The Synthesis Of Functionalized Ultrasmall Superparamagnetic Iron Nanoparticle And Application In Transplantated Islet Imaging In Vivo

Based on their versatile, biocompatible properties, superparamagnetic iron oxide (SPIO) or ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles are utilized for detecting and tracing cells or tumors in vivo. However, since the FDA approved first generation commercial products, Feridex? and Resovist?, discontinued manufacturing in2008and2009respectively, the problem of lacking a suitable clinical SPIO or USPIO agent is in urgent need to be solved. Here, we developed an innoxious and concise synthesis approach for a novel B-cell lymphoma (Bcl)-2monoclonal antibody-functionalized USPIO nanoparticle coated with an amphiphilic polymer (carboxylated polyethylene glycol monooleyl ether [OE-PEG-COOH]). These nanoparticles can be effectively internalized by beta cells and label primary islet cells, at relatively low iron concentration. The biocompatibility and cytotoxicity of these products were investigated by comparison with the commercial USPIO product, FeraSpin TM S. We also assessed the safe dosage range of the product. Although some cases showed a hypointensity change at the site of transplant, a strong magnetic resonance imaging (MRI) was detectable by a clinical MRI scanner, at field strength of3.0Tesla, in vivo, and the iron deposition/attached in islets was confirmed by Prussian blue and immunohistochemistry staining. It is noteworthy that based on our synthesis approach, in future, we could exchange the Bcl-2with other probes that would be more specific for the targeted cells and that would have better labeling specificity in vivo as Exendin-4-USPIO. The combined results point to the promising potential of the novel Bcl-2-functionalized PEG-USPIO as a molecular imaging agent for in vivo monitoring of islet cells or other cells.Part1Synthesis and Characterization of Bcl-2functionalized USPIOObjective:Preparation and research of the physicochemical characteristics of PEG-USPIO.Methods:PEG-USPIO was synthesized by a non-toxic synthesis method of inorganic iron complexes in oily circumstance. The morphology and structure of PEG-USPIO was characterized by transmission electron microscopy (TEM). The size of PEG-USPIO was measured by dynamic light scattering (DLS). The magnetic properties of PEG-USPIO were examined by vibrating sample magnetometer (VSM) and magnetic property measument system (MPMS).Results:PEG-USPIO was fabricated by non-toxic inorganic iron approach. The color of the USPIO is black brown and showed good distribution and steady. TEM shows the core sizes of nanoparticles are4～10nm. The DLS measurement indicates that the average hydrodynamic size of PEG-USPIO is about28nm. C13NMR confirmed that the-OH group of OE-PEG-OH was modified to-COOH group by the present of163ppm peak. The saturation magnetization of PEG-USPIO examined by VSM is38emu/g at2T compared to commercial product Feredex which is41emu/g. PEG-USPIO exhibits the typical property of superparamagnetic iron oxide in shortening T1and T2relaxation time with a gradually increasing superparamagnetic effect according to the increasing Fe concentration.Conclusions:Successfully synthesized PEG-USPIO demonstrates high stability, crystalline and magnetization.Part2Cytobiology study of INS-1, Beta-TC-6cells labeling withPEG-USPIOObjective:Study on the labeling effiency and biocompatibility of synthesized PEG-USPIO.Methods: Prussian blue staining and TEM were performed to detect uptaking of PEG-USPIO by INS-1cells. In vitro MRI and Prussian blue staining of gradient PEG-USPIO labeled cells was investigated to measure the label efficiency. MTT assays and flow cytometry were performed to evaluate the cytotoxicity corresponding to the biocompatibility of PEG-USPIO on ins-1cells. The labeled Beta-TC-6cells function was evaluated by Glucose-stimulated insulin release assays.Results:Prussian blue method and intracellular iron content assay reveal intracellular iron accumulation. In vitro Prussian blue assay show higher lebeling efficiency of PEG-USPIO than FereSpin S. No significant difference in morphology was observed between PEG-USPIO labeled; FereSpin S labeled and control cells. MTT assay demonstrate no significant difference in viability was evident with PEG-USPIO labeled and untreated cells until the iron concentration exceed30μg Fe/mL. The flow cytometry assay suggests that there was no significant difference in cellular apoptosis detection and ROS level until the iron concentration exceed30μg Fe/mL. There is no obviously difference in glucose-stimulated insulin release between PEG-USPIO labeled group and FereSpin S labeled group or between PEG-USPIO labeled and control group until the iron concentration exceed30μg Fe/mL,Conslusions: The newly synthesized PEG-USPIO demonstrates excellent biocompatibility and superparamagnetic behavior. Under30μg Fe/mL, PEG-USPIO could be efficiently internalized into INS-1cells, without affecting cell viability and function.Part3In vivo MRI tracking and morphological study of Bcl-2functionalized USPIO labeled mice islets transplantationObjective: To investigate the possibility of in vivo detection of PEG-USPIO labeled rodent islets in a clinical3.0-T MR scanner with30-mm mouse coil.Methods:0,1.25,2.5,5,10,20,30,40,60,80μg Fe/mL USPIO were compared for in vitro MRI scaning which was performed at a clinical3.0-T MR scanner respectively.150islets labeled with PEG-USPIO were transplanted beneath the renal capsule of C57BL/6mice. The transplanted tissue samples were sectioned to investigate with immunohistochemistry and Prussian blue staining immediately after MRI. In vivo MRI was performed1d,8d,15d,21d after syngenetic transplantation respectively.Results:primary islets labeled with PEG-USPIO demonstrate hypointense spot in T2-weighted image at the transplantation area, which can be detected by the clinical3.0-T MR scanner. Immunohistochemistry and Prussian blue staining confirm the transplanted location of PEG-USPIO labeled islet cells in the renal subcapsular area. Within21days after transplantation,150PEG-USPIO labeled islets can be detected by in vivo MRI, with signal faded over time.Conslusions: The newly synthesized functionalized PEG-USPIO can be used to efficiently label mice islets transplanted beneath the renal capsule of C57BL/6mice. The labeled islet grafts can be tracked in vivo by the clinical3.0-T MR scanner with mouse coil.Part4In vivo MRI tracking study of Exendin-4functionalized USPIOObjective: To investigate the possibility of in vivo detection of insulinoma-bearing mice by Exendin-4-USPIO injection via tail vein in a clinical3.0-T MR scanner with30-mm mouse coil.Methods:novel USPIO were synthesized by changing the targeting peptides to Exendin-4. The labeling efficiency and cytotoxity of this new product were assessed firstly. We centified the biodistribution of the USPIO by immunohistochemistry and Prussian blue staining assay. The exendin-4targeting property was verified by confocal microscope though the Qds connection. Systemic delivery of SPIO-exendin4into nude mice bearing s.c. insulinomas (derived from INS-1cells) leads to the accumulation of the nanoparticles in tumors, generating a strong magnetic resonance imaging contrast detectable by a clinical MRI scanner at field strength of3.0T In vivo. And the result were verified by immunohistochemistry and Prussian blue staining respectively.Results: A targeted superparamagnetic iron oxide nanoparticle using GLP-1analog-exendin-4which is conjugated to polyethylene glycol coated USPIO was synthesized successfully. The results demonstrated that exendin-4functionalized USPIO was able to specifically bind to and internalized by GLP-1R-expressing INS-1cells, with the higher labeling efficiency than non-targeted nanoparticles. Notably, exendin-4-USPIO could differentially label islets in pancreaticslices or beta cell grafts in vitro. Systemic delivery of exendin-4-USPIO into nude mice bearing s.c. insulinomas (derived from INS-1cells) leads to the accumulation of the nanoparticles in tumors, generating a strong magnetic resonance imaging contrast detectable by a clinical MRI scanner at field strength of3.0T, and the iron deposition in tumors was further confirmed by Prussian blue staining. Furthermore, preliminary biodistribution study indicated that exendin4-USPIO had a tendency to accumulate in pancreas. Toxicity assessments demonstrated good biocompatibility in vivo.Conslusions:The insulinoma grafts can be tracked in vivo by the clinical3.0-T MR scanner with mouse coil. The newly synthesized exendin-4-USPIO has potential as molecularly targeted imaging agents and possibly for future beta cell imaging.