Triggered liposomal release through a synthetic phosphatidylcholine analogue bearing a photocleavable moiety embedded within the sn-2 acyl chain

Article abstract of DOI:10.1002/chem.201304094

Liposomes represent promising carriers for drug delivery applications. To maximize this potential, there has been significant interest in developing liposomal systems encapsulating molecular cargo that are highly stable until their contents are released r

Full text of DOI:10.1002/chem.201304094

DOI: 10.1002/chem.201304094
Full Paper
&
Photocleavable Membranes
Triggered Liposomal Release through a Synthetic
Phosphatidylcholine Analogue Bearing a Photocleavable Moiety
Embedded within the sn-2 Acyl Chain
Andrew M. Bayer, Shahrina Alam, Samuel I. Mattern-Schain, and Michael D. Best*[a]
Abstract: Liposomes represent promising carriers for drug
delivery applications. To maximize this potential, there has
been significant interest in developing liposomal systems
encapsulating molecular cargo that are highly stable until
their contents are released remotely in a controlled manner.
Herein, we describe the design, synthesis, and analysis of
a photocleavable analogue of the ubiquitous lipid phos-
phoatidylcholine (PC) for the development of highly stable
and controllable photodisruptable membranes. Our strategy
was to develop a lipid that closely mimics the structure of
PC to optimize favorable properties including biocompatibil-
ity and stability of subsequent liposomes when mixed with
lipids possessing a broad range of physicochemical proper-
ties. Thus, NB-PC was designed, which contains a photocleav-
able 2-nitrobenzyl group embedded within the acyl chain at
the sn-2 position. Following the synthesis of NB-PC, lipo-
some disruption efficacy was evaluated through photolysis
studies involving the detection of nile red release. Studies
performed using a range of liposomes with different per-
centages of NB-PC, PC, phosphatidylethanolamine (PE), cho-
lesterol, and polyethylene glycol-PE (PEG-PE) demonstrated
minimal background release in controls, release efficacies
that correlate directly with the amount of NB-PC incorpora-
tion, and that release is only minimally impacted by the in-
clusion of the lipids PE and cholesterol that possess dispa-
rate properties. These results demonstrate that the NB-PC
system is a highly stable, flexible, and tunable system for
photoinitiated release from liposomal systems.
tions,[5] enzymes,[6] temperature,[7] and pH[8] changes have been
developed. As opposed to passive stimuli that are dependent
on internal factors, such as temperature and pH changes in
the local environment, light-mediated release is beneficial as it
can be utilized in an external manner with control over both
positioning and duration.
Introduction
The development of disease-specific delivery vesicles for drug
administration represents a critical emerging avenue for thera-
peutic intervention. Current research in this field faces several
obstacles, including rapid clearance of drugs and drug carriers
from the bloodstream, difficulty obtaining localization at the
diseased site, and challenges associated with efficient trigger-
ing of drug release at the diseased area. Liposomes have
emerged as promising biocompatible chaperones for medicinal
agents in which properties can be tuned to circumvent these
challenges.[1] For example, liposomal surfaces can be decorated
with polyethylene glycol (PEG) chains, creating “stealth” lipo-
somes with significantly enhanced circulation times.[2] In addi-
tion, chemical moieties can be introduced onto the liposome
surface to aid in the targeting of these containers to diseased
cells.[3] In order to maximize the potential for liposomal drug
delivery, it is of significant interest to control the release of mo-
lecular contents such that this occurs on a relatively quick
timescale at the target location. In the triggering of liposomal
content release, approaches exploiting light,[4] redox condi-
Light-mediated control of organic molecules is typically ach-
ieved using a photocleavable protecting group. There are
many existing photolabile groups, such as dithianes,[9] disul-
fides,[10] coumarin,[11] diazirine,[12] and 4-hydroxyphenacyl,[13] as
well as the 2-nitrobenzyl group,[14] the most widely studied
moiety in which near UV irradiation leads to decomposition of
the benzylic position to yield an aldehyde and a leaving group.
Several strategies have been reported involving disruption of
liposomes or photolabile amphiphiles. Zhang and Smith devel-
oped a phosphatidylethanolamine (PE) analogue bearing a ni-
trobenzyl protecting group on the amino moiety of the head-
group.[15] Upon irradiation of liposomes bearing 50 mol% PE
analogue, release was achieved, due to the phase change
driven by the preference of PE for the inverted hexagonal
phase. Nagasaki and co-workers utilized cationic amphiphiles
bearing two hydrophobic chains linked through a nitrobenzyl
group to enhance transfection efficiency of the pGL3 plasmid
into COS-1 cells.[16]
[a] A. M. Bayer, S. Alam, S. I. Mattern-Schain, Prof. M. D. Best
Department of Chemistry, University of Tennessee
1420 Circle Drive, Knoxville, TN 37996 (USA)
Fax: (+1)865-974-9332
Chandra and co-workers designed and synthesized several
amphiphiles containing a nitrobenzyl moiety separating a long
hydrophobic tail from a polar amino acid headgroup.[17] This
system was used for successful release of encapsulated fluores-
E-mail: mdbest@utk.edu
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201304094.
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cein from PC liposomes containing 5% photocleavable amphi-
phile. Similar amino acid-based amphiphiles were described by
Subramaniam and co-workers in which 6-bromo-7-hydroxycou-
marin-4-methyl (Bhc) was employed as both the hydrophobic
backbone and the photocleavable moiety.[18] Recently, Dong
and co-workers used polymerized vesicles containing lipids
bearing a cleavable moiety for release using two-photon ab-
sorption.[19] Photodisruption of liposomes has also been carried
out by dithiane lipids, as described by Wan et al.,[9] which re-
lease the phosphocholine headgroup under UV light. In addi-
tion, Liang and co-workers have incorporated the azobenzene
functional group in a silica-modified liposome, or cerasome, to
induce dye permeability induced through cis-to-trans isomer-
ism.[20] Lipid cross-linking initiated by irradiation has also been
employed as a strategy for photochemical membrane destabi-
lization and release.[21] It should be noted that synthetic lipids
bearing photocleavable moieties have also been developed as
caged analogues of biologically active lipids such as diacylgly-
cerol,[22] phosphatidic acid,[23] and phosphatidylinositol-(3,4,5)-
trisphosphate.[24]
Results and Discussion
Our design for a biologically inert photocleavable analogue
(NB-PC) of the common structural lipid PC is depicted in
Scheme 1A. This compound includes a 2-nitrobenzyl moiety
embedded within the sn-2 acyl chain of PC in order to gener-
ate a stable bilayer lipid in which membrane properties would
be significantly modulated through photocleavage. This acyl
chain also includes a succinyl linker to the PC headgroup as
well as a hexyl group to add hydrophobic character at the ter-
minus of the acyl chain. This was designed such that irradia-
tion would cause the breakage of the bond between the ben-
zylic position and amide nitrogen, releasing the hydrophobic
terminus of the acyl chain in the form of aldehyde 1, as well as
a shortened PC analogue bearing a polar terminal amide func-
tional group (2). Furthermore, it was envisaged that this re-
lease could stimulate the amide nitrogen to attack the succinyl
ester group, leading to the release of succinimide and thus the
further truncation of the lipid structure, potentially producing
the nonbilayer lipid lysophosphatidylocholine (LPC, 3).
While this prior work has resulted in a range of successful
strategies for triggered liposome release, further study is likely
needed to optimize properties
The synthesis of NB-PC was completed as described in
Scheme 2. Towards this end, Boc-(aminomethyl)nitrobenzoic
associated with delivery. In par-
ticular, the development of ana-
logues that bear close resem-
blance to robust and commonly
occurring lipids would seem to
be ideal for enhancing biocom-
patibility and thus stability
during circulation. For this
reason, we elected to pursue
a photocleavable analogue of
PC, since this lipid is the primary
component of cellular mem-
branes. Furthermore, our design
incorporates the photocleavable
group within a lipid acyl chain,
leaving the natural PC head-
group intact. In this way, our an-
alogues were designed to form
stable membrane bilayers with
naturally occurring aqueous in-
terfaces to maximize stability
and biocompatibility. Herein, we
describe the design and synthe-
sis of photocleavable PC ana-
logue NB-PC along with photo-
lysis studies aimed at character-
izing the release efficiency of
this compound when incorporat-
ed into liposomes composed of
different lipids that affect the
properties of the membrane bi-
Scheme 1. Approach for membrane disruption using photocleavable PC analogue NB-PC. a) Irradiation of NB-PC
is expected to directly produce aldehyde 1 and amide 2 through CꢀN bond breakage. Subsequent intramolecular
cyclization could also produce succinimide and LPC product 3. b) Cartoon depicting the disruption of membrane
structure upon photolysis of NB-PC.
layer.
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a 200 nm filter, and characteriza-
tion using dynamic light scatter-
ing (DLS). After this, the resulting
liposomes were irradiated with
350 nm light, during which nile
red emission at 612 nm was
tracked with excitation at
595 nm. As can be seen in
Figure 1, irradiation of NB-PC led
to a significant time-dependent
decrease (ꢁ80%) in nile red
emission, which was attributed
to fluorophore release over
a period of approximately one
hour. These data were then
fitted with a kinetic decay equa-
tion, providing figures including
fraction of original fluorescence
(y0), first order rate constant (k),
half-life (t1/2) fit parameters (A)
and correlation (R2) shown in
Table 1 (see Experimental Sec-
tion for further details). In con-
trol studies, liposomes com-
posed of DOPC that were irradi-
ated (light) and untreated (dark)
Scheme 2. Synthesis of NB-PC. 4-Aminomethylbenzoic acid (4) was protected with a trifluoroacetate group to 5,
nitrated to 6, and underwent protecting group exchange to 7 following a previously reported procedure.[17a] Cou-
pling with hexylamine to 8 was followed by chain extension through nucleophilic attack of succinic anhydride to
form photocleavable fatty acid 9. Finally, coupling onto the sn-2 chain of LPC (3) yielded NB-PC. TFAA=trifluoro-
acetic anhydride.
acid 7 was first synthesized from aminomethylbenzoic acid 4
as previously described[17a] through amine protection to 5, ni-
tration to 6 and protecting group exchange to 7. Next, an
amide bond coupling reaction with hexylamine was used to in-
troduce the hydrophobic terminus of compound 8. The succin-
yl linker was then introduced through deprotection of the Boc
protecting group of 8, followed by opening of succinic anhy-
dride to produce photocleavable fatty acid 9. Finally, this fatty
acid was introduced onto commercial palmitoyl-lysophosphati-
dylcholine through a coupling reaction in a manner similar to
a prior report.[25]
Following the completion of the synthesis of NB-PC, we set
out to characterize the efficacy of this compound for photoini-
tiated release of encapsulated cargo with properties similar to
typical drugs. In doing so, we exploited an assay involving the
release of nile red reported by Liang and co-workers
(Scheme 1B).[20] Nile red mimics the properties of common
drugs by inserting into the hydrophobic core of the membrane
bilayer interior, and thus the liposome solubilizes the encapsu-
Figure 1. Nile red release upon irradiation of liposomes containing 100%
NB-PC. Photocleavage led to significant release from NB-PC liposomes com-
pared to controls including NB-PC left in the dark and irradiation of DOPC
liposomes.
lated dye. Upon triggered release, the nile red is rendered in-
soluble, which can be detected by the decrease in emission of
the solution. In studies, one of the benefits of using an ana-
logue of the robust bilayer lipid PC is that this compound can
be incorporated into liposomes at a broad range of percent-
ages to form stable membranes. As such, Figure 1 shows the
release of nile red from liposomes composed entirely of either
commercially available dioleyl-PC (DOPC) or photocleavable
analogue NB-PC. In these studies, liposomes were generated
using standard procedures including hydration, extrusion with
as well as those composed of NB-PC that were not photolyzed
(dark) all led to minimal decreases in nile red emission (ꢁ16–
19%), confirming that release was directly caused by liposome
photolysis and that this only occurred in the presence of com-
pound NB-PC. These studies additionally indicate that photo-
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Table 1. Data resulting from curve fitting of release from NB-PC lipo-
somes and controls.
Table 2. Data resulting from curve fitting of release from liposomes con-
sisting of 10–100% NB-PC mixed with DOPE and DOPC.
Sample NB-PC DOPC y0
k
[sꢀ1
t1/2
[min]
A
R2
Sample NB-PC DOPE DOPC yo
k
[sꢀ1
t1/2
[min]
A
R2
[%]
[%]
]
[%]
[%]
[%]
]
Light
Light
Dark
Dark
100
0
100
0
0
100
0
0.2053 8.45E-04 13.7
0.8432 1.53E-03 7.6
0.8074 4.83E-04 23.9
0.8454 7.93E-04 14.6
0.8848 0.9628
0.1481 0.4233
0.1863 0.8597
0.1412 0.3813
Light
Light
Light
Light
Dark
Dark
Dark
Dark
10
25
50
100
10
25
50
50
50
0
50
50
50
0
40
25
0
0.6450 1.35E-03 8.6 0.3592 0.9569
0.5180 1.13E-03 10.2 0.4980 0.9614
0.3845 7.55E-04 15.3 0.6302 0.9853
0.2053 8.45E-04 13.7 0.8848 0.9628
0.8278 1.08E-03 10.7 0.1628 0.7013
0.8725 9.43E-04 12.2 0.1194 0.8070
0.8738 6.95E-04 16.6 0.1159 0.4473
0.8074 4.83E-04 23.9 0.1863 0.8597
100
0
40
25
0
50
100
bleaching of the dye is not occurring since light and dark con-
trols yielded comparable results.
0
With this successful result, we next set out to characterize
the effects of lipid composition on release efficacy to deter-
mine the flexibility of the system. In the next batch of studies,
we decided to include 50% dioleylphosphatidylethanolamine
(DOPE) in the liposomes, which may affect release due to the
nonbilayer properties of this lipid.[15,26] Thus, liposomes con-
taining DOPE fixed at 50%, varying NB-PC at 10, 25, and 50%,
and the remaining percentage filled in with DOPC were next
analyzed. Data from liposomes containing 100% NB-PC are
also included with these results for the sake of comparison. As
can be seen in Figure 2 and Table 2, the extent of nile red re-
lease was found to directly correlate with the percentage of
NB-PC included in the liposome, and dark controls once again
yielded minimal release. Inclusion of 10, 25 and 50% of NB-PC
resulted in ꢁ36, 48 and 62% decreases in nile red emission, re-
spectively, all lower than liposomes completely composed of
NB-PC (ꢁ80%). These data show that release can be carried
out using a broad range of percentages of NB-PC within lipo-
somes, and that the percentage can be used to tune the re-
lease properties of the vesicles. It should also be noted that
variations in the percentage of NB-PC did not have as signifi-
cant of an effect on background release, as dark controls
ranged from ꢁ12–19% emission decrease, indicating that in-
corporation of NB-PC does not destabilize the membrane bi-
layer.
We next set out to vary the percentage of DOPE in lipo-
somes used for studies, while keeping the percentage of NB-
PC fixed. This series of studies was explored to evaluate how
DOPE inclusion would affect release due to the nonbilayer
properties of this lipid, which may be pronounced after irradia-
tion of NB-PC and release of photolysis products, by increasing
the percentage of DOPE in the product liposomes. As can be
seen in Figure 3 and Table 3, the effect of DOPE variation was
less pronounced compared to that of NB-PC percentages. Lip-
osomes with fixed percentages of NB-PC (50%) and DOPE at 0,
10, 25, and 50% led to emission decreases of ꢁ68, 67, 66, and
62%, respectively. The extent of release in control liposomes
was comparable to prior studies involving the variation of NB-
PC. These data suggest that DOPE inclusion does not pro-
Figure 2. Nile red release upon irradiation of liposomes containing 10–100%
NB-PC mixed with DOPE and DOPC. Release efficacy was found to increase
directly with the percentage of NB-PC, while controls remained relatively
static.
Figure 3. Nile red release upon irradiation of liposomes containing 50% NB-
PC mixed with 0–50% DOPE along with DOPC. Release efficacy was not sig-
nificantly modified by large changes in the percentage of DOPE.
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Table 3. Data resulting from curve fitting of release from liposomes con-
sisting of 50% NB-PC mixed with 0–50% DOPE along with DOPC.
Table 4. Data resulting from curve fitting of release from liposomes con-
sisting of 50% NB-PC mixed with 0–50% cholesterol along with DOPC.
Sample NB-PC DOPE DOPC yo
k
[sꢀ1
t1/2
[min]
A
R2
Sample NB-PC Chol DOPC yo
k
[sꢀ1
t1/2
[min]
A
R2
[%]
[%]
[%]
]
[%]
[%] [%]
]
Light
Light
Light
Light
Light
Dark
Dark
Dark
Dark
Dark
50
50
50
50
100
50
50
50
50
100
0
10
25
50
0
50
40
25
0
0.3202 1.02E-03 11.3 0.7140 0.9877
0.3268 1.03E-03 11.2 0.7112 0.9844
0.3358 8.43E-04 13.7 0.7226 0.9616
0.3845 7.55E-04 15.3 0.6302 0.9853
0.2053 8.45E-04 13.7 0.8848 0.9628
0.8542 4.80E-04 24.1 0.1418 0.8591
0.8877 5.95E-04 19.4 0.1037 0.6430
0.8074 4.83E-04 23.9 0.1863 0.8597
0.8738 6.95E-04 16.6 0.1159 0.4473
0.8074 4.83E-04 23.9 0.1863 0.8597
Light
Light
Light
Light
Light
Dark
Dark
Dark
Dark
Dark
50
50
50
50
100
50
50
50
50
100
0
10
25
50
0
50
40
25
0
0.3202 1.02E-03 11.3 0.7140 0.9706
0.3024 1.00E-03 11.6 0.7350 0.9827
0.3189 9.42E-04 12.3 0.7241 0.9829
0.2821 9.73E-04 11.9 0.7808 0.9589
0.2053 8.45E-04 13.7 0.8848 0.9628
0.8542 4.80E-04 24.1 0.1418 0.8591
0.8718 5.58E-04 20.7 0.1168 0.5715
0.8564 4.97E-04 23.3 0.1281 0.5379
0.8707 3.07E-04 37.7 0.1211 0.6046
0.8074 4.83E-04 23.9 0.1863 0.8597
0
0
0
50
40
25
0
0
50
40
25
0
10
25
50
0
10
25
50
0
0
0
foundly affect triggered release and further point to the versa-
tility of this compound, since significant variation in the prop-
erties of the lipids incorporated into liposomes does not have
a great effect on release.
PEGylated lipids are commonly added to liposomes to in-
crease circulation in samples used for clinical applications. As
a result, we next sought to assess whether the inclusion of
such lipids affects release in this system. Towards this end, lipo-
somes composed of 15% distearoyl-PEG(2000) PE-amine
(DSPE-PEG(2000) amine), which is on the high end of amounts
typically used, and 85% NB-PC were compared to 100% NB-
PC liposomes. Both behaved similarly during irradiation and in
the dark, as can be seen in Figure 5 and Table 5. The PEG
sample showed fluorescence decreases of 79 and 14% in UV
light and in the dark, respectively, compared to 79 and 19%
for the pure NB-PC sample. Thus, the inclusion of PEGylated
lipids made little difference in controlled release in this system,
and thus this approach could be implemented to improve in
vivo survival in the pursuit of future medicinal applications.
Finally, with an effective release system in hand, we sought
to characterize the system further by determining the products
generated from membrane photocleavage. As previously dis-
cussed, irradiation of NB-PC was expected to directly generate
To further characterize the tolerance of this system for differ-
ent lipids with varying properties, we next sought to deter-
mine how release is affected by the inclusion of cholesterol,
which increases the fluidity and stability of membranes. As
shown by the data in Figure 4 and Table 4, inclusion of choles-
terol at 0, 10, 25 and 50%, with NB-PC fixed at 50%, led to
nile red emission decreases of ꢁ68, 70, 68 and 72%, respec-
tively. Thus, once again broad fluctuations in membrane prop-
erties caused by the inclusion of cholesterol did not significant-
ly affect the ability of NB-PC to induce the release of nile red
from liposomes.
Figure 4. Nile red release upon irradiation of liposomes containing 50% NB-
PC mixed with 0–50% cholesterol (CHOL) along with DOPC. Release efficacy
was not significantly modified by large changes in the percentage of choles-
terol.
Figure 5. Nile red release upon irradiation of liposomes containing NB-PC
compared to those containing 15% DSPE-PEG(2000) amine. Release efficacy
was not significantly modified by the addition of DSPE-PEG(2000) amine.
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produce significant differences in release, stealth liposomes are
a viable route for future usage of this compound. With this in-
formation, the stability and adaptability of the NB-PC system
provides a beneficial starting point for building a more com-
plex delivery system.
Table 5. Data resulting from curve fitting of release from liposomes con-
sisting of NB-PC alone or when mixed with DSPE-PEG(2000) amine.
Sample NB-PC PEG-PE yo
k
[sꢀ1
t1/2
A
R2
[%]
[%]
]
[min]
Light
Dark
100
85
100
85
0
15
0
0.2053 8.45E-04 13.7
0.2150 7.07E-04 16.3
0.8074 4.83E-04 23.9
0.8581 8.83E-04 13.1
0.8848 0.9628
0.8561 0.9700
0.1863 0.8597
0.1317 0.7304
Experimental Section
15
General experimental
Reagents and solvents were generally purchased from Acros, Al-
drich, or Fisher Scientific and used as received. Palmitoyl-lysophos-
phatidylcholine (3) and DSPE-PEG(2000) amine were purchased
from Avanti Polar Lipids, Inc. and (4-aminomethyl)benzoic acid was
purchased from Chem Impex International. Dry solvents were ob-
tained from a Pure Solv solvent delivery system purchased from In-
novative Technology, Inc. Column chromatography was performed
using 230–400 mesh silica gel purchased from Sorbent Technolo-
gies and a C18 (17%) reverse phase column (6 mL, 2 g) purchased
from Silicycle. NMR spectra were obtained using a Varian Mercury
300 MHz or Varian VNMRS 500 MHz spectrometer. Mass spectra
were obtained with a JEOL AccuTof DART or a Waters Quattro II
triple quadrupole spectrometer with high-resolution capabilities.
Optical rotations were measured with a PerkinElmer 241 Polarime-
ter using the sodium D line. Ultrapure water was purified via a Milli-
pore water system (ꢂ18 MWcm) triple water purification system.
4-(((Tert-butoxycarbonyl)amino)methyl)-3-nitrobenzoic acid (7) was
synthesized from (4-aminomethyl)benzoic acid (4) according to
a prior literature procedure.[17a] Detection of nile red emission de-
creases attributed to release were performed using a PerkinElmer
LS55 fluorescence spectrometer. Samples were irradiated with
a Rayonet Preparative Type RS photoreactor while suspended in
a cuvette with Pyrex as a filter.
compound 2 through traditional cleavage of the benzylic CꢀN
bond following nitrophenyl excitation (Scheme 1). Subsequent-
ly, the released amine of the terminal amide group of 2 could
potentially undergo nucleophilic attack of the proximal ester
linkage at the sn-2 position, which would liberate succinimide
and LPC (3). To evaluate this possibility, mass spectrometric
analysis of the products following irradiation of liposomes
composed of 100% NB-PC was performed using a JEOL Accu-
Tof DART instrument with an ESI source. The resulting mass
spectra indicated significant peaks corresponding to both PC-
amide 2 as well as LPC 3, providing evidence for the formation
of both of these products (see representative mass spectrum
in the Supporting Information).
Conclusion
Herein, we have described the design, synthesis, and analysis
of a novel photocleavable analogue of PC, NB-PC, which ena-
bles remote light-triggered release of hydrophobic compounds
from liposomes. Our strategy in this endeavor was to design
a compound that closely resembles the structure of PC, since
this is the most common lipid in cellular membranes and it
forms highly stable heterogeneous membranes when mixed
with a broad range of lipids with different properties. This was
done to enhance favorable properties including biocompatibil-
ity and stability, thus ensuring that nonspecific background re-
lease would be minimized and content release would be limit-
ed to membrane disruption caused by photolysis of NB-PC.
The data obtained from the release of nile red from NB-PC
liposomes indicate the benefits of this system. First, since NB-
PC can be incorporated into stable liposomes at any percent-
age, and release efficacy directly correlates with the amount of
NB-PC, this enables broad tunability of release properties to
achieve a desired outcome. On the other hand, major fluctua-
tions in NB-PC composition do not significantly affect back-
ground release rates, providing further evidence that any com-
position of NB-PC can be functional. Subsequent studies in-
volving fixed amounts of NB-PC with percentages of DOPE
and cholesterol ranging from 0–50% did not lead to major
modifications in liposome release efficacy, further demonstrat-
ing that the NB-PC system is highly stable and effective for
controlling release using liposomes composed of lipids with
widely varying physicochemical properties. The ultimate goal
of triggered drug release presents a number of technical chal-
lenges involving the logistics of delivering light to desired loca-
tions in living organisms, as well as other issues pertaining to
circulation and targeting. Since incorporation of PEG did not
tert-Butyl (4-(hexylcarbamoyl)-2-nitrobenzyl)carbamate (8)
To 1.67 g (5.64 mmol) of compound 7, dissolved in 350 mL of
chloroform, was added diisopropylethylamine (DIEA, 2.95 mL,
16.9 mmol), hydroxybenzotriazole (HOBt, 0.229 g, 1.69 mmol,), and
O-(benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophos-
phate (HBTU, 2.78 g, 7.33 mmol,). After 30 min, hexylamine
(2.24 mL, 16.9 mmol) was added. The reaction was then allowed to
stir overnight, after which it was washed with water, and the aque-
ous portion was extracted with chloroform (2ꢁ100 mL). The organ-
ic layers were then combined and washed with saturated sodium
chloride, dried with magnesium sulfate, filtered and concentrated
by rotary evaporation. Column chromatography using gradient elu-
tion of 30–50% ethyl acetate-hexanes gave 1.13 g of orange-
yellow product (53%). Rf =0.18 (25% ethyl acetate-hexanes);
1H NMR (300 MHz, CDCl3): d=8.33 (d, J=1.8 Hz, 1H), 7.92 (dd, J=
8.0, 1.9 Hz, 1H), 7.54 (d, J=8.1 Hz, 1H), 7.01 (t, J=5.9 Hz, 1H), 5.55
(t, J=6.5 Hz, 1H), 4.53 (d, J=6.5 Hz, 2H), 3.40 (q, J=7.0 Hz, 2H),
1.58 (m, 2H), 1.40 (s, 9H), 1.35–1.22 (m, 6H), 0.86 ppm (t, J=6.8 Hz,
3H); 13C NMR (75 MHz, CDCl3): d=165.01, 156.07, 147.69, 137.61,
135.15, 132.17, 130.89, 123.68, 80.16, 42.26, 40.51, 31.56, 29.51,
28.40, 26.75, 22.62, 14.10 ppm; HRMS-DART [MꢀH]ꢀ calcd for
C19H29N3O5, 378.2029; found 378.2018.
4-((4-(Hexylcarbamoyl)-2-nitrobenzyl)amino)-4-oxobutanoic
acid (9)
To compound 8 (1.19 g, 4.01 mmol), which was placed in an ice
bath, was added a solution of trifluoroacetic acid (TFA) in dichloro-
Chem. Eur. J. 2014, 20, 3350 – 3357
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methane (15 mL, 20%). After one hour of stirring, starting material
was no longer detected with TLC, and the solution was then con-
centrated and dried under high vacuum. To the resulting residue
was then added acetonitrile (40 mL), dry potassium carbonate
(1.67 g, 12.0 mmol), and succinic anhydride (0.442 g, 4.42 mmol),
and the mixture was allowed to stir at RT. After 24 h, citric acid
(50 mL, 10%) was added, and the reaction was extracted with
ethyl acetate (3ꢁ50 mL). The organic layers were then combined
and washed with saturated sodium chloride, dried with magnesi-
um sulfate, filtered, and concentrated using rotary evaporation.
Column chromatography with gradient elution of 50–100% ethyl
acetate–hexanes including 0.2% acetic acid gave product 10 as
a light yellow solid (893 mg, 59% yield). Rf =0.36 (10% methanol–
dichloromethane); 1H NMR (300 MHz, CD3OD): d=8.45 (d, J=
1.7 Hz, 1H), 8.04 (dd, J=8.1, 1.6 Hz, 1H), 7.68 (d, J=8.1 Hz, 1H),
4.68 (s, 2H), 3.35 (t, J=7.2 Hz, 2H), 2.64–2.50 (m, 4H), 1.59 (m, 2H),
1.44–1.24 (m, 6H), 0.89 ppm (t, 6.6 Hz, 3H); 13C NMR (75 MHz,
CD3OD): d=176.20, 175.10, 173.45, 149.33, 138.38, 135.85, 132.88,
131.05, 124.67, 74.12, 43.82, 41.22, 32.64, 30.29, 29.79, 27.74, 23.61,
14.36 ppm; HRMS-DART: [MꢀH]ꢀ calcd for C18H25N3O6, 378.1665;
found 378.1652.
with nile red in vials. To the appropriate lipid components corre-
sponding to each liposome sample was added ethanol-free chloro-
form (500 mL), and after brief vortexing, proper volumes of each
lipid were pipetted into a clean vial per calculations on a 5 mm,
500 mL total lipid scale, to obtain the desired molar percentage of
each component. Next, a solution of nile red was added per calcu-
lations on a 250 mm, 500 mL scale. As an example, for liposomes
composed of 50% NB-PC and 50% DOPE, ethanol-free chloroform
(500 mL) was added to separate vials containing NB-PC (4.9 mg),
DOPE (3.6 mg), and nile red (4.2 mg), producing stock solutions of
11, 9.7, and 26 mm concentrations, respectively. After 30 seconds
of vortexing, NB-PC (109.4 mL), DOPE (129.2 mL), and nile red
(4.74 mL) stock solutions were combined in a new vial. The chloro-
form was dried with a nitrogen stream, and the lipids were subse-
quently dried overnight under vacuum. The next day, the lipids
were hydrated with MilliQ purified water (500 mL), vortexed, and in-
cubated on a rotary evaporator at 608C for 3 sets of 20 min, with
vortexing after each set. Liposomes were then freeze-dried be-
tween a ꢀ408C dry ice bath and a 608C water bath for 10 cycles
and extruded through a 200 nm membrane for 21 passes using
a LiposoFast extruder (Avestin, Inc.), placing the uniform-sized vesi-
cles into a fresh vial. DLS scans were performed to confirm the for-
mation of stable liposomes.
1-Palmitoyl-2-(4-((4-(hexylcarbamoyl)-2-nitrobenzyl)amino)-
Next, 70 mL of this liposomal solution was diluted to 7 mL with ul-
trapure water. Two identical samples were made by placing 3 mL
of this dilute solution into quartz cuvettes and were sealed with
parafilm to minimize atmospheric exposure. After an initial fluores-
cence scan (lex =595 nm; lem =612 nm), one sample was irradiated
with 350 nm light while suspended and covered by Pyrex beakers
between four 350 nm bulbs in a Rayonet Preparative Type RS pho-
toreactor. The other sample was placed in a dark container. For
each fluorescence scan (every 5 min), the sample was removed
from the reactor or dark container and placed in the fluorimeter
for scanning. Total amounts of time plotted for release experiments
represent the amount of time the sample spent in the photoreac-
tor. Experiments were run at least four times each, including runs
with different batches of liposomes, and averaged to obtain the re-
sults shown in Figures 1–5, with error bars included to depict stan-
dard error. Data were then curve fit using an exponential decay
equation in SigmaPlot to obtain the values shown in Tables 1–5,
according to the function [Equation<(1)]:
4-oxobutanoyl)-sn-glycero-3-phosphocholine (NB-PC)
In a vial, compound 9 (211 mg, 0.556 mmol) was combined with di-
cyclohexylcarbodiimide (DCC, 0.86 mL, 0.556 mmol) and N,N-dime-
thylaminopyridine (DMAP, 0.068 g, 0.556 mmol) in 4 ꢂ-molecular
sieve-dried ethanol-free chloroform (2 mL) under argon, along with
crushed glass, similar to a method described by Rosseto and
Hadju.[27] After 30 min, palmitoyl-lysophosphatidylcholine (3, LPC,
69 mgg, 0.138 mmol) was added in one portion and argon atmos-
phere was then reestablished. After 6.5 h of sonication, Dowex
50Wx8 residue was added and the mixture was sonicated for
30 min before filtration through a fritted filter. After filtration and
concentration, normal phase column chromatography was carried
out through elution with 15% methanol–dichloromethane contain-
ing 0.2% acetic acid to remove any unreacted acid, followed by
65:25:4 chloroform–methanol–water to remove the product. Re-
verse phase chromatography using a C18 column with gradient
elution from water to methanol was then used to separate the
product from any residual dimethylaminopyridine and silica gel.
Residual water was removed by repeatedly concentrating with ace-
tonitrile. The solution was then concentrated to provide a slightly
yellow lipidlike substance in 65% yield. Rf =0.32 (65:25:4 chloro-
form-methanol-water); [a]2D2:5 +4.38 (c=2.9, CHCl3); 1H NMR
(500 MHz, 60% CDCl3-CD3OD) d=8.58 (s, 1H), 8.51 (s, 1H), 8.47–
8.40 (m, 1H), 8.11 (d, J=8.0 Hz, 1H,), 7.67 (d, J=7.8 Hz, 1H), 5.27–
5.20 (m, 1H), 4.73 (s, 2H), 4.36 (d, J=11.3 Hz, 1H), 4.27–4.15 (m,
3H), 4.08–3.97 (m, 2H), 3.58 (s, 2H), 3.45–3.37 (m, 2H), 3.27–3.16
(m, 9H), 2.80–2.53 (m, 4H), 2.32 (t, J=7.0 Hz, 2H), 1.63 (m, 4H),
1.43–1.22 (m, 30H), 0.95–0.85 ppm (m, 6H); 13C NMR (125 MHz,
60% CDCl3-CD3OD): d=174.45, 173.55, 172.81, 166.31, 148.43,
137.34, 135.37, 132.33, 130.35, 124.44, 71.53, 66.78, 64.23, 62.72,
59.48, 54.34, 41.16, 40.86, 34.34, 32.30, 31.93, 30.73, 30.68, 30.05,
30.02, 30.01, 29.99, 29.91, 29.87, 29.72, 29.66, 29.50, 27.11, 25.21,
22.94, 14.22 ppm; 31P NMR (125 MHz, 60% CDCl3–CD3OD): d=
ꢀ1.35 ppm, referenced to triphenyl phosphate at ꢀ17.70 ppm;
MS: [M+Na]+ calcd for C42H73N4O12P, 879.4855; found 879.4869.
Y ¼ yoþAeꢀkt
ð1Þ
in which k is the first order rate constant, t is time (in minutes), Y is
the % of initial fluorescence, and yo and A are fit parameters.
Values were tabulated in Tables 1–5, along with t1/2, the irradiation
half-life.
Acknowledgements
This material is based upon work supported by the National
Science Foundation under Grant Number CHE-0954297. We ac-
knowledge assistance from Dr. Shawn Campagna and Dr.
Liguo Song with the mass spectrometry-based analysis of
product formation.
Fluorescence-based liposome release studies
Keywords: controlled release
membranes · photocleavage
· drug delivery · lipids ·
Stock solutions were initiated by weighing out samples of DOPC,
DOPE, cholesterol, DSPE-PEG(2000) amine, and/or NB-PC along
Chem. Eur. J. 2014, 20, 3350 – 3357
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Received: October 18, 2013
Published online on February 25, 2014
Chem. Eur. J. 2014, 20, 3350 – 3357
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