Ternary Complex of Plasmid DNA with Protamine and γ-Polyglutamic Acid for Biocompatible Gene Delivery System (original) (raw)
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Ternary complexes of pDNA, polyethylenimine, and γ-polyglutamic acid for gene delivery systems
Biomaterials, 2009
We discovered a vector coated by γ-polyglutamic acid (γ-PGA) for effective and safe gene delivery. In order to develop a useful non-viral vector, we prepared several ternary complexes constructed with pDNA, polyethylenimine (PEI), and various polyanions, such as polyadenylic acid, polyinosinic–polycytidylic acid, α-polyaspartic acid, α-polyglutamic acid, and γ-PGA. The pDNA/PEI complex had a strong cationic surface charge and showed extremely
We discovered a vector coated by γ-polyglutamic acid (γ-PGA) for effective and safe gene delivery. In order to develop a useful non-viral vector, we prepared several ternary complexes constructed with pDNA, polyethylenimine (PEI), and various polyanions, such as polyadenylic acid, polyinosinic-polycytidylic acid, α-polyaspartic acid, α-polyglutamic acid, and γ-PGA. The pDNA/PEI complex had a strong cationic surface charge and showed extremely high transgene efficiency although it agglutinated with erythrocytes and had extremely high cytotoxicity. Those polyanions changed the positive ζ-potential of pDNA/PEI complex to negative although they did not affect the size. They had no agglutination activities and lower cytotoxicities but most of the ternary complexes did not show any uptake and gene expression; however, the pDNA/PEI/γ-PGA complex showed high uptake and gene expression. Most of the pDNA/PEI/γ-PGA complexes were located in the cytoplasm without dissociation and a few complexes were observed in the nuclei.
Biomacromolecules, 2003
Cationic polymers, such as poly-L-lysine (pLL) and polyethyleneimine (pEI), are receiving growing attention as vectors for gene therapy. They form polyelectrolyte complexes with DNA, resulting in a reduced size of the DNA and an enhanced stability toward nucleases. The major disadvantages of using both polymers for in vivo purposes are their cytotoxicity and, in the case of pEI, the fact that it's not biodegradable. In this work, we investigated the interaction between a series of cationic, glutamic acid based polymers and red blood cells. The MTT test was used to investigate the cytotoxicity of the complexes. The ability of the polymers to stabilize DNA toward nucleases was investigated. Transfection studies were carried out on Cos-1 cells. The results from the haemolysis studies, the haemagglutination studies, and the MTT assay show that the polymers are substantially less toxic than pLL and pEI. The polymers are able to protect the DNA from digestion by DNase I. The transfection studies show that the polymer-DNA complexes are capable of transfecting cells, most of them with poor efficiency compared to pEI-DNA complexes.
Colloids and Surfaces B: Biointerfaces, 2018
Polyethylenimine (PEI) has been extensively used for non-viral gene delivery. Increasing the molecular weight of PEI often improves transfection efficiency, but enhances cytotoxicity and non-specific interaction with plasma proteins, limiting its use in clinical applications. In this study, poly-L-glutamic acid (L-PGA) as an anionic polymer, was introduced to piperazine-modified PEI to improve its in vivo properties. The physicochemical properties, cytotoxicity, in vitro and in vivo tranfection efficiency of these carriers were evaluated. Conjugation of 50% of primary amines of PEI 25 kDa with piperazine in the presence of PGA 1% (PEI 25 Pip 50% /PGA 1%) could significantly increase transfection efficiency even in the presence of serum compared to PEI 25 kDa. Increasing the PGA content led to lower cytotoxicity of DNA/PEI 25 Pip 50% /PGA 1% triplexes. Systemic administration of triplexes in Balb/c mice resulted in significant enhancement of luciferase gene expression in brain, spleen, and liver compared to PEI 25 kDa. In a 30-day survival study, no significant changes were observed in mice body weights in DNA/PEI 25 Pip 50% /PGA 1% group. Moreover, this group exhibited a survival rate of 100% compared to 0% in mice receiving PEI 25 kDa. This novel PEI 25 Pip 50% /PGA 1% carrier could be used to overcome the serum inhibitory effects on gene expression in vivo, providing a promising gene delivery system for tissue-specific targeting.
Journal of Biotechnology, 2007
The cationic polylactic acid (PLA) nanoparticle has emerged as a promising non-viral vector for gene delivery because of its biocompatibility and biodegradability. However, they are not capable of prolonging gene transfer and high transfection efficiency. In order to achieve prolonged delivery of cationic PLA/DNA complexes and higher transfection efficiency, in this study, we used copolymer methoxypolyethyleneglycol-PLA (MePEG-PLA), PLA and chitosan (CS) to prepare MePEG-PLA-CS NPs and PLA-CS NPs by a diafiltration method and prepared NPs/DNA complexes through the complex coacervation of nanoparticles with the pDNA. The object of our work is to evaluate the characterization and transfection efficiency of MePEG-PLA-CS versus PLA-CS NPs. The MePEG-PLA-CS NPs have a zeta potential of 15.7 mV at pH 7.4 and size under 100 nm, while the zeta potential of PLA-CS NPs was only 4.5 mV at pH 7.4. Electrophoretic analysis suggested that both MePEG-PLA-CS NPs and PLA-CS NPs with positive charges could protect the DNA from nuclease degradation and cell viability assay showed MePEG-PLA-CS NPs exhibit a low cytotoxicity to normal human liver cells. The potential of PLA-CS NPs and MePEG-PLA-CS NPs as a non-viral gene delivery vector to transfer exogenous gene in vitro and in vivo were examined. The pDNA being carried by MePEG-PLA-CS NPs, PLA-CS NPs and lipofectamine could enter and express in COS7 cells. However, the transfection efficiency of MePEG-PLA-CS/DNA complexes was better than PLA-CS/DNA and lipofectamine/DNA complexes by inversion fluorescence microscope and flow cytometry. It was distinctively to find that the transfection activity of PEGylation of complexes was improved. The nanoparticles were also tested for their ability to transport across the gastrointestinal mucosa in vivo in mice. In vivo experiments showed obviously that MePEG-PLA-CS/DNA complexes mediated higher gene expression in stomach and intestine of BALB/C mice compared to PLA-CS/DNA and lipofectamine/DNA complexes. These results suggested that MePEG-PLA-CS NPs have favorable properties for non-viral gene delivery.
International Journal of Nanomedicine, 2008
In order to quantify the amount of ligands or poly(ethylene glycol) (PEG) on each vector, here we developed a system in which poly-L-glutamic acid (PLG) was used as surface modifi cation loading backbone, to which one PEG (MW 5000, 10000, 20000) or epidermal growth factor (EGF) was linked. The PLG conjugates can electro-statically adsorb upon DNA/ polycation complex with positive charge, and, the amount of EGF or PEG on the surface of complexes could be varied. We have made a series of complexes containing the various PLG conjugates and examined their physicochemical properties, and made a comparison of properties and transfection effi ciency between these complexes. EGF-and PEG-modifi ed complexes showed 10-25-folds higher cell transfection effi ciency than unmodifi ed complexes in medium with or without serum.
Journal of Biotechnology, 2007
The purpose of the study was to investigate the influence of cationic polymer structure on the formation of DNA-polycation complexes and their transfection activity. Primary, tertiary, and quaternary polyamines with molecular masses ranging from 8000 to 200,000 were investigated. DNA-cationic polymer interaction was characterized by low gradient viscometry, dynamic light scattering, circular dichroism, UV spectrometry, flow birefringence, DNA electrophoresis, and electron microscopy. Transfection activity of the complexes was evaluated by the expression of reporter gene (-galactosidase) and using synthetic FITC-labelled oligonucleotides. Complex formation was found to be dependent on the structure and molecular weight of the polymer and the ionic strength of the solution. Secondary DNA structure in complexes was not disrupted, and DNA was protected from protonation. Cell lines of different origin were used for testing of transfection activity of the complexes. The sensitivity of the cells to transfection was established to be highly dependent on the cell line. DNA-polycation complexes are non-toxic according to MTT. Polyallylamine, and polydimethylaminoethylmethacrylate were found to be the most promising polycations for gene delivery. Transfection efficacy of their complexes with DNA to T-98G cells reaches up to 90-100%. It was found that optimal molecular mass of polydimethylaminoethylmethacrylate is in the range of 8000-50,000 Da.
Macromolecular Bioscience, 2020
The main concept of gene therapy involves successful delivery of therapeutic genes into specific/diseased cells to replace the mutated ones. [1-3] DNA delivery is preferably carried out by vectors that are able to condense and protect the nucleic acid, facilitate cellular uptake and endosomal escape, and enable gene expression. [4,5] A large number of viral [6-8] and non-viral [9-17] vectors have been developed. Despite the positive results, viral vectors are often associated with a number of potential risks for the patient (high cytotoxicity, severe immune response, insertional mutagenesis, restoration of virulence resulting in additional diseases, etc.). [6-8] The non-viral vectors can be considerably safer and more biocompatible, but generally less efficient. [9-17] Polyplexes are promising non-viral gene delivery systems. [4,5,9-18] These are nanosized polyelectrolyte particles formed from natural or synthetic polymers and DNA. The complexation is based on electrostatic interactions; therefore, a necessary prerequisite is the presence of positively charged groups, such as amino groups, into the polymer chain. The type of the amino group could be essential for DNA/polymer interactions as well as for the biological relevance of the resulting polyelectrolyte complexes. The primary amino groups that are permanently charged at physiological pH possess high binding affinity and form the most stable complexes with DNA. [19-21] However, polymers with primary amines are usually associated with high toxicity. [21-23] The secondary and tertiary amines are able to be protonated to varying degrees depending on the pH of the medium and are responsible for the successful endosomal escape and transfection efficiency. [21,24] For example, the branched polyethylenimine (PEI) known as the "gold standard" is the most extensively studied polycation for gene delivery and transfection. [25,26] Its main advantage is the high content of amino groups, including primary, secondary, and tertiary, contributing to the effective condensation of the bulky DNA molecule. Moreover, the presence of secondary and tertiary amino groups provides high buffering capacity allowing polyplex particles to easily overcome the endo-lysosomal barrier through the so-called "proton sponge" effect, consisting of osmotic swelling and rupture of the endo-lysosomes, and Physicochemical characteristics and biological performance of polyplexes based on two identical copolymers bearing tertiary amino or quaternary ammonium groups are evaluated and compared. Poly(2-(dimethylamino) ethyl methacrylate)-b-poly(oligo(ethylene glycol) methyl ether methacrylate) block copolymer (PDMAEMA-b-POEGMA) is synthesized by reversible addition fragmentation chain transfer polymerization. The tertiary amines of PDMAEMA are converted to quaternary ammonium groups by quaternization with methyl iodide. The two copolymers spontaneously formed well-defined polyplexes with DNA. The size, zeta potential, molar mass, aggregation number, and morphology of the polyplex particles are determined. The parent PDMAEMA-b-POEGMA exhibits larger buffering capacity, whereas the corresponding quaternized copolymer (QPDMAEMA-b-POEGMA) displays stronger binding affinity to DNA, yielding invariably larger in size and molar mass particles bearing greater number of DNA molecules per particle. Experiments revealed that QPDMAEMA-b-POEGMA is more effective in transfecting pEGFP-N1 than the parent copolymer, attributed to the larger size, molar mass, and DNA cargo, as well as to the effective cellular traffic, which dominated over the enhanced ability for endo-lysosomal escape of PDMAEMA-b-POEGMA.
Secure and effective gene delivery system of plasmid DNA coated by polynucleotide
Journal of Drug Targeting, 2015
Polynucleotides are anionic macromolecules which are expected to transfer into the targeted cells through specific uptake mechanisms. So, we developed polynucleotides coating complexes of plasmid DNA (pDNA) and polyethylenimine (PEI) for a secure and efficient gene delivery system and evaluated their usefulness. Polyadenylic acid (polyA), polyuridylic acid (polyU), polycytidylic acid (polyC), and polyguanylic acid (polyG) were examined as the coating materials. pDNA/PEI/polyA, pDNA/PEI/polyU, and pDNA/PEI/polyC complexes formed nanoparticles with a negative surface charge although pDNA/PEI/polyG was aggregated. The pDNA/PEI/polyC complex showed high transgene efficiency in B16-F10 cells although there was little efficiency in pDNA/PEI/polyA and pDNA/PEI/polyU complexes. An inhibition study strongly indicated the specific uptake mechanism of pDNA/PEI/polyC complex.
Synthesis and characterization of polyaminoacidic polycations for gene delivery
Biomaterials, 2006
The properties as non viral gene vector of a protein-like polymer, the a,b-poly(N-2-hydroxyethyl)-D,L-aspartamide (PHEA) were exploited after its derivatization with 3-(carboxypropyl)trimethyl-ammonium chloride (CPTA) as molecule bearing a cationic group, in order to obtain stable polycations able to condense DNA. PHEA was firstly functionalized with aminic pendant groups by reaction with ethylenediamine (EDA) obtaining the a,b-poly(N-2-hydroxyethyl)(2-aminoethylcarbamate)-D,L-aspartamide (PHEA-EDA) copolymer. We demonstrated that polymer functionalization degree is easily modulable by varying reaction conditions, so allowing to produce two PHEA-EDA derivatives at different molar percentage of amine groups. Subsequently, the condensation reaction of PHEA-EDA copolymers with CPTA yielded a,b-poly(N-2-hydroxyethyl)(2-[3-(trimethylammonium chloride)propylamide]-amidoethylcarbamate)-D,L-aspartamide (PHEA-EDA-CPTA) polycation derivatives.