Composite Liquid Marbles as a Macroscopic Model System Representing Shedding of Enveloped Viruses via a Budding Mechanism

A model macroscopic system imitating entering of viruses into living cells is suggested. The system represents the contact of a composite (core-shell) liquid marble with hydrophobic/hydrophilic particles. Composite liquid marbles are water droplets coated with silicone oil armored with nanometric hydrophobic particles and served as an interfacial model of a living cell. Composite marbles swallowed hydrophilic polymer particles, but prevented hydrophobic particles from entering their core. Swallowing of hydrophilic particles by composite marbles resembles the budding of living cells by viruses. The interfacial mechanism of swallowing is suggested.

Viruses are the most abundant biological entities on earth, more than single-celled organisms and bacteria together. 1They also represent the largest and most genetically diverse reservoir of nucleic acid that can infect all forms of life.Currently,1.67 million unknown viruses are estimated to be circulating in animal reservoirs, demonstrating the potential for epidemics. 13][4] This envelope is partly composed of the cell membrane within which the virus replicated, and it contains proteins and carbohydrates. 4Viruses are tiny objects with a characteristic dimension of ca. 100 nm. 1 Thus, their experimental exploration is extremely challenging.In particular, the experimental study of the physics of the process of viral shedding occurring via viral budding 5 is a complicated task.
We suggest a macroscopic system imitating viral budding by living cells, namely we explored swallowing of solid particles by bi-liquid composite marbles.7][8] Liquid marbles are not hermetically coated by solid particles.3][24] The structure of the external interface of a composite liquid marble resembles that of living cell membranes, built of lipid bilayers (including cholesterols) and containing intrusions of globular proteins and fragments of the cytoskeleton, as depicted in Figure 1 a-b.In both of the composite marbles and living cells water is enveloped by a hydrophobic layer filled by heterogeneities.We do not exaggerate the physical similarity of the composite marbles and living cells, however we demonstrate that the core-shell marbles enable us to exemplify the budding of enveloped viruses within a macroscopic system.Thus, composite marbles supply an interfacial model of a living cell.
We studied the contact of composite marbles with four kinds of solid object of two different millimetrically-scaled sizes, namely: hydrophobic particles, hydrophilic particles, hydrophobic and hydrophilic particles coated with oil.Three very different scenarios of contact were observed: (i) pristine hydrophobic and oil-coated hydrophobic particles were attached to the surface of the liquid marble, (ii) hydrophilic particles were swallowed by the composite marbles and penetrated its core, at occurs when a living cell "absorbs" the enveloped virus; (iii) oil-coated hydrophilic particles accomplished the coating layer of the composite marbles, becoming attached to the marble in the nearest vicinity of its surface.All of these scenarios will be addressed below in detail.We pose a following general question: how do soft objects filled with water and coated with armored oil layers contact with entities possessing different hydrophilicity?

Composite marbles prepared as shown in
where , and primary diameter more like 30 nm are the radii ≅4.0  ≅0.5  ≅50  of the marble, PP and fumed fluorosilica particles respectively (see Figure 1).Thus, hydrophobic PP particles are small relative to the marble but large relative to the fumed flurosilica particles.For the purposes of brevity we label these PP particles as "small" ones, and the fumed flurosilica particles as the "colloidal" ones.The same spatial inter-relation generally keeps for "budding" of living cells by viruses, if R, a and b represent the characteristic dimensions of a living cell, virus particles and globular proteins respectively (see Figure 1).
The experimental findings evidence that the small hydrophobic PP particles behave as colloidal ones and do not penetrate into the marble volume, instead adhering to the composite marble surface.The physics of this attachment was explained in refs.6-8, when a smooth spherical particle of radius r comes to the liquid surface from air, the energy gain is given by: where is the Young contact angle inherent to the particle/liquid/air system and is the surface θ  γ tension of the liquid/air interface.It is seen from Eq.2, that the entire system containing the marble and particle lowers its energy by the particle adhering to the interface regardless of the radius of the particle.However, it is thermodynamically unfavorable for the hydrophobic PP particle to enter into the water volume, and it remains attached the composite marble surface as depicted in Figures 4a, 5a.hydrophilic particle) emerges, pushing the particle into the water core (see Figure 6).The force acts until the wetted area is increased, in other words until complete wetting of a hydrophilic  → particle by water.It seems reasonable to suggest that the similar interfacial mechanism governs entering of viruses into living cells.However, the study of the exact mechanism of virus budding of living cells remains a challenging experimental and theoretical task.It is noteworthy that the study of our model system only sheds light on the interfacial aspect of this mechanism.
In the case of oil-coated hydrophobic and hydrophilic particles the wetting scenario was different.Both of these particle types adjusted themselves to the coating layer of liquid marbles, thus accomplishing wrong word-not sure what you are trying to say? it as demonstrated in We also studied the contact of large hydrophobic and plasma hydrophilized ≈2.5  PP particles by the composite marble, illustrated in Figures 6a, b and c, d and the supporting Movies S8, S9 and S10.In this case the spatial scales are given by Eq. 4: The nature of the interfacial processes remained the same irrespective of the change in the spatial scales, namely: hydrophobic PP polymer particles did not penetrate into the composite marble core, whereas plasma hydrophilized PP particles were swallowed by the composite marble, as shown in Figure 7.The characteristic time of the "swallowing" was established experimentally as .τ≅???  We conclude that the composite (core-shell) marbles swallowed hydrophilic polymer particles, thus supplying the interfacial model of the processes occurring under budding of living cells by viruses.At the same time, the composite marbles prevented hydrophobic particles from entering their core.This conclusion is true for particles of various sizes.
26; the primary particle diameter is approx.30 nm.
A Nikon 1 v3 camera was used to capture images, movies of composite liquid marbles and the process of swallowing of different particles.

Polypropylene (PP) particles of two diameters (1 mm and 5 mm), supplied by
Sigma-Aldrich, were used as hydrophobic particles.The apparent water contact angle of pristine PP was θ =???

Methods
Hydrophobic glass substrates were prepared by coating of the cold plasma-treated microscope glass slides with a very thin layer of PDMS oil as described in our earlier work [35].The slides were coated with silicone oil with a spin coater (Laurell Technologies, WS-650MZ-23NPPB Spin Processor) at 5000 rpm.A 5 µL water drop was deposited using a micropipette on the oil-coated hydrophobic glass.The spreading parameter describing wetting of the silicone oil/water system is positive (for the detailed analysis of the spreading parameter see the discussion in ref. 25); thus, the silicone oil covers the water drop.Tilting of the glass substrate forced the water drop covered with silicone oil to roll to the Petri dish containing fumed fluorosilica particles, as shown in Figure 2. The particles adhere to the drop giving rise to a stable composite liquid marble as depicted in Figure 3.The process of composite liquid marble preparation, carried out under ambient conditions, is illustrated in the supplementary Movie S1.
The weight ratio of the components of the composite marbles, namely water/oil/particles was established by weighing as1/0.054/0.102.Polypropylene (PP) particles were plasma treated using an inductive radio frequency air plasma discharge with the following parameters: frequency was 13.56 MHz; air pressure was 0.5 Torr; power of the plasma discharge was 18 W; time span of irradiation was 300 s.The apparent water contact angle of plasma treated PP was .θ =???  Apparent contact angles were measured with a Rame-Hart goniometer at ambient conditions.

Figure 2 and 3 .
Movie S1 and described in detail in the Method section were transferred to hydrophobized glass slides.An image of the composite marble is shown in Figure Objects possessing very different wetting properties (in other words various surface energies) were placed in the vicinity of the composite marble, as shown in Figure 4 and pushed gently with a spatula towards the surface of it.Let us start with the millimetre-sized hydrophobic (polypropylene, PP) particles which were attached to the surface of the composite marble, as shown in Figures 4a, 5a and Movie S2.Let us introduce the spatial scales, inherent for this experimental situation, according to Eq.1:, Figures 4b, 5band Movie S4.The characteristic time of the "swallowing" was established

Figure 4c -
Figure 4c-d and Movies S6, S7.It was observed that oil-coated hydrophobic particles are

Figure 1 .Figure 2 .
Figure 1.Sketch demonstrating the interfacial resemblance of a composite liquid marble (a) and

Figure 6 .
Figure 6.Mechanism of penetration of hydrophilic particles into the bulk of a composite liquid

Figure 7 .
Figure 7. Attachment of oil-coated hydrophobic particle to a composite liquid marble: (a)