Alginate modified nanostructured calcium carbonate with enhanced delivery efficiency for gene and drug delivery

To improve the performance of nanostructured calcium carbonate in gene delivery, a hydrophilic polysaccharide, alginate, was added to calcium carbonate co-precipitation systems to form alginate/CaCO(3)/DNA nanoparticles. The size and ζ-potential of the nanoparticles were measured by a zetasizer. Due to the existence of alginate chains which retarded the growth of calcium carbonate based co-precipitates, the alginate/CaCO(3)/DNA nanoparticles exhibited a decreased size and enhanced stability in the aqueous solution. To evaluate the gene and drug co-delivery ability, doxorubicin hydrochloride (DOX), a water-soluble anticancer drug, was loaded in the nanoparticles to form alginate/CaCO(3)/DNA/DOX nanoparticles. The in vitro gene transfections mediated by different nanoparticles in 293 T cells and HeLa cells were carried out, using pGL3-Luc as a reporter plasmid. With an appropriate amount of alginate, the gene transfection efficiency of alginate modified nanoparticles could be significantly enhanced as compared with the nanoparticles without alginate modification for the gene delivery systems, as well as the gene and drug co-delivery systems. The study on in vitro cell inhibition effects showed that the cell viability decreased with increasing DOX amount loaded in alginate/CaCO(3)/DNA/DOX nanoparticles. The alginate modification is a useful strategy to improve the calcium carbonate co-precipitation technique for the preparation of gene and drug delivery systems, and the nanoparticles prepared in this study have promising applications in gene and drug delivery.     

Influence of the PsbA1/PsbA3, Ca(2+)/Sr(2+) and Cl(-)/Br(-) exchanges on the redox potential of the primary quinone Q(A) in Photosystem II from Thermosynechococcus elongatus as revealed by spectroelectrochemistry

Ca(2+) and Cl(-) ions are essential elements for the oxygen evolution activity of photosystem II (PSII). It has been demonstrated that these ions can be exchanged with Sr(2+) and Br(-), respectively, and that these ion exchanges modify the kinetics of some electron transfer reactions at the Mn?Ca cluster level (Ishida et al., J. Biol. Chem. 283 (2008) 13330-13340). It has been proposed from thermoluminescence experiments that the kinetic effects arise, at least in part, from a decrease in the free energy level of the Mn(4)Ca cluster in the S? state though some changes on the acceptor side were also observed. Therefore, in the present work, by using thin-layer cell spectroelectrochemistry, the effects of the Ca(2+)/Sr(2+) and Cl(-)/Br(-) exchanges on the redox potential of the primary quinone electron acceptor Q(A), E(m)(Q(A)/Q(A)(-)), were investigated. Since the previous studies on the Ca(2+)/Sr(2+) and Cl(-)/Br(-) exchanges were performed in PsbA3-containing PSII purified from the thermophilic cyanobacterium Thermosynechococcus elongatus, we first investigated the influences of the PsbA1/PsbA3 exchange on E(m)(Q(A)/Q(A)(-)). Here we show that i) the E(m)(Q(A)/Q(A)(-)) was up-shifted by ca. +38mV in PsbA3-PSII when compared to PsbA1-PSII and ii) the Ca(2+)/Sr(2+) exchange up-shifted the E(m)(Q(A)/Q(A)(-)) by ca. +27mV, whereas the Cl(-)/Br(-) exchange hardly influenced E(m)(Q(A)/Q(A)(-)). On the basis of the results of E(m)(Q(A)/Q(A)(-)) together with previous thermoluminescence measurements, the ion-exchange effects on the energetics in PSII are discussed.     

Gemini surfactant dimethylene-1,2-bis(tetradecyldimethylammonium bromide)-based gene vectors: a biophysical approach to transfection efficiency

Cationic liposomes have been proposed as biocompatible gene delivery vectors, able to overcome the barriers imposed by cell membranes. Besides lipids, other surfactant molecules have been successfully used in the composition of gene carriers. In the present work, we used a Gemini surfactant, represented by the general structure [C(14)H(29)(CH(3))(2)N(+)(CH(2))(2)N(+)(CH(3))(2)C(14)H(29)]2Br(-) and herein designated 14-2-14, to prepare cationic gene carriers, both as the sole component and in combination with neutral helper lipids, cholesterol and DOPE. The effectiveness of three Gemini-based formulations, namely neat 14-2-14, 14-2-14:Chol (1:1 molar ratio) and 14-2-14:Chol:DOPE (2:1:1 molar ratio), to mediate gene delivery was evaluated in DNA mixtures of +/- charge ratios ranging from 1/1 to 12/1. After ruling out cytotoxicity as responsible for the differences observed in the transfection competence, structural and physical properties of the vector were investigated, using several techniques. The size and surface charge density (zeta potential) of surfactant-based structures were determined by conventional techniques and the thermotropic behaviour of aqueous dispersions of surfactant/lipid/DNA formulations was monitored by fluorescence polarization of DPH and DPH-PA probes. The capacity of lipoplexes to interact with membrane-mimicking lipid bilayers was evaluated, using the PicoGreen assay and a FRET technique. Our data indicate inefficiency of the neat 14-2-14 formulation for gene delivery, which could result from the large dimensions of the particles and/or from its relative incompetence to release DNA upon interaction with anionic lipids. The addition of cholesterol or cholesterol and DOPE conferred to Gemini-based gene carrier transfection activity at specific ranges of +/- charge ratios. Fluorescence polarization data suggest that an order parameter within a specific range was apparently needed for complexes to display maximal transfection efficiency. The transfection-competent formulations showed to be efficiently destabilized by interaction with different anionic and zwitterionic bilayers, including those containing PS and cardiolipin. These data are discussed in terms of the potential of these formulations to address different intracellular targets.     

碳酸钙促进丙酮酸发酵过程中α-酮戊二酸的形成

在多重维生素营养缺陷型菌株光滑球拟酵母CCTCC M202019发酵生产丙酮酸的摇瓶和发酵罐实验中发现,CaCO₃的添加对发酵液中α-酮戊二酸（α-KG）的积累有重要影响。在维生素浓度不变且供氧充分的前提下,延迟CaCO₃添加时间可明显抑制α-KG的产生,并提高丙酮酸与α-KG的碳摩尔比(C_PYR/C_αKG);而增加培养基中的CaCO₃浓度会导致αKG积累的增加。用不同物质调节发酵液中pH的实验证实：在丙酮酸发酵过程中, Ca²⁺对αKG的积累起主要作用,CO₃^2-起辅助作用,两者对α-KG的积累具有协同效应。维持培养基中CaCO₃浓度不变,改变培养基中硫胺素的浓度,对αKG的积累,特别是对C_PYR/C_α-KG值没有影响;而增加培养基中生物素的浓度,则导致αKG的浓度不断上升且C_PYR/C_α-KG值不断下降。当有Ca2+存在时,胞内丙酮酸羧化酶的活性最高可提高40%,而丙酮酸脱氢酶系的活性没有明显变化。结果表明,丙酮酸发酵过程中α-KG的形成是由于CaCO₃促进了丙酮酸羧化反应,其中Ca²⁺可显著提高丙酮酸羧化酶的活性,而CO₃^2-则有可能作为丙酮酸羧化反应的底物。     

Enhanced cellular delivery and transfection efficiency of plasmid DNA using positively charged biocompatible colloidal gold nanoparticles

Efficient and safe nonviral gene delivery systems are a prerequisite for the clinical application of therapeutic genes. In this study, we report an enhancement of the transfection efficiency of plasmid DNA, via the use of positively charged colloidal gold nanoparticles (PGN). Plasmid DNA encoding for murine interleukin-2 (pVAXmIL-2) was complexed with PGN at a variety of ratios. The delivery of pVAXmIL-2 into C2C12 cells was dependent on the complexation ratios between PGN and the plasmid DNA, presented the highest delivery at a ratio of 2400:1. After complexation with DNA, PGN showed significantly higher cellular delivery and transfection efficiency than did the polyethylenimines (PEI) of different molecular weights, such as PEI25K (m.w. 25 kd) and PEI2K (m.w. 2 kd). PGN resulted in a cellular delivery of pVAXmIL-2 6.3-fold higher than was seen with PEI25K. The PGN/DNA complex resulted in 3.2- and 2.1-fold higher murine IL-2 protein expression than was seen in association with the PEI25K/DNA and PEI2K/DNA complexes, respectively. Following intramuscular administration, PGN/DNA complexes showed more than 4 orders of magnitude higher expression levels as compared to naked DNA. Moreover, the PGN/DNA complexes showed higher cell viability than other cationic nonviral vectors. Collectively, the results of this study suggest that the PGN/DNA complexes may harbor the potential for development into efficient and safe gene delivery vehicles.     

Intestinal bicarbonate secretion in marine teleost fish-source of bicarbonate, pH sensitivity, and consequences for whole animal acid-base and calcium homeostasis

Whole animal studies using seawater European flounder (Platichthys flesus) revealed that increasing intestinal [Ca(2+)] to 20 mM stimulated net HCO(3)(-) base secretion by 57%, but this was effectively balanced by an increase in net acid secretion, likely from the gills, to maintain whole animal acid-base status. Higher Ca(2+) concentrations (40 and 70 mM) in ambient seawater resulted in reduced plasma total CO(2). This indicates (1) imperfect acid-base compensation, and (2) that endogenous metabolic CO(2) is insufficient to fuel intestinal HCO(3)(-) secretion, under hyper-stimulated conditions. Bicarbonate secretion plays an important role in preventing calcium absorption by precipitating a large fraction of the imbibed calcium as CaCO(3). Indeed, under high Ca(2+) conditions (20 mM), up to 75% of the intestinal Ca(2+) is precipitated as CaCO(3) and then excreted. This is undoubtedly important in protecting the marine teleost kidney from the need for excessive calcium excretion and risk of renal stone formation. Using an in vitro pH-stat technique with the isolated intestinal epithelium, the replacement of serosal CO(2) with a HEPES buffered saline had no effect on HCO(3)(-) secretion, indicating that the endogenous supply of HCO(3)(-) from CO(2) hydration within epithelial cells is adequate for driving baseline secretion rates. Further, in vitro data demonstrated a stimulatory effect of low pH on intestinal HCO(3)(-) secretion. Thus, both luminal Ca(2+) and H(+) can regulate HCO(3)(-) secretion but the precise mechanisms and their potential interaction are currently unresolved.     

Calcium enhances the transfection potency of plasmid DNA-cationic liposome complexes

It is shown that calcium increases the in vitro transfection potency of plasmid DNA-cationic liposome complexes from 3- to 20-fold. The effect is Ca(2+) specific as other cations, such as Mg(2+) and Na(+), do not give rise to enhanced transfection and the effect can be inhibited by the presence of EGTA. It is shown that Ca(2+) increases cellular uptake of the DNA-lipid complexes, indicating that increased transfection potency arises from increased intracellular delivery of both cationic lipid and plasmid DNA in the presence of Ca(2+). In particular, it is shown that the levels of intact intracellular plasmid DNA are significantly enhanced when Ca(2+) is present. The generality of the Ca(2+) effect for enhancing complex-mediated transfection is demonstrated for a number of different cell lines and different cationic lipid formulations. It is concluded that addition of Ca(2+) represents a simple and useful protocol for enhancing in vitro transfection properties of plasmid DNA-cationic lipid complexes.     

Calcium enhances the transfection potency of stabilized plasmid-lipid particles

Previous work from this laboratory has shown that plasmid DNA can be encapsulated in small (70-nm-diameter) stabilized plasmid-lipid particles (SPLP) that consist of a single plasmid encapsulated within a bilayer lipid vesicle. SPLP preferentially transfect tumor tissue following intravenous administration. Although the levels of transgene expression in vivo are greater for SPLP than can be achieved with naked DNA or complexes, they are lower than may be required for therapeutic benefit. In the present work we examine whether Ca2+ can enhance the transfection potency of SPLP. It is shown that Ca2+ can enhance SPLP transfection potency in bovine hamster kidney cells by 60- to 100-fold when treated in serum containing medium and an additional 60-fold when serum is absent for the initial 10 min of the transfection period. When cells are treated with SPLP in the presence of Ca2+, there is a fivefold increase in intact plasmid in the cell. It is also shown that this Ca2+ effect involves the formation of calcium phosphate precipitates; however, these precipitates are not directly associated with the SPLP plasmid DNA. The ability of calcium phosphate to facilitate delivery of other macromolecules without direct association is also demonstrated by the release of large-molecular-weight dextrans from endosomal/lysosomal compartments in the presence of calcium phosphate. Finally, it is shown that, unlike naked DNA, SPLP transfection potency in the presence of calcium phosphate is not affected by nuclease activity.     

Low molecular weight polyethylenimine for efficient transfection of human hematopoietic and umbilical cord blood-derived CD34+ cells

With the emerging role of hematopoietic stem cells as potential gene and cell therapy vehicles, there is an increasing need for safe and effective nonviral gene delivery systems. Here, we report that gene transfer and transfection efficiency in human hematopoietic and cord blood CD34+ cells can be enhanced by the use of low molecular weight polyethylenimine (PEI). PEIs of various molecular weights (800-750,000) were tested, and our results showed that the uptake of plasmid DNA by hematopoietic TF-1 cells depended on the molecular weights and the N/P ratios. Treatment with PEI 2K (m.w. 2000) at an N/P ratio of 80/1 was most effective, increasing the uptake of plasmid DNA in TF-1 cells by 23-fold relative to Lipofectamine 2000. PEI 2K-enhanced transfection was similarly observed in hematopoietic K562, murine Sca-1+, and human cord blood CD34+ cells. Notably, in human CD34+ cells, a model gene transferred with PEI 2K showed 21,043- and 513-fold higher mRNA expression levels relative to the same construct transfected without PEI or with PEI 25 K, respectively. Moreover, PEI 2K-treated TF-1 and human CD34+ cells retained good viability. Collectively, these results indicate that PEI 2K at the optimal N/P ratio might be used to safely enhance gene delivery and transfection of hematopoietic and human CD34+ stem cells.     

pcDNA3．1表达载体转染对细胞生长的影响

为了探讨真核表达载体转染对细胞生长的影响,通过脂质体介导将pcDNA3.1( )表达载体DNA转染鼻咽癌细胞系HNE1,G418筛选后,Southern杂交鉴定稳定表达细胞株,以HNE1细胞为对照,观察pcDNA3.1( ）／HNE1克隆细胞的生物学特性;结果显示,在pcDNA3.1( )/HNE1阳性克隆中,一株细胞克隆培养过程中发生自溶性死亡,一株细胞生长明显受到抑制,另一株细胞生长无明显影响,揭示在宿主细胞中pcDNA3.1( )DNA与宿主基因组DNA发生了随机整合,从而表现不同的细胞生物学改变。     

Preparation and in vitro evaluation of novel lipopeptide transfection agents for efficient gene delivery

Gene therapy by delivery of nonviral expression vectors is highly desirable, due to their safety, stability, and suitability for production as bulk pharmaceuticals. However, low transfection efficiency remains a limiting factor in application on nonviral gene delivery. Despite recent advances in the field, there are still major obstacles to overcome. In an attempt to construct more efficient nonviral gene delivery vectors, we have designed a series of novel lipopeptide transfection agents, consisting of an alkyl chain, one cysteine, 1 to 4 histidine and 1 to 3 lysine residues. The lipopeptides were designed to facilitate dimerization (by way of the cysteine residues), DNA binding at neutral pH (making use of charged lysine residues), and endosomal escape (by way of weakly basic histidine residues). DNA/lipopeptide complexes were evaluated for their biophysical properties and transfection efficiencies. The number and identity of amino acids incorporated in the lipopeptide construct affected their DNA/lipopeptide complex forming capacity. As the number of lysine residues in the lipopeptide increased, the DNA complexes formed became more stable, had higher zeta potential (particle surface charge), and produced smaller mean particle sizes (typically 110 nm at a charge ratio of 5.0 and 240 nm at a charge ratio of 1.0). The effect of inclusion of histidines in the lipopeptide moiety had the opposite effect on complex formation to lysine, but was necessary for high transfection efficiency. In vitro transfection studies in COS-7 cells revealed that the efficiency of gene delivery of the luciferase encoding plasmid, pCMV-Luc, mediated by all the lipopeptides, was much higher than poly(L-lysine) (PLL), which has no endosomal escape system, and in two cases was slightly higher than that of branched polyethylenimine (PEI). Lipopeptides with at least two lysine residues and at least one histidine residue produced spontaneous transfection complexes with plasmid DNA, indicating that endosomal escape was achieved by incorporation of histidine residues. These low molecular weight peptides can be readily synthesized and purified and offer new insights into the mechanism of action of transfection complexes.     

Voltage-independent changes in L-type Ca(2+) current uncoupled from SR Ca(2+) release in cardiac myocytes

To determine the effect of voltage-independent alterations of L-type Ca(2+) current (I(Ca)) on the sarcoplasmic reticular (SR) Ca(2+) release in cardiac myocytes, we measured I(Ca) and cytosolic Ca(2+) transients (Ca(i)(2+); intracellular Ca(2+) concentration) in voltage-clamped rat ventricular myocytes during 1) an abrupt increase of extracellular [Ca(2+)] (Ca(o)(2+)) or 2) application of 1 microM FPL-64176, a Ca(2+) channel agonist, to selectively alter I(Ca) in the absence of changes in SR Ca(2+) loading. On the first depolarization in higher Ca(o)(2+), peak I(Ca) was increased by 46 +/- 6% (P < 0.001), but the increases in the maximal rate of rise of Ca(i)(2+) (dCa(i)(2+)/dt(max), where t is time; an index of SR Ca(2+) release flux) and the Ca(i)(2+) transient amplitude were not significant. Rapid exposure to FPL-64176 greatly slowed inactivation of I(Ca), increasing its time integral by 117 +/- 8% (P < 0.001) without significantly increasing peak I(Ca), dCa(i)(2+)/dt(max), or amplitude of the corresponding Ca(i)(2+) transient. Prolongation of exposure to higher Ca(o)(2+) or FPL-64176 did not further increase peak I(Ca) but greatly increased dCa(i)(2+)/dt(max), Ca(i)(2+) transient amplitude, and the gain of Ca(2+) release (dCa(i)(2+)/dt(max)/I(Ca)), evidently due to augmentation of the SR Ca(2+) loading. Also, the time to peak dCa(i)(2+)/dt(max) was significantly increased in the continuous presence of higher Ca(o)(2+) (by 37 +/- 5%, P < 0.001) or FPL-64176 (by 63 +/- 5%, P < 0.002). Our experiments provide the first evidence of a marked disparity between an increased peak I(Ca) and the corresponding SR Ca(2+) release. We attribute this to saturation of the SR Ca(2+) release flux as predicted by local control theory. Prolongation of the SR Ca(2+) release flux, caused by combined actions of a larger I(Ca) and maximally augmented SR Ca(2+) loading, might reflect additional Ca(2+) release from corbular SR.     

Mechanism of cell transfection with plasmid/chitosan complexes

Chitosan is useful as a non-viral vector for gene delivery. Although there are several reports supporting the use of chitosan for gene delivery, studies regarding effects on transfection and the chitosan-specific transfection mechanism remain insufficient. In this report, the level of expression with plasmid/chitosan was observed to be no less than that with plasmid/lipofectin complexes in SOJ cells. The transfection mechanism of plasmid/chitosan complexes as well as the relationship between transfection activity and cell uptake was analyzed by using fluorescein isothiocyanate-labeled plasmid and Texas Red-labeled chitosan. In regard to effects on transfection, there were several factors to affect transfection activity and cell uptake, for example: the molecular mass of chitosan, stoichiometry of complex, as well as serum concentration and pH of transfection medium. The level of transfection with plasmid/chitosan complexes was found to be highest when the molecular mass of chitosan was 40 or 84 kDa, ratio of chitosan nitrogen to DNA phosphate (N/P ratio) was 5, and transfection medium contained 10% serum at pH 7.0. We also investigated the transfection mechanism, and found that plasmid/chitosan complexes most likely condense to form large aggregates (5-8 microm), which absorb to the cell surface. After this, plasmid/chitosan complexes are endocytosed, and possibly released from endosomes due to swelling of lysosomal in addition to swelling of plasmid/chitosan complex, causing the endosome to rupture. Finally, complexes were also observed to accumulate in the nucleus using a confocal laser scanning microscope.     

Enhancement of transfection by physical concentration of DNA at the cell surface

Efficient DNA transfection is critical for biological research and new clinical therapies, but the mechanisms responsible for DNA uptake are unknown. Current nonviral transfection methods, empirically designed to maximize DNA complexation and/or membrane fusion, are amenable to enhancement by a variety of chemicals. These chemicals include particulates, lipids, and polymer complexes that optimize DNA complexation/condensation, membrane fusion, endosomal release, or nuclear targeting, which are the presumed barriers to gene delivery. Most chemical enhancements produce a moderate increase in gene delivery and a limited increase in gene expression. As a result, the efficiency of transfection and level of gene expression after nonviral DNA delivery remain low, suggesting the existence of additional unidentified barriers. Here, we tested the hypothesis that DNA transfection efficiency is limited by a simple physical barrier: low DNA concentration at the cell surface. We used dense silica nanoparticles to concentrate DNA-vector (i.e. DNA-transfection reagent) complexes at the surface of cell monolayers; manipulations that increased complex concentration at the cell surface enhanced transfection efficiency by up to 8.5-fold over the best commercially available transfection reagents. We predict that manipulations aimed at optimizing DNA complexation or membrane fusion have a fundamental physical limit; new methods designed to increase transfection efficiency must increase DNA concentration at the target cell surface without adding to the toxicity.     

Structural characterization and buffering capacity in relation to the transfection efficiency of biodegradable polyurethane

Inefficient release of polymer/DNA complexes from endocytic vesicles into the cytoplasm and the cytotoxic nature of cationic polymers are two of the primary causes of poor gene delivery. EG-polyurethane [poly(ethylene glycol)-PU, Poly 1], EGDM-polyurethane [poly(ethylene glycol), 2-(dimethylamino)ethylamine-PU, Poly 2], and MDEADM-polyurethane [N-methyldiethanolamine, 2-(dimethylamino)ethylamine-PU, Poly 3] were designed in this study to overcome these obstacles. The structural characteristics of polyurethanes and physicochemical properties of their formed complexes with DNA were determined to correlate their transfection efficiency. The results revealed that Poly 2 and Poly 3 could bind with plasmid DNA and yield positively charged complexes with a size required for transfection. Poly 3 showed the best in buffering capacity and its formed complexes with DNA could transfect COS-7 cells better than those of Poly 2 and Poly 1. This study reveals that the amine groups in the polymeric structure and the buffer capacity of a polymeric transfectant would affect its potential in DNA delivery. Also the size and binding properties of DNA and polymeric transfectants can be in correlation to the transfection efficiency of resulting DNA/polymer complexes.     

Characterization of cationic lipid DNA transfection complexes differing in susceptability to serum inhibition

Background

Cationic lipid DNA complexes based on DOTAP (1,2-dioleoyl-3-(trimethyammonium) propane) and mixtures of DOTAP and cholesterol (DC) have been previously optimized for transfection efficiency in the absence of serum and used as a non-viral gene delivery system. To determine whether DOTAP and DC lipid DNA complexes could be obtained with increased transfection effciency in the presence of high serum concentrations, the composition of the complexes was varied systematically and a total of 162 different complexes were analyzed for transfection efficiency in the presence and absence of high serum concentrations.

Results

Increasing the ratio of DOTAP or DC to DNA led to a dose dependent enhancement of transfection efficiency in the presence of high serum concentrations up to a ratio of approximately 128 nmol lipid/μg DNA. Transfection efficiency could be further increased for all ratios of DOTAP and DC to DNA by addition of the DNA condensing agent protamine sulfate (PS). For DOTAP DNA complexes with ratios of ≤ 32 nmol/μg DNA, peak transfection efficiencies were obtained with 4 μg PS/μg DNA. In contrast, increasing the amount of PS of DC complexes above 0.5 μg PS /μg DNA did not lead to significant further increases in transfection efficiency in the presence of high serum concentrations. Four complexes, which had a similar high transfection efficiency in cell culture in the presence of low serum concentrations but which differed largely in the lipid to DNA ratio and the amount of PS were selected for further analysis. Intravenous injection of the selected complexes led to 22-fold differences in transduction efficiency, which correlated with transfection efficiency in the presence of high serum concentrations. The complex with the highest transfection efficiency in vivo consisted of 64 nmol DC/ 16 μg PS/ μg DNA. Physical analysis revealed a predicted size of 440 nm and the highest zeta potential of the complexes analyzed.

Conclusions

Optimization of cationic lipid DNA complexes for transfection efficiency in the presence of high concentrations of serum led to the identification of a DC complex with high transduction efficiency in mice. This complex differs from previously described ones by higher lipid to DNA and PS to DNA ratios. The stability of this complex in the presence of high concentrations of serum and its high transduction efficiency in mice suggests that it is a promising candidate vehicle for in vivo gene delivery.     

(LYS)(16)-based reducible polycations provide stable polyplexes with anionic fusogenic peptides and efficient gene delivery to post mitotic cells

Extracellular stability, endocytic escape, intracellular DNA release and nuclear translocation of DNA are all critical properties of non-viral vector/DNA particles. We have evaluated a (Lys)(16)-based linear, reducible polycation (RPC) in combination with an acid-dependent, anionic fusogenic peptide for gene delivery to dividing and post-mitotic cells. The RPC was formed from Cys(Lys)(16)Cys monomers. Molecular weight was 24,000 Da, corresponding to an average of 10.5 peptide monomers per RPC. Non-reducible polylysine (PLL) (27,000 Da) and monomeric (Lys)(16) peptide were evaluated for comparison. (Lys)(16)/DNA particles were disrupted at fusogenic peptide concentrations well below those used for gene delivery. By contrast, RPC/DNA an PLL/DNA particles were stable in the presence of high concentrations of the anionic peptide. Addition of 10% serum virtually abolished the transfection ability of (Lys)(16)/DNA/fusogenic peptide particles, but had little effect on RPC/DNA/fusogenic peptide particles. RPC/DNA/fusogenic peptide particles were highly effective for gene delivery to both cell lines and post-mitotic corneal endothelium. PLL/DNA/fusogenic peptide particles were moderately effective on cell lines, but gave no gene delivery with corneal endothelial cells. We conclude that (Lys)(16)-based RPC/DNA/fusogenic peptide particles provide a gene delivery system which is potentially stable in the extracellular environment and, on reductive depolymerisation, can release DNA plasmids for nuclear translocation.     

Bead transfection of adherent cells

Bead transfection is a simple, rapid, efficient, and cost-effective method of gene transfer into adherent mammalian cells. It involves a brief incubation of the cells with glass beads in a solution containing the DNA to be transferred. We have optimized this technique using COS-7 (an SV40 transformed monkey kidney cell line) and a transient expression assay for chloramphenicol acetyl transferase (CAT). Stable transfection efficiency assessed using the selectable marker gene neomycin phosphotransferase (NEO^R) was 27% in COS-7 cells. As this technique delivers high transfection efficiency with little manipulation of the exogenous DNA and does not require the use of any viral sequences, it may be a useful alternative method of gene delivery in the development of gene therapy protocols.