Preparation of Multicompartment Lipid-Based Systems Based on

INVITED FEATURE ARTICLE pubs.acs.org/Langmuir

Preparation of Multicompartment Lipid-Based Systems Based on Vesicle Interactions Constantinos M. Paleos,* Dimitris Tsiourvas, and Zili Sideratou NCSR “Demokritos”, 15310 Aghia Paraskevi, Attiki, Greece ABSTRACT: Various strategies for constructing artificial multicompartment vesicular systems, which primitively mimic the structure of eukaryotic cells, are presented. These model systems are appropriate for addressing several issues such as the understanding of cell processes, the development of nanoreactors and novel multicompartment delivery systems for specific drug applications, the transport through bilayer membranes, and also hypothesizing on the evolution of eukaryotic cells as originating from the symbiotic association of prokaryotes.

1. INTRODUCTION Abiotically formed amphiphilic molecules form spherical vesicles16 through self-assembly, and the same or analogous amphiphiles also self-assemble, leading to the formation of singlecompartment prokaryotes or multicompartment eukaryotic cells.7,8 Because of common building blocks that are being employed for the formation of vesicles and cell membranes, they share some structural similarities. However, it must be stressed that vesicles, compared to cells, are relatively simple models of self-assembled molecules and that their similarities with cell membranes are at a primitive level only. The characteristic features9 exhibited by cells are molecular complexity, self-organization, interaction specificity, and multicompartment system formation. This latter property of multicompartmentalization primarily applies to eukaryotic cells.10,11 Thus, some analogies that can be detected between prokaryotes and single-compartment giant unilamellar vesicles (GUV)12 can possibly be found between eukaryotes and synthetic multicompartment vesicular systems. In the compartments or alternatively in the organelles of eukaryotic cells, which are isolated from the cytosol by bilayer membranes, highly sophisticated processes occur, such as selective compartmentalization, energy production and transfer, and material transport. However, not all of the above properties are exhibited by single-compartment prokaryotic cells or artificial single-compartment reactors. The realization of these diversified and complicated processes has triggered the interest of researchers, and the formation of artificial or synthetic cells has already been undertaken.13 Handling the complexities of building artificial cells14,15 is of the utmost importance, and toward this end, efforts should be directed. Because of the bilayer membranous structure of multicompartment vesicles, one may envisage that the physical and chemical properties of interacting vesicles would also affect their formation process. Considering these facts, the main emphasis of this article will be placed on discussing the structural features of participating entities and the conditions for reproducibly preparing these multicompartment vesicles. Fascinated by the exceptional complexity of processes occurring in living cells, which lead to the preparation of a r 2011 American Chemical Society

huge diversity of molecules, we believe that it would certainly be important if it was possible to approach their complexity and perfection in artificial multicompartment systems. Cell mimetics, extensively investigated by Menger and his collaborators16,17 as early as the 1990s, may be extended and may lead to (a) the development of sophisticated artificial nanoreactors1820 for the synthesis of specific compounds, although reactions occurring inside lipid-based multicompartment nanoreactors have not yet been investigated, (b) the development of novel, cocktail-type drug delivery systems2123 that could be appropriate for the encapsulation of noncompatible bioactive compounds, and (c) the formation of multicompartment systems that would further support the hypothesis24 of the evolution of eukaryotes from prokaryotes. In the following sections of this article, the strategies of the formation of multicompartment vesicles will be presented together with their properties. Also, in view of these characteristic properties, the prospective applications of multicompartment systems will be discussed.

2. MULTICOMPARTMENT SYSTEMS OBTAINED THROUGH ENCAPSULATION OF VESICLES IN THE INTERIOR OF GIANT VESICLES Several years ago, Zasadzinski et al.25 succeeded in the encapsulation of vesicles within giant vesicles, for which the term multicompartmental aggregate was coined. For the formation of this aggregate, a multistep process was followed, according to which the key step was the molecular recognition between streptavidin and biotin lipids. At the final step, the unrolling of multilamellar lipid tubules was taking place, leading to the formation of multicompartment systems. This process led to the encapsulation of vesicles within the bilayer of the cylinders, resulting in the formation of multicompartment systems that were named vesosomes. Received: July 15, 2011 Revised: October 10, 2011 Published: October 11, 2011 2337

dx.doi.org/10.1021/la2027187 | Langmuir 2012, 28, 2337–2346

Langmuir

INVITED FEATURE ARTICLE

Figure 2. Schematic representation of ethanol-induced IF vesicle formation through the interdigitationfusion of SUVs. Reproduced with permission from the authors of ref 28.

Figure 1. Schematic representation of the process for the encapsulation of vesicles in a lipid bilayer membrane. Reproduced with permission from ref 26. Copyright 2003 American Chemical Society.

It should be mentioned that the encapsulation of vesicles and colloids was also achieved by a procedure based on cochleate cylinders without the involvement of the recognition steps. Thus, in another investigation in the same laboratory,26 the addition of Ca2+ to 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) vesicles led to fusion that afforded multilamellar cochleate cylinders of anionic bilayers. At this stage, vesicles originating from neutral lipids or colloidal particles, which were not affected by Ca2+ or EDTA, were added to the dispersion of these cylinders. Complex formation between Ca2+ and EDTA resulted in the unrolling of the cylinders and the reclosure of the bilayers, leading to the formation of micrometer-sized DOPS giant vesicles encapsulating the added smaller vesicles or colloidal particles (Figure 1). For both methods, the formation of cochleate cylinders is the crucial step in obtaining multicompartment systems. Multicompartment vesicles were also prepared by using the interdigitationfusion method27,28 that was originally developed for the production of lipid vesicles of high internal volume. By analogy to the previously described method of producing multicompartment vesicles based on the coating of vesicles by cochleate cylinders, it was possible to encapsulate vesicles, colloids, and polymers by coating them with appropriately formed bilayer sheets. The method was based on the formation of a metastable phase of flat bilayer sheets that can be opened and closed under experimental conditions that do not disrupt other vesicles or chemically sensitive materials. For instance, by adding ethanol to 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) small unilamellar vesicles (SUVs) at temperatures below their main phasetransition temperature, Tm, the fusion of vesicles was induced, leading to the formation of a suspension of micrometer-sized bilayer sheets. The ethanol intercalating within the headgroups of the gel phase of DPPC lipids caused the interdigitation of their tails. Temperature increases above Tm resulted in the formation of giant vesicles. This process is referred to in the literature as the

interdigitationfusion (IF) method of producing IF vesicles, and it is shown pictorially in Figure 2. The method is experimentally simple, but it is not generally applicable; it applies to vesicles originating from phospholipids that can form an interdigitated bilayer, that is, the LβI phase. For complete interdigitation, the lipophilic chains of the lipids should be essentially straight in the all-trans conformation. Phospholipids with acyl chains containing permanent “kinks” due to cis double bonds, such as 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1-palmitoyl-2oleoyl-sn-glycero-3-phosphocholine (POPC), or egg phosphatidylcholine (EPC), cannot interdigitate and therefore do not form IF vesicles. Bilayer interdigitation is not also favored for phospholipids with small polar headgroups relative to the hydrophobic portion of the molecule, such as phosphatidylethanolamines.28 The interdigitationfusion method is best applied to phosphatidylcholines bearing fully saturated acyl chains, such as 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). DPPC or DSPC IF vesicle formulations exhibit high internal volumes that are typically greater than 20 μL/μmol of lipid. Encapsulation takes place by mixing a dispersion of 50 nm DSPC/CHOL (2:1) vesicles or colloids with the interdigitated DPPC sheets at room temperature.29 Heating the solution to 46 °C for 20 min results in the spontaneous encapsulation of vesicles at the same concentration as the surrounding solution as shown in Figure 3. The multicompartment systems obtained were also called vesosomes (i.e., lipid-based vesicular systems such as those originating from cochleate cylinders29). In addition, polymer lipids such as poly(ethylene glycol) lipids (PEG-2000 DPPE) that are commonly used to enhance the stability of vesicles can be incorporated into the interdigitated sheets employed for encapsulating vesicles. The number of bilayers and the size of the nanoparticles that are formed from interdigitated sheets were affected by the concentrations of cholesterol and ethanol.28 Figure 4 shows the structures formed from interdigitated sheets obtained from a 97.5:2.5 DPPC/CHOL dispersion of vesicles fused with 3 M ethanol. The sheets were added to a 50 mg/mL dispersion of 50 nm DSPC/CHOL vesicles and heated to 46 °C for 20 min. Freezefracture transmission electron microscopy images (TEM) show multilayer structures with large internal vesicles as well as smaller vesicles of 50 nm, which were all encapsulated inside the exterior membrane. As mentioned before, small vesicles were encapsulated at a concentration equal to that of the bulk solution. They can, however, be encapsulated within multiple bilayers in a single step. Indeed, by heating DPPC/CHOL interdigitated sheets, a multicompartment structure was obtained in a single step (Figure 4B). 2338

dx.doi.org/10.1021/la2027187 |Langmuir 2012, 28, 2337–2346

Langmuir Encapsulated vesicles have also been prepared from a variety of lipids to monitor the permeability, size, specific interactions, and so forth. It has been shown that the charge on the interior of the vesicles does not appear to change the encapsulation process. Finally, as far as the non-encapsulated vesicles are concerned they have been separated from the encapsulated materials by gentle centrifugation or even sedimentation if the size difference between the encapsulated particles and non-encapsulated vesicles is sufficiently large. The supernatant, once removed from the pelleted fraction, could be recycled to improve the overall efficiency of encapsulation.29 The size distribution of the so-obtained multicompartment vesicles is 0.53 μm. It is interesting that the multicompartment structure is retained even after size reduction by extrusion is performed by employing controlled pore size filters. In Figure 5A, the vesosomes shown were formed and then separated from the

Figure 3. Freezefracture TEM image of vesicles encapsulated in giant vesicles produced by adding a dispersion of 50 nm DSPC/CHOL (2:1) vesicles to interdigitated DPPC sheets and heating to 46 °C. Reproduced with permission from ref 29. Copyright 2002 American Chemical Society.


excess non-entrapped vesicles by centrifugation (∼2000 rpm), followed by extrusion through 0.4 μm filters. In Figure 5B, the vesosomes shown were first extruded through 0.4 μm filters and then separated by centrifugation to afford vesosomes that were 100200 nm in diameter. In both cases, 50 nm vesicles were encapsulated in the interior and remained intact.29 This property could be very important for the application of vesosomes as prospective drug delivery systems. A major problem in applying unilamellar vesicles as drug carriers has been the premature release of drugs in vivo. This may be due to vesicle degradation by enzymes or protein incorporation inside vesicle membranes, which significantly enhances bilayer permeability. This behavior has already been addressed in vesicles3033 by coating their exterior surface with poly(ethylene glycol) chains (PEGylation), which protects vesicles against macrophages and prolongs their circulation. However, PEGylation increases permeability and does not enhance drug retention.34,35 Another strategy for decreasing drug release would be to encapsulate unilamellar vesicles within another giant vesicle by utilizing multicompartment vesosomes.36 In this investigation, vesosomes extend drug retention during exposure to phospholipase A2 by several orders of magnitude compared to unilamellar vesicles of the same composition. This is achieved by preventing enzymes and/or proteins from reaching the encapsulated vesicles. The multicompartment structure of the vesosome also allowed for the optimization of the interior compartments and the external bilayer. Specifically, the external membrane provides a physical barrier against lipase, preventing the lipase from degrading the interior compartments. Also, by changing the composition of internal vesicle membranes it could further optimize drugs release in serum and possibly identify the components of serum responsible for the membrane degradation that leads to premature drug release. For example, sphingomyelinbased internal capsules would be resistant to phospholipase degradation even after the lipase has degraded the external barrier membrane. This encapsulationprotection concept could easily be applied to any colloidal delivery system that is susceptible to early degradation-induced drug leakage. For instance, the

Figure 4. (A) Freezefracture TEM image of a multicompartment structure formed by adding DSPC/CHOL (2:1) small vesicles to a solution of interdigitated sheets made of DPPC/CHOL (97.5:2.5 molar ratio, fused with 3 M ethanol) after heating to 46 °C. Typical structures formed from this lipid mixture had multiple small vesicle compartments inside one or more exterior bilayers. (B) Freezefracture TEM image of a one-step multicompartment structure made by heating interdigitated sheets made of DPPC/CHOL (97.5:2.5 molar ratio) fused with 3 M ethanol to 46 °C. Reproduced with permission from ref 29. Copyright 2002 American Chemical Society. 2339


Langmuir


Figure 5. Freezefracture TEM images of (A) a vesosome prepared, separated by centrifugation, and finally extruded through a 400 nm pore size filter and (B) a vesosome prepared, extruded through a 400 nm pore size filter, and finally separated by centrifugation. In both cases 50 nm vesicles were unaffected by the preparation processes and remained intact. Reproduced with permission from ref 29. Copyright 2002 American Chemical Society.

Figure 6. Mean average diameters obtained for control (() and multicompartment systems (9). Photon correlation spectroscopy measurements of 2:1, 5:1, 10:1, and 20:1 molar ratios of LUV/SUV obtained by simple mixing or by lipid film hydration with SUVs resulting in multicompartment vesicles (MCVs). Reproduced with permission from ref 22. Copyright 2007 Elsevier.

Figure 7. Schematic representation of a multicompartment vesicle (MCV) formed by lipid film (DMPC/CHOL) hydration with a dispersion of preformed SUVs (DOPC/DOPG/CHOL) and extrusion. Reproduced with permission from ref 22. Copyright 2007 Elsevier.

exterior membrane protects the interior vesicles from binding to streptavidin when the interior lipid bilayers are decorated with biotinylated lipids.36 When these results have been extended to the more complex environment of serum, it has been shown that vesosome drug release is slowed by 2 orders of magnitude compared to that of unilamellar vesicles. The vesosome also offers the flexibility to deliver more than one drug within a single carrier, which has been shown to be advantageous in chemotherapy.36,37 In addition, vesosomes, having a structure analogous to that exhibited by eukaryotes, protect their “organelles” from interacting in a hostile environment.36 In conclusion, the described strategy is a facile method for efficiently encapsulating vesicles, colloidal particles, or macromolecules with one or more bilayer membranes. The encapsulation process can entrap a diversity of vesicles varying in structure and size, and it should be feasible to expand this encapsulation to other complex and sensitive biological structures (DNA, DNA cationic lipid complexes, etc.).

3. MULTICOMPARTMENT SYSTEMS OBTAINED THROUGH THE BINDING OF VESICLES The characteristic feature of multicompartment systems that are described in this section is the “gluing” of vesicular compartments22 instead of coating the vesicles with a bilayer membrane, as was the case in the previous section. Thus, the multicompartment systems described in this section do not bear the protective bilayer coating, the absence of which will certainly affect their stability. The vesicular systems have a mean diameter of 200 nm and a narrow size distribution consisting of two different types of vesicles connected through a tight bilayer interface. For their preparation, (a) lipid films of DOPC/DOPG/CHOL (80:10:10 molar ratio) and DMPC/CHOL (90:10) were hydrated, (b) the DOPC/ DOPG/CHOL film was subsequently sonicated, affording small unilamellar vesicles (SUV), and (c) multicompartment vesicles (MCV) were prepared by the hydration of a DMPC/CHOL (90:10) lipid film using a previously prepared SUV suspension 2340


Langmuir


Figure 8. (A) Images showing the fluctuation and transformation of GV to OVV in stages. Images obtained at (a) the beginning and after (b) 40, (c) 50, (d) 80, (e) 120, and (f) 440 s. The bar is equal to 20 μm. (B) Images of the slow transformation of a GV to an OVV. (df) In this case, the transformation reversed at a point just before completion and halted for a while and then again proceeded to form an OVV. Images are taken at (a) the beginning and after (b) 6, (c) 6.5, (d) 7, (e) 27, (f) 38, (g) 73, and (h) 77 min. The bar indicates 10 μm. Reproduced with permission from ref 38. Copyright 2011 American Chemical Society.

consisting of DOPC/DOPG/CHOL at 2:1, 5:1, 10:1, and 20:1 lipid film/SUV molar ratios. The size of multicompartment vesicles was decreased by extrusion. Control vesicle systems were also prepared by simply mixing suspensions of SUV (DOPC/DOPG/ CHOL) and LUV (DMPC/CHOL) at the same LUV/SUV molar ratios and incubating at room temperature. The large unilamellar vesicles (LUVs) employed in these control vesicle systems consist of DMPC/CHOL and were prepared by extrusion at 40 °C. The giant vesicular aggregates that were initially formed, following repeated extrusions, were considerably reduced in size, and vesicle aggregates with a mean diameter of about 200 nm were obtained as shown in Figure 6. The diameters of LUV/SUV controls showed a linear increase that was dependent on LUV/ SUV molar ratios. This was not observed with multicompartment vesicles, which therefore suggests a more elaborate interaction between the two lipid bilayer populations.

The association between SUVs and the lipid bilayer achieved immediately upon hydration indicated that the hydration of lipid films with an SUV-containing aqueous phase does not lead to the encapsulation of the SUV. These results indicate the significance of hydrophobic interactions between the hydrated lipid film (DMPC/ CHOL) and the preformed SUV (DOPC/DOPG/CHOL) affording multicompartment vesicles as shown schematically in Figure 7. It is assumed that the interface connecting the two types of vesicles is held together by hydrophobic association, forming binding areas that may be rich in cholesterol. The mean diameter of the multicompartment vesicular system developed in this study is on the order of 200 nm, being therefore suitable for pharmaceutical applications that may involve systemic circulation. Multicompartment vesicles have therefore been developed that may be applied as combinatory chemotherapeutic systems. 2341


Langmuir


Table 1. GV-to-OVV Transformation by Incubation with Various Aqueous Solutions substance incubated with preformed GVs none (pure water) neutral phosphate buffer phthalate buffer

GVs transformed concentration

to OVVs (%)

0.04 mM

0 0

0.40 mM

20

0.40 mM

13

2.0 mM

0a

NaCl

0.60 mM

9

KI

0.60 mM

4

glucose

0.10 mM

7

0.75 mM 1.0 mM

5 0a

sucrose

1.0 mM

4

1,2-ethanediol

1.0 mM

5

dextran (MW ∼70 000)

0.036 wt %

0

a

Collapsed. Reproduced with permission from ref 38. Copyright 2011 American Chemical Society.

4. MULTICOMPARTMENT SYSTEMS OBTAINED BY INCUBATION WITH SELECTED LOW-MOLECULARWEIGHT COMPOUNDS Very recently, an experimentally convenient method was reported,38 according to which multicompartment vesicles were formed by incubating giant vesicles with selected compounds that induce their membrane transformation. Specifically, giant vesicles (GVs) were transformed to oligovesicular vesicles (OVVs) encapsulating one or more smaller GVs through incubation with a diluted aqueous solution of neutral phosphate buffer salts or glucose under mild conditions and at ambient temperature. Following the electroformation of giant vesicles, their membrane fluctuation was barely visible. However, the addition of the buffer caused wobbling of the membrane in approximately 40% of the GVs; subsequently, some GVs became flaccid and were transformed to OVVs as shown in Figure 8A. A portion of the external aqueous phase was engulfed and became the interior of the newly formed inner GV. Occasionally, the transformation was very slow, stopping before completion, and then it reversed from a discoid or even from a shape similar to that in Figure 8A (d) to form OVVs as shown in Figure 8B. However, it should be noted that the transformation from a formed OVV to GV was not observed. All of these morphological membrane transformations were reproducibly observed. In Table 1, the GV to OVV transformation is summarized under various conditions. The optimum concentration of the phosphate buffer for OVV formation was at around 0.40 mM. At low concentrations, the number of fluctuating GVs was smaller compared to the optimum concentration, and at a higher buffer concentration (2.0 mM), many GVs collapsed, probably because of high osmotic stress. An experiment using pure water instead of the buffer showed neither the fluctuation nor OVV formation, indicating that the deformation was not simply due to prolonged incubation. Other water-soluble substances were also found to be effective in inducing OVV formation. Among the compounds tested, neutral phosphate buffer was the most efficient agent, although salts of monovalent ions (NaCl or KI) or lowmolecular-weight nonionic species (glucose, sucrose, or 1,2ethanediol) also induced the transformation. It is interesting that

Figure 9. OVV obtained from a lipid mixture. GVs were formed from a POPC/POPG/CHOL (90:10:40) mixture incubated in a neutral phosphate buffer solution (0.40 mM, pH 7.0). The bar is equal to 20 μm. Reproduced with permission from ref 38. Copyright 2011 American Chemical Society.

rather low concentrations of these compounds at ambient temperature could trigger such large morphological changes in GVs. Considering the diversity of effective agents, the present fluctuation is likely to be an osmotic phenomenon. It is also of interest that the GV-to-OVV transformation also occurred with mixed lipids. Thus, incubation with dilute phosphate buffer was also effective with POPC/POPG (90:10), POPC/ CHOL (100:40), and POPC/POPG/CHOL (90:10:40) as shown in Figure 9. In all experiments, the transformation was essentially the same as the one obtained with egg PC in which 1518% of preformed GVs were transformed to OVVs. The insensitivity of the transformation to the incorporation of an anionic lipid in the GV membrane indicates that the interaction contribution between the membrane and the electrolyte ions to the transformation should be small. In this connection, it is very interesting to mention that this transformation coupled with the replacement of the external aqueous phase, may be used for the construction of OVVs with heterogeneous inner aqueous compartments.38 This system can be used, in principle, for the development of multicompartment systems incorporating a cocktail of drugs.

5. MULTICOMPARTMENT SYSTEMS FORMED THROUGH MOLECULAR RECOGNITION OF COMPLEMENTARY VESICLES In recent studies investigating interactions between vesicles bearing surface groups that can associate with each other, it was established that giant vesicles were obtained by incorporating in their aqueous cores smaller vesicles. Because of the appropriate structural features of the interacting vesicles, the commonly encountered steps of adhesion and fusion were followed by the formation of multicompartment systems.39 For instance, the interaction of vesicles functionalized with guanidinium groups with complementary counterparts bearing phosphate moieties led to the formation of multicompartment giant vesicles. Specifically, unilamellar vesicles of about 100 nm in diameter consisting of hydrogenated phosphatidylcholine (PC) and CHOL incorporating either dihexadecyl phosphate (DHP) or 1-[4(dihexadecylcarbamoyl)butyl] guanidinium p-toluenesulfonate (1) were allowed to interact.40 They spontaneously afforded large aggregates that in certain cases encapsulated smaller vesicles (i.e., they exhibited multicompartment structure). Strong binding 2342


Langmuir


Figure 10. Adhesion of vesicles through the molecular recognition of complementary moieties anchored in vesicular bilayers.

between the phosphate group of DHP lipid and the guanidinium group of 1 allowed experiments to be performed at low molar ratios of lipids relative to PC (1:19). Associated with this property is the interesting finding that the interaction of complementary vesicles is enhanced by the incorporation of cholesterol40 in their membranes. The presence of cholesterol results in the formation of the liquid ordered phase that appreciably enhances molecular recognition between vesicles. A diversity of interacting vesicles have been prepared on the basis of the same guanidinium-phosphate recognizable pair to study the process of multicompartment system formation further.41,42 In these experiments, mixed unilamellar vesicles were prepared by employing PC and CHOL as basic constituents. The vesicles also incorporated either octadecylguanidine hydrochloride (2), or N-[3-(octadecylamino)propyl] guanidine hydrochloride (3), or N-[3-(N,Ndioctadecylamino)propyl] guanidine hydrochloride (4) and DHP as recognizable molecules. Additionally, these interacting vesicles incorporated varying concentrations of PEGylated cholesterol (PEG-CHOL) (5) in order to study the potential inhibitory effect of PEG chains on the interaction effectiveness of vesicles. The first stage of vesicles adhesion, as pictorially shown in Figure 10, was followed by fusion, which finally led to the formation of larger particles under a non-leaking process. It was found that the degree of vesicles PEGylation affected the degree of fusion and multicompartment system formation. This behavior has been rationalized by the fact that the PEG polymeric chain that exhibits a high affinity for water at a relatively low concentration (015 wt %) depletes water located on vesicle interfaces. In this manner, an osmotic gradient that brings into contact vesicles or cell surfaces is created.43 2343


Langmuir


Figure 11. Phase contrast optical microscopy images of liposomal aggregates following the mixing of multilamellar PC/CHOL/DHP liposomes with complementary unilamellar PC/CHOL/PEG-CHOL/G-PEG-CHOL liposomes at (A, B) a 30% and (C, D) a 40% mol/mol guanidinium/ phosphate ratio. The bar in the lower right corner in D indicates 20 μm. Reproduced with permission from ref 44. Copyright 2009 Wiley-VCH Verlag.

On the basis of these results and with the aim of promoting the multicompartment character of these giant vesicles, further elaboration of the structural features and conditions of vesicle interaction was undertaken.44 Thus, in this case unilamellar vesicles consisting of PC, CHOL, and guanidinylated PEG cholesterol (G-PEG-CHOL) (6) were allowed to interact with multilamellar vesicles (400 nm diameter) bearing phosphate groups. In addition, the concentration of PEG chains on the interface of unilamellar vesicles (100 nm diameter) was enhanced by incorporating 5 at a concentration of 5% relative to cholesterol, affording the enhanced PEGylated vesicles. Thus, in these experiments, unilamellar guanidinylated vesicles consisting of a PC/CHOL/G-PEG-CHOL (19:9.5:1 lipid ratio) have interacted spontaneously with complementary multilamellar vesicles PC/CHOL/DHP (19:9.5:1) at various guanidinium/phosphate molar ratios. When enhanced PEGylated vesicles are employed (i.e., PC/CHOL/PEG-CHOL/G-PEG-

CHOL), a greater number of fused vesicles are observed at a 30% guanidinium/phosphate molar ratio, while at a 40% molar ratio, the size of fused multicompartment species was further increased as shown in Figure 11. The characteristic feature of these aggregates is their well-formed multicompartment nature. Multilamellar vesicles were also observed, apparently originating from multilamellar vesicles that have not interacted with unilamellar vesicles. Optical microscopy observations were in agreement with dynamic light scattering measurements, showing that the size of the aggregated particles obtained in both cases increased significantly, especially at a 40% guanidinium/phosphate molar ratio, where broad particle distributions were observed. Considerable fusion occurred when the enhanced PEGylated vesicles interacted with complementary multilamellar vesicles, as observed in fluorescence studies.44 In this connection, it should be noted that the PEG chain is not functioning as a mere spacer that bears the guanidinium group on its end and therefore 2344


Langmuir


Figure 12. Proposed mechanism depicting the steps for the formation of multicompartment systems in correspondence with phase contrast optical microscopy images. Reproduced with permission from ref 48. Copyright 2008 Elsevier.

facilitates its accessibility toward interaction with the phosphate group. It can crucially affect the fusion of cells or vesicles,45 which is dependent on the PEG-chain length, concentration, and whether it is anchored in the vesicular bilayer or dissolved in the aqueous phase.46 The initial step in vesicle adhesion occurs without significant bilayer disruption, and this is followed by fusion under a non-leaking process, forming multicompartment systems in the last stage. In the background of early results by Menger16,17 and Lehn,47 it was proposed24,48 that in the first step complementary vesicles interact, affording LUVs and GUVs. In the second step (i.e., after the formation of LUVs and GUVs), selective LUVGUV adhesion occurs,47 which is the starting point of two processes: (a) fusion to even larger vesicles and (b) the incorporation of large vesicles inside giant vesicles because of the destabilization of the bilayer, which is attributable to the action of the long PEG chains. The mechanistic steps of the process for the formation of multicompartment systems are shown pictorially in Figure 12 in relation to the corresponding phase contrast optical microscopy images. In this second step, multicompartment systems are formed. Specifically, the PEG-CHOL lipid has a synergistic role with respect to that of guanidinium-phosphate recognition facilitating multicompartment system formation. In fact, the long poly(ethylene glycol) chain, devoid of a guanidinium group at its end, further induces vesicles fusion44 with the simultaneous incorporation of relatively large vesicles in the interior of the giant ones. In light of these processes occurring between complementary vesicles, during which simple vesicles give rise to multicompartment membranous systems, one may be led to the hypothesis24 that these processes could have some type of analogy to those that had possibly occurred over millions years of evolution with communities of interacting entities, including prokaryotes from which eukaryotic cells were created. Being aware of the complexities of the living entities, as recently discussed by Martin et al.,49

our hypothesis provides a rationalization, at the membranous level only, on how lipid-based multicompartmentalization could have resulted in eukaryotes. It should also be noted that although eukaryotization, obtained through endosymbiosis and compartmentalization, is a widely accepted theory,10,50,51 it is still an area where extensive research is being conducted, primarily concerning the original cell that hosted the initial endosymbiosis.5254

6. CONCLUDING REMARKS AND OUTLOOK Strategies for the formation of bilayer membrane-based multicompartment systems are presented and aim to trigger interest, among others, in the preparation, characterization, and prospective applications of these lipid-based vesicular systems. Further research is required to apply these vesicular systems as drug carriers, which, because of their ability to incorporate a cocktail of drugs, could exhibit enhanced effectiveness. The construction of membrane-based multicompartment nanoreactors is still very far from realization, and such systems have not yet been reported in the literature. This is a significant but extremely difficult task and a great deal of effort is required for their development, which could lead to the production of a diversity of compounds that are difficult to obtain with conventional reactors. In this connection, mention should be made of the work of Jesorka et al.54 on nanotubevesicle networks in which the authors discuss aspects of applications involving the chemical kinetics and transport of materials in ultrasmall biomimetic media. Finally, by constructing multicompartment systems through the molecular recognition of complementary vesicles, we were led to a hypothesis of how multicompartmentalization resulted in artificial cell formation, which primitively mimics the membrane-based structure of eukaryotic cells. Although this hypothesis is associated with the issue of the origin of life, the first priority for the time being is to achieve the formation of systems, which, although non-living, will asymptotically approach the complexity and functionality of living cells. 2345


Langmuir

’ AUTHOR INFORMATION Corresponding Author

*Phone: +30-10-6503666. Fax: +30-10-6511766. E-mail: paleos@ chem.demokritos.gr.

’ ABBREVIATIONS (CHOL) cholesterol; (DHP) dihexadecyl phosphate; (DMPC) 1,2-dimyristoyl-sn-glycero-3-phosphocholine; (DOPC) 1,2-dioleoyl-sn-glycero-3-phosphocholine; (DOPG) 1,2-dioleoyl-sn-glycero3-phospho-(10 -rac-glycerol); (DOPS) 1,2-dioleoyl-sn-glycero-3phospho-L-serine; (DPPC) 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; (DSPC) 1,2-distearoyl-sn-glycero-3-phosphocholine; (EPC) egg phosphatidylcholine; (GUVs) giant unilamellar vesicles; (GVs) giant vesicles; (G-PEG-CHOL) guanidinylated PEG cholesterol; (PC) hydrogenated phosphatidylcholine; (IF) interdigitation fusion; (LUVs) large unilamellar vesicles; (MCVs) multicompartment vesicles; (OVVs) oligovesicular vesicles; (POPG) 1-palmitoyl2-oleoyl-sn-glycero-3-phospho-(10 -rac-glycerol); (POPC) 1-palmitoyl2-oleoyl-sn-glycero-3-phosphocholine; (PEG-CHOL) PEGylated cholesterol; (PEG-2000 DPPE) poly(ethylene glycol) lipids; (PEG) poly(ethylene glycol); (SUVs) small unilamellar vesicles; (TEM) transmission electron microscopy ’ REFERENCES (1) Summers, D. P.; Noveron, J.; Basa, R. C. B. Origins Life Evol. Biospheres 2009, 39, 127–140. (2) Hargreaves, W. R.; Mulvihill, S. J.; Deamer, D. W. Nature 1977, 266, 78–80. (3) Deamer, D. W.; Oro, J. BioSystems 1980, 12, 167–175. (4) Dworkin, J. P.; Deamer, D. W.; Sandford, S. A.; Allamandola, L. J. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 815–819. (5) Lazcano, A. Complexity, Self-Organization and the Origin of Life: The Happy Liaison? In Origins of Life: Self-Organization and/or Biological Evolution?; Gerin, M., Maurel, M.-C., Eds.; EDP Sciences: Cedex, France, 2009; Vol. 13, pp 1322. (6) Chen, I. A.; Walde, P.; Deamer, D.; Szostak, J. W. Cold Spring Harbor Perspect. Biol. 2010, 2, 1. (7) Segre, D.; Ben-Eli, D.; Deamer, D. W.; Lancet, D. Origins Life Evol. Biospheres 2001, 31, 119–145. (8) Monnard, P.-A.; Deamer, D. W. Anat. Rec. 2002, 268, 196–207. (9) Deamer, D. EMBO Rep. 2009, 10, S1–S4. (10) Martin, W. Philos. Trans. R. Soc. B 2010, 365, 847–855. (11) Luisi, P. L.; Walde, P.; Oberholzer, T. Curr. Opin. Colloid Interface Sci. 1999, 4, 33–39. (12) Walde, P.; Cosentino, K.; Engel, H.; Stano, P. ChemBioChem 2010, 11, 848–865. (13) Roodbeen, R.; Hest, J. C. M. BioEssays 2009, 31, 1299–1308. (14) Walde, P. BioEssays 2010, 32, 296–303. (15) Noireaux, V.; Maeda, Y. T.; Libchaber., A. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 3473–3480. (16) Menger, F. M.; Gabrielson, K. D. Angew. Chem., Int. Ed. Engl. 1995, 34, 2091–2106. (17) Menger, F. M.; Angelova, M. I. Acc. Chem. Res. 1998, 31, 789–797. (18) Renggli, K.; Baumann, P.; Langowska, K.; Onaca, O.; Bruns, N.; Meier, W. Adv. Funct. Mater. 2011, 21, 1241–1259. (19) Vriezema, D. M; Aragones, M. C.; Elemans, J. A. A. W.; Cornelissen, J. L. M.; Rowan, A. E.; Nolte, R. J. M. Chem. Rev. 2005, 105, 1445–1489. (20) Kim, T. K.; Meeuwissen, S. A.; Nolte, R. J. M.; van Hest, J. C. M. Nanoscale 2010, 2, 844–858. (21) Kisak, E. T.; Coldren, B.; Evans, C. A.; Boyer, C.; Zasadzinski, J. A. Curr. Med. Chem. 2004, 11, 1241–1253.


(22) Al-Jamal, W. T.; Kostarelos, K. Int. J. Pharm. 2007, 331, 182–185. (23) Hu, C.-M. J.; Aryal, S.; Zhang, L. Ther. Delivery 2010, 210, 323–334. (24) Paleos, C. M.; Tsiourvas, D. J. Mol. Recognit. 2006, 19, 60–67. (25) Walker, S. A.; Kennedy, M. T.; Zasadzinski, J. A. Nature 1997, 387, 61–64. (26) Evans, C. C.; Zasadzinski, J. Langmuir 2003, 19, 3109–3113. (27) Ahl, P. L.; Chen, L.; Perkins, W. R.; Minchey, S. R.; Boni, T. L.; Taraschi, T. F.; Janoff, A. S. Biochim. Biophys. Acta 1994, 1195, 237–244. (28) Ahl, P. L.; Perkins, W. R. Methods Enzymol. 2003, 367, 80–98. (29) Kisak, E. T.; Coldren, B.; Zasadzinski, J. A. Langmuir 2002, 18, 284–288. (30) Lasic, D. D.; Needham, D. Chem. Rev. 1995, 95, 2601–2628. (31) Needham, D.; Kim, D. H. H. Colloids Surf., B 2000, 18, 183–195. (32) Kaasgaard, T.; Mouritsen, O. G.; Jørgensen, K. Int. J. Pharm. 2001, 214, 63–65. (33) Webb, M. S.; Saxon, D.; Wong, F. M. P.; Lim, H. J.; Wang, Z.; Bally, M. B.; Choi, L. S. L.; Cullis, P. R.; Mayer, L. D. Biochim. Biophys. Acta 1998, 1372, 272–282. (34) Johnsson, M.; Edwards, K. Biophys. J. 2003, 85, 3839–3847. (35) Boyer, C.; Zasadzinski, J. A. ACS Nano 2007, 1, 176–182. (36) Zasadzinski, J. A.; Wong, B.; Forbes, N.; Braun, G.; Wu, G. Curr. Opin. Colloid Interface Sci. 2011, 16, 203–214. (37) Okumura, Y.; Nakaya, T.; Namai, H.; Urita, K. Langmuir 2011, 27, 3279–3282. (38) Paleos, C. M.; Tsiourvas, D.; Sideratou, Z. ChemBioChem 2011, 12, 510–521 and references cited therein. (39) Sideratou, Z.; Tsiourvas, D.; Paleos, C. M.; Tsortos, A.; Nounesis, G. Langmuir 2000, 16, 9186–9191. (40) Pantos, A.; Sideratou, Z.; Paleos, C. M. J. Colloid Interface Sci. 2002, 253, 435–442. (41) Pantos, A.; Sideratou, Z.; Tsiourvas, D.; Paleos, C. M.; Giatrellis, S.; Nounesis, G. Langmuir 2004, 20, 6165–6172. (42) Hui, S. W.; Kuhl, T. L.; Guo, Y. Q.; Israelachvili, J. Colloids Surf., B 1999, 14, 213–222. (43) Sideratou, Z.; Sterioti, N.; Tsiourvas, D.; Paleos, C. M. ChemPhysChem 2009, 10, 3083–3089. (44) Lentz, B. R. Chem. Phys. Lipids 1994, 73, 91–106. (45) K€asbauer, M.; Lasic, D. D.; Winterhalter, M. Chem. Phys. Lipids 1997, 86, 153–159. (46) Marchi-Artzner, V.; Gulik-Krzywicki, T.; Guedeau-Boudeville, M.-A.; Gosse, C.; Sanderson, J. M.; Dedieu, J.-C.; Lehn, J.-M. ChemPhysChem 2001, 2, 367–376. (47) Pantos, A.; Tsogas, I.; Paleos, C. M. Biochim. Biophys. Acta 2008, 1778, 811–823. (48) Lane, N.; Martin, W. Nature 2010, 467, 930–934. (49) Margulis, L. Symbiosis in Cell Evolution, 2nd ed.; W. H. Freeman: New York, 1993. (50) Hengeveld, R.; Fedonkin, M. A. Acta Biotheor. 2004, 52, 105–154. (51) Martin, W.; Koonin, E. V. Nature 2006, 440, 41–45. (52) Embley, T. M.; Martin, W. Nature 2006, 440, 623–630. (53) Poole, A. M.; Penny, D. BioEssays 2006, 29, 74–84. (54) Jesorka, A.; Orwar, O. Methods Enzymol. 2009, 464, 309–324.

2346


Preparation of Multicompartment Lipid-Based Systems Based on

Recommend Documents