| Background and Objective:Angiogenesis plays a crucial role in tumor growth, invasion, and metastasis. With the wide study in tumor angiogenesis at the molecular level, various molecular makers of tumor angiogenesis have been identified and well characterized. Tumor angiogenesis targeted molecular imaging is increasingly being recognized as a hotspot in the field of medical imaging in recent years. Ultrasonography (US) is a widely used imaging modality which offers high spatial resolution, allows noninvasive and inherently realtime imaging, and combines the advantages of easy manipulation, low expense and the absence of radiational injury. Moreover, the ultrasound contrast agent with a particle size of1-8μm could hardly leak out from the vascular and diffuse into the extravascular interstitial spaces, which is a real blood-pool agent without diffusion. Thus the contrast-enhanced ultrasound imaging does not have elevated background signal defects compared to other imaging modalities such as CT, MRI and PET, and the enhanced signals completely come from tumor vascular bed. Therefore, contrast enhanced ultrasound appears to be a promising method in tumor angiogenesis imaging. Some research indicated that many tumors rely on an angiogenic switch on the early stage. The kinase insert domain-containing receptor(KDR), as the main receptor of vascular endothelial growth factor, is one of the major regulators of angiogenesis. The activation of the VEGF/KDR pathway triggers multiple signaling networks that result in endothelial cell survival, mitogenesis, migration and vascular permeability, and KDR is essential for early tumor angiogenesis. In previous study, we have identified that the KDR up-regulated during breast tumor genesis and progression (from human normal breast tissue, simple epithelium hyperplasia, atypical hyperplasia and ductal carcinoma in situ to invasive ductal carcinoma), and KDR is overexpressed in particular in ductal carcinoma in situ and invasive ductal carcinoma. Our findings indicate that the KDR-targeted imaging of tumor angiogenesis may be an elegant approach for diagnosing breast cancer at an early stage. Additionally, the overexpression of KDR considered to be associated with tumor progression and poor prognosis in several tumors, including breast, ovarian, and pancreatic cancers. Therefore, the KDR-targeted molecular imaging of tumor angiogenesis may be particularly helpful for diagnosing tumors at early stages, evaluating tumor angiogenesis, estimating prognosis and minitoring anti-angiogenic treatment.Targeted ultrasound molecular imaging have been applied to characterize vascular thrombosis, inflammation, atherosclerosis and tumor angiogenesis, and preliminary results had been made. So far most of targeted ligands attached to surface of ultrasound microbubble are antibodys. Although antibodys can speficificly bind to attach to surface antigen of targeted cells, their obvious immunogenicity, high molecular weight and low sensitivity of imaging owing to the restriction of their surface modification sites has limited their applications in vivo molecular imaging. However, in our study, we choose small peptide K237as ligand of ultrasound microbubble. The small peptide K237which has a high degree affinity of KDR was isolated from phage display peptide library. A targeted contrast-enhanced ultrasound imaging contrast agent was created by attaching K237to the shell of microbubbles using biotin-streptavidin coupling chemistry. Its physical and chemical properties and biological activity were identified by in vitro experiments. The in vivo imaging of targeted contrast-enhanced ultrasound were conducted in nude mice model bearing human breast xenograft cancers to assess the feasibility of KDR-targeted ultrasound imaging of tumor angiogenesis.Methods1.Synthesis of Short peptidesPeptides which has a high affinity activy to KDR containing a sequence of HTMYYHHYQHHL and peptides with a crambled sequence was synthesized according to the methods in literatures.Peptides were purified by column chromatography and puritied by the method of high performance liquid chromatographic.2. Preparation of Dil-targted microbubblesTake a certain amount of DSPC、DPPE-Biot、DiI、PEG4000、pipoisson los ShaM (F188)add25ml distilled water in three-necked bottles according to Moore, stir in70℃water bath30min, cool to room temperature. The prepared lipid suspension to join50ml polypropylene pipe.the shear cutter head is inserted into the liquid level, the perfluoro propane2min saturated solutionwith certain shear rate, shear time, make package perfluoropropane gas lipid microspheres, packaging Yu Xilin bottle filling perfluoropropane gas confined, system the preparation of biotinylated Dil fluorescently labeled lipid microbubbles. Streptavidin was incubation with biotinylated lipid microbubbles for30min,after the spore purification, added biotin12peptide or a crambled sequence peptide,incubated for30min, after the spore purification, the targeted microbubble (MBp) and contrast microbubbles (MBc) were prepared and storaged in refrigerator at4℃.3.Physical properties identification of targeted microbubblesBiotinylated Dil labeled lipid microbubbles were prepared as above, then streptavidin was incubation with biotinylated lipid microbubbles, after the spore purification, added FITC-biotin12peptide, after the spore purification, the FITC targeted microbubble (FITC-MBp) were prepared.The shape and the distribution of and argeted microbubbles were observed by Light microscopy. The connection of FITC-peptide and the DiI-microbubbles were observed by fluorescence microscopy.4.Adhesion experiments of targeted microbubbles4.1.Adhesion experiments in vitro:The expression of KDR of the human colon cancer cell (LOVO)、human breast cancer cell(MDA-MB-231)and human colon cancer cell(LS174T) were detected by immune fluorescence and Western Blot. Targeted microbubbles (MBp) and control microbubbles (MBc) were respectively incubat with the three kinds of cells, the combination of microbubbbles was observed by fluorscence microscope.Count every random field of vision and cell of the combination of the total number of the cell,take10sets of data, take the mean and compare the differences of microbubbles that adhered on the different cells.4.2.Blocking experiments:Certain amount of K237were incubated with LOVO cells for30min before adding targeted microbubbles (MBp), calcute the numbers of microbubbles around cells every random field by fluoresctence microscope, take10sets of data and compare the differences of microbubbles to the noincubated LOVO group.4.3.Statistical analysis:Data were reported as mean±standard deviations by software SPSS13.0. Approximate F inspection Welch method for the analysis of variance were performed on attachment of targeted microbubbles in three kinds of cells, DunnetT3were performed between groups. Comparisons between targted microbubules and control microbubbles around LOVO by two independent t test sample. Comparisons between targted microbubules and blockings around LOVO by two independent t test sample.5. Animal preparation:Human breast cancer cell MDA-MB-231and human colon cancer cell LS174T were grown in1640medium with a high concentration of glucose (4.5g/L) and L-glutamine, and supplemented with10%fetal bovine serum and penicillin (100U/mL) and streptomycin (100ug/mL).36female6-8week-old nude mice in random order by subcutaneous injection of a suspension of either5.0x107/ml MDA-MB-231cells(n=18) or LS174T cells (n=18) in the right Breast region, observe tumors regularly.6. Tumor CEU imaging and image analysis 6.1.Division of laboratory animals:tumor group of MDA-MB-231nude mice (n=18) and LS174T nude mice (n=18) were randomly divided into two groups:12are for the targeting group,and the others are for the blocking group.6.2.Targeted ultrasound imaging:10days later,24murine tumor models were performed with CEU respectively by using MBp and MBc. After anesthesia mouse were fixed in homemade platform will probe put in on the support and adjust the probe to make good imaging。The ultrasound signal (video intensity, VI) from MBa and MBc were measured by second harmonic CEU imaging with pulsing interval time (PI) of ten seconds and a mechanical index (MI) of0.18, transmission frequency of7.0MHz and receiving frequency of14.0MHz. All experimental mice, the intravenous injection of2×108microbubbles were made in random order with30minutes interval. After the first picture of CEU imaging being taken, the microbubbles were destroyed by two to three seconds of continuous imaging with a high MI of1.9and the background-subtracted VI of tumor were measured.6.3.Blocking experiments in nude mouse:6murine tumor models of each group,0.2mlK237small peptides were injection15min before injection2.0×108/ml targeted microbubbles0.2ml, the imaging methods and imaging progress are as former.6.4.Imaging Analysis:video intensity of ultrasound images were analyzed by MCE software, the first image frame acoustic intensity value representing microbubbles in tissue and circulating concentrations of total, including the tumor tissue adhesion and in vivo blood circulating microbubbles, high mechanical index ultrasound after destruction of the sound intensity values for blood circulation in the microbubble concentration, the former is obtained by subtracting the latter adhesion in tissue of microbubbles concentration. Color coded images for tumor tissue and surroundings were obtained by color coding technology.6.5.Statistical analysis:Data are reported as mean±standard deviations by software SPSS13.0. One-way-ANOVA were performed on in vivo US imaging signals in animals imaged with MBp、MBc and blockings,heterogeneity of variance were performed by approximate F inspection Welch method for the analysis of variance. Multiple comparison of homogeneity of variance using LSD test, using DunnetT3test for heterogeneity of variance.7. Fluorescence imaging of living animals:All the mouse were peformed fluorescence imaging after contrast-enhanced ultrasound on the next day. Fluorescently labeled targeted microbubbles and isotype controls microbubbles were injected through tail vein in24murine tumor models (12in each group) after anaesthetized.The other nude mice were injected the small peptide K237before the injection of fluorescently labeled targeted microbubbles.The dose, concentration of microbubbles and time interval were according to contrast enhanced ultrasound. Adhesion of microbubbles in tumor were observed by KODAK Image Station4000MM Digital system, excitation light for549nm, emission wavelength for564nm.8.Examination of immunohistochemisty:Mouse were euthanized after fluorescence imaging, tumors and their surrounding tissues of nude mice were fixed in4%paraformaldehyde for immunohistochemical examination to test the expression of KDR. Results1.High performance liquid chromatography and mass spectrometry analysis: Synthetic peptide HPLC analysis peak of2522.9D determination value, and molecular weight2523.6D with basic theory, the content of about98.5%, synthetic success.2. Physical properties of microbubbles:The targeted microbubbles uniform distribution, no gathered phenomenon, single microbubble round structures in Light microscopy. The FITC-peptide linked well to the surface of DiI-microbubbles, which were observed with fluorescence microscopy.3. Performance texting of targeted microbubbles in vitro3.1. Adhesion experiments in vitro:The results of cell immunofluorescence and Western Blot show the LOVO cells were strong positive expression of KDR, MDA-MB-231cells positive for KDR expression, LS174T cells were KDR negative expression.There were many targeted microbubbles around LOVO cell in light microscopy, LOVO nuclei performance for bright blue fluorescence in fluorescence microscopy,there were many red targeted microbubbles around LOVO cell in fluorescence microscopy; There were a part of targeted microbubbles around MDA-MB-231cell in light microscopy, a part of targeted microbubbles around LOVO cell in fluorescence microscopy; there were little red targeted microbubbles around LS174T cell.3.2. Blocking experience:There were little targeted microbubbles around LOVO cell after certain amount of K237blocked in light microscopy, the red fluorescence intensity around LOVO cell were significantly weakened in fluorescence microscopy.3.3. Statistical analysis:In the targeted microbubbles,there was significant difference between three kinds of cells(P=0.000),in the control microbubbles,there was no significant difference between three kinds of cells(P=0.945).The adhesion of targeted microbubbles are significantly higher than the control microbubbles around LOVO cell (19.12±2.83.0.16±0.15, P=0.000).while the adhesion of targeted microbubbles around LOVO cell are significantly higher than targeted microbubbles around LS174T cell (19.12±2.83、0.30±0.23, P=0.000).There was significant difference afer K237blocking group and no blocking group of adhered targeted microbubbles around LOVO cell (3.22±2.24.19.12±2.83, P=0.000).4.Results for MCE imaging:In the tumor group of MDA-MB-231, the first frame of CEU images showed significant enhancement in the region of the internal tumor5min afer MBp injection. and no significant enhancement of MBc,in the tumor group of K237blocking there was no significant enhancement. In the tumor group of LS174T, the first frame of CEU images showed significant enhancement around the tumor, and no significant enhancement of MBc,in the tumor group of K237blocking there was no significant enhancement.5.Fluorescence imaging of living animals:6-10minutes after injection of fluorescently labeled MBp at the site the tumor of the two group all showed a bright red fluorescence,and there were no local red fluorescence appeared after the controlled microbubble and small peptide K237pre-injection of closed30min. 6.Results for examination of immunohistochemisty:It was indicated that the expression of KDR increasedr angiogenesis tissues in both MDA-MB-231and LS174T tumors by immunohistochemisty.7.Statistical analysis:In the tumor group of MDA-MB-231,there was significant difference of the VI in targeted microbubbles compared to the control microbubbles (38.11±11.75.12.52±5.23, P=0.000), about3times higer; The IV in tumor in blocking group, reduced obviously compared to the targeted microbubbles (38.11±11.75.13.77±4.35, P=0.000).In the tumor group of LS174T, There was significant difference of the VI in targeted microbubbles compared to the control microbubbles (30.18＋9.56、8.28±4.74, P=0.000), about3.6times higer; The IV in tumor in blocking group, reduced obviously compared to the targeted microbubbles,(30.18±9.56、9.23±3.44, P=0.000)Conclusions1. KDR-targeted microbubbles have high specificity to targeted cells in vitro.2.Targeted ultrasound imaging was produced by a small peptides specifically combined with endothelial and adhered to blood vessels of tumors.3. The targeted microbubbles may suitable for different types of solid tumor for targeted imaging, also may help in defining promising imaging targets for both early cancer detection and treatment monitoring of cancer using targeted contrast-enhanced US imaging. |
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