micromachines
Review

Recent Advances in Liposome-Based
Molecular Robots
Kan Shoji 1, *1
2*

and Ryuji Kawano 2, *Department of Mechanical Engineering, Nagaoka University of Technology, Kamitomioka 1603-1, Nagaoka,
Niigata 940-2188, Japan
Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology,
Naka-cho 2-24-16, Koganei, Tokyo 184-8588, Japan
Correspondence: kshoji@mech.nagaokaut.ac.jp (K.S.); rjkawano@cc.tuat.ac.jp (R.K.)

Received: 29 July 2020; Accepted: 19 August 2020; Published: 20 August 2020


Abstract: A molecular robot is a microorganism-imitating micro robot that is designed from themolecular level and constructed by bottom-up approaches. As with conventional robots, molecularrobots consist of three essential robotics elements: control of intelligent systems, sensors, and actuators,
all integrated into a single micro compartment. Due to recent developments in microfluidic
technologies, DNA nanotechnologies, synthetic biology, and molecular engineering, these individual
parts have been developed, with the final picture beginning to come together. In this review,
we describe recent developments of these sensors, actuators, and intelligence systems that canbe applied to liposome-based molecular robots. First, we explain liposome generation for thecompartments of molecular robots. Next, we discuss the emergence of robotics functions by using
and functionalizing liposomal membranes. Then, we discuss actuators and intelligence via theencapsulation of chemicals into liposomes. Finally, the future vision and the challenges of molecularrobots are described.
Keywords: molecular robot; giant unilamellar vesicles; synthetic ion channel; nanopore sensing;molecular motor; DNA computing

1. Introduction
An artificial cell is a cell-imitating artificial system that exhibits characteristics of living cells,
including evolution, self-reproduction, metabolization, and communication [1–3]. The developmentof artificial cells is one of the main objectives in the field of synthetic biology, and numerous
studies have been reported. For example, gene expression [4], metabolic networks [5,6], growth anddivision [7,8], adaptivity [9], communications [10,11], and motility [12] of artificial cells have beenreported. Although the recent developments of artificial cells successfully provided the potential forpractical applications [13,14], the functioning of artificial cells has not yet reached a practical level.
Hence, another concept called “molecular robotics” has been proposed, which aims to offer
practical uses in the fields of medicine, drug discovery, environmental science, food science, and energy
science [15–17]. In this concept, the essential robotics components that are intelligence systems [18],sensors [19], and actuators [20–23] are developed by integrating various technologies, such as DNA
nanotechnologies, synthetic biology, polymer chemistry, and robotics, and these components are
implemented into a microcompartment using bottom-up approaches. Although these elements havebeen individually developed, with performances that potentially offer practical uses, the integration ofthese elements into a single system is still challenging. To integrate these three elements into a single
robotics system, a compartment that can encapsulate these components is required. Giant unilamellar
vesicles (GUVs) [24,25], DNA capsules [26,27], gels [28,29], and polymersomes [30] have been proposed
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as structures that may act as such compartments. The compartment of a molecular robot must
be able to undergo membrane functionalization with membrane proteins and be able to facilitate
the encapsulation of molecules in order to implement intelligence systems, sensors, and actuators.
GUVs can, in particular, satisfy these requirements.
Here, we review recent advances in GUV-based molecular robot technologies, including methodsof GUV production, the emergence of sensors by the functionalization of liposomal membranes, and theemergence of actuation and intelligence by encapsulating macromolecules into GUVs. In addition,
we summarize the future outlook towards the integration of individual elements into a single
compartment and the manufacture of a liposome-based molecular robot.
2. Giant Unilamellar Vesicles (GUVs) Generation Using Microfluidic Devices
Unilamellar vesicles (UVs) consist of a single lipid bilayer that forms a spherical shell
shape in aqueous solution. UVs of 1–100 µm in diameter are called giant UVs (GUVs) and are
traditionally prepared as a simplified membrane model of a cell. For application as molecularrobots, GUVs must have a high stability, monodispersity of the droplet size, encapsulation efficiency,
and productivity. Conventional methods for GUV formation include the gentle hydration method [31]and electro-formation method [32]. However, the GUVs formed by these methods cannot be applied
to the development of molecular robots due to the lack of monodispersity in size and encapsulation
efficiency. Recently, to overcome these issues, numerous microfluidic techniques to form GUVs havebeen reported.
One such strategy involves a droplet transfer method via a microchannel [33,34]. In this method,water-in-oil emulsions (w/o emulsions) with phospholipids are first prepared, with GUVs being
subsequently produced by the transfer of the emulsions through a lipid monolayer formed at an
oil-water interface. To improve the yield and the monodispersity of the GUV size, microfabricationand microfluidic technologies have been applied. Because the uniformity of the GUV size is inrelation to that of the w/o emulsion size, optimal w/o emulsions are formed by microfluidic methods
instead of pipetting or vortexing. The Malmstadt group applied a T-junction microchannel to form
w/o emulsions [35]. The w/o emulsions were formed by flowing an aqueous solution and oil into
the T-junction microchannel. Then, the prepared droplet suspension was poured into the layered
solution of the oil/lipid mixture and the aqueous solution. Finally, GUVs were generated by the
droplet passing through the lipid monolayer at the oil-water interface. Because the experimental
conditions for generating the w/o emulsions—including the capillary number, flow rates, and channel
dimensions—allow the control of the droplet size, the monodispersity of the GUV size can beachieved. In addition, microfluidic channels for the on-chip generation of GUVs have also beenreported [36–38]. In these microfluidic channels, the formation of w/o emulsions and GUVs bytransferring w/o emulsions through the lipid monolayer are performed on the single microfluidic
chip. First, w/o emulsions were formed by a cross-junction microchannel. Then, the w/o emulsions
were transferred to the lipid monolayer prepared in the microchannels, facilitating the generation of
GUVs (Figure 1a). Another approach to improve the monodispersity and productivity of GUVs isa microcapillary-based centrifugal microchip [39,40]. In this approach, a water-filled glass capillary
was inserted into the oil layer of the oil-water layered microchamber. The droplets were effused fromthe capillary and transferred to the lipid monolayer by a centrifugal force. As a result, GUVs with ahigher throughput and uniformity in size were formed when compared with other droplet-transferred
methods, including microfluidic methods.
Another strategy to prepare GUVs using microfluidic technologies is by w/o/w emulsion template
methods [41,42]. In this strategy, w/o/w emulsions with lipid monolayers are initially prepared using
microfluidic channels, and the oil solvent is removed from the hydrophobic layer by the dewetting
phenomenon. As a result, a lipid bilayer forms and generates GUVs (Figure 1b). The Weitz group
first realized this strategy by using a mixture of toluene and chloroform as the organic solvent andevaporating it. The phospholipid concentration in the hydrophobic layer increases when the organic

Micromachines 2020, 11, 788

3 of 20solvents are evaporated, with a depletion force being generated due to excess phospholipid moleculesin the solvent. This induces the dewetting phenomenon. In this strategy, the method of dewetting
significantly impacts the productive efficiency of the GUVs. The Dekker group reported the use of
1-octanol as the oil phase in the w/o/w emulsion to give a quick and clean physical solvent-extraction
process [43]. On the other hand, the Huck group controlled the interfacial energies of w/o/w emulsion
systems precisely by adding surfactant in the outer water phase to provide a successful control of the
GUV formation processes, even in complex w/o/w systems including multicompartment emulsions [44].
Finally, a pulsed-jet flow method has been reported by the Takeuchi group [45,46]. In this method,
GUVs are generated by applying a pulsed-jet flow to planar lipid bilayers analogous to the formationof soap bubbles from a soap film. Because the planar lipid bilayer formed by the droplet contact
method (DCM) has a high stability [47,48], GUV generation is achievable without the rupture of thelipid bilayers. DCM is a droplet-based lipid bilayer formation method, where the lipid bilayers areformed by bringing into contact the lipid monolayers surrounding the droplets. Although in otherstrategies w/o emulsions are prepared before forming the GUVs, GUVs can be directly generated
from the lipid bilayers in this strategy. As a result, the encapsulation efficiency is higher than in otherstrategies where the formation of w/o emulsions by vortexing, pipetting, or using microfluidic channels
impacts on the encapsulation rate. In addition, by pre-forming asymmetric planar lipid bilayers andapplying a pulsed-jet flow, asymmetric GUVs can be generated [46].
Moreover, to efficiently encapsulate and reconstitute proteins into the GUVs and theliposomal membrane, a microfluidic pico-injection technology that is based on droplet pairing withelectro-coalescence has been applied to the bottom-up assembly of GUV-based artificial cells [49,50].
As stated above, numerous studies have reported on the improvement of the size-monodispersity,
encapsulation efficiency, and yield of GUVs in the past decade, and the related technologies are
maturing. In recent studies, DNA capsules [26,27] and polymersomes [30] were also proposed as
potential compartments of molecular robots. Although GUVs have conventionally been used as thecompartments of artificial cells, for molecular robots other materials may be considered that provide
comparable or superior functions to GUVs. In fact, polymersomes have a higher durability than
GUVs [51]. In addition, polymersomes can be efficiently prepared by a w/o/w emulsion template
method similar to that of GUVs (Figure 1c) [52,53]. Furthermore, pore-forming proteins can bereconstituted into polymeric membranes [54,55], with this system already in practical use as ananopore sequencer (MinION, Oxford Nanopore Technologies Ltd.). Because of these advantages ofpolymersomes, they have recently begun to be used as the compartments of artificial cell systems [56].
In the near future, it is likely that further research aimed towards the application of polymersomes andother capsule technologies to molecular robots will lead to their increased use as compartments.

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Figure 1. Schematic illustrations of giant unilamellar vesicles (GUV) and polymersome formation
Figure 1. Schematic illustrations of giant unilamellar vesicles (GUV) and polymersome formation
strategies. (a) The droplet transfer method. GUVs are formed by transferring w/o emulsions throughstrategies. (a) The droplet transfer method. GUVs are formed by transferring w/o emulsions throughthe lipid monolayer. Reproduced with permission from [38]. (b) w/o/w emulsion template method.
the lipid monolayer. Reproduced with permission from [38]. (b) w/o/w emulsion template method.
w/o/w emulsions are initially prepared by microfluidic channels. Then, GUVs are prepared by
w/o/w emulsions are initially prepared by microfluidic channels. Then, GUVs are prepared by dewetting
dewetting the oil solvent. Reproduced with permission from [41 (c) The w/o/w emulsion method canthe oil solvent. Reproduced with permission from [41] (c) The w/o/w emulsion method can also be
also be applied to the formation of polymersomes. (I) Bright-field microscope and (II) confocal
applied to the formation of polymersomes. (I) Bright-field microscope and (II) confocal microscopy
microscopy images of w/o/w emulsions. (III) The organic solvent is labeled with Nile Red. After
images of w/o/w emulsions. (III) The organic solvent is labeled with Nile Red. After evaporating of theevaporating of the organic solvent, aggregates of excess polymer either (IV) remain attached to theorganic
solvent, aggregates of excess polymer either (IV) remain attached to the polymersomes, or (V)polymersomes, or (V) occasionally detach from the polymersomes. Reproduced with permission fromoccasionallydetach from the polymersomes. Reproduced with permission from [52].
[52].

3. Functions of Liposomal Membranes
3. Functions of Liposomal Membranes
One of the advantages of applying GUVs as the compartments of molecular robots is the possibility
One of the advantages of applying GUVs as the compartments of molecular robots is theto add functions to the membrane by integrating proteins, peptides, and DNA structures. In addition,
possibility to add functions to the membrane by integrating proteins, peptides, and DNA structures.
theInsemi-permeabilityof the lipid membrane
canlipid
be exploited
to prepare
concentrated
encapsulation
addition, the semi-permeabilityof themembranecan betheexploited
to prepare
theof concentrated
chemicals orencapsulation
migration of of
GUVs.
In
this
section,we
explain
thefunctionsoflipid
membranes
chemicals or migration of GUVs. In this section, we explaintheappliedinmolecular
robotics
technologies.
functions of lipid membranes applied in molecular robotics technologies.
3.1.
Functionalization
3.1.
FunctionalizationofofLipid
LipidMembranes
MembranesThelipid membranes
membranesfor
formolecularmolecular
robotssensors,
Themainmainapplication
application ofof functionalized
functionalized lipid
robotsis is
as as
sensors,
sensing
Byintegrating
integratingthe
thelipid
lipidmembrane
membranewithsensingbeing
beingone
oneofofthe
theelemental
elemental robot
robot technologies.
technologies. By
withmembraneproteins,
peptides,synthesizedionchannels,andDNAnano-structures,themembranemembrane proteins, peptides, synthesizedchannels, and DNA nano-structures, the membrane is is
able
toto
sense
conditionsincluding
includingpH,
pH,
light
intensity,pressure,viscosity,able
sensetarget
targetmolecules
moleculesor
or external conditions
lightintensity,pressure,viscosity,
andtemperature.
lipidmembranes
membranesandandtheirtheirapplication
application
sensors.
andtemperature.Here,
Here,we
weexplain
explain functionalized
functionalized lipid
as as
sensors.

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3.1.1. Chemical Sensing
Ion channels, pore-forming proteins, and receptor proteins that are reconstituted into a cell
membrane play special roles in the transportation of ions and molecules and as the sensors of specificmolecules. Similarly for molecular robots, if these functions are embedded in the liposomal membraneat the interface between the robot interior and the exterior environment, they could act as sensors
to monitor external conditions. Synthetic ion channels and nanopore technologies have a potential
application in molecular robots for chemical detection because of their ability to transport ions andmolecules [20].
Synthetic ion channels are designed channel structures that can be inserted into the lipid bilayer
and that can control ion transportation through the channel depending on the specific molecules andvoltage, as well as other stimulations [57]. Hence, by analyzing changes in ion transportation, synthetic
ion channels function as chemical sensors. Although numerous studies about synthetic ion channels
have been performed since the 1980s by imitating and modifying motifs in nature, in this manuscript
we focus on recent studies about de novo-designed synthetic channels, including the use of peptides,organic compounds, metal–organic scaffolds, and DNA. Because synthetic ion channels were initiallydesigned by imitating pore-forming peptides, peptide-based synthetic ion channels have traditionallybeen studied by numerous groups. Recently, the DeGrado group reported on a de novo-designed
ion-selective four-helical bundle peptide that transports Zn2+ and Co2+ ions, but not Ca2+ , across lipid
membranes [58]. The Baker group also recently reported de novo-designed protein ion channels thatoffer a stable structure by designing a hydrogen bond network with membrane-exposed residues [59].
Furthermore, we recently reported a de novo-designed β-hairpin peptide that can form large nanopore
structures ranging from 1.6 to 6.2 nm, with the nanopore applied as a nanopore sensor that can detectdouble-stranded DNA (Figure 2a) [60].
The use of organic compounds also became one of the main ways to prepare synthetic ion channels
when π-stacks were established as a fundamentally new structural motif for building synthetic ionchannels. The Davis group demonstrated the transportation of Na+ ions by stacking G-quartets thatwere supported by π-stacking [61], while the Gong group successfully built an ion channel froma covalent, shape-persistent m-oligophenylethynyl (OPE) macrocycle [62]. Metal-organic channels
were introduced by the Kim group in 2008 [63]. Such ion channels were formed from Metal-Organic
Polyhedra-18 (MOP-18) that can transport protons and alkali-metal ions across lipid membranes.
Furukawa and our group also recently reported metal-organic cuboctahedra-based synthetic ionchannels that demonstrate two distinct conductance states, by embedding a single metal-organic
porous molecule with the geometry of an Archimedean cuboctahedron into an artificially reconstructed
lipid bilayer membrane (Figure 2b) [64].
Another strategy to develop synthetic ion channels utilizes DNA origami for the formation of
nanopore structures. The Simmel group first reported a synthetic ion channel constructed by DNA
origami techniques that could be inserted and fixed into the lipid bilayer (Figure 2c) [65]. In addition,
DNA nanopores of various pore sizes [66–68] that can provide sensing functions (including fortemperature [69], chemicals [70], and potential [71]) have recently been reported through the designand chemical modification of DNA sequences.
As mentioned above, various types of synthetic ion channels have been proposed, offering a
potential as sensors of liposome-based molecular robots. In particular, DNA nanopores would offer
advantages in sensing because several components of molecular robots are driven via the use of DNA
or RNA signals as the trigger, and DNA nanopores are able to sense such DNA signals. Furthermore,in addition to DNA nanopores, various lipid bilayer-assisted DNA origami nanostructures [72–75]
were also reported, and the functionalization of GUVs by DNA nanostructures would be expected.
In the future, evaluations and improvements in the stability and durability of these channels will be
required for their successful application in molecular robots as sensors.

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FigureSchematicillustrations
illustrationsofofsynthetic
syntheticion
ion channels.
channels. (a)ionchannelFigure2. 2.
Schematic(a)AAβ-hairpin-based
β-hairpin-basedsynthetic
syntheticion
channelthatforms
a β-barrelstructure
structureininaalipid
lipidbilayer.
bilayer. Cyan
Cyan lines
lines indicate
the
lipid
that
forms
a β-barrel
indicatewater
watermolecules,
molecules,and
and
the
lipid
molecules
wereomittedforclarity(excluding
phosphorus
atoms
asorangespheres).
Green
and
white
molecules were omitted for clarity (excluding phosphorus atoms as orange spheres). Green andspheres
indicate
the potassium
and chloride
ions, respectively.
Reproducedwith permission
from
white
spheres
indicate
the potassium
and chloride
ions, respectively.
Reproducedwith permission
[60].
(b)
A
metal-organic
cuboctahedra-based
syntheticion
channelthat
presents
twocurrent
states.
from [60]. (b) A metal-organic cuboctahedra-based synthetic ion channel that presents two current
Reproduced with permission from [64]. (c) A DNA origami nanopore that can be inserted into a lipid
states.
Reproduced with permission from [64]. (c) A DNA origami nanopore that can be inserted intobilayer. Red denotes a transmembrane stem; orange strands with orange ellipsoids indicate
a lipid bilayer. Red denotes a transmembrane stem; orange strands with orange ellipsoids indicatecholesterol-modified oligonucleotides. Reproduced with permission from [65].
cholesterol-modified oligonucleotides. Reproduced with permission from [65].

Nanoporetechnologies
offer
a potential
in designingchemicalsensors
for molecularNanoporetechnologies
alsoalso
offer
a potential
in designingchemicalsensors
for molecularrobotsrobots[76–80].
[76–80].
In
thistechnology,
pore-forming
transmembraneproteinsarereconstituted
into
the lipid
In this technology, pore-forming transmembrane proteins are reconstituted into the lipid bilayer,
and abilayer,andatargetmoleculepassesthroughthenanopore.
In
particular,
nanoporesensors
withtarget molecule passes through the nanopore. In particular, nanopore sensors with pore-forming proteins
pore-forming proteins such as α-hemolysin (αHL) [81] and Mycobacterium smegmatis porin A
such as α-hemolysin (αHL) [81] and Mycobacterium smegmatis porin A (MspA) [82] are commonly(MspA) [82] are commonly applied for the detection and analysis of single-stranded DNAs or RNAs.
applied for the detection and analysis of single-stranded DNAs or RNAs. On the other hand, various
On the other hand, various DNA nanotechnologies, including DNA computing [83] and DNA
DNA nanotechnologies, including DNA computing [83] and DNA origami [84], have been applied whenorigami [84], have been applied when manufacturing molecular robots. Therefore, nanopore
manufacturing molecular robots. Therefore, nanopore technologies have attracted much attention intechnologies have attracted much attention in sensing for molecular robots.
sensingNanopore
for molecularrobots.
sensingtechnologies are applied to evaluate nanopore-based chemical sensors. In thisNanoporesensingtechnologies
areobtained
applied by
to evaluate
nanopore-based
chemicalIn thismethod, chemical information
can bemeasuringthe change in thecurrentsensors.
when target
method,chemicalinformationcanbeobtainedbymeasuringthechangeinthecurrentwhen
targetmolecules translocate through the nanopores reconstituted in a planar bilayer lipid membrane
molecules translocate through the nanopores reconstituted in a planar bilayer lipid membrane (pBLM).

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Although the painting method [85] and the Montal–Mueller method [86] have been conventionallyused to prepare pBLMs, the stability and yield have been limited. In our group, we proposed nanopore
sensing platforms based on a droplet contact method (DCM) (Figure 3a) to conveniently and efficiently
perform pBLM formation and channel current measurements [48,87–92]. For example, the stabilityof the pBLM formed between two droplets was dramatically improved by utilizing a separator inwhich micro-scale holes were fabricated in a parylene film [48]. The fabrication of multiple chambers(Figure 3b) [48], a rotating system (Figure 3c) [87], and a microfluidic-based system (Figure 3d) [89]improved the throughput of the channel current recording. In recent developments, we developed
micro- and nano-electrode-based nanopore sensing platforms in which pBLMs can be formed by simply
pushing the electrode down into the layered bath solution of an oil/lipid mixture and an aqueous buffer
solution (Figure 3e) [91,92].
Because of our nanopore sensing platform having a higher membrane stability and throughput,
applications of DNA nanotechnologies to nanopore sensing were achieved. Namely, we successfully
created a logic gate [93] and detected specific microRNA [94,95] by combining DNA computing
techniques. In this logic gate, the AND logic operation was provided by amplification and transcription
from DNA to RNA using T7 RNA polymerase (T7RP). The calculation results were then detectedby nanopore sensing as the event frequency of RNA translocations (Figure 4a) [93]. For cases inwhich two input DNAs exist in the droplet with the template DNA and T7RP, defined as input (1 1),
two input DNAs form a duplex and the duplex hybridizes with the template DNA. Then, T7RP binds
to the promoter region and synthesizes a large amount of RNA as output 1. In the other cases (inputs(0 0), (0 1), and (1 0)), the input DNA cannot hybridize to the template DNA, resulting in output 0.
Furthermore, we also reported a simple detection method of multiple microRNAs (miRNAs) basedon DNA/miRNA hybridization and nanopore sensing (Figure 4b) [95]. In this method, two template
DNAs were designed to hybridize to miRNA-20a and miRNA-17-5p and construct a four-way junction.
When the four-way junction was formed by these DNAs and miRNAs, the four-way junction could nottranslocate through αHL nanopores and became stuck in the pores, resulting in a long blocking current
signal (Figure 4b). On the other hand, when both or one of the miRNAs were not present, the four-way
junction was not formed, and the dwell time of DNA or miRNA in the nanopore became much shorter
than in the case of the four-way junctions. Therefore, by analyzing the dwell times, this system can also
work as an AND gate. As shown above, a combination of nanopore technologies and DNA computing
could therefore facilitate the sensory function of molecular robots.
Micromachines 2020, 11, x FOR PEER REVIEW
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Figure 3. Cont.

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Figure 3. (a) A schematic illustration of the droplet contact method. (b) A multiple current recording
device inarearearrayed.
(a,b)
arearereproduced
withwithpermission
fromfrom[48].
in whichwhichinfinity-shaped
infinity-shapedchambers
chambers
arrayed.
(a,b)
reproduced
permission(c)high-throughput
channelcurrent
recordingdevicein inwhich[48].A(c)A high-throughput
channelcurrent
recordingdevicewhichlipid
lipidbilayers
bilayersare
areformed
formed andde-formed(d) AA microfluidic-based
microfluidic-based
de-formed byby rotatingrotating thethe chamber.
chamber. Reproduced
Reproduced with
with permission
permission fromfrom [87].
[87]. (d)multiple
lipidbilayer
bilayerformation.
formation.Lipid
Lipid
bilayers
formedby controllingthe water-oil
interfacemultiple lipid
bilayers
are areformedby controllingthe water-oil
interface
using
using
the
five-layered
microfluidic
channel.
Reproduced
withpermission
from
[90].
(e)
Lipid
the five-layered microfluidic channel. Reproduced with permission from [90]. (e) Lipid bilayer
bilayerformation
current
recordingusingusing
an Ag/AgClmicroelectrode.
The lipidThebilayerformationformation andandchannel
channelcurrent
recordingan Ag/AgClmicroelectrode.
lipidbilayercanbe controlled
manipulating
the microelectrode.
Droplets including
0, 0.1,
0.5, and 0,
1.00.1,µM0.5,
ssDNA
formation
can beby
controlled
by manipulating
the microelectrode.
Droplets
includingandwereprepared,andthechannelcurrents
were
continuously
measuredbymanipulating
theprobe.
1.0 μM ssDNA were prepared, and the channel currents were continuously measured by
Reproduced
permission
from [92].
Micromachines
2020, with
11,
x FOR
PEER
REVIEW
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manipulating
theprobe.
Reproducedwith permission from [92].

Figure 4. Cont.

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Figure 4.
4. ANDusing DNADNA computing
computing andand nanopore
nanopore technologies.
technologies. The
operation isis
FigureAND gates
gates using
The AND
AND operation
detectedasanincrease
of
(a)
the
event
frequency(reproduced
withpermission
from
[93])
and
(b)
the
detected as an increase of (a) the event frequency (reproduced with permission from [93]) and (b) the
dwell time
time (reproduced(reproduced with
with permission
permission fromfrom [94]).
[94]).
dwell

3.1.2. External Condition SensingToreleasesystemin ainGUV-based
drugdrugdelivery,external
condition-sensitive
GUVs,
To develop
developa adrug
drug
releasesystema GUV-based
delivery,external
condition-sensitive
includingto pH, light,
and light,
thermalgradients,
conventionallybeen developed
by developed
modifying the
GUVs, including
to pH,
andthermal have
gradients,
have conventionallybeen
byliposomal
membranes[96–100].
For
example,liposomes
formedbypalmitoyl
homocysteine,
which
can
modifying the liposomal membranes [96–100]. For example, liposomes formed by palmitoyl
releaseencapsulated
drugs
in areaswith a pHdrugs
lowerinthan
physiologicalreported byhomocysteine,
which canreleaseencapsulatedareasthewitha pH lower pH,
thanwere
the physiological
the
group
1980
[96]. Recently,
only
could
GUV not
rupturewith aGUV
subsequent
pH,Shinitzky
were reported
byintheShinitzkygroup innot
1980
[96].
Recently,
only could
rupturereleasewith aofdrugs be induced
pH-sensitive
GUVs
that consistedof oleic
membranefusionsubsequent
release ofby
drugs
be inducedby pH-sensitive
GUVs
thatacid:DOPE,
consisted ofbut
oleic
acid:DOPE,
but
could
as well[100].could
In addition,
the photo-sensitive
liposomes
offer a combinationof photodynamic
membranefusionas well [100].
In addition, thephoto-sensitive
liposomes offer
a combination
therapyand chemotherapy
drugs andoxygenspecies (Figure[101].
of photodynamic
therapy byandreleasing
chemotherapy
byproducing
releasing reactive
drugs andproducingreactive5a)oxygenThe
Schwille
group
recently
reversible
changes inreversible
trapped GUVs,
species(Figure5a) [101].
Thereported
SchwillepH-stimulatedgroup recently
reportedshape
pH-stimulated
shapewithout
theirwithout
free-standing
membraneschanges compromisingin trapped GUVs,
compromising
their[102].
free-standing membranes [102].
In another method, we reported nanopore
nanopore sensing-based
sensing-based viscosity
viscosity sensing
sensing [103].
[103]. In this sensing
system,canbe analyzed
by measuring
the channel
currentscurrentsof the hairpin
system, thetheviscosity
viscositycanbe analyzed
by measuring
the channelof theDNA-integrated
hairpin DNAαHL
nanopore.
In this sensor,
thechanneldistinctive
current current
signals (upper
integrated
αHL nanopore.
In thissensor,thecurrent
channelshows
currentthreeshows
three distinctive
signals
level,
level, andlowerlevel)
thatlevel)reflect
thereflect
three differentof hpDNA in(upperintermediatelevel, intermediatelevel,
and
lower
that
the threeorientations
different orientationsofthevestibuleand thephasebetween thesethreethesestatesthree
are continuously
observed.
hpDNA
in the[104],
vestibule[104],
andtransitions
the phase transitions
betweenstates are continuouslyBy
analyzingthe11,
frequency
ofREVIEW
the fluctuation,
the viscosity
be estimated5b).(Figure105b).
observed.
By
analyzing
frequencyof the fluctuation,
thecanviscosity
can be (Figure
estimated
Micromachines
2020,
x FORthe
PEER
of 20

Figure 5. Cont.

Micromachines 2020, 11, 788

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Figure 5.5. (a)(a)Schematic
Schematic
illustrationa photo-sensitive
liposome.
Near-infrared
light mediatedFigureillustrationof aofphoto-sensitive
liposome.
Near-infrared
light mediatedspecific
specific
drug
releaseandsynchronous
photodynamic
therapyandchemotherapy.
Reproducedwithdrug release and synchronous photodynamic therapy and chemotherapy. Reproduced with permission
permission
from[101].
(b)A
nanopore-based
viscosity
sensor.
Transitionsofcurrent
statesarefrom [101]. (b) A nanopore-based viscosity sensor. Transitions of current states are dependent on thedependentontheviscosity.
Reproduced
withpermissionfrom[103].
viscosity. Reproduced with permission from [103].

3.2. Use of Semi-Permeability of Lipid Membranes
Another advantage
advantage of
ofthe
thelipid
lipidmembranes
membranesin inmolecular
robotsis theirsemi-permeability.
molecular
robotsis theirsemi-permeability.
To
To
manufacture
cell-imitating
molecular
robots,itmustbepossibletoencapsulate
variousmolecules
manufacture cell-imitating molecular robots, it must be possible to encapsulate
into the GUVs
GUVs atataahighhighconcentration.
concentration.However,
However,GUVs
witha concentrated
inner
solution
could
GUVs
with
a concentrated
inner
solution
could
not
not
be
preparedbyconventional
GUV
formation
methods,
asshown
above.
Hence,
Fujiwara
andbe prepared by conventional GUV formation methods, as shown above. Hence, Fujiwara
Yanagisawa have
have proposed
proposed aa method
method toto concentrateconcentrate thethe inner
inner solution
solution using
using hypertonic
hypertonic conditions
conditions(Figuremethodwere
immersedin(Figure 6a)6a)[105].
[105]. In
In thisthismethod,
method,thetheGUVs
GUVspreparedpreparedby
bythe
thedroplet-transfer
droplet-transfer
methodwere
immersedtheoutersolutionwithahigherosmoticpressure
than
the
inner
solution.
Asaresult,theconcentrationsin the outer solution with a higher osmotic pressure than the inner solution. As a result, theofmacromoleculesin the GUVs were
thecontrolled
ratio of thebyosmoticpressure
innerconcentrationsof macromolecules
in controlled
the GUVs by
were
the ratioof the between
osmotic thepressureandouterthesolutions.
Furthermore,Anges–Koback
and Keating
have reported
a complete
budding
anda
betweeninner andouter solutions.
Furthermore,Anges–Koback
and Keating
havereported
asymmetricfusionofGUVs
toproducedaughtervesicles[106].
GUVs
encapsulatingapolyethylene
complete budding and asymmetric fusion of GUVs to produce daughter vesicles [106]. GUVs
glycol/dextran
prepared,glycol/dextran
and budding wasby andincreasing
thewasosmoticpressure.
encapsulating awere
polyethylene
wereinduced
prepared,budding
inducedby increasingIn
addition,thesemi-permeability
of
GUVs
was
exploitedinthemigrationof
GUVs.
We reported
the osmotic pressure.
a GUV
inducedby an osmoticofpressure
difference(Figure6b) migration[107]. We imitated
In movement
addition, thesemi-permeability
GUVs wasexploitedin theof GUVs.a cellWe
migrationinducedby osmoticpressuredifference,
which (Figure
is called6b)the[107].
“osmotic
enginereported a mechanism
GUV movement
inducedby an osmoticpressuredifference
We imitatedmodel”[108], andmechanism
applied thisinduced
mechanism
to GUVs.
In this model,
a spatial
gradientof theion “osmotic
channelsa cell migration
by osmoticpressuredifference,
which
is calledforms
ion concentrationdifference,
resultsto
in GUVs.
a net inflow
ofmodel,water atleadingedge ofandaengineanmodel”[108], and applied
thiswhichmechanism
In thisa thespatialgradiention
netoutflowof water
the trailing edge.
The GUVs
trappedtightlyinsidethe microchannel,
channels
forms
an ionatconcentrationdifference,
whichwere
results
in a net
inflowof waterat the leading
andsolutionwith different
salt concentrations
to thefrontwereand rearof thetightly
GUV. Asa result,
edgetheanda net outflow
of waterat the trailing flowed
edge. The
GUVs
trappedinsidethethewater passed
the lipid
froma lower to a flowed
higher salt
concentration,
andmicrochannel,
andthroughthe solution
withmembranedifferent salt
concentrations
to thefront and rearof theGUV moved in the direction of the lower salt concentration, owing to the osmotic pressure difference.

Micromachines 2020, 11, x FOR PEER REVIEW

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GUV. As a result, the water passed through the lipid membrane from a lower to a higher salt
concentration,
the GUV moved in the direction of the lower salt concentration, owing 11
to oftheMicromachines
2020,and11, 78820
osmotic pressure difference.

Figure 6. Applications of osmotic pressure to liposome-based molecular robots. (a) Control of the
Figure 6. Applications of osmotic pressure to liposome-based molecular robots. (a) Control ofconcentration of macromolecules in GUVs by an additive-free cell extract (AFCE). Reproduced withthe concentration of macromolecules in GUVs by an additive-free cell extract (AFCE). Reproduced
permission
fromfrom[105].
(b) (b)Osmotic-engine-driven
GUVs
with
with
permission[105].
Osmotic-engine-driven
GUVsininmicrochannels.
microchannels. Reproduced
Reproduced withpermission
from[107].
permission from [107].

4. Encapsulation in Giant Unilamellar Vesicles
Vesiclesthethelipid
membraneallowstheEncapsulation isis also
also aaspecificspecificcharacteristic
characteristicofofGUVs.
GUVs.Because
Becauselipid
membraneallowspermeationof waterandnon-polarized
small
the
permeationof waterandnon-polarized
smallmolecules,
molecules,butbutnotnot macromolecules
macromolecules oror polarizedmolecules, various
various molecules
molecules cancan bebe encapsulated
encapsulated into
into thethe GUVs
GUVs by adding them to the inner solution
molecules,
during GUV generation. In molecular robots, the encapsulated materials in the GUVs may be eitherof movement
movement andand intelligence.
intelligence.
drugs or materials that facilitate the functions of
4.1.
Actuators ofof Molecular
Molecular Robots
Robots
4.1. Actuators
One
To achieve
One ofof thethe mainmain functions
functions ofof thethe encapsulated
encapsulated materials
materials isis actuation.
actuation. To
achieve thethe concept
concept
accordingaccording toto whichwhich molecularmolecular robots
robots act
act asas microorganism-imitating
microorganism-imitating microrobots,
microrobots, thethe encapsulation
encapsulationof
The Hotani
Hotani groupof molecularmolecular motors
motors such
such asas kinesinkinesin andand myosin
myosin was
was performed.
performed. The
group reported
reportedmorphologicalchangesofG-actinencapsulated
GUVs
byincreasingthetemperature,withtheshapesmorphological changes of G-actin encapsulated GUVs by increasing the temperature, with the shapesofon theof GUVs
GUVs being
being dependent
dependent on
the type
type ofof actin-crosslinking
actin-crosslinking proteinsproteins due
due toto thethe organization
organization ofof theirtheirspecific
actin
networks[109,110].
The
Hayashigroup
alsoreportedthetransformationof
GUVs
inducedspecific actin networks [109,110]. The Hayashi group also reported the transformation of GUVs
bythe encapsulated
actin (Figure[111,112].
They successfully
deformed the
GUV byincreasing
inducedby the encapsulated
actin7a)(Figure7a) [111,112].
They successfully
deformedthe
GUV bytheconcentrationof the encapsulated
actin to a levelto that of tothethatcytoplasmof livingincreasing
the concentrationof the encapsulated
actin comparable
to a level comparableof the cytoplasm
cells.
In addition,
reversiblereversible
deformationsof GUVs ofbetweensphereshapeswereobtainedof living
cells. In addition,
deformations
GUVs spindle
betweenandspindleandsphereshapeswerebyadjustingthe osmotic
or by lightirradiationof fluorescent-labeledactin. Inactin.
a recent
obtainedby adjusting
the pressure
osmotic pressure
or bylight irradiationof fluorescent-labeled
In adevelopment,a controllable
and programmableamoeba-likemolecular
robot wasreportedby the Satorecent development,a controllable
and programmableamoeba-likemolecular
robot
was reportedbygroup(Figure7b)[113].
In
this
robot,
microtubes,
DNA-modified
kinesins,
anddouble-stranded
DNAs
the Sato group (Figure 7b) [113]. In this robot, microtubes, DNA-modified kinesins, and doubleare encapsulated into the GUVs, and linker DNAs with three cholesterol-modified DNAs are insertedmembrane-connected kinesins, and the shape of the membrane can be continuously changed.
Although it is still challenging to achieve a motility of molecular robots analogous to living
cells—such as migration and chemotaxis—because it is difficult to complexly align the proteins in the
GUVs like living cells, the switching techniques of activation and in-activation offer a potential
control of actuation
Micromachines
2020, 11, 788in molecular robots. As the next step, the conversion of the force generated
12 ofby20
molecular motors into the movement of GUVs must be researched, and an efficient mechanism forthis must be developed. Regarding other strategies, self-propelled techniques using bubbles, theinto the inner liposomal membrane as anchors. When connector DNAs exist in the GUVs, the kinesins
Marangoni effect, interfacial tension, and nanofiber growth have also been reported [114,115]. To
and the anchors are engaged. As a result, the microtubes can glide via the membrane-connected
improve the mobility of molecular robots, we propose that a combination of encapsulated molecularkinesins, and the shape of the membrane can be continuously changed.
motors and other technologies, such as self-propelled mechanisms, would be most efficient.

Figure 7.
7. Emergence
Emergence ofof GUV
GUV actuation
actuation by
by encapsulated
encapsulated molecules.
molecules. (a)(a) Reversible
Reversible deformations
deformations ofof
FigureGUVs
betweenspindleandsphereshapesbylightirradiationoffluorescent-labeledactin.
GUVs between spindle and sphere shapes by light irradiation of fluorescent-labeled actin. Reproduced
Reproduced
withfrom
permission
from[112]. (b) andControllable
and programmable
deformationof GUVs.
withpermission[112]. (b)Controllable
programmable
deformation of
GUVs. Reproduced
Reproduced
withfrom
permission
withpermission[113]. from [113].

Although it is still challenging to achieve a motility of molecular robots analogous to living
cells—such as migration and chemotaxis—because it is difficult to complexly align the proteins in the
GUVs like living cells, the switching techniques of activation and in-activation offer a potential control
of actuation in molecular robots. As the next step, the conversion of the force generated by molecularmotors into the movement of GUVs must be researched, and an efficient mechanism for this must
be developed. Regarding other strategies, self-propelled techniques using bubbles, the Marangoni
effect, interfacial tension, and nanofiber growth have also been reported [114,115]. To improve theMicromachines 2020, 11, 788

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mobility of molecular robots, we propose that a combination of encapsulated molecular motors andother technologies, such as self-propelled mechanisms, would be most efficient.
4.2. Intelligence of Molecular Robots
In robots, intelligence plays a significant role as the interface between sensors and actuators.
Once sensors installed in the membrane sense external signals, robots must determine a response.
In the field of molecular robotics, DNA computing technologies [83,116] and DNA-based calculation
systems, including isothermal cascade reactions of DNAs [117], are proposed as intelligence systems.
DNA computing is an area of natural computing first reported by Adleman in 1994 [83], based on
the principle that polynucleotides contain information encoded in the amino acid sequence, with thisinformation being capable of being transferred and/or copied using chemical and enzymatic reactions.
DNA computing was initially proposed as a parallel processing method that could calculate fasterthan conventional silicon-based computers. However, because of issues with low accuracy anda time-consuming data output process, the application of DNA computing is shifting towards
computation that can work in biological conditions. In fact, DNA computing-based calculation
methods in living cells have been reported (Figure 8a) [118,119]. To perform DNA computing andobtain the calculated result in molecular robots, the isothermal amplification of DNAs is needed.
The Komiya group has reported an isothermal cascade reaction that can exponentially amplify
pre-designed single-stranded DNA, ultimately aiming for the development of DNA-based computing
systems that can drive molecular robots [120]. Furthermore, the Sato group successfully performed theisothermal amplification of specific single-stranded DNAs in GUVs (Figure 8b) [121].
The emergence of intelligence in molecular robots still faces challenges because intelligence does
not emerge until it connects sensors and actuators and works as a controller. Clarification is required
regarding what signals will work for the input and output of molecular robots and for the subsequent
design of appropriate intelligence systems.
Micromachines 2020, 11, x FOR PEER REVIEW

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Figure 8. Cont.

Micromachines 2020, 11, 788

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Figure8.8. (a)(a) AADNA-based
DNA-basedcalculation
calculationnanodevice
nanodevicethat
thatcancanactactininliving
livingcells.
cells.Reproduced
Reproducedwith
with
Figurepermissionfrom[108].
(b)An
isothermal
amplification
circuitforspecificDNAmolecules
that
permission from [108]. (b) An isothermal amplification circuit for specific DNA molecules that isis
implementedininGUVs.
GUVs.Reproduced
Reproducedwith
withpermission
permissionfrom
from[110].
[110].
implemented

5.5.Conclusions
Conclusionsand
andFuture
FutureOutlook
OutlookIn
Inthisthisreview,
review,we
wefocusedfocusedon
onrecent
recentadvancesadvancesin
inliposome-based
liposome-basedmolecularmolecularrobots.
robots.We
Wedescribed
described
microfluidic
methodsofGUV
generation
andtheemergenceof
sensors,
actuators,
andmicrofluidic methods of GUV generation and the emergence of sensors, actuators, andintelligence
intelligencesystemsfunctionalization ofofliposomal
liposomalmembranes
membranesand
andencapsulationof chemicals
systems via
via thethe functionalization
thetheencapsulationof chemicals
into
into
GUVs.
To
date,
the
efficient
generation
of
GUVs
with
a
size-monodispersity
andchemical
GUVs. To date, the efficient generation of GUVs with a size-monodispersity and chemical
encapsulation
encapsulationability
abilityhas
hasbeenbeenachievedachievedby
byusingusingmicrofluidic
microfluidictechniques.
techniques.In
Inaddition,
addition,DNA
DNAcapsules
capsulesandpolymersomesofferapotentialasthecompartmentsofmolecular
robots.
Regardingthesensors,
and polymersomes offer a potential as the compartments of molecular robots. Regarding the sensors,
synthetic ion channels and nanopore sensing technologies are useful for sensing external chemicals.
In particular, recent developments of de novo-designed peptide, protein, and DNA channels can provide
stable structures with good size controllability, and these may attract attention for use as sensors ofmolecular robots. Nanopore sensing has recently offered specific chemical detection by combining
DNA computing techniques, and this system has also provided both sensory and intelligence functions.
Furthermore, viscosity measurements have been achieved by using hairpin DNA-integrated αHLnanopores. Regarding the actuators, encapsulated molecular motors can facilitate the deformation of
GUVs, with the actuation being controlled by specific stimulations. In the future, we will look towards
a mechanism in which the actuation of molecular motors will be efficiently converted into migratory
motion. It is proposed that the intelligent control of molecular robots can be achieved with DNA
computing technologies and the isothermal amplification of DNA. Because intelligence plays a role as
the interface between sensors and actuators, an intelligence system should be designed and optimized
for applications in molecular robots.
Finally, molecular robots are microorganism-imitating robots aimed towards a practical usage
in the fields of medicine, drug discovery, environmental science, food science, and energy sciencein a manner distinct from artificial cells. However, it is still challenging to integrate all the robotics

Micromachines 2020, 11, 788

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systems into a single compartment because it is necessary to design and construct each of the functions,
including sensors, actuators, and intelligence, so that they do not interfere with each other and can act
in a single GUV. In the present state, molecular robots have not shown a clear distinction from artificial
cells. As a field, we should aim to develop robotic systems that can provide superior functions andabilities to cells by combining various technologies that encompass not only synthetic biology but alsomechanical, electrical, chemical, and information engineering. We believe that the establishment ofmolecular robotics methods would offer a game-changing technology in these various fields.
Author Contributions: K.S. and R.K. wrote the manuscript and have read and agreed to publish this version ofmanuscript. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by KAKENHI (Grant No. 19K15418, 19H00901, and 15H00803) from the
MEXT Japan and the Leading Initiative for Excellent Young Researchers (LEADER) from the Japan Society for the
Promotion of Science.
Conflicts of Interest: The authors declare no conflict of interest.

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