However, in contrast to these reports, our study mainly focused on the feasibility of early evaluation of the efficacy of antiangiogenic drugs, so DOTA-G3CNGRC was not used for blocking experiments in our study.\u003c/p\u003e \u003cp\u003eStudies have shown that APN/CD13 is upregulated in the blood vessels of many solid tumors, and the expression of cancer cells, stroma and/or vasculature can be observed depending on the tumor type [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The formation of neovascularization is closely related to tumor growth, metastasis and invasion, so reducing the expression of APN/CD13 in tumor cells has a significant promoting effect on improving the therapeutic effect of antitumour drugs [\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. At present, a variety of antagonists targeting APN/CD13 have entered the clinical application or clinical trial stage, such as bestatin (ubenimex), amastatin, and probestin [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], among which ubenimex is the most commonly used in the clinic. Its mechanism of action may be through targeting the CD13/EMP3/FAK/NF-κB pathway, affecting autophagy and apoptosis, improving chemotherapy drug sensitivity, and thus reversing drug resistance [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Anti-tumour angiogenesis is currently a commonly used method, including many types of drugs, which have also been clinically applied [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Most patients may experience significant side effects for a period of time after usage, and resistance may develop subsequently [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Therefore, in addition to detecting serum tumor markers, imaging of a target should also be performed for direct evaluation, especially early assessment. Our findings introduced a new aspect to these approaches, but further research should be needed on various types of tumor models.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe Gd-DOTA-G3CNGRC probe prepared in this study showed a good affinity for CD13-positive tumor cells in vitro experiments, and a high T/NT ratio for APNCD13-positive graft tumors. After 1 week of anti-APN /CD13 treatment, although there was no significant change in the transverse diameter of the APN/ CD13-positive graft tumor, the uptake of the probe was significantly reduced. Therefore, we believed that Gd-DOTA-G3CNGRC magnetic resonance imaging can be used as an imaging method to evaluate the early efficacy of anti-angiogenesis studies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting Interests\u003c/h2\u003e \u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work supported by Science and Technology Department of Guizhou Province grant: No. [2019]1325.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAuthors in order:The author, Songsong Liu, participated in the research process, data collection, analysis and interpretation of data, drafting papers, statistical analysis;Sheng Han participated in the research process, data collection, analysis and interpretation of data, drafting papers, statistical analysis;Gongwei Jing participated in research process, data collection, analysis and interpretation of data, statistical analysis;Pinqin Wang participated in the research process, analysis and interpretation of MRI data, and supporting work;Yanteng Zhang participated in the research process, MRI data collection, statistical analysis,technical and material support;Ling Xiong participated in the research process, data collection, administration, technical and material support;Yingfang Zhang , participated in the research process, IC50 data collection, literature review and material support;Huasheng Qu participated in the research process, 99mTc-DOTA-G3CNGRCdata collection, literature review and material support;Bingxiu Ren participated in the design, implementation research, data collection, analysis and interpretation of data, drafting papers, critical review of the intellectual content of the manuscript, access to research funding.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThanks to all the staff of the central laboratory of the Third Affiliated Hospital of Zunyi Medical University (Zunyi First People's Hospital) for their selfless help in this study. Thanks for the funding of this study from Guizhou Science and Technology Department project: [2019] 1325.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data generated or analysed during this study are included in this published article [and its supplementary information files].\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eJiajie Kuai, Chenchen Han, Wei Wei. Potential Regulatory Roles of GRK2 in Endothelial Cell Activity and Pathological Angiogenesis. Frontiers in immunology 2021; 698424.\u003c/li\u003e\n\u003cli\u003eLuca Crippa, Mimma Bianco, Barbara Colombo, et al. A new chromogranin A-dependent angiogenic switch activated by thrombin. Blood 2013; 121:392-402.\u003c/li\u003e\n\u003cli\u003eLiu Chao, Castillo Alesha B. Targeting Osteogenesis-Angiogenesis Coupling for Bone Repair. Journal of the American Academy of Orthopaedic Surgeons 2018; 26 (7): e153-55.\u003c/li\u003e\n\u003cli\u003eArjan W. Griffioen and Andrew C. 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Transl Cancer Res 2023;12 (10): 2823-36.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"APN/CD13, Angiogenesis, Anti-angiogenesis, Asn-Gly-Arg (NGR) peptide, Gadolinium (III)","lastPublishedDoi":"10.21203/rs.3.rs-5274314/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5274314/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eAnti-angiogenesis has been recognized as a crucial strategy in anti-tumor therapy, and the early assessment of its efficacy is equally significant. In this study, we developed a magnetic resonance (MR) probe specifically targeting angiogenesis to facilitate targeted imaging for the early evaluation of anti-angiogenic effects.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWe synthesized DOTA-G3CNGRC, conjugated it with gadolinium (III), and subsequently evaluated the labeled probe in vitro. Tumor-bearing mouse models of HT-29 (CD13-negative expression) and HT-1080 (CD13-positive expression) were established, followed by MR Imaging conducted via intraperitoneal injection of the labeled probe and Gd-DOTA at a dosage of 0.5 mg/kg/day before and after ubenimex treatment over a consecutive period of seven days. The average pixel ratio of the transplanted tumor (target tissue, T) to the left hind leg (non-target tissue, NT) was determined using the region of interest technique (ROI), while changes in tumor size were meticulously recorded. Additionally, APN/CD13 expression levels in transplanted tumors were assessed both prior to and following treatment.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe labeling rate of probes was 88.99%. The IC50 of the probes was 61.66 nM. The T/NT ratio of HT-1080 was significantly higher than that of HT-29 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, n\u0026thinsp;=\u0026thinsp;5). After treatment, the T/NT value of HT-1080 transplanted tumors was significantly decreased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, n\u0026thinsp;=\u0026thinsp;5), accompanied by a significant decrease in CD13 expression and unapparent changes in tumor size ( \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.710, n\u0026thinsp;=\u0026thinsp;5).\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThe Gd-DOTA-G3CNGRC probes appeared potential for targeted MR imaging in assessing the early efficacy of anti-APN/CD13 drugs.\u003c/p\u003e","manuscriptTitle":"Early evaluation of anti-angiogenic effects with gadolinium (III) labeled APN/CD13 specific binding peptides magnetic resonance imaging","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-02 16:14:48","doi":"10.21203/rs.3.rs-5274314/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-02-03T19:44:38+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-02-03T11:16:48+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-01-16T15:57:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"37661188741371889851409168356116275129","date":"2025-01-15T22:25:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"188037398387051960542454789962382609728","date":"2025-01-09T11:34:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"40384775443946120610676996560858704556","date":"2025-01-04T07:14:48+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-11-13T16:26:44+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-11-07T13:41:53+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-11-07T11:44:47+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-11-04T09:03:07+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-10-16T08:48:37+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"15117c56-b884-45ee-841a-8be48e9e266d","owner":[],"postedDate":"December 2nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":40225223,"name":"Biological sciences/Cancer"},{"id":40225224,"name":"Biological sciences/Molecular biology"},{"id":40225225,"name":"Health sciences/Biomarkers"},{"id":40225226,"name":"Health sciences/Nephrology"},{"id":40225227,"name":"Health sciences/Oncology"}],"tags":[],"updatedAt":"2025-07-07T16:07:58+00:00","versionOfRecord":{"articleIdentity":"rs-5274314","link":"https://doi.org/10.1038/s41598-025-05905-1","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-07-01 15:58:35","publishedOnDateReadable":"July 1st, 2025"},"versionCreatedAt":"2024-12-02 16:14:48","video":"","vorDoi":"10.1038/s41598-025-05905-1","vorDoiUrl":"https://doi.org/10.1038/s41598-025-05905-1","workflowStages":[]},"version":"v1","identity":"rs-5274314","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5274314","identity":"rs-5274314","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}