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Oil Transfer Converts Phosphatidylcholine Vesicles into Non-Lamellar Lyotropic Liquid Crystalline Particles Isabelle Martiel, Stephan Handschin, Wye-Khay Fong, Laurent Sagalowicz, and Raffaele Mezzenga Langmuir, Just Accepted Manuscript • DOI: 10.1021/la504115a • Publication Date (Web): 08 Dec 2014 Downloaded from http://pubs.acs.org on December 13, 2014
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Langmuir
Oil Transfer Converts Phosphatidylcholine Vesicles into Non-Lamellar Lyotropic Liquid Crystalline Particles Isabelle Martiel,† Stephan Handschin,†,‡ Wye-Khay Fong,†,¶ Laurent Sagalowicz,§ and Raffaele Mezzenga∗,† Food and Soft Materials Science, Institute of Food, Nutrition & Health, ETH Zurich, Schmelzbergstrasse 9, CH-8092 Zurich, Switzerland, Scientific Center for Optical and Electron Microscopy (ScopeM), Auguste-Piccard-Hof 1, 8093 Zurich, Switzerland., Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, Victoria 3052, Australia., and Nestlé Research Center, Vers-Chez-Les-Blanc, CH-1000 Lausanne 26, Switzerland E-mail:
[email protected] Abstract
1
2
There is a need for the development of low-energy dispersion methods tailored to
3
the formation of phospholipid-based non-lamellar lyotropic liquid crystalline (LLC)
4
particles for delivery system applications. Here, facile formation of non-lamellar LLC
5
particles was obtained by simple mixing of a phosphatidylcholine (PC) liposome solu-
6
tion and an oil-in-water emulsion, with limonene or isooctane as an oil. The internal ∗
To whom correspondence should be addressed ETH Zurich ‡ ScopeM ¶ australia § Nestlé Research Center †
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structure of the particles was controlled by the PC-to-oil ratio, consistently with the
8
sequence observed in bulk phase. For the first time, reverse micellar cubosomes with
9
F m¯3m inner structure were produced. The size, morphology and inner structure of
10
the particles were characterized by small-angle X-ray scattering (SAXS), dynamic light
11
scattering (DLS) and freeze-fracture cryo scanning electron microscopy (cryo-SEM).
12
These findings pave the way to new strategies in low energy formulation of LLC deliv-
13
ery systems.
14
Introduction
15
Phosphatidylcholine (PC) is a ubiquitous, naturally occurring amphiphile which forms the
16
major components of cellular membranes. As such, it is well-known for its strong abil-
17
ity to self-assemble in lamellar structures in water, either as a lamellar bulk mesophase or
18
dispersed as vesicles, more commonly known as liposomes. PC-based liposomes are exten-
19
sively used as cell membrane models and as targeted drug delivery systems. 1 Disruption
20
of the planar arrangement of PC bilayers in lamellar structures results in the formation of
21
non-lamellar mesophases, such as the reverse hexagonal, reverse micellar cubic and reverse
22
micellar phases. 2 Formation of these non-lamellar mesophases can be triggered by the ad-
23
dition of a third apolar component, 2 for instance limonene, 3 cyclohexane, 4 diglycerides 5 or
24
α-tocopherol, 6 to form ternary PC/water/oil systems.
25
The most common ways of creating sub-micrometer PC based LLC particles in aqueous
26
formulations involve energy intense processes or the use and removal of cosolvents. High-
27
energy dispersion techniques such as ultrasonication 7 or microfluidisation 5 are often used.
28
However, efficient dispersion of non-lamellar PC mesophases by conventional high-energy dis-
29
persion methods is challenging compared to monoglyceride or phytantriol-based mesophases.
30
Kamo et al. showed that PC mesophase structures are not fully retained with high-energy
31
dispersion methods, 8 with formation of large amounts of vesicles in addition to the expected
32
non-lamellar structure. 2 With monoglyceride mesophases, high-energy dispersion processes 2 ACS Paragon Plus Environment
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are eased by partial or total melting of the mesophase at higher temperatures, which are
34
readily reached in the absence of external cooling. 7 PC-based mesophases remain stable up to
35
the boiling point of water, resulting in poorly dispersible phases (see direct ultrasonication of
36
a PC non-lamellar mesophase in the Supporting Information). PC-based LLC dispersions are
37
generally produced by a hydrotrope-like method 9 involving addition of water-miscible com-
38
ponents, such as ethanol and propylene glycol, to reduce the viscosity of the lipid mixture,
39
which is then dispersed by vigorous shaking and heat treatments. 5,6,10 Ethanol addition and
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heat treatments may however induce degradation of some sensitive loads, such as proteins.
41
This study exploits the ability of PC liposomes to dynamically incorporate hydropho-
42
bic compounds into the bilayer, thus triggering a transition to non-lamellar structures in
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dispersion by material transfer. Lamellar to non-lamellar transitions have been achieved in
44
matrices composed of monoglycerides and phytantriol by temperature variation, 7 electro-
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static screening 11,12 and specific interactions. 13–15 Material transfer has been shown to occur
46
between emulsion droplets and monoglyceride or phytantriol-based cubosomes or hexosomes,
47
and modify the inner structure of the particles, 16–18 thus, this dynamic, spontaneously oc-
48
curring approach was utilized in this study in order to create stable non-lamellar structures.
49
The approach presented here results in a facile and elegant low-energy dispersion method
50
to form non-lamellar PC dispersions, with a high conversion rate from dispersed lamellar
51
structures to non-lamellar particles. Two different methods were used to produce pure PC
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lamellar dispersions with different particle sizes and levels of energy input. Various oils and
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secondary emulsifiers were used in the oil-containing phase. Limonene, the oil component
54
mainly used in this study, is a natural terpenic oil, widespread as perfume ingredient or
55
flavour additive in foods and cosmetics, 19 but also a potent mesophase modifier. 2,20 The
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recently reported 3 reverse micellar cubic mesophase of face centered structure F m¯3m is
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dispersed here for the first time as sub-micrometer particles. By imaging these particles
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by freeze-fracture cryogenic scanning electron microscopy (cryo-SEM) and SAXS, the ar-
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rangement of monodisperse PC micelles in a face-centered cubic lattice inside the dispersed
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particles could be observed. The effect of a variety of parameters relevant for formulation
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purposes was studied: influence of the oil content and type, secondary emulsifier content and
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type, mixing conditions, as well as the reversibility and oil transfer mechanism.
63
Results and Discussion
64
Lamellar to non-lamellar transition in dispersion. Lamellar dispersions of pure PC
65
were obtained by two methods. The filtration method, also called extrusion, was used as
66
a relatively mild PC dispersion technique, producing relatively large particle sizes (Z-av.
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diameter 256 nm ± 3 nm, PDI 0.24 ± 0.02) with multilamellar structure (peak at q ' 0.1
68
Å−1 in Fig. 1f). Moderate ultrasonication was used as a harsher PC dispersion technique,
69
yielding reduced particle sizes (down to Z-av. diameter 111 nm ± 3 nm, PDI 0.27 ± 0.01) with essentially unilamellar structure (absence of peak in Fig. 1c).
10 μm
Intensityb(a.u.)
100 10 1
emulsion {20}
0.1 {10}
0.01 0.00
0.05
10 μm
a
0.15
0.20
PCb+bemulsion b PCb(sonicated)
{11}
c
0.10
-1
100 10
emulsion
1 0.1
{20} {10}
0.01
10 μm
{21}
qb(A )
1000
Intensityb(a.u.)
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0.00
0.05
{11} {10} 0.10
-1
{21}
a
PCb+bemulsion PCb+bTweenb80 d e PCb(filtered)
f
0.15
0.20
qb(A )
Figure 1: Right upper panel: SAXS patterns from: (a) emulsion with 10% limonene and 0.85% Tween 80, (b) hexosomes formed by mixing an ultrasonicated PC dispersion and the oil emulsion in ratio α = 0.36, (c) ultrasonicated PC dispersion. (b) was obtained by mixing (a) and (c). Right lower panel: SAXS patterns from: (d) hexosomes formed by mixing a filtered PC dispersion and the oil emulsion in ratio α = 0.36, (e) mixture of filtered PC dispersion and 0.85% Tween 80 solution, (f) filtered PC dispersion. (d) was obtained by mixing (a) and (f). Left panel: corresponding DIC micrographs. In (d) and (f), only the biggest particles are visible. The thick colored arrows symbolize mixing procedures. 70
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An oil-in-water (o/w) emulsion stabilized by a secondary emulsifier was added in con4 ACS Paragon Plus Environment
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filtered PC: large oignon-like lamellar particles
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Intensity (%)
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nonlamellar particles
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sonicated PC: small unilamellar vesicles 5
0 1
10
100
1000
10000
Diameter (nm)
Figure 2: Representative size distributions (DLS) from lamellar PC dispersions (plain lines) and hexagonal PC-limonene dispersions (dashed lines), obtained by filtering (blue lines) or sonicating (red lines) the PC dispersion.
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trolled amount to the pure PC dispersion. The two relevant parameters describing the
73
mixtures are the mass fraction of oil in the lipid phase (oil and PC), α, and the mass ratio
74
of secondary emulsifier to the lipids, β:
α=
moil moil + mPC
(1)
β=
m2nd emuls. . moil + mPC
(2)
75
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Upon addition of an adequate aliquot of limonene emulsion stabilized with Tween 80, a
77
change in the structure of the lamellar PC dispersion, for example to reverse hexagonal
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(Fig. 1b & d), as well as a change in the particle size distribution, which is dependent upon the
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processing method of the initial PC dispersion, were observed. For extruded PC dispersions,
80
an overall decrease of the average size was observed (Fig. 2, blue lines) presumably due to
81
the collapse of the multilamellar vesicles, while for ultrasonicated dispersions, the particle
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size tended to increase (Fig. 2, red lines). Both resulted in a narrowing in the distribution
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of particles size, indicating an increase in uniformity. Depending on the oil-to-PC ratio,
84
different inner structures were obtained as detailed in the following sections.
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Particle morphology. Visual sample observation and Differential Interference contrast 5 ACS Paragon Plus Environment
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optical microscopy (DIC, Fig. 1 left panel) confirmed DLS sizing results and the absence of
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large aggregates. The lamellar dispersions were imaged by cryo-TEM (Fig. 3) and cryo-SEM
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(Fig. S10 in Supporting Information). The filtered particles (Fig. 3b) appeared essentially
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multilamellar, with a relatively open structure and a large empty inner compartment. Some
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elongated vesicles reminiscent of so-called myelin figures and tubules of PC 21,22 were found.
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On the contrary, the sonicated liposomes (Fig. 3a) were mainly unilamellar and considerably
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smaller in size.
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Although cryo-TEM is to this day the standard method used in imaging non-lamellar LLC
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particles, 23–26 other electron microscopy techniques yielding topographic visualizations have
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proven useful. 27 Depending on the fracturing path, high pressure freeze-fracture cryo-SEM
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gives access to both the outer surface and the innner structure of the particles, the latter
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feature being particularly adequate for the characterization of micellar mesophases, 28–30 as
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initially shown by Delacroix et al. 5,31,32 on bulk F d¯3m phases. Hexagonal and bicontinuous
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cubic bulk phases were later imaged as well. 33 Koifman et al. have recently published cryo-
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SEM images of the bulk reverse micellar cubic phase in the PC/water/isooctane system. 3,30 In
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this case, the diameter of the reverse micelles was about 20 nm. High pressure cryo-SEM has
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been only scarcely used on dispersions. The single example reported so far is the investigation
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by Angelov et al. of protein loaded bicontinuous cubosomes. 34 One major advantage of high
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pressure freeze-fracture cryo-SEM over standard cryo-TEM for dispersions is that the size
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of visible particles is not limited in principle, whereas in cryo-TEM the thickness of the film
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imposes an upper size limit.
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Non-lamellar dispersions produced from PC dispersions filtered at 0.45 µm and limonene
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emulsions were imaged by cryo-SEM (Fig. 4). The biggest particles offered the most distinc-
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tive features, although smaller particles appeared also structured (as indicated by the black
110
arrows in Fig. 4c and Fig. S.10 in the Supporting Information). Vesicles were very rarely
111
observed (white arrow in Fig. 4c) and no oil droplet was found, implying a complete mixing
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of the lipid components.
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Figure 3: Cryo-TEM micrographs from pure PC dispersions prepared (a) by ultrasonication, (b) filtering and (c) PC dispersion after evaporation of the limonene from a PC-limonene hexagonal dispersion.
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Monoglyceride or phytatriol-based hexosomes usually display a defined hexagonal prism-
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like shape, with their inner structures consisting either of parallel straight cylinders 27,35
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or of concentric circular tubes which appear as curved striations in cryo-TEM images. 24,36
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The typical faceting into a hexagonal revolution solid 27,37 and the concentric inner structure
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of columnar hexagonal lyotropic mesophase dispersions was recently rationalised in terms
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of surface tension minimisation, elastic anisotropy and interfacial anchoring. 38 Here, the
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inner fracturing of hexagonal particles reveals arrays of channels that often bend along the
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particle shape, sometimes forming concentric structures (Fig. 4a-c and e). In some images,
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the fracture occurred perpendicular to the axis of the cylinders (Fig. 4d, inset), revealing the
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cross-section of the columnar hexagonal structure. The observed repeating unit is about 14
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(± 2) nm, which corresponds to the lattice parameter measured by SAXS of 13.15 nm. The
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prevalence of concentric structures suggests that this type of morphology may be favoured by
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the low-energy transition mechanism from an onion-like lamellar structure by oil diffusion,
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in addition to the previously mentioned interface stabilisation mechanisms. 24
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Reversed micellar cubosomes of F m¯3m structure were produced and characterized for the
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first time. Cryo-SEM clearly showed fracture along the (111) plane, as seen from the 3-fold
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symmetry of the fractured surface and the 6-fold symmetry of the Fast Fourier Transform
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(FFT) in that area (yellow inset in Fig. 4f). Fractures along the (100) plane were also ob7 ACS Paragon Plus Environment
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H2
f
I2 (Fm3m)
Figure 4: Freeze-fracture Scanning Electron Microscopy (cryo-SEM) images from various PClimonene dispersed non-lamellar LLC mesophases obtained from a PC dispersion filtered at 0.45 µm. First line (a-e): hexagonal phase (α = 0.30). Second line (f-h): micellar cubic with F m¯3m structure (α = 0.64). Scale bars are 200 nm. Black arrows point at small particles that appear nevertheless structured. The white arrow in (c) shows a small vesicle, which were very rarely observed. In (d), (f) and (g), insets show details of the black box area, or the not-to-scale Fourier Transform (FFT) of the area marked with a star of same color: white for (100) planes, showing 4-fold symmetry, and yellow for (111) plane, showing 6-fold symmetry.
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served, giving 4-fold symmetry in FFT (white insets in Fig. 4f and h). The distance between
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micelles in contact (fracture along the (100) plane) was about 16 nm, which corresponds to
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a lattice parameter of 23 nm. This is consistent with the lattice parameter measured by
134
SAXS of 23.4 nm. The inner structure of bigger particles appeared generally better ordered
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than that of smaller particles (black arrows in Fig. 4 and S.10 in Supporting Information).
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Comparison with the bulk phase. As previously reported for PC-based LLC disper-
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sions, 6 the lattice parameters in dispersion were noticeably larger than the lattice parameters
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observed in bulk phases in excess water, both for the pure hexagonal (Fig. 5A) and pure
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139
F m¯3m (Fig. 5B) phases. This probably reflects an enhanced water capacity in dispersion
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compared to the bulk phase. 20,39 In the F m¯3m phase from filtered dispersions, the 220 and
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311 reflections were not distinguishable when the initial PC dispersion was filtered down
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to 0.2 µm instead of only 0.45 µm, but the lattice parameter remained identical (Fig. S.9
143
in the Supporting Information). The lower number and increased relative broadness of the
144
reflections in the dispersions are linked with the smaller size of the liquid crystalline mon-
145
odomains in dispersion, which results in a lower coherence of scattered X-rays. As discussed
146
in the section about the influence of secondary emulsifier, the increase in lattice parameter
147
is partly due to the presence of secondary emulsifier 35,40 and partly to the confinement of the structure due to dispersion in small particles. Intensityy+a.u.4
A
{10} 1
0.1
{11} {20}
{21} {30}
{10} 0.05
{111}
10
0.10
0.15
-1
qy+A 4
0.20
reverseymicellarycubic Fm3m
{220}{311} {222}
1 0.1
reverseyhexagonal {21} H2
{20}
bulk
0.01
B
{11}
dispersed
0.00
Intensityy+a.u.4
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dispersed bulk
0.01 0.00
{111} 0.05
{220}
{222} {331}{422}
{311} 0.10
0.15
-1
{333+511} 0.20
qy+A 4
Figure 5: Comparison of dispersed (blue curves) and bulk (pink curves) phases in the hexagonal (A) and reverse micellar cubic (B) phases, in excess water conditions. The lamellar PC dispersions were prepared by filtering at 0.45 µm. The oil content is α = 0.38 in bulk and α = 0.31 in dispersion in A, α = 0.55 in bulk and α = 0.65 in dispersion in B. 148
149
Influence of the oil content. The oil content is the main leverage available to tune the
150
internal structure of the final LLC particles. The structural phase diagram of the mixtures
151
of PC lamellar dispersions and limonene emulsions was studied as a function of the limonene
152
content (α ratio), at fixed Tween 80 content β, for the two different PC dispersion modes.
153
Figure 6 shows the comparison of the respective phase diagrams in dispersion and in bulk, for
154
filtered and sonicated dispersions. Representative SAXS curves are given in the Supporting 9 ACS Paragon Plus Environment
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Information (Fig. S.9). The size of reverse micelles in the dispersed L2 phase was obtained
156
by form factor fitting 3 (Fig. S.7 in the Supporting Information).
157
As observed in other studies, 40 the phase sequence in dispersion and in bulk were identical,
158
although the phase boundaries in dispersion did not correspond exactly to the bulk phase
159
boundaries. This boundary shifting effect and the increase of lattice parameter were more
160
pronounced for the smaller particles produced by ultrasonication of the initial PC dispersion
161
than for the larger filtered particles (Fig. 6), which can be largely explained by the enhanced influence of the surface and secondary emulsifier. sonicated dispersion
Lα
H2
filtered dispersion
Lα
H2
bulk Lα
I2
L2 I2
H2
L2
I2
L2
26
LatticeRparameter,RRadiusRHnmI
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24 22 20 18
H2 H2+I2
16 14
I2
12 10 8
Lα
L2
H2+Lα
6 4 0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
α limonene =Rmlimonene/HmPC+mlimoneneI
Figure 6: Lower panel: Phase diagram PC-limonene in excess water in bulk phase (full symbols) and in dispersion at fixed Tween 80 content β = 0.04, produced from sonicated (open symbols) or filtered (half-filled symbols) PC dispersions. Diamonds represent the lattice parameter in the lamellar phase Lα , triangles in the reverse hexagonal phase H2 , squares in the reverse micellar cubic phase F m¯3m (circled squares indicate samples filtered only at 0.45 µm were the 220 and 311 reflections were distinguishable). Circles represent the micellar radius in the reverse micellar phase L2 . Upper panel: Corresponding phase boundaries in excess water, on the same axis as the lower graph. The hatched areas represent coexistences of the adjacent phases. 162
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The α ratio had a only limited influence on the final size of the particles (Table 1). In the
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case of filtered PC dispersions, a sharp decrease of the mean particle size was observed at 10 ACS Paragon Plus Environment
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lower α values, corresponding to the conversion of vesicles in hexosomes. For ultrasonicated
166
PC dispersions, the particle size slightly increased at this stage. At higher α values, the
167
particle size showed only a moderate increase with the α ratio, for both PC dispersion methods. Table 1: Size and polydispersity index (PDI) of PC/limonene LLC particles at various α and fixed Tween 80/(PC+oil) ratio (0.04 for filtered PC, 0.06 for sonicated PC), with standard deviation between brackets (n = 3). PC dispersion mode filtered
sonicated
α 0 0.24 0.31 0.48 0.65 0 0.30 0.47 0.61
Z-average diameter /nm 332 (± 4) 205 (± 6) 197 (± 5) 200 (± 5) 207 (± 6) 118 (± 4) 125 (± 4) 122 (± 4) 131 (± 5)
PDI 0.26 0.19 0.17 0.19 0.21 0.24 0.13 0.16 0.24
(± (± (± (± (± (± (± (± (±
0.01) 0.01) 0.02) 0.03) 0.01) 0.03) 0.01) 0.01) 0.01)
Phase Lα Lα + H2 H2 I2 I2 Lα H2 I2 L2
168
169
Influence of the secondary emulsifier. In order to produce non-lamellar dispersions,
170
it is necessary to include a secondary emulsifier which can stabilize the large interface area of
171
the particles. It has been shown that commonly used secondary surfactants such as Pluronic
172
F127 have an influence on the structure of the mesophase and particle size, by penetrating
173
the water channels in monoglyceride or phytantriol phases. 40–42 Analogously, in this study,
174
it was also found that the amount of Tween 80 influenced the inner structure and size of
175
the PC-based dispersed LLC particles, as well as the structure of the corresponding bulk
176
phase. The particle size was observed to decrease with increasing Tween 80 concentration
177
(Table 2), as reported for conventional dispersion methods. 16,40,41 Figure 7 shows that the
178
lattice parameter of the H2 phase increases with the fraction of Tween 80 in the water phase,
179
both in dispersed and bulk phases. The secondary surfactant also decreased the quality
180
of ordering in both dispersed and bulk phases, as seen from the decreasing intensity and
181
broadening of the hexagonal Bragg reflections (Fig. S.8 in the Supporting Information).
182
However, the lattice parameter extrapolated at β = 0 is still significantly higher than the
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183
lattice parameter in bulk phase, which shows that the presence of secondary emulsifier does
184
not fully account for the increased lattice parameter in dispersions compared to bulk phases.
185
The increase of lattice parameter and the shift of phase boundaries in dispersion were
186
even more marked in hexagonal particles produced from sonicated liposomes, which had the
187
smallest particle size (Table 1, Fig. 6 and 2). This is points out to a particle size effect,
188
namely the confinement effect on the self-assembled structure induced by dispersion in small particles, as reported in block-copolymer self-assemblies. 43,44 1 5 1 4
L a ttic e p a ra m e te r (n m )
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1 3 1 2 1 1 1 0 9 8 0 .0 0
0 .0 2
0 .0 4
β= m
0 .0 6 T 8 0
/(m
P C
0 .0 8
+ m
lim o n e n e
0 .1 0
0 .1 2
)
Figure 7: Lattice parameter of PC-limonene hexagonal phase, in bulk (full symbols) and in dispersion (open symbols), as a function of the Tween 80-to-lipids content ratio β. Dashed lines are guides for the eye. The corresponding SAXS curves are given in the Supporting Information, Fig. S8.
Table 2: Size and polydispersity index (PDI) of PC/limonene LLC particles at fixed α = 0.30 (H2 phase) and various Tween 80 contents, with standard deviation between brackets (n = 3), from a pure PC sonicated dispersion of Z-average diameter 111 (± 3) nm, PDI 0.27 (± 0.01). T. 80 concentration in emulsion /wt. % 0.5 0.75 1 1.25 1.5 2 3
β 0.018 0.027 0.036 0.045 0.054 0.072 0.109
Z-average diameter /nm 206 (± 2) 177 (± 2) 159 (± 1) 149 (± 1) 142 (± 2) 120 (± 1) 108 (± 1)
PDI 0.09 0.07 0.06 0.07 0.07 0.10 0.12
(± (± (± (± (± (± (±
0.01) 0.01) 0.03) 0.02) 0.01) 0.01) 0.01)
189
190
Reversibility and cycling. The lamellar to non-lamellar transition in dispersion by oil 12 ACS Paragon Plus Environment
Page 13 of 28
191
addition was found to be reversible by limonene evaporation. The same sample can be cycled
192
at least 4 times by addition of limonene emulsion and subsequent evaporation of limonene
193
(Fig. 8). However, the size of the particles did not revert to the initial value of the lamellar
194
dispersion, but remained similar to that of the non-lamellar particles. This is also visible
195
in cryoTEM pictures from the initial and recovered PC dispersions (Fig. 3a and c). It is
196
expected that the cycling of structural transitions by oil addition and removal cannot be
197
unlimited, since the accumulation of the secondary surfactant will in fine disrupt the inner structure of the particles.
A
{10}
2
{11}{20} {21}
IntensityfHa.u.L
10
0
EV. AD. EV.
Lα e H2 f
C1
10
AD. EV.
Lα g H2 h
C2
10
0.00
0.05
0.10
C1
0.15
AD.
0.20
qfHA L
3
10
H2
H2
b EV.
H2
H2
f EV. d EV. h g AD. e AD. c AD.
AD.
Lα
Lα
a 10
AD.
Lα c H2 d
10
B
Lα a H2 b
1
10
ZCavfdiameterfHnmL
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Langmuir
Lα
2
EV.f=fevaporation AD.f=femulsionfaddition
Lα 0
1
2 3 Cyclefnumber
4
Figure 8: A. SAXS curves from a pure PC dispersion prepared by ultrasonication (a), alternatively transformed to hexagonal and lamellar dispersions by successive limonene emulsion additions (AD., b, d, f, h) and complete evaporation steps (EV., c, e, g). B. Corresponding Z-average diameters D measured √ by DLS. Error bars graphically represent the polydispersity of the size distribution as ±D PDI. 198
13 ACS Paragon Plus Environment
Langmuir
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
199
Influence of the mixing conditions, oils and secondary emulsifiers. Apart from
200
the main parameters investigated in the previous sections, other conditions and components
201
can be used to yield LLC particles. The lamellar to non-lamellar structural transition was
202
obtained by adding, instead of an emulsion, the corresponding quantities of Tween 80 solution
203
and a drop of pure, non-emulsified limonene (SAXS data in Supporting Information, Fig. S.2)
204
and letting the closed vial stand in the dark. In the absence of agitation, the separated oil
205
phase disappeared within a few days and no aggregation of the structured particles was
206
visible. On the contrary, strong aggregation occurred when only the pure limonene drop
207
without any secondary emulsifier was added, or when the Tween 80 concentration in the
208
added 10%-limonene emulsion was below 0.25 wt.%. When adding solely a Tween 80 solution,
209
little or no change was observed in the vesicular dispersion structure and particle size, and
210
no non-lamellar structure appeared (Fig. 1d).
211
Transition to non-lamellar structures were obtained also with isooctane or cyclohexane
212
emulsions, but not with a medium chain triglycerides emulsion, which is consistent with
213
the bulk phase ternary behaviour PC/water/oil for these oils 2,3 (SAXS data in Supporting
214
Information, Fig. S.3).
215
The lamellar-to-non-lamellar transition was observed similarly by adding limonene emul-
216
sions stabilized with Pluronic F127, Tween 20 or casein in 150 M PBS buffer (SAXS data in
217
Supporting Information, Fig. S.4), while rapid aggregation took place in presence of acacia
218
gum stabilized emulsions. This difference may be due to the ability of the former secondary
219
surfactants to adsorb into the particles and efficiently stabilize them, 45–47 whereas acacia
220
gum might cause strong aggregation by non-absorbing depletion effects.
221
Transfer mechanism(s). As in acquainted interparticular mass transfer phenomena
222
studied in the literature, 16–18,48 the driving force for the transfer appears to be compositional
223
ripening. There are three possible mechanisms for the transfer of lipid molecules to take
224
place in the presently reported system: (i) transfer by collision between particles, 49 (ii)
225
micelle-mediated transfer by the secondary surfactant, 16,18,48 (iii) transfer by solubilization
14 ACS Paragon Plus Environment
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Langmuir
226
(or partitioning) of the lipid in the aqueous phase. 50 Preliminary time-resolved laboratory
227
SAXS and DLS experiments suggested that most of the transfer of material took place over
228
the first minutes after the mixing, followed by slower maturation of the structure (data in
229
Supporting Information, Figs. S.5 and S.6). The rapid clearing of large oil droplets after the
230
mixing implies that micelle-mediated and/or solubilization transfer are prevalent phenomena.
231
However, the disappearance of smaller lamellar particles in ultrasonicated dispersions (Fig. 2)
232
suggests that fusion of vesicles may also take place during the oil-induced phase transition. 12
233
The non-lamellar particles obtained remain considerably smaller than the limonene droplets
234
introduced, which suggests that the material transferred is predominantly limonene, while
235
the large and insoluble PC molecules can be considered as immobile. 17
236
Time-resolved sSAXS experiments revealed that hexosomes were formed within the first
237
minute after mixing (Fig. 9A), with a lattice parameter of 13.3 nm. The formation of the
238
hexagonal phase was followed from the intensity of the first H2 reflection at 3 different initial
239
concentrations of Tween 80, significantly above the critical micellar concentration of Tween
240
80. The peak intensity was normalized with respect to its plateau value and fitted with a
241
single exponential decay model to obtain the characteristic time constant of the transfer 16,48
242
(Fig. 9B). The transfer time constant was found to decrease with increasing Tween 80
243
concentration (inset in Fig. 9B), which strongly indicates that the transfer is at least partly
244
mediated by micelles.
245
Conclusion
246
This study demonstrated that it is possible to obtain non-lamellar PC-based particles by
247
simple addition of a carefully selected oil, for instance limonene, in a PC vesicle dispersion
248
produced by various routes, in presence of a secondary emulsifier. The inner structure of the
249
obtained LLC particles can be finely tuned by the oil-to-PC ratio, by the characteristics of
250
the initial PC dispersion and by the amount of secondary surfactant. Through this versatile
15 ACS Paragon Plus Environment
Langmuir
A
Intensity(-a.u.u
1
3c(PC(+(0.8c(Tween(80 0.1
200
Ti
120
m e(s
u
160
80
40
0.0
0.1
0.2
0.3
-1
0.4
q(-A u
B
1.0
0.8
Transfer(time(constant(-su
Relative(intensity(I/Imax
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 16 of 28
0.6
0.4
0.2
40 35 30 25 20 0.0
0.2
0.4
0.6
Tween(80(final(concentration(-cu
0.0 0
20
40
60
80
100
120
140
160
Time(-su
Figure 9: A. Time-resolved sSAXS curves from the mixture of 3% PC and 0.8% Tween 80 at α = 0.32. B. Evolution of the relative intensity of the first hexagonal peak I/Imax as a function of time, for 0.4% (green squares), 0.8% (red circles) and 2% Tween 80 (blue triangles) emulsions. Lines show the exponential decay fits. Inset: Time constant of the exponential decay model as a function of the final Tween 80 concentration. Error bars correspond to the standard deviation of the time constant the fitting procedure.
251
method, sub-micrometer F m¯3m reverse micellar cubosomes were reported for the first time,
252
and characterized by cryo-SEM.
253
By taking advantage of the easy dispersion of lamellar structures into vesicles, this new
254
strategy opens the way to a milder form of producing LLC delivery systems based on lamellar-
255
forming natural surfactants, of which the ubiquitous PC is the perfect example. The large
256
capacity of inner compartments and low content of vesicles in the final system appear promis-
257
ing. The presently reported route would be particularly suitable for applications as delivery
258
systems, where there is a strong need for low-energy processing, efficient entrapment and
259
controlled release of sensitive loads, while using better accepted, natural components. 16 ACS Paragon Plus Environment
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Langmuir
260
Methods
261
Materials. Soy bean phosphatidylcholine (PC) was purchased from Cargill, Germany,
262
under the product name Epikuron 200. Composition details are given in the Supporting
263
R Information. (R)-(+)-limonene, isooctane, polysorbate 80 (Tween 80), polysorbate 20
264
R (Tween 20), acacia gum and technical casein were purchased from Sigma-Aldrich. Medium-
265
R chain triglyceride (MCT) was provided by Cognis. Pluronic F127 is the tribloc copolymer
266
PEO106 -PPO70 -POE106 , produced by BASF, USA. MilliQ water was used for all sample and
267
solution preparation.
268
Preparation of dispersions. PC (about 100 mg) was dissolved in about 6 g chloroform
269
in a 100 ml balloon. The solvent was evaporated in a rotary evaporator at 45◦ C, 125 rpm
270
to form a dry lipid film on the wall of the round bottom flask. The film was hydrated with
271
a 3 to 4 mL water at 37◦ C by vortexing until complete dispersion. This coarse dispersion
272
was either (i) ultrasonicated for 3 min with a UP200S sonicator (200 W, 24 kHz, Hielscher,
273
Germany) set at 20% power, duty cycle 0.5, or (ii) filtered 10 times through a cellulose
274
acetate filter of 450 nm pore size. The sonicated PC dispersions and some of the filtered
275
PC dispersions were finally passed 10 times through a cellulose acetate filter of 200 nm
276
pore size. PC loss by filtering was characterized by Fourier-Transform Infrared Spectroscopy
277
(Varian 640 FTIR Spectrometer) in transmission mode using a liquid cell with CaF2 windows
278
separated by a 15 µm spacer (data in the Supporting Information, Fig. S1). Oil-in-water
279
(o/w) emulsions were prepared by ultrasonicating 10% oil phase in the aqueous solution of
280
secondary surfactant during 1 min at 50% power, 0.5 duty cycle, resulting in a milk-like
281
dispersion. Appropriate volumes of fine PC dispersion and o/w emulsion were pipetted into
282
a glass vial and homogenized by slight hand shaking. The vial was tightly closed to avoid
283
oil loss and let standing for a few hours prior to analysis. In cycling experiments, limonene
284
evaporation was carried out by leaving the dispersion overnight in an open vial under the
285
hood, under magnetic stirring. Concomitant water loss was monitored by weighing and
286
compensated. Bulk mesophase samples were prepared by mixing all the components in a 17 ACS Paragon Plus Environment
Langmuir
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287
glass vial, as previously reported. 2,3
288
Small-angle X-ray Scattering (SAXS). Laboratory SAXS measurements were per-
289
formed with a MicroMax-002+ microfocused X-ray machine (Rigaku), operating at 4 kW, 45
290
kV and 88 mA. The Kα X-ray radiation of wavelength λ = 1.5418 Å emitted at the Cu anode
291
is collimated through three pinholes of respective sizes 0.4, 0.3, and 0.8 mm. The scattered
292
intensity was collected on a two-dimensional Triton-200 X-ray detector (20 cm diameter,
293
200 µm resolution) normally for at least 30 min for bulk mesophases, respectively 2 hours
294
for dispersions. The scattering wave vector is defined as q = 4πsin(θ)/λ, where 2θ is the
295
scattering angle. The SAXS machine is equipped with two sample chambers with different
296
sample-to-detector distances, giving access to q ranges of 0.005 to 0.22 Å−1 and 0.01 to 0.44
297
Å−1 respectively. Silver behenate was used for q vector calibration. Scattered intensity data
298
were azimuthally averaged using SAXSgui software (Rigaku). Solid samples were loaded
299
in a Linkam hot stage with temperature control in a cell formed by two thin mica sheets
300
and a rubber o-ring 1 mm-spacer. Liquid samples were filled into 1.5 mm diameter quartz
301
capillaries, sealed with epoxy glue (UHU). The X-ray machine is thermostated at 20±0.1◦ C,
302
taken as room temperature.
303
Time resolved synchrotron SAXS mixing studies were performed using a previously re-
304
ported flowthrough setup coupled to a quartz capillary to enable time-resolved small-angle
305
X-ray scattering for structural elucidation in real time. 51 Briefly, mixing was conducted in a
306
thermostated glass vessel at T=37◦ C under constant magnetic stirring. A 3% PC dispersion
307
was prepared by sonicating Epikuron 200 in milliQ water. 5% limonene emulsions with vari-
308
ous concentrations of secondary stabilizer were used. The PC dispersion was drawn through
309
a 1.5 mm diameter quartz capillary mounted in the X-ray beam at a flow rate of approxi-
310
mately 10 mL/min to avoid beam damage, through silicone tubing (total volume
