Development Of Fe₃O₄-PLLA-PEG-PLLA Magnetic Microspheres In Supercritical Fluid And Their Biological Evaluation

Fe₃O₄-poly（L-lactide）-poly（ethylene glycol）-poly（L-lactide） magneticmicrospheres （Fe₃O₄-PLLA-PEG-PLLA MMPs） were prepared in a process ofsuspension-enhanced dispersion by supercritical CO₂（SpEDS）. The biologicalevaluation of MMPs was carried out at cellular levle, molecular level, andtoxicity test in vivo. The main work of this thesis can be summarized asfollows:1. The MMPs were prepared by SpEDS process, and their physicochemicalproperties were characterized. The resulting MMPs have a good sphericalshape and a little rough surface could be clearly found, and a mean particlesize of586nm. After coating with polymer, the particle size of the resultingMMPs is much bigger than that of the Fe₃O₄nanoparticles; however, thisparticle size is much smaller than that of the PLLA-PEG-PLLA microspheresprepared under the same conditions without Fe₃O₄nanoparticles. Moreover,FTIR spectra reveal that the SpEDS process was a typical physical process toprepare MMPs without chemical reaction. For Fe₃O₄-PLLA-PEG-PLLA MMPs,the Fe₃O₄content from TG-DSC and XPS measurements is4.29%and saturationmagnetization from VSM measurements is16.33emu/g.2. At the cellular level, an XTT assay was also performed fordetermination of cell viability. Results indicate that the iron-oxidenanoparticles would induce less cytotoxicity. Intracellular ROS levels wereinvestigated by measuring the oxidative conversion of2’,7’-dichlorofluorescindiacetate （DCFH-DA） to the fluorescent compound dichlorofluorescin （DCF）. TheROS generation indicate that the polymer-coated MMPs （2.0mg/mL） had ahigher toxicity than uncoated Fe₃O₄nanoparticles, which led to about20%loss of cell viability and an increase （0.2fold） in ROS generation, thedifferences were not statistically significant （p>0.05）. However, an oppositephenomenon was observed in tests of hemolysis, which showed that the MMPs displayed the weakest hemolytic activity, namely only about6%at thehighest concentration （20mg/mL）. This phenomenon reveals that polymer-coatedMMPs created less toxicity in red blood cells than uncoated Fe₃O₄nanoparticles. Furthermore, the NO production assay indicates that MMPswould elicit the immune-inflammation.3. At the molecular level, the MMPs were shown to be less genotoxic thanFe₃O₄nanoparticles by measuring the micronucleus （MN） frequency in CHO-K1cells.Furthermore, the mRNA expression of pro-inflammatory cytokines demonstrates thatpolymer-coated MMPs did not elicit a more intense secretion of pro-inflammatorycytokines than uncoated Fe₃O₄nanoparticles.4. Acute toxicity tests of MMPs show quite a low toxicity, with anLD50>1575.00mg/kg.The evidence of low toxicity presented in the results indicates that theFe₃O₄-PLLA-PEG-PLLA MMPs from the SpEDS process has a betterbiocompatibility than Fe₃O₄,and has great potential for use in biomedicalapplications.