Functional Magnetic Nanoparticles for Efficient Malaria DNA Vaccine Delivery

Functional magnetic nanoparticles for efficient malaria DNA vaccine delivery

Despite the numerous studies in the field of gene delivery over the last decades, much work remains to be done to deliver DNA for vaccination or therapeutic purposes. Due to the health and safety issues plaguing the use of viral vectors for therapeutic applications, there is an urgent need to develop an effective non-viral gene delivery system. These could involve physical methods including microinjection, electroporation, hydrodynamic pressure techniques, and particle bombardment; as well as chemical methods with polymeric or liposomal systems. Many modalities of gene delivery have proven to be inadequate for rapid and specific accumulation of active gene vectors on the target cells. For this reason, physical targeting by nucleic acid vectors associated with superparamagnetic particles under magnetic field (magnetofection) has recently been used for gene delivery, and has been shown to elevate any gene delivery vector up to several hundred times [1]. Magnetofection has been shown as an appropriate tool for rapid gene transfection at low dose in vitro and for site-specific in vivo applications [2, 3].

In this study, the delivery of malaria DNA vaccine encoded merozoite surface protein MSP1₁₉ (VR1020-PyMSP1₁₉) utilising magnetofection has been studied in vitro. The vector was composed of superparamagnetic nanoparticles (SPIONs) and PEI polymers that were prepared under different pH conditions of 4.0 and 7.0. The polyplexes with VR1020-PyMSP1₁₉ were stable in the aqueous media with average sizes of less than 150nm rendering them suitable for gene delivery purposes. To investigate whether pH condition of SPIONs-PEI complexes would affect the transfection efficiency of VR1020-PyMSP1₁₉ in vitro, COS-7 cell lines were transfected with the polyplexes in phosphate buffer at different PEI nitrogen to DNA phosphate (N/P) ratios. An SDS-PAGE assay was used to linearize PyMSP1₁₉ proteins derived in COS-7 cells and they were separated by electrophoresis depending on molecular weights. The SDS-PAGE was followed by Western Blot assay to visualize the expression of VR1020-PyMSP1₁₉. The vectors prepared at pH 4.0 demonstrated better DNA binding capacity, possibly due to the branched structure of polymers that facilitated some degree of protection over the DNA, while the more closed polymer structure at pH 7.0 exposed most of the DNA on the surface of the polyplexes. In addition, the cellular uptake of DNA complexed with SPIONs-PEI vectors at pH 4.0 was shown to increase significantly under the application of external magnetic field during the gene transfection process (Figure 1).

Cell viability studies were done via MTT assay to compare the in vitro cytotoxicity of SPIONs-PEI-DNA polyplexes with Lipofectamine 2000 (a cationic liposome) as the most effective non-viral reagent with consistently high transfection rates and slightly elevated toxicity due to four protonatable amines on its head group at physiological pH [4]. There was no obvious difference in cell toxicity between Lipofectamine 2000 and SPIONs-PEI-DNA polyplexes even at N/P ratios >15, however the SPIONs-PEI-DNA polyplexes showed dramatically higher gene expression than Lipofectamine. The cytotoxicity of SPIONs-PEI-DNA polyplexes was due to the high degree of polymer branches causing high positive charge density in the cell media. Increasing N/P ratio caused the reduction of cell viability, though the cell viability reached a plateau at approximately 60% for N/P ratios >15. These results indicated that SPIONs-PEI-DNA with N/P ratio of 10 was possibly optimal for gene transfection with low cytotoxicity while still maintaining relatively high gene transfection efficiency.

To validate the evidence of gene expression enhancement by magnetofection, fluorescent microscopy was also used to detect the expression of yellow fluorescence protein (YFP) plasmid combined with SPIONs-PEI and PEI polymer alone with N/P ratios of 5 and 10 to COS-7 cell lines. The observation with fluorescent microscopy (Figure 2) indicated that COS-7 cells transfected with SPIONs-PEI-DNA polyplexes under magnetic field exhibited significantly higher fluorescence compared to the cells transfected with PEI-DNA alone. The relatively higher level of gene expression at N/P of 10 may be due to the condensation of DNA into positively charged particles due to the higher amount of PEI, thus increasing the rate of their uptaking by the cells. In vivo studies to deliver DNA malaria vaccine with magnetofection are currently underway. 

Figure 1 Western Blot detection of PyMSP1₁₉. Lanes 1, 5, 10, 15, 20, 25 and 30 corresponding to different N/P ratios of SPIONs-PEI-DNA polyplexes with SPIONs-PEI prepared at pH 4.0

(a) Transfection under magnetic field; (b) Transfection in the absence of magnetic field.

Figure 2 Expression of yellow fluorescent gene (YFP) in COS-7 cells transfected with YFP. The upper row shows the effects of PEI polymer transfection alone; the lower row shows the effects of magnetofection using SPIONs-PEI-DNA polyplexes prepared with SPIONs-PEI complex at pH 4.0. The molar ratios of PEI nitrogen to DNA phosphate were 5 and 10, respectively.

References:

[1] Scherer, F., et al., Magnetofection: enhancing and targeting gene delivery by magnetic force in vitro and in vivo. Gene Therapy, 2002. 9(2): p. 102-109.

[2] Dobson, J., Gene therapy progress and prospects: magnetic nanoparticle-based gene delivery. Gene Therapy, 2006. 13(4): p. 283-287.

[3] Plank, C., et al., Magnetofection: Enhancing and targeting gene delivery with superparamagnetic nanoparticles and magnetic fields. Journal of Liposome Research, 2003. 13(1): p. 29-32.

[4] Djurovic, S., et al., Comparison of nonviral transfection and adeno-associated viral transduction on cardiomyocytes. Molecular Biotechnology, 2004. 28(1): p. 21-31.