Efficacy and cytotoxicity of cationic‐agent–mediated nonviral gene transfer into osteoblasts

Abstract

Ex vivo gene transfer into osteoblastic cells is an advantageous strategy for bone tissue engineering. This study investigated the efficacy and cytotoxicity of in vitro cationic-agent-mediated nonviral gene transfer into osteoblasts. Various cationic agents, lipid, gelatin, and polyethylenimine (PEI) were tested. Each was formulated in various concentrations to form a complex with plasmid DNA encoding red fluorescent protein. The cationic agent/DNA complexes were transfected into human fetal osteoblastic cell line and rat bone-marrow-derived primary osteoblasts, as well as NIH 3T3 fibroblast controls. Rat primary osteoblasts were transfected more with cationic lipid and PEI agents than with gelatin carrier, yielding transfection efficacy up to 18.1% and 12.7 %, respectively. In contrast, human fetal osteoblastic cell line was transfected more with cationic lipid and gelatin than with PEI. There was a positive correlation between the lipid and PEI doses and cytotoxicity. When the lipid and PEI were used to transfect the rat primary osteoblasts in a dose that yielded the highest transfection efficacy, cell survival rates decreased as low as 40%. When their transfection efficacies into primary osteoblasts were compromised at two thirds of the highest value, that is, 12.6% and 8.3% for the lipid and PEI, respectively, the cell survival rate was nearly 80%. Cationic gelatin was associated with cell survival rates over 60 % in any cell type, regardless of the doses tested. These results suggest that different types of osteoblastic cells may possess different ability to the uptake and expression of cationic-agent-bound DNA. There seemed to be agent-specific threshold doses that dropped the cell survival rate. Cationic-agent-mediated nonviral gene transfer into osteoblastic cells may be successful when the agent- and dose-dependent transfection efficacy and cytotoxicity are optimized.

Comparison of Transfection Efficiency of Nonviral Gene Transfer Reagents

“…Therefore, it is important to carefully characterize the most suitable transfection system for each cell type prior to functional studies in order to achieve maximal transgene expression. Transfection efficiencies for MC3T3-E1 preosteoblasts have been reported using cationic liposome [12], cationic polymer [13], cationic lipid, gelatin, and PEI [14], nanostructured calcium phosphates [15], and gold nanoparticles [16]. In the present study, the highest transfection efficiency in MC3T3-E1 cells using luciferase gene in the presence and absence of serum was seen with FuGENE HD (lipids and other components).…”

Bone Morphogenetic Protein-2 (BMP-2) and Vascular Endothelial Growth Factor (VEGF) Transfection to Human Periosteal Cells Enhances Osteoblast Differentiation and Bone Formation

“…Gene delivery by the lipofectamine reagent is efficient for a wide spectrum of eukaryotic cells; in some cells, the efficiency reaches more than 90% (31,32). Toxicity to the cells in this technique could be reduced by optimizing the amounts of plasmid and reagents.…”

Nanovector for Gene Transfection and MR Imaging of Mesenchymal Stem Cells

“…The condensation of anionic DNA into nanoparticles is an important prerequisite for gene delivery employing polymeric nanovectors. 31 To be efficient, the nanoparticles must bear a net positive charge. The protonable amino nitrogen atoms of PEI make the polymeric network an effective "proton sponge" at virtually any pH.…”