A new type of pH-sensitive phospholipid

Article abstract of DOI:10.1016/j.tetlet.2017.11.013

We describe the synthesis and characterization of a type of pH-sensitive pentaerythritol phospholipids, using a trialkoxybenzylidene acetal as the acid-labile moiety. This lipid was prepared by an eight-step synthesis via a latentiation strategy. Liposomes were prepared via the thin film extrusion method. The changes of liposomal sizes were measured by dynamic light scattering. Content release rates of the liposomes as a function of pH were monitored by using a calcein fluorescence dequenching assay. These results indicated that this new liposomal system was capable of releasing its contents under mildly acidic conditions. At last, in vitro cytotoxicity was assayed against three cell lines, suggesting this type of phospholipids was low toxic.

Full text of DOI:10.1016/j.tetlet.2017.11.013

Accepted Manuscript
A new type of pH-sensitive phospholipid
Li Zhan, Shuyuan Chang, Ting Liu, Shanbao Yu, Hui Li, Minjie Lu, Yong Zhu,
Yu Luo, Jiahui Yu, Fan Yang, Jie Tang
PII:
S0040-4039(17)31407-7
https://doi.org/10.1016/j.tetlet.2017.11.013
TETL 49456
DOI:
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
27 September 2017
1 November 2017
6 November 2017
Please cite this article as: Zhan, L., Chang, S., Liu, T., Yu, S., Li, H., Lu, M., Zhu, Y., Luo, Y., Yu, J., Yang, F.,
Tang, J., A new type of pH-sensitive phospholipid, Tetrahedron Letters (2017), doi: https://doi.org/10.1016/j.tetlet.
2017.11.013
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Tetrahedron Letters
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A new type of pH-sensitive phospholipid
Li Zhana, Shuyuan Changa, Ting Liua, Shanbao Yub, Hui Lib, Minjie Luc, Yong Zhuc, Yu Luoa,*, Jiahui Yua,
Fan Yanga and Jie Tanga
a School of Chemistry and Molecular Engineering, East China Normal University, 500 Dongchuan Road, Shanghai 200241, China
bPharmaBlock Sciences (Nanjing) Inc., 10 Xuefu Road, Nanjing 210032, China
cSuzhou Highfine Biotech. Co., Ltd., 32 Hongxi Road, Suzhou 215129, China
ARTICLE INFO
ABSTRACT
Article history:
Received
We describe the synthesis and characterization of
a type of pH-sensitive
pentaerythritol phospholipids, using a trialkoxybenzylidene acetal as the acid-labile
moiety. This lipid was prepared by an eight-step synthesis via a latentiation strategy.
Liposomes were prepared via the thin film extrusion method. The changes of
liposomal sizes were measured by dynamic light scattering. Content release rates of
the liposomes as a function of pH were monitored by using a calcein fluorescence
dequenching assay. These results indicated that this new liposomal system was
capable of releasing its contents under mildly acidic conditions. At last, in vitro
cytotoxicity was assayed against three cell lines, suggesting this type of phospholipids
was low toxic.
Received in revised form
Accepted
Available online
Keywords:
pH-sensitive liposome
pentaerythritol
acetal
phosphatidylcholine
drug delivery
2009 Elsevier Ltd. All rights reserved.
Since the discovery of liposomes by British haematologist
Bangham in 1961,1-3 liposomes have undergone intensive
investigation as drug delivery vehicles for over 50 years,
resulting in a number of successful applications for cancer
chemotherapy, fungal infections, and infection imaging. So far,
the liposomal drugs like Doxil and Ambisomes have reached the
market. Liposomes are most often composed of phospholipids,
especially phosphatidylcholine (PC), but may also include
phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and
cholesterol (Figure 1).
encapsulated drugs are widely reported in the literature. Among
these “smart” liposomes, low pH-sensitive liposomes have
attracted much interest.4-6 Ideally, these liposomes should be
relatively stable under the physiological pH but should be
triggered to release their contents upon exposure to decreased pH
at the targets. The rationale of pH-sensitive liposome is that the
decrease of pH occurs in many physiological and pathological
progressions such as endosome processing, tumor growth,
inflammation and myocardial ischemia. It has been found that
molecules internalized within cells via the endocytic pathway
encounter a significant pH drop from neutral to pH 5.9−6.0. The
pH could further reduce from 6.0 to 4.0 during progression from
late endosomes to lysosomes.7,8
O
O
O
O
P
O
O
P
n
n
NH3
N
O
O
O
O
O
O
Principally, pH-sensitive liposome system can be classified
into two different approaches: (1) formulations containing
polymorphic lipids or fusion proteins; (2) lipids containing an
acid-cleavable component. To date, a variety of pH-sensitive
linkers, such as hydrazones, vinyl ethers, and ortho esters have
been applied to pH-sensitive liposomes.9-14 Although acetals as
pH-sensitive moieties have been widely investigated in other
kinds of drug delivery systems,15-19 they were seldom used in pH-
sensitive liposomes. In addition, most phospholipids commonly
use glycerols as their skeletons. Herein, we describe a type of
n
O
n
O
O
O
PC
PE
O
O
O
O
O
n
n
O
O
n
O
P
O
O
N
O
O
P
O
OH
Spacer
O
O
n
O
OH
OH
O
pentaerythritol phosphatidylcholine (PEPC)
PG
Figure 1. Conventional phospholipids and pentaerythritol phospholipids
pentaerythritol
phosphatidylcholine
(PEPC),
using
pentaerythritols as the skeletons and acetals as pH-sensitive
moieties (Figure 1).
Since a pentaerythritol has four hydroxyls, two of them can be
acylated with long chain fatty acids. The other two can condense
with an appropriate aldehyde to construct an acetal. It was well
The most significant obstacles of conventional liposomes are
their rapid blood clearance, reticuloendothelial system
localization and lack of site-specific targeting. So far, the
development and application of novel targeting and/or release
triggering liposomes to improve the therapeutic index of

known that the structure of the aldehyde had a great effect on the
rate of hydrolysis.20 We first prepared three pentaerythritol
acetals (1, 2 and 3) according to reported methods, and examined
their hydrolysis rate by 1H NMR at pH 5.0.21 As shown in Figure
2, the compounds 1 and 2 were relatively stable at pH 5.0, but the
compound 3 exhibited an obvious degradation at this pH. Besides,
phenyl rings with more alkoxy groups could hydrolyze faster,
suggesting electron-donating groups could accelerate the
hydrolysis rate. These data were in accordance with others'
results that trimethoxybenzylidene acetals are more acid-
sensitive.15-19
large amount of starting materials remained. Since 7 was very
hydrophobic and had a low solubility in the aqueous media, lead
tetraacetate was a good alternative reagent. Thus, the oxidative
cleavage of the diol 7 with lead tetraacetate in dichloromethane
gave the desired aldehyde 8, which was reduced to afford the key
intermediate 9. The primary alcohol was then coupled with 2-
bromoethyl dichlorophosphate and hydrolyzed to yield the
bromide 10, which was further reacted with trimethylamine to
afford the target molecular 11 in good yields.
To further verify the pH-sensitivity of this spacer, another
pentaerythritol lipid 16, using an alkane as the spacer, was also
prepared. This lipid was assumed to be less acid-sensitive and
could serve as a control. The synthesis of this lipid was outlined
in Scheme 2.
O
O
OH
OH
O
O
O
O
O
O
O
O
1
O
O
OBn
12
12
12
12
HO
HO
O
O
OBn
O
O
OBn
O
O
OH
a
c
HO
HO
O
b
O
O
2
12
13
14
O
HO
HO
O
O
Br
O
O
O
O
N
O
O
P
O
d
12
12
O
e
O
O
O
O
O
O
O
O
O
O
P
O
O
3
O
O
O
P
Br
12
Cl
O
12
Cl
15
16
Figure 2. The hydrolysis rate of different pentaerythritol acetals at pH 5.0
Scheme 2. Reagents and conditions: (a) pentaerythritol, TsOH, DMF, 50%; (b) myristic acid, DCC, DMAP, CH2Cl2,
90%; (c) Pd/C, THF, H2 80%; (d) (i) Py, CH2Cl2; (ii) NaHCO3, H2O; (e) N(CH3 3, CHCl3, three steps 65%.
,
)
Thus, 1,3,5-trialkoxybenzene could serve as the best acid-
sensitive “spacer” in the pentaerythritol phospholipid. However,
the synthetic work of this type of acid-sensitive lipid did not
proceed smoothly at the beginning, because of the involvement
of a variety of functional groups. The acetal was acid-sensitive,
while the ester was base-sensitive. In addition, this benzylidene
acetal was also vulnerable to palladium-catalyzed hydrogenation.
Several synthetic routes were attempted, but failed to produce the
key intermediate 9 (Scheme 1). At last, a latentiation strategy was
employed. A terminal double bond was first introduced and then
transformed into a primary alcohol. The successful synthetic
route was outlined in Scheme 1.
2-Benzyloxy-1,1-dimethoxyethane was condensed with
pentaerythritol under acidic conditions to yield the acetal 12 in an
50% yield. Subsequently, 12 was coupled with myristic acid to
give the ester 13, which was then hydrogenated to afford the
primary alcohol 14 in good yields. At last, the compound 14 was
phosphorylated and hydrolyzed to yield the bromide 15, and then
subjected to nucleophilic substitution to produce the desired
compound 16 in moderate yields.
Since PEPC was designed for the acid-triggered drug delivery,
we were most interested in the following questions. (1) Could
PEPC form liposomes through self-assembly? (2) If PEPC
formed liposome, could acid trigger the content release?
O
O
O
O
O
a
b
HO
HO
O
O
c
OH
O
O
O
O
O
4
5
O
O
O
O
O
O
O
O
O
O
OH
OH
8
8
O
O
O
d
e
O
O
O
O
O
8
8
6
7
O
O
O
O
O
O
O
O
O
OH
8
8
O
O
O
H
O
f
8
8
g
O
O
O
O
O
P
O
O
Br
O
Cl
O
8
9
Cl
O
P
O
P
O
O
N
Br
O
O
O
O
O
O
8
8
h
O
O
O
O
O
O
O
O
O
O
Na
O
O
O
O
O
8
8
11
10
Scheme 1. Reagents and conditions: (a) allyl bromide, K2CO3, DMF, 85%; (b) pentaerythritol, TsOH, DMF,
92%; (c) capric acid, DCC, DMAP, CH2Cl2, 94%; (d) NMO, OsO4, acetone, 87%; (e) Pb(OAC)4, CH2Cl2; (f)
NaBH4, THF, MeOH, two steps 70%; (g) (i) Py, CHCl3; (ii) NaHCO3, H2O; (h) N(CH3)3, CHCl3, three steps
72%.
Thus, 2,6-dimethoxy-4-hydroxybenzaldehyde was reacted
with allyl bromide to give the compound 4 in a 85% yield, which
was condensed with pentaerythritol under acidic conditions to
yield the acetal 5 in an excellent yield. The acetal 5 was
subjected to acylation with capric acid to afford the ester 6.
However, the ozonolysis of the terminal double bond of 6 failed
to give the aldehyde 8. Alternatively, 6 was subjected to the
dihydroxylation with osmium tetraoxide and smoothly produced
the diol 7 in a 87% yield. The oxidative cleavage of the diol 7
with sodium periodate solution resulted in low yields, with a

3
Liposome of 11
Liposome of 11
Liposome of 16
Liposome of 16
Figure 3. Changes in the size of liposomes in response
to different pH
Figure 4. The calcein release from PEPC liposomes at different pH
To answer these questions, we first prepared empty liposomes
of 11 and 16, by conventional liposomal preparative methods.22
The average sizes of the vesicles were determined by dynamic
light scattering (DLS). The liposome of 11 had an average
diameter of 134 nm (PDI = 0.119), while the liposome of 16 had
an average diameter of 184 nm (PDI = 0.164). To study the acid-
sensitive process, the size destabilization of PEPC liposomes in
response to different pH values at different intervals was
determined using DLS measurements (Figure 3). Under acidic
conditions or neutral conditions, the liposomal size of 16
remained basically stable. However, the liposomal size of 11 was
stable at pH 7.4 but increased quickly with decreasing pH,
suggesting 11 was much more acid-sensitive than 16. This
phenomenon could be ascribed to the cleavage of the cyclic
acetal moiety. Namely, the acetal could be cleaved under mildly
acidic conditions and produce a hydrophobic unit, which was not
soluble in water. In addition, we also examined the hydrolysis of
compound 11 by 1H NMR at pH 4.5 (see the supporting
information). The results indicated that this compound was acid-
labile, indeed.
At last, in vitro cytotoxicity of compound 11 was tested by
MTT assays in A549, HepG2 and MGC803 cells. The cells
cultured after 72 h in the presence of 11 retained high cell
viability even at high concentrations (50 ug/mL) (Figure 5). This
result suggests that this amphiphilic lipid may be a safe drug
carrier in vivo.
Content release rates of PEPC liposomes as a function of pH
were monitored by using a calcein fluorescence dequenching
assay.14 As calcein encapsulated at self-quenching concentration
leaks into the extraliposomal solution, an increase in calcein
fluorescence occurs. Liposomes containing calcein (50 mM)
were incubated in buffer solutions of pH 4.5, 5.0, 6.0 or 7.4 at
37 °C and the time-dependent change in calcein fluorescence
monitored at 527 nm. The calcein release percentage at each time
point was calculated as a ratio versus 100% release by addition of
Triton X-100 to the liposome sample. In this study, liposomes
were modified with cholesterol to increase the stability. In the
new formulation, the ratio of PEPC to cholesterol was 2 to 1.
The calcein release from the liposome of 11 was obviously pH-
dependent (Figure 4). After 5 hours, an over 80% release was
observed at pH 4.5. However, no release occurred over 24 h at
pH 7.4. As for the compound 16, calcein release almost did not
occur over 24 h even at pH 4.5. These data show that acetal of
1,3,5-trialkoxybenzaldehyde is more acid-sensitive and prone to
hydrolysis than that of aliphatic aldehyde.
Figure 5. In vitro cell viabilities measured by the MTT assay after culture of the
cells with 11 as functions of different concentrations
In conclusion, we have successfully developed pH-responsive
liposomes comprising of a novel acid-labile cyclic acetal as the
pH-sensitive moiety and pentaerythritol as the skeleton. This
lipid could undergo self-association in aqueous media to form its
liposomes. The sizes of the liposomes increased with prolonged
time in acidic aqueous solution due to the hydrolysis of the acid-
labile acetal. Calcein release rates further confirmed the pH
sensitivity of this novel type of liposome. In addition, in vitro
cytotoxicity assay showed that this lipid had low cytotoxicity.
Thus, the present pH-sensitive liposomes can be used as a
potential smart drug carrier.

Acknowledgments
This research was financially supported by The National Key
Technology R&D Program (No. 2015BAK45B00). We also
thank the Laboratory of Organic Functional Molecules, the Sino-
French Institute of ECNU for supports.
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5
Research highlights
(1) A new type of pH-sensitive phospholipids with
an acetal as the acid-labile moiety.
(2) This lipid was prepared by an eight-step
synthesis via a latentiation strategy.
(3) This liposome could release its contents under
mildly acidic conditions.
(4) This type of phospholipids was low toxic.

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A new type of pH-sensitive phospholipid
Li Zhana, Shuyuan Changa, Ting Liua, Shanbao Yub, Hui Lib, Minjie Luc, Yong Zhuc, Yu Luoa,∗, Jiahui
Yua, Fan Yanga and Jie Tanga
O
P
O
N
O
O
O
O
R
R
O
O
O
O
+
O
H
O
pH 7.4
O
R = CH (CH )
3
2 8