Decaarginine-PEG-Artificial Lipid/DNA Complex for Gene Delivery: Nanostructure and Transfection Efficiency

2008 ◽  
Vol 8 (5) ◽  
pp. 2308-2315 ◽  
Author(s):  
Masahiko Furuhata ◽  
Radostin Danev ◽  
Kuniaki Nagayama ◽  
Yoshifumi Yamada ◽  
Hiroko Kawakami ◽  
...  
Keyword(s):  
Gene Delivery ◽  
Cellular Uptake ◽  
Plasmid Dna ◽  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
Distribution Analysis ◽  
Lipid Concentration ◽  
Uptake Mechanism ◽  
Transmission Electron ◽  
Green Fluorescent

Oligoarginine conjugates are highly efficient vectors for the delivery of plasmid DNA into cells. Decaarginine-conjugated lipid (Arg10-PEG-lipid) was synthesized and the effects of Arg10-PEG-lipid concentration at a fixed DNA concentration on transfection efficiency and the structure of the complexes were studied below and above critical micelle concentration (CMC), and at the lipid nitrogen/DNA phosphate (N/P) ratio corresponding to transfection, respectively. Arg10-PEG-lipid at the concentration below CMC showed stronger interaction with DNA by fluorescence intensity distribution analysis, and significantly higher luciferase and green fluorescent protein expression than that above CMC. A phase-contrast cryo-transmission electron microscope (cryo-TEM) experiment showed that the morphology of the complexes depended on the N/P ratio. At a low N/P ratio corresponding to that in transfection at a lipid concentration below CMC, a net-like structure developed in which plasmid DNA was involved. A further increase in the N/P ratio, a large fibrous nanostructure of complexes, was also observed. Without DNA, these structures were not obtained. The cellular uptake mechanism of complexes using flow cytometry with inhibitors suggested that complexes with two different morphologies showed similar cellular uptake and uptake mechanism, macropinocytosis. Differences in transfection efficiency of the complexes may be explained by a large fibrous nanostructure inhibiting the cellular internalization of complexes or the release of DNA from macropinosomes into cytoplasm. Arg10-PEG-lipid/DNA complexes formed a favorable nanostructure for gene delivery, depending on the N/P ratio in water.

scholarly journals Novel Histone-Based DNA Carrier Targeting Cancer-Associated Fibroblasts

Polymers ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 1695
Author(s):  
Alexey Kuzmich ◽  
Olga Rakitina ◽  
Dmitry Didych ◽  
Victor Potapov ◽  
Marina Zinovyeva ◽  
...  
Keyword(s):  
Gene Delivery ◽  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
Growth Factor Receptor ◽  
Surface Protein ◽  
Firefly Luciferase ◽  
Luciferase Assay ◽  
Cancer Associated Fibroblasts ◽  
Histone H2a ◽  
Green Fluorescent

Nuclear proteins, like histone H2A, are promising non-viral carriers for gene delivery since they are biocompatible, biodegradable, bear intrinsic nuclear localization signal, and are easy to modify. The addition of surface-protein-binding ligand to histone H2A may increase its DNA delivery efficiency. Tumor microenvironment (TME) is a promising target for gene therapy since its surface protein repertoire is more stable than that of cancer cells. Cancer-associated fibroblasts (CAFs) are important components of TME, and one of their surface markers is beta-type platelet-derived growth factor receptor (PDGFRβ). In this study, we fused histone H2A with PDGFRβ-binding peptide, YG2, to create a novel non-viral fibroblast-targeting DNA carrier, H2A-YG2. The transfection efficiency of histone complexes with pDNA encoding a bicistronic reporter (enhanced green fluorescent protein, EGFP, and firefly luciferase) in PDGFRβ-positive and PDGFRβ-negative cells was estimated by luciferase assay and flow cytometry. The luciferase activity, percentage of transfected cells, and overall EGFP fluorescence were increased due to histone modification with YG2 only in PDGFRβ-positive cells. We also estimated the internalization efficiency of DNA-carrier complexes using tetramethyl-rhodamine-labeled pDNA. The ligand fusion increased DNA internalization only in the PDGFRβ-positive cells. In conclusion, we demonstrated that the H2A-YG2 carrier targeted gene delivery to PDGFRβ-positive tumor stromal cells.

scholarly journals Delivery of Bioactive Gene Particles via Gelatin-Collagen-PEG-Based Electrospun Matrices

2021 ◽  
Vol 14 (7) ◽  
pp. 666
Author(s):  
Eleni Tsekoura ◽  
Porntipa Pankongadisak ◽  
Daniel Graf ◽  
Yaman Boluk ◽  
Hasan Uludağ
Keyword(s):  
Gene Delivery ◽  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
Electrospun Fiber ◽  
C2c12 Cells ◽  
Bone Morphogenetic Protein 2 ◽  
Target Cells ◽  
Fiber Mats ◽  
Mouse Osteoblast ◽  
Green Fluorescent

The fabrication of fiber mats via electrospinning has been adopted in the last decades to produce high quality scaffolds for tissue engineering. However, an effective combination of electrospinning methods with gene delivery therapies remains a challenge. In this study, we describe how the delivery of gene complexes via electrospun mats that contain different volumes of gelatin (Gel), collagen (Col), and polyethylene glycol (PEG) can affect gene expression by transfected cells. Non-viral complexes were formulated by using lipid modified polyethylenimine (PEI) polymer and plasmid DNAs (pDNA) like the reporter Green Fluorescent Protein (GFP) and the therapeutically relevant Bone Morphogenetic Protein-2 (BMP-2) and electrospuned after being mixed with different volumes of Gel-Col-PEG mats and delivered to human myoblast (C2C12) and mouse osteoblast cells (MC3T3). The entrapment of GFP complexes via different homogeneous electrospun fiber mats revealed that a high fraction of collagen in the mats affected the quality of the fibers and led to reduced transfection efficiency on target cells. On the other hand, the fabrication of double-layered mats that contained collagen without complexes as a first layer and gelatin-collagen-PEG with complexes as a second layer successfully induced GFP expression and ALP activity in C2C12 cells. We conclude that this study has established the advantage of formulating multilayered bioactive collagen-based mats for gene delivery applications.

scholarly journals Cationic Polybutyl Cyanoacrylate Nanoparticles for DNA Delivery

2009 ◽  
Vol 2009 ◽  
pp. 1-9 ◽  
Author(s):  
Jinghua Duan ◽  
Yangde Zhang ◽  
Wei Chen ◽  
Chengrong Shen ◽  
Mingmei Liao ◽  
...  
Keyword(s):  
Plasmid Dna ◽  
Hepg2 Cells ◽  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
Electrophoretic Analysis ◽  
Dna Encoding ◽  
Cell Viability Assay ◽  
Viability Assay ◽  
Low Cytotoxicity ◽  
Green Fluorescent

To enhance the intracellular delivery potential of plasmid DNA using nonviral vectors, we used polybutyl cyanoacrylate (PBCA) and chitosan to prepare PBCA nanoparticles (NPs) by emulsion polymerization and prepared NP/DNA complexes through the complex coacervation of nanoparticles with the DNA. The object of our work is to evaluate the characterization and transfection efficiency of PBCA-NPs. The NPs have a zeta potential of 25.53 mV at pH 7.4 and size about 200 nm. Electrophoretic analysis suggested that the NPs with positive charges could protect the DNA from nuclease degradation and cell viability assay showed that the NPs exhibit a low cytotoxicity to human hepatocellular carcinoma (HepG2) cells. Qualitative and quantitative analysis of transfection in HepG2 cells by the nanoparticles carrying plasmid DNA encoding for enhanced green fluorescent protein (EGFP-N1) was done by digital fluorescence imaging microscopy system and fluorescence-activated cell sorting (FACS). Qualitative results showed highly efficient expression of GFP that remained stable for up to 96 hours. Quantitative results from FACS showed that PBCA-NPs were significantly more effective in transfecting HepG2 cells after 72 hours postincubation. The results of this study suggested that PBCA-NPs have favorable properties for nonviral delivery.

331 OPTIMIZATION OF BRANCHED 25kDa POLYETHYLENIMINE FOR EFFICIENT GENE DELIVERY IN BOVINE FETAL FIBROBLASTS

2013 ◽  
Vol 25 (1) ◽  
pp. 313 ◽  
Author(s):  
D. O. Forcato ◽  
M. F. Olmos Nicotra ◽  
N. M. Ortega ◽  
A. P. Alessio ◽  
A. E. Fili ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Gene Delivery ◽  
Cell Viability ◽  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
Cost Effective ◽  
Experimental Conditions ◽  
Fetal Fibroblasts ◽  
Significant Difference ◽  
Green Fluorescent

Cost-effective, highly efficient, and noncytotoxic transfection of bovine fetal fibroblasts (BFF) has proven difficult to achieve by regular chemical and physical methods. The aims of this study were to evaluate transient transfection efficiency and toxicity of commercially available branched 25 kDa polyethylenimine (25 kDa PEI, Sigma-Aldrich, St. Louis, MO, USA) and to optimize the transfection conditions leading to high percentages of PEI-transfected fibroblasts with minimum cytotoxic effects. Bovine fetal fibroblast (BFF) cells were seeded a day before transfection in 24-well plates at a density of 3 × 104 cells per well in DMEM with antibiotics and 10% SFB. When 70 to 90% confluence was reached, cells were washed with PBS and incubated in DMEM without antibiotics or SFB. For the transfection-mix preparation, increasing amounts of plasmidic DNA (pZsGreen1; 2 to 6 µg) were added to 50 µL of DMEM without antibiotics or SFB, incubated for 5 min at room temperature, and complexed with 0.5 to 4 µg of PEI (from 1 mg mL–1 solution) in 50 µL of PBS for 10 min. This transfection mix was added to the cell cultures and, 2 h later, 500 µL of DMEM with antibiotics and 10% SFB was added to each well. Detection of green fluorescent protein (GFP) expression by flow cytometry (reported as percentage of green fluorescent cells) was performed 48 h after transfection. Results were analysed by ANOVA and Tukey test and expressed as mean ± SEM (P < 0.05). We found no significant difference between the percentage of GFP-positive cells transfected with 1 or 2 µg of 25 kDa PEI at 2 µg of DNA/well (15.2 ± 1.3 v. 16.9 ± 0.9%, respectively; P > 0.05), whereas cells transfected with 1 or 2 µg of low-molecular-weight PEI (2 kDa) showed extremely low transfection efficiencies. Increasing the DNA load up to 6 µg significantly enhanced cell transfection (3.5- and 6-fold comparing 2 µg v. 4 µg and 6 µg of DNA, respectively; P < 0.05) at 1 and 2 µg of 25 kDa PEI/well. In order to evaluate the cytotoxic effect of PEI, cell viability was determined using the MTT assay in 96-well plates (cells/well), with each condition scaled down to replicate the effect of 2 kDa or 25 kDa PEI in a 24-well plate. The MTT results (expressed as % of the control) indicated that PEI became cytotoxic at concentrations equivalent to 2 and 4 µg/well (54.7 ± 3.4 and 18.5 ± 5.7, respectively), whereas 1 µg/well produced a slight detrimental effect on cell viability (90.0 ± 2.6). No evidence of cytotoxicity was observed when the BFF were incubated with 0.5 µg/well of 25 kDa PEI and 1 or 2 µg/well of 2 kDa PEI. To study if a combination of low- and high-molecular-weight PEI could improve transfection efficiency and reduce toxicity, we tested a mixture (1 : 1) of 2 kDa and 25 kDa PEI. Even though the 1 : 1 mixture was less cytotoxic, the efficiency of gene delivery was not improved. We conclude that, under our experimental conditions, the highest percentage of GFP-expressing cells with good viability was obtained when 1 µg of 25 kDa PEI was added per well. Therefore, branched 25 kDa PEI transfection represents an efficient, simple, and cost-effective alternative for gene delivery in bovine fibroblast cells in culture.

scholarly journals Systemic AAV6-synapsin-GFP administration results in lower liver biodistribution, compared to AAV1&2 and AAV9, with neuronal expression following ultrasound-mediated brain delivery

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Danielle Weber-Adrian ◽  
Rikke Hahn Kofoed ◽  
Joseph Silburt ◽  
Zeinab Noroozian ◽  
Kairavi Shah ◽  
...  
Keyword(s):  
Gene Delivery ◽  
Intravenous Injection ◽  
Viral Vectors ◽  
Focused Ultrasound ◽  
Fluorescent Protein ◽  
Gfp Expression ◽  
Peripheral Organs ◽  
Neuronal Expression ◽  
Green Fluorescent ◽  
The Brain

AbstractNon-surgical gene delivery to the brain can be achieved following intravenous injection of viral vectors coupled with transcranial MRI-guided focused ultrasound (MRIgFUS) to temporarily and locally permeabilize the blood–brain barrier. Vector and promoter selection can provide neuronal expression in the brain, while limiting biodistribution and expression in peripheral organs. To date, the biodistribution of adeno-associated viruses (AAVs) within peripheral organs had not been quantified following intravenous injection and MRIgFUS delivery to the brain. We evaluated the quantity of viral DNA from the serotypes AAV9, AAV6, and a mosaic AAV1&2, expressing green fluorescent protein (GFP) under the neuron-specific synapsin promoter (syn). AAVs were administered intravenously during MRIgFUS targeting to the striatum and hippocampus in mice. The syn promoter led to undetectable levels of GFP expression in peripheral organs. In the liver, the biodistribution of AAV9 and AAV1&2 was 12.9- and 4.4-fold higher, respectively, compared to AAV6. The percentage of GFP-positive neurons in the FUS-targeted areas of the brain was comparable for AAV6-syn-GFP and AAV1&2-syn-GFP. In summary, MRIgFUS-mediated gene delivery with AAV6-syn-GFP had lower off-target biodistribution in the liver compared to AAV9 and AAV1&2, while providing neuronal GFP expression in the striatum and hippocampus.

A quantitative method for normalization of transfection efficiency using enhanced green fluorescent protein

2005 ◽  
Vol 342 (2) ◽  
pp. 341-344 ◽  
Author(s):  
Dineshkumar H. Dandekar ◽  
Manish Kumar ◽  
Jayashree S. Ladha ◽  
Krishna N. Ganesh ◽  
Debashis Mitra
Keyword(s):  
Green Fluorescent Protein ◽  
Quantitative Method ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
Green Fluorescent

scholarly journals Nucleofection Efficiency of Sheep Primary Fibroblasts Established from Refrigerated Skin

2018 ◽  
Vol 10 (4) ◽  
pp. 12
Author(s):  
Mahipal Singh ◽  
Xiaoling Ma
Keyword(s):  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
Genetically Engineered ◽  
Dermal Fibroblasts ◽  
Biologically Active ◽  
Cell Type ◽  
Gfp Expression ◽  
Primary Fibroblasts ◽  
Species Specific ◽  
Green Fluorescent

Dermal fibroblasts are useful for production of genetically engineered biologically active factors for development of cellular therapies and tissue engineering products for regenerative medicine. However, their transfection efficiencies using traditional non-viral methods are low and vary based on cell-type and species-specific differences. Using nucleofection technology, here we show that the transfection efficiency of primary fibroblasts established after 0-, 35-, and 65-days of postmortem storage of sheep skin tissues in a refrigerator was 59.49 % ± 9.66 %, 59.33 % ± 11.59 %, and 43.48 % ± 8.09 % respectively, as determined by analysis of green fluorescent protein (GFP) expression. 

Cholic Acid-Modified Polyethylenimine for Gene Delivery into Human Carcinoma Cells

2014 ◽  
Vol 1060 ◽  
pp. 3-6 ◽  
Author(s):  
Wanlop Weecharangsan ◽  
Orapan Paecharoenchai ◽  
Nattisa Niyomtham ◽  
Praneet Opanasopit ◽  
Boon-ek Yingyongnarongkul ◽  
...  
Keyword(s):  
Gene Delivery ◽  
Zeta Potential ◽  
Cholic Acid ◽  
Plasmid Dna ◽  
Gel Electrophoresis ◽  
Transfection Efficiency ◽  
Carcinoma Cells ◽  
Molar Ratio ◽  
Dna Complexes ◽  
Human Carcinoma

Polyethylenimine (PEI) was modified by cholic acid at a molar ratio of 1:1. Cholic acid (CA)-modified PEI (PEI-CA) were evaluated for formation of DNA complexes. PEI-CA/pEGFP plasmid DNA complexes were characterized for their size and zeta potential. Gel electrophoresis showed total retardation for PEI-CA/pEGFP complexes formed at weight ratios above 0.25. The particle size and zeta potential of the complexes at a polymer-to-DNA ratio of 0.5 were 295.3 nm and 30.5 mV, respectively. The transfection efficiency of PEI-CA/pEGFP complexes was comparable to unmodified PEI. Cytotoxicity result showed that PEI-CA had lower cytoxicity than PEI. This study suggests that PEI-CA has potential utility as a gene delivery carrier.

scholarly journals In Vitro Gene Delivery Mediated by Asialofetuin-Appended Cationic Liposomes Associated with γ-Cyclodextrin into Hepatocytes

2011 ◽  
Vol 2011 ◽  
pp. 1-13 ◽  
Author(s):  
Keiichi Motoyama ◽  
Yoshihiro Nakashima ◽  
Yukihiko Aramaki ◽  
Fumitoshi Hirayama ◽  
Kaneto Uekama ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Gene Delivery ◽  
Plasmid Dna ◽  
Transfection Efficiency ◽  
Cationic Liposomes ◽  
Nonviral Vector ◽  
Liposomal Membrane ◽  
Transfer Activity ◽  
Potential Use

The purpose of this study is to evaluate in vitro gene delivery mediated by asialofetuin-appended cationic liposomes (AF-liposomes) associating cyclodextrins (CyD/AF-liposomes) as a hepatocyte-selective nonviral vector. Of various CyDs, AF-liposomes associated with plasmid DNA (pDNA) and γ-cyclodextrin (γ-CyD) (pDNA/γ-CyD/AF-liposomes) showed the highest gene transfer activity in HepG2 cells without any significant cytotoxicity. In addition, γ-CyD enhanced the encapsulation ratio of pDNA with AF-liposomes, and also increased gene transfer activity as the entrapment ratio of pDNA into AF-liposomes was increased. γ-CyD stabilized the liposomal membrane of AF-liposomes and inhibited the release of calcein from AF-liposomes. The stabilizing effect of γ-CyD may be, at least in part, involved in the enhancing gene transfer activity of pDNA/γ-CyD/AF-liposomes. Therefore, these results suggest the potential use of γ-CyD for an enhancer of transfection efficiency of AF-liposomes.

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