2.1. Conformation of Lipid/Surfactant and Lipid/Surfactant/DNA Complexes
Fourier transformed infrared (FTIR) spectra obtained for DMPC/IMI_oxyC5_C10 and DMPC/IMI_oxyC5_C10/DNA complexes were dominated by vibrational bands of DMPC. The changes in vibrational modes reflect local effects at the molecular level, which in this certain type of structures are attributed to changes in structure and hydration level of lipid bilayers.
Fully-hydrated DMPC forms three main types of lamellar mesophases present in the studied temperature range [
25]. The phase transition sequence typical of DMPC Lβ′→Pβ′→Lα (gel→rippled gel→liquid crystalline) revealed upon increasing temperature, is manifested as discontinuous changes in band positions of CH
2 symmetric (~2850 cm
−1) and asymmetric (~2920 cm
−1) stretching vibrations [
26] and C=O stretching (~1734 cm
−1) vibrations [
27]. The exemplary FTIR absorption spectra from the CH
2/CH
3 stretching region collected for DMPC/IMI_oxyC5_C10 and DMPC/IMI_oxyC5_C10/DNA systems are presented in
Figure 2. The example of deconvoluted spectrum in the spectral range of CH
3/CH
2 symmetric and asymmetric stretching vibrations is presented in
Figure 3. The positions of CH
2 stretching modes increase upon rippled gel to liquid crystalline phase transition (the main transition is often referred to as melting of the hydrocarbon chains of phospholipid) (see
Figure 4). The spectral changes observed are directly connected with collective
trans-gauche conformational transitions in hydrophobic polymethylene chains of DMPC molecules. The discontinuous increase in wavenumbers (~2 cm
−1 and ~4 cm
−1) in the positions of bands characteristic of symmetric and asymmetric CH
2 stretching vibrations respectively, observed upon the heating of the samples, was in accordance with earlier observations [
26].
The increase in IMI_oxyC5_C10 surfactant concentration led to a significant decrease in the main transition (Pβ′→Lα) temperature (~24 °C for 150 mM DMPC and ~18 °C for 150 mM DMPC/30 mM IMI_oxyC5_C10) as well as to a broadening of the temperature range of transition as shown in
Figure 4. Moreover in the full temperature range a slight shift (by about 0.5–1 cm
−1) of the wavenumbers characterising both CH
2 stretching modes was noted with the increasing gemini surfactant concentration. These significant changes in phase behaviour of DMPC bilayers occurring with increasing surfactant concentration might be a result of internalisation of surfactant molecules into the bilayer structure. This mechanism leads to a main transition temperature shift to lower values as well as to a broadening of the transition temperature range observed for CH
2 symmetric and asymmetric stretching band positions.
Such phase transition behaviour was characteristic also of other lipid/surfactant mixed systems including those containing anionic or cationic surfactants (such as sodium dodecyl sulfate, dodecyltrimethylammonium bromide [
28] or (alcoxymethyl)dodecydimethylammonium chlorides [
29,
30]), zwitterionic surfactants (e.g., sulfobetaines [
31–
33]) as well as dicationic surfactants (alkane-α,ω-diyl-bis(dodecyldimethylammonium bromides) [
34], 1,1′-(1,6-hexan)bis-3- octyloxymethyl-imidazolium di-chloride [
35]).
The temperature dependence of band positions of CH
2 stretching vibrations for DMPC/IMI_oxyC5_C10/DNA complexes (
Figure 4) was comparable to that described above for DMPC/surfactant systems containing the same surfactant concentrations. Previous studies of cationic DMPC/DOTAP systems with a low DNA proportion observed that DNA insignificantly perturbs the global lipid organization or induced only a small disordering effect (with higher DNA concentrations) on the DMPC acyl chains [
36]. These observations suggest that only weak interactions take place between the lipid acyl chains and DNA, which is in very good agreement with our results.
Carbonyl groups of phospholipids present at the interface, between hydrophobic and hydrophilic parts of the bilayer, are capable of forming a hydrogen bond network with surrounding water molecules [
37,
38]. This fact leads to formation of two populations of carbonyl groups: water-bounded and unbounded. In the FTIR spectrum, these interactions are manifested by peak splitting into two bands, each connected with one of the described state of C=O group. Positions and intensities of those peaks vary for different lamellar mesophases (Lβ′ phase: ~1730.7 cm
−1—water-bounded state, ~1738.4 cm
−1—water-unbounded state; Pβ′ phase: ~1724 cm
−1—water-bounded, ~1742 cm
−1— water-unbounded; Lα phase: ~1721.5 cm
−1—water-bounded, ~1738 cm
−1—water-unbounded; as referred in [
27]) Therefore, the changes observed for C=O stretching vibrations provided information about the hydration level of bilayers. The hydration level of bilayers increases with increasing temperature [
26,
27]. For effective analysis of hydration changes, C=O stretching bands were localised using the first spectral momentum of the band (the centre of gravity) (
Figure 5). This method allows investigation of collective band changes treated as the effect of both, intensity and peak positions variations.
Two changes in the carbonyl stretching band position were observed in the spectrum of the reference system—150 mM DMPC water solution (
Figure 6). The observed shift in the
vCO band position towards higher wavenumbers (~0.6 cm
−1) reflects the appearance of pretransition (Lβ′→Pβ′) in the sample and the subsequent shift towards lower values (~1.5 cm
−1) is directly related to the main phase transition. The increase in the surfactant concentration in the systems studied led to a decrease in the transition temperature characterising the pretransition and the main transitions, and also to broadening of their temperature ranges. Spectral changes connected with the pretransition disappeared for two highest concentrations of gemini surfactant (15 mM and 30 mM IMI_oxyC5_C10). In the absorption spectra of these two systems only a single shift towards decreasing wavenumbers values ~0.7 cm
−1 was noted. Additionally, at high concentrations of the surfactant (7.5 mM, 15 mM and 30 mM IMI_oxyC5_C10) the carbonyl stretching band was shifted towards decreasing wavenumbers in the full temperature range.
The mechanism of pretransition depends on two events [
39]—the change in the tilt angle of hydrophobic chains and the changes in the effective area of hydrophilic head group as a result of increasing hydration, which in consequence leads to formation of a characteristic rippled structure. The presence of IMI_oxyC5_C10 surfactant in the bilayer structure may affect this mechanism in several ways. Electrostatic repulsion between the cationic head groups of the surfactant leads to an increase in the effective area of head groups and loosens up the packing of molecules in the bilayer. The increased orientation freedom may affect the collective hydrophobic chain tilt, and in consequence, the lateral stress of hydrophobic chains, responsible for formation of ripples, will be reduced. Moreover, the rippled gel phase stability depends on fluctuational correlations between adjacent bilayers [
39]. For higher surfactant concentrations (the two samples of the highest IMI_oxyC5_C10 surfactant concentration) the correlations may disappear as a consequence of electrostatic repulsions between bilayers [
39]. These structural disruptions are connected also with the increase in bilayer hydration. Facilitated water penetration into the bilayer interphase was observed as the C=O stretching band was shifted towards smaller wavenumbers upon increasing surfactant concentration.
Similarities between the FTIR spectra recorded for DMPC/IMI_oxyC5_C10/DNA and DMPC/IMI_oxyC5_C10 were also noted in the region of the stretching vibrations of the carbonyl group. In particular, the temperatures of phase transitions and the thermal ranges of these transitions were close. The only significant differences concerned the positions of the bands corresponding to the stretching vibrations of C=O in the liquid crystalline phase (Lα), for DMPC/IMI_oxyC5_C10/DNA as they appeared at by about 0.5 cm−1 lower wavenumbers than in the spectra of DMPC/IMI_oxyC5_C10.
Moreover for DMPC/IMI_oxyC5_C10/DNA lipoplexes, the characteristic shift of the C=O stretching band positions towards increasing wavenumbers (directly related to the pretransition) disappeared at 3 mM concentration of the surfactant in the system studied, and did not appear for any higher surfactant concentration (
Figure 6).
The FTIR spectra demonstrated also changes in the (PO
2)
− symmetric stretching region (
Figures 7 and
8). Phosphate groups are components of DMPC (the dominating population) as well as DNA molecules. Samples with the highest two concentrations of IMI_oxyC5_C10 surfactant (15 mM and 30 mM) show in full temperature range ~0.4 cm
−1 shift towards lower wavenumbers (
Figure 8). Moreover, for these samples, the temperature dependence of the (PO
2)
− stretching band position revealed the stepwise shift (~0.4 cm
−1 at around
T = 16 °C) upon temperature increase. These changes correspond to the shift of the C=O stretching band position towards decreasing wavenumbers observed for these samples.
Circular dichroism is the most popular spectroscopic method used for monitoring structural changes in the nucleic acid conformation. Therefore, circular dichroism was selected as the most sensitive method to determine the influence of IMI_oxyC5_C10 on DNA conformation. The CD spectra of pure DNA and lipoplexes with 0.05–5 mM of IMI_oxyC5_C10 are presented in
Figure 9. The spectrum of the pure DNA solution exhibits a positive band near 277 nm, a negative band near 245 nm and a crossover point near 260 nm, indicating a right-handed B-DNA form [
40,
41], which is a typical native double-stranded conformation of fully hydrated DNA.
The increased surfactant concentration slightly shifted the bands towards higher wavelength, for the negative band to 252 nm and for the positive band to 284 nm. The addition of the surfactant studied resulted in the interaction between the positively charged groups of the surfactant with the polyanionic DNA molecule, causing the exposure of hydrophobic parts of the surfactant to the solution.
The changes in the intensity of the CD bands can be assigned to changes in the hydration shell of the phosphate groups of DNA [
42]. The results obtained indicate that the DNA maintains the B-form up to the 2 mM concentration of IMI_oxyC5_C10 surfactant in the solution.
In complexes of DNA with dodecyltrimethylammonium bromide (DTAB), the almost identical shifts of the negative and positive bands in the CD spectrum were not accompanied by the conformational transition from B to A or B to Z form [
43]. Similar interactions between DNA and some polyamines, (e.g., spermine), observed in the CD spectra were described by Chang
et al.[
44]. Therefore, these changes can be attributed to the local perturbations in the DNA base geometry rather than to a change in the DNA helical structure. Higher concentration of IMI_oxyC5_C10 surfactant induces precipitation of IMI_oxyC5_C10/DNA lipoplexes. Unfortunately, due to the high absorption of polarised light in the UV range by phospholipids, the examination of DMPC/surfactant/DNA systems using circular dichroism was not possible.
2.2. Thermal Stability of Lipid/Surfactant and Lipid/Surfactant/DNA Complexes
The results of DSC studies of DMPC/dicationic surfactant and DMPC/dicationic surfactant/DNA systems are shown in
Figure 10, while the parameters characterising the phase transitions obtained from these data are summarised in
Table 1. The addition of IMI_oxyC5_C10 surfactant and DNA has a pronounced effect on the thermodynamic parameters of phase transitions observed for DMPC. This effect is however much stronger upon the surfactant addition. For the reference DMPC system, two phase transitions are observed and characterised by
Tonset = 14.8 °C and
Tonset = 24.2 °C corresponding to pretransition and main transition, respectively. These values are in agreement with the corresponding ones obtained for pure hydrated DMPC solutions [
45].
The influence of the dicationic surfactant on the thermodynamic parameters was significant. Even the smallest amount of IMI_oxyC5_C10 surfactant in the systems studied (0.75 mM) resulted in complete disappearance of the pretransition and a significant shift of the main phase transition temperature (ΔT = −0.7 °C). Simultaneously, a splitting of the main transition peak was observed. The first maximum can be assigned to the main transition process of DMPC/dicationic surfactant mixed phase, while the weak second peak corresponded to the main transition of pure DMPC. This effect disappears at IMI_oxyC5_C10 surfactant concentrations above 1.5 mM. With increasing concentration of IMI_oxyC5_C10 surfactant, a decrease in the main phase transition enthalpy and temperature was observed.
A decrease in enthalpy characterising the main phase transition in the systems with the highest surfactant concentrations (15 and 30 mM of IMI_oxyC5_C10) in relation to pure DMPC reached even 65% (ΔΔH = 10.8 kJ/mol) and 62% (ΔΔH = 10.3 kJ/mol). The addition of DNA to the pure phospholipid solution caused a small reduction in temperature (ΔT = −0.5 °C) and enthalpy of the main phase transition (ΔΔH = −0.6 kJ/mol).
The pretransition was detected only for the reference system DMPC (ΔH = −2.3 kJ/mol, Tonset = 14.8 °C and Tpeak = 16.1 °C) and for the system DMPC/DNA (ΔH = −3.6 kJ/mol; Tonset = 17.0 °C and Tpeak = 17.2 °C). After addition of the gemini surfactant, the pretransition is undetectable in the two series of samples studied (with and without DNA). These effects are also in full agreement with the earlier discussed FTIR results for these systems.
For the complexes of DMPC/IMI_oxyC5_C10/DNA, the decrease in enthalpy of the main phase transition was even a bit greater, while the shift of the characteristic temperatures was a bit smaller than for DMPC/IMI_oxyC5_C10 systems (see
Table 1). The temperature changes related to the incorporation of DNA and surfactant molecules into the lamellar phase have been observed for other systems of this type [
46].
2.3. Structural Parameters of Lipid/Surfactant/DNA Complexes
Figure 11 presents exemplary SAXS data recorded for DMPC/IMI_oxyC5_C10/DNA systems and d-spacing (d
001), calculated on the basis of scattering patterns. It should be noted that the concentration of DNA in the systems tested was relatively low in comparison to that in typical lipid/DNA model systems. The DNA concentration in the sample was chosen so that the ratio of negative charge of DNA polyanion (assuming a DNA mean chain length as 170 bp), to the positive charge of IMI_oxyC5_C10 surfactant ranged from 1.0 to 0.02. These values correspond to the typical range of DNA concentrations tested for the cationic carriers used in gene therapy.
Considering the low DNA concentration, we did not expect dramatic structural changes in the lipid matrix induced by the presence of DNA. The observed changes (about 0.4 nm) of the bilayer spacing were small in comparison with those in the systems not containing DNA. The observed increasing intensity of the diffraction peaks as a function of surfactant concentration also clearly shows the ability of the surfactant to promote organisation of lipid bilayers in the multilayer lamellar phase. Moreover, with increasing concentration of the surfactant in the system DMPC/IMI_oxyC5_C10/DNA (from 3 mM IMI_oxyC5_C10) besides the diffraction maxima
L1 and
L2 corresponding to the lattice constants d
001 and d
002, the SAXS curves reveal the third diffraction maximum,
L3 (see
Figure 11a). Detailed analysis of the d-spacing corresponding to this peak and its relation to the lattice constants d
001 and d
002, has clearly confirmed the presence of lamellar phase in the whole range of IMI_oxyC5_C10 concentrations studied in the systems of interest in this work. It should be emphasised that the above described structural changes manifested in the SAXS curves correlate well with the changes in the C=O stretching band positions as a function of the surfactant concentration (see
Figure 6). The correlations between SAXS and FTIR results support the conclusion that the presence of the surfactant promotes ordering of DMPC and DNA in the lamellar phase.
In previous studies conducted by Pullmannová
et al.[
34] or Munoz-Ubeda
et al.[
47], the diffraction peaks associated with the presence of DNA were also reported. In the system studied in this work, the DNA concentration was 10 times lower and the characteristic peak related to DNA-DNA packing [
43] was not observed. In the DMPC/IMI_oxyC5_C10/DNA system the increasing concentration of surfactant in the system caused a clearly visible decrease in the d-spacing value. Similar effects that the repeat distance d
001 decreases linearly with increasing molar ratio surfactant/phospholipid were observed for gemini surfactants-DOPE-DNA complexes [
34].
On the basis of the typical symmetry of diffraction patterns clearly showing the lamellar nature of the systems investigated, and on the basis of the previously described patterns of lipid-DNA interactions [
48–
51], a proposed scheme of DMPC/IMI_oxyC5_C10/DNA lipoplexes structure is shown in
Figure 12.