Synthesis and in vitro evaluation of glutamide-containing cationic lipids for gene delivery

A novel series of cationic amphiphiles based on dialkyl glutamides with cationic pyridinium head group were synthesized as potential gene delivery agents. Four cationic lipids with glutamide as linker and varying chain lengths were tested for their transfection efficiency in three cell lines. The DNA-lipid complexes were characterized for their ability to bind to DNA, protection from nuclease digestion, size, zeta-potential, and toxicity. All four lipids demonstrated efficient transfection in MCF-7, COS, and HeLa cells, and the reporter gene expression was much higher with DOPE as the helper lipid in the formulation when compared to cholesterol. Among these 14-carbon lipids, lipid 2 has shown the highest transfection efficiency, complete protection of DNA from nuclease digestion, and low toxicity. Interestingly, lipid 2 has also shown remarkable enhancement in transfection in the presence of serum.     

Novel cationic amphiphilic 1,4-dihydropyridine derivatives for DNA delivery

In order to find new efficient and safe agents for gene delivery, we have designed and synthesized nine novel single- and double-charged amphiphiles on the base of 1,4-dihydropyridine (1,4-DHP) ring. Some biophysical properties of the amphiphilic dihydropyridines and their complexes with DNA were examined. We investigated the transfer of beta-galactosidase gene into fibroblasts (CV1-P) and retinal pigment epithelial (D 4O7) cell lines in vitro. The structure-property relationships of the compounds were investigated in various ways. The net surface charges of 1,4-DHP liposomes were highly positive (25-49 mV). The double-charged compounds condensed DNA more efficiently than single-charged and the condensation increases with the increasing +/- charge ratio between the carrier and DNA. Double-charged compounds showed also buffering properties at endosomal pH and these compounds were more efficient in transfecting the cells, but transfection efficiency of amphiphiles was cell type-dependent. The length of alkyl chains in double-charged compounds affected the transfection efficacy. The most active amphiphile (compound VI) was double-charged and had two C(12) alkyl chains. At optimal charge ratio (+/- 4), it was 2.5 times more effective than PEI 25 and 10 times better than DOTAP, known efficient polymeric and liposomal transfection agents. Formulation of amphiphiles with DOPE did not change their activities. Our data demonstrate some important effects of amphiphile structure on biophysics and activity. The data also suggest that cationic amphiphilic 1,4-DHP derivatives may find use as DNA delivery system.     

Advantage of the ether linkage between the positive charge and the cholesteryl skeleton in cholesterol-based amphiphiles as vectors for gene delivery

Twelve novel cationic cholesterol derivatives with different linkage types between the cationic headgroup and the cholesteryl backbone have been developed. These have been tested for their efficacies as gene transfer agents as mixtures with dioleoyl phosphatidylethanolamine (DOPE). A pronounced improvement in transfection efficiency was observed when the cationic center was linked to the steroid backbone using an ether type bond. Among these, cholest-5-en-3b-oxyethane-N,N,N-trimethylammonium bromide (2a) and cholest-5-en-3b-oxyethane-N,N-dimethyl-N-2-hydroxyethylammonium bromide (3d) showed transfection efficiencies considerably greater than commercially available reagents such as Lipofectin or Lipofectamine. To achieve transfection, 3d did not require DOPE. Increasing hydration at the headgroup level for both ester- and ether-linked amphiphiles resulted in progressive loss of transfection efficiency. Transfection efficiency was also greatly reduced when a 'disorder'-inducing chain like an oleyl (cis-9-octadecenyl) segment was added to these cholesteryl amphiphiles. Importantly, the transfection ability of 2a with DOPE in the presence of serum was significantly greater than for a commercially available reagent, Lipofectamine. This suggests that these novel cholesterol-based amphiphiles might prove promising in applications involving liposome-mediated gene transfection. This investigation demonstrates the importance of structural features at the molecular level for the design of cholesterol-based gene delivery reagents that would aid the development of newer, more efficient formulations based on this class of molecules.     

Gene transfer efficacies of novel cationic amphiphiles with alanine, beta-alanine, and serine headgroups: a structure-activity investigation

Herein, we report on the relative in vitro efficacies of nine novel non-glycerol based cationic amphiphiles with increasing hydrophobic tails and the amino acids serine, alanine and beta-alanine as the headgroup functionalities (lipids 1-9, Scheme 1) in transfecting multiple cultured cells including CHO, COS-1, MCF-7, and HepG2. The gene transfer efficiencies of lipids 1-9 were evaluated using the reporter gene assays in all the four cell lines and the whole cell histochemical X-gal staining assays in representative CHO cells. In CHO, HepG2, and MCF-7 cells, cationic lipids with alanine (4-6) and beta-alanine (7-9) headgroups were found to be remarkably more transfection efficient than their serine headgroup counterparts (1-3). Most notably, in CHO, HepG2, and MCF-7 cells, in combination with cholesterol as auxiliary lipid, the transfection efficiencies of the cationic lipids with alanine and beta-alanine headgroups and myristyl and palmityl tails (lipids 4, 5, 7 and 8) were significantly higher (2-3-fold) than that of LipofectAmine-2000, a widely used commercially available liposomal tranfection vectors. Surprisingly, in COS-1 cells, although cationic lipids with beta-alanine headgroups (7-9) were strikingly transfection efficient (3-4-fold more efficacious than LipofectAmine-2000), the gene transfer properties of both their structural isomers (4-6) and their serine headgroup counterparts (1-3) were adversely affected. In summary, the present structure-activity investigation demonstrate that high gene delivery efficacies of cationic amphiphiles containing alanine or beta-alanine headgroups can get seriously compromised by substituting the alanine or beta-alanine with serine presumably due to the enhanced sensitivity of DNA associated with such serine-head-containing cationic lipids.     

Synthesis and optimization of cholesterol-based diquaternary ammonium Gemini Surfactant (Chol-GS) as a new gene delivery vector

Amongst a number of potential nonviral vectors, cationic liposomes have been actively researched, with both gemini surfactants and bola amphiphiles reported as being in possession of good structures in terms of cell viability and in vitro transfection. In this study, a cholesterol-based diquaternary ammonium gemini surfactant (Chol-GS) was synthesized and assessed as a novel nonviral gene vector. Chol-GS was synthesized from cholesterol by way of four reaction steps. The optimal efficiency was found to be at a weight ratio of 1:4 of lipid:DOPE (1,2-dioleoyl-L-alpha- glycero-3-phosphatidylethanolamine), and at a ratio of between 10:1~15:1 of liposome:DNA. The transfection efficiency was compared with commercial liposomes and with Lipofectamine, 1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE-C), and N-[1-(2,3-dioleoyloxy)propyl]- N,N,N-trimethylammonium chloride (DOTAP). The results indicate that the efficiency of Chol-GS is greater than that of all the tested commercial liposomes in COS7 and Huh7 cells, and higher than DOTAP and Lipofectamine in A549 cells. Confirmation of these findings was observed through the use of green fluorescent protein expression. Chol-GS exhibited a moderate level of cytotoxicity, at optimum concentrations for efficient transfection, indicating cell viability. Hence, the newly synthesized Chol-GS liposome has the potential of being an excellent nonviral vector for gene delivery.     

Cationic amphiphiles and the solubilization of cholesterol crystallites in membrane bilayers

Cationic amphiphiles used for transfection can be incorporated into biological membranes. By differential scanning calorimetry (DSC), cholesterol solubilization in phospholipid membranes, in the absence and presence of cationic amphiphiles, was determined. Two different systems were studied: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)+cholesterol (1:3, POPC:Chol, molar ratio) and 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-l-serine] (POPS)+cholesterol (3:2, POPS:Chol, molar ratio), which contain cholesterol in crystallite form. For the zwitterionic lipid POPC, cationic amphiphiles were tested, up to 7 mol%, while for anionic POPS bilayers, which possibly incorporate more positive amphiphiles, the fractions used were higher, up to 23 mol%. 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and DOTAP in methyl sulfate salt form (DOTAPmss) were found to cause a small decrease on the enthalpy of the cholesterol transition of pure cholesterol aggregates, possibly indicating a slight increase on the cholesterol solubilization in POPC vesicles. With the anionic system POPS:Chol, the cationic amphiphiles dramatically change the cholesterol crystal thermal transition, indicating significant changes in the cholesterol aggregates. For structural studies, phospholipids spin labeled at the 5th or 16th carbon atoms were incorporated. In POPC, at the bilayer core, the cationic amphiphiles significantly increase the bilayer packing, decreasing the membrane polarity, with the cholesterol derivative 3 beta-[N-(N',N'-dimethylaminoethane)-carbamoyl]-cholesterol (DC-chol) displaying a stronger effect. In POPS and POPS:Chol, DC-chol was also found to considerably increase the bilayer packing. Hence, exogenous cationic amphiphiles used to deliver nucleic acids to cells can change the bilayer packing of biological membranes and alter the structure of cholesterol crystals, which are believed to be the precursors to atherosclerotic lesions.     

Gene transfer effects on various cationic amphiphiles in CHO cells

Gene transfer is an important tool to explore genomic, cell biologic, or gene therapeutic research. In this paper we report that several cationic amphiphiles have the potential to efficiently deliver DNA into CHO cells, which is one of the cell lines considered to be important for production of proteins including therapeutic proteins. We have found that O,O′-ditetradecanoyl-N-(trimethylammonio acetyl) diethanolamine chloride (14Dea2), among 29 types of cationic amphiphiles tested, shows a transfection efficiency of more than 40% in CHO cells. In addition, the results from a series of hydrocarbon chains of varying lengths bound to a connector have shown that an optimal chain length is important for the efficient delivery of DNA into cells. Moreover, flow cytometer analysis has shown that 14Dea2 transfection leads to high levels of expression of the reporter gene (green fluorescent protein) in individual cells. These findings have suggested that 14Dea2 is able to effectively deliver a number of plasmids into a cell nucleus. Thus, our system might be a powerful tool for high efficiency gene transfer and production of high levels of recombinant protein.     

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.     

Cationic amphiphiles and the solubilization of cholesterol crystallites in membrane bilayers

Cationic amphiphiles used for transfection can be incorporated into biological membranes. By differential scanning calorimetry (DSC), cholesterol solubilization in phospholipid membranes, in the absence and presence of cationic amphiphiles, was determined. Two different systems were studied: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) + cholesterol (1:3, POPC:Chol, molar ratio) and 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-l-serine] (POPS) + cholesterol (3:2, POPS:Chol, molar ratio), which contain cholesterol in crystallite form. For the zwitterionic lipid POPC, cationic amphiphiles were tested, up to 7 mol%, while for anionic POPS bilayers, which possibly incorporate more positive amphiphiles, the fractions used were higher, up to 23 mol%. 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and DOTAP in methyl sulfate salt form (DOTAP^mss) were found to cause a small decrease on the enthalpy of the cholesterol transition of pure cholesterol aggregates, possibly indicating a slight increase on the cholesterol solubilization in POPC vesicles. With the anionic system POPS:Chol, the cationic amphiphiles dramatically change the cholesterol crystal thermal transition, indicating significant changes in the cholesterol aggregates. For structural studies, phospholipids spin labeled at the 5th or 16th carbon atoms were incorporated. In POPC, at the bilayer core, the cationic amphiphiles significantly increase the bilayer packing, decreasing the membrane polarity, with the cholesterol derivative 3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]-cholesterol (DC-chol) displaying a stronger effect. In POPS and POPS:Chol, DC-chol was also found to considerably increase the bilayer packing. Hence, exogenous cationic amphiphiles used to deliver nucleic acids to cells can change the bilayer packing of biological membranes and alter the structure of cholesterol crystals, which are believed to be the precursors to atherosclerotic lesions.     

Dynamics of lysosomal cholesterol in Niemann-Pick type C and normal human fibroblasts

The dynamics of endolysosomal cholesterol were investigated in Niemann-Pick type C (NPC) cells and in human fibroblasts treated with class 2 amphiphiles to mimic NPC cells. We showed through new approaches that the massive pools of endolysosomal cholesterol in these cells are not trapped but, rather, circulate to the cell surface at about the normal rate. This flux spared NPC and amphiphile-treated cells from disruption by the extraction of their plasma membrane cholesterol with cyclodextrin. Nocodazole, a microtubule-depolymerizing agent, reversed the resistance of NPC and U18666A-treated cells to cholesterol depletion, apparently by reducing the flux of endolysosomal cholesterol to the plasma membrane. Neither nocodazole nor bafilomycin A1 (an inhibitor of the vacuolar proton pump) acted in the same way as the NPC mutation or class 2 amphiphiles: both agents decreased plasma membrane cholesterol at the expense of the endolysosomal pool and both blocked the actions of the amphiphile, U18666A. Finally, the resistance of NPC cells to lysis by amphotericin B was shown not to reflect a reduction in plasma membrane cholesterol arising from a block in lysosomal cholesterol export but rather the diversion of the amphotericin B to cholesterol-rich endolysosomes. We conclude that the large pool of endolysosomal cholesterol in NPC and amphiphile-treated fibroblasts is dynamic and that its turnover, as in normal cells, is dependent on microtubules.     

Polycationic amphiphiles based on triethylenetetramine and their transfection efficacy

Novel polycationic amphiphiles derived from triethylenetetramine, a spermine analogue, containing cholesterol or dialkylglycerol residues as hydrophilic domains were synthesized. The amphiphiles and a helper lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) served for the preparation of cationic liposomes, physicochemical properties of which were evaluated. A comparative study of cytotoxicity and transfection efficiency demonstrated that the replacement of spermine by triethylenetetramine decreased transfection activity of cationic liposomes.     

In vitro gene transfer efficacies and serum compatibility profiles of novel mono-, di-, and tri-histidinylated cationic transfection lipids: a structure-activity investigation

Recently, we demonstrated that covalent grafting of an endosome-disrupting single histidine functionality in the headgroup region imparts high gene transfer properties to cationic amphiphiles (Kumar, V. V., et al. Gene Ther. 2003, 10, 1206-1215). However, whether covalent attachment of multiple histidine functionalities in the headgroup region are capable of further enhancing the gene transfer efficacies of cationic amphiphiles remains to be explored. To this end, herein, we report on the design, syntheses, physicochemical characterizations, in vitro gene transfer properties, and serum compatibilities of three novel nontoxic cationic transfection amphiphiles containing mono-, di-, and tri-histidine functionalities in their headgroup regions (lipids 1-3) in multiple cultured cells. Significantly, findings in both the reporter gene expression assay and the whole cell histochemical X-gal staining assay support the notion that there is no linear correlation between the in vitro transfection efficacies and the number of histidine functionalities in the polar headgroup regions for histidinylated cationic amphiphiles. The relative gene transfer efficiencies, as well as the serum compatibilities, of the present histidinylated cationic amphiphiles were found to be strikingly dependent on the medium of lipoplex formation. Most importantly, high serum compatibilities (up to 50% added serum) of the lipoplexes of lipids 1 and 3 make them promising nonviral transfection vectors for future systemic applications.     

Cholesterol movement in Niemann-Pick type C cells and in cells treated with amphiphiles

Cholesterol accumulates to massive levels in cells from Niemann-Pick type C (NP-C) patients and in cells treated with class 2 amphiphiles that mimic NP-C disease. This behavior has been attributed to the failure of cholesterol released from ingested low density lipoproteins to exit the lysosomes. However, we now show that the rate of movement of cholesterol from lysosomes to plasma membranes in NP-C cells is at least as great as normal, as was also found previously for amphiphile-treated cells. Furthermore, the lysosomes in these cells filled with plasma membrane cholesterol in the absence of lipoproteins. In addition, we showed that the size of the endoplasmic reticulum cholesterol pool and the set point of the homeostatic sensor of cell cholesterol were approximately normal in NP-C cells. The plasma membrane cholesterol pools in both NP-C and amphiphile-treated cells were also normal. Furthermore, the build up of cholesterol in NP-C lysosomes was not a physiological response to cholesterol overload. Rather, it appeared that the accumulation in NP-C lysosomes results from an imbalance in the brisk flow of cholesterol among membrane compartments. In related experiments, we found that NP-C cells did not respond to class 2 amphiphiles (e.g. trifluoperazine, imipramine, and U18666A); these agents may therefore act directly on the NPC1 protein or on its pathway. Finally, we showed that the lysosomal cholesterol pool in NP-C cells was substantially and preferentially reduced by incubating cells with the oxysterols, 25-hydroxycholesterol and 7-ketocholesterol; these findings suggest a new pharmacological approach to the treatment of NP-C disease.     

Enhancement of gene transfer using YIGSR analog of Tat-derived peptide

Cell penetrating peptide based gene carriers are notably known for low level of gene transfer. To remedy this, as laminin receptor (LR) has been previously linked to tumor metastasis, the LR-binding domain (YIGSR) as well as a scrambled sequence (SGIYR) were added to Tat-derived peptide sequence (YIGSR-Tat and SGIYR-Tat respectively). Peptides cellular uptake was assessed with high-LR (HT1080) and low-LR (HT29) cell lines by flow cytometry. Their ability to form complexes with DNA was examined using YOPRO-1 fluorescence assay and their transfection efficiencies evaluated using a luciferase reporter gene assay. DNA complexes were formed at (+/-) charge ratios as low as 2:1. While no conclusion could be drawn on the effect of YIGSR sequence on peptides uptake in both cell lines, a significant improvement in gene transfection in HT1080 cells was achieved using YIGSR-Tat compared to Tat and SGIYR-Tat. Additionally this increased efficiency was inhibited by excess free YIGSR. No significant difference in transfection efficiency was observed between Tat, SGIYR-Tat and YIGSR-Tat based complexes in HT29 cells. These studies demonstrate that attachment of receptor-binding ligand (YIGSR) to Tat-derived peptide can improve the efficiency of gene transfer in LR-positive cells (HT1080).     

Synthetic cationic amphiphiles for liposome-mediated DNA transfection

The compounds with efficient DNA transfection ability into eukaryotic cells were searched from various synthetic amphiphiles which have cationic heads and long saturated hydrocarbon tails. The efficiency of amphiphiles in gene transfer was examined by the transient expression of cytochrome b5 from its cDNA in COS cells. Among various synthetic amphiphiles, including N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride which is commercially available lipid, O,O'-didodecyl-N-[p-(2-trimethylammonioethyloxy)benzoyl]-(L) -glutamate bromide was highest in efficiency. The optimum condition for the amount of the amphiphile and DNA, and the incubation time were established to be 7.5-15 micrograms/22 mm dish and 1-10 micrograms/22 mm dish, and 48-72 h, respectively.     

Pyridinium cationic lipids in gene delivery: an in vitro and in vivo comparison of transfection efficiency versus a tetraalkylammonium congener

Cationic lipids provide a promising alternative to the use of viruses for delivering genes therapeutically. Among the several classes of lipidic vectors, those bearing a heterocyclic cationic head have shown important advantages, such as low cytotoxicity and improved efficiency across different cell lines. We recently reported a simple and efficient strategy for obtaining pyridinium cationic lipids, starting from pyrylium salts and primary amines. The present study is aimed to compare the cellular toxicity and transfection efficiency generated by the pyridinium polar head versus the tetramethylammonium one on several tumor cell lines and also in experimental animals, delivered via intratumor injections. Thus, the lead compound 1-(2,3-dioleoyloxypropyl)-2,4,6-trimethylpyridinium lipid (2Oc), coformulated with different helper lipids in various molar ratios, was tested against its ammonium congener DOTAP-a standard transfection reagent. The results revealed that when formulated with cholesterol at 1:1 molar ratio, the pyridinium lipid 2Oc was able to transfect several cancer cell lines with similar or better efficiency than its tetraalkylammonium congener DOTAP, while producing lower cytotoxicity. The NCI-H23 lung cancer cell line was found to be the most susceptible to be transfected. Therefore, we designed an in vivo assay based on this type of carcinoma in nude mice, which were injected intratumoral with 2Oc- and DOTAP-based lipoplexes. The red fluorescent protein reporter revealed that the pyridinium cationic lipid was superior to its tetraalkylammonium congener, transfecting the tissue on a higher area and with higher efficiency. These encouraging findings, together with the simple and efficient synthetic strategy, lay the foundation for further development of pyridinium lipids for gene therapy with improved transfection efficiency in vivo and even further reduced cytotoxicity.     

Characterization of the interplay between the main factors contributing to lipoplex-mediated transfection in cell cultures

Transfection efficiency of lipoplex-mediated gene delivery is multifactorial. However, the mode of interaction between the factors which affect transfection is not fully understood. To help fill this deficiency we evaluated the effect of the interplay between several variables that affect transfection efficiency in cell cultures. For this, we applied the Analysis of Variance Model with Fixed Effects and Repeated Measures to assess the data. The variables studied include: two different genes, Luc, and human growth hormone (hGH), in three different plasmids (two of which contain the luciferase (Luc) gene, but different promoter-enhancer regions (CMV and H19) and one plasmid coding hGH with a S16 promoter); three topoisoforms of pDNA (supercoiled (SC), open circular (OC), and closed circular (CC)); three cationic lipid compositions, all based on the monocationic lipid DOTAP (100% DOTAP, DOTAP/DOPE 1 : 1, and DOTAP/cholesterol 1 : 1, all ratios are mole ratios); two DNA-/L+ charge ratios (0.2 and 0.5); and two cell lines (NIH 3T3 and MBT-2). Our statistical analysis confirmed that the cell type, the gene used for transfection, the promoter type, the type of helper lipid, and DNA-/DOTAP+ charge ratio, all affect transfection efficiency in a statistically significant manner. The most efficient lipoplex formulation in both cell lines was that based on DOTAP (without helper lipid), having CC plasmid DNA. We suggest that for obtaining the most transfection-efficient lipoplex one should select the best topoisoform of pDNA for each particular cell type, and complex it with cationic liposomes having optimal lipid composition.     

Regulation of endoplasmic reticulum cholesterol by plasma membrane cholesterol

The abundance of cell cholesterol is governed by multiple regulatory proteins in the endoplasmic reticulum (ER) which, in turn, are under the control of the cholesterol in that organelle. But how does ER cholesterol reflect cell (mostly plasma membrane) cholesterol? We have systematically quantitated this relationship for the first time. We found that ER cholesterol in resting human fibroblasts comprised approximately 0.5% of the cell total. The ER pool rose by more than 10-fold in less than 1 h as cell cholesterol was increased by approximately 50% from below to above its physiological value. The curve describing the dependence of ER on plasma membrane cholesterol had a J shape. Its vertex was at the ambient level of cell cholesterol and thus could correspond to a threshold. A variety of class 2 amphiphiles (e.g., U18666A) rapidly reduced ER cholesterol but caused only minor alterations in the J-curve. In contrast, brief exposure of cells to the oxysterol, 25-hydroxycholesterol, elevated and linearized the J-curve, increasing ER cholesterol at all values of cell cholesterol. This finding can explain the rapid action of oxysterols on cholesterol homeostasis. Other functions have also been observed to depend acutely on the level of plasma membrane cholesterol near its physiological level, perhaps reflecting a cholesterol-dependent structural or organizational transition in the bilayer. Such a physical transition could serve as a set-point above which excess plasma membrane cholesterol is transported to the ER where it would signal regulatory proteins to down-regulate its further accumulation.     

Ethanol-based proliposome delivery systems of paclitaxel for in vitro application against brain cancer cells

In this study the anticancer activity of paclitaxel-loaded nano-liposomes on glioma cell lines was investigated. Soya phosphatidylcholine:cholesterol (SPC:Chol), hydrogenated soya phosphatidylcholine:cholesterol (HSPC:Chol) or dipalmitoylphosphatidylcholine:cholesterol (DPPC:Chol) in 1:1?mole ratio were used to prepare ethanol-based proliposomes. Following hydration of proliposomes, the size of resulting vesicles was subsequently reduced to nanometer scale via probe-sonication. The resulting formulations were characterized in terms of size, zeta potential and morphology of the vesicles, and entrapment efficiency of paclitaxel (PX) as well as the final pH of the preparations. DPPC-liposomes entrapped 35–92% of PX compared to 27–74% and 25–60% entrapped by liposomes made from SPC and HSPC formulations respectively, depending on drug concentration. The entrapment efficiency of liposomes was dependent on the lipid bilayer properties and ability of PX to modify surface charge of the vesicles. In vitro cytotoxicity studies revealed that PX-liposome formulations were more selective at inhibiting the malignant cells. The cytotoxicity of PX-liposomes was dependent on their drug-entrapment efficiency. This study has shown PX-liposomes generated from proliposomes have selective activity against glioma cell lines, and the synthetic DPPC phospholipid was most suitable for maximized drug entrapment and highest activity against the malignant cells in vitro.     

Isolation and characterization of Chinese hamster ovary cells defective in the intracellular metabolism of low density lipoprotein-derived cholesterol.

We have isolated clones of an established cell line which express defects in intracellular cholesterol metabolism. Chinese hamster ovary cells were mutagenized, and clones unable to mobilize low density lipoprotein (LDL)-derived cholesterol to the plasma membrane were selected. Biochemical analysis of two mutant clones revealed a phenotype characteristic of the lysosomal storage disease, Niemann-Pick type C. The mutant cell lines were found to be defective in the regulatory responses elicited by LDL-derived cholesterol. LDL-mediated stimulation of cholesterol esterification was grossly defective, and LDL suppression of 3-hydroxy-3-methylglutaryl-CoA reductase was impaired. However, the mutants modulated these activities normally in response to 25-hydroxycholesterol or mevalonate. The LDL-specific defects were predicated by the inability of these mutants to mobilize LDL-derived cholesterol from lysosomes. Cell fractionation studies showed that LDL-derived, unesterified cholesterol accumulated in the lysosomes of mutant cells to significantly higher levels than normal, commensurate with defective movement of cholesterol to other cellular membranes. Characterization of cell lines defective in intracellular cholesterol transport will facilitate identification of the gene(s) required for intracellular cholesterol movement and regulation.