Preparation Of Functional Magnetic Nanomaterials Based On Fe₃O₄ And Basic Research Of Their Biological Applications

In recent years,with the development of nano-materials science and technology,magnetic nano-materials based on Fe3O4 nanoparticles have attracted broad interest in the field of bioengineering and biomedicine,because Fe3O4 nanoparticles have small particle size,high sensitivity,low toxicity,stable performance,easy access to raw materials and other advantages.In this work,the magnetic Fe3O4 nanoparticles were used as the base materials,after surface modification by organic/inorganic materials they acted as antineoplastic drug carriers and fluorescent molecular probe carriers so as to construct the magnetic targeting drug delivery systems and intelligent magnetic probes.Their structures and application performances were studied and analyzed.The specific research contents are as follows:Firstly,the magnetic Fe3O4 nanoparticles with different functional groups(-OH,-COOH and-NH2)were prepared by coprecipitation method,high-temperature thermal decomposition method and hydrothermal method respectively.The nanoparticles were characterized by transmission electron microscopy(TEM),X-ray powder diffraction(XRD),fourier transform infrared spectroscopy(FT-IR),thermogravimetric analysis(TG)and vibrating sample magnetometer(VSM).The results show that the prepared magnetic Fe3O4 nanoparticles with the particle size in the range of 10-100nm have stable crystal structures and good magnetic responsive performance.The hydrophilic PEG/PEI@Fe3O4,PAA@Fe3O4,citrate-modified Fe3O4,and 1,6-hexamethylenediamine-modified Fe3O4 were homogeneously and stably dispersed in PBS buffer solution.The oleic acid modified hydrophobic Fe3O4 can form stable magnetic fluid in n-hexane.Secondly,based on the prepared magnetic Fe3O4 nanoparticles,the pH-sensitive magnetic targeting drug delivery systems were constructed by directly or indirectly loading the drugs:(1)PEG/PEI@Fe3O4-DOX,this drug delivery system was prepared by the reaction of a primary amine group on PEG/PEI@Fe3O4 with a 13-carbonyl of doxorubicin(DOX)to form a pH-sensitive imine linkage,the drug loading efficiency and loading content are 85.01%and 1.36%,respectively;(2)CMNPs-DOX,the trifluoroethyl ester terminated polyethylene glycol silane(APS-PEG-TFEE)was synthesized to connect bare Fe3O4 nanoparticles with DOX,the DOX loaded onto the carriers through electrostatic interaction,the drug loading efficiency and loading content are 76.19%and 3.26%,respectively;(3)Fe3O4@LDH-MTX,the drug carrier Fe3O4@LDH was prepared by hydrothermal method in one step,the drug intercalated into the lattice space of MgAl layered hydroxides(LDH)which can self-degrade in acid environment by host-guest exchange process,the drug loading efficiency and loading content are 91.78%and 18.36%,respectively;(4)CS/Fe3O4@mSiO2-DOX,the hydrophobic Fe3O4 nanoparticles were modified with mesoporous silica(mSiO2)which also acted as drug store warehouse,after DOX loading,the chitosan(CS)was employed to wrap the Fe3O4@mSiO2-DOX as the blocking agent to inhibit premature drug release,the drug loading efficiency and loading content are 93.60%and 29.30%,respectively.The physicochemical characterizations of the prepared pH-sensitive magnetic targeting drug delivery systems indicated that the delivery systems had suitable biological size,stable structure,good biocompatibility and magnetic responsive performance.Thirdly,the sustained-release properties of different pH-sensitive drug delivery systems were studied by simulating the microenvironment of normal tissue and tumor tissue with PBS buffer solution at different pH values.The results showed that the prepared drug delivery systems had good pH sensitivity,when pH<6.0,the loaded antitumor drug released rapidly in a short period of time to achieve the required dose to inhibit tumor growth followed by sustained release to maintain the dose,while only a small amount of drug released in the normal tissue microenvironment.In particular,the in vitro drug release kinetics studies of Fe3O4@LDH-MTX and CS/Fe3O4@mSiO2-DOX by Bhaskas,First-order and Higuchi equations showed that the drug release kinetics of Fe3O4@LDH-MTX is either by dissolution of LDH layer or ion-exchange process,and the ion-exchange process may be the main effect in the co-effect,and the drug release kinetics of CS/Fe3O4@mSiO2-DOX is rapid degradation and dissolution of "gatekeeper" CS,which are completely different with the pH-sensitive chemical bonds and electrostatic effects.In addition,WST-1 or MTT toxicity analysis showed that the prepared drug delivery carriers were almost free of biological toxicity,and the drug delivery systems had better inhibitory effect on the proliferation of tumor cells than individual drug molecules.Fourthly,the robust,inexpensive magnetic probe(RB-PEG-PAA-MNPs)was successfully prepared by the amidation reaction of PAA@Fe3O4 acted as a carrier with ammoniated rhodamine B(RB).The results of ion detection showed that RB-PEG-PAA-MNPs had high selectivity and sensitivity to Cr3+,even with the coexistence of competing ions(Ag+,Ca2+,Li+,NH4+,Sn2+,Pb2+,K+,Mg2+,Mn2+,Na+,Ni+,Fe2+,Cd2+,Cu2+,Fe3+,Co2+ and Zn2+),the probe still has high selectivity to Cr3+,and the limit of detection for Cr3+(LOD=0.131nM)is far less than the value(1.923μM)reported by the US Environmental Protection Agency.In addition,RB-PEG-PAA-MNPs could detect and remove Cr3+ in the samples in vitro,and the WST-1 toxicity analysis showed that the probes were almost non-toxic to HUVEC cells and had rapid living cells imaging effect.Finally,the Fe3O4@Au nanocomposites were prepared by reduction of gold chloride via zero-degree reaction in the presence of citrate-modified Fe3O4 nanoparticles,and the rhodamine B isothiocyanate(RBI)was supported on Fe3O4@Au by Au-S bond,the magnetic probe Fe3O4@Au-TA-RBI(abbreviated as probe)was prepared after hydrophilicity modification of mercaptoacetic acid(TA).The Au nanoparticles act as a fluorescent quenching agent for RBI,and TA is used as a surface modifier.The physical and chemical characterization showed that the prepared probe had stable structure,good hydrophilicity and magnetically responsive performance.In addition,the ion detection experiments in aqueous solution showed that the probe had high selectivity and sensitivity to Hg2+.In the presence of competing ions(Li+,Fe2+,Cd2+,Zn2+,Mg2+,Na+,K+,Mn2+,Ni2+,NH4+,Co2+,Ag+,Ca2+,Sn2+,Cu2+,Fe3+ and Pb2+),the probe still has high selectivity to Hg2+,and the limit of detection(LOD=3.03nM)was much less than the value(30nM)reported by World Health Organization.In particular,the cyclic detection test showed that the recovered probe by magnet was able to achieve the function of recycling by adding the RBI molecule.In addition,MTT toxicity analysis showed that the probe has good biocompatibility and live cell imaging function.