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Il_Farmaco-2005

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Il Farmaco 60 (2005) 701–710 http://france.elsevier.

com/direct/FARMAC/

Preparation and phase behaviour of surface-active pharmaceuticals: self-assembly of DNA and surfactants with membranes. Differential adiabatic scanning microcalorimetric study
Erhan Süleymanoglu a,b,* ˘
a

Biophysics Section, Department of Physical Chemistry of Drugs, Faculty of Pharmacy, J.A. Comenius University, Odbojarov 10, 83 232 Bratislava, The Slovak Republic b Department of Biophysics, The Slovak Academy of Sciences, Institute of Experimental Physics, Košice, The Slovak Republic Received 18 March 2005; accepted 3 May 2005 Available online 14 July 2005

Abstract Some energetics issues relevant to preparation and surface characterization of zwitterionic phospholipid–DNA self-assemblies, as alternative models of the currently used problematic lipoplexes are presented. Nucleic acid compaction capacities of Mg2+ and N-alkyl-N,N,Ntrimetylammonium ions (CnTMA, n = 12) were compared, with regard to surface interaction with unilamellar vesicles. Differential adiabatic scanning microcalorimetric measurements of synthetic phosphatidylcholine liposomes and calf thymus DNA and their ternary complexes with Mg2+ and C12TMA, were employed for deduction of the thermodynamic model describing their structural transitions. Small monodisperce and highly stable complexes are established after precompaction of DNA with detergent, followed by addition of liposomes. In contrast, divalent metal cation-mediated aggregation of vesicles either leads to heterogeneous multilamellar DNA–lipid arrangements, or to DNAinduced bilayer destabilization and lipid fusion. Possible dependence of the cellular internalization and gene transfection efficiency on the structure and physicochemical properties of DNA–Mg2+–liposomes or DNA–cationic surfactant–liposome systems is emphasized by proposing the structure of their molecular self-organizations with further implications in gene transfer research. © 2005 Elsevier SAS. All rights reserved.
Keywords: Zwitterionic liposomes–DNA self-organization; Surface active pharmaceuticals; Differential adiabatic scanning microcalorimetry; Phase behavior; Non-viral gene delivery

1. Introduction The molecular associations in mixed solutions of DNA with oppositely charged cosolutes-metal cations, cationic amphiphiles and macromolecules, have attracted research interest and efforts not only in terms of their physicochemical [1–3] and pharmaceutical relevance [4–6], but also due to their potential for applications in separation, purification and transfection of DNA [7,8].

* Corresponding author. G.Ü.E.F., The Central Laboratory, Department of Pharmaceutical Chemistry, Gazi Mahallesi, Polatli Caddesi, No: 115/5, Yenimahalle, 06560 Ankara, Turkey. Tel.: +90 312 211 1947; fax: +90 312 223 5018. http://erhan.boom.ru. E-mail address: erhan@atlas.cz (E. Süleymanoglu). ˘ 0014-827X/$ - see front matter © 2005 Elsevier SAS. All rights reserved. doi:10.1016/j.farmac.2005.05.010

Gene transfection is commonly used in biotechnology and has received considerable attention in biomedicine for curing genetic diseases [9,10]. Therapeutic gene transfer is achieved by employing viral [11] or non-viral [12–14], synthetic [15–17] or physical methods [18]. Research on human gene therapy faces certain hurdles due to the lack of suitable delivery tools for therapeutic nucleic acid transfer to target cells. Having considered the risky viral based delivery systems and problematic physical methods, nowadays much research effort is devoted to the design of artificial non-viral vehicles formed by association of nucleic acids with lipids (lipoplexes) or polymers (polyplexes). Whatever the approach is, in both designs, the objective is to achieve a stable packaging of genes to be transfected, to increase transgene expression and to improve their bioavailability, while decreasing their cytotoxicity. In

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this respect, the desired gene packaging becomes a physical pharmaceutics problem, requiring contributions from physicochemically oriented groups. There is an urgent need for optimized gene transfection methods capable of protecting the DNA from degradation via its route to gene expression [9,10]. Among these, lipidbased delivery method, has gained preference (http://www.wiley.co.uk/genetherapy/clinical/) in the light of the possibilities for performing simpler quality controls, as well as easier satisfaction of pharmaceutical requirements. Designing suitable lipoplex formulations requires systematic characterization of the DNA–lipid complexes as gene packaging systems for transfection [19]. From lipid-based experimental designs, various monolayer (Langmuir–Blodgett films) [20], black lipid membranes (BLMs) [21] and liposomal systems [10,13,14,22–25] are studied concerning their ability to bind nucleic acids. Using a broad range of spectroscopic, microscopic, thermodynamic, hydrodynamic, and other relevant techniques [26], a useful laboratory data have been collected, in search for a details regarding biophysical and colloidal factors determining the stability of various DNA–lipid self-assemblies. Since mainly electrostatic and hydrophobic interactions govern the formation of these self-assemblies, most of the designs are concerned with colloidal or interfacial electrified surface forces. Thus, mixed systems between lipids and surfactants can be employed as packaging agents for DNA delivery to the affected cells. Before switching to real in vitro transfection experiments with numerous gene reporter molecules, it is crucial first to achieve a stable DNA–lipid formulation with controllable properties. Measurable parameters of potential interest are phase behavior, morphology and structural characterization of DNA compaction with various condensing agents, such as, surfactants, charged and neutral polymers, metal ions, as well as mixtures of cationic and anionic macromolecules, and thermodynamically stable lipid vesicles. Thus, the molecular details of cationic lipid binding to DNA polyanion will be clarified. However, despite the collected research data, currently neither the energetics of these associations nor structure of the resultant complex is well understood. Following the interesting recent results of using cationic, small membrane-permeant molecules [27], as well as our own data on Mg2+ as bridging DNA with liposomes [28] we have focused on designs involving such agents as rapidly moving through model cellular and nuclear membranes. These could then bind nuclear DNA with high affinity. In the light of the latter proposal, we hypothesized that such molecules could affect the topology of bound DNA after associating with it so as to enable penetration of model cell membranes by this bound nucleic acid. Usually, the positive charge of these small molecules would complex with the negatively charged phosphate groups of DNA, forming a hydrophobic ion pair. The latter would then alter the solution properties of the complexed DNA by reducing its polarity, thus giving rise to membrane-permeant agents to trigger DNA transport through simulated cell membranes.

To explore new mechanisms for overcoming physical membrane barriers to the intracellular delivery of therapeutic nucleic acids (both DNA and RNA) it is worth studying the design of non-viral delivery tools that could form thermodynamically stable colloidal complexes with DNA, thus obviating the need for chemical conjugation of DNA to various ligands. Interestingly, these small cationic molecules would be able to form membrane-permeant ion pairs with the nucleic acid. In this respect, we have already reported our results with divalent metal cations, capable of complexing liposomes with DNA, followed by attachment to model cell membrane, destabilizing and internalizing through it, as an alternative lipoplex entry route to target cell via problematic receptor-mediated endocytic route. In our opinion, such membrane fluidity effects of various small cations deserve to be studied with respect to gene delivery vesicle designs, which potentially could enhance their transfection efficiencies, as suggested previously [28]. Hence, it is interesting to compare our previous results on divalent metal cations with other promising molecules, such as cationic surfactants for instance, which are well-known gene transfection enhancers [9]. Experimental designs of this sort focus on features such as influence of vesicle size and dose, surface charge and properties, and stability characteristics, which are to a major extent, thermodynamically governed processes. Unfortunately, little has been done so far to relate these physicochemical considerations to in vivo behavior of these dispersions. To understand the energetics of this important system, the thermodynamics of lipid and amphiphile-like ligand binding to DNA was studied and some preliminary results are presented here. Both naturally occurring lipids and amphiphile resembling model compounds were used, trying to compare the phase behavior of various ligands and their binding modes and their effect on the energetics of DNA–lipid complex formation. Assuming that alkylammonium ion and related forms form a relevant structure, their phase paramaters were compared with 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) concerning affinity for DNA binding, trying to find more clues from the correlation between DNA compaction and transfection efficiency obtained from previous studies of DNA associations with lipid dispersions and polycations with different chain length. Deducing from both theoretical and experimental studies will improve the current knowledge concerning molecular interactions and general biophysical chemistry of these promising formulations in terms of designing improved gene delivery systems.

2. Experimental 2.1. Materials DPPC, SSC (1.5 × 10–4 mol/l Na-citrate, 1.5 × 10–3 mol/l NaCl, pH 7.2) reagents were purchased from Sigma (St. Louis, MO, USA) and used without further purification. All other

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reagents were of analytical grade. Alkylamine aqueous solutions were stored in tightly sealed containers, to prevent their reactions with atmospheric CO2, as suggested [29]. Lipid and C12TMA solutions were mixed in polyethylene vials in desired ratios. Following solvent evaporation under nitrogen gas flow, the samples were evacuated at room temperature for couple of hours. Cationic detergent was dissolved in double distilled water and was added to the dry lipid, prior to measurement. 2.2. Methods 2.2.1. Preparation of polynucleotide solutions and concentration determinations Calf thymus DNA with MW of 8.6 MDa (= 13 kb), 42% GC, Tm = 87 °C, ~20 A260 units per mg DNA was used. Polinucleotide concentrations and molar ratios are based on the average nucleotide molecular weight of 308 [28]. 2.2.2. Preparation of liposomes Chromatographic tests for purity of the lipids were not performed, however, the purity of the lipid preparation was assured from the half-widths of their main phase transitions. 1.2 mM lipid in standard SSC buffer, pH 7.2 was used in all experiments and was stored at 4 °C. The formation of a thin layer of lipids of a 15 ml roundbottomed flask was achieved by a hand-shaking and hydration in particular buffer at around 70 °C. Vortexing of the lipid with the desired aqueous solution above the gel-toliquid crystalline phase transition of the lipid (Tm) for around 30 min resulted in multilamellar vesicles. Unilamellar vesicles (ULV) were obtained by extrusion of multilamellar vesicle (MLV) suspension through two stacked polycarbonate filters (Nucleopore, Inc.) of 100 nm pore size at around 60 °C. Repeated extrusion (10 times) through the extruder (Lipex Biomembranes, Inc., Vancouver, BC, Canada) created homogeneous vesicle suspension. This allowed the preparation of vesicles with a mean diameter of 90 nm and a trap volume in the range of 1.5–2.0 l/mol. 2.2.3. Preparation of liposome–nucleic acid mixtures Nucleic acid–lipid mixtures were prepared 1 h before microcalorimetric measurements by vigorous mixing of either phosphatidylcholine MLV or ULV dispersions and solvent, varying nucleic acid concentration and keeping DPPC concentration fixed. Control experiments of DNA–lipids in the absence of detergent or divalent cations, were performed in parallel. Phospholipid concentration employed was 0.3 mg/ml. The lipid samples were hydrated, as described above to form first MLV. The preparation of phosphatidylcholine ULV– calf thymus DNA complexes, was the same as in the case of MLVs, i.e. by mixing DNA solution with aqueous DPPC dispersion in the presence of Mg2+. The DNA concentration used throughout all experiments was 1.8 mM based on the above-mentioned assumption. A

freeze–thaw protocol was followed to ensure equal distribution of solutes between lamellae and adequate hydration of the lipids. Comparison with the case of liposomal preparations without employing freeze–thaw procedure showed no difference in terms of homogeneity of the suspension. This was done by placing the sample in a cryo-tube and freezing it, in liquid nitrogen for around 30 s. The cryo-tube was subsequently removed and was plunged into warm water (~60 °C). When the sample was thawed, the whole cycle of freeze–thawing was repeated six times. 2.2.4. Differential scanning calorimetry Calorimetric measurements were performed using Privalov type high sensitivity differential adiabatic scanning microcalorimeter DASM-4 (Biopribor, Pushchino, Russian Federation) with sensitivity higher than 4 × 10–6 cal/K and a noise level less than 5 × 10–7 W. Heating runs were performed with a scan rate of 0.5 K/min. The temperature at the maximum of the excess heat capacity curve was taken as the transition temperature Tm and the transition width DT1/2 was determined at the transition half-height. The calorimetric enthalpy DHcal of the transition was determined as the area under the excess heat capacity curve [28].

3. Results and discussion The presented work describes preliminary microcalorimetric measurements on designing and exploiting of surface active molecular assemblies of DNA, liposomes and N-alkylN,N,N-trimetylammonium ions (CnTMA, n = 12) and Mg2+, as a model supramolecular pharmaceutical formulation compacted for therapeutic gene transfection, or alternatively for use in DNA chromatography. Following the relevant proposal to mimic the way of action of much superior viruses, as compared to non-viral DNA carriers, for improvement of the transfection efficiencies of therapeutic gene delivery vehicles, our efforts concentrated on designing such tools, acting via membrane destabilization and fusion pathway instead of receptor-mediated route [30]. The employment of more naturally encountered surfaces is emphasized, as described in our recent study [28]. The advantages of using liposomes as compared to other lipid-containing drug carrier systems are well established [31]. Having considered the cytotoxicity problems of the currently employed cationic lipids, neutral phospholipid vesicles deserve to be studied in more detail as promising alternatives. Our current research focuses on a nanoscale complex formations between zwitterionic liposomes and DNA, mediated by various inorganic cations, acting as condensing agents. Our motivation for such an experimental design has come from recent reports on positive effects of divalent cations, such as Ca2+ and Mg2+ on transfection efficiency [32,33]. The phase behavior of complex formed between neutral lipid and calf thymus DNA in the presence of Mg2+ is pre-

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sented. N-alkyl-N,N,N-trimetylammonium ions were chosen as an amphiphilic system, whose surface properties are to be compared with Mg2+ with regard to ternary complex formation with liposomes and ability to compact DNA. Mg2+ is tested as a compaction agent in the presence of phospholipid vesicle suspensions, in the light of its role in numerous cellular events. Its high intracellular concentrations, as well as its well-known property of phosphate group transfer [34] makes it preferred natural divalent inorganic cation in comparison with commonly used synthetic polymers, which offer system versatility and large selection of polymer species, but are not encountered in biointerfaces. Ion transport, for instance, takes place by metal binding to cell membranes. Neutral phospholipids are interesting research subject not only in terms of the abovementioned preference of liposome gene delivery designers, but also concerning fundamental cell biology events. Since phosphatidylcholine moeity is a major constituent of the total phospholipid bilayer content, it is useful to study its interactions with various metals. Hence, the suggested phosphatidylcholine–Mg2+ binary mixture would then give further data on biological implications of metal ion control of cell membrane fluidity. N-alkyl-N,N,N-trimetylammonium ions (C12TMA) ions were selected due to their interesting but yet insufficiently studied effects as membrane destabilizing and lipid penetrating agent. Provided its interactions with model membranes are characterized sufficiently, a synthesis of new relevant quaternary bisammonium

Fig. 1. Thermotropic phase behavior of calf thymus DNA in complex with lipid in the presence of C12TMA and Mg2+, respectively. (1) DPPC–MLV; (2) equimolar mixture of DPPC–ULV with DNA: (3) DPPC–ULV–DNA complex by Mg2+ in 1:3:1 ratio; (4) DPPC–ULV–DNA complex in the presence of C12TMA; (5) DPPC–ULV–DNA mixture in the presence of C12TMA after heating of the sample; (6) DNA–Mg2+ binary mixture.

compounds and further studies on their effects on cell surfaces with biomedical profits will be stimulated [35]. Fig. 1 depicts DSC heating scans of DPPC vesicles and their ternary complexes with calf thymus DNA in the presence of either Mg2+ or C12TMA. The first curve (1) is a calibration mark starting with a typical DPPC multilayer phase transitions, with a pre-transition temperature peak at around 36 °C with DHcal of 3.9 kJ/mol and the gel-to-liquid crystal temperature at 41.9 °C (Table 1). The determined values are in a good agreement with those reported previously in the lipid database (LIPIDAT): http://lipidat.chemistry.ohiostate.edu. The signal after the lipid phase peaks is a base line with calibration mark (50 µW, DT = 4), obtained during the filling of both calorimetric cells with solvent. The next curves show the change in phase behavior of extruded unilamellar lipids upon their reaction with DNA in the presence of Mg2+ (2) and (3) in various ratios or C12TMA (4) and (5), respectively. Compared with pure DPPC, triple complexes of DPPC– ULV with DNA and Mg2+ in equimolar ratios possess broader lipid peak with decreased maximum. The pre-transition disappears (Fig. 1 (2, 3) and Table 1). Similar thermogram is obtained for this ternary complex in ratio of 1:3:1, however, with a shift of the second lipid–DNA phase to lower temperature at around 70 °C. The last endotherm belonging to free nucleic acid melting depicts a difference with that reported for plasmids, which show peaks at 60 °C attributed to linear and open-circle plasmid DNA. Another peak of such plasmid DNA phase is usually larger, seen at 80 °C and corresponds to supercoiled form of the plasmid [36]. This DSC scan of ternary complexation was preceded first by DPPC–ULV peak at 41.9 °C, by a second peak belonging to DPPC–DNA equimolar binary complex at 51.3 °C and a third minor peak of unbound DNA appearing at ~60 °C. This is a resolution profile of the thermograms (2) and (3), which results only after addition of Mg2+. The observed peak distribution indicates that the fraction of liposome-free DNA is less encountered than the bound DNA in the lipoplex. The liposome–DNA association results in the decrease of the DCp. Interestingly, no any DSC signal was detected for Mg2+–DPPC mixture (data not shown), which was observed previously turbidimetrically [28]. Addition of C12TMA to DPPC–ULV–DNA mixture results in broadening of the thermograms leading to difficulties of estimations of its onset point, peak top, and hence the endothermic effect. For these reasons, the quantitative evaluation of the surfactant effects should be done with precautions and are, therefore, not represented in Table 1. On the other hand, their properties reported here with respect to DNA–liposome

Table 1 Thermodynamics of DPPC–vesicle binding to calf thymus DNA in the presence of Mg2+ a DNA–lipid mixtures DPPC–MLV DPPC–ULV–Mg2+–DNA (1:1:1 ratio) DPPC–ULV–Mg2+–DNA (1:3:1 ratio)
a

Tp 36.05 – –

Tm 41.9 41.7 41.75

DT1/2 0.6 2.35 1.65

DHcal 31.9 10.8 9.7

DHvH 5501 1402 1998

R 172 129 206

Represents a mean of six independent DSC measurements. Each measurement was performed as described in Sections 2.1 and 2.2. r is estimated as: r = DH/DHcal.

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condensation is compared with that of Mg2+. This is done solely for testing the electrostatics and hydrophobicity reasoning with regard to the thermodynamics of phospholipid binding to DNA followed by its compaction. Moreover, DSC is a very sensitive way of studying certain changes in bilayer packing, with the presence of units interfering with chain packing in the bilayer causing a decrease in the temperature of the main transition [37]. The incorporation of a substance in a liposome bilayer has a more profound effect on the lipid phase transitions peaks. The shift or disappearance of pretransition or main phase transition can thus be used as an informative indicator of the existence and level of included materials in the leaflets. Thus, the entrapment of molecules in liposomes can be quantified by determining the change in temperature of the onset of the transition, as well as by measuring of the peak temperature as shifts in the transition temperature, rather than presenting them as abstract values (Table 1). The DSC scan (4) of Fig. 2, represents a thermogram of a complex formed between DPPC and DNA in the presence of C12TMA ions. It is interesting to study assemblies of this type, since surfactants are used for extraction of proteins from phospholipid membranes [38], which could also appear as nucleic acid–protein complexes, specifically or non-specifically attached to cell membrane fractions. These interactions can be approached by thermodynamic measurements. It is worth investigating their binding characteristics, as drugs usually form supramolecular complexes with proteins, membrane phospholipids and nucleic acids (DNA and RNA), and their subsequent release depends on the strength of binding and on the reversibility of this interaction. The evaluation of physicochemical stability of the samples with respect to temperature variations is crucial due to their thermodynamic profiles in terms of manufacturing issues, as recommended also for other relevant multiple emulsions [39]. Moreover, since the enthalpy of binding is a temperature dependent event [29], the effect of heating of the sample is emphasized (Fig. 1 (4)

Fig. 2. Phase behavior of the control samples used: (1) DNA–C12TMA binary mixture; (2) DNA in concentration used as in Sections 2.1 and 2.2; (3) DNA in doubled concentration; (4) DPPC–ULV–C12TMA binary mixture.

vs. (5). The single melting peak corresponding to the ternary complex formed between DPPC–C12TMA–DNA appears at approximately 33 °C. The melting behavior of the control sample DNA–C12TMA binary mixture (Fig. 2 (1)) is composed of two separate phases. The first situated at 47 °C belongs to DNA phase, the separate melting behavior of which is shown with two different concentrations on Fig. 2 (2) and (3), respectively. The second phase is a structural transition seen after 85 °C and continues until 100 °C, corresponding to complex of DNA bound to C12TMA. This type of melting behavior indicates the occurrence of aggregation reaction between the latter molecules. While, large enthalpy variations with temperature are compensated by the hydrophobic component of entropy, it is possible to estimate electrostatic and hydrophobic contributions to the enthalpy at various temperatures by applying additivity, as recently shown for alkylammonium binding to DNA by isothermal titration calorimetry [29]. Fig. 2 (2) is a DNA sample, prepared as described in Sections 2.1 and 2.2, while (3) is the same DNA sample in doubled concentration. These two thermograms show a typical melting behavior of DNA and its synthetic models, which in calorimetric determinations appear as a single peak. This highly cooperative process represents unwinding of the double stranded structure into two polynucleotide strands, which fold into separate chaotic globules. The equality of the heat capacities of the native and denatured DNA states is a typical feature of this process, which makes quantitative determination of thermal effects of melting, as well as free energy DG of stabilization of native DNA structure easier. This kind of structural transitions of DNA (Fig. 2 (2) and (3) are different from those of lipid phase behavior (Fig. 1 (1)). Lipids undergo another type of structural transitions, or gel-to-liquid crystalline transitions, which is also their main phase transition. In this respect, these are different from the abovementioned events in that in phospholipids the degree of cooperativity of their main phase transitions entirely depends not on the inner molecular but on interactions between molecules. For such systems, the effective Van’t Hoff enthalpy is greater than calorimetrically determined. In general, the degree of cooperativity of the is given as: DHvH = NDH, where N is the number of molecules in the cooperativity unit. Testing this kind of dependence on DNA concentration is crucial, as also shown by another relevant thermodynamic study [29]. In agreement with this reminding and with the mathematical model presented in the latter study, the prediction is that the position of the binding peak would be shifted upon changing the DNA concentration. Therefore, it is important to perform microcalorimetric scans at several nucleic acid concentrations at previously prepared DNA–ligand ratios. This is also seen for DPPC–Mg2+–DNA ternary complex in equimolar and 1:3:1 ratios, respectively (Fig. 1. (2) and (3)). DNA sample examined in two different concentrations already shows similar melting behavior, but with various peaks (Fig. 2 (2) and (3)), which supports the expectation of dependence of the results on nucleic acid concentration used. The lower DNA concentration in lipoplexes results in a little bit prolonged

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second peak, as compared with that of higher DNA amount (Fig. 1 (2) vs. (3)), as also indicated by the experimental enthalpies. However, to relate this effect to transfection efficiency of these lipoplexes still remains to be elucidated. Interestingly, this effect is less seen in the case of C12TMA–DNA binary mixtures, where the most reproducible results were obtained for their ratio of 1–1.8. In this case, the effect of heating and aggregation is more profound (Fig. 1 (4) and (5) and Fig. 2 (1)). Such dependence of the electrostatic component of binding Gibbs free energy and entropy on reactant concentration suggests the existence of an aggregation reaction, which was also observed in DNA–lipid complexes [28,29]. The Mg2+-ions at the equimolar amounts with DNA increases the Tm value by 33.7 °C, due to Mg2+-induced duplex stabilization. ULVs treated with the same Mg2+ concentration did not produce such a shift, which is normally detected spectroscopically [28]. Mg2+ induces the formation of substantial amount of circular DNA, suggesting that Mg2+ cations stabilize the interaction of polynucleotide cohesive ends, the effect being dependent on the concentration of MgCl2 and possibly being a sequence-specific event [40]. The formed circular molecules are stabilized by Mg2+, but are not covalently closed. Although, Mg2+ stabilizes end-to-end interactions, it is likely that a dynamic equilibrium exist between linear and circular fragments. C12TMA ions cause a decrease in lipid ordering by exerting a perturbation effect on the lipid bilayer structure. Trimethylammonium ions possess an intermediary position among CnTMA homologues, in terms of lipid ordering, as demonstrated by recent ESR studies [41]. This is characteristic biphasic behavior of these alkylammonium series, similar to other amphiphilic compounds, suggesting that the interaction of CnTMA molecules with lipid membranes has important biological implications. Moreover, other relevant ions, such as bis-quaternary ammonium ions (bis-A2+), e.g. alkane-bisalkylammonium ion, which is a divalent organic ion with two positively charged sites connected by a spacer chain and having also six side chains, has attracted research interest not only due to its biological properties as ion channel blocker of acetylcholine receptors, but also as a model compound for studying the cation effects on hydration of hydrophobic cations. In this respect, it is argued that if its hydrophobicity could be controlled precisely, it could have had a more significant value in a two-phase system, such as a membrane, indicating the importance of evaluating various alkylammonium species with different structures concerning this feature. C12TMA ions possess a hydrophilic positively charged ammonium group and a hydrophobic alkyl chain of 12 –CH2 groups. Due to this amphiphilic character these ions partition between aqueous and lipid phases. However, the observed cut-off effect of the whole series of alkylammonium ions in decreasing lipid order cannot be the result only of partition equilibrium between these two phases. This is also a concentration dependent effect possibly caused by lateral bilayer expansion due to positioning of such amphiphilic ions in the

region of the lipid leaflet, exerting an additional effect of amphiphile partitioning between aqueous and lipid phases [41]. Hydrophobically driven insertion of C12TMA ions into the phosphatidylcholine lipid layer brings about attractive electrostatic forces for DNA polyanion. In this respect, C12TMA ions affect the phospholipid vesicle in a similar way of Mg2+ adsorption onto their surface (Fig. 3) creating a positive liposomal interface, which can also be detected by their electrophoretic mobility measurements [42]. The structure of the interacting biopolymers in lipoplex formulations is a matter of controversy. Research evidence indicates that both DNA [43] and lipids [44] affects each other’s structural transitions during complex formation. Most of the interpretations for this self-assembly in terms of wellestablished polyelectrolyte theories for interactions between oppositely charged macromolecules are an oversimplified view of the real structures. It is likely that structures similar to cationic lipid–DNA complexes are formed [35,45]. In our opinion, kinetic vs. thermodynamic stability features govern this self-association in more complex way. Within this respect, the model considers the lipid–DNA structures as overlaying layers of DNA adsorbed onto lipid bilayers, after charge neutralization [28]. The process is governed by adsorbed cations (Me2+) on the surface of the zwitterionic lipids. The presence of such multilayers of alternating lipid–DNA assemblies is due to formation of condensed DNA as parallel arrangements between the lipid bilayers [46]. This is expected, due to the existence of 3-D correlation forces between the DNA-covered lipid layers, following DNA-driven formation of multilamellar liposomes from ULVs. Based on our own polyelectrolyte data, as well as on relevant measurements reported in the literature, Fig. 3 represents a proposal of the possible structures of C12TMA–DNA–liposomes and DNA–Mg2+-neutral liposomes ternary complexes. Fig. 3a suggests the possible structure of DNA–C12TMA– liposome complexes. Under the employed conditions, the initially relaxed DNA in solution is complexed by surfactant molecules, which adsorb on the nucleic acid surface forming a, micelle-like domains. C12TMA molecules also partition in lipid bilayer forming a swollen mixed bilayers. The latter can also lead to subsequent humpbacked vesicles with surfactant at regions of high curvature. Afterwards, the DNA in unfolded form is apparently adsorbed on the surface of surfactant– DPPC vesicles, as also suggested by a fluorescent study on neutral lipids, employing cationic surfactant [47]. Since surfactant molecules become incorporated into the liposome bilayer due to the partition equilibrium between bilayer and aqueous phase, normally the binding of C12TMA–DNA complexes to the vesicle surface through hydrophobic forces results in opening of the micelle-like domains and partitioning of C12TMA ions in the lipid bilayer. Hence, employment of cationic surfactant tend to form a fully relaxed DNA, which is bound with significant stability to plasma membranes, resulting in a difficult to internalize in a cell structure by endocytosis. In contrast, unilamellar phosphatidylcholine

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Fig. 3. Proposed structures of neutral liposome–detergent–DNA (a) and DPPC–Mg2+–neutral liposome (b) self-assemblies. Me2+ refers to inorganic divalent metal cations (modified version of the structures proposed originally by Clamme et al. [47] and Kuvitchkin and Sukhomudrenko [54]).

vesicles interact with DNA via a mechanism of nucleic acid helix-induced liposome aggregation and subsequent lipid fusion (Fig. 3b). A liposome with higher curvature could be formed, which is advantageous for further increasing the gene transfection efficacy. Thermotropic phase behavior reported in Figs. 1 and 2, probably represent several intermediary DNA–lipid structures [22]. Usually, several MLVs aggregate around a Mg2+-induced compacted DNA as grapes. ULVs follow DNA-induced fusion resulting in generation of liposome with preferable higher curvature. Even though the suggested structure remains to be confirmed by further structural analyses, such a local DNA unwinding is likely to occur, because of the spectroscopic evidence for significant disruption of the planar interactions between the bases [47,48]. The process is hydrophobically and electrostatically controlled, since liposomes aggregate and fuse, in the presence of oppositely charged particles [49]. In the currently presented system, zwitterionic liposomes fuse in the presence of

Mg2+. Hence, polyanions like DNA play an active role in adhesion, aggregation and fusion, by bridging two liposomes in close contact, with surface adsorbed metal cation-induced fusion. Since, a variety of possibilities exist regarding the structure of liposome–DNA formulations, it seems that, besides contributions due to charge neutralizations or relative lipid/DNA ratios, the absolute concentrations of the engaged system components play an additive role in thermodynamically preferred lipoplex structure formation. The major problem with nucleic acid delivery employing liposomes as a therapeutic gene vector is efficient escape from the clearance by the reticuloendotelial system (RES) [8,9,30,50,51]. The immune response is due to accumulation of serum proteins on the liposome surface, resulting in rapid removal primarily by liver, spleen, lymph nodes, and lung tissue, the process being referred to as opsonization. Usually neutral multilamellar and unilamellar vesicles follow a slower

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clearance rate than negatively and positively charged MLVs. ULVs are characterized with longer residence time than MLVs [9]. The unavoidable hurdle until now has been the final fate of liposome, which is engulfed by macrophages of the RES, via the lysosomal participation. Such fusion with lysosomes leads to destruction of liposomes through the action of phospholipases with subsequent release of vesicle’s content. Hence, efficient strategies to overcome opsonization of lipid vesicles have been designed, thus developing the concept of liposome targeting to specific target cells and tissues. Initially, conjugation of various polymers and other macromolecules for this purpose was highly cell type-dependent and ended with discouraging results. Therefore, besides the required knowledge on the influence of in vivo environment on both vesicle leakage and clearance rates, searching for improvement of the specificity of cell and tissue recognition becomes essential. The objective is to deduce alternative route of successful gene delivery avoiding the receptor-mediated intake. With this respect, we have worked on electrostatic and hydrophobic control of membrane destabilization. Despite the fact that often the fluidity of the phospholipid surface is described as disordered phase, a certain level of interfacial organization or lateral heterogeneity is present due to multilamellar nature of the vesicles with high degree of cooperativity. Hence, knowledge of its surface structure is crucial for designing efficient lipid-based gene delivery systems and suggesting new clues on their interactions with cellular surfaces, as well as internalization mechanisms of the entrapped therapeutic DNA through membranes. Although increased surface rigidity prolongs the circulatory half-time of lipid vehicles, the commonly employed phospholipids in drug delivery are at physiological temperature entirely or partly in the fluid state [52]. For proper understanding of the relationships between surface heterogeneity and lipid-carrier function it is important to distinguish between various levels of lipid lateral organization differing in their lifetimes and sizes. Lipid heterogeneity is a consequence of “solid–liquid” or “liquid–liquid” phase separation, as well as by thermal density fluctuations leading to the appearance of short lived gel-like microdomains in a fluid (liquid-crystalline) environment (Fig. 3). Their appearance implies function of low-ordered boundary regions, which in spite of their short lifetime and small size serve as sites of ion penetration and enzymatic reactions. They also may serve as nucleation centers preceding gross lipid phase separation induced by cations and are believed to play a role in fusion of the lipid carriers with a plasma membrane of living cells [52]. Numerous suggestions have been proposed for overcoming the membraneous barriers, which significantly inhibit the entry of therapeutic DNA to the nucleus. Certain cationic amphiphiles, such as those presented, able of free partitioning through cell membranes and rapidly localizing to specific nuclear chromosomal regions are being studied for their carrier potential [53]. The proposal that if such nucleic acid binding agents express their membrane transport properties cre-

ating a more hydrophobic DNA, then they could facilitate surpassing the membrane barriers that currently limit DNA delivery into the nucleus by non-viral vectors, remains to be tested. There are several potential ways, though which metal ions can increase gene transfection efficiency. Thus, their ability to partition rapidly through cell membranes entering the nucleus may confer novel intracellular trafficking pathways on complexed DNA. Fig. 4 shows the simplified possibility of the proposed DNA-mediated liposome fusion with target cell membranes. The suggested structure of the entrapped DNA [28] is based on Fig. 3b, originally described by Kuvitchkin and Suchomudrenko (1987) [54]. The model describes the aggregation of several vesicles resulting in fusion of ULVs, induced by polynucleotide chain unwinding. Thus, the desired highly fusogenic vesicle with higher curvature is formed. Mg2+ used, bridge the DNA polyanion to charge reversed liposomes accelerating further their membrane destabilizing properties. We hypothesized that based on these features of Mg2+ as a surface active compound, similarly to cationic peptides, an inorganic cation-mediated cell membrane destabilization could occur, governed by electrostatic and hydrophobic forces. As shown on Fig. 4, Mg2+ bring the neutral lipid vesicles with the encapsulated nucleic acid in close proximity with negatively charged target cell surface. When optimal combination of surface factors needed for bilayer destabilization is

Fig. 4. Proposed mechanism of Mg2+-induced cellular internalization of neutral liposome–DNA formulation.

E. Süleymanoglu / Il Farmaco 60 (2005) 701–710 ˘

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reached, coupled with suitable amphiphilicity, the DNAmediated fusion between zwitterionic liposomes and cell membrane occurs. This proposal does not, however, rule out the possibility of existence of another cellular internalization mechanism of neutral liposome–DNA complexes. Nevertheless, the described ability of free metal cations for both DNA and lipid binding, partitioning and permeating through cell membranes entering the nucleus [31,32], coupled with accelerated nuclear delivery of DNA, supported by helix-binding molecules [27], is consistent with the hypothesis that divalent metal cations can confer membrane-permeant properties on complexed DNA. The exact mechanism of nucleic acidexerted phospholipid fusion in the presence of metal cations is unknown. One possibility could be, that similarly to cationic peptides [30], the electrostatic screening of the hydration shell of inorganic cations compensates for the intervesicle repulsive forces. Once the opposing membranes are in close contact zone, the hydrophobic and electrostatic features of metal cation form a complex between liposome and cell phospholipids followed by lipid mixing (Fig. 4). Our proposal is further supported by recent results of Sato et al. (2003) [55] on Mg2+-induced DNA attachment to phospholipids. In addition, closely relevant work of describes DNA–Mg2+ nanoscale mixtures as potential non-viral vector for gene delivery [56]. Besides their membrane permeating properties, Mg2+ions employed could also increase the transfection efficiency in other respects. Nuclear stability and topology of genomic DNA seems to be one cellular mechanism for controlling gene transcription [27]. Metal cations may target complexed DNA via nuclear localization signals (NLS) to transcriptionally active chromatin regions. Thus, gene carrier-mediated nuclear targeting could represent a way of enhancing the gene expression levels. This suggestion is in agreement with recent reports on Ca2+ and Mg2+-induced increase of expression of the transfected genes [31,32]. Metal cations can govern the conformation of the transfected DNA, showing the role of nucleic acid topology control in gene transfection. Employment of naturally occurring divalent metal cations as oppose to other synthetic and potentially mutagenic molecules [27], deserves to be studied in more detail.

binding to cellular receptors and will further define the issue of nucleic acid receptors on cell surfaces.

Acknowledgments I thank Professor R.I. Zhdanov (V.N. Orekhovich Institute of Biomedical Chemistry, Russian Academy of Medical Sciences) for introducing me to the topic of nucleic acid– membrane interactions and for his help as my postgraduate supervisor. The hospitality of Professor P. Bálgavy (J.A. Comenius University-Bratislava) and Dr. J. Bagelova (Institute of Experimental Physics, The Slovak Academy of Sciences-Košice) is greatly acknowledged.

References
[1] R. Chang, Physical Chemistry for the Chemical and Biological Sciences, Chapter 16, Intermolecular Forces, University Science Books, Sausalito, California, 2000 (pp. 669–700). D. Leckband, J. Israelachvili, Intermolecular forces in biology, Q. Rev. Biophys. 34 (2001) 105–267. J.B.F.N. Engberts, M.J. Blandamer, Understanding organic reactions in water: from hydrophobic encounters to surfactant aggregates, Chem. Commun. (2001) 1701–1708. A.T. Florence, D. Attwood, Physicochemical Principles of Pharmacy, third ed, Palgrave, 1998. E.H. Kerns, L. Di, Physicochemical profiling: overview of the screens, Drug Discovery Today, Technologies 1 4 (2004) 343–348. P.J. Crowley, L.G. Martini, Formulation design: new drugs from old, Drug Discovery Today, Therapeutic Strategies 1 4 (2004) 537–542. A. Simmonds, M. Cunningham, The preparation of protein–nucleic acid conjugates, in: M. Aslam, A. Dent (Eds.), Bioconjugation: Protein Coupling Techniques for the Biomedical Sciences, Macmillan Reference Ltd, 1998, pp. 483–503. P.-G. de Gennes, Problems of DNA entry into a cell, Physica A (Amsterdam) 274 (1999) 1–7. A.J. Kirby, P. Camilleri, J.B.F.N. Engberts, M.C. Feiters, R.J.M. Nolte, O. Söderman, M. Bergsma, P.C. Bell, M.L. Fielden, P. García Rodríguez, A. Guédat, C. Kremer, C. McGregor, G. Perrin, M.C.P. Ronsin, van Eijk, Gemini surfactants: new synthetic vectors for gene transfection, Angew. Chem. Int. Ed. Engl. 42 (2003) 1448– 1457. N.S. Templeton, Gene and Cell Therapy-Therapeutic Mechanisms and Strategies, second ed, Marcel Dekker, 2003. N.A. Kootstra, I.M. Verma, Gene therapy with viral vectors, Annu. Rev. Pharmacol. Toxicol. 43 (2003) 413–439. R. Dass, Cytotoxicity issues pertinent to lipoplex-mediated gene therapy in-vivo, J. Pharm. Pharmacol. 54 (2002) 593–601. A.D. Miller, The problem with cationic liposome/micelle-based nonviral vector systems for gene therapy, Curr. Med. Chem. 10 (2003) 1195–1211. A.D. Miller, Nonviral liposomes, Methods Mol. Med. 90 (2004) 107–138. A.V. Kabanov, V.A. Kabanov, DNA complexes with polycations for the delivery of genetic material into cells, Bioconjug. Chem. 6 (1) (1995) 7–20 (Jan–Feb). C.L. Gebhart, A.V. Kabanov, Evaluation of polyplexes as gene transfer agents, J. Contr. Releas. 73 (2001) 401–416. K.B. Anwer, G. Rhee, S.K. Mendiratta, Recent progress in polymeric gene delivery systems, Crit. Rev. Ther. Drug Carrier Syst. 20 (4) (2003) 249–293.

[2] [3]

[4] [5] [6] [7]

[8] [9]

[10] [11]

4. Conclusions Neutral liposome–Mg2+–DNA formulations described here are highly dynamic structures, frequently changing their transfection characteristics, depending on particular laboratory protocol. On the other hand, these ternary complexes appear to be a reproducible lipoplex design, but their serum stability profiles are not yet well defined. While promising results are reported recently on their use [57], more immunological studies are needed for their in vivo evaluation. Nevertheless, the concept of metal-based pharmaceuticals [58], undertaken here, will open new insights for studying whether these supramolecular complexes follow the similar principles of

[12] [13]

[14] [15]

[16] [17]

710

E. Süleymanoglu / Il Farmaco 60 (2005) 701–710 ˘ [39] A. Baillet, P. Prognon, Analytical techniques for tracer method monitoring, in: J.-L. Grossiord, M. Seiller (Eds.), Multiple Emulsions: Structure, Properties and Applications, Éditions de Santé, Paris, 1998, pp. 225. [40] P.R. Dahlgren, Y.L. Lyubchenko, Atomic force microscopy study of the effects of Mg2+ and other divalent cations on the end-to-end DNA interactions, Biochemistry 41 (2002) 11372–11378. [41] J. Gallová, F. Sersen, F. Devinski, P. Balgavy, The perturbation effect of N-alkyl-N,N,N-trimetylammonium ions on a model membrane. A spin-label study, Acta Facultatis Pharmaceuticae Universitatis Comenianae, Tommus L (2003) 1–10. [42] A.A. Yaroslavov, O.Y. Udalykh, N.S. Melik-Nubarov, V.A. Kabanov, Y.A. Ermakov, V.A. Azov, F.M. Menger, Conventional and gemini surfactants embedded within bilayer membranes: contrasting behaviour, Chem. Eur. J. 7 (2001) 4835–4843. [43] Y.S. Tarahovsky, R.S. Khusainova, A.V. Gorelov, T.I. Nikolaeva, A.A. Deev, A.K. Dawson, G.R. Ivanitsky, DNA initiates polymorphic structural transitions in lecithin, FEBS Lett. 390 (1996) 133–136. [44] T. Akao, T. Fukumoto, H. Ihara, A. Ito, Conformational change in DNA induced by cationic bilayer membranes, FEBS Lett. 391 (1–2) (1996) 215–218. [45] C.R. Safinya, Structures of lipid–DNA complexes: supramolecular assembly and gene delivery, Curr. Opin. Struct. Biol. 11 49 (2001) 440–448. [46] W.M. Gelbart, R.F. Bruinsma, P.A. Pincus, V.A. Parsegian, DNAinspired electrostatics, Phys. Today (September 2000) 38–44. [47] J.P. Clamme, S. Bernacchi, G. Vuilleumier, G. Duportail, Y. Mély, Gene transfer by cationic surfactant is essentially limited by the trapping of the surfactant/DNA complexes onto the cell membrane: a fluorescence investigation, Bichim. Biophys. Acta 1467 (2000) 347– 361. [48] D. Llères, J.-M. Weibel, D. Heissler, G. Zuber, G. Duportail, Y. Mély, Dependence of the cellular internalization and transfection efficiency on the structure and physicochemical properties of cationic detergent/DNA/liposome, J. Gene Med. 6 (2004) 415–428. [49] S.M. Mel’nikov, R. Dias, Y.S. Mel’nikova, E.F. Marques, M.G. Miguel, B. Lindman, DNA conformational dynamics in the presence of catanionic mixtures, FEBS Lett. 453 (1999) 113–118. [50] P. Opanasopit, M. Nishikawa, M. Hashida, Factors affecting drug and gene delivery: effects of interaction with blood components, Crit. Rev. Ther. Drug Carrier Syst. 19 (3) (2002) 191–233. [51] H. Kamiya, H. Akita, H. Harashima, Pharmacokinetic and pharmacodynamic considerations in gene therapy, Drug Discov. Today 1 8 (21) (2003) 990–996. [52] L. Bergelson, A. Domb, The surface structure of lipid drug carriersinfluence on carrier–cell interaction, in: R.H. Müller, W. Mehnert (Eds.), Particle and Surface Characterization Methods, Medpharm GmbH Scientific Publishers, Stuttgart, Germany, 1997, pp. 169–184. [53] A. Asokan, M.J. Cho, Cytosolic delivery of macromolecules. 3. Synthesis and characterization of acid-sensitive bis-detergents, Bioconj, Chem. 15 (2004) 1166–1173. [54] V.V. Kuvitchkin, A.G. Sukhomudrenko, Interaction of natural and synthetic polynucleotides with liposomes in the presence of divalent actions, Biophysics, XXXII 4 (1987) 628–633 (In Russian). [55] Y. Sato, S.-I. Nomura, K. Yoshikawa, Enhanced uptake of giant DNA in cell-sized liposomes, Chem. Phys. Lett. 380 (2003) 279–285. [56] G. Bhakta, S. Mitra, A. Maitra, DNA encapsulated magnesium and manganous phosphate nanoparticles: potential non-viral vectors for gene delivery, Biomaterials 26 (2005) 2157–2163. [57] P. Esponda, M. Goldstein, S.S. Witkin, In vitro transfection of the human vas deferens using DNA–liposome and DNA–neutral lipid complexes, Fertility and Sterility 81 1 (2004) 171–175. [58] J. Mönkkönen, A. Urtti, Lipid fusion in oligonucleotide and gene delivery with cationic lipids, Adv. Drug Deliv. Rev. 34 (1998) 37–49.

[18] S. Mehier-Humbert, R.H. Guy, Physical methods for gene transfer: improving the kinetics of gene delivery into cells, Adv. Drug Deliv. Rev. 57 (5) (2005) 733–753. [19] M.G. Miguel, A.A.C.C. Pais, R.S. Dias, C. Leal, M. Rosa, B. Lindman, DNA–cationic amphiphile interactions, Colloids Surf. A: Physicochem. Eng. Aspects 228 (2003) 43–55. [20] T. Kunitake, Self-assemblies of biomembrane mimics, in: A. Baskin, W. Norde (Eds.), Physical Chemistry of Biological Interfaces, Marcel Decker, Inc., New York, Basel, 2000, pp. 283–305. [21] T. Hianic, A. Labajova, Electrostriction of supported lipid films at presence of cationic surfactants, surfactant–DNA and DNA–Mg2+ complexes, Bioelectrochemistry 58 (2002) 97–105. [22] F. Liu, L. Huang, Development of non-viral vectors for systemic gene delivery, J. Contr. Release 78 (2002) 259–266. [23] N.S. Templeton, Developments in liposomal gene delivery systems, Expert Opin. Biol. Ther. 1 (2001) 1–4. [24] N.S. Templeton, Liposomal delivery of nucleic acids in vivo, DNA Cell Biol. 12 (2002) 857–867. [25] A.S. Ulrich, Biophysical aspects of using liposomes as delivery vehicles, Bioscience Rep. 22 2 (2002) 129–149. [26] in: R.I. Mahato, S.W. Kim (Eds.), Pharmaceutical Perspectives of Nucleic Acid-Based Therapeutics, Taylor and Francis, 2002. [27] S. Fong, Y. Liu, T. Heath, P. Fong, D. Liggitt, R.J. Debs, Membranepermeant, DNA-binding agents alter intracellular trafficking and increase the transfection efficiency of complexed plasmid DNA, Mol. Ther. 10 4 (2004) 706–718. [28] E. Süleymanoglu, Self-assembling polyelectrolyte nanostructures for˘ mulated for therapeutic gene delivery, Mahidol University J. Pharm. Sci. 30 (2003) 37–51. [29] D. Matulis, I. Rouzina, V.A. Bloomfield, Thermodynamics of cationic lipid binding to DNA and DNA condensation: roles of electrostatics and hydrophobicity, J. Am. Chem. Soc. 124 (2002) 7331–7342. [30] G. Fujii, To fuse or not to fuse: the effects of electrostatic interactions, hydrophobic forces, and structural amphiphilicity on proteinmediated membrane destabilization, Adv. Drug Deliv. Rev. 20 38 (3) (1999) 257–277. [31] P.H. Stachl, Drug solubilization with organic solvents, surfactants and lipids, in: C.G. Wermuth (Ed.), The Practice of Medicinal Chemistry, second ed, Elsevier, 2003. [32] M.I. Lam, P.R. Cullis, Calcium enhances the transfection potency of plasmid DNA–cationic liposome complexes, Biochim. Biophys. Acta 1463 (2000) 279–290. [33] E.H. Chowdhury, K. Megumi, I. Harada, A.K. Kundu, A. Toshihiro, Dramatic effect of Mg2+ on transfecting mammalian cells by DNA/calcium phosphate precipitates, Anal. Biochem. 328 (2004) 96–97. [34] J.J.R. Fraústo da Silva, R.J.P. Williams, The Biological Chemistry of the Elements, The Inorganic Chemistry of Life, Chapter 9: The Biological Chemistry of Magnesium: Phosphate Metabolism, second ed, Oxford University Press, Oxford, New York, 2001 (251–276). [35] V. Majtán, L. Majtánova, Effect of new quaternary bisammonium compounds on the growth and cell surface hydrophobicity of Enterobacter cloaceae, Cent. Eur. J. Publ. Health 8 (2000) 80–82. [36] B.A. Lobo, S.A. Rogers, C.M. Wiethoff, S. Choosakoonkriang, S. Bogdanovich-Knipp, C.R. Middaugh, Characterization of cationic vector-based gene delivery vehicles using isothermal titration and differential scanning calorimetry, in: M.A. Findeis (Ed.), Methods in Molecular Medicine, Vol. 65, Nonviral Vectors for Gene Therapy, Humana Press Inc., Totowa, NJ, USA, 2001, pp. 319–348. [37] K.M.G. Taylor, D.Q.M. Craig, Physical methods of study: differential scanning calorimetry, in: V.P. Torchilin, V. Weissig (Eds.), Liposomes, second ed, Oxford University Press, 2003, pp. 79–101. [38] A. Chatterjee, S.P. Moulik, P.R. Majhi, S.K. Sanyal, Studies on surfactant–biopolymer interaction. I. Microcalorimetric investigation on the interaction of cetyltrimetylammonium bromide (CTAB) and sodium dodecylsulfate (SDS) with gelatin (Gn), lysozyme (Lz) and deoxyribonucleic acid (DNA), Biophys. Chem. 98 (2002) 313–327.

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