Polyamidoamine dendron-bearing lipids as a nonviral vector: influence of dendron generation

Recently, we demonstrated that octadecyl chains are important as alkyl chain moieties of polyamidoamine (PAMAM) dendron-bearing lipids for their serum-resistant transfection activity [Bioconjugate Chem.2007, 18, 1349-1354]. Toward production of highly potent vectors, we examined the influence of the generation of dendron moiety on transfection activity of PAMAM dendron-bearing lipids having two octadecyl chains. We synthesized dendron-bearing lipids with PAMAM G1, G2, and G3 dendrons, designated respectively as DL-G1-2C(18), DL-G2-2C(18), and DL-G3-2C(18). The DL-G2-2C(18) and DL-G3-2C(18) interacted with plasmid DNA effectively and formed stable lipoplexes with small sizes and spherical shape. However, DL-G1-2C(18) interacted with plasmid DNA less effectively and formed tubular-shaped lipoplexes with lower stability and larger size. Cells took up DL-G2-2C(18) and DL-G3-2C(18) lipoplexes efficiently, but cellular uptake of the DL-G1-2C(18) lipoplexes was less efficient. Nevertheless, DL-G1-2C(18) lipoplexes achieved 100-10?000 times higher levels of transgene expression, which was evaluated using luciferase gene as a reporter gene. Confocal scanning laser microscopic analysis of intracellular behaviors of the lipoplexes revealed that DL-G1-2C(18) lipoplexes generated free plasmid DNA molecules in the cytosol more effectively than other lipoplexes did. Moderate binding ability of DL-G1-2C(18) might be responsible for generation of lipoplexes which deliver plasmid DNA into cells, liberate it in the cytoplasm, and induce efficient transgene expression.     

Synthesis of novel cationic lipids having polyamidoamine dendrons and their transfection activity

We designed a novel type of cationic lipid, lipids with a cationic polar group in the polyamidoamine dendron, because these dendron-bearing lipids are expected to form complexes with plasmid DNA and achieve efficient transfection of cells by synergy of endosome buffering and membrane fusion with the endosome, both of which are useful for the promotion of the transfer of plasmid DNA from endosome to cytosol. Four kinds of lipids with polyamidoamine dendrons of first to fourth generations, DL-G1, DL-G2, DL-G3, and DL-G4, were synthesized. The lipid with a dendron of a higher generation exhibited greater ability to form lipoplexes with plasmid DNA, as estimated by agarose gel electrophoresis. While the DL-G1 lipoplex did not transfect CV1 cells, the lipoplexes containing the DL-G2, DL-G3, or DL-G4 could induce transfection of the cells, and their activity was elevated with increasing generation of the dendron. Addition of dioleoylphosphatidylethanolamine (DOPE), which is known to increase fusion ability of a lipid membrane, into the lipoplexes greatly enhanced their transfection activity. In addition, the comparison with DC-Chol-containing lipoplex, which is widely used as a nonviral vector, showed that the DL-G3-DOPE lipoplex exhibits more efficient transfections. These findings imply that these dendron-bearing lipids may form the basis for a novel family of cationic lipids for efficient gene delivery.     

Synthesis of poly(amidoamine) dendron-bearing lipids with poly(ethylene glycol) grafts and their use for stabilization of nonviral gene vectors

Recently, we developed a new type of cationic lipid that consists of an amine-terminated poly(amidoamine) dendron and two long alkyl groups. These dendron-bearing lipids achieved efficient gene transfection of cells through synergetic action of the proton sponge effect and membrane fusion in combination with fusogenic lipid dioleoylphosphatidylethanolamine. Using those dendron-bearing lipids as a base material, we developed in this study a functional component of gene vectors that stabilizes lipoplexes by multiple PEG chains and promotes gene transfection through the proton sponge effect. We combined a poly(ethylene glycol) (PEG, 550 Da) graft to each of four chain ends of the G2 dendron-bearing lipid (P4-DL). An analogous molecule having single PEG graft was also synthesized using the G0 dendron-bearing lipid (P1-DL). Inclusion of P4-DL decreased the size of the G3 dendron-bearing lipid-based lipoplexes more efficiently than P1-DL. In addition, P4-DL-containing lipoplexes exhibited two-orders-higher transfection efficiency than P1-DL-containing lipoplexes with the same PEG graft density. These results indicate the superiority of multiple attachments of PEG graft chains to a lipid for heightened ability to increase colloidal stability of lipoplexes while retaining their transfection activity. The lipoplexes stabilized by P4-DL were small, around 250 nm, and achieved efficient transfection in the presence of serum. Therefore, P4-DL and its analogues will form the basis for production of efficient nonviral vectors for in vivo use.     

Preparation of efficient gene carriers using a polyamidoamine dendron-bearing lipid: improvement of serum resistance

In a previous study, we developed a novel cationic lipid consisting of polyamidoamine dendron of third generation and two dodecyl chains, designated as DL-G3, which in combination with a fusogenic lipid dioleoylphosphatidylethanolamine (DOPE) achieves efficient transfection of CV1 cells by synergetic action of the proton sponge effect and membrane fusion. This study examines the effect of serum on the transfection activity of the DL-G3-DOPE-plasmid DNA lipoplexes. The transfection activity of a lipoplex with a composition optimized in the absence of serum decreased markedly in the presence of serum. However, the lipoplexes that induce efficient transfection in the presence of serum were obtainable by controlling the charge ratio of the primary amine of the DL-G3 to the phosphate group (N/P ratio) and DOPE content. The complex, which exhibited the highest transfection activity in the presence of serum, has a lower N/P ratio and higher DOPE content than that optimized in the absence of serum. Whereas disintegration of these complexes was induced by addition of heparin, which is a polysaccharide with negatively charged groups, the complex that retained transfection activity in the presence of serum required more negative charges of heparin for complex disintegration. That result implies its higher stability against negatively charged serum proteins. Comparison of the serum-resistant complex with some commercially available transfection reagents, such as Lipofectamine and SuperFect, indicates that the DL-G3 complex achieved more efficient transfection of these cells in the presence of serum.     

Evaluation of cationic assemblies constructed with amino acid based lipids for plasmid DNA delivery

We synthesized cationic lipids bearing lysine, histidine, or arginine as a cationic headgroup for use in gene transfer studies. The cationic assemblies formed from lysine- or arginine-type lipids gave unilamellar vesicles (approximately 100 nm diameter), whereas the morphology of the histidine-type lipids was tube-like. The competences of the cationic assemblies were sufficient to form lipoplexes, and the resulting lipoplexes were evaluated in terms of gene expression efficiencies with COS-7 cells. The lysine- or arginine-type lipids exhibited higher gene expression efficiencies than that of Lipofectamine2000, a conventional transgenic reagent, indicating that stable lipoplexes could be prepared between spherical cationic assemblies and plasmid DNA. The gene expression efficiency in relation to the cationic headgroup of the lipids was as follows: lysine > or = arginine > histidine. In addition, gene expression efficiency was enhanced by decreasing the length of the alkyl chain of the hydrophobic moiety. Unlike Lipofectamine2000, no reduction in transfection efficiency in the presence of fetal bovine serum was observed for the lipoplexes formed using synthetic cationic lipids. Moreover, the synthetic cationic lipids revealed remarkably low cytotoxicity compared with Lipofectamine2000. In conclusion, cationic assemblies formed from 1,5-ditetradecyl-N-lysyl-L-glutamate or 1,5-ditetradecyl-N-arginyl-L-glutamate can be used as an effective plasmid DNA delivery system.     

Phase behavior of cationic amphiphiles and their mixtures with helper lipid influences lipoplex shape,DNA translocation,and transfection efficiency

Cationic lipids are widely used for gene transfection, but their mechanism of action is still poorly understood. To improve this knowledge, a structure-function study was carried out with two pyridinium-based lipid analogs with identical headgroups but differing in alkyl chain (un)saturation, i.e., SAINT-2 (diC18:1) and SAINT-5 (diC18:0). Although both amphiphiles display transfection activity per se, DOPE strongly promotes SAINT-2-mediated transfection, but not that of SAINT-5, despite the fact that DOPE effectively facilitates plasmid dissociation from either lipoplex. This difference appears to correlate with membrane stiffness, dictated by the cationic lipid packing in the donor liposomes, which governs the kinetics of lipid recruitment by the plasmid upon lipoplex assembly. Because of its interaction with the relatively rigid SAINT-5 membranes, the plasmid becomes inappropriately condensed, which results in formation of structurally deformed lipoplexes. This structural deformation does not affect its cellular uptake but, rather, hampers plasmid translocation across endosomal and/or nuclear membranes. This is inferred from the observation that both lipoplexes effectively translocate much smaller oligonucleotides into cells. In fact, SAINT-5/DOPE-mediated transfection is greatly improved when, before lipoplex assembly, the plasmid is stabilized by condensation with polylysine. The results emphasize a role of the structural shape of the plasmid in gaining cytosolic/nuclear access. Moreover, it has been proposed that such a translocation is promoted when the lipoplex adopts the hexagonal phase, and data are presented that demonstrate that the lamellar SAINT-5/DOPE lipoplex adopts such a phase after its interaction with acidic phospholipid-containing membranes.     

Transfection activity of binary mixtures of cationic o-substituted phosphatidylcholine derivatives: the hydrophobic core strongly modulates physical properties and DNA delivery efficacy

A combination of two cationic lipid derivatives having the same headgroup but tails of different chain lengths has been shown to have considerably different transfection activity than do the separate molecules. Such findings point to the importance of investigating the hydrophobic portions of cationic amphiphiles. Hence, we have synthesized a variety of cationic phosphatidylcholines with unusual hydrophobic moieties and have evaluated their transfection activity and that of their mixtures with the original molecule of this class, dioleoyl-O-ethylphosphatidylcholine (EDOPC). Four distinct relationships between transfection activity and composition of the mixture (plotted as percent of the new compound added to EDOPC) were found, namely: with a maximum or minimum; with a proportional change; or with essentially no change. Relevant physical properties of the lipoplexes were also examined; specifically, membrane fusion (by fluorescence resonance energy transfer between cationic and anionic lipids) and DNA unbinding (measured as accessibility of DNA to ethidium bromide by electrophoresis and by fluorescence resonance energy transfer between DNA and cationic lipid), both after the addition of negatively charged membrane lipids. Fusibility increased with increasing content of second cationic lipid, regardless of the transfection pattern. However, the extent of DNA unbinding after addition of negatively charged membrane lipids did correlate with extent of transfection. The phase behavior of cationic lipids per se as well as that of their mixtures with membrane lipids revealed structural differences that may account for and support the hypothesis that a membrane lipid-triggered, lamellar-->nonlamellar phase transition that facilitates DNA release is critical to efficient transfection by cationic lipids.     

trans-2-Aminocyclohexanol-based amphiphiles as highly efficient helper lipids for gene delivery by lipoplexes

Lipidic amphiphiles equipped with the trans-2-aminocyclohexanol (TACH) moiety are promising pH-sensitive conformational switches (“flipids”) that can trigger a lipid bilayer perturbation in response to increased acidity. Because pH-sensitivity was shown to improve the efficiency of several gene delivery systems, we expected that such flipids could significantly enhance the gene transfection by lipoplexes. Thus a series of novel lipids with various TACH-based head groups and hydrocarbon tails were designed, prepared and incorporated into lipoplexes that contain the cationic lipid 1,2-dioleoyl-3-trimethylammonio-propane (DOTAP) and plasmid DNA encoding a luciferase gene. B16F1 and HeLa cells were transfected with such lipoplexes in both serum-free and serum-containing media. The lipoplexes consisting of TACH-lipids exhibited up to two orders of magnitude better transfection efficiency and yet similar toxicity compared to the ones with the conventional helper lipids 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or cholesterol. Thus, the TACH-lipids can be used as novel helper lipids for efficient gene transfection with low cytotoxicity.     

The alkyl moieties in wax esters and alkyl diacyl glycerols of sharks

The alkyl moieties in wax esters and alkyl diacyl glycerols from the liver of the dogfish, soupfin shark, and silky shark are almost exclusively saturated and monounsaturated, the main alkyl moieties being the C(16) and C(18) chains in both lipid classes. However, the alkyl moieties in wax esters occur in a wider range of chain lengths. The unsaturated alkyl moieties in the two classes of lipids are mixtures of isomers. The distribution of isomeric octadecenyl moieties in wax esters and alkyl diacyl glycerols is almost the same.     

Synthesis, characterization, and in vitro transfection activity of charge-reversal amphiphiles for DNA delivery

A series of charge-reversal lipids were synthesized that possess varying chain lengths and end functionalities. These lipids were designed to bind and then release DNA based on a change in electrostatic interaction with DNA. Specifically, a cleavable ester linkage is located at the ends of the hydrocarbon chains. The DNA release from the amphiphile was tuned by altering the length and position of the ester linkage in the hydrophobic chains of the lipids through the preparation of five new amphiphiles. The amphiphiles and corresponding lipoplexes were characterized by DSC, TEM, and X-ray, as well as evaluated for DNA binding and DNA transfection. For one specific charge-reversal lipid, stable lipoplexes of approximately 550 nm were formed, and with this amphiphile, effective in vitro DNA transfection activities was observed.     

In vitro cationic lipid-mediated gene delivery with fluorinated glycerophosphoethanolamine helper lipids.

There is a need for the development of nonviral gene transfer systems with improved and original properties. "Fluorinated" lipoplexes are such candidates, as supported by the remarkably higher in vitro and in vivo transfection potency found for such fluorinated lipoplexes as compared with conventional ones or even with PEI-based polyplexes (Boussif, O., Gaucheron, J., Boulanger, C., Santaella, C., Kolbe, H. V. J., Vierling, P. (2001) Enhanced in vitro and in vivo cationic lipid-mediated gene delivery with a fluorinated glycerophosphoethanolamine helper lipid. J. Gene Med. 3, 109-114). Here, we describe the synthesis of fluorinated glycerophosphoethanolamines (F-PEs), close analogues of dioleoylphosphatidylethanolamine (DOPE), and report on their lipid helper properties vs that of DOPE, as in vitro gene transfer components of fluorinated lipoplexes based on pcTG90, DOGS (Transfectam), or DOTAP. To evaluate the contribution of the F-PEs to in vitro lipoplex-mediated gene transfer, we examined the effect of including the F-PEs in lipoplexes formulated with these cationic lipids (CL) for various CL:DOPE:F-PE molar ratios [1:(1 - x):x with x = 0, 0.5 and 1; 1:(2 - y):y with y = 0, 1, 1.5, and 2], and various N/P ratios (from 10 to 0.8, N = number of CL amines, P = number of DNA phosphates). Irrespective of the F-PE chemical structure, of the colipid F-PE:DOPE composition, and of the N/P ratio, comparable transfection levels to those of their respective control DOPE lipoplexes were most frequently obtained when using one of the F-PEs as colipid of DOGS, pcTG90, or DOTAP in place of part of or of all DOPE. However, a large proportion of DOGS-based lipoplexes were found to display a higher transfection efficiency when formulated with the F-PEs rather than with DOPE alone while the opposite tendency was evidenced for the DOTAP-based lipoplexes. The present work indicates that "fluorinated" lipoplexes formulated with fluorinated helper lipids and conventional cationic lipids are very attractive candidates for gene delivery. It confirms further that lipophobicity and restricted miscibility of the lipoplex lipids with the endogenous lipids does not preclude efficient gene transfer and expression. Their transfection potency is rather attributable to their unique lipophobic and hydrophobic character (resulting from the formulation of DNA with fluorinated lipids), thus preventing to some extent DNA from interactions with lipophilic and hydrophilic biocompounds, and from degradation.     

In vitro lipofection with novel asymmetric series of 1,2-dialkoylamidopropane-based cytofectins containing single symmetric bis-(2-dimethylaminoethane) polar headgroups

Novel N,N'-diacyl-1,2-diaminopropyl-3-carbamoyl[bis-(2-dimethylaminoethane)] bivalent cationic lipids were synthesized and evaluated for in vitro transfection activity against a murine melanoma cell line. In the absence of the helper lipid DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), only the dioleoyl derivative 22 (1,2lb5) elicited transfection activity. The transfection activity of this lipid was reduced when formulated with DOPE. Contrary to that, the dimyristoyl derivative 19 (1,2lb2) mediated no activity when used alone but induced the highest levels of marker gene expression in the presence of DOPE. In an effort to correlate the transfection activity with cationic lipid structures, the physicochemical properties of cationic lipids in isolation and of lipoplexes were studied with surface tensiometry, photon correlation spectroscopy, gel electrophoresis mobility shift assay, and fluorescence techniques. In regard to the lipoplex properties, gel electrophoresis mobility shift assay and EtBr exclusion fluorescence assay revealed that the 1,2lb5 was the only lipid to associate and condense plasmid DNA, respectively. Photon correlation spectroscopy analysis found that 1,2lb5/DNA complexes were of relatively small size compared to all other lipoplexes. With respect to the properties of isolated lipids, Langmuir monolayer studies and fluorescence anisotropy on cationic lipid dispersions verified high two-plane elasticity and increased fluidity of the transfection competent dioleoyl derivative 1,2lb5, respectively. The results indicate that high transfection activity is mediated by cationic lipids characterized by an expanded mean molecular area, high molecular elasticity, and increased fluidity.     

DODAB:monoolein-based lipoplexes as non-viral vectors for transfection of mammalian cells

DNA/Cationic liposome complexes (lipoplexes) have been widely used as non-viral vectors for transfection. Neutral lipids in liposomal formulation are determinant for transfection efficiency using these vectors. In this work, we studied the potential of monoolein (MO) as helper lipid for cellular transfection. Lipoplexes composed of pDNA and dioctadecyldimethylammonium bromide (DODAB)/1-monooleoyl-rac-glycerol (MO) at different molar ratios (4:1, 2:1 and 1:1) and at different cationic lipid/DNA ratios were investigated. The physicochemical properties of the lipoplexes (size, charge and structure), were studied by Dynamic Light Scattering (DLS), Zeta Potential (ζ) and cryo-transmission electron microscopy (cryo-TEM). The effect of MO on pDNA condensation and the effect of heparin and heparan sulphate on the percentage of pDNA release from the lipoplexes were also studied by Ethidium Bromide (EtBr) exclusion assays and electrophoresis. Cytotoxicity and transfection efficiency of these lipoplexes were evaluated using 293T cells and compared with the golden standard helper lipids 1,2-dioleoyl-sn-glycero-3-hosphoethanolamine (DOPE) and cholesterol (Chol) as well as with a commercial transfection agent (Lipofectamine? LTX). The internalization of transfected fluorescently-labeled pDNA was also visualized using the same cell line. The results demonstrate that the presence of MO not only increases pDNA compactation efficiency, but also affects the physicochemical properties of the lipoplexes, which can interfere with lipoplex-cell interactions. The DODAB:MO formulations tested showed little toxicity and successfully mediated in vitro cell transfection. These results were supported by fluorescence microscopy studies, which illustrated that lipoplexes were able to access the cytosol and deliver pDNA to the nucleus. DODAB:MO-based lipoplexes were thus validated as non-toxic, efficient lipofection vectors for genetic modification of mammalian cells. Understanding the relation between structure and activity of MO-based lipoplexes will further strengthen the development of these novel delivery systems.     

Efficient gene transfection by bisguanylated diacetylene lipid formulations

We have previously shown that cationic cholesterol derivatives bearing guanidinium groups were efficient vectors for gene transfer. To further evaluate the potentiality of this novel class of cationic lipids, we undertook to study the transfection efficiency of guanidinium-based lipids with other hydrophobic moieties. Specifically, we synthesized a reagent where two guanidinium groups are linked to a diacetylene lipid which may provide the lipoplexes with favorable structural features. We report here that the cationic lipid bisguanidinium-diacetylene (BGDA) is highly efficient for in vitro gene transfection when formulated with dioleoylphosphatidyl ethanolamine (DOPE). We also show that liposomes composed of BGDA, DOPE, and a neutral diacetylene colipid, hydroxyethylenediacetylene (HEDA), are efficient for transfection. Thus, diacetylene-based lipids provide a novel scaffold for gene transfection and will be particularly useful for gaining new insights into the structure-activity relationships of the lipid/DNA complexes as they offer a means to study the effects of polymerizable domains.     

In vitro lipofection with novel series of symmetric 1,3-dialkoylamidopropane-based cationic surfactants containing single primary and tertiary amine polar head groups

A novel series of symmetric double-chained primary and tertiary 1,3-dialkoylamido monovalent cationic lipids were synthesized and evaluated for their transfection activities. In the absence of the helper lipid DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), only the primary and tertiary dioleoyl derivatives 1,3lmp5 and 1,3lmt5, respectively elicited transfection activity. This is a striking difference between symmetrical 1,2-diacyl glycerol-based monovalent cationic lipids that always found both dioleoyl and dimyristoyl analogues being efficient transfection reagents. In the presence of helper lipid, all cationic derivatives induced marker gene expression, except the dilauroyl analogues 1,3lmp1 and 1,3lmt1 that elicited no transfection activity. Combining electrophoretic mobility data of the lipoplexes at different charge ratios with transfection activity suggested two requirements for high transfection activity with monovalent double-chained cationic lipids, that is, binding/association of the lipid to the plasmid DNA and membrane fusion properties of the lipid layers surrounding the DNA.     

Phase behavior, DNA ordering, and size instability of cationic lipoplexes. Relevance to optimal transfection activity

Mechanisms of cationic lipid-based nucleic acid delivery are receiving increasing attention, but despite this the factors that determine high or low activity of lipoplexes are poorly understood. This study is focused on the fine structure of cationic lipid-DNA complexes (lipoplexes) and its relevance to transfection efficiency. Monocationic (N-(1-(2,3-dioleoyloxy)propyl),N,N,N-trimethylammonium chloride, N-(1-(2,3-dimyristyloxypropyl)-N,N-dimethyl-(2-hydroxyethyl)ammonium bromide) and polycationic (2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanammonium trifluoroacetate) lipid-based assemblies, with or without neutral lipid (1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine, 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine, cholesterol) were used to prepare lipoplexes of different L(+)/DNA(-) charge ratios. Circular dichroism, cryogenic-transmission electron microscopy, and static light scattering were used for lipoplex characterization, whereas expression of human growth hormone or green fluorescent protein was used to quantify transfection efficiency. All monocationic lipids in the presence of inverted hexagonal phase-promoting helper lipids (1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine, cholesterol) induced appearance of Psi(-) DNA, a chiral tertiary DNA structure. The formation of Psi(-) DNA was also dependent on cationic lipid-DNA charge ratio. On the other hand, monocationic lipids either alone or with 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine as helper lipid, or polycationic 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanammonium trifluoroacetate-based assemblies, neither of which promotes a lipid-DNA hexagonal phase, did not induce the formation of Psi(-) DNA. Parallel transfection studies reveal that the size and phase instability of the lipoplexes, and not the formation of Psi(-) DNA structure, correlate with optimal transfection.     

Novel imidazole-functionalized cyclen cationic lipids: Synthesis and application as non-viral gene vectors

A series of novel 1,4,7,10-tetraazacyclododecanes (cyclen)-based cationic lipids bearing histidine imidazole group 10a–10e were synthesized. These amphiphilic molecules have different hydrophobic tails (long chain, cholesterol or α-tocopherol) and various type of linking groups (ether, carbamate or ester). These molecules were used as non-viral gene delivery vectors, and their structure–activity relationships were investigated. As expected, the imidazole group could largely improve the buffering capabilities comparing to cyclen. The liposomes formed from 10 and dioleoylphosphatidyl ethanolamine (DOPE) could bind and condense plasmid DNA into nanoparticles with proper size and zeta-potentials. Comparing with Lipofectamine 2000, the formed lipoplexes gave lower transfected cells proportion, but higher fluorescence intensity, indicating their good intracellular delivering ability. Furthermore, results indicate that transfection efficiency of the cationic lipids is influenced by not only the hydrophobic tails but also the linking group. The cyclen-based cationic lipid with α-tocopherol hydrophobic tail and an ester linkage could give the highest transfection efficiency in the presence of serum.     

Cell transfection by DNA-lipid complexes — Lipoplexes

Nonviral vectors such as complexes of plasmid DNA with cationic lipids known as lipoplexes are considered as an attractive alternative to virus-based delivery systems. Unlike viruses, lipoplexes do not suffer from immunological and mutational hazards, though the efficiency of lipoplexes is often not sufficient for therapeutic purposes and require higher level of transfection than achieved until now. A number of critical steps responsible for transfection efficiency are discussed here. They include processes of lipoplexes formation, interaction with cell surface, their internalization into cell, and DNA release and delivery into the nucleus. All these processes should be thoroughly studied to be able to enhance the transfection efficacy.     

Structure Relationship of Cationic Lipids on Gene Transfection Mediated by Cationic Liposomes

The aim of this study was to investigate the transfection efficiency of cationic liposomes formulated with phosphatidylcholine (PC) and novel synthesized diethanolamine-based cationic lipids at a molar ratio of 5:1 in comparison with Lipofectamine™ 2000. Factors affecting transfection efficiency and cell viability, including the chemical structure of the cationic lipids, such as different amine head group (diamine and polyamine; and non-spermine and spermine) and acyl chain lengths (C14, C16, and C18) and the weight ratio of liposomes to DNA were evaluated on a human cervical carcinoma cell line (HeLa cells) using the pDNA encoding green fluorescent protein (pEGFP-C2). Characterizations of these lipoplexes in terms of size and charge measurement and agarose gel electrophoresis were performed. The results from this study revealed that almost no transfection was observed in the liposome formulations composed of cationic lipids with a non-spermine head group. In addition, the transfection efficiency of these cationic liposomes was in the following order: spermine-C14 > spermine-C16 > spermine-C18. The highest transfection efficiency was observed in the formulation of spermine-C14 liposomes at a weight ratio of 25; furthermore, this formulation was safe for use in vitro. In conclusion, cationic liposomes containing spermine head groups demonstrated promising potential as gene carriers.Key words: cationic lipids, cationic liposomes, gene transfection     

Lamellarity of cationic liposomes and mode of preparation of lipoplexes affect transfection efficiency.

Transfection of NIH-3T3 cells by a human growth hormone expression vector complexed with liposomes composed of N-(1-(2, 3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP) with or without helper lipids was studied. The transfection efficiency was dependent on the lamellarity of the liposomes used to prepare the lipoplexes. Multilamellar vesicles (MLV) were more effective than large unilamellar vesicles (LUV) of approximately 100 nm, irrespective of lipid composition. The optimal DNA/DOTAP mole ratio for transfection was 相似文献