An Initial Step of GAS-Containing Autophagosome-Like Vacuoles Formation Requires Rab7

Group A streptococcus (GAS; Streptococcus pyogenes) is a common pathogen that invades non-phagocytic human cells via endocytosis. Once taken up by cells, it escapes from the endocytic pathway to the cytoplasm, but here it is contained within a membrane-bound structure termed GAS-containing autophagosome-like vacuoles (GcAVs). The autophagosome marker GFP-LC3 associates with GcAVs, and other components of the autophagosomal pathway are involved in GcAV formation. However, the mechanistic relationship between GcAV and canonical autophagy is largely unknown. Here, we morphologically analyzed GcAV formation in detail. Initially, a small, GFP-LC3-positive GcAV sequesters each streptococcal chain, and these then coalesce into a single, large GcAV. Expression of a dominant-negative form of Rab7 or RNAi-mediated knockdown of Rab7 prevented the initial formation of small GcAV structures. Our results demonstrate that mechanisms of GcAV formation includes not only the common machinery of autophagy, but also Rab7 as an additional component, which is dispensable in canonical autophagosome formation.     

Fluorimetric detection of phospholipid vesicles bound to planar phospholipid membranes.

The first step in the fusion of two phospholipid membranes culminates in the aggregation of the two lipid bilayers. We have used a custom-built fluorimeter to detect multilamellar vesicles (liposomes) containing the fluorescent dye, 6-carboxyfluorescein (6-CF), bound to a planar lipid bilayer (BLM). Liposomes were added to one side of the BLM, and unbound vesicles were perfused out. This left a residual fluorescence from the BLM, but only when the membranes contained anionic lipids, and then only when millimolar levels of calcium were present. This residual fluorescence was consistently detected only when calcium was included in the buffer during the perfusion. This residual fluorescence originated from liposomes bound to the BLM. Breaking the BLM or lysing the adsorbed vesicles with distilled water abolished it. free 6-CF and/or calcium in the absence of liposomes resulted in no residual fluorescence. No residual fluorescence was detected when both the liposomes and the BLM were composed entirely of zwitterionic lipids. This was found to result from the insensitivity of the fluorimeter to a small number of liposomes adsorbed to the BLM. For this system, we conclude that calcium is necessary for both the initiation and maintenance of the state in which the vesicle membrane is bound to the planar bilayer when the membranes contain negatively charged lipids. This attachment is stronger than the interaction between zwitterionic membranes.     

Visible light-induced destabilization of endocytosed liposomes

The potential biomedical utility of the photoinduced destabilization of liposomes depends in part on the use of green to near infrared light with its inherent therapeutic advantages. The polymerization of bilayers can be sensitized to green light by associating selected amphiphilic cyanine dyes, i.e. the cationic 1,1'-dioctadecyl-3,3,3', 3'-tetramethylindocarbocyanine (DiI), or the corresponding anionic disulfonated DiI (DiI-DS), with the lipid bilayer. The DiI sensitization of the polymerization of 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine/1,2-bis[10-(2', 4'-hexadienoyloxy)-decanoyl]-sn-glycero-3-phosphocholine liposomes caused liposome destabilization with release of encapsulated aqueous markers. In separate experiments, similar photosensitive liposomes were endocytosed by cultured HeLa cells. Exposure of the cells and liposomes to 550 nm light caused a net movement of the liposome-encapsulated 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) from low pH compartment(s) to higher pH compartment(s). This suggests that photolysis of DiI-labelled liposomes results in delivery of the contents of the endocytosed liposomes to the cytoplasm. The release of HPTS into the cytoplasm appears to require the photoactivated fusion of the labelled liposomes with the endosomal membrane. These studies aid in the design of visible light sensitive liposomes for the delivery of liposome-encapsulated reagents to the cytoplasm.     

Endocytosis of liposomes by Entamoeba histolytica and transport of encapsulated molecules toward the cytoplasm

We have examined the interaction of a highly phagocytosing cell: Entamoeba histolytica with liposomes of different lipid compositions, and followed, by a semi-quantitative method, the intracellular fate of the entrapped molecules. Liposomes containing a small molecule, 6-carboxyfluorescein, are first phagocytosed. Then the encapsulated compound migrates from the vacuoles to the cytoplasm. Liposomes containing macromolecular substances, such as fluorescent albumin or ferritin, are also phagocytosed, but the encapsulated molecules remain within the vacuoles. We conclude that the transfer of carboxyfluorescein does not involve a fusion between liposomes and vacuoles, but more likely occurs via diffusion through membranes. The lipid composition of the liposomes does not affect phagocytosis of liposomes. In contrast, oleic acid greatly increases the transfer of carboxyfluorescein from vacuole to cytoplasm.     

Delivery of negatively charged liposomes into the atherosclerotic plaque of apolipoprotein E-deficient mouse aortic tissue

Liposomes have been used to diagnose and treat cancer and, to a lesser extent, cardiovascular disease. We previously showed the uptake of anionic liposomes into the atheromas of Watanabe heritable hyperlipidemic rabbits within lipid pools. However, the cellular distribution of anionic liposomes in atherosclerotic plaque remains undescribed. In addition, how anionic liposomes are absorbed into atherosclerotic plaque is unclear. We investigated the uptake and distribution of anionic liposomes in atherosclerotic plaque in aortic tissues from apolipoprotein E-deficient (ApoE^–/–) mice. To facilitate the tracking of liposomes, we used liposomes containing fluorescently labeled non-silencing small interfering RNA. Confocal microscopy analysis showed the uptake of anionic liposomes into atherosclerotic plaque and colocalization with macrophages. Transmission electron microscopy analysis revealed anionic liposomal accumulation in macrophages. To investigate how anionic liposomes cross the local endothelial barrier, we examined the role of clathrin-mediated endocytosis in human coronary artery endothelial cells (HCAECs) treated with or without the inflammatory cytokine tumor necrosis factor (TNF)-α. Pretreatment with amantadine, an inhibitor of clathrin-mediated endocytosis, significantly decreased liposomal uptake in HCAECs treated with or without TNF-α by 77% and 46%, respectively. Immunoblot analysis showed that endogenous clathrin expression was significantly increased in HCAECs stimulated with TNF-α but was inhibited by amantadine. These studies indicated that clathrin-mediated endocytosis is partly responsible for the uptake of liposomes by endothelial cells. Our results suggest that anionic liposomes target macrophage-rich areas of vulnerable plaque in ApoE^–/– mice; this finding may lead to the development of novel diagnostic and therapeutic strategies for treating vulnerable plaque in humans.     

Triggered release of doxorubicin following mixing of cationic and anionic liposomes

In many applications, an ability of liposomes to retain drug and then rapidly release it at some later time would be of benefit. In this work, we investigate the ability of cationic large unilamellar vesicles (LUV) to promote rapid release of doxorubicin from anionic LUV. It is shown that the addition of cationic liposomes containing cholesterol, dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylcholine (DSPC) and the cationic lipid N,N-dioleyl-N,N-dimethylammonium chloride (DODAC) to doxorubicin-containing LUV composed of cholesterol, DOPE, DSPC and the anionic lipid dioleoyphosphatidylglycerol (DOPG) can result in release of more than 90% of the drug in times of 30 s or less. Further, it is shown that these release characteristics are exquisitely dependent on the presence of DOPE and cholesterol. In the absence of DOPE, much slower release rates are observed, with maximum release levels of 50% after a 2-h incubation at 20 °C. Remarkably, threshold levels of more than 10 mol% cholesterol are required before any appreciable release is observed. [³¹P]NMR spectroscopy and freeze-fracture electron microscopy studies reveal that systems giving rise to rapid release of doxorubicin exhibit limited formation of inverted hexagonal (H_II) phase, suggesting that these lipids facilitate drug release by formation of local regions of non-bilayer structure. It is concluded that drug release triggered by mixing anionic and cationic liposomes could be of utility in drug delivery applications.     

The effect of the cholesterol content of small unilamellar liposomes on the fate of their lipid components in vivo

We have investigated the effect of the cholesterol content of small unilamellar liposomes composed of egg phosphatidylcholine (PC) and containing 6-carboxyfluorescein (6-CF) on the in-vivo fate of their radiolabelled PC (³H-PC) and tracer [1-¹⁴C]-cholesteryl oleate (¹⁴C-cholesteryl oleate) components. Chromatography of the blood plasma of mice at various times after injection with liposomes composed of equimolar amounts of PC and cholesterol (PCCHOL liposomes) showed a main peak (peak A) containing most ³H-PC, ¹⁴C-cholesteryl oleate and 6-CF and representing intact liposomes. With cholesterol- free liposomes (PC liposomes) on the other hand, there was increasing transfer of the two radiolabelled lipids from peak A to the subsequently eluted high density lipoproteins (HDL) (peak B) paralleled by increasing loss of liposomal stability as evidenced by 6-CF release. Studies on the rate of clearance of PCCHOL liposomes showed half-lives of 110 min (³H-PC) and 120 min (¹⁴C-cholesteryl oleate marker). Similar studies with PC liposomes revealed complex patterns of clearance evaluation of which was hampered by a number of observed or anticipated concurrent events: removal of liposomes by tissues, transfer of PC and cholesteryl oleate to HDL, clearance of HDL and donation of the two lipids by HDL to, or their exchange with lipids of, tissues.     

How are Nucleic Acids Released in Cells from Cationic Lipid-Nucleic Acid Complexes?

Abstract

Cationic lipid-nucleic acid complexes are widely used to deliver oligonucleotides, RNA and DNA into cells. Although much has been learned about the structure and forces that hold the complex together, an understanding of the mechanism of release of the nucleic acids from the complex into cells has been lacking. Recent studies have shown that anionic liposomes with compositions similar to the cytoplasmic face of the endosomal membrane are potent agents for inducing the rapid release of oligonucleotides and DNA from cationic lipid-nucleic acid complexes. Based upon these results, we propose that after the cationic lipid/nucleic complex is internalized by endocytosis it destabilizes the endosomal membrane. This destabilization induces flip-flop of anionic lipids from the cytoplasmic facing monolayer, which laterally diffuse into the complex and form a charge neutral ion-pair with the cationic lipids. This results in displacement of the nucleic acid from the cationic lipid and subsequent release of the nucleic acid into cytoplasm of the cell. We review the data that show the proposed mechanism accounts for a variety of observations on cationic lipid/nucleic acid complex-cell interactions.     

Cellular pathway of plasmids vectorized by cholesterol-based cationic liposomes.

We investigated by transmission electron microscopy the cellular route in tumor MCF7 cells of DNA labeled with digoxigenin, carried by cationic liposomes (Lip+) prepared from TMAEC-Chol [3 beta(N-(N',N',N'-trimethylaminoethane)-carbamoyl)cholesterol iodide] and TEAPC-Chol [3 beta(N-(N',N',N'-triethylaminopropane)-carbamoyl)cholesterol iodide], two cholesterol-based cationic lipids containing a quaternary ammonium. In a previous work we showed the pathway of cationic lipid/plasmid complexes from the beginning of endocytosis until their entry into the perinuclear area. Beyond this limit, unlabeled exogenous plasmids cannot be distinguished with nuclear DNA. This work dealt with the cellular fate of cationic liposome-vectorized plasmids labeled with digoxigenin using an immunogold procedure. Early after the beginning of transfection (30 min, 1 hr, 5 hr), gold particles were observed only in the cytoplasm and in endosome-like vesicles, whereas after 24 hr gold particles were densely present in the nucleus. These results demonstrate the nuclear localization of plasmids vectorized by the cationic liposomes used. The results are discussed in comparison with transfection efficiency measurements.     

Direct Cytosolic Delivery of Polar Cargo to Cells by Spontaneous Membrane-translocating Peptides

Direct cellular entry of potentially useful polar compounds into cells is prevented by the hydrophobic barrier of the membrane. Toward circumventing this barrier, we used high throughput screening to identify a family of peptides that carry membrane-impermeant cargos across synthetic membranes. Here we characterize the plasma membrane translocation of these peptides with polar cargos under a variety of conditions. The spontaneous membrane-translocating peptides (SMTPs) delivered the zwitterionic, membrane-impermeant dye tetramethylrhodamine (TAMRA) into cells even when the conditions were not permissive for endocytosis. They also delivered the larger, anionic membrane-impermeant dye Alexa Fluor 546 but did not deliver a quantum dot nanoparticle. Under all conditions, the SMTP-cargo filled the cytoplasm with a diffuse, non-punctate fluorescence that was partially excluded from the nucleus. d-Amino acid peptides behaved identically in vitro, ruling out proteolysis as an important factor in the diffuse cellular distribution. Thus, cytosolic delivery of SMTP-cargo conjugates is dominated by direct membrane translocation. This is in sharp contrast to Arg₉-TAMRA, a representative highly cationic, cell-penetrating peptide, which entered cells only when endocytosis was permitted. Arg₉-TAMRA triggered large scale endocytosis and did not appreciably escape the endosomal compartments in the 1-h timescales we studied. When injected into mice, SMTP-TAMRA conjugates were found in many tissues even after 2 h. Unconjugated TAMRA was rapidly cleared and did not become systemically distributed. SMTPs are a platform that could improve delivery of many polar compounds to cells, in the laboratory or in the clinic, including those that would otherwise be rejected as drugs because they are membrane-impermeant.     

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.     

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 N-terminal 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.     

箭舌豌豆根瘤液泡中细菌周膜来源的研究

电镜观察结果表明,幼龄箭舌豌豆根瘤侵染细胞的细胞质较少,中央是一些体积较大的液泡。细胞质中侵入线经常可见,由侵入线释放出来的细菌均有细菌周膜。这些细菌只位于细胞质中,不出现在液泡里面。成熟根瘤中的侵染细胞与此不同,它们中有大量的成熟侵染细胞,细胞质丰富,里面充满大量细菌,中央常有一个大液泡。当中央液泡发育到一定程度时,位于其附近的细菌可通过液泡膜内吞、液泡膜与细菌周膜融合及液泡膜破裂3种途径进入液泡,后一种途径常伴有寄主细胞质。液泡中的细菌绝大部分裸露在外,只有个别细菌具有细菌周膜且多位于液泡膜的破损处附近,因此细菌周膜可能是原来就有的。     

Functional characterization of an endosome-disruptive peptide and its application in cytosolic delivery of immunoliposome-entrapped proteins

Antibody-directed liposomes (immunoliposomes) are frequently used for targeted drug delivery. However, delivery of large biotherapeutic molecules (i.e. peptides, proteins, or nucleic acids) with immunoliposomes is often hampered by an inefficient cytosolic release of entrapped macromolecules after target cell binding and subsequent endocytosis of immunoliposomes. To enhance cytosolic drug delivery from immunoliposomes present inside endosomes, a pH-dependent fusogenic peptide (diINF-7) resembling the NH(2)-terminal domain of influenza virus hemagglutinin HA-2 subunit was used. Functional characterization of this dimeric peptide showed its ability to induce fusion between liposome membranes and leakage of liposome-entrapped compounds when exposed to low pH. In a second series of experiments, diINF-7 peptides were encapsulated in immunoliposomes to enhance the endosomal escape of diphtheria toxin A chain (DTA), which inhibits protein synthesis when delivered into the cytosol of target cells. Immunoliposomes targeted to the internalizing epidermal growth factor receptor on the surface of ovarian carcinoma cells (OVCAR-3) and containing encapsulated DTA did not show any cytotoxicity toward OVCAR-3 cells. Cytotoxicity was only observed when diINF-7 peptides and DTA were co-encapsulated in the immunoliposomes. Thus, diINF-7 peptides entrapped inside liposomes can greatly enhance cytosolic delivery of liposomal macromolecules by pH-dependent destabilization of endosomal membranes after cellular uptake of liposomes.     

Uptake of liposomes containing the photoprotein obelin by rat isolated adipocytes. Adhesion,endocytosis or fusion?

1. The uptake of liposomes containing the photoprotein obelin by rat isolated adipocytes was investigated with the aim of producing liposome–cell fusion, enabling obelin to be introduced into the cytoplasm of intact cells. 2. Incubation of liposomes containing obelin with rat isolated adipocytes resulted in a time-dependent uptake of entrapped obelin by the adipocytes. The uptake by adipocytes (at 2h) of liposomes prepared from phosphatidylcholine, phosphatidylcholine+phosphatidylserine (molar ratio 4:1) and phosphatidylcholine+N-octadecylamine (molar ratio 4:1) was approx. 6, 10 and 10% of original entrapped obelin per g dry wt. of adipocytes respectively. 3. During incubation with adipocytes some of the liposomes became permeable to Ca²⁺ ions, resulting in stimulation of obelin luminescence from within the liposomes. This increase in permeability to Ca²⁺ seemed to be the result of the interaction of liposomes with the cell membrane. 4. Approx. 50% of liposome uptake could be inhibited by cytochalasin B (500μm). This was consistent with this uptake being the result of endocytosis. The remaining uptake was probably the result of adhesion of liposomes to the cell membrane. 5. In an attempt to detect the presence of cytoplasmic obelin, after incubation of adipocytes with liposomes, a method of causing a rapid rise in cell-membrane permeability to Ca²⁺ was developed in which an anti-cell anti-body–complement reaction occurred at the cell membrane. There was no detectable transfer of active obelin into the cell cytoplasm. 6. After incubation of liposomes with adipocytes in the absence of bovine serum albumin, obelin luminescence from a small proportion of liposomes increased (approx. 1.5%) in response to anti-(5′-nucleotidase) antibody plus complement. 7. It was concluded that under the conditions of these experiments, (a) no detectable transfer (<0.1%) of liposome-entrapped obelin to the adipocyte cytoplasm had occurred, (b) an increase in liposome permeability to Ca²⁺ occurred during incubation with adipocytes, (c) at least 50% of liposome uptake by adipocytes was the result of endocytosis, presumably into secondary lysosomes, and the remaining uptake was apparently the result of loose attachment of liposomes to the cell surface, and (d) in the absence of bovine serum albumin, a portion of at least one surface antigen, the ectoenzyme 5′-nucleotidase, was transferred from the adipocyte membrane to the liposome membrane.     

Cationic liposomes formulated with DMPC and a gemini surfactant traverse the cell membrane without causing a significant bio-damage

Cationic liposomes have been intensively studied both in basic and applied research because of their promising potential as non-viral molecular vehicles. This work was aimed to gain more information on the interactions between the plasmamembrane and liposomes formed by a natural phospholipid and a cationic surfactant of the gemini family. The present work was conducted with the synergistic use of diverse experimental approaches: electro-rotation measurements, atomic force microscopy, ζ-potential measurements, laser scanning confocal microscopy and biomolecular/cellular techniques. Electro-rotation measurements pointed out that the interaction of cationic liposomes with the cell membrane alters significantly its dielectric and geometric parameters. This alteration, being accompanied by significant changes of the membrane surface roughness as measured by atomic force microscopy, suggests that the interaction with the liposomes causes locally substantial modifications to the structure and morphology of the cell membrane. However, the results of electrophoretic mobility (ζ-potential) experiments show that upon the interaction the electric charge exposed on the cell surface does not vary significantly, pointing out that the simple adhesion on the cell surface of the cationic liposomes or their fusion with the membrane is to be ruled out. As a matter of fact, confocal microscopy images directly demonstrated the penetration of the liposomes inside the cell and their diffusion within the cytoplasm. Electro-rotation experiments performed in the presence of endocytosis inhibitors suggest that the internalization is mediated by, at least, one specific pathway. Noteworthy, the liposome uptake by the cell does not cause a significant biological damage.     

Apolipoprotein E peptide-modified colloidal carriers: The design determines the mechanism of uptake in vascular endothelial cells

Supramolecular structures, particularly micelles and liposomes equipped with uptake-mediating address compounds, have attracted much attention as pharmaceutical formulations. Their development requires an understanding of the mechanism by which the carrier systems interact with and translocate into the target cells. We developed an apolipoprotein E-derived peptide, called A2, that efficiently translocates across cell membranes. Upon coupling of two palmitoyl chains (P2), the highly cationic sequence acquires detergent-like properties such as a strong tendency to self-associate and the ability to integrate into lipid bilayers. Confocal laser scanning microscopy and fluorescence activated cell sorting were used to compare the internalization of the fluorescence-labeled monomeric A2 with the uptake of the colloidal P2A2 micelles and P2A2-tagged liposomes into endothelial cells of blood vessels. Specific inhibitors of endocytosis were used to identify the underlying mechanisms. b.End3 and BAEC cells as example of endothelial cells of small capillaries and large vascular vessels, respectively, were examined. The uptake of monomeric A2 was characterized by poor cellular selectivity. A2 was efficiently internalized into both cell lines via at least two different mechanisms. Besides an endocytotic uptake route, a second passive pathway exists, that leads to a rapid distribution of A2 within the cytoplasm. Also liposomes tagged with P2A2 were non-selectively internalized into both b.End3 and BAEC cells. Their nonselective uptake was mediated by clathrin- and caveolin-independent endocytosis. In contrast, micellar P2A2 entered b.End3 cells via clathrin-mediated endocytosis, while no uptake of P2A2 into BAEC cells was observed. In conclusion, the specific clathrin-mediated uptake mode of P2A2 micelles might provide the basis for a blood brain barrier-specific targeting.     

Studies of membranotropic and fusogenic activity of two putative HCV fusion peptides

Over the past decades, membranotropic peptides such as positively charged cell-penetrating peptides (CPPs) or amphipathic antimicrobial peptides (AMPs) have received increasing interest in order to improve therapeutic agent cellular uptake.As far as we are concerned, we were interested in studying HCV fusion peptides as putative anchors. Two peptides, HCV6 and HCV7, were identified and conjugated to a fluorescent tag NBD and tested for their interaction with liposomes as model membranes. DSC and spectrofluorescence analyses demonstrate HCV7 propensity to insert or internalize in vesicles containing anionic lipids DMPG whereas no activity was observed with zwitterionic DMPC. This behavior could be explained by the peptide sequence containing a cationic arginine residue. On the contrary, HCV6 did not exhibit any membranotropic activity but was the only sequence able to induce liposomes' fusion or aggregation monitored by spectrofluorescence and DLS. This two peptides mild activity was related to their inefficient structuration in contact with membrane mimetics, which was demonstrated by CD and NMR experiments.Altogether, our data allowed us to identify two promising membrane-active peptides from E1 and E2 HCV viral proteins, one fusogenic (HCV6) and the other membranotropic (HCV7). The latter was also confirmed by fluorescence microscopy with CHO cells, indicating that HCV7 could cross the plasma membrane via an endocytosis process. Therefore, this study provides new evidences supporting the identification of HCV6 as the HCV fusion peptide as well as insights on a novel membranotropic peptide from the HCV-E2 viral protein.     

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