Modulation of a membrane lipid lamellar-nonlamellar phase transition by cationic lipids: A measure for transfection efficiency

Synthetic cationic lipids can be used as DNA carriers and are regarded to be the most promising non-viral gene carriers. For this investigation, six novel phosphatidylcholine (PC) cationic derivatives with various hydrophobic moieties were synthesized and their transfection efficiencies for human umbilical artery endothelial cells (HUAEC) were determined. Three compounds with relatively short, myristoleoyl or myristelaidoyl 14:1 chains exhibited very high activity, exceeding by ∼ 10 times that of the reference cationic derivative dioleoyl ethylPC (EDOPC). Noteworthy, cationic lipids with 14:1 hydrocarbon chains have not been tested as DNA carriers in transfection assays previously. The other three lipids, which contained oleoyl 18:1 and longer chains, exhibited moderate to weak transfection activity. Transfection efficiency was found to correlate strongly with the effect of the cationic lipids on the lamellar-to-inverted hexagonal, L_α → H_II, phase conversion in dipalmitoleoyl phosphatidylethanolamine dispersions (DPoPE). X-ray diffraction on binary DPoPE/cationic lipid mixtures showed that the superior transfection agents eliminated the direct L_α → H_II phase transition and promoted formation of an inverted cubic phase between the L_α and H_II phases. In contrast, moderate and weak transfection agents retained the direct L_α → H_II transition but shifted to higher temperatures than that of pure DPoPE, and induced cubic phase formation at a later stage. On the basis of current models of lipid membrane fusion, promotion of a cubic phase by the high-efficiency agents may be considered as an indication that their high transfection activity results from enhanced lipoplex fusion with cellular membranes. The distinct, well-expressed correlation established between transfection efficiency of a cationic lipid and the way it modulates nonlamellar phase formation of a membrane lipid could be useful as a criterion to assess the quality of lipid carriers and for rational design of new and superior nucleotide delivery agents.     

Growth of cationic lipid toward bilayer lipid membrane by solution spreading: scanning probe microscopy study

The growth of cationic lipid dioctadecyldimethylammonium bromide (DODAB) toward bilayer lipid membrane (BLM) by solution spreading on cleaved mica surface was studied by atomic force microscopy (AFM). Bilayer of DODAB was formed by exposing mica to a solution of DODAB in chloroform and subsequently immersing into potassium chloride solution for film developing. AFM studies showed that at the initial stage of the growth, the adsorbed molecules exhibited the small fractal-like aggregates. These aggregates grew up and expanded laterally into larger patches with time and experienced from monolayer to bilayer, finally a close-packed bilayer film (5.4+/-0.2 nm) was approached. AFM results of the film growth process indicated a growth mechanism of nucleation, growth and coalescence of dense submonolayer, it revealed the direct information about the film morphology and confirmed that solution spreading was an effective technique to prepare a cationic bilayer in a short time.     

Small interfering RNA delivery into the liver by cationic cholesterol derivative-based liposomes

Purpose: Previously, we reported that the cationic liposomes composed of a cationic cholesterol derivative, cholesteryl (2-((2-hydroxyethyl)amino)ethyl)carbamate (OH-C-Chol) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (termed LP-C), could deliver small interfering RNAs (siRNAs) with high transfection efficiency into tumor cells. In this study, to develop a liposomal vector for siRNA delivery in vivo, we prepared the poly(ethyleneglycol) (PEG)-modified cationic liposomes (LP-C-PEG) and evaluated their transfection efficiency in vitro and in vivo.

Materials and methods: We prepared LP-C-PEG/siRNA complexes (LP-C-PEG lipoplexes) formed in water or 50?mM NaCl solution, and evaluated their siRNA biodistribution and gene silencing effect in mice after intravenous injection.

Results: LP-C-PEG lipoplexes strongly exhibited in vitro gene silencing effects in human breast tumor MCF-7 cells as well as LP-C lipoplexes. In particular, formation of LP-C and LP-C-PEG lipoplexes in the NaCl solution increased the cellular association. When LP-C-PEG lipoplexes with Cy5.5-labeled siRNA formed in water or NaCl solution were injected into mice, accumulation of the siRNA was observed in the liver. Furthermore, injection of LP-C-PEG lipoplexes with ApoB siRNA could suppress ApoB mRNA levels in the liver and reduce very-low-density lipoprotein/low-density lipoprotein levels in serum compared with that after Cont siRNA transfection, although the presence of NaCl solution in forming the lipoplexes did not affect gene silencing effects in vivo.

Conclusions: LP-C-PEG may have potential as a gene vector for siRNA delivery to the liver.     

Gene delivery mediated by cationic liposomes: from biophysical aspects to enhancement of transfection

Cationic liposomes complexed with DNA have been used extensively as non-viral vectors for the intracellular delivery of reporter or therapeutic genes in culture and in vivo. However, the relationship between the features of the lipid-DNA complexes (`lipoplexes') and their mode of interaction with cells, the efficiency of gene transfer and gene expression remain to be clarified. To gain insights into these aspects, the size and zeta potential of cationic liposomes (composed of 1,2-dioleoyl-3- (trimethylammonium) propane (DOTAP) and its mixture with phosphatidylethanolamine (PE)), and their complexes with DNA at different (+/-) charge ratios were determined. A lipid mixing assay was used to assess the interaction of liposomes and lipoplexes with monocytic leukaemia cells. The use of inhibitors of endocytosis indicated that fusion of the cationic liposomes with cells occurred mainly at the plasma membrane level. However, very limited transfection of these cells was achieved using the above complexes. It is possible that the topology of the cationic liposome-DNA complexes does not allow the entry of DNA into cells through a fusion process at the plasma membrane. In an attempt to enhance transfection mediated by lipoplexes composed of DOTAP and its equimolar mixture with dioleoylphosphatidylethanolamine (DOPE) two different strategies were explored: (i) association of a targeting ligand (transferrin) to the complexes to promote their internalization, presumably by receptor-mediated endocytosis; and (ii) association of synthetic fusogenic peptides (GALA or the influenza haemagglutinin Nterminal peptide HA-2) to the complexes to promote endosomal destabilization and release of the genetic material into the cytoplasm. These strategies were effective in enhancing transfection in a large variety of cells, including epithelial and lymphoid cell lines, as well as human macrophages, especially with the use of optimized lipid/ DNA (+/-) charge ratios. Besides leading to high levels of transfection, the ternary complexes of cationic liposomes, DNA, and protein or peptide, have the advantages of being active in the presence of serum and being non-toxic. Moreover, such ternary complexes present a net negative charge and, thus, are likely to alleviate the problems associated with the use of highly positively charged complexes in vivo, such as avid complexation with serum proteins. Overall, the results indicate that these complexes, and their future derivatives, may constitute viable alternatives to viral vectors for gene delivery in vivo.     

Endocytosis in cellular uptake of drug delivery vectors: Molecular aspects in drug development

Drug delivery vectors are widely applied to increase drug efficacy while reducing the side effects and potential toxicity of a drug. They allow for patient-tailored therapy, dose titration, and therapeutic drug monitoring. A major part of drug delivery systems makes use of large nanocarriers: liposomes or virus-like particles (VLPs). These systems allow for a relatively large amount of cargo with good stability of vectors, and they offer multiple options for targeting vectors in vivo. Here we discuss endocytic pathways that are available for drug delivery by large nanocarriers. We focus on molecular aspects of the process, including an overview of potential molecular targets for studies of drug delivery vectors and for future solutions allowing targeted drug delivery.     

Gene delivery by lentivirus vectors

The capacity to efficiently transduce nondividing cells, shuttle large genetic payloads, and maintain stable long-term transgene expression are attributes that have brought lentiviral vectors to the forefront of gene delivery vehicles for research and therapeutic applications in a clinical setting. Our discussion initiates with advances in lentiviral vector development and how these sophisticated lentiviral vectors reflect improvements in safety, regarding the prevention of replication competent lentiviruses (RCLs), vector mobilization, and insertional mutagenesis. Additionally, we describe conventional molecular regulatory systems to manage gene expression levels in a spatial and temporal fashion in the context of a lentiviral vector. State of the art technology for lentiviral vector production by transient transfection and packaging cell lines are explicitly presented with current practices used for concentration, purification, titering, and determining the safety of a vector stock. We summarize lentiviral vector applications that have received a great deal of attention in recent years including the generation of transgenic animals and the stable delivery of RNA interference molecules. Concluding remarks address some of the successes in preclinical animals, and the recent transition of lentiviral vectors to human clinical trials as therapy for a variety of infectious and genetic diseases.     

Rapid delivery of small interfering RNA by biosurfactant MEL-A-containing liposomes

The downregulation of gene expression by RNA interference holds great potential for genetic analysis and gene therapy. However, a more efficient delivery system for small interfering RNA (siRNA) into the target cells is required for wide fields such as cell biology, physiology, and clinical application. Non-viral vectors are stronger candidates than viral vectors because they are safer and easier to prepare. We have previously used a new method for gene transfection by combining cationic liposomes with the biosurfactant mannosylerythritol lipid-A (MEL-A). The novel MEL-A-containing cationic liposomes rapidly delivered DNA (plasmids and oligonucleotides) into the cytosol and nucleus through membrane fusion between liposomes and the plasma membrane, and consequently, enhanced the gene transfection efficiency. In this study, we determined the efficiency of MEL-A-containing cationic liposomes for siRNA delivery. We observed that exogenous and endogenous protein expression was suppressed by approximately 60% at 24 h after brief (30 min) incubation of target cells with MEL-A-containing cationic liposome/siRNA complexes. Confocal microscopic analysis showed that suppression of protein expression was caused by rapid siRNA delivery into the cytosol. We found that the MEL-A-containing cationic liposomes directly delivered siRNA into the cytoplasm by the membrane fusion in addition to endocytotic pathway whereas Lipofectamine™ RNAiMax delivered siRNA only by the endocytotic pathway. It seems that the ability to rapidly and directly deliver siRNA into the cytosol using MEL-A-containing cationic liposomes is able to reduce immune responses, cytotoxicity, and other side effects caused by viral vectors in clinical applications.     

阳离子脂质体运载基因的跨膜机制

阳离子脂质体等非病毒载体以其制备简单、低毒性、低免疫原性、可生物降解等优点,成为近年来基因转运中的常用载体。理解阳离子脂质体运载基因的机制对阳离子脂质体的研究具有重要意义。从跨膜机制和信号调控的角度,介绍了脂质体/DNA复合体以特定构象避免细胞外基质中核酸酶的降解,跨越细胞膜进入细胞的过程;阐明了DNA在信号调控的作用下,逃离溶酶体并安全释放的机制;讨论了基因穿过核被膜进入到细胞核的方式,为进一步阐明阳离子脂质体运载基因的分子机制奠定基础。     

Development of wrapped liposomes: Novel liposomes comprised of polyanion drug and cationic lipid complexes wrapped with neutral lipids

Novel wrapped liposomes comprised of polyanion drug and cationic lipid complexes wrapped with neutral lipids were prepared using an efficient, innovative procedure. In this study, dextran fluorescein anionic (DFA) was used as an example of a polyanionic compound. During the process, neutral lipids accumulated around the complexes and eventually covered the complexes. The resulting liposomes were 120-140 nm in diameter and the encapsulation efficiency was up to 90%. In fetal bovine serum, DFA/cationic lipid complexes degraded rapidly but the wrapped liposomes were considerably more stable. Following intravenous administration to rats, DFA/cationic lipid complexes were rapidly eliminated whereas the wrapped liposomes exhibited a much longer blood half-life. These data suggest that DFA is located on the surface of the complexes, but DFA is present inside the wrapped liposomes. The drug-delivery properties of the wrapped liposomes established in the present study suggests that formulations based on this technology could offer important advantages for the administration of many types of drug including antisense oligonucleotides, plasmids and siRNAs which may therefore lead to improved therapeutic effectiveness of this range of drugs. The method of preparation of the wrapped liposomes is so simple that it should be straightforward to adapt to a manufacturing scale.     

Immunostimulation of dendritic cells by cationic liposomes

A nano-aggregate liposome-polycation-DNA (LPD), composed of a cationic lipid, protamine and plasmid DNA was found to effectively deliver a human papillomavirus (HPV)-E7 epitope antigen to the antigen presenting cells of the immune system, eliciting enhanced anti-tumor immune responses in mouse models of cervical carcinoma. Both the cationic liposome and plasmid DNA were essential for the full immunostimulation activity of LPD. Interestingly, cationic liposomes alone could stimulate the antigen presenting dendritic cells (DC) leading to the expression of co-stimulatory molecules, CD80 and CD86. However, cationic lipids could not stimulate DC for the expression of pro-inflammatory cytokines. Moreover, they were unable to enhance the expression of NF-κB, suggesting that dendritic cells stimulation by cationic lipids is signaled through an NF-κB independent mechanism. DC stimulation was specific to cationic lipids, the zwitterionic and anionic lipids showed little or no activity. The ability of different cationic lipids to stimulate the expression of co-stimulatory molecules on DC varied significantly. In general, the cationic lipids bearing ethyl phosphocholine head groups were better stimulants than their trimethylammonium counterparts. In case of the cationic lipids bearing trimethyl ammonium head groups, the ones bearing unsaturated or shorter saturated hydrophobic chains exhibited enhanced immunostimulatory activity. The LPS-induced TNF-α expression by dendritic cells was inhibited by active cationic lipids but not the inactive ones, suggesting the possible involvement of lipopolysaccharide binding protein (LBP) in cationic lipid mediated DC stimulation. Based on the structure-specific activation of dendritic cells by cationic lipids, a model for the immunostimulation of DC by such lipids is proposed.     

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.     

Novel nonviral vectors for gene delivery: Synthesis and applications

We have synthesized novel cationiclipids for gene delivery bearing an ester bond betweenthe lipid moiety and the polyamine head. We have foundthat an intramolecular rearrangement occurs duringpurification of one of the products. The rearrangementled to a cyclic lipopolyamine which was active for DNAgene transfer. The formation ofcyclization products depends on the spacer foundbetween the lipid and the polyamine. The introduction ofarginine in the linker position prevents the formation ofcyclic products. Linear as well as cyclicanalogues showed high-efficiency gene transfer whentested in vitro for luciferase gene expressionas compared to dioctadecylamidoglycyl spermineor lipofectamine and also in vivo in the Lewislung carcinoma model. The introduction of arginine in thelinker position promoted increased transfectionactivity, demonstrating that a diversity of linkers,such as short peptides or glycosides, can beintroduced into cationic lipids for targeted gene transfer.     

Involvement of lipid rafts in macrophage apoptosis induced by cationic liposomes

We have demonstrated that protein kinase Cδ (PKCδ) could be involved in macrophage apoptosis induced by cationic liposomes composed of stearylamine (SA-liposomes), but the detailed mechanism of how SA-liposomes activate PKCδ has remained unclear. In this paper, we clarified whether lipid rafts are involved in the PKCδ activation induced by SA-liposomes. Co-localization of SA-liposomes and Cholera toxin B subunit (CBT), which specifically binds to ganglioside GM1 on lipid rafts, was found by microscopic observation. The incorporation of SA-liposomes into lipid rafts was clearly inhibited by the pretreatment of cells with an agent, 2,6-di-O-methyl-α-cyclodextrin (DM-α-CD) which disrupts lipid rafts. Activation of PKCδ and externalization of phosphatidylserine induced by SA-liposomes were also suppressed by DM-α-CD, which extracts sphingolipids and proteins from lipid rafts. Reactive oxygen species (ROS) generation, which could be involved in the macrophage apoptosis, was also inhibited by DM-α-CD. Furthermore, apoptosis induced by SA-liposomes was clearly inhibited when the cells were pre-treated with DM-α-CD, but not nystatin, a cholesterol-sequestering agent that disrupt lipid rafts. These findings suggest that sphingolipids in lipid rafts are involved in the activation of PKCδ which leads to apoptosis induced by cationic liposomes, SA-liposomes.     

Characterization of the inhibitory effect of PEG-lipid conjugates on the intracellular delivery of plasmid and antisense DNA mediated by cationic lipid liposomes

Poly(ethylene glycol)-lipid (PEG-lipid) conjugates are widely used in the field of liposomal drug delivery to provide a polymer coat that can confer favorable pharmacokinetic characteristics on particles in the circulation. More recently these lipids have been employed as an essential component in the self-assembly of cationic and neutral lipids with polynucleic acids to form small, stable lipid/DNA complexes that exhibit long circulation times in vivo and accumulate at sites of disease. However, the presence of a steric barrier lipid might be expected to inhibit the transfection activity of lipid/DNA complexes by reducing particle-membrane contact. In this study we examine what effect varying the size of the hydrophobic anchor and hydrophilic head group of PEG-lipids has on both gene and antisense delivery into cells in culture. Lipid/DNA complexes were made using unilamellar vesicles composed of 5 mole% PEG-lipids in combination with equimolar dioleoylphosphatidylethanolamine and the cationic lipid dioleyldimethylammonium chloride. Using HeLa and HepG2 cells we show that under the conditions employed PEG-lipids had a minimal effect on the binding and subsequent endocytosis of lipid/DNA complexes but they severely inhibited active gene transfer and the endosomal release of antisense oligodeoxynucleotides into the cytoplasm. Decreasing the size of the hydrophobic anchor or the size of the grafted hydrophilic PEG moiety enhanced DNA transfer by the complexes.     

TACN-containing cationic lipids with ester bond: preparation and application in gene delivery

A series of novel cationic lipids based on 1,4,7-triazacyclononane (TACN) with different hydrophobic chains were synthesized via the formation of a biodegradable ester bond. These lipids were found to have good buffering capacity at the pH range of 5.0-6.5, which is similar to that of the acidic endosomal compartments. The liposomes formed from these lipids and DOPE could condense DNA into nanoparticles with proper sizes. In vitro experiments showed moderate to good gene transfection efficiency of the formed lipoplexes. The structure-activity relationships of this type of lipids were discussed.     

Cell Biological and Biophysical Aspects of Lipid-mediated Gene Delivery

Cationic lipids are conceptually and methodologically simple tools to deliver nucleic acids into the cells. Strategies based on cationic lipids are viable alternatives to viral vectors and are becoming increasingly popular owing to their minimal toxicity. The first-generation cationic lipids were built around the quaternary nitrogen primarily for binding and condensing DNA. A large number of lipids with variations in the hydrophobic and hydrophilic region were generated with excellent transfection efficiencies in vitro. These cationic lipids had reduced efficiencies when tested for gene delivery in vivo. Efforts in the last decade delineated the cell biological basis of the cationic lipid gene delivery to a significant detail. The application of techniques such as small angle X-ray spectroscopy (SAXS) and fluorescence microscopy, helped in linking the physical properties of lipid:DNA complex (lipoplex) with its intracellular fate. This biological knowledge has been incorporated in the design of the second-generation cationic lipids. Lipid-peptide conjugates (peptoids) are effective strategies to overcome the various cellular barriers along with the lipoplex formulations methodologies. In this context, cationic lipid-mediated gene delivery is considerably benefited by the methodologies of liposome-mediated drug delivery. Lipid mediated gene delivery has an intrinsic advantage of being a biomimetic platform on which considerable variations could be built to develop efficient in vivo gene delivery protocols.