一种可用于SiRNA转染的可降解的新型脂质聚合物

合成一种兼具阳离子脂质体和阳离子聚合物优点的可用于SiRNA的新型脂质聚合物载体.以低分子量的支链聚乙烯亚胺(BPEI,分子量600D)为基础,通过交联剂将油酸与之交联,形成PEI-交联剂-油酸为单元的高分子脂质聚合物.以稳定表达EGFP基因的HeLa-EGFP细胞为模型,比较脂质聚合物、高分子聚合物BPEI(MW 25KD)及阳离子脂质体Lipofectamine 2000介导的SiRNA转染效率,以MTT法检测转染后细胞毒性.将聚合物放置于中性环境37℃保温,检测在温育不同时间后结合FITC标记的寡核苷酸能力.脂质聚合物介导转染EGFP-SiRNA的细胞中,绿色荧光蛋白的信号下降了72.3%,下降幅度高于Lipofectamine 2000介导转粢的细胞,而BPEI介导的转染细胞中的荧光强度仅比空白对照下降20%左右.MTT结果显示在最适条件下,脂质聚合物的介导的SiRNA转染48h后,细胞存活率为94.87%,高于BPEI 25K和Lipofectamine 2000对照组(80.68%和64.87%).降解实验证实,脂质聚合物和BPEI25K相似,在与FITC标记的寡核苷酸结合后,使其荧光水平下降65%左右;在保温后,脂质聚合物对荧光的抑制能力逐步下降,在8h后其荧光强度与BPEI600对照组相似;BPEI 25K实验组在保温前后荧光强度无显著的变化.脂质聚合物是一种可降解的化合物,具有与Lipofectamine 2000相似的SiRNA转染效率,细胞毒性明显低于BPEI和Lipofectamine 2000.提示脂质聚合物兼具阳离子脂质体和阳离子聚合物的优点.     

磁性纳米颗粒作为基因转染载体的研究

通过扫描电子显微镜和Zeta电位仪对磁性纳米颗粒的形貌、粒径、表面电位等进行了表征。利用凝胶电泳阻滞试验分析磁性纳米颗粒与DNA的结合情况,研究磁性纳米颗粒对DNA的保护效果,运用MTT和流式细胞术分析磁性纳米颗粒对细胞的毒性。以绿色荧光蛋白基因为报告基因进行293T细胞的转染,研究磁性纳米颗粒与质粒DNA不同比例条件下对293T细胞的转染效率,并与脂质体(Lipofectamine2000)介导的转染进行比较分析。结果表明,磁性纳米颗粒与DNA可以稳定结合,可以保护DNA免受酶的消化作用,当磁性纳米颗粒与DNA比为1 1时,转染效率最高,优于脂质体(Lipotamine2000)介导的转染,且对细胞的毒害作用小于Lipotamine2000。     

蔗糖脂肪酸酯型阳离子脂质体的制备及基因转运性能研究

该文采用蔗糖脂肪酸酯(sucrose fatty acid esters,SEs)作为助脂质与季铵盐型阳离子脂质1,2-双-[N-十四烷氧酰胺乙基-N,N-二甲基碘化铵](CTA14)制备阳离子脂质体,测定了脂质体的粒径及Zeta电位,脂质体的平均粒径为210~230 nm,Zeta电位为50~65 mV。DNA延滞实验表明,蔗糖脂肪酸酯型脂质体能够有效压缩DNA。阳离子脂质体与绿色荧光蛋白基因(plasmid green fluorescent protein-N2,p GFP-N2)结合,形成脂质体/DNA复合物,通过载入人喉癌细胞(Hep-2)和人宫颈癌细胞(Hela),观察其转染效率和细胞毒性。结果表明,阳离子脂质与SEs以质量比1:1、2:1混合制备的脂质体均能高效转染Hep-2和Hela细胞。毒性实验显示,SEs对两种细胞的毒性很小,阳离子脂质单独存在时对癌细胞具有一定的细胞毒性,随着SEs加入量的增加,脂质体对的细胞毒性也明显减小。该文进一步证实了SEs能够作为助脂质用于基因载体系统进行基因转运。     

新型"脂质-HAP-DNA复合体"的制备和用于真核细胞基因转染研究

为寻找一种简单、经济、有效的DNA递送系统用于基因转染和基因治疗,制备了表面电荷为正电的纳米HAP,与表面电荷为负电的DNA结合形成DNA-HAP复合物,采用逆向蒸发法,用卵磷脂、DOPE和胆固醇制备成脂质体包封DNA-HAP复合物形成脂质-HAP-DNA复合体,脂质体和HAP对照,对所形成的脂质-HAP-DNA复合体(LHD)的特性、包封率、转染Hela细胞的效果进行初步检测研究。所获得的脂质-HAP-DNA复合体呈球形、平均粒径为643nm;平均包封率达11.67%,为中性脂质体;能有效转染真核细胞。该方法可作为提高基因转染效果的简单、经济、有效的手段之一,也为进一步提高非病毒载体的转染效率提供了一个思路。     

阳离子脂质体的转染机制及转染效率影响因素

阳离子脂质体是一种非常具有发展前景的基因载体。简要介绍了阳离子脂质体的结构特点;着重讨论了阳离子脂质体作为基因载体时介导基因转移的机制以及在转染过程中对基因转染效率产生影响的主要因素。     

阳离子脂质体的转染机制及转染效率影响因素

阳离子脂质体是一种非常具有发展前景的基因载体。简要介绍了阳离子脂质体的结构特点;着重讨论了阳离子脂质体作为基因载体时介导基因转移的机制以及在转染过程中对基因转染效率产生影响的主要因素。     

阳离子脂质体及其在体内基因转染中的应用

阳离子脂质体已经成为基因转移使用最广泛的载体之一。本文从阳离子脂质体的理化特性、质粒／阳性子脂质复合体与生物大分子的相互作用、质粒／脂质复合体的基因转移机制等方面,对阳离子脂质体及其在体内基因转染中的应用进行了综述。     

阳离子脂质体介导RNA干扰的效果评价

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。     

新型“脂质HAPDNA复合体”的制备和用于真核细胞基因转染研究

为寻找一种简单、经济、有效的DNA递送系统用于基因转染和基因治疗,制备了表面电荷为正电的纳米HAP,与表面电荷为负电的DNA结合形成DNA-HAP复合物,采用逆向蒸发法,用卵磷脂、DOPE和胆固醇制备成脂质体包封DNA-HAP复合物形成脂质HAP-DNA复合体, 脂质体和HAP对照,对所形成的脂质HAP-DNA复合体（LHD）的特性、包封率、转染Hela细胞的效果进行初步检测研究。所获得的脂质HAP-DNA复合体呈球形、平均粒径为643nm;平均包封率达11.67%,为中性脂质体;能有效转染真核细胞。该方法可作为提高基因转染效果的简单、经济、有效的手段之一,也为进一步提高非病毒载体的转染效率提供了一个思路。     

脂质体介导转染法的原理与应用

脂质体是磷脂分散在水中时形成的脂质双分子层 ,又称为人工生物膜。最初 ,人们只是运用脂质体模拟生物膜 ,研究膜的构造及功能 ,从而发现了膜的融合及内吞作用 ,因而可用作外源物质进入细胞的载体。相对于电穿孔法和磷酸钙共沉淀转染法 ,脂质体介导转染法简便易行 ,成本适中 ,具有较高的转染率和较小的细胞毒性。1 .脂质体的组成和制备1 .1　组成脂质体的脂类　　现有的商业化脂质体均为阳离子脂类与中性脂类的复合体 ,如ＬｉｐｏｆｅｃｔＡＭＩＮＥ、Ｌｉｐｏｆｅｃｔｉｎ等 ,中性脂类多为二油酰磷脂酰乙醇胺 (ＤＯＰＥ)。其中 ,阳离子脂类…     

Tumor-targeting magnetic lipoplex delivery of short hairpin RNA suppresses IGF-1R overexpression of lung adenocarcinoma A549 cells in vitro and in vivo

Liposomal magnetofection potentiates gene transfection by applying a magnetic field to concentrate magnetic lipoplexes onto target cells. Magnetic lipoplexes are self-assembling ternary complexes of cationic lipids with plasmid DNA associated with superparamagnetic iron oxide nanoparticles (SPIONs). Type1insulin-like growth factor receptor (IGF-1R), an important oncogene, is frequently overexpressed in lung cancer and mediates cancer cell proliferation and tumor growth. In this study, we evaluated the transfection efficiency (percentage of transfected cells) and therapeutic potential (potency of IGF-1R knockdown) of liposomal magnetofection of plasmids expressing GFP and shRNAs targeting IGF-1R (pGFPshIGF-1Rs) in A549 cells and in tumor-bearing mice as compared to lipofection using Lipofectamine 2000. Liposomal magnetofection provided a threefold improvement in transgene expression over lipofection and transfected up to 64.1% of A549 cells in vitro. In vitro, IGF-1R specific-shRNA transfected by lipofection inhibited IGF-1R protein by 56.1 ± 6% and by liposomal magnetofection by 85.1 ± 3%. In vivo delivery efficiency of the pGFPshIGF-1R plasmid into the tumor was significantly higher in the liposomal magnetofection group than in the lipofection group. In vivo IGF-1R specific-shRNA by lipofection inhibited IGF-1R protein by an average of 43.8 ± 5.3%; that by liposomal magnetofection inhibited IGF-1R protein by 43.4 ± 5.7%, 56.3 ± 9.6%, and 72.2 ± 6.8%, at 24, 48, and 72 h, respectively, after pGFPshIGF-1R injection. Our findings indicate that liposomal magnetofection may be a promising method that allows the targeting of gene therapy to lung cancer.     

Generation of magnetic nonviral gene transfer agents and magnetofection in vitro

This protocol details how to design and conduct experiments to deliver nucleic acids to adherent and suspension cell cultures in vitro by magnetic force-assisted transfection using self-assembled complexes of nucleic acids and cationic lipids or polymers (nonviral gene vectors), which are associated with magnetic (nano) particles. These magnetic complexes are sedimented onto the surface of the cells to be transfected within minutes by the application of a magnetic gradient field. As the diffusion barrier to nucleic acid delivery is overcome, the full vector dose is targeted to the cell surface and transfection is synchronized. In this manner, the transfection process is accelerated and transfection efficiencies can be improved up to several 1,000-fold compared with transfections carried out with nonmagnetic gene vectors. This protocol describes how to accomplish the following stages: synthesis of magnetic nanoparticles for magnetofection; testing the association of DNA with the magnetic components of the transfection complex; preparation of magnetic lipoplexes and polyplexes; magnetofection; and data processing. The synthesis and characterization of magnetic nanoparticles can be accomplished within 3-5 d. Cell culture and transfection is then estimated to take 3 d. Transfected gene expression analysis, cell viability assays and calibration will probably take a few hours. This protocol can be used for cells that are difficult to transfect, such as primary cells, and may also be applied to viral nucleic acid delivery. With only minor alterations, this protocol can also be useful for magnetic cell labeling for cell tracking studies and, as it is, will be useful for screening vector compositions and novel magnetic nanoparticle preparations for optimized transfection efficiency in any cell type.     

Efficient and gentle siRNA delivery by magnetofection

Abstract

Magnetic force combined with magnetic nanoparticles recently has shown potential for enhancing nucleic acid delivery. Achieving effective siRNA delivery into primary cultured cells is challenging. We compared the utility of magnetofection with lipofection procedures for siRNA delivery to primary and immortalized mammalian fibroblasts. Transfection efficiency and cell viability were analyzed by flow cytometry and effects of gene knockdown were quantified by real-time PCR. Lipofectamine 2000 and magnetofection achieved high transfection efficiencies comparable to similar gene silencing effects of about 80%; the cytotoxic effect of magnetofection, however, was significantly less. Magnetofection is a reliable and gentle alternative method with low cytotoxicity for siRNA delivery into difficult to transfect cells such as mammalian fibroblasts. These features are especially advantageous for functional end point analyses of gene silencing, e.g., on the metabolite level.     

Cardiac-targeting magnetic lipoplex delivery of SH-IGF1R plasmid attenuate norepinephrine-induced cardiac hypertrophy in murine heart

Recent studies have demonstrated a number of molecular mechanisms contributing to the initiation of cardiac hypertrophy response to pressure overload. IGF1R (insulin-like growth factor-1 receptor), an important oncogene, is overexpressed in hypertrophic heart and mediates the hypertrophic pathology process. In this study, we applied with liposomal magnetofection that potentiated gene transfection by applying an external magnetic field to enhance its transfection efficiency. Liposomal magnetofection provided high efficiency in transgene expression in vivo. In vivo, IGF1R-specific-shRNA (small-hairpin RNA) by magnetofection inhibited IGF1R protein expression by 72.2±6.8, 80.7±9.6 and 84.5±5.6%, at 24, 48 and 72 h, respectively, after pGFPshIGF1R injection, indicating that liposomal magnetofection is a promising method that allows the targeting of gene therapy for heart failure. Furthermore, we found that the treated animals (liposomal magnetofection with shIGF1R) showed reduced septal and posterior wall thickness, reduced HW:BWs (heart weight-to-body weights) compared with controls. Moreover, we also found that liposomal magnetofection-based shIGF1R transfection decreased the expression level of p-ERK (phosphorylated extracellular-signal-regulated kinase)1/2, p-AKT1 (phosphorylated protein kinase B1) compared with untreated hearts. These results suggested that liposomal magnetofection-mediated IGF1R-specific-shRNA may be a promising method, and suppression the IGF1R expression inhibited norepinephrine-induced cardiac hypertrophic process via inhibiting PI3K (phosphoinositide 3-kinase)/AKT pathway.     

Stearylated octaarginine and artificial virus-like particles for transfection of siRNA into primary rat neurons

RNA interference (RNAi) provides a powerful experimental tool for sequence-specific gene silencing, allowing efficient analysis of gene function in a multitude of cell types. However, application of RNAi in primary mammalian neurons has been limited by low-transfection efficiency and considerable toxicity of conventional transfection methods. In this study, we evaluated a peptide-mediated and a polymer/lipid-based cellular delivery method for siRNA into rat primary neurons and compared the results with a commonly used liposomal transfection reagent. Stearylated octaarginine (Stearyl-R8) was used as polypeptide and artificial virus-like particles (AVPs) were used as a combined liposomal-polymeric vector, since both reagents have been previously shown to successfully transfect DNA into cell lines. Stearyl-R8 and AVPs both promoted siRNA transfection into primary hippocampal neurons via the endosomal pathway. SiRNA-mediated gene silencing could be effectively induced in primary neuron cultures. In comparison with the commonly used cationic liposome transfection agent, both novel reagents were less detrimental to cell metabolic activity. We conclude that these novel transfection methods yield performances comparable to cationic liposome-mediated transfection for siRNA, while being less cytotoxic in primary neurons. Stearyl-R8 and AVPs may therefore represent novel and more cost-efficient alternatives to conventional siRNA-transfection reagents.     

Transfection efficiency and cytotoxicity of polyethyleneimine-coated magnetic iron oxide nanoparticles in rooster sperm cells

Since the morphology of the rooster spermatozoa is different to other animal spermatozoa, the aim of the current study was to investigate the transfection efficiency and cytotoxicity of polyethyleneimine (PEI) coated magnetic iron oxide nanoparticles (MION) on these cells. Liposome/nucleic acid (NA) complexes and PEI-coated MION linked to the labeled oligonucleotides were used. Viability and percentage of exogenous nucleic acid uptake of spermatozoa were measured by flow cytometry analyses. The results showed a significant increase in exogenous nucleic acid uptake by rooster spermatozoa (P < 0.001) when treated with MION-NA complexes in comparison to liposome/NA. There were no significant differences between efficiency of lipid-based transfection agent and MION (P > 0.05) during short incubation period. MION with or without magnetic field, did not show significant cytotoxicity while the lipid-based transfection agent significantly decreased (P < 0.05) the viability of rooster spermatozoa. Results of this study showed that magnetofection and lipofection were both effective methods which increased exogenous nucleic acid uptake by rooster spermatozoa. However, the magnetofection method was more successful in maintaining the cell's survival than lipofection method.     

Plasmid DNA transfection using magnetite cationic liposomes for construction of multilayered gene-engineered cell sheet

Modification of cellular functions by overexpression of genes is being increasingly practiced for tissue engineering. In the present study, we investigated whether transfection efficiency could be enhanced by magnetofection that involves the use of plasmid DNA (pDNA)/magnetite cationic liposomes (MCLs) complexes (pDNA/MCL) and magnetic force. The transfection efficiencies of the magnetofection technique by pDNA/MCL in fibroblasts and keratinocytes using reporter genes were 36- and 10-fold higher, respectively, than those of a lipofection technique by cationic liposomes. Moreover, in vitro construction of three-dimensional (3D) tissues is an important challenge. We recently proposed a novel technique termed "magnetic force-based tissue engineering" (Mag-TE) to produce 3D tissues. Since the fibroblasts after magnetofection incorporated both magnetite nanoparticles and pDNA, we investigated whether multilayered heterotypic cell sheets expressing transgene could be fabricated by Mag-TE. First, the fibroblasts were seeded onto an ultra-low attachment culture plate. When a magnet was placed under the plate, the cells accumulated at the bottom of the culture plate. After 24 h of culture, the transgene-expressing cells formed a multilayered cell sheet-like structure. These results indicated that MCLs are a potent biomanipulation tool for both gene transfer and 3D tissue construction, suggesting that these techniques are useful for tissue engineering.     

The magnetofection method: using magnetic force to enhance gene delivery

In order to enhance and target gene delivery we have previously established a novel method, termed magnetofection, which uses magnetic force acting on gene vectors that are associated with magnetic particles. Here we review the benefits, the mechanism and the potential of the method with regard to overcoming physical limitations to gene delivery. Magnetic particle chemistry and physics are discussed, followed by a detailed presentation of vector formulation and optimization work. While magnetofection does not necessarily improve the overall performance of any given standard gene transfer method in vitro, its major potential lies in the extraordinarily rapid and efficient transfection at low vector doses and the possibility of remotely controlled vector targeting in vivo.     

Gene delivery to respiratory epithelial cells by magnetofection

BACKGROUND: For the topical application of DNA vector complexes to the airways, specific extracellular barriers play a major role. In particular, short contact time of complexes with the cell surface caused by the mucociliary clearance hinders cellular uptake of complexes. The aim of this study was to evaluate the ability of magnetofection, a technique based on the principle of magnetic drug targeting, to overcome these barriers in comparison with conventional nonviral gene transfer methods such as lipofection and polyfection. METHODS: Experiments were carried out on permanent (16HBE14o-) and primary airway epithelial cells (porcine and human), and native porcine airway epithelium ex vivo. Transfection efficiency and dose-response relationship of magnetofection were examined by luciferase reporter gene expression. Sedimentation patterns and uptake of gene transfer complexes were characterized by fluorescence and electron microscopy, respectively. RESULTS: We show that (i) application of a magnetic field allows the magnetofectins to sediment and to enrich at the cell surface within a few minutes, (ii) magnetofection bears an improved dose-response relationship, (iii) magnetofection enhances transfection efficiency in both, permanent and primary airway epithelial cells, and (iv) magnetofection leads to significant transgene expression at very short incubation times in an ex vivo airway epithelium organ model. CONCLUSIONS: Magnetofection provides a potential novel method, which may overcome fundamental limitations of nonviral gene transfer to the airways. Due to the accelerated enrichment at the cell surface it may be of major interest for in vivo applications, where long-term incubation times at the target tissue are hardly achievable.     

Tissue distribution and cancer growth inhibition of magnetic lipoplex-delivered type 1 insulin-like growth factor receptor shRNA in nude mice

The targeted delivery of therapeutic genes into specific tissues, as well as the determination of the biological fate and potential toxicity of nanoparticles, remains a highly relevant challenge for gene-based therapies. Type 1 insulin-like growth factor receptor (IGF-1R), an important oncogene, is frequently over-expressed in lung cancer and mediates cancer cell proliferation as well as tumor growth. In our previous studies, we have successfully applied gene delivery mediated by commercially available nanoparticles (CombiMAG) under a magnetic field, which suppresses IGF-1R expression in a non-small cell lung cancer cell line (A549) in vitro. In the present study, we aimed to investigate the biological distribution and target tumor suppression of magnetofection, as well as its potential toxicity via CombiMAG-carrying plasmids expressing green fluorescent protein (GFP) and short hairpin RNAs (shRNAs) targeting IGF-1R (pGFPshIGF-1Rs) in tumor-bearing mice. The peak expression in various organs appeared 48 h after transfection. Transgene expression via magnetofection was 3-fold improvement than via lipofection. On the 30th day after injection, the tumor size and weight of the CombiMAG-treated group (789.32 ± 39.43 mm(3), 105.5 ± 6.1 mg) were significantly decreased compared with those of the lipofection group (893.83 ± 31.23 mm(3), 164.5 ± 9.1 mg; P< 0.05), and the suppression rate was ～36%. After a 30-day observation, the injection of CombiMAG did not cause any apparent toxicity. Therefore, IGF-1R shRNA nanoparticles can be valuable and safe delivery agents for RNA interference therapy to tumors in vivo.