Fabrication of core shell nanoparticles
Transmission electron microscopy revealed that we could obtain magnetic polymeric core/shell nanospheres, ie, mag-PEI nanoparticles, with high magnetic nanoparticle loading (). The size distribution was found to be narrow, as indicated in the histogram. The zeta potential determined by dynamic light scattering indicated that the mag-PEI nanoparticles had positive surface charges around 39.3 ± 1.9 mV.
Transmission electron microscopic image. (A) Mag-PEI and histogram showing size distribution. (B) Mag-PEI nanoparticles with high magnetic loading.
Gel retardation assay
DNA binding affinity and magnetoplex formation were confirmed using the gel retardation assay. One microgram of plasmid pGL3-basic containing the CMV promoter/enhancer was applied to a prepared magnetoplex with mag-PEI nanoparticles at different N/P ratios. Trailing of DNA disappeared in the gel at an N/P ratio of 0.8/1 (). The results showed that plasmid DNA was adsorbed onto the mag-PEI nanoparticle surface by electrostatic interaction, resulting in the magnetoplex. Our cationic mag-PEI nanoparticles could neutralize the negative charge of plasmid DNA and increase the mag-PEI nanoparticle-induced cationic properties of the magnetoplex, corresponding to the results of the dynamic light scattering analysis ().
Size and zeta potential of mag-PEI nanoparticle/DNA magnetoplex at N/P ratios of 0.4/1, 0.8/1, 1.6/1, 4.3/1, 8.7/1, and 17.5/1
The morphology and size of the magnetoplex were analyzed under atomic force microscopy at two different N/P ratios, ie, 0.8 and 4.3. Atomic force microscopy detected that the magnetoplex appearance was spherical, corresponding to the core structures, ie, mag-PEI nanoparticles (). It is likely that addition of more mag-PEI nanoparticles with N/P ratios in the range of 0.8/1–4.3/1 could improve the magnetoplex condensation. This result correlated well with size analyzed by dynamic light scattering (). However, magnetoplex distribution changed in response to changes in the N/P ratio, as shown at ratios of 0.8/1 and 4.3/1 (). As a result, use of excess mag-PEI nanoparticles caused aggregation of the magnetoplex (), which may have interrupted cell transfection. Therefore, the magnetoplex formed at an N/P ratio of 0.8/1 was selected for cell transfection in further studies.
Atomic force microscopy images of mag-PEI nanoparticles (A) mag-PEI nanoparticles forming magnetoplexes with DNA at N/P ratios of 0.81/1 (B) and 4.3/1 (C).
Size and zeta potential analysis
The size and zeta potential of the magnetic nanoparticles were determined at pH 7.4. During magnetoplex formation, a dynamic change in size and charge occurred at N/P ratios in the range of 0.4–17.5 (). The size of the mag-PEI/ DNA was larger than that of mag-PEI, indicating that adsorption of DNA had occurred on the particle surface. With a constant amount of DNA, the total charges at each N/P ratio were dependent on the amount of mag-PEI nanoparticles added to the DNA solution. At N/P ratios in the 0.4–1.6 range, the charges increased according to the amount of mag-PEI nanoparticles added. However, at N/P ratios in the range of 4.3–17.5, the excess amount of mag-PEI nanoparticles destabilized the complex, as indicated by a decrease in zeta potential.
Optimal transfection conditions and transfection efficiency
Gene transfection was investigated in the human LAN-5 neuroblastoma cell line. Cells were transfected with the magnetoplex at an optimal N/P ratio of 0.8. Gene transfection was performed by incubation of the magnetoplex with cells for 15, 30, 60, 120, and 180 minutes in the presence and absence of an external magnetic plate. Transfection via Lipofectamine 2000 and PolyMAG, two commercial transfection reagents, was carried out in the positive controls. Luciferase signals expressed in transfected cells were determined quantitatively. At all tested N/P ratios, the results confirm that magnetic-induced transfection was a very effective system for gene transfection (). Luciferase expression levels were enhanced when DNA transfections were stimulated under magnetic force using the magnetoFACTOR-96 plate. Our results show that incorporation of magnetic nanoparticles in polymeric-based vectors is an effective strategy to elevate the transfection signal and shorten the transfection time. The efficiency of gene transfection was increased through physical stimulation by an external magnetic field. Among the N/P ratios in the range of 0.4–17.5, the highest transfection efficiency was obtained at an N/P ratio of 0.8. This result indicates that transfection efficiency was affected by several physicochemical properties of the magnetoplex. With a low amount of mag-PEI nanoparticles (N/P ratio < 0.8), the DNA strands are not completely adsorbed onto the nanoparticles. Therefore, the DNA delivered into the cells is not properly protected and easily digested by intracellular enzymes. The N/P ratio of 0.8 is probably the optimal condition, including for size, zeta potential, and complex stability. Although at an N/P ratio of 1.6–4.3 the magnetoplex also has an appropriate size and zeta potential, it can also cause cell membrane damage due to the greater number of nanoparticles with a positive surface charge added to the system. Furthermore, the atomic force microscopy results indicated that the magnetoplex at an N/P ratio of 4.3 was agglomerated, which was an unsuitable condition for transfection. Therefore, to obtain high transfection efficiency, several factors needed to be compromised.
Transfection efficiency (A) and cytotoxicity (B) of mag-PEI nanoparticles at 15, 30, 60, 120, and 180 minutes in LAN-5 cells.
Unlike for PolyMAG, the results indicate that the increased transfection efficiency for mag-PEI nanoparticles is time-dependent. PolyMAG is a commercially available carrier enhancing the transfection signal within a short induction time, and expression levels are fairly constant at different incubation times. The difference in improvement of transfection over time is probably due to the difference in magnetic properties between PolyMAG and mag-PEI nanoparticles. PolyMAG has very strong magnetic properties, which strongly enforces cell internalization of particles into the cell within a short time. However, after 120 minutes of induction, the transfection efficiency obtained from mag-PEI nanoparticles was about the same level as that obtained from PolyMAG, and was increased after 180 minutes of induction time. Apparently, for LAN-5 cells, a magnetic-assisted transfection system is more effective than a liposome-based system like Lipofectamine 2000, and there was no statistically significant difference between cells transfected with and without a magnetic plate.
Evaluation of cytotoxicity
In this study, the toxicity of mag-PEI nanoparticles towards LAN-5 cells was investigated using the MTT assay. Cells were treated with the magnetoplex under the same conditions as the transfection procedures. The viability of LAN-5 cells after transfection was in the range of 80%–100% when incubated with magnetoplex at N/P ratios of 0.4/1, 0.8/1, 1.6/1, 4.3/1, 8.7/1, and 17.5/1 for 15, 30, 60, 120, and 180 minutes (). Viability of cells exposed to magnetic induction was lower than that of unexposed cells. However, the differences were not statistically significant. Therefore, this result verifies that the cytotoxicity of mag-PEI nanoparticles is very low, making these particles suitable for use in gene therapy.
Visualization of uptake of mag-PEI nanoparticles into LAN-5 cells was observed by confocal laser scanning microscopy. The RITC-labeled mag-PEI nanoparticle/DNA magnetoplex at an N/P ratio of 0.8/1 was incubated with the cells for 60 and 180 minutes. The incubations were done separately with and without external magnetic induction. At 24 hours after transfection, confocal laser scanning microscopy images revealed the degree of intensity of the magnetoplex entering into LAN-5 cells (). At both 60 and 180 minutes of incubation, the intensities were significantly increased when transfection was performed under magnetic induction. The results indicate that the magnetoplex distributed into the intracellular compartment, the cytoplasm, and the region of the nucleus. Internalization was confirmed by confocal Z-stack image scanning (data not shown). The result corresponded well with the luciferase activity in . This provides more evidence of acceleration of the transfection period through magnetoplex transfection in neuronal cells.
Confocal image of LAN-5 cells 24 hours after transfection. Cells incubated with or without a magnetic plate for (A) 60 minutes and (B) 180 minutes were used for investigation of the cellular uptake of mag-PEI nanoparticles.
cDNA synthesized from human brain medulla oblongata total RNA was used as a template for synthesizing the TPH-2 gene fragment. PCR was performed using the specific primers described in . The PCR product showed a specific band at 1.5 kilobases under an ultraviolet transilluminator (Syngene, Cambridge, UK). The band was cut and ligated into a pGEM®
-T vector (Promega). DNA sequencing verified that the isolated PCR product had 99.5% similarity to Homo sapiens
TPH-2 mRNA. The TPH-2 gene was then finally transferred into pGL3-CMV basic containing the CMV promoter/enhancer.19
The resulting plasmid was used for gene transfection into LAN-5 cells.
Role of mag-PEI nanoparticles as a carrier for TPH-2 expression
The aforementioned data indicate that mag-PEI nanoparticles are a promising carrier for magnetic-assisted transfection due to their effectiveness, with low cytotoxicity and a short transfection time. We are continuing to test mag-PEI nanoparticles at an N/P ratio of 0.8/1 as a carrier for delivery of the neuronal TPH-2 therapeutic gene into LAN-5 cells. The magnetic induction time was fixed at 60 minutes. After transfection, the cells were incubated for 24 hours and total RNA was isolated by the TRIzol reagent, as described earlier. Expression of the TPH-2 gene was measured by RT-PCR using isolated total RNA as a template. PCR products from the housekeeping gene, GAPDH, were used to normalize the gene expression values. As a result, mag-PEI nanoparticles showed efficiency in induction of TPH-2 expression comparable with that of PolyMAG (). Cells transfected with the mag-PEI nanoparticle/pGL3-CMV-TPH-2 magnetoplex under magnetic induction showed a signal that was 13 times stronger than that obtained without induction. We compared the effectiveness of mag-PEI nanoparticles for therapeutic gene delivery with that of a liposome-based system, ie, Lipofectamine 2000. The results show that the difference between TPH-2 expression in cells transfected with and without magnetic induction was not significantly different. Therefore, this study demonstrates the potential of our synthesized nanoparticle for magnet-assisted gene transfection. Mag-PEI nanoparticles successfully enhanced the transfection efficiency of TPH-2 gene delivery.
Figure 6 Semiquantitative reverse-transcriptase polymerase chain reaction result shows expression of the TPH-2 gene in LAN-5 24 hours after transfection by mag-PEI nanoparticles compared with positive control Lipofectamine 2000™ and PolyMAG, and negative (more ...)