Effective gene delivery with liposomal bubbles and ultrasound as novel non-viral system

We developed the novel liposomal bubbles (Bubble liposomes) containing ultrasound imaging gas, perfluoropropane. Bubble liposomes were made of pegylated liposomes and were smaller than conventional microbubbles. Bubble liposomes also had a function as imaging agents in cardiosonography. In addition, Bubble liposomes could deliver plasmid DNA into various types of cells in vitro without cytotoxicity by the combination of ultrasound. In vivo gene delivery, Bubble liposomes could deliver plasmid DNA into mouse femoral artery by the transdermally exposure of ultrasound. This transfection efficiency was more effectively than lipofection method. Interestingly, the gene expression was only observed at the site of ultrasound exposure. Therefore, we concluded that Bubble liposomes could be good tools to establish tissue-specific gene delivery system as well as ultrasound imaging agents.     

Effective gene delivery with liposomal bubbles and ultrasound as novel non-viral system

We developed the novel liposomal bubbles (Bubble liposomes) containing ultrasound imaging gas, perfluoropropane. Bubble liposomes were made of pegylated liposomes and were smaller than conventional microbubbles. Bubble liposomes also had a function as imaging agents in cardiosonography. In addition, Bubble liposomes could deliver plasmid DNA into various types of cells in vitro without cytotoxicity by the combination of ultrasound. In vivo gene delivery, Bubble liposomes could deliver plasmid DNA into mouse femoral artery by the transdermally exposure of ultrasound. This transfection efficiency was more effectively than lipofection method. Interestingly, the gene expression was only observed at the site of ultrasound exposure. Therefore, we concluded that Bubble liposomes could be good tools to establish tissue-specific gene delivery system as well as ultrasound imaging agents.     

Intracellular routing of plasmid DNA during non-viral gene transfer

Gene transfer using non-viral vectors is a promising approach for the safe delivery of therapeutic DNA in genetic and acquired human diseases. Whereas the lack of specific immune response favors the use of plasmid-cationic polymer complexes, the limited efficacy and short duration of transgene expression impose major hurdles in the application of non-viral gene delivery techniques. Here, we review the major cellular, metabolic and physico-chemical impediments that non-viral vectors encounter before plasmid DNA enters the nucleus. Following endocytosis of DNA-polycation complexes, a large fraction of the DNA is targeted to the lysosomes. Since the cytosolic release of heterologous DNA is a prerequisite for nuclear translocation, entrapment and degradation of plasmid DNA in endo-lysosomes constitute one of the major impediments to efficient gene transfer. Plasmid DNA that escapes the endo-lysosomal compartment encounters the diffusional and metabolic barriers of the cytoplasm, reducing greatly the number of intact plasmids that reach the nucleosol. Nuclear translocation of DNA requires either the disassembly of the nuclear envelope or active nuclear transport via the nuclear pore complex. A better understanding of the cellular and molecular basis of non-viral vector trafficking from the extracellular compartment into the nucleus may provide strategies to overcome those obstacles that limit the efficiency of gene delivery.     

Lipid-based emulsion system as non-viral gene carriers

The genetic materials for systemic administration meet a number of huddles before they reach the nucleus of the target cells, such as enzymatic degradation in the bloodstream, extravascularization around the target tissue, endocytosis by the target cells, and endosomal escape of the genes. Therefore, there have been tremendous needs of effective gene carriers that can deliver the genetic materials to the target site. Of numerous approaches, recent studies have demonstrated that the lipid-based emulsion systems have the high potential as non-viral gene carriers: 1 lipid emulsions are biocompatible because their major constituents are composed of the non-toxic oils and amphiphilic lipids; 2 the cationic lipid emulsions can form nano-sized complexes with negatively charged DNAs, through which the genetic materials can be protected from the enzymatic degradation in the body fluids; 3 The emulsion/DNA complexes are shown to be stable in the bloodstream since their surfaces are rarely recognized by the immune-related cells and serum proteins; and 4 the surfaces of the emulsion complexes are readily modified by varying the lipid composition. In this review, highlighted are the recent advances in the emulsion-based gene carriers.     

Viral and non-viral methods for gene transfer into skeletal muscle

Skeletal muscle is an important target for genetic manipulation and its stable post-mitotic nature allows the use of both integrating and non-integrating viral and non-viral vectors. Adeno-associated viral vectors and naked plasmid DNA are currently the vectors of choice for gene transfer into muscle. The last couple of years have seen major breakthroughs in the field of vector delivery systems, particularly those using the vascular route, such that gene therapy of muscular dystrophies and the use of muscle as a platform for the production of secreted proteins has become a clinical possibility.     

Polyethylenimine-based non-viral gene delivery systems.

Gene therapy has become a promising strategy for the treatment of many inheritable or acquired diseases that are currently considered incurable. Non-viral vectors have attracted great interest, as they are simple to prepare, rather stable, easy to modify and relatively safe, compared to viral vectors. Unfortunately, they also suffer from a lower transfection efficiency, requiring additional effort for their optimization. The cationic polymer polyethylenimine (PEI) has been widely used for non-viral transfection in vitro and in vivo and has an advantage over other polycations in that it combines strong DNA compaction capacity with an intrinsic endosomolytic activity. Here, we give some insight into strategies developed for PEI-based non-viral vectors to overcome intracellular obstacles, including the improvement of methods for polyplex preparation and the incorporation of endosomolytic agents or nuclear localization signals. In recent years, PEI-based non-viral vectors have been locally or systemically delivered, mostly to target gene delivery to tumor tissue, the lung or liver. This requires strategies to efficiently shield transfection polyplexes against non-specific interaction with blood components, extracellular matrix and untargeted cells and the attachment of targeting moieties, which allow for the directed gene delivery to the desired cell or tissue. In this context, materials, facilitating the design of novel PEI-based non-viral vectors are described.     

Pharmacodynamic approach to study the gene transfer process employing non-viral vectors

In the present work we set out to apply pharmacodynamic concepts derived from dose–response curves (Potency and Efficacy) to characterize the gene transfer efficiency of a vector:DNA complex. We employed two widely used vectors, the cationic lipid DOTAP (N,N,N-trimethyl 1-2-3-bis (1-oxo-9-octa-decenyl)oxy-(Z,Z)-1-propanaminium methyl sulfate) and the cationic polymer PEI (polyethylenimine, 800 kDa) to transfect several constructions of the green fluorescent protein cDNA. The analysis of dose–response curves indicated that in all cases the goodness-of-fit was > 0.99. Potency is a measure that provides information on gene activity per amount of DNA. Efficacy is a measure of maximum gene expression achievable using a specific vector:DNA complex, and depends on both the intrinsic efficacy of the gene (evaluated using different vectors to transfer the same gene construct) and on vector efficacy in DNA delivery (evaluated using a single vector to deliver different gene constructs). The results suggest that Potency and Efficacy are objective parameters for describing and comparing the goodness of vectors, as well as the intrinsic efficacy of a given gene construct. Furthermore, they are useful tools that may contribute to a better understanding of the mechanistic gene transfer process of each vector.     

Calcium phosphate nanoparticles as novel non-viral vectors for targeted gene delivery

Calcium phosphate nanoparticles present a unique class of non-viral vectors, which can serve as efficient and alternative DNA carriers for targeted delivery of genes. In this study we report the design and synthesis of ultra-low size, highly monodispersed DNA doped calcium phosphate nanoparticles of size around 80 nm in diameter. The DNA encapsulated inside the nanoparticle is protected from the external DNase environment and could be used safely to transfer the encapsulated DNA under in vitro and in vivo conditions. Moreover, the surface of these nanoparticles could be suitably modified by adsorbing a highly adhesive polymer like polyacrylic acid followed by conjugating the carboxylic groups of the polymer with a ligand such as p-amino-1-thio-beta-galactopyranoside using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride as a coupling agent. We have demonstrated in our studies that these surface modified calcium phosphate nanoparticles can be used in vivo to target genes specifically to the liver.     

Influence of lipid components on gene delivery by polycation liposomes: Transfection efficiency, intracellular kinetics and in vivo tumor inhibition

Transfection efficiency of non-viral gene vectors is influenced by many factors, including chemical makeup, cellular uptake pathway and intracellular delivery. To investigate the effect of lipid saturation on transfection efficiency of polycation liposomes (PCLs), a soybean phospholipids (SPL), egg phospholipids (EPL) and hydrogenated soybean phosphatidylcholine (HSPC) series was used to prepare PCLs. Testing these PCLs in a luciferase assay indicated that with increasing saturation (SPL相似文献   

A novel method to prepare liposomes containing amikacin.

This work describes a novel method to prepare liposomal amikacin composed of soyabean lecithin and cholesterol; these were also prepared using two other methods (cast film method and proliposome method). Encapsulation efficiency was evaluated. Liposomes prepared by the new method, which combines the method of preparing proliposomes with freeze-drying, had the highest encapsulation efficiency. The influence of drug to lipid ratio on the encapsulation efficiency was investigated. The in vitro efflux of amikacin from liposomes with different lecithin: cholesterol ratios was also investigated.     

星点设计优化非病毒载体前阳离子脂质体的制备工艺

目的 采用星点设计优化前阳离子脂质体的制备工艺并考察前阳离子脂质体的有关性质.方法 以薄膜分散膜挤压法制备前阳离子脂质体,以平均粒径、转染效率为指标,考察鱼精蛋白(Protamine)与质粒DNA的比例、脂质体骨架材料CHETA浓度、CHETA与质粒DNA的比例3个因素对制备条件的影响,将实验结果用数学方法处理并进行方程模型拟合,根据拟合方程及反应曲面图确定指标的最优值和各因素相对应的最佳取值范围.以LacZ为报告基因转染肝癌细胞HepG2,定量测定β-半乳糖苷酶活性和总蛋白含量,并计算转染效率.结果 影响平均粒径、转染效率的3个主要因子的最佳取值分别为:Protamine/DNA (2.5:1);CHETA (45%);CHETA/DNA (4:1).所制前阳离子脂质体形态近似于球体,平均粒径228.9±8 nm,多分散指数为0.122±0.02,Zeta电位为–25.08±2.5 mV,载基因前阳离子脂质体转染HepG2肝癌细胞转染效率为24.26±2.6 mU · mg-1.结论 星点设计应用简便、预测性好,制备的前阳离子脂质体符合设计要求.     

Non-linear pharmacodynamics in the transfection efficiency of a non-viral gene delivery system

Transfection efficiencies using LipofectAMINE varied by more than three orders of magnitude depending on the concentrations of lipid and plasmid DNA (pDNA) used to prepare the lipoplexes. When lipoplexes were formed at lower concentrations a striking positive but non-linear relationship was found between dose and transfection efficiency, while at higher (i.e., normally used) concentrations a linear relationship was maintained. To determine the contribution of intracellular pharmacokinetics (PK) and pharmacodynamics (PD) to the observed nonlinearity, we quantified pDNA in whole cells and nuclei by real-time PCR and compared the results with the transfection efficiencies. There was no significant difference in the efficiency of intracellular PK; however, a remarkable difference was observed in the efficiency of PD. Analysis of individual cells by confocal laser scanning microscopy (CLSM) revealed that the amount of nuclear-delivered pDNA was higher for lipoplexes prepared at the normal concentration (NCL) compared to those of lipoplexes prepared at low concentration (LCL). Moreover, the size of the NCL was larger than that of the LCL. Both the size of the lipoplex particle and the dose appear to contribute to the non-linear efficiency of PD. These results emphasize the need to control not only intracellular PK, but also PD for the rational development of non-viral gene delivery systems.     

Unraveling the intracellular efficacy of dextran-histidine polycation as an efficient nonviral gene delivery system

In this study, we attempted to elucidate the capability of a natural polymer dextran, by modification with histidine, to be an efficient, safe and promising nucleic acid delivery system in gene therapy. Physicochemical characterizations were performed to get an insight into the derivative. The efficiency of the derivative as a gene delivery vehicle was also studied in depth using fluorescence microscopy. Extensive efforts were made to have a better understanding of the cellular dynamics involved. The derivative proved itself to be 6.7-fold more excelling than PEI in its transfecting capability. Mechanisms underlying cellular internalization, vector unpacking, intranuclear localization and transgene expression were also investigated. The possibility of recruiting intracellular histone to promote the entry of the gene into the nucleus seemed promising. Our findings also explored the links that mediate the correlation between the uptake of the derivative and various endocytic pathways. The results thus obtained reflect the success of the entire journey of the synthesized delivery vehicle.     

Preparation and characterization of a novel nonviral gene transfer system: procationic-liposome-protamine-DNA complexes

Procationic-liposome-protamine-DNA (PLPD) vector, a novel nonviral gene delivery system, that may further adsorb transferrin (Tf) at its surface via electrostatic interactions to form Tf-PLPD, was prepared from soybean phosphatidylcholine (PC), cholesterol (Chol), and a kind of cholesterol derivative, CHETA(cholest-5-en-3-ol(3β)-[2-[[4-[(carboxymethyl)dithio]-1-iminobutyl] amino] ethyl] carba- mate) containing disulfide bond by film dispersion-filteration method. Central composite design was used to optimize the formulation. The presence of serum did not affect the transfection activity of PLPD or Tf-PLPD and the cell viability was not affected significantly when the cells were incubated with the complexes for 4 hr at 37°C. Compared with one kind of cationic liposomes(liposome-protamine-DNA), the PLPD had much less cytotoxicity to three hepar cell lines(including HepG2, SMMC7721, and Chang's normal heptocyte). The procationic lipoplex described here, combining the condensing effect of protamine and the targeting capability of Tf, was a perspective nonviral vector for gene delivery system.     

Dendrosome-dendriplex inside liposomes: as a gene delivery system

Dendrosomes are lipid vesicular entities containing entrapped dendrimer-DNA complexes and possessing low toxicity, acceptable transfection efficiency, and good in vivo tolerance. Herein, an attempt was made to explore the potential of dendrosomes as a gene delivery system combining the advantages of both polyamidoamine (PAMAM) dendrimer (nucleic acid condensation, facilitated endosomal release) and of non-cationic liposomes (increased cellular uptake, low cytotoxicity), and at the same time overcoming the drawbacks of these system (low encapsulation efficiency of non-cationic liposome and toxicity of dendrimers). Dendrosomes were assembled by loading optimized DNA-dendrimer complexes into liposomes prepared by solvating of dried lipid films made of DOPE/EggPC/Cholesterol (4.74:4.75:1.5 mole ratio). Dendrosomes were characterized in terms of size, zeta, encapsulation efficiency and the ability to protect the system from DNA degradation. The transfection efficiency and toxicity of the preparations were evaluated in HeLa cells using flow cytometry and CellTiter-Blue^® methods. The efficient transfection and low toxicity makes them an appealing alternative to be further explored for gene delivery in vivo.     

Multifunctional envelope-type nano device (MEND) as a non-viral gene delivery system

In this review, we describe a key role of octaarginine (R8) in developing our new concept of "Programmed Packaging", by which we succeeded in creating a multifunctional envelope-type nano device (MEND) as a non-viral gene-delivery system. This concept can be applied not only to nuclear targeting of plasmid DNA (pDNA) but also to cytosolic delivery of functional nucleic acids such as oligonucleotides or siRNA. This concept has been extended to other organelles such as mitochondria as a foundation for innovative nanomedicine. Finally, we discuss the rate-limiting step in gene delivery by comparing non-viral and viral gene delivery systems, which clearly indicates the importance of nuclear disposition of pDNA for efficient transfection.     

Angelica sinensis polysaccharide nanoparticles as novel non-viral carriers for gene delivery to mesenchymal stem cells

This study centers on the use of a nanoparticle based on the polysaccharide from Angelica sinensis (ASP) as an efficient and safe non-viral gene vector. After modification with branched low molecular weight polyethylenimine (1200 Da), the cationized ASP (cASP) was combined with the plasmid encoding transforming growth factor-beta 1 (TGF-β1) to form a spherical nano-scaled particle (i.e., cASP-pTGF-β1 nanoparticle). This nanoparticle was applied to transfect rat bone marrow mesenchymal stem cells and human umbilical cord mesenchymal stem cells. As a result, nanoparticles (cASP/pDNA weight ratio 10:1) had the greatest transfection efficiency in both cells, which was significantly higher than those of Lipofectamine2000 and PEI (25 kDa). This was in agreement with the findings of the semi-quantitative RT-PCR and live cell imaging. These nanoparticles were also less toxic than Lipofectamine2000 and PEI (25 kDa). Therefore, cASP could be a potential candidate for a novel non-viral gene vector.From the Clinical EditorThese authors demonstrate the use of a nanoparticle-based efficient and safe non-viral gene vector delivery system via a spherical nanoparticle based on a polysaccharide from Angelica sinensis, with parameters superior to Lipofectamine2000.     

Micro- and nanobubbles: A versatile non-viral platform for gene delivery

Micro- and nanobubbles provide a promising non-viral strategy for ultrasound mediated gene delivery. Microbubbles are spherical gas-filled structures with a mean diameter of 1–8 μm, characterised by their core–shell composition and their ability to circulate in the bloodstream following intravenous injection. They undergo volumetric oscillations or acoustic cavitation when insonified by ultrasound and, most importantly, they are able to resonate at diagnostic frequencies. It is due to this behaviour that microbubbles are currently being used as ultrasound contrast agents, but their use in therapeutics is still under investigation. For example, microbubbles could play a role in enhancing gene delivery to cells: when combined with clinical ultrasound exposure, microbubbles are able to favour gene entry into cells by cavitation. Two different delivery strategies have been used to date: DNA can be co-administered with the microbubbles (i.e. the contrast agent) or ‘loaded’ in purposed-built bubble systems – indeed a number of different technological approaches have been proposed to associate genes within microbubble structures. Nanobubbles, bubbles with sizes in the nanometre order of magnitude, have also been developed with the aim of obtaining more efficient gene delivery systems. Their small sizes allow the possibility of extravasation from blood vessels into the surrounding tissues and ultrasound-targeted site-specific release with minimal invasiveness. In contrast, microbubbles, due to their larger sizes, are unable to extravasate, thus and their targeting capacity is limited to specific antigens present within the vascular lumen. This review provides an overview of the use of microbubbles as gene delivery systems, with a specific focus on recent research into the development of nanosystems. In particular, ultrasound delivery mechanisms, formulation parameters, gene-loading approaches and the advantages of nanometric systems will be described.     

Saturable, non-Michaelis-Menten uptake of liposomes by the reticuloendothelial system.

Multilamellar vesicles (300-350 nm) were infused into the rat femoral vein at the rate of 4, 40 and 400 nmol phosphatidycholine min-1 for 6 h using [3H]inulin as an aqueous marker. The time courses of blood concentration of vesicles, normalized for infusion rate, were not superimposable, showing the non-linearity of liposome disposition in the blood circulation. These time courses of blood concentration were well fitted by a single Michaelis-Menten equation. On the other hand, the time courses of tissue content could not be so accommodated. Additionally, the observed relationship between the uptake of liposomes by the liver and their clearance from it and other organs differed essentially from a simulation based on Michaelis-Menten type saturable kinetics. Therefore, it is suggested that there is a time-dependent non-Michaelis-Menten type process in the phagocytosis of macrophages in the reticuloendothelial system.