(656a) 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-PyMSP1₁₉in 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.