CATIONIC POLYMER BASED GENE DELIVERY: UPTAKE AND INTRACELLULAR TRAFFICKING

COSMOS ◽  
2014 ◽  
Vol 10 (01) ◽  
pp. 17-24
Author(s):  
YOONKHEI HO ◽  
HENG-PHON TOO
Keyword(s):  
Gene Delivery ◽  
Transfection Efficiency ◽  
Cell Types ◽  
Endosomal Escape ◽  
Current Status ◽  
Uptake Mechanism ◽  
Nuclear Entry ◽  
Dna Complexes ◽  
Major Drawback ◽  
Gene Carriers

To date, low transfection efficiency remains the major drawback of polymer based gene delivery. Many cell types including stem cells, fibroblast and neurons are known to be poorly transfected with polymer based gene carriers and the high toxicity severely restrict their utility in gene delivery. Continual efforts are made to identify cellular barriers to efficient transfection as these carriers have low immunogenicity, ease of manufacturing and scalability. Here, we summarize the current status of understanding on uptake mechanism of polymer-DNA complexes (polyplexes), their endosomal escape, cytosolic transport and nuclear entry of pDNA.

scholarly journals Improved gene delivery to K-562 leukemia cells by lipoic acid modified block copolymer micelles

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Friederike Richter ◽  
Prosper Mapfumo ◽  
Liam Martin ◽  
Jana I. Solomun ◽  
Franziska Hausig ◽  
...  
Keyword(s):  
Gene Delivery ◽  
Lipoic Acid ◽  
Transfection Efficiency ◽  
Cell Types ◽  
Endosomal Escape ◽  
Leukemia Cell Line ◽  
Butyl Methacrylate ◽  
Suspension Cells ◽  
Cationic Micelles ◽  
K 562 Cells

AbstractAlthough there has been substantial progress in the research field of gene delivery, there are some challenges remaining, e.g. there are still cell types such as primary cells and suspension cells (immune cells) known to be difficult to transfect. Cationic polymers have gained increasing attention due to their ability to bind, condense and mask genetic material, being amenable to scale up and highly variable in their composition. In addition, they can be combined with further monomers exhibiting desired biological and chemical properties, such as antioxidative, pH- and redox-responsive or biocompatible features. By introduction of hydrophobic monomers, in particular as block copolymers, cationic micelles can be formed possessing an improved chance of transfection in otherwise challenging cells. In this study, the antioxidant biomolecule lipoic acid, which can also be used as crosslinker, was incorporated into the hydrophobic block of a diblock copolymer, poly{[2-(dimethylamino)ethyl methacrylate]101-b-[n-(butyl methacrylate)124-co-(lipoic acid methacrylate)22]} (P(DMAEMA101-b-[nBMA124-co-LAMA22])), synthesized by RAFT polymerization and assembled into micelles (LAMA-mic). These micelles were investigated regarding their pDNA binding, cytotoxicity mechanisms and transfection efficiency in K-562 and HEK293T cells, the former representing a difficult to transfect, suspension leukemia cell line. The LAMA-mic exhibited low cytotoxicity at applied concentrations but demonstrated superior transfection efficiency in HEK293T and especially K-562 cells. In-depth studies on the transfection mechanism revealed that transfection efficiency in K-562 cells does not depend on the specific oncogenic fusion gene BCR-ABL alone. It is independent of the cellular uptake of polymer-pDNA complexes but correlates with the endosomal escape of the LAMA-mic. A comparison of the transfection efficiency of the LAMA-mic with structurally comparable micelles without lipoic acid showed that lipoic acid is not solely responsible for the superior transfection efficiency of the LAMA-mic. More likely, a synergistic effect of the antioxidative lipoic acid and the micellar architecture was identified. Therefore, the incorporation of lipoic acid into the core of hydrophobic-cationic micelles represents a promising tailor-made transfer strategy, which can potentially be beneficial for other difficult to transfect cell types.

scholarly journals Synthesis, and Characterization, and Evaluation of Cellular Effects of the FOL-PEG-g-PEI-GAL Nanoparticles as a Potential Non-Viral Vector for Gene Delivery

2010 ◽  
Vol 2010 ◽  
pp. 1-10 ◽  
Author(s):  
S. Ghiamkazemi ◽  
A. Amanzadeh ◽  
R. Dinarvand ◽  
M. Rafiee-Tehrani ◽  
M. Amini
Keyword(s):  
Gene Delivery ◽  
Zeta Potential ◽  
Surface Charge ◽  
Cell Line ◽  
Graft Copolymer ◽  
Viral Vector ◽  
Transfection Efficiency ◽  
High Surface ◽  
Liver Cells ◽  
Dna Complexes

In this manuscript, we synthesized the potential non viral vector for gene delivery with proper transfection efficiency and low cytotoxicity. Polyethylenimine (PEI) is a well-known cationic polymer which has high positive surface charge for condensing plasmid DNA. However; it is highly cytotoxic in many cell lines because of the high surface charge, non-biodegradability and non-biocompatibility. To enhance PEI biodegradability, the graft copolymer “PEG-g-PEI” was synthesized. To target cancer liver cells, two targeting ligands folic acid and galactose (lactobionic acid) which are over expressed on human hepatocyte carcinoma were attached to graft copolymer and “FOL-PEG-g-PEI-GAL” copolymer was synthesized. Composition of this grafted copolymer was characterized using1H-NMR and FTIR spectra. The molecular weight and zeta potential of this copolymer was compared to PEI. The particle size and zeta potential of FOL-PEG-g-PEI-GAL/DNA complexes at various N/P ratio were measured using dynamic light scattering (DLS). Cytotoxicity of the copolymer was also studied in cultured HepG2 human hepatoblastoma cell line. The FOL-PEG-g-PEI-GAL/DNA complexes at various N/P ratios exhibited no cytotoxicity in HepG2 cell line compared to PEI 25K as a control. The novel copolymer showed enhanced biodegradability in physiological conditions in compared with PEI and targeted cultured HepG2 cells. More importantly, significant transfection efficiency was exhibited in cancer liver cells. Together, our results showed that “FOL-PEG-g-PEI-GAL” nanoparticals could be considered as a useful non-viral vector for targeted gene delivery.

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.

Peptide amphiphiles with multifunctional fragments promoting cellular uptake and endosomal escape as efficient gene vectors

2015 ◽  
Vol 3 (6) ◽  
pp. 1068-1078 ◽  
Author(s):  
Liang Luan ◽  
Qingbin Meng ◽  
Liang Xu ◽  
Zhao Meng ◽  
Husheng Yan ◽  
...  
Keyword(s):  
Gene Delivery ◽  
Cellular Uptake ◽  
Transfection Efficiency ◽  
Endosomal Escape ◽  
Peptide Amphiphiles ◽  
Gene Delivery Vectors ◽  
Efficient Gene ◽  
Gene Vectors ◽  
Lipofectamine 2000

A series of peptides containing multiple functional fragments were designed as gene-delivery vectors with transfection efficiency comparable to Lipofectamine 2000.

scholarly journals Interaction kinetics of peptide lipids-mediated gene delivery

2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Yinan Zhao ◽  
Tianyi Zhao ◽  
Yanyan Du ◽  
Yingnan Cao ◽  
Yang Xuan ◽  
...  
Keyword(s):  
Gene Delivery ◽  
Transfection Efficiency ◽  
Gene Transfection ◽  
Endosomal Escape ◽  
Late Endosomes ◽  
Key Factor ◽  
Interaction Kinetics ◽  
Dna Release

Abstract Background During the course of gene transfection, the interaction kinetics between liposomes and DNA is speculated to play very important role for blood stability, cellular uptake, DNA release and finally transfection efficiency. Results As cationic peptide liposomes exhibited great gene transfer activities both in vitro and in vivo, two peptide lipids, containing a tri-ornithine head (LOrn3) and a mono-ornithine head (LOrn1), were chosen to further clarify the process of liposome-mediated gene delivery in this study. The results show that the electrostatically-driven binding between DNA and liposomes reached nearly 100% at equilibrium, and high affinity of LOrn3 to DNA led to fast binding rate between them. The binding process between LOrn3 and DNA conformed to the kinetics equation: y = 1.663631 × exp (− 0.003427x) + 6.278163. Compared to liposome LOrn1, the liposome LOrn3/DNA lipoplex exhibited a faster and more uniform uptake in HeLa cells, as LOrn3 with a tri-ornithine peptide headgroup had a stronger interaction with the negatively charged cell membrane than LOrn1. The efficient endosomal escape of DNA from LOrn3 lipoplex was facilitated by the acidity in late endosomes, resulting in broken carbamate bonds, as well as the “proton sponge effect” of the lipid. Conclusions The interaction kinetics is a key factor for DNA transfection efficiency. This work provided insights into peptide lipid-mediated DNA delivery that could guide the development of the next generation of delivery systems for gene therapeutics.

Understanding why the same gene delivery vector behaves differently in different cell types is essential for developing more adaptable transfection systems

2021 ◽  
Author(s):  
Moataz Dowaidar
Keyword(s):  
Gene Delivery ◽  
High Efficiency ◽  
Transfection Efficiency ◽  
Cell Metabolism ◽  
New Technology ◽  
Cell Types ◽  
Viral Gene ◽  
Proliferation Capacity ◽  
Delivery Vector ◽  
Different Cell Types

Since their origin, non-viral gene delivery reagents have evolved into a variety of effective delivery reagents with a variety of components and designs, and are widely used in gene therapy and gene engineering. A flood of successful commercial gene delivery reagents has also developed, and PEI has emerged as the "gold standard" for the industry. On the other hand, their transfection efficiency must be enhanced and their cell toxicity must be reduced. In recent years, toxicity, efficiency and targeted investigations have progressed. In addition to creating and manufacturing reagents with reduced toxicity and higher efficiency, polypeptides that stimulate cell membrane perforation and tiny molecular compounds that can better compress pDNA, as well as various combinations with liposomes or polymer vectors, have demonstrated improved outcomes. However, most of these freshly created delivery vector reagents are still under investigation, and others require additional refinement to achieve high transfection efficiency and minimum toxicity. The processes behind the effects of various gene delivery reagents, genes, and drugs entering cells, as well as their transit, escape, and cell metabolism, are also unclear. This requires improving relevant research. Understanding why the same reagent reacts differently to different cell types is crucial to creating more adaptive transfection reagents for different cell lines. This is suggested because different cells have different growth cycles. Because of their weak proliferation capacity, primordial cells, for example, are harder to replicate.Artificial intelligence, real-world and virtual-world integration technology, big data, multiomics technology, and signal pathway research have all achieved substantial breakthroughs in recent years, and novel transfection reagents and drug delivery technologies are predicted to continue. It is worth examining how to take advantage of the scientific and high-efficiency benefits that new technology provides for research and how to solve the issues given by the in-depth examination of the selection and mechanism of action of novel composite materials in vector reagent creation.

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.

Chitosan Derivatives as Gene Carriers

2005 ◽  
Vol 288-289 ◽  
pp. 97-100 ◽  
Author(s):  
Tae Hee Kim ◽  
In Kyu Park ◽  
Su Il Kim ◽  
Hwan Jeong Jeong ◽  
Hee Seung Bom ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Chemical Modification ◽  
Review Paper ◽  
Transfection Efficiency ◽  
Gene Delivery System ◽  
Dna Complexes ◽  
Gene Carriers ◽  
Toxic Material ◽  
Cell Specificity ◽  
The Stability

Chitosan has been considered to be a good candidate for gene delivery system, since it is already known as a biocompatible, biodegradable, and low toxic material with high cationic potential. However, low specificity and low transfection efficiency of chitosan need to be overcome prior to clinical trial. In this review paper, chemical modification of chitosan for enhancement of cell specificity and transfection efficiency was explained. Also, chemical modification of chitosan for the stability of chitosan/DNA complexes was reviewed.

scholarly journals Design and Validation of a Process Based on Cationic Niosomes for Gene Delivery into Novel Urine-Derived Mesenchymal Stem Cells

Pharmaceutics ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 696
Author(s):  
Yerai Vado ◽  
Gustavo Puras ◽  
Melania Rosique ◽  
Cesar Martin ◽  
Jose Luis Pedraz ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Gene Delivery ◽  
Viral Vectors ◽  
Transfection Efficiency ◽  
Intracellular Localization ◽  
Cell Types ◽  
Functional Studies ◽  
Cellular Models ◽  
Cationic Niosomes

Background: Mesenchymal stem cells (MSCs) are stem cells present in adult tissues. They can be cultured, have great growth capacity, and can differentiate into several cell types. The isolation of urine-derived mesenchymal stem cells (hUSCs) was recently described. hUSCs present additional benefits in the fact that they can be easily obtained noninvasively. Regarding gene delivery, nonviral vectors based on cationic niosomes have been used and are more stable and have lower immunogenicity than viral vectors. However, their transfection efficiency is low and in need of improvement. Methods: We isolated hUSCs from urine, and the cell culture was tested and characterized. Different cationic niosomes were elaborated using reverse-phase evaporation, and they were physicochemically characterized. Then, they were screened into hUSCs for transfection efficiency, and their internalization was evaluated. Results: GPxT-CQ at a lipid/DNA ratio of 5:1 (w/w) had the best transfection efficiency. Intracellular localization studies confirmed that nioplexes entered mainly via caveolae-mediated endocytosis. Conclusions: In conclusion, we established a protocol for hUSC isolation and their transfection with cationic niosomes, which could have relevant clinical applications such as in gene therapy. This methodology could also be used for creating cellular models for studying and validating pathogenic genetic variants, and even for performing functional studies. Our study increases knowledge about the internalization of tested cationic niosomes in these previously unexplored cells.

scholarly journals Interaction Kinetics of Peptide Lipids-Mediated Gene Delivery

2019 ◽  
Author(s):  
Shubiao Zhang ◽  
Yinan Zhao ◽  
Yanyan Du ◽  
Yingnan Cao ◽  
Yang Xuan ◽  
...  
Keyword(s):  
Gene Delivery ◽  
Transfection Efficiency ◽  
Gene Transfection ◽  
Endosomal Escape ◽  
Late Endosomes ◽  
Key Factor ◽  
Interaction Kinetics ◽  
Dna Release

Abstract Background: During the course of gene transfection, the interaction kinetics between liposomes and DNA is speculated to play very important role for blood stability, cellular uptake, DNA release and finally transfection efficiency.Results: As cationic peptide liposomes exhibited great gene transfer activities both in vitro and in vivo, two peptide lipids, containing a tri-ornithine head (LOrn3) and a mono-ornithine head (LOrn1), were chosen to further clarify the process of liposome-mediated gene delivery in this study. The results show that the electrostatically-driven binding between DNA and liposomes reached nearly 100% at equilibrium, and high affinity of LOrn3 to DNA led to fast binding rate between them. The binding process between LOrn3 and DNA conformed to the kinetics equation: y = 1.663631 × exp(-0.003427x) + 6.278163. Compared to liposome LOrn1, the liposome LOrn3/DNA lipoplex exhibited a faster and more uniform uptake in Hela cells, as LOrn3 with a tri-ornithine peptide headgroup had a stronger interaction with the negatively charged cell membrane than LOrn1. The efficient endosomal escape of DNA from LOrn3 lipoplexes was facilitated by the acidity in late endosomes, resulting in broken carbamate bonds, as well as the “proton sponge effect” of the lipid.Conclusions: The interaction kinetics is a key factor for DNA transfection efficiency. This work provided insights into peptide lipid-mediated DNA delivery that could guide the development of the next generation of delivery systems for gene therapeutics.

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