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

Abstract

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.     

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.     

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.     

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.     

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.     

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.     

Ternary complexes comprising polyphosphoramidate gene carriers with different types of charge groups improve transfection efficiency

To understand the influence of charge groups on transfection mediated by polymer complexes, we have synthesized a series of biodegradable and cationic polyphosphoramidates (PPAs) with an identical backbone but different side chains. Our previous study showed that PPA with a spermidine side chain (PPA-SP) showed high transfection efficiency in culture, whereas PPAs with secondary, tertiary, and quaternary amino groups were significantly less efficient. To investigate whether the coexistence of 1 degrees amino charge groups with 3 degrees and 2 degrees amino charge groups in the DNA/polymer complexes would enhance their transfection efficiency, we evaluated a ternary complex system containing DNA and PPAs with 1 degrees amino groups (PPA-SP) and 3 degrees amino groups (PPA-DMA) and a quaternary complex system containing DNA and PPAs with 1 degrees and 2 degrees and 3 degrees amino groups (PPA-EA/PPA-MEA/PPA-DMA), respectively. Ternary complexes mediated 20 and 160 times higher transfection efficiency in COS-7 cells than complexes of DNA with PPA-SP or PPA-DMA alone, respectively. Similarly, quaternary complexes exhibited 8-fold higher transfection efficiency than PPA-EA/DNA complexes. The mechanism of enhancement in transfection efficiency by the mixture carriers appears to be unrelated to the particle size, zeta potential, or DNA uptake. The titration characterization and the transfection experiments using a proton pump inhibitor suggest that the enhancement effect is unlikely due to the slightly improved buffering capacity of the mixture over PPA-SP. This approach represents a simple strategy of developing polymeric gene carriers and understanding the mechanisms of polymer-mediated gene transfer.     

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.     

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 mediated by cationic liposomes: from biophysical aspects to enhancement of transfection

Cationic liposomes complexed with DNA have been used extensively as non-viral vectors for the intracellular delivery of reporter or therapeutic genes in culture and in vivo. However, the relationship between the features of the lipid-DNA complexes (`lipoplexes') and their mode of interaction with cells, the efficiency of gene transfer and gene expression remain to be clarified. To gain insights into these aspects, the size and zeta potential of cationic liposomes (composed of 1,2-dioleoyl-3- (trimethylammonium) propane (DOTAP) and its mixture with phosphatidylethanolamine (PE)), and their complexes with DNA at different (+/-) charge ratios were determined. A lipid mixing assay was used to assess the interaction of liposomes and lipoplexes with monocytic leukaemia cells. The use of inhibitors of endocytosis indicated that fusion of the cationic liposomes with cells occurred mainly at the plasma membrane level. However, very limited transfection of these cells was achieved using the above complexes. It is possible that the topology of the cationic liposome-DNA complexes does not allow the entry of DNA into cells through a fusion process at the plasma membrane. In an attempt to enhance transfection mediated by lipoplexes composed of DOTAP and its equimolar mixture with dioleoylphosphatidylethanolamine (DOPE) two different strategies were explored: (i) association of a targeting ligand (transferrin) to the complexes to promote their internalization, presumably by receptor-mediated endocytosis; and (ii) association of synthetic fusogenic peptides (GALA or the influenza haemagglutinin Nterminal peptide HA-2) to the complexes to promote endosomal destabilization and release of the genetic material into the cytoplasm. These strategies were effective in enhancing transfection in a large variety of cells, including epithelial and lymphoid cell lines, as well as human macrophages, especially with the use of optimized lipid/ DNA (+/-) charge ratios. Besides leading to high levels of transfection, the ternary complexes of cationic liposomes, DNA, and protein or peptide, have the advantages of being active in the presence of serum and being non-toxic. Moreover, such ternary complexes present a net negative charge and, thus, are likely to alleviate the problems associated with the use of highly positively charged complexes in vivo, such as avid complexation with serum proteins. Overall, the results indicate that these complexes, and their future derivatives, may constitute viable alternatives to viral vectors for gene delivery in vivo.     

Targeted delivery in breast cancer cells via iodine: nuclear localization sequence conjugate

The nonviral vector with iodine-nuclear localization sequence (namely, NLS-I) targeting breast cancer cells was fabricated. Ternary complexes were formed via charge interactions among NLS-I peptides, PEI 1800, and DNA, and we investigated their cellular internalization, nuclear accumulation as well as transfection efficiency. All the experiments were assessed by employing MCF-7 cells that express sodium/iodide symporter and HeLa cells that lack the expression of the symporter. In MCF-7 cells, cell internalization and nuclear accumulation of NLS-I was markedly increased compared to that in NLS. In addition, compared to that of the PEI1800/DNA complex, PEI1800/DNA/NLS-I complexes exhibited much enhanced luciferase reporter gene expression by up to 130-fold. By contrast, in HeLa cells, the evident improvements of cellular internalization, nuclear accumulation, and transfection efficiency by NLS-I were not observed. This study demonstrates an alternative method to construct a nonviral delivery system for targeted gene transfer into breast cancer cells.     

DODAB:monoolein-based lipoplexes as non-viral vectors for transfection of mammalian cells

DNA/Cationic liposome complexes (lipoplexes) have been widely used as non-viral vectors for transfection. Neutral lipids in liposomal formulation are determinant for transfection efficiency using these vectors. In this work, we studied the potential of monoolein (MO) as helper lipid for cellular transfection. Lipoplexes composed of pDNA and dioctadecyldimethylammonium bromide (DODAB)/1-monooleoyl-rac-glycerol (MO) at different molar ratios (4:1, 2:1 and 1:1) and at different cationic lipid/DNA ratios were investigated. The physicochemical properties of the lipoplexes (size, charge and structure), were studied by Dynamic Light Scattering (DLS), Zeta Potential (ζ) and cryo-transmission electron microscopy (cryo-TEM). The effect of MO on pDNA condensation and the effect of heparin and heparan sulphate on the percentage of pDNA release from the lipoplexes were also studied by Ethidium Bromide (EtBr) exclusion assays and electrophoresis. Cytotoxicity and transfection efficiency of these lipoplexes were evaluated using 293T cells and compared with the golden standard helper lipids 1,2-dioleoyl-sn-glycero-3-hosphoethanolamine (DOPE) and cholesterol (Chol) as well as with a commercial transfection agent (Lipofectamine? LTX). The internalization of transfected fluorescently-labeled pDNA was also visualized using the same cell line. The results demonstrate that the presence of MO not only increases pDNA compactation efficiency, but also affects the physicochemical properties of the lipoplexes, which can interfere with lipoplex-cell interactions. The DODAB:MO formulations tested showed little toxicity and successfully mediated in vitro cell transfection. These results were supported by fluorescence microscopy studies, which illustrated that lipoplexes were able to access the cytosol and deliver pDNA to the nucleus. DODAB:MO-based lipoplexes were thus validated as non-toxic, efficient lipofection vectors for genetic modification of mammalian cells. Understanding the relation between structure and activity of MO-based lipoplexes will further strengthen the development of these novel delivery systems.     

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.     

Cell transfection by DNA-lipid complexes — Lipoplexes

Nonviral vectors such as complexes of plasmid DNA with cationic lipids known as lipoplexes are considered as an attractive alternative to virus-based delivery systems. Unlike viruses, lipoplexes do not suffer from immunological and mutational hazards, though the efficiency of lipoplexes is often not sufficient for therapeutic purposes and require higher level of transfection than achieved until now. A number of critical steps responsible for transfection efficiency are discussed here. They include processes of lipoplexes formation, interaction with cell surface, their internalization into cell, and DNA release and delivery into the nucleus. All these processes should be thoroughly studied to be able to enhance the transfection efficacy.     

Preparation, characterization and transfection efficiency of cationic PEGylated PLA nanoparticles as gene delivery systems

The cationic polylactic acid (PLA) nanoparticle has emerged as a promising non-viral vector for gene delivery because of its biocompatibility and biodegradability. However, they are not capable of prolonging gene transfer and high transfection efficiency. In order to achieve prolonged delivery of cationic PLA/DNA complexes and higher transfection efficiency, in this study, we used copolymer methoxypolyethyleneglycol-PLA (MePEG-PLA), PLA and chitosan (CS) to prepare MePEG-PLA-CS NPs and PLA-CS NPs by a diafiltration method and prepared NPs/DNA complexes through the complex coacervation of nanoparticles with the pDNA. The object of our work is to evaluate the characterization and transfection efficiency of MePEG-PLA-CS versus PLA-CS NPs. The MePEG-PLA-CS NPs have a zeta potential of 15.7 mV at pH 7.4 and size under 100 nm, while the zeta potential of PLA-CS NPs was only 4.5 mV at pH 7.4. Electrophoretic analysis suggested that both MePEG-PLA-CS NPs and PLA-CS NPs with positive charges could protect the DNA from nuclease degradation and cell viability assay showed MePEG-PLA-CS NPs exhibit a low cytotoxicity to normal human liver cells. The potential of PLA-CS NPs and MePEG-PLA-CS NPs as a non-viral gene delivery vector to transfer exogenous gene in vitro and in vivo were examined. The pDNA being carried by MePEG-PLA-CS NPs, PLA-CS NPs and lipofectamine could enter and express in COS7 cells. However, the transfection efficiency of MePEG-PLA-CS/DNA complexes was better than PLA-CS/DNA and lipofectamine/DNA complexes by inversion fluorescence microscope and flow cytometry. It was distinctively to find that the transfection activity of PEGylation of complexes was improved. The nanoparticles were also tested for their ability to transport across the gastrointestinal mucosa in vivo in mice. In vivo experiments showed obviously that MePEG-PLA-CS/DNA complexes mediated higher gene expression in stomach and intestine of BALB/C mice compared to PLA-CS/DNA and lipofectamine/DNA complexes. These results suggested that MePEG-PLA-CS NPs have favorable properties for non-viral gene delivery.     

Salt ions and related parameters affect PEI–DNA particle size and transfection efficiency in Chinese hamster ovary cells

Transfection efficiency is directly associated with the expression level and quantity of recombinant protein after the transient transfection of animal cells. The transfection process can be influenced by many still-unknown factors, so it is valuable to study the precise mechanism and explore these factors in gene delivery. Polyethylenimine (PEI) is considered to have high transfection efficiency and endosome-disrupting capacity. Here we aimed to investigate optimal conditions for transfection efficiency by setting different parameters, including salt ion concentration, DNA/PEI ratio, and incubation time. We examined the PEI–DNA particle size using a Malvern particle size analyzer and assessed the transfection efficiency using flow cytometry in Chinese hamster ovary-S cells. Salt ions, higher amounts of PEI tended to improve the aggregation of PEI–DNA particles and the particle size of PEI–DNA complexes and the transfection efficiency were increased. Besides, the particle size was also found to benefit from longer incubation time. However, the transfection efficiency increased to maximum of 68.92 % at an incubation time of 10 min, but decreased significantly thereafter to 23.71 %, when incubating for 120 min (P < 0.05). Besides, PEI–DNA complexes formed in salt-free condition were unstable. Our results suggest DNA and PEI incubated in 300 mM NaCl at a ratio of 1:4 for 10 min could achieve the optimal transfection efficiency. Our results might provide guidance for the optimization of transfection efficiency and the industrial production of recombinant proteins.     

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

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

Targeting of polyplex to human hepatic cells by bio-nanocapsules, hepatitis B virus surface antigen L protein particles

We have previously demonstrated that lipoplex, a complex of cationic liposomes and DNA, could be targeted to human hepatic cells in vitro and in vivo by conjugation with bio-nanocapsules (BNCs) comprising hepatitis B virus (HBV) surface antigen L protein particles. Because the BNC-lipoplex complexes were endowed with the human hepatic cell-specific infection machinery from HBV, the complexes showed excellent specific transfection efficiency in human hepatic cells. In this study, we have found that polyplex (a complex of polyethyleneimine (PEI) and DNA) could form stable complexes with BNCs spontaneously. The diameter and ζ-potential of BNC-polyplex complexes are about 240 nm and +3.54 mV, respectively, which make them more suitable for in vivo use than polyplex alone. BNC-polyplex complexes with an N/P ratio (the molar ratio of the amine group of PEI to the phosphate group of DNA) of 40 showed excellent transfection efficiency in human hepatic cells. When acidification of endosomes was inhibited by bafilomycin A1, the complexes showed higher transfection efficiency than polyplex itself, strongly suggesting that the complexes escaped from endosomes by both fusogenic activity of BNCs and proton sponge activity of polyplex. Furthermore, the cytotoxicity is comparable to that of polyplex of the same N/P value. Thus, BNC-polyplex complexes would be a promising gene delivery carrier for human liver-specific gene therapy.