Preparation Of Iron Oxide Nanoparticles With Different Surface Modifications And Their Distribution In Biological Tissue

At present,controlling the interaction between nanoparticles and biological environment remains as a challenge.This work utilized different surface modifiers to coat IONPs nanoparticles to make them have good dispersibility and biocompatibility and other properties.Then differently modified IONPs were co-cultured with the cells or injected into the mouse brain.The interactions between the nanoparticles and biological tissues were studied in order to meet different applications in biomedical field.The research details are as follows:(1)Polyethylene glycol(PEG)and iron acetylacetone(Fe(acac)₃)were used as raw materials,PEG modified iron oxide nanoparticles(PEG-IONPs)with super paramagnetic properties,good dispersion,uniform morphology and uniform size were prepared by high temperature thermal decomposition method.The nanoparticles were incubated with PC-12cells,and the cytotoxicity of PEG-IONPs was assessed by CCK-8 assay.A low toxicity in the PEG-IONPs concentration range of 200-400?g/m L,and a cell growth promotion effect in the range of 25-100?g/m L were observed.PEG had been proved to have good biocompatibility and to promote cell proliferation.And then stereotaxic injections of PEG-IONPs into the substantia nigra of Sprague-Dawley rats were performed.The distribution of nanoparticles in rat brain was observed by inductively coupled plasma emission spectrometer(ICP-OES)and transmission electron microscopy(TEM).It was found that the iron contents were higher in the olfactory bulb,temporal lobe,prefrontal cortex and thalamus after the injection of PEG-IONPs for 24 hours and 7 days,PEG-IONPs were mostly distributed around the axonal membrane and dendritic membrane,and a small amount were distributed near the myelin membrane and mitochondrial membrane.(2)PEG/PEI-IONPs were synthesized using PEG and PEI as raw materials.Then folic acid(FA)was coupled to PEG/PEI-IONPs surface using EDC/NHS chemical method to obtain FA-IONPs.The characterization results are as follows:The average sizes of PEG/PEI-IONPs and FA-IONPs under TEM were 10.80�2.2 nm,12.66�1.9 nm,respectively.The hydrodynamic diameter as well as Zeta potential of PEG/PEI-IONPs and FA-IONPs were24.36 nm,+21.48 m V and 105.71 nm,-4.69 m V,respectively.The successful conjugation of FA was demonstrated by Zeta potential change,particle size change,and UV,FT-IR and X-Ray Photoelectron Spectroscopy(XPS)characterization results.In cooperation with Xiamen University,He La cells were cultured with PEG/PEI-IONPs,FA-IONPs and pure FA for 4 h,respectively.The mass spectra of the two groups of nanoparticles modified with FA showed more impurity peaks compared to the Fe⁺peak at m/z 56 for nanoparticles without FA.By comparing the laser desorption ionization spectra of pure FA,we could confirm that the impurity peaks obtained in the cell experiment came from FA.Then PEG/PEI-IONPs and FA-IONPs were incubated with lysosomal red dye(Lyso Tracker Red DND-99)for 1 h for staining.The stained cells were observed with a laser confocal microscope(CLSM)with nanometer spatial resolution and mass spectrometry imaging.The results shown that PEG/PEI-IONPs and FA-IONPs could enter cells in large quantities and were widely distributed in the lysosome.(3)High-temperature thermal decomposition was used to synthesize hydrophilic PEG-IONPs.Then by reacting with sodium citrate and chloroauric acid Au-Fe₃O₄ gold magnetic nanoparticles were obtained.From the TEM results,it can be seen that the Au-Fe₃O₄ NPs were about 40-60 nm in size with petal-like shape.The hydrodynamic diameter and Zeta potential are 78.8 nm and-9.7 m V,respectively.The Au-Fe₃O₄ NPs showed a smooth characteristic UV absorption peak at 568 nm,this characteristic peak range(500-600 nm)was in accordance with gold nanoparticles absorption peak.The result of X-Ray diffraction(XRD)characterization showed that the diffraction peaks of Au-Fe₃O₄corresponded to Au standard card(07-0484)and Fe₃O₄ standard card(28-0491).These characterization results proved the successful preparation of Au-Fe₃O₄ NPs.(4)Combined this work and previous studies by our group,we summarized and compared the results of the injection of different surface modified iron oxide nanoparticles(IONPs)into the substantia nigra of rats and their co-culture with cells,and the observed distributions of the nanoparticles in cells and brain tissue using TEM.Iron oxide nanoparticles modified with dimyristoylphosphatidylcholine(DMPC-IONPs)either injected into rat substantia nigra or co-cultured with cells,showed varying degrees of aggregation and endocytosis,and this may be due to the nature of DMPC as an important component of cell membranes.After a certain amount of biotin was injected in the cell culture media or brain tissue in advance,the IONPs modified with streptavidin(SA-IONPs)and the SA-IONPs modified with Fluorescein isothiocyanate(FITC-SA/PEI-IONPs)co-cultured with cells or injected into brain tissue could adhere to the cell membrane.This takes advantage of the strong binding force between biotin and SA,which allows the nanoparticles to stay on the cell membrane.IONPs modified with glutathione(GSH-IONPs)were injected into the murine brain,these nanoparticles appeared to be endocytosed in large amounts,and large numbers of nanoparticles are found in axons and cell bodies,it is very likely that they enter the cell through vesicle packaging that results in endocytosis,the GSH-IONPsreach axonal distant sites along with axoplasmic transport.IONPs co-modified with Arg-Gly-Asp and maleic anhydride(RDG/Mal-IONPs)and IONPs co-modified with bovine albumin and Mal(BSA/Mal-IONPs)injected into the rat brains were found onmembrane and in organelles including Golgi apparatus,mitochondria,endoplasmic reticulum,in contrast,Mal-IONPs were less distributed in these areas,RDG/Mal-IONPs,BSA/Mal-IONPs and Mal-IONPs were all in the form of aggregates,this is most likely to be related to the fact that biomolecules(such as albumin,etc.)in the organism wrap nanoparticles to form protein crowns.IONPs modified with chondroitin sulfate(CS-IONPs)tended to surround the extracellular space near the neuron cell body and synapse,and no obvious endocytosis were obserbed when they were injected into rat brains,this can be attributed to the fact that CS is an extracellular matrix,and there are many CS receptors on the cell membrane,allowing nanoparticles to stay near the cell membrane to avoid endocytosis.