Non-viral Gene Carriers Research Articles

Gellan gum blended PEI nanocomposites as gene delivery agents: Evidences from in vitro and in vivo studies

Branched Polyethylenimine, 25 kDa (PEI), was blended with gellan gum, an anionic heteropolysaccharide, for partial neutralization of its excess positive charge to form gellan gum-polyethylenimine (GP) nanocomposites (NCs). Subsequently, we manipulated the amount of gellan gum for obtaining a series of NCs and characterized them for their size, charge and morphology. Among all the NCs, one member, named GP3, showed the best transfection efficiency in tested cell lines in comparison with the rest of the series, PEI, Lipofectamine and other commercial transfection agents and also exhibited minimum cytotoxicity. It was found to transfect primary cells of mouse skin with better efficiency than PEI and Lipofectamine and was able to protect the plasmid DNA from nucleases and serum proteins present in the blood. GP3 exhibited efficient intracellular delivery of plasmid as revealed by confocal studies while its intracellular presence was also confirmed by the knockdown of GFP expression (using GFP specific siRNA) and JNKII by quantifying proteins in cell lysates and by western blotting and hybridization, respectively. In vivo cytotoxicity studies in Drosophila showed lack of induction of stress response in the exposed organisms. Further, exposed organisms did not show any developmental delay or mortality and no morphological defects were observed in the emerged flies. In vivo gene expression studies in Balb/c mice revealed maximum expression of luciferase enzyme in spleen. The study suggests that GP3 may act as an efficient non-viral gene carrier with diverse biomedical applications.

Gene delivery by pullulan derivatives in brain capillary endothelial cells for protein secretion

The blood-brain barrier (BBB) formed by brain capillary endothelial cells protects the brain against potentially harmful substances present in the circulation, but also restricts exogenous substances such as pharmacologically acting drugs or proteins from entering the brain. A novel and rather unchallenged approach to allow proteins to enter the brain is gene therapy based on delivery of genetic material into brain capillary endothelial cells. In theory in vivo transfection will allow protein expression and secretion from brain capillary endothelial cells and further into the brain. This would denote a new paradigm for therapy to transport proteins across the BBB. The aim of this study was to investigate the possibility to use brain capillary endothelial cells as factories for recombinant protein production. Non-viral gene carriers were prepared from pullulan, a polysaccharide, and spermine, a naturally occurring polyamine that were additionally conjugated with plasmid DNA. We were able to transfect rat brain endothelial cells (RBE4s) and human brain microvascular endothelial cells (HBMECs). Transfection of HBMECs with pullulan–spermine conjugated with plasmid DNA bearing cDNA encoding human growth hormone 1 (hGH1), led to secretion of hGH1 protein into the growth medium. Hence, the pullulan–spermine delivery system is a very promising method for delivering DNA to brain endothelial cells with potential for using these cells as factories for secretion of proteins.

Mechanism of the Introduction of Exogenous Genes into Cultured Cells Using DEAE-Dextran-MMA Graft Copolymer as a Non-Viral Gene Carrier. II. Its Thixotropy Property

From comparative investigations regarding the efficiency of introducing exogenous genes into cultured cells using DEAE-dextran and DEAE-dextran-MMA (methyl methacrylate ester) graft copolymer (2-diethylaminoethyl-dextranmethyl methacrylate graft copolymer; DDMC) as a nonviral carrier, we have confirmed that the gene transfection efficiency of DDMC is higher than that of DEAE-dextran. Comparative investigations in which DNA encoding luciferase (pGL3 control vector; Promega) was introduced into COS-7 cells derived from African green monkey kidney cells with and without the use of an incubator shaker were also carried out using various concentrations of DDMC. Without an incubator shaker, the transfection efficiency results were reversed, namely that the gene introduction efficiency of DDMC was inferior to that of DEAE-dextran. The aqueous solution of the cationic graft-copolymer displays thixotropic properties, which is why a strong shear stress is needed for it to flow and wet the cells. The reaction between DNA and DDMC is thought to be a Michaelis-Menten type complex formation reaction that can be described by the following equation: Complex amount = K1 (DNA concentration) (DDMC concentration).The complex formation reaction is thought to involve Coulomb forces between DDMC and DNA and is also significantly influenced by hydrogen bonding strength along with hydrophobic bonding strength due to the hydrophobicity of the grafted MMA sections.

The development of poly-L-arginine-coated liposomes for gene delivery

In this study, liposomes coated with cationic polymers, poly-L-arginine (PLA), were assessed as a promising gene transfer system in human cervical carcinoma (HeLa) cells and human hepatoma cell line (Huh7) cells. The liposomes were prepared using egg yolk phosphatidylcholine and sodium oleate in the molar ratio of 10:2 with an ultrasonic generator and then coated with PLA. The PLA-coated liposomes (PCLs) formed complexes with plasmid DNA encoding green fluorescent protein. The complexes were characterized by agarose gel electrophoresis and investigated for their transfection efficiency in HeLa and Huh7 cells. The data were compared with PLA/DNA complexes and the positive control Lipofectamine 2000™. The results showed that complete PCL/DNA complexes were formed at weight ratios of more than 0.05. Efficient gene transfer by PCLs was dependent on the cell type. The transfection efficiency of PCLs was about two times higher than that of PLA/DNA complexes in both HeLa cells and Huh7 cells. Cytotoxicity was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and showed that 80%–100% of both of the cells were viable after treating PCL/DNA complexes. The present results demonstrate that PCLs are a promising, nonviral gene carrier with low toxicity.

Gene therapy for C-26 colon cancer using heparin-polyethyleneimine nanoparticle-mediated survivin T34A.

BackgroundGene therapy provides a novel method for the prevention and treatment of cancer, but the clinical application of gene therapy is restricted, mainly because of the absence of an efficient and safe gene delivery system. Recently, we developed a novel nonviral gene carrier, ie, heparin-polyethyleneimine (HPEI) nanoparticles for this purpose.Methods and resultsHPEI nanoparticles were used to deliver plasmid-expressing mouse survivin-T34A (ms-T34A) to treat C-26 carcinoma in vitro and in vivo. According to the in vitro studies, HPEI nanoparticles could efficiently transfect the pGFP report gene into C-26 cells, with a transfection efficiency of 30.5% ± 2%. Moreover, HPEI nanoparticle-mediated ms-T34A could efficiently inhibit the proliferation of C-26 cells by induction of apoptosis in vitro. Based on the in vivo studies, HPEI nanoparticles could transfect the Lac-Z report gene into C-26 cells in vivo. Intratumoral injection of HPEI nanoparticle-mediated ms-T34A significantly inhibited growth of subcutaneous C-26 carcinoma in vivo by induction of apoptosis and inhibition of angiogenesis.ConclusionThis research suggests that HPEI nanoparticle-mediated ms-T34A may have a promising role in C-26 colon carcinoma therapy.

Innovative technology of engineering magnetic DNA nanoparticles for gene therapy

The present research study is a novel innovation materials technology to engineer magnetic DNA nanoparticles for gene therapy and to enhance the localisation and gene expression of a plasmid DNA via magnetic DNA nanoparticles. Spermine (Sm) was chemically introduced to the hydroxyl groups of dextran, a polysaccharide, to obtain cationised dextran (dextran-Sm). When Fe2+ solution was added to the mixture of dextran-Sm and a plasmid DNA, DNA nanoparticles were formed via metal coordination. In vitro release test of DNA from DNA nanoparticles revealed that the encapsulation of DNA in the nanoparticles caused the prolonged release of DNA. Significant gene expression was observed when DNA nanoparticles were applied for in vitro gene transfection compared with naked NK4 plasmid DNA. We conclude that the Fe2+-coordinated dextran-Sm conjugation is a promising way to enable the plasmid DNA to target to the cells to express specific gene as well as to enhance the localisation of the plasmid DNA. The present study clearly demonstrates the efficacy of DNA nanoparticles on enhancement of in vitro gene expression in cells by dextran-Sm-DNA nanoparticles as a non-viral gene carrier specifically where needed to track the in vivo localisation of the drug using physical equipment such as magnetic resonance images (MRI).

Gold/cationic polymer nano-scaffolds mediated transfection for non-viral gene delivery system

Gene delivery scaffolds based on DNA plasmid condensation with colloidal gold/cationic polymer were developed. The synthesized gold nanoparticles displayed spherical shape with an average size of 12nm under observation with a transmission electron microscope. Gold/cationic polymer nano-scaffolds were formed though electrostatic interaction yielding gold/polyethyleneimine (PEI), gold/chitosan and gold/chitosan/PEI complexes. Luciferase-encoding plasmid DNA was subsequently added and adsorbed on the prepared scaffolds to be used as a non-viral gene carrier. Physicochemical properties of DNA-binding scaffolds were examined including size and zeta potential. The results indicated that the sizes of gold/polymer based scaffolds were generally less than 400nm, and they carried positive charge on their surfaces. Gel retardation assay and atomic force microscopy confirmed the condensation of plasmid DNA on gold/polymer based scaffolds. Confocal fluorescent microscopy verified the presence of DNA in the cell using gold nano-scaffold as a carrier. Transfection efficiency assay using A549 and HeLa human cell lines revealed that gold/polymer based nano-scaffolds provided transfection efficiency approximately 10 times higher than polymeric-based gene carriers. This study proposed simple and practical approach, having the potential for use as an alternative gene carrier in non-viral gene delivery system.

Improved transfection efficiency of an aliphatic lipid substituted 2 kDa polyethylenimine is attributed to enhanced nuclear association and uptake in rat bone marrow stromal cell

Lipid substitutions of cationic polymers are actively explored to enhance the efficiency of nonviral gene carriers. We recently took this approach to develop a novel gene carrier by grafting linoleic acid (LA) to relatively biocompatible 2 kDa polyethylenimine (PEI2). The resulting polymer (PEI2LA) displayed improved transfection efficiency over the unmodified PEI2. The intracellular kinetics and distribution of the respective polyplexes were investigated in the present study to gain a better understanding of the role of lipid modification in intracellular trafficking of gene carriers. A Cy5-labeled plasmid DNA (pDNA) expressing the green fluorescent protein (GFP) was complexed with PEI2, PEI2LA, and 25 kDa polyethylenimine (PEI25) to transfect rat bone marrow stromal cells (BMSC). Subcellular fractionation was performed to measure the amount of nuclear associated pDNA. pDNA uptake, GFP-expression and nuclear-associated pDNA were measured by both flow cytometry and confocal laser scanning microscopy. PEI2LA mediated higher transgene expression and percentages of transfected cells than PEI25 and PEI2, respectively. There was a strong correlation between nuclear associated pDNA and transgene expression. PEI2LA polyplexes were significantly larger in size than PEI25. The amounts of pDNA associated with the nuclei were greater in PEI2LA than PEI25 polyplexes. The perinuclear pDNA distribution between GFP-expressing and nonGFP-expressing indicated that GFP-positive cells had a higher amount of pDNA associated with their nuclei. Improved transfection efficiency of PEI2LA was attributed to enhanced association with the nucleus, which may be a result of hydrophobic interaction between the lipid moieties on the modified lipopolymer and the nuclear membrane.

Bioreducible crosslinked low molecular weight branched PEI-PBLG as an efficient gene carrier

Low molecular weight polyethylenimine-poly(gamma-benzyl L-glutamate) (PEI-PBLG) was crosslinked by N,N′-cystamine-bisacrylamide (CBA) to get the polymer named as CBA-PEI-PBLG (CPP). CPP not only inherits PEI-PBLG’s amphiphilic advantages, but also possesses reducible properties. CPP can complex with DNA to form nanoparticles. CPP/DNA complex particles were characterized by particle size and zeta potential analysis. The result showed that the complex particles have suitable size and surface charges for gene delivery. And gel retardation assay also prove CBA-PEI-PBLG has proper condensing ability for DNA. CPP has good reducible property, and also has good biocompatibility because of introducing PBLG segment. The cytotoxicity of CPP was evaluated using MTT assay, and the results showed CPP has lower cytotoxicity compared with PEI 25 K. The transfection properties were characterized in different cells by using plasmid DNA as a reporter. CPP showed higher transfection efficiencies and lower cytotoxicity in HeLa cells. This was attributed to bioreducible and biocompatibility properties of the CPP. These results suggested that CPP is a promising low-toxic, highly effective non-viral gene carrier.

Disulfide-Containing Hyperbranched Polyethylenimine Derivatives via Click Chemistry for Nonviral Gene Delivery

Reducible disulfide-containing hyperbranched PEI-SS-HP was synthesized via click chemistry and evaluated as nonviral gene carrier. The structure of the polymers was confirmed by 1 H NMR and FTIR. It was shown that the PEI-SS-HP was able to bind plasmid DNA to positively charged nanoparticles. The reduction sensitivity of the PEI-SS-HP was confirmed by gel retardation assay in the presence of DTT mimicking an intracellular reductive environment. In vitro experiments revealed that the reducible PEI-SS-HP not only had much lower cytotoxicity, but also posed superior transfection activity as compared to non-degradable 25-kDa PEI. The results indicate that a reducibly degradable PEI-SS-HP can be a promising gene vector.

Co‐electrospun fibrous scaffold–adsorbed DNA for substrate‐mediated gene delivery

Incorporation of gene into electrospun nanofibers for localized gene transfection of target cells represents a robust platform for tissue regeneration. In this study, a new two-step approach was explored to immobilize DNA onto electrospun nanofibers for effective gene delivery, that is, nonviral gene vector of polyethylene glycol (PEG)-modified polyethylenimine (PEI) was incorporated into scaffolds by electrospinning and then target DNA was adsorbed onto the electrospun nanofibers via electrostatic interaction between DNA and PEI-PEG. PEI-PEG/DNA particles formed from the released DNA, and PEI-PEG had a uniform particle size of approximately 200 nm. This nanofiber-based gene delivery system exhibited high transfection efficiency, in which >65% of human embryonic kidney 293 cells and >40% of mesenchymal stem cells were transfected with green fluorescent protein gene. Compared with PEI, PEG modification of PEI had improved the biocompatibility and further increased the transfection efficiency. These results suggest that the combination of nonviral gene carrier with electrospun nanofibers could be used for localized gene delivery, which has multifold potential applications in tissue engineering or as an in vivo substrate for tissue regeneration.

Effects of Acid and Base on the Inductive Efficiency of Oligonucleotide on the Vesicle Formation from Single‐Chained Cationic Surfactant

AbstractRecently, we reported for the first time that oligonucleotide could induce single‐chained cationic surfactant molecules to aggregate into vesicles and the facilitative efficiency of oligonucleotide on vesicle formation was dependent on its size and sequence. In the present paper, we will continue to study the effects of acid and base on the facilitative efficiency of oligonucleotide on vesicle formation. It is found that proton ions show little effect on the facilitative efficiency while hydroxide ions make it decreased. Moreover, the percentage of oligonucleotide involved in vesicle formation in basic solution is much lower than that in acidic solution (which is almost equal to that in water). Since the structures and properties of DNA/amphiphile complex are very important for its application as nonviral gene carrier, this study may provide some helpful information for gene therapy.

Math1 Gene Transfer Based on the Delivery System of Quaternized Chitosan/Na-Carboxymethyl-<I>β</I>-Cyclodextrin Nanoparticles

Mammalian cochlear hair cells don't regenerate naturally after injury, which usually leave permanent hearing loss. Math1 gene is a positive regulator of hair cell differentiation during cochlear development and was proved to be very critical in hair cell regeneration in deaf animals. Generating new cochlear hair cells by forced Math1 expression may be a cure for hearing loss. However, satisfying gene delivering vectors in gene therapy are not available. We combined quaternized chitosan (QCS) with Na-carboxymethyl-beta-cyclodextrin (CM-beta-CD) as novel non-viral vector, which adsorbs pRK5-Math1-EGFP perfectly at the mass ratio of 4:1. In vitro cell transfection can reach a 40% transfect efficiency and relatively low cytotoxity than liposomes. These results suggest that QCS/CM-beta-CD nanoparticle complexes could be a novel non-viral gene carrier in further clinical application.

A serum-resistant polyamidoamine-based polypeptide dendrimer for gene transfection

A serum tolerant polycation gene vector, G 2 PAMAM-PGlu-G 1 PAMAMs (ALA), was designed, synthesized, characterized and evaluated. A honeycomb-like molecular structure model for mechanistic explanation of ALA was postulated and discussed. Designed as a star-shaped polyamidoamine (PAMAM)-based polypeptide dendrimer through peptide bond linkages, ALA was with non-toxic low generation G 2 PAMAM (G 2) as its central core, polyglutamate (PGlu)s as its star-shaped backbone branches and G 1 PAMAM (G 1)s as its branch grafts and peripheral terminals. IR, 1H NMR demonstrated its successful combination. As a gene carrier, ALA exhibited good DNA binding and condensation capacity with particle size (∼87 nm for N/P 40, ∼170 nm for N/P 30) and ζ-potential (∼16 mV for N/P 30–40), negligible cytotoxicity, exciting serum tolerant capacity and significant serum-promoted (serum-containing 56.6%>serum-free 32.7%), cell line dependent (Hek 293 > Bel 7402 > Hela), incubation period dependent (38 h > 18 h > 12 h > 9 h > 4 h > 2 h > 1 h) and sustained (peak transfection appeared at 30 h incubation) transfection efficiency. The presence of serum had not only no inhibition on, but also prominent promotion to, the transfection activity of ALA. All above features differentiated ALA clearly from most other serum-inhibitive nonviral gene carriers, and proved ALA the promising and challenging potential efficient gene vector for practical clinical application.

Chitosan-graft-spermine as a gene carrier in vitro and in vivo

Chitosan has been proposed as a non-viral gene carrier because of its biodegradable and biocompatible cationic polymeric properties. However, the transfection efficiency of chitosan-DNA complexes is still too low for clinical trials. To improve transfection efficiency, we prepared a chitosan-graft-spermine (CHI-g-SPE) copolymer by an imine reaction between periodate-oxidized chitosan and spermine. The CHI-g-SPE copolymer was complexed with plasmid DNA in various copolymer-DNA weight ratios, and the complexes were characterized. The CHI-g-SPE copolymer showed good DNA binding ability and high protection of DNA from nuclease attack. The CHI-g-SPE/DNA complexes had well-formed spherical shapes and a nanoscale size with homogenous size distribution. The CHI-g-SPE copolymer had low cytotoxicity and CHI-g-SPE/DNA complexes showed transfection efficiency that was enhanced over that of chitosan-DNA. Furthermore, aerosol delivery of CHI-g-SPE/GFP complexes showed higher GFP expression compared with chitosan/GFP complexes, without toxicity. Our results indicate that the CHI-g-SPE copolymer has potential as a gene carrier.

Polymeric Micelles Comprising Stearic Acid-Grafted Polyethyleneimine as Nonviral Gene Carriers

Non-viral vectors composed of biodegradable polymers or lipids have been considered as a safer alternative for gene carriers over viral vectors. Among some of the cationic polymers, polyethyleneimine (PEI) possess high pH-buffering capacity that can provide protection to nucleotides from acidic degradation and promotes endosomal and lysosomal release. However, it has been reported that cytotoxicity of PEI depends on the molecular weight of the polymer. Hence modifications of PEI structure for clinical application have been developed in order to reduce the cytotoxicity, or improve the insufficient transfection efficiency of lower molecular weight PEI. In this study, 10 k PEI was modified by grafting stearic acid (SA) and formulated to polymer micelles with positive surface charge and evaluated for pDNA delivery. The amine group on PEI was crosslinked with the carboxylic group of stearic acid by 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide (EDC) as linker. PEI-SA micelles were then prepared using oil in water (o/w) solvent evaporation method. The success of PEI-SA conjugation structure was confirmed with 1H NMR. The average diameter and zeta potential determined by photon correlation spectroscopy was 149.6 +/- 1.2 nm and 64.1 +/- 1.5 mV, respectively. These self-assemble positive charge micelles showed effective binding to pDNA for transfection. PEI-SA micelles exhibited lower cytotoxicity compared to that of PEI only, while flow cytometry analysis revealed PEI-SA/pEGFP complex provided 62% high EGFP expression. Luciferase activity also showed high transfection efficiency of PEI-SA micelles for weight ratio above 4.5 that was comparable to PEI only. These results demonstrated that stearic acid grafted PEI micelles can provide high transfection efficiency comparable to unmodified PEI, and exhibit low cytotoxicity. Stearic acid grafted PEI micelles can be promising polymer carriers in genetic therapy.

Pig transgenesis by Sleeping Beauty DNA transposition

Modelling of human disease in genetically engineered pigs provides unique possibilities in biomedical research and in studies of disease intervention. Establishment of methodologies that allow efficient gene insertion by non-viral gene carriers is an important step towards development of new disease models. In this report, we present transgenic pigs created by Sleeping Beauty DNA transposition in primary porcine fibroblasts in combination with somatic cell nuclear transfer by handmade cloning. Göttingen minipigs expressing green fluorescent protein are produced by transgenesis with DNA transposon vectors carrying the transgene driven by the human ubiquitin C promoter. These animals carry multiple copies (from 8 to 13) of the transgene and show systemic transgene expression. Transgene-expressing pigs carry both transposase-catalyzed insertions and at least one copy of randomly inserted plasmid DNA. Our findings illustrate critical issues related to DNA transposon-directed transgenesis, including coincidental plasmid insertion and relatively low Sleeping Beauty transposition activity in porcine fibroblasts, but also provide a platform for future development of porcine disease models using the Sleeping Beauty gene insertion technology.

Quantized Folding of Plasmid DNA Condensed with Block Catiomer into Characteristic Rod Structures Promoting Transgene Efficacy

Highly regulated folding of plasmid DNA (pDNA) through polyion complexation with the synthetic block catiomer, poly(ethylene glycol)-block-poly(L-lysine) (PEG-PLys), was found to occur in such a way that rod structures are formed with a quantized length of 1/2(n + 1) of the original pDNA length folding by n times. The folding process of pDNA was elucidated with regard to rigidity of the double-stranded DNA structure and topological restriction of the supercoiled closed-circular form, and a mechanism based on Euler's buckling theory was proposed. Folded pDNA exhibited higher gene expression efficiency compared to naked pDNA in a cell-free transcription/translation assay system, indicating that the packaging of pDNA into a polyion complex core surrounded by a PEG palisade is a promising strategy for constructing nonviral gene carrier systems. Extension of this finding may provide a reasonable model to further understand the packaging mechanism of supercoiled DNA structures in nature.

Development of cell-penetrating peptide-modified MPEG-PCL diblock copolymeric nanoparticles for systemic gene delivery

To develop a safe and efficient systemic non-viral gene vector, methoxy poly(ethylene glycol) (MPEG)/poly(ɛ-caprolactone) (PCL) diblock copolymers conjugated with a Tat analog through the ester or disulfide linkage were synthesized and their suitability as a systemic non-viral gene carrier evaluated. The physicochemical properties of the MPEG-PCL diblock copolymers were determined by GPC, 1H NMR and FT-IR spectroscopy. The particle sizes and in vitro (COS7 and S-180 cells) transfection efficiencies and cytotoxicity were evaluated. Furthermore, the luciferase activity was then determined in various tissues after intravenous injection of MPEG-PCL-SS-Tat/pCMV-Luc complex into mice bearing S-180 cells. The particle sizes of the MPEG-PCL-Tat copolymers with or without pDNA were about 40 and 60 nm, respectively. The luciferase activity in COS7 cells transfected with pCMV-Luc with MPEG-PCL-ester-Tat or MPEG-PCL-SS-Tat was higher than that with pDNA only. MPEG-PCL-SS-Tat greatly increased the transfection efficiency compared to MPEG-PCL-ester-Tat in COS7 and S-180 cells. In an in vitro cytotoxicity test MPEG-PCL-SS-Tat did not induce any remarkable cytotoxicity. In an in vivo experiment, the synthesized MPEG-PCL-SS-Tat copolymers promoted the delivery and expression of pDNA into tumor tissue in tumor-bearing mice. In conclusion, this vector might be applicable as a tumor-targeting non-viral systemic gene carrier in the clinical setting.

Cationic nanoemulsions as non-viral vectors for plasmid DNA delivery

Non-viral gene carriers have been extensively investigated as alternatives to viral vectors for therapeutic gene delivery. Many cationic lipid carriers including liposomes, emulsions, and solid lipid nanoparticles are used to transfer plasmid DNA. Stable nanoemulsions were prepared and modified by conjugating fatty acids with cationic amino acids including lysine, arginine, and histidine with the help of carbodiimide. Concentrations of crosslinker and amino acids were optimized to obtain the maximal surface potential. The zeta potential and size distribution of the cationic nanoemulsions were measured using photon correlation spectroscopy. The morphology of nanoemulsion-DNA complexes was examined by transmission electron microscopy. The transfection efficiencies and cytotoxicity of three cationic nanoemulsions were evaluated using 3T3 fibroblast cells. The maximal zeta potentials of lysine-, arginine-, and histidine-modified nanoemulsions were 50, 43, and 7 mV, respectively. The transfection efficiencies of amino acid-modified nanoemulsions were in the order of lysine > arginine > histidine. Low cytotoxicities of these three amino acid-modified nanoemulsions were observed. A facile and inexpensive in situ modification for producing cationic nanoemulsions was developed. The results show the potential of amino acid-modified cationic nanoemulsions as non-viral vectors for gene delivery.