Preparation Of Gelatinase-targeted And USPIO-loaded Nanoparticles And Study Of Distribution In Mice

Objective:To prepare gelatinase-targeted and USPIO-loaded nanoparticles, and measure parameters in physics and chemistry, explore their stability in vitro and distribution in mice, and investigate their potential use as MRI contrast agents to position lymph node metastasis in tumor models.Methods:Oil-soluble USPIO particle (-6nm in diameter) was synthesized of ferrous acetylacetonate by high temperature decomposition. To synthesize mPEG-Pep-PCL, amidation reactions were used to combine with PEG, gelatinase-targeted substrate peptides and PCL. A self-assembly method was used to construct USPIO-loaded hydrophilic nanoparticles. Their diameter, polydispersity, Zeta potential, Fe concentration, and stability in vitro were analyzed. In vivo study, fresh blood, liver, spleen and kidney of mice were collected at definite time points in following 72h after intravenously administration of nanoparticles. A measure system of Phenanthroline was used to determine Fe content in blood, liver, spleen and kidney. A kind of hollow nanoparticles with amino termination was also constructed by a self-assembly method. After marked with NIR-797 (a kind of near infrared dyes), these new nanocomplexes were administrated intravenously. Their distribution in mice was detected by NIRF imaging in following 72h after administration.Results:In TEM observation, oil-soluble USPIO nanoparticles were uniform spherical particles, with 5-10nm in diameter. The structure character of mPEG-Pep-PCL/mPEG-PCL was confirmed by NMR analysis. Their estimated number-average molecular weight was samely about 12000Da. USPIO-loaded hydrophilic nanoparticles were prepared with polymer mPEG-Pep-PCL/mPEG-PCL. Their hydrodynamic size was respectively 102.0nm vs 106.4nm, with polydispersity index 0.044 vs 0.090, Zeta potential -0.27mV vs -0.97mV, and Fe concentration 77.7ug/ml vs 84.5ug/ml. Within 12 weeks after gelatinase-targeted particles preparation, particle size fluctuated between 100nm and 106nm, and polydispersity index mainly kept below 0.15. After intravenous administration of hydrophilic nanoparticle (2mg Fe/kg, body weight), PK software 3p87 was used to analyze the variation character of plasma Fe concentration, which was closer to two-compartment model with a functional equation C=Ae-α·t+Be-β·t).Parameters A, α, B and β were respectively 28.48ug/ml vs 34.62ug/ml,0.434h-1 vs 0.539h-1,15.36ug/ml vs 14.34ug/ml, and 0.027h-1 vs 0.026h-1. Half life of distribution phase t,/2(a) and half life of elimination phase t1/2(β) were respectively 1.60h vs 1.29h, and 25.95h vs 26.61h. Within 72h after intravenous administration, both kinds of nanoparticles were mainly distributed in the liver. Hydrodynamic size of these two nanoparticles, whose surface ending was amino and was marked with NIR-797, was 127.5nm vs 126.7nm, with polydispersity index 0.086 vs 0.104 respectively and Zeta potential -22.60mV vs-29.19mV respectively. The signal of NIRF was detectable in the liver even at 72h after intravenous administration.Conclusions:By a self-assembly method, we successfully prepared gelatinase-targeted and USPIO-loaded nanoparticles, with uniform size (about 100nm in particle diameter) and good stability in aqueous phase. After intravenous administration in mice, the variation of plasma Fe concentration was following two-compartment model, and nanoparticles were mainly distributed and metabolized in the liver. With the result of initial experiment in tumor models, it was confirmed that the gelatinase-targeting function is the only cause of differences seen in tumor MR imaging between the two kinds of nanoparticles.