DOI 10.1002/bies.200900106
Review article
Synthetic cells and organelles:
compartmentalization strategies
Renée Roodbeen and Jan C. M. van Hest*
Institute for Molecules and Materials, Radboud University Nijmegen, Nijmegen, The Netherlands
The recent development of RNA replicating protocells
and capsules that enclose complex biosynthetic cascade
reactions are encouraging signs that we are gradually
getting better at mastering the complexity of biological
systems. The road to truly cellular compartments is still
very long, but concrete progress is being made. Compartmentalization is a crucial natural methodology to
enable control over biological processes occurring
within the living cell. In fact, compartmentalization has
been considered by some theories to be instrumental in
the creation of life. With the advancement of chemical
biology, artificial compartments that can mimic the cell
as a whole, or that can be regarded as cell organelles,
have recently received much attention. The membrane
between the inner and outer environment of the compartment has to meet specific requirements, such as semipermeability, to allow communication and molecular
transport over the border. The membrane can either be
built from natural constituents or from synthetic polymers, introducing robustness to the capsule.
Keywords: artificial organelles; chemical biology; liposomes;
minimal cells; polymersomes
Introduction
The structural and functional complexity of the cellular
environment has been a source of inspiration and fascination
over the years for researchers active in the molecular life
sciences. Recently, fascination has been gradually replaced
by a more daring attitude to mimic the cell and its organelles,
which is one of the important challenges of the emerging
discipline of chemical biology. In particular, the development of
a synthetic cell is something that grasps the imagination of
researchers since it directly relates to crucial questions
regarding the definition and the origin of life, both of which are
still under much debate.(1,2) From a molecular science point of
view, useful definitions of life have been proposed by Luisi and
Ruiz Mirazo, in which criteria are given on how a minimal cell
should be constructed. Luisi, for example, has defined life as
follows: ‘‘A system which is spatially defined by a semi-
*Correspondence to: J. C. M. van Hest, Institute for Molecules and Materials,
Radboud University Nijmegen, Heyendaalseweg 135, 6525 AJ Nijmegen, The
Netherlands.
E-mail: [email protected]
BioEssays 31:1299–1308, ß 2009 Wiley Periodicals, Inc.
permeable compartment of its own making and which is selfsustaining by transforming external energy/nutrients by its
own process of components production.’’(1) In fact, both
definitions point in the direction of a system in which
compartmentalization in a semi-permeable capsule plays a
crucial role. They therefore support the autopoiesis model,
which was originally described by Varela et al.(3) The term
‘‘autopoiesis’’ means self-reproduction and stipulates that
the formation of a cellular compartment is mandatory for the
creation of life. The cell has a semi-permeable membrane as
a boundary in which the self-sustaining reactions occur. Due
to the semi-permeable boundary, nutrients can diffuse into
the cell, where they are processed to maintain the cell. This
autopoiesis model is the basis for most synthetic cell research
and contrasts somewhat with the RNA world model, which
was introduced by Gilbert in 1986.(4) In the latter theory, RNA
began self-replication in a prebiotic soup and produced, over
time, proteins and DNA;(5) a boundary or compartment was
only introduced later on in the evolutionary process.
The development of synthetic organelles has a more
pragmatic character to it. Similar to research on synthetic
cells, compartments are created that host biological processes. These compartments should preferably be able to
communicate with the outside environment, which requires
that the membranes are semi-permeable. Contrary to
synthetic cell research, the main aim is not to create selfsustaining or replicating systems, but rather to construct
robust bioreactors, which, for example, can be applied in
biotechnology or drug delivery for the production of bioactive
ingredients. Compartmentalization therefore specifically
functions as a method to protect the biological process from
undesired influences from outside the capsule and to keep the
different elements of the biological process close together. As
a result, the choice of the materials for the membranes of
synthetic cells and organelles is based on different grounds;
the synthetic cell membrane should preferably be (re)created
by the processes occurring within the compartment, whereas
the synthetic organelle membrane should predominantly be
stable and semi-permeable. Although synthetic cells and
organelles have been developed independently of each other,
both fields have reached the point that similarities between the
two lines of research have become obvious. The two
approaches can therefore be compared with each other
and possibly become more integrated in the near future. This
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R. Roodbeen and J. C. M. van Hest
review gives an overview of the different approaches that
have been undertaken to create synthetic cells, followed by
methods of construction of artificial organelles. Finally, a
perspective is given where these two fascinating lines of
research could meet in the near future.
Synthetic cells
Several names for synthetic cells are in use, for example, the
(synthetic) protocell, the artificial cell, or the minimal cell.(6–10)
Occasionally these terms are used interchangeably and can
lead to confusion, especially between the minimal cell and the
artificial cell as they do not necessarily mean the same thing.
A minimal cell has the minimal requirements to be called
living. An artificial cell, made from artificial components, is not
necessarily designed to be living or minimal but, for example,
to be used as a biocompatible drug delivery system.(11) To
create a minimal cell two approaches can be used and are
classified as top-down and bottom-up; the latter is also known
as the reconstruction method. The top-down approach is
directed toward the elimination of all cellular components that
are not essential for a cell to be living. As long as the cell is
capable of self-maintenance, reproduction, and evolvability, it
can be regarded as being alive.(9) This notion has led to the
search for the minimal genome within the field of molecular
biology. Based on the mapping out all of the gene products
that are necessary for DNA transcription and translation,
protein biosynthesis, and lipid metabolism(9,10) (Fig. 1), an
estimated 150 genes are predicted as the minimal number. A
practical implementation of the top-down approach is that the
minimal genome can be introduced in a host cell, which takes
care of the regulation and execution of the gene transcription
and translation processes. Upon cell division, newly programmed synthetic cells are thus formed. This genome
transplantation approach has recently been demonstrated by
the Venter group.(12)
The second approach, the bottom-up or reconstruction
approach, involves the recreation of a copy of extant cells,
which deals with the assembly of all the necessary
biomolecules in a compartment to recreate elements of a
functional cell. This molecular approach has to start at an
even more simplified level since this minimal cell has to be
assembled totally from its basic building blocks; this involves
more than top-down adjustments made on the genetic level. It
is therefore foreseen that the complexity of the minimal
cell, created using the reconstruction approach, can only
be gradually increased when a better understanding of the
systems is obtained. The minimal cell approaches that have
been described until now therefore focus on investigating
the (re)production of one of the basic elements of the cell: the
cell membrane components, its genetic information, or a
limited set of proteins. In fact, since these systems are still far
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Figure 1. Schematic representation of all the compounds required
for genome transcription and replication. (Reprinted with permission
from Ref.(10) Copyright Nature Publishing Group.)
away from a natural minimal cell, the terminology artificial or
synthetic cell seems to be more appropriate. Some examples
of these three categories are discussed in the following
sections.
Membrane reproduction
One of the first reconstruction approaches to the synthetic cell
was focused on the reproduction of the compartment that
encapsulates the bioactive ingredients. In particular, vesicles
composed of amphiphilic bilayers have been one topic of
investigation since these structures can be regarded as
mimetic structures for the cell membrane. Methods to create
systems that are capable of procuring their own amphiphiles,
and hence their own compartment, are therefore highly
interesting since it can be seen as a first step toward a selfreplicating system.(6–10,13) After initial studies directed at
micelle reproduction,(14,15) the first example of a vesicle that
could reproduce itself was given by Walde et al. in 1994.(6) A
chemical approach, in which the fatty oleic and caprylic acids
were used for vesicle formation, was chosen. When a solution
of a fatty acid precursor (the corresponding anhydrides of the
fatty acid) was added to these vesicles, hydrolysis of the
precursor resulted in the formation of extra fatty acid and
therefore in the growth of the vesicle. The interesting part of
this reaction was that the vesicles catalyzed their own
formation; when some vesicles were already present, the
entire process took place quicker and without a lag
phase.(16,17) Furthermore, vesicle fission was also observed,
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R. Roodbeen and J. C. M. van Hest
which demonstrates that with this method vesicle compartments can reproduce themselves. Similar observations were
made for the more biomimetic phospholipid-based liposomes.(18,19) Instead of using intrinsically reactive anhydrides,
it was also proven to be possible to trigger lipid and vesicle
formation photochemically.(20)
In another approach, a biocatalytic route was followed to
modify the vesicle composition.(21) Giant vesicles from 1palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC)
and palmitoyl coenzyme A (P-CoA) were made in the
presence of sn-glycerol-3-phosphate (G3P) and were subsequently injected with the enzyme G3P-acetyl transferase
(G3P-AT). This enzyme couples P-CoA and G3P together
and forms a lipid, which was then inserted into the vesicular
membrane. During the reaction, the vesicle shrank and
intravesicular liposome formation was observed as a result of
the changed composition of the membrane.
Compartmentalized polynucleotide
production
Much research has been devoted to the encapsulation and
replication of DNA or RNA inside a compartment (Fig. 2).(22–28)
To be successful in this goal, the reagents necessary to build
up the polynucleotides should be able to pass the membrane
and enter the compartment, but the products synthesized
should be maintained inside. This therefore requires semipermeable membranes. One of the first syntheses of an
oligonucleotide (polyA from ADP by the enzyme PNPase) was
performed in oleic-acid-based vesicles. In this case, the
synthesis of PolyA relied on the encapsulation of PNPase and
the diffusion of ADP from the extravesicular solution into
the vesicle. It was shown that polyA was not able to
leave the vesicle. Furthermore, vesicle reproduction by the
addition of oleic anhydride, as discussed in the previous
section, was compatible with polyA synthesis. The same
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concept was also demonstrated with template-directed
production of RNA.(28)
A closer look into the permeability of vesicles by an
investigation into the efficiency of a non-enzymatic copying
process of an encapsulated dC15 template as a function of
vesicle composition was performed by Mansy et al.(23) For this
templating to occur, activated nucleotides had to be able to
cross the membrane. The authors showed that the permeability of the membrane could be increased when it was
composed of a mixture of short-chain saturated fatty acids,
their corresponding alcohols, and glycerol monoesters
(decanoic acid, decanol, and the glycerol monoester of
decanoic acid, respectively). The interesting fact about this
mixture is that these amphiphiles can be regarded as prebiotic
and, therefore, can be seen as logical constituents of a
protocell membrane. Indeed, template-directed synthesis was
observed, although when permeability was increased too
much, the templating reaction took place more slowly since
diffusion of the nucleotides out of the compartment was also
enhanced. The polynucleotides were, however, always
retained in the capsule. This experiment furthermore shows
that a heterotrophic model, in which protocells have to obtain
nutrients from the outside instead of making themselves, is
plausible.
In a number of cases, investigations were mainly directed
toward the replication process of polynucleotides inside a
compartment. These experiments were performed in vesicles, such as phospholipid-based liposomes, which lacked the
permeability of the above-mentioned examples. To be
functional all the necessary components had, therefore, to
be simultaneously encapsulated inside the liposome. Oberholzer et al.(24) replicated an RNA strand using all four
nucleosides and the enzyme Qb-replicase in an oleate
vesicle. Reproduction of the vesicle compartment was once
again achieved by the addition of oleic anhydride. Even
though an RNA template can now be replicated in a
reproducing vesicle, there is still a long way to go toward
Figure 2. The heterotrophic model of cell development. Left: Protocell containing an internal template in a medium filled with amphiphiles and
activated nucleotides. Middle: The protocell grows after insertion of the amphiphile, the internal template is (non-enzymatically) replicated.
Right: Intrinsic or extrinsic physical forces cause the enlarged protocell to undergo fission to afford two separate protocells, both containing the
internal template. (Reprinted with permission from Ref.(23) Copyright Nature Publishing Group.)
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the formation of a minimal cell since the RNA replication
described above requires enzymes, which are not produced
inside the vesicle. In addition, the information in the RNA does
not contribute to its own reproduction or the reproduction of
the vesicle.
Besides RNA replication, DNA replication has also been
achieved.(25,26) The formation of a giant liposome in which a
cholesterol-DNA (15-mer) conjugate was inserted was
reported by Shohda and Sugawara.(25) A 100-mer DNA
template was encapsulated and hybridized with the 15-mer
primer. The simultaneously encapsulated nucleotides and
DNA polymerase allowed new DNA strands (100-mers) to be
formed. Since the encapsulation of these components is
based on statistics, most vesicles did not contain all
components. It was indeed observed that only 0.1% of the
liposomes showed functional PCR, which underlines the
complexity of this approach.(26)
Protein production in vesicles
Modern cells are strongly dependent on protein activity, which
suggests that protein synthesis was also part of the processes
occurring within the first living cells. For minimal cell formation
the aim is to create vesicles that are able to synthesize
proteins. So far most research has been focused on lipid
vesicles and the model protein, green fluorescent protein
(GFP), to allow facile detection of protein synthesis inside the
liposome.
In an early example, POPC liposomes were used to
synthesize a simple poly(Phe) peptide from polyU mRNA with
a cell-free peptide synthesis extract containing both the 30S
and 50S ribosomal subunits.(29) In a similar approach Yu
et al.(30) reported the use of egg phosphatidylcholine-based
vesicles as protein production vessel. In this case, a plasmid
containing the GFPmut1 gene was encapsulated with the
other components necessary for protein synthesis. This was
achieved by injecting an aqueous solution of these components onto a lipid film of the amphiphiles, followed by vortexing
to stimulate vesicle formation. With flow cytometry, fluorescence originating from GFP could be detected. It was also
observed, however, that only a small amount of liposomes
were active, which is not surprising since a complex mixture of
compounds has to be encapsulated and the transport of
reagents over the liposomal membrane is not possible.
An improved liposomal protein expression system was
reported by Murtas et al.(31) who used a well-defined, cell-free
minimal protein synthesis set composed of 36 enzymes, 70S
ribosomes, tRNAs, and cofactors (Pure Systems), which was
co-encapsulated with a plasmid coding for enhanced GFP
(eGFP). The liposomes were made from POPC and varied in
diameter from 600 nm to 2 mm. In this case it was difficult to
estimate the exact fraction of active liposomes. Experiments
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R. Roodbeen and J. C. M. van Hest
were also performed in which the phospholipid-forming
enzymes sn-glycerol-3-phosphate acyltransferase and lysophosphatidic acid acyltransferase were produced. These
enzymes are involved in the formation of POPC and can
therefore, in principle, be utilized for the self-replication of the
vesicle. It was shown that both enzymes were synthesized
and active in the vesicle, although their production capacity
was low. This observation was explained by the fact that both
enzymes have different optimal operation conditions and
therefore no morphological changes could be induced in the
liposomal compartments.(32)
The size dependence of the liposomes on efficient protein
expression was also investigated.(33) In theory, the cell should
have a diameter of at least 200–300 nm to harbor all
the necessary components, namely, DNA/RNA, ribosomes,
a complete tRNA set, and proteins. Interestingly, however,
100 nm POPC liposomes containing the Pure Systems cellfree protein synthesis set and the plasmid for eGFP were still
capable of protein synthesis. This cannot be explained by a
pure statistical encapsulation, which means that during
vesicle formation an interaction between the compounds to
be encapsulated and the liposomal structure had to take
place.
Although it is surprising from a statistical point of view that
some protein synthesis activity is measured with the small
liposomes, in general the problem of the above-mentioned
systems remains the lack of permeability of the POPC
vesicles. This therefore requires all the necessary components to be present inside the vesicle from the start to allow
protein production, which results in low yields being obtained
since, statistically, the chances are small that a liposome
encapsulates all the necessary components.
An elegant approach to circumvent this problem was
described by Noireaux and Libchaber.(34,35) Instead of
expressing only GFP inside L-a-lecithin liposomes, by using
a cell-free E. coli expression extract, they coexpressed a poreforming protein, a-hemolysin. This protein forms a heptamer
in the membrane and allows molecules up to 3 kDa to pass
through. The expression of eGFP inside the vesicle could now
be continued for up to 4 days since energy and nutrients
could be transported from the outside into the vesicular
compartment.
Artificial organelles
As described in the previous section, synthetic cell membranes have exclusively been composed of low molecular
weight, vesicle-forming amphiphilic molecules such as
phospholipids or fatty acids. This is a logical choice since
these compounds can be found in the membrane of living
cells and are presumed to have been part of protocell
structures. Furthermore, such membranes are quite often
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R. Roodbeen and J. C. M. van Hest
semi-permeable by themselves, which fulfills an important
requirement for the functioning of a synthetic cell. Probably
the most important rationale for the use of low molecular
weight amphiphiles as membrane constituents is the fact that
they can be made by a simple (bio)synthetic process. The
constant production of membrane-forming molecules in close
proximity to synthetic cells can, in principle, lead to uptake by
the membrane, followed by the formation of new compartments by a fission process, which is one of the prerequisites
for the occurrence of self-replication. The fact that these
membranes are fluid-like and therefore not very robust is of no
real importance for this fundamental line of research.
The main focus of research on artificial organelles is to
create nanometer-sized reactors with biological and, in
particular, enzymatic activity. The above-mentioned liposomes can of course be used for this purpose, however,
since the focus is not on reproduction, these capsules can
also be made from constituents other than low molecular
weight amphiphiles. Many examples are reported of artificial
organelles based on polymeric capsules, which are in most
cases polymersomes. These are vesicles that have a
polymeric membrane with an insoluble middle and a soluble
outer layer and are generally built from amphiphilic block
copolymers of the AB or ABA type (Fig. 3).(36–40) On bringing
these polymers into contact with water they spontaneously
form vesicles, encapsulating the aqueous medium. Their
formation process is therefore quite similar to the low
Review article
molecular weight amphiphile vesicles, which probably also
explains their popularity in the artificial organelle community.
Polymersomes are, due to their thicker membrane, much
more stable and thus more robust than vesicles based on
small organic molecules. The main disadvantage of the
polymersome structure is that it has a greatly decreased
permeability. Polymersomes are generally closed shells that
shield their lumen from the outside environment; however, in
rare cases intrinsically porous polymersomes have been
reported. For example, the AB block copolymer polystyreneblock-polyisocyanoalanine-(2-thiophene-3-yl-ethyl)amide
(PS-PIAT, Fig. 3) forms porous vesicles in water due to the
imperfect stacking of the molecules in the membrane,
which allows the diffusion of small molecules in and out of
the capsule.(41) In most other cases membrane proteins are
inserted into the polymeric shell to induce permeability.
Polymersomes that harbor such a transmembrane channel
protein are sometimes also referred to as synthosomes.(42)
Another method to create intrinsically porous polymer
capsules is based on the layer-by-layer (LbL) deposition
approach (Fig. 4). As a starting point, a dissolvable scaffold
is used upon which alternating layers of positively and
negatively charged polyelectrolytes [most often the cationic
poly(allylamine hydrochloride) (PAH) and the anionic polystyrene sulfonate (PSS), Fig. 3] are deposited.(43–46) After
sufficient layers are added, the scaffold is dissolved and a
hollow particle is obtained. Since the polyelectrolyte layers
Figure 3. Different polymers used for capsule formation. a: Polymethyl oxazoline-poly(dimethyl siloxane)-polymethyl oxazoline (PMOXAPDMS-PMOXA) and (b) PS-PIAT, both applied in polymersome formation; (c) polyallylamine (left) and polystyrene sulfonate (right), applied in the
LbL deposition method.
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Figure 4. The LbL technique to create hollow semi-permeable capsules. a–d: The initial steps involve stepwise film formation by repeated
exposure of the templates to polyelectrolytes of alternating charge. e: After the desired number of polyelectrolyte layers is deposited, the core is
dissolved and yields free polyelectrolytic hollow shells (f). (Reproduced with permission from Ref.(60) Copyright Wiley-VCH Verlag GmbH & Co.
KGaA.)
create a semi-permeable membrane, the final structure is a
porous nanoreactor. The attractiveness of this approach is
that, due to the templating procedure, well-defined capsules
are made; however, this multistep formation process is less
biomimetic than polymersome formation.
Although potential applications of artificial organelles are
obvious, at the moment this field of research is still mostly in
the conceptual stage, which means that researchers are
primarily focused on increasing the level of complexity of
biological processes that can be encapsulated within the
synthetic compartment. In the next section this development
is discussed in more detail.
Monofunctional bioreactors
The simplest type of artificial organelle that can be created
consists of a compartment that contains one type of functional
protein. This, for example, was demonstrated for polymethyl
oxazoline-poly(dimethyl
siloxane)-polymethyl
oxazoline
(PMOXA-PDMS-PMOXA, Fig. 3) polymersomes in which
the enzyme Cu,Zn superoxide dismutase was encapsulated.
Although this polymersome can be regarded as a closed-shell
system, the substrate O
2 is small enough to reach the
enzyme by diffusing across the membrane.(47) In this case
there is therefore no need for the additional introduction of a
channel protein. When intrinsically permeable polymer
capsules are used, the use of a channel protein is not
required. This was shown for LbL capsules that were filled
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with urease, an enzyme that catalyzes the decomposition of
urea to form carbonate anions.(48,49) In this method, PAH/PSS
polyelectrolyte capsules were exposed to a urease-containing
water/ethanol mixture. This solvent mixture permeabilized the
capsule walls and allowed the urease molecules to enter
the capsule’s lumen. After removal of the ethanol, the ureasecontaining capsules were shown to be active. In addition to
urease, horseradish peroxidase (HRP) was also encapsulated inside the inner compartment of hollow LbL capsules.(50)
Via a slightly modified procedure HRP could be trapped in the
polymer membrane rather than inside the lumen.(51)
Intrinsically porous PS-PIAT polymersomes were used for
the encapsulation of the lipase Candida antarctica lipase B
(CalB). When a profluorescent substrate was added to the
aqueous medium outside of the capsules, activity was
observed by the occurrence of fluorescence inside the
polymersomes. The activity of encapsulated CalB was
also probed using flow cytometry.(52) With this technique
active nanoreactors could be separated from non-active
capsules, which resulted in a completely active population of
bioreactors.
A final intriguing example of a monofunctional artificial
organelle was reported by Graff et al.(27) and involved the
creation of a DNA-loading device. For this purpose they
incorporated the bacterial channel-forming membrane protein
LamB in a capsule composed of the PMOXA-PDMS-PMOXA
tri-block copolymer. LamB is a trimeric membrane protein that
triggers l-phage to release its DNA into the vesicle
(schematically depicted in Fig. 5). Transmission electron
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R. Roodbeen and J. C. M. van Hest
Figure 5. Schematic representation of DNA injection into a LamB
containing polymersome by bacteriophage lambda. (Reprinted with
permission from Ref.(27).)
microscopy (TEM) was used to show that the bacteriophage
was indeed bound to the polymersome. Actual DNA injection
by the phage was demonstrated after isolation of the
polymersomes. The preserved functionality of LamB in an
artificial environment is noteworthy since it effectively fools a
phage into recognizing a synthetic polymer vesicle as though
it were a cell.
Multifunctional bioreactors
The possible functions of artificial organelles are greatly
increased when, besides the functional enzyme, a membrane
protein is also introduced. In addition to the fact that they
induce permeability for small molecules, membrane proteins
can also be selective in their transport abilities and are
responsive to external stimuli. Graff et al.(53) reported the
synthesis of POPC vesicles, which were stabilized by the UVinduced cross-linking reaction of n-butyl methacrylate and
ethylene glycol dimethacrylate within the POPC bilayer
(Fig. 6). b-Lactamase, an enzyme that hydrolyzes ampicillin
to ampicillinoic acid, was encapsulated inside these vesicles.
Since the permeability of the POPC membrane for ampicillin
is very low, the outer membrane protein F (OmpF), a channel
protein isolated from E. coli, was inserted in the liposomal
bilayer. This membrane protein yields an aqueous channel
that allows passive diffusion of metabolites with molecular
masses of <400 g/mol. The incorporation of OmpF was
confirmed by measuring the b-lactamase-catalyzed hydrolysis of extravesicularly added ampicillin to ampicillinoic acid.
This concept was also translated to PMOXA-PDMSPMOXA polymersomes.(54) It was found that OmpF channel
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Figure 6. Schematic representation of a cross-linked POPC liposome, which contains OmpF in its membrane to allow ampicillin to
enter the vesicle. Encapsulated b-lactamase hydrolyzes ampicillin to
ampicillinoic acid. (Reprinted with permission from Ref.(53).)
proteins, after insertion inside the polymer vesicles, maintained their activity because the polymer was surprisingly
flexible and could accommodate the smaller protein structure.
Thus, b-lactamase was encapsulated inside the polymer
vesicle. When ampicillin was added outside the capsules, the
activity of the enzyme could be measured, which demonstrates that the channel proteins functioned effectively. Since
the OmpF channel is responsive to membrane voltages,
the capsules could be closed by applying a transmembrane
potential of 100 mV. By lowering the potential, the channels
could be reopened. The same OmpF functional capsule was
also used to encapsulate the pH-dependent enzyme acid
phosphatase.(55)
A mutant of FhuA, a larger channel protein, has also been
incorporated into the same type of polymersomes.(56) In this
case the polymersomes were loaded with HRP and catalytic
activity was observed when both types of proteins were
present.
A drawback of inserting membrane proteins into an
artificial membrane is that in a normal cell the proteins have
a preferred position, that is, it is known which terminus should
be in the cytoplasm and which terminus is exposed to the
extracellular fluid. When membrane proteins are inserted in a
(symmetrical) artificial membrane this information is lost and
the proteins suffer a loss of function. An asymmetric tri-block
copolymer that could direct the orientation of membrane
proteins was therefore created.(57) With the polymer
PEO-PDMS-PMOXA [PEO: poly(ethylene oxide)] indeed
asymmetric membranes were formed. Aquaporin 0, a
homotetramer with six transmembrane regions and five
loops, was inserted inside the asymmetric vesicles. In
contrast to liposomes and symmetric polymersomes where
insertion is a random process, physiological insertion was
observed in the asymmetric polymersomes.
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Besides encapsulation of enzymes inside the lumen of the
polymersome, it also proved to be possible to encapsulate
enzymes in the polymer membrane, or even to anchor
them onto the surface of the polymersome.(58–60) These
three procedures were also combined, which allowed
for the positional assembly of three different enzymes into
one polymersome nanoreactor (Fig. 7).(61) The three enzymes
that were assembled were Cal B, glucose oxidase (GOx), and
HRP. These enzymes were shown to work together in a
cascade fashion for the conversion of glucose monoacetate.
What is remarkable is the fact that upon encapsulation a more
than statistical inclusion of the enzymes was observed. This is
similar to Luisi’s report on liposomal artificial cells.(33) Capsule
formation and encapsulation are therefore processes that, in
some cases, seem to be cooperative.
With LbL capsules, two-step cascade reactions could also
be established by incorporating both GOx and HRP.(62–64)
With this method it was shown to be possible to create an
organelle inside an organelle, both of which were functionalized with different enzymes. This was achieved by first
creating a calcium carbonate scaffold containing HRP. On this
scaffold, a polymer layer was introduced by the LbL technique
and a second scaffold, containing GOx, was introduced on top
of the LbL capsule. After a second round of LbL deposition
and finally removal of the scaffolds, the capsule-in-capsule
geometry, with two functional enzymes, was obtained.(65)
An elegant experiment was performed based on a
liposome or polymersome, in which parts of the cellular
machinery for the biosynthesis of ATP were reconstituted
(Fig. 8).(66,67) Bacteriorhodopsin (BR) is a light-driven
Figure 7. Schematic representation of a multistep reaction performed in a PS-PIAT polymersome. 1: Monoacetylated glucose is
deprotected by CalB, which is embedded in the polymersome membrane. 2: In the inner aqueous compartment, GOx oxidizes glucose to
gluconolactone generating a molecule of hydrogen peroxide. 3:
Hydrogen peroxide is used by HRP to convert ABTS to ABTSþ.
The HRP is tethered to the polymersome surface. (Reproduced with
permission from Ref.(61) Copyright Wiley-VCH Verlag GmbH & Co.
KGaA.)
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R. Roodbeen and J. C. M. van Hest
Figure 8. Schematic overview of the ATP synthetic system reconstituted with both bacteriorhodopsin (BR) and F0F1-ATP synthase.
(Reproduced with permission from Ref.(67) Copyright 2005 American
Chemical Society.)
transmembrane proton pump, which was embedded in the
polymersome membrane to build and maintain a proton
gradient over it. This gradient fueled the action of F0F1-ATP
synthase, the rotary motor protein that catalyzes the
phosphorylation of ADP to produce ATP. Again the orientation
of BR in the polymersome membrane is of crucial importance.
BR has to enable an influx of protons into the vesicle to drive
the formation of ATP. Fine tuning of the insertion process
finally gave the researchers the ability to direct the position of
the BR channel protein. This biomimetic polymersome
thus successfully replicated the biosynthesis of ATP and
demonstrated the feasibility of performing biosynthesis in
polymersomes.
Finally, a system that comes very close to the concept of
the artificial organelle, in which a polymersome nanoreactor
was introduced into a living cell, was developed by the group
of Meier.(68) This integration was achieved by labeling the
outer surface of trypsin-loaded PMOXA-PDMS-PMOXA
polymersomes with polyguanylic acid (polyG), a signaling
moiety that is recognized by certain macrophages and
triggers internalization.(69) These vesicles were internalized
in macrophages and, after internalization, all regular cellular
trypsin activity was inhibited by the addition of specific
trypsin inhibitors to the cell growth medium. In spite of these
inhibitors, trypsin activity was detected in macrophages
containing the polymersome nanoreactors.
BioEssays 31:1299–1308, ß 2009 Wiley Periodicals, Inc.
R. Roodbeen and J. C. M. van Hest
Conclusions
Although the concept of synthetic cells and organelles has
been around for many years, it has recently drawn increased
attention. This is a logical result of the emergence of the new
research field of chemical biology, in which scientists combine
their knowledge of chemistry and biology in an intricate
manner. Materials scientists who work on capsule formation
find an interesting application for their assembly techniques in
the creation of artificial organelles. On the other hand,
biologists aim to reduce the number of biological building
blocks necessary to recreate a cell. It is also clear that
research on both synthetic cells and artificial organelles is still
in its conceptual phase. The building of a synthetic cell has
mainly been limited to a modular approach in which the
multiple aspects of a synthetic cell are only addressed one at a
time. The challenge in the near future is to integrate these
different approaches to add a new level of complexity to the
molecular systems, in which the replication of genetic
information, protein synthesis, and capsule reproduction
are combined. Even more daring is to introduce appropriate
feedback mechanisms that switch on and off the different
processes that occur within the synthetic cellular environment. This is of crucial importance for the living cell and has
been mostly ignored in the current approaches.
In the case of artificial organelles research, the application
of these systems as bioreactors is foreseeable, although the
real benefits of artificial organelles compared to other catalytic
devices still have to be unambiguously demonstrated. A real
breakthrough can be expected when artificial cells and
organelles are merged. The introduction of artificial organelles in living and synthetic cells opens up the possibility of
incorporating unnatural functionality into biological systems. If
self-replicating polymersomes can be designed, this class of
artificial organelles potentially turns into a robust synthetic
cellular structure. Of course, this entirely synthetic, bottom-up
approach to biology will require many hurdles to be taken and
it seems to be less straightforward than the top-down
approach, which is focused on the construction of a synthetic
genome. The reward of the bottom-up approach is that
researchers are not fully constrained by the boundaries of
biological systems and are not limited to the choice of mere
biological building blocks, as is the case of the top-down
synthetic cell approach. It is clear that future research on
artificial cells and organelles will confront us with many
exciting and sometimes unexpected results and will aid us to
obtain a better understanding of the principles of life.
References
1. Luisi PL. 1998. About various definitions of life. Orig Life Evol Biosph 28:
613–22.
BioEssays 31:1299–1308, ß 2009 Wiley Periodicals, Inc.
Review article
2. Ruiz-Mirazo K, Pereto J, Moreno A. 2004. A universal definition of life:
autonomy and open ended evolution. Orig Life Evol Biosph 34: 323–46.
3. Varela FJ, Maturana HR, Uribe R. 1974. Autopoiesis: the organization of
living systems, its characterization and a model. BioSystems 5: 187–95.
4. Gilbert W. 1986. Origin of life – the RNA world. Nature 319: 618.
5. Dworkin JP, Lazcano A, Miller SL. 2003. The roads to and from the RNA
world. J Theor Biol 222: 127–34.
6. Walde P, Wick R, Fresta M, et al. 1994. Autopoietic self-reproduction of
fatty-acid vesicles. J Am Chem Soc 116: 11649–54.
7. Szostak JW, Bartel DP, Luisi PL. 2001. Synthesizing life. Nature 409:
387–90.
8. Luisi PL. 2002. Towards the engineering of minimal living cells. Anat Rec
268: 208–14.
9. Luisi PL, Ferri F, Stano P. 2006. Approaches to semi-synthetic minimal
cells. Naturwissenschaften 93: 1–12.
10. Forster AC, Church GM. 2006. Towards synthesis of a minimal cell. Mol
Syst Biol 45: 1–10.
11. Leduc PR, Wong MS, Ferreira PM, et al. 2007. Towards an in vivo
biologically inspired nanofactory. Nat Nanotechnol 2: 1–6.
12. Gibson DG, Benders GA, Andrews-Pfannkoch C, et al. 2008. Complete
chemical synthesis, assembly and coning of a Mycoplasma genitalium
genome. Science 319: 1215–20.
13. Pohorille A, Deamer D. 2002. Artificial cells, prospects for biotechnology.
Trends Biotechnol 20: 123–8.
14. Luisi PL, Varela FJ. 1989. Self-replicating micelles – a chemical version
of a minimal autopoeitic system. Orig Life Evol Biosph 19: 633–43.
15. Bachmann PA, Walde P, Luisi PL, et al. 1991. Self-replicating micelles –
aqueous micelles and enzymatically driven reaction in reverse micelles.
J Am Chem Soc 113: 8204–9.
16. Blöchliger E, Blocher M, Walde P, et al. 1998. Matrix effect in the size
distribution of fatty acid vesicles. J Phys Chem B 102: 10383–90.
17. Rasi S, Mavelli F, Luisi PL. 2003. Cooperative micelle binding and matrix
effect in oleate vesicle formation. J Phys Chem B 107: 14068–76.
18. Berclaz N, Müller M, Walde P, et al. 2001. Matrix effect of vesicle
formation as investigated by cryotransmission electron microscopy.
J Phys Chem B 105: 1056–64.
19. Macı́a J, Solé RV. 2007. Synthetic turing protocells: vesicle selfreproduction through symmetry breaking instabilities. Phil Trans R
Soc B 362: 1821–9.
20. DeClue MS, Monnard P-A, Bailey JA, et al. 2009. Nucleobase mediated,
photocatalytic vesicle formation from an ester precursor. J Am Chem Soc
131: 931–3.
21. Wick R, Luisi PL. 1996. Enzyme-containing liposomes can endogenously
produce membrane-constituting lipids. Chem Biol 3: 277–85.
22. Walde P, Goto A, Monnard P, et al. 1994. Oparins reactions revisited –
enzymatic-synthesis of poly(adenylic acid) in micelles and selfreproducing vesicles. J Am Chem Soc 116: 7541–7.
23. Mansy SS, Schrum JP, Krishnamurthy M, et al. 2008. Template-directed
synthesis of a genetic polymer in a model protocell. Nature 454: 122–6.
24. Oberholzer T, Wick R, Luisi PL, et al. 1995. Enzymatic RNA replication in
self-reproducing vesicles – an approach to a minimal cell. Biochem
Biophys Res Commun 207: 250–7.
25. Shohda K, Sugawara T. 2006. DNA polymerization on the inner surface of
a giant liposome for synthesizing an artificial cell model. Soft matter 2:
402–8.
26. Oberholzer T, Albrizio M, Luisi PL. 1995. Polymerase chain reaction in
liposomes. Chem Biol 2: 677–82.
27. Graff A, Sauser M, van Gelder P, et al. 2002. Virus-assisted loading of
polymer nanocontainer. Proc Natl Acad Sci USA 99: 5064–8.
28. Monnard P-A, Luptak A, Deamer W. 2007. Models of primitive cellular
life: polymerases and templates in liposomes. Phil Trans R Soc Lond B
362: 1741–50.
29. Oberholzer T, Nierhaus KH, Luisi PL. 1999. Protein expression in
liposomes. Biochem Biophys Res Commun 261: 238–41.
30. Yu W, Sato K, Wakabayashi M, et al. 2001. Synthesis of functional protein
in liposome. J Biosci Bioeng 92: 590–3.
31. Murtas G, Kuruma Y, Bianchini P, et al. 2007. Protein synthesis in
liposomes with a minimal set of enzymes. Biochem Biophys Res Commun
363: 12–7.
1307
Review article
32. Kuruma Y, Stano P, Ueda T, et al. 2009. A synthetic biology approach to
the construction of membrane proteins in semi-synthetic minimal cells.
Biochim Biophys Acta 1788: 567–74.
33. Pereira de Souza T, Stano P, Luisi PL. 2009. The minimal size of
liposome-based model cells brings about a remarkably enhanced entrapment and protein synthesis. ChemBioChem 10: 1056–63.
34. Noireaux V, Libchaber A. 2004. A vesicle bioreactor as a step
toward an artificial cell assembly. Proc Natl Acad Sci USA 101: 17669–74.
35. Noireaux V, Bar-Ziv R, Godefroy J, et al. 2005. Toward an artificial cell
based on gene expression in vesicles. Phys Biol 2: 1–8.
36. Discher BM, Won YY, Ege DS, et al. 1999. Polymersomes: tough vesicles
made from diblock copolymers. Science 284: 1143–6.
37. Discher BM, Hammer DA, Bates FS, et al. 2000. Polymer vesicles in
various media. Curr Opin Colloid Interface Sci 5: 125–31.
38. Discher DE, Eisenberg A. 2002. Polymer vesicles. Science 297: 967–73.
39. Discher DE, Ahmed F. 2006. Polymersomes. Annu Rev Biomed Eng 8:
323–41.
40. Olsen BD, Segalman RA. 2008. Self-assembly of rod-coil block
copolymers. Mater Sci Eng R 62: 37–66.
41. Vriezema DM, Hoogboom J, Velonia K, et al. 2003. Vesicles
and polymerized vesicles from thiophene-containing rod-coil block
copolymers. Angew Chem Int Ed 42: 772–6.
42. Onaca O, Nallani M, Ihle S, et al. 2006. Functionalized nanocompartments (synthosomes): limitations and prospective applications in industrial biotechnology. Biotechnol J 1: 1–11.
43. Shi XY, Shen MW, Mohwald H. 2004. Polyelectrolyte multilayer
nanoreactors toward the synthesis of diverse nanostructured materials.
Prog Polym Sci 29: 987–1019.
44. Decher G, Hong JD, Schmitt J. 1992. Buildup of ultrathin multilayer films
by a self-assembly process. 3. Consecutively alternating adsorption of
anionic and cationic polyelectrolytes on charged surfaces. Thin solid films
210: 831–5.
45. Sukhorukov G, Donath E, Davis S, et al. 1998. Stepwise polyelectrolyte
assembly on particle surfaces: a novel approach to colloid design. Polym
Adv Technol 9: 759–67.
46. Donath E, Sukhorukov GB, Caruso F, et al. 1998. Novel hollow polymer
shells by colloid-templated assembly of polyelectrolytes. Angew Chem
Int Ed 37: 2202–5.
47. Axthelm F, Casse O, Koppenol WH, et al. 2008. Antioxidant nanoreactor
based on superoxide dismutase encapsulated in superoxide-permeable
vesicles. J Phys Chem B 112: 8211–7.
48. Antipov AA, Sukhorukov GB, Mohwald H. 2003. Influence of the ionic
strength on the polyelectrolyte multilayers’ permeability. Langmuir 19: 2444–8.
49. Antipov AA, Shchukin D, Fedutik Y, et al. 2003. Urease-catalyzed
carbonate precipitation inside the restricted volume of polyelectrolyte
capsules. Macromol Rapid Commun 24: 274–7.
50. Ghan R, Shutava T, Patel A, et al. 2004. Enzyme-catalyzed polymerization of phenols within polyelectrolyte microcapsules. Macromolecules 37:
4519–24.
51. Shutava T, Zheng ZG, John V, et al. 2004. Microcapsule modification
with peroxidase-catalyzed phenol polymerization. Biomacromolecules 5:
914–21.
1308
R. Roodbeen and J. C. M. van Hest
52. Nallani M, Woestenenk R, De Hoog HPM, et al. 2009. Sorting catalytically active polymersome nanoreactors by flow cytometry. Small 5: 1138–
43.
53. Graff A, Winterhalter M, Meier W. 2001. Nanoreactors from polymerstabilized liposomes. Langmuir 17: 919–23.
54. Nardin C, Widmer J, Winterhalter M, et al. 2001. Amphiphilic
block copolymer nanocontainers as bioreactors. Eur Phys J E 4:
403–10.
55. Broz P, Driamov S, Ziegler J, et al. 2006. Toward intelligent nanosize
bioreactors: a pH-switchable, channel-equipped, functional polymer
nanocontainer. Nano Lett 6: 2349–53.
56. Nallani M, Benito S, Onaca O, et al. 2006. A nanocompartment system
(synthosome) designed for biotechnological applications. J Biotechnol
123: 50–9.
57. Stoenescu R, Graff A, Meier W. 2004. Asymmetric ABC-triblock copolymer membranes induce a directed insertion of membrane proteins.
Macromol Biosci 4: 930–5.
58. Opsteen JA, Brinkhuis RP, Teeuwen RLM, et al. 2007. ‘‘Clickable’’
polymersomes. Chem Commun 30: 3136–8.
59. van Dongen SFM, Nallani M, Schoffelen S, et al. 2008. A block copolymer for functionalisation of polymersome surfaces. Macromol Rapid
Commun 29: 321–5.
60. Vriezema DM, Garcia PML, Oltra NS, et al. 2007. Positional assembly of
enzymes in polymersome nanoreactors for cascade reactions. Angew
Chem Int Ed 46: 7378–82.
61. van Dongen SFM, Nallani M, Cornelissen JJLM, et al. 2009. A threeenzyme cascade reaction through positional assembly of enzymes in a
polymersome nanoreactor. Chem Eur J 15: 1107–14.
62. Balabushevich NG, Sukhorukov GB, Larionova NI. 2005. Polyelectrolyte multilayer microspheres as carriers for bienzyme system: preparation
and characterization. Macromol Rapid Commun 26: 1168–72.
63. Mak WC, Bai J, Chang XY, et al. 2009. Matrix-assisted colloidosome
reverse-phase layer-by-layer encapsulating biomolecules in hydrogel
microcapsules with extremely high efficiency and retention stability.
Langmuir 25: 769–75.
64. Mak WC, Cheung KY, Trau D. 2008. Diffusion controlled and temperature
stable microcapsule reaction compartments for high-throughput microcapsule-PCR. Adv Funct Mater 18: 2930–7.
65. Kreft O, Prevot M, Mohwald H, et al. 2007. Shell-in-shell microcapsules: a
novel tool for integrated, spatially confined enzymatic reactions. Angew
Chem Int Ed 46: 5605–8.
66. Steinberg-Yfrach G, Rigaud J-L, Durantini EN, et al. 1998. Light-driven
production of ATP catalysed by F0F1-ATP synthase in an artificial photosynthetic membrane. Nature 392: 479–82.
67. Choi HJ, Montemagno CD. 2005. Artificial organelle: ATP synthesis from
cellular mimetic polymersomes. Nano Lett 5: 2538–42.
68. Ben-Haim N, Broz P, Marsch S, et al. 2008. Cell-specific integration of
artificial organelles based on functionalized polymer vesicles. Nano Lett 8:
1368–73.
69. Broz P, Benito SM, Saw C, et al. 2005. Cell targeting by a generic
receptor-targeted polymer nanocontainer platform. J Controlled Release
102: 475–88.
BioEssays 31:1299–1308, ß 2009 Wiley Periodicals, Inc.