Endocytosis of Coacervates into Liposomes

Recent studies have shown that the interactions between condensates and biological membranes are of functional importance. Here, we study how the interaction between complex coacervates and liposomes as model systems can lead to wetting, membrane deformation, and endocytosis. Depending on the interaction strength between coacervates and liposomes, the wetting behavior ranged from nonwetting to engulfment (endocytosis) and complete wetting. Endocytosis of coacervates was found to be a general phenomenon: coacervates made from a wide range of components could be taken up by liposomes. A simple theory taking into account surface energies and coacervate sizes can explain the observed morphologies. Our findings can help to better understand condensate–membrane interactions in cellular systems and provide new avenues for intracellular delivery using coacervates.

For the modification of microscopy slides, we used poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG, SuSoS AG), µ-slides with 18 wells and 6 channels (No. 1.5, polymer coverslip, Ibidi GmbH). For both two types of slides, the surface was cleaned using a plasma cleaner, then adding 30 μL/100 μL of a 0.1 mg/mL PLL-g-PEG solution, which dissolved in 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH = 7.4), to each well/channel, covering it with the lid and incubating slides at room temperature for 24 h. Finally, slides were washed with MQ water and dried with compressed N2.

b. polyU/polyA labelling
The polyU and polyA were labelled with AlexaFluor647-hydrazide using a periodate oxidation reaction. Take polyU for an example, 72.5 μL of polyU (20 mg/mL), 14.5 µL of nuclease free water, 3.33 µL of 3 M sodium acetate (pH = 5.2), and 10 µL of 25 mM sodium periodate (in nuclease free water, freshly prepared on the day) were added into a 1.5 mL microcentrifuge tube (1.5 mL, Eppendorf). The mixture was incubated on ice for 50 min. Subsequently, the polyU was spun down (14,000 g at 4 ºC for 10 min, twice) by using Amicon spin concentrators (30 kDa). After that, the concentrated polyU was mixed with 25 nmol AlexaFluor647-hydrazide dye and 100 mM sodium acetate (pH = 5.2). The mixture was incubated at 4 ºC for at least 6h/overnight in the dark, after which the labelled polyU was purified with several rounds of sample concentration using an Amicon spin concentrator (30 kDa) and diluting with nuclease free water during the concentration process. The purity was checked by 1% agarose gel electrophoresis and the concentration was calculated by using the Nanodrop. The concentration of labelled polyU and polyA were 21.2 mg/mL and 4.0 mg/mL, respectively. The labelled DNA oligonucleotide polyG11 was dissolved in nuclease-free water at a concentration of 100

c. Stock solutions
μM. In addition, the following stock solutions were also prepared in nuclease-free water: polyA (6.4 mg/mL), polyC (6.4 mg/mL), polyU (10 mg/mL), torula yeast RNA (3.4 mg/mL), GFP-K72 (140 µM). Lipid stock solutions in CHCl3 were prepared by drying the purchased lipid solution in the flask using a rotary evaporator and then redissolving the dried lipid in the desired amount of CHCl3 to a final concentration of 50 mg/mL for POPG and POPC and 25 mg/mL for DOTAP. ATTO-488-DOPE, ATTO-655-DOPE and cholesterol powders were dissolved in CHCl3 at a concentration of 1 mg/mL, 1 mg/mL and 60 mg/mL, respectively. All the solutions were stored at −20 °C, except Tris-HCl, MgCl2, NaCl, glucose and sucrose, which were stored at 4 °C. All lipid solutions were transferred to HPLC vials and kept under argon.

d. Preparation of coacervates
The liposomes were prepared based on the water-in-oil (W/O) emulsion transfer method, as has been reported previously. 3  μL of the precipitated liposomes suspension was collected through a hole pierced using a needle at the bottom of the microcentrifuge tube. The obtained liposomes suspension was further centrifuged at 5,000 rpm (2,348 ×g) at 20 °C for 5 min. The supernatant was removed from the microcentrifuge tube as far as possible whilst not damaging the newly formed pellet, which was then redispersed in 20-100 μL of the outer solution. Finally, dispersed liposomes with 5-50 μm in diameter were obtained, as shown in Figure S1.

e. Preparation of coacervates
Typically, coacervates were prepared by first mixing NaCl, Tris-HCl, MgCl2, MQ, glucose and the desired type of negatively charged polyU, polyC, polyA, RNA, ATP or pGlu in a microcentrifuge tube (0.

f. Confocal microscopy experiments
Samples of interaction between coacervates and liposomes for the microscopy experiments were prepared in microcentrifuge tubes. Normally, 20 µL of a freshly prepared coacervate or liposome dispersion was added directly to a modified μ-slide chamber for checking the formation and concentration. Then, the liposomes were diluted with the outer solution to a suitable concentration and the diluted liposomes were added to the μ-slide channel (6×). The slide was placed onto the microscope and the coacervate droplets were then added from one side of the channel.
Images and videos were obtained by using a Leica TCS Sp8X confocal microscope, equipped with

g. Critical salt concentrations
The critical salt concentration of single-phase coacervates was measured on a microplate reader (Tecan Spark), equipped with a microinjector, as described elsewhere. 4 Briefly, turbidity of a coacervate solution with a total starting volume of 100 µL (CNaCl = 0 M) was monitored as a function of the concentration of NaCl at a wavelength of 600 nm and a temperature of 25 ± 0.5 ℃ in 96-well plates (Greiner Bio-one, clear flat-bottom wells) by titration with NaCl (0.3-3.0 M) in 2 or 5 µL steps. Samples were incubated for 10 min at test temperature. After each injection step, the samples were mixed by shaking for 3 s, followed by equilibration for 5 s, and shaken for another 3 s before every readout. The critical point was calculated by extrapolating the first-order derivative at the inflection point to zero turbidity. Note that this critical salt concentration does not take into account ions from other sources than the added NaCl, and the actual critical ionic strength may be slightly higher.

h. Measuring the surface charge of coacervates and liposomes
Zeta potential measurements were conducted on a Malvern DLS-Zetasizer. Coacervates were formed in similar buffer conditions to those for the interaction with liposomes experiments (50 mM Tris-HCl at pH = 7.4, 5 mM MgCl2, 300 mM glucose, and/or 50 mM NaCl). The positively charged and negatively charged molecules for forming the coacervates were each diluted 2 to 10 times. After coacervates were formed, samples were injected into a disposable folded capillary cell (DTS1070) and measured at 25 °C. Three measurements were taken, each consisting of 100 runs. For the measurements of liposomes, they were diluted 3 to 8 times after formation and the other test methods and conditions were the same as for the coacervates.

i. Supported lipid bilayer formation
Supported lipid bilayers (SLBs) were prepared following the vesicle fusion method described in Kurniawan et al. 5  mM NaCl) at 2 mg/mL using a vortex. The lipid mixture was sonicated using a probe sonicator (MSE Soniprep 150) at 4 µm amplitude, 15 s on/off, on ice for 10 cycles. Right before SLB formation, the vesicles were diluted to 0.5 mg/mL in SLB buffer and extruded through a 100 nm polycarbonate filter (Avestin) to form monodisperse small unilamellar vesicles (SUVs). To form the SLB, a mica disk (Nano-Tec) was glued to a glass coverslip. Several layers of mica were peeled off using scotch tape. The clean mica surface was activated using a plasma cleaner (Diener Electronics Femto) at full power and time for 2 cycles. A small PDMS ring was placed around the mica disk onto the glass coverslip and the mica was incubated with 400 µL of 0.5 mg/mL lipids for 20 min at room temperature. After incubation, the remaining SUVs were washed away by carefully adding and removing 500 µl of SLB buffer for a total of eight times, taking care to always keep the SLB in buffer. Presence of lipids at the mica surface was tested using confocal microscopy (Leica SP8 Liachroic), as shown in Figure S14.

j. Contact angle measurement
For the contact angle measurements, spermine/polyU (13:1) coacervate droplets (condensed phase) were deposited on SLBs with various DOTAP compositions (0-50 wt%). The contact angle was determined using an FTA1000 Drop Shape Instrument (First Ten Angstroms). Briefly, spermine/polyU (13:1) coacervate droplets were prepared in 5 mL microcentrifuge tubes (5 mL, Eppendorf) and incubated at 30 °C for 5 min for droplet formation. Subsequently, the coacervate droplets were spun down in a centrifuge (Eppendorf 5810R) at 4,000 rpm (841 ×g), 10 min, at 30 °C. The top dilute phase was carefully removed and the bottom condensed phase was stored at 30 °C until use. Using a blow dryer, the metal stage on the drop shape instrument that holds the sample was preheated to around 28-30 °C. The previously prepared SLB was placed into a preheated custom-made glass box and preheated SLB buffer was added to completely cover the slide. The PDMS ring was removed carefully and the box was placed on the metal stage. Using a plastic spatula and a fine point needle, a droplet of condensed phase was placed on top of the submerged SLB.
Images were taken at 0, 5 and 10 min, respectively. The temperature of the buffer was monitored using a temperature probe. The temperature was kept around 28 °C by heating the stage as needed. Contact angles were determined using Image J (with contact angle plugins), 6 contact angle = 180 -average Theta (average of left and right Theta). As the droplets need time to equilibrate, we analyzed the images at 10 min, which as shown in Figure S15. Tables   Table S1. Structures of the lipids used in this study.  Table S3. Zeta potential of different combinations of liposomes, zeta potential and critical salt concentrations (CSC) of different types of coacervate droplets. Turbidity measurements and zeta potential were performed in triplicate and error represents the standard deviation (n = 3).

Note to Figures S4-S6
We note that the wrapping times of coacervates can vary significantly. While wrapping was complete in 15 seconds in Figure 1e, in one other case, it took nearly 20 minutes. It appears that the coacervates that are wrapped slowly spend a substantial time in a partially wrapped state ( Figure S6b). Possibly, the strength of the interaction between the coacervate droplet and the membrane is weaker than in the example shown in Figure 1e. This could occur in the same sample, because not all coacervates and liposomes have the same surface charge: Figure S7 and Table S3 indicate that the zeta potentials of both coacervates and liposomes have a distribution with a 10-50% standard deviation. Indeed, we also observed coacervates that remained in a partially wetted state in the sample containing 20% DOTAP, likely because their interaction was not strong enough to ensure full wrapping. Finally, it is also possible that the number of coacervates interacting with the same liposome simultaneously has an effect on the wrapping time: adhering coacervates could lead to an increase of the effective membrane tension and slow down wrapping.
We quantified the changes of the membrane area upon endocytosis, and found that it remains to a good approximation constant during the engulfment of a single coacervate droplet: relative perimeter changes in Figure 1e, S6a and S6b: -1%, +2%, +3% (±8%), respectively.
However, when multiple coacervates (about 20) had entered the liposome by endocytosis, the membrane area was significantly smaller, as can be seen in Figure S4d-e (final perimeter change: -12 ± 8%). We do note that these images are 2D cross sections of a 3D liposome. Slight shifting of the focal plane could also lead to changes in the apparent perimeter or area. Figure

Note to Figure S7
Changing the spermine/polyU ratio not only changes the surface charge, but possibly also the material properties of the coacervates. We therefore determined their critical salt concentration (CSC, Figure S7c), as an indirect measure of the expected changes in the interfacial tension, viscosity and density of the coacervates. The CSC decreases with increasing polyU content, suggesting that coacervates with a higher polyU content have a lower interfacial tension, viscosity and density, and may therefore be 'softer'. As softer droplets are more prone to spreading and require stronger adhesion energy to achieve successful internalization, the formation of endosomes may happen for slightly different interaction strengths and droplet sizes for other condensates.  Figure S9: Images of liposome channel with droplets partially encapsulate by lipids or forming an endocytosis structure. (cf. Fig. 2b,c,f,g,h). All scale bars represent 10 µm.

Note to Figure S10
We found that endocytosis and other wetting phenomena can occur for a wide range of liposome sizes (diameters between 7 and 22 µm) and coacervate sizes (diameters between 0.9 and 7.7 µm). To provide more insight into the suggested size effect of endocytosis, we analyzed the sizes of the coacervate droplets that displayed endocytosis and partial wetting in all samples with liposomes containing 20% DOTAP and coacervates composed of spermine/polyU 13:1 and 7:1 (for example shown in Figure S10c,h). We found that endocytosis occurs for coacervates that are on average slightly smaller, while partial wetting occurs for larger coacervates (see histogram below) However, there is substantial overlap between both distributions and the difference between the averages is less than a standard deviation. This is likely the result of the distribution of surface charge of both coacervates and liposomes ( Figure S7-8, Table   S3), which implies that the strength of the interaction is not identical for all coacervates and liposomes in a single sample.

Supplementary theory of coacervate-liposome interactions a. Droplet shapes
One of the crucial findings of Kusumaatmaja et al. is that the shape of a large droplet wrapped by an elastic membrane is determined in analogy to the shape of a droplet at the interface of two other liquids. 7 This result is based on the rational that the bending energy depends only marginally on the droplet radius, while the area of the droplet and the contact area increase quadratically with the droplet radius. The balance of the related surface tensions thus dominates the droplet shape. The angle that the droplet forms at the contact line is then given by Neumann's law: We distinguish five shape types. The parameter regimes in which the different shapes are found are as follows.

b. Impact of the liposome size on the effective membrane tension
To derive the impact of the liposome size on the effective membrane tension, we first consider the limiting case of an infinite, planar membrane. Following the argument by Kusumaatmaja et al., 7 the bending energy is neglected for sufficiently large droplets. The membrane energy thus only contains surface tension terms.
To describe the shape of the droplet and the membrane, we use the spherical cap approximation. The energy difference, , between a droplet that wets and deforms the membrane and a spherical coacervate droplet adjacent to a planar membrane reads: with , and the surface and membrane tensions as depicted on Figure 4a in the main text. 0 (or coac ) denotes the radius of the spherical droplet. The radii , and the angles , are schematically depicted in Figure S16a, Eq. S8 is equivalently written as:  In the second step, we consider a finite liposome with an initially spherical shape with radius (or lipo ).
in Figure S16b shows schematically a droplet that wets and deforms a liposome with the radii , , and the angles , , . The radius of the contact line is denoted as . The geometry of the system implies the following relations:

Supplementary movie captions
Movie S1. This video is a Z-stack rotation along the Y axis, the screen shots are shown in Figure S5 g and h.
Movie S2. We show the zoom in part of the white dashed square area in Figure S5g video, which is extracted from Movie S1.
Movie S3. This video recorded the coacervate droplets spermine/polyU (13/1) that were not engulfed by POPC/DOTAP (20 wt%) liposomes were dissolved and coacervate droplets that were engulfed by liposomes Movie S6. This video was recorded from the same sample as Movie S5, but in a different position, and the size of the coacervates and liposomes is different from Movie S5. In this video, the replay speed is 50 times faster than the recorded experiment (a time stamp is shown at the bottom left, the total time is 22 min and 5 s).