We demonstrated the effective transgene carriers using PEI-decorated superparamagnetic iron oxide nanoparticles (SPION) as well as chemically-conjugated chondroitin sulfate-PEI copolymers (CP). Thus, in this study, CP was used to coat onto the surface of SPION (CPIO) for magneto-gene delivery systems. With an assisted magnetic field, the trasfection efficiencies of CPIO/DNA magnetoplexes were superior to a gold standard, PEI, and a commerically-available Magnetofection™ reagent, PolyMAG. In miRNA delivery, the results showed CPIO/pMIRNA-128 indeed expressed miRNA-128 higher with the assisted magnetic field than without.
Introduction: Gene therapy shows great potential in treatment of a wide range of tumors, especially in glioblastomas because of its innovative strategies and high specificity. The treatment of glioblastomas includes strategies to deliver tumor-suppressor genes, suicide genes, immune response-induced cytokine genes, and conditionally replicating oncolytic viruses. However, successful gene therapy processes need to construct a strict delivery system to ensure target genes can be delivered to the diseased tissue and to avoid naked genes being degraded. In this study, to develop an ideal gene delivery systems, CP was coated onto SPION (CPIO) through electrostatic interactions to prepare a novel magneto-gene carrier. CPIO was utilized to load DNA and form a magnetoplex that could further enhance gene transfection efficiency with an assisted magnetic field.
Experimental Methods: The different w/w ratios of CPIO/pDNA were prepared to test DNA condensation efficiencies. The in vitro cell tests of CPIO/DNA were done in U87-MG in the presence of an external magnetic field. The internalization of CPIO/pDNA with or without the magnetic field was analized using flow cytometry and CLSM. For miRNA delivery, we constructed a pDNA containing miRNA-128 precursor sequence and trasfected it into U87 cells. The miRNA expression was tested using a miRNA plate assay kit and FISH. To demonstrate the enhanced accumulation of CPIO/DNA at the tumor site, we labeled pDNA with a Cy5 dye and traced its fluorescence intensity using an U87-xenograft nude mouse model.
Results and Discussion: CPIO/Cy5-DNA at the w/w ratio of 5 was chosen to compare with or without magnetic field condition for best cellular uptake into U87 cells. From the fluorescent magnetoplexes, we clearly observed more fluorescent spots of FITC-CPIO and Cy5-DNA in the cytoplasm as the magnetic field was applied. In flow cytometric results, magnetoplex with an assisted magnetic field showed better internalization efficiency than without.
Magnetofection-mediated microRNA expression was visualized via fluorescence in situ hybridization (FISH). The red fluorescence of Cy5-labled probe was observed in the cytoplasm of U87 cells. This result indicates that CPIO as well as PolyMag could successfully transfect pMIRNA-128 in U87 cells. The extracted RNA from transfected cells was detected by a streptavidin-HRP conjugate and chemiluminecscent substrate. The CPIO and PolyMag without the magnetic field increased miRNA expression level of ~1.5 fold as compared with the control group. In the presence of the magnetic field, the miRNA expression level of CPIO/pDNA was ~1.5 fold higher than that without the assisted magnetic field and ~2.6 fold higher than the control group.
Kaohsiung Medical University (KMU-DT103007); The Ministry of Science and Technology of Taiwan (MOST103-2320-B-037-012-MY3)