Shubiao Zhang

Transfection efficiency of cationic lipids with different hydrophobic domains in gene delivery.

The structure of cationic lipids is a major factor for their transfection activity. A cationic lipid generally contains four functional domains: a hydrophilic headgroup, a linker, a backbone domain, and a hydrophobic domain. The structure of the hydrophobic domain determines the phase transition temperature and the fluidity of the bilayer and influences the stability of liposomes, the DNA protection from nucleases, the endosomal escape, the DNA release from complex, and the nuclear penetration. Also, toxicity of the lipids is influenced by the hydrophobic domain. The compounds used for gene delivery are classified according to the structure of the hydrophobic domain as follows: aliphatic chains, steroid domain, and fluorinated domain. In this review, we summarized recent research results concerning the structures of the hydrophobic domain, in order to find the effect of the hydrophobic domain on transfection efficiency. Understanding these would be very important for scientists to prepare novel cationic lipids and design novel formulations with high transfection efficiency.

The Headgroup Evolution of Cationic Lipids for Gene Delivery

Cationic lipids are one of the most widely used nonviral vectors for gene delivery and are especially attractive because they can be easily synthesized and extensively characterized. Additionally, they can best facilitate the elucidation of structure-activity relationships by modifying each of their constituent domains. The polar hydrophilic headgroups enable the condensation of nucleic acids by electrostatic interactions with the negatively charged phosphate groups of the genes, and further govern transfection efficiency. The headgroups of cationic lipids play a crucial role for gene delivery; they can be quaternary ammoniums, amines, aminoacids or peptides, guanidiniums, heterocyclic headgroups, and some unusual headgroups. This review summarizes recent research results concerning the nature (such as the structure and shape of cationic headgroup) and density (such as the number and the spacing of cationic headgroup) of head functional groups for improving the design of efficient cationic lipids to overcome the critical barriers of in vitro and in vivo transfection.

Hybrids of Nonviral Vectors for Gene Delivery

Gene transfer has been a critical step for gene therapy. Viral vectors and nonviral vectors are commonly used for gene transfer. It has been shown that nonviral vectors have the advantage over viral ones, as they are nonimmunogenic, easy to produce, and not oncogenic. However, they have the major limitation of inefficient transfection; the hybridized usage of nonviral vectors may provide a partial solution. This article reviews the hybrids (different materials used in one delivery system) of nonviral vectors for gene delivery including hybrids between cationic lipids and helper lipids, conjugates of peptides or targeting moieties and lipids, and hybrids of cationic liposomes and polymers.

Non-viral vectors for the mediation of RNAi

Though the delivery of siRNA into cells, tissues or organs remains to be a big obstacle for its applications, recently siRNA therapeutics has developed rapidly and already there are clinical trials ongoing or planned. Some non-viral vectors have attracted much more attention and shown the great potential for combating the delivery obstacle. As a novel class of lipid like materials lipidoids have the advantages of easy synthesis and large library of compounds. Cell penetrating peptides and chitosans have been used for the delivery of bioactive molecules for many years, but they are showing great promise for the delivery of siRNA. The hybrids of inorganic particles and the conjugates of siRNA have indicated the complex utilization different materials may provide another solution to the delivery problem. The most exciting thing is some clinical trials are undergoing, which provokes the hope of real curing method by using RNAi mediated by some non-viral vectors.

Synthesis and biological activity of carbamate-linked cationic lipids for gene delivery in vitro.

We have introduced a convenient synthesis method for carbamate-linked cationic lipids. Two cationic lipids N-[1-(2,3-didodecylcarbamoyloxy)propyl]-N,N,N-trimethylammonium iodide (DDCTMA) and N-[1-(2,3-didodecyl carbamoyloxy)propyl]-N-ethyl-N,N-dimethylammonium iodide (DDCEDMA), with identical length of hydrocarbon chains, alternative quaternary ammonium heads, carbamate linkages between hydrocarbon chains and quaternary ammonium heads, were synthesized for liposome-mediated gene delivery. Liposomes composed of DDCEDMA and DOPE in 1:1 ratio exhibited a lower zeta potential as compared to those made of pure DDCEDMA alone, which influences their DNA-binding ability. pGFP-N2 plasmid was transferred by cationic liposomes formed from the above cationic lipids into Hela and Hep-2 cells, and the transfection efficiency of some of cationic liposomes was superior or parallel to that of two commercial transfection agents, Lipofectamine2000 and DOTAP. Combined with the results of the agarose gel electrophoresis and transfection experiment, the DNA-binding ability of cationic lipids was too strong to release DNA from complex in the transfection, which could lead to relative low transfection efficiency and high cytotoxicity.

The biological routes of gene delivery mediated by lipid-based non-viral vectors

Cationic lipid/DNA complexes (lipoplexes) represent an attractive alternative to viral vectors for cell transfection in vitro and in vivo but still suffer from relatively low efficiency. Comprehension of the interactions between vectors and DNA as well as cellular pathways and mechanisms in DNA entry into cells and ultimately nuclei will lead to the design of better adapted non-viral vectors for gene therapy applications. Here, some recent developments in the field on the pathways and mechanisms involved in lipoplex-mediated transfection are discussed. The techniques that are widely used to study the mechanism of gene delivery are also discussed.

Peptide-based cationic liposome-mediated gene delivery

Introduction: As an important type of nonviral gene delivery vector, peptide-based cationic liposomes have shown many advantages over other cationic liposomes, such as good biodegradability, excellent biocompatibility and targeting ability to cells, and great potential application in improving the delivery of gene therapeutics. Areas covered: This article reviews the research progress on peptide-based cationic liposomes for gene delivery, including the structure characteristics of peptide lipids, the development of peptide cationic liposomes and three special types of peptide cationic liposomes: peptide-based Gemini lipids, lipitoids and lipids with cholesterol hydrophobic group. This review hopes to provide some suggestions on the design of peptide cationic lipids and insight into their development trend in the field of gene delivery. Expert opinion: As peptide-based cationic liposomes still hold some limitations, future research needs to select suitable peptide heads and investigate the surface modification of peptide cationic liposomes, in order to facilitate targeting and reduce cytotoxicity.

Tri-peptide cationic lipids for gene delivery

Several novel tri-peptide cationic lipids were designed and synthesized for delivering DNA and siRNA. They have tri-lysine and tri-ornithine as head groups, carbamate group as linker and 12 and 14 carbon atom alkyl groups as tails. These tri-peptide cationic lipids were prepared into cationic liposomes for the study of the physicochemical properties and gene delivery. Their particle size, Zeta potential and DNA-binding were characterized to show that they were suitable for gene transfection. The further results indicate that these lipids can transfer DNA and siRNA very efficiently into NCI-H460 and Hep-2 tumor cells. The selected lipid, CDO14, was able to deliver combined siRNAs against c-Myc and VEGF for silencing distinct oncogenic pathways in lung tumors of mice, with little in vitro and in vivo toxicity.

Novel Gemini cationic lipids with carbamate groups for gene delivery

To obtain efficient non-viral vectors, a series of Gemini cationic lipids with carbamate linkers between headgroups and hydrophobic tails were synthesized. They have the hydrocarbon chains of 12, 14, 16 and 18 carbon atoms as tails, designated as G12, G14, G16 and G18, respectively. These Gemini cationic lipids were prepared into cationic liposomes for the study of the physicochemical properties and gene delivery. The DNA-bonding ability of these Gemini cationic liposomes was much better than their mono-head counterparts (designated as M12, M14, M16 and M18, respectively). In the same series of liposomes, bonding ability declined with an increase in tail length. They were tested for their gene-transferring capabilities in Hep-2 and A549 cells. They showed higher transfection efficiency than their mono-head counterparts and were comparable or superior in transfection efficiency and cytotoxicity to the commercial liposomes, DOTAP and Lipofectamine 2000. Our results convincingly demonstrate that the gene-transferring capabilities of these cationic lipids depended on hydrocarbon chain length. Gene transfection efficiency was maximal at a chain length of 14, as G14 can silence about 80 % of luciferase in A549 cells. Cell uptake results indicate that Gemini lipid delivery systems could be internalised by cells very efficiently. Thus, the Gemini cationic lipids could be used as synthetic non-viral gene delivery carriers for further study.

Cationic liposomes as carriers for gene delivery: Physico-chemical characterization and mechanism of cell transfection

The inter-correlations among the parameters that influence the transfection efficiency of liposome-DNA complexes (lipoplexes) as carriers in gene therapy, optimum procedures for transfection and mechanism of cell transfection were investigated. The morphology, size and ζ-potential of liposomes and lipoplexes were measured by atomic force microscopy (AFM) and dynamic light scattering techniques. The lipoplexes formation was tested by using gel electrophoresis. Transfection efficiency was assessed using luciferase and green fluorescence protein reporter plasmids, respectively. It was found that transfection efficiency markedly depended on liposome to DNA weight ratio, lipoplexes morphology and size. The intracellular trafficking of exogenous DNAs was observed by confocal laser microscopy. After 4 h incubation, DNAs delivered by Lipofectamine 2000 reached the nucleus with a much higher frequency than 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). It was concluded that lipoplexes with filaments contributing more than globular obtained higher transfection efficiency with the maximum expression of 500000 RLU/mg luciferase in HeLa cells, for they are easy to release pDNA after endocytosis. These results implied that by further modifying the chemical features of the lipid vectors and controlling their biological behaviors, more efficient lipid delivery vectors may be achieved. Key words : Cationic liposomes, gene delivery, transfection efficiency, AFM, intracellular trafficking.