Preparation And Property Research Of Drug And Gene Carriers Based On Superparamagnetic Nanoparticles

About a century ago, the German bacteriologist Paul Ehrlich proposed theconcept of targeted therapy, which can delivery the drugs to the particular cells ortissues under the action of the certain guide mechanism. Moreover, thus it canvirtually increase the concentration of drug in the diseased area to achieve bettertherapeutic effects and reduce the toxic side effects on normal cells. Now the targetedtherapy is mainly applied to treat the malignant tumors. The National Cancer Instituteof U.S. National Institutes of Health （NIH） defines it as: targeted therapy is atreatment method, which can make drugs or other substances identify and attack thespecific cancer cells, with no damage to the normal cells. Therefore, it can effectiverycure cancers. We can say the targeted drug delivery system can provide an idealadministration route for treating cancer. The magnetic targeting refers to deliveringthe drugs or gene vector to targeted site under the guidance of an external magneticfield. It improves the current technology of drug delivery and gene transfection. Dueto the advantages of easy synthesis, small size, low toxicity, and uniquesuperparamagnetism, the superparamagnetic magnetite nanoparticles （SPION） havebeen widely applied to biology, and they have also been approved by the UnitedStates Food and Drug Administration （FDA） for disease imaging. In recent years,some investigations show the potential application value of magnetite nanoparticles intargeted delivery of various drugs. The superparamagnetic magnetite nanoparticlescould be incorporated within the drug carrier systems to facilitate the manipulationand delivery of the drug-loaded nanocarriers towards a desired area by externallylocalized magnetic steering. Therefore, to deliver the drugs and genes bysuperparamagnetic magnetite nanoparticles have great clinical potential.The combination of superparamagnetic magnetite nanoparticles and amphiphilicblock copolymers MPEG-PLGA can be used for anticancer drug delivery. Amphiphilic block copolymers can freely form nanomicelles with core-shell structurewhen its concentration is above the critical micelle concentration （CMC）. They have anumber of unique features, such as nanoscale size, biocompatible, biodegradablility,no immunogenicity, functional core and easy fabrication. The micelles have ahydrophobic inner core, which serves as a potent nanocontainer for hydrophobiccompounds through hydrophobic interactions, making them suitable as nanocarriersfor encapsulation, and delivery of water insoluble agents, including anticancer drugs,such as evodiamine （EVO）, paclitaxel （PTX）, camptothecin, and superparamagneticmagnetite nanoparticles, and it can improve their solubility in aqueous solutions. Inaddition, tailor-designed hydrophilic shell surrounding the micellar core caneffectively prevent their aggregation, precipitation, clearance by reticuloendothelialsystem and consequently prolong their blood circulation. Therefore, theself-assembled core-shell structures of the amphiphilic block copolymers as a drugdelivery system can greatly enhance solubility of insoluble antitumor drugs, improvetheir therapeutic efficacy in cancer chemotherapy. These micelles are of great value inbiomedicine application.The combination of superparamagnetic magnetite nanoparticles withpolyethylenimine and PLGA can be used for gene delivery. Polyethylenimine （PEI）,as a non-viral vector, has been applied to gene-delivery system since1995. Due to itsadvantages of aggregating plasmid DNA and escaping from the lysosome through theproton-sponge mechanism, it becomes one of the most promising and extensivelystudied gene carriers. PEI has very high cationic charge density, which can absorb thenegatively charged DNA molecules to form nanoparticle complexes, and has beenshown to be able to interact with the negatively charged cell membranes andinternalize itself into the cell through endocytosis. PEI has also been shown to protectcombined DNA from enzyme digestion. Nowadays, many researches have been doneto large number of branch PEI, such as25k bPEI（average MW,25,000） as genecarriers for gene delivery of DNA. It has high transfection efficiency. However, itsultrahigh cytotoxicity and aggregation in the bloodstream severely restrict its clinicalapplication. To achieve high gene transfection efficiency and reduce this cytotoxicity, PEI is modified by blocking primary amine groups through a coupling reactionbetween the amine group of PEI and COOH-activated PLGA nanosphere with Fe₃O₄nano particles in their core. This gene nanocarrier may provide a new strategy for thedevelopment of gene delivery vectors.In this paper, we mainly apply the magnetic targeting of superparamagneticmagnetite magnetite nanoparticles to deliver the anticancer drugs and genes, andfurther discuss on the bioactivity of these two nanocarriers. We have achieved a seriesof attractive results as follow:1. We have developed a magnetic nanocarrier with magnetic targeting abilitymainly applies to the targeted delivery of hydrophobic drugs. The amphiphilic blockcopolymer MPEG–PLGA [methoxy poly （ethylene glycol）-poly（d,l-lactide-coglycolide）] can self-assembly into the micelles with core-shell structurewith the solvent evaporation technique under vigorously sonication. Bothsuperparamagnetic iron oxide nanoparticles （SPION） and anticancer drug evodiaminewere loaded in the hydrophobic core, and the hydrophilic shell made the micelleshave good solubility in water, and then could be stably present in the aqueous solution.At the same time, we obtain the satisfactory drug-loading content （8.61±0.73%）, anda relatively high encapsulation efficiency （40.36±3.42%）. We characterized thephysical properties of the SPION-evodiamine-loaded nanocarrier, such as size,morphology, drug release and targeting etc. Besides, we performed the in vitroexperiments of drug absorption, cytotoxicity and apoptosis mechanisms by Humancervical carcinoma—HeLa cell lines. The tumor models of Kunming mice were usedfor evaluating the antitumor activity of SPION-evodiamine-loaded nanocarrier in vivo.Our results show great improvement in targeting delivery anticancer drugs andantitumor efficacy in vitro and vivo, and with good security. Especially in vivoantitumor experiments, it shows that the antitumor activity of theSPION-evodiamine-loaded nanocarrier is much higher than the free evodiamine. It isvery important for the future clinical application. In conclusion, this targeting strategywill open up a new avenue for designing enhanced drug delivery system in cancertherapy. 2. In this study we have prepared poly （D,L-lactide-co-glycolide）（PLGA）nanosphere with Fe₃O₄nanoparticles in their core, modify PEI to the surface ofPLGA/Fe₃O₄nanosphere via the coupled reaction between the amino of PEI andcarboxyl of PLGA （50/50）. The magnetic microsphere was characterized by DLS,Zeta Potential Analyzer, and FTIR, The result shows the particle size ofPEI-PLGA/Fe₃O₄nanosphere was279.2±19.5nm and bigger than PLGA/Fe₃O₄nanosphere185.4±4.9nm. Both of them have narrow polydispersity. The zetapotential of PEI-PLGA/Fe₃O₄nanosphere was22.67±2.29mV, while thePLGA/Fe₃O₄nanosphere was-20.03±2.61mV. It indicated that PEI was connected tothe surface of PLGA/Fe₃O₄nanoparticles by covalent linkage. The cytotoxicity ofPEI-PLGA/Fe₃O₄nanosphere was evaluated by MTT, and the transfection efficiencyof the nanosphere was measured using GFP plasmid DNA. Furthermore, thePEI-PLGA/Fe₃O₄nanosphere had a substantially lower cytotoxicity and a highertransfection activity than PEI polyplexes. These results demonstrated thatPEI-PLGA/Fe₃O₄nanosphere was an attractive genetic vector.