Arginine-rich cross-linking peptides with different SV40 nuclear localization signal content as vectors for intranuclear DNA delivery

The major barriers for intracellular DNA transportation by cationic polymers are their toxicity, poor endosomal escape and inefficient nuclear uptake. Therefore, we designed novel modular peptide-based carriers modified with SV40 nuclear localization signal (NLS). Core peptide consists of arginine, histidine and cysteine residues for DNA condensation, endosomal escape promotion and interpeptide cross-linking, respectively. We investigated three polyplexes with different NLS content (10?mol%, 50?mol% and 90?mol% of SV40 NLS) as vectors for intranuclear DNA delivery. All carriers tested were able to condense DNA, to protect it from DNAase I and were not toxic to the cells. We observed that cell cycle arrest by hydroxyurea did not affect transfection efficacy of NLS-modified carriers which we confirmed using quantitative confocal microscopy analysis. Overall, peptide carrier modified with 90?mol% of SV40 NLS provided efficient transfection and nuclear uptake in non-dividing cells. Thus, incorporation of NLS into arginine-rich cross-linking peptides is an adequate approach to the development of efficient intranuclear gene delivery vehicles.     

DNA transfer into human lung cells is improved with Tat-RGD peptide by caveoli-mediated endocytosis

Cell lines and primary cells exhibit varying degrees of resistance to DNA transfection strategies. In this study, we employed the synthetic peptide Tat-RGD (TR), composed of the HIV-1 derived translocation peptide Tat fused to the integrin binding RGD motif, as a tool for improving DNA transfer into pulmonary cells. Binding experiments between DNA and TR and cytotoxicity measurements of TR treated cells were undertaken to optimize DNA and TR concentrations for transfection. Addition of a complex of TR and DNA (TRD) to A549 cells yielded significant transgene expression. When TRD was combined with Lipofectamine (TRDL), the expression was increased by 5-fold over Lipofectamine (DL) and by approximately 30-fold over TRD-mediated transfections. Also, in primary smooth muscle cells (SMC) and fibroblasts (FB) derived from pulmonary arteries, an increase in TRDL-mediated transfection efficiency was observed by a factor of approximately 2 and approximately 3 over that of DL. Laser scanning confocal microscopy for visualizing TR-dependent DNA uptake demonstrated that the internalization of TRDL complexes is linked to caveoli in the plasma membrane. Interfering with caveoli formation by methyl-b-cyclo-dextrin drastically decreased the transfection efficiency by TR. In conclusion, the Tat-RGD peptide mediates efficient gene delivery in human pulmonary cells, in particular when combined with a standard cationic lipid based transfection reagent. The enhancement of DNA uptake by Tat-RGD is suggested to be mediated by caveoli-dependent endocytosis.     

新型非病毒三元复合DNA载体的研究

基因治疗是未来临床医学最具潜力的治疗方式,目前阻碍临床基因治疗发展的主要因素是缺乏安全和高效的基因载体,因此研究理想的非病毒转基因载体具有重要的意义.构建了由质粒DNA(D)-抗DNA抗体(A)-阳离子脂质体(C)组成的三元复合纳米基因载体(DAC),研究表明,三组分在磷酸缓冲液中可通过分子组装形成复合纳米胶束,DAC在细胞培养中表现出显著高效的基因表达,DAC在血管平滑肌细胞中的基因转染效率比不含抗DNA抗体的二元组合(DC)高4倍,比不含阳离子脂质体的二元组合(DA)约高11倍.激光共聚焦荧光显微观察证明,DAC细胞摄取量和DNA进入细胞核的量均明显高于对照组,而DC二元组合(不含抗DNA抗体)的DNA很少进入细胞核,细胞在DAC存在下生长正常.未发现细胞毒性.研究结果提示,DAC的作用机理主要是三元复合胶束中DNA的装载量比二元载体大得多,抗DNA抗体与阳离子脂质体的协同作用明显有利于DNA被细胞摄取和胞吞,从而提高了基因的转染和表达.     

Gene transfection efficiency of tracheal epithelial cells by DC-chol-DOPE/DNA complexes.

We evaluated the transfection efficiency of five different cationic liposome/plasmid DNA complexes, during the in vitro gene transfer into human epithelial tracheal cell lines. A dramatic correlation between the transfection efficiency and the charge ratio (positive charge of liposome to negative charge of DNA) has been found. DC-Chol-DOPE was found to be the most effective liposome formulation. Therefore, a morphological and structural analysis of DC-Chol-DOPE liposomes and DC-Chol-DOPE/DNA complexes, has been performed by transmission electron microscopy (TEM) and by confocal laser scanning microscopy (CLSM), respectively. The process of interaction between DC-Chol-DOPE/DNA complexes and human epithelial tracheal cells has been studied by CLSM. These results raise some issues for in vivo gene therapy.     

An insight into the gene delivery mechanism of the arginine peptide system: role of the peptide/DNA complex size

Cationic peptides have been used successfully to transfer macromolecules into living cells. Previously, we have reported a short arginine peptide-based gene delivery system. However, the mechanisms that allow arginine peptides to promote gene delivery yet remain unknown. In the present study, we investigated the effect of the arginine peptide/DNA complex size on the transfection efficiency. After combining peptides with DNA, a 400 nm complex was observed. As the incubation time was increased, the complex grew larger, reaching 6 microm after 1 h of incubation. Transfection and cellular uptake efficiency were likewise investigated for the effects of the different sizes of complexes. Large complexes were found to be advantageous for transfection. However, better internalization efficiency was found with small complexes, indicating that the amount of peptide/DNA complexes taken up by cells is not the rate-limiting step in the final transfection efficiency. The intracellular path of the peptide/DNA complex was studied using fluorescent labeling and confocal microscopy. In the early stages of transfection, complexes were observed only on the cell surface, and these complexes migrated into cytoplasm however, after 6 h, the presence of complexes in the perinuclear region was noted. We were able to detect colocalization of green and red fluorescence in both the cytoplasm and the nucleus. These results suggest that peptide/DNA complexes reach the nucleus as associated complexes.     

A model for non-viral gene delivery: through syndecan adhesion molecules and powered by actin

BACKGROUND: Cell transfection requires cationic DNA complexes and heparan sulfate proteoglycans (HSPGs) at the cell surface. Syndecans are transmembrane HSPGs that are ubiquitously expressed on adherent cells. Their polyanionic heparan sulfate moieties are bound at the distal end of their ectodomain, thus facilitating interaction with large cationic particles. METHODS: We propose a model for cell entry involving syndecans as receptors for the DNA complexes by comparing transfection with bacteria uptake and using drug inhibition experiments along with confocal microscopy. RESULTS: When combined with results from the literature, our data suggest the following sequence of events: after initial particle binding, gradual electrostatic zippering of the plasma membrane onto the particle is sustained by lateral diffusion of syndecan molecules that cluster into cholesterol-rich rafts. Clustering in turn triggers PKC activity and linker protein-mediated actin binding to the cytoplasmic tail of the syndecans. Resulting tension fibers and a growing network of cortical actin may then pull the particle into the cell. CONCLUSIONS: Diversion of integrin- and syndecan-mediated cell adhesion processes for particle engulfment appears to be widely exploited by animals (chylomicrons), by pathogens (bacteria, viruses) and, as suggested here, by non-viral vectors.     

Possible mechanism of polycation liposome (PCL)-mediated gene transfer

A novel gene transfer system utilizing polycation liposomes (PCLs), obtained by modifying liposomes with cetyl polyethylenimine (PEI), was previously developed (Gene Ther. 7 (2002) 1148). PCLs show notable transfection efficiency with low cytotoxicity. However, the mechanism of PCL-mediated gene transfer is still unclear. In this study, we examined the intracellular trafficking of PCL-DNA complexes by using HT1080 cells, fluorescent probe-labeled materials, and confocal laser scan microscopy. We found that the PCL-DNA complexes were taken up into cells by the endosomal pathway, since both cellular uptake of the complex and gene expression were blocked by wortmannin, an inhibitor of this pathway. We also observed that the plasmid DNA and cetyl PEI complex became detached from the PCL lipids and was preferentially transferred into the nucleus in the form of the complex, whereas the PCL lipids remained in the cytoplasmic area, possibly in the endosomes. In fact, nigericin, which dissipates the pH gradient across the endosomal membrane, inhibited the detachment of lipids from the PCL-DNA complex and subsequent gene expression. Taken together, our data indicate the following mechanism for gene transfer by PCLs: PCLs effectively transfer DNA to endosomes and release cetyl PEI-DNA complexes into the cytosol. Furthermore, cetyl PEI also contributes to gene entry into the nucleus.     

Transfection of cells using flow-through electroporation based on constant voltage

Electroporation is a high-efficiency and low-toxicity physical gene transfer method. Classical electroporation protocols are limited by the small volume of cell samples processed (less than 10(7) cells per reaction) and low DNA uptake due to partial permeabilization of the cell membrane. Here we describe a flow-through electroporation protocol for continuous transfection of cells, using disposable devices, a syringe pump and a low-cost power supply that provides a constant voltage. We show transfection of cell samples with rates ranging from 40 μl min(-1) to 20 ml min(-1) with high efficiency. By inducing complex migrations of cells during the flow, we also show permeabilization of the entire cell membrane and markedly increased DNA uptake. The fabrication of the devices takes 1 d and the flow-through electroporation typically takes 1-2 h.     

On the possible involvement of bovine serum albumin precursor in lipofection pathway

Protein factors involved in lipofection pathways remain elusive. Using avidin-biotin affinity chromatography and mass finger printing analysis technique, herein we report the identification of a 70 kDa size protein (bovine serum albumin precursor, BSAP) which binds strongly with lipoplexes and may play role in lipofection pathway. Using multiple cultured animal cells and three structurally different cationic transfection lipids, we show that the efficiencies of liposomal transfection vectors get significantly enhanced (by ~2.5- to 5.0-fold) in cells pre-transfected with lipoplexes of reporter plasmid construct encoding BSAP. Findings in the cellular uptake experiments in A549 cells cultured in DMEM supplemented with 10% (w/w) BODIPY-labelled BSAP are consistent with the supposition that BSAP enters cell cytoplasm from the cell culture medium (DMEM supplemented with 10% FBS) used in lipofection. Cellular uptake studies by confocal microscopy using BODIPY-labelled BSAP and FITC-labelled plasmid DNA revealed co-localization of plasmid DNA and BSAP within the cell cytoplasm and nucleus. In summary, the present findings hint at the possible involvement of BSAP in lipofection pathway.     

Transition from a normal to inverted cylinder for an amidine-bearing lipid/pDNA complex and its excellent transfection

A cationic lipid (TRX) having an amidine headgroup was synthesized, and a lipoplex (i.e., plasmid DNA+lipid complex) was prepared from the mixture of TRX and two other neutral colipids. Small-angle X-ray scattering showed that the addition of DNA induced a structural transition from normal to inverted hexagonally packed cylinders. Transmission electron microscopy showed a threadlike micelle was transformed to a spherical micelle about 50-200 nm in diameter by adding DNA. A combination of these results leads to the conclusion that the complexed DNA is hexagonally packed (or condensed) into spherical aggregates. The size and morphology are believed suitable for endocytosis uptake or vesicle fusion. The complex made from pDNA (pEGFP-C1) and TRX was transfected to Hep G2. Flow cytometry and confocal microscopy showed that the present system expressed green fluorescence protein (GFP) more than a conventional transfection reagent. Additionally, TRX was less cytotoxic than other transfection reagents. This paper presents the attractive possibility of the amidine group for a transfection device.     

Recombinant fusion proteins TAT-Mu, Mu and Mu-Mu mediate efficient non-viral gene delivery

BACKGROUND: The inherent ability of certain peptides or proteins of viral, prokaryotic and eukaryotic origin to bind DNA was used to generate novel peptide-based DNA delivery protocols. We have developed a recombinant approach to make fusion proteins with motifs for DNA-binding ability, Mu and membrane transduction domains, TAT, and tested them for their DNA-binding, uptake and transfection efficiencies. In one of the constructs, the recombinant plasmid was designed to encode the Mu moiety of sequence MRRAHHRRRRASHRRMRGG in-frame with TAT of sequence YGRKKRRQRRR to generate TAT-Mu, while the other two constructs, Mu and Mu-Mu, harbor a single copy or two copies of the Mu moiety. METHODS: Recombinant his-tag fusion proteins TAT-Mu, Mu and Mu-Mu were purified by overexpression of plasmid constructs using cobalt-based affinity resins. The peptides were characterized for their size and interaction with DNA, complexed with plasmid pCMVbeta-gal, and shown to transfect MCF-7, COS and CHOK-1 cells efficiently. RESULTS: Recombinant fusion proteins TAT-Mu, Mu and Mu-Mu were cloned and overexpressed in BL21(DE3)pLysS with greater than 95% purity. The molecular weight of TAT-Mu was determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) to be 11.34 kDa while those of Mu and Mu-Mu were 7.78 and 9.83 kDa, respectively. Live uptake analysis of TAT-Mu, Mu and Mu-Mu as DP (DNA+peptide) or DPL (DNA+peptide+lipid) complexes into MCF-7 cells, followed by immunostaining and laser scanning confocal microscopy, demonstrated that the complexes are internalized very efficiently and localized in the nucleus. DNA:peptide complexes (DP) transfect MCF-7, COS and CHOK-1 cells. The addition of cationic liposomes enhances the uptake of the ternary complexes (DPL) further and also brings about 3-7-fold enhancement in reporter gene expression compared to DP alone. CONCLUSIONS: Recombinant proteins that are heterologous fusions, having DNA-binding domains and nuclear localization epitopes, generated in this study have considerable potential to facilitate DNA delivery and enhance transfection. The domains in these fusion proteins would be promising in the development of non-viral gene delivery vectors particularly in cells that do not divide.     

Gene delivery by dendrimers operates via a cholesterol dependent pathway

Understanding the cellular uptake and intracellular trafficking of dendrimer–DNA complexes is an important prerequisite for improving the transfection efficiency of non-viral vector-mediated gene delivery. Dendrimers are synthetic polymers used for gene transfer. Although these cationic molecules show promise as versatile DNA carriers, very little is known about the mechanism of gene delivery. This paper investigates how the uptake occurs, using an endothelial cell line as model, and evaluates whether the internalization of dendriplexes takes place randomly on the cell surface or at preferential sites such as membrane rafts. Following extraction of plasma membrane cholesterol, the transfection efficiency of the gene delivered by dendrimers was drastically decreased. Replenishment of membrane cholesterol restored the gene expression. The binding and especially internalization of dendriplexes was strongly reduced by cholesterol depletion before transfection. However, cholesterol removal after transfection did not inhibit expression of the delivered gene. Fluorescent dendriplexes co-localize with the ganglioside GM1 present into membrane rafts in both an immunoprecipitation assay and confocal microscopy studies. These data strongly suggest that membrane cholesterol and raft integrity are physiologically relevant for the cellular uptake of dendrimer–DNA complexes. Hence these findings provide evidence that membrane rafts are important for the internalization of non-viral vectors in gene therapy.     

Transfection-mediated cell synchronization: acceleration of G1-S phase transition by gamma irradiation.

We have previously provided evidence that the uptake of DNA into cells is cell cycle specific following transfection. We show here that, immediately after transfection, successfully transfected cells are greatly enriched for cells in early G1 or G0 phase and that, upon removal of the DNA precipitates, cells progress through G1 and enter S phase in a synchronous fashion. We also demonstrate that this approach can be utilized in meaningful cell-cycle experiments, and we show that gamma irradiation accelerates the G1-S phase transition in a cell line with a functionally inactive p53 protein.     

Enhanced nuclear import and transfection efficiency of TAT peptide-based gene delivery systems modified by additional nuclear localization signals

Cellular uptake and nuclear localization are two major barriers in gene delivery. In order to evaluate whether additional nuclear localization signals (NLSs) can improve gene transfection efficiency, we introduced different kinds of NLSs to TAT-based gene delivery systems to form three kinds of complexes, including TAT-PV/DNA, TAT/DNA/PV, and TAT/DNA/HMGB1. The DNA binding ability of different vectors was evaluated by agarose gel electrophoresis. The in vitro transfections mediated by different complexes under different conditions were carried out. The cells treated by different complexes were observed by confocal microscopy. The MTT assay showed that all complexes did not exhibit apparent cytotoxicity in both HeLa and Cos7 cell lines even at high N/P ratios. The luciferase reporter gene expression mediated by TAT-PV/DNA complexes exhibited about 200-fold enhancement as compared with TAT/DNA complexes. Confocal study showed that, except TAT/DNA/PV, all other complexes exhibited enhanced nuclear accumulation and cellular uptake in both HeLa and Cos7 cell lines. These results indicated that the introduction of nuclear localization signals could enhance the transfection efficacy of TAT-based peptides, implying that the TAT peptide-based vectors demonstrated here have promising potential in gene delivery.     

Transfection efficiency boost of cholesterol-containing lipoplexes

Most lipid formulations require cholesterol for successful transfection, but the precise reason remains to be more clearly understood. Here, we have studied the effect of cholesterol on the transfection efficiency (TE) of lipoplexes in vitro. Addition of cholesterol to highly effective DC-Chol-DOPE/DNA lipoplexes increases TE, with 40mol% cholesterol yielding about 10-fold improvement. The transfection mechanisms of cholesterol-containing lipoplexes have been investigated by combining dynamic light scattering, synchrotron small angle X-ray scattering, laser scanning confocal microscopy and transfection efficiency measurements. Our results revealed that cholesterol-containing lipoplexes enter the cells partially by membrane fusion and this mechanism accounts for efficient endosomal escape. We also found evidence that formulations with high cholesterol content are not specifically targeted to metabolic degradation. These studies will contribute to rationally design novel delivery systems with superior transfection efficiency.     

Quantitative aspects of endocytic activity in lipid-mediated transfections.

Variation in transfection efficiency observed in different cell-types is poorly understood. To investigate the influence of endocytic activity on lipid-mediated transfections, we have monitored both the processes in 12 different cell-types. The endocytic activity shows a strong positive correlation (P < 0.01), with transfection efficiency. Treatment with wortmannin resulted in cell-type-dependent inhibition of transfection. Studies on M-phase cells by confocal microscopy show that compared to interphase cells, uptake of cationic liposomes was substantially reduced. In addition, transfection efficiency of cells in mitotic phase was inhibited by >70% compared to controls. Our study based on several cell-types demonstrates for the first time that quantitative aspects of endocytosis have decisive influence on the overall process of lipid-mediated transgene expression.     

Sequence-specific Methyltransferase-Induced Labelling (SMILing) of plasmid DNA for studying cell transfection

Plasmid DNA (pUC19 and pBR322) was sequence-specifically, covalently labelled with Cy3 fluorophores using a newly synthesised N-adenosylaziridine cofactor and the DNA methyltransferase M.TaqI. The fluorescently labelled plasmids were used for transfection of mammalian cells and their intracellular distribution was visualised by epifluorescence and confocal fluorescence microscopy. Although these prokaryotic plasmids do not contain nuclear import sequences, translocation into the nuclei was observed.     

Lipoplex formulation of superior efficacy exhibits high surface activity and fusogenicity, and readily releases DNA

Lipoplexes containing a mixture of cationic phospholipids dioleoylethylphosphatidylcholine (EDOPC) and dilauroylethylphosphatidylcholine (EDLPC) are known to be far more efficient agents in transfection of cultured primary endothelial cells than are lipoplexes containing either lipid alone. The large magnitude of the synergy permits comparison of the physical and physico-chemical properties of lipoplexes that have very different transfection efficiencies, but minor chemical differences. Here we report that the superior transfection efficiency of the EDLPC/EDOPC lipoplexes correlates with higher surface activity, higher affinity to interact and mix with negatively charged membrane-mimicking liposomes, and with considerably more efficient DNA release relative to the EDOPC lipoplexes. Observations on cultured cells agree with the results obtained with model systems; confocal microscopy of transfected human umbilical artery endothelial cells (HUAEC) demonstrated more extensive DNA release into the cytoplasm and nucleoplasm for the EDLPC/EDOPC lipoplexes than for EDOPC lipoplexes; electron microscopy of cells fixed and embedded directly on the culture dish revealed contact of EDLPC/EDOPC lipoplexes with various cellular membranes, including those of the endoplasmic reticulum, mitochondria and nucleus. The sequence of events outlining efficient lipofection is discussed based on the presented data.     

The impact of DNA topology on polyplex uptake and transfection efficiency in mammalian cells

The effect of DNA vector topology when complexed to poly-l-lysine (PLL) and its quantification in transfection efficiency has not been fully addressed even though it is thought to be of importance from both production and regulatory viewpoints. This study investigates and quantifies cell uptake followed by transfection efficiency of PLL:DNA complexes (polyplexes) in Chinese hamster ovary (CHO) cells and their dependence on DNA topology. PLL is known for its ability to condense DNA and serve as an effective gene delivery vehicle. Characterization of PLL conjugated to a 6.9 kb plasmid was carried out. Dual labeling of both the plasmid DNA (pDNA) and PLL enabled quantitative tracking of the complexed as well as dissociated elements, within the cell, and their dependence on DNA topology. Polyplex uptake was quantified by confocal microscopy and image analysis. Supercoiled (SC) pDNA when complexed with PLL, forms a polyplex with a mean diameter of 139.06 nm (±0.84% relative standard error [RSE]), whereas open circular (OC) and linear-pDNA counterparts displayed mean diameters of 305.54 (±3.2% RSE) and 841.5 nm (±7.2% RSE) respectively. Complexes containing SC-pDNA were also more resistant to nuclease attack than its topological counterparts. Confocal microscope images reveal how the PLL and DNA remain bound post transfection. Quantification studies revealed that by 1 h post transfection 61% of SC-pDNA polyplexes were identified to be associated with the nucleus, in comparison to OC- (24.3%) and linear-pDNA polyplexes (3.5%) respectively. SC-pDNA polyplexes displayed the greatest transfection efficiency of 41% which dwarfed that of linear-pDNA polyplexes of 18.6%. Collectively these findings emphasize the importance of pDNA topology when complexed with PLL for gene delivery with the SC-form being a key pre-requisite.