DNA transfer into vascular smooth muscle using fusigenic Sendai virus (HJV)-liposomes

Manipulation of the genetic machinery of cells both in vitro and in vivo is becoming an ever more important means of elucidating pathways of molecular and cellular biochemistry. In addition, gene therapy has been proposed as a novel and potentially powerful treatment for both inherited and acquired diseases. Successful gene transfer and gene blockade generally depend on high efficiency delivery of exogenous DNA or RNA into living cells, and much effort has therefore been focused on the development of methods for achieving this delivery in a safe and effective manner. We describe here our application of fusigenic Sendai virus (HVJ)-liposome technology toward the effective delivery of DNA into vascular smooth muscle cells (VSMC) in cell culture. Cellular uptake and intracellular distribution of oligodeoxynucleotide (ODN) after transfection with HVJ-liposome complexes was characterized using fluorescent (FITC)-labeled ODN, and the biologic effect of HVJ-liposome mediated transfection was demonstrated via inhibition of DNA synthesis in cultured VSMC using antisense ODN against basic fibroblast growth factor.     

Enhanced delivery of cell-penetrating peptide-peptide nucleic acid conjugates by endosomal disruption

Improvement of cellular uptake and cellular localization is still one of the main obstacles to the development of antisense-antigene therapeutics, including peptide nucleic acid (PNA). Cell-penetrating peptides (CPPs) such as Tat peptide and polyarginine have been widely used to improve the cellular uptake of PNA and other antisense agents. Cellular uptake of most CPP conjugates occurs mainly through endocytotic pathways, and most CPP conjugate is retained in the endosomal compartments of the cell. Several methods to induce endosome disruption have been shown to improve the bioavailability of CPP conjugates to the cytosol and/or nucleus by facilitating escape from the endosomal compartments. Here we describe protocols for the delivery of CPP-PNA conjugates to adherent cultured cells using photodynamic treatment (photochemical internalization), Ca2+ treatment or chloroquine treatment to potentiate the antisense effects of CPP-PNA conjugates through increased release of CPP conjugates into the cytoplasm. This protocol, consisting of CPP-mediated delivery assisted by an endosome-disruption agent, allows the delivery of the CPP-PNA conjugates to the nucleus and/or cytosol of cultured cells. The endosome-disruption treatment improves the nuclear antisense effects of CPP-PNA conjugates by up to two orders of magnitude using 24-hour delivery.     

Efficient transfection of primary zebrafish fibroblasts by nucleofection

Although various gene delivery techniques are available, their application in zebrafish cell cultures has not been extensively studied. Here, we report that nucleofection of zebrafish primary embryonic fibroblasts results in higher transfection efficiency in comparison to other non-viral gene delivery methods. The transfection was performed using green fluorescent protein (GFP) gene constructs of a different size. Greatest DNA uptake was obtained with 4.9-kb plasmid, resulting in 43% GFP positive cells. Nucleofection with 7.4-kb pH2B-GFP plasmid followed by geneticin (G418) selection was successfully used to establish a cell line expressing nuclear histone 2B-GFP fusion protein. Efficient transfection of zebrafish fibroblasts by nucleofection offers a non-viral technique of plasmid delivery and can be used to overexpress genes of interest in these cells.     

Identification of a Short Cell-Penetrating Peptide from Bovine Lactoferricin for Intracellular Delivery of DNA in Human A549 Cells

Cell-penetrating peptides (CPPs) have been shown to deliver cargos, including protein, DNA, RNA, and nanomaterials, in fully active forms into live cells. Most of the CPP sequences in use today are based on non-native proteins that may be immunogenic. Here we demonstrate that the L5a CPP (RRWQW) from bovine lactoferricin (LFcin), stably and noncovalently complexed with plasmid DNA and prepared at an optimal nitrogen/phosphate ratio of 12, is able to efficiently enter into human lung cancer A549 cells. The L5a CPP delivered a plasmid containing the enhanced green fluorescent protein (EGFP) coding sequence that was subsequently expressed in cells, as revealed by real-time PCR and fluorescent microscopy at the mRNA and protein levels, respectively. Treatment with calcium chloride increased the level of gene expression, without affecting CPP-mediated transfection efficiency. Zeta-potential analysis revealed that positively electrostatic interactions of CPP/DNA complexes correlated with CPP-mediated transport. The L5a and L5a/DNA complexes were not cytotoxic. This biomimetic LFcin L5a represents one of the shortest effective CPPs and could be a promising lead peptide with less immunogenic for DNA delivery in gene therapy.     

Phosphate-buffered saline-based nucleofection of primary endothelial cells

Although various nonviral transfection methods are available, cell toxicity, low transfection efficiency, and high cost remain hurdles for in vitro gene delivery in cultured primary endothelial cells. Recently, unprecedented transfection efficiency for primary endothelial cells has been achieved due to the newly developed nucleofection technology that uses a combination of novel electroporation condition and specific buffer components that stabilize the cells in the electrical field. Despite superior transfection efficiency and cell viability, high cost of the technology has discouraged cardiovascular researchers from liberally adopting this new technology. Here we report that a phosphate-buffered saline (PBS)-based nucleofection method can be used for efficient gene delivery into primary endothelial cells and other types of cells. Comparative analyses of transfection efficiency and cell viability for primary arterial, venous, microvascular, and lymphatic endothelial cells were performed using PBS. Compared with the commercial buffers, PBS can support equally remarkable nucleofection efficiency to both primary and nonprimary cells. Moreover, PBS-mediated nucleofection of small interfering RNA (siRNA) showed more than 90% knockdown of the expression of target genes in primary endothelial cells. We demonstrate that PBS can be an unprecedented economical alternative to the high-cost buffers or nucleofection of various primary and nonprimary cells.     

Insights into the mechanism of magnetofection using PEI-based magnetofectins for gene transfer

BACKGROUND: Gene delivery by the use of magnetic forces, so-called magnetofection, has been shown to enhance transfection efficiency of viral and non-viral systems up to several-hundred-fold. For this purpose gene carriers, such as polyethylenimine (PEI), are associated with superparamagnetic nanoparticles and complexed with plasmid DNA. Gene delivery is targeted by the application of a magnetic field. METHODS: To investigate the underlying mechanism, we studied the impact of the applied magnetic field on the transfection process of PEI-coated superparamagnetic iron oxide gene vectors (magnetofectins) using various cell lines. In particular, we addressed the question whether accelerated sedimentation of magnetofectins is the driving force or if the magnetic field itself directly influences the endocytic processing of the magnetofectins. The cellular uptake mechanism of magnetofectins was studied by electron microscopy and transfection experiments in the presence of various inhibitors that operate at different steps of endocytosis. RESULTS: In this study we could show that cellular uptake of magnetofectins proceeds obviously by endocytosis. Cellular uptake of magnetofectins behaves almost analogously as compared with PEI polyplexes. Besides unspecific endocytosis, apparently clathrin-dependent as well as caveolae-mediated endocytic uptake is involved. CONCLUSIONS: The magnetic field itself does not alter the uptake mechanism of magnetofectins. Obviously, the magnetic forces lead to an accelerated sedimentation of magnetofectins on the cell surface and do not directly affect the endocytic uptake mechanism. So further improvement of magnetic field application could lead to efficient targeting of gene expression into the desired organ and tissue in vivo.     

Novel Fatty Acid Modifications of Transportan 10

Cell-penetrating peptides (CPPs) are able to efficiently internalize into cells and can therefore be used as vectors for non-viral cellular delivery of different cargoes. Previous studies have shown that hydrophobic modifications of different CPPs can increase their transfection efficiency dramatically. In this study we have modified the cell penetrating-peptide transportan 10 (TP10) with a variety of hydrophobic molecules to determine the role of hydrophobicity in the uptake of these molecules. The results can be used to synthesize more efficient delivery vectors. To evaluate how these constructs are able to transport cargoes into cells we used 2′-OMe splice correcting oligonucleotides. Non-covalent peptide-cargo complexes were formed and their transfection efficiency was measured using a luciferase readout system. The hydrophobicity of the novel modifications was correlated with their biological efficacy. We determined the most efficient range of hydrophobicity for TP10 analogs for delivering oligonucleotides into cells. In order to assess how the transfection efficacy of these particles is dependent on their size the hydrodynamic diameter of the formed nanoparticles was measured using dynamic light scattering. These findings will be used to develop highly efficient non-viral gene therapy vectors.     

Cellular fate of a modular DNA delivery system mediated by silica nanoparticles

Development of efficient molecular medicines, including gene therapeutics, RNA therapeutics, and DNA vaccines, depends on efficient means of transfer of DNA or RNA into the cell. Potential problems, including toxicity and immunogenicity, surrounding viral methods of DNA delivery have necessitated the use of nonviral, synthetic carriers. To better design synthetic carriers, or transfection reagents, the modular design of viruses has inspired a modular approach to DNA and RNA delivery. Each modular component can be designed to circumvent each of the many barriers. The modular approach will allow modification of individual components for a specific application. By utilizing a dense silica nanoparticle to form a ternary complex, transfection efficiency of a DNA-transfection reagent complex was increased by a factor of approximately 10 by concentrating the DNA at the surface of cells. Surface modification of the silica nanoparticles allowed determination of the cellular uptake mechanism with only minor alteration of transfection efficiency. Nanoparticles are internalized by an endosome-lysosomal route followed by perinuclear accumulation. The modification mechanism confirms that surface modification of the modular system can allow specific moieties to be incorporated into the modular system without significant alteration of the transfection efficiency. By showing that the modular system based upon concentration of DNA at the level of the cell can be used to increase transfection efficiency, we have shown that further modification of the system may better target DNA delivery and overcome other barriers of DNA expression.     

Cell-penetrating peptides: Application in vaccine delivery

Recent years have seen a surge in interest in cell-penetrating peptides (CPP) as an efficient means for delivering therapeutic targets into cellular compartments. The cell membrane is impermeable to hydrophilic substances yet linking to CPP can facilitate delivery into cells. Thus the unique translocatory property of CPP ensures they remain an attractive carrier, with the capacity to deliver cargoes in an efficient manner having applications in drug delivery, gene transfer and DNA vaccination. Fundamental for an effective vaccine is the delivery of antigen epitopes to antigen-presenting cells, ensuing processing and presentation and induction of an immune response. Vaccination with proteins or synthetic peptides incorporating CTL epitopes have proven limited due to the failure for exogenous antigens to be presented efficiently to T cells. Linking of antigens to CPP overcomes such obstacles by facilitating cellular uptake, processing and presentation of exogenous antigen for the induction of potent immune responses. This review will encompass the various strategies for the delivery of whole proteins, T cell epitopes and preclinical studies utilizing CPP for cancer vaccines.     

Optimization of gene delivery to HEK293T cells by microporation using a central composite design methodology

Microporation is an efficient method for delivering plasmid DNA molecules into cultured cells. Herein, we present the optimization of gene delivery by microporation using a Central Composite Design methodology. It was given relevance not only to the transfection efficiency but also to the cell recovery. Different amounts of DNA (1 and 3 μg) mainly affected cell viabilities and cell recoveries, which decrease from 93 to 76% and from 47 to 25% respectively, when higher DNA quantity is used. With this work we suggest an easy methodology to improve transfection of mammalian cells underlining the feasibility to achieve 60% of gene delivery efficiencies whilst recovering 50% of cells, with 90% of viability.     

Transfection efficiency and uptake process of polyplexes in human lung endothelial cells: a comparative study in non-polarized and polarized cells

BACKGROUND: Following systemic administration, polyplexes must cross the endothelium barrier to deliver genes to the target cells underneath. To design an efficient gene delivery system into lung epithelium, we evaluated capture and transfection efficiencies of DNA complexed with either Jet-PEI (PEI-polyplexes) or histidylated polylysine (His-polyplexes) in human lung microvascular endothelial cells (HLMEC) and tracheal epithelial cells. METHODS: After optimizing growth conditions to obtain a tight HLMEC monolayer, we characterized uptake of polyplexes by flow cytometry and evaluated their transfection efficiency. Polyplexes were formulated as small particles. YOYO-labelled plasmid fluorescence intensity and luciferase activity were used as readouts for uptake and gene expression, respectively. RESULTS: PEI-polyplexes were more efficiently taken up than His-polyplexes by both non-polarized (2-fold) and polarized HLMEC (10-fold). They were mainly internalized by a clathrin-dependent pathway whatever the cell state. In non-polarized cells, His-polyplexes entered also mainly via a clathrin-dependent pathway but with an involvement of cholesterol. The cell polarization decreased this way and a clathrin-independent pathway became predominant. PEI-polyplexes transfected more efficiently HLMEC than His-polyplexes (10(7) vs. 10(5) relative light units (RLU)/mg of proteins) with a more pronounced difference in polarized cells. In contrast, no negative effect of the cell polarization was observed with tracheal epithelial cells in which both polyplexes had comparable efficiency. CONCLUSIONS: We show that the efficiency of polyplex uptake by HLMEC and their internalization mechanism are polymer-dependent. By contrast with His-polyplexes, the HLMEC polarization has little influence on the uptake process and on the transfection efficiency of PEI-polyplexes.     

Developing a Novel Gene-Delivery Vector System Using the Recombinant Fusion Protein of Pseudomonas Exotoxin A and Hyperthermophilic Archaeal Histone HPhA

Non-viral gene delivery system with many advantages has a great potential for the future of gene therapy. One inherent obstacle of such approach is the uptake by endocytosis into vesicular compartments. Receptor-mediated gene delivery method holds promise to overcome this obstacle. In this study, we developed a receptor-mediated gene delivery system based on a combination of the Pseudomonas exotoxin A (PE), which has a receptor binding and membrane translocation domain, and the hyperthermophilic archaeal histone (HPhA), which has the DNA binding ability. First, we constructed and expressed the rPE-HPhA fusion protein. We then examined the cytotoxicity and the DNA binding ability of rPE-HPhA. We further assessed the efficiency of transfection of the pEGF-C1 plasmid DNA to CHO cells by the rPE-HPhA system, in comparison to the cationic liposome method. The results showed that the transfection efficiency of rPE-HPhA was higher than that of cationic liposomes. In addition, the rPE-HPhA gene delivery system is non-specific to DNA sequence, topology or targeted cell type. Thus, the rPE-HPhA system can be used for delivering genes of interest into mammalian cells and has great potential to be applied for gene therapy.     

Characterization of Ku70(2)-NLS as bipartite nuclear localization sequence for non-viral gene delivery

Several barriers have to be overcome in order to achieve gene expression in target cells, e.g. cellular uptake, endosomal release and translocation to the nucleus. Nuclear localization sequences (NLS) enhance gene delivery by increasing the uptake of plasmid DNA (pDNA) to the nucleus. So far, only monopartite NLS were analysed for non-viral gene delivery. In this study, we examined the characteristics of a novel bipartite NLS like construct, namely NLS Ku70. We synthesized a dimeric structure of a modified NLS from the Ku70 protein (Ku70(2)-NLS), a nuclear transport active mutant of Ku70(2)-NLS (s1Ku70(2)-NLS) and a nuclear transport deficient mutant of Ku70(2)-NLS (s2Ku70(2)). We examined the transfection efficiency of binary Ku70(2)-NLS/DNA and ternary Ku70(2)-NLS/PEI/DNA gene vector complexes in vitro by using standard transfection protocols as well as the magnetofection method. The application of Ku70(2)-NLS and s1Ku70(2)-NLS increased gene transfer efficiency in vitro and in vivo. This study shows for the first time that the use of bipartite NLS compounds alone or in combination with cationic polymers is a promising strategy to enhance the efficiency of non-viral gene transfer.     

Transfection of myeloid leukaemia cell lines is distinctively regulated by fibronectin substratum

Gene transfer into haematopoietic cells is a challenging approach. The extracellular matrix component fibronectin has been known to modulate the cell cycle dynamics, viability and differentiation of leukaemia cells. Thus, our aim was to investigate the influence of fibronectin substratum on the liposomal transfection of myeloid leukaemia cell lines. Liposomal transfection was performed with K562 and HL-60 as representative lines of transfection-competent and -incompetent myeloid leukaemia cells, respectively. Flow cytometry analyses were performed to determine transfection efficiency monitored by green fluorescent protein (GFP) expression and to assess cell viability and cell cycle status. Quantitation of GFP gene expression and DNA uptake was assayed by real time PCR. The current data showed that the adhesion to fibronectin deteriorated the transfection of K562 cells. In contrary, it enhanced the delivery of plasmid DNA into HL-60 cells. Correspondingly, the adhesion to fibronectin influenced the transfection efficiency mainly by modulating the intracellular presence of plasmid DNA. The cell cycle and viability which is regulated by fibronectin had a minor impact on the success of gene delivery. This phenomenon may be considered as an important factor which may modulate the potential gene transfer approaches for myeloid leukaemia.     

Polyamidoamine-grafted multiwalled carbon nanotubes for gene delivery: synthesis, transfection and intracellular trafficking

Functionalized multiwalled carbon nanotubes (f-MWNTs) are of great interest and designed as a novel gene delivery system. In this paper, we presented synthesis of polyamidoamine-functionalized multiwalled carbon nanotubes (PAA-g-MWNTs) and their application as a novel gene delivery system. The PAA-g-MWNTs, obtained from amide formation between PAA and chemically oxidized MWNTs, were stable in aqueous solution and much less toxic to cells than PAA and PEI 25KDa. More importantly, PAA-g-MWNTs showed comparable or even higher transfection efficiency than PAA and PEI at optimal w/w ratio. Intracellular trafficking of Cy3-labeled pGL-3 indicated that a large number of Cy3-labeled pGL-3 were attached to nucleus membrane, the majority of which was localized in nucleus after incubation with cells for 24 h. We have demonstrated that PAA modification of MWNTs facilitate higher DNA uptake and gene expression in vitro. All these facts suggest potential application of PAA-g-MWNTs as a novel gene vector with high transfection efficiency and low cytotoxicity.     

The agony of choice: how to find a suitable CPP for cargo delivery

Successful and effective cellular delivery remains a main obstacles in the medical field. The use of cell‐penetrating peptides (CPPs) has become one of the most important tools for the internalisation of a wide range of molecules including pharmaceuticals. It is still difficult to choose one CPP for one biological application because there is no ubiquitous CPP meeting the diverse requirements. In our case, we are looking for a suitable CPP to deliver the pro‐apoptotic KLA peptide (KLAKLAKKLAKLAK) by a simple co‐incubation strategy. For that reason, we selected three different cell lines (fibroblastic, cancerous and macrophagic cells) and studied the uptake and subcellular localisation of six different CPPs alone as well as mixed with the KLA peptide. Furthermore, we used the CPPs with a carboxyamidated or a carboxylated C‐terminus and analysed the impact of the C‐termini on internalisation and cargo delivery. We could clearly showed that the cellular CPP uptake is not only dependent on the used CPP and cell line but also highly affected by its chemical nature of the C‐terminus (uptake: carboxyamidated CPPs > carboxylated CPPs) and can influence its cellular localisation. We successfully delivered the KLA peptide in the three cell lines and learned that here as well, the C‐terminus is crucial for an effective peptide delivery. Finally, we induced apoptosis in mouse leukaemic monocyte macrophage (RAW 264.7) and in human breast adenocarcinoma (MCF‐7) cells using the mixture of amidated MPG peptide : KLA and in african green monkey kidney fibroblast (Cos‐7) cells using carboxylated integrin peptide : KLA. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Charge density and molecular weight of polyphosphoramidate gene carrier are key parameters influencing its DNA compaction ability and transfection efficiency

A series of polyphosphoramidates (PPAs) with different molecular weights (MWs) and charge densities were synthesized and examined for their DNA compaction ability and transfection efficiency. A strong correlation was observed between the transfection efficiency of PPA/DNA nanoparticles and the MW and net positive charge density of the PPA gene carriers in three different cell lines (HeLa, HEK293, and HepG2 cells). An increase in MW and net positive charge density of PPA carrier yielded higher DNA compaction capacity, smaller nanoparticles with higher surface charges, and higher complex stability against challenges by salt and polyanions. These favorable physicochemical properties of nanoparticles led to enhanced transfection efficiency. PPA/DNA nanoparticles with the highest complex stability showed comparable transfection efficiency as PEI/DNA nanoparticles likely by compensating the low buffering capacity with higher cellular uptake and affording higher level of protection to DNA in endolysosomal compartment. The differences in transfection efficiency were not attributed by any difference in cytotoxicity among the carriers, as all nanoparticles showed a minimal level of cytotoxicity under the transfection conditions. Using PPA as a model system, we demonstrated the structural dependence of transfection efficiency of polymer gene carrier. These results offer more insights into nanoparticle engineering for nonviral gene delivery.     

Protein delivery by the cell-penetrating peptide YTA2

In most cases, the transport of cell-penetrating peptide (CPP) with a cargo molecule over the plasma membrane requires a cross-linking of the cargo molecule to the peptide. Lately, a method of cargo delivery, coincubation with CPP, has been applied. We have studied uptake and toxicity of the CPP, YTA2, in the Bowes human melanoma cell line and human MDA-MB-231 breast cancer cell line and compared the results with known cell-penetrating peptides. The results show that fluoresceinyl YTA2 is taken up by the Bowes cells with 3.23 nmol/mg protein and shows low membrane toxicity to the cells with an EC50 of 60 microM. Furthermore, we show that YTA2 is capable of delivering cargo proteins, such as beta-galactosidase and tetramethyl rhodamine iso-thiocyanate (TRITC) labeled streptavidin into cells by coincubation. The delivery of TRITC-labeled streptavidin was quantified to 42.4 pmol streptavidin/mg protein. The delivery of proteins into the cells by mere coincubation is an advantage, since the chemical coupling between the CPP and the cargo molecule, which adds time-consuming synthesis and purification steps, can be omitted. In addition, the flexibility in CPP cargo delivery is increased.     

Protamine-modified DDAB lipid vesicles promote gene transfer in the presence of serum

Cationic lipid vesicle-mediated gene transfer has become common for in vitro gene delivery. However, the transfection efficiency is often impaired by serum. DDAB (dimethyldioctadecyl ammonium bromide) lipid vesicle-mediated gene transfer, which we previously reported, has the same problem. To overcome this obstacle, we here report a novel transfection vehicle using protamine-modified DDAB lipid vesicles. While free protamine was simply added to the DNA/lipid complex in the previous study, in the present method the protamine is chemically conjugated to stearic acid and incorporated into DDAB lipid vesicles. Gene transfer was not significantly inhibited in 10% serum-containing medium by this method for the transfection of cultured cells. Protamine-modified DDAB lipid vesicles also enhanced virus transduction efficiency in the presence of serum using a replication-defective retroviral vector. Furthermore, the vesicles allowed efficient gene transfer for avian embryos in vivo. These results indicate that the method is useful for the production of transgenic animals and gene therapy.     

Development of efficient transient transfection systems for introducing antisense oligonucleotides into human epithelial skin cells

Systemic treatment with antisense oligonucleotides is confounded by the dual problems of potential cytotoxicity of antisense oligonucleotides and carrier molecules such as cationic lipids. Treatment of pathologic conditions affecting the skin may avoid these problems to a large degree due to local application. The success of antisense strategies has been limited by the poor uptake of the transfection reagent and inadequate intracellular compartmentalization. Human skin epithelial cells, therefore, are attractive experimental tools for testing both in vitro and in vivo antisense therapies. In the present study, we determined commercially available liposomes which reproducibly induced a nontoxic increase of oligonucleotide uptake in cultured SZ95 sebocytes and keratinocytes. The final protocol for SZ95 sebocytes was a 4-hour incubation with DOTAP in a 2:1 (w/w) lipid/oligonucleotide ratio in serum-free medium. The fluorescein-labeled (ATCG)(5) random oligonucleotide molecules were detected within the nucleus. The optimum transfection system for primary keratinocytes was poly-L-ornithine (12 microg/ml) in a medium without bovine pituitary extract over 4 hours. The uptake of the oligonucleotide increased in the presence of the polycation and oligonucleotide molecules were localized in the cytoplasm of keratinocytes. Oligonucleotide transfection with the help of cationic lipids did not affect the expression of androgen receptor and of the house-keeping gene beta-actin. Thus, cationic lipids are useful for delivery of antisense oligonucleotides into skin cells in vitro and may be used for topical application on animal and human skin.