Stephanie
Tassler
a,
Dorota
Pawlowska
a,
Christopher
Janich
b,
Bodo
Dobner
b,
Christian
Wölk
b and
Gerald
Brezesinski
*a
aMax Planck Institute of Colloids and Interfaces, Science Park Potsdam-Golm, Am Mühlenberg 1, 14476 Potsdam, Germany. E-mail: brezesinski@mpikg.mpg.de
bMartin-Luther-University (MLU) Halle-Wittenberg, Institute of Pharmacy, Wolfgang-Langenbeck-Straße 4, 06120 Halle (Saale), Germany
First published on 7th February 2018
The influence of the chain composition on the physical–chemical properties will be discussed for five transfection lipids containing the same lysine-based head group. For this purpose, the chain composition will be gradually varied from saturated tetradecyl (C14:0) and hexadecyl (C16:0) chains to longer but unsaturated oleyl (C18:1) chains with double bonds in the cis configuration. In this work, we investigated the lipids as Langmuir monolayers at the air–water-interface in the absence and presence of calf thymus DNA applying different techniques such as infrared reflection absorption spectroscopy (IRRAS) and grazing incidence X-ray diffraction (GIXD). The replacement of saturated tetradecyl (C14:0) and hexadecyl (C16:0) chains by unsaturated oleyl (C18:1) chains increases the fluidity of the lipid monolayer: TH10 < TT10 < OH10 < OT10 < OO10 resulting in a smaller packing density. TH10 forms the stiffest and OO10 the most fluid monolayer in this structure–property study. OO10 has a higher protonation degree compared to the saturated lipids TT10 and TH10 as well as to the hybrids OT10 and OH10 because of a better accessibility of the amine groups. Depending on the bulk pH, different scenarios of DNA coupling to the lipid monolayers have been proposed.
Besides structural parameters such as the head group, the spacer and the backbone, the chain composition is crucial for the formation of liposomes and lipoplexes, and finally for the cellular uptake. The structure of the hydrophobic domain determines the phase transition temperature and the fluidity of the bilayer, further it influences the stability of liposomes, the DNA protection from nucleases, the endosomal escape, the DNA release from the lipoplex and the nuclear penetration. Especially the number and the length of aliphatic chains play a crucial role. While single-chain lipids tend to form micelles, they transfect poorly compared to their double- and triple-chain derivatives.8,9 Furthermore, the length of the aliphatic chain is important and determines, together with the size of the head group and the nature of the backbone and spacer, the overall molecule shape. There are many different opinions in the literature considering the minimum chain lengths, which is required for gene transfection (C10,10 C1211,12 or C1413). Aliphatic chains varying from C5 to C25 have been investigated, with the conclusion that the transfection efficiency is not a linear function of the length. A few groups are convinced that C14 is the best choice for efficient transfection, but obviously not for all lipid composites.12–14 For gemini lipids C18 turned out to be a suitable chain length.15,16 Further, the toxicity of lipids is influenced by the hydrophobic domain.17 Generally, it was found that the cytotoxicity increases with decreasing chain length. Shorter-chain lipids have lower phase transition temperatures, which prohibits them from developing stable liposomes.13,18 In contrast, lipids with longer aliphatic chains increase the rigidity of bilayers, which is necessary to form stable liposomes.11 In the liquid-crystalline state, longer saturated chains can be very flexible (gauche conformation) due to rotational freedom of each single bond.19 However, under physiological conditions they are usually in the gel state with chain segments in all-trans conformation. Except a few authors,20,21 most groups report that unsaturated C18:1 chains are frequently the best choice for good transfection.22–25 Oleyl chains can promote the endosomal escape due to the enhancement of membrane fluidity of the transfection complexes.10 Furthermore, they allow strong anchoring in the membrane and have a good miscibility with unsaturated helper lipids like DOPC and DOPE.26
The effect of asymmetry is another variable influencing the transfection efficiency of cationic lipids. There are two possibilities to introduce asymmetry. One way is to choose a helper lipid with a chain length different from the cationic lipid or to introduce a mismatch in the cationic lipid itself by using different chains. For the first scenario it was found that a high level of asymmetry (C18:1 cationic lipid, C12:0 helper lipid) as well as high symmetry (C16:0 cationic lipid, C16:0 helper lipid) results in high transfection rates.27 In the case of the asymmetry being based on a mismatch within the cationic lipid itself, no clear trend was described in the literature. Some research groups claim that asymmetry enhances the transfection efficiency28–30 but a few groups report contrary findings.31,32 Our research goal is to correlate the structure of malonic acid diamide lipids with the physical–chemical properties and the interaction with DNA. Such data can be used for future correlations with the biological activity.33 To enable a systematic study, lipids with different chain patterns have been synthesized,34–36 namely TT (C14:0, C14:0), TH (C14:0, C16:0), OT (C18:1, C14:0), OH (C18:1, C16:0) and OO (C18:1, C18:1). In this paper, the physical–chemical properties of lipids with the head group type 10 (see Fig. 1) and the above described alkyl chain patterns have been examined in 2D model systems (Langmuir monolayers). The focus is set on the lipid self-assembling as well as the complex formation with DNA. Consequently, this article gives information on how to adjust physical–chemical parameters of transfection lipids by variation of the alkyl chain patterns to influence the coupling of DNA.
The lipids containing an oleyl chain have been synthesized from oleylamine with technical grade.38
Milli-Q Millipore water with a specific resistance of 18.2 MΩ cm was taken for all measurements and sample preparations. All chemicals used for the preparation of the bromide ion based buffers were purchased from Sigma Aldrich. The buffer had a constant concentration of 2 mM bromide anions. The pH 3 was adjusted by taking 1,4-diazabicyclo(2,2,2)octane (pKa1 = 4.2, pKa2 = 8.2), and pH 10 was obtained by using piperazine (pKa1 = 5.7, pKa2 = 9.8). The deoxyribonucleic acid sodium salt from calf thymus was purchased from Sigma Aldrich (Type 1, CAS: 73049-39-5). A stock solution of 1 mM ct-DNA was prepared freshly in 1 mM NaCl solution by gently stirring in the fridge (5 °C) overnight. The molar mass of DNA refers to a monomer containing one charge per phosphate moiety with 10% of hydration, therefore, M ∼ 370 g mol−1.39 The DNA solutions were diluted with water or the corresponding bromide ion based buffer to a concentration of 0.1 mM directly before the experiments.
The oleyl chain was introduced for two reasons. First, because it is known for a high transfection efficiency, and second, it ensures a sufficient miscibility with the commonly used helper lipid DOPE.
The malonic acid/lysine backbone of TT10, TH10, OT10, OH10 and OO10 is biocompatible and ensures low cytotoxicity resulting in a low immune response. Due to this backbone, the lipids exhibit a peptide-like character. Head group 10 has a linear ethylene diamine spacer and two primary amine groups (Fig. 1). The pKa value increases from 5 to 5.6 for TH10 to OO10, respectively, indicating that their readiness to act as proton donors decreases with increasing chain fluidity.37 This can also influence the interaction with DNA.
The π–A-isotherm of the TT10, TH10, OT10, OH10 and OO10 Langmuir monolayers on water at 20 °C are presented in Fig. 2A. Water saturated with CO2 has a pH value of approximately 5.8, therefore, the lipids are less charged than on the pH 3 buffer and exhibit weaker electrostatic repulsions. Hence, they have lower LE–LC transition pressures. The saturated lipids TT10 and TH10 are in the liquid-condensed phase state already at close to zero pressures (re-sublimation). Their π–A-isotherms are almost identical. The hybrid OH10 is in the LC phase as well, but the molecular area of OH10 is larger compared with TT10 and TH10. OT10 and OO10 are in the liquid-expanded phase state, but OT10 seems to be in a coexistence between LE and LC of 5 mN m−1 and 40 mN m−1. OO10 has the largest molecular area and is in the LE state.
On the pH 10 buffer (Fig. S1B, ESI†), the lipids are completely unprotonated. Only OO10 is in the LE phase state, while the saturated TH10 and the hybrid OH10 are in the LC phase state. Because of the saturated tetradecyl chain TH10 achieves a tighter packing and occupies a less area than the oleyl chain containing OH10.
For the lipid TT10, three Bragg peaks can be correlated with an oblique chain lattice, namely Qxy = 1.19 Å−1, Qxy = 1.34 Å−1 and Qxy = 1.53 Å−1. The reflections could be indexed as (1,0), (0,1) and (1,−1). Since their Qz values are in the ratio Q3z = Q2z + Q1z, an oblique unit cell with a distortion equal to 0.29 has been determined. Very similar Bragg peaks were found for TH10 and OH10 as well, namely Qxy = 1.2 Å−1, Qxy = 1.36 Å−1 and Qxy = 1.53 Å−1, and Qxy = 1.19 Å−1, Qxy = 1.39 Å−1 and Qxy = 1.53 Å−1, respectively. But since the signal intensity for OH10 is very low, it was not possible to determine the corresponding Qz values. We can only assume an oblique unit cell, because the three peaks found are at very similar positions compared with TT10 and TH10.
Compared to TT10, TH10 has two extra CH2 groups resulting in slightly stronger van der Waals interactions. As discussed before, TH10 forms a stiffer monolayer due to a tighter molecular packing. Therefore, the lattice parameters of TH10 (t = 35.3°, A0 = 20.6 Å2, Axy = 25.3 Å2, and distortion d = 0.28) are a bit smaller than for TT10 (t = 38.5°, A0 = 21 Å2, Axy = 25.7 Å2 and d = 0.29).
TT10 and TH10 have a high tilt angle, which can be explained by the large area requirement of the head group. In order to compensate the large head group areas the chains are strongly tilted. Subsequently, the cross-sectional areas per chain are a bit higher than the typical values for free rotating chains (A0 ∼ 20 Å2), because these strongly tilted chains are less tightly packed (see also the increased wavenumbers of CH2 stretching vibrations of TT10 (Fig. 2B)). The in-plane area Axy, which describes the real in-plane area occupied by one chain at the surface, is consistent with the area per molecule of the corresponding π–A-isotherms (Fig. 2A).
The GIXD pattern of OT10 reveals several Bragg peaks. The three Braggs peaks at Qxy = 1.19 Å−1, Qxy = 1.39 Å−1 and Qxy = 1.52 Å−1 can be associated with an oblique chain structure, but, as for OH10, the signal intensity was too low for the determination of the corresponding Bragg rods.
Besides the Bragg peaks assigned to the chain lattice, there are additional Bragg peaks in the diffraction pattern of TT10, TH10, OH10 and OT10 indicating further 2D alignments. The Bragg peak at Qxy = 1.61 Å−1 gives a real spacing distance of 3.9 Å, which is a typical value for the distance between the Cα carbons in a β-sheet structure. These transfection lipids contain a lysine moiety in the head group, which results in a peptide-mimic character. Therefore, this peak can be indexed as Cα–Cα spacing. The hypothesis is supported by the Bragg peaks found at Qxy = 1.31 Å−1, indicating intermolecular hydrogen bonds. Qxy = 1.31 Å−1 gives a real spacing distance of 4.8 Å, which is a typical value for strand separation in β-sheets due to their hydrogen bonds (strand-NH⋯OC-opposite strand). TT10, TH10, OH10 and OT10 have a high ability to form intra- and intermolecular hydrogen bonds between the primary amine and carbonyl groups. Since the intramolecular hydrogen bonds and the hydrogen bonds with water molecules from the subphase are not ordered, they do not cause Bragg peaks. Consequently, the observed Bragg peaks in the GIXD pattern are associated with intermolecular hydrogen bonds between different TT10, TH10, OH10 and OT10 molecules, respectively. The Bragg peak at Qxy = 0.59 Å−1 (d = 10.6 Å) indicates ordering of the head groups.
The same two Bragg peaks, namely Qxy = 1.31 Å−1 and Qxy = 1.61 Å−1, have been found for lipid 7, which is a representative of the first generation of our malonic acid amide lipids,51,52 but the low Qxy range was not investigated for lipid 7. Therefore, the lattices could be very similar.
Moreover, in the GIXD pattern of TT10 and OT10 two Bragg peaks at Qxy = 1.52 Å−1 (degenerated peak) and Qxy = 1.68 Å−1 (non-degenerated peak) have been observed. The Bragg rods have a FWHM of 0.17 Å−1. By using the Scherrer formula, to calculate the length of the scattering unit, one obtains Lz equal to 32.5 Å, which is too large for a lipid molecule. It might be that the lipids TT10 and OT10 were not completely dissolved in the solution (chloroform/methanol 3
:
1). In this case, lipid microcrystals could be present at the surface. The crystals are most probably made of lipid layers and double layers. Since no Scherrer rings or any bending of contour plots are visible, the signals have to come from oriented crystals that give a spot-like signal rather than from a powder-like structure where the Scherrer rings are expected. Probably only the in-plane cross-section of the double layer can be seen so no 3D structures contribute to the signal. The presence of crystals is the most reasonable explanation. The two Bragg peaks are indicative of a rectangular unit cell. Both peaks are located at the horizon characterizing a lattice of non-tilted chains. Lz = 32.5 Å corresponds to interdigitated chains of two layers. The packing in such interdigitated layers is very tight (A0 ∼ 18.5 Å2) and corresponds to the herringbone chain packing mode.
Besides the indexed Bragg peaks there are other peaks in the GIXD patterns of TH10, OH10 and OT10, which are marked in grey (Fig. 3B–D). Similar peaks were found for a highly ordered head group structure of a sugar-unit containing the phospholipid GPI-fragment, namely GlcNα1-6myoIno-1-phosphodistearylglycerol.53,54 The chains were strongly tilted (43°). In the present case of the transfection lipids, the observed Bragg peaks could also indicate a sub-gel phase,55–57 which is formed by the ordering of whole molecules instead of only chains. However, the signal intensity is very weak compared to the other Bragg peaks. Most likely, the van der Waals interactions cannot be optimized due to the large spacing between the head groups ordered by a hydrogen bonding network.
The resulting increase in the surface pressure (Δπ = π60min − πinitial) due to the adsorption of ct-DNA after 60 min is given in Table 1. The initial pressure was in all cases ∼8 mN m−1.
ct-DNA containing bromide ion based buffer | Δπ [mN m−1] of TH10 monolayer | Δπ [mN m−1] of OO10 monolayer |
---|---|---|
pH 3 | 0.4 | 0 |
pH 7 | 11.3 | 12.2 |
pH 10 | 8 | 6.4 |
The protonation state of DNA and the cationic lipids explain the strong increase of the surface pressure at pH 7. The negatively charged DNA is attracted by the weakly protonated cationic lipids due to electrostatic interactions between the phosphate diester backbone (PO2−) of DNA and the amine groups (R-NH3+) in the lipid head group. The adsorbed DNA requires a large area. Even at pH 10, at which the cationic lipids are unprotonated, a large amount of negatively charged DNA is attracted to the interface and occupies space, which results in an increasing surface pressure for both TH10 and OO10. Almost no increase in the surface pressure is detected for the TH10 and OO10 monolayers at pH 3 (Table 1). The monolayers of protonated TH10 and OO10 are not influenced by the presence of weakly charged DNA.
As expected, the unsaturated lipid OO10 is more fluid than the saturated derivative TH10. DNA has a fluidising effect on the saturated lipid TH10 at pH 3 and pH 7. At pH 10, the monolayer remains in the LC phase state with chains in all-trans conformation. Surprisingly, the wavenumbers even decrease in the presence of DNA. It seems like adsorbing and penetrating DNA compresses the TH10 monolayer leading to a tighter packing (Fig. 5C).
![]() | ||
Fig. 5 Possible scenarios of DNA adsorption to the lipid monolayers at different pH values. (A) pH 3, (B) pH 7 and (C) pH 10. |
At pH 7, the wavenumbers are intermediate, which either means that some chain segments are in the gauche conformation whereas other chain segments remain in the all-trans conformation, or that there are patches of lipids in LE and LC states as suggested in Fig. 5B. The idea of coexisting LE and LC phases is supported by the strong increase in the surface pressure as shown in the adsorption isotherms (ΔπTH10 = 11.3 mN m−1 and ΔπOO10 = 12.2 mN m−1). Comparing the wavenumbers for OO10 between 20 and 25 mN m−1 (Fig. 4B), it seems that OO10 is more fluid without the DNA than with the DNA adsorbed. This implies that DNA penetrates the monolayer and compresses the lipids, which results in lower wavenumbers. Upon further compressing by the barriers, the penetrated DNA seems to be squeezed out at 27 mN m−1, because the wavenumbers increase by about 2 cm−1.
At pH 3, both monolayers are in the LE phase state at low surface pressures, but upon compression TH10 undergoes a transition to the LC state. The adsorbed DNA changes the wavenumbers only marginally. Since the increase in the surface pressure observed in the adsorption isotherms for TH10 and OO10 at pH 3 is also negligible, it might be that DNA does not penetrate and influence the lipid monolayers. More likely, the DNA is coordinated by the protonated head groups underneath the monolayer (Fig. 5A). In order to get deeper insights into the interaction between ct-DNA and the cationic lipids, the amount of attached DNA has been estimated.
The intensity plots of the νasym PO2− band versus the area per molecule of the corresponding lipids are given in Fig. 6 for TH10 and OO10. Here, the increase in chain unsaturation results in a higher amount of DNA attached to the lipid monolayer. The smaller packing density of the OO10 monolayer assists the adsorption of DNA at pH 3 and pH 7. On DNA containing pH 10 buffer, the performance of both lipids is very weak. The relative amount of attached DNA is negligible. Upon compression, the little amount of adsorbed DNA is squeezed out from the monolayer, indicating weak electrostatic interactions. Further, the shift of the νasym PO2− band to higher wavenumbers (1260 cm−1) indicates dehydration of the DNA phosphate diester groups and the absence of hydrogen bonds (see ESI,† Fig. S8). On DNA containing pH 3 and pH 7 buffers, the νasym PO2− band is at 1220 cm−1 which implies stronger hydration of the DNA phosphate groups and the presence of hydrogen bonds. Interestingly, the slope of the linear fit differs dramatically for TH10 and OO10 on the DNA containing pH 7 buffer. With further compression the intensity of the phosphate band for TH10 strongly increases, while it slightly decreases for OO10. Here it looks like DNA is squeezed out from the OO10 monolayer as already assumed in the discussion regarding the phase state. For TH10 and OO10, a small amount of attached DNA introduces further adsorption of DNA to the lipid monolayer (Manning condensation58). The negatively charged polyelectrolyte would only go to the interfaces, if the interaction with the interfaces is more attractive than the interactions between the polyelectrolyte and subphase. The adsorption of DNA to the lipid monolayer is a competition between an attractive surface potential and the entropic repulsion, which keeps the polyelectrolyte in the subphase. The electrostatic interactions depend on the charge density of DNA and the lipids,59 the salt concentration60 and the pH value.61 In case a DNA chain segment adsorbs at the charged monolayer, the system loses translation energy equal to the thermic energy (kB·T, where kB is the Boltzmann constant and T the temperature), but increases the entropy due to the released counter ions (Z·kB·T, where Z is the charge of the counter ion). As a result, the surface charge decreases and subsequently an equilibrium of adsorption and desorption will be established. For polyelectrolytes this phenomenon is described as the Manning condensation.58 A weakly charged monolayer at the interface will attract only a small amount of DNA inducing further attraction of DNA to the interface. Additionally, the adsorbed DNA affects the potential of the electrical double layer in the way that the surface pH changes in accordance with the Boltzmann equation. This leads to an increase in the protonation degree of the TH10 and OO10 monolayers and consequently to further binding of DNA chain segments.62 This explains why there is still a large amount of DNA attached to TH10 and OO10 at pH 7, where their protonation degrees are rather small.
Despite the higher protonation degree of OO10, the charge densities of TH10 and OO10 are comparable at 30 mN m−1on the bromide ion based buffer at pH 3.37 Both lipids are in the same phase state (liquid-expanded), but TH10 is closer to the phase transition to the liquid-condensed phase state (πtr = 35 mN m−1, ESI,† Fig. S1). Therefore, the TH10 monolayer is already better ordered than the OO10 monolayer. This order seems to disturb the interactions with DNA in the case of TH10, while the flexibility of the fluid OO10 monolayer benefits the interaction with DNA. More DNA is attached to the OO10 monolayer at pH 3 (Fig. 6).
Arb. units | Arbitrary units |
π–A-isotherm | Surface pressure–area-isotherm |
ct-DNA | Calf thymus deoxyribonucleic acid |
DOPE | 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine |
GIXD | Grazing incidence X-ray diffraction |
IRRAS | Infrared reflection absorption spectroscopy |
LC | Liquid-condensed phase with chains in all-trans conformation |
LE | Liquid-expanded phase state with chains in gauche conformation |
OH10 | N-(2-Aminoethyl)-N′-{6-amino-1-[N-(9Z)-octadec-9-enylamino]-1-oxohexan-(2S)-2-yl}-2-hexadecylpropandiamide |
OO10 | N-(2-Aminoethyl)-N′-{6-amino-1-[N-(9Z)-octadec-9-enylamino]-1-oxohexan-(2S)-2-yl}-2-[(9Z)-octadec-9-enyl]propandiamide |
OT10 | N-(2-Aminoethyl)-N′-{6-amino-1-[N-(9Z)-octadec-9-enylamino]-1-oxohexan-(2S)-2-yl}-2-tetradecylpropandiamide |
TH10 | N-(2-Aminoethyl)-N′-[6-amino-1-oxo-1-(N-tetradecylamino)hexan-(2S)-2-yl]-2-hexadecylpropandiamide |
TT10 | N-(2-Aminoethyl)-N′-[6-amino-1-oxo-1-[(N-tetradecylamino)hexan-(2S)2-yl]]-2-tetradecylpropandiamid |
TRXF | Total reflection X-ray fluorescence |
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c8cp00047f |
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