Influence of Polymer Architecture and Molecular Weight of Poly(2-(dimethylamino)ethyl methacrylate) Polycations on Transfection Efficiency and Cell Viability in Gene Delivery

2011 ◽  
Vol 12 (12) ◽  
pp. 4247-4255 ◽  
Author(s):  
Christopher V. Synatschke ◽  
Anja Schallon ◽  
Valérie Jérôme ◽  
Ruth Freitag ◽  
Axel H. E. Müller
Keyword(s):  
Molecular Weight ◽  
Gene Delivery ◽  
Cell Viability ◽  
Transfection Efficiency ◽  
Polymer Architecture ◽  
Ethyl Methacrylate

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.

Zinc ion coordination significantly improved the transfection efficiency of low molecular weight polyethylenimine

2019 ◽  
Vol 7 (4) ◽  
pp. 1716-1728 ◽  
Author(s):  
Huapan Fang ◽  
Lin Lin ◽  
Jie Chen ◽  
Jiayan Wu ◽  
Huayu Tian ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Gene Delivery ◽  
Delivery System ◽  
Transfection Efficiency ◽  
Zinc Ion ◽  
Low Molecular Weight ◽  
Gene Delivery System ◽  
System P

A zinc ion coordination-contained polycationic gene delivery system.

Diol glycidyl ether-bridged low molecular weight PEI as potential gene delivery vehicles

2015 ◽  
Vol 3 (13) ◽  
pp. 2660-2670 ◽  
Author(s):  
Qian Guo ◽  
Yan-Hong Liu ◽  
Miao-Miao Xun ◽  
Ji Zhang ◽  
Zheng Huang ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Gene Delivery ◽  
Ring Opening ◽  
Transfection Efficiency ◽  
Glycidyl Ether ◽  
Ring Opening Polymerization ◽  
Low Molecular Weight ◽  
Delivery Vehicles ◽  
Gene Delivery Vehicles

PEI 600-based polymers were synthesized via ring-opening polymerization and exhibited much better transfection efficiency and biocompatibility than PEI 25 kDa.

Highly Branched Poly(β-amino esters) for Non-Viral Gene Delivery: High Transfection Efficiency and Low Toxicity Achieved by Increasing Molecular Weight

2016 ◽  
Vol 17 (11) ◽  
pp. 3640-3647 ◽  
Author(s):  
Yongsheng Gao ◽  
Jian-Yuan Huang ◽  
Jonathan O’Keeffe Ahern ◽  
Lara Cutlar ◽  
Dezhong Zhou ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Gene Delivery ◽  
Transfection Efficiency ◽  
Viral Gene ◽  
Amino Esters ◽  
High Transfection Efficiency ◽  
Low Toxicity ◽  
Viral Gene Delivery

scholarly journals Amino Acid-Linked Low Molecular Weight Polyethylenimine for Improved Gene Delivery and Biocompatibility

Molecules ◽  
2020 ◽  
Vol 25 (4) ◽  
pp. 975 ◽  
Author(s):  
Xiao-Ru Wu ◽  
Ji Zhang ◽  
Ju-Hui Zhang ◽  
Ya-Ping Xiao ◽  
Xi He ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Gene Delivery ◽  
Transfection Efficiency ◽  
Gel Permeation Chromatography ◽  
Viral Gene ◽  
Low Molecular Weight ◽  
Ph Buffering ◽  
Gene Delivery Vectors ◽  
Viral Gene Delivery

The construction of efficient and low toxic non-viral gene delivery vectors is of great significance for gene therapy. Herein, two novel polycations were constructed via Michael addition from low molecular weight polyethylenimine (PEI) 600 Da and amino acid-containing linkages. Lysine and histidine were introduced for the purpose of improved DNA binding and pH buffering capacity, respectively. The ester bonds afforded the polymer biodegradability, which was confirmed by the gel permeation chromatography (GPC) measurement. The polymers could well condense DNA into nanoparticles and protect DNA from degradation by nuclease. Compared with PEI 25 kDa, these polymers showed higher transfection efficiency, lower toxicity, and better serum tolerance. Study of this mechanism revealed that the polyplexes enter the cells mainly through caveolae-mediated endocytosis pathway; this, together with their biodegradability, facilitates the internalization of polyplexes and the release of DNA. The results reveal that the amino acid-linked low molecular weight PEI polymers could serve as promising candidates for non-viral gene delivery.

scholarly journals A Low-Molecular-Weight Polyethylenimine/pDNA-VEGF Polyplex System Constructed in a One-Pot Manner for Hindlimb Ischemia Therapy

Pharmaceutics ◽  
2019 ◽  
Vol 11 (4) ◽  
pp. 171 ◽  
Author(s):  
Xiaoshuang Guo ◽  
Zihan Yuan ◽  
Yang Xu ◽  
Xiaotian Zhao ◽  
Zhiwei Fang ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Gene Delivery ◽  
High Efficiency ◽  
Transfection Efficiency ◽  
Low Molecular Weight ◽  
Physical Interaction ◽  
Hindlimb Ischemia ◽  
One Pot

Peripheral arterial disease (PAD) is often characterized by continued reduction in blood flow supply to limbs. Advanced therapeutic strategies like gene therapy could potentially be applied to limb ischemia therapy. However, developing a gene delivery system with low toxicity and high efficiency remains a great challenge. In this study, a one-pot construction was used to integrate vector synthesis and polyplex fabrication simultaneously in a simple and robust manner. We fabricated an interpenetrating gene delivery network through the physical interaction between low-molecular-weight polyethylenimine (PEI 1.8 kDa) and plasmid DNA (pDNA) and the chemical bonding between PEI and glutaraldehyde (GA), which was named the glutaraldehydelinked-branched PEI (GPEI) polyplex. The final GPEI polyplex system was pH-responsive and biodegradable due to the imine linkage and it could successfully deliver desired vascular endothelial growth factor (VEGF) pDNA. Compared with PEI (25 kDa)/pDNA polyplexes, GPEI polyplexes showed lower cytotoxicity and higher transfection efficiency both in vitro and in vivo. In addition, we demonstrated that GPEI polyplexes could efficiently promote the formation of new capillaries in vivo, which may provide a practicable strategy for clinical hindlimb ischemia therapy in the future.

scholarly journals Lower-Molecular-Weight Chitosan-Treated Polyethyleneimine: a Practical Strategy For Gene Delivery to Mesenchymal Stem Cells

2018 ◽  
Vol 50 (4) ◽  
pp. 1255-1269 ◽  
Author(s):  
Minchen Liu ◽  
Lu Zhang ◽  
Qingqing Zhao ◽  
Xinchi Jiang ◽  
Luyao Wu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Molecular Weight ◽  
Mesenchymal Stem Cells ◽  
Gene Delivery ◽  
Laser Scanning ◽  
Intracellular Transport ◽  
Transfection Efficiency ◽  
A549 Cells ◽  
Intracellular Location ◽  
Tgf Β1

Background/Aims: Genetic modification of mesenchymal stem cells (MSCs) is an essential requirement for their use as a delivery vehicle. To achieve higher transfection efficiency and better reproducibility than previously synthesized chitosan (100 kDa)-polyethylenimine (PEI; 1200 Da), we synthesized a low molecular weight PEI (1200 Da)-grafted chitosan (50 kDa) (CP). Methods: Safety of CP/DNA or PEI (25 kDa)/DNA was evaluated by an MTT assay using A549 cells or MSCs and a zebrafish embryo model. Effects of CP/DNA on the characteristics of MSCs were evaluated using flow cytometry. Additionally, a pGL3 plasmid was used to investigate the transfection efficiency of PEI (25 kDa), chitosan (100 kDa)-PEI (1200 Da), and CP with different N/P mass ratios on A549 cells and MSCs. Furthermore, CP/pGL3 was used to investigate the effect of serum on transfection, and intracellular transport was assessed by observing the intracellular location of DNA using laser scanning confocal microscopy. In addition, the effect of endocytosis on transfection efficiency was evaluated using A549 cells pre-treated with different inhibitors. Investigations related to analysis of transfection efficiency were all performed using the BCA protein assay to standardize the data. Furthermore, TGF-β1-and CXCR4-expressing plasmids were applied to evaluate the gene transfer efficiency of CP, including its effects on the osteogenic differentiation and migratory ability of MSCs. Results: The safety evaluation demonstrated that CP/DNA had significantly lower toxicity than PEI (25 kDa)/DNA. Additionally, DNA entered MSCs transfected by CP without changing their properties, while the examination of intracellular transport demonstrated that CP/pGL3 was internalized rapidly into MSCs. Furthermore, studies of the internalization mechanism showed that CP/pGL3 complexes entered the cells through caveolae-mediated endocytosis, thereby suggesting that the CP coating helped DNA enter A549 cells without the requirement for receptors. Compared to PEI (25 kDa), the interference of serum on transfection was reduced significantly with the use of CP in both A549 cells and MSCs. To evaluate the effects of gene delivery using the constructed CP complex and the possibility of obtaining gene-engineered MSCs, TGF-β1- and CXCR4-expressing plasmids were successfully delivered into MSCs, confirming their ability to induce osteogenesis and change the migratory ability of MSCs, respectively. Conclusion: These results demonstrated that CP could be used to deliver genes into MSCs and could potentially be used in gene therapy based on MSCs.

scholarly journals The impact of anionic polymers on gene delivery: how composition and assembly help evading the toxicity-efficiency dilemma

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Friederike Richter ◽  
Katharina Leer ◽  
Liam Martin ◽  
Prosper Mapfumo ◽  
Jana I. Solomun ◽  
...  
Keyword(s):  
Gene Delivery ◽  
Cell Viability ◽  
Colloidal Stability ◽  
Serum Proteins ◽  
Transfection Efficiency ◽  
Genetic Material ◽  
Endosomal Escape ◽  
Shielding Effect ◽  
Viral Gene ◽  
The Impact

AbstractCationic polymers have been widely studied for non-viral gene delivery due to their ability to bind genetic material and to interact with cellular membranes. However, their charged nature carries the risk of increased cytotoxicity and interaction with serum proteins, limiting their potential in vivo application. Therefore, hydrophilic or anionic shielding polymers are applied to counteract these effects. Herein, a series of micelle-forming and micelle-shielding polymers were synthesized via RAFT polymerization. The copolymer poly[(n-butyl acrylate)-b-(2-(dimethyl amino)ethyl acrylamide)] (P(nBA-b-DMAEAm)) was assembled into cationic micelles and different shielding polymers were applied, i.e., poly(acrylic acid) (PAA), poly(4-acryloyl morpholine) (PNAM) or P(NAM-b-AA) block copolymer. These systems were compared to a triblock terpolymer micelle comprising PAA as the middle block. The assemblies were investigated regarding their morphology, interaction with pDNA, cytotoxicity, transfection efficiency, polyplex uptake and endosomal escape. The naked cationic micelle exhibited superior transfection efficiency, but increased cytotoxicity. The addition of shielding polymers led to reduced toxicity. In particular, the triblock terpolymer micelle convinced with high cell viability and no significant loss in efficiency. The highest shielding effect was achieved by layering micelles with P(NAM-b-AA) supporting the colloidal stability at neutral zeta potential and completely restoring cell viability while maintaining moderate transfection efficiencies. The high potential of this micelle-layer-combination for gene delivery was illustrated for the first time.

scholarly journals Lignin-Based Nonviral Gene Carriers Functionalized by Poly[2-(Dimethylamino)ethyl Methacrylate]: Effect of Grafting Degree and Cationic Chain Length on Transfection Efficiency

Biomolecules ◽  
2022 ◽  
Vol 12 (1) ◽  
pp. 102
Author(s):  
Xiaohong Liu ◽  
Hui Yin ◽  
Xia Song ◽  
Zhongxing Zhang ◽  
Jun Li
Keyword(s):  
Gene Delivery ◽  
Chain Length ◽  
Cell Lines ◽  
Transfection Efficiency ◽  
Graft Copolymers ◽  
Gene Transfection ◽  
Structural Factors ◽  
Ethyl Methacrylate ◽  
Branched Structure ◽  
Grafting Degree

Lignin is a natural renewable biomass resource with great potential for applications, while its development into high value-added molecules or materials is rare. The development of biomass lignin as potential nonviral gene delivery carriers was initiated by our group through the “grafting-from” approach. Firstly, the lignin was modified into macroinitiator using 2-bromoisobutyryl bromide. Then cationic polymer chains of poly[2-(dimethylamino)ethyl methacrylate] (PDMAEMA) were grown from the lignin backbone using atom transfer radical polymerization (ATRP) to yield lignin-PDMAEMA graft copolymers (LPs) with branched structure. To gain a deep understanding of the relationship between the nonviral gene transfection efficiency of such copolymers and their structural and compositional factors, herein eight lignin-based macroinitiators with different modification degrees (MDs, from 3.0 to 100%) were synthesized. Initiated by them, a series of 20 LPs were synthesized with varied structural factors such as grafting degree (GD, which is equal to MD, determining the cationic chain number per lignin macromolecule), cationic chain length (represented by number of repeating DMAEMA units per grafted arm or degree of polymerization, DP) as well as the content of N element (N%) which is due to the grafted PDMAEMA chains and proportional to molecular weight of the LPs. The in vitro gene transfection capability of these graft copolymers was evaluated by luciferase assay in HeLa, COS7 and MDA-MB-231cell lines. Generally, the copolymers LP-12 (N% = 7.28, MD = 36.7%, DP = 13.6) and LP-14 (N% = 6.05, MD = 44.4%, DP = 5.5) showed good gene transfection capabilities in the cell lines tested. Overall, the performance of LP-12 was the best among all the LPs in the three cell lines at the N/P ratios from 10 to 30, which was usually several times higher than PEI standard. However, in MDA-MB-231 at N/P ratio of 30, LP-14 showed the best gene transfection performance among all the LPs. Its gene transfection efficiency was ca. 11 times higher than PEI standard at this N/P ratio. This work demonstrated that, although the content of N element (N%) which is due to the grafted PDMAEMA chains primarily determines the gene transfection efficiency of the LPs, it is not the only factor in explaining the performance of such copolymers with the branched structure. Structural factors of these copolymers such as grafting degree and cationic chain length could have a profound effect on the copolymer performance on gene transfection efficiency. Through carefully adjusting these factors, the gene transfection efficiency of the LPs could be modulated and optimized for different cell lines, which could make this new type of biomass-based biomaterial an attractive choice for various gene delivery applications.

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