Exploring the Lipid World Hypothesis: A Novel Scenario of Self-Sustained Darwinian Evolution of the Liposomes

Amphiphilic macromolecules, for example, phospholipids, being released in an aqueous environment inevitably ascend and concentrate at the water-air interface. After reaching a critical concentration, the phospholipids undergo self-assembly into the Langmuir layer and bilayer, micelles, and liposomes at the water-air interface, where these assemblies are affected by solar UV radiation. The UV radiation decomposes all the assemblies into separate phospholipid molecules, forming a new Langmuir layer. This cycle of self-assembly/destruction is repeated every day when the Sun comes up, with a half-life of any assembly of about 12 h (nighttime). Provided that during the nighttime liposomogenesis the liposomes encapsulate a solute heavier than the surrounding media, for example, a solute of amino acids or sugars, those heavy liposomes will descend from a water-air interface to the bottom of the pool and become protected from UV by water. Every nighttime will add new heavy liposomes to the liposomal congregation at the bottom of the pool. The submerged liposomes, being protected from the damaging UV radiation, acquire the longevity necessary for autocatalytic replication of amphiphiles and, through their mutation and the selection of those amphiphilic assemblies, achieve the highest membrane stability.

We want to start with a quotation from Martin Gerard Rutten's work (Rutten, 1957), whose notion is extremely important for our hypothesis. Rutten writes:

Geologic science is mainly built upon the Principle of Actualism. That implies that the same natural processes, found now in operation in the atmosphere, hydrosphere, and lithosphere, have been in operation throughout most of geologic history. Intensities varied, both in time and geographically, but no other, mysterious processes can be postulated in Earth's history as long as one bases this history on an actualistic conception.

We add that regarding the origin of life on Earth, the following natural processes should also be considered: solar UV radiation, day/night cycle, gravity, and the natural formation of amphiphiles and bilayer vesicles in aqueous media. While the presence of the first three processes is evident, the fourth process, particularly the formation of phospholipid bilayer vesicles, requires substantiation.

The classical view on phospholipid membrane assembly suggests that phospholipid synthesis is catalyzed by membrane-bound enzymes, which themselves require preexisting membranes (Szathmáry, 1999; Bhattacharya et al., 2019) and enzymes (Exterkate et al., 2018) for function, which put the problem into the “chicken or egg” category. However, enzyme-free natural reactions leading to fatty acids synthesis and self-assembled vesicles are described for various experimental conditions (e.g., Bonhorst et al., 1948; Gebicki and Hicks, 1973; Simoneit, 1995; Apel et al., 2002), including the abiotic synthesis of protocell-like vesicles (Budin et al., 2009). The abiotic synthesis of hydrocarbons was suggested under natural conditions of hydrothermal fields (Proskurowski et al., 2008) and was shown in experiments with hydrothermal reactions of pyruvic acid (Hazen and Deamer, 2007). Recent observations have shown that experimental conditions similar to natural, for example, at soda lakes and hydrothermal oceanic vents, facilitate the enzyme-free synthesis of phospholipids in water (Liu et al., 2020). (Note: the extraterrestrial origin of amphiphilic molecules is out of the scope of the hypothesis, though such a source is obvious [e.g., Deamer and Pashley, 1989].)

Based on this discovery (Liu et al., 2020), we can further suggest that when phospholipid molecules are continuously released into water, they inevitably form a thin film at the air-water interface, the so-called Langmuir monolayer (Kaganer et al., 1999; Giner-Casares et al., 2014). Once the surface of the water becomes saturated with phospholipids, a spontaneous formation of a floating phospholipid bilayer and bilayer vesicles, or liposomes, encapsulating the surrounding media takes place (Ivkov, 1986; Marsh, 2012; Lombardo et al., 2015; Su et al., 2020). The formation of liposomes, or liposomogenesis, is an entirely physical process (Walde, 2006) and occurs spontaneously (Edwards et al., 1996; Lasic et al., 2001; Antonietti and Förster, 2003; Phapal et al., 2017) or under experimental conditions (Bangham et al., 1965); the liposomogenesis may include a micellar stage (Kahana and Lancet, 2021).

The hydrocarbon chains in lipid bilayers are packed to a density equal to or less than that of pure water (Sheetz and Chan, 1972; Nagle and Wilkinson, 1978; Dill and Flory, 1981). Therefore, the liposomes remain at the air-water interface (Andre et al., 1991; Ivanova et al., 1991; Launois-Surpas et al., 1992; Kulkarni and Brown, 1994; Raneva et al., 1995; Heurtault et al., 2003; Pichot et al., 2013; Staton and Dungan, 2018; Bai et al., 2019) where they can be affected by the solar UV radiation.

Solar UV radiation is universally considered an important force for life's origin on Earth. However, multiple views on the consequences of this force vary wildly. For example, Oparin considered the effect of UV to be destructive for life matter (Oparin, 1941), and this view is still upheld (Hazen, 2005; Messerotti and Chela-Flores, 2006; Todd et al., 2021). On the other hand, Haldane (1929) writes, “when ultraviolet light acts on a mixture of water, carbon dioxide, and ammonia, a wide variety of organic substances are made, including sugars and apparently some of the materials from which proteins are built up.” Harold C. Urey (1952), deliberating the Oparin-Haldane hypothesis, also concludes that “the effects of electrical discharges on chemical substances leave no doubt that many compounds would be formed due to the absorption of ultraviolet.” A creative synthetic nature of UV for the transition from abiotic matter to biotic was one of the ideas behind Stanley Miller's famous experiment: “Electrical discharge was used to form free radicals instead of ultraviolet light because quartz absorbs wavelengths short enough to cause photo-dissociation of the gases” (Miller, 1953).

Nevertheless, the opinions regarding the UV effect on phospholipid bilayers and vesicles are univocal. Starting with the first studies on the electric breakdown of phospholipid membranes by UV (Potapenko et al., 1972; Putvinskij et al., 1977; Putvinsky et al., 1979; Vladimirov et al., 1986), it was confirmed in numerous experiments that UV decomposes liposomal phospholipid bilayers into separate phospholipid molecules (e.g., Mandal and Chatterjee, 1980; Murphy, 1983; Agarwal et al., 1987; Bose et al., 1989; Pelle et al., 1990; De et al., 1993; Budai et al., 2008; Mazari et al., 2009; Pashkovskaya et al., 2010; Pires et al., 2019). It should be noted that in one experimental study a UV-induced formation of bilayer vesicles was observed (Veronese et al., 1998), although the UV source used was lacking the shortwave spectrum (<300 nm) typical for primitive Earth (Miller and Urey, 1959).

Extrapolations from the historical evolution of the solar UV luminosity indicate that at the time of primitive Earth, the UV radiation was of a shorter wavelength (Miller and Urey, 1959) and had an intensity of 3–4 orders of magnitude higher than at present (Zahnle and Walker, 1982; Cockell, 1998, 2000). Oparin (1974) wrote:

The action of shortwave UV light or some other energy source upon the dissolved organic substances in the primitive ocean created a thermodynamic equilibrium. Numerous calculations have shown that under these conditions, nothing like the concentrated ‘primordial soup’ could appear, since under the action of UV the decomposition processes had to proceed much faster than the synthesis.

Therefore, the destructive effects of UV on prebiotic and biotic compounds have always been considered the major limiting factor for life originated on the water surface of early Earth (Sagan, 1973; Oparin, 1974; Cleaves and Miller, 1998; Messerotti and Chela-Flores, 2006; Björn et al., 2015; Todd et al., 2021). How can phospholipid vesicles, which are formed and tend to concentrate at the air-water interface, be spared from being dismantled into individual phospholipid molecules by strong UV radiation? Can we come up with a model of phospholipid vesicle protection from UV?

Martin Rutten gave a simple solution in his book The Origin of Life by Natural Causes:

But how can the floating liposomes submerge into deeper layers of water?

It is well known that the liposomal entrapment of either lighter or heavier solutions than the aqueous media in which the liposomes are suspended results in the formation of either floating or submerging liposomes. Irrespective of the composition of the entrapped solution, the heavy liposomes would sink away from the air-water interface to deeper layers of water (Chakrabarti et al., 1990, 1992; Veiro and Cullis, 1990; Kernen et al., 1992; Shin et al., 2005). As a matter of fact, loading liposomes with an aqueous media of variable specific gravity is a common approach in liposome separation techniques (e.g., Chakrabarti et al., 1990; Li et al., 2006; Schulz et al., 2009; Bian and De Camilli, 2019).

From the revolutionary Miller-Urey experiments (Miller, 1953; Miller and Urey, 1959) followed by many others (for review, see Lazcano and Bada, 2003), we know that, in addition to amino acids, numerous molecules heavier than water, for example simple sugars (Bada, 2004) and even simple peptides (Parker et al., 2014), could be continuously synthesized in the prebiotic Earth atmosphere. These Miller-Urey molecules (MUm) may come into contact with the Langmuir layer and be encapsulated during liposomogenesis at the water-air interface during nighttime. The presence of MUm in the liposomal interior may change the liposomal buoyancy from a positive to a negative. The liposomes then may descend from the water-air interface. Additionally, the already formed liposomes can extract MUm from the surrounding media during the nighttime; such extraction was observed in experiments (de Souza et al., 2014), even against the concentration gradient of these molecules (Sugiyama et al., 2020), and this phenomenon is thought to be relevant to the origin of life (de Souza et al., 2014).

Another source of heavy elements for the heavy liposomogenesis is plausible. Solutes of hydrogen sulfide, ferric iron, along with a variety of molecules, for example B, Zn, Mn, P, S, are assumed to be naturally present in primordial water (Ranjan et al., 2018; Damer and Deamer, 2020; Van Kranendonk et al., 2021). These Natural Primordial Heavy molecules (NPHm) could be carried by the upward water motion to the surface. That process can coincide with the liposomogenesis during nighttime, or NPHm can be extracted and concentrated by already formed liposomes by the mechanisms mentioned above (de Souza et al., 2014; Sugiyama et al., 2020).

While the liposomal entrapment of heavier molecules, coming from either the atmosphere or the bottom of the pool, is a random event, the behavior of heavy liposomes is not. It is governed by the laws of physics according to the newly acquired gravimetric properties (Chakrabarti et al., 1990, 1992; Veiro and Cullis, 1990). The liposomes that entrap the solution with a specific gravity heavier than that of the surrounding media must inevitably submerge and congregate at the bottom of a pool, where they become protected from the damaging UV radiation by the water.

To survive the distraction from UV radiation, a liposome vesicle formed at the water surface must submerge during the dark hours of the nighttime to a depth where the UV intensity will be sufficiently attenuated due to absorption in water.

How deep should a liposome be submerged in water to be protected from UV? In other words, what is the water thickness sufficient to attenuate UV (≥99%)?

UV attenuation strongly depends on the water composition, particularly on the concentration and type of solutes (Ranjan et al., 2022). While pure fresh water is largely transparent for UV (e.g., Su and Yeh, 1996; Ranjan et al., 2022), Cleaves and Miller demonstrated that UV flux is attenuated down to 1% of the initial value by just 2 mm of ocean water (Cleaves and Miller, 1998). Similar results were obtained in experiments with sedimental waters: the UV light is extinguished to 1% of the incident level by about 1 mm of sedimental waters (Garcia-Pichel and Bebout, 1996), or by water with a 2.5 g/L solution of FeCl₃ (Gómez et al., 2007). While an accurate extrapolation from the above experiments to the prebiotic conditions is difficult, let us assume that the necessary “safe” depth is about 10 mm.

How long would it take for a liposome to submerge to that depth to be protected from UV?

A terminal (settling) velocity of an object gravitationally falling in the water is determined by the balance between the gravitational force, $F_g$, buoyancy force, $F_b$, and the frictional force, $F_d$. For a spherical vesicle, $F_g=\frac{4}{3}\pi {\rho }_{v}g{R}^3$, $F_b=\frac{4}{3}\pi {\rho }_{w}g{R}^3$, and $F_d=6\pi \mu Rv$, where ${\rho }_v$ and ${\rho }_w$ are the vesicle and water mass densities, respectively, R and v are the vesicle radius and the velocity, and $\mu$ is the water dynamic viscosity coefficient. From that, the settling velocity is given by:

Taking a typical water viscosity $\mu =10^{-3}kg{m}^{-1}{s}^{-1}$, obtain in practical units:

Assume that the vesicles continuously submerge for the 12 h of nighttime darkness. The submersion depth versus the vesicle radius is shown in Fig. 3 for “light” vesicles with a relative specific density of ${\rho }_v∕{\rho }_w=1.1$ and “heavy” vesicles with a relative specific density of ${\rho }_v∕{\rho }_w=2.0$.

We see that light vesicles with a radius greater than 3 μm are able to submerge at a depth of 10 mm during the nighttime, while for heavy vesicles, the minimum required radius is lower, about 1 μm. The vesicles that are too small or too light will not have enough time to submerge to that depth and will be destroyed by UV when the Sun comes up. Larger and heavier vesicles will submerge to that “safe” depth and will be protected from UV.

The important point is that the terminal velocity is proportional to the square of the radius of liposomes; thus the submerging time is inversely proportional to that. That would substantially increase the survivability of larger liposomes.

Therefore, based on these calculations and experimental observations (Garcia-Pichel and Bebout, 1996; Cleaves and Miller, 1998; Ranjan et al., 2022; Gómez et al., 2007), we can suggest that (1) nighttime (12 h) is sufficient for the submergence of heavy liposomes in primordial water to a depth of about 10 mm, which should offer complete protection from the damaging UV radiation, and (2) the negative buoyancy of liposomes can constitute a fitness feature under UV selective pressure that favors heavier/larger liposomes.

At this point, we would like to change the dwelling of the hypothesized events from the bottomless ocean (hydrothermal vents) to the localized fields emerging above sea level, such as hot springs, boiling pools, geysers, fumaroles, and shallow aqueous pools associated with these hydrothermal fields (Mulkidjanian, 2011; Damer and Deamer, 2015; Deamer, 2017). In this new scenario, all submerging heavy liposomes will not be scattered in the water depth; but eventually, all appear spatially in the same plane, that is, congregate at the bottom of a pool and become protected from the damaging UV radiation during daytime.

What could be the longevity of liposomes if the UV destruction force is removed from the scenario?

Experimental observation showed that liposomes could maintain their integrity in the nonfrozen aqueous media from 60 days (Hsieh et al., 2002; Kim et al., 2004) to a year (Sakai et al., 2000; Zana, 2005; Tokuno et al., 2016), even at +40°C (Sakai et al., 2000). Therefore, we can hypothesize similar longevity of prebiotic liposomes, which allows the hypothesis to apply an important concept of the Lipid World model: autocatalytic replication of amphiphiles, their mutation, and selection of the fittest amphiphilic assemblies. (Note: amphiphilic assemblies' replication, mutations, and selections can occur any time after the emergence of such assemblies since these processes occur in nanoseconds' time range [Kahana and Lancet, 2021; Kahana et al., 2022]; however, the evolved novelties will not survive beyond 12 h at the water-air interface.)

It is important to note that the above longevity of liposomes was observed with a single species of amphiphiles in the laboratory, which is an unlikely scenario for the prebiotic conditions. Numerous analyses suggested a heterogeneous repertoire of amphiphiles under prebiotic conditions (Chen et al., 2004; Zhu and Szostak, 2009; Szostak, 2011); furthermore, the heterogeneous amphiphiles showed the most advantages in a simulation study with the GARD model (Lancet et al., 2018).

A compositional design of amphiphilic micelles and the liposomal membrane was elaborated in the model of heterogeneous amphiphilic assemblies forming a mutually catalytic network (Segré et al., 2000, 2001), based on the concept pioneered by Kauffman (1971, 1993). Catalytic self-replication of amphiphilic assemblies was analyzed theoretically (Farmer et al., 1986; Kauffman, 1986; Segré et al., 1998b) and was shown in experiments (Lee et al., 1997). The model of noncovalent amphiphilic assemblies acting as transporters of compositional information and capable of undergoing replication, mutation, selection, and evolution were elucidated in numerous contributions by Lancet's group (e.g., Segré et al., 1998a, 1998b, 2000, 2001; Segré and Lancet, 2000; Markovitch and Lancet, 2011). Particularly, the detailed behavior of liposomes' constituents, in light of the origin of life and evolution, was analyzed by Lancet and coauthors in a simulation study using the metabolic graded autocatalysis replication domain (M-GARD) model (Lancet et al., 2018).

It is logical to suggest that among the inherited variations of amphiphilic assemblies certain variations will constitute the most resilient membrane design and facilitate maximum liposomal longevity in the environment. Alternatively, other variations of amphiphilic assemblies would fail to provide lasting membrane integrity, resulting in liposomal breakdown and amphiphiles returning to the water-air interface.

Our hypothesis suggests that the heavy liposomes that acquired a resilient membrane would be aggregated at the bottom of a pool, staying in physical contact with each other for a long time. It is well-known that physical contact between liposomes resulted in liposomal fusion/growth (Nir et al., 1983; Connor et al., 1984; Noguchi and Takasu, 2001; Deshpande et al., 2019), exchange of contents and membranes (Hanczyc and Szostak, 2004; Chan et al., 2008; Yang et al., 2009; Hardy et al., 2015) and fission/split (Döbereiner et al., 1993; Deshpande et al., 2018; Penič et al., 2020).

Since the above mechanistic processes assume an exchange of compositional information between liposomes, it is plausible that this physical interaction can also contribute to transmissible variations and selection of amphiphilic assemblies/liposomes by the processes described by Lancet's group in GARD models.

In theory, continuous variations in these two sets of compositional information must have the following consequences for the fate of the heavy liposomes:

Therefore, our model extends the GARD/Lipid World scenario (Segré et al., 2001; Lancet et al., 2018) and explains how one of the counterparts of multistage processes—liposomes—can emerge first, survive, and propagate for a long time in abundance by themselves, thereby circumventing the obstacle regarding the spontaneous occurrence in time and space of several probable events.

While the next evolutionary step—a co-emergence of RNA precursor molecules, via the endogenous or exogenous pathways—is out of the scope of our hypothesis, we would like to sketch plausible scenarios.

In the endogenous scenario, we can appeal to autocatalytic chemical networks, a phenomenon that the founding contributions (e.g., Kahana and Lancet, 2021) and our model consider among major requirements for early protocell survival and evolution. There are indications that RNA monomers can be synthesized within liposomes, via the same processes that underline autocatalytic replication, mutation, and selection of amphiphilic assemblies (Xavier et al., 2020). This pathway appears to be parsimonious and does not require any further steps.

As to the exogenous pathways, we can appeal to many models and experimental observations on the spontaneous synthesis of RNA precursors under prebiotic conditions: in the atmosphere (e.g., Oró and Kimball, 1961, 1962; Nguyen et al., 2015; Becker et al., 2016; Ferus et al., 2017; Rodriguez et al., 2019); at the water-air interface (e.g., Nam et al., 2018); and in water (e.g., Cafferty et al., 2016; Joyce and Szostak, 2018).

However, within the exogenous pathways a question arises: What is the probability of interaction between dispersed RNA precursors and liposomes? We believe that in our model there are two scenarios with this probability being very high.

First scenario: Considering the continuous synthesis of phospholipids in primordial water (Liu et al., 2020) and Langmuir monolayer formation (Kaganer et al., 1999; Giner-Casares et al., 2014), the RNA precursors that are synthesized in the atmosphere, or at the water-air interface, would come in contact with Langmuir monolayer. During the nighttime liposomogenesis, these precursors would be encapsulated and, together with other heavy molecules, would serve as initial heavy cargo for the submergence of liposomes.

Second scenario: Since RNA precursors have a higher specific gravity than water, for example, a ribose (Zhao and Wang, 2021), these precursors must come in contact with the water surface and, over time, descend into the water. What would be a hypothetical depth-point where RNA precursors would be concentrated? It is the bottom of the pool, which, in our model, already housed the heavy liposomes. We can further invoke the observation of spontaneous extraction of molecules from surrounding media by liposomes (de Souza et al., 2014; Sugiyama et al., 2020).

Providing the retention of a heavy content and continuous autocatalytic replication, mutation, and selection of amphiphilic assemblies toward membrane stability, as elucidated in numerous contributions by Lancet's group, we can hypothesize extended longevity of the heavy liposomes and the longevity of the population of heavy liposomes hence much greater. Since all heavy liposomes are in the same plane exchanging content (Hanczyc and Szostak, 2004; Chan et al., 2008; Yang et al., 2009; Hardy et al., 2015), an accidental assembly of RNA precursors into functional molecules became plausible, especially considering the lipid-assisted synthesis of RNA-like polymers (Rajamani et al., 2008).

The hypothesis can be tested by following experiments.

In this work, we offer a new model of the self-sustained Darwinian evolution of liposomes. The model can be viewed as an extension of the Lipid World hypothesis, to which it adds the contribution of the following ever-present natural events we believe to be essential for the origin of life:

Vladimir Subbotin: Initial conceptualization, formal analysis, visualization, writing—original draft, writing—review and editing.

Gennady Fiksel: Elaboration of the hypothesis, methodology, terminal velocity model, validation, mathematical analysis, writing—original draft, writing—review and editing.

The authors thank two anonymous reviewers for their constructive comments, critiques, and insights.