Alkyl chain moieties of polyamidoamine dendron-bearing lipids influence their function as a nonviral gene vector

We recently developed a novel family of cationic lipids consisting of a polyamidoamine (PAMAM) dendron and two dodecyl chains. Their transfection activity increases with increasing generation of the dendron moiety [Takahashi et al. (2003) Bioconjugate Chem. 14, 764-773]. In the present study, to elucidate the effect of hydrophobic tail moieties of the dendron-bearing lipids, two kinds of PAMAM G3 dendron-bearing lipids were synthesized with different alkyl lengths, DL-G3-2C18 and DL-G3-2C12. Their functions as gene vectors were compared. Irrespective of their different alkyl chain lengths, these dendron-bearing lipids formed complexes with plasmid DNA with similar efficiency. However, their complex sizes differed markedly: DL-G3-2C18 lipoplexes exhibited much smaller diameters than DL-G3-2C12 lipoplexes. Interaction of the lipoplexes with heparin revealed that the DL-G3-2C18 lipoplexes required more heparin than DL-G3-2C12 lipoplexes to cause dissociation of plasmid DNA from the lipoplexes. Although the DL-G3-2C12 lipoplexes and DL-G3-2C18 lipoplexes transfected CV1 cells with similar efficiency in the absence of serum, only the latter retained high transfection activity in the presence of serum. These results indicate that hydrophobic interaction of alkyl chain moieties plays an important role in the increment of stability and the serum-resistant transfection activity for dendron-bearing lipid lipoplexes.     

Synthesis of novel cationic lipids having polyamidoamine dendrons and their transfection activity

We designed a novel type of cationic lipid, lipids with a cationic polar group in the polyamidoamine dendron, because these dendron-bearing lipids are expected to form complexes with plasmid DNA and achieve efficient transfection of cells by synergy of endosome buffering and membrane fusion with the endosome, both of which are useful for the promotion of the transfer of plasmid DNA from endosome to cytosol. Four kinds of lipids with polyamidoamine dendrons of first to fourth generations, DL-G1, DL-G2, DL-G3, and DL-G4, were synthesized. The lipid with a dendron of a higher generation exhibited greater ability to form lipoplexes with plasmid DNA, as estimated by agarose gel electrophoresis. While the DL-G1 lipoplex did not transfect CV1 cells, the lipoplexes containing the DL-G2, DL-G3, or DL-G4 could induce transfection of the cells, and their activity was elevated with increasing generation of the dendron. Addition of dioleoylphosphatidylethanolamine (DOPE), which is known to increase fusion ability of a lipid membrane, into the lipoplexes greatly enhanced their transfection activity. In addition, the comparison with DC-Chol-containing lipoplex, which is widely used as a nonviral vector, showed that the DL-G3-DOPE lipoplex exhibits more efficient transfections. These findings imply that these dendron-bearing lipids may form the basis for a novel family of cationic lipids for efficient gene delivery.     

Synthesis of poly(amidoamine) dendron-bearing lipids with poly(ethylene glycol) grafts and their use for stabilization of nonviral gene vectors

Recently, we developed a new type of cationic lipid that consists of an amine-terminated poly(amidoamine) dendron and two long alkyl groups. These dendron-bearing lipids achieved efficient gene transfection of cells through synergetic action of the proton sponge effect and membrane fusion in combination with fusogenic lipid dioleoylphosphatidylethanolamine. Using those dendron-bearing lipids as a base material, we developed in this study a functional component of gene vectors that stabilizes lipoplexes by multiple PEG chains and promotes gene transfection through the proton sponge effect. We combined a poly(ethylene glycol) (PEG, 550 Da) graft to each of four chain ends of the G2 dendron-bearing lipid (P4-DL). An analogous molecule having single PEG graft was also synthesized using the G0 dendron-bearing lipid (P1-DL). Inclusion of P4-DL decreased the size of the G3 dendron-bearing lipid-based lipoplexes more efficiently than P1-DL. In addition, P4-DL-containing lipoplexes exhibited two-orders-higher transfection efficiency than P1-DL-containing lipoplexes with the same PEG graft density. These results indicate the superiority of multiple attachments of PEG graft chains to a lipid for heightened ability to increase colloidal stability of lipoplexes while retaining their transfection activity. The lipoplexes stabilized by P4-DL were small, around 250 nm, and achieved efficient transfection in the presence of serum. Therefore, P4-DL and its analogues will form the basis for production of efficient nonviral vectors for in vivo use.     

Preparation of efficient gene carriers using a polyamidoamine dendron-bearing lipid: improvement of serum resistance

In a previous study, we developed a novel cationic lipid consisting of polyamidoamine dendron of third generation and two dodecyl chains, designated as DL-G3, which in combination with a fusogenic lipid dioleoylphosphatidylethanolamine (DOPE) achieves efficient transfection of CV1 cells by synergetic action of the proton sponge effect and membrane fusion. This study examines the effect of serum on the transfection activity of the DL-G3-DOPE-plasmid DNA lipoplexes. The transfection activity of a lipoplex with a composition optimized in the absence of serum decreased markedly in the presence of serum. However, the lipoplexes that induce efficient transfection in the presence of serum were obtainable by controlling the charge ratio of the primary amine of the DL-G3 to the phosphate group (N/P ratio) and DOPE content. The complex, which exhibited the highest transfection activity in the presence of serum, has a lower N/P ratio and higher DOPE content than that optimized in the absence of serum. Whereas disintegration of these complexes was induced by addition of heparin, which is a polysaccharide with negatively charged groups, the complex that retained transfection activity in the presence of serum required more negative charges of heparin for complex disintegration. That result implies its higher stability against negatively charged serum proteins. Comparison of the serum-resistant complex with some commercially available transfection reagents, such as Lipofectamine and SuperFect, indicates that the DL-G3 complex achieved more efficient transfection of these cells in the presence of serum.     

trans-2-Aminocyclohexanol-based amphiphiles as highly efficient helper lipids for gene delivery by lipoplexes

Lipidic amphiphiles equipped with the trans-2-aminocyclohexanol (TACH) moiety are promising pH-sensitive conformational switches (“flipids”) that can trigger a lipid bilayer perturbation in response to increased acidity. Because pH-sensitivity was shown to improve the efficiency of several gene delivery systems, we expected that such flipids could significantly enhance the gene transfection by lipoplexes. Thus a series of novel lipids with various TACH-based head groups and hydrocarbon tails were designed, prepared and incorporated into lipoplexes that contain the cationic lipid 1,2-dioleoyl-3-trimethylammonio-propane (DOTAP) and plasmid DNA encoding a luciferase gene. B16F1 and HeLa cells were transfected with such lipoplexes in both serum-free and serum-containing media. The lipoplexes consisting of TACH-lipids exhibited up to two orders of magnitude better transfection efficiency and yet similar toxicity compared to the ones with the conventional helper lipids 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or cholesterol. Thus, the TACH-lipids can be used as novel helper lipids for efficient gene transfection with low cytotoxicity.     

Phase behavior, DNA ordering, and size instability of cationic lipoplexes. Relevance to optimal transfection activity

Mechanisms of cationic lipid-based nucleic acid delivery are receiving increasing attention, but despite this the factors that determine high or low activity of lipoplexes are poorly understood. This study is focused on the fine structure of cationic lipid-DNA complexes (lipoplexes) and its relevance to transfection efficiency. Monocationic (N-(1-(2,3-dioleoyloxy)propyl),N,N,N-trimethylammonium chloride, N-(1-(2,3-dimyristyloxypropyl)-N,N-dimethyl-(2-hydroxyethyl)ammonium bromide) and polycationic (2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanammonium trifluoroacetate) lipid-based assemblies, with or without neutral lipid (1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine, 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine, cholesterol) were used to prepare lipoplexes of different L(+)/DNA(-) charge ratios. Circular dichroism, cryogenic-transmission electron microscopy, and static light scattering were used for lipoplex characterization, whereas expression of human growth hormone or green fluorescent protein was used to quantify transfection efficiency. All monocationic lipids in the presence of inverted hexagonal phase-promoting helper lipids (1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine, cholesterol) induced appearance of Psi(-) DNA, a chiral tertiary DNA structure. The formation of Psi(-) DNA was also dependent on cationic lipid-DNA charge ratio. On the other hand, monocationic lipids either alone or with 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine as helper lipid, or polycationic 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanammonium trifluoroacetate-based assemblies, neither of which promotes a lipid-DNA hexagonal phase, did not induce the formation of Psi(-) DNA. Parallel transfection studies reveal that the size and phase instability of the lipoplexes, and not the formation of Psi(-) DNA structure, correlate with optimal transfection.     

Lamellarity of cationic liposomes and mode of preparation of lipoplexes affect transfection efficiency.

Transfection of NIH-3T3 cells by a human growth hormone expression vector complexed with liposomes composed of N-(1-(2, 3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP) with or without helper lipids was studied. The transfection efficiency was dependent on the lamellarity of the liposomes used to prepare the lipoplexes. Multilamellar vesicles (MLV) were more effective than large unilamellar vesicles (LUV) of approximately 100 nm, irrespective of lipid composition. The optimal DNA/DOTAP mole ratio for transfection was 相似文献   

Novel N,N '-diacyl-1,3-diaminopropyl-2-carbamoyl bivalent cationic lipids for gene delivery--synthesis, in vitro transfection activity, and physicochemical characterization

Novel N,N'-diacyl-1,3-diaminopropyl-2-carbamoyl bivalent cationic lipids were synthesized and their physicochemical properties in lamellar assemblies with and without plasmid DNA were evaluated to elucidate the structural requirements of these double-chained pH-sensitive surfactants for potent non-viral gene delivery and expression. The highest in vitro transfection efficacies were induced at +/-4:1 by the dimyristoyl, dipalmitoyl and dioleoyl derivatives 1,3lb2, 1,3lb3 and 1,3lb5, respectively, without inclusion of helper lipids. Transfection activities were reduced in the presence of either 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine alone or in combination with cholesterol for all derivatives except 1,3lb5, which maintained reporter gene expression levels at +/-4:1 and yielded increased lipofection activity at a lower charge ratio of +/-2:1. Ethidium bromide displacement indicated efficient plasmid DNA binding and compaction by the transfection-competent analogs. Dynamic light-scattering and electrophoretic mobility studies revealed lipoplexes of the active lipids with large particle sizes (mean diameter>or=500 nm) and zeta potentials with positive values (low ionic strength) or below neutrality (high ionic strength). Langmuir film balance studies showed high in-plane elasticity of these derivatives in isolation. In agreement with the monolayer experiments, fluorescence polarization studies verified the fluid nature of the highly transfection-efficient amphiphiles, with gel-to-liquid crystalline phase transitions below physiological temperature. The active compounds also interacted with endosome-mimicking vesicles to a greater extent than the poorly active derivative 1,3lb4, as revealed by fluorescence resonance energy transfer experiments. Taken together, the results suggest that well-hydrated and highly elastic cationic lipids with increased acyl chain fluidity and minimal cytotoxicity elicit high transfection activity.     

Biophysical characterization of anionic lipoplexes

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

In vitro cationic lipid-mediated gene delivery with fluorinated glycerophosphoethanolamine helper lipids.

There is a need for the development of nonviral gene transfer systems with improved and original properties. "Fluorinated" lipoplexes are such candidates, as supported by the remarkably higher in vitro and in vivo transfection potency found for such fluorinated lipoplexes as compared with conventional ones or even with PEI-based polyplexes (Boussif, O., Gaucheron, J., Boulanger, C., Santaella, C., Kolbe, H. V. J., Vierling, P. (2001) Enhanced in vitro and in vivo cationic lipid-mediated gene delivery with a fluorinated glycerophosphoethanolamine helper lipid. J. Gene Med. 3, 109-114). Here, we describe the synthesis of fluorinated glycerophosphoethanolamines (F-PEs), close analogues of dioleoylphosphatidylethanolamine (DOPE), and report on their lipid helper properties vs that of DOPE, as in vitro gene transfer components of fluorinated lipoplexes based on pcTG90, DOGS (Transfectam), or DOTAP. To evaluate the contribution of the F-PEs to in vitro lipoplex-mediated gene transfer, we examined the effect of including the F-PEs in lipoplexes formulated with these cationic lipids (CL) for various CL:DOPE:F-PE molar ratios [1:(1 - x):x with x = 0, 0.5 and 1; 1:(2 - y):y with y = 0, 1, 1.5, and 2], and various N/P ratios (from 10 to 0.8, N = number of CL amines, P = number of DNA phosphates). Irrespective of the F-PE chemical structure, of the colipid F-PE:DOPE composition, and of the N/P ratio, comparable transfection levels to those of their respective control DOPE lipoplexes were most frequently obtained when using one of the F-PEs as colipid of DOGS, pcTG90, or DOTAP in place of part of or of all DOPE. However, a large proportion of DOGS-based lipoplexes were found to display a higher transfection efficiency when formulated with the F-PEs rather than with DOPE alone while the opposite tendency was evidenced for the DOTAP-based lipoplexes. The present work indicates that "fluorinated" lipoplexes formulated with fluorinated helper lipids and conventional cationic lipids are very attractive candidates for gene delivery. It confirms further that lipophobicity and restricted miscibility of the lipoplex lipids with the endogenous lipids does not preclude efficient gene transfer and expression. Their transfection potency is rather attributable to their unique lipophobic and hydrophobic character (resulting from the formulation of DNA with fluorinated lipids), thus preventing to some extent DNA from interactions with lipophilic and hydrophilic biocompounds, and from degradation.     

Synthesis, characterization, and in vitro transfection activity of charge-reversal amphiphiles for DNA delivery

A series of charge-reversal lipids were synthesized that possess varying chain lengths and end functionalities. These lipids were designed to bind and then release DNA based on a change in electrostatic interaction with DNA. Specifically, a cleavable ester linkage is located at the ends of the hydrocarbon chains. The DNA release from the amphiphile was tuned by altering the length and position of the ester linkage in the hydrophobic chains of the lipids through the preparation of five new amphiphiles. The amphiphiles and corresponding lipoplexes were characterized by DSC, TEM, and X-ray, as well as evaluated for DNA binding and DNA transfection. For one specific charge-reversal lipid, stable lipoplexes of approximately 550 nm were formed, and with this amphiphile, effective in vitro DNA transfection activities was observed.     

Transfection with fluorinated lipoplexes based on new fluorinated cationic lipids and in the presence of a bile salt surfactant

The synthesis of two fluorinated cationic lipids, which are analogues of frequently used synthetic gene carrier agents (including the cationic 2,3-dioleoyloxy-N-[2-(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminium (DOSPA) component of the commercially available liposomal Lipofectamine), and the disintegration and DNA accessibility (evaluated by the ethidium bromide (BET) intercalation assay) as well as the in vitro transfection efficacy of cationic lipoplexes formulated with these new lipids in conjunction with conventional or fluorinated helper lipids, in the absence or presence of sodium taurocholate (STC), a powerful anionic bile salt detergent, is reported. A higher stability, with respect to the STC lytic activity and DNA accessibility, of the fluorinated cationic lipoplexes as compared with their respective lipofectamine-based ones was demonstrated. Indeed, while the Lipofectamine lipoplexes were fully disintegrated at a [STC]/[lipid] molar ratio of 2000, only 40-60% of the DNA intercalation sites of the lipoplexes based on the fluorinated analogue of DOSPA were accessible to ethidium bromide. A higher transfection potential in the presence of STC was further found for the lipoplexes formulated with the fluorinated analogue of DOSPA as compared with the Lipofectamine preparation. For a STC concentration of 7.5 mM, lipofection mediated with these fluorinated lipoplexes was significantly higher (nearly 30- to 50-fold, p < 0.05) than with the Lipofectamine ones. These results confirm the remarkable transfection potential of fluorinated lipoplexes.     

Phase behavior of cationic amphiphiles and their mixtures with helper lipid influences lipoplex shape,DNA translocation,and transfection efficiency

Cationic lipids are widely used for gene transfection, but their mechanism of action is still poorly understood. To improve this knowledge, a structure-function study was carried out with two pyridinium-based lipid analogs with identical headgroups but differing in alkyl chain (un)saturation, i.e., SAINT-2 (diC18:1) and SAINT-5 (diC18:0). Although both amphiphiles display transfection activity per se, DOPE strongly promotes SAINT-2-mediated transfection, but not that of SAINT-5, despite the fact that DOPE effectively facilitates plasmid dissociation from either lipoplex. This difference appears to correlate with membrane stiffness, dictated by the cationic lipid packing in the donor liposomes, which governs the kinetics of lipid recruitment by the plasmid upon lipoplex assembly. Because of its interaction with the relatively rigid SAINT-5 membranes, the plasmid becomes inappropriately condensed, which results in formation of structurally deformed lipoplexes. This structural deformation does not affect its cellular uptake but, rather, hampers plasmid translocation across endosomal and/or nuclear membranes. This is inferred from the observation that both lipoplexes effectively translocate much smaller oligonucleotides into cells. In fact, SAINT-5/DOPE-mediated transfection is greatly improved when, before lipoplex assembly, the plasmid is stabilized by condensation with polylysine. The results emphasize a role of the structural shape of the plasmid in gaining cytosolic/nuclear access. Moreover, it has been proposed that such a translocation is promoted when the lipoplex adopts the hexagonal phase, and data are presented that demonstrate that the lamellar SAINT-5/DOPE lipoplex adopts such a phase after its interaction with acidic phospholipid-containing membranes.     

Cell transfection by DNA-lipid complexes — Lipoplexes

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

Highly fluorinated lipospermines for gene transfer: synthesis and evaluation of their in vitro transfection efficiency

Fluorinated double-chain lipospermines (one or both of these chains being ended by a highly fluorinated tail of various length) which are close analogues of DOGS (Transfectam) were designed as synthetic vectors for gene delivery. For N/P ratios (N = number of amine functions of the lipid; P = number of DNA phosphates) from 0.8 to 10, these lipospermines condensed DNA, with or without the use of DOPE, to form fluorinated lipoplexes. The efficiency of the fluorinated lipoplexes to transfect lung epithelial A549 cells was significantly higher than that of the DOGS lipoplexes. No specific cell toxicity was evidenced for the fluorinated lipoplexes as compared to that of the DOGS ones. The palette of structural elements explored allowed to determine those required for efficient transfection, highlighting the importance of highly fluorinated chains, the unique properties of unsaturated double-chain lipids and of the use of DOPE as helper lipid on transfection.     

Lipid composition of cationic nanoliposomes implicate on transfection efficiency

Abstract

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

Probing the in vitro mechanism of action of cationic lipid/DNA lipoplexes at a nanometric scale

Cationic lipids are used for delivering nucleic acids (lipoplexes) into cells for both therapeutic and biological applications. A better understanding of the identified key-steps, including endocytosis, endosomal escape and nuclear delivery is required for further developments to improve their efficacy. Here, we developed a labelling protocol using aminated nanoparticles as markers for plasmid DNA to examine the intracellular route of lipoplexes in cell lines using transmission electron microscopy. Morphological changes of lipoplexes, membrane reorganizations and endosomal membrane ruptures were observed allowing the understanding of the lipoplex mechanism until the endosomal escape mediated by cationic lipids. The study carried out on two cationic lipids, bis(guanidinium)-tris(2-aminoethyl)amine-cholesterol (BGTC) and dioleyl succinyl paramomycin (DOSP), showed two pathways of endosomal escape that could explain their different transfection efficiencies. For BGTC, a partial or complete dissociation of DNA from cationic lipids occurred before endosomal escape while for DOSP, lipoplexes remained visible within ruptured vesicles suggesting a more direct pathway for DNA release and endosome escape. In addition, the formation of new multilamellar lipid assemblies was noted, which could result from the interaction between cationic lipids and cellular compounds. These results provide new insights into DNA transfer pathways and possible implications of cationic lipids in lipid metabolism.     

On the possible involvement of bovine serum albumin precursor in lipofection pathway

Protein factors involved in lipofection pathways remain elusive. Using avidin-biotin affinity chromatography and mass finger printing analysis technique, herein we report the identification of a 70 kDa size protein (bovine serum albumin precursor, BSAP) which binds strongly with lipoplexes and may play role in lipofection pathway. Using multiple cultured animal cells and three structurally different cationic transfection lipids, we show that the efficiencies of liposomal transfection vectors get significantly enhanced (by ~2.5- to 5.0-fold) in cells pre-transfected with lipoplexes of reporter plasmid construct encoding BSAP. Findings in the cellular uptake experiments in A549 cells cultured in DMEM supplemented with 10% (w/w) BODIPY-labelled BSAP are consistent with the supposition that BSAP enters cell cytoplasm from the cell culture medium (DMEM supplemented with 10% FBS) used in lipofection. Cellular uptake studies by confocal microscopy using BODIPY-labelled BSAP and FITC-labelled plasmid DNA revealed co-localization of plasmid DNA and BSAP within the cell cytoplasm and nucleus. In summary, the present findings hint at the possible involvement of BSAP in lipofection pathway.     

Physical Characterization of Gemini Surfactant-Based Synthetic Vectors for the Delivery of Linear Covalently Closed (LCC) DNA Ministrings

In combination with novel linear covalently closed (LCC) DNA minivectors, referred to as DNA ministrings, a gemini surfactant-based synthetic vector for gene delivery has been shown to exhibit enhanced delivery and bioavailability while offering a heightened safety profile. Due to topological differences from conventional circular covalently closed (CCC) plasmid DNA vectors, the linear topology of LCC DNA ministrings may present differences with regards to DNA interaction and the physicochemical properties influencing DNA-surfactant interactions in the formulation of lipoplexed particles. In this study, N,N-bis(dimethylhexadecyl)-α,ω-propanediammonium(16-3-16)gemini-based synthetic vectors, incorporating either CCC plasmid or LCC DNA ministrings, were characterized and compared with respect to particle size, zeta potential, DNA encapsulation, DNase sensitivity, and in vitro transgene delivery efficacy. Through comparative analysis, differences between CCC plasmid DNA and LCC DNA ministrings led to variations in the physical properties of the resulting lipoplexes after complexation with 16-3-16 gemini surfactants. Despite the size disparities between the plasmid DNA vectors (CCC) and DNA ministrings (LCC), differences in DNA topology resulted in the generation of lipoplexes of comparable particle sizes. The capacity for ministring (LCC) derived lipoplexes to undergo complete counterion release during lipoplex formation contributed to improved DNA encapsulation, protection from DNase degradation, and in vitro transgene delivery.