Solid-phase synthesis of 89 polyamine-based cationic lipids for DNA delivery to mammalian cells

The ability of non-viral gene delivery systems to overcome extracellular and intracellular barriers is a critical issue for future clinical applications of gene therapy. In recent years much effort has been focused on the development of a variety of DNA carriers, and cationic liposomes have become the most common non-viral gene delivery system. Solid-phase synthesis was used to produce three libraries of polyamine-based cationic lipids with diverse hydrophobic tails. These were characterised, and structure-activity relationships were determined for DNA binding and transfection ability of these compounds when formulated as cationic liposomes. Two of the cationic lipids produced high-efficiency transfection of human cells. Surprisingly, these two compounds were from the library with two headgroups and one aliphatic tail, a compound class regarded as detergent-like and little investigated for transfection. These cationic lipids are promising reagents for gene delivery and illustrate the potential of solid-phase synthesis methods for lipoplex discovery.     

Polymers for DNA delivery

Nucleic acid delivery has many applications in basic science, biotechnology, agriculture, and medicine. One of the main applications is DNA or RNA delivery for gene therapy purposes. Gene therapy, an approach for treatment or prevention of diseases associated with defective gene expression, involves the insertion of a therapeutic gene into cells, followed by expression and production of the required proteins. This approach enables replacement of damaged genes or expression inhibition of undesired genes. Following two decades of research, there are two major methods for delivery of genes. The first method, considered the dominant approach, utilizes viral vectors and is generally an efficient tool of transfection. Attempts, however, to resolve drawbacks related with viral vectors (e.g., high risk of mutagenicity, immunogenicity, low production yield, limited gene size, etc.), led to the development of an alternative method, which makes use of non-viral vectors. This review describes non-viral gene delivery vectors, termed "self-assembled" systems, and are based on cationic molecules, which form spontaneous complexes with negatively charged nucleic acids. It introduces the most important cationic polymers used for gene delivery. A transition from in vitro to in vivo gene delivery is also presented, with an emphasis on the obstacles to achieve successful transfection in vivo.     

Recent progress in cationic polymeric gene carriers for cancer therapy

In recent years,various carriers for gene delivery nave been developed for biomedical applications.Among all kinds of gene carriers,cationic polymeric carriers for delivery therapeutic gene as non-viral carriers have received growing interests due to their improved high transfection efficiency with the relative safety.In particular,the advancement of novel polymeric gene carriers has gained much progress in the development of effective anticancer therapy.Herein,this review focused on the development of cationic polymeric carriers for cancer therapy,including polyethylenimine(PEI),polyamidoamine(PAMAM) dendrimers,polylysine(PLL),chitosan and modified cationic polymers.And recent progresses in the development of novel polymeric carriers for gene delivery,such as targeted gene carriers,responsive gene carriers and multifunctional gene carriers,were summarized.Finally,the future perspectives in the development of novel polymeric carriers for delivery gene were presented.     

含氟高分子基因载体

阳离子高分子被广泛应用为非病毒类基因载体,但这类高分子材料的转染效率与细胞毒性之间通常存在"恶性"关联,即获得高转染效率时往往会伴随严重的细胞毒性.如何制备兼具高效、低毒特点的高分子载体是成功实施基因治疗的关键.含氟高分子是一类具有独特理化性质的高分子,能够在低电荷密度条件下与核酸形成稳定的复合物,从而实现高效、低毒的基因转染.含氟功能基团可帮助阳离子高分子改善复合物稳定性、细胞内吞、内涵体逃逸、胞内核酸释放等多个环节,从而赋予了含氟高分子在基因递送过程中的氟效应.该专论系统地总结了含氟高分子基因载体的研究,介绍了含氟高分子的基因递送性能、作用机理以及在基因治疗、基因编辑中的应用,并对含氟高分子载体的未来发展进行了展望.     

金属配合物作为核酸释放的载体

基因治疗是指利用一种载体将健康的基因载入细胞替换致病的基因.由基因缺陷导致的人类疾病达1200多种,最合理的选择是采用基因替换的方法进行治疗.基因治疗的关键问题是解决"使用何种载体才能安全有效地将治疗基因载入靶细胞".非病毒基因载体主要是一些有机阳离子物种,一直受到极大重视;近年来,磷酸钙、纳米粒子和金属配合物释放核酸的功能也开始受到关注.本文总结了金属配合物作为非病毒基因载体使用的研究进展,希望由此理解配合物释放核酸的优势和不足之处.     

Microfluidic approaches for gene delivery and gene therapy

Recent advances in microfluidics have created new and exciting prospects for gene delivery and therapy. The micro-scaled environment within microfluidic systems enables precise control and optimization of multiple processes and techniques used in gene transfection and the production of gene and drug transporters. Traditional non-viral gene transfection methods, such as electroporation, microinjection and optical gene transfection, are improved from the use of innovative microfluidic systems. Additionally, microfluidic systems have also made the production of many viral and non-viral vectors controlled, automated, and reproducible. In summary, the development and application of microfluidic systems are producing increased efficiency in gene delivery and promise improved gene therapy results.     

Novel cationic 6-lauroxyhexyl lysinate modified poly(lactic acid)-poly(ethylene glycol) nanoparticles enhance gene transfection

The leading principle of non-viral delivery systems for gene therapy is to mediate high levels of gene expression with low cytotoxicity. Nowadays, biodegradable nanoparticles formulated with poly(lactic acid)-poly(ethylene glycol) (PLA-PEG) were wildly developed. However, the relative lower gene transfection efficiency and higher cytotoxicity still remained critical problems. To address these limitations, PLA-PEG nanoparticles have been composited with other components in their formulation. Here, a novel cationic lipid, 6-lauroxyhexyl lysinate (LHLN), was fabricated onto PLA-PEG nanoparticles as a charge modifier to improve the transfection efficiency and cytotoxicity. The obtained cationic LHLN modified PLA-PEG nanoparticles (LHLN-PLA-PEG NPs) could condense pDNA thoroughly via electrostatic force, leading to the formation of the LHLN-PLA-PEG NPs/pDNA complexes (NPs/DNA complexes). The nanoparticles obtained have been characterized in relation to their physicochemical and biological properties, and the results are extremely promising in terms of low cell toxicity and high transfection efficiency. These results indicated that the novel cationic LHLN modified PLA-PEG nanoparticles could enhance gene transfection in vitro and hold the potential to be a promising non-viral nanodevice.     

Lipothiophosphoramidates for gene delivery: critical role of the cationic polar headgroup

When considering a family of cationic lipids designed for gene delivery, the nature of the cationic polar head probably has a great influence on both the transfection efficacy and toxicity. Starting from a cationic lipothiophosphoramidate bearing a trimethylammonium headgroup, we report herein the impact on gene transfection activity of the replacement of the trimethylammonium moiety by a trimethylphosphonium or a trimethylarsonium group. A series of three different human epithelial cell lines were used for the experimental transfection studies (HeLa, A549 and 16HBE14o(-)). The results basically showed that such structural modifications of the cationic headgroup can lead to a high transfection efficacy at low lipid/DNA charge ratios together with a low cytotoxicity. It thus appears that the use of a trimethylarsonium cationic headgroup for the design of efficient gene carriers, which was initially proposed in the lipophosphoramidate series, can be extended to other series of cationic lipids and might therefore have great potential for the development of novel non-viral vectors in general.     

Chemoenzymatic synthesis and high-throughput screening of an aminoglycoside-polyamine library: identification of high-affinity displacers and DNA-binding ligands

Chemoenzymatic parallel synthesis and high-throughput screening were employed to develop a multivalent aminoglycoside-polyamine library for use as high-affinity cation-exchange displacers and DNA-binding ligands. Regioselective lipase-catalyzed acylation, followed by chemical aminolysis, was used to generate vinyl carbonate and vinyl carbamate linkers, respectively, of the aminoglycosidic cores. These were further derivatized with polyamines, leading to library generation. A parallel batch-displacement assay was employed to identify the efficacy of the library candidates as potential displacers for protein purification. Using this approach, low-molecular-mass displacers with affinities higher than those previously observed have been identified. The aminoglycoside-polyamine library was also screened for DNA binding efficacy using an ethidium bromide displacement assay. These highly cationic molecules exhibited strong DNA-binding properties and may have potential for enhanced gene delivery.     

Cationic nanoemulsions as non-viral vectors for plasmid DNA delivery

Non-viral gene carriers have been extensively investigated as alternatives to viral vectors for therapeutic gene delivery. Many cationic lipid carriers including liposomes, emulsions, and solid lipid nanoparticles are used to transfer plasmid DNA. Stable nanoemulsions were prepared and modified by conjugating fatty acids with cationic amino acids including lysine, arginine, and histidine with the help of carbodiimide. Concentrations of crosslinker and amino acids were optimized to obtain the maximal surface potential. The zeta potential and size distribution of the cationic nanoemulsions were measured using photon correlation spectroscopy. The morphology of nanoemulsion-DNA complexes was examined by transmission electron microscopy. The transfection efficiencies and cytotoxicity of three cationic nanoemulsions were evaluated using 3T3 fibroblast cells. The maximal zeta potentials of lysine-, arginine-, and histidine-modified nanoemulsions were 50, 43, and 7 mV, respectively. The transfection efficiencies of amino acid-modified nanoemulsions were in the order of lysine > arginine > histidine. Low cytotoxicities of these three amino acid-modified nanoemulsions were observed. A facile and inexpensive in situ modification for producing cationic nanoemulsions was developed. The results show the potential of amino acid-modified cationic nanoemulsions as non-viral vectors for gene delivery.     

Vinyl polymers as non-viral gene delivery carriers: current status and prospects

Since the first application of polymers as non-viral gene delivery systems in 1965 by Vaheri and Pagano using functionalised dextran (A. Vaheri and J. S. Pagano, "Infectious poliovirus RNA: a sensitive method of assay", Virology 1965, 27, 434-6), a large number of different polymers have been developed, studied and compared for application as DNA carriers. Vinyl-based polymers are one type of polymers that have gained considerable interest. The interest in developing this particular type of polymer is partly related to the straightforward way in which large amounts of these polymers can be prepared by radical (co)polymerisation. This opens up a path for establishing a wide range of structure-property relations using polymer libraries. The present review aims to give an overview of past and ongoing research using vinyl-based gene delivery systems. The application of cationic, neutral and zwitterionic polymers as DNA carriers is summarised and discussed. [structure: see text] Chemical structure of DEAE-functionalised dextran.     

Effective gene delivery into primary dendritic cells using synthesized PDMAEMA-iron oxide nanocubes

Dendritic cells (DCs) have been a target of vaccine delivery, gene therapy, and cancer immunotherapy. However, gene delivery to primary DCs using traditional non-viral molecules has been a difficult challenge. Herein we have developed a gene delivery system to primary DCs using magnetic iron oxide nanocubes (MCs) coated with cationic polymer under the induction of a magnetic field. The MCs were coated with positively charged polymer, poly(2-dimethylamino) ethyl methacrylate (MCs-PD) before the plasmid gene (pMAX-GFP) was adsorbed on their surfaces. Three different sizes (15, 40 and 90 nm) of MCs were synthesized, and subsequently, PDMAEMA was assembled onto the MC surfaces (MCs-PD). MCs-PD exhibited zeta potentials of +23 to +26 mV, and the obtained particles showed superparamagnetic character with saturation magnetization of 17–66 emu/g. The MCs-PD of 10–100 μg/mL showed low toxicity on bone marrow-derived dendritic cells (BMDCs) in MTT assay, and they were well taken up by BMDCs under a magnetic field. Moreover, the particles with small size exhibited the enhanced plasmid transfection efficiency without the activation of BMDCs. The MCs-PD could be a promising non-viral gene delivery system that helps to manipulate primary DCs in vitro, which will be beneficial for cell-based immunotherapy.     

Correlation between structure and transfection efficiency: a study of DC-Chol--DOPE/DNA complexes

The supramolecular structural nature of some cationic liposomes-DNA complexes, currently used as vehicles in non-viral gene delivery, has been elucidated by recent X-ray diffraction experiments. The relationship between the chemico-physical properties of these self-assembled structures and their transfection efficiency is extensively studied. Here we report a first comprehensive structural study by using energy dispersive X-ray diffraction, of the complex DC-Chol--DOPE/DNA (3beta[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol dioleoylphosphatidylethanolamine/DNA), which has been classified as one of the most effective in in-vivo experiments. Our results show that DC-Chol--DOPE/DNA lipoplexes have a columnar inverted hexagonal structure, which is not influenced by the cationic liposome/DNA charge ratio. The transfection efficiency of C6 rat glioma cells by DC-Chol--DOPE/DNA lipoplexes and the toxicity of lipoplexes to cells are dramatically affected by cationic liposome/DNA weight ratio.It seems therefore that the lipoplex structures have not any influence on transfection efficiency and toxicity in our experimental system.     

脂质纳米颗粒的制备及在基因治疗中的应用

基因治疗已经成为人类治疗疾病的一种重要手段.然而,为了将基因药物用于临床,需要更加复杂的递送系统.脂质纳米颗粒(LNPs)系统是目前领先的非病毒递送系统,在治疗诊断学方面取得了许多令人鼓舞的进展,其具有实现基因药物临床治疗应用的潜力.由于LNPs纳米尺寸的优势及类脂化合物的生物相容性和生物降解性,LNPs能够克服阻碍基...     

Branched and linear poly(ethylene imine)-based conjugates: synthetic modification, characterization, and application

Poly(ethylene imine)s (PEIs) are widely used in different applications, but most extensively investigated as non-viral vector systems. The high ability of cationic PEIs to complex and condense negatively charged DNA and RNA combined with their inherent proton sponge behavior accounts for the excellent efficiency in gene delivery. Further chemical modifications of the polymer expand the application potential, primarily aiming at increased transfection efficiency, cell selectivity and reduced cytotoxicity. Improvements in the synthesis of tailor-made PEIs in combination with new in-depth analytical techniques offer the possibility to produce highly purified polymers with defined structures. The contemporary strategies towards linear and branched poly(ethylene imine)s with modified surface characteristics, PEI-based copolymers as well as conjugates with bioactive molecules will be discussed. In this regard, the versatile branched PEIs have been successfully modified in a statistical manner, whereas the linear counterparts open avenues to design and synthesize well-defined architectures, in order to exploit their high potential in gene delivery.     

Cloud computing approaches to accelerate drug discovery value chain

Continued advancements in the area of technology have helped high throughput screening (HTS) evolve from a linear to parallel approach by performing system level screening. Advanced experimental methods used for HTS at various steps of drug discovery (i.e. target identification, target validation, lead identification and lead validation) can generate data of the order of terabytes. As a consequence, there is pressing need to store, manage, mine and analyze this data to identify informational tags. This need is again posing challenges to computer scientists to offer the matching hardware and software infrastructure, while managing the varying degree of desired computational power. Therefore, the potential of "On-Demand Hardware" and "Software as a Service (SAAS)" delivery mechanisms cannot be denied. This on-demand computing, largely referred to as Cloud Computing, is now transforming the drug discovery research. Also, integration of Cloud computing with parallel computing is certainly expanding its footprint in the life sciences community. The speed, efficiency and cost effectiveness have made cloud computing a 'good to have tool' for researchers, providing them significant flexibility, allowing them to focus on the 'what' of science and not the 'how'. Once reached to its maturity, Discovery-Cloud would fit best to manage drug discovery and clinical development data, generated using advanced HTS techniques, hence supporting the vision of personalized medicine.     

Synthesis of poly(beta-amino ester)s with thiol-reactive side chains for DNA delivery

The safe and efficient delivery of DNA remains the major barrier to the clinical application of non-viral gene therapy. Here, we present novel, biodegradable polymers for gene delivery that are capable of simple graft modification and demonstrate the ability to respond to intracellular conditions. We synthesized poly(beta-amino ester)s using a new amine monomer, 2-(pyridyldithio)-ethylamine (PDA). These cationic, degradable polymers contain pyridyldithio functionalities in the side chains that react with high specificity toward thiol ligands. This reactivity is demonstrated using both mercaptoethylamine (MEA) and the thiol peptide RGDC, a ligand that binds with high affinity to certain integrin receptors. These two polymer derivatives displayed strong DNA binding as determined using electrophoresis and dye exclusion assays. In addition, the MEA-based polymer and plasmid DNA were shown to self-assemble into cationic complexes with effective diameters as low as 100 nm. Furthermore, this DNA binding ability was substantially reduced in response to intracellular glutathione concentrations, which may aid in DNA unpackaging inside the cell. These complexes also displayed low cellular toxicity and were able to mediate transfection at levels comparable to PEI in human hepatocellular carcinoma cells. These results suggest that PDA-based poly(beta-amino ester)s may serve as a modular platform for polymer-mediated gene delivery.     

基于门舒特金反应的酶触发自降解高分子载体及其在基因输送中的应用

用邻位苄基溴与双胺进行门舒特金反应,合成了2种线性的季铵盐阳离子聚合物.其中,含有酚基酯键的阳离子聚合物,一旦进入细胞后,可以在细胞内的酯酶催化下快速水解,使得聚合物自降解断裂为不带电的非季铵盐小分子,从而快速释放DNA,最终达到提高转染效率的目的.通过对复合物纳米颗粒的粒径和电势测定,证明了这2种阳离子聚合物都能够有效地结合DNA形成表面带正电的复合物纳米颗粒.凝胶阻滞电泳实验表明,所合成的阳离子聚合物都能稳定地包裹DNA.而在酯酶条件下,含有酚基酯键的阳离子聚合物可以发生降解,使得纳米复合物释放出DNA.同时,含有酚基酯键的阳离子聚合物由于其独特的可降解性,相比于PEI,降低了细胞毒性.在体外细胞转染实验中,2种阳离子聚合物都有较好的转染效果.其中酯酶响应的载体在高N/P下依然表现出较高的转染效率,说明该阳离子载体能够在细胞内有效降解并释放出DNA.     

含双Cyclen阳离子脂质的合成及与DNA的相互作用

设计合成了一类新型的阳离子脂质小分子化合物1.化合物1含有双cyclen亲水端和甾体疏水基团.凝胶电泳实验显示,含胆固醇和皂素的双大环多胺阳离子脂质化合物1与二油酰磷脂酰乙醇胺( DOPE)形成的阳离子脂质体能够与DNA有效作用,在氮磷比为6时即可完全包裹DNA,具有作为非病毒基因载体的潜在应用价值,为设计新型阳离子脂...     

Higher-throughput screening for Caco-2 permeability utilizing a multiple sprayer liquid chromatography/tandem mass spectrometry system

In the current drug discovery environment, higher-throughput analytical assays have become essential to keep pace with the screening demands for drug metabolism and pharmacokinetics (DMPK) attributes. This has been dictated by advances primarily in chemical procedures, notably combinatorial and parallel syntheses, which has resulted in many-fold increases in the number of compounds requiring DMPK evaluation. Because of its speed and specificity, liquid chromatography/tandem mass spectrometry (LC/MS/MS) has become the dominant technology for sample analysis in the DMPK screening assays. For higher-throughput assays, analytical speed as well as other factors such as method development, data processing, quality control, and report generation, must be optimized. The four-way multiplexed electrospray interface (MUX), which allows for the analysis of four LC eluents simultaneously, has been adopted to maximize the rate of sample introduction into the mass spectrometer. Generic fast-gradient HPLC methods that are suitable for approximately 80% of the new chemical entities encountered have been developed. In-house-written software programs have been used to streamline information flow within the system, and for quality control by automatically identifying analytical anomalies. By integrating these components together with automated method development and data processing, a system capable of screening 100 compounds per week for Caco-2 permeability has been established.