Novel cationic lipids possessing protonated cyclen and imidazolium salt for gene delivery

In this study, two novel protonated cyclen and imidazolium salt-containing cationic lipids differing only in their hydrophobic region (cholesterol or diosgenin) have been designed and synthesized for gene delivery. Cationic liposomes were easily prepared from each of these lipids individually or from the mixtures of each cationic lipid and dioleoylphosphatidyl ethanolamine (DOPE). Several studies including DLS, gel retardation assay, ethidium bromide intercalation assay, and TEM demonstrated that these amphiphilic molecules are able to bind and compact DNA into nanometer particles that could be used as non-viral gene delivery agents. Our results from in vitro transfection showed that in association with DOPE, two cationic lipids can induce effective gene transfection in HEK293 cells. Furthermore, the gene transfection efficiencies of two cationic lipids were dramatically increased in the presence of calcium ion (Ca²⁺). It is notable that the gene transfection abilities of two cationic lipids were maintained in the presence of 10% serum. Besides, different cytotoxicity was found for two lipoplexes. This study demonstrates that the title cationic lipids have large potential to be efficient non-viral gene vector.     

Cationic transfection lipids in gene therapy: successes,set-backs,challenges and promises

The clinical success of gene therapy is critically dependent on the development of efficient and safe gene delivery reagents, popularly known as "Transfection Vectors". The transfection vectors commonly used in gene therapy are mainly of two types: viral and non-viral. The efficiencies of viral transfection vectors are, in general, superior to their non-viral counterparts. However, the myriads of potentially adverse immunogenic aftermaths associated with the use of viral vectors are increasingly making the non-viral gene delivery reagents as the vectors of choice. Among the existing arsenal of non-viral gene delivery reagents, the distinct advantages associated with the use of cationic transfection lipids include their: (a) robust manufacture; (b) ease in handling & preparation techniques; (c) ability to inject large lipid:DNA complexes and (d) low immunogenic response. The present review will highlight the successes, set-backs, challenges and future promises of cationic transfection lipids in non-viral gene therapy.     

Biotinylated Cyclen‐Contained Cationic Lipids as Non‐Viral Gene Delivery Vectors

A series of 1, 4, 7, 10‐tetraazacyclododecane (cyclen)‐based cationic lipids, namely 5a–c bearing a biotin moiety and a variety of end groups (cholesterol, diosgenin, and α‐tocopherol) via biodegradable carbamate bond linkage were prepared and applied as non‐viral gene delivery vectors. The liposomes formed from 5 and dioleoylphosphatidylethanolamine could bind and condense plasmid DNA into nanoparticles with appropriate size and zeta potentials. All biotinylated cyclen cationic lipids showed higher cell viability than commercially available lipofectamine 2000 even at high N/P ratios, while their transfection efficiency was relatively lower. Further, results indicate that among the three lipids, α‐tocopherol‐containing compound 5c has higher DNA‐binding ability, lower cytotoxicity, and higher transfection efficiency. Transfection in two different cell lines revealed that these lipoplexes have higher gene delivery efficiency toward tumor cells.     

Cationic lipid vectors for plasmid DNA delivery

Successful gene therapy depends on efficient gene transfer vectors. Viral vectors and non-viral vectors have been investigated extensively. Cationic lipids are non-viral vectors, which resemble traditional pharmaceuticals, display little immunogenicity, and have no potential for viral infection. However, toxicity and low transfection efficiency are two barriers limiting the clinical applications of cationic lipids. Over the last decade, hundreds of cationic lipids have been synthesized to address these problems. In this brief review, we summarized recent research results concerning the structures of DNA/liposomes complexes, some important strategies used to design different classes of cationic lipids, and use of disulfide cationic lipids in plasmid DNA delivery.     

Current status of non-viral gene therapy for CNS disorders

ABSTRACT

Introduction: Viral and non-viral vectors have been used as methods of delivery in gene therapy for many CNS diseases. Currently, viral vectors such as adeno-associated viruses (AAV), retroviruses, lentiviruses, adenoviruses and herpes simplex viruses (HHV) are being used as successful vectors in gene therapy at clinical trial levels. However, many disadvantages have risen from their usage. Non-viral vectors like cationic polymers, cationic lipids, engineered polymers, nanoparticles, and naked DNA offer a much safer option and can therefore be explored for therapeutic purposes.

Areas covered: This review discusses different types of viral and non-viral vectors for gene therapy and explores clinical trials for CNS diseases that have used these types of vectors for gene delivery. Highlights include non-viral gene delivery and its challenges, possible strategies to improve transfection, regulatory issues concerning vector usage, and future prospects for clinical applications.

Expert opinion: Transfection efficiency of cationic lipids and polymers can be improved through manipulation of molecules used. Efficacy of cationic lipids is dependent on cationic charge, saturation levels, and stability of linkers. Factors determining efficacy of cationic polymers are total charge density, molecular weights, and complexity of molecule. All of the above mentioned parameters must be taken care for efficient gene delivery.     

Gene transfer efficacies of serum-resistant amino acids-based cationic lipids: dependence on headgroup, lipoplex stability and cellular uptake

Serum is a major obstacle to efficient cationic liposome-mediated gene transfection. In this paper, three alkaline amino acids based cationic lipids including lysinylated cholesterol (lipid 1), histidinylated cholesterol (lipid 2) and argininylated cholesterol (lipid 3) were used as non-viral gene vectors. The physicochemical properties such as size, Zeta potential, stability and cellular uptake of the lipoplexes formed from lipids 1-3 as well as the transfection efficacies with or without serum were investigated. The results demonstrated that lipid 1 and lipid 3 showed good properties in lipoplex stability and cellular uptake. Interestingly, lipid 3-based liposome showed serum-enhanced effect on the gene transfection. The transfection efficiency of lipid 1 and lipid 3 was remarkably higher than that of lipid 2. Moreover, they exhibited 10-20-fold more efficaciously than the control, 1,2-dioleoyloxy-3-(trimethylammonio)-propane (DOTAP) liposome in serum-containing media. The data suggested the strong effect of the type of the headgroup on gene transfection. The lysine/arginine derivative cationic lipids could be promising nonviral vectors for gene delivery in vivo.     

非病毒载体在肿瘤基因治疗领域的研究进展

随着肿瘤基因治疗领域的研究进展，临床应用逐渐增多。载体是癌症基因治疗的主要难题。当前广泛使用的病毒载体存在的安全问题越来越受到人们的重视，已经有多种非病毒载体用于肿瘤基因治疔，如：裸DNA直接注射、阳离子脂质、阳离子聚合物。研究非病毒载体的目标是：它能像靶向的合成病毒载体那样对肿瘤组织表现出高度特异性；具有很高的转染效率；潜在的安全性问题能够被控制。     

阳离子脂质材料及其在基因转染中的应用

目的通过对已报道的阳离子脂质材料的结构及其应用的综述,为该类基因转染载体的合理设计和进一步应用提供借鉴。方法对已有的各种阳离子脂质材料的结构及基因转染特性进行分析,并探讨其对基因转染的影响。结果阳离子脂质材料在基因转染中有着巨大的应用潜力,特定的脂质材料结构赋予其在基因转染中不同的效能。结论对阳离子脂质材料结构特征的综合分析,为进一步合理构建新的高效阳离子脂质体基因递送载体提供了思路。     

Structure-activity relationship in cationic lipid mediated gene transfection

Non-viral synthetic vectors for gene delivery represent a safer alternative to viral vectors. Their main drawback is the low transfection efficiency, especially in vivo. Among the non-viral vectors currently in use, the cationic liposomes composed of cationic lipids are the most common. This review discusses the physicochemical properties of cationic lipids, the formation, macrostructure and specific parameters of the corresponding formulated liposomes, and the effect of all these parameters on transfection efficiency. The optimisation of liposomal vectors requires both the understanding of the biological variables involved in the transfection process, and the effect of the structural elements of the cationic lipids on these biological variables. The biological barriers relevant for in vitro and in vivo transfection are identified, and solutions to overcome them based on rational design of the cationic lipids are discussed. The review focuses on the relationship between the structure of the cationic lipid and the transfection activity. The structure is analysed in a modular manner. The hydrophobic domain, the cationic head group, the backbone that acts as a scaffold for the other domains, the linkers between backbone, hydrophobic domain and cationic head group, the polyethyleneglycol chains and the targeting moiety are identified as distinct elements of the cationic lipids used in gene therapy. The main chemical functionalities used to built these domains, as well as overall molecular features such as architecture and geometry, are presented. Studies of structure-activity relationships of each cationic lipid domain, including the authors', and the trends identified by these studies, help furthering the understanding of the mechanism governing the formation and behaviour of cationic liposomes in gene delivery, and therefore the rational design of new improved cationic lipids vectors capable of achieving clinical significance.     

The design of cationic lipids for gene delivery

Synthetic gene delivery vectors are gaining increasing importance in gene therapy as an alternative to recombinant viruses. Among the various types of non-viral vectors, cationic lipids are especially attractive as they can be prepared with relative ease and extensively characterised. Further, each of their constituent parts can be modified, thereby facilitating the elucidation of structure-activity relationships. In this forward-looking review, cationic lipid-mediated gene delivery will mainly be discussed in terms of the structure of the three basic constituent parts of any cationic lipid: the polar headgroup, hydrophobic moiety and linker. Particular emphasis will be placed on recent advances in the field as well as on our own original contributions. In addition to reviewing critical physicochemical features (such as headgroup hydration) of monovalent lipids, the use of headgroups with known nucleic-acid binding modes, such as linear and branched polyamines, aminoglycosides and guanidinium functions, will be comprehensively assessed. A particularly exciting innovation in linker design is the incorporation of environment-sensitive groups, the intracellular hydrolysis of which may lead to more controlled DNA delivery. Examples of pH-, redox- and enzyme-sensitive functional groups integrated into the linker are highlighted and the benefits of such degradable vectors can be evaluated in terms of transfection efficiency and cationic lipid-associated cytotoxicity. Finally, possible correlations between the length and type of hydrophobic moiety and transfection efficiency will be discussed. In conclusion it may be foreseen that in order to be successful, the future of cationic lipid-based gene delivery will probably require the development of sophisticated virus-like systems, which can be viewed as "programmed supramolecular systems" incorporating the various functions required to perform in a chronological order the different steps involved in gene transfection.     

阳离子脂质体用做基因传递载体的研究进展

 基因治疗的难题之一在于研制安全有效的基因传递载体。常用的基因传递载体分为病毒载体和非病毒载体两类,其中,非病毒载体中的阳离子脂质体因具有低毒性与免疫原性、生物相容性好、易于制备等优点而受到广泛关注,具有良好的应用前景。近年来对阳离子脂质体载体的研究主要集中在对其传递基因机制的考察、各种影响其转基因效率的因素的探求、应用各种方法研制安全性和转染活性更佳的新型阳离子脂质体等方面。文中从转基因特点、传递机制、常用的制备材料、影响转基因效率的因素、近年来出现的新型阳离子脂质体等方面综述了此类基因传递载体的研究进展。     

Chitosan-DNA nanoparticles as non-viral vectors in gene therapy: strategies to improve transfection efficacy.

Currently, the major drawback of gene therapy is the gene transfection rate. The two main types of vectors that are used in gene therapy are based on viral or non-viral gene delivery systems. The viral gene delivery system shows a high transfection yield but it has many disadvantages, such as oncogenic effects and immunogenicity. However, cationic polymers, like chitosan, have potential for DNA complexation and may be useful as non-viral vectors for gene therapy applications. Chitosan is a natural non-toxic polysaccharide, it is biodegradable and biocompatible, and protects DNA against DNase degradation and leads to its condensation. The objective of this paper was to summarize the state of the art in gene therapy and particularly the use of chitosan to improve the transfection efficiency in vivo and in vitro.     

非病毒基因递送载体的研究进展

目前,基因药物的递送成为药学研究的热点,基因递送载体主要包括病毒载体和非病毒载体。非病毒基因载体的毒性低,生物相容性好,转染效率高,具有潜在的临床应用价值。本文就靶向递送基因载体、多功能基因载体、同时载基因与化疗药物的载体、智能基因载体和脂质体等非病毒基因递送载体的研究进展做一综述。     

Asymmetric 1-alkyl-2-acyl phosphatidylcholine: a helper lipid for enhanced non-viral gene delivery

Rationally designed asymmetrical alkylacyl phosphatidylcholines (APC) have been synthesized and evaluated as helper lipids for non-viral gene delivery. A long aliphatic chain (C22-C24) was introduced at the 1-position of glycerol backbone, a branched lipid chain (C18) at the 2-position, and a phosphocholine head group at the 3-position. The fusogenicity of APC depends on the length and degree of saturation of the alkyl chain. Cationic lipids were formulated with APC as either lipoplexes or nanolipoparticles, and evaluated for their stability, transfection efficiency, and cytotoxicity. APC mediated high in vitro transfection efficiency, and had low cytotoxicity. Small nanolipoparticles (less than 100 nm) can be obtained with APC by applying as low as 0.1% PEG-lipid. Our study extends the type of helper lipids that are suitable for gene transfer and points the way to improve non-viral nucleic acid delivery system other than the traditional cationic lipids optimization.     

The role of the disulfide group in disulfide-based polymeric gene carriers

Background: An essential prerequisite for successful gene therapy is the development of safe and efficient gene delivery carriers. For this purpose, cationic polymers have been widely studied as non-viral carriers, but they generally suffer from low transfection efficiency and/or high cytotoxicity. To address these problems, disulfide-based cationic polymers have been designed as intelligent gene carriers that are capable of inducing highly efficient gene transfection with low cytotoxicity. Objective: The present review discusses the effects of the disulfide linker on the gene delivery properties of cationic polymers in relation to various gene delivery barriers. Methods: The literature regarding the gene delivery barriers encountered by polymeric gene delivery is reviewed and discussed in relation to the presence of the disulfide moiety in these gene carriers. Conclusions: The presence of disulfide linkages in cationic polymers can in many aspects favorably influence the gene delivery properties, such as increasing DNA binding ability, enabling de-shielding of ‘stealth’ (PEG) groups, fine-tuning of the buffer capacity for enhanced endosomal escape, improving carrier-unpacking and decreasing cytotoxicity. Therefore, disulfide-based cationic polymers are promising candidates for the next generation of non-viral carriers.     

脂质-鱼精蛋白-DNA复合物的构建及其对细胞的体外转染

目的研究新型非病毒载体脂质-聚阳离子-DNA（LPD）复合物的制备方法及其对体外细胞的转染率。方法用薄膜-挤压法制备空白阳离子脂质体，与鱼精蛋白-DNA复合物在室温孵育后，得到LPD；用透射电镜观察其形态，用激光粒度仪测定其粒径和zeta电位；LPD与DNA酶I溶液在37 ℃下孵育不同时间后，用琼脂糖凝胶电泳观察其降解情况；用荧光法测定其包封率；用X-gal染色法考察了LPD对张氏（Chang）肝细胞，HepG2肝癌细胞和SMMC-7721肝癌细胞的转染率。结果LPD的形态近似于球体，平均粒径为143.5 nm，平均zeta电位为+32.6 mV；37 ℃下核酸酶作用2 h后，LPD中的DNA几乎无降解；平均包封率为93.42%；LPD对张氏（Chang）肝细胞、HepG2肝癌细胞和SMMC-7721肝癌细胞的转染率分别为(69±6)%，(43±7)%和(96.2±1.8)%。结论LPD是一种制备工艺简单、体外稳定性好、转染率高，具有应用潜力的非病毒载体系统。     

Barriers to Non-Viral Vector-Mediated Gene Delivery in the Nervous System

Efficient methods for cell line transfection are well described, but, for primary neurons, a high-yield method different from those relying on viral vectors is lacking. Viral transfection has several drawbacks, such as the complexity of vector preparation, safety concerns, and the generation of immune and inflammatory responses when used in vivo. However, one of the main problems for the use of non-viral gene vectors for neuronal transfection is their low efficiency when compared with viral vectors. Transgene expression, or siRNA delivery mediated by non-viral vectors, is the result of multiple processes related to cellular membrane crossing, intracellular traffic, and/or nuclear delivery of the genetic material cargo. This review will deal with the barriers that different nanoparticles (cationic lipids, polyethyleneimine, dendrimers and carbon nanotubes) must overcome to efficiently deliver their cargo to central nervous system cells, including internalization into the neurons, interaction with intracellular organelles such as lysosomes, and transport across the nuclear membrane of the neuron in the case of DNA transfection. Furthermore, when used in vivo, the nanoparticles should efficiently cross the blood-brain barrier to reach the target cells in the brain.     

Structure-function relationship research of glycerol backbone-based cationic lipids for gene delivery

Transfection activities of two series of synthetic glycerol backbone-based cationic lipids were studied as gene delivery carriers. The variable length of hydrocarbon chains, diverse quaternary ammonium heads, different linkage, as well as alternative anion combined with them allowed to find how these factors affect cationic lipids on their gene delivery performance. The structure-function relationship of the synthetic glycerol backbone-based cationic lipids was discussed, and the transfection efficiency of some of the cationic liposomes was superior or parallel to that of two commercial transfection agents.     

基于聚乙烯亚胺的非病毒基因载体研究进展

如今,越来越多的非病毒基因载体被应用于基因传递中,聚乙烯亚胺（PEI）作为一种重要的阳离子聚合非病毒基因传递载体受到广泛的关注。针对如何克服其生物不可降解性和细胞毒性并进一步提高转染效率的问题,笔者通过文献综合介绍了一些新的策略,并具体对以PEI为基础的新型载体的合成、新型体外转染方法的建立以及体内应用等方面的研究进展进行综述。