Efficiency Of Plasmid DNA Delivery Research Articles

Synthesis and Transfection Efficiency of Disulfide Policationic Amphiphiles

The synthesis of new polycationic amphiphiles containing a disulfide group in their structure has been carried out. Cationic liposomes were formed on the basis of the obtained compounds and helper lipid 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine, which demonstrated the absence of toxicity to HEK293 and HeLa cells and high delivery efficiency of fluorescently labeled oligodeoxyribonucleotide. The efficiency of plasmid DNA delivery depended on the cell line and structure of the amphiphile, with liposomes based on tetracationic amphiphiles being the most effective transfectants. These liposomes may be used for in vitro transfection of eukaryotic cells as well as for further in vivo biological tests.

Lipid nanoparticle-mediated CRISPR/Cas9 gene editing and metabolic engineering for anticancer immunotherapy

Metabolic engineering of the tumor microenvironment has emerged as a new strategy. Lactate dehydrogenase A (LDHA) is a prominent target for metabolic engineering. Here, we designed a cationic lipid nanoparticle formulation for LDHA gene editing. The plasmid DNA delivery efficiency of our lipid nanoparticle formulations was screened by testing the fluorescence of lipid nanoparticles complexed to plasmid DNA encoding green fluorescence protein (GFP). The delivery efficiency was affected by the ratios of three components: a cationic lipid, cholesterol or its derivative, and a fusogenic lipid. The lipid nanoparticle designated formulation F3 was complexed to plasmid DNA co-encoding CRISPR-associated protein 9 and LDHA-specific sgRNA, yielding the lipoplex, pCas9-sgLDHA/F3. The lipoplex including GFP-encoding plasmid DNA provided gene editing in HeLa-GFP cells. Treatment of B16F10 tumor cells with pCas9-sgLDHA/F3 yielded editing of the LDHA gene and increased the pH of the culture medium. pCas9-sgLDHA/F3 treatment activated the interferon-gamma and granzyme production of T cells in culture. In vivo, combining pCas9-sgLDHA/F3 with immune checkpoint-inhibiting anti-PD-L1 antibody provided a synergistic antitumor effect and prolonged the survival of tumor model mice. This study suggests that combining metabolic engineering of the tumor microenvironment with immune checkpoint inhibition could be a valuable antitumor strategy.

Сравнительный анализ эффективности общедоступных методов трансфекции модельных клеточных линий для задач биотехнологии

Transient gene expression is one of the most common methods in molecular biology, equally relevant for basic research projects and biotechnological industries. Despite the existence of commercial transfection systems, which afford high transfection efficiency and high expression levels of reporter genes, expanding such systems to industrial scales is often problematic due to high costs of the reagents. The well-described methods of cationic and calcium-phosphate transfection are accessible and ensure reproducible results at much lower costs. This study is aimed at comparative validation of calcium phosphate and cationic (polyethylenimine-based) transfection protocols along with the commercially available TurboFect reagent for mono- and cotransfections on a panel of commonly used cell lines including HEK293T, Huh7, BHK-21, CHO and MRC5. The efficiency of transfection with plasmid constructs encoding different fluorescent proteins was measured by flow cytometry. Of all the tested methods, calcium phosphate transfection afforded the highest efficiency of plasmid DNA delivery in all the cell lines except BHK21, for which the PEI method turned out to be more efficient than calcium phosphate transfection, and CHO, for which both methods showed comparable efficiency.

Autophagy inhibitors enhance biomolecular delivery efficiency of extracellular vesicles

In recent years, extracellular vesicles (EVs) were loaded with therapeutic molecules to be delivered to recipient cells, so the possibility of utilizing EVs for drug delivery has been investigated in various models. Nonetheless, most EVs are degraded through the autophagy pathway after being up-taken by recipient cells, resulting in a low delivery efficiency. Here we introduced a strategy to overcome inefficient delivery of EVs. We demonstrated that autophagy inhibitors, used for reducing lysosomal degradation of EVs, enhanced the protein or plasmid DNA delivery efficiency of EVs in recipient cells without influencing the uptake of EVs by recipient cells. Moreover, autophagy inhibitors could also improve gene-editing efficiency of EV-loaded CRISPR/Cas9 system.

Peptide-Mediated Gene Transfer into Marine Purple Photosynthetic Bacteria.

Use of photosynthetic organisms is one of the sustainable ways to produce high-value products. Marine purple photosynthetic bacteria are one of the research focuses as microbial production hosts. Genetic transformation is indispensable as a biotechnology technique. However, only conjugation has been determined to be an applicable method for the transformation of marine purple photosynthetic bacteria so far. In this study, for the first time, a dual peptide-based transformation method combining cell penetrating peptide (CPP), cationic peptide and Tat-derived peptide (dTat-Sar-EED) (containing D-amino acids of Tat and endosomal escape domain (EED) connected by sarcosine linkers) successfully delivered plasmid DNA into Rhodovulum sulfidophilum, a marine purple photosynthetic bacterium. The plasmid delivery efficiency was greatly improved by dTat-Sar-EED. The concentrations of dTat-Sar-EED, cell growth stage and recovery duration affected the efficiency of plasmid DNA delivery. The delivery was inhibited at 4 °C and by A22, which is an inhibitor of the actin homolog MreB. This suggests that the plasmid DNA delivery occurred via MreB-mediated energy dependent process. Additionally, this peptide-mediated delivery method was also applicable for E. coli cells. Thus, a wide range of bacteria could be genetically transformed by using this novel peptide-based transformation method.

Amphiphilic small peptides for delivery of plasmid DNAs and siRNAs.

Although various delivery systems for nucleic acids have been reported, development of an efficient and non-toxic delivery carrier is still a key subject for gene therapy. To find new efficient delivery carriers for nucleic acids, we synthesized amphiphilic peptides composed of a guanidino group, an oleyl group, and a cysteine. We prepared both linear and branched types of peptides and found that the linear peptides were superior to the branched peptides as nucleic acid carriers. Our study also suggested that the intermolecular cysteine disulfides might allow the linear peptides to form the optimal particle sizes with nucleic acids for cellular uptake. The incorporation of a benzoyl group to the linear peptide gave rise to smaller, less suitable particle size with plasmid DNA, which greatly reduced the efficiency of plasmid DNA delivery. On the other hand, the benzoyl modification maintained the optimal particle size with siRNA, and interestingly it significantly enhanced the siRNA delivery. The higher efficiency is because the hydrophobicity from the benzoyl group might assist in interacting with the hydrophobic cell membrane. This demonstrates that a small structural change can modulate the preference of the carriers. Our study may provide an insight designing efficient delivery carriers.

A comparison of plasmid DNA delivery efficiency and cytotoxicity of two cationic diblock polyoxazoline copolymers

Cationic polymers as non-viral gene delivery carriers are widely used because of their strong condensing properties and long-term safety, but acute cytotoxicity is a persistent challenge. In this study, two types of polyplexes were prepared by co-formulating plasmid DNA and two cationic diblock copolymers PABOXA5-b-PMOXA33-PA (primary amine) and PABOXA5-b-PMOXA33-TA (tertiary amine) to check their transfection efficacies in HeLa cells and HEK293T cells, respectively. The plasmid DNA/PABOXA5-b-PMOXA33-PA polyplex showed higher transfection efficacy compared to the plasmid DNA/PABOXA5-b-PMOXA33-TA polyplex under an N/P ratio of 40. Both polymers exhibited low toxicity, attributed to the shielding effect of a hydrophilic, noncharged block. Mechanistic insight into differential transfection efficiencies of the polymers were gained by visualization and comparison of the condensates via transmission electron and atomic force microscopy. The results provide information suited for further structure optimization of polymers that are aimed for targeted gene delivery.

Insight into the Role of Physicochemical Parameters in a Novel Series of Amphipathic Peptides for Efficient DNA Delivery

Amphipathic peptides constitute a class of molecules with the potential to develop as efficient and safer alternatives to viral and other nonviral vectors for intracellular delivery of therapeutics. These peptides can be useful for nucleic acid delivery and hence promise to have pharmaceutical application, particularly in gene therapy. In order to design novel amphipathic peptides and improve their efficiency of therapeutic cargo delivery, one needs to understand the role of the physicochemical properties of the peptide. There are very few reports in the literature where the physicochemical properties of the peptide have been correlated with efficiency of plasmid DNA delivery. In the present work we hunted out a naturally occurring amphipathic peptide termed Mgpe-1 (derived from HUMAN Protein phosphatase 1E) as a possible novel DNA delivery agent. We systematically altered the physicochemical parameters of this peptide to further enhance its DNA delivery efficiency. We changed its amphipathicity (from secondary to primary), the total charge (from +6 to +9), hydrophobicity, and the amino acid composition (lysine and serines to arginine; substitution of tryptophan) and studied which of these alterations affect DNA delivery efficiency. Our results showed that although Mgpe-1 exhibited very strong cellular uptake, its plasmid DNA delivery efficiency was poor. The presence of nine arginines improved the DNA delivery efficiency, and the effect was observed in both the primary and the secondary amphipathic variants. We further observed that the presence of tryptophan was important but not essential and the effect of its removal was stronger in the case of the secondary amphipathic peptide. However, increase in total hydrophobicity of the peptide led to a fall in transfection efficiency in the primary amphipathic peptide whereas the secondary amphipathic peptide having the same chemical composition was almost unaffected by this change. The primary amphipathic peptides with high positive charge and low hydrophobicity formed colloidally stable polyplexes with DNA and avoided a major impediment in DNA delivery, namely, the aggregation of polyplexes and cytotoxicity. The secondary amphipathic variants by virtue of the positional arrangement of the amino acids led to formation of polyplexes with partly hydrophilic surfaces which prevented aggregation and controlled particle size irrespective of the hydrophobicity. Two variants in the series Mgpe-3 and Mgpe-4 having nine positive charges with less hydrophobicity showed high transfection efficiency in multiple cell lines along with serum stability and much less cytotoxicity and promise to be novel and efficient DNA delivery vectors.

Structural rearrangements and chemical modifications in known cell penetrating peptide strongly enhance DNA delivery efficiency

Amphipathic peptides with unusual cellular translocation properties have been used as carriers of different biomolecules. However, the parameters which control the delivery efficiency of a particular cargo by a peptide and the selectivity of cargo delivery are not very well understood. In this work, we have used the known cell penetrating peptide pVEC (derived from VE-cadherin) and systematically changed its amphipathicity (from primary to secondary) as well as the total charge and studied whether these changes influence the plasmid DNA condensation ability, cellular uptake of the peptide–DNA complexes and in turn the efficiency of DNA delivery of the peptide. Our results show that although the efficiency of DNA delivery of pVEC is poor, modification of the same peptide to create a combination of nine arginines along with secondary amphipathicity improves its plasmid DNA delivery efficiency, particularly in presence of an endosomotropic agent like chloroquine. In addition, presence of histidines along with 9 arginines and secondary amphipathicity shows efficient DNA delivery with low toxicity even in absence of chloroquine in multiple cell lines. We attribute these enhancements in transfection efficiency to the differences in the mechanism of complex formation by the different variants of the parent peptide which in turn are related to the chemical nature of the peptide itself. These results exhibit the importance of understanding the physicochemical parameters of the carrier and complex in modulating gene delivery efficiency. Such studies can be helpful in improving peptide design for delivery of different cargo molecules.

129. Effect of Molecular Weight on Plasmid DNA Delivery Efficiency of Palmitic Acid Substituted Polyethylenimine

129. Effect of Molecular Weight on Plasmid DNA Delivery Efficiency of Palmitic Acid Substituted Polyethylenimine

Transgene expression in mononuclear muscle cells not infiltrating inflammatory cells following intramuscular plasmid gene electrotransfer

In situ electroporation-assisted intramuscular plasmid DNA delivery offers high efficiency for therapeutic protein replacement. Expression may be impaired by an immune response against the plasmid or transgenic protein. Expression of the transgene in non-muscle cells may increase the immune response. Gene transfer efficiency and phenotypic identification of intramuscular transgene-expressing mononuclear cells was studied following electroporation-mediated plasmid delivery. Plasmids expressing beta-galactosidase (pVR1012-betagal) or enhanced green fluorescent protein (eGFP) (pVR1012-eGFP) were electrotransferred into rat tibialis anterior muscles. Both transfection efficiency and the inflammatory response were determined in pVR1012-betagal-injected muscles by beta-galactosidase and haematoxylin and eosin staining of muscles 7 days post-plasmid injection. Muscles injected with pVR1012-eGFP were stained for CD3, CD68 and desmin at 24 and 48 h post-injection to determine whether mononuclear cells expressing eGFP were of immune or myogenic origin. With electroporation, beta-galactosidase expression was significantly enhanced by up to ten-fold compared to plasmid injection without electroporation. A large area of regenerating muscle fibres and inflammatory cell infiltration was found in electroporated plasmid-injected muscle. No eGFP expression was found in CD3- or CD68-positive cells. Small mononuclear cells expressing eGFP showed negative staining for CD3 and CD68, but all stained positive for desmin. In situ electroporation enhanced transfection efficiency of plasmid DNA delivery into muscle. Alongside its advantage for improving gene transfer, electroporation led to an increased inflammatory response and muscle damage. Mononuclear cells in muscle were transfected with plasmid and expressed the transgene. These cells were of myogenic origin with no evidence of transgene expression in infiltrating inflammatory cells.

Influence of Polycation Molecular Weight on Poly(2-dimethylaminoethyl methacrylate)-Mediated DNA Delivery In Vitro

Establishing clear structure-property-transfection relationships is a critical step in the development of clinically relevant polymers for nonviral gene therapy. In this study, we determined the influence of poly(2-dimethylaminoethyl methacrylate) (PDMAEMA) molecular weight on cytotoxicity, DNA binding, and in vitro plasmid DNA delivery efficiency in human brain microvascular endothelial cells (HBMEC). Conventional free radical polymerization was used to synthesize PDMAEMA with weight-average molecular weights ranging from 43,000 to 915,000 g/mol. MTT and LDH assays revealed that lower molecular weight PDMAEMA (M(w) = 43,000 g/mol) was slightly less toxic than higher molecular weights (M(w) > 112,000 g/mol) and that the primary mode of toxicity was cellular membrane destabilization. An electrophoretic gel shift assay revealed that all PDMAEMA molecular weights completely bound with plasmid DNA. However, heparin competitive binding experiments revealed that higher molecular weight PDMAEMA (M(w) = 915,000 g/mol) had a greater binding affinity toward plasmid DNA than lower molecular weight PDMAEMA (M(w) = 43,000 g/mol). The molecular weight of PDMAEMA was found to have a dramatic influence on transfection efficiency, and luciferase reporter gene expression increased as a function of increasing molecular weight. However, cellular uptake of polyplexes was determined to be insensitive to PDMAEMA molecular weight. In addition, our data did not correlate polyplex size with transfection efficiency. Collectively, our data suggested that the intracellular fate of the polyplexes, which involves endosomal release and DNase resistance, is more important to overall transfection efficiency than barriers to entry, such as polyplex size.

Relationship between the Extent of Lipid Substitution on Poly(l-lysine) and the DNA Delivery Efficiency

Poly(L-lysine) (PLL) is a commonly used polymer for nonviral gene delivery. However, the polymer exhibits significant toxicity and is not very effective for transgene expression. To enhance the gene delivery efficiency of the polymer, we imparted an amphiphilic property to PLL by substituting approximately 10% of epsilon-NH2 with several endogenous lipids of variable chain lengths (lipid carbon chain ranging from 8 to 18). Lipid-modified PLL with high molecular weight (approximately 25 vs 4 kDa) was found to be more effective in delivering plasmid DNA intracellularly in clinically relevant bone marrow stromal cells (BMSC). For lipid-substituted 25 kDa PLL, a correlation between the extent of lipid substitution and the plasmid DNA delivery efficiency was obtained. Additionally, transgene expression by BMSC significantly increased (20-25%) when amphiphilic PLLs were used for plasmid delivery as compared to native PLL and the commercial transfection agent Lipofectamine-2000. The transfection efficiency of the polymers was positively correlated with the extent of lipid substitution. The amphiphilic polymers were able to modify the cells up to 7 days after transfection, after which the expression was decreased to background levels within 1 week. We conclude that lipid-substituted PLL can be used effectively as a nonviral carrier for DNA, and the extent of lipid substitution was an important determinant of gene delivery.

Enhanced in vivo transgene expression and immunogenicity from plasmid vectors following electrostimulation in rodents and primates

Safe and efficient methods for in vivo delivery of transgenes of interest must be developed so that the promise of these therapies can be practically used in the clinic. In this work, we describe the use of electrostimulation to enhance the in vivo efficiency of plasmid DNA delivery. The method was optimized to work over a range of moderate frequencies, utilizing low field strengths and simple symmetrical waveforms. After studying several parameters of delivery in mice, we demonstrate how this methodology can be employed to significantly improve both gene expression (over 16-fold) and the immunogenicity of HIV-1 vaccines (over 28-fold) compared to naked DNA in non-human primates. Compared to an efficient viral Ad5 vector system, the gene expression levels of DNA + electrostimulation were surprisingly within a factor of four of the viral delivery system.

The potential of topical DNA vaccines adjuvanted by cytokines

To improve the efficacy of DNA immunization epidermal Langerhans cells are attractive targets to deliver antigen-encoding plasmid DNA. Topical vaccination with naked plasmid DNA has been shown to induce immune responses, and their potency might be improved by chemical and physical methods aimed to enhance the efficiency of plasmid DNA delivery into the skin. Cytokines have also been evaluated as adjuvants for DNA vaccines because they influence the host immune response. This review focuses on the action of several cytokines tested as molecular adjuvants for DNA vaccines and the combination of them with the DermaVir Patch vaccine. DermaVir vaccine, topically administered under a patch, consists of a plasmid DNA that is chemically formulated into a nanoparticle to support vaccine delivery into epidermal Langerhans cells and to induce antigen-specific memory T cells.

Full deacylation of polyethylenimine dramatically boosts its gene delivery efficiency and specificity to mouse lung.

High-molecular-mass polyethylenimines (PEIs) are widely used vectors for nucleic acid delivery. We found that removal of the residual N-acyl moieties from commercial linear 25-kDa PEI enhanced its plasmid DNA delivery efficiency 21 times in vitro, as well as 10,000 times in mice with a concomitant 1,500-fold enhancement in lung specificity. Several additional linear PEIs were synthesized by acid-catalyzed hydrolysis of poly(2-ethyl-2-oxazoline), yielding the pure polycations. PEI87 and PEI217 exhibited the highest efficiency in vitro: 115-fold and 6-fold above those of the commercial and deacylated PEI25s, respectively; moreover, PEI87 delivered DNA to mouse lung as efficiently as the pure PEI25 but at a lower concentration and with a 200-fold lung specificity. These improvements stem from an increase in the number of protonatable nitrogens, which presumably results in a tighter condensation of plasmid DNA and a better endosomal escape of the PEI/DNA complexes. As a validation of the potential of such linear, fully deacylated PEIs in gene therapy for lung diseases, systemic delivery in mice of the complexes of a short interfering RNA (siRNA) against a model gene, firefly luciferase, and PEI25 or PEI87 afforded a 77% and 93% suppression of the gene expression in the lungs, respectively. Furthermore, a polyplex of a siRNA against the influenza viral nucleocapsid protein gene and PEI87 resulted in a 94% drop of virus titers in the lungs of influenza-infected animals.

Efficient transient expression system based on square pulse electroporation and in vivo luciferase assay of fertilized fish eggs

Electroporation mediated DNA transfer into fish eggs has been improved by using a train of square pulses. Fertilized eggs of African catfish ( Clarias gariepinus), zebrafish ( Brachydanio rerio) and rosy barb ( Barbus conchonius) were dechorionated enzymatically followed by application of pulses. Efficiency of plasmid DNA delivery was significantly increased by applying multiple pulses on dechorionated eggs. Optimization of physical parameters such as field strength, pulse width and pulse numbers resulted in reproducible transient expression in 25–50% of embryos and larvae by using the firefly luciferase and the E. coli β-galactosidase (lacZ) genes both driven by CMV IE1 promoter. Temporal luceferase expression was assayed using both qualitative (sheet film) and quantitative (scintillation counting) methods in developing embryos and fry in vivo. Spatial expression of lacZ was assayed by histochemical staining. A number of embryos revealed foreign gene product also localised in the vegetal pole of the embryo.