The effectiveness, cytotoxicity, and intracellular trafficking of nonviral vectors for gene delivery to bone mesenchymal stem cells
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Abstract:
Nonviral gene delivery that enables exogenous gene expression in bone mesenchymal stem cells could accelerate clinical application of cell-based gene therapy. This study systematically investigated and compared the potential of polyethylenimine and Lipofectamine 2000 as gene carriers to modify bone mesenchymal stem cells including transfection efficiency, cytotoxicity, intracellular trafficking as well as cell membrane damage and apoptosis/necrosis. Polyethylenimine at its optimal N/P ratio of 10 demonstrated the same toxic effects but lower transfection efficiency (17.1% vs 39.5%) compared to Lipofectamine. Intracellular trafficking resulted in over 80% of bone mesenchymal stem cells that were able to take up polyethylenimine polyplexes, but only 20.69% showed nuclear uptake; however, for Lipofectamine, about half bone mesenchymal stem cells were found to uptake lipoplexes but about 30% displayed nuclear localization. Moreover, the percentages of nuclear localization of both vectors were in close relationship with their transfection efficiency. We concluded that for bone mesenchymal stem cell transfection, polyethylenimine displayed high cellular uptake but Lipofectamine was more effective in delivering genes into the nucleus, which was likely the underlying basis for a more efficient gene expression. Further structure modification of polyethylenimine such as improving its nuclear entry ability will eventually make it a better candidate for bone mesenchymal stem cells’ in vitro gene delivery.Keywords:
Lipofectamine
Polyethylenimine
In this study, the optimal dose of Lipofectamine 3000 and Turbofect to transfect adherent cell lines such as CHO-K1 and HEK293 cells in comparison with non-adherent H9T-cells with pEGFP-N1 and pCDH was identified.Lipofectamine 3000 is a new transfection reagent which is claimed to be more efficient than other transfection reagents like Turbofect. Transfection efficiency could be affected by the nature of target cell line and vector.Transfection efficiency and cytotoxicity of each reagent was identified by using flow cytometry and XTT assay, respectively.Lipofectamine 3000 was more efficient in transfecting pCDH, while Turbofect was more efficient in separate transfection of CHO-K1 and HEK293 with pEGFP-N1. Lipofectamine 3000 could be cytotoxic in transfecting H9T-cells with pCDH. Also, H9T-cells were not sufficiently transfected with each plasmid vector by using each Lipofectamine 3000 and Turbofect. Turbofect had less cytotoxicity effect on all three cell lines than Lipofectamine 3000.Transfection of suspended cells like H9T-cells by using Lipofectamine 3000 and Turbofect would not result in sufficient transfection.Lipofectamine 3000 is the best choice for transfection of CHO-K1 and HEK293 with pCDH while Turbofect is preferably used in transfecting these cell lines with pEGFP-N1 (Tab. 1, Fig. 2, Ref. 26).
Lipofectamine
HEK 293 cells
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Objective: To transfect siRNA into murine macrophage by different methods, and comparison of different transfection methods. Method: The cells were transfected by different methods with lipofectamine,Roche transfection reagent,electroporation and lentiviral vectors,The analysis of transfection efficiency of different methods by flow cytometer,levels of target mRNA and cell viability were determined. Result: The efficiency of Lentivirus-mediated siRNA transfection is the highest,up to 34. 75 ± 5. 30%,and the RNAi effect is best; Followed by Roche transfection reagent and electroporation,but electroporation cell viability lowest; Lipofectamine difficult to transfected,only 12. 17 ± 1. 53%,but cell viability of Lipofectamine was greatest. Conclusion: Through the comparison of the four kinds of murine macrophage transfection methods,the advantages and disadvantages of four kinds of transfection methods was acquired,and provides the basis for murine macrophage transfection.
Lipofectamine
Viability assay
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The enhance effect of oligonucleotide transfection mediated by cationic liposomes (Lipofectin, LipofectAmine, FuGENE6 and DM-R1E-C) were investagated using flow cytometry in Hela cells. Two 19-mer molecules, phosphodiester oligonucleotide (ODN19) and phosphorohieoate oligonucleotides (SODN19), were employed, which are labeled with Fluorescein isothiocyanate(FITC) and have the same sequence. The results show that, for the unmodified ODN19 molecules, LipofectAmine and DMRIE-C can obviously improve the transfection, but no improving effect were observed in other two liposome. For the S-ODN19 transfection, all of the 4 calionic liposomes possess the observed ability to enhance the transfection, of which the LipofectAmine was more efficient than three others. The mean FITC density (5203.11) of LipofectAmine mediated transfection was several decades times comparable to the control cultures without mediated liposome. The improving effect of liposomes mediated transfection was ranked as LipofectAmine FuGENE6 DMRIE-C lipofectin. Meanwhile. The experiment also suggested that, when transfection procedure was performed for about 4 hours, a high efficiency can be expected in FuGENE6 mediated oligonucleotides transfection.
Lipofectamine
HeLa
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Polyethylenimine (PEI), a commercially available gene transfection reagent, is a promising nonviral vector due to its inherent ability to efficiently condense genetic materials and its successful transfection performance in vitro. However, its low transfection efficiency in vivo, along with its high cytotoxicity, limit any further applications in gene therapy. To enhance the gene transfection performance and reduce the cytotoxicity of linear polyethylenimine, pseudopolyrotaxane PEI25k/CD and the polyrotaxanes PEI25k/CD-PA and PEI25k/CD-PB were prepared and their transfection efficiencies were then evaluated. The pseudopolyrotaxane PEI25k/CD exhibited better transfection efficiency and lower cytotoxicity than the transfection reagent linear PEI25k, even in the presence of serum. It also showed a remarkably higher cell viability, similar DNA protecting capability, and better DNA decondensation and release ability, and could be useful for the development of novel and safe nonviral gene delivery vectors for gene therapy.
Polyethylenimine
Viability assay
Biocompatibility
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[Objective] The research aimed to provide references for the research on non-viral vector cationic lipids.[Method] With cationic liposome Lipofectamine 2000 as vector,the transfection efficiency of Lipofectamine 2000 to different cells(HeLa cell,B16 cell,MCF-7 cell and SW-480 cell) were studied through DNA delay test and plasmid transfection test and it was compared with that of other transfection reagents(Sofast and DOTAP).And the effects of cell types on the transfection efficiency of Lipofectamine 2000 were discussed.[Result] With the increasing of the proportion of transfection reagent in the complex,DNA delay action was strengthened.When the mass ratio of Lipofectamine 2000 to DNA was increased to 5:1,only a few DNA moved out from the origin site.The transfection efficiency of Lipofectamine 2000 to SW-480 cell was obviously higher than other 3 kinds of cell types.The transfection effeiciency of Lipofectamine 2000 on B16 cell line was better than that of Sofast and that of Sofast and DOTAP was equivalent.[Conclusion] Lipofectamine 2000 had high transfection efficiency to most cells,but it was not suitable for all types of cells.
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HeLa
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We simultaneously tested the transfection efficiency of NT2/D1 and HeLa cells with Lipofectamine (Life Technologies) and Effectene (Qiagen) transfection reagents using the pCH110 eukaryotic assay vector, which contains the lacZ reporter gene. Under our culture conditions for NT2/D1 and HeLa cells, Effectene transfection efficiency could be augmented by simply increasing the amount of plasmid DNA 1.5-3 times above the recommended concentration without any visible cytotoxicity. With the Lipofectamine reagent, optimal transfection efficiency was obtained for both cell lines within the recommended concentrations, but at the top of the range. The results indicate that optimization of the transfection process should include plasmid DNA concentrations above the levels suggested by the manufacturers, in order to accomplish the highest transfection efficiency.
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HeLa
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Objective:To optimize the Lipofectamine 2000 transfection conditions for rat BHK cells and Chinese hamster BRL cells using β-Gal vectors.Methods: Lipofectamine 2000 were mixed in varied ratios with plasmid DNA to transfect the rat BHK cells and Chinese hamster BRL cells on a 96-well plate.Successfully transfected cells were stained blue due to expression of β-Gal degradable substrates(X-gal),enabling estimation of the transfection efficacy by counting the blue cells under microscope.ANOVA was performed for statistical test.Results: The maximal efficacy was(43.2±9.3)% in BHK cells when β-Gal vector and Lipofectamine 2000 were mixed in a ratio of 0.27∶0.8(μg∶μL),and was(5.7±1.3)% in BRL cells when β-Gal vector and Lipofectamine 2000 were mixed in a ratio of 0.10∶0.3(μg∶μL).Conclusion: Lipofectamine 2000 appears to be useful for the transfection of mammal cells;however,the transfection conditions should be optimized for individual cell types.
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Chinese hamster
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The aim of the present study was to improve transfection efficiency using different combinations of cationic liposomes, linear polyethylenimine and DNA. A novel gene delivery system (lipopolyplex) was developed by premixing cationic liposomes containing cholesterol or oligoamine modified cholesterol (derivative I-III) and linear polyethyleneimines (PEIs) following addition of plasmid at three different C/P ratios. The resultant complexes were characterized for their size, zeta potential and ability of DNA condensation. Luciferase reporter gene was used for determination of transfection efficiency in Neuro2A cells. Mean particle size of prepared complexes was less than 200 nm and they showed positive surface charge. The transfection efficiency of vectors was reduced by increasing in carrier concentration/plasmid DNA ratio (C/P ratio) while gene expression of cationic liposome or PEI was increased at higher C/P ratios. Complexes composed of PEI 2.5 or 250 kDa and liposome containing derivative I had the highest transfection activity. Furthermore, non-viral vectors described in this study showed low cytotoxicity. The results show that small and large molecular weight linear PEI in combination with liposome have little toxicity and may enhance transfection efficiency. Keywords: Cationic liposome, Cytotoxicity, Gene delivery, Lipopolyplex, Polyethylenimine, Transfection, Cholesterol derivatives, Nanostructures, Non-viral gene delivery, Nanocomplexes
Polyethylenimine
Cationic polymerization
Zeta potential
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Objective The purpose of this study is to investigate the optimal conditions for DNA transfection with polyethylenimine (PEI).Methods The influences of various factors on the transfection efficiency of 25 kDa linear PEI were investigated to get optimal conditions.Results PEI could fully condense DNA with a PEL/DNA ratio≥2,and the optimal mixing time was 10 mi- nutes.But the presence of serum significantly lowered the efficiency of PEI.The increasing PEI/ DNA ratio could raise transfection efficiency.The optimal density of 293T cells for transfection was 80%.Conclusion The factors affecting the transfection efficiency of PEI were studied,and the optimal conditions were identified.
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Objective:To improve efficiency of transgenes by albumin microbubble,which is a non-virus gene delivery vector.Method:Polyethylenimine coated albumin microbubble(PAMB) was prepared by sonicating the mixture of human albumin,polyethylenimine and glucose.The transgene efficiency of polyethylenimine,polyethylenimine +albumin,PAMB and Lipofectamine 2000 was compared by detecting the positive cell ratio of green fluorescent protein(GFP) using flow cytometry.Results:The average efficiency of transgene by polyethylenimine,polyethylenimine+albumin,PAMB and Lipofectamine 2000 was(13.75±4.88)%,(5.20± 1.15)%,(49.17±6.75)% and(53.72±5.69)%,respectively.The efficiency of PAMB group was higher than that in polyethylenimine group(P 0.05),however,there was no statistical significance between PAMB group and Lipofectamine 2000 group(P 0.05).Conclusion:Albumin microbubble modified by polyethylenimine is highly effective on transgene,and it is a useful non-virus gene delivery vector.This study may provide a new method for gene transfection in vivo.
Polyethylenimine
Lipofectamine
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