PVP-SPIO For Targeting Transplanted Mice Islets: Synthesis And In Vivo Detection By Magnetic Resonance Imaging

Diabetes mellitus(DM),which affects millions of people worldwide,is characterized by abnormally high levels of glucose in blood caused by either absolute (type 1 DM) or relative(type 2 DM) insulin deficiency due to the destruction of pancreatic P-cells by T cells of the immune system or decreased insulin sensitivity respectively.By 2010 the number of people with diabetes is expected to exceed 350 million.Late diabetic complications will cause considerable morbidity in 5-10%of these patients and place an enormous burden on society.Islet cell replacement is considered as the optimal treatment recently.However the success of this approach is hampered by the absence of reliable methods to follow the fate of transplanted islets non-invasively.In vivo molecular imaging seems to be the most appropriate technique to achieve this goal in small animals and eventually in humans.Such measures would potentially allow the assessment of islet engraftment and the early recognition of graft loss,leading to greater improvements in islet graft survival and function.The term molecular imaging can be broadly defined as the in vivo characterization and measurement of biologic processes at the cellular and molecular level.Three modalities have demonstrated applicability in the near future:bioluminescence imaging (BLI),magnetic resonance imaging(MRI) and positron emission tomography(PET).A main advantage of using MRI is its high spatial resolution and the ability to extract more than one measurement parameter at a given imaging session.Superparamagnetic iron oxide nanoparticles(SPIO) have demonstrated their utility as an important tool for enhancing magnetic resonance contrast,allowing researchers to monitor not only anatomical changes,but physiological and molecular changes as well.Recently,stem and progenitor cells of neural and non-neural origin have been magnetically labeled using SPIO in order to track them by MRI in cell therapy approaches for CNS and cardiovascular disorders.In this study,we have developed method of high temperature hydrolysis of chelate metal alkoxide complexes to coat iron oxide nanoparticles with biocompatible polymer, polyvinylpyrrolidone(PVP).Synthesized PVP-SPIO nanoparticles have demonstrated excellent biocompatibility and superparamagnetic behavior.The imaging of 100 PVP-SPIO labeled islets in mice renal subcapsular model of transplantation under a clinical 3.0-Tesla MR scanner showed high spatial resolution in vivo.These results indicated the great potential applications of PVP-SPIO as MRI contrast agent might be possible for monitoring the transplanted islet grafts in the clinical management of diabetes in the near future.Part 1 Synthesis and Characterization of PVP-SPIOObjective:Preparation and research of the physicochemical characteristics of PVP-SPIO. Methods:PVP-SPIO was synthesized by high-temperature hydrolysis of chelate metal alkoxide complexes in liquid polyol.The morphology and structure of PVP-SPIO was characterized by transmission electron microscopy(TEM),high-resolution transmission electron microscopy(HRTEM) and X-ray powder diffraction(XRD).The size of PVP-SPIO was measured by dynamic light scattering(DLS).Fourier transmission infrared spectra(FTIR) were performed by FTIR spectrometer.The magnetic properties of PVP-SPIO were examined by vibrating sample magnetometer(VSM).Results:PVP-SPIO was fabricated by high-temperature polyol approach.TEM shows the core sizes of nanoparticles are 5～10 nm.The HRTEM image reveals that there are two kinds of the lattice fringes with lattice spacing of about 0.25 and 0.29 nm corresponding to the {220} and the {311} plane of SPIO,respectively.The DLS measurement indicates that the average hydrodynamic size of PVP-SPIO is about 45 nm. The XRD pattern and FTIR analysis further confirm the formation of PVP-SPIO.The saturation magnetization of PVP-SPIO examined by VSM is 63emu/g at 2T.PVP-SPIO exhibits the typical property of superparamagnetic iron oxide in shortening T₁ and T₂ relaxation time with a gradually increasing superparamagnetic effect according to the increasing Fe concentration.Conclusions:Successfully synthesized PVP-SPIO demonstrates high stability, crystalline and magnetization. Part 2 Cytobiology study of Beta-TC-6 cells labeling with PVP-SPIOObjective:Study on the labeling effiency and biocompatibility of synthesized PVP-SPIO.Methods:Prussian blue staining and TEM were performed to detect uptaking of PVP-SPIO by Beta-TC-6 cells.In vitro MRI and prussian blue staining of gradient PVP-SPIO labeled cells was investigated to measure the label efficiency.MTT assays,growth curves and flow cytometry were performed to evaluate the cytotoxicity corresponding to the biocompatibility of PVP-SPIO on Beta-TC-6 cells.The labeled Beta-TC-6 cells function was evaluated by Glucose-stimulated insulin release assays.Results:Prussian blue method and TEM reveal intracellular iron accumulation.In vitro MRI show higher lebeling efficiency of PVP-SPIO than Feridex.No significant difference in morphology was observed between PVP-SPIO labeled,Feridex labeled and control cells.MTT assay demonstrate no significant difference in viability was evident with PVP-SPIO labeled and untreated cells.The flow cytometry assay suggests that there was no significant difference in cellular apoptosis detection and ROS level. There is no difference in glucose-stimulated insulin release between PVP-SPIO labeled group and Feridex labeled group or between PVP-SPIO labeled and control group.Conslusions:The newly synthesized PVP-SPIO demonstrates excellent biocompatibility and superparamagnetic behavior.PVP-SPIO could be efficiently internalized into Beta-TC-6 cells,without affecting cell viability and function. Part 3 In vivo MRI tracking and morphological study of PVP-SPIO labeled mice islets transplantationObjective:To investigate the possibility of in vivo detection of PVP-SPIO labeled mice islets in a clinical 3.0-T MR scanner with 30-mm mouse coil.To find an optimal sequence and parameter for MRI of PVP-SPIO labeled islets for the following in vivo study.Methods:0.5×10⁵,1×10⁵,2×10⁵,4×10⁵,8×10⁵,1.6×10⁶,3.2×10⁶,6.4×10⁶ Beta-TC-6 cells were respectively collected after labeling with PVP-SPIO(100μg Fe/mL) for 24h.In vitro MRI of cell pellets was performed at a clinical 3.0-T MR scanner.2×10⁵ Beta-TC-6 cells labeled with PVP-SPIO were transplanted beneath the renal capsule of C57BL/6 mice tracked with in vivo MRI.The transplanted tissue samples were sectioned to investigate with immunohistochemistry and prussian blue staining immediately after MRI.100 islets labeled with PVP-SPIO were transplanted beneath the renal capsule of C57BL/6 mice.In vivo MRI was performed 1d,8d,15d after syngenetic transplantation respectively.Results:Beta-TC-6 cells labeled with PVP-SPIO demonstrate hypointense spot in T₂-weighted image at the transplantation area,which can be detected by the clinical 3.0-T MR scanner.Immunohistochemistry and prussian blue staining confirm the transplanted location of PVP-SPIO labeled islet cells in the renal subcapsular area. Within 15 days after transplantation,100 PVP-SPIO labeled islets can be detected by in vivo MRI,with signal faded over time. Conslusions:The newly synthesized PVP-SPIO can be used to efficiently label mice islets transplanted beneath the renal capsule of C57BL/6 mice.The labeled islet grafts can be tracked in vivo by the clinical 3.0-T MR scanner with mouse coil.Part 4 Functional graft imaging of PVP-SPIO labeled Beta-TC-6 cells with in vivo MRIObjective:To establish the oxidative injury transplantation model of Beta-TC-6 cells.We have adapted for monitoring the function of grafts noninvasively with MRI.Methods:To determine the presence of apoptosis and oxidative stress,MTT assays and flow cytometry were performed after Beta-TC-6 cells treated with H₂O₂.Normal group and oxidative injury group of Beta-TC-6 cells labeled with PVP-SPIO were transplanted beneath the renal capsule of C57BL/6 mice.In vivo MRI was performed days 1 after transplantation respectively.The signal changing on T_amap weight image was analyzed according to the R₂ Ratio=(R_{2 Graft}/R_{2 Kidney})×100%.The transplanted tissue samples were sectioned to investigate with TUNEL assay.Results:MTT assays and flow cytometry demonstrate that H₂O₂ induced apoptosis of Beta-TC-6 cells and reduced the cell viability in a dose-dependent manner.The R₂ Ratio on in vivo MRI for graft in normal group is significantly higher than oxidative injury group induced by H₂O₂(P<0.05).The number of apoptosis of TUNEL positive cells is obviously lower in normal group than in oxidative injury group.Conslusions:The newly synthesized PVP-SPIO in combination with our model of mice islet transplantation,can be used to monitoring the function of grafts noninvasively with the clinical 3.0-T MR scanner with mouse coil.