J. Phys. Chem. B 2008, 112, 14655–14664
14655
Vesicle Behavior: In Search of Explanations† Pier Luigi Luisi,* Tereza Pereira de Souza, and Pasquale Stano Biology Department, UniVersity of RomaTre; Viale G. Marconi 446, 00146 Rome, Italy ReceiVed: April 2, 2008; ReVised Manuscript ReceiVed: June 24, 2008
In this paper we will present a series of experiments in the general field of surfactant aggregates and, in particular, in vesicle chemistry, which have found no definitive explanation until now. These experiments concern vesicle self-reproduction (in particular, the so-called matrix effect); the interaction between vesicle and RNA, where RNA appears capable of discriminating between vesicles differing slightly in size; the fusion of oppositely charged vesicles, which brings about unexpected behavior of size distribution; and some aspects of local concentration inside vesicles, which still lack clarification in terms of local versus overall concentration. The theoretical and experimental implications of this not yet understood behavior will be discussed, emphasizing that progress in the field must face the difficulty of applying thermodynamics to these kinetically trapped systems, and the general difficulty of understanding how kinetic and thermodynamic factors interplay with each other. Introduction In the course of the past few years of work with vesicles as model compartments for the study of minimal cells, we discovered a few unexpected phenomena for which we did not find, and we have not found yet, a complete satisfactory mechanistic interpretation. They concern (i) some aspects of self-reproduction of vesicles, in particular those related to the so-called matrix effect; (ii) the selective interaction of vesicles with RNA; (iii) the fusion of vesicles having opposite charges; (iV) the incorporation of multiple chemicals inside the same vesicle; and (V) the issue of local concentration of a solute incorporated in the water pool of vesicles. In this paper, we would like to describe these phenomena, proposing some possible explanations for them, but mostly with the aim of challenging colleagues and students of liposome chemistry to provide and debate with us alternative mechanistic interpretations. 1. The “Matrix Effect” It is well-known that fatty acids form different self-assembled aggregates in aqueous solution, mainly depending on the pH of the solution, which affects the protonation state of fatty acids and consequently the most stable packing regime.1-3 At high pH (>9.5) micelles are formed, whereas at intermediate pH (7.5 - 9.0), fatty acids form vesicles. Lower pH values induce the precipitation of fatty acids. It should be said that the figures given above are approximate, and that the ionic strength and the fatty acid chain length are additional factors that must be considered. In addition, there are pH regions where micelles and vesicles coexist.4 The formation of vesicles at intermediate pH has been explained by invoking the formation of a dimer between protonated and unprotonated carboxylate groups, due to an unusually high pKa of fatty acids, when assembled in bilayers.1,5,6 One simple way to produce a suspension of fatty acid vesicles in water is to add an aliquot of concentrated fatty acid salt to a † Part of the “Janos H. Fendler Memorial Issue”. * Author for correspondence. E-mail:
[email protected]; Phone and Fax (+39) 06 5733 6329.
pH 8.5 buffered solution, for example, by adding sodium oleate micelles to bicine buffer. Oleic acid/oleate vesicles (oleate vesicles for simplicity) will be formed within a short time,7,8 as shown by the turbidity increase with time. The size distribution of the resulting vesicles is broad, typically ranging from 30 nm radius to more than 1 µm, with several morphologies, as demonstrated by electron microscopy and dynamic light scattering (DLS) studies. Suppose now we perform the same addition of oleate micelles to a buffered solution containing preformed oleate (or phosphatidylcholine) vesicles with a narrow size distribution, say around 50 nm (for example, vesicles prepared by the extrusion technique), so that the concentration of preformed vesicles is doubled. As shown in Figure 1A,B, two main effects characterize this process: (i) the increase of vesicle formation rate, as evident from the turbidity versus time profiles (Figure 1A); and (ii) the size distribution that is not broader but strongly resembling the size distribution of preformed vesicles. We have called this phenomenon the “matrix effect”,7-10 to indicate that the preformed vesicles exert a kind of matrix for the newly formed ones. The prerequisite for this phenomenon is, expectedly, a strong interaction between the preformed vesicles and the added fresh oleate. If the added oleate makes vesicles by its own accord, there is no influence of the ones on the others. More particularly, it can be qualitatively understood that a key factor is the ratio between two competitive rates: the rate of undisturbed vesicle formation starting from micelles, and the rate of binding of added surfactant to the preformed vesicles. In fact, a matrix effect is observed when oleate micelles are added to oleate vesicles, or when oleate micelles are added to 1-palmitoyl-2oleoyl-sn-glycero-3-phosphatidylcholine (POPC) liposomes, but not when POPC (ethanol solution) is added to POPC liposomes; in this last case, the velocity of the formation of POPC liposomes is so large, that no interaction with the existing POPC liposomes can take place, and there is no matrix effect. Extensive studies have been carried out in the case of excess oleate micelles added to preformed POPC vesicles. For example, the matrix effect still exists up to micelles/vesicles ratios of 100:1 (molar ratio between oleate and POPC),8 and if oleate
10.1021/jp8028598 CCC: $40.75 2008 American Chemical Society Published on Web 08/27/2008
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Figure 1. Self-reproduction of oleate vesicles and the matrix effect. (A) Turbidity vs time profile of (a) 3 mM oleate vesicle formation in bicine buffer; (b) 1.5 mM oleate vesicle formation in the presence of 1.5 mM preformed “100 nm” oleate vesicles. (B) DLS size distributions: (a) oleate vesicles as formed spontaneously in buffer; (b) oleate vesicle as formed in the presence of preformed “100 nm” oleate vesicles; (c) size distribution of preformed “100 nm” oleate vesicles before addition of the oleate micelles. (C) Freeze-fracture electronmicrographs taken 40 s after the addition of oleate micelles to preformed oleate vesicles. Characteristic “twin-vesicles” are observed. The bar represents 200 nm. Data adapted from Rasi;11 micrographs reproduced, with permission, from Stano et al.12
micelles are repetitively added (two times) to an initial population of POPC vesicles (final micelles/vesicles ) 3:1).11 In all cases, a population of mixed oleate/POPC vesicles arises, whose mean size is strongly biased toward the size of initially present vesicles. Electronmicrographs, recorded during the transitory phase of the micelles-to-vesicles transformation,7,12 show the presence of characteristic structures, recently dubbed “twin vesicles” (see Figure 1C), which may shed light on the mechanism of the matrix effect. Finally, other groups have studied experimentally and theoretically the system described in this paragraph. In particular, Szostak and co-workers have reported a stopped-flow kinetic analysis of micelle addition to preformed vesicles,13 whereas Ueno and co-workers have used size exclusion chromatography in order to analyze in greater detail the vesicle size distribution after the addition of micelles.14 The matrix effect is related to the self-reproduction of vesicles. As shown in Figure 2, the addition of micelles to preformed vesicles can be regarded as a feeding process, where preformed vesicles uptake building block molecules in a sort of “growth”, and then divide in two (or more) new vesicles having approximately the same size of the parent ones. In contrast to that, if preformed vesicles are not present, micelles undergo a spontaneous micelles-to-vesicles transformation, with the result of a heterogeneous population of vesicles with different size and morphology. Thanks to the matrix effect, in other words, not only do we observe a vesicle self-reproduction process, but we also assist to the approximate conservation of size in the next generations of vesicles. As we have already emphasized,15 the fact that, under certain conditions, which are not so stringent, a duplication of a given size of the vesicles can be obtained is interesting also from the point of view of prebiotic chemistry. In fact, this seems to
Luisi et al.
Figure2. Possiblepathwaysofvesicleself-reproduction(growth-division). A preformed vesicle (on the left) uptakes surfactants or surfactant precursors from the environment and undergoes a transformation. (a) A second layer of oleate membrane is formed by interaction of preexisting vesicle and oleate micelles or monomers. A process of vesicle “translocation” may occur, leading to two daughter vesicles. The curvature, initially imposed by the size of preformed vesicle, is somehow maintained throughout the division. (b) Oleate micelles or oleate monomers bind to a specific point of the preformed vesicles, following a cooperative process, so that a budded vesicle forms. We suppose that this vesicle relaxes to a more symmetric structure, thanks to lipid diffusion. The high-curvature region is therefore taken up by the two-lobe vesicle, which ultimately divides into two similar vesicles. The dashed line indicates the possible existence of a membrane. (c) The membrane building blocks, in the form of micelles or monomer, are taken up by the preformed vesicle, which grow in a nonspherical way. After elongation, the two vesicle lobes will form two new vesicles of similar size.
provide a mechanism by which a given size can be repeatedly made over and over by simple addition of fresh surfactant. In the current approach, the external addition of fresh surfactant (in the form of micelles) is a model for the formation of fresh surfactants from an in situ prebiotic reaction. It follows that the matrix effect would, in principle, allow the formation of particles with approximately constant dimension (and hence constant physicochemical and biochemical properties) over time. This would be a duplication brought about by mere physical forces, prior to the development of biochemical machinery to permit a more precise and regulated duplication. A general review on the importance of surfactant assemblies in an origins of life scenario, discussed also by Deamer,16 has been recently published.17 1.1. Discussion and Questions. First, it is important to briefly remark on the use of terms such as self-reproduction in the case of vesicles. By self-reproduction we mean a process where a certain molecular or supramolecular structure makes a copy of itself by capturing some molecular building blocks present in the environment, transforming them into its own structural constituents. The consequent growth may bring the system to an unstable state, so that division occurs. It is important to point out that this process leads to two (or more) new structures that are structurally similar (but not identical) to the parent one. This case differs from the more familiar example of replication, as in the case of DNA, where exact molecular copies are generated. The vesicle behavior under the conditions of the matrix effect is unexpected and surprising. At first sight, there is no reason to expect the “catalytic effect”, namely, why and how the presence of a tiny percentage of preformed POPC vesicles should induce a strong acceleration of the formation of oleate vesicles. Likewise, there is no obvious explanation of the fact that, under certain conditions, the size of the newly formed vesicles should be the same or very similar to the size of the preformed, pre-existing vesicles. We and others have tackled
Vesicle Behavior: In Search of Explanations these questions, but, until now, no satisfactorily explanation of these two effects has been formulated. In particular, it must be explained what are the factors that control the conservation of vesicle size and how the self-reproduction proceeds at the molecular or supramolecular level. The explanation must take into account the available data, which concern basically the kinetics of vesicle growth and the twin vesicles geometry, which probably represent the intermediate of the process. In general, the mechanism of vesicle self-reproduction has been explained qualitatively as follows: the preformed vesicles uptake new membrane-forming compound (added to the systems in the form of micelles), then grow, reach an unstable state, and consequently divide.7,10 The first growing step has been described as micellar coating by Chen and co-wokers,13 and as monomer uptake by Rogerson and co-wokers.18 In both cases, no inferences are done on the geometry of the intermediate. It is probably reasonable to assume that, following the uptake of building blocks, there should be a break of the initial spherical geometry in favor of a distorted vesicle, which takes the path to division. At this point, the basic question is, why is the division occurring? Why not a process of pure vesicle growth? Our tentative answer is based on the consideration that the structure that precedes the vesicle division should be nonspherical. In such geometry, the surface-to-volume ratio (S/V) is higher than in the spherical case, and this state must have been necessarily reached through unbalanced surface and volume increases, which depart from the spherical growth. In spherical growth, if the surface increases by a factor R, the volume must increase by a factor R3/2. In order to reach a state with higher S/V, the parent vesicle must increase its surface faster than its volume. Freeze-fracture electron micrographs, on the other hand, show possible intermediates (i.e., a vesicle with high S/V) in the form of “twin vesicles”, characteristic two-lobe structures found in the transitory phase of the self-reproduction process. This architecture may suggest how to complete the mechanistic description of the matrix effect, and to find an explanation of the size conservation. In fact, in addition to the favorable S/V value, twin vesicles have two lobes of approximately similar size, which can be originated by the mechanism depicted in Figure 2. In the first case (Figure 2, path a), concentric bilamellar or oligolamellar vesicles split via a mechanism of translocation, already described for giant vesicles by Wick et al.,19 and by Menger and Gabrielson.20 Notice that the precursor of twin vesicles in such mechanism has high S/V, despite its quasispherical geometry, due to higher lamellarity. In the alternative cases, the growth process does not occur spherically. In path b (Figure 2), a locally cooperative binding of micelles or monomer on the site of initial binding occurs, i.e., a budded structure, which may collapse to a more symmetrical shape, giving rise to the twin structure. In path c (Figure 2), the growth is nonspherical in the sense that an elongated vesicle is created, which ultimately divides into two new vesicles. In all cases (paths a, b, and c), however, the similar size of the twin vesicle lobes may account for the constancy of size between parent vesicle and daughter vesicles. As already said, this is just a qualitative picture of how things may happen. The challenge to the field is to possibly transform this, or alternative qualitative interpretation, into a solid theoretical, quantitative framework. All this was relative to the size problems of the matrix effect, and does not say much about the kinetic effect, namely, the observed enhancement of the formation rate of oleate vesicles in the presence of a small amount of POPC. Of course the two
J. Phys. Chem. B, Vol. 112, No. 46, 2008 14657 effects must be correlated to one another, but again, the challenge is to conceive a theoretical explanation well grounded on the physical characteristic and thermodynamics of these systems. In particular, it does not appear to be easy to conceive a mechanism by which a tiny initial fraction of POPC liposomes may “catalyze” the formation of a large excess of a different surfactant. Clearly the presence of mixed vesicles must be the determining factor, so that the incoming oleate monomers may be rapidly taken up, yielding other mixed vesicles; however, more studies and an in depth analysis appear necessary to explain this kind of unexpected catalytic effect. There is another interesting aspect related to the growth and multiplication of vesicles, and that is when we are in the presence of entrapped solute molecules. In particular, what is the fate of water-soluble compounds during the matrix effect process? This question too has not yet found an exhaustive answer. In 2001, it was shown that, when ferritin, an iron rich protein, is entrapped inside preformed vesicles, and oleate micelles are added, the average number of ferritin molecules inside the vesicles decreases, as expected for a redistribution of such molecules among the daughter vesicles.9,21 Detailed statistical analyses were not performed, however, and actually it is unclear whether such redistribution follows statistical laws or whether there is deviation from the normal behavior. Recently, Long and co-workers have reported on related aspects by using giant vesicles.22 Moreover, a key objective, related also to studies on the origin of life, is to extend such studies to multiple interacting solutes, such as in the case of a metabolic pathway. We have used water-in-oil emulsion droplets to study the division of compartments with ongoing complex chemical reactions (the expression of fluorescent protein),23 but similar studies based on vesicles are missing. Some theoretical studies on vesicles have also been carried out by Bolton and Wattis,24,25 and more recently by Bozˇicˇ,26 who interestingly introduce the hydraulic permeability in the analysis. More generally, this presentation of the matrix effect behavior has made clear a number of quite open questions, which are interesting and probably important both from the point of view in the general field of surfactant aggregates, and because of their possible biological implications in early compartments and cellular model systems. We will be back to this second point later on in this paper. 2. Size of Vesicles Interacting with RNA Both RNA and vesicles are considered very important elements in early life, but somehow their mutual interaction has not obtained much attention in the literature. Some particular aspects of the interaction between vesicles and RNA have been considered by Yarus’ group. By applying selection and amplification cycles, these authors were able to detect lipid-binding RNA molecules on the surface of phospholipid vesicles,27-30 extending their analysis to the modification of bilayer properties by supramolecular RNA complexes. Recent studies have emphasized the consequences of a heterogeneous vesicle population containing or not containing RNA on the competition between vesicles.31 RNA is a negatively charged polyelectrolyte, and may interact preferentially with cationic vesicles. In one particular series of experiments in our group, we investigated the possible influence of the vesicle size on the interaction with a mixture of t-RNA.32 At this aim, “doped” POPC vesicles, containing 3.5 mol% of cetyltrimethylammo-
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Figure 3. Interaction between 0.5 mM positively charged vesicles (POPC/CTAB 96.5/3.5) and 1.2 µM t-RNA. (A) Turbidity vs time profile of interaction between charged vesicles of different sizes and t-RNA: (a) 160 nm vesicles; (b) 80 nm vesicles. Large vesicles react with t-RNA, whereas small vesicles do not. (B) DLS size distribution of positively charged vesicles before and after t-RNA addition. “160 nm” vesicles (B, top, curve a), after addition of t-RNA, produce (reversibly) vesicle aggregates (curve b) of large size, where the electrostatic charge of CTAB has been almost neutralized by t-RNA. In contrast, “80 nm” vesicles (B, bottom, curve c), remain unchanged (curve d) after interaction with t-RNA. Adapted from Thomas and Luisi.32
nium bromide (CTAB) were prepared by classical extrusion methods in two distinct families having 80 and 160 nm diameter, respectively, and allowed to interact with t-RNA. Quite surprisingly, the outcome of this interaction depends sharply on the vesicle size. As shown in Figure 3, the addition of t-RNA to 160 nm (diameter) vesicles leads to large aggregates (>1 µm), which does not occur when smaller vesicles (80 nm) are used. In other words, in this size range, a 2-fold increase in size dramatically changes the manner of interaction between cationic vesicles and t-RNA. Moreover, it has been shown that the formation of vesicle clusters by the bridging action of t-RNA is reVersible, and that the vesicles maintain their initial size in the cluster, which is formed when the vesicle charge is approximately neutralized by t-RNA. Additional experiments with a heterogeneous vesicle population, built by mixing monodisperse vesicles of different sizes, suggest that the threshold for switching on the process of clustering is around 100 nm. This observation, together with the absence of aggregation of cholesterol-containing large vesicles, suggests that electrostatics alone does not suffice to explain the RNA/cationic vesicles interaction. 2.1. Discussion and Questions. The unexpected behavior in this case, and the question to be answered, is why and how vesicles, differing by just a factor of 2 in diameter, react very differently with RNA. The size, in this context, operates as a switcher for deciding whether vesicles undertake the RNAinduced clustering pathway or not. One can see this behavior within the larger framework of the interaction between cationic vesicles and polyelectrolytes of opposite charge. Here, one first step should involve the binding of t-RNA to the charged vesicles, and the second step should be the vesicle aggregation in the case of the larger size. Concerning the binding, and the fact that t-RNA is kept constant in this experiments, a first possible explanation may involve different charge densities on the surface of vesicles with different size. However, small vesicles doped with higher CTAB concentrations (e.g., 5 mol%) do not aggregate in the presence
Luisi et al. of RNA,32 and therefore charge density does not appear to be the key factor. In addition to electrostatic forces, steric and hydrophobic forces should be considered. In particular, membrane curvature (1/radius) may affect the stability of t-RNAvesicles adducts. The second step, i.e. the cluster formation by RNA bridging, appears also to be regulated by electrostatic forces. In fact, it has been found that the precipitation occurs when the opposite charges of the RNA-vesicles bridged particles are approximately balanced. However, the selectivity observed is probably connected to other factors, which remain to be clarified. Another interesting aspect of these experiments concerns the question of whether and to what extent the bound t-RNA may eventually become entrapped in the interior of vesicles. In general, the entrapment of biomolecular systems inside vesicles is a well-studied field, and is based on the fact that the permeability through phospholipid and fatty acid membranes is very low for high molecular weight compounds. In fact, since single enzymes,33 complex multienzymatic systems,34 and even complete transcription-translation ribosomal-enzymatic machinery has been trapped successfully inside vesicles without leaking outside.35-37 Also the entrapment of nucleic acid and enzymes after dehydrating-rehydrating steps is well described in the literature.38 The study of RNA-vesicle interaction was not focused on verifying the possible entrapment of RNA inside vesicles. Further research in this field may be of great relevance, especially if a selective mechanism will be found. This is not unreasonable, considering that large vesicles are removed by the clustering mechanism described above, and the remaining small vesicles may then eventually internalize bound RNA. There are other questions arising from these preliminary observations. For example, is this effect specific to t-RNA, or general for all kind of RNA? Is there a limit for the molecular weight/length of RNA? Do DNA and/or other charged biopolymers have similar effects? In conclusion, the combination of two well-studied systems, such as vesicles and RNA, can lead to new unexpected behavior, with deserves careful investigation since these two systems have great relevance in the origin of life scenario; see also the work by Szostak, Bartel, and Luisi in 2001.39 3. Fusion between Oppositely Charged Vesicles The third issue that, in our opinion, deserves discussion is the fusion of vesicles. Vesicle fusion is interesting per se since it is one of the fundamental reactive paths of vesicles, together with growth and division. In addition to this basic aspect, the fusion among vesicles can be seen as an important mechanism for the emergence of novel properties in the origin of life scenarios, as a kind of model for symbiogenesis, by which two complex systems share their constitution and bring about a novel system at higher degree of biocomplexity. Generally, as is wellknown, the low permeability of membranes can represent an obstacle to the development of self-sustaining, compartmentalized biochemical networks (Figure 4A). Fusion between vesicles can be achieved by a mechanism of mutual vesicle binding and reaction. In order to occur, strong hydration forces must be overcome.40,41 In fact, the presence of tightly bound water and counterions, together with the head groups’ repulsion, represent a formidable energy barrier to bilayer interactions at very short distances. In living cells, membrane fusion is a highly regulated and mediated event, which requires complex biochemical machineries.
Vesicle Behavior: In Search of Explanations
Figure 4. Vesicle fusion. (A) Fusion between vesicles as a way to productively combine several different compartments, with consequent increase of molecular complexity and as a way to circumvent the problem of low membrane permeability. Notice that when oppositely charged vesicles fuse together, they produce new vesicles that are less reactive than the initial one, so that the fusion process, in this case, is accompanied by a self-regulatory feedback. (B) An experimental case of fusion between oppositely charged vesicles: oleate vesicles and DDAB vesicles were mixed at different ratios, and the resultant populations of vesicles monitored via DLS (oleate/DDAB ) 100/0: thick solid line; 99.06/0.04: dashed line; 90/10: dotted line; 75/25 dashed-dotted line; 59/41: thin solid line). Very interestingly, at a particular oleate/DDAB ratio (59/41), a very narrow size distribution is obtained, even if the starting distribution was broad and the corresponding vesicles were stable for at least three months. Adapted from Thomas and Luisi.47
Experimentally, vesicle fusion has been achieved basically in three ways: (i) by depletion mechanism (typically by adding poly(ethylene glycol) (PEG) to neutral and charged vesicles); (ii) by bridging mechanisms, i.e., the fusion between vesicles of the same charge induced by a fusogenic agent of opposite charge (for example, the Ca2+-induced fusion of phosphatidylserine vesicles); and (iii) by direct reaction between oppositely charged vesicles. The latest approach is particularly relevant since it is expected (in contrast with the first two cases) that a strong attraction force between the membranes might facilitate the fusion. Moreover, the new vesicles produced by this mechanism have a damped net charge density, with the consequence of generating a spontaneous feedback loop, which inhibits further unspecific reactions (see comment to Figure 4A). Very interestingly, however, the number of reports on the fusion of oppositely charged vesicles is very small.42-44 In addition, as we have pointed out a few years ago,45 the term “fusion” is often used in a rather confused and controversial way. We believe that this term should be restricted to the case in which there is a real fusion and mixing between the water pools of the compartments. The simple coming together of vesicles, as induced, for example, by the addition of calcium ions, is not a sufficient evidence of a real fusion; it may simply indicate a binding, followed by the separation of the vesicles to the initial state.45 A good example of fusion is that described by Pantazatos and MacDonald.46 Let us go now to the experiments that do not have yet an interpretation. We have studied the direct interaction among positively (V+) and negatively charged vesicles (V-), and in this context we have observed a puzzling behavior in the reaction among dimethyldidodecyl ammonium bromide (DDAB) and oleic acid/oleate vesicles.47 It must be preliminarily noted that, in the described experimental conditions, the size distribution of each of these two families of vesicles is rather broad. When
J. Phys. Chem. B, Vol. 112, No. 46, 2008 14659 the two populations are mixed at different ratios, precipitation occurs in a narrow range around 1:1 mixing, whereas, in the excess of one of the two surfactants, stable vesicles are observed. If the size distributions of the resulting stable vesicles are investigated, a rather striking observation can be made. In particular, when the ratio [DDAB]/[oleic acid + oleate] is around 0.4, a very sharp size distribution with a peak centered at about 100 nm (Figure 4B) characterizes the resulting catanionic vesicles, which were stable for at least three months.47 More recently, we have reinvestigated this reaction, by using water-soluble probes with the aim of detecting possible fusion events. The system is composed of oleic acid-based anionic vesicles (V-) and DDAB-based cationic vesicles (V+). In order to increase their stability and entrapment efficiency, POPC has been used as “helper” lipid, in molar fractions of 20% and 50% for V- and V+, respectively. In order to reveal the possible fusion between the oppositely charged vesicles, we have employed a classical fluorescence assay, based on the terbium/ dipicolinic acid fluorescent complex. Preliminary data indicate that vesicle reactivity is correctly described by a landscape of possible reactions, where fusion is rarely the unique process. For example, we were unable to observe fusion without simultaneous release of entrapped components, and, in general, fusion yields are rather low (
