Ethidium Bromide Intercalation Assay Research Articles

Comparison of cationic liposome and PAMAM dendrimer for delivery of anti-Plk1 siRNA in breast cancer treatment

Delivery of negatively charged, high molecular weight and unstable siRNA is difficult. The present study describes the development and comparison of cationic liposomes (CLs) and polyamidoamine (PAMAM) dendrimer generation 4 (PG4) nanocarriers of gene for cancer therapy. CLs and PG4 were complexed with anticancer siRNA (siPlk1) to form siPlk1-CLs lipoplex and siPlk1-PG4 dendriplex. siPlk1-CLs/PG4 complexes were characterized for average particle size, zeta potential, fluorescence and integrity of siPlk1 by agarose gel electrophoresis, ethidium bromide intercalation assay, circular dichroism, protection against RNase and stability in serum. The complexation of CLs/siPlk1 and PG4/siPlk1 were at a 100/1 and 2/1 charge ratio respectively. The CLs and PG4 were effective in protecting siPlk1 from RNase activity, also they enhanced the siPlk1 serum stability. Additionally, siPlk1-CLs and siPlk1-PG4 were evaluated by cell culture studies. In vitro anticancer activity study using MCF-7 cells showed that siPlk1-CLs and siPlk1-PG4 causes nearly similar cell death. Both siPlk1-CLs and siPlk1-PG4 resulted in enhanced cellular uptake of siPlk1 in MDA-MB-231 cells compared to naked siPlk1 solution. Cell cycle analysis suggested that increased cell population arrest in subG1 phase by siPlk1-CLs and siPlk1-PG4 compared to naked siPlk1 solution. These observations suggested that CLs and PG4 can be a potential carrier for siPlk1 delivery in breast cancer treatment.

Spacer structure and hydrophobicity influences transfection activity of novel polycationic gemini amphiphiles

Three novel polycationic gemini amphiphiles with different spacers were developed and evaluated in terms of their physiochemical properties and transfection efficiencies. Cationic liposomes formed by these amphiphiles and the helper lipid DOPE were able to successfully condense DNA, as shown by gel mobility shift and ethidium bromide intercalation assays. Transfection activity of the liposomes was superior to Lipofectamine® 2000 and was dependent on spacer structure, hydrophobicity, and nucleic acid type (pDNA or siRNA). We demonstrated that the cationic liposomes 2X6/DOPE and 2X7/DOPE are potential non-toxic vehicles for gene delivery.

High transfection efficiency promoted by tailor-made cationic tri-block copolymer-based nanoparticles.

Syntheses of PDMAEMA-b-PβAE-b-PDMAEMA block copolymers for an extremely effective non-viral vector. The block copolymer PDMAEMA3000-b-PβAE12000-b-PDMAEMA3000-based polyplexes at 100/1N/P ratio exhibits between 40- and 60-fold higher transgene expression in HeLa and COS-7 cell lines than commonly used polymeric non-viral vectors, namely branched PEI (known as the gold standard) and TurboFect™ (commercial available).

Cationic modified gold nanoparticles show enhanced gene delivery in vitro

Abstract Gold nanoparticles (AuNPs) can be suitably engineered for applications in gene delivery, and as carriers of peptides and proteins. They present innovative strategies in gene therapy for the treatment of genetic as well as acquired diseases. Optimisation of AuNPs in binding and the eventual release of the therapeutic agent in a controlled manner are crucial steps in the pathway to effective drug or gene therapy. In this study, we propose the formulation of cationic polymer [chitosan (Chit) and poly-l-lysine (PLL)] and cysteine (Cys)-coated AuNPs. These cationic AuNPs displayed good stability and resistance to aggregation under physiological conditions. All functionalised formulations remained in the size range 15.7–186 nm with narrow particle distributions and low polydispersity index (<0.9). Band shift and ethidium bromide intercalation assays determined the complexation of pDNA to the functionalised AuNP (FAuNP). Au-Chit/pDNA exhibited a significant 126% (p<0.01) cell viability in HepG2 cells, followed by Au-PLL/pDNA at 104% in HeLa cells and Au-cysteine/pDNA at 91.8% (p<0.001) in Caco-2 cells. Significant transgene activities in all cell lines were noted with enhanced gene expression for the FAuNP complexes over the cationic complexes on their own. The results of this study suggest that the low cytotoxicity and promising transgene activity of these FAuNPs, coupled with the ability to parametrically control particle size and surface properties, make these formulations suitable nanocarriers for drugs and genes.

169. Polymer Functionalized Gold Nanoparticles in Gene Delivery In Vitro

Non-viral gene delivery approaches have been extensively studied as a basic tool for intracellular gene transfer and gene therapy especially for genetic aberrations including cancer. Gold nanoparticles have attracted strong biomedical interest for drug/gene delivery due to their low toxic nature, surface plasmon resonance and capability of increasing the stability of the payload. In the present study the synthesis of photoluminescent nanoparticles consisting of a gold core coated with polyethyleneimine, poly-L-lysine, cysteine and chitosan is reported. These functionalized gold nanoparticles (FAuNPs) were investigated at different pH values and ionic strength to identify the optimum conditions to produce stable monodisperse nanoparticles. FAuNPs showed good stability at low ionic strength which is important for the flexibility of the polymer chain. All nanoparticle/polymer formulations showed spherical particles in the size range 11.9-195 nm with narrow particle distributions and low PDI (<1.2). Nanoparticle and pDNA complexation was efficiently demonstrated in the band shift and ethidium bromide intercalation assays respectively, with serum nuclease digestion revealing partial protection of theResults: Our novel PEGylated PLGA nanoparticles (without siRNA) showed higher accumulation and extended residence time in the inflamed foot of lipopolysaccharide (LPS)-induced mouse model of chronic inflammation. TNF-α-siRNA-NPs were 176.6±15.8 nm in diameter, with a zeta potential of -25.2±2.0 mV and an entrapment efficiency of 31±4%. The nanoparticles were stable in simulated biological medium. In vitro, less than 40% of the siRNA was released with 72 h at 37oC. Finally, TNF-α-siRNA-NPs significantly decreased TNF-α release from J774A.1 cells.complexed plasmid DNA (pCMV-luc). MTT cytotoxicity experiments indicated that the FAuNPs elicited a dose dependent cytotoxic effect in four mammalian cell lines (HepG2, HEK293, HeLa and Caco2). Au-PEI/pDNA maintained over 80% cell viability across all cell lines, while the Au-cys/pDNA exhibited a significant 91.8% (p<0.001) in Caco2 cells, Au-Chit/pDNA 126% (p<0.01) in HepG2 cells and Au-PLL/pDNA 104% in Hela cells. Transfection studies were accomplished using the luciferase reporter gene assay. Results showed that the FAuNPs produced greater transgene activity than the cationic polymer/DNA complexes on their own. This was evident for the Au-PEI/pDNA complex which produced a 12 fold increase in the HEK293 cells and a 9 fold increase in the HepG2 cells, compared to the PEI/pDNA complexes. The results of this study suggest that FAuNP's low cytotoxicity coupled with the ability to parametrically control particle size and surface properties, make these nanoparticles suitable non-viral gene delivery vectors. However further engineering and modifications of the FAuNPs may be required to enable in vivo gene delivery.

Cyclen‐Based Cationic Lipids Containing Carbamate Linkages as Efficient Gene Delivery Vectors with Low Toxicity

AbstractTwo novel cationic lipids based on protonated cyclen and steroid (cholesterol or diosgenin) moieties with carbamate linkage have been designed and synthesized for gene delivery. Cationic liposomes were easily prepared from each of these lipids individually or from the mixtures of each cationic lipid and dioleoylphosphatidyl ethanolamine (DOPE). Several studies including dynamic light scattering, gel retardation assay, ethidium bromide intercalation assay, and transmission electron microscopy (TEM) imaging demonstrate that these amphiphilic molecules are able to bind and compact DNA into nanoparticles which may act as nonviral gene delivery agents. Results from in vitro transfection show that in association with (DOPE), two cationic lipids can induce effective gene transfection in HEK 293, A549, and H460 cells. Especially, in tumor cells (A549 and H460), the gene transfection efficiency of two cationic lipids were found to be higher than that of commercially available Lipofectamine 2000. The lipoplexes formed from both cationic lipids were found to have low cytotoxicity in three cell lines even at high N/P ratios.

Cationic Lipids Containing Cyclen and Ammonium Moieties as Gene Delivery Vectors

In this study, two novel cationic lipids containing protonated cyclen and quaternary ammonium moieties were designed and synthesized as non-viral gene delivery vectors. The structures of the two lipids differ in their hydrophobic region (cholesterol or diosgenin). Cationic liposomes were easily prepared from the lipids individually or from the mixtures of each cationic lipid and dioleoylphosphatidylethanolamine. Several studies including DLS, gel retardation assay, and ethidium bromide intercalation assay suggest that these amphiphilic molecules are able to bind and compact DNA into nanometer particles which can be used as non-viral gene delivery agents. Our results from in vitro transfection show that in association with dioleoylphosphatidylethanolamine, two cationic lipids can induce effective gene transfection in human embryonic kidney 293 cells, although the gene transfection efficiencies of two cationic lipids were found to be lower than that of lipofectamine 2000(TM) . Besides, different cytotoxicity was found for two lipoplexes. This study demonstrates that the title cationic lipids have large potential to be efficient non-viral gene vectors.

Novel cationic lipids possessing protonated cyclen and imidazolium salt for gene delivery

In this study, two novel protonated cyclen and imidazolium salt-containing cationic lipids differing only in their hydrophobic region (cholesterol or diosgenin) have been designed and synthesized for gene delivery. Cationic liposomes were easily prepared from each of these lipids individually or from the mixtures of each cationic lipid and dioleoylphosphatidyl ethanolamine (DOPE). Several studies including DLS, gel retardation assay, ethidium bromide intercalation assay, and TEM demonstrated that these amphiphilic molecules are able to bind and compact DNA into nanometer particles that could be used as non-viral gene delivery agents. Our results from in vitro transfection showed that in association with DOPE, two cationic lipids can induce effective gene transfection in HEK293 cells. Furthermore, the gene transfection efficiencies of two cationic lipids were dramatically increased in the presence of calcium ion (Ca 2+). It is notable that the gene transfection abilities of two cationic lipids were maintained in the presence of 10% serum. Besides, different cytotoxicity was found for two lipoplexes. This study demonstrates that the title cationic lipids have large potential to be efficient non-viral gene vector.

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.

Gene vectors based on DOEPC/DOPE mixed cationic liposomes: a physicochemical study

A double approach, experimental and theoretical, has been followed to characterize from a physicochemical standpoint the compaction process of DNA by means of cationic colloidal aggregates. The colloidal vectors are cationic liposomes constituted by a mixture of a novel cationic lipid, 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (chloride salt) (DOEPC) and a zwitterionic lipid, the 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE). A wide variety of high precision experimental techniques have been used to carry out the analysis: electrophoretic mobility, small-angle X-ray scattering (SAXS), cryogenic transmission electron microscopy (cryo-TEM) and fluorescence spectroscopy (ethidium bromide intercalation assays). On the other hand, a theoretical model that considers the renormalization of charges of both the polyelectrolyte and the colloidal aggregates sheds light as well on the characteristics of the compaction process. This global information reveals that the compaction of DNA by the cationic liposomes is mostly driven by the strong electrostatic interaction among the positively charged surfaces of the colloidal aggregates and the negatively charged DNA, with a potent entropic component. DOEPC/DOPE liposomes are mostly spherical, with a mean diameter of around 100 nm and a bilayer thickness of 4.4 nm. From a morphological viewpoint, an appreciable amount of multilamellar structures has been found not only on the lipoplexes but also on the parent liposomes. The isoneutrality of the lipoplexes is found at liposome/DNA mass ratios that decrease with the molar fraction of cationic lipid in the mixed liposome (α). This liposome composition has a clear effect as well on the lipoplex structure, which goes from an inverted hexagonal phase (HII), usually related to improved cell transfection efficiency, at low cationic lipid molar fraction (α ≈ 0.2), to a lamellar structure (Lα) when the cationic lipid content in the mixed liposomes increases (α ≥ 0.4), irrespective of the lipoplex charge ratio. On the other hand, a theoretical complexation model is employed to determine the net charge of the lipoplexes studied in this work, by using renormalized charges. The model allows us to confirm and predict the experimental isoneutrality conditions as well as to determine the maximum magnitude of this charge as a function of the composition of the resulting lipoplexes.

Transfection efficiencies of PAMAM dendrimers correlate inversely with their hydrophobicity

Dendriplexes were characterized by ethidium bromide intercalation assay and their transfection efficiency was studied using HEK 293 cells and human mesenchymal stem cells. PAMAM G4 showed a higher transfection efficiency than PAMAM G3–G6, G4-OH, G4-25% or G4-50% dendrimers. Substitution of OH groups for the NH 2 surface groups rendered the dendrimer unable to form dendriplexes and to transfect cells. Partial (25%) substitution of CH 3 groups for the NH 2 groups markedly impaired transfection; 50% substitution decreased the ability of PAMAM G4 to transfect threefold. It was concluded that increased hydrophobicity decreased the ability of dendrimers to transfect. PAMAM G4-50% is highly hydrophobic and forms micelles in solution, which can transfect pGFP. The results of ethidium bromide intercalation assays, ANS fluorescence studies and transfection efficiencies of PAMAM dendrimers were correlated. Subsequently, we constructed a neurotrophin-encoding plasmid and studied its delivery to mesenchymal stem cells using PAMAM G4 dendrimer and Lipofectamine 2000. Lipofectamine 2000 was a more effective carrier (18.5%) than PAMAM G4 dendrimer (1.2%).

Tailor‐Made Poly(amidoamine)s for Controlled Complexation and Condensation of DNA

A set of polymer carriers for DNA delivery was synthesized by combining monodisperse, sequence-defined poly(amidoamine) (PAA) segments with poly(ethylene oxide) (PEO) blocks. The precise definition of the PAA segments provides the possibility of correlating the chemical structure (monomer sequence) with the resulting biological properties. Three different PAA-PEO conjugates were synthesized by solid-phase supported synthesis, and the cationic nature of the PAA segments was systematically varied. This allows for the tailoring of interactions with double-stranded plasmid DNA (dsDNA). The potential of the PAA-PEO conjugates as non-viral vectors for gene delivery is demonstrated by investigating the dsDNA complexation and condensation properties. Depending on the applied carrier, a transition in polyplex (polymer-DNA ion complex) structures is observed. This reaches from extended ring-like structures to highly compact toroidal structures, where supercoiling of the DNA is induced. An aggregation model is proposed that is based on structural investigations of the polyplexes with atomic force microscopy (AFM) and dynamic light scattering (DLS). While the cationic PAA segment mediates primarily the contact of the carrier to the dsDNA, the PEO block stabilizes the polyplex and generates a "stealth" aggregate, as was suggested by Zeta potentials that were close to zero. The controlled aggregation leads to stable, single-plasmid complexes, and stabilizes the DNA structure itself. This is shown by ethidium bromide intercalation assays and DNase digestion assays. The presented PAA-PEO systems allow for the formation of well-defined single-plasmid polyplexes, preventing hard DNA compression and strongly polydisperse polyplexes. Moreover carrier polymers and the resulting polyplexes exhibit no cytotoxicity, as was shown by viability tests; this makes the carriers potentially suitable for in vivo delivery applications.

In vitro transfection of plasmid DNA by cationized gelatin prepared from different amine compounds

The objective of this paper is to compare the in vitro transfection efficiency of a luciferase plasmid DNA using cationized gelatin prepared from different amine compounds. The compounds used here were ethylenediamine, putrescine, spermidine and spermine, chemically introduced to the carboxyl group of gelatin for the cationization. Complexation of the cationized gelatin with the plasmid DNA was performed by simply mixing the two materials at various N+/P− mixing ratios (the molar number ratio of amino groups of gelatin to the phosphate groups of DNA) in aqueous solution. Gel retardation studies revealed that the formation of cationized-gelatin–plasmid DNA complexes depended on the N+/P− mixing ratio. The stronger interaction of plasmid DNA with the cationized gelatin of spermine compared to the other cationized gelatins was observed by an ethidium bromide intercalation assay and Scatchard binding analysis. When the transfection efficiency of plasmid DNA complexed with the various cationized gelatins at different N+/P− mixing ratios was evaluated for mouse L929 fibroblasts, the highest transfection efficiency was observed for the complex prepared from the cationized gelatin of spermine at a N+/P− mixing ratio of 2. The present study indicates that there is an optimal N+/P− mixing ratio and a type of amine compound or cationization extent of cationized gelatin to enhance the transfection efficiency of plasmid DNA.

Biophysical and Transfection Studies of an Amine-Modified Poly(vinyl alcohol) for Gene Delivery

Novel, multifunctional polymers remain an attractive objective for drug delivery, especially for hydrophilic macromolecular drugs candidates such as peptides, proteins, RNA, and DNA. To facilitate intracellular delivery of DNA, new amine-modified poly(vinyl alcohol)s (PVAs) were synthesized by a two-step process using carbonyl diimidazole activated diamines to produce PVAs with different degrees of amine substitution. The resulting polymers were characterized using NMR, thermogravimetric analysis (TGA), and gelpermation chromatography (GPC). Atomic force microscopy (AFM), dynamic light scattering photon correlation spectroscopy (PCS), and zeta-potential were used to investigate polyplexes of DNA with PVA copolymers. These studies suggest an influence of the polycation structure on the morphology of condensed DNA in polyplexes. Significant differences were observed by changing both the degrees of amine substitution and the structure of the PVA backbone, demonstrating that both electrostatic and hydrophobic interactions affect DNA condensation. DNA condensation measured by an ethidium bromide intercalation assay showed a higher degree of condensation with pDNA with increasing degrees of amine substitution and more hydrophobic functional groups. These findings are in line with transfection experiments, in which a good uptake of these polymer DNA complexes was noted, unfortunately, with little endosomal escape. Co-administration of chloroquine resulted in increased endosomal escape and higher transfection efficiencies, due to disruption of the endosomal membrane. In this study, the structural requirements for DNA complexation and condensation were characterized to provide a basis for rational design of nonviral gene delivery systems.

Surface Charge Density Determines the Efficiency of Cationic Gemini Surfactant Based Lipofection

The efficiencies of the binary liposomes composed of 1,2-dimyristoyl- sn-glycero-3-phosphocholine and cationic gemini surfactant, (2S,3R)-2,3-dimethoxy-1,4-bis( N-hexadecyl- N, N-dimethylammonium)butane dibromide as transfection vectors, were measured using the enhanced green fluorescent protein coding plasmid and COS-1 cells. Strong correlation between the transfection efficiency and lipid stoichiometry was observed. Accordingly, liposomes with X SR−1 ≥ 0.50 conveyed the enhanced green fluorescent protein coding plasmid effectively into cells. The condensation of DNA by liposomes with X SR−1 > 0.50 was indicated by static light scattering and ethidium bromide intercalation assay, whereas differential scanning calorimetry and fluorescence anisotropy of diphenylhexatriene revealed stoichiometry dependent reorganization in the headgroup region of the liposome bilayer, in alignment with our previous Langmuir-balance study. Surface charge density and the organization of positive charges appear to determine the mode of interaction of DNA with (2S,3R)-2,3-dimethoxy-1,4-bis( N-hexadecyl- N, N-dimethylammonium)butane dibromide/1,2-dimyristoyl- sn-glycero-3-phosphocholine liposomes, only resulting in DNA condensation when X SR−1 > 0.50. Condensation of DNA in turn seems to be required for efficient transfection.

Effect of DNA/liposome mixing ratio on the physicochemical characteristics, cellular uptake and intracellular trafficking of plasmid DNA/cationic liposome complexes and subsequent gene expression

In order to identify the important factors involved in cationic liposome-mediated gene transfer, in vitro transfection efficiencies by plasmid DNA complexed with DOTMA/DOPE liposomes at different DNA/liposome mixing ratios were evaluated using four types of cultured cells with respect to their physicochemical properties. Significant changes were observed in the particle size and zeta potential of the complexes as well as in their structures, assessed by atomic force microscopy, which depended on the mixing ratio. In transfection experiments, except for RAW 264.7 cells (mouse macrophages), efficient gene expression was obtained in MBT-2 cells (mouse bladder tumor), NLH3T3 cells (mouse fibroblasts) and HUVEC (human umbilical vein endothelial cells) at an optimal ratio of 1:5, 1:7.5 or 1:5, respectively. On the other hand, cellular uptake of the [ 32P]DNA/liposome complexes increased in all cell types with an increase in the mixing ratio, which was not reflected by the transfection efficiency. The cellular damage determined by MTT assay was minimal even at the highest DNA/liposome ratio (1:10), indicating that the lower gene expression level at the higher ratio was not due to cytotoxicity induced by the complex. An ethidium bromide intercalation assay showed that the release of plasmid DNA from the complex, following the addition of negatively charged liposomes, was restricted as the mixing ratio increased. Furthermore, confocal microscopic studies using HUVEC showed that the 1:5 complexes exhibited a dispersed distribution in the cytoplasm whereas a punctuate intracellular distribution was observed for the 1:10 complexes. This suggests that there was a significant difference in intracellular trafficking, probably release from the endosomes or lysosomes, of the plasmid DNA/cationic liposome complexes between these mixing ratios. Taken together, these findings suggest that the DNA/liposome mixing ratio significantly affects the intracellular trafficking of plasmid DNA complexed with the cationic liposomes, which is an important determinant of the optimal mixing ratio in cationic liposome-mediated transfection.

Intracellular Accumulation of Secreted Proteoglycans Inhibits Cationic Lipid-mediated Gene Transfer: CO-TRANSFER OF GLYCOSAMINOGLYCANS TO THE NUCLEUS

Molecules secreted by potential target cells may interfere with cationic lipid-mediated gene transfer. This has been studied using human lung fibroblasts and human epidermoid lung cancer cells. Secreted cell medium components caused a substantial decrease both in the uptake of cationic lipid-DNA complexes (2-4-fold) and in reporter gene expression (100-1000-fold). Metabolic labeling of the cell medium showed that especially [35S]sulfate-labeled macromolecules competed with DNA for binding to the cationic lipid. Release of DNA from the cationic lipid by cell medium components was demonstrated by an ethidium bromide intercalation assay. In the presence of the cationic lipid, the secreted macromolecules were internalized by the cells. By enzymatic digestions, it was shown that the competing macromolecules consist of chondroitin/dermatan sulfate and heparan sulfate proteoglycans and that the effects on transfection were mediated by the polyanionic glycosaminoglycan portion of the proteoglycan. Accordingly, pretreatment of cell medium with the polycationic peptide protamine sulfate abrogated the inhibitory effects on gene transfer. Fluorescence microscopy studies revealed that heparan sulfate, internalized as a complex with cationic lipids, accumulated in the cell nuclei. These results support the view that the lack of specificity of this type of gene transfer vehicle is a major hindrance to efficient and safe in vivo administration.