Presence Of Trehalose Research Articles

Bioactivity of bone resorptive factor loaded on osteoconductive matrices: Stability post-dehydration

Since calcium phosphate cements were proposed two decades ago, extensive research has been realized to develop and improve their properties. They have proved their efficiency as bone graft substitutes and their ability to incorporate and release drugs. However, to date, all ‘resorbable’ osteoconductive synthetic biomaterials are in fact simply soluble. In order to investigate a synthetic material capable of inducing osteoclast remodelling post-implantation, a formulation of calcium phosphate cement loaded with a pro-resorptive cytokine (RANKL) was studied. Many prior release studies on calcium phosphates did not confirm that the matrix had no detrimental effect on the molecule to be released during storage prior to use or that bioactivity was maintained during storage. In this report, the stability of our protein was tested after loading onto the cement, and various regimens to improve stability were compared. The presence of trehalose was shown to stabilize the bioactivity of RANKL adsorbed to brushite cement. The reduction of both moisture and oxygen in the storage vessel improved osteoclastogenic potential of the matrix compared with that stored in ambient atmosphere and temperature. No loss in activity was observed over the study period for the loaded matrix stored in dry nitrogen.

Molecular mechanism for the effects of trehalose on β-hairpin folding revealed by molecular dynamics simulation

Recent work has shown that trehalose can facilitate and inhibit protein folding, but little is known about the molecular basis of these effects. Molecular-level insights into how the osmolyte affects protein folding are of significance for the rational design of small molecular additives for enhancing or hindering the folding of proteins. To investigate the molecular mechanisms of the facilitation and inhibition effects of trehalose on protein folding, molecular dynamics (MD) simulation of a β-hairpin peptide (Trp-Arg-Tyr-Tyr-Glu-Ser-Ser-Leu-Glu-Pro-Glu-Pro-Asp) in different trehalose concentrations (0–0.26 mol/L) is performed using an all-atom model. It is found that at a proper trehalose concentration (0.065 mol/L), the peptide folds faster than that in water, but it cannot fold to the β-hairpin at higher trehalose concentrations. Free energy landscape analysis indicates the presence of three intermediate states in both pure water and in 0.065 mol/L trehalose, but the potential energy barriers in the folding pathway decrease greatly in 0.065 mol/L trehalose, so the peptide folding is facilitated. Moreover, at this trehalose concentration, there is a favorable balance between the peptide backbone hydrogen bonds (H-bonds) and the peptide-trehalose H-bonds, leading to the stabilization of the folded peptide. At higher trehalose concentrations, however, trehalose molecules cluster in the peptide region and interact with the peptide via many H-bonds that prevent the peptide from folding to its native structure. The energy landscape analysis indicates that the potential energy barriers increase so greatly that the peptide cannot overcome it, getting trapped in a local free energy basin. The work reported herein has elucidated the molecular mechanism of the peptide folding in the presence of trehalose.

Desiccation Tolerance of Adult Stem Cells in the Presence of Trehalose and Glycerol

Development of protocols for storing desiccated cells at ambient temperatures offers tremendous economic and practical advantages over traditional storage procedures like cryopreservation and freeze-drying. As a first step for developing such procedures for adult stem cells, we have measured the post-rehydration membrane integrity (PRMI) of two passages, Passage-0 (P0) and Passage-1 (P1), of human adipose-derived stem cells (ASCs). ASCs were dried using a convective stage at three different drying rates (slow, moderate and rapid) in D-PBS with trehalose (50 mM) and glycerol (384 mM). ASCs were incubated in the drying media for 30 mins prior to drying at the prescribed rate on the convective stage for 30 mins. After drying, the ASCs were stored for 48 hrs in three different conditions: i) at ambient temperature, ii) in plastic bags at ambient temperature and iii) in vacuum sealed plastic bags at ambient temperature. PRMI was assessed after incubating the rehydrated ASCs with stromal medium for a further 48 hrs. Our measurements show that the PRMI of ASCs was: i) higher when ASCs were dried slowly; ii) increased when they were stored in vacuum as opposed to at ambient or in plastic bags; and iii) decreased with increasing passage of ASCs, i.e. under similar drying and storage conditions P0 ASCs had higher PRMI than P1 ASCs. Our results suggest that the best PRMI (37% for P0 ASCs and ~14% for P1 ASCs) can be achieved when the ASCs were dried slowly and stored in vacuum.

Concentration- and dehydration-dependent structural transitions in poly- l-lysine

The structural changes in poly- l-lysine (PLL), investigated as a function of simultaneous changes in concentration and pH, revealed a transition from right handed α-helical to left handed poly- l-proline II (PP II)-type helical structure. The PPII-type structure of PLL in solution state was not influenced by the addition of sugars (trehalose or sucrose). The structure of PLL changes from PPII-type in solution state to β-sheet in the dehydrated film state. In the presence of trehalose at PLL–trehalose mass ratio of 1:0.5, the structure of PLL changes from PPII-type in solution to a mixture of α-helix + β-sheet in the dehydrated film state. In the presence of trehalose at PLL–trehalose mass ratio of 1:1, the PPII-type structure of PLL in solution is maintained in the dehydrated film state.

RETRACTED: The cryoprotective effect of trehalose supplementation on boar spermatozoa quality

In order to improve boar sperm quality during frozen-thawed process, the influence of the presence of trehalose on success of cryopreservation of boar sperm were investigated. We evaluated freeze-thawing tolerance of boar spermatozoa in a base cooling extender with the addition of different trehalose concentrations (0, 25, 50, 100 and 200mmol/l), and tried to determine the optimum concentration of trehalose. We chose sperm motility, acrosome integrity, membrane integrity and cryocapacitation as parameters to evaluate cryopreservation capacity of boar spermatozoa. We obtained the best results for 100mmol/l trehalose-supplemented extenders, with values of 49.89% for motility, 66.52% for acrosome integrity and 44.61% for membrane integrity, while freeze-thawing tolerance was diminished significantly for 200mmol/l of trehalose. Before and after capacitation, the CTC score for semen diluted by extender containing 100mmol/l trehalose was 3.68% and 43.82%, respectively. In conclusion, trehalose could confer a greater cryoprotective capacity to boar spermatozoa. Trehalose-supplementation with 100mmol/l concentration in basic extender could significantly improve sperm motility, membrane integrity and acrosome integrity parameters, and reduce boar spermatozoa cryocapacitation during the cryopreservation process.

Long term storage of virus templated fluorescent materials for sensing applications

Wild type, mutant, and chemically modified Cowpea mosaic viruses (CPMV) were studiedfor long term preservation in the presence and absence of cryoprotectants. Viral complexeswere reconstituted and tested via fluorescence spectroscopy and a UV/vis-based RNaseassay for structural integrity. When viruses lyophilized in the absence of cryoprotectantwere rehydrated and RNase treated, UV absorption increased, indicating that the capsidswere damaged. The addition of trehalose during lyophilization protected capsid integrityfor at least 7 weeks. Measurements of the fluorescence peak maximum of CPMV lyophilizedwith trehalose and reconstituted also indicate that the virus remained intact. Microarraybinding assays indicated that CPMV particles chemically modified for use as a fluorescenttracer were intact and retained binding specificity after lyophilization in the presence oftrehalose. Thus, we demonstrate that functionalized CPMV nanostructures can bestored for the long term, enabling their use in practical sensing applications.

Optimising biocatalyst design for obtaining high transesterification activity by α-chymotrypsin in non-aqueous media

BackgroundEnzymes are often used in organic solvents for catalyzing organic synthesis. Two enzyme preparations, EPRP (enzyme precipitated and rinsed with n-propanol) and PCMC (protein coated microcrystals) show much higher activities than lyophilized powders in such systems. Both preparations involve precipitation by an organic solvent. The clear understanding of why these preparations show higher catalytic activity than lyophilized powders in organic solvents is not available.ResultsIt was found that EPRPs of α-chymotrypsin prepared by precipitation with n-propanol in the presence of trehalose contained substantial amount of trehalose (even though trehalose alone at these lower concentrations was not precipitated by n-propanol). The presence of trehalose in these EPRPs resulted in much higher transesterification rates (45.2 nmoles mg-1min-1) as compared with EPRPs prepared in the absence of trehalose (16.6 nmoles mg-1min-1) in octane. Both kinds of EPRPs gave similar initial transesterification rates in acetonitrile. Use of higher concentrations of trehalose (when trehalose alone also precipitates out), resulted in the formation of PCMCs, which showed higher transesterification rates in both octane and acetonitrile. SEM analysis showed the relative sizes of various preparations. Presence of trehalose resulted in EPRPs of smaller sizes.ConclusionThe two different forms of enzymes (EPRP and PCMC) known to show higher activity in organic solvents were found to be different only in the way the low molecular weight additive was present along with the protein. Therefore, the enhancement in the transesterification activity in EPRPs prepared in the presence of trehalose was due to: (a) better retention of essential water layer for catalysis due to the presence of the sugar. This effect disappeared where the reaction media was polar as the polar solvent (acetonitrile) is more effective in stripping off the water from the enzyme; (b) reduction in particle size as revealed by SEM. In the case of PCMC, the enhancement in the initial rates was due to an increase in the surface area of the biocatalyst since protein is coated over the core material (trehalose or salt).It is hoped that the insight gained in this work would help in a better understanding for designing high activity biocatalyst preparation of non-aqueous media.

Thermodynamics and mechanism of cutinase stabilization by trehalose

Trehalose has been widely used to stabilize cellular structures such as membranes and proteins. The effect of trehalose on the stability of the enzyme cutinase was studied. Thermal unfolding of cutinase reveals that trehalose delays thermal unfolding, thus increasing the temperature at the midpoint of unfolding by 7.2 degrees . Despite this stabilizing effect, trehalose also favors pathways that lead to irreversible denaturation. Stopped-flow kinetics of cutinase folding and unfolding was measured and temperature was introduced as experimental variable to assess the mechanism and thermodynamics of protein stabilization by trehalose. The main stabilizing effect of trehalose was to delay the rate constant of the unfolding of an intermediate. A full thermodynamic analysis of this step has revealed that trehalose induces the phenomenon of entropy-enthalpy compensation, but the enthalpic contribution increases more significantly leading to a net stabilizing effect that slows down unfolding of the intermediate. Regarding the molecular mechanism of stabilization, trehalose increases the compactness of the unfolded state. The conformational space accessible to the unfolded state decreases in the presence of trehalose when the unfolded state acquires residual native interactions that channel the folding of the protein. This residual structure results into less hydrophobic groups being newly exposed upon unfolding, as less water molecules are immobilized upon unfolding.

2P-129 トレハロース存在下でのマグネシウムパラクリスタルの形成(筋肉(筋蛋白質・収縮),第46回日本生物物理学会年会)

2P-129 トレハロース存在下でのマグネシウムパラクリスタルの形成(筋肉(筋蛋白質・収縮),第46回日本生物物理学会年会)

Enhancement of hydrophilic drug loading and release characteristics through micellization with new carboxymethyldextran-PEG block copolymers of tunable charge density

The micellization of a model cationic drug, diminazene diaceturate (DIM) and a series of new diblock copolymers, carboxymethyldextran-poly(ethylene glycols) (CMD-PEG), were evaluated as a function of the ionic charge density or degree of substitution (DS) of the carboxymethyldextran block and the molar ratio, [+]/[−], of positive charges provided by the drug to negative charges provided by CMD-PEG. Micelles ([+]/[−] = 2) incorporated up to 64% (w/w) DIM and ranged in hydrodynamic radius ( R H) from 36 to 50 nm, depending on the molecular weight and DS of CMD-PEG. The critical association concentration (CAC) was on the order of 15–50 mg/L for CMD-PEG of DS > 60%, and ca. 100 mg/L for CMD-PEG of DS ∼ 30%. The micelles were stable upon storage in solution for up to 2 months and after freeze-drying in the presence of trehalose. They remained intact within the 4 < pH < 11 range and for solutions of pH 5.3, they resisted increases in salinity up to ∼0.4 M NaCl in the case of CMD-PEG of high DS. However, micelles of DIM and a CMD-PEG of low DS (30%) disintegrated in solutions containing more than 0.1 M NaCl, setting a minimum value to the DS of copolymers useful in in vivo applications. Sustained in vitro DIM release was observed for micelles of CMD-PEG of high DS ([+]/[−] = 2).

Cell partitioning during the directional solidification of trehalose solutions

Previous studies have demonstrated that ice/cell interaction influences post thaw viability and specific cryoprotective agents can affect those interactions. Trehalose, a disaccharide, has been shown to have a protective benefit during conventional slow freezing. Existing theories have been put forth to explain the protective benefit of trehalose during desiccation and vitrification, but these theories do not explain the protective benefit observed during conventional freezing protocols. The overall objective of this investigation was to characterize cell/ice interactions in the presence of trehalose using non-planar freezing conditions. To that end, lymphoblasts suspended in phosphate buffered saline solution with various levels of trehalose (0, 10, 100, and 300 mM) were frozen on a directional solidification stage. The partitioning of cells into the interdendritic space or engulfment by an advancing dendrite was determined as a function of velocity and solution composition. For a given temperature gradient, the fraction of cells entrapped into the interdendritic region increased with increasing velocity. With small additions of trehalose (10 mM), the velocity at which cells were entrapped in the interdendritic region increased. At high trehalose concentrations (100, 300 mM), interface morphology was significantly different and cells were engulfed by the advancing interface. Dehydration of cells in the region shortly before and after the interface was significant and depended upon of the type of interaction experienced by the cell (entrapped vs. engulfed). These studies suggest that one potential mechanism for the action of trehalose involves changing the ice/cell interactions during conventional slow freezing.

Negative staining across holes: Application to fibril and tubular structures

The negative staining technique, when used with holey carbon support films, presents superior imaging conditions than is the case when samples are adsorbed to continuous carbon films. A demonstration of this negative staining approach is presented, using ammonium molybdate in combination with trehalose, applied to several fibrillar and tubular samples. Fibrils formed from the amyloid-β peptide and the protease inhibitor pepstain A spread very well unsupported across holes and the different polymorphic fibril forms can be readily assessed. However, tubular forms of amyloid-β have a tendency to be flattened, due to surface tension forces prior to and during specimen drying. Sub-fibril assembly forms and D-banded rat tail type 1 collagen fibres are presented. The air-dried collagen images produced are shown to contain almost as much detail as those obtainable by cryo-negative staining. Fragile DNA and DNA-protein nanotubes are also shown to yield superior quality images to those produced on continuous carbon films. The iron-storage protein, frataxin, creates elongated oligomeric assemblies, containing bound ferrihydrite microcrystals. The iron particles within these flexuous oligomers can be defined in the presence of ammonium molybdate, but they are more readily demonstrated if the frataxin is spread across holes in the presence of trehalose alone. The samples used here serve to show the likely benefit obtainable from negative staining across holes for a range of other fibrillar and tubular samples in biology, medicine and nanobiotechnology.

Volume recovery, surface properties and membrane integrity of Lactobacillus delbrueckii subsp. bulgaricus dehydrated in the presence of trehalose or sucrose

Although the practical importance of adding sugars before drying is well known, the mechanism of protection of bacteria by sugars is not clear. The response of the dehydrated micro-organisms to rehydration is analysed in terms of structural and functional changes, and correlated with their potentiality to grow in rich media. These aspects are related with the membrane integrity and the metabolic state of the rehydrated bacteria, measured by means of surface properties and permeability. To attain this objective, Lactobacillus delbrueckii subsp. bulgaricus was dehydrated in the presence and in the absence of sucrose and trehalose. The bacterial response upon rehydration was investigated by determining: (i) the lag time of the bacterial growing in rich media, (ii) the restoration of the surface properties and the cellular volume and (iii) the membrane integrity. Lactobacillus delbrueckii subsp. bulgaricus was grown in MRS at 37 degrees C overnight [De Man et al. (1960)J Appl Bacteriol 23, 130] and then dehydrated for 10, 20 and 30 min at 70 degrees C in a vacuum centrifuge. The lag time of micro-organisms was determined by optical density changes after rehydration. The surface properties were determined by measuring the zeta potential of the bacteria suspended in aqueous solution. The cellular volume recovery was measured, after stabilization in saline solution, by light scattering and by the haematocrit method [Alemohammad and Knowles (1974)J Gen Microbiol 82, 125]. Finally, the membrane integrity has been determined by using specific fluorescent probes [SYTO 9 and propidium iodide, (PI)] that bind differentially depending on the integrity of the bacterial membrane. The lag time of Lact. delbrueckii subsp bulgaricus, dehydrated by heat in the presence of sucrose or trehalose and after that rehydrated, was significantly shortened, when compared with that obtained for bacteria dried in the absence of sugars. In these conditions, trehalose and sucrose maintained the zeta potential and the cell volume close to the control (nondried) cells. However, the membrane integrity, measured with fluorescent probes, was maintained only when cells were dehydrated for 10 min in the presence of sugars. For larger times of dehydration, the membrane integrity was not preserved, even in the presence of sugars. When the micro-organisms are dehydrated in the absence of protectants, the membrane damage occurs with a decrease in the absolute value of the zeta potential and a decrease in the cellular volume recovered after rehydration. In contrast, when the zeta potential and the cellular volume are restored after rehydration to that corresponding to nondried cells, the micro-organisms are able to recover and grow with a reduced lag time. This can only be achieved when the dehydration is carried out in the presence of sugars. At short dehydration times, the response is associated with the preservation of the membrane integrity. However, for longer times of dehydration the zeta potential and volume recovery occurs in the presence of sugars in spite of a severe damage at membrane level. In this condition, cells are also recovered. In conclusion, to predict the ability of growing after dehydration, other bacterial structural parameters besides membrane integrity, such as zeta potential and cellular volume, should be taken into account. The correlation of the lag time with the surface and permeability properties is of practical importance because the correlation of these two parameters with cell viability, allow to determine the potential bacterial capacity to grow in a rich medium after the preservation procedure, without necessity of performing a kinetic curve of growth, which is certainly time-consuming.

Neutron scattering and HPLC study on l-ascorbic acid and its degradation

The present paper shows a systematic dynamic and kinetic study on l-ascorbic acid and its degradation at high temperature. The neutron scattering study allows, through the behavior of quasi-elastic neutron scattering (QENS) spectra, to characterize the diffusive dynamics of l-ascorbic acid in water mixtures. Ascorbic acid undergoes degradation process at high temperature, but the presence of trehalose in solution markedly avoids ascorbic acid loss enhancing its t 1/2 (half life time), as determined by high performance liquid chromatography (HPLC).

Identification of trehalose dimycolate (cord factor) in Mycobacterium leprae

Glycolipids of Mycobacterium leprae obtained from armadillo tissue nodules infected with the bacteria were analyzed. Mass spectrometric analysis of the glycolipids indicated the presence of trehalose 6,6′-dimycolate (TDM) together with trehalose 6-monomycolate (TMM) and phenolic glycolipid-I (PGL-I). The analysis showed that M. leprae-derived TDM and TMM possessed both α- and keto-mycolates centering at C78 in the former and at C81 or 83 in the latter subclasses, respectively. For the first time, MALDI-TOF mass analyses showed the presence of TDM in M. leprae.

C-Phycocyanin Hydration Water Dynamics in the Presence of Trehalose: An Incoherent Elastic Neutron Scattering Study at Different Energy Resolutions

We present a study of C-phycocyanin hydration water dynamics in the presence of trehalose by incoherent elastic neutron scattering. By combining data from two backscattering spectrometers with a 10-fold difference in energy resolution we extract a scattering law S( Q, ω) from the Q-dependence of the elastic intensities without sampling the quasielastic range. The hydration water is described by two dynamically different populations—one diffusing inside a sphere and the other diffusing quasifreely—with a population ratio that depends on temperature. The scattering law derived describes the experimental data from both instruments excellently over a large temperature range (235–320 K). The effective diffusion coefficient extracted is reduced by a factor of 10–15 with respect to bulk water at corresponding temperatures. Our approach demonstrates the benefits and the efficiency of using different energy resolutions in incoherent elastic neutron scattering over a large angular range for the study of biological macromolecules and hydration water.

Molecular adaptation strategies to high temperature and thermal denaturation mechanism of the D‐trehalose/D‐maltose‐binding protein from the hyperthermophilic archaeon Thermococcus litoralis

The D-trehalose/D-maltose-binding protein (TMBP), a monomeric protein of 48 kDa, is one component of the trehalose and maltose uptake system. In the hyperthermophilic archaeon T. litoralis this is mediated by a protein-dependent ATP-binding cassette system transporter. The gene coding for a thermostable TMBP from the archaeon T. litoralis has been cloned, and the recombinant protein has been expressed in E. coli. The recombinant TMBP has been purified to homogeneity and characterized. It exhibits the same functional and structural properties as the native one. In fact, it is highly thermostable and binds both trehalose and maltose with high affinity. In this work we used differential scanning calorimetry studies together with a detailed analysis, at the molecular level, of the three-dimensional protein structure to shed light on the basis of the high thermostability exhibited by the recombinant TMBP from the archaeon T. litoralis. The obtained data suggest that the presence of trehalose does not change the overall mechanism of the denaturation of this protein but it selectively modifies the stability of the TMBP structural domains.

Role of trehalose in resistance to macrophage killing: study with a tps1/tps1 trehalose-deficient mutant of Candida albicans

Accumulation of trehalose by yeast is an important protective mechanism against different stress conditions. This study examined the effect of trehalose on several growth features, as well as its association with the intracellular survival of yeasts exposed to macrophages. A tps1/tps1 mutant and its parental counterpart, CAI4, exhibited similar growth rates and preserved their dimorphic conversion and agglutination ability. However, electron-microscopy of cell-wall architecture showed a partial loss of material from the outer cell-wall layer in the tps1/tps1 mutant. Flow-cytometry revealed that the mutant had lower auto-fluorescence levels and a higher fluorescein isothiocynate staining efficiency. When co-cultured with macrophages, a slight reduction in binding to macrophages and slower ingestion kinetics were revealed for the tps1/tps1 mutant, but these did not interfere significantly with the amount of yeast ingested by macrophages after co-incubation for 2 h. Under the same conditions, CAI4 cells were more resistant to macrophage killing than was the tps1 null mutant, provided that the macrophages had been stimulated previously with interferon-gamma. Measurement of trehalose content and the anti-oxidant activities of yeast cells recovered after phagocytosis revealed that the trehalose content and the glutathione reductase activity were increased only in CAI4 cells, whereas levels of catalase activity were increased similarly in both strains. These results suggest that the presence of trehalose in Candida albicans is a contributory factor that protects the cell from injury caused by macrophages.

The Natural Osmolyte Trehalose Is a Positive Regulator of the Heat-Induced Activity of Yeast Heat Shock Transcription Factor

In Saccharomyces cerevisiae, the intracellular concentration of trehalose increases rapidly in response to many environmental stresses, including heat shock. These high trehalose levels have been correlated with tolerance to adverse conditions and led to the model that trehalose functions as a chemical cochaperone. Here, we show that the transcriptional activity of Hsf1 during the heat shock response depends on trehalose. Strains with low levels of trehalose have a diminished transcriptional response to heat shock, while strains with high levels of trehalose have an enhanced transcriptional response to heat shock. The enhanced transcriptional response does not require the other heat-responsive transcription factors Msn2/4 but is dependent upon heat and Hsf1. In addition, the phosphorylation levels of Hsf1 correlate with both transcriptional activity and the presence of trehalose. These in vivo results support a new role for trehalose, where trehalose directly modifies the dynamic range of Hsf1 activity and therefore influences heat shock protein mRNA levels in response to stress.

Trehalose Enhances Transgene Expression Mediated by DNA‐PEI Complexes

Using enhancers to improve the transfection efficiency of polyethylenimine (PEI) can circumvent the needs of chemical modifications as well as subsequent purification and characterization of the modified PEI. In this study, we found that incorporating trehalose into the transfection reagent could improve the transgene expression mediated by DNA-PEI complexes. Such enhancements were not observed when trehalose was replaced by other disaccharides. In an effort to explore the mechanisms, we examined how the timing of trehalose treatments and the durations of trehalose affected the percentages of cells expressing green fluorescent protein and the levels of intracellular ethidium monoazide labeled plasmid. Treatments with trehalose for 5-120 min prior to transfection could cause drops in transfection efficiency by 30-50%; such treatments, however, hardly affected the amounts of intracellular plasmid, indicating that the preexistence of intracellular trehalose could reduce transfection efficiency without lowering the endocytic activity. The transfection efficiency remained almost unchanged when the transfected cells were treated with trehalose after the removal of transfection reagents, indicating that trehalose had minimal effects on the machinery of protein synthesis. Despite the enhanced transgene expression, the presence of trehalose during transfection showed inhibitory effects on the internalization of DNA-PEI complexes. Additionally, the extent of enhancement in transgene expression strongly depended on the duration of trehalose. As the above observations suggested, only during the transfection process when complexes and trehalose coexisted, trehalose became an effective enhancer of transgene expression mediated by DNA-PEI complexes possibly by affecting the mechanisms of intracellular trafficking.