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1.
Byk Gerardo Dubertret Catherine Schwartz Bertrand Frederic Marc Jaslin Gabrielle Rangara Ravi Scherman Daniel 《International journal of peptide research and therapeutics》1997,4(4-6):263-267
Summary We have synthesized novel cationic lipids for gene delivery bearing an ester bond between the lipid moiety and the polyamine
head. We have found that an intramolecular rearrangement occurs during purification of one of the products. The rearrangement
led to a cyclic lipopolyamine which was active for DNA gene transfer. The formation of cyclization products depends on the
spacer found between the lipid and the polyamine. The introduction of arginine in the linker position prevents the formation
of cyclic products. Linear as well as cyclic analogues showed high-efficiency gene transfer when tested in vitro for luciferase
gene expression as compared to dioctadecylamidoglycyl spermine or lipofectamine and also in vivo in the Lewis lung carcinoma
model. The introduction of arginine in the linker position promoted increased transfection activity, demonstrating that a
diversity of linkers, such as short peptides or glycosides, can be introduced into cationic lipids for targeted gene transfer. 相似文献
2.
Gerardo Byk Catherine Dubertret Bertrand Schwartz Marc Frederic Gabrielle Jaslin Ravi Rangara Daniel Scherman 《Letters in Peptide Science》1997,4(4-6):263-267
We have synthesized novel cationiclipids for gene delivery bearing an ester bond betweenthe lipid moiety and the polyamine head. We have foundthat an intramolecular rearrangement occurs duringpurification of one of the products. The rearrangementled to a cyclic lipopolyamine which was active for DNAgene transfer. The formation ofcyclization products depends on the spacer foundbetween the lipid and the polyamine. The introduction ofarginine in the linker position prevents the formation ofcyclic products. Linear as well as cyclicanalogues showed high-efficiency gene transfer whentested in vitro for luciferase gene expressionas compared to dioctadecylamidoglycyl spermineor lipofectamine and also in vivo in the Lewislung carcinoma model. The introduction of arginine in thelinker position promoted increased transfectionactivity, demonstrating that a diversity of linkers,such as short peptides or glycosides, can beintroduced into cationic lipids for targeted gene transfer. 相似文献
3.
Melittin enables efficient vesicular escape and enhanced nuclear access of nonviral gene delivery vectors 总被引:6,自引:0,他引:6
Ogris M Carlisle RC Bettinger T Seymour LW 《The Journal of biological chemistry》2001,276(50):47550-47555
Entry of exogenously applied DNA into the cytoplasm and subsequent transport into the nucleus are major cellular barriers for nonviral gene delivery vectors. To overcome these barriers, we have covalently attached the cationic peptide melittin to poly(ethylenimine) (PEI). This conjugate condensed DNA into small, discrete particles (<100 nm in diameter), and the membrane lytic activity of melittin enabled efficient release of the DNA into the cytoplasm, as monitored by fluorescence microscopy and flow cytometry. Compared with PEI, the transfection activity was strongly increased within a broad range of cell lines and types tested, including different tumor cell lines but also primary hepatocytes and human umbilical vein endothelial cells. The early onset of gene expression (within 4 h, reaching maximal values after 12 h) and the high reporter gene expression achieved in slowly dividing or confluent cells suggested a further role of melittin after releasing the DNA into the cytoplasm. Intracytoplasmic microinjection of melittin-containing PEI.DNA complexes into fibroblasts produced 40% cellular frequency of reporter gene expression that was inhibitable by co-injection of wheat germ agglutinin, whereas simple PEI.DNA complexes showed only 10%. These data suggest that melittin enables release of nonviral gene transfer particles into the cytoplasm and also enhances their transport into the nucleus, possibly via the cationic cluster KRKR near the C terminus of the peptide. 相似文献
4.
Yun Wu Zhengzheng Fei L. James Lee Barbara E. Wyslouzil 《Biotechnology and bioengineering》2010,105(4):834-841
DNA/polyethylenimine (PEI) polyplexes are an important class of nonviral vectors. Although the conventional preparation method, bulk mixing, is straightforward, the formation of the DNA/PEI polyplexes is not well controlled. This work explores coaxial electrohydrodynamic spraying (EHDS) as a novel, alternative method to produce DNA/PEI polyplexes in a more controlled manner. Both pGFP/PEI and pSEAP/PEI polyplexes were produced by EHDS with a coaxial needle setup. The size of the polyplexes was determined using dynamic light scattering, and their ability to transfect NIH 3T3 cells was observed by using an inverted fluorescence microscope (pGFP) or quantified by measuring the activity level of alkaline phosphatase (pSEAP). At nitrogen to phosphate ratio (N/P) of 6.7, the polyplexes produced by coaxial EHDS had delivery efficiencies up to 2.6 times higher than those produced by bulk mixing. The N/P ratio and the structure of the EHDS used to make the polyplexes were crucial factors in determining the delivery efficiency. Biotechnol. Bioeng. 2010. 105: 834–841. © 2009 Wiley Periodicals, Inc. 相似文献
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Efficient in vivo gene delivery by the negatively charged complexes of cationic liposomes and plasmid DNA 总被引:3,自引:0,他引:3
We examined changes in zeta potential (the surface charge density, zeta) of the complexes of liposome (nmol)/DNA (microg) (L/D) formed in water at three different ratios (L/D=1, 10 and 20) by changing the ionic strength or pH to find an optimum formulation for in vivo gene delivery. At high DNA concentrations, zeta of the complexes formed in water at L/D=10 was significantly lowered by adding NaCl (zeta=+8.44+/-3.1 to -27.6+/-3.5 mV) or increasing pH from 5 (zeta=+15.3+/-1.0) to 9 (zeta=-22.5+/-2.5 mV). However, the positively charged complexes formed at L/D=20 (zeta=+6.2+/-3.5 mV) became negative as NaCl was added at alkaline pH as observed in medium (zeta=-19.7+/-9.9 mV). Thus, the complexes formed in water under the optimum condition were stable and largely negatively charged at L/D=1 (zeta=-58.1+/-3.9 mV), unstable and slightly positively charged at L/D=10 (zeta=+8.44+/-3.7 mV), and unstable and largely positively charged at L/D=20 (zeta=+24.3+/-3.6 mV). The negatively charged complexes efficiently delivered DNA into both solid and ascitic tumor cells. However, the positively charged complexes were very poor in delivering DNA into solid tumors, yet were efficient in delivering DNA into ascitic tumors grown in the peritoneum regardless of complex size. This slightly lower gene transfer efficiency of the negatively charged complexes can be as efficient as the positively charged ones when an injection is repeated (at least two injections), which is the most common case for therapy regimes. The results indicate that optimum in vivo lipofection may depend on the site of tumor growth. 相似文献
7.
为构建一种具有广泛适用性的原核启动子报告系统,以质粒pFLX107为骨架,通过多克隆位点替换和序列改造,构建出基于lacZ基因和pUC复制子的pFGH系列报告载体,然后以lacZ基因缺失株MC4100为宿主菌筛选背景活性最低的质粒作为最终的报告系统,并利用诱导型启动子araBAD和组成型启动子rpsM分别对其进行测试。结果显示,在所构建的pFGH系列质粒中,pFGH06的背景活性显著低于同系列其他质粒,在28 ℃培养条件下甚至显著低于低拷贝参考质粒pRCL的活性 (P<0.01)。进一步的评估测试显示,质粒pFGH06可用于诱导型启动子或组成型启动子的克隆及活性测定,且在模拟应用于启动子筛选时,通过蓝白斑筛选即可实现对目标启动子的完全识别。与已报道的原核启动子报告系统相比,pFGH06具有体积小、克隆位点多、背景活性可调、对启动子筛选识别效率高等优点,具有广泛的应用前景。 相似文献
8.
Non-viral vectors represent an important alternative in gene delivery. Among these vectors, cationic liposomes are widely studied, because of their ability to form stable complexes with DNA fragments (lipoplexes). In the present work, we report on the characterization by electron spin resonance (ESR) spectroscopy and zeta potential measurements of cationic liposomes and of their complexes with oligonucleotides. Liposomes were made with a zwitterionic lipid, DOPE, and a cationic lipid, either DOTAP or DC-Chol. Oligonucleotides were the 20-base single strand polyA, the 20-base single strand polyT, and the corresponding double strand dsAT. The zeta potential as a function of the oligonucleotide/lipid+ ratio gave an S-shaped titration curve. Well-defined surface potential changes took place upon charge compensation between the cationic lipid heads and the phosphate groups on the oligonucleotides. The inversion point depended on the specific system under study. The bilayer properties and the changes that occurred with the incorporation of DNA fragments were also monitored by ESR spectroscopy of appropriately tailored spin probes. For all the systems investigated, the ESR spectra showed that no major alteration took place after lipoplex formation and molecular packing remained substantially unchanged. Both zeta potential and ESR measurements were in favor of an external mode of packing of the lipoplexes. 相似文献
9.
Non-viral vectors represent an important alternative in gene delivery. Among these vectors, cationic liposomes are widely studied, because of their ability to form stable complexes with DNA fragments (lipoplexes). In the present work, we report on the characterization by electron spin resonance (ESR) spectroscopy and zeta potential measurements of cationic liposomes and of their complexes with oligonucleotides. Liposomes were made with a zwitterionic lipid, DOPE, and a cationic lipid, either DOTAP or DC-Chol. Oligonucleotides were the 20-base single strand polyA, the 20-base single strand polyT, and the corresponding double strand dsAT. The zeta potential as a function of the oligonucleotide/lipid+ ratio gave an S-shaped titration curve. Well-defined surface potential changes took place upon charge compensation between the cationic lipid heads and the phosphate groups on the oligonucleotides. The inversion point depended on the specific system under study. The bilayer properties and the changes that occurred with the incorporation of DNA fragments were also monitored by ESR spectroscopy of appropriately tailored spin probes. For all the systems investigated, the ESR spectra showed that no major alteration took place after lipoplex formation and molecular packing remained substantially unchanged. Both zeta potential and ESR measurements were in favor of an external mode of packing of the lipoplexes. 相似文献
10.
Berchel M Le Gall T Couthon-Gourvès H Haelters JP Montier T Midoux P Lehn P Jaffrès PA 《Biochimie》2012,94(1):33-41
Lipophophoramidates constitute a class of synthetic vectors which were especially designed for gene delivery. In this family of compounds, the phosphorus functional group links two lipid chains to a spacer ended by a polar headgroup. Such vectors, which can readily be obtained, offer an alternative to the numerous examples of glycerolipid-based vectors that have been more exhaustively studied. Since the pioneering work describing this series of synthetic vectors, several chemical modifications have been proposed with the aim of correlating the molecular structure with the gene transfection efficacy. It has indeed been observed that some modifications which may be considered as minor at first glance, actually have important consequences on both the transfection efficacy and cytotoxic side effects. We herein discuss the modification of the structure of lipophosphoramidates, in particular of their lipidic part and of the nature of the cationic polar head which may be constituted by a trimethylammonium, trimethylphosphonium or trimethylarsonium motif. We also report that, as well as the in vitro transfection efficacy which governs the selection of the most promising vectors for in vivo studies, other aspects related to the synthetic pathway must be also considered for the development of new synthetic vectors (such as modularity of the synthesis, scaling-up). 相似文献
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Matsingou C Hatziantoniou S Georgopoulos A Dimas K Terzis A Demetzos C 《Chemistry and physics of lipids》2005,138(1-2):1-11
Labd-13(E)-ene-8alpha,15-diol (1) and its derivative labd-13(E)-ene-8alpha-ol-15-yl-acetate (2) are water insoluble biological active molecules and their structures were elucidated using NMR and X-ray techniques. Differential scanning calorimetry (DSC) was applied to study the thermal effects of 1 and 2 on DPPC bilayers. Liposomes composed of egg phosphatidylcholine/dipalmytoylphosphatidylglycerol (9:0.1 molar ratio) were prepared by the thin-film hydration method and were used for incorporating 1 and 2. Free and liposomal 1 and 2 were tested for their activity against human cancer cell lines using the sulphorhodamine B assay. The effect of 1 and 2 on DPPC bilayers caused abolition of the pre-transition temperature, lowering of the main phase transition and reduction of the transition enthalpy only in the presence of cholesterol. The liposomes that have been designed and developed offer high incorporation efficiency; 62.4% (0.369 drug/lipid molar ratio) and 99.7% (0.661 drug/lipid molar ratio) for 1 and 2, respectively. Liposomal 2 showed growth-inhibiting activity against the majority of the tested cell lines. 相似文献
13.
The construction of live oral carriers based on attenuated Salmonella strains as vectors offers a new approach to vaccine development. We have constructed a set of plasmid vectors which have the thyA gene of Escherichia coli (encoding thymidylate synthetase) as the marker for selection and maintenance of plasmid clones. The thyA system offers an alternative to antibiotic-resistance selection markers. It can be easily adapted to a particular host-vector combination since thyA chromosomal mutations can be readily introduced by trimethoprim selection. We also describe the construction of thyA-based plasmids with the Vibrio cholerae rfb genes (encoding O-antigen biosynthesis of the Inaba serotype). These have been found to be useful in the construction of candidate bivalent cholera-typhoid vaccines. 相似文献
14.
Efficient and non‐toxic gene delivery by anionic lipoplexes based on polyprenyl ammonium salts and their effects on cell physiology
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Monika Rak Anna Ochałek Ewa Bielecka Joanna Latasiewicz Katarzyna Gawarecka Jolanta Sroka Jarosław Czyż Katarzyna Piwowarczyk Marek Masnyk Marek Chmielewski Tadeusz Chojnacki Ewa Swiezewska Zbigniew Madeja 《The journal of gene medicine》2016,18(11-12):331-342
15.
将带有绿色荧光蛋白(GFP)报告基因的真核表达质粒与阳离子聚合物聚乙亚胺(PEI)结合,用肝癌细胞株CM7221实验,研究其转染效率及可能引起的细胞毒性;进一步用此PEI/DNA复合物转染小鼠皮肤组织,通过报告基因检测,研究转染基因的表达位置及持续表达时间。结果发现,低分子量PEI介导的细胞转染效率最高可达550%,转染效率与PEI结构无关,但是随着分子量的增加,转染活性略有下降。同时,随着分子量的增加,PEI对细胞的毒性也相应的加大;动物皮肤转染实验显示,转染24h后,GFP基因在皮肤组织的毛囊、汗腺、皮脂腺等处高效表达,表达可持续7天。表明低分子量PEI是低毒性、高转染效率的有用非病毒转染载体,能够在动物皮肤组织中进行基因转移,这对皮肤疾病的基因治疗具有潜在的应用价值。 相似文献
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Solid lipid nanoparticle (SLN), nanostructured lipid carriers (NLC) and hybrid nanoparticles, have gained increasing interest as drug delivery systems because of their potential to load and release drugs from the Biopharmaceutical classification system (BCS) of class II (low solubility and high permeability) and of class IV (low solubility and low permeability). Lipid properties (e.g. high solubilizing potential, biocompatibility, biotolerability, biodegradability and distinct route of absorption) contribute for the improvement of the bioavailability of these drugs for a set of administration routes. Their interest continues to grow, as translated by the number of patents being field worldwide. This paper discusses the recent advances on the use of SLN, NLC and lipid-polymer hybrid nanoparticles for the loading of lipophilic, poorly water-soluble and poorly permeable drugs, being developed for oral, topical, parenteral and ocular administration, also discussing the industrial applications of these systems. A review of the patents filled between 2014 and 2017, concerning the original inventions of lipid nanocarriers, is also provided. 相似文献
19.
Effect of phospholipid composition on the surface potential of liposomes and the activity of enzymes incorporated 总被引:2,自引:0,他引:2
1. Microsomes of rat liver and brain and mitochondria of rat liver and guinea-pig brown adipose tissue were solubilized with the nonionic detergent Lubrol-WX and the solubilized material was incorporated into liposomes of various phospholipid composition. In proteoliposomes thus formed the kinetics of arylsulphatase, glycerol-3-phosphate dehydrogenase, monoamine oxidase and acetylcholinesterase were measured. 2. It was shown that the apparent Km values of arylsulphatase and glycerol-3-phosphate dehydrogenase were higher in liposomes prepared with negatively charged phospholipids and lower in liposomes containing positively charged organic amines, as compared with th Km value of enzymes incorporated into liposomes prepared from phosphatidylcholine alone. The opposite was true for monoamine oxidase and acetylcholinesterase, i.e. enzymes possessing cationic substrates. Phospholipid composition did not essentially influence the activity of the enzymes extrapolated for infinite substrate concentration (V values). 3. As compared with proteoliposomes made from phosphatidylcholine, the binding constant (Ka) of 8-anilino-1-naphthalene sulphonate was higher when the vesicles contained acidic phospholipids or bis(hexadecanyl)phosphate and lower when they contained organic amines. 4. A correlation between changes of the surface potential calculated from Ka values of anilino-naphthalene sulphonate and variations in apparent Km values of the four enzymes under investigation indicates that the activity of membrane-bound enzymes may be modulated by charged phospholipids due to decreasing or increasing substrate concentration in the unstirred layer, as predicted from the Boltzmann distribution. 相似文献
20.
Adeno-associated viral vectors as agents for gene delivery: application in disorders and trauma of the central nervous system 总被引:4,自引:0,他引:4
The use of viral vectors as agents for gene delivery provides a direct approach to manipulate gene expression in the mammalian central nervous system (CNS). The present article describes in detail the methodology for the injection of viral vectors, in particular adeno-associated virus (AAV) vectors, into the adult rat brain and spinal cord to obtain reproducible and successful transduction of neural tissue. Surgical and injection procedures are based on the extensive experience of our laboratory to deliver viral vectors to the adult rat CNS and have been optimized over the years. First, a brief overview is presented on the use and potential of viral vectors to treat neurological disorders or trauma of the CNS. Next, methods to deliver AAV vectors to the rat brain and spinal cord are described in great detail with the intent of providing a practical guide to potential users. Finally, some data on the experimental outcomes following AAV vector-mediated gene transfer to the adult rat CNS are presented as is a brief discussion on both the advantages and limitations of AAV vectors as tools for somatic gene transfer. 相似文献