(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.     

Exploration of peptide motifs for potent non-viral gene delivery highly selective for dividing cells

BACKGROUND: The immunogenicity of viral DNA vectors is an important problem for gene therapy. The use of peptide motifs for gene delivery would largely overcome this problem, and provide a simple, safe and powerful approach for non-viral gene therapy. METHODS: We explored the functional properties of two motifs: the (Lys)(16) motif (for binding and condensing DNA, and probably also nuclear translocation of plasmids) and the fusogenic peptide motif of influenza virus (for acid-dependent endocytic escape of peptide/DNA particles). The physical properties and gene delivery efficiencies of (Lys)(16)-containing peptides in combination with free fusogenic peptide were evaluated, and compared with a single composite peptide incorporating both moieties. Post-mitotic corneal endothelial cells and growth-arrested HeLa were included, so as not to neglect the question of nuclear translocation of plasmids. RESULTS: The fusogenic moiety in the composite peptide was able to adopt an alpha-helical configuration unhindered by the (Lys)(16) moiety, and retained acid-dependent fusogenic properties. The composite peptide gave remarkably high levels of gene delivery to dividing cell lines. However, in marked contrast to (Lys)(16)/DNA complexes plus free fusogenic peptide, the composite peptide was completely ineffective for gene delivery to post-mitotic and growth-arrested cells. CONCLUSIONS: Attachment of the fusogenic peptide to (Lys)(16) appears to block (Lys)(16)-mediated nuclear translocation of plasmid, but not fusogenic peptide mediated endocytic escape. This strengthens the experimental basis for (Lys)(16)-mediated nuclear translocation of plasmids, and provides a single peptide with potent gene delivery properties, restricted to dividing cells. This property is potentially useful in experimental biology and clinical medicine.     

Regional hydrodynamic gene delivery to the rat liver with physiological volumes of DNA solution

BACKGROUND: The major barrier to the clinical application of hydrodynamic gene delivery to the liver is the large volume of fluid required using standard protocols. Regional hydrodynamic gene delivery via branches of the portal vein has not previously been reported, and we have evaluated this approach in a rat model. METHODS: The pGL3 plasmid with the luciferase reporter gene was used at 50 micro g/ml in isotonic solutions, and was administered with a syringe pump for precise control of the hydrodynamic conditions evaluated. Gene expression was individually measured in six anatomically distinct liver lobes. The effect of systemic chloroquine to promote endocytic escape and a (Lys)(16)-containing peptide to condense the DNA into approximately 100-nm nanoparticles was also evaluated. RESULTS: Hydrodynamic conditions for excellent gene delivery were obtained by using 3-ml volumes ( approximately 12 ml/kg) of isotonic DNA solution delivered at 24 ml/min to the right lateral lobe ( approximately 20% of the liver mass). Under these conditions, >95% of gene delivery usually occurred in the targeted right lateral lobe. Outflow obstruction was essential for gene delivery, both at optimal and at very low levels of hydrodynamic gene delivery. The use of systemic chloroquine to promote endocytic escape did not augment hydrodynamic gene delivery, while condensation of DNA in non-ionic isotonic solutions (5% dextrose) to nanoparticles of approximately 100 nm completely abolished gene delivery. CONCLUSIONS: Regional hydrodynamic gene delivery via a branch of the portal vein offers a physiological model of liver gene therapy, for experimental and clinical application.     

A smart nanoassembly consisting of acid-labile vinyl ether PEG-DOPE and protamine for gene delivery: preparation and in vitro transfection

The conception of a modular designed and viruslike nonviral vector has been presented for gene delivery. Recently, we constructed a new smart nanoassembly (SNA) with multifunctional components that was composed of a condensed core of pDNA with protamine sulfate (PS) and a dioleoyl phosphatidylethanolamine (DOPE)-based lipid envelope containing poly(ethylene glycol)--vinyl ether--DOPE (PVD). SNAs with mPEG 2000 (SNAs1) or mPEG 5000 (SNAs2) loading PS/DNA were prepared by the lipid film hydration technique. The particle size was about 160 nm for SNAs1 and 240 nm for SNAs2 loading PS/DNA (10:1 w/w), and the zeta potential was about 4 mV for two SNAs. The in vitro release experiment indicated that PVD possessed a good ability for self-dePEGylation, which could result in the recovery of an excellent fusogenic capacity of DOPE at low pH. SNAs showed a higher transfection efficiency and much lower cytotoxicity than did Lipofectamine 2000 on HEK 293, HeLa, and COS-7 cells. The cellular uptake and subcellular localization demonstrated that the superior transfection efficiency of SNAs could result from the fact that the DOPE-based lipid envelope containing PVD increased PS/DNA in the cytoplasm, and protamine enhanced the nuclear delivery or overcame the nuclear membrane barrier. These results implied that the PVD-based nanoassembly loading PS/DNA could be a promising gene delivery system.     

Targeting alphavbeta3 and alpha5beta1 for gene delivery to proliferating VSMCs: synergistic effect of TGF-beta1

TGF-beta1 levels increase after vascular injury and promote vascular smooth muscle cell (VSMC) proliferation. We define a nonviral gene delivery system that targets alphavbeta3 and alpha5beta1 integrins that are expressed on proliferating VSMCs and strongly induced by TGF-beta1. A 15-amino acid RGDNP-containing peptide from American Pit Viper venom was linked to a Lys(16) peptide as vector (molossin vector) and complexed with Lipofectamine or fusogenic peptide for delivery of luciferase or beta-galactosidase reporter genes to primary cultures of human, rabbit, and rat VSMCs. Preincubation of VSMCs with TGF-beta1 for 24 h, but not with PDGF-BB, interferon-gamma, TNF-alpha, nor PMA, increased alphavbeta3 and alpha5beta1 expressions on VSMCs and enhanced gene delivery of molossin vector. Thus beta-galactosidase activity increased from 35 +/- 5% (controls) to 75 +/- 5% after TGF-beta1 treatment, and luciferase activity increased fourfold over control values. Potential use of this system in vessel bypass surgery was examined in an ex vivo rat aortic organ culture model after endothelial damage. Molossin vector system delivered beta-galactosidase to VSMCs in the vessel wall that remained for up to 12 days posttransfection. The molossin vector system, when combined with TGF-beta1, enhances gene delivery to proliferating VSMCs and might have clinical applications for certain vasculoproliferative diseases.     

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.     

Bio‐electrospraying: A potentially safe technique for delivering progenitor cells

Bio‐electrospraying is fast becoming an attractive tool for in situ cell delivery into scaffolds for tissue engineering applications, with several cell types been successfully electrosprayed. Bone marrow derived mesenchymal progenitor/stem cells (BMSC), which are an important cell source for tissue engineering, have not been explored in detail and the effect of electrospraying on their “stemness” is not known. This study therefore investigates the effects of electrospraying on BMSC viability, proliferation, and multilineage differentiation potential. Electrospraying a BMSC suspension at flow rate of 6 mL/h and voltages of 7.5–15 kV could successfully generate a continuous, stable and linearly directed electrospray of cells. Morphological observation, trypan blue tests and alamar blue based metabolic assays revealed about 88% of these electrosprayed cells were viable, and proliferated at rates similar to native BMSCs. However, at higher voltages, electrospraying became unstable and reduced cell viability, possibly due to electrical or thermal damage to the cells. BMSCs electrosprayed at 7.5 kV also retained their multipotency and could be successfully differentiated into adipogenic, chondrogenic, and osteogenic lineages, demonstrating similar morphology and gene expression levels as induced native BMSCs. These results indicate that bio‐electrospraying could be safely used as a progenitor/stem cell delivery technique for tissue engineering and regenerative medicine applications. Biotechnol. Bioeng. 2010;106: 690–698. © 2010 Wiley Periodicals, Inc.     

Poly(β-amino ester)-nanoparticle mediated transfection of retinal pigment epithelial cells in vitro and in vivo

A variety of genetic diseases in the retina, including retinitis pigmentosa and leber congenital amaurosis, might be excellent targets for gene delivery as treatment. A major challenge in non-viral gene delivery remains finding a safe and effective delivery system. Poly(beta-amino ester)s (PBAEs) have shown great potential as gene delivery reagents because they are easily synthesized and they transfect a wide variety of cell types with high efficacy in vitro. We synthesized a combinatorial library of PBAEs and evaluated them for transfection efficacy and toxicity in retinal pigment epithelial (ARPE-19) cells to identify lead polymer structures and transfection formulations. Our optimal polymer (B5-S5-E7 at 60 w/w polymer:DNA ratio) transfected ARPE-19 cells with 44±5% transfection efficacy, significantly higher than with optimized formulations of leading commercially available reagents Lipofectamine 2000 (26±7%) and X-tremeGENE HP DNA (22±6%); (p<0.001 for both). Ten formulations exceeded 30% transfection efficacy. This high non-viral efficacy was achieved with comparable cytotoxicity (23±6%) to controls; optimized formulations of Lipofectamine 2000 and X-tremeGENE HP DNA showed 15±3% and 32±9% toxicity respectively (p>0.05 for both). Our optimal polymer was also significantly better than a gold standard polymeric transfection reagent, branched 25 kDa polyethyleneimine (PEI), which achieved only 8±1% transfection efficacy with 25±6% cytotoxicity. Subretinal injections using lyophilized GFP-PBAE nanoparticles resulted in 1.1±1×10(3)-fold and 1.5±0.7×10(3)-fold increased GFP expression in the retinal pigment epithelium (RPE)/choroid and neural retina respectively, compared to injection of DNA alone (p?=?0.003 for RPE/choroid, p<0.001 for neural retina). The successful transfection of the RPE in vivo suggests that these nanoparticles could be used to study a number of genetic diseases in the laboratory with the potential to treat debilitating eye diseases.     

The polar region consecutive to the HIV fusion peptide participates in membrane fusion

The fusion peptide of HIV-1 gp41 is formed by the 16 N-terminal residues of the protein. This 16-amino acid peptide, in common with several other viral fusion peptides, caused a reduction in the bilayer to hexagonal phase transition temperature of dipalmitoleoylphosphatidylethanolamine (T(H)), suggesting its ability to promote negative curvature in membranes. Surprisingly, an elongated peptide corresponding to the 33 N-terminal amino acids raised T(H), although it was more potent than the 16-amino acid fusion peptide in inducing lipid mixing with large unilamellar liposomes of 1:1:1 dioleoylphosphatidylethanolamine/dioleoylphosphatidylcholine/choleste rol. The 17-amino acid C-terminal fragment of the peptide can induce membrane fusion by itself, if it is anchored to a membrane by palmitoylation of the amino terminus, indicating that the additional 17 hydrophilic amino acids contribute to the fusogenic potency of the peptide. This is not solely a consequence of the palmitoylation, as a random peptide with the same amino acid composition with a palmitoyl anchor was less potent in promoting membrane fusion and palmitic acid itself had no fusogenic activity. The 16-amino acid N-terminal fusion peptide and the longer 33-amino acid peptide were labeled with NBD. Fluorescence binding studies indicate that both peptides bind to the membrane with similar affinities, indicating that the increased fusogenic activity of the longer peptide was not a consequence of a greater extent of membrane partitioning. We also determined the secondary structure of the peptides using FTIR spectroscopy. We find that the amino-terminal fusion peptide is inserted into the membrane as a beta-sheet and the 17 C-terminal amino acids lie on the surface of the membrane, adopting an alpha-helical conformation. It was further demonstrated with the use of rhodamine-labeled peptides that the 33-amino acid peptide self-associated in the membrane while the 16-amino acid N-terminal peptide did not. Thus, the 16-amino acid N-terminal fusion peptide of HIV inserts into the membrane and, like other viral fusion peptides, lowers T(H). In addition, the 17 consecutive amino acids enhance the fusogenic activity of the fusion peptide presumably by promoting its self-association.     

Specific gene transfer mediated by lactosylated poly-L-lysine into hepatoma cells.

Plasmid DNA/glycosylated polylysine complexes were used to transfer in vitro a luciferase reporter gene into human hepatoma cells by a receptor-mediated endocytosis process. HepG2 cells which express a galactose specific membrane lectin were efficiently and selectively transfected with pSV2Luc/lactosylated polylysine complexes in a sugar dependent manner: i) HepG2 cells which do not express membrane lectin specific for mannose were quite poorly transfected with pSV2Luc/mannosylated polylysine complexes, ii) HeLa cells which do not express membrane lectin specific for galactose were not transfected with pSV2Luc/lactosylated polylysine complexes. The transfection efficiency of HepG2 cells with pSV2Luc/lactosylated polylysine complexes was greatly enhanced either in the presence of chloroquine or in the presence of a fusogenic peptide. A 22-residue peptide derived from the influenza virus hemagglutinin HA2 N-terminal polypeptide that mimics the fusogenic activity of the virus, was selected. In the presence of the fusogenic peptide, the luciferase activity in HepG2 cells was 10 fold larger than that of cells transfected with pSV2Luc/lactosylated polylysine complexes in the presence of chloroquine.     

Imaging in solution of (Lys)(16)-containing bifunctional synthetic peptide/DNA nanoparticles for gene delivery

The physical properties of non-viral vector/DNA nanoparticles in physiological aqueous solution are poorly understood. A Fluid Particle Image Analyser (FPIA), normally used for analysis of industrial and environmental fluids, was used to visualise individual (Lys)(16)-containing peptide/DNA particles. Eight (Lys)(16)-containing synthetic peptides were used to generate peptide/DNA particles at a constant + to - charge ratio of 2.8:1 with 10 microg/ml of plasmid DNA in phosphate buffered saline. Dynamic Light Scattering (DLS) and gene delivery studies were also performed. We present the first images of non-viral vector/DNA nanoparticles in physiological aqueous solution, together with precise measurements of individual particle size and shape in solution and, for the first time, an accurate measure of particle number. Particle size and shape, particle number, and efficiency for gene delivery varied markedly with different peptides. Under standard conditions for in vitro gene delivery, we estimate approximately 60 peptide/DNA nanoparticles per target cell, each containing approximately 70,000 plasmids. This novel capacity to image individual vector/DNA nanoparticles in solution and to count them accurately will enable a more precise assessment of non-viral gene delivery systems, and a more quantitative interpretation of gene delivery experiments.     

Low molecular weight disulfide cross-linking peptides as nonviral gene delivery carriers

Cross-linking peptides have been developed by inserting multiple Cys residues into a 20 amino acid condensing peptide that polymerizes through disulfide bond formation when bound to DNA resulting in small, highly stable DNA condensates that mediate efficient in vitro gene transfer [McKenzie et al. (2000) J. Biol. Chem. 275, 9970-9977]. In the present study, a minimal peptide of four Lys and two terminal Cys residues was found to substitute for Cys-Trp-(Lys)(17)-Cys, resulting in DNA condensates with similar particle size and gene expression in HepG2 cells. Substitution of His for Lys residues resulted in an optimal peptide of Cys-His-(Lys)(6)-His-Cys that, in addition to the attributes described above, also provided buffering capacity to enhance in vitro gene expression in the absence of chloroquine. The reported structure-activity relationships systematically explore peptides with combinations of Lys, Cys, and His residues resulting in low molecular weight peptides with improved gene transfer properties.     

Transient non-viral cutaneous gene delivery in burn wounds

BACKGROUND: Gene transfer to burn wounds could present an alternative to conventional and often insufficient topical and systemic application of therapeutic agents to aid in wound healing. The goals of this study were to assess and optimize the potential of transient non-viral gene delivery to burn wounds. METHODS: HaCaT cells were transfected with luciferase or beta-galactosidase transgene using either pure plasmid DNA (pDNA) or complexed with Lipofectamine 2000, FuGENE6, or DOTAP-Chol. Expression was determined by bioluminescence and fluorescence. Forty male Sprague-Dawley rats received naked pDNA, lipoplexes, or carrier control intradermally into either unburned skin, superficial, partial, or full-thickness scald burn. Animals were sacrificed after 24 h, 48 h, or 7 days, and transgene expression was assessed. RESULTS: Gene transfer to HaCaT cells showed the overall highest expression for DOTAP/Chol (77.85 ng luciferase/mg protein), followed by Lipofectamine 2000 (33.14 ng luciferase/mg protein). pDNA-derived gene transfer to superficial burn wounds showed the highest expression among burn groups (0.77 ng luciferase/mg protein). However, lipoplex-derived gene transfer to superficial burns and unburned skin failed to show higher expression. CONCLUSIONS: Lipofectamine 2000 and DOTAP/Chol lipoplex showed significantly enhanced gene transfer, whereas no transfection was detectable for naked DNA in vitro. In contrast to the in vitro study, naked DNA was the only agent with which gene delivery was successful in experimental burn wounds. These findings highlight the limited predictability of in vitro analysis for gene delivery as a therapeutic approach.     

Epigenetic states of donor cells significantly affect the development of somatic cell nuclear transfer (SCNT) embryos in pigs

The type and pattern of epigenetic modification in donor cells can significantly affect the developmental competency of somatic cell nuclear transfer (SCNT) embryos. Here, we investigated the developmental capacity, gene expression, and epigenetic modifications of SCNT embryos derived from porcine bone marrow‐derived mesenchymal stem cells (BMSCs) and fetal fibroblasts (FFs) donor cells compared to embryos obtained from in vitro fertilization (IVF). Compared to FFs, the donor BMSCs had more active epigenetic markers (Histone H3 modifications: H3K9Ac, H3K4me3, and H3K4me2) and fewer repressive epigenetic markers (H3K9me3, H3K9me2, and DNA methyltransferase 1). Embryos derived from BMSC nuclear‐transfer (BMSC‐NT embryos) and IVF embryos had significantly higher cleavage and blastocyst rates (BMSC‐NT: 71.3 ± 3.4%, 29.1 ± 2.3%; IVF: 69.2 ± 2.2%, 30.2 ± 3.3%; respectively) than FF‐NT embryos (58.1 ± 3.4%, 15.1 ± 1.5%, respectively). Bisulfite sequencing revealed that DNA methylation at the promoter regions of NANOG and POU5F1 was lower in BMSC‐NT embryos (30.0%, 9.8%, respectively) than those in FF‐NT embryos (34.2%, 28.0%, respectively). We also found that BMSC‐NT embryos had more H3K9Ac and less H3K9me3 and 5‐methylcytosine than FF‐NT embryos. In conclusion, our finding comparing BMSCs versus FFs as donors for nuclear transfer revealed that differences in the initial epigenetic state of donor cells have a remarkable effect on overall nuclear reprogramming of SCNT embryos, wherein donor cells possessing a more open chromatin state are more conducive to nuclear reprogramming.     

Efficient gene transfer by histidylated polylysine/pDNA complexes.

Plasmid/polylysine complexes, which are used to transfect mammalian cells, increase the uptake of DNA, but plasmid molecules are sequestered into vesicles where they cannot escape to reach the nuclear machinery. However, the transfection efficiency increases when membrane-disrupting reagents such as chloroquine or fusogenic peptides, are used to disrupt endosomal membranes and to favor the delivery of plasmid into the cytosol. We designed a cationic polymer that forms complexes with a plasmid DNA (pDNA) and mediates the transfection of various cell lines in the absence of chloroquine or fusogenic peptides. This polymer is a polylysine (average degree of polymerization of 190) partially substituted with histidyl residues which become cationic upon protonation of the imidazole groups at pH below 6.0. The transfection efficiency was optimal with a polylysine having 38 +/- 5% of the epsilon-amino groups substituted with histidyl residues; it was not significantly impaired in the presence of serum in the culture medium. The transfection was drastically inhibited in the presence of bafilomycin A1, indicating that the protonation of the imidazole groups in the endosome lumen might favor the delivery of pDNA into the cytosol.     

Essential roles of the nitric oxide (no)/cGMP/protein kinase G type-Iα (PKG-Iα) signaling pathway and the atrial natriuretic peptide (ANP)/cGMP/PKG-Iα autocrine loop in promoting proliferation and cell survival of OP9 bone marrow stromal cells

Inappropriate signaling conditions within bone marrow stromal cells (BMSCs) can lead to loss of BMSC survival, contributing to the loss of a proper micro-environmental niche for hematopoietic stem cells (HSCs), ultimately causing bone marrow failure. In the present study, we investigated the novel role of endogenous atrial natriuretic peptide (ANP) and the nitric oxide (NO)/cGMP/protein kinase G type-Iα (PKG-Iα) signaling pathway in regulating BMSC survival and proliferation, using the OP9 BMSC cell line commonly used for facilitating the differentiation of HSCs. Using an ANP-receptor blocker, endogenously produced ANP was found to promote cell proliferation and prevent apoptosis. NO donor SNAP (S-nitroso-N-acetylpenicillamine) at low concentrations (10 and 50 μM), which would moderately stimulate PKG activity, protected these BMSCs against spontaneous apoptosis. YC-1, a soluble guanylyl cyclase (sGC) activator, decreased the levels of apoptosis, similar to the cytoprotective effects of low-level NO. ODQ (1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one), which blocks endogenous NO-induced activation of sGC and thus lowers endogenous cGMP/PKG activity, significantly elevated apoptotic levels by 2.5- and three-fold. Pre-incubation with 8-Bromo-cGMP or ANP, which bypass the ODQ block, almost completely prevented the ODQ-induced apoptosis. A highly-specific PKG inhibitor, DT-3, at 20, and 30 μM, caused 1.5- and two-fold increases in apoptosis, respectively. ODQ and DT-3 also decreased BMSCs proliferation and colony formation. Small Interfering RNA gene knockdown of PKG-Iα increased apoptosis and decreased proliferation in BMSCs. The data suggest that basal NO/cGMP/PKG-Iα activity and autocrine ANP/cGMP/PKG-Iα are necessary for preserving OP9 cell survival and promoting cell proliferation and migration.     

阳离子脂质体介导基因转染肿瘤细胞

使用基因转运载体运载肿瘤细胞进行转染是基因治疗的关键环节之一。Lipo-fectamine2000和DOTAP作为商品转染试剂,具有较高的转染效率。为了进一步发掘其作为基因转运载体的应用潜力,该文研究了Lipofectamine2000和DOTAP的粒径、Zeta电位及形态,并分别与绿色荧光蛋白基因（pGFP—N2）、荧光素酶基因（pGL3）结合,形成脂质体／DNA复合物,通过载入人喉癌细胞（Hep-2）和人肺癌细胞（NCI—H460）,考察了其转染效率和细胞毒性。结果表明,脂质体Lipofectamine2000与DOTAP都能有效压缩DNA,形成复合物。Lipofectamine2000与DOTAP井目比,转染效率高,与DNA最佳转染比例范围为2：1～4：1。毒性实验显示,在N／P大于3／l时,Lipofectamine2000与DOTAP对癌细胞具有一定的细胞毒性。细胞种类对脂质体的转染效率有很大影响,Lipo—fectamine2000对Hep－2细胞的转染效率比NcI—H460高。     

Efficient non-viral gene delivery mediated by nanostructured calcium carbonate in solution-based transfection and solid-phase transfection

Among different non-viral gene delivery methods, the technique of co-precipitation of Ca(2+) with DNA in the presence of inorganic anions is an attractive option because of the biocompatibility and biodegradability. In this study, nano-sized CaCO(3)/DNA co-precipitates for gene delivery were prepared. The effect of Ca(2+)/CO(3)(2-) molar ratio on the gene delivery was investigated. The mechanism of the transfection mediated by CaCO(3)/DNA co-precipitates was studied by treatment of the cells with chloroquine, wortmannin and cytochalasin D, respectively. The in vitro gene transfections in different cells were carried out for both solution-based transfection and solid-phase transfection. The gene expression of the calcium carbonate based approach is strongly affected by the Ca(2+)/CO(3)(2-) ratio because the size of CaCO(3)/DNA co-precipitates is mainly determined by the Ca(2+)/CO(3)(2-) ratio. In addition, the encapsulation efficiency of DNA increases with decreasing Ca(2+)/CO(3)(2-) ratio. With a suitable Ca(2+)/CO(3)(2-) ratio, CaCO(3)/DNA co-precipitates could effectively mediate gene transfection with the expression levels higher than that of Lipofectamine 2000 in the presence of serum. The mechanism study shows that CaCO(3)/DNA co-precipitates are internalized via endocytosis of the cells and macropinocytosis is the main route of internalization. Compared with the solution-based transfection, CaCO(3)/DNA co-precipitates in the solid-phase transfection exhibit a lower gene expression level. The calcium carbonate based approach has great potential in gene delivery.     

A physicochemical approach for predicting the effectiveness of peptide-based gene delivery systems for use in plasmid-based gene therapy.

Novel synthetic peptides, based on carrier peptide analogs (YKAKnWK) and an amphipathic peptide (GLFEALLELLESLWELLLEA), have been formulated with DNA plasmids to create peptide-based gene delivery systems. The carrier peptides are used to condense plasmids into nanoparticles with a hydrodynamic diameter (DH) ranging from 40 to 200 nm, which are sterically stable for over 100 h. Size and morphology of the carrier peptide/plasmid complex have been determined by photon correlation spectroscopy (PCS) and transmission electron microscopy (TEM), respectively. The amphipathic peptide is used as a pH-sensitive lytic agent to facilitate release of the plasmid from endosomes after endocytosis of the peptide/plasmid complex. Hemolysis assays have shown that the amphipathic peptide destabilizes lipid bilayers at low pH, mimicking the properties of viral fusogenic peptides. However, circular dichroism studies show that unlike the viral fusion peptides, this amphipathic peptide loses some of its alpha-helical structure at low pH in the presence of liposomes. The peptide-based gene delivery systems were tested for transfection efficiency in a variety of cell lines, including 14-day C2C12 mouse myotubes, using gene expression systems containing the beta-galactosidase reporter gene. Transfection data demonstrate a correlation between in vitro transfection efficiency and the combination of several physical properties of the peptide/plasmid complexes, including 1) DNA dose, 2) the zeta potential of the particle, 3) the requirement of both lytic and carrier peptides, and 4) the number of lysine residues associated with the carrier peptide. Transfection data on 14-day C2C12 myotubes utilizing the therapeutic human growth hormone gene formulated in an optimal peptide gene delivery system show an increase in gene expression over time, with a maximum in protein levels at 96 h (approximately 18 ng/ml).     

Development of an effective gene delivery system: a study of complexes composed of a peptide-based amphiphilic DNA compaction agent and phospholipid

We recently described a basic technology to efficiently combine compacted DNA with phospholipids and hydrophobic peptides, to produce homogenous complexes that are completely resistant to nuclease. We have developed this technology further to form gene delivery complexes that transfect cells effectively in vitro. In addition to plasmid DNA, the complexes contained two basic components: (i) a DNA compacting peptide (-CGKKKFKLKH), either conjugated to lipid or extended to contain (WLPLPWGW-) and (ii) either phosphatidylethanolamine or phosphatidylcholine. Complexes containing a 5.5-fold charge equivalence (peptide charge/DNA charge) of WLPLPWGWCGKKKFKLKH and 5 nmol dimyristoleoylphosphatidylethanolamine/µg DNA produced the highest luciferase gene expression, exceeding 1 × 10⁹ relative light units/s/mg protein (>3 µg luciferase per mg protein). These complexes transfected OVCAR-3, COS-7 and HeLa cells at either similar or superior levels when compared to polyethylenimine or lipofectamine complexes. With green fluorescent protein reporter gene, >50% of HeLa cells were positive 30 h after addition of these complexes. Furthermore, these optimal complexes were the least sensitive to pre-treatment of cells with chloroquine, indicating efficient endosomal escape. Our results indicated that self-assembling complexes of plasmid DNA, amphiphilic peptide and phosphatidylethanolamine are highly effective non-viral gene delivery systems.