Novel spiropyran amphiphiles and their application as light-responsive liquid crystalline components

Article abstract of DOI:10.1021/jp403840m

Light-responsive materials formed by liquid crystalline lipids in water have potential application to drug delivery through inclusion of photochromic additives such as spiropyran. A series of novel analogues of spiropyran (SP) have been synthesized with an SP headgroup that possess a C₈ (SP-OC), C₁₂ (SP-L), and C₁₆ (SP-P) tail to probe the influence of the length of the hydrophobic tail on their physicochemical properties and effect on behavior in liquid crystal matrices with a view to application as stimulus-responsive elements on ultraviolet irradiation. In addition, compounds possessing an oleyl (SP-OL) and phytanyl (SP-PHYT) tail, to mimic those of the parent reverse bicontinuous cubic (V₂) phase forming lipids, glyceryl monooleate (GMO) and phytantriol, were also prepared. The photochromic compounds were characterized by their melting points and photophysical behavior in solution using techniques including hot stage microscopy (HSM), differential scanning calorimetry (DSC), and UV-visible spectroscopy. Their effect on the equilibrium nanostructure of bulk V ₂ phases and phase-switching kinetics after exposure to UV light was assessed using small-angle X-ray scattering (SAXS). The melting point of the SP derivatives decreased linearly with increasing chain length, which suggests that interactions between the head groups governed their melting point, rather than the van der Waals interactions between the tails. Changing the R group did not influence the equilibrium rate constants for the isomerization of SP. Phase transition temperatures of liquid crystalline (LC) matrices were influenced significantly by incorporation of the SP derivatives and were greatest when the photochromic compound possessed an intermediate tail length substituent compared to the short alkyl or bulkier moieties. The level of disruption of lipid packing, and hence phase structure, were dependent on the duration of UV exposure.

Full text of DOI:10.1021/jp403840m

Article
pubs.acs.org/JPCB
Novel Spiropyran Amphiphiles and Their Application as Light-
Responsive Liquid Crystalline Components
Kristian J. Tangso,† Wye-Khay Fong,† Tamim Darwish,‡ Nigel Kirby,§ Ben J. Boyd,*,†
and Tracey L. Hanley*,‡
†Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University (Parkville Campus), 381
Royal Parade, Parkville, Victoria 3052, Australia
‡Australian Nuclear Science and Technology Organization, Locked Bag 2001, Kirrawee DC, NSW 2232, Australia
§SAXS/WAXS Beamline, Australian Synchrotron, Clayton, Victoria, Australia
S
* Supporting Information
ABSTRACT: Light-responsive materials formed by liquid
crystalline lipids in water have potential application to drug
delivery through inclusion of photochromic additives such as
spiropyran. A series of novel analogues of spiropyran (SP)
have been synthesized with an SP headgroup that possess a C8
(SP-OC), C12 (SP-L), and C16 (SP-P) tail to probe the
influence of the length of the hydrophobic tail on their
physicochemical properties and effect on behavior in liquid
crystal matrices with a view to application as stimulus-
responsive elements on ultraviolet irradiation. In addition, compounds possessing an oleyl (SP-OL) and phytanyl (SP-PHYT)
tail, to mimic those of the “parent” reverse bicontinuous cubic (V2) phase forming lipids, glyceryl monooleate (GMO) and
phytantriol, were also prepared. The photochromic compounds were characterized by their melting points and photophysical
behavior in solution using techniques including hot stage microscopy (HSM), differential scanning calorimetry (DSC), and UV−
visible spectroscopy. Their effect on the equilibrium nanostructure of bulk V2 phases and phase-switching kinetics after exposure
to UV light was assessed using small-angle X-ray scattering (SAXS). The melting point of the SP derivatives decreased linearly
with increasing chain length, which suggests that interactions between the head groups governed their melting point, rather than
the van der Waals interactions between the tails. Changing the R group did not influence the equilibrium rate constants for the
isomerization of SP. Phase transition temperatures of liquid crystalline (LC) matrices were influenced significantly by
incorporation of the SP derivatives and were greatest when the photochromic compound possessed an intermediate tail length
substituent compared to the short alkyl or bulkier moieties. The level of disruption of lipid packing, and hence phase structure,
were dependent on the duration of UV exposure.
additives, and solvent composition.7−14 The nanostructure, in
INTRODUCTION
■
turn, is what dictates the rate at which drug is released,13,15,16
Lipid-based liquid crystalline systems are attracting increasing
and transitions between phases are of interest for triggering
release of incorporated active ingredients.
There are several variables that may be used for triggering
interest as a means of controlled release drug delivery. When
amphiphilic lipids are present in excess water, they can self-
assemble into thermodynamically stable liquid crystalline
phases (often termed “mesophases” or just “phases”).1−6 The
phase transitions in situ after administration, including
temperature,13 salt, dilution, and pH.17,18 Fong et al.13
nonlamellar mesophases of most current interest in drug
demonstrated that temperature-stimulated systems can provide
delivery systems are the reversed bicontinuous cubic (V2) phase
drug release on demand. However, in practice there is potential
and the reversed hexagonal (H2) phase. Their ability to
for accidental activation of drug release in cases where direct
solubilize hydrophilic, hydrophobic, and amphiphilic drugs
application of temperature is used to stimulate the phase
make them excellent candidates for use as drug delivery
change, presenting a major limitation. A possible alternative
could be to utilize additives that can induce a phase change in
response to a more selective external stimulus such as
light.19−21
matrices (Figure 1). The most commonly studied lipids for
forming V2 phases in excess water are glyceryl monooleate
(GMO) a dietary lipid, and phytantriol, an ingredient
commonly used in the cosmetic industry; the chemical
structures are illustrated in Figure 1.
Lipid packing within the mesophase structure is a key
determinant of the overall nanostructure formed in water and
can be influenced by lipid concentration, temperature, pressure,
Received: April 18, 2013
Revised: June 7, 2013
Published: August 3, 2013
© 2013 American Chemical Society
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have synthesized a series of spirobenzopyran derivatives where
this equilibrium photochemical and physicochemical behavior
was established in solution.22,31,32 This structural change can be
utilized to alter lipid packing, and studies have shown that
spiropyran is able to induce phase changes in LC matrices.1
Fong et al.1 hypothesized that attachment of an alkyl group to
the spiropyran moiety “anchors” the dye into the bilayer
structure, thereby improving its ability to disrupt the
nanostructure of the mesophase. Incorporation of a laurate
derivative of SP (SP-L) into the V2 phase formed by phytantriol
in excess water induced a reversible change between the V2 and
H2 phases on UV irradiation, whereas underivatized SP had no
effect.
It is not known whether the laurate tail is the optimal
hydrophobic group to enable anchoring in the mesophase
bilayer and disruption of structure on irradiation. To this end,
the influence of the hydrophobic group on the physicochemical
properties and behavior of spiropyran derivatives in LC
matrices was investigated. Novel photochromic amphiphiles
possessing C8 (SP-OC) and C16 (SP-P) tails were prepared to
complement the previous studies using C12 (SP-L), which was
resynthesized for the current studies. Amphiphiles possessing
oleyl (SP-OL) and phytanyl (SP-PHYT) tails were also
synthesized to mimic those of the “parent” V2 phase forming
lipids, glyceryl monooleate (GMO) and phytantriol. The
analogues of spiropyran, illustrated in Figure 2, were all
assessed for their ability to influence the equilibrium
nanostructure of the LC matrices and their efficiency for
disruption of lipid packing and subsequent induction of phase
switching upon UV irradiation.
Figure 1. (Top) Chemical structures for phytantriol and glyceryl
monooleate (GMO). (Bottom) Structure of the reversed bicontinuous
cubic phase (left), the reversed hexagonal phase (center), and inverse
micelles (right), indicating compartments within the nanostructure
which can encapsulate hydrophilic and hydrophobic drugs.
Rendering liquid crystals light responsive by incorporating
photochromic compounds that alter lipid packing upon
irradiation may yield novel light-activated “on demand” drug
delivery systems with potential to provide a selective and
noninvasive approach to delivery in tissues that are not
amenable to direct heat activation, such as the posterior section
of the eye. Materials such as azobenzenes, spirooxazines, and
spiropyrans are families of organic photochromic compounds
that undergo reversible isomerization in response to UV
irradiation and can be employed to disrupt lipid packing within
the LC structure.22 Although azobenzenes have shown great
potential as versatile and tunable components for nano-
devices,23−25 their toxicity remains under question. In contrast,
the cytotoxicity of spiropyrans has been tested, and they were
found to be safe for application in bionanosensing.26
Consequently, spiropyrans have recently been of interest for
the development of light responsive liquid crystalline nanoma-
terials.27
MATERIALS AND METHODS
■
Synthesis. 1-(2-Hydroxyethyl)-3,3-dimethylindolino-6′-ni-
trobenzopyrylospiran (spiropyran alcohol, SP-OH) was pur-
chased from Tokyo Chemical Industry Co., Ltd. (Tokyo,
Japan). Lauric and palmitic acid (98% deuterated) were
generously provided by the National Deuteration Facility
(Lucas Heights, NSW, Australia). 4-(Dimethylamino)pyridine
(DMAP), N,N′-dicyclohexylcarbodiimide (DCC), and hydro-
genated lauric, palmitic, octanoic, and oleic acids were obtained
from Sigma-Aldrich (St. Louis, MO). Phytanic acid was a
generous gift from CSIRO (Melbourne, Australia). These
ingredients were used without further purification. Thin layer
chromatography (TLC) was performed on Fluka analytical
silica gel aluminum sheets (25 F254) and Davisil silica gel
LC60 Å (40−63 μm) was used for flash column chromatog-
raphy (FCC). Both materials were purchased from Sigma-
Spiropyrans reversibly switch between the colorless, non-
polar, closed “spiro” form (SP) and the colored, zwitterionic,
open “merocyanine” form (MC) when illuminated with white
and UV light, respectively28−32 (Figure 2). Drummond et al.
Figure 2. Equilibrium reaction that exists between the stable spiro isomer (SP) and the merocyanine form (MC) in solution. The structures of the R
group for each SP derivative are indicated in the legend.
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Aldrich (St. Louis, MO). Details of the experimental procedure
for the synthesis of SP derivatives, which employed standard
DCC/DMAP coupling of the acid to the free alcohol on SP-
OH, are described in the Supporting Information.
SAXS/WAXS beamline at the Australian Synchrotron (AS).
The holder was allowed to equilibrate at 28 °C for at least 30
min before acquiring the first scattering pattern, with 10 min
equilibration at each 2.5 0.1 °C temperature increment from
28 to 75 °C. Radiation with wavelength (λ) of 1.0322 Å (12
keV) was selected and an acquisition time of 0.5 s was used.
The 2D scattering patterns were collected on a 1 M Pilatus
detector, which covered the q range of interest from 0.0021 to
0.0986 Å where q = 4π sin(θ/λ) and θ is the scattering angle.
GMO-based bulk phase samples were packed into quartz
glass capillaries (Capillary Tube Supplies Ltd., Germany) with a
path length of 2.0 mm, sealed with wax, and then inserted into a
thermostated metal heating block controlled by a Peltier system
accurate to 0.1 °C. The samples were introduced to the beam
of a Bruker Nanostar SAXS camera, with pinhole collimation
for point focus geometry. The instrument source was a copper
rotating anode (0.3 m filament) operating at 45 keV and 110
mA, fitted with Montel mirrors, resulting in Cu Kα radiation
wavelength 1.54 Å. The SAXS camera was fitted with a Vantec
2000 2D detector (effective pixel size 550 μm) which was
located 700 mm from the sample to provide a q-range of
0.008−0.35 Å−1. Samples were equilibrated for 15 min
(previously determined as an appropriate equilibration period
these sample in this apparatus with incremental temperature
changes), and scattering patterns were collected over 15 min
under vacuum to minimize air scattering. Samples were heated
stepwise from 20 to 65 °C at 3 °C increments.
Nuclear Magnetic Resonance (NMR) Spectroscopy. 1H
2
NMR (400 MHz), 13C NMR (100.6 MHz), and H NMR
(102.6 MHz) spectra were recorded on a Bruker 400 MHz
1
instrument at ca. 25 °C. In H NMR spectra, chemical shifts
(ppm) were referenced to residual solvent protons (7.26 ppm
in CDCl3). In 13C NMR spectra, chemical shifts (ppm) were
referenced to the carbon signal of the deuterated solvent (77.0
ppm in CDCl3). NMR spectra of the SP derivatives are shown
in the Supporting Information.
Hot Stage Microscopy (HSM). As a preliminary technique,
hot stage microscopy (HSM) was used to determine the
melting point of the SP derivatives. A small sample of each
compound was heated at a rate of 5 °C/min using a Mettler
Toledo FP82HT hot stage fitted with a Mettler Toledo FP90
central processor temperature controller. The melting point
range was observed under a Zeiss Axiolab E microscope.
Differential Scanning Calorimetry (DSC). To confirm
the hot stage microscopic observations, the melting points were
also studied using DSC. A Perkin-Elmer DSC 7 fitted with a
Thermal Analysis Controller 7/DX was used to measure the
transfer of heat energy (enthalpy, ΔH) to or from the sample
(2−6 mg) as the temperature was raised from 25 to 120 °C at a
rate of 10 °C/min.
UV−Visible Spectroscopy. UV−visible absorption spectra
were measured on a Varian Cary-50 Bio spectrophotometer
(Melbourne, Australia) fitted with a Peltier temperature control
cell from 200 to 800 nm at a scan rate of 600 nm s−1 and kinetic
measurements at 540 nm. Solutions for UV−visible measure-
ments were prepared to concentrations of 2.0 × 10−4 M in
methanol. White light and UV illuminations were conducted
using a white light torch and a 12 LED ultraviolet 380 nm
torch, respectively.
Lipids. Myverol 18-99K from Kerry Ingredients (Almere,
The Netherlands) was used as a substitute for GMO for its
relatively low cost and similar phase behavior to pure GMO.33
The composition of Myverol 18-99K has been described in
detail previously.34 Phytantriol was a gift from DSM Nutritional
Products Ltd., Germany, with a minimum purity of 95%. Milli-
Q grade water purified through a Milli-pore system was used
throughout this study.
Preparation of Bulk Liquid Crystalline Samples (“Bulk
Phases”). Various concentrations (mol % relative to lipid) of
each SP derivative were dissolved in lipid. The aqueous phase,
either Milli-Q water or phosphate buffer solution (PBS)
controlled at pH 7.4, and lipid component (2:1, v:v) were
thoroughly mixed by heating transiently to approximately 60
°C and vortex mixing to homogeneity for at least three cycles.
Bulk phases were then left to equilibrate on a tube roller at 25
°C for at least 48 h prior to analysis.
Structural Characterization of Bulk Phases Using
Small-Angle X-ray Scattering (SAXS). Equilibrium Phase
Diagrams. For comparison of phase transition temperatures
and the generation of phase diagrams, two different SAXS
setups were utilized for phytantriol (Figure 6) and GMO
(Figure 7) systems, respectively.
Dynamic Irradiation Experiments. Time-resolved irradi-
ation experiments using synchrotron SAXS were conducted to
assess the efficiency and reversibility of phase transitions
induced by these SP derivatives.35
Bulk sample matrices prepared in PBS (pH ∼7.4) containing
varying concentrations (0.5, 1.0, or 1.5 mol %) of photo-
chromic additive were loaded into 1 mm quartz capillaries and
irradiated with UV light (60 mW, 375 nm) at different exposure
times: 15, 30, or 60 s. The UV light was delivered to the
samples using an EXFO Acticure 4000 UV lamp and fiber optic
cable, with the end of the fiber optic cable positioned
approximately 5 cm from the sample at a slight tangent to
the X-ray beam. The irradiation studies were conducted at
hutch temperature (∼30 °C) at the SAXS/WAXS beamline at
the Australian Synchrotron. The 2D scattering patterns were
acquired every 2 s for the first 2 min, then every minute until
the system reverted back to the “starting phase”, using a 1 M
Pilatus detector (active area 169 × 179 mm2 with a pixel size of
172 μm) which was located 956 mm from the sample position.
The computer software ScatterBrain Analysis was used to
reduce 2D scattering patterns to the one-dimensional scattering
function I(q). The d-spacing of the liquid crystalline lattice is
derived from Bragg’s law (2d sin θ = nλ), where n is an integer,
λ is the wavelength, and θ is the scattering angle). Since the
scattering profiles of the V2, H2, and L2 phases have been well
characterized using SAXS, observing the difference in relative
positions of the Bragg peaks, correlated by the Miller indices of
known phases, we can confirm the phase identity of the system
being analyzed. The V2D phase has the Bragg reflections
√2:√3:√4:√6:√8:√9, etc., while the H2 phase can be
identified by the Bragg reflections 1:√3:√4, etc. The L2 is
identified by a singular characteristic broad peak. The absolute
location of the peaks allows for the calculation of mean lattice
parameter, a, of the matrices, from the corresponding
interplanar distance, d (d = 2π/q), using the appropriate
scattering law for the phase structure. For cubic phases, a =
Phytantriol-based bulk phase samples were loaded into a flat
stainless steel metal plate (96 sample positions with a thickness
of 0.5 mm) sealed on both sides with Kapton tape, which was
attached to a thermostated heating block and mounted on the
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d(h2 + k2 + l2)1/2, while for the H2 phase, a = 4d/3(h2 + k2)1/2
where h, k, and l are the Miller indices for the particular
structure present. Since the L2 phase shows only one broad
peak, d is termed the characteristic distance.36
,
were not birefringent when observed under crossed polarized
microscope, precluding the likelihood of lamellar structure.
On the other hand, the oleyl and phytanyl derivatives do
appear to present a bulky, convoluted structure and
compromised packing might reasonably be expected with
consequent lower melting points. A number of deuterated
versions of the SP derivatives were prepared with a view to their
use in future small-angle neutron scattering studies. To provide
a more complete data set, their behavior has been pooled and
described with that of the hydrogenated derivatives. The
deuterated compounds demonstrated similar behavior in their
melting point to their hydrogenated equivalents, with a slightly
higher onset melting temperature.
Photophysical Behavior of SP Derivatives. The rate at
which the SP headgroup switches between the closed and open
form in response to UV and visible irradiation is indicative of
their photochromic behavior in solution. In the absence of UV
irradiation, a chemical equilibrium exists between the open
merocyanine form (MC) of spiropyran and the closed spiro
form (SP), as depicted in Figure 2, with the latter isomer being
more abundant. When a solution of spiropyran is excited with
UV light, the equilibrium between the two isomers is shifted to
favor MC, which is made evident when the solution strengthens
in color as the concentration of MC increases. After irradiation,
MC switches back to SP. This process can easily be followed by
the visible absorption of MC as a function of time.
RESULTS AND DISCUSSION
■
Synthesis of the SP Derivatives. Five SP derivatives were
successfully synthesized and are detailed in Figure 2. Full
synthesis details are contained in the Supporting Infornation.
1H and 13C NMR spectroscopy was used to characterize the
synthesized derivatives. As no uncharacteristic peaks were
observed in the 1H and 13C NMR spectra for the hydrogenated
SP derivatives and the 2H NMR spectra for the deuterated form
of selected SP moieties, the estimated purity of the synthesized
spiropyrans is >98% (see Darwish et al., Supporting
Information, Table SI1, for comparison, where chemical shifts
for n-propanol spiropyran are listed).28
Melting Points of the SP Derivatives. DSC thermograms
of the SP derivatives are illustrated in the Supporting
Information. The melting points of the SP derivatives decreased
approximately linearly with increasing chain length (Figure 3).
The observed equilibrium rate constants for the isomer-
ization of photochromic compounds in methanol were found to
be comparable (Table 1, Figure 5). This implies that the R
group on the structure of the SP derivative does not
significantly influence the isomerization rate, and thus it can
be deduced that changes in bulk phase structure during
irradiation would not be due to differences in the photophysical
properties of the SP derivative, but is directly indicative of
disruption of structure caused by geometric changes in the
structure of the additive. An example of the spectral changes of
SP-P in methanol at 25 °C immediately after UV irradiation
and white light illumination and a description of how the
isomerization rates were calculated can be found in the
Supporting Information. For SP-L the rate of isomerization in
the V2 liquid crystalline matrices has been previously shown to
be the same as in methanol.1
Figure 3. Influence of chain length/size on the melting point of SP-
OC, SP-L, SP-P, SP-OL, and SP-PHYT determined by hot stage
microscopy (circles) and differential scanning calorimetry (triangles).
The melting points of the deuterated compounds are denoted by
unfilled symbols. Plotted values represent the “onset” melting
temperature.
Effect of SP Derivatives on the Equilibrium Nano-
structure of Bulk Phytantriol and GMO Reverse
Bicontinuous Cubic Phases. In agreement with previous
studies on SP-L,1 incorporation of SP compounds into bulk V2
phases prepared using phytantriol (Figure 6) and GMO (Figure
7) lowered the transition temperature to the H2 phase to
varying degrees in the absence of UV irradiation. Determi-
nation of the phase transition temperature is an important
consideration in designing the optimal formulation that
requires a minimal concentration of photochromic compound,
possesses a phase transition temperature close to physiological
temperature, and requires minimal UV exposure.
The bulk phytantriol-based cubic phase containing SP-OC
(Figure 6A) existed as V2 phase up to 5 mol % of the additive
and there was no significant change in the phase transition
temperatures with increasing concentration of photochromic.
This suggests that the C8 tail has insignificant impact on the
lipid packing within the LC and a large amount of the additive
might be necessary to disrupt the liquid crystalline structure on
irradiation.
This rather counterintuitive finding was in agreement for both
independent methods (HSM and DSC). Usually increasing the
chain length in a homologous series leads to an increase in
melting point due to increased intermolecular van der Waals
forces between the molecules. The interesting trend shown in
Figure 3 therefore suggests that interactions between the head
groups rather than the hydrophobic tails might be governing
the melting point. Space-filling models for the derivatives are
illustrated in Figure 4. It is not immediately apparent why the
longer chain alkyl derivatives necessarily might pack less
strongly than the short octanoate derivative, because the chain
is quite separate from the head structure in this representation
and neighboring chains might be expected to interact more
extensively than the C8 derivative. It is therefore likely that the
chain adopts a bulky conformation and disrupts the hydrogen
bonding between adjacent headgroups. In the case of similar
amphiphiles with sugar head groups, regioisomers and chirality
has also been shown to provide seemingly anomalous melting
behavior, but in those compounds it is clear that they form
lamellar structures in the solid state.37 The neat SP derivatives
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Figure 4. Space-filling models of the spiropyran derivatives in the closed form prior to UV irradiation (top panel) and the open form after exposure
to UV light (bottom panel) arranged in their lowest energy state, as predicted in Chem3D Pro 12.0.
Table 1. Observed Equilibrium Rate Constants for
Isomerization of SP Derivatives in Methanol at 2.0 × 10−4
M
a
(mean s.d., n = 1−3)
k1 (after white light)
×10−4 s−1
k2 (after UV light) ×10−4
s−1
photochromic
SP-OC (h)
SP-L (h)
○
8.2
●
7.3
6.1 0.5
7.0
5.1 0.1
4.2
SP-L (d)
△
▲
SP-P (h)
7.1 1.4
11.7
5.0 0.05
7.7
SP-P (d)
□
+
■
+
SP-OL (h)
SP-PHYT (h)
17.7
8.3
×
12.8
×
9.1
a
Symbol key included in support of Figure 6.
Figure 5. First-order growth and decay of MC form after exposure to 5
min white and UV light, respectively; absorbances measured at 540
nm. Refer to Table 1 for what symbols correspond to.
In comparison, SP-L, which possessed an intermediate length
C12 tail, caused a slight lowering of the phase transition
temperature when incorporated into the phytantriol LC matrix
(Figure 6B). This was apparent at >1 mol % of SP-L, where the
V2 structure coexisted with the H2 phase near physiological
temperature. It can be inferred that when incorporated at these
concentrations SP-L would provide sufficient lipid packing
disruption to induce V2 → H2 phase transition at close to ∼37
°C on application of UV irradiation. This is consistent with the
behavior observed in the study reported by Fong et al.1 At >1.5
mol % the V2 phase did not exist without the coexisting H2
phase, and hence there is an upper limit for the amount that
could be added without compromising the structure of the
“initial phase”.
SP-P has a slightly longer tail (C16) than SP-L, but displayed
similar phase behavior (Figure 6C) with the phase transition
temperatures being suppressed to close to physiological
temperature in the presence of 1.5 mol % of the palmitate
derivative.
Incorporation of SP analogues with an unsaturated (oleyl)
and highly branched (phytantriol) tail into the phytantriol-
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Figure 6. Effect of the spiropyran derivatives on the equilibrium nanostructure of bulk phytantriol-based V2 phases upon heating in the absence of
UV irradiation. Bulk phases were heated stepwise from 20 to 65 °C at 3 °C increments and LC structures characterized by SAXS. Panels A−E are
temperature vs concentration phase diagrams of bulk phytantriol LC containing different amounts of photochromic additive, SP-OC, SP-L, SP-P, SP-
●
OL, and SP-PHYT, respectively. Estimated phase boundary lines are drawn to outline regions where samples exists as V2 phase ( ), coexisting V2
⧫
■
▲
and H2 phases ( ), H2 phase ( ), and coexisting H2 and L2 phases (★) and L2 phase ( ). Samples were prepared either in Milli-Q water (filled
shapes) or in PBS at pH 7.4 (unfilled shapes).
the phase transition temperatures in the presence of the SP
derivatives were suppressed by approximately 5 °C compared
to GMO alone, no significant differences between the transition
temperatures were observed on addition of the five SP
derivatives (Figure 7). It should be noted that the transition
temperatures for GMO alone are ∼10 °C higher than for
phytantriol, and hence the sensitivity to small amounts of the
additives may be reduced.
These experiments indicate that most of the systems
introduced here have the potential to undergo a phase
transition on irradiation of the photochromic additive with
UV light when incorporated at an appropriate concentration.
Phase-Switching Kinetics of Phytantriol and GMO
Cubic Phases Using with Photochromic Compounds
after Exposure to UV Light. As recently reported,1 SP-L was
able to induce reversible isothermal phase switching of cubic
phase to the H2 phase in response to UV irradiation.
As mentioned earlier, in addition to identification of the
phase present in a given sample, information related to its
lattice parameter (LP) can also be deduced from SAXS
scattering profiles.36 In the case where heat is generated on
irradiation, such as NIR irradiation in the presence of gold
nanorods, the equilibrium LP versus temperature plots can be
used as a calibration plot to derive the “apparent temperature”
felt by the matrix which is a consequence of the disruption of
lipid packing due to temperature in the dynamic experi-
ments.38,39
In the case of photochromic compounds and the effect of UV
irradiation on their ability to disrupt lipid packing, the degree of
disruption can by analogy be considered as an “equivalent
temperature” Tequiv. Consequently, from the temperature scans
conducted to produce the phase behavior diagrams of the LC
Figure 7. Influence of photochromic additive on the phase behavior of
GMO-based bulk cubic phase upon heating in the absence of UV
irradiation. Bulk phases were heated stepwise from 20 to 65 °C at 3 °C
increments and LC structures characterized by SAXS.
based LC resulted in a dramatic lowering of the phase transition
temperatures. In both cases, a very small fraction of additive
(<0.5 mol %) suppressed the phase transition temperatures to
well below physiological temperature (Figure 6D,E). At >0.5
mol % SP-PHYT the cubic phase did not exist at the lowest
temperature studied. This supports the hypothesis that a tail
with a similar structure to the parent lipid, phytantriol, would
enhance the miscibility between the two components and,
consequently, SP-PHYT provided the most disruption to the
lipid packing in the LC matrix.
In the case of GMO, the effect of the different SP derivative
was only studied at a single concentration (1 mol %). Although
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systems loaded with the photochromic compounds, LP versus
temperature calibration plots were also generated; an example
for SP-OC is shown in the Supporting Information. Using these
calibration curves, the equivalent temperature was extrapolated
for each scattering profile obtained at every time point
throughout the dynamic irradiation experiments. Therefore,
the phase-switching kinetics across changes in experimental
variables can be compared using Tequiv versus time profiles, as
illustrated in Figure 8. It must be noted again that it is not the
Table 2. Efficiency of SP Derivatives To Induce Phase
Switching in Bulk Phytantriol-Based LC upon UV
Irradiation, and Comparison of Time (s) To Reach
Maximum Equivalent Temperature (Tequiv) and Respective
Mesophase(s) Reached for Samples Loaded with Varying
Concentration of Photochromic Additive at pH ∼7.4 and
Different UV Exposure Times
UV
exposure
time (s)
max
Tequiv
(°C)
time to
max Tequiv
(s)
concn
final
mesophase
photochromic (mol %)
SP-OC
0.5
1.0
0.5
1.0
1.5
0.5
1.0
1.5
1.0
1.5
0.5
1.0
1.5
15
30
60
15
30
60
15
30
60
15
30
60
15
30
60
15
30
60
15
30
60
15
30
60
15
30
60
15
30
60
15
30
60
15
30
60
15
30
60
43.0
59.5
64.5
39.2
50.8
59.7
35.3
55.3
68.8
47.3
55.3
58.8
34.1
55.2
56.7
41.8
61.1
68.5
36.3
51.9
−
15
26
47
25
40
60
9
V2 + H2
H2
L2
V2
H2
H2
SP-L
V2 + H2
H2
19
45
12
27
50
19
25
56
23
27
57
16
27
−
H2 + L2
H2
H2
L2
V2 + H2
H2 + L2
L2
SP-P
V2 + H2
H2
Figure 8. Effect of different UV exposure times on the phase-switching
kinetics of phytantriol-based LC matrices containing 1 mol % SP-OC
as representative of profiles providing the data in Table 2 for the other
derivatives.
H2 + L2
H2
H2
L2
58.8
18
H2
−
−
−
−
−
−
H2
thermal motion that is inducing a change in nanostructure, but
rather the disruption to the matrix caused by photoisomeriza-
tion of the spiropyran moieties in response to UV irradiation.
The duration of UV exposure has been shown to influence
the extent to which phase the LC matrix transitions to, as well
as its phase-switching kinetics (Figure 8). The 1 mol % SP-OC
phytantriol-based LC system, in this example, gave rise to a
mixed V2 + H2 after only 15 s of UV “on”, and after 30 s of UV
exposure the LC transitioned completely to the H2 phase, and
relaxed back to the initial phase on removal of UV light. As
depicted in Table 2, prolonged UV exposure can lead to phase
transitions from V2 to H2 and even up to the L2 phase,
indicating significant capacity in the isomerization of the SP
derivatives to provide a range of disruption ability depending
on the degree of isomerization.
SP-OL
29.3
40.1
50.7
44.0
56.9
60.2
44.7
62.0
68.9
38.8
54.1
−
25
35
60
13
23
54
20
25
56
25
30
−
H2
L2
H2
H2
L2
SP-PHYT
H2
H2
L2
H2
H2
−
H2
52.5
13
−
−
−
−
−
−
The selection of the optimal light-responsive system was
based on rapid switching to the H2 phase, and possessing
maximum Tequiv above and closest to physiological temperature
(∼37 °C). The sample containing 1 mol % SP-L therefore met
the desired properties of the optimal light-activated LC system.
The phytantriol structure possesses a C13 side chain with a kink.
The similarity in hydrophobic length between phytantriol and
the C12 alkyl tail rationalizes the proposal that SP-L is best
matched structurally to occupy space within the phytantriol
bilayer structure and provide the greatest disorder to the lipid
packing upon UV irradiation. Further modeling of this
mechanism is essential to gain a better understanding as to
why the various spiropyran derivatives display different lipid
phase-switching kinetics.
delivery, and similar experiments are underway to explore
spiropyran−LC systems that reversibly switch from a slow
releasing matrix to fast releasing matrix upon UV irradiation
(e.g., H2 or L2 to V2).16 As UV penetration through biological
material is generally limited, preliminary studies are also in
progress that utilize NIR light as an alternate light source for its
greater penetration depth (up to ∼50 mm).40 Upconverting
nanoparticles (UCNPs) emit UV light in response to NIR light
exposure. These particles can be incorporated into the optimal
spiropyran-containing LC matrices to selectively induce phase
transitions.
The photoresponsive LC systems introduced here have
shown to be promising candidates for light-activated drug
10209
dx.doi.org/10.1021/jp403840m | J. Phys. Chem. B 2013, 117, 10203−10210

The Journal of Physical Chemistry B
Article
(15) Lee, K. W. Y.; Nguyen, T.-H.; Hanley, T.; Boyd, B. J. Int. J.
Pharm. 2009, 365, 190−199.
CONCLUSION
■
Novel amphiphilic spiropyran derivatives with different hydro-
phobic tail structures were synthesized. For the alkylated
derivatives, their melting points showed an inverse dependence
with tail length, indicating that the headgroup interactions
dominate the solid-state behavior. The photophysical behavior
in methanol was invariant with hydrophobe structure. Inclusion
of the photochromic additives into both phytantriol- and
GMO-based bulk V2 phases lowered the phase transition
temperature to the H2 phase. The prolonged irradiation of
these systems with UV-light-induced phase switching from the
V2 → H2 → L2 phases. Spiropyran laurate (SP-L) represents a
novel effective photochromic additive for imparting light
responsiveness to lipid-based liquid crystalline drug delivery
systems.
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ASSOCIATED CONTENT
* Supporting Information
Details on the synthesis of the spiropyran derivatives, H, H,
and 13C NMR spectroscopy characterizations (including
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spectroscopy, and SAXS studies. This material is available free
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AUTHOR INFORMATION
Corresponding Author
*E-mail: ben.boyd@monash.edu (B.J.B.); tracey.hanley@ansto.
gov.au (T.L.H.).
Notes
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The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors acknowledge the Australian Institute of Nuclear
Science and Engineering for funding this project (ALN-
GRA11161). K.J.T. also thanks AINSE for support in the
form of an Honours stipend and W.-K.F. thanks AINSE for a
PGRA scholarship. B.J.B. acknowledges the Australian Research
Council for a Future Fellowship. Part of this work was
conducted at the SAXS/WAXS beamline at the Australian
Synchrotron, Victoria, Australia.
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