Synthesis Of Gd(OH)₃Nanoparticles And Experimental Studies Of MR Imaging

Objective:To synthesize a novel gadolinum-loaded nanorods as a molecular imagingcontrast agent for magnetic resonance imaging.And to study the toxiciy, relax-ativity, ditribution in normal mice,MR imaging of carcinoma of prostate inmice with this novel cntrast media.Material and Methods:(1) The monodispersed Gd(OH)₃nanorods were fabricated by one-pothydrothermal method. Typically, Gd(NO₃)₃·6H₂O (10mmol) was dissolvedinto deionized water (20mL) to form a homogeneous solution. A NaOHaqueous solution (2.5M) was added into above solution under vigorouslymagnetic stirring through continuous titration until the pH value reached12.The final volume was then adjusted to45mL by adding deionized water andthe resulting solution was sealed to a Teflon-lined stainless-steel autoclave (50mL). After reaction for24h at180℃, the autoclave was cooled to ambienttemperature naturally.(2)MTT reduction assays were carried out to quantifiedthe cytotoxicity of Gd(OH)₃nanorods. In a typical procedure, cells (HepG2andMCF-7cell lines) were cultured in96-well plates as a density of5000per wellfor12h to allow the cells to attach. Subsequently, serial dilutions of differentnanorod formulations were added to the culture medium. At the end of theincubation time, the medium containing nanorods were removed, and cellsamples were treated with MTT for another4h, which was followed by theaddition of dimethyl sulfoxide (DMSO) to dissolve the formazan crystals.Bio-Rad model-680microplate reader was applied to measure the absorbanceat a wavelength of570nm (corrected for background absorbance at490nm). Six replicates were done for each treatment group and percent viability wasnormalized to cell viability in the absence of nanorods.In a typical experiment,HepG2cells with a density of2×10₄were plated in a12-well plate for4h toallow the cells to attach. After the cells were washed twice by coolphosphate-buffered saline (PBS), nanorods (200μg/mL) were added to the cellculture medium. After incubating for48h, the cells were washed again withPBS several times to remove the remaining nanorods, and then observed underan Olympus BX-51optical system microscopy.Mice were sacrificed30daysafter administration for the histology studies. The tissues (heart, spleen, liver,lung, and kidney)were collected from two groups (contro and test group).Invitro T₁-weighted MR imaging was acquired by using a1.5T clinical MRIinstrument. Dilutions of Gd(OH)₃nanorods in PBS buffer containing1%agarose with expected different concentrations as contrast agent were placed ina series of4.0mL Eppendorf tubes for T₁-weighted MR imaging and fixed in10%neutral buffered formalin. For in vivo MR imaging, Gd(OH)₃nanorodswere dispersed in0.9wt%NaCL solution, then injected into the mice throughthe tail vein.(3)12mice were randomly divided into2groups and administeredwith two contrast agents: Gd-DTPA and Gd(OH)₃.1.5Tesla MR seanner wereused. The Plain T₁WI SE sequences were performed.The repeated T₁WI SEsequences were then Performed15min,30min, l h,2h,4h,6h,8h,12h,18h and24h after the intravenous injection of Gd-DTPA and Gd(OH)₃. Thesignal intensity of the liver,and renal cortex in the pre-and post-contrast T₁WIwas measured using a region of interesting with2mm₂.(4)The prostatecarcinoma cell were inoculated subeutaneously in mice to produce prostatecarcinoma model.12mice were randomly divided into two groups andadministered with Gd-DTPA and Gd(OH)₃. MR T₂WI FSE sequence and T₁WISE sequences before and after the intravenous injection of two contrast agentswere performed.The scan parameters and the time points were the same as last part. The T₁WI signal intensity of the tumor was measured and the value ofsignal to noise ratio (SNR) was calculated.Results:(1) For the fabrication of Gd(OH)₃nanorods, a vigorous hydrothermalmethod has been introduced to perform the experiment. Scanning electronmicroscopy (SEM) image for the pure Gd(OH)₃nanorods confirm that thesample consists of monodisperse rod-like nanoparticles with a mean length of100nm, an average diameter of15nm, as well as smooth surface.(2) MTTassay showde the cell viability results, revealing that more than90%cells weresurvived even after48h of incubation with the nanorods. The mice of the testand control groups were sacrificed30days after administration. No tissuedamage or any other adverse effect associated with the administration of theGd(OH)₃nanorods were observed. The mass magnetic susceptibility value ofthe as-prepared nanorods was determined to be8.9×10^-5emu/g Oe. The T₁WIimages were evaluated at a1.5T human clinical scanner and the relaxivity r1value of the nanorods calculated from the slope of the plot of the relaxation rate(1/T₁) as a function of Gd3+concentration was determined to be12.3mM^-1S^-1.Along with the strength of the applied magnetic field increased, ideal linearcorrelation between the magnetization and the applied magnetic field in thenanorods was obtained, demonstrating that Gd(OH)₃nanorods possessedparamagnetism.(3) The signal value of T₁WI of the liver and kidney was notstastistically different when no enhacement. The signal value of T₁WI of theliver was stastistically different at the time point of15min,30min, l h,2h,4h,6h,8h,12h,18h and24h among two groups. The signal value of T₁WI of thekidney was not stastistically different when no enhacement. The signal value ofT₁WI of the kidney was stastistically different at the time Point of15min,30min,l h,2h,4h,6h,8h,12h,18h and24h among two groups.(4) Themodel of the prostate carcinoma was successfully obtained in12mice. All the prostate carcinoma were detected by MR and the range of the tumor size wasfrom15mm to20mm. The tumor is low signal on T₁WI and high signal onT₂WI. The signal value of T₁WI of the tumor was not stastistically differentwhen no enhacement. The signal value of T₁WI of the tumor was stastisticallydifferent at the time Point of15min,30min,l h,2h,4h,6h,8h,12h,18h and24h among two groups. The SNR of the tumor was not stastistically differentwhen no enhacement and15min.The SNR of the tumor was stastisticallydifferent at the time point of30min,l h,2h,4h,6h,8h,12h,18h and24hamong two groups.Conclusions:In summary, we have synthesized monodisperse Gd(OH)₃nanorodsthrough a facile and scalable hydrothermal route. Detailed characterizationshave been performed to evaluate the properties of the nanorods. Cell-cytotoxicity assay and post-injection histology analysis further demonstrate theexcellent biocompatibility of the nanorods, indicating the feasibility for diseasediagnosis and chemotherapy. MRI measurement studies of the Gd(OH)₃nanorods revealed a higher T₁-weight relaxation rate compared withcommercial Gd-DTPA complex. In addition, the capability of nanorods ascontrast agents for MR imaging has been evaluated through preliminary in vivocharacterization. The advantage of the Gd(OH)₃for the malignant prostatetumor of mice is very apparent. Due to these advantages, the well-constructedGd(OH)₃nanorods are promising for MR imaging, showing more potentials forfurther clinical application. The specific functionalization of the as-preparednanorods for specific targeting is in progress, and research extended for moredetailed in vivo live animal systems are also undergoing.