The electrostatic repulsion between the phosphodiester anionic charges of nucleic acids (NA) and the negatively-charged headgroups of cell membrane phospholipids hinders naked NA to permeate the plasma membrane [1, 2]. Additionally, nucleases present in the cells and in biological fluids enzymatically degrade NA, limiting their biofunctionality. Although many alternative methods have been developed to deliver NA to cells, factors such as versatility [3, 4], applicability [5, 6] and efficiency [7, 8] have discouraged their disseminated use in gene therapy.
Given these limitations, the intracellular delivery of genetic material can only be achieved through the use of physical, biological or chemical methods that promote gene insertion into cells. Physical methods have generally low
Cationic liposomes are spherical vesicles composed of one or more cationic lipid or phospholipid bilayers [23, 24]. They include both cationic and neutral surfactants in their composition and may differ in size , lamellarity  or charge . The cationic amphiphiles (which are mainly of synthetic nature) share two common features: the net cationic charge on the hydrophilic headgroup, and the hydrophobic tail that anchors the molecule to the liposome lipid bilayer . The chemical structure of the cationic lipids varies markedly and each molecule can have a single (monovalent surfactant) or multiple cationic charges (multivalent surfactant) . The neutral
The driving force for lipoplex formation is the electrostatic interaction between the net positive charge of the cationic liposomes and the negatively charged DNA at an optimal ratio (+/-). This fact also enables the resulting complex to adsorb to the negatively charged cell surface [33-36]. After adsorption, cellular uptake of the complexed DNA facilitates intracellular DNA delivery and subsequent transgene expression . In the case of DODAB/MO formulations, in which DODAB acts as a monovalent cationic surfactant and MO as
The high structural dependence of the system on MO content and temperature [39, 40] could reveal itself useful for optimizing lipoplex resistance against deleterious interactions with biological fluids and cell components, while remaining biocompatible and efficient as delivery agent. The presence of MO in these formulations also reduces the net positive charge necessary for successful NA complexation, thus reducing transfection-associated cytotoxicity . In summary, a multidisciplinary approach to lipofection vectors will lead to the development of formulations with the most appropriate characteristics. Careful design of liposomal composition is essential for overcoming biological barriers, in order to achieve optimal transfection efficiency
2. Cationic lipid-mediated gene transfection
2.1. DODAB:MO liposomes
When assessing the potential of a new lipofection reagent, it is fundamental to study the physicochemical properties of the base liposomal formulation, to better adjust lipoplex morphology (lipoplex size, charge ratio (+/-), fluidity and structure) for optimal transfection conditions. In this way, the behaviour of the lipofection reagent (resistance to extracellular components, cytotoxicity)
MO was first proposed as
The use of MO in liposomal formulations brings other advantages apart from the fluidization of DODAB’s membranes. MO is a natural-occurring neutral surfactant that has the particularity of forming two inverted bicontinuous cubic phases (QIID and QIIG) in excess water [61, 62]. It possesses a single unsaturated acyl chain (C18:1) attached to a glycerol headgroup . Its tendency to form inverted bicontinuous cubic phases has been explored in the past for different applications such as protein crystallization [64, 65] or matrix for gel electrophoresis , and justifies the structural richness of the liposomal system formed with DODAB .
The aggregation behaviour of concentrated DODAB/MO mixtures has been studied through different techniques including phase scan imaging (Fig. 1) that reveals a two-region phase diagram consisting of either DODAB or MO enriched zones . If ΧDODAB ≥ 0.5, bilayer-based structures dominate (Fig. 1A’, 1B’) and their size and fluidity depend on the molar composition of the mixture, with DODAB gel phase appearing as hydrated crystals . When ΧDODAB < 0.5, aggregates are dominated by densely packed cubic-oriented particles, visible as a cubic isotropic phase (Q) associated with high MO contents (Fig. 1D’, 1E’) .
This dual phase behaviour of DODAB/MO lipid mixtures confers a structural complexity to the system that extends itself to lipoplex organization, which can be fine-tuned to suit the biological application. Additionally, results show that MO has a similar effect on aggregate morphology than an increase in temperature, which can be modulated to produce formulations more suitable for gene transfection [39, 40].
2.2. Role of MO as helper lipid in pDNA/DODAB/MO lipoplexes
The incubation of nucleic acids with DODAB/MO mixtures or other cationic vesicle formulation leads to the formation of lipoplexes [67, 68]. The electrostatic interaction between opposite charges is the key factor that determines the adsorption of the cationic vesicles to the DNA molecules, a transient state that ends when a critical cationic vesicle concentration is reached. This leads to the disruption of the lipid vesicles which allows the formation of highly organized structures where the DNA molecules are tightly condensed between adjacent bilayers – the so-called lipoplexes [69-71]. The excess of cationic lipid is required for lipoplex binding to the cell surface but any subsequent addition of cationic lipid to the complex does not enhance DNA delivery and only increases toxicity in the exposed cells .
Lipoplexes such as the pDNA/DODAB/MO system can be directly visualized by techniques such as cryo-TEM imaging (Fig. 2), which also gives information on the structural properties of the system (size, compactation, organization) . Cryo-TEM imaging reveals that pDNA/DODAB/MO lipoplexes present the same dual phase diagram as obtained for DODAB/MO lipid mixtures . pDNA/DODAB/MO lipoplexes at ΧDODAB > 0.5 (Fig. 2A) exhibit a multilamellar structure consisting of stacked alternating lipid bilayers and pDNA monolayers. The analysis using Fast Fourier Transforms (FFT) corroborates this observation, by denoting a mono-orientated organization pattern at repeating distances of about 5 nm (Fig. 2A’’, 2A’’’ and 2A’’’’) .
In contrast, pDNA/DODAB/MO lipoplexes at ΧDODAB ≤ 0.5 (Fig. 2B) show high-curvature zones where lipid bilayers intercross each other with pDNA monolayers stacked between them. These high-curvature zones have been interpreted as MO-rich domains that alternate with DODAB-rich domains presenting multilamellar organization. The FFT diagrams show that these MO-rich domains possess a distinct structural organization with bi-orientated patterns in angles of 90% between them, consistent with the existence of cubic inverted bicontinuous mesophases (Fig. 2B’’, 2B’’’ and 2B’’’’) .
The DODAB/MO aggregate organization influences the final structural properties of the resulting pDNA/DODAB/MO lipoplexes, with MO content having a dramatic effect on how DNA is condensed and protected within the membrane.
By definition, a
Dioleoylphosphatidylethanolamine (DOPE) is the most established
This evidence has motivated the search for new
MO is another promising alternative to common
The fluidizing effect of MO contributes favourably to the complexation efficiency of DNA, quickening lipoplex formation . At the same time, the formed inverted bicontinuous cubic mesophases improve the resistance of aggregates to extracellular component destabilization, thereby potentially enhancing transfection efficiency . MO-based aggregates induce a relatively low cytotoxicity level, which further reinforces its use as a new
2.3. Recent progress in gene delivery with cationic lipids
The quest for the perfect cationic liposome formulation has been based on empirical testing of novel surfactant molecules that had never been previously used for NA delivery [91-93]. The only goals for candidate molecules are the attainance of high transfection efficiency with low cytotoxicity [94, 95].
After the first generation of cationic lipids based on double-chain surfactants with plain ammonium headgroups (DODAB, DOTAP, DOTMA or DMRIE) [96, 97], soon came cationic lipids with poly-ammonium and multivalent functional radicals (DOGS, DOSPA). The latter exhibited higher transfection efficiencies but also higher cytotoxicity due to the immunogenicity of the cationic ammonium headgroups [98, 99]. This negative effect was balanced with the appearance of
Polyethylene glycol (PEG)-based lipids emerged as interesting hydrophilic polymer-based surfactants that could provide steric stability to cationic liposomes, increasing lipoplex lifetime in the bloodstream and also decreasing the toxic effects observed
Inclusion of pH-sensitive molecules in the formulations has been shown to improve transgene expression by favouring DNA release from the endosomal compartment. Examples of pH-sensitive molecules used in non-viral gene delivery include polyhistidine, dioleoyldimethylammonium propane (DODAP) or cholesteryl hemisuccinate (CHEMS) [112, 113].
Another major breakthrough with impact in gene therapy was the possibility of specific cell targeting by liposomes. Amphiphiles with hydrophilic headgroups could be chemically linked to molecules such as folate, transferrin or the epidermal growth factor that potentiate specific delivery to cancer cells, markedly increasing the therapeutic benefits achieved with lipoplexes, with little secondary effects [114-118].
More recently, cationic lipids with amino acid headgroup (serine, alanine) [119, 120] and sugar-based cationic lipids (D-galactose) have appeared as promising families of cationic surfactants [121, 122]. Small molecular weight peptides (glutamate, cysteine) augment the hydrophilicity of the lipoplex surface, as with small surface sugars (galactose, mannose) that additionally allow targetability of the lipoplexes.
3. Lipoplex interaction with extracellular milieu
3.1. Resistance to components of biological fluids
An effective delivery system must confer stability to complexed NA in physiological conditions [123, 124]. Systemic delivery of NA requires a stealth carrier that protects NA from indiscriminate interaction with complement and coagulation pathways that lead to rapid removal from blood circulation of the lipoplexes by opsonization [125-127]. pDNA/DODAB/MO lipoplexes were therefore tested regarding their sensitivity when simulating their interaction with the body (temperature, salt, exposure to serum, nucleases and membrane lipases), to be validated for systemic applications .
Fig. 4 shows the variation of free pDNA fraction after incubation of pDNA/DODAB:MO lipoplexes (2:1, 1:1 and 1:2) with different constituents of the plasma. Increasing the temperature from 25C to physiological temperature (37C) leads to a reduced but visible release of pDNA from the lipoplex, more evident for lower MO contents. The gel phase of DODAB (ΧDODAB> 0.5) is clearly more disturbed by incubation at higher temperature than the liquid-crystalline phase of DODAB/MO lipid mixtures (ΧDODAB ≤ 0.5). This tendency is maintained upon NaCl addition at physiological concentration (150mM), showing the protective role of MO upon the electrostatic imbalance provoked by salt addition.
DODAB/MO formulations with varying MO content behave very differently when exposed to serum (Fig. 4). Serum may strongly interfere with lipoplexes, both
Some authors have managed to transiently overcome this inhibitory effect of serum on lipofection by increasing the charge ratio (+/-) of cationic liposome to DNA [130, 131]. Significantly enhanced gene transfer has also been achieved by pre-incubating the delivery system with serum proteins prior to NA complexation [132, 133].
3.2. Lipoplex adhesion to the cell surface
The adsorption and uptake of lipoplexes may be affected by the presence of proteoglycans at the plasma cell membrane surface. It is therefore important to study how lipoplexes interact with these extracellular matrix components during cell transfection. Association of lipoplexes with negative polyelectrolytes free in solution might also be useful to evaluate eventual loss of pDNA at the cell surface .
Proteoglycans (membrane receptors consisting of a protein core and one or more anionic glycosaminoglycan chains including heparin, dermatan and chondroitin sulphates) were identified as the mediating agents for cationic liposome/DNA cellular uptake both
When the lipoplexes interact with heparin and heparin sulphate, the negative charge of the polyelectrolytes determines NA release from the lipoplex through the same type of cooperative process that is responsible for lipoplex formation [136-138].
On subjecting pDNA/DODAB:MO (2:1 and 1:1) lipoplexes to increasing amounts of heparin (HEP), the improved resistance and stability of the lipoplexes obtained with increasing amounts of MO could be confirmed (Fig. 5). The fact that the system with higher MO content (ΧDODAB= 0.5) shows enhanced resistance to heparin relatively to pDNA/DODAB:MO (2:1) lipoplexes suggests that pDNA dissociation is mainly dependent on structural properties (Fig. 2) rather than physicochemical properties of the lipoplexes.
4. Modulation of cell behaviour by lipoplexes
In addition to the efficiency of MO based lipoplexes, patient tolerability is determinant for therapeutic application of these systems.
The cell lines presented here are routinely used for toxicity studies and are commercially available. The human Embryonic Kidney (HEK) 293 cell line was originally derived from human embryonic kidney cells grown in tissue culture, from which 293T cell line is derived. BJ5ta cells are normal human foreskin fibroblasts immortalized with telomerase. Murine cell lines L929 and C2C12 are fibroblasts and myoblasts, respectively.
Another aspect to be taken into account is the possibility that the liposomes and lipoplexes may differently affect parameters such as metabolism, cell membrane structure and chemistry, cell proliferation and mobility. For a comprehensive study, a minimum of three different methodologies, monitoring at least two of these parameters, should be used. From our own results, it was observable that the metabolism of L929 and C2C12 cells was more pronouncedly affected by the contact with DODAB:MO liposomes compared to the other cell lines, especially with a lipid concentration ≥ 20 µg/ml (Fig. 6). At these higher concentrations, DODAB:MO (1:1) induced lower levels of cytotoxicity in all the cell types, which probably reflects the higher content of MO and concomitant lower content of cationic lipid. Interestingly, the cell membrane integrity assay did not reveal such obvious, concentration-dependent variations in cytotoxicity (Fig. 7). The results obtained with the proliferation test (Fig. 8) were quite concordant with those from the metabolic assay (Fig. 6), indicating again the L929 and C2C12 cells as more sensitive, while BJ5ta proliferation was clearly increased when incubated with up to 20 µg/ml lipid (Fig. 8).
Lipoplexes prepared from these liposomal formulations, at concentrations typically used in transfection experiments, constantly leading to slightly lower viability rates compared to the base DODAB:MO liposomes (data not shown).
The fact that MO-based aggregates cause reduced levels of cytotoxicity for concentrations typically used on transfection assays, reinforcing the use of MO as a new
4.2. Cellular uptake and intracellular trafficking
In spite of extensive efforts to unravel the
Different mechanisms for complex internalization have been proposed, in particular for lipoplexes and polyplexes. Endocytosis at the plasma membrane may be clathrin-dependent or -independent. Clathrin-independent mechanisms include fusion of lipoplexes with the plasma membrane, phagocytosis, macropinocytosis and caveolae-mediated uptake .
Intracellular trafficking of lipoplexes can be followed by co-localization studies of labeled particle components and dyes, or antibodies that recognize cell organelles or molecules playing a role in the process (e.g. clathrin coating endocytic pits in the plasma membrane) [139, 145] (Fig. 9). Cell lines harboring mutations in some of these molecules may also be used to evaluate their importance for the internalization process of specific formulations. The use of inhibitors of endocytosis has also been widely used but has two major limitations: the significant toxicity induced by the inhibitors themselves and the evidence corroborating that internalization can be simultaneously mediated by different pathways.
The endosomal escape is thought to be the major limitation for efficient gene transfection . A number of strategies have been explored to enhance NA endosomal release. For example, the incorporation of a non-lamellar forming lipid such as DOPE that disrupts the endosome membrane or inclusion of a pH-dependent molecule that senses the acidification in the endosome compartment leading to disruption of its membrane .
Modulation of the endosomal escape during lipoplex intracellular trafficking was replicated by exposing pDNA/DODAB:MO (2:1, 1:1 and 1:2) lipoplexes to acidic conditions in the presence of increasing amounts of hydrochloric acid (pH ranging from 7.5 to 2.5) (Fig. 10). The percentage of released DNA steadily increased upon milieu acidification from pH 7.4 to 4.5, which is the pH range typical in the endosome. This trend correlates negatively with the MO content in the formulation, suggesting that MO’s inverted bicontinuous cubic structures may protect more efficiently the lipoplex structure in this environment. More stringent acidification of the environment (pH 4.5 to pH 2.5) inverts the release tendency, which can be related to degradation of naked pDNA in solution.
Lipoplex charge ratio (+/-) also affects the intensity of pDNA release. Using the same DODAB:MO base formulation, increasing charge ratio (+/-) seems to prevent pDNA release from the lipoplex. This effect was already visible in the destabilization of pDNA/DODAB/MO lipoplexes by plasma constituents such as serum and salt, and probably reflects more efficient pDNA condensation in presence of excess cationic lipid.
Increasing ammonium/phosphate ratio carries the risk of increased cytotoxicity. One possible solution may be using increasing amounts of MO in lipoplex formulation for better protection of pDNA integrity without imposing major toxic effects to the target cell.
Non-viral vectors, although less toxic than viral vectors, may still elicit a strong, nonspecific immune response. Toxicity frequently results from characteristics of the encapsulating polymer or lipid such as the length, saturation, or branching of the polymer. Efforts to reduce the toxicity of nonviral vectors have largely resulted in attempts to make the vectors more biodegradable and biocompatible. Many of the aforementioned systems (i.e. triggered release with disulfides, PEG copolymers) incorporated more biologically active components, thereby reducing the elicited immune response. For example, the incorporation in liposomes of molecules known to suppress the production of the cytokine tumor necrosis factor (TNF-α), as compared to lipoplex alone, succeeded in maintain its levels low while achieving comparable levels of transgene expression . Another method explored by Tan  significantly reduced toxicity through the sequential injection of liposome and later of DNA, as opposed to using formed lipoplexes. With this approach, cytokine levels (IL-12, TNF-α) were reduced by greater than 80% compared to lipoplex delivery . Thus, significant advances have been made towards decreasing the toxicity of these non-viral vectors. Interestingly, DODAB:MO based liposomes and lipoplexes were found to induce production of low levels of TNF-α by macrophages, comparable or lower than DOTMA/DOPE and DOTMA/cholesterol lipoplexes (data not shown) .
4.3. Transfection efficiency
Transfection efficiency of plasmid DNA can be directly evaluated by detecting the protein encoded by the reporter gene. Examples of reporter genes are: green fluorescent protein (GFP) and similar, detectable by techniques as microscopy or flow cytometry; β-galactosidase, whose activity can be evaluated by a colorimetric assay; luciferase, whose activity can be measured with a luminometer, after a substrate is converted into a luminescent form by luciferase. In Figure 11 is depicted an experiment that allows to identify the effect on transfection efficiency of varying the content of MO in the liposomal formulations, lipid:DNA charge ratio in the lipoplexes and also the quantity of pDNA added to the cells, as pDNA dosage is known to affect transfection efficiency. It can be observed that the incorporation of MO in the liposomes resulted in a transfection efficiency improvement when compared to the cationic lipid DODAB alone. When using 1 μg DNA/well, the transfection levels of pDNA/DODAB:MO systems are of the same order of magnitude as Lipofectamine™ LTX. For a lower MO content (pDNA/DODAB:MO (2:1) formulation), a dose effect response (0.5 μg and 1 μg of pDNA) was observed. For higher MO content (pDNA/DODAB:MO (1:1) formulation), the transfection efficiencies remained constant at both CRs. This result strengthens the role of MO as
The identification of the most important formulation parameters and how they influence macromolecule delivery and bioactivity will give direction towards the development of novel therapeutic solutions. The morphology and structure of the lipoplex is influenced by the surrounding environment and the chemical nature of its constituents. Physicochemical properties of the systems define the course of most events when lipoplex interact with the body, tissues and cells. The effectiveness of vector internalization, its intracellular trafficking and successful transgene expression in target cells, is directly dependent on the
A good lipofection system must protect NA from deleterious interaction with biological fluids and cell components, while remaining biocompatible and efficient as delivery agent. In summary, with this work we intend to demonstrate that MO can be used safely and efficiently as
This work was supported by FCT research project PTDC/QUI/69795/2006, which is co-funded by the program COMPETE from QREN with co-participation from the European Community fund FEDER; CFUM [PEst-C/FIS/UI0607/2011]; CBMA [Pest C/BIA/UI4050/2011]; J.P.N. Silva holds a PhD Grant (SFRH/BD/46968/2008); A. C.N. Oliveira holds a PhD grant (SFRH/BD/68588/2010).
American Association of Pharmaceutical Scientists Journal, Patil S. D. Rhodes D. G. Burgess D. J. -Based D. N. A. Therapeutics Delivery D. N. A. Systems-A Comprehensive. Review 2005 E61 E77
Journal of Physics A: Mathematical and General, Levin Y. Arenzon J. J. Kinetics of. Charge Inversion. 2003 5857 5863
Expert Opinion on Drug Delivery, Pitt W. G. Husseini G. A. Staples B. J. Ultrasonic Drug. Delivery-A General. Review 2004
Ultrasound in Medicine and Biology Miller D. L. Dou C. Induction of. Apoptosis in. Sonoporation Ultrasonic Gene. Transfer 2009 144 154
Automatika, Rebersek M. Miklavcic D. Advantages Disadvantages of. Different Concepts. of Electroporation. Pulse Generation. 2011 12 19
Gehl, J., Electroporation Theory and Methods, Perspectives for Drug Delivery, Gene Therapy and Research.Acta Physiologica Scandinavica, 2003 437 447
André, F.M., et al., Variability of Naked DNA Expression After Direct Local Injection- The Influence of the Injection Speed.Gene Therapy, 2006 1619 1627
Gene Transfer and Therapy. Gene Therapy, Herweijer H. Wolff J. A. Progress Prospects-Naked D. N. A. Gene Transfer. Therapy 2003 453 458
Machida, C.A., Viral Vectors for Gene Therapy- Methods and Protocols.st ed, ed. H. Press. 2003New Jersey (U.S.A). 606.
Heiser, W.C., Gene Delivery to Mammalian Cells- Viral Gene Transfer Techniques.st ed, ed. H. Press. 2004New Jersey (U.S.A.). 584.
Arima, H., Polyfection ad Nonviral Gene Transfer Method- Design of Novel Nonviral Vector using Ciclodextrin.Journal of the Pharmaceutical Society of Japan, 2004 451 464
Eliyahu H. Barenholz Y. Domb A. J. Polymers for. D. N. A. Delivery Molecules. 2005 34 64
Proceedings of the National Academy of Sciences U.S.A., Felgner P. L. et al. Lipofection-A Procedure. Highly Efficient. Lipid-mediated D. N. A-transfection Procedure. 1987 7413 7417
Pampinella, F., et al., Analysis of Differential Lipofection Efficiency in Primary and Established Myoblasts.Molecular Therapy, 2002 161 169
Felgner, P.L., et al., Nomenclature for Synthetic Gene Delivery Systems.Human Gene Therapy, 1997 511 512
Biotechnology Progress, Douglas K. L. Toward Development. of Artificial. Viruses for. Gene Therapy. A. Comparative Evaluation. of Viral. Non-Viral Transfection. 2008 871 883
Stone, D., et al., Viral Vectors for Gene Delivery and Gene Therapy Within the Endocrine System.Journal of Endocrinology, 2000 103 118
Florea, B.I., et al., Transfection Efficiency and Toxicity of Polyethylenimine in Differentiated Calu-3 and Nondifferentiated COS-1 Cell Cultures.American Association of Pharmaceutical Scientists Journal, 2002 1 11
Lee, M., Apoptosis Induced by PolyethylenimineDNA Complex in Polymer Mediated Gene Delivery.Bulletin of the Korean Chemical Society, 2007 95 98
Biochemical and Biophysical Research Communications, Egilmez N. K. Iwanuma Y. Bankert R. B. Evaluation And. Optimization Of. Different Cationic. Liposome Formulations. 1996 169 173
Choi, W., et al., Low Toxicity Of Cationic Lipid-based Emulsion For Gene Transfer.Biomaterials, 2004 5893 5903
Yingyongnarongkul, B., et al., High Transfection Efficiency and Low Toxicity Cationic Lipids with Aminoglycerol-Diamine Conjugate.Bioorganic & Medicinal Chemistry, 2009 176 188
st ed, ed. C. Press. Lasic D. D. Barenholz Y. Nonmedical Applications. of-From Liposomes. Gene Delivery. Diagnostics to. Ecology 1996Boca Raton (USA).
Lasic D. D. Liposomes Science. Medicine 1996 34 43
Gonçalves, E., R.J. Debs, and T.D. Heath, The Effect of Liposome Size on the Final Lipid-DNA Ratio of Cationic Lipoplexes.Biophysical Journal, 2004 1554 1563
Zuidam, N.J., et al., Lamellarity of Cationic Liposomes and Mode of Preparation of Lipoplexes Affect Transfection Efficiency.Biochimica et Biophysica Acta, 1999 207 220
Claessens, M.M.A.E., et al., Charged Lipid Vesicles Effects of Salts on Bending Rigidity, Stability,and Size.Biophysical Journal, 2004 3882 3893
Labas, R., et al., Nature as a Source of Inspiration for Cationic Lipid Synthesis.Genetica, 2010 153 168
Ahmad, A., et al., New Multivalent Cationic Lipids Reveal Bell Curve for Transfection Efficiency Versus Membrane Charge Density Lipid-DNA Complexes for Gene Delivery.Journal of Gene Medicine, 2005 739 748
Zuhorn, I.S., et al., Phase Behavior Of Cationic Amphiphiles And Their Mixtures With Helper Lipids.Biophysical Journal, 2002 2096 2108
st ed, ed. Elsevier. Lasic D. D. Papahadjopoulos D. Medical Applications. of Liposomes. 1998Amsterdam (Netherlands). 795.
Madeira, C., et al., Liposome Complexation Efficiency Monitored by FRET- Effect of Charge Ratio, Helper Lipid and Plasmid Size.European Biophysical Journal, 2007 609 620
Zelphati, O., et al., Stable and Monodisperse Lipoplex Formulations forGene Delivery. Gene Therapy, 1998 1272 1282
Gershon, H., et al., Mode of Formation and Structural Features of DNA-Cationic Liposome Complexes Used for Transfection.Biochemistry, 1993 7143 7151
Akao, T., et al., Conformational Change in DNA Induced by Cationic Bilayer Membranes.Federation of European Biochemical Societies Letters, 1996 215 218
Dan, N., Formation of Ordered Domains in Membrane-Bound DNA.Biophysical Journal, 1996 1267 1272
Cruz, M.T.G., et al., Kinetic Analysis of the Initial Steps Involved in Lipoplex-Cell Interactions.Biochimica et Biophysica Acta, 2001 136 151
Silva, J.P.N., et al., DODAB:Monoolein-based Lipoplexes as Non-viral Vectors for Transfection of Mammalian Cells.Biochimica et Biophysica Acta, 2011 2440 2449
Silva, J.P.N., P.J.G. Coutinho, and M.E.C.D.R. Oliveira, Characterization of Mixed DODAB-Monoolein Aggregates Using Nile Red as a Solvatochromic and Anisotropy Fluorescent Probe.Journal of Photochemistry and Photobiology A: Chemistry, 2009 32 39
Journal of Colloid and Interface Science, Oliveira I. M. S. C. et al. Aggregation Behavior. of Aqueous. Dioctadecyldimethylammonium Bromide. Monoolein Mixtures. A. Multitechnique Investigation. on the. Influence of. Composition Temperature 2011 206 217
Silva, J.P.N., P.J.G. Coutinho, and M.E.C.D.R. Oliveira, Characterization of Monoolein-Based Lipoplexes Using Fluorescence Spectroscopy.Journal of Fluorescence, 2008 555 562
Molecular Pharmaceutics, Kearns M. D. Donkor A. M. Savva M. Structure-Transfection Activity. Studies of. Novel Cationic. Cholesterol-Based Amphiphiles. 2008 128 139
Biophysical Journal, Tarahovsky Y. S. Koynova R. Mac R. C. Donald D. N. A. Release from. Lipoplexes by. Anionic Lipids. Correlation with. Lipid Mesomorphism. Interfacial Curvature. Membrane Fusion. 2004 1054 1064
Koynova, R., et al., Lipoplex Formulation of Superior Efficacy Exhibits High Surface Activity and Fusogenicity, and Readily Releases DNA.Biochimica et Biophysica Acta, 2007 375 386
PT104158, I.N.d.P. Industrial, Editor. Real-Oliveira M. E. C. D. et al. Aplicação da. Monooleína como. Novo Lípido. Adjuvante em. Lipofecção in. Portuguese Patent. n. P. 2011 1 27
WO2010/020935 A2, W.I.P. Organization, Editor. Real-Oliveira M. E. C. D. et al. Use of. as Monoolein a. New Auxiliary. Lipid in. Lipofection in. International Patent. n. W. 2010 1 27
Tsuruta, L.R., M.M. Lessa, and A.M. Carmona-Ribeiro, Interactions Between Dioctadecyldimethylammonium Chloride or Bromide Bilayers in Water.Langmuir, 1995 2938 2943
Nascimento, D.B., et al., Counterion Effects on Properties of Cationic Vesicles.Langmuir, 1998 7387 7391
Okuyama, K., et al., Molecular and Crystal Structure of the Lipid-Model Amphiphile Dioctadecyldimethylammonium Bromide Monohydrate.Bulletin of the Chemical Society of Japan, 1988 1485 1490
Langmuir, Feitosa E. Brown W. Fragment Vesicle Structures. in Sonicated. Dispersions of. Dioctadecyldimethylammonium Bromide. 1997 4810 4816
Schulz, P.C., et al., Phase Behaviour of the Dioctadecylammonium Bromide-Water System.Journal of Thermal Analysis, 1998 49 62
Chemistry and Physics of Lipids, Feitosa E. Barreleiro P. C. A. Olofsson G. Phase Transition. in Dioctadecyldimethylammonium. Bromide Chloride Vesicles. Prepared by. Different Methods. 2000 201 213
Benatti, C.R., et al., Structural and Thermal Characterization of Dioctadecyldimethylammonium Bromide Dispersions by Spin Labels.Chemistry and Physics of Lipids, 2001 93 104
Proverbio, Z.E., P.C. Schulz, and J.E. Puig, Aggregation of the Aqueous Dodecyltrimethylammonium Bromide- Didodecyldimethylammonium Bromide System at Low Concentration.Colloid and Polymer Science, 2002 1045 1052
Feitosa, E., et al., Cationic Liposomes in Mixed Didodecyldimethylammonium Bromide and Dioctadecyldimethylammonium Bromide Aqueous Dispersions Studied by Differential Scanning Calorimetry, Nile Red Fluorescence and Turbidity.Langmuir, 2006 3579 3585
Chemistry and Physics of Lipids, Feitosa E. Karlsson G. Edwards K. Unilamellar Vesicles. Obtained by. Simply Mixing. Dioctadecyldimethylammonium Chloride. Bromide with. Water 2006 66 74
Journal of Physical Chemistry B, Rodriguez-Pulido A. et al. Theoretical A. Experimental Approach. to the. Compaction Process. of D. N. A. by Dioctadecyldimethylammonium. Bromide-Zwitterionic Mixed. Liposomes 2009 15648 15661
Mel’nikov, Y.S., S.M. Mel’nikova, and J.E. Lofroth, Physico-chemical Aspects of the Interaction between DNA and Oppositely Charged Mixed Liposomes.Biophysical Chemistry, 1999 125 141
Hungerford, G., et al., Domain Formation in DODAB-Cholesterol Mixed Systems Monitored Via Nile Red Anisotropy.Journal of Fluorescence, 2005 835 840
Hungerford, G., et al., Interaction of DODAB with Neutral Phospholipids and Cholesterol Studied Using Fluorescence Anisotropy.Journal of Photochemistry and Photobiology A: Chemistry, 2006 99 105
Journal de Physique II, Briggs J. Chung H. Caffrey M. The-Composition Temperature. Phase Diagram. Mesophase Structure. Characterization of. the-Water Monoolein. System 1996 723 751
Czeslik, C., et al., Temperature- and Pressure-Dependent Phase Behavior of Monoacylglycerides Monoolein and Monoelaidin.Biophysical Journal, 1995 1423 1429
Vacklin, H., et al., The Bending Elasticity of 1-Monoolein upon Relief of Packing Stress.Langmuir, 2000 4741 4748
Current Opinion in Structural Biology, Caffrey M. Lipid’s A. Eye View. of Membrane. Protein Crystallization. in Mesophases. 2000 486 497
Journal of Structural Biology, Caffrey M. Membrane Protein. Crystallization 2003 108 132
Carlsson, N., et al., Bicontinuous Cubic Phase of Monoolein and Water as Medium for Electrophoresis of Both Membrane-bound Probes and DNA.Langmuir, 2006 4408 4414
st ed, ed. J.W.S. Incorporated. Dias R. Lindman B. Interactions D. N. A. with Polymers. Surfactants 2008Hoboken (USA). 425.
Bielke W. Erbacher C. Nucleic Acid. Transfection 1st. ed ed. S. Verlag 2010Berlin (Germany). 316.
Proceedings of the National Academy of Sciences U.S.A., Hofland H. E. J. Shepard L. Sullivan S. M. Formation of. Stable Cationic. Lipid D. N. A. Complexes 1996 7305 7309
Oberle, V., et al., Lipoplex Formation under Equilibrium Conditions Reveals a Three-Step Mechanism.Biophysical Journal, 2000 1447 1454
Faraday Discussions, Barreleiro P. C. A. May R. P. Lindman B. Mechanism of. Formation-Cationic of. D. N. A. Vesicle Complexes. 2002 191 201
Masotti, A., et al., Comparison of Different Commercially Available Cationic Liposome-DNA Lipoplexes Parameters Influencing Toxicity and Transfection Efficiency.Colloids and Surfaces B: Biointerfaces, 2009 136 144
Hui, S.W., et al., The Role of Helper Lipids In Cationic Liposome-Mediated Gene Transfer.Biophysical Journal, 1996 590 599
Biochimica et Biophysica Acta, Farhood H. Serbina N. Huang L. The Role. of Dioleoyl. Phosphatidylethanolamine in. Cationic Liposome. Mediated Transfer. 1995 289 295
Biochimica et Biophysica Acta, Hirsch-Lerner D. et al. Effect of. Helper ‘‘. Lipid’’ on. Lipoplex Electrostatics. 2005 71 84
Ciani, L., et al., DOTAP-DOPE and DC-Chol-DOPE Lipoplexes for Gene Delivery Zeta Potential Measurements and Electron Spin Resonance Spectra.Biochimica et Biophysica Acta, 2004 70 79
Esposito, C., et al., The Analysis of Serum Effects on Structure, Size and Toxicity of DDAB-DOPE and DC-Chol-DOPE Lipoplexes Contributes to Explain their Different Transfection Efficiency.Colloids and Surfaces B: Biointerfaces, 2006 187 192
Maitani, Y., et al., Cationic Liposome (DC-Chol-DOPE 1-2) and a Modified Ethanol Injection Method to Prepare Liposomes, Increased Gene Expression.International Journal of Pharmaceutics, 2007 33 39
Journal of Physical Chemistry B, Rodriguez-Pulido A. et al. Physicochemical A. Characterization of. the Interaction. between-Chol D. C. Cationic D. O. P. E. Liposomes D. N. A. 2008 12555 12565
Smisterová, J., et al., Molecular Shape of the Cationic Lipid Controls the Structure of Cationic Lipid-DOPE-DNA Complexes and the Efficiency of Gene Delivery.Journal of Biological Chemistry, 2001 47615 47622
Yuan X. B. Non-Viral Gene. Therapy 1st. ed ed. Intech 2011Rijeka (Croatia). 707.
Pharmaceutical Research, Kiefer K. Clement J. Garidel P. Transfection Efficiency. Cytotoxicity of. Non-viral Gene. Transfer Reagents. in Human. Smooth Muscle. Endothelial Cells. 2004 1009 1017
Tenchov, B.G., R.C. MacDonald, and D.P. Siegel, Cubic Phases in Phosphatidylcholine-Cholesterol Mixtures- Cholesterol as Membrane Fusogen.Biophysical Journal, 2006 2508 2516
st Edition). 1st ed, ed. E.A. Press. Huang L. Hung M. Wagner E. Nonviral Vectors. for Gene. Therapy-Part I. (1st Edition. 1999California (U.S.A.). 442.
nd Edition). 2nd ed, ed. E.A. Press. Huang L. Hung M. Wagner E. Nonviral Vectors. for Gene. Therapy-Part I. (2nd Edition. 2005California (U.S.A.). 377.
Molecular Pharmaceutics, Koynova R. Wang L. Mac R. C. Donald Cationic. Phospholipids Forming. Cubic-Lipoplex Phases. Structure Transfection Efficiency. 2008 739 744
Proceedings of the National Academy of Sciences U.S.A., Koynova R. Wang L. Mac R. C. Donald An. Intracellular-Nonlamellar Lamellar. Phase Transition. Rationalizes the. Superior Performance. of Some. Cationic Lipid. Transfection Agents. 2006 14373 14378
Biophysical Journal, Koynova R. Mac R. C. Donald Mixtures. of Cationic. Lipid-Ethylphosphatidylcholine O. with Membrane. Lipids D. N. A. 2003 2449 2465
Koynova, R.,Lipid Phases Eye View to Lipofection- Cationic Phosphatidylcholine Derivatives as Efficient DNA Carriers for Gene Delivery. Lipid Insights, 2008 41 59
Biochimica et Biophysica Acta, Tenchov B. G. et al. Modulation of. a. Membrane Lipid. Lamellar-Nonlamellar Phase. Transition by. Cationic-A Lipids. Measure for. Transfection Efficiency. 2008 2405 2412
st ed, ed. S. Verlag. Taira K. Kataoka K. Niidome T. Non-viral Gene. Therapy-Gene Design. Delivery 2005Tokyo (Japan). 490.
Wickstrom, E., Clinical Trials of Genetic Therapy with Antisense DNA and DNA Vectors.st ed, ed. M.D. Incorporated. 1998New York (U.S.A). 421.
Templeton, N.S., Gene and Cell Therapy: Therapeutic Mechanisms and Strategies.nd ed, ed. M.D. Incorporated. 2005New York (U.S.A.). 896.
st ed, ed. S. Verlag. Villiers M. M. Aramwit P. Kwon G. S. Nanotechnology in. Drug Delivery. 2009Berlin (Germany). 663.
Lamprecht, A., Nanotherapeutics- Drug Delivery Concepts in Nanoscience.st ed, ed. P. Stanford. 2009Danvers (USA). 293.
Rose, J.K., Liposomal Transfection of Nucleic Acids into Animal Cells, in U.S.Patent n. 005279833, U.S.I.P. Organization, Editor. 1994 1 13
Patent n. 005651981, U.S.I.P. Organization, Editor. Ashley G. W. Mac R. C. Donald Shida M. Cationic Phospholipids. for Transfection. in U. S. 1997 1 14
W.I.P. Organization, Editor. Niedzinski E. J. Bennet M. Multi-Functional Polyamines. for Delivery. of-Active Biologically. Polynucleotides in. International Patent. n. 2003 1 37
W.I.P. Organization, Editor. Barenholz Y. Simberg D. Sphingolipids Polyakylamine. Conjugates for. Use in. Transfection in. International Patent. n. 2004 1 49
Reszka, R., Cholesterol Derivative for Liposomal Gene Transfer, in U.S.Patent n. 005888821, U.S.I.P. Organization, Editor. 1999 1 4
Mori H. Nishikawa N. Glycerol Derivatives. in U. S. Patent n. 00522179 Organization U. S. I. P. Editor 1993 1 14
Chemical Communications, Camilleri P. et al. Novel A. Class of. Cationic Gemini. Surfactants Showing. Efficient In. Vitro Gene. Transfection Properties. 2000 1253 1254
Kirby, A.J., et al., Gemini Surfactants: New Synthetic Vectors for Gene Transfection.Angewandte Chemie International Edition, 2003 1448 1457
Sekhon B. S. Gemini . Dimeric Surfactants. Resonance 2004 42 49
Current Gene Therapy, Wettig S. D. Verrall R. E. Foldvari M. Gemini-A Surfactants. New Family. of Building. Blocks for. Non-Viral Gene. Delivery Systems. 2008 9 23
Bombelli, C., et al., Gemini Surfactant Based Carriers in Gene and Drug Delivery.Current Medicinal Chemistry, 2009 171 183
Palmer, L.R., et al., Transfection Properties of Stabilized Plasmid-Lipid Particles Containg Cationic PEG Lipids.Biochimica et Biophysica Acta, 2003 204 216
Buyens, K., et al., Elucidating the Encapsulation of Short Interfering RNA in PEGylated Cationic Liposomes.Langmuir, 2009 4886 4891
Kim, J.Y., et al., The Use of PEGylated Liposomes to Prolong Circulation Lifetimes of Tissue Plasminogen Activator.Biomaterials, 2009 5751 5756
Harvie, P., F.M.P. Wong, and M.B. Bally, Use of Poly(ethylene glycol)-Lipid Conjugates to Regulate the Surface Attributes and Transfection Activity of Lipid-DNA Particles.Journal of Pharmaceutical Sciences, 2000 652 663
Veronese, F.M., PEGylated Protein Drugs: Basic Science and Clinical Applications (Milestones in Drug Therapy).st ed, ed. B. Basel. 2009Basel (Switzerland). 297.
Sakaguchi, N., et al., The Correlation Between Fusion Capability and Transfection Activity in Hybrid Complexes of Lipoplexes and pH-Sensitive Liposomes.Biomaterials, 2008 4029 4036
Wasungu, L., et al., Transfection Mediated by pH-Sensitive Sugar-Based Gemini Surfactants; Potential for In Vivo Gene Therapy Applications.Journal of Molecular Medicine, 2006 774 784
Patent n. 006749863, U.S.I.P. Organization, Editor. Chang E. H. Xu L. Pirollo K. Targeted Liposome. Gene Delivery. in U. S. 2004 1 25
Patent n. 005830878, U.S.I.P. Organization, Editor. Gorman C. M. Mc Clarrinon M. Cationic-D Lipid. Complexes N. A. for Gene. Targeting in. U. S. 1998 1 24
Current Drug Delivery, Hattori Y. Maitani Y. Folate-Linked-Based Lipid. Nanoparticle for. Targeted Gene. Delivery 2005 243 252
Petrak, K., Essential Properties ofDrug-Targeting Delivery Systems. Drug Discovery Today, 2005 1667 1673
Pharmaceutical Research, Russ V. Wagner E. Cell Tissue Targeting. of Nucleic. Acids for. Cancer Gene. Therapy 2007 1047 1057
Yang, P., et al., Enhanced Gene Expression in Epithelial Cells Transfected With Aminoacid-Substituted Gemini Nanoparticles.European Journal of Pharmaceutics and Biopharmaceutics, 2010 311 320
Molecular Membrane Biology, Rosa M. et al. Pre-Condensation D. N. A. with an. Amino Acid-based. Cationic-A Amphiphile. Viable Approach. for-Based Liposome. Gene Delivery. 2008 23 34
Letrou-Bonneval, E., et al., Galactosylated Multimodular Lipoplexes for Specific Gene Transfer into Primary Hepatocytes.Journal of Gene Medicine, 2008 1198 1210
Higuchi, Y., et al., Effect of the Particle Size of Galactosylated Lipoplex on Hepatocyte-Selective Gene Transfection after Intraportal Administration.Biological and Pharmaceutical Bulletin, 2006 1521 1523
Molecular Membrane Biology, Audouy S. Hoekstra D. Cationic-Mediated Lipid. Transfection In. Vitro In Vivo. . Review 2001 129 143
Bihan, O.L., et al., Probing the In Vitro Mechanism of Action of Cationic Lipid-DNA Lipoplexes at a Nanometric Scale.Nucleic Acids Research, 2010 1 15
rd ed, ed. I. Healthcare. Gregoriadis G. Liposome-I-Liposome Technology. Preparation Related Techniques. 2007New York (USA). 660.
Gregoriadis, G., Liposome Technology- II- Entrapment of Drugs and Other Materials Into Liposomes.rd ed, ed. I. Healthcare. 2007New York (USA). 424.
Gregoriadis, G., Liposome Technology- III- Interactions of Liposomes with the Biological Milieu.rd ed, ed. I. Healthcare. 2007New York (USA). 464.
EP2335687 A2, E.P. Office, Editor. Real-Oliveira M. E. C. D. et al. Use of. as Monoolein a. New Auxiliary. Lipid in. Lipofection in. European Patent. n. E. 2011 1 27
Zuhorn, I.S., et al., Interference of Serum with Lipoplex-cell Interaction.Biochimica et Biophysica Acta, 2002 25 36
Gene Therapy, Yang J. P. Huang L. Overcoming the. Inhibitory Effect. of Serum. on Lipofection. by Increasing. the Charge. Ratio of. Cationic Liposome. to D. N. A. 1997 950 960
Biochimica et Biophysica Acta, Zhang Y. Anchordoquy T. J. The Role. of Lipid. Charge Density. in the. Serum Stability. of Cationic. Lipid-D N. A. Complexes 2004 143 157
Molecular Therapy, Conwell C. C. Liu F. Huang L. Gene Transfer. Activity is. Enhanced Significantly. by Allowing. Cationic Polymer. to Interact. With Serum. Proteins Prior. to D. N. A. Addition 2005p. S80
Arpke, R.W. and P.W. Cheng, Characterization of Human Serum Albumin-Facilitated Lipofection Gene Delivery Strategy.Journal of Cell Science & Therapy, 2011 108 114
Mounkes, L.C., et al., Proteoglycans Mediate Cationic Liposome-DNA Complex-Based Gene Delivery In Vitro and In Vivo.Journal of Biological Chemistry, 1998 26164 26170
Wiethoff, C.M., et al., The Potential Role of Proteoglycans in Cationic Lipid-mediated Gene Delivery.Journal of Biological Chemistry, 2001 32806 32813
Silva, M.E. and C.P. Dietrich, Structure of Heparin.Journal of Biological Chemistry, 1975 6841 6846
Proceedings of the National Academy of Sciences U.S.A, Rosenberg R. D. Armand G. Lam L. Structure-Function Relationships. of Heparin. Species 1978 3065 3069
Mascotti, D.P. and T.M. Lohman, Thermodynamics of Charged Oligopeptide-Heparin Interactions.Biochemistry, 1995 2908 2915
Journal of Biological Chemistry, Zuhorn I. S. Kalicharan R. Hoekstra D. Lipoplex-Mediated Transfection. of Mammalian. Cells Occurs. Through the. Cholesterol-Dependent-Mediated Clathrin. Pathway of. Endocytosis 2002 18021 18028
Habib, N.A., Cancer Gene Therapy- Past Achievements and Future Challenges.st ed, ed. K.A. Publishers. 2002New York (U.S.A.). 458.
European Journal of Pharmaceutical Sciences, Ilarduya C. T. Sun Y. Düzgünes N. Gene Delivery. by Lipoplexes. Polyplexes 2010 159 170
Public Library of Science ONE, Resina S. Prevot P. Thierry A. R. Physico-Chemical Characteristics. of Lipoplexes. Influence Cell. Uptake Mechanisms. Transfection Efficacy. 2009 e6058
Ferrari, M.E., et al., Trends in Lipoplex Physical Properties Dependent on Cationic Lipid Structure, Vehicle and Complexation Procedure do ot Correlate with Biological Activity.Nucleic Acids Research, 2001 1539 1548
Hoekstra, D., et al., Gene Delivery by Cationic Lipids In and Out of an Endosome.Biochemical Society Transactions, 2007 68 71
Molecular Therapy, Elouahabi A. Ruysschaert J. Formation Intracellular Trafficking. of Lipoplexes. Polyplexes 2005 336 347
Bhushan B. Handbook of. Nanotechnology 2nd. ed ed. S. Verlag 2010Berlin (Germany). 1968.
Caracciolo, G., et al., Efficient Escape from Endosomes Determines the Superior Efficiency of Multicomponent Lipoplexes.Journal of Physical Chemistry B, 2009 4995 4997
Liu, F., et al., Effect of Non-Ionic Surfactants on the Formation of DNA-Emulsion Complexes and Emulsion-Mediated Gene Transfer.Pharmaceutical Research, 1996 1642 1646
Tan, Y., et al., Sequential Injection of Cationic Liposome and Pasmid DNA Effectively Transfects the Lung with Minimal Inflammatory Toxicity.Molecular Therapy, 2001 673 682
Yasuda, S., et al., Comparison of the Type of Liposome Involving Cytokine Production Induced by Non-CpG Lipoplex in Macrophages.Molecular Pharmaceutics, 2010 533 542