Inclusion of a single-tail amino acid-based amphiphile in a lipoplex formulation: effects on transfection efficiency and physicochemical properties

Effects of the addition of a cationic amino acid-based synthetic amphiphile, arginine N-lauroyl amide dihydrochloride (ALA), to a lipid-based transfection formulation have been investigated. It is shown that the inclusion of ALA results in a substantial enhancement of the transfection capability of lipoplexes prepared with liposomes of 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine and cholesterol, which themselves mediate highly efficient transfection. A possible explanation for the increased biological activity is that ALA adsorbed to the surface of the DNA-lipid complexes is involved in triggering internalization. However, in order to identify possible additional factors underlying the enhanced transfection efficiency, the physical properties of formulations with and without ALA were characterized using cryo-transmission electron microscopy, dynamic light scattering, and an ethidium bromide intercalation assay. ALA seems to have limited influence on the initial internal structure of the complexes and the protection of DNA, but its presence is found to decrease the average effective size of the dispersed particles; this change in size may be important in improving the biological activity. Furthermore, ALA can act to influence the transfection efficiency of the formulation by promoting the release of DNA following internalization in the transfected cells.     

DNA pre-condensation with an amino acid-based cationic amphiphile. A viable approach for liposome-based gene delivery

A study related to the development and characterization of a new gene delivery system was performed. The approach consists in both the pre-condensation of plasmid DNA with an arginine-based cationic surfactant, arginine–N-lauroyl amide dihydrochloride (ALA), which was found not to be toxic, and the incorporation of the blood protein transferrin (Tf) into the formulations.Two cationic liposome formulations were used, one composed of a mixture of dioleoyl trimethylammoniopropane and cholesterol (DOTAP:Chol) and the other a pH sensitive formulation constituted of DOTAP, Chol, Dioleoyl phosphatidylethanolamine (DOPE) and cholesteryl hemisuccinate (CHEMS).Particles with different ALA/DNA and cationic lipid/DNA charge ratios were produced and a physicochemical characterization of the systems developed was performed. DNA conformational changes in the presence of ALA were studied by Circular Dichroism (CD) and the ALA binding to DNA was followed by surface tension measurements. Insight into the structure and morphology of the various ALA-complexes (complexes composed of ALA, DNA, Tf and liposomes) was obtained by cryogenic-Transmission Electron Microscopy (cryo-TEM) and the sizes of the ALA-complexes were determined through Photon Correlation Spectroscopy (PCS). We found that the transfection capacity of these systems is directly related with the presence of ALA and the lipidic composition. Complexes based on the pH sensitive liposome formulation present better transfection profiles. The correlation between the inner structure, density and size of the ALA-complexes and their biological activity is discussed. Overall, we demonstrate that the presence of ALA improves the transfection efficiency when conjugated with cationic liposome systems.     

DNA pre-condensation with an amino acid-based cationic amphiphile. A viable approach for liposome-based gene delivery

A study related to the development and characterization of a new gene delivery system was performed. The approach consists in both the pre-condensation of plasmid DNA with an arginine-based cationic surfactant, arginine-N-lauroyl amide dihydrochloride (ALA), which was found not to be toxic, and the incorporation of the blood protein transferrin (Tf) into the formulations.Two cationic liposome formulations were used, one composed of a mixture of dioleoyl trimethylammoniopropane and cholesterol (DOTAP:Chol) and the other a pH sensitive formulation constituted of DOTAP, Chol, Dioleoyl phosphatidylethanolamine (DOPE) and cholesteryl hemisuccinate (CHEMS).Particles with different ALA/DNA and cationic lipid/DNA charge ratios were produced and a physicochemical characterization of the systems developed was performed. DNA conformational changes in the presence of ALA were studied by Circular Dichroism (CD) and the ALA binding to DNA was followed by surface tension measurements. Insight into the structure and morphology of the various ALA-complexes (complexes composed of ALA, DNA, Tf and liposomes) was obtained by cryogenic-Transmission Electron Microscopy (cryo-TEM) and the sizes of the ALA-complexes were determined through Photon Correlation Spectroscopy (PCS). We found that the transfection capacity of these systems is directly related with the presence of ALA and the lipidic composition. Complexes based on the pH sensitive liposome formulation present better transfection profiles. The correlation between the inner structure, density and size of the ALA-complexes and their biological activity is discussed. Overall, we demonstrate that the presence of ALA improves the transfection efficiency when conjugated with cationic liposome systems.     

Structure of transfection-active histone H1/DNA complexes

Relationships between the structure of transfecting complexes of histone H1 and DNA and their transfection efficiency were studied. Transfection activity proved to be connected to complex aggregates. Low speed centrifugation of the complexes resulted in loss of the transfection activity. The complexes/aggregates were active with high efficiency in a broad range of weight input ratios r _i (0.1<r _i<30). Using atomic force microscopy (AFM), the complexes were imaged at negative, nearly electroneutral and positive charge conditions. Electroneutral complexes at r _i=1 showed a multitude of different complex forms. Fibrillar, network-like and branched structures were frequently present in one complex. Strongly positive charged complexes had a toroidal appearance. All these different forms contributed to the high transfection efficiency. Cellular uptake is supposed to be by phagocytosis.     

Serum decreases the size of Metafectene-and Genejammer-DNA complexes but does not affect significantly their transfection activity in SCCVII murine squamous cell carcinoma cells

Cationic liposome-DNA (lipoplexes) or polymer-DNA (polyplexes) complexes have been used to deliver therapeutic genes, both in vitro and in vivo. However, gene transfer by these non-viral vectors is usually inhibited by biological milieu. A relatively high efficiency of transfection could be achieved in human oral cancer cells transfected with the polycationic liposome, Metafectene, and the polyamine reagent, GeneJammer, in the presence of 60% fetal bovine serum (FBS) (Konopka et al., Cell. Mol. Biol. Lett. 10 (2005) 455–470). Here, we examined the efficacy of these vectors to deliver β-galactosidase (β-gal), luciferase and Herpes Simplex Virus thymidine kinase (HSV-tk) genes to SCCVII murine squamous cell carcinoma cells, which are used to generate an orthotopic murine model of oral cancer. We also evaluated the hydrodynamic size and zeta potential of the vectors and the effect of FBS and mouse serum (up to 60%) on the size of Metafectene and GeneJammer complexes with the pCMV.Luc plasmid. Our results indicate that Metafectene and GeneJammer are highly effective in transfecting SCCVII cells. Approximately 60–70% of SCCVII cells transfected with pCMV.lacZ were positive for β-gal staining. The expression of β-galactosidase was essentially not affected by serum. Mouse serum (20–60%) reduced both Metafectene-and GeneJammer-mediated luciferase expression by ∼30–45%, while FBS did not affect transfection efficiency. The delivery of the HSV-tk gene by Metafectene or GeneJammer in the presence of 0% or 60% FBS, followed by GCV treatment for 6 days, resulted in over 90% cytotoxicity. The mean diameters of the DNA complexes of Metafectene and GeneJammer decreased significantly as a function of the serum concentration. The reduction in the size of the lipoplexes and polyplexes by serum was essentially not inhibitory to transfection of SCCVII cells. This is in contrast to previous hypotheses that serum-induced decrease in the size of lipoplexes is the primary cause of serum inhibition of transfection.     

Enhancing transfection efficiency using polyethylene glycol grafted polyethylenimine and fusogenic peptide

This study presents a new formulation method for improving DNA transfection efficiency using a fusogenic peptide and polyethylene glycol grafted polyethylenimine. Succinimidyl succinate polyethylene glycol (PEG-SSA) was conjugated with polyethylenimine (PEI). PEI is well known for a good endosomal escaping and DNA condensing agent. The positively charged synthetic fusogenic peptide, KALA, was coated on the negatively charged PEG-g-PEI/DNA and PEI/DNA complexes. The KALA/PEI/DNA complexes exhibited aggregation behavior at higher KALA coating amounts with an effective diameter of around 1,000 nm. However, the KALA/PEG-g-PEI/DNA complexes were 100–300 nm in size with a surface zeta-potential (ζ) value of about +20 mV. The conjugated PEG molecules suppressed any KALA-mediated inter-particle aggregation, and thereby improved the transfection efficiency. Consequently, the transfection efficiency of the KALA/PEG-g-PEI/DNA complexes was obtained by utilizing both the fusogenic activity of KALA and the steric repulsion effect of PEC.     

In Vivo Gene Transfer to the Mouse Nasal Cavity Mucosa Using a Stable Cationic Lipid Emulsion

We evaluate a new cationic emulsion as a mucosal gene carrier and elucidate the relationship between the transfection efficiency and the stability of the carrier/DNA complex. A cationic lipid emulsion was formulated with soybean oil and 1,2-dioleoyl-sn-glycero-3-trimethylammonium-propane (DOTAP) as major components and was used to transfer genes to the epithelial cells of the mouse nasal cavity via intranasal instillation. Correlation between the transfection efficiency and the stability of the carrier/DNA complex was investigated by measuring the carrier size changes and by observing the degree of DNA protection against DNase I digestion in the presence of heparin. The cationic emulsion showed at least 3 times better transfection activity than the liposomal carriers in nasal mucosae. The cationic emulsion was stable in the presence of heparin whereas the liposomal carriers became very unstable. Unlike DNA in liposome/DNA complexes, DNA in the emulsion/DNA complex was resistant to heparin exchange and DNase I digestion. The cationic emulsion was more effective in delivering DNA to nasal mucosae than commercially available liposomal carriers. The transfection activities of the lipid carriers in nasal cavity mucosae are in agreement with the stability of the lipid carriers and their complexes with DNA.     

Biophysical characterization of anionic lipoplexes

Transfection efficiency of liposomal gene delivery vectors depends on an optimal balance in the electro-chemical and structural properties of the transfection-capable complexes. We have recently reported a novel anionic lipoplex DNA delivery system composed of a ternary complex of endogenous occurring non-toxic anionic lipids, physiological Ca²⁺ cations, and plasmid DNA encoding a gene of interest with high transfection efficiency and low toxicity. In this work, we investigate the electro-chemical and structural properties anionic lipoplexes and compare them with those of Ca²⁺-DNA complexes. Biophysical characterization is used to explain the transfection efficiency of anionic lipoplexes in mammalian CHO-K1 cells. Circular dichroism and fluorescence spectroscopy showed that the plasmid DNA underwent conformational transition from native B-DNA to Z-DNA due to compaction and condensation upon Ca²⁺-mediated complexation with anionic liposomes. Zeta potential measurements and gel electrophoresis studies demonstrated that Ca²⁺ interaction with plasmid DNA during the formation of lipoplexes also led to increased association of supercoiled plasmid DNA with the lipoplexes, leading to charge neutralization which is expected to facilitate transfection. However, even 10-fold higher concentrations of Ca²⁺ alone (in the absence of the anionic liposomes) were unable to induce these changes in plasmid DNA molecules. A model explaining the possible mechanism of anionic lipoplex formation and the correlation of high transfection efficiency to biophysical properties was proposed. These studies confirm the utility of biophysical studies to identify optimal formulation conditions to design efficient liposomal gene delivery vectors.     

Mechanisms of Lipoplex Formation: Dependence of the Biological Properties ofTransfection Complexes on Formulation Procedures

Phospholipid-DNA complexes were made of the cationic triester derivative of phosphatidylcholine, EDOPC (1,2-dioleoyl- sn-glycero-3-ethylphosphocholine), by varying conditions of complex formation, in particular, the rate and direction of mixing, as well as by changing the mode of dispersing the lipid (extrusion or vortexing). The biological effects of variations in the formulation procedure were assessed by measuring transfection activity and cell association in cultures of BHK cells. Formulation procedures generally had little effect on cell association, but had marked effects on transfection efficiency. Transfection varied from effectively nil to extremely efficient with what appeared to be modest changes in formulation procedure. Formulation procedures also had significant effects on average sizes and size distributions of lipoplexes as determined by dynamic light scattering. Among the four possibilities of rapid or slow mixing combined with the two possible directions of mixing, slow addition of DNA to lipid gave results that differed significantly from the other three modes. In the case of vortexed lipid, the latter procedure was much less satisfactory than the other three, whereas in the case of extruded lipid, it was the only mode that produced satisfactory transfection. The factors that determine the difference in lipoplex properties can be identified as both geometric and physical. The geometric factor has to do with the symmetries of the participating units. There are three physical factors that are critical: the difference in vesicle stability upon interaction with DNA, the time dependence of interdiffusion of the components relative to that of vesicle rupture, and difference in input concentrations. These factors determine lipoplex size and, as already also shown by others, lipoplex size influences transfection efficiency.     

Gene delivery by dendrimers operates via a cholesterol dependent pathway

Understanding the cellular uptake and intracellular trafficking of dendrimer–DNA complexes is an important prerequisite for improving the transfection efficiency of non-viral vector-mediated gene delivery. Dendrimers are synthetic polymers used for gene transfer. Although these cationic molecules show promise as versatile DNA carriers, very little is known about the mechanism of gene delivery. This paper investigates how the uptake occurs, using an endothelial cell line as model, and evaluates whether the internalization of dendriplexes takes place randomly on the cell surface or at preferential sites such as membrane rafts. Following extraction of plasma membrane cholesterol, the transfection efficiency of the gene delivered by dendrimers was drastically decreased. Replenishment of membrane cholesterol restored the gene expression. The binding and especially internalization of dendriplexes was strongly reduced by cholesterol depletion before transfection. However, cholesterol removal after transfection did not inhibit expression of the delivered gene. Fluorescent dendriplexes co-localize with the ganglioside GM1 present into membrane rafts in both an immunoprecipitation assay and confocal microscopy studies. These data strongly suggest that membrane cholesterol and raft integrity are physiologically relevant for the cellular uptake of dendrimer–DNA complexes. Hence these findings provide evidence that membrane rafts are important for the internalization of non-viral vectors in gene therapy.     

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.     

Interference of serum with lipoplex-cell interaction: modulation of intracellular processing

We have investigated the mechanism of lipoplex-mediated transfection, employing a dialkyl pyridinium surfactant (SAINT-2), and using serum as a modulator of complex stability and processing. Particle size and stability determine lipoplex internalization, the kinetics of intracellular processing, and transfection efficiency. Clustered SAINT-2 lipoplexes are obtained in the absence of serum (-FBS lipoplexes), but not in its presence (+FBS lipoplexes), or when serum was present during lipoplex formation [FBS], conditions that mimic potential penetration of serum proteins. The topology of DNA in [FBS] lipoplexes shifts from a supercoiled, as in -FBS lipoplexes, to a predominantly open-circular conformation, and is more prone to digestion by DNase. Consistently, atomic force microscopy revealed complexes with tubular extensions, reflecting DNA that protrudes from the lipoplex surface. Interestingly, the internalization of [FBS] lipoplexes is approximately three-fold higher than that of -FBS and +FBS lipoplexes, yet their transfection efficiency is approximately five-fold lower. Moreover, in contrast to -FBS and +FBS complexes, [FBS] complexes were rapidly processed into the late endosomal/lysosomal degradation pathway. Intriguingly, transfection by [FBS] complexes is greatly improved by osmotic rupture of endocytic compartments. Our data imply that constraints in size and morphology govern the complex' ability to interact with and perturb cellular membranes, required for gene release. By extrapolation, we propose that serum may regulate these parameters in an amphiphile-dependent manner, by complex 'penetration' and modulation of DNA conformation.     

Zeta potential of transfection complexes formed in serum-free medium can predict in vitro gene transfer efficiency of transfection reagent

We have tested the zeta potential (zeta, the surface charge density) of transfection complexes formed in serum-free medium as a rapid and reliable technique for screening transfection efficiency of a new reagent or formulation. The complexes of CAT plasmid DNA (1 microgram) and DC-chol/DOPE liposomes (3-20 nmol) were largely negatively charged (zeta=-15 to -21 mV), which became neutral or positive as 0.5 microgram or a higher amount of poly-L-lysine (PLL, MW 29300 or MW 204000) was added (-3.16+/-3.47 to +6.04+/-2.23 mV). However, the complexes of CAT plasmid DNA (1 microgram) and PLL MW 29300 (0.5 microgram or higher) were neutral or positively charged (-3.22+/-2.3 to +6.55+/-0.64 mV), which remained the same as 6.6 nmol of the liposomes was added. The complexes formed between two positively charged compounds, PLL MW 29300 (0.5 microgram) and the liposomes (3-20 nmol), were as closely positively charged as DNA/PLL or DNA/liposomes/PLL complexes (+3.31+/-0.41 to 7.16+/-1.0 mV). These results indicate that PLL determined the overall charge of the DNA/liposome/PLL ternary complexes. The complexes formed with histone (0.75 microgram or higher) were also positively charged, whose transfection activity was as high as PLL MW 29300. However, the complexes formed with protamine or PLL MW 2400 remained negatively charged. These observations are in good agreement with the transfection activity of the formulation containing each polycationic polymer. The presence of PLL MW 29300 did not change the hydrodynamic diameter of DNA/liposome/PLL complexes (d(H)=275-312 nm). The complexes made of different sizes of PLL (MW 2400 and 204000) also did not significantly change their size. This suggests that DNA condensation may not be critical. Therefore, zeta of the transfection complex can predict the transfection efficiency of a new formulation or reagent.     

High-frequency Protoplast-transfection of Streptomyces parvulus 2297 with Actinophage R4 DNA

High frequency transfection of Streptomyces parvulus with actinophage R4 DNA was performed by modifying the procedure of protoplast transformation of S. coelicolor A3(2) with SCP2 plasmid DNA [Bibb et al., Nature, 274, 398 (1978)]. Optimum conditions for protoplast transfection included the presence of 16~24% (w/v) polyethyleneglycol 4000, and the maximum efficiency of transfection was 3 × 10^?5 per phage DNA molecule. This value was at least 100 times higher than the efficiency of previously reported transfection systems in Streptomyces.     

An insight into the gene delivery mechanism of the arginine peptide system: role of the peptide/DNA complex size

Cationic peptides have been used successfully to transfer macromolecules into living cells. Previously, we have reported a short arginine peptide-based gene delivery system. However, the mechanisms that allow arginine peptides to promote gene delivery yet remain unknown. In the present study, we investigated the effect of the arginine peptide/DNA complex size on the transfection efficiency. After combining peptides with DNA, a 400 nm complex was observed. As the incubation time was increased, the complex grew larger, reaching 6 microm after 1 h of incubation. Transfection and cellular uptake efficiency were likewise investigated for the effects of the different sizes of complexes. Large complexes were found to be advantageous for transfection. However, better internalization efficiency was found with small complexes, indicating that the amount of peptide/DNA complexes taken up by cells is not the rate-limiting step in the final transfection efficiency. The intracellular path of the peptide/DNA complex was studied using fluorescent labeling and confocal microscopy. In the early stages of transfection, complexes were observed only on the cell surface, and these complexes migrated into cytoplasm however, after 6 h, the presence of complexes in the perinuclear region was noted. We were able to detect colocalization of green and red fluorescence in both the cytoplasm and the nucleus. These results suggest that peptide/DNA complexes reach the nucleus as associated complexes.     

DNA binding,prominent photonuclease activity and antibacterial PDT of cobalt(II) complexes of phenanthroline based photosensitizers

Abstract

The chemistry of Co(II) complexes showing efficient light induced DNA cleavage activity, binding propensity to calf thymus DNA and antibacterial PDT is summarized in this article. Complexes of formulation [Co(mqt)(B)₂]ClO₄ 1–3 where mqt is 4-methylquinoline-2-thiol and B is N,N-donor heterocyclic base, viz. 1,10-phenanthroline (phen 1), dipyrido[3,2-d:2′,3′-f]quinoxaline (dpq 2) and dipyrido[3,2-a:2′,3′-c]phenazine (dppz 3) have been prepared and characterized. The DNA-binding behaviors of these three complexes were explored by absorption spectra, viscosity measurements and thermal denaturation studies. The DNA binding constants for complexes 1, 2 and 3 were determined to be 1.6?×?10³?M^?1, 1.1?×?10⁴?M^?1 and 6.4?×?10⁴?M^?1 respectively. The experimental results suggest that these complexes interact with DNA through groove binding mode. The complexes show significant photocleavage of supercoiled (SC) DNA proceeds via a type-II process forming singlet oxygen as the reactive species. Antimicrobial photodynamic therapy was studied using photodynamic antimicrobial chemotherapy (PACT) assay against E. coli and all complexes exhibited significant reduction in bacterial growth on photoirradiation.     

Lipid composition of cationic nanoliposomes implicate on transfection efficiency

Abstract

Cationic liposome (CL)-DNA complexes (lipoplexes) have appeared as leading nonviral gene carriers in worldwide gene therapy clinical trials. Arriving at therapeutic dosages requires the full understanding of the mechanism of transfection. However, using CLs to deliver therapeutic nucleic acids and drugs to target organs have some problems, including low transfection efficiency. The aim of this study was developing novel CLs containing four neutral lipids; cholesterol, 1,2-dioleoyl phosphatidylethanolamine, distearoylphosphatidylcholine and dipalmitoylphosphatidylcholine as a helper lipid and dimethyl dioctadecyl ammonium bromide as a cationic lipid to increase transfection efficiency. We have investigated the correlation between number of lipid composition and transfection efficiency. The morphology, size and zeta potential of liposomes and lipoplexes were measured and lipoplexes formation was monitored by gel retardation assay. Transfection efficiency was assessed using firefly luciferase reporter assay. It was found that transfection efficiency markedly depended on liposome to plasmid DNA (pDNA) weight ratio, lipid composition and efficiency of pDNA entrapment. High transfection efficiency of plasmid by four component lipoplexes was achieved. Moreover, lipoplexes showed lower transfection efficiency and less cytotoxicity compared to Lipofectamine?. These results suggest that lipid composition of nanoliposomes is an important factor in control of their physical properties and also yield of transfection.     

Transfecting pDNA to E. coli DH5α using bovine serum albumin nanoparticles as a delivery vehicle

We describe the formulation of bovine serum albumin nanoparticles (BSA‐NPs) by the coacervation method using surfactants. Plasmids (pUC18, pUC18egfp and pBBR1MCS‐2) isolated from E. coli were incorporated into the BSA matrix by incubating in albumin solution prior to formulation of NPs. Plasmid incorporation was calculated by % yield, entrapment efficiency, DNA loading capacity and release of entrapped DNA by comparing with blank NPs. BSA‐DNA binding studies were carried out by using fluorescence spectroscopy and Fourier Transform Infra Red Spectroscopy (FT‐IR). The surface charge distribution of the NPs loaded with plasmid was calculated using zeta potential. The photoluminescence of BSA‐NPs was quenched when loaded with pDNA, confirming the interaction of DNA with BSA. Altogether, these results provide evidences for the excellent DNA carrying efficiency of BSA‐NPs without loss of plasmid's integrity. The NPs were used to transfect E. coli DH5α strain lacking ampicillin resistance. They, however, showed ampicillin resistance subsequent to transfection with plasmid encoding ampicillin resistance gene. Effect of transfection was confirmed by confocal microscopy and by the isolation of the plasmid by agarose gel electrophoresis from the transfected bacterial culture. This study clearly demonstrates the efficacy of BSA‐NPs as delivery vehicle for pDNA transfection. Copyright © 2014 John Wiley & Sons, Ltd.     

The Biogenesis of the Photosynthetic Apparatus and the Activity of Chlorophyll Biosynthesis in a Plastome Mutant of Sunflower

It was demonstrated that, in the phenotypically colorless leaves of a sunflower (Helianthus annuusL.) plastome mutant with a heavily reduced level of chlorophyll, all pigment–protein complexes of the photosynthetic apparatus typical for the wild type were present. However, the ratio between them was changed. During aging of the mutant leaves, pigment–protein complexes of photosystem I were destroyed first followed by those of photosystem II. Chlorophyll a/b-containing light-harvesting complex II turned out to be the most stable. This conforms to an increased content of lutein and violaxanthin in mutant leaves. A synchrony of the decreases in the chlorophyll and 5-aminolevulinic acid (ALA) contents throughout all ontogenetic stages of the colorless mutant leaves made it possible to suggest that a decrease in the synthesis and resynthesis of chlorophyll during the formation and development of such leaves is caused by the inhibition of an initial stage of this process, namely, the biosynthesis of ALA molecules. The activity of the enzymes converting ALA into protochlorophyllide did not limit chlorophyll biosynthesis. Possible mechanisms controlling the synthesis of ALA destined for chlorophyll formation are discussed.     

Quaternary complexes composed of plasmid DNA/protamine/fish sperm DNA/stearic acid grafted chitosan oligosaccharide micelles for gene delivery

Quaternary complexes with condensed core of plasmid DNA, protamine, fish sperm DNA and shell of stearic acid grafted chitosan oligosaccharide (CSO-SA), were prepared. The CSO-SA could self-assemble to form nano-sized micelles in aqueous solution and demonstrated excellent internalization ability of tumor cells. Dynamic light scattering (DLS) measurement and transmission electrostatic microscope (TEM) images showed that quaternary complexes had spherical shape with about 25 nm number average diameter, and the size of quaternary complexes was smaller than that of CSO-SA micelles and CSO-SA micelles/plasmid DNA binary complexes. The transfection efficiencies of quaternary complexes on HEK293 and MCF-7 cells increased with incubation time, and were significantly higher than that of CSO-SA micelles/plasmid DNA binary complexes. The optimal transfection efficiency of quaternary complexes on HEK293 and MCF-7 cells measured by flow cytometer after 96 h was 23.82% and 41.43%, respectively. Whereas, the transfection efficiency of Lipofectamine? 2000 on HEK293 and MCF-7 cells after 96 h was 32.45% and 33.23%, respectively. The data of luciferease activity measurement showed that the optimal ratio of plasmid DNA:fish sperm DNA:protamine:CSO-SA was 1:1:5:5. The results indicated that the present quaternary complexes were potential non-viral gene delivery system.