review
Drug Delivery
Nanocapsules for Programmed Neurotransmitter Release:
Toward Artificial Extracellular Synaptic Vesicles
Keitaro Sou,* Duc Long Le, and Hirotaka Sato
Nanocapsules present a promising platform for delivering chemicals and
biomolecules to a site of action in a living organism. Because the biological
action of the encapsulated molecules is blocked until they are released from
the nanocapsules, the encapsulation structure enables triggering of the
topical and timely action of the molecules at the target site. A similar mecha-
nism seems promising for the spatiotemporal control of signal transduction
triggered by the release of signal molecules in neuronal, metabolic, and
immune systems. From this perspective, nanocapsules can be regarded as
practical tools to apply signal molecules such as neurotransmitters to inter-
vene in signal transduction. However, spatiotemporal control of the payload
release from nanocapsules persists as a key technical issue. Stimulus-
responsive nanocapsules that release payloads in response to external input
of physical stimuli are promising platforms to enable programmed payload
release. These programmable nanocapsules encapsulating neurotransmit-
ters are expected to lead to new insights and perspectives related to artificial
extracellular synaptic vesicles that might provide an experimental and thera-
peutic strategy for neuromodulation and nervous system disorders.
1. Introduction
Molecules such as neurotransmitters, cytokines, lipids, pep-
tides, proteins, RNAs, nitric oxide, and carbon monoxide play
important roles in communication between cells through
signal transduction in living organisms.[1–5] These signal mole­
cules are fundamentally reserved in intracellular vesicles and
are released to extracellular space as vesicles (exosomes) or
molecules (signal molecules) as necessary. Once the target cells
sense the vesicles or signal molecules, the signal is introduced
into intracellular space to induce biological events through a
signal transduction pathway. Such signal transduction using
exosomes and signal molecules enables remote communica-
tion between cells.
Dr. K. Sou
Research Institute for Science and Engineering
Waseda University
3-4-1 Ohkubo, Shinjuku, Tokyo 169-8555, Japan
E-mail: soukei@aoni.waseda.jp
D. L. Le, Prof. H. Sato
School of Mechanical and Aerospace Engineering
Nanyang Technological University
50 Nanyang Avenue, Singapore 639798, Singapore
The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/10.1002/smll.201900132.
DOI: 10.1002/smll.201900132
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The human nervous system has a
complicated signal transduction network
in which excitatory or inhibitory signal
molecules called neurotransmitters are
essential for nerve excitation and inhibi-
tion.[1,6,7] Based on evidence that injec-
tion of external excitatory and inhibitory
neurotransmitter molecule solutions acts
transiently on the neuronal system,[8,9]
the development of neurotransmitter-
delivery systems is key to the practical
application of the neurotransmitter mole­
cules. Organic electronic devices have
been developed to support such delivery
systems.[10–12] Such electronic devices
capable of releasing neurotransmitters
are attractive to modify signal transduc-
tion in the nervous system experimentally
and therapeutically. However, the input
of an electrical signal might present diffi-
culties in the nervous system because the
electronic signal influences the neuronal
function.[13,14] Moreover, the insertion and
implantation of invasive devices in neuronal tissues involve a
high risk of damaging the nervous system mechanically. Cur-
rent strategies for clinical neurostimulation are limited either
by high invasiveness, low precision, or poor depth of penetra-
tion. Consequently, further technical efforts are being aimed at
the development of non-invasive systems that enable precise
neurotransmitter release in deep tissues. Nanoparticles are
anticipated for use as potent tools to achieve this goal.[15,16]
From a broader perspective, a drug-delivery system using
nanoparticles can be expected to provide useful knowledge
for the development of neurotransmitter-delivery systems. For
drug-delivery applications, lipid vesicles called liposomes are
the most popular nanocapsules.[17–19] Since liposomes were first
reported in the 1960s, there has been a considerable number
of research featuring liposomes as a drug-delivery method.
The main target has been the delivery of conventional anti-
cancer drugs safely and effectively to tumors. For this purpose,
poly(ethylene glycol)-modified liposomes encapsulating doxo-
rubicin (DOXIL) have been approved for clinical application.[20]
Furthermore, liposomes have been anticipated for use as bio-
compatible and biodegradable capsules to encapsulate proteins,
nucleic acids, and biomolecules for biomedical applications,
such as artificial blood cells and gene delivery.[21–25]
Recently, we reported for the first time our demonstration
of neurotransmitter-loaded thermosensitive liposomes trig-
gering on-demand muscle relaxation in a living organism.[26]
The neurotransmitter-loaded liposomes, which have structures
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resembling synaptic vesicles encapsulating a neurotransmitter

in a lipid bilayer membrane, can be regarded as artificial extra- Keitaro Sou is an adjunct
cellular synaptic vesicles that enable modification of the signal associate professor at Waseda
transduction by replacing the function of synaptic vesicles. As University. He received his
described herein, we specifically examine the nanocapsules for Ph.D. in chemistry from
programmed neurotransmitter releases for application as artifi- Waseda University in 2000.
cial extracellular synaptic vesicles. He was research associate,
assistant professor, and
associate professor at ARISE
(1999–2009), and associate
2. Neurotransmitters and Synaptic Vesicles
professor at WABIOS
Neurons communicate information via a structure called a syn- (2014–2017), Waseda
apse. Actually, “excitatory synapses” excite neurons receiving University, and a visiting
signal and “suppressive synapses” suppress the excitement. The researcher at University of Texas Health Science Center
signal transduction is triggered by excitatory or inhibitory signal at San Antonio (2001, 2003). His research focuses on the
molecules called neurotransmitters. Numerous neurotransmit- development of self-assembled functional materials for
ters have been identified,[27] a major of which are typically water- bioscience and biomedical applications.
soluble molecules having a dissociable group, as presented in
Table  1. These molecules act as excitatory or inhibitory neuro- Duc Long Le is a Ph.D. student
transmitters to neurons. They are secreted mainly from neurons at the School of Mechanical
in the central nervous system (CNS), consisting of the brain and and Aerospace Engineering,
spinal cord, the peripheral nervous system (PNS) consisting of Nanyang Technological
nerves and ganglia outside of the brain and spinal cord in bilat- University. He received his
eral animals, and the invertebrate neuromuscular junction. B.E. degree at the School of
Acetylcholine, the first neurotransmitter discovered, was Mechanical and Aerospace
identified in 1914. Biogenic amines such as norepineph- Engineering from Nanyang
rine, dopamine, and serotonin, and amino acids such as Technological University in
γ-aminobutyric acid (GABA), glycine, glutamate (glutamic acid), 2017. His current research
and aspartate (aspartic acid) are known neurotransmitters. Pep- focuses on thermosensitive
tides such as substance P, met-enkephalin (endorphin), and nanovesicles for drug delivery
oxytocin are known neuropeptides. in cyborg insects.
Generally, the neurotransmitter molecules are decomposed
rapidly by enzymes in biological environments. For example, Hirotaka Sato is an asso-
acetylcholine released in biological fluids is decomposed rap- ciate professor at Nanyang
idly by acetylcholine esterase.[28] It is noteworthy that, by some Technological University. He
process, the neurons can store such biodegradable neurotrans- received his Ph.D. in chem-
mitters stably. Actually, the neurotransmitters are encapsu- istry from Waseda University
lated within a synaptic vesicle that is an organelle with a lipid in 2005. He was a research
bilayer membrane (Figure  1A). The neurotransmitters are associate at Waseda University
released as necessary to the extracellular space between axon (2005–2006), postdoctoral
terminal and dendrite (20–40 nm synaptic cleft) from the syn- fellow at the University of
aptic vesicles. The release of the neurotransmitters is induced Michigan (2007) and at the
by fusion of synaptic vesicles with neuron cell membrane (exo- University of California at
cytosis).[29] The fusion of synaptic vesicles with a neuronal cell Berkeley (2008–2011),
membrane is triggered in a Ca2+-dependent manner where the and assistant professor at Nanyang Technological
voltage-dependent calcium channel on the neuronal membrane University (2011–2017). His research focuses on MEMS,
controls the influx of Ca2+ into the axon terminal.[30] The neu- insect–machine hybrid system, and electrochemistry.
rotransmitters released to the synaptic cleft bind with receptors
on the dendrite to induce excitation or inhibition of neuronal
systems. The neurotransmitter action is transient because of
its short biological half-life. Consequently, the encapsulation 3. Brain Delivery Using Stimuli-Responsive
structure of neurotransmitters is regarded as a sophisticated Nanoparticles
strategy to escape from biological decomposition in biological
environments and to induce the transient excitation or inhibi- Brain is one of the major target organs for drug delivery using
tion to neurons as necessary. For understanding of this bio- nanoparticles.[16,31] The blood capillary provides an access route
logical mechanism, neurotransmitter-loaded nanocapsule is a to brain for endogenous and drug-delivery molecules. However,
reasonable structure as an artificial synaptic vesicle to deliver the brain capillary has a tight junction between endothelial cells
neurotransmitters stably to extracellular space around a syn- called blood–brain barrier (BBB) creating a restrictive barrier
apse (Figure 1B). between CNS and blood circulation. Consequently, an issue

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Table 1.  Major neurotransmitters.

Neurotransmitter Structure Functions Secretion sites

Acetylcholine O Excitatory to vertebrate skeletal muscles; CNS; PNS; invertebrate neuromuscular
CH3 excitatory or inhibitory at other sites junction
N
H3C O CH3
CH3
Biogenic amines
Norepinephrine HO Excitatory or inhibitory CNS; PNS

NH2
HO
OH
Dopamine HO Generally excitatory CNS; PNS

NH2
HO

Serotonin HO Generally inhibitory CNS

NH2

N
H
Histamine HN Generally excitatory CNS

N NH2
Amino acids
γ-Aminobutyric acid (GABA) Inhibitory CNS; invertebrate neuromuscular junction
H2N COOH
Glycine Inhibitory CNS
H2N COOH
Glutamate, glutamic acid COOH Excitatory CNS; invertebrate neuromuscular junction

H2 N COOH
Aspartate, aspartic acid COOH Excitatory CNS

COOH
H2N
Neuropeptides
Substance P Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met Excitatory CNS; PNS
Met-enkephalin (endorphin) Tyr-Gly-Gly-Phe-Met Generally inhibitory CNS
Oxytocin Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly Generally inhibitory CNS

to be verified is whether the nanoparticles can cross the BBB.
In fact, nanoparticles reach CNS during systemic circulation
through intracellular and/or intercellular pathways (Figure 2A).
The BBB plays a role to transport endogenous nanoparticles
into CNS from blood circulation. Lipoproteins (i.e., low-density
lipoprotein, LDL), endogenous nanoparticles for the systemic
delivery of lipids to cells, could cross the BBB via receptor-
mediated transcytosis through intracellular pathway.[32,33] The
receptor-mediated transcytosis mechanism provides a feasible
strategy for active delivery of drug-delivery nanoparticles across
the BBB. To this end, surface-modified nanoparticles with
affinity ligand molecules to the overexpressing receptors (i.e.,
LDL receptor,[34,35] transferrin receptor[36,37]) on endothelial cells
composing BBB facilitate the nanoparticle delivery into CNS
across the BBB. Another intracellular route is adsorptive tran-
scytosis. Positively charged substances adsorb on the negatively
charged endothelial cell surface in an electrostatic manner. In
adsorptive transcytosis pathway, the endothelial cells uptake the
adsorbed cationic substances by endocytosis and release them
into the brain interstitial fluid by exocytosis. Polycationic pro-
teins and cationic peptides have been investigated to develop
drug-delivery systems based on adsorptive transcytosis.[38]

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Figure 1.  Signal transduction at synapse mediated by neurotransmitter molecules released from A) native synaptic vesicles and B) neurotransmitter-
loaded nanocapsules (artificial extracellular synaptic vesicles).
Nanoparticles modified with cationic proteins, cationic pep-
tides, cationic polymers, and cationic amino acids are possible
nanovectors to deliver drugs and genes to brain cells in CNS
across the BBB.[39,40] Some small molecules such as lipophilic
molecules, amino acids, and glucose are known to cross the
BBB by diffusion or transport protein pathway. These specific
molecules which cross the BBB would be delivered into CNS
by release from circulating nanoparticles in the brain capillary.
For passive delivery, the circulation time of nanoparticles
is an important factor to access the CNS. Generally, optimal
nanoparticle size for prolonged circulation is in the range less
than 200 nm. For brain delivery across the BBB, nanoparticles
ranging 20–100 nm are used for prolonged circulation.[41,42] It
is recognized that passive nanoparticle delivery through tight
junctions between endothelial cells is still a challenge in normal
brain. Exceptionally, the capillaries in circumventricular organs
such as pineal body have fenestrations which allow high perme-
ability unlike the BBB. Electron microscopic studies have dem-
onstrated the presence of transendothelial pores (30–80 nm in
diameter) in the circumventricular organs.[43–45] These fenes-
trated capillaries in the circumventricular organs are remark-
able pathways to access CNS by passive delivery. In addition,
the evidence that brain injury such as cerebral ischemia and
brain trauma, and neurodegenerative diseases, including Par-
kinson’s disease, Alzheimer disease, and epilepsy, cause leaky
compromised BBB due to disruption of tight junction may
open passive delivery pathway to the site of action.[16,31]
After delivering the neurotransmitters using nan­ocapsules
into the nervous system, such as brain, a key technical issue
is how to control the release of neurotransmitters, precisely
and on demand, from the nanocapsules at a region of interest.
A salient consideration in the release of signal mole­ cules
including neurotransmitters in living cells is that these signal
molecules are released from the cells as necessary in response
to biological and environmental stimuli. The released mole­
cules therefore act as signal molecules to trigger biological
events through a signal transduction pathway. Such a concept
of release and subsequent action with the system has been
introduced into the development of drug-delivery systems.
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Nanocapsules of various types such as liposomes, micelles,
polymeric nanoparticles, silica nanoparticles, and magnetic
nanoparticles have been developed for drug-delivery applica-
tions.[46–48] Particularly, stimulus-responsive nanocapsules
which release their cargos in response to external physical
and chemical stimuli are promising platforms to enable on-
demand release of encapsulated drugs from the nanocapsules
(Figure 2B).[49–51]
Stimuli-responsive nanoparticles to endogenous stimuli such
as pH, redox, and hypoxia have been studied for drug delivery
and stimuli-responsive drug release for cancer therapy.[48,52–54]
Since these endogenous stimuli systems depend on physiolog-
ical conditions, precise spatiotemporal control of the payload
release would be difficult. On the other hand, exogenous stimuli
such as heat, light, ultrasound, and magnetic field enable pro-
grammed payload release from stimuli-responsive nanoparticles,
as these external signals can be applied with controlled time,
intensity, and region in an intervening manner. Among the exog-
enous stimuli, heat is an attractive option thanks to its versatility
and minimal invasion to biological tissues. Thermosensitive
liposomes are therefore promising biocompatible platforms that
can be programmed for release of cargo by heat stimulus.[55–57]
4. Thermosensitive Liposomes
Nanocapsules with lipid bilayer membranes enable liposome
encapsulation of neurotransmitters. The neurotransmitter-
loaded liposomes are simply prepared by the hydration of
lipids in an aqueous neurotransmitter solution.[26] The size of
liposomes is controllable to be 50–200 nm by using conventional
techniques such as an extrusion method by passing through a
membrane filter with nanopores.[58] Surface modification of
liposomes with poly(ethylene glycol) chain achieves prolonged
circulation time for passive delivery.[18–20] Also, the conjugation
chemistry to attach the specific ligand molecules onto liposome
surfaces for active delivery has been well established so far.[59,60]
Liposomes are formed by self-assembly through non-covalent
intramolecular interaction between lipid molecules in aqueous
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Figure 2.  Brain delivery with stimulus-responsive nanoparticles. A) Possible routes for nanoparticle brain delivery across the blood–brain barrier (BBB).
Normal brain capillary has a tight junction between the endothelial cells called BBB, creating a restrictive barrier between central nervous system
(CNS) and blood circulation. Nanoparticles cross the BBB through intracellular pathway as receptor-mediated transcytosis or adsorptive transcytosis.
Small molecules such as lipophilic molecules, amino acids, and glucose released from nanoparticles in the brain capillary can cross the BBB through
diffusion or transport protein pathway. Brain injury and neurodegenerative diseases possibly cause leaky capillaries by BBB dysfunction (compromised
BBB). Circumventricular organs have fenestrated capillaries with transendothelial pores (30–80 nm in diameter).[43–45] These leaky capillaries provide
intercellular route for passive delivery of nanoparticles and drug-delivery molecules into CNS. B) On-demand payload release from stimulus-responsive
nanoparticles responding to external input. The release of the payload triggers the biological actions of the payload.

phase. Therefore, the strategy to control the payload release
from liposomes is based on the regulation of the molecular
assembly state of the lipid bilayer membrane. Liposomes of
which the molecular assembling state is responsive to heat
stimulus are typically called temperature-sensitive, tempera-
ture-responsive, or thermosensitive liposomes.[55–57] A typical
bilayer membrane composed of saturated diacyl phospho-
cholines show a gel to liquid-crystalline phase transition. The
temperature triggering the phase transition is called the gel to
liquid-crystalline phase transition temperature (Tm). Figure 3 A
shows that Tm is correlated linearly with the reciprocal of hydro-
carbon chain length in terms of the number of carbon atoms
(n) in a range of n = 9–24 (R2 = 0.999).[61] Tm is approximated by
the following Equation (1):
Tm = − 1948.9 (1/n ) + 162.8 (1)
The thermotropic phase transition of the lipid bilayer mem-
brane is a dynamic phenomenon accompanying the marked
change in molecular assembling state. At Tm, the conformation
of carboncarbon bonds in hydrocarbon chains of the lipid
molecule changes from a trans form to a gauche form, dras-
tically altering the molecular packing state of the lipid mole­
cule.[62,63] It is noteworthy that the membrane permeability of
the lipid bilayer membrane is increased at Tm by the formation
of a grain boundary defect between the solid and liquid phases
(Figure 3B).[55] As the formation of the grain boundary defect
in lipid bilayer membrane is facilitated by mixing lipids with
different Tm, liposomes composed of 1,2-dipalmitoyl-sn-glycero-
3-phosphocholine (DPPC, Tm  = 41 °C) and 1,2-distearoyl-sn-
glycero-3-phosphocholine (DSPC, Tm  = 55 °C) have been
investigated in an early study.[64] Although the drug release is
enhanced, it is generally understood that the release efficiency

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Figure 3.  Temperature-dependent payload release from thermosensitive liposomes with different gel to liquid-crystalline phase transition temperatures
(Tm). A) Correlation of the main phase transition temperature of bilayer membrane composed of fully hydrated, saturated diacyl phosphocholine and
the reciprocal of hydrocarbon chain length in the number of carbon atoms, n. The inset portrays the chemical structure of the saturated diacyl phospho-
cholines. The presented data were collected from published data.[61] B) Change of membrane permeability on thermosensitive liposomes at Tm. Mixed
state of solid and liquid phases at around Tm leads to the grain boundary defect in the bilayer membrane that permits payload release. C) Schematics
showing payload release from thermosensitive liposomes by heat stimulus. Encapsulation of hypertonic solution accelerates the payload release. D)
Representative profiles of payload release from thermosensitive liposomes composed by saturated diacyl phosphocholine with different hydrocarbon
chain length by heat stimulus. Adapted with permission.[68] Copyright 2016, American Chemical Society.

is not high. This would be caused by the instability of the grain
boundary defect. Later, Needham et al. found that the incor-
poration of lysolecithin, monoacyl phospholipid, enhances the
payload release by stabilizing the defect formed by phase transi-
tion.[65] By mixing 10 mol% of 1-stearoyl-2-hydroxy-sn-glycero-
3-phosphocholine (MSPC) to liposomes composed of DPPC and
1,2-distealoyl-sn-glycero-3-phosphoethnolamine with covalently
attached poly(ethylene glycol) (PEG-DSPE), the permeability of
doxorubicin through the membrane is increased tenfold com-
pared with the liposomes without MSPC.[66] The mechanism is
that the MSPC accumulates in the grain boundary defect and
forms stable nanopore (≈10 nm).[55,67] Although the efficiency
of payload release from thermosensitive liposomes without
grain boundary defect stabilizer by heating above Tm is not so
high, increasing the osmotic pressure of the inner aqueous
phase of liposomes markedly enhances the payload release
from liposomes (Figure 3C). Thermosensitive liposomes encap-
sulating hypertonic solution (550 mOsm L−1) rapidly release the
cargo above the phase transition temperature in physiological
osmotic pressure (300 mOsm L−1).[57] In our recent investiga-
tion, approximately 45% of GABA was released from DPPC
liposomes encapsulating hypertonic 550 mm GABA solution
for the first 10 s during incubation at 45 °C (4.5% s−1).[26] Sub-
sequently, there was a minor release of GABA for 5 min at
45 °C (approximately 1.6% min−1). The disordered liposome
membrane above Tm allowed water to flow into the liposomes
to compensate for the osmotic pressure gap between the inside
and the outside of the liposomes, which rapidly pushed the
encapsulated GABA out. When the osmotic pressure gap is
cancelled by releasing the GABA, the release rate slows down to
a great extent. The threshold temperature that triggers the pay-
load release corresponding to Tm of the phosphocholines is pro-
grammable as a factor of the hydrocarbon chain length of phos-
phocholines (Figure 3D).[68] Mixtures of lipids with different Tm
can be regarded as varying Tm at a narrower temperature scale
depending on the mixing ratio of the lipids.[69] It is desirable
for the thermosensitive liposomes used for therapeutic applica-
tions to retain the neurotransmitters in liposomes at tempera-
tures lower than the human body temperature (around 37 °C).
When the temperature is elevated above the body temperature
by externally applying thermal energy, the neurotransmitter is
released at a threshold temperature. It should be noted that the
survival rate of the normal cells decreases when the tempera-
ture exceeds 43 °C.[70] Thus, the appropriate target temperature
to release neurotransmitters from liposomes is in the range of
37 to 43 °C. The threshold temperature at 39–40 °C on DPPC
liposomes (n = 16 in Figure 3D) is in an optimum temperature
range to release neurotransmitters for therapeutic applications.

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Because most neurotransmitters are water-soluble small mole-
cules, as shown in Table 1, neurotransmitters of many kinds can
be encapsulated stably in the inner aqueous phase of liposomes.
Moreover, the small molecules are ideal cargo for efficient
release through the small grain boundary defect in the bilayer
membrane. Consequently, these thermosensitive liposomes
can be a common platform for programmed neurotransmitter
release with heat stimulation. Noteworthy exceptions are hydro-
phobic or amphipathic molecules embedded in lipid membranes
and macromolecules with low membrane permeability.
5. Programmed Payload Release from
Nanocapsules
Typical payload release profiles from conventional liposomes
are in the category of sustained release, which is a slow release
of contents over an extended period of time.[71,72] For example,
the drug release from liposomes composed of DSPC and PEG-
DSPE is below 15% per day for at least 4 days in plasma at
37 °C.[71] An example of a system requiring sustained release
is insulin delivery. Insulin is a peptide hormone that is secreted
naturally from β-cells on islets of Langerhans. Once the func-
tion of pancreatic islets to secrete the insulin is disordered,
the blood glucose level becomes elevated, resulting in diabetes
mellitus. Although the injection of insulin is effective to treat
diabetes, a salient difficulty is maintaining an effective insulin
level in blood. Pharmaceutical devices for “sustained release”
of insulin have been developed to overcome this difficulty.[46,73]
Further advanced glucose-responsive insulin delivery systems
have been proposed to treat diabetes.[74,75]
Stimulus-sensitive liposomes enable burst release and
multiple release responding to the input of physical stimuli
(Figure 4A). Burst release is an attractive profile to treat cancer
cells with anti-cancer drugs.[57,76,77] A higher dose of anti-cancer
drugs is effective to kill the cancer cells, but dosages have been
constrained by the side effects of the drugs in conventional
systemic delivery. Moreover, when weak drug treatment is
repeated, cancer cells acquire drug resistance and become more
difficult to deal with. However, using stimuli-sensitive nanocap-
sules, the drug release site can be restricted by local heating at
the target site. Consequently, the local drug concentration can
be increased rapidly at the target tumor site. This local therapy
enables minimization of the systemic side effects and maximi-
zation of the drug action at the tumor site. For development
of the signal molecule release system in neurons, on-demand
stepwise release is a practical profile. Organic electronic devices
have achieved on-demand precise release of neurotransmit-
ters responding to the external electronic input. Further
combination with biosensors for neurotransmitters enables
auto-regulated neuromodulation as an organic electronic bio-
mimetic neuron.[78]
On-demand release of payload from a nanoplatform is a
recent advanced technology intended for development of drug-
delivery systems in which stimulus-responsive nanomaterials
are applicable for on-demand control of the drug release.[79–90]
The light source energy is useful for the remote and rapid con-
trol of physical stimulus input at a target region.[57,80,82,85–88,90]
As the photothermal materials convert the light energy to heat
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stimulus, thermosensitive nanomaterials enable the on-demand
payload release by laser irradiation (Figure 4B).[57,82,85–88,90]
Radio frequency,[79] alternative magnetic fields,[81] and ultra-
sound[83,84,89] also allow non-invasive heating that can trigger
on-demand drug release (Figure 4C). These nanomaterials
developed for drug-delivery systems are potent platforms for
on-demand neurotransmitter release in nervous tissues in
combination with non-invasive focal heating of tissues in a con-
trolled fashion.
6. Toward Artificial Extracellular Synaptic Vesicles
Despite the importance of neurotransmitter delivery to mod-
ulate the nervous system for development of therapeutic
strategies for nervous system disorders, reports of studies
elucidating the on-demand release of neurotransmitters from
nanoparticles are still scarce among the relevant literature. An
early study using mesoporous silica nanospheres proposed a
stimulus-responsive controlled release of neurotransmitters
and drug molecules with chemically removable surface-derivat-
ized cadmium sulfide nanoparticle caps.[91] In this system, the
release of drug molecules from mesoporous silica nanospheres
is triggered by adding disulfide bond-reducing molecules such
as dithiothreitol and mercaptoethanol in vitro. Nanoparticles
have been considered for encapsulation of neurotransmitters of
several kinds such as dopamine,[92–101] leucine-enkephalin,[102]
glutamate,[103] neuropeptide substance P,[104] acetylcholine,[105]
adenosine,[106] and GABA.[26,107–109] Nevertheless, few in vitro
studies have succeeded in the on-demand release of neuro-
transmitters.[103] A recent study finally demonstrated for the
first time that neurotransmitter-loaded nanocapsules composed
of thermosensitive liposomes can trigger on-demand muscle
relaxation in living organisms by heat stimulus.[26] Although the
study was limited to the effect of a single stimulus on muscle
relaxation by the burst release of inhibitory neurotransmitter
(GABA) from thermosensitive liposomes, the report described
extension to a stepwise payload release system by controlling
the heating temperature and time.
GABA plays an important role in the mechanism and
treatment of epilepsy.[110] Experimental and clinical evidence
indicates that drugs that increase synaptic GABA are potent
anticonvulsants. Actually, GABA-loaded nanocapsules can
provide an invasive GABA release system for experimenta-
tion and for the treatment of epileptic seizures. Introduction
of a further precise and rapid heating system is expected to
be necessary to enable on-demand stepwise payload release
in target tissues such as the brain. Photothermal stimulus by
irradiation of laser to light to heat conversion materials is a
potent approach to achieving rapid on-demand cargo release
from thermosensitive nanoparticles. Near infrared dyes,
porphyrin-derivatives, and gold nanoparticles are combined
with thermosensitive nanocapsules as light-to-heat conver-
sion materials.
Moreover, because signal transduction is regulated by the
release of inhibitory neurotransmitters and excitatory neu-
rotransmitters, selective release of inhibitory and excitatory
neurotransmitters from nanocapsules remains as a technical
challenge. Chitgupi et al. reported an advanced system for
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Figure 4.  Programmed payload release from nanocapsules by physical stimulus. A) Representative profiles of payload release responding to the
stimulus input. Arrows indicate the time points at which the stimulus is applied. B) On-demand payload release from thermosensitive liposomes by
irradiation of near infrared (NIR) light (980 nm wavelength) in HeLa cells labeled with a temperature-sensitive fluorescent molecule (ER thermo yellow).
Right: Representative fluorescence images of ER thermo yellow (upper panel) and calcein (lower panel) in HeLa cells during heating experiments.
Fluorescence intensity of ER thermo yellow decreases concomitantly with increasing temperature, that of calcein is increased when calcein is released
from the liposomes. The elapsed time is presented at the top left of each image. Reproduced with permission.[57] Copyright 2015, Royal Society of
Chemistry. C) On-demand payload release from thermosensitive liposomes by irradiation of high-intensity focused ultrasound (HIFU). Irradiation of
HIFU produces heating at the topical region. Reproduced with permission.[83] Copyright 2015, American Chemical Society.

selective and selectable light-triggered cargo release from
liposomes containing purpurin-phospholipid (Pur-P) or pyroph-
eophorbide-phospholipid (Pyr-P) by irradiation of lasers having
different wavelengths (Figure  5A).[111] In this system, Pur-P is
excited to produce heat by the irradiation of a 655 nm laser.
However, the excitation wavelength for Pyr-P is 690 nm. There-
fore, by irradiation with a laser of 655 or 690 nm wavelength,
selective cargo release from liposomes containing Pur-P or Pyr-P
can be achieved (Figure 5B). Light at wavelengths in the near-
infrared region (650–950 nm wavelength) presents benefits as
an input signal to allow for deeper penetration into tissues than
that which can be achieved using light in the ultraviolet-visible
range.[112,113] Photothermal payload release from thermosensi-
tive liposomes can be achieved by encapsulated near-infrared
dyes without using chemically modified special lipids with
chromophores.[82,114–119] In fact, IR825, a photothermal dye,
absorbs near-infrared light of around 825 nm and converts the
light energy to heat. Thermosensitive liposomes embedding
IR825 in their bilayer membrane release the payload by irradia-
tion using a 808 nm near-infrared laser (Figure 5C).[115] Simi-
larly, cyanine dyes such as indocyanine green (ICG, 805 nm),[82]
IR820 (820 nm),[116] cypate (788 nm),[117] IR780 (780 nm),[118]
DiR (748 nm),[119] and DiD (644 nm)[120] have been used to
release payloads from thermosensitive liposomes using near-
infrared laser irradiation (Figure 5D). The absorption wave-
length of cyanine dyes is controllable by their chemical structure,
for example, the absorption shifts to longer wavelengths
with extension of the polymethine chain comparing DiR and
DiD. Water-soluble ICG and IR820 are encapsulated into an
aqueous core of liposomes; others are embedded in a lipid

Small 2019, 15, 1900132 1900132  (8 of 12) © 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.advancedsciencenews.com www.small-journal.com

Figure 5.  Selective payload release from nanocapsules by irradiation of near-infrared laser light of different wavelengths. A) Structure of porphyrin–
phospholipids and absorbance of liposomes containing 2 mol% porphyrin–phospholipids in PBS. Laser irradiation wavelengths used to trigger the
payload release are indicated by arrows. Reproduced with permission.[111] Copyright 2018, American Chemical Society. B) Selective, selectable light-
triggered release of doxorubicin (Dox) or basic orange (BO) from liposomes containing porphyrin–phospholipids. Indicated liposome mixtures were
subjected to laser irradiation to release liposomal cargo with a 665 nm laser (upper) or 690 nm laser (lower). Reproduced with permission.[111] Copyright
2018, American Chemical Society. C) Schematic illustration showing the structure of thermosensitive stealth liposome containing IR825 and release of
Dox from the liposomes by irradiation of near-infrared light. Reproduced with permission.[115] Copyright 2015, John Wiley & Sons Inc. D) Commercially
available cyanine dyes with different absorption wavelengths that are used to trigger photothermal payload release from liposomes by near-infrared
laser irradiation.

bilayer membrane similarly to IR825. This knowledge can be
extended to the selective release of neurotransmitters from
thermosensitive liposomes embedded near infrared dyes with
different excitation wavelength in a lipid bilayer membrane.
Such a selective release system would enable experimental and
therapeutic neuromodulation by the release of excitatory or
inhibitory neurotransmitters from nanocapsules substituting
the function of synaptic vesicles as artificial extracellular artifi-
cial vesicles (Figure 6).
7. Outlook and Perspective
Neurodegenerative diseases such as Parkinson’s disease
and Alzheimer’s disease are increasingly problematic for
human health. The cause of Parkinson’s disease is generally
unknown, but the dopamine level in the corpus striatum is
lowered because of progressive substantia nigra degenera-
tion.[121] Although a further understanding of the mecha-
nisms for nanoparticle uptake across the BBB is expected,
systemic intravenous administration is a feasible approach
to deliver nanoparticles into the nervous system, such as the
brain.[16,31–42] For example, systemic intravenous administra-
tion of the neurotransmitter dopamine-loaded PLGA nano-
particles causes markedly increased levels of dopamine and
its metabolites in the striatum of parkinsonian rats.[97] A more
recent study has demonstrated an inhibitory neurotransmitter
GABA-loaded thermosensitive liposome injected intramuscu-
larly triggers on-demand muscle relaxation responding to heat
stimulation.[26]

Small 2019, 15, 1900132 1900132  (9 of 12) © 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.advancedsciencenews.com
Figure 6.  Conceptual illustration of artificial extracellular synaptic vesicles used to modify the nervous system by on-demand selective release of excita-
tory or inhibitory neurotransmitter from nanocapsules.
This evidence underscores the fact that nanoparticles encap-
sulating excitatory and inhibitory neurotransmitters function
as artificial extracellular synaptic vesicles in living organisms.
Further development of programmable nanocapsules for on-
demand selective and stepwise release of neurotransmitters can
be expected to lead to breakthroughs for experimental and ther-
apeutic approaches to nervous system disorders. In parallel,
developing a noninvasive technique to apply a stimulus accu-
rately to the nanocapsules in deep tissues is important. Because
nanocapsules can co-encapsulate the photothermal conversion
agents with neurotransmitters, heat stimulus with a focus on
the nanocapsules is feasible using light energy. Taken together,
evidence from earlier studies shows that efficient and noninva-
sive experimental and therapeutic application of neurotransmit-
ters for nervous system disorders can be achieved by further
advancement of nanocapsules that enable precise programmed
neurotransmitter release on demand.
Acknowledgements
This work was partly supported by JSPS KAKENHI (JP16H03844) and the
Singapore Ministry of Education (MOE2017-T2-2-067, RG143/16 (S)).
Conflict of Interest
The authors declare no conflict of interest.
Small 2019, 15, 1900132
www.small-journal.com
Keywords
drug delivery, liposomes, nanocapsules, neurotransmitters, synaptic
vesicles
Received: January 8, 2019
Revised: February 20, 2019
Published online: March 19, 2019
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