J Korean Assoc Oral Maxillofac Surg.  2013 Jun;39(3):112-119. 10.5125/jkaoms.2013.39.3.112.

Enhanced bone morphogenic protein adenoviral gene delivery to bone marrow stromal cells using magnetic nanoparticle

• Lee JT
• Jung JW
• Choi JY
• Kwon TG

• KMID: 1430478
• DOI: http://doi.org/10.5125/jkaoms.2013.39.3.112

Abstract

OBJECTIVES
This study investigated the question of whether adenoviral magnetofection can be a suitable method for increasing the efficacy of gene delivery into bone marrow stromal cell (BMSC) and for generation of a high level of bone morphogenic protein (BMP) secretion at a minimized viral titer.
MATERIALS AND METHODS
Primary BMSCs were isolated from C57BL6 mice and transduced with adenoviral vectors encoding beta galactosidase or BMP2 and BMP7. The level of BMP secretion, activity of osteoblast differentiation, and cell viability of magnetofection were measured and compared with those of the control group.
RESULTS
The expression level of beta galactosidase showed that the cell transduction efficiency of AdLacZ increased according to the increased amount of magnetic nanoparticles. No change in cell viability was observed after magnetofection with 2 microL of magnetic nanoparticle. Secretion of BMP2 or BMP7 was accelerated after transduction of AdBMP2 and 7 with magnetofection. AdBMP2 adenoviral magnetofection resulted in up to 7.2-fold higher secretion of BMP2, compared with conventional AdBMP2-transduced BMSCs. Magnetofection also induced a dramatic increase in secretion of BMP7 by up to 10-fold compared to the control. Use of only 1 multiplicity of infection (moi) of magnetofection with adenoviral transduction of AdBMP2 or AdBMP7 resulted in significantly higher transgene expression compared to 20 moi of conventional adenoviral transduction.
CONCLUSION
Magnetic particle-mediated gene transudation is a highly efficient method of gene delivery to BMSCs. Magnetofection can lower the amount of viral particles while improving the efficacy of gene delivery.

Keyword

Magnetics; Nanoparticles; Adenovirus; Mesenchymal stromal cells

MeSH Terms

Adenoviridae
Animals
beta-Galactosidase
Bone Marrow
Cell Survival
Magnetics
Magnets
Mesenchymal Stromal Cells
Mice
Nanoparticles
Osteoblasts
Transgenes
Virion
beta-Galactosidase

Figure

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  Fig. 1 Efficiency of gene transfer with adenoviral magnetofection compared to conventional adenoviral transduction. The evaluation of expression of β galactosidase with X-gal staining involved counting the number of blue nuclei/field. β galactosidase expression increased significantly according to the use of magnetofection (A) and increased viral titer of adLacZ (B). Magnetofection (2 µL of magnetic particle) revealed at least 400 times' higher efficiency compared to conventional adenoviral transduction. (×100)(moi: multiplicity of infection)

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  Fig. 2 Dependence on varying magnetofectin (magnetic nanoparticle) dose at constant multiplicity of infection (moi) (AdLacZ 200 moi). The efficiency of the transduction was potentiated according to the increased amount of magnetic particles without significant cell apoptosis. (×100)

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  Fig. 3 BMP2 protein in conditioned media of bone marrow stromal cell (BMSC) transduced with AdBMP2 with/without magnetic nanoparticle. BMSCs were transduced with the indicated individual AdBMP2 titer. The conditioned media were prepared by culturing cells in serum-free medium for 24 hours. Levels of BMP2 were measured using specific ELISA kits.(moi: multiplicity of infection).

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  Fig. 4 BMP7 protein in conditioned media of bone marrow stromal cell (BMSC) transduced with AdBMP7 with/without magnetic nanoparticle. BMSCs were transduced with the indicated individual AdBMP7 titer. The conditioned media were prepared and measured using a method similar to BMP2. The application of magnetofection accelerated BMP7 secretion from BMSC.(moi: multiplicity of infection).

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  Fig. 5 Alkaline phosphatase (ALP) activity of transduced bone marrow stromal cell (BMSC) with different multiplicity of infection (moi) or inclusion of magnetofection (A) and assayed for mineralization by ALP staining after culturing for 8 days in mineralizing medium (B).(Mag: magnetofection)

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  Fig. 6 Evaluation of cell viability. (A) Magnetofection with 2 µL of magnetic particle in adenoviral gene transfer did not change cell viability significantly (P>0.05). The comparison of the relative toxicity caused by magnetofection was evaluated with 3-(4,5-Dimethylthiazol-2-yl)-2.5-diphenyltetra-zoliumbromide (MTT) assay. (B) Magnetofection with high amount (10 µL) of magnetic particles and high dose of AdBMP2 (200 moi) triggered cell apoptosis. (×100)(OD: optical density, moi: multiplicity of infection, BMSC: bone marrow stromal cell, BMP: bone morphogenic protein)

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  Fig. 7 Schematic illustration of adenoviral magnetofection to bone marrow stromal cell (BMSC). Magnetic force directs magnetic particle-viral complex to target BMSC.

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