Light-triggered hydrophilic drug release from liposomes through removal of a photolabile protecting group

Article abstract of DOI:10.1039/C8RA08584F

The antibiotic penicillin G cannot be completely incorporated into hydrophobic lipid-membranes owing to its hydrophilicity. Through modification with a hydrophobic and photolabile protecting group, penicillin G was effectively incorporated into liposomes and released by photoirradiation at 365?nm.

Full text of DOI:10.1039/C8RA08584F

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Light-triggered hydrophilic drug release from
liposomes through removal of a photolabile
Cite this: RSC Adv., 2019, 9, 166
protecting group†
a
Yuya Goto,a Masafumi Ueda,ab Kouta Sugikawa,
Kazuma Yasuharac
a
*
and Atsushi Ikeda
Received 17th October 2018
Accepted 11th December 2018
The antibiotic penicillin G cannot be completely incorporated into hydrophobic lipid-membranes owing to
its hydrophilicity. Through modification with a hydrophobic and photolabile protecting group, penicillin G
was effectively incorporated into liposomes and released by photoirradiation at 365 nm.
DOI: 10.1039/c8ra08584f
rsc.li/rsc-advances
molecules was determined to be log Pow z 1.9.14 Therefore, if
the log Pow of the guest molecules can be controlled, the
Introduction
amount of drug released can be varied. For example, a dimer of
a coumarin derivative (log Pow > 3) was reverted to monomers
in the lipid membrane by photoirradiation at 254 nm and the
monomer (log Pow ¼ 1.78) was released.15 In this work, we used
penicillin G (1)16,17 (log Pow ¼ 1.85)18 as an example of
a hydrophilic drug. We report the synthesis of 1 protected with
the hydrophobic photolabile protecting group 2-nitro-3-
naphthalenemethanol (NNM)19–21 (Scheme 1), and the release
of the penicillin G from liposomes by photocleavage of the
protecting group (Scheme 2).
The ability to regulate the release of an appropriate dose of
drug at a specic location with the correct timing is very
important for liposomal drug-delivery systems used in selec-
tive tissue targeting.1–5 Drug release can be controlled by pH,
thermal, enzymatic, or photochemical triggers. Many research
groups have reported liposomal drug-delivery systems that use
the liposome collapse or the considerable distortion of the
membrane that results from these triggers to affect release.6–10
In contrast, several groups have reported drug-delivery
systems that use the introduction and elimination of a guest
molecule protecting group to control release. For example,
hydrophilic guest molecules can be incorporated into hydro-
phobic lipid membranes using hydrophobic protecting
groups. In lipid membrane-incorporated guest molecules
(LMIGs), the incorporation or leakage percentages of guest
molecules are predicted by the hydrophobicity of the guest
molecules. Schwarzenbach et al. directly determined the
liposome–water distribution ratio (log Klipw) of phenols and
phenoxides and reported that the log Klipw values of phenols
are very similar to the octanol–water partition coefficient
(log Pow).11–13 In contrast, we investigated the relationship
between the equilibrium and the log Pow for some small model
guest molecules with a p-moiety.14 Our results indicated that
many of the guest molecules with log Pow < 1.9 leaked from the
lipid membranes, while most of the guest molecules with
log Pow > 1.9 did not. That is, the threshold for leakage of guest
aDepartment of Applied Chemistry, Graduate School of Engineering, Hiroshima
University, 1-4-1 Kagamiyama, Higashi-Hiroshima 739-8527, Japan
bSchool of Science, Kitasato University, 1-15-1 Kitasato, Minami-ku, Sagamihara,
Kanagawa 252-0373, Japan
cGraduate School of Materials Science, Nara Institute of Science and Technology, 8916-
5 Takayama, Ikoma, Nara 630-0192, Japan
† Electronic supplementary information (ESI) available: Experimental procedures,
1H NMR, MS/MS and UV-vis absorption spectra. See DOI: 10.1039/c8ra08584f
Scheme 1 (a) Synthesis of NNM-1 and (b) photocleavage of NNM-1.
166 | RSC Adv., 2019, 9, 166–171
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To conrm the generation of 1, we measured the electrospray
ionisation mass spectroscopy (ESI-MS) (Fig. 2). Before photo-
irradiation, 1 was not observed in the ESI-MS spectrum of NNM-
1 as a fragment (Fig. 2a). An ambiguous ion at m/z 345.09 was
found to be a fragment of NNM-1 because the same peak was
observed as a product ion of m/z 520 in the ESI-MS/MS for
fragments generated by collision induced dissociation
(Fig. S3†). However, only trace amounts of ions of 1 were
observed in the sample of NNM-1 before the photoirradiation at
365 nm. In contrast, aer photoirradiation, ions of 1 were
detected in both the positive and negative modes, while only
Scheme 2 Schematic illustration of (a) NNM-1 with a conjugated
photolabile protecting group inside the lipid membrane and (b) 1
outside the lipid membrane as a result of photocleavage of the pro-
tecting group through photoirradiation at 365 nm, and the structure of
DMPC.
Results and discussion
Photocleavage of the protecting group in NNM-1 in DMSO
To conrm the photocleavage of the protecting group in NNM-1
(log Pow ¼ 4.13),13 we measured the 1H NMR and UV-vis
absorption spectra of NNM-1 in DMSO aer photoirradiation
at 365 nm for 24 h. As shown in Fig. 1 and S2,† NNM moiety 1H
NMR peaks in NNM-1 (Fig. 1b; blue circles) were no longer
detected aer photoirradiation at 365 nm, and new peaks
appeared (Fig. 1c). Because these peaks (Fig. 1c; red circles) are
similar to those of the sodium salt of 1 (Fig. 1a; red circles), the
photocleavage of NNM-1 and generation of 1 were suggested as
shown in Scheme 1b. Because of the disagreement in the
chemical shis of the 1H NMR peaks in Fig. 1a and c, we
attempted to prepare 1. However, 1 decomposed when treated
with an HCl solution, as it is unstable in acidic conditions.22
Fig. 1 Partial 1H NMR spectra (400 MHz, DMSO-d6, 25 ꢀC) of (a) Fig. 2 ESI-MS spectra of NNM-1 (a) before (positive ion mode), (b)
sodium salt of 1 and (b and c) NNM-1 (b) before and (c) after photo- after (positive ion mode), and (c) after (negative ion mode) photo-
irradiation at 365 nm for 24 h (red circles: 1, blue circles: NNM-1).
irradiation at 365 nm for 24 h in CHCl3.
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Table 1 Average hydrodynamic diameters (Dhy, nm) of LMI-NNM-1
before and after photoirradiation determined by dynamic light scat-
tering at 25 ꢀ
C
Average Dhy/nm
PDIa
Before photoirradiation
Aer photoirradiation
66.3
68.3
0.103
0.087
a
PDI: polydispersity index.
from lipid membranes.14 In contrast, NNM-1 which leaked out
of the liposomes, precipitated as a result of being insoluble in
water. To determine the concentration of NNM-1 in LMI-NNM-
1, the residue was dissolved in CDCl3 containing ethanol (0.4
mM) as an internal standard, to measure the 1H NMR spec-
trum aer lyophilisation of the LMI-NNM-1 solution (Fig. S5†).
From the peak intensities, [NNM-1]/[DMPC] was calculated to
be 3.4 mol%. Because the initial ratio of [NNM-1]/[DMPC] was
10 mol%, the residual NNM-1 ([NNM-1]/[DMPC] ¼ 6.6 mol%)
was not incorporated into the liposomes and precipitated. The
precipitate was removed from the aqueous solution by ltra-
tion to give LMI-NNM-1. The incorporation of a high propor-
tion of NNM-1 in the lipid membrane was a result of the
hydrophobicity of NNM-1 (log Pow ¼ 4.13).14 The average
hydrodynamic diameter (Dhy) of LMI-NNM-1 was almost
unchanged following photoirradiation, suggesting that the
liposome morphology did not change aer the photocleavage
of NNM-1 (Table 1).
Fig. 3 Time-dependent UV-vis spectral changes of NNM-1 (inset:
incubation time-dependence of the absorbance at 410 nm) upon
photoirradiation at 365 nm (1.1 mW cmꢁ2) for 0 (red line), 1 (orange
line), 2 (yellow line), 3 (green line), 6 (blue line) and 12 (purple line) h
([NNM-1] ¼ 0.40 mM).
trace amounts of ions of NNM-1 were observed (Fig. 2c). These
results clearly show that NNM-1 was almost completely cleaved
aer the photoirradiation. Therefore, the disagreement in the
1H NMR peaks between Fig. 1a and c, is thought to arise from
Fig. 1a showing the spectrum of the sodium salt of 1.
As shown in Fig. 3, the UV-vis absorption of NNM-1 changed
aer photoirradiation at 365 nm in DMSO. The spectrum aer
12 h was similar to that of the sodium salt of 1 in H2O (Fig. S4†),
and was consistent with a previous result. The absorption
changes of NNM-1 were saturated aer 12 h of photoirradiation.
However, the absorbance decreased aer 3 h of photo-
irradiation. This result suggests that compound 1 decomposed,
and the emergence of peaks of some degradation products
supported this. The structures of the degradation products
could not be determined as they were only produced in small
amounts.
Photocleavage of the protecting group in NNM-1 in liposomes
To determine the saturation time of photoirradiation, UV-vis
absorption spectra were measured. The absorption of NNM-1
under both dark conditions and photoirradiation was measured
for the LMI-NNM-1 solution (Fig. 4). Under dark conditions, the
UV-vis absorption spectrum showed only marginal change
(Fig. 4a). This result indicates that, (i) NNM-1 did not decom-
pose in the absence of photoirradiation and (ii) the LMI-NNM-1
solution was stable for 1 d. Aer photoirradiation, the absorp-
tion intensity decreased and then saturated aer 12 h. Peak
broadening was observed in a similar manner to NNM-1 in
DMSO (Fig. 3 and 4b). This result suggests that NNM-1 was
Determination of leakage percentages of 1 and NNM-1 from
liposomes
Lipid membrane-incorporated 1 and NNM-1 (LMI1 and LMI- almost completely photocleaved aer 12 h.
NNM-1, respectively) were prepared using 1,2-dimyristoyl-sn-
To conrm whether NNM-1 and cleaved 1 were present in the
glycero-3-phosphocholine (DMPC, Scheme 2) and a premixing lipid membrane or bulk water, we measured the 1H NMR
method (Scheme S1†).15,23,24 LMI1 and LMI-NNM-1 were ob- spectra of LMI-NNM-1 aer photoirradiation for 12 h. The peak
tained by dissolving DMPC and the guest molecules (1 or intensities in Fig. 5 and S6† were normalised relative to the
NNM-1) in chloroform, followed by concentration of the value of DMSO (0.4 mM) as an internal standard. The leakage
resulting mixture to give a residue that was extracted with percentage of 1 was found to be 74%, based on the peak
water. Because compound 1 does not precipitate at this intensities of 1 relative to the DMSO peak. In contrast, no peaks
concentration, all 1 present existed either in the lipid were observed for the LMI-NNM-1 solution (Fig. 5b). It is well
membrane or in bulk solution. Therefore, the leakage known that lipids peaks and those belonging to guest molecules
percentage of 1 from liposomes was calculated to be 82%, are not detected for LMIGs as a consequence of peak broad-
based on the 1H NMR spectrum, which indicated that 18% of 1 ening resulting from the formation of the liposomes.25–30
was residual in the lipid membrane ([1]/[DMPC] ¼ 10 mol%). Therefore, the absence of peaks belonging to NNM-1 suggests
The leakage of 1 (log Pow ¼ 1.85) is consistent with previous that all of the NNM-1 was completely incorporated into the
ndings that many guest molecules with log Pow < 1.9 leak liposomes.
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aureus) ATCC 25923. The minimum inhibitory concentration
(MIC) of LMI-NNM-1 aer photoirradiation at 365 nm for 12 h
(0.23 mg mLꢁ1) was much lower than that measured before
photoirradiation (>1.82 mg mLꢁ1). This indicates that the anti-
microbial activity of LMI-NNM-1 aer photoirradiation was
higher than that before photoirradiation. Consequently, the
penicillin G 1 released from the lipid membranes was able to
function as an antimicrobial agent. However, the MIC value was
higher than that of pristine penicillin G sodium salt (0.03 mg
mLꢁ1) because the leakage percentage of 1 was found to be 74%,
and 1 released to the inner aqueous phase of the vesicles did not
act as an antimicrobial agent.
Conclusions
The leakage percentage of pristine penicillin G 1 (log Pow ¼ 1.85)
from lipid membranes was found to be 82%. In contrast, the
majority of a sample of 1 was incorporated into the hydrophobic
lipid membrane following modication with NNM as a hydro-
phobic protecting group. Consequently, the leakage of NNM-1
(log Pow ¼ 4.13) was minimal. Aer photoirradiation at 365 nm,
deprotected 1 was released from the lipid membrane and the
leakage percentage from NNM-1 incorporated in the lipid
membrane was 74%. Because the leakage percentage of 1 from
the liposomes was 82%, the release of 1 by photoirradiation was
considered to have proceeded efficiently.
Experimental
Materials
Fig. 4 Time-dependent UV-vis spectral changes of liposome-NNM-1
mixture (a) after 0 (black line) and 24 h (red line) incubation under dark
conditions and (b) upon photoirradiation at 365 nm (1.1 mW cmꢁ2) for
0 (red line), 1 (orange line), 2 (yellow line), 3 (green line), 6 (blue line)
and 12 (purple line) h (inset: incubation time-dependence of the
absorbance at 257 nm).
Penicillin G sodium salt and 1,2-dimyristoyl-sn-glycero-3-phos-
phocholine (DMPC) were purchased from Tokyo Chemical
Industry Co., Ltd. (Tokyo, Japan) and Funakoshi Co., Ltd
(Tokyo, Japan), respectively. NNM19 was synthesized according
to previously reported methods.
Synthesis of NNMBr
A solution of NNM (100.0 mg, 0.49 mmol) in dichloromethane
(5 mL) was added to triethylamine (101.2 mg, 1.00 mmol) and
methylsulfonyl chloride (85.9 mg, 0.75 mmol) in an ice bath.
The reaction mixture was then stirred in the ice bath for 30 min.
Aer 30 min, the reaction mixture was extracted with
dichloromethane and washed with saturated sodium hydrogen
carbonate solution and saturated sodium chloride solution. The
organic layer was then dried with MgSO4 and ltered. Aer
evaporating in vacuo, the crude product was dissolved in THF (5
mL) and lithium bromide (338 mg, 3.9 mmol) was added. The
reaction mixture was stirred at room temperature for 16 h. Aer
evaporating in vacuo, the crude product was extracted with
dichloromethane and washed with saturated sodium hydrogen
carbonate solution and saturated sodium chloride solution. The
organic layer was dried with MgSO4 and ltered. The product
was dried under vacuum. Yellow solid, 55.3 mg (42%). 1H NMR
(400 MHz, CDCl3) d 8.65 (s, 1H), 8.01–7.90 (m, 3H), 7.71–7.65 (tt,
J ¼ 4.6 Hz, 2H) 5.04 (s, 2H) ppm.
Fig. 5 Partial 1H NMR spectra (400 MHz, D2O, 25 ꢀC) of (a) liposomes-
1 mixture ([1]/[DMPC] ¼ 10 mol%) and liposomes-NNM-1 mixture (b)
before and (c) after photoirradiation at 365 nm for 12 h ([NNM-1]/
[DMPC] ¼ 10 mol%), (red circles: 1 in bulk water).
Antimicrobial activity of LMI-NNM-1 before and aer
photoirradiation
We determined the antimicrobial activity of LMI-NNM-1 before
and aer photoirradiation toward Staphylococcus aureus (S.
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Preparation of lipid-membrane-incorporated 1 and NNM-1
using the premixing method
Conflicts of interest
There are no conicts to declare.
An appropriate amount of a mixture of DMPC (6.00 mmol) and 1
or NNM-1 ([DMPC] : [1 or NNM-1] ¼ 10 : 1 mol molꢁ1) was
dissolved in chloroform. The solvent was evaporated under
a gentle stream of nitrogen, followed by a period under vacuum
to remove trace solvent. The resulting thin lipid lms were
hydrated on the wall of the vial above the phase transition
temperature with an appropriate amount of water or D2O (1.5
mL). The hydrated materials were subjected to eight freeze–
thaw cycles (ꢁ195 and +50 ꢀC) to give unilamellar vesicles,
which were extruded 11 times through 0.05 mm pores using
a LiposoFast™ Mini-extruder (Avestin, Ottawa, ON, USA) above
the phase transition temperature.
Acknowledgements
This work was supported by a JSPS KAKENHI Grant-in-Aid for
Scientic Research (B) (Grant No. JP16H04133) and a Grant-in-
Aid for Challenging Exploratory Research (Grant No.
JP16K13982). We thank Sarah Dodds, PhD, from Edanz Group
(www.edanzediting.com/ac) for editing
manuscript.
a
dra of this
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