lipopeptides
Cavalli, S.
Citation
Cavalli, S. (2007, January 25). Chemistry, structural insight and applications of β-sheet forming lipopeptides. Retrieved from https://hdl.handle.net/1887/9452
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Self-Assembly of Amphiphilic Lipopeptides
Abstract. The self-assembly behaviour of a series of amphiphilic lipopeptides, ALPs, was studied. The formation of elongated fibers upon deposition of lipopeptide monolayers on solid support (mica) was visualized by AFM. Furthermore, the lipopeptides aggregated in water with irregular shape and size. Circular dichroism studies of ALP samples clearly displayed a β-pleated sheet folding of the peptide domain having six to eight amino acid residues. Mixing octa-ALP with DOPC resulted in the formation of spherical structures and elongated fibers as observed with electron microscopy. This study showed that the formation of well-defined assemblies with a regular size and morphology is rather difficult to achieve. The complications encountered in the preparation of uniform lipid vesicles decorated with β-sheet oligopeptides could be overcome using a post-modification method of pre-formed liposome directly at the surface. Monolayers of hexa- and octa-ALP have been shown to template the mineralization of calcium carbonate at the air-water interface (Chapter 3).
Therefore the preparation of defined aggregates of the ALPs could lead to new templates for biomineralization in solution.
4.1 Introduction
Peptides have been considered particularly attractive as simple and versatile molecular self-assembling building blocks for the construction of nanostructures, especially due to their capability to respond to external signals (i.e. responsiveness to solvent, pH, temperature, electronic or photonic energy and metals).1 Challenging is to design, a priori, molecules that self-assemble in a predictive manner into targeted three-dimensional supramolecular structures. The way in which molecules self-assemble can be programmed at the molecular level by varying their interaction energies and shape. This is especially the case with peptides, for which much is already known about their secondary structural folding and stability.2 The self-assembly of peptides, proteins and artificial peptide-mimetics into nanoarchitectures with a large variety of morphologies such as tapes,3 tubes,4 hydrogels,5 and spheres4,6 has attracted much attention as an highly efficient approach for the construction of novel functional nanomaterials. Moreover, modification of peptides with one or more alkyl tail has been shown to facilitate the formation of various supramolecular organizations as ribbons,7 twisted ribbons8 and fibers,9 enabling the fabrication of an astonishing number of different nanomaterials that can have important applications in biotechnology and bioengineering.10
Chart 4.1. The amphiphilic lipopeptides, ALPs (1a-c). Three ALP’s, each exhibiting a different length of the peptidic part, (Leu-Glu)2-,(Leu-Glu)3- and (Leu-Glu)4, termed tetra-, hexa- and octa-ALP, respectively (cf. 1a-c).
In this Chapter, the self-assembly of a series of amphiphilic lipopeptides, ALPs (Chart 4.1) is discussed. The ALPs (1a-c) are composed of amphiphilic oligopeptide moieties with alternating hydrophobic and hydrophilic amino acid residues, (Leu-Glu)n , interlinked by a succinyl moiety to the phospholipid 1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine (DOPE). The ALPs, each exhibiting a different length of the peptidic part, (Leu-Glu)2-, (Leu-Glu)3- and (Leu-Glu)4 and termed tetra-, hexa- and octa-ALP, respectively (cf. 1a, 1b and 1c in Chart 4.1), represent a first example of highly ordered peptide-based amphiphiles with a unique hybrid character. On the one hand, the alternating hydrophilic and hydrophobic oligopeptide head groups have the tendency to form crystalline structures, as discussed in Chapter 2 and previously reported by Rapaport et al.11 On the other hand, ALPs exhibit flexibility, attributed mostly to the fluid-like character typical of unsaturated lipid
n
HN N H
NH2
O HO O
O O
O O O
O P O OH O
NH O
O 1 (a: n=2; b: n=3; c: n=4)
tails. A related lipid, 1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolcholine (DOPC), easily forms vesicles in aqueous solution due to its fluid-like property, amphiphilicity and molecular structure. Thus, the conjugated phospholipid tail (DOPE) was introduced to facilitate the formation of vesicular aggregates upon dispersion in aqueous media. However, with the hybrid nature of the ALPs, particularly challenging is the ability to obtain three-dimensional structures, which could exhibit on their surface a β-sheet organization of the peptide domain similar to the well-defined two-dimensional β-sheet architecture found for the monolayers at the air-water interface (Figure 4.1). The series of ALPs was therefore studied to seek the proper length of the peptide, necessary to generate well-ordered β-sheet domains in aggregates with defined size and shape.
3D ß-sheet
decorated vesicles ß-sheet 3D ß-sheet tapes
2D ß-sheet monolayer air water
3D ß-sheet
decorated vesicles ß-sheet 3D ß-sheet tapes
2D ß-sheet monolayer air water 2D ß-sheet monolayer
air water
Figure 4.1. Schematic representation of 3D assembled structures exhibiting on their surface the well-defined 2D β-sheet architecture of the lipopeptide monolayer.
Notably, as monolayers of hexa- and octa-ALP have been shown to template the mineralization of calcium carbonate (Chapter 3), it is envisioned that these type of aggregates could also act as three-dimensional templates in solution.12 Controlled self-assembly of oligopeptides with a β-sheet structure allows a periodic presentation of functional groups on a surface, which can nucleate mineral crystallization.13 As reorientation of functional groups in the monolayers was demonstrated to be fundamental for templating a new crystal phase at the surface14 (Chapter 3), the influence of a curvature, due to the formation of vesicular aggregates, on the orientation of the β-sheet and consequently on the interaction between the peptide and the mineral phase (i.e. CaCO3), could be of interest for a better understanding of
the nucleation mechanism in the bulk of the solution. Furthermore, well-defined β-sheet assemblies may be used to prepare bio-inspired organic scaffolds that could induce inorganic crystal growth on their surface. Such new biomaterials may find a variety of applications (i.e. for bone tissue regeneration15 or as semiconductors at the nanometer-scale).16
4.2 Results and discussions
4.2.1 Fiber formation at the air-water interface
As discussed in Chapter 2, grazing incidence X-ray diffraction (GIXD) measurements performed at the air-water interface revealed the formation of two-dimensional β-sheet domains in monolayers composed of hexa- or octa-ALP.
Furthermore, in the case of octa-ALP, GIXD analysis also showed that the monolayer collapsed upon compression into well-defined β-sheet multilayers. Insights on these structures were provided by atomic force microscopy (AFM) and the results are reported in the first part of this chapter.
An octa-ALP monolayer, formed on a water subphase by covering 80%
(see experimental section) of the surface,17 was transferred from the air-water interface onto freshly cleaved mica by slow draining of the water underneath the monolayer. AFM imaging showed the presence of tens of micrometers elongated helicoidally objects (10-37 nm in height) aligned on the mica surface (see Appendix 3). Tape-like peptide-based aggregates have been already observed in solution.3,18 In addition, Hartgerink et al.19 have shown that also alkylated peptides are able to form fibres. Furthermore, the majority of the tapes appeared to be twisted, which could be attributed to the intrinsic chiral nature of the individual peptides constituting these structures.7a The chirality of molecular building blocks have been proven to play an important role in the self-assembly processes. Interestingly, many nanofibers self-assembled from a wide range of peptides with various degrees of left-handed helical twist.20 As an example, the innate right-handed twist of the β-stranded peptide backbone of an eight-residue peptide, FKFEFKFE (KFE8),21 led to the formation of left-handed double-helical ribbons of regular pitch at the nanometer-scale.
The calculated length of an eight residue peptide in an extended β-strand conformation is ~ 2.8 nm. In the case of the octa-ALP ~ 1.4 nm should be added if the linker (composed of the succinyl group and the hydrophilic head group of the phospholipids, see Chart 4.1) is also in an extended conformation, as discussed in detail previously (Chapter 2 ).
Figure 4.2. The calculated length of an eight residue peptide in an extended β-strand conformation is ~ 2.8 nm.
For the other dimensions, 0.9 nm in height (with the amide bonds in the peptide backbone parallel to the plane) and 0.47-0.48 nm in width per strand are expected.
Moreover, only if counting for the β-sheet peptide subunit, structures with dimensions of 0.9 nm in height and 0.47-0.48 nm in width per strand are expected (Figure 4.2).7a To these values ~ 1.8 nm could be added, which corresponds to the length of the stretched lipid tails.
Taking into account the GIXD data, tabulated in Appendix 1, a thickness of 1.6-1.8 nm for the octa-ALP monolayer and 2.6-3.0 nm for the multilayers were found, corresponding to a bilayer. The data relative to the thickness of the multilayer is lower than the value found by AFM. However, one should be aware that the data obtained by the GIXD experiments correspond to the crystalline part of the film only. No information is available about amorphous domains using this technique. It is therefore not surprising that thicker aggregates were detected by AFM, which probably correspond to multilayered assemblies.
The voltage in the friction image is higher relative to the background, indicating that the side which is exposed to the contact with the tip is hydrophilic, therefore it was hypothesized that these twisted ribbon-like aggregates expose the peptide architecture at their outer face with the lipid tails in the internal core of the tapes, as proposed in the schematic representation in Figure 4.3.
In some regions of the deposited film, holes with a depth of 11.5-12.7 nm were found, which allowed an estimation of the thickness of the ALP layer deposit on the mica support.
The height of the film obtained by the AFM observations is slightly lower compared to the thickness of the monolayer found analyzing the GIXD data. In this case, a possible explanation could be related to the fact that the film aligned flat on the mica surface, while at the air-water interface the monolayer tended to bend (Chapter 2).
NH O HN
O
HO O NH
O HN
O
HO O NH O
HN O
HO O
NH2 NH
O HN
O
HO O HN
HO O NH
HN
HO O NH
HN
HO O NH
HN
HO O NH O O
O O
O O
O O H2N
0.5 nm
2.8 nm
0.9 nm
Figure 4.3. Schematic representation of ALP assemblies, showing an hydrophobic internal core of the fibers, which expose the hydrophilic peptide at their outer surface. Peptide (black), succinyl and DOPE head group (gray) and DOPE tails (white).
4.2.2 Self-Assembly in Aqueous Solution
Studies on the aggregation of ALPs (1a-c) upon dispersion in aqueous solutions were performed using different techniques for the preparation of the assemblies. The aggregates in solution were studied by circular dichroism (CD) to gain information about the folding of the peptide domain. Furthermore, dynamic light scattering (DLS) and transmission electron microscopy (TEM) provided insight on the size and shape of the assemblies.
Solutions of ALPs (1a-c) were prepared in 1/1 (v/v) chloroform/methanol mixture.
Several methods were used to disperse the ALPs in aqueous solutions, including dialysis and hydration of lipid films. Only after extrusion through a 200 nm track-etch membrane some spherical aggregates were observed. All other preparation procedures resulted in aggregates with irregular morphology and size, indicating the difficulty to prepare in a reproducible manner well-defined assemblies. A possible explanation could be related to the hydrophilic/hydrophobic block ratio.22 Furthermore, the formation of rigid β-sheet domains could interfere with the assembly into spherical aggregates. However, CD studies on these samples were performed to investigate the secondary structure of the peptide domain, revealing that tetra-ALP did not form β-sheets. In contrast hexa-ALP started to adopt a β-sheet conformation, which appeared predominant in the case of octa-ALP (data not shown).
Moreover, the influence of the lipid moiety was studied by measuring the CD spectrum of a solution of octapeptide H2N-(Leu-Glu)4-NH2 (2) in phosphate buffer (pH = 5.5), which showed that the peptide existed only in a random coil conformation. Thus, the lipid tail induced the β-sheet folding by self-assembly, even though the aggregates were irregular in shape.
It is envisioned that dilution of the concentration of ALPs using a co-lipid could help in the preparation of vesicles. 1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolcholine (DOPC),
which is able to form vesicles efficiently, was used as co-lipid in order to obtain liposomes with β-sheet domains at their surface. It was expected that dilution of the peptide head group concentration would facilitate the formation of vesicular aggregates. Therefore, lipid mixtures using different composition of octa-ALP (1c) and DOPC were prepared, as listed in Table 4.1.23
Table 4.1. Lipid composition of the different samples. All samples were prepared using a total lipid concentration of 1 mM.
Sample Composition
A octa-ALP (1c)
B 50 mol% octa-ALP (1c) mixed with 50 mol% DOPC C 10 mol% octa-ALP (1c) mixed with 90 mol% DOPC
D DOPC E 10 mol% octapeptide (2) mixed with 90 mol% DOPC
DOPC and octapeptide (2)were used as controls. Lipid mixtures were dissolved in 1/1 (v/v) chloroform/methanol mixture and dried under vacuum. The films were hydrated with a 50 mM phosphate buffer (pH = 5.5) and the resulting turbid solutions were submitted to several cycles of freezing and thawing followed by sonication.24 First, the influence of the DOPC on the secondary structure of octa-ALP was investigated using circular dichroism (Figure 4.4). The CD spectra of all samples were characterized by a maximum around 202 nm and a minimum at 218 nm, demonstrating the presence of β-pleated sheet assemblies. Thus, dilution of octa-ALP with DOPC did not disturb the formation of a well-defined secondary structure. As expected, DOPC did not show any CD signal in the region characterized by amide bonds transitions. In the case of the octapeptide 2, a random coil signal was expected (in agreement with former studies). However, no signal was observed, probably due to the very low concentration of the peptide (only 10 mol% mixed to DOPC). Remarkably, when the same concentration of octa-ALP was used, indication of folding into β-sheets was already visible by CD. This demonstrates that small oligopeptides with an alternating hydrophilic-hydrophobic amino acid sequence need to be “forced” in order to adopt a β-sheet conformation, for example by conjugation of lipid tails or by incorporation into an aggregate.
Figure 4.4.CD spectra in 50 mM phosphate buffer (pH = 5.5) of (2) octa-ALP (sample A); (]) 50 mol%
octa-ALP mixed with 50 mol% DOPC (sample B); (') 10 mol% octa-ALP mixed with 90 mol% DOPC (sample C); (u) DOPC (sample D); () 10 mol% octapeptide mixed with 90 mol% DOPC (sample E). Samples were prepared using a total lipid concentration of 1mM. In the case of CD spectra of sample A and C values were corrected for the path length.
Information about the size of the aggregates was obtained by Dynamic Light Scattering (DLS) measurements (Figure 4.5).25 In the case of pure octa-ALP (sample A) the main broad population was around 240 nm. A smaller population with a diameter of 21 nm was also observed as well as few larger aggregates (>2 µm). An equimolar mixture of octa-ALP and DOPC (sample B) resulted in a smaller average diameter compared to octa- ALP alone. Lowering the ratio even further to 10 mol% of octa-ALP (sample C), resulted in even smaller aggregates. In this case the main population was around 25 nm with a “sholder”
of bigger aggregates at around 101 nm. DOPC (sample D) formed quit uniform vesicles with smal diameters (30 nm). These result indicate that in general the increase of DOPC resulted in the formation of smaller aggregates. The amphiphilic nature of the peptide was revealed when octapeptide 2 was mixed with DOPC. An increase in size was observed compared to pure DOPC, indicative of partitioning of the peptide in the membrane.
200 210 220 230 240 250 260
-50 0 50 100 150
Ellipticity (a. u.)
λ (nm)
Figure 4.5.Size distributions obtained from DLS measurements. Rh is the hydrodynamic radius. sample A;26 Rh = 20.74, 240.6 and 2307 nm (polydispersity index = 0.03, 0.26 and 0.00, respectively); sample B, Rh = 101.0 and 1761 nm (polydispersity index = 0.60 and 0.07, respectively); sample C, Rh = 24.91, 117.3 and 2153 nm (polydispersity index = 0.28, 0.08 and 0.01, respectively); sample D; Rh = 30.53 (polydispersity index = 0.173) and sample E, Rh = 151.9 (polydispersity index = 0.30).
10 100 1000
0 1 2 3 4 5 6
% intensity
Rh (nm) Sample A
10 100 1000
0 1 2 3 4 5 6
Sample E
% intensity
Rh (nm)
10 100 1000
0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5
% intensity
Rh (nm) Sample B
10 100 1000
0 1 2 3 4 5 6
% intensity
Rh (nm) Sample C
10 100 1000
0 1 2 3 4 5 6 7
Sample D
% intensity
Rh (nm)
The size and morphology was subsequently studied by transmission electron microscopy (TEM).
Figure 4.6. TEM images of octa-ALP (sample A). Samples were negatively stained with 2% (v/v) phosphotungstic acid (PTA). In the inset an enlargement of an image taken in a different region of the grid is shown. Scale bar 100 nm.
Figure 4.7. TEM images of sample B, containing 50 mol% octa-ALP mixed with 50 mol% DOPC. Samples were negatively stained with 2% (v/v) phosphotungstic acid (PTA). Scale bar 100 nm.
Two different forms were observed in the case of octa-ALP (sample A), as shown in
A
A B
fibrous structures. The average thickness of ∼ 25 nm indicates that these fibers are composed of multilayers of the octa-ALP, similar to the fibers observed at the air-water interface (see section 4.2.1).
TEM imaging of sample B, containing equimolar amount of octa-ALP and DOPC, revealed again the coexistence of vesicular aggregates (diameter between ∼ 50-100 nm) and long filaments (∼ 25 nm wide), which seemed to be more curved compared to the fibers observed in the case of sample A (Figure 4.7). This might be caused by the increase in flexibility induced by the DOPC.
Figure 4.8. TEM images of (A, B and C) sample C, containing 10 mol% octa-ALP in DOPC; (D) DOPC (sample D). Samples were negatively stained with 2% (v/v) phosphotungstic acid (PTA). Scale bar 100 nm.
Lowering the octa-ALP content to 10 mol% resulted in a transition to more spherical and oval objects (Figure 4.8). Beside a few long tapes ∼ 50 nm wide, also many short
B
A
C D
elongated oval objects, ∼ 70-200 nm in length and ∼ 30-50 nm in width, were observed, which tended to aggregate into bigger oval structures.
As expected, DOPC formed vesicles with size ranging from 25-100 nm in diameter (Figure 4.8, D), in agreement with the DLS measurements. Similar vesicular objects were also found in the case of sample E (not shown).
4.3 Conclusions
In conclusion, octa-ALP (1c) formed multilayered β-sheet tape-like structures upon deposition of the monolayer from the air-water interface on solid support (mica). Tentatively, a model is proposed, in which octa-ALP assembled as bilayer into tapes with an hydrophobic internal core and the hydrophilic peptide at their outer surface. These multilayers subsequently formed long twisted fibers. Mixtures with different ratios of octa-ALP and DOPC formed a variety of different aggregation types. Both vesicular-type aggregates and fibrous structures coexisted. CD analysis revealed that these aggregates clearly displayed a β-pleated sheet folding of the peptide domain. The outcome of this study showed that the formation of well-defined assemblies is rather difficult to achieve. The tendency to form rigid β-sheet arrays probably complicate the self-assembly process. The complications encountered here in the preparation of lipid vesicles decorated by β-sheet peptides could be overcome by using a functionalized lipid already inserted in a pre-formed liposome and conjugate the (Leu-Glu)4 peptide motif directly at the surface. An example of this approach is given in the following Chapter (Chapter 5, Section 5.3). As the monolayers of ALPs have been shown to template the mineralization of calcium carbonate at the surface (Chapter 3), the preparation of such types of aggregates could lead to new templates for biomineralization in solution.
4.4 Experimental Section
General materials and methods. All reagents and solvents were commercial products purchased from Sigma-Aldrich B.V. or Biosolve B.V. and used as received. Lipids were purchased from Lipoid and Avanti Polar Lipids, Inc. Milli-Q water with a resistance of more than 18.2 MΩ/cm was provided by a Millipore Milli-Q filtering system with filtration trough a 0.22 µm Millipak filte. Phosphate buffer was prepared mixing aqueous 5 mM Na2HPO42·H2O with aqueous 5 mM NaH2PO42·H2O to the desired pH (pH = 5.5).
Synthesis. ALPs were synthesized as described in the experimental section of Chapter 2.
Circular dichroism (CD). CD spectra were measured with a Jobin Yvon CD6 spectrometer. CD measurements were performed using mainly 0.2 or 1 mm path round cuvettes. Spectra were recorded from 260 nm to 190 nm with an integration time of 4.0 seconds and a bandpass of 2.0 nm. The CD spectra are shown
as an average of 5 runs and are presented as raw data. Spectra of water or PBS were used as baselines. Cuvettes were cleaned with chloroform, ethanol, water and finally acetone and dried with a N2 flow.
Atomic Force Microscopy (AFM). AFM was performed in collaboration with dr. Silvina Federman (Ben Gurion University of the Negev, Beer-Sheva, Israel). AFM topography scans were made in contact and friction mode in air using a PicoSPM (Molecular Imaging, Tempe, AZ). Samples were prepared using a milli-Q filled mini Teflon® trough (diameter = 4.24 cm) by spreading 17 µl of a solution of octa-ALP in 1/9 v/v TFA/CHCl3 (c = 0.19 mg/ml) resulting in 80% coverage of the surface of the trough (area = 28.27 1016 Å2). The amount of molecules to spread was calculated taking into account the limiting MmA (obtained by extrapolation of the steepest part of the π-A isotherm, 215 Å2/molecule). After 10 min the water was removed with a syringe from the bottom side, resulting in monolayer deposition onto two freshly cleaved mica positioned at the bottom of the trough. Samples were left drying overnight in a closed Petri dish. This procedure was performed in duplo.
Protocol for the preparation of the lipid aggregates. Several methods were used to disperse the ALPs in water including dialysis and hydration of a lipid films followed by sonication or extrusion. In the case of the dialysis method: ALPs were first dissolved in 1/9 (v/v) trifluoroacetic acid/methanol mixture to a total lipid concentration of 1 mM. To these solutions, a 2 fold volume of milli-Q water was added slowly. The turbid solutions were subsequently dialyzed against water to remove the excess of organic solvents using membrane dialysis Spectra/Por with a membrane pore size of 1000 g/mol. The water was exchanged three times in a total period of 24 hrs. After dialysis samples were directly analysed by TEM and CD spectroscopy. The size of the particles was measured by Dynamic Light Scattering (DLS) or Photon Correlation Spectroscopy (PCS). In the case of the method of the hydration of lipid films: ALPs were first dissolved in 1/1 (v/v) chloroform/methanol mixture. The solvent mixture was evaporated under a flow of N2 and then under vacuum for 10 min. Next, the lipid films were hydrated for 1 h at 40 °C with milli-Q water to a total lipid concentration of 1 mM. Samples were then separated in three different portions and analyzed directly or placed in a sonicator bath (Branson 5200) at room temperature (RT) for about 20 min until a clear solution appeared. The third portion of the samples was extruded through a 0.2 µm membrane using a mini-extruder (avanti polar lipids). Subsequently, samples were analyzed as already described above. Lipid mixtures using different ratios of octa-ALP and DOPC were prepared as listed in Table 4.1 and homogeneously dissolved in 1/1 v/v methanol/chloroform to a total lipid concentration of 1 mM. The solvent was evaporated under a flow of N2 and then under vacuum for 10 min. Next, the lipid films were hydrated for 1 h at 40 °C with a 50 mM phosphate buffer (pH = 5.5) to a total lipid concentration of 1 mM. The samples were vortexed resulting in a turbid suspension and submitted to 5 cycles of freeze-thaw followed by sonication at RT for 2-4 hrs until a clear solution appeared. Then the samples were analyzed as described above.
Photon Correlation Spectroscopy (PCS). Photon Correlation Spectroscopy (PCS) was used to measure the size of the aggregates in solution, employing a Zetasizer 3000 HSA, Melvern Instruments.
Temperature = 25.0 °C, Viscosity = 0.890 cP; Angle = 90.0 deg RI medium = 1.33; RI particle 1.50 + Abs. 0.00.
Dynamic Light Scattering (DLS). Hydrodynamic radii were measured at ambient temperature by dynamic light scattering using a DynaPro 99 instrument (Protein Solutions, Lakewood, NJ). The laser wavelength was 824 nm and the scattering angle was 90˚.
Transmission Electron Microscopy (TEM). TEM was conducted on a JEOL 1010 instrument with an accelerating voltage of 60 kV. Samples for TEM were prepared by placing a drop of each solution on carbon-coated copper grids. After 10 min the excess of solvent was removed from the edge of the grid. The
samples were negatively stained for 5 min using 2% (v/v) phosphotungstic acid (PTA). Negative images are shown in order to retain image quality.
4.5 References and notes
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17 When the monolayer was transferred by Langmuir-Blodgett dipping on mica, no features were visible by AFM, probably due to the uniform distribution of the film on the mica surface. However, attempts to transfer onto mica the monolayer at lower π by vertical dipping failed. For this reason, a lower surface coverage (80%) was chosen and samples were prepared by deposition of the Langmuir monolayer onto mica as described in the experimental section.
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22 Antonietti, M.; Föster, S. Adv. Mater. 2003, 15, 1323-1333 (see also Appendix 4). Castile, J. D.; Taylor, K. M.
Int. J. Pharm. 1999, 188, 87-95. (A slightly modification of the freeze-thaw procedure described in the paper was used for the preparation of the aggregates studied in the chapter).
23 since both DOPC and DOPE have the dioleoyil tail a phase separation in the mixed system was not expected.
24 Castile, J. D.; Taylor, K. M. G. Int. J. Pharm. 1999, 188, 87-95. (A slight modification of the freeze-thaw procedure described in the paper was used for the preparation of the aggregates studied in the chapter).
25 For comparison Photon Correlation Spectroscopy (PCS) was also used to measure the size of the aggregates in solution. Size distributions obtained from PCS measurements of sample A: cumulant Z average = 312.4 nm (polydispersity index = 0.830); sample B: cumulant Z average = 58.5 nm (polydispersity index = 0.570); control C: cumulant Z average = 474.2 (polydispersity index = 0.996); control D: cumulant Z average = 77.4 (polydispersity index = 0.560) and control E: cumulant Z average = 146.6 (polydispersity index = 0.805).
26 In this case, the sample was diluted.
