Cholesterol domains in cationic lipid/DNA complexes improve transfection

The interaction between the cationic lipid DOTAP and cholesterol is examined in high cholesterol formulations by differential scanning calorimetry (DSC). Preparation of liposomes above 66 mol% cholesterol results in formulations that exhibit a calorimetric transition for anhydrous cholesterol at 38-40 °C. The enthalpy of this transition progressively increases at higher cholesterol contents, and is not detected below 66 mol% cholesterol. Furthermore, the enthalpy changes indicate that the composition of the non-domain forming portion containing DOTAP saturated with cholesterol is relatively constant above 66 mol% cholesterol. Greater transfection efficiency in the presence of 50% serum is observed at the formulations with high cholesterol contents where anhydrous cholesterol domains are detected by DSC. Although formulations possessing higher cholesterol exhibited a greater resistance to serum-induced aggregation, maintenance of small particle size does not appear to be responsible for the enhanced transfection efficiency. Additional studies quantifying albumin binding suggest that cholesterol domains in the lipid/DNA complex do not bind protein, and this may enable these moieties to enhance transfection by facilitating membrane fusion.     

The role of lipid charge density in the serum stability of cationic lipid/DNA complexes

To evaluate the role of lipid charge density in the serum stability of DOTAP-Chol/DNA complexes (lipoplexes), lipid-DNA interactions, extent of aggregation, supercoil content, and in vitro transfection efficiency of lipoplexes were investigated. In general, higher serum concentration destabilized, and increasing molar charge ratio of DOTAP to negatively charged phosphates in the DNA (DOTAP(+)/DNA(-)) stabilized lipoplexes in serum as assessed by the criteria used in this study. The increase of cholesterol content led to increased serum stability, and DOTAP:Chol (mol/mol 1:4)/DNA lipoplex with DOTAP(+)/DNA(-) ratio 4 was the most serum stable formulation of all the formulations examined, and maintained lipid-DNA interactions, did not aggregate and exhibited high in vitro transfection efficiency in 50% (v/v) serum. The increased stability of this formulation could not be explained by the decreased charge density of the lipid component. Furthermore, no single parameter examined in the study could be used to consistently predict the in vitro transfection efficiency of lipoplexes in serum. Surprisingly, no correlation between the maintenance of supercoiled DNA content and in vitro transfection efficiency was found in the study.     

Aphidicolin-resistant polyomavirus and subgenomic cellular DNA synthesis occur early in the differentiation of cultured myoblasts to myotubes.

Small DNA viruses have been historically used as probes of cellular control mechanisms of DNA replication, gene expression, and differentiation. Polyomavirus (Py) DNA replication is known to be linked to differentiation of may cells, including myoblasts. In this report, we use this linkage in myoblasts to simultaneously examine (i) cellular differentiation control of Py DNA replication and (ii) an unusual type of cellular and Py DNA synthesis during differentiation. Early proposals that DNA synthesis was involved in the induced differentiation of myoblasts to myotubes were apparently disproved by reliance on inhibitors of DNA synthesis (cytosine arabinoside and aphidicolin), which indicated that mitosis and DNA replication are not necessary for differentiation. Theoretical problems with the accessibility of inactive chromatin to trans-acting factors led us to reexamine possible involvement of DNA replication in myoblast differentiation. We show here that Py undergoes novel aphidicolin-resistant net DNA synthesis under specific conditions early in induced differentiation of myoblasts (following delayed aphidicolin addition). Under similar conditions, we also examined uninfected myoblast DNA synthesis, and we show that soon after differentiation induction, a period of aphidicolin-resistant cellular DNA synthesis can also be observed. This drug-resistant DNA synthesis appears to be subgenomic, not contributing to mitosis, and more representative of polyadenylated than of nonpolyadenylated RNA. These results renew the possibility that DNA synthesis plays a role in myoblast differentiation and suggest that the linkage of Py DNA synthesis to differentiation may involve a qualitative cellular alteration in Py DNA replication.     

Zinc improves gene transfer mediated by DNA/cationic polymer complexes

Background

The weak efficiency of plasmid transfer into the cytosol remains one of the major limiting factors to achieve an efficient transfection with DNA/cationic polymer complexes. We found that divalent metal Zn²⁺ can improve the polyfection efficiency, especially with DNA/histidylated polylysine (His‐pLK) complexes.

Methods and results

The supplementation of the transfection medium with 250 µM ZnCl₂ increased the polyfection of human hepatocarcinoma (HepG2) cells with a plasmid encoding EGFP complexed with pLK, polyethyleneimine and His‐pLK. Zn²⁺ is more efficient on DNA/His‐pLK complexes: the number of EGFP‐positive cells increased from 1% to more than 40%. This phenomenon is selective to Zn²⁺ because no effect was obtained with other divalent cations. The effect of zinc varies from cell to cell. The binding of Zn²⁺ to histidyl residues might increase zinc endosomal concentration favoring membrane fusion. Flow cytometry and confocal microscopy studies clearly indicate that with His‐pLK, the plasmid is better delivered in the cytosol as well as in the cell nucleus in zinc‐treated cells. An investigation conducted with the histidine‐rich peptide H5WYG showed that zinc inhibits membrane permeabilization but promotes membrane fusion as evidenced by resonance energy transfer.

Conclusions

Data reported here imply that the addition of zinc ions in the transfection medium can trigger an increase of the fusion of endosomes containing polyplexes which is more effective in the presence of histidine‐rich molecules. Consequently, the amount of plasmid in the cytosol available to reach the nucleus is increased leading to an improvement of polyfection. Copyright © 2002 John Wiley & Sons, Ltd.

     

Cellular uptake and delivery monitoring of liposome/DNA complexes during in vitro transfection of CFTR gene

The cellular uptake and distribution of cationic liposomes Dc-Chol/DOPECFTR gene complexes were assessed by electronic and confocal laser scanner microscopy (CLSM) for the CFTR gene transfer to human adenocarcinoma and tracheal epithelial cell lines. Cationic lipid forms unilamellar and multilamellar vesicles capable of rapid and efficient transport of gene into target cells. The number of fluorescent complexes was increasing with time in cells up to 6 hours showing a punctate and homogeneous DNA distribution in the cytoplasmatic and nuclear compartments, including the nucleolus. No significant difference in the biochemical and cellular behavior was observed between the investigated system and other systems previously tested. This study adds new insights into the CFTR cationic liposome-mediated gene delivery.     

Characterization of cationic lipid DNA transfection complexes differing in susceptability to serum inhibition

Background

Cationic lipid DNA complexes based on DOTAP (1,2-dioleoyl-3-(trimethyammonium) propane) and mixtures of DOTAP and cholesterol (DC) have been previously optimized for transfection efficiency in the absence of serum and used as a non-viral gene delivery system. To determine whether DOTAP and DC lipid DNA complexes could be obtained with increased transfection effciency in the presence of high serum concentrations, the composition of the complexes was varied systematically and a total of 162 different complexes were analyzed for transfection efficiency in the presence and absence of high serum concentrations.

Results

Increasing the ratio of DOTAP or DC to DNA led to a dose dependent enhancement of transfection efficiency in the presence of high serum concentrations up to a ratio of approximately 128 nmol lipid/μg DNA. Transfection efficiency could be further increased for all ratios of DOTAP and DC to DNA by addition of the DNA condensing agent protamine sulfate (PS). For DOTAP DNA complexes with ratios of ≤ 32 nmol/μg DNA, peak transfection efficiencies were obtained with 4 μg PS/μg DNA. In contrast, increasing the amount of PS of DC complexes above 0.5 μg PS /μg DNA did not lead to significant further increases in transfection efficiency in the presence of high serum concentrations. Four complexes, which had a similar high transfection efficiency in cell culture in the presence of low serum concentrations but which differed largely in the lipid to DNA ratio and the amount of PS were selected for further analysis. Intravenous injection of the selected complexes led to 22-fold differences in transduction efficiency, which correlated with transfection efficiency in the presence of high serum concentrations. The complex with the highest transfection efficiency in vivo consisted of 64 nmol DC/ 16 μg PS/ μg DNA. Physical analysis revealed a predicted size of 440 nm and the highest zeta potential of the complexes analyzed.

Conclusions

Optimization of cationic lipid DNA complexes for transfection efficiency in the presence of high concentrations of serum led to the identification of a DC complex with high transduction efficiency in mice. This complex differs from previously described ones by higher lipid to DNA and PS to DNA ratios. The stability of this complex in the presence of high concentrations of serum and its high transduction efficiency in mice suggests that it is a promising candidate vehicle for in vivo gene delivery.     

Molecular shape of the cationic lipid controls the structure of cationic lipid/dioleylphosphatidylethanolamine-DNA complexes and the efficiency of gene delivery

Pyridinium amphiphiles, abbreviated as SAINT, are highly efficient vectors for delivery of DNA into cells. Within a group of structurally related compounds that differ in transfection capacity, we have investigated the role of the shape and structure of the pyridinium molecule on the stability of bilayers formed from a given SAINT and dioleoylphosphatidylethanolamine (DOPE) and on the polymorphism of SAINT/DOPE-DNA complexes. Using electron microscopy and small angle x-ray scattering, a relationship was established between the structure, stability, and morphology of the lipoplexes and their transfection efficiency. The structure with the lowest ratio of the cross-sectional area occupied by polar over hydrophobic domains (SAINT-2) formed the most unstable bilayers when mixed with DOPE and tended to convert into the hexagonal structure. In SAINT-2-containing lipoplexes, a hexagonal topology was apparent, provided that DOPE was present and complex assembly occurred in 150 mm NaCl. If not, a lamellar phase was obtained, as for lipoplexes prepared from geometrically more balanced SAINT structures. The hexagonal topology strongly promotes transfection efficiency, whereas a strongly reduced activity is seen for complexes displaying the lamellar topology. We conclude that in the DOPE-containing complexes the molecular shape and the nonbilayer preferences of the cationic lipid control the topology of the lipoplex and thereby the transfection efficiency.     

Characterization of DNA/lipid complexes by fluorescence resonance energy transfer

Fluorescence resonance energy transfer (FRET) is a potential method for the characterization of DNA-cationic lipid complexes (lipoplexes). In this work, we used FRET models assuming a multilamellar lipoplex arrangement. The application of these models allows the determination of the distance between the fluorescent intercalator on the DNA and a membrane dye on the lipid, and/or the evaluation of encapsulation efficiencies of this liposomal vehicle. The experiments were carried out in 1,2-dioleoyl-3-trimethylammonium-propane/pUC19 complexes with different charge ratios. We used 2-(3-(diphenylhexatrienyl)propanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (DPH-PC) and 2-(4,4-difluoro-5-octyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (BODIPY-PC) as membrane dyes, and ethidium bromide (EtBr) and BOBO-1 as DNA intercalators. In cationic complexes (charge ratios (+/-) >or= 2), we verified that BOBO-1 remains bound to DNA, and FRET occurs to the membrane dye. This was also confirmed by anisotropy and lifetime measurements. In complexes with all DNA bound to the lipid (charge ratio (+/-) = 4), we determined 27 A as the distance between the donor and acceptor planes (half the repeat distance for a multilamellar arrangement). In complexes with DNA unbound to the lipids (charge ratio (+/-) = 0.5 and 2), we calculated the encapsulation efficiencies. The presented FRET methodology is, to our knowledge, the first procedure allowing quantification of lipid-DNA contact.     

Cellular uptake of phosphorothioate oligonucleotide facilitated by cationic porphyrin: a microfluorescence study

Cationic 5,10,15,20-tetrakis (1-methyl-4-pyridyl) porphyrin was tested as a delivery agent for oligonucleotides. By using fluorescence microimaging, it has been shown that complexation of the porphyrin to the phosphorothioate analog of dT(15) labeled by rhodamine enabled its nonendocytic penetration into the cell and regular distribution in the cytoplasm and preferentially into the nucleus. Time-resolved microfluorescence spectroscopy revealed that the oligonucleotide integrity was kept. A small fraction of the porphyrin molecules seems to undergo change of the binding mode after internalization, probably due to duplex formation between the oligonucleotide and its cellular target sequences, or due to dissociation of the porphyrin from the oligonucleotide and subsequent interactions in the cellular environment.     

Self-promoted cellular uptake of peptide/DNA transfection complexes

The designed alpha-helical amphipathic peptide LAH4 assembles several properties, which makes it an interesting candidate as a gene-delivery vehicle. Besides being short and soluble in aqueous solutions, LAH4 presents cationic residues, which allow for efficient complexation of DNA. In addition, this peptide is poorly hemolytic at neutral pH, while it is able to destabilize biological membranes in acidic conditions. In this study, the structure of the peptide/DNA transfection complex was examined by circular dichroism and solid-state nuclear magnetic resonance spectroscopies and the thermodynamics of its formation and disassembly was monitored in a quantitative manner as a function of pH by isothermal titration calorimetry. Notably, the number of peptides within the complex considerably decreases upon acidification of the medium. This observation has direct and important consequences for the mechanism of action because the acidification of the endosome results in high local concentrations of free peptide in this organelle. Thus, these peptides become available to interact with the endosomal membranes and thereby responsible for the delivery of the transfection complex to the cytoplasm. When these data are taken together, they indicate a dual role of the peptide during the transfection process, namely, DNA complexation and membrane permeabilization.     

Intracellular visualization of BrdU-labeled plasmid DNA/cationic liposome complexes.

Difficulties in specific detection of transfected DNA in cells represent an important limitation in the study of the gene transfer process. We studied the cellular entry and fate of a plasmid DNA complexed with a cationic lipid, Vectamidine (3-tetradecylamino-N-tert-butyl-N'-tetradecylpropionamidine) in BHK21 cells. To facilitate its detection inside the cells, bromodeoxyuridine (BrdU) was incorporated into plasmid DNA under conditions that minimize plasmid alteration. BrdU was localized in cells incubated with Vectamidine/BrdU-labeled plasmid DNA complexes by immunogold labeling and electron microscopy (EM). Labeling was predominantly associated with aggregated liposome structures at the surface of and inside the cells. EM observations of cells transfected with Vectamidine/DNA complexes showed that the liposome/DNA aggregates accumulate in large vesicles in the cell cytosol. On the other hand, using rhodamine-labeled Vectamidine and revealing BrdU with FITC-conjugated antibodies permitted simultaneous detection in the cells of both components of the complexes with confocal laser scanning microscopy. The DNA and lipids co-localized at the surface of and inside the cells, indicating that the complex is internalized as a whole. Our results show that the BrdU-labeled plasmid DNA detection system can be a useful tool to visualize exogenous DNA entry into cells by a combination of electron and confocal microscopy.     

Cellular uptake and cytotoxicity of octahedral rhenium cluster complexes

Cellular uptake behavior of a novel class of octahedral rhenium cluster compounds, hexahydroxo complexes K₄[{Re₆S₈}(OH)₆] · 8H₂O (1) and K₄[{Re₆Se₈}(OH)₆] · 8H₂O (2), was evaluated in human cervical adenocarcinoma HeLa cells. Confocal microscopy and flow cytometry studies demonstrated that rhenium cluster 1 was not internalized into cell, while rhenium cluster 2 was. Conjugation of a polymer to rhenium cluster 1, namely the derivative K₄[{Re₆S₈}(OH)₅L] (3) (L is amphiphilic diblock copolymer MPEG550-CH₂CONH-GlyPheLeuGlyPheLeu-COO⁻), considerably enhanced cellular uptake in a concentration-dependent manner and was predominantly localized in the cytoplasm and nucleus upon incubation time. The uptake of rhenium cluster 2 was mediated by energy-dependent endocytosis, whereas rhenium cluster 3 was directly ingested into cells by cell-fusion-like mechanism. According to the cytotoxicity evaluation test, both rhenium clusters 2 and 3 did not exhibit acute cytotoxic effects up to 50 μM, at the practical concentration level of biological applications. It is, therefore, expected that the rhenium cluster complexes can be promising potential candidates as diagnostic agents for medical treatment.     

Cellular uptake and metabolism of DNA frayed wires

DNA frayed wires are a novel, multistranded form of DNA that arises from interactions between single-stranded oligodeoxyribonucleotides with the general sequence d(N(x)G(y)) or d(G(y)N(x)), where y > 10 and x > 5. Frayed wires exhibit greater stability with respect to thermal and chemical denaturation than single- or double-stranded DNA molecules and, thus, may have potential usefulness for DNA drug delivery. However, the stability and uptake of frayed wires have not been investigated in biological systems. Our objective was to examine the cellular uptake and stability of frayed wires in cultured hepatic cells. In these studies, the parent oligonucleotide d(A(15)G(15)) was used to form DNA frayed wires (DNA(FW)) while a random 30-mer oligonucleotide was used as the control nonaggregated DNA (DNA(SS)). Uptake and metabolism studies of DNA(FW) were performed in cultured human hepatoma, HepG2 cells and compared to DNA(SS). Our results indicate that DNA(FW) are not cytotoxic and that their intracellular uptake in HepG2 cells is 2-3.5-fold greater than that of DNA(SS) within the first 2 h (p < 0.05). Similarly, nuclear localization of DNA(FW) is 10-13-fold higher than that of DNA(SS) (p < 0.05). As both internalized and extracellular DNA(FW) appear to be more stable in vitro than DNA(SS), the enhanced uptake may be due to either increased stability or enhanced intracellular transport. These studies also indicate that uptake of DNA(FW) likely occurs via active processes such as receptor-mediated endocytosis similar to mechanisms which have been proposed for DNA(SS). The internalization pathways of DNA(FW) may differ somewhat from that of DNA(SS) insofar as chloroquine does not appear to alter DNA(FW) uptake and degradation, as is the case with DNA(SS).     

Stimulation of mechano-growth factor expression by myofibrillar proteins in murine myoblasts and myotubes

Mechano-growth factor (MGF) is a product of alternative splicing of the insulin-like growth factor 1 (IGF-1) mRNA. MGF is known to stimulate myoblast proliferation and to protect neurons and cardiomyocytes from apoptosis. MGF expression is dramatically increased in response to mechanical stimuli and tissue damage. The mechanisms of induction of MGF expression are as yet imperfectly understood. There is certain evidence that some protein factors able to stimulate MGF synthesis in normal myoblasts are released from damaged muscle. This study was undertaken to explore the nature of these protein inductors of MGF expression and to investigate the mechanism of their action. We report here that myofibrillar fraction of skeletal muscle homogenate activated MGF expression in murine myoblasts and myotubes in culture. The expression of another splice form of IGF-1 gene, IGF-1Ea, was also stimulated by myofibrils. Three myofibrillar proteins able to stimulate MGF synthesis were isolated. These proteins were identified by MALDI and immunoblotting as myomesin, myosin-binding protein C, and titin. The activation of MGF expression was associated with the increase of cAMP level in the cells. Inhibitor of adenylyl cyclase dideoxyadenosine arrested stimulation of MGF synthesis by all three myofibrillar proteins.     

Cellular uptake of cationic gadolinium-DOTA peptide conjugates with and without N-terminal myristoylation

Cellular and nuclear uptake of dual labelled conjugates could be of great value for chemotherapy and cancer diagnostics. Therefore we designed conjugates in which gadolinium (Gd)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), a contrast agent for magnetic resonance imaging and fluorescein isothiocyanate (FITC), a fluorescence marker were coupled to membrane translocation sequences (MTS). The MTSs we employed were the third helix of the Antennapedia homeodomain, the HIV-1 Tat peptide and the N-myristoylated HIV-1 Tat peptide. We used confocal laser scanning microscopy, fluorescence activated cell sorting, magnetic resonance imaging (MRI) and viability tests to examine the cellular and nuclear uptake of these conjugates into U373 glioma cells, as well as their cytotoxic effects. We found that the Antennapedia conjugate was taken up by no more than 20% of the cells. The HIV-1 Tat conjugate showed even lower uptake into less than 3% of cells. Interestingly, N-myristoylation of the HIV-1 Tat conjugate drastically improved its cellular uptake. Up to 70% of cells showed cellular and nuclear uptake of the N-myristoylated HIV-1 Tat conjugate. Conjugate cytotoxicity appears to correlate with cellular uptake.     

Cellular uptake and distribution of cobalt complexes of fluorescent ligands

The development of complexes that allow the monitoring of the release and distribution of fluorescent models of anticancer drugs initially bound to cobalt(III) moieties is reported. Strong quenching of fluorescence upon ligation to cobalt(III) was observed for both the carboxylate- and the hydroximate-bound fluorophores as was the partial return of fluorescence following addition of ascorbate and cysteine. The extent of the increase in the fluorescence intensity observed following addition of these potential reductants is indicative of the fluorophore being displaced from the complex by the action of ascorbate or cysteine, by ligand exchange. The cellular distribution of the fluorescence revealed that coordination to cobalt can dramatically alter the subcellular distribution of a bound fluorophore. This work shows that fluorescence can be an effective means of monitoring these agents in cells, and of determining their sites of activation. The results also reveal that the cytotoxicity of such agents correlates with their uptake and distribution patterns and that these are influenced by the types of ligands attached to the complex.     

Phosphatidylserine directly and positively regulates fusion of myoblasts into myotubes

Cell membrane consists of various lipids such as phosphatidylserine (PS), phosphatidylcholine (PC), and phosphatidylethanolamine (PE). Among them, PS is a molecular marker of apoptosis, because it is located to the inner leaflet of plasma membrane generally but it is moved to the outer leaflet during programmed cell death. The process of apoptosis has been implicated in the fusion of muscle progenitor cells, myoblasts, into myotubes. However, it remained unclear whether PS regulates muscle cell differentiation directly. In this paper, localization of PS to the outer leaflet of plasma membrane in proliferating primary myoblasts and during fusion of these myoblasts into myotubes is validated using Annexin V. Moreover, we show the presence of PS clusters at the cell–cell contact points, suggesting the importance of membrane ruffling and PS exposure for the myogenic cell fusion. Confirming this conclusion, experimentally constructed PS, but not PC liposomes dramatically enhance the formation of myotubes from myoblasts, thus demonstrating a direct positive effect of PS on the muscle cell fusion. In contrast, myoblasts exposed to PC liposomes produce long myotubes with low numbers of myonuclei. Moreover, pharmacological masking of PS on the myoblast surface inhibits fusion of these cells into myotubes in a dose-dependent manner.     

Nanostructures of complexes formed by calf thymus DNA interacting with cationic surfactants

Synchrotron small-angle X-ray scattering was used to study the nanostructures of the complexes formed by calf thymus DNA interacting with cationic lipids (or surfactants) of didodecyldimethylammonium bromide (DDAB), cetyltrimethylammonium bromide (CTAB), and their mixture with a zwitterionic lipid of 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (PHGPC). The effects of lipid/DNA ratios, DNA chain flexibility, lipid topology, and neutral lipid mixing on the nanostructures of DNA-lipid complexes were investigated. The complexes between double-stranded DNA (dsDNA) and double-tailed DDAB formed a bilayered lamellar structure, whereas the complexes between dsDNA and single-tailed CTAB preferred a structure of 2D hexagonal close packing of cylinders. With single stranded DNA (ssDNA) interacting with CTAB, the complexes showed a Pm3n cubic structure due to the different chain flexibility between dsDNA and ssDNA. The lipid molecules bound by rigid dsDNA like to form cylindrical micelles, whereas lipids bound to flexible ssDNA could form spherical or short cylindrical micelles. The addition of the neutral single-chained PHGPC lipids to the CTAB lipids could induce a structural transition of dsDNA-lipid complexes from a 2D hexagonal to a multi-bilayered lamellar structure. The parallel DNA strands were intercalated in the water layers of lamellar stacks of the mixed lipid bilayers. The DNA-DNA spacing depended on the ratios of charged lipid to neutral lipid, and charged lipid to DNA, respectively.