Article pubs.acs.org/accounts
Molecular Recognition and Organizational and Polyvalent Effects in Vesicles Induce the Formation of Artificial Multicompartment Cells as Model Systems of Eukaryotes Constantinos M. Paleos* and A. Pantos National Center For Scientific Research “Demokritos”, 15310 Aghia Paraskevi, Attiki, Greece CONSPECTUS: Researchers have become increasingly interested in the preparation and characterization of artificial cells based on amphiphilic molecules. In particular, artificial cells with multiple compartments are primitive mimics of the structure of eukaryotic cells. Endosymbiotic theory, widely accepted among biologists, states that eukaryotic cells arose from the assembly of prokaryotic cells inside other cells. Therefore, replicating this process in a synthetic system could allow researchers to model molecular and supramolecular processes that occur in living cells, shed light on mass and energy transport through cell membranes, and provide a unique, isolated space for conducting chemical reactions. In addition, such structures can serve as drug delivery systems that encapsulate both bioactive and nonbiocompatible compounds. In this Account, we present various coating, incubation, and electrofusion strategies for forming multicompartment vesicle systems, and we are focusing on strategies that rely on involving molecular recognition of complementary vesicles. All these methods afforded multicompartment systems with similar structures, and these nanoparticles have potential applications as drug delivery systems or nanoreactors for conducting diverse reactions. The complementarity of interacting vesicles allows these artificial cells to form, and the organization and polyvalency of these interacting vesicles further promote their formation. The incorporation of cholesterol in the bilayer membrane and the introduction of PEG chains at the surface of the interacting vesicles also support the structure of these multicompartment systems. PEG chains appear to destabilize the bilayers, which facilitates the fusion and transport of the small vesicles to the larger ones. Potential applications of these well-structured and reproducibly produced multicompartment systems include drug delivery, where researchers could load a cocktail of drugs within the encapsulated vesicles, a process that could enhance the bioavailability of these substances. In addition, the production of artificial cells with multiple compartments provides a platform where researchers could carry out individual reactions in small, isolated spaces. Such a reactive space can avoid problems that occur when the environment can be destructive to reactants or products or when a diverse set of compounds difficult to obtain in a conventional reactor space are produced. Our work on these artificial cells with multicompartment structures also led us to formulate a hypothesis on the processes that possibly generated eukaryotic cells. We hope both that our research efforts will excite interest in these nanoparticles and that this research could lead to systems designed for specific scientific and technological applications and further insights into the evolution of eukaryotic cells.
1. INTRODUCTION Preparation and characterization of artificial cells1,2 based on amphiphilic molecules are areas of growing scientific and technological interest. Specifically, artificial cells that exhibit multicompartment character primitively mimic the structure of eukaryotic cells.3,4 The latter were formed by the symbiosis of prokaryotic cells inside other cells according to the now widely accepted endosymbiotic theory.5 In principle, it can be anticipated that inside artificial multicompartment cells processes take place and compounds can be produced that may be analogous to the ones synthesized in eukaryotic cells. It is possible therefore to safely assume that molecular and supramolecular structural analogies between artificial and eukaryotic cells can analogously affect their properties as well as their synthetic capabilities. In this context, artificial cells due to their relative simplicity can be used as models that can shed © 2014 American Chemical Society
light on processes of living cells including mass and energy transport through bilayer membranes or can be applied for conducting reactions requiring unique multicompartment environment. Most importantly, artificial cells can be employed as drug delivery systems encapsulating a cocktail of bioactive nonbiocompatible compounds. An experimental proof for the assumed analogies between eukaryotic and artificial cells was set forth as early as 1977 by Deamer.6 Specifically, abiotically formed amphiphilic molecules self-assemble and form vesicles,7−10 while the same or analogous amphiphiles can also self-assemble leading to the formation of single compartment prokaryotic or multicompartment eukaryotic cells.11,12 Due to common building blocks that Received: November 5, 2013 Published: April 15, 2014 1475
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evolutionary events. It postulates that membrane-bound compartments of eukaryotic cells, that is, mitochondria, which are the energy producing organelles, and chloroplasts, which are the organelles where photosynthesis takes place in plant cells, were formed from ancient bacteria. These bacteria, through symbiosis,36 adhered first to an ancient proto-eukaryotic cell and were encapsulated through endocytosis4,37 becoming organelles. In this connection, it has to be noted that symbiosis is living together of organisms of different species following a recognition step, while endosymbiosis, which is a topological condition, is a kind of symbiosis where one partner lives inside of another. Symbiosis is not an evolutionary process, but it refers to physiological, temporal, or topological association with environmentally determined fates.35 On the other hand, symbiogenesis implies the appearance of new tissues, new organs, or new features that resulted from prolonged symbiotic association. The molecular and genetic similarities between modern mitochondria with α-proteobacteria and modern chloroplasts with cyanobacteria resulted in a widespread acceptance of endosymbiosis among biologists. The prevailing model asserts that the host cell, which had a nucleus, was attacked by ancient bacteria, which eventually became mitochondria. Thus, the genesis of mitochondria occurred after the formation of the nucleus and did not play a mechanistic role in the formation of nucleus.4 The presence of the nucleus is the feature that uniquely defines the eukaryotic cells and distinguishes them from bacteria. It is therefore assumed that the origin of the bacterial cell is the origin of life itself, whereas serial endosymbiotic theory describes the subsequent origin of cells bearing nuclei by the process of symbiogenesis.35
have been employed for the formation of vesicles and cells, they basically share structural similarities. Specifically, the characteristic features9 exhibited by eukaryotic cells and to a much lesser extent by vesicles are molecular complexity, self-organization, interaction specificity, and multicompartmentalization. The latter property is only shared by eukaryotic cells,10,13 while there exist closer analogies between prokaryotes and single compartment giant unilamellar vesicles, GUV.2 In the compartments or alternatively in the organelles of eukaryotic cells, isolated from the cytosol by bilayer membranes, highly sophisticated processes occur; for instance, energy is produced in mitochondria, which is transferred through bilayer membranes. In fact, it is multicompartmentalization inherent with isolation from the surrounding environment that makes feasible for several processes to take place. The realization of these diversified and complicated processes has triggered interest of researchers to prepare artificial or synthetic cells. Handling the complexities for building artificial cells1,14,15 is of the outmost importance, and toward this end efforts should be directed. Having a lifelong interest in organized molecular assemblies, including polymerization in thermotropic liquid crystalline media, to which I was introduced by Prof. M. M. Labes and subsequently inspired by the work of Prof J. M. Lehn on supramolecular systems and specifically on his pioneering papers dealing with interactions between complementary vesicles,20,21 I dedicate to both of them this Account. Following my introduction to this field, I investigated micellar, liposomal, and interfacial system polymerizations, the results of which have been presented in original papers and discussed in my early reviews.16−19 Furthermore, being triggered by the pioneering work of Ringsdorf22 on investigating organization models, surface recognition, and biomembrane dynamics and also cell mimetic studies by Menger,23,24 I focused my research interests on modeling cell−cell interactions by investigating vesicle−vesicle,25−27 vesicle−dendrimer, and cell−dendrimer interactions.28−30 The incentives for undertaking these adventures, highlighting various aspects of multicompartment systems,31−34 stem from the interest to prepare effective drug delivery systems employing vesicular systems and dendritic polymers and also on attempting to discover links with endosymbiosis. This process was hypothesized to provide an explanation for the formation of multicompartment eukaryotic cells. In this Account, we will emphasize those properties of interacting vesicular systems that induce the formation of artificial multicompartment cells. These properties are molecular complementarity between interacting vesicles, organization and polyvalency effects of the interacting moieties incorporated in these self-assembled species, and transport abilities of the latter through the bilayer membrane of their complementary counterparts. Before entering the main theme of our Account, it is appropriate to present a brief discussion of endosymbiotic theory.
3. FORMATION OF MULTICOMPARTMENT LIPID-BASED SYSTEMS The main strategies for the formation of multicompartment lipid-based systems are (a) multicompartment vesicles formed through coating of vesicles by multilamellar lipid tubules or interdigitated DPPC sheets, (b) multicompartment system formation through incubation with certain ionic and nonionic compounds, (c) multicompartment system formation based on vigorous electrof usion, and (d) multicompartment systems formed through molecular recognition of complementary vesicles. Among these strategies the fourth one will be discussed in more detail since the steps of this method bear analogies with those that possibly occurred during endosymbiosis. In fact, it is the endosymbiosis that we are interested to mimic in our experiments. According to the first strategy, a modular-type process was followed for the formation of multicompartment vesicles, which, however, was most likely impossible under prebiotic conditions. Established methods were employed leading to the formation of the modules, which subsequently participated in usually convenient processes and afforded multicompartment vesicles. Thus, in 1997 Zasadzinski et al.,38 in a letter to Nature, reported a procedure by which they achieved the encapsulation of vesicles inside the aqueous core of giant ones, for which they have coined the term “vesosomes”. The formation of vesosomes is based on molecular recognition of vesicles mediated by biotin−streptavidin complex formation. The vesicular aggregates obtained, ranging in size from 0.3 to 1.0 mm, were encapsulated within an outer bilayer by interaction with cochleate cylinders, which are biotin-functionalized multilamellar lipid tubules formed spontaneously by negatively
2. ENDOSYMBOSIS: A THEORY OF EUKARYOTIC CELL EVOLUTIONRELATED TERMS The mode of creation of multicompartment eukaryotic cells from prokaryotes, which occurred about four billion years ago, has been explained by the serial endosymbiotic theory (SET), which was proposed by Lynn Margulis35 in the 1960s. Great effort was made by Margulis for her theory to be widely accepted since endosymbiosis is among the most important 1476
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Figure 1. Interacting model of complementary vesicles.
choline (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-[phosphorac-(1-glycerol)] (POPG), and cholesterol at a weight ratio of 18:2:1 were electrofused, multiple vesicles were transformed to a single structure as monitored in real time with optical microscopy. When voltage increased, the number of fused vesicles increased. When PEG was not encapsulated inside vesicles, the shape of the outermost membrane was mostly spherical following fusion whereas various shapes, such as tori and elongated tubes, were observed when 3 mM PEG 6000 (2.5% wt/wt) was incorporated. In the absence of PEG, excess membrane invaginated into the vesicle to form a multivesicular structure, whereas in the presence of PEG, the extent of membrane invagination was relatively small, causing excess membrane in the outermost shell to obtain various shapes. Fused vesicles containing PEG often acquired an elongated shape and finally resulted in a budded shape following neck formation. Employing the previous background on multicompartment systems formation, we were led to carefully investigate the nature and structural features of aggregates obtained following molecular recognition of complementary vesicles. Specifically, to investigate at first whether the obtained aggregates exhibit multicompartment structures and second to elucidate the factors that induce and promote their formation. In molecular recognition experiments between vesicles, their membranes usually consisted of phosphatidylcholine (PC), cholesterol (CHOL), protective poly(ethylene glycol) (PEG) coating, and always complementary moieties at their surface for accomplishing effective molecular adhesion. Such a model of an interacting complementary pair of vesicles is shown in Figure 1. The preferred complementary pair for achieving strong binding of vesicles was the guanidinium−phosphate complementary pair.33 The selected pair of complementary vesicles was therefore prepared by anchoring dihexadecyl phosphate, DHP (1), in the bilayer of one kind of vesicles and 1-[4dihexadecylcarbamoyl)butyl] guanidinium p-toluenesulfonate (2) in complementary vesicles, which were allowed to interact at environmental temperature.44 Guanidinium and phosphate groups, decorating the surface of the vesicles, adhere and fuse forming spontaneously multicompartment systems.31 To further establish that interaction of vesicles afforded multicompartment systems, various complementary vesicle pairs have been prepared, based on PC and CHOL and incorporating guanidinium and phosphate groups in their bilayer.45,46 Thus, one type of vesicles incorporated octadecylguanidine hydrochloride (3), N-[3-(octadecylamino)propyl]
charged phospholipids, such as phosphatidylserine, in the presence of Ca2+ ions. At the final stage, unrolling of the cochleate cylinders took place, leading to the encapsulation of the small vesicles and formation of multicompartment systems. This tedious and costly method has however been simplified by unrolling of cochleate cylinders without the involvement of the biotin−streptavidin recognition stage.39 Multicompartment vesicles were also formed by the interdigitation−fusion method,40,41 in a manner analogous to one described above involving cochleate cylinders. This strategy was based on the formation of a metastable phase, consisting of flat bilayer sheets, which can be opened and closed under conditions that do not disrupt the vesicles or the colloids present in the system. Specifically, at the first stage, addition of ethanol to dipalmitoylphosphatidylcholine (DPPC) small unilamellar vesicles (SUVs), at temperatures below their main phase transition, Tm, induced fusion and led to a suspension of micrometer-sized bilayer sheets with an interdigitation of the tails. Increasing temperature above Tm results in the formation of giant multicompartment vesicles, which were also named “vesosomes”.40,41 Giant liposomes are in general susceptible to transformations including fusion, fission, birthing, and healing, which mimic those of living cells. These transformations are characterized as cytomimetic processes and are triggered either by adding certain compounds or by temperature change, as primarily investigated by Menger et al.23,24 Recently a method to transform giant unilamellar vesicles (GUVs) to multicompartment lipid systems was also reported.42 Thus, GUVs were incubated with selected compounds such as an aqueous solution of neutral phosphate buffer or glucose, at ambient temperature, which transformed them to oligovesicular vesicles (OVVs), encapsulating one or more smaller GUVs. The optimum concentration of phosphate buffer for GUV to OVV transformation was about 0.40 mM, while at a high buffer concentration (2.0 mM) many GUVs collapsed, probably due to the high osmotic stress. It is worth noting that such a large morphological change results in GUVs at low concentrations (0.1−1.0 mM) and at ambient temperature. Taking into account the diversity of effective agents this fluctuation should be likely osmotic. In the domain of giant unilamellar vesicle transformations belongs the elegant investigation by Yomo et al.43 Coupling of fusion (leading to multivesicular systems) and budding was observed, depending on the experimental conditions. When GUVs consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho1477
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multilamellar vesicles (400 nm diameter) bearing phosphate groups were interacted. In addition, PEG chain concentration at the interface of the unilamellar vesicles was enhanced by 5% relative to cholesterol, affording the so-called enhanced PEGylated vesicles. Therefore, unilamellar guanidinylated vesicles consisting of PC/CHOL/G-PEG-CHOL (19:9.5:1) lipid ratio were interacted with complementary multilamellar vesicles of PC/CHOL/DHP (19:9.5:1) at various guanidinium/phosphate molar ratios. With these enhanced PEGylated vesicles, a greater number of fused vesicles was observed at 30% guanidinium/phosphate molar ratio, while the size of fused multicompartment species was further increased at 40% molar ratio as shown Figure 2. Fusion was increased when the guanidine hydrochloride (4), or N-[3-(N,N dioctadecylamino)propyl] guanidine hydrochloride (5), while the other type incorporated DHP (1). The role of PEG chains on the effectiveness of vesicles interaction was assessed by varying the concentration of PEGylated cholesterol, PEG-CHOL (6). It was found that the degree of vesicle PEGylation affected the degree of fusion and also multicompartment system formation. This was attributed to the high affinity of PEG polymeric chains for water, which leads to water depletion at vesicle interfaces. An osmotic gradient is therefore established,47 which brings in contact the interacting vesicles.
Figure 2. Multicompartment giant vesicles observed with phase contrast optical microscopy following mixing of multilamellar PC/ CHOL/DHP liposomes with complementary unilamellar PC/CHOL/ PEG-CHOL/G-PEG-CHOL liposomes at 40% mol/mol guanidinium/phosphate ratio. The bar at the right lower corner indicates 20 μm. Reproduced with permission from ref 48. Copyright 2009 WileyVCH Verlag.
enhanced PEGylated vesicles interacted with complementary multilamellar vesicles, as observed with fluorescence studies.46 It should be noted that PEG chain not only acts as a spacer of the guanidinium group, but also affects the fusion of vesicles or cells,49 depending on PEG chain length, its concentration, and whether it is anchored in vesicle bilayers or is freely dissolved in the aqueous phase.50 Adhesion of vesicles occurs without significant bilayer disruption, which is followed by fusion under a nonleaking process and formation of multicompartment systems in the last stage. Multicompartment system formation following the interaction of complementary vesicles is not a unique property for vesicles covered with guanidinium and phosphate groups at their external surface. Thus, in an analogous study,51 the recognizable amphiphiles 5,5-didodecylbarbituric acid, DBA (8), and 9-hexadecyladenine, HA (9), were incorporated in vesicles based on hydrogenated PC and CHOL at a PC/CHOL 2:1 molar ratio. In this case, however, hydrogen-bonding binding between the complementary moieties of these lipids was relatively weak, and therefore, they were incorporated at a high molar content relative to PC, that is, recognizable lipid/PC = 1. Following interaction of the complementary vesicles, multicompartment aggregates were obtained, which exhibit structural analogies to the ones previously discussed. Taking into account the above results, we have proposed27,32 a mechanistic scheme, Figure 3, for rationalizing the formation of multicompartment aggregates. Thus, interacting complementary vesicles afford large unilamellar vesicles (LUVs) and giant unilamellar vesicles (GUVs). Subsequently selective LUV−GUV adhesion can occur as established in an analogous
For promotion of the multicompartment character of these giant vesicles, the composition of the interacting vesicle bilayers was modified.48 Unilamellar vesicles consisting of PC, CHOL, and guanidinylated PEG cholesterol, G-PEG-CHOL (7), and 1478
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ment prokaryotes from which multicompartment eukaryotic cells were generated. In the following section, we will highlight the property of molecular complementarity, which is of crucial importance in the interaction of cells and vesicles, as affected by molecular organization and polyvalency effects toward the favorable construction of both eukaryotes and multicompartment systems. Furthermore, encapsulation of the organelles in cells or vesicles, through penetration or disruption of bilayer or endocytosis, is also a crucial property for the creation of eukaryotic cells and multicompartment systems.
investigation by Lehn et al.21 Following the formation of this adhesion-type aggregate, encapsulation of smaller vesicles inside larger ones is achieved through multiple steps. This process is in line with snapshot images obtained by phase contrast optical microscopy, Figure 3. Vesicle transport through bilayer membranes is apparently induced by the destabilization of bilayers due to the presence of the long PEG chains.49,50 Investigating these biomimetic processes taking place between complementary vesicles, we may hypothesize27 that they could bear some remote analogies, at least at the level of membranous structure and organization, to the processes that possibly occurred over millions years of evolution between communities of interacting entities, including single compart-
4. ORGANIZATIONAL AND POLYVALENT EFFECTS AMPLIFY MOLECULAR COMPLEMENTARITY OF INTERACTING VESICLES INDUCING MULTICOMPARTMENT SYSTEMS FORMATION Amphiphilic molecules existing in the prebiotic world gave rise through appropriate self-assembly to the formation of prokaryotes. The same or analogous amphiphiles under analogous conditions self-assemble in the laboratory forming SUVs, which can be considered as simple models52−55 of prokaryotes. In fact, as already mentioned, Deamer and his coworkers6 performed significant experiments by preparing under
Figure 3. Mechanistic scheme for the formation of multicompartment vesicles as related to images observed by phase contrast optical microscopy. 1479
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living cells. Through this endeavor, it is also possible to find links between the processes that led to eukaryotic cells generation and artificial cell formation. The investigations are however at an early stage, and further work is required for applying these nanoparticles as drug carriers, which due to their potential to incorporate a cocktail of drugs could exhibit enhanced effectiveness. Also, the construction of membranebased multicompartment nanoreactors is a very complicated task compared with conventional reactors, but the existing prospects in conducting a diversity of processes justify the undertaking of this effort. Furthermore, by construction of artificial cells through the strategy of molecular recognition of complementary vesicles, it is possible to model and hypothesize on the processes that generated eukaryotic cells. Although with this hypothesis we could tackle the issue of the origin of life, it is also an extremely important endeavor to achieve the formation of artificial cells, which could asymptotically approach the complexity and functionality of living cells.
simulated prebiotic conditions phospholipids, which are constituents of the membranes of prokaryotes and eukaryotes as well as of vesicular systems. An inherent property of cells for symbiosis is molecular complementarity, which operates as the driving force for binding of cells and the ultimate encapsulation of the guest inside the host cell. In an analogous manner, mimicking the process for endosymbiotic creation of eukaryotes, formation of artificial multicompartment systems was induced by properly functionalizing the external surface of the interacting vesicles with complementary moieties in order to ensure their adhesion.31 In this connection, it has early been established in a seminal work56 that binding of interacting species is enhanced as the degree of organization of the interacting moieties is increased. In fact the binding interaction of guanidinium and phosphate complementary system is enhanced by factors of 102−104 when this pair is transferred from bulk water to the bilayer of vesicles.56 The significant role of organization in enhancing binding of vesicles was also established when the complementary moieties, for instance, guanidinium and phosphate moieties, were further organized within the vesicular bilayer due to the incorporation of cholesterol.44 Thus, according to an established phase diagram,57,58 at cholesterol concentrations exceeding 25 mol % with respect to PC, in which these experiments were performed, the bilayer is in the liquid-ordered phase. This phase is simultaneously fluid and ordered; that is, it exhibits properties that are crucial for inducing interaction between recognizable molecules.44 It is also critical that the recognizable guanidinium and phosphate-based lipids are incorporated at a low molar ratio relative to PC (1:19), and therefore their presence does not appreciably perturb the molecular organization of the PC−CHOL bilayer. Apparently, molecular organization coupled with fluidity of the recognizable lipids in the liquid−ordered phase results in an enhanced binding of vesicles. This enhancing role of cholesterol in vesicle binding, which was observed by optical studies, was further established44 by isothermal titration calorimetry (ITC). The role of cholesterol in vesicle recognition is also evident from the reaction rates, becoming approximately four times faster in the presence of cholesterol. Furthermore, in synergy with complementarity and organization, the polyvalent effects,59,60 discussed in an excellent review by Whitesides,59 operating in biological systems also apply and enhance binding of interacting vesicles for the formation of artificial cells. This is in line with the fact that a significant number of closely located recognizable groups of one vesicle can simultaneously be accessed by its complementary vesicle, therefore greatly enhancing binding. As exposed57,58 strong adhesion between vesicles is an effective starting stage for proceeding to multicompartment system formation.
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AUTHOR INFORMATION
Corresponding Author
*Phone: +30-10-6503666. Fax: +30-10-6511766. E-mail:
[email protected]. Notes
The authors declare no competing financial interest. Biographies Constantinos M. Paleos obtained his B.S. in Chemistry from Athens University and his Ph.D. from Drexel University in Philadelphia. In 1973, after three years with Amoco Chemicals and Motor Oil, he joined the Institute of Physical Chemistry at NCSR “Demokritos”, where he was elected Director for the periods 1994−1999 and 2001− 2007. Currently he is consultant to this Institute. In 1991 and 1992, he was visiting professor at Louis Pasteur University, Strasbourg. His research activities focus in the area of “Nanomaterials of Supramolecular Organized Structure” including the synthesis and characterization of liquid crystals, molecular recognition of functional liposomes, preparation and characterization of multifunctional dendritic polymers, multicompartment lipid-based systems, design and preparation of drug and gene delivery carriers, and molecular transporters. Dr Alexandros Pantos is a project Chemist at Dendrigen SA, Greece. He is an expert in organic synthesis, preparation, and characterization of functional liposomes and dendritic polymers. He was a postdoctoral fellow at the Institute of Physical Chemistry, NCSR “Demokritos”, in a Program supervised by GSRT, Greece. He graduated from the Chemistry Department, University of Athens, Greece, and obtained his Ph.D. from the same University in conjunction with the “Laboratory of Nanomaterials of Organized Supramolecular Structure” at “Demokritos”. His research interests are focused on modeling cell interactions through the preparation and interactions of functional liposomes and dendritic polymers with final objective the development of effective drug delivery systems.
5. CONCLUDING REMARKS AND OUTLOOK The strategy induced by molecular recognition of vesicles for the formation of multicompartment artificial cells is primarily discussed in this Account. The process and self-assembled multicompartment aggregates obtained bear some analogies to symbiotic association of cells giving rise to eukaryotes. Primary objectives for the formation of these artificial cells are their application as drug delivery systems and also as nanoreactors for conducting reactions analogous to the ones conducted in
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DEDICATION
Dedicated to Prof. M. M. Labes, Temple University, Philadelphia, PA, USA, and to Prof. J. M. Lehn, ISIS, Louis Pasteur University F-67083 Strasbourg Cedex. 1480
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