Stimuli-responsive lipidic cubic phase: Triggered release and sequestration of guest molecules

Article abstract of DOI:10.1002/chem.201405580

New stimuli-responsive nanomaterials, made up of host-guest lipidic cubic phases (LCPs) are presented. These biocompatible, stable, transparent and water-insoluble LCPs are composed of monoolein (MO) as a neutral host, and small amounts of one of three ju

Full text of DOI:10.1002/chem.201405580

DOI: 10.1002/chem.201405580
Communication
&
Drug Delivery
Stimuli-Responsive Lipidic Cubic Phase: Triggered Release and
Sequestration of Guest Molecules
Nelli Rahanyan-Kꢀgi,[a] Simone Aleandri,[a] Chiara Speziale,[b] Raffaele Mezzenga,[b] and
Ehud M. Landau*[a]
tion into the lipid bilayer, and amphiphilic ones to the interface
Abstract: New stimuli-responsive nanomaterials, made up
between the bilayer and the aqueous compartments, thus ren-
of host–guest lipidic cubic phases (LCPs) are presented.
dering LCPs ideal molecular carriers.[9] Entrapment of guest
These biocompatible, stable, transparent and water-insolu-
molecules within the LCP protects them from chemical and
ble LCPs are composed of monoolein (MO) as a neutral
physical degradation.[10] Based on their phase diagrams, they
host, and small amounts of one of three judiciously de-
can coexist stably with any amount of excess water,[2] implying
signed and synthesized designer lipids as guest that pre-
that they are water insoluble. Finally, LCPs are soft solids that
serve the structure and stability of LCPs, but render them
can be manipulated to adopt any shape. The combination of
specific functionalities. Efficient pH- and light-induced
these material properties is unique, and offers advantages in
binding, release and sequestration of hydrophilic dyes are
their utilization as biomaterials for various applications.
demonstrated. Significantly, these processes can be per-
Stimuli-responsive chemical systems have been used in
formed sequentially, thereby achieving both temporal and
a number of areas of science and technology.[11] In the field of
dosage control, opening up the possibility of using such
drug release, such systems enable control of drug distribution
LCPs as effective carriers to be used in drug delivery appli-
in response to exogenous (e.g., temperature, light) or endoge-
cations. Specifically, because of the inherent optical trans-
nous (e.g., pH, redox) stimuli.[12] The materials used respond
parency and molecular isotropy of LCPs they can be envis-
either to a specific physical incitement, or are made up of mo-
aged as light-induced drug carriers in ophthalmology. The
lecular building blocks that undergo chemical transformations
results presented here demonstrate the potential of mo-
such as protonation, cleavage or conformational change. They
lecular design in creating new functional materials with
have potential therapeutic advantage when continuous release
predicted operating mode.
of active ingredients might be toxic,[13] and an “on/off” switch-
ing should provide control over effectiveness of the therapy.
Responsive drug delivery systems used to date include lipo-
Lipidic cubic phases (LCPs) are nano-compartmentalized bio-
materials composed of specific lipids and water that form ther-
modynamically stable, nontoxic, and biocompatible gels.[1,2]
The structure of LCPs is akin to an ordered molecular sponge
consisting of a lipid bilayer that is curved in three dimen-
sions,[3] surrounded by two identical, yet nonintersecting aque-
ous channels.[4] These materials are optically transparent and
isotropic by molecular symmetry. Moreover, they are bicontinu-
ous, and enable diffusion in both compartments. Compartmen-
talization of the LCPs can be utilized to introduce either hydro-
philic, lipophilic, or amphiphilic molecules.[5–8] Hydrophilic com-
pounds are embedded in proximity of the lipids’ head groups
or in the aqueous channels, whereas lipophilic molecules parti-
somes, micelles, polymer nanoparticles, dendrimers, and inor-
ganic nanoparticles. Despite great advances in material science
and technology, a number of problems still persist, including
toxicity, stability, biocompatibility and biodegradability, as well
as efficient targeting to the diseased tissue. Synthetic polymer-
ic systems often exhibit toxicity issues, which may limit the
utility of these materials in vivo, since only nontoxic molecules
and macromolecules can be used.[1] Biocompatibility and bio-
degradability are practical problems that are often encoun-
tered with inorganic nanoparticles such as Au–Ag and gold
nanorods. Moreover, in most drug-targeting systems less than
approximately 5% of the active compound reaches the dis-
eased site.[11]
To overcome these problems, designer host–guest lipidic
cubic phases are presented as alternative delivery and seques-
tration systems that can be triggered by external stimuli. Diffu-
sion of embedded molecules within LCP depends, among
others, on the size and polarity of the molecules. In the case of
hydrophilic drugs, modification of lipids’ head groups allows
external control over drug release. For example, incorporation
of a negatively charged lipid results in slower release of posi-
tively charged molecules from the cubic phase.[14] Stimuli-re-
sponsive drug delivery systems that use various external stimu-
li have been designed, including light,[15] pH and salt concen-
[a] Dr. N. Rahanyan-Kꢀgi,+ Dr. S. Aleandri,+ Prof. E. M. Landau
Department of Chemistry, University of Zꢁrich
Winterthurerstrasse 190, 8057 Zꢁrich (Switzerland)
E-mail: ehud.landau@oci.uzh.ch
[b] C. Speziale, Prof. R. Mezzenga
Department of Health Science and Technology, ETH Zꢁrich
Schmelzbergstrasse 9, 8092 Zꢁrich (Switzerland)
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201405580. It contains full experimental de-
tails.
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tration.[16] Fong et al. used spiropyran laurate to form light-re-
sponsive LCPs as drug-delivery systems that photoisomerize re-
versibly following irradiation, thereby switching from the in-
verted bicontinuous cubic (fast release) to the inverted hexag-
onal liquid crystal structures (slow release).[15] Negrini et al. de-
veloped a pH-sensitive LCP that can be used for release of
molecules in the gastrointestinal tract, while preventing the re-
lease in the stomach environment.[16]
from MO-based LCPs doped with lipid 3 (denoted herewith as
MO/3, composed of 99/1%, w/w, in which the molar ratio be-
tween lipid 3 and MG is 84:1) was investigated at various pH
conditions. The p-nitro benzoic acid head group (pKaffi3.6) ren-
ders lipid 3, and the ensuing LCPs pH sensitive. As shown in
Figure 2, deprotonation of the carboxylate head group at pH 7
results in binding of the positively charged MG, in contrast to
the pure MO LCP, the head groups of which are neutral. Upon
successive decrease of the pH from 7 to 5, 3 and 2.5, the car-
boxylate head group is progressively protonated, resulting in
decreased binding between lipid 3 and MG, and leading to an
increase of MG release. At pH 5 approximately 95% of lipid 3
is ionized, whereas at pH 3 and 2.5, that is, below the pKa
value of the p-nitro benzoic acid head group, MG release is
faster than with pure MO-based LCPs at pH 7 (Figure 2), and
MO-based LCPs at pH 2.5 (Figure S3 in the Supporting Informa-
tion).
Herein, we present a host–guest system composed of mono-
olein (MO) as the host lipid, and one of three designer synthet-
ic lipids as the guest. These complex systems form stimuli-re-
sponsive, functional LCPs that are either light or pH sensitive.
Lipids 1 and 2 were designed in order to obtain light-respon-
sive biomaterials, whereas lipid 3 (Figure 1) is utilized in pH-
sensitive formulations.[10] MO’s glycerol head group is localized
at the interface between the lipid bilayer and the aqueous
compartment.[17] Addition of small amounts of the designer
guest lipids results in modified LCPs with functionalized head
groups that exhibit electrostatic interaction with hydrophilic
molecules residing in the aqueous compartments. Conditions
are established for the triggered release and sequestration of
two hydrophilic dyes, the anionic crocein orange G (CO), and
the cationic methylene green zinc chloride double salt (MG),
upon interaction with LCPs doped with the guest lipids.
Triggered release of MG was demonstrated in a dynamic ex-
periment shown in Figure 3. Initially MG is bound within the
LCP at pH 7. After four hours the pH of the overlay was de-
The current work explores two regimes: 1) triggered release
from the LCPs using pH-sensitive lipid 3 and light-sensitive
lipid 2; 2) sequestration into the LCPs using light-sensitive lipid
1. In the release experiment, specially designed stainless steel
holders were filled with 15 mg of LCP containing the solubi-
lized dye.[18] The surface area of the LCP was flattened with
a spatula and the holder was placed in a spectroscopic cuv-
ette. LCPs were overlaid with 1 mL of various aqueous solu-
tions and the time-dependent concentration of the dye re-
leased into the overlay was evaluated (by using Eq. (1), see the
Supporting Information) at different pH values (MQ water at
pHffi7, 20 mm NaCl, and 20 mm citric acid at pH values of 2.5,
3, and 5) for LCPs containing the pH-sensitive lipid 3, and fol-
lowing exposure to UV light for LCPs with the light-sensitive
lipid 2. Release of the positively charged dye MG (0.24 mm in
LCPs) from the aqueous compartments of pure MO LCP, and
Figure 2. Percentages of MG release as a function of time from the aqueous
compartments of the following LCPs into the overlay: MO, pH 7 (white
squares); MO/3, pH 7 (black triangles); MO/3, pH 5 (white circles); MO/3,
pH 3 (white triangles); MO/3 pH 2.5 (black squares).
Figure 1. Structures of the synthetic guest lipids 1, 2, and 3 (additives), and of the host lipid MO.
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pH 7, is effectively released upon addition of 20 mm of NaCl,
as the carboxylate moieties are shielded by the salt, thereby re-
ducing the electrostatic attraction to MG.[19]
These results are in excellent agreement with the control of
release by host–guest electrostatic interactions in cubic phases
reported recently by Negrini et al.[20] Small angle X-ray scatter-
ing (SAXS) experiments (Figure 5) performed on LCPs doped
with guest lipid 3 under identical conditions to those imple-
mented in the triggered-release experiments demonstrate that
addition of the synthetic guest lipid does not affect the phase
identity of the MO/H2O system, which is Pn3m throughout.[2]
The symmetry of the LCP is unchanged after 24 h, at both
pH 7 and 3. The unit-cell parameters are 101.0, 102.3 and
102.2 ꢂ, respectively (Table S1 in the Supporting Information).
Photochemical triggering of drug release represents a power-
ful alternative to pH, provided that the medium is transparent.
LCPs are ideal for these purposes, as they are optically trans-
parent and isotropic gels.[8] Incorporation of designer light-re-
sponsive lipids[21,22] into LCPs was therefore envisaged. Lipids
containing o-nitrobenzyl ester moieties are particularly well
suited to modulate chemical reactivity because they undergo
rapid cleavage reactions when exposed to UV (ffi360 nm)
light.[23] Towards this goal, photochromic lipid additive 2 was
incorporated into LCPs (0.1%, w/w). Photoinduced cleavage of
lipid 2 with UV light was characterized spectroscopically (Fig-
ure S2 in the Supporting Information). Lipid 2 (0.1%, w/w of
total lipid content) was incorporated into an MO-based LCP,
and the release characteristics of CO were assessed. As shown
in Figure 6 (black triangles), efficient binding of the negatively
charged CO to the positively charged ammonium head group
of 2 is affected at pH 7 (the molar ratio between 2 and CO is
1:1). Exposure of the lipid 2-doped LCP to UV light for 15 min
initiates a photochemical cleavage of the of nitrobenzyl
group,[24] yielding a negatively charged carboxylate head
group located at the lipid–water interface of the LCP. The re-
sulting release of CO, which is due to electrostatic repulsion, is
shown in Figure 6 (white circles). Further exposure to UV radia-
tion for 15 min after 4 and 6 h results in additional release of
CO. In order to ascertain that CO release is due to the UV-trig-
gered photocleavage of the lipid’s head group, and is not in-
duced by temperature change or any other accompanying re-
action that may take place, a control experiment was carried
out using the positively charged oleyl amine (0.1%, w/w) as
additive. Lacking light-responsive groups, no release was ob-
Figure 3. Percentages of MG release as a function of time from the aqueous
compartment of MO/3 LCP into overlay.
Figure 4. Percentages of MG release as a function of time from the LCP’s
aqueous compartments into the overlay: MO/3, pH 7 (white circles); MO/3,
pH 7, 20 mm NaCl (black triangles).
creased to 5, causing partial release, and after an additional
two hours the pH was decreased to 3, resulting in an addition-
al step-like release profile.
The effect of ionic strength on dye binding and release is
shown in Figure 4. MG, bound to a LCP doped with lipid 3 at
Figure 5. SAXS data on MO/3 LCPs under different conditions: a) immediately after preparation; b) after 24 h at pH 7; c) after 24 h at pH 3.
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Figure 7. Percentages of MG sequestrated as a function of time from the
overlay to the aqueous compartments of LCPs: MO (white circles); MO/
1 (black circles). The UV light was turned on for 15 min after 2, 4 and 6 h, re-
spectively.
Figure 6. Percentages of CO release as a function of time from the aqueous
compartments of MO/2 LCP into the overlay: without irradiation (black trian-
gles); with UV irradiation (white circles). The UV light was turned on for
15 min after 2, 4 and 6 h, respectively.
and 106.0 ꢂ, respectively (Figure S5 and Table S1 in the Sup-
porting Information).
served even after 24 h (Figure S1 in the Supporting Informa-
tion).
The results presented here demonstrate the potential of mo-
lecular design in achieving new, stimuli-responsive nanomateri-
als. Small amounts of judiciously designed and synthesized
lipid additives can be incorporated into MO-based LCPs with-
out destabilizing them, while rendering them functional in
binding, release and sequestration. In this investigation we
present pH and light as external stimuli. In the case of pH,
stable LCPs enable the controlled release of hydrophilic drugs
from the aqueous channels of the mesophases into the sur-
rounding environment. In the case of light, these systems can
specifically release from, or sequester into LCPs. Significantly,
these processes can be performed sequentially, thereby achiev-
ing both temporal and dosage control. Due to the thermody-
namic stability of fully hydrated LCPs with any amount of
excess water, that is, their absolute insolubility in water, and to
their soft-gel consistency, we envisage the possibility of apply-
ing such nanostructured biomaterials to specific locations in
vivo, thereby achieving exquisite spatial control of drug deliv-
ery. Moreover, the use of light as an external stimulus[24] has
many advantages in comparison with other stimuli: it is milder
than acids or bases, and variation of intensity, wavelength and
duration can provide a high level of pharmacological control.[11]
LCPs are ideal in this respect, as they are optically transparent
and isotropic by molecular symmetry, and can thus be envis-
aged as light-induced drug carriers in ophthalmology. Finally,
because of the ability of LCPs to incorporate molecules of vir-
tually any polarity or charge, the strategy presented here is
general, that is, molecule independent, and shows great prom-
ise for biomedical applications.
Addition of lipid 2 (0.1%, w/w) to MO induces a cubic Pn3m
(characteristic for the MO/H2O system under these conditions)
to cubic Ia3d phase transition, as shown by SAXS (Figure S4
and Table S1 in the Supporting Information). This transition
occurs before and after UV irradiation, with unit-cell parame-
ters of 157.0 and 157.3 ꢂ, respectively. Based on the critical
packing parameter (CPP),[1] this transition may be due to the
larger polar head group area of lipid 2, which leads to a smaller
CPP and to the observed Ia3d cubic symmetry. Importantly,
upon hydration of the Ia3d LCP for 24 h the phase reverts to
the Pn3m symmetry (a=105.7 ꢂ), as expected for an MO cubic
phase in excess water.
UV-induced control of the head-group structure and charge
was shown to be effective in sequestration of charged mole-
cules from the environment into LCPs. Sequestration experi-
ments were conducted using the neutral lipid 1 embedded as
additive in LCP (0.01%, w/w). In this experiment the stainless
steel holders were filled with 15 mg of MO/1 LCP, the surface
area of the LCPs was flattened with a spatula and the holder
was placed in a spectroscopic cuvette overlaid with an aque-
ous solution containing 9.6 mm MG. The molar ratio between
the light-sensitive lipid 1 embedded in the LCP and MG in the
overlay was 1:1. Sequestration of MG from the overlay into the
doped and pure LCPs was identical prior to UV illumination, as
both LCPs are composed of neutral lipids. Following exposure
to UV light, the head group of lipid 1 is converted to a nega-
tively charged carboxylate, which is shown to sequester the
positively charged dye (Figure 7). This effect can be repeated,
resulting in sequential sequestration of the dye. As shown by
SAXS, addition of the synthetic guest lipid 1 does not affect
the phase identity of LCPs, which is Pn3m throughout. The
symmetry of the LCP is unchanged after UV treatment or after
incubation for 24 h, with unit-cell parameters of 103.1, 103.3
Acknowledgements
This work was partially supported by Polish–Swiss joint Re-
search Programme grant PSPB-079/2010 to E.M.L.
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Keywords: drug delivery · lipids · lipidic cubic phase · stimuli-
responsive lipids · triggered release
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Received: October 9, 2014
Published online on December 15, 2014
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