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ArticleLong-Term Stable Liposome Modified by PEG-Lipid in Natural
SeawaterKayano Izumi, Jiajue Ji, Keiichiro Koiwai, and Ryuji Kawano*
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sı Supporting Information*

ABSTRACT: This paper describes the stabilization of liposomes
using a PEGylated lipid, N-(methylpolyoxyethylene oxycarbonyl)1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt
(DSPE-PEGs), and the evaluation of the survival rate in natural
seawater for future environmental applications. Liposomes innatural seawater were first monitored by confocal microscopy, andthe stability was compared among different lengths and theintroduction ratio of DSPE-PEGs. The survival rate increased withan increase in the PEG ratio. In addition, the survival rate indifferent cationic solutions (Na+, K+, Mg2+, and Ca2+ solutions)
was studied to estimate the effects of the DSPE-PEG introduction.
We propose that these variations in liposome stability are due tothe cations, specifically the interaction between the poly(ethylene glycol) (PEG) chains and divalent ions, which contribute to
making it difficult for cations to access the lipid membrane. Our studies provide insights into the use of PEG lipids and may offer a
promising approach to the fabrication of liposomal molecular robots using different natural environments.

1. INTRODUCTION
A cell-sized liposome consisting of a lipid bilayer has beenstudied as a simple cell model.1 It has the role of a cell-likecompartment for gene expression2 or protein production3 in
the field of synthetic biology. Practical applications such as
drug delivery have been expected in the usage of liposomes.4,5
It can effectively transport the drug to the diseased target byprotecting the drug from degradation through encapsulation.
Recently, the liposome has also attracted attention as the bodyof a molecular robot.6−8 A molecular robot is a biomoleculardevice consisting of sensors as perception, circuits as
information processing, and actuators as vehicle functions, allintegrated into a confined liposome with more focus on anengineering perspective. This robot aims to systematicallycontrol the molecular system or extend the living system rather
than the artificial cells that focus on mimicking cells. The main
objective of developing robots is to use them as biocompatible
tools in biological environments. Chen et al. developed
artificial β-cells using liposomes aiming for medical applications.9 The cell can sense glucose in the outer solution, drive
glucose-triggered multiple molecular circuits, and releaseinsulin-like living β-cells.
Although the applications of molecular robots have been
mainly considered for use in biomedical fields, the robot also
has the potential to be used in a natural environment. For
example, the molecular robot will be used in natural seawater
to monitor the water quality in aquaculture such as prawns.10
To apply the liposome in a natural environment, long-term
stability is a significant problem to be solved. The stabilizing© XXXX The Authors. Published byAmerican Chemical Society

strategy of liposomes is broadly classified into three categories
using nontoxic materials. (1) The gelation of the inner solution
or the construction of the undercoats such as the cytoskeletonlike network has been attempted to prepare a stable
liposome.11−13 The gel network inside the liposome forms
by temperature regulation11,12 or light irrigation,13 mechanically reinforcing the liposome structure. (2) Incorporation ofcholesterol into the membrane is another strategy tomechanically stabilize the liposome. Cholesterols align alonglipid chains due to the amphipathic structure and form a wellpacked membrane,14 stabilizing the bilayer configuration.15 (3)Covering the membrane surface with polymers such aspoly(ethylene glycol) (PEG) is another strategy for stabilization as the nontoxic material. PEG is introduced to themembrane by being tagged with lipid heads and forms ashielding layer, inhibiting the adhesion of macromolecules that
destroy liposomes.16−18
The stability of liposomes in natural seawater is still poorly
understood. Natural seawater is an electrolyte aqueous solution
containing a variety of components: ions, amino acids, andproteins from living organisms at the molecular level.19−22 To
Received: December 25, 2023
Accepted: January 10, 2024A

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ArticleFigure 1. Conceptual diagram of stability improvement of the modified liposome. (A) Liposome stability was considered to be disrupted by cationsand (B) to be improved by PEGs blocking the ions attacking the membrane.

improve liposome stability, the resistance to the ions should be
essential because the ions are the most abundant molecules
except for H2O in natural seawater.20 Previous studies havereported that lipid bilayers interact with ions and change their
physical properties.23−27 We consider that the interaction
makes liposomes unstable, which should be solved to improve
the stability.
We reported here the improvement of the long-term stabilityof liposomes by using poly(ethylene glycol) (PEG) because
PEGylated lipid has the potential to inhibit the ion access tothe membrane (Figure 1). We employed microscopic
observation for the evaluation of the effects on the PEGs in
the stabilization for more than 3 days. We first compared theliposome stability in buffer solution and natural seawater from
the Miura peninsula in Japan. To evaluate the ionic effects on
the membrane, we used different ionic solutions such as Na+,
K+, Mg2+, and Ca2+ with Cl−. Finally, we considered themolecular mechanism of the ionic effects on the liposome
membranes.

Table 1. Composition of the Inner/Outer Solution of the
Liposomea
in/out

label

inner

inner buffer

outernatural seawater
buffer solution
480 mM Na+
buffer
10 mM K+ buffer
60 mM Mg2+
buffer
200 mM Mg2+
buffer
10 mM Ca2+
buffer
200 mM Ca2+
buffercomponents
1000 mM sucrose, 0.1 mM calcein, and 50 mM
Tris-HCl
natural seawater sampled at the Misaki
peninsula1000 mM glucose and 50 mM Tris-HCl
480 mM NaCl, 40 mM glucose, and 50 mM
Tris-HCl
10 mM KCl, 980 mM glucose, and 50 mM
Tris-HCl
60 mM MgCl2, 830 mM glucose, and 50 mM
Tris-HCl
200 mM MgCl2, 400 mM glucose, and 50 mM
Tris-HCl
10 mM CaCl2, 970 mM glucose, and 50 mM
Tris-HCl
200 mM CaCl2, 400 mM glucose, and 50 mM
Tris-HCla

Eight solutions were prepared. The amount of inclusions in natural
seawater was estimated approximately at 1000 mM (sample N = 8)
using an osmometer (VAPRO vapor pressure osmometer Model 560,
ELI Tech Group Inc.), and the osmolality between the inner andouter was adjusted by glucose to be isotonic, approximately 1000 mM(see also the Supporting Information text).

2. EXPERIMENTAL SECTION
2.1. Reagents. In this study, the following chemicals wereused: 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC;NOF corporation, Japan), 1,2-dioleoyl-sn-glycero-3-phospho(1′-rac-glycerol) (sodium salt) (DOPG; Avanti Polar Lipids,AL), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt) (Rhodamine
PE; Avanti Polar Lipids, AL), liquid paraffin (FUJIFILM Wako
Pure Chemical Corporation, Japan), calcein (Sigma-Aldrich
Co., LCC, MO), glucose (FUJIFILM Wako Pure Chemical
Corporation, Japan), sucrose (FUJIFILM Wako Pure Chemical Corporation, Japan), tris(hydroxymethyl)aminomethane(Nacalai Tesque Inc., Japan), hydrochloric acid (FUJIFILM
Wako Pure Chemical Corporation, Japan), sodium hydroxide(FUJIFILM Wako Pure Chemical Corporation, Japan), N(methylpolyoxyethylene oxycarbonyl)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 2000, sodium salt (DSPEPEG2000; NOF Corporation, Japan), and N-(methylpolyoxyethylene oxycarbonyl)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 5000, sodium salt (DSPE-PEG5000; NOF
Corporation, Japan). 100 mM Tris-HCl was prepared fromtris(hydroxymethyl) aminomethane (pH 7, adjusted with somehydrochloric acids and sodium hydroxide).
2.2. Preparation of Solutions. An inner solution andeight outer solutions were prepared to evaluate the liposome
stability (Table 1): Natural seawater was sampled from theMiura peninsula in the Kanagawa prefecture. The seawater was
filtered through a 0.45 μm filter to remove large contaminants.
The inner solution was prepared so that the osmolality was thesame as that of the seawater (1000 mmol/kg).3 Six ionicbuffers containing representative seawater cations (Na+, K+,
Mg2+, and Ca2+)19,20 were prepared to discuss the effects ofcations23−27 on liposome stability. Chloride was used because
the chloride ions have little effect on the membrane properties
to neutralize the solution,26 and the osmolality was adjusted by
glucose to be isotonic.
2.3. Preparation of Different Membrane Compositions. Polymers were used with the expectation that theywould act as liposome stabilizers. They form shield-likehydrophilic layers on the surfaces and prevent protein
interaction with the membrane.16−18 The structural differenceof the PEG chains, such as length and ratio, was estimated in
how it affects the stability of liposomes (see also the
Supporting Information text). DSPE-PEG2000 and DSPEPEG5000, which are specifically used in medical studies,28−30
were prepared to investigate the stabilizing effect of PEG
length (Tables 2 and S1). In addition, the PEG mixture ofB

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ArticleTable 2. Lipid Composition of the Membranes with Different PEG Lengths or Ratiosa
membrane composition*label

DSPE-PEG [%]POPC [%]

POPC
5 mol % DSPE-PEG2000
5 mol % DSPE-PEG5000
5 mol % DSPE-PEG2000/5000
20 mol % DSPE-PEG5000

05
5total 5
20

100
95
95
95
80

notes
lipid only

the mixture of DSPE-PEG2000/DSPE-PEG5000 = 1:1 (mol/mol)also prepared by fivefold lipidsaDifferent compositions of membranes were prepared (see also the Supporting Information text and Table S1). For each composition, + 1 mol %DOPG was further added to avoid liposomes adhering to each other and +0.1 mol % rhodamine PE was also added to recognize liposome
membranes easily.

DSPE-PEG2000/DSPE-PEG5000 = 1:1 (mol/mol) (DSPEPEG2000/5000) was prepared as expected to form a bumpyhydrophilic layer and uniquely affect liposome stability.
Various molar ratios of DSPE-PEG5000/POPC were also
prepared to investigate the contribution of liposome stability(Tables 2 and S1).
2.4. Preparation of Liposomes Using Droplet Transfer. Liposomes with different membrane compositions as
described in Section 2.3 were prepared by using the droplet
transfer method.31 Briefly, a 1.28 mM lipid mixture for 200 μL
of chloroform was poured into a 1.5 mL centrifuge tube andsettled to remove chloroform unless otherwise explained.
Dried lipids were dissolved by 125 μL of liquid paraffin using a
40 kHz ultrasonic cleaner MSC-2 (AZ ONE Corporation,
Japan) at 70 °C for 10 min. To form water−oil (W/O)
emulsions, 25 μL of 1000 mM sucrose, 0.1 mM calcein, and 50
mM Tris-HCl dissolved in MQ (inner) were added to thetube, and the contents were mixed by tapping 40 times. 100 μLof the mixture was slowly added to 200 μL of 1000 mM
glucose and 50 mM Tris-HCl which was dissolved in MQ(buffer solution) in another centrifuge tube to form a lipid
monolayer at both the inner-oil and oil-outer interfaces. It wasthen centrifuged at 9000g for 20 min using a CT15E centrifuge(Hitachi Koki Co. Ltd., Japan). As the w/o emulsions
transferred from the oil phase to the outer solution, a lipid
bilayer membrane was formed at the interfaces, resulting in theformation of liposomes at the bottom of the tube without
internal leakage. The pellet of the liposomes was extracted, and500 μL of a buffer solution was added to the centrifuge tube.
The solution was pipetted, and 250 μL of the mixture wasadded to a new centrifuge tube. The mixture was centrifugedagain at 6000g for 10 min to remove small lipid aggregates.
The pellet of the liposomes was extracted, and 200 μL of theouter solution (Table 1) was added to the tube.
2.5. Observation and Analysis of Liposome Stability.
To observe the liposomes, a 3.0 mm hole was punched on a
5.0 mm-thick silicone rubber sheet and an observationchamber was made by gluing it on the cover glass. The
chamber was coated with 0.1% bovine serum albumin (BSA)
solution for 30 min to avoid unexpected rupture of theliposomes by attaching the chamber. After coating, 15 μL of
liposome solution was added to the chamber (Figure S1A).
The chamber was then sealed with another cover glass andsettled for more than 30 min to sink liposomes to the bottom,
and a large image of the whole chamber was taken for eachmeasurement. An AX confocal microscope (Nikon Corp.,
JAPAN) equipped with a 40× water objective lens (NA 1.25,
Nikon Corp., JAPAN) was used for the visualization. All
experiments were performed at room temperature (RT). The
liposomes were stained by rhodamine for the membrane andcalcein for the inner solution. We confirmed that the liposomes
were evenly distributed throughout the chamber and extracted
an in-focus 1329 × 1329 μm2 square unit area for the analysis
because liposomes distributed at the edge of the chamber wereout of focus.
On the day of liposome preparation, we took the preimage
and calculated liposome productivity.
productivity [%]total liposome area in pre took image=
total analytical area (1329 × 1329 m 2)

(1)

Based on the results, we diluted the liposome concentration to
occupy 5−30% of the square area by adding outer solutions. In
our preliminary experiments, the fluorescents were drasticallyphotobleached after one or two measurements. We interpreted
that the fluorescents in the liposomes were completely
photobleached and hardly recovered.32 We stored the diluted
liposome solution in a 1.5 mL light-protected centrifuge tube
at RT and resampled the liposomes for each measurement(Figure S1B). Liposomes were pipetted 50 times before eachmeasurement to disperse bottom-sinking liposomes.
The stability of the liposomes was assessed by the survival
rate.
survival rate [%] =total liposome area on day a
total liposome area on day 0(2)

where day a is the number of elapsed days defined by the
preparation day as Day 0. The significant difference between
the two membrane compositions was also evaluated by using
the p-value of Welch’s t test. If the p-value was less than 0.05,
then, we defined it as statistically different. Images were taken
for a maximum of 14 days: the day of liposome preparation(Day 0), Day 1, Day 2, Day 3, Day 7, and Day 14. If we could
not detect calcein, we confirmed whether the liposomes reallydisappeared or only the calcein leaked by differential
interference contrast imaging or rhodamine. To better
understand which factor mainly improved the liposome
stability, we also examined the PEG configuration on themembrane and the size difference of the liposomes at eachmembrane composition (Supporting Information text). For
comparison, we used the data from Day 3 unless otherwise
stated, and at least three samples were prepared for eachcondition to ensure reproducibility.

3. RESULTS
3.1. Stability of Liposomes with/without DSPE-PEG
Evaluated in Buffer Solution and Natural Seawater.
POPC or DSPE-PEG/POPC liposomes were prepared using a
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ArticleFigure 2. Liposomes on Day 3 with different membrane compositions. POPC, DSPE-PEG2000, DSPE-PEG5000, and a mixture of DSPEPEG2000/DSPE-PEG5000 = 1:1 (mol/mol) (DSPE-PEG2000/5000) were prepared in (A) buffer solution and (B) natural seawater. POPC and 5
and 20 mol % DSPE-PEG5000 liposomes were prepared in (C) buffer solution and (D) natural seawater. The inner solution of the liposomes wascolored green by calcein fluorescence.

Figure 3. PEG properties and the survival rate. (A, B) Average survival rate of 5 mol % DSPE-PEG2000, DSPE-PEG5000, and DSPE-PEG2000/
5000 in (A) buffer solution and (B) natural seawater. (C, D) Average survival rate of POPC and 5 and 20 mol % DSPE-PEG5000 liposomes in (C)buffer solution and (D) natural seawater. The horizontal axis shows the number of days after liposome preparation, and the vertical axis shows thesurvival rate. *p < 0.05 and **p < 0.01. Trials N ≥ 3 for each.

droplet transfer method.31 Initially, we expected that theaddition of DSPE-PEG to the POPC membrane might reduce
the productivity of liposome generation due to the longhydrophilic PEG chains. The addition of DSPE-PEG did not
significantly affect productivity, even when the PEG length waschanged from PEG2000 to PEG5000 (Figure S2A). However,
the molar ratio of DSPE-PEG/POPC did have an impact onliposome productivity, and 30 mol % DSPE-PEG resulted ininsufficient liposome generation (Figure S2B). As a result, weused the DSPE-PEG content to a maximum of 20 mol % in thesubsequent experiments. Next, we investigated the temporalstability of the liposomes in both buffer solution and natural
seawater from the Miura peninsula in Japan using confocal
microscopy over a period of 3 days. During the period, theliposome solution was protected from light and stored at RT.
Figure 2 illustrates the microscopic observations of liposomeswith different lengths of the PEG chains (Figure 2A,B) anddifferent molar ratios of DSPE-PEG/POPC (Figure 2C,D)
after 3 days. Notably, the number of surviving liposomes in theseawater was significantly lower compared with the buffer.
Figure 3 provides a quantitative analysis of the mean survival
rate over 3 days. The data on Day 3 were used for theD

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ArticleFigure 4. Liposome stability in four cationic solutions. Average survival rate of POPC and 5 and 20 mol % PEG liposomes in (A) 480 mM sodiumion (Na+), (B) 10 mM potassium ion (K+), (C) 60 mM magnesium ion (Mg2+), and (D) 10 mM calcium ion (Ca2+) buffers. Each ionicconcentration was adjusted the same as natural seawater. (E) Average survival rate of POPC in monovalent or divalent solution. Average survival
rate of POPC and 5 and 20 mol % PEG liposomes in (F) monovalent ions (Na+ and K+) and (G) divalent ions (Mg2+ and Ca2+). *p < 0.05 and**p < 0.01. Trials N ≥ 3 for each.

subsequent evaluation. Among all the membrane conditions,
POPC liposomes exhibited the lowest survival rate, decreasing
to approximately 18% in the buffer and 9% in the seawater on
Day 3. In contrast, liposomes containing DSPE-PEG showed
greater stability in both the buffer and the seawater. The lengthof the PEG chain had no significant effect on the stability,
whereas the molar ratio affected the stability in both of them
(Figure 3). In the buffer solution, the survival rate of liposomeswith the 20 mol % DSPE-PEG system was approximately
threefold higher than in the POPC system (on Day 3 in Figure
3C). Even though a similar difference could not be detectedwith significance (p < 0.05) in the case of seawater, the stability
in the seawater was also improved by increasing the molar
ratio; the mean survival rate in the 20 mol % DSPE-PEG
system was fivefold higher than in the POPC system (on Day 3
in Figure 3D). The presence of DSPE-PEG seemed to be amore substantial improvement in the temporal stability within
the seawater compared to that of the buffer (Figure 3C,D).

We next checked the size of the liposome because it is an
important parameter and should affect the stability.33,34 The
size distribution was then evaluated by the interquartile range(IQR) of the liposome radius. In all conditions, the IQR
converged 0.7 < IQR < 3.4 μm (see also the Supporting
Information Text and Figure S3), suggesting that the size did
not affect the stability in this experiment. We also investigatedthe effect of DSPE itself, without the PEG chain, on theliposome stability because DSPE is a saturated lipid, and it
should be possible to form a more rigid membrane than
POPC. The survival rate of liposomes of DSPE/POPC (2:8
mol/mol) without PEG showed a similar tendency to that of
POPC liposomes (Figure S4), indicating that the PEG coating
mainly contributed to the liposome stability. The PEG
configuration on the membrane was then estimated based on
the Flory radius because the conformation of PEGs on themembrane should affect the liposome stability (see also the
Supporting Information Text and Table S2). The conformations of 5 and 20 mol % DSPE-PEG were brush and denseE

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ArticleFigure 5. Liposome stability in high-divalent solutions. Average survival rate of POPC and 5 and 20 mol % PEG liposomes in (A−C) Mg2+ and(D−F) Ca2+. (G−I) Average survival rate of POPC and 5 and 20 mol % PEG liposomes in 200 mM divalent ions. *p < 0.05 and **p < 0.01. Trials
N ≥ 3 for each.

with lipid heads and water molecules and then reduce thedipole potential of the membrane.23−27 To investigate theeffects of cations contained in the seawater, we evaluatedliposome stability in electrolyte solutions that contain one of
the cations, Na+ , K +, Mg 2+, or Ca 2+, with a similar
concentration found in nature (Table 1).20 Notably, theorder of survival rates exhibited a consistent trend even among
different ion species (Na+ and K+) in the case of monovalentions (Figure 4A,B). In the divalent cations (Ca2+ and Mg2+),
the survival rate of 20 mol % DSPE-PEG liposomes was thehighest (Figure 4C,D). The influence of cation valence on
liposome stability was significant and is depicted in Figure 4E−
G. In the case of POPC liposomes, the survival rate wassubstantially lower in divalent solutions compared to that inmonovalent ones (Figure 4E). The presence of DSPE-PEG led
to a more substantial improvement in stability within divalentsolutions compared with monovalent systems (Figure 4F,G).
The difference in survival rate between POPC and the moststable membrane condition on Day 3 was the largest in
divalent solutions (threefold in Mg2+ with p < 0.05 andfourfold in Ca2+ with p < 0.05) than in monovalent solutions(onefold in Na+ with no significant difference and twofold in
K+ with p < 0.05). Interestingly, the survival rate of 20 mol %DSPE-PEG liposomes in divalent solutions on Day 3 wasbrush, implying that these conformations of PEGs may
contribute to improving the liposome stability. Since experiments up to this point were conducted for 3 days, we next
examined the longer, 14-day stability. The survival rate ofliposomes with 20 mol % DSPE-PEG on Day 14 was 3% evenin the seawater (Figure S5). These findings indicate thatincorporating DSPE-PEG into the liposomes improves theirtemporal stability, particularly in natural seawater, and that amolar ratio of 20 mol % DSPE-PEG offers the best results forenhancing liposome survival.
3.2. Liposome Stability Decreased by Cations
Contained in Natural Seawater. 3.2.1. Monovalent andDivalent Cations Decreased Liposome Stability. Although
the stability of liposomes in natural seawater tended to belower compared to that in the buffer solution, we observed that
DSPE-PEG/POPC liposomes appeared to improve stability inboth environments compared to POPC liposomes. Interestingly, the introduction of DSPE-PEG seemed to have a more
effective effect on stability in the seawater. In our experiments,
the buffer solution contained only glucose and tris-HCl,leading us to hypothesize that the PEG chains might mitigate
interactions between cations and the lipid membrane. Natural
seawater contains a higher concentration of various ions,
including monovalent and divalent cations, which can interact
F

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ArticleFigure 6. Schematic diagram of liposome stabilization/destabilization. Liposome stabilization of (A) POPC, (B) 5 mol % DSPE-PEG, (C) 20 mol% DSPE-PEG with low-affinity ions or in low-ionic solution, and (D) 20 mol % DSPE-PEG in high-ionic solution.

comparable to that in monovalent ones. These insightful
findings underscore the greater influence of divalent ions in
reducing liposome stability while highlighting the effectivenessof DSPE-PEG introduction in enhancing stability against theseions.
3.2.2. PEG Liposomes Were More Stable in High-Cationic
Solutions than in Low-Cationic Ones. To study further theeffect of divalent ions, we conducted stability tests of liposomes
under identical concentrations of Mg2+ and Ca2+ solutions(200 mM) as depicted in Figure 5. The presence of DSPEPEG enhanced the stability: the survival rate of 5 mol % DSPEPEG liposomes closely resembled that of low-ionic solutions,while 20 mol % DSPE-PEG liposomes exhibited even higher
stability than that of low-ionic conditions (Figure 5B,C,E,F).
The difference between POPC and 20 mol % DSPE-PEG
liposomes was striking, showing an eightfold increase in Mg2+
with p < 0.01 and an impressive fivefold increase in Ca2+ with p< 0.05. Contrary to our initial expectations, there did not
emerge a significant difference between the two cations (Figure5G−I).

were enhanced with increasing DSPE-PEG ratio and in thecase of the high-cationic solution (Figure 6B−D). Since theformation of PEG−cation complexes has been experimentallyand theoretically studied,38−40 the PEG−cation complexes
should also form on the surface of liposomes, resulting in theformation of a rigid shield-like layer with the PEG−cation
complex.
For the actual implementation of the liposome-based
molecular robot in natural seawater, several uncertainties
remain. Even if ions are the main components, natural seawater
also contains a wide variety of organic components such asamino acids21 and fatty acids,22 and they have the potential tointeract with the membrane, change the properties, and affectthe liposome stability. For instance, serine, which is an aminoacid found in natural seawater,21 can interact with the lipid
membrane and change the membrane rigidity.41 Other
environmental conditions such as pH and temperature should
also be considered in further investigation.

4. DISCUSSION
Here, we attempted to improve the temporal stability of theliposomes with a DSPE-PEG/POPC membrane in natural
seawater. The stability of the liposomes improved withincreasing the DSPE-PEG ratio (Figure 3C,D), and thestability was affected by the types of cations contained in theseawater (Figure 4E−G). In the case of the liposome
consisting of the POPC membrane itself, the stability of theliposome tended to decrease with higher concentrations ofcations (Figure 5A,D). In contrast, the stability was improved
with the introduction of 20 mol % DSPE-PEG into the POPC
membrane even in the higher ionic solutions (Figure 5C,F).
We further found that the introduction of the DSPE-PEG
improved the stability against the divalent cations, although thedivalent ions such as Mg2+ and Ca2+ remarkably decreased thestability of the POPC liposome (Figure 5).
Based on the results, the effects of the DSPE-PEG
introduction are attempted to be explained based on the
PEG chain configuration as shown in Figure 6. First, cations
could decrease the stability of the POPC liposome byinteracting with the membranes (Figure 6A).23−27 The
divalent ions, which have larger hydrated radii than those of
the monovalent ions, are considered to have a stronger binding
ability and a slower exchange rate with oxygen moieties (Table
S3).35−38 The ions should alter the membrane properties bybinding to lipid oxygens, and the binding strengths could affectthe disruption of lipid alignment, thus destabilizing liposomes.
Next, DSPE-PEGs improve liposome stability by forming ashield-like layer that prevents the cations from accessing deep
inside the PEG layer as previously reported.16−18 The effects

5. CONCLUSIONSIn conclusion, we discussed the membrane composition atwhich liposomes with PEG-lipid are stable in natural seawater.
The liposome stability was evaluated by a microscopic
observation. It turned out that the high-density PEG liposomes
were the most stable in natural seawater for 3 days ofobservation, and some of them can live for 14 days. Factors forliposome destabilization were specified using various ionicsolutions, which indicates that the divalent ions caused thedestabilization. To date, liposome stability has been mainly
investigated and discussed in the medical field.42−44 In thisstudy, liposome stability in natural seawater was evaluated for
future environmental applications. Our study can contribute tofabricating liposomal molecular robots for employing various
natural environments.

■

ASSOCIATED CONTENT

sı Supporting Information*

The Supporting Information is available free of charge athttps://pubs.acs.org/doi/10.1021/acsomega.3c10346.
Membrane composition for evaluation of liposome
productivity, theoretical PEG conformation, physicalproperties of the cations, evaluation processes ofliposomes, liposome productivity, size distribution of
the liposome, correlation between the lipid type andliposome stability, and liposome stability on Day 14(PDF)G

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AUTHOR INFORMATION(4) Kraft, J. C.; Freeling, J. P.; Wang, Z.; Ho, R. J. Emerging
Research and Clinical Development Trends of Liposome and Lipid
Nanoparticle Drug Delivery Systems. J. Pharm. Sci. 2014, 103 (1),29−52.
(5) Yuan, Z.; Das, S.; Lazenby, R. A.; White, R. J.; Park, Y. C.
Repetitive drug releases from light-activatable micron-sized liposomes.
Colloids Surf., A 2021, 625, 126778 DOI: 10.1016/j.colsurfa.2021.126778.
(6) Hagiya, M.; Konagaya, A.; Kobayashi, S.; Saito, H.; Murata, S.
Molecular Robots with Sensors and Intelligence. Acc. Chem. Res. 2014,47 (6), 1681−1690.
(7) Murata, S.; Konagaya, A.; Kobayashi, S.; Saito, H.; Hagiya, M.
Molecular Robotics: A New Paradigm for Artifacts. New Gener.
Comput. 2013, 31 (1), 27−45.
(8) Shoji, K.; Kawano, R. Recent Advances in Liposome-Based
Molecular Robots. Micromachines 2020, 11 (9), No. 788,
DOI: 10.3390/mi11090788.
(9) Chen, Z.; Wang, J.; Sun, W.; Archibong, E.; Kahkoska, A. R.;
Zhang, X.; Lu, Y.; Ligler, F. S.; Buse, J. B.; Gu, Z. Synthetic beta cells
for fusion-mediated dynamic insulin secretion. Nat. Chem. Biol. 2018,
14 (1), 86−93.
(10) Koiwai, K.; Koyama, T.; Tsuda, S.; Toyoda, A.; Kikuchi, K.;
Suzuki, H.; Kawano, R. Single-cell RNA-seq analysis reveals penaeid
shrimp hemocyte subpopulations and cell differentiation process. eLife
2021, 10, No. e66954, DOI: 10.7554/eLife.66954.
(11) Petralito, S.; Paolicelli, P.; Nardoni, M.; Trilli, J.; Di Muzio, L.;
Cesa, S.; Relucenti, M.; Matassa, R.; Vitalone, A.; Adrover, A.;
Casadei, M. A. Gelation of the internal core of liposomes as a strategy
for stabilization and modified drug delivery I. Physico-chemistry
study. Int. J. Pharm. 2020, 585, No. 119467.
(12) Peng, Z.; Wada, K.; Shimba, K.; Miyamoto, Y.; Yagi, T.
Formation of Agarose-supported Liposomes by Polymer-assisted
Method toward Biosensor. IEEJ Trans. Electr. Electron. Eng. 2021, 141(5), 646−653.
(13) Wang, Y.; Tu, S.; Pinchuk, A. N.; Xiong, M. P. Active drug
encapsulation and release kinetics from hydrogel-in-liposome nanoparticles. J. Colloid Interface Sci. 2013, 406, 247−255.
(14) Doole, F. T.; Kumarage, T.; Ashkar, R.; Brown, M. F.
Cholesterol Stiffening of Lipid Membranes. J. Membr. Biol. 2022, 255(4−5), 385−405.
(15) Liu, P.; Chen, G.; Zhang, J. A Review of Liposomes as a Drug
Delivery System: Current Status of Approved Products, Regulatory
Environments, and Future Perspectives. Molecules 2022, 27 (4),
No. 1372, DOI: 10.3390/molecules27041372.
(16) Le, N. T. T.; Cao, V. D.; Nguyen, T. N. Q.; Le, T. T. H.; Tran,
T. T.; Hoang Thi, T. T. Soy Lecithin-Derived Liposomal Delivery
Systems: Surface Modification and Current Applications. Int. J. Mol.
Sci. 2019, 20 (19), No. 4706, DOI: 10.3390/ijms20194706.
(17) Jin, J.; Han, Y.; Zhang, C.; Liu, J.; Jiang, W.; Yin, J.; Liang, H.
Effect of grafted PEG chain conformation on albumin and lysozyme
adsorption: A combined study using QCM-D and DPI. Colloids Surf.,
B 2015, 136, 838−844.
(18) Marruecos, D. F.; Kastantin, M.; Schwartz, D. K.; Kaar, J. L.
Dense Poly(ethylene glycol) Brushes Reduce Adsorption andStabilize the Unfolded Conformation of Fibronectin. Biomacromolecules 2016, 17 (3), 1017−1025.
(19) Duxbury, A. C.; Mackenzie, F. T.; Byrne, R. H. Seawater;
Encyclopedia Britannica Inc., 2023.
(20) Nguyen, T. V. Preparation of Artificial Sea Water (ASW) for
Culturing Marine Bacteria. In Protocols for Marine Biotechnology;
AUT, 2018.
(21) Hubberten, U.; Lara, R. J.; Kattner, G. Amino acid compositionof seawater and dissolved humic substances in the Greenland Sea.
Mar. Chem. 1994, 45 (1−2), 121−128.
(22) Slowey, J. F.; Jeffrey, L. M.; Hood, D. W. The fatty-acid content
of ocean water. Geochim. Cosmochim. Acta 1962, 26 (6), 607−616.
(23) Yang, J.; Calero, C.; Bonomi, M.; Marti, J. Specific Ion Bindingat Phospholipid Membrane Surfaces. J. Chem. Theory Comput. 2015,
11 (9), 4495−4499.

Corresponding Author

Ryuji Kawano − Department of Biotechnology and Life
Science, Tokyo University of Agriculture and Technology,
Tokyo 184-8588, Japan; orcid.org/0000-0001-65230649; Email: rjkawano@cc.tuat.ac.jp
Authors

Kayano Izumi − Department of Biotechnology and Life
Science, Tokyo University of Agriculture and Technology,
Tokyo 184-8588, Japan
Jiajue Ji − Department of Biotechnology and Life Science,
Tokyo University of Agriculture and Technology, Tokyo 1848588, Japan
Keiichiro Koiwai − Laboratory of Genome Science, Tokyo
University of Marine Science and Technology, Tokyo 1088477, Japan
Complete contact information is available at:https://pubs.acs.org/10.1021/acsomega.3c10346
Author Contributions

R.K. and K.K. conceived the original idea. K.K., K.I., and J.J.
collected natural seawater. K.I. and J.J. conducted experiments
and analyzed the data. K.I. and R.K. wrote the entire article. All
authors have approved the final version of the article.
Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS
This research was partially supported by JSPS KAKENHI,
grant numbers 21K19786 (R.K. and K.K.) and 21H05229(R.K.), and JST-CREST JPMJCR21B2 (R.K.). This work wasalso supported by the Support for Pioneering Research
Initiated by the Next Generation of FLOuRISH Institute,Tokyo University of Agriculture and Technology granted by
the Ministry of Education, Culture, Sports, Science andTechnology, Japan (K.I.), and by the proposal-based Project
Fund by the WISE program (K.I.). The authors thank Prof. T.
Inoue of Johns Hopkins University for constructive discussions.

■

ABBREVIATIONSPEG, poly(ethylene glycol); DSPE-PEG, N-(methylpolyoxyethylene oxycarbonyl)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt; POPC, 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine; DOPG, 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (sodium salt); rhodamine PE, 1,2dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine
rhodamine B sulfonyl) (ammonium salt); W/O, water−oil;
BSA, bovine serum albumin; RT, room temperature■

ArticleREFERENCES(1) Walde, P. Building artificial cells and protocell models:
experimental approaches with lipid vesicles. BioEssays 2010, 32 (4),296−303.
(2) Saito, H.; Kato, Y.; Le Berre, M.; Yamada, A.; Inoue, T.;
Yosikawa, K.; Baigl, D. Time-Resolved Tracking of a Minimum Gene
Expression System Reconstituted in Giant Liposomes. ChemBioChem
2009, 10 (10), 1640−1643.
(3) Noireaux, V.; Libchaber, A. A vesicle bioreactor as a step toward
an artificial cell assembly. Proc. Natl. Acad. Sci. U.S.A. 2004, 101 (51),17669−17674.
H

https://doi.org/10.1021/acsomega.3c10346
ACS Omega XXXX, XXX, XXX−XXXACS Omega

http://pubs.acs.org/journal/acsodf(24) Binder, H.; Zschörnig, O. The effect of metal cations on thephase behavior and hydration characteristics of phospholipid
membranes. Chem. Phys. Lipids 2002, 115 (1−2), 39−61.
(25) Seto, H.; Yamada, T. Quasi-elastic neutron scattering study ofthe effects of metal cations on the hydration water between
phospholipid bilayers. Appl. Phys. Lett. 2020, 116 (13), No. 133701,
DOI: 10.1063/1.5144012.
(26) Melcrová, A.; Pokorna, S.; Pullanchery, S.; Kohagen, M.;
Jurkiewicz, P.; Hof, M.; Jungwirth, P.; Cremer, P. S.; Cwiklik, L. The
complex nature of calcium cation interactions with phospholipid
bilayers. Sci. Rep. 2016, 6, No. 38035.
(27) Friedman, R. Membrane-Ion Interactions. J. Membr. Biol. 2018,
251 (3), 453−460.
(28) Ashok, B.; Arleth, L.; Hjelm, R. P.; Rubinstein, I.; Ö nyüksel, H.
In vitro characterization of PEGylated phospholipid micelles for
improved drug solubilization: effects of PEG chain length and PC
incorporation. J. Pharm. Sci. 2004, 93 (10), 2476−2487.
(29) Rydberg, H. A.; Yanez Arteta, M.; Berg, S.; Lindfors, L.;
Sigfridsson, K. Probing adsorption of DSPE-PEG2000 and DSPEPEG5000 to the surface of felodipine and griseofulvin nanocrystals.
Int. J. Pharm. 2016, 510 (1), 232−239.
(30) Sou, K.; Endo, T.; Takeoka, S.; Tsuchida, E. Poly(ethylene
glycol)-Modification of the Phospholipid Vesicles by Using the
Spontaneous Incorporation of Poly(ethylene glycol)-Lipid into the
Vesicles. Bioconjugate Chem. 2000, 11 (3), 372−379.
(31) Pautot, S.; Frisken, B. J.; Weitz, D. A. Engineering asymmetricvesicles. Proc. Natl. Acad. Sci. U.S.A. 2003, 100 (19), 10718−10721.
(32) Fujiwara, S.; Shoji, K.; Watanabe, C.; Kawano, R.; Yanagisawa,
M. Microfluidic Formation of Honeycomb-Patterned Droplets
Bounded by Interface Bilayers via Bimodal Molecular Adsorption.
Micromachines 2020, 11 (7), No. 701, DOI: 10.3390/mi11070701.
(33) Pandur, Ž .; Dogsa, I.; Dular, M.; Stopar, D. Liposome
destruction by hydrodynamic cavitation in comparison to chemical,
physical and mechanical treatments. Ultrason. Sonochem. 2020, 61,
No. 104826.
(34) The Giant Vesicle Book; Dimova, R.; Marques, C., Eds.; CRC
Press, 2020.
(35) Tansel, B.; Sager, J.; Rector, T.; Garland, J.; Strayer, R. F.;
Levine, L.; Roberts, M.; Hummerick, M.; Bauer, J. Significance of
hydrated radius and hydration shells on ionic permeability during
nanofiltration in dead end and cross flow modes. Sep. Purif. Technol.
2006, 51 (1), 40−47.
(36) Helm, L.; Merbach, A. E. Inorganic and Bioinorganic Solvent
Exchange Mechanisms. Chem. Rev. 2005, 105 (6), 1923−1959.
(37) Umebayashi, Y.; Yamaguchi, T. Electrolyte Solution Maruzen;
2013.
(38) Fang, Y.; Giesecke, M.; Furo, I. Complexing Cations byPoly(ethylene oxide): Binding Site and Binding Mode. J. Phys. Chem.
B 2017, 121 (9), 2179−2188.
(39) Kamiyama, Y.; Shibata, M.; Kanzaki, R.; Fujii, K. Lithium-ion
coordination-induced conformational change of PEG chains in ionicliquid-based electrolytes. Phys. Chem. Chem. Phys. 2020, 22 (10),5561−5567.
(40) Ohki, T.; Harada, M.; Okada, T. Structural and Thermodynamic Aspects of Ionic Solvation in Concentrated AqueousPoly(ethylene glycol). J. Phys. Chem. B 2007, 111 (25), 7245−7252.
(41) Kanwa, N.; De, S. K.; Maity, A.; Chakraborty, A. Interaction ofaliphatic amino acids with zwitterionic and charged lipid membranes:
hydration and dehydration phenomena. Phys. Chem. Chem. Phys.
2020, 22 (6), 3234−3244.
(42) Beltrán-Gracia, E.; López-Camacho, A.; Higuera-Ciapara, I.;
Velázquez-Fernández, J. B.; Vallejo-Cardona, A. A. Nanomedicine
review: clinical developments in liposomal applications. Cancer
Nanotechnol. 2019, 10 (1), No. 11, DOI: 10.1186/s12645-0190055-y.
(43) Yingchoncharoen, P.; Kalinowski, D. S.; Richardson, D. R.
Lipid-Based Drug Delivery Systems in Cancer Therapy: What Is
Available and What Is Yet to Come. Pharmacol. Rev. 2016, 68 (3),701−787.

Article(44) Chen, C.; Han, D.; Cai, C.; Tang, X. An overview of liposome
lyophilization and its future potential. J. Controlled Release 2010, 142(3), 299−311.

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