Effects of physicochemical characteristics of poly(2-(dimethylamino)ethyl methacrylate)-based polyplexes on cellular association and internalization.

The cationic polymer poly(2-(dimethylamino)ethyl methacrylate) (p(DMAEMA)) is able to efficiently bind and condense DNA and to mediate transfection of a variety of cell types. In this study, fluorescence activated cell sorting (FACS), confocal laser fluorescence microscopy (CSLM) and electron microscopy (EM) techniques were used to investigate in vitro the cellular interaction of p(DMAEMA)-based polyplexes with human ovarian carcinoma cells (OVCAR-3). Cellular association and subsequent internalization only occurred when the polyplexes exhibited a positive zeta potential. Small-sized polyplexes have an advantage over large-sized complexes regarding cellular entry. The effect of the presence of tertiary amine groups versus the presence of quatenary amine groups was evaluated by comparing p(DMAEMA) with its quaternary ammonium analogue poly(2-(trimethylamino)ethyl methacrylate) (p(TMAEMA)). The combined cellular interaction and transfection results suggest that the latter polymer does not have an intrinsic endosomal escape property, in contrast to the 'proton sponge' effect proposed for p(DMAEMA). PEGylation of p(DMAEMA) effectively shielded the surface charge and yielded a notably lower degree of cellular interaction. Data on the effects of the presence of endocytosis inhibitors and an endosome-disruptive peptide in the culture medium on the cellular interaction and transfection activity of p(DMAEMA)-based polyplexes support endocytosis as being the principal pathway for intracellular delivery of plasmid. Both the CLSM and EM studies did not reveal the presence of polyplexes or plasmid outside the endocytic vesicles or within the nucleus, suggesting that intracellular trafficking from the endosomes to the nucleus is a very inefficient process.

Association and dissociation characteristics of polymer/DNA complexes used for gene delivery.

PURPOSE: The DNA association/dissociation properties of water-soluble cationic methacrylate polymers with closely related structures (poly(2-dimethylamino)ethyl methacrylate) [p(DMAEMA)], poly(2-(trimethylamino)ethyl methacrylate chloride) [p(TMAEMA)]) and the frequently used transfectant poly(L-lysine) were studied to gain a better insight into their transfection characteristics. METHODS: Association of DNA with different polymers and dissociation of the complexes, achieved by adding an excess of anionic polymers or salt, were studied by using spectroscopic techniques (fluorescence, circular dichroism (CD)), agarose gel electrophoresis and an enzymatic assay (DNase I treatment). The transfection efficiency of the polyplexes was evaluated in tissue culture with OVCAR-3 cells. RESULTS: Plasmid DNA complexed with either poly(L-lysine) or p(DMAEMA) was protected against digestion by DNase I. Fluorescence and CD spectroscopy as well as gel electrophoresis revealed that p(DMAEMA) with a relatively high molecular weight and poly(L-lysine) have similar DNA association/dissociation characteristics. Therefore, differences in transfection potential of the polyplexes cannot be ascribed to differences in binding characteristics, but are probably caused by other factors. As compared with the other polymers, p(TMAEMA) has a high affinity for DNA as was concluded from the observation that poly(aspartic acid) was unable to fully dissociate complexes containing this polymer. This fact might very well explain the low transfection efficiency of these polyplexes. p(DMAEMA) with a relatively low molecular weight probably has a low affinity for DNA, which might explain both the formation of DNA aggregates (psi-DNA) and the low transfection potential obtained when using this polymer. CONCLUSIONS: DNA association/dissociation studies shed light on the preferred characteristics of polymeric transfectants.

Chiral DNA packaging in DNA-cationic liposome assemblies.

Recent studies have indicated that the structural features of DNA-lipid assemblies, dictated by the lipid composition and cationic lipid-to-DNA ratio, critically affect the efficiency of these complexes in acting as vehicles for cellular delivery of genetic material. Using circular dichroism we find that upon binding DNA, positively-charged liposomes induce a secondary conformational transition of the DNA molecules from the native B form to the C motif. Liposomes composed of positively-charged and neutral 'helper' lipids, found to be particularly effective as transfecting agents, induce - in addition to secondary conformational changes - DNA condensation into a left-handed cholesteric-like phase. A structural model is presented according to which two distinct, yet inter-related modes of DNA packaging coexist within such assemblies. The results underline the notion that subtle changes in the components of a supramolecular assembly may substantially modulate the interplay of interactions which dictate its structure and functional properties.

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

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

Effect of DNA topology on the transfection efficiency of poly((2-dimethylamino)ethyl methacrylate)-plasmid complexes.

In this paper the effect of the topology of plasmid DNA (supercoiled, open-circular and linear) on its binding characteristics with the polymeric transfectant poly((2-dimethylamino)ethyl methacrylate) was studied. The formed polyplexes were also evaluated for their transfection properties in vitro in two different cell lines. Anion-exchange chromatography was used for the separation of supercoiled and open-circular plasmid from a plasmid stock solution. Linear plasmids were prepared by endonucleases that cleaved the plasmid either in the promoter region or in a region not specific for expression (ampicillin resistance region). Plasmid DNA was also heat-denatured for 6 h at 70 degrees C, resulting in DNA mainly in the open-circular and oligomeric forms. The transfection of two different cell lines was dependent on the topology of the DNA in the order supercoiled>open-circular approximately heat-denatured>linear DNA prepared by cleaving in the nonspecific region>linear DNA prepared by cleaving in the promoter region. No differences in the size of the complexes or in the quenching of the DNA-intercalating fluorophore acridine orange were found as function of the topology. However, circular dichroism spectroscopy revealed differences between the topological plasmid species, both in the free form and in the presence of excess of cationic polymer.

Characterization of DNA-lipid complexes commonly used for gene delivery.

Cationic liposomes are used to deliver genes into cells. Here we describe some poorly understood basic features of DNA-lipid complexes (lipoplexes), especially the electrostatics, stability and DNA structure of lipoplexes, and their effects on transfection (lipofection). Use of the lipophilic, pH-sensitive fluorophore 4-heptadecyl-7-hydroxycoumarin, in combination with Gouy-Chapman calculations, showed that cationic liposomes had a large positive surface potential (180-240 mV) and a high pH (10-11.5) at the location of the probe on the liposomal surface in 20 mM Hepes buffer (pH 7.4). Other electrostatic characteristics were also found, such as the existence of protonable groups of cationic or helper lipids or salt bridges between those. Addition of DNA resulted in neutralization of cationic lipids, which was lower than expected and depending on the type of lipid and the DNA/cationic lipid ratio. The liposomes containing DOTAP (N-(1-(2,3-dioleoyloxy)propyl)-N,N, N-trimethylammonium chloride) were unstable upon dilution, probably due to the high critical micellar concentration of DOTAP, 7x10(-5) M. Large instability expressed as continuous size increase was demonstrated by the time-dependent static changes in light-scattering monitored following the mixing of cationic liposomes and DNA at DNA/cationic lipid molar ratios between 0.2 and 0.8. Addition of cationic liposomes composed of 100% DOTAP or DOTAP/DOPE (1/1) liposomes, induced instantaneous transition of the plasmid DNA from the B- toward a partial C-type conformation as shown by circular dichroism (CD) spectroscopy and at certain conditions Psi--DNA could be found as well. The Psi--DNA is characterized by inter-helical interaction between parallel helices. The highest lipofection was obtained under conditions of lipoplex instability, and when the DNA was partially dehydrated and had a partial Psi-- structure.

Influence of the antiallergic drug oxatomide and derivatives on membrane structures: relation with inhibition of calcium influx in rat basophilic leukemia cells.

Oxatomide is an H1 antihistaminic drug that also inhibits mediator release from mast cells. From previous studies, it appeared that inhibition of the influx of extracellular calcium is the major cause of this inhibition of exocytosis. Here, we explored the role of drug-membrane interactions in the inhibition of mediator release. We investigated the effects on phase transition and fluidity of artificial membranes. All compounds studied distorted the phase transition in L-alpha-dipalmitoylphosphatidylcholine liposomes, which correlated with the drug-induced increase in membrane fluidity measured by fluorescence anisotropy of the bilayer interacting probe 1-[4-(trimethylamino)-phenyl]-6-phenylhexa-1,3,5-triene. Erythrocytes were used to study membrane effects on a cellular level. The hypotonic-induced haemolysis of erythrocytes was inhibited by the drugs. Compounds which increased membrane fluidity of liposomes to a greater extent were also more active in decreasing haemolysis. Drug-induced disturbance of the membranes is related to their effect on the activity of store-operated Ca2+ channels. The activity of these channels in rat basophilic leukemia cells, assayed as 45Ca2+ influx, was most effectively inhibited by oxatomide derivatives, thereby inducing a more rigid membrane structure. Small changes in molecular structure affect the activity of the drugs and these structure-activity relations are discussed.

Electrostatic and structural properties of complexes involving plasmid DNA and cationic lipids commonly used for gene delivery.

UNLABELLED: The present study is aimed to characterize the interactions between plasmid DNA and cationic, large unilamellar vesicles, 110+/-20nm in size, composed of lipids commonly used for transfections including DOTAP/DOPE (mole ratio 1/1), DOTAP/DOPC (mole ratio 1/1), 100% DOTAP, or DC-CHOL/DOPE (mole ratio 1/1). [ ABBREVIATIONS: DOTAP, N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine; DC-CHOL, 3 beta-[N-(N',N'-dimethylaminoethane)carbamoyl] cholesterol]. A novel approach of combining Gouy-Chapman calculations and fluorescence measurements of the pH at the surface of lipid assemblies by the fluorophore 4-heptadecyl-7-hydroxycoumarin showed that electrostatic parameters played a key role in the instantaneous formation of the DNA-lipid complexes upon addition of different amounts of plasmid DNA to cationic liposomes in 20 mM Hepes buffer (pH 7.4). Addition of large amounts of plasmid DNA leads to neutralization of 60% of the protonated DC-CHOL in DC-CHOL/DOPE (1/1) assemblies and 80% of the DOTAP in lipid assemblies. The characterization of these electrostatic parameters of the complexes suggests better and closer surrounding of plasmid DNA by lipids when DOPE is present. Time-dependent static light-scattering measurements monitored the formation of complexes and also showed that these complexes were highly unstable with respect to size at DNA/cationic lipid molar ratios between 0.2 and 0.8.

Electrostatic parameters of cationic liposomes commonly used for gene delivery as determined by 4-heptadecyl-7-hydroxycoumarin.

UNLABELLED: Cationic liposomes are used to deliver genes into cells in vitro and in vivo. The present study is aimed to characterize the electrostatic parameters of cationic, large unilamellar vesicles, 110 +/- 20 nm in size, composed of DOTAP/DOPE (mole ratio 1/1), DOTAP/DOPC (mole ratio 1/1), 100% DOTAP, DMRIE/DOPE 1/1, or DC-CHOL/DOPE (mole ratio 1/1). { ABBREVIATIONS: DOTAP, N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine; DMRIE, 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide; DC-CHOL, 3beta[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol}. The cationic liposomes had a large positive surface potential and a high pH at the liposomal surface in 20 mM Hepes buffer (pH 7.4) as monitored by the pH-sensitive fluorophore 4-heptadecyl-7-hydroxycoumarin. In contrast to DOTAP and DMRIE which were 100% charged, DC-CHOL in DC-CHOL/DOPE (1/1) liposomes was only about 50% charged in 20 mM Hepes buffer (pH 7.4). This might result in an easier dissociation of bilayers containing DC-CHOL from the plasmid DNA (which is necessary to enable transcription), in a decrease of the charge on the external surfaces of the liposomes or DNA-lipid complexes, and in an increase in release of the DNA-lipid complex into the cytosol from the endosomes. Other electrostatic characteristics found were that the primary amine group of DOPE in cationic liposomes dissociated at high (> 7.9) pHbulk and that a salt bridge was likely between the quaternary amine of DOTAP or DMRIE and the phosphate group of DOPE or DOPC, but not between the tertiary amine of DC-CHOL and the phosphate group of DOPE. The liposomes containing DOTAP were unstable upon dilution, probably due to the high critical aggregation concentration of DOTAP, 7 X 10(-5) M. This might also be a mechanism of the dissociation of bilayers containing DOTAP from the plasmid DNA.

Gamma-irradiation damage to liposomes differing in composition and their protection by nitroxides.

The present study aims to determine the effect of bilayer composition on oxidative damage and the protection against it in lipid multicomponent membranes. Irradiation damage in 200-nm liposomes and the protection provided by the nitroxide radicals, 2,2,6,6-tetramethylpiperidine-1-oxyl (Tempo) and 4-hydroxy-2,2,6,6-tetramethylpiperidine--1-oxyl (Tempol) were assessed by monitoring several chemical and physical parameters. Liposomes were prepared in four different lipid compositions (mole ratios), DPPC:DPPG 10:1; DPPC:DPPG:cholesterol 10:1:4; EPC:EPG 10:1; and EPC:EPG:cholesterol 10:1:4, and gamma-irradiated with a dose of 32 kGy. Lipid degradation was determined by HPLC and GC analyses, whereas size and differential scanning calorimetry measurements were used to monitor physical changes in the liposomal dispersions. The results indicate that: (1) addition of 5 mM Tempo or Tempol, or freezing of the sample inhibited radiation-induced lipid degradation; (2) Tempo and Tempol caused neither physical nor chemical changes in the liposomal dispersions; and (3) both nitroxides prevented or reduced some of the radiation-induced changes in thermotropic characteristics of the liposomes, preventing a shift in the temperature of the maximum of the main phase transition.

Gamma-irradiation of liposomes composed of saturated phospholipids: effect of bilayer composition, size, concentration and absorbed dose on chemical degradation and physical destabilization of liposomes.

Liposomes composed of dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG), or mixtures of these two phospholipids were exposed to gamma-irradiation in an air environment. Disappearance of the mother compounds was monitored by HPLC analysis. Plotting of the logarithmic values of residual DPPC or DPPG concentration versus irradiation dose resulted in straight lines. The slopes of these lines (overall degradation constants) depended on the type of phospholipids, concentration of the liposomes and the size of the liposomes. Under the chosen conditions, addition of DPPG in DPPC-liposomes did not affect the degradation rate constant of DPPC and vice versa. The presence of phosphate buffer (pH 7.4), pH or presence of sodium chloride did not affect the irradiation damage either. Minor changes were found upon analysis of total fatty acids by GLC and upon measurement of water soluble phosphate compounds. These changes were less pronounced than the changes monitored by HPLC of phospholipids, because the HPLC analysis monitored the overall degradation of the liposomal phospholipids. Thin-layer chromatography/fast atom bombardment mass spectrometry (TLC/FAB-MS) analysis of irradiated and non-irradiated DPPC or DPPG provided information on the structure of several degradation products. Degradation routes which include these degradation products are proposed. Gamma-irradiation neither affected the size of the liposomes nor the bilayer rigidity as determined by dynamic light scattering and fluorescence anisotropy of the probe 1,6-diphenyl-1,3,5-hexatriene (DPH), respectively. However, upon gamma-irradiation, changes in the melting characteristics of the liposomes were found by differential scanning calorimetry (DSC) measurements. The pre-transition melting enthalpy of the liposomal bilayer decreased or disappeared and the main-transition broadened. The changes found in DSC scans correlated qualitatively well with the changes recorded after HPLC analysis of phospholipids.

Gamma-irradiation of non-frozen, frozen, and freeze-dried liposomes.

PURPOSE: The aim of this work was to investigate the possibilities and limitations of gamma-irradiation as a sterilisation method for nonfrozen, frozen, and freeze-dried liposomes. METHODS: Liposomes with an average size of 0.2 micron were irradiated with doses up to about 5 x 10(4) Gy in a nitrogen atmosphere. RESULTS: Phospholipids in dipalmitoylphosphatidycholine/dipalmitoylphosphatidylglycerol (DPPC/DPPG) 10/1-liposomes and egg phosphatidylcholine/egg phosphatidylglycerol (EPC/EPG) 10/1-liposomes in 10 mM phosphate buffer (pH 7.4) without trehalose degraded considerably upon gamma-irradiation. Irradiation damage was reduced in the presence of 10% trehalose added as a cryoprotectant, but trehalose reacted with species induced by gamma-irradiation as demonstrated by large decreases in pH. Both pH decrease and oxidative damage of EPC/EPG 10/1-liposomes were strongly dependent on the physical state during irradiation (nonfrozen, frozen or freeze-dried). No changes in liposomal size were found upon gamma-irradiation, and hardly any change was seen in bilayer rigidity. Differences in the gel-to-liquid phase transition of DPPC/DPPG 10/1-liposome dispersions before and after gamma-irradiation were small in the presence of 10% trehalose, but larger in the absence of trehalose. CONCLUSION: The degradation of trehalose limits the use of freezing or freeze-drying liposome dispersions as a way to minimise irradiation damage.

Physical (in) stability of liposomes upon chemical hydrolysis: the role of lysophospholipids and fatty acids.

As a consequence of chemical hydrolysis of liposomal phospholipids the organization of the lipid assembly can change from a lamellar into a micellar system. Different approaches provided evidence for this conversion: 31P-NMR analysis, turbidity measurements and ultracentrifugation experiments. Two conditions have to be met before this conversion can take place: (1) the liposomes must pass through a gel-to-liquid crystalline phase-transition during a heating or cooling run, and (2) the degree of chemical hydrolysis must exceed a critical hydrolysis percentage (or the phospholipid bilayer must contain critical amounts of lysophospholipid and fatty acid). As monitored by turbidity measurements, this critical level of hydrolysis and the relative change depended on the chain length and on the head group of the liposomal phospholipids. It does not depend on concentration, pH, storage temperature or on size of the liposomes within the experimental range. Addition of cholesterol to bilayers composed of dipalmitoylphosphatidylcholine prevents the lamellar to micellar transformation. Fluorescence anisotropy measurements of the lipophilic probe 1,6-diphenyl-1,3,5-hexatriene in 0.18-microns dipalmitoylphosphatidylcholine/dipalmitoylphosphatidylglycerol (10:1)-liposomes indicated that behavior of the probe below and above the phase-transition temperature was not affected by chemical hydrolysis, or even by formation of micelles. However, the phase-transition temperature range broadened and shifted towards higher temperatures upon hydrolysis.

Chemical hydrolysis of phospholipids.

Hydrolysis kinetics of phospholipids in liposomes composed of dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine/cholesterol (DPPC/CHOL) 10/4 (molar ratio) and egg phosphatidylcholine (EPC) at pH 4.0 and different temperatures could be described by Arrhenius curves without breaks. However, the Arrhenius curves for the hydrolysis of liposomal DPPC and distearoylphosphatidylcholine (DSPC) under the same conditions were biphasic. A break was observed in the curves extending over a broad range before and after the known Tm of each of these phospholipids in liposomes (42 and 56 degrees C, respectively). The activation energy (Ea) for the hydrolysis of liposomal DPPC and DSPC below the Tm was substantially larger than the Ea for liposomal DMPC, DPPC/CHOL 10/4, and EPC and decreased when DPPC was mixed with CHOL in a 10/4 molar ratio. Hardly any influence of the presence of alpha-tocopherol, cryoprotectants (glucose, trehalose, sucrose, and propylene glycol), and the major hydrolysis products lysophospholipids and fatty acids or of the absence of sodium chloride on the hydrolysis kinetics of DPPC at pH 4.0 and 30 degrees C was observed. Changes in fatty acid chains and size did not influence the hydrolysis rate constant (kobs) of liposomal phospholipids at pH 4.0 and 30 degrees C either. The only effects of uncharged compounds on the kobs of liposomal DPPC at pH 4.0 and 30 degrees C were found upon mixing with a high concentration of the detergent Triton X-100 or palmitic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Sterilization of liposomes by heat treatment.

Autoclaving of liposomes composed of egg phospholipids or saturated phospholipids, the latter sometimes combined with cholesterol, was performed in an isotonic acetate buffer (pH 4.0) or Hepes buffer (pH 7.4). After a standard autoclaving cycle (15 min, 121 degrees C), no change could be observed in pH, size, and extent of oxidation. Dependent on the experimental conditions, a minor or substantial increase in the fraction of hydrolyzed phospholipids was found. After a sterilization cycle, pronounced leakage was found for a water-soluble, encapsulated compound (calcein) and for an amphiphilic compound (doxorubicin). Lipophilic, liposome bilayer-associated compounds [N-trifluoroacetyldoxorubicin-14-valerate (AD-32) and alpha-tocopherol] remained in the liposomes after autoclaving. However, substantial degradation of AD-32 was observed. Under proper conditions liposomes without or with thermostable, lipophilic drugs can be sterilized by autoclaving. However, the hydrolysis of phospholipids can pose a problem, as hydrolysis kinetics depend on the pH used. In the chosen circumstances the autoclaving cycle caused massive loss of hydrophilic, nonbilayer interacting compounds; under those conditions "free" drug removal or drug encapsulation should be performed after the autoclaving step.

Hydrolysis of partially saturated egg phosphatidylcholine in aqueous liposome dispersions and the effect of cholesterol incorporation on hydrolysis kinetics.

Hydrolysis kinetics of partially hydrogenated egg phosphatidylcholine (PHEPC) were studied as a function of pH, temperature, buffer concentration, ionic strength, and the effect of cholesterol incorporation. Results showed that PHEPC has a maximum stability at around pH 6.5. General acid base catalysis was observed for acetate, HEPES and Tris buffers. Increasing the ionic strength of the buffer solutions did not influence the hydrolysis kinetics. The relationship between the observed hydrolysis rate constants and the temperature could adequately be described by the Arrhenius equation. Incorporation of cholesterol did not affect the hydrolysis kinetics. This result indicates that the hydrolysis kinetics of PHEPC do not depend on the changes in bilayer rigidity induced by cholesterol incorporation. Cholesterol is stable under the experimental conditions used in this study; no changes were observed in cholesterol concentration over the experimental time interval.