共查询到20条相似文献,搜索用时 15 毫秒
1.
Inoh Y Furuno T Hirashima N Kitamoto D Nakanishi M 《Biochemical and biophysical research communications》2011,(3):635-640
The downregulation of gene expression by RNA interference holds great potential for genetic analysis and gene therapy. However, a more efficient delivery system for small interfering RNA (siRNA) into the target cells is required for wide fields such as cell biology, physiology, and clinical application. Non-viral vectors are stronger candidates than viral vectors because they are safer and easier to prepare. We have previously used a new method for gene transfection by combining cationic liposomes with the biosurfactant mannosylerythritol lipid-A (MEL-A). The novel MEL-A-containing cationic liposomes rapidly delivered DNA (plasmids and oligonucleotides) into the cytosol and nucleus through membrane fusion between liposomes and the plasma membrane, and consequently, enhanced the gene transfection efficiency. In this study, we determined the efficiency of MEL-A-containing cationic liposomes for siRNA delivery. We observed that exogenous and endogenous protein expression was suppressed by approximately 60% at 24 h after brief (30 min) incubation of target cells with MEL-A-containing cationic liposome/siRNA complexes. Confocal microscopic analysis showed that suppression of protein expression was caused by rapid siRNA delivery into the cytosol. We found that the MEL-A-containing cationic liposomes directly delivered siRNA into the cytoplasm by the membrane fusion in addition to endocytotic pathway whereas Lipofectamine™ RNAiMax delivered siRNA only by the endocytotic pathway. It seems that the ability to rapidly and directly deliver siRNA into the cytosol using MEL-A-containing cationic liposomes is able to reduce immune responses, cytotoxicity, and other side effects caused by viral vectors in clinical applications. 相似文献
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考察自制的肽型阳离子脂质体CDO14作为RNA转染载体的细胞毒性及其运载si RNA进行RNA干扰的效果。通过MTT法检测脂质体对稳定表达荧光素酶的肺癌A549(Luc-A549)细胞的毒性。以脂质体为载体将荧光素酶si RNA(Luc-si RNA)转染至Luc-A549细胞内,用发光仪检测转染细胞内荧光素酶含量,BCA法检测细胞内总蛋白含量。在裸鼠腋下接种Luc-A549细胞,成瘤后尾静脉注射Luc-si RNA和脂质体的复合物,利用活体成像系统检测模型小鼠体内荧光素酶的表达量。细胞毒性实验表明,自制脂质体的毒性与商品脂质体DOTAP相近,低于商品脂质体Lipo2000;细胞转染实验表明自制脂质体作为基因转染载体的转染效率高于DOTAP;体内转染实验表明CDO14作为载体转染效果优于DOTAP。结果表明,肽型阳离子脂质体CDO14具有毒性小、转染效率高等优点,有望作为转染载体用于基因治疗。 相似文献
3.
Tianzhi Yang Tracy Bantegui Kaitlynn Pike Raymond Bloom Roger Phipps 《Journal of liposome research》2014,24(4):270-279
One of the biggest challenges for small interfering RNAs (siRNAs) as therapeutic agents is their insufficient cellular delivery efficiency. We developed long circulating and cationic liposomes to improve the cell uptake and inhibitory effectiveness of siRNA on the expression of vascular endothelial growth factor (VEGF) in cancer cells. SiRNA liposomes were obtained by polyelectrolyte complexation between negatively charged siRNA and positively charged liposome prepared by a hydration method. Gel electrophoresis was used to evaluate the loading efficiency of siRNA on the cationic liposome. The optimized siRNA liposomes were observed to be spherical in shape and had smooth surfaces with particle sizes of 167.7?±?2.0?nm and zeta potentials of 4.03?±?0.69?mV, which had no significant change when stored at 4?°C for three months. Fluorescence-activated cell sorting studies and confocal laser scanning images indicated that the cationic liposomes significantly increased the uptake of fluorescence-labeled siRNA in cancer cells. Effects of the siRNA on the inhibition of VEGF were tested by measuring concentrations of VEGF in cell culture media via an enzyme-linked immunosorbent assay and intracellular VEGF levels using a western blotting method. The liposomal siRNA was significantly effective at inhibiting the expression of VEGF in lung, liver and breast cancer cells. Optimal liposomes could effectively deliver siRNA into cancer cells and inhibit VEGF as a therapy agent. 相似文献
4.
Michael J. Bennett Michael H. Nantz Rajiv P. Balasubramaniam Dieter C. Gruenert Robert W. Malone 《Bioscience reports》1995,15(1):47-53
Cationic liposome transfection reagents are useful for transferring polynucleotides into cells, and have been proposed for human pulmonary gene therapy. The effect of adding cholesterol to cationic lipid preparations has been tested by first formulating the cationic lipid N-[1-(2,3-dioleoyloxy)propyl-N-[1-(2-hydroxy)ethyl]-N,N-dimethyl ammonium iodide (DORI) with varying amounts of dioleoylphos-phatidylethanolamine (DOPE) and cholesterol. Cholesterol was found to enhance lipid-mediated transfection in both the respiratory epithelial cells and mouse fibroblasts. These findings will facilitate nucleic acid transfection of many cell types including differentiated epithelial cell monolayers, and therefore may be useful for examining gene regulation in various cell types and for developing pulmonary gene therapy.Abbreviations (DORI)
N-[1-(2,3-dioleoyloxy)propyl]-N-[1-(2-hydroxy)ethyl]-N,N-dimethyl ammonium iodide
- (DOPE)
dioleoylphosphatidylethanolamine
- (DOTMA)
N-[1-(2,3-dioleoyloxy) propyl]-N,N,N-trimethyl ammonium chloride
- (Mem)
Eagle's modified essential medium
- (DMEM)
Dulbecco's Modified Eagle's Medium 相似文献
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The effects of liposomes on apoptosis in macrophages were evaluated from DNA content and DNA fragmentation. Cationic liposomes composed of different kinds of cationic lipids induced apoptosis in mouse splenic macrophages and the macrophage-like cell line, RAW264.7 cells. Generation of reactive oxygen radicals from macrophages treated with cationic liposomes was detected using flow cytometry, and further apoptosis was inhibited by the addition of oxidant scavenger, N-acetylcysteine. From these findings, the production of reactive oxygen species may be important in the regulation of apoptosis induced by cationic liposomes. 相似文献
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Veeravagu Murugaiah William Zedalis Gary Lavine Klaus Charisse Muthiah Manoharan 《Analytical biochemistry》2010,401(1):61-67
Gene silencing induced by short interfering RNA (siRNA) has proven to be useful in genomic research and has great potential for therapeutic applications; however, siRNAs are not readily bioavailable. Cationic liposomes offer effective protection of drug product from nucleases and enable distribution to desired target organs. The amount of siRNA in the formulation must be determined accurately. We have developed a stability-indicating, ion-pair, reversed-phase high-performance liquid chromatography method to separate and accurately quantitate two siRNA duplexes in a liposome without sample pretreatment. The gradient mobile phase system consisted of 385 mM hexafluoro-2-propanol, 14.5 mM triethylamine, and 5% methanol (mobile phase A) and 385 mM hexafluoro-2-propanol, 14.5 mM triethylamine, and 90% methanol (mobile phase B). The column used was an XBridge C18 column (50 × 2.1 mm i.d., 2.5 μm particle size), and separation was performed at 60 °C. Quantitation was achieved with ultraviolet (UV) detection at 260 nm. Linearity was established for the single strands of both siRNA duplexes for concentrations ranging from 10 to 110 μg/ml. Accuracy of the method was determined by replicate analysis (n = 5) at four concentrations (R2 > 0.996 and relative standard deviations [RSDs] of 1-4%). The use of an ion-pairing reagent that is compatible with mass spectrometry detection makes this method amenable to liquid chromatography-mass spectrometry (LC-MS) impurity profiling. 相似文献
9.
Porter CD 《The journal of gene medicine》2002,4(6):622-633
Background
Retroviral particles that are inappropriately enveloped can transduce target cells if pre‐associated with cationic liposomes. This study optimises and addresses the mechanism of liposome‐enhanced gene delivery, and explores the potential for such agents to compensate for fusion deficiency associated with chimaeric envelope proteins.Methods
Particles bearing wild‐type, chimaeric or no envelope proteins were complexed with DOTAP or DC‐Chol/DOPE cationic liposomes and added to target cells for various times. Particle binding was determined by detection of cell‐associated capsid protein and infectivity was measured histochemically.Results
Stable association of cationic liposomes with retrovirus particles significantly enhanced their binding rate to target cells in proportion to the increase of transduction kinetics for infectious virus. Binding of virus was equivalent with or without envelope protein and/or virus receptor, indicating that a non‐specific interaction precedes receptor recognition. Non‐infectious combinations were rescued by the intrinsic fusogenicity of the cationic liposomes, which enabled entry of the viral core, but left subsequent events unaltered. The optimised transduction rate with non‐enveloped particles and DOTAP approached that of amphotropic‐enveloped virus in some cases, although the effect was target‐cell‐dependent. DC‐Chol/DOPE was less potent at direct fusion but was able to enhance 600‐fold the receptor‐dependent action of chimaeric envelopes that were deficient in fusion by virtue of the addition of targeting domains.Conclusions
These data have implications for the development of retroviral vector targeting strategies from the perspectives of the specificity of target cell interaction and compensating for chimaeric envelope fusion deficiency. Copyright © 2002 John Wiley & Sons, Ltd.10.
D Lesage A Cao D BrianeN Lievre R Coudert M RaphaelJ.L Salzmann E Taillandier 《生物化学与生物物理学报:生物膜》2002,1564(2):393-402
This paper reports results concerning the transfection of gliosarcoma cells 9L using an original cholesterol-based cationic liposome as carrier. This cationic liposome was prepared from triethyl aminopropane carbamoyl cholesterol (TEAPC-Chol) and a helper lipid, dioleoyl phosphatidyl ethanolamine (DOPE). The used concentration of liposome was not cytotoxic as revealed by the MTT test. TEAPC-Chol/DOPE liposomes allowed the plasmids encoding reporter genes to enter the nucleus as observed both by electron microscopy and functionality tests using fluorescence detection of green fluorescent protein (GFP) and luminometric measurements of luciferase activity. By changing the cationic lipid/DNA molar charge ratio, optimal conditions were determined. Further, improvement of the transfection level has been obtained by either precondensing plasmid DNA with poly-l-lysine or by adding polyethylene glycol (PEG) in the transfection medium. The optimal conditions determined are different depending on whether the transfection is made with cells in culture or with tumors induced by subcutaneous (s.c.) injection of cells in Nude mice. For in vivo assays, a simple method to overcome the interference of haemoglobin with the chemiluminescence intensity of luciferase has been used. These results would be useful for gaining knowledge about the potential for the cationic liposome TEAPC-Chol/DOPE to transfect brain tumors efficiently. 相似文献
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Hattori Y Kawakami S Suzuki S Yamashita F Hashida M 《Biochemical and biophysical research communications》2004,317(4):992-999
The present study investigated the potency of the mannosylated cationic liposomes (Man liposomes) that we have developed in novel DNA vaccine carrier. Ovalbumin (OVA) was selected as a model antigen for vaccination; accordingly, OVA-encoding pDNA (pCMV-OVA) was constructed to evaluate DNA vaccination. The potency of the Man liposome/pCMV-OVA complex was compared with naked pCMV-OVA and that complexed with DC-Chol liposomes. In cultured mouse peritoneal macrophages, MHC class I-restricted antigen presentation of the Man liposome/pCMV-OVA complex was significantly higher than that of naked pCMV-OVA and that complexed with DC-Chol liposomes. After intravenous administration, OVA mRNA expression and MHC class I-restricted antigen presentation on CD11c+ cells and inflammatory cytokines, such as TNF-alpha, IL-12, and IFN-gamma, that can enhance the Th1 response of the Man liposome/pCMV-OVA complex were higher than that of naked pCMV-OVA and that complexed with DC-Chol liposomes. Also, the spleen cells from mice immunized by intravenous administration of the Man liposome/pCMV-OVA complex showed the highest proliferation response and IFN-gamma secretion. These findings suggest that the targeted delivery of DNA vaccine by Man liposomes is a potent vaccination method for DNA vaccine therapy. 相似文献
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Background
A variety of synthetic carriers, such as cationic polymers and lipids, have been used as nonviral carriers for small interfering RNA (siRNA) delivery. Although siRNA polyplexes and lipoplexes exhibited good gene silencing efficiencies, they often showed serious cytotoxicities, which are not useful for clinical applications. A double‐stranded RNA binding cellular protein with highly specific siRNA binding property and noncytotoxicity was used for siRNA delivery.Methods
A double‐stranded RNA binding domain (dsRBD) of human double‐stranded RNA activated protein kinase R was genetically produced and utilized to complex siRNA for intracellular delivery. For characterization of the siRNA/dsRBD complexes, decomplexation assay and RNase protection assay were performed. Cytotoxicity and target gene inhibition ability were also examined using human carcinoma cell lines.Results
The recombinantly produced polypeptide dsRBD exhibited its inherent binding activity for siRNA without sequence specificity, and the siRNA/dsRBD complexes protected siRNA from degradation by ribonucleases. Green fluorescent protein (GFP) siRNA/dsRBD complexes showed prominent down‐regulation of a target GFP gene, when an endosomal escape function was supplemented by addition of a fusogenic peptide, KALA, in the formulation.Conclusions
The results suggest that dsRBD‐based protein carriers could be successfully applied for a wide range of therapeutic siRNAs for intracellular gene inhibition without showing any cytotoxicity. Copyright © 2009 John Wiley & Sons, Ltd. 相似文献13.
Poly(ethylene glycol)-lipid (PEG-lipid) conjugates are widely used in the field of liposomal drug delivery to provide a polymer coat that can confer favorable pharmacokinetic characteristics on particles in the circulation. More recently these lipids have been employed as an essential component in the self-assembly of cationic and neutral lipids with polynucleic acids to form small, stable lipid/DNA complexes that exhibit long circulation times in vivo and accumulate at sites of disease. However, the presence of a steric barrier lipid might be expected to inhibit the transfection activity of lipid/DNA complexes by reducing particle-membrane contact. In this study we examine what effect varying the size of the hydrophobic anchor and hydrophilic head group of PEG-lipids has on both gene and antisense delivery into cells in culture. Lipid/DNA complexes were made using unilamellar vesicles composed of 5 mole% PEG-lipids in combination with equimolar dioleoylphosphatidylethanolamine and the cationic lipid dioleyldimethylammonium chloride. Using HeLa and HepG2 cells we show that under the conditions employed PEG-lipids had a minimal effect on the binding and subsequent endocytosis of lipid/DNA complexes but they severely inhibited active gene transfer and the endosomal release of antisense oligodeoxynucleotides into the cytoplasm. Decreasing the size of the hydrophobic anchor or the size of the grafted hydrophilic PEG moiety enhanced DNA transfer by the complexes. 相似文献
14.
Non-viral vectors such as cationic lipids are capable of delivering nucleic acids, including genes, siRNA or antisense RNA
into cells, thus potentially resulting in their functional expression. These vectors are considered as an attractive alternative
for virus-based delivery systems, which may suffer from immunological and mutational hazards. However, the efficiency of cationic-mediated
gene delivery, although often sufficient for cell biological purposes, runs seriously short from a therapeutics point of view,
as realizing this objective requires a higher level of transfection than attained thus far. To develop strategies for improvement,
there is not so much a need for novel delivery systems. Rather, better insight is needed into the mechanism of delivery, including
lipoplex–cell surface interaction, route of internalization and concomitant escape of DNA/RNA into the cytosol, and transport
into the nucleus. Current work indicates that a major obstacle involves the relative inefficient destabilization of membrane-bounded
compartments in which lipoplexes reside after their internalization by the cell. Such an activity requires the capacity of
lipoplexes of undergoing polymorphic transitions such as a membrane destabilizing hexagonal phase, while cellular components
may aid in this process. A consequence of the latter notion is that for development of a novel generation of delivery devices,
entry pathways have to be triggered by specific targeting to select delivery into intracellular compartments which are most
susceptible to lipoplex-induced destabilization, thereby allowing the most efficient release of DNA, a minimal requirement
for optimizing non-viral vector-mediated transfection.
Dedicated to Prof. K. Arnold on the occasion of his 65th birthday. 相似文献
15.
Unique features of a pH-sensitive fusogenic peptide that improves the transfection efficiency of cationic liposomes 总被引:1,自引:0,他引:1
Futaki S Masui Y Nakase I Sugiura Y Nakamura T Kogure K Harashima H 《The journal of gene medicine》2005,7(11):1450-1458
BACKGROUND: One of the critical steps in intracellular gene delivery using cationic liposomes is the endosomal escape of the plasmid/liposome complexes to the cytosol. The addition of GALA, a pH-sensitive fusogenic peptide, is a promising method to accelerate this step in order to enhance the expression of the desired proteins. Detailed studies on the methods of enhancement would broaden the horizon of its application. METHODS: Using representative commercially available cationic liposomes (Lipofectin, Lipofectamine, and Lipofectamine 2000), the effects of GALA on transfection efficiency were studied by luciferase assay and confocal microscopic observations. RESULTS: A concentration-dependent increase in the transfection efficiency was observed for GALA. Addition of 0.1 microM GALA to the plasmid/liposome complex significantly increased the transfection efficiency, especially in the case of Lipofectin, but higher concentration of GALA decreased transfection efficiency. Successful reduction in the liposomal dosage was attained by employing GALA while maintaining a high transfection efficiency. Interestingly, although the transfection efficiency was higher in the presence of GALA, a lower amount of the plasmid DNA was taken up by the cells. Confocal microscopic observations of the rhodamine-labeled plasmid did not show a significant difference in the cellular localization among cells incubated in the presence or absence of GALA, suggesting that a slight increase in GALA-induced release of the plasmid to the cytosol may cause a significant change in the transfection efficiency. CONCLUSION: The unique features of GALA to mediate improved transfection efficiencies were identified. 相似文献
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Reza K. Oskuee Alexander Philipp Ali Dehshahri Ernst Wagner Mohammad Ramezani 《The journal of gene medicine》2010,12(9):729-738
Background
Carboxyalkylation of branched 25 kDa polyethylenimine (PEI) was considered to reduce the positive surface charge of the polymer without reducing its ‘proton sponge’ buffering capacity, and to provide alkylene domains for hydrophobic interactions, thus generating optimized novel PEI carriers for efficient delivery of small interfering RNA (siRNA).Methods
Substitution of PEI was evaluated in the range of 6 to > 50 mole percentage of primary amines. Additionally, variation of the carboxyalkyl chain (one to 15 methylene groups) was explored to modulate the carrier hydrophobicity. Carriers were characterized in their buffering capacity, capability of siRNA polyplex formation, and cytotoxicity. Marker gene‐silencing efficacy was evaluated using Neuro2A‐eGFPLuc neuroblastoma cells.Results
Carboxyalkylation strongly reduced cytotoxicity of PEI and improved siRNA mediated luciferase gene knockdown. An optimum silencing activity was observed at an alkylcarboxylation degree of 6–9 mole percentage of primary amines and with a broad range of carboxyalkylene chains (containing one to 15 methylene groups). Strongly enhanced gene‐silencing efficacy also was observed when the biocompatible polymers were separately added at 1 h after transfection with tolerated doses of standard PEI25/siRNA polyplexes.Conclusions
Carboxyalkylation of branched 25 kDa PEI resulted in polymers with strongly reduced cytotoxicity and improved silencing efficacy. Mechanistic studies demonstrated that the presence of a surplus of free carboxyalkylated polymer is responsible for the improved siRNA delivery. Copyright © 2010 John Wiley & Sons, Ltd. 相似文献20.
Multivalent methionine‐functionalized biocompatible block copolymers for targeted small interfering RNA delivery and subsequent reversal effect on adriamycin resistance in human breast cancer cell line MCF‐7/ADR 下载免费PDF全文
Yang Wu Wei Zhang Tinghan Li Rong Ma Dan Chen Junying Zhang Jianzhong Wu Jinhai Tang 《The journal of gene medicine》2017,19(8)