磁性纳米颗粒作为载体在基因转染中的研究进展

磁性纳米颗粒具有很强的结合、浓缩与保护DNA的作用,具有超顺磁性、较高的安全性和低的免疫原性,可以结合大片段DNA,在外加磁场的作用下可实现安全、高效的基因靶向性运输,提高外源基因的转染效率。由于磁性纳米颗粒的独特性质,使得其作为非病毒载体在基因治疗中的应用进展迅速。我们简要介绍磁性纳米材料的特点、种类及结构,磁性纳米基因载体的特点,以及磁性纳米颗粒作为载体在基因转染中的应用情况。     

硅纳米颗粒作为基因转染载体的研究

通过不同浓度的NaCl、NaI修饰硅纳米颗粒,用琼脂糖凝胶电泳分析硅纳米颗粒与DNA结合力及对DNA的保护作用,同时用绿色荧光蛋白基因作报告基因,以硅纳米颗粒作为基因转染的载体,转染HT1080细胞。经电镜观察证实硅纳米颗粒进入细胞内;硅纳米颗粒与DNA结合后,能对DNA起保护作用;并且硅颗粒作为基因转染的载体,将绿色荧光蛋白基因导入HT1080细胞,用荧光显微镜观察到发绿色荧光的细胞。结果表明,硅纳米颗粒可作为基因转染的载体。     

猪肾细胞脂质磁转染系统的构建与表征

磁性纳米基因载体是一种非病毒基因载体,经过功能性基团修饰后能够连接阳离子转染剂构建细胞转染系统。本文将磁转染技术结合常用的脂质体转染,形成了一种新型动物体细胞转染方法,即称脂质磁转染(Liposomal magnetofection,LMF)。这将为体细胞克隆培育转基因动物提供稳定遗传的细胞系。为构建脂质磁性纳米基因载体复合物系统,本研究利用一种磁性纳米基因载体通过分子自组装与脂质阳离子转染剂结合,用于携带外源基因转染动物体细胞。通过原子力显微镜(AFM)观测、ζ电位-粒度等分析表征手段,研究磁性纳米基因载体的形貌、粒径分布、负载及浓缩DNA的方式。结果表明,通过猪肾(PK)细胞的LMF实验,与脂质体(Lipofectamine2000)介导的转染比较,具有较高的转染率,更重要的是克服了脂质体转染瞬时表达的缺陷。MTT细胞毒性试验结果也显示该方法具有较低的细胞毒性。因此LMF是一种切实可行的高效低毒性的细胞转染方法。     

程序组装多功能化纳米基因载体

基因治疗成功的关键之一是采用安全高效的载体递送基因。多功能化的非病毒基因载体可克服转染过程中的多种屏障,提高转染效率。通过科研实践和文献查阅,本文总结出实现载体多功能化的三种程序组装方式,即层层自组装、共聚物自组装和脂质掺入,并对近年来国内外通过程序组装构建多功能非病毒基因载体的研究进展做一简要综述。     

量子点在基因转染中的应用研究进展

量子点因其独特的纳米尺寸效应、光学特性和生物相容性,既能作为纳米载体与目的基因结合,又能作为纳米荧光标记物跟踪记录其在转染过程中的位置,给基因工程的发展带来了新的契机。在阐述量子点用于基因转染的优势、标记基因的方法等基础上,作者系统综述了量子点在基因转染中的应用,并对其发展趋势和应用前景进行了展望。     

纳米基因载体在植物遗传转化中的应用

纳米基因载体已成功地应用于生物医学领域并显示了优越的转染效率、良好的生物相容性和有效的基因保护作用。近年来,纳米颗粒作为基因载体在植物转导中的应用潜力越来越受到关注,也为植物遗传工程提供了新的可能性。在阐述纳米载体的特性、在植物细胞中的转导机制及转导优势的基础上,重点讨论了纳米载体在植物转基因中的应用,并对其前景进行了展望。     

纳米颗粒作为基因载体的应用

随着纳米技术的发展,纳米颗粒因具有较高的转染效率、良好的靶向性及有效的基因保护作用而被用作基因载体。简要介绍了磁性纳米颗粒、硅纳米颗粒及阳离子多聚物颗粒等的研究进展。     

无机纳米粒子作为基因载体的研究进展

转染是将具生物功能的核酸转移、运送到细胞内,并使其在细胞内维持生物功能的过程。作为现代生物化学和分子生物学中的一种主要技术手段,转染对于基因治疗有重要的意义。无机纳米粒子作为基因载体受到人们日益广泛的关注,其具有易于制备,可进行多样化的表面修饰等多种优势。本文将概述无机纳米粒子作为基因载体的现状及其对基因表达的影响。     

基于纳米基因载体的动植物遗传转化研究进展

转基因技术在动植物优良新品种的培育中发挥着重要作用,而随着纳米生物技术的发展,基于纳米材料构建基因载体的动植物转基因技术,对于发展动植物转基因新方法以及加速转基因种质材料的大规模制备、优良新品种的培育进程具有更为重要的意义。综述了纳米基因载体的种类与性质,并结合动植物遗传育种的研究进展,分析了纳米基因载体相比于其他载体的特点及优势,同时,重点阐述了基于纳米基因载体的基因转染技术的基本原理和操作过程,及其在动植物遗传转化中的应用,以期为动植物基因工程改造提供新思路。     

壳聚糖纳米粒介导基因转染的影响因素及改性修饰

壳聚糖带正电荷,可与带负电荷的DNA结合形成纳米级的多聚复合物(纳米粒)。作为一种基因载体,壳聚糖对DNA具有很好的结合和保护作用,对生物体无毒、相容性好,被广泛应用于基因转染及基因预防和治疗中。壳聚糖的主要缺点是转染效率较低,但对其进行改性或修饰后,有可能提高其转染效率。     

Fabrication of functionalized double-lamellar multifunctional envelope-type nanodevices using a microfluidic chip with a chaotic mixer array

Multifunctional envelope-type nanodevices (MENDs) are very promising non-viral gene delivery vectors because they are biocompatible and enable programmed packaging of various functional elements into an individual nanostructured liposome. Conventionally MENDs have been fabricated by complicated, labor-intensive, time-consuming bulk batch methods. To avoid these problems in MEND fabrication, we adopted a microfluidic chip with a chaotic mixer array on the floor of its reaction channel. The array was composed of 69 cycles of the staggered chaotic mixer with bas-relief structures. Although the reaction channel had very large Péclet numbers (>10(5)) favorable for laminar flows, its chaotic mixer array led to very small mixing lengths (<1.5 cm) and that allowed homogeneous mixing of MEND precursors in a short time. Using the microfluidic chip, we fabricated a double-lamellar MEND (D-MEND) composed of a condensed plasmid DNA core and a lipid bilayer membrane envelope as well as the D-MEND modified with trans-membrane peptide octaarginine. Our lab-on-a-chip approach was much simpler, faster, and more convenient for fabricating the MENDs, as compared with the conventional bulk batch approaches. Further, the physical properties of the on-chip-fabricated MENDs were comparable to or better than those of the bulk batch-fabricated MENDs. Our fabrication strategy using microfluidic chips with short mixing length reaction channels may provide practical ways for constructing more elegant liposome-based non-viral vectors that can effectively penetrate all membranes in cells and lead to high gene transfection efficiency.     

Massive calcium-activated endocytosis without involvement of classical endocytic proteins

We describe rapid massive endocytosis (MEND) of >50% of the plasmalemma in baby hamster kidney (BHK) and HEK293 cells in response to large Ca transients. Constitutively expressed Na/Ca exchangers (NCX1) are used to generate Ca transients, whereas capacitance recording and a membrane tracer dye, FM 4-64, are used to monitor endocytosis. With high cytoplasmic adenosine triphosphate (ATP; >5 mM), Ca influx causes exocytosis followed by MEND. Without ATP, Ca transients cause only exocytosis. MEND can then be initiated by pipette perfusion of ATP, and multiple results indicate that ATP acts via phosphatidylinositol-bis 4,5-phosphate (PIP(2)) synthesis: PIP(2) substitutes for ATP to induce MEND. ATP-activated MEND is blocked by an inositol 5-phosphatase and by guanosine 5'-[γ-thio]triphosphate (GTPγS). Block by GTPγS is overcome by the phospholipase C inhibitor, U73122, and PIP(2) induces MEND in the presence of GTPγS. MEND can occur in the absence of ATP and PIP(2) when cytoplasmic free Ca is clamped to 10 μM or more by Ca-buffered solutions. ATP-independent MEND occurs within seconds during Ca transients when cytoplasmic solutions contain polyamines (e.g., spermidine) or the membrane is enriched in cholesterol. Although PIP(2) and cholesterol can induce MEND minutes after Ca transients have subsided, polyamines must be present during Ca transients. MEND can reverse over minutes in an ATP-dependent fashion. It is blocked by brief β-methylcyclodextrin treatments, and tests for involvement of clathrin, dynamins, calcineurin, and actin cytoskeleton were negative. Therefore, we turned to the roles of lipids. Bacterial sphingomyelinases (SMases) cause similar MEND responses within seconds, suggesting that ceramide may be important. However, Ca-activated MEND is not blocked by reagents that inhibit SMases. MEND is abolished by the alkylating phospholipase A(2) inhibitor, bromoenol lactone, whereas exocytosis remains robust, and Ca influx causes MEND in cardiac myocytes without preceding exocytosis. Thus, exocytosis is not prerequisite for MEND. From these results and two companion studies, we suggest that Ca promotes the formation of membrane domains that spontaneously vesiculate to the cytoplasmic side.     

Mechanistic analysis of massive endocytosis in relation to functionally defined surface membrane domains

A large fraction of endocytosis in eukaryotic cells occurs without adaptors or dynamins. Here, we present evidence for the involvement of lipid domains in massive endocytosis (MEND) activated by both large Ca transients and amphipathic compounds in baby hamster kidney and HEK293 cells. First, we demonstrate functional coupling of the two MEND types. Ca transients can strongly facilitate detergent-activated MEND. Conversely, an amphipath with dual alkyl chains, ditridecylphthalate, is without effect in the absence of Ca transients but induces MEND to occur within seconds during Ca transients. Ca transients, like amphipaths, enhance the extraction of lipids from cells by β-cyclodextrins. Second, we demonstrate that electrical and/or optical signals generated by selected membrane probes are nearly insensitive to MEND, suggesting that those probes segregate into membrane domains that are not taken up by MEND. Triphenylphosphoniums are increasingly excluded from domains that internalize as the carbon chain length increases from 4 to 12. The small cationic membrane dye, FM 4-64, binds well to domains that internalize, whereas a closely related dye with a larger hydrophobic moiety, di-4-ANEPPDHQ (ANEPPDHQ) is excluded. Multiple carrier-type ionophores and a small amphipathic anion, niflumic acid, are also excluded. Probes with modest MEND sensitivity include the hydrophobic anion, dipicrylamine, carbonyl cyanide m-chlorophenylhydrazone, and NBD-phosphatidylethanolamine. Third, we demonstrate that large Ca transients can strongly enhance the extracellular binding of several membrane probes, monitored electrically or optically, consistent with a more disordered membrane with more amphipath-binding sites. Fluorescence shifts of ANEPPDHQ report increased disorder of the extracellular monolayer after large Ca transients, consistent with an increased propensity of the membrane to phase separate and vesiculate. Collectively, the results indicate that >50% of the outer monolayer is ordered and can be selectively internalized during MEND responses initiated by two very different cell perturbations.     

Microbial dormancy improves development and experimental validation of ecosystem model

Climate feedbacks from soils can result from environmental change followed by response of plant and microbial communities, and/or associated changes in nutrient cycling. Explicit consideration of microbial life-history traits and functions may be necessary to predict climate feedbacks owing to changes in the physiology and community composition of microbes and their associated effect on carbon cycling. Here we developed the microbial enzyme-mediated decomposition (MEND) model by incorporating microbial dormancy and the ability to track multiple isotopes of carbon. We tested two versions of MEND, that is, MEND with dormancy (MEND) and MEND without dormancy (MEND_wod), against long-term (270 days) carbon decomposition data from laboratory incubations of four soils with isotopically labeled substrates. MEND_wod adequately fitted multiple observations (total C–CO₂ and ¹⁴C–CO₂ respiration, and dissolved organic carbon), but at the cost of significantly underestimating the total microbial biomass. MEND improved estimates of microbial biomass by 20–71% over MEND_wod. We also quantified uncertainties in parameters and model simulations using the Critical Objective Function Index method, which is based on a global stochastic optimization algorithm, as well as model complexity and observational data availability. Together our model extrapolations of the incubation study show that long-term soil incubations with experimental data for multiple carbon pools are conducive to estimate both decomposition and microbial parameters. These efforts should provide essential support to future field- and global-scale simulations, and enable more confident predictions of feedbacks between environmental change and carbon cycling.     

New directions in liposome gene delivery

The history of liposomes, progress in liposome gene delivery, and future directions are discussed. Specific characteristics of liposomes and DNA:liposome complexes have been identified that are essential for optimal delivery and gene expression. Of particular interest are the requirements for increased delivery and high levels of gene expression in vivo. At present, significant efforts are focused towards achieving specific delivery and gene expression in target organs and tissues.     

Exploring N-imidazolyl-O-carboxymethyl chitosan for high performance gene delivery

Chitosan shows good biocompatibility and biodegradability, but the poor water solubility and low transfection efficiency hinder its applications as a gene delivery vector. We here report the detailed synthesis and characterization of a novel ampholytical chitosan derivative, N-imidazolyl-O-carboxymethyl chitosan (IOCMCS), used for high performance gene delivery. After chemical modification, the solubility of the resulting polymer is enhanced, and the polymer is soluble in a wide pH range (4-10). Gel electrophoresis study reveals the strong binding ability between plasmid DNA and the IOCMCS. Moreover, the IOCMCS does not induce remarkable cytotoxicity against human embryonic kidney (HEK293T) cells. The cell transfection results with HEK293T cells using the IOCMCS as gene delivery vector demonstrate the high transfection efficiency, which is dependent on the degree of imidazolyl substitution. Therefore, the IOCMCS is a promising candidate as the DNA delivery vector in gene therapy due to its high solubility, high gene binding capability, low cytotoxicity, and high gene transfection efficiency.     

Production of high-titer transmission-defective RNA virus-based episomal vector using tangential flow filtration

In recent years, viral vector based in vivo gene delivery strategies have achieved a significant success in the treatment of genetic diseases. RNA virus-based episomal vector lacking viral glycoprotein gene (ΔG-REVec) is a nontransmissive gene delivery system that enables long-term gene expression in a variety of cell types in vitro, yet in vivo gene delivery has not been successful due to the difficulty in producing high titer vector. The present study showed that tangential flow filtration (TFF) can be effectively employed to increase the titer of ΔG-REVec. Concentration and diafiltration of ΔG-REVec using TFF significantly increased its titer without loss of infectious activity. Importantly, intracranial administration of high titer vector enabled persistent transgene expression in rodent brain.     

Nanosecond Electroporation: Another Look

As the medical field moves from treatment of diseases with drugs to treatment with genes, safe and efficient gene delivery systems are needed to make this transition. One such safe, non-viral, and efficient gene delivery system is electroporation (electrogenetherapy). Exciting discoveries using electroporation could make this technique applicable to drug and vaccine delivery in addition to gene delivery. Typically milli and microsecond pulses have been used for electroporation. Recently, the use of nanosecond electrical pulses (10-300 ns) at very high magnitudes (10-300 kV/cm) has been studied for direct DNA transfer to the nucleus in vitro. This article reviews the work done using high-intensity nanosecond pulses, termed as nanosecond electroporation (nsEP), in electroporation gene delivery systems.     

Well-controlled cationic water-soluble phospholipid polymer-DNA nanocomplexes for gene delivery

The facile synthesis of biocompatible and nontoxic gene delivery vectors has been the focus of research in recent years due to the high potential in treating genetic diseases. 2-Methacryloxyethyl phosphorylcholine (MPC) copolymers were recently studied for their ability to produce nontoxic and biocompatible materials. The synthesis of well-defined and water-soluble MPC polymer based cationic vectors for gene delivery purposes was therefore attractive, due to the potential excellent biocompatibility of the resulting copolymers. Herein, cationic MPC copolymers of varying architectures (block versus random) were produced by the reversible addition--fragmentation chain transfer (RAFT) polymerization technique. The copolymers produced were evaluated for their gene delivery efficacy in the presence and absence of serum. It was found that copolymer architectures and molecular weights do affect their gene delivery efficacy. The statistical copolymers produced larger particles, and showed poor gene transfection efficiency as compared to the diblock copolymers. The diblock copolymers served as efficient gene delivery vectors, in both the presence and absence of serum in vitro. To the best of our knowledge, this is the first report where the effect of architecture of MPC based copolymer on gene delivery efficacy has been studied.     

Nonviral gene therapy targeting cardiovascular system

The goal of gene therapy is either to introduce a therapeutic gene into or replace a defective gene in an individual's cells and tissues. Gene therapy has been urged as a potential method to induce therapeutic angiogenesis in ischemic myocardium and peripheral tissues after extensive investigation in recent preclinical and clinical studies. A successful gene therapy mainly relies on the development of the gene delivery vector. Developments in viral and nonviral vector technology including cell-based gene transfer will further improve transgene delivery and expression efficiency. Nonviral approaches as alternative gene delivery vehicles to viral vectors have received significant attention. Recently, a simple and safe approach of gene delivery into target cells using naked DNA has been improved by combining several techniques. Among the physical approaches, ultrasonic microbubble gene delivery, with its high safety profile, low costs, and repeatable applicability, can increase the permeability of cell membrane to macromolecules such as plasmid DNA by its bioeffects and can provide as a feasible tool in gene delivery. On the other hand, among the promising areas for gene therapy in acquired diseases, ischemic cardiovascular diseases have been widely studied. As a result, gene therapy using advanced technology may play an important role in this regard. The aims of this review focus on understanding the cellular and in vivo barriers in gene transfer and provide an overview of currently used chemical vectors and physical tools that are applied in nonviral cardiovascular gene transfer.