Introduction: Gene therapy has been greatly progressed to treat number of diseases such as inherit disorder, cancer etc. More recently, a number of strategies have been investigated to improve the low transfection efficacy such as electroporation[1]. Previously, our group has proposed a novel strategy for enhancement of cytoplasmic protein delivery called freeze concentration[2]. Upon freezing, water is frozen into ice crystals which lead to increase in the concentration of solutes results in phase separation. Spontaneous ice nucleation will occur and ice grows in all directions when solution is super cooled at -5 to -45 ̊C. The excluded solutes are inevitably concentrated into an unfrozen portion. This phenomenon effectively enhances the adsorption of protein complex to the cells located in the concentrated phase. Herein, we describe the use of freeze concentration phenomenon for effective gene transfection.
Materials and Methods: The pEGFP-N1 vector was used containing a green fluorescent protein and transformed in E.coli DH5α competent cells. Plasmids were collected and purified using Geno pure plasmid midi kit (Roche, Germany). The plasmid was dissolved in ultrapure water at 0.6 mg/mL. Lipofectamine (7.5 µL) and plasmid (2.5 µg) were used for the transfection and was incubated for 5 min to form carrier-DNA complex. One million L929 cells were suspended in 1mL commercial DMSO cryoprotectant (10% v/v) with DNA loaded lipofectamine in 1.9 mL vial were frozen at -80 ̊C for a one day. Cryoprotectant was added in order to prevent from freezing damage. The solutions were thawed and washed with PBS (-) 3 times. The unfrozen and frozen cells were observed by using confocal laser scanning microscope (CLSM).
Results and Discussions: In order to evaluate the cellular uptake and transfection efficiency during freezing, we investigated the EGFP fluorescence by CLSM. As shown in figure, the L929 cells expressed more EGFP fluorescence by using freeze thaw method.
In contrast, a very weak fluorescence was observed in the case of unfrozen state. The cell viability after freeze thaw was maintained over 90%. Moreover, a quantitative comparison of pEGFP delivery was assessed by measuring the intensity of fluorescence using CLSM.
This figure indicated that transfection efficiency of EGFP positive cells was 3 times higher than unfrozen EGFP positive cells using lipofectamine as a carrier. It is more likely that the plasmid DNA was concentrated around the cell membrane and could increase the adsorption and internalization to the cells. These results suggested that freeze concentration can facilitate gene transfection due to the enhanced concentration of DNA located across the peripheral cell membrane.
Conclusions: In this study, we developed a novel methodology called freeze concentration for efficient gene transfection. Freeze concentration phenomenon is expected to be an effective delivery approach for plasmid DNA to improve transfection and expression efficiency.
References:
[1] J.A. Kim, W.G. Lee and N.C. Jung, “ Enhanced electro-mediated gene delivery using carrier genes”, Bioelectrochemistry Vol. 78 Jun. 2010.
[2] S. Ahmed, F. Hayashi, T. Nagashima and K. Matsumura, “ Protein cytoplasmic delivery using polyampholyte nanoparticles and freeze concentration”, Biomaterials Vol. 35 Aug. 2014.