Transfection For Eukaryotic Cell Lines With Polyethylenimine Coated Albumin Microbubbles

Objective:To improve the transgene efficiency of albumin microbubbles,we investigated a method of transgene with polyethylenimine coated albumin microbubbles.Method:(1) Albumin microbubbles (AMB) were prepared by sonicating the mixture of human albumin, manose and glucose; (2) Air filled in albumin microbubbles was exchanged with perfluoropropane and polyethylene glycol (PEG) was added as one gradient of microbubbles to improve the stability of albumin microbubbles; (3) Polyethylenimine (PEI) coated albumin microbubbles (PAMB) was prepared by sonicating the mixture of human albumin, PEI, polyethylene glycol (PEG), glucose, manose and perfluoropropane. Through transfecting CHO cell line with plasmid pIRSE2-EGFP, the optimal weight ratio of albumin and PEI was insured by measureing the percentage of GFP with flow cytometry; (4) Mean particle diameter and surface potential of AMB and PAMB were determined by photon cross correlation spectroscopy with a NANOPHOX particle size analysis system and ZetaProbe 7020; (5) To identify PAMB combined with pDNA, PAMB and pDNA with addition of SYBR green were mixed in serum-free Opti-MEM?Ⅰmedium in an Eppendorf tube. When SYBR green was combined with plasmid DNA, it could be excited by blue light and emitted green fluorescence. Thereafter, if PAMB combined with pDNA, it could emit green fluorescence. Green microbubbles could be inspected with a fluorescence microscrope; (6) Through transfecting CHO, 293T, M231, 293, SK-Hep-1, SKBR-3, MCF-7 and COS cell lines with plasmid pIRSE2-EGFP, we compared the transgene efficiency of PEI, albumin+PEI, Lipofectamine 2000 and PAMB; (7) The cytotoxicity and cell proliferation ablity of PEI, Lipofectamine 2000 and PAMB were measured by cell counting kit-8. Cell proliferation index was calculated by the OD value prior to Transfection, and 0 h, 24 h, 48 h, 72 h posttransfection / OD value prior to transfection; (8) COS cell line was transfected by AMB, SonoVue and PAMB mediated by ultrasound with different ultrasound (U) intensity and time; (9) The effect of ultrasound on microbubbles transgene was investigated. After CHO cell sections were prepared, reagents were added as follows in each group:①o nly NA-Green (a kind of DNA fluorescence dye which can not enter cells actively);②AMB+DNA+NA-Green;③AMB+DNA+NA-Green+U(1.0 W/cm2, 30 S);④P AMB+ NA-Green;⑤PAMB+DNA+NA-Green;⑥P AMB+DNA+ NA-Green+U (1.0 W/cm2, 30 S). After incubation at 37℃, 5% CO2 for 6 h; the medium was removed. The sections were rinsed for three times and immobilized with 1% paraformaldehyde, then were inspected with confocal microscoup and photos were taken.Results:(1) The half time of albumin microbubbles was extended from half an hour or several days to 6 months after air that filled in albumin microbubbles was exchanged with perfluoropropane and polyethylene glycol (PEG) was added as one gradient of microbubbles; (2) The optimal weight ratio of albumin and PEI was 125:1, which was obtained through transfecting CHO cell line with plasmid pIRSE2-EGFP, and the highest transfection efficiency of PAMB was about 55% on CHO cell line; (3) The particle size analysis showed the average particle diameter of AMB and PAMB was about 1400nm and 600nm, and the surface potentials of PAMB and AMB were -44.56±0.75 mV and -59.28±3.48 mV, respectively; (4) AMB could not combine with pDNA and only emitted plenty of fluorescence without outlook of microbubbles, PAMB combined with pDNA emitted fluorescence; (5) The transgene efficiency in PAMB group was higher than that in PEI group and albumin + PEI group in each cell line (P<0.01) and there was no statistical difference between PAMB group and Lipofectamine 2000 group; (6) Cytotoxicity of PAMB was lower than PEI and Lipofectamine 2000(P < 0.01), the proliferation index in no treatment group and PAMB was higher than that in PEI and Lipofectamine 2000 group (P < 0.01), there was no statistical difference PAMB group and no treatment group (P>0.05); (7) COS cells were transfected with AMB, SonoVue and PAMB mediated by ultrasound with different ultrasound intensity and time, the results showed that the transfection efficiency of PAMB was higher than the efficiency of AMB and SonoVue (P < 0.01), there was no statistical difference between AMB and SonoVue, and there were no statistical differences between different ultrasound intensity and time, and compared with PAMB alone; (8) Destruction of AMB and SonoVue mediated by ultrasound could increase the permeability of membrane, plasmid DNA enter cells through these micropores, while PAMB could bring plasmid DNA into cells actively.Conlusion:In summary, PAMB is very useful and low toxicity gene delivery method with high transfection efficiency in vitro. Further studies are necessary to examine the detailed mechanism of the effect of ultrasound on PAMB and transfection efficiency in vivo.