Near-infrared light-sensitive liposomes for controlled release

Article abstract of DOI:10.1039/c6ra18702a

A photoresponsive amphiphilic lipid molecule with phosphatidylcholine as the headgroup and a near-infrared (NIR) light-responsive unit on the hydrophobic moiety was synthesized. Liposomes constituting the NIR-responsive lipid, cholesterol and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, were constructed and applied as a photocontrolled release system. The precisely controlled release of the loaded cargo was demonstrated by adjusting the irradiation parameters and content of the photoresponsive species. The low cytotoxicity and good biocompatibility of the photoresponsive lipid was demonstrated by MTT assay. This study provides us with a new choice for developing a photoresponsive drug delivery system.

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Near-infrared light-sensitive liposomes for controlled release
Yawei Sun*, Yanyun Ji, Haiyan Yu, Dong Wang, Meiwen Cao and Jiqian Wang*
Received 00th January 20xx,
Accepted 00th January 20xx
A photoresponsive amphiphilic lipid molecule with phosphatidylcholine as the headgroup and a near-infrared (NIR) light-
responsive unit on the hydrophobic moiety were synthesized. Liposomes constituting the NIR-responsive lipid, cholesterol
and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, were constructed and applied as a photocontrolled release system.
The precisely controlled release of the loaded cargo was demonstrated by adjusting the irradiation parameters and
content of the photoresponsive species. The low cytotoxicity and good biocompatibility of the photoresponsive lipid was
demonstrated by the MTT assay. This study provides us a new choice for developing a photoresponsive drug delivery
system.
DOI: 10.1039/x0xx00000x
www.rsc.org/
prepared, their photoresponsiveness, release behaviour, and
biocompatibility were systematically studied.
Introduction
Numerous nanoscale drug delivery vehicles have been developed1
for preclinical and clinical studies. The control over the release of
encapsulated drugs is the key to achieve desired success.2–4
Liposomes represent one of the most widely explored drug delivery
carrier. Stimuli responsiveness liposome-based drug delivery
systems has attracted scientists’ attention because they are easy-
to-prepare and possess good biocompability.5–9
Experimental
Materials
All of the reagents were purchased from Aldrich and Alfa Aesar Ltd
in China. The solvents were purchased from Beijing Chemical
Reagent Company and used without further purification. Deionized
Millipore Milli-Q water (18.2 MΩ·cm-1) was used in all of the
experiments. The photoresponsive lipids were synthesized using the
protocols shown in Figure 1 and Figure 2, the crucial intermediates
and final products were purified by silica column chromatography.
The cell line NIH3T3 was purchased from the Cell Bank of the
Shanghai Institute for Biological Science, Chinese Academy of
Sciences (Shanghai, China).
Photo irradiation can act as a viable strategy to externally control
the release of the payloads. The photoactivation of liposomes can
be realized by destabilizing the stability of the lipid bilayers.
Photoinduced
polymerization,10
isomerization,11
and
photodegradation12 have been applied to trigger cargo release from
liposomes. However, most of these photoresponsive moieties were
ultraviolet (UV)-responsive, the limited tissue penetration ability
has restricted their applications. Recently, near-infrared (NIR) light
was applied to trigger the on-demand release from drug-
encapsulated cargos.13–20 NIR light is more suitable for biomedical
appplication as it can penetrate deeper into tissues (up to 10 cm)
with less photodamage to cells and tissues comparing to UV or
visible light. Though numerous NIR responsive drug carriers were
reported in the past years, the incorporation of NIR responsive unit
into liposome was seldom reported.18
Instruments and Methods
Proton nuclear magnetic resonance (1H NMR) spectra were
recorded on a Bruker AMX 400 Spectrometer (400 MHz) at 298 K
using partially deuterated solvents and the internal standard
tetramethylsilane (TMS). Mass spectrometry (MS) spectra were
obtained on an Agilent 6510 Q-TOF. UV measurements were
performed on a Shimadzu UV2450 Spectrophotometer at 25°C.
Fluorescence measurements were obtained on a Hitachi F2500
fluorescence spectrophotometer at 25°C. UV irradiation was
performed on an AuLight CEL-HXF300 xenon light source with a UV
band filter from 350–380 nm, and the modulation of power
intensity was realized by adjusting the input voltage. NIR irradiation
was performed using a Ti:sapphire regenerative amplifier (Hogan
Inc) running at a 1 kHz repetition rate, which generated about 170
fs pulses at 808 nm. The laser beam was focused on the sample cell
with a spot size of about 6 mm and an average power of 4 W. The
cryo-sample was imaged with JEOL JEM-1400 TEM under 120 kV.
The images were recorded on a Gatan multiscan CCD and processed
with Digital Micrograph. DLS measurements were performed on a
Malvern Zetasizer Nano ZS with a 173° scattering angle in a 45 μL
quartz cuvette at 25°C, and all of the results were presented as the
average of at least three repeats.
Here, we designed a novel photolabile amphiphilic lipid by
incorporating the NIR light-responsive 6-bromo-7-hydroxy-4-
hydroxycoumarin (Bhc) unit into the lipid acyl chain.21 The
phosphocholine (PC) group was selected as the hydrophilic moiety
since the PC-type lipid is the primary component of cellular
membranes.22 As a control, the amphiphilic lipid-containing UV-
responsive unit was synthesized and investigated. Liposomes
constituting photoresponsive amphiphilic lipids with and without 1-
palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
(POPC)
were
Address here. Centre for Bioengineering and Biotechnology, China University of
Petroleum (East China) 66 Changjiang West Road, yawsun@upc.edu.cn,
jqwang@upc.edu.cn
Electronic Supplementary Information (ESI) available: Cryo-TEM of the liposome
before and after irradiation, synthesis data, 1H NMR and MS spectra of compounds
are available free of charge. See DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
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Liposome Preparation
2 mmol lipid (photo-responsive lipid, POPC and cholesterol) was
well dissolved in a mixture solvent consisting 9 mL CHCl3 and 1 mL
MeOH in a 50 mL round-bottom flask. The solvent was removed by
rotary evaporation under reduced pressure to form a thin layer on
the flask’s wall. The flask was kept under vacuum overnight to
remove the residue organic solvent. Then 5 mL HEPES buffer (pH
7.4) containing 10 mmol·L−1 HEPES, 100 mmol·L−1 NaCl, 1 mmol·L−1
EDTA, and 100 mmol·L−1 5(6)-carboxyfluorescein (CF) were added,
and the flask was shaken in a water bath (60°C). After all of the lipid
films were dispersed, the suspension was transferred to a plastic
centrifuge tube and subjected to freeze-thaw cycles by alternately
placed in liquid nitrogen (−195.6°C) and a water bath (60°C) for four
cycles to disrupt the large multilamellar vesicles and to anneal any
structure effects.23 Liposomes of the desired size were obtained by
30 times extrusion through a 100 nm polycarbonate membrane
mounted in the mini-extruder (Avanti Polar Lipids) at 25°C. Non-
entrapped carboxyfluorescein (CF) was removed by eluting through
a Sephadex G100 column with HEPES buffer as the eluent. The
liposome-containing fraction was collected and kept in the dark.
Photocontrolled Release
Figure 1. Synthesis route of Bhc-PC and NB-PC. i) palmitoyl-
lysophosphatidylcholine, DCC, DMAP, CH2Cl2, 24 h. ii) 1-
bromohexane, K2CO3, acetone, reflux, 6 h; iii) NaBH4, THF, 1 h; iv)
glutaric anhydride, triethylamine, CH2Cl2, 12 h.
The photoactive lipid was synthesized by covalently conjugating a
26-28
photocleavable Bhc with lyso PC.
Cleavage of the Bhc group by
Release of the entrapped CF molecule from vesicles was studied by
fluorescence dequenching at 25°C. 2, 4The aqueous solution of CF-
loaded vesicles was stored in a 1 mm quartz cuvette and exposed to
the NIR laser (808 nm, 4W) or UV light (50 mW·cm-2) with a band
path filter (350–380 nm) at different time points. After irradiation,
the sample was incubated at 25°C for additional 5 min in the dark.
The change in fluorescence emission intensity at 517 nm (excited at
UV light or NIR laser induced destabilization of the lipid membrane
and the consequent release of the encapsulated cargo. As shown in
Figure 1, the Bhc-PC (compound 3) and NB-PC (2-nitrobenzyl
containing PC, compound 8) were obtained by coupling the
photoresponsive
carboxylic
acid
and
palmitoyl-
lysophosphatidylcholine (LPC, compound 2) via esterification (Fig.
1a). The Bhc-containing carboxylic acid (compound 1) group was
synthesized according to
carboxylic acid was synthesized from 5-hydroxy-2-
a
previous study.29 NB-containing
492 nm) was tracked on
a Hitachi F2500 fluorescence
spectrophotometer, as a function of irradiation time. The release of
CF (%) was calculated using the following equation:
7
nitrobenzaldehyde through esterification to 5, reduction to 6,
followed by opening of glutaric anhydride to the photocleavable
carboxylic acid 7, in a yield of 53% (Fig. 1b). All of the intermediates
and final products were purified by column chromatography. Their
molecular structures and purities were confirmed by 1H NMR and
electrospray ionization mass spectrometry (ESI-MS).
Liposomes of 100–200 nm in diameter exhibited a prolonged
circulation half-life in the blood and excellent enhanced
permeability and retention (EPR) effects toward solid tumours. In
our study, these unilamellar liposomes with desired size were
ꢂꢃ − ꢂꢄ
ꢂ∞ − ꢂꢄ
ꢀ
ꢁ
CF release % =
where I0 is initial fluorescence intensity, It is fluorescence intensity
at time t, and I∞ is fluorescence intensity when all CF molecules
were released from the vesicles, which occurred upon addition of
0.4% (the final concentration) Triton X-100 and heating for an
additional 30 min. As 0.4% Triton X-100 had negative effects on the
fluorescence intensity of CF (20% suppression), the I∞ value was
corrected accordingly. The procedure for the control experiment
was similar to the method outlined above.
prepared by extrusion through
a polycarbonate membrane.
Cytotoxicity against Mammalian Cells
Cholesterol was added to improve the tendency for liposome
formation by improving hydrophobic interactions in the bilayer, it
could increase the fluidity and stability of the liposome.30-33 As
shown in Table 1, liposomes containing 33% cholesterol, varying
percentages of Bhc-PC (22%, 44%, 66%) and POPC were prepared.
The NB-PC liposome was used as the control and the POPC
cholesterol-based liposome was selected as the blank.
The cytotoxicity of photoresponsive liposomes in mammalian cells
was determined with the MTT assay. 24, 25 Briefly, NIH3T3 mouse
embryonic fibroblasts were pre-seeded on sterilized 96-well plates
at a density of 2 × 104 cells in 100 μL medium containing 10%
HEPES. Then the plates were incubated for 24 h at 37°C in an
atmosphere containing 5% CO2. Aliquots (100 μL) of different
concentrations of liposome solutions were added to the wells (well
without cells served as controls), and the cells were incubated for
an additional 24 h. Subsequently, a 20 μL aliquot of MTT solution (5
mg·mL−1) was added to each well and the plates were incubated for
4 h at 37°C. The precipitated formazan was dissolved in 200 μL
DMSO. The absorbance at 570 nm was measured using a microplate
autoreader (Molecular Devices, M2e).Cell survival percent was
expressed as the percent ratio of NIH3T3 cells without liposome.
Results and discussion
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Figure 2. Cryo-TEM image of liposome. a) Liposome 3 before NIR
irradiation; b) Liposome 3 after NIR irradiation; c) Liposome 6
before UV irradiation; d) Liposome 6 after UV irradiation (bar = 100
nm).
Figure 4. a) Hydrodynamic diameter distributions of liposome 3
before and after NIR irradiation; b) Hydrodynamic diameter
distributions of liposome 6 before and after UV irradiation; c)
Hydrodynamic diameter distributions of liposome 1 (balck open
△
square, □) and 2 before (blue open uptriangle, ) and after NIR
irradiation (red open circle, ○ and cyano open downtriangle ), and
liposomes 4 and 5 before (megenta solid square and navy open
diamond )and after UV irradiation (dark yellow solid circle● and
wine open star ); d) Hydrodynamic diameter distributions of the
POPC
66%
44%
22%
-
Cholesterol
34%
Bhc-PC
NB-PC
▽
█
Control
-
22%
44%
66%
-
-
-
◇
☆
Liposome 1
Liposome 2
Liposome 3
Liposome 4
Liposome 5
Liposome 6
34%
blank liposome before and after NIR irradiation.
34%
-
34%
-
44%
22%
-
34%
22%
44%
66%
34%
-
34%
-
Table 1. The mole-composition of cholesterol, Bhc-PC, NB-PC, and
POPC in the liposomes
Figure 5. a) Release profiles of CF from liposomes containing Bhc-PC
upon 808 nm laser irradiation (4 W); b) Release profiles of CF from
liposomes containing Bhc-PC upon UV irradiation (50 mW/cm2); c)
Release profiles of CF from liposomes containing NB-PC upon UV
irradiation (50 mW/cm2); d) Release profiles of CF from liposomes
Figure 3. a) Photolysis route of Liposome 3 upon NIR irradiation containing NB-PC upon 808 nm laser irradiation (4 W).
(808 nm, 4W); b) ESI-MS data of photolyzed product from Bhc-PC;
Cryo-TEM was used to characterize the morphology of the
c) Fluorescence emission spectra (λ = 380 nm) of liposome 3 liposomes. The microscopic images indicated that liposomes had a
exposed to NIR light (808 nm, 4W).
diameter of about 100 nm (Fig. 2a, 2c). Introduction of the photo
responsive unit into the lipid structure did not affect the liposome’s
stability and morphology. The elongated or tube-shaped liposomes
implies the possible segration of cholesterol and lipid components.
Liposomes constituted of different ratio of photo-responsive lipid
(Liposome 1, 2, 4 and 5)exhibited similar morphology (Fig. S1).
Moreover, the cryo-TEM images showed an obvious contrast
between the periphery and central area of the sphere, indicating
liposome formation. After light irradiation, the liposomes
disappeared and aggregates around 20 nm were observed (Figs.2b,
2d, 4a, 4b). We presumed that these aggregates consisted of
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cleaved species of the photoresponsive lipid and cholesterol. The
morphology of the POPC-containing liposome (liposomes 1, 2, 4, 5)
was preserved after irradiation (Figs. 4c, 4d, S1), suggesting that the
POPC maintains the basic frame of the liposome.
To assess the photolysis behavior of Bhc-PC, Liposomes 3
without CF was exposed to NIR light. The ESI-MS results was utilized
to study the photolysis pathway. Upon light irradiation, the signal of
3A and the hydrolysized product 2 was observed in ESI-MS, which
coincide with our assumption. Compound 3B was not detected due
to its poor solubility in aqueous solution (Fig. 3b). Photolysis of
Liposome 3 under NIR light irradiation was also evidenced by
changes in fluorescence emission intensity of the coumarin groups.
Figure 3c shows the fluorescence emission spectra of liposome 3
recorded as a function of cumulative NIR exposure time. The
Figure 6. MTT assay of liposomes 1–6 and the blank liposome.
To evaluate the cytotoxicity of the photoresponsive liposomes,
the MTT assay was performed. As shown in Figure 6, the metabolic
activity of the cells was slightly higher after incubation with Bhc-PC-
containing liposomes (liposome 1–3) for 48 h. Similar results were
observed when liposomes containing 22% and 44% (liposomes 4
and 5, respectively) of NB-PC were utilized, but slightly lower
metabolic activity of the cells was noted as the NB-PC content
increased to 66% (liposome 6). An acceptable level of cell viability (>
75%) was observed. Taken together, these findings demonstrate
that liposomes containing Bhc-PC exhibit low toxicity towards
mammalian cells.
emission intensity gradually decreased as
a function of NIR
irradiation time, indicating the generation of compound 3B, which
was insoluble in aqueous medium and had partly quenched
fluorescence. The photolysis of Liposome 6 was also investigated by
UV-vis absorption, the decreased absorption at 320 nm and
increased absorption around 270 nm and 370 nm indicated the
disappearing of o-NB and formation of 2-nitrosobenzaldehyde (Fig.
S2a). ESI-MS results illustrate the main constituents is compound 7A
and 2 (Fig. S2c), which meet the results of UV-vis absorption.
After confirming the efficiency of the photocleavage, we
characterized the release profiles of the liposomes under light
irradiation. The water-soluble fluorophore CF was selected to mimic
the drug encapsulated in the interior cavity of the liposome, and its
release was examined by the increase of fluorescence intensity at
517 nm. We observed that the encapsulated CF was smoothly
released from Bhc-PC containing liposomes (liposomes 1–3) upon
NIR irradiation, and release was accelerated when the Bhc-PC
content in the liposome was increased (Fig. 5a). A 60% release was
observed after 1 h of irradiation. As a comparative experiment, CF
release kinetics were examined under UV irradiation, the release
demonstrated a relatively steep trend at the start of irradiation that
reached a plateau in a short period of time (Fig. 5b). This could be
attributed to the fact that the NIR two-photon process is slow and
less efficient than the photocleavage obtained with UV irradiation.
These findings demonstrate that the Bhc-containing liposome
exhibited responsiveness to NIR laser as well as UV light, and its on-
demand release can be regulated by irradiation time as well as
liposome composition.
Conclusions
A novel photoresponsive PC-type lipid molecule with a Bhc
moiety on the acyl chain was designed and synthesized. The
large unilamellar liposome composed of cholesterol and the
photoresponsive lipid, POPC, encapsulated the hydrophilic
molecule in the interior cavity of the liposome, and released
the cargo upon NIR and UV irradiation. The release behaviors
were controlled either by adjusting the percentage of
photoresponsive lipid or by irradiation time and intensity,
demonstrating an important controlled release process. The
combination of being easy-to-prepare, being NIR-responsive,
and having low toxicity in cells indicates that these liposomes
may be developed into efficient tools for drug and gene
delivery.
We also studied the release of CF from NB-PC containing
liposomes (Liposomes 4-6) under UV and NIR irradiation. As
expected, release ender UV irradiation gradually increased with
time and plateaued after 15 min of UV irradiation (Fig. 5c), which
was consistent with changes in the UV-visible absorption spectra
(Fig. S2). The release of CF from liposomes 1–3 was barely observed
when they were irradiated with NIR (Fig. 5d), due to the low two-
photon absorption cross section of 2-nitrobenzyl. The results
illustrates the Bhc-containing liposome have better performance
than NB-PC containing liposome upon NIR irradiation.
Acknowledgements
This work was supported by the National Natural Science
Foundation of China under grant numbers 21573288,
21573287 and 21503276, and the Fundamental Research
Funds for the Central Universities (27R15CX04061A and
27R14CX05040A).
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A 6-Bromo-7-hydroxy-4-hydroxymethylcoumarin containing amphiphilic lipid was
synthesized and applied as near-infrared light triggered controlled release system.