1. Tarbiyat modares university 2. Tarbiyat modares university 3. Tarbiyat modares university
Abstract
Introduction
Gene therapy is one of several approaches used to treat incurable diseases and genetic disorders. the success of this approach requires the development of innovative gene delivery vectors. research efforts have focused on the development of safe and effective viral and non-viral vector systems. although viral vectors have high transduction efficiency, they are immunogenicity to target cells, toxicity and enzymatic degradation. non-viral delivery systems are being pursued to facilitate therapeutic gene transfer in the clinic. these carriers, such as cationic polymers (spermine, polyethylene amine, etc.), typically interact with anionic dna via charged forces, condensing the long, string- like dna molecules into compact, nano-sized particles that are suitable for cellular uptake. dextran–spermine-based polycations were found to be highly effective in transfecting cells in vitro. however, low transfection efficiencies (as compared to viral vectors) and the inability to target specific cells or tissues still remained unsolved. in order to overcome these difficulties, magnetofection was developed to enhance the delivery of nucleic acids associated with magnetic nanoparticles. magnetofection is a novel and highly efficient method of transfecting cells in culture. in the present study, superparamagnetic iron oxide nanoparticles coated with spermin/dextran were prepared. these surface cationic magnetic spermin/dextran -iron oxide nanoparticles can potentially enhance magnetofection efficiency to improve gene delivery systems.
Methods
Superparamagnetic iron oxide nanoparticles (mnps) were prepared and coated with spermin/dextran. these nanoparticles were characterized using fourier transform infrared spectroscopy (ft-ir), vibrating sample magnetometry (vsm) and dynamic light scattering (dls). the morphology of the particles was studied by scanning electron microscopey (sem). the ability and stability of spermin/dextran -iron oxide nanoparticles conjugates to bind to dna was studied by a gel retardation and serum stability assay respectively.
human embryonic kidney 293 cells line (hek293) were cultured in dulbeccos modified eagle medium: nutrient mixture f-12 (dmem/f12). cell viability was determined using the mtt assay. luciferase assay was performed by luminometer to measure the transfection.
Results
Data was showed the size of the complexes was small enough to uptake by cell, and gel electrophoresis results indicated that the weight rate of the complex was found equal to 10 w/w (polycation/dna/mnp). the data obtained from gel retardation as well as serum stability showed that it was able to form complexes with the pdna, while free pdna was completely digested with intracellular dnases. mtt assay showed that our spermin/dextran- pdna -mnp did not showed high toxicity. the result of luminometer showed that complex spermin/dextran- pdna –mnp magnetization enhanced luciferase activity and transfection rate in compared with complex spermin/dextran- pdna.
Conclusion
Spermin-dextran nanomaterials are able to deliver nucleic acid in cell culture. to increase the transfer efficiency of this nanocomplex, magnetic nanoparticles are joined to this polymer and formed spermine / dextran-mnp. studying spermine/dextran- pdna -mnp in human hek 293 cells suggests that these particles are less cytotoxic. according to our results surface cationic magnetic spermin/dextran -iron oxide nanoparticle was stable and enhanced magnetofection efficiency. these results showed the potential usefulness of using magnetofection with less toxicity and high performance compared with the common viral method.
