Achievements and open questions in the self-reproduction of vesicles

and synthetic minimal cells

Pasquale Stano and Pier Luigi Luisi*

Received 14th July 2009, Accepted 2nd March 2010

First published as an Advance Article on the web 4th May 2010

DOI: 10.1039/b913997d

Supramolecular chemistry was enriched, about twenty years ago, by the discovery of the

self-reproduction of micelles and vesicles. The dynamic aspects and complexity of these systems

makes them good models for biological compartments. For example, the self-reproduction of

vesicles suggests that the growth in size and number of a vesicle population resembles the pattern

of living cells in several aspects, but it take place solely due to physical forces. Several reports

demonstrate that reverse micelles, micelles, sub-micrometric and giant vesicles can self-reproduce,

generating new particles at the expenses of a suitable precursor. Recently, similar studies are in

progress on more complex vesicle-based systems, namely semi–synthetic minimal cells. These are

artificial cell–like compartments that are built by filling liposomes with the minimal number of

biomolecules, such as DNA, ribosomes, enzymes, etc., in order to construct a living cell in the

laboratory. This approach aims to investigate the minimal requirements for molecular systems

in order to display some living properties, while it finds relevance in origins of life studies and in

synthetic (constructive) biology.

1. Introduction

In addition to their use in biophysics, biotechnology, and

medicine,1 synthetic compartments such as micelles, reverse

micelles and vesicles have often been used as a model of

biological compartments. Lipid vesicles (liposomes), in

particular, due to their structural similarity with living cells,

are not only considered the best model for cellular membranes,

but also important tools for experimental investigations on the

origins of life.2 It has been proposed that fatty acid vesicles,

which form spontaneously by self-assembly, may have played

a significant role in prebiotic times, being essential in the

transition from non-living molecules to early living systems.3

Vesicles provide several advantages in such primitive

scenarios; in addition to their evident structural similarity

with modern cells, for example the semi–permeable character

of the boundary, they also protect entrapped material from

external perturbations and create a microenvironment that

may differ from the external one. However, one of the main

reasons for their key role in origins of life scenarios is indeed

Biology Department, University of RomaTre, Italy.E-mail: luisi@mat.ethz.ch; Fax: (+39) 06 57336329;Tel: (+39) 06 57336329

Pasquale Stano

Pasquale Stano graduatedfrom the University of Pisa,where he worked on dinuclearsupramolecular Schiff-basecomplexes and the kineticsand mechanism of amineexchange. In 2002 he joinedLuisi’s group at ETH Zurichworking on the use of lipo-somes as drug delivery agents.Since 2004 he has worked onliposome reactivity and on theuse of liposomes as cell modelsat the ‘‘Enrico Fermi’’ Centre(Rome), and then as aResearch Fellow at the

Biology Department of the University of RomaTre (Rome).Additional interests are in small-peptide catalysis and on the useof liposomes in biotechnology.

Pier Luigi Luisi

Professor Pier Luigi Luisigraduated from the Universityof Pisa (Scuola NormaleSuperiore) in 1963. Afterinitial studies on macro-molecular science at Lenin-grad, Uppsala, Strasburg,Eugene, he joined the ETHZurich in 1970, where hebecame Professor of Macro-molecular Chemistry (1980)and then SupramolecularChemistry (1984). He workedon enzymes in reverse micellesand on the self-reproduction ofsupramolecular structures.

Since 2002 he holds the chair of Biophysics at the Universityof RomaTre (Rome). He is the coordinator of two internationalprojects on the construction of semi-synthetic minimal cells.His research focuses on synthetic biology, origin of life, self-organization, autopoiesis, and emergent properties.

This journal is �c The Royal Society of Chemistry 2010 Chem. Commun., 2010, 46, 3639–3653 | 3639

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the discovery of their self-reproduction, which is the topic of

this article. In addition to vesicles, it has been shown that

micelles,4 reverse micelles4a,5 and water-in-oil droplets6 also

undergo a self-reproduction process, and all in all, these

observations have broadened our current knowledge on

compartments’ reactivity in the last 20 years.

Recent trends in synthetic biology are related to these

topics, since the initial studies on self-reproduction of vesicles

have flowed into more complex approaches regarding the

construction of cell-like systems (often called artificial,

synthetic or semi–synthetic cells). These advances may find

future applications in biotechnology, so that they will nicely

complement and have great importance in basic research. In

fact, the construction (synthesis) of cells can be seen as a way

of directing observations of the emergence of life (perhaps

‘‘limping life’’7) from non living molecules. In fact, in the

self-reproduction of vesicles and in the construction of self-

reproducing synthetic living cells, emphasis is given to the

self-organization and out-of-equilibrium character of certain

molecular assemblies and of their transformations, and to

their dynamic relationships with the environment. In these

systems, the self-reproduction or cell–like properties can be

observed at a level which differs from that of constituent

molecules, i.e., they can be seen as emergent properties.

For some years after the discovery of vesicle self-reproduction

not enough attention has been given to these studies, but we

are now witnessing some attitude changes in the last years,

thanks to the (re)appraisal of a systemic view of (bio)chemical

processes, that has also pervaded chemistry as shown in the

recent European ‘‘systems chemistry’’ program. All this has

also caused a renewed interest for autopoiesis,8 which is the

theoretical framework for understanding the logic underlying

the construction of minimal self-reproducing systems, as

simple vesicles, micelles, or semi–synthetic cells.

In this review, we will describe the major achievements in

self-reproducing vesicles; experiments on self-reproduction of

micelle and reverse micelles will be only shortly mentioned

(the interested reader may refer to recently published reviews9).

Then we will describe the attempts to construct semi–synthetic

minimal cells that could be able to self-reproduce and to

display minimal living properties, as self-maintenance and

evolvability.

Before starting, however, there is a note on semantics. We

will use the term ‘‘self-reproduction’’ dealing with micelles,

vesicles, and minimal cells, and we will avoid the term

‘‘self-replication’’. In fact, as noted earlier,10 self-replication

refers to an exact replica of the original, as in the case of DNA

structure. ‘‘Self-reproduction’’ is more appropriate in the case

of vesicles or cells, where new particles form from pre-existing

ones, and the process is not strictly controlled by molecular

complementarity. New particles are not necessarily identical to

each other or with respect to the previous ones.

2. Autopoietic self-reproduction

The theory autopoiesis (i.e. self-production) deals with the

logic of cellular life. Introduced by Humberto Maturana and

Francisco Varela in the Seventies,8a autopoiesis identifies the

main activity of the cell as the maintenance of its own dynamic

and structural organization. This occurs despite the large

number of transformations taking place inside its boundary

and involving all elementary components of the cell (enzymes,

metabolites, RNA, etc). Self–maintenance is possible since the

cell regenerates from the inside all components (boundary

molecules included) that are being transformed and/or

disposed of. This is possible thanks to a network of processes

that produces all components, that in turn generate the

processes that produce such components, and so on

(Fig. 1a).8b The living cell, an autopoietic unit, is a thermo-

dynamic open system that exchanges matter and energy with

its environment. By definition, in autopoietic systems, and

therefore in living cells, the result of any transformation and

interactions among the system’s components is the production

of components that regenerate the system’s processes and so

on, following a circular organization—this property is called

operational closure.8a,b Autopoiesis is not restricted to merely a

descriptive statement of the living. It might indeed be used as a

theoretical framework to realize complex chemical and

biochemical systems obeying, possibly at different degrees,

the foundations of autopoietic theory. The question is then

whether and at what extent is it possible to design and

construct autopoieitic (molecular) systems in the laboratory.

In Fig. 1b, a cartoon represents a minimal autopoietic

system. The drawing is very general, and it can be easily

adapted to real molecular systems. An autopoietic system

sustains itself by transforming external components

(here indicated as P) into the elements (S) of the autopoietic

system, which self-organize into the autopoietic unit. The

transformation, here indicated by a simple process (P - S)

Fig. 1 Autopoiesis and minimal autopoietic systems. (a) The circular

logic of autopoiesis as minimal life. The process of living is seen as

cyclic, one in which the internally produced molecular components

assemble into the self-bounded functional structure, which generates

the microenvironment reaction (metabolic) network, which then

produces the molecular components. . . and so on. The system

exchange energy and matter with the external environment. (b) A

minimal autopoietic system is constituted by a self-bounded system,

which can uptake a precursor P from the environment, transform it by

one or more reaction(s) into the boundary element S, which can also

undergo a degradative process to W. Depending on the relative rates

of these processes, the autopoietic system can grow, stay in a homeo-

static state, or die. Redrawn after.8b

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occurs within the system, i.e. within the self-generating boundary

that separates and distinguishes the system from the environ-

ment. Together with the constructive/anabolic step (P - S),

there is a destructive/catabolic step (S - W) that transforms

the elements of the autopoietic system into different forms. In

molecular terms, the molecule P is the precursor of molecule S,

which is recruited by the autopoietic system as a functional/

functional component of its organization, and that it is

eventually converted into the waste product W. Thus, an

autopoietic system, working out of equilibrium, continuously

uptakes from—and releases components to—the environment

with the only result of maintaining its own organization

and structure. More precisely, structural components are

continuously renewed by the two concurrent anabolic and

catabolic processes; however, despite this continual turnover

of components, the whole organization of the autopoietic unit

does not change.

A simple phenomenological kinetic analysis is also shown in

Fig. 1b. The two processes (P - S, and S - W) proceed with

rates vP and vD, here indicated in their pseudo-first order form.

If the rate of S production exceeds the decay rate (vP–vD 4 0)

and the autopoietic unit grows; we will soon see that this

‘‘growth’’ is related to the autopoietic self-reproduction. When

vP–vD = 0, the two rates are equal, and this case represents a

homeostatic stationary state, with no net changes despite the

two concurrent reactions. In the third case, vP–vD o 0

the production rate is overwhelmed by the decay rate, so that

the autopoietic unit collapses (or dies) by gradual consump-

tion of its components. Several experimental studies have been

devoted to growth and self-reproduction, whereas there is only

one report on autopoietic homeostasis.11

In this article, the discussion will be focused on the first case,

when the particles show a tendency to grow.

The autopoietic mechanism

It is clear that growth alone brings about a physical enlarge-

ment of the autopoietic system, without the necessity of self-

reproduction. Self-reproduction implies growth and division,

since two or more systems must originate from the initial one.

This is possible only if a critical state is reached after growth,

and if such a state allows a spontaneous rearrangement,

generating new systems that are structurally and functionally

similar to the initial one.

The general mechanism for the self-reproduction of supra-

molecular structures such as micelles, reverse micelles and

vesicles follows the schemes shown in Fig. 1b and 2a. A

precursor binds to the surface of the structure, it is trans-

formed into the boundary-forming component by an inter-

facial or internalized reaction, bringing about an increase in

the number of molecules of the particle (Fig. 2a). For micelles

and reverse micelles (Fig. 2b–c, respectively), which are

assemblies at the thermodynamical equilibrium,12a,b the size

growth modifies the (i) aggregation number or (ii) the water-

to-surfactant ratio (indicated as w0 = [water]/[surfactant]),

respectively. Due to this forced change, the structure becomes

unstable in those given conditions, and rearranges to the most

stable state by division or fragmentation in smaller micelles or

reverse micelles. Vesicles (Fig. 2d), on the other hand, are very

often not at the thermodynamical equilibrium,12c,d being

recognized as metastable states in many surfactants’ phase

diagrams. Despite their metastable nature, however, vesicles

can exist for a very long time (up to several months). The fact

that large and small vesicles coexists in vesicle samples

suggests that the increased size is not the key factor for

stimulating a vesicle division after vesicle growth. Therefore,

the process of vesicles growth-division certainly involves more

complex pathways which depend on the way in which a certain

‘‘critical’’ state is reached. This means that thermodynamics

alone does not determine the vesicle transformation, and that

kinetics constrains have to be taken into account. Interest-

ingly, a general theoretical interpretation of the observed

behavior has not been formulated yet.

In other words, self-reproduction of micelles, reverse

micelles and vesicles do not stem directly from the autopoietic

theory, but emerges from the physico–chemical nature of these

self-assembled structures.

There are some consequences that derive from the general

mechanism of Fig. 2a. The first regards the general stoichio-

metry of such a process, which involves a non linear growth

of the particle number. In fact, in a pure division mechanism,

the increase in particle number follows a geometrical

progression (1, 2, 4, 8, . . . 2N after N divisions), as in cellular

division. Such a process can be defined as ‘‘autocatalytic’’,

because the overall production rate of new particles depends

ultimately on the number of already existing ones (see Box 1

for a comment on the nature of autocatalysis in such

processes).

Fig. 2 General mechanism for the self-reproduction of surfactant

based supramolecular structures (a). When a suitable precursor is

added to the particles, it can passively diffuse into the particle

boundaries, and there it can then be transformed into the boundary–

forming compound S. As a result, the particle size increases, and this

may bring about destabilization, and consequent division into two or

more new particles. The typical structures of micelles (b), reverse

micelles (c), and vesicles (d) are shown (drawn not to scale). Micelles

and vesicles form in aqueous phase, whereas reverse micelles in apolar

solvent. Micelles and reverse micelles have typical size of few nano-

metres, whereas vesicles’ size span from 30 nm to more than 50 mm.

Reproduced, with permission, from Ref. 46.

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Table 1 collects the experimental strategies used for

self-reproduction, classified according to the type of supra-

molecular aggregate and to the nature of precursors P. With

the exception of the recent reports from Sugawara and

coworkers,13 self-reproduction has been carried out by using

fatty acid amphiphiles, since they readily self-assemble in

different forms, depending on the solvent and on the pH of

the solution. Fatty acids form micelles or vesicles in aqueous

phase, at high and intermediate pH, respectively; they also

form reverse micelles in apolar solvents.

Thus in order to generate fatty acids, i.e., the building

blocks S shown in Fig. 2a, different chemical transformations

have been exploited (i.e., the P - S reaction). This is the case

for protonation of the corresponding soaps (Table 1, line 1), or

the alkaline hydrolysis of fatty acid esters and anhydrides

(line 2 and 3), or through the oxidation of the corresponding

long chain primary alcohol (line 4). Sugawara et al. introduced

a non fatty acid self-reproducing system, where the building

block S is an ad hoc designed bola amphiphile (line 5), formed

by a condensation reaction between an aromatic aldehyde and

an amine, to form a Schiff’s base.13

3. Self-reproduction of micelles and reverse

micelles

It was 1990 when the autopoietic approach was applied for the

first time to chemical systems,5a shortly preceded by a

programmatic script from Luisi and Varela.14

Reverse micelles (Fig. 2c) are supramolecular structures that

form spontaneously when certain surfactants are dispersed in

an apolar phase (the ‘‘oil’’), in the presence of a small amount

of aqueous phase (e.g. 0.1–0.5% v/v).

Reverse micelles meet several criteria for being potential

autopoietic particles. As the precursor P can be solubilized in

oil, the micelle can uptake it, and transform it into the

boundary-forming molecule S by means of a reaction

catalyzed by internalized compounds.

Three different approaches have been used to produce the

reverse micelles’ forming surfactant, which was in all cases

octanoic acid. In the first one,5b octanoic acid octyl ester

was hydrolyzed to octanoate and octyl alcohol by LiOH,

solubilized within reverse micelles. Using triglycerids as

precursors of fatty acids, it was shown that lipase-containing

reverse micelles can grow and divide by means of an enzymatic

hydrolysis.4a The permanganate–based oxidation of 1-octanol

to octanoic acid4a also provides a route to autopoietic

self-reproduction.

In contrast to reverse micelles, normal micelles form in

aqueous media, and can be considered self-bounded hydro-

phobic compartments (Fig. 2b). Initial studies4a aimed to

achieve micelles self-reproduction by using the permanganate

oxidation described above. Octanoic acid micelles were loaded

with 1-octanol, so that the precursor P in this case was

embedded within the micelle. After the total conversion of

Table 1 List of precursors used for the self-reproduction of supra-molecular fatty acids structures

# Precursor Vesicles MicellesReversemicelles

1 Fatty acidcarboxylate in formof micelles

18b,26,29,30,36 — —

2 Fatty acid methylor ethyl esters

20 4b 4a,5

3 Fatty acidanhydrides

21,33 — —

4 Long chain primaryalcohol

— 4a 4a

5 Ad hoc designed bolaamphiphile

13a — —

a Non-fatty acid giant vesicles.

Box 1. Autocatalysis and phase-transfer in

microheterogeneous systems

In the context of micelles and vesicle self-reproduction,

original work (see Table 1) has referred to such processes

as autocatalytic. The phenomenon of ‘‘autocatalysis’’ is,

in its more general meaning, the catalysis of a reaction by

the products. Let us indicate as P the surfactant precursor

that can be chemically converted into the surfactant S; and

as Sn a self-assembled structure derived from n molecules S

(see also Fig. 1b and 2). P is typically present as a separate

phase, since it is sparingly soluble in water.

When the process is decomposed into elementary steps, P

molecules are firstly absorbed by the aggregate Sn,

then—taking advantage by favourable exposure to aqueous

phase—are transformed into S molecules, so that the

aggregate Sn can grow (e.g., to give Sn+k), and eventually

divide. From this viewpoint, Sn acts as a microhetero-

geneous phase that solubilizes P so that it can react with

the reactant (generally present in the aqueous phase, e.g.,

hydroxide ions). Clearly, the amount of P solubilized by

Sn increases as Sn similarly increases its transformation

rate. Though it cannot be defined as a catalyst in strict

sense, Sn accelerates the reaction rate by increasing the

concentration of reacting Pmolecules and so acts in general

terms as a phase–transfer catalyst.

This mechanism, being based on phase transport, would

work with other substrates too—for instance, water-

insoluble compounds that are firstly solubilized into Sn,

then transformed and the products released in the medium.

On the other hand, the overall process is designed so that

the product S, derived from P, is actually the building block

of Sn, and the overall reaction can be indeed ideally

described as nP+Sn - 2Sn, i.e., an autocatalytic one.

In other words, the term ‘‘autocatalysis’’ is used in this

context in a broad sense, and it does not refer to an increase

of the rate constant for the transformation of P into S.

Sigmoid concentration–vs.–time curves have been

recorded experimentally by following these reactions; and

different models have been used to describe and simulate

numerically the course of micelle and vesicle self-

reproduction.15a,31,44

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1-octanol into octanoic acid by means of permanganate

oxidation, the micelle concentration increased by a factor of

1.4. Next studies on micelle self-reproduction were designed in

a way to closely simulate the general autopoietic mechanism

shown above. Ethyl caprylate, a water–insoluble ester, was

stratified over an alkaline solution, either in the absence or in

the presence of pre-formed caprylate micelles.4b Let us start by

illustrating the first case (absence of pre-formed micelles).

When ethyl caprylate is overlaid on a NaOH solution, the

formation of caprylate by alkaline hydrolysis is very sluggish,

taking place at the macroscopic interface between the ‘‘oil’’

and the aqueous solution. At the beginning, the concentration

of caprylate in water increases slowly with time, and its value is

below the caprylate critical micelle concentration (in these

conditions, c.m.c. B100 mM). Caprylate molecules slowly

accumulate in the water phase until the moment when the

c.a.c. is reached, and the first micelles form. This path

represents the spontaneous onset of micelles, that would act

as autopoietic units. Once micelles form, they can uptake ethyl

caprylate molecules, so that their hydrolysis, occurs at the

micellar interface. As a consequence of such in situ production

of new caprylate molecules, the micelles grow and divide. Since

more micelles form, more ethyl caprylate is taken up and

hydrolyzed, and so on, establishing an autocatalytic auto-

poietic self-reproduction. The caprylate concentration vs. time

profile clearly shows a sharply sigmoidal shape indicating an

autocatalytic mechanism. In particular, in the first 30 h,

caprylate concentration increases slowly, then suddenly

increases to give rise to a sort of step function, deriving from

a geometrical progression of micelle concentration. The over-

all rate of ethyl caprylate hydrolysis increases by a factor 900.

Once all ethyl caprylate is converted into caprylate, the process

stops, and the system becomes homogeneous. If micelles are

present from the beginning, the initial lag phase simply

decreases or disappears because the mechanism of micelle

self-reproduction can start immediately, without the need of

slowly reaching the c.m.c.

A short remark must be made on the word ‘‘catalysis’’ when

applied to these systems. In fact, it has been argued15 that this

term cannot be used properly in such context, since it generally

refers to the decrease of the activation energy of key reactions.

In this context, the ‘‘catalytic’’ effect comes from the

tremendous increase of microscopic interface, which is a sort

of ‘‘physical catalysis’’.

The use of micelles (and reverse micelles) as model

autopoietic systems was soon overcome by the interest in

vesicle self-reproduction. Quite recently, however, micelle-

based systems have again attracted the interest of researchers

in Los Alamos (US) and in Europe, who aim to develop the

so-called ‘‘Los Alamos Bug’’16, a chemical system embedded

in micelles which should perform a series of reactions leading

ultimately to the self-reproduction of the whole structure.

Moreover, new studies have been recently published on the

self-reproduction of water-in-oil microdroplets,6 that is inter-

esting because such synthetic compartments have recently

found successful application in molecular evolution and

library screening and microfluidics.17

To summarize, the studies on self-reproduction of supra-

molecular aggregates started with reverse micelles and soon

after with micelles. These systems are characterized by a fast

molecular dynamics and are generally considered equilibrium

systems; as a consequence, micellar growth is followed by a

spontaneous rearrangement (i.e., formation of new micelles)

since large micelles are not stable under those experimental

conditions.

4. Self-reproduction of vesicles

The first studies on self-reproduction of vesicles were carried out

immediately after the results achieved with micelles. In contrast

to these smaller compartments, however, vesicles are generally

not considered equilibrium structures, and therefore it can be

argued that their self-reproduction requires a more complex

explanation, which is not yet available, although some mecha-

nistic hypotheses have been proposed and recently discussed.18

4.1 Conventional fatty acid vesicles

Here we will refer to vesicles with sizes well below 0.5–1 mm as

conventional vesicles (as opposite to ‘‘giant’’ ones), whose

self-reproduction studies are summarized in Table 2. All works

have been carried out with fatty acid vesicles and mixed

vesicles (phospholipids/fatty acids), whereas we record only

one study done on cationic systems.19 A reason for such choice

is due to the distinctive properties of fatty acids. Fatty acid

vesicles are rather dynamical systems when compared with

phospholipids. For example they have a relatively high critical

aggregation concentration (c.a.c.) and differ in several aspects

with phospholipid vesicles, for example in permeability,

stability, interaction with other lipids. The second feature

stems from the recognition that fatty acid vesicles are, to date,

the most plausible candidate to play the role of prebiotic

compartments.3

To understand the chemistry of fatty acid assemblies, their

ionization pattern must be first examined. We have reported a

short introduction to these systems in Box 2.

Table 2 Original reports on conventional vesicles self-reproduction

Year Description Precursor Reference

1991 Attempt to vesicle self-reproductionby reconstituting a lipid-synthesispathway catalyzed by four enzymes

G3P,acylCoAs,CDP-choline

41a

1993 Attempts to self-reproduce oleatevesicles by lipase-catalysis

Ethyl oleate 20

19941994 Self-reproduction of oleate vesicles

by alkaline hydrolysis of oleicanhydride (and simultaneousinternalized polymerisation of ADPto poly(A) or RNA replication)

Oleicanhydride

21

19951998 Self-reproduction of POPC and

oleate vesicles by oleatemicelle-to-vesicle conversion

Oleatemicelles

26

19992004 Self-reproduction of POPC/DDAB

vesiclesDDAB 19

G3P: glycerol-3-phosphate, AcylCoAs: acyl-coenzyme-A, CDP-choline:

cytidine-diphospho-choline, ADP: adenosine diphosphate, Poly(A):

poly(adenilate), POPC: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidyl-

choline, DDAB: didodecyl-dimethyl-ammonium bromide

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In order to observe an autopoietic self-reproduction of

fatty acid vesicles, water–insoluble precursors, as fatty acid

ethyl and methyl esters, and fatty acid anhydrides were

successfully employed in the first studies, dated in the

early Nineties. An initial attempt was based on the lipase-

catalyzed hydrolysis of ethyl oleate (the precursor), to give

oleate. The overall system was designed in order to act as a

bioreactor that could uptake ethyl oleate from the environ-

ment, transform it into the boundary forming molecule

(oleate), grow its membranous surface, and divide.20 Unfortu-

nately, the process was rather slow, being complete in about

200 h; and a clear demonstration of growth-division steps was

not provided.

Fatty acid anhydrides were employed soon after21 as

alternative fatty acid precursors. In this case a chemical

transformation (alkaline hydrolysis mainly at pH 8.5) was

sufficient to drive the in situ formation of fatty acids. The

overall system, then, foresaw the binding of hydrophobic

anhydride to the fatty acid bilayer of vesicles, so that the

reactive event (e.g., the anhydride hydrolysis) occurred

at the interface between the membrane and the solution

(see Fig. 2a).

Experimental evidences—based on turbidity, chemical

analysis, and electron microscopy—demonstrate that the

formation of oleic acid vesicles from oleic anhydride follows

the general pattern of autopoietic self-reproduction, as in the

case of micelles. In particular, the sigmoidal concentration vs.

time profile is observed also with vesicles, and the presence or

the absence of pre-formed vesicles at the beginning of the

anhydride hydrolysis produce typical patterns that can be

ascribed to an autocatalytic process, i.e., vesicles ‘‘catalyze’’

their own formation.21a–c As remarked before, this sort of

catalysis is a physical catalysis, based on the absorption of

precursors on the surface of the self-reproducing particle.

Next, there has been a shift from the heterogeneous

(two-phases) system based on oleic anhydride or oleate esters

in favour of a homogenous one, based on the spontaneous

micelles–to–vesicles transition, typical of fatty acids. This

approach, in fact, allows real-time photometric analysis such

as spectroturbidimetry, fluorescence measurements, and light

scattering.

It is in fact possible to add a small aliquot of concentrated

oleate micelles (pH 4 10) to a buffered solution (pH 8.5),

containing—or not—pre-formed oleate vesicles. At this lower

pH, oleate molecules, assembled essentially as micelles, will

finally reach the association state typical of the lamellar phase,

i.e. form vesicles. The mechanism may involve a rearrange-

ment of the micelles themselves, or the involvement of

monomeric oleate molecules, always present in the solution.

Irrespective of the detailed mechanism, fatty acid micelles act

as precursors of fatty acid vesicles, as in the case of anhydride

or ester hydrolysis. Notice however, that the generation of the

membrane-forming molecule (the oleate) does not involve the

break of strong covalent bonds, as in the case of ester or

anhydride hydrolysis. Nevertheless, investigations on this

homogeneous system allowed a more detailed analysis of

vesicle self-reproduction, which was hindered by the more

complex biphasic mechanism based on the use of water-

insoluble precursors. It is therefore clear that such an

approach has been studied in greater detail.

Box 2. Self-assembly of fatty acids in aqueous

solution

The assembly of fatty acids in aqueous solution shows

a characteristic behavior due to the carboxylic group

ionization degree. It has been shown3 that fatty acid

bilayers can be formed only at intermediate pH, where

the carboxylate and the acid form coexist. ‘‘Fatty acid

vesicles’’, therefore, are mixed vesicles formed by the

carboxylic and the carboxylate forms of fatty acids

(we will refer to them simply as ‘‘fatty acids vesicles’’, for

brevity). At the two sides of the intermediate pH range

where vesicles are stable, there is the phase separation at

low pH (where the acid is fully protonated), and the

formation of micelles at high pH, where fatty acids are

fully ionized. The stability of fatty acid bilayers has been

explained by the formation of hydrogen bonds22 between

the carboxylate and the carboxylic acid heads of partially

ionized fatty acids, that corresponds to a reduction of head

groups repulsion, and to a formal shift of the surfactant

parameter from conical to quasi-cylindrical shape, that

according to classical treatment,23 allows the formation of

the lamellar phase, and therefore of vesicles.

It is interesting to note that the approximately equimolar

presence of acid and base form in fatty acid vesicles occurs

typically at pH values from 7 to 9.6, indicating that the pKa

of fatty acids in their assembled form has been shifted by

around 3 pH units when compared to carboxylic acids,

e.g., acetic acid (pKa 4.7). This has been explained by a

proton–sequestering electrostatic effect exerted by the poly-

anionic surface of fatty acid membranes. In other words,

carboxylates are stronger bases when organized as a poly-

anionic surface. They become fully protonated at pH 7 and

release all the protons at pH 10.22

A simplified account on fatty acid assemblies states

therefore that, above a critical concentration, two kinds

of organized molecular systems exist, depending on the pH

of the solution. At an intermediate pH (generally between

7 and 9), the lamellar state prevails, whereas micelles form

at higher pH. These two forms can be obtained simply by

changing the pH of the solution, i.e., in a fully reversible

way. Recent reports have however re-evaluated the simplistic

pH-morphology model, by measuring—via electron spin

resonance—the coexistence of mixed systems, for example

vesicles and other micelle-like forms, at intermediate pH.24

Although the simple interpretative model may suffice for a

rough description of fatty acid behavior in aqueous

solution, it must be recognized that the effect of counter

ions, co-solvents, co-surfactants, temperature and concen-

tration can play a major role in determining the structure

and the stability of fatty acid assemblies. Recent reviews

point out the complexity of fatty acid phase diagram.3i,25

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4.2 The ‘‘matrix’’ effect

What is the matrix effect? The use of oleate micelles instead

of oleic anhydride, as surfactant precursors, was introduced in

1998 by Bloechliger et al.26a In this work it was discovered that

the micelle-to-vesicle transformation was accelerated by the

presence of pre-formed vesicles, and autocatalysis was evident

from the sigmoidal turbidity–time profile. Moreover, detailed

electron microscopy and light scattering analysis surprisingly

indicated that at the end of the self-reproduction a rather

homogeneous distribution of vesicle sizes was obtained, and

that this size corresponded to that of pre-formed vesicles.

This was in contrast to the broad size distribution that

characterized the spontaneous micelles-to-vesicles trans-

formation of oleate micelles, when carried out in the absence

of pre-formed vesicles (Fig. 3a). These observation were

ascribed to an effect of the ‘‘matrix’’ (the pre-existing vesicles)

on the formation of new oleate vesicles from oleate micelles.

Therefore, the matrix effect is a sort of kinetic and size-

controlling effect exerted by the pre-existing vesicles on the

formation of new vesicles. Clearly, pre-formed vesicles interact

with freshly added oleate micelles, by providing an

alternative (and faster) pathway when compared with the

unassisted micelles-to-vesicle transformation in the absence

of pre-formed ones.

In principle, the uptake of boundary–forming compounds

by pre-formed vesicles could also bring about vesicle growth,

which would correspond to autopoietic growth. The fact that

vesicle sizes do not increase significantly means that a

fragmentation process is also present, and we can identify

the overall process as a self-reproductive act.

Lonchin et al.26b extended the previous observations by

showing that pre-formed phospholipid vesicles (made of

POPC = 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine),

act similarly; i.e., oleate micelles can be added to POPC

liposomes to give vesicle self-reproduction. Moreover, POPC

liposomes act as powerful ‘‘matrix’’ structures, since a matrix

effect is even present at 1 : 1000 POPC vesicle/oleate micelle

molar ratio, suggesting a very strong interaction between

pre-formed phospholipid vesicles and freshly added oleate.

This behavior is certainly favoured by the easy and fast

insertion of fatty acids into phosphatidylcholine membranes.27

It is however unclear how a very small amount of pre-formed

POPC vesicles may tremendously influence oleate vesicle

formation.

Investigating the mechanism of the matrix effect. As con-

sequence of these initial observations, a large amount of work

has followed, mainly for mechanistic purposes, based on

different techniques like electron microscopy, dynamic light

scattering, turbidity, fluorescence, stopped-flow, and chromato-

graphic separations (see Table 3) as well as theoretical

modeling.28

In a couple of 2001 papers,29a,b the strategy was to label

pre-formed vesicles with water-soluble compounds, and to

follow the distribution of such probes after the self-reproduction.

Vesicles from POPC as well as oleate were loaded with ferritin,

which can be easily visualized by electron microscopy. Oleic

anhydride or oleate micelles were then added and the initial

state (pre-formed vesicles) and the final one (product of the

reaction) were carefully analyzed by means of cryo-

transmission electron microscopy (cryo-TEM), so that vesicles

were characterized by their size and ferritin content. The aim

of the experiment was to follow the size distribution of

vesicles, and the numerical distribution of ferritin inside

vesicles. Two extreme cases can be depicted theoretically

(Fig. 4): (a) the formation of new vesicles (from oleate micelles

or oleic anhydride) follows an independent path, without

interaction with pre-formed ferritin–containing vesicles, or

(b) the formation of new vesicles follows an interaction

Fig. 3 Self-reproduction of oleate vesicles and the matrix effect. In

the first experimental setup (a), oleic anhydride is added to buffer

solution to record its hydrolysis and consequent spontaneous vesicle

formation of oleate vesicles. Alternatively, oleic anhydride is added to

a suspension of pre-formed oleate vesicles, to induce vesicle self-

reproduction. Electron microscopy analysis shows that in the absence

of pre-formed vesicles, a broadly sized vesicle population is produced

(histogram 1). In contrast, when pre-formed vesicles having a narrow

size distribution are present (histogram 2), the new vesicles have a size

distribution very close to the size distribution of pre-formed ones

(histogram 3). This has been called ‘‘a matrix effect’’. Turbidimetric

kinetic analysis indicate that preformed vesicles increase the rate of

vesicle formation when oleate micelles are added to pre-formed

POPC vesicles (b). Oleate micelles slowly transform into vesicles

(curve 1). This process is greatly accelerated by the presence of

pre-formed vesicles (curve 2). Redrawn after Bloechliger et al.,26a

and Lonchin et al.26b

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between the precursor(s) and the pre-formed ferritin–containing

vesicles, which growth (b1) or growth and divide (b2). For the

mechanism (a), the average number of trapped ferritin

molecules should not change significantly, and the size of

ferritin containing vesicle should also remain constant.

Moreover, at the end of the process, several ‘‘empty’’ vesicles

are expected to be found. In mechanism (b1) it is expected that

the average ferritin content does not change, and that the size

of ferritin–containing vesicles increases. In mechanism (b2) it

is instead foreseeable that the average number of ferritin

molecules inside vesicles decreases, and that the size of such

vesicles does not differ substantially from the pre-existing

vesicles (the matrix effect). The experimental results indicate

that when oleic anhydride (5 fold excess) is added to

pre-formed ferritin–containing oleate vesicles, vesicle growth

and de novo formation of vesicles are the most abundant

processes. Thus, in this case, mechanisms (a) and (b1) operate.

In contrast, when oleate micelles (25 fold excess) are added to

POPC vesicles, only some small empty vesicles are found,

whereas ferritin–containing vesicles were characterized by a

clear reduction of the average number of ferritin molecules per

vesicles, indicating that vesicles divide and ferritin is distri-

buted among daughter vesicles. In this case, mechanism (b2) is

dominant.

Rasi et al.,29c,d by using turbidity and DLS analysis, studied

the matrix–effect for differently sized vesicles, and demon-

strated that the conservation of size is particularly evident for

POPC vesicles after addition of oleate micelles, whereas if

pre-formed vesicles are fatty acid vesicles they mainly display a

growth dynamic. Further analysis was performed in order to

extract number–weighted size distribution data from raw DLS

results, showing that the number of vesicle increases after

self-reproduction.29e

These results were successively expanded by the report of

Cheng and Luisi,29f who have shown that 135 nm vesicles

undergo the process of self-reproduction faster than smaller

ones (65 nm), by a rate factor of about three.

Ueno and coworkers also investigated the effect of

pre-formed lecithin vesicles on the vesicle formation from

oleate micelles.30 Thanks to size exclusion chromatography

(SEC), the authors were able to separate vesicles of different

sizes, suggesting that new small vesicles were indeed formed in

addition to vesicles with the same size as the initial ones. From

this study the authors suggested an alternative mechanism to

explain data on the addition of oleate micelles to pre-formed

lecithin vesicles. In this alternative mechanism, pre-formed

vesicles were transitorily solubilized by oleate micelles, which

act as a detergent (to give mixed micelles). The final state

reached by this process was however the vesicular one.

Detailed kinetic studies have been carried out by Chen and

Szostak,18b as well as Walde and Robinson et al.,29g both

employing stopped–flow techniques.

Chen and Szostak18b studied fatty acid vesicle growth based

on FRET analysis. To this aim, fluorescent dyes were firstly

incorporated into pre-formed oleate vesicles. When oleate

molecules inserted into pre-existing vesicle membrane the

‘‘dilution’’ of the dyes, which brought about a decrease in

the FRET signal, was expected. It was shown that vesicle

growth followed a single exponential profile for low micelles-

to-vesicles ratio (o0.4), whereas a two–phase kinetic profile

was recorded at higher ratios. The first fast process has been

described as vesicle ‘‘coating’’ by micelles, with consequent

rearrangement (incorporation/growth). The second slow

process was instead ascribed to an independent micelle-to-

vesicle transformation (without participation of pre-existing

vesicles), which occurred when ‘‘excess’’ micelles were present,

and pre-formed vesicles had been already coated.

The addition of oleate, linoleate and caprilate micelles to

1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) was

investigated by Walde and Robinson and coworkers, who

used stopped–flow turbidimetry. In this case, data were

interpreted by following the model of fast equilibrium between

Table 3 Further studies of vesicle self-reproduction

Year Description Reference

2001 Cryo-TEM studies of ferritin-containing POPCand oleate vesicles self-reproduce

29a,b

2003 DLS and turbidity studies on oleate vesicleself-reproduction

29c–f

20042004 Vesicle self-reproduction studies via vesicle SEC

separation and analysis30

20052004 Stopped-flow fluorescence and turbidimetry

studies of oleate vesicle self-reproduction18b,29g

20062006 ffEM study of oleate vesicle self-reproduction 29h

cryo-TEM: cryo-transmission electronmicroscopy, POPC: 1-palmitoyl-

2-oleoyl-sn-glycero-3-phosphatidylcholine, DLS: dynamic light

scattering, SEC: size exclusion chromatography (gel filtration

chromatography), ffEM: freeze-fracture electron microscopy

Fig. 4 Ferritin–containing vesicles and the mechanism of vesicle self-

reproduction. New vesicles, formed after the addition of precursors to

ferritin–containing vesicles, can follow an independent path (a), where

new empty vesicles should be found. If the precursor is taken up by

pre-formed vesicles (b1, b1), these may grow without dividing (b1), as

revealed by the fact that the average number of entrapped ferritin

should be approximately constant, and due to their larger size. If, on

the other hand, a division occurs (b2), the average number of

entrapped ferritin should decrease, and the size of ferritin–containing

vesicles should not increase significantly. Experimental data suggest

the dominance of path (b2). Data taken from Berclaz et al.29a,b

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fatty acid micelles and monomeric fatty acid molecules. The

authors demonstrated that initial rates were substantially

unaffected by the micelles-to-vesicles ratio, and suggested

that monomers were taken up by pre-formed vesicles.

Fluorescein–released measurements (also confirmed in

ref. 29h) indicated that the solutes entrapped inside vesicles

was not significantly released during the process, suggesting

that the mechanism of vesicle solubilization30 was unlikely to

be the main process.

Perhaps, one of the most interesting pieces of evidence for

the mechanism of the matrix effect in vesicle self-reproduction

is a recent report that describes possible intermediates of the

self-reproduction process.29h Freeze-fracture electron micro-

graphs of samples frozen immediately after oleate micelles

addition to pre-formed oleate vesiscles, show a relatively

abundant proportion (20%) of typical dimeric structures,

called ‘‘twin vesicles’’ (Fig. 5a), whereas such structures are

not present at the end of the process.

To summarize, although there are no doubts on the inter-

action of micelles with pre-formed vesicles, the detailed

pathway that follows remains elusive. In particular, one open

question is about the geometry and morphology of vesicles. If

the vesicle growth follows a spherical symmetry, the internal

volume has to increase together with the surface growth. This

means that solvent must enter into the growing vesicle in order

to keep the surface-to-volume ratio constant. If, on the other

hand, the volume growth is hindered, the vesicle growth

consequently becomes non-spherical, and the surface increase

overcomes volume increase.18a The two mechanisms require

different additional specifications. Whereas in the second case

the division might be caused by a physical instability related to

the non-spherical shape, in the first case it is difficult to explain

how a spontaneous shape transition may take place starting

from a spherical symmetry. Another important issue is the

influence of flip-flop rate of fatty acids on the relaxation of

membrane excess curvature probably caused by insertion of

oleate molecules in the outer membrane leaflet. This may cause

a morphological change that favours vesicle destabilization.

Theoretical studies and modeling. Theoretical studies on

vesicle self-reproduction and the matrix effect have been

reported. In particular, Svetina’s physical analysis18c,d focuses

on the relationship among the membrane bending modulus, its

hydraulic permeability and spontaneous curvature, as well as

the rate of membrane growth, looking for optimal conditions

for vesicle self-reproduction. This ultimately suggests that

vesicles with optimal physical parameters have selective

advantages for self-reproduction. Fanelli and McKane18e

developed a model based on irreversible thermodynamics by

carefully taking into account the membrane energy in their

description of vesicle instability. Bolton and Wattis,31a–c on

the other hand, have developed a microscopic model based on

a novel generalization of the Becker-Doring equations of

nucleation, which describes the stepwise growth of vesicles,

and that reproduces the experimentally observed kinetics quite

well. Kinetic models and stochastic simulations have been

reported by Mavelli,28a,f whereas different models for

self-reproducing systems have been proposed by Sole and

coworkers.31d

4.3 Giant vesicles

Giant vesicles (GVs), thanks to their distinctive large size

(4 10 mm) are easily visualized by optical microscopy, and

knowledge about their formation, stability, and reactivity can

be gained by direct observation, often facilitated by the use of

fluorescent dyes that mark the vesicle lumen and/or the

membrane. Despite this obvious advantage, there is a limited

number of reports dealing with self-reproduction of GVs. This

can be due to two reasons. Firstly, the GVs preparation

methods require a fine control of experimental conditions—

such as the electroformation,32a the controlled swelling,32b,c

and others32c,d—and often do not allow the use of buffers at

high ionic strengths. Secondly, it is questionable whether the

reactivity of giant vesicles mirrors that of the submicrometric

vesicles. The very different size implies a different membrane

curvature and different surface-to-volume ratios, so that forces

and dynamics in GVs can be in a very different range when

compared with smaller vesicles.

A first attempt to self-reproduce GVs was reported in

1994–95 by Wick et al.33 Similarly to the approach used for

small vesicles, oleic anhydride was added to oleic acid giant

vesicles. Optical microscopy analysis revealed that—although

with difficult reproducibility—two processes may take place in

GVs systems. The first one belonged to the class of non–

symmetrical growth, since self-reproduction proceeded as a

budding from the parent vesicle, followed by division. The

second one followed an initial formation of a bilamellar vesicle

(inclusion vesicle), that evolved symmetrically by enlarging the

external membrane, with a final expulsion of the internal

vesicle, a phenomenon called ‘‘translocation’’, and it was

analogous to that described by Menger.34 To date, there is

no simple physical explanation of such a mechanism.

As recently reported by Sugawara and coworkers13, GVs

self-reproduction can also be achieved with non-fatty acid

Fig. 5 Freeze–fracture electron microscopy of oleate vesicles (taken

after 40 s from the addition of oleate micelles) reveals that ‘‘twin

vesicles’’ can be the actual intermediate of the self-reproduction. Twin

vesicles are not present at the end of the reaction. Adapted from

Stano et al.29h Several intermediates have been observed in the case of

the transformations occurring to oleate giant vesicles after the addition

of oleate micelles: (1) budding mechanism;33a,b (2) translocation;33a,b

(3) evagination;36b (4) tubular growth and division.36a

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vesicles. In this new approach, ad hoc designed pro-surfactants

are used to set up a rather complex scheme, that involves either

chemical reactions or supramolecular assembly (Fig. 6). The

membrane is composed of a bola surfactant S, whose

precursors are two building blocks A and B (A brings an

aldehyde function, B an amine, and the bola amphiphile S is

formed by tail-to-tail Schiff base binding of A and B). Notice

that, in contrast to the approaches with fatty acids, the

‘‘P - S’’ transformation is now a bond-forming reaction.

The building block B, as well as a catalyst C are present inside

a pre-formed GV, and a precursor A0 is added to the system.

As A0 enters into the GV, it is transformed into A by means of

the catalyst C, and finally A and B react to produce S, the

bilayer–forming compound. However, as indicated by the

experimental observation, S molecules self-assemble inside

the GV to give rise to an internalized new vesicle (inclusion

vesicle), that translocates outside. The overall process brings

about two vesicles starting from one. This system is interesting

since the formation of the surfactant S can occur only inside

the parent GV. However, since the catalyst C is not

reproduced, the process will ultimately stop due to dilution

among the newly formed vesicles. We will come back to this

issue in the final section of this paper. The final note about

Sugawara’s report is its relevance for systems chemistry,35

intended as a fully–synthetic system of reacting and self-

assembling molecules.

More recently, a new report on GVs self-reproduction has

appeared, based on the addition of fatty acid micelles

(oleate or myristoleate micelles) to multilamellar GVs.36a

Zhu and Szostak show that such kinds of vesicles may grow

by elongation, transforming into a fragile cylindrical GV that

can be fragmented by mild mechanical agitation. Moreover, it

has been shown that the entire process can be repeated several

times, providing a route to a self-reproducing cycle for

vesicles. When RNA is inserted into GVs, self-reproduction

occurs with almost no content leakage. From a conceptual

point of view, it is remarkable that the non-spherical vesicle

growth easily brings about mechanical instability, which

determines the vesicle fragmentation in several smaller

(and spherical or spheroidal) vesicles. Clearly, in this case

the vesicle surface also grows faster than its volume. Insightful

control experiments carried out with permeable buffer species

(ammonium acetate) suggest that the slower volume growth

observed with non-permeable buffers (e.g., bicine) can be due

to the emergence of osmotic gradients, that counteract the

swelling of vesicles, and therefore favor the departure from

spherical growth.

The direct observation of fatty acids (as monomers or in the

form of vesicles) interacting with lecithin unilamellar GVs has

also been reported by Peterlin et al.,36b who recorded first an

initial increase of the vesicle size, then the appearance of

unexpected patterns, like evagination of small satellite vesicles

connected to the mother vesicle by narrow necks. Tubular

protrusions were also observed.

In conclusion, GVs perfectly fulfil the requirements

for a direct observation of a variety of transformations

(morphological changes, fusion, growth, self-reproduction),

they also have the additional possibility of following fluores-

cent markers. The available reports, however, show a rather

complex and strongly conditions–dependent pattern that may

be very different from what has been proposed in the case of

submicrometric unilamellar vesicles. It is important to note,

for example, that the curvature of GVs membranes is almost

negligible when compared with that of conventional (small)

vesicles. It follows that local changes of membrane composi-

tion or curvature, arising from localized uptake, or from

fluctuations, may direct the morphological changes and the

reactivity. Further studies are certainly needed to deepen our

knowledge on such systems.

5. Towards semi–synthetic cells and their

self-reproduction

Both in its importance for supramolecular chemistry and in

studies of the origins of life, vesicles self-reproduction has

prompted the development of more complex models of

minimal self-reproducing systems, namely the construction

of vesicle–based cell–like systems, with the final aim of

creating living cells in the laboratory. At this aim, chemists,

physicists and biologists collaborate in a convergent way to

design and build the first man–made cell, displaying all or

some living properties. These constructs, which are called

protocells, artificial cells, minimal cells, synthetic cells or

semi-synthetic cells, are the subject of flourishing research into

the origins of life and synthetic biology communities. Despite

the different names (that somehow reflect different facets of

this research) there is a common feature that keep these

apparently diverse approaches together. It is the quest

for chemical systems, based on microcompartments

Fig. 6 Self-reproduction of giant vesicles (GVs) (Sugawara and

coworkers13). The membrane of GVs is composed by the surfactant S

(a Schiff base), derived by the condensation of the precursors A

(an aldehyde) and B (an amine). The aldehyde A, in turn, derives from

the acetal A0, which is converted into A by the help of the catalyst C,

present only inside GVs. After the synthesis of enough S molecules,

they form a smaller GV that is thereafter expelled outside the ‘‘mother

GV’’. Such mechanisms, called ‘‘translocation’’ have been observed in

similar systems.33,34

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(typically vesicles) that are organized at the structural and

functional levels and that display cell–like behavior.

There are several groups which are actively involved in this

field. In addition to our research line based on the semi–

synthetic approach,7,10c,21a,37 a large amount of work has been

done recently by Jack Szostak at Harvard on fatty acid

vesicles,2e,18b,36a,38a by David Deamer at the University of

California on several aspects of vesicle chemistry38b–d and by

Tetsuya Yomo at the University of Osaka on giant

multilamellar vesicles (see below). With the name of ‘‘Los

Alamos bug’’,16,38e,f Steen Rasmussen and coworkers have

defined a system, currently under investigation, based on

micelles that should produce their own components on the

basis of a minimal reaction network embedded within the

surface of micelles. The possibility of communication between

artificial and natural cells have been recently investigated.38g

Despite growing interest in studies on reactions and bio-

molecular systems inside vesicles, only a few have specifically

addressed the point of self-reproduction of the whole

structure, or at least of the synthesis of the boundary mole-

cules. We limit ourselves to the discussion of these few

attempts, by shortly introducing the strategies and the results.

The semi–synthetic approach (Fig. 7) consists of

vesicle–based assemblies, composed of the minimal number

of biochemical components, such as enzymes, DNA genes and

RNAs, which are placed together in synthetic compartments

(vesicles). Thanks to the fact that transformation and

functions can be carried out by well-known biomolecules

(enzymes, ribosomes, etc.) it is expected that the semi–

synthetic approach could really bring about the construction

of artificial cells in the near future. On the other hand, systems

based on simpler molecules (and possibly prebiotically

plausible) would have the advantage of being better models

of early cells and shed light on the transition from the

non–living to the living world.

The philosophy behind minimal cells lies again in the

autopoietic theory. In particular, emphasis is placed on the

need for a cellular system of minimal complexity that has an

internal chemical network producing itself according to the

operational closure of autopoietic systems. Clearly, this

‘‘minimal’’ requirement depends on the chemical nature of

the network, i.e., a biochemical network based on modern

translation necessarily requires a large number of subunits, but

nevertheless it accomplishes only a few, simple functions.

Therefore, semi–synthetic minimal cells should not be seen

as structuralmodels of early cells (this is clearly not compatible

with the nature of the enzymes, DNA, etc, used to construct

them), rather they should be seen as minimal functional

models. Here we mean that the minimal cell should have the

minimal number of functions required for its autopoietic self-

maintenance, self-reproduction, and evolvability. Autopoiesis,

on the other hand, is a necessary but not sufficiently a

condition for life, and therefore the claim to construct ‘‘living’’

cells has been subjected to deep discussions.8b,c,11c

There are already several reports on complex enzymatic

reactions inside vesicles (see Table 4), ranging from polymeri-

sation of ADP to form poly(A),21d,39a RNA replication,21a,39b

polymerase chain reaction,39c mRNA translation,39d DNA

elongation39e and DNA transcription.39f Some of these reac-

tions have been already carried out simultaneously to vesicle

self-reproduction,21a,d achieved with the method of anhydride

hydrolysis (see asterisk labeled entries in Table 4). More

complex reactions, as the protein expression inside lipid

vesicle,40 are now possible by the semi–synthetic approach.

Also the explicit ‘‘population approach’’ highlighted in the

works by Yomo and coworkers is noteworthy.39b,40a,c,d,f

Fig. 7 A cartoon illustrating the construction of semi–synthetic

minimal cell from isolated compounds. DNA, enzymes, ribosomes,

etc. can be entrapped within lipid vesicles, giving rise to complex

systems that may synthesize proteins, enzymes, and originate minimal

living functions. Low molecular-weight compounds are either

provided from the beginning or added from outside. Reproduced with

minor modifications from,45 with permission.

Table 4 Compartmentalized reactions carried out in vesicles

Year Description Reference

1994 ADP polymerization to poly/A, catalyzed byPNPase (*)

21d,39a

19941995 RNA replication in small vesicles (*) 21a,c1995 Polymerase chain reaction (PCR) in small vesicles 39c1999 mRNA translation in small vesicles 39d2001 DNA transcription in large and giant vesicles 39f20012003 GFP synthesis in small, large and giant vesicles 40a–c2004 Cascade proteins (T7 RNA polymerase and GFP)

expression in large and giant vesicles40d

2004 Proteins (hemolysin and GFP) expression in giantvesicles

40e

20062007 GFP synthesis with purified enzymes (PURE-

SYSTEMs) in small, sub-micrometric, large andgiant vesicles

40f,g,i

20072009 Membrane proteins (GPAT and LPAAT)

expression in submicrometric vesicles by usingPURESYSTEMs, and lipid biosynthesis insidevesicles

40h,41c

2008 Coupled synthesis of Qb replicase inside vesiclesand replication of RNA, and synthesis ofb-galactosidase

39b

(*) Works where internalized reactions occurred together with vesicle

(shell) self-reproduction, ADP: adenosine diphosphate, PNPase:

polynucleotide phosphorilase, GFP: green fluorescent protein, T7: a

kind of promoter sequence, PURESYSTEMs: a kit of 36 purified

enzymes, t-RNAs and ribosomes of known composition, GPAT:

glycerol-3-phosphate 1-acyltransferase, EC [2.3.1.15], LPAAT:

lysophosphatidic acid 2-acyltransferase, EC [2.3.1.51].

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Once it is possible to express functional proteins inside

vesicles, cell–like systems can in principle be realized, for

example implementing some interesting functions inside

vesicles, such as nutrient uptake, replication of components,

cell growth, or lipid synthesis.

Is it possible to achieve self-reproduction of semi–synthetic

minimal cells?

In order to accomplish complete self-reproduction, minimal

cells must undergo the so-called core-and-shell self-reproduction

(Fig. 8). By ‘‘core’’ self-reproduction we mean the reproduction—

from within—of all components present in the minimal cell

(enzymes, genes, etc.). By ‘‘shell’’ self-reproduction we mean a

process where the vesicle membrane (that encloses the core

components and physically defines the minimal cell) uptakes

membrane–forming molecules, with a consequent growth and

division. Ideally, the ‘‘shell’’ self-reproduction should occur by

incorporating the in situ generated lipids (generated by the

internal metabolic network). For a sustainable self-reproduction,

the redistribution of the core and shell components among the

two, or more, new compartments that arise from the division

of the parent should be well balanced, in order to avoid the

lack of key components. In this eventuality, the newborn cells

will not have all the required functions, and will die. The same

happens when at least one required component is not

produced among the cycles of growth–division, or in the

limited cases of shell reproduction without core components

reproduction. Sooner or later, there will be cells that will miss

such components, and die (the so-called ‘‘death by dilution’’).

Examples of the latter scenario can be found in early

works.21a,d

The core-and-shell self-reproduction, as can be easily under-

stood, is a rather far and difficult goal, but there have been

attempts to carry out studies by dividing this complex task

into simpler sub-tasks, so that the final results might be

achieved by stepwise increasing our knowledge and technical

ability.

The very first attempt to accomplish a sort of shell

self-reproduction, was carried out in 1991 by Schmidli et al.,41a

who described the reconstitution—in lecithin liposomes—of

the whole four-enzymes route to carry out the biosynthesis of

lecithin itself starting from water–soluble precursors. This

system was clearly a way to achieve self-reproduction of

vesicles by enzymatic transformation of precursors P into

the membrane–forming compound S (a lecithin). Clearly, the

catalysts of such a transformation (the four enzymes) were not

supposed to be reproduced during the process of lipid

synthesis, so that—even if successful—this approach would

have brought about death by dilution after a certain

number of self-reproduction cycles. In the work of Schmidli

et al., the low overall yield did not allow the imitation of

the general scheme indicated in Fig. 2a, but it was possible

to demonstrate that the four enzymes could sequentially

accomplish the synthesis of short chain lecithins inside

liposomes. A first possible advance to achieve core-and-shell

self-reproduction based on the system of Schmidli et al.41a

was to insert the machinery needed to produce in situ

the enzymes required for the synthesis of lipids into lipid

vesicles. In this way, enzymes were produced, which in

turn produced lipids, and core-and-shell self-reproduction

could be sustained—at least partially. In fact, the core

self-reproduction was not complete, since the machinery that

produced the enzymes did not reproduce itself. Even if this

approach was not completely satisfying from the theoretical

standpoint, it represented an advance in the realization of

minimal cell.

The biochemical machinery that synthesizes the enzymes

needed for lipid production, is the full set of genes, ribosomes,

enzymes and tRNAs required to perform in vitro transcription

and translation processes. Therefore, the starting components

are: (a) the genes codifying for the enzyme responsible for

this transformation; (b) the whole set of macromolecular

components required to synthesize these enzymes from the

corresponding genes; (c) water–soluble precursors (Fig. 8).

Recently, the Schimdli’s approach has been investigated by

our group,40h,41b,c limited however to the first two enzymes,

which convert glycerol-3-phosphate to phosphatidic acid. Two

genes, codifying for the first two enzymes of lipid-salvage-

pathway (namely the transformation of glycerol-3-phosphate

to diacyl phosphatidic acid) are introduced into lipid vesicles,

together with the PURESYSTEMs, a set of purified

enzymes,42 ribosomes, and low molecular-weight compounds,

in order to produce the two enzymes within vesicles. In

this work, as in the more recent report on fatty acid

synthase,41d the low yield of lipid production inside vesicles

has strongly limited the possibility of observing any signi-

ficant morphological transformation based on the scheme of

Fig. 2a.

It is certainly important to continue research in these

directions, because the synthesis of cell boundary from

within is a fundamental step towards the construction of

autopoietic cells.

Fig. 8 Schematic illustration of core-and-shell self-reproduction in

synthetic or semi–synthetic systems. A set of precursors Pk are added

to minimal autopoietic vesicle systems, composed of core components

Ci and shell components Sj. The functional and structural integration

of Ci and Sj is the autopoietic network that transforms Pk into the

components Ci + Sj. Actually, the autopoietic network produces its

own component, in a way that is globally autocatalytic. Since the

redistribution of components is subjected to stochastic fluctuations,

after a series of growth–division, one of the vesicles may be missing at

least one component, for example one of the Ci components, becoming

non–functional. This event leads to a non–reproducing system (dead).

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6. Conclusions and future perspectives

In this review we have resumed the main results on self-

reproduction of vesicles, micelles and reverse micelles, discussing

the experimental results within the framework of autopoiesis.

These results are particularly noteworthy since they demonstrate

not only that autopoietic cycles exist, but also that autopoietic

cycles may start spontaneously according to physical laws.

Vesicle self-reproduction represents a breakthrough in the

field of supramolecular chemistry for at least three important

reasons. The first is related to the discovery of a new reactive

path in vesicle systems, that enriches the list of known vesicle

processes (fusion, aggregation, fission, swelling and shrinking,

interaction with polymers and bio-polymers, etc.), so that a

deeper understanding of vesicles has been achieved. The

second is instead linked to the field of the origin of life, due

to the observation that fatty acid vesicles are—to date—the

most plausible candidate for prebiotic compartments, and

therefore the discovery of their self-reproduction further

supports origins of life scenarios, and strengthens the role of

compartmentalized and compartmentalizing systems. The

third reason is that it accounts for the great relevance of

vesicles self-reproduction on the realization of minimal living

systems in the lab, and its importance in demonstrating that

life can be reconstructed starting from a finite and known

number of non-living components. As with other emergent

properties, life becomes a property of a molecular system when

a certain degree of complexity is reached, accompanied by a

well defined and specific self-organization pattern. Autopoiesis

and more in particular autopoietic self-reproduction play central

roles in the design and construction of minimal living cells.

Some unclear points persist, and scientific debate on these is

still lively. Fatty acid systems dominate the experimental

landscape of published work, and only recently have other

systems been investigated.13 An extension of self-reproduction

studies to other surfactant (anionic, neutral and cationic19) can

favor a better understanding by comparing their behavior. It

will also be very important to investigate possible mechanisms

by carrying out experiments specifically designed to test a

working hypothesis. The theoretical approach to vesicle self-

reproduction still lacks a coherent view on the interplay

between thermodynamics and kinetics in these systems. A

deeper mechanistic investigation into the morphological

changes that accompany the growth-division process of

‘‘small’’ vesicles, as has been recently done for GVs36a would

be highly valuable. Further studies may be focused on some

factors that might affect self-reproduction capacity or mecha-

nism, such as the membrane rigidity, vesicle size and composi-

tion, internal content, electro-osmotic gradients, phase

transition temperature, etc. All these factors may give insights

into the self-reproduction mechanism. Another unclear

(yet very important) aspect concerns the fate of water–soluble

compounds entrapped in the parent vesicles, following the act

of growing and dividing. Will trapped compound be evenly

distributed among daughter vesicles? This and other complex

aspects of vesicle self-reproduction are of course related to the

construction of minimal living cells.

In this more complex system, the open questions certainly

overcome current knowledge. From a general viewpoint, the

ultimate goal (coupled core-and-shell self-reproduction) in

minimal cell research is certainly difficult to achieve, and

therefore a stepwise approach can be useful to address the

different problems associated with this challenging project.

For example, the self-reproduction of core components is an

intermediate stage which can be reached by developing a

molecular network that produces itself. A recent example of

autocatalytic production of genetic material inside liposomes

has been provided by Yomo and coworkers,39b who designed

a messenger RNA that codifies for Qb replicase. When

the messenger RNA and the ribosomal machinery are

coentrapped inside liposomes, Qb replicase is then synthesized,

and replicates the messenger RNA, that in turn leads to more

Qb replicase and so on, in an autocatalytic fashion. This is an

example of (partial) self-reproduction of core components.

However, several questions remain open. We have seen that

a quantitative description of the vesicle self-reproduction

process is still missing, as well as the clarification about its

path within the space of vesicle possible shapes. It is also

important to define the vesicle self-reproduction in terms of

dynamics between pre-forming vesicles, precursors, and environ-

ment (the important of buffer species has been only recently

emphasized36a), so that—under proper conditions—it will

become possible to demonstrate that vesicle self-reproduction

may also occur in chemically different systems, sharing common

properties with fatty acid ones. On the other hand, the require-

ments for the accomplishment of the first self-reproducing

minimal cell are still rather demanding, in terms of scientific

understanding and technical skills, and the achievement of a first,

simple model would already be a considerable success. Further

investigations could focus on the physics of division, also

considering stochastic redistribution of minimal cell components

among the new compartments, or on the identification of the

number of generations that can be sustained before the collapse

of the minimal metabolism organization.

The concept of minimal cells, closely related to vesicle

self-reproduction, and more in general to autopoietic organi-

zation, has been recently recognized as part of the chemical

edge of synthetic biology43 and systems chemistry.35 The

flourishing of studies in this field witnesses a sort of shared

confidence that these are experimentally accessible targets.

Acknowledgements

This work has been funded by the SYNTHCELLS project

(Approaches to the Bioengineering of Synthetic Minimal

Cells, EU Grant #FP6–043359), by the Human Frontiers

Science Program (RGP0033/2007-C), by the Italian Space

Agency (Grant Nr. I/015/07/0), and within the COST Systems

Chemistry CM0703 Action. Parts of this paper have been

adapted from more extensive discussions on self-reproduction

of supramolecular structures.9 Graphical abstract: courtesy of

Paolo Carrara (Uniroma3).

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30 (a) S. Chungcharoenwattana and M. Ueno, Chem. Pharm. Bull.,2004, 52, 1058–1062; (b) S. Chungcharoenwattana and M. Ueno,Chem. Pharm. Bull., 2005, 53, 260–262; (c) S. Chungcharoenwattana,H. Kashiwagi andM.Ueno,Colloid Polym. Sci., 2005, 283, 1180–1189.

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32 (a) M. I. Angelova and D. D. Dimitrov, Faraday Discuss. Chem.Soc., 1986, 81, 303–311; (b) K. Akashi, H. Miyata, H. Itoh andK. Kinoshita Jr., Biophys. J., 1996, 71, 3242–3250; (c) N. Magome,T. Takemura and K. Yoshikawa, Chem. Lett., 1997, 205–206;(d) Giant Vesicles, ed. P. L. Luisi and P. Walde, John Wiley &Sons, Chichester, 2000.

33 (a) R. Wick, P. Walde and P. L. Luisi, J. Am. Chem. Soc., 1995,117, 1435–1436; (b) R. Wick, P. Walde and P. L. Luisi,

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34 F. M. Menger and K. Gabrielson, J. Am. Chem. Soc., 1994, 116,1567–1568.

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36 (a) T. F. Zhu and J. W. Szostak, J. Am. Chem. Soc., 2009, 131,5705–5713; (b) P. Peterlin, V. Arrigler, K. Kogej, S. Svetina andP. Walde, Chem. Phys. Lipids, 2009, 159, 67–76.

37 (a) P. L. Luisi, Anat. Rec., 2002, 268, 208–214; (b) S. Islas,A. Becerra, P. L. Luisi and A. Lazcano, Origins Life Evol.Biosphere, 2004, 34, 243–256; (c) P. L. Luisi, T. Oberholzer andA. Lazcano, Helv. Chim. Acta, 2002, 85, 1759–1777.

38 (a) S. S. Mansy and J. W. Szostak, Cold Spring Harbor Symp.Quant. Biol., 2009, LXXIV, 1–8; (b) P.-A. Monnard, A. Luptakand D. W. Deamer, Philos. Trans. R. Soc. London, Ser. B, 2007,362, 1741–1750; (c) T. Namani and D. W. Deamer, Origins LifeEvol. Biosphere, 2008, 38, 329–341; (d) S. E. Maurer,D. W. Deamer, J. M. Boncella and P.-A. Monnard, Astrobiology,2009, 9, 979–987; (e) C. Knutson, G. Benko, T. Rocheleau,F. Mouffouk, J. Maselko, L. Chen, A. P. Shreve andS. Rasmussen, Artif. Life, 2008, 14, 189–201; (f) M. S. DeClue,P.-A. Monnard, J. A. Bailey, S. E. Maurer, G. E. Collis,H.-J. Ziock, S. Rasmussen and J. M. Boncella, J. Am. Chem.Soc., 2009, 131, 931–933; (g) P. M. Gardner, K. Winzer andB. G. Davis, Nat. Chem., 2009, 1, 377–383.

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40 (a) W. Yu, K. Sato, M. Wakabayashi, T. Nakatshi, E. P.Ko-Mitamura, Y. Shima, I. Urabe and T. Yomo, J. Biosci.Bioeng., 2001, 92, 590–593; (b) T. Oberholzer and P. L. Luisi,J. Biol. Phys., 2002, 28, 733–744; (c) S. M. Nomura, K. Tsumoto,T. Hamada, K. Akiyoshi, Y. Nakatani and K. Yoshikawa,ChemBioChem, 2003, 4, 1172–1175; (d) K. Ishikawa, K. Sato,Y. Shima, I. Urabe and T. Yomo, FEBS Lett., 2004, 576,387–390; (e) V. Noireaux and A. Libchaber, Proc. Natl. Acad.Sci. U. S. A., 2004, 101, 17669–17674; (f) T. Sunami, K. Sato,T. Matsuura, K. Tsukada, I. Urabe and T. Yomo, Anal. Biochem.,2006, 357, 128–136; (g) G. Murtas, Y. Kuruma, P. Bianchini,A. Diaspro and P. L. Luisi, Biochem. Biophys. Res. Commun.,2007, 363, 12–17; (h) Y. Kuruma, Origins Life Evol. Biosphere,2007, 37, 409–413; (i) T. de Souza, P. Stano and P. L. Luisi,ChemBioChem, 2009, 10, 1056–1063.

41 (a) P. K. Schmidli, P. Schurtenberger and P. L. Luisi, J. Am. Chem.Soc., 1991, 113, 8127–8130; (b) P. Luci, ETH-Z Dissertation Nr.15108, Zurich, 2003; (c) Y. Kuruma, P. Stano, T. Ueda andP. L. Luisi, Biochim. Biophys. Acta, Biomembr., 2009, 1788,567–574; (d) G. Murtas, Syst. Synth. Biol., 2009, DOI: 10.1007/s11693-009-9048-1.

42 Y. Shimizu, A. Inoue, Y. Tomari, T. Suzuki, T. Yokogawa,K. Nishikawa and T. Ueda, Nat. Biotechnol., 2001, 19, 751–755.

43 (a) P. L. Luisi, C. Chiarabelli and P. Stano, Origins Life Evol.Biosphere, 2006, 36, 605–616; (b) P. L. Luisi, Chem. Biodiversity,2007, 4, 603–621.

44 (a) Y. Chizmadzhew, M. Maestro and F. Mavelli, Chem. Phys.Lett., 1994, 22, 656–662; (b) J. Billingham and P. V. Coveney,J. Chem. Soc., Faraday Trans., 1994, 90, 1953–1959.

45 C. Chiarabelli, P. Stano and P. L. Luisi, Curr. Opin. Biotechnol.,2009, 20, 492–497.

46 P. Stano, Syst. Synth. Biol., 2010, DOI: 10.1007/s11693-010-9054-3.

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