The Impacts of Sortase A and the 4'-Phosphopantetheinyl Transferase Homolog Sfp on Streptococcus mutans Extracellular Membrane Vesicle Biogenesis.

Extracellular membrane vesicles (EMVs) are produced by many Gram-positive organisms, but information regarding vesiculogenesis is incomplete. We used single gene deletions to evaluate the impacts on Streptococcus mutans EMV biogenesis of Sortase A (SrtA), which affects S. mutans EMV composition, and Sfp, a 4'-phosphopantetheinyl transferase that affects Bacillus subtilis EMV stability. ΔsrtA EMVs were notably larger than Δsfp and wild-type (WT) EMVs. EMV proteins identified from all three strains are known to be involved in cell wall biogenesis and cell architecture, bacterial adhesion, biofilm cell density and matrix development, and microbial competition. Notably, the AtlA autolysin was not processed to its mature active form in the ΔsrtA mutant. Proteomic and lipidomic analyses of all three strains revealed multiple dissimilarities between vesicular and corresponding cytoplasmic membranes (CMs). A higher proportion of EMV proteins are predicted substrates of the general secretion pathway (GSP). Accordingly, the GSP component SecA was identified as a prominent EMV-associated protein. In contrast, CMs contained more multi-pass transmembrane (TM) protein substrates of co-translational transport machineries than EMVs. EMVs from the WT, but not the mutant strains, were enriched in cardiolipin compared to CMs, and all EMVs were over-represented in polyketide flavonoids. EMVs and CMs were rich in long-chain saturated, monounsaturated, and polyunsaturated fatty acids, except for Δsfp EMVs that contained exclusively polyunsaturated fatty acids. Lipoproteins were less prevalent in EMVs of all three strains compared to their CMs. This study provides insight into biophysical characteristics of S. mutans EMVs and indicates discrete partitioning of protein and lipid components between EMVs and corresponding CMs of WT, ΔsrtA, and Δsfp strains.

Enhanced purification coupled with biophysical analyses shows cross-ß structure as a core building block for Streptococcus mutans functional amyloids.

Streptococcus mutans is an etiologic agent of human dental caries that forms dental plaque biofilms containing functional amyloids. Three amyloidogenic proteins, P1, WapA, and Smu_63c were previously identified. C123 and AgA are naturally occurring amyloid-forming fragments of P1 and WapA, respectively. We determined that four amyloidophilic dyes, ThT, CDy11, BD-oligo, and MK-H4, differentiate C123, AgA, and Smu_63c amyloid from monomers, but non-specific binding to bacterial cells in the absence of amyloid precludes their utility for identifying amyloid in biofilms. Congo red-induced birefringence is a more specific indicator of amyloid formation and differentiates biofilms formed by wild-type S. mutans from a triple ΔP1/WapA/Smu_63c mutant with reduced biofilm forming capabilities. Amyloid accumulation is a late event, appearing in older S. mutans biofilms after 60 hours of growth. Amyloid derived from pure preparations of all three proteins is visualized by electron microscopy as mat-like structures. Typical amyloid fibers become evident following protease digestion to eliminate non-specific aggregates and monomers. Amyloid mats, similar in appearance to those reported in S. mutans biofilm extracellular matrices, are reconstituted by co-incubation of monomers and amyloid fibers. X-ray fiber diffraction of amyloid mats and fibers from all three proteins demonstrate patterns reflective of a cross-ß amyloid structure.

Transfection of plasmid DNA by nanocarriers containing a gemini cationic lipid with an aromatic spacer or its monomeric counterpart.

This study performed a biophysical characterization (electrochemistry, structure and morphology) and assessment of the biological activity and cell biocompatibility of GCL/DOPE-pDNA lipoplexes comprised of plasmid DNA and a mixed lipid formed by a DOPE zwitterionic lipid and a gemini cationic lipid N-N'-(1,3-phenylene bis (methylene)) bis (N,N-dimethyl-N-(1-dodecyl) ammonium dibromide (12PH12) containing an aromatic spacer or its monomeric counterpart surfactant, N-benzyl-N,N-dimethyl-N-(1-dodecyl) ammonium bromide (12PH). Electrochemical results reveal that i) the gemini cationic lipid (12PH12) and the plasmid pDNA yield effective charges less than their nominal charges (+2 and -2/bp, respectively) and that ii) both vectors (12PH12/DOPE and 12PH/DOPE) could compact pDNA and protect it from DNase I degradation. SAXS and cryo-TEM experiments indicate the presence of a lamellar lyotropic liquid crystal phase represented as alternating layers of mixed lipid and plasmid. Transfection efficiency (by FACS and luminometry) and cell viability assay in COS-7 cells, performed with two plasmid DNAs (pEGFP-C3 and pCMV-Luc VR1216), confirm the goodness of the proposed formulations (12PH12/DOPE and 12PH/DOPE) to transport genetic material, with efficiencies and biocompatibilities comparable to or better than those exhibited by the control Lipofectamine 2000*. In conclusion, although major attention has been paid to gemini cationic lipids in the literature, due to the large variety of modifications that their structures may support to improve the biological activity of the resulting lipoplexes, it is remarkable that the monomeric counterpart surfactant with an aromatic group analyzed in the present work also exhibits good biological activity. The in vitro results reported here indicate that the optimum formulations of the gene vectors studied in this work efficiently transfect plasmid DNA with very low toxicity levels and, thus, may be used in forthcoming in vivo experiments.

A biophysical study of gene nanocarriers formed by anionic/zwitterionic mixed lipids and pillar[5]arene polycationic macrocycles.

The use of divalent cations as mediators between anionic lipids (ALs) and nucleic acids has been explored for several years in gene therapy. However, a promising anionic lipid system which could surpass the outcomes of current cationic lipids (CLs) has not been found yet. One plausible reason for such poor efficiencies may be the impossibility of AL-DNA lipoplexes mediated by divalent cations to reach charge inversion, in contrast with the usual behavior of CL-DNA lipoplexes. In the present study, divalent bridge-cations have been replaced by a multivalent positively charged macrocycle in order to see whether charge reversal is reached and how this fact may improve transfection efficiency (TE). For that purpose, an extensive biophysical and biochemical study has been carried out on lipoplexes constituted by a mixture of: (i) an anionic lipid DOPG (sodium salt of 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol)); (ii) a zwitterionic lipid DOPE (1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine), which acts as a neutral helper lipid at physiological pH 7.4; (iii) a plasmid DNA (pDNA); and (iv) a polycationic macrocycle, pillar[5]arene (P10+), with the role of bridging the electrostatic interaction between the anionic mixed lipids and the pDNA, also negatively charged. The studies have been done at several DOPG molar compositions (α) and pillar[5]arene concentrations. Electrochemical experiments (zeta potential and gel electrophoresis) have revealed that, interestingly, DOPG/DOPE-P10+-pDNA lipoplexes show a charge inversion. Both studies have indicated that, at [P10+] ≥ 15 µM, pDNA is efficiently compacted by DOPG/DOPE mixed lipids, using P10+ as a bridge between the negative charge of the AL and anionic pDNA. SAXS diffractograms have shown the presence of two lyotropic liquid crystal phases: an inverted hexagonal one (H) found at low composition (α = 0.2), and a lamellar one (Lα) at medium composition (α = 0.5). Cryo-TEM and AFM experiments have confirmed these structures. Transfection and cell viability experiments using COS-7 cells in the presence of serum have reported moderate-to-high transfection levels and good cell viability results. The whole ensemble of the biophysical and biochemical results of the DOPG/DOPE-P10+-pDNA lipoplex indicates that this system may open up a novel and very promising route in the anionic non-viral gene vectors field.

Efficient Cellular Knockdown Mediated by siRNA Nanovectors of Gemini Cationic Lipids Having Delocalizable Headgroups and Oligo-Oxyethylene Spacers.

The use of small interfering RNAs (siRNAs) to silence specific genes is one of the most promising approaches in gene therapy, but it requires efficient nanovectors for successful cellular delivery. Recently, we reported liposomal gene carriers derived from a gemini cationic lipid (GCL) of the 1,2-bis(hexadecyl dimethyl imidazolium) oligo-oxyethylene series ((C16Im)2(C2H4O)nC2H4 with n = 1, 2, or 3) and 1,2-dioleyol phosphatidylethanolamine as highly efficient cytofectins for pDNA. On the basis of the satisfactory outcomes of the previous study, the present work focuses on the utility of coliposomes of these gemini lipids with the biocompatible neutral lipid mono oleoyl glycerol (MOG) as highly potent vectors for siRNA cellular transport in the presence of serum. The (C16Im)2(C2H4O)nC2H4/MOG-siRNA lipoplexes were characterized through (i) a physicochemical study (zeta potential, cryo-transmission electron microscopy, small-angle X-ray scattering, and fluorescence anisotropy) to establish the relationship between size, structure, fluidity, and the interaction between siRNA and the GCL/MOG gene vectors and (ii) a biological analysis (flow cytometry, fluorescence microscopy, and cell viability) to report the anti-GFP siRNA transfections in HEK 293T, HeLa, and H1299 cancer cell lines. The in vitro biological analysis confirms the cellular uptake and indicates that a short spacer, a very low molar fraction of GCL in the mixed lipid, and a moderate effective charge ratio of the lipoplex yielded maximum silencing efficacy. At these experimental conditions, the siRNA used in this work is compacted by the GCL/MOG nanovectors by forming two cubic structures (Ia3d and Pm3n) that are correlated with excellent silencing activity. These liposomal nanocarriers possess high silencing activity with a negligible cytotoxicity, which strongly supports their practical use for in vivo knockdown studies.

Anionic/Zwitterionic Lipid-Based Gene Vectors of pDNA.

The use of anionic lipids (ALs) as non-viral gene vectors depicts a promising alternative to cationic lipids (CLs) since they are more biocompatible and present lower levels of phagocytosis by macrophages. Several experimental methods, such as electrophoretic mobility (ζ-potential), gel electrophoresis, small-angle X-ray scattering (SAXS), fluorescence and confocal fluorescence microscopies (FM and CFM), flow assisted cell sorting-flow cytometry (FACS-FCM), and cell viability/cytotoxicity assays can be used for a complete physicochemical and biochemical characterization of lipoplexes formed by an AL, a zwitterionic lipid (ZL), and a plasmid DNA (pDNA), their electrostatic interaction being necessarily mediated by divalent cations, such as Ca(2+). In the present chapter, we summarize the protocols optimized for the mentioned characterization techniques.

Polycationic Macrocyclic Scaffolds as Potential Non-Viral Vectors of DNA: A Multidisciplinary Study.

The potential of lipoplexes constituted by the DNA pEGFP-C3 (encoding green fluorescent protein), polycationic calixarene-based macrocyclic vector (CxCL) with a lipidic matrix (herein named TMAC4), and zwitterionic lipid 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) as nontoxic DNA vectors has been analyzed from both biophysical and biochemical perspectives. For that purpose, several experimental methods, such as zeta potential (PALS methodology), agarose gel electrophoresis, small-angle X-ray scattering (SAXS), transmission electronic cryo-microscopy (cryo-TEM), atomic force microscopy (AFM), fluorescence microscopy, and cytotoxicity assays have been used. The electrochemical study shows that TMAC4 has 100% of its nominal charge available, whereas pDNA presents an effective negative charge that is only 10% that of its nominal one. PALS studies indicate the presence of three populations of nanoaggregates in TMAC4/DOPE lipid mixtures, with sizes of approximately 100, 17, and 6 nm, compatible with liposomes, oblate micelles, and spherical micelles, respectively, the first two also being detected by cryo-TEM. However, in the presence of pDNA, this mixture is organized in Lα multilamellar structures at all compositions. In fact, cryo-TEM micrographs show two types of multilamellar aggregation patterns: cluster-type at low and moderate CxCL molar fractions in the TMAC4/DOPE lipid mixture (α = 0.2 and 0.5), and fingerprint-type (FP), which are only present at low CxCL molar fraction (α = 0.2). This structural scenario has also been observed in SAXS diffractograms, including the coexistence of two different phases when DOPE dominates in the mixture. AFM experiments at α = 0.2 provide evidence that pDNA makes the lipid bilayer more deformable, thus promoting a potential enhancement in the capability of penetrating the cells. In fact, the best transfection perfomances of these TMAC4/DOPE-pDNA lipoplexes have been obtained at low CxCL molar fractions (α = 0.2) and a moderate-to-high effective charge ratio (ρeff = 20). Presumably, the coexistence of two lamellar phases is responsible for the better TE performance at low α.

A delocalizable cationic headgroup together with an oligo-oxyethylene spacer in gemini cationic lipids improves their biological activity as vectors of plasmid DNA.

Lipoplex nano-aggregates constituted of plasmid DNA (pDNA) pEGFP-C3 and mixed cationic liposomes, consisting of several percentages of a gemini cationic lipid (GCL) of the 1,2-bis(hexadecyl imidazolium) oxyethylene series, referred to as (C16Im)2(C2O)n, with oxyethylene spacers (n = 1, 2 or 3) between the imidazolium cationic groups and the DOPE zwitterionic helper lipid, have been characterized by various biophysical and biological approaches carried out at several GCL compositions (α), and either the mass or the effective charge ratio of the lipoplex. The electrochemical study by ζ-potential confirms that the three GCLs yield a 10% lower effective charge than the nominal one, while compacted pDNA yields only a 25% effective negative charge. The SAXS study reveals, irrespective of the spacer length (n) and effective charge ratio (ρeff), the presence of two lamellar structures, i.e., one (Lα,main) in the whole GCL composition and another (Lα,DOPE,rich) with higher periodicity values that coexists with the previous one at low GCL composition (α = 0.2). The cryo-TEM analysis shows two types of multilamellar structures consisting of cationic lipidic bilayers with pDNA sandwiched between them: a cluster-type (C-type) at low α = 0.2 and a fingerprint-type (FP-type) at α≥ 0.5, both with similar interlamellar spacing (d) in agreement with the Lα,main structure determined by SAXS. Transfection efficacies (TEs) of each lipid mixture were determined in four different cell lines (HEK293T, HeLa, Caco-2 and A549) at several α and ρeff values in the absence and presence of serum (FBS). The optimized formulations (α = 0.2 and ρeff = 2.0) substantially transfect cells much better than a commercial transfection reagent, Lipofectamine 2000 and previously studied efficient lipoplexes containing other cationic head groups or spacers both in the absence and presence of serum. The activity of optimized formulations may be attributed to the combination of several factors, such as: (a) the fusogenic character of DOPE which results in higher fluidity of the lipoplexes at α = 0.2, (b) the coexistence of two lamellar structures at α = 0.2 that synergizes the TE of these lipid vectors, and mainly (c) the higher biocompatibility of the GCLs reported in this work due to the presence of two imidazolium cationic groups together with an oligo-oxyethylene spacer. The length of the spacer in the GCL seems to have less impact, although (C16Im)2(C2O)n/DOPE-pDNA lipoplexes with n = 1 and 3 show higher gene transfection than n = 2. All the optimum formulations reported herein are all highly efficient with negligible levels of toxicity, and thus, may be considered as very promising gene vectors for in vivo applications.

Ca(2+)-mediated anionic lipid-plasmid DNA lipoplexes. Electrochemical, structural, and biochemical studies.

Several experimental methods, such as zeta potential, gel electrophoresis, small-angle X-ray scattering, gene transfection, fluorescence microscopy, flow cytometry, and cell viability/cytotoxicity assays, have been used to analyze the potential of anionic lipids (AL) as effective nontoxic and nonviral DNA vectors, assisted by divalent cations. The lipoplexes studied are those comprised of the green fluorescent protein-encoding plasmid DNA pEGFP-C3, an anionic lipid as 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG) or 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), and a zwitterionic lipid, the 1,2-dioleoyl-sn -glycero-3-phosphatidylethanolamine (DOPE, not charged at physiological pH). The studies have been carried on at different liposome and lipoplex compositions and in the presence of a variety of [Ca2+]. Electrochemical experiments reveal that DOPG/DOPE and DOPS/DOPE anionic liposomes may compact more effectively pDNA at low molar fractions (with an excess of DOPE) and at AL/pDNA ratios ≈20. Calcium concentrations around 15-20 mM are needed to yield lipoplexes neutral or slightly positive. From a structural standpoint, DOPG/DOPE-Ca2+-pDNA lipoplexes are self-assembled into a HIIc phase (inverted cylindrical micelles in hexagonal ordering with plasmid supercoils inside the cylinders), while DOPS/DOPE-Ca2+-pDNA lipoplexes show two phases in coexistence: one classical HIIc phase which contains pDNA supercoils and one Lα phase without pDNA among the lamellae, i.e., a lamellar stack of lipidic bilayers held together by Ca2+ bridges. Transfection and cell viability studies were done with HEK293T and HeLa cells in the presence of serum. Lipoplexes herein studied show moderate-to-low transfection levels combined with moderate-to-high cell viability, comparable to those yield by Lipofectamine2000*, which is a cationic lipid (CL) standard formulation, but none of them improve the output of typical CL gen vectors, mostly if they are gemini or dendritic. This fact would be indicating that, nowadays, lipofection via anionic lipids and divalent cations as mediators still needs to enhance transfection levels in order to be considered as a real and plausible alternative to lipofection through improved CLs-based lipoplexes.

Cationic gemini lipids containing polyoxyethylene spacers as improved transfecting agents of plasmid DNA in cancer cells.

Lipoplex nano-aggregates have been analyzed through biophysical characterization (electrostatics, structure, size and morphology), and biological studies (transfection efficiency and cell viability) in five cancer cell lines. Lipoplexes were prepared from pEGFP-C3 plasmid DNA (pDNA) and mixed liposomes, constituted by a zwitterionic lipid (DOPE) and a gemini cationic lipid (GCL) synthesized in this work, [bis(hexadecyl dimethyl ammonium) oxyethylene], referred to as (C16Am)2(C2O)n, (where n is the oxyethylene spacer length, n = 1, 2 or 3, between the ammonium heads). Cryo-TEM micrographs show nano-aggregates with two multilamellar structures, a cluster-type (at low-to-medium GCL composition) and a fingerprint-type that coexists with the cluster-type at medium GCL composition and appears alone at high GCL composition. SAXS diffractograms show that these lipoplexes present three lamellar structures, two of them coexisting at low and high GCL composition. The optimized transfection efficiency (TE) of pDNA was higher for lipoplexes containing GCLs with a longer (n = 3) or shorter (n = 1) polyoxyethylene spacer, at high GCL composition (α = 0.7) with low charge ratio (ρeff = 2). In the all cancer cell lines studied, the TE of the optimized formulations was much better than those of both lipofectamine 2000 and lipoplexes with GCLs of the bis(hexadecyl dimethyl ammonium) alkane series recently reported. Probably, (a) the coexistence of two lamellar structures at high GCL composition synergizes the TE of these lipid vectors, (b) the orientation of the polyoxyethylene region in (C16Am)2(C2O)3/DOPE may occur in such a way that the spacing between two cationic heads becomes smaller than that in (C16Am)2(C2O)2/DOPE which is poor in terms of TE, and (c) the synergistic interactions between serum proteins and (C16Am)2(C2O)n/DOPE-pDNA lipoplexes containing a polyoxyethylene spacer improve TE, especially at high GCL content. Lipoplexes studied here show very low levels of toxicity, which confirm them as improved vectors of pDNA in gene therapy.

Effects of a delocalizable cation on the headgroup of gemini lipids on the lipoplex-type nanoaggregates directly formed from plasmid DNA.

Lipoplex-type nanoaggregates prepared from pEGFP-C3 plasmid DNA (pDNA) and mixed liposomes, with a gemini cationic lipid (CL) [1,2-bis(hexadecyl imidazolium) alkanes], referred as (C16Im)2Cn (where Cn is the alkane spacer length, n = 2, 3, 5, or 12, between the imidazolium heads) and DOPE zwitterionic lipid, have been analyzed by zeta potential, gel electrophoresis, SAXS, cryo-TEM, fluorescence anisotropy, transfection efficiency, fluorescence confocal microscopy, and cell viability/cytotoxicity experiments to establish a structure-biological activity relationship. The study, carried out at several mixed liposome compositions, α, and effective charge ratios, ρeff, of the lipoplex, demonstrates that the transfection of pDNA using CLs initially requires the determination of the effective charge of both. The electrochemical study confirms that CLs with a delocalizable positive charge in their headgroups yield an effective positive charge that is 90% of their expected nominal one, while pDNA is compacted yielding an effective negative charge which is only 10-25% than that of the linear DNA. SAXS diffractograms show that lipoplexes formed by CLs with shorter spacer (n = 2, 3, or 5) present three lamellar structures, two of them in coexistence, while those formed by CL with longest spacer (n = 12) present two additional inverted hexagonal structures. Cryo-TEM micrographs show nanoaggregates with two multilamellar structures, a cluster-type (at low α value) and a fingerprint-type, that coexist with the cluster-type at moderate α composition. The optimized transfection efficiency (TE) of pDNA, in HEK293T, HeLa, and H1299 cells was higher using lipoplexes containing gemini CLs with shorter spacers at low α value. Each lipid formulation did not show any significant levels of toxicity, the reported lipoplexes being adequate DNA vectors for gene therapy and considerably better than both Lipofectamine 2000 and CLs of the 1,2-bis(hexadecyl ammnoniun) alkane series, recently reported.

How does the spacer length of cationic gemini lipids influence the lipoplex formation with plasmid DNA? Physicochemical and biochemical characterizations and their relevance in gene therapy.

Lipoplexes formed by the pEGFP-C3 plasmid DNA (pDNA) and lipid mixtures containing cationic gemini surfactant of the 1,2-bis(hexadecyl dimethyl ammonium) alkanes family referred to as C16CnC16, where n=2, 3, 5, or 12, and the zwitterionic helper lipid, 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) have been studied from a wide variety of physical, chemical, and biological standpoints. The study has been carried out using several experimental methods, such as zeta potential, gel electrophoresis, small-angle X-ray scattering (SAXS), cryo-TEM, gene transfection, cell viability/cytotoxicity, and confocal fluorescence microscopy. As reported recently in a communication (J. Am. Chem. Soc. 2011, 133, 18014), the detailed physicochemical and biological studies confirm that, in the presence of the studied series lipid mixtures, plasmid DNA is compacted with a large number of its associated Na+ counterions. This in turn yields a much lower effective negative charge, qpDNA−, a value that has been experimentally obtained for each mixed lipid mixture. Consequently, the cationic lipid (CL) complexes prepared with pDNA and CL/DOPE mixtures to be used in gene transfection require significantly less amount of CL than the one estimated assuming a value of qDNA−=−2. This drives to a considerably lower cytotoxicity of the gene vector. Depending on the CL molar composition, α, of the lipid mixture, and the effective charge ratio of the lipoplex, ρeff, the reported SAXS data indicate the presence of two or three structures in the same lipoplex, one in the DOPE-rich region, other in the CL-rich region, and another one present at any CL composition. Cryo-TEMand SAXS studies with C16CnC16/DOPE-pDNA lipoplexes indicate that pDNA is localized between the mixed lipid bilayers of lamellar structures within a monolayer of ∼2 nm. This is consistent with a highly compacted supercoiled pDNA conformation compared with that of linear DNA. Transfection studies were carried out with HEK293T, HeLa, CHO, U343, and H460 cells. The α and ρeff values for each lipid mixture were optimized on HEK293T cells for transfection, and using these values, the remaining cells were also transfected in absence (-FBS-FBS) and presence (-FBS+FBS) of serum. The transfection efficiency was higher with the CLs of shorter gemini spacers (n=2 or 3). Each formulation expressed GFP on pDNA transfection and confocal fluorescence microscopy corroborated the results. C16C2C16/DOPE mixtures were the most efficient toward transfection among all the lipid mixtures and, in presence of serum, even better than the Lipofectamine2000, a commercial transfecting agent. Each lipid combination was safe and did not show any significant levels of toxicity. Probably, the presence of two coexisting lamellar structures in lipoplexes synergizes the transfection efficiency of the lipid mixtures which are plentiful in the lipoplexes formed by CLs with short spacer (n=2, 3) than those with the long spacer (n=5, 12).

Why is less cationic lipid required to prepare lipoplexes from plasmid DNA than linear DNA in gene therapy?

The most important objective of the present study was to explain why cationic lipid (CL)-mediated delivery of plasmid DNA (pDNA) is better than that of linear DNA in gene therapy, a question that, until now, has remained unanswered. Herein for the first time we experimentally show that for different types of CLs, pDNA, in contrast to linear DNA, is compacted with a large amount of its counterions, yielding a lower effective negative charge. This feature has been confirmed through a number of physicochemical and biochemical investigations. This is significant for both in vitro and in vivo transfection studies. For an effective DNA transfection, the lower the amount of the CL, the lower is the cytotoxicity. The study also points out that it is absolutely necessary to consider both effective charge ratios between CL and pDNA and effective pDNA charges, which can be determined from physicochemical experiments.