Synthesis Of Ultrasmall Manganese-doped Iron Oxide Nanoparticles For T₁/T₂ Dual-contrast Magnetic Resonance Imaging

Objectives:T₁ or T₂ single-contrast enhanced magnetic resonance imaging?MRI?has been proved some inherent defects that may cause diagnostic errors,while longitudinal relaxation time?T₁?and transverse relaxation time?T₂??T₁/T₂?dual-contrast enhanced MRI can provide complementary information and effectively improve the accuracy of diagnosis.Contrast agent?CA?,regarded as the key of MRI,is one of the most simple and effective way to achieve dual-contrast enhanced MRI,which has attracted extensive attention of researchers.Previous reports have shown that ultrasmall manganese-doped iron oxide nanoparticles?Mn-IONPs?exist excellent longitudinal relaxation?r₁?,but their transverse relaxation?r₂?is relatively weak.Based on this situation,our research group want to improve the r₂ of Mn-IONPs through surface modification or structural transformation while maintaining good r₁,so as to construct a kind of CA that can realize T₁/T₂ dual-contrast MRI.Materials and Methods:1.Synthesis and characterization of iron-eruciate and manganese-oleate complexes:Ferric chloride?FeCl₃·6H₂O?and manganese chloride?MnCl₂·4H₂O?were used as precursors to react with erucic acid and oleic acid in methanol solution of NaOH to prepare target products,and the structure was observed using fourier transform infrared spectroscopy?FTIR?.2.Synthesis and characterization of Mn-IONPs:Hydrophobic Mn-IONPs were obtained by thermal decomposition of iron-eruciate and manganese-oleate complexes in benzyl ether.And the properties of Mn-IONPs were identified using transmission electron microscope?TEM?,high resolution transmission electron microscopy?HRTEM?,X-ray diffraction?XRD?,and superconducting quantum interference device?SQUID?.3.Synthesis and characterization of poly??-amino ester?-b-poly?ethylene glycol??PAE-PEG?:With an ice bath,the precursors consist of methoxy poly?ethylene glycol?acrylate?mPEG-A?,1,3-Bis?4-Piperidyl?Propane?TDP?,and 1,6-Hexanediol Diacrylate?HDD?formed amphiphilic polymer PAE-PEG through Michael addition reaction.Next,the properties of PAE-PEG were characterized using proton nuclear magnetic resonance?H¹NMR?and FTIR,and the micellization-demicellization behavior of the pH-sensitive PAE-PEG was investigated by fluorescence spectroscopy using pyrene as a probe.4.Surface modification of Mn-IONPs and characterization:After coating with1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy?polyethylene glycol?-2000]?DSPE-PEG?and PAE-PEG,and ligand exchange with TMAH,the hydrophilic Mn-IONPs@DSPE,Mn-IONPs@PAE and Mn-IONPs-TMAH were obtained.Next,the component of these nanoparticles were characterized using inductively coupled plasma emission spectrometer?ICP?.5.Cytotoxicity of Mn-IONPs@DSPE:The cytotoxicity of Mn-IONPs@DSPE was carried out with HepG2 cell lines through cocultured with Mn-IONPs@DSPE at different concentrations and different time,and then detected cell viability with the help of CCK-8.6.In vitro MR imaging of Mn-IONPs@DSPE and Mn-IONPs-TMAH:A series concentrations of Mn-IONPs@DSPE and Mn-IONPs-TMAH solutions were prepared and imaged using a 3.0 T MR with the following sequences:FSE T₁WI,T₁ mapping,FSE T₂WI,and T₂ mapping.7.In vitro MR imaging of Mn-IONPs@PAE:Mn-IONPs@DSPE solutions with a series concentration of Mn-IONPs@PAE at different pH?7.4,7.0,6.5,and 6.0?were prepared and imaged using a 3.0 T MR with the sequences of FSE T₁WI,T₁ mapping,FSE T₂WI,and T₂mapping.8.In vivo MR imaging of Mn-IONPs@DSPE:The in vivo MR imaging of Mn-IONPs was carried out with C57BL/6 mice.The FSE T₁WI and FSE T₂WI images of mice liver were acquired using a special rat coil and a 3.0 T clinical GE MRI scanner before and after injection of the Mn-IONPs@DSPE?1:20?with 5 mg[Fe+Mn]per kilogram body weight.Results:1.Synthesis and characterization of iron-eruciate and manganese-oleate complexes:The obtained iron-eruciate was a red to black gelatinous substance,while manganese-oleate presented reddish brown.FTIR showed that the characteristic spectra of iron-eruciate appeared at 1582,1557,and 1433 cm^-1,and that of manganese oleate were 1606,1549 and1423 cm^-1,which were consistent with previous report indicating that the obtained products were target substance.2.Synthesis and characterization of Mn-IONPs:The TEM images and corresponding distribution graphs of the obtained nanoparticles showed that these nanoparticles were of a size 3.0±0.28 nm with a narrow distribution and good uniformity.The crystal lattice fringes shown in the HRTEM images demonstrated the high crystallinity of the Mn-IONPs,and the measured interplanar distances of 2.56 and 2.97?were equivalent to the?311?and?220?lattice planes of Fe₃O₄,respectively.Compared with the spinel Fe₃O₄ powder diffraction data?JCPDS card no.01-1111?,these diffraction peaks in XRD shifted slightly toward that of MnFe₂O₄?JCPDS card no.10-0319?,which can be attributed to the doped manganese.The hysteresis loop showed that the saturation magnetization?Ms?of Mn-IONPs was 24.45 emu g^-1.Meanwhile,the negligible coercivity and remanence when the external magnetic field were absent indicate their superparamagnetic behavior.3.Synthesis and characterization of PAE-PEG:The pH-sensitive PAE-PEG was successfully prepared through Michael addition reaction.And the results of ¹H-NMR were consistent with that of previous report.The transformation from micellization to demicellization appeared at the pH of 7.0,proving its pH-sensitivity.4.Surface modification of Mn-IONPs and characterization:Hydrophobic Mn-IONPs have been successfully modified with DSPE-PEG,PAE-PEG and TMAH to be excellent hydrophilic.And the results of ICP showed that the molar ratio of Mn/Fe is 0.2,proving that the Mn²⁺was successfully doped into Fe₃O₄.5.Cytotoxicity of Mn-IONPs@DSPE:The cytotoxicity of Mn-IONPs@DSPE was carried out with HepG2 cell lines at the help of CCK-8.The results showed insignificant toxicity with a cell viability greater than 80%at a concentration less than 50?g/mL for 12 h and 24 h demonstrating that Mn-IONPs@DSPE have a good biocompatibility.6.In vitro MR imaging of Mn-IONPs@DSPE and Mn-IONPs-TMAH:The Mn-IONPs@DSPE and Mn-IONPs-TMAH solutions showed concentration-dependent signal enhancement.With the concentration increasing,the signal intensity tended to be brighter in T₁WI but gradually grew darker in T₂WI.The linear fitting of the concentration and 1/T₁ or1/T₂ showed that r₁ values of Mn-IONPs@DSPE 1:1,Mn-IONPs@DSPE 1:5,Mn-IONPs@DSPE 1:20,Mn-IONPs@DSPE 1:40 were 2.4,3.5,7.1,and 7.8 mM^-1s^-1,respectively,and the r₂ values were 199.5,149.7,120.9,and 94.8 mM^-1s^-1,respectively.And the r₁ and r₂ of Mn-IONPs-TMAH are 3.7 and 36.9 mM^-1s^-1,respectively.7.In vitro MR imaging of Mn-IONPs@PAE:The Mn-IONPs@PAE solutions showed concentration-and pH-dependent signal enhancement.With the concentration increasing,the signal intensity tended to be brighter in T₁WI but gradually grew darker in T₂WI.As the pH decends,the signal in T₁WI decrease while that in T₂WI increase,indicating the pH-sensitivity of Mn-IONPs@PAE.8.In vivo MR imaging of Mn-IONPs@DSPE:The axial T₁WI and T₂WI MR images of mice liver acquired preinjection and postinjection?0,10,30,60,and 120 min?of Mn-IONPs@DSPE.The significantly brighter T₁WI MR images and darker T₂WI MR images suggest good r₁ and r₂ values of Mn-IONPs@DSPE in vivo.Conclusions:1.The monodisperse,ultrasmall,superparamagnetic Mn-IONPs with an average size of3.0±0.28 nm were successfully synthesized by thermal decomposition of home-made iron-eruciate and manganese-oleate complexes.2.After coating with DSPE-PEG,the Mn-IONPs@DSPE showed great signal enhancement,especially,Mn-IONPs@DSPE 1:20 showed great r₁(7.1 mM^-1s^-1)and r₂(120.9mM^-1s^-1),making it a better candidate for T₁/T₂ dual-contrast mMRI.3.The nanoparticles modified with pH-sensitive PAE-PEG existed excellent hydrophilic and significant pH response in vitro,which was expected to be a pH-sensitive CA for imaging in vivo.