| BackgroundAs one of the best noninvasive and convenient imaging technique applied in clinical diagnosis, magnetic resonance imaging (MRI) provides competitive advantages over other techniques, such as intrinsically high spatial resolution, less exposure to radiation, rapid in vivo acquisition of images, and a long effective imaging window. It can provide high-quality3D images of soft tissues and deliver images with high anatomic resolution to visualize drug delivery, monitor biological process and follow functional change in vivo. Over the last decade, a particularly active area of research in the MRI field is the development of MRI contrast agents with high relaxivity, biocompatibility, and selectivity for targeting organs and tissues. The MRI contrast agent is a kind of the chemical agent which was used to increase the MR signal contrast between the normal tissue and diseased tissue. By using the MRI contrast agents can improve the sensibility and contrast for MR imaging. For example, superparamagnetic iron oxide (SPIO) nanoparticles, which induce the dark contrast enhancement in T2-weighted MR images, have been used as negative contrast agents and are clinically applied in ranged from nontargeted detection of diseases by accumulating at certain tissues to targeted detection of biomolecular markers in cells. In addition, chelated complexes of paramagnetic metal ions such as Gd3+and Mn2+have a marked effect on spin-lattice relaxation of surrounding water protons and lead to bright contrast enhancement in T1-weighted MR imaging (positive contrast agents). More than30%of all current MRI examinations use paramagnetic Gd3+complexes as contrast enhancing agents. However, there are some serious problems for metal ions contrast agents, for instance, the Nephrogenic Systemic Fibrosis (NSF) increased because of the gadolinium contrast agents using in patients with renal insufficiency. Retrospective study found since the first reported NSF case was observed on using gadodiamide in humans in1997, NSF has appeared in parallel with our increased use of gadodiamide-enhanced MRI. That caused wide public concern. Alternatively, NSF could be a toxic reaction to free Gd that is liberated from gadodiamide. Free Gd is highly toxic, in particular in its ionic form (Gd3+). Because of the longer half-life of contrast Gd-based media in patients with nephropathy, the gadodiamide contrast agents were restricted for patients with severe renal insufficiency. To prevent those problems, investigaters obtain the small dose and low exposed free ions by increased the stability of paramagnetic metal ions contrast agents, which are effective relaxation, to reduce the risk of NSF. However, we hope to compound a novel T1contrast agent with excellent biological compatibility, high safety performance, great image quality, long circulation and drain dasily, such as the stable free radical class compound for contrast of MRI. This research was expected to compound a novel absence metal ions contrast agents(PA-TEMPO) that possess high water solubility, long in vivo lifetimes and high1H water relaxivities(rl) with T1relaxation, the polyacefylene (PA) was used as carrier and2,2,6,6-Tetramethylpiperidinooxy(TEMPO) as magnetic substance, the PEG chains may increasing hydrophilic and long circulation in vivo. PA was Linked Folic Acid (FA) and Fluorescein (FI) for modifying organic free radical. Eventually, the novel targeted MRI contrast agent (PA-TEMPO-FI-FA) labeled FA and FI was obtained, which was used in early tumor diagnosis.ObjectivesTo synthesize a novel free metal organic radical MR contrast agents labeled FA and FI (PA-TEMPO-FI-FA), and utilizing nude mouse model with Hela cells tumor, to discuss the feasibility and imaging characteristic of this macromolecular contrast agent in vivo for targeting tumor.Chapter1:The application and research progress of magnetic resonance imagingChapter2:Synthesis and characterization of the novel free metal organic radical MR contrast agents2.1Materials and Methods2.1.1Synthesize of acetylene directives containing TEMPO (PA-TEMPO)Propynylamine,succinic anhydride,4-hydroxy-2,2,6,6-tetramethyl-piperidinooxy, and poly(ethylyene glycol)(Mw:2000)(PEG2K) were used to synthesize the Acetylene directives containing TEMPO (PA-TEMPO).2.1.2Synthesize of FI-modified PA-TEMPO (PA-TEMPO-FI), both FA-and FI-modified PA-TEMPO (PA-TEMPO-FI-FA)Reaction was occured in PA-TEMPO with FI and FA, to synthesize the macromolecular targeted contrast agent PA-TEMPO-FI and PA-TEMPO-FI-FA.2.1.3Initial study of the physico-chemical property of PA-TEMPO as a novel MR contrast agentTEMPO containing acetylene, poly (ethylene glycol)(PEG) containing acetylene and Acetylene directives containing TEMPO (PA-TEMPO) were analyzed with8100-infrared spectrometer (FT-IR) and Nuclear Magnetic Resonance Spectrometer. 2.2ResultsAcetylene monomer1and2were characterized by spectroscopic methods and satisfactory analysis data corresponding to their molecular structures were obtained. PA-TEMPO gave satisfactory analysis data corresponding to expected molecular structure, which was obtained by polymerization of acetylene monomer1and2.Chapter3:The evaluation of the novel organic radical MR contrast agentsSection1:In vitro studies of the novel MR organic radical contrast agent3.1Materials and Methods3.1.1The cytotoxicity assay of the PA-TEMPO, PA-TEMPO-FI and PA-TEMPO-FI-FA in vitroThe measure of relative Hela cell viability (%) using3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay:Hela cellswere routinely culture in full media at37℃in a5%C02,95%air humidified atmosphere. Then, the cells were seeded in96-well tissue culture plates(6×103cells/well)and continued incubating with full media containing PA-TEMEPO, PA-TMEPO-FI or PA-TEMPO-FI-FA solution in different concentrations respectively. After24h incubation,10ul of MTT dye solution(5mg/ml of D-hanks solution)Was added to each well. After4h incubation at37℃, the medium was removed and formazan crystals were solubilised with150ul of dimethylsulphoxide(DMSO)and the solution Was vigorously mixed to dissolve the reacted dye. After1Omin,the absorbance of each well was read on a microplate reader(BioTech ELX800instamaents)at490nm. The control group contained cells and cell culture medium without PA-TEMPO or PA-TEMPO-FI-FA. The relative cell viability(%) was calculated by [[OD]test/[OD]sample control]/[OD] control x100%. Statistical analysis:SPSS20.0statistical package was used as analyzing software. Two—way classification ANOVA was used to analyze the difference of Hela cell viability(%) after incubated with full media containing PA-TEMPO, PA-TEMPO-FI or PA-TEMPO-FI-FA solution in different concentrations. P<0.05was considered as significant difference.3.1.2T1signal intensity measurement and Tl relaxation rate calculation of PA-TEMPO, PA-TEMPO-FI and PA-TEMPO-FI-FA in vitroMR phantom studies were conducted using Phosphate buffer saline (PBS) suspension of PA-TEMPO, PA-TEMPO-FI and PA-TEMPO-FI-FA compounds with varying concentrations(1,3,5,10mM). The samples were placed in a plastic rack to obtain T1weighted image using a3.0T MR system and scanned with SE T1WI and T1mapping sequence. Then averageT1signal intensity and T1values of each sample were measured on post-processed T1map images and T1relaxation rate Was calculated. The slope of the line provides the molar r1of PA-TEMPO, PA-TEMPO-FI and PA-TEMPO-FI-FA.3.1.3In vitro fluorescence imaging of cells uptakeHELA cells (1×104cells/well) were seeded on glass cover slips in24-well plates at37℃and5%CO2and then allowed to adhere to the plates overnight. PA-TEMPO-FI or PA-TEMPO-FI-FA solution was added at concentration of3mM and incubated at37℃. After2h of incubation, the cells were fixed in a4%formaldehyde/PBS solution (pH7.2). Nucleus was stained with DAPI and washed with PBS three times. Fixed cells were imaged under fluorescence microscope (Olympus BX51, JP). Fluorescent images were acquired on an IVIS Lumina XR system with the same parameters.3.1.4In vitro cellular MR ImagingHela cells (2×104cells/well) were seeded in6-well plates at37℃and5%CO2and then incubated for24h. PA-TMEPO-FI or PA-TEMPO-FI-FA solution was added at concentration of0-5MM and incubated at37℃. After4h of incubation, labeled cells (5×105) were dispersed and suspended by500uL of0.1%agarose gel. The samples were placed in a plastic rack to obtain T1weighted image using a3.0T MR system and scanned with SE T1WI sequence. Then average T1signal intensity and r1of each sample were measured。Section2:In vivo studies of the novel MR organic radical contrast agent3.2Materials and Methods3.2.1Establishment of tumor-bearing nude mice:For the tumor model,5×106cells in100μL PBS were injected subcutaneously into the right shoulder back of4-6weeks old female BALB/c nude mice.3.2.2In vivo and ex vivo optical imaging study of the targeted MR contrast agent PA-TEMPO-FI-FA in tumor-bearing nude miceIn experiment, PA-TEMPO-FI-FA aqueous solution (0.5mL,2.5mmol) was administered into the bloodstream of each mouse through tail vein injection. The fluorescent images were then performed in a dark room on mice with imaging system. We obtained MR images before and after the intravenous administration at different time points. To confirm the bio-distribution of PA-TEMPO-FI-FA in different organs obtained from the NIR imaging, the mice were sacrificed at3h post-injection. Different organs were separated and washed by saline and assembled for ex vivo fluorescence imaging.3.2.3In vivo MR imaging of the targeted MR contrast agent PA-TEMPO-FI-FA in tumor-bearing nude miceThe mice bearing tumor (n=5) were anesthetized with an intraperitoneal injection of pentobarbital sodium (60mg/kg)10minutes before the MR studies. In vivo MR imaging was performed using a clinical3.0T MR scanner (Signa Excite, GE Healthcare) equipped with a mouse-imaging coil. Coronal T1-weighted SE (TR/TE=540/10.7ms, FOV=12cm, matrix=320×224, NEX=4, thickness/interval=3.0/0.5mm). MR images were taken at different time points before and after administration of contrast agents. The total0.5mL of saline (2.5mmol, pH7.2) containing PA-TEMPO-FI-FA was administered via tail vein. We obtained MR images before and2hours after the intravenous administration.The MR data were digitally transferred from a PACS workstation to a personal computer and processed with MATLAB software (The Math-Works, Natick, MA, USA) and software developed in house using Microsoft Visual C++. ROIs that contained the entire tumor were drawn in each section of the T1-weithed images. Using the software developed in house, the data acquired from each slice were summated to derive the pixel-by-pixel SI values for the entire tumor. A baseline pixel histogram of a tumor was created from MR images obtained before the injection of the PA-TEMPO-FI-FA solution to establish the standard deviation (SD) of the average tumor signal in the absence of solution. Subsequently, serial images were spatially coregistered using a cross-correlation routine, and the tumor ROI mask was copy to each time point. Pixels with an MR signal intensity increase SD above the baseline tumor signal were considered enhanced pixels. The enhance fraction within the whole tumor for each individual mouse was determined by the number of enhanced pixels divided by the number of total pixels within the tumor. Paired-Samples t Test Was used to analyze the difference of signal intensity before and after injecting with PA-TEMPO-FI-FA solution. P<0.05Was considered as significant difference.3.2.4Fluorescence immunohistochemistryTumor-bearing mice were sacrificed24h after MR imaging studies, HeLa tumoral biopsies were fixed in10%formalin and embedded in paraffin. The frozen section were observed for the fluorescence imaging in the tumor.3.2.5Acute toxicity experience of PA-TEMPO-FI-FA for nomal miceMice were dissected at2days after intravenous administration of saline or PA-TEMPO-FI-FA (dosage:2.5mmol) for control group and treatment group, respectively. General aspects of mice were observed in two day, the major organs were immediately dissected and fixed in a4%formaldehyde/PBS solution (pH7.4) for at least48h. The tissues were processed routinely into paraffin, sectioned and stained with hematoxylin and eosin (H&E) per standard clinical laboratory protocols.3.3Results and Discussion3.3.1Evaluation of cellular cytotoxicity for the novel organic radical MR contrast agentWe used the MTT viability assay to assess the cytotoxicity of the PA-TEMPO, PA-TMEPO-FI and PA-TEMPO-FI-FA. Cells without any treatment were used as negative controls. The cytotoxicity of three were similar when the concentration was below5mM, and the cells viability was above80%; The cells exposed to PA-TEMPO higher the8mM/mL concentration manifested significantly reduced viability compared with the control cells. However, PA-TEMPO-FI-FA did not show any significant effect on cell viability for all concentrations, establishing the little cytotoxicity, indication of their excellent biocompatibility in vitro. The difference of Hela cell viability (%) after incubated with full media containing PA-TEMPO, PA-TEMPO-FI or PA-TEMPO-FI-FA suspension in different concentrations are statistically significant(P<0.05).3.3.2Evaluation of T1signal intensity and T1relaxation rate for the novel organic radical MR contrast agentThe MR imaging signal intensity increased with the increasing of the PA-TEMPO, PA-TEMPO-FI or PA-TEMPO-FI-FA concentration. The PA-TEMPO, PA-TEMPO-FI exhibited a slight increased positive contrast in the T1-weighted MR image than that of PA-TEMPO-FI-FA with the same concentration. The1H water relaxivities r1of the PA-TEMPO and PA-TEMPO-FI-FA in PBS were measured under different concentration. The results showed that The plots of the reciprocal of the1H relaxation rate of water (1/Tl) versus the samples concentration were linear for all of the agents. The slopes of the plots indicated that under physiological conditions (PBS, pH7.2), the1H water relaxivities of PA-TEMPO, PA-TEMPO-FI are slightly higher than PA-TEMPO-FI-FA.3.3.3Evaluation of cells uptake for the novel organic radical MR contrast agentThe biological process for the uptake of PA-TEMPO-FI and PA-TEMPO-FI-FA by cancer cells was also demonstrated by fluorescence microscopy. Cells without any treatment were used as negative controls. The cell nucleus was stained blue with DAPI. The strong green fluorescence around the cell nucleus labeled with the blue fluorescence for PA-TEMPO-FI-FA demonstrated that a significant increase in uptake compared with PA-TEMPO-FI, which had been taken up relatively a small amount by the HeLa cells. The results confirmed the folic acid (FA), which possed active targeting ability for FA-expressing cancer celles, might contribute to the uptake of PA-TEMPO-FI-FA.3.3.4Evaluation of cellular MR imaging for the novel organic radical MR contrast agent in vitroTo further determine the potential utility of PA-TEMPO-FI-FA, in vitro MR imaging studies were performed. All MR images of HeLa cells incubated with different concentration of PA-TMEPO-FI or PA-TEMPO-FI-FA for4h, cells without any treatment were used as negative controls. The T1WI image showed slight enhancement in HeLa cells with the concentration at5mM for the PA-TEMPO-FI, but significant enhancement for the PA-TEMPO-FI-FA above3mM. A quantitative analysis of the MR images revealed that the SNRs of HeLa cells treated with the PA-TEMPO-FI-FA were significantly higher than that of the corresponding cells treated with culture medium alone or the same concentration of PA-TEMPO-FI. Overall, the in vitro cell imaging experiments demonstrated that the synthetic organic radical contrast agent could realize in vitro MR image enhancement, and the PA-TEMPO-FI-FA might be used for targeted MR imaging of FAR-expressing cancer cells, which was owned significantly increased contrast enhancement.3.3.5The distribution of the novel organic radical MR contrast agent (PA-TEMPO-FI-FA) in tumor-bearing nude miceThe whole-body optical imaging was performed at different time points after administered PA-TEMPO-FI-FA via tail vein. At30min, the whole body of the nude mice was displayed abundant fluorescent light widely, except tumor. The distribution of fluorescent light was changed at1.5h, and it was focused on tumor. However, there were litter fluorescent light focused on other area after2h. These in vivo and ex vivo optical imaging results indicate that the targeted PA-TEMPO-FI-FA can aggregate in tumor excess-expressing FAR for long time.3.3.6The MRI of the novel organic radical MR contrast agent (PA-TEMPO-FI-FA) in tumor-bearing nude miceThe MR imaging was performed at different time points after administered PA-TEMPO-FI-FA via tail vein. It could been observed increased T1signal intensity in the tumor area, and peak value was appeared at2h post-injection. SPSS20.0statistical package was used as analyzing software. Paired—Samples T Test was used to analyze. The signal intensities of the tumor location on T1-weighted images at2h after tail vein injection of PA-TEMPO-FI-FA solution were significantly higher than that of the pre-contrast groups(P<0.05). In vivo results clearly showed that the PA-TEMPO-FI-FA contrast agent had excellent in vivo performance. It had high targeting efficiently to FAR-expressing tumor, with significant higher contrast enhancement in MRI.3.3.7Fluorescence immunohistochemistry To confirm that the in-vivo injected PA-TEMPO-FI-FA was localized in tumor areas, we performed fluorescence microscopy for tumor tissues after imaging. The results showed that PA-TEMPO-FI-FA with green fluorescence was observe in tumor cells, which confirmed that PA-TEMPO-FI-FA had been uptaked and accumulated in tumor cells.3.3.8Acute toxicity experience of PA-TEMPO-FI-FA for nomal miceNo adverse drug reactions were observed for mices in2days. Mices were dissected in2days after intravenous administration of PA-TEMPO-FI-FA (dosage:2.5mmol). Six representative organs including brain, heart, liver, spleen, lung, and kidney were no apparent histopathological abnormalities or lesions for the groups treated with PA-TEMPO-FI-FA compared with those of the control group, which further confirmed the low toxicity and good biocompatibility of the novel contrast agent.ConclusionsThe novel organic radical MR contrast agent----PA-TEMPO-FI-FA are tumor-targeted based on the paramagnetic TEMPO-type organic radical possessing T1relaxation. |
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